Clinical and molecular characterization of a novel pathogenic AIFM1 E336K mutation connecting mitochondrial dysfunction and neurodegeneration

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In this work, we identified and comprehensively characterized a novel hemizygous AIFM1 mutation, c1006G > A (E336K), in male patients with a progressive childhood onset hereditary axonal sensorimotor polyneuropathy inherited in an X-linked recessive pattern, accompanied by sensorineural hearing loss but without cognitive impairment. Their clinical phenotype was consistent with Charcot-Marie-Tooth disease type 4 (CMTX4). Patient-derived fibroblasts exhibited reduced AIF protein stability despite preserved mRNA levels, impaired growth under OXPHOS-dependent conditions, decreased basal respiration, and altered assembly of mitochondrial respiratory supercomplexes. These defects were accompanied by reduced CHCHD4 abundance and decreased mitochondrial mass. Biochemical analyses of the purified E336K protein revealed compromised FAD retention, decreased thermal stability, impaired NADH affinity, destabilization of the charge-transfer complex required for AIF:CHCHD4 interaction, and a shift in coenzyme preference toward NADPH. Structurally, the substitution of Glu336 with Lys remodels the electrostatic environment of the NADH-binding cleft, thereby impairing redox function and weakening CHCHD4 binding. Despite these defects, the E336K mutation preserved DNA binding, nuclease activity, and binding to nuclear partners, although parthanatos induction was attenuated in patient fibroblasts. Collectively, these molecular alterations converge on disrupted mitochondrial bioenergetics and dynamics, providing a direct mechanistic link to the patient’s neurodegenerative course. These findings advance our understanding of AIFM1-related mitochondrial disorders and establish a framework for future precision molecular therapies. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The human apoptosis-inducing factor (hAIF), encoded by the AIFM1 gene on chromosome Xq25, is a mitochondrial flavoprotein with dual roles in energy metabolism and caspase-independent programmed cell death [ 44 ]. Under physiological conditions, AIF is anchored to the inner mitochondrial membrane, where it functions as a FAD-dependent NADH oxidoreductase essential for mitochondrial morphology, redox homeostasis, and the biogenesis and stability of oxidative phosphorylation system (OXPHOS) complexes, particularly complexes I and III [ 12 , 51 ]. Structurally, AIF comprises an N-terminal mitochondrial targeting sequence, a central oxidoreductase domain with FAD- and NADH-binding motifs, and a C-terminal domain required for NADH-dependent dimerization and apoptotic activity [ 19 ]. These modular domains allow AIF to act as an NADH/NAD + redox sensor, switching from a metabolic supporter to a death effector in response to cellular stress. NADH binding and its oxidation by the AIF’s flavin cofactor induce the formation of a stable isoalloxazine rd :NAD + charge-transfer complex (CTC) with FAD, triggering conformational rearrangements that displace the regulatory C‑terminal loop (residues 509–559), promoting dimerization, and facilitating interaction with CHCHD4, a central component of the mitochondrial disulfide relay system [ 6 , 19 , 21 , 35 ]. Through this long-lived interaction, reduced and dimeric AIF supports the import and folding of CHCHD4-dependent substrates, thereby supporting respiratory chain assembly, including complex I and complex IV subunits [ 9 , 25 , 39 ]. Disruption of this pathway leads to mitochondrial dysfunction, redox imbalance, and, in animal models, embryonic lethality [ 12 , 28 , 39 ]. Beyond its metabolic function, AIF mediates large-scale DNA fragmentation and chromatin condensation in response to severe genotoxic stress, in a caspase-independent process known as parthanatos [ 5 , 44 ]. This apoptogenic function requires the proteolytic cleavage and nuclear translocation of AIF, events favored by redox-sensitive conformational changes in the protein upon NADH depletion [ 32 , 44 ]. AIF, thus, lies at the intersection of mitochondrial bioenergetics and programmed cell death pathways. Pathogenic variants in AIFM1 were first reported in 2010, with the identification of a hemizygous deletion (ΔR201) in a male patient with severe mitochondrial encephalopathy [ 20 ]. Since then, an expanding phenotypic spectrum has been linked to AIFM1 mutations, X-linked Charcot-Marie-Tooth disease type 4 (CMTX4/COWCK) [ 37 , 48 ] (also referred to as Cowchock syndrome, after the author who first described a family with this phenotype in 1985) [ 10 ], combined oxidative phosphorylation deficiency 6 (COXPD6) [ 4 ], and auditory neuropathy spectrum disorder (DFNX5) [ 53 ] (Table 1 and S1). Most pathogenic variants cluster within the FAD- and NADH-binding domains pointing to a central role of redox balance impairment in disease pathogenesis. Nonetheless, most variants remain poorly characterized at the functional and molecular levels and the mechanistic basis underlying their pathogenicity are unknown. Table 1 Summary of previously reported pathogenic AIFM1 variants associated to Charcot-Marie-Tooth disease type 4 (CMTX4/COWCHOCK) Mutation Disease onset Progression Phenotypes Domain Protein level OXPHOS activity Redox activity Cell death Ref. COWCHOCK syndrome T141I 4.5y Slow Childhood-onset cerebellar ataxia with auditory neuropathy, axonal sensorimotor neuropathy and cardiomyopathy FAD-binding --- --- --- --- [ 22 ] M171I 5-11y Slow Childhood-onset slowly progressive axonal motor and sensory neuropathy, lower limb weakness and atrophy, steppage gait, pes cavus, absence of CNS symptoms or cognitive impairment. FAD-binding --- Abnormal mitochondrial morphology and accumulation in nerve and muscle suggest mitochondrial dysfunction ---- ---- [ 48 ] F210L 14y Slow Distal muscle atrophy and weakness (particularly ankle flexors), absent ankle reflexes, and mild sensory impairment. No involvement of central nervous system or other organs. FAD-binding Normal Misassemble of Complex I and Complex III (~ 80% reduction) ---- No effect; normal interaction with HSP70 (inhibitor of AIF nuclear translocation) [ 24 ] F210S 16-18m Slow, but severe progression Early-onset axonal polyneuropathy with exclusive motor fiber involvement. Sensory nerves were unaffected. Cognitive function and cranial nerves were normal. Severe distal weakness and muscle atrophy with mild to moderate proximal weakness. FAD-binding Reduced (normal mRNA expression) Mitochondrial fragmentation and reduced interconnectivity suggest mitochondrial dysfunction --- Not altered [ 41 ] G262S 1y Slow Mitochondrial encephalomyopathy characterized by ataxia (sensory and cerebellar), hearing loss, cognitive impairment, visual loss, distal muscle weakness, and neuropathy. FAD-binding Strong reduction; less stability Reduced Complex IV activity, partial reduction of other complexes Reduced NADH affinity, increased NADH oxidation, destabilized CTC ---- [ 4 , 43 ] L311V 10y Slow Severe axonal sensorimotor neuropathy, distal muscle atrophy and weakness, pes cavus, sensorineural hearing loss, cognitive decline, and scoliosis NADH-binding Predicted destabilization ---- ---- --- [ 49 ] G399S 2y Slow Cerebellar ataxia and atrophy and dysarthria, sensorineural hearing loss, intellectual disability, axonal peripheral neuropathy, and mood and behavioral disorders. NADH-binding Normal --- --- --- [ 34 ] Table 1 . Continued M340T 5 y Slow Childhood-onset cerebellar ataxia with auditory neuropathy, axonal sensorimotor neuropathy NADH-binding Reduced Reduced Complex I, II and IV. ΔΨ m decreased. --- Reduced, impaired [ 7 , 22 , 52 ] I433M 5y,13y Rapid, Slow Neurodegenerative syndrome with hearing loss, ataxia, neuropathy, and optic atrophy. FAD-binding --- --- --- --- [ 46 ] E493V Early age Slow Axonal motor and sensory neuropathy, bilateral sensorineural hearing loss, cognitive impairment, distal muscle wasting and weakness (lower > upper limbs) C-terminal Normal level but reduced stability No affected Increased NADH oxidation, destabilized CTC Enhanced [ 37 , 43 , 47 ] H457Y 3y, 4y Slow Complex progressive movement disorder. Features included cerebellar ataxia, disabling cerebellar tremor, sensorineural hearing loss, sensory polyneuropathy, language, fine motor delays, dystonia and optic nerve hypoplasia C-terminal --- --- --- --- [ 45 ] NA: not available; d: days; m: months; y, year; ---: not studied; ΔΨ m : mitochondrial membrane potential Structural analyses of selected pathogenic variants –including ΔR201, V243L, G308E, G338E, R422Q, R451Q– have revealed decreased flavin affinity, compromised protein stability, impaired CTC formation and defective OXPHOS activity (Table 1 and S1). Clinically, these mutations often present as early-onset, progressive neuromuscular syndromes, with features such as peripheral neuropathy, ataxia, cerebellar hypoplasia, optic atrophy, and sensorineural hearing loss (Table 1 and S1). Notably, several pathogenic variants (F210L/S, R422Q, V243L, G308E, G338E) retain apoptotic competence in vitro –preserving DNA-binding and nuclear translocation– while displaying markedly reduced redox activity, suggesting that mitochondrial dysfunction, rather than impaired apoptosis, is the principal driver of disease (Table 1 and S1). Here, we report the identification and functional characterization of a novel AIFM1 missense variant, E336K, in a male patient exhibiting a neurodegenerative phenotype consistent with Cowchock syndrome. This E336K mutation is located in the AIF NADH-binding domain, where residue E336 lies within a highly conserved structural region essential for NADH coordination and redox cycling [ 19 , 43 ]. We have comprehensively characterized this mutation from an integrated, multi-level perspective –spanning longitudinal clinical and neurological follow-up, molecular and cellular functional studies, and structural– biophysical analyses. By directly linking the patient’s clinical evolution to the underlying molecular defect, we elucidate the pathogenic mechanism of the E336K variant and establish a model for genotype–phenotype correlations in AIFM1-related disorders. This integrative approach deepens our understanding of AIF biology and surely will contribute to facilitate rational developments in future precision molecular therapies. Material and Methods Patients and clinical evaluations Clinical data were collected from a family originating from northern Spain (Aragón) spanning three generations with a phenotype consistent with CMTX4. Two brothers were clinically affected; their mother and the daughter of one affected patient were also carriers. Genetic testing results were available for all family members except for one clinically affected brother. This relative presented clinically with a polyneuropathy; however, his evaluations were not included, as he declined follow-up and further investigations. For one of the affected patients, complete clinical information was obtained, and longitudinal follow-up was carried out by the Neuromuscular Unit at Hospital Universitario Miguel Servet from June 2016 to June 2024. In May 2023, the patient was enrolled in the research project Flavoenzymes in Health, Diseases and Drug Discovery conducted by the University of Zaragoza (Hospital Miguel Servet, Department of Biochemistry, Molecular and Cell Biology, Faculty of Sciences, and the Institute for Biocomputation and Complex Systems Physics). As part of this study, blood and skin tissue samples were obtained, the latter through a skin biopsy. No muscle tissue was collected, as the patient declined to undergo muscle biopsy. From the electronic medical record, the following evaluations were retrieved: laboratory tests: Complete blood count (CBC), chemistry, coagulation, lactic acid, autoantibodies, vitamin E, creatine kinase (CK), long-chain fatty acids, thyroid hormone. Brain and acoustic nerve MRI, brainstem auditory evoked potentials (BAEPs), serial audiometries, echocardiogram and Holter monitoring, as well as multidisciplinary assessments by cardiology, and otorhinolaryngology services, in addition to annual follow-up by neurology. A neurophysiological study was performed to confirm the neuropathy phenotype using a Synergy 28 system. Motor and sensory nerve conduction studies were conducted in the median and ulnar nerves of the upper limbs. In the lower limbs, motor conduction studies were carried out in the peroneal and tibial nerves, and sensory studies in the superficial peroneal and sural nerves. The assessment was completed with an electromyographic evaluation, as shown in Table 2 . Table 2 Neurophysiological findings in patient holding the E336K AIF mutation Nerve/muscle Study type Findings Clinical Interpretation Nerve Sural R/L Sensory No response Severe sensory impairment Nerve Superficial peroneal R/L Sensory No response Severe sensory impairment Nerve Median sensory R Sensory Lat 3.45 ms | Amp 1.8 µV | Vel 46.4 m/s Moderate sensory neuropathy Nerve Ulnar sensory R Sensory Lat 2.55 ms | Amp 4.7 µV | Vel 47.1 m/s Moderate sensory neuropathy Nerve Median motor R Motor Lat 4.30–8.40 ms | Amp 7.8–8.2 mV | Vel 59.8 m/s Mild to moderate motor neuropathy Nerve Ulnar motor R Motor Lat 3.35–9.55 ms | Amp 11.0–7.6 mV | Vel 65.4 / 44.4 m/s Mild motor neuropathy Nerve Common peroneal R/L Motor No response Severe motor neuropathy Nerve Tibial R/L Motor No response Severe motor neuropathy Muscle Tibialis anterior R EMG Normal IA, no spontaneous activity, Mixed I recruitment Axonal damage, no denervation, altered recruitment Muscle Gastrocnemius med. R EMG Normal IA, no spontaneous activity, Mixed I recruitment Axonal damage, no denervation, altered recruitment Muscle Tibialis anterior L EMG Normal IA, no spontaneous activity, Mixed I recruitment Axonal damage, no denervation, altered recruitment Muscle Gastrocnemius med. L EMG Normal IA, no spontaneous activity, Mixed I recruitment Axonal damage, no denervation, altered recruitment Muscle Vastus medialis R EMG Normal IA, no spontaneous activity, Mixed II recruitment Axonal damage, no denervation, altered recruitment MuscleVastus medialis L EMG Normal IA, no spontaneous activity, Mixed II recruitment Axonal damage, no denervation, altered recruitment Muscle Biceps R EMG Normal IA, no spontaneous activity, Mixed III recruitment Axonal damage, no denervation, altered recruitment Muscle Abductor digiti minimi (ulnar) R EMG Normal IA, no spontaneous activity, Mixed III recruitment Axonal damage, no denervation, altered recruitment Summary of motor and sensory nerve conduction studies and electromyographic evaluation. The results demonstrate a severe, length-dependent, axonal sensorimotor polyneuropathy, consistent with Charcot-Marie-Tooth disease type X4 (CMTX4, Cowchock syndrome). EMG: electromyography; IA: insertion activity. Written informed consent was obtained from the patient for participation in the study, for the performance of the skin biopsy, and for publication of the results Genetic/Mutational analysis A comprehensive exome sequencing analysis was performed on the DNA sample extracted from peripheral blood, with the aim of identifying genomic variants in 53 genes associated with Charcot-Marie-Tooth disease and related hereditary sensory-motor neuropathies (NIMGenetics). The panel used for library preparation was designed using Ion AmpliSeq™ Exome technology (Life Technologies), capturing > 97% of CCDS (> 19,000 genes, > 198,000 exons, > 85% of alterations responsible for genetic diseases) and flanking splice regions (5 bp). It has a size of ~ 33 Mb and comprises a total of 293,903 amplicons. Library sequencing was performed on a next-generation high-throughput sequencer, the Ion Proton™ (Life Technologies). In the study conducted, a mean coverage depth of 79 was obtained. A total of 1177 amplicons covering the selected genes in this panel were analyzed. 96% of the amplicons included in the study had a read depth greater than 20X. The obtained sequences were aligned to the reference genome (build 37 of the human genome, Hg19) using the TMAP –Ion-Alignment software. The aligned and filtered sequences, according to specific quality criteria, were analyzed to identify nucleotide variations with respect to the reference genome (VariantCaller). Variant annotation was performed with ION Reporter (Life Technologies). The analysis focuses on identifying variants located in exonic regions or splice regions, which imply a change at the protein level (missense or nonsense mutations, and nucleotide insertions, deletions, or indels), and which are present in a frequency higher than 40% of the reads. The list of identified variants was evaluated using information from databases ( http://www.ncbi.nlm.nih.gov/SNP/ , http://www.1000genomes.org , http://evs.gs.washington.edu/EVS , http://https://www.ncbi.nlm.nih.gov/clinvar/ , https://varsome.com/ , https://franklin.genoox.com/clinical-db/home , https://databases.lovd.nl/shared/genes ). Furthermore, the functional effect of genomic variations classified as pathogenic was assessed using 9 prediction systems (SIFT, PROVEAN, PolyPhen2, MutationTaster, MutationAssessor, LRT, FATHMM, MetaSVM, and CONDEL) included in the ALAMUT analysis package ( http://www.interactive-bioware.com ) and the ANNOVAR package ( http://www.openbioinformatics.org/annovar/ ). Finally, the association of the identified mutations with OMIM syndromes was evaluated. Sanger sequencing of the genomic fragment chrX:129271006–129271226 was performed on the DNA familial samples (brother, Labgenetics; mother and daughter, NIMGenetics) to identify the AIFM1 variant c.1006G > A; p.Glu336Lys. Cell culture Control and mutant human fibroblasts were obtained from skin biopsies from a healthy donor and a patient with X-linked Charcot-Marie-Tooth disease carrying a single mutation, c.1006G > A (p. Glu336Lys), in the AIF gene, respectively. Skin biopsies were collected with informed consent in accordance with institutional ethical guidelines and under approved protocols. Biopsy samples were sterilized with ethanol, rinsed with sterile PBS and mechanically disaggregated into ~ 1 mm 2 pieces using forceps and a scalpel. The tissue fragment was then transferred to 15 mL corning tubes containing 5 mL of high-glucose DMEM supplemented with 20% fetal bovine serum (FBS, GIBCO), 1% penicillin-streptomycin (GIBCO), and 1 mL of collagenase I (4 mg/mL). Samples were incubated overnight at 37°C in a 5% CO 2 atmosphere. Following enzymatic digestion, cells and residual tissue were centrifuged at 1500 g for 10 minutes. The resulting pellet was washed with sterile PBS and centrifuged three times under the same conditions. Finally, the pellet was resuspended in high-glucose DMEM supplemented with 20% FBS and seeded in 60 mm culture dishes. Primary cultures were immortalized at passage 4 by transduction with the lentiviral plasmid pLOX-Ttag-iresTK (Tronolab). Once exponential growth was established, the FBS concentration in the culture medium was reduced to 10%. Growth measurements Growth rate in galactose-containing medium was determined by seeding 5*10 4 cells per well on 12-wells plates with 2 mL of either high-glucose DMEM supplemented with 10% FBS, or glucose-free DMEM supplemented with 0.9 mg /mL galactose,1 mM sodium pyruvate, and 10% FBS. Cells were incubated at 37°C for 5 days, and cell numbers were recorded every 24 hours. Cell Viability Assays The relative growth of different cell lines in response to oxidative phosphorylation (OXPHOS) inhibitors was evaluated using the MTT reduction assay, following the method described by Mosmann T.[ 30 ]. Briefly, 1 × 10⁴ cells per well were plated in 96-well flat-bottom plates and cultured in galactose-containing medium in the presence of two concentrations of rotenone (2 and 10 nM), antimycin A (2 and 10 nM), or sodium azide (20 and 100 µM) for 48 h at 37°C. After drug exposure, cells were incubated with fresh medium containing 1 mg/mL MTT for 4 h at 37°C in a humidified atmosphere. Formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm using a microplate reader. Results were expressed as percentages relative to untreated control cells. All experiments were performed at least in triplicate. Oxygen consumption measurements Endogenous and maximal O 2 consumption in intact cells was measured using an Oxytherm Clark-type electrode (Hansatech), as previously described [ 23 ], with minor modifications [ 1 ]. Evaluation of protein expression For preparation of total cell protein extracts, cells were harvested from 60 mm-diameter culture plates, washed twice with PBS, and lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 5 mM EDTA, 1% Triton X-100, 0.5% Sodium Deoxycholate, 50 mM NaCl) containing 1x cOmplete™ Protease Inhibitor Cocktail (Roche). Steady-state levels of proteins, including AIF, CHCHD4 or subunits of the mitochondrial respiratory chain complexes were estimated by loading 60 µg of total protein per lane onto SDS-PAGE gels. Proteins were separated on 12% acrylamide/bisacrylamide gels and electroblotted onto PVDF membranes. Western blotting was performed using specific primary antibodies against human AIF (SIGMA), CHCHD4 (Proteintech), β-actin (SIGMA), as well as antibodies targeting complex I (anti-NDUFA9, Invitrogen), complex II (anti-70 kDa subunit, SDHA, Invitrogen), complex III (anti-Core1, Invitrogen) and complex IV (anti-COXI, Invitrogen). Detection was carried out using HRP-conjugated secondary antibodies (anti-mouse or anti-rabbit, Invitrogen) and the Pierce™ ECL Western Blotting Substrate (Thermo Scientific). Native polyacrylamide electrophoresis analysis of respiratory supercomplexes Mitochondria were isolated from cultured cell lines according to Schägger [ 42 ], with minor modifications [ 2 ]. Digitonin-solubilized mitochondrial proteins (100 µg) were separated by native PAGE. The assembly of respiratory supercomplexes was analysed by BN-PAGE using commercial native 3–12% acrylamide gradient gels (Novex). Mitochondrial Superoxide production and mitochondrial mass analysis To assess mitochondrial ROS production and mitochondrial mass, cells were stained with either MitoSOX™ red (5 µM, Invitrogen) or MitoTracker™ Green FM (200 nM, Invitrogen) for 30 min at 37 ºC. Mean fluorescence intensity was measured by flow cytometry using a FACSCalibur cytometer (BD Biosciences), and data were analyzed using FlowJo Software, version 10.8.1. Quantitative PCR determination of mRNA transcripts Total RNA was isolated from 5*10 6 cells using TRIzol™ reagent (Invitrogen), and 1 µg of RNA was reverse transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche). Transcript levels of human AIF, SOD2 and CHCHD4 were quantified by qPCR using a LightCycler System (Roche) with the LightCycler Fast-Start DNA MasterPLUS SYBR Green I Kit (Roche), following the manufacturer’s recommendations. Transcript levels were normalized to Actb mRNA (NM_007393). Primer sequences are provided in Table S4. AIF induced cell death analysis AIF-mediated cell death was analyzed by simultaneous annexin-V and 7AAD staining and flow cytometry, after cell death induction with MNNG as previously described [ 5 ]. Briefly, control and mutant cells were grown to a 70% confluence, and exposed to MNNG (0.5 µM) for 20 min. After treatment, the medium was replaced, and cells were incubated for 24 h at 37°C in a humidified atmosphere with 5% CO 2 . Cells were then collected, stained with annexin-V-FITC and 7-AAD (Sigma) for 10 min in annexin binding buffer (140 mM NaCl, 2.5 mM CaCl 2 , 10 mM HEPES/NaOH, pH 7.4), and analyzed FACSCalibur flow cytometer (BD Biosciences, Madrid, Spain). Similar to previous sections, collected data were further processed by using FlowJo Software, version 10.8.1. Recombinant protein expression and purification Constructs for WT, AIF ∆77 and AIF Δ101 and overexpression protocols were previously reported [ 33 , 38 , 47 ]. The genes encoding the human AIF mutation E336K Δ77 and E336K Δ101 were obtained by site-directed mutagenesis from GenScript® and subsequently subcloned between the NcoI and NdeI sites of the pET-28a(+) expression vector. WT AIF as well as both the apoptotic E336K ∆101 and soluble mitochondrial E336K ∆77 variants, along with CHCHD4 (UniProtKB Q8N4Q1) were heterologously expressed as recombinant proteins carrying an N-terminal removable His 6 -tag (CACCAT) using the pET28a(+) expression vector. Expression was performed in Escherichia coli C41 (DE3) strain, except for CHCHD4, which was expressed in Shuffle T7 cells to enhance disulfide bond formation. The production of E336K Δ77 was performed by growing transformed cells in 2xYT medium containing 30 mg/L kanamycin (Sigma-Aldrich), supplemented with 8 mg/L of riboflavin (Sigma-Aldrich) at 37°C and 180 r.p.m. until OD 600nm ~ 0.5. Then, cultures were induced with 0.5 mM IPTG (Glentham life sciences) and incubated 24h in the same conditions. For AIF ∆101 , cells were grown as previously described [ 38 ]. After the cells were harvested, both E336K ∆77 and E336K Δ101 variants were purified as previously reported for the apoptotic WT ∆101 form [ 38 ]. CHCHD4 production and purification was performed as formerly described [ 16 ]. WT AIF concentrations were determined using the molar absorptivity coefficient previously reported [ 19 ]. For E336K Δ101 this value was estimated by protein denaturation by boiling for 5 minutes at 100 ºC, followed by quantification of the released FAD after a 3 minutes centrifugation. The extinction coefficients, ε 452 nm , for WT Δ101 and E336K Δ101 variants were 13.7, and 14,6 M − 1 ·cm − 1, respectively. CHCHD4 concentrations were calculated through its theoretical ε 280 nm value (13.3 mM − 1 ·cm − 1 ) obtained from the ProtParam tool (ExPASy). All proteins were stored in 50 mM potassium phosphate, pH 7.4 at -80 ºC. Molecular weight determination by size exclusion chromatography The AIF Δ101 variants, in the absence or presence of 2 mM of NADH and/or CHCHD4, were loaded onto a Superdex 200 increase 10/300 GL (Cytiva) column attached to an ÄKTA go system (Cytiva). AIF Δ101 :CHCHD4 mixtures (1:3 molar ratio regards to AIF) were preincubated in 50 mM phosphate buffer pH 7.4, without or with NADH, for 10 min at RT before loading onto the column. Protein elution was carried out in 50 mM phosphate buffer, 150 mM NaCl, pH 7.4, at a flow rate of 0.4 mL/min. Column calibration was performed with the gel filtration calibration kit (Cytiva) containing 6 proteins in the 13.7–440 kDa range. Chromatograms were analyzed using a set of Gaussian functions. Spectroscopic characterization UV-Visible spectra were recorded in CARY 3500 (Agilent). Circular dichroism (CD) spectra were acquired in a thermostated Chirascan (Applied Photophysics Ltd.) at 25°C in 50 mM potassium phosphate buffer, pH 7.4 (150 mM ionic strength) in the absence and presence of a 100-fold molar excess of NADH. Near-UV/Vis CD spectra were collected using 20 µM AIF Δ101 in a 1 cm-pathlength cuvette, while Far-UV CD spectra were acquired using 5 µM AIF Δ101 in a 0.1 cm-pathlength cuvette. Fluorescence spectra were acquired in a thermostated Cary Eclipse Fluorescence spectrophotometer (Agilent) using 2µM AIF Δ101 in a 1cm-pathlength cuvette. Flavin fluorescence emission spectra were acquired in the 480-600nm range upon excitation at 450 nm. Emission spectra of aromatic residues were collected from 300 to 600nm upon excitation at 280 nm. Thermal denaturation assays Thermal denaturation curves were monitored by FAD fluorescence emission, near-UV/Vis CD and far UV-CD. Curves were monitored from 15°C to 90°C with scan rates of 1 ºC/min and 1.5 ºC/min respectively for fluorescence and CD assays, both in presence or absence of a 100-fold excess of NADH. For flavin fluorescence, measures were carried out with 2 µM AIF Δ101 in a 1 cm-pathlength cuvette with excitation at 450 nm. For far-UV and near-UV/Vis, curves were monitored with 5 µM AIF Δ101 in a 0.1 cm-pathlength cuvette or 20 µM AIF Δ101 in a 1 cm-pathlength cuvette, respectively. Experimental data were roughly normalized to values between 0 and 1 and globally fitted to a two-step (native (N) ↔ unfolded (U)) or three-step unfolding model (native (N) ↔ intermediate (I) ↔ unfolded (U)) using the following equations [ 40 ]: \(\:{S}_{obs}=\:\frac{{S}_{N}+\:{m}_{N}T+\left({S}_{U}+{m}_{U}T\right){e}^{-\left(\varDelta\:G/RT\right)}}{1+{e}^{-\left(\varDelta\:G/RT\right)}}\) Eq. 1 \(\:{S}_{obs}=\:\frac{{S}_{N}+\:{m}_{N}T+\left({S}_{I}+{m}_{I}T\right){e}^{-\left(\varDelta\:{G}_{1}/RT\right)}+\left({S}_{U}+{m}_{U}T\right){e}^{-\left((\varDelta\:{G}_{1}+\varDelta\:{G}_{2})/RT\right)}}{1+{e}^{-\left(\varDelta\:{G}_{1}/RT\right)}+{e}^{-\left((\varDelta\:{G}_{1}+\varDelta\:{G}_{2})/RT\right)}}\) Eq. 2 in which S obs is the measured protein signal at a given temperature (T), S x (S N , S I and S U ) represents the y-intercept of native, intermediate and unfolded protein at 0K and m x (m N , m I and m U ) is the slope of the native, intermediate and unfolded states, respectively. The Stabilization Gibbs energy depends on temperature according to \(\:\varDelta\:G=\varDelta\:H\left(1-\frac{1}{{T}_{m}}\right)+{\varDelta\:C}_{P}\left(T-{T}_{m}-T\text{ln}\frac{T}{{T}_{m}}\right)\) , where ΔH is the unfolding enthalpy, T m is the midtransition temperature, ΔC P is the unfolding heat capacity change, and R is the ideal gas constant. Kinetics measurements The steady-state diaphorase activity of AIF ∆101 was measured in a Cary 100 spectrophotometer (Agilent). The measurements were carried out in air saturated 50 mM potassium phosphate buffer, pH 7.4 at 25°C, using NADH or NADPH as the substrate donor and 95 µM dichlorophenolindophenol (DCPIP, \(\:\varDelta\:{\epsilon\:}_{620nm}\) = 21 mM −1 cm −1 ) as acceptor [ 19 ]. Initial reaction rates at different NADH concentrations were fitted to the Michaelis-Menten equation to determine the kinetic constants: \(\:\frac{\nu\:}{\text{e}}=\frac{{k}_{\text{c}\text{a}\text{t}}\:\left[NAD\right(P\left)H\right]}{{K}_{m}^{NADH}+\left[NAD\right(P\left)H\right]}\) Eq. 3 \(\:\frac{\nu\:}{\text{e}}=\frac{{k}_{\text{c}\text{a}\text{t}}/{K}_{m}^{NAD\left(P\right)H}\left[NAD\right(P\left)H\right]}{1+\frac{{k}_{\text{c}\text{a}\text{t}}/{K}_{m}^{NAD\left(P\right)H}\left[NAD\right(P\left)H\right]}{{k}_{\text{c}\text{a}\text{t}}}}\) Eq. 4 In which v represents the initial velocity, e is the enzyme concentration, \(\:{K}_{m}^{NAD\left(P\right)H}\) stands for the Michaelis constant for the NAD(P)H, k cat is the turnover number of the enzyme and \(\:{k}_{\text{c}\text{a}\text{t}}/{K}_{m}^{NAD\left(P\right)H}\:\) is the enzyme catalytic efficiency. The reactivity of the CTC towards molecular oxygen was examined by full reduction of AIF ∆101 with NADH (1:1.5 ratio relative to AIF ∆101 ), in both presence or absence of CHCHD4 (1:3 ratio relative to AIF) in 50 mM potassium phosphate buffer, pH 7.4 and 25 ºC as previously reported[ 38 ]. Reoxidation was monitored using a Cary 100 spectrophotometer (Agilent). A SX18.MV stopped-flow spectrophotometer (Applied Photophysics Ltd.) was used to investigate the reductive half-reaction of AIF ∆101 variants upon mixing with increasing concentrations of NADH (0.03-10 mM) in both absence and presence of CHCHD4 (1:3 ratio regards to AIF). Measurements were collected using PDA and monochromator detectors in air-saturated 50 mM potassium phosphate buffer, pH 7.4, at 25°C. Observed rate constants ( k obs ) for HT were determined by global fitting of the spectra or exponential fitting of single-wavelength traces, assuming one-step process using Pro-K and ProData-XD software. The averaged k obs at each NADH concentration were then non-linearly fitted to the equation that describes the formation of an enzyme:substrate complex prior to the HT event: \(\:{k}_{\text{o}\text{b}\text{s}}=\frac{{k}_{\text{H}\text{T}}NADH}{{K}_{\text{d}}^{NADH}+NADH}+{k}_{\text{r}\text{e}\text{v}}\) Eq. 4 In which k HT and k rev represent the HT constant and its reverse reaction, respectively and \(\:{K}_{\text{d}}^{NADH}\) stands for the dissociation constant of the transient AIF ∆101 :NADH complex. Isothermal titration calorimetry (ITC) ITC assays were performed using an Auto-iTC200 (Malvern) thermostated at 25 25 ºC for CypA, 15ºC for dsDNA or 10ºC for CHCHD4. Typically, 100 µM CHCHD4, CypA or dsDNA were used to titrate ~ 10 µM AIF ∆101 . Solutions were degassed at 15°C for 3 min before each assay. A sequence of 2 µL injections of titrant solution every 150 s was programmed, and the stirring speed was set to 750 rpm. The association constant ( K a ), the enthalpy of binding (ΔH), and the binding stoichiometry (N) were estimated through non-linear least-squares regression of the experimental data using a single ligand binding site model implemented in Origin 7.0 (OriginLab). The dissociation constant ( K d ), the free energy change (Δ G ), and the entropy change (Δ S ) were calculated from basic thermodynamic relationships. Electrophoretic-mobility-shift-assays (EMSAs) DNA retardation assays were carried out as formerly described [ 33 ]. Briefly, 500 ng of GeneRuler 100 bp dsDNA ladder (Thermo Scientific) were incubated with 6 µg of AIF ∆101 for 30 min at 25°C in 50 mM potassium phosphate buffer, pH 7.4 and subsequently mixed with 6x DNA loading dye (Thermo Fisher Scientific. The samples were resolved by electrophoresis in 1% agarose gel stained with SYBR™ Safe. The electrophoresis was carried out for 1h at 90V. Nuclease activity assays Assays were performed as previously described [ 33 ]. Briefly, 250 ng of a double-stranded supercoiled pcDNA3.1 plasmid were incubated at 30 ºC with 250 ng of purified AIF ∆101 for 1 or 5 minutes in 20 mM Tris, pH 8.0 supplemented with 0.1 mM CaCl 2 and 1 mM MgCl 2 . The samples were subsequently mixed with 6x DNA loading dye (Thermo Fisher Scientific), heated for 10 minutes at 65°C, and loaded onto a 0.7% agarose gel with SYBR™ Safe. The electrophoresis was carried out for 1 h at 90 V. Crystallization and structure determination of the E336K ∆101 variant Crystallization was carried out using hanging-drop vapor diffusion at 20°C, mixing E336K ∆101ox protein solution (10 mg/ml) with reservoir solution (18% PEG 6K, 0.1 M Tris-HCl pH 8.5, 0.2 M Li₂SO₄) [ 19 ]. Crystals were cryoprotected with 20% glycerol and flash-cooled in liquid nitrogen. X-ray diffraction data were collected at 100 K on the BL13-XALOC beamline (ALBA Synchrotron) using a Pilatus3 X 6M detector. Data were processed with XDS [ 27 ] and SCALA [ 15 ] from the CCP4 package [ 50 ], and the structure was solved by molecular replacement using Phaser [ 29 ], with AIF ∆101ox (PDB 4BV6) as the search model. Model building and refinement used Coot [ 14 ], phenix.refine [ 3 ], and REFMAC [ 31 ], yielding a final model containing residues 127–610, one FAD molecule, one glycerol, and 239 water molecules. Residues 546–558 and the last three C-terminal residues were unresolved. Data collection and refinement statistics are provided in Table S5. Data and statistical analysis Data were analyzed and shown using StatView 5.0 (SAA Institute, Mesa, AZ, USA), SigmaPlot (Systat. Software Inc.), Origin 7.0 (OriginLab) and Pro-K (Applied Photophysics Ltd.) and PyMol [ 11 ]. Results were displayed as mean ± SD and statistically analyzed by Fisher’s PLSD post hoc test from ANOVA. In all cases, differences were considered statistically significant at p ≤ 0.05. Results Clinical findings Patients and Clinical Evaluation We report the case of a 64-year-old male from Aragón, Spain, with a progressive sensorimotor polyneuropathy. Genetic testing confirmed a diagnosis of X-linked hereditary sensorimotor polyneuropathy (CMTX4/COWCK), associated with a hemizygous mutation c.1006G > A (p. Glu336Lys) in the AIFM1 gene (Xq26.1). Family history revealed that his mother and daughter are carriers of the same mutation, and his brother is also affected. Natural History and Clinical Progression The patient was born in 1961. Since childhood, he exhibited hypotonia, generalized areflexia, and pes cavus, for which he underwent orthopedic surgery at the age of 8. He also developed progressive bilateral sensorineural hearing loss, requiring hearing aids by age 25. By age 41, hearing loss reached 90% bilaterally. A right cochlear implant (Nucleus 7) was placed at age 57 with partial benefit. He underwent cataract surgery at age 38. Motor disability followed a slowly progressive course. At the age of 32, he discontinued playing tennis due to fatigue and weakness in the lower limbs. At 38, he required unilateral walking support. He was evaluated at the neuromuscular unit of Hospital Universitario Miguel Servet at the age of 41 (in 2003). Severe gait ataxia was documented. Neurological exam showed visual acuity of 0.8 bilaterally, global weakness graded 4/5 in upper limbs and pelvic girdle, and 1–2/5 in foot flexors/extensors, with marked distal amyotrophy producing a "stork leg" appearance and post-surgical clubfoot. He had global areflexia, preserved thermal sensitivity, and reduced tactile and pain sensation in a glove-and-stocking pattern. Joint position and vibration senses were abolished in lower limbs and decreased distally in upper limbs. Mild finger-to-nose ataxia was present; lower limb ataxia was severe and disabling. No truncal ataxia. Cardiovascular exam was normal. Associated symptoms included oscillopsia and nystagmus diagnosed by ophthalmology. Metabolic testing was normal. Brain Magnetic Resonance Imaging (MRI) and cardiological evaluation were unremarkable. A heterozygous GAA repeat expansion (250 repeats) in FRDA/X25 led to a provisional diagnosis of Friedreich's ataxia. As compound heterozygosity was suspected, further studies were initiated. By age 45 (2007), he needed a wheelchair for distances > 50 meters. Manual dexterity declined significantly, affecting eating and typing. He required assistance with all ADLs. Arm strength was preserved (5/5), though clumsy fine motor control was noted. Pansensory hypoesthesia in lower limbs and reduced sensation in arms. In 2009, FRDA repeat expansion testing was repeated, excluding Friedreich’s ataxia. GDAP1 testing (2010) was negative for pathogenic variants. At age 55 (2016), genetic revaluation revealed a hemizygous c.1006G > A (p. Glu336Lys) mutation in exon 10 of AIFM1 (NM_004208.4) (Fig. 1 A). This change results in the substitution of glutamic acid with lysine at position 336 of the protein (p.Glu336Lys), generating a missense mutation. This variant has not been reported in population databases (gnomAD v4.1.0, exomeAD). The results of bioinformatics analysis systems for predicting the effect of mutations indicate, in 7 out of 9 prediction tools used (PROVEAN, SIFT, PolyPhen2, LRT, MutationTaster, MutationAssessor, and CONDEL), that this is a deleterious variant. Currently, this variant is reported in the ClinVar database (ID: 641733) as a likely pathogenic variant (SCV000934480.7) and as a variant of uncertain significance (SCV004036880.1, SCV002765018.3). Using ACMG criteria, this variant is classified as likely pathogenic (PP3, PP5, PM1, PM2)[ 36 ].This new mutation confirmed Cowchock syndrome, being the clinical status of the patient stable but severely disabled. Patient was wheelchair-bound, with severe impairments in fine motor skills, worsened by deafness and oscillopsia. Occasional choking episodes with aspiration and self-limited vestibular symptoms were reported. He developed muscle contractures that were refractory to all muscle relaxants and showed a fluctuating course. Repeated brain MRI and vestibulocochlear nerve imaging were unremarkable. Visual evoked potentials revealed a delayed P100 latency at 153 ms, while brainstem auditory evoked potentials were bilaterally absent. Identification of the AIFM1 mutation prompted In 2017, the patient continued to exhibit profound motor and sensory impairment, with complete dependence in activities of daily living (ADLs) except for feeding. Communication was limited to lip-reading. Riboflavin therapy was maintained. By 2019, the cochlear implant was functioning effectively. The patient remained physically stable, ambulating short distances with bilateral support and driving long distances. Manual dexterity was slower but preserved. Muscle strength was graded 5/5 in the upper limbs and approximately 4+/5 in the hands. Distal tactile, vibratory, and proprioceptive hypoesthesia persisted, without evidence of clinical progression. He reported mood instability characterized by depressive and irritable episodes, as well as sleep disturbances. Riboflavin was reduced to 50 mg twice daily due to gastrointestinal intolerance. Family genetic studies confirmed that his mother, and daughter were carriers of the AIFM1 variant. His brother was also clinically affected, with a phenotype consistent with polyneuropathy. Annual follow-ups from 2021 to 2024 documented persistent chronic shoulder pain interfering with sleep, together with a slow progression of motor and sensory deficits. Manual skills gradually declined, with frequent dropping of objects and difficulty fastening buttons. At home, he ambulated with a cane and demonstrated brachial strength of 4 + distally and 5/5 proximally. Distal sensory loss remained evident, with absent deep tendon reflexes (0/4). Cognitive function was preserved, and mood remained stable. A graphical timeline showing the progression of the patient’s disability is presented in (Fig. 1 B). The following complementary studies were carried out to aid in the phenotypic characterization of the patient: i) Neurophysiological evaluation (EMG/NCS) demonstrated a severe, length-dependent, axonal sensorimotor polyneuropathy without signs of active denervation. ii) Sensory nerve conduction studies revealed absent responses in the sural and superficial peroneal nerves bilaterally, and reduced amplitudes with delayed latencies in the median and ulnar nerves, consistent with moderate sensory neuropathy. iii) Motor conduction studies showed absent responses in the common peroneal and tibial nerves bilaterally, and reduced amplitudes with mild to moderate slowing in the median and ulnar nerves. iv) Electromyographic examination revealed mixed recruitment patterns without spontaneous activity. The detailed findings of the study are summarized in (Tables 2 and 3 ). These results are consistent with a severe axonal sensorimotor neuropathy, supporting the clinical diagnosis of Charcot-Marie-Tooth disease type X4 (CMTX4, Cowchock syndrome), subsequently confirmed by genetic testing AIFM1 mutation. Table 3 Audiometric findings before and after cochlear implantation in patient holding the E336K AIF mutation Parameter Pre-implant Post-implant (Right Cochlear Implant) Hearing thresholds Severe-to-profound bilateral sensorineural hearing loss (≈ 90% by age 41) Clear improvement in free-field thresholds Hearing aids Required since age 25, with limited benefit Not required, replaced by CI Sentence comprehension Severely impaired ≈ 90% sentence comprehension with implant Speech recognition Minimal, lip-reading required Significantly improved with combination of lip-reading + implant Left ear outcome Profound hearing loss, no functional benefit Persistent profound hearing loss, no functional benefit Overall functional outcome Severe bilateral anacusis Partial functional recovery with right CI Summary of the most relevant parameters, including hearing thresholds, speech comprehension, and functional outcomes. Marked improvement was observed after right cochlear implantation, with partial recovery of auditory performance despite persistent profound hearing loss in the left ear. CI: cochlear implant. Impact of the E336K mutation on OXPHOS performance To investigate the impact of the E336K mutation on AIF cellular functions, immortalized fibroblast lines were established from skin biopsies of the affected individual and a healthy control. To analyze whether the mutation alters transcriptional regulation or protein stability, AIF expression was assessed at both the mRNA and protein levels using quantitative RT-PCR and Western blot analysis, respectively. As shown in Fig. 2 A and 2 B, although the relative expression levels of AIF mRNA in mutant fibroblasts were comparable to those of control cells, the amount of AIF protein was significantly reduced, suggesting decreased stability of the mutant protein. Given AIF’s role in mitochondrial biogenesis and function, we next analyzed the impact of the mutation on OXPHOS performance. Mutant fibroblasts exhibited significantly impaired growth when cultured in galactose medium compared to glucose (Fig. 2 C), indicating defective OXPHOS capacity. This impairment was confirmed by direct measurements of oxygen consumption in intact cells, which revealed an average ~ 50% reduction in basal respiration compared to controls (Fig. 2 D). Results of cell growth sensitivity to different inhibitors of respiratory complexes shown in Fig. 2 E suggest that this reduction might be mainly due to defects in mitochondrial complexes I and III function as mutant cells display significantly more sensitivity than control cells to rotenone and antimycin A, specific inhibitors of these complexes. To further investigate this, the structural organization of mitochondrial respiratory complexes and supercomplexes (SCs) was analyzed by native electrophoresis followed by Western Blot (Fig. 2 F), where mutant cells displayed reduced incorporation of complex III into supercomplexes (SCs), accompanied by an increase in both its free dimeric form (CIII₂) and in CIII–CIV assemblies. This is consistent with defective respirasome formation or stability, possibly linked to reduced levels of CI. To better understand the molecular basis for mitochondrial dysfunction and disease associated with the E336K mutation in AIF, we assessed the expression of CHCHD4 -the physiological mitochondrial inner membrane partner of AIF [ 21 , 35 ]- both at the mRNA and protein levels. The mutation of AIF negatively affects to CHCHD4 both mRNA and protein expression, as its relative levels are significantly reduced compared to those of control cells (Fig. 3 A and 3 B). In line with these observations, mitochondrial mass levels, evaluated both by MitoTracker™ Green staining followed by flow cytometry and mtDNA copy number quantification (Fig. 3 C) were significantly reduced in E336K cells. To further investigate the effects of this impairment on the biogenesis of the OXPHOS system, the steady-state levels of mitochondrial respiratory complexes subunits were quantified on whole cell extract, showing a significant decrease of the relative levels of complex III and IV subunits in mutant cells when compared to those of control fibroblasts (Fig. 3 D). Thus, the levels of UQCRC1(CIII) normalized by actin, were reduced by approximately 50%, while the CO1 (CIV) signal decreased to ~ 20% of that observed in control cells. Although not statistically significant, the relative levels of CI subunits were also reduced, whereas SDHA, a CII subunit, remained unchanged. Finally, we investigated whether these alterations affect mitochondrial ROS production, which could potentially affect the disease phenotype. As illustrated in Fig. 4 A, the production of mitochondrial superoxide, quantified by flow cytometry following cell staining with MitoSOX, was significantly lower in mutant cells compared to controls. Although this observation seems in contradiction with the mitochondrial dysfunction, this difference was abolished when ROS levels were normalized to mitochondrial mass (Fig. 4 B). Moreover, mitochondrial superoxide dismutase (SOD2) mRNA expression was significantly decreased in mutant cells (Fig. S1 A), indicating that the reduced ROS signal is not due to compensatory upregulation of mitochondrial antioxidant defenses. In summary, the E336K mutation reduces AIF protein stability, impairs OXPHOS capacity through defective assembly of respiratory supercomplexes, decreases CHCHD4 expression, and lowers mitochondrial mass, altogether leading to compromised mitochondrial function. The E336K mutation alters redox properties, conformational dynamics, and CHCHD4 interaction in AIF To better understand the molecular basis of the pathogenicity associated with the E336K substitution here identified, we further characterized the recombinant mutant protein using the synthetic constructs E336K ∆77 and E336K ∆101, which serve as a realistic model of, respectively, the soluble mitochondrial and apoptotic mutant isoforms studied in patient cells [ 19 , 47 ]. Both mutant isoforms were purified to homogeneity, but with markedly different outcomes. E336K ∆101 exhibited a UV-visible absorption spectrum comparable to that of the WT protein, displaying the characteristic flavin bands I and II at 451 and 380 nm, respectively, along with a shoulder at 467 nm (Fig. S2A). These features indicate that the flavin cofactor remained in the oxidized state and was properly incorporated into the protein. In contrast, protein form E336K ∆77 progressively lost the flavin cofactor during purification and handling, ultimately showing a strong tendency to precipitate. Due to the low purification yield and the reduced ability of the mitochondrial E336K ∆77 isoform to retain the FAD cofactor, subsequent analyses focused on the apoptotic E336K ∆101 variant. This mutant oxidized form, E336K ∆101ox , displayed an apparent molecular weight (appMW) of ~ 52 kDa, consistent with a monomeric state as determined by size exclusion chromatography (Fig. 5 A and 5 C). Additionally, its CD spectral properties were comparable to those of WT, indicating that the mutation does not affect overall protein folding (Fig. S2B). In contrast, the mutant’s NADH reduced form, E336K ∆101rd , showed significant alteration in its spectroscopic properties, including attenuation of the fluorescence quantum yield of at least one tryptophan and changes in the shape of the far-CD signals (Fig. S2 B-D). Moreover, upon NADH incubation, the mutant protein eluted as three peaks with appMW of ~ 150, ~128 and ~ 65 kDa (Fig. 5 B and 5 D), corresponding to a dimeric form, a dimer-monomer transition due to partial reoxidation, and a monomeric form, respectively [ 19 ]. These results indicate that the E336K ∆101 mutant retains its ability to form dimers under reducing conditions, although the dimers exhibit diminished stability and a slightly less compact dimeric assembly compared to the WT protein. To explore the effects of the E336K mutation on AIF conformational stability, we determined the thermal stability of this mutant protein in the presence and absence of NADH (Table S2, Fig. S2E-F). The results indicate that the mutation negatively impacts the thermal stability of AIF ∆101ox (Fig. S2E, Table S2), as evidenced by a decrease of ~ 5–6 degrees in both T m s associated with the unfolding process when compared with WT ∆101ox ,, while the unfolding mechanism remains unchanged. However, when evaluating the thermal stability of E336K in the presence of NADH, the coenzyme does not destabilize the mutant, in stark contrast to the WT behavior, being the two identified T m s close to those of E336K ox and of NADH reduced WT ∆101 . Altogether, these findings suggest structural and dynamic differences between the WT and the E336K mutant, both in the absence of the coenzyme as well as upon its binding and flavin reduction. In view of these observations, we also investigated the impact of this mutation on AIF redox properties using different biochemical approaches. E336K ∆101 exhibited a strong decrease in coenzyme affinity regarding WT ∆101 , with a K m NADH value ~ 6-fold higher than that of WT ∆101 (Table 4 and Fig. S3A). In contrast, the turnover number ( k cat ) remained similar, resulting in a ~ 3-fold reduction in catalytic efficiency relative to WT ∆101 . Notably, the mutant displayed both a ~ 2-fold higher turnover number and a ~ 23-fold greater affinity when compared with WT ∆101 for NADPH compared to NADH (Table 4 and Fig. S3B). Consequently, the E336K ∆101 variant was substantially more efficient at oxidizing NADPH than the WT protein, with an estimated ~ 1000-fold increase in the K m NADPH / k cat , thereby markedly reducing the specificity for NADH over NADPH compared to the WT ∆101 . Table 4 Steady-state kinetic parameters of AIF∆101 variants with NADH and NADPH as hydride donors Variants NADH NADPH Specificity \(\:{\varvec{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}\) (s − 1 ) \(\:{\varvec{K}}_{\varvec{m}}^{\varvec{N}\varvec{A}\varvec{D}\varvec{H}}\) (mM) \(\:{\varvec{k}}_{\varvec{c}\varvec{a}\varvec{t}}/{\varvec{K}}_{\varvec{m}}^{\varvec{N}\varvec{A}\varvec{D}\varvec{H}}\) (s − 1 mM − 1 ) \(\:{\varvec{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}\) (s − 1 ) \(\:{\varvec{K}}_{\varvec{m}}^{\varvec{N}\varvec{A}\varvec{D}\varvec{P}\varvec{H}}\) (mM) \(\:{\varvec{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}/{\varvec{K}}_{\mathbf{m}}^{\varvec{N}\varvec{A}\varvec{D}\varvec{P}\varvec{H}}\) (s − 1 mM − 1 ) NADH/NADPH efficiency WT ∆101 2.0 ± 0.1 0.5 ± 0.1 4.0 ± 1 0.02 ± 0.01 0.5 ± 0.1 0.04 ± 0.02 75 E336K ∆101 2.5 ± 0.1 2.8 ± 0.5 0.9 ± 0.2 4.9 ± 0.1 0.12 ± 0.01 40 ± 4 0.0225 Assays were performed using DCPIP as hydride acceptor at 25 ºC in 50 mM potassium phosphate, pH 7.4. (n = 3, mean ± SD); Table 5 Pre-steady state kinetic parameters of AIF∆101 variants with NADH as hydride donor Variants Pre-steady-sate CTC \(\:{\varvec{k}}_{\mathbf{H}\mathbf{T}}\) (s − 1 ) \(\:{\varvec{K}}_{\varvec{d}}^{\varvec{N}\varvec{A}\varvec{D}\varvec{H}}\) (mM) \(\:{\varvec{k}}_{\mathbf{H}\mathbf{T}}/{\varvec{K}}_{\mathbf{d}}^{\varvec{N}\varvec{A}\varvec{D}\varvec{H}}\) (s − 1 mM − 1 ) Half-life (min) WT ∆101 1.1 ± 0.1 4.3 ± 0.1 0.30 + 0.03 20 E336K ∆101 2.9 ± 0.1 13.4 ± 0.1 0.20 ± 0.01 NS Assays were performed at 25 ºC in 50 mM potassium phosphate, pH 7.4. (n = 3, mean ± SD); NS: not stabilized under assayed conditions. To assess the mutation impact on hydride transfer (HT) from the NADH coenzyme to the FAD cofactor, we performed stopped-flow transient analysis (Table 4 and Fig. S3C). In E336K ∆101 , as in WT ∆101 , the complete FAD reduction was accompanied by a progressive formation of isoalloxazine:NAD + CTC, as in WT ∆101 and with similar spectral intensity, suggesting a comparable extent of CTC stabilization (Fig. 5 E-F). However, unlike the native protein –where the CTC had a half-life time of 20 minutes– E336K ∆101 did not reach full reduction at stoichiometric molar coenzyme concentrations. This incomplete reduction impeded CTC stabilization and precluded assessment of its reactivity towards O 2 . Furthermore, the mutant exhibited faster HT (~ 2-fold increase) and lower coenzyme affinity (~ 2-fold decrease), while maintaining similar HT efficiency (Table 4 ). These results demonstrate that the E336K mutation significantly compromises the redox properties of AIF by decreasing NADH binding affinity and destabilizing the CTC. Considering previous findings and the relevance of the AIF dimer stabilization in forming a long-lived complex with CHCHD4 [ 9 , 21 , 39 ], we investigated the impact of the mutation on this interaction using ITC (Fig. 5 G and Table S3). As previously reported for WT ∆101ox [ 38 ], no heat exchange was detected when titrating E336K ∆101ox with CHCHD4, indicating a lack of specific binding under assayed conditions. In the presence of NADH, the E336K ∆101rd mutant was able to bind CHCHD4, but with slightly lower affinity than WT ∆101rd ( K d ~ 5-fold lower than for WT ∆101rd :CHCHD4 complex) and with notable differences in thermodynamic contributions to the binding. The WT ∆101rd :CHCHD4 interaction was mainly driven by a strong favorable enthalpic contribution, typical of specific binding, while the entropic term was unfavorable. In contrast, the E336K∆ 101rd :CHCHD4 complex showed a weaker binding enthalpic contribution and even a favorable entropic term, suggesting that non-specific forces may play a more prominent role. This altered thermodynamic profile could impair the formation of a functional E336K ∆101rd :CHCHD4 complex, which is crucial for mitochondrial homeostasis [ 9 , 21 ]. Impact of E336K mutation on AIF structure To investigate the structural impact of the E336K mutation, the X-ray crystal structure of E336K ox was resolved and evaluated in the context of structures for monomeric WT Δ101ox and dimeric WT Δ101rd :NAD + [ 19 ] (Fig. 6 A). Superposition of WT Δ101ox and E336K ox revealed a high degree of structural similarity (r.m.s.d. = 0.21 Å for 451 atoms), with both structures sharing similar overall fold. Comparison with WT Δ101rd : NAD + (r.m.s.d. = 1.06 Å for 400 atoms) showed that the conformational rearrangements of the β-hairpin and regulatory C-loop associated with coenzyme binding were absent in E336K ox . This suggests that the E336K substitution, in agreement with the monomeric conformation observed by size exclusion chromatography, does not promote permissive dimer formation as reported for other AIF variants [ 8 ]. In WT Δ101ox and WT Δ101rd :NAD + structures, the side chain of E336 adopts a similar orientation (Fig. 6 B). Notably, in the WT Δ101rd :NAD + structure, the E336 carboxylate oxygen atoms (O2B and O3B) hydrogen bond to the hydroxyl groups of the ribityl of the adenine nucleotide moiety of NAD + (sitting at 2.8 and 3.5 Å respectively) [ 19 ]. Substitution of E336 with lysine may disrupt such interaction, as the positively charged and longer lysine side chain might preclude an orientation conducive to coenzyme hydrogen bonding (Fig. 6 B). Furthermore, as shown in Fig. 6 C, the mutation modifies the electrostatic landscape of the environment of residue 336, particularly in the cleft where the ribose of the adenine nucleotide moiety of the coenzyme binds. This surface becomes more positive and potentially eager to accommodate the 2’-phosphorylated ribose form of the coenzyme, NADPH. This is exemplified in Fig. 6 D, where based on the WT Δ101rd :NAD + structure a model for the E336K Δ101rd :NADP + interaction is presented, envisaging the interaction between the 2′-P of NADPH and residues K336 and K342. Such differences in surface charge distribution between the WT and the mutant may correlate with observed stability and enzymatic activity alterations, and are particularly in line with E336K preference for using NADPH as hydride donor (Table 4 ). Impact of the E336K mutation on AIF-mediated cell death and nuclear interactions As mentioned, besides its role in mitochondrial function and biogenesis, AIF also participates in a caspase-independent cell death pathway known as parthanatos [ 44 ]. To analyze whether the E3336K variant alters this function, control and mutant fibroblasts were exposed to the alkylating agent MNNG for 20 minutes. After 24 hours, cell death was quantified by flow cytometry using annexin V-FITC and 7-AAD staining. As shown in Fig. 7 A, both control and mutant cells underwent cell death; however, the percentage of dead cells was significantly lower in cultures expressing the E336K mutant (Fig. 7 B). Consistently, MTT assays revealed greater growth inhibition in mutant cells compared to controls, with viabilities of 22% and 45.9%, respectively. These results indicate that although the E336K mutation does not abolish AIF-mediated cell death, its efficiency in fibroblasts appears reduced (Fig. S1 B). Upon apoptotic-stimuli, AIF translocates from the mitochondria to the nucleus, where it is proposed to form a DNA-degradosome complex –through interactions with proteins such as the endonuclease CypA– essential for the caspase-independent apoptotic pathway[ 5 , 33 ]. To assess the impact of the E336K mutation on AIF’ s interactions with its nuclear partners, we performed ITC using dsDNA and CypA as ligands (Fig. 7 C, Fig. S4C-F and Table S3). Both WT and E336K Δ101 proteins exhibited similar binding affinities toward dsDNA and CypA, with K d in the micromolar range for both interactions. The thermodynamic profiles of the interactions with dsDNA were also comparable between variants and predominantly entropy-driven, consistent with non-specific electrostatic interactions previously described for WT AIF. EMSA further confirmed similar DNA-binding behavior, with both variants exhibiting comparable levels of DNA retention in the gels (Fig. 7 D). These results suggest that the E336K mutation does not significantly affect AIF's ability to bind DNA. Notably, some differences were observed in the interaction with CypA. WT Δ101 exhibited a strong enthalpy-driven interaction with a modest positive entropy change, indicative of a specific well-defined binding interface [ 17 , 18 , 33 ]. In contrast, the pathological variant showed a slightly weaker enthalpic contribution (Fig. 7 C and Table S3), potentially reflecting minor conformational rearrangements upon complex formation. Given the recently reported intrinsic nuclease activity of AIF [ 33 ], we investigated the impact of the E336K mutation on AIF-mediated DNA degradation. The E336K mutation does not suppress the intrinsic DNA-cleaving capability of AIF when using purified plasmid DNA as substrate., exhibiting an activity comparable to that of the WT protein (Fig. 7 E). In summary, these findings demonstrate that the E336K mutation does not abolish AIF’s capacity to trigger cell death or to interact with nuclear partners such as DNA and CypA, although it may modestly reduce the efficiency of parthanatos induction and subtly affect the thermodynamics of CypA binding. Discussion This study provides the first integrated clinical, cellular and molecular mechanistic characterization of the novel pathogenic AIFM1 variant E336K. This combined approach significantly broadens the mechanistic and phenotypic spectrum of AIF-related disease. Our data indicate that the pathological impact of E336K is primarily driven by loss of AIF’s pro-survival, mitochondria-supporting, function rather than by impairment of its pro-death role [ 6 , 44 , 51 ]. Clinically, the patient presented with progressive axonal sensorimotor neuropathy and sensorineural hearing loss, while cognition remained preserved –a phenotype consistent with CMTX4/Cowchock syndrome and well within the established spectrum of AIFM1–related disorders. These findings underscore both the diagnostic challenges associated with AIFM1 mutations and the importance of variant-specific functional analyses to refine genotype–phenotype correlations [ 6 , 43 , 51 ]. At the cellular level, patient-derived fibroblasts displayed reduced AIF protein, impaired OXPHOS capacity, and defective organization of respiratory supercomplexes. Notably, CHCHD4 expression and mitochondrial mass were decreased, coherently linking the cellular phenotype to the AIF–CHCHD4 axis, which sustains the import, oxidative folding, and assembly of specific respiratory components [ 9 , 16 , 21 , 35 , 39 ]. Crystallographic analysis provides a structural rationale: substitution of Glu336 with Lys remodels the electrostatic potential of the coenzyme binding pocket, precisely at the site of the adenosine-ribose moiety of NADH –a key region for stabilizing the charge transfer complex (CTC) and mediating redox-driven conformational changes in AIF [ 19 , 43 ]. These defects converge on a primary disruption of AIF’s pro-survival role in mitochondrial maintenance and respiratory chain biogenesis, offering a unifying structural explanation for the patient’s biochemical phenotype [ 8 , 19 , 43 , 47 ]. Collectively, these alterations decrease AIF’s ability to sense mitochondrial NADH redox state and to sustain the conformational ensembles that underwrite its pro-survival functions [ 8 , 47 ]. Of particular relevance are the results of the systematic analysis of AIF’s interaction network with physiological partners. Mechanistically, the reduced dimeric form of AIF interacts with CHCHD4 to support mitochondrial disulfide relay and respiratory chain assembly [ 9 , 21 , 39 ]. While E336K retained the ability to dimerize, the resulting dimers were less stable and less compact. Moreover, its interaction with CHCHD4 displayed markedly reduced affinity and altered thermodynamic signatures, indicating a weakened and less specific interacting interface. These molecular changes provide a direct link to the observed cellular phenotypes –decreased CHCHD4 levels, reduced mitochondrial mass, and disorganized supercomplexes– and offer a cohesive explanation for the OXPHOS insufficiency observed in patient-derived fibroblasts. In contrast, the apoptogenic functions of AIF were largely preserved: DNA binding, CypA interaction, and nuclease activity remained intact, while parthanatos induction was only attenuated, possibly due to the observed impact of mutation on mutant stability. Interestingly, a closely located pathogenic variant (M340T) has been associated with a similar clinic-biological dissociation –dominant mitochondrial dysfunction with preserved apoptotic competence– further reinforcing this mechanistic theme within the AIFM1 spectrum [ 52 ]. From a translational perspective, these findings highlight the need to integrate structure-function studies into the interpretation of AIFM1 variants, particularly in X-linked neuropathies with hearing loss. Therapeutic strategies based on cofactor supplementation may hold promise, especially in cases with impaired FAD retention and stability, as observed in this study. The shift toward NADPH utilization further suggests a possible compensatory route that could be therapeutically leveraged. More broadly, targeting NADH-driven allosteric regulation, stabilizing the CTC, or enhancing the CHCHD4 pathway robustness represent concrete, testable avenues for future intervention. In summary, our work provides the first comprehensive clinical-to-molecular characterization of an AIFM1 mutation. The E336K mutation exemplifies a recurring principle in AIFM1-related disease: variants that disrupt the NADH-sensing/redox cycle primarily erode mitochondrial-supporting functions while leaving apoptotic roles relatively intact. By reshaping the coenzyme-binding electrostatics, destabilizing the NADH-dependent CTC, and weakening CHCHD4 engagement, E336K triggers a pathogenic cascade that explains the selective vulnerability of high-energy tissues such as peripheral nerves, the inner ear, and muscle. This work expands the clinical and mechanistic spectrum of AIFM1 disease, refines the interpretation of AIFM1 variants, and provides a foundation for exploring mechanism-based therapeutic strategies, including cofactor supplementation and targeted interventions aimed at stabilizing AIF-CHCHD4 function. Declarations Ethics approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki. Ethical approval C.P.-C.I. PI18/224 was obtained from Comité de Ética de la Investigación de la Comunidad Autónoma de Aragón (CEICA), Zaragoza, Spain. Written informed consent for participation in this study was obtained from the patient. Consent for publication All authors have approved the manuscript. Competing interests The authors declare no competing interests Supplementary information The only version contains supplementary material available: Supplementary Figure S1 to Figure S4 and supplementary Table S1 to Table S5. Funding This work was funded by the Spanish State Research Agency and by FEDER (MCIN/AEI-FEDER, Grants PID2022-136369NB-I00, Grant PID2021-124354NB-I00), as well as by the Gobierno de Aragón, grant number “Grupo de Referencia E35_17R” to M.M-M., M.M, P.F.-S., R.M.-L. and P.F. and grant number “LMP220_21” to P.F.-S. and R.M.-L. The authors would like to acknowledge Servicios Generales de Apoyo a la Investigación (SAI), University of Zaragoza, for their support, as well as at BIFI-University of Zaragoza for providing instrumentation. Author Contribution Conceptualization: P.F and R.M-L; Data curation: P.F., R.M.-L., M.M.J., A.V.-C., P.F.-S., M.M and M.B.; Funding acquisition: P.F., R.M.-L, M.M. and P.F.-S.; Investigation: R.M.-L, M.P, R. G-V. and P. F-S (cell culture, respiration and viability analysis), J. M.-B. and C. R-Y (Cytometer measurements), M.F., O.S., M.M.-B. and P.F (protein production and molecular characterization); M.F and M.M.-J (crystallization and structure determination) M.D.M. (genetic analysis) and M.B. (clinical studies). Writing original draft: P.F., R.M.-L., M.F., O.S, M.M-J. and M.B. Review & editing: all authors. All authors have given approval to final version of the manuscript. Data Availability The structural data for the AIF E336K variant generated in this study by X-ray crystallography have been deposited in the Protein Data Bank under accession code PDB 9SZQ. References Acin-Perez R, Bayona-Bafaluy MP, Bueno M, Machicado C, Fernandez-Silva P, Perez-Martos A, Montoya J, Lopez-Perez MJ, Sancho J, Enriquez JA. An intragenic suppressor in the cytochrome c oxidase I gene of mouse mitochondrial DNA. Hum Mol Genet. 2003;12:329–39. 10.1093/hmg/ddg021 . Acin-Perez R, Fernandez-Silva P, Peleato ML, Perez-Martos A, Enriquez JA. Respiratory active mitochondrial supercomplexes. Mol Cell. 2008;32:529–39. 10.1016/j.molcel.2008.10.021 . Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A, Zwart PH, Adams PD. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr. 2012;68:352–67. 10.1107/S0907444912001308 . Ardissone A, Piscosquito G, Legati A, Langella T, Lamantea E, Garavaglia B, Salsano E, Farina L, Moroni I, Pareyson D et al. (2015) A slowly progressive mitochondrial encephalomyopathy widens the spectrum of AIFM1 disorders. Neurology 84: 2193–2195 10.1212/WNL.0000000000001613 Artus C, Boujrad H, Bouharrour A, Brunelle MN, Hoos S, Yuste VJ, Lenormand P, Rousselle JC, Namane A, England P al. AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX. EMBO J. 2010;29:1585–99. 10.1038/emboj.2010.43 . Bano D, Prehn JHM. Apoptosis-Inducing Factor (AIF) in Physiology and Disease: The Tale of a Repented Natural Born Killer. EBioMedicine. 2018;30:29–37. 10.1016/j.ebiom.2018.03.016 . Bogdanova-Mihaylova P, Alexander MD, Murphy RP, Chen H, Healy DG, Walsh RA, Murphy SM. Clinical spectrum of AIFM1-associated disease in an Irish family, from mild neuropathy to severe cerebellar ataxia with colour blindness. J Peripher Nerv Syst. 2019;24:348–53. 10.1111/jns.12348 . Brosey CA, Ho C, Long WZ, Singh S, Burnett K, Hura GL, Nix JC, Bowman GR, Ellenberger T, Tainer JA. (2016) Defining NADH-Driven Allostery Regulating Apoptosis-Inducing Factor. Structure: 10.1016/j.str.2016.09.012 Brosey CA, Shen R, Tainer JA. (2025) NADH-bound AIF activates the mitochondrial CHCHD4/MIA40 chaperone by a substrate-mimicry mechanism. EMBO J. © 2025. The Author(s). City. Cowchock FS, Duckett SW, Streletz LJ, Graziani LJ, Jackson LG. X-linked motor-sensory neuropathy type-II with deafness and mental retardation: a new disorder. Am J Med Genet. 1985;20:307–15. 10.1002/ajmg.1320200214 . Delano WL. PyMOL: an open-source molecular graphics tool. CCP4 Newsl Protein Crystallogr. 2002;40:82–92. Delavallée L, Mathiah N, Cabon L, Mazeraud A, Brunelle-Navas MN, Lerner LK, Tannoury M, Prola A, Moreno-Loshuertos R. Baritaud M (2020) Mitochondrial AIF loss causes metabolic reprogramming, caspase-independent cell death blockade, embryonic lethality, and perinatal hydrocephalus. Mol Metab: 101027 Doi 10.1016/j.molmet.2020.101027 Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA. PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 2004;32:W665–667. 10.1093/nar/gkh381 . Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66:486–501. 10.1107/S0907444910007493 . Evans P. Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr. 2006;62:72–82. 10.1107/S0907444905036693 . Fagnani E, Cocomazzi P, Pellegrino S, Tedeschi G, Scalvini FG, Cossu F, Da Vela S, Aliverti A, Mastrangelo E, Milani M. CHCHD4 binding affects the active site of apoptosis inducing factor (AIF): Structural determinants for allosteric regulation. Structure. © 2024 The Author(s). City: Published by Elsevier Inc; 2024. pp. 594–602. e594. Farina B, Di Sorbo G, Chambery A, Caporale A, Leoni G, Russo R, Mascanzoni F, Raimondo D, Fattorusso R, Ruvo M et al. (2017) Structural and biochemical insights of CypA and AIF interaction. Sci Rep 7: 1138 10.1038/s41598-017-01337-8 Farina B, Sturlese M, Mascanzoni F, Caporale A, Monti A, Di Sorbo G, Fattorusso R, Ruvo M, Doti N. Binding mode of AIF(370–394) peptide to CypA: insights from NMR, label-free and molecular docking studies. Biochem J. 2018;475:2377–93. 10.1042/BCJ20180177 . Ferreira P, Villanueva R, Martinez-Julvez M, Herguedas B, Marcuello C, Fernandez-Silva P, Cabon L, Hermoso JA, Lostao A, Susin SA al. Structural insights into the coenzyme mediated monomer-dimer transition of the pro-apoptotic apoptosis inducing factor. Biochemistry. 2014;53:4204–15. 10.1021/bi500343r . Ghezzi D, Sevrioukova I, Invernizzi F, Lamperti C, Mora M, D'Adamo P, Novara F, Zuffardi O, Uziel G, Zeviani M. Severe X-linked mitochondrial encephalomyopathy associated with a mutation in apoptosis-inducing factor. Am J Hum Genet. 2010;86:639–49. 10.1016/j.ajhg.2010.03.002 . Hangen E, Feraud O, Lachkar S, Mou H, Doti N, Fimia GM, Lam NV, Zhu C, Godin I, Muller Ket al, et al. Interaction between AIF and CHCHD4 Regulates Respiratory Chain Biogenesis. Mol Cell. 2015;58:1001–14. 10.1016/j.molcel.2015.04.020 . Heimer G, Eyal E, Zhu X, Ruzzo EK, Marek-Yagel D, Sagiv D, Anikster Y, Reznik-Wolf H, Pras E, Oz Levi Det al, et al. Mutations in AIFM1 cause an X-linked childhood cerebellar ataxia partially responsive to riboflavin. Eur J Paediatr Neurol. 2018;22:93–101. 10.1016/j.ejpn.2017.09.004 . Hofhaus G, Shakeley RM, Attardi G. Use of polarography to detect respiration defects in cell cultures. Methods Enzymol. 1996;264:476–83. 10.1016/s0076-6879(96)64043-9 . Hu B, Wang M, Castoro R, Simmons M, Dortch R, Yawn R, Li J. A novel missense mutation in AIFM1 results in axonal polyneuropathy and misassembly of OXPHOS complexes. Eur J Neurol. 2017;24:1499–506. 10.1111/ene.13452 . Joza N, Susin SA, Daugas E, Stanford WL, Cho SK, Li CY, Sasaki T, Elia AJ, Cheng HY, Ravagnan Let al, et al. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature. 2001;410:549–54. 10.1038/35069004 . Jurrus E, Engel D, Star K, Monson K, Brandi J, Felberg LE, Brookes DH, Wilson L, Chen J, Liles K et al. (2018) Improvements to the APBS biomolecular solvation software suite. Protein Sci 27: 112–128 10.1002/pro.3280 Kabsch W. Xds. Acta Crystallogr D Biol Crystallogr. 2010;66:125–32. Doi 10.1107/S0907444909047337. Klein JA, Longo-Guess CM, Rossmann MP, Seburn KL, Hurd RE, Frankel WN, Bronson RT, Ackerman SL. The harlequin mouse mutation downregulates apoptosis-inducing factor. Nature. 2002;419:367–74. 10.1038/nature01034 . McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr. 2007;40:658–74. 10.1107/S0021889807021206 . Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. City: J Immunol Methods; 1983. pp. 55–63. Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997;53:240–55. 10.1107/S0907444996012255 . Mussulini BHM, Maruszczak KK, Draczkowski P, Borrero-Landazabal MA, Ayyamperumal S, Wnorowski A, Wasilewski M, Chacinska A. (2025) MIA40 suppresses cell death induced by apoptosis-inducing factor 1. EMBO Rep. © 2025. The Author(s). City. Novo N, Romero-Tamayo S, Marcuello C, Boneta S, Blasco-Machin I, Velazquez-Campoy A, Villanueva R, Moreno-Loshuertos R, Lostao A. Medina M (2023) Beyond a platform protein for the degradosome assembly: The Apoptosis-Inducing Factor as an efficient nuclease involved in chromatinolysis. PNAS Nexus 2: pgac312 10.1093/pnasnexus/pgac312 Pandolfo M, Rai M, Remiche G, Desmyter L, Vandernoot I. Cerebellar ataxia, neuropathy, hearing loss, and intellectual disability due to AIFM1 mutation. Neurol Genet. 2020;6:e420. 10.1212/NXG.0000000000000420 . Reinhardt C, Arena G, Nedara K, Edwards R, Brenner C, Tokatlidis K, Modjtahedi N. AIF meets the CHCHD4/Mia40-dependent mitochondrial import pathway. Biochim Biophys Acta Mol Basis Dis. 2020;1866:165746. 10.1016/j.bbadis.2020.165746 . Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405–24. 10.1038/gim.2015.30 . Rinaldi C, Grunseich C, Sevrioukova IF, Schindler A, Horkayne-Szakaly I, Lamperti C, Landoure G, Kennerson ML, Burnett BG, Bonnemann Cet al, et al. Cowchock syndrome is associated with a mutation in apoptosis-inducing factor. Am J Hum Genet. 2012;91:1095–102. 10.1016/j.ajhg.2012.10.008 . Romero-Tamayo S, Laplaza R, Velazquez-Campoy A, Villanueva R, Medina M, Ferreira P. (2021) W196 and the β-Hairpin Motif Modulate the Redox Switch of Conformation and the Biomolecular Interaction Network of the Apoptosis-Inducing Factor. Oxid Med Cell Longev 2021: 6673661 10.1155/2021/6673661 Salscheider SL, Gerlich S, Cabrera-Orefice A, Peker E, Rothemann RA, Murschall LM, Finger Y, Szczepanowska K, Ahmadi ZA, Guerrero-Castillo Set al, et al. AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5. EMBO J. 2022;41:e110784. Sancho J (2013) The stability of 2-state, 3-state and more-state proteins from simple spectroscopic techniques… plus the structure of the equilibrium intermediates at the same time. Arch Biochem Biophys 531: 4–13 Doi 10.1016/j.abb.2012.10.014. Sancho P, Sanchez-Monteagudo A, Collado A, Marco-Marin C, Dominguez-Gonzalez C, Camacho A, Knecht E, Espinos C, Lupo V. A newly distal hereditary motor neuropathy caused by a rare AIFM1 mutation. Neurogenetics. 2017;18:245–50. 10.1007/s10048-017-0524-6 . Schagger H. Native electrophoresis for isolation of mitochondrial oxidative phosphorylation protein complexes. Methods Enzymol. 1995;260:190–202. 10.1016/0076-6879(95)60137-6 . Sevrioukova IF. Structure/Function Relations in AIFM1 Variants Associated with Neurodegenerative Disorders. J Mol Biol. 2016;428:3650–65. 10.1016/j.jmb.2016.05.004 . Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Costantini P, Loeffler M et al. (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397: 441–446 10.1038/17135 Tunyi J, Abreu NJ, Tripathi R, Mathew MT, Mears A, Agrawal P, Thakur V, Rezai AR, Reyes EL. (2023) Deep Brain Stimulation for the Management of AIFM1-Related Disabling Tremor: A Case Series. Pediatr Neurol. © 2023 Elsevier Inc, City, pp 47–50. Vasquez A, Schimmenti LA, Demirel N, Rabatin AE, Fischer CR, Pinto MV, Boesch RP, Selcen D. Novel AIFM1 Variant in 2 Siblings With Sensorineural Hearing Loss and Cerebellar Ataxia. Neurol Genet. 2024;10:e200216. 10.1212/NXG.0000000000200216 . Villanueva R, Romero-Tamayo S, Laplaza R, Martinez-Olivan J, Velazquez-Campoy A, Sancho J, Ferreira P, Medina M. Redox- and Ligand Binding-Dependent Conformational Ensembles in the Human Apoptosis-Inducing Factor Regulate Its Pro-Life and Cell Death Functions. Antioxid Redox Signal. 2019;30:2013–29. 10.1089/ars.2018.7658 . Wang B, Li X, Wang J, Liu L, Xie Y, Huang S, Pakhrin PS, Jin Q, Zhu C, Tang B et al. (2018) A novel AIFM1 mutation in a Chinese family with X-linked Charcot-Marie-Tooth disease type 4. Neuromuscul Disord. © 2018 Elsevier B.V, City, pp 652–659. Wang C, Lin Z, Yuan Z, Tang T, Fan L, Liu Y, Wu X. Whole-exome sequencing detected a novel AIFM1 variant in a Han-Chinese family with Cowchock syndrome. Hereditas. 2023;160:22. 10.1186/s41065-023-00282-z . Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011;67:235–42. 10.1107/S0907444910045749 . Wischhof L, Scifo E, Ehninger D, Bano D. AIFM1 beyond cell death: An overview of this OXPHOS-inducing factor in mitochondrial diseases. EBioMedicine. 2022;83:104231. 10.1016/j.ebiom.2022.104231 . Zhao Y, Lin Y, Wang B, Liu F, Zhao D, Wang W, Ren H, Wang J, Xu Z, Yan C. al (2023) A Missense Variant in AIFM1 Caused Mitochondrial Dysfunction and Intolerance to Riboflavin Deficiency. Neuromolecular Med. © 2023. The Author(s), under exclusive licence to Springer Science + Business Media, LLC, part of Springer Nature., City, pp 489–500. Zong L, Guan J, Ealy M, Zhang Q, Wang D, Wang H, Zhao Y, Shen Z, Campbell CA, Wang F al. Mutations in apoptosis-inducing factor cause X-linked recessive auditory neuropathy spectrum disorder. J Med Genet. 2015;52:523–31. 10.1136/jmedgenet-2014-102961 . Additional Declarations No competing interests reported. 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10:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8316196/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8316196/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12964-026-02862-8","type":"published","date":"2026-04-09T15:57:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":102909922,"identity":"4409f785-ff9d-40d7-903d-bc37e83820e1","added_by":"auto","created_at":"2026-02-18 09:57:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":57133,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic confirmation and clinical course of the CMTX4 case. \u003cstrong\u003e(A)\u003c/strong\u003e Chromatogram showing the m.1006G\u0026gt;A mutation within the AIFM1 gene in the patient. (\u003cstrong\u003eB\u003c/strong\u003e) Timeline showing the disability progression in patient with CMTX4. Each symptom is plotted according to the time of appearance, illustrating the chronological sequence and clinical evolution over the course of the illness.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8316196/v1/03dd102eabeeccb36d9c3678.jpg"},{"id":102909927,"identity":"605d73d7-136a-4224-8ca1-bbcbf28a5251","added_by":"auto","created_at":"2026-02-18 09:57:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90006,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the AIF E336K mutation on OXPHOS performance in immortalized fibroblasts. Evaluation of relative expression of AIF at mRNA (\u003cstrong\u003eA\u003c/strong\u003e) and protein (\u003cstrong\u003eB\u003c/strong\u003e) levels. (\u003cstrong\u003eC) \u003c/strong\u003eDoubling time ratio for WT and E336K immortalized fibroblasts in galactose versus glucose containing media. (\u003cstrong\u003eD) \u003c/strong\u003eOxygen consumption rate in intact cells for WT and E336K fibroblasts. (\u003cstrong\u003eE)\u003c/strong\u003e Differential effect of OXPHOS inhibitors on WT \u003cem\u003evs\u003c/em\u003e E336K cells viability as measured by the MTT reduction method. (\u003cstrong\u003eF)\u003c/strong\u003e Immunodetection of assembled supercomplexes in digitonin-permeabilized mitochondria from WT and E336K fibroblasts separated by BNGE and probed with specific antibodies for CI (anti-NDUFB6), CIII (anti-Core1), CIV (anti-Co1) and CII (anti-SDHA). Data are expressed as mean±SD of the mean (n≥3 in all cases). Asterisks indicate p\u0026gt;0.05.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8316196/v1/8df4496a840f3f3015fda470.jpg"},{"id":102909921,"identity":"f744753d-d114-40c4-93be-be3c23b058c5","added_by":"auto","created_at":"2026-02-18 09:57:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":84190,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of the AIF E333K mutation in mitochondrial biogenesis. Measurement of relative expression of CHCHD4 at mRNA (\u003cstrong\u003eA\u003c/strong\u003e) and protein (\u003cstrong\u003eB\u003c/strong\u003e) levels in WT and E336K fibroblasts. (\u003cstrong\u003eC\u003c/strong\u003e) Evaluation of mitochondrial mass by flow cytometry (left and central panels) and of mtDNA copy number by qPCR (right panel) in WT and E336K cells. (\u003cstrong\u003eD) \u003c/strong\u003eRelative amount of steady-state mitochondrial complexes subunits measured by western blot. Cell protein extracts were separated by SDS-PAGE and probed with specific antibodies for actin, CI (anti-NDUFS3), CIII (anti-Core1), CIV (anti-Co1) and CII (anti-SDHA). The amount of each protein was calculated relative to actin (right panel).\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8316196/v1/ffe28cf285331a295af5f250.jpg"},{"id":102909942,"identity":"9d497e45-6226-471d-b42d-02de25a4bd61","added_by":"auto","created_at":"2026-02-18 09:57:22","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":26823,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the AIF E336K mutation on mitochondrial ROS production. (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eEvaluation of mitochondrial superoxide production by flow cytometry after MitoSOX\u003csup\u003eTM\u003c/sup\u003e staining. (\u003cstrong\u003eB\u003c/strong\u003e) Mitochondrial ROS production normalized by mitochondrial mass in WT and E336K immortalized fibroblasts.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8316196/v1/a32b768bd4115d19b3e4a4c4.jpg"},{"id":102909851,"identity":"27ac2764-288d-42ec-b9f0-64d158f8073e","added_by":"auto","created_at":"2026-02-18 09:57:05","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":146849,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of E336K mutation on AIF molecular and redox properties. Influence on the stability of AIF complexes with CHCHD4. Elution profiles of WT\u003csub\u003eΔ101\u003c/sub\u003e (\u003cstrong\u003eA-B\u003c/strong\u003e) and E336K\u003csub\u003eΔ101\u003c/sub\u003e (\u003cstrong\u003eC-D\u003c/strong\u003e) variants in the absence and presence of a 3-fold molar excess of CHCHD4 and 2 mM NADH (A, C without NADH; and B, D with NADH). Control elution profiles of free WT\u003csub\u003eΔ101\u003c/sub\u003e, E336K\u003csub\u003eΔ101\u003c/sub\u003e, and CHCHD4 are shown as continuous blue, light blue and dark cyan lines, respectively. Elution profiles of AIF\u003csub\u003eΔ101\u003c/sub\u003e:CHCHD4 mixtures are depicted as continuous dark grey lines. Gaussian analyses identify distinct populations corresponding to free AIF\u003csub\u003eΔ101 \u003c/sub\u003evariants and CHCHD4 (colored dashed lines), and to AIF\u003csub\u003eΔ101\u003c/sub\u003e:CHCHD4 complexes (dark grey dashed lines). (\u003cstrong\u003eE-F\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSpectral evolution of flavin reduction by NADH for WT\u003csub\u003eΔ101\u003c/sub\u003e and E336K\u003csub\u003eΔ101\u003c/sub\u003e. Insets displayed simulated spectral species from global fitting to a single step (A→B) kinetic model. Initial spectra before NADH addition are shown in dark blue and light blue respectively for WT\u003csub\u003eΔ101 \u003c/sub\u003eand E336K\u003csub\u003eΔ101\u003c/sub\u003e. (\u003cstrong\u003eG\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eThermodynamic dissection of the interaction of NADH reduced WT\u003csub\u003eΔ101\u003c/sub\u003e and E336K\u003csub\u003eΔ101 \u003c/sub\u003eAIF variants with CHCHD4, as assessed by ITC. Gibbs free energy (Δ\u003cem\u003eG\u003c/em\u003e), enthalpy (Δ\u003cem\u003eH\u003c/em\u003e) and entropy contributions (-TΔ\u003cem\u003eS\u003c/em\u003e) are shown in blue, green and red bars, respectively. For ITC AIF\u003csub\u003eΔ101\u003c/sub\u003e:CHCHD4 binding assays, reduced forms of AIF\u003csub\u003eΔ101\u003c/sub\u003e variants were obtained by premixing the oxidized proteins and NADH at a 1:100 ratio.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8316196/v1/4385de9028363e3ec4f18460.jpg"},{"id":102909880,"identity":"bc7819ce-f6ea-4860-be7c-519204fc77cd","added_by":"auto","created_at":"2026-02-18 09:57:09","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":87898,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of the E336K mutation on the structural properties of AIF. (\u003cstrong\u003eA\u003c/strong\u003e) Overall structure of E336K AIF\u003csub\u003eΔ101ox\u003c/sub\u003e (PDB ID: 9SZQ), colored according to domain type: NADH binding (yellow), FAD binding (raspberry red) and C-terminal (blue). The side chain of K336 is depicted as cyan C atom balls and the FAD cofactor as orange sticks.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eStructural comparison of the point mutation site in E336K\u003csub\u003eΔ101ox \u003c/sub\u003e(colors as in A, PDB ID: 9SZQ), WT\u003csub\u003eΔ101ox \u003c/sub\u003e(white, PDB 4BV6) and WT\u003csub\u003eΔ101rd\u003c/sub\u003e:NAD\u003csup\u003e+\u003c/sup\u003e (grey, PDB ID: 4BUR, chain A). In the WT\u003csub\u003eΔ101rd\u003c/sub\u003e:NAD\u003csup\u003e+ \u003c/sup\u003ecomplex, the NAD\u003csup\u003e+ \u003c/sup\u003ecoenzyme is shown as sticks with pink carbons. The side chain of residue 336 is shown as sticks, cyan when mutated, and white and grey respectively for WT\u003csub\u003eΔ101\u003c/sub\u003e and WT\u003csub\u003eΔ101rd\u003c/sub\u003e:NAD\u003csup\u003e+\u003c/sup\u003e. Dashed lines indicate the hydrogen bonds formed by E336 with the ribose of the adenine moiety of the coenzyme in the WT\u003csub\u003eΔ101rd\u003c/sub\u003e:NAD\u003csup\u003e+\u003c/sup\u003e complex. (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSurface electrostatic potential of WT\u003csub\u003eΔ101\u003c/sub\u003e (left) and the E336K\u003csub\u003eΔ101\u003c/sub\u003e (right). Electrostatic surface potentials were calculated at pH 7.4 using APBS and PDB2PQR [11, 13, 26]. (\u003cstrong\u003eD\u003c/strong\u003e) Potential binding mode of the 2′P-adenine nucleotide moiety of NADP⁺ in a putative E336K\u003csub\u003eΔ101rd\u003c/sub\u003e:NADP⁺ structural model. K342 and K336 are displayed in sticks with carbon atoms in cyan.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8316196/v1/fbb07a6dda9639ec1ec09ad5.jpg"},{"id":102909982,"identity":"0ceffb68-6556-4cee-8379-f14b38b7e930","added_by":"auto","created_at":"2026-02-18 09:57:30","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":83733,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of E336K mutation on AIF apoptotic function. (\u003cstrong\u003eA-B\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eMNNG induced cell death in WT and E336K fibroblasts. Cell death was assessed by flow cytometry after simultaneous staining with Annexin V-FITC and 7-ADD in cells treated with MNNG. (\u003cstrong\u003eA\u003c/strong\u003e) Representative dot plots illustrating staining patterns in treated \u003cem\u003evs\u003c/em\u003e untreated WT and E336K cells. (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eQuantification of cell death after 20 minutes of MNNG exposure, 24 h prior to analysis. Data are presented as mean ± SD from three independent experiments. (\u003cstrong\u003eC\u003c/strong\u003e) Thermodynamic dissection of the interactions of AIF\u003csub\u003eΔ101ox \u003c/sub\u003evariants\u003csub\u003e \u003c/sub\u003ewith CypA and dsDNA as assessed by ITC. Gibbs free energy (Δ\u003cem\u003eG\u003c/em\u003e), enthalpy (Δ\u003cem\u003eH\u003c/em\u003e) and entropy contributions (-TΔ\u003cem\u003eS\u003c/em\u003e) are shown in blue, green and red bars, respectively. (\u003cstrong\u003eD\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eEffect of E336K mutation in DNA retention by AIF. Electrophoretic mobility shift assays (EMSAs) were performed using 6 µg of WT\u003csub\u003eΔ101\u003c/sub\u003e or its E336K\u003csub\u003eΔ101 \u003c/sub\u003evariant with 500 ng of 100 bp dsDNA ladder (Thermo Scientific). Incubations were carried out for 30 min at 25 ºC in 50 mM potassium phosphate buffer, pH 7.4. Mixtures were resolved on a 1 % agarose gel electrophoresis and visualized using SYBR™ Safe. As a control, the dsDNA ladder (D) was incubated without the protein under the same conditions. (\u003cstrong\u003eE\u003c/strong\u003e) Time course of the E336K mutation effect on AIF nuclease activity. A total of 250 ng of double-stranded supercoiled pcDNA3.1 plasmid was incubated with 250 ng of WT\u003csub\u003eΔ101\u003c/sub\u003e or E336K\u003csub\u003eΔ101 \u003c/sub\u003ein 20 mM Tris/HCl buffer, pH 8.0, supplemented with 0.1 mM CaCl\u003csub\u003e2\u003c/sub\u003e and 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e at 30 ºC for either 1 or 5 minutes. As a control, the plasmid was incubated without the protein under the same conditions. Samples were separated by electrophoresis on a 0.7 % agarose gel and visualized by SYBR™ Safe.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8316196/v1/ceef2ea309a4fc527661c882.jpg"},{"id":106809347,"identity":"9906e5a4-1930-4055-9383-e696e8841b2c","added_by":"auto","created_at":"2026-04-13 16:09:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2334543,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8316196/v1/cb588141-a17c-45e3-9fc1-60ad0d863306.pdf"},{"id":102909847,"identity":"645ffc04-aa21-4646-8da8-c63365b19eec","added_by":"auto","created_at":"2026-02-18 09:57:04","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1975099,"visible":true,"origin":"","legend":"","description":"","filename":"SIActaNeuropathologicaFerrerMrefsformatted.docx","url":"https://assets-eu.researchsquare.com/files/rs-8316196/v1/0ba18dad813c73d12d07a618.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Clinical and molecular characterization of a novel pathogenic AIFM1 E336K mutation connecting mitochondrial dysfunction and neurodegeneration","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe human apoptosis-inducing factor (hAIF), encoded by the AIFM1 gene on chromosome Xq25, is a mitochondrial flavoprotein with dual roles in energy metabolism and caspase-independent programmed cell death [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Under physiological conditions, AIF is anchored to the inner mitochondrial membrane, where it functions as a FAD-dependent NADH oxidoreductase essential for mitochondrial morphology, redox homeostasis, and the biogenesis and stability of oxidative phosphorylation system (OXPHOS) complexes, particularly complexes I and III [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStructurally, AIF comprises an N-terminal mitochondrial targeting sequence, a central oxidoreductase domain with FAD- and NADH-binding motifs, and a C-terminal domain required for NADH-dependent dimerization and apoptotic activity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These modular domains allow AIF to act as an NADH/NAD\u0026thinsp;+\u0026thinsp;redox sensor, switching from a metabolic supporter to a death effector in response to cellular stress. NADH binding and its oxidation by the AIF\u0026rsquo;s flavin cofactor induce the formation of a stable isoalloxazine\u003csub\u003erd\u003c/sub\u003e:NAD\u003csup\u003e+\u003c/sup\u003e charge-transfer complex (CTC) with FAD, triggering conformational rearrangements that displace the regulatory C‑terminal loop (residues 509\u0026ndash;559), promoting dimerization, and facilitating interaction with CHCHD4, a central component of the mitochondrial disulfide relay system [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Through this long-lived interaction, reduced and dimeric AIF supports the import and folding of CHCHD4-dependent substrates, thereby supporting respiratory chain assembly, including complex I and complex IV subunits [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Disruption of this pathway leads to mitochondrial dysfunction, redox imbalance, and, in animal models, embryonic lethality [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBeyond its metabolic function, AIF mediates large-scale DNA fragmentation and chromatin condensation in response to severe genotoxic stress, in a caspase-independent process known as parthanatos [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This apoptogenic function requires the proteolytic cleavage and nuclear translocation of AIF, events favored by redox-sensitive conformational changes in the protein upon NADH depletion [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. AIF, thus, lies at the intersection of mitochondrial bioenergetics and programmed cell death pathways.\u003c/p\u003e \u003cp\u003ePathogenic variants in AIFM1 were first reported in 2010, with the identification of a hemizygous deletion (ΔR201) in a male patient with severe mitochondrial encephalopathy [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Since then, an expanding phenotypic spectrum has been linked to AIFM1 mutations, X-linked Charcot-Marie-Tooth disease type 4 (CMTX4/COWCK) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] (also referred to as Cowchock syndrome, after the author who first described a family with this phenotype in 1985) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], combined oxidative phosphorylation deficiency 6 (COXPD6) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and auditory neuropathy spectrum disorder (DFNX5) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S1). Most pathogenic variants cluster within the FAD- and NADH-binding domains pointing to a central role of redox balance impairment in disease pathogenesis. Nonetheless, most variants remain poorly characterized at the functional and molecular levels and the mechanistic basis underlying their pathogenicity are unknown.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of previously reported pathogenic AIFM1 variants associated to Charcot-Marie-Tooth disease type 4 (CMTX4/COWCHOCK)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"11\" nameend=\"c11\" namest=\"c1\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMutation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eDisease onset\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003eProgression\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003ePhenotypes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eDomain\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eProtein level\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eOXPHOS activity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eRedox activity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eCell death\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eRef.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"11\" nameend=\"c11\" namest=\"c1\"\u003e \u003cp\u003eCOWCHOCK syndrome\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT141I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.5y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eChildhood-onset cerebellar ataxia with auditory neuropathy, axonal sensorimotor neuropathy and cardiomyopathy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFAD-binding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM171I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5-11y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eChildhood-onset slowly progressive axonal motor and sensory neuropathy, lower limb weakness and atrophy, steppage gait, pes cavus, absence of CNS symptoms or cognitive impairment.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFAD-binding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAbnormal mitochondrial morphology and accumulation in nerve and muscle suggest mitochondrial dysfunction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF210L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eDistal muscle atrophy and weakness (particularly ankle flexors), absent ankle reflexes, and mild sensory impairment. No involvement of central nervous system or other organs.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFAD-binding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMisassemble of Complex I and Complex III (~\u0026thinsp;80% reduction)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNo effect; normal interaction with HSP70 (inhibitor of AIF nuclear translocation)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF210S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16-18m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlow, but severe progression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eEarly-onset axonal polyneuropathy with exclusive motor fiber involvement. Sensory nerves were unaffected. Cognitive function and cranial nerves were normal. Severe distal weakness and muscle atrophy with mild to moderate proximal weakness.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFAD-binding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eReduced (normal mRNA expression)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMitochondrial fragmentation and reduced interconnectivity suggest mitochondrial dysfunction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNot altered\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG262S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eMitochondrial encephalomyopathy characterized by ataxia (sensory and cerebellar), hearing loss, cognitive impairment, visual loss, distal muscle weakness, and neuropathy.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFAD-binding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eStrong reduction; less stability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eReduced Complex IV activity, partial reduction of other complexes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eReduced NADH affinity, increased NADH oxidation, destabilized CTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL311V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSevere axonal sensorimotor neuropathy, distal muscle atrophy and weakness, pes cavus, sensorineural hearing loss, cognitive decline, and scoliosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNADH-binding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePredicted destabilization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG399S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eCerebellar ataxia and atrophy and dysarthria, sensorineural hearing loss, intellectual disability, axonal peripheral neuropathy, and mood and behavioral disorders.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNADH-binding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"11\" nameend=\"c11\" namest=\"c1\"\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Continued\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM340T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eChildhood-onset cerebellar ataxia with auditory neuropathy, axonal sensorimotor neuropathy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNADH-binding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eReduced\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eReduced Complex I, II and IV. ΔΨ\u003csub\u003em\u003c/sub\u003e decreased.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eReduced, impaired\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI433M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5y,13y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRapid, Slow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eNeurodegenerative syndrome with hearing loss, ataxia, neuropathy, and optic atrophy.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFAD-binding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE493V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEarly age\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eAxonal motor and sensory neuropathy, bilateral sensorineural hearing loss, cognitive impairment, distal muscle wasting and weakness (lower\u0026thinsp;\u0026gt;\u0026thinsp;upper limbs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC-terminal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNormal level but reduced stability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo affected\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eIncreased NADH oxidation, destabilized CTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eEnhanced\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH457Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3y, 4y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eComplex progressive movement disorder. Features included cerebellar ataxia, disabling cerebellar tremor, sensorineural hearing loss, sensory polyneuropathy, language, fine motor delays, dystonia and optic nerve hypoplasia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC-terminal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003eNA: not available; d: days; m: months; y, year; ---: not studied; ΔΨ\u003csub\u003em\u003c/sub\u003e: mitochondrial membrane potential\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eStructural analyses of selected pathogenic variants \u0026ndash;including ΔR201, V243L, G308E, G338E, R422Q, R451Q\u0026ndash; have revealed decreased flavin affinity, compromised protein stability, impaired CTC formation and defective OXPHOS activity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S1). Clinically, these mutations often present as early-onset, progressive neuromuscular syndromes, with features such as peripheral neuropathy, ataxia, cerebellar hypoplasia, optic atrophy, and sensorineural hearing loss (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S1). Notably, several pathogenic variants (F210L/S, R422Q, V243L, G308E, G338E) retain apoptotic competence \u003cem\u003ein vitro\u003c/em\u003e \u0026ndash;preserving DNA-binding and nuclear translocation\u0026ndash; while displaying markedly reduced redox activity, suggesting that mitochondrial dysfunction, rather than impaired apoptosis, is the principal driver of disease (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S1).\u003c/p\u003e \u003cp\u003eHere, we report the identification and functional characterization of a novel AIFM1 missense variant, E336K, in a male patient exhibiting a neurodegenerative phenotype consistent with Cowchock syndrome. This E336K mutation is located in the AIF NADH-binding domain, where residue E336 lies within a highly conserved structural region essential for NADH coordination and redox cycling [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. We have comprehensively characterized this mutation from an integrated, multi-level perspective \u0026ndash;spanning longitudinal clinical and neurological follow-up, molecular and cellular functional studies, and structural\u0026ndash; biophysical analyses. By directly linking the patient\u0026rsquo;s clinical evolution to the underlying molecular defect, we elucidate the pathogenic mechanism of the E336K variant and establish a model for genotype\u0026ndash;phenotype correlations in AIFM1-related disorders. This integrative approach deepens our understanding of AIF biology and surely will contribute to facilitate rational developments in future precision molecular therapies.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and clinical evaluations\u003c/h2\u003e \u003cp\u003eClinical data were collected from a family originating from northern Spain (Arag\u0026oacute;n) spanning three generations with a phenotype consistent with CMTX4. Two brothers were clinically affected; their mother and the daughter of one affected patient were also carriers. Genetic testing results were available for all family members except for one clinically affected brother. This relative presented clinically with a polyneuropathy; however, his evaluations were not included, as he declined follow-up and further investigations.\u003c/p\u003e \u003cp\u003e For one of the affected patients, complete clinical information was obtained, and longitudinal follow-up was carried out by the Neuromuscular Unit at Hospital Universitario Miguel Servet from June 2016 to June 2024. In May 2023, the patient was enrolled in the research project \u003cem\u003eFlavoenzymes in Health, Diseases and Drug Discovery\u003c/em\u003e conducted by the University of Zaragoza (Hospital Miguel Servet, Department of Biochemistry, Molecular and Cell Biology, Faculty of Sciences, and the Institute for Biocomputation and Complex Systems Physics). As part of this study, blood and skin tissue samples were obtained, the latter through a skin biopsy. No muscle tissue was collected, as the patient declined to undergo muscle biopsy.\u003c/p\u003e \u003cp\u003eFrom the electronic medical record, the following evaluations were retrieved: laboratory tests: Complete blood count (CBC), chemistry, coagulation, lactic acid, autoantibodies, vitamin E, creatine kinase (CK), long-chain fatty acids, thyroid hormone. Brain and acoustic nerve MRI, brainstem auditory evoked potentials (BAEPs), serial audiometries, echocardiogram and Holter monitoring, as well as multidisciplinary assessments by cardiology, and otorhinolaryngology services, in addition to annual follow-up by neurology. A neurophysiological study was performed to confirm the neuropathy phenotype using a Synergy 28 system. Motor and sensory nerve conduction studies were conducted in the median and ulnar nerves of the upper limbs. In the lower limbs, motor conduction studies were carried out in the peroneal and tibial nerves, and sensory studies in the superficial peroneal and sural nerves. The assessment was completed with an electromyographic evaluation, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNeurophysiological findings in patient holding the E336K AIF mutation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNerve/muscle\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStudy type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFindings\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eClinical Interpretation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNerve Sural R/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSensory\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo response\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSevere sensory impairment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNerve Superficial peroneal R/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSensory\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo response\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSevere sensory impairment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNerve Median sensory R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSensory\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLat 3.45 ms | Amp 1.8 \u0026micro;V | Vel 46.4 m/s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModerate sensory neuropathy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNerve Ulnar sensory R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSensory\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLat 2.55 ms | Amp 4.7 \u0026micro;V | Vel 47.1 m/s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModerate sensory neuropathy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNerve Median motor R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMotor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLat 4.30\u0026ndash;8.40 ms | Amp 7.8\u0026ndash;8.2 mV | Vel 59.8 m/s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMild to moderate motor neuropathy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNerve Ulnar motor R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMotor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLat 3.35\u0026ndash;9.55 ms | Amp 11.0\u0026ndash;7.6 mV | Vel 65.4 / 44.4 m/s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMild motor neuropathy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNerve Common peroneal R/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMotor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo response\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSevere motor neuropathy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNerve Tibial R/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMotor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo response\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSevere motor neuropathy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle Tibialis anterior R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEMG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal IA, no spontaneous activity, Mixed I recruitment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAxonal damage, no denervation, altered recruitment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle Gastrocnemius med. R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEMG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal IA, no spontaneous activity, Mixed I recruitment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAxonal damage, no denervation, altered recruitment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle Tibialis anterior L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEMG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal IA, no spontaneous activity, Mixed I recruitment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAxonal damage, no denervation, altered recruitment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle Gastrocnemius med. L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEMG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal IA, no spontaneous activity, Mixed I recruitment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAxonal damage, no denervation, altered recruitment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle Vastus medialis R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEMG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal IA, no spontaneous activity, Mixed II recruitment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAxonal damage, no denervation, altered recruitment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscleVastus medialis L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEMG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal IA, no spontaneous activity, Mixed II recruitment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAxonal damage, no denervation, altered recruitment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle Biceps R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEMG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal IA, no spontaneous activity, Mixed III recruitment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAxonal damage, no denervation, altered recruitment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle Abductor digiti minimi (ulnar) R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEMG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal IA, no spontaneous activity, Mixed III recruitment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAxonal damage, no denervation, altered recruitment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eSummary of motor and sensory nerve conduction studies and electromyographic evaluation. The results demonstrate a severe, length-dependent, axonal sensorimotor polyneuropathy, consistent with Charcot-Marie-Tooth disease type X4 (CMTX4, Cowchock syndrome). EMG: electromyography; IA: insertion activity.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e Written informed consent was obtained from the patient for participation in the study, for the performance of the skin biopsy, and for publication of the results\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenetic/Mutational analysis\u003c/h3\u003e\n\u003cp\u003eA comprehensive exome sequencing analysis was performed on the DNA sample extracted from peripheral blood, with the aim of identifying genomic variants in 53 genes associated with Charcot-Marie-Tooth disease and related hereditary sensory-motor neuropathies (NIMGenetics). The panel used for library preparation was designed using Ion AmpliSeq\u0026trade; Exome technology (Life Technologies), capturing\u0026thinsp;\u0026gt;\u0026thinsp;97% of CCDS (\u0026gt;\u0026thinsp;19,000 genes, \u0026gt;\u0026thinsp;198,000 exons, \u0026gt;\u0026thinsp;85% of alterations responsible for genetic diseases) and flanking splice regions (5 bp). It has a size of ~\u0026thinsp;33 Mb and comprises a total of 293,903 amplicons. Library sequencing was performed on a next-generation high-throughput sequencer, the Ion Proton\u0026trade; (Life Technologies). In the study conducted, a mean coverage depth of 79 was obtained. A total of 1177 amplicons covering the selected genes in this panel were analyzed. 96% of the amplicons included in the study had a read depth greater than 20X. The obtained sequences were aligned to the reference genome (build 37 of the human genome, Hg19) using the TMAP \u0026ndash;Ion-Alignment software. The aligned and filtered sequences, according to specific quality criteria, were analyzed to identify nucleotide variations with respect to the reference genome (VariantCaller). Variant annotation was performed with ION Reporter (Life Technologies). The analysis focuses on identifying variants located in exonic regions or splice regions, which imply a change at the protein level (missense or nonsense mutations, and nucleotide insertions, deletions, or indels), and which are present in a frequency higher than 40% of the reads. The list of identified variants was evaluated using information from databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/SNP/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/SNP/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.1000genomes.org\u003c/span\u003e\u003cspan address=\"http://www.1000genomes.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://evs.gs.washington.edu/EVS\u003c/span\u003e\u003cspan address=\"http://evs.gs.washington.edu/EVS\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://https://www.ncbi.nlm.nih.gov/clinvar/\u003c/span\u003e\u003cspan address=\"http://https://www.ncbi.nlm.nih.gov/clinvar/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://varsome.com/\u003c/span\u003e\u003cspan address=\"https://varsome.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://franklin.genoox.com/clinical-db/home\u003c/span\u003e\u003cspan address=\"https://franklin.genoox.com/clinical-db/home\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://databases.lovd.nl/shared/genes\u003c/span\u003e\u003cspan address=\"https://databases.lovd.nl/shared/genes\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Furthermore, the functional effect of genomic variations classified as pathogenic was assessed using 9 prediction systems (SIFT, PROVEAN, PolyPhen2, MutationTaster, MutationAssessor, LRT, FATHMM, MetaSVM, and CONDEL) included in the ALAMUT analysis package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.interactive-bioware.com\u003c/span\u003e\u003cspan address=\"http://www.interactive-bioware.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the ANNOVAR package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.openbioinformatics.org/annovar/\u003c/span\u003e\u003cspan address=\"http://www.openbioinformatics.org/annovar/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Finally, the association of the identified mutations with OMIM syndromes was evaluated. Sanger sequencing of the genomic fragment chrX:129271006\u0026ndash;129271226 was performed on the DNA familial samples (brother, Labgenetics; mother and daughter, NIMGenetics) to identify the AIFM1 variant c.1006G\u0026thinsp;\u0026gt;\u0026thinsp;A; p.Glu336Lys.\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eControl and mutant human fibroblasts were obtained from skin biopsies from a healthy donor and a patient with X-linked Charcot-Marie-Tooth disease carrying a single mutation, c.1006G\u0026thinsp;\u0026gt;\u0026thinsp;A (p. Glu336Lys), in the AIF gene, respectively. Skin biopsies were collected with informed consent in accordance with institutional ethical guidelines and under approved protocols. Biopsy samples were sterilized with ethanol, rinsed with sterile PBS and mechanically disaggregated into ~\u0026thinsp;1 mm\u003csup\u003e2\u003c/sup\u003e pieces using forceps and a scalpel. The tissue fragment was then transferred to 15 mL corning tubes containing 5 mL of high-glucose DMEM supplemented with 20% fetal bovine serum (FBS, GIBCO), 1% penicillin-streptomycin (GIBCO), and 1 mL of collagenase I (4 mg/mL). Samples were incubated overnight at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Following enzymatic digestion, cells and residual tissue were centrifuged at 1500 g for 10 minutes. The resulting pellet was washed with sterile PBS and centrifuged three times under the same conditions. Finally, the pellet was resuspended in high-glucose DMEM supplemented with 20% FBS and seeded in 60 mm culture dishes. Primary cultures were immortalized at passage 4 by transduction with the lentiviral plasmid pLOX-Ttag-iresTK (Tronolab). Once exponential growth was established, the FBS concentration in the culture medium was reduced to 10%.\u003c/p\u003e\n\u003ch3\u003eGrowth measurements\u003c/h3\u003e\n\u003cp\u003eGrowth rate in galactose-containing medium was determined by seeding 5*10\u003csup\u003e4\u003c/sup\u003e cells per well on 12-wells plates with 2 mL of either high-glucose DMEM supplemented with 10% FBS, or glucose-free DMEM supplemented with 0.9 mg /mL galactose,1 mM sodium pyruvate, and 10% FBS. Cells were incubated at 37\u0026deg;C for 5 days, and cell numbers were recorded every 24 hours.\u003c/p\u003e\n\u003ch3\u003eCell Viability Assays\u003c/h3\u003e\n\u003cp\u003eThe relative growth of different cell lines in response to oxidative phosphorylation (OXPHOS) inhibitors was evaluated using the MTT reduction assay, following the method described by Mosmann T.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Briefly, 1 \u0026times; 10⁴ cells per well were plated in 96-well flat-bottom plates and cultured in galactose-containing medium in the presence of two concentrations of rotenone (2 and 10 nM), antimycin A (2 and 10 nM), or sodium azide (20 and 100 \u0026micro;M) for 48 h at 37\u0026deg;C. After drug exposure, cells were incubated with fresh medium containing 1 mg/mL MTT for 4 h at 37\u0026deg;C in a humidified atmosphere. Formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm using a microplate reader. Results were expressed as percentages relative to untreated control cells. All experiments were performed at least in triplicate.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOxygen consumption measurements\u003c/h2\u003e \u003cp\u003eEndogenous and maximal O\u003csub\u003e2\u003c/sub\u003e consumption in intact cells was measured using an Oxytherm Clark-type electrode (Hansatech), as previously described [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], with minor modifications [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEvaluation of protein expression\u003c/h3\u003e\n\u003cp\u003eFor preparation of total cell protein extracts, cells were harvested from 60 mm-diameter culture plates, washed twice with PBS, and lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 5 mM EDTA, 1% Triton X-100, 0.5% Sodium Deoxycholate, 50 mM NaCl) containing 1x cOmplete\u0026trade; Protease Inhibitor Cocktail (Roche). Steady-state levels of proteins, including AIF, CHCHD4 or subunits of the mitochondrial respiratory chain complexes were estimated by loading 60 \u0026micro;g of total protein per lane onto SDS-PAGE gels. Proteins were separated on 12% acrylamide/bisacrylamide gels and electroblotted onto PVDF membranes. Western blotting was performed using specific primary antibodies against human AIF (SIGMA), CHCHD4 (Proteintech), β-actin (SIGMA), as well as antibodies targeting complex I (anti-NDUFA9, Invitrogen), complex II (anti-70 kDa subunit, SDHA, Invitrogen), complex III (anti-Core1, Invitrogen) and complex IV (anti-COXI, Invitrogen). Detection was carried out using HRP-conjugated secondary antibodies (anti-mouse or anti-rabbit, Invitrogen) and the Pierce\u0026trade; ECL Western Blotting Substrate (Thermo Scientific).\u003c/p\u003e\n\u003ch3\u003eNative polyacrylamide electrophoresis analysis of respiratory supercomplexes\u003c/h3\u003e\n\u003cp\u003eMitochondria were isolated from cultured cell lines according to Sch\u0026auml;gger [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], with minor modifications [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Digitonin-solubilized mitochondrial proteins (100 \u0026micro;g) were separated by native PAGE. The assembly of respiratory supercomplexes was analysed by BN-PAGE using commercial native 3\u0026ndash;12% acrylamide gradient gels (Novex).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Superoxide production and mitochondrial mass analysis\u003c/h2\u003e \u003cp\u003eTo assess mitochondrial ROS production and mitochondrial mass, cells were stained with either MitoSOX\u0026trade; red (5 \u0026micro;M, Invitrogen) or MitoTracker\u0026trade; Green FM (200 nM, Invitrogen) for 30 min at 37 \u0026ordm;C. Mean fluorescence intensity was measured by flow cytometry using a FACSCalibur cytometer (BD Biosciences), and data were analyzed using FlowJo Software, version 10.8.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative PCR determination of mRNA transcripts\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from 5*10\u003csup\u003e6\u003c/sup\u003e cells using TRIzol\u0026trade; reagent (Invitrogen), and 1 \u0026micro;g of RNA was reverse transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche). Transcript levels of human AIF, SOD2 and CHCHD4 were quantified by qPCR using a LightCycler System (Roche) with the LightCycler Fast-Start DNA MasterPLUS SYBR Green I Kit (Roche), following the manufacturer\u0026rsquo;s recommendations. Transcript levels were normalized to \u003cem\u003eActb mRNA\u003c/em\u003e (NM_007393). Primer sequences are provided in Table S4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAIF induced cell death analysis\u003c/h2\u003e \u003cp\u003eAIF-mediated cell death was analyzed by simultaneous annexin-V and 7AAD staining and flow cytometry, after cell death induction with MNNG as previously described [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Briefly, control and mutant cells were grown to a 70% confluence, and exposed to MNNG (0.5 \u0026micro;M) for 20 min. After treatment, the medium was replaced, and cells were incubated for 24 h at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were then collected, stained with annexin-V-FITC and 7-AAD (Sigma) for 10 min in annexin binding buffer (140 mM NaCl, 2.5 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 10 mM HEPES/NaOH, pH 7.4), and analyzed FACSCalibur flow cytometer (BD Biosciences, Madrid, Spain). Similar to previous sections, collected data were further processed by using FlowJo Software, version 10.8.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRecombinant protein expression and purification\u003c/h2\u003e \u003cp\u003eConstructs for WT, AIF\u003csub\u003e∆77\u003c/sub\u003e and AIF\u003csub\u003eΔ101\u003c/sub\u003e and overexpression protocols were previously reported [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The genes encoding the human AIF mutation E336K\u003csub\u003eΔ77\u003c/sub\u003e and E336K\u003csub\u003eΔ101\u003c/sub\u003e were obtained by site-directed mutagenesis from GenScript\u0026reg; and subsequently subcloned between the NcoI and NdeI sites of the pET-28a(+) expression vector. WT AIF as well as both the apoptotic E336K\u003csub\u003e∆101\u003c/sub\u003e and soluble mitochondrial E336K\u003csub\u003e∆77\u003c/sub\u003e variants, along with CHCHD4 (UniProtKB Q8N4Q1) were heterologously expressed as recombinant proteins carrying an N-terminal removable His\u003csub\u003e6\u003c/sub\u003e-tag (CACCAT) using the pET28a(+) expression vector. Expression was performed in \u003cem\u003eEscherichia coli\u003c/em\u003e C41 (DE3) strain, except for CHCHD4, which was expressed in Shuffle T7 cells to enhance disulfide bond formation.\u003c/p\u003e \u003cp\u003eThe production of E336K\u003csub\u003eΔ77\u003c/sub\u003e was performed by growing transformed cells in 2xYT medium containing 30 mg/L kanamycin (Sigma-Aldrich), supplemented with 8 mg/L of riboflavin (Sigma-Aldrich) at 37\u0026deg;C and 180 r.p.m. until OD\u003csub\u003e600nm\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;0.5. Then, cultures were induced with 0.5 mM IPTG (Glentham life sciences) and incubated 24h in the same conditions. For AIF\u003csub\u003e∆101\u003c/sub\u003e, cells were grown as previously described [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. After the cells were harvested, both E336K\u003csub\u003e∆77\u003c/sub\u003e and E336K\u003csub\u003eΔ101\u003c/sub\u003e variants were purified as previously reported for the apoptotic WT\u003csub\u003e∆101\u003c/sub\u003e form [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCHCHD4 production and purification was performed as formerly described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. WT AIF concentrations were determined using the molar absorptivity coefficient previously reported [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For E336K \u003csub\u003eΔ101\u003c/sub\u003e this value was estimated by protein denaturation by boiling for 5 minutes at 100 \u0026ordm;C, followed by quantification of the released FAD after a 3 minutes centrifugation. The extinction coefficients, ε\u003csub\u003e452 nm\u003c/sub\u003e, for WT\u003csub\u003eΔ101\u003c/sub\u003e and E336K\u003csub\u003eΔ101\u003c/sub\u003e variants were 13.7, and 14,6 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;cm\u003csup\u003e\u0026minus;\u003c/sup\u003e1, respectively. CHCHD4 concentrations were calculated through its theoretical ε\u003csub\u003e280 nm\u003c/sub\u003e value (13.3 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) obtained from the ProtParam tool (ExPASy). All proteins were stored in 50 mM potassium phosphate, pH 7.4 at -80 \u0026ordm;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMolecular weight determination by size exclusion chromatography\u003c/h2\u003e \u003cp\u003eThe AIF\u003csub\u003eΔ101\u003c/sub\u003e variants, in the absence or presence of 2 mM of NADH and/or CHCHD4, were loaded onto a Superdex 200 increase 10/300 GL (Cytiva) column attached to an \u0026Auml;KTA go system (Cytiva). AIF\u003csub\u003eΔ101\u003c/sub\u003e:CHCHD4 mixtures (1:3 molar ratio regards to AIF) were preincubated in 50 mM phosphate buffer pH 7.4, without or with NADH, for 10 min at RT before loading onto the column. Protein elution was carried out in 50 mM phosphate buffer, 150 mM NaCl, pH 7.4, at a flow rate of 0.4 mL/min. Column calibration was performed with the gel filtration calibration kit (Cytiva) containing 6 proteins in the 13.7\u0026ndash;440 kDa range. Chromatograms were analyzed using a set of Gaussian functions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSpectroscopic characterization\u003c/h2\u003e \u003cp\u003eUV-Visible spectra were recorded in CARY 3500 (Agilent). Circular dichroism (CD) spectra were acquired in a thermostated Chirascan (Applied Photophysics Ltd.) at 25\u0026deg;C in 50 mM potassium phosphate buffer, pH 7.4 (150 mM ionic strength) in the absence and presence of a 100-fold molar excess of NADH. Near-UV/Vis CD spectra were collected using 20 \u0026micro;M AIF\u003csub\u003eΔ101\u003c/sub\u003e in a 1 cm-pathlength cuvette, while Far-UV CD spectra were acquired using 5 \u0026micro;M AIF\u003csub\u003eΔ101\u003c/sub\u003e in a 0.1 cm-pathlength cuvette. Fluorescence spectra were acquired in a thermostated Cary Eclipse Fluorescence spectrophotometer (Agilent) using 2\u0026micro;M AIF\u003csub\u003eΔ101\u003c/sub\u003e in a 1cm-pathlength cuvette. Flavin fluorescence emission spectra were acquired in the 480-600nm range upon excitation at 450 nm. Emission spectra of aromatic residues were collected from 300 to 600nm upon excitation at 280 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThermal denaturation assays\u003c/h2\u003e \u003cp\u003eThermal denaturation curves were monitored by FAD fluorescence emission, near-UV/Vis CD and far UV-CD. Curves were monitored from 15\u0026deg;C to 90\u0026deg;C with scan rates of 1 \u0026ordm;C/min and 1.5 \u0026ordm;C/min respectively for fluorescence and CD assays, both in presence or absence of a 100-fold excess of NADH. For flavin fluorescence, measures were carried out with 2 \u0026micro;M AIF\u003csub\u003eΔ101\u003c/sub\u003e in a 1 cm-pathlength cuvette with excitation at 450 nm. For far-UV and near-UV/Vis, curves were monitored with 5 \u0026micro;M AIF\u003csub\u003eΔ101\u003c/sub\u003e in a 0.1 cm-pathlength cuvette or 20 \u0026micro;M AIF\u003csub\u003eΔ101\u003c/sub\u003e in a 1 cm-pathlength cuvette, respectively. Experimental data were roughly normalized to values between 0 and 1 and globally fitted to a two-step (native (N) \u0026harr; unfolded (U)) or three-step unfolding model (native (N) \u0026harr; intermediate (I) \u0026harr; unfolded (U)) using the following equations [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:{S}_{obs}=\\:\\frac{{S}_{N}+\\:{m}_{N}T+\\left({S}_{U}+{m}_{U}T\\right){e}^{-\\left(\\varDelta\\:G/RT\\right)}}{1+{e}^{-\\left(\\varDelta\\:G/RT\\right)}}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;1\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:{S}_{obs}=\\:\\frac{{S}_{N}+\\:{m}_{N}T+\\left({S}_{I}+{m}_{I}T\\right){e}^{-\\left(\\varDelta\\:{G}_{1}/RT\\right)}+\\left({S}_{U}+{m}_{U}T\\right){e}^{-\\left((\\varDelta\\:{G}_{1}+\\varDelta\\:{G}_{2})/RT\\right)}}{1+{e}^{-\\left(\\varDelta\\:{G}_{1}/RT\\right)}+{e}^{-\\left((\\varDelta\\:{G}_{1}+\\varDelta\\:{G}_{2})/RT\\right)}}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;2\u003c/p\u003e \u003cp\u003ein which S\u003csub\u003eobs\u003c/sub\u003e is the measured protein signal at a given temperature (T), S\u003csub\u003ex\u003c/sub\u003e (S\u003csub\u003eN\u003c/sub\u003e, S\u003csub\u003eI\u003c/sub\u003e and S\u003csub\u003eU\u003c/sub\u003e) represents the y-intercept of native, intermediate and unfolded protein at 0K and m\u003csub\u003ex\u003c/sub\u003e (m\u003csub\u003eN\u003c/sub\u003e, m\u003csub\u003eI\u003c/sub\u003e and m\u003csub\u003eU\u003c/sub\u003e) is the slope of the native, intermediate and unfolded states, respectively. The Stabilization Gibbs energy depends on temperature according to \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varDelta\\:G=\\varDelta\\:H\\left(1-\\frac{1}{{T}_{m}}\\right)+{\\varDelta\\:C}_{P}\\left(T-{T}_{m}-T\\text{ln}\\frac{T}{{T}_{m}}\\right)\\)\u003c/span\u003e\u003c/span\u003e, where ΔH is the unfolding enthalpy, T\u003csub\u003em\u003c/sub\u003e is the midtransition temperature, ΔC\u003csub\u003eP\u003c/sub\u003e is the unfolding heat capacity change, and R is the ideal gas constant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eKinetics measurements\u003c/h2\u003e \u003cp\u003eThe steady-state diaphorase activity of AIF\u003csub\u003e∆101\u003c/sub\u003e was measured in a Cary 100 spectrophotometer (Agilent). The measurements were carried out in air saturated 50 mM potassium phosphate buffer, pH 7.4 at 25\u0026deg;C, using NADH or NADPH as the substrate donor and 95 \u0026micro;M dichlorophenolindophenol (DCPIP, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varDelta\\:{\\epsilon\\:}_{620nm}\\)\u003c/span\u003e\u003c/span\u003e= 21 mM\u003csup\u003e\u0026minus;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e) as acceptor [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Initial reaction rates at different NADH concentrations were fitted to the Michaelis-Menten equation to determine the kinetic constants:\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\nu\\:}{\\text{e}}=\\frac{{k}_{\\text{c}\\text{a}\\text{t}}\\:\\left[NAD\\right(P\\left)H\\right]}{{K}_{m}^{NADH}+\\left[NAD\\right(P\\left)H\\right]}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;3\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\nu\\:}{\\text{e}}=\\frac{{k}_{\\text{c}\\text{a}\\text{t}}/{K}_{m}^{NAD\\left(P\\right)H}\\left[NAD\\right(P\\left)H\\right]}{1+\\frac{{k}_{\\text{c}\\text{a}\\text{t}}/{K}_{m}^{NAD\\left(P\\right)H}\\left[NAD\\right(P\\left)H\\right]}{{k}_{\\text{c}\\text{a}\\text{t}}}}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;4\u003c/p\u003e \u003cp\u003eIn which \u003cem\u003ev\u003c/em\u003e represents the initial velocity, \u003cem\u003ee\u003c/em\u003e is the enzyme concentration, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{m}^{NAD\\left(P\\right)H}\\)\u003c/span\u003e\u003c/span\u003e stands for the Michaelis constant for the NAD(P)H, \u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e is the turnover number of the enzyme and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{k}_{\\text{c}\\text{a}\\text{t}}/{K}_{m}^{NAD\\left(P\\right)H}\\:\\)\u003c/span\u003e\u003c/span\u003eis the enzyme catalytic efficiency.\u003c/p\u003e \u003cp\u003eThe reactivity of the CTC towards molecular oxygen was examined by full reduction of AIF\u003csub\u003e∆101\u003c/sub\u003e with NADH (1:1.5 ratio relative to AIF\u003csub\u003e∆101\u003c/sub\u003e), in both presence or absence of CHCHD4 (1:3 ratio relative to AIF) in 50 mM potassium phosphate buffer, pH 7.4 and 25 \u0026ordm;C as previously reported[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Reoxidation was monitored using a Cary 100 spectrophotometer (Agilent).\u003c/p\u003e \u003cp\u003eA SX18.MV stopped-flow spectrophotometer (Applied Photophysics Ltd.) was used to investigate the reductive half-reaction of AIF\u003csub\u003e∆101\u003c/sub\u003e variants upon mixing with increasing concentrations of NADH (0.03-10 mM) in both absence and presence of CHCHD4 (1:3 ratio regards to AIF). Measurements were collected using PDA and monochromator detectors in air-saturated 50 mM potassium phosphate buffer, pH 7.4, at 25\u0026deg;C. Observed rate constants (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e) for HT were determined by global fitting of the spectra or exponential fitting of single-wavelength traces, assuming one-step process using Pro-K and ProData-XD software. The averaged \u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e at each NADH concentration were then non-linearly fitted to the equation that describes the formation of an enzyme:substrate complex prior to the HT event:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:{k}_{\\text{o}\\text{b}\\text{s}}=\\frac{{k}_{\\text{H}\\text{T}}NADH}{{K}_{\\text{d}}^{NADH}+NADH}+{k}_{\\text{r}\\text{e}\\text{v}}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;4\u003c/p\u003e \u003cp\u003eIn which \u003cem\u003ek\u003c/em\u003e\u003csub\u003eHT\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003erev\u003c/sub\u003e represent the HT constant and its reverse reaction, respectively and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{\\text{d}}^{NADH}\\)\u003c/span\u003e\u003c/span\u003e stands for the dissociation constant of the transient AIF\u003csub\u003e∆101\u003c/sub\u003e:NADH complex.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIsothermal titration calorimetry (ITC)\u003c/h2\u003e \u003cp\u003eITC assays were performed using an Auto-iTC200 (Malvern) thermostated at 25 25 \u0026ordm;C for CypA, 15\u0026ordm;C for dsDNA or 10\u0026ordm;C for CHCHD4. Typically, 100 \u0026micro;M CHCHD4, CypA or dsDNA were used to titrate\u0026thinsp;~\u0026thinsp;10 \u0026micro;M AIF\u003csub\u003e∆101\u003c/sub\u003e. Solutions were degassed at 15\u0026deg;C for 3 min before each assay. A sequence of 2 \u0026micro;L injections of titrant solution every 150 s was programmed, and the stirring speed was set to 750 rpm. The association constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e), the enthalpy of binding (ΔH), and the binding stoichiometry (N) were estimated through non-linear least-squares regression of the experimental data using a single ligand binding site model implemented in Origin 7.0 (OriginLab). The dissociation constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e), the free energy change (Δ\u003cem\u003eG\u003c/em\u003e), and the entropy change (Δ\u003cem\u003eS\u003c/em\u003e) were calculated from basic thermodynamic relationships.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eElectrophoretic-mobility-shift-assays (EMSAs)\u003c/h2\u003e \u003cp\u003eDNA retardation assays were carried out as formerly described [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Briefly, 500 ng of GeneRuler 100 bp dsDNA ladder (Thermo Scientific) were incubated with 6 \u0026micro;g of AIF\u003csub\u003e∆101\u003c/sub\u003e for 30 min at 25\u0026deg;C in 50 mM potassium phosphate buffer, pH 7.4 and subsequently mixed with 6x DNA loading dye (Thermo Fisher Scientific. The samples were resolved by electrophoresis in 1% agarose gel stained with SYBR\u0026trade; Safe. The electrophoresis was carried out for 1h at 90V.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eNuclease activity assays\u003c/h2\u003e \u003cp\u003eAssays were performed as previously described [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Briefly, 250 ng of a double-stranded supercoiled pcDNA3.1 plasmid were incubated at 30 \u0026ordm;C with 250 ng of purified AIF\u003csub\u003e∆101\u003c/sub\u003e for 1 or 5 minutes in 20 mM Tris, pH 8.0 supplemented with 0.1 mM CaCl\u003csub\u003e2\u003c/sub\u003e and 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e. The samples were subsequently mixed with 6x DNA loading dye (Thermo Fisher Scientific), heated for 10 minutes at 65\u0026deg;C, and loaded onto a 0.7% agarose gel with SYBR\u0026trade; Safe. The electrophoresis was carried out for 1 h at 90 V.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCrystallization and structure determination of the E336K\u003csub\u003e∆101\u003c/sub\u003e variant\u003c/h2\u003e \u003cp\u003eCrystallization was carried out using hanging-drop vapor diffusion at 20\u0026deg;C, mixing E336K\u003csub\u003e∆101ox\u003c/sub\u003e protein solution (10 mg/ml) with reservoir solution (18% PEG 6K, 0.1 M Tris-HCl pH 8.5, 0.2 M Li₂SO₄) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Crystals were cryoprotected with 20% glycerol and flash-cooled in liquid nitrogen. X-ray diffraction data were collected at 100 K on the BL13-XALOC beamline (ALBA Synchrotron) using a Pilatus3 X 6M detector. Data were processed with XDS [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and SCALA [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] from the CCP4 package [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], and the structure was solved by molecular replacement using Phaser [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], with AIF\u003csub\u003e∆101ox\u003c/sub\u003e (PDB 4BV6) as the search model. Model building and refinement used Coot [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], phenix.refine [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and REFMAC [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], yielding a final model containing residues 127\u0026ndash;610, one FAD molecule, one glycerol, and 239 water molecules. Residues 546\u0026ndash;558 and the last three C-terminal residues were unresolved. Data collection and refinement statistics are provided in Table S5.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eData and statistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed and shown using StatView 5.0 (SAA Institute, Mesa, AZ, USA), SigmaPlot (Systat. Software Inc.), Origin 7.0 (OriginLab) and Pro-K (Applied Photophysics Ltd.) and PyMol [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Results were displayed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and statistically analyzed by Fisher\u0026rsquo;s PLSD post hoc test from ANOVA. In all cases, differences were considered statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eClinical findings\u003c/h2\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003ePatients and Clinical Evaluation\u003c/h2\u003e \u003cp\u003eWe report the case of a 64-year-old male from Arag\u0026oacute;n, Spain, with a progressive sensorimotor polyneuropathy. Genetic testing confirmed a diagnosis of X-linked hereditary sensorimotor polyneuropathy (CMTX4/COWCK), associated with a hemizygous mutation c.1006G\u0026thinsp;\u0026gt;\u0026thinsp;A (p. Glu336Lys) in the \u003cem\u003eAIFM1\u003c/em\u003e gene (Xq26.1). Family history revealed that his mother and daughter are carriers of the same mutation, and his brother is also affected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eNatural History and Clinical Progression\u003c/h2\u003e \u003cp\u003eThe patient was born in 1961. Since childhood, he exhibited hypotonia, generalized areflexia, and pes cavus, for which he underwent orthopedic surgery at the age of 8. He also developed progressive bilateral sensorineural hearing loss, requiring hearing aids by age 25. By age 41, hearing loss reached 90% bilaterally. A right cochlear implant (Nucleus 7) was placed at age 57 with partial benefit. He underwent cataract surgery at age 38.\u003c/p\u003e \u003cp\u003eMotor disability followed a slowly progressive course. At the age of 32, he discontinued playing tennis due to fatigue and weakness in the lower limbs. At 38, he required unilateral walking support. He was evaluated at the neuromuscular unit of Hospital Universitario Miguel Servet at the age of 41 (in 2003). Severe gait ataxia was documented. Neurological exam showed visual acuity of 0.8 bilaterally, global weakness graded 4/5 in upper limbs and pelvic girdle, and 1\u0026ndash;2/5 in foot flexors/extensors, with marked distal amyotrophy producing a \"stork leg\" appearance and post-surgical clubfoot. He had global areflexia, preserved thermal sensitivity, and reduced tactile and pain sensation in a glove-and-stocking pattern. Joint position and vibration senses were abolished in lower limbs and decreased distally in upper limbs. Mild finger-to-nose ataxia was present; lower limb ataxia was severe and disabling. No truncal ataxia. Cardiovascular exam was normal. Associated symptoms included oscillopsia and nystagmus diagnosed by ophthalmology.\u003c/p\u003e \u003cp\u003eMetabolic testing was normal. Brain Magnetic Resonance Imaging (MRI) and cardiological evaluation were unremarkable. A heterozygous GAA repeat expansion (250 repeats) in FRDA/X25 led to a provisional diagnosis of Friedreich's ataxia. As compound heterozygosity was suspected, further studies were initiated.\u003c/p\u003e \u003cp\u003eBy age 45 (2007), he needed a wheelchair for distances\u0026thinsp;\u0026gt;\u0026thinsp;50 meters. Manual dexterity declined significantly, affecting eating and typing. He required assistance with all ADLs. Arm strength was preserved (5/5), though clumsy fine motor control was noted. Pansensory hypoesthesia in lower limbs and reduced sensation in arms.\u003c/p\u003e \u003cp\u003eIn 2009, FRDA repeat expansion testing was repeated, excluding Friedreich\u0026rsquo;s ataxia. GDAP1 testing (2010) was negative for pathogenic variants.\u003c/p\u003e \u003cp\u003eAt age 55 (2016), genetic revaluation revealed a hemizygous c.1006G\u0026thinsp;\u0026gt;\u0026thinsp;A (p. Glu336Lys) mutation in exon 10 of \u003cem\u003eAIFM1\u003c/em\u003e (NM_004208.4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This change results in the substitution of glutamic acid with lysine at position 336 of the protein (p.Glu336Lys), generating a missense mutation. This variant has not been reported in population databases (gnomAD v4.1.0, exomeAD). The results of bioinformatics analysis systems for predicting the effect of mutations indicate, in 7 out of 9 prediction tools used (PROVEAN, SIFT, PolyPhen2, LRT, MutationTaster, MutationAssessor, and CONDEL), that this is a deleterious variant. Currently, this variant is reported in the ClinVar database (ID: 641733) as a likely pathogenic variant (SCV000934480.7) and as a variant of uncertain significance (SCV004036880.1, SCV002765018.3). Using ACMG criteria, this variant is classified as likely pathogenic (PP3, PP5, PM1, PM2)[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].This new mutation confirmed Cowchock syndrome, being the clinical status of the patient stable but severely disabled. Patient was wheelchair-bound, with severe impairments in fine motor skills, worsened by deafness and oscillopsia. Occasional choking episodes with aspiration and self-limited vestibular symptoms were reported. He developed muscle contractures that were refractory to all muscle relaxants and showed a fluctuating course. Repeated brain MRI and vestibulocochlear nerve imaging were unremarkable. Visual evoked potentials revealed a delayed P100 latency at 153 ms, while brainstem auditory evoked potentials were bilaterally absent. Identification of the AIFM1 mutation prompted\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn 2017, the patient continued to exhibit profound motor and sensory impairment, with complete dependence in activities of daily living (ADLs) except for feeding. Communication was limited to lip-reading. Riboflavin therapy was maintained.\u003c/p\u003e \u003cp\u003eBy 2019, the cochlear implant was functioning effectively. The patient remained physically stable, ambulating short distances with bilateral support and driving long distances. Manual dexterity was slower but preserved. Muscle strength was graded 5/5 in the upper limbs and approximately 4+/5 in the hands. Distal tactile, vibratory, and proprioceptive hypoesthesia persisted, without evidence of clinical progression.\u003c/p\u003e \u003cp\u003eHe reported mood instability characterized by depressive and irritable episodes, as well as sleep disturbances. Riboflavin was reduced to 50 mg twice daily due to gastrointestinal intolerance.\u003c/p\u003e \u003cp\u003eFamily genetic studies confirmed that his mother, and daughter were carriers of the AIFM1 variant. His brother was also clinically affected, with a phenotype consistent with polyneuropathy.\u003c/p\u003e \u003cp\u003eAnnual follow-ups from 2021 to 2024 documented persistent chronic shoulder pain interfering with sleep, together with a slow progression of motor and sensory deficits. Manual skills gradually declined, with frequent dropping of objects and difficulty fastening buttons. At home, he ambulated with a cane and demonstrated brachial strength of 4\u0026thinsp;+\u0026thinsp;distally and 5/5 proximally. Distal sensory loss remained evident, with absent deep tendon reflexes (0/4). Cognitive function was preserved, and mood remained stable.\u003c/p\u003e \u003cp\u003eA graphical timeline showing the progression of the patient\u0026rsquo;s disability is presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe following complementary studies were carried out to aid in the phenotypic characterization of the patient: i) Neurophysiological evaluation (EMG/NCS) demonstrated a severe, length-dependent, axonal sensorimotor polyneuropathy without signs of active denervation. ii) Sensory nerve conduction studies revealed absent responses in the sural and superficial peroneal nerves bilaterally, and reduced amplitudes with delayed latencies in the median and ulnar nerves, consistent with moderate sensory neuropathy. iii) Motor conduction studies showed absent responses in the common peroneal and tibial nerves bilaterally, and reduced amplitudes with mild to moderate slowing in the median and ulnar nerves. iv) Electromyographic examination revealed mixed recruitment patterns without spontaneous activity. The detailed findings of the study are summarized in (Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results are consistent with a severe axonal sensorimotor neuropathy, supporting the clinical diagnosis of Charcot-Marie-Tooth disease type X4 (CMTX4, Cowchock syndrome), subsequently confirmed by genetic testing AIFM1 mutation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAudiometric findings before and after cochlear implantation in patient holding the E336K AIF mutation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePre-implant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePost-implant (Right Cochlear Implant)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHearing thresholds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSevere-to-profound bilateral sensorineural hearing loss (\u0026asymp;\u0026thinsp;90% by age 41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClear improvement in free-field thresholds\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHearing aids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRequired since age 25, with limited benefit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNot required, replaced by CI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSentence comprehension\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeverely impaired\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026asymp;\u0026thinsp;90% sentence comprehension with implant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpeech recognition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMinimal, lip-reading required\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSignificantly improved with combination of lip-reading\u0026thinsp;+\u0026thinsp;implant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeft ear outcome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProfound hearing loss, no functional benefit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePersistent profound hearing loss, no functional benefit\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOverall functional outcome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSevere bilateral anacusis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePartial functional recovery with right CI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eSummary of the most relevant parameters, including hearing thresholds, speech comprehension, and functional outcomes. Marked improvement was observed after right cochlear implantation, with partial recovery of auditory performance despite persistent profound hearing loss in the left ear. CI: cochlear implant.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eImpact of the E336K mutation on OXPHOS performance\u003c/h2\u003e \u003cp\u003eTo investigate the impact of the E336K mutation on AIF cellular functions, immortalized fibroblast lines were established from skin biopsies of the affected individual and a healthy control. To analyze whether the mutation alters transcriptional regulation or protein stability, AIF expression was assessed at both the mRNA and protein levels using quantitative RT-PCR and Western blot analysis, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, although the relative expression levels of AIF mRNA in mutant fibroblasts were comparable to those of control cells, the amount of AIF protein was significantly reduced, suggesting decreased stability of the mutant protein. Given AIF\u0026rsquo;s role in mitochondrial biogenesis and function, we next analyzed the impact of the mutation on OXPHOS performance. Mutant fibroblasts exhibited significantly impaired growth when cultured in galactose medium compared to glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating defective OXPHOS capacity. This impairment was confirmed by direct measurements of oxygen consumption in intact cells, which revealed an average\u0026thinsp;~\u0026thinsp;50% reduction in basal respiration compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Results of cell growth sensitivity to different inhibitors of respiratory complexes shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE suggest that this reduction might be mainly due to defects in mitochondrial complexes I and III function as mutant cells display significantly more sensitivity than control cells to rotenone and antimycin A, specific inhibitors of these complexes. To further investigate this, the structural organization of mitochondrial respiratory complexes and supercomplexes (SCs) was analyzed by native electrophoresis followed by Western Blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), where mutant cells displayed reduced incorporation of complex III into supercomplexes (SCs), accompanied by an increase in both its free dimeric form (CIII₂) and in CIII\u0026ndash;CIV assemblies. This is consistent with defective respirasome formation or stability, possibly linked to reduced levels of CI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo better understand the molecular basis for mitochondrial dysfunction and disease associated with the E336K mutation in AIF, we assessed the expression of CHCHD4 -the physiological mitochondrial inner membrane partner of AIF [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]- both at the mRNA and protein levels. The mutation of AIF negatively affects to CHCHD4 both mRNA and protein expression, as its relative levels are significantly reduced compared to those of control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In line with these observations, mitochondrial mass levels, evaluated both by MitoTracker\u0026trade; Green staining followed by flow cytometry and mtDNA copy number quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) were significantly reduced in E336K cells. To further investigate the effects of this impairment on the biogenesis of the OXPHOS system, the steady-state levels of mitochondrial respiratory complexes subunits were quantified on whole cell extract, showing a significant decrease of the relative levels of complex III and IV subunits in mutant cells when compared to those of control fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Thus, the levels of UQCRC1(CIII) normalized by actin, were reduced by approximately 50%, while the CO1 (CIV) signal decreased to ~\u0026thinsp;20% of that observed in control cells. Although not statistically significant, the relative levels of CI subunits were also reduced, whereas SDHA, a CII subunit, remained unchanged.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, we investigated whether these alterations affect mitochondrial ROS production, which could potentially affect the disease phenotype. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the production of mitochondrial superoxide, quantified by flow cytometry following cell staining with MitoSOX, was significantly lower in mutant cells compared to controls. Although this observation seems in contradiction with the mitochondrial dysfunction, this difference was abolished when ROS levels were normalized to mitochondrial mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Moreover, mitochondrial superoxide dismutase (SOD2) mRNA expression was significantly decreased in mutant cells (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA), indicating that the reduced ROS signal is not due to compensatory upregulation of mitochondrial antioxidant defenses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, the E336K mutation reduces AIF protein stability, impairs OXPHOS capacity through defective assembly of respiratory supercomplexes, decreases CHCHD4 expression, and lowers mitochondrial mass, altogether leading to compromised mitochondrial function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eThe E336K mutation alters redox properties, conformational dynamics, and CHCHD4 interaction in AIF\u003c/h2\u003e \u003cp\u003eTo better understand the molecular basis of the pathogenicity associated with the E336K substitution here identified, we further characterized the recombinant mutant protein using the synthetic constructs E336K\u003csub\u003e∆77\u003c/sub\u003e and E336K\u003csub\u003e∆101,\u003c/sub\u003e which serve as a realistic model of, respectively, the soluble mitochondrial and apoptotic mutant isoforms studied in patient cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Both mutant isoforms were purified to homogeneity, but with markedly different outcomes. E336K\u003csub\u003e∆101\u003c/sub\u003e exhibited a UV-visible absorption spectrum comparable to that of the WT protein, displaying the characteristic flavin bands I and II at 451 and 380 nm, respectively, along with a shoulder at 467 nm (Fig. S2A). These features indicate that the flavin cofactor remained in the oxidized state and was properly incorporated into the protein. In contrast, protein form E336K\u003csub\u003e∆77\u003c/sub\u003e progressively lost the flavin cofactor during purification and handling, ultimately showing a strong tendency to precipitate. Due to the low purification yield and the reduced ability of the mitochondrial E336K\u003csub\u003e∆77\u003c/sub\u003e isoform to retain the FAD cofactor, subsequent analyses focused on the apoptotic E336K\u003csub\u003e∆101\u003c/sub\u003e variant.\u003c/p\u003e \u003cp\u003eThis mutant oxidized form, E336K\u003csub\u003e∆101ox\u003c/sub\u003e, displayed an apparent molecular weight (appMW) of ~\u0026thinsp;52 kDa, consistent with a monomeric state as determined by size exclusion chromatography (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Additionally, its CD spectral properties were comparable to those of WT, indicating that the mutation does not affect overall protein folding (Fig. S2B). In contrast, the mutant\u0026rsquo;s NADH reduced form, E336K\u003csub\u003e∆101rd\u003c/sub\u003e, showed significant alteration in its spectroscopic properties, including attenuation of the fluorescence quantum yield of at least one tryptophan and changes in the shape of the far-CD signals (Fig. S2 B-D). Moreover, upon NADH incubation, the mutant protein eluted as three peaks with appMW of ~\u0026thinsp;150, ~128 and ~\u0026thinsp;65 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), corresponding to a dimeric form, a dimer-monomer transition due to partial reoxidation, and a monomeric form, respectively [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These results indicate that the E336K\u003csub\u003e∆101\u003c/sub\u003e mutant retains its ability to form dimers under reducing conditions, although the dimers exhibit diminished stability and a slightly less compact dimeric assembly compared to the WT protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore the effects of the E336K mutation on AIF conformational stability, we determined the thermal stability of this mutant protein in the presence and absence of NADH (Table S2, Fig. S2E-F). The results indicate that the mutation negatively impacts the thermal stability of AIF\u003csub\u003e∆101ox\u003c/sub\u003e (Fig. S2E, Table S2), as evidenced by a decrease of ~\u0026thinsp;5\u0026ndash;6 degrees in both T\u003csub\u003em\u003c/sub\u003es associated with the unfolding process when compared with WT\u003csub\u003e∆101ox\u003c/sub\u003e,, while the unfolding mechanism remains unchanged. However, when evaluating the thermal stability of E336K in the presence of NADH, the coenzyme does not destabilize the mutant, in stark contrast to the WT behavior, being the two identified T\u003csub\u003em\u003c/sub\u003es close to those of E336K\u003csub\u003eox\u003c/sub\u003e and of NADH reduced WT\u003csub\u003e∆101\u003c/sub\u003e. Altogether, these findings suggest structural and dynamic differences between the WT and the E336K mutant, both in the absence of the coenzyme as well as upon its binding and flavin reduction. In view of these observations, we also investigated the impact of this mutation on AIF redox properties using different biochemical approaches. E336K\u003csub\u003e∆101\u003c/sub\u003e exhibited a strong decrease in coenzyme affinity regarding WT\u003csub\u003e∆101\u003c/sub\u003e, with a \u003cem\u003eK\u003c/em\u003em\u003csup\u003eNADH\u003c/sup\u003e value\u0026thinsp;~\u0026thinsp;6-fold higher than that of WT\u003csub\u003e∆101\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig. S3A). In contrast, the turnover number (\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e) remained similar, resulting in a\u0026thinsp;~\u0026thinsp;3-fold reduction in catalytic efficiency relative to WT\u003csub\u003e∆101\u003c/sub\u003e. Notably, the mutant displayed both a\u0026thinsp;~\u0026thinsp;2-fold higher turnover number and a\u0026thinsp;~\u0026thinsp;23-fold greater affinity when compared with WT\u003csub\u003e∆101\u003c/sub\u003e for NADPH compared to NADH (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig. S3B). Consequently, the E336K\u003csub\u003e∆101\u003c/sub\u003e variant was substantially more efficient at oxidizing NADPH than the WT protein, with an estimated\u0026thinsp;~\u0026thinsp;1000-fold increase in the \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e\u003csup\u003eNADPH\u003c/sup\u003e/\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e, thereby markedly reducing the specificity for NADH over NADPH compared to the WT\u003csub\u003e∆101\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSteady-state kinetic parameters of AIF∆101 variants with NADH and NADPH as hydride donors\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariants\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eNADH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eNADPH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSpecificity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{k}}_{\\mathbf{c}\\mathbf{a}\\mathbf{t}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(s\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{K}}_{\\varvec{m}}^{\\varvec{N}\\varvec{A}\\varvec{D}\\varvec{H}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(mM)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{k}}_{\\varvec{c}\\varvec{a}\\varvec{t}}/{\\varvec{K}}_{\\varvec{m}}^{\\varvec{N}\\varvec{A}\\varvec{D}\\varvec{H}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(s\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emM\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{k}}_{\\mathbf{c}\\mathbf{a}\\mathbf{t}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(s\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{K}}_{\\varvec{m}}^{\\varvec{N}\\varvec{A}\\varvec{D}\\varvec{P}\\varvec{H}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(mM)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{k}}_{\\mathbf{c}\\mathbf{a}\\mathbf{t}}/{\\varvec{K}}_{\\mathbf{m}}^{\\varvec{N}\\varvec{A}\\varvec{D}\\varvec{P}\\varvec{H}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(s\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emM\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eNADH/NADPH\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eefficiency\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWT\u003c/b\u003e\u003csub\u003e\u003cb\u003e∆101\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE336K\u003c/b\u003e\u003csub\u003e\u003cb\u003e∆101\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e40\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0225\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003eAssays were performed using DCPIP as hydride acceptor at 25 \u0026ordm;C in 50 mM potassium phosphate, pH 7.4. (n\u0026thinsp;=\u0026thinsp;3, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD);\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePre-steady state kinetic parameters of AIF∆101 variants with NADH as hydride donor\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariants\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003ePre-steady-sate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCTC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{k}}_{\\mathbf{H}\\mathbf{T}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(s\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{K}}_{\\varvec{d}}^{\\varvec{N}\\varvec{A}\\varvec{D}\\varvec{H}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(mM)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{k}}_{\\mathbf{H}\\mathbf{T}}/{\\varvec{K}}_{\\mathbf{d}}^{\\varvec{N}\\varvec{A}\\varvec{D}\\varvec{H}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(s\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emM\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eHalf-life\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(min)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWT\u003c/b\u003e\u003csub\u003e\u003cb\u003e∆101\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1 \u0026plusmn; 0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.3 \u0026plusmn; 0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.30\u0026thinsp;+\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE336K\u003c/b\u003e\u003csub\u003e\u003cb\u003e∆101\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eAssays were performed at 25 \u0026ordm;C in 50 mM potassium phosphate, pH 7.4. (n\u0026thinsp;=\u0026thinsp;3, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD); NS: not stabilized under assayed conditions.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo assess the mutation impact on hydride transfer (HT) from the NADH coenzyme to the FAD cofactor, we performed stopped-flow transient analysis (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig. S3C). In E336K\u003csub\u003e∆101\u003c/sub\u003e, as in WT\u003csub\u003e∆101\u003c/sub\u003e, the complete FAD reduction was accompanied by a progressive formation of isoalloxazine:NAD\u003csup\u003e+\u003c/sup\u003e CTC, as in WT\u003csub\u003e∆101\u003c/sub\u003e and with similar spectral intensity, suggesting a comparable extent of CTC stabilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F). However, unlike the native protein \u0026ndash;where the CTC had a half-life time of 20 minutes\u0026ndash; E336K\u003csub\u003e∆101\u003c/sub\u003e did not reach full reduction at stoichiometric molar coenzyme concentrations. This incomplete reduction impeded CTC stabilization and precluded assessment of its reactivity towards O\u003csub\u003e2\u003c/sub\u003e. Furthermore, the mutant exhibited faster HT (~\u0026thinsp;2-fold increase) and lower coenzyme affinity (~\u0026thinsp;2-fold decrease), while maintaining similar HT efficiency (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results demonstrate that the E336K mutation significantly compromises the redox properties of AIF by decreasing NADH binding affinity and destabilizing the CTC.\u003c/p\u003e \u003cp\u003eConsidering previous findings and the relevance of the AIF dimer stabilization in forming a long-lived complex with CHCHD4 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], we investigated the impact of the mutation on this interaction using ITC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and Table S3). As previously reported for WT\u003csub\u003e∆101ox\u003c/sub\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], no heat exchange was detected when titrating E336K\u003csub\u003e∆101ox\u003c/sub\u003e with CHCHD4, indicating a lack of specific binding under assayed conditions. In the presence of NADH, the E336K\u003csub\u003e∆101rd\u003c/sub\u003e mutant was able to bind CHCHD4, but with slightly lower affinity than WT\u003csub\u003e∆101rd\u003c/sub\u003e (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e ~ 5-fold lower than for WT\u003csub\u003e∆101rd\u003c/sub\u003e:CHCHD4 complex) and with notable differences in thermodynamic contributions to the binding. The WT\u003csub\u003e∆101rd\u003c/sub\u003e:CHCHD4 interaction was mainly driven by a strong favorable enthalpic contribution, typical of specific binding, while the entropic term was unfavorable. In contrast, the E336K∆\u003csub\u003e101rd\u003c/sub\u003e:CHCHD4 complex showed a weaker binding enthalpic contribution and even a favorable entropic term, suggesting that non-specific forces may play a more prominent role. This altered thermodynamic profile could impair the formation of a functional E336K\u003csub\u003e∆101rd\u003c/sub\u003e:CHCHD4 complex, which is crucial for mitochondrial homeostasis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImpact of E336K mutation on AIF structure\u003c/h3\u003e\n\u003cp\u003eTo investigate the structural impact of the E336K mutation, the X-ray crystal structure of E336K\u003csub\u003eox\u003c/sub\u003e was resolved and evaluated in the context of structures for monomeric WT\u003csub\u003eΔ101ox\u003c/sub\u003e and dimeric WT\u003csub\u003eΔ101rd\u003c/sub\u003e:NAD\u003csup\u003e+\u003c/sup\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Superposition of WT\u003csub\u003eΔ101ox\u003c/sub\u003e and E336K\u003csub\u003eox\u003c/sub\u003e revealed a high degree of structural similarity (r.m.s.d. = 0.21 \u0026Aring; for 451 atoms), with both structures sharing similar overall fold. Comparison with WT\u003csub\u003eΔ101rd\u003c/sub\u003e: NAD\u003csup\u003e+\u003c/sup\u003e (r.m.s.d. = 1.06 \u0026Aring; for 400 atoms) showed that the conformational rearrangements of the β-hairpin and regulatory C-loop associated with coenzyme binding were absent in E336K\u003csub\u003eox\u003c/sub\u003e. This suggests that the E336K substitution, in agreement with the monomeric conformation observed by size exclusion chromatography, does not promote permissive dimer formation as reported for other AIF variants [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn WT\u003csub\u003eΔ101ox\u003c/sub\u003e and WT\u003csub\u003eΔ101rd\u003c/sub\u003e:NAD\u003csup\u003e+\u003c/sup\u003e structures, the side chain of E336 adopts a similar orientation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Notably, in the WT\u003csub\u003eΔ101rd\u003c/sub\u003e:NAD\u003csup\u003e+\u003c/sup\u003e structure, the E336 carboxylate oxygen atoms (O2B and O3B) hydrogen bond to the hydroxyl groups of the ribityl of the adenine nucleotide moiety of NAD\u003csup\u003e+\u003c/sup\u003e (sitting at 2.8 and 3.5 \u0026Aring; respectively) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Substitution of E336 with lysine may disrupt such interaction, as the positively charged and longer lysine side chain might preclude an orientation conducive to coenzyme hydrogen bonding (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Furthermore, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, the mutation modifies the electrostatic landscape of the environment of residue 336, particularly in the cleft where the ribose of the adenine nucleotide moiety of the coenzyme binds. This surface becomes more positive and potentially eager to accommodate the 2\u0026rsquo;-phosphorylated ribose form of the coenzyme, NADPH. This is exemplified in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, where based on the WT\u003csub\u003eΔ101rd\u003c/sub\u003e:NAD\u003csup\u003e+\u003c/sup\u003e structure a model for the E336K\u003csub\u003eΔ101rd\u003c/sub\u003e:NADP\u003csup\u003e+\u003c/sup\u003e interaction is presented, envisaging the interaction between the 2\u0026prime;-P of NADPH and residues K336 and K342. Such differences in surface charge distribution between the WT and the mutant may correlate with observed stability and enzymatic activity alterations, and are particularly in line with E336K preference for using NADPH as hydride donor (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eImpact of the E336K mutation on AIF-mediated cell death and nuclear interactions\u003c/h2\u003e \u003cp\u003eAs mentioned, besides its role in mitochondrial function and biogenesis, AIF also participates in a caspase-independent cell death pathway known as parthanatos [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. To analyze whether the E3336K variant alters this function, control and mutant fibroblasts were exposed to the alkylating agent MNNG for 20 minutes. After 24 hours, cell death was quantified by flow cytometry using annexin V-FITC and 7-AAD staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, both control and mutant cells underwent cell death; however, the percentage of dead cells was significantly lower in cultures expressing the E336K mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Consistently, MTT assays revealed greater growth inhibition in mutant cells compared to controls, with viabilities of 22% and 45.9%, respectively. These results indicate that although the E336K mutation does not abolish AIF-mediated cell death, its efficiency in fibroblasts appears reduced (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpon apoptotic-stimuli, AIF translocates from the mitochondria to the nucleus, where it is proposed to form a DNA-degradosome complex \u0026ndash;through interactions with proteins such as the endonuclease CypA\u0026ndash; essential for the caspase-independent apoptotic pathway[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. To assess the impact of the E336K mutation on AIF\u0026rsquo; s interactions with its nuclear partners, we performed ITC using dsDNA and CypA as ligands (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, Fig. S4C-F and Table S3). Both WT and E336K\u003csub\u003eΔ101\u003c/sub\u003e proteins exhibited similar binding affinities toward dsDNA and CypA, with \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e in the micromolar range for both interactions. The thermodynamic profiles of the interactions with dsDNA were also comparable between variants and predominantly entropy-driven, consistent with non-specific electrostatic interactions previously described for WT AIF. EMSA further confirmed similar DNA-binding behavior, with both variants exhibiting comparable levels of DNA retention in the gels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). These results suggest that the E336K mutation does not significantly affect AIF's ability to bind DNA.\u003c/p\u003e \u003cp\u003eNotably, some differences were observed in the interaction with CypA. WT\u003csub\u003eΔ101\u003c/sub\u003e exhibited a strong enthalpy-driven interaction with a modest positive entropy change, indicative of a specific well-defined binding interface [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In contrast, the pathological variant showed a slightly weaker enthalpic contribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and Table S3), potentially reflecting minor conformational rearrangements upon complex formation.\u003c/p\u003e \u003cp\u003eGiven the recently reported intrinsic nuclease activity of AIF [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], we investigated the impact of the E336K mutation on AIF-mediated DNA degradation. The E336K mutation does not suppress the intrinsic DNA-cleaving capability of AIF when using purified plasmid DNA as substrate., exhibiting an activity comparable to that of the WT protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eIn summary, these findings demonstrate that the E336K mutation does not abolish AIF\u0026rsquo;s capacity to trigger cell death or to interact with nuclear partners such as DNA and CypA, although it may modestly reduce the efficiency of parthanatos induction and subtly affect the thermodynamics of CypA binding.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides the first integrated clinical, cellular and molecular mechanistic characterization of the novel pathogenic AIFM1 variant E336K. This combined approach significantly broadens the mechanistic and phenotypic spectrum of AIF-related disease. Our data indicate that the pathological impact of E336K is primarily driven by loss of AIF\u0026rsquo;s pro-survival, mitochondria-supporting, function rather than by impairment of its pro-death role [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Clinically, the patient presented with progressive axonal sensorimotor neuropathy and sensorineural hearing loss, while cognition remained preserved \u0026ndash;a phenotype consistent with CMTX4/Cowchock syndrome and well within the established spectrum of AIFM1\u0026ndash;related disorders. These findings underscore both the diagnostic challenges associated with AIFM1 mutations and the importance of variant-specific functional analyses to refine genotype\u0026ndash;phenotype correlations [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAt the cellular level, patient-derived fibroblasts displayed reduced AIF protein, impaired OXPHOS capacity, and defective organization of respiratory supercomplexes. Notably, CHCHD4 expression and mitochondrial mass were decreased, coherently linking the cellular phenotype to the AIF\u0026ndash;CHCHD4 axis, which sustains the import, oxidative folding, and assembly of specific respiratory components [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Crystallographic analysis provides a structural rationale: substitution of Glu336 with Lys remodels the electrostatic potential of the coenzyme binding pocket, precisely at the site of the adenosine-ribose moiety of NADH \u0026ndash;a key region for stabilizing the charge transfer complex (CTC) and mediating redox-driven conformational changes in AIF [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. These defects converge on a primary disruption of AIF\u0026rsquo;s pro-survival role in mitochondrial maintenance and respiratory chain biogenesis, offering a unifying structural explanation for the patient\u0026rsquo;s biochemical phenotype [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Collectively, these alterations decrease AIF\u0026rsquo;s ability to sense mitochondrial NADH redox state and to sustain the conformational ensembles that underwrite its pro-survival functions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOf particular relevance are the results of the systematic analysis of AIF\u0026rsquo;s interaction network with physiological partners. Mechanistically, the reduced dimeric form of AIF interacts with CHCHD4 to support mitochondrial disulfide relay and respiratory chain assembly [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. While E336K retained the ability to dimerize, the resulting dimers were less stable and less compact. Moreover, its interaction with CHCHD4 displayed markedly reduced affinity and altered thermodynamic signatures, indicating a weakened and less specific interacting interface. These molecular changes provide a direct link to the observed cellular phenotypes \u0026ndash;decreased CHCHD4 levels, reduced mitochondrial mass, and disorganized supercomplexes\u0026ndash; and offer a cohesive explanation for the OXPHOS insufficiency observed in patient-derived fibroblasts. In contrast, the apoptogenic functions of AIF were largely preserved: DNA binding, CypA interaction, and nuclease activity remained intact, while parthanatos induction was only attenuated, possibly due to the observed impact of mutation on mutant stability. Interestingly, a closely located pathogenic variant (M340T) has been associated with a similar clinic-biological dissociation \u0026ndash;dominant mitochondrial dysfunction with preserved apoptotic competence\u0026ndash; further reinforcing this mechanistic theme within the AIFM1 spectrum [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom a translational perspective, these findings highlight the need to integrate structure-function studies into the interpretation of AIFM1 variants, particularly in X-linked neuropathies with hearing loss. Therapeutic strategies based on cofactor supplementation may hold promise, especially in cases with impaired FAD retention and stability, as observed in this study. The shift toward NADPH utilization further suggests a possible compensatory route that could be therapeutically leveraged. More broadly, targeting NADH-driven allosteric regulation, stabilizing the CTC, or enhancing the CHCHD4 pathway robustness represent concrete, testable avenues for future intervention.\u003c/p\u003e \u003cp\u003eIn summary, our work provides the first comprehensive clinical-to-molecular characterization of an AIFM1 mutation. The E336K mutation exemplifies a recurring principle in AIFM1-related disease: variants that disrupt the NADH-sensing/redox cycle primarily erode mitochondrial-supporting functions while leaving apoptotic roles relatively intact. By reshaping the coenzyme-binding electrostatics, destabilizing the NADH-dependent CTC, and weakening CHCHD4 engagement, E336K triggers a pathogenic cascade that explains the selective vulnerability of high-energy tissues such as peripheral nerves, the inner ear, and muscle. This work expands the clinical and mechanistic spectrum of AIFM1 disease, refines the interpretation of AIFM1 variants, and provides a foundation for exploring mechanism-based therapeutic strategies, including cofactor supplementation and targeted interventions aimed at stabilizing AIF-CHCHD4 function.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e The study was conducted in accordance with the Declaration of Helsinki. Ethical approval C.P.-C.I. PI18/224 was obtained from Comit\u0026eacute; de \u0026Eacute;tica de la Investigaci\u0026oacute;n de la Comunidad Aut\u0026oacute;noma de Arag\u0026oacute;n (CEICA), Zaragoza, Spain. Written informed consent for participation in this study was obtained from the patient.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003e All authors have approved the manuscript.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eSupplementary information\u003c/h2\u003e \u003cp\u003eThe only version contains supplementary material available:\u003c/p\u003e \u003cp\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e to Figure S4 and supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e to Table S5.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was funded by the Spanish State Research Agency and by FEDER (MCIN/AEI-FEDER, Grants PID2022-136369NB-I00, Grant PID2021-124354NB-I00), as well as by the Gobierno de Arag\u0026oacute;n, grant number \u0026ldquo;Grupo de Referencia E35_17R\u0026rdquo; to M.M-M., M.M, P.F.-S., R.M.-L. and P.F. and grant number \u0026ldquo;LMP220_21\u0026rdquo; to P.F.-S. and R.M.-L. The authors would like to acknowledge \u003cem\u003eServicios Generales de Apoyo a la Investigaci\u0026oacute;n\u003c/em\u003e (SAI), University of Zaragoza, for their support, as well as at BIFI-University of Zaragoza for providing instrumentation.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: P.F and R.M-L; Data curation: P.F., R.M.-L., M.M.J., A.V.-C., P.F.-S., M.M and M.B.; Funding acquisition: P.F., R.M.-L, M.M. and P.F.-S.; Investigation: R.M.-L, M.P, R. G-V. and P. F-S (cell culture, respiration and viability analysis), J. M.-B. and C. R-Y (Cytometer measurements), M.F., O.S., M.M.-B. and P.F (protein production and molecular characterization); M.F and M.M.-J (crystallization and structure determination) M.D.M. (genetic analysis) and M.B. (clinical studies). Writing original draft: P.F., R.M.-L., M.F., O.S, M.M-J. and M.B. Review \u0026amp; editing: all authors. All authors have given approval to final version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe structural data for the AIF E336K variant generated in this study by X-ray crystallography have been deposited in the Protein Data Bank under accession code PDB 9SZQ.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAcin-Perez R, Bayona-Bafaluy MP, Bueno M, Machicado C, Fernandez-Silva P, Perez-Martos A, Montoya J, Lopez-Perez MJ, Sancho J, Enriquez JA. An intragenic suppressor in the cytochrome c oxidase I gene of mouse mitochondrial DNA. Hum Mol Genet. 2003;12:329\u0026ndash;39. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/hmg/ddg021\u003c/span\u003e\u003cspan address=\"10.1093/hmg/ddg021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAcin-Perez R, Fernandez-Silva P, Peleato ML, Perez-Martos A, Enriquez JA. Respiratory active mitochondrial supercomplexes. Mol Cell. 2008;32:529\u0026ndash;39. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molcel.2008.10.021\u003c/span\u003e\u003cspan address=\"10.1016/j.molcel.2008.10.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAfonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A, Zwart PH, Adams PD. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr. 2012;68:352\u0026ndash;67. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1107/S0907444912001308\u003c/span\u003e\u003cspan address=\"10.1107/S0907444912001308\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArdissone A, Piscosquito G, Legati A, Langella T, Lamantea E, Garavaglia B, Salsano E, Farina L, Moroni I, Pareyson D et al. (2015) A slowly progressive mitochondrial encephalomyopathy widens the spectrum of AIFM1 disorders. Neurology 84: 2193\u0026ndash;2195 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1212/WNL.0000000000001613\u003c/span\u003e\u003cspan address=\"10.1212/WNL.0000000000001613\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArtus C, Boujrad H, Bouharrour A, Brunelle MN, Hoos S, Yuste VJ, Lenormand P, Rousselle JC, Namane A, England P al. AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX. EMBO J. 2010;29:1585\u0026ndash;99. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/emboj.2010.43\u003c/span\u003e\u003cspan address=\"10.1038/emboj.2010.43\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBano D, Prehn JHM. Apoptosis-Inducing Factor (AIF) in Physiology and Disease: The Tale of a Repented Natural Born Killer. EBioMedicine. 2018;30:29\u0026ndash;37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ebiom.2018.03.016\u003c/span\u003e\u003cspan address=\"10.1016/j.ebiom.2018.03.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBogdanova-Mihaylova P, Alexander MD, Murphy RP, Chen H, Healy DG, Walsh RA, Murphy SM. Clinical spectrum of AIFM1-associated disease in an Irish family, from mild neuropathy to severe cerebellar ataxia with colour blindness. J Peripher Nerv Syst. 2019;24:348\u0026ndash;53. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/jns.12348\u003c/span\u003e\u003cspan address=\"10.1111/jns.12348\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrosey CA, Ho C, Long WZ, Singh S, Burnett K, Hura GL, Nix JC, Bowman GR, Ellenberger T, Tainer JA. (2016) Defining NADH-Driven Allostery Regulating Apoptosis-Inducing Factor. Structure: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.str.2016.09.012\u003c/span\u003e\u003cspan address=\"10.1016/j.str.2016.09.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrosey CA, Shen R, Tainer JA. (2025) NADH-bound AIF activates the mitochondrial CHCHD4/MIA40 chaperone by a substrate-mimicry mechanism. EMBO J. \u0026copy; 2025. The Author(s). City.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCowchock FS, Duckett SW, Streletz LJ, Graziani LJ, Jackson LG. X-linked motor-sensory neuropathy type-II with deafness and mental retardation: a new disorder. Am J Med Genet. 1985;20:307\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ajmg.1320200214\u003c/span\u003e\u003cspan address=\"10.1002/ajmg.1320200214\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelano WL. PyMOL: an open-source molecular graphics tool. CCP4 Newsl Protein Crystallogr. 2002;40:82\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelavall\u0026eacute;e L, Mathiah N, Cabon L, Mazeraud A, Brunelle-Navas MN, Lerner LK, Tannoury M, Prola A, Moreno-Loshuertos R. Baritaud M (2020) Mitochondrial AIF loss causes metabolic reprogramming, caspase-independent cell death blockade, embryonic lethality, and perinatal hydrocephalus. Mol Metab: 101027 Doi \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molmet.2020.101027\u003c/span\u003e\u003cspan address=\"10.1016/j.molmet.2020.101027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDolinsky TJ, Nielsen JE, McCammon JA, Baker NA. PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 2004;32:W665\u0026ndash;667. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkh381\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkh381\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEmsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66:486\u0026ndash;501. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1107/S0907444910007493\u003c/span\u003e\u003cspan address=\"10.1107/S0907444910007493\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEvans P. Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr. 2006;62:72\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1107/S0907444905036693\u003c/span\u003e\u003cspan address=\"10.1107/S0907444905036693\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFagnani E, Cocomazzi P, Pellegrino S, Tedeschi G, Scalvini FG, Cossu F, Da Vela S, Aliverti A, Mastrangelo E, Milani M. CHCHD4 binding affects the active site of apoptosis inducing factor (AIF): Structural determinants for allosteric regulation. Structure. \u0026copy; 2024 The Author(s). City: Published by Elsevier Inc; 2024. pp. 594\u0026ndash;602. e594.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarina B, Di Sorbo G, Chambery A, Caporale A, Leoni G, Russo R, Mascanzoni F, Raimondo D, Fattorusso R, Ruvo M et al. (2017) Structural and biochemical insights of CypA and AIF interaction. Sci Rep 7: 1138 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-017-01337-8\u003c/span\u003e\u003cspan address=\"10.1038/s41598-017-01337-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarina B, Sturlese M, Mascanzoni F, Caporale A, Monti A, Di Sorbo G, Fattorusso R, Ruvo M, Doti N. Binding mode of AIF(370\u0026ndash;394) peptide to CypA: insights from NMR, label-free and molecular docking studies. Biochem J. 2018;475:2377\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1042/BCJ20180177\u003c/span\u003e\u003cspan address=\"10.1042/BCJ20180177\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerreira P, Villanueva R, Martinez-Julvez M, Herguedas B, Marcuello C, Fernandez-Silva P, Cabon L, Hermoso JA, Lostao A, Susin SA al. Structural insights into the coenzyme mediated monomer-dimer transition of the pro-apoptotic apoptosis inducing factor. Biochemistry. 2014;53:4204\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/bi500343r\u003c/span\u003e\u003cspan address=\"10.1021/bi500343r\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhezzi D, Sevrioukova I, Invernizzi F, Lamperti C, Mora M, D'Adamo P, Novara F, Zuffardi O, Uziel G, Zeviani M. Severe X-linked mitochondrial encephalomyopathy associated with a mutation in apoptosis-inducing factor. Am J Hum Genet. 2010;86:639\u0026ndash;49. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ajhg.2010.03.002\u003c/span\u003e\u003cspan address=\"10.1016/j.ajhg.2010.03.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHangen E, Feraud O, Lachkar S, Mou H, Doti N, Fimia GM, Lam NV, Zhu C, Godin I, Muller Ket al, et al. Interaction between AIF and CHCHD4 Regulates Respiratory Chain Biogenesis. Mol Cell. 2015;58:1001\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molcel.2015.04.020\u003c/span\u003e\u003cspan address=\"10.1016/j.molcel.2015.04.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeimer G, Eyal E, Zhu X, Ruzzo EK, Marek-Yagel D, Sagiv D, Anikster Y, Reznik-Wolf H, Pras E, Oz Levi Det al, et al. Mutations in AIFM1 cause an X-linked childhood cerebellar ataxia partially responsive to riboflavin. Eur J Paediatr Neurol. 2018;22:93\u0026ndash;101. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ejpn.2017.09.004\u003c/span\u003e\u003cspan address=\"10.1016/j.ejpn.2017.09.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHofhaus G, Shakeley RM, Attardi G. Use of polarography to detect respiration defects in cell cultures. Methods Enzymol. 1996;264:476\u0026ndash;83. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0076-6879(96)64043-9\u003c/span\u003e\u003cspan address=\"10.1016/s0076-6879(96)64043-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu B, Wang M, Castoro R, Simmons M, Dortch R, Yawn R, Li J. A novel missense mutation in AIFM1 results in axonal polyneuropathy and misassembly of OXPHOS complexes. Eur J Neurol. 2017;24:1499\u0026ndash;506. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/ene.13452\u003c/span\u003e\u003cspan address=\"10.1111/ene.13452\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoza N, Susin SA, Daugas E, Stanford WL, Cho SK, Li CY, Sasaki T, Elia AJ, Cheng HY, Ravagnan Let al, et al. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature. 2001;410:549\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/35069004\u003c/span\u003e\u003cspan address=\"10.1038/35069004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJurrus E, Engel D, Star K, Monson K, Brandi J, Felberg LE, Brookes DH, Wilson L, Chen J, Liles K et al. (2018) Improvements to the APBS biomolecular solvation software suite. Protein Sci 27: 112\u0026ndash;128 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/pro.3280\u003c/span\u003e\u003cspan address=\"10.1002/pro.3280\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKabsch W. Xds. Acta Crystallogr D Biol Crystallogr. 2010;66:125\u0026ndash;32. Doi 10.1107/S0907444909047337.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlein JA, Longo-Guess CM, Rossmann MP, Seburn KL, Hurd RE, Frankel WN, Bronson RT, Ackerman SL. The harlequin mouse mutation downregulates apoptosis-inducing factor. Nature. 2002;419:367\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature01034\u003c/span\u003e\u003cspan address=\"10.1038/nature01034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr. 2007;40:658\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1107/S0021889807021206\u003c/span\u003e\u003cspan address=\"10.1107/S0021889807021206\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. City: J Immunol Methods; 1983. pp. 55\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997;53:240\u0026ndash;55. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1107/S0907444996012255\u003c/span\u003e\u003cspan address=\"10.1107/S0907444996012255\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMussulini BHM, Maruszczak KK, Draczkowski P, Borrero-Landazabal MA, Ayyamperumal S, Wnorowski A, Wasilewski M, Chacinska A. (2025) MIA40 suppresses cell death induced by apoptosis-inducing factor 1. EMBO Rep. \u0026copy; 2025. The Author(s). City.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNovo N, Romero-Tamayo S, Marcuello C, Boneta S, Blasco-Machin I, Velazquez-Campoy A, Villanueva R, Moreno-Loshuertos R, Lostao A. Medina M (2023) Beyond a platform protein for the degradosome assembly: The Apoptosis-Inducing Factor as an efficient nuclease involved in chromatinolysis. PNAS Nexus 2: pgac312 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/pnasnexus/pgac312\u003c/span\u003e\u003cspan address=\"10.1093/pnasnexus/pgac312\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePandolfo M, Rai M, Remiche G, Desmyter L, Vandernoot I. Cerebellar ataxia, neuropathy, hearing loss, and intellectual disability due to AIFM1 mutation. Neurol Genet. 2020;6:e420. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1212/NXG.0000000000000420\u003c/span\u003e\u003cspan address=\"10.1212/NXG.0000000000000420\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReinhardt C, Arena G, Nedara K, Edwards R, Brenner C, Tokatlidis K, Modjtahedi N. AIF meets the CHCHD4/Mia40-dependent mitochondrial import pathway. Biochim Biophys Acta Mol Basis Dis. 2020;1866:165746. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbadis.2020.165746\u003c/span\u003e\u003cspan address=\"10.1016/j.bbadis.2020.165746\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405\u0026ndash;24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/gim.2015.30\u003c/span\u003e\u003cspan address=\"10.1038/gim.2015.30\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRinaldi C, Grunseich C, Sevrioukova IF, Schindler A, Horkayne-Szakaly I, Lamperti C, Landoure G, Kennerson ML, Burnett BG, Bonnemann Cet al, et al. Cowchock syndrome is associated with a mutation in apoptosis-inducing factor. Am J Hum Genet. 2012;91:1095\u0026ndash;102. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ajhg.2012.10.008\u003c/span\u003e\u003cspan address=\"10.1016/j.ajhg.2012.10.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomero-Tamayo S, Laplaza R, Velazquez-Campoy A, Villanueva R, Medina M, Ferreira P. (2021) W196 and the β-Hairpin Motif Modulate the Redox Switch of Conformation and the Biomolecular Interaction Network of the Apoptosis-Inducing Factor. Oxid Med Cell Longev 2021: 6673661 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2021/6673661\u003c/span\u003e\u003cspan address=\"10.1155/2021/6673661\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalscheider SL, Gerlich S, Cabrera-Orefice A, Peker E, Rothemann RA, Murschall LM, Finger Y, Szczepanowska K, Ahmadi ZA, Guerrero-Castillo Set al, et al. AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5. EMBO J. 2022;41:e110784.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSancho J (2013) The stability of 2-state, 3-state and more-state proteins from simple spectroscopic techniques\u0026hellip; plus the structure of the equilibrium intermediates at the same time. Arch Biochem Biophys 531: 4\u0026ndash;13 Doi 10.1016/j.abb.2012.10.014.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSancho P, Sanchez-Monteagudo A, Collado A, Marco-Marin C, Dominguez-Gonzalez C, Camacho A, Knecht E, Espinos C, Lupo V. A newly distal hereditary motor neuropathy caused by a rare AIFM1 mutation. Neurogenetics. 2017;18:245\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10048-017-0524-6\u003c/span\u003e\u003cspan address=\"10.1007/s10048-017-0524-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchagger H. Native electrophoresis for isolation of mitochondrial oxidative phosphorylation protein complexes. Methods Enzymol. 1995;260:190\u0026ndash;202. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0076-6879(95)60137-6\u003c/span\u003e\u003cspan address=\"10.1016/0076-6879(95)60137-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSevrioukova IF. Structure/Function Relations in AIFM1 Variants Associated with Neurodegenerative Disorders. J Mol Biol. 2016;428:3650\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jmb.2016.05.004\u003c/span\u003e\u003cspan address=\"10.1016/j.jmb.2016.05.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSusin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Costantini P, Loeffler M et al. (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397: 441\u0026ndash;446 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/17135\u003c/span\u003e\u003cspan address=\"10.1038/17135\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTunyi J, Abreu NJ, Tripathi R, Mathew MT, Mears A, Agrawal P, Thakur V, Rezai AR, Reyes EL. (2023) Deep Brain Stimulation for the Management of AIFM1-Related Disabling Tremor: A Case Series. Pediatr Neurol. \u0026copy; 2023 Elsevier Inc, City, pp 47\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVasquez A, Schimmenti LA, Demirel N, Rabatin AE, Fischer CR, Pinto MV, Boesch RP, Selcen D. Novel AIFM1 Variant in 2 Siblings With Sensorineural Hearing Loss and Cerebellar Ataxia. Neurol Genet. 2024;10:e200216. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1212/NXG.0000000000200216\u003c/span\u003e\u003cspan address=\"10.1212/NXG.0000000000200216\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillanueva R, Romero-Tamayo S, Laplaza R, Martinez-Olivan J, Velazquez-Campoy A, Sancho J, Ferreira P, Medina M. Redox- and Ligand Binding-Dependent Conformational Ensembles in the Human Apoptosis-Inducing Factor Regulate Its Pro-Life and Cell Death Functions. Antioxid Redox Signal. 2019;30:2013\u0026ndash;29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1089/ars.2018.7658\u003c/span\u003e\u003cspan address=\"10.1089/ars.2018.7658\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang B, Li X, Wang J, Liu L, Xie Y, Huang S, Pakhrin PS, Jin Q, Zhu C, Tang B et al. (2018) A novel AIFM1 mutation in a Chinese family with X-linked Charcot-Marie-Tooth disease type 4. Neuromuscul Disord. \u0026copy; 2018 Elsevier B.V, City, pp 652\u0026ndash;659.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang C, Lin Z, Yuan Z, Tang T, Fan L, Liu Y, Wu X. Whole-exome sequencing detected a novel AIFM1 variant in a Han-Chinese family with Cowchock syndrome. Hereditas. 2023;160:22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s41065-023-00282-z\u003c/span\u003e\u003cspan address=\"10.1186/s41065-023-00282-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011;67:235\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1107/S0907444910045749\u003c/span\u003e\u003cspan address=\"10.1107/S0907444910045749\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWischhof L, Scifo E, Ehninger D, Bano D. AIFM1 beyond cell death: An overview of this OXPHOS-inducing factor in mitochondrial diseases. EBioMedicine. 2022;83:104231. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ebiom.2022.104231\u003c/span\u003e\u003cspan address=\"10.1016/j.ebiom.2022.104231\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Y, Lin Y, Wang B, Liu F, Zhao D, Wang W, Ren H, Wang J, Xu Z, Yan C. al (2023) A Missense Variant in AIFM1 Caused Mitochondrial Dysfunction and Intolerance to Riboflavin Deficiency. Neuromolecular Med. \u0026copy; 2023. The Author(s), under exclusive licence to Springer Science\u0026thinsp;+\u0026thinsp;Business Media, LLC, part of Springer Nature., City, pp 489\u0026ndash;500.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZong L, Guan J, Ealy M, Zhang Q, Wang D, Wang H, Zhao Y, Shen Z, Campbell CA, Wang F al. Mutations in apoptosis-inducing factor cause X-linked recessive auditory neuropathy spectrum disorder. J Med Genet. 2015;52:523\u0026ndash;31. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1136/jmedgenet-2014-102961\u003c/span\u003e\u003cspan address=\"10.1136/jmedgenet-2014-102961\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8316196/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8316196/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMutations in the AIFM1, which encodes the apoptosis-inducing factor (AIF), are associated with a broad spectrum of neurometabolic disorders, yet their pathogenic mechanisms remains incompletely defined. In this work, we identified and comprehensively characterized a novel hemizygous AIFM1 mutation, c1006G\u0026thinsp;\u0026gt;\u0026thinsp;A (E336K), in male patients with a progressive childhood onset hereditary axonal sensorimotor polyneuropathy inherited in an X-linked recessive pattern, accompanied by sensorineural hearing loss but without cognitive impairment. Their clinical phenotype was consistent with Charcot-Marie-Tooth disease type 4 (CMTX4). Patient-derived fibroblasts exhibited reduced AIF protein stability despite preserved mRNA levels, impaired growth under OXPHOS-dependent conditions, decreased basal respiration, and altered assembly of mitochondrial respiratory supercomplexes. These defects were accompanied by reduced CHCHD4 abundance and decreased mitochondrial mass. Biochemical analyses of the purified E336K protein revealed compromised FAD retention, decreased thermal stability, impaired NADH affinity, destabilization of the charge-transfer complex required for AIF:CHCHD4 interaction, and a shift in coenzyme preference toward NADPH. Structurally, the substitution of Glu336 with Lys remodels the electrostatic environment of the NADH-binding cleft, thereby impairing redox function and weakening CHCHD4 binding. Despite these defects, the E336K mutation preserved DNA binding, nuclease activity, and binding to nuclear partners, although parthanatos induction was attenuated in patient fibroblasts. Collectively, these molecular alterations converge on disrupted mitochondrial bioenergetics and dynamics, providing a direct mechanistic link to the patient\u0026rsquo;s neurodegenerative course. These findings advance our understanding of AIFM1-related mitochondrial disorders and establish a framework for future precision molecular therapies.\u003c/p\u003e","manuscriptTitle":"Clinical and molecular characterization of a novel pathogenic AIFM1 E336K mutation connecting mitochondrial dysfunction and neurodegeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-18 09:54:52","doi":"10.21203/rs.3.rs-8316196/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-17T16:25:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-13T17:27:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75648397577853670592924151529931422002","date":"2026-01-05T08:51:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-29T08:36:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275098685081691990434009846957535047336","date":"2025-12-16T08:13:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-14T01:56:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-12T09:59:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-12T09:57:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Communication and Signaling","date":"2025-12-09T09:44:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"978db322-196e-452b-babc-f50c8fb198b9","owner":[],"postedDate":"February 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:06:54+00:00","versionOfRecord":{"articleIdentity":"rs-8316196","link":"https://doi.org/10.1186/s12964-026-02862-8","journal":{"identity":"cell-communication-and-signaling","isVorOnly":false,"title":"Cell Communication and Signaling"},"publishedOn":"2026-04-09 15:57:25","publishedOnDateReadable":"April 9th, 2026"},"versionCreatedAt":"2026-02-18 09:54:52","video":"","vorDoi":"10.1186/s12964-026-02862-8","vorDoiUrl":"https://doi.org/10.1186/s12964-026-02862-8","workflowStages":[]},"version":"v1","identity":"rs-8316196","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8316196","identity":"rs-8316196","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}