Chaperone Mediated Autophagy is deficient in Spinal Motoneurons of ALS patients with TDP-43 proteinopathy

Chaperone Mediated Autophagy is deficient in Spinal Motoneurons of ALS patients with TDP-43 proteinopathy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chaperone Mediated Autophagy is deficient in Spinal Motoneurons of ALS patients with TDP-43 proteinopathy Daniel Garrigos, Marta Martinez-Morga, Ana Pombero, Raquel García-Lopez, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7444163/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Feb, 2026 Read the published version in Acta Neuropathologica Communications → Version 1 posted 9 You are reading this latest preprint version Abstract Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of motor neurons (MNs), ultimately resulting in paralysis and respiratory failure within 3 to 5 years of onset. Fewer than 10% of ALS cases are familial (fALS), while the vast majority are sporadic (sALS) with an unknown etiology. A pathological hallmark of ALS is the accumulation of misfolded TDP-43 protein aggregates within MNs. Although TDP-43 is known to be degraded via chaperone-mediated autophagy (CMA), the status of CMA activity in sALS has not been previously explored. To investigate this, we analyzed CMA in human spinal cord tissue by assessing the expression of LAMP2A, a key lysosomal receptor and marker of CMA activity. In control samples, spinal cord MNs exhibited robust LAMP2A expression. In contrast, MNs from sALS patients showed a marked reduction in LAMP2A levels, coinciding with the presence of TDP-43 pathology. Notably, analysis of LC3, a marker of macroautophagy, revealed no significant differences in expression between control and sALS MNs. Interestingly, MNs within the Onuf’s nucleus, a population known to be resistant to degeneration in ALS, retained normal LAMP2A expression and did not exhibit TDP-43 aggregation in sALS cases. These findings demonstrated that CMA is essential for the clearance of TDP-43 in spinal cord MNs and that its dysfunction may contribute to the pathogenesis of sALS. Furthermore, the high dependence of spinal cord MNs on CMA activity may underlie their selective vulnerability to degeneration when CMA is impaired, and highlight CMA enhancement as a promising therapeutic strategy to restore proteostasis and prevent MN degeneration in ALS. motor neurodegeneration sALS differential vulnerability to neurodegeneration chaperone mediated autophagy human motoneurons Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons (MNs) in the primary motor cortex and spinal cord (SC) [ 16 , 74 ]. Most patients succumb within 3–5 years due to progressive palsy and respiratory failure [ 63 ]. The global prevalence and incidence are estimated at 4.1–8.4 per 100,000 individuals and 0.6–3.8 per 100,000 person-years, respectively[ 53 ], with approximately 30,000 deaths reported annually [ 71 ]. ALS arises from a complex interplay of genetic and environmental factors [ 59 ], and manifests in familial (fALS) and sporadic (sALS) forms [ 90 ]. More than 20 genetic mutations have been implicated, with C9ORF72, FUS, TARDBP , and SOD1 being the most frequently involved [ 74 ]. Regardless of etiology or site of onset, ALS is a predominantly MN-selective neurodegenerative disorder for reasons that remain unclear. This selectivity has been attributed to several intrinsic features of MNs, including their large size, high metabolic demands, dependence on mitochondrial integrity, vulnerability to excitotoxicity, disrupted intracellular calcium homeostasis, and impaired ubiquitin–proteasome system function [ 15 , 66 , 74 ]. A bottom-up pathogenic model, retrograde neuropathy originating at neuromuscular terminals, may account for the preferential degeneration of spinal MNs and the potential for trans-synaptic involvement of the primary motor cortex [ 64 , 66 ]. Moreover, differences in neuromuscular junction architecture may underlie the relative resistance of certain MN subpopulations, such as oculomotor and Onuf’s nuclei, although this remains experimentally unverified [ 15 ]. Current therapeutic options for ALS remain limited. Riluzole, the first approved drug, extends survival by only 6–19 months [ 2 ]. Edaravone received FDA approval in 2017 [ 89 ], but failed to show efficacy in an independent clinical trial in Italy [ 55 ]. More recently, antisense oligonucleotide therapies have been developed for fALS with SOD1 mutations [ 79 ]. Tofersen, approved in SOD1 fALS following a phase III trial, did not yield significant clinical improvement but showed encouraging biomarker responses, including reductions in cerebrospinal fluid SOD1 protein and plasma neurofilament light chain (NfL) levels [ 60 ]. Similarly, Relyvrio, initially approved for slowing ALS progression, was later withdrawn after a failed phase III trial [ 4 , 67 ]. Given the absence of effective treatments for ALS, there is an urgent need for further preclinical research and improved drug-target identification. Among the cellular pathways increasingly implicated in ALS pathogenesis, autophagy plays a central role in maintaining protein homeostasis and cellular integrity [ 48 , 81 , 86 ]. This multi-step process involves the lysosomal degradation and recycling of intracellular components, including aberrant proteins and damaged organelles [ 81 ]. Three types of autophagy are described in mammals: macroautophagy (MA), microautophagy, and chaperone-mediated autophagy (CMA). While MA and microautophagy involve bulk degradation and direct lysosomal delivery of cargo, respectively [ 1 ], CMA selectively targets proteins bearing a KFERQ-like motif. These are recognized by the HSC70 chaperone and delivered to the lysosomal receptor LAMP2A, the key limiting factor for CMA in the neurons [ 14 , 42 , 72 , 76 ]. Notably, TDP-43, a nuclear RNA-binding protein involved in RNA processing, genome integrity, and mRNA metabolism [ 5 , 31 , 65 ], contains the KFERQ-like motif [ 47 ]. In approximately 95% of ALS cases, TDP-43 is mislocalized, forming phosphorylated and ubiquitinated cytoplasmic aggregates that are hallmark features of spinal MN pathology [ 12 , 19 , 23 , 65 ]. Although these aggregates are prominent in sALS, mutations in TARDBP and C9orf72 can also lead to TDP-43 proteinopathy [ 75 ]. Clearance of TDP-43 aggregates is critical to mitigate their cytotoxicity and has been linked to both the ubiquitin–proteasome system and MA [ 24 , 65 , 85 ]. Moreover, reducing TDP-43 levels in ALS mouse models improves motor deficits, suggesting that motoneuronal dysfunction may be at least partially reversible[ 43 ]. It has been proved in experimental models that MA activation, for instance through mTOR inhibition, enhances TDP-43 turnover and cell viability [ 32 , 33 ]. Given the role of TDP-43 in ALS and its potential recognition by CMA, we investigated LAMP2A expression, a key marker of CMA in human SCs [ 47 ]. We analyzed SC tissue from six control subjects (n = 6; 3 females, 3 males) and ten sALS patients (n = 10; 6 females, 4 males), which exhibited varying degrees of TDP-43 proteinopathy. In control SCs, we observed intense LAMP2A expression in MNs across all regional levels. In contrast, sALS SC samples showed a marked reduction in LAMP2A expression in MNs, both in early pathological stages, characterized by nuclear TDP-43 clearance and granular cytoplasmic aggregates, and in advanced stages with dense cytoplasmic TDP-43 inclusions. Interestingly, Onuf’s nucleus MNs, which are relatively spared in ALS [ 15 , 57 ], displayed strong LAMP2A expression and lacked TDP-43 pathology. These findings suggest that CMA dysfunction may contribute to the selective vulnerability of MNs in ALS and underscore a potential protective role of preserved CMA activity in resistant MN populations. M&M Human tissue processing Human control samples without neurological disorder (n = 6 SC) were obtained from anonymous donations through the Anatomy Innovation Service of Miguel Hernandez University Medical School, which provides administrative and ethical support, with the approval of the Institutional Review Board. Samples and data from patients included in this study (n = 10 SC) were provided by the Biobank IMIB (National Registry of Biobanks B. 0000859) (PT20/00109), integrated in the Platform ISCIII Biobanks and Biomodels and they were processed following standard operating procedures with the appropriate approval of the Ethics and Scientific Committees. Voluntary donations were obtained from patients included in the Phase I and II of clinical trial nº EudraCT:2006-00309612, NCT00855400 and EC 07/90762, NCT01254539 (Supplementary Table 1). Tissue preparation and Immunohistochemical staining Spinal cords were fixed in 10% formalin (Sigma-Aldrich, Germany) for 5 days at room temperature. Following fixation, the SC was transversely trimmed into tissue slides (TS) 5–7 mm thick and labelled in a rostro-caudal order into progressive cervical, thoracic, lumbar, or sacral regions (Supplementary Fig. 1). TS underwent a progressive dehydration process in ethanol, followed by butanol and were subsequently embedded in paraffin. Transversal sections, 7–10 µm thick, were then obtained from four selected TS at each SC region, covering the entire region, and mounted on microscopy slides (MS) in 20 parallel series (Supplementary Fig. 1). Spinal cords were processed to paraffin embedding and sectioning sections following the protocol described in Supplementary Fig. 1. MS were processed by Hematoxylin-Eosin (H&E, serie 1), Cresyl violet (CV, serie 2), and immunohistochemistry (series 3–11). We conducted immunohistochemical analysis on subsequent parallel series of MS from cervical segments C3-6 (n = 20), thoracic segments T8-11 (n = 20), lumbar segments L2-5 (n = 20), and sacral segments S1-4 (n = 20) of control and sALS SCs. To minimize experimental bias in the data of ALS patients, T2–T6 segments were excluded because 7 patients had received intraspinal autologous graft of mononuclear bone marrow cells (MNBMc) in the T3–T5 [ 11 ], intrathecal MNBMc graft, or intrathecal administration of saline solution, as placebo group (clinical trials (CT) NCT00855400 and NCT04849065, Phase I and II; Supplementary Table 1). Finally, one patient was not included in Phase II CT and was classified as a not treated ALS patient (Supplementary Table 1). Our previous pathological data from the Phase I CT showed no significant modifications in non-experimental segments[ 10 ], therefore, the rostral and caudal segments relative to T2–T7 were considered affected in accordance with the natural progression of the disease in each patient. Immunohistochemistry procedures were described in Blanquer et al., [ 10 ]. Briefly, sections were treated with primary antibodies, diluted in EnVision FLEX Antibody Diluent (DAKO, Denmark), 24 hours at 4°C (Supplementary Table 2). Following primary antibody incubation, sections were incubated 2 hours with the appropriate biotinylated secondary antibody (Supplementary Table 2). Then, sections were incubated with Avidin–Biotin Complex for 1 h (ABC kit, Vector Laboratories CA-94010). For colorimetric detection (brown), the tissue was incubated with 1% 3,3'-Diaminobenzidine (DAB; Vector Laboratories SK-4100) and 0.0018% H2O2 in PBS. For double immunohistochemistry with anti-TDP 43/anti-ChAT and anti-TDP-43/anti-LAMP2A, anti-TDP-43 was incubated with 1% 3,3'-Diaminobenzidine (DAB; Vector Laboratories SK-4100), 0.025% ammonium nickel sulfate hexahydrate, and 0.0018% H2O2 in PBS for colorimetric detection (black). For double immunochemistry with anti-GFAP/anti-IBA1, anti-IBA1 was processed to obtain a black color. Sections series processed in parallel without primary or secondary antibodies did not show any specific or nonspecific labeling (Supplementary Fig. 2). Finally, the sections were dehydrated and mounted in Eukitt (O.Kindler GmbH and CO, Freiburg). Microscopy and statistical analysis Images were captured using an optical microscope (Leica CTR6000) and were utilized to assess the percentage of cellular area expressing various markers, including LAMP2A, LC3, and GBA (see below). After identifying MNs in ventro-medial and ventro-lateral columns (where more MNs were identified in ALS patients), the ImageJ program was employed to quantify the percentage of area expressing LAMP2A, LC3, and GBA relative to the total cell area (n = 20 MNs/marker in each group). Statistical analysis comparing sALS and control samples was performed using Sigmaplot v11.0 software. The data were presented as mean values ± standard error (SE), and pairwise comparisons between sALS and control samples (attending: gender, segmental level and MNs column) were conducted using the Student’s t-test. Graphs and statistical visualizations were generated using GraphPad Prism version 10 (GraphPad Software, San Diego, CA). A significance level of p < 0.05 was considered statistically significant, with *p < 0.05, **p < 0.01, and ***p < 0.001 denoting different levels of significance. Results Spinal motoneurons exhibit elevated expression levels of LAMP2A. Given that TDP-43 is a potential substrate for CMA [ 37 , 65 ], we investigated LAMP2A expression, the rate-limiting component of CMA [ 72 ], in spinal MNs. To investigate LAMP2A expression in the human SC, we conducted immunohistochemical analyses on sections from C3–C4, T8–T9, L3–L4, and S2–S3 segments (n = 10 slides per segment) obtained from control SC (n = 6; Supplementary Table 1). MNs were identified based on their distinct morphological features observed with H&E and CV staining, as well as their immunoreactivity for choline acetyltransferase (ChAT) (Fig. 1 a). In parallel slides we observed robust and specific LAMP2A expression in MNs across all SC segments (Fig. 1 b–g). The immunoreactivity exhibited a puncta perinuclear pattern within the MN cytoplasm, consistent with lysosomal membrane localization of LAMP2A [ 76 ] (Fig. 1 e-h). Additionally, most MNs also contained lipofuscin granules that were immunonegative for both ChAT and LAMP2A (Fig. 1 d, e; Supplementary Fig. 3a–d). In contrast, LAMP2A immunoreactivity was markedly lower in other neuronal populations, including Clarke’s column neurons (TCNs; Fig. 1 i, j; Supplementary Fig. 3e) and dorsal horn sensory neurons (SNs; Fig. 1 k; Supplementary Fig. 3e). In TCNs, LAMP2A-positive puncta were distributed throughout the cytoplasm rather than showing a perinuclear concentration as seen in MNs (Fig. 1 i, j). Additionally, glial cells (GCs) exhibited minimal LAMP2A immunoreactivity (Supplementary Fig. 3d, f–h). Then, we explored potential age- and sex-related differences in CMA by comparing SC samples from Control 1 and 6, derived from 64- and 62-year-old females, respectively, with Control 3 and 5, obtained from 53-year-old males (Fig. 2 a). Our analysis did not reveal significant differences in LAMP2A expression among these SC samples. Notably, despite the male samples being approximately 10 years younger than the female samples, LAMP2A expression levels were comparable. Furthermore, comparison of SC samples from Control 1 and 6 with Control 4 (a 70-year-old female, almost 10 years older) also showed no substantial differences in LAMP2A expression (data not shown). Therefore, do not reveal age-related or sex-specific differences in LAMP2A expression in MNs. These findings indicate that spinal MNs selectively express high levels of LAMP2A, suggesting heightened CMA activity. Spinal motoneurons of sALS patients exhibit low expression levels of LAMP2A. To analyze CMA activity in spinal MNs of sALS patients, we performed LAMP2A immunohistochemistry on sections from C3-C4, T8-T9, L3-L4, and S2-S3 segments of sALS patient’s SC (n = 10; Supplementary Table 1). In sALS SCs, cervical, lumbar, and sacral segments exhibited a greater number of detectable MNs based on morphological and immunohistochemical criteria (a mean of 9–12 MNs/section, 12–17 MN/section, and 5–10 MN/section, respectively) compared to thoracic segments (0–3 MN/section). Consequently, we increased in our study the number of thoracic slides (n = 20) to ensure at least 20 thoracic MNs were analyzed in each patient, along with 20 MNs from other SC regions (n = 10 from each segment), which were then compared with the same number of control MNs. In all cases of sALS SC, LAMP2A immunopositivity was notably weak in most of the MNs (Fig. 2 , 3 ). While the majority of MNs from sALS patients showed a significantly reduced expression of LAMP2A (Fig. 1 a, b; 3 a–e), a small subset (approximately 5–10% of MNs) exhibited peripheral or normal cytoplasmic distribution of LAMP2A-positive puncta (Fig. 2 d, e). These findings suggest a decrease in CMA activity in sALS MNs relative to controls. In contrast, spinal MNs from the patient ALS40 displayed a moderate reduction in LAMP2A expression in approximately 60% of MNs (Fig. 2 f, g). Macroautophagy is preserved in sALS patients’ motor neurons In animal models of ALS, impaired MA and autophagosome formation have been reported [ 9 , 20 , 24 , 32 , 33 , 73 ]. Therefore, to evaluate MA function in our human SC sections, we examined the expression of the autophagosome marker microtubule-associated protein light chain 3 (LC3) using immunohistochemistry [ 39 ]. Moreover, in selective MA, a specific interaction between p62 and LC3 is necessary to mediate the autophagic degradation of p62-positive structures [ 91 ]. The detection of p62-positive aggregates serves as an indicator of MA deficiency in tissues [ 91 ]. Consequently, p62 expression was also analyzed in our samples. Lastly, to investigate lysosomal formation and distribution in MNs we assessed the expression of the lysosomal enzyme glucocerebrosidase (GBA) [ 8 ]. Abundant immunopositive LC3 puncta aggregates were detected in the cytoplasm of both controls (Fig. 3 f) and sALS spinal MNs (Fig. 3 g-j). GBA immunolabeling was observed as a puncta pattern dispersed in the cytoplasm with higher perinuclear density (Fig. 3 k-p). To compare CMA, MA and lysosomal formation, we quantified immunopositive puncta of LAMP2A, LC3B, and GBA in SC MNs where the nucleus was clearly detected and in nonconsecutive sections to ensure that we did not count the same neuron multiple times. We selected 10 MNs of the antero-lateral MNs column in two control SC (Controls 1 and 3; 5 MNs at cervical and 5 MNs at lumbar segments), as well as 10 MNs of antero-lateral MNs column in two patients’ SC, ALS 24 and 47 (5 MNs at cervical and 5 MNs at lumbar segments) (Fig. 3 a-p). Our quantitative analysis demonstrated that LAMP2A expression is significantly reduced in sALS MNs, while autophagosomes formation detected by LC3 puncta and the number of lysosomes related to the GBA expression showed no significant differences (Fig. 3 q). Furthermore, to confirm the specificity of the increased LAMP2A immunoreactivity in control SC MNs, we observed no qualitative differences in LC3 expression between MNs and TCN in control samples (Fig. 3 r, s). The analysis of P62-protein intracytoplasmic deposits in all the parallel series processed by P62 immunohistochemistry detected only 5 MNs in lumbar sections of ALS 40 patient SC (Fig. 3 t), where also TDP-43 deposits appeared in the parallel series (Fig. 3 u). All other control and sALS SC samples showed no p62 deposits. These results further support that MA is not significantly affected in human sALS MNs. Analysis of LAMP2A, LC3, GBA, and p62 expression in MNs from control and ALS patient samples did not reveal any significant sex- or age-related differences in expression levels. However, in ALS 40, increased LAMP2A expression and p62 accumulation were observed in a subset of MNs, suggesting the possibility of a distinct etiological variant of sALS in this patient. Glial cells increase the expression of LAMP2A in sALS spinal cords In control SC tissue, glial cells in both white and gray matter exhibit weak LAMP2A expression (Fig. 4 a; Supplementary Fig. 3d, f, g). In contrast, LAMP2A-immunopositive glial cells of sALS SC were observed in the anterior horn gray matter and were more abundant in the lateral and medial corticospinal tracts (lcst and mcst; Fig. 4 b–c). High-magnification images of the lcst revealed intensely LAMP2A-expressing cells with reactive astroglial morphology [ 40 , 84 ], interspersed among LAMP2A-negative axonal fascicles (Fig. 4 d–f). GFAP immunostaining highlighted the typical morphology of reactive astrocytes (Supplementary Fig. 4a–c), which in some cases displayed disrupted cytoplasmic processes and somatic vacuolization (Supplementary Fig. 4c). This phenomenon, known as clasmatodendrosis [ 87 ], was first described by Ramón y Cajal in 1913 [ 18 ]. To determine if this increase of LAMP2A is due to the inflammation associated with the axonal neurodegeneration resulting from degeneration of primary cortical MNs, we studied the presence of immunocompetent cells infiltration using IBA1 and CD68 immunohistochemistry. LAMP2A expression in astroglial cells was colocalized within lcst and mcst, alongside IBA1-positive microglia (Supplementary Fig. 4d-f). There was also a notable infiltration of CD68-immunopositive cells, which appeared to be concentrated within perivascular spaces (Fig. 4 g, h; Supplementary Fig. 4d, e). IBA1-positive microglial cells were also observed surrounding axons within these tracts (Supplementary Fig. 4f). Therefore, the significant increase of LAMP2A expression in glial cells may be due to an inflammatory process in cortico-spinal tracts as a consequence of axonal degeneration. Moreover, high-power pictures from the anterior horn showed the presence of CD68 immunopositive cells between MNs (Fig. 4 i), suggesting that an inflammatory process in the gray matter may also be associated with a reactive increase of LAMP2A expression in some glial cells. Double immunodetection using LAMP2A and IBA1 showed the distribution of LAMP2A-positive astroglia and IBA1-positive microglia in the cortico-spinal tracts (Supplementary Fig. 4f). The analysis of glial activation by LAMP2A expression in lateral and medial cortico-spinal tracts of sALS MNs did not show any age- or sex-specific differences when comparing expression levels in SC sections. TDP-43 proteinopathy in LAMP2A deficient sALS motoneurons. Spinal MNs from control SC exhibited TDP-43 immunoexpression primarily in the cell nucleus (Fig. 5 ). We performed ChAT (Fig. 5 a) and double ChAT/TDP-43 immunohistochemistry (Fig. 5 b, c), identifying colocalization of TDP-43 nuclear expression in ChAT-positive MNs (Fig. 5 b, c). Additionally, we conducted double immunohistochemistry in another section series to investigate the localization of TDP-43 and LAMP2A in spinal MNs (Fig. 5 d-g). Some TDP-43 immunopositive puncta were detected in the cytoplasm (Fig. 5 e), occasionally in continuity with its nuclear expression (Fig. 5 f), suggesting a nucleo-cytoplasmic transfer of this protein [ 5 ]. These cytoplasmic TDP-43-positive colocalized with LAMP2A-positive puncta (Fig. 5 d, e). The observed colocalization of TDP-43 and LAMP2A in the cytoplasm of control MNs further supports the involvement of CMA in TDP-43 cytoplasmic clearance. Nuclear depletion and cytoplasmic deposits of TDP-43 in motor system neurons and glia have been described in ALS patients and animal models [ 12 , 19 ]. As mentioned before, it has also been reported that TDP-43 is a substrate of CMA [ 37 , 44 , 54 , 65 ]. Therefore, we explored if LAMP2A deficient MNs of our sALS patients presented TDP-43 proteinopathy. We performed TDP-43 immunohistochemistry on sections of parallel series as described previously: C3-4, T8-9, L3-4, and S2-3 SC segments (n = 10 slides in each segment) of sALS patients SC (n = 10; see Supplementary Table 1). Anti-TDP-43 immunohistochemistry was counterstained with Cresyl violet, showing different degrees of proteinopathy in sALS spinal MNs. Most MNs in the spinal anterior gray matter showed TDP-43 proteinopathy (90–95% of MNs) (Fig. 6 a-g, k). We classified TDP-43 cytoplasmic inclusions according to Kon et al. [ 49 ] into three categories: 1) fine punctate granules scattered diffusely in the cytoplasm (DPSC) (Fig. 6 k), 2) round inclusions (RIs) about 1–15 µm in diameter (Fig. 6 g), and 3) skein-like inclusions (SLIs) (Fig. 6 d-f). Predominant nuclear immunolocalization and punctate granules in the cytoplasm, similar to those observed in control MNs, were observed in 3–5% of sALS MNs (Fig. 6 , i). Additionally, 30–35% of MNs showed only partial or total depletion of nuclear TDP-43 immunopositive granules, with DPSC (Figs. 6 -k), but without important cytoplasmic deposits, which has been described as the initial proteinopathy in relation to stress granules in ALS MNs [ 49 , 61 ]. In the rest of the analyzed MNs (60%), they showed nuclear clearance accompanied by cytoplasmic deposits, together with strong degenerative cytoplasmic vacuolization in colocalization with TDP-43 aggregates, in agreement with Martin [ 58 ] (Fig. 6 -g). Moreover, the normal cisternal pattern of Nissl bodies in the cytoplasm (Fig. 6 -j) disappeared in sALS MNs showing TDP-43 aggregates and was replaced by a granular, scattered pattern intermixed with DPSC of TDP-43 (Fig. 6 , g, k). In contrast, anterior horn interneurons showed normal nuclear TDP-43 expression (Fig. 6 ). To analyze the co-expression of TDP-43 and LAMP2A in the patient’s MNs, another parallel series of sections were processed by double immunohistochemistry using anti-TDP-43 and anti-LAMP2A antibodies (Fig. 7 ). First, the reduction of LAMP2A expression in sALS MNs was clear, being especially evident in MNs with RIs (Fig. 7 , b). Second, in most sALS MNs with reduction of LAMP2A expression, nuclear clearance and DPSC of TDP-43 was clearly detected (Fig. 7 ). In addition, the distribution and typology of TDP-43 aggregates in LAMP2A-depleted MNs were heterogeneous and sometimes combined in the same cell, detecting a mixture of DPSC with round aggregates and SLIs. TDP-43 filiform inclusions were localized into MNs dendritic proximal segments (Fig. 7 , f, i, j). Moreover, some MNs showed TDP-43 filaments across the cellular membrane (Fig. 7 , h), and TDP-43 filaments were detected in the interstitial space (Fig. 7 ), suggesting the possibility of transmembrane trafficking and raising the possibility of transcellular transmission of misfolded TDP-43 protein. Interestingly, ALS40 SC MNs showed a reduced degree of TDP-43 proteinopathy. Actually, although more than 40% of MNs showed TDP-43 cytoplasm aggregates (Supplementary Fig. 5, a-c), more than 30% of MNs showed only nuclear depletion of TDP-43, and another 30% normal localization of this protein (Supplementary Fig. 5). These findings suggest that CMA is more active in this patient, as described previously (Fig. 2 , g). Accordingly, we examined the expression of LAMP2A in ALS40 SC (Supplementary Fig. 5, h). Our analysis revealed that the ALS 40 SC MNs exhibited higher LAMP2A expression than the other sALS SC, which may account for the reduced TDP-43 pathology. Quantification of LAMP2A expression in ALS40 SC MNs was higher than in control SC MNs (Supplementary Fig. 5). The analysis of TDP-43 expression, and TDP-43/LAMP2A co-expression in sALS MNs, with the exception of ALS40, did not show any sex-specific differences when comparing expression levels in sections from SC (Supplementary Table 1). As was described above, ALS40, although with more LAMP2A expression and less degree of TDP-43 proteinopathy, has the faster evolution: 11 months in comparison to the rest with a mean of 27,4 months (Supplementary Table 1). This may suggest a different pathogenic mechanism underlying the MN degeneration and reflects the heterogeneity of the disease evolution in each patient. Onuf’s nucleus motoneurons exhibit high expression levels of LAMP2A in sALS spinal cords. In the sacral levels of sALS patients, Onuf’s nucleus was identified due to the presence of a significant number of ChAT-positive MNs in the anterior horn, between anterolateral and anteromedial columns (Fig. 8 -c). While surrounding spinal MNs in the anterior horn showed a cytoplasm with a degenerative vacuolar profile, Onuf’s MNs appeared without cytoplasmic pathology (Fig. 8 , c, h). Moreover, Onuf’s MNs exhibited strong LAMP2A expression, with punctate lysosomal immunoreactivity comparable to that of control SC MNs (Fig. 8 d). In contrast, spinal MNs in the same segment showed markedly reduced LAMP2A expression, with sparse puncta predominantly localized at the cell periphery (Fig. 8 e). We have studied the localization of TDP-43 protein in Onuf’s MNs of sALS patients in combination with LAMP2A expression or Nissl staining. In Onuf’s MNs, TDP-43 was predominantly localized in the nucleus of LAMP2A-positive MNs, similar to control MNs (Fig. 8 , g), and the normal cisternal pattern of Nissl bodies in the cytoplasm was detected (Fig. 8 , i). These findings support that high CMA activity is crucial to maintain Onuf’s MNs alive in sALS patients and reinforces the possibility that CMA in SC MNs may play a role in preventing TDP-43 proteinopathy and MNs vulnerability of sALS. Discussion Our results show for the first time that human SC MNs exhibit high levels of LAMP2A expression, suggesting that MNs require increased CMA activity to function properly. On the other hand, SC samples from sALS patients showed reduced LAMP2A expression in their MNs. These findings may explain the selective vulnerability of MNs observed in prior studies, which has been attributed to structural and functional differences unique to MNs [ 64 , 66 , 82 ]. Despite the reduced LAMP2A expression in sALS MNs, MA appeared unaffected compared to control MNs, as evidenced by the presence of autophagosomes detected by robust LC3B-positive cytoplasmic puncta pattern. GBA expression was unchanged, indicating normal lysosomal activity, including autophagosomes. TDP-43 was primarily localized in the nucleus of control spinal MNs, but in sALS MNs, it was absent from the nucleus and accumulated in the cytoplasm. Notably, Onuf’s nucleus in sALS patients showed higher LAMP2A expression and protection from TDP-43 cytoplasmic pathology. Finally, ALS40-specific differences further support the role of LAMP2A activation in preventing or delaying TDP-43 proteinopathy in sALS patients, also highlighting the heterogeneity of the etiology and pathology of this disease. CMA is selectively upregulated in spinal cord MNs CMA is a selective autophagic pathway that targets proteins with a lysosomal targeting motif, such as the pentapeptide chain KFERQ [ 47 ]. The chaperone Hsc70 binds to KFERQ-containing proteins, transporting them to the lysosomal surface, where they interact with LAMP2A before being unfolded and degraded in the lumen. CMA degrades approximately 30–35% of cytosolic proteins, impacting critical processes such as lipid and glucose metabolism, DNA repair, cellular reprogramming, stress responses, and immunological function [ 47 , 52 ]. Impairment of CMA has been associated with several age-related diseases, including neurodegenerative disorders, atherosclerosis, metabolic diseases, and cancer [ 21 , 24 , 26 , 27 , 52 , 56 ]. The elevated CMA activity in spinal MNs could be attributed to their large size and high firing rates, consistent with Henneman’s size principle, which states that the size of a neuron is a crucial determinant in reaching the threshold for action potential firing [ 35 ]. Fast-fatigable motor units are more vulnerable to ALS, whereas slow motor units, which are more resistant, may help to reinnervate motor endplates [ 64 , 66 ]. These findings suggest that the functional properties of MNs may influence their vulnerability to ALS. This functional activity necessitates the regulation of ion channels, receptor proteins, and ion buffering mechanisms, all of which contribute to the need for enhanced proteostasis control. Additionally, due to their highly polarized nature MNs require efficient protein production and degradation pathways to survive. As mentioned earlier, CMA has been implicated in neurodegenerative diseases, contributing to the degradation of pathogenic proteins such as α-synuclein and tau [ 14 , 17 , 22 , 24 , 29 , 36 , 52 ]. Loss of CMA function leads to significant changes in the neuronal proteome, disrupting essential neuronal functions and promoting neurodegeneration [ 14 , 76 ]. Moreover, the specificity of high requirements of CMA function in MNs could explain their heightened vulnerability to CMA impairment compared to other cell types, providing a potential mechanism for the selective neurodegeneration of MNs in sALS. Interestingly, Khawaja et al. [ 46 ] reported that CMA activity declines with age across most organs and cell types, with a more marked reduction observed in males. This decline is often associated with a reduced number of lysosomes functionally competent for CMA, suggesting that sex-specific differences in CMA activity may influence tissue vulnerability to age-related degenerative processes. Jacob et al. [ 38 ] reviewed the mechanisms underlying sexual dimorphism in ALS, emphasizing sex-specific heterogeneity in both genetic and non-genetic mechanisms across experimental models and patient cohorts. Within this framework, the apparent accelerated decline of CMA observed in males, reflected by LAMP2A expression levels comparable to those of females approximately ten years older, may underlie the sex-biased prevalence and distinct clinical phenotypes of ALS. While age- and sex-associated reductions in CMA have been reported in murine models, further studies are required to validate these patterns in human SC MNs. In the present study, the limited sample size precluded definitive conclusions regarding age- or sex-related differences in LAMP2A expression in SC MNs. CMA is downregulated in sporadic ALS Spinal MNs Spinal cord sections from sALS patients demonstrated reduced LAMP2A expression in MNs; however, approximately 5–10% of MNs exhibited only a partial reduction in LAMP2A levels. This observation may reflect intrinsic heterogeneity in LAMP2A expression among MNs or indicate a progressive decline in its expression, potentially contributing to, or resulting from, the differential vulnerability of MN populations based on their functional and structural properties.(as reviewed in Ovsepian et al. [ 66 ]). While MA has been well studied in ALS [ 24 , 37 , 77 , 80 , 83 ], CMA remains less explored. Our analysis revealed that LC3 expression was comparable between control and sALS spinal MNs, suggesting preserved MA function in sALS. Despite its importance in neuronal homeostasis, MA does not appear to have cell-specific requirements in MNs, as no substantial differences in LC3 expression were observed between control MNs and other spinal neurons. Although MA impairments have been reported in ALS models, our findings suggest its role in disease pathophysiology may be secondary, with experimental activation potentially mitigating TDP-43 proteinopathy rather than addressing a primary defect [ 24 ]. A study by Arosio et al. [ 3 ] reported TDP-43 proteinopathy with reduced levels of Hsc70 in lymphomonocytes of sALS patients without change in LAMP2A. In contrast, our results demonstrate reduced LAMP2A expression specifically in sALS MNs, reflecting fundamental differences in cellular expression and autophagy requirements across cell types. Mutations in the LAMP2 gene cause Danon disease, an X-linked lysosomal storage disorder characterized by cardio-myopathy and cognitive dysfunction. The pathological hallmark of this disease is the accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscles [ 28 , 30 ]. The cognitive dysfunction seen in humans with Danon disease suggests a critical role of LAMP-2 in brain function [ 76 ]. These cognitive abnormalities are likely due to hippocampal dysfunction, associated with altered lysosomal activity, including the accumulation of p62-positive aggregates, autophagic vacuoles, and lipid storage within neurons. Notably, in agreement with our results, Rothaug et al. [ 76 ] reported that the absence of LAMP2 did not appear to affect MA in the brain cells under physiological or starvation conditions. In Danon disease MNs degeneration has not been observed [ 28 , 30 ], which may be attributed to the cell-type-specific role of LAMP2B, the isoform mutated in Danon disease [ 50 , 92 ], or to the limited lifespan of patients, which may preclude the manifestation of MN degeneration. The selective accumulation of TDP-43 in CMA-deficient MNs, in the absence of other CMA substrate accumulation, may reflect the compensatory degradation of these proteins via alternative pathways, such as endosomal microautophagy (eMI), which has been shown to process KFERQ-like motif–containing proteins [ 47 , 93 ]. Although we did not observe major alterations in macroautophagy or lysosomal distribution in MNs from patients with sALS, investigating the role of eMI in human MNs may help clarify the specificity of TDP-43 pathology in sALS. Interestingly, LAMP2A levels were significantly higher in glial cells of sALS patients. The upregulation of LAMP2A in glial cells may be a reactive response to inflammatory processes occurring in the anterior horn and corticospinal tracts [ 70 , 93 ]. Axonal degeneration is known to trigger inflammation and oxidative stress in glial cells, leading to activation of NFE2L2, which in turn increases LAMP2A expression and enhances CMA activity [ 68 ]. Indeed, this inducible regulation of LAMP2A, and consequently of CMA activity, by NFE2L2 (coding NRF2 antioxidant transcription factor) is not sufficient to overcome the low LAMP2A expression observed in sALS MNs, despite their elevated oxidative stress [ 15 , 66 , 74 ]. Bono et al. [ 13 ] reviewed the possible role of alterations of antioxidant response in ALS neurons and glia by the activation of KEAP1-NRF2 without conclusive information on the primary cause of ALS. Interestingly, increased autophagy was reported by Ryu et al., [ 78 ] underlining clasmatodendrosis as an autophagic death of astrocytes (reviewed in Balaban et al. [ 6 ]). Although Guise et al. [ 34 ] found no significant differences in LAMP2A expression between ALS and control MNs using laser-guided tissue dissection, our results suggest that it may be due to the inclusion of perineuronal astroglial fragments in the dissected samples, which exhibit strong LAMP2A activation and could mask the reduced LAMP2A in MNs. This supports the notion that the alteration in LAMP2A expression in sALS MNs is a primary, constitutive, cell-autonomous defect. CMA in Onuf's Nucleus MNs and selective vulnerability in sALS Our results also provide evidence that Onuf’s nucleus in sALS patients retains LAMP2A expression. Slow motor units are then more protected, and they reinnervate the end-plate left by faster, fatigable motor units after cell death [ 64 , 66 ]. Interestingly, our work is the first to show that LAMP2A expression is not deficient in the Onuf’s MNs of sALS patients. The mechanisms by which Onuf’s nucleus remains spared in ALS and other neurodegenerative diseases, but impaired in others, remains unknown. Recently, RNA-seq analysis of mice Onuf's nucleus extracted by laser microdissection revealed that matrix metalloproteinase-9 (MMP9), an inflammatory biomarker that is highly expressed by ALS-affected spinal MNs, is not overexpressed in Onuf's neurons [ 41 ]. This MMP9 may represent a factor to reduce EN1 transference from interneurons to MNs, decreasing the paracrine neurotropism of this factor [ 51 ]. This, along with proper CMA functioning, could lead to protection of this nucleus against ALS-mediated cell death. The localization of EN1 in spinal and Onuf’s MNs may shed some light on these mechanisms. The differential CMA activation likely contributes to the protective mechanisms underlying the selective preservation of Onuf's MNs in sALS. We propose that maintained CMA function in these MNs protects against TDP-43 proteinopathy and subsequent cell death. TDP-43 and autophagy in sALS Our findings demonstrate colocalization of LAMP2A and TDP-43 in the cytoplasm of control MNs, suggesting that CMA may facilitate cytoplasmic TDP-43 clearance. This supports the hypothesis that CMA dysfunction contributes to TDP-43 proteinopathy in sALS MNs. In TDP-43 proteinopathy, alterations in the localization and function of other proteins have been described, including ribonucleoprotein K. The binding of ribonucleoprotein K with Nrf2 transcript was associated with an impaired translation of Nrf2 mRNA, leading to an insufficient antioxidant response and motoneuron degeneration [ 62 ]. Wang et al. [ 88 ] and Barmada et al. [ 7 ] observed that stimulating autophagy by increasing LC3 levels in mouse neurons and human MNs derived from iPSCs improved TDP-43 clearance and reduced protein toxicity. However, TDP-43 aggregation may also result from defects in other protein degradation systems, such as CMA or the ubiquitin-proteasome system [ 25 , 45 ]. Our data demonstrate that human sALS spinal MNs show no alterations in MA, indicating that MA dysfunction is not a primary pathological feature of sALS. Although alterations of genes promoting TDP-43 proteinopathy have been linked to dysregulation of MA in in vitro cells and animal models [ 20 ], our patients did not have a family history of ALS or showed C9ORF72 genetic mutations (data from patient’s clinical record), which could potentially underlie MA dysfunction. Moreover, it has described a mutual negative interaction between TDP-43 cytoplasmic aggregates and MA mechanisms [ 73 ]. Actually, Park et al. [ 69 ] reported a reduction in MA in yeast as a result of TDP-43-induced toxicity following its overexpression. These findings suggest a potential reciprocal inhibition between MA and TDP-43 proteinopathy that requires further investigation in human MNs. Conclusion Our study shows for the first time a high CMA activity in healthy human spinal motor neurons, as evidenced by elevated LAMP2A levels. However, sALS patients show a significant decrease in LAMP2A expression that correlates with cytoplasmic aggregates and the nuclear clearance of TDP-43. Notably, MA markers such as LC3 and the lysosomal enzyme GBA remain unchanged. CMA is preserved in Onuf’s nucleus MNs. Enhanced LAMP2A expression in glial cells indicates a maintained antioxidant response in other brain cells. These findings strongly implicate CMA impairment as a key determinant of selective MN vulnerability in ALS. Collectively our results provide novel insights into pathological protein accumulation mechanisms and highlight CMA enhancement as a promising therapeutic strategy to restore proteostasis and prevent neurodegeneration in ALS. Abbreviations a= axon AF = Anterior funiculus. AGH = Anterior gray horn. ALS= Amyotrophic lateral sclerosis. ChAT: choline acetyltransferase CMA= Chaperone-mediated autophagy. CV= Cresyl violet. DPSC= Fine punctate granules scattered diffusely in the cytoplasm fALS: Familiar amyotrophic lateral sclerosis. GBA= Lysosomal enzyme glucocerebrosidase. GCs= Glial cells. H&E= Hematoxylin-Eosin. LC3= Autophagosome marker microtubule-associated protein light chain 3. lcst = Lateral corticospinal trac. LF= Lateral funiculus. Lf = Lipofuscin. MA= macroautophagy. mcst = Medial corticospinal trac. MMP9= Matrix metalloproteinase-9. MNs = Motoneurons. MNBMc: Mononuclear bone marrow cells. MS= microscopy slides. N = Neuronal nucleus. PF = Posterior funiculus. PGH = Posterior gray horn. Rls: Round inclusions S = Neuronal soma. sALS: sporadic amyotrophic lateral sclerosis. SC= spinal cord. SLIs= skein-like inclusions. SNs = Sensory neurons. TCNs = Clarke's thoracic column neurons. TDP-43= Trans-activation response DNA-binding protein. TS= Tissue slides. V = Vacuoles. Declarations Generative AI and AI-assisted technologies in the writing process During the preparation of this work the authors used Chat-GPT to streamline some parts of the text. After using this tool, the authors reviewed and edited the content as needed and took full responsibility for the content of the publication. Data availability statement The datasets used and analysed during the current study are available from the corresponding author on reasonable request. - Consent to Publish declaration: not applicable - Ethics approval and consent to participate The Anatomy Innovation Service of Miguel Hernandez University Medical School provides administrative and ethical support, with the approval of the Institutional Review Board. Samples and data from patients included in this study were provided by the Biobank IMIB (National Registry of Biobanks B. 0000859) (PT20/00109), integrated in the Platform ISCIII Biobanks and Biomodels and they were processed following standard operating procedures with the appropriate approval of the Ethics and Scientific Committees. Voluntary donations were obtained from patients included in the Phase I and II of clinical trial nº EudraCT:2006-00309612, NCT00855400 and EC 07/90762, NCT01254539. Acknowledgements We would first like to thank the patients who participated in the clinical trials and donated their tissues, making it possible to accurately complete the pathological study of the effects of the therapy applied in each trial arm. We want to particularly acknowledge the patients and the Biobank IMIB (PT20/00109) integrated in the Platform ISCIII Biobanks and Biomodels for their collaboration and the Anatomical Innovation Unit of the UMH. Funding This work was funded by the following projects: Spanish State Research Agency, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (Grant Numbers SEV-2017-0723), the Spanish Ministerio de Ciencia e Innovación grant numbers SAF2017-83702-R and PID2020-11817RB-I00 and the Generalitat Valenciana (program Prometeo II, Grant Number 2018/041). This work has been partially funded by the Instituto de Salud Carlos III (ISCIII) through the RICORS Project 'RD21/0017/0017; RD21/0017/0001; TERAV' supported by the Next Generation EU Program (Recovery, Transformation and Resilience Plan) RV was supported by fellowship Ramon y Cajal (RYC) 2019-027520-I funded by Ministerio de Ciencia, Innovación y Universidades (MCIU) and Agencia Estatal de Investigación (AEI) MCIU/AEI/10.13039/501100011033, as “European Social Fund (ESF) Investing in your future”. Competing interests The authors report no competing interests. 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Supplementary Files SupplementaryTable1.dox.docx SupplementaryTable2.dox.docx SuplFig1.tiff SuplFig2.tiff SuplFig3.tiff SuplFig4.tiff SuplFig5.tif Cite Share Download PDF Status: Published Journal Publication published 04 Feb, 2026 Read the published version in Acta Neuropathologica Communications → Version 1 posted Editorial decision: Revision requested 24 Sep, 2025 Reviews received at journal 24 Sep, 2025 Reviews received at journal 13 Sep, 2025 Reviewers agreed at journal 03 Sep, 2025 Reviewers agreed at journal 03 Sep, 2025 Reviewers invited by journal 02 Sep, 2025 Editor assigned by journal 27 Aug, 2025 Submission checks completed at journal 27 Aug, 2025 First submitted to journal 24 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Alicante. Spain.","correspondingAuthor":true,"prefix":"","firstName":"Salvador","middleName":"","lastName":"Martínez","suffix":""}],"badges":[],"createdAt":"2025-08-24 05:08:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7444163/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7444163/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40478-026-02238-6","type":"published","date":"2026-02-04T15:58:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90982914,"identity":"4f7bf951-2158-4d39-b6f3-9242a35f94e6","added_by":"auto","created_at":"2025-09-10 09:32:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21626796,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChAT-positive cells and LAMP2A expression in control motor neurons (MNs). \u003c/strong\u003e(a) Distribution of ChAT-positive MNs in the lumbar spinal cord. (b–k) LAMP2A immunoreactivity in neurons of the control lumbar spinal cord. (b) Low-magnification image showing the localization of gray and white matter regions in spinal cord sections. (c) High LAMP2A expression was selectively observed in MNs of the anterior gray horn (AGH) (arrows). (d–h) MNs in the anterior gray horn exhibited a distinct, highly LAMP2A-immunopositive puncta pattern in the perinuclear cytoplasm (arrows in f and g). (i–j) Neurons of Clarke’s thoracic column showed no LAMP2A immunostaining, or only scattered puncta expression localized to the cell body periphery (arrows). (k) Sensory neurons in the dorsal horn did not show LAMP2A immunoreactivity (arrow). Abbreviations: AF = Anterior funiculus, AGH = Anterior gray horn, lcst = Lateral corticospinal tract, Lf = Lipofuscin, MNs = Motoneurons, PF = Posterior funiculus, PGH = Posterior gray horn, S = Neuronal soma, N = Neuronal nucleus, n = Neuronal nucleolus, SN = Sensory neuron, TCN = Clarke's thoracic column neurons. Scale bar: A: 300 μm; B: 3 mm; C: 300 μm; D: 120 μm; E: 60 μm; F, G: 20 μm; H: 160 μm; I, J, K: 20 μm.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/cb95035a92f06509d2e178bb.png"},{"id":90981398,"identity":"35590f60-8b8c-4a38-932c-68f344b67d26","added_by":"auto","created_at":"2025-09-10 09:24:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42096481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of LAMP2A in healthy and sALS motor neurons (MNs). \u003c/strong\u003e(a) Ten MNs representing spinal motor neurons from two control spinal cords, showing high expression of LAMP2A in the perinuclear region (Control 1: cells 1, 3, 5, 7, and 9; Control 3: cells 2, 4, 6, 8, and 10). (b) Ten distinct spinal MNs from two different sALS spinal cords, displaying low expression of LAMP2A (ALS 47: cells 1, 3, 5, 7, and 9; ALS24: cells 2, 4, 6, 8, and 10). (c–d) Low-magnification images showing weak LAMP2A expression in MNs (arrows) and strong LAMP2A expression in glial cells (arrowheads) within the anterior gray horn. (e) In some sALS MNs, LAMP2A-positive puncta are localized at the periphery of the cytoplasm (arrows). (f–g) Images of MNs in sALS spinal cords exhibit nearly normal localization of LAMP2A-positive puncta (arrows), along with strong LAMP2A expression in glial cells (arrowheads). Abbreviations: S = Neuronal soma; N = Neuronal nucleus. Scale bar: A, B, C: 60 μm; D: 60 μm; E: 30 μm; F, G: 60 μm.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/6450734ba815ef20b82284c2.png"},{"id":90981400,"identity":"39ea55f5-feda-46f6-b6c9-df32ec0fe061","added_by":"auto","created_at":"2025-09-10 09:24:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9302380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential expression of LAMP2A, LC3 and GBA markers in control and sALS spinal MNs.\u003c/strong\u003e (a-e) Presence of perinuclear expression of LAMP2A in control MNs (A) but not in sALS (b-e). (f-j) Autophagosomes detected by LC3 puncta aggregates are present in both control (f) and sALS neurons (g-j). (k-p) GBA positive neurons show elevated perinuclear density in control (k) and sALS neurons (m-p). (q) LAMP2A expression is significantly reduced in sALS MNs (***p\u0026lt;0.001) while remaining unaltered in LC3 and GBA positive cells. (r-s) Microphotographs showing LC3 expression in thoracic column neurons of sALS spinal cord. (t-u) Localization of p62 (t) and TDP-43 (u) deposits in sALS lumbar spinal MNs. Scale bar: (a-p) 40 μm; R,S: 40 μm; t, u: 40 μm.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/1a0ac644a22e5d77f162a4ea.png"},{"id":90982912,"identity":"67c7cfa2-e31a-4f4f-915f-9b135cb31480","added_by":"auto","created_at":"2025-09-10 09:32:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":19681150,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLAMP2A and CD68 inflammation marker expression in glial cells. \u003c/strong\u003e(a) Control spinal cord section showing the absence of LAMP2A immunolocalization in the white matter. (b-f) LAMP2A positive astroglial cells in the lateral corticospinal tract of sALS spinal cord sections (arrows). Corticospinal axonal profiles are identified (a). Some LAMP2A positive glial cells showed cellular processes evolving axonal profiles (f, arrowheads). (g, h) CD68 expression as a marker for inflammatory cells. Perivascular infiltration of CD68 positive cells is predominant in the lateral corticospinal tract. (i) Inflammatory cells (CD68 immunopositive) are also detected in the anterior gray matter (arrows). LF: Lateral funiculus; MN: Motoneuron; PGH = Posterior gray horn. a = axon. Scale bars: a: 3 mm; b: 2 mm; c: 150 μm; d: 60 μm; e: 30 μm; f: 30 μm. g: 2 mm; h: 200 μm; i: 30 μm.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/4ac1c3ec7de2a40ebd0c81e8.png"},{"id":90981402,"identity":"1e073fdc-0269-4d03-a001-17cb09c00cdb","added_by":"auto","created_at":"2025-09-10 09:24:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":18833866,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-expression of LAMP2A and TDP-43 protein in healthy neurons.\u003c/strong\u003e (a) ChAT positive neurons in the AGH specifically identify MNs. (b, c) Co-expression of TDP-43 protein predominantly in the nucleus (black staining) and ChAT protein in the cytoplasm (brown staining). (d, e) Co-expression of LAMP2A (brown staining) and TDP-43 (black staining), showing the close cytoplasmic colocalization of TDP-43 and LAMP2A positive zones (arrows). (f, g) Co-expression of ChAT (brown staining) and TDP-43 (black staining). Arrow in (f) identifies nucleo-cytoplasmic connection of TDP-43 protein. (g) Cytoplasmic colocalization of TDP-43 (large arrows) and LAMP2A (small arrows) positive zones. Lf = Lipofuscin. N: nucleus. Scale bars: a: 600 μm; b: 30 μm; c: 60 μm; d, f: 50 μm; g: 30 μm.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/d96bff5caab7b149ba3d6d44.png"},{"id":90981416,"identity":"17c87c6d-9501-4894-a890-40b457e2c195","added_by":"auto","created_at":"2025-09-10 09:24:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":33367702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTDP-43 immunostaining in sALS MNs shows proteinopathy as nuclear clearance and cytoplasmic deposits.\u003c/strong\u003e (a-c) MNs in the AGH are identified by their large size in Nissl’s staining. Most of the MNs showed nuclear clearance and TDP-43 cytoplasmic aggregates and large vacuoles in their cytoplasm (arrows). (d) High power image of two MNs identified in c, showing large cytoplasmic vacuoles in contact with TDP deposits, (e-g) Images of MNs in sALS spinal cord to illustrate nuclear clearance and TDP-43 aggregates (arrows) in close contact with cytoplasmic vacuoles. (f, g) The cisternal pattern of Nissl bodies in the cytoplasm exhibits a scattered granular pattern (arrowheads). (h) MN showing nuclear localization of TDP-43. (i) MN displaying normal nuclear localization of TDP-43 in the nucleus and fine punctate granules scattered diffusely in the cytoplasm (arrows) with normal cisternal pattern of Nissl bodies (arrowheads). (j) MN showing normal localization of TDP-43 in the nucleus without cytoplasmic deposits. (k) MNs showing only TDP-43 nuclear clearance, with TDP-43 cytoplasmic punctate aggregates (arrows) also exhibited Nissl cistern distortion (arrowheads). (m) Interneurons in the AGH show nuclear localization of TDP-43 (arrow) and normal pattern of Nissl cisterns (arrowheads). AF = Anterior Fasciculus. AGH = Anterior gray horn. CV = Cresyl violet. N= nucleus. V = Vacuoles. Scale bars: a: 600 μm; b, c: 350 μm; d, e: 80 μm; f, g, h: 20 μm; i, j: 30 μm; k: 15 μm; m: 20 μm.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/12149ce55a711e5665741cb0.png"},{"id":90981472,"identity":"3dc88a21-3b8e-4e98-954f-9dbae9856cbd","added_by":"auto","created_at":"2025-09-10 09:24:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":24329508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-expression of TDP-43 and LAMP2A shows nuclear clearance and aberrant cytoplasmic deposits of TDP-43, together with low LAMP2A expression. \u003c/strong\u003e(a) sALS MNs showing TDP-43 nuclear clearance (large arrowhead), round inclusions (RIs) (small arrowhead) and skein-like inclusions (SLIs) (arrow) in their cytoplasms, with very low LAMP2A expression. (b) sALS MN showing TDP-43 round inclusions (RIs) (arrowhead) with very low LAMP2A expression in the cytoplasm (small arrows); interneurons (large arrow) show normal nuclear localization of TDP-43, with some punctate expression in the cytoplasm. (c) RLIs in the cytoplasm of MNs (arrows) and weak expression of LAMP2A. (d) Nuclear clearance and fine punctate granules scattered diffusely in the cytoplasm (DPSC) (arrowhead) and SLIs in another MN (arrow). (e) sALS MN showing RLIs (arrows) and filiform aggregates in the soma and dendrites of MNs (arrowhead). (f) sALS MN showing filiform aggregates in the soma (arrows) and dendrites (arrowheads). (g, h) MNs showing RLIs, SLIs TDP-43 inclusions and filiform aggregates across the cellular membrane (arrows). (i) sALS MN showing SLIs in the soma (arrow) and filiform aggregates in the dendrites (arrowhead). (j) TDP-43 filiform aggregate into a dendrite (arrowheads). (k) TDP-43 filiform aggregate in the interstitial space (arrowhead). (m) sALS MN showing nuclear clearance and DPSC (arrows). N= Nucleus. S = Soma. Scale bars: a: 60 μm; b: 40μm; c, d: 60 μm; e: 40 μm; f: 25 μm; g, h, i: 30 μm; j, k: 15 μm; m: 40 μm.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/ffbc44a62aeb0eb963e117d0.png"},{"id":90981470,"identity":"b6267ab9-e91c-4498-abaa-3f36b57945d6","added_by":"auto","created_at":"2025-09-10 09:24:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":16070660,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of Onuf’s nucleus through ChAT immunostaining and its co-expression of LAMP2A and TDP-43.\u003c/strong\u003e (a-c) ChAT positive MNs with cresyl violet counterstaining. (b, c) High power microphotograph showing an ChAT positive spinal MN with cytoplasmic vacuolization (arrow) and an Onuf`s ChAT positive MN without structural pathology. (d) High expression of LAMP2A in sALS Onuf’s MNs. (e) Spinal MNs close to Onuf’s MNs showed low and predominantly peripheral expression of LAMP2A (arrows). (f, g) Nuclear localization of TDP-43 in Onuf’s MNs showing LAMP2A highly expressed in the MNs perinuclear somatic area. (H) Spinal MNs near to Onuf’s MNs (arrowhead) show cytoplasmic vacuolization (arrow). (I) Nissl bodies’ staining with cresyl violet and TDP-43 immunostaining in sALS Onuf’s MNs, showing normal cisternal structures (arrows). AGH = Anterior gray horn. AF = Anterior funiculus. N= Nucleus. S = Soma. Scale bars: a: 300 μm; b: 150 μm; c: 70 μm; d: 40 μm; e: 30 μm; f: 200 μm; g: 70 μm; h: 60 μm; i: 40 μm.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/4f135026c0f22d00a99be19a.png"},{"id":102234926,"identity":"a142d929-9d6f-4b4e-acd1-3944f0a0186c","added_by":"auto","created_at":"2026-02-09 16:14:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":167466624,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/44791187-19a0-469e-aa1a-85a80a7c7b75.pdf"},{"id":90981452,"identity":"dff73155-71c5-4a68-a5c8-927ca5129c30","added_by":"auto","created_at":"2025-09-10 09:24:20","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2925554,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.dox.docx","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/f0894ad8440ef25a578bcecd.docx"},{"id":90981456,"identity":"9778cc40-d0c3-4045-85df-0ce21beec5db","added_by":"auto","created_at":"2025-09-10 09:24:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2925168,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.dox.docx","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/1c0aacbbc875576e6f860dfb.docx"},{"id":90981427,"identity":"e5ba7a07-ca85-4448-a24d-476d8fed2f33","added_by":"auto","created_at":"2025-09-10 09:24:19","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":578406,"visible":true,"origin":"","legend":"","description":"","filename":"SuplFig1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/13f66af9ae46f00019356537.tiff"},{"id":90981459,"identity":"ad98fc82-8472-4e14-833e-ff5ecbdbe99a","added_by":"auto","created_at":"2025-09-10 09:24:20","extension":"tiff","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1845694,"visible":true,"origin":"","legend":"","description":"","filename":"SuplFig2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/7919c69f93e25851f4ec49c3.tiff"},{"id":90982910,"identity":"f094e5a6-0df7-4cbb-aaf3-b69e87b265fa","added_by":"auto","created_at":"2025-09-10 09:32:19","extension":"tiff","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3246540,"visible":true,"origin":"","legend":"","description":"","filename":"SuplFig3.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/01aa749c31b893cff590032b.tiff"},{"id":90982917,"identity":"2cad2233-80ba-4487-ba91-4169104be862","added_by":"auto","created_at":"2025-09-10 09:32:21","extension":"tiff","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3614516,"visible":true,"origin":"","legend":"","description":"","filename":"SuplFig4.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/16acaabfdf3611fe575ae008.tiff"},{"id":90981424,"identity":"7a36ba29-1d3c-4896-82f0-dbb9f4b7d87c","added_by":"auto","created_at":"2025-09-10 09:24:19","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":9447028,"visible":true,"origin":"","legend":"","description":"","filename":"SuplFig5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7444163/v1/77d04a586d0b3317d429103b.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chaperone Mediated Autophagy is deficient in Spinal Motoneurons of ALS patients with TDP-43 proteinopathy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons (MNs) in the primary motor cortex and spinal cord (SC) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Most patients succumb within 3\u0026ndash;5 years due to progressive palsy and respiratory failure [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The global prevalence and incidence are estimated at 4.1\u0026ndash;8.4 per 100,000 individuals and 0.6\u0026ndash;3.8 per 100,000 person-years, respectively[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], with approximately 30,000 deaths reported annually [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. ALS arises from a complex interplay of genetic and environmental factors [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], and manifests in familial (fALS) and sporadic (sALS) forms [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. More than 20 genetic mutations have been implicated, with \u003cem\u003eC9ORF72, FUS, TARDBP\u003c/em\u003e, and \u003cem\u003eSOD1\u003c/em\u003e being the most frequently involved [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRegardless of etiology or site of onset, ALS is a predominantly MN-selective neurodegenerative disorder for reasons that remain unclear. This selectivity has been attributed to several intrinsic features of MNs, including their large size, high metabolic demands, dependence on mitochondrial integrity, vulnerability to excitotoxicity, disrupted intracellular calcium homeostasis, and impaired ubiquitin\u0026ndash;proteasome system function [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. A bottom-up pathogenic model, retrograde neuropathy originating at neuromuscular terminals, may account for the preferential degeneration of spinal MNs and the potential for trans-synaptic involvement of the primary motor cortex [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Moreover, differences in neuromuscular junction architecture may underlie the relative resistance of certain MN subpopulations, such as oculomotor and Onuf\u0026rsquo;s nuclei, although this remains experimentally unverified [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCurrent therapeutic options for ALS remain limited. Riluzole, the first approved drug, extends survival by only 6\u0026ndash;19 months [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Edaravone received FDA approval in 2017 [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e], but failed to show efficacy in an independent clinical trial in Italy [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. More recently, antisense oligonucleotide therapies have been developed for fALS with SOD1 mutations [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Tofersen, approved in SOD1 fALS following a phase III trial, did not yield significant clinical improvement but showed encouraging biomarker responses, including reductions in cerebrospinal fluid SOD1 protein and plasma neurofilament light chain (NfL) levels [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Similarly, Relyvrio, initially approved for slowing ALS progression, was later withdrawn after a failed phase III trial [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Given the absence of effective treatments for ALS, there is an urgent need for further preclinical research and improved drug-target identification.\u003c/p\u003e\u003cp\u003eAmong the cellular pathways increasingly implicated in ALS pathogenesis, autophagy plays a central role in maintaining protein homeostasis and cellular integrity [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. This multi-step process involves the lysosomal degradation and recycling of intracellular components, including aberrant proteins and damaged organelles [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Three types of autophagy are described in mammals: macroautophagy (MA), microautophagy, and chaperone-mediated autophagy (CMA). While MA and microautophagy involve bulk degradation and direct lysosomal delivery of cargo, respectively [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], CMA selectively targets proteins bearing a KFERQ-like motif. These are recognized by the HSC70 chaperone and delivered to the lysosomal receptor LAMP2A, the key limiting factor for CMA in the neurons [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNotably, TDP-43, a nuclear RNA-binding protein involved in RNA processing, genome integrity, and mRNA metabolism [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], contains the KFERQ-like motif [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In approximately 95% of ALS cases, TDP-43 is mislocalized, forming phosphorylated and ubiquitinated cytoplasmic aggregates that are hallmark features of spinal MN pathology [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Although these aggregates are prominent in sALS, mutations in TARDBP and C9orf72 can also lead to TDP-43 proteinopathy [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Clearance of TDP-43 aggregates is critical to mitigate their cytotoxicity and has been linked to both the ubiquitin\u0026ndash;proteasome system and MA [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Moreover, reducing TDP-43 levels in ALS mouse models improves motor deficits, suggesting that motoneuronal dysfunction may be at least partially reversible[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. It has been proved in experimental models that MA activation, for instance through mTOR inhibition, enhances TDP-43 turnover and cell viability [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGiven the role of TDP-43 in ALS and its potential recognition by CMA, we investigated LAMP2A expression, a key marker of CMA in human SCs [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. We analyzed SC tissue from six control subjects (n\u0026thinsp;=\u0026thinsp;6; 3 females, 3 males) and ten sALS patients (n\u0026thinsp;=\u0026thinsp;10; 6 females, 4 males), which exhibited varying degrees of TDP-43 proteinopathy. In control SCs, we observed intense LAMP2A expression in MNs across all regional levels. In contrast, sALS SC samples showed a marked reduction in LAMP2A expression in MNs, both in early pathological stages, characterized by nuclear TDP-43 clearance and granular cytoplasmic aggregates, and in advanced stages with dense cytoplasmic TDP-43 inclusions. Interestingly, Onuf\u0026rsquo;s nucleus MNs, which are relatively spared in ALS [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], displayed strong LAMP2A expression and lacked TDP-43 pathology. These findings suggest that CMA dysfunction may contribute to the selective vulnerability of MNs in ALS and underscore a potential protective role of preserved CMA activity in resistant MN populations.\u003c/p\u003e"},{"header":"M\u0026M","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eHuman tissue processing\u003c/h2\u003e\u003cp\u003e Human control samples without neurological disorder (n\u0026thinsp;=\u0026thinsp;6 SC) were obtained from anonymous donations through the Anatomy Innovation Service of Miguel Hernandez University Medical School, which provides administrative and ethical support, with the approval of the Institutional Review Board. Samples and data from patients included in this study (n\u0026thinsp;=\u0026thinsp;10 SC) were provided by the Biobank IMIB (National Registry of Biobanks B. 0000859) (PT20/00109), integrated in the Platform ISCIII Biobanks and Biomodels and they were processed following standard operating procedures with the appropriate approval of the Ethics and Scientific Committees. Voluntary donations were obtained from patients included in the Phase I and II of clinical trial n\u0026ordm; EudraCT:2006-00309612, NCT00855400 and EC 07/90762, NCT01254539 (Supplementary Table\u0026nbsp;1).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTissue preparation and Immunohistochemical staining\u003c/h3\u003e\n\u003cp\u003eSpinal cords were fixed in 10% formalin (Sigma-Aldrich, Germany) for 5 days at room temperature. Following fixation, the SC was transversely trimmed into tissue slides (TS) 5\u0026ndash;7 mm thick and labelled in a rostro-caudal order into progressive cervical, thoracic, lumbar, or sacral regions (Supplementary Fig.\u0026nbsp;1). TS underwent a progressive dehydration process in ethanol, followed by butanol and were subsequently embedded in paraffin. Transversal sections, 7\u0026ndash;10 \u0026micro;m thick, were then obtained from four selected TS at each SC region, covering the entire region, and mounted on microscopy slides (MS) in 20 parallel series (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eSpinal cords were processed to paraffin embedding and sectioning sections following the protocol described in Supplementary Fig.\u0026nbsp;1. MS were processed by Hematoxylin-Eosin (H\u0026amp;E, serie 1), Cresyl violet (CV, serie 2), and immunohistochemistry (series 3\u0026ndash;11). We conducted immunohistochemical analysis on subsequent parallel series of MS from cervical segments C3-6 (n\u0026thinsp;=\u0026thinsp;20), thoracic segments T8-11 (n\u0026thinsp;=\u0026thinsp;20), lumbar segments L2-5 (n\u0026thinsp;=\u0026thinsp;20), and sacral segments S1-4 (n\u0026thinsp;=\u0026thinsp;20) of control and sALS SCs. To minimize experimental bias in the data of ALS patients, T2\u0026ndash;T6 segments were excluded because 7 patients had received intraspinal autologous graft of mononuclear bone marrow cells (MNBMc) in the T3\u0026ndash;T5 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], intrathecal MNBMc graft, or intrathecal administration of saline solution, as placebo group (clinical trials (CT) NCT00855400 and NCT04849065, Phase I and II; Supplementary Table\u0026nbsp;1). Finally, one patient was not included in Phase II CT and was classified as a not treated ALS patient (Supplementary Table\u0026nbsp;1). Our previous pathological data from the Phase I CT showed no significant modifications in non-experimental segments[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], therefore, the rostral and caudal segments relative to T2\u0026ndash;T7 were considered affected in accordance with the natural progression of the disease in each patient.\u003c/p\u003e\u003cp\u003eImmunohistochemistry procedures were described in Blanquer et al., [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Briefly, sections were treated with primary antibodies, diluted in EnVision FLEX Antibody Diluent (DAKO, Denmark), 24 hours at 4\u0026deg;C (Supplementary Table\u0026nbsp;2). Following primary antibody incubation, sections were incubated 2 hours with the appropriate biotinylated secondary antibody (Supplementary Table\u0026nbsp;2). Then, sections were incubated with Avidin\u0026ndash;Biotin Complex for 1 h (ABC kit, Vector Laboratories CA-94010). For colorimetric detection (brown), the tissue was incubated with 1% 3,3'-Diaminobenzidine (DAB; Vector Laboratories SK-4100) and 0.0018% H2O2 in PBS. For double immunohistochemistry with anti-TDP 43/anti-ChAT and anti-TDP-43/anti-LAMP2A, anti-TDP-43 was incubated with 1% 3,3'-Diaminobenzidine (DAB; Vector Laboratories SK-4100), 0.025% ammonium nickel sulfate hexahydrate, and 0.0018% H2O2 in PBS for colorimetric detection (black). For double immunochemistry with anti-GFAP/anti-IBA1, anti-IBA1 was processed to obtain a black color. Sections series processed in parallel without primary or secondary antibodies did not show any specific or nonspecific labeling (Supplementary Fig.\u0026nbsp;2). Finally, the sections were dehydrated and mounted in Eukitt (O.Kindler GmbH and CO, Freiburg).\u003c/p\u003e\n\u003ch3\u003eMicroscopy and statistical analysis\u003c/h3\u003e\n\u003cp\u003eImages were captured using an optical microscope (Leica CTR6000) and were utilized to assess the percentage of cellular area expressing various markers, including LAMP2A, LC3, and GBA (see below). After identifying MNs in ventro-medial and ventro-lateral columns (where more MNs were identified in ALS patients), the ImageJ program was employed to quantify the percentage of area expressing LAMP2A, LC3, and GBA relative to the total cell area (n\u0026thinsp;=\u0026thinsp;20 MNs/marker in each group). Statistical analysis comparing sALS and control samples was performed using Sigmaplot v11.0 software. The data were presented as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE), and pairwise comparisons between sALS and control samples (attending: gender, segmental level and MNs column) were conducted using the Student\u0026rsquo;s t-test. Graphs and statistical visualizations were generated using GraphPad Prism version 10 (GraphPad Software, San Diego, CA). A significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant, with *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 denoting different levels of significance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSpinal motoneurons exhibit elevated expression levels of LAMP2A.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that TDP-43 is a potential substrate for CMA [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], we investigated LAMP2A expression, the rate-limiting component of CMA [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], in spinal MNs.\u003c/p\u003e\u003cp\u003eTo investigate LAMP2A expression in the human SC, we conducted immunohistochemical analyses on sections from C3\u0026ndash;C4, T8\u0026ndash;T9, L3\u0026ndash;L4, and S2\u0026ndash;S3 segments (n\u0026thinsp;=\u0026thinsp;10 slides per segment) obtained from control SC (n\u0026thinsp;=\u0026thinsp;6; Supplementary Table\u0026nbsp;1). MNs were identified based on their distinct morphological features observed with H\u0026amp;E and CV staining, as well as their immunoreactivity for choline acetyltransferase (ChAT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In parallel slides we observed robust and specific LAMP2A expression in MNs across all SC segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u0026ndash;g). The immunoreactivity exhibited a puncta perinuclear pattern within the MN cytoplasm, consistent with lysosomal membrane localization of LAMP2A [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-h). Additionally, most MNs also contained lipofuscin granules that were immunonegative for both ChAT and LAMP2A (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e; Supplementary Fig.\u0026nbsp;3a\u0026ndash;d).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast, LAMP2A immunoreactivity was markedly lower in other neuronal populations, including Clarke\u0026rsquo;s column neurons (TCNs; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei, j; Supplementary Fig.\u0026nbsp;3e) and dorsal horn sensory neurons (SNs; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek; Supplementary Fig.\u0026nbsp;3e). In TCNs, LAMP2A-positive puncta were distributed throughout the cytoplasm rather than showing a perinuclear concentration as seen in MNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei, j). Additionally, glial cells (GCs) exhibited minimal LAMP2A immunoreactivity (Supplementary Fig.\u0026nbsp;3d, f\u0026ndash;h).\u003c/p\u003e\u003cp\u003eThen, we explored potential age- and sex-related differences in CMA by comparing SC samples from Control 1 and 6, derived from 64- and 62-year-old females, respectively, with Control 3 and 5, obtained from 53-year-old males (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Our analysis did not reveal significant differences in LAMP2A expression among these SC samples. Notably, despite the male samples being approximately 10 years younger than the female samples, LAMP2A expression levels were comparable. Furthermore, comparison of SC samples from Control 1 and 6 with Control 4 (a 70-year-old female, almost 10 years older) also showed no substantial differences in LAMP2A expression (data not shown). Therefore, do not reveal age-related or sex-specific differences in LAMP2A expression in MNs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese findings indicate that spinal MNs selectively express high levels of LAMP2A, suggesting heightened CMA activity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpinal motoneurons of sALS patients exhibit low expression levels of LAMP2A.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo analyze CMA activity in spinal MNs of sALS patients, we performed LAMP2A immunohistochemistry on sections from C3-C4, T8-T9, L3-L4, and S2-S3 segments of sALS patient\u0026rsquo;s SC (n\u0026thinsp;=\u0026thinsp;10; Supplementary Table\u0026nbsp;1). In sALS SCs, cervical, lumbar, and sacral segments exhibited a greater number of detectable MNs based on morphological and immunohistochemical criteria (a mean of 9\u0026ndash;12 MNs/section, 12\u0026ndash;17 MN/section, and 5\u0026ndash;10 MN/section, respectively) compared to thoracic segments (0\u0026ndash;3 MN/section). Consequently, we increased in our study the number of thoracic slides (n\u0026thinsp;=\u0026thinsp;20) to ensure at least 20 thoracic MNs were analyzed in each patient, along with 20 MNs from other SC regions (n\u0026thinsp;=\u0026thinsp;10 from each segment), which were then compared with the same number of control MNs. In all cases of sALS SC, LAMP2A immunopositivity was notably weak in most of the MNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). While the majority of MNs from sALS patients showed a significantly reduced expression of LAMP2A (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u0026ndash;e), a small subset (approximately 5\u0026ndash;10% of MNs) exhibited peripheral or normal cytoplasmic distribution of LAMP2A-positive puncta (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e). These findings suggest a decrease in CMA activity in sALS MNs relative to controls. In contrast, spinal MNs from the patient ALS40 displayed a moderate reduction in LAMP2A expression in approximately 60% of MNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, g).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eMacroautophagy is preserved in sALS patients’ motor neurons\u003c/h3\u003e\n\u003cp\u003eIn animal models of ALS, impaired MA and autophagosome formation have been reported [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Therefore, to evaluate MA function in our human SC sections, we examined the expression of the autophagosome marker microtubule-associated protein light chain 3 (LC3) using immunohistochemistry [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Moreover, in selective MA, a specific interaction between p62 and LC3 is necessary to mediate the autophagic degradation of p62-positive structures [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. The detection of p62-positive aggregates serves as an indicator of MA deficiency in tissues [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. Consequently, p62 expression was also analyzed in our samples. Lastly, to investigate lysosomal formation and distribution in MNs we assessed the expression of the lysosomal enzyme glucocerebrosidase (GBA) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAbundant immunopositive LC3 puncta aggregates were detected in the cytoplasm of both controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef) and sALS spinal MNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-j). GBA immunolabeling was observed as a puncta pattern dispersed in the cytoplasm with higher perinuclear density (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek-p).\u003c/p\u003e\u003cp\u003eTo compare CMA, MA and lysosomal formation, we quantified immunopositive puncta of LAMP2A, LC3B, and GBA in SC MNs where the nucleus was clearly detected and in nonconsecutive sections to ensure that we did not count the same neuron multiple times. We selected 10 MNs of the antero-lateral MNs column in two control SC (Controls 1 and 3; 5 MNs at cervical and 5 MNs at lumbar segments), as well as 10 MNs of antero-lateral MNs column in two patients\u0026rsquo; SC, ALS 24 and 47 (5 MNs at cervical and 5 MNs at lumbar segments) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-p). Our quantitative analysis demonstrated that LAMP2A expression is significantly reduced in sALS MNs, while autophagosomes formation detected by LC3 puncta and the number of lysosomes related to the GBA expression showed no significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eq). Furthermore, to confirm the specificity of the increased LAMP2A immunoreactivity in control SC MNs, we observed no qualitative differences in LC3 expression between MNs and TCN in control samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003er, s).\u003c/p\u003e\u003cp\u003eThe analysis of P62-protein intracytoplasmic deposits in all the parallel series processed by P62 immunohistochemistry detected only 5 MNs in lumbar sections of ALS 40 patient SC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003et), where also TDP-43 deposits appeared in the parallel series (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eu). All other control and sALS SC samples showed no p62 deposits. These results further support that MA is not significantly affected in human sALS MNs.\u003c/p\u003e\u003cp\u003eAnalysis of LAMP2A, LC3, GBA, and p62 expression in MNs from control and ALS patient samples did not reveal any significant sex- or age-related differences in expression levels. However, in ALS 40, increased LAMP2A expression and p62 accumulation were observed in a subset of MNs, suggesting the possibility of a distinct etiological variant of sALS in this patient.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eGlial cells increase the expression of LAMP2A in sALS spinal cords\u003c/h2\u003e\u003cp\u003eIn control SC tissue, glial cells in both white and gray matter exhibit weak LAMP2A expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea; Supplementary Fig.\u0026nbsp;3d, f, g). In contrast, LAMP2A-immunopositive glial cells of sALS SC were observed in the anterior horn gray matter and were more abundant in the lateral and medial corticospinal tracts (lcst and mcst; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u0026ndash;c). High-magnification images of the lcst revealed intensely LAMP2A-expressing cells with reactive astroglial morphology [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e], interspersed among LAMP2A-negative axonal fascicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed\u0026ndash;f). GFAP immunostaining highlighted the typical morphology of reactive astrocytes (Supplementary Fig.\u0026nbsp;4a\u0026ndash;c), which in some cases displayed disrupted cytoplasmic processes and somatic vacuolization (Supplementary Fig.\u0026nbsp;4c). This phenomenon, known as clasmatodendrosis [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e], was first described by Ram\u0026oacute;n y Cajal in 1913 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine if this increase of LAMP2A is due to the inflammation associated with the axonal neurodegeneration resulting from degeneration of primary cortical MNs, we studied the presence of immunocompetent cells infiltration using IBA1 and CD68 immunohistochemistry. LAMP2A expression in astroglial cells was colocalized within lcst and mcst, alongside IBA1-positive microglia (Supplementary Fig.\u0026nbsp;4d-f). There was also a notable infiltration of CD68-immunopositive cells, which appeared to be concentrated within perivascular spaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, h; Supplementary Fig.\u0026nbsp;4d, e). IBA1-positive microglial cells were also observed surrounding axons within these tracts (Supplementary Fig.\u0026nbsp;4f). Therefore, the significant increase of LAMP2A expression in glial cells may be due to an inflammatory process in cortico-spinal tracts as a consequence of axonal degeneration. Moreover, high-power pictures from the anterior horn showed the presence of CD68 immunopositive cells between MNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei), suggesting that an inflammatory process in the gray matter may also be associated with a reactive increase of LAMP2A expression in some glial cells. Double immunodetection using LAMP2A and IBA1 showed the distribution of LAMP2A-positive astroglia and IBA1-positive microglia in the cortico-spinal tracts (Supplementary Fig.\u0026nbsp;4f).\u003c/p\u003e\u003cp\u003eThe analysis of glial activation by LAMP2A expression in lateral and medial cortico-spinal tracts of sALS MNs did not show any age- or sex-specific differences when comparing expression levels in SC sections.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTDP-43 proteinopathy in LAMP2A deficient sALS motoneurons.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSpinal MNs from control SC exhibited TDP-43 immunoexpression primarily in the cell nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We performed ChAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) and double ChAT/TDP-43 immunohistochemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c), identifying colocalization of TDP-43 nuclear expression in ChAT-positive MNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c). Additionally, we conducted double immunohistochemistry in another section series to investigate the localization of TDP-43 and LAMP2A in spinal MNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-g). Some TDP-43 immunopositive puncta were detected in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee), occasionally in continuity with its nuclear expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef), suggesting a nucleo-cytoplasmic transfer of this protein [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These cytoplasmic TDP-43-positive colocalized with LAMP2A-positive puncta (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, e). The observed colocalization of TDP-43 and LAMP2A in the cytoplasm of control MNs further supports the involvement of CMA in TDP-43 cytoplasmic clearance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNuclear depletion and cytoplasmic deposits of TDP-43 in motor system neurons and glia have been described in ALS patients and animal models [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. As mentioned before, it has also been reported that TDP-43 is a substrate of CMA [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Therefore, we explored if LAMP2A deficient MNs of our sALS patients presented TDP-43 proteinopathy. We performed TDP-43 immunohistochemistry on sections of parallel series as described previously: C3-4, T8-9, L3-4, and S2-3 SC segments (n\u0026thinsp;=\u0026thinsp;10 slides in each segment) of sALS patients SC (n\u0026thinsp;=\u0026thinsp;10; see Supplementary Table\u0026nbsp;1). Anti-TDP-43 immunohistochemistry was counterstained with Cresyl violet, showing different degrees of proteinopathy in sALS spinal MNs. Most MNs in the spinal anterior gray matter showed TDP-43 proteinopathy (90\u0026ndash;95% of MNs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-g, k). We classified TDP-43 cytoplasmic inclusions according to Kon et al. [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] into three categories: 1) fine punctate granules scattered diffusely in the cytoplasm (DPSC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek), 2) round inclusions (RIs) about 1\u0026ndash;15 \u0026micro;m in diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg), and 3) skein-like inclusions (SLIs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-f). Predominant nuclear immunolocalization and punctate granules in the cytoplasm, similar to those observed in control MNs, were observed in 3\u0026ndash;5% of sALS MNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, i). Additionally, 30\u0026ndash;35% of MNs showed only partial or total depletion of nuclear TDP-43 immunopositive granules, with DPSC (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-k), but without important cytoplasmic deposits, which has been described as the initial proteinopathy in relation to stress granules in ALS MNs [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In the rest of the analyzed MNs (60%), they showed nuclear clearance accompanied by cytoplasmic deposits, together with strong degenerative cytoplasmic vacuolization in colocalization with TDP-43 aggregates, in agreement with Martin [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-g). Moreover, the normal cisternal pattern of Nissl bodies in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-j) disappeared in sALS MNs showing TDP-43 aggregates and was replaced by a granular, scattered pattern intermixed with DPSC of TDP-43 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, g, k). In contrast, anterior horn interneurons showed normal nuclear TDP-43 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo analyze the co-expression of TDP-43 and LAMP2A in the patient\u0026rsquo;s MNs, another parallel series of sections were processed by double immunohistochemistry using anti-TDP-43 and anti-LAMP2A antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). First, the reduction of LAMP2A expression in sALS MNs was clear, being especially evident in MNs with RIs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, b). Second, in most sALS MNs with reduction of LAMP2A expression, nuclear clearance and DPSC of TDP-43 was clearly detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In addition, the distribution and typology of TDP-43 aggregates in LAMP2A-depleted MNs were heterogeneous and sometimes combined in the same cell, detecting a mixture of DPSC with round aggregates and SLIs. TDP-43 filiform inclusions were localized into MNs dendritic proximal segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, f, i, j). Moreover, some MNs showed TDP-43 filaments across the cellular membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, h), and TDP-43 filaments were detected in the interstitial space (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), suggesting the possibility of transmembrane trafficking and raising the possibility of transcellular transmission of misfolded TDP-43 protein.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eInterestingly, ALS40 SC MNs showed a reduced degree of TDP-43 proteinopathy. Actually, although more than 40% of MNs showed TDP-43 cytoplasm aggregates (Supplementary Fig.\u0026nbsp;5, a-c), more than 30% of MNs showed only nuclear depletion of TDP-43, and another 30% normal localization of this protein (Supplementary Fig.\u0026nbsp;5). These findings suggest that CMA is more active in this patient, as described previously (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, g). Accordingly, we examined the expression of LAMP2A in ALS40 SC (Supplementary Fig.\u0026nbsp;5, h). Our analysis revealed that the ALS 40 SC MNs exhibited higher LAMP2A expression than the other sALS SC, which may account for the reduced TDP-43 pathology. Quantification of LAMP2A expression in ALS40 SC MNs was higher than in control SC MNs (Supplementary Fig.\u0026nbsp;5).\u003c/p\u003e\u003cp\u003eThe analysis of TDP-43 expression, and TDP-43/LAMP2A co-expression in sALS MNs, with the exception of ALS40, did not show any sex-specific differences when comparing expression levels in sections from SC (Supplementary Table\u0026nbsp;1). As was described above, ALS40, although with more LAMP2A expression and less degree of TDP-43 proteinopathy, has the faster evolution: 11 months in comparison to the rest with a mean of 27,4 months (Supplementary Table\u0026nbsp;1). This may suggest a different pathogenic mechanism underlying the MN degeneration and reflects the heterogeneity of the disease evolution in each patient.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOnuf\u0026rsquo;s nucleus motoneurons exhibit high expression levels of LAMP2A in sALS spinal cords.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the sacral levels of sALS patients, Onuf\u0026rsquo;s nucleus was identified due to the presence of a significant number of ChAT-positive MNs in the anterior horn, between anterolateral and anteromedial columns (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e-c). While surrounding spinal MNs in the anterior horn showed a cytoplasm with a degenerative vacuolar profile, Onuf\u0026rsquo;s MNs appeared without cytoplasmic pathology (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, c, h). Moreover, Onuf\u0026rsquo;s MNs exhibited strong LAMP2A expression, with punctate lysosomal immunoreactivity comparable to that of control SC MNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). In contrast, spinal MNs in the same segment showed markedly reduced LAMP2A expression, with sparse puncta predominantly localized at the cell periphery (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe have studied the localization of TDP-43 protein in Onuf\u0026rsquo;s MNs of sALS patients in combination with LAMP2A expression or Nissl staining. In Onuf\u0026rsquo;s MNs, TDP-43 was predominantly localized in the nucleus of LAMP2A-positive MNs, similar to control MNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, g), and the normal cisternal pattern of Nissl bodies in the cytoplasm was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, i). These findings support that high CMA activity is crucial to maintain Onuf\u0026rsquo;s MNs alive in sALS patients and reinforces the possibility that CMA in SC MNs may play a role in preventing TDP-43 proteinopathy and MNs vulnerability of sALS.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results show for the first time that human SC MNs exhibit high levels of LAMP2A expression, suggesting that MNs require increased CMA activity to function properly. On the other hand, SC samples from sALS patients showed reduced LAMP2A expression in their MNs. These findings may explain the selective vulnerability of MNs observed in prior studies, which has been attributed to structural and functional differences unique to MNs [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Despite the reduced LAMP2A expression in sALS MNs, MA appeared unaffected compared to control MNs, as evidenced by the presence of autophagosomes detected by robust LC3B-positive cytoplasmic puncta pattern. GBA expression was unchanged, indicating normal lysosomal activity, including autophagosomes. TDP-43 was primarily localized in the nucleus of control spinal MNs, but in sALS MNs, it was absent from the nucleus and accumulated in the cytoplasm. Notably, Onuf\u0026rsquo;s nucleus in sALS patients showed higher LAMP2A expression and protection from TDP-43 cytoplasmic pathology. Finally, ALS40-specific differences further support the role of LAMP2A activation in preventing or delaying TDP-43 proteinopathy in sALS patients, also highlighting the heterogeneity of the etiology and pathology of this disease.\u003c/p\u003e\n\u003ch3\u003eCMA is selectively upregulated in spinal cord MNs\u003c/h3\u003e\n\u003cp\u003eCMA is a selective autophagic pathway that targets proteins with a lysosomal targeting motif, such as the pentapeptide chain KFERQ [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The chaperone Hsc70 binds to KFERQ-containing proteins, transporting them to the lysosomal surface, where they interact with LAMP2A before being unfolded and degraded in the lumen. CMA degrades approximately 30\u0026ndash;35% of cytosolic proteins, impacting critical processes such as lipid and glucose metabolism, DNA repair, cellular reprogramming, stress responses, and immunological function [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Impairment of CMA has been associated with several age-related diseases, including neurodegenerative disorders, atherosclerosis, metabolic diseases, and cancer [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe elevated CMA activity in spinal MNs could be attributed to their large size and high firing rates, consistent with Henneman\u0026rsquo;s size principle, which states that the size of a neuron is a crucial determinant in reaching the threshold for action potential firing [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Fast-fatigable motor units are more vulnerable to ALS, whereas slow motor units, which are more resistant, may help to reinnervate motor endplates [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. These findings suggest that the functional properties of MNs may influence their vulnerability to ALS. This functional activity necessitates the regulation of ion channels, receptor proteins, and ion buffering mechanisms, all of which contribute to the need for enhanced proteostasis control. Additionally, due to their highly polarized nature MNs require efficient protein production and degradation pathways to survive. As mentioned earlier, CMA has been implicated in neurodegenerative diseases, contributing to the degradation of pathogenic proteins such as α-synuclein and tau [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Loss of CMA function leads to significant changes in the neuronal proteome, disrupting essential neuronal functions and promoting neurodegeneration [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Moreover, the specificity of high requirements of CMA function in MNs could explain their heightened vulnerability to CMA impairment compared to other cell types, providing a potential mechanism for the selective neurodegeneration of MNs in sALS.\u003c/p\u003e\u003cp\u003eInterestingly, Khawaja et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] reported that CMA activity declines with age across most organs and cell types, with a more marked reduction observed in males. This decline is often associated with a reduced number of lysosomes functionally competent for CMA, suggesting that sex-specific differences in CMA activity may influence tissue vulnerability to age-related degenerative processes. Jacob et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] reviewed the mechanisms underlying sexual dimorphism in ALS, emphasizing sex-specific heterogeneity in both genetic and non-genetic mechanisms across experimental models and patient cohorts. Within this framework, the apparent accelerated decline of CMA observed in males, reflected by LAMP2A expression levels comparable to those of females approximately ten years older, may underlie the sex-biased prevalence and distinct clinical phenotypes of ALS. While age- and sex-associated reductions in CMA have been reported in murine models, further studies are required to validate these patterns in human SC MNs. In the present study, the limited sample size precluded definitive conclusions regarding age- or sex-related differences in LAMP2A expression in SC MNs.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCMA is downregulated in sporadic ALS Spinal MNs\u003c/h2\u003e\u003cp\u003eSpinal cord sections from sALS patients demonstrated reduced LAMP2A expression in MNs; however, approximately 5\u0026ndash;10% of MNs exhibited only a partial reduction in LAMP2A levels. This observation may reflect intrinsic heterogeneity in LAMP2A expression among MNs or indicate a progressive decline in its expression, potentially contributing to, or resulting from, the differential vulnerability of MN populations based on their functional and structural properties.(as reviewed in Ovsepian et al. [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]).\u003c/p\u003e\u003cp\u003eWhile MA has been well studied in ALS [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e], CMA remains less explored. Our analysis revealed that LC3 expression was comparable between control and sALS spinal MNs, suggesting preserved MA function in sALS. Despite its importance in neuronal homeostasis, MA does not appear to have cell-specific requirements in MNs, as no substantial differences in LC3 expression were observed between control MNs and other spinal neurons. Although MA impairments have been reported in ALS models, our findings suggest its role in disease pathophysiology may be secondary, with experimental activation potentially mitigating TDP-43 proteinopathy rather than addressing a primary defect [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA study by Arosio et al. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] reported TDP-43 proteinopathy with reduced levels of Hsc70 in lymphomonocytes of sALS patients without change in LAMP2A. In contrast, our results demonstrate reduced LAMP2A expression specifically in sALS MNs, reflecting fundamental differences in cellular expression and autophagy requirements across cell types.\u003c/p\u003e\u003cp\u003eMutations in the \u003cem\u003eLAMP2\u003c/em\u003e gene cause Danon disease, an X-linked lysosomal storage disorder characterized by cardio-myopathy and cognitive dysfunction. The pathological hallmark of this disease is the accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscles [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The cognitive dysfunction seen in humans with Danon disease suggests a critical role of LAMP-2 in brain function [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. These cognitive abnormalities are likely due to hippocampal dysfunction, associated with altered lysosomal activity, including the accumulation of p62-positive aggregates, autophagic vacuoles, and lipid storage within neurons. Notably, in agreement with our results, Rothaug et al. [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e] reported that the absence of LAMP2 did not appear to affect MA in the brain cells under physiological or starvation conditions. In Danon disease MNs degeneration has not been observed [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], which may be attributed to the cell-type-specific role of LAMP2B, the isoform mutated in Danon disease [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e], or to the limited lifespan of patients, which may preclude the manifestation of MN degeneration.\u003c/p\u003e\u003cp\u003eThe selective accumulation of TDP-43 in CMA-deficient MNs, in the absence of other CMA substrate accumulation, may reflect the compensatory degradation of these proteins via alternative pathways, such as endosomal microautophagy (eMI), which has been shown to process KFERQ-like motif\u0026ndash;containing proteins [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. Although we did not observe major alterations in macroautophagy or lysosomal distribution in MNs from patients with sALS, investigating the role of eMI in human MNs may help clarify the specificity of TDP-43 pathology in sALS.\u003c/p\u003e\u003cp\u003eInterestingly, LAMP2A levels were significantly higher in glial cells of sALS patients. The upregulation of LAMP2A in glial cells may be a reactive response to inflammatory processes occurring in the anterior horn and corticospinal tracts [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. Axonal degeneration is known to trigger inflammation and oxidative stress in glial cells, leading to activation of NFE2L2, which in turn increases LAMP2A expression and enhances CMA activity [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Indeed, this inducible regulation of LAMP2A, and consequently of CMA activity, by NFE2L2 (coding NRF2 antioxidant transcription factor) is not sufficient to overcome the low LAMP2A expression observed in sALS MNs, despite their elevated oxidative stress [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Bono et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] reviewed the possible role of alterations of antioxidant response in ALS neurons and glia by the activation of KEAP1-NRF2 without conclusive information on the primary cause of ALS. Interestingly, increased autophagy was reported by Ryu et al., [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e] underlining clasmatodendrosis as an autophagic death of astrocytes (reviewed in Balaban et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]). Although Guise et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] found no significant differences in LAMP2A expression between ALS and control MNs using laser-guided tissue dissection, our results suggest that it may be due to the inclusion of perineuronal astroglial fragments in the dissected samples, which exhibit strong LAMP2A activation and could mask the reduced LAMP2A in MNs.\u003c/p\u003e\u003cp\u003eThis supports the notion that the alteration in LAMP2A expression in sALS MNs is a primary, constitutive, cell-autonomous defect.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCMA in Onuf's Nucleus MNs and selective vulnerability in sALS\u003c/h2\u003e\u003cp\u003eOur results also provide evidence that Onuf\u0026rsquo;s nucleus in sALS patients retains LAMP2A expression. Slow motor units are then more protected, and they reinnervate the end-plate left by faster, fatigable motor units after cell death [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Interestingly, our work is the first to show that LAMP2A expression is not deficient in the Onuf\u0026rsquo;s MNs of sALS patients. The mechanisms by which Onuf\u0026rsquo;s nucleus remains spared in ALS and other neurodegenerative diseases, but impaired in others, remains unknown. Recently, RNA-seq analysis of mice Onuf's nucleus extracted by laser microdissection revealed that matrix metalloproteinase-9 (MMP9), an inflammatory biomarker that is highly expressed by ALS-affected spinal MNs, is not overexpressed in Onuf's neurons [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This MMP9 may represent a factor to reduce EN1 transference from interneurons to MNs, decreasing the paracrine neurotropism of this factor [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. This, along with proper CMA functioning, could lead to protection of this nucleus against ALS-mediated cell death. The localization of EN1 in spinal and Onuf\u0026rsquo;s MNs may shed some light on these mechanisms. The differential CMA activation likely contributes to the protective mechanisms underlying the selective preservation of Onuf's MNs in sALS. We propose that maintained CMA function in these MNs protects against TDP-43 proteinopathy and subsequent cell death.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eTDP-43 and autophagy in sALS\u003c/h2\u003e\u003cp\u003eOur findings demonstrate colocalization of LAMP2A and TDP-43 in the cytoplasm of control MNs, suggesting that CMA may facilitate cytoplasmic TDP-43 clearance. This supports the hypothesis that CMA dysfunction contributes to TDP-43 proteinopathy in sALS MNs.\u003c/p\u003e\u003cp\u003eIn TDP-43 proteinopathy, alterations in the localization and function of other proteins have been described, including ribonucleoprotein K. The binding of ribonucleoprotein K with Nrf2 transcript was associated with an impaired translation of Nrf2 mRNA, leading to an insufficient antioxidant response and motoneuron degeneration [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWang et al. [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e] and Barmada et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] observed that stimulating autophagy by increasing LC3 levels in mouse neurons and human MNs derived from iPSCs improved TDP-43 clearance and reduced protein toxicity. However, TDP-43 aggregation may also result from defects in other protein degradation systems, such as CMA or the ubiquitin-proteasome system [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our data demonstrate that human sALS spinal MNs show no alterations in MA, indicating that MA dysfunction is not a primary pathological feature of sALS. Although alterations of genes promoting TDP-43 proteinopathy have been linked to dysregulation of MA in \u003cem\u003ein vitro\u003c/em\u003e cells and animal models [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], our patients did not have a family history of ALS or showed \u003cem\u003eC9ORF72\u003c/em\u003e genetic mutations (data from patient\u0026rsquo;s clinical record), which could potentially underlie MA dysfunction. Moreover, it has described a mutual negative interaction between TDP-43 cytoplasmic aggregates and MA mechanisms [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Actually, Park et al. [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] reported a reduction in MA in yeast as a result of TDP-43-induced toxicity following its overexpression. These findings suggest a potential reciprocal inhibition between MA and TDP-43 proteinopathy that requires further investigation in human MNs.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study shows for the first time a high CMA activity in healthy human spinal motor neurons, as evidenced by elevated LAMP2A levels. However, sALS patients show a significant decrease in LAMP2A expression that correlates with cytoplasmic aggregates and the nuclear clearance of TDP-43. Notably, MA markers such as LC3 and the lysosomal enzyme GBA remain unchanged. CMA is preserved in Onuf\u0026rsquo;s nucleus MNs. Enhanced LAMP2A expression in glial cells indicates a maintained antioxidant response in other brain cells. These findings strongly implicate CMA impairment as a key determinant of selective MN vulnerability in ALS. Collectively our results provide novel insights into pathological protein accumulation mechanisms and highlight CMA enhancement as a promising therapeutic strategy to restore proteostasis and prevent neurodegeneration in ALS.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ea= axon\u003c/p\u003e\n\u003cp\u003eAF = Anterior funiculus.\u003c/p\u003e\n\u003cp\u003eAGH = Anterior gray horn.\u003c/p\u003e\n\u003cp\u003eALS= Amyotrophic lateral sclerosis.\u003c/p\u003e\n\u003cp\u003eChAT: choline acetyltransferase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCMA= Chaperone-mediated autophagy.\u003c/p\u003e\n\u003cp\u003eCV= Cresyl violet.\u003c/p\u003e\n\u003cp\u003eDPSC= Fine punctate granules scattered diffusely in the cytoplasm\u003c/p\u003e\n\u003cp\u003efALS: Familiar amyotrophic lateral sclerosis.\u003c/p\u003e\n\u003cp\u003eGBA= Lysosomal enzyme glucocerebrosidase.\u003c/p\u003e\n\u003cp\u003eGCs= Glial cells.\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E= Hematoxylin-Eosin.\u003c/p\u003e\n\u003cp\u003eLC3= Autophagosome marker microtubule-associated protein light chain 3.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;lcst = Lateral corticospinal trac.\u003c/p\u003e\n\u003cp\u003eLF= Lateral funiculus.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Lf = Lipofuscin.\u003c/p\u003e\n\u003cp\u003eMA= macroautophagy.\u003c/p\u003e\n\u003cp\u003emcst = Medial corticospinal trac.\u003c/p\u003e\n\u003cp\u003eMMP9= Matrix metalloproteinase-9.\u003c/p\u003e\n\u003cp\u003eMNs = Motoneurons.\u003c/p\u003e\n\u003cp\u003eMNBMc: Mononuclear bone marrow cells.\u003c/p\u003e\n\u003cp\u003eMS= microscopy slides.\u003c/p\u003e\n\u003cp\u003eN = Neuronal nucleus.\u003c/p\u003e\n\u003cp\u003ePF = Posterior funiculus.\u003c/p\u003e\n\u003cp\u003ePGH = Posterior gray horn.\u003c/p\u003e\n\u003cp\u003eRls: Round inclusions\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eS = Neuronal soma.\u003c/p\u003e\n\u003cp\u003esALS: sporadic amyotrophic lateral sclerosis.\u003c/p\u003e\n\u003cp\u003eSC= spinal cord.\u003c/p\u003e\n\u003cp\u003eSLIs= skein-like inclusions.\u003c/p\u003e\n\u003cp\u003eSNs = Sensory neurons.\u003c/p\u003e\n\u003cp\u003eTCNs = Clarke's thoracic column neurons.\u003c/p\u003e\n\u003cp\u003eTDP-43= Trans-activation response DNA-binding protein. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTS= Tissue slides.\u003c/p\u003e\n\u003cp\u003eV = Vacuoles.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eGenerative AI and AI-assisted technologies in the writing process\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the authors used Chat-GPT to streamline some parts of the text. After using this tool, the authors reviewed and edited the content as needed and took full responsibility for the content of the publication.\u003c/p\u003e\n\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e-\u0026nbsp;Consent to Publish declaration: not applicable\u003c/p\u003e\n\u003cp\u003e-\u0026nbsp;Ethics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe Anatomy Innovation Service of Miguel Hernandez University Medical School provides administrative and ethical support, with the approval of the Institutional Review Board. Samples and data from patients included in this study were provided by the Biobank IMIB (National Registry of Biobanks B. 0000859) (PT20/00109), integrated in the Platform ISCIII Biobanks and Biomodels and they were processed following standard operating procedures with the appropriate approval of the Ethics and Scientific Committees. Voluntary donations were obtained from patients included in the Phase I and II of clinical trial n\u0026ordm; EudraCT:2006-00309612, NCT00855400 and EC 07/90762, NCT01254539.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe would first like to thank the patients who participated in the clinical trials and donated their tissues, making it possible to accurately complete the pathological study of the effects of the therapy applied in each trial arm. We want to particularly acknowledge the patients and the Biobank IMIB (PT20/00109) integrated in the Platform ISCIII Biobanks and Biomodels for their collaboration and the Anatomical Innovation Unit of the UMH.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was funded by the following projects:\u0026nbsp;Spanish State Research Agency, through the \u0026ldquo;Severo Ochoa\u0026rdquo; Programme for Centres of Excellence in R\u0026amp;D (Grant Numbers SEV-2017-0723), the Spanish Ministerio de Ciencia e Innovaci\u0026oacute;n grant numbers SAF2017-83702-R and PID2020-11817RB-I00 and the Generalitat Valenciana (program Prometeo II, Grant Number 2018/041). This work has been partially funded by the Instituto de Salud Carlos III (ISCIII) through the RICORS Project \u0026apos;RD21/0017/0017; RD21/0017/0001; TERAV\u0026apos; supported by the Next Generation EU Program (Recovery, Transformation and Resilience Plan)\u003c/p\u003e\n\u003cp\u003eRV was supported by fellowship Ramon y Cajal (RYC) 2019-027520-I funded by Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades (MCIU) and Agencia Estatal de Investigaci\u0026oacute;n (AEI) MCIU/AEI/10.13039/501100011033, as \u0026ldquo;European Social Fund (ESF) Investing in your future\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors report no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdrakhmanov A, Gogvadze V, Zhivotovsky B (2020) To Eat or to Die: Deciphering Selective Forms of Autophagy. 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Nat Genet 54:1305\u0026ndash;1319. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41588-022-01148-2\u003c/span\u003e\u003cspan address=\"10.1038/s41588-022-01148-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"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":"acta-neuropathologica-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anec","sideBox":"Learn more about [Acta Neuropathologica Communications](https://actaneurocomms.biomedcentral.com/)","snPcode":"40478","submissionUrl":"https://submission.springernature.com/new-submission/40478/3","title":"Acta Neuropathologica Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"motor neurodegeneration, sALS, differential vulnerability to neurodegeneration, chaperone mediated autophagy, human motoneurons","lastPublishedDoi":"10.21203/rs.3.rs-7444163/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7444163/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of motor neurons (MNs), ultimately resulting in paralysis and respiratory failure within 3 to 5 years of onset. Fewer than 10% of ALS cases are familial (fALS), while the vast majority are sporadic (sALS) with an unknown etiology. A pathological hallmark of ALS is the accumulation of misfolded TDP-43 protein aggregates within MNs. Although TDP-43 is known to be degraded via chaperone-mediated autophagy (CMA), the status of CMA activity in sALS has not been previously explored. To investigate this, we analyzed CMA in human spinal cord tissue by assessing the expression of LAMP2A, a key lysosomal receptor and marker of CMA activity. In control samples, spinal cord MNs exhibited robust LAMP2A expression. In contrast, MNs from sALS patients showed a marked reduction in LAMP2A levels, coinciding with the presence of TDP-43 pathology. Notably, analysis of LC3, a marker of macroautophagy, revealed no significant differences in expression between control and sALS MNs. Interestingly, MNs within the Onuf\u0026rsquo;s nucleus, a population known to be resistant to degeneration in ALS, retained normal LAMP2A expression and did not exhibit TDP-43 aggregation in sALS cases. These findings demonstrated that CMA is essential for the clearance of TDP-43 in spinal cord MNs and that its dysfunction may contribute to the pathogenesis of sALS. Furthermore, the high dependence of spinal cord MNs on CMA activity may underlie their selective vulnerability to degeneration when CMA is impaired, and highlight CMA enhancement as a promising therapeutic strategy to restore proteostasis and prevent MN degeneration in ALS.\u003c/p\u003e","manuscriptTitle":"Chaperone Mediated Autophagy is deficient in Spinal Motoneurons of ALS patients with TDP-43 proteinopathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-10 09:23:34","doi":"10.21203/rs.3.rs-7444163/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-25T02:22:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T17:31:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-13T22:55:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93745439480367881734630995845364631202","date":"2025-09-03T15:51:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"142624748205692698075401097479030750347","date":"2025-09-03T08:25:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-03T00:54:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-28T00:19:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-28T00:17:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Neuropathologica Communications","date":"2025-08-24T04:52:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-neuropathologica-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anec","sideBox":"Learn more about [Acta Neuropathologica Communications](https://actaneurocomms.biomedcentral.com/)","snPcode":"40478","submissionUrl":"https://submission.springernature.com/new-submission/40478/3","title":"Acta Neuropathologica Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"db6c30b2-8d34-4915-a951-52bc37b021dd","owner":[],"postedDate":"September 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:08:08+00:00","versionOfRecord":{"articleIdentity":"rs-7444163","link":"https://doi.org/10.1186/s40478-026-02238-6","journal":{"identity":"acta-neuropathologica-communications","isVorOnly":false,"title":"Acta Neuropathologica Communications"},"publishedOn":"2026-02-04 15:58:50","publishedOnDateReadable":"February 4th, 2026"},"versionCreatedAt":"2025-09-10 09:23:34","video":"","vorDoi":"10.1186/s40478-026-02238-6","vorDoiUrl":"https://doi.org/10.1186/s40478-026-02238-6","workflowStages":[]},"version":"v1","identity":"rs-7444163","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7444163","identity":"rs-7444163","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}