Kaempferol enhances ER-mitochondria coupling and protects motor neurons from ER stress and mitochondrial dysfunction in C9ORF72- ALS

Kaempferol enhances ER-mitochondria coupling and protects motor neurons from ER stress and mitochondrial dysfunction in C9ORF72- ALS | 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 Kaempferol enhances ER-mitochondria coupling and protects motor neurons from ER stress and mitochondrial dysfunction in C9ORF72- ALS Federica Pilotto, Paulien Smeele, Olivier Scheidegger, Rim Diab, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5190511/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Feb, 2025 Read the published version in Acta Neuropathologica Communications → Version 1 posted 9 You are reading this latest preprint version Abstract Repeat expansions in the C9ORF72 gene are a frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Considerable progress has been made in identifying C9ORF72 -mediated disease and resolving its underlying etiopathogenesis. The contributions of intrinsic mitochondrial deficits as well as chronic endoplasmic reticulum stress to the development of the C9ORF72-linked pathology is well established, Nevertheless, to date, no cure or effective therapy is available, and thus attempts to find a potential drug target, have received increasing attention. Here, we investigated the mode of action and therapeutic effect of a naturally occurring dietary flavanol, kaempferol in preclinical rodent and human models of C9ORF72 -ALS. Notably, kaempferol treatment of C9ORF72 -ALS human patient-derived motor neurons/neurons, resolved mitochondrial deficits, promoted resiliency against severe ER stress and conferred neuroprotection. Treatment of symptomatic C9ORF72 mice with kaempferol, normalized mitochondrial calcium uptake, restored mitochondria function, and diminished ER stress. Importantly, in vivo, chronic kaempferol administration ameliorated pathological motor dysfunction and inhibited motor neuron degeneration, thus highlighting the translational potential of kaempferol. Lastly, in silico modelling identified a novel kaempferol target and mechanistically the neuroprotective mechanism of kaempferol is through the iP3R-VDAC1 pathway via the modulation of GRP75 expression. Thus, kaempferol holds great promise for the treatment of neurodegenerative diseases where both mitochondrial and ER dysfunction are causally linked to the pathophysiology. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Current research has highlighted that dysregulated ER stress and associated unfolded protein response (UPR) signaling are the leading cellular impairments contributing to the main pathogenic mechanism in neurodegeneration [1–4]. In the context of amyotrophic lateral sclerosis (ALS), motor neurons are selectively prone to ER stress, influencing disease manifestation and kinetics[5–7]. Importantly, a hexanucleotide repeat expansion (HRE) in the first intronic region of the C9ORF72 gene accounts for over 40% of all known familial and 10% of known sporadic forms of ALS. In healthy individuals, approximately 20- 24 copies of the GGGGCC HRE are observed within the first intron of the C9ORF72 gene. However, in disease, this GGGGCC sequence is expanded and can range from hundreds to thousands of repeats. This specific intronic HRE leads to the development of three mutually non-exclusive pathological hallmarks, eventually resulting in the appearance of a spectrum disorder; C9ORF72 ALS/frontal temporal dementia (FTD). Firstly, the HRE has been shown to cause haploinsufficiency of the C9ORF72 gene, leading to reduced C9ORF72 RNA and protein expression. Secondly, there is a gain of toxic function due to the bidirectional transcription of the GGGGCC HRE resulting in the production of toxic G4C2 and G2C4 repeat RNA species. Lastly, there is a gain of toxic function via the non-ATG mediated RAN translation of repeat RNAs to produce five different toxic dipeptide repeat proteins (Poly(GA), Poly(GR), Poly(GP), Poly(PR), and Poly(PA)), which act on and damage multiple cellular processes [8–10]. C9ORF72 induced pluripotent stem cells (iPSC)-derived motor neurons, exhibit increased endoplasmic reticulum (ER) stress, together with reduced mitochondrial membrane potential, concomitant impairment in calcium homeostasis and reduced levels of the antiapoptotic protein Bcl-2 [6,7,11]. Additionally, C9ORF72 is majorly a cytosolic protein, which has also been shown to localize to the mitochondrial inner membrane, where it has a function in controlling the process of oxidative phosphorylation, thus maintaining, and regulating cellular energy production and balance [12]. Thus, emerging evidence suggests that ER stress signaling and mitochondrial dysfunction are intricately associated with the pathophysiological manifestations linked to C9ORF72 -ALS. One likely region of membrane contacts where the mitochondria are reversibly tethered to the ER are regions termed “Mitochondria-associated membranes (MAMs),” which play a crucial regulatory role in the supply of calcium from the ER to mitochondria. These sites are critical for the rapid calcium uptake by mitochondria through voltage-dependent anion channels (VDACs) located at the outer mitochondrial membrane (OMM) [13]. Moreover, MAMs have been implicated in the regulation of calcium homeostasis, mitochondrial function, autophagy, apoptosis, and glucose homeostasis [14]. Within the context of ALS, mutations within MAM localized and functioning proteins such as VAPB and Sig1R are known to cause familial ALS (fALS). The point mutation P56S in VAPB P56S causes increased binding to the mitochondrial protein tyrosine phosphatase interacting protein 51 (PTPIP51), which causes reduced ER-mitochondria contacts, enhanced Ca 2+ release from the ER and aberrant MAM morphology, and mitochondrial dysfunction [15]. A recent study from our group revealed the existence of an ER stress-mediated adaptive response in C9ORF72 patient-derived motoneurons (MNs), which was observed by the early yet transient increase in the expression of the MAM-localized chaperone GRP75. Transient GRP75 expression augmented ER-mitochondrial association, boosting mitochondrial function, and sustaining cellular bioenergetics during the initial stage of disease, thereby neutralizing early mitochondrial deficits. The gradual emergence of Poly(GA) aggregates led to the sequestration of GRP75, causing a subsequent loss of function at the MAM, resulting in mitochondrial Ca 2+ uptake impairments and mitochondrial dysfunction in both human patient-derived and rodent motor neurons [7]. Similarly, another recent study showed that dysfunctional ER-mitochondria signaling and the disruption of VAPB-PTPIP51 tethers in C9ORF72 patient-derived neurons and mutant C9orf72 transgenic mice occurred presymptomatically and contributed to the pathogenic process. These impairments were linked to the expression of DPRs, which disrupted the VAPB-PTPIP51 interaction at the ER-mitochondria contacts and that may involve activation of glycogen synthase kinases-3β (GSK3β), a known negative regulator of VAPB-PTPIP51 binding. Compounds harboring the potential to simultaneously target both ER stress and mitochondrial function hold great promise as future therapeutic targets in neurodegenerative diseases and more so in ALS. Kaempferol is a naturally occurring dietary flavanol, present in fruits and vegetables and has been shown to possess ER stress-inhibitory activity in cultured mammalian cells [23], anti-neuroinflammatory activity in rat models of ischemic stroke [24], mitochondrial Ca 2+ uniporter channel activator [25] and ameliorated energy metabolism and symptomatic behavior in a rat model of traumatic brain injury [26]. We here identify that kaempferol exerts a neuroprotective effect on C9ORF72 ALS neurons by inhibiting ER stress and sustaining mitochondrial function. Notably, in C9ORF72 -ALS human patient-iPSC-derived motor neurons and directly fibroblast-converted neurons kaempferol counteracted mitochondrial deficits and provided neuroprotection in the presence of elevated ER stress, thus indicating the probable translational potential of kaempferol. Additionally, in vivo, treatment with kaempferol in the symptomatic rodent model of C9ORF72 -ALS was successful in reducing pathological hallmarks and concomitantly delaying the progression of behavioral pathology. Lastly, in silico modeling identified a novel prominent binding affinity of kaempferol to chaperone GRP75 and mechanistically the neuroprotective mechanism of kaempferol is through the iP3R-VDAC1 pathway via the modulation of GRP75 and ATP5J expression. EXPERIMENTAL PROCEDURES Mice strains The C9-500 BAC mouse line (FVB/NJ-Tg(C9orf72)500Lpwr/J) carrying a human C9ORF72 gene under a human promoter with ~ 500 hexanucleotide repeats described in (Liu et al., 2016) [ 16 , 17 ] was purchased from Jackson Laboratory (RRID: IMSR_JAX:029099) and kept in heterozygosis crossed with (non-carrier) mice FVB/NJ (Janvier labs, SC-FVBN-F). Our colony displays an acute phenotype, which is observed in 25–30% of females with a median life span of 105 days whereas the remaining female and male mice exhibit a slow-progressing phenotype with females having a median life span of approximately 250 days and males 260 days. Long-range PCR was regularly done to identify repeat length-matched cohorts. Kaempferol treatment included both genders and only slow-progressing mice. All behavior and survival assays were performed in repeat length-matched (700–800 repeats). Animal care, housing, ethical experimental usage, and procedures were in accordance with the Swiss Veterinary Law guidelines, and the study was approved by the Animal Commission of Canton of Bern, Switzerland, license number BE-35/17, BE-82/18 In vivo/ in vitro drug treatments Kaempferol (Sigma-Aldrich, K0133) at 10mg/kg was administered via i.p. injections daily to both wild type ( WT) and C9-500 for the first two weeks, followed by every alternate day administration chronically until end-stage P240. WT and C9-500 mice were treated every alternate day with Salubrinal (Enzo life sciences, 270-428-M005) at a dosage of 1mg/Kg or saline as control. iMNs were treated with Tunicamycin (TU) (1 µg/ml) for 24h before further analysis. For Kaempferol (KMP) treatments, iMNs were treated with 10 µg/ml kaempferol for 2h or 48h. Reverse Rotarod and Hanging wire test Rotarod apparatus (Ugo Basile, Comerio, Italy) was used to assess general motor performance as well as motor coordination and not only endurance as usually assessed by common accelerated rotarod protocols, during the light phase of the 12h light/12h dark cycle. For the inverse rotarod, the rod accelerated from 15 to 33 rpm in 10 seconds, then 33 rpm to 15 rpm in 10 seconds, followed by inversing the direction of the rod and a repeat of the same procedure. One trial lasted a maximum of 110 seconds, with 3 min resting in between trials. Three trials every 10 days were performed for overexpressing C9-500 animals and respective controls, while initially, KMP-treated animals were tested for 4 consecutive days. Mice were trained on the rotating rod at a fixed speed for 3 days before the baseline recording. All measurements were done blindly without knowing the genotype of the mice. The hanging wire test was performed as previously described [ 18 ]. Briefly: mice were placed on a cage top and once the animal was stable on top of the grid, the cage top was gently inverted, and the latency of the first fall is recorded. All the animals are placed on the inverted cage top each time they fall to assess the average number of falls. The test last for 120 seconds. iPSC differentiation into motor neurons (iMNs) The healthy and C9ORF72- ALS iPSCs were obtained from iPSC bank (Biomedicum Stem Cell Center, GoEditStem platform, HiLIFE, Helsinki, Finland and the iPSC Core, Cedar Sinai, USA). iPSCs were cultured in GeltrexTM (ThermoFischer) coated plates in mTeSRTM1 (StemCell technologies) media. MN differentiation was performed as previously described [ 19 , 20 ] with some modifications. Human iPSCs were dissociated to single cells using Accutase (StemCell technologies) and seeded at 3X106 onto 10cm plate with N2B27 differentiation medium (Advanced DMEM/F12:Neurobasal (1:1) medium, 1% Pen/strep (Gibco), 1% GlutaMAX (Gibco), 0.1mM 2-mercaptoethanol (Gibco), 1X B27 supplement (Gibco), 1X N2 supplement (Gibco), supplemented with 10ng/ml basic fibroblast growth factor ((StemCell technologies), 20 µM SB431542 (StemCell technologies), 0.1 µM LDN193189 (StemCell technologies), 3 µM CHIR99021 (StemCell technologies), 10 µM L-Ascorbic Acid (L-AA; Sigma) and 1X Revitacell supplement (Gibco)) to initiate the formation of embryoid bodies (EBs). On day 2 media patterning of EBs was induced by the addition of media supplemented with 100nM all-trans retinoic acid (RA; sigma) and 500nM Smoothened Agonist (SAG; StemCell technologies). EBs were pelleted and fed with fresh media on every alternate day until day 14. 10ng/ml Brain derived neurotrophic factor (BDNF; StemCell technologies) was added from day 7 while 10 µM DAPT (StemCell technologies) was added from day 9. EBs were dissociated using trypsin on day 16 and triturated with ice cold cell trituration and wash medium (1X PBS (Gibco), 0.45% Glucose, 0.1% Bovine Serum Albumin (BSA; Sigma), 2mM MgCl 2 , 0.8mM EDTA (Invitrogen), 2.5% Fetal Bovine Serum (FBS; Sigma), 1X N2 supplement, 1X B27 supplement and DNAse). Triturated EBs were then plated on poly-ornithine/laminin (Sigma) coated plates in MN feeding medium (Neurobasal medium (Gibco), 1X glutaMAX, 1X Non-essential amino acid (NEAA, Gibco), 0.1mM 2-mercapthoethanol, 1X N2 supplement, 1X Pen/strep, 1X B27 supplement, 10ng/ml glial cell derived neurotrophic factor (GDNF; StemCell technologies), BDNF 10ng/ml, 10ng/ml insulin-like growth factor (IGF-1; StemCell technologies), 10ng/ml Ciliary neurotrophic factor (CNTF; StemCell technologies), 100nM RA and 10 µM AA and kept at incubator at 37°C and 5% CO2 for further maturation. Generation of direct fibroblast-induced neurons (dNs) Fibroblast collection: skin punches were collected, and fibroblasts were grown in fibroblast media (10% FBS (Gibco), 1% anti-anti (Gibco) in DMEM Glutamax (Gibco)) for up to 1 month, passaging once every 1–2 weeks. In addition, this study used a fibroblast sample from the NINDS Repository, as well as clinical data. NINDS Repository sample numbers corresponding to the samples used are: AG08620. Patient skin-derived fibroblasts were directly reprogramed into neural progenitor cells (NPCs) as previously described [ 50 ]. Briefly, 100,000–200,000 fibroblasts were seeded into a fibronectin (5 µg/mL, Millipore) coated 6-well plate and cultured in a 37°C, 5% CO 2 incubator. Next day, fibroblasts were transduced with a retroviruses for SOX2, cMyc, KLF4, and OCT3/4. 24 h later virus was removed, and media replaced with fresh fibroblast media (10% FBS (Gibco), 1% anti-anti (Gibco) in DMEM Glutamax (Gibco)). After a 24-h rest period, media of transduced fibroblasts was changed to a neuralizing media (1% B27 (Gibco), 1% N2 (GIBCO), 1% anti-anti (Gibco), 20 ng/mL FGF2 (peprotech), 20 ng/mL EGF (peprotech), and 5 µg/mL heparin (Sigma)). Cells were cultured in this media until converted into NPC. NPCs were cultured in (1% B27 (Gibco), 1% N2 (Gibco), 1% anti-anti (Gibco), 20 ng/mL FGF2 (peprotech)). Induced direct neuron generation (dNeus): patient and healthy fibroblasts were directly converted to neurons using small molecules as previously described [ 28 ]. Briefly, 10 cm plates were coated overnight with polyornithine (10 µg/mL, Sigma) in borate buffer. Next day, plates were washed with DPBS and coated with laminin (5 µg/mL, Invitrogen) and fibronectin (2.5 µg/mL, Millipore Sigma) in DMEM/F12 at 37°C for 2 h. Fibroblast cells (850,000) were seeded onto the plates in culture medium for 1 day. The cells were transferred to neuronal induction medium (DMEM/F12: Neurobasal (Gibco) [1:1] with 0.5% N-2 (Gibco), 1% B-27 (Gibco), 100 µM cAMP (Sigma), and 20 ng/mL bFGF (Peprotech)) with the following chemicals: VPA (0.5 mM, Sigma), CHIR99021 (3 µM, Axon medchem), repsox (1 µM, BioVision), forskolin (10 µM, Tocris), SP600125 (10 µM, Sigma), GO6983 (5 µM, Sigma) and Y-27632 (5 µM, Sigma). Half the medium was changed after 3 days with fresh induction medium. On the fifth day, cells were switched to neuronal maturation medium (DMEM/F12: Neurobasal [1:1] with 0.5% N-2 (Gibco), 1% B-27 (Gibco), 100 µM cAMP (Sigma), 20 ng/mL bFGF (Peprotech), 20 ng/mL BDNF (Gibco) and 20 ng/mL GDNF (Gibco)) with the following chemicals: CHIR99021 (3 µM), forskolin (10 µM) and SP600125 (10 µM). Immunofluorescence on iMNs iMNs plated on coverslips were fixed using 4% paraformaldehyde PFA for 15min and blocked for 1h with 3% bovine serum albumin (BSA) and 0.1% TritonX-100 in phosphate buffered saline PBS. After blocking, neurons were incubated with the following primary antibody: anti-BiP (Abcam, ab21685, 1:500), goat anti-ChAT (Millipore, AB144P, 1:500), chicken anti-MAP2 (Sigma Aldrich, AB15452, 1:500) (Thermo fisher scientific, Invitrogen PA5-29202, 1:500) in blocking buffer overnight at 4°C. After washing three times with PBS, cells were incubated in blocking buffer with Alexa Fluor fluorescently labeled secondary antibodies and DAPI for 1h at room temperature. Cells were then washed with PBS and mounted on glass slides. Images were acquired with a confocal microscope Olympus FluoViewTM FV1000 (Olympus) fitted with a 20X or 40x air objective and 60x immersion oil objective. Immunofluorescence and immunohistochemistry of rodent tissue Mice were transcardially perfused with 4% PFA in 1X PBS; brain, cerebellum and lumbar spinal cord were isolated and kept overnight at 4°C in the same fixative solution, followed by 30% sucrose in PBS for cryoprotection until samples were used. After embedding in Tissue Tek O.C.T compound (Bio system, 4583). Spinal cord (50 µm), sections were cut using a cryostat. Antibodies used for immunofluorescence were: rabbit anti-GRP78/BiP (1:500, Abcam, ab21685), mouse anti-KDEL/BiP (1:500, Enzo Life Science, SPA-827), rabbit anti-P i -EIF2α (1:25, Cell Signaling, 3597L), goat-anti ChAT (1:1000, Millipore, AB144P), mouse anti-GRP75 (1:200, Abcam, ab2799), rabbit anti-GRP75 (1:200, Abcam, ab53098), rabbit anti-GFAP (1:500, Abcam, ab7260), mouse anti-8-hydroxy-guanosine (8-OHdG), (1:500, Abcam, ab62623) mouse anti-NeuN clone 60 (1:1000, Millipore, MAB377). Heat-mediated antigen retrieval was performed using Sodium citrate buffer 10mM pH 6 for GRP75 and ATP5J staining. Sections were kept for 2h in PBS solution containing 0.05% Triton X-100 and 10% normal donkey serum (NDS, Jackson immunoresearch, 017-000-121) after the antibodies were applied in PBS, 3% normal donkey serum (NDS), 0.05% Triton X-100, and incubated overnight (for brain) and for two days for spinal cord at 4°C. Sections were then briefly washed with PBS and incubated for 120 min at room temperature, with appropriate combinations of secondary antibodies from Invitrogen. Spinal cord sections for immunohistochemistry were processed as follows: sections were treated with heat-mediated antigen retrieval using sodium citrate buffer 10mM pH 6 and immersed in 3% H2O2 in PBS for 20 min to block endogenous peroxidase activity. This was followed by a blocking step in PBS containing 0.05% Triton X-100 and 10% NDS and incubated overnight at 4° with goat anti-ChAT (1:500, Millipore, AB144P) antibody diluted in the same blocking solution. The next day the sections were incubated with the appropriate biotinylated secondary antibody (1:500) followed by 1h incubation in PBS solution containing biotin-avidin complex (1:100, Vector Labs), finally, the 3,3′-diaminobenzidine (DAB) reaction was developed. The glass slides were dehydrated via ascending concentrations of ethanol and rinsed in xylene before being cover slipped. Images were acquired using an Olympus microscope (BX51). Muscles histology Mice were deeply anesthetized with isoflurane and decapitated before the gastrocnemius muscles were dissected. Muscles were quickly frozen in liquid nitrogen-cooled isopentane. 8µm thick sections were cut on a cryostat and placed directly onto coverslips. For hematoxylin & eosin (H&E) staining, muscle sections were stained with Mayers hematoxylin for 5min, followed by three 10min washes in deionized H2O. Muscles were then placed in eosin G for 1min, then rinsed in 70% ethanol. The tissue was then dehydrated in a series of ethanol washes (70%, 95%, 100%), rinsed in xylene, then mounted on a glass slide. NADH staining was performed by incubating muscle sections in 0.2M Tris buffer containing Nitrotetrazolium Blue (Sigma) and β-nicotinamide adenine dinucleotide (NADH, Sigma) for 30min at 37°C. Following three washes in deionized H2O, coverslips were mounted onto a glass slide. ATP staining pH4.3 and 9.4 were performed by incubating the slides in pre incubating solution pH4.3 per 10min at 4°, containing 0.1M Na acetate buffer with 10mM EDTA. Follow by three 5min washes in deionized H20, to be then incubated in the incubation solution: for pH4.3 containing sodium acetate 0.1M, EDTA 0.1M pH8 and for pH9.4 containing 0.1M glycine (Sigma, 50046), 5mg ATP and DTT in NaCl buffer with CaCl2 per 30min at 37°C. Coverslips were then washed with deionized H2O 3 times every 5min, incubated with Cobalt chloride 2% 3 times per 1min at room temperature, rinsed with deionized water and incubated with ammonium sulfide 1% at room temperature for 30 second. Coverslips were rinsed under running water and dehydrated in a series of ethanol washes (70%, 95%, 100%), rinsed in xylene, then mounted on a glass slide. Imaging and image analysis Confocal images were acquired using a Leica SP5 (Leica Microsystems) fitted with a 20X, 40 and 63X oil objective, Leica SP8 (Leica Microsystems) fitted with a 63X oil objective, or Olympus Fluoview 1000-BX61 (Olympus, Tokyo) microscope, fitted with a 20X, 40X air objective or 60X immersion oil objective. All images were processed using Imaris software version 7.6.3 or Fiji. For the analysis of BiP, P i -EIF2α, GRP75, GFAP and ATP5J labeling intensities, data were acquired using identical confocal settings, with signals at the brightest cells being non-saturated, and that background levels outside motor neuron pools were still detectable. Images were analyzed quantitatively using FiJi or Imaris. Signal intensity values for the antigen of interest were calculated over several consecutive lumbar spinal cord Z-stack spaced 0.5 µm, after background subtraction from every different channel. For the calculation of motor neuron percentages, Choline Acetyltransferase (ChAT) positive motor neurons were examined and signal intensity values for the antigen of interest within these ChAT positive motor neurons were calculated in 3–4 animals. Lowest signals had values of below 50 and high-intensity neurons exhibited labeling values up to 255. Signal values below 50 in the case of BiP in WT animals were counted as basal expression. To count the motor neuron numbers, a cell counter plugin from Fiji was used. Imaris software was used to reconstruct the 3D isosurface of Poly(GA) aggregates volume. Fiji software was employed to analyze the mitochondria-ER contacts and sphericity for 3view EM images. Proximity ligation assay (PLA) in perfused mouse spinal cord tissue PLA was adapted from Gomes et al., [ 21 ]. In short, 50 µm free-floating spinal cord sections were mounted on a frost slide (Huberlab, 10.0120.04) and air-dried. Slides were rinsed in 0.01% Triton X-100 in 1× PBS per 12 minutes followed by 3 washes in 1X PBS (3 per 5 minutes). Slides were kept in a humid chamber and blocking solution was added (Duolink PLA probe kit, DUO92008, Merck) for 1h at 37°C. Subsequently, slides were incubated with the following primary antibodies (rabbit anti-IP3R, ABCAM, ab5804; mouse anti-GRP75, ABCAM, ab53098; rabbit anti-GRP75, ABCAM, ab227215; rabbit anti-ATP5J, Thermo Fisher Scientific, PA5-29202) in a humidity chamber for 2 nights at 4°C. Slides were rinsed in buffer A (Duolink In Situ Wash Buffers, Fluorescence DUO82049) (3 times every 5 minutes) and incubated with PLA probes (Probe Anti-mouse MINUS, DUO92004 and Probe anti-rabbit PLUS, DUO92002 ) at a working concentration of 1:10 for 1h at 37°C. Ligation (Duolink In Situ Detection Reagents Red, DUO92008) was performed at 37°C per 45 minutes followed by 3 washes in buffer A (3 per 5 minutes). The amplification (Duolink In Situ Detection Reagents Red, DUO92008) step was performed at 37°C in a dark humidity chamber per 100 minutes. Finally, sections were rinsed in buffer B (Duolink In Situ Wash Buffers, Fluorescence DUO82049) (2 per 10 minutes) followed by a third wash in 0.001% buffer B. Slides were left to dry and coverslips were mounted with Duolink In Situ Mounting Medium with DAPI (DUO82040), and edges were sealed with nail polish. Sections were kept at -20 overnight before confocal imaging. Colorimetric staining of mitochondria complexes Mice were rapidly perfused with PBS, fresh spinal cord was removed, embedded in O.C.T compound, and quickly frozen on dry ice. 20 µm thick sections were cut with a cryostat and transferred onto an adhesive glass slide. Sections were incubated for 30 min at 37 C in freshly prepared appropriate complex histochemistry media: (1) Complex I: 1.23mg/ml (1.5 mM) Nitroblue tetrazolium (NBT; N6876, Sigma) and 0.625 mg/ml NADH (N8129, Sigma) were mixed in PBS, pH = 7.4. Complex IV: 0.5mg/ml 3,3’-diaminobenzidine tetrahydrochloride (DAB, D7304, Sigma), 1mg/ml cytochrome c (C2506, Sigma), and approximately 2µg/ml (a few crystals) bovine catalase (C9322, Sigma) were mixed in PBS, pH = 7.4. After the staining of each complex, sections were washed 3×10 min in PBS before being dehydrated for 4min in 70% ETOH, 4min in 90% ETOH, 10 min in 100% ETOH and 10 min in Xylol and mounted with Eukitt. Images were acquired using an Olympus microscope (BX51). Mitochondrial calcium imaging Mitochondrial calcium imaging for cortical neurons was adapted from [ 22 ]. In brief, cells were incubated for 45 min with the staining solution containing: 156mM NaCl, 3mM KCl, 2mM MgSO 4 , 1.25mM KH 2 PO 4 , 10mM D-glucose, 2mM CaCl 2 and 10mM HEPES pH 7.35, 5 µg/ml (w/v) Fluo-4, AM, 10 µM Verapamil. After a brief wash with Ca 2+ free HBSS, cells were incubated for 10 min with the intracellular solution containing: 6mM NaCl, 130mM KCl, 7.8mM MgCl 2 , 1mM KH 2 PO 4 , 0.4mM CaCl 2 , 2mM EGTA, 10mM HEDTA, 2mM malate, 2mM glutamate, 2mM ADP, 20mM HEPES pH 7.1, 25 µg/ml (w/v) digitonin and 1 µM thapsigargin. An Olympus Fluoview 1000-BX61 (Olympus, Tokyo) microscope fitted with a 40x water immersion objective was used to acquire images every 20 seconds, the first 60 seconds were considered as baseline, afterwards 50mM KCl was added to the imaging solution to depolarize the cells. Neurons were treated 10 µg/ml of kaempferol for 1h prior staining solution passage, moreover to maintain their effect the KMP was also added to the staining solution before starting the imaging for a total time of treatment of 1h and 45min. Images were analyzed using Fiji, multiple ROI were chosen inside the cytosol of the cells and the fluorescence intensity was calculated over the different frame. To calculate ΔF, the median intensity values are divided by the average of the first 60 seconds of recording (F0) per single ROI. Statistical Analysis Statistical significances throughout the paper were evaluated by two-tailed, paired and unpaired Student’s t test and one or two-way ANOVA. Post ANOVA Bonferroni, Sidak, Tukey’s test was used to evaluate statistical significance throughout the paper as indicated in the respective figure legend. Values are expressed as mean ± standard error of the mean (SEM). Error bars: SEM *P < 0.05, **P < 0.01, ***P < 0.001. RESULTS Kaempferol protects human iPSC-derived motor neurons (iMNs) and fibroblast-induced neurons (iNs) We started the study by evaluating the neuroprotective action of kaempferol, a compound known to act on UPR and oxidative stress. Firstly, we generated human C9ORF72 iPSC-derived motor neurons (iMNs) as previously described [ 7 ]. iPSCs from two healthy controls, three C9ORF72 lines, and one isogenic control were differentiated into motor neurons (iMNs), see ( Suppl. Table 1 ) for demographic details and ( Suppl. Figure 1 ) for iMN molecular characterization. Since Ca 2+ homeostasis is a crucial determinant for neuronal function, and Ca 2+ buffering by mitochondria majorly occurs at the ER–mitochondria contact sites, which ultimately is important for ATP production, we first employed seahorse assay to measure mitochondrial function. Seahorse assay measures key parameters of mitochondrial function by directly measuring the oxygen consumption rate (OCR) and a representative example from control and diseased C9ORF72 line, C9(1) is presented pre- and post-kaempferol treatment (Fig. 1 A). Notably, all three C9ORF72 patient-derived iMNs revealed impaired mitochondrial function as observed by the reduced basal respiration and ATP production compared to the healthy control treatment of iMNs with kaempferol for 48 hours (h) rescued mitochondrial dysfunction by normalizing basal respiration (Fig. 1 B) and ATP levels (Fig. 1 C). Note the near normalized ATP levels in mutation-corrected Iso-C9 (3) patient line, compared to the uncorrected C9 (3) patient line, suggesting that the observed mitochondrial dysfunction is causally linked to the disease mutation. Next, we tested whether kaempferol would also protect mutant iMNs from ER stress and thus sustain neuroprotection. To this end, we induced strong ER stress by treating iMNs with Tunicamycin (TU) for 24h. In parallel, iMNs were dually treated with TU and kaempferol for 24h ( scheme Fig. 1 D). TU treatment led to the induction of ER stress as observed by increased BiP expression in both control and C9ORF72 patient-derived iMNs. Notably, dual treatment with TU and kaempferol for 24h inhibited the observed ER stress response, indicating that kaempferol even in the presence of ER stress activators can effectively counteract the onset of ER stress (Fig. 1 E). We next examined whether kaempferol treatment could protect iMNs from degeneration due to ER stress. TU treatment for 24h led to a substantial loss of iMNs with nearly 60% of iMNs being dead in both control and diseased conditions. In contrast, dually treated iMNs exhibited negligible neuronal death, indicating that kaempferol can confer neuroprotection in conditions associated with strong ER stress (Fig. 1 F). Given the prominent role of aging in neurodegeneration, we next generated neurons via the direct reprogramming of fibroblasts to neurons (dNs), as previously described by us 7 , thus maintaining aging and epigenetic signatures of the donor. See ( Suppl. Table 1 ) for demographic details. We treated dNs with TU and kaempferol for 24 hours ( scheme Fig. 1 G), which similar to our experiments in iMNs led to significant death in controls and C9ORF72 dNs, however, the dual treatment together with kaempferol counteracted ER-stress induced neuronal death irrespective of their genotype (Fig. 1 H). In conclusion, kaempferol treatment restores mitochondrial function, and provides neuroprotection against ER stress, thereby alleviating cellular dysfunction in human C9ORF72-patient neurons. Kaempferol treatment protects motor neurons and reduces pathological aggregates in a rodent model. Next, we determined whether kaempferol treatment exerted a neuroprotective effect on motor neurons in C9-500 model of C9ORF72 -ALS. This mouse model displays premature ER stress, mitochondrial dysfunction as well as the striking presence of RNA foci, pTDP43 aggregates, and robust accumulation of DPRs, all of which are pathological hallmarks of C9ORF72 -ALS [ 7 , 27 – 29 ]. We performed chronic kaempferol treatment from P138 until P240, an age-corresponding to the end stage of the disease [ 18 , 27 ]. Firstly, counting the number of ChAT immunostained spinal motor neurons revealed that chronic kaempferol treatment revealed a substantially high number of preserved motor neurons compared to C9-500 untreated mice. Notably, the treatment led to a comparable number of mutant motor neurons as those observed in WT mice, suggestive of sustained neuroprotection. In comparison, a ~ 40% reduction in motor neuron numbers, indicative of ongoing motor neuron degeneration, was observed in untreated saline injected C9-500 spinal cord (Fig. 2 A). Poly(GA) inclusions are prominent pathological hallmarks, indicative of progressing disease and their expression is dependent on P i -eIF2α signaling, which is induced during unfolded protein response (UPR) and/or integrated stress response (ISR) [ 33 – 35 ]. We longitudinally assessed from the presymptomatic stage (P60) until the symptomatic stage (P150), the presence of Poly(GA) inclusion within spinal motor neurons of C9-500 mice. Immunostaining against N-terminal Poly(GA), revealed that 15.1 ± 3.4% of Poly(GA) aggregates were > 1 µm 3 at P60, however, this fraction of the larger Poly(GA) aggregates increased with age, coinciding with the symptomatic phase (32.9 ± 2.8% > 1 µm 3 at P125, and 50.8 ± 2.1% > 1 µm 3 at P150). Note the complete lack of Poly(GA) immunopositivity in WT motor neurons (Fig. 2 B, graph bottom ). Since sustained motor neuron numbers after kaempferol treatment indicated a neuroprotective action of kaempferol, thus we examined the accumulation of Poly(GA) aggregates within motor neurons as an indicator of motor neuron function. Immunostaining against Poly(GA) and quantification revealed widespread accumulation of Poly(GA) within saline-treated C9-500 spinal motor neurons, however, the average numbers as well as the fraction of large Poly(GA) aggregates within individual motor neurons were dramatically reduced within chronically kaempferol treated spinal motor neurons (Fig. 2 C). Astrogliosis is a typical hallmark of many neurodegenerative diseases as well as ALS, thus we stained lumbar spinal cord sections for the glial fibrillary acidic protein (GFAP), to assess if KMP has an effect in reducing neuroinflammatory events. While C9-500 control animals show a widespread astrogliosis within the ventral horn of the spinal cord, KMP chronically treated C9-500 mice display a reduction in GFAP intensity (Fig. 2 D). As C9ORF72 -linked ALS also affects the muscles, mainly the neuromuscular junctions, we also assessed the overall muscle structure. Muscle histopathological analyses via H&E staining of saline and kaempferol-treated C9-500 mice revealed the presence of atrophic fibers, small fiber size, with central nuclei and angular muscle fibers, indicative of muscle fiber degeneration and denervation in P240 C9-500 saline-treated muscle Gastrocnemius (GC) muscle. In contrast, kaempferol-treated mice displayed regular GC muscle fiber size and negligible signs of atrophy (Fig. 2 E). Moreover, we evaluated muscle fiber types via NADH staining, which revealed that C9-500 muscles had nearly lost groups of all NADH-positive myocytes, indicative of impaired muscle function. In contrast, kaempferol-treated mutant C9-500 muscles maintained a strong NADH staining within groups of myocytes reflecting the presence of varied fiber types and sustained muscle function in treated C9-500 mice (Fig. 2 F). Overall, this data suggests that the systemic administration of kaempferol can elicit a beneficial and neuroprotective effect on both spinal cord and muscles as observed by the reduced accumulation of toxic Poly(GA) aggregates, and preserved motor neuron numbers and muscle function. Kaempferol acts on the ER stress pathway and inhibits UPR signaling in vivo. Based on the very promising neuroprotective findings observed after kaempferol treatment in C9-500 mice, we next focused on its mode of action on specific cellular impairments. We tested whether kaempferol, known to possess ER stress inhibitory activity in cultured mammalian cells [ 23 ] could also reduce ER stress in vivo in the C9-500 mouse model. The early phase of ER stress was assessed by immunolabeling the C9-500 mouse spinal cord sections with antibodies against the luminal ER protein BiP/GRP78. Age-related longitudinal quantification of BiP expression revealed that at P30, C9-500 motor neurons expressed similar levels of BiP as in WT; but as from P60 on, a gradual increase in BiP expression in mutant motor neurons was observed. P80 onward, significantly high BiP levels, indicative of ER stress progression was observed (Fig. 3 A). We further assessed whether ER stress evolved into UPR by measuring the appearance of P i -eIF2α immunoreactivity in ChAT-positive ventral horn motor neurons. At P60, when BiP levels slightly increase, we found no indication of UPR signaling, however, an abrupt transition to UPR signaling was observed at P125, whereby nearly 50% of motor neurons displayed strong P i -eIF2α immunolabeling (53.8 ± 3.8%), and nearly all motor neurons displayed P i -eIF2α immunopositivity close to end stage at P200 (75 ± 4.9%), confirming an advanced UPR signaling in the majority of C9-500 motor neurons (Fig. 3 B). Note the complete lack of P i -eIF2α immunolabeling within WT motor neurons. Next, we assessed ER stress status in WT and C9-500 mice, which were injected with 10mg/kg of kaempferol, which was again started at P138, a timepoint after the onset of UPR signaling and lasted until late end-stage; P240 (scheme for treatment and analyses Fig. 3 C). Probing for UPR marker P i -eIF2α, we found a prominent expression within mutant C9-500 motor neurons at P240, which was restricted to ChAT-positive spinal ventral horn motor neurons in mutant condition. However, kaempferol treatment completely inhibited the expression of P i -eIF2α indicating that kaempferol acts on the UPR signaling pathway (Fig. 3 D). We next assessed the effect of ER stress inhibitor; Salubrinal on C9-500 spinal motor neurons [ 36 ]. Chronic treatment with Salubrinal as expected also reduced UPR signaling as observed by diminished P i -eIF2α immunopositivity with P180 C9-500 motor neurons (Fig. 3 E). Kaempferol restores mitochondria electron transport chain function in C9-500 mice. Besides ER stress, mitochondrial dysfunction due to C9ORF72 haploinsufficiency a well as the expression of specific DPRs such as PolyGR [ 6 , 7 , 12 , 37 ] has been causally implicated in C9ORF72 -linked ALS pathology. Thus, we assessed whether kaempferol treatment could rescue the observed mitochondrial dysfunction, which has been shown to protect neuronal mitochondria from loss of mitochondrial transmembrane electric potential caused by oxidative stress as well as excessive mitochondrial fission in neuronal models of excitotoxicity and ischemic stroke [ 38 , 39 ]. We initially evaluated oxidative stress in spinal motoneurons, given its role in inducing nuclear and mitochondrial DNA damage. This assessment was performed using the oxidative DNA damage marker, 8-hydroxy-2′-deoxyguanosine (8-OHdG), detected via a specific antibody (REF David’s paper). To quantify the expression of this marker, we set a threshold of 60 arbitrary units (a.u.) of intensity to distinguish between low and high-expressing motoneurons. Strikingly, KMP-treated C9-500 animals displayed a significant reduction in high-expressing motoneurons compared to saline-treated animals ( C9-500 saline: 88% vs. C9-500 + KMP: 21%) (Fig. 4 A). Since the primary function of mitochondria is energy production in the form of an elevated ATP/ADP ratio, we focused on assessing whether deficits in electron transport chain (ETC) function was observed in C9-500 spinal motor neurons. To this end, we used a colorimetric assay to assess Complex I, and IV activity in wildtype ( WT ) and C9-500 spinal cord after kaempferol treatment. Complex I activity was measured via the NADH dehydrogenase catalyzed redox reaction in which NADH was oxidized and nitroblue tetrazolium (NBT) was reduced. The blue/purple color so formed due to NBT reduction faithfully correlates with the amount of endogenous NADH dehydrogenase activity present within cells. This blue/purple color was specifically and prominently reduced within the soma of P240 C9-500 motor neurons as compared to WT motor neurons. Notably, kaempferol treatment of C9-500 mice normalized Complex I levels to WT levels (Fig. 4 B). Subsequently, we analyzed the activity of Complex IV by determining the intensity of cytochrome c oxidation, which is detected by the brown coloring produced by the oxidation of diaminobenzidine (DAB). Like Complex I, also reduced Complex IV activity was observed within mutant C9-500 motor neurons, and kaempferol treatment restored this deficit to normal WT levels (Fig. 4 C). While treatment with Salubrinal unlike kaempferol treatment did not improve behavioral symptoms in C9-500 mice, it did reduce UPR signaling, therefore we also explored whether Salubrinal harbors the potential to ameliorate or restore normal mitochondrial function. Treatment with Salubrinal did not affect the observed deficits in mitochondrial ETC levels and both Complex I (Fig. 4 D) and Complex IV levels remained reduced in Salubrinal-treated mutant motor neurons (Fig. 4 E). Taken together these results suggest that kaempferol acts on both cellular organelles, reducing ER stress and promoting mitochondrial function, while Salubrinal works selectively on inhibiting the ER stress/UPR pathway. A short treatment regime with kaempferol selectively ameliorates behavioral hallmarks in symptomatic C9-500 mice. Based on the observed neuroprotective action of kaempferol in the CNS, we assessed whether kaempferol treatment could rescue the behavioral defects observed in C9-500 mouse model of C9ORF72 -ALS. This mouse model displays premature ER stress, mitochondrial dysfunction as well as striking presence of RNA foci, pTDP43 aggregates, and robust accumulation of DPRs, all of which are pathological hallmarks of C9ORF72 -ALS [ 7 , 27 – 29 ]. A short treatment with Kaempferol at 10mg/kg was performed for two weeks via daily intraperitoneal (i.p.) injections [ 30 ] as from P138-P153, an age corresponding to the appearance of the pathology-associated behavioral phenotype in C9-500 mice [ 7 , 18 ]. The reverse rotarod and hanging wire tests were performed pre-kaempferol treatment to obtain baseline measurement, followed by four consecutive measurements per week post-kaempferol treatment for half of the cohort of mice, whereas the other half received saline (scheme Fig. 5 A). Reverse rotarod assay on the C9-500 mice cohort pre-kaempferol treatment revealed a striking deficit in motor performance compared to WT mice as observed by the dramatic decline in latency to fall measured in seconds. Notably after one week of kaempferol treatment C9-500 mice presented markedly improved motor coordination and balance ( Supplementary video 1 and Fig. 5 B, left graph ). We further continued to chronically treat the mice every alternate day with kaempferol until the late stage of the disease; P190, which revealed that the ameliorated rotarod performance was sustained (Fig. 5 B, right graph ). Similarly, the hanging wire test revealed that after two weeks of kaempferol treatment, the average number of falls within the two minutes was reduced compared to the performance of the same cohort of mice pre-kaempferol treatment (Fig. 5 C). Likewise, the time taken to first fall improved slightly between pre- and post-kaempferol treatment, suggesting that kaempferol treatment led to an overall amelioration in muscle endurance and function (Fig. 5 D). We next assessed whether another compound; Salubrinal which is known to inhibit ER stress and has been shown to ameliorate ALS-associated pathological symptoms in other familial ALS mouse models [ 5 , 31 , 32 ], could also have a beneficial outcome in C9-500 mice. Like kaempferol treatment regime, Salubrinal treatment was performed on symptomatic P145 C9-500 mice lasting until P170 (Fig. 5 E). This treatment led to no improvement in the reverse rotarod performance (Fig. 5 F). Moreover, hanging wire tests revealed no significant amelioration in muscle endurance or stamina as measured by number of falls within two minutes ( Fig. 5 G) or time to the first fall (Fig. 5 H). It is noteworthy that unlike studies done on SOD1G93A mice, wherein Salubrinal treatment was neuroprotective [ 5 ], in the C9-500 mice model, we found no protective effect of Salubrinal, indicative of a likely more complex interplay between various cellular impairments ranging from mitochondrial dysfunction to proteostasis and autophagy thus, leading to an accumulation of toxic events resulting in the development of the symptomatic pathology. Kaempferol potentially elicits its action by binding to GRP75. We sought to understand how kaempferol could dually act on inhibiting ER stress while sustaining mitochondrial function. We hypothesized that kaempferol likely exerts its effect at mitochondrial-associated membranes (MAM), representing regions of contact between the ER and mitochondria. MAM constitutes regions of ER membranes that are reversibly tethered to mitochondria. These membranes are involved in the import of specific lipids from the ER to mitochondria and the regulation of calcium homeostasis, mitochondrial function, autophagy, and apoptosis [ 40 , 41 ]. We performed an in-silico search for potential kaempferol interactors, which likely function at the MAM. The structure of kaempferol was uploaded to CB-Dock ( http://clab.labshare.cn/cb-dock/php/dockingresult.php ) for analysis of the docking potential of kaempferol to MAM-located molecules. These analyses led to the identification of GRP75 (75-kDA glucose-regulated protein) as being the strongest binding partner of kaempferol as indicated by the vina score. GRP75 is a major mitochondrion located chaperone, which interacts with the component of both the mitochondrial quality control system and MAMs, thus playing a key role in mitochondrial homeostasis (Fig. 6 A). The crystal structure of GRP75 revealed a significant interaction of kaempferol with the nucleotide-binding domain (NBD) of GRP75 (Fig. 6 B). We further assessed if other molecules could show similar interaction with GRP75 and found that 17-AAG had the second highest Vina score for binding to GRP75 (Fig. 6 C) and strongly docked to the NBD site of GRP75 (Fig. 6 D). Further, analyses revealed that a substantial number of overlapping human proteins were targeted by both kaempferol and 17-AAG (Fig. 6 E and Suppl. Table 2 ). However, literature analysis revealed that 17-AAG indirectly increases the expression level of GRP75 [ 42 ], while inhibiting HSP90 chaperone family, thereby impairing protein folding of multiple client substrates and autophagy that is impaired in C9ORF72 -ALS. In contrast, kaempferol, reduces UPR signaling by augmenting the expression of ER folding chaperones [ 23 , 43 ] and enhances mitochondrial Ca2 + uptake, by a yet unidentified mechanism involving the binding to the mitochondrial uniporter, in traumatic brain injury model (TBI) [ 44 ]. Interestingly, no binding affinity of Salubrinal to GRP75 was observed (data not shown). Since a previous study from us has shown reduced GRP75 expression at the MAM in C9-500 mice and its pathological sequestration by Poly(GA) aggregates within neurons, therefore we focussed on assessing whether kaempferol could exert neuroprotection via modulating GRP75, thus counteracting both ER and mitochondrial deficits. Kaempferol enhances IP3R-VDAC1 interactions, promoting optimal mitochondrial function via GRP75. Based on our in-silico screening, we hypothesized that the mechanism behind kaempferol-mediated neuroprotection could be via its interaction with GRP75 at the MAM. Since GRP75 serves as a scaffold, bringing iP3R-VDAC1 in proximity, we firstly, assessed iP3R-VDAC1 interactions via proximity ligation assay (PLA). Chronic, kaempferol treatment from symptomatic stage until the end stage of disease revealed increased iP3R-VDAC1 interactions versus saline-treated mutant spinal motor neurons of C9-500 mice (Fig. 7 A). Since saline-treated mutant C9-500 motor neurons revealed dramatically reduced iP3R-VDAC1 interactions compared to WT motor neurons, and this reduced interaction was normalized after kaempferol treatment, indicated that kaempferol could potentially attenuate mitochondrial dysfunction by promoting optimal Ca 2+ uptake by mitochondria. Thus, we examined the physiological process of Ca 2+ uptake by mitochondria, which majorly occurs at the ER–mitochondria contact sites via iP3R-VDAC1. To this end, cortical neurons from WT and C9-500 neonates were cultured and we measured mitochondrial Ca 2+ uptake. Fluo-4AM was combined with an intracellular buffer that eliminated cytosolic and ER Ca 2+ signals [ 22 ], thereby enabling specifically the measurement of mitochondrial Ca 2+ uptake. A striking deficit was observed in mitochondrial Ca 2+ uptake in C9-500 cortical neurons as observed by significantly reduced Ca 2+ transients compared WT cortical neurons. These deficits in Ca 2+ uptake by mutant mitochondria were efficiently eliminated by treating cortical neurons with kaempferol, confirming our finding that kaempferol elicits its effect at the MAM, modulating the mitochondrial uptake of Ca 2+ (Fig. 7 B). We next measured GRP75 expression in spinal motor neurons to assess whether kaempferol modulates GRP75 expression. Motor neurons from both WT and C9-500 mice treated with kaempferol displayed augmented GRP75 expression (Fig. 7 C). Of note the degree of enhanced GRP75 expression was much higher in mutant C9-500 motor neurons, as they present dramatically reduced GRP75 expression at late stages of the disease. To underpin the molecular mechanism associated with the beneficial effect of GRP75 on the mitochondria, we co-immunoprecipitated GRP75 from P125, C9-500 and WT ventral spinal cord, followed by mass spectrometric (MS) analyses. The interactome of GRP75 revealed known interactions such as those with VDAC1 as well as other interactions shared between the two genotypes ( Suppl. Table 3 ). We focused on GRP75 interactions, which were strongly present in the C9-500 spinal cord. Within the top five GRP75 interacting proteins, as measured semi-quantitatively via peptide match score summation (PMSS), was mitochondrial ATP synthase-coupling factor 6 (ATP5J), (Fig. 7 D). ATP5J produces ATP from ADP in the presence of a proton gradient across the mitochondrial membrane, generated by the ETC of the respiratory chain, we validated the interaction between ATP5J and GRP75 in C9-500 motor neurons using PLA. Endogenous GRP75 interactions with ATP5J was observed in both WT and C9-500 motor neurons, albeit strongly at P125 mutant motor neurons. However, akin to reduced iP3R-VDAC1 interactions at P240, ATP5J-GRP75 were much lower in late-stage mutant motor neurons, indicative of advanced mitochondrial dysfunction and degeneration (Fig. 7 E). As kaempferol treatment augmented GRP75 expression, we examined ATP5J expression in response to kaempferol treatment. While ATP5J levels were reduced in P240 motor neurons, likely reflecting the reduced GRP75 expression, spinal motor neurons treated with kaempferol even at end stage presented high ATP5J immunoreactivity, reflecting an overall beneficial effect of kaempferol on mitochondria via the GRP75-ATP5J pathway (Fig. 7 F) DISCUSSION ALS is a multifactorial disease, with complex overlapping clinical symptoms, and a fatal outcome within 3–5 years of diagnosis [ 45 , 46 ]. Several studies have indicated that ER stress plays a pivotal role in the pathophysiology of ALS [ 2 , 47 ], thus targeting ER stress is an interesting objective for therapeutic intervention. Besides, ER stress, mitochondrial dysfunction associated with oxidative stress, mitochondrial Ca 2+ uptake deficits and bioenergetic deficits is closely associated with ALS pathophysiology [ 48 – 51 ], therefore identifying compounds that can ameliorate both ER and mitochondrial impairments in ALS is of extreme importance to the field of ALS. In this study, we identified and explored the potential of a dietary flavanol kaempferol as a modulator of both ER stress response as well as mitochondria function in ALS. Focussing on the most common genetic form of ALS, C9ORF72 -linked ALS, we show that kaempferol harbours the potential to attenuate ER stress and UPR signaling in vivo in symptomatic C9-500 rodent motor neurons as well as in vitro in human C9ORF72 patient-derived iMNs. Moreover, we provide compelling evidence for the ability of kaempferol to restore ATP production and to normalize mitochondrial function in C9ORF72 ALS. This is noteworthy, as previous compounds such as Salubrinal have been shown to interfere only with the PERK/ P i -eIF2α -UPR pathway [ 5 , 10 , 52 ], or by suppressing the activity of integrated stress response, upstream of the PERK/ P i -eIF2α -UPR pathway [ 29 ]. Moreover, drugs like Salubrinal or guanabenz enhance the persistent translation inhibition by P i -eIF2α, thereby, accelerating neurodegeneration in a model of prion disease [ 53 ] or in mutant male SOD1 mice [ 54 ]. In addition, both these compounds elicit toxicity on other organelles such as nephrotoxicity observed after chronic Salubrinal administration [ 55 ] or blood pressure dysregulation after guanabenz administration [ 56 ]. These studies highlight the unmet need for a drug candidate with the ability to reduce UPR signaling in affected neurons, without side effects. In this context, kaempferol and its derivatives are natural dietary phytochemicals, lacking evident toxicity, and are known to exhibit antioxidant, anti-inflammatory, anticancer, and neuroprotective activity [ 57 ]. Notably, we found that treatment with kaempferol initiated after the onset of pathological symptoms was able to abrogate intrinsic mitochondrial deficits present in C9ORF72 neurons. These data are remarkable, as kaempferol normalized mitochondrial ATP levels in human C9ORF72 motor neurons, an important determinant of overall adequate neuronal energy supply and optimal function. While Salubrinal as expected, did inhibit ER stress, it did not ameliorate mitochondrial function either in vivo in C9-500 motor neurons nor in vitro in human iMNs. On the contrary, Salubrinal has been shown to promote cell death in cancer by activating the ISR and inducing mitochondrial oxidative stress, thereby irreversibly damaging mitochondria. Further, Salubrinal treatment in glucose-deprived conditions leads to the upregulation of mitochondrial ROS, thereby inducing mitochondrial stress and dysfunction [ 58 ]. Previous studies have shown that kaempferol alleviates oxidative stress and apoptosis during lung ischemia-reperfusion injury, by enhancing mitochondrial membrane potential and inhibiting the opening of mitochondrial permeability transition pores [ 59 ]. Importantly, kaempferol has been shown to control mitochondrial calcium regulation by directly activating the mitochondrial calcium uniporter (MCU) in a concentration-dependent manner. Even at a low concentration such as 1µM of kaempferol treatment, practically doubled the uptake of mitochondrial Ca²⁺ [ 60 ]. Therefore, based on our findings, it is very likely that kaempferol treatment counteracts mitochondrial dysfunction as well as mitochondrial Ca²⁺ uptake deficits in C9ORF72 -ALS [ 6 , 7 ], thus promoting optimal mitochondrial function and neuroprotection. Kaempferol has been shown to modulate key signaling pathways involved in neurodegeneration and neuroinflammation, such as the PI3K/Akt, MAPK/ERK, and NF-κB pathways, nevertheless, further research is necessary to decipher the underlying mechanisms of action, to optimize dosage schedules, and assess the safety and efficacy of this intervention in human clinical trials. Of note, administration of kaempferol via the i.p. route at symptomatic ages in C9-500 mice attenuated the accumulation of large Poly(GA) aggregates, increased the number of surviving motor neurons, and improved motor behavior. We believe that these findings enhance the predictive value that kaempferol could be beneficial in patients with C9ORF72 -ALS and in other familial forms of ALS, where ER stress and mitochondrial impairments occur together with mutant protein inclusions. In other neurodegenerative diseases such as Alzheimer´s disease, kaempferol treatment inhibited β-A structure formation by hindering amyloid fibril elongation [ 61 ]. Yet in another study treatment with kaempferol derivative; kaempferide revealed the enhanced expression of the brain-derived neurotrophic factor (BDNF), which in turn augmented the phosphorylation of transcription factor cAMP, thus promoting synaptic plasticity [ 62 ]. In Parkinson´s disease, kaempferol ameliorated nigrostriatal dopaminergic neuron lesions; by inhibiting interleukin (IL) 1β, IL-6, and TNFα production, highlighting the anti-inflammatory activity of kaempferol [ 63 ]. The mechanism via which kaempferol elicits its anti-ER stress properties remains unclear. However, studies have shown that kaempferol inhibits the expression of BiP, thereby inhibiting the activation of UPR sensors PERK, ATF6, and IRE1 [ 23 ]. These findings fit well with our observations of reduced BiP and P i -eIF2α expression after kaempferol treatment. Our data from in silico modeling identified that kaempferol strongly binds to GRP75; a chaperone located within the mitochondria, but is majorly involved at the MAM, functioning as a structural scaffold protein for the iP3R-VDAC1 channel. This channel is the most prominent channel at the MAM promoting optimal transfer of Ca 2+ from the ER to the mitochondria. GRP75 expression levels are progressively reduced in C9-500 mice, and those reductions coincide with the onset of UPR signaling and the appearance of large Poly(GA) inclusions within motor neurons [ 7 ]. Our PLA data suggests that within the C9-500 spinal motor neurons, kaempferol enhances iP3R-VDAC1 interactions, which aids in the optimal mitochondrial Ca 2+ uptake, important for the proper ETC process. Of note, our data also revealed the ability of kaempferol to enhance the expression of GRP75 and its interacting partner ATP5J in motor neurons, suggesting that kaempferol could act on process regulating protein translation. Interestingly, like quercetin yet another flavanol, kaempferol might positively modulate gene expression[ 16 , 17 ]. Moreover, kaempferol could positively influence GRP75 expression at a translational level by inhibiting UPR signaling, thus removing the global translational block. Therefore, kaempferol might have a complex and multifaceted impact on the protein translation process, which may contribute to its pharmacological activity and potential therapeutic applications. However, further experiments are needed to dissect how kaempferol impacts GRP75 expression and iP3R-VDAC1 function in the context of C9ORF72 -ALS. In view of the translation potential of our findings, we measured ER stress and mitochondrial function in human C9ORF72 patient-derived iMNs and found that kaempferol not only ameliorated ER stress and restored normal mitochondrial function in three different C9ORF72 -patient iMN lines, but also provided neuroprotection by promoting iMNs survival in the presence of ER stress. To the best of our knowledge, no previous drug compound has been reported to have a protective effect in C9-500 mice by dually ameliorating ER stress and mitochondrial function. Concomitantly, the reduction in large Poly(GA) aggregates, sustained motor neuron survival as well as improved muscle and behavioral phenotype, highlights the beneficial potential of Kaempferol in the C9-500 mice, when treatment was initiated at disease signs onset. This is a clinically relevant time point for the initiation of therapy in human patients, as usually, they do not receive a diagnosis before symptoms onset. Additionally, we identified a novel mode of action of kaempferol, via the regulation of GRP75 expression and function. These observations suggest that kaempferol is a likely strong candidate for the prevention/slowing down of disease pathology in familial ALS cases. In conclusion, kaempferol rescues motor neuron survival in vitro and in vivo by regulating the ER stress response and ameliorating intrinsic mitochondrial function, which are key pathways implicated in the pathophysiology of C9ORF72 -ALS. Although the precise mode of action of Kaempferol remains incomplete, our data suggests that one likely pathway involves the interaction with the nucleotide-binding domain of GRP75, thus enhancing/stimulating GRP75 activity. Hence, kaempferol is a promising compound for diseased motor neurons, meriting further study as a promising drug candidate in ALS. Abbreviations Unfolded Protein response (UPR) amyotrophic lateral sclerosis (ALS) frontal temporal dementia (FTD) induced pluripotent stem cells (iPSC) glycogen synthase kinases-3β (GSK3β) Mitochondria-associated membranes (MAMs) protein tyrosine phosphatase interacting protein 51 (PTPIP51) motoneurons (MNs) iPSC-derived motor neurons (iMNs) direct reprogramming of fibroblasts to neurons (dNs) integrated stress response (ISR) Declarations ETHICS DECLARATIONS: Animal experiments: The study was approved by the Animal Commission of Canton of Bern, Switzerland, license number BE-35/17, BE-82/18 Human fibroblast-derived cells: Cells were anonymized and provided to us under an MTA from respective consortia. Culturing of cells only did not require internal review board (IRB) approval Consent for publication All authors have approved the manuscript and agree with its submission. Conflicts of interests The authors declare that they have no competing interests. AUTHOR CONTRIBUTIONS: F.P., S.S. conceived the study and wrote the manuscript. F.P., P.S., R.D., M.S., and O.S. J.A.S.D. performed experiments and analyzed data. O.S., H.P., and S.S. provided reagents. S.S. supervised the overall project, all authors read and commented on the manuscript. ACKNOWLEDGEMENTS: We thank Pentti Tienari, Helsinki University Central Hospital, Finland, Biomedicum Stem Cell Center, GoEditStem platform, HiLIFE, Helsinki, Finland; Bhuvaneish T. Selvaraj and Siddharthan Chandran, UK Dementia Research Institute, University of Edinburgh, UK, Euan MacDonald Centre for MND Research, University of Edinburgh, UK for providing iPSC cell lines. We thank Manfred Heller, Proteomic Core Facility, Department for BioMedical Research, University of Bern for Mass spectrometry. We also thank Nicolas Charlet-Berguerand, Institut de Génétique et de Biologie Moléculaire et Cellulaire (INSERM U964, CNRS UMR7104 for Poly(GA) antibody, Angelina Oestmann and Maria Essers for maintaining mouse colonies and helping with behavioral assays. FUNDING: This study was funded by the SPINAL CORD INJURIES/DISEASE RESEARCH PROGRAM (SCIDRP), University of Missouri, Missouri, USA, European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement #725825), Swiss National Science Foundation, Swiss Foundation for Research on Muscle Diseases, and E-rare grant (CALSER) to S.S. References Halliday M, Mallucci GR (2014) Targeting the Unfolded Protein Response in Neurodegeneration: A New Approach to Therapy. Neuropharmacology 76:169–174. 10.1016/j.neuropharm.2013.08.034 Hetz C, Saxena SER (2017) Stress and the Unfolded Protein Response in Neurodegeneration. 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Supplementary Files Suppl.Table1.pdf Supplementary Table 1 Demographic information about iPSC and fibroblast lines from C9ORF72-ALS/FTD patients and healthy control (CTRL) employed to generate iMNs and dNs. SupplTable23.pdf Supplementary Table 2 List of overlapping human proteins targeted by both kaempferol and 17-AAG Supplementary Table 3 The interactome of GRP75 as revealed by mass spectrometry analyses, note the strong interaction between the established association of GRP75 with VDAC1. Suppl1.eps Supplementary Figure 1 Representative images: 2-week-old iMNs derived from Control (1) immunolabeled for the neuronal marker MAP2 and the motor neuron marker ChAT. Scale bars: 30 μm, zoom: 15 μm. Suppl.video1.mp4 Supplementary video Inverse rotarod measurement showing ameliorated motor coordination in C9-500 mice after Kaempferol treatment PilottoetalGraphicalabstractcopylegend.tif Cite Share Download PDF Status: Published Journal Publication published 01 Feb, 2025 Read the published version in Acta Neuropathologica Communications → Version 1 posted Editorial decision: Revision requested 14 Nov, 2024 Reviews received at journal 13 Nov, 2024 Reviews received at journal 10 Nov, 2024 Reviewers agreed at journal 30 Oct, 2024 Reviewers agreed at journal 28 Oct, 2024 Reviewers invited by journal 18 Oct, 2024 Editor assigned by journal 07 Oct, 2024 Submission checks completed at journal 07 Oct, 2024 First submitted to journal 01 Oct, 2024 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5190511","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":378240375,"identity":"fbf0d4ec-ed22-49f4-bbe8-41ffdfc00c4a","order_by":0,"name":"Federica Pilotto","email":"","orcid":"","institution":"University Hospital of Bern","correspondingAuthor":false,"prefix":"","firstName":"Federica","middleName":"","lastName":"Pilotto","suffix":""},{"id":378240376,"identity":"b992ad1d-3934-472e-8400-974270f3cff0","order_by":1,"name":"Paulien Smeele","email":"","orcid":"","institution":"Department of Physical Medicine \u0026 Rehabilitation, University of Missouri, Columbia, MO, USA","correspondingAuthor":false,"prefix":"","firstName":"Paulien","middleName":"","lastName":"Smeele","suffix":""},{"id":378240377,"identity":"42eaebe2-7785-4f7c-8adf-901d7efd81a2","order_by":2,"name":"Olivier Scheidegger","email":"","orcid":"","institution":"University Hospital of Bern","correspondingAuthor":false,"prefix":"","firstName":"Olivier","middleName":"","lastName":"Scheidegger","suffix":""},{"id":378240378,"identity":"8c2ebeab-a8bb-4f26-980c-69a005431fe6","order_by":3,"name":"Rim Diab","email":"","orcid":"","institution":"University Hospital of Bern","correspondingAuthor":false,"prefix":"","firstName":"Rim","middleName":"","lastName":"Diab","suffix":""},{"id":378240379,"identity":"8ddc853b-5717-4170-b004-af1500d66e87","order_by":4,"name":"Martina Schobesberger","email":"","orcid":"","institution":"University Hospital of Bern","correspondingAuthor":false,"prefix":"","firstName":"Martina","middleName":"","lastName":"Schobesberger","suffix":""},{"id":378240380,"identity":"5c4faef9-69a1-477e-8115-1a8c9064dec8","order_by":5,"name":"Julieth Andrea Sierra-Delgado","email":"","orcid":"","institution":"Department of Physical Medicine \u0026 Rehabilitation, University of Missouri, Columbia, MO, USA","correspondingAuthor":false,"prefix":"","firstName":"Julieth","middleName":"Andrea","lastName":"Sierra-Delgado","suffix":""},{"id":378240381,"identity":"d21c4bd6-740a-46bf-8637-c7023289a11f","order_by":6,"name":"Smita Saxena","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYFACxgY46wGDAYR1gIGBGZ+WRpgeZgMitSCsYZNAEsWtRX7a4fYHP/7YJfZLNz+rulFQZ88vkfvwAEOFdWIDDi0GtxMbG3vbko0l5xwzu51jcDhx5ox0gwMMZ9Jxa5FObGzgbWCWM7iRANJyIMHgRhrDAca2wzi1yM8G2vLnTz2P/Y30b8U5BnX2EC3/cGthADqsmYftsJyBRI4Zc44BM+MGsJYG3FpAfpkt23bcWOJGTrE02C89zxgOJBxLN8btsPQHH9/8qU7sn5G+8XPOH2CIsacxf/hQYy2L02HYQQJpykfBKBgFo2AUoAEAZeFeE0PplJ8AAAAASUVORK5CYII=","orcid":"","institution":"Department of Physical Medicine \u0026 Rehabilitation, University of Missouri, Columbia, MO, USA","correspondingAuthor":true,"prefix":"","firstName":"Smita","middleName":"","lastName":"Saxena","suffix":""}],"badges":[],"createdAt":"2024-10-02 03:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5190511/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5190511/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40478-025-01927-y","type":"published","date":"2025-02-01T15:58:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71870836,"identity":"0f8417f3-b859-445b-af15-393913a61075","added_by":"auto","created_at":"2024-12-19 10:18:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":315795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKMP restores mitochondria impairments and reduces tunicamycin-induced ER stress in C9ORF72 neurons\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Seahorse graph plotted for oxygen consumption rate (OCR) on y-axis with time on X-axis. Representative traces of mitochondrial respiration from Ctrl(1) and C9(1) iMNs, showing mitochondrial functional deficits in C9ORF72 patient line at one week, but those deficits are normalized after KMP treatment. (\u003cstrong\u003eb\u003c/strong\u003e) Mitochondrial oxygen consumption rate analysis on iMNs reveals impairments in basal respiration in all three C9ORF72 patient lines compared to healthy control or respective isogenic control. Basal respiration is restored to control level after KMP treatment (Unpaired t-test: CNTRL (1) vs C9 (1) ***; CNTRL (1) vs C9 (1) + KMP n.s.; CNTRL (2) vs C9 (2) ***; CNTRL (2) vs C9 (2) + KMP n.s.; Iso-C9 (3) vs C9 (3) *; Iso-C9 (3) vs C9 (3) + KMP *). The experiment was performed in triplicates from three different batches of conversion. (\u003cstrong\u003ec\u003c/strong\u003e) Mitochondria oxygen consumption rate analysis on iMNs reveals impairments in ATP production in all C9ORF72 patient lines compared to healthy control or respective isogenic. ATP production is restored to the control level after KMP treatment (Unpaired t-test: CNTRL (1) vs C9 (1) **; CNTRL (1) vs C9 (1) + KMP * CNTRL (2) vs C9 (2) ***; CNTRL (2) vs C9 (2) + KMP *; Iso-C9 (3) vs C9 (3) *; Iso-C9 (3) vs C9 (3) + KMP n.s.). Experiment was performed in triplicates from three different batches of conversion. (\u003cstrong\u003ed\u003c/strong\u003e) Schematic depiction of the experimental timeline for iMNs Tunicamycin (TU) treatment and TU treatment combined with 24h of KMP. (\u003cstrong\u003ee\u003c/strong\u003e) Representative confocal images of BiP immunostaining in iMNs, quantitative analysis revealed an increase in BiP expression after 24h TU treatment, whereas ER stress was completely inhibited after TU+KMP treatment. (Unpaired t-test: CNTRL (1-2) vs C9 (1.2) n.s. Iso-C9 (3) vs C9 (3) n.s.; CNTRL (1-2) vs CNTRL (1-2)+ TU***; C9 (1-2) vs C9 (1-2)+ TU***; Iso-C9 (3) vs Iso-C9 (3)+ TU ***; C9(3) vs C9 (3)+ TU ***; CNTRL (1-2)+ TU vs CNTRL (1-2)+ TU+KMP ***; C9 (1-2)+ TU vs C9 (1-2)+ TU+KMP***; Iso-C9 (3) )+ TU vs Iso-C9 (3) )+ TU+KMP ***; C9 (3) + TU vs C9 (3)+ TU )+ KMP***). Scale bars: 100 μm. (\u003cstrong\u003ef\u003c/strong\u003e) CNTRL and C9 iMNs treated with TU displayed significant cell death, whereas the addition of KMP for 24h was found to be neuroprotective (TU: CNTRL (1) 41% alive vs 59% dead; C9 (1) 45% alive vs 55% dead; C9 (2) 35% alive vs 65% dead; TU+KMP: CNTRL (1) 96% alive vs 4% dead; C9 (1) 93% alive vs 7% dead; C9 (2) 81% alive vs 19% dead). Scale bars: 10 μm. (\u003cstrong\u003eg\u003c/strong\u003e) Schematic depiction of the experimental timeline for dNs generation and Tunicamycin (TU) treatment and TU treatment combined with 24h of KMP. (\u003cstrong\u003eh\u003c/strong\u003e) CNTRL and C9 dNs treated with TU displayed significant cell death, whereas those treated with KMP for 24h were resilient to ER stress-induced neurodegeneration.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5190511/v1/ab46fb2ccaaf4345629c7c2e.png"},{"id":71870845,"identity":"16919518-c5cc-41b1-b22a-adf7e2056219","added_by":"auto","created_at":"2024-12-19 10:18:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3035161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaempferol promotes motor neuron survival and reduces pathology progression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Immunohistochemical staining for MN marker ChAT and quantitative analyses. reveals significant differences in motor neuron numbers between \u003cem\u003eC9-500\u003c/em\u003e mice treated with KMP until the end stage of the disease at P240 and the saline group (Unpaired t test: \u003cem\u003eC9-500\u003c/em\u003e + saline mean±SEM 21.75±1.097 vs \u003cem\u003eC9-500 + \u003c/em\u003eKMP mean±SEM 32.48±1.127, t=6.455, P\u0026lt;0.0001). Scale bar = 150 μm. (\u003cstrong\u003eb\u003c/strong\u003e) ChAT-positive \u003cem\u003eC9-500\u003c/em\u003e MNs from \u003cem\u003eC9-500\u003c/em\u003e mice show in an age-dependent manner the accumulation of greater than 1µm\u003csup\u003e3\u003c/sup\u003e poly-GA aggregates. (MN numbers: P60 \u003cem\u003eC9-500\u003c/em\u003e: n=42; P125 \u003cem\u003eC9-500\u003c/em\u003e: n=47; P150 \u003cem\u003eC9-500\u003c/em\u003e: n=38; from 4 mice/genotype/age). Scale bar: 30 μm. (\u003cstrong\u003ec\u003c/strong\u003e) Quantitative analyses of Poly(GA) aggregates reveal a significant reduction in the number of aggregates \u003cem\u003ein C9-500\u003c/em\u003e mice treated with KMP (Unpaired t-test: \u003cem\u003eC9-500\u003c/em\u003e \u003cem\u003e+ \u003c/em\u003esaline: n=19 motor neurons vs \u003cem\u003eC9-500\u003c/em\u003e \u003cem\u003e+ \u003c/em\u003eKMP; n= 22 motor neurons, t=3.110, P=0.0035**). Quantitative analysis of PolyGA aggregate volume reveals a significant decrease in \u0026gt;1 μm\u003csup\u003e3\u003c/sup\u003e aggregates within spinal MN after KMP treatment (Unpaired t-test: \u003cem\u003eC9-500\u003c/em\u003e \u003cem\u003e+ \u003c/em\u003esaline: n=19 motor neurons vs \u003cem\u003eC9-500\u003c/em\u003e \u003cem\u003e+ \u003c/em\u003eKMP; n= 22 motor neurons, t=3.632, P=0.0011**). n = 4-5 mice/genotype/treatment. Scale bar: 10 μm. (\u003cstrong\u003ed\u003c/strong\u003e) Representative confocal images of GFAP staining and quantitative analysis. Note the reduced astrogliosis in \u003cem\u003eC9-500\u003c/em\u003e animals treated with KMP (Unpaired t-test: \u003cem\u003eC9-500\u003c/em\u003e \u003cem\u003e+ \u003c/em\u003esaline vs \u003cem\u003eC9-500\u003c/em\u003e \u003cem\u003e+ \u003c/em\u003eKMP (t=5.789, P\u0026lt;0.0001***). (\u003cstrong\u003ee\u003c/strong\u003e) Representative images of muscle sections with staining for H\u0026amp;E from \u003cem\u003eC9-500\u003c/em\u003e mice treated with saline and KMP. Blue arrowheads in the H\u0026amp;E images point to small and degenerating fibers that are not present in \u003cem\u003eC9-500\u003c/em\u003e mice treated with KMP. Scale bar: 100 μm. (\u003cstrong\u003ef\u003c/strong\u003e) Representative images of muscle sections with staining NADH from \u003cem\u003eC9-500\u003c/em\u003e mice treated with saline and KMP. Red arrows in the NADH staining point to small and degenerating fibers that are not present in \u003cem\u003eC9-500\u003c/em\u003e mice treated with KMP. Moreover, the NADH staining presents pale fibres in \u003cem\u003eC9-500\u003c/em\u003e saline animals while in KMP-treated \u003cem\u003eC9-500\u003c/em\u003e mice, a strong checkboard pattern is present. Scale bar: 150 μm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5190511/v1/bbd4bce174f5918b398b3ac3.png"},{"id":71872271,"identity":"f4ba5724-2e7a-4bb2-9c44-c75bc1c02633","added_by":"auto","created_at":"2024-12-19 10:26:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1399400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eC9-500\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e motor neurons exhibit early and progressive ER stress and UPR signaling, which is attenuated by kaempferol treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a)Representative images immunolabeled for ER stress marker BiP in \u003cem\u003eWT \u003c/em\u003eand \u003cem\u003eC9-500\u003c/em\u003e spinal cord, reveals an increase in BiP intensity within ChAT-positive mutant motor neurons at P80. The graph on the right depicts quantitative analyses of BiP expression, showing an age-dependent increase within C9-500 motor neurons as measured in arbitrary units (a.u.). (Unpaired t-test, motor neuron numbers: at P30 \u003cem\u003eWT\u003c/em\u003e, n=18 vs \u003cem\u003eC9-500,\u003c/em\u003e n=18, t=0.6159, df=34 n.s. P=0.5421; at P60 \u003cem\u003eWT,\u003c/em\u003e n=29 vs \u003cem\u003eC9-500\u003c/em\u003e, n=28, t=3.353, df=55 **P=0.0015; at P80 \u003cem\u003eWT\u003c/em\u003e, n=27 vs \u003cem\u003eC9-500\u003c/em\u003e, n=38, t=5.741, df=63,***P\u0026lt;0.0001; at P125 \u003cem\u003eWT\u003c/em\u003e, n=21 vs \u003cem\u003eC9-500\u003c/em\u003e, n=39, t=4.804, df=58 ***P\u0026lt;0.0001; at P150 \u003cem\u003eWT\u003c/em\u003e, n=41 vs \u003cem\u003eC9-500\u003c/em\u003e, n=53, t=12.74, df=92, ***P\u0026lt;0.0001; at P200 \u003cem\u003eWT\u003c/em\u003e, n=58 vs \u003cem\u003eC9-500\u003c/em\u003e, n=57, t=27.11, df=113, **P=0.0078 ). Scale bar = 30 μm, n= 4-5 mice/genotype/age. (\u003cstrong\u003eb\u003c/strong\u003e) Representative images depicting the appearance of UPR signaling in C9-500 motor neurons as measured by the presence of phosphorylated eIF2α (P\u003csub\u003ei\u003c/sub\u003e-eIF2α) from P125, which encompasses 78% ± 3.403 motor neurons by P200. Number of ChAT+ve motor neurons analysed at P60 \u003cem\u003eWT\u003c/em\u003e, n=85 \u0026amp; \u003cem\u003eC9-500\u003c/em\u003e, n=94, at P125 \u003cem\u003eWT\u003c/em\u003e, n=82 \u0026amp; \u003cem\u003eC9-500\u003c/em\u003e, n=91, at P200 \u003cem\u003eWT\u003c/em\u003e, n=192 \u0026amp; \u003cem\u003eC9-500\u003c/em\u003e, n=151). Scale bar = 20 μm, n= 3 mice/genotype/age. (\u003cstrong\u003ec\u003c/strong\u003e) Schematic depiction of the experimental timeline for in vivo treatment of symptomatic \u003cem\u003eC9-500\u003c/em\u003e mice with kaempferol (KMP) until the end stage of disease P240. Initial short treatment lasting for two weeks was performed by daily intraperitoneal (i.p.,) injection of kaempferol, followed by injection every alternate day up to P240. (\u003cstrong\u003ed\u003c/strong\u003e) Representative images showing the activation of UPR via the expression of P\u003csub\u003ei\u003c/sub\u003e-eIF2α between saline-treated KMP-treated \u003cem\u003eC9-500\u003c/em\u003e mice. Quantitative analyses of P\u003csub\u003ei\u003c/sub\u003e-eIF2α expression reveal a significant reduction in UPR levels in \u003cem\u003eC9-500\u003c/em\u003e motor neurons after KMP treatment compared to saline controls. One-way ANOVA: P\u0026lt;0.0001*** F=48.47, Sidak’s multiple comparisons: \u003cem\u003eWT\u003c/em\u003e + saline vs \u003cem\u003eWT + \u003c/em\u003eKMP n.s. \u003cem\u003eC9-500\u003c/em\u003e + saline vs \u003cem\u003eC9-500\u003c/em\u003e + KMP***. Scale bar = 30 μm. (\u003cstrong\u003ee\u003c/strong\u003e) Scheme showing Sal training schedule. Representative images of P\u003csub\u003ei\u003c/sub\u003e-Eif2α from saline and Sal-treated \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e animals. (\u003cstrong\u003ef\u003c/strong\u003e) Quantitative analyses show reduced expression of P\u003csub\u003ei\u003c/sub\u003e-eIF2α. (One-way ANOVA, F= 306.6, P\u0026lt;0,0001, Sidak’s multiple comparison test: \u003cem\u003eWT\u003c/em\u003e vs \u003cem\u003eWT \u003c/em\u003e+ Sal, n.s.; \u003cem\u003eC9-500\u003c/em\u003e vs \u003cem\u003eC9-500\u003c/em\u003e +Sal,***). Scale bar = 20 μm, n= 3 animals per genotype/group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5190511/v1/d17e12342e9a5716828f80e7.png"},{"id":71872273,"identity":"3a6dc68c-b0f6-4f0d-bc65-209a88cd1511","added_by":"auto","created_at":"2024-12-19 10:26:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2839675,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaempferol treatment ameliorates mitochondrial function in mutant \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC9-500\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e motor neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Representative confocal images showing 8-OHdG expression levels within motor neurons. Quantification analyses of intensity and relative percentage of MNs expressing high and low levels of oxidative stress (low expressing MNs: \u003cem\u003eWT\u003c/em\u003e saline 93%, \u003cem\u003eWT\u003c/em\u003e + KMP 91%, \u003cem\u003eC9-500\u003c/em\u003e saline 12%,\u003cem\u003e C9-500\u003c/em\u003e + KMP 88%; high expressing MNs: \u003cem\u003eWT\u003c/em\u003esaline 7%, \u003cem\u003eWT\u003c/em\u003e + KMP 9%, \u003cem\u003eC9-500\u003c/em\u003e + saline 88%,\u003cem\u003e C9-500\u003c/em\u003e + KMP 21%. Chi square test: \u003cem\u003eWT\u003c/em\u003e saline vs \u003cem\u003eC9-500\u003c/em\u003e + saline; P\u0026lt;0.0001***; \u003cem\u003eC9-500\u003c/em\u003e + saline vs \u003cem\u003eC9-500\u003c/em\u003e + KMP: P\u0026lt;0.0001***) Scale bars: 50 μm.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Representative images showing Complex I activity via the NADH dehydrogenase catalyzed redox reaction within motor neurons, traced with dotted red lines. Quantification analyses of staining intensity for Complex I of the mitochondria respiratory chain show that KMP treatment significantly improves Complex I activity within \u003cem\u003eC9-500\u003c/em\u003e motor neurons. Note the negligible blue coloration for Complex I within motor neurons (Unpaired t-test, Complex I: \u003cem\u003eC9-500\u003c/em\u003e + saline n=45 vs \u003cem\u003eC9-500\u003c/em\u003e + KMP n=21, t=16.32, P\u0026lt;0.0001. Scale bars: 30 μm. (\u003cstrong\u003ec\u003c/strong\u003e) Representative images showing Complex IV activity via cytochrome c oxidation, traced with dotted black lines. Quantification analyses of staining intensity for the Complex IV of the mitochondria respiratory chain reveal that KMP treatment significantly improves Complex IV activity within \u003cem\u003eC9-500\u003c/em\u003e motor neurons. Note the pale-yellow coloration for Complex IV within motor neurons, indicative of a dysfunction in the mitochondrial respiratory chain. (Unpaired t-test, Complex IV: \u003cem\u003eC9-500\u003c/em\u003e + saline n=21 vs C9-500 + KMP n=29, t=8.449, P\u0026lt;0.0001). Scale bars: 30 μm. (\u003cstrong\u003ed\u003c/strong\u003e) Representative images of mitochondrial respiratory Complex I staining within \u003cem\u003eC9-500\u003c/em\u003e spinal motor neurons, traced with red dotted lines. Note the non-significant changes in Complex I levels in the spinal cord of \u003cem\u003eC9-500\u003c/em\u003e treated with ER stress inhibitor salubrinal (Complex I, One-way ANOVA: F= 28.35, P\u0026lt;0,0001, Sidak’s multiple comparison test: \u003cem\u003eWT\u003c/em\u003e vs \u003cem\u003eW T\u003c/em\u003e+ Sal, n.s.; \u003cem\u003eC9-500\u003c/em\u003e vs \u003cem\u003eC9-500\u003c/em\u003e + Sal, n.s.Scale bars: 100 μm. (\u003cstrong\u003ee\u003c/strong\u003e) Representative images of mitochondrial respiratory Complex IV staining within \u003cem\u003eC9-500\u003c/em\u003e spinal motor neurons, traced with black dotted lines. No changes in Complex IV levels in the spinal cord of \u003cem\u003eC9-500\u003c/em\u003e treated with Salubrinal are observed. (Complex IV One-way ANOVA: F= 25.72, P\u0026lt;0,0001, Sidak’s multiple comparison test: \u003cem\u003eWT\u003c/em\u003evs \u003cem\u003eWT \u003c/em\u003e+ Sal, n.s.; \u003cem\u003eC9-500\u003c/em\u003e vs \u003cem\u003eC9-500 \u003c/em\u003e+ Sal, n.s.).Scale bars: 100 μm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5190511/v1/54e9e9e9f7d3d929e0915bf8.png"},{"id":71872274,"identity":"1e641207-128e-44e3-8f7c-af2a684537e0","added_by":"auto","created_at":"2024-12-19 10:26:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":70126,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaempferol but not salubrinal trt. rescues behavioral phenotype in symptomatic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC9-500\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Schematic depiction of the experimental timeline for in vivo treatment of symptomatic \u003cem\u003eC9-500\u003c/em\u003e mice with kaempferol (KMP). Initial short treatment lasting for two weeks was performed by daily intraperitoneal (i.p.,) injection of kaempferol. This was followed by injection every alternate day up to P190. (\u003cstrong\u003eb\u003c/strong\u003e) Reverse rotarod plotted as latency to fall, \u003cem\u003eC9-500\u003c/em\u003e mice treated with KMP show significant improvement in motor performance compared to \u003cem\u003eC9-500\u003c/em\u003e saline treated group. Note the substantial improvement in motor performance to near normal \u003cem\u003eWT\u003c/em\u003elevels, already one week after KMP treatment, which is sustained after two weeks of KMP treatment as well as until late disease stage at P190. (Number of animals: 10 \u003cem\u003eWT \u003c/em\u003e+ saline; 10 \u003cem\u003eC9-500\u003c/em\u003e + saline; 10 \u003cem\u003eWT\u003c/em\u003e + KMP; and 11 \u003cem\u003eC9-500\u003c/em\u003e + KMP). Two-way ANOVA: interaction P=\u0026lt;0.0001***, F (15,216)=21.95, time P\u0026lt;0.0001*** F (5,216)=197.2, treatment P\u0026lt;0.0001***, F (3,216)=580.1, Bonferroni post hoc. \u003cem\u003eWT\u003c/em\u003e + saline vs \u003cem\u003eC9-500\u003c/em\u003e+ saline ***; \u003cem\u003eC9-500\u003c/em\u003e + saline vs \u003cem\u003eC9-500\u003c/em\u003e + KMP ***. (\u003cstrong\u003ec\u003c/strong\u003e) Hanging wire test showing muscle performance in \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e mice before and after KMP treatment. The total number of falls within a period of 2 minutes were significantly higher in \u003cem\u003eC9-500\u003c/em\u003e mice group than \u003cem\u003eWT \u003c/em\u003emice cohort. However, continuous KMP treatment significantly reduced the number of falls within \u003cem\u003eC9-500\u003c/em\u003eafter KMP treated group as compared to \u003cem\u003eC9-500\u003c/em\u003e before KMP treatment (Number of animals: 10 \u003cem\u003eWT\u003c/em\u003e pre-trt., 10 \u003cem\u003eC9-500\u003c/em\u003e pre-trt.; 10 \u003cem\u003eWT\u003c/em\u003e+ KMP; and 10 \u003cem\u003eC9-500\u003c/em\u003e + KMP). One way ANOVA: P\u0026lt;0.0001*** F=18.15, Sidak’s multiple comparisons: \u003cem\u003eWT\u003c/em\u003e pre-trt. vs \u003cem\u003eC9-500\u003c/em\u003e pre-trt. ***; \u003cem\u003eC9-500\u003c/em\u003e pre-trt. vs \u003cem\u003eC9-500\u003c/em\u003e + KMP (2 weeks) ***. (\u003cstrong\u003ed\u003c/strong\u003e) Hanging wire test showing muscle performance in \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e mice before and after KMP treatment. No significant difference was observed in the \u003cem\u003eC9-500\u003c/em\u003e mice cohort in the timing to first fall after one week of KMP treatment, but significant improvement was observed after 2 weeks of KMP treatment. number of falls, One way ANOVA: P\u0026lt;0.0001*** F=13.84, Sidak’s multiple comparisons: \u003cem\u003eWT\u003c/em\u003epre-trt. vs \u003cem\u003eC9-500\u003c/em\u003e pre-trt. ***; \u003cem\u003eC9-500\u003c/em\u003e pre-trt. vs \u003cem\u003eC9-500\u003c/em\u003e+ KMP (2 weeks) n.s. Time to first fall; One way ANOVA: P\u0026lt;0.0001***, F= 9.246, Sidak’s multiple comparisons: WT pre-trt. vs \u003cem\u003eC9-500\u003c/em\u003e pre-trt. **; \u003cem\u003eC9-500\u003c/em\u003epre-trt. vs \u003cem\u003eC9-500\u003c/em\u003e + KMP n.s. (\u003cstrong\u003ee\u003c/strong\u003e) Schematic depiction of the experimental timeline for in vivo treatment of symptomatic \u003cem\u003eC9-500\u003c/em\u003e mice with salubrinal (Sal), which was performed by intraperitoneal (i.p,) injection of Sal every alternate day to avoid nephrotoxicity in mice. Treatment was stopped at P170, following the animal ethics guidelines as no beneficial effect of the treatment was observed. (\u003cstrong\u003ef\u003c/strong\u003e) Inverse Rotarod test presented as latency to fall, does not show significant improvement in \u003cem\u003eC9-500\u003c/em\u003e mice after Sal treatment (Two-way ANOVA: interaction F (9,92)=0.9044, n.s.; Age F(3,92)=5.3**.; treatment F(3,92)=120***, Sidak’s multiple comparisons test \u003cem\u003eC9-500\u003c/em\u003e + saline vs \u003cem\u003eC9-500 \u003c/em\u003e+ Sal: P140 n.s; P150 n.s.; P160 n.s.; P170 n.s.). n=6 \u003cem\u003eWT\u003c/em\u003e + saline, 6 \u003cem\u003eWT\u003c/em\u003e + Sal, n=8 \u003cem\u003eC9-500\u003c/em\u003e+ saline, n= 8 \u003cem\u003eC9-500\u003c/em\u003e + Sal. (\u003cstrong\u003eg\u003c/strong\u003e) Hanging wire test performed at P140 and P170, showing no significant improvement in \u003cem\u003eC9-500\u003c/em\u003e animals after Sal treatment in the average number of falls within a period of 2 minutes. (One way ANOVA no. of falls: F=34.60***, Sidak’s multiple comparison at P140: \u003cem\u003eWT\u003c/em\u003e + saline vs \u003cem\u003eC9-500 \u003c/em\u003e+ saline ***, \u003cem\u003eWT\u003c/em\u003e + Sal vs \u003cem\u003eC9-500\u003c/em\u003e + Sal ***, \u003cem\u003eC9-500\u003c/em\u003e + saline vs \u003cem\u003eC9-500\u003c/em\u003e + Sal n.s.; at P170: \u003cem\u003eWT\u003c/em\u003e + saline vs \u003cem\u003eC9-500\u003c/em\u003e + saline ***, \u003cem\u003eWT\u003c/em\u003e + \u003cem\u003eSal\u003c/em\u003e vs \u003cem\u003eC9-500\u003c/em\u003e+ Sal ***, \u003cem\u003eC9-500\u003c/em\u003e + saline vs \u003cem\u003eC9-500\u003c/em\u003e + Sal n.s.). (\u003cstrong\u003eh\u003c/strong\u003e) Hanging wire test performed at P140 and P170, showing no significant improvement in the time taken to first fall is observed in \u003cem\u003eC9-500\u003c/em\u003eanimals after Sal treatment (One way ANOVA average time to first fall: F=31.96,***, Sidak’s multiple comparison at P140: WT + saline vs \u003cem\u003eC9-500 \u003c/em\u003e+ saline***, \u003cem\u003eWT\u003c/em\u003e + Sal vs \u003cem\u003eC9-500\u003c/em\u003e + Sal ***, \u003cem\u003eC9-500\u003c/em\u003e + Saline vs \u003cem\u003eC9-500\u003c/em\u003e + Sal n.s.; at P170: \u003cem\u003eWT\u003c/em\u003e + saline vs \u003cem\u003eC9-500\u003c/em\u003e + saline ***, \u003cem\u003eWT\u003c/em\u003e + Sal vs \u003cem\u003eC9-500\u003c/em\u003e + Sal ***, \u003cem\u003eC9-500\u003c/em\u003e + saline vs \u003cem\u003eC9-500\u003c/em\u003e + Sal n.s.).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5190511/v1/2f16da81d70d973f258e6f75.png"},{"id":71870839,"identity":"82b28518-d168-425b-8036-ecef130671e6","added_by":"auto","created_at":"2024-12-19 10:18:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1571006,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn silico modeling reveals preferential Kaempferol binding to the nucleotide-binding domain of GRP75.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Table depicting Vina scores, and cavity information of the docking simulation pose for GRP75 nucleotide-binding domain (NBD) and Kaempferol (Kmp).\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eb\u003c/strong\u003e) The structure of Kmp was uploaded to CB-Dock (http://clab.labshare.cn/cbdock/php/dockingresult.php) for analysis of the docking potential with GRP75. The crystal structure of the human GRP75 nucleotide-binding domain (PDB ID: 6NHK), the active site is colored white (carbon), red (oxygen), blue (nitrogen), and yellow (sulphur). The crystal pose of the ligand KMP in the cavity sized 3793 is colored white (hydrogen), grey (carbon), and red (oxygen). (\u003cstrong\u003ec\u003c/strong\u003e) Table depicting Vina scores, and cavity information of the docking simulation pose for GRP75 nucleotide-binding domain (NBD) and 17-AAG. (\u003cstrong\u003ed\u003c/strong\u003e) The crystal structure of the human GRP75 nucleotide-binding domain (PDB ID: 6NHK), the active site is colored white (carbon), red (oxygen), blue (nitrogen), and yellow (sulphur). The crystal pose of the ligand 17-AAG in the cavity sized 3793 is colored white (hydrogen), grey (carbon), and red (oxygen). Note the comparatively lower Vina score for 17-AAG binding to GRP75-NBD compared to that of KMP within the same pocket, suggesting a stronger fit of KMP in the NBD domain of GRP75. (e) Venn diagram depicting the number of overlapping human proteins that are targeted by both KMP and 17-AAG. The PharmMapper database was used to predict the targets of Kmp and 17-AAG. This mapping predicted a large overlap of targets shared (cut-off z-score: 0.5) by the two compounds, including HSP90A. Both ligands did not target GRP75. See Suppl. Table 1 for the complete list of overlapping targets.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5190511/v1/a62c25d2bfdd078cad27441b.png"},{"id":71870841,"identity":"16e1c9c6-19f2-4147-aec8-e9b1becf8867","added_by":"auto","created_at":"2024-12-19 10:18:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":773099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaempferol enhances IP3R and VDAC1 association promoting mitochondrial Ca2+ uptake\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u0026nbsp;\u003c/strong\u003eRepresentative images of proximity ligation assay (PLA) between IP3R and VDAC1 in \u003cem\u003eC9-500\u003c/em\u003e + saline and \u003cem\u003eC9-500\u003c/em\u003e + KMP treated mice, showing a notable increase in the number of puncta in \u003cem\u003eC9-500\u003c/em\u003e + KMP treated animals (One- way ANOVA: F= 100.0, P\u0026lt;0.0001***, Sidak’s multiple comparison test: WT + saline vs \u003cem\u003eC9-500\u003c/em\u003e + saline, t=5.303, ***; \u003cem\u003eC9-500\u003c/em\u003e + saline vs \u003cem\u003eC9-500\u003c/em\u003e + KMP, t=16.31, ***). Scale bar = 10 μm, n = 3-4 mice per genotype/treatment.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Baseline and stimulated mitochondrial Ca2+ uptake traces in \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e cortical neurons (dotted lines) and after KMP treatment (bold lines). KMP treatment significantly improves Ca2+ uptake in the mitochondria of \u003cem\u003eC9-500\u003c/em\u003e cortical neurons (number of neurons \u003cem\u003eWT\u003c/em\u003e: 22, \u003cem\u003eC9-500\u003c/em\u003e: 24, \u003cem\u003eWT\u003c/em\u003e+KMP:21, \u003cem\u003eC9-500\u003c/em\u003e+KMP: 21; multiple t-test 100 seconds \u003cem\u003eC9-500\u003c/em\u003e mean=0.967, \u003cem\u003eC9-500\u003c/em\u003e + KMP mean=2.285, P\u0026lt;0.0001). (\u003cstrong\u003ec\u003c/strong\u003e) Representative confocal images of spinal cord immunolabeled for GRP75, shows a significant increase in \u003cem\u003eC9-500\u003c/em\u003e and \u003cem\u003eWT\u003c/em\u003e motor neurons treated with KMP (One-way ANOVA F= 102.8, P\u0026lt;0.0001***, Sidak’s multiple comparison test: \u003cem\u003eWT\u003c/em\u003e saline vs \u003cem\u003eC9-500\u003c/em\u003e saline t=6.203, ***; \u003cem\u003eC9-500\u003c/em\u003e saline vs \u003cem\u003eC9-500\u003c/em\u003e+KMP t=16.42, ***). Scale bars: 30 μm. (\u003cstrong\u003ed\u003c/strong\u003e) List of proteins found to specifically interact with GRP75 in \u003cem\u003eC9-500\u003c/em\u003e ventral spinal cord but not in \u003cem\u003eWT\u003c/em\u003e after mass spectroscopy (MS) analysis of immunoprecipitation for GRP75. Specifically, in red are highlighted GRP75 and VDAC1 as an internal control of the experiment (present in both \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e samples), in green is highlighted ATP5J (ATP synthase factor 6 of the mitochondria), whose interaction with GRP75 was detected in \u003cem\u003eC9-500\u003c/em\u003e ventral spinal cord lysates, which are enriched for motor neurons. (\u003cstrong\u003ee\u003c/strong\u003e) PLA between ATP5J and GRP75 in \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e at P125 and P200, showing notable increase in number of puncta in \u003cem\u003eC9-500\u003c/em\u003e at P125 when motor neurons express high levels of GRP75 (Unpaired t-test P125 \u003cem\u003eWT\u003c/em\u003e vs \u003cem\u003eC9-500\u003c/em\u003e, t=25.28, ***; P200 \u003cem\u003eWT\u003c/em\u003e vs \u003cem\u003eC9-500\u003c/em\u003e, t=3.58, ***). (\u003cstrong\u003ef\u003c/strong\u003e) Representative confocal images of \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e motor neurons stained for GRP75 and ATP5J. Motor neurons expressing high levels of GRP75 also express high levels of ATP5J. Right: Q.A of ATP5J expression across different disease stages: One-way ANOVA F=95.75, P\u0026lt;0.0001***; Sidak’s multiple comparison test: P30 \u003cem\u003eWT\u003c/em\u003e n= 26 vs P30 \u003cem\u003eC9-500\u003c/em\u003e n=46, t=0.4823, n.s.; P125 \u003cem\u003eWT\u003c/em\u003e n=42 vs P125 \u003cem\u003eC9-500\u003c/em\u003e n=59, t=16.29***, P200 \u003cem\u003eWT\u003c/em\u003e n= 27 vs P200 \u003cem\u003eC9-500\u003c/em\u003e n=27, t=3.795***. Scale bars: 30 μm. (\u003cstrong\u003eg\u003c/strong\u003e) Representative images of ATP5J staining depicting the increased intensity of ATP5J expression in \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e treated with KMP (One-way ANOVA: F= 90.48, P\u0026lt;0.0001***, Sidak’s multiple comparison test: \u003cem\u003eWT\u003c/em\u003e saline vs \u003cem\u003eWT\u003c/em\u003e+KMP t=4.928, ***; \u003cem\u003eC9-500\u003c/em\u003e saline vs \u003cem\u003eC9-500\u003c/em\u003e + KMP t=15.55, ***). Scale bars: 10 μm.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5190511/v1/d979f739bedc0026a52f83ae.png"},{"id":75351313,"identity":"8cd6b59a-ac37-4547-8d11-74e62846311b","added_by":"auto","created_at":"2025-02-03 16:09:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11945451,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5190511/v1/3ab55951-22bb-4ecc-b5e0-d9a3339726bd.pdf"},{"id":71870846,"identity":"f7a849b3-c16c-4b60-911f-1bf98fe63506","added_by":"auto","created_at":"2024-12-19 10:18:33","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":99938,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDemographic information about iPSC and fibroblast lines from C9ORF72-ALS/FTD patients and healthy control (CTRL) employed to generate iMNs and dNs.\u003c/p\u003e","description":"","filename":"Suppl.Table1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5190511/v1/229170264c6a52ae7584e45c.pdf"},{"id":71872522,"identity":"15c04d44-4727-4f6a-8855-8fb65a320d0b","added_by":"auto","created_at":"2024-12-19 10:34:32","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":609188,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eList of overlapping human proteins targeted by both kaempferol and 17-AAG\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe interactome of GRP75 as revealed by mass spectrometry analyses, note the strong interaction between the established association of GRP75 with VDAC1.\u003c/p\u003e","description":"","filename":"SupplTable23.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5190511/v1/3a98864d2bebcdb41783688c.pdf"},{"id":71872523,"identity":"9c7e08bf-790f-4cf0-a397-d88803060757","added_by":"auto","created_at":"2024-12-19 10:34:33","extension":"eps","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17582010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative images: 2-week-old iMNs derived from Control (1) immunolabeled for the neuronal marker MAP2 and the motor neuron marker ChAT. Scale bars: 30 μm, zoom: 15 μm.\u003c/p\u003e","description":"","filename":"Suppl1.eps","url":"https://assets-eu.researchsquare.com/files/rs-5190511/v1/5bbbe556b50c1e51872edd78.eps"},{"id":71870847,"identity":"22ea534c-83a3-41e1-8710-58ecf57873dc","added_by":"auto","created_at":"2024-12-19 10:18:34","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":49757026,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary video\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInverse rotarod measurement showing ameliorated motor coordination in \u003cem\u003eC9-500\u003c/em\u003e mice after Kaempferol treatment\u003c/p\u003e","description":"","filename":"Suppl.video1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5190511/v1/6d0a26ec18265b87463bd847.mp4"},{"id":71870842,"identity":"402e6c1e-016b-42c8-bfe9-9c16b179063a","added_by":"auto","created_at":"2024-12-19 10:18:32","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":4303872,"visible":true,"origin":"","legend":"","description":"","filename":"PilottoetalGraphicalabstractcopylegend.tif","url":"https://assets-eu.researchsquare.com/files/rs-5190511/v1/1f8349c6054f116147cfb4d4.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Kaempferol enhances ER-mitochondria coupling and protects motor neurons from ER stress and mitochondrial dysfunction in C9ORF72- ALS","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCurrent research has highlighted that dysregulated ER stress and associated unfolded protein response (UPR) signaling are the leading cellular impairments contributing to the main pathogenic mechanism in neurodegeneration [1\u0026ndash;4]. In the context of amyotrophic lateral sclerosis (ALS), motor neurons are selectively prone to ER stress, influencing disease manifestation and kinetics[5\u0026ndash;7]. Importantly, a hexanucleotide repeat expansion (HRE) in the first intronic region of the C9ORF72 gene accounts for over 40% of all known familial and 10% of known sporadic forms of ALS. In healthy individuals, approximately 20- 24 copies of the GGGGCC HRE are observed within the first intron of the \u003cem\u003eC9ORF72\u0026nbsp;\u003c/em\u003egene. However, in disease, this GGGGCC sequence is expanded and can range from hundreds to thousands of repeats. This specific intronic HRE leads to the development of three mutually non-exclusive pathological hallmarks, eventually resulting in the appearance of a spectrum disorder; \u003cem\u003eC9ORF72\u003c/em\u003e ALS/frontal temporal dementia (FTD). Firstly, the HRE has been shown to cause haploinsufficiency of the \u003cem\u003eC9ORF72\u003c/em\u003e gene, leading to reduced \u003cem\u003eC9ORF72\u003c/em\u003e RNA and protein expression. Secondly, there is a gain of toxic function due to the bidirectional transcription of the GGGGCC HRE resulting in the production of toxic G4C2 and G2C4 repeat RNA species. Lastly, there is a gain of toxic function via the non-ATG mediated RAN translation of repeat RNAs to produce five different toxic dipeptide repeat proteins (Poly(GA), Poly(GR), Poly(GP), Poly(PR), and Poly(PA)), which act on and damage multiple cellular processes [8\u0026ndash;10].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC9ORF72\u003c/em\u003e induced pluripotent stem cells (iPSC)-derived motor neurons, exhibit increased endoplasmic reticulum (ER) stress, together with reduced mitochondrial membrane potential, concomitant impairment in calcium homeostasis and reduced levels of the antiapoptotic protein Bcl-2 [6,7,11]. Additionally, C9ORF72 is majorly a cytosolic protein, which has also been shown to localize to the mitochondrial inner membrane, where it has a function in controlling the process of oxidative phosphorylation, thus maintaining, and regulating cellular energy production and balance [12]. Thus, emerging evidence suggests that ER stress signaling and mitochondrial dysfunction are intricately associated with the pathophysiological manifestations linked to \u003cem\u003eC9ORF72\u003c/em\u003e-ALS. One likely region of membrane contacts where the mitochondria are reversibly tethered to the ER are regions termed \u0026ldquo;Mitochondria-associated membranes (MAMs),\u0026rdquo; which play a crucial regulatory role in the supply of calcium from the ER to mitochondria. These sites are critical for the rapid calcium uptake by mitochondria through voltage-dependent anion channels (VDACs) located at the outer mitochondrial membrane (OMM) [13]. Moreover, MAMs have been implicated in the regulation of calcium homeostasis, mitochondrial function, autophagy, apoptosis, and glucose homeostasis [14]. Within the context of ALS, mutations within MAM localized and functioning proteins such as VAPB and Sig1R are known to cause familial ALS (fALS). The point mutation P56S in \u003cem\u003eVAPB\u003csup\u003eP56S\u003c/sup\u003e\u003c/em\u003e causes increased binding to the mitochondrial protein tyrosine phosphatase interacting protein 51 (PTPIP51), which causes reduced ER-mitochondria contacts, enhanced Ca\u003csup\u003e2+\u003c/sup\u003e release from the ER and aberrant MAM morphology, and mitochondrial dysfunction [15].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA recent study from our group revealed the existence of an ER stress-mediated adaptive response in \u003cem\u003eC9ORF72\u003c/em\u003e patient-derived motoneurons (MNs), which was observed by the early yet transient increase in the expression of the MAM-localized chaperone GRP75. Transient GRP75 expression augmented ER-mitochondrial association, boosting mitochondrial function, and sustaining cellular bioenergetics during the initial stage of disease, thereby neutralizing early mitochondrial deficits. The gradual emergence of Poly(GA) aggregates led to the sequestration of GRP75, causing a subsequent loss of function at the MAM, resulting in mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake impairments and mitochondrial dysfunction in both human patient-derived and rodent motor neurons [7]. Similarly, another recent study showed that dysfunctional ER-mitochondria signaling and the disruption of VAPB-PTPIP51 tethers in \u003cem\u003eC9ORF72\u0026nbsp;\u003c/em\u003epatient-derived neurons and mutant \u003cem\u003eC9orf72\u003c/em\u003e transgenic mice occurred presymptomatically and contributed to the pathogenic process. These impairments were linked to the expression of DPRs, which disrupted the VAPB-PTPIP51 interaction at the ER-mitochondria contacts and that may involve activation of glycogen synthase kinases-3\u0026beta; (GSK3\u0026beta;), a known negative regulator of VAPB-PTPIP51 binding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompounds harboring the potential to simultaneously target both ER stress and mitochondrial function hold great promise as future therapeutic targets in neurodegenerative diseases and more so in ALS.\u0026nbsp;Kaempferol is\u0026nbsp;a naturally occurring dietary flavanol,\u0026nbsp;present in fruits and vegetables and has been shown to possess ER stress-inhibitory activity in cultured mammalian cells [23], anti-neuroinflammatory activity in rat models of ischemic stroke [24], mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uniporter channel activator [25] and ameliorated energy metabolism and symptomatic behavior in a rat model of traumatic brain injury [26]. We here identify that kaempferol exerts a neuroprotective effect on \u003cem\u003eC9ORF72\u003c/em\u003e ALS neurons by inhibiting ER stress and sustaining mitochondrial function. Notably, in \u003cem\u003eC9ORF72\u003c/em\u003e-ALS human patient-iPSC-derived motor neurons and directly fibroblast-converted neurons kaempferol counteracted mitochondrial deficits and provided neuroprotection in the presence of elevated ER stress, thus indicating the probable translational potential of kaempferol. Additionally, in vivo, treatment with kaempferol in the symptomatic rodent model of \u003cem\u003eC9ORF72\u003c/em\u003e-ALS was successful in reducing pathological hallmarks and concomitantly delaying the progression of behavioral pathology. Lastly, in silico modeling identified a novel prominent binding affinity of kaempferol to chaperone GRP75 and mechanistically the neuroprotective mechanism of kaempferol is through the iP3R-VDAC1 pathway via the modulation of GRP75 and ATP5J expression.\u0026nbsp;\u003c/p\u003e"},{"header":"EXPERIMENTAL PROCEDURES","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eMice strains\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe \u003cem\u003eC9-500\u003c/em\u003e BAC mouse line (FVB/NJ-Tg(C9orf72)500Lpwr/J) carrying a human \u003cem\u003eC9ORF72\u003c/em\u003e gene under a human promoter with ~\u0026thinsp;500 hexanucleotide repeats described in (Liu et al., 2016) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] was purchased from Jackson Laboratory (RRID: IMSR_JAX:029099) and kept in heterozygosis crossed with (non-carrier) mice FVB/NJ (Janvier labs, SC-FVBN-F). Our colony displays an acute phenotype, which is observed in 25\u0026ndash;30% of females with a median life span of 105 days whereas the remaining female and male mice exhibit a slow-progressing phenotype with females having a median life span of approximately 250 days and males 260 days. Long-range PCR was regularly done to identify repeat length-matched cohorts. Kaempferol treatment included both genders and only slow-progressing mice. All behavior and survival assays were performed in repeat length-matched (700\u0026ndash;800 repeats). Animal care, housing, ethical experimental usage, and procedures were in accordance with the Swiss Veterinary Law guidelines, and the study was approved by the Animal Commission of Canton of Bern, Switzerland, license number BE-35/17, BE-82/18\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo/ in vitro drug treatments\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eKaempferol (Sigma-Aldrich, K0133) at 10mg/kg was administered via i.p. injections daily to both wild type (\u003cem\u003eWT)\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e for the first two weeks, followed by every alternate day administration chronically until end-stage P240. \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e mice were treated every alternate day with Salubrinal (Enzo life sciences, 270-428-M005) at a dosage of 1mg/Kg or saline as control. iMNs were treated with Tunicamycin (TU) (1 \u0026micro;g/ml) for 24h before further analysis. For Kaempferol (KMP) treatments, iMNs were treated with 10 \u0026micro;g/ml kaempferol for 2h or 48h.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReverse Rotarod and Hanging wire test\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eRotarod apparatus (Ugo Basile, Comerio, Italy) was used to assess general motor performance as well as motor coordination and not only endurance as usually assessed by common accelerated rotarod protocols, during the light phase of the 12h light/12h dark cycle. For the inverse rotarod, the rod accelerated from 15 to 33 rpm in 10 seconds, then 33 rpm to 15 rpm in 10 seconds, followed by inversing the direction of the rod and a repeat of the same procedure. One trial lasted a maximum of 110 seconds, with 3 min resting in between trials. Three trials every 10 days were performed for overexpressing \u003cem\u003eC9-500\u003c/em\u003e animals and respective controls, while initially, KMP-treated animals were tested for 4 consecutive days. Mice were trained on the rotating rod at a fixed speed for 3 days before the baseline recording. All measurements were done blindly without knowing the genotype of the mice. The hanging wire test was performed as previously described [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Briefly: mice were placed on a cage top and once the animal was stable on top of the grid, the cage top was gently inverted, and the latency of the first fall is recorded. All the animals are placed on the inverted cage top each time they fall to assess the average number of falls. The test last for 120 seconds.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eiPSC differentiation into motor neurons (iMNs)\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe healthy and \u003cem\u003eC9ORF72-\u003c/em\u003eALS iPSCs were obtained from iPSC bank (Biomedicum Stem Cell Center, GoEditStem platform, HiLIFE, Helsinki, Finland and the iPSC Core, Cedar Sinai, USA). iPSCs were cultured in GeltrexTM (ThermoFischer) coated plates in mTeSRTM1 (StemCell technologies) media. MN differentiation was performed as previously described [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] with some modifications. Human iPSCs were dissociated to single cells using Accutase (StemCell technologies) and seeded at 3X106 onto 10cm plate with N2B27 differentiation medium (Advanced DMEM/F12:Neurobasal (1:1) medium, 1% Pen/strep (Gibco), 1% GlutaMAX (Gibco), 0.1mM 2-mercaptoethanol (Gibco), 1X B27 supplement (Gibco), 1X N2 supplement (Gibco), supplemented with 10ng/ml basic fibroblast growth factor ((StemCell technologies), 20 \u0026micro;M SB431542 (StemCell technologies), 0.1 \u0026micro;M LDN193189 (StemCell technologies), 3 \u0026micro;M CHIR99021 (StemCell technologies), 10 \u0026micro;M L-Ascorbic Acid (L-AA; Sigma) and 1X Revitacell supplement (Gibco)) to initiate the formation of embryoid bodies (EBs). On day 2 media patterning of EBs was induced by the addition of media supplemented with 100nM all-trans retinoic acid (RA; sigma) and 500nM Smoothened Agonist (SAG; StemCell technologies). EBs were pelleted and fed with fresh media on every alternate day until day 14. 10ng/ml Brain derived neurotrophic factor (BDNF; StemCell technologies) was added from day 7 while 10 \u0026micro;M DAPT (StemCell technologies) was added from day 9. EBs were dissociated using trypsin on day 16 and triturated with ice cold cell trituration and wash medium (1X PBS (Gibco), 0.45% Glucose, 0.1% Bovine Serum Albumin (BSA; Sigma), 2mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.8mM EDTA (Invitrogen), 2.5% Fetal Bovine Serum (FBS; Sigma), 1X N2 supplement, 1X B27 supplement and DNAse). Triturated EBs were then plated on poly-ornithine/laminin (Sigma) coated plates in MN feeding medium (Neurobasal medium (Gibco), 1X glutaMAX, 1X Non-essential amino acid (NEAA, Gibco), 0.1mM 2-mercapthoethanol, 1X N2 supplement, 1X Pen/strep, 1X B27 supplement, 10ng/ml glial cell derived neurotrophic factor (GDNF; StemCell technologies), BDNF 10ng/ml, 10ng/ml insulin-like growth factor (IGF-1; StemCell technologies), 10ng/ml Ciliary neurotrophic factor (CNTF; StemCell technologies), 100nM RA and 10 \u0026micro;M AA and kept at incubator at 37\u0026deg;C and 5% CO2 for further maturation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eGeneration of direct fibroblast-induced neurons (dNs)\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFibroblast collection: skin punches were collected, and fibroblasts were grown in fibroblast media (10% FBS (Gibco), 1% anti-anti (Gibco) in DMEM Glutamax (Gibco)) for up to 1 month, passaging once every 1\u0026ndash;2 weeks. In addition, this study used a fibroblast sample from the NINDS Repository, as well as clinical data. NINDS Repository sample numbers corresponding to the samples used are: AG08620. Patient skin-derived fibroblasts were directly reprogramed into neural progenitor cells (NPCs) as previously described [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Briefly, 100,000\u0026ndash;200,000 fibroblasts were seeded into a fibronectin (5 \u0026micro;g/mL, Millipore) coated 6-well plate and cultured in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. Next day, fibroblasts were transduced with a retroviruses for SOX2, cMyc, KLF4, and OCT3/4. 24 h later virus was removed, and media replaced with fresh fibroblast media (10% FBS (Gibco), 1% anti-anti (Gibco) in DMEM Glutamax (Gibco)). After a 24-h rest period, media of transduced fibroblasts was changed to a neuralizing media (1% B27 (Gibco), 1% N2 (GIBCO), 1% anti-anti (Gibco), 20 ng/mL FGF2 (peprotech), 20 ng/mL EGF (peprotech), and 5 \u0026micro;g/mL heparin (Sigma)). Cells were cultured in this media until converted into NPC. NPCs were cultured in (1% B27 (Gibco), 1% N2 (Gibco), 1% anti-anti (Gibco), 20 ng/mL FGF2 (peprotech)). Induced direct neuron generation (dNeus): patient and healthy fibroblasts were directly converted to neurons using small molecules as previously described [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Briefly, 10 cm plates were coated overnight with polyornithine (10 \u0026micro;g/mL, Sigma) in borate buffer. Next day, plates were washed with DPBS and coated with laminin (5 \u0026micro;g/mL, Invitrogen) and fibronectin (2.5 \u0026micro;g/mL, Millipore Sigma) in DMEM/F12 at 37\u0026deg;C for 2 h. Fibroblast cells (850,000) were seeded onto the plates in culture medium for 1 day. The cells were transferred to neuronal induction medium (DMEM/F12: Neurobasal (Gibco) [1:1] with 0.5% N-2 (Gibco), 1% B-27 (Gibco), 100 \u0026micro;M cAMP (Sigma), and 20 ng/mL bFGF (Peprotech)) with the following chemicals: VPA (0.5 mM, Sigma), CHIR99021 (3 \u0026micro;M, Axon medchem), repsox (1 \u0026micro;M, BioVision), forskolin (10 \u0026micro;M, Tocris), SP600125 (10 \u0026micro;M, Sigma), GO6983 (5 \u0026micro;M, Sigma) and Y-27632 (5 \u0026micro;M, Sigma). Half the medium was changed after 3 days with fresh induction medium. On the fifth day, cells were switched to neuronal maturation medium (DMEM/F12: Neurobasal [1:1] with 0.5% N-2 (Gibco), 1% B-27 (Gibco), 100 \u0026micro;M cAMP (Sigma), 20 ng/mL bFGF (Peprotech), 20 ng/mL BDNF (Gibco) and 20 ng/mL GDNF (Gibco)) with the following chemicals: CHIR99021 (3 \u0026micro;M), forskolin (10 \u0026micro;M) and SP600125 (10 \u0026micro;M).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eImmunofluorescence on iMNs\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eiMNs plated on coverslips were fixed using 4% paraformaldehyde PFA for 15min and blocked for 1h with 3% bovine serum albumin (BSA) and 0.1% TritonX-100 in phosphate buffered saline PBS. After blocking, neurons were incubated with the following primary antibody: anti-BiP (Abcam, ab21685, 1:500), goat anti-ChAT (Millipore, AB144P, 1:500), chicken anti-MAP2 (Sigma Aldrich, AB15452, 1:500) (Thermo fisher scientific, Invitrogen PA5-29202, 1:500) in blocking buffer overnight at 4\u0026deg;C. After washing three times with PBS, cells were incubated in blocking buffer with Alexa Fluor fluorescently labeled secondary antibodies and DAPI for 1h at room temperature. Cells were then washed with PBS and mounted on glass slides. Images were acquired with a confocal microscope Olympus FluoViewTM FV1000 (Olympus) fitted with a 20X or 40x air objective and 60x immersion oil objective.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence and immunohistochemistry of rodent tissue\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMice were transcardially perfused with 4% PFA in 1X PBS; brain, cerebellum and lumbar spinal cord were isolated and kept overnight at 4\u0026deg;C in the same fixative solution, followed by 30% sucrose in PBS for cryoprotection until samples were used. After embedding in Tissue Tek O.C.T compound (Bio system, 4583). Spinal cord (50 \u0026micro;m), sections were cut using a cryostat. Antibodies used for immunofluorescence were: rabbit anti-GRP78/BiP (1:500, Abcam, ab21685), mouse anti-KDEL/BiP (1:500, Enzo Life Science, SPA-827), rabbit anti-P\u003csub\u003ei\u003c/sub\u003e-EIF2α (1:25, Cell Signaling, 3597L), goat-anti ChAT (1:1000, Millipore, AB144P), mouse anti-GRP75 (1:200, Abcam, ab2799), rabbit anti-GRP75 (1:200, Abcam, ab53098), rabbit anti-GFAP (1:500, Abcam, ab7260), mouse anti-8-hydroxy-guanosine (8-OHdG), (1:500, Abcam, ab62623) mouse anti-NeuN clone 60 (1:1000, Millipore, MAB377). Heat-mediated antigen retrieval was performed using Sodium citrate buffer 10mM pH 6 for GRP75 and ATP5J staining. Sections were kept for 2h in PBS solution containing 0.05% Triton X-100 and 10% normal donkey serum (NDS, Jackson immunoresearch, 017-000-121) after the antibodies were applied in PBS, 3% normal donkey serum (NDS), 0.05% Triton X-100, and incubated overnight (for brain) and for two days for spinal cord at 4\u0026deg;C. Sections were then briefly washed with PBS and incubated for 120 min at room temperature, with appropriate combinations of secondary antibodies from Invitrogen.\u003c/p\u003e \u003cp\u003eSpinal cord sections for immunohistochemistry were processed as follows: sections were treated with heat-mediated antigen retrieval using sodium citrate buffer 10mM pH 6 and immersed in 3% H2O2 in PBS for 20 min to block endogenous peroxidase activity. This was followed by a blocking step in PBS containing 0.05% Triton X-100 and 10% NDS and incubated overnight at 4\u0026deg; with goat anti-ChAT (1:500, Millipore, AB144P) antibody diluted in the same blocking solution. The next day the sections were incubated with the appropriate biotinylated secondary antibody (1:500) followed by 1h incubation in PBS solution containing biotin-avidin complex (1:100, Vector Labs), finally, the 3,3\u0026prime;-diaminobenzidine (DAB) reaction was developed. The glass slides were dehydrated via ascending concentrations of ethanol and rinsed in xylene before being cover slipped. Images were acquired using an Olympus microscope (BX51).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMuscles histology\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMice were deeply anesthetized with isoflurane and decapitated before the gastrocnemius muscles were dissected. Muscles were quickly frozen in liquid nitrogen-cooled isopentane. 8\u0026micro;m thick sections were cut on a cryostat and placed directly onto coverslips. For hematoxylin \u0026amp; eosin (H\u0026amp;E) staining, muscle sections were stained with Mayers hematoxylin for 5min, followed by three 10min washes in deionized H2O. Muscles were then placed in eosin G for 1min, then rinsed in 70% ethanol. The tissue was then dehydrated in a series of ethanol washes (70%, 95%, 100%), rinsed in xylene, then mounted on a glass slide. NADH staining was performed by incubating muscle sections in 0.2M Tris buffer containing Nitrotetrazolium Blue (Sigma) and β-nicotinamide adenine dinucleotide (NADH, Sigma) for 30min at 37\u0026deg;C. Following three washes in deionized H2O, coverslips were mounted onto a glass slide. ATP staining pH4.3 and 9.4 were performed by incubating the slides in pre incubating solution pH4.3 per 10min at 4\u0026deg;, containing 0.1M Na acetate buffer with 10mM EDTA. Follow by three 5min washes in deionized H20, to be then incubated in the incubation solution: for pH4.3 containing sodium acetate 0.1M, EDTA 0.1M pH8 and for pH9.4 containing 0.1M glycine (Sigma, 50046), 5mg ATP and DTT in NaCl buffer with CaCl2 per 30min at 37\u0026deg;C. Coverslips were then washed with deionized H2O 3 times every 5min, incubated with Cobalt chloride 2% 3 times per 1min at room temperature, rinsed with deionized water and incubated with ammonium sulfide 1% at room temperature for 30 second. Coverslips were rinsed under running water and dehydrated in a series of ethanol washes (70%, 95%, 100%), rinsed in xylene, then mounted on a glass slide.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eImaging and image analysis\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eConfocal images were acquired using a Leica SP5 (Leica Microsystems) fitted with a 20X, 40 and 63X oil objective, Leica SP8 (Leica Microsystems) fitted with a 63X oil objective, or Olympus Fluoview 1000-BX61 (Olympus, Tokyo) microscope, fitted with a 20X, 40X air objective or 60X immersion oil objective. All images were processed using Imaris software version 7.6.3 or Fiji. For the analysis of BiP, P\u003csub\u003ei\u003c/sub\u003e-EIF2α, GRP75, GFAP and ATP5J labeling intensities, data were acquired using identical confocal settings, with signals at the brightest cells being non-saturated, and that background levels outside motor neuron pools were still detectable. Images were analyzed quantitatively using FiJi or Imaris. Signal intensity values for the antigen of interest were calculated over several consecutive lumbar spinal cord Z-stack spaced 0.5 \u0026micro;m, after background subtraction from every different channel. For the calculation of motor neuron percentages, Choline Acetyltransferase (ChAT) positive motor neurons were examined and signal intensity values for the antigen of interest within these ChAT positive motor neurons were calculated in 3\u0026ndash;4 animals. Lowest signals had values of below 50 and high-intensity neurons exhibited labeling values up to 255. Signal values below 50 in the case of BiP in \u003cem\u003eWT\u003c/em\u003e animals were counted as basal expression. To count the motor neuron numbers, a cell counter plugin from Fiji was used. Imaris software was used to reconstruct the 3D isosurface of Poly(GA) aggregates volume. Fiji software was employed to analyze the mitochondria-ER contacts and sphericity for 3view EM images.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProximity ligation assay (PLA) in perfused mouse spinal cord tissue\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePLA was adapted from Gomes et al., [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In short, 50 \u0026micro;m free-floating spinal cord sections were mounted on a frost slide (Huberlab, 10.0120.04) and air-dried. Slides were rinsed in 0.01% Triton X-100 in 1\u0026times; PBS per 12 minutes followed by 3 washes in 1X PBS (3 per 5 minutes). Slides were kept in a humid chamber and blocking solution was added (Duolink PLA probe kit, DUO92008, Merck) for 1h at 37\u0026deg;C. Subsequently, slides were incubated with the following primary antibodies (rabbit anti-IP3R, ABCAM, ab5804; mouse anti-GRP75, ABCAM, ab53098; rabbit anti-GRP75, ABCAM, ab227215; rabbit anti-ATP5J, Thermo Fisher Scientific, PA5-29202) in a humidity chamber for 2 nights at 4\u0026deg;C. Slides were rinsed in buffer A (Duolink In Situ Wash Buffers, Fluorescence DUO82049) (3 times every 5 minutes) and incubated with PLA probes (Probe Anti-mouse MINUS, DUO92004 and Probe anti-rabbit PLUS, DUO92002 ) at a working concentration of 1:10 for 1h at 37\u0026deg;C. Ligation (Duolink In Situ Detection Reagents Red, DUO92008) was performed at 37\u0026deg;C per 45 minutes followed by 3 washes in buffer A (3 per 5 minutes). The amplification (Duolink In Situ Detection Reagents Red, DUO92008) step was performed at 37\u0026deg;C in a dark humidity chamber per 100 minutes. Finally, sections were rinsed in buffer B (Duolink In Situ Wash Buffers, Fluorescence DUO82049) (2 per 10 minutes) followed by a third wash in 0.001% buffer B. Slides were left to dry and coverslips were mounted with Duolink In Situ Mounting Medium with DAPI (DUO82040), and edges were sealed with nail polish. Sections were kept at -20 overnight before confocal imaging.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eColorimetric staining of mitochondria complexes\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMice were rapidly perfused with PBS, fresh spinal cord was removed, embedded in O.C.T compound, and quickly frozen on dry ice. 20 \u0026micro;m thick sections were cut with a cryostat and transferred onto an adhesive glass slide. Sections were incubated for 30 min at 37 C in freshly prepared appropriate complex histochemistry media: (1) Complex I: 1.23mg/ml (1.5 mM) Nitroblue tetrazolium (NBT; N6876, Sigma) and 0.625 mg/ml NADH (N8129, Sigma) were mixed in PBS, pH\u0026thinsp;=\u0026thinsp;7.4. Complex IV: 0.5mg/ml 3,3\u0026rsquo;-diaminobenzidine tetrahydrochloride (DAB, D7304, Sigma), 1mg/ml cytochrome c (C2506, Sigma), and approximately 2\u0026micro;g/ml (a few crystals) bovine catalase (C9322, Sigma) were mixed in PBS, pH\u0026thinsp;=\u0026thinsp;7.4. After the staining of each complex, sections were washed 3\u0026times;10 min in PBS before being dehydrated for 4min in 70% ETOH, 4min in 90% ETOH, 10 min in 100% ETOH and 10 min in Xylol and mounted with Eukitt. Images were acquired using an Olympus microscope (BX51).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial calcium imaging\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMitochondrial calcium imaging for cortical neurons was adapted from [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In brief, cells were incubated for 45 min with the staining solution containing: 156mM NaCl, 3mM KCl, 2mM MgSO\u003csub\u003e4\u003c/sub\u003e, 1.25mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 10mM D-glucose, 2mM CaCl\u003csub\u003e2\u003c/sub\u003e and 10mM HEPES pH 7.35, 5 \u0026micro;g/ml (w/v) Fluo-4, AM, 10 \u0026micro;M Verapamil. After a brief wash with Ca\u003csup\u003e2+\u003c/sup\u003e free HBSS, cells were incubated for 10 min with the intracellular solution containing: 6mM NaCl, 130mM KCl, 7.8mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.4mM CaCl\u003csub\u003e2\u003c/sub\u003e, 2mM EGTA, 10mM HEDTA, 2mM malate, 2mM glutamate, 2mM ADP, 20mM HEPES pH 7.1, 25 \u0026micro;g/ml (w/v) digitonin and 1 \u0026micro;M thapsigargin. An Olympus Fluoview 1000-BX61 (Olympus, Tokyo) microscope fitted with a 40x water immersion objective was used to acquire images every 20 seconds, the first 60 seconds were considered as baseline, afterwards 50mM KCl was added to the imaging solution to depolarize the cells. Neurons were treated 10 \u0026micro;g/ml of kaempferol for 1h prior staining solution passage, moreover to maintain their effect the KMP was also added to the staining solution before starting the imaging for a total time of treatment of 1h and 45min. Images were analyzed using Fiji, multiple ROI were chosen inside the cytosol of the cells and the fluorescence intensity was calculated over the different frame. To calculate ΔF, the median intensity values are divided by the average of the first 60 seconds of recording (F0) per single ROI.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eStatistical significances throughout the paper were evaluated by two-tailed, paired and unpaired Student\u0026rsquo;s t test and one or two-way ANOVA. Post ANOVA Bonferroni, Sidak, Tukey\u0026rsquo;s test was used to evaluate statistical significance throughout the paper as indicated in the respective figure legend. Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Error bars: SEM *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eKaempferol protects human iPSC-derived motor neurons (iMNs) and fibroblast-induced neurons (iNs)\u003c/h2\u003e \u003cp\u003eWe started the study by evaluating the neuroprotective action of kaempferol, a compound known to act on UPR and oxidative stress. Firstly, we generated human \u003cem\u003eC9ORF72\u003c/em\u003e iPSC-derived motor neurons (iMNs) as previously described [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. iPSCs from two healthy controls, three C9ORF72 lines, and one isogenic control were differentiated into motor neurons (iMNs), see (\u003cb\u003eSuppl. Table\u0026nbsp;1\u003c/b\u003e) for demographic details and (\u003cb\u003eSuppl. Figure\u0026nbsp;1\u003c/b\u003e) for iMN molecular characterization. Since Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis is a crucial determinant for neuronal function, and Ca\u003csup\u003e2+\u003c/sup\u003e buffering by mitochondria majorly occurs at the ER\u0026ndash;mitochondria contact sites, which ultimately is important for ATP production, we first employed seahorse assay to measure mitochondrial function. Seahorse assay measures key parameters of mitochondrial function by directly measuring the oxygen consumption rate (OCR) and a representative example from control and diseased \u003cem\u003eC9ORF72\u003c/em\u003e line, C9(1) is presented pre- and post-kaempferol treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Notably, all three \u003cem\u003eC9ORF72\u003c/em\u003e patient-derived iMNs revealed impaired mitochondrial function as observed by the reduced basal respiration and ATP production compared to the healthy control treatment of iMNs with kaempferol for 48 hours (h) rescued mitochondrial dysfunction by normalizing basal respiration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and ATP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Note the near normalized ATP levels in mutation-corrected \u003cem\u003eIso-C9 (3)\u003c/em\u003e patient line, compared to the uncorrected \u003cem\u003eC9 (3)\u003c/em\u003e patient line, suggesting that the observed mitochondrial dysfunction is causally linked to the disease mutation. Next, we tested whether kaempferol would also protect mutant iMNs from ER stress and thus sustain neuroprotection. To this end, we induced strong ER stress by treating iMNs with Tunicamycin (TU) for 24h. In parallel, iMNs were dually treated with TU and kaempferol for 24h (\u003cb\u003escheme\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). TU treatment led to the induction of ER stress as observed by increased BiP expression in both \u003cem\u003econtrol\u003c/em\u003e and \u003cem\u003eC9ORF72\u003c/em\u003e patient-derived iMNs. Notably, dual treatment with TU and kaempferol for 24h inhibited the observed ER stress response, indicating that kaempferol even in the presence of ER stress activators can effectively counteract the onset of ER stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). We next examined whether kaempferol treatment could protect iMNs from degeneration due to ER stress. TU treatment for 24h led to a substantial loss of iMNs with nearly 60% of iMNs being dead in both control and diseased conditions. In contrast, dually treated iMNs exhibited negligible neuronal death, indicating that kaempferol can confer neuroprotection in conditions associated with strong ER stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Given the prominent role of aging in neurodegeneration, we next generated neurons via the direct reprogramming of fibroblasts to neurons (dNs), as previously described by us\u003csup\u003e7\u003c/sup\u003e, thus maintaining aging and epigenetic signatures of the donor. See (\u003cb\u003eSuppl. Table\u0026nbsp;1\u003c/b\u003e) for demographic details. We treated dNs with TU and kaempferol for 24 hours (\u003cb\u003escheme\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), which similar to our experiments in iMNs led to significant death in controls and \u003cem\u003eC9ORF72\u003c/em\u003e dNs, however, the dual treatment together with kaempferol counteracted ER-stress induced neuronal death irrespective of their genotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). In conclusion, kaempferol treatment restores mitochondrial function, and provides neuroprotection against ER stress, thereby alleviating cellular dysfunction in human C9ORF72-patient neurons.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eKaempferol treatment protects motor neurons and reduces pathological aggregates in a rodent model.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we determined whether kaempferol treatment exerted a neuroprotective effect on motor neurons in \u003cem\u003eC9-500\u003c/em\u003e model of \u003cem\u003eC9ORF72\u003c/em\u003e-ALS. This mouse model displays premature ER stress, mitochondrial dysfunction as well as the striking presence of RNA foci, pTDP43 aggregates, and robust accumulation of DPRs, all of which are pathological hallmarks of \u003cem\u003eC9ORF72\u003c/em\u003e-ALS [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We performed chronic kaempferol treatment from P138 until P240, an age-corresponding to the end stage of the disease [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Firstly, counting the number of ChAT immunostained spinal motor neurons revealed that chronic kaempferol treatment revealed a substantially high number of preserved motor neurons compared to \u003cem\u003eC9-500\u003c/em\u003e untreated mice. Notably, the treatment led to a comparable number of mutant motor neurons as those observed in \u003cem\u003eWT\u003c/em\u003e mice, suggestive of sustained neuroprotection. In comparison, a\u0026thinsp;~\u0026thinsp;40% reduction in motor neuron numbers, indicative of ongoing motor neuron degeneration, was observed in untreated saline injected \u003cem\u003eC9-500\u003c/em\u003e spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Poly(GA) inclusions are prominent pathological hallmarks, indicative of progressing disease and their expression is dependent on P\u003csub\u003ei\u003c/sub\u003e-eIF2α signaling, which is induced during unfolded protein response (UPR) and/or integrated stress response (ISR) [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. We longitudinally assessed from the presymptomatic stage (P60) until the symptomatic stage (P150), the presence of Poly(GA) inclusion within spinal motor neurons of \u003cem\u003eC9-500\u003c/em\u003e mice. Immunostaining against N-terminal Poly(GA), revealed that 15.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4% of Poly(GA) aggregates were \u0026gt;\u0026thinsp;1 \u0026micro;m\u003csup\u003e3\u003c/sup\u003e at P60, however, this fraction of the larger Poly(GA) aggregates increased with age, coinciding with the symptomatic phase (32.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8% \u0026gt; 1 \u0026micro;m\u003csup\u003e3\u003c/sup\u003e at P125, and 50.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1% \u0026gt; 1 \u0026micro;m\u003csup\u003e3\u003c/sup\u003e at P150). Note the complete lack of Poly(GA) immunopositivity in \u003cem\u003eWT\u003c/em\u003e motor neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cb\u003egraph bottom\u003c/b\u003e). Since sustained motor neuron numbers after kaempferol treatment indicated a neuroprotective action of kaempferol, thus we examined the accumulation of Poly(GA) aggregates within motor neurons as an indicator of motor neuron function. Immunostaining against Poly(GA) and quantification revealed widespread accumulation of Poly(GA) within saline-treated \u003cem\u003eC9-500\u003c/em\u003e spinal motor neurons, however, the average numbers as well as the fraction of large Poly(GA) aggregates within individual motor neurons were dramatically reduced within chronically kaempferol treated spinal motor neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Astrogliosis is a typical hallmark of many neurodegenerative diseases as well as ALS, thus we stained lumbar spinal cord sections for the glial fibrillary acidic protein (GFAP), to assess if KMP has an effect in reducing neuroinflammatory events. While \u003cem\u003eC9-500\u003c/em\u003e control animals show a widespread astrogliosis within the ventral horn of the spinal cord, KMP chronically treated \u003cem\u003eC9-500\u003c/em\u003e mice display a reduction in GFAP intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). As \u003cem\u003eC9ORF72\u003c/em\u003e-linked ALS also affects the muscles, mainly the neuromuscular junctions, we also assessed the overall muscle structure. Muscle histopathological analyses via H\u0026amp;E staining of saline and kaempferol-treated \u003cem\u003eC9-500\u003c/em\u003e mice revealed the presence of atrophic fibers, small fiber size, with central nuclei and angular muscle fibers, indicative of muscle fiber degeneration and denervation in P240 \u003cem\u003eC9-500\u003c/em\u003e saline-treated muscle Gastrocnemius (GC) muscle. In contrast, kaempferol-treated mice displayed regular GC muscle fiber size and negligible signs of atrophy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Moreover, we evaluated muscle fiber types via NADH staining, which revealed that \u003cem\u003eC9-500\u003c/em\u003e muscles had nearly lost groups of all NADH-positive myocytes, indicative of impaired muscle function. In contrast, kaempferol-treated mutant \u003cem\u003eC9-500\u003c/em\u003e muscles maintained a strong NADH staining within groups of myocytes reflecting the presence of varied fiber types and sustained muscle function in treated \u003cem\u003eC9-500\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Overall, this data suggests that the systemic administration of kaempferol can elicit a beneficial and neuroprotective effect on both spinal cord and muscles as observed by the reduced accumulation of toxic Poly(GA) aggregates, and preserved motor neuron numbers and muscle function.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKaempferol acts on the ER stress pathway and inhibits UPR signaling in vivo.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on the very promising neuroprotective findings observed after kaempferol treatment in \u003cem\u003eC9-500\u003c/em\u003e mice, we next focused on its mode of action on specific cellular impairments. We tested whether kaempferol, known to possess ER stress inhibitory activity in cultured mammalian cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] could also reduce ER stress in vivo in the \u003cem\u003eC9-500\u003c/em\u003e mouse model. The early phase of ER stress was assessed by immunolabeling the \u003cem\u003eC9-500\u003c/em\u003e mouse spinal cord sections with antibodies against the luminal ER protein BiP/GRP78. Age-related longitudinal quantification of BiP expression revealed that at P30, \u003cem\u003eC9-500\u003c/em\u003e motor neurons expressed similar levels of BiP as in WT; but as from P60 on, a gradual increase in BiP expression in mutant motor neurons was observed. P80 onward, significantly high BiP levels, indicative of ER stress progression was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We further assessed whether ER stress evolved into UPR by measuring the appearance of P\u003csub\u003ei\u003c/sub\u003e-eIF2α immunoreactivity in ChAT-positive ventral horn motor neurons. At P60, when BiP levels slightly increase, we found no indication of UPR signaling, however, an abrupt transition to UPR signaling was observed at P125, whereby nearly 50% of motor neurons displayed strong P\u003csub\u003ei\u003c/sub\u003e-eIF2α immunolabeling (53.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8%), and nearly all motor neurons displayed P\u003csub\u003ei\u003c/sub\u003e-eIF2α immunopositivity close to end stage at P200 (75\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9%), confirming an advanced UPR signaling in the majority of \u003cem\u003eC9-500\u003c/em\u003e motor neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Note the complete lack of P\u003csub\u003ei\u003c/sub\u003e-eIF2α immunolabeling within \u003cem\u003eWT\u003c/em\u003e motor neurons. Next, we assessed ER stress status in \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e mice, which were injected with 10mg/kg of kaempferol, which was again started at P138, a timepoint after the onset of UPR signaling and lasted until late end-stage; P240 (scheme for treatment and analyses Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Probing for UPR marker P\u003csub\u003ei\u003c/sub\u003e-eIF2α, we found a prominent expression within mutant \u003cem\u003eC9-500\u003c/em\u003e motor neurons at P240, which was restricted to ChAT-positive spinal ventral horn motor neurons in mutant condition. However, kaempferol treatment completely inhibited the expression of P\u003csub\u003ei\u003c/sub\u003e-eIF2α indicating that kaempferol acts on the UPR signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). We next assessed the effect of ER stress inhibitor; Salubrinal on \u003cem\u003eC9-500\u003c/em\u003e spinal motor neurons [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Chronic treatment with Salubrinal as expected also reduced UPR signaling as observed by diminished P\u003csub\u003ei\u003c/sub\u003e-eIF2α immunopositivity with P180 \u003cem\u003eC9-500\u003c/em\u003e motor neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKaempferol restores mitochondria electron transport chain function in C9-500 mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBesides ER stress, mitochondrial dysfunction due to \u003cem\u003eC9ORF72\u003c/em\u003e haploinsufficiency a well as the expression of specific DPRs such as PolyGR [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] has been causally implicated in \u003cem\u003eC9ORF72\u003c/em\u003e-linked ALS pathology. Thus, we assessed whether kaempferol treatment could rescue the observed mitochondrial dysfunction, which has been shown to protect neuronal mitochondria from loss of mitochondrial transmembrane electric potential caused by oxidative stress as well as excessive mitochondrial fission in neuronal models of excitotoxicity and ischemic stroke [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. We initially evaluated oxidative stress in spinal motoneurons, given its role in inducing nuclear and mitochondrial DNA damage. This assessment was performed using the oxidative DNA damage marker, 8-hydroxy-2\u0026prime;-deoxyguanosine (8-OHdG), detected via a specific antibody (REF David\u0026rsquo;s paper). To quantify the expression of this marker, we set a threshold of 60 arbitrary units (a.u.) of intensity to distinguish between low and high-expressing motoneurons. Strikingly, KMP-treated \u003cem\u003eC9-500\u003c/em\u003e animals displayed a significant reduction in high-expressing motoneurons compared to saline-treated animals (\u003cem\u003eC9-500\u003c/em\u003e saline: 88% vs. \u003cem\u003eC9-500\u003c/em\u003e\u0026thinsp;+\u0026thinsp;KMP: 21%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Since the primary function of mitochondria is energy production in the form of an elevated ATP/ADP ratio, we focused on assessing whether deficits in electron transport chain (ETC) function was observed in \u003cem\u003eC9-500\u003c/em\u003e spinal motor neurons. To this end, we used a colorimetric assay to assess Complex I, and IV activity in wildtype (\u003cem\u003eWT\u003c/em\u003e) and \u003cem\u003eC9-500\u003c/em\u003e spinal cord after kaempferol treatment. Complex I activity was measured via the NADH dehydrogenase catalyzed redox reaction in which NADH was oxidized and nitroblue tetrazolium (NBT) was reduced. The blue/purple color so formed due to NBT reduction faithfully correlates with the amount of endogenous NADH dehydrogenase activity present within cells. This blue/purple color was specifically and prominently reduced within the soma of P240 \u003cem\u003eC9-500\u003c/em\u003e motor neurons as compared to \u003cem\u003eWT\u003c/em\u003e motor neurons. Notably, kaempferol treatment of \u003cem\u003eC9-500\u003c/em\u003e mice normalized Complex I levels to WT levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Subsequently, we analyzed the activity of Complex IV by determining the intensity of cytochrome c oxidation, which is detected by the brown coloring produced by the oxidation of diaminobenzidine (DAB). Like Complex I, also reduced Complex IV activity was observed within mutant \u003cem\u003eC9-500\u003c/em\u003e motor neurons, and kaempferol treatment restored this deficit to normal \u003cem\u003eWT\u003c/em\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). While treatment with Salubrinal unlike kaempferol treatment did not improve behavioral symptoms in \u003cem\u003eC9-500\u003c/em\u003e mice, it did reduce UPR signaling, therefore we also explored whether Salubrinal harbors the potential to ameliorate or restore normal mitochondrial function. Treatment with Salubrinal did not affect the observed deficits in mitochondrial ETC levels and both Complex I (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and Complex IV levels remained reduced in Salubrinal-treated mutant motor neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Taken together these results suggest that kaempferol acts on both cellular organelles, reducing ER stress and promoting mitochondrial function, while Salubrinal works selectively on inhibiting the ER stress/UPR pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eA short treatment regime with kaempferol selectively ameliorates behavioral hallmarks in symptomatic C9-500 mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on the observed neuroprotective action of kaempferol in the CNS, we assessed whether kaempferol treatment could rescue the behavioral defects observed in \u003cem\u003eC9-500\u003c/em\u003e mouse model of \u003cem\u003eC9ORF72\u003c/em\u003e-ALS. This mouse model displays premature ER stress, mitochondrial dysfunction as well as striking presence of RNA foci, pTDP43 aggregates, and robust accumulation of DPRs, all of which are pathological hallmarks of \u003cem\u003eC9ORF72\u003c/em\u003e-ALS [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A short treatment with Kaempferol at 10mg/kg was performed for two weeks via daily intraperitoneal (i.p.) injections [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] as from P138-P153, an age corresponding to the appearance of the pathology-associated behavioral phenotype in \u003cem\u003eC9-500\u003c/em\u003e mice [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The reverse rotarod and hanging wire tests were performed pre-kaempferol treatment to obtain baseline measurement, followed by four consecutive measurements per week post-kaempferol treatment for half of the cohort of mice, whereas the other half received saline (scheme Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Reverse rotarod assay on the \u003cem\u003eC9-500\u003c/em\u003e mice cohort pre-kaempferol treatment revealed a striking deficit in motor performance compared to \u003cem\u003eWT\u003c/em\u003e mice as observed by the dramatic decline in latency to fall measured in seconds. Notably after one week of kaempferol treatment \u003cem\u003eC9-500\u003c/em\u003e mice presented markedly improved motor coordination and balance (\u003cb\u003eSupplementary video 1\u003c/b\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cb\u003eleft graph\u003c/b\u003e). We further continued to chronically treat the mice every alternate day with kaempferol until the late stage of the disease; P190, which revealed that the ameliorated rotarod performance was sustained (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cb\u003eright graph\u003c/b\u003e). Similarly, the hanging wire test revealed that after two weeks of kaempferol treatment, the average number of falls within the two minutes was reduced compared to the performance of the same cohort of mice pre-kaempferol treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Likewise, the time taken to first fall improved slightly between pre- and post-kaempferol treatment, suggesting that kaempferol treatment led to an overall amelioration in muscle endurance and function (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). We next assessed whether another compound; Salubrinal which is known to inhibit ER stress and has been shown to ameliorate ALS-associated pathological symptoms in other familial ALS mouse models [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], could also have a beneficial outcome in \u003cem\u003eC9-500\u003c/em\u003e mice. Like kaempferol treatment regime, Salubrinal treatment was performed on symptomatic P145 \u003cem\u003eC9-500\u003c/em\u003e mice lasting until P170 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). This treatment led to no improvement in the reverse rotarod performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Moreover, hanging wire tests revealed no significant amelioration in muscle endurance or stamina as measured by number of falls within two minutes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) or time to the first fall (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). It is noteworthy that unlike studies done on SOD1G93A mice, wherein Salubrinal treatment was neuroprotective [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], in the \u003cem\u003eC9-500\u003c/em\u003e mice model, we found no protective effect of Salubrinal, indicative of a likely more complex interplay between various cellular impairments ranging from mitochondrial dysfunction to proteostasis and autophagy thus, leading to an accumulation of toxic events resulting in the development of the symptomatic pathology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKaempferol potentially elicits its action by binding to GRP75.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe sought to understand how kaempferol could dually act on inhibiting ER stress while sustaining mitochondrial function. We hypothesized that kaempferol likely exerts its effect at mitochondrial-associated membranes (MAM), representing regions of contact between the ER and mitochondria. MAM constitutes regions of ER membranes that are reversibly tethered to mitochondria. These membranes are involved in the import of specific lipids from the ER to mitochondria and the regulation of calcium homeostasis, mitochondrial function, autophagy, and apoptosis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. We performed an in-silico search for potential kaempferol interactors, which likely function at the MAM. The structure of kaempferol was uploaded to CB-Dock (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://clab.labshare.cn/cb-dock/php/dockingresult.php\u003c/span\u003e\u003cspan address=\"http://clab.labshare.cn/cb-dock/php/dockingresult.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for analysis of the docking potential of kaempferol to MAM-located molecules. These analyses led to the identification of GRP75 (75-kDA glucose-regulated protein) as being the strongest binding partner of kaempferol as indicated by the vina score. GRP75 is a major mitochondrion located chaperone, which interacts with the component of both the mitochondrial quality control system and MAMs, thus playing a key role in mitochondrial homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The crystal structure of GRP75 revealed a significant interaction of kaempferol with the nucleotide-binding domain (NBD) of GRP75 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). We further assessed if other molecules could show similar interaction with GRP75 and found that 17-AAG had the second highest Vina score for binding to GRP75 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) and strongly docked to the NBD site of GRP75 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Further, analyses revealed that a substantial number of overlapping human proteins were targeted by both kaempferol and 17-AAG (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE \u003cb\u003eand Suppl. Table\u0026nbsp;2\u003c/b\u003e). However, literature analysis revealed that 17-AAG indirectly increases the expression level of GRP75 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], while inhibiting HSP90 chaperone family, thereby impairing protein folding of multiple client substrates and autophagy that is impaired in \u003cem\u003eC9ORF72\u003c/em\u003e-ALS. In contrast, kaempferol, reduces UPR signaling by augmenting the expression of ER folding chaperones [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and enhances mitochondrial Ca2\u003csup\u003e+\u003c/sup\u003e uptake, by a yet unidentified mechanism involving the binding to the mitochondrial uniporter, in traumatic brain injury model (TBI) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Interestingly, no binding affinity of Salubrinal to GRP75 was observed (data not shown). Since a previous study from us has shown reduced GRP75 expression at the MAM in \u003cem\u003eC9-500\u003c/em\u003e mice and its pathological sequestration by Poly(GA) aggregates within neurons, therefore we focussed on assessing whether kaempferol could exert neuroprotection via modulating GRP75, thus counteracting both ER and mitochondrial deficits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKaempferol enhances IP3R-VDAC1 interactions, promoting optimal mitochondrial function via GRP75.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on our in-silico screening, we hypothesized that the mechanism behind kaempferol-mediated neuroprotection could be via its interaction with GRP75 at the MAM. Since GRP75 serves as a scaffold, bringing iP3R-VDAC1 in proximity, we firstly, assessed iP3R-VDAC1 interactions via proximity ligation assay (PLA). Chronic, kaempferol treatment from symptomatic stage until the end stage of disease revealed increased iP3R-VDAC1 interactions versus saline-treated mutant spinal motor neurons of \u003cem\u003eC9-500\u003c/em\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Since saline-treated mutant \u003cem\u003eC9-500\u003c/em\u003e motor neurons revealed dramatically reduced iP3R-VDAC1 interactions compared to \u003cem\u003eWT\u003c/em\u003e motor neurons, and this reduced interaction was normalized after kaempferol treatment, indicated that kaempferol could potentially attenuate mitochondrial dysfunction by promoting optimal Ca\u003csup\u003e2+\u003c/sup\u003e uptake by mitochondria. Thus, we examined the physiological process of Ca\u003csup\u003e2+\u003c/sup\u003e uptake by mitochondria, which majorly occurs at the ER\u0026ndash;mitochondria contact sites via iP3R-VDAC1. To this end, cortical neurons from \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e neonates were cultured and we measured mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake. Fluo-4AM was combined with an intracellular buffer that eliminated cytosolic and ER Ca\u003csup\u003e2+\u003c/sup\u003e signals [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], thereby enabling specifically the measurement of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake. A striking deficit was observed in mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake in \u003cem\u003eC9-500\u003c/em\u003e cortical neurons as observed by significantly reduced Ca\u003csup\u003e2+\u003c/sup\u003e transients compared \u003cem\u003eWT\u003c/em\u003e cortical neurons. These deficits in Ca\u003csup\u003e2+\u003c/sup\u003e uptake by mutant mitochondria were efficiently eliminated by treating cortical neurons with kaempferol, confirming our finding that kaempferol elicits its effect at the MAM, modulating the mitochondrial uptake of Ca\u003csup\u003e2+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). We next measured GRP75 expression in spinal motor neurons to assess whether kaempferol modulates GRP75 expression. Motor neurons from both \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e mice treated with kaempferol displayed augmented GRP75 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Of note the degree of enhanced GRP75 expression was much higher in mutant \u003cem\u003eC9-500\u003c/em\u003e motor neurons, as they present dramatically reduced GRP75 expression at late stages of the disease. To underpin the molecular mechanism associated with the beneficial effect of GRP75 on the mitochondria, we co-immunoprecipitated GRP75 from P125, \u003cem\u003eC9-500\u003c/em\u003e and \u003cem\u003eWT\u003c/em\u003e ventral spinal cord, followed by mass spectrometric (MS) analyses. The interactome of GRP75 revealed known interactions such as those with VDAC1 as well as other interactions shared between the two genotypes (\u003cb\u003eSuppl. Table\u0026nbsp;3\u003c/b\u003e). We focused on GRP75 interactions, which were strongly present in the \u003cem\u003eC9-500\u003c/em\u003e spinal cord. Within the top five GRP75 interacting proteins, as measured semi-quantitatively via peptide match score summation (PMSS), was mitochondrial ATP synthase-coupling factor 6 (ATP5J), (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). ATP5J produces ATP from ADP in the presence of a proton gradient across the mitochondrial membrane, generated by the ETC of the respiratory chain, we validated the interaction between ATP5J and GRP75 in \u003cem\u003eC9-500\u003c/em\u003e motor neurons using PLA. Endogenous GRP75 interactions with ATP5J was observed in both \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eC9-500\u003c/em\u003e motor neurons, albeit strongly at P125 mutant motor neurons. However, akin to reduced iP3R-VDAC1 interactions at P240, ATP5J-GRP75 were much lower in late-stage mutant motor neurons, indicative of advanced mitochondrial dysfunction and degeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). As kaempferol treatment augmented GRP75 expression, we examined ATP5J expression in response to kaempferol treatment. While ATP5J levels were reduced in P240 motor neurons, likely reflecting the reduced GRP75 expression, spinal motor neurons treated with kaempferol even at end stage presented high ATP5J immunoreactivity, reflecting an overall beneficial effect of kaempferol on mitochondria via the GRP75-ATP5J pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eALS is a multifactorial disease, with complex overlapping clinical symptoms, and a fatal outcome within 3\u0026ndash;5 years of diagnosis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Several studies have indicated that ER stress plays a pivotal role in the pathophysiology of ALS [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], thus targeting ER stress is an interesting objective for therapeutic intervention. Besides, ER stress, mitochondrial dysfunction associated with oxidative stress, mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake deficits and bioenergetic deficits is closely associated with ALS pathophysiology [\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], therefore identifying compounds that can ameliorate both ER and mitochondrial impairments in ALS is of extreme importance to the field of ALS. In this study, we identified and explored the potential of a dietary flavanol kaempferol as a modulator of both ER stress response as well as mitochondria function in ALS. Focussing on the most common genetic form of ALS, \u003cem\u003eC9ORF72\u003c/em\u003e-linked ALS, we show that kaempferol harbours the potential to attenuate ER stress and UPR signaling in vivo in symptomatic \u003cem\u003eC9-500\u003c/em\u003e rodent motor neurons as well as in vitro in human \u003cem\u003eC9ORF72\u003c/em\u003e patient-derived iMNs. Moreover, we provide compelling evidence for the ability of kaempferol to restore ATP production and to normalize mitochondrial function in \u003cem\u003eC9ORF72\u003c/em\u003e ALS. This is noteworthy, as previous compounds such as Salubrinal have been shown to interfere only with the PERK/ P\u003csub\u003ei\u003c/sub\u003e-eIF2α -UPR pathway [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], or by suppressing the activity of integrated stress response, upstream of the PERK/ P\u003csub\u003ei\u003c/sub\u003e-eIF2α -UPR pathway [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Moreover, drugs like Salubrinal or guanabenz enhance the persistent translation inhibition by P\u003csub\u003ei\u003c/sub\u003e-eIF2α, thereby, accelerating neurodegeneration in a model of prion disease [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] or in mutant male SOD1 mice [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In addition, both these compounds elicit toxicity on other organelles such as nephrotoxicity observed after chronic Salubrinal administration [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] or blood pressure dysregulation after guanabenz administration [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. These studies highlight the unmet need for a drug candidate with the ability to reduce UPR signaling in affected neurons, without side effects. In this context, kaempferol and its derivatives are natural dietary phytochemicals, lacking evident toxicity, and are known to exhibit antioxidant, anti-inflammatory, anticancer, and neuroprotective activity [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, we found that treatment with kaempferol initiated after the onset of pathological symptoms was able to abrogate intrinsic mitochondrial deficits present in \u003cem\u003eC9ORF72\u003c/em\u003e neurons. These data are remarkable, as kaempferol normalized mitochondrial ATP levels in human \u003cem\u003eC9ORF72\u003c/em\u003e motor neurons, an important determinant of overall adequate neuronal energy supply and optimal function. While Salubrinal as expected, did inhibit ER stress, it did not ameliorate mitochondrial function either in vivo in \u003cem\u003eC9-500\u003c/em\u003e motor neurons nor in vitro in human iMNs. On the contrary, Salubrinal has been shown to promote cell death in cancer by activating the ISR and inducing mitochondrial oxidative stress, thereby irreversibly damaging mitochondria. Further, Salubrinal treatment in glucose-deprived conditions leads to the upregulation of mitochondrial ROS, thereby inducing mitochondrial stress and dysfunction [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Previous studies have shown that kaempferol alleviates oxidative stress and apoptosis during lung ischemia-reperfusion injury, by enhancing mitochondrial membrane potential and inhibiting the opening of mitochondrial permeability transition pores [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Importantly, kaempferol has been shown to control mitochondrial calcium regulation by directly activating the mitochondrial calcium uniporter (MCU) in a concentration-dependent manner. Even at a low concentration such as 1\u0026micro;M of kaempferol treatment, practically doubled the uptake of mitochondrial Ca\u0026sup2;⁺ [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Therefore, based on our findings, it is very likely that kaempferol treatment counteracts mitochondrial dysfunction as well as mitochondrial Ca\u0026sup2;⁺ uptake deficits in \u003cem\u003eC9ORF72\u003c/em\u003e-ALS [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], thus promoting optimal mitochondrial function and neuroprotection. Kaempferol has been shown to modulate key signaling pathways involved in neurodegeneration and neuroinflammation, such as the PI3K/Akt, MAPK/ERK, and NF-κB pathways, nevertheless, further research is necessary to decipher the underlying mechanisms of action, to optimize dosage schedules, and assess the safety and efficacy of this intervention in human clinical trials.\u003c/p\u003e \u003cp\u003eOf note, administration of kaempferol via the i.p. route at symptomatic ages \u003cem\u003ein C9-500\u003c/em\u003e mice attenuated the accumulation of large Poly(GA) aggregates, increased the number of surviving motor neurons, and improved motor behavior. We believe that these findings enhance the predictive value that kaempferol could be beneficial in patients with \u003cem\u003eC9ORF72\u003c/em\u003e-ALS and in other familial forms of ALS, where ER stress and mitochondrial impairments occur together with mutant protein inclusions. In other neurodegenerative diseases such as Alzheimer\u0026acute;s disease, kaempferol treatment inhibited β-A structure formation by hindering amyloid fibril elongation [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Yet in another study treatment with kaempferol derivative; kaempferide revealed the enhanced expression of the brain-derived neurotrophic factor (BDNF), which in turn augmented the phosphorylation of transcription factor cAMP, thus promoting synaptic plasticity [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In Parkinson\u0026acute;s disease, kaempferol ameliorated nigrostriatal dopaminergic neuron lesions; by inhibiting interleukin (IL) 1β, IL-6, and TNFα production, highlighting the anti-inflammatory activity of kaempferol [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mechanism via which kaempferol elicits its anti-ER stress properties remains unclear. However, studies have shown that kaempferol inhibits the expression of BiP, thereby inhibiting the activation of UPR sensors PERK, ATF6, and IRE1 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These findings fit well with our observations of reduced BiP and P\u003csub\u003ei\u003c/sub\u003e-eIF2α expression after kaempferol treatment. Our data from in silico modeling identified that kaempferol strongly binds to GRP75; a chaperone located within the mitochondria, but is majorly involved at the MAM, functioning as a structural scaffold protein for the iP3R-VDAC1 channel. This channel is the most prominent channel at the MAM promoting optimal transfer of Ca\u003csup\u003e2+\u003c/sup\u003e from the ER to the mitochondria. GRP75 expression levels are progressively reduced in \u003cem\u003eC9-500\u003c/em\u003e mice, and those reductions coincide with the onset of UPR signaling and the appearance of large Poly(GA) inclusions within motor neurons [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Our PLA data suggests that within the \u003cem\u003eC9-500\u003c/em\u003e spinal motor neurons, kaempferol enhances iP3R-VDAC1 interactions, which aids in the optimal mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake, important for the proper ETC process. Of note, our data also revealed the ability of kaempferol to enhance the expression of GRP75 and its interacting partner ATP5J in motor neurons, suggesting that kaempferol could act on process regulating protein translation. Interestingly, like quercetin yet another flavanol, kaempferol might positively modulate gene expression[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Moreover, kaempferol could positively influence GRP75 expression at a translational level by inhibiting UPR signaling, thus removing the global translational block. Therefore, kaempferol might have a complex and multifaceted impact on the protein translation process, which may contribute to its pharmacological activity and potential therapeutic applications. However, further experiments are needed to dissect how kaempferol impacts GRP75 expression and iP3R-VDAC1 function in the context of \u003cem\u003eC9ORF72\u003c/em\u003e-ALS.\u003c/p\u003e \u003cp\u003eIn view of the translation potential of our findings, we measured ER stress and mitochondrial function in human \u003cem\u003eC9ORF72\u003c/em\u003e patient-derived iMNs and found that kaempferol not only ameliorated ER stress and restored normal mitochondrial function in three different \u003cem\u003eC9ORF72\u003c/em\u003e-patient iMN lines, but also provided neuroprotection by promoting iMNs survival in the presence of ER stress. To the best of our knowledge, no previous drug compound has been reported to have a protective effect in \u003cem\u003eC9-500\u003c/em\u003e mice by dually ameliorating ER stress and mitochondrial function. Concomitantly, the reduction in large Poly(GA) aggregates, sustained motor neuron survival as well as improved muscle and behavioral phenotype, highlights the beneficial potential of Kaempferol in the \u003cem\u003eC9-500\u003c/em\u003e mice, when treatment was initiated at disease signs onset. This is a clinically relevant time point for the initiation of therapy in human patients, as usually, they do not receive a diagnosis before symptoms onset. Additionally, we identified a novel mode of action of kaempferol, via the regulation of GRP75 expression and function. These observations suggest that kaempferol is a likely strong candidate for the prevention/slowing down of disease pathology in familial ALS cases.\u003c/p\u003e \u003cp\u003eIn conclusion, kaempferol rescues motor neuron survival in vitro and in vivo by regulating the ER stress response and ameliorating intrinsic mitochondrial function, which are key pathways implicated in the pathophysiology of \u003cem\u003eC9ORF72\u003c/em\u003e-ALS. Although the precise mode of action of Kaempferol remains incomplete, our data suggests that one likely pathway involves the interaction with the nucleotide-binding domain of GRP75, thus enhancing/stimulating GRP75 activity. Hence, kaempferol is a promising compound for diseased motor neurons, meriting further study as a promising drug candidate in ALS.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eUnfolded Protein response (UPR)\u003c/p\u003e\n\u003cp\u003eamyotrophic lateral sclerosis (ALS)\u003c/p\u003e\n\u003cp\u003efrontal temporal dementia (FTD)\u003c/p\u003e\n\u003cp\u003einduced pluripotent stem cells (iPSC)\u003c/p\u003e\n\u003cp\u003eglycogen synthase kinases-3\u0026beta; (GSK3\u0026beta;)\u003c/p\u003e\n\u003cp\u003eMitochondria-associated membranes (MAMs)\u003c/p\u003e\n\u003cp\u003eprotein tyrosine phosphatase interacting protein 51 (PTPIP51)\u003c/p\u003e\n\u003cp\u003emotoneurons (MNs)\u003c/p\u003e\n\u003cp\u003eiPSC-derived motor neurons (iMNs)\u003c/p\u003e\n\u003cp\u003edirect reprogramming of fibroblasts to neurons (dNs)\u003c/p\u003e\n\u003cp\u003eintegrated stress response (ISR)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eETHICS DECLARATIONS:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal experiments:\u0026nbsp;\u003c/strong\u003eThe study was approved by the Animal Commission of Canton of Bern, Switzerland, license number BE-35/17, BE-82/18\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman fibroblast-derived\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;cells:\u0026nbsp;\u003c/strong\u003eCells were anonymized and provided to us under an MTA from respective consortia. Culturing of cells only did not require internal review board (IRB) approval\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll authors have approved the manuscript and agree with its submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS:\u0026nbsp;\u003c/strong\u003eF.P., S.S. conceived the study and wrote the manuscript. F.P., P.S., R.D., M.S., and O.S. J.A.S.D. performed experiments and analyzed data. O.S., H.P., and S.S. provided reagents. S.S. supervised the overall project, all authors read and commented on the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS:\u0026nbsp;\u003c/strong\u003eWe thank Pentti Tienari, Helsinki University Central Hospital, Finland, Biomedicum Stem Cell Center, GoEditStem platform, HiLIFE, Helsinki, Finland; Bhuvaneish T. Selvaraj and Siddharthan Chandran, UK Dementia Research Institute, University of Edinburgh, UK, Euan MacDonald Centre for MND Research, University of Edinburgh, UK for providing iPSC cell lines. We thank Manfred Heller, Proteomic Core Facility, Department for BioMedical Research, University of Bern for Mass spectrometry. We also thank Nicolas Charlet-Berguerand, Institut de G\u0026eacute;n\u0026eacute;tique et de Biologie Mol\u0026eacute;culaire et Cellulaire (INSERM U964, CNRS UMR7104 for Poly(GA) antibody, Angelina Oestmann and Maria Essers for maintaining mouse colonies and helping with behavioral assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING:\u0026nbsp;\u003c/strong\u003eThis study was funded by the SPINAL CORD INJURIES/DISEASE RESEARCH PROGRAM (SCIDRP), University of Missouri, Missouri, USA, European Research Council (ERC) under the European Union\u0026apos;s Horizon 2020 research and innovation program (grant agreement #725825), Swiss National Science Foundation, Swiss Foundation for Research on Muscle Diseases, and E-rare grant (CALSER) to S.S.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHalliday M, Mallucci GR (2014) Targeting the Unfolded Protein Response in Neurodegeneration: A New Approach to Therapy. 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Int J Mol Sci 20:491. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms20030491\u003c/span\u003e\u003cspan address=\"10.3390/ijms20030491\" 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":"","lastPublishedDoi":"10.21203/rs.3.rs-5190511/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5190511/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRepeat expansions in the \u003cem\u003eC9ORF72\u003c/em\u003e gene are a frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Considerable progress has been made in identifying \u003cem\u003eC9ORF72\u003c/em\u003e-mediated disease and resolving its underlying etiopathogenesis. The contributions of intrinsic mitochondrial deficits as well as chronic endoplasmic reticulum stress to the development of the C9ORF72-linked pathology is well established, Nevertheless, to date, no cure or effective therapy is available, and thus attempts to find a potential drug target, have received increasing attention. Here, we investigated the mode of action and therapeutic effect of a naturally occurring dietary flavanol, kaempferol in preclinical rodent and human models of \u003cem\u003eC9ORF72\u003c/em\u003e-ALS. Notably, kaempferol treatment of \u003cem\u003eC9ORF72\u003c/em\u003e-ALS human patient-derived motor neurons/neurons, resolved mitochondrial deficits, promoted resiliency against severe ER stress and conferred neuroprotection. Treatment of symptomatic \u003cem\u003eC9ORF72\u003c/em\u003e mice with kaempferol, normalized mitochondrial calcium uptake, restored mitochondria function, and diminished ER stress. Importantly, in vivo, chronic kaempferol administration ameliorated pathological motor dysfunction and inhibited motor neuron degeneration, thus highlighting the translational potential of kaempferol. Lastly, in silico modelling identified a novel kaempferol target and mechanistically the neuroprotective mechanism of kaempferol is through the iP3R-VDAC1 pathway via the modulation of GRP75 expression. Thus, kaempferol holds great promise for the treatment of neurodegenerative diseases where both mitochondrial and ER dysfunction are causally linked to the pathophysiology.\u003c/p\u003e","manuscriptTitle":"Kaempferol enhances ER-mitochondria coupling and protects motor neurons from ER stress and mitochondrial dysfunction in C9ORF72- ALS","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-19 10:18:27","doi":"10.21203/rs.3.rs-5190511/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-14T16:25:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-13T22:12:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-10T05:46:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2302050864905184992402329508744713510","date":"2024-10-30T14:40:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8101420117274133931800414249480669071","date":"2024-10-29T01:53:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-18T13:56:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-07T07:37:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-07T07:37:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Neuropathologica Communications","date":"2024-10-02T03:41:41+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":"b0e6c7a4-af19-473f-9aaf-bb02c0894ce5","owner":[],"postedDate":"December 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-03T16:03:23+00:00","versionOfRecord":{"articleIdentity":"rs-5190511","link":"https://doi.org/10.1186/s40478-025-01927-y","journal":{"identity":"acta-neuropathologica-communications","isVorOnly":false,"title":"Acta Neuropathologica Communications"},"publishedOn":"2025-02-01 15:58:03","publishedOnDateReadable":"February 1st, 2025"},"versionCreatedAt":"2024-12-19 10:18:27","video":"","vorDoi":"10.1186/s40478-025-01927-y","vorDoiUrl":"https://doi.org/10.1186/s40478-025-01927-y","workflowStages":[]},"version":"v1","identity":"rs-5190511","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5190511","identity":"rs-5190511","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}