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

Pilotto, Federica; Smeele, Paulien Hermine; Scheidegger, Olivier; Diab, Rim; Schobesberger, Martina; Sierra-Delgado, Julieth Andrea; Saxena, Smita

doi:10.1186/s40478-025-01927-y

Research
Open access
Published: 01 February 2025

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

Federica Pilotto^3,4,
Paulien Hermine Smeele^1,2,
Olivier Scheidegger³,
Rim Diab⁴,
Martina Schobesberger⁴,
Julieth Andrea Sierra-Delgado^1,2 &
…
Smita Saxena^1,2,4

Acta Neuropathologica Communications volume 13, Article number: 21 (2025) Cite this article

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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 are 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, 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 treating neurodegenerative diseases where both mitochondrial and ER dysfunction are causally linked to the pathophysiology.

Graphical abstract

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 [1,2,3,4]. In the context of amyotrophic lateral sclerosis (ALS), motor neurons (MNs) are selectively prone to ER stress, influencing disease manifestation and kinetics [5,6,7]. 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 (G₄C₂) HRE are observed within the first intron of the C9ORF72 gene. However, in disease, this G₄C₂ 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 G₄C₂ HRE, which produces toxic G₄C₂ and G₄C₂ 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 [8,9,10].

C9ORF72 induced pluripotent stem cells (iPSC)-derived motor neurons (iMNs), exhibit increased endoplasmic reticulum (ER) stress, together with reduced mitochondrial membrane potential, concomitant impairment in calcium (Ca²⁺) 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 Ca²⁺ from the ER to mitochondria. These sites are critical for the rapid Ca²⁺ 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 Ca²⁺ homeostasis, mitochondrial function, autophagy, apoptosis, and glucose homeostasis [14]. Within the context of ALS, mutations, 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 Ca²⁺ 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 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 Ca²⁺ uptake impairments and mitochondrial dysfunction in both human patient-derived and rodent MNs [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, a known negative regulator of VAPB-PTPIP51 binding [16].

Therapeutic agents 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 (KMP) 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 [17, 18]and mice [19], anti-neuroinflammatory activity in rat models of ischemic stroke [20], mitochondrial Ca²⁺ uniporter channel activator [21] and ameliorated energy metabolism and symptomatic behavior in a rat model of traumatic brain injury [22]. While KMP has been predominantly studied as a potential tumor suppressor [19, 23,24,25], higher intake of KMP and other flavanols, was shown to be inversely correlated with the incidence of Alzheimer’s disease [26], emphasizing the neuroprotective potential of KMP in neurodegenerative diseases.

We here identify that KMP exerts a neuroprotective effect on C9ORF72 ALS neurons by sustaining mitochondrial function and inhibiting ER stress. Notably, in C9ORF72-ALS human patient-iMNs and directly fibroblast-converted neurons, KMP counteracted mitochondrial deficits and provided neuroprotection in the presence of elevated ER stress, thus indicating the probable translational potential of KMP. Additionally, in vivo, treatment with KMP 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 KMP to chaperone GRP75 and mechanistically the neuroprotective mechanism of KMP 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) [27, 28] 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. KMP 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

KMP (Sigma-Aldrich, K0133) at 10 mg/kg was administered via i.p. injections daily to both wild type (WT) and C9-500 starting at symptomatic age P138 for the first two weeks, followed by every alternate day KMP administration until end-stage P240. WT and C9-500 mice were treated every alternate day with Salubrinal (Sal) (Enzo life sciences, 270-428-M005) at a dosage of 1 mg/Kg or saline starting at symptomatic age P145 until P180. iMNs or dNs were treated with Tunicamycin (TU) (1 µg/ml) for 24 h before further analysis. For KMP treatments, iMNs were treated with 10 µg/ml KMP for 2 h or 48 h.

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 endurance as usually assessed by common accelerated rotarod protocols, during the light phase of the 12 h light/12 h dark cycle. For the inverse rotarod, the protocol involved sequential acceleration (from 15 to 33 rpm in 10 s) and deceleration (from 33 to 15 rpm in 10 s) followed by the immediate inversion of the direction of the rod, and the same rotation protocol was again followed. One trial lasted a maximum of 110 s, with 3 min (min) resting in between trials. Three trials every 10 days were performed for 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 [29]. 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 was recorded. All the animals are placed on the inverted cage top each time they fall to assess the average number of falls. The test lasts for 120 s.

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 mTeSR (StemCell technologies) media. MN differentiation was performed as previously described [30, 31] with some modifications. Human iPSCs were dissociated to single cells using Accutase (StemCell technologies) and seeded at 3X106 onto 10 cm plate with N2B27 differentiation medium (Advanced DMEM/F12:Neurobasal (1:1) medium, 1% Pen/strep (Gibco), 1% GlutaMAX (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 1X B27 supplement (Gibco), 1X N2 supplement (Gibco), supplemented with 10 ng/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 100 nM all-trans retinoic acid (RA; sigma) and 500 nM Smoothened Agonist (SAG; StemCell technologies). EBs were pelleted and fed with fresh media on every alternate day until day 14. 10 ng/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), 2 mM MgCl₂, 0.8 mM 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), 0.1 mM 2-mercapthoethanol, 1X N2 supplement, 1X Pen/strep, 1X B27 supplement, 10 ng/ml glial cell derived neurotrophic factor (GDNF; StemCell technologies), BDNF 10 ng/ml, 10 ng/ml insulin-like growth factor (IGF-1; StemCell technologies), 10 ng/ml Ciliary neurotrophic factor (CNTF; StemCell technologies), 100 nM 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 [32]. 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% CO₂ incubator. The next day, fibroblasts were transduced with retroviruses for SOX2, cMyc, KLF4, and OCT3/4. 24 h later virus was removed, and the media was replaced with fresh fibroblast media (10% FBS (Gibco), 1% anti-anti (Gibco) in DMEM Glutamax (Gibco)). After a 24-h rest period, the 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. The 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 a 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 15 min and blocked for 1 h 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 a blocking buffer with Alexa Fluor fluorescently labeled secondary antibodies and DAPI for 1 h 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 40 × air objective and 60 × 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 10 mM pH 6 for GRP75 and ATP5J staining. Sections were kept for 2 h 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 the 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 10 mM 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 1 h 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 adding the coverslip. 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. 8 μm 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 5 min, followed by three 10 min washes in deionized H2O. Muscles were then placed in eosin G for 1 min and then rinsed in 70% ethanol. The tissue was then dehydrated in a series of ethanol washes (70%, 95%, 100%), rinsed in xylene, and then mounted on a glass slide. NADH staining was performed by incubating muscle sections in 0.2 M Tris buffer containing Nitrotetrazolium Blue (Sigma) and β-nicotinamide adenine dinucleotide (NADH, Sigma) for 30 min at 37 °C. Following three washes in deionized H2O, coverslips were mounted onto a glass slide. ATP staining pH 4.3 and 9.4 were performed by incubating the slides in pre-incubating solution pH4.3 per 10 min at 4°, containing 0.1 M Na acetate buffer with 10 mM EDTA. Followed by three 5 min washes in deionized H₂0, to be then incubated in the incubation solution: for pH 4.3 containing sodium acetate 0.1 M, EDTA 0.1 M pH8 and pH 9.4 containing 0.1 M glycine (Sigma, 50,046), 5 mg ATP and DTT in NaCl buffer with CaCl2 per 30 min at 37 °C. Coverslips were then washed with deionized H2O 3 times every 5 min, incubated with Cobalt chloride 2% 3 times per 1 min at room temperature, rinsed with deionized water, and incubated with ammonium sulfide 1% at room temperature for 30 s. Coverslips were rinsed under running water, dehydrated in a series of ethanol washes (70%, 95%, 100%), rinsed in xylene, and 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, 8-OHdG and ATP5J labeling intensities, data were acquired using identical confocal settings, with signals at the brightest cells being non-saturated, and background levels outside MN 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 MN percentages, Choline Acetyltransferase (ChAT) positive MNs were examined and signal intensity values for the antigen of interest within these ChAT positive MNs 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 MN numbers, a cell counter plugin from Fiji was used. Imaris software was used to reconstruct the 3D isosurface of Poly(GA) aggregates volume.

Proximity ligation assay (PLA) in perfused mouse spinal cord tissue

PLA was adapted from Gomes et al., [33]. 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 1X PBS per 12 min followed by 3 washes in 1X PBS (each wash was for 5 min). Slides were kept in a humid chamber and blocking solution was added (Duolink PLA probe kit, DUO92008, Merck) for 1 h at 37 °C. Subsequently, slides were incubated with the following primary antibodies (1:200, rabbit anti-IP3R, ABCAM, ab5804; 1:100, mouse anti-GRP75, ABCAM, ab53098; 1:100, rabbit anti-GRP75, ABCAM, ab227215; 1:200 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 min) and incubated with PLA probes (Probe Anti-mouse MINUS, DUO92004 and Probe anti-rabbit PLUS, DUO92002) at a working concentration of 1:10 for 1 h at 37 °C. Ligation (Duolink In Situ Detection Reagents Red, DUO92008) was performed at 37 °C per 45 min followed by 3 washes in buffer A (3 per 5 min). The amplification (Duolink In Situ Detection Reagents Red, DUO92008) step was performed at 37 °C in a dark humidity chamber for 100 min. Finally, sections were rinsed in buffer B (Duolink In Situ Wash Buffers, Fluorescence DUO82049) (2 per 10 min) 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: 1.23 mg/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: 0.5 mg/ml 3,3′-diaminobenzidine tetrahydrochloride (DAB, D7304, Sigma), 1 mg/ml cytochrome c (C2506, Sigma), and approximately 2 μg/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 4 min in 70% ETOH, 4 min 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 Ca²⁺ imaging for cortical neurons was adapted from [34]. In brief, cells were incubated for 45 min with the staining solution containing: 156 mM NaCl, 3 mM KCl, 2 mM MgSO₄, 1.25 mM KH₂PO₄, 10 mM D-glucose, 2 mM CaCl_2, and 10 mM HEPES pH 7.35, 5 µg/ml (w/v) Fluo-4, AM, 10 µM Verapamil. After a brief wash with Ca²⁺ free HBSS, cells were incubated for 10 min with the intracellular solution containing: 6 mM NaCl, 130 mM KCl, 7.8 mM MgCl₂, 1 mM KH₂PO₄, 0.4 mM CaCl₂, 2 mM EGTA, 10 mM HEDTA, 2 mM malate, 2 mM glutamate, 2 mM ADP, 20 mM 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 40 × water immersion objective was used to acquire images every 20 s, the first 60 s were considered as the baseline, afterward 50 mM KCl was added to the imaging solution to depolarize the cells. Neurons were treated with 10 µg/ml of KMP for 1 h before 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 1 h and 45 min. Images were analyzed using Fiji, multiple regions of interest (ROI) were chosen inside the cytosol of the cells and the fluorescence intensity was calculated over the different frames. To calculate ΔF, the median intensity values are divided by the average of the first 60 s of recording (F0) per ROI.

CB-Dock analyses and PharmaMapper

CB-Dock is a computational docking tool specifically designed for molecular docking tasks, focusing on the flexible docking of small molecules into protein binding sites. CB-Dock uses a cavity-detection algorithm to identify potential binding pockets on a protein's surface, eliminating the need for manually specifying binding sites. Its name is derived from "cavity-based docking," for automatically identifying and targeting protein binding sites. The receptor (GRP75) protein structure was downloaded from the Protein Data Bank (PDB) and the ligand KMP or 17-AAG representing the small molecule intended for docking was downloaded in MOL2 format. Both files were uploaded on the CB-Dock platform, where the cavity-detection algorithm identified binding sites (pockets) on the protein structure. These pockets are ranked based on size and accessibility, and the software optimizes the ligand's conformation within the binding site to achieve the best fit and a Vina score is given. The Vina score is a numerical value generated by AutoDock Vina, and the score is expressed in kcal/mol and reflects the estimated free energy of binding. A more negative Vina score indicates stronger predicted binding affinity, meaning the ligand is more likely to bind tightly to the receptor. PharmaMapper was used to identify potential protein targets for a given small molecule; KMP and 17-AAG by comparing the molecule's structure against a large database of 3D pharmacophore models derived from known protein structures.

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, and Tukey’s test were 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 iMNs and fibroblast-induced neurons (dNs)

We started the study by evaluating the neuroprotective action of KMP, a compound known to act on UPR and oxidative stress pathways. Firstly, we generated human C9ORF72 iMNs as previously described [7]. iPSCs from two healthy controls, three C9ORF72 lines, and one isogenic control were differentiated into iMNs, see (Table 1) for demographic details and (Suppl. Figure 1) for iMN molecular characterization. Since Ca²⁺ homeostasis is a crucial determinant for neuronal function, and Ca²⁺ buffering by mitochondria majorly occurs at the ER-mitochondria contact sites, which is important for ATP production, we first employed the 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-KMP treatment (Fig. 1A). 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 KMP for 48 h rescued mitochondrial dysfunction by normalizing basal respiration (Fig. 1B) and ATP levels (Fig. 1C). 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 KMP would 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 24 h. In parallel, iMNs were dually treated with TU and KMP for 24 h (scheme Fig. 1D). 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 KMP for 24 h inhibited the observed ER stress response, indicating that KMP even in the presence of ER stress activators can effectively counteract the onset of ER stress (Fig. 1E). We next examined whether KMP treatment could protect iMNs from degeneration due to ER stress. TU treatment for 24 h 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 KMP can confer neuroprotection in conditions associated with strong ER stress (Fig. 1F). 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 the aging and epigenetic signatures of the donor. See (Table 1) for demographic details. We treated dNs either with TU for 24 h or KMP for 48 h, followed for the last 24 h of culture with co-treatment with TU (scheme Fig. 1G), which was similar to our experiments in iMNs led to significant death in controls and C9ORF72 dNs, however, the dual treatment together with KMP counteracted ER-stress induced neuronal death irrespective of their genotype (Fig. 1H). In conclusion, KMP treatment restores mitochondrial function, and provides neuroprotection against ER stress, thereby alleviating cellular dysfunction in human C9ORF72-patient neurons.

Table 1 Description of the human iPSC lines used in the study: demographic information about iPSC and fibroblast lines from C9ORF72-ALS/FTD patients and healthy control (CTRL) employed to generate iMNs and dNs

Full size table

Kaempferol treatment protects MNs and reduces pathological aggregates in a rodent model

Next, we determined whether KMP treatment exerted a neuroprotective effect on MNs in the 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, 35–37]. We performed chronic KMP treatment from P138 until P240, an age-corresponding to the end stage of the disease [29, 35]. Firstly, counting the number of ChAT immunostained spinal MNs revealed that the number of MNs was substantially higher in C9-500 mice treated with KMP than in saline-treated, control mice. Notably, the treatment led to a comparable number of mutant MNs as those observed in WT mice, suggestive of sustained neuroprotection. In comparison, a ~ 40% reduction in MN numbers, indicative of ongoing MN degeneration, was observed in the spinal cord of saline-injected mice (Fig. 2A). 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) [38–40]. We longitudinally assessed from the presymptomatic stage (P60) until the symptomatic stage (P150), the presence of Poly(GA) inclusion within spinal MNs of C9-500 mice. Immunostaining against N-terminal Poly(GA), revealed that 15.1 ± 3.4% of Poly(GA) aggregates were > 1 µm³ at P60, however, this fraction of the larger Poly(GA) aggregates increased with age, coinciding with the symptomatic phase (32.9 ± 2.8% > 1 µm³ at P125, and 50.8 ± 2.1% > 1 µm³ at P150). Note the complete lack of Poly(GA) immunopositivity in WT MNs (Fig. 2B, graph bottom). Since sustained MN numbers after KMP treatment indicated a neuroprotective action of KMP, thus we examined the accumulation of Poly(GA) aggregates within MNs as an indicator of improved neuronal homeostasis. Immunostaining against Poly(GA) and quantification revealed widespread accumulation of Poly(GA) inclusions within saline-treated C9-500 spinal MNs, however, the average numbers, as well as the fraction of large Poly(GA) aggregates within individual MNs, were dramatically reduced within chronically KMP treated C9-500 mice (Fig. 2C). 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 widespread astrogliosis within the ventral horn of the spinal cord, KMP-treated C9-500 mice display a reduction in GFAP intensity (Fig. 2D). 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 KMP-treated C9-500 mice revealed the presence of atrophic fibers, small fiber size, with central nuclei and angular muscle fibers, indicative of muscle fiber deficits and denervation in P240 C9-500 saline-treated muscle gastrocnemius (GC) muscle. In contrast, KMP-treated mice displayed regular GC muscle fiber size and negligible signs of atrophy (Fig. 2E). 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 muscle deficits. In contrast, KMP-treated mutant C9-500 muscles maintained a strong NADH staining within groups of myocytes reflecting the presence of varied fiber types and sustained muscle fiber composition in treated C9-500 mice (Fig. 2F). Overall, this data suggests that the systemic administration of KMP 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 MN numbers and reduce muscle fiber deficits.

Kaempferol acts on the ER stress pathway and inhibits UPR signaling in vivo

Based on the very promising neuroprotective findings observed after KMP treatment in C9-500 mice, we next focused on its mode of action on specific cellular impairments. We tested whether KMP, known to possess ER stress inhibitory activity in cultured mammalian cells [17] 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 MNs expressed similar levels of BiP as in WT; but as from P60 on, a gradual increase in BiP expression in mutant MNs was observed. P80 onward, significantly high BiP levels, indicative of ER stress progression was observed (Fig. 3A). We further assessed whether ER stress evolved into UPR by measuring the appearance of P_i-eIF2α immunoreactivity in ChAT-positive ventral horn MNs. At P60, when BiP levels slightly increased, we found no indication of UPR signaling, however, an abrupt transition to UPR signaling was observed at P125, whereby nearly 50% of MNs displayed strong P_i-eIF2α immunolabeling (53.8 ± 3.8%), and nearly all MNs 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 MNs (Fig. 3B). Note the complete lack of P_i-eIF2α immunolabeling within WT MNs. Next, we assessed ER stress status in WT and C9-500 mice, which were injected with 10 mg/kg of KMP, 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. 3C). Probing for UPR marker P_i-eIF2α, we found a prominent expression within mutant C9-500 MNs at P240, which was restricted to ChAT-positive spinal ventral horn MNs in mutant condition. However, KMP treatment completely inhibited the expression of P_i-eIF2α indicating that KMP acts on the UPR signaling pathway (Fig. 3D). We next assessed the effect of ER stress inhibitor; Sal on C9-500 spinal MNs [41]. Chronic treatment with Sal as expected also reduced UPR signaling as observed by diminished P_i-eIF2α immunopositivity with P180 C9-500 MNs (Fig. 3E).

Kaempferol restores mitochondria electron transport chain function in C9-500 mice

Besides ER stress, mitochondrial dysfunction due to C9ORF72 haploinsufficiency as well as the expression of specific DPRs such as PolyGR [6, 7, 12, 42] has been causally implicated in C9ORF72-linked ALS pathology. Thus, we assessed whether KMP 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 [43, 44]. We initially evaluated oxidative stress in spinal MNs, 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 [45]. 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 MNs. Strikingly, KMP-treated C9-500 animals displayed a significant reduction in high-expressing MNs compared to saline-treated animals (C9-500 saline: 88% vs. C9-500 + KMP: 21%) (Fig. 4A). 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 MNs. To this end, we used a colorimetric assay to assess Complex I, and IV activity in wildtype (WT) and C9-500 spinal cord after KMP 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 MNs as compared to WT MNs. Notably, KMP treatment of C9-500 mice normalized Complex I levels to WT levels (Fig. 4B). 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 MNs, and KMP treatment restored this deficit to normal WT levels (Fig. 4C). While treatment with Sal reduced UPR signaling, therefore we also explored whether Sal harbors the potential to ameliorate or restore normal mitochondrial function. Treatment with Sal did not affect the observed deficits in mitochondrial ETC levels and both Complex I (Fig. 4D) and Complex IV levels remained reduced in Sal-treated mutant MNs (Fig. 4E). Taken together these results suggest that KMP acts on both cellular organelles, reducing ER stress and promoting mitochondrial function, while Sal 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 KMP in the CNS, we assessed whether KMP treatment could rescue the behavioral defects observed in C9-500 mouse model of C9ORF72-ALS. A short treatment with KMP at 10 mg/kg was performed for two weeks via daily intraperitoneal (i.p.) injections [46] as from P138-P153, an age corresponding to the appearance of the pathology-associated behavioral phenotype in C9-500 mice [7, 29]. The reverse rotarod and hanging wire tests were performed pre-KMP treatment to obtain baseline measurement, followed by four consecutive measurements per week post-KMP treatment for half of the cohort of mice, whereas the other half received saline (scheme Fig. 5A). Reverse rotarod assay on the C9-500 mice cohort pre-KMP 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 KMP treatment C9-500 mice presented markedly improved motor coordination and balance (Supplementary video 1 and Fig. 5B, left graph). We further continued to chronically treat the mice every alternate day with KMP until the late stage of the disease; P190, which revealed that the ameliorated rotarod performance was sustained (Fig. 5B, right graph). Similarly, the hanging wire test revealed that after two weeks of KMP treatment, the average number of falls within the two minutes was reduced compared to the performance of the same cohort of mice pre-KMP treatment (Fig. 5C). Likewise, the time taken to first fall improved slightly between pre- and post-KMP treatment, suggesting that KMP treatment led to an overall amelioration in muscle endurance and function (Fig. 5D). We next assessed whether another compound; Sal which is known to inhibit ER stress and has been shown to ameliorate ALS-associated pathological symptoms in other familial ALS mouse models [5, 47, 48], could also have a beneficial outcome in C9-500 mice. Like KMP treatment regime, Sal treatment was performed on symptomatic P145 C9-500 mice lasting until P170 (Fig. 5E). This treatment led to no improvement in the reverse rotarod performance (Fig. 5F). Moreover, hanging wire tests revealed no significant amelioration in muscle endurance or stamina as measured by number of falls within two minutes (Fig. 5G) or time to the first fall (Fig. 5H). It is noteworthy that unlike studies done on SOD1-G93A mice, wherein Sal treatment was neuroprotective [5], in the C9-500 mice model, we found no protective effect of Sal, 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 KMP could dually act on inhibiting ER stress while sustaining mitochondrial function. We hypothesized that KMP 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 Ca²⁺ homeostasis, mitochondrial function, autophagy, and apoptosis [49, 50]. We performed an in-silico search for potential KMP interactors, which likely function at the MAM. The structure of KMP was uploaded to CB-Dock (http://clab.labshare.cn/cb-dock/php/dockingresult.php) for analysis of the docking potential of KMP to MAM-located molecules. These analyses led to the identification of GRP75 (75-kDA glucose-regulated protein) as being the strongest binding partner of KMP 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. 6A). The crystal structure of GRP75 revealed a significant interaction of KMP with the nucleotide-binding domain (NBD) of GRP75 (Fig. 6B). 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. 6C) and strongly docked to the NBD site of GRP75 (Fig. 6D). Further, analyses revealed that a substantial number of overlapping human proteins were targeted by both KMP and 17-AAG (Fig. 6E and Suppl. Table 1). However, literature analysis revealed that 17-AAG indirectly increases the expression level of GRP75 [51], while inhibiting HSP90 chaperone family, thereby impairing protein folding of multiple client substrates and autophagy that is impaired in C9ORF72-ALS. KMP enhances mitochondrial Ca²⁺ uptake, by a yet unidentified mechanism involving the binding to the mitochondrial uniporter, in traumatic brain injury model (TBI) [52]. Interestingly, no binding affinity of Sal 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 KMP could exert neuroprotection via modulating GRP75 expression or mode of action, 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 KMP-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, KMP treatment from symptomatic stage until the end stage of disease revealed increased iP3R-VDAC1 interactions versus saline-treated mutant spinal MNs of C9-500 mice (Fig. 7A). Since saline-treated mutant C9-500 MNs revealed dramatically reduced iP3R-VDAC1 interactions compared to WT MNs, and this reduced interaction was normalized after KMP treatment, indicated that KMP could potentially attenuate mitochondrial dysfunction by promoting optimal Ca²⁺ uptake by mitochondria. Thus, we examined the physiological process of Ca²⁺ 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 Ca²⁺ uptake. Fluo-4AM was combined with an intracellular buffer that eliminated cytosolic and ER Ca²⁺ signals [34], thereby enabling specifically the measurement of mitochondrial Ca²⁺ uptake. A striking deficit was observed in mitochondrial Ca²⁺ uptake in C9-500 cortical neurons as observed by significantly reduced Ca²⁺ transients compared WT cortical neurons. These deficits in Ca²⁺ uptake by mutant mitochondria were efficiently eliminated by treating cortical neurons with KMP, confirming our finding that KMP elicits its effect at the MAM, modulating the mitochondrial uptake of Ca²⁺ (Fig. 7B). We next measured GRP75 expression in spinal MNs to assess whether KMP modulates GRP75 expression. MNs from both WT and C9-500 mice treated with KMP displayed augmented GRP75 expression (Fig. 7C). Of note the degree of enhanced GRP75 expression was much higher in mutant C9-500 MNs, 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 2). 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. 7D). 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 MNs using PLA. Endogenous GRP75 interactions with ATP5J was observed in both WT and C9-500 MNs, albeit strongly at P125 mutant MNs. However, akin to reduced iP3R-VDAC1 interactions at P240, ATP5J-GRP75 were much lower in late-stage mutant MNs, indicative of advanced mitochondrial dysfunction and degeneration (Fig. 7E). As KMP treatment augmented GRP75 expression, we examined ATP5J expression in response to KMP treatment. While ATP5J levels were reduced in P240 MNs, likely reflecting the reduced GRP75 expression, spinal MNs treated with KMP even at end stage presented high ATP5J immunoreactivity, reflecting an overall beneficial effect of KMP on mitochondria via the GRP75-ATP5J pathway (Fig. 7F).

Discussion

ALS is a multifactorial disease, with complex overlapping clinical symptoms, and a fatal outcome within 3–5 years of diagnosis [53, 54]. Several studies have indicated that ER stress plays a pivotal role in the pathophysiology of ALS [2, 55], thus targeting ER stress is an interesting objective for therapeutic intervention. Besides, ER stress, mitochondrial dysfunction associated with oxidative stress, mitochondrial Ca²⁺ uptake deficits and bioenergetic deficits is closely associated with ALS pathophysiology [56–59], therefore identifying compounds that can alleviate both mitochondrial and ER impairments in ALS is extremely important to the field of ALS. In this study, we identified and explored the potential of a dietary flavanol KMP 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 KMP harbours the potential to attenuate ER stress and UPR signaling in vivo in symptomatic C9-500 rodent MNs as well as in vitro in human C9ORF72 patient-derived iMNs. Moreover, we provide compelling evidence for the ability of KMP to restore ATP production and normalize mitochondrial function in C9ORF72 ALS. This is noteworthy, as previous compounds such as Sal have been shown to interfere only with the PERK/ P_i-eIF2α -UPR pathway [5, 10, 60], or by suppressing the activity of integrated stress response, upstream of the PERK/ P_i-eIF2α -UPR pathway [37]. Moreover, drugs like Sal or guanabenz enhance the persistent translation inhibition by P_i-eIF2α, thereby, accelerating neurodegeneration in a model of prion disease [61] or in mutant male SOD1 mice [62]. In addition, both these compounds elicit toxicity on other organelles such as nephrotoxicity observed after chronic Sal administration [63] or blood pressure dysregulation after guanabenz administration [64]. 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, KMP and its derivatives are natural dietary phytochemicals, lacking evident toxicity, and are known to exhibit antioxidant, anti-inflammatory, anticancer, and neuroprotective activity [65].

Notably, we found that treatment with KMP initiated after the onset of pathological symptoms was able to abrogate intrinsic mitochondrial deficits present in C9ORF72 neurons. These data are remarkable, as KMP normalized mitochondrial ATP levels in human C9ORF72 iMNs, an important determinant of overall adequate neuronal energy supply and optimal function. While Sal as expected, did inhibit ER stress, it did not ameliorate mitochondrial function either in vivo in C9-500 MNs or in vitro in human iMNs. On the contrary, Sal has been shown to promote cell death in cancer by activating the ISR and inducing mitochondrial oxidative stress, thereby irreversibly damaging mitochondria. Further, Sal treatment in glucose-deprived conditions leads to the upregulation of mitochondrial ROS, thereby inducing mitochondrial stress and dysfunction [66]. Our finding that KMP ameliorated mitochondrial dysfunction and inhibited ER stress may be linked to its ability to regulate Ca²⁺ signaling, especially as a key process that links these ER-mitochondria interactions. The transfer of Ca²⁺ from the ER to mitochondria via the MAMs may lead to either pro-survival or pro-death effects, likely contributing to elevated mitochondrial ATP generation to cope with increased energy demands during mitochondrial stress. Conversely, enhanced mitochondrial Ca²⁺ may promote apoptosis [67]. Previous studies have shown that KMP alleviates oxidative stress and apoptosis during lung ischemia–reperfusion injury, by enhancing mitochondrial membrane potential and inhibiting the opening of mitochondrial permeability transition pores [68]. Importantly, KMP has been shown to control mitochondrial Ca²⁺ regulation by directly activating the mitochondrial Ca²⁺ uniporter (MCU) in a concentration-dependent manner. Even at a low concentration such as 1 μM of KMP treatment, practically doubled the uptake of mitochondrial Ca²⁺ [69]. Moreover, recent, studies, have also identified a mitochondria-mediated control of the UPR signaling. PIGBOS; a microprotein localized to the mitochondrial outer membrane controls UPR responses during stress. PIGBOS interacts with the ER protein CLCC1 at MAM contact sites and changes in PIGBOS expression led to enhanced UPR and increased cell death [70]. Therefore, based on our findings, it is very likely that KMP treatment harbors the ability to counteract mitochondrial Ca²⁺ uptake deficits and dysfunction in C9ORF72-ALS [6, 7], thus reducing mitochondrial stress and promoting optimal mitochondrial function and neuroprotection. KMP 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 KMP 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 MNs, and improved motor behaviour. We believe that these findings enhance the predictive value that KMP 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, KMP treatment inhibited β-A structure formation by hindering amyloid fibril elongation [71]. Yet in another study treatment with KMP 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 [72]. In Parkinson´s disease, KMP ameliorated nigrostriatal dopaminergic neuron lesions; by inhibiting interleukin (IL) 1β, IL-6, and TNFα production, highlighting the anti-inflammatory activity of KMP [73].

The mechanism via which KMP elicits its anti-ER stress properties remains unclear. However, studies have shown that KMP inhibits the expression of BiP, thereby inhibiting the activation of UPR sensors PERK, ATF6, and IRE1 [17]. These findings fit well with our observations of reduced BiP and P_i-eIF2α expression after KMP treatment. Our data from in silico modeling identified that KMP 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 Ca²⁺ 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 MNs [7]. Our PLA data suggests that within the C9-500 spinal MNs, KMP enhances iP3R-VDAC1 interactions, which aids in the optimal mitochondrial Ca²⁺ uptake, important for the proper ETC process. Of note, our data also revealed the ability of KMP to enhance the expression of GRP75 and its interacting partner ATP5J in MNs, suggesting that KMP could act on the process regulating protein translation. Interestingly, like quercetin yet another flavanol, KMP might positively modulate gene expression [27, 28]. However, further experiments are needed to dissect how KMP impacts GRP75 expression and iP3R-VDAC1 function in the context of C9ORF72-ALS.

Given the translation potential of our findings, we measured ER stress and mitochondrial function in human C9ORF72 patient-derived iMNs and found that KMP 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, and improved muscle and behavioral phenotype highlight the beneficial potential of KMP 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 KMP, via the regulation of GRP75 expression and function. These observations suggest that KMP is a likely strong candidate for the prevention/slowing down of disease pathology in familial ALS cases and could support future combinatorial therapies such as the dual treatment with the approved medication Riluzole that reduces excitotoxicity in ALS.

In conclusion, KMP 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 KMP 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, KMP is a promising compound for diseased MNs, meriting further study as a promising drug candidate in ALS.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

ALS:: Amyotrophic lateral sclerosis
a.u.:: Arbitrary units
Ca²⁺ :: Calcium
dNs:: Direct reprogramming of fibroblasts to neurons
FTD:: Frontal temporal dementia
iPSC:: Induced pluripotent stem cells
ISR:: Integrated stress response
iMNs:: IPSC-derived motor neurons
KMP:: Kaempferol
Min:: Minutes
MAMs:: Mitochondria-associated membranes
MNs:: Motor neurons
PTPIP51:: Protein tyrosine phosphatase interacting protein 51
Sal:: Salubrinal
TU:: Tunicamycin
UPR:: Unfolded Protein response

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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 and all Saxena lab members for support and discussions.

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.

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Authors and Affiliations

Department of Physical Medicine and Rehabilitation, University of Missouri, Columbia, MO, USA
Paulien Hermine Smeele, Julieth Andrea Sierra-Delgado & Smita Saxena
NextGen Precision Health, University of Missouri, Columbia, MO, USA
Paulien Hermine Smeele, Julieth Andrea Sierra-Delgado & Smita Saxena
Institut Neuromyogène, Pathophysiology and Genetics of the Neuron and Muscle, Inserm U1315, CNRS, Université Claude Bernard Lyon I, UMR 5261, 69008, Lyon, France
Federica Pilotto & Olivier Scheidegger
Department of Neurology, Inselspital University Hospital, Bern, Switzerland
Federica Pilotto, Rim Diab, Martina Schobesberger & Smita Saxena

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Federica Pilotto
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Paulien Hermine Smeele
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Olivier Scheidegger
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Rim Diab
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Martina Schobesberger
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Julieth Andrea Sierra-Delgado
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Contributions

F.P., S.S. conceived the study and wrote the manuscript. F.P., performed experiments and data analyses with contributions from P.H.S., R.D., and J.A.S.D. M.S. and O.J. performed muscle histology and analyses. O.S. and S.S. provided reagents. S.S. supervised the overall project, all authors read and commented on the manuscript.

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Correspondence to Smita Saxena.

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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.

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Supplementary Information

40478_2025_1927_MOESM1_ESM.eps

Supplementary material 1: Figure S1. 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.

40478_2025_1927_MOESM2_ESM.pdf

Supplementary material 2: Table S1. List of overlapping human proteins targeted by both kaempferol and 17-AAG. Supplementary Table 2: The interactome of GRP75 as revealed by mass spectrometry analyses, note the strong interaction between the established association of GRP75 with VDAC1.

Supplementary material 3: Video: Inverse rotarod measurement showing ameliorated motor coordination in C9-500 mice after Kaempferol treatment

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Pilotto, F., Smeele, P.H., Scheidegger, O. et al. Kaempferol enhances ER-mitochondria coupling and protects motor neurons from mitochondrial dysfunction and ER stress in C9ORF72-ALS. acta neuropathol commun 13, 21 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40478-025-01927-y

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Received: 02 October 2024
Accepted: 06 January 2025
Published: 01 February 2025
DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40478-025-01927-y

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

Abstract

Graphical abstract

Introduction

Experimental procedures

Mice strains

In vivo/in vitro drug treatments

Reverse rotarod and hanging wire test

iPSC differentiation into motor neurons (iMNs)

Generation of direct fibroblast-induced neurons (dNs)

Immunofluorescence on iMNs

Immunofluorescence and immunohistochemistry of rodent tissue

Muscles histology

Imaging and image analysis

Proximity ligation assay (PLA) in perfused mouse spinal cord tissue

Colorimetric staining of mitochondria complexes

Mitochondrial calcium imaging

CB-Dock analyses and PharmaMapper

Statistical analysis

Results

Kaempferol protects human iMNs and fibroblast-induced neurons (dNs)

Kaempferol treatment protects MNs and reduces pathological aggregates in a rodent model

Kaempferol acts on the ER stress pathway and inhibits UPR signaling in vivo

Kaempferol restores mitochondria electron transport chain function in C9-500 mice

A short treatment regime with kaempferol selectively ameliorates behavioral hallmarks in symptomatic C9-500 mice

Kaempferol potentially elicits its action by binding to GRP75

Kaempferol enhances IP3R-VDAC1 interactions, promoting optimal mitochondrial function via GRP75

Discussion

Data availability

Abbreviations

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Animal experiments

Human fibroblast-derived cells

Consent for publication

Competing interests

Additional information

Publisher's Note

Supplementary Information

40478_2025_1927_MOESM1_ESM.eps

40478_2025_1927_MOESM2_ESM.pdf

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