Early-Stage Treatment with Withaferin A Reduces Levels of Misfolded Superoxide Dismutase 1 and Extends Lifespan in a Mouse Model of Amyotrophic Lateral Sclerosis
Approximately 20 % of cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Recent studies have shown that Withaferin A (WA), an inhibitor of nuclear factor-kappa B activity, was efficient in reducing disease phenotype in a TAR DNA binding protein 43 transgenic mouse model of ALS. These findings led us to test WA in mice from 2 transgenic lines expressing different ALS-linked SOD1 mutations, SOD1G93A and SOD1G37R. Intraperitoneal administration of WA at a dosage of 4 mg/kg of body weight was initiated from postnatal day 40 until end stage in SOD1G93A mice, and from 9 months until end stage in SOD1G37R mice. The beneficial effects of WA in the SOD1G93A mice model were accompanied by an alleviation of neuroinflammation, a decrease in levels of misfolded SOD1 species in the spinal cord, and a reduction in loss of motor neurons resulting in delayed disease progression and mortality. Interestingly, WA treatment triggered robust induction of heat shock protein 25 (a mouse ortholog of heat shock protein 27), which may explain the reduced level of misfolded SOD1 species in the spinal cord of SOD1G93A mice and the decrease of neuronal injury responses, as revealed by real-time imaging of biophotonic SOD1G93A mice expressing a luciferase transgene under the control of the growth-associated protein 43 promoter. These results suggest that WA may represent a potential lead compound for drug development aiming to treat ALS.
Key WordsALS Neuroinflammation Withaferin A SOD1G93A SOD1G37R
Amyotrophic lateral sclerosis (ALS) is a fatal progressive degenerative disorder characterized by progressive muscle weakness, muscle atrophy, and eventual paralysis, leading to death within 2–5 years. About 5–10 % of patients inherit the disease, typically in an autosomal dominant manner [familial ALS (FALS)]. In 20 % of FALS, missense mutations have been identified in the gene coding for superoxide dismutase 1 (SOD1) [1, 2, 3].Various hypotheses have been proposed to explain the toxicity of SOD1 mutants, including protein aggregation [4, 5], oxidative stress , mitochondrial dysfunction , and excitotoxicity . TAR DNA binding protein 43 (TDP-43) is another protein detected in pathological inclusions of ALS and cases of frontotemporal lobar degeneration with ubiquitin inclusions [9, 10]. Dominant mutations in TARDBP, which codes for TDP-43, have been reported by several groups as a primary cause of ALS [11, 12, 13, 14, 15, 16], and may account for ~3.0 % of cases of FALS and ~1.5 % of sporadic cases.
Previously, we showed that treatment of a TDP-43 transgenic mouse model of ALS with Withaferin A (WA), an inhibitor of nuclear factor-kappa B (NF-кB) activity, ameliorated disease symptoms and pathological phenotypes such as reduction of denervated neuromuscular junctions and attenuation of neuroinflammation . These findings led us to test WA in mice from 2 transgenic lines expressing different ALS-linked SOD1 mutations, SOD1G93A and SOD1G37R. Importantly, recent studies by Frakes et al.  have demonstrated that in a SOD1G93A mouse model of ALS, motor neuron death involves activated microglia in a NF-κB dependent manner. WA is a steroid lactone found in the medicinal plant Withania somnifera. Semipurified root extract of W. somnifera consisting of withanolides and withanosides reversed behavioral deficits, plaque pathology, and accumulation of β-amyloid peptides and oligomers in the brains of amyloid precursor protein/presenilin-1 Alzheimer’s disease transgenic mice . WA exhibits a variety of beneficial effects, including antitumor, anti-inflammatory, and immunomodulatory properties . In addition, WA may act as an inducer of heat shock proteins (Hsps) .
Here, we investigated the effects of WA treatment on disease progression and pathological changes in 2 ALS mouse models expressing either SOD1G93A or SOD1G37R mutants. We report that when started early in disease pathogenesis, at time of onset of initial motor function deficits [22, 23], treatment with WA significantly extended the lifespan of SOD1G93A and SOD1G37R mice. WA treatment was associated with a reduction of neuronal stress, attenuated inflammation, upregulation of Hsp25 (mouse ortholog of Hsp27) and Hsp70, and a decrease in levels of misfolded SOD1 species.
Materials and Methods
Generation of Glial Fibrillary Acidic Protein–luciferase (luc)/SOD1G93A and Growth-associated Protein-43–luc/Green Fluorescent Protein/SOD1G93A Transgenic Mice
The transgenic glial fibrillary acidic protein (GFAP)–luciferase (luc) mice (FVB/N background) were obtained from Caliper (Caliper Life Sciences, Hopkinton, MA, USA). As previously described , the GFAP–luc mice were crossed with the transgenic SOD1G93A transgenic mice (C57/BL6; The Jackson Laboratory, Bar Harbor, ME, USA) to generate double transgenic GFAP–luc/SOD1G93A mice [25, 26]. The genotyping was performed as previously described . The presence of GFAP–luc transgene was assessed by polymerase chain reaction (PCR) with HotStar Taq Master mix Kit (Qiagen, Mississauga, ON, Canada) in 15 mM MgCl2 PCR buffer with the following primers: 5′GAAATGTCCGTTCGGTTGGCAGAAGC and 5′CCAAAACCGTGATGGAATGGAACAACA. The presence of the SOD1G93A mutant transgene was assessed by PCR as previously described . To confirm that the transgene copy number of SOD1G93A was not altered in the mice used for this study, the genomic SOD1 levels were evaluated by quantitative reverse transcriptase PCR using genomic DNA isolated from tail tissue. Analysis of the mouse housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase was used for normalization purposes. Oligoprimer pairs (used at concentration of 300 nm) were designed by GeneTool 2.0 software (Biotools Inc., Edmonton, AB, Canada) and their specificity was verified by blast in the GenBank database.
5′-GGCGCAGTAGGCAAGGTGGT and 5′-CAGCAGGATGCTCTCCAGTTC .
All experimental procedures were approved by the animal care ethics committee of Laval University and were in accordance with The Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care.
Analysis of Clinical Symptoms
The onset of weight loss was determined at the time when mice started to exhibit a decline of body weight after reaching a peak. The survival was defined as the loss of righting reflex (the age when the animal could not right itself within 30 s when placed on its side). Measurements of body weight and the loss of hind limb reflex were used to score the clinical onset of disease in SOD1G93A mice, as previously described . The SOD1G93A reflex score and body weight were measured every 2 days, beginning at 45 days. Scoring was performed in a blind manner by animal technicians who had no information about the genotype but had experience in grading SOD1G93A mice paralysis.
In Vivo Bioluminescence Imaging
As previously described, images were gathered using IVIS 200 Imaging System (Xenogen, Alameda, CA, USA) [24, 31]. Twenty minutes prior to the imaging session the mice received an intraperitoneal (i.p.) injection of D-luciferine, a luciferase substrate (150 mg/kg; Xenogen) dissolved in 0.9 % saline. The mice were then anesthetized with 2 % isoflurane in 100 % oxygen at a flow rate of 2 L/min and placed in the heated, light-tight imaging chamber. Images of lumbar spinal cord region of interest were collected using high sensitivity charge-coupled device camera with wavelengths ranging from 300 to 600 nm. Exposition time for imaging was 1 min using different fields of view and a F/1 lens aperture. The bioluminescence emission was normalized and displayed in physical units of surface radiance, photons/s/cm2/steradian [31, 32]. The light output was quantified by determining the total number of photons emitted per second using Living Image 4.1 acquisition and imaging software (PerkinElmer, Waltham, MA, USA). Region-of-interest measurements on the images were used to convert surface radiance (photons/s/cm2/steradian) to source flux or total flux of photons expressed in photons/s.
Administration of WA
WA was obtained from Enzo Life sciences (Farmingdale, NY, USA). WA was first dissolved in dimethyl sulfoxide (DMSO) and diluted in 0.9 % saline. The final concentration of DMSO was 10 %. The drug was made fresh every 2 weeks and was protected from light. Male and female transgenic mice and their transgenic littermates were divided randomly into the following 2 groups (n =15 per group): 1) transgenic controls, which received vehicle (0.9 % saline with 10 % DMSO); and 2) the transgenic WA treatment group, which received an i.p. injection of WA at a rate of 4 mg/kg body weight, twice a week. The treatment was initiated at early symptomatic stage (40 days of age) as recently proposed by Vinsant et al. [22, 23].
Tissue Collection and Immunofluorescence Microscopy
Mice were anesthetized by i.p. injection of chloral hydrate (10 mg/ml) and transcardially perfused with 30 ml 0.9 % NaCl, followed by ice-cold phosphate buffered saline (PBS) 1× buffered 4 % paraformaldehyde at pH 7.4. Tissue samples were then postfixed overnight in 4 % paraformaldehyde and equilibrated in phosphate-buffered 20 % sucrose. Spinal cords were cut at a thickness of 25 μm. The double immunofluorescence analysis was performed according to the following procedure. After 1–2 h air drying, sections were blocked in PBS containing 10 % goat serum and 0.25 % Triton X-100 for 30 min. Spinal cord sections were incubated using primary antibodies: 1 : 500 rabbit polyclonal antiglial fibrillary acidic protein (Dako, Carpinteria, CA, USA), 1 : 500 rabbit anti-ionized calcium binding adaptor molecule-1 (Iba-1; Wako Chemicals USA, Richmond, VA, USA), 1 : 50 rabbit polyclonal cyclic adenosine monophosphate-dependent transcription factor (ATF)-3 (Santacruz Biotechnology, Santa Cruz, CA, USA), and 1 : 500 mouse monoclonal neuronal nuclear antigen (Millipore, Temecula, CA, USA). Slides were washed in PBS containing 5 % goat serum and 0.25 % triton X-100, and incubated with the appropriate fluorescent-conjugated secondary antibodies (Alexa; Molecular Probes, Eugene, OR, USA) for 2 h at room temperature. A final wash was performed in PBS and slides were coverslipped with Fluoromount medium (Electron Microscopy Sciences, Hatfield, PA, USA).
Stereological Counts of Motor Neurons
Sections of horizontal spinal cord were Nissl stained to identify motor neurons in the lumbar spinal cord. The L3–L5 spinal cord sections were individually traced with a 40× microscopic observation and sampled under 400× magnification. The density of labeled cells was estimated by the optical fractionator method using Stereo Investigator software (MBF Biosciences, Williston, ND, USA). The counting parameters were the distance between counting frames (150 μm), the counting frame size (150 μm × 150 μm), the dissector height (10 μm), and the guard zone thickness (1 μm). Motor neurons were identified based on: 1) anatomic location (ventral horn/laminae 9); 2) presence of a distinct nucleolus within the plane of the optical dissector; and 3) a cross-sectional area ≥250 μm2. Results are expressed as the total number of motor neuron/mm3.
Immunoprecipitation and Western Blotting
At postnatal day 120, spinal cords were dissected out, rapidly frozen in liquid nitrogen, and stored at –80 °C for cytokine array, immunoprecipitation and Western blot analysis. Whole protein lysates from mouse spinal cords were extracted by homogenization of the tissues in TNG-T lysis buffer (50 mM Tris–HCl pH: 7.4; 100 mM NaCl; 10 % glycerol; 1 % Triton X), sonicated and centrifuged for 20 min at 9000 g at 4 °C. Blots were immunostained overnight at 4 °C with primary antibodies, Hsp25/27 (rabbit polyclonal antibody 1 : 2500; Cell Signaling, Danvers, MA, USA), Hsp70, (rabbit polyclonal antibody, clone D69, detects endogenous level of total HSP70 protein, at dilution of 1 : 1000; Cell Signaling), Hsf-1 (rat monoclonal antibody Ab-1, clone 4B4, 1 : 1000; Thermo Scientific, Waltham, MA, USA), Iba-1 (1 : 1000; Wako Chemicals), Toll-like receptor 2 (AB16894, 1 : 1000; Abcam, Cambridge, MA, USA). As previously described , immunoprecipitation experiments for misfolded SOD1 were done using the Dynabeads standard protocol (Invitrogen, Carlsbad, CA, USA). Briefly, Dynabeads were washed and coated with the mouse monoclonal antimisfolded SOD1 antibody B8H10 (2 h at room temperature), washed with PBS with Tween 20 and bovine serum albumin/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid–PBS and incubated overnight with 300 μg spinal cord lysate protein at 4 °C with rotation. After incubation, the beads were washed and fractioned on 14 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
The expression profile of inflammatory cytokines were performed with a mouse cytokine antibody array (Raybio Mouse Inflammation Antibody Array 1, Cat#AAM- INF-1; RayBiotech, Norcross, GA, USA) as previously described in detail . Protein samples were obtained by homogenization of WA-injected and vehicle-injected SOD1G93A spinal cord (n =3) at P120 in 1× cell lysis buffer with protease inhibitor cocktail (#P8340; Sigma, St. Louis, MO, USA) included in the RayBiotech kit. After extraction, samples were spun down at 13,000 rpm for 10 min at 4 °C and supernatant was used for the experiment. For each group (3 mice/group) samples were pooled together and incubated with the array membrane overnight at 4 °C. After washing in the washing buffer (included in the RayBiotech kit), membranes were incubated with biotin-conjugated antibodies overnight. Signal detection was performed according to the RayBiotech protocol, by exposing membranes to x-ray film (Biomax MR1; #8701302; Kodak, Rochester, NY, USA), and the obtained results analyzed using ImageJ software . Data are expressed in arbitrary units relative to appropriate positive control. Statistical analysis was performed by using a 2-tailed unpaired Student’s t test.
Flow Cytometry Analysis
Blood was collected from the submandibular vein of WA- and vehicle-injected mice at 112 and 125 days, as previously described , and sent for flow cytometry analyses (Centre hospitalier de l’Université Laval Hospital Research Institute’s Core Flow Cytometry Laboratory). The panel of antibodies (all from BD Biosciences, San Jose, CA, USA) used to evaluate the leukocytes from mice included CD4 (APC Rat Anti-Mouse CD4, clone RM4-5), CD8 (PE-CF594 Rat Anti-Mouse CD8a, Clone 53-6.7), CD25 (FITC Rat Anti-Mouse CD25, clone 7D4), CD45 (V500 Rat Anti-Mouse CD45,clone 3O-f11), FoxP3 (V450 Rat Anti-Mouse FOXP3, clone MF23), interleukin (IL)-4 (PE-Cy 7 rat Anti-Mouse IL-4, clone 11B11), and IL-10 ( PE Rat Anti-Mouse IL-10, clone JEs5-16E3). Samples were analyzed on a flow cytometer (BD LSRII; BD Biosciences) by a blinded individual.
Data were analyzed using Prism 5.0 (Graph Pad Software, La Jolla, CA, USA). Behavioral data were computed by performing 2-way analyses of variance (except when specified) followed by Bonferroni post-tests and survival data using Mantel–Cox log-rank tests. Optical densities for Iba-1 and GFAP staining, as well as quantification of Western blots, were analyzed by Image J followed by a 2-tailed Student’s t test.
WA Extends Survival in Transgenic Mice Overexpressing SOD1G93A or SOD1G37R Mutants
Reduction of Early Neuronal Injury Response Biophotonic Signals by WA Treatment in GAP-43–luc/gfp/SOD1G93A Mice
Treatment with WA extended survival in 2 different SOD1 mutant mouse models. Therefore, by using a live imaging approach and a cell type-specific reporter mouse, we further investigated potential therapeutic mechanisms and cellular targets. To visualize the effects of WA treatment in SOD1G93A mice in real time we took advantage of the GAP-43–luc/gfp reporter mice, recently generated and validated in our laboratory . Importantly, the results of our recent study revealed that the GAP-43 biophotonic signals imaged from the spinal cords of live SOD1G93A mice may serve as a valid biomarker to assess early neuronal injury response in SOD1 mutant-mediated disease . Moreover, immunofluoresence analysis revealed almost perfect co-relation between GAP-43-driven gfp transgene and ATF-3, known to be upregulated in injured and/or stressed neurons [35, 36, 37]. Double transgenic GAP-43–luc/gfp/SOD1G93A mice were generated by crossing heterozygous mice carrying the mutant SOD1G93A transgene with the heterozygous GAP-43–luc/gfp mice co-expressing reporter transgene, luc, and gfp, driven by the murine GAP-43 promoter. In this mouse model, an upregulation of GAP-43 (luciferase expression detectable as a bioluminescence/photon emission and gfp expression detectable by confocal microscopy) can be followed longitudinally in live animals using bioluminescence/biophotonic imaging and a high sensitivity/high resolution charge-coupled device camera.
WA Reduced the Level of the Misfolded SOD1 Species and Induced Upregulation of Hsps in SOD1G93A Mice
Neuroprotective Effects of WA in SOD1G93A Mice
Based on our the aforementioned results, we next examined whether WA treatment attenuated the loss of spinal motor neurons in SOD1G93A mice. Cryosections of the lumbar spinal cord (L3–L4) from 120-day-old SOD1G93A mice were Nissl-stained and cells with diameters >25 μm (motor neurons) were quantified . We found 32 % loss of motor neurons in the lumbar spinal cord of SOD1G93A mice when compared with wild-type mice at postnatal day 120 (SOD1G93A: 33.0 ± 1.5; wild-type: 48.3 ± 0.3; p ≤0.01) (Fig. 3F,G). In contrast, there was only 12 % loss of motor neurons at postnatal day 120 in SOD1G93A mice treated with WA when compared with wild-type mice (42.7 ± 0.9 motor neurons; n =3; p ≤0.05). Thus, early WA treatment led to a ~30 % increase of motor neuron survival at postnatal day 120 to the end stage of disease (Fig. 3G).
WA Treatment Suppressed Neuroinflammatory Signals in SOD1G93A Mice
WA Alters Cytokine Profiles in Spinal Cord Without Affecting Proliferation and Polarization of Peripheral Immune Cell Population
No Beneficial Outcome with Late Initiation of WA Treatment
Finally, to assess the effects of late treatment initiation (postnatal day 90) with WA on cytokines profiles, we evaluated the levels of different pro- and anti-inflammatory cytokines in the spinal cord of WA- and vehicle-treated mice at postnatal day 120. Our results confirmed an altered cytokine profile but, surprisingly, we observed alterations in levels of both anti- and proinflammatory groups of cytokines. Quantitative analysis revealed major changes in the levels of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6, between the 2 experimental groups (Fig. 7E–G). Remarkably, quantitative analysis showed a significant increase in the levels of anti-inflammatory cytokines in WA-treated group (Fig. 7H–J). In addition, levels of macrophage-CSF was also significantly increased in the WA treatment group with no significant change in levels of GM-CSF and granulocyte-CSF (Fig. 7K–M).
We next analysed the effect of late WA treatment on the population of T lymphocytes in the blood of SOD1G93A mice by flow cytometry. There was no significant difference in the population of Tregs, or IL-10 and IL-4 levels in blood between WA-treated and control animals (Fig. 7N–P), and quantitative analysis revealed no significant changes in the populations of CD4+, CD8+, and CD11b + at the periphery (Fig. 7Q–S). Taken together, our results suggest that when administered at advanced disease stage, WA was unable to induce significantly Hsp25 and Hsp70. Surprisingly, late initiation of WA treatment increased both anti- and proinflammatory cytokine levels in the spinal cord tissue. The late WA treatment did not have significant impact on the peripheral immune cells/immune response.
Our previous study revealed the beneficial effects of WA, including a reduction of inflammation and amelioration of motor deficits in a mouse model of ALS based on overexpression of the human TDP-43 transgene . Here, we report that WA treatment conferred neuroprotective effects with extension of lifespan in 2 mouse models of ALS with overexpression of different mutant SOD1 (SOD1G93A or SOD1G37R) (Fig. 1). WA was effective only when treatment was initiated early in disease pathogenesis, at the time of onset of motor function deficits, as recently reported by Vinsant et al. [22, 23].
Our analyses of SOD1G93A mice suggest that WA may exert protective effects through multiple pathways. It is well established that WA exerts potent anti-inflammatory effects [71, 72, 73], and our results confirmed that WA can reduce neuroinflammation in SOD1G93A mice when treatment is initiated at early stage of disease. For instance, we took advantage of double transgenic GFAP–luc/SOD1G93A mice in which astrocyte activation can be visualized throughout disease progression . The results of our in vivo imaging revealed an attenuation of astrogliosis by WA treatment at 8–10 weeks of age and then at 17 and 18 weeks of age in SOD1G93A mice (Fig. 4A,B). Immunofluorescence microscopy and immunoblotting further confirmed a decrease in GFAP and of Iba-1 signals in 17-week-old SOD1G93A mice treated with WA (Fig. 4C,D). As activated astrocytes and microglia can produce a variety of cytokines, with some having harmful effects , we further determined the effect of WA treatment on cytokine expression pattern in the spinal cord of SOD1G93A mice. Interestingly, early WA treatment resulted in a significant increase in the levels of IL-10 in lumbar spinal cord of SOD1G93A mice at 120 days of age (Fig. 5D). IL-10 is known to confer beneficial effects in several neuroinflammatory disease models, including experimental autoimmune encephalomyelitis, traumatic or excitotoxic spinal cord injuries, stroke, and Parkinson’s disease [74, 75, 76, 77, 78]. Conversely, WA caused a downregulation in the level of GM-CSF in spinal cord of SOD1G93A mice (Fig. 5H). GM-CSF is a proinflammatory cytokine, upregulated in various neurological disorders, such as Alzheimer’s disease, vascular dementia, and multiple sclerosis [79, 80, 81]. Thus, the reduction of inflammation by WA treatment in SOD1G93A mice may be owing, in part, to an upregulation of anti-inflammatory cytokine IL-10 and by a downregulation of proinflammatory cytokine GM-CSF. Moreover, we carried out FACS analysis of the blood to examine the effect of WA treatment on the lymphocyte population, specifically Tregs. A previous study on SOD1G93A mice revealed that the numbers of Treg cells are increased at early slowly progressing stages, augmenting IL-4 expression, and are then decreased when the disease rapidly accelerates, possibly through the loss of FoxP3 expression . In patients with ALS, the numbers of Tregs and expression levels for FOX-3 and IL-4 were inversely correlated with disease progression rates . However, our FACS analysis revealed no effect of WA on the number of Tregs in the blood or on thee levels of IL-10 or IL-4 (Fig. 6F–I). Taken together, our data provided no evidence of protective inflammatory responses through a modulation of peripheral Tregs.
The levels of misfolded SOD1 species in the spinal cord have been used as a valuable indicator of disease progression . Immunotherapeutic approaches aiming to reduce the levels of misfolded SOD1 species have been effective in delaying disease onset and progression in SOD1G93A mice [33, 35]. An upregulation of Hsps with the ensuing reduction in levels of misfolded SOD1 may constitute another mechanism by which WA may confer neuroprotection in SOD1G93A mice. As shown in Fig. 3 (C,D), WA treatment significantly increased the amount of Hsp25 (a mouse ortholog of Hsp27) in the spinal cord of SOD1G93A mice, which is line with a report that WA is an inducer of Hsps. Many reports have shown that Hsp27/25 protects against neuronal damage induced by FALS-related SOD1 mutant [46, 47, 83, 84, 85, 86]. Moreover, Hsp27/25 was found to inhibit the in vitro aggregation of SOD1G93A proteins . Thus, an upregulation of Hsp25 in WA-treated SOD1G93A mice may explain, in part, the reduction in levels of misfolded SOD1 species as determined by immunoprecipitation with the specific B8H10 antibody (Fig. 3A,B) and increased number of surviving motor neurons (Fig. 3F,G).
The combined results revealed an effective therapeutic effect of WA when treatment is initiated at onset of motor deficits in SOD1G93A mice, which has recently been reassessed to be at 30–40 days of age according to leaded grid test and treadmill gait analysis [22, 23]. However, when WA treatment was initiated at a later stage of disease (90 days of age), at a time coincident with detection of motor neuron death [22, 23], there was no beneficial effect on the survival of SOD1G93A mice (Fig. 7A). As shown in Fig. 7B, when administered after disease onset, WA lost its ability to upregulate Hsp25 and Hsp70. Interestingly, previous work by Maatkamp et al.  revealed that in SOD1G93A mutant mice, a decrease in Hsp25 protein expression precedes degeneration of large motor neurons. Taken together, these data suggest that a therapeutic intervention for ALS based on WA medication (and possibly some other therapeutic approaches) would need to be initiated early in the pathogenic process at time when cellular responses to stress or to inflammatory signals are still adequate. For instance, a late-onset initiation of WA administration in SOD1G93A mice caused increases in both anti- and proinflammatory cytokines (Fig. 7E–M), suggesting a marked deregulation of immune system responses at a late stage of disease.
If started at early disease stage, WA should be effective in attenuating deleterious neuroinflammatory responses and in conferring neuroprotection partly through an upregulation of Hsp25 and reduction of misfolded protein species. WA is a steroid lactone present in a medicinal plant, W. somnifera, which has been used for centuries in Ayurvedic medicine. The therapeutic effects of WA in various ALS mouse models suggest that WA should be considered as a promising lead compound for drug development aiming to treat ALS.
This work was supported by the Canadian Institutes of Health Research (CIHR), the Amyotrophic Lateral Sclerosis Society of Canada, and the Muscular Dystrophy Association, USA. We gratefully thank Christine Bareil, Geneviève Soucy, and Sophie Vachon for their technical help. J.K. holds a Senior Scholarship Award from Fonds de recherche du Québec en Santé (FRQS). J.-P.J. holds a Canada Research Chair Tier 1 in mechanisms of neurodegeneration.
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