Olesoxime delays muscle denervation, astrogliosis, microglial activation and motoneuron death in an ALS mouse model

Neuropharmacology 62 (2012) 2346e2353

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Olesoxime delays muscle denervation, astrogliosis, microglial activation and motoneuron death in an ALS mouse model C. Sunyach a, b, M. Michaud c, T. Arnoux c, N. Bernard-Marissal a, b, J. Aebischer a, b, V. Latyszenok c, C. Gouarné c, C. Raoul a, b, R.M. Pruss c, T. Bordet c, B. Pettmann a, b, * a b c

INSERM-Avenir team, Mediterranean Institute of Neurobiology, Parc Scientifique de Luminy, 163, route de Luminy, 13273 Marseille Cedex 9, France Université de la Méditerranée, UMR S901, Aix-Marseille 2, 13009, France TROPHOS, Parc Scientifique de Luminy, Case 931, 13288 Marseille Cedex 9, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 November 2011 Received in revised form 31 January 2012 Accepted 13 February 2012

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The pathology is mimicked to a striking degree in transgenic mice carrying familial ALS-linked SOD1 gene mutations. Olesoxime (TRO19622), a novel neuroprotective and reparative compound identified in a high-throughput screen based on motoneuron (MN) survival, delays disease onset and improves survival in mutant SOD1G93A mice, a model for ALS. The present study further analyses the cellular basis for the protection provided by olesoxime at the neuromuscular junctions (NMJ) and the spinal cord. Studies were carried out at two disease stages, 60 days, presymptomatic and 104 days, symptomatic. Cohorts of wild type and SOD1G93A mice were randomized to receive olesoxime-charged food pellets or normal diet from day 21 onward. Analysis showed that olesoxime initially reduced denervation from 60 to 30% compared to SOD1G93A mice fed with control food pellets while at the symptomatic stage only a few NMJs were still preserved. Immunostaining of cryostat sections of the lumbar spinal cord with VAChT to visualize MNs, GFAP for astrocytes and Iba1 for microglial cells showed that olesoxime strongly reduced astrogliosis and microglial activation and prevented MN loss. These studies suggest that olesoxime exerts its protective effect on multiple cell types implicated in the disease process in SOD1G93A mice, slowing down muscle denervation, astrogliosis, microglial activation and MN death. A Phase 3 clinical study in ALS patients will determine whether olesoxime could be beneficial for the treatment of ALS. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Amyotrophic Lateral Sclerosis Therapeutics Motoneuron Neuromuscular junctions Astrogliosis Microgliosis

1. Introduction Amyotrophic Lateral Sclerosis (ALS) is a progressive muscle and motoneuron (MN) wasting condition leading irrevocably to paralysis and death within 2e5 years from diagnosis. Treatment is confined to the only FDA-approved drug, Riluzole, whose effect was demonstrated almost 20 years ago and shows a significant albeit small effect on survival, extending the lifespan of patients with ALS by about 3e6 months (Lacomblez et al., 1996). There is therefore continued urgent requirement for new drugs to help clinicians and patients fight against muscle denervation and MN death, to improve quality of life and delay fatal outcome. The clinical and neuropathological hallmarks of ALS, including selective spinal MN degeneration and glial activation * Corresponding author. INSERM-Avenir team, The Mediterranean Institute of Neurobiology, INMED, 163, route de Luminy, 13273 Marseille Cedex 09, France. Tel.: þ33 491828198; fax: þ33 491828105. E-mail address: [email protected] (B. Pettmann). 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2012.02.013

are mimicked to a striking degree in transgenic mice carrying mutations in the SOD1 gene, which are responsible for 20% of the familial forms of the disease (Bruijn et al., 2004; Kato, 2008). These mouse models e particularly the SOD1G93A model allow investigators to decipher the presymptomatic sequence of molecular and cellular events leading to muscle weakness, paralysis and death. The SOD1G93A mice develop a disease with a highly reproducible and robust pathological time line (Kanning et al., 2010). It is hence a precious tool to test drug candidates not only for their impact on disease onset and progression but also to scrutinize the cellular basis of their potential protective effect. Through a phenotypic cell-based screening approach using MN survival as a relevant read out, we previously identified olesoxime (Cholest-4-en-3-one, oxime; TRO19622), a novel neuroprotective and reparative compound (Bordet et al., 2007). In vitro, olesoxime promoted motor neuron survival in the absence of trophic support in a dose-dependent manner. In vivo, olesoxime rescued motor neurons from axotomy-induced cell

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death in neonatal rats and promoted nerve regeneration following sciatic nerve crush in mice (Bordet et al., 2007). In a first preclinical study exploring the therapeutic potential of olesoxime in SOD1G93A mice we reported a 15-days delay in the onset of body weight loss in olesoxime-treated mice ((Bordet et al., 2007); Fig. 5a), along with a significant delay of approximately 11 days between weeks 14 and 21 in the decline in muscle strength ((Bordet et al., 2007); Fig. 5b). In addition, treatment with olesoxime also resulted in a significant increase in lifespan. Olesoxime-treated SOD1G93A mice lived 10% longer (13 days) than did vehicle-treated controls (Bordet et al., 2007). Altogether these results suggested that olesoxime acts on effectors governing early disease process rather than disease progression. Therefore, the present work extends our initial study by evaluating the protection provided by olesoxime at the neuromuscular junctions (NMJs) and the spinal cord, our goal being to better understand the cellular basis underlying the previously reported beneficial effect of the drug.

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2. Materials and methods 2.1. Animals All experiments with animals were approved by the National Ethics Committee on Animal Experimentation, and were performed in compliance with the European Community and National Directives for the Care and Use of Laboratory Animals. Transgenic SOD1G93A mice (line G1H) (Gurney et al., 1994) were obtained from Transgenic Alliance. SOD1G93A mice were maintained as hemizygotes by crossing transgenic males with non-transgenic females of the same B6/SJL genetic background (Jackson laboratories). Progeny were genotyped by PCR as previously described (Raoul et al., 2002). SOD1G93A copy numbers were regularly monitored by qPCR as previously described (Mead et al., 2011).

2.2. Drug, treatment and analysis paradigm Olesoxime (Cholest-4-en-3-one, oxime; TRO19622) was provided by Trophos SA (Marseille, France). To determine olesoxime effect on disease manifestations and evolution in SOD1G93A mice, four experimental groups were formed. Genderbalanced wild type and transgenic mice were randomly assigned to receive olesoxime-charged food pellets (Ole) or control diet (chow) (ALTROMIN, Lage,

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Fig. 1. Olesoxime transiently preserves neuromuscular junctions in SOD1G93A mice gastrocnemius muscle. Representative illustrations of innervated and denervated motor endplates (a) identified by staining of post-synaptic compartment with a-bungarotoxin (red); and axons with anti-neurofilament antibodies (green). NMJs were considered completely or partially innervated when endplates appeared in yellow (co-localization of pre- and post-synaptic compartments) or denervated when presynaptic compartment was absent (red). Quantitative analysis of innervated NMJs at D60 (b) and D104 (c) in control (chow-) and olesoxime (Ole-) treated wild type (WT) and SOD1G93A animals. Data are shown as percentage of innervated endplates. Scale bar represents 50 mm. Each column shows average  S.E.M. (n ¼ 5e8; * P < 0.05, ** P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Olesoxime protects SOD1G93A motoneurons. Illustrations of VAChT staining of lumbar spinal cord sections (a) showing MNs with distinct nucleoli (insert) in chow- and Oletreated wild type (WT) and SOD1G93A mice. Number of axons in ventral root (L5) of sciatic nerve of mice at D104, counted on sections stained with toluidine blue (b). Histogram shows mean values  S.E.M. (n ¼ 8). (c) MNs were counted on 56 sections of lumbar spinal cord from D104 mice. Graph shows mean values per animals  S.E.M (n ¼ 8; **P < 0.01).

Germany; control diet for rodents: C1000; the same diet is enriched with 600 mg of olesoxime/kg of food pellets) from day 21 (D21) onward. Body weight was recorded weekly. Five and eight animals of each group were sacrificed at D60 and at D104 respectively. Satellite mice (n ¼ 4) were kept to assess drug concentration (see below) in plasma, spinal cord and brain collected at D60 and D104. 2.3. Assessment of olesoxime plasma, cerebral and spinal concentrations At sacrifice (D60 and D104), blood was collected into lithium-heparin tubes and centrifuged at 400 g to obtain plasma. Samples were stored frozen (20  C) until analysis. Brain and spinal cord were dissected immediately after intracardiac PBS perfusion. Cerebral, spinal and plasma concentrations of olesoxime were

determined by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) detection. 2.4. Immunohistochemical analyses Mice were anaesthetized and the right leg gastrocnemius muscle was immediately dissected out, weighed and frozen in cold isopentane. After cauterization, mice were perfused transcardiacally with Sorenson’s buffer 0.2 M followed by 4% paraformaldehyde (Electron Microscopy Sciences). The ventral roots of the sciatic nerve (L4-L6) were dissected and fixed overnight in 4% paraformadehyde in Sorenson’s buffer at 4  C. Spinal cord and left leg gastrocnemius muscle were retrieved and incubated in 20% sucrose in PBS overnight before embedding in Optimal Cutting

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Temperature compound (OCT, CML). For all subsequent analyses, quantifications were performed blindly regarding the genotype and the treatment group.

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Fig. 3. Olesoxime rescues motoneurons from death triggered by FasL but not by LIGHT. Mouse MNs were cultured for 24 h and then incubated with 100 ng/ml of FasL (a) or human sLIGHT (b) alone or together with 10 mM olesoxime in 0.5% DMSO containing 0.1% dialysed BSA. DMSO was used as a control. MN survival was determined 48 h later and expressed relative to non-treated cells. Graph shows mean values  S.E.M. (n ¼ 3, independent experiments with triplicates; ns: P > 0.05, ***P < 0.001).

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2.4.1. Spinal cord analysis Eighteen-micrometer-thick spinal cord cryosections were collected onto Superfrost Plus Slides (CML), washed with PBS and incubated for 1e2 h with PBS supplemented with 4% BSA, 4% heat-inactivated goat serum and 0.1% Triton X100. Sections were then incubated with the appropriate primary antibody overnight: rabbit anti-VAChT 1/1000 (Sigma-Aldrich), mouse anti-GFAP 1/500 (MAB 360, Millipore), rabbit anti-Iba1 1/500 (Waco) antibodies. Spinal cord sections were washed 3 with PBS, incubated with secondary biotinylated antibody (Invitrogen), washed 3 with PBS and incubated in Vectastain peroxidase/DAB detection system following the manufacturer’s instruction (Dako). Sections were washed in PBS, followed by a last wash in distilled water and mounted in Mowiol mounting medium. Pictures were taken with an Apotome Axio Imager Z2 microscope (Zeiss) using 20 and 60 objectives. Immunoquantification was performed using AxioVision software. 2.4.2. Muscle NMJ counting Thirty five-micrometer-thick longitudinal sections were collected onto Superfrost Plus Slides. Tissue sections were dried and permeabilized in blocking solution (5% BSA in PBS, 0.5% Triton X-100) at 37  C for 2 h. Rabbit polyclonal antineurofilament 145 (Chemicon) antibodies were diluted (1/1000) in the same blocking solution and incubated at 4  C, overnight. Anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen) was applied on sections together with a-bungarotoxin-tetramethylrhodamine conjugate (Invitrogen) both at a 1/1000 dilution in PBS, 1% BSA and incubated 2 h at room temperature before washing and

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Fig. 4. Exposure to olesoxime reduces microglial activation in SOD1G93A lumbar spinal cord. Illustrations of lumbar spinal cord sections stained with anti-Iba1 antibody (a, c). Number of Iba1-positive cells per ventral horn of lumbar spinal cord section at D60 (b) and D104 (d) in chow- and Ole-treated wild type (WT) and SOD1G93A mice. Columns represent average of n ¼ 3e5 (D60) and n ¼ 8 (D104) mice of each group, with 20 ventral horns counted per animal. Results are expressed  S.E.M. (**P < 0.01).

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Fig. 5. Olesoxime decreases astrocytes activation in SOD1G93A lumbar spinal cord. Astrocytosis assessed by GFAP immunostaining of ventral horns of the spinal cord lumbar part (a, c). Number of GFAP-positive cells per ventral horn of lumbar spinal cord section at D60 (b) and D104 (d) in chow- and Ole-treated wild type (WT) and SOD1G93A mice. Columns represent average of n ¼ 3e5 (D60) and n ¼ 8 (D104) mice of each group, with 20 ventral horns counted per animal. Results are expressed  S.E.M. (*P < 0.05; **P < 0.01).

mounting in Mowiol. Specimens were examined under an Axio Imager Z2 microscope (Zeiss) to visualize stained end-plates. Innervated or denervated end-plates were counted on apoptome 35 mm Z-stacks as described (Fischer et al., 2004). 2.4.3. Histological analysis of spinal motor axons (L4-L6) After overnight fixation as described above, each sample was post-fixed in 1% osmium tetroxide in Sorenson’s buffer overnight at room temperature, dehydrated in serial alcohol solutions, and embedded in Epon resin (Electron Microscopy Sciences). Cross sections (1 mm-thick) were prepared and stained with 1% Toluidine blue. Axonal counting was performed on ventral roots of the nerve section using a semi-automated digital image processing program (Image J). 2.4.4. Histological analysis of gastrocnemius muscle Ten-micrometer-thick transverse gastrocnemius muscle cryosections were stained with eosine and hematoxylin. Muscle fiber areas were analyzed using an adapted tool from Image J manually corrected before surface area determination. 2.5. Motoneuron culture Motoneurons (MNs) were isolated from E12.5 CD1 embryos spinal cord as described (Arce et al., 1999) and modified by (Raoul et al., 2002) using iodixanol density gradient centrifugation. MNs were plated on poly-ornithine/laminin-coated wells at a density of 1500 cells/cm2 in the presence of neurotrophic factors (0.1 ng/ ml glial-derived neurotrophic factor (GDNF), 1 ng/ml brain-derived neurotrophic factor (BDNF) and 10 ng/ml ciliary neurotrophic factor (CNTF) in complete

Neurobasal medium (Arce et al., 1999) (Invitrogen). Surviving neurons were counted directly using a phase-contrast microscope. 2.6. Statistical analysis Statistical significance was determined by unpaired two-tailed t-test or by a oneway analysis of variance (ANOVA) followed by Newman-Keuls post hoc tests. All analyses were performed with GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Results are expressed as means  SEM values. P values less than 0.05 were considered significant.

3. Results 3.1. Evaluation of animal exposure to olesoxime To optimize the therapeutic exposure, mice were treated from weaning to days 60 (D60) or 104 (D104) with the compound formulated into food pellets at a loading dose of 600 mg/g (Magalon et al., 2011). To control the exposure of the animals to the drug, we assessed olesoxime concentration in plasma and central nervous system tissue (brain and spinal cord). The concentrations observed (Table 1) were very similar to those described in the previously

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published survival studies in SOD1G93A mice (Bordet et al., 2007) that received a dose of 30 mg/kg/day by subcutaneous injection. At the end point (D104), no significant differences were observed in the body weight of wild type or SOD1G93A animals, regardless of the treatment they received (Suppl Fig. 1a). 3.2. Olesoxime transiently maintains NMJ integrity in SODG93A mice The elimination of the neuromuscular junction (NMJ), more specifically of the post-synaptic apparatus, is one of the earliest events in the ALS disease process. It is followed by axonal degeneration and late-onset degeneration of motor neuron cell bodies. Taking the co-localisation of presynaptic neurofilament labeling with a-bungarotoxin post-synaptic stainingas a criteria of innervated endplates (Fig. 1a; left panel) and the absence of colocalisation as denervation (Fig. 1a; right panel), we quantified the innervation of the gastrocnemius muscle in D60 presymptomatic animals (Fig. 1b). In wild type mice, whether treated or not with olesoxime, virtually 100% of the muscle end-plates were innervated. As previously reported in transgenic animals (Fischer et al., 2004), only 40% terminal axons showed end plate innervation in chow-treated transgenic animals. Strikingly, in olesoximeexposed mice, 60% of the NMJs remained innervated indicating that in the early phase of the disease, before classical motor symptom onset, olesoxime significatively delays denervation of the gastrocnemius muscle (Fig. 1b). Examination of the stained muscle sections did not provide evidence of sprouting, indicating that olesoxime prevents denervation rather than promotes reinnervation. In symptomatic D104 animals (Fig. 1c), no changes were observed in the neuromuscular connections of wild type animals regardless of the treatment. In transgenic mice fed on control diet, the proportion of NMJs showing overlapping staining dropped to 24%, indicating the progression of the disease. Thirtythree percent of end-plates were innervated in olesoxime-treated SOD1G93A animals, showing a modest but still significant protection of NMJ integrity (Fig. 1c). Gastrocnemius muscle weight in SOD1G93A animals was found to be similar in olesoxime- or chowtreated animals (supp Fig. 1b) and although a marked increase in the proportion of small caliber muscle fibers was documented in transgenic animals versus wild type, no difference was observed between chow- or olesoxime-treated groups (Suppl Fig. 1c). Altogether these results indicate that despite the marked preservation of NMJs by olesoxime observed at early stage (D60), only modest (NMJ) or no (muscle weight and muscle fiber area) beneficial effects were seen on gastrocnemius atrophy at D104. 3.3. Olesoxime attenuates MN loss Denervation at endplates in the muscle is followed by a dyingback process of the motor axons and MN death (Fischer et al., 2004; Frey et al., 2000). We proceeded therefore to first analyze motor axons in ventral roots of the sciatic nerve, then measure MN survival. As Fischer and colleagues (Fischer et al., 2004) reported that pathological changes in the ventral roots were not seen until 80 Table 1 Plasma, brain and spinal cord olesoxime concentrations. Mice

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days, we only looked at axon numbers and calibers at D104. In the ventral root of the sciatic nerve corresponding to the L5 region of the spinal cord, a 50% loss of axons was observed in transgenic mice (Fig. 2b). Olesoxime did not protect large axons from degeneration nor prompt regeneration at D104 (Fig. 2b). MN survival was estimated by counting VAChT-positive neurons (Fig. 2a). A loss of 35% of MNs was observed in SOD1G93A mice as compared to their wild type littermates (Fig. 2c). Interestingly olesoxime-treated transgenic animals had 20% more anterior horn MNs in the lumbar spinal cord than chow-fed mice (Fig. 2c). These results suggest that although not efficient in preserving axons at this stage, olesoxime treatment partially prevented the degeneration of MNs soma. In the past and recently, we have identified two MN-selective death pathways, dependent on FasL (Raoul et al., 2002) and LIGHT (Aebischer et al., 2011) respectively. FasL triggers cell death via the mitochondrial apoptotic pathway, while LIGHT-triggered death operates through a novel and distinct pathway which is independent of the mitochondria. We have therefore tested the capacity of olesoxime to protect MNs in vitro from death induced by FasL or soluble LIGHT. Remarkably, olesoxime completely rescued MNs from FasL-induced death but was ineffective against death triggered by sLIGHT (Fig. 3). These results indicate that olesoxime protects MNs that specifically die through the mitochondrial pathway. 3.4. Long term treatment reduces microglial and astrocytes activation In addition to MN loss and muscle disconnection, evidence of reactive microglia and astrocytes has been observed in motor regions of the CNS in ALS-linked transgenic mice (Fischer et al., 2004; Henkel et al., 2009; Turner and Talbot, 2008) and in sporadic and familial ALS patients (Haidet-Phillips et al., 2011; Schiffer et al., 1996). Reactive astrocytes and microglia are characterized by the upregulation of Iba1 (Ionized calcium binding adaptor molecule 1) and GFAP (Glial Fibrillary Acidic Protein) quite early in the disease process, before overt loss of MNs occurs (Fischer et al., 2004; Saxena et al., 2009). We therefore looked at the effect of olesoxime on microglial and astroglial activation at presymptomatic (D60) and symptomatic (D104) stages of the disease. Lumbar sections of the spinal cord anterior horn adjacent to those used to evaluate MN death were labeled with Iba1 and GFAP to count microglial cells and astrocytes. In D60 and D104 wild type mice, microglia are present, but in a so called “surveying state” with a low expression of Iba1 (Fig. 4a and c). At presymptomatic stage (D60), their SOD1G93A littermates present an increased number of microglial cells (Fig. 4a and b), which have changed their morphology (Fig. 4a, inserts) and express higher levels of Iba1. Olesoxime exerts a modest but not significant effect on microglial activation, reducing the number of Iba1 positive cells by 13%. Interestingly, at symptomatic stage (D104) the number of Iba1-labeled amoeboid cells increases in chow-treated transgenic mice (Fig. 4c and d) but remains almost unchanged in drug-treated animals as compared to D60 (compare Fig. 4b and d). This reflects a 29% decrease between olesoxime-treated and untreated transgenic groups. Observations on astrogliosis appeared to be very similar. At D60, SOD1G93A mice already showed signs of astrocytosis as the number of GFAP-stained cells with thick soma and processes, a morphology typical of reactive astrocytes, increased (Fig. 5a and inserts, b). In olesoximefed transgenic mice the number of these cells was slightly but significantly reduced (15%). As expected, astrogliosis increased further by D104 in transgenic untreated mice, but the number of activated astrocytes observed in SOD1G93A that had received the drug was 25% less, remaining at a level similar to that observed at

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D60 (Fig. 5b and d). These results show that olesoxime, although not significantly preventing early onset of astrogliosis and microgliosis, prevents or substantially delays further progression of these phenomena. 4. Discussion The present report analyses the cellular basis underlying the protection provided by olesoxime in a mouse model of ALS. Our results clearly show that olesoxime exerts its protective effect on multiple aspects of disease development: it displays a robust but transient protection of neuromuscular connections and retards astrogliosis, microglial activation and MN death. Poor motor performances eventually ending in paralysis leading to respiratory failure and death in ALS are the consequences of the early event of muscle nerve disconnection. Preserving muscle innervation is therefore an important goal for therapeutic strategies against ALS. Delays in the decline of motor performance in SOD1G93A mice on a test of motor coordination, the grid test, due to olesoxime treatment have already been reported (Bordet et al., 2007) and we show here that the transient maintenance of neuromuscular junctions supports these improved motor performances. Degeneration of MNs remains the important feature accounting for the rapid progression seen in ALS. We show here that olesoxime prevents death of MNs in SOD1G93A mice, but to a limited extent only (20%). MNs can be classified in different subtypes (Kanning et al., 2010) that are not equal in their sensitivity to different death inducers. In this respect we have previously demonstrated that they display intrinsic differences in their susceptibility to different death pathways. In particular, we showed that Fas ligand triggers an MN-restricted death pathway (Raoul et al., 2002) to which SOD1G93A MNs present an increased susceptibility (Raoul et al., 2006). The functional involvement of the Fas death pathway in MN degeneration has since been confirmed in vivo in ALS mouse models (Locatelli et al., 2007; Petri et al., 2006). Interestingly, we recently deciphered another MN-selective death pathway, which is dependent on LIGHT (Aebischer et al., 2011) and elevated levels of LIGHT were detected in the spinal cord of patients with sporadic ALS. Thus, a contribution of the LIGHT-induced death pathway was proposed in human ALS pathogenesis (Aebischer et al., 2012). Importantly, activation of the Fas- and LIGHTinduced death pathways were shown to be additive (Aebischer et al., 2011), suggesting two distinct MN populations. In the present work we show that olesoxime completely rescues MNs from FasL-induced death but is ineffective against death triggered by sLIGHT. As olesoxime has been shown to target the mitochondria, this differential effect of olesoxime is in accordance with the distinct signaling pathways triggered by FasL, dependent on the mitochondria (Raoul et al., 2002), and LIGHT, independent of the mitochondria (Aebischer et al., 2011; Bordet et al., 2007).While reinforcing our previous reports on in vitro neuroprotection of olesoxime (Bordet et al., 2007), this observation may also contribute to the only partial protection of SOD1G93A MNs we demonstrated in vivo. Overall the results obtained on different neuronal systems including MNs (Bordet et al., 2007; Bordet et al., 2010) (and the present study) point to a direct effect of olesoxime on MNs. As MNs have been shown to be a primary determinant of disease onset and of an early phase of disease progression in SOD1G93A (Boillee et al., 2006a), our results could account for the fact that the major beneficial effect of the drug is a delayed onset (Bordet et al., 2007). While a full understanding of the targets and mechanism of action of olesoxime is incomplete, our previous study showed that olesoxime targets outer mitochondrial membrane proteins: TSPO

(Translocator Protein 18 kD) and VDAC (Voltage-Dependent Anion channel) (Bordet et al., 2007). Interestingly, Israelson and colleagues (Israelson et al., 2010) recently demonstrated that SODG93A directly inhibits VDAC conductance at the mitochondrial outer membrane in SOD1G93A mice. Since VDAC is a key player in mitochondria-mediated apoptosis (Shoshan-Barmatz et al., 2008), it is likely that olesoxime neuroprotection reported both in vitro (Bordet et al., 2007) and in vivo involves uptake into mitochondria via TSPO and then direct interaction with VDAC1 ((Bordet et al., 2007; Martin et al., 2011) and present report). Maintenance of neuromuscular junction integrity may also be due to olesoxime’s ability to promote neurite outgrowth by enhancing microtubule dynamics (Rovini et al., 2010). These targets and actions could be responsible for the beneficial effects of olesoxime on glial cells as well as neurons. Activated glial cells, a component of neuroinflammation, are a common characteristic of neurodegenerative disorders. In the present study we report that in SODG93A mice, activation of glial cells occurs early (D60) and well before MN death. This observation is in agreement with previous observations in this mouse model (Fischer et al., 2004; Saxena et al., 2009). In recent years, the role of glia in the pathology of ALS as well as the interaction between astrocytes, microglia and MNs have been under intense scrutiny as new potential therapeutic targets. It is now well accepted that the surrounding cells are active players in MN dysfunction and death (Boillee et al., 2006a; Boillee et al., 2006b; Yamanaka et al., 2008) in mouse models of the disease as well as in FALS and in SALS (HaidetPhillips et al., 2011). Long term treatment with olesoxime, although not preventing a first stage of glial activation, blocks the progression of the number of Iba1-positive and GFAP-positive glial cells, an action that could limit glia toxicity toward motor neurons. Indeed, it has been shown in mouse models, FALS and SALS, that oxidative stress plays a major role through production of ROS and NO by glial cells (for review, (Barber and Shaw, 2010)). Olesoxime has been reported to be protective against trophic factor deprivation of MNs (Bordet et al., 2007) and target deprivation of neurons of the dorsal lateral geniculate nucleus (Martin et al., 2011). As both conditions lead to rapid generation of reactive oxygen species (ROS) (Martin et al., 2011; Estevez et al., 1998), we could propose that in SOD1G93A mice, olesoxime attenuates MN loss by reducing NO and superoxide production by glia. Alternatively (but not excluding the previous hypothesis), protection of MNs could lead to a reduction of activated astrocytes and microglia in the spinal cord. In conclusion, the present report, along with our previous studies, demonstrates that olesoxime not only promotes survival of MNs but also attenuates activation of surrounding glial cells also involved in the disease process. Thus olesoxime is a promising therapeutic candidate for such a multifactorial disease as ALS and potentially other neurodegenerative diseases. Data from a recently conducted clinical study in ALS patients and an ongoing study in patients with spinal muscular atrophy will determine whether olesoxime could be beneficial for the treatment of these motor neuron diseases. Conflict of interest VL, TA, MM, CG, RMP & TB are employees of Trophos SA. Acknowledgments This work was supported by the European Union under the 7th Framework Programme for RTD through the MitoTarget project (Grant Agreement HEALTH-F2-2008223388) and by the “Institut National de la Santé et de la Recherche Médicale” (Inserm). CS, NB and JA were supported by EC, Ministère de l’Enseignement

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