Oxidative stress in skeletal muscle stimulates early expression of Rad in a mouse model of amyotrophic lateral sclerosis

Free Radical Biology & Medicine 48 (2010) 915–923

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Oxidative stress in skeletal muscle stimulates early expression of Rad in a mouse model of amyotrophic lateral sclerosis Benoît Halter a,b,1, José-Luis Gonzalez de Aguilar a,b,⁎,1, Frédérique Rene a,b, Susanne Petri c, Bastien Fricker a,b, Andoni Echaniz-Laguna a,b,d, Luc Dupuis a,b, Yves Larmet a,b, Jean-Philippe Loeffler a,b a

INSERM U692, Laboratoire de Signalisations Moléculaires et Neurodégénérescence, Faculté de Médecine, Université de Strasbourg, 67085 Strasbourg, France UMRS692, Université de Strasbourg, 67085 Strasbourg, France Department of Neurology and Clinical Neurophysiology, Medical School of Hannover, Hannover, Germany d Département de Neurologie, Hôpitaux Universitaires de Strasbourg, Strasbourg, France b c

a r t i c l e

i n f o

Article history: Received 24 June 2009 Revised 18 November 2009 Accepted 6 January 2010 Available online 14 January 2010 Keywords: Amyotrophic lateral sclerosis Denervation Ischemia–reperfusion Mutant SOD1 Oxidative stress Ras-related associated with diabetes Skeletal muscle Tempol Free radicals

a b s t r a c t Motor neuron degeneration and progressive muscle atrophy characterize amyotrophic lateral sclerosis (ALS) in humans and related mutant superoxide dismutase-1 (SOD1) transgenic mice. Our previous microarray studies on ALS muscle revealed strong up-regulation of Ras-related associated with diabetes (Rad), an inhibitor of voltage-gated calcium channels. The mechanisms controlling Rad expression in disease are unknown. We analyzed Rad expression in skeletal muscle from ALS patients and animal models and investigated whether it is regulated by oxidative stress. In mutant SOD1 mice, Rad up-regulation preceded motor symptoms and markedly increased as disease progressed. Increased Rad expression was also obtained in surgically denervated muscle. No clinical signs of denervation were seen in asymptomatic mice, however. We therefore suspected that muscular mutant SOD1 toxicity causes precocious Rad up-regulation. We confirmed the accumulation of reactive oxygen species (ROS) at asymptomatic stages, coincident with the rise in Rad expression. By subjecting muscle to ischemia–reperfusion, we observed ROS accumulation and Rad overexpression. The cell-permeative antioxidant Tempol inhibited the stimulatory effect of ischemia– reperfusion. Tempol also reduced Rad up-regulation after experimental denervation. Our study provides strong evidence for the implication of oxidative stress in modulating Rad expression, in association with the initiation and progression of ALS muscle atrophy. © 2010 Elsevier Inc. All rights reserved.

Amyotrophic lateral sclerosis (ALS) is an adult-onset degenerative disease characterized by selective loss of upper and lower motor neurons, progressive muscle atrophy, paralysis, and death. About 90% of cases present with unknown etiology and are so-called sporadic, in opposition to those resulting from genetic disorders, which are for the most part dominantly inherited [1]. Twenty percent of familial cases are caused by missense mutations in the gene encoding Cu/Znsuperoxide dismutase (SOD1), a free radical-scavenging enzyme that protects cells against oxidative stress [2]. The way by which these mutations trigger ALS is still debated. Indeed, it is thought that the disease arises from the interplay of multiple toxic mechanisms that affect not only motor neurons but also nonneuronal neighboring cells Abbreviations: AChRα, acetylcholine receptor α subunit; ALS, amyotrophic lateral sclerosis; NMJ, neuromuscular junction; Rad, Ras-related associated with diabetes; ROS, reactive oxygen species; SOD1, Cu/Zn-superoxide dismutase; Tempol, 4-hydroxy2,2,6,6-tetramethylpiperidine-1-oxyl. ⁎ Corresponding author. INSERM U692, Laboratoire de Signalisations Moléculaires et Neurodégénérescence, Faculté de Médecine, Université de Strasbourg, 67085 Strasbourg, France. Fax: +33 368 853065. E-mail address: [email protected] (J.-L. Gonzalez de Aguilar). 1 These authors contributed equally to this work. 0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2010.01.014

[3]. The stress produced by the aberrant accumulation of reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide, hydroxyl radical, and peroxynitrite, is commonly postulated as a major feature of the pathological process [4]. The ordered sequence of the pathological events required to trigger ALS also remains obscure. Nevertheless, it is increasingly clear that the degeneration of distal axons and neuromuscular junctions (NMJs) begins very early in the disease, long before the death of the parent cell bodies [5–9]. Such a pattern of progression may explain why genetic and pharmacological interventions aimed at protecting spinal motor neuron cell bodies were unable to prevent muscle denervation, weakness, and death in animal models of motor neuron disease [10–14]. In contrast, muscle-derived GDNF, which is a potent survival factor for motor neurons, stabilized NMJs, reduced motor impairment, and increased overall life span in two animal models of familial ALS [15,16]. More interestingly, identical results were obtained when isoforms of IGF-I were specifically expressed in muscle as a means to locally repair muscle function [17,18]. In view of these findings, deciphering the molecular actors involved in the early stages of muscle atrophy, before death of motor neurons occurs, becomes a central issue for the understanding

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of the pathogenesis of ALS and the development of effective therapeutic approaches. Using high-density oligonucleotide microarrays, we recently analyzed gene expression in skeletal muscle from a transgenic mouse model of mutant SOD1-linked ALS and found molecular markers of the muscle atrophy occurring during disease [19]. One of the most significant changes we observed was a high increase in the expression of Ras-related associated with diabetes (Rad), which is a small GTP-binding protein of the RGK family, including Rad, Rem, Rem2, and Gem/Kir [20]. Rad was initially identified as being overexpressed in skeletal muscle of patients with type 2 diabetes mellitus [21], although subsequent studies did not confirm these findings [22]. Based on in vitro studies, Rad has been endowed with a variety of yet incompletely understood biological functions, including inhibition of insulin-stimulated glucose uptake [23], modulation of cytoskeleton remodeling [24], and inhibition of voltage-gated calcium channel activity [25]. Except for a few sparse observations on the expression of Rad [20,26,27], the nature of its regulation under physiological or pathological conditions remains to be explored. Our goal in this study was to identify mechanisms pathologically relevant to ALS that could underlie changes in the expression of Rad. To this end, we measured the expression of Rad in skeletal muscle from ALS patients and related animal models, as well as under various in vivo and in vitro experimental conditions. In particular, we investigated whether Rad is regulated by oxidative stress triggered per se by hind-limb ischemia–reperfusion or indirectly, by surgically induced muscle denervation. By addressing these questions, we provide new insight into the molecular factors involved in the initiation and progression of muscle atrophy in ALS. Materials and methods Animals Transgenic FVB/N males expressing the murine G86R SOD1 mutation were obtained in our animal facility and genotyped as described [28]. Transgenic C57BL/6 males with the human G93A SOD1 mutation were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were maintained at 23°C with a 12h light/dark cycle and had water and regular rodent chow ad libitum. We had previously determined the progression of disease symptoms in G86R mice according to a clinical rating scale going from score 4 to 0 [13]. Score 4 is given to asymptomatic G86R mice, relative to their wild-type littermates. Score 3 corresponds to an alteration in hind-limb extension when the animal is hung by the tail. Score 2 is given when any slight alteration in the locomotion is observed. Score 1 represents an asymmetrical or symmetrical paralysis of the limbs. Score 0 corresponds to the stage at which the animal is unable to roll over within 10 s after being pushed on its back. For this study, gastrocnemii were dissected from G86R mice at 60 and 75 days of age (score 4 or asymptomatic); at 90 days of age, when about 40% of the animals present with altered hind-limb extension reflexes (score 3 or preparalyzed); and at the onset of hind-limb paralysis at 105–107 days of age (score 1). G93A mice were used at the onset of paralysis. Nontransgenic male littermates served as controls. To induce peripheral nerve injury, wild-type animals were anesthetized with 1 mg/kg body wt ketamine chlorhydrate and 0.5 mg/kg body wt xylazine. Then, the sciatic nerve was exposed at the midthigh level and crushed with a fine forceps for 30 s or sectioned 3 mm long with microscissors. The skin incision was sutured, and the animals were allowed to recover. For ischemia–reperfusion experiments, the hind limb of wild-type animals, anesthetized as indicated above, was carefully exposed by incising the skin and trussed up at the hip joint. After 2 h of ischemia, the tourniquet was cut, and the hind limb was allowed to reperfuse for 3 h. The hind limb contralateral to the lesion served as

control. To reduce oxidative stress during ischemia–reperfusion, we injected intraperitoneally two consecutive doses of 250 and 125 mg/ kg of the cell-permeative antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol; Sigma–Aldrich, St Quentin, France), 10 min before cutting the tourniquet and after 1 h of reperfusion, respectively. To reduce oxidative stress in axotomized mice, we injected Tempol intraperitoneally at 250 mg/kg/day for 5 days. A solution of 0.9% NaCl was used as vehicle. Experiments followed current European Union regulations (Directive 86/609/EEC) and were performed under the supervision of authorized investigators. Patients We studied biopsies from the left deltoid muscle in five patients with sporadic ALS (three male and two female patients) with a mean age of 59 years (range, 50–70 years). According to the El Escorial criteria, all patients had definite ALS [29]. The mean disease duration was 9 months (range, 7–30), and the mean ALSFRS-R score was 40 (range, 30–47) at the time of muscle biopsy. The control group included five patients subjected to a standard surgical diagnosis procedure without significant neurological history. All patients gave written informed consent before biopsy. Tissues were immediately frozen in liquid nitrogen and stored at −80°C until use. The study was approved by the ethical committee of the Hospital of Strasbourg. Quantitative RT-PCR Total RNA was prepared following standard protocols. Briefly, each frozen sample was placed into a tube containing a 5-mm stainless steel bead. The samples were kept on ice while 1 ml Trizol reagent (Invitrogen, Groningen, The Netherlands) was added, and homogenization was performed in a TissueLyser (Qiagen, Valencia, CA, USA) at 30 Hz for 3 min twice. RNA was extracted with chloroform/ isopropyl alcohol/ethanol and stored at − 80°C until use. One microgram of total RNA was used to synthesize cDNA using Iscript reverse transcriptase (Bio-Rad Laboratories, Marnes La Coquette, France) and oligo(dT) primer as specified by the manufacturer. Gene expression was measured using the SYBR green reagent (2 × SYBR Green Supermix; Bio-Rad Laboratories) following the manufacturer's instructions on a Bio-Rad iCycler. PCR was performed under optimized conditions: 95°C denaturing for 5 min, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Primers (Eurogentec, Seraing, Belgium) were as follows: Rad, forward, 5′-ACAAGGGCAGCTTTGAGAAA-3′, reverse, 5′-GCTGCTGATGTCTCGATGAA-3′; acetylcholine receptor α subunit (AChRα), forward, 5′-CCACAGACTCAGGGGAGAAG-3′, reverse, 5′AACGGTGGTGTGTGTTGATG-3′; and 18S, forward, 5′-CGTCTGCCCTATCAACTTTCG-3′, reverse, 5′-TTCCTTGGATGTGGTAGCCG-3′. Relative quantification was achieved by calculating the ratio between the cycle number (Ct) at which the signal crossed a threshold set within the logarithmic phase of the gene of interest and that of the 18s reference gene. Ct values were the means of duplicates. Western blot Muscle samples were homogenized in phosphate-buffered saline containing 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, and 1% protease inhibitor cocktail. Homogenates were then boiled for 5 min and sonicated for 20 s. After centrifugation (15,000 g for 20 min at 4°C), equal amounts of protein, according to the bicinchoninic acid assay, were separated on a 13% SDS–polyacrylamide gel. Separated proteins were then electrotransferred to nitrocellulose membranes and subjected to reversible staining with 0.1% Ponceau S in 5% acetic acid to ensure homogeneous transfer. Immunostaining was performed with a rabbit polyclonal anti-Rad antiserum [21] (kindly provided by Dr. C.R. Kahn, Joslin Diabetes Center, Boston, MA, USA) diluted 1/2000 and horseradish peroxidase-conjugated goat anti-

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rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted 1/2000. Blots were developed by enhanced chemiluminescence detection. In situ hybridization

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sciatic notch level. An anode needle was inserted at the base of the tail. The active recording needle electrode was inserted into the medial part of the gastrocnemius. The reference recording needle electrode was inserted over the Achilles tendon. The myoelectric signal was bandpass filtered (2 Hz–5 kHz) to eliminate artifacts. An initial negative deflection and biphasic waveform indicated recording at the motor point.

In situ hybridization histochemistry was performed as previously described [30,31]. Gastrocnemius sections 12 μm thick were cut perpendicular to the muscle axis on a cryostat at – 20°C, thawmounted on poly-L-lysine (Sigma–Aldrich)-coated slides, fixed in 4% phosphate-buffered formaldehyde for 5 min, and stored in 96% ethanol at 4°C until use. The Rad oligonucleotide probe (5′-GATAGAACGGTCATATGTGTGCCCTGCTGCTTCTGCTTCAG-3′; Eurogentec) was 3′-end labeled with [α-35S]dATP (Perkin–Elmer, Zaventem, Belgium). Only labels between 250,000 and 350,000 cpm/μl of the eluate were used for the experiments. Slides were hybridized for 48 h at 42°C with labeled probe diluted in hybridization buffer (50% formamide, 4 × SSC, 5 × Denhardt's solution, and 10% dextran sulfate) to a final concentration of 0.07 pmol/ml. After being washed in 1 × SSC for 30 min at 56°C, sections were dipped in NTB 2 nuclear track emulsion (Kodak, Stuttgart, Germany) or exposed to Biomax X-ray film (Kodak) for 4 weeks. Dipped sections were counterstained with hematoxylin/ eosin, dehydrated, and coverslipped. For negative control, a 200-fold excess of unlabeled oligonucleotides was added to the radioactive probe and applied to the adjacent section, leading to a complete suppression of the signal (not shown). For semiquantitative analysis of Rad mRNA expression, optical density readings of individual muscle fibers were taken on film autoradiograms (five muscle fibers per specimen, respectively, not shown). Absolute values of radioactivity were determined from a 14C plastic standard (Amersham, Freiburg, Germany) exposed on the same sheet of film using ImageJ software.

Data are expressed as the means ± SEM. Statistical analysis was accomplished using the Student t test or ANOVA followed by Tukey's multiple comparisons test (PRISM version 4.0b; GraphPad, San Diego, CA, USA). Differences at P b 0.05 were considered significant.

Immunostaining

Results

Animals were anesthetized as indicated above and intracardially perfused with 4% phosphate-buffered formaldehyde. Spinal cords were dissected, further fixed for 24 h at 4°C, and cryoprotected in 20% sucrose solution. The lumbar spinal cord region corresponding to segments L3–L5 was cut on a cryostat into sections 20 μm thick and labeled by indirect immunofluorescence using goat anti-choline acetyltransferase (Chemicon International, Hampshire, UK) diluted 1/50 and FITC-coupled donkey anti-goat IgG diluted 1/500 (Jackson ImmunoResearch Laboratories). Motor neuron counts were performed in the ventral horns of seven nonadjacent sections per animal using a Nikon microscope at a 200 × magnification.

Skeletal muscle fibers strongly up-regulate Rad during the course of ALS

Electromyography Recordings were obtained with a standard EMG apparatus (Dantec, Les Ulis, France) in accordance with the guidelines of the American Association of Electrodiagnostic Medicine. Mice were anesthetized as indicated above and kept under a heating lamp to maintain a physiological muscle temperature (±31°C). An ordinary concentric needle electrode (No. 9013S0011, diameter 0.3 mm; Medtronic, Minneapolis, MN, USA) was inserted into the muscle to be explored, and a monopolar needle electrode (No. 9013R0312, diameter 0.3 mm; Medtronic) was inserted into the back of the mouse to ground the system. To measure spontaneous activity, each gastrocnemius was monitored for at least 2 min. Spontaneous activity was differentiated from voluntary activity by visual and auditory inspection. Only spontaneous activity with a peak-to-peak amplitude of at least 50 μV was considered to be significant. To determine compound muscle action potentials, amplitudes (mV) from the left and right muscle-evoked responses were measured and averaged. Supramaximal square pulses, of 0.2 ms duration, were delivered through a needle electrode to the sciatic nerve at the

Oxidative stress assessment Detection of ROS was assessed in situ using the hydroethidine fluorescent assay [32]. Hydroethidine (Invitrogen Molecular Probes, Eugene, OR, USA) enters cells freely and is oxidized to ethidium derivatives, which bind to DNA, thus leading to an enhancement of fluorescence. Oxidized hydroethidine was visualized using a Nikon G2A fluorescence filter block, with an excitation filter of 510–560 nm, a dichroic mirror of 575 nm, and a barrier filter of 590 nm. Hydroethidine was prepared in DMSO and stored at − 20°C until use. Mice were injected intraperitoneally with 200 μl of a solution of 1 μg/μl hydroethidine in 0.9% saline containing 20% DMSO. After 20 min, gastrocnemii were dissected and shock frozen in melting isopenthane. Ten-micrometer-thick cryostat sections were extemporaneously observed in a Nikon microscope at a 200 × magnification and photographed under the same exposure time. Statistics

Based on our microarray analysis of the gene expression changes occurring in muscle from ALS mice, we had previously detected increased expression of Rad in a mix of hind-limb G86R muscles, including gastrocnemius and soleus [19]. In this study, we extend these data and show that Rad expression is highly up-regulated in gastrocnemius during the course of disease (Fig. 1A). At 75 days of age, when mice do not present with any clinical sign of motor impairment [13], Rad mRNA levels were about sixfold higher in G86R mice than in wild-type littermates (Fig. 1B). At 90 days of age, when muscle atrophy becomes apparent, Rad expression experienced a very dramatic increase but tended to decline in completely paralyzed animals (Fig. 1A). To test whether these observations result from an artifact related to SOD1 transgene insertion, we analyzed Rad expression in paralyzed gastrocnemius from G93A mice, another transgenic line showing histopathological characteristics quasiidentical to those of G86R mice [33], as well as in deltoid biopsies from patients with sporadic ALS. Rad mRNA levels significantly increased under both conditions (Fig. 1C). Most importantly, Rad protein content, virtually nonexistent in controls, was strongly upregulated in G93A mice and ALS patients (Fig. 1D). Previous studies had reported increased Rad expression only within myogenic progenitor cells in response to regenerative processes [27]. Contrasting with this observation, our in situ hybridization experiments performed in paralyzed gastrocnemius from symptomatic G86R mice showed Rad mRNA abundantly and regularly distributed throughout the muscle fibers, compared to wild-type littermates (Figs. 1E and 1F). Altogether, these findings indicate that Rad up-regulation in ALS skeletal muscle starts at the asymptomatic stage and then highly increases as the animals get sick, affecting the totality of the atrophic muscle fibers.

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mRNA levels, which peaked at 7 days postaxotomy and declined thereafter (Fig. 2A). Compared to the expression of the typical denervation marker AChRα, we found that Rad mRNA levels, although fluctuating, increased by twofold as early as 5 h postaxotomy, when the induction of AChRα expression had not happened yet (Fig. 2B). Rad up-regulation was noticeable in all denervated, atrophic muscle fibers (Figs. 2C and 2D), mirroring the situation in paralyzed G86R muscle. Taken together, these findings reveal that Rad is a precocious responsive gene stimulated by denervation. Time course of denervation in G86R mice To elucidate whether 75-day-old G86R gastrocnemius suffers from early denervation that could explain increased Rad expression, we followed the time course of several cellular and physiological parameters indicative of denervation. The number of choline acetyltransferase-positive motor neurons in the ventral horns of the lumbar spinal cord showed no cell loss at 75 days of age, compared to the dramatic 60% decrease in the amount of motor neurons that was observed in paralyzed G86R mice (Figs. 3A and 3B). Then, we focused on the physiology of the neuromuscular system using an electromyography approach. The amplitude of the compound muscle action potential, a reduction of which reflects a decrease in the number of functional motor units, remained unchanged in 75-day-old G86R mice, compared to wild-type littermates. It tended to decrease slightly

Fig. 1. Up-regulation of Rad in mutant SOD1 mice and patients with sporadic ALS. (A) Time course of Rad expression in gastrocnemius, as determined by quantitative RT-PCR. Results are shown as fold changes relative to wild-type littermates at each indicated age. (B) Relative Rad mRNA levels in gastrocnemius from wild-type and G86R mice at 75 days of age (n = 5–13 mice per group in (A) and (B); ⁎P b 0.05). Rad (C) mRNA and (D) protein levels in paralyzed gastrocnemius from G93A mice (left) and in deltoid biopsies from ALS patients (right), compared to wild-type mice and healthy subjects (Ct), respectively (n = 5 mice and 5 patients per group; ⁎P b 0.05). Each band in the Western blots represents one individual. (E) Representative photomicrographs showing Rad mRNA location on cross sections of gastrocnemius from wild-type (top) and G86R (bottom) mice, as determined by in situ hybridization. Sections are counterstained with thionin dye. Scale bar, 20 μm. (F) Semiquantitative analysis of film autoradiograms of Rad mRNA expression as shown in (E) (n = 3 mice per group; ⁎P b 0.05).

Muscle denervation stimulates Rad expression Because denervation is a key process inducing muscle atrophy in ALS, we investigated whether Rad up-regulation occurs in surgically denervated muscle. The time course of Rad expression in gastrocnemius after acute sciatic nerve axotomy showed a strong increase in

Fig. 2. Up-regulation of Rad in response to denervation. (A) Rad and AChRα mRNA levels in gastrocnemius after sciatic nerve axotomy, as determined by quantitative RTPCR. Results are shown as fold changes relative to the side contralateral to the lesion at each indicated time. (B) Relative Rad and AChRα mRNA levels are specifically shown at 5 and 24 h postaxotomy, respectively (n = 4 mice per group in (A) and (B)). (C) Representative photomicrographs showing Rad mRNA location on cross sections of gastrocnemius from control (top) and axotomized (bottom) mice, as determined by in situ hybridization. Sections are counterstained with thionin dye. Scale bar, 20 μm. (D) Semiquantitative analysis of film autoradiograms of Rad mRNA expression as shown in (C) (n = 3 mice per group; ⁎P b 0.05).

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Fig. 3. Characterization of denervation in G86R mice. (A) Quantification of the number of motor neurons in the ventral horns of the lumbar spinal cord from wild-type and G86R mice at the indicated ages, as determined by choline acetyltransferase immunolabeling. (B) Representative photomicrographs showing choline acetyltransferase staining on cross sections of the lumbar spinal cord from 75-day-old (top) and paralyzed (bottom) G86R mice. Scale bar, 250 μm. (C) Amplitude of compound muscle action potential (CMAP) in gastrocnemius from wild-type and G86R mice at the indicated ages. (D) Percentage of mice presenting with spontaneous denervation activity in at least one muscle territory of the gastrocnemius. (E) Representative electromyograms showing isolated fibrillations (top) and overt fasciculations (bottom) in denervated G86R muscle. (F) Relative AChRα mRNA levels in gastrocnemius from wild-type and G86R mice at the indicated ages (n = 5–10 mice per group in (A), (C), (D), and (F); ⁎P b 0.05).

at 80 days of age and fell to the half in paralyzed G86R gastrocnemius (Fig. 3C). Similarly, none of the 75-day-old G86R mice presented with abnormal spontaneous electrical activity, which would have reflected the typical response of muscle to loss of innervation. Altered electromyograms were observed in approximately 25% of G86R mice at 80 days of age and in all the cohort of animals with overt hind-limb paralysis (Figs. 3D and 3E). Along with this, the expression of AChRα, of which an increase is commonly considered a marker of denervation, was stimulated in gastrocnemius from 90-day-old and paralyzed G86R mice but unaltered at 75 days of age (Fig. 3F). Altogether, these findings strongly suggest that denervation does not represent a major feature in the early stages of disease. Ischemia–reperfusion of hind-limb muscles stimulates Rad expression The above results indicate that the increase in Rad expression in early disease stages must respond to a stimulus distinct from overt denervation. Because mutant SOD1 has been shown to accumulate in ALS mouse skeletal muscle [34] and trigger oxidative damage [35,36], we suspected ROS of causing precocious Rad up-regulation. Using C2C12 myoblasts, we found that treatment with increasing doses of hydrogen peroxide induced a significant increase in Rad mRNA levels (Supplementary Material 1), which supports a direct effect of ROS on Rad expression. Then, to re-create an in vivo condition characterized by increased production of ROS, dissociated from motor neuron degeneration, we subjected hind-limb muscles

to 2 h of ischemia followed by 3 h of reperfusion. Of note, recent evidence supports that hydroethidine can be oxidized by superoxide anions to form 2-hydroxyethidium as a specific product and by other ROS, such as hydrogen peroxide, hydroxyl radical, and peroxynitrite, to form ethidium. Analysis by HPLC and fluorescence microscopy has determined that the detection of 2-hydroxyethidium, as a more realistic measure of superoxide anions, is confounded, because its emission spectrum overlaps the emission of ethidium, which is the result of the action of a mixture of ROS [37,38]. Therefore, we interpreted our hydroethidine measures as an indicator of the global accumulation of various oxygen-derived species, not only superoxide anions. Under these conditions, high amounts of ROS were generated in gastrocnemius after ischemia– reperfusion (Fig. 4A). A massive increase in Rad mRNA levels was concomitantly observed, compared to both nonlesioned and shamoperated gastrocnemii (Fig. 4B). To test whether these observations result from an artifact linked to a pressing effect of the tourniquet on the sciatic nerve, we directly crushed this nerve in another series of animals and measured Rad mRNA levels 5 h postoperation. Such a manipulation did not affect Rad expression (inset in Fig. 4B). Interestingly, accumulation of ROS was also noticeable as early as 75 days of age in G86R mice (Fig. 4C), coincident with the rise in Rad expression (Fig. 1A). In all, these findings show that an ischemia– reperfusion stress is, in itself, sufficient to trigger Rad up-regulation and point to ROS as a candidate factor in the regulation of Rad at early stages of ALS.

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Tempol after ischemia–reperfusion. Tempol is effective in scavenging a wide variety of reactive intermediates [40], so that it is likely that other ROS could be involved more directly in regulating Rad expression. Interestingly, the accumulation of ROS has also been detected in muscle in association with the atrophy induced by loss of innervation [41]. We thus attempted to reproduce the effect of Tempol on our denervated muscle paradigm. The increase in Rad expression observed in gastrocnemius 5 days after sciatic nerve axotomy was partially reversed by Tempol. In contrast, the induction

Fig. 4. Up-regulation of Rad in response to oxidative stress. (A) Representative photomicrographs showing hydroethidine labeling in cross sections of gastrocnemius from control (left) and ischemic–reperfused (right) mice. Scale bar, 40 μm. (B) Rad mRNA levels in gastrocnemius after 2 h of ischemia followed by 3 h of reperfusion (I-R), as determined by quantitative RT-PCR. The inset shows Rad mRNA levels in gastrocnemius 5 h after sciatic nerve crush (n = 6 mice per group; ⁎P b 0.05). (C) Representative photomicrographs showing hydroethidine labeling in cross sections of gastrocnemius from wild-type (left) and 75-day-old G86R (right) mice. Scale bar, 40 μm.

The antioxidant Tempol reverses muscle Rad up-regulation in response to ischemia–reperfusion or denervation To establish a causative relationship between the accumulation of ROS and the up-regulation of Rad, we stimulated its expression by ischemia–reperfusion and administered the cell-permeative antioxidant Tempol [39]. Under these conditions, we found a very significant inhibition of the stimulatory effect of the ischemia–reperfusion stress on gastrocnemius Rad mRNA levels (Fig. 5A). To gain insight into the nature of the radical species involved in the up-regulation of Rad expression after ischemia–reperfusion, we addressed the question whether Tempol could decrease injury-induced Rad expression by reducing peroxynitrite levels. Excess peroxynitrite induces protein tyrosine nitration, which is an indicator of oxidative stress, as occurs for example after ischemia–reperfusion. We reproduced the same experimental conditions as those used to restore basal Rad expression by Tempol in response to ischemia–reperfusion and tested the effect of Tempol on protein tyrosine nitration, by means of the immunological detection of 3-nitrotyrosine in Western blot (Supplementary Material 2). We found that ischemia–reperfusion made several 3-nitrotyrosine-immunoreactive bands appear or induced an increase in the intensities of several bands already present. However, Tempol hardly affected the observed pattern of increased protein tyrosine nitration. Thus, we suggest that, at least under our experimental conditions, protein tyrosine nitration does not seem to be involved in the regulation leading to the restoration of basal Rad expression by

Fig. 5. Inhibitory effect of Tempol on Rad up-regulation. (A) Relative Rad mRNA levels in gastrocnemius after 2 h of ischemia followed by 3 h of reperfusion, in the absence (Ct) or presence of two consecutive injections of 250 and 125 mg/kg Tempol. (B) Relative Rad (top) and AChRα (bottom) mRNA levels in gastrocnemius from sciatic nervetransected mice in the absence (Ct) or presence of daily injections of 250 mg/kg Tempol for 5 days (n = 5 or 6 mice per group in (A) and (B); ⁎P b 0.05 versus corresponding contralateral, #P b 0.05 versus ipsilateral Ct).

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of AChRα, typically present upon denervation, remained unchanged (Fig. 5B). Altogether, these findings demonstrate the direct involvement of ROS as a stimulatory factor for the expression of Rad. Discussion In this study, we show that muscle ROS are directly involved in triggering overexpression of Rad. This newly identified factor was upregulated in skeletal muscle affected by the chronic degenerative condition ALS, in both mice and patients, as well as after treating C2C12 myoblasts with hydrogen peroxide. In addition, the antioxidant Tempol was able to reduce Rad overexpression in in vivo experimental settings characterized by acute oxidative stress, including hindlimb ischemia–reperfusion and nerve axotomy-induced muscle denervation. The up-regulation of Rad was intimately associated with muscle atrophy, because it took place within all the atrophied muscle fibers of the gastrocnemius in end-stage diseased G86R mice and after surgical sectioning of the sciatic nerve. Strikingly, no signs of denervation were found in G86R mice during the first steps of Rad up-regulation, which preceded in fact the alteration of the electrical properties of the muscles and the increase in AChRα expression that typically characterize denervation in ALS. Identifying the cause of enhanced Rad expression should therefore provide new clues for the understanding of the initiatory mechanisms of disease. Oxidative stress has been postulated to occur early in ALS skeletal muscle. By crossing G93A mice with mice containing an antioxidant response element reporter, Kraft and collaborators [35] showed the activation of antioxidant mechanisms in gastrocnemius earlier than in spinal cord and long before motor impairment. In addition, recent studies revealed that muscle-restricted expression of mutant G93A SOD1 was sufficient to induce oxidative damage and muscle atrophy [36]. We were able to detect the accumulation of ROS in skeletal muscle from asymptomatic G86R mice, and this was coincident with the initial rise in Rad expression, which strongly suggests that these events could be interconnected. To further address this question, we took advantage of an ischemia–reperfusion paradigm, which allowed us to dissociate toxic mechanisms occurring intrinsically within skeletal muscle from the influence of the progressive motor neuron degeneration as seen in ALS mice. The restoration of circulation after a period of ischemia results in oxidative damage through the accumulation of ROS [42,43]. We demonstrated

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here that the return of blood supply to skeletal muscle after 2 h of ischemia triggered both ROS accumulation and Rad overexpression. Most importantly, the administration of the cell-permeative antioxidant Tempol, which is known to scavenge a wide variety of reactive intermediates [40,44,45], reversed very significantly the stimulatory effect on Rad expression obtained after reperfusion. These findings, together with the fact that hydrogen peroxide was able to up-regulate Rad in C2C12 myoblasts, indicate that oxidative stress is, in itself, sufficient to stimulate Rad expression. We can therefore contend that the accumulation of ROS found early in G86R muscle does up-regulate Rad at presymptomatic stages of ALS. It is known that muscle atrophy induced by loss of innervation is associated with an increase in the generation of ROS [41]. Our treatment of sciatic nerve-axotomized mice with the antioxidant Tempol significantly reduced Rad overexpression in denervated muscle. These findings, first, corroborate the presence of oxidative stress during denervation-induced muscle atrophy and, second, further reinforce the stimulatory effect of ROS on Rad expression. Keeping this in mind, it is noteworthy that the up-regulation of Rad in G86R mice underwent a dramatic increase at the onset of apparent denervation. We speculate that the increase in the up-regulation of Rad after progression toward overt disease can be the result of the oxidative stress accumulated from both local mutant SOD1 toxicity and loss of innervation (Fig. 6). Growing evidence indicates that RGK GTPases, including Rad, are potent inhibitors of voltage-dependent calcium channel currents via the interaction with auxiliary CaVβ subunits, which form part of the multiprotein complexes of many voltage-gated calcium channels [20,46]. Auxiliary CaVβ subunits are important for the cell surface trafficking of the pore-forming subunits, as well as for the modulation of calcium current amplitude and channel gating activity [47]. Interestingly, increasing Rad expression in several cell lines resulted in nuclear sequestration of β subunits and inhibition of channel activity [25,48]. Similarly, overexpression of the RGK GTPase Rem in cultured myotubes reduced the number of functional L-type calcium channels at the plasma membrane and negatively regulated excitation–contraction coupling, the process whereby muscle cells convert electrical stimuli to mechanical responses [49]. On the other hand, suppression of Rad expression in mice induced cardiac hypertrophy [50] and ventricular tachycardia through up-regulation of the expression of L-type calcium channels [51]. Taken as a whole, these

Fig. 6. Schematic representation of the hypothetical mechanisms leading to Rad overexpression and muscle dysfunction in mutant SOD1-linked ALS mice. In presymptomatic animals, at about 75 days of age (left), endogenous mutant SOD1 toxicity starts to stimulate Rad expression. In symptomatic animals, at about 90 days of age (right), both endogenously generated and denervation-dependent oxidative stress exacerbate Rad up-regulation. Once accumulated, it is speculated that Rad may interfere with muscle function by perturbing excitation–contraction coupling (EC).

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findings strongly suggest that the aberrant expression of Rad may be pathologically relevant. Indeed, the lack of β1a subunits in voltagegated dihydropyridine receptors from skeletal muscle, as occurs in null-mutant mice and in the zebrafish mutant relaxed, triggered excitation–contraction uncoupling and subsequent lethal muscle paralysis [52,53]. These observations let us envisage that, by increasing its expression in response to oxidative stress, as we observed in this study, Rad could contribute to skeletal muscle dysfunction and paralysis in ALS (Fig. 6). Our recent studies showed that mild uncoupling of mitochondrial respiration in skeletal muscle was sufficient to profoundly affect NMJ stability and function and to induce distal degeneration of motor neurons [54]. In line with these findings, the analysis of Rad expression during the course of disease in mutant SOD1 mice has shown that skeletal muscle suffers from pathological modifications that do not necessarily result, at least at early stages of disease, from motor neuron injury but are inherent to the presence of oxidative stress within the muscle fibers. In addition, our study validated Rad up-regulation in muscle biopsies from patients with sporadic ALS, which is the most frequent form of the disease. In the absence of a validated biomarker, measuring the expression of Rad could represent a surrogate marker of the initiation and extent of the oxidative stress characteristic of the disease. Despite substantial evidence supporting the use of antioxidants for patients with ALS, no significant benefit has been obtained so far [55]. We hope that gaining new insight into the targets of the oxidative stress contributing to the disease may be of help in developing more effective antioxidant therapies. Acknowledgments We express our gratitude to the patients and their families. We thank A. Picchinenna and M.J. Ruivo for excellent technical assistance. We also thank Dr. C.R. Kahn (Joslin Diabetes Center, Boston, MA, USA), for providing anti-Rad antiserum, and Dr. J. Zoll (Service de Physiologie et d'Explorations Fonctionnelles, Centre Hospitalier Régional Universitaire de Strasbourg, Strasbourg, France) for helping with the ischemia–reperfusion experiments. We are also indebted to Dr. N. Deroide (Service de Physiologie et d'Explorations Fonctionnelles Multidisciplinaires, Hôpital Lariboisiere, Paris, France) for his aid with the human biopsy experiments. The study was supported in part by the Association Française contre les Myopathies (AFM) and Association pour la Recherche sur la Sclérose Latérale Amyotrophique. B. Halter is the recipient of a grant from the Région Alsace, Association pour la Recherche et le Développement de Moyens de Lutte contre les Maladies Neurodégénératives, and AFM. J.L. Gonzalez de Aguilar is the recipient of a Chaire INSERM/Université de Strasbourg. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.freeradbiomed.2010.01.014. References [1] Gros-Louis, F.; Gaspar, C.; Rouleau, G. A. Genetics of familial and sporadic amyotrophic lateral sclerosis. Biochim. Biophys. Acta 1762:956–972; 2006. [2] Rosen, D. R.; Siddique, T.; Patterson, D.; Figlewicz, D. A.; Sapp, P.; Hentati, A.; Donaldson, D.; Goto, J.; O'Regan, J. P.; Deng, H. X., et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362:59–62; 1993. [3] Boillée, S.; Vande Velde, C.; Cleveland, D. W. ALS: a disease of motor neurons and their nonneuronal neighbors. Neuron 52:39–59; 2006. [4] Barber, S. C.; Mead, R. J.; Shaw, P. J. Oxidative stress in ALS: a mechanism of neurodegeneration and a therapeutic target. Biochim. Biophys. Acta 1762: 1051–1067; 2006. [5] Azzouz, M.; Leclerc, N.; Gurney, M.; Warter, J. M.; Poindron, P.; Borg, J. Progressive motor neuron impairment in an animal model of familial amyotrophic lateral sclerosis. Muscle Nerve 20:45–51; 1997.

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