Amyotrophic lateral sclerosis: all roads lead to Rome

Journal of Neurochemistry, 2007, 101, 1153–1160

REVIEW

doi:10.1111/j.1471-4159.2006.04408.x

Amyotrophic lateral sclerosis: all roads lead to Rome Jose-Luis Gonzalez de Aguilar,*, Andoni Echaniz-Laguna,*, ,à Anissa Fergani,*, Fre´de´rique Rene´,*, Vincent Meininger,§ Jean-Philippe Loeffler*, and Luc Dupuis*, *Inserm, U692, Laboratoire de Signalisations Molećulaires et Neurode´geńe´rescence, Strasbourg, France Universite´ Louis Pasteur, Faculte´ de Me´decine, UMRS692, Strasbourg, France àCHU Strasbourg, De´partement de Neurologie, Strasbourg, France §Hoˆpital de la Pitie´-Salpeˆtrie`re, ALS National Referral and Coordinating Centre, Paris, France

Abstract Amyotrophic lateral sclerosis (ALS) is the most frequent adultonset motor neuron disease characterized by degeneration of upper and lower motor neurons, generalized weakness and muscle atrophy. Most cases of ALS appear sporadically but some forms of the disease result from mutations in the gene encoding the antioxidant enzyme Cu/Zn superoxide dismutase (SOD1). Several other mutated genes have also been found to predispose to ALS including, among others, one that encodes the regulator of axonal retrograde transport dynactin. As all roads lead to the proverbial Rome, we discuss here how distinct molecular pathways may converge to the same final result that is motor neuron death. We critically review the basic research on SOD1-linked ALS to propose a pioneering

Main clinical and neuropathological features of ALS

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease or Lou Gehrig’s disease, is the most common adult-onset motor neuron disease. Described in 1869 by the French neurologist Jean-Martin Charcot, the primary disease hallmark is the selective and progressive degeneration of the neurons in the corticospinal tracts. Dysfunction of upper motor neurons (or cortical motor neurons) leads to spasticity and hyper-reflexia while that of lower motor neurons (in the brainstem and spinal cord) triggers generalized weakness, muscle atrophy and paralysis (Rowland 1998). Failure of the respiratory muscles is generally the fatal event, occurring within 1–5 years of onset. ALS is a disease of mature adults, with a median age of onset of 55 years. Due to its uniform lethality, ALS appears as a rare disease (prevalence of 4–6

model of a ‘systemic’ form of the disease, causally involving multiple cell types, either neuronal or non-neuronal. Contrasting this, we also postulate that other neuron-specific defects, as those triggered by dynactin dysfunction, may account for a primary motor neuron disease that would represent ‘pure’ neuronal forms of ALS. Identifying different disease subtypes is an unavoidable step toward the understanding of the physiopathology of ALS and will hopefully help to design specific treatments for each subset of patients. Keywords: amyotrophic lateral sclerosis, dynactin, dynein, superoxide dismutase 1, systemic disease, vascular endothelial growth factor. J. Neurochem. (2007) 101, 1153–1160.

per 100 000 each year), although its incidence is of one to two per 100 000 each year. Most cases (90%) are classified as sporadic ALS (SALS), as they are not associated with a documented family history. The remainder 10% are inherited and referred to as familial ALS. Sporadic and familial forms Received October 2, 2006; revised manuscript received November 13, 2006; accepted November 21, 2006. Address correspondence and reprint requests to Jean-Philippe Loeffle, Inserm U692, Laboratoire de Signalisations Molećulaires et Neurode´geńe´rescence, Universite´ Louis Pasteur, Faculte´ de Me´decine, 11 rue Humann, 67085 Strasbourg, France. E-mail: loeffl[email protected] Abbreviations used: ALS, amyotrophic lateral sclerosis; HRE, hypoxia responsive element; mSOD1, mutant SOD1; SALS, sporadic ALS; SETX, senataxin; SOD1, Cu/Zn superoxide dismutase; VEGF, vascular endothelial growth factor.

Ó 2007 The Authors Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 1153–1160

1153

1154 J.-L. Gonzalez de Aguilar et al.

are clinically and pathologically similar, suggesting a common pathogenesis (Bruijn et al. 2004). However, the precise cause for most cases is still unknown, and there is no effective remedy to stop the course of the disease. Only riluzole, a compound with anti-glutamate activity, is able to slightly prolong the survival (Lacomblez et al. 1996). Several pathological hallmarks are found in the spinal cord of autopsied ALS patients. These include the atrophy of dying motor neurons, with notable swelling of the perikarya and proximal axons and the presence of Bunina bodies, spheroids and strands of ubiquitinated material in degenerating axons and in cell somas. Reactive gliosis often accompanies this motor neuron pathology. Because degeneration and death of motor neurons appear as both the central issue and final endpoint of the disease, it was long hypothesized that specific-pathogenic mechanisms originating in these cells should be responsible for the whole pathological process. However, recent studies have postulated that not only motor neurons but also other non-neuronal cell types may exert pathogenic influences and thus contribute to the disease onset. Indeed, whether motor neurons in ALS die from a complex interaction between multiple factors or because of the manifestation of a unique, yet unidentified genetic–biochemical abnormality is a matter of controversy. The aim of this review is to revisit some basic research to support the notion that, as all roads lead to the proverbial Rome, disparate molecular pathways may lead to the same final destination (i.e. motor neuron death), and propose a preliminary classification of ALS into different subtypes on the basis of their underlying pathophysiological mechanisms. Complexity arises: genetics and animal models of ALS

To date, six mutated genes have been reported to cause or predispose to ALS (for review see (Pasinelli and Brown 2006) or (Gros-Louis et al. 2006)). They encode cytosolic SOD1 (Rosen et al. 1993), alsin (Hadano et al. 2001; Yang et al. 2001), dynactin (Puls et al. 2003; Munch et al. 2004), angiogenin (Greenway et al. 2006), senataxin (SETX) (Chen et al. 2004) and VAPB (synaptobrevin/VAMP (vesicle-associated membrane protein)-associated protein B) (Nishimura et al. 2004). SETX shows strong homology with DNA/RNA helicases, which suggests a potential function in RNA processing (Chen et al. 2004). On the other hand, VAPB has been involved in the unfolded protein response as well as in inositol metabolism (Kanekura et al. 2006). As the data concerning SETX and VAPB is scarce, we will not discuss these further. SOD1 is a cytosolic Cu/Zn-binding protein involved in antioxidant resistance. More than 100 mutations in sod1 have been reported to occur in ALS patients, with no clear mutational hot spot. Further strengthening the complex nature of the disease is that the same sod1 mutation does not

necessarily cause a homogenous phenotype, but is rather able to induce different clinical presentations within the same family (Orrell et al. 1997; Mase et al. 2001; Rezania et al. 2003). Historically, the discovery of sod1 mutations led to the generation of the first animal models of ALS. Several mouse lines were generated that overexpress ubiquitously mutant SOD1 (mSOD1) at levels sufficient to induce a motor neuron disease closely resembling human ALS (Gurney et al. 1994; Ripps et al. 1995; Wong et al. 1995; Deng et al. 2006). The clinical features observed in these mice are summarized in Table 1. Several mutations in als2, which encodes a protein called alsin, were shown to be linked to a rare juvenile form of ALS primarily characterized by upper motor neuron involvement (Hadano et al. 2001; Yang et al. 2001). Alsin possesses guanine nucleotide exchange factor homology domains known to activate small G-proteins of the Ras superfamily. Mice knocked out for als2 were generated (Cai et al. 2005; Devon et al. 2006; Hadano et al. 2006; Yamanaka et al. 2006) (Table 1) that showed upper motor neuron defects with little, if any, lower motor neuron involvement (Yamanaka et al. 2006). This pathological phenotype is more consistent with the occurrence of spastic paraplegia, and hence mutant alsin mice should be rather considered as a model for this disease than for ALS. A subset of familial and sporadic forms of ALS has been recently associated with mutations in the gene encoding dynactin, an activator of the molecular motor dynein (Puls et al. 2003, 2005; Munch et al. 2004). Earlier studies on mice had reported that motor neuron-restricted overexpression of dynamitin, another subunit of the dynein complex, induces disassembling of dynactin and hence dynein dysfunction. Interestingly, these transgenic mice show a motor neuron disease reminiscent of ALS, with decreased strength and endurance, muscle denervation and motor neuron loss (LaMonte et al. 2002). Follow-up studies revealed the generation of two mouse lines, called legs at odd angles (loa) and cramped (Cra), that bear heterozygous mutations in the gene encoding the dynein heavy chain and also develop an age-dependent degeneration of motor neurons associated with muscle histological abnormalities (Hafezparast et al. 2003). More recently, mutations in ang, which encodes the potent inducer of neovascularization angiogenin, were found to be linked to several familial ALS and SALS cases (Greenway et al. 2006). These findings are reminiscent of that observed by Oosthuyse et al. (2001), who reported that mice with a deletion in the hypoxia responsive element (HRE) of the promoter of the gene encoding vascular endothelial growth factor (VEGF), another angiogenic factor, exhibit a motor neuron disease closely resembling ALS (Table 1). As the discovery of these so called VEGF d/ d mice, Lambrechts et al. (2003) showed an association between specific haplotypes in the VEGF gene promoter and ALS, thus suggesting that certain genetic variants of vegf


In the case of mSOD1 mice, not all data have been obtained from all the mouse strains generated but mostly include those from SOD1(G93A) and SOD1(G86R) mice. To avoid multiple references, a review article has been cited. +, presence of the anomaly ), absence of the anomaly ALS, amyotrophic lateral sclerosis; SOD, Cu/Zn superoxide dismutase; mSOD, mutant SOD; ND, not determined.

a

(Bruijn et al. 2004) (Hafezparast et al. 2003) (LaMonte et al. 2002) (Oosthuyse et al. 2001) (Cai et al. 2005; Devon et al. 2006; Hadano et al. 2006; Yamanaka et al. 2006) + ) ) ) ) + ND ND ND + + ND ND ND ) + ND ND + +/) + + ND + ) + + + + ) + ND + ND ) + ND + + ) + + ND + ND + ND + + )

Ventral root axon Motor Muscle Wallerian Muscle neuron Ubiquitin Astrocyte Microglial Muscle fiber type degeneration number degeneration staining proliferation proliferation weakness atrophy switching (sciatic nerve) decrease

+ + + + ) Mutant SOD1a Mutant dynein (loa/Cra) Dynamitin overexpressors VEGF d/d Als2)/)

Table 1.

Summary of the pathological features in different ALS animal models

Upper motor neuron Pre-mature signs death References

Amyotrophic lateral sclerosis: all roads lead to Rome 1155

could predispose to the disease. However, more recent studies have failed to confirm this association (Van Vught et al. 2005; Chen et al. 2006). The studies mentioned above illustrate the multiple and complex nature of ALS. In this review, taking one step further, we intend to define two subtypes of the disease according to the dissection of the underlying pathophysiological mechanisms in the corresponding animal models. We will focus on SOD1-linked ALS viewed as a ‘systemic’ form of the disease, and on dynein/dynactin-linked ALS considered as a ‘pure’ motor neuron form. The inclusion in one or another subtype of the motor neuron disease observed in the VEGF d/d mice will be also discussed. SOD1-linked ALS: a systemic disease with prominent motor unit failure?

mSOD1 mice are the most widely studied animal models of ALS. A large consensus exists now on the fact that mutations in SOD1 confer a gain of function to the mutant enzyme. This notion was early established when it was reported that mice knocked out for wild-type SOD1 do not display motor neuron disease (Reaume et al. 1996). In addition, decreasing the levels of endogenous wild-type SOD1 in mSOD1 mice does not modify the course of motor neuron loss (Bruijn et al. 1998). In turn, increasing the levels of wild-type SOD1 in these same animals accelerates motor neuron loss and exacerbates the overall phenotype (Jaarsma et al. 2000; Deng et al. 2006). There has been a flurry of research during the last 10 years detailing how mSOD1 is able to generate, instead of fighting against oxidative stress and hence trigger motor neuron death. Because mSOD1 can induce motor neuron apoptosis, and several apoptotic markers have been localized in the motor neurons of mSOD1 mice (for review see Guegan and Przedborski 2003), it has long been thought that the unique expression of mSOD1 in motor neurons was sufficient to trigger ALS, that is, mSOD1 acts as a ‘cell-autonomous’ insult for motor neurons. However, this hypothesis remains controversial. Indeed, two different groups early demonstrated that the targeted expression of mSOD1 only in motor neurons does not provoke the disease in transgenic mice (Pramatarova et al. 2001; Lino et al. 2002). To evaluate whether the absolute number of cells expressing mSOD1, or their nature, is important for the disease, Clement et al. (2003) generated chimeric animals harboring different proportions of wild-type and mSOD1 expressing cells. The more wild-type cells the mice had the longer they survived irrespective of these cells being motor neurons or not. In addition, wild-type motor neurons presented with ubiquitin deposits in animals with a high proportion of cells expressing mSOD1 while, in contrast, mutant motor neurons lacked such deposits when the number of surrounding mSOD1expressing cells was low. These findings show that,



independently of the considered cell type, it is the ratio between wild-type and mSOD1 expressing cells rather than motoneuronal expression of mSOD1 that determines ALS. Contrasting this notion, Jaarsma and collaborators have recently claimed that Thy1-driven mSOD1 expression is sufficient to trigger ALS (Jaarsma et al. 2006). In fact, knocking down mSOD1 only in motor neurons but not in other cells increases modestly the overall lifespan of the animals (Boillee et al. 2006), thus suggesting that the motoneuronal expression of mSOD1 would contribute only mildly to the pathology. These results raise a key question: if motor neurons are not the critical cells that trigger SOD1-linked ALS, which other cell(s) could be involved? Astrocytes, microglial and muscle cells as well as Schwann cells and oligodendrocytes could in principle be implicated. Although the targeted expression of mSOD1 only in astrocytes proved to be insufficient to trigger motor neuron disease (Gong et al. 2000), these cells could be a good guess. Indeed, astrocytic proliferation is prominent in mSOD1 mice and in ALS patients, and it has been demonstrated that activated astrocytes secrete proteins that are toxic for motor neurons such as nerve growth factor (Pehar et al. 2004, 2005). As far as microglial cells are concerned, recent studies have shown that the genetic ablation of mSOD1 in the macrophage/microglia lineage yields an overall increase in survival of mSOD1 mice (Boillee et al. 2006). Nevertheless, one should be cautious when interpreting these results as it cannot be excluded that such an ablation in the macrophage lineage, which includes not only microglia but also monocytes and other tissueresiding macrophages such as Kupffer cells in the liver, could have offered overall protection in other tissues and organs. Myocytes may also be considered as a potential contributor to the pathophysiology of SOD1-linked ALS. Indeed, muscle atrophy is one of the earliest events detectable in these animals (Brooks et al. 2004), followed by the fragmentation of the neuromuscular junction, retrograde axonal degeneration and lastly motor neuron death (Fischer et al. 2004). This dying back pattern of degeneration suggests that certain muscle abnormalities precede motor neuron death rather than resulting from it. Such abnormalities include increased nutrient uptake, modified expression of genes involved in carbohydrate and lipid metabolism, increased mitochondrial uncoupling protein-3 content, decreased ATP levels and altered mitochondrial respiration (Dupuis et al. 2003, 2004a,b, 2006) were also observed in mSOD1 muscles. In all, these findings show that mSOD1 mice display significant muscle energy defects long before presenting with motor neuron degeneration. Functional evidence for an active role of muscle in ALS came from studies by Dobrowolny et al. (2005), who showed that a genetic intervention targeted at muscle fibers is sufficient to significantly delay the disease. These authors generated transgenic mice expressing insulin-like growth

factor-1, a well-characterized anabolic hormone, in skeletal muscle using an isoform which is locally active but does not enter the circulation. Crossing these animals with mSOD1 mice yielded a remarkable increase of lifespan. In keeping with this, ALS symptoms appear in mice only when expressing mSOD1 in motor neurons, astrocytes and muscles at the same time (Wang et al. 2005), which suggests, although do not formally prove, that skeletal muscle contributes to ALS physiopathology. It should be noted that, however, a recent study by Kaspar and collaborators demonstrated a lack of effect of muscle-specific silencing of mSOD1 in mSOD1 mouse disease (Kaspar et al. 2006). If muscle participates to motor neuron degeneration, the most reasonable explanation would be that ‘toxic’ signals originating from skeletal muscle could destroy the neuromuscular junctions and subsequently provoke a retrograde axonopathy leading to motor neuron death. This notion is consistent with early data by Kong and Xu (1999), who showed that peripheral axotomy is able to slow motor neuron degeneration in mSOD1 mice. More recently, our own studies on the protein Nogo-A have brought new insights on the complex relationships between muscles and motor neurons in ALS. Nogo-A is a well-known repellent for axonal regeneration synthesized by oligodendrocytes in the CNS (Schwab 2004; Fergani et al. 2005). In ALS, Nogo-A is also selectively expressed, through yet unknown mechanisms, in muscles of mSOD1 mice and SALS patients (Dupuis et al. 2002). Furthermore, the levels of expression of Nogo-A in muscle are correlated with the functional status of the patients: the more Nogo-A is expressed, the more severe the disease is (Jokic et al. 2005). Most importantly, knocking out Nogo-A increased mSOD1 mouse lifespan and protected motor neurons, whereas overexpressing Nogo-A in adult mouse muscle fibers led to neuromuscular junction instability and nerve terminal retraction (Jokic et al. 2006), which strongly suggests that Nogo-A might be a major molecular player underlying neuromuscular junction destabilization in ALS. The picture arising from the different studies on mSOD1 mice is that different cell types orchestrate motor neuron death. Recent evidence further suggests that, beyond the neuromuscular system, the disease in mSOD1 mice is systemic. Indeed, energy homeostasis in two strains of mSOD1 mice has been shown to be strongly imbalanced, leading to body weight deficiency. This ‘systemic’ pathology is characterized by an increase in the basal metabolic rate in mSOD1 mice as compared with control littermates, and is associated with decreased energy stores and changes in plasma levels of several hormones and metabolites. Interestingly, compensating this energy deficit in mSOD1 mice by increasing the energy content of the diet extends lifespan and rescues motor neurons (Dupuis et al. 2004a). Overall, we can conclude that SOD1-linked ALS is not a pure motor neuron disease but rather a systemic affection implicating several



Fig. 1 Cu/Zn superoxide dismutase 1-linked amyotrophic lateral sclerosis viewed as a systemic pathology with prominent motor unit failure. In mutant Cu/Zn superoxide dismutase 1 mice, several cell types are involved in the triggering of motor neuron degeneration. These include motor neurons themselves but also astrocytes, microglia and skeletal muscle fibers.

tissues and cells, which all contribute to motor neuron degeneration (Fig. 1). Dynein/dynactin-linked ALS: a pure motor neuron disease?

Dynein is a microtubule cytoplasmic motor protein activated by dynactin. The dynein/dynactin complex has multiple basic cellular functions, including vesicular transport and cell division. Dynein is also involved in neuron-specific activities such as retrograde transport in the axon (Levy and Holzbaur 2006). In contrast to what is observed in SOD1-linked ALS, several seminal studies have shown that dynein/dynactin perturbations trigger motor neuron disease through a neuronautonomous mechanism. Post-natal overexpression of dynamitin only in motor neurons, by using the Thy-1 promoter, is sufficient to trigger their death (LaMonte et al. 2002; Hafezparast et al. 2003). Similarly, motoneuronal overexpression of an ALS-linked mutant dynactin is also sufficient to induce motor neuron degeneration. These results therefore suggest that an alteration of the dynein/dynactin complex of motoneuronal origin is enough to induce motor neuron disease. It should be noted, however, that it is not known at the present time whether the recently described ALSassociated mutations loa and Cra present in the dynein heavy chain also lead to such a ‘motor neuron only’ effect. How do these alterations of the dynein/dynactin complex cause motor neuron degeneration? Loa homozygous pups display severely compromised migration of the axons of facial motor neurons, present with an abnormal branching and elongation of limb nerves and die shortly after birth (Hafezparast et al. 2003). Furthermore, cultured motor neurons isolated from loa/loa embryos exhibit decreased

Fig. 2 Dynein/dynactin-linked amyotrophic lateral sclerosis viewed as a motor neuron-specific pathology. In mice with dynein dysfunction, motor neurons show altered retrograde transport accounting by itself for the subsequent motor neuron death and amyotrophic lateral sclerosis pathology.

retrograde transport rates (Hafezparast et al. 2003), thus demonstrating that dynein is critical for motor neuron development and survival. The mechanisms underlying the neuron-specific toxic effects of mutant dynactin are currently being unraveled. Indeed, ALS-associated mutations in the dynactin subunit p150glued induce its accumulation, impair dynactin function and trigger motor neuron death. Inhibiting this aggregation by overexpression of the chaperone heatshock protein 70 prevented neuronal death, suggesting that the aggregates were the toxic species (Levy et al. 2006). Overall, we postulate that the specific defects in the dynein/ dynactin complex restricted to motor neurons are able by themselves to trigger ALS, and thus represent a ‘pure’ motor neuron form of the disease (Fig. 2). VEGF d/d mice as a model of ALS: pure motor neuron disease or systemic affection?

VEGF d/d mice were initially designed to display lower induction of vegf under hypoxic conditions but, later on, were surprisingly found to develop a motor neuron disease with characteristically decreased baseline levels of VEGF in brain and spinal cord (Oosthuyse et al. 2001). What are the mechanisms of motor neuron death in these mice? HRE deletion in the promoter region of vegf impairs vascular perfusion of nervous tissue in VEGF d/d mice, and this defect in vegf leads to an absence of VEGF up-regulation following hypoxia (Oosthuyse et al. 2001). Thus, altered vascular perfusion of motor neurons could be one of the possible mechanisms leading to their death. Alternatively, the loss of the intrinsic neurotrophic properties of VEGF could be another one. Lowering VEGF in mSOD1 mice and ischemic mouse models enhances motor neuron loss (Lambrechts et al. 2003). In turn, increasing the sensitivity of motor



neurons to VEGF by motorneuronal overexpression of its receptor leads to increased survival of mSOD1 mice (Storkebaum et al. 2005). Summarizing, it is plausible that lowered VEGF levels may alone provoke motor neuron death in VEGF d/d mice (Bogaert et al. 2006) but the nature of the cells in which HRE deletion is necessary to trigger ALS is still elusive. In this setting, it would be of interest to determine whether the targeted vegf HRE deletion in motor neurons is sufficient to trigger disease. As a result, VEGFlinked ALS cannot be ascribed for the moment to either the ‘neuronal’ or ‘systemic’ form of the disease. When in Rome: future challenges in ALS

The study of the animal models of ALS has shed light on several abnormal molecular pathways that all lead to the same fatal end that is motor neuron death. It may be envisioned that ALS is not a single disease but rather a syndrome and as such it may be subdivided into different subtypes. mSOD1 mice present with a systemic process in which multiple cell types are involved in the triggering of motor neuron death. Thus, SOD1-linked ALS may represent a ‘systemic’ subtype of the disease. Alternatively, mutant dynein/dynactin mice present with motor neuron abnormalities (i.e., altered axonal transport), which seem sufficient to provoke neuronal death. As a consequence, dynein/dynactinlinked ALS may represent a ‘pure’ motor neuron subtype of the disease. Interestingly, it was recently shown that dynein mutations, as earlier reported for neurofilament overexpression (Couillard-Despres et al. 1998), paradoxically attenuate motor neuron disease and extend lifespan of mSOD1 mice (Kieran et al. 2005; Teuchert et al. 2006). An attractive way to put all the pieces of the puzzle together would be to hypothesize that the defect in retrograde transport observed in loa and Cra mice could attenuate a putative mSOD1driven toxic signal originating from skeletal muscle and provoking motor neuron death (Kong and Xu 1999). Further exhaustive work is obviously needed to test this hypothesis. A major challenge in ALS research is to determine to which extent the results obtained from animal models are translatable to the patients. In this respect, it is of interest to note that most of recent ALS clinical trials have failed, in part because their rationale was based on data from mSOD1 mice. One should also keep in mind that mutation in sod1 account for only 2% of all ALS cases. However, several clinical reports have shown that 30–60% of the ALS population presents with an hypermetabolic trait (Desport et al. 1999, 2000, 2001, 2005) reminiscent of what is observed in mSOD1 mice (Dupuis et al. 2004a). Although these findings may appear a priori contradictory, they suggest that at least a subset of ALS patients may present with a ‘systemic’ form of the disease, even in the absence of SOD1 mutations. Defining subtypes of ALS based on pathophysiological mechanisms is therefore of major importance because, if

different pathways can induce motor neuron disease, multiple therapeutic targets can also be envisaged. Subtyping ALS may thus become a first step in the quest to generate specific treatments for each population of patients. Acknowledgements The INSERM-U692 laboratory is supported by Association Française contre les Myopathies (AFM), Association pour la Recherche sur la Scle´rose Late´rale Amyotrophique (ARS), Fondation pour la Recherche sur le Cerveau (FRC), Fondation pour la Recherche Me´dicale (FRM) and Association pour la Recherche et le De´veloppement de Moyens de Lutte contre les Maladies Neurode´geńe´ratives (AREMANE). A. F. is supported by grants from Re´gion Alsace, Association pour l’Etude de la Culture d’Embryons et des The´rapeutiques des Maladies du Syste`me Nerveux (ACE) and FRM. A. E.-L. is supported by a grant from ARS. The authors thank the staff of the laboratory for the numerous initial discussions in the preparation of this review.

References Bogaert E., Damme P. V., Van Den Bosch L. and Robberecht W. (2006) Vascular endothelial growth factor in amyotrophic lateral sclerosis and other neurodegenerative diseases. Muscle Nerve 34, 391–405. Boillee S., Yamanaka K., Lobsiger C. S., Copeland N. G., Jenkins N. A., Kassiotis G., Kollias G. and Cleveland D. W. (2006) Onset and progression in inherited ALS determined by motor neurons and microglia. Science 312, 1389–1392. Brooks K. J., Hill M. D., Hockings P. D. and Reid D. G. (2004) MRI detects early hindlimb muscle atrophy in Gly93Ala superoxide dismutase-1 (G93A SOD1) transgenic mice, an animal model of familial amyotrophic lateral sclerosis. NMR Biomed. 17, 28–32. Bruijn L. I., Houseweart M. K., Kato S., Anderson K. L., Anderson S. D., Ohama E., Reaume A. G., Scott R. W. and Cleveland D. W. (1998) Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1. Science 281, 1851–1854. Bruijn L. I., Miller T. M. and Cleveland D. W. (2004) Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu. Rev. Neurosci. 27, 723–749. Cai H., Lin X., Xie C. et al. (2005) Loss of ALS2 function is insufficient to trigger motor neuron degeneration in knock-out mice but predisposes neurons to oxidative stress. J. Neurosci. 25, 7567–7574. Chen Y. Z., Bennett C. L., Huynh H. M. et al. (2004) DNA/RNA helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4). Am. J. Hum. Genet. 74, 1128–1135. Chen W., Saeed M., Mao H. et al. (2006) Lack of association of VEGF promoter polymorphisms with sporadic ALS. Neurology 67, 508– 510. Clement A. M., Nguyen M. D., Roberts E. A. et al. (2003) Wild-type nonneuronal cells extend survival of SOD1 mutant motor neurons in ALS mice. Science 302, 113–117. Couillard-Despres S., Zhu Q., Wong P. C., Price D. L., Cleveland D. W. and Julien J. P. (1998) Protective effect of neurofilament heavy gene overexpression in motor neuron disease induced by mutant superoxide dismutase. Proc. Natl Acad. Sci. USA 95, 9626–9630. Deng H. X., Shi Y., Furukawa Y. et al. (2006) Conversion to the amyotrophic lateral sclerosis phenotype is associated with intermolecular linked insoluble aggregates of SOD1 in mitochondria. Proc. Natl Acad. Sci. USA 103, 7142–7147.



Desport J. C., Preux P. M., Truong T. C., Vallat J. M., Sautereau D. and Couratier P. (1999) Nutritional status is a prognostic factor for survival in ALS patients. Neurology 53, 1059–1063. Desport J. C., Preux P. M., Truong C. T., Courat L., Vallat J. M. and Couratier P. (2000) Nutritional assessment and survival in ALS patients. Amyotroph. Lateral Scler. Other Motor Neuron Disord. 1, 91–96. Desport J. C., Preux P. M., Magy L., Boirie Y., Vallat J. M., Beaufrere B. and Couratier P. (2001) Factors correlated with hypermetabolism in patients with amyotrophic lateral sclerosis. Am. J. Clin. Nutr. 74, 328–334. Desport J. C., Torny F., Lacoste M., Preux P. M. and Couratier P. (2005) Hypermetabolism in ALS: correlations with clinical and paraclinical parameters. Neurodegen. Dis. 2, 202–207. Devon R. S., Orban P. C., Gerrow K. et al. (2006) Als2-deficient mice exhibit disturbances in endosome trafficking associated with motor behavioral abnormalities. Proc. Natl Acad. Sci. USA 103, 9595– 9600. Dobrowolny G., Giacinti C., Pelosi L., Nicoletti C., Winn N., Barberi L., Molinaro M., Rosenthal N. and Musaro A. (2005) Muscle expression of a local Igf-1 isoform protects motor neurons in an ALS mouse model. J. Cell. Biol. 168, 193–199. Dupuis L., Gonzalez de Aguilar J. L., di Scala F. et al. (2002) Nogo provides a molecular marker for diagnosis of amyotrophic lateral sclerosis. Neurobiol. Dis. 10, 358–365. Dupuis L., di Scala F., Rene F., de Tapia M., Oudart H., Pradat P. F., Meininger V. and Loeffler J. P. (2003) Up-regulation of mitochondrial uncoupling protein 3 reveals an early muscular metabolic defect in amyotrophic lateral sclerosis. FASEB J. 17, 2091–2093. Dupuis L., Oudart H., Rene F., Gonzalez de Aguilar J. L. and Loeffler J. P. (2004a) Evidence for defective energy homeostasis in amyotrophic lateral sclerosis: benefit of a high-energy diet in a transgenic mouse model. Proc. Natl Acad. Sci. USA 101, 11 159– 11 164. Dupuis L., Gonzalez de Aguilar J. L., Oudart H., de Tapia M., Barbeito L. and Loeffler J. P. (2004b) Mitochondria in amyotrophic lateral sclerosis: a trigger and a target. Neurodegen. Dis. 1, 245–254. Dupuis L., Gonzalez de Aguilar J. L., Echaniz-Laguna A. and Loeffler J. P. (2006) Mitochondrial dysfunction in amyotrophic lateral sclerosis also affects skeletal muscle. Muscle Nerve 34, 253–254. Fergani A., Dupuis L., Jokic N., Larmet Y., de Tapia M., Rene F., Loeffler J. P. and Gonzalez de Aguilar J. L. (2005) Reticulons as markers of neurological diseases: focus on amyotrophic lateral sclerosis. Neurodegen. Dis. 2, 185–194. Fischer L. R., Culver D. G., Tennant P., Davis A. A., Wang M., Castellano-Sanchez A., Khan J., Polak M. A. and Glass J. D. (2004) Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man. Exp. Neurol. 185, 232–240. Gong Y. H., Parsadanian A. S., Andreeva A., Snider W. D. and Elliott J. L. (2000) Restricted expression of G86R Cu/Zn superoxide dismutase in astrocytes results in astrocytosis but does not cause motoneuron degeneration. J. Neurosci. 20, 660–665. Greenway M. J., Andersen P. M., Russ C. et al. (2006) ANG mutations segregate with familial and ‘sporadic’ amyotrophic lateral sclerosis. Nat. Genet. 38, 411–413. Gros-Louis F., Gaspar C. and Rouleau G. A. (2006) Genetics of familial and sporadic amyotrophic lateral sclerosis. Biochim. Biophys. Acta 1762, 956–972. Guegan C. and Przedborski S. (2003) Programmed cell death in amyotrophic lateral sclerosis. J. Clin. Invest. 111, 153–161. Gurney M. E., Pu H., Chiu A. Y. et al. (1994) Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264, 1772–1775.

Hadano S., Hand C. K., Osuga H. et al. (2001) A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2. Nat. Genet. 29, 166–173. Hadano S., Benn S. C., Kakuta S. et al. (2006) Mice deficient in the Rab5 guanine nucleotide exchange factor ALS2/alsin exhibit agedependent neurological deficits and altered endosome trafficking. Hum. Mol. Genet. 15, 233–250. Hafezparast M., Klocke R., Ruhrberg C. et al. (2003) Mutations in dynein link motor neuron degeneration to defects in retrograde transport. Science 300, 808–812. Jaarsma D., Haasdijk E. D., Grashorn J. A., Hawkins R., van Duijn W., Verspaget H. W., London J. and Holstege J. C. (2000) Human Cu/ Zn superoxide dismutase (SOD1) overexpression in mice causes mitochondrial vacuolization, axonal degeneration, and premature motoneuron death and accelerates motoneuron disease in mice expressing a familial amyotrophic lateral sclerosis mutant SOD1. Neurobiol. Dis. 7, 623–643. Jaarsma D., Teuling E. and Hoogenraad C. C. (2006) Neuron-specific expression of mutant SOD1 is sufficient to cause motoneuron disease in mice Soc. Neurosci. Abstr. 508, 1. Jokic N., Gonzalez de Aguilar J. L., Pradat P. F. et al. (2005) Nogo expression in muscle correlates with amyotrophic lateral sclerosis severity. Ann. Neurol. 57, 553–556. Jokic N., Gonzalez de Aguilar J. L., Dimou L., Lin S., Fergani A., Ruegg M. A., Schwab M. E., Dupuis L. and Loeffler J. P. (2006) The neurite outgrowth inhibitor Nogo-A promotes denervation in an amyotrophic lateral sclerosis model. EMBO Rep. 7, 1162–1167. Kanekura K., Nishimoto I., Aiso S. and Matsuoka M. (2006) Characterization of amyotrophic lateral sclerosis-linked P56S mutation of vesicle-associated membrane protein-associated protein B (VAPB/ALS8). J. Biol. Chem. 281, 30 223–30 233. Kaspar B. K., Kim S. H., Miller T. M., Hester M., Umapati P., Arnson H., Rizo L., Mendell J. R., Gage F. H. and Cleveland D. W. (2006) Muscle is not a primary target in ALS as revealed by gene therapy in muscle of ALS Mice Soc. Neurosci. Abstr. 508, 3. Kieran D., Hafezparast M., Bohnert S., Dick J. R., Martin J., Schiavo G., Fisher E. M. and Greensmith L. (2005) A mutation in dynein rescues axonal transport defects and extends the life span of ALS mice. J. Cell. Biol. 169, 561–567. Kong J. and Xu Z. (1999) Peripheral axotomy slows motoneuron degeneration in a transgenic mouse line expressing mutant SOD1 G93A. J. Comp. Neurol. 412, 373–80. Lacomblez L., Bensimon G., Leigh P. N., Guillet P. and Meininger V. (1996) Dose-ranging study of riluzole in amyotrophic lateral sclerosis. Amyotrophic Lateral Sclerosis/Riluzole Study Group II. Lancet 347, 1425–1431. Lambrechts D., Storkebaum E., Morimoto M. et al. (2003) VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death. Nat. Genet. 34, 383– 394. LaMonte B. H., Wallace K. E., Holloway B. A., Shelly S. S., Ascano J., Tokito M., Van Winkle T., Howland D. S. and Holzbaur E. L. (2002) Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration. Neuron 34, 715–727. Levy J. R. and Holzbaur E. L. (2006) Cytoplasmic dynein/dynactin function and dysfunction in motor neurons. Int. J. Dev. Neurosci. 24, 103–111. Levy J. R., Sumner C. J., Caviston J. P. et al. (2006) A motor neuron disease-associated mutation in p150Glued perturbs dynactin function and induces protein aggregation. J. Cell. Biol. 172, 733–745. Lino M. M., Schneider C. and Caroni P. (2002) Accumulation of SOD1 mutants in postnatal motoneurons does not cause motoneuron pathology or motoneuron disease. J. Neurosci. 22, 4825–4832.



Mase G., Ros S., Gemma A. et al. (2001) ALS with variable phenotypes in a six-generation family caused by leu144phe mutation in the SOD1 gene. J. Neurol. Sci. 191, 11–18. Munch C., Sedlmeier R., Meyer T. et al. (2004) Point mutations of the p150 subunit of dynactin (DCTN1) gene in ALS. Neurology 63, 724–726. Nishimura A. L., Mitne-Neto M., Silva H. C. et al. (2004) A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am. J. Hum. Genet. 75, 822–831. Oosthuyse B., Moons L., Storkebaum E. et al. (2001) Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat. Genet. 28, 131– 138. Orrell R. W., Habgood J. J., Gardiner I., King A. W., Bowe F. A., Hallewell R. A., Marklund S. L., Greenwood J., Lane R. J. and deBelleroche J. (1997) Clinical and functional investigation of 10 missense mutations and a novel frameshift insertion mutation of the gene for copper-zinc superoxide dismutase in UK families with amyotrophic lateral sclerosis. Neurology 48, 746–751. Pasinelli P. and Brown R. H. (2006) Molecular biology of amyotrophic lateral sclerosis: insights from genetics. Nat. Rev. Neurosci. 7, 710– 723. Pehar M., Cassina P., Vargas M. R., Castellanos R., Viera L., Beckman J. S., Estevez A. G. and Barbeito L. (2004) Astrocytic production of nerve growth factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis. J. Neurochem. 89, 464–473. Pehar M., Vargas M. R., Cassina P., Barbeito A. G., Beckman J. S. and Barbeito L. (2005) Complexity of astrocyte-motor neuron interactions in amyotrophic lateral sclerosis. Neurodegen. Dis. 2, 139– 146. Pramatarova A., Laganiere J., Roussel J., Brisebois K. and Rouleau G. A. (2001) Neuron-specific expression of mutant superoxide dismutase 1 in transgenic mice does not lead to motor impairment. J. Neurosci. 21, 3369–3374. Puls I., Jonnakuty C., LaMonte B. H. et al. (2003) Mutant dynactin in motor neuron disease. Nat. Genet. 33, 455–456. Puls I., Oh S. J., Sumner C. J. et al. (2005) Distal spinal and bulbar muscular atrophy caused by dynactin mutation. Ann. Neurol. 57, 687–694. Reaume A. G., Elliott J. L., Hoffman E. K. et al. (1996) Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury. Nat. Genet. 13, 43–47.

Rezania K., Yan J., Dellefave L., Deng H. X., Siddique N., Pascuzzi R. T., Siddique T. and Roos R. P. (2003) A rare Cu/Zn superoxide dismutase mutation causing familial amyotrophic lateral sclerosis with variable age of onset, incomplete penetrance and a sensory neuropathy. Amyotroph. Lateral Scler. Other Motor Neuron Disord. 4, 162–166. Ripps M. E., Huntley G. W., Hof P. R., Morrison J. H. and Gordon J. W. (1995) Transgenic mice expressing an altered murine superoxide dismutase gene provide an animal model of amyotrophic lateral sclerosis. Proc. Natl Acad. Sci. USA 92, 689–693. Rosen D. R., Siddique T., Patterson D. et al. (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59–62. Rowland L. P. (1998) Diagnosis of amyotrophic lateral sclerosis. J. Neurol. Sci. 160(Suppl 1), S6–24. Schwab M. E. (2004) Nogo and axon regeneration. Curr. Opin. Neurobiol. 14, 118–124. Storkebaum E., Lambrechts D., Dewerchin M. et al. (2005) Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rat model of ALS. Nat. Neurosci. 8, 85–92. Teuchert M., Fischer D., Schwalenstoecker B., Habisch H. J., Bockers T. M. and Ludolph A. C. (2006) A dynein mutation attenuates motor neuron degeneration in SOD1(G93A) mice. Exp. Neurol. 198, 271–274. Van Vught P. W., Sutedja N. A., Veldink J. H. et al. (2005) Lack of association between VEGF polymorphisms and ALS in a Dutch population. Neurology 65, 1643–1645. Wang J., Xu G., Slunt H. H., Gonzales V., Coonfield M., Fromholt D., Copeland N. G., Jenkins N. A. and Borchelt D. R. (2005) Coincident thresholds of mutant protein for paralytic disease and protein aggregation caused by restrictively expressed superoxide dismutase cDNA. Neurobiol. Dis. 20, 943–952. Wong P. C., Pardo C. A., Borchelt D. R., Lee M. K., Copeland N. G., Jenkins N. A., Sisodia S. S., Cleveland D. W. and Price D. L. (1995) An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron 14, 1105–1116. Yamanaka K., Miller T. M., McAlonis-Downes M., Chun S. J. and Cleveland D. W. (2006) Progressive spinal axonal degeneration and slowness in ALS2-deficient mice. Ann. Neurol. 60, 95–104. Yang Y., Hentati A., Deng H. X. et al. (2001) The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis. Nat. Genet. 29, 160–165.
