Molecular pathways of motor neuron injury in ... - Semantic Scholar

REVIEWS

Molecular pathways of motor neuron injury in amyotrophic lateral sclerosis Laura Ferraiuolo, Janine Kirby, Andrew J. Grierson, Michael Sendtner and Pamela J. Shaw Abstract | Amyotrophic lateral sclerosis (ALS) is a genetically diverse disease. At least 15 ALS-associated gene loci have so far been identified, and the causative gene is known in approximately 30% of familial ALS cases. Less is known about the factors underlying the sporadic form of the disease. The molecular mechanisms of motor neuron degeneration are best understood in the subtype of disease caused by mutations in superoxide dismutase 1, with a current consensus that motor neuron injury is caused by a complex interplay between multiple pathogenic processes. A key recent finding is that mutated TAR DNA-binding protein 43 is a major constituent of the ubiquitinated protein inclusions in ALS, providing a possible link between the genetic mutation and the cellular pathology. New insights have also indicated the importance of dysregulated glial cell–motor neuron crosstalk, and have highlighted the vulnerability of the distal axonal compartment early in the disease course. In addition, recent studies have suggested that disordered RNA processing is likely to represent a major contributing factor to motor neuron disease. Ongoing research on the cellular pathways highlighted in this Review is predicted to open the door to new therapeutic interventions to slow disease progression in ALS. Ferraiuolo, L. et al. Nat. Rev. Neurol. 7, 616–630 (2011); doi:10.1038/nrneurol.2011.152

Introduction

Academic Neurology Unit, Sheffield Institute for Translational Neuroscience, Department of Neuroscience, School of Medicine and Biomedical Sciences, University of Sheffield, 385A Glossop Road, Sheffield S10 2HQ, UK (L. Ferraiuolo, J. Kirby, A. J. Grierson, P. J. Shaw). The Institute for Clinical Neurobiology, Versbacher Strasse 5, University of Würzburg, 97078 Würzburg, Germany (M. Sendtner). Correspondence to: P. J. Shaw pamela.shaw@ sheffield.ac.uk

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of devastating impact that causes injury and death of lower motor neurons in the brainstem and spinal cord, and of upper motor neurons in the motor cortex. The resulting progressive failure of the neuromuscular system usually culminates in death from respiratory failure. The worldwide incidence of approximately two per 100,000 individuals is fairly uniform, except for a few high-incidence foci, such as the Kii peninsula and Guam. The mean age of onset is 55–60 years, and the disease more commonly affects men than women. The average survival from symptom onset is approximately 3 years, although a proportion of patients have a slower disease course.1 ALS was traditionally considered to be a pure motor disorder. However, recent findings in subsets of patients with ALS have highlighted the involvement of sensory and spino cerebellar pathways, as well neuronal groups within the substantia nigra and the hippocampal dentate granule layer.2 ALS is, therefore, regarded as a multisystem dis order in which the motor neurons tend to be affected earli est and most severely. The major gross and microscopic pathological changes are highlighted in Box 1.2–9 The pathogenic processes underlying ALS are multi factorial and, at present, not fully determined. This Review discusses recent advances in our understanding of the neurodegenerative process, relating predominantly to the subtype of disease caused by mutations in the gene encoding superoxide dismutase 1 (SOD1). Newly Competing interests The authors declare no competing interests.

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identified genes associated with ALS have important roles in RNA processing, endosomal trafficking and protein degradation pathways. We highlight the complex interplay between multiple mechanisms, including genetic factors, oxidative stress, excitotoxicity and protein aggregation, as well as impairments in RNA processing, axonal transport and mitochondrial function (Figure 1). We also discuss the increasingly recognized role of neighboring glial cells in motor neuron injury,10 the involvement of particular molecular signaling pathways in motor neuron survival and cell death,11–14 and the emerging concept that the neuromuscular junction and distal axonal compartment are early and important aspects of disease patho physiology.15 Finally, we consider why motor neurons are particularly susceptible to cell death in ALS, and suggest links between cellular pathogenic pathways and key clinical features of the disease. Mouse models of ALS harboring mutant SOD1 (mSOD1 models), discussed in this article, have been used extensively for preclinical drug testing, with many therapies reported to ameliorate the disease course. However, the utility of these mouse models for the development of human therapies has been questioned owing to poor results in clinical trials (Box 2).16,17

Pathogenic mechanisms Genetic factors ALS is most commonly a sporadic disease. 5–10% of cases are familial and usually of autosomal dominant inheritance (Table 1). The identification of the genetic subtypes of ALS has established key pathogenic mechanisms, which www.nature.com/nrneurol

© 2011 Macmillan Publishers Limited. All rights reserved

A M YOT R O P H I C L AT E R A L S C L E R O S I S are applicable not only to the minority of cases that carry familial ALS (FALS) mutations, but also to sporadic ALS (SALS) more broadly. The FALS-associated gene variants so far identified (Table 1) have provided important clues about the underlying molecular mechanisms in the disease, as discussed below. In addition, candidate gene analysis in patients with SALS has identified polymorphisms in the genes encoding vascular endothelial growth factor, hereditary hemochromatosis protein and paraxonase enzymes PON1–3, as well as copy number variation in the survival motor neuron genes SMN1 and SMN2, as potential risk factors for ALS.18 These findings have not, however, been consistently replicated in different populations. Similarly, recent genome-wide association studies have identified several potential ALS susceptibility loci, including FGGY,19 inositol 1,4,5-trisphosphate receptor type 2 (ITPR2)20 and dipeptidyl-peptidase 6 (DPP6).21 These results have yet to be validated in additional independent populations. In 2010, two separate groups reported the association of SALS with single nucleotide polymorphisms on chromosome 9p21,22,23 which is also an identified susceptibility locus for FALS and frontotemporal dementia24 (see Note added in proof ). Intermediate-length polyglutamine expansions in ataxin‑2 have recently been identified as a risk factor for sporadic ALS.25 A detailed discussion of genetic factors in ALS is beyond the scope of this article and has been reviewed elsewhere.26

Oxidative stress Oxidative stress causes structural damage and changes in redox-sensitive signaling. It arises from an imbalance between the generation and removal of reactive oxygen species (ROS), and/or from a reduction in the ability of the biological system to remove or repair ROS-induced damage. The accumulation of oxidative stress in nonreplicating neurons during aging may be an important factor that tips the balance from an ability to cope with a toxic insult, such as the presence of a disease-causing mutation, into a vicious circle of cellular injury that culminates in neuronal death and onset of neurodegeneration in middle or later life. Particular interest has been shown in the role of oxidative stress in ALS, because mutations in SOD1, which encodes a major antioxidant protein, account for 20% of FALS cases.27 Biosamples from patients with ALS—including cerebrospinal fluid (CSF), serum and urine—show eleva tion of markers of free radical damage. 28–30 Moreover, elevated levels of oxidative damage to proteins,31 lipids32 and DNA33 have been found in postmortem tissue from SALS and SOD1-related FALS cases. Oxidative damage to RNA species has also been documented in mSOD1 mouse models as well as the human CNS.34 In the murine model, mRNA oxidation is primarily found in motor neurons and oligodendrocytes, occurs in the early presymptomatic disease stages, and is associated with decreased expression of the encoded proteins. Some mRNA species seem to be more susceptible to oxidation, including those involved in the mitochondrial electron transport chain, protein biosynthesis, folding and degradation pathways,

Key points ■■ Multiple cellular events contribute to the pathobiology of amyotrophic lateral sclerosis (ALS), including oxidative stress, mitochondrial dysfunction, excitotoxicity, protein aggregation, impaired axonal transport, neuroinflammation, and dysregulated RNA signaling ■■ TAR DNA-binding protein 43 is a major constituent of the ubiquitinated protein inclusions found in surviving motor neurons in most forms of ALS ■■ Glial pathology and disruption of glial cell–motor neuron communication contribute to neurodegeneration and the propagation of motor neuron injury ■■ Understanding the links between molecular changes and clinical features of the disease should guide future therapeutic efforts ■■ Degenerative changes in motor neurons seem to affect the health of the distal axonal compartment at an early stage of disease, highlighting an important neuroprotective target

Box 1 | Pathological changes in amyotrophic lateral sclerosis Gross pathological changes ■■ Atrophy of the precentral gyrus ■■ Shrinkage, sclerosis and pallor of the corticospinal tracts ■■ Thinning of the spinal ventral roots and hypoglossal nerves ■■ Atrophy of the somatic and bulbar muscles

Microscopic changes ■■ Depletion of ≥50% of the spinal cord motor neurons ■■ Diffuse astrocytic gliosis of spinal gray matter ■■ Atrophic and basophilic changes in surviving motor neurons ■■ Presence in surviving lower motor neurons of ubiquitinated inclusion bodies with thread-like, skein-like or compact morphology ■■ Variable depletion of giant pyramidal neurons (Betz cells) in the motor cortex ■■ Variable astrocytic gliosis in the gray matter of the motor cortex and underlying subcortical white matter ■■ Evidence of microglial activation in pathologically affected areas ■■ Cytoplasmic aggregate inclusions within glial cells8,9

myelination, the cytoskeleton, and the tricarboxylic acid cycle and glycolysis pathways.34 Oxidative damage occurs in cellular and murine models of SOD1-related ALS (reviewed elsewhere35) and, interestingly, the SOD1 protein itself seems to be particularly susceptible to oxidative post-translational modification.36 Recently developed cellular models of mutant TAR DNA-binding protein 43 (TDP-43)-related ALS indicate that the presence of this mutant protein also induces oxidative stress in motor neuronal cell lines.37 Sources of oxidative stress in ALS have been mostthoroughly investigated in mSOD1 models, in which activation of several aberrant oxidative reactions have been proposed (as reviewed previously 35). However, mutant SOD1 that is enzymatically inactive due to depletion of copper loading is still capable of causing motor neuron degeneration,38 and recent evidence suggests that mSOD1 may cause oxidative stress by mechanisms beyond its cata lytic activity. Specifically, mSOD1 in microglia increases NADPH oxidase (NOX)-mediated superoxide production by locking Rac1 into its active state in the NOX complex, resulting in prolongation of ROS production. 39 NOX2 expression is increased in mSOD1 mice and in the CNS

NATURE REVIEWS | NEUROLOGY

VOLUME 7 | NOVEMBER 2011 | 617 © 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS Reduced lactate release

Motor neuron

Apoptosis

Astrocytes Lactate efflux transporters

Ca2+

Pro-NGF

ROS Altered RNA processing

p75 NGF receptor Complement activation

Excitotoxicity

BAX

NO PGE2

Misfolded proteins

C1q Autophagy

PGE2

IL

NO Inflammation

Impaired axonal transport

Glutamate transporters

Mitochondrial dysfunction

IL MCP-1

Proteasome impairment IL

ER stress

Microglia

Distal axonopathy

Figure 1 | Molecular mechanisms of motor neuron injury in ALS. ALS is a complex disease involving activation of several cellular pathways in motor neurons, and dysregulated interaction with neighboring glial cells. Microglia activate an inflammatory cascade via secretion of MCP‑1 and other cytokines. Astrocytes contribute to motor neuron injury through various mechanisms, including release of inflammatory mediators such as NO and PGE2; reduced expression and activity of the glutamate reuptake transporter EAAT2; reduced lactate release; and activation of pro-NGF–p75 receptor signaling. Motor neurons might also undergo transcriptional dysregulation and abnormal RNA processing which, together with overproduction of ROS, contribute to aberrant protein folding. Aberrant proteins can form aggregates, leading to proteasome impairment and ER stress, and ultimately activating autophagy and apoptotic pathways. Mitochondrial impairment and dysregulation of calcium handling are two major components of motor neuron injury that also lead to activation of the apoptotic cascade. Impaired axonal transport may contribute to an energy deficit in the distal axon and the dying-back axonopathy that is observed in ALS. Motor neurons can produce and secrete complement subunits that are important signals of cellular stress to neighboring cells. Abbreviations: ALS, amyotrophic lateral sclerosis; EAAT2, excitatory amino acid transporter 2; ER, endoplasmic reticulum; IL, interleukin; MCP‑1, monocyte chemoattractant protein 1; NGF, nerve growth factor; NO, nitric oxide; PGE2, prostaglandin E2; ROS, reactive oxygen species.

of patients with ALS, and survival of SOD1G93A mice is extended by knockout of Nox1 or Nox2.40,41 The transcription factor nuclear erythroid 2‑related factor 2 (NRF2) is a master regulator of the antioxidant response. This factor binds and upregulates antioxidant response element (ARE) genes following oxidative stress. Recent evidence has emerged that NRF2–ARE signaling may be dysregulated in mSOD1 models of ALS,11 and in the CNS of patients with ALS.42 Multiple studies have shown that oxidative stress interacts with, and potentially exacerbates, other patho physiological processes that contribute to motor neuron injury, including excitotoxicity,43 mitochondrial impairment,44 protein aggregation,45 endoplasmic reticulum (ER) stress,46 and alterations in signaling from astrocytes 618 | NOVEMBER 2011 | VOLUME 7

and microglia.47,48 Thus, effective alleviation of oxidative stress could potentially ameliorate multiple facets of the pathobiology of motor neuron degeneration. A metaanalysis of therapeutic interventions tested in mSOD1 mice up to 2007 concluded that antioxidant therapies were the most effective class of drug at improving survival.49 Antioxidants have not yet shown benefit in patients with ALS, although the reported trials have often been of suboptimal design.50

Mitochondrial dysfunction Mitochondria have a central role in intracellular energy production, calcium homeostasis and control of apoptosis. Several lines of evidence implicate mitochondrial dysfunction in ALS pathogenesis (Figure 2). In mSOD1 www.nature.com/nrneurol


A M YOT R O P H I C L AT E R A L S C L E R O S I S mice, the mutated protein aggregates in vacuoles in the mitochondrial intermembrane space,51 and shows an age-dependent increase in adherence to the mito chondrial outer membrane, which is postulated to lead to organelle dysfunction selectively in the spinal cord by impeding protein import.52–54 Defective respiratory chain function associated with oxidative damage to mito chondrial proteins and lipids has been found in tissue from patients with ALS55 and in mSOD1 models.56 Thus, dysregulated energy metabolism is likely to contribute to motor neuron dysfunction in ALS. Calcium buffering is impaired in mitochondria purified from the CNS of mSOD1 mice,57 and could increase the susceptibility of motor neurons to the altered calcium homeostasis associated with glutamate-mediated exci toxicity (discussed below). Furthermore, ER stress, which is predicted to disrupt ER–mitochondria calcium exchange, has been reported in models of ALS.58 Motor neuron cell death in ALS is thought to involve the activation of caspases and apoptosis, and damage to mitochondrial function is likely to contribute to this process.59 Altered mitochondrial morphology has been observed in skeletal muscle and spinal motor neurons from patients with ALS.60 Interestingly, in some mSOD1 mouse models, mitochondrial vacuolation occurs during the presympto matic disease stage, suggesting an early event in the pathophysiological cascade of motor neuron injury.51 Mitochondrial morphology is also altered in primary motor neurons and NSC-34 cells (a murine motor neuronal cell line) expressing mutant SOD1.61 Axonal transport of mitochondria is impaired in experimental models of ALS, and it is possible that a reduction in the mitochondrial content of the distal axon, combined with impaired mitochondrial function, leads to the dying-back axonopathy that is seen in ALS.62,63 Mitochondria represent an attractive target for ALS therapy development, and the novel mitochondrion-targeted neuroprotective compound olexisome64 is currently in a phase III clinical trial for ALS.

Excitotoxicity Glutamate is the main excitatory neurotransmitter in the CNS and exerts its effects through an array of ionotropic and metabotropic postsynaptic receptors. The excitatory signal is terminated by removal of glutamate from the synaptic cleft by glutamate reuptake transporters, the most abundant of which is excitatory amino acid transporter 2 (EAAT2; also known as SLC1A2 or GLT1). Excitotoxicity, the neuronal injury resulting from excessive activation of glutamate receptors, may be caused by increased synaptic levels of glutamate, or by increased sensitivity of the postsynaptic neuron to glutamate, resulting from alterations in neuronal energy homeostasis or glutamate receptor expression.65 Disruption of intracellular calcium homeostasis, with secondary activation of proteolytic and ROS-generating enzyme systems, and perturbation of mitochondrial function and ATP production are key components of excitotoxicity.66 α-Amino‑3-hydroxy‑5-methyl‑4-isoxazolepropionic acid (AMPA) receptors mediate much of the routine

Box 2 | The mutant SOD1 mouse model Many therapies have been reported to ameliorate disease in the mouse model of amyotrophic lateral sclerosis (ALS) that harbors mutant superoxide dismutase 1 (SOD1). However, the utility of this mouse model for the development of human therapies has been questioned owing to poor results in clinical trials.16,17 Many interventions reported as positive have shown mild effects on the disease course, which might simply represent ‘biological noise’ in the mouse model. An important concept in preclinical trials in mouse models that requires clarification is the definition of ‘onset’ and ‘progression’ of disease. A current challenge lies in distinguishing which therapies are truly delaying the onset of disease versus prolonging the compensatory preclinical period, in which the disease process is ongoing in the absence of clinical signs of motor dysfunction. In mutant SOD1 mice, various abnormalities are present well before the onset of clinical signs, often in the first few postnatal days. These abnormalities include behavioral motor changes,187 motor neuron electrophysiological abnormalities,188,189 neuropathological changes such as mitochondrial swelling and vacuolization, 190 and transcriptome changes reflecting an attempt to increase energy provision in motor neurons.131 A recently described mutant SOD1 zebrafish model shows evidence of chronic neuronal stress from early embryogenesis.191 The zebrafish model has several strengths that complement murine models of ALS. For example, this model is relatively inexpensive and is useful for high-throughput screening to identify pharmacological and genetic modifiers of disease phenotype. The transparency of the zebrafish embryos is advantageous for the imaging of motor neurons in an in vivo system.

fast excitatory glutamatergic neurotransmission in the CNS. Motor neurons are especially vulnerable to AMPAmediated excitotoxicity, including distal axonal injury.67,68 The calcium permeability of the AMPA receptor complex is largely determined by the GluR2 subunit, which is post-transcriptionally edited at the Gln/Arg site 586 in the second transmembrane domain, making the receptor complex calcium-impermeable.69 Cell-specific features of motor neurons, including low expression of both GluR269 and calcium-buffering proteins,70 render these neurons vulnerable to AMPA receptor-mediated toxicity. A body of evidence implicates excitotoxicity as a mechanism contributing to motor neuron injury in ALS, although clear evidence that it is a primary disease mecha nism is lacking. In some patients with ALS, levels of CSF glutamate are elevated,71 and the expression and activity of EAAT2 are reduced in pathologically affected areas of the CNS,72–74 although whether this is a cause or a consequence of neuronal loss is unclear.75 Electrophysiological studies in humans have shown hyperexcitability of the motor system in the presymptomatic76 or early stages77 of ALS. Evidence suggests that the calcium permeability of AMPA receptors in the spinal ventral horn may be dysregulated by abnormal editing of the GluR2 AMPA receptor subunit.78 In addition, a recently identified ALSlinked gene encodes d‑amino acid oxidase (DAO). This enzyme is responsible for the oxidative deamination of d‑amino acids, one of which—d-serine—is an activator and co-agonist of N‑methyl‑d-aspartate (NMDA) receptors.79 Mutations in DAO could potentially contribute to excitotoxic motor neuron injury. Several findings in the mSOD1 model also point towards a role for excitotoxicity in ALS pathology, including altered electrophysiological properties and increased sensitivity of motor neurons to excitotoxicity,80 altered AMPA receptor subunit expression, reduced expression



REVIEWS Table 1 | Genes associated with familial ALS Genetic subtype

Chromosomal locus

Gene

Onset/ inheritance

Reference

21q22

Superoxide dismutase 1 (SOD1)

Adult/AD

Rosen (1993)27

ALS4

9q34

Senataxin (SETX)

Juvenile/AD

Chen et al. (2004)164

ALS6

16p11.2

Fused in sarcoma (FUS)

Adult/AD

Kwiatkowski et al. (2009)192 Vance et al. (2009)193

ALS9

14q11.2

Angiogenin (ANG)

Adult/AD

Greenway et al. (2006)161

ALS10

1p36.2

TAR DNA-binding protein (TARDBP)

Adult/AD

Sreedharan et al. (2008)89

Oxidative stress ALS1 RNA processing

Endosomal trafficking and cell signaling ALS2

2q33

Alsin (ALS2)

Juvenile/AR

Yang et al. (2001)102

ALS11

6q21

Polyphosphoinositide phosphatase (FIG4)

Adult/AD

Chow et al. (2009)108

ALS8

20q13.3

Vesicle-associated membrane protein-associated protein B (VAPB)

Adult/AD

Nishimura et al. (2004)104

ALS12

10p13

Optineurin (OPTN)

Adult/AD and AR

Maruyama et al. (2010)105

d-amino

Adult/AD

Mitchell et al. (2010)79

Glutamate excitotoxicity ND

12q24

acid oxidase (DAO)

Ubiquitin/protein degradation ND

9p13–p12

Valosin-containing protein (VCP)

Adult/AD

Johnson et al. (2010)99

ALSX

Xp11

Ubiquilin 2 (UBQLN2)

Adult/X-linked

Deng et al. (2011)101

17q21

Microtubule-associated protein tau (MAPT)

Adult/AD

Hutton et al. (1998)120

ALS5

15q15–q21

Spatacsin (SPG11)

Juvenile/AR

Orlacchio et al. (2010)184

ALS–FTD

9p13.3

σ Non-opioid receptor 1 (SIGMAR1)

Adult/AD Juvenile/AR

Luty et al. (2010)185 Al-Saif et al. (2011)186

ALS–FTD

9q21–q22

Chromosome 9 open reading frame 72 (C9ORF72)

Adult/AD

Hosler et al. (2000)24 Renton et al. (2011)196 De Jesus-Hernandez et al. (2011)197

ALS3

18q21

Unknown

Adult/AD

Hand et al. (2002)194

ALS7

20ptel–p13

Unknown

Adult/AD

Sapp et al. (2003)195

Cytoskeleton ALS–dementia–PD Other genes

Unknown genes

Abbreviations: AD, autosomal dominant; ALS, amyotrophic lateral sclerosis; AR, autosomal recessive; FTD, frontotemporal dementia; PD, Parkinson disease.

and activity of EAAT2,81 increased glutamate efflux from spinal cord nerve terminals,82 a reduction in the motor neuron inhibitory–excitatory synaptic ratio,83 and loss of regulation by astrocytes of the expression of GluR2 by neighboring motor neurons.84 Ameliorating excitotoxicity is the only strategy that has so far slowed disease progression in ALS. Riluzole, which has several effects including inhibition of pre synaptic glutamate release,85 causes a modest increase in survival.86 However, other antiglutamate agents including gabapentin, lamotrigine, topiramate and talampanel have not been effective in clinical trials.87 The reasons for this are uncertain, although a possible explanation is that riluzole has multiple potential neuroprotective effects, some of which extend beyond modification of glutamate excitatory neurotransmission.85 620 | NOVEMBER 2011 | VOLUME 7

Protein aggregation Pathological protein aggregates, identified as compact or skein-like ubiquitinated inclusions, are a cardinal feature of ALS.4–6 The identification of TDP-43 as the major protein constituent of these inclusions initiated a major shift in our understanding of the pathobiology of ALS.7 Under normal conditions, TDP-43 is predominantly localized in the nucleus, and loss of nuclear TDP-43 staining is seen in most cells containing TDP-43-positive cytoplasmic inclusions.7 TDP-43 inclusions are not restricted to motor neurons, and it seems that cytoplasmic redistribution of TDP-43 is an early pathogenic event in ALS.88 In 2008, mutations in TARDBP, the gene encoding TDP-43, were discovered in several FALS pedigrees, thereby consolidating the evidence for TDP-43 dysfunction in ALS and establishing www.nature.com/nrneurol


A M YOT R O P H I C L AT E R A L S C L E R O S I S a

b AMPA receptors lacking the GluR2 subunit

Glutamate

Ca2+

Endoplasmic reticulum stress

Cytochrome c Ca2+ MPTP Caspase activation

ROS Ca2+

Lack of buffering proteins

Electron transport chain mSOD1

Apoptosis

ROS

Mitochondrial membrane depolarization Lipid peroxidation ATP production

mSOD1

Figure 2 | Mitochondrial dysfunction in ALS. a | Studies on postmortem tissue and animal models of ALS have indicated a decrease in the activity of the complexes that form the mitochondrial electron transport chain, which may be caused by oligomers of mSOD1 associating with mitochondria. These oligomers could to lead to alterations in the mitochondrial redox state, damage to the mitochondrial protein import machinery, and sequestration of the antiapoptotic factor Bcl‑2. Loss of EAAT2, and increased expression of calcium-permeable AMPA receptors lacking the edited form of the GluR2 subunit, leads to elevated intracellular calcium in motor neurons. High intracellular calcium concentrations may result in a toxic shift of calcium from the endoplasmic reticulum to the mitochondria, leading to excitotoxicity. Defective electron transport chain activity and calcium homeostasis are thought to underlie aberrant ROS generation. b | Together, these pathways result in depolarization of the mitochondrial membrane potential, reduced production of ATP, increased peroxidation of mitochondrial membrane lipids, opening of the MPTP channel, and initiation of apoptosis with release of cytochrome c into the cytoplasmic compartment. Abbreviations: ALS, amyotrophic lateral sclerosis; AMPA, α‑amino‑3-hydroxy‑5-methyl‑4-isoxazole propionic acid; EAAT2, excitatory amino acid transporter 2; MPTP, mitochondrial permeability transition pore; mSOD1, mutated superoxide dismutase 1; ROS, reactive oxygen species.

this protein as a crucial player in both sporadic and familial disease.89 Neurofilament-rich hyaline conglomerate inclusions are observed in the perikaryon or proximal dendrites of spinal cord motor neurons in some ALS cases, particularly those with SOD1 mutations.5 Increased expression of phosphorylated neurofilament epitopes in the cell body of motor neurons is also seen.90 Altered stoichio metry and phosphorylation of neurofilaments may have a critical effect on the functioning of the axonal compartment of the motor neuron. Small eosinophilic Bunina bodies containing cystatin C are seen in motor neurons in up to 85% of cases,6,91 although the significance of these inclusions remains uncertain. SOD1 inclusions are found in motor neurons of patients with FALS, as well as in mouse and cellular models expressing SOD1 mutations.92 Monoclonal antibodies that are specific for epitopes of misfolded SOD1 strongly label inclusions in motor neurons of patients with SOD1 FALS,93 and seem to label similar structures in some patients with SALS.94 Similarly, cytoplasmic inclusions containing mutant fused in sarcoma (FUS) protein have been observed in some patients with FUS-related FALS.95,96 Proteins found in aggregates in ALS provide several important clues about the disease pathogenesis. Loss of nuclear TDP-43 and/or aggregation of the protein in cytoplasmic inclusions may be key pathogenic processes in both SALS and FALS. The neurofilamentous

pathology suggests that neurofilament dysfunction is important in some forms of ALS. The increase in phosphorylated neurofilament epitopes in motor neuron peri karya may contribute to the observed slowing of axonal neurofilament transport.97,98 Other genetic variants provide further evidence that dysregulated protein processing contributes to the pathophysiology of ALS. Exome sequencing identified missense mutations in the valosin-containing protein (VCP) gene in five patients with FALS. 99 VCP is an evolutionarily conserved AAA+ ATPase that is important in multiple biological processes including cell signaling, protein homeostasis, organelle biogenesis and autophagy 100 through its role in identifying ubiquiti nated proteins in multimeric complexes and mediating their proteasomal degradation. Mutations in ubiquilin 2 (UBQLN2), which encodes a cytosolic ubiquitin-like protein, have recently been found to be associated with a dominantly inherited X‑linked subtype of ALS.101 The identified missense mutations lead to impairment of the protein degradation pathway in a cellular model.

Dysregulated endosomal trafficking Endocytosis is the process by which extracellular mol ecules are captured at the cell surface membrane and taken into the cell. The cargo is then delivered to its final destination via a complex organelle system termed the endosomal network. Dysregulation of this system



REVIEWS has been implicated in several genetic subtypes of ALS. Mutations in the alsin gene are associated with a form of autosomal recessive juvenile-onset ALS (ALS2).102 Alsin is a guanine nucleotide exchange factor for the small GTPase protein Rab5 and is involved in endosomal fusion and trafficking, as well as neurite outgrowth. 103 In cellular models, loss of alsin function causes a reduction in endosomal mobility, increased conversion of endosomes to lysosomes, and increased degradation of cargoes such as internalized glutamate receptors.103 Rare ALS-associated mutations further implicate dysregulated endosomal trafficking as an important pathophysiological mechanism. These mutations occur in the following genes: vesicle associated membrane protein-associated protein B (VAPB),104 optineurin,105 a component of the endosomal sorting complex required for transport (ESCRTIII), charged multivesicular pro tein 2B (CHMP2B),106,107 and VCP (described above), which forms a complex with the endocytotic protein clathrin.99,100 Heterozygous mutations in the FIG4 gene have been found in a few FALS pedig rees. 108 FIG4 encodes polyphosphoinositide phosphatase, which controls the cellular abundance of phosphatidylinositol 3,5-bisphosphate, a signaling lipid that mediates retro grade trafficking of endosomal vesicles to the Golgi apparatus.109 Whether the deleterious variants of FIG4 have a d ominant-negative effect or cause a partial loss of function remains unclear. Motor neurons may be particularly susceptible to disruptions in this network because their long axonal processes result in a high demand for turnover of membrane components.

Impaired axonal transport Axonal pathology is a key feature of ALS, and might, therefore, have a crucial role in the pathophysiology of the disease. Motor neurons are highly polarized cells with long axons, and axonal transport is required for delivery of essential components—such as RNA, proteins and o rganelles—to the axonal compartment, which includes synaptic structures at the neuromuscular junction (NMJ). The principal machinery for axonal transport uses microtubule-dependent kinesin and cytoplasmic dynein molecular motors, which mediate transport towards the NMJ (anterograde transport) and towards the cell body (retrograde transport), respectively. In mSOD1 mice, defective axonal transport occurs early in the disease process,62,110–112 supporting the hypothesis that dysregulation of axonal transport and the axonal compartment play a part in the pathophysio logy of ALS. Mutant SOD1 impairs both anterograde and retrograde transport of several cargoes. The defects seem to be cargo-specific, as only anterograde transport of mitochondria is disrupted.62,110–112 The mechanisms underlying defective axonal transport in mSOD1 models are unknown, but are likely to involve several pathways. Impaired mitochondrial function (discussed above) leads to decreased axonal mitochondrial transport,62,113 which could result in defective transport of other cargoes because of the energy requirement of axonal transport. Tumor necrosis factor, which 622 | NOVEMBER 2011 | VOLUME 7

is elevated in mSOD1 mice, 114 disrupts kinesin function via a mechanism involving p38 mitogen-activated protein kinase (p38 MAPK),115 activation of which has been demonstrated in models of ALS.116,117 Excitotoxic damage by glutamate is thought to contribute to motor neuron injury in ALS. In cultured neurons, glutamate reduces axonal transport of neurofilaments via activation of protein kinases that phosphorylate neurofilament proteins.97,118 Lastly, evidence suggests that damage to axonal transport cargoes might cause dysregulation of their binding to, or release from, molecular motors. For example, neurofilament transport is negatively regulated by phosphorylation, and p38 MAPK and cyclin-dependent kinase 5—two protein kinases that phosphorylate neurofilaments in vivo—are activated in ALS mouse models.98,117–119 Further support for the involvement of axonal transport defects and cytoskeletal dysfunction in ALS is provided by the identification of variants in the genes encoding microtubule-associated protein tau (which is associated with ALS, dementia and parkinsonism),120 neurofilament heavy chain and peripherin in ALS cases.121,122 Evidence from both patients and disease models supports the concept that ALS is a dying-back axono pathy.123,124 Axonal transport defects are likely to con tribute to the dying-back process, and in particular defects in anterograde axonal transport and mitochondrial dysf unction may combine to cause energy depletion specifically in the distal axon.62

Neuroinflammation Activated microglia and infiltrating lymphocytes indicate an inflammatory component in the CNS pathology of ALS.125 Proinflammatory mediators including monocyte chemoattractant protein 1 and IL‑8126 are present in the CSF of patients with ALS, and biochemical indices of immune-response activation are present in the blood.127 Reduced counts of CD4+CD25+ regulatory T (TREG) cells and monocytes (CD14+ cells) are detected early in ALS, suggesting recruitment of these cells to the CNS early in the neurodegenerative process. TREG cells interact with CNS microglia, attenuating neuroinflammation by stimulating secretion of anti-inflammatory cytokines.128 Consistent with this scenario, double transgenic mice carrying mSOD1 and lacking CD4 develop a more aggressive ALS phenotype, which is reversible by bone marrow transplantation.129 Neuroinflammation is closely linked to activation of the immune response. A recent study identified CD40L—a ligand expressed by T cells that activates the immune response when bound by CD40 on antigen-presenting cells—as a promising therapeutic target. Intraperitoneal injection of a CD40L-specific monoclonal antibody, MR1, in SOD1G93A mice delayed the onset of disease, extended survival, and reduced CNS astrogliosis and microglial activation.130 The reported activation of the complement system in both the mSOD1 mice131,132 and in human biosamples133 may trigger an adaptive immune response, involving antigen-presenting cells, T cells and B cells, ultimately leading to inflammation. www.nature.com/nrneurol


A M YOT R O P H I C L AT E R A L S C L E R O S I S Astrocyte activation plays a central part in inflammation, and mSOD1 astrocytes secrete inflammatory mediators, including prostaglandin E2, leukotriene B4, and nitric oxide under both basal and activated conditions.134 Moreover, co-cultures of human embryonic stem cell-derived motor neurons and either rodent or human astroc ytes expressing mSOD1 showed motor neuron toxicity that could be ameliorated by inhibiting prosta glandin D2 receptors or NOX2.135,136 Taken together, these findings suggest that mSOD1 astrocytes are intrinsically more likely to enter an activated proinflammatory state than are their wild-type counterparts.

Endoplasmic reticulum stress Intracellular inclusions related to accumulation of misfolded or unfolded proteins are a pathological hallmark of ALS. Protein misfolding elicits the ER stress response pathway. The initial unfolded-protein response (UPR)137 involves the recognition of aberrant proteins by ER‑resident chaperones that correct protein folding. In addition, non-functional proteins can cause suppression of general translation and ER‑associated protein degradation. 138 Although these mechanisms are initially cytoprotective, prolonged activation can trigger apoptotic signaling.139 Several lines of evidence implicate ER stress as an early feature of motor neuron injury in ALS. Protein disulphide isomerase (PDI), an ER‑resident chaperone and a marker of the UPR, is activated in mSOD1 mice, where it colocalizes with mSOD1 inclusions,140 and in biosamples from patients with SALS.141 PDI and other UPR-induced proteins are upregulated prior to disease onset in mSOD1 rodents, suggesting that ER stress is involved in the early stages of motor neuron injury.141 Markers of ER stress are also upregulated in the CSF and spinal cord of patients with SALS.141 In mSOD1 mice, gene expression profiles of motor neurons innervating fast fatigable fibers, which are vulner able to disease, or fast fatigue-resistant and slow fibers, which are more resistant to the disease, indicated that vulnerable motor neurons displayed differential upregu lation of several UPR markers, and this response preceded muscle denervation.142 Similar changes eventually occurred in disease-resistant motor neurons after a delay of 25–30 days.142 Nerve crush experiments showed that vulnerable motor neurons were selectively more prone than resistant motor neurons to UPR activation.142 Exposure of NSC-34 cells and primary spinal motor neurons to CSF from patients with ALS leads to clear evidence of ER stress, including expression of UPR markers, ER fragmentation and caspase‑12 activation.143 The CSF constituents that are responsible for these changes have not yet been identified. Although UPR activation is thought to be cytoprotective, at least in the initial phases of cellular stress, 139 a surprising increase in survival was observed in mSOD1 mice lacking a key UPR transcription factor, X‑boxbinding protein 1,144 accompanied by increased activation of ER‑associated protein degradation, enhanced autophagy and decreased mSOD1 aggregation. 145 The

protective role of autophagy in ALS is also supported by the evidence that CHMP2B mutations, which disrupt autophagy, are found in some ALS cases.106,107 Mutations in VAPB are a rare cause of FALS.104 VAPB is a type II integral ER membrane protein involved in multiple cellular processes, including intracellular trafficking, lipid transport and the UPR.146 Identified mutations result in conformational changes that lead to VAPB aggregation and increased ER stress.147

Dysregulated transcription and RNA processing RNA processing was first implicated in motor neuron degeneration by the identification of mutations in SMN1 as a cause of spinal muscular atrophy.148 The SMN protein functions in the assembly of small nuclear ribonucleoproteins, which are important for pre-mRNA splicing.149 Gene expression profiling demonstrated transcriptional repression in NSC-34 cells stably expressing SOD1G93A and in isolated motor neurons from SOD1G93A mice with late-stage disease.11,131 Identification of TDP-43, a ubiquitously expressed RNA–DNA binding protein, as a major component of the ubiquitinated inclusions in ALS7 focused attention on altered RNA processing as an important potential pathophysiological mechanism in this disease (Figure 3). TDP-43 is a predominantly nuclear protein that is implicated in multiple aspects of RNA processing, including transcriptional regulation, alternative splicing and microRNA (miRNA) processing. TDP-43-positive cytoplasmic inclusions are present in both neuronal and glial cells in cases of ALS, although not in mSOD1-related or FUS-related ALS.150 This difference may signify an alternative neurodegenerative cascade in these genetic subtypes of ALS. Mutations in TARDBP are responsible for 4% of FALS cases and 1.5% of SALS cases. 150 The majority of mutations reside in exon 6, which encodes the glycine-rich domain involved in protein–protein interactions and possibly also nuclear transport. Expres sion of mutant TDP-43 in cultured cells leads to a predominantly cytoplasmic localization of the protein, particularly in cytoplasmic stress granules.151 FUS is an RNA–DNA-binding protein that is involved in transcriptional regulation, RNA and miRNA processing, and mRNA transport. The FUS gene is mutated in 4% of FALS cases and less than 1% of SALS cases.150 Most mutations are located in exons 13–15, which encode the RGG-rich region and the nuclear localization signal. These mutations disrupt the transportin-mediated nuclear localization of FUS, and induce the formation of FUS-containing cytoplasmic stress granules.152,153 Whether motor neuron injury is caused by loss of normal nuclear functions of TDP-43 and FUS in RNA processing (Figure 3), or by toxic gains of function, or both is unknown. TDP-43 and FUS contain two RNA recognition domains—structures that are common to many RNA-interacting proteins, including those that are involved in mRNA transport. TDP-43 and FUS may form part of such RNA transport complexes and, when mutated, could thereby contribute to motor neuron injury through loss of axonal mRNA transport. Alternatively,



REVIEWS Transcriptional regulation ■ Mutant SOD1 is associated with transcriptional repression ■ TDP-43 self-regulates gene expression and has many other targets, including ACRV1 and APOA2 ■ FUS acts as a transcriptional activator in oncogenic fusions and regulates NF-κB and SPI-1 ■ SETX is an RNA helicase and component of ribonucleoprotein complexes ■ ANG regulates ribosomal RNA transcription

Stress granules TDP-43 and FUS are found in cytoplasmic stress granules

Alternative splicing TDP-43 is known to regulate the splicing of multiple transcripts, e.g. CFTR and SMN

DNA hnRNA

mRNA transport TDP-43 binds to and stabilizes NFL mRNA, allowing spatio-temporal translation of mRNA targets

mRNA

MicroRNA processing TDP-43 and FUS are found in complex with Drosha, implicating a role in microRNA processing

Nucleus–cytoplasm shuttling TDP-43 and FUS are found primarily in the nucleus but are also present in the cytoplasm

Figure 3 | Emerging evidence for dysregulation of RNA processing in ALS. TDP-43 is implicated in multiple aspects of RNA processing including transcriptional regulation, alternative splicing and microRNA processing. Most pathogenic mutations in its gene, TARDBP, reside in exon 6, which encodes the glycine-rich domain involved in protein–protein interactions and, potentially, nuclear transport. FUS is an RNA–DNA-binding protein involved in transcriptional regulation, RNA and microRNA processing, and mRNA transport. Most mutations are located in exons 13–15, which encode the RGG-rich region and the nuclear localization signal. These mutations disrupt the transportin-mediated nuclear localization of FUS, and induce the formation of FUS-containing cytoplasmic stress granules. TDP-43 and FUS contain two RNA recognition domains and may participate in RNA transport complexes. ANG acts as a transfer RNA-specific ribonuclease and regulates ribosomal RNA transcription. Mutations in ANG and SEXT are thought to result in loss of function of the encoded proteins in regulating transcription. In SOD1-related ALS, RNA processing is also dysregulated. Abbreviations: ACRV1, acrosomal vesicle protein 1; ALS, amyotrophic lateral sclerosis; ANG, angiogenin; APOA2, apolipoprotein A-II; CFTR, cystic fibrosis transmembrane conductance regulator; FUS, fused in sarcoma; hnRNA, heterogeneous nuclear RNA; NF-κB, nuclear factor-κB; NFL, neurofilament light polypeptide; SETX, senataxin; SMN, survival motor neuron protein; SOD1, superoxide dismutase 1; TDP-43, TAR DNA-binding protein 43.

decreased nuclear expression and function of these proteins could disrupt various aspects of RNA processing, including pre-mRNA splicing, nuclear mRNA export, mRNA sorting to distinct cytoplasmic compartments, and/or processing of noncoding RNAs. Over 4,000 RNA-binding targets of TDP-43 have recently been identified. These targets include RNA transcripts of the neurodegeneration-related genes FUS, VCP and TARDBP, and are enriched for transcripts encoding proteins involved in RNA metabolism, synaptic function and CNS develop ment.154 The recognition motif (TG)6, which occurs in both exonic and intronic sequences, is enriched in TDP-43 targets.154 Three recent studies using ultraviolet cross-linking and immunoprecipitation (CLIP) analysis applied to brain and spinal cord tissue from mouse models and humans have 624 | NOVEMBER 2011 | VOLUME 7

investigated the RNA-binding targets of TDP-43.155–157 The results have provided evidence for RNA splicing changes in CNS tissue that showed features of TDP43-related proteinopathy or that lacked TDP-43. These reports demonstrate that TDP-43 binds to a multitude of target RNA molecules—approximately 30% of the mouse transcriptome. Binding occurs most commonly to long UG‑rich sequences in introns, and less frequently to noncoding RNA molecules or to the 3' untranslated region of mRNA. The high proportion of intronic binding sites points to a nuclear function of TDP-43. Exceptionally long introns are prominent in transcripts from the brain, which might explain in part the neuronal vulnerability to loss of TDP-43 function. Blocking Tardbp43 expression with antisense oligonucleotides in adult mouse striatum altered the expression levels of 601 mRNA transcripts and changed the splicing patterns of 965 mRNA transcripts, including those corresponding to genes that are relevant to neurodegeneration, such as FUS, progranulin and choline acetyltransferase.155 A further recent study indicates that mutant TDP-43 dysregulates alternative splicing of mRNA.158 Fibroblast cell lines derived from patients with TARDBP-related ALS showed the expected loss of nuclear expression of TDP-43, together with widespread changes in RNA splicing, including changes in transcripts of other RNAprocessing genes and genes that have been previously implicated in ALS. The authors concluded that splicing dysregulation associated with loss of nuclear TDP-43 is likely to contribute to the pathophysiology of ALS. Several in vivo models of TDP-43 dysfunction, including knockout models and models overexpressing the wild-type and mutant forms of the protein, have recently been generated in mouse, rat, Drosophila melanogaster, zebrafish (Danio rerio) and Caenorhabditis elegans. A comprehensive account of these models is beyond the scope of this article, and is provided in two recent reviews.159,160 A note of caution relating to genetic studies is that although gene structures are often similar between species, intronic sequences show great variability. Given the key role of TDP-43 in binding to long intronic sequences, the intraspecies variability in introns may hinder accurate modeling of human TDP-43 proteinopathies in other species, including rodents.157 Further evidence of dysfunctional RNA metabolism in ALS emerges from the presence of mutations in angiogenin (ANG)161 and the DNA–RNA helicase senataxin (SETX) in some cases. ANG, the expression of which is increased during hypoxia to promote angiogenesis, also acts as a transfer RNA-specific ribonuclease and regulates ribosomal RNA transcription.162 A proposed mechanism by which ANG normally prevents cell death is inhibition of the translocation of apoptosis-inducing factor into the nucleus.163 Mutations in ANG are likely to have a deleterious effect through loss of function, as overexpression of ANG extends the lifespan of mSOD1 mice.162 SETX autosomal dominant mutations are associated with juvenile-onset FALS.164 The SETX protein is predicted to be a component of large ribonucleoprotein complexes, with roles in maintaining DNA repair in www.nature.com/nrneurol


A M YOT R O P H I C L AT E R A L S C L E R O S I S response to oxidative stress, and RNA processing.164 The mechanisms by which mutant SETX causes ALS remain to be determined. Additional evidence that dysregulated RNA processing may contribute to motor neuron injury in ALS arises from the detection of biomarkers of RNA oxidation in human ALS and mSOD1 mice,34 and the transcriptional repression within motor neurons that occurs in the presence of mSOD1.11,131

The role of non-neuronal cells An important recent concept is that motor neuron death in ALS is a non-cell-autonomous process, in which neighboring glial cells have a crucial role. In chimeric mice expressing mSOD1 in specific cell types, normal motor neurons developed signs of ALS pathology when surrounded by mSOD1-expressing glia.165 The proportion of non-neuronal cells that lacked the transgene correlated positively with the proportion of surviving mSOD1-expressing motor neurons and the lifespan of the chimeric mice. To determine the contribution of astrocytes and microglia to this finding, double transgenic mice were generated expressing the Cre–Lox recombination system to exclude mSOD1 from motor neurons or cells of the myeloid lineage.166 A note of caution about this experimental approach is that the time point at which Cre–Lox recombination becomes effective in these models is not known. Transgenic mice in which the mutant allele, encoding SOD1 G37R, had been deleted from motor neurons showed delayed disease onset, but no alteration in the disease course once it had been initiated. When mSOD1 was eliminated from microglia, disease onset was not altered, but disease progression was slowed by nearly 50%. Thus, the onset of the disease and its sub sequent progression and propagation are likely to represent two separate phases of disease, highlighting distinct possibilities for therapeutic intervention. Although targeted expression of mSOD1 in astrocytes fails to produce an ALS phenotype,167 selective silencing of the mutant gene in astrocytes substantially slows disease progression in mSOD1 mice.168 Rodent primary astrocytes expressing mSOD1 have toxic effects on cultured primary motor neurons and both embryonic mouse and human stem cell-derived motor neurons.135,169 This finding indicates that astrocytes expressing mSOD1 exert toxic effects or are unable to provide the required trophic support for motor neurons. Conflicting results were obtained when targeted expression of mSOD1 was limited to motor neurons in murine models.170,171 The amount of SOD1 expressed seems to be crucial in triggering motor neuron injury and disease onset. Mice lacking the SOD1G37R mutation in oligodendrocytes show a more aggressive disease course than do mice in which oligodendrocytes express mSOD1, and this phenotype is accompanied by reduced expression of the neurotrophic factor insulin-like growth factor 1.172 Expression of SOD1G93A exclusively in Schwann cells does not lead to motor neuron pathology, and increasing mSOD1 expression in Schwann cells does

not exacerbate the disease phenotype.173 Thus, although mSOD1 expression in oligodendrocytes and Schwann cells alters the properties of these cells, it does not seem to be sufficient to cause motor neuron degeneration. A better understanding of the crosstalk between glia and motor neurons should inform therapeutic efforts to ameliorate motor neuron injury in ALS. Towards this goal, a recent study examined how astrocyte properties change in the presence of mSOD1 and contribute to motor neuron degeneration.174 Reduced metabolic support from lactate release and activation of the pronerve growth factor–p75 receptor signaling pathway were identified as key components of astrocyte toxicity to motor neurons. Elimination of the astrocyte toxicity could be achieved by increasing lactate provision to motor neurons, depleting NGF levels, or blocking the p75 receptor.

Motor neuron vulnerability The selective vulnerability of particular neuronal groups to the neurodegenerative process is a key feature of ALS and other neurodegenerative disorders. The reason why motor neurons are particularly susceptible to injury in the presence of mutations that affect ubiquitously distributed proteins, such as SOD1 and TDP-43, is not completely understood. The cell-specific features of motor neurons that may predispose these cells to age-related degeneration have been reviewed elsewhere175,176 and are summarized in Box 3. Important properties are likely to include the large cell size, including long axons and large terminal arbors. This feature has implications for intracellular transport, energy metabolism and the requirement for mitochondrial and cytoskeletal support, as well as the regulation of mRNA distribution for protein synthesis. The neurons that are vulnerable to injury in ALS have particular sensitivity to glutamatergic toxicity via AMPA receptor activation. These neurons, unlike most other neuronal groups, have high expression levels of calcium-permeable AMPA receptors that lack the GluR2 subunit,69 and low levels of cytosolic calcium-buffering proteins. 70 Vulnerable motor neurons also seem to have a high threshold for mounting a protective heat shock response,176 increased sensitivity to ER stress,142 and mitochondrial features that predispose the cells to oxidative damage and calcium overload.177,178 Emerging evidence suggests that motor neurons that are more resistant to the disease process, such as fast fatigue-resistant motor neurons in the spinal cord and the oculomotor neurons in the brainstem, differ from the vulnerable spinal cord motor neurons in some of these key properties.69,70,175,176 In SOD1-related ALS in humans, the motor neurons that survive the disease process show upregulation of genes that promote cell survival—namely, those encoding the phosphatidylinositol 3-kinase and the phosphatase and tensin homolog–protein kinase B pathway.12 Increased understanding of the properties of neurons that are more resistant to the disease process in ALS is likely to uncover strategies to boost intrinsic neuroprotective defense mechanisms.



REVIEWS Box 3 | Why are motor neurons vulnerable in amyotrophic lateral sclerosis? Large cell size and long, large-volume axonal compartment ■■ High reliance on a robust cytoskeleton and axonal transport mechanisms ■■ High metabolic demands High reliance on optimal mitochondrial function ■■ Increased generation of reactive oxygen species (ROS) and a tendency towards oxidative stress ■■ Possible motor neuron-specific and/or spinal cord-specific mitochondrial properties High physiological reliance on signaling through ROS and reactive nitrogen species ■■ High intrinsic oxidative stress Vulnerability to excitotoxicity and dysregulation of intracellular calcium homeostasis ■■ High expression of calcium-permeable α‑amino‑3-hydroxy‑5-methyl‑4-isoxazole propionic acid (AMPA) receptors lacking the GluR2 subunit ■■ Low expression of calcium-buffering proteins ■■ High reliance on efficient synaptic glutamate reuptake transport mechanisms Reduced capacity for heat shock response and chaperone activity Possibly reduced proteasome function High physiological expression of particular proteins, such as superoxide dismutase 1 (SOD1) ■■ Greater vulnerability to toxicity in the presence of mutant SOD1

From molecular to clinical features In this section, we discuss four striking aspects of the clinical phenotype that are relevant to the pathophysio logical mechanisms described above. Appreciating such links between mechanistic and clinical findings could lead to improved therapeutic strategies for ALS.

Muscle cramps and fasciculations Muscle cramps, which can precede other symptoms by years, and fasciculations (visible spontaneous contractions of motor units) are prominent clinical features of ALS. They may result from increased excitatory drive to motor neurons caused by dysregulation of glutamatergic neurotransmission, reduced inhibitory interneuronal mechanisms or persistent inward Na+ conductance in motor axons.179 Age of disease onset A noteworthy clinical feature of FALS is that although the mutant protein has been present throughout life, the disease only manifests after several decades. This feature implies that the motor system can initially compensate for the cellular stress caused by the mutant protein but, with age, the homeostatic mechanisms become overwhelmed and irreversible motor neuron damage ensues. Several important changes occur in the aging CNS, including deterioration of mitochondrial function, antioxidant defense, key signaling pathways and astrocyte properties.180 An important avenue of future investigation will be to identify the effects of aging on motor neurons and glia, which may help in developing therapeutic strategies to boost intrinsic neuroprotective mechanisms that were effective for many years prior to disease onset. 626 | NOVEMBER 2011 | VOLUME 7

Spread of disease ALS often starts focally and asymmetrically in the upper limb, lower limb or bulbar territories, with progressive spread of injury to contiguous groups of motor neurons, such that the clinical features often show an anatomically ‘logical’ progression. This pattern of spread implies that motor neuron injury propagates from an initial nidus of pathology to neighboring, healthy neurons. As discussed above, glia may have an important role in the propagation of motor neuron injury. In addition, CSF from patients with ALS patients is toxic to cultured motor neurons, at least under some experimental conditions,181 and so might also play a part in the spread of disease. These mechanistic findings relating to glia and the CSF highlight potential therapeutic targets to interrupt disease propagation, which represents an important area of future research. Energy metabolism The contribution of mitochondrial dysfunction and oxidative stress to motor neuron degeneration are likely to converge and cause neuronal energy depletion, perhaps affecting the distal axon most severely. Several features of dysregulation of energy metabolism, including a hypermetabolic profile, hyperlipidemia and weight loss, are found in patients with ALS, which may reflect an attempt to compensate for failing mitochondrial energy generation within the motor system.182

Conclusions The mechanisms of motor neuron degeneration in ALS are complex and multifactorial. Mutations in at least 15 genes can cause FALS, and the genetic basis of 70% of FALS cases remains to be found. We are still in the early stages of understanding the genetic and environmental causes of SALS. Much of our current understanding of disease biology has come from studying the subtype of ALS associated with mutations in SOD1, in which a complex interplay between multiple pathophysiological mechanisms culminates in motor neuron injury (Figure 1). SOD1related FALS does not seem to cause TDP-43 proteino pathy, indicating that the pathophysiological mechanisms in this subtype of ALS are different from those in other subtypes. The relative importance of different pathways will vary according to the subgroup of the patient, and a crucial task for the future is to subclassify ALS more accurately, which will improve prognostic prediction and therapeutic targeting. Interestingly, a recent report has reopened the possibility of a viral contribution to SALS,183 with the finding that transcripts of a human endogenous retrovirus (HERV‑K) polymerase are increased in the brains of patients with ALS compared with several control groups. Further work is required to replicate these results and confirm their specificity for SALS. The identification of TDP-43 as a major constituent of the cytoplasmic inclusions that are found in most patients with ALS has been a major step towards connecting postmortem pathology with pathophysiological mechanisms. Further research is needed to determine why ubiquiti nated inclusions containing mutated TDP-43 and FUS are www.nature.com/nrneurol


A M YOT R O P H I C L AT E R A L S C L E R O S I S found in SALS and FALS patients without mutations in the corresponding genes, and how these alterations link to motor neuron injury. Therapies aimed at preventing the dying-back axonopathy and improving glial support for motor neurons are likely to be important avenues for future therapeutic endeavor. For some newly discovered genes associated with ALS, such as those encoding spatacsin184 and the σ non-opioid intracellular receptor 1,185,186 the molecular mechanisms of motor neuron injury will be unraveled as the normal functions of these proteins become better understood.

Note added in proof An intronic expansion in the gene called chromosome 9 open reading frame 72 has recently been identified as the underlying cause of 9p21-linked SALS and FALS,196,197 which are discussed in the ‘Genetic factors’ section of this article. 1.

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Review criteria Articles were selected on the basis of personal knowledge of the authors and the following PubMed searches: “(ALS OR amyotrophic lateral sclerosis)” or “(MND OR motor neuron disease)” together with one of the following search terms: “genetics”, “oxidative stress”, “mitochondria”, “excitotoxicity”, “glutamate”, “protein aggregation”, “cytoskeleton”, “axonal transport”, “inflammation”, “astrocytes”, “glia”, “microglia”, “endoplasmic reticulum”, “transcription”, “RNA”, “SOD1”, “TARDBP”, “TDP43”, “FUS”, “alsin”, “FIG4”, “senataxin”, “angiogenin”, “VAPB”, “optineurin”, “VCP”. Reference lists of the identified papers were examined for further relevant articles. The search was limited to full-text articles published in English during the past 15 years. The final selection was based on importance and relevance as judged by the contributing authors.

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