Amyotrophic Lateral Sclerosis

Introduction

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig disease and motor neuron disease (MND), is a progressive neurological disease characterized by degeneration of both upper and lower motor neurons. The term ALS was first introduced by Charcot in 1874, and was based on correlations he made from clinical and post-mortem observations. Although recognizable as a distinct clinical entity, much remains to be discovered about ALS. Using modern tools of advanced cell biology and molecular genetics, the complex molecular mechanisms of ALS are gradually being unravelled.

Based on what is now known, ALS should perhaps be considered a syndrome rather than a single disease entity. The clinical hallmark of ALS is the presence of progressive degeneration of both upper and lower motor neurons. Upper motor neurons are located mainly in layer V of the motor cortex, and are known as Betz cells. Axons from upper motor neurons project to lower motor neurons located in the motor nuclei of the brainstem and in the anterior horn of the spinal cord. The lower motor neurons project to skeletal muscles.

Upper motor neuron degeneration leads to increased muscle stiffness (spasticity) and impaired fine movements. Deep tendon reflexes are increased in amplitude. Lower motor neuron degeneration leads to wasting and weakness, often with prominent muscle twitching (fasciculations) and reduced or absent deep tendon reflexes. See also Cerebral Cortex, Motor Output from the Brain and Spinal Cord, and Motor System Organization

In ALS, motor neurons innervating all the voluntary muscles are affected, with the exception of eye muscles, which are seldom involved, and the bladder, which may be affected late in the illness. Involvement of lower cranial nerves, innervating the tongue and swallowing apparatus leads to difficulty with speech and in swallowing. When the disease begins in this region, it is considered to be ‘bulbar’ in onset. Spinal onset disease refers to development of the first symptoms in the arms or legs.

Epidemiology and Clinical Features

The frequency of ALS is relatively consistent within populations of European extraction. A meta-analysis of five different population-based registers in Europe has shown that the crude annual incidence rate of ALS in the European population is 2.16 per 100 000 person-years (95% CI=2.0 to 2.3). The incidence is higher among men (3.0 per 100 000 person-years; 95% CI=2.8 to 3.3) than among women (2.4 per 100 000 person-years; 95% CI=2.2 to 2.6). Disease occurrence decreases rapidly after 80 years of age.

ALS is less common in populations of mixed ancestral origin. It is not known whether the age of onset or clinical features differ across all ancestral populations, as few detailed studies have been performed in populations of non-Caucasian origin. The male to female ratio is 1.5:1 in most studies, although in familial ALS, the ratio is closer to unity; and in a single study from a population of mixed ancestries, the ratio was reversed in those of African descent. Spinal onset ALS is more common among men compared to women, particularly in the 70–80 year age group, and bulbar onset disease is commoner in later life for both males and females.

The condition progresses across anatomic segments and is generally asymmetric at onset. Death occurs in majority of the cases within 3–5 years from the onset of the first symptoms. The majority of those with ALS die from respiratory failure. See also Epidemiological Tools

ALS and Cognitive Function

ALS is associated with cognitive impairment in a significant proportion of cases. A subgroup (18–20%) meets the criteria for frontotemporal dementia (ALS-FTD), typically a frontal variant with marked executive dysfunction and behavioural change. Progressive language deficits have also been reported. Up to 50% of all patients with ALS show evidence of milder cognitive and behavioural impairment which may progress to dementia in some cases. The presence of ALS-dementia is a negative prognostic indicator for survival. See also Frontal Lobe Disorders

Motor neuron degeneration

Macroscopic examination of the brain and spinal cord in ALS shows pallor of the corticospinal tracts, with evidence of scarring to which Charcot referred as ‘sclerosis’ (meaning hardening). Microscopic examination of the anterior horns of the spinal cord shows loss of motor neurons with evidence of glial proliferation (gliosis). A number of intracellular inclusions have been identified in motor neurons from the brain and spine (Table 1). Many of these inclusions can be stained using specific antibodies to particular proteins. In ALS, most inclusions stain form ubiquitin and TAR DNA-binding protein-43 (TDP-43) and immunostaining for the latter is also present in glia. TDP-43 inclusions are now recognized as a common characteristic of most ALS patients, with the exception of patients with familial ALS caused by mutations in the superoxide dismuatase (SOD1) gene. Interestingly, similar TDP-43 inclusions are seen in brains from patients with some forms of frontotemporal dementia, suggesting a pathologic overlap between the two conditions.

Table 1. Inclusions found in the neurons of patients with amyotrophic lateral sclerosis

Type of inclusion	Location	Staining	Significance
Spheroid	α Motor neuron	Silver	Neurofilament
Conglomerate	Pyramidal Betz cell	Silver	Neurofilament plus
Bunina body	α Motor neuron	Haematoxylin and eosin	Unclear
Eosinophilic	α Motor neuron	Haematoxylin and eosin	Neurofilament plus
Skein	Subcortical neuron	Antiubiquitin antibody	Ubiquitin positive

Modern clinical, imaging and pathological studies have indicated that neurodegeneration in ALS is not confined to motor neurons. There is also evidence of the involvement of other systems, pathways and tracts within the central nervous system (CNS) in ALS. Nonmotor pathways in the frontal and temporal lobes can be involved, leading to altered cognitive function, and there is emerging evidence of involvement of the autonomic nervous system. See also Frontal Lobe Disorders

Genetics

Although the aetiology is not well understood, most researchers agree that ALS is a complex genetic condition. Approximately 5–10% of ALS is known to be familial with a Mendelian pattern of inheritance, and a total of 12 genes and loci of major effect have been identified to date (Table 2). Mutations in SOD1 remain the most prevalent, accounting for up to 20% of all familial ALS within the United States. However, the discovery of mutations in two different DNA (deoxyribonucleic acid)-/RNA (ribonucleic acid)-binding proteins, TDP-43 and FUS (fused in sarcoma) / TLS (translated in liposarcoma), has opened exciting new avenues of research. Although the exact roles of TDP-43 and FUS/TLS require further clarification, both are known to be multifunctional proteins that have been implicated in several steps of gene expression and regulation, including transcription, RNA splicing, RNA transport and translation. FUS andTDP-43 are also thought to be involved in the processing of small regulatory RNAs (microRNAs) and in RNA maturation and splicing. Interestingly, mutations in another RNA regulatory protein, ANG (angiogenin, ribonuclease, RNase A family), also cause ALS, and mutations in progranulin, which is functionally similar to ANG, have been causally implicated in frontotemporal dementia. See also RNA Editing and Human Disorders, and RNA Processing and Human Disorders

Table 2. Known causative genes/loci in familial ALS

Name	Gene	Locus	Protein
Note: VAMP, vesicle-associated membrane protein.
ALS1	SOD1	21q22.1	Cu/Zn superoxide dismutase
ALS2	ALS2	2q33-35	Alsin
ALS3		18q21
ALS4	SETX	9q34	Senataxin
ALS5		15q15-q22
ALS6	FUS	16q15-q22	FUS
ALS7		20ptel
ALS8	VAPB	20q13.33	VAMP-associated protein
ALS9	ANG	14q11	Angiogenin
ALS10	TARDBP	1q36	TAR DNA-binding-protein-43
ALS-FTD		9q21-22
ALS-FTD		9p13.2-21,3

Mutations in TDP-43 account for 5–10% of familial ALS and mutations in FUS for up to 5% of the familial cases. Mutations in ANG account for up to 1% of cases. By investigating how RNA processing is disrupted, the discovery of mutations in TDP-43, FUS and ANG may in turn help to identify other important genes and biochemical pathways that are central to ALS pathogenesis, and by extension, may lead to the development of new generations of therapeutics.

Ninety per cent of the people with ALS have no known family history of the condition and are classified as having sporadic ALS (SALS). Twin studies in SALS have estimated the genetic contribution to be between 35% and 85%. The existence of a genetic contribution towards the neurodegenerative process in sporadic ALS is further corroborated by the higher-than-expected frequency of other neurodegenerative diseases (e.g. Parkinson disease and dementia) in the kindreds of some people with sporadic ALS. This implies an overlap between ALS and some of the commoner neurodegenerations, and suggests the existence of groups of susceptibility genes that increase the overall risk of neurodegeneration within kindreds. The evolving evidence that neurodegenerative diseases may be caused by disruption of RNA processing and regulation is consistent with this hypothesis. It may also be the case that the genes of major effect for some forms of neurodegeneration may also be acting as susceptibility genes in their wild-type (nonmutant) forms. For example, mutations in the gene progranulin can cause frontotemporal dementia, but certain nonpathogenic variants in the coding sequence of the gene are also associated with an increased risk of ALS.

It is likely that detailed clinical and genetic studies of families with a high burden of neurodegeneration will provide further insights into this overlap and will, in time, help to identify new groups of susceptibility genes. See also Genotype-Phenotype Relationships

To date, attempts to directly identify the complex genetic basis for sporadic ALS by finding important susceptibility genes have met with limited success. A large number of case controlled association studies of candidate gene studies have been performed and a number of ‘at risk’ genes identified. In many cases, replication of genetic associations has proven to be difficult. Notwithstanding, a list of ‘susceptibility’ genes can be constructed using the criteria of at least one replication in a second cohort. These genes can be segregated into functional categories, as listed in Table 3. The relative contribution of each gene to disease susceptibility is low, rarely exceeding an odds ratio of 2.0, and the mechanism by which this risk is conferred remains to be elucidated.

Table 3. Known susceptibility genes for sporadic ALS

Gene	Functional significance
Oxidative stress
SOD1	Cytoplasmic antioxidant soluble form may become neurotoxic
HFE (human hemochromatosis protein)	Regulator of iron metabolism
Cytoskeleton, microtubule, axonal transport
MAPT (microtubule-associated protein tau)	Microtubule protein disruption, involved in other neurodegenerative diseases
NEFH (neurofilament, heavy polypeptide)	Neurofilament protein, mutations alter axonal transport
PRPH (peripherin)	Intermediate filament, transgenic mice develop motor neuron degeneration
DCT1 (divalent cation transporter 1)	Disruption in dynein/dynactin complex alters axonal transport, produces phenotype in mice
Metabolism
PON 1-3 (paroxonase 1-3)	Paroxonases are important detoxifying enzymes. Association in five different populations, but different haplotypes implicated in different ancestral populations
Progranulin	Gene of major effect in FTD. Coding variations associated with ALS in some populations, similar in function to angiogenin
DNA/RNA repair
ANG (angiogenin, ribonuclease, RNase A family, 5)	RNA ribonuclease and hypoxia responsive agent; overlap in function with vascular endothelial growth factor (VEGF) and progranulin
APEX	DNA repair enzyme
SMN1 (survival of motor neuron-1), SMN2	Affects RNA splicing, gene of major effect in spinal muscular atrophy
TDP-43	RNA regulator
Unknown
9p13.2-21,3	Also links to familial ALS-FTD
Excitotoxicity
UNC13A	Associated with control of glutamate release

Genome-wide association studies (GWAS) represent another approach to identifying genetic susceptibility in a disease population. GWAS differs from a candidate gene approach, as there is no a priori hypothesis of involvement of any individual gene or locus. There are many methodological challenges to GWAS, particularly in the study of rare diseases like ALS, as power is conferred by size of the study population (cases and controls). The statistical methodology must also be stringent to compensate for multiple testing and populations must be stratified to account for variables associated with ancestral origin.

In the recent past, several GWAS have been performed on sporadic ALS. The findings have been disappointing. Although a small number of possible genes have been identified in each of the GWAS, all of the studies to date have lacked power because of their sample sizes, and the ‘hits’ have been accordingly difficult to replicate in a second population. A number of international consortia have been convened to address these problems, and further genes and pathways will undoubtedly be uncovered with increasingly effective collaborations and with the advent of the next generation of bioinformatic technologies, including whole genome sequencing. See also Genetic Epidemiology of Complex Traits, Human Population Genetics: Drift and Migration, and Linkage and Association Studies

Environmental Exposures

Environmental exposures are also thought to be of importance in ALS. Various environmental risk factors have been suggested, including a lifetime of intensive sport or physical exertion, smoking and active service in the US armed forces. Many studies have been confounded by faulty design, inadequate matching of controls and because of the relative rarity of the disease by insufficient numbers. Matching environmental exposures with genetic susceptibility has also been challenging because ALS is a rare disease. And although direct evidence is lacking in ALS, it is likely that some environmental exposures can alter genetic programming by epigenetic mechanisms, as has been described in other conditions including some cancers. See also Epigenetic Variation in Humans

Pathophysiology

Because of the availability of transgenic animal models, most efforts to understand ALS pathogenesis over the past 15 years have focused on mutations in SOD1. But despite considerable progress, no clear consensus has emerged as to how SOD1 mutations lead to premature death of motor neurons. Using crossbred transgenic animals expressing a mutant form of human SOD1 in some cell types but not in others (‘chimeric mice’), it has been established that the disease onset occurs in neurons where mutant SOD1 gains a toxic function. The rate and severity of disease progression is determined by surrounding astrocytes containing mutant protein. And once the motor neurons become injured, local activation of the innate immune response occurs within the CNS by recruitment of microglial cells, which in turn leads to further damage to motor neurons. See also Astrocytes and Brain Signalling, and Microglia

Various pathways have been implicated in the toxic gain-of-function of mutant SOD1. These include the ability of misfolded mutant proteins to alter mitochondrial function, the activation of stress pathways within the endoplasmic reticulum, the development of axonal transport defects and the excessive production of extracellular superoxide radicals. There is also some evidence that wild-type SOD1 can become misfolded and may be secreted into the extracellular space where it is injurious to neighbouring cells, leading to a neurotoxic cascade. See also Axonal Transport and the Neuronal Cytoskeleton, and Oxidative Stress

Many questions remain about the SOD1 transgenic mouse and its relevance to human ALS. Not all SOD1 mutations are toxic in laboratory animals. The A4V mutation, which is associated with a rapidly progressive disease in humans, does not produce a phenotype in mice. Neurons from mSOD1 mice do not exhibit mislocalized -terminal TDP-43 fragments or TDP-43 hyperphosphorylated cytosolic TDP-43, as is the case in human ALS. It is being increasingly recognized that the SOD1 animal model is an incomplete, albeit useful, model of human ALS. Transgenic mice incorporating mutations in TDP-43, FUS and ANG are eagerly awaited, as they may help to further elucidate the most important pathogenic cascades in ALS. See also Transgenic Mice

Management of ALS

Although a curative treatment for ALS is not yet available, appropriate management can have a major influence on patient care, quality of life and survival. Regularly updated guidelines for management of ALS are published by the European ALS Consortium. Early recognition of cognitive impairment using standardized neuropsychological testing can be helpful to patients and carers. It is also important for clinical management and research purposes. Attendance at a specialist ALS clinic can increase both quality of life and survival, as can the early introduction of supportive therapies such as noninvasive ventilation. As malnutrition is a negative prognostic indicator, nutritional supplementation is recommended. See also Respiratory Failure and Assisted Respiration

Clinical Trials and Drug Development

To date, the many advances in understanding the complexity of ALS have not translated into effective therapies. At present, Riluzole, a glutamate anatagonist, is the only pharmacological agent that alters the course of the disease, albeit with modest effect. Over the past 15 years, a plethora of promising therapies, identified in preclinical studies, have proved unsuccessful in human trials. The failure of translation from preclinical to clinical has led to an appropriate re-assessment of the validity of the SOD1 mouse as a disease model; the recognition of the importance of gender differences in mice; and of the important effects of subtle differences in genetic backgrounds among laboratory animals. Because these factors had been under-appreciated in the past, early preclinical studies may have been confounded. An increased stringency is now demanded of preclinical studies.

In humans, the failure of some promising compounds in Phases II and III trials may also have been a reflection of the paucity of preclinical data regarding the biological activity of the therapy, and/or a failure to identify the appropriate dose range for testing. In such cases, the failure has been one of trial design rather than a failure of the compound.

Going forward, effective translation from preclinical to clinical studies will require extensive knowledge about the drug activity, bioavailability and efficacy in both the preclinical and clinical settings, and proof of biological activity in the target tissue. Additionally, there is an urgent need for reliable biomarkers of disease activity for ALS.

Conclusions

The ALS syndrome is a complex and heterogeneous one. The condition can no longer be viewed as a pure motor system degeneration. Clinical, pathological and genetic overlaps with other neurodegenerative conditions, notably frontotemporal dementia have been identified. Recent advances in epidemiology, genetics and bioinformatics have begun to uncover the complex pathophysiological processes involved. Although effective disease modifying pharmacological therapies remain elusive, good clinical management can improve survival and quality of life. Clinical biomarkers of disease onset and progression are urgently required to facilitate the development of novel therapeutics based on sound biological principles.