Molecular Mechanisms of Amyotrophic Lateral Sclerosis

Introduction

Amyotrophic lateral sclerosis (ALS) is recognized as the most common form of adult-onset motor neuron disease. This progressive, fatal neurodegenerative disorder occurs in approximately two persons per 100,000. Since the initial description of the symptoms and associated pathology in 1874, considerable insights into the genetic, molecular, and biochemical mechanisms of ALS have been gained. The pathological hallmark of ALS is the death of pyramidal motor neurons of the corticospinal pathway in the motor cortex and spinal column. This leads to a myriad of clinical symptoms, such as muscle weakness, muscle atrophy, and spasticity. Considerable variability in site of onset, rate of progression, and survival time occurs in ALS patients and underscores the overall heterogeneity of the disease, which can result from multiple etiologies. This suggests that ALS may represent a collection of disorders that produce similar pathological and clinical phenotypes. The purpose of this chapter is to discuss various mechanisms of ALS, understand how these mechanisms contribute to selective motor neuron vulnerability/degeneration, discuss mechanistic insights gained from ALS model systems, and develop a systematic view of how these mechanisms converge to produce the disease phenotype.

Broadly, ALS can be separated into two categories based on etiology. The vast majority of cases are classified as sporadic ALS (SALS) and are of unknown cause. Approximately 5–10% of all ALS cases are the result of inherited genetic abnormalities and are thus classified as familial ALS (FALS). Although FALS cases are a small fraction of the overall ALS population and are further stratified on the basis of the underlying genetic mutation, considerable insight into disease mechanisms have been gained by studying these rare forms of ALS. As we will emphasize throughout this review, FALS and FALS model systems have proven highly valuable for the study of both FALS and SALS. These monogenic disease-causing variants provide an approach to understand how a triggering event (in this case a genetic abnormality) can produce a cascade of molecular events that ultimately lead to motor neuron degeneration. Our approach is thus to discuss individual mechanisms associated with ALS, combining evidence from research on SALS, FALS, and ALS model systems. This overview will serve as groundwork for building a systematic framework that examines how the interplay of various mechanisms lead to ALS and opportunities these mechanisms present for therapeutic intervention.

RNA Metabolism

Altered RNA Splicing, Transport, and Translation

While most of the genetic alterations of RNA binding proteins impact their subcellular distribution and accumulation into SGs and/or pathologic inclusions (see below), we will focus here on specific effects of disease-causing mutations in TDP-43 and FUS on RNA metabolism ( Fig. 4.1 ). RNA binding proteins have diverse roles in the cell and function within many nuclear substructures and the cytoplasm. At present, the vast majority of evidence for impaired RNA processing in ALS has come from studies of TDP-43 and FUS. However, many other RNA binding proteins linked to ALS interact with TDP-43 and/or FUS, and therefore likely impact RNA metabolism. Since both TDP-43 and FUS bind RNA/DNA ( Fig. 4.1 ), determining the specific binding sequences and effects on gene expression were crucial to understanding how these proteins contribute to cell death in ALS. Using CLIP-SEQ, TDP-43 was shown to bind over 6000 RNA targets in the brain, approximately 30% of the transcriptome. TDP-43 binding to long introns (>100 kb) is required for the normal maturation and splice site selection of immature mRNA species. TDP-43 binding to the 3′-UTR of mRNAs may impact on stability or transport, whereas binding to long noncoding RNAs (ncRNAs) may influence their regulatory roles. Splicing of many RNA targets of TDP-43 is altered in ALS spinal cord tissue.

Likewise, FUS binds over 5500 RNA targets in the brain, and its binding pattern to long introns suggests that FUS remains bound to pre-mRNAs until splicing is complete. Loss of FUS function results in changes of the splicing pattern or abundance in over 1000 RNAs. All three members of the FET gene family (FUS, EWSR1, and TAF-15) are implicated in ALS, suggesting that the functions of these proteins on global RNA splicing and metabolism are particularly important for motor neurons. In addition, both TDP-43 and FUS bind to long ncRNAs (lncRNA) and influence their function and subcellular localization ( Fig. 4.1 ). Both proteins, e.g., bind NEAT1, a lncRNA core component of nuclear paraspeckles, which function in cell stress responses and in the nuclear retention of hyperedited RNAs. FUS directly regulates NEAT1 levels and decreasing FUS levels leads to reduced numbers of paraspeckles. At the same time, FUS-positive inclusions contain other paraspeckle proteins, suggesting that pathological changes in FUS levels or function impair normal paraspeckle formation/function, thereby altering cellular homeostatic responses and increasing motor neuron vulnerability to degeneration.

Finally, both TDP-43 and FUS exhibit neuron-specific functions that further implicate them in neurodegenerative diseases. Both localize to dendrites in response to neuronal activity. Disease-causing mutations that result in functional impairments and protein mis-localization therefore likely impact on synaptic structure and function via loss of localized translation of specific mRNAs. Given the remarkable functional diversity of RNA binding proteins such as TDP-43 and FUS, it is unsurprising that mutations that affect their structure and function confer numerous aberrant changes in the regulation of gene expression. Motor neurons, in particular, seem acutely vulnerable to alterations in the levels of ALS-linked RNA binding proteins by either loss of function, gain of toxic function, or both. While much insight into the sequence targets, processing functions, and subcellular associations of ALS-associated RNA binding proteins have been gained in recent years, determining which of these properties contribute directly to motor neuron vulnerability/degeneration remains an unanswered question with considerable therapeutic implications.

Adenosine Deaminase Acting on RNA 2

Further evidence for motor neuron-specific defects in RNA processing have come from studies of RNA editing of the GluA2 subunit of the l-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. Adenosine deaminase acting on RNA 2 (ADAR2)-mediated conversion of adenosine to inosine (A-to-I editing) of the GluA2 pre-mRNA results in replacement of glutamine with arginine in the translated protein. This editing normally occurs in all motor neurons and results in expression of Ca ²⁺ -impermeable AMPA receptors. ADAR2 levels, and consequently A-to-I editing, are dramatically reduced in SALS motor neurons. The resultant enhancement of AMPA Ca ²⁺ permeability leads to increased motor neuron vulnerability and the development of TDP-43 pathology.

MicroRNAs

Over the past decade, microRNAs (miRNAs) have been identified as significantly impacting overall gene expression by modulating the stability and/or translational repression of target mRNAs. miRNAs are short noncoding RNAs (~22 nucleotides) and over 1000 have been identified in humans, constituting a large class of regulators of gene expression. Approximately 60% of all mammalian mRNAs are predicted targets of miRNAs. miRNA binding to mRNA targets results in reduced protein expression from the specific bound mRNA. A single miRNA may have many mRNA transcript targets, therefore impacting on the expression of a large number of genes. Various types of cellular stress impact on the levels of miRNAs and therefore regulate how a cell responds to stress. As we further describe below, stress responses during ALS represent an important pathogenic mechanism and therefore miRNAs likely contribute to the overall ability of a cell to properly respond to acute or chronic stress conditions that exist during ALS.

Recent studies have examined miRNA changes in ALS patients and model systems, including the G93A SOD1 transgenic mouse model, circulating monocytes, skeletal muscle, and lumbar spinal cord tissue from ALS patients, and serum from FALS patients and premanifest carriers of genetic mutations. Altered levels of miRNAs may have significant impact on gene expression during ALS ( Fig. 4.2 ). Perhaps due to differences in models, cell/tissue types, and analytical methods used in these studies, a common set of miRNA alterations was not observed. However, two studies detected increased levels of miR-146a and miR-155 in microglial cells. In addition, increases in miR-146a were detected in two studies using either peripheral monocytes or spinal cord tissue from ALS patients. The results support further studies on miRNA changes in ALS using standardized approaches and model systems, ideally in large collaborative research efforts. Pathway analysis of genes modulated by the miRNAs altered in ALS will provide important information regarding mechanistic pathways regulated by miRNAs during ALS and possibly very early changes in these pathways. In addition, miRNAs may correlate with the rate of ALS disease progression, as miR-206 levels have been associated with the rate of disease progression in the G93A SOD1 mouse model. Finally, both TDP-43 and FUS (and likely other RNA binding proteins implicated in ALS) impact miRNA biogenesis, linking ALS disease-causing mutations to miRNA-regulated gene expression.

Protein Aggregation and Toxicity

The abnormal aggregation of proteins into inclusion bodies in motor neurons is a well-known pathological feature of both SALS and FALS. Early characterizations of inclusions defined their filamentous, skein-like morphology, along with their eosinophilic core (and, hence, proteinaceous composition). Subsequent work revealed a variety of characteristic inclusion types in ALS motor neurons, including larger skein-like inclusions reactive for ubiquitin, smaller filamentous inclusions containing neurofilament proteins, dense spheroids with a Lewy body-like appearance (compact inclusions), and Bunina bodies, small granular inclusions of lysosomal origin. Since the identification of inclusions as a pathological hallmark of ALS, considerable effort has been devoted to determining the protein constituents and neurotoxic mechanisms.

Superoxide Dismutase 1

The discovery that point mutations in the superoxide dismutase 1 ( SOD1 ) gene can produce familial forms of ALS (approximately 20% of all FALS cases ) led to the identification of mutant, misfolded SOD1 protein within inclusions. Subsequent work has shown that SOD1 pathology can also occur in SALS cases. SOD1 catalyzes the conversion of the superoxide anion into hydrogen peroxide and molecular oxygen via the cyclical reduction and oxidation of copper. Despite altered enzymatic activity in some FALS-linked SOD1 mutant proteins, existing evidence suggests that loss of SOD1 antioxidant function does not contribute to SOD1-linked FALS. Notably, disease progression and severity are not correlated with mutant SOD1 enzymatic activity in human patients and mice lacking the SOD1 gene develop normally and do not develop motor deficits, in contrast to mutant-SOD1-expressing mice that develop progressive motor abnormalities and SOD1 aggregates. FALS-linked mutant SOD1 aggregates into insoluble amyloid-like fibrils in vitro, in transgenic mutant-SOD1-expressing mice, and in FALS patients. The mutant SOD1 aggregation is driven by mutation-induced misfolding, which can result from the protein sequence itself, reduced capacity to bind metal ions, or both.

While human ALS patients and transgenic G93A SOD1 mice develop insoluble, ubiquitinated SOD1 aggregates in motor neurons, current models implicate soluble, misfolded SOD1 as the toxic species, similar to models proposed in other aggregating proteins, such as amyloid beta. Soluble, misfolded SOD1 can form oligomeric pore structures and exerts many deleterious effects within the cell. The protein is capable of inducing endoplasmic reticulum (ER) stress, which overwhelms the cell’s capacity to provide normal clearance of cytoplasmic proteins and can ultimately lead to apoptotic cell death. Mutant SOD1 also aberrantly accumulates in the mitochondrial intermembrane space, impairing normal mitochondrial function. Where the mutation reduces metal ion binding, mutant SOD1 also disrupts calcium homeostasis, enhancing susceptibility to cellular stress. More recent work suggests that cells also secrete misfolded SOD1, which can then seed the aggregation SOD1 (mutant or wild-type native) in adjacent cells via a prion-like process, providing a possible mechanism for spread of disease. Therefore, mutant SOD1 may induce intracellular mechanisms of cell death and propagate spread of disease via cell–cell communication pathways.