Decoding ALS: From Genes to Mechanism

Non-cell-autonomous toxicity: the neighborhood matters

Like the other major neurodegenerative diseases, all of the genes whose mutations cause ALS are expressed in multiple cell types. Indeed, it is now clear that ALS arises in part by non-cell-autonomous mechanisms. In other words, disease arises from a combination of mutant damage within motor neurons and their glial partners, rather than from damage exclusively in neurons.

For mutant SOD1, this concept is underscored by studies in mice revealing that high expression levels of mutant SOD1 in all motor neurons is not sufficient for early-onset disease¹⁸. Conversely, reduction of mutant SOD1 synthesis in motor neurons is of no benefit in slowing the rate of disease progression after onset even when applied pre-symptomatically^19–21. Thus, ALS is a disease not just of the motor neuron but the motor system, which is comprised of the motor neuron and intimately associated cells of several types.

The critical role of glia in ALS

The importance of glial cells in motor neuron degeneration and death emerged from studies in which mutant SOD1 synthesis was silenced in microglia, astrocytes or oligodendrocyte precursors. Microglia, the innate immune cells of the nervous system, become activated in all instances of ALS (Fig. 2b). Synthesis by microglia of mutant SOD1 is a key determinant of rapid disease progression, as determined by selectively silencing the mutant gene within microglia²¹ or using cell grafting to replace mutant-expressing microglia with normal ones²². Consistent with these findings, inhibiting nuclear factor kappa B (NFκB) suppresses this neuroinflammatory component of microglial toxicity to co-cultured motor neurons²³.

An additional mechanism of mutant SOD1 damage produced by microglia is counterintuitive: stimulation of excessive extracellular production of superoxide (O⁻₂)²⁴. Misfolded mutant SOD1 can associate with Rac1, a small GTPase that controls the activation of NADPH oxidase, a complex that produces superoxide (Fig. 2b). Thus, instead of its normal function of removing intracellular superoxide, mutant SOD1 may drive microglia to produce high levels of extracellular superoxide.

Disturbance in microglial function has also recently emerged as a potential contributor to the subtype of ALS associated with mutations in C9ORF72. Recognition that C9ORF72 mutation results in decreased expression levels in ALS patients⁸ has led to speculation that loss of C9ORF72 protein function may contribute to disease. The protein encoded by C9ORF72 is a probable guanine exchange factor for one or more not-yet-identified G proteins. Its inactivation in mice results in abnormal microglia and age-related neuroinflammation, providing further evidence that non-cell-autonomous, microglial-mediated inflammation may contribute to ALS^25–27.

A critical contribution to pathogenesis from mutant SOD1 is driven by oligodendrocytes, the cells that myelinate the axons of upper motor neurons and the initial axonal segments of lower motor neurons. Reduction in mutant SOD1 synthesis early in oligodendrocyte maturation produced a more striking delay in disease onset²⁸ than did similar suppression of mutant synthesis by motor neurons^18,21. Oligodendroglia also support motor neuron function by direct supply to the axon of the energy metabolite lactate through the action of the monocarboxylate transporter 1 (MCT1) (Fig. 2c). Mutant SOD1 impairs expression of MCT1 by oligodendrocytes in mouse models of ALS²⁹. Similar reduction in MCT1 accumulation is found in sporadic ALS²⁹, consistent with a non-cell-autonomous role of reduced oligodendroglial energy supply as a general component of ALS pathogenesis.

A third glial cell type, the astrocyte, provides nutrients, buffering ions, recycling of the neurotransmitter glutamate, and the final layer of the blood brain barrier. Selective reduction in mice of mutant SOD1 synthesis by astrocytes slowed disease onset³⁰ or progression²⁰. This delayed progression was accompanied by delayed microglial activation, demonstrating a functional crosstalk between mutant astrocytes and microglia.

One of the early-proposed mechanisms underlying ALS was glutamate excitotoxicity, the excessive firing of motor neurons derived from failure to rapidly remove synaptic glutamate (Fig. 2d). Astrocytes limit motor neuron firing through their rapid recovery of glutamate, a function mediated by the EAAT2 glutamate transporter (Fig. 2d). Loss of EAAT2 has been observed in both SOD1 mutant rodent models^16,31 and familial and sporadic ALS patient samples³². The resulting failure to quickly clear synaptic glutamate triggers repetitive action potential firing, a corresponding increase in calcium influx, and endoplasmic reticulum (ER) and mitochondrial stress whose calcium storage abilities become overwhelmed.

Astrocytes also protect motor neurons from excitotoxic damage via release of an unidentified soluble factor(s) that induces motor neurons to upregulate the glutamate receptor subunit GluR2³³. Incorporation of GluR2 subunits into glutamate receptors reduces their calcium permeability, providing protection from excitotoxicity by decreasing influx of calcium into neurons. Astrocytes expressing mutant SOD1 fail to regulate GluR2 expression in co-cultured neurons, thereby increasing their vulnerability to excitotoxic damage³³.

Beyond excitotoxicity, multiple teams have used in vitro co-cultures of motor neurons and astrocytes (or astrocyte-conditioned media) to show that astrocytes expressing ALS-linked mutations produce a diffusible toxicity to motor neurons^34–38 (Fig. 4d). There is, however, no consensus as to the identity of the toxic species. Even more provocatively, astrocytes from familial or sporadic ALS patients (produced from autopsy samples by direct isolation of astrocytes³⁸ or isolation of neuronal precursor cells that can subsequently be converted into astrocytic precursors and then into astrocytes³⁷) are toxic to co-cultured normal motor neurons. The latter finding³⁷ is especially provocative as it indicates that neuronal precursor cells in the affected portions of sporadic ALS patient tissue have already acquired damage and this damage is retained following multiple divisions in cell culture and subsequent differentiation. A crucial unsettled controversy remains as to whether toxicity from sporadic ALS-derived astrocytes is³⁷ or is not³⁸ mediated by changes in SOD1.

Perhaps most importantly, a non-cell-autonomous contribution of astrocytes to ALS-like disease has been demonstrated in rodents expressing mutant SOD1, in which cervical transplantation of lineage-restricted astrocyte precursors resulted in delayed progression of disease³⁹.

Multiple ALS-linked genes induce ER stress or impair protein degradation machinery

ER stress has been implicated broadly in ALS (Fig. 2a). Initial evidence arose from studies of mutant SOD1, with evidence for misfolded SOD1 binding to the cytoplasmic surface of the integral membrane ER protein derlin-1⁴⁰. This binding led to inhibition of ER-associated degradation (ERAD), the pathway for degradation of misfolded proteins extracted from the ER. Moreover, relieving ER stress delays disease progression in an animal model of disease⁴¹.

There is now overwhelming evidence that disruption of the two major protein clearance pathways, the ubiquitin-proteasome system and autophagy, can be central components of disease mechanism in ALS (Fig. 2a). Multiple ALS-causing mutations are in genes whose products are directly involved in protein degradation, including ubiquilin 2⁴² and p62 (encoded by SQSTM1)⁴³, both of which function as adapters to bring polyubiquitinated proteins to the proteasome or autophagosome for degradation. Mutations have also been reported in optineurin⁴⁴, a proposed receptor for autophagy⁴⁵, and valosin-containing protein (VCP)⁴⁶, which functions in ERAD and sorting of endosomal proteins. Additional studies have reported FTD-linked mutations in CHMP2B⁴⁷, which has been implicated in autophagosome maturation and/or endosomal cargo sorting/degradation. ALS-linked mutations are also found in VAPB⁴⁸, which functions in the unfolded protein response for delivery of ER-ejected substrates to the proteasome. A preponderance of biochemical evidence from mice expressing mutant SOD1 has demonstrated decreased activities of the proteasome pre-symptomatically in lumbar spinal cords⁴⁹ or after sustained expression of mutant SOD1 in a cultured neuronal line⁵⁰.

Axonal disorganization and disrupted transport in ALS

Axonal cytoskeletal disorganization, especially of neurofilaments, has long been a conspicuous feature of both familial and sporadic ALS (Fig. 2e). As the most asymmetric cells in nature (with some axons more than a meter long), motor neurons have a crucial requirement for axonal transport to deliver the components synthesized in the cell bodies to axons and synapses. ALS-linked SOD1 mutants have been demonstrated to slow both anterograde⁵¹ and retrograde^52,53 routes months before neurodegeneration. Indeed, reduction in retrograde transport by mutation in dynactin⁵⁴, an activator of the retrograde motor cytoplasmic dynein⁵⁵, provokes human motor neuron disease.

The peculiar architecture of neurons also presents a challenge for these cells to change local gene expression at the synapse in response to neuronal input or change in the synaptic environment. To meet this challenge, neurons transport all of the necessary components for translation (e.g., mRNA, ribosomes and translation factors) to distal sites for local protein synthesis⁵⁶. The spatial distribution of mRNAs depends upon proper microtubule-dependent transport of neuronal RNA transport granules and other factors, and is regulated by several RNA-binding proteins associated with ALS, including TDP-43, FUS and hnRNPA1. ALS-causing mutations in TDP-43 impair the axonal transport of RNA granules in Drosophila and cultured neurons, including motor neurons derived from ALS patients⁵⁷.

A prion-like spread in inherited ALS

Prion-like, templated conversion of a natively folded protein into a misfolded version of itself is now recognized as a prominent feature for cell-to-cell spread of aggregates in neurodegenerative disease. Examples include templated α-synuclein, Aβ, and tau misfolding in Parkinson’s and Alzheimer’s diseases and in chronic brain injury (reviewed in ref⁵⁸). Evidence for similar templated toxicity has emerged for misfolded SOD1^59,60, with wild type SOD1 exacerbating toxicity from mutant SOD1 in mice⁶¹. Prion-like propagation and disease development initiated focally has been demonstrated after injection of mutant SOD1-containing lysates into mutant SOD1-expressing mice⁶². This finding replicates the correlation of focal initiation and spreading in both familial and sporadic ALS patients⁶³.

To date, prion-like propagation has not been achieved in rodents without co-existence of a pre-existing, weakly active mutant ALS gene; it is unclear whether this challenges a prion-like spread model for sporadic ALS or raises the possibility that there must be a pre-existing sensitivity in these patients to facilitate such spreading. Coupled with recognition that misfolded mutant SOD1 can be secreted by motor neurons or astrocytes⁶⁴, potentially by the newly discovered pathway of unconventional secretion of misfolded proteins as an adaptation to proteasome dysfunction⁶⁵, stochastic focal initiation provides a plausible mechanism for age-dependent disease onset and subsequent spread. With most instances of ALS marked by aggregated TDP-43 rather than SOD1, an unresolved question is whether TDP-43 also exhibits templated misfolding that can spread cell-to-cell.

Phase separation of RNA-binding proteins gives rise to membrane-less organelles

RNA metabolism transpires in complex RNA-protein assemblies, which themselves may coalesce into a variety of membrane-less organelles, such as nucleoli and stress granules. Interestingly, these membrane-less organelles behave as complex liquids that arise by phase separation, a process in which protein-laden RNAs separate from the surrounding aqueous nucleoplasm or cytoplasm in a manner akin to the separation of oil and vinegar⁸⁰. It was recently discovered that phase separation is mediated by low complexity sequence domains (LCDs) present in RNA-binding proteins such as TDP-43, FUS and hnRNPA1^81–83 (Fig. 3a–b). Whereas the assembly of membrane-less organelles is biologically advantageous (e.g., stress granule formation), phase transition also presents a risk, since RNA-binding proteins within the liquid phase are highly concentrated, with fibrillization-prone LCDs placed in close proximity. Indeed, ALS-causing mutations are frequently found in the LCDs of TDP-43⁸⁴, FUS⁸⁵ and hnRNPA1⁷². As a consequence, these mutations alter the dynamics of membrane-less organelles and also accelerate fibrillization, resulting in the formation of pathological amyloid-like fibrils that deposit in the cell bodies and neuropil^81–83,86 (Fig. 3c).

Mutations in the LCDs of at least six different hnRNPs result in a clinicopathological spectrum that ranges from ALS/FTD to IBM (Fig. 3a and Box 1). It should be noted that some disease-causing mutations in RNA-binding proteins do not impact the LCD. For example, some ALS-causing mutations in FUS and hnRNPA1 disturb the nuclear localization sequence (NLS) and result in accumulation of these proteins in the cytoplasm^70,71,73. Phase transition by LCD-containing RNA-binding proteins is exquisitely concentration dependent⁸², and it is likely that increased accumulation of FUS and hnRNPA1 in the cytoplasm as a consequence of mutations impacting their respective NLSs is sufficient to drive excess phase separation, as evidenced by hyperassembly of stress granules in cells derived from patients with the respective mutations^72,87.

RNA metabolism defects in ALS

There are multiple potential consequences of disturbed phase transitions by disease-associated RNA-binding proteins, including (1) altered material properties of RNA granules, which can impair the biological processes served by these structures; (2) the generation of oligomers or amyloid-like fibrils that have toxic properties; and (3) a partial or complete loss of the normal function of key RNA-binding proteins⁸⁸. With respect to this last point, a long-recognized feature of ALS histopathology is redistribution of TDP-43 from the nucleus to the cytoplasm⁷. Similar redistribution is observed for FUS and hnRNPA1 when disease mutations occur in these genes^70,71,73. This redistribution may reflect a cytoplasmic sink produced by poorly dynamic cytoplasmic RNA granules, the deposition of amyloid-like fibrils, defects in nucleocytoplasmic trafficking, and potentially other mechanisms.

The culmination of this redistribution is depletion of RNA-binding proteins from the nucleus that has the potential to cause a relative loss of nuclear function. A long-recognized nuclear function of TDP-43 is regulation of alternative splicing⁸⁹. Experimental depletion of TDP-43 in rodents was found to alter hundreds of splicing events in the brain, resulting in depletion of several RNAs encoding synaptic proteins⁷⁵. Additionally, loss of nuclear TDP-43 generates usage of cryptic splice sites⁹⁰ that, in general, may lower levels of correctly spliced protein-encoding mRNAs. Furthermore, the fact that TDP-43 autoregulates its synthesis⁷⁵ establishes the possibility of a feed-forward mechanism amplifying the impact of partial loss of TDP-43 function.

Loss of FUS or hnRNPA1 from the adult nervous system produces defects analogous to that of loss of TDP-43, although different subsets of mRNAs are associated with depletion of each of these RNA-binding proteins^76,77. An important unanswered question is to what extent does ALS caused by other genetic perturbations, especially C9ORF72-related ALS (see below), also involve disturbances in RNA biology that intersect mechanistically with mutations in TDP-43, FUS and hnRNPA1.

Regulatory RNA biogenesis and function in ALS

Both TDP-43 and FUS are components of macromolecular complexes that generate small non-coding RNAs (microRNAs) that function in RNA silencing. Loss of TDP-43 or FUS results in reduced expression of microRNAs in model systems, including Drosophila models and iPS-derived motor neurons from patients with TDP-43 mutations, suggesting a possible role for altered RNA silencing in ALS⁹¹. microRNAs contribute to the maintenance of neuromuscular junctions, suggesting that motor neurons may be especially sensitive to disturbances in microRNA biogenesis^92,93. Indeed, global downregulation of microRNAs has been reported in motor neurons from patients with sporadic ALS⁹⁴, although the role of reduced microRNAs in pathogenesis remains to be established⁹⁴. Nonetheless, the expression of regulatory RNAs appears to be robustly and consistently altered in the serum of ALS patients, and this may present an opportunity for biomarker development⁹⁵.

A contribution to disease from reduced C9ORF72 function?

Three non-exclusive mechanisms by which expanded GGGGCC repeats may cause C9-ALS/FTD have been proposed (Fig. 4). Decreased C9ORF72 expression levels in C9-ALS/FTD patients^104,105 has led to speculation that loss of C9ORF72 protein may contribute to disease. The function of the C9ORF72 protein is poorly understood, but it contains a conserved DENN domain and can function as a guanine nucleotide exchange factor for several Rab proteins in experimental systems¹⁰⁶. In cultured cells and in zebrafish, depletion of endogenous C9orf72 may exacerbate the toxicity of aggregation-prone proteins such as polyglutamine-expanded ataxin-2¹⁰⁷. However, knockdown of endogenous C9orf72 mRNA with antisense oligonucleotides was well tolerated in mice and did not result in behavioral or motor impairments¹⁰⁸. Furthermore, conditional knockout of C9orf72 in mouse brain did not cause appreciable motor neuron or other degenerative phenotypes, with no evidence of hallmark ALS/FTD pathologies¹⁰⁹. Mice with complete C9orf72 knockout in all tissues also developed and aged normally without motor neuron disease, but, interestingly, developed progressive splenomegaly and lymphadenopathy^25–27,110. These mice were found to have abnormal macrophages and microglia, as well as age-related neuroinflammation^25,110 and signs of autoimmunity²⁷, raising the possibility of a non-cell-autonomous inflammatory contribution to C9-ALS/FTD.

Nevertheless, the dominant inheritance pattern of C9-ALS/FTD, the absence of ALS/FTD patients with null alleles or missense mutations in C9ORF72, and the absence of neurodegeneration in C9orf72 knockout mice argue against loss of function of C9ORF72 as the sole driver of disease. Indeed, most empirical evidence points to gain-of-toxic-function as the primary mechanism driving neurodegeneration in C9-ALS/FTD. For example, AAV-mediated delivery to the brain of a construct expressing expanded GGGGCC repeats elicits neurodegeneration¹¹¹, although the nature of the toxic species in C9-ALS/FTD remains unclear. 

Toxic gain of function from accumulation of repeat-containing RNA

The initial description of the C9ORF72 mutation included evidence that RNA foci containing the GGGGCC repeat accumulate in the brain and spinal cord of C9-ALS/FTD patients⁸ (Fig. 1b). Soon thereafter it was appreciated that C9ORF72 is bidirectionally transcribed, and that both sense- (GGGGCC) and antisense- (CCCCGG) containing foci accumulate in affected cells^112–114. The accrual of pathological RNA foci in C9-ALS/FTD is reminiscent of pathological RNA foci observed in myotonic dystrophy types 1 and 2 and fragile X tremor ataxia syndrome, which are also caused by pathological repeat expansions in non-coding regions¹⁰². In these diseases, the accumulated repeat-containing RNA sequesters RNA-binding proteins involved in splicing, leading to defects in RNA splicing that underlie some aspects of pathogenesis¹⁰². Similarly, a number of RNA-binding proteins bind to expanded GGGGCC and GGCCCC repeats in vitro, and rare co-localization with RNA foci has been observed for several of these proteins in patient tissue^115–119.

Simple model systems have illustrated functional consequences of sequestration of some hexanucleotide repeat-binding proteins, for example Purα and RANGAP, but the contribution of these interactions to disease remains uncertain^118,120. Notably, GGGGCC (but not CCCCGG) repeats can adopt a stable secondary structure known as a G-quadruplex, which may contribute to the persistence of this RNA species as well as its ability to reach distal neurites and associate with RNA-binding proteins in transport granules and potentially interfere with local translation^121–123.

Toxic gain of function from accumulation of dipeptide repeats

Substantial evidence has now accrued implicating a second mode of gain-of-toxic-function in C9-ALS/FTD; specifically, toxicity from DPR proteins produced by repeat-associated non-AUG (RAN) translation. This type of unconventional translation occurs in the absence of an initiating AUG codon¹²⁴ and may rely upon secondary structure formed by repeat-expanded RNA^124,125. In C9-ALS/FTD, this unconventional translation occurs in all reading frames from both sense and antisense transcripts, and results in the production of five DPR proteins: glycine-alanine (GA) and glycine-arginine (GR) from sense GGGGCC transcripts, proline-arginine (PR) and proline-alanine (PA) from antisense GGCCCC transcripts, and glycine-proline (GP) from both sense and antisense transcripts^{112–114,126}. It is now appreciated that all of these DPRs are produced in C9-ALS/FTD patients and account for the ubiquitin- and p62-positive, but TDP-43-negative neuronal cytoplasmic and intranuclear inclusions that are found widely in the brain and spinal cord^{112,114,117,126,127} (Fig. 1b).

Issues of when, where, and how much expression of each DPR species occurs in patient brain remain to be clarified. Multiple reports have described DPR deposition in C9-ALS/FTD patient brain, and in some instances an inverse relationship between the burden of DPRs and neurodegeneration has been described^126,128,129. These studies have been based on examination of post-mortem brain at end-stage disease and have relied upon detection of large inclusions by immunohistochemistry, an approach that is likely to under-represent the pathological burden of soluble DPRs. Nevertheless, the apparent discrepancy between the burden of DPR deposition (greatest in the cerebellum) and the severity of neurodegeneration (greatest in the motor cortex and spinal cord) need to be resolved in order to understand the role of DPRs in disease.

Experiments conducted in model systems have provided evidence that at least some DPR species are toxic in cultured cells and animal models of disease, although the DPR expression levels used to produce short-term toxicity have sometimes been quite high. The arginine-containing peptides (GR and PR) in particular appear to be most toxic. For example, the addition of GR or PR peptides to cultured cells results in their uptake and accumulation in nucleoli, defects in RNA processing, and subsequent cell death¹³⁰. Similarly, independent expression of each of the five DPR species in cultured neurons revealed GR and PR to be very toxic, whereas similar levels of PA, GA and PG were well tolerated. Consistent with these observations, Drosophila engineered to express each of the five DPR species have consistently shown that GR and PR are very toxic in neuronal tissue, whereas GA is modestly toxic and GP and PA appear to be non-toxic^131–133. That said, other investigations have reported toxicity associated with GA expression in cell culture^134–136 and in AAV-mediated delivery to mouse brain¹³⁶, albeit in the latter instance the GA-mediated toxicity accompanied its accumulation to just under 2% of total brain protein. Additionally, the efforts reported thus far to model DPR toxicity have used short repeat (<100) lengths. Unsettled too is how the properties of those short DPRs compare with the RAN translation products in patients, which may be much larger.

A defect in nucleocytoplasmic trafficking

Whereas the nature of the toxic gain of function remains an open question, there is converging evidence that one downstream consequence of the C9ORF72 mutation is impaired nucleocytoplasmic trafficking. A comprehensive, unbiased screen for genetic modifiers of GGGGCC repeat-expanded toxicity in Drosophila identified 18 genes centered on the nuclear pore complex and nucleocytoplasmic trafficking¹³³. A separate unbiased screen for genetic modifiers of PR toxicity in yeast also identified numerous genes encoding components of the nuclear pore complex and effectors of nucleocytoplasmic trafficking¹³⁷. A third study focused on the nucleocytoplasmic transport factor RANGAP, which binds GGGGCC RNA. In these experiments, genes encoding RANGAP and other nucleocytoplasmic transport factors were identified as modifiers of GGGGCC repeat-expanded toxicity in Drosophila¹²⁰. Consistent with these results, morphological abnormalities were found in nuclear envelope architecture in cell and animal models of disease as well as in the brain of C9-ALS/FTD patients. Moreover, defects in nucleocytoplasmic transport of RNA and proteins were found in induced pluripotent stem cell (iPSC)-derived neurons from C9-ALS/FTD patients^120,133,137.

Approaches to therapy for C9-ALS/FTD

The relative contribution of the various proposed modes of toxicity to the eventual development of C9-ALS/FTD remains an important unanswered question that will influence strategies for therapeutic intervention. For example, efforts are underway to impede RAN translation with small molecules, but the success of such an approach will depend on the role of DPRs in disease. Irrespective of the primary basis for toxic gain of function, however, the mutant C9ORF72 gene itself presents an attractive target for therapeutic intervention. For example, antisense oligonucleotides are able to reverse pathological features present in iPSC-derived neurons^108,138,139 or fibroblasts¹⁰⁸ from C9-ALS/FTD patients. Indeed, iPSC-derived neuronal and glial cells may prove to be a useful model system in which to develop approaches to mitigate mutant C9ORF72-related toxicity even before the basis of toxicity is fully elucidated.

Therapeutic efforts will also be aided by the recent development of transgenic mouse models expressing human C9ORF72 with ~450 hexanucleotide repeats that recapitulate aspects of the molecular pathology^26,140–142, neuropsychological deficits^142,26 and the motor phenotype¹⁴² of C9-ALS/FTD. Particularly promising is the finding that pathological abnormalities can be reversed, and development of neuropsychological deficits delayed, by single-dose infusion of an antisense oligonucleotide that induces catalytic degradation of hexanucleotide-containing RNAs, without exacerbating reduction in RNAs encoding the C9ORF72 protein²⁶.