Old versus New Mechanisms in the Pathogenesis of ALS

Abstract

Amyotrophic Lateral Sclerosis (ALS) is recognized as a very complex disease. As we have learned in the past 20 years from studies in patients and in models based on the expression of mutant SOD1, ALS is not a purely motor neuron disease as previously thought. While undoubtedly motor neurons are lost in patients, a number of alterations in those cell-types that interact functionally with motor neurons (astrocytes, microglia, muscle fibers, oligodendrocytes) take place even long before onset of symptoms. At the same time, disturbance of several, only partly inter-related physiological functions play some role in the onset and progression of the disease. Traditionally, mitochondrial damage and oxidative stress, excitotoxicity, neuroinflammation, altered axonal transport, ER stress, protein aggregation and defective removal of toxic proteins have been considered as key factors in the pathogenesis of ALS, with the relatively recent addition of disturbances in RNA metabolism. This complexity makes the search for an effective treatment extremely difficult and prompts further studies to reveal other possible, previously unappreciated aspects of the pathogenesis of ALS. In this review, we focus on previous knowledge on ALS mechanisms as well as new facets emerging from studies on genetic ALS patients and models that may both provide precious information for a novel therapeutic approach.

Keywords: ALS; RNA metabolism; amyotrophic lateral sclerosis; motor neuron; protein aggregation.

Figure 1

Converging pathogenic mechanisms triggered by RNA‐related mutant genes. A. FUS and TDP‐43 have a central role in different steps of RNA related pathway. While they are mainly localized in the nucleus, where they act as transcriptional and splicing regulators, both FUS and TDP‐43 are also involved in cytoplasmic mRNA transport and local translation. ALS mutations alter their normal cellular distribution, relocating both proteins in cytoplasmic protein aggregates and/or stress granules. This causes both a loss of their normal, and mainly nuclear functions, and a gain of toxic function by protein aggregates in the cytoplasm. In particular, mutated FUS and TDP‐43 affect the proper splicing regulation of thousands of RNA targets. This might also be a result of sequestration of snRNPs and SMN splicing complexes in the cytosol, which in turn leads to their depletion from nuclear gems. Further, mutated FUS and TDP‐43 alter stress granule dynamics, thus impairing stress granules‐mediated translational repression in condition of stress. Local translation into distal neuronal processes and neuromuscular junctions (MNJ) might also be affected, as TDP‐43 plays a role in the axonal transport of defined target mRNAs for local translation. Finally, ALS‐associated mutation in FUS and TDP‐43 affect nuclear protein import, as in the case of C9orf72. B. The transcription of mutant C9orf72 gene lead to the expression of both sense and antisense C9orf72 RNA transcripts containing expanded G4C2 (and C4G2) repeat, which can affect normal cellular pathway in numerous manners. First, the C9orf72 RNA transcripts accumulate in the nucleus as RNA foci which interact and sequester different RNA binding proteins, leading to alterations in their normal cellular functions. Interaction between G4C2 repeat expansion and nucleolin causes nucleolar stress and defects in rRNA biogenesis. Similarly, the hexanucleotide repeat binds several splicing factors, such as hnRNP H, hnRNP A3, SRSF2, thus impairing splicing regulation of hundreds of genes. Second, expanded repeats impact on the control of nucleo‐cytoplasmic trafficking of both mRNAs and proteins by targeting mRNA export adaptors and the nuclear pore complex machinery. As a consequence, mRNA translation is affected. Protein translation might also be impaired by sequestration of translational regulators, such as initiation and elongation factors, which in turn might lead to impairment in stress granules‐mediated translation repression. Finally, bypassing normal surveillance mechanisms in an obscure way, expanded RNAs translocate from nucleus to cytoplasm, where they are translated through an unconventional process independent from the presence of an upstream ATG (RAN translation). The ribosomal reading of the sense and antisense transcripts lead to the expression of five different poly‐dipeptide repeat proteins (DPRs). DPRs accumulate in cytosolic inclusions that affect cell viability by unknown mechanism, but they also target nuclear RNA processing and protein transport leading to a detrimental loop of RNA dysfunction.

Figure 2

Protein aggregation‐related toxicity in ALS. SOD1, TDP‐43 and FUS may share toxicity mediated by protein aggregates that form both as a consequence of gene mutations and/or as a result of oxidative or ER stress. In the case of FUS and TDP‐43, this process can be enhanced by their localization into stress granules. Correct protein handling by the Ubiquitin Proteasome System (UPS), as well as proper aggregate removal by the autophagic machinery can be hampered by the same aggregated proteins, but also by dysfunction/mutations in proteins that regulate these processes. As a result, aggregates accumulate over time and can propagate into neighboring cells following their extracellular release, which is due to discharge of seeds of aggregates by dying cells, or mediated by exosomes. Inclusions of Dipeptide Repeat Proteins (DPRs) that are formed by an aberrant translation of the C9orf72 expanded repeat, are thought to share the same toxic mechanisms as the other aggregation‐prone ALS factors.