ALS Genetics: Gains, Losses, and Implications for Future Therapies

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder caused by the loss of motor neurons from the brain and spinal cord. The ALS community has made remarkable strides over three decades by identifying novel familial mutations, generating animal models, elucidating molecular mechanisms, and ultimately developing promising new therapeutic approaches. Some of these approaches reduce the expression of mutant genes and are in human clinical trials, highlighting the need to carefully consider the normal functions of these genes and potential contribution of gene loss-of-function to ALS. Here, we highlight known loss-of-function mechanisms underlying ALS, potential consequences of lowering levels of gene products, and the need to consider both gain and loss of function to develop safe and effective therapeutic strategies.

Keywords: ALS; C9ORF72; FUS; OPTN; SOD1; TARDBP; TBK1; TDP-43; gain of function; loss of function.

Figure 1:. ASOs Targeting Mutant ALS Genes

A. ASOs targeting mRNA from three ALS genes—SOD1, C9ORF72, and FUS—for degradation have been administered to human ALS patients. These ASOs are at various stages of development, as indicated. B. ASOs are delivered broadly throughout the central nervous system after injection into the cerebrospinal fluid at the base of the spinal cord (intrathecal administration).

Figure 2:. C9ORF72

A. G₄C₂ repeat expansions in a non-coding region of C9ORF72 are hypothesized to cause ALS by at least one of three main mechanisms: 1) RNA foci-mediated toxicity, 2) dipeptide repeat protein (DPR)-mediated toxicity, and/or 3) reduced levels of C9ORF72 protein. B. C9ORF72 forms a complex with SMCR8 and WDR41 and plays a role in autophagy initiation via Rab1a-dependent recruitment of the ULK1 complex to phagophore assembly sites. C. The C9ORF72/SMCR8/WDR41 complex acts as a GDP/GTP exchange factor for Rab8a and Rab39b GTPases, which are involved in autophagy. D. TBK1 interacts with the C9ORF72/SMCR8/WDR41 complex and regulates autophagy by phosphorylating SMCR8. E. C9orf72 knockout mice and Tbk1 dendritic cell knockout mice have similar phenotypes affecting the immune system.

Figure 3:. TDP-43

A. Under physiological conditions, TDP-43 predominantly localizes to the nucleus and plays a role in RNA metabolism. B. In most cases of ALS, TDP-43 is depleted from the nucleus and forms cytoplasmic aggregates. TDP-43 pathology may contribute to ALS through 1) a loss of normal function in the nucleus and/or 2) a toxic GOF via formation of cytoplasmic aggregates. C. Nuclear TDP-43 binds to STMN2 pre-mRNA transcripts and suppresses inclusion of the cryptic exon 2a. This mRNA isoform encodes functional STMN2, a microtubule-associated protein involved in neurite outgrowth and repair. D. Reduced levels of nuclear TDP-43 cause inclusion of cryptic exon 2a and premature polyadenylation of STMN2 mRNA, resulting in decreased levels of STMN2 and impaired axonal regeneration in vitro.

Figure 4:. TBK1 and OPTN

A. TBK1 phosphorylates several autophagy receptors including OPTN. Phosphorylation of OPTN by TBK1 increases its interactions with ATG8 proteins/LC3 and ubiquitinated cargo. B. TBK1 plays a role in inflammatory pathways by acting downstream of proteins that sense bacterial lipopolysaccharides and viral RNA/DNA. OPTN promotes TBK1 activation in response to viral RNA. These pathways result in expression of proinflammatory cytokines and type I interferons. C. Several proteins, including TBK1 and OPTN, have been shown to inhibit RIPK1-dependent inflammation and cell death. D. Tbk1^+/− mice with reduced myeloid Tak1 expression and mice with oligodendrocyte or myeloid-specific knockout of Optn exhibit neuroinflammatory and ALS/FTD-like phenotypes.

Figure 5:. Next-Generation Therapies: Targeting LOF and GOF Effects

A. G₄C₂ repeat expansions in C9ORF72 cause production of sense and antisense RNA foci and DPRs, as well as reduced levels of C9ORF72, all of which may contribute to disease. A combination therapy that targets G₄C₂ repeat-containing RNAs and increases levels of C9ORF72 could mitigate both the LOF and toxic GOF effects resulting from this mutation. B. TDP-43 is depleted from the nucleus and forms cytoplasmic inclusions in most ALS cases. Nuclear depletion of TDP-43 alters mRNA metabolism and results in decreased levels of STMN2, which may contribute to ALS pathogenesis. A combination therapy that targets toxic cytoplasmic TDP-43 and restores STMN2 levels through gene therapy or an ASO that blocks cryptic exon inclusion could be more beneficial to patients than either treatment alone.