A matter of life and death.

CRISPR-Cas9 Screens Identify the RNA Helicase DDX3X as a Repressor of C9ORF72 (GGGGCC)n Repeat-Associated Non-AUG Translation

The human genome contains over one million short tandem repeats. Expansion of a subset of these repeat tracts underlies over fifty human disorders, including common genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72), polyglutamine-associated ataxias and Huntington disease, myotonic dystrophy, and intellectual disability disorders such as Fragile X syndrome. In this Review, we discuss the four major mechanisms by which expansion of short tandem repeats causes disease: loss of function through transcription repression, RNA-mediated gain of function through gelation and sequestration of RNA-binding proteins, gain of function of canonically translated repeat-harbouring proteins, and repeat-associated non-AUG translation of toxic repeat peptides. Somatic repeat instability amplifies these mechanisms and influences both disease age of onset and tissue specificity of pathogenic features. We focus on the crosstalk between these disease mechanisms, and argue that they often synergize to drive pathogenesis. We also discuss the emerging native functions of repeat elements and how their dynamics might contribute to disease at a larger scale than currently appreciated. Lastly, we propose that lynchpins tying these disease mechanisms and native functions together offer promising therapeutic targets with potential shared applications across this class of human disorders. Expansion of short tandem repeats can impair RNA and protein function and cause diseases through four main mechanisms: transcription repression, RNA gelation and sequestration of RNA-binding proteins, protein gain of function, and repeat-associated non-AUG toxic translation. Synergy between these mechanisms exacerbates disease, but also offers promising therapeutic targets.

Molecular mechanisms underlying nucleotide repeat expansion disorders.

p53 is a central regulator driving neurodegeneration caused by C9orf72 poly(PR).

Stress Granules (SGs) are membraneless cytoplasmic RNA granules, which contain translationally stalled mRNAs, associated translation initiation factors and multiple RNA-binding proteins (RBPs). They are formed in response to various stresses and contribute to reprogramming of cellular metabolism to aid cell survival. Because of their cytoprotective nature, association with translation regulation and cell signaling, SGs are an essential component of the integrated stress response pathway, a complex adaptive program central to stress management. Recent advances in SG biology unambiguously demonstrate that SGs are heterogeneous in their RNA and protein content leading to the idea that various SG subtypes exist. These SG variants are formed in cell type- and stress-specific manners and differ in their composition, dynamics of assembly and disassembly, and contribution to cell viability. As aberrant SG dynamics contribute to the formation of pathological persistent SGs that are implicated in neurodegenerative diseases, the biology of different SG subtypes may be directly implicated in neurodegeneration. Here, we will discuss mechanisms of SG formation, their subtypes, and potential contribution to health and disease.

Stress granule subtypes: an emerging link to neurodegeneration

A GGGGCC hexanucleotide repeat expansion within the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense repeat-containing transcripts undergo repeat-associated non-AUG-initiated translation to produce five dipeptide proteins (DPRs). The polyGR and polyPR DPRs are extremely toxic when expressed in Drosophila neurons. To determine the mechanism that mediates this toxicity, we purified DPRs from the Drosophila brain and used mass spectrometry to identify the in vivo neuronal DPR interactome. PolyGR and polyPR interact with ribosomal proteins, and inhibit translation in both human iPSC-derived motor neurons, and adult Drosophila neurons. We next performed a screen of 81 translation-associated proteins in GGGGCC repeat-expressing Drosophila to determine whether this translational repression can be overcome and if this impacts neurodegeneration. Expression of the translation initiation factor eIF1A uniquely rescued DPR-induced toxicity in vivo, indicating that restoring translation is a potential therapeutic strategy. These data directly implicate translational repression in C9orf72 repeat-induced neurodegeneration and identify eIF1A as a novel modifier of C9orf72 repeat toxicity.

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C9orf72 arginine-rich dipeptide proteins interact with ribosomal proteins in vivo to induce a toxic translational arrest that is rescued by eIF1A.

Nucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. Unconventional translation (RAN translation) of C9orf72 repeats generates dipeptide repeat proteins that can cause neurodegeneration. We performed a genetic screen for regulators of RAN translation and identified small ribosomal protein subunit 25 (RPS25), presenting a potential therapeutic target for C9orf72-related amyotrophic lateral sclerosis and frontotemporal dementia and other neurodegenerative diseases caused by nucleotide repeat expansions.

RPS25 is required for efficient RAN translation of C9orf72 and other neurodegenerative disease-associated nucleotide repeats

G4C2 repeat expansions within the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeats undergo repeat-associated non-ATG translation to generate toxic dipeptide repeat proteins. Here, we show that insulin/IGF signalling is reduced in fly models of C9orf72 repeat expansion using RNA sequencing of adult brain. We further demonstrate that activation of insulin/IGF signalling can mitigate multiple neurodegenerative phenotypes in flies expressing either expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR. Levels of poly-GR are reduced when components of the insulin/IGF signalling pathway are genetically activated in the diseased flies, suggesting a mechanism of rescue. Modulating insulin signalling in mammalian cells also lowers poly-GR levels. Remarkably, systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Overall, our data suggest that modulation of insulin/IGF signalling could be an effective therapeutic approach against C9orf72 ALS/FTD.

Idoia Glaria

Papers

C9orf72 arginine-rich dipeptide proteins interact with ribosomal proteins in vivo to induce a toxic translational arrest that is rescued by eIF1A.

RPS25 is required for efficient RAN translation of C9orf72 and other neurodegenerative disease-associated nucleotide repeats

Enhanced insulin signalling ameliorates C9orf72 hexanucleotide repeat expansion toxicity in Drosophila.