Amyotrophic lateral sclerosis caused by hexanucleotide repeat expansions in C9orf72: from genetics to therapeutics

Abstract

GGGGCC repeat expansions in C9orf72 are a common genetic cause of amyotrophic lateral sclerosis in people of European ancestry; however, substantial variability in the penetrance of the mutation, age at disease onset, and clinical presentation can complicate diagnosis and prognosis. The repeat expansion is bidirectionally transcribed in the sense and antisense directions into repetitive RNAs and translated into dipeptide repeat proteins, and both accumulate in the cortex, cerebellum, and the spinal cord. Furthermore, neuropathological aggregates of phosphorylated TDP-43 are observed in motor cortex and other cortical regions, and in the spinal cord of patients at autopsy. C9orf72 repeat expansions can also cause frontotemporal dementia. The GGGGCC repeat induces a complex interplay of loss-of-function and gain-of-function pathological mechanisms. Clinical trials using antisense oligonucleotides to target the GGGGCC repeat RNA have not been successful, potentially because they only target a single gain-of-function mechanism. Novel therapeutic approaches targeting the DNA repeat expansion, multiple repeat-derived RNA species, or downstream targets of TDP-43 dysfunction are, however, on the horizon, together with the development of diagnostic and prognostic biomarkers.

Figure 1.. C9orf72 gene structure, transcripts, and isoforms and repeat expansion detection methods.

Schematic representation of the chromosome 9p21 genomic locus with the C9orf72 gene containing 11 exons and the (CCCCGG)_n repeat located between exons 1a and 1b. Coding exons are shown in blue, non-coding exons in grey. Key genetic variants defining the risk founder haplotype (rs147211831, rs3849942 and rs117204439) are shown relative to the C9orf72 genomic locus with their risk alleles shown in capital in red and the reference (non-risk) alleles shown in green. Note that the founder haplotype has been observed with a variable number of repeat units; however, the median number of repeat units was found to be 12. The number of repeat units determines the pathogenic nature of the repeat. Alternative splicing leads to the generation of three transcripts (V1-V3). The repeat is located in the first intron of V1 and V3 and in the promoter region of V2. Translation of V2 and V3 generates a long C9orf72 protein isoform of 481 amino acids. V1 is predicted to encode a short C9orf72 protein isoform of 222 amino acids but its relevance remains unclear.

Figure 2.. C9ALS/FTD-related neuropathology.

A. Normal motor neuron showing nuclear TDP-43 by immunofluorescence (antibody to TDP-43 is in green channel and DAPI is in blue channel). B. ALS motor neuron showing loss of nuclear TDP-43 and aggregation in the cytoplasm by immunofluorescence (antibody to TDP-43 is in green channel and DAPI is in blue channel). C. Sense-RNA foci in the nucleus of a motor neuron with loss of nuclear TDP and cytoplasmic aggregates by co-immunofluorescence-in situ hybridization (antibody to TDP-43 is in green channel, probes complementary to the sense RNA foci containing GGGGCC-repeats is in red channel and DAPI is in blue channel). The red signal in the nucleolus is non-specific. D. Multiple antisense-RNA foci in the nucleus of a motor neuron with loss of nuclear TDP and cytoplasmic aggregates by co-immunofluorescence-in situ hybridization (antibody to TDP-43 is in green channel, probes complementary to the antisense RNA foci containing GGCCCC-repeats is in red channel and DAPI is in blue channel). The signal in the peripheral regions of the nucleolus is unique to antisense foci. E-I. Neurons showing aggregated dipeptide repeat (DPR) polypeptides encoded by the GGGGCC repeat expansion in C9orf72. DPRs are visualized by immunohistochemistry with antibodies specific to each of the DPRs. Since RNA from the repeat is transcribed in both the sense and antisense direction, translation of the repeat results in 5 different DPRs (polyGA, polyGR, polyGP in the sense direction and polyPR, polyPA and polyGP in the antisense direction.

Figure 3.. Recent advances in C9ALS/FTD disease mechanisms.

The C9orf72 repeat expansion mutation can exert pathogenesis through gain of functions from the repeat DNA, sense and antisense repeat RNA and the translated DPRs, and loss of function of the C9orf72 protein. Recently reported key effects of the different entities and the impact of these interactions are detailed, with the links and converging pathologies highlighted, demonstrating the diversity of cellular pathways affected in disease. The Repeat DNA sequesters DAXX leading to global chromatin remodelling and transcriptional changes, including reduced C9orf72 expression. Sense and antisense repeat RNA form foci which sequester RBPs and impact RNA splicing, nuclear pore integrity, translation using phenylalanine and the integrated stress response. The dipeptide repeat proteins (DPRs) all form inclusions (cytoplasmic > nuclear/neuritic) in patient brain. The most toxic polyGR/PR disrupt LLPS/membraneless organelles (nucleolus, RNA granules, nuclear pore), bind microtubules and mitochondria; these effects results impair genomic stability, RNA splicing and transport, translation, nucleus-cytoplasm and neuritic transport, oxidative stress. PolyGA is the most aggregation prone DPR, sequestering other DPRs, nucleus-cytoplasm transport and proteosome components and chaperones, disrupting nucleus-cytoplasm transport and protein clearance. C9orf72 haploinsufficiency causes loss of the C9orf72 protein and its role in membrane trafficking, impacting autophagy, lysosomes, the cytoskeleton, nuclear-cytoplasm transport, lipid metabolism, and synapse function; through these it can casues immune cell dysfunction (interferon response) and exacerbate DPR toxicity.