Rachel Coneys

Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-β1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-β1 followed by COL6A1. Knockdown of TGF-β1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-β1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD.

Here we report a conserved transcriptomic signature of reduced fatty acid and lipid metabolism gene expression in a Drosophila model of C9orf72 repeat expansion, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), and in human postmortem ALS spinal cord. We performed lipidomics on C9 ALS/FTD Drosophila, induced pluripotent stem (iPS) cell neurons and postmortem FTD brain tissue. This revealed a common and specific reduction in phospholipid species containing polyunsaturated fatty acids (PUFAs). Feeding C9 ALS/FTD flies PUFAs yielded a modest increase in survival. However, increasing PUFA levels specifically in neurons of C9 ALS/FTD flies, by overexpressing fatty acid desaturase enzymes, led to a substantial extension of lifespan. Neuronal overexpression of fatty acid desaturases also suppressed stressor-induced neuronal death in iPS cell neurons of patients with both C9 and TDP-43 ALS/FTD. These data implicate neuronal fatty acid saturation in the pathogenesis of ALS/FTD and suggest that interventions to increase neuronal PUFA levels may be beneficial.

repeat expansion in C9orf72. The repeats undergo bidirectional transcription to produce sense and antisense repeat RNA species, which are translated into dipeptide repeat proteins (DPRs). As toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs, targeting both strands may provide the most effective therapeutic strategy. CRISPR-Cas13 systems mature their own guide arrays, allowing targeting of multiple RNA species from a single construct. We show CRISPR-Cas13d variant CasRx effectively reduces overexpressed C9orf72 sense and antisense repeat transcripts and DPRs in HEK cells. In C9orf72 patient-derived iPSC-neuron lines, CRISPR-CasRx reduces endogenous sense and antisense repeat RNAs and DPRs and protects against glutamate-induced excitotoxicity. AAV delivery of CRISPR-CasRx to two distinct C9orf72 repeat mouse models significantly reduced both sense and antisense repeat-containing transcripts. This highlights the potential of RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.

Abstract We report a conserved transcriptomic signature of reduced fatty acid and lipid metabolism gene expression in human post-mortem ALS spinal cord and a Drosophila model of the most common genetic cause of FTD/ALS, a repeat expansion in C9orf72 . To investigate lipid alterations, we performed lipidomics on C9FTD/ALS iPSC-neurons and post-mortem FTLD brain tissue. This revealed a common and specific reduction in phospholipid species containing polyunsaturated fatty acids (PUFAs). To determine whether this PUFA deficit contributes to neurodegeneration, we fed C9FTD/ALS flies PUFAs, which yielded a modest increase in survival. However, increasing PUFA levels specifically in neurons of the C9orf72 flies, by overexpressing fatty acid desaturase enzymes, led to a substantial extension of lifespan. Neuronal overexpression of fatty acid desaturases also suppressed stressor induced neuronal death in C9FTD/ALS patient iPSC-neurons. These data implicate neuronal fatty acid saturation in the pathogenesis of FTD/ALS and suggest that interventions to increase PUFA levels specifically within neurons will be beneficial.

Abstract Induced pluripotent stem cell (iPSC) models are powerful tools for neurodegenerative disease modelling, as they allow mechanistic studies in a human genetic environment and they can be differentiated into a range of neuronal and non-neuronal cells. However, these models come with inherent challenges due to line-to-line and clonal variability. To combat this issue, the iPSC Neurodegenerative Disease Initiative (iNDI) has generated an iPSC repository using a single clonal reference line, KOLF2.1J, into which disease-causing mutations and revertants are introduced via gene editing. Here we describe the generation and validation of lines carrying the most common causative mutation for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), a repeat expansion in the C9orf72 gene, for the iNDI collection of neurodegenerative iPSC models. We demonstrate that these C9orf72 knock-in lines differentiate efficiently into neurons and display characteristic C9orf72 -associated pathologies, including reduced C9orf72 levels and the presence of dipeptide repeat proteins (DPRs) and RNA foci, which increase in abundance over time in culture. These pathologies are not present in revertant cells lacking the repeat expansion. These repeat expansion and revertant cell lines are now available to academic and for-profit institutions through the JAX iPS cell repository and will help to facilitate and standardise iPSC-based ALS/FTD research.