Azzouz, M. | bioRxiv Science

bioRxiv2021

C9orf72-derived poly-GA DPRs undergo endocytic uptake in iNPC-derived astrocytes and spread to motor neurons

Dipeptide repeat proteins (DPRs) are aggregation-prone polypeptides encoded by the pathogenic G4C2 repeat expansion in the C9orf72 gene, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). In this study, we focus on the role of poly-GA DPRs in disease spread. We demonstrate that recombinant poly-GA oligomers can directly convert into solid-like aggregates and form characteristic &[beta]-sheet fibrils in vitro. To dissect the process of cell-to-cell DPR transmission, we closely follow the fate of poly-GA DPRs in either their oligomeric or fibrillized form after administration in the cell culture medium. We observe that poly-GA DPRs are taken up via dynamin-dependent and - independent endocytosis, eventually converging at the lysosomal compartment and leading to axonal swellings in neurons. We then use a co-culture system to demonstrate astrocyte-to- motor neuron DPR propagation, showing that astrocytes may internalise and release aberrant peptides in disease pathogenesis. Overall, our results shed light on the mechanisms of poly- GA cellular uptake and cell-to-cell propagation, suggesting lysosomal impairment as a possible feature underlying the cellular pathogenicity of these DPR species.

neuroscience↗

bioRxiv2021

A cell-penetrant peptide blocking C9ORF72-repeat RNA nuclear export suppresses neurodegeneration

Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), a spectrum of incurable debilitating neurodegenerative diseases. Here, we report a novel ALS/FTD drug concept with in vivo and in vitro therapeutic activity in preclinical models of C9ORF72-ALS/FTD. Our data demonstrate that supplementation or oral administration of a cell-penetrant peptide, which competes with the SRSF1:NXF1 interaction, confers neuroprotection by inhibiting the nuclear export of pathological C9ORF72-repeat transcripts in various models of disease including primary neurons, patient-derived motor neurons and Drosophila. Our drug-like rationale for disrupting the nuclear export of microsatellite repeat transcripts in neurological disorders provides a promising alternative to conventional small molecule inhibitors often limited by poor blood-brain barrier penetrance.

molecular biology↗

bioRxiv2021

Safety and efficacy of C9ORF72-repeat RNA nuclear export inhibition in amyotrophic lateral sclerosis

BackgroundLoss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to progressive paralysis and death. Dysregulation of thousands of RNA molecules with roles in multiple cellular pathways hinders the identification of ALS-causing alterations over downstream changes secondary to the neurodegenerative process. How many and which of these pathological gene expression changes require therapeutic normalisation remains a fundamental question. MethodsHere, we investigated genome-wide RNA changes in C9ORF72-ALS patient-derived neurons and Drosophila, as well as upon neuroprotection taking advantage of our gene therapy approach which specifically inhibits the SRSF1-dependent nuclear export of pathological C9ORF72-repeat transcripts. This is a critical study to evaluate (i) the overall safety and efficacy of the partial depletion of SRSF1, a member of a protein family involved itself in gene expression, and (ii) a unique opportunity to identify neuroprotective RNA changes. ResultsOur study demonstrates that manipulation of 362 transcripts out of 2,257 pathological changes in C9ORF72-ALS patient-derived neurons is sufficient to confer neuroprotection upon partial depletion of SRSF1. In particular, expression of 90 disease-altered transcripts is fully reverted upon neuroprotection leading to the characterisation of a human C9ORF72-ALS disease-modifying gene expression signature. These findings were further investigated in vivo in diseased and neuroprotected Drosophila transcriptomes, highlighting a list of 21 neuroprotective changes conserved with 16 human orthologues in patient-derived neurons. We also functionally validated the high therapeutic potential of one of these disease-modifying transcripts, demonstrating that inhibition of ALS-upregulated human KCNN1-3 (Drosophila SK) voltage-gated potassium channel orthologs mitigates degeneration of human motor neurons as well as Drosophila motor deficits. ConclusionsStrikingly, manipulating the expression levels of a small proportion of RNAs is sufficient to induce a therapeutic effect, further indicating that the SRSF1-targeted gene therapy approach is safe in the above preclinical models as it does not disrupt globally gene expression. The efficacy of this intervention is also validated at genome-wide level with therapeutically-induced RNA changes involved in the vast majority of biological processes affected in C9ORF72-ALS. Finally, the identification of a characteristic signature with key RNA changes modified in both the disease state and upon neuroprotection also provides potential new therapeutic targets and biomarkers.

systems biology↗