Axonal transport defects in C9orf72 ALS/FTD: the role of dipeptide repeat proteins

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are adult-onset neurodegenerative diseases that are characterized by the degeneration of motor neurons in the spinal cord, brainstem, and motor cortex, or neurons in the frontal and anterior temporal cortex, respectively. In recent years, it has become evident that ALS and FTD belong to the same disease spectrum as these two diseases share several clinical, neuropathological, and genetic features. In 2011, a non-coding GGGGCC (G4C2) repeat expansion in the C9orf72 gene has been identified as the most common genetic cause of both ALS and FTD (C9-ALS/FTD). At present, three non-mutually exclusive pathological mechanisms have been proposed to underlie the neurotoxicity induced by the C9orf72 repeat expansion. The first is a loss-of-function mechanism due to decreased C9orf72 mRNA and protein expression observed in C9-ALS/FTD patients. The second is an RNA gain-of-function scenario caused by the accumulation of expanded repeat transcripts that sequester numerous RNA-binding proteins. The third is a protein gain-of-function mechanism mediated by the generation of dipeptide repeat (DPR) proteins via non-ATG mediated translation of the expanded repeat transcripts. This repeat-associated non-ATG (RAN) translation occurs in all reading frames of sense and antisense transcripts, giving rise to five different DPR proteins. Of those, the arginine-rich DPRs - poly-PR and poly-GR - are known to be highly toxic in numerous disease models and hamper multiple cellular processes. Axonal transport is a highly regulated process whereby motor proteins travel along microtubules to deliver cargoes and is essential for neuronal homeostasis and survival. Inefficient microtubule-based transport in axons has been linked with several adult-onset neurodegenerative disorders, including ALS. Multiple studies showed altered trafficking of cargos across different ALS disease models; however, how this process is affected by disease-causing mutations remains largely unexplored. In this thesis, we aimed to gain insights into the mechanisms that underlie axonal transport defects in the context of C9-ALS/FTD. First, we established an in vitro C9orf72 disease model using human iPSC-derived spinal motor neurons (sMNs) and investigated the effect of the G4C2 repeat expansion on the intracellular trafficking of cargos. We observed reduced transport of organelles in iPSC-derived motor neurons from C9-ALS/FTD patients, and this defect was rescued by CRISPR/Cas9-mediated correction of the G4C2 repeat expansion. Next, we investigated the potential contribution of DPRs to the axonal transport defects observed in the C9orf72-derived sMNs. We evaluated whether DPRs are sufficient to alter the transport of organelles by treating control sMNs with synthetic peptides or by transgenic expression in Drosophila fly neurons. We showed that exposure to poly-GR and poly-PR is sufficient to recapitulate intracellular transport defects in the absence of other C9orf72-disease mechanisms both in vitro and in vivo. Finally, we evaluated how DPRs impair transport. By combining multiple approaches, we found that arginine-rich DPRs interact with microtubules and motor proteins. Moreover, we made use of reconstituted in vitro transport assays and demonstrated that the arginine-rich DPRs directly impede the motility of dynein and kinesin-1 motor complexes along microtubules. We observed that arginine-rich DPRs bind to the unstructured tubulin tails of tubulin and act as "molecular roadblocks" facilitating arrest or detachment of motor proteins. Altogether the data collected in this thesis provide evidence that axonal transport is altered in is C9-ALS/FTD and provide new insights into the toxic role of arginine-rich DPRs produced by the repeat expansion. We unravel a possible mechanism by which axonal transport is hampered in C9-ALS/FTD, which implies inhibitory interactions of arginine-rich DPRs with the components of the axonal transport machinery. While additional studies are required to determine the overall contribution of axonal transport to disease pathogenesis, our findings provide mechanistic details that could stimulate the development of novel therapeutic strategies.

Publication year:2021

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