Molecular mechanisms of axonal transport impairment in C9orf72-associated neurodegenerative disease

Understanding the etiology of neurodegenerative diseases and developing therapeutic strategies to tackle these conditions has become an increasingly important challenge for biomedical research. Neurons, with their extended axons and dendrites, rely on the integrity of the microtubule cytoskeleton and efficient axonal transport by the microtubule-based motors kinesin and dynein to maintain cell shape and function. Perturbation of the axonal transport machinery has been associated with various neurodegenerative diseases, including Alzheimer’s disease, and certain forms of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS or motor neuron disease). However, the cellular mechanisms that lead to impaired transport in these diseases are poorly understood. In this work I focus on the most common heritable form of ALS/FTD, which is caused by an expansion in the gene C9orf72. Previous experiments indicated that the arginine-rich dipeptide repeats (DPRs) produced from the C9orf72 expansion can disrupt many cellular processes, including intracellular transport. However, the mechanism by which axonal transport is dysregulated in C9orf72- ALS/FTD and whether this occurs by direct or indirect inhibition by arginine-rich DPRs remained unknown. Here, I show using in vitro assays that dynein is recruited to the surface of liquid droplets consisting of arginine-rich DPRs and RNA, and stabilises these structures. I also demonstrate that the large, extended shape of dynein might be responsible for the motor’s recruitment to the droplet surface. These data raise the possibility that sequestration of dynein complexes by arginine-rich DPR/RNA condensates, or stabilisation of these structures by dynein, contributes to C9orf72-ALS/FTD. I also use in vitro motility assays to show that the movement of purified kinesin-1 and dynein complexes along microtubules is directly inhibited by arginine-rich DPRs. I go on to demonstrate that expression of these peptides in the wing nerves of live adult Drosophila impedes axonal cargo trafficking. Accumulations of arginine-rich DPRs on the microtubule tracks lead to increased motor pausing and detachment in vitro. Arrest of transport is more frequently observed in patient-derived neurons and control neurons exposed to arginine-rich DPRs, lending support to the physiological relevance of my in vitro findings. The findings of my genetic interaction studies in Drosophila indicate that impaired microtubule- based transport contributes to the toxicity of arginine-rich DPRs. Additional experiments show that arginine-rich DPRs directly bind the C-terminal tail of tubulin and, in addition to impeding motor movement, dysregulate growth, nucleation and bundling of microtubules in vitro. Collectively, this work expands our understanding of the molecular mechanisms contributing to C9orf72-ALS/FTD. The discovery of microtubule-related processes that are perturbed by arginine-rich DPRs also paves the way for novel therapeutic strategies, including preventing the binding of arginine-rich DPRs to motors or the C-terminal tails of microtubules