Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by dying-back degeneration of upper and lower motor neurons. The most common known cause of familial and sporadic forms of ALS as well as frontotemporal dementia (FTD) is the GGGGCC hexanucleotide repeat expansion (HRE) in C9ORF72 (C9). Our laboratory and others recently discovered that impaired nucleocytoplasmic transport (NCT) is a fundamental and early pathogenic event in C9-ALS that requires stress granule formation. However, downstream effects of impaired NCT are unclear. Recent studies have shown that autophagosome biosynthesis occurs in the distal axon followed by retrograde transport of autophagic vesicles (AVs) to the soma as they mature, providing a potential link between axon transport (AT) and autophagy, two mechanisms well known to be involved early in ALS pathophysiology. In Drosophila expressing 30 GGGGCC repeats (30R), we have found an accumulation of p62 and lysosomes, suggesting that impaired regulation of autophagy and lysosomes may be a pathogenic mechanism for C9-ALS. Further, we have found preliminary evidence of a reduction in retrograde autophagosome transport in 30R Drosophila. Consistent with this, preliminary experiments in iPS motor neurons (iPSNs) derived from patients with C9-ALS showed an accumulation of lysosomes in axons.
Specific Aim 1 will further characterize axon transport of multiple cargo in 30R Drosophila and C9 iPSNs using live cell imaging methods.
Specific Aim 2 will examine the interrelation between axon transport, autophagy and lysosomal function and determine if rescuing autophagy can rescue axon transport deficits of AVs. Finally, preliminary fly data shows that Mitf/TFEB, a transcription factor regulating autophagy and lysosomes, is mislocalized to the cytoplasm in 30R Drosophila, indicating that impaired nucleocytoplasmic transport may lead to impaired autophagy and lysosome regulation.
Specific Aim 3 will address the hypothesis that impaired nucleocytoplasmic transport is upstream of impaired axon transport defects and disruptions in autophagy. By using powerful parallel approaches in Drosophila, allowing precise genetic manipulation of AT and autophagy, and iPSNs derived from patients with C9-ALS, allowing experimental manipulation of human cells with the disease, this proposal will investigate detailed mechanistic pathways of axon transport and regulation of autophagy and lysosomes in C9-ALS. Results from these studies will not only aid our understanding of the pathogenesis and treatment strategies of ALS, but they will also further our understanding of the axonal biology of autophagy, important in all neurodegenerative diseases.
Amyotrophic lateral sclerosis (ALS) is a devastating, fatal neurodegenerative disorder with no known cure. A recently discovered mutation in C9ORF72, now recognized as the most common cause of ALS, is providing new insights into fundamental cellular pathways that are disrupted in ALS. This proposal aims to pinpoint the mechanisms underlying impaired axonal transport of autophagic vesicles and lysosomes in C9ORF72 ALS, and test therapeutic strategies for reversing these defects.
