INVESTIGATING THE ROLE OF SNAP29 AND THE AUTOPHAGY PATHWAY IN DROSOPHILA MELANOGASTER MODELS OF C9ORF72 - LINKED ALS/FTD.

It is now well established that the autophagy pathway, responsible for clearing damaged proteins and organelles, progressively declines in normal aging and in neurodegenerative diseases, leading to toxic protein aggregate accumulation. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating neurodegenerative diseases, share overlapping clinical manifestations, pathogenic mechanisms, and genetic risk factors. Notably, a hexanucleotide repeat expansion of G4C2 in the non-coding region of the C9orf72 gene is frequently observed in patients with ALS and FTD. This repeat expansion is known to contribute to disease through a dual mechanism: loss of function of the C9orf72 gene and/or toxic gain of function mediated by the repeat RNA and toxic dipeptide repeats (DPRs) generated from the G4C2 expansion itself. While G4C2 toxicity has been implicated in impairing various cellular processes, its effects on the autophagy pathway remain poorly understood. In this study, we broadly aimed to investigate the effects of G4C2 repeats on the autophagy pathway. Firstly, we observed a decline in the nuclear localization of Mitf/TFEB, a key regulator of autophagy and lysosomal biogenesis, in response to G4C2 toxicity, consistent with previous findings. Furthermore, we found that motoneuronal G4C2 toxicity led to the age-associated accumulation of ref(2)P/p62, an autophagy cargo receptor, although this accumulation did not precede the onset of locomotor deficits. Interestingly, G4C2 toxicity did not significantly disrupt autophagosome maturation or later stages of autophagy, despite its high toxicity levels in vivo. These findings suggest that G4C2 toxicity alone may not severely impair the autophagy pathway and, in turn, that disruption in degradative autophagy may not represent an important disease mechanism contributing to G4C2 toxicity in vivo. Secondly, we investigated the possible role of Snap29, a Soluble N-ethylmaleimide-sensitive factor Attachment protein Receptor (SNARE) protein involved in autophagosome-lysosome fusion, in modulating G4C2-induced toxicity. Through targeted downregulation of Snap29, we discovered that Snap29 promotes G4C2-induced toxicity in the fly eye. However, this effect was not recapitulated in motor neurons, despite the fact that motoneuronal Snap29 downregulation did suppress G4C2-induced accumulation of ref(2)P/p62 in the heads of adult flies. Thirdly, through a comprehensive series of genetic interaction studies, we explored the effects of downregulating various genes involved in autophagosome biogenesis, cargo sequestration, lysosomal function, and other cellular pathways interacting with autophagy. In sheer contrast to Snap29, downregulation of genes involved in autophagosome-lysosome fusion and lysosomal genes enhanced G4C2 toxicity, emphasizing the importance of lysosome-mediated degradation in mitigating G4C2-induced damage. Conversely, downregulation of genes involved in autophagosome biogenesis and cargo sequestration suppressed G4C2 toxicity, implicating early steps of the autophagy pathway in G4C2-mediated neurotoxicity. Intriguingly, our subsequent investigations uncovered a novel aspect of G4C2-induced toxicity: the promotion of Atg1-dependent extracellular release of ref(2)P at the larval neuromuscular junction, indicating the involvement of secretory autophagy. Secretory autophagy, an emerging branch of the autophagy pathway, remains poorly understood, particularly in the context of neurodegeneration. This intriguing discovery highlights the need for further exploration of secretory autophagy and its implications for G4C2 toxicity and neurodegenerative diseases.