C9orf72-associated G4C2 hexanucleotide repeat expression in Drosophila mushroom bodies causes age-dependent TDP-43 pathology and dementia relevant phenotypes mediated in part by the glypican Dlp/GPC6

Hexanucleotide repeat expansions (HREs) in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet the age-, sex-, repeat-length-, and circuit-specific influence on the pathology of neurons remains incompletely understood. Here, we established a Drosophila model of C9orf72 ‑associated dementia by expressing G4C2 repeats in mushroom body neurons (MBNs), a brain region critical for memory, locomotion, and sleep. Expression of 44X G4C2 repeats ((G4C2) 44X ) led to axonal thinning, accumulation of Repeat Associated Non-AUG (RAN) translated GR‑GFP dipeptide repeats (DPRs), premature nuclear‑to‑cytoplasmic mislocalization of endogenous TDP‑43, increased caspase and a loss of presynaptic active zones (AZs) during aging. Behaviorally, (G4C2) 44X expression caused defects in locomotion and spontaneous alternation behaviors and fragmentation of sleep architecture during aging in a sex‑dependent manner, recapitulating core features of FTD. Surprisingly, the shorter (G4C2) 12X repeat, traditionally considered a control, also produced detectable RAN translation and intermediate phenotypes in aging MBNs, suggesting that length- and tissue- associated factors modulate repeat toxicity. We further identified a reduction of the glypican Dally-like protein (Dlp) in (G4C2) 44X during aging, suggesting Wnt‑related signaling disruptions linked to TDP‑43 proteinopathies. Restoring Dlp expression in MBNs mitigated locomotor and spontaneous alternation behaviors, and loss of presynaptic AZs. In contrast, axonal thinning and TDP‑43 mislocalization were not significantly improved by restoring Dlp, suggesting that multiple mechanisms contribute to G4C2‑induced toxicity. Supporting our findings in Drosophila MBNs, suppression of GPC6, a human ortholog of Dlp, by CRISPRi in TDP-43 knock-down iNeurons identified GPC6, as a significant contributor to TDP-43-dependent synaptic loss. Together, our findings reveal an aging‑sensitive, circuit‑specific model of C9orf72 ‑associated neurodegenerative phenotypes and highlight potential roles for DPR accumulation and Dlp/GPC6 dependent synaptic loss in FTD pathomechanisms.