<i>Drosophila</i> aux orchestrates the phosphorylation-dependent assembly of the lysosomal V-ATPase in glia and contributes to SNCA/α-synuclein degradation

Glia contribute to the neuropathology of Parkinson disease (PD), but how they react opposingly to be beneficial or detrimental under pathological conditions, like promoting or eliminating SNCA/α-syn (synuclein alpha) inclusions, remains elusive. Here we present evidence that aux (auxilin), the <i>Drosophila</i> homolog of the PD risk factor GAK (cyclin G associated kinase), regulates the lysosomal degradation of SNCA/α-syn in glia. Lack of glial GAK/aux increases the lysosome number and size, regulates lysosomal acidification and hydrolase activity, and ultimately blocks the degradation of substrates including SNCA/α-syn. Whereas SNCA/α-syn accumulates prominently in lysosomes devoid of glial aux, levels of injected SNCA/α-syn preformed fibrils are further enhanced in the absence of microglial GAK. Mechanistically, aux mediates phosphorylation at the serine 543 of Vha44, the V₁ C subunit of the vacuolar-type H⁺-translocating ATPase (V-ATPase), and regulates its assembly to control proper acidification of the lysosomal milieu. Expression of Vha44, but not the Vha44 variant lacking S543 phosphorylation, restores lysosome acidity, locomotor deficits, and DA neurodegeneration upon glial aux depletion, linking this pathway to PD. Our findings identify a phosphorylation-dependent switch controlling V-ATPase assembly for lysosomal SNCA/α-syn degradation in glia. Targeting the clearance of glial SNCA/α-syn inclusions via this lysosomal pathway could potentially be a therapeutic approach to ameliorate the disease progression in PD.<b>Abbreviation</b>: aux: auxilin; GAK: cyclin G associated kinase; LTG: LysoTracker Green; LTR: LysoTracker Red; MR: Magic Red; PD: Parkinson disease; SNCA/a-syn: synuclein alpha; V-ATPase: vacuolar-type H⁺-translocating ATPase.