The impact of VPS35 D620N mutation on alternative autophagy and its reversal by estrogen in Parkinson's disease

VPS35 plays a key role in neurodegenerative processes in Alzheimer's disease and Parkinson's disease (PD). Many genetic studies have shown a close relationship between autophagy and PD pathophysiology, and specifically, the PD-causing D620N mutation in VPS35 has been shown to impair autophagy. However, the molecular mechanisms underlying neuronal cell death and impaired autophagy in PD are debated. Notably, increasing evidence suggests that Rab9-dependent "alternative" autophagy, which is driven by a different molecular mechanism that driving ATG5-dependent "conventional" autophagy, also contributes to neurodegenerative process. In this study, we investigated the relationship between alternative autophagy and VPS35 D620N mutant-related PD pathogenesis. We isolated iPSCs from the blood mononuclear cell population of two PD patients carrying the VPS35 D620N mutant. In addition, we used CRISPR-Cas9 to generate SH-SY5Y cells carrying the D620N variant of VPS35. We first revealed that the number of autophagic vacuoles was significantly decreased in ATG5-knockout Mouse Embryonic Fibroblast or ATG5-knockdown patient-derived dopaminergic neurons carrying the VPS35 D620N mutant compared with that of the wild type VPS35 control cells. Furthermore, estrogen, which activates alternative autophagy pathways, increased the number of autophagic vacuoles in ATG5-knockdown VPS35 D620N mutant dopaminergic neurons. Estrogen induces Rab9 phosphorylation, mediated through Ulk1 phosphorylation, ultimately regulating alternative autophagy. Moreover, estrogen reduced the apoptosis rate of VPS35 D620N neurons, and this effect of estrogen was diminished under alternative autophagy knockdown conditions. In conclusion, alternative autophagy might be important for maintaining neuronal homeostasis and may be associated with the neuroprotective effect of estrogen in PD with VPS35 D620N.