Objective: To evaluate the pathogenic mechanisms for auxilin-mediated Parkinson’s disease (PD).

Background: Auxilin participates in uncoating of clathrin-coated vesicles (CCVs), thereby facilitating synaptic vesicle (SV) re-generation in presynaptic termini. Loss-of-function mutations of auxilin (PARK19) cause early-onset parkinsonism. Here, we utilized auxilin-knockout (KO) mice to elucidate the mechanisms through which auxilin deficiency, and more broadly clathrin-uncoating deficits lead to parkinsonism.

Method: We performed behavioral and histochemical evaluations to understand if auxilin KO mice develop PD-like abnormalities. To get an unbiased insight into the mechanisms, we performed proteomic analysis of the whole-brain, synaptosomes, and CCVs in auxilin-KO and WT mice. Taking clues from proteomics, we evaluated dopamine release and reuptake kinetics invivo in the dorsal striatum using fast-scan cyclic voltammetry. Dopamine transporters (DAT) were evaluated by immunostaining and ex-vivo imaging with a dicholoropane probe. The SVs, CCVs, autophagosomes and axonal membrane in the dorsal striatum were evaluated using electron microscopy.

Results: We demonstrate that auxilin KO mice display the cardinal features of PD, including progressive motor deficits, synucleinopathy, nigral dopaminergic loss, and neuroinflammation. Through unbiased proteomic and neurochemical analysis, we determined disrupted dopamine homeostasis in auxilin KO mice. We measured dopamine reuptake kinetics in vivo and confirmed that they were decreased, potentially due to dopamine transporter (DAT) misrouting into axonal membrane deformities of the dorsal striatum. We also showed that elevated synaptic autophagy and defective SV protein sorting contribute to ineffective dopamine sequestration and homeostasis.

Conclusion: Parkinsonian pathology in auxilin deficiency begin with a disruption of clathrin uncoating leading to fewer functional SVs for neurotransmitter filling. While these deficits occur at all synapses, it is particularly detrimental to nigrostriatal dopaminergic synapses due to the toxicity of cytosolic dopamine, dopaminergic axonal deformity, and aberrant DAT trafficking mediated dysfunction in dopamine reuptake. Thus, investigating auxilin loss-of-function mutations have also helped decipher the mechanisms for dopaminergic specific vulnerability of PD. This study also enhances our understanding of how SV endocytosis deficits lead to PD.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/presynaptic-dopamine-compartmentalization-defects-initiate-auxilin-mediated-parkinsonism/