Exploring Dopamine Transporter Recycling and Trafficking Defects in Parkinson's Disease

Yale University, New Haven CT

Investigators

Abstract

PROJECT SUMMARY Parkinson's Disease (PD) is characterized by the progressive loss of dopaminergic (DA) neurons within the substantia nigra (SN), resulting in debilitating motor impairments. To develop therapeutic interventions that can be administered prior to extensive DA neuron loss, early pathomechanisms at the DA synapse must be explored. Central to PD pathology is the dysregulation of dopamine neurotransmission, which is in part mediated by the dopamine transporter (DAT). Interestingly, DAT surface levels are controlled by the endolysosomal system, which includes several proteins mutated in familial and sporadic PD. For example, loss-of-function (LOF) mutations in the clathrin uncoating chaperone, auxilin, cause early-onset PD, whereas the D620N mutation in VPS35, a retromer protein, causes familial PD that is phenotypically similar to sporadic PD. My lab's study of auxilin knockout (KO) mice suggests that disruptions in DAT function and trafficking precede DA neurodegeneration, which is further supported by changes in DAT expression and function in VPS35 D620N knockin (KI) mice. Determining whether DAT dynamics are impaired across genetic etiologies of PD will be pivotal to the field's understanding of early PD pathology and DA vulnerability. This study will test the hypothesis that DAT recycling and trafficking defects occur in forms of PD involving endolysosomal mutations by using cutting-edge live and super-resolution imaging techniques and in vitro PD models. To measure DAT endo- and exocytosis with high temporal resolution in Aim 1, I developed a DAT-pHluorin that I will express in primary SN DA neurons from auxilin KO and VPS35 D620N KI mice. To account for species differences, I will also validate my results in induced pluripotent stem cell (iPSC)-derived DA neurons from PD patients with auxilin LOF and VPS35 D620N mutation. I hypothesize that auxilin PD models will display delayed DAT vesicle reacidification after endocytosis, and both auxilin and VPS35 PD models will exhibit reduced magnitudes and rates of DAT reinsertion following endocytosis, signifying impaired DAT recycling, due to distinct mechanisms. To further explore these mechanisms, Aim 2 will utilize DAT-PRIME imaging, which fluorescently labels surface DAT in live cells, to track DAT post-endocytic fate in the aforementioned models. Using stimulated emission depletion microscopy paired with immunofluorescence staining, I will assess the colocalization of DAT with various endolysosomal markers to understand the role of auxilin and VPS35 in controlling post-endocytic fate of DAT. I predict that loss of clathrin uncoating by auxilin will stall DAT trafficking at the early endosome stage, leading to reduced DAT trafficking to recycling endosomes and retromer compartments. On the other hand, VPS35 D620N neurons are likely to show enhanced lysosomal trafficking of DAT due to retromer LOF. This study will shed light on mechanisms underlying early DA dysfunction in PD and provides me with the opportunity to further my scientific training through mentorship by experts in synaptic biology, neuronal cell culture, and microscopy.