LAUSR

Parkinson’s disease (PD), the most common neurodegenerative movement disorder, affects about 2% of the population over the age of 65. That percentage increases to about 5% in people over the age of 85 (Lang & Lozano, 1998a,b). PD is clinically characterized by its motor symptoms including resting tremor, rigidity, bradykinesia, and postural instability. Neuropathologically, PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta resulting in reduced levels of dopamine in the striatum (Lang & Lozano, 1998a). Dopaminergic cell loss is also accompanied by the accumulation of intracellular inclusions termed Lewy bodies, composed of aggregated protein, of which a-synuclein is a major component (Lang & Lozano, 1998a; Spillantini et al., 1997). Currently, no disease-modifying treatments for PD exist, and symptomatic treatments do not offer long-term disease control. The lack of disease-modifying treatments can be attributed to the fact that themechanism(s) by which dopaminergic neurons die remain unclear. Although PD is largely an idiopathic disease, 5–10% of cases are hereditary. To date, mutations in at least 13 genes have been shown to cause familial PD, including autosomal dominant mutations in the vacuolar protein sorting 35 ortholog (VPS35, PARK17) gene (Hernandez, Reed, & Singleton, 2016; Vilari~ no-G€ uell et al., 2011; Zimprich et al., 2011). A single missense mutation in VPS35 (D620N) was originally reported in 2011 in Swiss and Austrian families by two independent groups. Since its initial discovery, the D620N mutation has been shown to be present in PD subjects worldwide (Vilari~ no-G€ uell et al., 2011; Zimprich et al., 2011). Although the D620N mutation has been shown to clearly segregate with disease, several other rare putative pathogenic mutations in VPS35 have been identified (i.e. P316S, R524W, L774M, etc.) but their disease association remains unclear. Clinically, VPS35-associated PD presents like that of idiopathic PD being progressive, tremor-predominant and responsive to levodopa treatment. The neuropathological characteristics of VPS35-associated PD have yet to be established (Williams, Chen, & Moore, 2017). The VPS35 protein functions as a subunit of the retromer complex, a heteropentameric complex composed of VPS35, VPS26, VPS29, and a SNX-BAR dimer, to facilitate endosome-to-Golgi complex and endosome-to-plasma membrane transport of transmembrane protein cargo (Williams et al., 2017). Interestingly, the D620N mutation in VPS35 does not disrupt its interaction with other components of the retromer or disrupt the transport of canonical protein cargo. Studies from our laboratory have demonstrated that overexpression of D620N VPS35 induces toxicity in primary rodent cortical neurons and in vivo using viral-mediated gene transfer (Tsika et al., 2014). Additionally, overexpression of the D620N mutant has been shown to induce neurodegeneration in a Drosophila model (H. S. Wang et al., 2014). However, the molecular mechanism through which the D620N mutation in VPS35 acts to induce neurodegeneration in experimental models and human disease remains unclear. To date, three cellular mechanisms have been identified that are disrupted by the D620Nmutation in VPS35. Those mechanisms include WASH complex binding defects, AMPA receptor trafficking defects, and impaired mitochondrial function and dynamics (Williams et al., 2017). Briefly, the D620N mutation has been shown by two independent groups to impair binding to the WASH complex (McGough et al., 2014; Zavodszky et al., 2014). The WASH complex is an accessory protein complex that can bind to a small percentage of the retromer and facilitate the formation of F-actin patches that help guide cargo for transport (Williams et al., 2017). Interestingly, impaired WASH complex binding results in macroautophagy defects through abnormal trafficking of the autophagy receptor ATG9A (Zavodszky et al., 2014). The D620N mutation has also been shown to impair AMPA receptor trafficking both through overexpression of D620N VPS35 and depletion of VPS35 protein (Munsie et al., 2014; Tian et al., 2015). When overexpressed, the D620N mutation causes defects in the trafficking of GluR1 which causes downstream synaptic transmission defects (Munsie et al., 2014). Interestingly, neurons with depletion of VPS35 also exhibit abnormal synaptic transmission and trafficking of both GluR1 and GluR2 (Tian et al., 2015). Finally, the D620N mutation has also been implicated in mitochondrial dysfunction by increasing mitochondrial fragmentation when overexpressed through abnormal recycling of the fission factor, DLP1 (W. Wang et al., 2016). Additionally, VPS35 knockout in mice is reported to impair mitochondrial fusion through reducing the levels of fusion factor, MFN2 (Tang et al., 2015). While intriguing, none of these disrupted pathways have been shown yet to directly impact neurodegeneration therefore leaving the tantalizing possibility that the true cause of D620N VPS35-induced neurotoxicity has yet to be identified. With this in mind, we are actively using several different methods to dissect the protein interactome of VPS35 (WT and D620N) to determine differential interactors that may underlie D620N VPS35-induced neurodegeneration. In addition to the above proposed cellular mechanisms of D620N VPS35-induced dysfunction,