Role of retromer complex in mediating neurotoxicity induced by Parkinson's disease-linked LRRK2

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder and mutations in at least twelve genes cause familial forms of PD. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of autosomal dominant PD, whereas common genetic variation at the LRRK2 locus is associated with increased PD risk. At present, the downstream cellular pathways critical for mediating LRRK2-induced neurodegeneration remain poorly defined. We have identified a novel interaction of LRRK2 with the dominant PD-linked gene product, VPS35. VPS35 functions as a key component of the retromer complex which mediates retrograde transport of protein cargo from endosomes to the trans-Golgi network or plasma membrane. Previous studies suggest that familial LRRK2 mutants induce a retromer deficiency in mammalian cells and mouse brain, with VPS35 overexpression rescuing neurotoxicity induced by mutant LRRK2 in primary neuronal and Drosophila models. A critical validation of this potential mechanism is currently lacking in neuronal and rodent LRRK2 models. To extend these prior studies, we have evaluated the functional interaction of LRRK2 and VPS35 using a variety of approaches. We observe the robust interaction and colocalization of FLAG-tagged LRRK2 and V5-tagged VPS35 in human cells. Familial PD mutations in either protein have a limited effect on their interaction whereas functional mutations support a stronger interaction of VPS35 with kinase-inactive LRRK2. Domain mapping suggests that the N- and C-terminal regions of LRRK2 can support the interaction with VPS35. We find that VPS35 does not serve as a substrate of LRRK2 kinase activity in vitro, and familial VPS35 mutations fail to alter LRRK2 kinase activity in cells. We demonstrate that familial mutations in LRRK2 fail to alter the steady-state levels of endogenous retromer subunits when overexpressed in human cell lines or rat primary cortical neurons. Furthermore, we find no evidence for altered retromer protein levels in distinct brain regions of human G2019S LRRK2 transgenic mice or in G2019S mutant and idiopathic PD brains relative to control brains. Finally, we show that VPS35 heterozygosity in mice does not increase the susceptibility of dopaminergic neurons to degeneration induced by the adenoviral-mediated expression of human G2019S LRRK2 in the nigrostriatal pathway. We are currently exploring the impact of LRRK2 mutations on retromer cargo sorting and protein interactions in neuronal and rodent models. Our data reveal an interaction of LRRK2 with VPS35 but so far fail to provide clear evidence for a retromer deficiency induced by mutant LRRK2 in PD-relevant models.