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Vacuolar protein sorting 35 (VPS35) is a core component of the retromer complex that regulates protein recycling from maturing endosomes to the trans-Golgi network or plasma membrane. A pathogenic substitution in VPS35 (D620N) leads to autosomal-dominant, clinically-typical, late-onset Parkinson’s disease (PD). Initial studies on the effects of the D620N on neuronal functions of retromer have been prone to overexpression effects. Here, we modeled VPS35 D620N parkinsonism in a knock-in mouse that expresses mutant protein at endogenous levels and used it to examine mutant effects on protein trafficking and glutamate synapse function. In cultured neurons and brain tissue from VKI mice, we show that the levels, interaction, and localization of retromer core subunits (VPS35 and VPS26) are maintained; however, VPS35 binding to the WASH complex subunit FAM21 is decreased by the D620N mutation in brain lysate. In cultured neurons, the mutation results in an accumulation of VPS35-FAM21-positive clusters and recycling endosomes in dendrites, increased glutamate release and increased post-synaptic AMPA-receptor surface expression, without alterations to dendritic morphology or synapse numbers. Glutamate transmission phenotypes were less severe in homozygous mutant cells, possibly due to compensatory or aberrant downregulation of presynaptic VGluT1. LRRK2 kinase is another protein implicated in late-onset PD that interacts physically and functionally with VPS35. PD-causing mutations of LRRK2 increase its kinase activity. We hypothesized D620N expression would increase LRRK2 kinase activity, representing a convergence of pathogenic mechanisms between PD proteins. D620N expression increased LRRK2 kinase activity, which was robustly inhibited by the selective LRRK2 inhibitor MLi-2. LRRK2 kinase inhibition exerted genotype-specific effects on post-synaptic receptor expression, mimicking PD-like phenotypes in WT cells, and having opposing effects in mutant cells. The interpretation of mutant effects was obscured by mutant-specific effects of the solubilization vehicle, Captisol, on electrophysiological readouts. This novel knock-in mouse improves our understanding of the early neurochemical and cellular effects of the D620N mutation in neurons. These observations provide candidate pathophysiological pathways that may drive eventual transition to late-stage parkinsonism in VPS35 families, and support the emerging synaptopathy model of neurodegeneration. These findings suggest caution should be employed regarding widespread use of LRRK2 kinase inhibition in non-LRRK2 PD. |
