Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition

Abstract

Vacuolar protein sorting 35 (VPS35) regulates neurotransmitter receptor recycling from endosomes. A missense mutation (D620N) in VPS35 leads to autosomal-dominant, late-onset Parkinson’s disease. Here, we study the basic neurobiology of VPS35 and Parkinson’s disease mutation effects in the D620N knock-in mouse and the effect of leucine-rich repeat kinase 2 (LRRK2) inhibition on synaptic phenotypes. The study was conducted using a VPS35 D620N knock-in mouse that expresses VPS35 at endogenous levels. Protein levels, phosphorylation states, and binding ratios in brain lysates from knock-in mice and wild-type littermates were assayed by co-immunoprecipitation and western blot. Dendritic protein co-localization, AMPA receptor surface expression, synapse density, and glutamatergic synapse activity in primary cortical cultures from knock-in and wild-type littermates were assayed using immunocytochemistry and whole-cell patch clamp electrophysiology. In brain tissue, we confirm VPS35 forms complexes with LRRK2 and AMPA-type glutamate receptor GluA1 subunits, in addition to NMDA-type glutamate receptor GluN1 subunits and D2-type dopamine receptors. Receptor and LRRK2 binding was unaltered in D620N knock-in mice, but we confirm the mutation results in reduced binding of VPS35 with WASH complex member FAM21, and increases phosphorylation of the LRRK2 kinase substrate Rab10, which is reversed by LRRK2 kinase inhibition in vivo. In cultured cortical neurons from knock-in mice, pRab10 is also increased, and reversed by LRRK2 inhibition. The mutation also results in increased endosomal recycling protein cluster density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate transmission, and GluA1 surface expression. LRRK2 kinase inhibition, which reversed Rab10 hyper-phosphorylation, did not rescue elevated glutamate release or surface GluA1 expression in knock-in neurons, but did alter AMPAR traffic in wild-type cells. The results improve our understanding of the cell biology of VPS35, and the consequences of the D620N mutation in developing neuronal networks. Together the data support a chronic synaptopathy model for latent neurodegeneration, providing phenotypes and candidate pathophysiological stresses that may drive eventual transition to late-stage parkinsonism in VPS35 PD. The study demonstrates the VPS35 mutation has effects that are independent of ongoing LRRK2 kinase activity, and that LRRK2 kinase inhibition alters basal physiology of glutamate synapses in vitro.

The Parkinson’s Disease related mutant VPS35 (D620N) amplifies the LRRK2 response to endolysosomal stress

“…However, we can recapitulate two robust findings associated with the VPS35 (D620N) mutation i.e. diminished interaction between retromer and the WASH complex and activation of LRRK2 [19, 20, 23]. This second facet is the main focus of our study, as it reflects a direct connection between two pieces of the Parkinson’s Disease jigsaw.…”

“…Many suggestions have been made for the patho-physiological role of VPS35 (D620N) that use known retromer-dependent pathways as their inspiration. In addition, a persuasive link between VPS35 and LRRK2 has been established, in that VPS35 (D620N) results in hyperactivation of LRRK2 [19, 20].…”

VPS35 and retromer dysfunction in Parkinson's disease

“…Additionally, human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons harbouring the D620N mutation were also observed to have an increase in GluR1 cluster intensity. Supporting these findings, a recent study by Kadgein et al [45] observed GluR1 association with VPS35 in neurons derived from a D620N VPS35 knockin mouse model. While previous studies have used overexpression of VPS35 variants, D620N VPS35 knockin mice provide a more physiologically relevant model, expressing VPS35 at endogenous levels.…”

“…Notably, VPS35 mRNA is detected in the mammalian brain where it is highly expressed in frontal cortex, hippocampus, striatum and cerebellum, and shows highest expression in neurons and oligodendrocytes [43]. Moreover, VPS35 is required for various cellular mechanisms, as evidenced by defects in WASH complex binding, AMPA receptor sorting, the autophagy-lysosomal pathway, and mitochondrial dynamics and activity induced by the PDlinked D620N mutation [44][45][46][47]. Interestingly, while the D620N mutation does not disrupt the interaction of VPS35 with other subunits of the retromer complex, there is some discordance within the literature surrounding the mechanism of VPS35-dependent neurodegeneration.…”

Beware of Misdelivery: Multifaceted Role of Retromer Transport in Neurodegenerative Diseases

“…Although limited evidence is available, several studies claim that the disorder in retromer function by PD-related VPS35 mutations may affect synaptic function (Figure 3). In mouse primary cortical neurons, the presence of D620N VPS35 was less frequently present in dendritic spines than WT VPS35, and D620N VPS35 tended to form clusters with FAM21 in EEs (Munsie et al, 2015;Kadgien et al, 2021). In the synaptic nerve terminal, VPS35 participates in the cell surface recycling of GluA1, dopamine D1 receptor (D1R), and dopamine transporter (DAT), and thus, D620N VPS35 might increase the surface expression of these receptors, thereby producing chronic stress in neuronal circuits (Munsie et al, 2015;Wu et al, 2017;Kadgien et al, 2021).…”