Journal Article

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

Kadgien, Chelsie A., Kamesh, Anusha, Milnerwood, Austen J.

2021

Overview

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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) / Amino acids / Analysis / Animals / Basal ganglia / Biomedical and Life Sciences / Biomedicine / Brain / Cells, Cultured / Central nervous system diseases / Dendrites - metabolism / Dopamine / Dopamine D2 receptors / Dopamine Receptor / Dopamine receptors / Electrophysiology / Endosomes / Endosomes - physiology / Gain of Function Mutation / Gene Knock-In Techniques / Genetic aspects / Genotype & phenotype / Glutamate / Glutamate receptors / Glutamatergic transmission / Glutamic Acid - physiology / Glutamic acid receptors (ionotropic) / Immunocytochemistry / Immunoprecipitation / Kinases / Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 - antagonists & inhibitors / Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 - physiology / Localization / LRRK2 protein / Lysates / Medical research / Medicine, Experimental / Mice / Mice, Inbred C57BL / Miniature Postsynaptic Potentials - physiology / Missense mutation / Movement disorders / Mutation / Mutation, Missense / Nerve Tissue Proteins - metabolism / Nervous system / Neural networks / Neurodegeneration / Neurodegenerative diseases / Neurology / Neurons / Neurophysiology / Neurosciences / NMDA / Parkinson Disease - genetics / Parkinson's disease / Patch-Clamp Techniques / Phenotypes / Phosphorylation / Physiological aspects / Physiology / Point Mutation / Protein Binding / Protein Interaction Mapping / Protein turnover / Proteins / Psychopharmacology / rab GTP-Binding Proteins - metabolism / Receptor density / Receptors, AMPA - metabolism / Recombinant Proteins - genetics / Recombinant Proteins - metabolism / Synapses / Synapses - metabolism / Vesicular Transport Proteins - genetics / Vesicular Transport Proteins - physiology / α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors