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Altered striatal dopamine levels in Parkinson's disease VPS35 D620N mutant transgenic aged mice.

ABSTRACT: Vacuolar protein sorting 35 (VPS35) is a major component of the retromer complex that mediates the retrograde transport of cargo proteins from endosomes to the trans-Golgi network. Mutations such as D620N in the VPS35 gene have been identified in patients with autosomal dominant Parkinson's disease (PD). However, it remains poorly understood whether and how VPS35 deficiency or mutation contributes to PD pathogenesis; specifically, the studies that have examined VPS35 thus far have differed in results and methodologies. We generated a VPS35 D620N mouse model using a Rosa26-based transgene expression platform to allow expression in a spatial manner, so as to better address these discrepancies. Here, aged (20-months-old) mice were first subjected to behavioral tests. Subsequently, DAB staining analysis of substantia nigra (SN) dopaminergic neurons with the marker for tyrosine hydroxylase (TH) was performed. Next, HPLC was used to determine dopamine levels, along with levels of its two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the striatum. Western blotting was also performed to study the levels of key proteins associated with PD. Lastly, autoradiography (ARG) evaluation of [³H]FE-PE2I binding to the striatal dopamine transporter DAT was carried out. We found that VPS35 D620N Tg mice displayed a significantly higher dopamine level than NTg counterparts. All results were then compared with that of current VPS35 studies to shed light on the disease pathogenesis. Our model allows future studies to explicitly control spatial expression of the transgene which would generate a more reliable PD phenotype.

SUBMITTER: Vanan S

PROVIDER: S-EPMC7706192 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Altered striatal dopamine levels in Parkinson's disease VPS35 D620N mutant transgenic aged mice.

Vanan Sarivin S Zeng Xiaoxia X Chia Sook Yoong SY Varnäs Katarina K Jiang Mei M Zhang Ke K Saw Wuan Ting WT Padmanabhan Parasuraman P Yu Wei-Ping WP Zhou Zhi-Dong ZD Halldin Christer C Gulyás Balázs B Tan Eng-King EK Zeng Li L

Molecular brain 20201201 1

Vacuolar protein sorting 35 (VPS35) is a major component of the retromer complex that mediates the retrograde transport of cargo proteins from endosomes to the trans-Golgi network. Mutations such as D620N in the VPS35 gene have been identified in patients with autosomal dominant Parkinson's disease (PD). However, it remains poorly understood whether and how VPS35 deficiency or mutation contributes to PD pathogenesis; specifically, the studies that have examined VPS35 thus far have differed in re ...[more]

PMID: 33261640

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Project description: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.

Dataset Information

Altered striatal dopamine levels in Parkinson's disease VPS35 D620N mutant transgenic aged mice.

Publications

Altered striatal dopamine levels in Parkinson's disease VPS35 D620N mutant transgenic aged mice.

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