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.

中文翻译：

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VPS35 D620N 突变敲入小鼠神经元中的内体运输和谷氨酸突触活性增加，并且对 LRRK2 激酶抑制具有抵抗力

液泡蛋白分选 35 (VPS35) 调节内体中神经递质受体的再循环。VPS35 中的错义突变 (D620N) 会导致常染色体显性遗传的迟发性帕金森病。在这里，我们研究了 D620N 敲入小鼠中 VPS35 和帕金森病突变效应的基础神经生物学以及富含亮氨酸重复激酶 2 (LRRK2) 抑制对突触表型的影响。该研究是使用内源水平表达 VPS35 的 VPS35 D620N 敲入小鼠进行的。通过免疫共沉淀和蛋白质印迹测定了敲入小鼠和野生型同窝小鼠脑裂解物中的蛋白质水平、磷酸化状态和结合比率。使用免疫细胞化学和全细胞膜片钳电生理学分析来自敲入和野生型同窝小鼠的原代皮层培养物中的树突蛋白共定位、AMPA受体表面表达、突触密度和谷氨酸突触活性。在脑组织中，我们确认VPS35除了与NMDA型谷氨酸受体GluN1亚基和D2型多巴胺受体外，还与LRRK2和AMPA型谷氨酸受体GluA1亚基形成复合物。在 D620N 敲入小鼠中，受体和 LRRK2 的结合没有改变，但我们证实突变导致 VPS35 与 WASH 复合体成员 FAM21 的结合减少，并增加 LRRK2 激酶底物 Rab10 的磷酸化，体内 LRRK2 激酶抑制可逆转这一现象。在培养的敲入小鼠皮层神经元中，pRab10 也增加，并通过 LRRK2 抑制而逆转。该突变还导致内体回收蛋白簇密度（VPS35-FAM21 共簇和 Rab11 簇）、谷氨酸传输和 GluA1 表面表达增加。LRRK2 激酶抑制可逆转 Rab10 过度磷酸化，但不能挽救敲入神经元中谷氨酸释放或表面 GluA1 表达升高，但确实改变了野生型细胞中的 AMPAR 流量。这些结果提高了我们对 VPS35 细胞生物学以及 D620N 突变对神经元网络发育的影响的理解。这些数据共同支持潜在神经退行性变的慢性突触病模型，提供可能推动 VPS35 PD 最终转变为晚期帕金森病的表型和候选病理生理应激。该研究表明，VPS35 突变的影响与正在进行的 LRRK2 激酶活性无关，并且 LRRK2 激酶抑制会改变体外谷氨酸突触的基础生理学。