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.

中文翻译：

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帕金森病 VPS35 D620N 突变转基因老年小鼠纹状体多巴胺水平的改变

液泡蛋白分选 35 (VPS35) 是逆向复合体的主要组成部分，它介导货物蛋白质从内体到反式高尔基体网络的逆行运输。已经在常染色体显性遗传帕金森病 (PD) 患者中发现了 VPS35 基因中的 D620N 等突变。然而，人们对 VPS35 缺陷或突变是否以及如何导致 PD 发病机制知之甚少。具体而言，迄今为止对 VPS35 进行检查的研究在结果和方法上有所不同。我们使用基于 Rosa26 的转基因表达平台生成了 VPS35 D620N 小鼠模型，以允许以空间方式表达，以便更好地解决这些差异。在这里，老年（20 个月大）的小鼠首先接受了行为测试。随后，使用酪氨酸羟化酶 (TH) 标记物对黑质 (SN) 多巴胺能神经元进行 DAB 染色分析。接下来，使用 HPLC 测定纹状体中的多巴胺水平及其两种代谢物 3,4-二羟基苯乙酸 (DOPAC) 和高香草酸 (HVA) 的水平。还进行了蛋白质印迹以研究与 PD 相关的关键蛋白质的水平。最后，对 [3H]FE-PE2I 与纹状体多巴胺转运蛋白 DAT 的结合进行放射自显影 (ARG) 评估。我们发现 VPS35 D620N Tg 小鼠的多巴胺水平明显高于 NTg 小鼠。然后将所有结果与当前 VPS35 研究的结果进行比较，以阐明疾病的发病机制。