The identification of multiple genes linked to Parkinson's disease (PD) invites the question as to how they may co-operate. We have generated isogenic cell lines that inducibly express either wild-type or a mutant form of the retromer component VPS35 (D620N), which has been linked to PD. This has enabled us to test proposed effects of this mutation in a setting where the relative expression reflects the physiological occurrence. We confirm that this mutation compromises VPS35 association with the WASH complex, but find no defect in WASH recruitment to endosomes, nor in the distribution of lysosomal receptors, cation-independent mannose-6-phosphate receptor and Sortilin. We show VPS35 (D620N) enhances the activity of the Parkinson’s associated kinase LRRK2 towards RAB12 under basal conditions. Furthermore, VPS35 (D620N) amplifies the LRRK2 response to endolysosomal stress resulting in enhanced phosphorylation of RABs 10 and 12. By comparing different types of endolysosomal stresses such as the ionophore nigericin and the membranolytic agent l-leucyl-l-leucine methyl ester, we are able to dissociate phospho-RAB accumulation from membrane rupture.

中文翻译：

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帕金森病相关突变体 VPS35 (D620N) 放大 LRRK2 对内溶酶体应激的反应

与帕金森病 (PD) 相关的多个基因的鉴定引发了一个问题：它们如何合作。我们已经生成了可诱导表达与 PD 相关的逆转录体成分 VPS35 (D620N) 的野生型或突变体形式的同基因细胞系。这使我们能够在相对表达反映生理发生的环境中测试该突变的拟议影响。我们确认该突变损害了 VPS35 与 WASH 复合物的关联，但未发现 WASH 募集至内体的缺陷，也未发现溶酶体受体、阳离子非依赖性甘露糖 6-磷酸受体和分拣蛋白的分布缺陷。我们发现 VPS35 (D620N) 在基础条件下增强帕金森病相关激酶 LRRK2 对 RAB12 的活性。此外，VPS35 (D620N) 放大 LRRK2 对内溶酶体应激的反应，从而增强 RAB 10 和 12 的磷酸化。通过比较不同类型的内溶酶体应激，例如离子载体尼日利亚霉素和膜溶解剂 L-亮氨酰-L-亮氨酸甲酯，我们发现能够将磷酸化 RAB 积累与膜破裂分离。