VPS35 as a therapeutic target in Parkinson's disease

修改项目摘要/摘要部分 帕金森病（PD）的一个关键病理特征是α-突触核蛋白（αSyn）的寡聚和聚集形式在细胞内包涵体中的积累。VPS 35是一个参与多种蛋白质降解途径的逆转录亚基，包括αSyn的降解。VPS 35突变是晚发型常染色体显性PD的原因。VPS 35蛋白在脑中的慢病毒过表达减弱αSyn转基因小鼠中αSyn蛋白的积累。鉴于这些趋同的数据，我们建议增加VPS 35的水平作为PD的潜在疾病修饰策略。首先，我们的目标是在基于αSyn的PD小鼠模型中验证一种使用药理学伴侣靶向VPS 35的新型治疗方法。选择药理学分子伴侣是因为它们能够增加啮齿动物脑中的VPS 35水平。我们的研究开始于药理学伴侣R55的研究，R55通过结合VPS 39-VPS 35 retromer亚基之间的活性结合位点来稳定retromer复合物并增加细胞系中的VPS 35水平。我们产生了一个小的R55启发的化合物库，该化合物被设计为增加逆转录稳定性和亲脂性，具有更大的血脑屏障（BBB）渗透性，并且我们鉴定了先导化合物1，3苯基双胍基腙（2a）。我们评估了2a在Neuro 2A细胞中的体外功效、在WT小鼠中的体内功效和在肌萎缩性侧索硬化症（ALS）小鼠模型中的功效。我们有初步的数据表明，我们的先导化合物在体内增加黑质中的VPS 35水平。我们现在建议测试该化合物在体内促进αSyn清除的能力，并评估其潜在的神经保护作用。我们将评估AAV-A53 T-αSyn小鼠模型中的神经保护作用，并通过在该小鼠模型以及神经分化的SH-SY 5 Y神经母细胞瘤细胞中的分析，评估不同αSyn清除途径在2a保护作用中的作用。拟议的研究将通过提供这种特定药物的关键翻译验证和更广泛的这种新的治疗策略，具有重要的翻译意义。

Modified Project Summary/Abstract Section A key pathological feature of Parkinson disease (PD) is the accumulation of oligomeric and aggregated forms of alpha-synuclein (αSyn) in intracellular inclusions. VPS35 is a retromer subunit involved in multiple protein degradation pathways, including degradation of αSyn. Mutations in VPS35 are a cause of late-onset autosomal dominant PD. Lentiviral overexpression of VPS35 protein in the brain attenuates the accumulation of αSyn protein in αSyn transgenic mice. Given these converging data, we propose to pharmacologically increase levels of VPS35 as a potential disease-modifying strategy for PD. First, we aim to validate a novel therapeutic approach using pharmacological chaperones to target VPS35 in an αSyn based mouse model of PD. The pharmacological chaperones were selected for their ability to increase VPS35 levels in the brain of rodents. Our investigation started from studies of a pharmacological chaperone, R55, that stabilizes the retromer complex and increase VPS35 levels in cell lines by binding an active binding site between VPS39-VPS35 retromer subunits. We generated a small library of R55-inspired compounds designed to increase retromer stability and lipophilicity with greater blood brain barrier (BBB) penetration and we identified the lead compound, 1,3 phenyl bis-guanylhydrazone (2a). We assessed 2a efficacy in vitro in Neuro2A cells, in vivo in WT mice and in an Amyotrophic Lateral Sclerosis (ALS) mouse model. We have preliminary data demonstrating that our lead compound increases VPS35 levels in the substantia nigra in vivo. We now propose to test the ability of this compound in vivo to promote αSyn clearance and to assess its potential neuroprotective effects. We will assess for neuroprotection in the AAV-A53T-αSyn mouse model, and also will assess the roles of different αSyn clearance pathways in the protective effects of 2a through analyses in this mouse model as well as in neuronally differentiated SH-SY5Y neuroblastoma cells. The proposed studies will have important translational implications by providing key translational validation of this specific agent and more broadly of this novel therapeutic strategy.