Using Primary Fibroblasts and iPSC-Derived Neurons from Patients with AP-4-associated Hereditary Spastic Paraplegia to Support an Unbiased Autophagy-based Phenotypic Screening for Novel Therapeutic Targets

该提案旨在确定治疗AP-4相关遗传性痉挛性截瘫（HSP）的新治疗靶点，HSP是一组与自噬功能障碍相关的疾病。AP-4相关的HSP是一种进行性神经退行性疾病，其特征在于全面发育迟缓、智力迟钝、癫痫发作和进行性痉挛，导致通常在10岁之前的早期失肌。这项为期两年的博士后项目提案提出了一种协调的实验方法，用于在AP-4相关HSP的患者来源的成纤维细胞和iPSC来源的神经元中开发基于自噬的表型小分子筛选。选择作为主要、正交和次要试验的细胞表型得到文献证据和稳健初步数据的充分支持。拟议的实验描述了一种逐步的方法，通过ATG 9A易位（主要测定），APP易位，ATG 9A介导的自噬体形成和基于转录组的信号转导通路表征（正交测定）以及AP-4缺陷的神经元特异性表型的校正（次要测定）来测量AP-4功能。这个实验设计是嵌入在一个全面的研究计划，并承诺高度的可译性。AP-4相关的HSP作为具有缺陷性自噬的疾病的范例疾病模型。提出的筛选还可能揭示治疗其他形式的HSP和其他与儿童期自噬缺陷和成年期神经退行性疾病相关的神经退行性疾病的机制。

This proposal aims to identify novel therapeutic targets for the treatment of AP-4-associated hereditary spastic paraplegia (HSP), a group of diseases associated with a autophagy dysfunction. AP-4-associated HSP is a progressive neurodegenerative disorder characterized by global developmental delay, mental retardation, epileptic seizures and progressive spasticity leading to early loss of ambulation, usually before the age of 10 years. This proposal for a two-year postdoctoral project presents a coordinated experimental approach to developing an autophagy-based phenotypic small molecule screening in patient-derived fibroblasts and iPSC-derived neurons of AP-4-associated HSP. The cellular phenotypes selected as primary, orthogonal and secondary assays are well supported by evidence from the literature and robust preliminary data. The proposed experiments describe a stepwise approach that measures AP-4 function by ATG9A translocation (primary assay), APP translocation, ATG9A-mediated autophagosome formation and transcriptome-based characterization of signaling pathways (orthogonal assays), as well as correction of neuron-specific phenotypes of AP-4 deficiency (secondary assays). This experimental design is embedded in a comprehensive research program and promises a high a high degree of translatability. AP-4-associated HSP serves as a paradigmatic disease model for a disease with defective autophagy. The proposed screens may also uncover mechanisms to treat other forms of HSP and other neurodegenerative diseases associated with defective autophagy in childhood and neurodegenerative diseases in adulthood.