The heterogeneity of protein-rich inclusions and its significance in neurodegeneration is poorly understood. Standard patient-derived iPSC models develop inclusions neither reproducibly nor in a reasonable time frame. Here, we developed screenable iPSC “inclusionopathy” models utilizing piggyBac or targeted transgenes to rapidly induce CNS cells that express aggregation-prone proteins at brain-like levels. Inclusions and their effects on cell survival were trackable at single-inclusion resolution. Exemplar cortical neuron α-synuclein inclusionopathy models were engineered through transgenic expression of α-synuclein mutant forms or exogenous seeding with fibrils. We identified multiple inclusion classes, including neuroprotective p62-positive inclusions versus dynamic and neurotoxic lipid-rich inclusions, both identified in patient brains. Fusion events between these inclusion subtypes altered neuronal survival. Proteome-scale α-synuclein genetic- and physical-interaction screens pinpointed candidate RNA-processing and actin-cytoskeleton-modulator proteins like RhoA whose sequestration into inclusions could enhance toxicity. These tractable CNS models should prove useful in functional genomic analysis and drug development for proteinopathies.

中文翻译：

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快速 iPSC 包涵体病模型揭示了 α-突触核蛋白包涵体的形成、后果和分子亚型


富含蛋白质的包涵体的异质性及其在神经退行性变中的重要性知之甚少。标准的患者来源 iPSC 模型既不能重复地产生包涵体，也不能在合理的时间范围内产生包涵体。在这里，我们开发了可筛选的 iPSC“包涵体病”模型，利用 piggyBac 或靶向转基因快速诱导在脑样水平表达易聚集蛋白的 CNS 细胞。包涵体及其对细胞存活的影响可在单包涵体分辨率下进行追踪。示例皮质神经元 α-突触核蛋白包涵体病模型是通过转基因表达 α-突触核蛋白突变形式或外源性播种原纤维来设计的。我们确定了多个包涵体类别，包括神经保护性 p62 阳性包涵体与动态和神经毒性脂质富集包涵体，两者都是在患者大脑中发现的。这些包涵体亚型之间的融合事件改变了神经元存活。蛋白质组规模的 α-突触核蛋白遗传和物理相互作用筛选确定了候选 RNA 加工和肌动蛋白-细胞骨架调节剂蛋白，如 RhoA，其螯合到包涵体中可能会增强毒性。这些易于处理的 CNS 模型应该被证明在蛋白质病的功能基因组分析和药物开发中有用。