PDF(Pubmed)
Abstract：
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.
摘要：
富含蛋白质的内含物的异质性及其在神经变性中的意义知之甚少。标准患者衍生的iPSC模型既不能重复也不能在合理的时间范围内形成内含物。这里,我们利用piggyBac或靶向转基因快速诱导中枢神经系统细胞,在脑样水平上表达聚集倾向蛋白,开发了可筛选的iPSC“包涵体病”模型。包涵体及其对细胞存活的影响在单包涵体分辨率下是可跟踪的。示例性皮质神经元α-突触核蛋白包涵体病模型通过α-突触核蛋白突变体形式的转基因表达或与原纤维的外源接种来工程改造。我们确定了多个包含类，包括神经保护性p62阳性内含物与动态和神经毒性富含脂质的内含物，两者都在患者大脑中发现。这些包涵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蛋白，如RhoA，其螯合到内含物中可以增强毒性。这些可处理的CNS模型应被证明可用于蛋白质病的功能基因组分析和药物开发。
