KCNJ2 inhibition mitigates mechanical injury in human brain organoids

Traumatic brain injury (TBI) strongly correlates with neurodegenerative disease. However, it remains unclear which neurodegenerative mechanisms are intrinsic to the brain itself and which strategies most potently mitigate these processes, particularly in individuals genetically predisposed to neurodegeneration. We developed a high-intensity ultrasound platform to inflict mechanical injury to iPSC-derived cortical organoids. Mechanically injured organoids elicit several classic hallmarks of TBI including neuron death, tau phosphorylation, and TDP-43 phosphorylation and nuclear egress. We found that deep layer excitatory neurons were particularly vulnerable to injury and that TDP-43 proteinopathy promotes loss-of-function and cell death. Injured organoids derived from C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) patients displayed exacerbated TDP-43 dysfunction. Using genome-wide CRISPR interference screening, we identified a mechanosensory channel, KCNJ2, whose inhibition potently mitigated neurodegenerative processes in vitro and in vivo, including in C9ORF72 ALS/FTD organoids. Thus, targeting KCNJ2 may reduce acute neuron death after brain injury, and we present a scalable, genetically-flexible cerebral organoid model that may enable identification of additional modifiers downstream of mechanical stress. Overall design: HIFU and sham treated organoids were collected 24 hours after injury and processed for bulk RNA sequencing创伤性脑损伤（TBI）与神经退行性疾病密切相关，但目前仍尚不明确哪些神经退行性机制是大脑自身固有的，又有哪些策略能够最有效地抑制此类进程，尤其是在具有神经退行性疾病遗传易感性的人群中。我们开发了一套高强度超声平台，用于对诱导多能干细胞（iPSC）来源的大脑皮层类器官施加机械损伤。经机械损伤的类器官会呈现出TBI的多项经典特征，包括神经元死亡、tau蛋白磷酸化、TDP-43蛋白磷酸化及其核迁出。我们发现，深层兴奋性神经元对损伤尤为易感，且TDP-43蛋白病会促进神经元功能丧失与细胞死亡。源自C9ORF72型肌萎缩侧索硬化/额颞叶痴呆（ALS/FTD）患者的损伤类器官，其TDP-43功能障碍表现得更为严重。我们通过全基因组CRISPR干扰筛选，鉴定出一种机械敏感离子通道KCNJ2，抑制该通道可在体外与体内模型中有效缓解神经退行性进程，包括在C9ORF72型ALS/FTD类器官中。因此，靶向KCNJ2或可减少脑损伤后的急性神经元死亡；同时我们构建了一套可扩展、遗传可操控的大脑皮层类器官模型，该模型可用于鉴定机械应激下游的其他调控因子。实验整体设计：于损伤后24小时收集经高强度聚焦超声（HIFU）处理与假处理的类器官，进行批量RNA测序分析。