Human VCP mutant ALS/FTD microglia display immune and lysosomal phenotypes independently of GPNMB

Background: Microglia play crucial roles in mediating neuronal homeostasis but have been implicated in contributing to amyotrophic lateral sclerosis (ALS). However, the role of microglia in ALS remains incompletely understood. Methods: Here, we generated highly enriched cultures of VCP mutant microglia derived from human induced pluripotent stem cells (hiPSCs) to investigate their cell autonomous and non-cell autonomous roles in ALS pathogenesis. We used RNA-sequencing, proteomics and functional assays to study hiPSC derived VCP mutant microglia and their effects on hiPSC derived motor neurons and astrocytes. Results: Transcriptomic, proteomic and functional analyses revealed immune and lysosomal dysfunction in VCP mutant microglia. Stimulating healthy microglia with inflammatory inducer lipopolysaccharide (LPS) showed partial overlap with VCP mutant microglia in their reactive transformation. LPS-stimulated VCP mutant microglia displayed differential activation of inflammatory pathways compared with LPS-stimulated healthy microglia. Conserved gene expression changes were identified between VCP mutant microglia, SOD1 mutant mice microglia, and postmortem ALS spinal cord microglial signatures, including increased expression of the transmembrane glycoprotein GPNMB. While knockdown of GPNMB affected inflammatory and phagocytosis processes in microglia, this was not sufficient to ameliorate cell autonomous phenotypes in VCP mutant microglia. Secreted factors from VCP mutant microglia were sufficient to activate the JAK-STAT pathway in hiPSC derived motor neurons and astrocytes. Conclusions: VCP mutant microglia undergo cell autonomous reactive transformation involving immune and lysosomal dysfunction that partially recapitulate key phenotypes of microglia from ALS models and post mortem tissue and are independent of GPNMB. VCP mutant microglia elicit non cell autonomous responses in motor neurons and astrocytes involving the JAK-STAT pathway. To study differences in ALS or LPS treated microglia and their effects on other CNS cell types we differentiated microglia from human iPSC lines from VCP mutant ALS or healthy control backgrounds and conducted RNA sequencing of microglia monocultures or astrocytes or motor neurons treated with microglia conditioned media. To investigate the role of GPNMB in microglia we conducted RNA sequencing of samples treated with either an siRNA targeting GPNMB or a scrambled control siRNA.

背景：小胶质细胞（microglia）在介导神经元稳态中发挥关键作用，同时也被证实与肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）的发病相关。然而，小胶质细胞在ALS中的具体作用仍未完全阐明。
研究方法：本研究通过人类诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）构建了高纯度的VCP突变型小胶质细胞培养体系，以探究其在ALS发病过程中的细胞自主与非细胞自主调控作用。我们采用RNA测序、蛋白质组学及功能实验，对hiPSC来源的VCP突变型小胶质细胞，以及其对hiPSC来源的运动神经元和星形胶质细胞的影响进行研究。
研究结果：转录组学、蛋白质组学及功能分析显示，VCP突变型小胶质细胞存在免疫与溶酶体功能异常。用炎性诱导剂脂多糖（lipopolysaccharide, LPS）刺激健康小胶质细胞，其反应性转化特征与VCP突变型小胶质细胞存在部分重叠。与LPS刺激的健康小胶质细胞相比，LPS刺激的VCP突变型小胶质细胞的炎性通路激活模式存在显著差异。我们在VCP突变型小胶质细胞、SOD1突变小鼠小胶质细胞以及ALS患者死后脊髓组织的小胶质细胞特征中，发现了保守的基因表达变化，包括跨膜糖蛋白GPNMB的表达上调。尽管敲低GPNMB可影响小胶质细胞的炎性反应与吞噬过程，但不足以改善VCP突变型小胶质细胞的细胞自主表型。VCP突变型小胶质细胞分泌的因子可激活hiPSC来源的运动神经元与星形胶质细胞中的JAK-STAT通路。
研究结论：VCP突变型小胶质细胞会发生以免疫与溶酶体功能异常为特征的细胞自主反应性转化，该表型部分重现了ALS模型动物及死后组织中小胶质细胞的关键病理特征，且该过程不依赖GPNMB。VCP突变型小胶质细胞可对运动神经元和星形胶质细胞引发涉及JAK-STAT通路的非细胞自主应答。
为探究ALS相关或LPS处理的小胶质细胞及其对中枢神经系统其他细胞类型的影响，我们分别从VCP突变型ALS患者及健康对照的hiPSC系中诱导分化小胶质细胞，并对小胶质细胞单一培养体系，或经小胶质细胞条件培养基处理的星形胶质细胞、运动神经元开展RNA测序。
为探究GPNMB在小胶质细胞中的功能，我们对转染靶向GPNMB的小干扰RNA（small interfering RNA, siRNA）或错配对照siRNA的样本进行了RNA测序。