Membralin deficiency dysregulates astrocytic glutamate homeostasis leading to ALS-like impairment

Mechanisms underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS) are yet unclear. Specific deletion of the ER-component membralin in astrocytes manifested postnatal motor defects and lethality in mice, causing the accumulation of extracellular glutamate through reducing the glutamate transporter EAAT2. Restoring EAAT2 levels in membralin KO astrocytes limited astrocyte-dependent excitotoxicity in motor neurons. Transcriptomic profiles from mouse astrocytic membralin KO motor cortex indicateed significant perturbation in KEGG pathway components related to ALS, including downregulation of Eaat2 and upregulation of Tnfrsf1a. Changes in gene expression with membralin deletion also overlapped with mouse ALS models and reactive astrocytes. Our results shown that activation of TNF receptor (TNFR1)-NFkB pathway known to suppress Eaat2 transcription was upregulated with membralin deletion. Further, reduced membralin and EAAT2 levels correlated with disease progression in spinal cord from SOD1-mutant mouse models, and reductions in membralin/EAAT2 were observed in human ALS spinal cord. Importantly, overexpression of membralin in SOD1G93A astrocytes decreased TNFR1 levels and increased EAAT2 expression, and improved motor neuron survival. Importantly, upregulation of membralin in SOD1G93A mice significantly prolonged mouse survival. Together, our study provides a mechanism for ALS pathogenesis where membralin limits glutamatergic neurotoxicity, suggesting that modulating membralin has potential in ALS therapy. Motor cortex mRNA profiles from 22-day old wild type (WT) and membralin astrocyte conditional knockout (Astro-mem KO) were generated by RNAseq. Five biological repeats were included per genotype.肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）的运动神经元变性潜在机制目前尚未阐明。星形胶质细胞中内质网（endoplasmic reticulum, ER）组分膜联蛋白（membralin）的特异性敲除，可导致小鼠出现产后运动功能缺陷并致死，其通过降低谷氨酸转运体EAAT2的表达，引发细胞外谷氨酸蓄积。在膜联蛋白敲除（membralin KO）的星形胶质细胞中恢复EAAT2水平，可减轻星形胶质细胞介导的运动神经元兴奋性毒性。小鼠星形胶质细胞膜联蛋白敲除模型的运动皮层转录组谱显示，与ALS相关的KEGG通路组分出现显著扰动，包括Eaat2下调与Tnfrsf1a上调。膜联蛋白缺失引发的基因表达变化，同时与小鼠ALS模型及反应性星形胶质细胞的基因表达谱存在重叠。本研究结果表明，已知可抑制Eaat2转录的肿瘤坏死因子受体1（tumor necrosis factor receptor 1, TNFR1）-NF-κB通路在膜联蛋白缺失时被激活上调。进一步研究发现，在SOD1突变型小鼠模型的脊髓组织中，膜联蛋白与EAAT2的水平降低与疾病进展相关；且在人类ALS患者的脊髓组织中也观察到膜联蛋白与EAAT2的表达下调。值得注意的是，在SOD1G93A星形胶质细胞中过表达膜联蛋白，可降低TNFR1水平并上调EAAT2表达，同时改善运动神经元存活能力。同样重要的是，在SOD1G93A小鼠中上调膜联蛋白的表达，能够显著延长小鼠生存期。综上，本研究阐明了ALS发病的一项潜在机制：膜联蛋白可限制谷氨酸能神经毒性，这提示调控膜联蛋白的表达有望成为ALS的治疗策略。本研究通过RNA测序（RNA-seq）获取了22日龄野生型（wild type, WT）与星形胶质细胞条件性敲除膜联蛋白（Astro-mem KO）小鼠的运动皮层mRNA表达谱，每组基因型设置5次生物学重复。