Membralin deficiency dysregulates astrocytic glutamate homeostasis, leading to ALS-like impairment
Lu-Lin Jiang, … , Timothy Y. Huang, Huaxi Xu
Lu-Lin Jiang, … , Timothy Y. Huang, Huaxi Xu
Published May 21, 2019
Citation Information: J Clin Invest. 2019;129(8):3103-3120. https://doi.org/10.1172/JCI127695.
View: Text | PDF
Research Article Cell biology Neuroscience

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

Abstract

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 indicated 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 show that activation of the TNF receptor (TNFR1) NFκB 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. Our study provided a mechanism for ALS pathogenesis where membralin limited glutamatergic neurotoxicity, suggesting that modulating membralin had potential in ALS therapy.

Authors

Lu-Lin Jiang, Bing Zhu, Yingjun Zhao, Xiaoguang Li, Tongfei Liu, Juan Pina-Crespo, Lisa Zhou, Wenxi Xu, Maria J. Rodriguez, Haiyang Yu, Don W. Cleveland, John Ravits, Sandrine Da Cruz, Tao Long, Dongxian Zhang, Timothy Y. Huang, Huaxi Xu

×

Figure 6

TNFR1/NFκB signaling mediates EAAT2 reductions resulting from membralin deletion.

Options: View larger image (or click on image) Download as PowerPoint
TNFR1/NFκB signaling mediates EAAT2 reductions resulting from membralin ...
(A) qRT-PCR analysis of Eaat2 mRNA from motor cortex in WT and Astro–mem-KO animals, normalized to 18s rRNA. (B) TNFR1 levels were assessed by immunoblot in spinal cord from WT and mem-KO animals. Quantification depicts normalized TNFR1 levels (WT mean set to 1.0). (C) Nuclear NFκB localization was examined in P3 WT and mem-KO spinal cord. Number of NFκB-positive nuclei was determined in n = 3 animals/genotype. Scale bar: 100 μm. Arrows indicate nuclear NFκB. (D) WT or mem-KO astrocytes were transfected with 50 nM control or TNFR1 siRNA and stained for GFAP (green), NFκB (red), or DAPI. Scale bar: 50 μm. (E) Quantification of NFκB-positive nuclei shown in D (normalized to DAPI; scale bar: 50 μm). (F) Mem-KO astrocytes transfected with control or TNFR1 siRNA were immunoblotted for EAAT2, TNR1, or actin as indicated. Adjacent graphs depict TNFR1 (left graph) or EAAT2 (right graph) levels. All quantification graphs represent mean ± SE; statistical significance was determined by unpaired Student’s t test (significance in F was subjected to paired t test analysis), with the exception of E, which was subjected to 1-way ANOVA/Tukey’s multiple comparison. *P < 0.05, **P < 0.01.