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.
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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

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Figure 7

Reduced membralin levels correlate with reductions in EAAT2 in ALS mouse models and human ALS spinal cord.

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Reduced membralin levels correlate with reductions in EAAT2 in ALS mouse...
(A) Lysates from spinal cord from SOD1G85R (black) or SOD1G37R (red) animals at pre-onset, onset, and end-stage time points (n = 2 animals) were immunoblotted for EAAT2, membralin, or actin as indicated. (B) EAAT2 monomer (left graph) or dimer (right graph) levels were plotted in comparison to membralin levels in SOD1G85R (black) and SOD1G37R (red) animals. (C) Lysates from control, fALS, and sALS patient spinal cord samples were immunoblotted for EAAT2 and membralin. (D) Relative membralin and EAAT2 monomer/dimer band intensities were normalized to actin (control sample mean value set to 1.0). Significance values were determined by 1-way ANOVA/Dunnett’s multiple comparison. *P <0.05, ***P < 0.001. (E) EAAT2 monomer (upper graph) or dimer (lower graph) levels were plotted in comparison to membralin levels in control (black), fALS (red) or sALS (orange) human spinal cord samples. (F) Human spinal cord samples from control, sALS or fALS were subjected to membralin or EAAT2 staining as indicated. Gray matter regions within the tissues are indicated by dotted gray lines. Scale bar: 300 μm. (G) Cell lysates from WT (n = 6 pups) or SOD1G93A (n = 6 pups) astrocytes were transduced with control or membralin AAV vectors and immunoblotted for the components indicated. (H) EAAT2 monomer and dimer band intensities in immunoblots from G were normalized to actin and plotted with respect to WT/AAV control transduced astrocytes (set to 1.0, mean ± SE). Significant values were determined by 1-way ANOVA/Tukey’s multiple comparison. *P < 0.05. (I) Matched SOD1G93A astrocyte cultures transduced with control (black) and membralin (blue) AAV vectors were evaluated for EAAT2 and TNFR1 levels analyzed by paired t tests, showing a significant increase of EAAT2 and decreased TNFR1 levels with membralin transduction relative to controls. R2 values (goodness of fit), and 2-tailed significance values for the linear correlation plots shown in B and E were determined by Pearson correlation analysis.