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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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

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Figure 4

Restoration of EAAT2 levels suppresses astroglial neurotoxicity with membralin deletion.

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Restoration of EAAT2 levels suppresses astroglial neurotoxicity with mem...
(A) Restoring EAAT2 expression levels in mem-KO astrocytes. Cell lysates from mem-KO astrocytes transduced with control or GFAP-EAAT2-HA AAV vectors or nontransduced WT astrocytes were immunoblotted for EAAT2 or actin as indicated. (B) Murine WT motor neurons were cocultured with mem-KO astrocytes transduced with control or EAAT2-HA AAV constructs for 7 days. Neurons and astrocytes were stained and visualized for GFAP (purple), HA (red), SMI32 (green), and DAPI (blue). Scale bar: 50 μm. (C) SMI32-stained motor neurons from B were subject to neurite branch quantification for number (left) and length (right). Each dot represents the mean of the motor neurons from individual fields, from n = 4 independent experiments (mean ± SE). (D) Conditioned media from mem-KO astrocytes transduced with control AAV (upper panels) or EAAT2-HA AAV (lower panels) were applied to mouse motor neurons for 24 hours, where neurons were stained for SMI32 (green), active caspase 3 (red), and DAPI (blue). Scale bar: 50 μm. Number of active caspase 3–stained motor neurons (normalized to DAPI) were quantified in the adjacent graph (each dot represents the mean value from individual cover glass from n = 4 independent experiments, mean ± SE). Significance values in C and D were determined by Student’s t test. *P < 0.05, ***P < 0.001.