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 2

Membralin deletion in astrocytes results in accumulation of neurotoxic components in the conditioned media.

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Membralin deletion in astrocytes results in accumulation of neurotoxic c...
(A–C) Mem-KO astrocytes induce motor neuron impairment. (A) WT or mem-KO astrocytes were cocultured with ES-derived (Hb9::GFP) WT motor neurons with SMI32-stained neurites (green), and astrocytes (GFAP, red) were visualized in cocultures. DAPI is indicated in blue. Scale bar: 50 μm. (B) SMI32-stained neurites in motor neurons from A were skeletonized using Imaris software. (C) Distribution of branch number (upper graph) and primary filament length (lower graph) outputs are presented. (D) WT motor neurons (stained with SMI32, green) derived from mouse ES cells were exposed to conditioned media from WT or mem-KO astrocytes for 24 hours. Caspase 3 activation (red) was visualized for neuronal toxicity. Scale bar: 50 μm. Quantification depicts SMI32/caspase 3 colocalization normalized to DAPI (mean ± SE, n = 30 for WT and n = 49 cover glasses for KO from more than 3 independent experiments). (E) Conditioned media collected from WT (black bars) and mem-KO (red bars) astrocytes was subjected to analysis by GC-MS; absolute values (50 μM) (right graph) and log2 (fold change; left graph) in metabolites were characterized over WT (log2[mem-KO/WT]). (F) Glutamate in conditioned media from WT (n = 7 biological repeats) and mem-KO astrocytes (n = 7 biological repeats) was normalized relative to WT samples (set to 1.0, mean ± SE). (G) Lysates from cultured WT (black, n = 10 biological repeats) and mem-KO (red, n = 8 biological repeats) astrocytes were subject to immunoblot analysis for the indicated components normalized to actin (WT mean set to 1.0, mean ± SE is shown). Statistical significance values in C, D, F, and G were determined by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.