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 August 1, 2019; First 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 1

Membralin deletion in astrocytes manifests motor defects and early postnatal lethality.

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Membralin deletion in astrocytes manifests motor defects and early postn...
(A) WT and membralin whole-body deletion (mem-KO) animals at P3 (reshown in Supplemental Figure 1B). Relative body weight of WT (black), mem heterozygous (gray), and homozygous KO (red) animals at P3 (WT animals set to 1.0). Bottom graph shows Kaplan-Meier survival curves for WT (black), whole-body mem-KO (red), and Nestin-Cre mem-KO (Nestin-Cre) animals (orange). (B) SMI32 (motor neurofilaments), TUNEL (apoptosis), GFAP (astrocytes), and IBA1 (microglia) staining in spinal cord from P3 WT and KO animals. Scale bars: 200 μm. Graphs indicate quantified motor neuron number, cell death, and astroglial/microglial staining area (mean ± SE). GFAP and IBA1 staining was compared with WT (normalized to 1.0 AU). (C) Schematic of transgenic membralin Tg overexpression mouse lines under Prion-mem (black) or Hb9-mem (red) promoters. Right: Kaplan-Meier survival curve of mem-KO lines crossed with Prion-mem Tg (Prion-mem, black) or Hb9-mem Tg (Hb9-mem, red) lines. (D) Membralin expression in WT, mem-KO, and mem-KO/Prion-mem Tg spinal cord was examined by immunoblot. (E) Schematic of mouse Cre-driver lines crossed with memfl/fl lines to generate cell-specific membralin deletion strains. (F) Survival curves of Cre lines crossed with memfl/fl. GFAP-Cre (red) resulted in early postnatal lethality. (G) Sprawling motor reflex with tail suspension in WT (memfl/fl), or memfl/fl × GFAP-Cre lines (Astro–mem-KO) at P15. Astrocytic Cre expression in Astro–mem-KO animals was determined by Cre (brown) and GFAP (purple) staining in spinal cord. Scale bars: 60 μm. (H) Histological examination of motor cortex from P20 WT or Astro–mem-KO lines stained for the components indicated. Scale bars: 50 μm. (I) IBA1, GFAP, or CD68 staining area was quantified (n = 3/genotype, WT = 1.0). Significance in A, B, and I was calculated by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.