Inhibition of astrocyte hemichannel improves recovery from spinal cord injury
Chao Zhang, … , Naomi L. Sayre, Jean X. Jiang
Chao Zhang, … , Naomi L. Sayre, Jean X. Jiang
Published March 8, 2021
Citation Information: JCI Insight. 2021;6(5):e134611. https://doi.org/10.1172/jci.insight.134611.
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Research Article Therapeutics

Inhibition of astrocyte hemichannel improves recovery from spinal cord injury

Abstract

Spinal cord injury (SCI) causes severe disability, and the current inability to restore function to the damaged spinal cord leads to lasting detrimental consequences to patients. One strategy to reduce SCI morbidity involves limiting the spread of secondary damage after injury. Previous studies have shown that connexin 43 (Cx43), a gap junction protein richly expressed in spinal cord astrocytes, is a potential mediator of secondary damage. Here, we developed a specific inhibitory antibody, mouse-human chimeric MHC1 antibody (MHC1), that inhibited Cx43 hemichannels, but not gap junctions, and reduced secondary damage in 2 incomplete SCI mouse models. MHC1 inhibited the activation of Cx43 hemichannels in both primary spinal astrocytes and astrocytes in situ. In both SCI mouse models, administration of MHC1 after SCI significantly improved hind limb locomotion function. Remarkably, a single administration of MHC1 30 minutes after injury improved the recovery up to 8 weeks post-SCI. Moreover, MHC1 treatment decreased gliosis and lesion sizes, increased white and gray matter sparing, and improved neuronal survival. Together, these results suggest that inhibition of Cx43 hemichannel function after traumatic SCI reduces secondary damage, limits perilesional gliosis, and improves functional recovery. By targeting hemichannels specifically with an antibody, this study provides a potentially new, innovative therapeutic approach in treating SCI.

Authors

Chao Zhang, Zhao Yan, Asif Maknojia, Manuel A. Riquelme, Sumin Gu, Grant Booher, David J. Wallace, Viktor Bartanusz, Akshay Goswami, Wei Xiong, Ningyan Zhang, Michael J. Mader, Zhiqiang An, Naomi L. Sayre, Jean X. Jiang

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Figure 1

MHC1 antibody binds Cx43 and inhibits the opening of Cx43 hemichannels.

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MHC1 antibody binds Cx43 and inhibits the opening of Cx43 hemichannels. ...
(A and B) Fixed parental HeLa cells or HeLa cells stably transfected with Cx43 were incubated with MHC1 antibody and then labeled with HRP-conjugated anti-human IgG (A) or rhodamine-conjugated anti-human IgG and counterstained with DAPI (B). Scale bars: 50 μm (A), 30 μm (B). (C) The binding affinity of MHC1 and IgG control to Cx43 peptide was determined by ELISA. n = 4. (D) Kinetics of MHC1 binding to the Cx43 extracellular domain peptide (N′-CFLSRPTEKTI) as assessed using an Octet RED96. (E) Parental HeLa cells or HeLa cells stably transfected with Cx43 were incubated with EGTA to remove extracellular Ca2+ ([Ca2+ ]0) in the absence or presence of MHC1 (66.7 nM) or control IgG (66.7 nM) before dye uptake assay with 15-minute treatment of 50 μM ethidium bromide (EtBr). (F) Primary astrocytes isolated from rat cortical brain were incubated for 3 or 24 hours with MHC1 (66.7 nM) or CBX (100 μM) before scrape-loading dye transfer assay was performed with Lucifer yellow (1%) and rhodamine dextran (1%) for 5 minutes. The level of EtBr dye uptake in E and dye transfer in F was determined by fluorescence microcopy and quantified by NIH ImageJ software. Scale bar: 200 μm (E and F). Data are presented as mean ± SEM of 3 independent experiments; each experiment had 2–3 repeats (2–3 wells). Michaelis-Menten equation was used in statistical analysis model (C). General linear model was used in statistical analysis (E and F). ***P < 0.001; ****P < 0.0001.