Inhibition of astrocyte hemichannel improves recovery from spinal cord injury

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

doi:10.1172/jci.insight.134611

Cited by 31 publications

(20 citation statements)

References 65 publications

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“…A monoclonal Cx43(M1) antibody targeting the second extracellular loop domains of Cx43 was originally generated by Abmart (Tulsa, OK, USA) and described previously ( Zhang et al, 2021 ). Briefly, mice were immunized with a Cx43 extracellular domain peptide, and after functional characterization of the hybridoma clones, genes that encode the antibody heavy and light chain variable region were cloned from the mouse hybridoma cell line HC1 by reverse transcription quantitative PCR (RT-qPCR), using a combination of a group of cloning PCR primers.…”

Section: Methodsmentioning

confidence: 99%

Connexin hemichannels with prostaglandin release in anabolic function of bone to mechanical loading

Zhao

Riquelme

Guda

et al. 2022

eLife

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Mechanical stimulation, such as physical exercise, is essential for bone formation and health. Here, we demonstrate the critical role of osteocytic Cx43 hemichannels in anabolic function of bone in response to mechanical loading. Two transgenic mouse models, R76W and Δ130–136, expressing dominant-negative Cx43 mutants in osteocytes were adopted. Mechanical loading of tibial bone increased cortical bone mass and mechanical properties in wild-type and gap junction-impaired R76W mice through increased PGE2, endosteal osteoblast activity, and decreased sclerostin. These anabolic responses were impeded in gap junction/hemichannel-impaired Δ130–136 mice and accompanied by increased endosteal osteoclast activity. Specific inhibition of Cx43 hemichannels by Cx43(M1) antibody suppressed PGE2 secretion and impeded loading-induced endosteal osteoblast activity, bone formation and anabolic gene expression. PGE2 administration rescued the osteogenic response to mechanical loading impeded by impaired hemichannels. Together, osteocytic Cx43 hemichannels could be a potential new therapeutic target for treating bone loss and osteoporosis.

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Section: Methodsmentioning

confidence: 99%

Connexin hemichannels with prostaglandin release in anabolic function of bone to mechanical loading

Zhao

Riquelme

Guda

et al. 2022

eLife

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“…In this work, we developed a novel all-optical assay to probe Ca 2+ uptake through normal or pathological HCs, which are emerging drug targets for a variety of pathological conditions [41][42][43][44][45][46][47][48][49]61,62], using cytosolic GCaMP6s fluorescence intensity variations as a readout. This choice was dictated by different considerations.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, the opening of wild type (wt) Cx HCs is associated with an impressive list of pathological conditions that affect a major proportion of the world population, including ischemia/stroke, Alzheimer's disease, epilepsy, liver fibrosis and cirrhosis, nonalcoholic steatohepatitis, inflammation and others [41][42][43][44][45][46]. Of note, inhibition of Cx HCs in animal models in vivo exerted anticonvulsant effects [47], lowered the degree of liver fibrosis induced by administration of thioacetamide [48] and improved recovery from spinal cord injury [49].…”

Section: Introductionmentioning

confidence: 99%

“…The Ca 2+ uptake assay we developed provides a highly sensitive readout of Cx HC activity, as detailed hereafter. Furthermore, the assay is also suitable to evaluate the efficacy of different HC inhibitors that are attracting a growing attention as potential therapeutics [41][42][43][44][45][46][47][48], including antibodies targeting the extracellular domain of HCs [49,[59][60][61][62].…”

Section: Introductionmentioning

confidence: 99%

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A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels

Nardin

Tettey-Matey

Donati

et al. 2022

IJMS

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Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measurements. Here, we present an all-optical assay based on fluorometric measurements of ionized calcium (Ca2+) uptake with a Ca2+-selective genetically encoded indicator (GCaMP6s) that permits the optical tracking of cytosolic Ca2+ concentration ([Ca2+]cyt) changes with high sensitivity. We exemplify use of the assay in stable pools of HaCaT cells overexpressing human Cx26, Cx46, or the pathological mutant Cx26G45E, under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on). We demonstrate the usefulness of the assay for the characterization of new monoclonal antibodies (mAbs) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same methodology can be extended seamlessly to high-throughput high-content platforms to screen other kinds of inhibitors and/or to probe HCs expressed in primary cells and microtissues.

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“…Primary injury is usually triggered by mechanical damage of the spinal cord, while secondary injury can be caused by the delayed sequences of complex biochemical as well as cell processes ( Feng et al, 2021 ). Altogether, primary and secondary injuries result in irreversible neuronal injury, eventually culminating in unfavorable functional recovery after SCI ( Zhang et al, 2021 ). Hence, to prevent irreversible injury by targeting specific molecules is a feasible therapeutic regimen against SCI.…”

Section: Introductionmentioning

confidence: 99%

Blockage of ERCC6 Alleviates Spinal Cord Injury Through Weakening Apoptosis, Inflammation, Senescence, and Oxidative Stress

Zou

Zhang²,

Zhang³

et al. 2022

Front. Mol. Biosci.

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Objective: Spinal cord injury (SCI) is a devastating disease resulting in lifelong disability, but the molecular mechanism remains unclear. Our study was designed to observe the role of excision repair cross-complementing group 6 (ERCC6) following SCI and to determine the underlying mechanism.Methods: SCI mouse models and LPS-induced microglia cell models were established. ERCC6 expression was blocked by ERCC6-siRNA-carrying lentivirus. Nissl staining was utilized for detecting neuronal damage, and apoptosis was analyzed with TUNEL and Western blotting (apoptotic markers). Immunofluorescence was used for measuring macrophage markers (CD68 and F4/80) and astrocyte and microglia markers (GFAP and Iba-1). Pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) were measured via ELISA. Senescent cells were estimated via SA-β-Gal staining as well as Western blot (senescent markers p21 and p27). Oxidative stress was investigated by detecting the expression of 4-HNE, Nrf2, and Keap1, and intracellular ROS levels.Results: ERCC6 expression was remarkably upregulated both in the spinal cord of SCI mice and LPS-induced microglia cells. ERCC6 deficiency alleviated neuronal damage and apoptosis. Macrophage infiltration and inflammatory response were suppressed by si-ERCC6 treatment. Moreover, ERCC6 blockage ameliorated astrocyte and microglia activation and cell senescence in the damaged spinal cord. Excessive oxidative stress was significantly decreased by ERCC6 knockdown in SCI.Conclusion: Collectively, ERCC6 exerts crucial functions in mediating physiological processes (apoptosis, inflammation, senescence, and oxidative stress), implying that ERCC6 might act as a prospective therapeutic target against SCI.

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Inhibition of astrocyte hemichannel improves recovery from spinal cord injury

Cited by 31 publications

References 65 publications

Connexin hemichannels with prostaglandin release in anabolic function of bone to mechanical loading

Connexin hemichannels with prostaglandin release in anabolic function of bone to mechanical loading

A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels

Blockage of ERCC6 Alleviates Spinal Cord Injury Through Weakening Apoptosis, Inflammation, Senescence, and Oxidative Stress

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