Small Interference RNA Targeting Connexin-43 Improves Motor Function and Limits Astrogliosis After Juvenile Traumatic Brain Injury.
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ABSTRACT:
SUBMITTER: Ichkova A
PROVIDER: S-EPMC6566476 | biostudies-literature | 2019 Jan-Dec
REPOSITORIES: biostudies-literature
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Small Interference RNA Targeting Connexin-43 Improves Motor Function and Limits Astrogliosis After Juvenile Traumatic Brain Injury.
Ichkova Aleksandra A Fukuda Andrew M AM Nishiyama Nina N Paris Germaine G Obenaus Andre A Badaut Jerome J
ASN neuro 20190101
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Project description:BackgroundIn the central nervous system (CNS), connexin 43 (Cx43) is mainly expressed in astrocytes and regulates astrocytic network homeostasis. Similar to Cx43 overexpression, abnormal excessive opening of Cx43 hemichannels (Cx43Hcs) on reactive astrocytes aggravates the inflammatory response and cell death in CNS pathologies. However, the role of excessive Cx43Hc opening in intracerebral hemorrhage (ICH) injury is not clear.MethodsHemin stimulation in primary cells and collagenase IV injection in C57BL/6J (B6) mice were used as ICH models in vitro and in vivo. After ICH injury, the Cx43 mimetic peptide Gap19 was used for treatment. Ethidium bromide (EtBr) uptake assays were used to measure the opening of Cx43Hcs. Western blotting and immunofluorescence were used to measure protein expression. qRT-PCR and ELISA were used to determine the levels of cytokines. Coimmunoprecipitation (Co-IP) and the Duolink in situ proximity ligation assay (PLA) were applied to measure the association between proteins.ResultsIn this study, Cx43 expression upregulation and excessive Cx43Hc opening was observed in mice after ICH injury. Delayed treatment with Gap19 significantly alleviated hematoma volume and neurological deficits after ICH injury. In addition, Gap19 decreased inflammatory cytokine levels in the tissue surrounding the hematoma and decreased reactive astrogliosis after ICH injury in vitro and in vivo. Intriguingly, Cx43 transcriptional activity and expression in astrocytes were significantly increased after hemin stimulation in culture. However, Gap19 treatment downregulated astrocytic Cx43 expression through the ubiquitin-proteasome pathway without affecting Cx43 transcription. Additionally, our data showed that Gap19 increased Yes-associated protein (YAP) nuclear translocation. This subsequently upregulated SOCS1 and SOCS3 expression and then inhibited the TLR4-NFκB and JAK2-STAT3 pathways in hemin-stimulated astrocytes. Finally, the YAP inhibitor, verteporfin (VP), reversed the anti-inflammatory effect of Gap19 in vitro and almost completely blocked its protective effects in vivo after ICH injury.ConclusionsThis study provides new insight into potential treatment strategies for ICH injury involving astroglial Cx43 and Cx43Hcs. Suppression of abnormal astroglial Cx43 expression and Cx43Hc opening by Gap19 has anti-inflammatory and neuroprotective effects after ICH injury.
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Project description:BackgroundConnexin 43 (Cx43), the most widely expressed gap junction protein, is associated with a number of physiological and pathological conditions. Many functions of Cx43 have been shown to be independent of gap junction formation and only require the expression of Cx43 C-terminal fragments. Recent evidence demonstrated that naturally occurring C-terminal isoforms can be generated via internal translation.FindingsHere, we confirm that C-terminal domains of Cx43, particularly the major 20-kDa isoform, can be independently generated and regulated by internal translation of the same single GJA1 gene transcript that encodes full-length Cx43. Through direct RNA transfection experiments, we provide evidence that internal translation is not due to a bona fide cap-independent IRES-mediated mechanism, as upstream ribosomal scanning or translation is required. In addition to the mTOR pathway, we show for the first time, using both inhibitors and cells from knockout mice, that the Mnk1/2 pathway regulates the translation of the main 20-kDa isoform.ConclusionsInternal translation of the Cx43 transcript occurs but is not cap-independent and requires translation upstream of the internal start codon. In addition to the PI3K/AKT/mTOR pathway, the major 20-kDa isoform is regulated by the Mnk1/2 pathway. Our results have major implications for past and future studies describing gap junction-independent functions of Cx43 in cancer and other pathological conditions. This study provides further clues to the signalling pathways that regulate internal mRNA translation, an emerging mechanism that allows for increased protein diversity and functional complexity from a single mRNA transcript.
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