Targeting connexin 43 provides anti-inflammatory effects after intracerebral hemorrhage injury by regulating YAP signaling

Abstract

Background In 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. Methods Hemin 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. Results In 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. Conclusions This 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.

YAP prevents premature senescence of astrocytes and cognitive decline of Alzheimer's disease through regulating CDK6 signaling

“…We also note that one previous study has shown that the nuclear YAP was remarkably decreased in neurons under AD pathology due to the sequestration into cytoplasmic amyloid beta aggregates, which indicates YAP‐dependent neuronal necrosis in AD (Tanaka et al., 2020 ). However, in our studies, we found that YAP was mainly expressed in astrocytes, which was consistent with previous studies (L. Huang et al., 2020 ; Z. Huang et al., 2016 ; Z. Huang et al., 2016 ; C. Xie et al., 2020 ; H. Yu et al., 2020 ). The different observations may result from different samples (human patients versus mouse) or brain regions (occipital lobe of cortex versus hippocampus) or different YAP antibody (sc‐15407, Santa Cruz Biotechnology versus ab205270, Abcam or WH0010413M1, Sigma).…”

Mechanisms of Connexin Mimetic Peptides

“…The first Gap19 iteration, with a molecular weight of around 1100 Da and net charge of +3, was not attached with a specific internalization sequence, rather it was assumed the "KKFK" sequence permitted plasma membrane translocation as previously described [156]. Later, a TAT-bound Gap19 (TAT-19) was shown to have anti-inflammatory and neuroprotective effects [157]. TAT-Gap19 (Cx43), as well as TAT-GAP24 (same L2 region in Cx32), both block hemichannels reducing acetaminophen induced liver injury [108], limiting Ca 2+ activated hemichannel activation, and reducing arrythmias in human heart tissues [158].…”

“…There is conflicting information on whether Cx43 PKC-phosphorylation leads to enhanced or reduced GJ coupling [78,157]. Phosphorylation of Cx43 by PKC is reported primarily at Cx43-S368, though five other sites are also reported to be altered, thus it is possible combinations of these phosphorylation sites may occur [78].…”

Mechanisms of Connexin Regulating Peptides

“…The first Gap19 iteration, with a molecular weight of around 1100 Da and net charge of +3, was not attached with a specific internalization sequence, rather it was assumed the “KKFK” sequence permitted plasma membrane translocation as previously described [ 157 ]. Later, a TAT-bound Gap19 (TAT-19) was shown to have anti-inflammatory and neuroprotective effects [ 158 ]. TAT-Gap19 (Cx43), as well as TAT-GAP24 (same L2 region in Cx32), both block hemichannels reducing acetaminophen-induced liver injury [ 111 ], limiting Ca 2+ activated hemichannel activation, and reducing arrhythmias in human heart tissues [ 159 ].…”