Astrocyte-derived exosome-transported GJA1-20k attenuates traumatic brain injury in rats.
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ABSTRACT:
SUBMITTER: Feng J
PROVIDER: S-EPMC10150843 | biostudies-literature | 2023 Apr
REPOSITORIES: biostudies-literature
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Astrocyte-derived exosome-transported GJA1-20k attenuates traumatic brain injury in rats.
Feng Jiugeng J Zou Shufeng S Yang Xuanyong X Wang Zhigang Z Jiang Bixia B Hou Tingting T Duan Jian J Hong Tao T Chen Wei W
Chinese medical journal 20230405 7
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Project description:Cardiac hypertrophy (CH) is characterized by an increase in cardiomyocyte size, and is the most common cause of cardiac-related sudden death. A decrease in gap junction (GJ) coupling and mitochondrial dysfunction are important features of CH, but the mechanisms of decreased coupling and energy impairment are poorly understood. It has been reported that GJA1-20k has a strong tropism for mitochondria and is required for the trafficking of connexin 43 (Cx43) to cell-cell borders. In this study, we investigated the effects of GJA1-20k on Cx43 GJ coupling and mitochondrial function in the pathogenesis of CH. We performed hematoxylin-eosin (HE) and Masson staining, and observed significant CH in 18-week-old male spontaneously hypertensive rats (SHRs) compared to age-matched normotensive Wistar-Kyoto (WKY) rats. In cardiomyocytes from SHRs, the levels of Cx43 at the intercalated disc (ID) and the expression of GJA1-20k were significantly reduced, whereas JAK-STAT signaling was activated. Furthermore, the SHR rats displayed suppressed mitochondrial GJA1-20k and mitochondrial biogenesis. Administration of valsartan (10 mg· [Formula: see text] d-1, i.g., for 8 weeks) prevented all of these changes. In neonatal rat cardiomyocytes (NRCMs), overexpression of GJA1-20k attenuated Ang II-induced cardiomyocyte hypertrophy and caused elevated levels of GJ coupling at the cell-cell borders. Pretreatment of NRCMs with the Jak2 inhibitor AG490 (10 µM) blocked Ang II-induced reduction in GJA1-20k expression and Cx43 gap junction formation; knockdown of Jak2 in NRCMs significantly lessened Ang II-induced cardiomyocyte hypertrophy and normalized GJA1-20k expression and Cx43 gap junction formation. Overexpression of GJA1-20k improved mitochondrial membrane potential and respiration and lowered ROS production in Ang II-induced cardiomyocyte hypertrophy. These results demonstrate the importance of GJA1-20k in regulating gap junction formation and mitochondrial function in Ang II-induced cardiomyocyte hypertrophy, thus providing a novel therapeutic strategy for patients with cardiomyocyte hypertrophy.
| S-EPMC8115281 | biostudies-literature
Project description:Connexin 43 (Cx43, encoded by GJA1) is a cell-cell communication gap junction protein expressed in all organ systems. It was recently found that GJA1 mRNA undergoes alternative translation to generate N-terminal truncated isoforms, of which GJA1-20k is the most abundant. Here we report a surprising finding that, unlike full length GJA1-43k, GJA1-20k has a strong tropism for mitochondria. Exploring function, we found that GJA1-20k appears to be an organelle chaperone and that overexpression of GJA1-20k is sufficient to rescue mitochondrial localization to the cell periphery upon exposure to hydrogen peroxide, which effectively limits the network fragmentation that occurs with oxidative stress. By high-resolution fluorescent imaging and electron microscopy, we determined that GJA1-20k is enriched at the interface between mitochondria and microtubules, appearing to load organelles for transport. Mutagenesis experiments revealed that although the microtubule-binding domain (MTBD) in GJA1-20k is not necessary for protein localization to mitochondria, the MTBD is essential for GJA1-20k to facilitate mitochondrial transport and maintain mitochondrial localization at the periphery. These results reveal an unexpected role for the alternatively translated isoform of the Cx43 gap junction protein, GJA1-20k, which is to facilitate microtubule-based mitochondrial transport and to maintain mitochondrial network integrity during cellular stress.
| S-EPMC5682029 | biostudies-literature
Project description:Connexin 43 (Cx43), a product of the GJA1 gene, is a gap junction protein facilitating intercellular communication between cardiomyocytes. Cx43 protects the heart from ischemic injury by mechanisms that are not well understood. GJA1 mRNA can undergo alternative translation, generating smaller isoforms in the heart, with GJA1-20k being the most abundant. Here, we report that ischemic and ischemia/reperfusion (I/R) injuries upregulate endogenous GJA1-20k protein in the heart, which targets to cardiac mitochondria and associates with the outer mitochondrial membrane. Exploring the functional consequence of increased GJA1-20k, we found that AAV9-mediated gene transfer of GJA1-20k in mouse hearts increases mitochondrial biogenesis while reducing mitochondrial membrane potential, respiration, and ROS production. By doing so, GJA1-20k promotes a protective mitochondrial phenotype, as seen with ischemic preconditioning (IPC), which also increases endogenous GJA1-20k in heart lysates and mitochondrial fractions. As a result, AAV9-GJA1-20k pretreatment reduces myocardial infarct size in mouse hearts subjected to in vivo ischemic injury or ex vivo I/R injury, similar to an IPC-induced cardioprotective effect. In conclusion, GJA1-20k is an endogenous stress response protein that induces mitochondrial biogenesis and metabolic hibernation, preconditioning the heart against I/R insults. Introduction of exogenous GJA1-20k is a putative therapeutic strategy for patients undergoing anticipated ischemic injury.
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Project description:The Connexin43 gap junction gene GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.
| S-EPMC8492060 | biostudies-literature
Project description:RationaleDelivery of Cx43 (connexin 43) to the intercalated disc is a continuous and rapid process critical for intercellular coupling. By a pathway of targeted delivery involving microtubule highways, vesicles of Cx43 hemichannels are efficiently trafficked to adherens junctions at intercalated discs. It has also been identified that actin provides rest stops for Cx43 forward trafficking and that Cx43 has a 20 kDa internally translated small C terminus isoform, GJA1-20k (Gap Junction Protein Alpha 1- 20 kDa), which is required for full-length Cx43 trafficking, but by an unknown mechanism.ObjectiveWe explored the mechanism by which the GJA1-20k isoform is required for full-length Cx43 forward trafficking to intercalated discs.Methods and resultsUsing an in vivo Adeno-associated virus serotype 9-mediated gene transfer system, we confirmed in whole animal that GJA1-20k markedly increases endogenous myocardial Cx43 gap junction plaque size at the intercalated discs. In micropatterned cell pairing systems, we found that exogenous GJA1-20k expression stabilizes filamentous actin without affecting actin protein expression and that GJA1-20k complexes with both actin and tubulin. We also found that filamentous actin regulates microtubule organization as inhibition of actin polymerization with a low dose of latrunculin A disrupts the targeting of microtubules to cell-cell junctions. GJA1-20k protects actin filament from latrunculin A disruption, preserving microtubule trajectory to the cell-cell border. For therapeutic implications, we found that prior in vivo Adeno-associated virus serotype 9-mediated gene delivery of GJA1-20k to the heart protects Cx43 localization to the intercalated discs against acute ischemic injury.ConclusionsThe internally translated GJA1-20k isoform stabilizes actin filaments, which guides growth trajectories of the Cx43 microtubule trafficking machinery, increasing delivery of Cx43 hemichannels to cardiac intercalated discs. Exogenous GJA1-20k helps to maintain cell-cell coupling in instances of anticipated myocardial ischemia.
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Project description:Traumatic brain injury (TBI) is a major global burden of health. As an accepted inflammatory mediator, high mobility group box 1 (HMGB1) is found to be effective in facilitating neurogenesis and axonal regeneration. SH3RF2 (also known as POSHER), an E3 ligase SH3 domain-containing ring finger 2, belongs to the SH3RF family of proteins. Here, we aimed to investigate the role of redox states of HMGB1 on neurite outgrowth and regeneration both in vitro and in vivo. In this study, distinct recombinant HMGB1 redox isoforms were used. Sequencing for RNA-seq and data analysis were performed to find the potential downstream target of nonoxid-HMGB1 (3S-HMGB1). Protein changes and distribution of SH3RF2 were evaluated by western blot assays and immunofluorescence. Lentivirus and adeno-associated virus were used to regulate the expression of genes. Nonoxid-HMGB1-enriched exosomes were constructed and used to treat TBI rats. Neurological function was evaluated by OF test and NOR test. Results demonstrated that nonoxid-HMGB1 and fr-HMGB1, but not ds-HMGB1, promoted neurite outgrowth and axon elongation. RNA-seq and western blot assay indicated a significant increase of SH3RF2 in neurons after treated with nonoxid-HMGB1 or fr-HMGB1. Notably, the beneficial effects of nonoxid-HMGB1 were attenuated by downregulation of SH3RF2. Furthermore, nonoxid-HMGB1 ameliorated cognitive impairment in rats post-TBI via SH3RF2. Altogether, our experimental results suggest that one of the promoting neurite outgrowth and regeneration mechanisms of nonoxid-HMGB1 is mediated through the upregulated expression of SH3RF2. Nonoxid-HMGB1 is an attractive therapeutic candidate for the treatment of TBI.
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Project description:In response to traumatic brain injury, a subpopulation of cortical astrocytes is activated, resulting in acquisition of stem cell properties, known as reactive astrocytes-derived progenitor cells (Rad-PCs). However, the underlying mechanisms remain largely unknown during this process. In this study, we examined the role of N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, in Rad-PCs after cortical stab injury in adult rats. Immunohistochemical analysis showed that in the cerebral cortex of normal adult rats, NDRG2 was exclusively expressed in astrocytes. After liu cortical injury, the expression of NDRG2 was significantly elevated around the wound and most cells expressing NDRG2 also expressed GFAP, a reactive astrocyte marker. Importantly, NDRG2-expressing cells were co-labeled with Nestin, a marker for neural stem cells, some of which also expressed cell proliferation marker Ki67. Overexpression of NDRG2 further increased the number of NDRG2/Nestin double-labeling cells around the lesion. In contrast, shRNA knockdown of NDRG2 decreased the number of NDRG2+/Nestin+ cells. Intracerebroventricular administration of stab-injured rats with a Notch antagonist, DAPT, led to a significant decrease in Nestin+/NDRG2+ cells around the injured boundary, but did not affect NDRG2+ cells. Moreover, overexpression or knockdown of NDRG2 led to up- and down-regulation of the expression of Notch intracellular domain NICD and Notch target gene Hes1, respectively. Taken together, these results suggest that NDRG2 may play a role in controlling the formation of Rad-PCs in the cerebral cortex of adult rats following traumatic injury, and that Notch signaling pathway plays a key role in this process.
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Project description:Connexin-43 (Cx43) gap junctions provide intercellular coupling, which ensures rapid action potential propagation and synchronized heart contraction. Alterations in Cx43 localization and reductions in gap junction coupling occur in failing hearts, contributing to ventricular arrhythmias and sudden cardiac death. Recent reports have found that an internally translated Cx43 isoform, GJA1-20k, is an auxiliary subunit for the trafficking of Cx43 in heterologous expression systems. Here, we have created a mouse model by using CRISPR technology to mutate a single internal translation initiation site in Cx43 (M213L mutation), which generates full-length Cx43, but not GJA1-20k. We found that GJA1M213L/M213L mice had severely abnormal electrocardiograms despite preserved contractile function, reduced total Cx43, and reduced gap junctions, and they died suddenly at 2 to 4 weeks of age. Heterozygous GJA1M213L/WT mice survived to adulthood with increased ventricular ectopy. Biochemical experiments indicated that cytoplasmic Cx43 had a half-life that was 50% shorter than membrane-associated Cx43. Without GJA1-20k, poorly trafficked Cx43 was degraded. The data support that GJA1-20k, an endogenous entity translated independently of Cx43, is critical for Cx43 gap junction trafficking, maintenance of Cx43 protein, and normal electrical function of the mammalian heart.
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Project description:Mitochondrial dysfunction is a hallmark of secondary neuroinflammatory responses and neuronal death in spinal cord injury (SCI). Even though mitochondria-based therapy is an attractive therapeutic option for SCI, the efficacy of transplantation of allogeneic mitochondria in the treatment of SCI remains unclear. Herein, we determined the therapeutic effects of mitochondrial transplantation in the traumatic SCI rats. Compressive SCI was induced by applying an aneurysm clip on the T10 spinal cord of rats. A 100-μg bolus of soleus-derived allogeneic mitochondria labeled with fluorescent tracker was transplanted into the injured spinal cords. The results showed that the transplanted mitochondria were detectable in the injured spinal cord up to 28 days after treatment. The rats which received mitochondrial transplantation exhibited better recovery of locomotor and sensory functions than those who did not. Both the expression of dynamin-related protein 1 and severity of demyelination in the injured cord were reduced in the mitochondrial transplanted groups. Mitochondrial transplantation also alleviated SCI-induced cellular apoptosis and inflammation responses. These findings suggest that transplantation of allogeneic mitochondria at the early stage of SCI reduces mitochondrial fragmentation, neuroapoptosis, neuroinflammation, and generation of oxidative stress, thus leading to improved functional recovery following traumatic SCI.
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