Downregulation of Src-kinase and glutamate-receptor phosphorylation after traumatic brain injury.
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ABSTRACT:
SUBMITTER: Park Y
PROVIDER: S-EPMC3790935 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Both brain native inflammatory cells and infiltrated peripheral white blood cells (WBCs) are primary participants in the brain inflammatory damage post-TBI. Metabotropic glutamate receptor 5 (mGluR5) has been reported to regulate microglias and astrocytes to affect inflammation after TBI, but its effect on modulating infiltrated peripheral WBCs remains unclear. In a mouse moderate TBI model, we found that mGluR5 knockout (KO) significantly reduced neutrophil infiltration and inflammatory cytokine expression in the brain at 24 hours post TBI, which was accompanied by improved neurological dysfunction. Further investigation indicated that mGluR5 KO reduced the permeability of blood-brain barrier (BBB), the entrance for neutrophils to enter brain, and markedly decreased the mRNA levels of neutrophil-associated chemokines in brain tissue, including CXCL1, CXCL2, CCL2, CCL4 and CCL5. Using brain microvascular endothelial cells (BMECs), neutrophils and a BBB model in vitro, we confirmed the inhibitory effect of mGluR5 deficiency on neutrophil infiltration and demonstrated that blockade of protein kinase C (PKC) signaling was involved in it. These results provide insight into the role of mGluR5 in the regulation of inflammation in the acute phase of TBI, which may provide novel clues for TBI therapy.
| S-EPMC5577182 | biostudies-literature
Project description:MicroRNA-491-5p (miR-491-5p) plays an important role in regulating cell proliferation and migration; however, the effect of miR-491-5p on neovascularization after traumatic brain injury remains poorly understood. In this study, a controlled cortical injury model in C57BL/6 mice and an oxygen-glucose deprivation model in microvascular endothelial cells derived from mouse brain were established to simulate traumatic brain injury in vivo and in vitro, respectively. In the in vivo model, quantitative real-time-polymerase chain reaction results showed that the expression of miR-491-5p increased or decreased following the intracerebroventricular injection of an miR-491-5p agomir or antagomir, respectively, and the expression of miR-491-5p decreased slightly after traumatic brain injury. To detect the neuroprotective effects of miR-491-p, neurological severity scores, Morris water maze test, laser speckle techniques, and immunofluorescence staining were assessed, and the results revealed that miR-491-5p downregulation alleviated neurological dysfunction, promoted the recovery of regional cerebral blood flow, increased the number of lectin-stained microvessels, and increased the survival of neurons after traumatic brain injury. During the in vitro experiments, the potential mechanism of miR-491-5p on neovascularization was explored through quantitative real-time-polymerase chain reaction, which showed that miR-491-5p expression increased or decreased in brain microvascular endothelial cells after transfection with an miR-491-5p mimic or inhibitor, respectively. Dual-luciferase reporter and western blot assays verified that metallothionein-2 was a target gene for miR-491-5p. Cell counting kit 8 (CCK-8) assay, flow cytometry, and 2?,7?-dichlorofluorescein diacetate (DCFH-DA) assay results confirmed that the downregulation of miR-491-5p increased brain microvascular endothelial cell viability, reduced cell apoptosis, and alleviated oxidative stress under oxygen-glucose deprivation conditions. Cell scratch assay, Transwell assay, tube formation assay, and western blot assay results demonstrated that miR-491-5p downregulation promoted the migration, proliferation, and tube formation of brain microvascular endothelial cells through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor pathway. These findings confirmed that miR-491-5p downregulation promotes neovascularization, restores cerebral blood flow, and improves the recovery of neurological function after traumatic brain injury. The mechanism may be mediated through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway and the alleviation of oxidative stress. All procedures were approved by Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, China (approval No. 2020-304) on June 22, 2020.
| S-EPMC8504397 | biostudies-literature
Project description:Traumatic brain injury (TBI) is considered as the most common cause of disability and death, and therefore an effective intervention of cascade pathology of secondary brain injury promptly can be a potential therapeutic direction for TBI prognosis. Further study of the physiological mechanism of TBI is urgent and important. Phosphoglycerate mutase 5 (Pgam5), a mitochondrial protein, mediate mitochondrial homeostasis, cellular senescence, and necroptosis. This study evaluated the effects of Pgam5 on neurological deficits and neuroinflammation of controlled cortical impact-induced TBI mouse model in vivo and LPS + ATP-induced microglia model in vitro. Pgam5 was overexpressed post-TBI. Pgam5 depletion reduced pyroptosis-related molecules and improved microglia activation, neuron damage, tissue lesion, and neurological dysfunctions in TBI mice. RNA-seq analysis and molecular biology experiments demonstrated that Pgam5 might regulate inflammatory responses by affecting the post-translational modification and protein expression of related genes, including Nlrp3, caspase1, Gsdmd, and Il-1β. In microglia, Pgam5-sh abrogated LPS + ATP-induced Il-1β secretion through Asc oligomerization-mediated caspase-1 activation, which was independent of Rip3. The data demonstrate the critical role Pgam5 plays in nerve injury in the progression of TBI, which regulates Asc polymerization and subsequently caspase1 activation, and thus reveals a fundamental mechanism linking microglial inflammasome activation to Asc/caspase1-generated Il-1β-mediated neuroinflammation. Thus, our data indicate Pgam5 worsens physiological and neurological outcomes post-TBI, which may be a potential therapeutic target to improve neuroinflammation after TBI.
| S-EPMC8511105 | biostudies-literature
Project description:Blood-brain barrier (BBB) disruption is a serious pathological consequence of traumatic brain injury (TBI), for which there are limited therapeutic strategies. Tissue inhibitor of metalloproteinase-2 (TIMP2), a molecule with dual functions of inhibiting MMP activity and displaying cytokine-like activity through receptor binding, has been reported to inhibit VEGF-induced vascular hyperpermeability. Here, we investigate the ability of TIMP2 to ameliorate BBB disruption in TBI and the underlying molecular mechanisms. Both TIMP2 and AlaTIMP2, a TIMP2 mutant without MMP-inhibiting activity, attenuated neurological deficits and BBB leakage in TBI mice; they also inhibited junctional protein degradation and translocation to reduce paracellular permeability in human brain microvascular endothelial cells (ECs) exposed to hypoxic plus inflammatory insult. Mechanistic studies revealed that TIMP2 interacted with α3β1 integrin on ECs, inhibiting Src activation-dependent VE-cadherin phosphorylation, VE-cadherin/catenin complex destabilization, and subsequent VE-cadherin internalization. Notably, localization of VE-cadherin on the membrane was critical for TIMP2-mediated EC barrier integrity. Furthermore, TIMP2-mediated increased membrane localization of VE-cadherin enhanced the level of active Rac1, thereby inhibiting stress fiber formation. All together, our studies have identified an MMP-independent mechanism by which TIMP2 regulates EC barrier integrity after TBI. TIMP2 may be a therapeutic agent for TBI and other neurological disorders involving BBB breakdown.
| S-EPMC10849766 | biostudies-literature
Project description:We hypothesize that the primary mechanism for removal of glutamate from the extracellular space is altered after traumatic brain injury (TBI). To evaluate this hypothesis, we initiated TBI in adult male rats using a 2.0 atm lateral fluid percussion injury (LFPI) model. In the ipsilateral cortex and hippocampus, we found no differences in expression of the primary glutamate transporter in the brain (GLT-1) 24 h after TBI. In contrast, we found a decrease in glutamate uptake in the cortex, but not the hippocampus, 24 h after injury. Because glutamate uptake is potently regulated by protein kinases, we assessed global serine-threonine protein kinase activity using a kinome array platform. Twenty-five kinome array peptide substrates were differentially phoshorylated between LFPI and controls in the cortex, whereas 19 peptide substrates were differentially phosphorylated in the hippocampus (fold change ≥ ± 1.15). We identified several kinases as likely to be involved in acute TBI, including protein kinase B (Akt) and protein kinase C (PKC), which are well-characterized modulators of GLT-1. Exploratory studies using an inhibitor of Akt suggest selective activation of kinases in LFPI versus controls. Ingenuity pathway analyses of implicated kinases from our network model found apoptosis and cell death pathways as top functions in acute LFPI. Taken together, our data suggest diminished activity of glutamate transporters in the prefrontal cortex, with no changes in protein expression of the primary glutamate transporter GLT-1, and global alterations in signaling networks that include serine-threonine kinases that are known modulators of glutamate transport activity.
| S-EPMC5198172 | biostudies-literature
Project description:Traumatic brain injury (TBI) induces a series of epigenetic changes in brain tissue, among which histone modifications are associated with the deterioration of TBI. In this study, we explored the role of histone H3 modifications in a weight-drop model of TBI in rats. Screening for various histone modifications, immunoblot analyses revealed that the phosphorylation of histone H3 serine 10 (p-H3S10) was significantly upregulated after TBI in the brain tissue surrounding the injury site. A similar posttraumatic regulation was observed for phosphorylated extracellular signal-regulated kinase (p-ERK), which is known to phosphorylate H3S10. In support of the hypothesis that ERK-mediated phosphorylation of H3S10 contributes to TBI pathogenesis, double immunofluorescence staining of brain sections showed high levels and colocalization of p-H3S10 and p-ERK predominantly in neurons surrounding the injury site. To test the hypothesis that inhibition of ERK-H3S10 signaling ameliorates TBI pathogenesis, the mitogen-activated protein kinase-extracellular signal-regulated kinase kinase (MEK) 1/2 inhibitor U0126, which inhibits ERK phosphorylation, was administered into the right lateral ventricle of TBI male and female rats via intracerebroventricular cannulation for 7 days post trauma. U0126 administration indeed prevented H3S10 phosphorylation and improved motor function recovery and cognitive function compared to vehicle treatment. In agreement with our findings in the rat model of TBI, immunoblot and double immunofluorescence analyses of brain tissue specimens from patients with TBI demonstrated high levels and colocalization of p-H3S10 and p-ERK as compared to control specimens from non-injured individuals. In conclusion, our findings indicate that phosphorylation-dependent activation of ERK-H3S10 signaling participates in the pathogenesis of TBI and can be targeted by pharmacological approaches.
| S-EPMC9557206 | biostudies-literature
Project description:Traumatic brain injury-induced acute lung injury (TBI-ALI) is a serious complication after brain injury for which predictive factors are lacking. In this study, we found significantly elevated blood glutamate concentrations in patients with TBI or multiple peripheral trauma (MPT), and patients with more severe injuries showed higher blood glutamate concentrations and longer durations of elevated levels. Although the increase in amplitude was similar between the two groups, the duration was longer in the patients with TBI. There were no significant differences in blood glutamate concentrations in the patients with MPT with regard to ALI status, but the blood glutamate levels were significantly higher in the patients with TBI-ALI than in those without ALI. Moreover, compared to patients without ALI, patients with TBI showed a clearly enhanced inflammatory response that was closely correlated with the blood glutamate levels. The blood glutamate concentration was also found to be a risk factor (adjusted odds ratio, 2.229; 95% CI, 1.082-2.634) and was a better predictor of TBI-ALI than the Glasgow Coma Scale (GCS) score. These results indicated that dramatically increased blood glutamate concentrations were closely related to the occurrence of TBI-ALI and could be used as a predictive marker for "at-risk" patients.
| S-EPMC5511144 | biostudies-literature
Project description:BackgroundComplex changes in the brain microenvironment following traumatic brain injury (TBI) can cause neurological impairments for which there are few efficacious therapeutic interventions. The reactivity of astrocytes is one of the keys to microenvironmental changes, such as neuroinflammation, but its role and the molecular mechanisms that underpin it remain unclear.MethodsMale C57BL/6J mice were subjected to the controlled cortical impact (CCI) to develop a TBI model. The specific ligand of AXL receptor tyrosine kinase (AXL), recombinant mouse growth arrest-specific 6 (rmGas6) was intracerebroventricularly administered, and selective AXL antagonist R428 was intraperitoneally applied at 30 min post-modeling separately. Post-TBI assessments included neurobehavioral assessments, transmission electron microscopy, immunohistochemistry, and western blotting. Real-time polymerase chain reaction (RT-PCR), siRNA transfection, and flow cytometry were performed for mechanism assessments in primary cultured astrocytes.ResultsAXL is upregulated mainly in astrocytes after TBI and promotes astrocytes switching to a phenotype that exhibits the capability of ingesting degenerated neurons or debris. As a result, this astrocytic transformation promotes the limitation of neuroinflammation and recovery of neurological dysfunction. Pharmacological inhibition of AXL in astrocytes significantly decreased astrocytic phagocytosis both in vivo and in primary astrocyte cultures, in contrast to the effect of treatment with the rmGas6. AXL activates the signal transducer and activator of the transcription 1 (STAT1) pathway thereby further upregulating ATP-binding cassette transporter 1 (ABCA1). Moreover, the supernatant from GAS6-depleted BV2 cells induced limited enhancement of astrocytic phagocytosis in vitro.ConclusionOur work establishes the role of AXL in the transformation of astrocytes to a phagocytic phenotype via the AXL/STAT1/ABCA1 pathway which contributes to the separation of healthy brain tissue from injury-induced cell debris, further ameliorating neuroinflammation and neurological impairments after TBI. Collectively, our findings provide a potential therapeutic target for TBI.
| S-EPMC8264993 | biostudies-literature
Project description:Remodeling of the remnant neuronal network after brain injury possibly mediates spontaneous functional recovery; however, the mechanisms inducing axonal remodeling during spontaneous recovery remain unclear. Here, we show that altered ?-aminobutyric acid (GABA) signaling is crucial for axonal remodeling of the contralesional cortex after traumatic brain injury. After injury to the sensorimotor cortex in mice, we found a significant decrease in the expression of GABA(A)R-?1 subunits in the intact sensorimotor cortex for 2 weeks. Motor functions, assessed by grid walk and cylinder tests, spontaneously improved in 4 weeks after the injury to the sensorimotor cortex. With motor recovery, corticospinal tract (CST) axons from the contralesional cortex sprouted into the denervated side of the cervical spinal cord at 2 and 4 weeks after the injury. To determine the functional implications of the changes in the expression of GABA(A)R-?1 subunits, we infused muscimol, a GABA R agonist, into the contralesional cortex for a week after the injury. Compared with the vehicle-treated mice, we noted significantly inhibited recovery in the muscimol-treated mice. Further, muscimol infusion greatly suppressed the axonal sprouting into the denervated side of the cervical spinal cord. In conclusion, recovery of motor function and axonal remodeling of the CST following cortical injury requires suppressed GABA(A)R subunit expression and decreased GABAergic signaling.
| S-EPMC3101813 | biostudies-other
Project description:The bone marrow-derived cell (BMDC)-associated inflammatory response plays a key role in the development of acute lung injury (ALI). Activation of adenosine A2A receptor (A2AR) is generally considered to be antiinflammatory, inhibiting BMDC activities to protect against ALI. However, in the present study, we found that in a mouse model of neurogenic ALI induced by severe traumatic brain injury (TBI), BMDC A2AR exerted a proinflammatory effect, aggravating lung damage. This is in contrast to the antiinflammatory effect observed in the mouse oleic acid-induced ALI model (a nonneurogenic ALI model.) Moreover, the A2AR agonist CGS21680 aggravated, whereas the antagonist ZM241385 attenuated, the severe TBI-induced lung inflammatory damage in mice. Further investigation of white blood cells isolated from patients or mouse TBI models and of cultured human or mouse neutrophils demonstrated that elevated plasma glutamate after severe TBI induced interaction between A2AR and the metabotropic glutamate receptor 5 (mGluR5) to increase phospholipase C-protein kinase C signaling, which mediated the proinflammatory effect of A2AR. These results are in striking contrast to the well-known antiinflammatory and protective role of A2AR in nonneurogenic ALI and indicate different therapeutic strategies should be used for nonneurogenic and neurogenic ALI treatment when targeting A2AR.
| S-EPMC3620361 | biostudies-literature