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^{Downregulation of microRNA-9-5p promotes synaptic remodeling in the chronic phase after traumatic brain injury}.

ABSTRACT: The level of microRNA-9-5p (miRNA-9-5p) in brain tissues is significantly changed in the chronic phase after traumatic brain injury (TBI). However, the effect of miRNA-9-5p on brain function after TBI has not been elucidated. In this study, we used a controlled cortical impact (CCI) model to induce TBI in Sprague-Dawley rats. Brain microvascular endothelial cells (BMECs), astrocytes, and neurons were extracted from immature Sprague-Dawley rats and cocultured to reconstruct the neurovascular unit (NVU) in vitro. The results showed that downregulation of miRNA-9-5p in the chronic phase contributed to neurological function recovery by promoting astrocyte proliferation and increasing the release of astrocyte-derived neurotrophic factors around injured brain tissues after TBI. A dual-luciferase reporter assay validated that miRNA-9-5p was a post-transcriptional modulator of thrombospondin 2 (Thbs-2), and downregulation of miRNA-9-5p promoted Thbs-2 expression in astrocytes. Furthermore, we verified that Thbs-2 can promote Notch pathway activation by directly binding to Jagged and Notch. Through in vitro experiments, we found that the expression of synaptic proteins and the number of synaptic bodies were increased in neurons in the NVU, which was constructed using astrocytes pretreated with miRNA-9-5p inhibitor. Moreover, we also found that downregulation of miRNA-9-5p promoted Thbs-2 expression in astrocytes, which activated the Notch/cylindromatosis/transforming growth factor-β-activated kinase 1 pathway in neurons and promoted the expression of synaptic proteins, including post-synaptic density protein 95 and synaptotagmin. Based on these results, miRNA-9-5p may be a new promising prognostic marker and treatment target for TBI.

SUBMITTER: Wu J

PROVIDER: S-EPMC7790831 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:OBJECTIVE:This study investigated the role of microRNAs generated from adipose tissue macrophages (ATMs) during adipose tissue remodeling induced by pharmacological and nutritional stimuli. METHODS:Macrophage-specific Dicer knockout (KO) mice were used to determine the roles of microRNA generated in macrophages in adipose tissue remodeling induced by the β3-adrenergic receptor agonist CL316,243 (CL). RNA-seq was performed to characterize microRNA and mRNA expression profiles in isolated macrophages and PDGFRα+ adipocyte stem cells (ASCs). The role of miR-10a-5p was further investigated in cell culture, and in adipose tissue remodeling induced by CL treatment and high fat feeding. RESULTS:Macrophage-specific deletion of Dicer elevated pro-inflammatory gene expression and prevented CL-induced de novo beige adipogenesis in gonadal white adipose tissue (gWAT). Co-culture of ASCs with ATMs of wild type mice promoted brown adipocyte gene expression upon differentiation, but co-culture with ATMs of Dicer KO mice did not. Bioinformatic analysis of RNA expression profiles identified miR-10a-5p as a potential regulator of inflammation and differentiation in ATMs and ASCs, respectively. CL treatment increased levels of miR-10a-5p in ATMs and ASCs in gWAT. Interestingly, CL treatment elevated levels of pre-mir-10a in ATMs but not in ASCs, suggesting possible transfer from ATMs to ASCs. Elevating miR-10a-5p levels inhibited proinflammatory gene expression in cultured RAW 264.7 macrophages and promoted the differentiation of C3H10T1/2 cells into brown adipocytes. Furthermore, treatment with a miR-10a-5p mimic in vivo rescued CL-induced beige adipogenesis in Dicer KO mice. High fat feeding reduced miR-10a-5p levels in ATMs of gWAT, and treatment of mice with a miR-10a-5p mimic suppressed pro-inflammatory responses, promoted the appearance of new white adipocytes in gWAT, and improved systemic glucose tolerance. CONCLUSIONS:These results demonstrate an important role of macrophage-generated microRNAs in adipogenic niches and identify miR-10a-5p as a key regulator that reduces adipose tissue inflammation and promotes therapeutic adipogenesis.

Project description:IntroductionAxonal injury results in long-term neurological deficits in traumatic brain injury (TBI) patients. Apolipoprotein E (ApoE) has been reported to activate intracellular adaptor protein Disabled-1 (Dab1) phosphorylation via its interaction with ApoE receptors. The Dab1 pathway acts as a regulator of axonal outgrowth and growth cone formation in the brain.AimsWe hypothesized that ApoE may alleviate axonal injury and regulate axonal regeneration via the Dab1 pathway after TBI.ResultsIn this study, we established a model of controlled cortical impact (CCI) to mimic TBI in vivo. Using diffusion tensor imaging to detect white matter integrity, we demonstrated that APOE-deficient mice exhibited lower fractional anisotropy (FA) values than APOE+/+ mice at 28 days after injury. The expression levels of axonal regeneration and synapse plasticity biomarkers, including growth-associated protein 43 (GAP43), postsynaptic density protein 95 (PSD-95), and synaptophysin, were also lower in APOE-deficient mice. In contrast, APOE deficiency exerted no effects on the levels of myelin basic protein (MBP) expression, oligodendrocyte number, or oligodendrocyte precursor cell number. Neurological severity score (NSS) and behavioral measurements in the rotarod, Morris water maze, and Y maze tests revealed that APOE deficiency caused worse neurological deficits in CCI mice. Furthermore, Dab1 activation downregulation by the ApoE receptor inhibitor receptor-associated protein (RAP) or Dab1 shRNA lentivirus attenuated the beneficial effects of ApoE on FA values, GAP43, PSD-95, and synaptophysin expression, and neurological function tests. Additionally, the effects of ApoE on axonal regeneration were further validated in vitro. In a mechanical scratch injury model of primary cultured neurons, recombinant ApoE protein treatment enhanced axonal outgrowth and growth cone formation in injured neurons; however, these effects were attenuated by Dab1 shRNA, consistent with the in vivo results.ConclusionCollectively, these data suggest that ApoE promotes axonal regeneration partially through the Dab1 pathway, thereby contributing to functional recovery following TBI.

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^{Downregulation of microRNA-9-5p promotes synaptic remodeling in the chronic phase after traumatic brain injury}.

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Downregulation of microRNA-9-5p promotes synaptic remodeling in the chronic phase after traumatic brain injury.

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OmicsDI is part of the ELIXIR infrastructure

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^{Downregulation of microRNA-9-5p promotes synaptic remodeling in the chronic phase after traumatic brain injury}.