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TMEM175 mediates Lysosomal function and participates in neuronal injury induced by cerebral ischemia-reperfusion.

ABSTRACT: As the main organelles for the clearance of damaged proteins and damaged organelles, the function of lysosomes is crucial for maintaining the intracellular homeostasis of long-lived neurons. A stable acidic environment is essential for lysosomes to perform their functions. TMEM175 has been identified as a new K+ channel that is responsible for regulating lysosomal membrane potential and pH stability in neurons. This study aimed to understand the role of TMEM175 in lysosomal function of neurons and neuronal injury following cerebral ischemia-reperfusion (I/R). A middle-cerebral-artery occlusion/reperfusion (MCAO/R) model was established in adult male Sprague-Dawley rats in vivo, and cultured neurons were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic ischemia-reperfusion (I/R) injury in vitro. We found that the protein level of TMEM175 decreased after cerebral I/R injury and that TMEM175 overexpression ameliorated MCAO/R-induced brain-cell death and neurobehavioral deficits in vivo. Furthermore, these results were recapitulated in cultured neurons. Acridine orange (AO) staining, as well as LysoSensor Green DND-189, cathepsin-B (CTSB), and cathepsin-D (CTSD) activities, showed that TMEM175 deficiency inhibited the hydrolytic function of lysosomes by affecting lysosomal pH. In contrast, TMEM175 upregulation reversed OGD/R-induced lysosomal dysfunction and impaired mitochondrial accumulation in cultured neurons. TMEM175 deficiency induced by cerebral I/R injury leads to compromised lysosomal pH stability, thus inhibiting the hydrolytic function of lysosomes. Consequently, lysosomal-dependent degradation of damaged mitochondria is suppressed and thereby exacerbates brain damage. Exogenous up-regulation of TMEM175 protein level could reverse the neuronal lysosomal dysfunction after ischemia-reperfusion.

SUBMITTER: Zhang M

PROVIDER: S-EPMC7429711 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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TMEM175 mediates Lysosomal function and participates in neuronal injury induced by cerebral ischemia-reperfusion.

Zhang Mengling M Lu Haifeng H Xie Xueshun X Shen Haitao H Li Xiang X Zhang Yunhai Y Wu Jiang J Ni Jianqiang J Li Haiying H Chen Gang G

Molecular brain 20200815 1

As the main organelles for the clearance of damaged proteins and damaged organelles, the function of lysosomes is crucial for maintaining the intracellular homeostasis of long-lived neurons. A stable acidic environment is essential for lysosomes to perform their functions. TMEM175 has been identified as a new K<sup>+</sup> channel that is responsible for regulating lysosomal membrane potential and pH stability in neurons. This study aimed to understand the role of TMEM175 in lysosomal function o ...[more]

PMID: 32799888

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Project description:Stroke and subsequent cerebral ischemia/reperfusion (I/R) injury is a frequently occurring disease that can have serious consequences in the absence of timely intervention. Circular RNAs (circRNAs) in association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression. However, whether circRNAs have a role in cerebral I/R injury pathogenesis, especially soon after onset, is unclear. In this study, we used the SD rat middle cerebral artery occlusion (MCAO) model of stroke to examine the role of circRNAs in cerebral I/R injury. We used high-throughput sequencing (HTS) to compare the expression levels of circRNAs in cerebral cortex tissue from MCAO rats during the occlusion-reperfusion latency period 3 hours after I/R injury with those in control cerebral cortices. Our sequencing results revealed that expression levels of 44 circRNAs were significantly altered after I/R, with 16 and 28 circRNAs showing significant up- and down-regulation, respectively, relative to levels in control cortex. We extended these results in vitro in primary cultured neuron cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R) using qRT-PCR to show that levels of circ-camk4 were increased in OGD/R neurons relative to control neurons. Bioinformatics analyses predicted that several miRNAs could be associated with circ-camk4 and this prediction was confirmed in a RNA pull-down assay. KEGG analysis to predict pathways that involve circ-camk4 included the glutamatergic synapse pathway, MAPK signaling pathway, and apoptosis signaling pathways, all of which are known to be involved in brain injury after I/R. Our results also demonstrate that levels of the human homolog to circ-camk4 (hsa-circ-camk4) are elevated in SH-SY5Y cells exposed to OGD/R treatment. Overexpression of hsa-circ-camk4 in SH-SY5Y cells significantly increased the rate of cell death after OGD/R, suggesting that circ-camk4 may play a key role in progression of cerebral I/R injury.

Project description:AimsChemokine receptor 7 (CXCR7) exerts protective effects on the brain. MicroRNAs (miRNAs) are involved in cerebral ischemia/reperfusion (I/R) injury, but their involvement in CXCR7-mediated brain protection is unknown. In this study, we investigated the role of miRNAs in CXCR7-mediated brain protection.MethodsCXCR7 levels in peripheral blood samples from patients with acute ischemic stroke (AIS) and ischemic penumbra area brain tissues from middle cerebral artery occlusion (MCAO) rats after recanalization were measured. An miRNA microarray analysis was performed to examine the expression of miRNAs caused by CXCR7 knockdown in ischemic penumbra area brain tissue in middle cerebral artery occlusion-reperfusion rats and to predict corresponding downstream target genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed the most enriched pathways. A dual-luciferase reporter assay confirmed the direct regulation of miR-182 on the target gene TCF7L2. The correlation between TCF7L2 and CXCR7/miR-182 was verified using rescue assays.ResultsCXCR7 expression was upregulated in MCAO rats and mechanical thrombectomy patients with AIS compared to that in controls. The motor and sensory functions of MCAO rats with CXCR7 knockdown further decreased, and the infarct volume and cerebral edema increased. miRNA microarray data showed that seven miRNAs were differentially expressed after shRNA-CXCR7 treatment. The dual-luciferase reporter assay confirmed that miR-182 directly targeted the TCF7L2 gene. Rescue assays confirmed that TCF7L2 is downstream of CXCR7/miR-182. KEGG pathway analysis showed that the Hippo pathway may be a key pathway in CXCR7 upregulation and plays a role in protecting the brain after interventional surgery. Animal experiments have shown that CXCR7-mediated cerebral I/R injury promotes the phosphorylation of key molecules YAP and TAZ in the Hippo pathway.ConclusionCXCR7 protects against cerebral I/R injury, possibly via the miR-182/TCF7L2/Hippo pathway. These results indicate that CXCR7 affects cerebral ischemia-reperfusion injury through miRNA regulation and downstream pathways.

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TMEM175 mediates Lysosomal function and participates in neuronal injury induced by cerebral ischemia-reperfusion.

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TMEM175 mediates Lysosomal function and participates in neuronal injury induced by cerebral ischemia-reperfusion.

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