RNA-Seq analysis of knocking out the neuroprotective proton-sensitive GPR68 on basal and acute ischemia-induced transcriptome changes and signaling in mouse brain.
RNA-Seq analysis of knocking out the neuroprotective proton-sensitive GPR68 on basal and acute ischemia-induced transcriptome changes and signaling in mouse brain.
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DOI：
10.1096/fj.202002511r
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发表时间：
2021-04
期刊：
影响因子：
--
通讯作者：
Zha XM
中科院分区：
文献类型：
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作者：
Zhou G;Wang T;Zha XM
脑酸信号在生理和疾病条件下都发挥着重要作用。大脑中一个关键的神经元代谢型质子受体是GPR68，它参与海马长时程增强(LTP)，并在酸性和缺血条件下发挥神经保护作用。在这里，为了更好地了解GPR68在大脑中的功能，我们在小鼠身上进行了mRNA-Seq分析。首先，我们研究了假手术动物以确定基线表达。与野生型(WT)相比，GPR68−/−(KO)大脑在错误折叠蛋白结合、对有机环化合物的反应和内质网伴侣复合体等基因本体论(GO)方面下调了基因。接下来，我们研究了短暂性大脑中动脉闭塞(TMCAO)后的表达谱。TMCAO上调的基因聚集到与细胞因子/趋化因子相关的功能和免疫反应，而tMCAO下调的基因聚集到通道活动和突触信号。对于质子敏感受体，tMCAO下调ASIC1a，上调GPR4和GPR65，但对ASIC2、PAC和GPR68无影响。GPR68缺失不会改变这些质子受体的表达，无论是在基线还是在缺血后。最后，我们进行了基线和tMCAO后差异基因的GeneVenn分析。缺血可上调三种血红蛋白基因H2-AA、PpBP、Sigless和Tagln在WT中的表达，而在KO中则不表达。免疫组织化学染色显示，tMCAO诱导的血红蛋白定位于神经元。Western印迹分析进一步表明，Hb的诱导依赖于GPR68。综上所述，这些数据表明，GPR68在基线水平的缺失扰乱了伴侣功能和细胞信号反应，并暗示了血红蛋白介导的抗氧化机制在缺血时GPR68依赖的神经保护中的贡献。
Brain acid signaling plays important roles in both physiological and disease conditions. One key neuronal metabotropic proton receptor in the brain is GPR68, which contributes to hippocampal long-term potentiation (LTP) and mediates neuroprotection in acidotic and ischemic conditions. Here, to gain greater understanding of GPR68 function in the brain, we performed mRNA-Seq analysis in mice. First, we studied sham-operated animals to determine baseline expression. Compared to wild-type (WT), GPR68−/− (KO) brain downregulated genes which are enriched in Gene Ontology (GO) terms of misfolding protein binding, response to organic cyclic compounds, and endoplasmic reticulum chaperone complex. Next, we examined the expression profile following transient middle cerebral artery occlusion (tMCAO). tMCAO-upregulated genes cluster to cytokine/chemokine-related functions and immune responses while tMCAO-downregulated genes cluster to channel activities and synaptic signaling. For proton-sensitive receptors, tMCAO downregulated ASIC1a, upregulated GPR4 and GPR65, but had no effect on ASIC2, PAC, or GPR68. GPR68 deletion did not alter the expression of these proton receptors, either at baseline or after ischemia. Lastly, we performed GeneVenn analysis of differential genes at baseline and post-tMCAO. Ischemia upregulated the expression of three hemoglobin genes, along with H2-Aa, Ppbp, Siglece, and Tagln, in WT but not in KO. Immunostaining showed that tMCAO-induced hemoglobin localized to neurons. Western blot analysis further showed that hemoglobin induction is GPR68-dependent. Together, these data suggest that GPR68 deletion at baseline disrupts chaperone functions and cellular signaling responses and imply a contribution of hemoglobin-mediated antioxidant mechanism to GPR68-dependent neuroprotection in ischemia.