RNA‐Seq analysis of knocking out the neuroprotective proton‐sensitive GPR68 on basal and acute ischemia‐induced transcriptome changes and signaling in mouse brain

Abstract

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 that 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 and 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 GPR68dependent. 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.

Physiological relevance of proton-activated GPCRs

“…Authors speculated that in mild acidosis, PKC activation via OGR1 provides protective effects, while in severe acidosis, other protons sensors, such as ASICs and the proton-activated chloride channel (PAC), are active and cause further damage. RNA sequencing data from brains collected from Gpr68 knockout mice subjected to transient middle cerebral artery occlusion do not show changes in the expression of ASICs or PAC [158]. However, Gpr68 deletion caused changes in three genes encoding hemoglobin and a few genes already associated with neuroprotection.…”

GPR68 Contributes to Persistent Acidosis-Induced Activation of AGC Kinases and Tyrosine Phosphorylation in Organotypic Hippocampal Slices

“…However, GPR68 deletion only has partial effect on either pPKASS or pAktSS signals, suggesting that additional effectors are involved. GPR65, another proton-sensitive GPCR, has only limited expression in the brain, both in overall expression level and the number of GPR65 expressing cells (Vollmer et al, 2016 ; Sato et al, 2020 ; Zhou et al, 2021 ). GPR65 thus is unlikely to alter phosphorylation at a global (i.e., whole slice) level.…”

“…Brain expresses several types of proton receptors, including the acid-sensing ion channel−1a (ASIC1a) and−2 (ASIC2), proton-activate chloride channel (PAC; TMEM206), GPR4, GPR65, and GPR68 (Wemmie et al, 2002 ; Askwith et al, 2004 ; Xiong et al, 2004 ; Zha et al, 2009 ; Sherwood et al, 2011 ; Du et al, 2014b ; Price et al, 2014 ; Wei et al, 2015 ; Guyenet et al, 2016 ; Hosford et al, 2018 ; Ullrich et al, 2019 ; Yang et al, 2019 ; Sato et al, 2020 ; Wenzel et al, 2020 ; Zhou et al, 2021 ). ASIC1a, PAC, and GPR68 exhibit neuronal expression in the brain.…”

Biglycan as a potential regulator of tumorgenicity and immunogenicity in K-RAS-transformed cells

“…Bioinformatics was performed according to the procedures described in previous studies. 51 Reference genome and gene model annotation files were directly downloaded from genome website browser (NCBI/UCSC/Ensembl). To quantify gene expression levels, HTSeq v0.6.1 was used to count the read numbers mapped of each gene.…”