Abstract
Persistent acidosis occurs in ischemia and multiple neurological diseases. In previous studies, acidic stimulation leads to rapid increase in intracellular calcium in neurons. However, it remains largely unclear how a prolonged acidosis alters neuronal signaling. In our previous study, we found that GPR68-mediated PKC activities are protective against acidosis-induced injury in cortical slices. Here, we first asked whether the same principle holds true in organotypic hippocampal slices. Our data showed that 1-h pH 6 induced PKC phosphorylation in a GPR68-dependent manner. Go6983, a PKC inhibitor worsened acidosis-induced neuronal injury in wild type (WT) but had no effect in GPR68−/− slices. Next, to gain greater insights into acid signaling in brain tissue, we treated organotypic hippocampal slices with pH 6 for 1-h and performed a kinome profiling analysis by Western blot. Acidosis had little effect on cyclin-dependent kinase (CDK) or casein kinase 2 activity, two members of the CMGC family, or Ataxia telangiectasia mutated (ATM)/ATM and RAD3-related (ATR) activity, but reduced the phosphorylation of MAPK/CDK substrates. In contrast, acidosis induced the activation of CaMKIIα, PKA, and Akt. Besides these serine/threonine kinases, acidosis also induced tyrosine phosphorylation. Since GPR68 is widely expressed in brain neurons, we asked whether GPR68 contributes to acidosis-induced signaling. Deleting GPR68 had no effect on acidosis-induced CaMKII phosphorylation, attenuated that of phospho-Akt and phospho-PKA substrates, while abolishing acidosis-induced tyrosine phosphorylation. These data demonstrate that prolonged acidosis activates a network of signaling cascades, mediated by AGC kinases, CaMKII, and tyrosine kinases. GPR68 is the primary mediator for acidosis-induced activation of PKC and tyrosine phosphorylation, while both GPR68-dependent and -independent mechanisms contribute to the activation of PKA and Akt.
Highlights
Protons regulate multiple biological processes, including energy metabolism, synaptic plasticity, and neuronal survival in the brain (Chesler, 2003; Huang Y. et al, 2015)
To focus on chronic acidosis-induced signaling without the confounding factor of neuronal injury, we first performed a time course analysis of neuronal injury in response to pH 6 in organotypic hippocampal slices
We focused on phospho-PKC substrates (pPKCSS) and pCaMKIIα because previous studies have demonstrated their importance in acidosis-induced injuries (Katsura et al, 1999; Mattiazzi et al, 2007; Wang et al, 2020b). pH 6 induced time-dependent increase in pPKCSS and pCaMKIIα signals (Figure 7)
Summary
Protons regulate multiple biological processes, including energy metabolism, synaptic plasticity, and neuronal survival in the brain (Chesler, 2003; Huang Y. et al, 2015). A persistent increase in extracellular proton concentration, or acidosis, in the brain occurs in diseases including ischemic stroke, recurrent seizures, and traumatic brain injury. Most of the neuronal study focuses on acid-induced acute rise of calcium (Xiong et al, 2004; Yermolaieva et al, 2004; Zha et al, 2006; Wang et al, 2020b). The majority of acidosis-induced, calcium-independent, signaling was discovered in non-neuronal cells. Revealing how persistent pH reduction alters signaling cascades in neuronal tissue will provide crucial insights into our understanding of acid signaling in multiple diseases
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