The role of RPTPz in neuronal intracellular pH homeostasis and in crosstalk to adjacent astrocytes

Abstract

Receptor protein tyrosine phosphatase ζ (RPTPζ) is a transmembrane protein almost exclusively expressed in the CNS regulates cells adhesion and signaling during development. RPTPζ ‘s close homolog RPTPγ, senses extracellular CO₂/HCO₃⁻ and modulates acid-base transport in response to basolateral Δ[CO₂] and Δ[HCO₃⁻] in the kidney. Here, we investigate if RPTPζ similarly defends against acid-base disturbances in the brain. We hypothesize that metabolic (MAc) or respiratory (RAc) acidosis ↑RPTPζ phosphatase activity to regulate the signal cascades that defend cells against intracellular pH (pHi) decreases. ↑[CO₂]ₒ or ↓[HCO₃⁻]ₒ that stimulate RPTPζ phosphatase activity could ↑acid-extrusion or ↓acid-loading rates, both of which would minimize the ↓pHi during MAc or RAc, and may promote adaptation during a subsequent acidosis. RT-PCR cloning from co-cultures of mouse hippocampal (HC) neurons and astrocytes, and pup or adult mouse HC tissue identifies six expressed RPTPζ variants. Three previously validated variants: NM_001081306, NM_001311064, and NM_001361349 and three variants formerly identified as hypothetical assemblies: XM_006505013, XM_006505014, and XM_006505015. Staining in HC co-cultures and pup or adult HC tissue sections determined that RPTPζ exclusively expresses in MAP2-positive neurons and not GFAP-positive astrocytes. pH-sensitive microfluorimetry on HC co-cultures subject to dual RAc (8% CO₂/22 mM HCO₃⁻) or MAc (5% CO₂/14 mM HCO₃⁻) pulses show wild-type (WT) neurons exhibit consistent baseline pHi ((pHi)Ctrl) values when perfused with 5% CO₂/22 mM HCO₃⁻ (pH 7.4) buffer compared to RPTPζ–/– neurons that exhibit more variable and more acidic mean (pHi)Ctrl. Moreover, in response to RAc, RPTPζ–/– neurons exhibit slower maximum rates of acidification ((dpHi/dt)early) during the first RAc, and larger ΔpHi during both RAc pulses compared to WT. In response to dual MAc, pulses, RPTPζ–/– neurons exhibit faster (dpHi/dt)early during both MAc pulses compared to WT neurons, and faster (dpHi/dt)2early vs. (dpHi/dt)1early. MAc-induced (dpHi/dt)1early = (dpHi/dt)2early in WT neurons. ΔpHi is larger in RPTPζ–/– vs. WT neurons during both MAc pulses, but is not different between Δ(pHi)1 vs. Δ(pHi)2. RPTPζ–/– astrocytes exhibit more acidic (pHi)Ctrl values, larger ΔpHi and slower (dpHi/dt)early during both RAc pulses vs. WT, with no change in acidification rate between the first and second RAc pulse. MAc-exposed RPTPζ –/– astrocytes do not exhibit substantially greater ΔpHi vs. WT astrocytes, but do present faster (dpHi/dt)early during both MAc pulses. Our data indicate that both RPTPζ –/– HC neurons and astrocytes are less able to defend against RAc or MAc compared to WT. However, because we do not detect RPTPζ –/– expression in astrocytes our results highlight a potential RPTPζ –/–-mediated neuron–astrocyte crosstalk that modulates acid extrusion in the astrocytes. R01-DK113197, DK128315 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.