Protein phosphatase role in adenosine A1 receptor-induced AMPA receptor trafficking and rat hippocampal neuronal damage in hypoxia/reperfusion injury

Abstract

Adenosine signaling via A1 receptor (A1R) and A2A receptor (A2AR) has shown promise in revealing potential targets for neuroprotection in cerebral ischemia. We recently showed a novel mechanism by which A1R activation with N(6)-cyclopentyl adenosine (CPA) induced GluA1 and GluA2 AMPA receptor (AMPAR) endocytosis and adenosine-induced persistent synaptic depression (APSD) in rat hippocampus. This study further investigates the mechanism of A1R-mediated AMPAR internalization and hippocampal slice neuronal damage through activation of protein phosphatase 1 (PP1), 2A (PP2A), and 2B (PP2B) using electrophysiological, biochemical and imaging techniques. Following prolonged A1R activation, GluA2 internalization was selectively blocked by PP2A inhibitors (okadaic acid and fostriecin), whereas inhibitors of PP2A, PP1 (tautomycetin), and PP2B (FK506) all prevented GluA1 internalization. Additionally, GluA1 phosphorylation at Ser831 and Ser845 was reduced after prolonged A1R activation in hippocampal slices. PP2A inhibitors nullified A1R-mediated downregulation of pSer845-GluA1, while PP1 and PP2B inhibitors prevented pSer831-GluA1 downregulation. Each protein phosphatase inhibitor also blunted CPA-induced synaptic depression and APSD. We then tested whether A1R-mediated changes in AMPAR trafficking and APSD contribute to hypoxia-induced neuronal injury. Hypoxia (20 min) induced A1R-mediated internalization of both AMPAR subunits, and subsequent normoxic reperfusion (45 min) increased GluA1 but persistently reduced GluA2 surface expression. Neuronal damage after hypoxia-reperfusion injury was significantly blunted by pre-incubation with the above protein phosphatase inhibitors. Together, these data suggest that A1R-mediated protein phosphatase activation causes persistent synaptic depression by downregulating GluA2-containing AMPARs; this previously undefined role of A1R stimulation in hippocampal neuronal damage represents a novel therapeutic target in cerebral ischemic damage.

The Purine Salvage Pathway and the Restoration of Cerebral ATP: Implications for Brain Slice Physiology and Brain Injury

“…In the context of brain slice electrophysiology these actions include: adenosine-mediated internalisation of the inhibitory adenosine A 1 and A 3 receptors, but not the excitatory A 2A receptor, inhibition of neurotransmitter release, in particular of glutamate, and endocytosis of glutamate AMPA receptors [28][29][30][31][32]. These effects would dramatically shift the balance of excitation and inhibition in the slice preparation and be far removed from the in vivo situation where extracellular levels of adenosine are kept at a very low levels (typically submicromolar [33]).…”

UBE3A-mediated PTPA ubiquitination and degradation regulate PP2A activity and dendritic spine morphology

“…The regulatory B subunit belongs to one of four families containing PR55/B (B55), PR61/B′ (B56), PR48/ PR72/PR130/B″, or PR93/PR110/B‴, and determines the substrate specificity and enzymatic activity of PP2A (13). In the nervous system, PP2A is crucial for neuronal growth and differentiation, cytoskeleton assembly, dendritic spine morphology, and synaptic plasticity (14,15). However, it remains largely unknown how regulatory factors function together to modulate PP2A activity in vivo.…”

Upregulation of AMPA receptor GluA1 phosphorylation by blocking adenosine A1 receptors in the male rat forebrain

“…Early studies found that A 1 receptors physically interacted with GluA1 subunits in hippocampal neurons (Chen et al, ). In rat hippocampal slices, prolonged A 1 receptor activation with CPA reduced GluA1 phosphorylation at S845 and S831 and induced GluA1 endocytosis and synaptic depression (Chen et al, ; Stockwell et al, ). These data suggest the hippocampus as another brain region where a significant inhibitory tone of A 1 receptors on S845 phosphorylation exists.…”

“…With regard to the linkage to AMPA receptors, dopamine D 1 signaling has been well characterized to be positively coupled to GluA1 S845 phosphorylation as aforementioned. However, whether A 1 receptors regulate S845 phosphorylation has been less studied to date (Chen et al, 2014;Hobson et al, 2013;Stockwell, Chen, Niazi, Nosib, & Cayabyab, 2016).…”