Interactions of AMPAR-Adenosine Receptor-Equilibrative Nucleoside Transporter in the Hippocampus: Implication for Stroke
Date
2015-02-18
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Degree Level
Doctoral
Abstract
The activation of presynaptic adenosine A1 receptors (A1Rs) is known to cause profound synaptic depression during hypoxia/cerebral ischemic insults, but postsynaptic function of A1Rs are still unclear. The goal of the current study is to provide a more comprehensive view of adenosinergic signaling. Firstly, I established that A1Rs and GluA2-containing AMPARs formed stable protein complexes from hippocampal brain homogenates and cultured hippocampal neurons. In contrast, adenosine 2A receptors (A2ARs) did not co-precipitate or colocalize with GluA2-containing AMPARs. Secondly, by using different approaches I have confirmed that prolonged stimulations of A1Rs with the agonist CPA was found to cause adenosine-induced persistent synaptic depression (APSD) in hippocampal brain slices, and APSD levels were blunted by inhibiting clathrin-mediated endocytosis of GluA2 with the Tat-GluA2-3Y peptide. This was initially demonstrated by biotinylation assays and membrane fractionation, in which prolonged CPA incubation showed a significant depletion of both GluA2 and GluA1 surface expression from hippocampal brain slices and cultured hippocampal neurons. In contrast, Tat-GluA2-3Y peptide or dynamin inhibitor Dynasore prevented CPA-induced GluA2 and GluA1 internalization. Additionally, confocal imaging analysis confirmed that functional A1Rs, but not A2ARs, are required for clathrin-mediated endocytosis of AMPARs in hippocampal neurons. Pharmacological inhibitors and shRNA knockdown of p38 MAPK and JNK were found to prevent A1R-mediated internalization of GluA2 but not GluA1 subunits. However, Tat-GluA2-3Y peptide or A1R antagonist DPCPX can prevent the hypoxia-mediated internalization of both GluA2 and GluA1. Finally, in the pial vessel disruption (PVD) cortical stroke model, reduced hippocampal GluA2, GluA1, and A1R surface expression and synaptic depression have been shown in hippocampal slices from a unilateral cortical lesioned brain compared to sham brain, which is consistent with our previous results of AMPAR downregulation and decreased probability of transmitter release. The PVD-lesioned brains also displayed increased hippocampal neurodegeneration compared to sham brains. Taken together, these results indicate a previously unknown mechanism that A1R-induced persistent synaptic depression involves clathrin-mediated GluA2 and GluA1 internalization in hypoxia/cerebral ischemia.
Both equilibrative nucleoside transporters (ENTs) and A1Rs are widely expressed in the hippocampus, and regulate extracellular adenosine level and induce synaptic depression, respectively, during cerebral ischemia. However, the cellular mechanisms that control the cell surface expression of ENTs and A1Rs in the brain remain poorly resolved. Since ENTs contain consensus sites for Casein Kinase 2 (CK2) phosphorylation, I tested the hypothesis that ENT and A1R interactions and CK2 inhibition are involved in A1R-dependent downregulation of ENT surface expression during hypoxia. Coimmunoprecipitation from rat hippocampal brain homogenates and confocal imaging microscopy of primary cultured hippocampal neurons revealed physical associations of ENTs with A1Rs, but not with A2ARs. Using whole lysates and membrane fractions from hippocampal brain slices and a phospho-specific antibody to immunoprecipitate the phosphoSerine254-ENT1 (pSer254-ENT1, a known CK2 target), I then determined that ENT1 was constitutively phosphorylated. Several CK2 inhibitors (TBB, DMAT, and DRB), but not the ENT1-selective inhibitor (NBTI) reduced pSer254-ENT1 level in whole hippocampal lysates. DRB also decreased, while CK2 activator spermine increased, the surface expression of pSer254-ENT1 in biotinylation assays of hippocampal brain slices. Moreover, biotinylation of cultured hippocampal neurons revealed that ENT1 and ENT2 surface expression was downregulated by CK2 or ENT inhibitors and by A1R agonist CPA, but not in the presence of A1R antagonist DPCPX. Pretreatments of hippocampal slices with CK2 or ENT blockers also enhanced hypoxia-mediated downregulation of ENT and A1R surface expression. These results indicate that CK2-induced and A1R-linked ENT trafficking represents an important regulatory mechanism of hypoxic/ischemic hippocampal brain damage.
The high prevalence of neurodegenerative disorders that accompany memory deficits occurs in the elderly, including stroke and Alzheimer’s disease, and it is also known that extracellular levels of adenosine are enhanced in aged brains. To determine whether the mechanisms we previously identified for A1R-mediated AMPAR internalization also contribute to dysfunction in synaptic plasticity in aged brains, I compared surface levels of AMPARs from hippocampal slices of young (1 month) and old (7-12 months) animals. I found that surface expression of AMPARs decreased in aged hippocampus. To study changes in synaptic plasticity, I then performed electrophysiological studies to compare chemically-induced long term potentiation (cLTP) in the hippocampus of young and old rats. Consistent with the biochemical results, I demonstrated that aged hippocampal slices displayed impaired cLTP, which suggests that aging impaired synaptic plasticity by promoting decreased surface expression of AMPARs. Next, I evaluated the surface levels of AMPARs before and after cLTP in young and old hippocampus to determine whether basal clathrin-mediated endocytosis of AMPARs contributes to impairments in cLTP. Following the cLTP induction, brain slices were analyzed biochemically. Under basal conditions, I showed that young brains contained higher levels of surface-expressed AMPARs compared to older brains. To test the hypothesis that this difference in baseline AMPAR surface expression contributes to cLTP deficits and was likely due to increased rate of endocytosis associated with enhanced adenosine tone in aged brains, I demonstrated with the use of two blockers of endocytosis pathways (Tat-GluA2-3Y peptide and Dynasore) that cLTP could be similarly enhanced in the young and older brains. Therefore, these results indicate that increased adenosinergic signaling in aged brains leads to increased endocytosis of AMPARs and impaired synaptic plasticity.
Together, these data suggest that interactions of AMPAR-A1R-equilibrative nucleoside transporter in the hippocampus regulate glutamatergic synaptic transmission, and enhanced A1R signaling increases both neurodegeneration in ischemic conditions and synaptic impairments in ischemic and aged brains.
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Keywords
AMPAR, adenosine receptors, equilibrative nucleoside transporter, stroke
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Physiology
Program
Physiology