Regulation of proteins involved in synaptic plasticity by Ca2+ and cAMP: novel insights into Ca3

Kwai, Kwai Abel Alier (2003) Regulation of proteins involved in synaptic plasticity by Ca2+ and cAMP: novel insights into Ca3. PhD thesis, University of Glasgow.

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Elevation of intracellular Ca2+ and cAMP are key triggering events leading to hippocampal long-term potentiation (LTP, a form of synaptic plasticity) induction. In this study, the aim was to compare the ability of elevated Ca2+ influx (achieved via the Ca2+ ionophore, A23187) or increased intracellular cAMP levels (achieved via adenylate cyclase activator, forskolin) to modulate the expression or activation of selected proteins involved in LTP, with emphasis on the CAS region of the hippocampus. The proteins investigated include: alphaCaMKII, MAP2, beta-activin, Pyk2 and MAPK. They have all been implicated in LTP in CA1 and dentate gyrus (DG) regions of the hippocampus. To test the hypothesis that Ca2+ influx or cAMP elevation might activate Pyk2 and MAPK, the levels of phosphorylated Pyk2 and MAPK were measured in acute rat hippocampal slice preparations after exposure to A23187 (5muM) or forskolin (50muM). Using an immunoprecipitation assay, the levels of phosphorylated Pyk2 were increased in the presence of A23187 with a peak effect around 10 minutes. When the CA1 and CA3 regions of the hippocampus were investigated separately, phosphorylation of Pyk2 was achieved in both regions after exposure to A23187, suggesting a key role for Ca2+ in both regions. Staurosporine, a general PKC inhibitor, and chelerythrine, a PKMepsilon inhibitor, were compared for their ability to attenuate the effect of A23187. Exposure of acute hippocampal slices to chelerythrine or staurosporine prior to A23187 application resulted in the reduced phosphorylation of Pyk2, suggesting that PKMepsilon and novel PKC may be involved in Pyk2 activation by Ca2+. Application of forskolin to acute slices resulted in the reduced activation of Pyk2 below basal level, suggesting that cAMP inhibits Pyk2. MAPK was phosphorylated for more than 20 minutes in the presence of either A23187 or forskolin suggesting an important role for Ca2+ and cAMP in the activation of MAPK in acute hippocampal slices. However, when CA1 and CA3 regions were investigated separately, A23187 and forskolin only activated MAPK in the CA1 region, with no effect in the CA3 region, hence suggesting a different mode of activation of MAPK in the two regions. The effect of elevation of intracellular Ca2+ and cAMP via A23187 and forskolin respectively, on the expression of alphaCaMKII, MAP2 and beta-activin protein were studied in organotypic slice cultures of rat hippocampus by immunocytochemistry or western blot. The levels of MAP2 expression were increased 4 hours after forskolin treatment, but were unaffected by A23187 treatment. Conversely, the levels of alphaCaMKII expression were increased 4 hours after A23187 treatment, but were unaffected by forskolin. The regulation of the expression of these proteins was the same in the CA3 region as in the CA1 and dentate gyrus of the hippocampus. While rapamycin reduced the basal levels of MAP2 expression, it did not affect the ability of either forskolin or A23187 to enhance MAP2 or alphaCaMKII levels. These results suggest that cAMP and Ca2+ differentially modulate the expression of these two plasticity-related genes, and that translational enhancement via the mammalian target of rapamycin kinase is not involved in these effects. The expression of beta-activin was enhanced in the presence of A23187 or forskolin in both the CA1 and CA3 regions, again suggesting a similar pathway in both regions, and that both increased intracellular Ca2+ and cAMP levels can participate in the regulated expression of this protein. These results suggest that Ca2+ and cAMP mediate distinct components of neurochemical changes that underlie LTP maintenance. The proteins monitored in this study showed clear differences in their response to these two second messengers. However, in general the alterations in protein expression in the CA3 region corresponded with those in the CA1 and DG regions, suggesting that the pathways regulating the expression of a given protein may not be cell-specific.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Neurosciences.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Morris, Prof. Brian
Date of Award: 2003
Depositing User: Enlighten Team
Unique ID: glathesis:2003-71219
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 27 May 2021 08:46

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