Phosphorylation sites on specific neuronal proteins can control the mode of synaptic vesicle exocytosis and thereby regulate synaptic transmission

Singh, Deeba (2017) Phosphorylation sites on specific neuronal proteins can control the mode of synaptic vesicle exocytosis and thereby regulate synaptic transmission. Doctoral thesis, University of Central Lancashire.

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Synaptic vesicles (SVs) can exocytose via Full fusion (FF) or by Kiss-and-Run (KR) mechanisms. In this thesis, synaptosomes prepared from adult rat cerebrocortex demonstrated that these modes can be switched by regulating the intracellular calcium levels and/or protein phosphorylation reactions. The stimuli employed were: 30 mM K+ (HK), 1 mM 4-aminopyridine (4AP) and 5 M ionomycin (ION) together with 5 mM Ca2+. In this model employed, myosin-II and dynamins can regulate the closure of the fusion pore of the readily releasable pool (RRP) of SVs during KR but are independent of each other’s actions.
In biochemical assays, synaptosomes were maximally labelled with FM2-10 dye such that both the RRP and the RP were loaded and such terminals were subsequently evoked to release the dye and this was compared to the endogenous release of glutamate (GLU). Results show that the rise in [Ca2+]i at the active zone produced by HK5C activates PKC isoforms, which in turn cause phosphorylation of myosin-II and dynamins. It is hypothesised that dynamins are active at relatively lower increases in [Ca2+]i that do not activate PKCs whereas myosin-II is active at relatively higher increases in [Ca2+]i that activate certain PKCs. Such activation of PKC stimulates myosin-II -but inhibits the action of dynamins – and it is this active protein that can close the fusion pore. ION5C, however, activates dynamin(s) but not myosin-II, and under such conditions dynamin can close the fusion pore. This dynamin-dependent KR mode is independent of clathrin because this is not perturbed by the blockade of clathrin action using the drug, pitstop2TM and the HK5C evoked myosin-II-dependent KR is independent of both clathrin and dynamin. Therefore, the KR mechanism described, herein, is distinct from ultra-fast endocytosis that has both a dynamin and clathrin dependence. Pre-treatment of synaptosomes with DYN and/or pitstop2TM prior to the initial pre-stimulation with HK5C inhibits all dynamin and clathrin dependent processes so that SVs that recycle after the HK5C pre-stimulation would be perturbed if they had such a requirement. Indeed, following this treatment some SVs were not released by ION5C, although, the 4AP5C evoked GLU release is not affected and it would appear that the 4AP5C sensitive pool (the RRP) can still release following blockade of dynamin and clathrin. This demonstrates that the RRP underwent KR during the first pre-stimulus (actually via a myosin-II- dependent process) and was available to release again during the second round, and again this cannot be via ultra-fast endocytosis.
The role of cAMP and PKA pathways in regulating the modes was investigated. Adenylate cyclase activation by forksolin inhibits the release of the RP but switches all the RRP vesicles to KR when evoked by 4AP5C. Adenylate cyclase inhibition by 9-Cyclopentyl-Adenine (9-Cp-Ade) has no effect on GLU release or exocytotic mode. Forskolin’s action on the RP is due to a specific increase in cAMP, because pre-treatment of synaptosomes with 9-Cp-Ade before forskolin prevents the forkskolin action. The PKA activator, Sp-5,7-dichloro-cBIMPs (cBIMPS) does not affect evoked GLU release although it does switch some of the RP to a KR mode whereas inhibiting PKA with KT 5720 has not effect on GLU release but induces the RRP to undergo FF. Clearly, forskolin’s action on both the RP and the RRP SVs is distinct from PKA activation and it may work through activating the exchange protein directly activated by cAMP (EPAC). Previous experiments have showed that inhibition of protein phosphatase 2A (PP2A) by okadaic acid (OA) switches the RRP to FF for all stimuli, but OA pre-treatment before forskolin failed to prevent 4AP5C switching some SVs in the RRP from FF to KR. This suggests that an adenylate cyclase pathway can override the OA-sensitive pathway.
The Seahorse extracellular flux analyser was used to measure various mitochondrial respiration parameters (basal, ATP production, spare capacity, maximum respiration, proton leakage and non-mitochondrial respiration). Employing 0.3 and 3 M phenyl arsine oxide appears to perturb the spare respiratory capacity, whilst 0.1 M did not. This indicated that we were previously using too high a concentration of this drug to study modes of exocytosis. Such a result led to the testing of the drugs employed in this study on exocytosis to check that they did no produce a non-specific effect on the bioenergetics of the synaptosomes: 9-Cp-Ade, Blebb, Go 6983, cBIMPs, forskolin or OA did not change the mitochondrial respiratory parameters indicating that any exocytotic effects shown were specific.
The specific phosphorylation of Ser-778, Ser-774 and Ser-795 on dynamin 1 was investigated using well characterized commercial antibodies and western blotting. It was concluded that the phosphorylation of Ser-774 and Ser-778 showed no correlation with dynamin’s activity towards regulating closure of the fusion pore although these sites may well correlate with clathrin dependent endocytosis and bulk endocytosis. However, the phosphorylation of Ser-795 on dynamin may play an important role in the inhibition of dynamin’s activity towards closing the fusion pore during KR. Phosphorylation on Ser-795 may be under the regulation of PKC as demonstrated using a PKC activator, phorbol myristate acetate, and an inhibitor, Go 6983. Therefore, endogenous PKC activation may phosphorylate this site when a very high Δ[Ca2+]i is achieved at the active zone. It was difficult to obtain a conclusion as to which PKC isoform could regulate the mode of SV exocytosis by phosphorylation of Ser-795 on dynamin due to the low phosphorylation levels obtained whilst employing Go 6983 and Go 6976.

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