Dynamins and myosin-II regulate the distinct modes of synaptic vesicle exocytosis in mature cerebrocortical nerve terminals and this involves calcium dependent phosphorylations.

Bhuva, Dilip (2015) Dynamins and myosin-II regulate the distinct modes of synaptic vesicle exocytosis in mature cerebrocortical nerve terminals and this involves calcium dependent phosphorylations. Doctoral thesis, University of Central Lancashire.

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Synaptic vesicle (SV) exocytosis is vital to maintaining neuronal transmission at chemical synapses and defects in this processes has been linked to various psychiatric and neuronal disorders. Further, distinct modes of exocytosis have been implicated in post-synaptic plasticity and these latter processes maybe compromised in many neurodegenerative disorders. Therefore, it is vitally important to elucidate the machinery involved in SV exocytosis and decode the regulatory pathways for distinct modes of SV exocytosis. Herein, synaptosomes, pinched off nerve terminals, prepared from cerebral cortex of adult male Wistar rats were used as a model system to investigate these processes. Especially, A. Ashton had previously demonstrated the existence of KR mode of exocytosis in these synaptosomes and determined that the distinct modes can be regulated by adjusting the activity of various kinases and phosphatases.

Synaptosomes were maximally labelled with 100 µM FM2-10 dye such that all the releasable vesicles, from readily releasable pool (RRP) and reserve pool (RP), were loaded with the dye. The exocytosis of the dye was then studied by employing various secretagogues (high K+ {HK}, 4-aminopyridine {4AP} or ionomycin {ION}) in the presence of 5 mM [Ca2+]e; these stimuli only induced a single round of release. This dye release was then directly compared to Glu release from terminals treated identically (more than 80% of these synaptosomes are glutamatergic), and differences between dye and Glu release were studied following various drug treatments.

The results show that the inhibition of dynamins can increase the FM2-10 dye release during certain stimulation conditions (4AP5C and ION5C; where 5C represents 5 mM [Ca2+]e) without changing the Glu released indicating that dynamin(s) are required for the closure of the fusion pore (and therefore KR) during the employment of these stimuli. However experiments involving blockade of the ATPase activity of non-muscle myosin-II suggest that myosin-II may also be able to regulate the fusion pore, independent of dynamin-I, when a different stimulus (HK5C) is employed. The three stimuli employed here produced distinct kinetics for changes in [Ca2+]i and suggest that dynamin-I may only be able to regulate KR mode of exocytosis when the Δ[Ca2+]i is relatively lower (overall Δ[Ca2+]i < 140 nM) and that myosin-II replaces dynamin-I in this function when these Ca2+ changes are relatively higher (overall Δ[Ca2+]i < 140 nM).

In order to investigate the phosphoregulatory pathways of these two phospho-proteins (dynamins and myosin-II) the activity of various enzymes including protein phosphatase (PP) 2A, PP1, calcineurin and PKC protein kinase C (PKC) was modulated externally. The data indicate that the properties of dynamin-I and myosin-II can be regulated by phosphoregulation induced by PKC and this induces their function in the KR mode of exocytosis. When the Δ[Ca2+]i is lower (overall Δ[Ca2+]i < 140 nM), the relevant PKC remains deactivated and dynamin-I can continue to close the fusion pore of exocytosing vesicles thereby causing KR. On the other hand if the overall Δ[Ca2+]i is greater than 140 nM then relevant PKCs are activated which will then phosphorylate dynamin-I and myosin-II rendering the former inactive and the latter active such that myosin-II can now replace dynamin-I in closing the fusion pore. Western blot analysis revealed that dynamin-I is dephophorylated at Ser 795 residue by PP2A, and that this residue can be phosphorylated by Ca2+ activated PKC as increase in phosphorylation of Ser 795 (by blockade of PP2A) or by supramaximal stimulation of PKC by active phorbol esters leads to a switch in the RRP to a FF mode, it would appear that this site may be important for defining the mode of exocytosis. Activated PKC can also phosphorylate myosin-II but the phosphorylation sites on the myosin-II oligomer that play such a role remain be determined.

These significant new findings help establish that SVs can switch between modes of exocytosis and that there are specific proteins implicated in this process. Clearly, further work may reveal the importance of these processes for synaptic plasticity and whether certain psychiatric or neuronal diseases could be explained by perturbation of these distinct modes of exocytosis.

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