SNAP-25 and Cdk5 as exocytotic regulators : Consequences for synaptic function and insulin release

The process by which cells release substances through fusion of vesicles with the plasma membrane is called exocytosis. Regulated exocytosis needs to be tightly controlled in order to respond to the large variation in stimuli and demands for release of neurotransmitters, peptides and hormones. Disturbances in release underlie a multitude of neuropsychiatric disorders and hormonal imbalances. This thesis aimed at exploring exocytotic regulatory mechanisms, focusing on protein isoforms and phosphorylation. Central for exocytosis is the formation of a trans-membrane SNARE protein complex, consisting of VAMP2, Syntaxin 1 and SNAP-25. The in vivo importance of two SNAP-25 isoforms was investigated in a transgenic mouse model in which SNAP-25b expression was replaced with supplementary SNAP-25a, thereby retaining physiological levels of SNAP-25. SNAP-25a is expressed early in development whereas SNAP-25b is the dominant isoform in adult brain. The SNAP-25b null mutants exist in two versions, unfloxed with a neo cassette retained and reduced levels of SNAP-25 expression, and floxed with the selection gene excised. Both models demonstrated reduced short-term plasticity in the hippocampus and developed seizures with accompanying changes in neuropeptide expression. Unfloxed mouse mutants had a more severe phenotype with developmental defects and early lethality. Adult floxed mouse mutants show cognitive impairments with increased anxiety and deficits in spatial learning and with time, morphological changes in the hippocampus appeared. Cdk5 was initially identified as a kinase important for neurite outgrowth but is emerging as a regulator of cell signaling. For activity Cdk5 is dependent on its activator proteins, p35 and p39. We investigated the roles of Cdk5 and the activators in synaptic vesicle and secretory granule exocytosis. In the neuroblastoma-glioma cell line NG108-15, Cdk5 and p35 proteins were preferentially distributed to soluble pools. p39 was expressed at low levels and primarily present in membrane and cytoskeletal fractions. Recordings of postsynaptic membrane potentials in co-cultures with NG108-15 cells and differentiating muscle cells demonstrated that a dominant negative Cdk5 mutant inhibited spontaneous transmitter release. Overexpression of either Cdk5 activator p35 or p39 increased both the number of functional synapses and the frequency of spontaneous neurotransmitter release. Insulin release from the pancreatic beta cell is important for regulating blood glucose levels and in Type II diabetes this process is impaired. We have previously identified Cdk5 as an enhancer of beta cell exocytosis and now show that both Cdk5 activators are expressed in mouse primary beta cells and associate with membranes. Munc18-1 is a Cdk5 substrate essential for neuronal exocytosis participating in SNARE complex assembly. Capacitance recordings using phosphorylation mutants showed that Munc18-1 is a substrate of Cdk5/p39, promoting Ca2+-dependent beta cell exocytosis. Munc18-2 is preferentially expressed in cells not expressing Munc18-1. We demonstrated that Munc18-2 is expressed in pancreatic beta cells, in addition to Munc18-1. Munc18-2 showed a more cytosolic localization and Munc18-1 was selectively translocated to the plasma membrane after glucose-stimulation. Phosphorylation mutants of Munc18-1 and Munc18-2 showed different subcellular localization compared to wt proteins, implicating that phosphorylation is important for Munc18 cycling. Thus, we have shown that in vivo the switch from SNAP-25a to SNAP-25b is essential for developing and maintaining accurate synaptic performance in plastic brain areas. Cdk5 promotes exocytosis in both neuronal and pancreatic beta cells, in the latter via the p39 activator. Furthermore, we have identified Munc18 proteins as Cdk5 substrates in beta cells and demonstrated a late role for Cdk5 phosphorylation of Munc18-1 in exocytosis