Cholesterol and sphingolipids are abundant in neuronal membranes, where they help the organisation of the membrane microdomains involved in major roles such as axonal and dendritic growth, and synapse and spine stability. The aim of this study was to analyse their roles in presynaptic physiology. We first confirmed the presence of proteins of the exocytic machinery (SNARES and Ca v 2.1 channels) in the lipid microdomains of cultured neurons, and then incubated the neurons with fumonisin B (an inhibitor of sphingolipid synthesis), or with mevastatin or zaragozic acid (two compounds that affect the synthesis of cholesterol by inhibiting HMG-CoA reductase or squalene synthase). The results demonstrate that fumonisin B and zaragozic acid efficiently decrease sphingolipid and cholesterol levels without greatly affecting the viability of neurons or the expression of synaptic proteins. Electron microscopy showed that the morphology and number of synaptic vesicles in the presynaptic boutons of cholesterol-depleted neurons were similar to those observed in control neurons. Zaragozic acid (but not fumonisin B) treatment impaired synaptic vesicle uptake of the lipophilic dye FM1-43 and an antibody directed against the luminal epitope of synaptotagmin-1, effects that depended on the reduction in cholesterol because they were reversed by cholesterol reloading. The time-lapse confocal imaging of neurons transfected with ecliptic SynaptopHluorin showed that cholesterol depletion affects the post-depolarisation increase in fluorescence intensity. Taken together, these findings show that reduced cholesterol levels impair synaptic vesicle exocytosis in cultured neurons.

Neuronal calcium sensor 1 (NCS-1) belongs to a family of EF-hand calcium-binding proteins and is mainly expressed in neurons and neuroendocrine cells, where it causes facilitation of neurotransmitter release through unknown mechanisms. The yeast homologue of NCS-1 has been demonstrated to interact with and regulate the activity of yeast phosphatidylinositol 4-OH kinase β (PI4Kβ). However, in neurons and neurosecretory cells NCS-1 has not unequivocally been shown to interact with PI4Kβ. Here we have compared the subcellular distribution of NCS-1 and PI4Kβ and investigated whether they are capable of forming complexes. In neurons, both proteins are widely distributed and are present in perikarya and, to a lesser extent, in nerve terminals. A consistent portion of NCS-1 and PIK4β is cytosolic,whereas a portion of both proteins appears to be associated with the membranes of the endoplasmic reticulum and the Golgi complex. Very small amounts of NCS-1 and PI4Kβ are present in synaptic vesicles. Our results further demonstrate that in neurosecretory cells, endogenous NCS-1 and PIK4βinteract to form a complex that can be immunoisolated from membrane as well as from cytosolic fractions. Moreover, both proteins can be recruited to membranes when cells are treated with nucleotide receptor agonists known to increase polyphosphoinositide turnover and concomitantly induce exocytosis of secretory vesicles. Finally, in PC12 cells overexpressing NCS-1, the amount of PI4Kβ associated with the membranes is increased concomitantly with the increased levels of NCS-1 detected in the same membrane fractions. Together,these findings demonstrate that mammalian NCS-1 and PI4Kβ interact under physiological conditions, which suggest a possible role for NCS-1 in the translocation of PI4Kβ to target membranes.

Neuronal calcium sensor-1 (NCS-1) and its non-mammalian homologue,frequenin, have been implicated in a spectrum of cellular processes, including regulation of stimulated exocytosis of synaptic vesicles and secretory granules (SGs) in neurons and neuroendocrine cells and regulation of phosphatidylinositol 4-kinase beta activity in yeast. However, apart from these intriguing putative functions, NCS-1 and frequenin are relatively poorly understood. Here, the distribution, dynamics and function of NCS-1 were studied using PC12 cells that stably express NCS-1-EYFP (NCS-1 fused to enhanced yellow fluorescent protein) or that stably overexpress NCS-1. Fluorescence and electron microscopies show that NCS-1-EYFP is absent from SGs but is present on small clear organelles, some of which are just below the plasma membrane. Total internal reflection fluorescence microscopy shows that NCS-1-EYFP is associated with synaptic-like microvesicles (SLMVs) in growth cones. Overexpression studies show that NCS-1 enhances exocytosis of synaptotagmin-labeled regulated secretory organelles (RSOs) under basal conditions and during stimulation by UTP. Significantly, these studies implicate NCS-1 in the enhancement of both basal and stimulated phosphoinositide-dependent exocytosis of RSOs in PC12 cells, and they show that NCS-1 is distributed strategically to interact with putative targets on the plasma membrane and on SLMVs. These studies also reveal that SLMVs undergo both fast directed motion and highly hindered diffusive motion in growth cones, suggesting that cytoskeletal constituents can both facilitate and hinder SLMV motion. These results also reveal interesting similarities and differences between transport organelles in differentiated neuroendocrine cells and neurons.

SNARE (Soluble N-ethyl-maleimide sensitive factor Attachment protein Receptor) proteins assemble in tight core complexes, which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and are distributed in distinct subcellular compartments. The molecular mechanisms underlying sorting of SNAREs to their physiological sites of action are still poorly understood. Here have we analyzed the transport of syntaxin1A in epithelial cells. In line with previous data we found that syntaxin1A is not transported to the plasma membrane, but rather is retained intracellularly when overexpressed in MDCK and Caco-2 cells. Its delivery to the cell surface is recovered after munc-18-1 cotransfection. Furthermore, overexpression of the ubiquitous isoform of munc-18, munc-18-2, is also capable of rescuing the transport of the t-SNARE. The interaction between syntaxin 1A and munc-18 occurs in the biosynthetic pathway and is required to promote the exit of the t-SNARE from the Golgi complex. This enabled us to investigate the targeting of syntaxin1A in polarized cells. Confocal analysis of polarized monolayers demonstrates that syntaxin1A is delivered to both the apical and basolateral domains independently of the munc-18 proteins used in the cotranfection experiments. In search of the mechanisms underlying syntaxin 1A sorting to the cell surface, we found that a portion of the protein is included in non-ionic detergent insoluble complexes. Our results indicate that the munc-18 proteins represent limiting but essential factors in the transport of syntaxin1A from the Golgi complex to the epithelial cell surface. They also suggest the presence of codominant apical and basolateral sorting signals in the syntaxin1A sequence.