O Mundigl

v- and t-SNAREs in neuronal exocytosis: A need for additional components to define sites of release

Synaptic vesicle recycling is a specialized form of membrane recycling which takes place in all cells between early endosomes and the plasmalemma. Synaptic vesicles exocytosis is highly regulated and occurs only at presynaptic active zones. In contrast, exocytosis of endosome-derived vesicles of the housekeeping recycling pathway takes place constitutively and throughout the cell surface. Since v- and t-SNAREs play a key role in membrane interactions leading to fusion, unique v- and t-SNAREs may be implicated in synaptic vesicle exocytosis. It was found, however, that the same v-SNAREs of the synaptobrevin family are found both on synaptic vesicles and on endosome-derived vesicles which undergo constitutive fusion. Likewise, t-SNAREs which act as plasmalemmal receptors for synaptic vesicles are not restricted to synaptic active zones. Thus, v- and t-SNAREs interactions may define which organelles can fuse with the plasmalemma, but require additional components to define properties of the exocytotic reaction which are specific for distinct classes of secretory organelles.

Formation of synaptic vesicles

Synaptic vesicles (SVs) are specialized secretory organelles used for the fast and focal signaling between nerve cells. They are small and homogeneous in size (50 nm), and contain non-peptide neurotransmitters such as glutamate, gamma-aminobutyric acid (GABA) and acetylcholine. The exocytosis of SVs occurs at low rates in resting nerve terminals and is greatly stimulated by depolarization-induced Ca2+ influx. Following exocytosis, SV membranes are rapidly retrieved, refilled locally with neurotransmitters and reused for the assembly of new SVs. Over the past few years, significant progress has been made in characterizing the molecular composition of SVs. From these studies, we know that SVs share a conserved set of membrane proteins with transport vesicles involved in other pathways. Furthermore, these findings have provided us with a new understanding about the evolutionary origin of SVs from recycling vesicles present in all cells.

Mechanisms of synaptogenesis in hippocampal neurons in primary culture

To improve our understanding of the mechanisms which regulate the formation and the functional maturation of synaptic contacts between neurons, we used hippocampal neurons maintained in primary cultures as experimental system. In this model, which offers several advantages for the study of neuronal development and synaptogenesis, we investigated some of the cellular mechanisms underlying the formation of presynaptic and postsynaptic compartments.

Traffic of synaptic vesicle proteins in polarized and nonpolarized cells

SUMMARY Neurons have at least two pathways of regulated secretion, which involve two classes of secretory organelles: typical synaptic vesicles (SVs) and large dense-core vesicles. Large dense-core vesicles store and secrete peptide neurotransmitters and amines, and may be seen as the neuronal counterpart of secretory granules of endocrine cells. SVs are highly specialized secretory organelles, which store and secrete non-peptide hormones and play a dominant role in the fast, point-to-point signalling typical of the nervous system. Microvesicles that share a variety of biochemical and functional similarities with SVs (synaptic-like microvesicles) have recently been described in endocrine cells. SVs and synaptic-like microvesicles are closely related to vesicular carriers of the receptor-mediated recycling pathway. They undergo repeated cycles of exo-endocytosis, which are thought to involve endosomal intermediates. In mature neurons, SVs are concentrated in axon endings. To gain insight into the mechanisms responsible for SV targeting, we have studied the traffic of SV proteins in both endocrine cells and developing hippocampal neurons in primary culture at different stages of differentiation. Additionally, the distribution of the SV protein synaptophysin, when expressed by transfection in fibroblastic cells or in polarized epithelial cells (MDCK cells), was investigated. SV proteins are already present in developing neurons at stages preceding the establishment of neuronal polarity. As axons and dendrites form, SV proteins are found in both types of processes, although they become progressively more concentrated in the axon. Throughout these developmental stages SVs undergo active exo-endocytotic recycling. The nonpolarized distribution of SV proteins is observed even at stages when the transferrin receptor, a protein that is present in epithelial cells only at the basolateral surface, is already restricted to dendrites. This indicates that, in immature neurons, SV proteins are not selectively targeted to axons and that the accumulation in axons may at least partially result from a specific retention. In agreement with this finding, synaptophysin, when transfected into MDCK cells, was targeted to both the basolateral and the apical plasma membrane. Brefeldin A, a fungal metabolite that induces a modification of the steady-state localization of endosomal proteins in a variety of cell types, was found to have a different effect on the distribution of SV proteins in dendrites and in axons. Taken together, these observations support the existence of two separate endosomal systems in axons and dendrites, which have differential properties, are enriched in different proteins, and may be related to the basolateral and apical endosomes of epithelial cells.