Poster Sessions

CB -34

Joshua Park

H. Lou, A. Sarcon, T. Adams, N. Cawley, P. Loh

CARBOXYPEPTIDASE E CYTOPLASMIC TAIL MEDIATES DOCKING/EXOCYTOSIS OF SYNAPTIC VESICLES

Activity-dependent secretion of neurotransmitters and neuropeptides is essential for brain function. Previously, we reported that Carboxypeptidase E knock-out (CPE-KO) mice had impaired glutamate neurotransmission as evidenced by the lack of the b-wave in their retinograms. In this study, we show the absence of high K+stimulated secretion of glutamate from cultured primary embryonic hypothalamic neurons (E16) and adult mouse synaptosomes isolated from CPE-KO mice, raising the possibility that CPE may be present on glutamate vesicles and play a role in mediating release of these vesicles. Indeed, subcellular fractionation studies revealed that a synaptophysin-enriched synaptic vesicle fraction from mouse brain contained CPE and the vesicular glutamate transporter, Vglut2, but not a dense-core granule-specific protein marker, chromogranin A (CgA). Moreover, intact synaptic vesicles were found to bind the extreme C-terminal CPE antibodies, indicating the existence of a population of synaptic vesicles which contains transmembrane CPE with a cytoplasmic tail. Electron microscopic analysis of 100 hypothalamic synaptic densities revealed that 40% of synapses had no docked synaptic vesicles at the presynaptic membrane in CPE-KO mice versus none in WT mice, implicating that in some neurons, CPE is involved in synaptic vesicle docking, possibly mediated by its cytoplasmic tail. In in vitro GST pulldown assays, GST-CPEC10 bound various proteins necessary for vesicle docking and exocytosis: Rim1 and Rab27A, from brain and PC12 cell cytosol. A role of the CPE cytoplasmic tail (CPEC10) in exocytosis of RFP-synaptophysin labeled, CgA negative synaptic vesicles were demonstrated by TIRF microscopic studies. Expression of GFP-tagged CPEC10 in PC12 cells diminished high K+ stimulated exocytosis of RFP-synaptophysin labeled vesicles. From all the observations in this study, we propose that the vesicular CPE cytoplasmic tail plays a critical new role in the recruitment of a protein complex necessary for docking/exocytosis of certain types of synaptic vesicles such as the glutaminergic vesicles in the hypothalamus.