The intracellular sorting and packaging of pro-neuropeptides, prohormones and neurotrophins to the granules of the regulated secretory pathway (RSP) is essential for processing, storage and release of active proteins and peptides in the neuroendocrine cell. Processing of these proproteins takes place in the secretory granules and therefore the proprotein converting enzymes (PC1/3, PC2 and carboxypeptidase E) must also be sorted into these organelles. We have investigated the sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin), proinsulin and brain derived neurotrophic factor (BDNF) to the RSP. Such studies will lead to the better understanding of diseases related to defects in hormone and neuropeptide targeting such as; obesity, diabetes, memory and learning. Previously we have shown that carboxypeptidase E (CPE) has a non-enzymatic role, acting as a binding protein to facilitate the sorting of prohormones such as POMC into the granules of the RSP. Using a CPE knockout (KO) mouse model, we showed that 50% of newly synthesized POMC in primary cultures of the pituitary anterior lobe cells was degraded and suggests that in the absence of efficient sorting to the granules of the RSP due to the lack of CPE, POMC was targeted for degradation. However, some of the remaining POMC was sorted into the RSP. A candidate for a compensatory sorting receptor is Secretogranin III (SgIII) which has been shown to bind POMC in precipitation assays. Analysis of pituitary extracts show that the full length 65 kDa form, which was the form shown to bind POMC, is increased in the KO pituitary suggesting that this increase may facilitate sorting of the residual POMC into the RSP. The mechanism regulating the formation of large dense-core secretory granules (LDCGs) into which the prohomones are packaged is another major focus of our research. We previously showed that chromogranin A (CgA) plays a critical role in the control of LDCG formation and that the mechanism was through the control of granule protein degradation within the Golgi by regulating the level of a protease inhibitor. We identified the inhibitor as protease nexin-1 (PN-1) which expression was up-regulated transcriptionally by CgA (or a fragment of CgA). PN-1 prevented granule protein degradation and increased LDCG formation when up-regulated, but when reduced in its expression by PN-1 antisense-RNA, the proteins were degraded and LDCGs were not made. We hypothesized that CgA might be signaling the cell to increase the levels of PN-1 in response to reduced LDCGs after stimulated secretion. To this end we have identified a 26 amino acid C-terminal fragment of CgA in secretion medium, which we named serpinin, that was able to enhance PN-1 transcription and granule biogenesis in 6T3 cells, an endocrine cell line that normally lacks LDCGs. Serpinin was elevated in the medium after high K+ stimulation of AtT-20 cells. We propose that serpinin binds to a cognate receptor on the plasma membrane to cause signaling to the nucleus to enhance PN-1 mRNA transcription. Thus we have discovered a new CgA-derived peptide, serpinin, which is co-secreted with POMC-derived hormones upon stimulation of pituitary corticotrophs that signals replenishment of LDCGs by transcriptionally up-regulating the expression of a protease inhibitor, PN-1. PN-1 then stabilizes granule proteins resulting in increased LDCG biogenesis. Interestingly, in addition to PN-1, we found that the water channel protein, Aquaporin 1 (AQP1) was also up-regulated in its expression by CgA. We have now demonstrated that AQP1 is present in LDCGs of endocrine tissues and appears to play an integral role in the formation and/or function of the RSP. When down-regulated in its expression in AtT20 cells by antisense-RNA, the number of LDCGs decreased by 66% and in pulse-chase studies there was a defect in regulated secretion of the endogenous ACTH hormone, as well as increased degradation of newly synthesized granule proteins such as POMC and CPE at the Golgi apparatus. This indicated that AQP1 is necessary for maintaining hormone secretion and granule biogenesis in corticotrophs. Indeed, AQP1 knockout mice showed decreased circulating ACTH relative to control mice, thereby establishing for the first time an important intracellular role of this water channel in endocrine function in vivo. We have also investigated the mechanism of post-Golgi transport and delivery of hormone and BDNF vesicles to the plasma membrane for activity-dependent secretion which is critical for endocrine function and synaptic plasticity. We showed that the CPE cytoplasmic tail on these secretory granules plays an important role in their transport. Overexpression of the CPE cytoplasmic tail in the cytoplasm to compete with the endogenous tail diminished localization of endogenous POMC, BDNF and fluorescence-tagged CPE in the processes of an endocrine cell line, AtT20; and hippocampal neurons. In live hippocampal neurons, primary pituitary and AtT20 cell images, overexpression of the CPE tail inhibited the movement of BDNF- and POMC/CPE-containing vesicles to the processes, respectively. S-tagged CPE tail pulled down endogenous microtubule-based motors, dynactin (p150), dynein and KIF1A/KIF3A from cytosol of AtT20 and brain cells. Finally, overexpression of the CPE tail inhibited the regulated secretion of ACTH from AtT20 cells. Thus this study has uncovered a novel mechanism whereby the vesicular CPE cytoplasmic tail plays an important role in anchoring regulated secretory pathway POMC/ACTH and BDNF vesicles to microtubule-based motors for transport and activity-dependent secretion in endocrine cells and neurons. We recently showed that transmembrane CPE is not only associated with large dense core vesicles, but also with glutamate-containing synaptic vesicles in mouse hypothalamus. High K+ stimulated release of glutamate from cultured hippocampal neurons was absent in CPE-KO mice. Furthermore, electron microscopic analysis of 100 hypothalamic synaptic densities revealed that 40% of the synapses had no docked synaptic vesicles at the presynaptic density in CPE-KO mice in contrast to 15% for the WT mice, implicating that in some neurons, CPE may be involved in synaptic vesicle docking, possibly mediated by its cytoplasmic tail. In vitro GST pulldown assays using both brain and PC12 cell cytosol, GST tagged CPEC10, the CPE cytoplasmic tail, bound Rab27A and Rim1, molecules necessary for synaptic vesicle docking to the plasma membrane. These results suggest that the vesicular CPE cytoplasmic tail plays role in recruiting Rab27A and Rim1 necessary for docking and subsequent exocytosis of a subpopulation of synaptic vesicles in the hypothalamus. We have studied the role of CPE in the nervous system in vivo. We showed that CPE KO mice that are very obese lacked the anorexigenic neuropeptide, CART, in the hypothalamus. These animals over-eat, thus providing further evidence linking decrease of this neuropeptide to the cause of obesity. We demonstrated deficiencies in several behavioral assays including the Morris water maze and object preference tests indicating a problem with learning and memory in CPE KO mice. Indeed electrophysiological measurements have showed a defect in the generation of long term potentiation (LTP) in hippocampal slices of these mice. The major cause for this defect is due to the total degeneration of neurons in the CA3 region of the hippocampus. This was evident only in 4 week and older CPE-KO mice. Three week old KO animals were normal, suggesting that CPE is important in maintaining survival of CA3 neurons which are enriched in this enzyme. When CPE was overexpressed in hippocampal neurons, they were protected from apoptosis after induced oxidative stress using hydrogen peroxide. Thus, CPE has a novel neuroprotective role in adult hippocampal neurons.