Molecules that govern the formation, integrity and function of the hippocampus remain an important area of investigation. Here we show that absence of the proneuropeptide processing enzyme, carboxypeptidase E (CPE) in CPE knock-out (KO) mice had a profound effect on memory, synaptic physiology and the cytoarchitecture of the hippocampus in these animals. Adult CPE-KO mice displayed deficits in memory consolidation as revealed by the water-maze, object preference and social transmission of food preference tests. These mice also showed no evoked long termpotentiation. Additionally, CPE-KO mice at 4 weeks of age and older, but not at 3 weeks of age, exhibited marked degeneration specifically of the pyramidal neurons in the hippocampal CA3 region which normally expresses high levels of CPE. Immunohistochemistry revealed that the neuronal marker, NeuN, was reduced, while the glial marker, GFAP, was increased, characteristic of gliosis in the CA3 area of CPE-KO mice. Calbindin staining indicated early termination of the mossy fibers before reaching the CA1 region in these mice. Thus, absence of CPE leads to degeneration of the CA3 neurons and perturbation of the cytoarchitecture of the hippocampus. Ex vivo studies showed that overexpression of CPE in cultured hippocampal neurons protected them against H 2 O 2 oxidativestress induced cell death. These findings taken together indicate that CPE is essential for the survival of adult hippocampal CA3 neurons to maintain normal cognitive function.

YP.Obese carboxypeptidase E knockout mice exhibit multiple defects in peptide hormone processing contributing to low bone mineral density. Am J Physiol Endocrinol Metab 299: E189 -E197, 2010. First published May 11, 2010; doi:10.1152/ajpendo.00516.2009.-Carboxypeptidase E (CPE) is a prohormone/proneuropeptide processing enzyme, and mice bearing CPE mutations exhibit an obese and diabetic phenotype. Studies on CPE knockout (KO) mice revealed poor prohormone processing, resulting in deficiencies in peptide hormones/neuropeptides such as insulin, gonadotropin-releasing hormone, and cocaineand amphetamine-regulated transcript (CART). Here, we show that CPE KO mice, an obese animal model, have low bone mineral density (BMD) accompanied by elevated plasma CTX-1 (carboxy-terminal collagen crosslinks), and osteocalcin, indicators of increased bone turnover. Receptor activator for NF-B ligand (RANKL) expression was elevated ϳ2-fold relative to osteoprotegerin in the femur of KO animals, suggesting increased osteoclastic activity in the KO mice. In the hypothalamus, mature CART, a peptide involved in eating behavior and implicated in bone metabolism, was undetectable. The melanocortin and neuropeptide Y (NPY) systems in the hypothalamus have also been implicated in bone remodeling, since MC4R KO and NPY KO mice have increased BMD. However, reduction of ␣-MSH, the primary ligand of MC4R by up to 94% and the lack of detectable NPY in the hypothalamus of CPE KO do not recapitulate the singlegene KO phenotypes. This study highlights the complex physiological interplay between peptides involved in energy metabolism and bone formation and furthermore suggests the possibility that patients, bearing CPE and CART mutations leading to inactive forms of these molecules, may be at a higher risk of developing osteoporosis. CARBOXYPEPTIDASE E (CPE) is a processing enzyme that is highly expressed in endocrine cells and peptidergic neurons (17,19). It functions to cleave carboxy-terminally extended lysine and arginine residues from peptide hormone and neuropeptide intermediates to form bioactive peptides in the regulated secretory pathway (RSP). In addition to its enzymatic function, CPE has been shown to facilitate trafficking of several prohormones into the granules of the RSP (10, 26). Recently, live-cell imaging and coimmunoprecipitation studies demonstrated a role for its cytoplasmic carboxyl terminus in the transport of peptidergic vesicles via interaction with dynactin, an anterograde microtubule-based motor protein complex (27,28). The involvement of CPE in multiple cellular functions would suggest that deficiencies in CPE would lead to many pathologies. Indeed, the CPE knockout (KO) mouse exhibits multiple endocrinopathies leading to diabetes, infertility, and obesity (7).During our initial characterization of the phenotype of the CPE KO mice, which included physical and biochemical measurements as well as behavioral tests (7), we observed unexpectedly that bone mineral density (BMD) measurements of the CPE KO mice were lower th...

A direct link between receptor glycosylation and activation following natural ligand interaction has not been observed. Here, we discover a membrane sialidase-controlling mechanism that depends on ligand binding to its receptor to induce enzyme activity which targets and desialylates the receptor and, consequently, causes the induction of receptor dimerization and activation. We also identify a specific sialyl alpha-2,3-linked beta-galactosyl sugar residue of TrkA tyrosine kinase receptor, which is rapidly targeted and hydrolyzed by the sialidase. Trk-expressing cells and primary cortical neurons following stimulation with specific neurotrophic growth factors express a vigorous membrane sialidase activity. Neuraminidase inhibitors, Tamiflu, BCX1812, and BCX1827, block sialidase activity induced by nerve growth factor (NGF) in TrkA-PC12 cells and by brain-derived neurotrophic factor (BDNF) in primary cortical neurons. In contrast, the neuraminidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, specific for plasma membrane ganglioside Neu3 and Neu2 sialidases has no inhibitory effect on NGF-induced pTrkA. The GM1 ganglioside specific cholera toxin subunit B applied to TrkA-PC12 cells has no inhibitory effect on NGF-induced sialidase activity. Neurite outgrowths induced by NGF-treated TrkA-PC12 and BDNF-treated PC12(nnr5) stably transfected with TrkB receptors (TrkB-nnr5) cells are significantly inhibited by Tamiflu. Our results establish a novel mode of regulation of receptor activation by its natural ligand and define a new function for cellular sialidases.

Carboxypeptidase E (CPE) is involved in maturation of neuropeptides and sorting of brain-derived neurotrophic factor (BDNF) to the regulated pathway for activity-dependent secretion from CNS neurons. CPE knock-out (CPE-KO) mice have many neurological deficits including learning and memory. Here, we analyzed the dendritic arborization and spine morphology of CPE-KO mice to determine a possible correlation of defects in such structures with the neurological deficits observed in these animals. Analysis of pyramidal neurons in layer V of cerebral cortex and in hippocampal CA1 region in 14 week old CPE-KO mice showed more dendritic complexity compared to wild type (WT) mice. There were more dendritic intersections and more branch points in CPE-KO versus WT neurons. Comparison of pyramidal cortical neurons in 6 week versus 14 week old WT mice showed a decrease in dendritic arborization, reflecting the occurrence of normal dendritic pruning. However, this did not occur in CPE-KO neurons. Furthermore, analysis of spine morphology demonstrated a significant increase in the number of D-type spines regarded as non-functional in the cortical neurons of CPE-KO animals. Our findings suggest that CPE is an important novel player in mediating appropriate dendritic patterning and spine formation in CNS neurons.