Carbamazepine Prevents Hippocampal Neurodegeneration in Mice Lacking the Neuroprotective Protein, Carboxypetidase E.

Carboxypeptidase E (CPE) has recently been described as a neuroprotective protein, and in mice devoid of CPE, a complete loss of the hippocampal CA3 neurons is observed. The pattern of loss is characteristic of that caused by status epilepticus. We therefore set out to determine when this loss occurred, what might induce it and if it could be prevented. We found that the hippocampus was intact in 4 week old CPE knock out (KO) mice that had not undergone weaning. However, weaning of 2 or 3 week old CPE KO mice, which involves maternal separation (emotional stress) and ear tagging and tail snipping for genotyping (physical stress), resulted in degeneration of the CA3 neurons by 3 and 4 weeks of age, respectively, while the wild-type mice were unaffected. Moreover, the physical stress caused a more severe neurodegeneration phenotype than the emotional stress of the maternal separation alone. Daily treatment with carbamazepine, an antiepileptic agent, in 2 week old CPE KO mice for 2 weeks prevented the neurodegeneration, despite the weaning process at 3 weeks. No further neurodegeneration was observed 3 weeks post weaning in carbamazepine treated mice. These results showed that degeneration of the CA3 neurons in the hippocampus, previously observed in 6 week old CPE KO mice, is not due to a developmental defect, but caused by physical and emotional stress during the weaning process. This degeneration was prevented by carbamazepine suggesting that the stress associated with weaning caused epileptic-like events in the CPE KO mice.

Obese carboxypeptidase E knockout mice exhibit multiple defects in peptide hormone processing contributing to low bone mineral density.

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 cocaine- and 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-kappaB ligand (RANKL) expression was elevated approximately 2-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 alpha-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 single-gene 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 knockout mice exhibit abnormal dendritic arborization and spine morphology in central nervous system neurons.

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 knockout (CPE-KO) mice have many neurological deficits, including deficits in 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 with wild type (WT) mice. There were more dendritic intersections and more branch points in CPE-KO vs. WT neurons. Comparison of pyramidal cortical neurons in 6- vs. 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 nonfunctional 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.

Absence of carboxypeptidase E leads to adult hippocampal neuronal degeneration and memory deficits.

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-term potentiation. 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) oxidative-stress 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.

Trypanosome trans-sialidase mediates neuroprotection against oxidative stress, serum/glucose deprivation, and hypoxia-induced neurite retraction in Trk-expressing PC12 cells.

Trypanosome trans-sialidase (TS) is a sialic acid-transferring enzyme and a novel ligand of tyrosine kinase (TrkA) receptors but not of neurotrophin receptor p75NTR. Here, we show that TS targets TrkB receptors on TrkB-expressing pheochromocytoma PC12 cells and colocalizes with TrkB receptor internalization and phosphorylation (pTrkB). Wild-type TS but not the catalytically inactive mutant TSDeltaAsp98-Glu induces pTrkB and mediates cell survival responses against death caused by oxidative stress in TrkA- and TrkB-expressing cells like those seen with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). These same effects are not observed in Trk deficient PC12(nnr5) cells, but are re-established in PC12(nnr5) cells stably transfected with TrkA or TrkB, are partially blocked by inhibitors of tyrosine kinase (K-252a), mitogen-activated protein/mitogen-activated kinase (PD98059) and completely blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K). Both TrkA- and TrkB-expressing cells pretreated with TS or their natural ligands are protected against cell death caused by serum/glucose deprivation or from hypoxia-induced neurite retraction. The cell survival effects of NGF and BDNF against oxidative stress are significantly inhibited by the neuraminidase inhibitor, Tamiflu. Together, these observations suggest that trypanosome TS mimics neurotrophic factors in cell survival responses against oxidative stress, hypoxia-induced neurite retraction and serum/glucose deprivation.

Dependence of neurotrophic factor activation of Trk tyrosine kinase receptors on cellular sialidase.

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.

Trypanosome trans-sialidase targets TrkA tyrosine kinase receptor and induces receptor internalization and activation.

Trypanosome trans-sialidase (TS) is a sialic acid-transferring enzyme that hydrolyzes alpha2,3-linked sialic acids and transfers them to acceptor molecules. Here we show that a highly purified recombinant TS derived from T. cruzi parasites targets TrkA receptors on TrkA-expressing PC12 cells and colocalizes with TrkA internalization and phosphorylation (pTrkA). Maackia amurensis lectin II (MAL-II) and Sambucus nigra lectin (SNA) block TS binding to TrkA-PC12 cells in a dose-dependent manner with subsequent inhibition of TS colocalization with pTrkA. Cells treated with lectins alone do not express pTrkA. The catalytically inactive mutant TSDeltaAsp98-Glu also binds to TrkA-expressing cells, but is unable to induce pTrkA. TrkA-PC12 cells treated with a purified recombinant alpha2,3-neuraminidase (Streptococcus pneumoniae) express pTrkA. Wild-type TS but not the mutant TSDeltaAsp98-Glu promotes neurite outgrowth in TrkA-expressing PC12 cells. In contrast, these effects are not observed in TrkA deficient PC12nnr5 cells but are reestablished in PC12nnr5 cells stably transfected with TrkA and are significantly blocked by inhibitors of tyrosine kinase (K-252a) and MAP/MEK protein kinase (PD98059). Together these observations suggest for the first time that hydrolysis of sialyl alpha2,3-linked beta-galactosyl residues of TrkA receptors plays an important role in TrkA receptor activation, sufficient to promote cell differentiation (neurite outgrowth) independent of nerve growth factor.