A carboxypeptidase E (CPE) knockout (KO) mouse was generated by deletion of exons 4 and 5 from the CPE gene, and its phenotype was characterized. KO mice became obese by 10-12 wk of age and reached 60-80 g by 40 wk. At this age, body fat content was more than double that in the wild-type (WT) controls. The null animals consumed more food overall, were less physically active during the light phase of the light-dark cycle, and burned fewer calories as fat than WT littermates. Fasting levels of glucose and insulin-like immunoreactivity in plasma were elevated in both male and female KO mice at approximately 20 wk; males recovered fully and females partially from this state by 32 wk. At this time, insulin-like immunoreactivity in the plasma, identified as proinsulin, was 50-100 times higher than that of the WT animals. The KO mice showed impaired glucose clearance and were insulin resistant. High levels of leptin and no circulating fully processed cocaine- and amphetamine-related transcript, a peptide that is responsive to leptin-induced feedback inhibition of feeding, were found in serum. The KO mice were subfertile and showed deficits in GnRH processing in the hypothalamus. Behavioral analyses revealed that KO animals showed diminished reactivity to stimuli and had reduced muscle strength and coordination, as well as visual placing and toe-pinch reflexes. These data demonstrate that CPE KO mice display a wide range of neural and endocrine abnormalities and suggest that CPE may have additional physiological roles beyond those ascribed to peptide processing and sorting of prohormones in cells.

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.

Voltage-gated K + channels function in macromolecular complexes with accessory subunits to regulate brain function. Here, we describe a peptidyl-prolyl cis-trans isomerase NIMAinteracting 1 (Pin1)-dependent mechanism that regulates the association of the A-type K + channel subunit Kv4.2 with its auxiliary subunit dipeptidyl peptidase 6 (DPP6), and thereby modulates neuronal excitability and cognitive flexibility. We show that activity-induced Kv4.2 phosphorylation triggers Pin1 binding to, and isomerization of, Kv4.2 at the pThr 607-Pro motif, leading to the dissociation of the Kv4.2-DPP6 complex. We generated a novel mouse line harboring a knock-in Thr607 to Ala (Kv4.2TA) mutation that abolished dynamic Pin1 binding to Kv4.2. CA1 pyramidal neurons of the hippocampus from these mice exhibited altered Kv4.2-DPP6 interaction, increased A-type K + current, and reduced neuronal excitability. Behaviorally, Kv4.2TA mice displayed normal initial learning but improved reversal learning in both Morris water maze and lever press paradigms. These findings reveal a Pin1mediated mechanism regulating reversal learning and provide potential targets for the treatment of neuropsychiatric disorders characterized by cognitive inflexibility.

Although Netos are considered auxiliary subunits critical for kainate receptor (KAR) function, direct evidence for their regulation of native KARs is limited. As Neto KAR regulation is GluK subunit/Neto isoform specific, such regulation must be determined in cell-type specific contexts. We demonstrate Neto1/2 expression in somatostatin- (SOM), cholecystokinin/cannabinoid receptor 1- (CCK/CB1), and parvalbumin- (PV) containing interneurons. KAR-mediated excitation of these interneurons is contingent upon Neto1 as kainate yields comparable effects in Neto2 knockouts and wildtypes, but fails to excite interneurons or recruit inhibition in Neto1 knockouts. In contrast, presynaptic KARs in CCK/CB1 interneurons are dually regulated by both Neto1 and Neto2. Neto association promotes tonic presynaptic KAR activation dampening CCK/CB1 interneuron output and loss of this brake in Neto mutants profoundly increases CCK/CB1 interneuron-mediated inhibition. Our results confirm that Neto1 regulates endogenous somatodendritic KARs in diverse interneurons and demonstrate Neto regulation of presynaptic KARs in mature inhibitory presynaptic terminals.

Previous reports identified two peptides that mimic the action of neuroprotective proteins derived from astrocytes. These peptides, NAPVSIPQ and SALLRSIPA, prevent neuronal cell death produced by electrical blockade, N-methyl-D-aspartate, and ␤-amyloid peptide (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). In the present study, all Damino acid peptides of NAPVSIPQ and SALLRSIPA were synthesized and compared respectively to the corresponding all L-amino acid peptides. In rat cerebral cortical test cultures cotreated with 1 M tetrodotoxin, the D-amino acid peptides produced similar potency and efficacy for neuroprotection as that observed for their respective L-amino acid peptides. Since all these peptides tested individually exhibited attenuation of efficacy at concentrations of Ͼ10 pM, combinations of these peptides were tested for possible synergies. Equimolar D-NAPVSIPQ and D-SALLRSIPA combination treatment produced potent neuroprotection (EC 50 , 0.03 fM) that did not attenuate with increasing concentrations. Similarly, the combination of L-NAPVSIPQ and D-SALLRSIPA also had high potency (EC 50 , 0.07 fM) without attenuation of efficacy. Combined administration of peptides was tested in a model of fetal alcohol syndrome and in a model of learning impairment: apolipoprotein E knockout mice. Intraperitoneal administration of D-NAPV-SIPQ plus D-SALLRSIPA to pregnant mice (embryonic day 8) attenuated fetal demise after treatment with an acute high dose of alcohol. Furthermore, oral administration of D-NAPVSIPQ plus D-SALLRSIPA significantly increased fetal survival after maternal alcohol treatment. Apolipoprotein E knockout mice injected with D-NAPVSIPQ plus D-SALLRSIPA showed improved performance in the Morris water maze. These studies suggest therapeutic potential for the combined administration of neuroprotective peptides that can act through a mechanism independent of chiral recognition.