Delayed O-methylation of L-DOPA in MB-COMT-deficient mice after oral administration of L-DOPA and carbidopa

1.Catechol-O-methyltransferase (COMT) is involved in the O-methylation of l-DOPA, dopamine, and other catechols. The enzyme is expressed in two isoforms: soluble (S-COMT), which resides in the cytoplasm, and membrane-bound (MB-COMT), which is anchored to intracellular membranes. 2.To obtain specific information on the functions of COMT isoforms, we studied how a complete MB-COMT deficiency affects the total COMT activity in the body, peripheral l-DOPA levels, and metabolism after l-DOPA (10mg kg(-1)) plus carbidopa (30mg kg(-1)) administration by gastric tube in wild-type (WT) and MB-COMT-deficient mice. l-DOPA and 3-O-methyl-l-DOPA (3-OMD) levels were assayed in plasma, duodenum, and liver. 3.We showed that the selective lack of MB-COMT did not alter the total COMT activity, COMT enzyme kinetics, l-DOPA levels, or the total O-methylation of l-DOPA but delayed production of 3-OMD in plasma and peripheral tissues.Peer reviewe

Alterations in the polysialylated neural cell adhesion molecule and retinal ganglion cell density in mice with diabetic retinopathy

AIM: To investigate the impact of polysialylated neural cell adhesion molecule (PSA-NCAM) on the survival of retinal ganglion cells (RGCs) in the experimentally induced diabetes in mice. METHODS: Diabetes was induced in 2.5 months old Swiss Webster mice by intraperitoneal injection of streptozotocin (STZ, 90 mg/kg) once daily for two consecutive days. Examination of the proteins of interest in the retinas from diabetic mice at 2mo after diabetes induction was performed using immunohistochemistry and Western blot analysis. RGCs were counted in the wholemounted retinas, and Brn3a marker was used. RESULTS: Examination of retinas from diabetic mice at 2mo after diabetes induction revealed a considerable reduction in RGC density. Our experiments also demonstrated a redistribution of PSA-NCAM in the retina of diabetic animals. PSA-NCAM immunoreactivity was diminished in the inner part of the retina where RGCs were located. In contrast, an enhanced PSA-NCAM immunoreactivity was detected in the outer layers of the retina. PSA-NCAM signal was co-localized with glial fibrillary acidic protein immunoreactivity in the Müller cell branches. Previous studies have shown that matrix metalloproteinase-9 (MMP-9) is responsible for the reduction in PSA-NCAM levels in neuronal cells. The reduced levels of PSA-NCAM in inner layers (nerve fiber layer, ganglion cell layer) were accompanied by the increased expression of MMP-9. In contrast, in the outer retinal layers, the expression of MMP-9 was much less pronounced. CONCLUSION: MMP-9 induces PSA-NCAM shedding in the inner part of the retina and the decreased level of PSA-NCAM in the inner part of the retina might be, at least in part, responsible for the loss of RGCs in diabetic mice

Generation of membrane-bound catechol-O-methyl transferase deficient mice with disctinct sex dependent behavioral phenotype.

PMID: 28195063Catechol-O-methyltransferase (COMT) has two isoforms: soluble (S-COMT), which resides in the cytoplasm, and membrane-bound (MB-MT), anchored to intracellular membranes. COMT is involved in the O-methylation of L-DOPA, dopamine and other catechols. The exact role of MB-COMT is still mostly unclear. We wanted to create a novel genetically modified mouse model that specifically lacks MB-COMT activity and to study their behavioral phenotype. MB-COMT knock-in mutant mice were generated by introducing two point mutations in exon 2 of the Comt gene (ATGCTG->GAGCTC disabling the function of the P2 promoter and allowing only the P1-regulated S-COMT transcription. The first mutation changes methionine to glutamic acid whereas the second one does not affect coding. The expression of the two COMT isoforms, total COMT activity in several areas of the brain and peripheral tissues and extracellular dopamine concentrations after L-DOPA (10 mg/kg) and carbidopa (30 mg/kg) subcutaneous administration were assessed. A battery of behavioral tests was performed to compare MB-COMT deficient mice and their wild type littermates of both sexes. MB-COMT deficient mice were seemingly normal, bred usually and had unaltered COMT activity in the brain and periphery despite a complete lack of the MB-COMT protein. MB-COMT deficient male mice showed higher extracellular dopamine levels than their wild-type littermates in the striatum, but not in the mPFC. In addition, the MB-COMT deficient male mice exhibited a distinct endophenotype characterized by schizophrenia-related behaviors like aggressive behavior and reduced prepulse inhibition. They also had prolonged immobility in the tail suspension test. Both sexes were sensitized to acute pain and had normal motor activity but disturbed short-term memory. Hence the behavioral phenotype was not limited to schizophrenia-related endophenotype and some behavioural findings were not sex-dependent. Our findings indicate that MB-COMT is critical for behavior, and its function in COMT-dependent brain areas cannot be entirely substituted by the remaining S-COMT.Peer reviewe

Depressiooni-sarnane fenotüüp ja häiritud rakusisene signaaliülekanne närviraku adhesioonimolekuli (NCAM)-defitsiitsetel hiirtel

Väitekirja elektroonilisest versioonist puuduvad publikatsioonide täistekstid.Recent hypothesis of the pathogenesis of depression links the development of this disease with reduced brain plasticity. In the central nervous system, plasticity and connectivity in the brain are mediated by the neural cell adhesion molecule (NCAM) and its polysialylated form PSA-NCAM. Therefore, according to the plasticity theory of depression, NCAM may have a crucial role in the development of this condition. NCAM is able to bind a series of counter-receptors including fibroblast growth factor receptor (FGFR), and through the binding, activate the downstream signalling pathways, which all lead to the activation of cyclic-AMP-response element binding protein (CREB). The aim of this study was to investigate whether mice with the constitutive deficiency in NCAM exhibited dysfunctional neuronal plasticity and whether it results in the depressive-like phenotype. By using NCAM-deficient mice, NCAM interaction partners and downstream signalling pathways were studied in detail. Also was investigated, whether a synthetic peptide FGL, which mimics the actions of NCAM, is able to reverse emerged disturbances and restore the activation of intracellular signalling cascades in these animals. The results showed that NCAM deficient mice do have depression-like phenotype accompanied with reduced adult hippocampal neurogenesis. Also was seen the reduction in the activation of NCAM interaction partner FGFR and in downstream signalling pathways like calcium-calmodulin dependent kinases II and IV (CaMKII and IV) and CREB in NCAM-deficient mice. FGL peptide was able to ameliorate the signs of depressive-like behaviour, increase the neurogenesis and furthermore, FGL peptide restored the activation levels of FGFR, CaMKII, CaMKIV and CREB. The ability of FGL to modulate the levels of phosphorylated interaction partners and intracellular signalling pathways might partly explain antidepressant-like properties of the peptide.Üks viimaseid hüpoteese depressiooni patogeneesis seob selle haiguse tekkemehhanismid vähenenud aju plastilisusega. Kesknärvisüsteemis on peamisteks aju plastilisuse kujundajateks närviraku adhesioonimolekul (NCAM) ja tema polüsiaalhappega seotud vorm (PSA-NCAM). Sellest lähtuvalt võiks olla NCAM seotud depressiooni tekkemehhanismidega. Eelnevalt on näidatud, et NCAM on võimeline seostuma ka paljude rakupinna molekulidega ja sealtkaudu vahendama rakusisest signaaliülekannet. Üheks olulisemaks interaktsioonipartneriks NCAM’le on fibroblastide kasvufaktori retseptor (FGFR). Omavahelise seostumise kaudu käivitavad nad mitmeid signaalradu, mis kõik viivad transkriptsioonifaktor CREB aktiveerumiseni. Antud töö eesmärkideks oli selgitada, kas hiirtel, kellel puuduvad kõik NCAM isovormid, võiks esineda depressiooni-sarnane fenotüüp, muutused täiskasvanuea neurogeneesis ja häireid NCAM interaktsioonipartnerite ja NCAM-vahendatud rakusiseste signaalradade aktivatsioonis. Samuti hinnata FGL peptiidi toimet esinevatele muutustele. Töö tulemused näitasid, et NCAM-defitsiitsetel hiirtel esineb depressiooni-sarnane fenotüüp, millega kaasneb ka langenud täiskasvanuea hipokampaalne neurogenees. Samuti esinesid muutused FGFR, kaltsium-kalmoduliin sõltuvate kinaaside II ja IV (CaMKII ja IV) ning CREB aktivatsioonis nendel loomadel. FGL peptiid oli võimeline kõrvaldama kõiki esinevaid muutusi. Kuna FGL peptiid on võimeline aktiveerima NCAM interaktsioonipartnerit, FGFR’i ja alanevaid signaalradu, võiks see osaliselt seletada ka tema antidepressiivset efekti

DNA Methylation Regulates Cocaine-Induced Behavioral Sensitization in Mice

The behavioral sensitization produced by repeated cocaine treatment represents the neural adaptations underlying some of the features of addiction in humans. Cocaine administrations induce neural adaptations through regulation of gene expression. Several studies suggest that epigenetic modifications, including DNA methylation, are the critical regulators of gene expression in the adult central nervous system. DNA methylation is catalyzed by DNA methyltransferases (DNMTs) and consequent promoter region hypermethylation is associated with transcriptional silencing. In this study a potential role for DNA methylation in a cocaine-induced behavioral sensitization model in mice was explored. We report that acute cocaine treatment caused an upregulation of DNMT3A and DNMT3B gene expression in the nucleus accumbens (NAc). Using methylated DNA immunoprecipitation, DNA bisulfite modification, and chromatin immunoprecipitation assays, we observed that cocaine treatment resulted in DNA hypermethylation and increased binding of methyl CpG binding protein 2 (MeCP2) at the protein phosphatase-1 catalytic subunit (PP1c) promoter. These changes are associated with transcriptional downregulation of PP1c in NAc. In contrast, acute and repeated cocaine administrations induced hypomethylation and decreased binding of MeCP2 at the fosB promoter, and these are associated with transcriptional upregulation of fosB in NAc. We also found that pharmacological inhibition of DNMT by zebularine treatment decreased cocaine-induced DNA hypermethylation at the PP1c promoter and attenuated PP1c mRNA downregulation in NAc. Finally, zebularine and cocaine co-treatment delayed the development of cocaine-induced behavioral sensitization. Together, these results suggest that dynamic changes of DNA methylation may be an important gene regulation mechanism underlying cocaine-induced behavioral sensitization