Osalise või täieliku NCAM valgu puudulikkusega hiirte fenotüüp

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Sünaptiline plastilisus on aju plastilisuse üks oluline osa, mis võimaldab ajul pidevalt luua ja eemaldada rakkudevahelisi kontakte. Aju plastilisus, mis hõlmab närvikoe erinevaid morfoloogilisi muutusi, on oluline faktor nii kognitiivsetele võimetele nagu õppimine, mälu, aistingud ja teadvus kui ka meeleoluseisunditele. Neuronaalsete rakkude adhesioonimolekul (NCAM) ja tema polüsialüülitud vorm (PSA-NCAM) on valgud, mis on üheks peamisteks sünaptilise plastilisuse kujundajateks. Eelnevad uuringud kinnitavad, et NCAM molekulid on võimelised seostuma nii omavahel kui ka teiste erinevate rakupinna valkudega. Sel moel osaleb NCAM/PSA-NCAM erinevate signaalradade käivitamises ja regulatsioonis. Üheks olulisemaks NCAMi interaktsioonipartneriks on fibroblastide kasvufaktori retseptor 1 (FGFR1). Käesoleva uurimistöö eesmärgiks oli selgitada, kuidas osaline (NCAM+/-) või täielik (NCAM-/-) NCAM/PSA-NCAMi puudumine hiirtel mõjutab nende loomade fenotüüpi; vaadeldi, kas NCAM-/- hiirte ärevuskäitumine võib olla mõjutatud nende halvenenud kognitiivsetest võimetest. Selgitamaks, milline signaalrada vastutab kognitiivsete ja milline depressioonisarnase käitumise eest, uuriti nendel hiirtel NCAM-vahendatud signaalradu, peamisi interaktsioonipartnereid ning serotonergilist süsteemi. Antud töö tulemused kinnitasid, et NCAM-/- hiirtel esines depressioonisarnane käitumine ning olid vähenenud kognitiivsed võimed. NCAM+/- hiirtel esines samuti depressioonisarnane käitumine, kuid nende kognitiivsed võimed ei olnud kontrollhiirtega võrreldes muutunud. Selgus ka, et NCAM-/- hiirte käitumine ärevuskatsetes on mõjutatud nende halvenenud kognitiivsetest võimetest. Leiti, et nii NCAM+/- kui NCAM-/- hiirtel esinesid muutused FGFR1 aktivatsioonis, kuid erinevalt NCAM-/- hiirtest ei kaasnenud NCAM+/- hiirtel FGFR1 aktivatsiooni langusega kaltsium-kalmoduliinist sõltuvate kinaaside II ja IV (CaMKII ja IV) ning transkriptsioonifaktori CREB aktivatsiooni muutust, mis võibki olla nende loomade käitumusliku fenotüübi põhjus. Ühtlasi leiti, et NCAM puudulikkus mõjutab serotoniini transporteri ekspressiooni ning oletatavalt just vähenenud serotoniini transporteri ekspressioon võib olla nendel loomadel ilmnenud depressioonisarnase käitumise põhjuseks.Synaptic plasticity is one of the main factors responsible for brain plasticity. For sense and consciousness, emotional behaviour and for cognitive processes, such as learning and memory, continuous activity-induced remodelling of neuronal circuits is required. Neural cell adhesion molecule (NCAM) and its polysialyted form (PSA-NCAM) are the most significant key players in synaptic plasticity, providing stable connections between cells and at the same time, remodelling synaptic networks. It has been shown that NCAM is capable of binding to a number of proteins on the cell surface including the NCAM molecule itself on the opposing cell surface. Thus, many downstream signal pathways are dependent on NCAM/PSA-NCAM-mediated cellular signalling. One of the major interaction partners of NCAM is fibroblast growth factor receptor 1 (FGFR1). The aim of this study was to explore the effect of partial (NCAM+/-) or complete (NCAM-/-) deficiency in NCAM/PSA-NCAM on the behavioural phenotype of mice and to illuminate how disrupted cognitive abilities influence NCAM-/- mice in anxiety tests. The intracellular pathway responsible for the cognitive dysfunction in the mice and those pathways implicated in the reduced ability of the mice to cope with stress were investigated by examining the main NCAM pathways and NCAM interaction partners and also alterations in the serotonergic system. The data show that NCAM-/- mice demonstrate a depression-like behaviour with altered cognitive functions. NCAM+/- mice demonstrate a depression-like behaviour without alterations in cognitive functions. The data also show that the impaired cognition of NCAM-/- mice affect their behaviour in anxiety tests. We found that both NCAM+/- and NCAM-/- mice showed reduced phosphorylation of the FGFR1 but in contrast to NCAM-/- mice, the observed behavioural phenotype in NCAM+/- mice was not associated with alterations in the basal levels of the transcription factor CREB or phosphorylated Ca2+-calmodulin-dependent protein kinase II and IV (CaMKII and CaMKIV).We also found that reduced levels of NCAM/PSA-NCAM led to alterations in the expression of the serotonin transporter. Thus, the observed depression-like phenotype in NCAM+/- or NCAM-/- mice may be caused by a reduction in expression of the serotonin transporter

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

Enhanced Cognition and Neurogenesis in miR-146b Deficient Mice

The miR-146 family consists of two microRNAs (miRNAs), miR-146a and miR-146b, which are both known to suppress a variety of immune responses. Here in this study, we show that miR-146b is abundantly expressed in neuronal cells, while miR-146a is mainly expressed in microglia and astroglia of adult mice. Accordingly, miR-146b deficient (Mir146b-/-) mice exhibited anxiety-like behaviors and enhanced cognition. Characterization of cellular composition of Mir146b-/- mice using flow cytometry revealed an increased number of neurons and a decreased abundancy of astroglia in the hippocampus and frontal cortex, whereas microglia abundancy remained unchanged. Immunohistochemistry showed a higher density of neurons in the frontal cortex of Mir146b-/- mice, enhanced hippocampal neurogenesis as evidenced by an increased proliferation, and survival of newly generated cells with enhanced maturation into neuronal phenotype. No microglial activation or signs of neuroinflammation were observed in Mir146b-/- mice. Further analysis demonstrated that miR-146b deficiency is associated with elevated expression of glial cell line-derived neurotrophic factor (Gdnf) mRNA in the hippocampus, which might be at least in part responsible for the observed neuronal expansion and the behavioral phenotype. This hypothesis is partially supported by the positive correlation between performance of mice in the object recognition test and Gdnf mRNA expression in Mir146b-/- mice. Together, these results show the distinct function of miR-146b in controlling behaviors and provide new insights in understanding cell-specific function of miR-146b in the neuronal and astroglial organization of the mouse brain