Development of depression-like behavior and altered hippocampal neurogenesis in a mouse model of chronic neuropathic pain.

Chronic-pain patients often suffer from depression. In rodent models of neuropathic pain, animals develop depression-like and anxiety behaviors, indicating a relationship between chronic pain and affective disorders. However, the underlying neurobiological mechanisms linking chronic pain and depression are not yet fully understood. Neurogenesis in the hippocampus is a fundamental process related to brain plasticity. Reduced neurogenesis has been associated with the development of mood disorders and cognitive impairments. The current study aims to elucidate the underlying long-term changes in brain plasticity induced by neuropathic pain in mice at a time point when depression-like behavior has already developed. Furthermore, our focus is set on alterations in neurogenesis in the hippocampus. We found that manifestation of anxiety- and depressive-like behavior as well as cognitive impairment co-occur with decreased survival of newly generated cells but not with impaired proliferative activity or reduced number of immature neurons in the dentate gyrus area of the hippocampus. Moreover, we detected an impairment of differentiation of newly generated cells into mature calbindin-positive neurons, accompanied with a shift towards increased differentiation into astroglial cells. These findings indicate that a reduction in mature functional neurons, rather than reduced proliferation or neuronal progenitor cells, are the long-term changes in hippocampal plasticity that manifest in neuropathic pain conditions after depression-like behavior has developed.

Glucocorticoid Receptor Stimulation Resulting from Early Life Stress Affects Expression of DNA Methyltransferases in Rat Prefrontal Cortex.

Early life stress initiates long-term neurobiological changes that affect stress resilience and increased susceptibility to psychopathology. Maternal separation (MS) is used to cause early life stress and it induces profound neurochemical and behavioral changes that last until adulthood. The molecular pathways of how MS affects the regulation of DNA methyltransferases (Dnmt) in brain have not been entirely characterized. We evaluated MS effects on Dnmt1, Dnmt3a and Dnmt3b expression, DNMT enzyme activity and glucocorticoid receptor (GR) recruitment to different Dnmt loci in the prefrontal cortex (PFC) of Wistar rats. We found increased plasma corticosterone levels after MS that were associated with induced Dnmt expression and enzyme activity in rat PFC at post-natal day 15 (PND15). Chromatin immunoprecipitation showed increased binding of GR at the Dnmt3b promoter after MS, suggesting that genomic signaling of GR is an important regulatory mechanism for the induced Dnmt3b expression and DNMT activity. Although GR also binds to Dnmt3a promoter and a putative regulatory region in intron 3 in rat PFC, its expression after maternal separation may be influenced by other mechanisms. Therefore, GR could be a link between early life stress experience and long-term gene expression changes induced by aberrant DNA methylation.

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.

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 (10 mg kg-1) plus carbidopa (30 mg 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.

Prolyl endopeptidase is involved in the degradation of neural cell adhesion molecules in vitro.

Membrane-associated glycoprotein neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) play an important role in brain plasticity by regulating cell-cell interactions. Here, we demonstrate that the cytosolic serine protease prolyl endopeptidase (PREP) is able to regulate NCAM and PSA-NCAM. Using a SH-SY5Y neuroblastoma cell line with stable overexpression of PREP, we found a remarkable loss of PSA-NCAM, reduced levels of NCAM180 and NCAM140 protein species, and a significant increase in the NCAM immunoreactive band migrating at an apparent molecular weight of 120 kDa in PREP-overexpressing cells. Moreover, increased levels of NCAM fragments were found in the concentrated medium derived from PREP-overexpressing cells. PREP overexpression selectively induced an activation of matrix metalloproteinase-9 (MMP-9), which could be involved in the observed degradation of NCAM, as MMP-9 neutralization reduced the levels of NCAM fragments in cell culture medium. We propose that increased PREP levels promote epidermal growth factor receptor (EGFR) signaling, which in turn activates MMP-9. In conclusion, our findings provide evidence for newly-discovered roles for PREP in mechanisms regulating cellular plasticity through NCAM and PSA-NCAM.

Pharmacological approach for targeting dysfunctional brain plasticity: Focus on neural cell adhesion molecule (NCAM).

Brain plasticity refers to the ability of the brain to undergo functionally relevant adaptations in response to external and internal stimuli. Alterations in brain plasticity have been associated with several neuropsychiatric disorders, and current theories suggest that dysfunctions in neuronal circuits and synaptogenesis have a major impact in the development of these diseases. Among the molecules that regulate brain plasticity, neural cell adhesion molecule (NCAM) and its polysialylated form PSA-NCAM have been of particular interest for years because alterations in NCAM and PSA-NCAM levels have been associated with memory impairment, depression, autistic spectrum disorders and schizophrenia. In this review, we discuss the roles of NCAM and PSA-NCAM in the regulation of brain plasticity and, in particular, their roles in the mechanisms of depression. We also demonstrate that the NCAM-mimetic peptides FGL and Enreptin are able to restore disrupted neuronal plasticity. FGL peptide has also been demonstrated to ameliorate the symptoms of depressive-like behavior in NCAM-deficient mice and therefore, may be considered a new drug candidate for the treatment of depression as well as other neuropsychiatric disorders with disrupted neuroplasticity.

NCAM-deficient mice show prominent abnormalities in serotonergic and BDNF systems in brain - Restoration by chronic amitriptyline.

Mood disorders are associated with alterations in serotonergic system, deficient BDNF (brain-derived neurotrophic factor) signaling and abnormal synaptic plasticity. Increased degradation and reduced functions of NCAM (neural cell adhesion molecule) have recently been associated with depression and NCAM deficient mice show depression-related behavior and impaired learning. The aim of the present study was to investigate potential changes in serotonergic and BDNF systems in NCAM knock-out mice. Serotonergic nerve fiber density and SERT (serotonin transporter) protein levels were robustly reduced in the hippocampus, prefrontal cortex and basolateral amygdala of adult NCAM(-)(/-) mice. This SERT reduction was already evident during early postnatal development. [(3)H]MADAM binding experiments further demonstrated reduced availability of SERT in cell membranes of NCAM(-)(/-) mice. Moreover, the levels of serotonin and its major metabolite 5-HIAA were down regulated in the brains of NCAM(-)(/-) mice. NCAM(-)(/-) mice also showed a dramatic reduction in the BDNF protein levels in the hippocampus and prefrontal cortex. This BDNF deficiency was associated with reduced phosphorylation of its receptor TrkB. Importantly, chronic administration of antidepressant amitriptyline partially or completely restored these changes in serotonergic and BDNF systems, respectively. In conclusion, NCAM deficiency lead to prominent and persistent abnormalities in brain serotonergic and BDNF systems, which likely contributes to the behavioral and neurobiological phenotype of NCAM(-/-) mice.

PSA modification of NCAM supports the survival of injured retinal ganglion cells in adulthood.

Neural cell adhesion molecule (NCAM) is known as the cell surface glycoprotein, and it belongs to the immunoglobulin superfamily of adhesion molecules. Polysialic acid (PSA) is a carbohydrate attached to NCAM via either of two specific sialyltransferases: ST8SiaII and ST8SiaIV. Polysialylated neural cell adhesion molecule (PSA-NCAM) mediates cell interactions, plays a role in axon growth, migration, synaptic plasticity during development and cell regeneration. Some evidence has shown that PSA-NCAM supports the survival of neurons. It was demonstrated that PSA-NCAM is present in abundance in the retina during development and in adulthood. The aim of this study was to investigate whether PSA-NCAM promotes retinal ganglion cell (RGC) survival in transgenic mice with deficiencies in sialyltransferases or NCAM or after the administration of endoneuraminidase (Endo-N). RGC injury was induced by intravitreal administration of kainic acid (KA). These studies showed that injection of Endo-N after 14 days enhances the toxicity of KA to RGCs in wild-type (WT) mice by 18%. In contrast, in knockout mice (ST8SiaII-/-, ST8SiaIV-/-, NCAM-/-), survival of RGCs after KA injury did not change. Deficiencies of either ST8SiaII or ST8SiaIV did not influence the level of PSA-NCAM in the adult retina, however, in neonatal animals, decreased levels of PSA-NCAM were observed. In knockout ST8SiaII-/- adults, a reduced number of RGCs was detected, whereas in contrast, increased numbers of RGCs were noted in NCAM-/- mice. In conclusion, these data demonstrate that PSA-NCAM supports the survival of injured RGCs in adulthood. However, the role of PSA-NCAM in the adult retina requires further clarification.

Schizophrenia-like phenotype of polysialyltransferase ST8SIA2-deficient mice.

Posttranslational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is crucial for nervous system development and brain plasticity. PolySia attachment is catalyzed by the polysialyltransferases (polySTs) ST8SIA2 and ST8SIA4, two enzymes with distinct but also common functions during neurodevelopment and in the adult brain. A growing body of evidence links aberrant levels of NCAM and polySia as well as variation in the ST8SIA2 gene to neuropsychiatric disorders, including schizophrenia. To investigate whether polyST deficiency might cause a schizophrenia-like phenotype, St8sia2 (-/-) mice, St8sia4 (-/-) mice and their wildtype littermates were assessed neuroanatomically and subjected to tests of cognition and sensorimotor functions. St8sia2 (-/-) but not St8sia4 (-/-) mice displayed enlarged lateral ventricles and a size reduction of the thalamus accompanied by a smaller internal capsule and a highly disorganized pattern of fibers connecting thalamus and cortex. Reduced levels of the vesicular glutamate transporter VGLUT2 pointed towards compromised glutamatergic thalamocortical input into the frontal cortex of St8sia2 (-/-) mice. Both polyST-deficient lines were impaired in short- and long-term recognition memory, but only St8sia2 (-/-) mice displayed impaired working memory and deficits in prepulse inhibition. Furthermore, only the St8sia2 (-/-) mice exhibited anhedonic behavior and increased sensitivity to amphetamine-induced hyperlocomotion. These results reveal that reduced polysialylation in St8sia2 (-/-) mice leads to pathological brain development and schizophrenia-like behavior. We therefore propose that genetic variation in ST8SIA2 has the potential to confer a neurodevelopmental predisposition to schizophrenia.

Maternal separation is associated with DNA methylation and behavioural changes in adult rats.

Early life stress is known to promote long-term neurobiological changes, which may underlie the increased risk of psychopathology. Maternal separation (MS) is used as an early life stressor that causes profound neurochemical and behavioural changes in the pups that persist into adulthood. However, the exact mechanism of how MS alters these behavioural changes is not yet understood. Epigenetic modifications, such as DNA methylation, are critical regulators of persistent gene expression changes and may be related to behavioural disorders. The aim of the present study was to investigate whether early life stress on rats could alter cocaine-induced behavioural sensitisation in adulthood via aberrant DNA methylation. We have three main findings: (1) MS increased DNA methyltransferases (DNMTs) expression in the nucleus accumbens (NAc) of infant and adult rats; (2) MS induced DNA hypomethylation on a global level in the NAc, and hypermethylation of the promoter regions of the protein phosphatase 1 catalytic subunit (PP1C) and adenosine A2Areceptor (A2AR) genes, which was associated with their transcriptional downregulation in the NAc; (3) MS-induced molecular changes paralleled an increased response to cocaine-induced locomotor activity and exploratory behaviour in adult rats. Thus, our results suggest that stressful experiences in early life may create a background, via aberrant DNA methylation, which promotes the development of cocaine-induced behavioural sensitisation in adulthood.

Partial reduction in neural cell adhesion molecule (NCAM) in heterozygous mice induces depression-related behaviour without cognitive impairment.

The neural cell adhesion molecule (NCAM) plays an important role in brain plasticity. Using mice deficient in all isoforms of NCAM we have previously demonstrated that constitutive deficiency in the NCAM gene (NCAM-/-) resulted in cognitive impairment, anhedonic behaviour and a reduced ability to cope with stress. This was accompanied by reduced basal phosphorylation of the fibroblast growth factor receptor 1 (FGFR1) and reduced phosphorylation of calcium-calmodulin kinase (CaMK) II and IV and cAMP response element binding protein (CREB). The present study was aimed to investigate how partial deficiency in NCAM in mice (NCAM+/-) affected phenotype. We found that NCAM+/- mice showed a longer period of immobility in the tail suspension test, increased latency to feed in the novelty-suppressed feeding test and reduced preference for sucrose in sucrose preference test. Both NCAM+/- and NCAM-/- mice showed reduced extinction of contextual fear. In contrast to NCAM-/- mice, NCAM+/- mice did not demonstrate memory impairment in either object recognition or contextual fear conditioning tests. Levels of phosphorylated FGFR1 in the hippocampus and prefrontal/frontal cortex of NCAM+/- mice were partially reduced and no changes in the phosphorylation of CaMKII, CaMKIV or CREB in the hippocampus were found. We conclude that a constitutive partial reduction in NCAM proteins results in a behavioural phenotype related to depression without impairment in cognitive functions, also affecting the level of FGFR1 phosphorylation without major alterations in CaMKII and CaMKIV intracellular signalling. Partial reduction in FGFR1 phosphorylation might explain the observed behavioural phenotype in NCAM+/- mice.

Repeated citalopram administration counteracts kainic acid-induced spreading of PSA-NCAM-immunoreactive cells and loss of reelin in the adult mouse hippocampus.

Systemic or intracerebral administration of kainic acid in rodents induces neuronal death followed by a cascade of neuroplastic changes in the hippocampus. Kainic acid-induced neuroplasticity is evidenced by alterations in hippocampal neurogenesis, dispersion of the granule cell layer and re-organisation of mossy fibres. Similar abnormalities are observed in patients with temporal lobe epilepsy and, therefore, kainic acid-induced hippocampal neuroplasticity might mimic pathological mechanisms leading to the formation of 'epileptic brain' in patients with temporal lobe epilepsy. Previous studies have demonstrated that selective serotonin re-uptake inhibitor antidepressants might reduce the severity of seizures in epileptic patients and reduce neuronal death in laboratory animal models of kainic acid-induced neurotoxicity. In the present study, we investigated whether kainic acid-induced neuroplasticity in mice is modulated by the repeated administration of citalopram, a selective serotonin reuptake inhibitor. We found that at the histopathological level, repeated citalopram treatment counteracted the kainic acid-induced neuronal loss and dispersion of young granule neurons expressing the polysialylated neural cell adhesion molecule within the granule cell layer of the hippocampus. Citalopram also counteracted the downregulation of reelin on both mRNA and protein levels induced by kainic acid administration. Our findings indicate that repeated administration of citalopram is able to prevent kainic acid-induced abnormal brain plasticity and thereby prevent the formation of an epileptic phenotype.

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.

NCAM-mimetic, FGL peptide, restores disrupted fibroblast growth factor receptor (FGFR) phosphorylation and FGFR mediated signaling in neural cell adhesion molecule (NCAM)-deficient mice.

Neural cell adhesion molecule (NCAM) is a membrane-bound glycoprotein expressed on the surface of neuronal and glial cells. Previous in vitro studies have demonstrated that NCAM promotes neuronal functions largely via three main interaction partners: the fibroblast growth factor receptor (FGFR), a member of Src family of tyrosine kinases, Fyn and Raf1 kinase which all activate different intracellular signaling pathways. The objective was to clarify, which signaling pathways are being disrupted in NCAM knockout mice and whether FGL peptide is able to restore observed disruptions. Therefore we compared the levels of phosphorylation of FGFR1, Src kinase Fyn, Raf1 kinase, MAP kinases, Akt kinase and calcium/calmodulin-dependent kinases II and IV (CaMKII and CaMKIV) in the hippocampus of NCAM knockout mice to their wild-type littermates. The data of our study show that mice constitutively deficient in all isoforms of NCAM have decreased basal phosphorylation levels of FGFR1 and CaMKII and CaMKIV. Furthermore, NCAM-mimetic, FGL peptide, is found to be able to restore FGFR1, CaMKII and CaMKIV phosphorylation levels and thereby mimic the interactions of NCAM at this receptor in NCAM deficient mice. Also, we found that Fyn(Tyr530), Raf1, MAP kinases and Akt kinase phosphorylation in adult animals is not affected by NCAM deficiency but interestingly, we found an over-expression of another cell adhesion molecule L1. We conclude that in NCAM deficient mice FGFR1-dependent signaling is disrupted and it can be restored by FGL peptide.

Depression-like behaviour in neural cell adhesion molecule (NCAM)-deficient mice and its reversal by an NCAM-derived peptide, FGL.

The neural cell adhesion molecule (NCAM) plays a pivotal role in brain plasticity. Brain plasticity itself has a crucial role in the development of depression. The aim of this study was to analyze whether NCAM-deficient (NCAM(-/-)) mice exhibit depression-like behaviour and whether a peptide termed FGL, derived from the NCAM binding site for the fibroblast growth factor (FGF) receptor, is able to reverse the depression-like signs in NCAM(-/-) mice. Our study showed that NCAM(-/-) mice demonstrated increased freezing time in the tail-suspension test and reduced preference for sucrose consumption in the sucrose preference test, reduced adult neurogenesis in the dentate gyrus and reduced levels of the phosphorylated cAMP response element-binding protein (pCREB) in the hippocampus. FGL administered acutely or repeatedly reduced depression-like behaviour in NCAM(-/-) mice without having an effect on their wild-type littermates. Repeated administration of FGL enhanced survival of the newly born neurons in NCAM(-/-) mice and increased the levels of pCREB in both NCAM(+/+) and NCAM(-/-) mice. In conclusion, our data demonstrate that NCAM deficiency in mice results in a depression-like phenotype which can be reversed by the acute or repeated administration of FGL. The results also suggest a role of the deficit in NCAM signalling through the FGF receptor in depression.

Dysregulated CREB signaling pathway in the brain of neural cell adhesion molecule (NCAM)-deficient mice.

The neural cell adhesion molecule (NCAM) mediates cell-cell interactions and plays an important role in processes associated with neural plasticity, including learning and memory formation. It has been shown that mice deficient in all isoforms of NCAM (NCAM-/- mice) demonstrate impairment in long-term plasticity at multiple hippocampal synapses, disrupted spatial learning, and impaired contextual and auditory-cued fear conditioning. The formation of long-term memory is associated with activation of transcription factor CREB (cAMP response element binding protein). The aims of this study were to investigate NCAM-mediated signaling transduction pathways and the levels of the phosphorylated (Ser133) active form of the CREB in the brain structures (the pre- and frontal cortex, basolateral amygdala, and hippocampus) involved in the memory formation in NCAM-deficient mice. Immunohistochemical analysis revealed reduced levels of pCREB in the prefrontal cortex (PFC), frontal cortex (FC), CA3 subregion of the hippocampus (CA3) and basolateral nucleus of amygdala (BLA) in NCAM-/- mice. NCAM-/- mice had also reduced levels of the phosphorylated CaMKII and CaMKIV in PFC/FC and the hippocampus, which are the downstream signaling molecules of NCAM. The levels of non-phosphorylated kinases did not differ from those seen in the wild-type mice. These results provide evidence that NCAM deficiency results in the dysregulation of CREB-mediated signaling pathways in the brain regions, which is related to the formation of memory.