Differential brain expression pattern of Sez6 alternative splicing isoform with deleted transmembrane domain.

Seizure-related gene 6 (Sez6) is a transmembrane protein specifically localized on neuronal dendrites and responsible for dendritic branching and synapse formation. Alternative splicing produces three isoforms of Sez6 mRNAs: the dominant isoform encodes a transmembrane-type protein, whereas the two recessive isoforms encode transmembrane and secretory proteins. In the present study, to clarify the differential functions of these isoforms, the expression patterns resulting from Sez6 splicing isoforms were investigated in the mouse brain as well as in cultured neurons. The whole brains were sliced into coronal sections of 1-mm thickness, and brain areas were punched out from these coronal sections. The mRNA levels of each Sez6 isoform in the prefrontal cortex, cingulate cortex, striatum, hippocampus, and amygdala, where Sez6 expression has been reported previously, were analyzed using a qPCR technique, and primary neurons cultured under different treatment conditions were assessed in terms of increased Sez6 gene expression. Our results show that the splicing patterns of Sez6 were modulated in a brain area-specific manner. In particular, the striatum showed a characteristic splicing pattern of recessive isoforms. Moreover, neuronal activation by convulsant drug stimulation increased recessive isoforms like the dominant isoform in cultured cortical neurons at 5 or 10 days in vitro. In conclusion, alternative splicing of Sez6, as well as of other proteins expressed specifically in the brain, results in brain area-specific expression patterns. Furthermore, the alternative splicing of Sez6 may be modulated by drugs that elevate Sez6 gene expression.

Effects of post-weaning social isolation on social behaviors and oxytocinergic activity in male and female rats.

AIMS: Post-weaning social deprivation is known to induce behavioral and neuronal alterations associated with anxiety and stress responses in adulthood. However, the effects of social deprivation on the development of sociability are poorly understood. We examined the effects of social deprivation on subsequent social behaviors and oxytocinergic activity using socially-isolated (approximately two months post-weaning) male and female rats. MAIN METHODS: The behaviors were analyzed using a social preference test and a social approach test. Immunohistochemical investigations were conducted in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) to examine the effects of social isolation on oxytocinergic activity in these regions. Oxytocinergic activity was measured by quantifying the number of oxytocin neurons expressing Fos following exposure to a novel conspecific. In all of the experiments of this study, ovariectomized females were used for social stimuli. KEY FINDINGS: The behavioral results show that isolation-reared females, but not males, displayed impaired social preference and decreased social approach towards ovariectomized females, compared with the pair-reared group, suggesting low priority of processing social versus non-social stimuli and low motivation for contact with a stranger, respectively. The immunohistochemical results show that social isolation decreased both the number and the ratio of Fos-positive cells in oxytocin neurons in the PVN in females, but not in males, following exposure to ovariectomized females. In the SON, the Fos-positive ratio was decreased in isolation-reared females, but not in males, compared with the pair-reared group. SIGNIFICANCE: Post-weaning social isolation changed social behaviors and oxytocinergic activity in female rats, suggesting that in female rats post-weaning social experiences contribute to the development of sociability. These findings could impact the treatment of social dysfunction in humans.

Glutamate transporter EAAT2: regulation, function, and potential as a therapeutic target for neurological and psychiatric disease.

Glutamate is the predominant excitatory neurotransmitter in the central nervous system. Excitatory amino acid transporter 2 (EAAT2) is primarily responsible for clearance of extracellular glutamate to prevent neuronal excitotoxicity and hyperexcitability. EAAT2 plays a critical role in regulation of synaptic activity and plasticity. In addition, EAAT2 has been implicated in the pathogenesis of many central nervous system disorders. In this review, we summarize current understanding of EAAT2, including structure, pharmacology, physiology, and functions, as well as disease relevancy, such as in stroke, Parkinson's disease, epilepsy, amyotrophic lateral sclerosis, Alzheimer's disease, major depressive disorder, and addiction. A large number of studies have demonstrated that up-regulation of EAAT2 protein provides significant beneficial effects in many disease models suggesting EAAT2 activation is a promising therapeutic approach. Several EAAT2 activators have been identified. Further understanding of EAAT2 regulatory mechanisms could improve development of drug-like compounds that spatiotemporally regulate EAAT2.

Restored glial glutamate transporter EAAT2 function as a potential therapeutic approach for Alzheimer's disease.

Glutamatergic systems play a critical role in cognitive functions and are known to be defective in Alzheimer's disease (AD) patients. Previous literature has indicated that glial glutamate transporter EAAT2 plays an essential role in cognitive functions and that loss of EAAT2 protein is a common phenomenon observed in AD patients and animal models. In the current study, we investigated whether restored EAAT2 protein and function could benefit cognitive functions and pathology in APPSw,Ind mice, an animal model of AD. A transgenic mouse approach via crossing EAAT2 transgenic mice with APPSw,Ind. mice and a pharmacological approach using a novel EAAT2 translational activator, LDN/OSU-0212320, were conducted. Findings from both approaches demonstrated that restored EAAT2 protein function significantly improved cognitive functions, restored synaptic integrity, and reduced amyloid plaques. Importantly, the observed benefits were sustained one month after compound treatment cessation, suggesting that EAAT2 is a potential disease modifier with therapeutic potential for AD.

Small-molecule activator of glutamate transporter EAAT2 translation provides neuroprotection.

Glial glutamate transporter EAAT2 plays a major role in glutamate clearance in synaptic clefts. Several lines of evidence indicate that strategies designed to increase EAAT2 expression have potential for preventing excitotoxicity, which contributes to neuronal injury and death in neurodegenerative diseases. We previously discovered several classes of compounds that can increase EAAT2 expression through translational activation. Here, we present efficacy studies of the compound LDN/OSU-0212320, which is a pyridazine derivative from one of our lead series. In a murine model, LDN/OSU-0212320 had good potency, adequate pharmacokinetic properties, no observed toxicity at the doses examined, and low side effect/toxicity potential. Additionally, LDN/OSU-0212320 protected cultured neurons from glutamate-mediated excitotoxic injury and death via EAAT2 activation. Importantly, LDN/OSU-0212320 markedly delayed motor function decline and extended lifespan in an animal model of amyotrophic lateral sclerosis (ALS). We also found that LDN/OSU-0212320 substantially reduced mortality, neuronal death, and spontaneous recurrent seizures in a pilocarpine-induced temporal lobe epilepsy model. Moreover, our study demonstrated that LDN/OSU-0212320 treatment results in activation of PKC and subsequent Y-box-binding protein 1 (YB-1) activation, which regulates activation of EAAT2 translation. Our data indicate that the use of small molecules to enhance EAAT2 translation may be a therapeutic strategy for the treatment of neurodegenerative diseases.

Increased glial glutamate transporter EAAT2 expression reduces epileptogenic processes following pilocarpine-induced status epilepticus.

Several lines of evidence indicate that glutamate plays a crucial role in the initiation of seizures and their propagation; abnormal glutamate release causes synchronous firing of large populations of neurons, leading to seizures. In the present study, we investigated whether enhanced glutamate uptake by increased glial glutamate transporter EAAT2, the major glutamate transporter, could prevent seizure activity and reduce epileptogenic processes. EAAT2 transgenic mice, which have a 1.5-2 fold increase in EAAT2 protein levels as compared to their non-transgenic counterparts, were tested in a pilocarpine-induced status epilepticus (SE) model. Several striking phenomena were observed in EAAT2 transgenic mice compared with their non-transgenic littermates. First, the post-SE mortality rate and chronic seizure frequency were significantly decreased. Second, neuronal degeneration in hippocampal subfields after SE were significantly reduced. Third, the SE-induced neurogenesis and mossy fiber sprouting were significantly decreased. The severity of cell loss in epileptic mice was positively correlated with that of mossy fiber sprouting and chronic seizure frequency. Our results suggest that increased EAAT2 expression can protect mice against SE-induced death, neuropathological changes, and chronic seizure development. This study suggests that enhancing EAAT2 protein expression is a potential therapeutic approach.

Riluzole produces distinct anxiolytic-like effects in rats without the adverse effects associated with benzodiazepines.

In this study, we investigated the anxiolytic-like effect of riluzole using three different innate anxiety models in rats. In the elevated plus-maze test, riluzole significantly increased the time spent in, and entries into, the open arm after 60 min administration. This finding was supported by results obtained from light/dark and open-field tests. The magnitude of the anxiolytic-like effects of riluzole in each of the behavioral models was similar to those produced by a benzodiazepine, diazepam, suggesting that riluzole has a robust anxiolytic-like activity in rats. To clarify the involvement of sodium channels in this anxiolytic activity, we examined the effect of a co-administered sodium channel activator, veratrine. The anxiolytic-like action of riluzole was diminished by veratrine in the elevated plus-maze, light/dark and open-field tests. Based on these results, it is suggested that the anxiolytic mechanism of riluzole is clearly distinct from that of diazepam. In addition, to examine whether riluzole directly and non-selectively affected the GABA(A)-benzodiazepine receptor complex, we performed three behavioral tests (footprint analysis, Y-maze test and the ethanol-induced sleeping time test) that are closely related to the GABA(A)-benzodiazepine pathways. In contrast to diazepam, riluzole produced no significant effects in these tests. Here, we provide the first report demonstrating that riluzole produces distinct anxiolytic-like effects in rats without the adverse effects associated with benzodiazepines.

Rhotekin modulates differentiation of cultured neural stem cells to neurons.

Rhotekin is a downstream signal of Rho and is expressed in the central nervous system. However, the physiological role of rhotekin in the development of neural stem cells (NSCs) into neurons is unknown. In this study, we knocked down the expression of rhotekin protein with small interfering RNA (siRNA) in the NSCs and in neural differentiated cells and measured cell proliferation, differentiation, neurite length, and survival. By using immunocytochemistry and Western blot, the production of rhotekin was observed in NSCs and neuronal cells. Furthermore, rhotekin production was increased in accordance with neural differentiation. Rhotekin knock-down reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) activity and increased the cell death 72 hr after transfection in neurons. On the other hand, in NSCs, rhotekin knock-down increased MTT activity and the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells. In the present study, we demonstrated that rhotekin is required for maintenance and survival of neurons and positively regulates differentiation and neurite outgrowth. Moreover, we found that rhotekin is produced in NSCs and that the role of rhotekin is to regulate cell proliferation negatively. In conclusion, these results suggest that rhotekin is one of the key molecules in the differentiation of NSCs into neurons.

Dexamethasone indirectly induces Ndrg2 expression in rat astrocytes.

N-myc downstream-regulated gene 2 (Ndrg2) has been associated with cell proliferation, differentiation, and apoptosis. Ndrg2 expression in the brain is induced by glucocorticoid treatment or chronic stress in vivo. It has been postulated that glucocorticoid-induced Ndrg2 expression in astrocytes is regulated by the glucocorticoid response element half-site (GRE1/2) upstream of the Ndrg2 transcription site. Here we examined the mechanisms of dexamethasone-induced Ndrg2 expression in rat astrocytes. Ndrg2 mRNA expression in primary astrocytes was significantly increased after 24 hr of exposure to dexamethasone in a concentration-dependent manner. Dexamethasone-induced Ndrg2 mRNA and protein expression was blocked by pretreatment with RU486, a glucocorticoid receptor antagonist. Moreover, dexamethasone-induced Ndrg2 mRNA expression was reduced by pretreatment with the protein synthesis inhibitor cycloheximide. The Ndrg2 reporter assay showed that deletion of a putative GRE1/2, located upstream of Ndrg2, did not affect induction by dexamethasone. A region between -755 and -701 bp from the transcription start site was shown to regulate induction by dexamethasone using promoter constructs progressively deleted from the 5' to 3' ends. This region contained the predicted transcription factor binding sites for early B-cell factor 1 (Ebf1), nuclear factor-κB (NFκB), and paired box gene 5 (Pax5). Mutation at the NFκB- or Pax5-binding site, but not the Ebf1 binding site, abolished dexamethasone-induced promoter activation. These results indicate that Ndrg2 expression was indirectly induced by dexamethasone at the DNA level, potentially by the binding of NFκB or Pax5 to the transcription factor binding sites, and GRE1/2 was not involved in this induction.

Riluzole rapidly attenuates hyperemotional responses in olfactory bulbectomized rats, an animal model of depression.

Growing evidence indicates that the glutamatergic neurotransmitter system is central to the neurobiology and treatment of depression. Riluzole, a drug currently used to slow the progression of amyotrophic lateral sclerosis (ALS), directly affects the glutamatergic system. In this study, we investigated the effects of riluzole in olfactory bulbectomy (OBX) rats, an animal model of depression. The olfactory bulbs in rats were removed by suction. The emotionality of rats was measured by scoring their responses to given stimuli, i.e., attack, startle, struggle, and fight responses. The OBX rats chronically treated with vehicle for 7 days at 14 days following surgery showed significant increases in emotionality responses. Single (1st day administration) and subchronic (7th day administration) riluzole treatment (1-10 mg/kg, po) significantly and dose-dependently reduced hyperemotional responses in OBX rats. Both single and subchronic riluzole treatment (10 mg/kg, po) had no significant effects on the emotional responses in sham operated rats. In addition, we demonstrated that single riluzole treatment (10 mg/kg, po) significantly decreased extracellular glutamate levels in medial prefrontal cortex of OBX rats by in vivo microdialysis. We provide the first experimental evidence that riluzole rapidly attenuated hyperemotional responses in OBX rats, an animal model of depression.

Neuroserpin is expressed in early stage of neurogenesis in adult rat hippocampus.

In the adult rat hippocampal formation, neurogenesis occurs in the dentate gyrus subgranular zone (SGZ). We used laser capture microdissection and an antidepressant-related genes microarray to analyze gene expression profiles of cells from the SGZ and from the outermost granule cell layer. Of the differentially expressed genes in the SGZ, we focused on neuroserpin, which is highly expressed in the adult rat SGZ. Neuroserpin immunoreactivity was present in cells positive for NeuN (postmitotic cell marker) and Tuj1 (immature neuron marker) but not in cells positive for calbindin (mature neuron marker). Although neuroserpin is expressed during late stage of neurogenesis in development, our results suggest that neuroserpin may play some roles in early stage of neurogenesis in adult rat hippocampus.

Gamma knife radiosurgery for arteriovenous malformations: the Furukawa experience.

The Furukawa experience treating 534 patients with cerebral arteriovenous malformations using gamma knife radiosurgery (GKRS) is summarized. By repeating radiosurgery for any residual nidus after the first GKRS, the rates of cumulative complete obliteration 7 years after this initial GKRS, according to four volume categories (< or =1, 4 > or = >1, 10 > or = > 4, > 10cm3), were 92, 89, 68 and 43%, respectively. Bleeding after GKRS was observed in 8.1% of the patients and was more frequently seen in patients with a large nidus and history of bleeding two or more times before GKRS. Cyst formation was recognized in 4.7% of patients, two thirds of which required some form of surgical intervention. Refinement of the total GKRS system contributed to earlier and more effective nidus obliteration.

Gene expression profiling reveals complex changes in the olfactory bulbectomy model of depression after chronic treatment with antidepressants.

We investigated the effects of antidepressants on the gene expression profile and behavior of olfactory-bulbectomized (OBX) rats. Removal of the main olfactory bulbs in rats alters neuronal function in brain areas involved in emotional regulation, resulting in maladaptive behavioral patterns similar to the symptoms of patients with depression. Previously, we found that OBX-induced behavioral and neuronal abnormalities were completely rescued by chronic treatment with SNC80, an opioid delta agonist, as well as with classical monoaminergic antidepressants. Thus, to determine the basis for this effect, we analyzed gene expression in OBX rat frontal cortex using a GeneChip rat Genome oligonucleotide array after imipramine or SNC80 treatment. We found that imipramine and SNC80 induced the following systematic changes in OBX rats: zinc ion binding; hydrolase activity, acting on acid anhydrides, in phosphorus-containing anhydrides; protein serine/threonine kinase activity; N-acetyltransferase activity; protein modification process; regulation of cellular process; and regulation of neurotransmitter levels. Defining the roles of candidate neuronal systems in antidepressant-induced neural changes are likely to transform the course of research on the biological basis of mood disorders.

Ubiquitin ligase Kf-1 is involved in the endoplasmic reticulum-associated degradation pathway.

Kf-1 was first identified as a gene showing enhanced expression in the cerebral cortex of a sporadic Alzheimer's disease patient. To date, however, the functional properties of Kf-1 protein remain unknown. In this study, immunohistochemical analysis showed that Kf-1 immunoreactivity was detected in rat hippocampus and cerebral cortex neurons. Interestingly, it was colocalized with endoplasmic reticulum (ER) marker. To investigate the specific function of Kf-1 protein, we generated Myc tagged wild type Kf-1 (Myc-Kf-1WT) and RING finger domain deletion mutant of Kf-1 (Myc-Kf-1DeltaR), and then transfected in HEK293 cells. Myc-Kf-1WT displayed a reticular pattern typical of ER localization, with large perinuclear aggregates and colocalized with ER marker, calnexin. Myc-Kf-1WT facilitated ubiquitination of endogenous proteins, whereas Myc-Kf-1DeltaR did not show ubiquitin ligase activity. In addition, we found that Kf-1 interacted with components of the ER-associated degradation (ERAD) pathway, including Derlin-1 and VCP. Taken together, these properties suggest that Kf-1 is an ER ubiquitin ligase involved in the ERAD pathway.

Prg1 is regulated by the basic helix-loop-helix transcription factor Math2.

Math2 (NEX-1/NeuroD6) is a member of the basic helix-loop-helix transcription factor family and is involved in neuronal differentiation and maturation. In this study, we identified the genes targeted by Math2 using DNA microarrays and cultured rat cortical cells transfected with Math2. Of the genes regulated by Math2, we focused on plasticity-related gene 1 (Prg1). Prg1 expression induced by Math2 was confirmed in cultured rat cortical cells and PC12 cells analyzed by real-time quantitative PCR. In the promoter region of rat Prg1, we identified four E-boxes [designated -E1 to -E4 (CANNTG)] recognized by the basic helix-loop-helix transcription factor. Using chromatin immunoprecipitation assays, we found that Math2 directly bound to at least one of these E-boxes. The Prg1 reporter assay showed that -E1 was critical for the regulation of Math2-mediated Prg1 expression. Investigation of the functional roles of Math2 and Prg1 in PC12 cells revealed that 72 h after transfection with either Math2 or Prg1, neurite length and number were significantly induced. Co-transfection with Prg1-siRNA completely inhibited Math2-mediated morphological changes. Our results suggest that Math2 directly regulates Prg1 expression and that the Math2-Prg1 cascade plays an important role in neurite outgrowth in PC12 cells.

Antidepressant-like effects of the delta-opioid receptor agonist SNC80 ([(+)-4-[(alphaR)-alpha-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-methoxyphenyl)methyl]-N,N-diethylbenzamide) in an olfactory bulbectomized rat model.

The responses of olfactory bulbectomized (OBX) rats to antidepressant treatment are similar to those of depressed patients since chronic administration of an antidepressant reverses OBX-induced behavioral and physiological changes. Previously, using several animal models, it was demonstrated that single treatment with delta-opioid receptor agonists produced an antidepressant-like effect. This study examined the antidepressant effects resulting from subchronic exposure for 8 days to the delta-opioid receptor agonist SNC80 in an OBX rat model of depression. The olfactory bulbs were removed by suction. The emotionality of rats was measured by scoring their responses to given stimuli, i.e., attack, startle, struggle, and fight responses. The OBX rats chronically treated with vehicle for 7 days at 14 days following surgery showed a significant increase in emotionality score and a decrease in the time spent and entries in the open arm of a plus-maze. In the case of OBX rats, these changes were dose- and time-dependently reversed by chronic SNC80 treatment (1-10 mg/kg, s.c.) for 7 days, as same as desipramine (10 mg/kg, i.p.). Moreover, the concentration of 5-HT and its metabolite 5-HIAA in the frontal cortex, hippocampus, and amygdala were decreased in OBX rats, and these changes were also normalized by SNC80 treatment, rather than desipramine treatment. In addition, SNC80 also significantly reversed the loss of TH-positive cells produced by OBX in the dorsal raphe. In conclusion, we demonstrated that subchronic SNC80 treatment could completely reverse OBX-induced behavioral abnormalities and defects in serotonergic function.

Identification and expression of frizzled-3 protein in rat frontal cortex after antidepressant and electroconvulsive treatment.

The biological basis for the therapeutic mechanisms of depression are still unknown. While performing EST (expressed sequence tag) analysis to identify some molecular machinery responsible for the antidepressant effect, we determined the full-length nucleotide sequence of rat frizzled-3 protein (Frz3) cDNA. Interestingly, Northern blot analysis demonstrated that elevated levels of Frz3 were expressed continually from embryonic day 20.5 to postnatal 4 weeks in developing rat brain. In adult rat brain, Frz3 mRNA was expressed predominantly in the cerebral cortex and hypothalamus and moderately in the hippocampus. Using real-time quantitative PCR, we demonstrated that chronic treatment with two different classes of antidepressants, imipramine and sertraline, reduced Frz3 mRNA expression significantly in rat frontal cortex. Electroconvulsive treatment (ECT) also reduced Frz3 expression. In contrast, antidepressants and ECT failed to reduce Frz2 expression. Additionally, chronic treatment with the antipsychotic drug haloperidol did not affect Frz3 expression. Recently, the Frz/Wingless protein pathway has been proposed to direct a complex behavioral phenomenon. In conclusion, the Frz3-mediated signaling cascade may be a component of the molecular machinery targeted by therapeutics commonly used to treat depression.

Ndrg2 promotes neurite outgrowth of NGF-differentiated PC12 cells.

Ndrg2 is a member of the N-myc downstream-regulated genes. Thus far, two different isoforms of rat Ndrg2 protein, Ndrg2S and Ndrg2L, have been identified. Recently, we have identified rat Ndrg2 as a novel target molecule of antidepressants and ECT. The functional role of Ndrg2 in the central nervous system, however, remains unclear. In the present study, we examined the expression of endogenous Ndrg2, cellular localization of transfected Ndrg2 protein, and morphological changes resulting from overexpression of Ndrg2 in NGF-differentiated PC12 cells. Neurites began to sprout 1-2 days after exposure to NGF; subsequent neurite growth continued for 5 days. During this time, we evaluated Ndrg2 mRNA expression by real-time quantitative PCR and found that expression significantly increased in a time-dependent manner. Interestingly, V5-conjugated Ndrg2S and Ndrg2L proteins expressed in NGF-differentiated PC12 specifically localized to cell surface membranes and growth cones. Moreover, Ndrg2S and Ndrg2L overexpression promoted neurite elongation in NGF-differentiated PC12 cells. In conclusion, our findings offer novel insights into the physiological roles of Ndrg2 in the central nervous system.

Expression of Ndrg2 in the rat frontal cortex after antidepressant and electroconvulsive treatment.

Although the therapeutic action of antidepressants most likely involves the regulation of serotonergic and noradrenergic signal transduction, no consensus has been reached concerning their precise molecular or cellular mechanisms of action. In the present study, we demonstrated that chronic treatment with a tricyclic antidepressant (imipramine) and a selective serotonin reuptake inhibitor (sertraline) reduced the expression of Ndrg2 mRNA and protein in the rat frontal cortex. Ndrg2 is a member of the N-Myc downstream-regulated genes. Interestingly, repeated ECT also significantly decreased Ndrg2 expression in this region of the brain. These data suggest that Ndrg2 may be a common functional molecule that is decreased after antidepressant treatment and ECT. Although, the functional role of Ndrg2 in the central nervous system remains unclear, our findings suggest that Ndrg2 may be associated with treatment-induced adaptive neural plasticity in the brain, a chronic target of antidepressant action. In conclusion, we have identified Ndrg2 as a candidate target molecule of antidepressants and ECT.