Junfa Li

Fluoxetine increases the activity of the ERK-CREB signal system and alleviates the depressive-like behavior in rats exposed to chronic forced swim stress.

Our previous research indicates that the extracellular signal-regulated kinase (ERK)-cyclic AMP-responsive-element-binding protein (CREB) signal system may be involved in the molecular mechanism of depression. The present study further investigated the effect of antidepressant fluoxetine on the ERK-CREB signal system and the depressive-like behaviors in rats. Fluoxetine was administrated to either naive rats or stressed rats for 21 days. The results showed that chronic forced swim stress induced depressive-like behaviors and decreased the levels of P-ERK2, P-CREB, ERK1/2 and CREB in hippocampus and prefrontal cortex. Fluoxetine alleviated the depressive-like behaviors and reversed the disruptions of the P-ERK2 and P-CREB in stressed rats. Fluoxetine also exerted mood-elevating effect and increased the levels of the P-ERK2 and P-CREB in naive rats. These results suggest that the ERK-CREB signal system may be the targets of the antidepressant action of fluoxetine and participate in the neuronal mechanism of depression.

Neurogranin/RC3 Enhances Long-Term Potentiation and Learning by Promoting Calcium-Mediated Signaling

In neurons, neurogranin (Ng) binds calmodulin (CaM), and its binding affinity is reduced by increasing Ca2+, phosphorylation by PKC, or oxidation by oxidants. Ng concentration in the hippocampus of adult mice varied broadly (Ng+/+, ∼160-370 and Ng+/-, ∼70-230 pmol/mg); the level in Ng+/+ mice is one of the highest among all neuronal CaM-binding proteins. Among Ng+/- mice, but less apparent in Ng+/+, a significant relationship existed between their hippocampal levels of Ng and performances in the Morris water maze. Ng-/- mice performed poorly in this task; they also displayed deficits in high-frequency-induced long-term potentiation (LTP) in area CA1 of hippocampal slices, whereas low-frequency-induced long-term depression was enhanced. Thus, compared with Ng+/+ mice, the frequency-response curve of Ng-/- shifted to the right. Paired-pulse facilitation and synaptic fatigue during prolonged stimulation at 10 Hz (900 pulses) were unchanged in Ng-/- slices, indicating their normal presynaptic function. Measurements of Ca2+ transients in CA1 pyramidal neurons after weak and strong tetanic stimulations (100 Hz, 400 and 1000 msec, respectively) revealed a significantly greater intracellular Ca2+ ([Ca2+]i) response in Ng+/+ compared with Ng-/- mice, but the decay time constants did not differ. The diminished Ca2+ dynamics in Ng-/- mice are a likely cause of their decreased propensity to undergo LTP. Thus, Ng may promote a high [Ca2+]i by a “mass-action” mechanism; namely, the higher the Ng concentration, the more Ng-CaM complexes will be formed, which effectively raises [Ca2+]i at any given Ca2+ influx. This mechanism provides potent signal amplification in enhancing synaptic plasticity as well as learning and memory.

The FBP Interacting Repressor Targets TFIIH to Inhibit Activated Transcription

FUSE-binding protein (FBP) binds the single-stranded far upstream element of active c-myc genes, possesses potent transcription activation and repression domains, and is necessary for c-myc expression. A novel 60 kDa protein, the FBP interacting repressor (FIR), blocked activator-dependent, but not basal, transcription through TFIIH. Recruited through FBPs nucleic acid-binding domain, FIR formed a ternary complex with FBP and FUSE. FIR repressed a c-myc reporter via the FUSE. The amino terminus of FIR contained an activator-selective repression domain capable of acting in cis or even in trans in vivo and in vitro. The repression domain of FIR targeted only TFIIHs p89/XPB helicase, required at several stages in transcription, but not factors required for promoter selection. Thus, FIR locks TFIIH in an activation-resistant configuration that still supports basal transcription.

The vasopressin 1b receptor is prominent in the hippocampal area CA2 where it is unaffected by restraint stress or adrenalectomy

The vasopressin 1b receptor (Avpr1b) is one of two principal receptors mediating the behavioral effects of vasopressin (Avp) in the brain. Avpr1b has recently been shown to strongly influence social forms of aggression in mice and hamsters. This receptor appears to play a role in social recognition and motivation as well as in regulating the hypothalamic-pituitary-adrenal axis. Most of these studies have been performed in knockout mice, a species in which the localization of the Avpr1b has not been described, thus precluding correlations with the behaviors. We performed in situ hybridization histochemistry (ISHH) with specific probes and found especially prominent expression within the CA2 pyramidal neurons of the hippocampus, with much lower expression in the hypothalamic paraventricular nucleus and amygdala. Reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed expression in those as well other areas in which the ISHH was not sensitive enough to detect labeled cells (e.g. piriform cortex, septum, caudate-putamen and lower brainstem areas). Mouse Avpr1b transcript levels were not altered in the CA2 field by restraint stress or adrenalectomy. Finally, ISHH and RT-PCR showed expression of the Avpr1b gene in the rat and human hippocampi as well. We suggest that the CA2 field may form or retrieve associations (memories) between olfactory cues and social encounters.

The depressive-like behaviors are correlated with decreased phosphorylation of mitogen-activated protein kinases in rat brain following chronic forced swim stress

In the present study, 40 Sprague-Dawley rats were divided into forced swim stress group and controls, with 20 rats in each group (10 for behavioral tests, 10 for protein detection). The forced swim stress group received swim stress for 14 consecutive days, and the controls were stress-free. After stress, 20 rats were tested for behavioral observation using body weight gain, open field, elevated plus-maze and saccharin preference test, and 20 rats were decapitated for protein detection. The extracellular signal-regulated kinase (Erk) and phospho-Erk (P-Erk) in the hippocampus and prefrontal cortex were determined using western blot. It was found that the body weight gain of stressed animals during the 7 stressed days and the 14 stressed days was significantly decreased compared to that of controls. Stressed animals spent less time in open arms and longer time in closed arms. The stressed animals demonstrated decreased locomotor activity and increased grooming in open field. The saccharin solution intake and the ratio of saccharin solution intake to total liquid intake were both decreased in the stressed group. Stressed animals showed decreased P-Erk2 and decreased ratio of P-Erk2 to total Erk2 in the hippocampus and prefrontal cortex, but their Erk1/2 was increased in the prefrontal cortex with no change in hippocampus. The saccharin solution intake positively correlated with the P-Erk2 in the hippocampus and negatively correlated with the Erk2 in the prefrontal cortex. In conclusion, chronic forced swim stress was a good animal model of depression, and it induced depressive-like behavior and decreased P-Erk2 in the hippocampus and prefrontal cortex in rats. The depressive-like behaviors were correlated with decreased phosphorylation of Erk, which suggested that the dysfunction of Erk activity might be one of biological mechanisms underlying depression induced by stress.

N-Methyl-d-aspartate Induces Neurogranin/RC3 Oxidation in Rat Brain Slices

Neurogranin/RC3 (Ng), a postsynaptic neuronal protein kinase C (PKC) substrate, binds calmodulin (CaM) at low level of Ca2+. In vitro, rat brain Ng can be oxidized by nitric oxide (NO) donors and by oxidants to form an intramolecular disulfide bond with resulting downward mobility shift on nonreducing SDS-polyacrylamide gel electrophoresis. The oxidized Ng, as compared with the reduced form, is a poorer substrate of PKC but like the PKC-phosphorylated Ng has a lower affinity for CaM than the reduced form. To investigate the physiological relevance of Ng oxidation, we tested the effects of neurotransmitter,N-methyl-d-aspartate (NMDA), NO donors, and other oxidants such as hydrogen peroxide and oxidized glutathione on the oxidation of this protein in rat brain slices. Western blot analysis showed that the NMDA-induced oxidation of Ng was rapid and transient, it reached maximum within 3–5 min and declined to base line in 30 min. The response was dose-dependent (EC50 ∼100 μm) and could be blocked by NMDA-receptor antagonist 2-amino-5-phosphonovaleric acid and by NO synthase inhibitorN G-nitro-l-arginine methyl ester and N G-monomethyl-l-arginine. Ng was oxidized by NO donors, sodium nitroprusside,S-nitroso-N-acetylpenicillamine, andS-nitrosoglutathione, and H2O2 at concentrations less than 0.5 mm. Oxidation of Ng in brain slices induced by sodium nitroprusside could be reversed by dithiothreitol, ascorbic acid, or reduced glutathione. Reversible oxidation and reduction of Ng were also observed in rat brain extracts, in which oxidation was enhanced by Ca2+ and the oxidized Ng could be reduced by NADPH or reduced glutathione. These results suggest that redox of Ng is involved in the NMDA-mediated signaling pathway and that there are enzymes catalyzing the oxidation and reduction of Ng in the brain. We speculate that the redox state of Ng, similar to the state of phosphorylation of this protein, may regulate the level of CaM, which in turn modulates the activities of CaM-dependent enzymes in the neurons.

Downregulation of miR-181b in mouse brain following ischemic stroke induces neuroprotection against ischemic injury through targeting heat shock protein A5 and ubiquitin carboxyl-terminal hydrolase isozyme L1

Understanding the molecular mechanism of cerebral hypoxic preconditioning (HPC)‐induced endogenous neuroprotection may provide potential therapeutic targets for ischemic stroke. By using bioinformatics analysis, we found that miR‐181b, one of 19 differentially expressed miRNAs, may target aconitate hydratase (ACO2), heat shock protein A5 (HSPA5), and ubiquitin carboxyl‐terminal hydrolase isozyme L1 (UCHL1) among 26 changed protein kinase C isoform‐specific interacting proteins in HPC mouse brain. In this study, the role of miR‐181b in oxygen–glucose deprivation (OGD)‐induced N2A cell ischemic injury in vitro and mouse middle cerebral artery occlusion (MCAO)‐induced cerebral ischemic injury in vivo, and its regulation of ACO2, HSPA5, and UCHL1 were further determined. We found that miR‐181b expression levels significantly decreased in mouse brain following MCAO and in OGD‐treated N2A cells. Up‐ and downregulation of miR‐181b by transfection of pre‐ or anti‐miR‐181b could negatively regulate HSPA5 and UCHL1 (but not ACO2) protein levels as well as N2A cell death and programmed cell death in OGD‐treated N2A cells. By using a T7 promoter‐driven control dual luciferase assay, we confirmed that miR‐181b could bind to the 3′‐untranslated rergions of HSPA5 and UCHL1 mRNAs and repress their translations. miR‐181b antagomir reduced caspase‐3 cleavage and neural cell loss in cerebral ischemic cortex and improved neurological deficit of mice after MCAO. In addition, HSPA5 and UCHL1 short interfering RNAs (siRNAs) blocked anti‐miR‐181b‐mediated neuroprotection against OGD‐induced N2A cell injury in vitro. These results suggest that the downregulated miR‐181b induces neuroprotection against ischemic injury through negatively regulating HSPA5 and UCHL1 protein levels, providing a potential therapeutic target for ischemic stroke.

Phosphorylation of p38 MAPK mediates hypoxic preconditioning-induced neuroprotection against cerebral ischemic injury via mitochondria translocation of Bcl-xL in mice

Hypoxic preconditioning (HPC) initiates intracellular signaling pathway to provide protection, but the role of p38 mitogen-activated protein kinase (p38 MAPK) in HPC-induced neuroprotection against cerebral ischemic injuries is a matter of debate. In this study, we found that HPC could reduce 6h middle cerebral artery occlusion (MCAO)-induced infarct volume, edema ratio and cell apoptosis, as well as enhancing the up-regulated p38 MAPK phosphorylation (P-p38 MAPK) levels in the peri-infarct region of mice after 6h MCAO. However, intracerebroventricular injection of p38 MAPK inhibitor SB203580 abolished this HPC-induced neuroprotection. HPC significantly increased the translocation of anti-apoptotic Bcl-2-related protein Bcl-xL from the cytosol to the mitochondria in the peri-infarct region of MCAO mice. Interestingly, the results of reciprocal immunoprecipitation showed that Bcl-xL and P-p38 MAPK were coimmunoprecipitated reciprocally only in the peri-infarct region of HPC and MCAO treated mice, while Bcl-xL and total p38 (T-p38 MAPK), not P-p38 MAPK, could be coimmunoprecipited by each other in the brain of normal control mice. In addition, we found SB203580 significantly decreased P-p38 MAPK levels, and inhibited HPC-induced mitochondria translocation of Bcl-xL in the brain of HPC and MCAO treated mice. Taken together, our findings suggested that P-p38 MAPK mediates HPC-induced neuroprotection against cerebral ischemic injury via mitochondria translocation of Bcl-xL, which might be a key anti-cell apoptotic mechanism of HPC.

Identification of protein kinase C isoforms involved in cerebral hypoxic preconditioning of mice

Recently, accumulated studies have suggested that protein kinases C (PKC) play a central role in the development of ischemic-hypoxic preconditioning (I/HPC) in the brain. However, which types of PKC isoforms might be responsible for neuroprotection is still not clear, especially when the systematic investigation of PKC isoform-specific changes in brain regions was rare in animals with ischemic-hypoxic preconditioning. By using Western blot, we have demonstrated that the levels of cPKC betaII and gamma membrane translocation were increased in the early phase of cerebral hypoxic preconditioning. In this study, we combined the Western blot and immunostaining methods to investigate the effects of repetitive hypoxic exposure (H1-H4, n = 6 for each group) on membrane translocation and protein expression of several types of PKC isoforms, both in the cortex and hippocampus of mice. We found that the increased membrane translocation of nPKCepsilon (P < 0.05, versus normoxic H0) but not its protein expression levels in both the cortex and hippocampus during development of cerebral HPC in mice. However, there were no significant changes in both membrane translocation and protein expression levels of nPKCdelta, theta, eta, mu, and aPKC iota/lambda, zeta in these brain areas after hypoxic preconditioning. Similarly, an extensive subcellular redistribution of cPKCbetaII, gamma, and nPKCepsilon was observed by immunostaining in the cortex after three series of hypoxic exposures (H3). These results indicate that activation of cPKCbetaII, gamma, and nPKCepsilon might be involved in the development of cerebral hypoxic preconditioning of mice.

Enhanced protein expressions of sortilin and p75NTR in retina of rat following elevated intraocular pressure-induced retinal ischemia.

Elevated introcular pressure (IOP)-induced retinal neuron ischemic death includes an early phase of necrosis and prolonged phase of apoptosis. We used this ischemic model to observe the changes of sortilin and p75(NTR) protein expressions in rat retina. The results of Western blot analysis showed the expression of p75(NTR) at the band of 75 (mature form), 60 (non-glycosylated pieces) and 50 kDa (ectodomain shedding pieces), and the expression of sortilin at the 95 and 90 kDa (the mature form). The protein expressions of p75(NTR) (60 and 50 kDa pieces) and sortilin (90 kDa) increased significantly (p < 0.05) at days 3, 5 and 7 after retinal ischemia. This effect was also confirmed by immunofluorescence staining. Sortilin was primarily present in cell membrane of the ganglion cells layer (GCL) and large ganglion cell bodies by immunofluorescence labeling. There was little expression of p75(NTR) in the normal retina, while expression increased extensively in GCL, inner plexiform layer (IPL) and inner nuclear layer (INL) after retinal ischemia. p75(NTR) was shown to co-localize with neurofilament in the axons of neuronal cells by double-labeling. These results suggested that the protein expressions of 60 and 50 kDa forms of p75(NTR), and the 90 kDa mature form of sortilin increased in ischemia-induced retinal neuron of rats.