GADD45B in the ventral hippocampal CA1 modulates aversive memory acquisition and spatial cognition.

AIMS: This study was designed to investigate the role of growth arrest and DNA damage-inducible ß (GADD45B) in modulating fear memory acquisition and elucidate its underlying mechanisms. MAIN METHODS: Adeno-associated virus (AAV) that knockdown or overexpression GADD45B were injected into ventral hippocampal CA1 (vCA1) by stereotactic, and verified by fluorescence and Western blot. The contextual fear conditioning paradigm was employed to examine the involvement of GADD45B in modulating aversive memory acquisition. The Y-maze and novel location recognition (NLR) tests were used to examine non-aversive cognition. The synaptic plasticity and electrophysiological properties of neurons were measured by slice patch clamp. KEY FINDINGS: Knockdown of GADD45B in the vCA1 significantly enhanced fear memory acquisition, accompanied by an upregulation of long-term potentiation (LTP) expression and intrinsic excitability of vCA1 pyramidal neurons (PNs). Conversely, overexpression of GADD45B produced the opposite effects. Notably, silencing the activity of vCA1 neurons abolished the impact of GADD45B knockdown on fear memory development. Moreover, mice with vCA1 GADD45B overexpression exhibited impaired spatial cognition, whereas mice with GADD45B knockdown did not display such impairment. SIGNIFICANCE: These results provided compelling evidence for the crucial involvement of GADD45B in the formation of aversive memory and spatial cognition.

Inactivation of Zona Incerta Blocks Social Conditioned Place Aversion and Modulates Post-traumatic Stress Disorder-Like Behaviors in Mice.

Zona incerta (ZI), a largely inhibitory subthalamic region connected with many brain areas, has been suggested to serve as an integrative node for modulation of behaviors and physiological states, such as fear memory conditioning and aversion responses. It is, however, unclear whether ZI regulated the repeated social defeat stress (RSDS)-induced social conditioned place aversion (CPA) and post-traumatic stress disorder (PTSD)-like behaviors. In this study, the function of ZI was silenced via bilateral injection of tetanus toxin light chain (Tet-tox), a neurotoxin that completely blocks the evoked synaptic transmissions, expressing adeno-associated viruses (AAVs). We found ZI silencing: (1) significantly blocked the expression of RSDS-induced social CPA with no effect on the innate preference; (2) significantly enhanced the anxiety level in mice experienced RSDS with no effect on the locomotion activity; (3) altered the PTSD-associated behaviors, including the promotion of spatial cognitive impairment and the preventions of PPI deficit and social avoidance behavior. These effects were not observed on non-stressed mice. In summary, our results suggest the important role of ZI in modulating RSDS-induced social CPA and PTSD-like behaviors.

Morphine reverses the effects of 1-methyl-4-phenylpyridinium in PC12 cells through activating PI3K/Akt.

AIM OF THE STUDY: Parkinson's disease (PD) is a neurodegenerative disorder. It is caused by the degeneration of dopaminergic neurons and the dopamine (DA) deletion in the substantia nigra pars compacta (SNpc). Morphine elevates the level of dopamine in the mesolimbic dopamine system and plays a role in alleviating PD symptoms. However, the molecular mechanism is still unclear. The aim of the study is to investigate the mechanism on morphine alleviating PD symptoms. MATERIALS AND METHODS: The viability of PC12 cells was measured by using MTT assay. The expressions of tyrosine hydroxylase (TH), thioredoxin-1 (Trx-1), CyclinD1 and Cyclin-dependent kinase5 (Cdk5) were detected by Western Blot. RESULTS: In present study, we found that morphine increased the cell viability in PC12 cells. 1-methyl-4-phenylpyridi-nium (MPP+) reduced the cell viability and TH expression, which were reversed by morphine. MPP+ decreased the expressions of Trx-1, CyclinD1, Cdk5, which were restored by morphine. Moreover, the role of morphine in restoring the expressions of Trx-1, CyclinD1 and Cdk5 decreased by MPP+ was abolished by LY294002, phosphatidylinositol-3-kinase (PI3K)/Akt inhibitor. CONCLUSIONS: These results suggest that morphine reverses effects induced by MPP þ through activating PI3K/Akt pathway.

Downregulation of thioredoxin-1 in the ventral tegmental area delays extinction of methamphetamine-induced conditioned place preference.

BACKGROUND: Drug addiction is characterized by compulsive drug use and relapse. Thioredoxin-1 is emerging as an important modulator involved in the cellular protective response against a variety of toxic stressors. Previous study has reported that thioredoxin-1 overexpression prevents the acquisition of methamphetamine-conditioned place preference. Here, we aimed to investigate the effect of thioredoxin-1 on methamphetamine-conditioned place preference extinction and the possible mechanism. METHODS: (a) An extinction procedure in mice was employed to investigate the effect of thioredoxin-1 on the extinction of methamphetamine-conditioned place preference. After the acquisition of methamphetamine-conditioned place preference, mice underwent the following procedures: the injection of thioredoxin-1 small interfering RNA in the ventral tegmental area followed by the post-conditioned place preference test, four days of extinction training followed by four days of recovery after surgery. (b) The levels of thioredoxin-1, dopamine D1 receptor, tyrosine hydroxylase, phosphorylated extracellular regulated kinase, and phosphorylated cyclic adenosine monophosphate response element binding protein were examined by using Western blot analysis. RESULTS: Thioredoxin-1 downregulation in the ventral tegmental area delayed methamphetamine-conditioned place preference extinction. The expression of thioredoxin-1 was decreased in the ventral tegmental area of mice in control and negative groups after methamphetamine-conditioned place preference extinction, but not in the thioredoxin-1 siRNA group. The levels of dopamine D1 receptor, tyrosine hydroxylase, phosphorylated extracellular regulated kinase, and phosphorylated cyclic adenosine monophosphate response element binding protein were decreased in the ventral tegmental area, nucleus accumbens, and prefrontal cortex of mice in the control and negative groups after methamphetamine-conditioned place preference extinction, but were inversely increased in thioredoxin-1 siRNA group. CONCLUSIONS: The results suggest that downregulation of thioredoxin-1 in the ventral tegmental area may delay methamphetamine-conditioned place preference extinction by regulating the mesocorticolimbic dopaminergic signaling pathway.

Thioredoxin-1 downregulation in the nucleus accumbens promotes methamphetamine-primed reinstatement in mice.

Relapse of drug abuse after abstinence is a major challenge to the treatment of addicts. Thioredoxin-1 (Trx-1) is an important regulator of neuroprotection, and inhibits morphine-induced hyperlocomotion, reward and withdrawal signs, as well as blocks methamphetamine (METH)-induced conditioned place preference (CPP). The nucleus accumbens (NAc) is essential for relapse like behavior in reinstatement animal models. In the present study, we aimed to investigate the role of Trx-1 in the NAc in METH-primed reinstatement by using a reinstatement procedure in mice. Adeno-associated virus vectors expressing shRNA-mTrx-1 (AAV-shRNA-mTrx-1) were bilaterally microinjected into the NAc after METH-CPP extinction. The results showed that Trx-1 downregulation in the NAc promoted the reinstatement of METH-CPP. We also examined the expression of N-methyl-D-asparate (NMDA) receptor 2B subunit (GluN2b), the levels of phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated cAMP-response element binding protein (p-CREB) in the NAc by western blot analysis, and found that the GluN2b expression, p-ERK and p-CREB levels were increased in the NAc in response to low-dose METH in AAV-shRNA-mTrx-1 mice, but were not changed in control and AAV-vehicle mice. These data indicate that the increased GluN2b expression, and p-ERK and p-CREB levels in the NAc of AAV-shRNA-mTrx-1 mice may be responsible for the METH-primed reinstatement. Thus, we suggest that downregulation of Trx-1 in the NAc may make mice more sensitive to METH reinstatement.

The role of thioredoxin-1 in resisting methamphetamine-induced rewarding effect.

Methamphetamine (METH) is a highly addictive drug of abuse which induces behavioral sensitization and rewarding effects. Thioredoxin-1 (Trx-1) is a redox protein and plays roles in regulating activity of transcription factor, such as cAMP responsive element-binding protein (CREB), AP-1, p53, is emerging as an important modulator of neuronal function. It has been reported that Trx-1 is involved in morphine dependence. In this study, we examined the rewarding effect after METH administration by conditioned place preference (CPP) of mice, and detected the levels of dopamine and the activity of cAMP responsive element-binding protein (CREB), the expressions of ΔFosB and cyclin-dependent kinase 5 (CDK5) in the ventral tegmental area (VTA) and nucleus accumbens (NAc) in mice. Our results showed that the expression of METH-CPP was occluded in Trx-1 overexpression transgenic (TG) mice. The increase of dopamine level induced by METH was not further higher in Trx-1 TG mice. METH decreased the expression of Trx-1 which was restored in TG mice. The activity of CREB and the expressions of ΔFosB and CDK5 were increased by METH in wile-type mice, which were not further increased in TG mice. These results suggest that overexpression of Trx-1 may occlude the CPP induced by METH through regulating the activity of CREB and the expression of ΔFosB.

MiR-99a suppresses proliferation, migration and invasion of esophageal squamous cell carcinoma cells through inhibiting the IGF1R signaling pathway.

miR-99a is down-regulated in esophageal squamous cell carcinoma (ESCC), however the role and underlying mechanism are still unknown. We aim to explore the role and mechanism of miR-99a down-regulation in ESCC. The expression of miR-99a in ESCC tissues and cell lines was detected by Human miRNA Microarrays and Real-time PCR. The effects of miR-99a on cell proliferation, migration and invasion were determined by Cell Counting Kit-8 (CCK-8) assay, transwell migration and invasion assay. Target gene of miR-99a were analyzed by target prediction software and validated by Real-time PCR and Western blotting assay. Our microarray results and four Gene Expression Omnibus (GEO) datasets showed lower expression level of miR-99a in ESCC tissues. Overexpression of miR-99a using mimics significantly suppressed cell proliferation, and decreased expressions of CCND1, CCNA2 and CCNE1. We also found that enhanced miR-99a significantly inhibited migration, invasion and epithelial-mesenchymal transition (EMT) of ESCC cells, and down-regulated EMT associated transcription factor Slug, and MMPs including MMP2, MMP7 and MMP13. TargetScan predicted insulin-like growth factor 1 receptor (IGF1R) as the cadidate target gene of miR-99a, and western blotting confirmed the negative correlation between miR-99a and IGF1R. Importantly, we further found that knockdown of IGF1R also significantly inhibited the proliferation, migration, invasion and slug-induced EMT of ESCC cells, and reduced the cell cycle regulatory proteins and MMPs. In conclusion, our findings suggested that loss of miR-99a in ESCC promoted the tumor cell proliferation, migration, invasion and slug-induced EMT through activating IGF1R signaling pathway.

Thioredoxin-1 Increases Survival in Sepsis by Inflammatory Response Through Suppressing Endoplasmic Reticulum Stress.

Sepsis is the main cause of death in critically ill patients, pathogenesis of which is still unclear. The nuclear factor κB (NF-κB) inflammatory signal pathway mediated by endoplasmic reticulum stress is involved in sepsis. Thioredoxin-1 (Trx-1) is an important protein of regulating oxidative stress. It plays a crucial role in the anti-oxidation, anti-apoptosis, and anti-inflammation. However, the role and the mechanism of Trx-1 in sepsis have not been extensively studied. In the present study, we showed that the survival was longer in sepsis induced by cecal ligation and puncture in Trx-1 overexpression transgenic (Tg) mice compared with wild-type mice. Wet/dry lung weight ratio was decreased in Trx-1 Tg mice. The levels of TNF-α and IL-1ß in plasma and lung tissue were inhibited in Tg mice. The expressions of glucose-regulated protein 78, inositol-requiring enzyme 1α (IRE1α), tumor necrosis factor receptor-associated factor 2, C/EBP homologous protein, NF-κB, and inhibitors of NF-κBα were increased in lung tissue. More importantly, the overexpression of Trx-1 in transgenic mice suppressed NF-κB inflammatory signal pathway by inhibiting the activation of molecules involved in ER stress. Our results suggest that Trx-1 may play protective role in extending survival in sepsis by regulating inflammatory response through suppressing ER stress.