Role of hippocampal ß-adrenergic and glucocorticoid receptors in the novelty-induced enhancement of fear extinction.

Fear extinction forms a new memory but does not erase the original fear memory. Exposure to novelty facilitates transfer of short-term extinction memory to long-lasting memory. However, the underlying cellular and molecular mechanisms are still unclear. Using a classical contextual fear-conditioning model, we investigated the effect of novelty on long-lasting extinction memory in rats. We found that exposure to a novel environment but not familiar environment 1 h before or after extinction enhanced extinction long-term memory (LTM) and reduced fear reinstatement. However, exploring novelty 6 h before or after extinction had no such effect. Infusion of the ß-adrenergic receptor (ßAR) inhibitor propranolol and glucocorticoid receptor (GR) inhibitor RU486 into the CA1 area of the dorsal hippocampus before novelty exposure blocked the effect of novelty on extinction memory. Propranolol prevented activation of the hippocampal PKA-CREB pathway, and RU486 prevented activation of the hippocampal extracellular signal-regulated kinase 1/2 (Erk1/2)-CREB pathway induced by novelty exposure. These results indicate that the hippocampal ßAR-PKA-CREB and GR-Erk1/2-CREB pathways mediate the extinction-enhancing effect of novelty exposure. Infusion of RU486 or the Erk1/2 inhibitor U0126, but not propranolol or the PKA inhibitor Rp-cAMPS, into the CA1 before extinction disrupted the formation of extinction LTM, suggesting that hippocampal GR and Erk1/2 but not ßAR or PKA play critical roles in this process. These results indicate that novelty promotes extinction memory via hippocampal ßAR- and GR-dependent pathways, and Erk1/2 may serve as a behavioral tag of extinction.

Stress within a restricted time window selectively affects the persistence of long-term memory.

The effects of stress on emotional memory are distinct and depend on the stages of memory. Memory undergoes consolidation and reconsolidation after acquisition and retrieval, respectively. Stress facilitates the consolidation but disrupts the reconsolidation of emotional memory. Previous research on the effects of stress on memory have focused on long-term memory (LTM) formation (tested 24 h later), but the effects of stress on the persistence of LTM (tested at least 1 week later) are unclear. Recent findings indicated that the persistence of LTM requires late-phase protein synthesis in the dorsal hippocampus. The present study investigated the effect of stress (i.e., cold water stress) during the late phase after the acquisition and retrieval of contextual fear memory in rats. We found that stress and corticosterone administration during the late phase (12 h) after acquisition, referred to as late consolidation, selectively enhanced the persistence of LTM, whereas stress during the late phase (12 h) after retrieval, referred to as late reconsolidation, selectively disrupted the restabilized persistence of LTM. Moreover, the effects of stress on the persistence of LTM were blocked by the corticosterone synthesis inhibitor metyrapone, which was administered before stress, suggesting that the glucocorticoid system is involved in the effects of stress on the persistence of LTM. We conclude that stress within a restricted time window after acquisition or retrieval selectively affects the persistence of LTM and depends on the glucocorticoid system.

[Effects of HO-1 gene expression on proliferation of imatinib resistant CML cells].

OBJECTIVE: To investigate the effect of heme oxygenase-1 (HO-1) expression on cell growth and apoptosis in imatinib resistant chronic myeloid leukemia (CML) cells (K562/A02-IM), and explore the relationship between HO-1 gene and CML. METHODS: The expression of HO-1 in 20 drug-resistant CML patients was detected by RT-PCR. Different concentrations of hemin were used to induce HO-1 expression of K562/A02-IM, HO-1 expression at different time was detected by RT-PCR and Western blot analysis. Cell apoptosis was detected by Annexin V/PI staining, and MTT assay was used to detect viability of K562/A02-IM cells after induction or inhibition of HO-1 gene by hemin and zinc protoporphyrin (ZPP). RESULTS: RT-PCR showed that HO-1 was expressed in the bone marrow mononuclear cells (BMMNCs). When treated with hemin at different concentrations (0, 10, 20, 40 µmol/L) for 16 h, the expression of HO-1 in K562/A02-IM was increased in a dose-dependent manner, and peaked at 20 µmol/L of hemin for 16 h. The apoptosis rates were (17.61 ± 0.01)%, (12.13 ± 0.11)%, (7.94 ± 0.03)% and (4.62 ± 0.15)% at 0,10, 20 and 40 µmol/L of hemin respectively for 16 h and were (14.7 ± 0.05)%, (8.1 ± 0.07)% and (16.3 ± 0.13)% at 20 µmol/L of hemin treatment for 8,16, and 24 h respectively. Hemin induced apoptosis of K562/A02-IM cells in a dose-dependent manner. The expression of HO-1 was induced in K562/A02-IM cells in a dose-dependent manner, and the survival of K562/A02-IM cells was significantly increased as compared to that of control group. When HO-1 was inhibited by ZPP, the cells survival was sharply decreased compared to that of the control group (P < 0.05). CONCLUSION: HO-1 was expressed in the BMMNCs. It is a kind of molecules whose expression can be induced and can promote the growth of drug-resistant cells. Inhibition of HO-1 expression probably be used for the treatment of drug-resistant CML.

Over-expression of aldehyde dehydrogenase-2 protects against H2O2-induced oxidative damage and apoptosis in peripheral blood mononuclear cells.

AIM: To construct an eukaryotic expression vector containing the aldehyde dehydrogenase-2 (ALDH2) gene, and determine whether transfection with the ALDH2 gene can provide protection against hydrogen peroxide-induced oxidative damage, as well as attenuate apoptosis or cell death in human peripheral blood mononuclear cells (PBMCs). METHODS: The ALDH2 gene was cloned from human hepatocytes by RT-PCR. The eukaryotic expression vector containing the gene was constructed and then transfected into PBMCs via liposomes. RT-PCR, indirect immunofluorescence assay, and Western blot were used to evaluate the expression of the transgene in target cells. MTT assay and flow cytometry were used to detect the effects of ALDH2 on PBMCs damaged by hydrogen peroxide (H2O2). The level of intracellular reactive oxygen species (ROS) was determined by fluorescence spectrophotometry. RESULTS: The eukaryotic expression vector pcDNA3.1/myc-His-ALDH2 was successfully constructed and transfected into PBMCs. RT-PCR results showed higher mRNA expression of ALDH2 in the gene-transfected group than in the two control groups (empty vector-transfected group and negative control). Indirect immunofluorescence assay and Western blot indicated distinct higher protein expression of ALDH2 in the gene-transfected group. The cell survival rate against H2O2-induced oxidative damage was higher in the ALDH2 gene-transfected group. Moreover, apoptosis rates in gene-transfected PBMCs incubated with 50 and 75 µmol/L H2O2 decreased by 7% and 6%, respectively. The generation of intracellular ROS was also markedly downregulated. CONCLUSION: ALDH2 gene transfection can protect PBMCs against H2O2-induced damage and attenuate apoptosis, accompanied with a downregulation of intracellular ROS. ALDH2 functions as a protector against oxidative stress.

[Study on the effect of cell proliferation and anti-oxidative damage of aldehyde dehydrogenase-2 gene transfected into K562 cells].

OBJECTIVE: To construct a eukaryotic expression vector containing aldehyde dehydrogenase-2 (ALDH2) gene and investigate the effects and its possible mechanisms of ALDH2 gene on cell proliferation and anti-oxidative damage in the K562 cells. METHODS: An eukaryotic expression vector containing the ALDH2 gene cloned from human hepatocytes was constructed and transfected into K562 cells by liposome. RT-PCR and Western blot were used to evaluate the expression of ALDH2. MTT assay was used to check the cell proliferation and trypan blue exclusion to check K562 cells damage induced by hydrogen peroxide (H2O2). RT-PCR and fluorescence spectrophotometry were used to determine the expression of heme oxygenase-1 (HO-1) and the generation of intracellular reactive oxygen species (ROS) respectively. RESULTS: RT-PCR and Western blot analysis showed distinct higher ALDH2 protein expression in gene transfected group. The latter group had a higher cell proliferation (P < 0.05) and survival rate against H2O2 induced-oxidative damage, being increased by 7.8 times (IC(50) was 12.3 µmol/L and 1.4 µmol/L for K562-pcDNA3.1-ALDH2 and control cells, respectively, P < 0.01). The HO-1 mRNA expression and the generation of intracellular ROS were downregulated at a specific concentration of H2O2 in the ALDH2 gene transfected group. CONCLUSION: ALDH2 gene transfection can protect K562 cells against oxidative damage, and the downregulation of HO-1 expression and intracellular ROS may be involved in this process.