Pretreatment with Lovastatin Improves Depression-Like Behavior After Traumatic Brain Injury Through Activation of the AMPK Pathway.

BACKGROUND: The beneficial effect of pretreatment with statins on traumatic brain injury (TBI)-induced depression and anxiety and its mechanism of action remain unclear. In this study, we combined epidemiological and experimental animal data to clarify this issue. METHODS: We used the Taiwan National Health Insurance database to identify patients who were diagnosed with TBI from 2000 to 2013 and compared patients with and without statin treatment matched by age, sex, and underlying comorbidities in a 1:1 ratio. The risk of developing depression and/or anxiety was compared between patients with and without a statin using Cox proportional hazards regression. We also used a rat model to assess the effect of lovastatin pretreatment on neurobehavioral and neuropathological changes following TBI. RESULTS: The risk of developing depression was lower in the 41,803 patients in the statin cohort than nonstatin cohort (adjusted hazard ratio, 0.91 [95% confidence interval, 0.83-0.99]). In animal models, the lovastatin group had significantly reduced infarct volume, decreased immobility time and latency to eat, a reduced number of Fluoro- Jade-positive cells and levels of glial fibrillary acidic protein and tumor necrosis factor-alpha, and increased adenosine monophosphate -activated protein kinase (AMPK) and its upstream kinase liver kinase B1 in the hippocampal dentate gyrus. These effects were blocked in AMPK inhibitor-pretreated TBI rats. CONCLUSIONS: Our epidemiological data showed that a decreased risk of depression was associated with statin pretreatment, which was supported by an animal study. The underlying mechanism for this appears to involve AMPK activation in the statin pretreatment-induced alleviation of TBI.

High-frequency ultrasound imaging for monitoring the function of meningeal lymphatic system in mice.

The meningeal lymphatic system drains the cerebrospinal fluid from the subarachnoid space to the cervical lymphatic system, primarily to the deep cervical lymph nodes. Perturbations of the meningeal lymphatic system have been linked to various neurologic disorders. A method to specifically monitor the flow of meningeal lymphatic system in real time is unavailable. In the present study, we adopted the high-frequency ultrasound (HFUS) with 1,1'diocatadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-loaded microbubble and FePt@PLGA nanoparticle contrast agents to evaluate the flow of the meningeal lymphatic system in 2-month-old mice. Statistical analysis was performed to identify changes of HFUS signals among the microbubbles, FePt@PLGA nanoparticles, and saline control groups. Approximately 15 min from the start of intracerebroventricular injection of contrast agents, their signals were evident at the deep cervical lymph nodes and lasted for at least 60 min. These signals were validated on the basis of the presence of DiI and Fe signals in the deep cervical lymph nodes. Ligation of afferent lymphatic vessels to the deep cervical lymph nodes eliminated the HFUS signals. Moreover, ablation of lymphatic vessels near the confluence of sinuses decreased the HFUS signals in the deep cervical lymph nodes. Glioma-bearing mice that exhibited reduced lymphatic vessel immunostaining signals near the confluence of sinuses had lowered HFUS signals in the deep cervical lymph nodes within 60 min. The proposed method provides a minimally invasive approach to monitor the qualities of the meningeal lymphatic system in real time as well as the progression of the meningeal lymphatic system in various brain disease animal models.

Modulation of methamphetamine memory reconsolidation by neural projection from basolateral amygdala to nucleus accumbens.

Drug-associated conditioned cues promote subjects to recall drug reward memory, resulting in drug-seeking and reinstatement. A consolidated memory becomes unstable after recall, such that the amnestic agent can disrupt the memory during the reconsolidation stage, which implicates a potential therapeutic strategy for weakening maladaptive memories. The basolateral amygdala (BLA) involves the association of conditioned cues with reward and aversive valences and projects the information to the nucleus accumbens (NAc) that mediates reward-seeking. However, whether the BLA-NAc projection plays a role in drug-associated memory reactivation and reconsolidation is unknown. We used methamphetamine (MeAM) conditioned place preference (CPP) to investigate the role of BLA-NAc neural projection in the memory reconsolidation. Two weeks before CPP training, we infused adeno-associated virus (AAV) carrying the designer receptor exclusively activated by designer drugs (DREADD) or control constructs. We infused clozapine-N-oxide (CNO) after the recall test to manipulate the neural activity of BLA-NAc projections in mice. We found that after recall, DREADD-mediated inhibition of BLA neurons projecting to the NAc core blunted consolidated MeAM-associated memory. Inhibition of BLA glutamatergic nerve terminals in the NAc core 1 h after recall disrupted consolidated MeAM-associated memory. However, inhibiting this pathway after the time window of reconsolidation failed to affect memory. Furthermore, under the condition without memory retrieval, DREADD-mediated activation of BLA-NAc core projection was required for amnesic agents to disrupt consolidated MeAM-associated memory. Our findings provide evidence that the BLA-NAc pathway activity is involved in the post-retrieval processing of MeAM-associated memory in CPP.

A Selective Histone Deacetylase Inhibitor Induces Autophagy and Cell Death via SCNN1A Downregulation in Glioblastoma Cells.

Glioblastoma multiforme (GBM) is a grade IV, highly malignant brain tumor. Because of the heterogeneity of GBM, a multitarget drug is a rational strategy for GBM treatment. Histone deacetylase inhibitors (HDACis) regulate the expression of numerous genes involved in cell death, apoptosis, and tumorigenesis. We found that the HDAC4/HDAC5 inhibitor LMK235 at 0.5 µM significantly reduced the cell viability and colony formation of patient-derived, temozolomide-resistant GBM P#5 TMZ-R, U-87 MG, and T98G cells. Moreover, LMK235 also significantly increased TUBA acetylation, which is an indicator of HDAC inhibition. Interestingly, LMK235 induced MAP1LC3 robust readout and puncta accumulation but did not enhance PARP1 cleavage or the proportion of annexin V-positive cells, suggesting that LMK235-induced cell death occurred via autophagy activation. Further RNA-seq analysis after LMK235 treatment showed that 597 different expression genes compared to control. After bioinformatic analysis by KEGG and STRING, we focused on 34 genes and validated their mRNA expression by qPCR. Further validation showed that 2 µM LMK235 significantly reduced the mRNA and protein expression of SCNN1A. Cell viability of SCNN1A-silenced cells were reduced, but cells were rescued while treated with an autophagy inhibitor bafilomycin A1. Conclusively, SCNN1A plays a role in LMK235-induced autophagy and cell death in GBM cells.

A heat shock protein 90 inhibitor reduces oncoprotein expression and induces cell death in heterogeneous glioblastoma cells with EGFR, PDGFRA, CDK4, and NF1 aberrations.

AIMS: Glioblastoma (GBM) is a highly malignant brain tumor. After treatment with the first-line drug temozolomide, only 50% of patients are responsive. Recent literature shows that the difficulty in treating GBM is mainly due to the heterogeneity of its four major cellular states, which are characterized by differences in EGFR, PDGFRA, CDK4, and NF1. Therefore, development of a multitarget drug is a potential strategy for treating heterogeneous GBM. MAIN METHODS: In this study, the antitumor ability of a potent heat shock protein 90 inhibitor, NVP-AUY922 (AUY922), was evaluated in GBM cell lines (U-87 MG and T98G cells) and patient-derived GBM cell lines [P#5 and P#5 temozolomide-resistant (TMZ-R) cells]. KEY FINDINGS: We found that AUY922 significantly reduced cell viability and colony formation in four GBM cell lines. AUY922 also significantly induced apoptosis by increasing PARP1 cleavage and the number of annexin V-positive cells. The autophagy indicators as MAP1LC3B cleavage and MAP1LC3B puncta were increased after AUY922 treatment. AUY922-induced cell death could be partially reversed by pharmacological inhibition of either apoptotic inhibitor or autophagy inhibitor. Moreover, AUY922 reduced the mRNA and protein expressions of EGFR, PDGFRA, CDK4, and NF1, which contribute to the four cellular state subtypes in GBM cells. In addition, the downstream signaling proteins of these four proteins, AKT/p-AKT, MAPK/p-MAPK, and BRAF, were downregulated after AUY922 treatment. SIGNIFICANCE: Taken together, AUY922 led to GBM cell death via apoptosis and autophagy, and reduced the mRNA and protein expression of EGFR, PDGFRA, CDK4, and NF1in heterogeneous GBM cells.

Erratum: Cilostazol eliminates radiation-resistant glioblastoma by re-evoking big conductance calcium-activated potassium channel activity.

[This corrects the article on p. 1148 in vol. 11, PMID: 33948351.].

Disulfiram Sensitizes a Therapeutic-Resistant Glioblastoma to the TGF-ß Receptor Inhibitor.

Despite neurosurgery following radiation and chemotherapy, residual glioblastoma (GBM) cells develop therapeutic resistance (TR) leading to recurrence. The GBM heterogeneity confers TR. Therefore, an effective strategy must target cancer stem cells (CSCs) and other malignant cancer cells. TGF-ß and mesenchymal transition are the indicators for poor prognoses. The activity of aldehyde dehydrogenases (ALDHs) is a functional CSC marker. However, the interplay between TGF-ß and ALDHs remains unclear. We developed radiation-resistant and radiation-temozolomide-resistant GBM models to investigate the underlying mechanisms conferring TR. Galunisertib is a drug targeting TGF-ß receptors. Disulfiram (DSF) is an anti-alcoholism drug which functions by inhibiting ALDHs. The anti-tumor effects of combining DSF and Galunisertib were evaluated by in vitro cell grow, wound healing, Transwell assays, and in vivo orthotopic GBM model. Mesenchymal-like phenotype was facilitated by TGF-ß in TR GBM. Additionally, TR activated ALDHs. DSF inhibited TR-induced cell migration and tumor sphere formation. However, DSF did not affect the tumor growth in vivo. Spectacularly, DSF sensitized TR GBM to Galunisertib both in vitro and in vivo. ALDH activity positively correlated with TGF-ß-induced mesenchymal properties in TR GBM. CSCs and mesenchymal-like GBM cells targeted together by combining DSF and Galunisertib may be a good therapeutic strategy for recurrent GBM patients.

Cilostazol eliminates radiation-resistant glioblastoma by re-evoking big conductance calcium-activated potassium channel activity.

In spite of radio- and chemotherapy, glioblastoma (GBM) develops therapeutic resistance leading to recurrence and poor prognosis. Therefore, understanding the underlying mechanisms of resistance is important to improve the treatment of GBM. To this end, we developed a radiation-resistant cell model by exposure to consecutive periods of irradiation. Simultaneously, single high-dose irradiation was introduced to determine "when" GBM developed consecutive irradiation-induced resistance (CIIR). We found that CIIR promoted TGF-ß secretion, activated pro-survival Akt, and downregulated p21 in a p53-independent manner. Furthermore, CIIR upregulated multidrug-resistant proteins, resulting in temozolomide resistance. CIIR GBM also enhanced cell mobility and accelerated cell proliferation. The big-conductance calcium-activated potassium channel (BK channel) is highly expressed and activated in GBM. However, CIIR diminishes BK channel activity in an expression-independent manner. Cilostazol is a phosphodiesterase-3 inhibitor for the treatment of intermittent claudication and was able to reverse CIIR-induced BK channel inactivation. Paxilline, a BK channel blocker, promoted cell migration and proliferation in parental GBM cells. In contrast, Cilostazol inhibited CIIR-induced cell motility, proliferation, and the ability to form tumor spheres. Moreover, we established a radiation-resistant GBM in vivo model by intracranially injecting CIIR GBM cells into the brains of NOD/SCID mice. We found that Cilostazol delayed tumor in vivo growth and prolonged survival. As such, inactivation of the BK channel assists GBM in developing radiation resistance. Accordingly, restoring BK channel activity may be an effective strategy to improve therapeutic efficacy, and cilostazol could be repurposed to treat GBM.

Regulation of stress-provoked aggressive behavior using endocannabinoids.

Reactive impulsive aggression is characterized by outbursts of rage and violence when subjects encounter threatening stressful events. Although impulsive aggression and violence create a high-cost burden on health and society, relatively little is known about treatment. Early adolescent social isolation (SI) alters brain development and functions. It induces hyper-excitatory in the ventral hippocampus (vHip) to promote acute stress-provoked outbursts of aggression, referred to as impulsive aggression, in mouse models. Cannabinoid type 1 receptors (CB1Rs) act on presynaptic sites and suppress neurotransmitter release into synapses. Given that CB1R activation inhibits neurotransmitter releases and modulates excitatory network activity, we tested the hypothesis that CB1R activation reduces impulsive aggression in SI mice through decreasing excitatory activity in the vHip. Here, we report that CB1R agonists, WIN-552122 (WIN) or arachidonylcyclopropylamide (ACPA), ameliorated acute stress-provoked attack behavior in the resident-intruder test without affecting general locomotion activity. Increasing endocannabinoids (eCBs) by inhibiting degradation enzymes in the vHip reduced impulsive aggression, and the effect was blunted by administration of AM251, a CB1R antagonist. Acute stress in SI mice induced c-Fos expression, a marker of neuronal activation, on vHip neurons projecting to the ventromedial hypothalamus (VMH), a well-known brain area that controls attack behavior. eCB augmentation inhibited c-Fos expression in VMH-projecting vHip neurons surrounded by CB1Rs. These results suggest that enhancing eCB signaling in order to activate CB1Rs suppresses impulsive aggression via suppressing vHip→VMH neural activity and point to a role of CB1R activation in ameliorating impulsive aggression in adults who have had adverse experiences during early adolescence.

Inhibition of Sonic Hedgehog Signaling Suppresses Glioma Stem-Like Cells Likely Through Inducing Autophagic Cell Death.

Glioblastoma (GBM) often recurs after radio- and chemotherapies leading to poor prognosis. Glioma stem-like cells (GSCs) contribute to drug resistance and recurrence. Thus, understanding cellular mechanism underlying the growth of GSCs is critical for the treatment of GBM. Here GSCs were isolated from human U87 GBM cells with magnetic-activated cell sorting (MACS) using CD133 as a marker. The CD133+ cells highly expressed sonic hedgehog (Shh) and were capable of forming tumor spheroids in vitro and tumor in vivo. Athymic mice received intracranial injection of luciferase transduced parental and CD133+ GBM cells was utilized as orthotopic GBM model. Inhibited Shh by LDE225 delayed GBM growth in vivo, and downregulated Ptch1 and Gli1. CD133+ cell proliferation was more sensitive to inhibition by LDE225 than that of CD133- cells. Treatment with LDE225 significantly reduced CD133+-derived tumor spheroid formation. Large membranous vacuoles appeared in the LDE225-treated cells concomitant with the conversion of LC3-I to LC3-II. In addition, LDE225-induced cell death was mitigated in the presence of autophagy inhibitor 3-methyladenine (3-MA). Tumor growth was much slower in Shh shRNA-knockdown mice than in control RNA-transfected mice. Conversely, tumor growth was faster in Shh overexpressed mice. Furthermore, combination of LDE225 and rapamycin treatment resulted in additive effect on LC3-I to LC3-II conversion and reduction in cell viability. However, LDE225 did not affect the phosphorylated level of mTOR. Similarly, amiodarone, an mTOR-independent autophagy enhancer, reduced CD133+ cell viability and tumor spheroid formation in vitro and exhibited anti-tumor activity in vivo. These results suggest that Shh inhibitor induces autophagy of CD133+ cells likely through mTOR independent pathway. Targeting Shh signal pathway may overcome chemoresistance and provide a therapeutic strategy for patients with malignant gliomas.

MicroRNA-206 Regulates Stress-Provoked Aggressive Behaviors in Post-weaning Social Isolation Mice.

When facing stressful conditions, some people tend to be impulsively aggressive whereas others are not. However, the causes and underlying mechanisms remain elusive. It has been reported that acute stress induces outbursts of aggression in post-weaning social isolation (SI) mice but not in group housing (GH) mice. Here we report epigenetic regulation of impulsive aggression in SI mice. At post-natal day 21, mice were randomly assigned to GH or SI groups. We found that SI mice exhibited a higher level of microRNA 206 (miR-206) compared with GH mice. Intra-hippocampal injection of AM206, an antagomir of miR-206, decreased stress-induced attack behavior in SI mice and increased BDNF expression. Moreover, BDNF expression was required for AM206 effects on the reduction of aggression. On the other hand, miR-206 overexpression in GH mice induced attack behavior. Intranasal administration of AM206 rather than a scramble control significantly reduced attack behavior and depression-like behavior in SI mice. Our results suggest that miR-206 mediates development of maladaptive impulsive aggression in early life adversity and that its antagomir could potentially be a therapeutic target against stress-exacerbated aggressive behavior.

TIAM2S as a novel regulator for serotonin level enhances brain plasticity and locomotion behavior.

TIAM2S, the short form of human T-cell lymphoma invasion and metastasis 2, can have oncogenic effects when aberrantly expressed in the liver or lungs. However, it is also abundant in healthy, non-neoplastic brain tissue, in which its primary function is still unknown. Here, we examined the neurobiological and behavioral significance of human TIAM2S using the human brain protein panels, a human NT2/D1-derived neuronal cell line model (NT2/N), and transgenic mice that overexpress human TIAM2S (TIAM2S-TG). Our data reveal that TIAM2S exists primarily in neurons of the restricted brain areas around the limbic system and in well-differentiated NT2/N cells. Functional studies revealed that TIAM2S has no guanine nucleotide exchange factor (GEF) activity and is mainly located in the nucleus. Furthermore, whole-transcriptome and enrichment analysis with total RNA sequencing revealed that TIAM2S-knockdown (TIAM2S-KD) was strongly associated with the cellular processes of the brain structural development and differentiation, serotonin-related signaling, and the diseases markers representing neurobehavioral developmental disorders. Moreover, TIAM2S-KD cells display decreased neurite outgrowth and reduced serotonin levels. Moreover, TIAM2S overexpressing TG mice show increased number and length of serotonergic fibers at early postnatal stage, results in higher serotonin levels at both the serum and brain regions, and higher neuroplasticity and hyperlocomotion in latter adulthood. Taken together, our results illustrate the non-oncogenic functions of human TIAM2S and demonstrate that TIAM2S is a novel regulator of serotonin level, brain neuroplasticity, and locomotion behavior.

The Ventral Hippocampus Controls Stress-Provoked Impulsive Aggression through the Ventromedial Hypothalamus in Post-Weaning Social Isolation Mice.

Impulsively aggressive individuals may suddenly attack others when under stress, but the neural circuitry underlying stress-provoked aggression is poorly understood. Here, we report that acute stress activates ventral hippocampus (vHip) neurons to induce attack behavior in post-weaning socially isolated mice. Chemogenetic inhibition of vHip neural activity blunts stress-provoked attack behavior, whereas chemogenetic activation promotes it. The activation of cell bodies in vHip neurons projecting into the ventromedial hypothalamus (VMH) induces attack behavior, suggesting that the vHip-VMH projection contributes to impulsive aggression. Furthermore, optogenetic inhibition of vHip glutamatergic neurons blocks stress-provoked attacks, whereas optogenetic activation of vHip glutamatergic neurons drives attack behavior. These results show direct evidence that vHip-VMH neural circuitry modulates attack behavior in socially isolated mice.

Mechanism underlying NMDA blockade-induced inhibition of aggression in post-weaning socially isolated mice.

When faced with stressful conditions, people with a tendency toward impulsive aggression may suddenly hurt others. We have previously shown that the blockade of NMDA receptors (NMDARs) within the ventral hippocampus (VH) produces anti-aggressive effects. However, little is known about the mechanism for tamping down stress-provoked attack behavior. Here, we report that expression of brain-derived neurotrophic factor (BDNF) after inhibition of NMDARs in the VH is required for blunting stress-provoked attack behavior in post-weaning socially isolated mice. Administration of NMDAR antagonist MK-801 decreased the phosphorylated eukaryotic elongation factor 2 (p-eEF2) and increased BDNF expression in the VH. Infusion of eEF2 kinase inhibitor NH125 to the VH decreased attack behavior and increased BDNF expression. Knockdown of BDNF in the VH blocked the anti-aggressive effect of MK-801 and NH125. Furthermore, MK-801 rapidly increased the activity of protein phosphatase 2A (PP2A). Intra-VH infusion of PP2A inhibitor okadaic acid blocked the anti-aggressive effects of MK-801. These results suggest that blockade of NMDAR reduces attack behavior through increasing PP2A activity leading to dephosphorylation of eEF2 and an increase in BDNF expression. Our findings indicate that the enhancement of BDNF expression is beneficial for preventing impulsive aggression in at-risk beings.

Impact of social relationships on Alzheimer's memory impairment: mechanistic studies.

Alzheimer's disease (AD) is characterized by progressive memory and neuronal loss culminating in cognitive impairment that not only affects a person's living ability but also becomes a society's as well as a family's economic burden. AD is the most common form of dementia in older persons. It is expected that the number of people with AD dementia will increase dramatically in the next 30 years, projecting to 75 million in 2030 and 131.5 million in 2050 worldwide. So far, no sufficient evidence is available to support that any medicine is able to prevent or reverse the progression of the disease. Early studies have shown that social environment, particularly social relationships, can affect one's behavior and mental health. A study analyzing the correlation between loneliness and risk of developing AD revealed that lonely persons had higher risk of AD compared with persons who were not lonely. On the other hand, it has been reported that we can prevent cognitive decline and delay the onset of AD if we keep mentally active and frequently participate in social activities. In this review, we focus on the impact of social behaviors on the progression of cognitive deficit in animal models of AD with a particular emphasis on a mechanistic scheme that explains how social isolation exacerbates cognitive impairment and how social interaction with conspecifics rescues AD patients' memory deficit.

Synergistic inhibition of tumor growth by combination treatment with drugs against different subpopulations of glioblastoma cells.

BACKGROUND: Glioma stem cells (GSCs) contribute to tumor recurrence and drug resistance. This study characterizes the tumorigenesis of CD133+ cells and their sensitivity to pharmacological inhibition. METHODS: GSCs from human U87 and rat C6 glioblastoma cell lines were isolated via magnetic cell sorting using CD133 as a cancer stem cell marker. Cell proliferation was determined using the WST-1 assay. An intracranial mouse model and bioluminescence imaging were used to assess the effects of drugs on tumor growth in vivo. RESULTS: CD133+ cells expressed stem cell markers and exhibited self-renewal and enhanced tumor formation. Minocycline (Mino) was more effective in reducing the survival rate of CD133+ cells, whereas CD133- cells were more sensitive to inhibition by the signal transducer and activator of transcription 3 (STAT3) inhibitor. Inhibition of STAT3 decreased the expression of CD133+ stem cell markers. The combination of Mino and STAT3 inhibitor synergistically reduced the cell viability of glioma cells. Furthermore, this combination synergistically suppressed tumor growth in nude mice. CONCLUSION: The results suggest that concurrent targeting of different subpopulations of glioblastoma cells may be an effective therapeutic strategy for patients with malignant glioma.

Co-housing reverses memory decline by epigenetic regulation of brain-derived neurotrophic factor expression in an animal model of Alzheimer's disease.

Co-housing with a company exerts profound effects on memory decline in animal model of Alzheimer's disease (AD). Recently, we found that APP/PS1 mice of 9-month-old improved their memories after co-housing with wide-type mice for 3months by increasing hippocampal brain-derived neurotrophic factor (BDNF) expression. However, the mechanism of how co-housing could induce BDNF expression remains elusive. Here we examined epigenetic changes in the mouse hippocampus that accompanied the co-housing-induced memory improvement. We found that the level of histone deacetylase 2 (HDAC2), but not that of HDAC1, was significantly lower in the memory improved mice than in the control and memory un-improved APP/PS1 mice after co-housing. Knockdown of Hdac2 resulted in a higher freezing response after co-housing. Conversely, over-expression of HDAC2 blocked co-housing-induced memory improvement. The level of Bdnf exon IV mRNA increased significantly after knockdown of Hdac2. ChIP assay revealed a decreased occupancy of HDAC2 in the promoter region of Bdnf exon IV of memory improved mice but not memory un-improved and control APP/PS1 mice. Consistently, the acetylation of histone 3 on Lys 9 (H3K9) and histone 4 on Lys12 (H4K12) increased significantly in the promoter region of Bdnf exon IV. These results suggest HDAC2 expression is reduced after co-housing resulting in a decreased occupancy of HDAC2 and increased histone H3K9 and H4K12 acetylation in the promoter region of Bdnf exon IV, leading to increased BDNF expression in the hippocampus that improves memory.

Involvement of protein phosphatases in the destabilization of methamphetamine-associated contextual memory.

Destabilization refers to a memory that becomes unstable when reactivated and is susceptible to disruption by amnestic agents. Here we delineated the cellular mechanism underlying the destabilization of drug memory. Mice were conditioned with methamphetamine (MeAM) for 3 d, and drug memory was assessed with a conditioned place preference (CPP) protocol. Anisomycin (ANI) was administered 60 min after the CPP retrieval to disrupt reconsolidation. We found that destabilization of MeAM CPP after the application of ANI was blocked by the N-methyl-d-aspartate receptor (NMDAR) antagonist MK-801 and the NR2B antagonist ifenprodil (IFN) but not by the NR2A antagonist NVP-AAM077 (NVP). In addition, decrease in the phosphorylation of GluR1 at Serine845 (p-GluR1-Ser845), decrease in spine density, and a reduction in the AMPAR/NMDAR ratio in the basolateral amygdala (BLA) were reversed after the MK-801 treatment. The effect of ANI on destabilization was prevented by the protein phosphatase 2B (calcineurin, CaN) inhibitors cyclosporine A (CsA) and FK-506 and the protein phosphatase 1 (PP1) inhibitors calyculin A (CA) and okadaic acid (OA). These results suggest that memory destabilization involves the activation of NR2B-containing NMDARs, which in turn allows the influx of Ca(2+) Increased intracellular Ca(2+) stimulates CaN, leading to the dephosphorylation and inactivation of inhibitor 1 and the activation of PP1. PP1 then dephosphorylates p-GluR1-Ser845 to elicit AMPA receptor (AMPAR) endocytosis and destabilization of the drug memory.

Epigenetic regulation of BDNF in the learned helplessness-induced animal model of depression.

Major depressive disorder (MDD), one of the most common mental disorders, is a significant risk factor for suicide and causes a low quality of life for many people. However, the causes and underlying mechanism of depression remain elusive. In the current work, we investigated epigenetic regulation of BDNF in the learned helplessness-induced animal model of depression. Mice were exposed to inescapable stress and divided into learned helplessness (LH) and resilient (LH-R) groups depending on the number they failed to escape. We found that the LH group had longer immobility duration in the forced swimming test (FST) and tail suspension tests (TST), which is consistent with a depression-related phenotype. Western blotting analysis and enzyme-linked immunosorbent assay (ELISA) revealed that the LH group had lower BDNF expression than that of the LH-R group. The LH group consistently had lower BDNF mRNA levels, as detected by qPCR assay. In addition, we found BDNF exon IV was down-regulated in the LH group. Intraperitoneal injection of imipramine or histone deacetylase inhibitors (HDACi) to the LH mice for 14 consecutive days ameliorated depression-like behaviors and reversed the decrease in BDNF. The expression of HDAC5 was up-regulated in the LH mice, and a ChIP assay revealed that the level of HDAC5 binding to the promoter region of BDNF exon IV was higher than that seen in other groups. Knockdown of HDAC5 reduced depression-like behaviors in the LH mice. Taken together, these results suggest that epigenetic regulation of BDNF by HDAC5 plays an important role in the learned helplessness model of depression.

α-Carboline derivative TJY-16 inhibits tumor growth by inducing G2/M cell cycle arrest in glioma cells.

BACKGROUND: Glioblastoma multiforme (GBM) is the most lethal primary brain tumors which remains difficult to cure despite advances in surgery, radiotherapy and chemotherapy. Therefore, the development of new drug is urgently needed. α-carboline derivatives were usually isolated from marine animals such as Britannia marine tunicate Dendrodoa grossularia and Indonesian ascidian Polycarpa aurata. In this study, we have synthesized several α-carboline compounds and examined their anti-glioma activities. RESULTS: We report that among α-carboline derivatives TJY-16 (6-acetyl-9-(3,4,5-trimethoxybenzyl)-9H-pyrido[2,3-b] indole) is the most potent α-carboline analog to induce glioma cell death with IC50 value of around 50 nM. TJY-16 decreased cell viability of glioma cells in a concentration- and time-dependent manner. Trypan blue exclusion assay showed that the reduction of cell viability was due to both cell growth inhibition and cell death. Flow cytometric analysis showed that TJY-16 induced G2/M cell cycle arrest followed by induction of sub-G1 phase. Hoechst staining detected the apoptotic features such as nuclear shrinkage and DNA condensation. Western blot analysis showed the increased level of cleaved caspase-3. The activation of caspase-8 and depolarization of mitochondrial membrane potential (ΔΨm) indicated that both extrinsic and intrinsic apoptotic pathways were involved in TJY-16-induced apoptosis. TJY-16 effectively inhibited tumor growth and induced caspase-3 activation in the xenograft tumor model of U87 glioma cells. CONCLUSIONS: Our results suggest that TJY-16 may kill glioma cells by inducing G2/M cell cycle arrest followed by apoptosis. Thus, TJY-16 is a promising agent for the treatment of malignant gliomas.