Activation of mTORC1 Signaling Cascade in Hippocampus and Medial Prefrontal Cortex Is Required for Antidepressant Actions of Vortioxetine in Mice.

BACKGROUND: Although thought of as a multimodal-acting antidepressant targeting the serotonin system, more molecules are being shown to participate in the antidepressant mechanism of vortioxetine. A previous report has shown that vortioxetine administration enhanced the expression of rapamycin complex 1 (mTORC1) in neurons. It has been well demonstrated that mTORC1 participates in not only the pathogenesis of depression but also the pharmacological mechanisms of many antidepressants. Therefore, we speculate that the antidepressant mechanism of vortioxetine may require mTORC1. METHODS: Two mouse models of depression (chronic social defeat stress and chronic unpredictable mild stress) and western blotting were first used together to examine whether vortioxetine administration produced reversal effects against the chronic stress-induced downregulation in the whole mTORC1 signaling cascade in both the hippocampus and medial prefrontal cortex (mPFC). Then, LY294002, U0126, and rapamycin were used together to explore whether the antidepressant effects of vortioxetine in mouse models of depression were attenuated by pharmacological blockade of the mTORC1 system. Furthermore, lentiviral-mTORC1-short hairpin RNA-enhanced green fluorescence protein (LV-mTORC1-shRNA-EGFP) was adopted to examine if genetic blockade of mTORC1 also abolished the antidepressant actions of vortioxetine in mice. RESULTS: Vortioxetine administration produced significant reversal effects against the chronic stress-induced downregulation in the whole mTORC1 signaling cascade in both the hippocampus and mPFC. Both pharmacological and genetic blockade of the mTORC1 system notably attenuated the antidepressant effects of vortioxetine in mice. CONCLUSIONS: Activation of the mTORC1 system in the hippocampus and mPFC is required for the antidepressant actions of vortioxetine in mice.

Salt-inducible kinase 1-CREB-regulated transcription coactivator 1 signalling in the paraventricular nucleus of the hypothalamus plays a role in depression by regulating the hypothalamic-pituitary-adrenal axis.

Elucidating the molecular mechanism underlying the hyperactivity of the hypothalamic-pituitary-adrenal axis during chronic stress is critical for understanding depression and treating depression. The secretion of corticotropin-releasing hormone (CRH) from neurons in the paraventricular nucleus (PVN) of the hypothalamus is controlled by salt-inducible kinases (SIKs) and CREB-regulated transcription co-activators (CRTCs). We hypothesised that the SIK-CRTC system in the PVN might contribute to the pathogenesis of depression. Thus, the present study employed chronic social defeat stress (CSDS) and chronic unpredictable mild stress (CUMS) models of depression, various behavioural tests, virus-mediated gene transfer, enzyme-linked immunosorbent assay, western blotting, co-immunoprecipitation, quantitative real-time reverse transcription polymerase chain reaction, and immunofluorescence to investigate this connection. Our results revealed that both CSDS and CUMS induced significant changes in SIK1-CRTC1 signalling in PVN neurons. Both genetic knockdown of SIK1 and genetic overexpression of CRTC1 in the PVN simulated chronic stress, producing a depression-like phenotype in naive mice, and the CRTC1-CREB-CRH pathway mediates the pro-depressant actions induced by SIK1 knockdown in the PVN. In contrast, both genetic overexpression of SIK1 and genetic knockdown of CRTC1 in the PVN protected against CSDS and CUMS, leading to antidepressant-like effects in mice. Moreover, stereotactic infusion of TAT-SIK1 into the PVN also produced beneficial effects against chronic stress. Furthermore, the SIK1-CRTC1 system in the PVN played a role in the antidepressant actions of fluoxetine, paroxetine, venlafaxine, and duloxetine. Collectively, SIK1 and CRTC1 in PVN neurons are closely involved in depression neurobiology, and they could be viable targets for novel antidepressants.

Hippocampal miR-206-3p participates in the pathogenesis of depression via regulating the expression of BDNF.

As a widely-known neuropsychiatric disorder, the exact pathogenesis of depression remains elusive. MiRNA-206 (miR-206) is conventionally known as one of the myomiRs and has two forms: miR-206-3p and miR-206-5p. Recently, miR-206 has been demonstrated to regulate the biosynthesis of brain-derived neurotrophic factor (BDNF), a very popular target involved in depression and antidepressant responses. Here we assumed that miR-206 may play a role in depression, and various methods including the chronic social defeat stress (CSDS) model of depression, quantitative real-time reverse transcription PCR, western blotting, immuofluorescence and virus-mediated gene transfer were used together. It was found that CSDS robustly increased the level of miR-206-3p but not miR-206-5p in the hippocampus. Both genetic overexpression of hippocampal miR-206-3p and intranasal administration of AgomiR-206-3p induced not only notable depressive-like behaviors but also significantly decreased hippocampal BDNF signaling cascade and neurogenesis in naïve C57BL/6J mice. In contrast, both genetic knockdown of hippocampal miR-206-3p and intranasal administration of AntagomiR-206-3p produced significant antidepressant-like effects in the CSDS model of depression. Furthermore, it was found that the antidepressant-like effects induced by miR-206-3p inhibition require the hippocampal BDNF-TrkB system. Taken together, hippocampal miR-206-3p participates in the pathogenesis of depression by regulating BDNF biosynthesis and is a feasible antidepressant target.

Two resveratrol analogs, pinosylvin and 4,4'-dihydroxystilbene, improve oligoasthenospermia in a mouse model by attenuating oxidative stress via the Nrf2-ARE pathway.

Two synthesized resveratrol analogs from our laboratory, namely pinosylvin (3,5-dihydroxy-trans-stilbene, PIN) and 4,4'-dihydroxystilbene (DHS), have been carefully evaluated for treatment of oligoasthenospermia. Recent studies have demonstrated that PIN and DHS improved sperm quality in the mouse. However, the mechanism of action of PIN and DHS on oligoasthenospermia remains unknown. Herein, we investigated the mechanistic basis for improvements in sperm parameters by PIN and DHS in a mouse model of oligoasthenospermia induced by treatment with busulfan (BUS) at 6 mg/kg b.w.. Two weeks following busulfan treatment, mice were administered different concentrations of PIN or DHS daily for 2 consecutive weeks. Thereafter, epididymal sperm concentration and motility were determined, and histopathology of the testes was performed. Serum hormone levels including testosterone (T), luteinizing hormone (LH), and follicle stimulating hormone (FSH) were measured using corresponding specific enzyme-linked immunosorbent assay (ELISA) kits. Testicular mRNA expression profiles were determined by RNA sequencing analysis. These findings were validated by quantitative real-time PCR, western blotting and ELISA. Both PIN and DHS improved the epididymal sperm concentration and motility, enhanced testosterone levels, and promoted testicular morphological recovery following BUS treatment. PIN treatment was found to significantly reduce oxidative stress via the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE)-dependent antioxidant, glutathione peroxidase 3. DHS treatment significantly reduced oxidative stress via the Nrf2-ARE-dependent antioxidants glutathione S-transferase theta 2 and glutathione S-transferase omega 2. In summary, PIN and DHS ameliorated oligoasthenospermia in this mouse model by attenuating oxidative stress via the Nrf2-ARE pathway.

Andrographolide Exerts Significant Antidepressant-Like Effects Involving the Hippocampal BDNF System in Mice.

BACKGROUND: Major depressive disorder is a worldwide neuropsychiatric disorder associated with various symptoms, but current antidepressants used in clinical practice have various side effects and high failure rates. Andrographolide is the main bioactive ingredient of Andrographis paniculata and exhibits numerous pharmacological actions. This study aimed to evaluate the antidepressant-like effects of andrographolide in male C57BL/6J mice. METHODS: The antidepressant-like effects of andrographolide in mice were explored in a forced swim test, tail suspension test, and chronic unpredictable mild stress model of depression. Western blotting and immunofluorescence were further performed to assess the effects of chronic unpredictable mild stress and andrographolide on the brain-derived neurotrophic factor signalling cascade and hippocampal neurogenesis. Moreover, a pharmacological inhibitor (K252a) and a lentiviral-short hairpin RNA (LV-TrkB-shRNA) were used to clarify the antidepressant-like mechanism of andrographolide. RESULTS: Andrographolide exhibited antidepressant-like potential in the forced swim test and tail suspension test without influencing the locomotor activity of mice. Repeated andrographolide treatment not only produced significant antidepressant-like effects in the chronic unpredictable mild stress model but also prevented the decreasing effects of chronic unpredictable mild stress on hippocampal brain-derived neurotrophic factor signalling and neurogenesis in mice. Importantly, blockade of the hippocampal brain-derived neurotrophic factor system by K252a and TrkB-shRNA fully abolished the antidepressant-like effects of andrographolide in mice. CONCLUSIONS: Andrographolide exerts antidepressant-like effects in mice via promoting the hippocampal brain-derived neurotrophic factor signalling cascade.

The Anti-dementia Effects of Donepezil Involve miR-206-3p in the Hippocampus and Cortex.

Alzheimer's disease (AD) is a most serious age-related neurodegenerative disorder accompanied with significant memory impairments in this world. Recently, microRNAs (miRNAs) have been reported to be invlolved in the pathophysiology of AD. Previous studies have shown that miRNA-206 (miR-206) is implicated in the pathogenesis of AD via suppressing the expression of brain-derived neurotrophic factor (BDNF) in the brain. Here, we examined the miR-206-3p and miR-206-5p expression in the hippocampus and cortex of Abeta precursor protein (APP)/presenilin-1 (PS1) transgenic mice treated with donepezil, a drug approved for treating AD in clinic. We found that the expression of miR-206-3p was significantly up-regulated in the hippocampus and cortex of APP/PS1 mice, while donepezil administration significantly reversed this dysfunction. In addition, enhancing the miR-206-3p level by the usage of AgomiR-206-3p significantly attenuated the anti-dementia effects of donepezil in APP/PS1 mice. Together, these results suggested that miR-206-3p is involved in the anti-dementia effects of donepezil, and could be a novel pharmacological target for treating AD.

Antidepressant-Like Effects of GM1 Ganglioside Involving the BDNF Signaling Cascade in Mice.

BACKGROUND: Depression is a serious psychiatric disorder that easily causes physical impairments and a high suicide rate. Monosialotetrahexosylganglioside is a crucial ganglioside for the central nervous system and has been reported to affect the function of the brain derived neurotrophic factor system. This study is aimed to evaluate whether monosialotetrahexosylganglioside has antidepressant-like effects. METHODS: Antidepressant-like effects of monosialotetrahexosylganglioside were assessed in the chronic social defeat stress model of depression, and various behavioral tests were performed. Changes in the brain derived neurotrophic factor signaling pathway after chronic social defeat stress and monosialotetrahexosylganglioside treatment were also investigated. A tryptophan hydroxylase inhibitor and brain derived neurotrophic factor signaling inhibitors were used to determine the antidepressant mechanisms of monosialotetrahexosylganglioside. RESULTS: Monosialotetrahexosylganglioside administration significantly reversed the chronic social defeat stress-induced reduction of sucrose preference and social interaction in mice and also prevented the increased immobility time in the forced swim test and tail suspension test. In addition, monosialotetrahexosylganglioside completely ameliorated the stress-induced dysfunction of brain derived neurotrophic factor signaling cascade in the hippocampus and medial prefrontal cortex, 2 regions closely involved in the pathophysiology of depression. Furthermore, the usage of brain derived neurotrophic factor signaling cascade inhibitors, K252a and anti-brain derived neurotrophic factor antibody, each abolished the antidepressant-like effects of monosialotetrahexosylganglioside, while the usage of a serotonin system inhibitor did not. CONCLUSIONS: Taken together, our findings suggest that monosialotetrahexosylganglioside indeed has antidepressant-like effects, and these effects were mediated through the activation of brain derived neurotrophic factor signaling cascade.

The salt-inducible kinases inhibitor HG-9-91-01 exhibits antidepressant-like actions in mice exposed to chronic unpredictable mild stress.

Major depressive disorder is a frequently occurring neuropsychiatric disorder throughout the world. However, the limited and delayed therapeutic efficacy of monoaminergic medications has led to intensive research efforts to develop novel antidepressants. We have previously demonstrated that hippocampal salt-inducible kinase 2 (SIK2) plays a role in the pathogenesis of depression via regulating the downstream CREB-regulated transcription coactivator 1 (CRTC1)-cAMP response element-binding protein (CREB)-brain derived neurotrophic factor (BDNF) pathway. HG-9-91-01 is a potent and selective inhibitor of salt-inducible kinases (SIKs). The present study aims to explore whether HG-9-91-01 has antidepressant-like actions in male C57BL/6J mice. The chronic unpredictable mild stress (CUMS) model of depression, various behavioral tests, western blotting, co-immunoprecipitation, immunofluorescence, stereotactic infusion, and viral-mediated genetic knockdown were used together. It was found that hippocampal infusion of HG-9-91-01 induced significant antidepressant-like effects in the CUMS model, accompanied with preventing the enhancement of CUMS on the hippocampal SIK2 expression and cytoplasmic translocation of CRTC1. HG-9-91-01 treatment also reversed the decreasing effects of CUMS on the BDNF signaling cascade and adult neurogenesis in the hippocampus. Moreover, the antidepressant-like actions of HG-9-91-01 in mice required the hippocampal CRTC1-CREB-BDNF pathway. In conclusion, HG-9-91-01 has potential of being a novel antidepressant candidate.

Hippocampal F3/Contactin plays a role in chronic stress-induced depressive-like effects and the antidepressant actions of vortioxetine in mice.

Depression is a very prevalent psychiatric disorder which threats nearly one in six of the population in this world. To date, the pathogenesis of depression remains elusive and is thought to depend on multiple factors in which chronic stress is critical. Currently, it has been demonstrated that besides monoaminergic dysfunction, depression is accompanied by several other important pathological phenomena such as impaired neurogenesis and decreased brain-derived neurotrophic factor (BDNF)-cAMP response element binding protein (CREB) signaling cascade in the hippocampus. F3/Contactin is a cell-adhesion molecule which has been reported to correlate with hippocampal neurogenesis and BDNF-CREB signaling. Here we assumed that F3/Contactin may be implicated in depression, and various methods including western blotting, immunofluorescence, virus-mediated gene transfer and chronic stress models of depression were adopted together. It was found that both chronic restraint stress (CRS) and chronic social defeat stress (CSDS) significantly decreased the expression of F3/Contactin in the hippocampus. Adeno-associated virus (AAV)-mediated over-expression of hippocampal F3/Contactin notably prevented the CRS-induced and CSDS-induced depressive-like behaviors in mice. Moreover, hippocampal F3/Contactin over-expression also fully reversed the CRS-induced and CSDS-induced dysfunction in the hippocampal BDNF-CREB signaling and neurogenesis of mice. Furthermore, administration of vortioxetine, a multimodal-acting antidepressant, fully ameliorated the inhibitory actions of both CRS and CSDS on the hippocampal F3/Contactin expression. In contrast, AAV-mediated knockdown of hippocampal F3/Contactin significantly abolished the protecting effects of vortioxetine against CRS and CSDS. Collectively, hippocampal F3/Contactin is implicated in depression and could be a novel antidepressant target.

Hippocampal overexpression of chordin protects against the chronic social defeat stress-induced depressive-like effects in mice.

Depression is a serious and worldwide neuropsychiatric disesase, and developing novel antidepressant targets beyond the monoaminergic systems is now popular and necessary. Bone morphogenetic protein (BMP) signals modulate numerous developmental, physiological, and homeostatic processes. The functions of BMPs are also regulated by secreted extracellular antagonists such as chordin and noggin. Chordin has abundant expression in adult brain, and may play critical role in the central nervous system. In this study, the chronic social defeat stress (CSDS) model of depression, various behavioral tests, western blotting, quantitative real-time reverse transcription PCR, immunohistochemistry, recombinant mouse chordin protein and AAV-Chordin-EGFP were together used to explore the role of chordin in the pathogenesis of depression. It was found that CSDS significantly decreased the expression of chordin in the hippocampus but not other related brain regions. Moreover, both pharmacological and genetic overexpression of hippocampal chordin fully protected against the CSDS-induced depressive-like effects in mice. Collectively, hippocampal chordin could be a novel antidepressant target, and this study further highlights the importance of the hippocampal BMP system in the pathophysiology of depression.

Hippocampal Salt-Inducible Kinase 2 Plays a Role in Depression via the CREB-Regulated Transcription Coactivator 1-cAMP Response Element Binding-Brain-Derived Neurotrophic Factor Pathway.

BACKGROUND: Developing novel pharmacological targets beyond monoaminergic systems is now a popular strategy for finding new ways to treat depression. Salt-inducible kinase (SIK) is a kinase that regulates the nuclear translocation of cyclic adenosine monophosphate response element binding protein (CREB)-regulated transcription coactivator (CRTC) by phosphorylation. Here, we hypothesize that dysfunction of the central SIK-CRTC system may contribute to the pathogenesis of depression. METHODS: Chronic social defeat stress (CSDS) and chronic unpredictable mild stress (CUMS) models of depression, various behavioral tests, viral-mediated gene transfer, Western blotting, coimmunoprecipitation, quantitative real-time reverse transcription polymerase chain reaction, and immunohistochemistry were used in this study (for in vivo studies, n = 10; for in vitro studies, n = 5). RESULTS: Both CSDS and CUMS markedly increased the expression of hippocampal SIK2, which reduced CRTC1 nuclear translocation and binding of CRTC1 and CREB in the hippocampus. Genetic overexpression of hippocampal SIK2 in naïve mice simulated chronic stress, inducing depressive-like behaviors in the forced swim test, tail suspension test, sucrose preference test, and social interaction test, as well as decreasing the brain-derived neurotrophic factor signaling cascade and neurogenesis in the hippocampus. In contrast, genetic knockdown and knockout of hippocampal SIK2 protected against CSDS and CUMS, exerting significant antidepressant-like effects that were mediated via the downstream CRTC1-CREB-brain-derived neurotrophic factor pathway. Moreover, fluoxetine, venlafaxine, and mirtazapine all significantly restored the effects of CSDS and CUMS on the hippocampal SIK2-CRTC1 pathway, which was necessary for their antidepressant actions. CONCLUSIONS: The hippocampal SIK2-CRTC1 pathway is involved in the pathogenesis of depression, and hippocampal SIK2 could be a novel target for the development of antidepressants.

A partition-type tubular scaffold loaded with PDGF-releasing microspheres for spinal cord repair facilitates the directional migration and growth of cells.

The best tissue-engineered spinal cord grafts not only match the structural characteristics of the spinal cord but also allow the seed cells to grow and function in situ. Platelet-derived growth factor (PDGF) has been shown to promote the migration of bone marrow stromal cells; however, cytokines need to be released at a steady rate to maintain a stable concentration in vivo. Therefore, new methods are needed to maintain an optimal concentration of cytokines over an extended period of time to effectively promote seed cell localization, proliferation and differentiation. In the present study, a partition-type tubular scaffold matching the anatomical features of the thoracic 8-10 spinal cord of the rat was fabricated using chitosan and then subsequently loaded with chitosan-encapsulated PDGF-BB microspheres (PDGF-MSs). The PDGF-MS-containing scaffold was then examined in vitro for sustained-release capacity, biocompatibility, and its effect on neural progenitor cells differentiated in vitro from multilineage-differentiating stress-enduring cells (MUSE-NPCs). We found that pre-freezing for 2 hours at -20°C significantly increased the yield of partition-type tubular scaffolds, and 30 µL of 25% glutaraldehyde ensured optimal crosslinking of PDGF-MSs. The resulting PDGF-MSs cumulatively released 52% of the PDGF-BB at 4 weeks in vitro without burst release. The PDGF-MS-containing tubular scaffold showed suitable biocompatibility towards MUSE-NPCs and could promote the directional migration and growth of these cells. These findings indicate that the combination of a partition-type tubular scaffold, PDGF-MSs and MUSE-NPCs may be a promising model for the fabrication of tissue-engineered spinal cord grafts.

Mycobacterium marinum Infection in Zebrafish and Microglia Imitates the Early Stage of Tuberculous Meningitis.

Mycobacterium tuberculosis (M. tuberculosis) invading and activating microglia causes the most serious subtypes of tuberculosis called tubercular meningitis. However, the developmental process of tubercular meningitis, especially the early phase, is poorly understood due to lacking well-established and well-accepted visible models in vitro and in vivo. Here, consistent with one recent report, we found Mycobacterium marinum (M. marinum) invade the zebrafish brain and subsequently cause granuloma-like structures. We further showed that M. marinum, which shares similar characteristics with M. tuberculosis, can invade microglia and replicate in microglia, which subsequently promote the secretion of pro-inflammatory cytokines such as IL-1ß, IL-6, and TNF-α. M. marinum infection in microglia can also promote autophagy, which conversely limits the replication of M. marinum. Thus, pharmacological activation of autophagy by rapamycin could prevent M. marinum replication. Our study provides in vivo and in vitro models to study underlying pathogenic mechanisms of tubercular meningitis by using M. marinum. Our results also showed that activation of autophagy could be a meaningful way to prevent tubercular meningitis.

Lentiviral Vector-Mediated p27kip1 Expression Facilitates Recovery After Spinal Cord Injury.

Traumatic spinal cord injury (SCI) causes tissue loss and associated neurological dysfunction attributable to both mechanical damage and secondary biochemical and physiological responses. Upregulation of cell cycle proteins occurs in both neurons and glia after SCI and may contribute to these changes. Increased cell cycle protein is associated with neuronal and oligodendroglial apoptosis, reactive astrogliosis, glial scar formation, and microglial activation. Here, using lentiviral vectors (LV), we induced the expression of the cyclin-dependent kinase (CDK) inhibitor p27kip1 in the lesioned spinal cord of adult rat. Treatment with LV-p27kip1 significantly reduced the expression of cell cycle proteins and improved functional recovery. In addition, p27kip1 overexpression also reduced lesion volume, decreased astrocytic reactivity, attenuated microglial activation, reduced cell death, and improved the local microenvironment. We suggest that these effects reflect the ability of p27kip1 to inhibit cell cycle pathways. Thus, the present study provides further support for the therapeutic potential of cell cycle inhibitors in the treatment of SCI.

Synthesis and evaluation of aroylthiourea derivatives of 4-ß-amino-4'-O-demethyl-4-desoxypodophyllotoxin as novel topoisomerase II inhibitors.

A novel series of aroylthiourea derivatives of 4-ß-amino-4'-O-demethyl-4-desoxy- podophyllotoxin were synthesized. Their cytotoxicities against three cancer cell lines were investigated by MTT assay. The kDNA decatenation assay indicated that compounds 5a, 5f, 5h and 5l inhibited topoisomerase II-mediated kDNA decatenation. DNA flow cytometric analysis revealed that compound 5a induced cell cycle arrest at G2/M phase in HCT-116 cell line.