Disfunction of dorsal raphe nucleus-hippocampus serotonergic-HTR3 transmission results in anxiety phenotype of Neuroplastin 65-deficient mice.

Anxiety disorders are the most common psychiatric condition, but the etiology of anxiety disorders remains largely unclear. Our previous studies have shown that neuroplastin 65 deficiency (NP65-/-) mice exhibit abnormal social and mental behaviors and decreased expression of tryptophan hydroxylase 2 (TPH2) protein. However, whether a causal relationship between TPH2 reduction and anxiety disorders exists needs to be determined. In present study, we found that replenishment of TPH2 in dorsal raphe nucleus (DRN) enhanced 5-HT level in the hippocampus and alleviated anxiety-like behaviors. In addition, injection of AAV-NP65 in DRN significantly increased TPH2 expression in DRN and hippocampus, and reduced anxiety-like behaviors. Acute administration of exogenous 5-HT or HTR3 agonist SR57227A in hippocampus mitigated anxiety-like behaviors in NP65-/- mice. Moreover, replenishment of TPH2 in DRN partly repaired the impairment of long-term potentiation (LTP) maintenance in hippocampus of NP65-/- mice. Finally, we found that loss of NP65 lowered transcription factors Lmx1b expression in postnatal stage and replenishment of NP65 in DRN reversed the decrease in Lmx1b expression of NP65-/- mice. Together, our findings reveal that NP65 deficiency induces anxiety phenotype by downregulating DRN-hippocampus serotonergic-HTR3 transmission. These studies provide a novel and insightful view about NP65 function, suggesting an attractive potential target for treatment of anxiety disorders.

Neuroplastin 65 deficiency reduces amyloid plaque formation and cognitive deficits in an Alzheimer's disease mouse model.

Introduction: Alzheimer's disease (AD) is characterized by increasing cognitive dysfunction, progressive cerebral amyloid beta (Aß) deposition, and neurofibrillary tangle aggregation. However, the molecular mechanisms of AD pathologies have not been completely understood. As synaptic glycoprotein neuroplastin 65 (NP65) is related with synaptic plasticity and complex molecular events underlying learning and memory, we hypothesized that NP65 would be involved in cognitive dysfunction and Aß plaque formation of AD. For this purpose, we examined the role of NP65 in the transgenic amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD. Methods: Neuroplastin 65-knockout (NP65-/-) mice crossed with APP/PS1 mice to get the NP65-deficient APP/PS1 mice. In the present study, a separate cohort of NP65-deficient APP/PS1 mice were used. First, the cognitive behaviors of NP65-deficient APP/PS1 mice were assessed. Then, Aß plaque burden and Aß levels in NP65-deficient APP/PS1 mice were measured by immunostaining and western blot as well as ELISA. Thirdly, immunostaining and western blot were used to evaluate the glial response and neuroinflammation. Finally, protein levels of 5-hydroxytryptamin (serotonin) receptor 3A and synaptic proteins and neurons were measured. Results: We found that loss of NP65 alleviated the cognitive deficits of APP/PS1 mice. In addition, Aß plaque burden and Aß levels were significantly reduced in NP65-deficient APP/PS1 mice compared with control animals. NP65-loss in APP/PS1 mice resulted in a decrease in glial activation and the levels of pro- and anti-inflammatory cytokines (IL-1ß, TNF-α, and IL-4) as well as protective matrix YM-1 and Arg-1, but had no effect on microglial phenotype. Moreover, NP65 deficiency significantly reversed the increase in 5-hydroxytryptamine (serotonin) receptor 3A (Htr3A) expression levels in the hippocampus of APP/PS1 mice. Discussion: These findings identify a previously unrecognized role of NP65 in cognitive deficits and Aß formation of APP/PS1 mice, and suggest that NP65 may serve as a potential therapeutic target for AD.

Inhibiting 5-hydroxytryptamine receptor 3 alleviates pathological changes of a mouse model of Alzheimer's disease.

Extracellular amyloid beta (Aß) plaques are main pathological feature of Alzheimer's disease. However, the specific type of neurons that produce Aß peptides in the initial stage of Alzheimer's disease are unknown. In this study, we found that 5-hydroxytryptamin receptor 3A subunit (HTR3A) was highly expressed in the brain tissue of transgenic amyloid precursor protein and presenilin-1 mice (an Alzheimer's disease model) and patients with Alzheimer's disease. To investigate whether HTR3A-positive interneurons are associated with the production of Aß plaques, we performed double immunostaining and found that HTR3A-positive interneurons were clustered around Aß plaques in the mouse model. Some amyloid precursor protein-positive or ß-site amyloid precursor protein cleaving enzyme-1-positive neurites near Aß plaques were co-localized with HTR3A interneurons. These results suggest that HTR3A -positive interneurons may partially contribute to the generation of Aß peptides. We treated 5.0-5.5-month-old model mice with tropisetron, a HTR3 antagonist, for 8 consecutive weeks. We found that the cognitive deficit of mice was partially reversed, Aß plaques and neuroinflammation were remarkably reduced, the expression of HTR3 was remarkably decreased and the calcineurin/nuclear factor of activated T-cell 4 signaling pathway was inhibited in treated model mice. These findings suggest that HTR3A interneurons partly contribute to generation of Aß peptide at the initial stage of Alzheimer's disease and inhibiting HTR3 partly reverses the pathological changes of Alzheimer's disease.

Artemisinin protects DPSC from hypoxia and TNF-α mediated osteogenesis impairments through CA9 and Wnt signaling pathway.

Dental pulp stem cells (DPSCs) possess the ability of multi-lineage differentiation, and are excellent sources of tissue engineering and regenerative medicine. Oxygen concentration and inflammation are two critical environmental factors that affect the osteogenic differentiation of DPSCs. We aimed to study the role of the antimalarial drug artemisinin on the osteogenic differentiation of human DPSCs under the hypoxia and inflammation conditions. We demonstrated that hypoxia (5% O2) and inflammation (20 ng/mL TNF-α), alone or in combination, significantly diminished in vitro cell survival and increased apoptotic rates. Notably, hypoxia and TNF-α exerted accumulative effect in suppressing the osteogenic differentiation of DPSCs, as evidenced by reduced expression levels of osteogenesis-associated genes including ALP, RUNX2 and OCN in osteogenic condition, as well as reduced mineral nodules formation as indicated by alizarin red staining. Artemisinin at the dose of 40 µM markedly reversed the suppression in cell survival caused by hypoxia or inflammation, and reduced apoptotic rates and the expressions of pro-apoptotic proteins. Additionally, artemisinin restored osteogenic differentiation of DPSCs under the hypoxia or/and inflammation conditions. Moreover, the beneficial effect of artemisinin was dependent on upregulated expression of CA9 and CA9-mediated antioxidant responses, as CA9 knockdown abolished the protective role of artemisinin on DPSC osteogenesis. Furthermore, while hypoxia or/and inflammation significantly inactivated the Wnt/ß-catenin signaling in DPSCs, additional exposure to artemisinin re-activated this pathway to promote osteogenic differentiation of DPSCs. Our results provide novel insight on the link between artemisinin and DPSC osteogenesis, and suggest promising artemisinin-based strategies for better dentin/pulp tissue engineering.

Effects of primary microglia and astrocytes on neural stem cells in in vitro and in vivo models of ischemic stroke.

Transplantation of neural stem cells (NSCs) can protect neurons in animal stroke models; however, their low rates of survival and neuronal differentiation limit their clinical application. Glial niches, an important location of neural stem cells, regulate survival, proliferation and differentiation of neural stem cells. However, the effects of activated glial cells on neural stem cells remain unclear. In the present study, we explored the effects of activated astrocytes and microglia on neural stem cells in vitro stroke models. We also investigated the effects of combined transplantation of neural stem cells and glial cells after stroke in rats. In a Transwell co-culture system, primary cultured astrocytes, microglia or mixed glial cells were exposed to glutamate or H2O2 and then seeded in the upper inserts, while primary neural stem cells were seeded in the lower uncoated wells and cultured for 7 days. Our results showed that microglia were conducive to neurosphere formation and had no effects on apoptosis within neurospheres, while astrocytes and mixed glial cells were conducive to neurosphere differentiation and reduced apoptosis within neurospheres, regardless of their pretreatment. In contrast, microglia and astrocytes induced neuronal differentiation of neural stem cells in differentiation medium, regardless of their pretreatment, with an exception of astrocytes pretreated with H2O2. Rat models of ischemic stroke were established by occlusion of the middle cerebral artery. Three days later, 5 × 105 neural stem cells with microglia or astrocytes were injected into the right lateral ventricle. Neural stem cell/astrocyte-treated rats displayed better improvement of neurological deficits than neural stem cell only-treated rats at 4 days after cell transplantation. Moreover, neural stem cell/microglia-, and neural stem cell/astrocyte-treated rats showed a significant decrease in ischemic volume compared with neural stem cell-treated rats. These findings indicate that microglia and astrocytes exert different effects on neural stem cells, and that co-transplantation of neural stem cells and astrocytes is more conducive to the recovery of neurological impairment in rats with ischemic stroke. The study was approved by the Animal Ethics Committee of Tongji University School of Medicine, China (approval No. 2010-TJAA08220401) in 2010.

Electroacupuncture promotes peripheral nerve regeneration after facial nerve crush injury and upregulates the expression of glial cell-derived neurotrophic factor.

The efficacy of electroacupuncture in the treatment of peripheral facial paralysis is known, but the specific mechanism has not been clarified. Glial cell-derived neurotrophic factor (GDNF) has been shown to protect neurons by binding to N-cadherin. Our previous results have shown that electroacupuncture could increase the expression of N-cadherin mRNA in facial neurons and promote facial nerve regeneration. In this study, the potential mechanisms by which electroacupuncture promotes nerve regeneration were elucidated through assessing the effects of electroacupuncture on GDNF and N-cadherin expression in facial motoneurons of rabbits with peripheral facial nerve crush injury. New Zealand rabbits were randomly divided into a normal group (normal control, n = 21), injury group (n = 45) and electroacupuncture group (n = 45). Model rabbits underwent facial nerve crush injury only. Rabbits in the electroacupuncture group received facial nerve injury, and then underwent electroacupuncture at Yifeng (TE17), Jiache (ST6), Sibai (ST2), Dicang (ST4), Yangbai (GB14), Quanliao (SI18), and Hegu (LI4; only acupuncture, no electrical stimulation). The results showed that in behavioral assessments, the total scores of blink reflex, vibrissae movement, and position of apex nasi, were markedly lower in the EA group than those in the injury group. Hematoxylin-eosin staining of the right buccinator muscle of each group showed that the cross-sectional area of buccinator was larger in the electroacupuncture group than in the injury group on days 1, 14 and 21 post-surgery. Toluidine blue staining of the right facial nerve tissue of each group revealed that on day 14 post-surgery, there was less axonal demyelination and fewer inflammatory cells in the electroacupuncture group compared with the injury group. Quantitative real time-polymerase chain reaction showed that compared with the injury group, N-cadherin mRNA levels on days 4, 7, 14 and 21 and GDNF mRNA levels on days 4, 7 and 14 were significantly higher in the electroacupuncture group. Western blot assay displayed that compared with the injury group, the expression of GDNF protein levels on days 7, 14 and 21 were significantly upregulated in the electroacupuncture group. The histology with hematoxylin-eosin staining and Nissl staining of brainstem tissues containing facial neurons in the middle and lower part of the pons exhibited that on day 7 post-surgery, there were significantly fewer apoptotic neurons in the electroacupuncture group than in the injury group. By day 21, there was no significantly difference in the number of neurons between the electroacupuncture and normal groups. Taken together, these results have confirmed that electroacupuncture promotes regeneration of peripheral facial nerve injury in rabbits, inhibits neuronal apoptosis, and reduces peripheral inflammatory response, resulting in the recovery of facial muscle function. This is achieved by up-regulating the expression of GDNF and N-cadherin in central facial neurons.

Ginsenoside Rg1 protects against neurodegeneration by inducing neurite outgrowth in cultured hippocampal neurons.

Ginsenoside Rg1 (Rg1) has anti-aging and anti-neurodegenerative effects. However, the mechanisms underlying these actions remain unclear. The aim of the present study was to determine whether Rg1 affects hippocampal survival and neurite outgrowth in vitro after exposure to amyloid-beta peptide fragment 25-35 (Aß25-35), and to explore whether the extracellular signal-regulated kinase (ERK) and Akt signaling pathways are involved in these biological processes. We cultured hippocampal neurons from newborn rats for 24 hours, then added Rg1 to the medium for another 24 hours, with or without pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) family or Akt signaling pathways for a further 24 hours. We then immunostained the neurons for growth associated protein-43, and measured neurite length. In a separate experiment, we exposed cultured hippocampal neurons to Aß25-35 for 30 minutes, before adding Rg1 for 48 hours, with or without Akt or MAPK inhibitors, and assessed neuronal survival using Hoechst 33258 staining, and phosphorylation of ERK1/2 and Akt by western blot analysis. Rg1 induced neurite outgrowth, and this effect was blocked by API-2 (Akt inhibitor) and PD98059 (MAPK/ERK kinase inhibitor), but not by SP600125 or SB203580 (inhibitors of c-Jun N-terminal kinase and p38 MAPK, respectively). Consistent with this effect, Rg1 upregulated the phosphorylation of Akt and ERK1/2; these effects were reversed by API-2 and PD98059, respectively. In addition, Rg1 significantly reversed Aß25-35-induced apoptosis; this effect was blocked by API-2 and PD98059, but not by SP600125 or SB203580. Finally, Rg1 significantly reversed the Aß25-35-induced decrease in Akt and ERK1/2 phosphorylation, but API-2 prevented this reversal. Our results indicate that Rg1 enhances neurite outgrowth and protects against Aß25-35-induced damage, and that its mechanism may involve the activation of Akt and ERK1/2 signaling.

Transplantation of neural stem cells overexpressing glial cell line-derived neurotrophic factor enhances Akt and Erk1/2 signaling and neurogenesis in rats after stroke.

BACKGROUND: Our previous studies have indicated that the beneficial effects of grafting neural stem cells (NSCs) overexpressing glial cell line-derived neurotrophic factor (GDNF) in rats after stroke. However, the underlying mechanisms are highly debatable. In this study, we investigated whether neurogenesis, Akt, and extracellular signal-regulated kinase 1/2 (Erk1/2) signaling were involved in this process. METHODS: Transient ischemic stroke were induced by occluding middle cerebral artery for 2 hours and reperfusion. At 3 days after reperfusion, GDNF/NSCs, NSCs, and vehicle were administered. Immunohistochemical staining was used to evaluate neurogenesis by nestin antibody; phosphorylation of Akt and Erk1/2 was investigated by Western blotting analysis. RESULTS: Transplantation of GDNF/NSCs and NSCs significantly increased nestin-positive cells compared to control group (vehicle) from 1 to 7 weeks after reperfusion, and GDNF/NSCs showed stronger effect than NSCs at 2 and 3 weeks after reperfusion. Meanwhile, enhanced phosphorylation level of Erk1/2 was observed in the GDNF/NSCs and NSCs groups compared with control group, and phosphorylation level of Erk1/2 in GDNF/NSCs group was remarkably higher than that of NSCs group at any given time. In contrast, expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), known as inhibitor of Erk1/2 signaling, was significantly decreased in the GDNF/NSCs and NSCs groups compared with the control group. Moreover, much enhanced and prolonged phosphorylation level of Akt of GDNF/NSCs group was detected compared with control and NSCs group. CONCLUSION: Grafting GDNF/NSCs enhances neurogenesis and activates Akt and Erk1/2 signaling, that may provide the potential for GDNF/NSCs in stroke treatment.

Ginsenoside Rb1 regulates the expressions of brain-derived neurotrophic factor and caspase-3 and induces neurogenesis in rats with experimental cerebral ischemia.

AIM OF THE STUDY: Recent studies have revealed that ginsenoside Rb1 (GRb1) is neuroprotective for cerebral ischemia. However, the mechanism underlying of this function is unclear. We assessed whether this neuroprotective effect of GRb1 was mediated by the levels of brain-derived neurotrophic factor (BDNF), by the levels of caspase-3 proteins and by induced neurogenesis in rats following transient cerebral ischemia or not. MATERIALS AND METHODS: Cerebral ischemia was prepared by a 2 h occlusion of the middle cerebral artery and reperfusion, followed by infusion of GRb1 (40 mg/kg) and saline (GRb1 and ischemia groups, respectively). All rats were sacrificed at 3 and 12 h, 1, 2, 3, 5, and 10 days after reperfusion. Normal and sham-operated rats were used in control group. Modified Neurological Severity Scores (mNSS) test and hematoxylin and eosin staining were respectively performed to evaluate neurological function and histological feature. Immunohistochemistry was used to identify intrinsic neurogenesis by nestin antibody. Western blotting was used to detect BDNF and caspase-3 protein content. RESULTS: GRb1 infusion after cerebral ischemia significantly promoted recoveries of neurological functions at 3 and 5 days after reperfusion compared to ischemic rats. The number of nestin-positive cells was apparently increased after GRb1 infusion compared to ischemia rats at given time. Moreover, BDNF was significantly increased in GRb1-treated rats compared to ischemia rats at different time points. In contrast, GRb1 infusion after the onset of reperfusion, caspase-3 at a given time was significantly reduced compared to ischemia rats, but still significantly increased compared to control rats. CONCLUSIONS: Promotion of the neurogenesis and regulation of the expressions of BDNF and caspase-3 may be involved in GRb1-induced neuroprotection against cerebral ischemia.

[Effects of Ginsenoside RB1 on neural cell apoptosis and expressions of Bcl-2 and Bax in rats following subjected to cerebral ischemia-reperfusion].

OBJECTIVE: To observe the effects of Ginsenoside Rb1 (GRb1) on neuronal cell apoptosis and the expressions of Bcl-2 and Bax in rats after cerebral ischemia-reperfusion so as to investigate the neuroprotective mechanism of GRb1. METHDOS: The model of cerebral ischemia-reperfusion was established by occluding rat middle cerebral artery for 2 h. The rats were randomly divided into two groups: ischemia-reperfusion group (I/R group) and GRb1 treat group (GRb1 group). GRb1 (40 mg/kg, i.p.) was administered immediately to rats after the onset of reperfusion. Two groups were further subdivided 7 subgroups according to various reperfusion time (3 h, 12 h, 1 d, 2 d, 3 d, 5 d and 10 d, n=4 per time point). HE staining was used to observe histological features. TUNEL and immunohistochemical method were used to analyze the cell apoptosis and expressions of Bcl-2 and Bax, respectively. RESULTS: Compared with I/R group, GRb1 reduced pathological changes, and decreased the number of apoptotic neural cells (P<0.05 on 12 h, 1 d, 2 d and 3 d) and up-regulated the number of Bcl-2 positive cells (P<0.05 on 12 h, 1 d, 3 d, 5 d and 10 d), and meanwhile down-regulated the number of Bax positive cells (P<0.05 on 3 h, 12 h, 1 d, 2 d, 3 d, 5 d and 10 d) in the ipsilateral hemisphere. CONCLUSION: The neuroprotective effect of GRb1 on cerebral ischemia-reperfusion injury is related to inhibit neuronal apoptosis and to up-regulate the expression of Bcl-2 with down-regulating the expression of Bax.