α2,3- and α2,6-linked sialic acids are important for cell binding and replication of Newcastle disease virus in chicken primary neuronal cells.

Velogenic Newcastle disease virus (NDV) causes encephalitis and severe neurological disorders in avian species. Sialic acid (SA) has been considered as a receptor for NDV infection and determining tissue tropism. The neurotropic mechanism of NDV in birds is completely unknown. Here we have investigated the role of viral receptor SA in neurotropism of NDV in chickens. We determined that α2,3- and α2,6-linked SA receptors were implicated in NDV encephalitis and viral binding to primary neuronal cells using immunohistofluorescence and virus-cell binding assay. Both SA receptors were found to co-localize with velogenic strain F48E9 in neuropathological lesions of chicken brains after infection through intraocular-nasal routes. The replication of velogenic F48E9 in primary neuronal cells was more efficient than that of lentogenic strain LaSota. The virus-neuronal cell binding capabilities of both the velogenic and the lentogenic strains have no difference. Furthermore, the cell-binding capability and the replication of both strains were significantly decreased by pretreatment with sialidases in neuronal cells. These results demonstrated that α2,3- and α2,6-linked SA receptors were important for the initiation of NDV infection in the chicken nervous system. This study should provide preliminary evidence for a better understanding of the neurotropism of NDV in chickens.

Cytotoxicity of synthetic cannabinoids on primary neuronal cells of the forebrain: the involvement of cannabinoid CB1 receptors and apoptotic cell death.

The abuse of herbal products containing synthetic cannabinoids has become an issue of public concern. The purpose of this paper was to evaluate the acute cytotoxicity of synthetic cannabinoids on mouse brain neuronal cells. Cytotoxicity induced by synthetic cannabinoid (CP-55,940, CP-47,497, CP-47,497-C8, HU-210, JWH-018, JWH-210, AM-2201, and MAM-2201) was examined using forebrain neuronal cultures. These synthetic cannabinoids induced cytotoxicity in the forebrain cultures in a concentration-dependent manner. The cytotoxicity was suppressed by preincubation with the selective CB1 receptor antagonist AM251, but not with the selective CB2 receptor antagonist AM630. Furthermore, annexin-V-positive cells were found among the treated forebrain cells. Synthetic cannabinoid treatment induced the activation of caspase-3, and preincubation with a caspase-3 inhibitor significantly suppressed the cytotoxicity. These synthetic cannabinoids induced apoptosis through a caspase-3-dependent mechanism in the forebrain cultures. Our results indicate that the cytotoxicity of synthetic cannabinoids towards primary neuronal cells is mediated by the CB1 receptor, but not by the CB2 receptor, and further suggest that caspase cascades may play an important role in the apoptosis induced by these synthetic cannabinoids. In conclusion, excessive synthetic cannabinoid abuse may present a serious acute health concern due to neuronal damage or deficits in the brain.

Treatment of microglia with Anti-PrP monoclonal antibodies induces neuronal apoptosis in vitro.

Previous reports highlighted the neurotoxic effects caused by some motif-specific anti-PrPC antibodies in vivo and in vitro. In the current study, we investigated the detailed alterations of the proteome with liquid chromatography-mass spectrometry following direct application of anti-PrPC antibodies on mouse neuroblastoma cells (N2a) and mouse primary neuronal (MPN) cells or by cross-linking microglial PrPC with anti-PrPC antibodies prior to co-culture with the N2a/MPN cells. Here, we identified 4 (3 upregulated and 1 downregulated) and 17 (11 upregulated and 6 downregulated) neuronal apoptosis-related proteins following treatment of the N2a and N11 cell lines respectively when compared with untreated cells. In contrast, we identified 1 (upregulated) and 4 (2 upregulated and 2 downregulated) neuronal apoptosis-related proteins following treatment of MPN cells and N11 when compared with untreated cells. Furthermore, we also identified 3 (2 upregulated and 1 downregulated) and 2 (1 upregulated and 1 downregulated) neuronal apoptosis-related related proteins following treatment of MPN cells and N11 when compared to treatment with an anti-PrP antibody that lacks binding specificity for mouse PrP. The apoptotic effect of the anti-PrP antibodies was confirmed with flow cytometry following labelling of Annexin V-FITC. The toxic effects of the anti-PrP antibodies was more intense when antibody-treated N11 were co-cultured with the N2a and the identified apoptosis proteome was shown to be part of the PrPC-interactome. Our observations provide a new insight into the prominent role played by microglia in causing neurotoxic effects following treatment with anti-PrPC antibodies and might be relevant to explain the antibody mediated toxicity observed in other related neurodegenerative diseases such as Alzheimer.

Cubeben induces autophagy via PI3K-AKT-mTOR pathway to protect primary neurons against amyloid beta in Alzheimer's disease.

Autophagy is a lysosomal degradative process by which it recycles cytosolic components and degrades protein aggregates inside cells. Here, we identified cubeben as an inducer of autophagy in primary neuronal cells. Autophagy induction was accompanied the upregulation of autophagic proteins like Beclin, ATG5, ATG12 and lipidation of LC3-II in primary neuronal cells. Cubeben induces autophagy by the inhibition of PI3K-AKT pathway in a dose dependent manner. Constitutive active P110α abrogates cubeben induced autophagic induction in primary neuronal cells. Furthermore, cubeben inhibits amyloid beta induced toxicity in primary neuronal cells. Thus our data suggests that cubeben as a potential anti-Alzheimer therapeutic lead.

Alborixin clears amyloid-ß by inducing autophagy through PTEN-mediated inhibition of the AKT pathway.

Imbalance in production and clearance of amyloid beta (Aß) is the primary reason for its deposition in Alzheimer disease. Macroautophagy/autophagy is one of the important mechanisms for clearance of both intracellular and extracellular Aß. Here, through screening, we identified alborixin, an ionophore, as a potent inducer of autophagy. We found that autophagy induced by alborixin substantially cleared Aß in microglia and primary neuronal cells. Induction of autophagy was accompanied by up regulation of autophagy proteins BECN1/Beclin 1, ATG5, ATG7 and increased lysosomal activities. Autophagy induced by alborixin was associated with inhibition of the phosphoinositide 3-kinase (PI3K)-AKT pathway. A knock down of PTEN and consistent, constitutive activation of AKT inhibited alborixin-induced autophagy and consequent clearance of Aß. Furthermore, clearance of Aß by alborixin led to significant reduction of Aß-mediated cytotoxicity in primary neurons and differentiated N2a cells. Thus, our findings put forward alborixin as a potential anti-Alzheimer therapeutic lead. Abbreviations: Aß: amyloid beta; ALB: alborixin; ATG: autophagy-related; BECN1: beclin 1; DAPI: 4, 6-diamidino-2-phenylindole; DCFH-DA: 2,7-dichlorodihydrofluorescein diacetate; fAß: fibrillary form of amyloid beta; GFAP: glial fibrillary acidic protein; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule-associated protein 2; MTOR: mechanistic target of rapamycin kinase; PTEN: phosphatase and tensin homolog; ROS: reactive oxygen species; SQSTM1: sequestosome 1; TMRE: tetramethylrhodamine, ethyl ester.

Anti-migraine and anti-depression activities of Tianshu capsule by mediating Monoamine oxidase.

BACKGROUND: Tianshu capsule(TSC)is a Chinese patent medicine. It's widely used to treat migraine clinically in China. AIM OF THE STUDY: In the present study, we investigated anti-migraine and anti-depression activities of TSC using in vivo animal models together with in vitro studies to investigate the mechanism of action. MATERIALS AND METHODS: Nitroglycerin (NTG) -induced migraine rat model, rat was given a subcutaneous injection of the NTG suspension (10 mg/kg) once a week for 5 weeks. Behavioral observation was carried out and brain tissues were sampled to determine levels of 5-hydroxytryptamine (5-HT), dopamine (DA), norepinephrine (NE), monoamine oxidase A (MAO-A), monoamine oxidase B (MAO-B) and tyrosine hydroxylase (TH) by ELISA, in order to evaluate the effect of TSC on migraine progression. Tail suspension test and forced swim test were carried in order to evaluate the effect of TSC on depression progression. RESULTS: TSC treatments decreased scratch head times significantly in a rat migraine model. Meanwhile, TSC suppressed activities of MAO-A and MAO-B, up-regulated 5-HT, DA and NE expressions in brain tissues. Tail suspension test showed a decrease of immobility time in TSC groups. Furthermore, TSC increased climb times, up-regulated activities of 5-HT, DA and NE in forced swim test mice. Additionally, TSC could attenuate the reduction of 5-HT, DA and NE induced by corticosterone in primary neuronal cells. CONCLUSION: TSC could effectively prevent depression, one of the most frequent comorbidities in migraine. It may provide a new target for treating migraine.