Inhibition of the Transforming Growth Factor-β Signaling Pathway Confers Neuroprotective Effects on Beta-Amyloid-Induced Direct Neurotoxicity and Microglia-Mediated Neuroinflammation

Piper sarmentosum Roxb. confers neuroprotection on beta-amyloid (Aβ)-induced microglia-mediated neuroinflammation and attenuates tau hyperphosphorylation in SH-SY5Y cells

Transforming growth factor β (TGF-β) signaling pathway is a major pathway in cellular processes such as cell growth, apoptosis, and cellular homeostasis. The signaling pathway activated by 17β-estadiol (E2) appeared to inhibit the TGF-β signaling pathway by cross-talk with the TGF-β components in estrogen receptor (ER) positive cells. In this study, we examined the inhibitory effects of endocrine disrupting chemicals (EDCs), including 4-nonylphenol (NP), 4-otylphenol (OP), bisphenol A (BPA), and benzophenon-1 (BP-1), in the TGF-β signaling pathway in BG-1 ovarian cancer cells expressing estrogen receptors (ERs). The transcriptional and translational levels of TGF-β related genes were examined by reverse transcription-PCR (RT-PCR), Western blot analysis, and xenograft mouse models of ovarian cancer cells. As a result, treatment with NP, OP, and BPA induced the expressions of SnoN, a TGF-β pathway inhibitor, and c-Fos, a TGF-β target transcription factor. Treatment with NP, BPA, and BP-1 resulted in decreased phosphorylation of Smad3, a downstream target of TGF-β. These results indicate that NP and BPA may stimulate the proliferation of BG-1 cells via inhibition of the TGF-β signaling pathway. In a xenograft mouse model, transplanted BG-1 ovarian cancer cells showed significantly decreased phosphorylation of Smad3 and increased expression of SnoN in the ovarian tumor masses following treatment with E2, NP, or BPA. In parallel with an in vitro model, the expressions of these TGF-β signaling pathway were similarly regulated by NP or BPA in a xenograft mouse model. These results support the fact that the existence of an unproven relationship between EDCs/ER-α and TGF-β signaling pathway and a further study are required in order to verify more profound and distinct mechanism(s) for the disturbance of the TGF-β signaling pathway by diverse EDCs.

Epilepsy is a prevalent neurological disorder and it is a significant health risk, affecting >50 million people worldwide. The development of novel and appropriate strategies is required for ameliorating the progression and/or limiting the detrimental consequences of epilepsy. In the current study, kainic acid (KA), a neurotoxin, was used to induce seizures in mice. The flavonoid quercetin has recently been reported to have neuroprotective effects. Therefore, the effects of quercetin on KA-induced epilepsy and the potential underlying molecular mechanisms were examined. It was noted that quercetin attenuated the KA-induced seizure score and proinflammatory cytokine production, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and activation of nuclear factor κB (NF-κB) in mice. Quercetin attenuated KA-induced proinflammatory cytokine (TNF-α and IL-1β) release from microglia cells, as well as activation of NF-κB and ionized calcium binding adapter molecule 1 in microglia cells. Therefore, quercetin inhibited KA-induced epilepsy by microglia cell inactivation and the production of NF-κB, TNF-α and IL-1β.

Deregulated lncRNA MAGI2-AS3 in Alzheimer's disease attenuates amyloid-β induced neurotoxicity and neuroinflammation by sponging miR-374b-5p.

Previous studies indicate that astrocytes are the brain cells that express acidic fibroblast growth factor (aFGF) and that the expression is increased upon activation. However, there has been no study investigating the significance of this phenomenon. Here we report that aFGF treatment of IFNγ-stimulated human astrocytes, and LPS/IFNγ-stimulated human microglia, enhances their secretion of inflammatory cytokines and other materials toxic to human neuroblastoma SH-SY5Y cells. The mechanism of aFGF enhancement involves stimulation of the receptor FGFR2 IIIb. We show by RT-PCR that this receptor, but not other FGF receptors, is robustly expressed by astrocytes and microglia. We establish by Western blotting, and immunohistochemistry on postmortem human brain tissue that the FGFR2 IIIb protein is expressed by both of these glial cell types. We blocked the inflammatory stimulant action of aFGF by transfecting microglia and astrocytes with a small inhibitory RNA (siRNA) to FGFR2 IIIb as well as by removal of aFGF using an anti-aFGF antibody. Treatment with bFGF in combination with the stimulants was without effect, but together with aFGF, it partially counteracted the action of aFGF, indicating that it may be a weak antagonist of FGFR2 IIIb. The inflammatory effect was also attenuated by treatment with inhibitors of protein kinase C, Src tyrosine kinase, and MEK-1/2 indicating the involvement of these intracellular pathways. Our data suggest that inhibition of expression or release of aFGF could have therapeutic potential by inhibiting inflammation in neurodegenerative diseases such as Alzheimer disease where many neuroinflammatory molecules are prominently expressed.

Autophagy is a degradation pathway for the turnover of dysfunctional organelles or aggregated proteins in cells. Extracellular accumulation of β-amyloid peptide has been reported to be a major cause of Alzheimer's disease (AD) and large numbers of autophagic vacuoles accumulate in the brain of AD patient. However, how autophagic process is involved in Aβ-induced neurotoxicity and how Aβ peptide is transported into neuron and metabolized is still unknown. In order to study the role of autophagic process in Aβ-induced neurotoxicity, EGFP-LC3 was over-expressed in SH-SY5Y cells (SH-SY5Y/pEGFP-LC3). It was found that treatment with Aβ25-35, Aβ1-42 or serum-starvation induced strong autophagy response in SH-SY5Y/pEGFP-LC3. Confocal double-staining image showed that exogenous application of Aβ1-42 in medium caused the co-localization of Aβ1-42 with LC3 in neuronal cells. Concomitant treatment of Aβ with a selective α7nAChR antagonist, α-bungarotoxin (α-BTX), enhanced Aβ-induced neurotoxicity in SH-SY5Y cells. On the other hand, nicotine (nAChR agonist) enhanced the autophagic process and also inhibited cell death following Aβ application. In addition, nicotine but not α-BTX increased primary hippocampal neuronal survival following Aβ treatment. Furthermore, using Atg7 siRNA to inhibit autophagosome formation in an early step or α7nAChR siRNA to knockdown α7nAChR significantly enhanced Aβ-induced neurotoxicity. Confocal double-staining image shows that nicotine treatment in the presence of Aβ enhanced the co-localization of α7nAChR with autophagosomes. These results suggest that α7nAChR may act as a carrier to bind with eAβ and internalize into cytoplasm and further inhibit Aβ-induced neurotoxicity via autophagic degradation pathway. Our results suggest that autophagy process plays a neuroprotective role against Aβ-induced neurotoxicity. Defect in autophagic regulation or Aβ-α7nAChR transport system may impair the clearance of Aβ and enhance the neuronal death.

Emerging data support that plant food based isoflavones have ameliorating effects on a variety of neurodegenerative diseases including Parkinson's disease (PD). Our previous investigation revealed that dietary isoflavones including genistein (GEN), daidzein (DAI), and equol (EQL; a gut microbial metabolite of DAI) showed promising blood-brain barrier permeability and anti-neuroinflammatory activity in murine microglial BV2 cells. However, the neuroprotective effects of EQL against neurotoxins induced toxicity in PD related models remains unclear. Herein, EQL, along with GEN and DAI, were evaluated for their cytoprotective effect in a non-contact co-culture model with LPS-BV2-conditioned media and human neuroblastoma SH-SY5Y cells. In addition, their neuroprotective effects against PD related neurotoxins including 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+) induced cytotoxicity were evaluated in SH-SY5Y cells. Furthermore, EQL was evaluated for its neuroprotective effects against MPP+ induced neurotoxicity using in vivo PD model including Caenorhabditis elegans lifespan assay. DAI (10μM) and EQL (10 and 20μM) showed cytoprotective effects by decreasing LPS-BV2-conditioned media induced cytotoxicity in SH-SY5Y cells by 29.2, 32.4 and 27.2%, respectively. EQL (10 and 20μM) also showed neuroprotective effects by decreasing 6-OHDA and MPP+ induced cytotoxicity in SH-SY5Y cells by 30.6-34.5 and 17.9-18.9%, respectively. Additionally, data from the in vivo assay supported EQL's neuroprotective effect as it increases survival of C. elegans exposed to MPP+ from 72 to 108h. Our findings support a growing body of evidence of the neuroprotective effects of dietary isoflavones and further studies are warranted to elucidate their mechanisms of action.

Fucosterol exerts protection against amyloid β-induced neurotoxicity, reduces intracellular levels of amyloid β and enhances the mRNA expression of neuroglobin in amyloid β-induced SH-SY5Y cells

In Alzheimer's disease, accumulation of beta amyloid (Aβ) triggers amyloidogenesis and hyperphosphorylation of tau protein leading to neuronal cell death. Piper sarmentosum Roxb. (PS) is a traditional medicinal herb used by Malay to treat rheumatism, headache and boost memory. It possesses various biological effects, such as anti-cholinergic, anti-inflammatory, anti-oxidant and anti-depressant-like effects. The present study aimed to investigate neuroprotective properties of PS against Aβ-induced neurotoxicity and to evaluate its potential mechanism of action. Neuroprotective effects of hexane (HXN), dichloromethane (DCM), ethyl acetate (EA) and methanol (MEOH) extracts from leaves (L) and roots (R) of PS against Aβ-induced neurotoxicity were investigated in SH-SY5Y human neuroblastoma cells. Cells were pre-treated with PS for 24 h followed by 24 h of induction with Aβ. The neuroprotective effects of PS were studied using cell viability and cellular reactive oxygen species (ROS) assays. The levels of extracellular Aβ and tau proteins phosphorylated at threonine 231 (pT231) were determined. Gene and protein expressions were assessed using qRT-PCR analyses and western blot analyses, respectively. Hexane extracts of PS (LHXN and RHXN) protected SH-SY5Y cells against Aβ-induced neurotoxicity, and decreased levels of extracellular Aβ and phosphorylated tau (pT231). Although extracts of PS inhibited Aβ-induced ROS production, it was unlikely that neuroprotective effects were simply due to the anti-oxidant capacity of PS. Further, mechanistic study suggested that the neuroprotective effects of PS might be due to its capability to regulate amyloidogenesis through the downregulation of BACE and APP. These findings suggest that hexane extracts of PS confer neuroprotection against Aβ- induced neurotoxicity in SH-SY5Y cells by attenuating amyloidogenesis and tau hyperphosphorylation. Due to its neuroprotective properties, PS might be a potential therapeutic agent for Alzheimer's disease.

This in vitro investigation was designed to examine potential neuroprotection by dicaffeoylquinic acids (diCQAs) extracted from a traditional Chinese medicinal herb herba erigerontis and their effects against the toxicity induced by β-amyloid peptide (Aβ25-35 ). The neuroblastoma SH-SY5Y cell line was treated with Aβ or 3, 4-diCQA, 3, 5-diCQA or 4, 5-diCQA. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction was assayed by spectrophotometrical method, lipid peroxidation (malondialdehyde) on the basis of the level of thiobarbituric acid-reactive substance, the activity of superoxide dismutase by the xanthine oxidase procedure, the frequency of apoptosis by flow cytometry, and the levels of α3 and α7 nicotinic acetylcholine receptor (nAChR) subunit proteins by Western blotting. When the cells were exposed to Aβ25-35 , MTT reduction declined, oxidative stress and apoptosis were enhanced, and the expression of α3 and α7 nAChR subunit proteins was lowered. Expression of the α7 nAChR subunit protein was increased by all three diCQAs, and the level of α3 was increased by 3, 5-diCQA and 4, 5-diCQA. Significantly, pretreatment with diCQAs attenuated the neurotoxic effects of Aβ25-35 , a neuroprotective effect in which the upregulation of α7 and α3 nAChR may be involved. The diCQAs exert a protective effect on Aβ-induced neurotoxicity in SH-SY5Y cells and a potential underlying mechanism involving stimulation of nAChRs.

Alzheimer disease is characterized by neuronal loss and brain plaques of extracellular amyloid β (Aβ), but the means by which Aβ may induce neuronal loss is not entirely clear. Although high concentrations of Aβ (μM) can induce direct toxicity to neurons, we find that low concentration (nM) induce neuronal loss through a microglia-mediated mechanism. In mixed neuronal-glial cultures from rat cerebellum, 250 nM Aβ1-42 (added as monomers, oligomers or fibers) induced about 30% loss of neurons between 2 and 3 days. This neuronal loss occurred without any increase in neuronal apoptosis or necrosis, and no neuronal loss occurred with Aβ42-1. Aβ greatly increased the phagocytic capacity of microglia and induced phosphatidylserine exposure (an "eat-me" signal) on neuronal processes. Blocking exposed phosphatidylserine by adding annexin V or an antibody to phosphatidylserine or inhibiting microglial phagocytosis by adding either cytochalasin D (to block actin polymerization) or cyclo(RGDfV) (to block vitronectin receptors) significantly prevented neuronal loss. Loss of neuronal synapses occurred in parallel with loss of cell bodies and was also prevented by blocking phagocytosis. Inhibition of phagocytosis prevented neuronal loss with no increase in neuronal death, even after 7 days, suggesting that microglial phagocytosis was the primary cause of neuronal death induced by nanomolar Aβ.

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders. It is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra. The intricate neurotoxic mechanisms underlying dopaminergic neurdegeneration still need to be elucidated. Nowadays, more and more emerging studies connect the pathogenic roles of glial cells in PD. Pro-inflammatory cytokines, such as interlukin (IL)-1β and tumor necrosis factor (TNF)-α were elevated in PD brains. In addition, the microglial activation is demonstrated to mediate the neurodegeneration process in PD. In this current experiment, we took the advantages of co-culture models of human neuroblastoma SH-SY5Y cell line and microglia–like THP-1 cell line; and studies the effect of MPP+ treatment on mono-cultured neurons and co-cultured neurons. In addition, we assessed the potential neuroprotection effects of antioxidants vitamin C and vitamin E on MPP+ and microglia-mediated neurotoxicity. Results from MTT assay showed that MPP+ treatment increased SH-SY5Y cell death in a dose-dependent manner. Differentiated SH-SY5Y cells are less sensitive to MPP+ treatment. Western blot analysis indicated that decreased phosphorylation of p38 is involved in both differentiation of SH-SY5Y and protected cells from MPP+ toxicity. Furthermore, neither vitamin C nor vitamin E effectively protects neurons from MPP+ insults. When compared with the mono-culture neurons, treatment of MPP+ significantly increased cell death in 24 hr in the co-culture group. However, there is no significant difference in cell viabilities between mono-culture and co-culture groups after 48 hr and 72 hr of MPP+ treatment. In addition, in co-culture system, vitamin C and vitamin E administration did not significantly protect neurons from MPP+ insult. Our results suggest that the microglia-neuron interaction might be a short term effect, and that oxidative stress may not be a primary cause of either MPP+-induced or microglia-mediated neurotoxicity in this event.

For 10 years, research has focused on signaling pathways controlling translation to explain neuronal death in Alzheimer Disease (AD). Previous studies demonstrated in different cellular and animal models and AD patients that translation is down-regulated by the activation of double-stranded RNA-dependent protein kinase (PKR). Among downstream factors of PKR, the Fas-associated protein with a death domain (FADD) and subsequent activated caspase-8 are responsible for PKR-induced apoptosis in recombinant virus-infected cells. However, no studies have reported the role of PKR in death receptor signaling in AD. The aim of this project is to determine physical and functional interactions of PKR with FADD in amyloid-beta peptide (Abeta) neurotoxicity and in APP(SL)PS1 KI transgenic mice. In SH-SY5Y cells, results showed that Abeta42 induced a large increase in phosphorylated PKR and FADD levels and a physical interaction between PKR and FADD in the nucleus, also observed in the cortex of APP(SL)PS1 KI mice. However, PKR gene silencing or treatment with a specific PKR inhibitor significantly prevented the increase in pT(451)-PKR and pS(194)-FADD levels in SH-SY5Y nuclei and completely inhibited activities of caspase-3 and -8. The contribution of PKR in neurodegeneration through the death receptor signaling pathway may support the development of therapeutics targeting PKR to limit neuronal death in AD.

Objectives: Schizophrenia is a common psychiatric disease with its complex neurobiology. The current knowledge about the neurobiology of schizophrenia is based on dopaminergic and glutamatergic dysregulation of the central nervous system. The facts that all current antipsychotic drugs act on dopaminergic D2 receptor antagonism and glutamatergic N-Methyl D-Aspartate (NMDA) receptor antagonists reveal schizophrenia-like behavioral and structural alteration in rodents and humans are the major evidences of these hypotheses. However, they are not sufficient to explain the disease neurobiology and lead novel therapeutics. In recent years, it has been shown that the functions of certain intracellular pathways are disrupted in schizophrenia, especially Akt/glycogen synthase kinase 3beta (GSK-3β)/β-catenin signaling pathway. Certain studies showed that GSK-3β inhibition accompanied to the effects of antipsychotic drugs and specific inhibition of the GSK-3β might be a valuable approach for the novel treatments for schizophrenia. Agmatine is an endogenous amine that interacts with many receptors and enzymes including adrenergic, glutamatergic, cholinergic, serotonergic and imidazoline receptors and nitric oxide synthase enzyme. The limited number of studies showed conflicting results about the role of agmatine on schizophrenia and there was no study to evaluate the intracellular molecular mechanisms of agmatine`s schizophrenia-related effects. Herein, we aimed to investigate the effects of agmatine on MK-801 induced neurotoxicity and the disruption of the Akt/GSK-3β/β-catenin signaling pathway in SH-SY5Y human neuroblastoma cells. Methods: SH-SY5Y cells were grouped as the medium, agmatine (50μM), agmatine (100μM), olanzapine (10μM), MK-801 (100μM), MK-801+Agmatine (50μM), MK-801+Agmatine (100μM) and MK-801+Olanzapine (10μM). Real-time cell analysis (RTCA), real-time polymerase chain reaction (Rt-PCR) and western blot (WB) were used for monitoring the cell viabilities, quantifying gene and protein expressions, respectively. Cell viabilities monitored for the 24 hours and comparative viabilities (%) were calculated at the 6th, 12th and 24th hours of treatments. Akt, GSK-3β and β-Catenin gene expressions phosphorylated (p) and non-phosphorylated protein expressions were investigated at the 24th hour of treatments. Results: In RTCA, MK-801 administration decreased cell viabilities at the 6th (p<0.01) and 24th (p<0.001) hours compared to medium in cells. Agmatine (50μM) and agmatine (100μM) pre-treatments increased the cell viabilities at the 6th (p<0.05), 12th (p<0.01), and 24th (p<0.05) hours while olanzapine at 24th (p<0.05) compared to MK-801. In Rt-PCR, MK-801 administration increased GSK-3B decreased CTNNB1 gene expressions (p<0.001) compared to the medium. Agmatine (50μM) and olanzapine reversed (p<0.001) the effects of MK-801 for both gene expressions while agmatine (100μM) reversed (p<0.001) only GSK-3B. In WB, MK-801 administration decreased the p-GSK-3β/GSK-3β ratio compared to medium while only agmatine (50μM) increased the ratio compared to MK-801 administration. Conclusion: Herein, it has been shown that agmatine had a neuroprotective effect on MK-801 induced cell toxicity in SH-SY5Y cells. Our results have suggested a low dose (50μM) agmatine shows antipsychotic like effects on Akt/GSK-3β/β-catenin signaling pathway in SH-SY5Y cells bot, not high dose (100μM). We have suggested that these doses dependent regulatory effects of agmatine might be a possible explanation for its conflicting role in schizophrenia.

BackgroundOne hallmark of Alzheimer disease is microglial activation. Therapeutic approaches for this neurodegenerative disease include the modulation of microglial cells. α1-antitrypsin (A1AT) has been shown to exert anti-inflammatory effects on macrophages and lung epithelial cells and an inhibition of calpain activity in neutrophil granulocytes. Nothing is known about the effect of A1AT on microglial-mediated neuroinflammation. Our aim was to investigate the effect of A1AT on amyloid-β (Aβ)- and LPS-treated microglial cells in vitro with respect to cytokine production, stress pathways, cell viability, phagocytotic abilities and the underlying mechanisms.MethodsPrimary microglial cells were isolated from Swiss Webster mouse embryos on embryonic day 13.5. Cytokines in the supernatants of treated primary microglial cells were analyzed with ELISAs, and accumulated nitrite was detected with Griess reagents. Intracellular stress pathways were investigated in cell lysates using western blotting. Intracellular calcium levels were detected in BV-2 microglial cells loaded with the Ca2+-sensitive (fluorescent) dye Fluo-4. Calpain activity in primary microglial cells was assessed by using a calpain activity assay. Cell viability of Aβ-treated microglial cells was analyzed using MTT assay. Phagocytosis of Aβ was evaluated with western blot analysis.ResultsUpon co-administration, A1AT reduced pro-inflammatory mediators induced by LPS or Aβ. Interestingly, we detected a reduction in calpain activity and in the concentration of intracellular calcium that might mediate the anti-inflammatory effects of A1AT. Inhibition of the classic activation pathways, such as phosphorylation of mitogen-activated protein kinases or activation of protein kinase A were excluded as a mechanism of A1AT-mediated effects. In addition, A1AT increased the viability of Aβ-treated microglial cells and reduced Aβ phagocytosis.ConclusionsWe provide evidence on the mechanism of action of A1AT on microglial-mediated neuroinflammation in vitro. Our in vitro data indicate that A1AT treatment modulates microglial cells in inflammatory conditions and that this modulation is due to an inhibition of calpain activity and intracellular calcium levels. The underlying mechanisms of the effects observed here are promising for future therapeutic strategies and should thus be further pursued in transgenic mouse models of Alzheimer disease.