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Shanshan W, Hongying M, Jingjing F, Rui Y. Metformin and buparlisib synergistically induce apoptosis of non-small lung cancer (NSCLC) cells via Akt/FoxO3a/Puma axis. Toxicol In Vitro 2024; 97:105801. [PMID: 38479708 DOI: 10.1016/j.tiv.2024.105801] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Accepted: 02/26/2024] [Indexed: 04/16/2024]
Abstract
Non-small cell lung cancer (NSCLC) is a global health issue lacking effective treatments. Buparlisib is a pan-PI3K inhibitor that shows promising clinical results in treating NSCLC. However, chemoresistance is inevitable and hampers the application of buparlisib. Studies show that a combination of phytochemicals and chemotherapeutics enhances its effectiveness. Here, we evaluated the role of metformin, an agent with multiple pharmacological properties, in enhancing the anti-tumour activities of buparlisib against NSCLC cells. Our results showed that metformin and buparlisib synergistically inhibited cell viability, migration, and invasion of NSCLC cells. In addition, co-treatment of metformin and buparlisib also induced cell cycle arrest and cell death in NSCLC cells. Mechanistically, metformin and buparlisib repressed Mcl-1 and upregulated Puma in NSCLC cells in a p53-independent manner. Moreover, they inhibited the PI3K/Akt signalling pathway, leading to activation of the FoxO3a/Puma signalling in NSCLC cells. Our findings suggest that combined treatment of metformin and buparlisib might provide a promising strategy for treating NSCLC.
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Affiliation(s)
- Wang Shanshan
- The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang 315020, China
| | - Ma Hongying
- The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang 315020, China
| | - Fang Jingjing
- The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang 315020, China
| | - Yu Rui
- Department of Biochemistry and Molecular Biology, and Zhejiang Key Laboratory of Pathophysiology, Medical School of Ningbo University, Ningbo, Zhejiang 315020, China.
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2
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Lei T, Yang Z, Li H, Qin M, Gao H. Interactions between nanoparticles and pathological changes of vascular in Alzheimer's disease. Adv Drug Deliv Rev 2024; 207:115219. [PMID: 38401847 DOI: 10.1016/j.addr.2024.115219] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2023] [Revised: 02/16/2024] [Accepted: 02/20/2024] [Indexed: 02/26/2024]
Abstract
Emerging evidence suggests that vascular pathological changes play a pivotal role in the pathogenesis of Alzheimer's disease (AD). The dysfunction of the cerebral vasculature occurs in the early course of AD, characterized by alterations in vascular morphology, diminished cerebral blood flow (CBF), impairment of the neurovascular unit (NVU), vasculature inflammation, and cerebral amyloid angiopathy. Vascular dysfunction not only facilitates the influx of neurotoxic substances into the brain, triggering inflammation and immune responses but also hampers the efflux of toxic proteins such as Aβ from the brain, thereby contributing to neurodegenerative changes in AD. Furthermore, these vascular changes significantly impact drug delivery and distribution within the brain. Therefore, developing targeted delivery systems or therapeutic strategies based on vascular alterations may potentially represent a novel breakthrough in AD treatment. This review comprehensively examines various aspects of vascular alterations in AD and outlines the current interactions between nanoparticles and pathological changes of vascular.
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Affiliation(s)
- Ting Lei
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, West China School of Pharmacy, Mental Health Center and National Chengdu Center for Safety Evaluation of Drugs, West China Hospital, Sichuan University, Chengdu 610041, China
| | - Zixiao Yang
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, West China School of Pharmacy, Mental Health Center and National Chengdu Center for Safety Evaluation of Drugs, West China Hospital, Sichuan University, Chengdu 610041, China
| | - Hanmei Li
- School of Food and Biological Engineering, Chengdu University, Chengdu 610106, China
| | - Meng Qin
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, West China School of Pharmacy, Mental Health Center and National Chengdu Center for Safety Evaluation of Drugs, West China Hospital, Sichuan University, Chengdu 610041, China.
| | - Huile Gao
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, West China School of Pharmacy, Mental Health Center and National Chengdu Center for Safety Evaluation of Drugs, West China Hospital, Sichuan University, Chengdu 610041, China.
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Vyas P, Tulsawani R, Vohora D. Dual Targeting by Inhibition of Phosphoinositide-3-Kinase and Mammalian Target of Rapamycin Attenuates the Neuroinflammatory Responses in Murine Hippocampal Cells and Seizures in C57BL/6 Mice. Front Immunol 2021; 12:739452. [PMID: 34887852 PMCID: PMC8650161 DOI: 10.3389/fimmu.2021.739452] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2021] [Accepted: 10/04/2021] [Indexed: 11/25/2022] Open
Abstract
Emerging evidence suggests the association of seizures and inflammation; however, underlying cell signaling mechanisms are still not fully understood. Overactivation of phosphoinositide-3-kinases is associated with both neuroinflammation and seizures. Herein, we speculate the PI3K/Akt/mTOR pathway as a promising therapeutic target for neuroinflammation-mediated seizures and associated neurodegeneration. Firstly, we cultured HT22 cells for detection of the downstream cell signaling events activated in a lipopolysaccharide (LPS)-primed pilocarpine (PILO) model. We then evaluated the effects of 7-day treatment of buparlisib (PI3K inhibitor, 25 mg/kg p.o.), dactolisib (PI3K/mTOR inhibitor, 25 mg/kg p.o.), and rapamycin (mTORC1 inhibitor, 10 mg/kg p.o.) in an LPS-primed PILO model of seizures in C57BL/6 mice. LPS priming resulted in enhanced seizure severity and reduced latency. Buparlisib and dactolisib, but not rapamycin, prolonged latency to seizures and reduced neuronal loss, while all drugs attenuated seizure severity. Buparlisib and dactolisib further reduced cellular redox, mitochondrial membrane potential, cleaved caspase-3 and p53, nuclear integrity, and attenuated NF-κB, IL-1β, IL-6, TNF-α, and TGF-β1 and TGF-β2 signaling both in vitro and in vivo post-PILO and LPS+PILO inductions; however, rapamycin mitigated the same only in the PILO model. Both drugs protected against neuronal cell death demonstrating the contribution of this pathway in the seizure-induced neuronal pyknosis; however, rapamycin showed resistance in a combination model. Furthermore, LPS and PILO exposure enhanced pAkt/Akt and phospho-p70S6/total-p70S6 kinase activity, while buparlisib and dactolisib, but not rapamycin, could reduce it in a combination model. Partial rapamycin resistance was observed possibly due to the reactivation of the pathway by a functionally different complex of mTOR, i.e., mTORC2. Our study substantiated the plausible involvement of PI3K-mediated apoptotic and inflammatory pathways in LPS-primed PILO-induced seizures and provides evidence that its modulation constitutes an anti-inflammatory mechanism by which seizure inhibitory effects are observed. We showed dual inhibition by dactolisib as a promising approach. Targeting this pathway at two nodes at a time may provide new avenues for antiseizure therapies.
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Affiliation(s)
- Preeti Vyas
- Department of Pharmacology, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India
| | - Rajkumar Tulsawani
- Defense Institute of Physiology & Allied Science, Defense Research and Development Organization, New Delhi, India
| | - Divya Vohora
- Department of Pharmacology, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India
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Ding XW, Li R, Geetha T, Tao YX, Babu JR. Nerve growth factor in metabolic complications and Alzheimer's disease: Physiology and therapeutic potential. Biochim Biophys Acta Mol Basis Dis 2020; 1866:165858. [PMID: 32531260 DOI: 10.1016/j.bbadis.2020.165858] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2020] [Revised: 05/11/2020] [Accepted: 06/02/2020] [Indexed: 02/07/2023]
Abstract
As the population ages, obesity and metabolic complications as well as neurological disorders are becoming more prevalent, with huge economic burdens on both societies and families. New therapeutics are urgently needed. Nerve growth factor (NGF), first discovered in 1950s, is a neurotrophic factor involved in regulating cell proliferation, growth, survival, and apoptosis in both central and peripheral nervous systems. NGF and its precursor, proNGF, bind to TrkA and p75 receptors and initiate protein phosphorylation cascades, resulting in changes of cellular functions, and are associated with obesity, diabetes and its complications, and Alzheimer's disease. In this article, we summarize changes in NGF levels in metabolic and neuronal disorders, the signal transduction initiated by NGF and proNGF, the physiological and pathophysiological relevance, and therapeutic potential in treating chronic metabolic diseases and cognitive decline.
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Affiliation(s)
- Xiao-Wen Ding
- Department of Nutrition, Dietetics, and Hospitality Management, Auburn University, Auburn, AL 36849, USA
| | - Rongzi Li
- Department of Nutrition, Dietetics, and Hospitality Management, Auburn University, Auburn, AL 36849, USA
| | - Thangiah Geetha
- Department of Nutrition, Dietetics, and Hospitality Management, Auburn University, Auburn, AL 36849, USA; Boshell Metabolic Diseases and Diabetes Program, Auburn University, Auburn, AL 36849, USA
| | - Ya-Xiong Tao
- Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA.
| | - Jeganathan Ramesh Babu
- Department of Nutrition, Dietetics, and Hospitality Management, Auburn University, Auburn, AL 36849, USA; Boshell Metabolic Diseases and Diabetes Program, Auburn University, Auburn, AL 36849, USA.
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Cai Z, Hu X, Tan R, Feng Y, Sun M, Ma N, Li X, Huang L, An J, Ge Q, Lu H. Neuroprotective effect of green tea extractives against oxidative stress by enhancing the survival and proliferation of PC12 cells. Mol Cell Toxicol 2019. [DOI: 10.1007/s13273-019-0042-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
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Zhou T, Wang H, Shen J, Li W, Cao M, Hong Y, Cao M. The p35/CDK5 signaling is regulated by p75NTR in neuronal apoptosis after intracerebral hemorrhage. J Cell Physiol 2019; 234:15856-15871. [PMID: 30770557 DOI: 10.1002/jcp.28244] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2018] [Revised: 01/02/2019] [Accepted: 01/10/2019] [Indexed: 01/24/2023]
Abstract
The p75 neurotrophin receptor (p75NTR), a member of tumor necrosis factor receptor superfamily, involves in neuronal apoptosis after intracerebral hemorrhage (ICH). It has been previously demonstrated that phosphorylation of p35 is a crucial factor for fighting against the proapoptotic p25/CDK5 signaling in neuronal apoptosis. Then, in ICH models of rats and primary cortical neurons, we found that the expressions of p75NTR, p-histone H1 (the kinase activity of CDK5), p25, Fas-associated phosphatase-1 (FAP-1), and phosphorylated myocyte enhancer factor 2D (p-MEF2D) were enhanced after ICH, whereas the expression of p35-Thr(138) was attenuated. Coimmunoprecipitation analysis indicated several interactions as follows: p35/p25 and CKD5, p75NTR and p35, as well as p75NTR and FAP-1. After p75NTR or FAP-1 depletion with double-stranded RNA interference in PC12 cells, the levels of p25 and p-histone H1 were attenuated, whereas p35-Thr(138) was elevated. Considering p75NTR has no effect of dephosphorylation, our results suggested that p75NTR might promote the dephosphorylation of p35-Thr(138) via interaction with FAP-1, and the p75NTR/p35 complex upregulated p25/CDK5 signaling to facilitate the neuronal apoptosis following ICH. So, in the study, we aimed to provide a theoretical and experimental basis that p75NTR could be regulated to reduce neuronal apoptosis following ICH for potential clinical treatment.
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Affiliation(s)
- Tingting Zhou
- Department of Neurology, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, People's Republic of China
| | - Hongmei Wang
- Department of Neurology, Nantong Rich Hospital, Nantong, Jiangsu Province, People's Republic of China
| | - Jiabing Shen
- Department of Neurology, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, People's Republic of China
| | - Wanyan Li
- Department of Neurology, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, People's Republic of China
| | - Maosheng Cao
- Department of Neurology, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, People's Republic of China
| | - Yao Hong
- Department of Neurology, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, People's Republic of China
| | - Maohong Cao
- Department of Neurology, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, People's Republic of China
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7
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Inhibition of TNF-α-induced neuronal apoptosis by antidepressants acting through the lysophosphatidic acid receptor LPA1. Apoptosis 2019; 24:478-498. [DOI: 10.1007/s10495-019-01530-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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8
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Mousavi SH, Bakhtiari E, Hosseini A, Jamialahmadi K. Protective effects of glucosamine and its acetylated derivative on serum/glucose deprivation-induced PC12 cells death: Role of reactive oxygen species. Res Pharm Sci 2018; 13:121-129. [PMID: 29606966 PMCID: PMC5842483 DOI: 10.4103/1735-5362.223794] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Finding products with antiapoptotic activities has been one of the approaches for the treatment of neurodegenerative disorders. Serum/glucose deprivation (SGD) has been used as a model for the investigation of the molecular mechanisms of neuronal ischemia. Recent studies indicated that glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) have many pharmacological effects including antioxidant activities. The present study aimed to investigate the protective effects of GlcN and GlcNAc against SGD-induced PC12 cells injury. The PC12 cells were pretreated with GlcN and GlcNAc for 2 h, and then exposed to SGD for 6, 12 and 24 h. Cell viability was evaluated by MTT assay. The level of intracellular reactive oxygen species (ROS) was determined by flow cytometry using 2',7'- dichlorofluorescin diacetate (DCFH-DA) as a probe. SGD condition caused a significant reduction in cell survival after 6, 12, and 24 h (P < 0.001). Pretreatment with GlcN and GlcNAc (0.6-20 mM) increased cell viability following SGD insult. A significant increase in cell apoptosis was observed in cells under SGD condition after 12 h (P < 0.001). Pretreatment with GlcN and GlcNAc (5-20 mM) decreased apoptosis following SGD condition after 12 h. SGD resulted in a significant increase in intracellular ROS production after 12 h. Pretreatment with both amino sugars at concentrations of 10 to 20 mM could reverse the ROS increment. Results indicated that GlcN and GlcNAc had a cytoprotective property against SGD-induced cell death via anti-apoptosis and antioxidant activities, suggesting that these aminosugers have the potential to be used as novel therapeutic agents for neurodegenerative disorders.
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Affiliation(s)
- Seyed Hadi Mousavi
- Medical Toxicology Research center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, I.R. Iran.,Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, I.R. Iran
| | - Elham Bakhtiari
- Eye Research Center, Mashhad University of Medical Sciences, Mashhad, I.R. Iran.,Research Center for Patient Safety, Mashhad University of Medical Sciences, Mashhad, I.R. Iran
| | - Azar Hosseini
- Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, I.R. Iran
| | - Khadijeh Jamialahmadi
- Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, I.R. Iran.,Department of Medical Biotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, I.R. Iran
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The Cstf2t Polyadenylation Gene Plays a Sex-Specific Role in Learning Behaviors in Mice. PLoS One 2016; 11:e0165976. [PMID: 27812195 PMCID: PMC5094787 DOI: 10.1371/journal.pone.0165976] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2016] [Accepted: 10/20/2016] [Indexed: 11/19/2022] Open
Abstract
Polyadenylation is an essential mechanism for the processing of mRNA 3′ ends. CstF-64 (the 64,000 Mr subunit of the cleavage stimulation factor; gene symbol Cstf2) is an RNA-binding protein that regulates mRNA polyadenylation site usage. We discovered a paralogous form of CstF-64 called τCstF-64 (Cstf2t). The Cstf2t gene is conserved in all eutherian mammals including mice and humans, but the τCstF-64 protein is expressed only in a subset of mammalian tissues, mostly testis and brain. Male mice that lack Cstf2t (Cstf2t-/- mice) experience disruption of spermatogenesis and are infertile, although female fertility is unaffected. However, a role for τCstF-64 in the brain has not yet been determined. Given the importance of RNA polyadenylation and splicing in neuronal gene expression, we chose to test the hypothesis that τCstF-64 is important for brain function. Male and female 185-day old wild type and Cstf2t-/- mice were examined for motor function, general activity, learning, and memory using rotarod, open field activity, 8-arm radial arm maze, and Morris water maze tasks. Male wild type and Cstf2t-/- mice did not show differences in learning and memory. However, female Cstf2t-/- mice showed significantly better retention of learned maze tasks than did female wild type mice. These results suggest that τCstf-64 is important in memory function in female mice. Interestingly, male Cstf2t-/- mice displayed less thigmotactic behavior than did wild type mice, suggesting that Cstf2t may play a role in anxiety in males. Taken together, our studies highlight the importance of mRNA processing in cognition and behavior as well as their established functions in reproduction.
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Bou Khalil R, Khoury E, Koussa S. Linking multiple pathogenic pathways in Alzheimer's disease. World J Psychiatry 2016; 6:208-214. [PMID: 27354962 PMCID: PMC4919259 DOI: 10.5498/wjp.v6.i2.208] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/01/2016] [Revised: 04/24/2016] [Accepted: 05/10/2016] [Indexed: 02/05/2023] Open
Abstract
Alzheimer's disease (AD) is a chronic neurodegenerative disorder presenting as progressive cognitive decline with dementia that does not, to this day, benefit from any disease-modifying drug. Multiple etiologic pathways have been explored and demonstrate promising solutions. For example, iron ion chelators, such as deferoxamine, are a potential therapeutic solution around which future studies are being directed. Another promising domain is related to thrombin inhibitors. In this minireview, a common pathophysiological pathway is suggested for the pathogenesis of AD to prove that all these mechanisms converge onto the same cascade of neuroinflammatory events. This common pathway is initiated by the presence of vascular risk factors that induce brain tissue hypoxia, which leads to endothelial cell activation. However, the ensuing hypoxia stimulates the production and release of reactive oxygen species and pro-inflammatory proteins. Furthermore, the endothelial activation may become excessive and dysfunctional in predisposed individuals, leading to thrombin activation and iron ion decompartmentalization. The oxidative stress that results from these modifications in the neurovascular unit will eventually lead to neuronal and glial cell death, ultimately leading to the development of AD. Hence, future research in this field should focus on conducting trials with combinations of potentially efficient treatments, such as the combination of intranasal deferoxamine and direct thrombin inhibitors.
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Wilkins HM, Swerdlow RH. TNFα in cerebral ischemia: another stroke against you? J Neurochem 2015; 132:369-72. [PMID: 25656781 PMCID: PMC4339020 DOI: 10.1111/jnc.13028] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2014] [Accepted: 12/29/2014] [Indexed: 11/29/2022]
Abstract
Read the full article ‘Rapid mitochondrial dysfunction mediates TNF‐alpha‐induced neurotoxicity' on page 443.
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Affiliation(s)
- Heather M Wilkins
- The Alzheimer's Disease Center and the Departments of Neurology, Molecular and Integrative Physiology, and Biochemistry and Molecular Biology, University of Kansas School of Medicine, Kansas City, Kansas, USA
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Doll DN, Rellick SL, Barr TL, Ren X, Simpkins JW. Rapid mitochondrial dysfunction mediates TNF-alpha-induced neurotoxicity. J Neurochem 2015; 132:443-51. [PMID: 25492727 DOI: 10.1111/jnc.13008] [Citation(s) in RCA: 113] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2014] [Revised: 11/20/2014] [Accepted: 11/24/2014] [Indexed: 01/20/2023]
Abstract
Tumor necrosis factor alpha (TNF-α) is known to exacerbate ischemic brain injury; however, the mechanism is unknown. Previous studies have evaluated the effects of TNF-α on neurons with long exposures to high doses of TNF-α, which is not pathophysiologically relevant. We characterized the rapid effects of TNF-α on basal respiration, ATP production, and maximal respiration using pathophysiologically relevant, post-stroke concentrations of TNF-α. We observed a reduction in mitochondrial function as early as 1.5 h after exposure to low doses of TNF-α, followed by a decrease in cell viability in HT-22 cells and primary neurons. Subsequently, we used the HT-22 cell line to determine the mechanism by which TNF-α causes a rapid and profound reduction in mitochondrial function. Pre-treating with TNF-R1 antibody, but not TNF-R2 antibody, ameliorated the neurotoxic effects of TNF-α, indicating that TNF-α exerts its neurotoxic effects through TNF-R1. We observed an increase in caspase 8 activity and a decrease in mitochondrial membrane potential after exposure to TNF-α which resulted in a release of cytochrome c from the mitochondria into the cytosol. These novel findings indicate for the first time that an acute exposure to pathophysiologically relevant concentrations of TNF-α has neurotoxic effects mediated by a rapid impairment of mitochondrial function. This study focuses on the neurotoxic mechanism of a pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α). We demonstrate a prompt mitochondrial dysfunction followed by nerve cell loss after exposure to TNF-α. These studies may provide evidence that the immune system can rapidly and adversely affect brain function and that TNF-α signaling may be a target for neuroprotection.
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Affiliation(s)
- Danielle N Doll
- Neurobiology and Anatomy, West Virginia University, Morgantown, West Virginia, USA; Center for Neuroscience, Morgantown, WV, USA; Center for Basic and Translational Stroke Research, Morgantown, WV, USA
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Ke K, Shen J, Song Y, Cao M, Lu H, Liu C, Shen J, Li A, Huang J, Ni H, Chen X, Liu Y. CDK5 Contributes to Neuronal Apoptosis via Promoting MEF2D Phosphorylation in Rat Model of Intracerebral Hemorrhage. J Mol Neurosci 2014; 56:48-59. [DOI: 10.1007/s12031-014-0466-5] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2014] [Accepted: 11/10/2014] [Indexed: 12/22/2022]
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14
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Guo H, Kong S, Chen W, Dai Z, Lin T, Su J, Li S, Xie Q, Su Z, Xu Y, Lai X. Apigenin Mediated Protection of OGD-Evoked Neuron-Like Injury in Differentiated PC12 Cells. Neurochem Res 2014; 39:2197-210. [DOI: 10.1007/s11064-014-1421-0] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2014] [Revised: 08/05/2014] [Accepted: 08/19/2014] [Indexed: 12/17/2022]
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Sanchez A, Tripathy D, Yin X, Luo J, Martinez JM, Grammas P. Sunitinib enhances neuronal survival in vitro via NF-κB-mediated signaling and expression of cyclooxygenase-2 and inducible nitric oxide synthase. J Neuroinflammation 2013; 10:93. [PMID: 23880112 PMCID: PMC3726353 DOI: 10.1186/1742-2094-10-93] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2013] [Accepted: 07/18/2013] [Indexed: 01/12/2023] Open
Abstract
BACKGROUND Angiogenesis is tightly linked to inflammation and cancer. Regulation of angiogenesis is mediated primarily through activation of receptor tyrosine kinases, thus kinase inhibitors represent a new paradigm in anti-cancer therapy. However, these inhibitors have broad effects on inflammatory processes and multiple cell types. Sunitinib is a multitarget receptor tyrosine kinase inhibitor, which has shown promise for the treatment of glioblastoma, a highly vascularized tumor. However, there is little information as to the direct effects of sunitinib on brain-derived neurons. The objective of this study is to explore the effects of sunitinib on neuronal survival as well as on the expression of inflammatory protein mediators in primary cerebral neuronal cultures. METHODS Primary cortical neurons were exposed to various doses of sunitinib. The drug-treated cultures were assessed for survival by MTT assay and cell death by lactate dehydrogenase release. The ability of sunitinib to affect NF-κB, COX2 and NOS2 expression was determined by western blot. The NF-κB inhibitors dicoumarol, SN50 and BAY11-7085 were employed to assess the role of NF-κB in sunitinib-mediated effects on neuronal survival as well as COX2 and NOS2 expression. RESULTS Treatment of neuronal cultures with sunitinib caused a dose-dependent increase in cell survival and decrease in neuronal cell death. Exposure of neurons to sunitinib also induced an increase in the expression of NF-κB, COX2 and NOS2. Inhibiting NF-κB blunted the increase in cell survival and decrease in cell death evoked by sunitinib. Treatment of cell cultures with both sunitinib and NF-κB inhibitors mitigated the increase in COX2 and NOS2 caused by sunitinib. CONCLUSIONS Sunitinib increases neuronal survival and this neurotrophic effect is mediated by NF-κB. Also, the inflammatory proteins COX2 and NOS2 are upregulated by sunitinib in an NF-κB-dependent manner. These data are in agreement with a growing literature suggesting beneficial effects for inflammatory mediators such as NF-κB, COX2 and NOS2 in neurons. Further work is needed to fully explore the effects of sunitinib in the brain and its possible use as a treatment for glioblastoma. Finally, sunitinib may be useful for the treatment of a range of central nervous system diseases where neuronal injury is prominent.
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Affiliation(s)
- Alma Sanchez
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, TX, USA
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Christov A, Ottman JT, Grammas P. Vascular inflammatory, oxidative and protease-based processes: implications for neuronal cell death in Alzheimer's disease. Neurol Res 2013; 26:540-6. [PMID: 15265271 DOI: 10.1179/016164104225016218] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022]
Abstract
A substantial literature demonstrates activation of inflammatory processes in the Alzheimer's disease (AD) brain and an association between inflammation and oxidative stress. We have shown that brain microvessels from AD patients express high levels of inflammatory proteins and that these proteins evoke release of the neurotoxic protease thrombin from brain endothelial cells. The objective of this study was to determine the effects of inflammatory proteins on brain endothelial cell reactive oxygen species generation, protease release and cell apoptosis. Also, the effects of inflammatory proteins on neuronal reactive oxygen species generation, injury and apoptosis were assessed. Treatment of cultured brain endothelial cells with inflammatory proteins (LPS, IL-1beta, IL-6, IFN-gamma, TNF-alpha) resulted in a significant increase (p < 0.01) in intracellular levels of reactive oxygen species by 1 h. Inflammatory proteins also caused release of tissue plasminogen activator and increased apoptosis by 24 h in these cells. In cultured neurons, inflammatory proteins caused an increase in reactive oxygen species, membrane fluidity, and apoptosis by 24 h, as detected by fluorescent microscopy. Taken together, these data support the hypothesis that vascular inflammatory, oxidative and protease-based processes contribute to neuronal cell death, and suggest that therapies targeted at these mediators and processes could be effective in AD.
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Affiliation(s)
- Alexander Christov
- Department of Pathology and the Oklahoma Center for Neuroscience, University of Oklahoma Health Science Center, Oklahoma City, OK, USA
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Tripathy D, Sanchez A, Yin X, Luo J, Martinez J, Grammas P. Thrombin, a mediator of cerebrovascular inflammation in AD and hypoxia. Front Aging Neurosci 2013; 5:19. [PMID: 23675346 PMCID: PMC3648692 DOI: 10.3389/fnagi.2013.00019] [Citation(s) in RCA: 65] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2013] [Accepted: 04/09/2013] [Indexed: 12/16/2022] Open
Abstract
Considerable evidence implicates hypoxia and vascular inflammation in Alzheimer's disease (AD). Thrombin, a multifunctional inflammatory mediator, is demonstrable in the brains of AD patients both in the vessel walls and senile plaques. Hypoxia-inducible factor 1α (HIF-1α), a key regulator of the cellular response to hypoxia, is also upregulated in the vasculature of human AD brains. The objective of this study is to investigate inflammatory protein expression in the cerebrovasculature of transgenic AD mice and to explore the role of thrombin as a mediator of cerebrovascular inflammation and oxidative stress in AD and in hypoxia-induced changes in brain endothelial cells. Immunofluorescent analysis of the cerebrovasculature in AD mice demonstrates significant (p < 0.01–0.001) increases in thrombin, HIF-1α, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases (MMPs), and reactive oxygen species (ROS) compared to controls. Administration of the thrombin inhibitor dabigatran (100 mg/kg) to AD mice for 34 weeks significantly decreases expression of inflammatory proteins and ROS. Exposure of cultured brain endothelial cells to hypoxia for 6 h causes an upregulation of thrombin, HIF-1α, MCP-1, IL-6, and MMP2 and ROS. Treatment of endothelial cells with the dabigatran (1 nM) reduces ROS generation and inflammatory protein expression (p < 0.01–0.001). The data demonstrate that inhibition of thrombin in culture blocks the increase in inflammatory protein expression and ROS generation evoked by hypoxia. Also, administration of dabigatran to transgenic AD mice diminishes ROS levels in brain and reduces cerebrovascular expression of inflammatory proteins. Taken together, these results suggest that inhibiting thrombin generation could have therapeutic value in AD and other disorders where hypoxia, inflammation, and oxidative stress are involved.
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Affiliation(s)
- Debjani Tripathy
- Garrison Institute on Aging, Department of Neurology, Texas Tech University Health Sciences Center Lubbock, TX, USA
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Zhou QB, Duan CZ, Jia Q, Liu P, Li LY. Baicalin attenuates focal cerebral ischemic reperfusion injury by inhibition of protease-activated receptor-1 and apoptosis. Chin J Integr Med 2013; 20:116-22. [DOI: 10.1007/s11655-013-1441-7] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2012] [Indexed: 12/20/2022]
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20
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The Role of HSPA12B in Regulating Neuronal Apoptosis. Neurochem Res 2013; 38:311-20. [DOI: 10.1007/s11064-012-0922-y] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2012] [Revised: 10/26/2012] [Accepted: 11/08/2012] [Indexed: 10/27/2022]
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Abstract
Alzheimer's disease (AD) is a neurodegenerative disease with no available disease-modifying drugs. However, it has been postulated that neurovascular damage is a primary occurrence in this disease. Neurovascular damage is the result of the presence of cardiovascular risk factor generating hypoxia, oxidative stress, and metabolic changes that activate the endothelial cells of the brain microvasculature in order to respond to the stress by the development of angiogenesis. This endothelial activation could lead to a secretion of many proinflammatory cytokines and growth factors, such as thrombin. Heparin and related oligosaccharides have been shown to be efficient in the improvement of symptoms of AD. Their efficacy may be limited by their nonselective inhibitory effect of thrombin's activity. Direct thrombin inhibitors, such as dabigatran, might be efficient in the treatment of patients with AD because of their high selectivity for thrombin's activity inhibition while having a safer side effects profile than heparin.
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Affiliation(s)
- Bou Khalil Rami
- Psychiatric Hospital of the Cross, Saint Joseph University, Beirut, Lebanon.
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Tripathy D, Sanchez A, Yin X, Martinez J, Grammas P. Age-related decrease in cerebrovascular-derived neuroprotective proteins: effect of acetaminophen. Microvasc Res 2012; 84:278-85. [PMID: 22944728 PMCID: PMC3483357 DOI: 10.1016/j.mvr.2012.08.004] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2012] [Revised: 07/17/2012] [Accepted: 08/17/2012] [Indexed: 12/19/2022]
Abstract
As the population ages, the need for effective methods to maintain brain function in older adults is increasingly pressing. Vascular disease and neurodegenerative disorders commonly co-occur in older persons. Cerebrovascular products contribute to the neuronal milieu and have important consequences for neuronal viability. In this regard vascular derived neuroprotective proteins, Such as vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), and pituitary adenylate cyclase activating peptide (PACAP) are important for maintaining neuronal viability, especially in the face of injury and disease. The objective of this study is to measure and compare levels of VEGF, PEDF and PACAP released from isolated brain microvessels of Fischer 344 rats at 6, 12, 18, and 24 months of age. Addition of acetaminophen to isolated brain microvessels is employed to determine whether this drug affects vascular expression of these neuroprotective proteins. Experiments on cultured brain endothelial cells are performed to explore the mechanisms/mediators that regulate the effect of acetaminophen on endothelial cells. The data indicate cerebrovascular expression of VEGF, PEDF and PACAP significantly decreases with age. The age-associated decrease in VEGF and PEDF is ameliorated by addition of acetaminophen to isolated brain microvessels. Also, release of VEGF, PEDF, and PACAP from cultured brain endothelial cells decreases with exposure to the oxidant stressor menadione. Acetaminophen treatment upregulates VEGF, PEDF and PACAP in brain endothelial cells exposed to oxidative stress. The effect of acetaminophen on cultured endothelial cells is in part inhibited by the selective thrombin inhibitor hirudin. The results of this study suggest that acetaminophen may be a useful agent for preserving cerebrovascular function. If a low dose of acetaminophen can counteract the decrease in vascular-derived neurotrophic factors evoked by age and oxidative stress, this drug might be useful for improving brain function in the elderly.
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Affiliation(s)
- Debjani Tripathy
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas
| | - Alma Sanchez
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas
| | - Xiangling Yin
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas
| | - Joseph Martinez
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas
| | - Paula Grammas
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas
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Protective Effect of Scutellaria litwinowii Extract on Serum/Glucose-Deprived Cultured PC12 Cells and Determining the Role of Reactive Oxygen Species. J Toxicol 2012; 2012:413279. [PMID: 22888343 PMCID: PMC3409551 DOI: 10.1155/2012/413279] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2012] [Accepted: 06/24/2012] [Indexed: 11/18/2022] Open
Abstract
Considering the wide, positive reporting of the role of reactive oxygen species in ischemic brain injury, searching for antioxidant drugs within herbal remedies is logical. In this study, the protective effects of Scutellaria litwinowii Bornm. & Sint. on cell viability and reactive oxygen species production in cultured PC12 cells were investigated under serum/glucose-deprivation-induced cell death. After cells were seeded overnight, they were then deprived of serum/glucose for 24 h. Cells were treated with different concentrations of S. litwinowii extract (7.75–250 μg/mL). Cell viability was quantitated by MTT assay, and intracellular reactive oxygen species production was measured by flow cytometry. Serum/glucose-deprivation induced significant cell death after 24 h (P < 0.001). Treatment with S. litwinowii (7.75–250 μg/mL) reduced serum/glucose deprivation-induced cytotoxicity in PC12 cells after 24 h. A significant increase in intracellular reactive oxygen species production was seen following serum/glucose deprivation (P < 0.001). S. litwinowii (62 and 125 μg/mL, P < 0.01) treatment reversed the increased reactive oxygen species production following ischemic insult. This demonstrates that S. litwinowii extract protects PC12 cells against serum/glucose-deprivation-induced cell death by antioxidant mechanisms, which indicates the potential therapeutic application of S. litwinowii in managing cerebral ischemic and neurodegenerative disorders.
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Pigment epithelium-derived factor (PEDF) protects cortical neurons in vitro from oxidant injury by activation of extracellular signal-regulated kinase (ERK) 1/2 and induction of Bcl-2. Neurosci Res 2011; 72:1-8. [PMID: 21946416 DOI: 10.1016/j.neures.2011.09.003] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2011] [Revised: 09/07/2011] [Accepted: 09/12/2011] [Indexed: 12/31/2022]
Abstract
Mitigating oxidative stress-induced damage is critical to preserve neuronal function in diseased or injured brains. This study explores the mechanisms contributing to the neuroprotective effects of pigment epithelium-derived factor (PEDF) in cortical neurons. Cultured primary neurons are exposed to PEDF and H₂O₂ as well as inhibitors of phosphoinositide-3 kinase (PI3K) or extracellular signal-regulated kinase 1/2 (ERK1/2). Neuronal survival, cell death and levels of caspase 3, PEDF, phosphorylated ERK1/2, and Bcl-2 are measured. The data show cortical cultures release PEDF and that H₂O₂ treatment causes cell death, increases activated caspase 3 levels and decreases release of PEDF. Exogenous PEDF induces a dose-dependent increase in Bcl-2 expression and neuronal survival. Blocking Bcl-2 expression by siRNA reduced PEDF-induced increases in neuronal survival. Treating cortical cultures with PEDF 24 h before H₂O₂ exposure mitigates oxidant-induced decreases in neuronal survival, Bcl-2 expression, and phosphorylation of ERK1/2 and also reduces elevated caspase 3 level and activity. PEDF pretreatment effect on survival is blocked by inhibiting ERK or PI3K. However, only inhibition of ERK reduced the ability of PEDF to protect neurons from H₂O₂-induced Bcl-2 decrease and neuronal death. These data demonstrate PEDF-mediated neuroprotection against oxidant injury is largely mediated via ERK1/2 and Bcl-2 and suggest the utility of PEDF in preserving the viability of oxidatively challenged neurons.
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Yi H, Lee SJ, Lee J, Myung CS, Park WK, Lim HJ, Lee GH, Kong JY, Cho H. Sphingosylphosphorylcholine attenuated β-amyloid production by reducing BACE1 expression and catalysis in PC12 cells. Neurochem Res 2011; 36:2083-90. [PMID: 21674237 DOI: 10.1007/s11064-011-0532-0] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2011] [Accepted: 06/08/2011] [Indexed: 11/30/2022]
Abstract
Abnormal accumulation of β-amyloid (Aβ) is the main characteristic of Alzheimer's disease (AD) brain and Aβ peptides are generated from proteolytic cleavages of amyloid precursor protein (APP) by β-site APP-converting enzyme 1 (BACE1) and presenilin 1 (PS1). Sphingosylphosphorylcholine (SPC), a choline-containing sphingolipid, showed suppressive effect on Aβ production in PC12 cells which stably express Swedish mutant of amyloid precursor protein (APPsw). SPC (> 3 μM) significantly lowered the accumulation of Aβ40/42 and the expression of BACE1. However, the transcriptions of other APP processing enzymes like ADAM10 and PS1 were not affected by the SPC addition. Meanwhile, phosphocholine (PC) or other lysophospholipids, such as lysophosphatidylcholine (LPC), lysophosphatidic acid (LPA), sphingosyl-1-phosphate (S1P), did not alter BACE1 expression. Down-regulatory effect of SPC on BACE1 expression appeared to be mediated by NF-κB which is known to suppress the trans-activation of BACE1 promoter in PC12 cells. Here, the nuclear tanslocation of NF-κB was enhanced by SPC treatment in immune-fluorescent image analysis and NF-κB reporter assay. Furthermore, the catalytic activities of BACE1 and BACE2 were dose-dependently inhibited by SPC displaying IC₅₀ values of 2.79 μM and 12.05 μM, respectively. Overall, these data suggest that SPC has the potential to ameliorate Aβ pathology in neurons by down-regulating the BACE1-mediated amyloidogenic pathway.
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Affiliation(s)
- Hyoseok Yi
- Pharmacology Research Center, Korea Research Institute of Chemical Technology, Yuseong, Daejeon 305-343, Korea
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Grammas P. Neurovascular dysfunction, inflammation and endothelial activation: implications for the pathogenesis of Alzheimer's disease. J Neuroinflammation 2011; 8:26. [PMID: 21439035 PMCID: PMC3072921 DOI: 10.1186/1742-2094-8-26] [Citation(s) in RCA: 311] [Impact Index Per Article: 22.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2011] [Accepted: 03/25/2011] [Indexed: 02/07/2023] Open
Abstract
Alzheimer's disease (AD) is an age-related disorder characterized by progressive cognitive decline and dementia. Alzheimer's disease is an increasingly prevalent disease with 5.3 million people in the United States currently affected. This number is a 10 percent increase from previous estimates and is projected to sharply increase to 8 million by 2030; it is the sixth-leading cause of death. In the United States the direct and indirect costs of Alzheimer's and other dementias to Medicare, Medicaid and businesses amount to more than $172 billion each year. Despite intense research efforts, effective disease-modifying therapies for this devastating disease remain elusive. At present, the few agents that are FDA-approved for the treatment of AD have demonstrated only modest effects in modifying clinical symptoms for relatively short periods and none has shown a clear effect on disease progression. New therapeutic approaches are desperately needed. Although the idea that vascular defects are present in AD and may be important in disease pathogenesis was suggested over 25 years ago, little work has focused on an active role for cerebrovascular mechanisms in the pathogenesis of AD. Nevertheless, increasing literature supports a vascular-neuronal axis in AD as shared risk factors for both AD and atherosclerotic cardiovascular disease implicate vascular mechanisms in the development and/or progression of AD. Also, chronic inflammation is closely associated with cardiovascular disease, as well as a broad spectrum of neurodegenerative diseases of aging including AD. In this review we summarize data regarding, cardiovascular risk factors and vascular abnormalities, neuro- and vascular-inflammation, and brain endothelial dysfunction in AD. We conclude that the endothelial interface, a highly synthetic bioreactor that produces a large number of soluble factors, is functionally altered in AD and contributes to a noxious CNS milieu by releasing inflammatory and neurotoxic species.
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Affiliation(s)
- Paula Grammas
- Garrison Institute on Aging, and Department of Neurology, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.
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Tripathy D, Yin X, Sanchez A, Luo J, Martinez J, Grammas P. Cerebrovascular expression of proteins related to inflammation, oxidative stress and neurotoxicity is altered with aging. J Neuroinflammation 2010; 7:63. [PMID: 20937133 PMCID: PMC2965134 DOI: 10.1186/1742-2094-7-63] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2010] [Accepted: 10/11/2010] [Indexed: 12/17/2022] Open
Abstract
Background Most neurodegenerative diseases are age-related disorders; however, how aging predisposes the brain to disease has not been adequately addressed. The objective of this study is to determine whether expression of proteins in the cerebromicrovasculature related to inflammation, oxidative stress and neurotoxicity is altered with aging. Methods Brain microvessels are isolated from Fischer 344 rats at 6, 12, 18 and 24 months of age. Levels of interleukin (IL)-1β and IL-6 RNA are determined by RT-PCR and release of cytokines into the media by ELISA. Vessel conditioned media are also screened by ELISA for IL-1α, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α, (TNFα), and interferon γ (IFNγ). Immunofluorescent analysis of brain sections for IL-1β and IL-6 is performed. Results Expression of IL-1β and IL-6, both at RNA and protein levels, significantly (p < 0.01) decreases with age. Levels of MCP-1, TNFα, IL-1α, and IFNγ are significantly (p < 0.05-0.01) lower in 24 month old rats compared to 6 month old animals. Immunofluorescent analysis of brain vessels also shows a decline in IL-1β and IL-6 in aged rats. An increase in oxidative stress, assessed by increased carbonyl formation, as well as a decrease in the antioxidant protein manganese superoxide dismutase (MnSOD) is evident in vessels of aged animals. Finally, addition of microvessel conditioned media from aged rats to neuronal cultures evokes significant (p < 0.001) neurotoxicity. Conclusions These data demonstrate that cerebrovascular expression of proteins related to inflammation, oxidative stress and neurotoxicity is altered with aging and suggest that the microvasculature may contribute to functional changes in the aging brain.
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Affiliation(s)
- Debjani Tripathy
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas, USA
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Sanchez A, Wadhani S, Grammas P. Multiple neurotrophic effects of VEGF on cultured neurons. Neuropeptides 2010; 44:323-31. [PMID: 20430442 PMCID: PMC2879433 DOI: 10.1016/j.npep.2010.04.002] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/19/2009] [Revised: 03/03/2010] [Accepted: 04/06/2010] [Indexed: 01/17/2023]
Abstract
A large literature demonstrates the multifunctional nature of vascular endothelial growth factor (VEGF). Though initially characterized as an endothelial cell-specific factor, recent studies reveal that VEGF has numerous effects on diverse cell types in the brain including neurons. The objective of this study is to examine the effects of VEGF in cultured cortical neurons on survival, p38 mitogen-activated protein kinase (p38 MAP kinase) activity, pro- and anti-apoptotic protein expression and on release of neurotrophic and neurotoxic factors. The results show that VEGF dose-dependently enhances the survival of neurons in culture. VEGF decreases active caspase 3 levels and increases expression of the anti-apoptotic protein Bcl-2. VEGF decreases phosphorylated p38 MAP kinase level and activity in cortical neurons. In addition to modulating survival/death pathways in cortical neurons, VEGF also regulates release of proteins that affect neuronal viability. VEGF causes a dose-dependent release of the neurotrophic protein pigment epithelial-derived factor (PEDF), while significantly decreasing release of the neurotoxic protein amyloid beta. The VEGF-mediated decrease in amyloid beta is dependent on a functional Flt-1 receptor and is inhibited by dicoumarol, a multifunctional inhibitor of stress-activated protein kinase (SAPK)/JNK and NFkappaB pathways. Taken together, these data demonstrate that the neurotrophic effects of VEGF are likely mediated directly by increasing survival and decreasing apoptotic proteins and signals as well as indirectly by modulating release of proteins that affect neuronal viability.
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Affiliation(s)
- Alma Sanchez
- Garrison Institute on Aging and Department of Neurology Texas Tech University Health Sciences Center, Lubbock, Texas
| | - Suchin Wadhani
- Garrison Institute on Aging and Department of Neurology Texas Tech University Health Sciences Center, Lubbock, Texas
| | - Paula Grammas
- Garrison Institute on Aging and Department of Neurology Texas Tech University Health Sciences Center, Lubbock, Texas
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Yin X, Wright J, Wall T, Grammas P. Brain endothelial cells synthesize neurotoxic thrombin in Alzheimer's disease. THE AMERICAN JOURNAL OF PATHOLOGY 2010; 176:1600-6. [PMID: 20150433 DOI: 10.2353/ajpath.2010.090406] [Citation(s) in RCA: 78] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Alzheimer's disease (AD) is characterized by neuronal death; thus, identifying neurotoxic proteins and their source is central to understanding and treating AD. The multifunctional protease thrombin is neurotoxic and found in AD senile plaques. The objective of this study was to determine whether brain endothelial cells can synthesize thrombin and thus be a source of this neurotoxin in AD brains. Microvessels were isolated from AD patient brains and from age-matched controls. Reverse transcription-PCR demonstrated that thrombin message was highly expressed in microvessels from AD brains but was not detectable in control vessels. Similarly, Western blot analysis of microvessels showed that the thrombin protein was highly expressed in AD- but not control-derived microvessels. In addition, high levels of thrombin were detected in cerebrospinal fluid obtained from AD but not control patients, and sections from AD brains showed reactivity to thrombin antibody in blood vessel walls but not in vessels from controls. Finally, we examined the ability of brain endothelial cells in culture to synthesize thrombin and showed that oxidative stress or cell signaling perturbations led to increased expression of thrombin mRNA in these cells. The results demonstrate, for the first time, that brain endothelial cells can synthesize thrombin, and suggest that novel therapeutics targeting vascular stabilization that prevent or decrease release of thrombin could prove useful in treating this neurodegenerative disease.
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Affiliation(s)
- Xiangling Yin
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
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Involvement of ERK1/2 Pathway in Neuroprotection by Salidroside Against Hydrogen Peroxide-Induced Apoptotic Cell Death. J Mol Neurosci 2009; 40:321-31. [DOI: 10.1007/s12031-009-9292-6] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2009] [Accepted: 09/09/2009] [Indexed: 10/20/2022]
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Zhao P, Leonoudakis D, Abood ME, Beattie EC. Cannabinoid receptor activation reduces TNFalpha-induced surface localization of AMPAR-type glutamate receptors and excitotoxicity. Neuropharmacology 2009; 58:551-8. [PMID: 19654014 DOI: 10.1016/j.neuropharm.2009.07.035] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2009] [Revised: 07/28/2009] [Accepted: 07/29/2009] [Indexed: 01/08/2023]
Abstract
After injury or during neurodegenerative disease in the central nervous system (CNS), the concentration of tumor necrosis factor alpha (TNFalpha) rises above normal during the inflammatory response. In vitro and in vivo, addition of exogenous TNFalpha to neurons has been shown to induce rapid plasma membrane-delivery of AMPA-type glutamate receptors (AMPARs) potentiating glutamatergic excitotoxicity. Thus the discovery of drug targets reducing excess TNFalpha-induced AMPAR surface expression may help protect neurons after injury. In this study, we investigate the neuroprotective role of the CB1 cannabinoid receptor using quantitative immunofluorescent and real-time video microscopy to measure the steady-state plasma membrane AMPAR distribution and rate of AMPAR exocytosis after TNFalpha exposure in the presence or absence of CB1 agonists. The neuroprotective potential of CB1 activation with TNFalpha was measured in hippocampal neuron cultures challenged by an in vitro kainate (KA)-mediated model of Excitotoxic Neuroinflammatory Death (END). Here, we demonstrate that CB1 activation blocks the TNFalpha-induced increase in surface AMPARs and protects neurons from END. Thus, neuroprotective strategies which increase CB1 activity may help to reduce the END that occurs as a result of a majority of CNS insults.
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MESH Headings
- Animals
- Benzoxazines/pharmacology
- Cannabinoid Receptor Agonists
- Cell Death/drug effects
- Cell Death/physiology
- Cell Membrane/drug effects
- Cell Membrane/physiology
- Cells, Cultured
- Exocytosis/drug effects
- Exocytosis/physiology
- Hippocampus/drug effects
- Hippocampus/physiology
- Kainic Acid/toxicity
- Morpholines/pharmacology
- Naphthalenes/pharmacology
- Neurons/drug effects
- Neurons/physiology
- Neuroprotective Agents/pharmacology
- Neurotoxins/toxicity
- Rats
- Rats, Sprague-Dawley
- Receptor, Cannabinoid, CB1/agonists
- Receptor, Cannabinoid, CB1/metabolism
- Receptor, Cannabinoid, CB2/metabolism
- Receptors, AMPA/metabolism
- Receptors, Cannabinoid/metabolism
- Time Factors
- Tumor Necrosis Factor-alpha/metabolism
- Video Recording
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Affiliation(s)
- Pingwei Zhao
- Forbes Norris ALS/MDA Research Center, California Pacific Medical Center Research Institute, 475 Brannan St., Suite 220, San Francisco, CA 94107, USA
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32
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Sanchez A, Tripathy D, Grammas P. RANTES release contributes to the protective action of PACAP38 against sodium nitroprusside in cortical neurons. Neuropeptides 2009; 43:315-20. [PMID: 19497618 PMCID: PMC2726654 DOI: 10.1016/j.npep.2009.05.002] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/03/2009] [Revised: 05/12/2009] [Accepted: 05/13/2009] [Indexed: 12/21/2022]
Abstract
Pituitary adenylate cyclase activating polypeptide (PACAP), a promising neuroprotective peptide, plays an important role during development of the nervous system and in regeneration after injury. PACAP directly promotes survival via multiple signaling systems in neurons. This neuropeptide also has immuno-modulatory properties and can regulate the expression of various inflammatory mediators such as chemokines in nonneuronal cells. Chemokines and their G protein-coupled receptors are widely distributed in the brain, suggesting important functions for these inflammatory proteins in the CNS. The ability of brain endothelial cells and glia to release chemokines has been well documented, whether neurons are also a source for these mediators is unclear. The objective of this study is to determine whether PACAP38 affects expression of regulated on activation normal T expressed and secreted (RANTES) and macrophage inflammatory protein 1-alpha (MIP-1alpha) in cultured neurons and if these chemokines contribute to the neuroprotective effect of PACAP38. The data show that incubation of neuronal cultures with both PACAP38 and sodium nitroprusside (SNP) reduces the neuronal cell death evoked by SNP alone. PACAP38 dose-dependently increases immunodetectable levels of both RANTES and MIP-1alpha released in the media by cultured neurons. Co-treatment with a neutralizing antibody to RANTES decreases the PACAP38-mediated protection against SNP. Although RANTES treatment of neurons increased MIP-1alpha levels in the media and MIP-1alpha supports neuronal survival in unstressed cultures, MIP-1alpha does not protect neurons from SNP-induced toxicity. Furthermore, co-treatment with a MIP-1alpha neutralizing antibody did not affect PACAP38-induced protection against SNP. These results show that the protective effect of PACAP38 on cultured neurons is mediated, in part, by release of RANTES. The ability of PACAP to directly enhance neuronal survival through multiple intracellular signaling pathways as well as via the release of neuroprotective mediators such as RANTES highlights its utility as a potential therapeutic agent for the treatment of neurodegenerative diseases.
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Affiliation(s)
- Alma Sanchez
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas
| | - Debjani Tripathy
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas
| | - Paula Grammas
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, Texas
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Involvement of PGE2 and PGDH but not COX-2 in thrombin-induced cortical neuron apoptosis. Neurosci Lett 2009; 452:172-5. [PMID: 19383433 DOI: 10.1016/j.neulet.2009.01.045] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2008] [Revised: 12/11/2008] [Accepted: 01/16/2009] [Indexed: 12/14/2022]
Abstract
The pathways that contribute to thrombin-induced neuron death have been incompletely defined. Induction of cyclooxygenase 2 (COX-2), the enzyme that catalyzes the first step in prostaglandin synthesis, promotes neuronal injury. PGE2, a downstream product of COX-2 metabolism, is neurotoxic in vitro and in vivo, and is thought to be the bioactive mediator responsible for COX-2 neurotoxicity. The objective of this study is to determine the ability of thrombin to affect PGE2 metabolism in cultured neurons. The data show that in thrombin-induced apoptosis of cultured neurons, PGE2 release increases when COX-2 is absent, and is regulated by prostaglandin dehydrogenase (PGDH), a key enzyme that degrades PGE2. NS398, a COX-2 specific inhibitor, protects neurons against thrombin toxicity, by inducing active PGDH. These data implicate PGDH in thrombin-mediated neuronal cell death.
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Rao HV, Thirumangalakudi L, Grammas P. Cyclin C and cyclin dependent kinases 1, 2 and 3 in thrombin-induced neuronal cell cycle progression and apoptosis. Neurosci Lett 2008; 450:347-50. [PMID: 19103257 DOI: 10.1016/j.neulet.2008.12.018] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2008] [Revised: 10/31/2008] [Accepted: 12/06/2008] [Indexed: 01/05/2023]
Abstract
The extent to which neurons proceed into the cell cycle and the mechanisms whereby cell cycle re-entry leads to apoptosis vary in response to agonists. We previously showed upregulation of early G1 regulators in thrombin-treated neurons yet neurons did not proceed to S phase but to apoptosis. The objective of this study is to explore mechanisms which might prevent S phase entry and promote apoptosis in thrombin-treated neurons. Cultured rat brain neurons are exposed to thrombin (200nM) for 30min to 4.5h and the expression of cyclin C, cyclin dependent kinases (cdk1, cdk2, cdk3, cdk8) and the cell cycle inhibitor p27 assessed. Our data show a simultaneous decrease of both cyclin C and cdk3 proteins soon after thrombin treatment. The decrease in cyclin C also correlates with decreases in cdk1 and cdk2, at both mRNA and protein levels. There is no change in expression of cdk8 or the cell cycle inhibitor p27 in response to thrombin treatment. These results suggest that decreases in G1-S regulators cyclin C and cdks 3, cdk2 and cdk1 in response to thrombin could make conditions unfavorable for S phase entry and favor neuronal apoptosis.
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Affiliation(s)
- Haripriya Vittal Rao
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, 3601 4th Street Stop 9424, Lubbock, TX 79430, USA
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FILIP ROSANA, FERRARO GRACIELA, MANUELE MARIAGABRIELA, ANESINI CLAUDIA. ILEX BRASILIENSIS:PHYTOCHEMICAL COMPOSITION AND MECHANISM OF ACTION AGAINST THE PROLIFERATION OF A LYMPHOMA CELL LINE. J Food Biochem 2008. [DOI: 10.1111/j.1745-4514.2008.00196.x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
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Yu S, Liu M, Gu X, Ding F. Neuroprotective effects of salidroside in the PC12 cell model exposed to hypoglycemia and serum limitation. Cell Mol Neurobiol 2008; 28:1067-78. [PMID: 18481168 PMCID: PMC11515027 DOI: 10.1007/s10571-008-9284-z] [Citation(s) in RCA: 85] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2008] [Accepted: 04/29/2008] [Indexed: 10/22/2022]
Abstract
The hypoglycemia and serum limitation-induced cell death in cultured PC12 cells represents a useful in vitro model for the study of brain ischemia and neurodegenerative disorders. Salidroside is a phenylpropanoid glycoside isolated from Rhodiola rosea L., a traditional Chinese medicinal plant, and has displayed a broad spectrum of pharmacological properties. In this study, MTT assay, Hoechst 33342 staining, and flow cytometry with annexin V/PI staining collectively showed that pretreatment with salidroside attenuated, in a dose-dependent manner, cell viability loss, and apoptotic cell death in cultured PC12 cells induced by hypoglycemia and serum limitation. RT-PCR, Western blot analysis, and enzymatic colorimetric assay indicated the changes in expression levels of Bcl-2, Bax, and caspase3 in PC12 cells on exposure to hypoglycemia and serum limitation with and without salidroside pretreatment, respectively. Rhodamine 123 staining and flow cytometry with 2',7'-Dichlorofluorescin diacetate staining revealed the changes in the mitochondrial membrane potential and radical oxygen species (ROS) production in PC12 cells on exposure to hypoglycemia and serum limitation with and without salidroside pretreatment, respectively. The experimental results suggest that salidroside protects the PC12 cells against hypoglycemia and serum limitation-induced cytotoxicity possibly by the way of the modulation of apoptosis-related gene expression, the restoration of the mitochondrial membrane potential, and the inhibition of the intracellular ROS production. Our findings might raise a possibility of potential therapeutic applications of salidroside for preventing and treating cerebral ischemic and neurodegenerative diseases.
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Affiliation(s)
- Shu Yu
- Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, JS 226001 People’s Republic of China
| | - Mei Liu
- Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, JS 226001 People’s Republic of China
| | - Xiaosong Gu
- Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, JS 226001 People’s Republic of China
| | - Fei Ding
- Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, JS 226001 People’s Republic of China
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Sanchez A, Chiriva-Internati M, Grammas P. Transduction of PACAP38 protects primary cortical neurons from neurotoxic injury. Neurosci Lett 2008; 448:52-5. [PMID: 18938212 DOI: 10.1016/j.neulet.2008.10.021] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2008] [Revised: 09/25/2008] [Accepted: 10/01/2008] [Indexed: 10/21/2022]
Abstract
Neurotrophic factors such as pituitary adenylate cyclase activating polypeptide (PACAP38) are promising therapeutics for neurodegenerative diseases. However, delivery of trophic factors into brain neurons remains a challenge. The objective of this study is to determine whether adeno-associated virus (AAV) can mediate PACAP38 gene delivery into neurons in vitro and if transduction of AAV/PACAP38 into cortical neurons protects cells against neurotoxic insult. Primary cortical neuronal cultures are transduced with rAAV/PACAP38/GFP and cell survival against the nitric oxide releasing neurotoxin sodium nitroprusside (SNP) determined. GFP expression, a surrogate marker for successful transduction, is detected using fluorescent microscopy. The results show expression of GFP transgene and AAV capsid proteins in neurons. PACAP38 transduction significantly increases cell survival of neurons exposed to SNP. These results support the feasibility of using AAV-mediated delivery of PACAP38 to enhance neuronal survival and suggest that AAV-delivered PACAP38 maybe a therapeutic strategy for neurodegenerative diseases.
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Affiliation(s)
- Alma Sanchez
- Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
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Sanchez A, Rao HV, Grammas P. PACAP38 protects rat cortical neurons against the neurotoxicity evoked by sodium nitroprusside and thrombin. ACTA ACUST UNITED AC 2008; 152:33-40. [PMID: 18682263 DOI: 10.1016/j.regpep.2008.07.004] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2008] [Revised: 07/02/2008] [Accepted: 07/08/2008] [Indexed: 10/21/2022]
Abstract
Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 is a multifunctional anti-inflammatory and anti-apoptotic neuropeptide widely distributed in the nervous system. The objective of this study is to determine whether PACAP38 is neuroprotective against sodium nitroprusside (SNP) and thrombin, two mechanistically distinct neurotoxic agents. Treatment of primary cortical neuronal cultures with 1 mM SNP for 4 h causes neuronal cell death that is significantly reduced by 100 nM PACAP38. PACAP38 down-regulates SNP-induced cell cycle protein (cyclin E) expression and up-regulates p57(KIP2), a cyclin-dependent kinase inhibitor as well as the anti-apoptotic protein Bcl-2. Similarly, neuronal death induced by 100 nM thrombin or the thrombin receptor activating peptide (TRAP 6) is reduced by PACAP38 treatment. Thrombin-stimulated cell cycle protein (cdk4) expression is decreased by PACAP38 while PACAP38 inhibits thrombin-mediated reduction of p57(KIP2). However, the decrease in Bcl-2 evoked by thrombin is not affected by PACAP38. Finally, both SNP and thrombin (or TRAP) increase caspase 3 activity, an effect that is decreased by PACAP38. These data show that PACAP38 supports neuronal survival in vitro suppressing cell cycle progression and enhancing anti-apoptotic proteins. Our results support the possibility that PACAP could be a useful therapeutic agent for reducing neuronal cell death in neurodegenerative diseases.
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Affiliation(s)
- Alma Sanchez
- Department of Neuropsychiatry, Texas Tech University Health Sciences Center, Lubbock TX 79430, USA
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Larsen EC, Hatcher JF, Adibhatla RM. Effect of tricyclodecan-9-yl potassium xanthate (D609) on phospholipid metabolism and cell death during oxygen-glucose deprivation in PC12 cells. Neuroscience 2007; 146:946-61. [PMID: 17434680 PMCID: PMC2041837 DOI: 10.1016/j.neuroscience.2007.02.022] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2007] [Revised: 02/05/2007] [Accepted: 02/08/2007] [Indexed: 02/02/2023]
Abstract
Alterations in lipid metabolism play an integral role in neuronal death in cerebral ischemia. Here we used an in vitro model, oxygen-glucose deprivation (OGD) of rat pheochromocytoma (PC12) cells, and analyzed changes in phosphatidylcholine (PC) and sphingomyelin (SM) metabolism. OGD (4-8 h) of PC12 cells triggered a dramatic reduction in PC and SM levels, and a significant increase in ceramide. OGD also caused increases in phosphatidylcholine-phospholipase C (PC-PLC) and phospholipase D (PLD) activities and PLD2 protein expression, and reduction in cytidine triphosphate:phosphocholine cytidylyltransferase-alpha (CCTalpha, the rate-limiting enzyme in PC synthesis) protein expression and activity. Phospholipase A2 activity and expression were unaltered during OGD. Increased neutral sphingomyelinase activity during OGD could account for SM loss and increased ceramide. Surprisingly, treatment with PC-PLC inhibitor tricyclodecan-9-yl potassium xanthate (D609) aggravated cell death in PC12 cells during OGD. D609 was cytotoxic only during OGD; cell death could be prevented by inclusion of sera, glucose or oxygen. During OGD, D609 caused further loss of PC and SM, depletion of 1,2-diacylglycerol (DAG), increase in ceramide and free fatty acids (FFA), cytochrome c release from mitochondria, increases in intracellular Ca2+ ([Ca2+]i), poly-ADP ribose polymerase (PARP) cleavage and phosphatidylserine externalization, indicative of apoptotic cell death. Exogenous PC during OGD in PC12 cells with D609 attenuated PC, SM loss, restored DAG, attenuated ceramide levels, decreased cytochrome c release, PARP cleavage, annexin V binding, attenuated the increase in [Ca2+]i, FFA release, and significantly increased cell viability. Exogenous PC may have elicited these effects by restoring membrane PC levels. A tentative scheme depicting the mechanism of action of D609 (inhibiting PC-PLC, SM synthase, PC synthesis at the CDP-choline-1,2-diacylglycerol phosphocholine transferase (CPT) step and causing mitochondrial dysfunction) has been proposed based on our observations and literature.
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Affiliation(s)
- E. C. Larsen
- Department of Neurological Surgery, University of Wisconsin, Madison, WI
| | - J. F. Hatcher
- Department of Neurological Surgery, University of Wisconsin, Madison, WI
| | - Rao Muralikrishna Adibhatla
- Department of Neurological Surgery, University of Wisconsin, Madison, WI
- Cardiovascular Research Center, University of Wisconsin, Madison, WI
- Neuroscience Training Program, University of Wisconsin, Madison, WI
- Veterans Administration Hospital, Madison, WI
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40
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Rao HV, Thirumangalakudi L, Desmond P, Grammas P. Cyclin D1, cdk4, and Bim are involved in thrombin-induced apoptosis in cultured cortical neurons. J Neurochem 2007; 101:498-505. [PMID: 17254021 DOI: 10.1111/j.1471-4159.2006.04389.x] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Thrombin, a multifunctional serine protease, is neurotoxic in vitro and in vivo. Thrombin has been shown to be increased in Alzheimer's disease (AD) and other neuropathological conditions and could be a mediator of pathological neuronal cell death in the brain. The mechanisms of thrombin-induced neuronal cell death are incompletely understood. The objective of this study is to explore mechanisms that contribute to thrombin-induced neuronal apoptosis focusing on the role of cell cycle regulators and the pro-apoptotic protein Bim (Bcl-2-interacting mediator of cell death) in this process. Our data show that thrombin treatment of primary cerebral cortical cultures results in dose-dependent apoptotic cell death. Exposure of neuronal cultures to thrombin leads to induction of cell cycle proteins cyclin D1 and cyclin E, at both mRNA and protein levels. In addition, thrombin treatment causes the appearance of cyclin-dependent kinase 4 (cdk4) and expression of the pro-apoptotic protein Bim. Inhibition of cdk4 prevents both induction of Bim expression and thrombin-induced neuronal apoptosis. These data demonstrate that thrombin-induced apoptosis proceeds via cell cycle activation involving cdk4 resulting in induction of Bim. Thus, cell cycle proteins could be therapeutic targets in diseases such as AD where thrombin has been implicated.
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Affiliation(s)
- Haripriya Vittal Rao
- Garrison Institute on Aging and Department of Neuropsychiatry and Behavioral Sciences, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA
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Semmler A, Frisch C, Debeir T, Ramanathan M, Okulla T, Klockgether T, Heneka MT. Long-term cognitive impairment, neuronal loss and reduced cortical cholinergic innervation after recovery from sepsis in a rodent model. Exp Neurol 2007; 204:733-40. [PMID: 17306796 DOI: 10.1016/j.expneurol.2007.01.003] [Citation(s) in RCA: 180] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2006] [Revised: 12/17/2006] [Accepted: 01/08/2007] [Indexed: 10/23/2022]
Abstract
Sepsis is a disease with a high and growing prevalence worldwide. Most studies on sepsis up to date have been focused on reduction of short-term mortality. This study investigates cognitive and neuroanatomical long-term consequences of sepsis in a rat model. Sepsis was induced in male Wistar rats weighing 250-300 g by an i.p. injection of bacterial lipopolysaccharide (LPS, 10 mg/kg). Three months after complete recovery from sepsis, animals showed memory deficits in the radial maze and changes in open field exploratory patterns but unaffected inhibitory avoidance learning. Behavioral findings were matched by sepsis-induced loss of neurons in the hippocampus and the prefrontal cortex on serial sections after NeuN-staining and reduced cholinergic innervation in the parietal cortex measured by immunoradiography of vesicular acetylcholine transporter (VAChT). Together these results suggest that sepsis can induce persistent behavioral and neuroanatomical changes and warrant studies of the neurological long-term consequences of sepsis in humans.
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Affiliation(s)
- Alexander Semmler
- Department of Neurology, Sigmund-Freud-Strasse 25, University Hospital Bonn, 53105 Bonn, Germany.
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42
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Kajta M, Trotter A, Lasoń W, Beyer C. Impact of 17beta-estradiol on cytokine-mediated apoptotic effects in primary hippocampal and neocortical cell cultures. Brain Res 2006; 1116:64-74. [PMID: 16949056 DOI: 10.1016/j.brainres.2006.07.105] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2006] [Revised: 04/26/2006] [Accepted: 07/28/2006] [Indexed: 11/18/2022]
Abstract
Estrogens are developmental regulators of mitochondrial apoptotic pathway in the central nervous system, but little is known about their involvement in cytokine-induced apoptosis. In the present study, we evaluated effects of 17beta-estradiol on pro-inflammatory cytokine- and staurosporine-mediated activation of caspase-3 and LDH-release in primary neuronal/glial cell cultures of mouse hippocampal and neocortical cells at different stages of their development in vitro. Enzyme activities were determined with colorimetric methods 6 h, 14 h, 24 h, and 48 h after exposure to the apoptotic agents. Biochemical data were supported at the cellular level by Hoechst 33342 and MAP-2 stainings, which were carried out 48 h after the treatment. Cytokines (co-treatment with Il-1beta and TNFalpha; 1 ng/ml) increased caspase-3 activity in the hippocampal and neocortical cells up to over 200% of control values, and these effects were mostly observed on 2 and 7 days in vitro (DIV). Moderate, but significant cytokine-mediated increase in LDH-release was demonstrated in both tissues, especially on 7 and 12 DIV. Estradiol (100 nM) inhibited the activation of caspase-3 at early stage of development (2 DIV) in the hippocampal, but not in the neocortical cultures. The cytokine-induced activation of caspase-3 and LDH-release was inhibited by estradiol in estrogen receptor-independent way. These data point to a possible role of estrogens as non-estrogen receptor-related inhibitors of cytokine-activated apoptotic pathway in the developing central nervous system.
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Affiliation(s)
- Malgorzata Kajta
- Department of Anatomy and Cell Biology, University of Ulm, Albert-Einstein-Alle 11, 89061 Ulm, Germany.
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43
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Lee DY, Park KW, Jin BK. Thrombin induces neurodegeneration and microglial activation in the cortex in vivo and in vitro: proteolytic and non-proteolytic actions. Biochem Biophys Res Commun 2006; 346:727-38. [PMID: 16777064 DOI: 10.1016/j.bbrc.2006.05.174] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2006] [Accepted: 05/24/2006] [Indexed: 12/27/2022]
Abstract
The present study evaluated the role of thrombin and its receptors in neurodegeneration and microglial activation. Immunocytochemical evidence indicated that intracortical injection of thrombin resulted in a significant loss of neurons and the activation of microglia in the rat cortex in vivo. Reverse transcription PCR and double-label immunocytochemistry further demonstrated the early and transient expression of pro-inflammatory cytokines and neurotoxic factors as well as their colocalization within activated microglia. The thrombin-induced loss of cortical neurons was partially blocked by N(G)-nitro-L-arginine methyl ester hydrochloride, a nitric oxide synthase inhibitor, and by NS-398, a cyclooxygenase-2 inhibitor, indicating that the activation of microglia is involved in the neurotoxicity of thrombin in the cortex in vivo. In addition, thrombin activated cortical microglia in culture, as indicated by the expression of several pro-inflammatory cytokines and produced cell death in microglia-free, neuron-enriched cortical cultures. However, agonist peptides for thrombin receptors, including protease-activated receptor-1 (SFLLRN), -3 (TFRGAP), and -4 (GYPGKF), failed to activate microglia and were not neurotoxic in culture. Intriguingly, morphological and biochemical evidence indicated that thrombin-induced neurotoxicity but not microglial activation was prevented by hirudin, a specific inhibitor of thrombin. Collectively, the present data suggest that a non-proteolytic activity of thrombin activates microglia and that the proteolytic activity mediates its neurotoxicity.
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Affiliation(s)
- Da Yong Lee
- Neuroscience Graduate Program and Brain Disease Research Center, Ajou University School of Medicine, Suwon 443-721, Republic of Korea
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44
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Yang ES, Park JW. Regulation of nitric oxide-induced apoptosis by sensitive to apoptosis gene protein. Free Radic Res 2006; 40:279-84. [PMID: 16484044 DOI: 10.1080/10715760500511500] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Sensitive to apoptosis gene (SAG) protein, a novel zinc RING finger protein that protects mammalian cells from apoptosis by redox reagents, is a metal chelator and a potential reactive oxygen species (ROS) scavenger, but its antioxidant properties have not been completely defined. Nitric oxide (NO), a radical species produced by many types of cells, is known to play a critical role in many regulatory processes, yet it may also participate in collateral reactions at higher concentrations, leading to cellular oxidative stress. In this report, we demonstrate that modulation of SAG expression in U937 cells regulates NO-induced apoptosis. When we examined the protective role of SAG against NO-induced apoptosis with U937 cells transfected with the cDNA for SAG, a clear inverse relationship was observed between the amount of SAG expressed in target cells and their susceptibility to apoptosis. We also observed the significant decrease in the endogenous production of ROS and oxidative DNA damage in SAG-overexpressed cells compared to control cells upon exposure to NO. These results suggest that SAG plays an important protective role in NO-induced apoptosis, presumably, through regulating the cellular redox status.
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Affiliation(s)
- Eun Sun Yang
- School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702701, Korea
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45
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Semmler A, Okulla T, Sastre M, Dumitrescu-Ozimek L, Heneka MT. Systemic inflammation induces apoptosis with variable vulnerability of different brain regions. J Chem Neuroanat 2006; 30:144-57. [PMID: 16122904 DOI: 10.1016/j.jchemneu.2005.07.003] [Citation(s) in RCA: 251] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2005] [Accepted: 07/08/2005] [Indexed: 11/23/2022]
Abstract
During severe sepsis several immunological defence mechanisms initiate a cascade of inflammatory events leading to multi-organ failure including septic encephalopathy and ultimately death. To assess the reaction and participation of parenchymal brain cells during endotoxaemia, the present study evaluates micro- and astroglial activation, expression of the inducible nitric oxide synthase (iNOS) pro- and antiapoptotic protein levels Bax and Bcl-2, and apoptosis. Male Wistar rats received 10 mg/kg lipopolysaccharide (LPS) or vehicle intraperitoneally and were sacrificed for brain collection at 4, 8 or 24 h after induction of experimental sepsis. One group of animals received 10 mg/kg of the NOS inhibitor N-monomethyl-L-arginine (L-NMMA) intraperitoneally 1 day before and during the experiment. Immunohistochemical evaluation revealed a sepsis-induced, time-dependent increase in the immunoreactivity of iNOS, glial fibrillary acidic protein (GFAP) and activated microglia (ED-1), paralleled by a time-dependent increase of apoptotic brain cells marked by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL), an increase of Bax-positive cells and a decrease of Bcl-2-positive cells. Evaluation of different brain regions revealed that the hippocampus is the most vulnerable region during experimental sepsis. iNOS-inhibition with L-NMMA significantly reduced the number of apoptotic cells in hippocampus, midbrain and cerebellum. In addition, it reduced the increase of the proapoptotic protein Bax in all examined brain regions and reduced the decrease of Bcl-2-positive cells in the hippocampus. We therefore conclude, that peripheral inflammation leads to a profound glial activation, the generation of nitric oxide and changes of Bax and Bcl-2 protein regulation critical for apoptosis.
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46
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Li MH, Jang JH, Surh YJ. Nitric oxide induces apoptosis via AP-1-driven upregulation of COX-2 in rat pheochromocytoma cells. Free Radic Biol Med 2005; 39:890-9. [PMID: 16140209 DOI: 10.1016/j.freeradbiomed.2005.05.015] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/07/2005] [Revised: 05/05/2005] [Accepted: 05/05/2005] [Indexed: 10/25/2022]
Abstract
Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin synthesis, is induced in many cells by numerous inflammatory mediators, including nitric oxide (NO). Upregulation of COX-2 expression has been implicated in the pathophysiology of neuronal cell death. In the present study, we have found that the NO-induced upregulation of COX-2 via activation of activator protein-1 (AP-1) signaling leads to apoptotic cell death. Cultured rat pheochromocytoma (PC12) cells treated with sodium nitroprusside (SNP), a NO-releasing compound, exhibited marked induction of COX-2 expression, which was associated with apoptotic cell death as evidenced by internucleosomal DNA fragmentation, cleavage of poly(ADP-ribose) polymerase, activation of caspase-3, accumulation of p53, increased Bax/Bcl-XL ratio, and dissipation of mitochondrial membrane potential. In addition to the upregulation of COX-2 expression, SNP treatment led to activation of AP-1. Pretreatment of PC12 cells with c-fos antisense oligonucleotide abolished the NO-induced increase in DNA binding of AP-1 and upregulation of COX-2 expression. Furthermore, pretreatment with a selective COX-2 inhibitor (SC58635) rescued the PC12 cells from the apoptotic cell death induced by NO. Similar results were obtained when the NO-induced upregulation of COX-2 expression was blocked by the siRNA interference. These results suggest that excessive NO production during inflammation induces apoptosis in PC12 cells through AP-1-mediated upregulation of COX-2 expression.
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Affiliation(s)
- Mei-Hua Li
- National Research Laboratory of Molecular Carcinogenesis and Chemoprevention, College of Pharmacy, Seoul National University, Shinlim-dong, Kwanak-ku, Seoul 151-742, South Korea
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Chang CJ, Shih WL, Yu FL, Liao MH, Liu HJ. Apoptosis induced by bovine ephemeral fever virus. J Virol Methods 2005; 122:165-70. [PMID: 15542140 DOI: 10.1016/j.jviromet.2004.08.016] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2004] [Revised: 08/18/2004] [Accepted: 08/18/2004] [Indexed: 10/26/2022]
Abstract
The potential significance of bovine ephemeral fever virus (BEFV)-induced apoptosis and involved viral molecules was fully unknown. In the present study, evidence is provided demonstrating that bovine ephemeral fever virus induces apoptosis in several cell lines. Five types of assays for apoptosis were used in examining BEFV-infected cells. (1) Assay for DNA fragmentation, (2) nuclear staining with acridine orange, (3) ELISA detection of cytoplasmic histone-associated DNA fragment, (4) terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labelling (TUNEL) assay of BEFV-infected cells, (5) observation of blebbing of the plasma membrane and the formation of apoptotic bodies of apoptic cells by scanning electron microscope. The level of lactate dehydrogenase (LDH) in BEFV-infected cells was increased significantly after 20-25 h post-infection. Caspases-2, -3, -4, -6, -8, -9, and -10 were activated in BEFV-infected BHK-21 cells. To determine further whether BEFV-induced apoptosis was caspase-dependent, the effect of the tripeptide pan-ICE (caspase) inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyketone on the inhibition of apoptosis in BEFV-infected BHK-21 cells, was investigated. Apoptosis could be blocked by the caspase inhibitor (Z-VAD-fmk), indicating that BEFV induces caspase-dependent apoptosis in cultured cells.
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Affiliation(s)
- Chia J Chang
- Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan, ROC
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48
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Ehling R, Gassner C, Lutterotti A, Strasser-Fuchs S, Kollegger H, Kristoferitsch W, Reindl M, Berger T. Genetic variants in the tumor necrosis factor receptor II gene in patients with multiple sclerosis. ACTA ACUST UNITED AC 2004; 63:28-33. [PMID: 14651520 DOI: 10.1111/j.1399-0039.2004.00166.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Common genetic variants have been shown to influence disease susceptibility, disease course, or both in multiple sclerosis (MS). Several studies have suggested a role for tumor necrosis factor-alpha (TNF-alpha) in the pathogenesis of MS. Recently, it has been reported that the TNF receptor (TNFR) II plays an essential role in the pathology and progression of experimental autoimmune encephalomyelitis, an animal model of MS. To investigate whether TNFR II polymorphisms influence susceptibility and/or clinical progression of MS, genomic DNA of 321 samples of the Austrian Genetics in MS study group and DNA of 174 platelet donors, who served as healthy controls, were genotyped for five polymorphic sites in the TNFR II gene: exon 6 nucleotide (nt) 676*T-->G, exon 6 nt 783*G-->A (both are associated with non-conserved amino acid substitution), exon 10 nt 1663*G-->A, exon 10 nt 1668*T-->G, and exon 10 nt 1690*T-->C (all of which are located in the 3' non-coding region of the gene). We found a significant association between exon 10 nt 1668*T-->G polymorphism and susceptibility to MS. The other investigated nucleotide substitutions were not associated with susceptibility to or clinical parameters in MS.
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Affiliation(s)
- R Ehling
- Department of Neurology, University of Innsbruck, Anichstrasse, Innsbruck, Austria
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Mhatre M, Nguyen A, Kashani S, Pham T, Adesina A, Grammas P. Thrombin, a mediator of neurotoxicity and memory impairment. Neurobiol Aging 2004; 25:783-93. [PMID: 15165703 DOI: 10.1016/j.neurobiolaging.2003.07.007] [Citation(s) in RCA: 81] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2002] [Revised: 06/25/2003] [Accepted: 07/29/2003] [Indexed: 01/19/2023]
Abstract
Thrombin has been found in neuritic plaques in Alzheimer's disease (AD). Also, traumatic brain injury, where neurons are exposed to high thrombin levels, is associated with an increased incidence of AD. Our objective was to determine the effects of thrombin administered in vivo on cognitive function and neuropathology. Rats were trained using a radial eight-arm maze and then thrombin (25 or 100 nM, 0.25 microl/h, 28 days) or vehicle was delivered via intracerebroventricular infusion. Animals that received 100 nM thrombin demonstrated cognitive impairments including deficits in reference memory and an increase in task latency. Also, significant neuropathology was detected in these animals such as enlargement of cerebral ventricles, an increased number of TUNEL-positive cells, astrogliosis, and an increase in the immunoreactivity for phosphorylated neurofilament, and apolipoprotein-E fragments. Thrombin-induced changes in cognitive function and ventricular enlargement were inhibited by hirudin. These findings demonstrate that thrombin is a mediator of neurotoxicity and cognitive deficits and suggest that inhibition of thrombin may be a treatment strategy for AD- or head trauma-associated cognitive deficits.
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Affiliation(s)
- Molina Mhatre
- Department of Pathology, University of Oklahoma Health Sciences Center, 975 NE 10th Street, Oklahoma City, OK 73104, USA
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Peeraully MR, Jenkins JR, Trayhurn P. NGF gene expression and secretion in white adipose tissue: regulation in 3T3-L1 adipocytes by hormones and inflammatory cytokines. Am J Physiol Endocrinol Metab 2004; 287:E331-9. [PMID: 15100092 DOI: 10.1152/ajpendo.00076.2004] [Citation(s) in RCA: 98] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The sympathetic nervous system plays a central role in lipolysis and the production of leptin in white adipose tissue (WAT). In this study, we have examined whether nerve growth factor (NGF), a target-derived neurotropin that is a key signal in the development and survival of sympathetic neurons, is expressed and secreted by white adipocytes. NGF mRNA was detected by RT-PCR in the major WAT depots of mice (epididymal, perirenal, omental, mesenteric, subcutaneous) and in human fat (subcutaneous, omental). In mouse WAT, NGF expression was observed in mature adipocytes and in stromal vascular cells. NGF expression was also evident in 3T3-L1 cells before and after differentiation into adipocytes. NGF protein, measured by ELISA, was secreted from 3T3-L1 cells, release being higher before differentiation. Addition of the sympathetic agonists norepinephrine, isoprenaline, or BRL-37344 (beta(3)-agonist) led to falls in NGF gene expression and secretion by 3T3-L1 adipocytes, as did IL-6 and the PPARgamma agonist rosiglitazone. A substantial decrease in NGF expression and secretion occurred with dexamethasone. In contrast, LPS increased NGF mRNA levels and NGF secretion. A major increase in NGF mRNA level (9-fold) and NGF secretion (<or=40-fold) in 3T3-L1 adipocytes occurred with TNF-alpha. RT-PCR showed that the genes encoding the p75 and trkA NGF receptors were expressed in mouse WAT. These results demonstrate that white adipocytes secrete NGF (an adipokine), NGF synthesis being influenced by several factors with TNF-alpha having a major stimulatory effect. We suggest that NGF is a target-derived neurotropin and an inflammatory response protein in white adipocytes.
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Affiliation(s)
- Muhammad R Peeraully
- Neuroendocrine and Obesity Biology Unit, Liverpool Centre for Nutritional Genomics, School of Clinical Sciences, University of Liverpool, UK
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