Inhibiting Cell Cycle Progression Reduces Nitric Oxide- and Amyloid Beta-Induced Neurotoxicity in Pc12 Cells and Bv2 Microglia: a Mechanism Involves BNIP3

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Project title

Code/計畫編號

NSC97-2311-B019-004-MY3

Translated Name/計畫中文名

抑制細胞週期減少一氧化氮和Amyloid-beta造成之神經毒性---涉及BNIP3之可能機制

Project Coordinator/計畫主持人

Shye-Jye Tang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=1672747

Year

2008

Start date/計畫起

01-08-2008

Expected Completion/計畫迄

01-07-2009

Bugetid/研究經費

920千元

ResearchField/研究領域

生物科學

"急速的人口老化及全球人平均壽命的增長，失智人口也隨年齡增加而有逐年上升的趨勢，失智人口當中以阿茲海默症(Alizheimer＇s disease, AD)族群居多。AD便是一種隨著年齡增長腦神經退化的現象。在致病性上包含形成腦部plaques和 amyloid beta peptide (Aβ), activated microglia, reactive astrocytes, neurofibrillary tangles 和dystrophic neurites。由過量累積Aβ,是AD致病的主要原因。微膠細胞為腦部之巨噬細胞,是產生腦部發炎和發炎引起的神經退化的主要細胞。由Aβ產生之微膠細胞之發炎反應在產生AD中扮演重要的角色。這發炎反應包括分泌細胞激素和放出一氧化氮。 Cyclin-dependent kinases (CDKs)參予細胞週期的調控。研究顯示CDK抑制劑可產生神經細胞保護作用,抑制神經細胞活化和微膠細胞產生之發炎反應。在AD病人中發現不正常活化細胞週期相關基因,但是現象與AD產生之神經退化作用之關係,正進行研究中。在我們先前的研究中顯示,細胞週期抑制劑Olomoucine 和Roscovitine可抑制BV2 細胞生長、發炎反應和一氧化氮的釋放。同時Olomoucine可抑制NO產生之細胞凋亡。經 microarray分析,顯示Olomoucine可抑制BNIP3表現,一具促進細胞凋亡之基因。發現 Olomoucine處理後,可減少BNIP3表現及NO促進cytochrome c釋放。這結果顯示,經抑制細胞週期,可降低BNIP3表現而抑制NO產生之細胞凋亡。 BV2微膠細胞可由LPS和Aβ促使發炎反應,而NO進而產生細胞凋亡。PC12細胞經神經生長因子刺激後,可分化成神經細胞,且NO和Aβ可造成這細胞凋亡。計畫中,將採用這兩細胞株,探討NO和Aβ造成之細胞凋亡與BNIP3之關係。細胞週期的調控與NO和Aβ造成之粒腺體失活和細胞凋亡之關係,並不明瞭。計畫中將以三年時間探討下列主題: (1) NO和Aβ造成之細胞凋亡是否涉及細胞週期相關基因 (2)細胞週期抑制劑可否經抑制BNIP3,而促進NO和Aβ造成之細胞凋亡的細胞存活 (3)大量表現cyclin D1或E2F1 可促進BNIP3表現,而增加NO和Aβ造成之細胞凋亡 (4)細胞週期抑制劑可否經抑制 HIF-1活化而減少BNIP3表現,而促進NO和Aβ造成之細胞凋亡的細胞存活 (5)使用BNIP3 的 interference RNA 或以dominant-negative mutant BNIP3ΔTM抑制BNIP3功能,並測定 NO和Aβ造成之細胞凋亡。這計畫將可研究是否經抑制細胞週期而減少BNIP3 表現,可降低神經細胞經 NO 和Aβ 造成之細胞凋亡。這發現將可提出一新的神經保護機制和用於AD 的治療。" "Alzheimer’s disease (AD) is a chronic neuro-degenerative disease characterized by memory loss and dementia. AD remains the most common cause of dementia in all age groups. The pathological hallmarks include extracellular plaques containing amyloid beta peptide (Aβ), activated microglia, reactive astrocytes, neurofibrillary tangles and dystrophic neurites. Excessive accumulation of Aβ has been proposed as a pivotal event in the pathogenesis of AD. Microglia are resident brain macrophages, and are the key cells in brain inflammation and inflammatory neurodegeneration. Initiation and maintenance of pro-inflammatory reactions elicited by Aβ in the microglia lineage have been reported to play a crucial role in AD. The pro-inflammatory reactions include the secretion of cytokines and the release nitric oxide (NO). The cyclin-dependent kinase (CDK) family of enzymes is responsible for the orderly progression of cells through the various phases of the cell cycle. CDK inhibitors have been shown to promote neuroprotection, reduce astroglial scar formation and microglial activation, as well as attenuate reactive astrogliosis and the microglia induced inflammatory responses. Cell cycle-related molecules are up-regulated in postmitotic neurons within affected brain regions during AD. However, it is unclear whether deregulation of cell cycle events contributes to neurodegeneration in AD. In our previous study, we have demonstrated that Olomoucine and Roscovitine inhibit cell proliferation and diminish NO production and cytokine gene expression in LPS-stimulated mouse microglial BV2 cells. In addition, Olomoucine reduces LPS-induced cell death resulting from decrease of NO generated by LPS stimulation. Moreover, Olomoucine promote survival after treatment of SNAP, a NO donor. We also demonstrate that mitochrondrial dysfunction was observed after LPS and SNAP stimulation. We used cDNA microarray analysis to show that Olomoucine strongly reduces expression of Bcl-2/adenovirus E1B 19kDa-interacting protein 3 (BNIP3) in BV2 cells. After Olomoucine pre-treatment, the expression of BNIP3 was down-regulated and reduced cytochrome c release after SNAP treatment. These findings suggest that inhibition of cell cycle progression is capable of reducing BNIP3 expression and preventing NO-induced apoptosis. BV2 cells exhibit pro-inflammatory responses by LPS or Aβ stimulation and the cells are showed apoptosis by treatment of NO. PC12 cells are able to be differentiated into neuronal cells by stimulation of nerve growth factor. In addition, apoptosis are demonstrated by treatment of NO and Aβ in PC12 cells. In order to investigate the relationship between BNIP3 and NO- or Aβ-induced apoptosis, we will use BV2 microglial cells and PC12 neuronal cells as cell line models. Whether regulation of cell cycle may involve the NO- and Aβ-induced mitochondrial dysfunction and apoptosis in neuronal cells is undocumented. The overall objective in this three-year research proposal will find out the following subjects: To understand the mechanisms responsible for neuronal cell death in vitro, we will investigate (1) whether cell cycle proteins are implicated in NO- and Aβ-induced apoptosis of these cells (2) whether the CDK inhibitors, Olomoucine or Roscovitine, can affect neuronal cell survival by down-regulation of the expression of BNIP3 in NO- and Aβ-induced apoptosis (3) whether overexpression of cyclinD1 or E2F1 may promote NO- and Aβ-induced apoptosis resulted from enhancing BNIP3 expression (4) whether NO- and Aβ-induced BNIP3 expression abolished by pretreatment with CDK inhibitors is mediated by reduction hypoxia-inducible factor-1 (HIF-1) activation (5) whether knock-down expression of BNIP3 by interference RNA or inhibition of BNIP3 function by dominant-negative mutant BNIP3ΔTM may block neuronal death evoked by NO or Aβ. In this project, we will demonstrate that NO- and Aβ-induced apoptosis of neuronal cells proceeds through an induction of BNIP3, which, in turn, can be specifically attenuated by inhibiting cell cycle progression. Our finding may propose a novel neuroprotective mechanism and a potentially useful treatment for AD."

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Inhibiting Cell Cycle Progression Reduces Nitric Oxide- and Amyloid Beta-Induced Neurotoxicity in Pc12 Cells and Bv2 Microglia: a Mechanism Involves BNIP3

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