Altered Glial Calcium Signaling in Neurodegeneration

神经退行性变中神经胶质钙信号的改变

• 批准号: 6758246
• 负责人: VERA GOLOVINA
• 金额: $ 29.79万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6758246
• 关键词: Alzheimer' s disease; Downs syndrome; amyloid proteins; antisense nucleic acid; astrocytes; biological signal transduction; calcium binding protein; calcium channel; calcium flux; calcium transporting ATPase; disease /disorder model; endoplasmic reticulum; glia; immunocytochemistry; inositol phosphates; interleukin 1; laboratory mouse; neural degeneration; polymerase chain reaction; western blottings

DESCRIPTION (provided by applicant): A common feature of Alzheimer's disease (AD) and Down's syndrome (DS) is a cascading microglia/astrocyte activation, characterized by the abundant production of proinflammatory cytokines, such as interleukin-1beta (IL-1beta). IL-1beta stimulates astrocytes to synthesize and release neuroactive agents such as S100beta, a cytokine that fosters neuronal dysfunction and death by raising intracellular free Ca2+concentration ([Ca2+]cyt). IL-1beta also upregulates expression and processing of beta-amyloid precursor protein (beta-APP) leading to increased production of amyloid beta-protein (A-beta) that is thought to play a major role in the pathogenesis of AD. Both, IL-1beta and A-beta increase [Ca2+]cyt in astrocytes by augmenting Ca2+ influx. The mechanisms underlying Ca2+ dysregulation, although unknown, may be causally implicated in reactive changes of astrocytes leading to neurotoxicity. They may involve activation of store- and/or receptor-operated Ca2+ entry (SOCE and ROCE, respectively) through TRPC-encoded store- and receptor-operated Ca2+ channels (SOCs, ROCs). The goal of this program is to test the hypothesis that dysregulation of Ca 2+homeostasis with elevated [Ca2+]cyt and ER Ca2+concentration ([Ca2+]ER), in IL-1beta or A-beta-treated astrocytes and in astrocytes from trisomy 16 (Ts16) mouse, an animal model of DS and AD, is the result of upregulated TRPC expression and enhanced SOCE and/or ROCE. The Specific Aims are: 1. To investigate how acute and chronic (24 hr) IL-1beta - and A-beta-treatment affects Ca2+ homeostasis in freshly dissociated and primary cultured mouse cortical astrocytes using Ca2+-sensitive fluorochromes. SOCE will be probed by measuring the rates and magnitudes of SOCE-evoked changes in [Ca2+]cyt, and [Ca2+]ER. 2. To identify the molecular mechanisms responsible for IL-1beta- and Abeta-induced global and local Ca2+ signaling in astrocytes. To determine whether IL-1beta and A-beta affect expression and distribution of TRPC channels and ER Ca2+ transporters and receptors (SERCA2b, IP3 and ryanodine receptors) by using PCR, immunoblotting and high spatial resolution immunocytochemistry. 3. To determine whether inhibition of TRPC-encoded SOCs and/or ROCs down-regulates S 100beta and beta-APP expression in IL-1beta - and A-beta-treated cells and in Ts16 astrocytes (Ts16 mice and DS humans overexpress betaAPP and S100beta). Antisense oligos for TRPC(1-7) genes and Ca2+-free media will be used to eliminate Ca2+ influx. Expression of S100beta and beta-APP will be identified by immunoblotting. Determining the mechanisms of IL-1beta - and A-beta-induced Ca2+influx in astrocytes could lead to novel therapeutic strategies for AD.

描述（由申请人提供）：阿尔茨海默氏病（AD）和唐氏综合症（DS）的一个共同特征是层叠的小胶质细胞/星形胶质细胞激活，其特征是促炎细胞因子的大量产生，例如interleuukinkin-1Beta（Il-1beta）。 IL-1BETA刺激星形胶质细胞合成和释放神经活性剂，例如S100BETA，S100BETA是一种通过提高细胞内游离CA2+浓度来促进神经元功能障碍和死亡的细胞因子（[[CA2+] CYT）。 IL-1BETA还上调了β-淀粉样蛋白前体蛋白（β-APP）的表达和处理，从而导致淀粉样蛋白β-蛋白质（A-BETA）的产生增加，这被认为在AD的发病机理中起主要作用。通过增加Ca2+流入，IL-1BETA和A-BETA在星形胶质细胞中增加了[Ca2+] Cyt。 CA2+失调的基础机制虽然未知，但可能与导致神经毒性的星形胶质细胞的反应性变化有关。它们可能涉及通过TRPC编码的商店和受体操作的Ca2+通道（SOC，ROCS）激活储存和/或受体操作的Ca2+进入（分别为SOCE和ROCE）。 The goal of this program is to test the hypothesis that dysregulation of Ca 2+homeostasis with elevated [Ca2+]cyt and ER Ca2+concentration ([Ca2+]ER), in IL-1beta or A-beta-treated astrocytes and in astrocytes from trisomy 16 (Ts16) mouse, an animal model of DS and AD, is the result of upregulated TRPC expression and enhanced SOCE和/或ROCE。具体目的是：1。用于研究急性和慢性（24小时）IL-1BETA和A-BETA处理如何使用Ca2+敏感的荧光素化的新鲜分离和原发性小鼠皮质星形胶质细胞的Ca2+稳态。 SOCE将通过测量[Ca2+] Cyt和[Ca2+] ER的SOCE诱发变化的速率和大小来探测。 2。确定负责IL-1Beta和Abeta诱导的星形胶质细胞中全球和局部CA2+信号传导的分子机制。确定IL-1BETA和A-BETA是否会通过使用PCR，免疫印迹和高空间分辨率的免疫细胞化学化学，影响TRPC通道以及ER CA2+转运蛋白和受体（SERCA2B，IP3和Ryanodine受体）的表达和分布。 3。确定对TRPC编码的SOC和/或ROC的抑制是否会在IL-1Beta和A-Beta处理的细胞以及TS16星形胶质细胞中（TS16小鼠和DS人体过度表达BetaApp和S100beta）中的S 100beta和Beta-App表达。 TRPC（1-7）基因和无CA2+培养基的反义寡聚将用于消除Ca2+流入。 S100BETA和Beta-App的表达将通过免疫印迹来识别。确定星形胶质细胞中IL-1Beta和A-Beta诱导的Ca2+涌入的机制可能会导致AD的新型治疗策略。