Altered Glial Calcium Signaling in Neurodegeneration

神经退行性变中神经胶质钙信号的改变

基本信息

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
项目类别：
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

项目摘要

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）的一个共同特征是级联小胶质细胞/星形胶质细胞激活，其特征是促炎细胞因子的大量产生，例如白细胞介素-1β（IL-1β）。 IL-1β 刺激星形胶质细胞合成和释放神经活性剂，如 S100β，这是一种通过提高细胞内游离 Ca2+ 浓度 ([Ca2+]cyt) 促进神经元功能障碍和死亡的细胞因子。 IL-1β 还上调 β-淀粉样前体蛋白 (β-APP) 的表达和加工，导致淀粉样β-蛋白 (A-β) 的产生增加，而 A-β 被认为在 AD 的发病机制中发挥着重要作用。 IL-1β 和 A-β 均通过增加 Ca2+ 流入来增加星形胶质细胞中的 [Ca2+] 细胞色素。 Ca2+ 失调的机制虽然未知，但可能与星形胶质细胞的反应性变化导致神经毒性有关。它们可能涉及通过 TRPC 编码的钙池和/或受体操作的 Ca2+ 通道（SOC、ROC）激活钙池和/或受体操作的 Ca2+ 进入（分别为 SOCE 和 ROCE）。该计划的目标是检验这样的假设：在 IL-1beta 或 A-beta 处理的星形胶质细胞中以及在来自16 三体 (Ts16) 小鼠是 DS 和 AD 的动物模型，是 TRPC 表达上调和 SOCE 和/或 ROCE 增强的结果。具体目标是： 1. 使用 Ca2+ 敏感荧光染料研究急性和慢性（24 小时）IL-1β 和 A-β 治疗如何影响新鲜分离和原代培养的小鼠皮质星形胶质细胞中的 Ca2+ 稳态。将通过测量 SOCE 引起的 [Ca2+]cyt 和 [Ca2+]ER 变化的速率和幅度来探测 SOCE。 2. 确定星形胶质细胞中 IL-1beta 和 Abeta 诱导的全局和局部 Ca2+ 信号传导的分子机制。通过使用 PCR、免疫印迹和高空间分辨率免疫细胞化学来确定 IL-1β 和 A-β 是否影响 TRPC 通道以及 ER Ca2+ 转运蛋白和受体（SERCA2b、IP3 和兰尼碱受体）的表达和分布。 3. 确定抑制 TRPC 编码的 SOC 和/或 ROC 是否会下调 IL-1β 和 A-β 处理的细胞以及 Ts16 星形胶质细胞（Ts16 小鼠和 DS 人类过度表达 betaAPP 和S100测试版）。 TRPC(1-7) 基因的反义寡核苷酸和无 Ca2+ 培养基将用于消除 Ca2+ 流入。 S100beta和beta-APP的表达将通过免疫印迹来鉴定。确定星形胶质细胞中 IL-1β 和 A-β 诱导的 Ca2+ 内流的机制可能会带来新的 AD 治疗策略。