Mechanism of CREB dysregulation in Alzheimer brain

描述(由申请人提供)： 阿尔茨海默病(AD)的特征是认知功能障碍和进行性神经变性。阿尔茨海默病的病理特征包括A型斑块的积聚、神经纤维缠结的形成和突触功能障碍。小胶质细胞是大脑中驻留的巨噬细胞，通过减少A？负荷起到保护作用。然而，过度激活的小胶质细胞释放的细胞因子和自由基会导致神经元损伤。受损的神经元通过向小胶质细胞发出求救信号来促进自身的死亡。这项建议通过体外和体内研究，密切关注暴露于激活的小胶质细胞的神经元中的分子事件。神经元中信号通路和基因表达模式的改变被认为在AD的病理过程中起着重要作用。在这个模型中，转录因子在信号通路和基因表达之间起着至关重要的作用。环磷酸腺苷反应元件结合蛋白(CREB)是一种核转录因子，可增强认知、记忆形成和神经元存活。已知CREB在AD大脑中下调。然而，CREB的治疗靶向是一个挑战，因为它的作用范围很广。转录因子通过上游信号以上下文和细胞类型依赖的方式被引导到适当的启动子。我们已经确定了生长因子刺激的激活CREB的信号通路，以及氧化应激干扰培养神经元CREB功能的机制。在最近对AD死后样本和阿尔茨海默氏症转基因小鼠的研究中，我们发现A？产生的氧化应激导致海马区CREB表达下降是一种晚期事件。但在CREB含量下降之前，CREB功能持续下调。我们初步研究的以下两个关键发现表明，炎症是AD进展的早期事件，干扰CREB功能。(1)A？激活的人小胶质细胞条件培养液可降低CREB调节的人神经前体细胞(NPC)来源神经元中BDNF的表达。(Ii)与Aβ激活的小胶质细胞共同培养的CREB功能保护神经元的恢复。炎症的保护性和神经退行性途径可以在转录水平上描绘出来。炎症介导的CREB异常调节发生在以下位置：(1)细胞因子和活性氧减少CREB的磷酸化/活化。(Ii)炎症激活c-jun、STAT-1和NF-βB，这些转录因子与CREB竞争有限的辅活化子、CBP和p300。(Iii)炎症可将CREB导向神经功能所需途径以外的途径。(Iv)炎症反应产生的蛋白质在功能上拮抗CREB靶蛋白，促进神经元存活。我们的假设是，慢性神经炎的致病成分，与氧化应激协同作用，下调了CREB介导的AD大脑中神经保护基因的转录。这一假说将在与小胶质细胞共培养的人神经前体细胞来源的神经元和三重转基因阿尔茨海默病小鼠中进行验证，方法包括激光捕获显微解剖、基于网络基序的分析和基于设计的体视学，其具体目的如下：目的1.确定A？激活的小胶质细胞和星形胶质细胞对培养的人神经前体细胞来源神经元CREB表达下调的机制。目的2.确定神经元CREB下调是三重转基因阿尔茨海默病(3xTg-AD)小鼠脑内神经炎症的致病成分：治疗策略可以针对转录因子及其网络产生深远的有益影响。我们的实验室有15年的经验和专业知识，可以在炎症和氧化应激过程中竞争转录因子的平行激活背景下研究CREB功能。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is characterized by cognitive dysfunction and progressive neurodegeneration. Hallmarks of AD pathology include accumulation of A¿-containing plaques, formation of neurofibrillary tangles and synaptic dysfunction. Microglia, the resident macrophages of the brain, play a protective role by reducing A¿ load. However, cytokines and free radicals released from hyperactivated microglia cause neuronal damage. Injured neurons facilitate their own demise by sending out distress signals to microglia. This proposal takes a close look at the molecular events in neurons exposed to activated microglia by in vitro and in vivo studies. Altered signaling pathways and orchestrated gene expression patterns in neurons are known to play significant roles in causing AD pathology. In this model, transcription factors provide a crucial link between signaling pathways and gene expression. Cyclic AMP response element binding protein (CREB), a nuclear transcription factor, enhances cognition, memory formation and neuronal survival. CREB is known to be downregulated in the AD brain. However, therapeutically targeting CREB is a challenge because of its broad spectrum actions. Transcription factors are directed to appropriate promoters in a context and cell-type dependent manner by upstream signals. We have characterized the growth factor-stimulated signaling pathways that activate CREB and the mechanism through which oxidative stress interferes with CREB function in cultured neurons. In a recent study with AD post-mortem samples and Alzheimer's transgenic mice, we identified the decrease of hippocampal CREB expression by A¿-generated oxidative stress as a late event. But the decrease in CREB content is preceded by persistent downregulation of CREB function. The following two critical findings from our preliminary studies suggest that inflammation, an early event in the progression of AD, interferes with CREB function. (i) CREB-regulated BDNF expression in human neuroprogenitor cell (NPC)- derived neurons was decreased by conditioned medium from A¿-activated human microglia. (ii) Restoration of CREB function protected neurons cocultured with A¿-activated microglia. Protective and neurodegenerative pathways of inflammation can be delineated at the transcriptional level. Inflammation-mediated CREB dysregulation takes place at the following sites: (i) Cytokines and reactive oxygen species decrease CREB phosphorylation/activation. (ii) Inflammation activates c-jun, STAT-1 and NF-?B, the transcriptions factors that compete with CREB for the limited pool of coactivators, CBP and p300. (iii) Inflammation can direct CREB to pathways other than those needed for neuronal function. (iv) Proteins produced in response to inflammation functionally antagonize CREB target proteins that promote neuronal survival. Our hypothesis is that the pathogenic component of chronic neuroinflammation, acting synergistically with oxidative stress, downregulates CREB-mediated transcription of neuroprotective genes in the AD brain. This hypothesis will be tested in human neuroprogenitor cell derived-neurons, cocultured with microglia and in a triple transgenic Alzheimer's mice using novel methodologies including laser capture microdissection, network motif-based analysis and design-based stereology with the following Specific Aims: Aim 1. To determine the mechanism of CREB downregulation in cultured human neuroprogenitor cell-derived neurons exposed to A¿-activated microglia and astrocytes. Aim 2. To identify neuronal CREB downregulation as a pathogenic component of neuroinflammation in triple transgenic Alzheimer's (3XTg-AD) mouse brain: Therapeutic strategies can target transcription factors and their network for profound beneficial effects. Our lab has 15 years of experience and the expertise to examine CREB function in the context of parallel activation of competing transcription factors during inflammation and oxidative stress.