CaMKI, Apoptosis and Cholinergic Neurons in Aging and AD

描述（申请人提供）：基底前脑胆碱能神经元（BFCN）系统与学习、记忆和注意力的过程密切相关。在人脑中，BFCN选择性地易受老年痴呆症等神经退行性疾病的影响。我们实验室最近的初步观察表明，AD的BFCN显示钙调蛋白激酶I （CaMKI）基因的表达降低，凋亡蛋白fas相关死亡结构域（FADD）的免疫反应性增加。研究表明，CaMKI下调可导致神经元凋亡。因此，CaMKI在AD BFCN中的表达下调可能与这些神经元的凋亡有关。我们还观察到，在人类的正常衰老过程中，BFCN显示出钙结合蛋白calbinin - d28k （CB）的大量损失，在阿尔茨海默病中进一步减少，在阿尔茨海默病中退化的BFCN似乎是那些在正常衰老过程中失去这种蛋白质的人。我们假设CaMKI在衰老和AD BFCN中下调导致促凋亡和/或抗凋亡基因上调，上述基因表达的变化可能与年龄相关的CB丢失有关，这是在这些神经元中最早观察到的变化，并可能导致钙的失调。本提案中的实验旨在使用更新颖的方法扩展我们以前研究的范围。本实验将验证单细胞激光捕获显微镜结合定量实时PCR分析的假设：A.衰老和阿尔茨海默病的BFCN会显示CaMKI和环AMP反应元件结合蛋白（CREB）基因表达减少，促凋亡基因表达增加和/或抗凋亡基因表达减少；B.上述基因表达的改变主要发生在cb阴性BFCN中；C.利用小抑制RNA降低小鼠BFCN中CaMKI的表达，会导致凋亡相关基因的类似改变；D.使用腺相关病毒载体增加CB表达会导致CaMKI的上调。该结果将对老化和神经系统疾病中BFCN的选择性消耗具有重要意义。它们也将构成未来旨在确定上述基因改变的原因和影响的更全面研究的基础。

DESCRIPTION (provided by applicant): The basal forebrain cholinergic neuronal (BFCN) system is intimately involved in the processes of learning, memory and attention. Within the human brain, the BFCN are selectively vulnerable in neurodegenerative diseases that afflict the elderly, such as Alzheimer's disease (AD). Recent preliminary observations from our laboratory indicate that the BFCN in AD display decreased expression of the gene for calmodulin kinase I (CaMKI) and increased immunoreactivity for the apoptotic protein Fas-associated death domain (FADD). It has been shown that CaMKI downregulation causes apoptosis in neurons. Thus, the downregulation of CaMKI expression in AD BFCN may be associated with apoptosis in these neurons. We have also observed that the BFCN display a substantial loss of the calcium binding protein calbindin-D28K (CB) in the course of normal aging in the human and a further decrease in Alzheimer's disease and that the BFCN which degenerate in Alzheimer's disease appear to be those which lose this protein in the course of normal aging. We hypothesize that downregulation of CaMKI in aging and AD BFCN results in upregulation of pro-apoptotic and / or downregulation of anti- apoptotic genes, and that changes in the expression of the above genes may be related to the age-related loss of CB, which is the earliest change observed in these neurons and may result in calcium dysregulation. The experiments in this proposal are intended to expand the scope of our previous studies using more novel methodologies. The proposed experiments will test the hypotheses that using single cell laser capture microscopy combined with quantitative real time PCR analysis : A. The BFCN in aging and Alzheimer's disease will display reduced expression of genes for CaMKI and the cyclic AMP response element binding protein (CREB), increased expression of pro-apoptotic genes and / or decreased expression of anti-apoptotic genes; B. altered expression of the above genes will occur primarily in CB-negative BFCN; C. decreased expression of CaMKI in cultured mouse BFCN using small inhibitory RNA will result in similar alterations in apoptosis- related genes; and D. increased expression of CB using adeno-associated viral vectors will result in upregulation of CaMKI. The results will have important implications for selective depletion of BFCN in aging and neurological diseases. They will also form the basis of more comprehensive future studies aimed at determining the causes and effects of the alterations of the above genes.