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。在人类大脑内，BFCN选择性地容易患上困扰老年人的神经退行性疾病，如阿尔茨海默病(AD)。我们实验室最近的初步观察表明，BFCN在AD中表现为钙调蛋白激酶I(CaMKI)基因表达减少，而凋亡蛋白Fas相关死亡结构域(FADD)的免疫反应增强。已有研究表明，CaMKI的下调会导致神经元的凋亡。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，在人类正常衰老过程中，BFCN显示出钙结合蛋白Calbindin-D28K(CB)的大量丢失，并在阿尔茨海默病中进一步减少，而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丢失该蛋白的BFCN。我们推测，在衰老和AD BFCN中，CaMKI的下调导致促凋亡基因的上调和/或抗凋亡基因的下调，并且上述基因的表达变化可能与年龄相关的CB的丢失有关，CB是这些神经元中观察到的最早的变化，可能导致钙调节失调。这项建议中的实验旨在使用更新颖的方法扩大我们之前的研究范围。拟议的实验将验证使用单细胞激光捕获显微镜结合实时定量PCR分析的假设：a.衰老和阿尔茨海默病的BFCN将显示CaMKI和环状AMP反应元件结合蛋白(CREB)基因的表达减少，促凋亡基因的表达增加和/或抗凋亡基因的表达减少；b.上述基因的改变主要发生在CB阴性的BFCN中；c.使用小抑制RNA降低培养的小鼠BFCN中CaMKI的表达将导致与凋亡相关的基因类似的变化；用腺相关病毒载体增加CB的表达将导致CaMKI的上调。这一结果将对BFCN在衰老和神经系统疾病中的选择性耗竭具有重要意义。它们还将构成未来更全面的研究的基础，旨在确定上述基因变化的原因和影响。

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.