Calcineurin and Biobehavioral Markers of Brain Aging

描述(申请人提供)：我们先前的工作表明，老年大鼠海马区蛋白磷酸酶活性增加，并有助于与年龄相关的突触强度和认知能力的改变。这项拟议的研究调查了蛋白磷酸酶钙调神经磷酸酶是否与其他几个脑老化生物标志物显著相互作用，包括钙通道功能增强和基因表达改变。在这个项目的每个目标中，利用重组病毒(腺病毒和慢病毒)操纵在正常大鼠的海马区和海马区培养的钙调神经磷酸酶的活性。第一个特定目的是检测钙调神经磷酸酶是否引起电压敏感钙通道的增龄性变化(即L型钙通道电流增加和N型钙通道基因表达水平降低)。此外，还将探讨钙调神经磷酸酶与血管干细胞的分子相互作用。第二个目的是测试钙调神经磷酸酶活性的增加是否会导致全球基因表达的类似衰老的变化，这是通过基因芯片进行评估的。钙调神经磷酸酶/核因子-AT转录途径的具体作用将使用含有有效的NFAT抑制剂Vivit的重组病毒来探索。第三个目的是使用重组病毒来测试钙调神经磷酸酶/核因子-AT途径是否与Morris游泳任务中年龄相关的记忆缺陷有关。在完成行为训练后，将从这些大鼠制备完整的和部分分离的海马片，以确定钙调神经磷酸酶/核因子-AT通路是否也与年龄相关的突触强度、可塑性(即长期增强和长期抑制)以及L型血管干细胞活性的改变有关。此外，将从拉链切片中提取单个神经元和胶质细胞，利用基因微阵列技术检测单细胞基因表达谱。

DESCRIPTION (provided by applicant): Our previous work indicates that protein phosphatase activity is increased in hippocampus of aged rats and contributes to age-related alterations in synaptic strength and cognition. The proposed studies investigate whether the protein phosphatase calcineurin interacts significantly with several other brain aging biomarkers, including increased Ca2+ channel function and altered gene expression. In each aim of this project, calcineurin activity in hippocampal cultures and in hippocampus of intact rats, is manipulated using recombinant viruses (adeno- and lentivirus). The first specific aim tests whether constitutively active calcineurin causes aging-like changes in voltage sensitive Ca2+ channels (VSCC) (i.e. an increase in L-type VSCC currents and a decrease in N-type VSCC mRNA levels). Molecular interactions of calcineurin with VSCCs also will be explored. The second aim tests whether increased calcineurin activity leads to aging-like changes in global gene expression, as assessed using gene microarrays. The specific role of the calcineurin/NF-AT transcriptional pathway will be explored using recombinant virus containing a potent NFAT inhibitor, VIVIT. The third aim uses recombinant viruses to test whether the calcineurin/NF-AT pathway contributes to age-related memory deficits on the Morris swim task. After completion of behavioral training, intact and partially dissociated hippocampal slices will be prepared from these rats to determine whether the calcineurin/NF-AT pathway also is responsible, in part, for age-related alterations in synaptic strength, plasticity (i.e. long-term potentiation and long-term depression), and L-type VSCC activity. Furthermore, individual neurons and glia will be harvested from zipper slices to examine single cell gene expression profiles using gene microarray technology.