Calcineurin and Biobehavioral Markers of Brain Aging

描述（由申请人提供）：我们之前的工作表明，老年大鼠海马中蛋白磷酸酶活性增加，并有助于突触强度和认知的年龄相关改变。拟议的研究调查蛋白磷酸酶钙调磷酸酶是否与其他几种脑衰老生物标志物显著相互作用，包括增加的Ca2+通道功能和改变的基因表达。在本项目的每个目标中，使用重组病毒（腺病毒和慢病毒）操纵海马体培养物和完整大鼠海马体中的钙调磷酸酶活性。第一个特定目的是测试组成型活性钙调磷酸酶是否引起电压敏感Ca2+通道（VSCC）的衰老样变化（即l型VSCC电流增加和n型VSCC mRNA水平降低）。钙调磷酸酶与VSCCs的分子相互作用也将被探讨。第二个目的是测试钙调磷酸酶活性的增加是否会导致整体基因表达的衰老样变化，如使用基因微阵列进行评估。钙调磷酸酶/NF-AT转录途径的具体作用将利用含有有效的NFAT抑制剂VIVIT的重组病毒进行探索。第三个目的是使用重组病毒来测试钙调神经磷酸酶/NF-AT通路是否有助于莫里斯游泳任务中与年龄相关的记忆缺陷。完成行为训练后，将从这些大鼠身上制备完整的和部分分离的海马切片，以确定钙调神经磷酸酶/NF-AT通路是否也部分负责突触强度、可塑性（即长期增强和长期抑制）和l型VSCC活性的年龄相关改变。此外，单个神经元和胶质细胞将从拉链切片中收获，使用基因微阵列技术检查单细胞基因表达谱。

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.