DOPAMINE AND VESICULAR MONOAMINE TRANSPORTERS IN AGING

衰老过程中运动和认知功能障碍的发展可能部分与中枢多巴胺(DA)神经传递的紊乱有关。在分子水平上，DA信号受多种大分子的动态调节，包括生物合成酶、降解酶、分泌蛋白、离子通道、突触前和突触后受体和转运体。DA功能的各种异常被认为是各种神经退行性疾病、精神病理学和药物成瘾的原因。神经营养因子等多种神经因子支持DA神经元的发育和神经可塑性。这些分子的改变或任何表达和调节的受损都可能导致DA神经元的年龄相关性脆弱性。质膜多巴胺转运体(DAT)和囊泡单胺转运体(VMAT2)是正常DA所必需的 神经传递。DAT终止DA的作用并维持细胞外DA水平，而VMAT2将胞质DA加载到囊泡内，随后囊泡释放DA，并维持细胞内DA水平。细胞内或细胞外高浓度的DA和/或DA代谢物对纹状体突触前多巴胺能终末有毒性作用。因此，这两种转运蛋白功能的严密调控平衡的扰动可能使神经元容易受到各种侮辱的损害，如生长因子缺乏或在不同寿命水平暴露于环境毒素。虽然DAT mRNAs和结合位点的变化与衰老和帕金森病有关，但还没有研究与正常衰老相关的突触前DA终末转运蛋白功能、调节和亚细胞分布的相关变化。本项目的中心目标是了解DAT和VMAT2功能调节在衰老过程中的神经适应性变化，并验证DAT和VMAT2的表达可以预测DA终末选择性易损性的假设。结合最新的生化和分子方法与动物核心概述的动物模型，本项目建议系统地确定(A)与正常衰老相关的DAT和VMAT2表达和调控的进展，以及(B)在环境侮辱条件下与年龄相关的DAT和VMAT2表达和调控的变化 (C)生长因子缺乏(GDNF和BDNF杂合转基因动物)下DAT和VMAT2表达和调控的早期适应性变化，以及与衰老过程中DA神经元脆弱性的关系。为了实现这一点，许多与转运蛋白功能相关的因变量将被量化，包括1)表达，转运蛋白活性，亚细胞分布和2) 转运蛋白的磷酸化状态，PP2Ac/α-突触核蛋白-DAT结合。这些研究将有助于我们了解DA神经元在正常衰老过程中的代偿性变化，并可能为老年神经退行性疾病的药物干预寻找新的分子靶点。

The development of motor and cognitive dysfunction during aging may be, in part associated with perturbations of central dopamine (DA) neurotransmission. At the molecular level, DA signaling is dynamically regulated by a diverse set of macromolecules including biosynthetic enzymes, degradative enzymes, secretory proteins, ion channels, pre- and post synaptic receptors and transporters. Various abnormalities in DA function have been postulated as causes of various types of neurodegenerative diseases, psychopathology and drug addiction. Several neuronal factors such as neurotrophic factors support development and neuroplasticity of DA neurons. Alterations or any compromised expression and regulation of these molecules may leads to age-related vulnerability of DA neurons. The plasma membrane dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) are essential for normal DA neurotransmission. DAT terminates the action of DA and maintain the extracellular levels of DA, whereas VMAT2 loads cytoplasmic DA into to vesicles and subsequent vesicular DA release as well as maintain intracellular cytoplasmic DA levels. High concentrations of DA and/or DA-metabolites in either intracellular cytoplasmic or elevated extracellular DA are documented to be toxic to striatal presynaptic dopaminergic terminals. Thus perturbation of the tightly regulated balance of these two transporters function may predispose the neurons to damage by variety of insults, such as growth factor deficiency or exposure to environmental toxins at different levels of life-span. While changes in DAT mRNAs and binding sites have been implicated in aging and Parkinson's disease, there are no studies investigating relevant alterations in transporter function, regulation and sub-cellular distribution in pre-synaptic DA terminals that are associated with normal aging. The central goal of this project is to comprehend the neuroadaptive changes in DAT and VMAT2 functional regulation during aging and test the hypothesis that the expression of DAT and VMAT2 can predict the selective vulnerability of DA terminals. Combining state-of art biochemical and molecular approaches with the animal model outlined in animal core, this project proposes to determine systematically (a) the progression of changes in the expression and regulation DAT and VMAT2 associated with normal aging and (b) age-related alterations in the expression and regulation of DAT and VMAT2 under the conditions of environmental insults such as exposure to methamphetamine, and (c) the early adaptive changes in the expression and regulation of DAT and VMAT2 under growth factor deficiency (GDNF, and BDNF heterozygous transgenic animals) and the relationship to DA neuron vulnerability during aging processes. To accomplish this a number of dependent variables related to transporter function will be quantified, including 1) expression, transporter activity, sub-cellular distribution and 2) phosphorylation state of transporter, PP2Ac/alpha-synuclein -DAT association. These studies will aid in our understanding the compensatory changes occurring in DA neurons during normal aging process and could identify novel molecular targets for pharmacological intervention in age related neurodegenerative diseases.