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）神经传递的扰动有关。在分子水平上，DA信号传导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蛋白。DA功能的各种异常已被假定为各种类型的神经退行性疾病、精神病理学和药物成瘾的原因。一些神经元因子如神经营养因子支持DA神经元的发育和神经可塑性。这些分子的改变或任何受损的表达和调节可能导致DA神经元的年龄相关的脆弱性。质膜多巴胺转运体（DAT）和囊泡单胺转运体（VMAT 2）是正常DA的必需物质 神经传递DAT终止DA的作用并维持细胞外DA水平，而VMAT 2将细胞质DA加载到囊泡中并随后囊泡DA释放以及维持细胞内细胞质DA水平。高浓度的DA和/或DA-代谢物在细胞内的细胞质或升高的细胞外DA被证明是有毒的纹状体突触前多巴胺能末梢。因此，这两种转运蛋白功能的严格调节平衡的扰动可能使神经元易于受到各种损伤的损害，例如生长因子缺乏或在不同水平的寿命中暴露于环境毒素。虽然DAT mRNA和结合位点的变化与衰老和帕金森病有关，但没有研究调查与正常衰老相关的突触前DA末端中转运蛋白功能、调节和亚细胞分布的相关改变。本项目的中心目标是了解衰老过程中DAT和VMAT 2功能调节的神经适应性变化，并验证DAT和VMAT 2的表达可以预测DA终末的选择性脆弱性的假设。本项目将最先进的生物化学和分子生物学方法与动物核心中概述的动物模型相结合，系统地确定（a）与正常衰老相关的DAT和VMAT 2的表达和调节的变化进展和（B）在环境损伤条件下DAT和VMAT 2的表达和调节的年龄相关改变 例如暴露于甲基苯丙胺，和（c）在生长因子缺乏（GDNF和BDNF杂合转基因动物）下DAT和VMAT 2的表达和调节的早期适应性变化以及在衰老过程中与DA神经元脆弱性的关系。为了实现这一点，将量化与转运蛋白功能相关的许多因变量，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，PP 2Ac/α-突触核蛋白-DAT缔合。这些研究将有助于我们了解DA神经元在正常衰老过程中发生的代偿性变化，并可能为年龄相关的神经退行性疾病的药物干预确定新的分子靶点。

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.