Characterization of Non-motor Phenotypes in Parkinson's Disease

1.开发一种新的α-突触核蛋白转基因小鼠品系，其在中脑多巴胺能神经元中选择性表达转基因。 PD研究的进展受到缺乏基因工程小鼠模型的阻碍，这些模型在中脑中发展出黑质纹状体DA神经元的PD样进行性变性。几组设计过表达WT、A53 T和A30 P α-突触核蛋白的转基因小鼠，但这些小鼠均未显示出明显的中脑DA神经元损失，这可能是由于黑质纹状体DA神经元中转基因表达较弱所致。为了克服这一技术挑战，我们建议开发一种条件性α-突触核蛋白转基因小鼠模型，其中α-突触核蛋白被设计为在中脑DA神经元中高度表达。为了实现这一目标，我们已经开发了一种新的PITX 3启动子控制下的tTA转基因小鼠系。PITX 3（pituitary homeobox 3）选择性表达于中脑DA能神经元，包括黑质腹侧被盖区（ventral tegmental area，VTA）和黑质腹侧被盖区（substantia nigra pars rectata，SNc）的DA能神经元。PITX 3的表达从胚胎第12.5天开始，一直持续到成年。因此，我们对该项目的具体目标是： 目的1：建立和鉴定PITX 3启动子控制下的条件性α-突触核蛋白WT和A53 T转基因小鼠; 目标二：目的探讨α-突触核蛋白A53 T突变导致中脑DA能神经元功能障碍和变性的细胞和分子机制。 为了在黑质纹状体DA神经元中选择性表达α-突触核蛋白A53 T突变，我们通过靶DNA重组方法将四环素反式激活因子（tTA）编码序列插入PITX 3基因位点，构建了一个新的tTA小鼠品系。将tTA cDNA插入PITX 3基因的最后一个编码外显子，并在PITX 3基因的终止密码子和tTA基因的起始密码子之间插入一个内部核糖体进入位点（IRES）序列。这种基因修饰将导致PITX 3和tTA双顺反子信使RNA在PITX 3基因座的控制下转录。我们已经成功地开发了两个品系的PITX 3-IRES-tTA基因敲入小鼠。为了确定tTA在这些动物中的表达模式，我们将PITX 3-IRES-tTA小鼠与四环素操纵子控制的GFP转基因小鼠Tg（tetO-HIST 1H 2BJ/GFP）47 Efu/J系杂交。如预期的，GFP的表达限于双转基因小鼠中的中脑DA神经元。然后，我们将α-突触核蛋白A53 T小鼠与PITX 3-IRES-tTA小鼠杂交，以在中脑DA神经元中选择性表达这种PD连锁显性突变。PITX 3-IRES-tTA/A53 T双转基因小鼠较小，在1月龄时表现出直立活动减少以及步态异常。正如最初设计的那样，转基因仅由中脑DA神经元表达，该神经元由对人α-突触核蛋白特异性的抗体揭示。在中脑DA能神经元的索马胞体及其终止于纹状体的轴突终末和脑的其它区域中观察到人α-突触核蛋白免疫反应性。 已有研究表明黑质纹状体DA能神经元的功能障碍在PD的发病机制中起重要作用。然而，PD相关的基因突变如何影响黑质纹状体DA神经元的功能和存活在体内还没有得到很好的研究。在这里，我们已经成功地开发了新的转基因小鼠，选择性地表达PD连锁显性基因突变的中脑DA神经元。我们的初步数据表明，α-突触核蛋白A53 T突变在中脑DA神经元中的过度表达导致早在1月龄的运动活动显著减少和明显的步态异常。DA神经元在1月龄时没有明显的变性，表明这些神经元在调节运动活动方面可能有功能改变。我们将继续监测PITX 3-IRES-tTA/A53 T双转基因小鼠在不同年龄段的运动行为和病理变化的进展。我们还将应用神经化学和电生理学研究这些小鼠多巴胺传递的潜在缺陷。对原代培养的中脑DA能神经元进行基因表达阵列和细胞生物学研究，将有助于阐明导致DA能神经元功能障碍和变性的分子和细胞途径。 2.在JAX中沉积α-突触核蛋白转基因小鼠供公众访问。 JAX小鼠数据库012450 C57BL/6J-Tg（tetO-SNCA）1Cai/J JAX小鼠数据库012442 库存Tg（tetO-SNCA* A53 T）E2 Cai/J 3. GSK R&D中国获得PITX 3/A53 T转基因小鼠许可。

1. Development of a new line of alpha-synuclein transgenic mice that selectively express the transgene in the midbrain dopaminergic neurons. The progress of PD research is hindered by the lack of genetically engineered mouse models that develop PD-like progressive degeneration of nigrostriatal DA neurons in the midbrain. Several groups designed transgenic mice over-expressing WT, A53T, and A30P alpha-synuclein, but none of these mice show clear loss of midbrain DA neurons, which is likely resulted from a weaker expression of transgene in the nigrostriatal DA neurons. To overcome this technical challenge, we propose to develop a conditional alpha-synuclein transgenic mouse model in which alpha-synuclein is designed to highly express in the midbrain DA neurons. To achieve this goal, we have developed a new line of tTA transgenic mice under the control of PITX3 promoter. PITX3, pituitary homeobox 3, is selectively expressed in midbrain DA neurons, including DA neurons in substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). The expression of PITX3 starts from embryonic day 12.5 and stays through adulthood. Thus, our specific aims for this project are: Aim 1: To generate and characterize conditional alpha-synuclein WT and A53T transgenic mice under the control of PITX3 promoter; Aim 2: To investigate the cellular and molecular mechanisms of alpha-synuclein A53T mutation that cause dysfunction and degeneration of midbrain DA neurons. To selectively express alpha-synuclein A53T mutation in nigrostriatal DA neurons, we have generated a new line of tetracycline transactivator (tTA) mice by inserting tTA coding sequence into the PITX3 gene locus via target DNA recombination approach. The tTA cDNA was introduced into the last coding exon of PITX3 gene and a copy of internal ribosomal entry site (IRES) sequence was inserted between the stop codon of PITX3 gene and the start codon of tTA gene. This genetic modification would lead to transcription of PITX3 and tTA bicistronic messenger RNA under the control of PITX3 gene locus. We have successfully developed two lines of PITX3-IRES-tTA knock-in mice. To determine the expression pattern of tTA in these animals, we crossed PITX3-IRES-tTA mice with a line of tetracycline operator-controlled GFP transgenic mice Tg (tetO-HIST1H2BJ/GFP) 47Efu/J. As expected, the expression of GFP was restricted to midbrain DA neurons in the double transgenic mice. We then crossed alpha-synuclein A53T mice with PITX3-IRES-tTA mice to selectively express this PD-linked dominant mutation in midbrain DA neurons. PITX3-IRES-tTA/A53T double transgenic mice were smaller and displayed decreased rearing activity as well as gait abnormalities at 1 month of age. As initially designed, the transgene was only expressed by midbrain DA neurons revealed by an antibody specific to human alpha-synuclein. The human alpha-synuclein immuno-reactivity was observed in the soma of midbrain DA neurons and at their axonal terminals ending at the striatum and other areas of the brain. Previous studies have demonstrated that dysfunction of nigrostriatal DA neurons plays a key role in the pathogenesis of PD. However, how the PD-related genetic mutations affect the function and survival of nigrostriatal DA neurons in vivo is not well studied. Here we have successfully developed novel lines of transgenic mice that selectively express PD-linked dominant genetic mutations in the midbrain DA neurons. Our preliminary data indicated that over-expression of alpha-synuclein A53T mutation in midbrain DA neurons caused a significant decrease of motor activities and obvious gait abnormalities at as early as 1 month of age. There was no apparent degeneration of DA neurons at 1 month of age, indicating a likely functional alteration of these neurons in regulating motor activities. We will keep monitoring the progression of motor behavioral and pathological changes of PITX3-IRES-tTA/A53T double transgenic mice at different ages. We will also apply neurochemical and electrophysiological studies on potential deficiency in dopamineric transmission in these mice. Gene expression array and cell biology studies on primary cultured midbrain DA neurons will help to define the molecular and cellular pathways leading to dysfunction and degeneration of DA neurons. 2. Deposition of alpha-synuclein transgenic mice in JAX for public access. JAX Mouse Database 012450 C57BL/6J-Tg(tetO-SNCA)1Cai/J JAX Mouse Database 012442 STOCK Tg(tetO-SNCA*A53T)E2Cai/J 3. license of PITX3/A53T transgenic mice by GSK R&D China.