Characterization of Non-motor Phenotypes in Parkinson's Disease

1.建立了在中脑多巴胺能神经元选择性表达α-突触核蛋白转基因小鼠的新品系。 由于缺乏基因工程小鼠模型，导致中脑黑质纹状体DA神经元出现帕金森病样进行性变性，阻碍了帕金森病研究的进展。几个小组设计了过表达WT、A53T和A30Pα-突触核蛋白的转基因小鼠，但这些小鼠中脑DA神经元都没有明显的丢失，这可能是由于转基因在黑质纹状体DA神经元中表达较弱所致。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，在该模型中，α-突触核蛋白被设计为在中脑DA神经元中高表达。为了实现这一目标，我们开发了PITX3启动子控制下的TTA转基因小鼠新品系。PITX3，即垂体同源盒3，选择性表达于中脑DA神经元，包括黑质致密部(SNC)和腹侧被盖区(VTA)的DA神经元。PITX3从胚胎12.5天开始表达，并持续到成年期。因此，我们对这一项目的具体目标是： 目的：建立和鉴定PITX3启动子控制下的条件性α-突触核蛋白WT和A53T转基因小鼠； 目的：探讨α-突触核蛋白A53T突变导致中脑DA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神经元中选择性地表达α-突触核蛋白A53T突变，我们通过靶DNA重组的方法将四环素反式激活因子(TTA)编码序列插入到PITX3基因位点，从而获得了一个新的四环素反式激活因子(TTA)小鼠品系。将TTA基因插入到PITX3基因的最后一个编码外显子中，在PITX3基因的终止密码子和TTA基因的起始密码子之间插入一份内部核糖体进入位点(IRES)序列。这种基因修饰将导致PITX3和TTA双顺反子信使RNA在PITX3基因座控制下的转录。我们已经成功培育了两个PITX3-IRES-TTA敲入小鼠系。为了确定TTA在这些动物中的表达模式，我们将PITX3-IRES-TTA小鼠与四环素操纵子控制的GFP转基因小鼠TG(Teto-HIST1H2BJ/GFP)47Efu/J杂交，正如预期的那样，GFP在双转基因小鼠中的表达仅限于中脑DA神经元。然后，我们将A53T小鼠与PITX3-IRES-TTA小鼠杂交，选择性地在中脑DA神经元中表达这种与PD相关的显性突变。PITX3-IRES-TTA/A53T双转基因小鼠个头较小，1月龄时饲养能力下降，步态异常。按照最初的设计，转基因只在中脑DA神经元中表达，这种表达是由一种针对人α-突触核蛋白的抗体揭示的。人α-突触核蛋白免疫反应在中脑DA神经元胞体及其轴突终末纹状体和脑内其他区域观察到。 已有研究表明黑质纹状体DA神经元功能障碍在帕金森病的发病机制中起关键作用。然而，PD相关基因突变如何影响黑质纹状体DA神经元的功能和存活还没有得到很好的研究。在这里，我们成功地开发了新的转基因小鼠系，它们选择性地在中脑DA神经元中表达与PD相关的显性基因突变。我们的初步数据表明，早在1月龄时，中脑DA神经元中α-突触核蛋白A53T突变的过度表达就导致了运动活动的显着降低和明显的步态异常。1月龄时DA神经元未见明显变性，提示这些神经元调节运动活动的功能可能发生了改变。我们将继续监测不同年龄PITX3-IRES-TTA/A53T双转基因小鼠的运动行为和病理变化。我们还将对这些小鼠的多巴胺传递的潜在缺陷进行神经化学和电生理学研究。对原代培养的中脑DA神经元进行基因表达谱和细胞生物学研究，将有助于明确导致DA神经元功能障碍和变性的分子和细胞途径。 2.将α-突触核蛋白转基因小鼠存放在JAX中以供公众使用。 JAX鼠标数据库012450 C57BL/6J-TG(日语)1CAI/J JAX鼠标数据库012442股票TG(TETO-SNCA*A53T)E2Cai/J 3.葛兰素史克研发人员中国获得PITX3/A53T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.