Characterization of Non-motor Phenotypes in Parkinson's Disease

帕金森病非运动表型的特征

• 批准号: 8552521
• 负责人: Huaibin Cai
• 金额: $ 20.49万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552521
• 关键词: Address; Adult; Affect; Age; Age-Months; Aging; Alpha-Synuclein transgenic mouse; Animals; Antibodies; Anxiety; Area; Attention; Behavioral; Bradykinesia; Brain; Cellular biology; China; Clinical; Code; Cognition Disorders; Cognitive; Complementary DNA; Corpus striatum structure; Data; Databases; Deposition; Development; Disease; Embryo; Emotional; Emotional disorder; Exons; Functional disorder; Gait abnormality; Gene Expression; Gene Mutation; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genetically Engineered Mouse; Goals; Hippocampus (Brain); Homeobox; Impaired cognition; Initiator Codon; Knock-in Mouse; Lead; Licensing; Link; Messenger RNA; Midbrain structure; Modification; Molecular; Monitor; Motor; Motor Activity; Mus; Mutant Strains Mice; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathogenesis; Pathway interactions; Pattern; Phenotype; Pituitary Gland; Play; Research; Rest Tremor; Site; Substantia nigra structure; Symptoms; System; Terminator Codon; Tetracyclines; Trans-Activators; Transgenes; Transgenic Mice; Ventral Tegmental Area; alpha synuclein; cognitive function; design; disability; dopaminergic neuron; frontal lobe; in vivo; interest; mouse model; neurochemistry; neuronal cell body; novel; pars compacta; promoter; research and development; selective expression; synuclein, alpha (non A4 component of amyloid precursor) protein, human; transgene expression; transmission process

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.

1。在中脑多巴胺能神经元中有选择地表达转基因的新的α-核蛋白转基因小鼠的新系。 缺乏基因工程的小鼠模型的阻碍，这些模型会导致中脑中骨纹状体DA神经元的PD样渐进性变性。几个组设计了过表达WT，A53T和A30Pα-核蛋白的转基因小鼠，但是这些小鼠均未显示出中脑DA神经元的明显丧失，这可能是由于黑粒细胞点dA神经元中转基因表达较弱所致。为了克服这一技术挑战，我们建议开发有条件的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，我们在PITX3启动子的控制下开发了一系列TTA转基因小鼠系列。 PITX3，垂体同源物3在中脑DA神经元中有选择地表达，包括黑质Nigra pars compacta（SNC）和腹侧段区域（VTA）的DA神经元。 PITX3的表达始于胚胎第12.5天，并一直贯穿成年。因此，我们对该项目的具体目标是： 目标1：在PITX3启动子的控制下产生和表征有条件的α-突触核蛋白WT和A53T转基因小鼠； 目的2：研究α-突触核蛋白A53T突变的细胞和分子机制，导致中脑DA神经元的功能障碍和变性。 为了选择性地表达黑质纹状体DA神经元中的α-突变突变，​​我们通过通过目标DNA重组方法将TTA编码序列插入PITX3基因位点，从而生成了新的四环素反式反式激活器（TTA）小鼠。将TTA cDNA引入了PITX3基因的最后一个编码外显子中，并将内部核糖体入口位点（IRES）序列的副本插入PITX3基因的终止密码子和TTA基因的起始密码子之间。这种遗传修饰将导致PITX3基因基因座的控制下PITX3和TTA Bicistronic Messenger RNA的转录。我们已经成功地开发了两条PITX3-IRES-TTA敲入小鼠。为了确定这些动物中TTA的表达模式，我们将PITX3-IRES-TTA小鼠与四环素算子控制的GFP转基因小鼠TG（TETO-HISTO-HIST1H2BJ/GFP）47EFU/J。不出所料，在双转基因小鼠中，GFP的表达仅限于中脑DA神经元。然后，我们用PITX3-IRES-TTA小鼠越过α-突触核蛋白A53T小鼠，在中脑DA神经元中有选择地表达这种PD连接的显性突变。 PITX3-IRES-TTA/A53T双转基因小鼠较小，饲养活动降低以及1个月大时的步态异常。正如最初设计的那样，转基因仅由中脑DA神经元表达，该神经元被人α-突触核蛋白的抗体揭示。在中脑DA神经元的体内及其轴突末端，在纹状体和大脑的其他区域末端观察到人α-突触核蛋白免疫反应性。 先前的研究表明，骨纹状体DA神经元功能障碍在PD发病机理中起关键作用。然而，对PD相关的遗传突变如何影响体内骨纹状体DA神经元的功能和存活。在这里，我们成功地开发了新型的转基因小鼠系，这些小鼠有选择地表达中脑DA神经元中的PD连接的主要基因突变。我们的初步数据表明，中脑DA神经元中α-核蛋白A53T突变的过表达导致运动活动的显着降低和最早在1个月大时的步态异常。在1个月大时，DA神经元没有明显的变性，表明这些神经元在调节运动活动时可能会发生功能改变。我们将继续监测不同年龄段的Pitx3-IRES-TTA/A53T双转基因小鼠的运动行为和病理变化的进展。我们还将对这些小鼠多巴胺传播的潜在缺乏应用神经化学和电生理研究。基因表达阵列和细胞生物学研究对原发性培养的中脑DA神经元的研究将有助于定义分子和细胞途径，导致DA神经元的功能障碍和变性。 2。在JAX中的α-突触核蛋白转基因小鼠的沉积，以供公众进入。 JAX鼠标数据库012450 C57BL/6J-TG（TETO-SNCA）1CAI/J JAX鼠标数据库012442股票TG（TETO-SNCA*A53T）E2CAI/J 3。gsk R＆D中国的PITX3/A53T转基因小鼠的许可。