Characterization of Non-motor Phenotypes in Parkinson's Disease

帕金森病非运动表型的特征

• 批准号: 8736659
• 负责人: Huaibin Cai
• 金额: $ 16.49万
• 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/8736659
• 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.开发新的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。 由于缺乏基因工程小鼠模型来发展中脑黑质纹状体 DA 神经元的 PD 样进行性变性，PD 研究的进展受到阻碍。几个小组设计了过度表达 WT、A53T 和 A30P α-突触核蛋白的转基因小鼠，但这些小鼠均未表现出中脑 DA 神经元的明显损失，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，我们开发了受 PITX3 启动子控制的新 tTA 转基因小鼠品系。 PITX3（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 的表达从胚胎第 12.5 天开始，一直持续到成年期。因此，我们这个项目的具体目标是： 目标1：生成并表征PITX3启动子控制下的条件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 神经元中选择性表达 α-突触核蛋白 A53T 突变，我们通过靶 DNA 重组方法将 tTA 编码序列插入 PITX3 基因位点，生成了新的四环素反式激活子 (tTA) 小鼠品系。将tTA cDNA引入PITX3基因的最后一个编码外显子，并在PITX3基因的终止密码子和tTA基因的起始密码子之间插入内部核糖体进入位点(IRES)序列的拷贝。这种基因修饰将导致 PITX3 和 tTA 双顺反子信使 RNA 在 PITX3 基因位点的控制下转录。我们已成功培育出两株 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 小鼠与一系列四环素操作员控制的 GFP 转基因小鼠 Tg (tetO-HIST1H2BJ/GFP) 47Efu/J 杂交。正如预期的那样，GFP 的表达仅限于双转基因小鼠的中脑 DA 神经元。然后，我们将 α-突触核蛋白 A53T 小鼠与 PITX3-IRES-tTA 小鼠杂交，以在中脑 DA 神经元中选择性表达这种与 PD 相关的显性突变。 PITX3-IRES-tTA/A53T 双转基因小鼠体型较小，1 月龄时表现出饲养活动减少和步态异常。正如最初设计的那样，转基因仅在中脑 DA 神经元中表达，这是由人类 α-突触核蛋白特异性抗体揭示的。在中脑 DA 神经元的胞体及其终止于纹状体和大脑其他区域的轴突末端观察到人类 α-突触核蛋白免疫反应性。 既往研究表明黑质纹状体DA神经元功能障碍在PD的发病机制中发挥着关键作用。然而，PD相关基因突变如何影响体内黑质纹状体DA神经元的功能和存活尚未得到充分研究。在这里，我们成功开发了新的转基因小鼠品系，它们在中脑 DA 神经元中选择性表达与 PD 相关的显性基因突变。我们的初步数据表明，中脑 DA 神经元中 α-突触核蛋白 A53T 突变的过度表达早在 1 个月大时就导致运动活动显着下降和明显的步态异常。 1 个月大时，DA 神经元没有明显的退化，表明这些神经元在调节运动活动方面可能发生功能改变。我们将持续监测PITX3-IRES-tTA/A53T双转基因小鼠在不同年龄阶段的运动行为和病理变化进展。我们还将应用神经化学和电生理学研究来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 神经元进行基因表达阵列和细胞生物学研究将有助于确定导致 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沉积在 JAX 中以供公众访问。 JAX 小鼠数据库 012450 C57BL/6J-Tg(tetO-SNCA)1Cai/J JAX 小鼠数据库 012442 STOCK Tg(tetO-SNCA*A53T)E2Cai/J 3. GSK中国研发中心的PITX3/A53T转基因小鼠授权。