Establishment of inducible astrocyte specific p38 MAPK knockout mouse line

诱导型星形胶质细胞特异性p38 MAPK敲除小鼠系的建立

• 批准号: 8822444
• 负责人: SHAOHUA YANG
• 金额: $ 18.25万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8822444
• 关键词: Alzheimer' s Disease; Area; Astrocytes; Attenuated; Biological Process; Brain; Breeding; Cell physiology; Central Nervous System Diseases; Cicatrix; Complement Factor B; Down-Regulation; Elements; Embryo; Exposure to; Filament; Gene Expression; Genes; Genetic Recombination; Glial Fibrillary Acidic Protein; Hypertrophy; Hypoxia; Immune response; In Vitro; Infarction; Inflammation; Inflammatory; Inflammatory Response; Ischemic Stroke; Knock-out; Knockout Mice; Laboratories; MAP Kinase Gene; MAPK14 gene; Mediating; Mental Depression; Microglia; Middle Cerebral Artery Occlusion; Modeling; Molecular; Multiple Sclerosis; Mus; Neuroglia; Neurons; Nuclear; Parkinson Disease; Peripheral; Play; Process; Proteins; Recovery; Recovery of Function; Research; Role; STAT protein; Signal Pathway; Signal Transduction; Spinal cord injury; Stress; Stroke; Tamoxifen; Transgenes; Transgenic Mice; Traumatic Brain Injury; Up-Regulation; astrogliosis; attenuation; axon regeneration; axonal sprouting; cytokine; cytotoxic; in vivo; nerve stem cell; nervous system disorder; neural precursor cell; novel; painful neuropathy; promoter; protein expression; public health relevance; response; subventricular zone; synaptogenesis

DESCRIPTION (provided by applicant): Astrocytes respond to all forms of CNS insult by a process commonly referred as reactive astrogliosis characterized as hypertrophy of cellular processes and upregulation of intermediated filament proteins such as glial fibrillary acidic protein (GFAP). Compelling evidence has indicated that reactive astrogliosis is not simply an all-or-none phenomenon but, rather, a finely tuned continuum of molecular, cellular, and functional changes that range from subtle alternations in gene expression to scar formation. Recent research findings have also suggested that reactive astrogliosis may exert both beneficial and detrimental effects in a temporal dependent manner regulated by specific molecular signaling cascades. Increasing evidence has indicated that p38 plays a role in many other biological functions at the CNS. The activation of p38 signaling releases pro-inflammatory cytokines that are known to be involved in various CNS disorders including ischemic stroke, Alzheimer's disease, Parkinson's disease, multiple sclerosis, neuropathic pain and depression. The role of p38 MAPK in peripheral immune and inflammatory response has been extensive studied. However, the action of p38 in astrogliosis, the major component of neuroinflammatory response in CNS, has largely unknown. Our preliminary study demonstrated an activation of p38 signaling in reactive astrogliosis in the glial scar area in a mouse ischemic stroke model. Our laboratory has established a novel transgenic mouse line (hGFAP Cre/p38 loxP) in which the hGFAP promoter-driven Cre transgene deletes the floxed p38�ene and a downregulation of GFAP expression was found in primary astrocytes derived from the transgenic mice. Consistently, attenuation of reactive astrogliosis was observed upon p38 inhibition in primary astrocyte cultures. Reduction of ischemic stroke induced astrogliosis was observed in the hGFAP cre / p38 loxp mice as compared with wild type littermates. As Cre-mediated recombination is irreversible, embryonic expression in neural precursor cells directed by the GFAP promoter have resulted in substantial Cre-mediated recombination in mature neurons as well as glial cell in all GFAP-Cre mice characterized to date. In addition, the constitutive p38 knockout will not be able to study the role of p38 in astrogliosis in a temporal dependent manner. Therefore, there is a clear need of an inducible astrocyte specific p38 knockout mouse line. In this application, we proposed to establish an inducible astrocyte specific p38 knockout mouse line, characterize the inducible gene recombination and p38 knockout in the established transgenic mouse line, and determine the spatial and temporal dependent action of p38 knockout on glial scar formation and functional recovery after ischemic stroke. Specific Aim 1 is to establish the inducible astrocyte specific p38 knockout, hGFAP Cre-ERT2 / p38 loxP, mouse line. Specific Aim 2 is to determine the spatial and temporal dependent action of p38 knockout on glial scar formation and functional recovery after ischemic stroke using the established hGFAP Cre-ERT2 / p38 loxP mouse line. We predict that, in the established hGFAP Cre-ERT2 / p38 loxP mouse line, tamoxifen inducible gene recombination will be achieved predominantly in astrocyte with a minimal in the neural stem cell at subventricular zone. We expect that induction of p38 MAPK knockout in astrocyte after ischemic stroke attenuates glial scar formation, ameliorates the inhibitory action of glial scar on axonal sprouting and synaptogenesis, and enhances functional recovery.

描述(申请人提供)：星形胶质细胞对所有形式的中枢神经系统损伤的反应，通常被称为反应性星形胶质细胞增生症，其特征是细胞突起肥大和中间丝蛋白上调，如胶质纤维酸性蛋白(GFAP)。令人信服的证据表明，反应性星形胶质细胞增生症不是一个简单的要么全有要么全无的现象，而是分子、细胞和功能变化的微调连续体，范围从基因表达的细微变化到疤痕的形成。最近的研究结果还表明，反应性星形胶质细胞增生症可能在特定的分子信号级联调控下，以时间依赖的方式发挥有益和有害的影响。越来越多的证据表明，p38在中枢神经系统的许多其他生物学功能中发挥着作用。P38信号的激活释放了促炎细胞因子，已知这些细胞因子参与了多种中枢神经系统疾病，包括缺血性中风、阿尔茨海默病、帕金森病、多发性硬化症、神经病理性疼痛和抑郁症。P38MAPK在外周免疫和炎症反应中的作用已被广泛研究。然而，作为中枢神经系统炎症反应的主要成分，p38在星形胶质细胞增生症中的作用在很大程度上还不清楚。我们的初步研究表明，在小鼠缺血性中风模型中，p38信号在胶质瘢痕区域的反应性星形胶质细胞增生中激活。我们实验室建立了一个新的转基因小鼠系(hGFAP Cre/p38loxP)，在该品系中，由hGFAP启动子驱动的Cre转基因缺失了p38�基因，并在转基因小鼠的原代星形胶质细胞中发现了GFAP表达的下调。在原代培养的星形胶质细胞中，通过抑制p38蛋白表达，可以观察到反应性星形胶质细胞增生的减弱。与野生型相比，hGFAP cre/p38loxP小鼠缺血性卒中诱导的星形胶质细胞增生减少。由于Cre介导的重组是不可逆的，在GFAP启动子引导的神经前体细胞中的胚胎表达已导致所有GFAP-Cre小鼠成熟神经元和神经胶质细胞中大量Cre介导的重组。此外，结构性的p38基因敲除将不能以时间依赖的方式研究p38在星形胶质细胞增生症中的作用。因此，显然需要一种可诱导的星形胶质细胞特异性p38基因敲除小鼠系。在这项应用中，我们建议建立一种可诱导的星形胶质细胞特异性p38基因敲除小鼠系，对所建立的转基因小鼠系中的可诱导基因重组和p38基因敲除进行表征，并确定p38基因敲除在缺血性卒中后胶质瘢痕形成和功能恢复中的时空依赖性作用。目的1建立可诱导的星形胶质细胞特异性p38基因敲除小鼠系hGFAP CRE-ERT2/p38loxP。具体目的2是利用已建立的hGFAP Cre-ERT2/p38loxP小鼠系，研究p38基因敲除在缺血性卒中后胶质瘢痕形成和功能恢复中的时空依赖性作用。我们预测，在已建立的hGFAP CRE-ERT2/p38loxP小鼠系中，他莫昔芬诱导的基因重组将主要在星形胶质细胞中实现，在脑室下区的神经干细胞中很少。我们期望在缺血性卒中后诱导星形胶质细胞p38MAPK基因敲除可以减少胶质瘢痕的形成，改善胶质瘢痕对轴突萌发和突触形成的抑制作用，促进功能恢复。