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: 8920175
• 负责人: SHAOHUA YANG
• 金额: $ 21.9万
• 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 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8920175
• 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; Health; 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; 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（由申请方提供）：星形胶质细胞通过通常称为反应性星形胶质细胞增生的过程对所有形式的CNS损伤作出反应，其特征为细胞突起肥大和中间丝蛋白（如胶质细胞酸性蛋白（GFAP））上调。令人信服的证据表明，反应性星形胶质细胞增生不是一个简单的全有或全无的现象，而是一个微调的连续的分子，细胞和功能的变化，从基因表达的微妙变化疤痕形成。最近的研究结果也表明，反应性星形胶质细胞增生可能发挥有益和有害的影响，在特定的分子信号级联调节的时间依赖性的方式。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作用。p38信号传导的激活释放促炎性细胞因子，其已知参与各种CNS病症，包括缺血性中风、阿尔茨海默病、帕金森病、多发性硬化、神经性疼痛和抑郁症。p38 MAPK在外周免疫和炎症反应中的作用已被广泛研究。然而，p38在星形胶质细胞增生中的作用，CNS中神经炎症反应的主要成分，在很大程度上是未知的。我们的初步研究表明，在小鼠缺血性中风模型中，在胶质瘢痕区域的反应性星形胶质细胞增生中p38信号被激活。本实验室建立了一种新的转基因小鼠系（hGFAP Cre/p38 loxP），其中hGFAP启动子驱动的Cre转基因删除了floxed p38 ′ ene，并在转基因小鼠的原代星形胶质细胞中发现GFAP表达下调。一致地，在原代星形胶质细胞培养物中，在p38抑制后观察到反应性星形胶质细胞增生减弱。与野生型同窝小鼠相比，在hGFAP cre / p38 loxp小鼠中观察到缺血性中风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，因此由GFAP启动子指导的神经前体细胞中的胚胎表达导致迄今为止表征的所有GFAP-Cre小鼠中的成熟神经元以及神经胶质细胞中的大量Cre介导的重组。此外，组成性p38基因敲除将无法研究p38在星形胶质细胞增生中的作用，在一个时间依赖性的方式。因此，显然需要诱导型星形胶质细胞特异性p38敲除小鼠系。在本申请中，我们提出建立一个诱导型星形胶质细胞特异性p38基因敲除小鼠系，表征诱导型基因重组和p38基因敲除在建立的转基因小鼠系，并确定空间和时间依赖性的作用p38基因敲除对胶质瘢痕形成和功能恢复缺血性中风后。具体目的一是建立星形胶质细胞特异性p38基因敲除小鼠hGFAP Cre-ERT 2/ p38 loxP系。具体目标2是使用已建立的hGFAP Cre-ERT 2/ p38 loxP小鼠系确定p38敲除对缺血性卒中后胶质瘢痕形成和功能恢复的空间和时间依赖性作用。我们预测，在已建立的hGFAP Cre-ERT 2/ p38 loxP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实现最小。我们预期缺血性卒中后星形胶质细胞中p38 MAPK基因敲除的诱导可减少胶质瘢痕形成，改善胶质瘢痕对轴突发芽和突触发生的抑制作用，并增强功能恢复。