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Cell Specific RIPK3 signaling after traumatic brain injury in mice

小鼠脑外伤后细胞特异性 RIPK3 信号转导

基本信息

批准号：
10377444
负责人：
MICHAEL J WHALEN
金额：
$ 42.43万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10377444
关键词：
Acute Aging Apoptosis Astrocytes Behavior Bone Marrow Brain Brain Injuries CASP8 gene CD95 Antigens Cell Death Cell Fraction Cells Cessation of life Chimera organism Chronic Chronic Phase Clinical Clinical Trials Cognitive deficits Cortical Contusions Cre lox recombination system Data Detergents Disease Down-Regulation Endothelial Cells Endothelium Etiology Event Experimental Models Fluorescence-Activated Cell Sorting Gene Expression Genetic Goals HMGB1 gene Hippocampus (Brain)Histology Human Immune Immunoprecipitation Impairment Inflammasome Inflammation Inflammation Mediators Inflammatory Injury Interleukin-1 beta Knock-out Knockout Mice Lead Lesion Link MAP Kinase Gene MAP3K7 gene Mediating Methodology Microglia Modeling Molecular Motor Mus Necrosis Nerve Degeneration Neurodegenerative Disorders Neurologic Dysfunctions Neurological outcome Neuronal Injury Neurons Outcome Pathway interactions Peripheral Phosphorylation Phosphotransferases Population Protein Kinase Protein-Serine-Threonine Kinases Proteins Publishing Quality of life RIPK1 gene RIPK3 gene Reagent Regulation Resolution Role Signal Transduction Survivors Systemic disease TBI Patients TBK1 gene TLR4 gene TNF gene TNFRSF1A gene TRADD gene Tamoxifen Testing Traumatic Brain Injury Tumor Necrosis Factor Receptor Tumor-infiltrating immune cells Ubiquitination Up-Regulation Western Blotting Work age related neurodegeneration antagonist brain cell brain tissue cell type cognitive function controlled cortical impact experimental study functional outcomes genetic regulatory protein grasp improved improved outcome inhibitor injured macrophage morris water maze motor deficit multicatalytic endopeptidase complex neuron loss new therapeutic target novel object recognition p38 Mitogen Activated Protein Kinase proteostasis response stroke model therapeutic target tool

项目摘要

Receptor interacting protein kinases (RIPK)-1 and -3 are serine-threonine kinases that regulate apoptosis, necrosis, and inflammation. RIPKs mediate programmed necrosis via RIPK1-RIPK3-MLKL necrosome assembly, and apoptosis via the ripoptosome involving FLIP, caspase-8, TRADD, and RIPK3, among others. RIPK1 inhibitors are currently in human clinical trials for several systemic diseases. In contrast, RIPK3 has been less studied in part because clinically acceptable RIPK3 inhibitors are not yet available. Building on our prior studies showing improved neurological outcome in mice deficient in TNF and Fas receptor (upstream activators of RIPK3), we used genetic tools to interrogate a possible role for RIPK3 in a mouse controlled cortical impact (CCI) model. Mice lacking RIPK3 had improved motor and cognitive function after CCI, and greater protection in cognitive function tests than RIPK1 kinase dead or MLKL knockout mice, suggesting a unique role for RIPK3- independent of necroptosis because acute neuronal cell death or lesion volume was not reduced by RIPK3 knockout. Using immunopanning to isolate specific brain cell populations, we found the highest levels of RIPK3 expression in endothelium and immune cells. RIPK3 KO mice had reduced HMGB1 release after CCI, reduced interleukin-1 beta processing in brain tissue and endothelial cells, and maintained K48 ubiquitination of neuronal proteins and brain TBK1 levels- mechanisms that have previously been shown to modulate outcome after CCI and contribute to neurodegeneration in aging-related neurodegenerative diseases. We hypothesize that RIPK3 signaling in endothelial and immune cells induces neurological dysfunction after cerebral contusion by activating multiple acute inflammation pathways and is a potential therapeutic target to improve outcome. To test this hypothesis we propose three specific aims: Aim 1, to identify upstream mechanisms regulating RIPK3 activation in specific brain cell types after CCI in mice, using immunopanning and FACS to isolate specific brain cell types; Aim 2, to define cell-specific functional roles for RIPK3 in brain vs. peripheral immune cells using bone marrow chimeras and inducible/conditional RIPK3 knockout mice; and Aim 3, to identify mechanisms downstream of RIPK3 that might contribute to neurodegeneration in the chronic period after TBI.

受体相互作用蛋白激酶 (RIPK)-1 和 -3 是调节细胞凋亡的丝氨酸-苏氨酸激酶，坏死、炎症。 RIPK 通过 RIPK1-RIPK3-MLKL 坏死体介导程序性坏死通过涉及 FLIP、caspase-8、TRADD 和 RIPK3 等的核糖体进行组装和凋亡。 RIPK1 抑制剂目前正在进行针对多种系统性疾病的人体临床试验。相比之下，RIPK3 研究较少，部分原因是尚无临床可接受的 RIPK3 抑制剂。建立在我们的先前的研究表明，缺乏 TNF 和 Fas 受体（上游）的小鼠的神经系统结果得到改善 RIPK3 激活剂），我们使用遗传工具来探究 RIPK3 在小鼠控制的小鼠中的可能作用皮质影响（CCI）模型。缺乏 RIPK3 的小鼠在 CCI 后运动和认知功能得到改善，并且在认知功能测试中比 RIPK1 激酶死亡或 MLKL 敲除小鼠具有更大的保护作用，这表明 RIPK3 的独特作用-独立于坏死性凋亡，因为急性神经元细胞死亡或病变体积不受影响 RIPK3 敲除可减少。使用免疫淘选来分离特定的脑细胞群，我们发现内皮细胞和免疫细胞中 RIPK3 表达水平最高。 RIPK3 KO 小鼠 HMGB1 减少 CCI 后释放，减少脑组织和内皮细胞中白细胞介素 1 β 的加工，并维持神经元蛋白的 K48 泛素化和大脑 TBK1 水平 - 先前已显示的机制调节 CCI 后的结果并促进衰老相关神经退行性疾病的神经退行性变疾病。我们假设内皮细胞和免疫细胞中的 RIPK3 信号传导可诱导神经系统损伤通过激活多种急性炎症途径来治疗脑挫裂伤后的功能障碍，是一种潜在的治疗方法治疗目标以改善结果。为了检验这一假设，我们提出了三个具体目标：目标 1，使用 CCI 确定小鼠 CCI 后特定脑细胞类型中调节 RIPK3 激活的上游机制免疫淘选和流式细胞仪分离特定的脑细胞类型；目标 2，定义细胞特定的功能角色使用骨髓嵌合体和诱导/条件 RIPK3 比较大脑与外周免疫细胞中的 RIPK3 基因敲除小鼠；目标 3，确定 RIPK3 下游可能有助于 TBI 后慢性期的神经退行性变。