Cell Specific RIPK3 signaling after traumatic brain injury in mice

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， 坏死和发炎。RIPKs通过RIPK1-RIPK3-MLKL核小体介导程序性坏死 装配，以及通过涉及FliP、caspase-8、Tradd和RIPK3等核糖体的凋亡。 RIPK1抑制剂目前正在对几种系统性疾病进行人体临床试验。相比之下，RIPK3拥有 研究较少，部分原因是临床上还没有可接受的RIPK3抑制剂。建立在我们的 先前的研究表明，缺乏肿瘤坏死因子和Fas受体的小鼠的神经结局有所改善(上游 RIPK3的激活剂)，我们使用遗传工具来询问RIPK3在受控小鼠中可能扮演的角色 皮质冲击(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，RIPK1激酶死亡或MLKL基因敲除小鼠的保护作用更强，这表明 RIPK3的独特作用-不依赖于坏死性下垂，因为急性神经细胞死亡或损伤体积不是 被RIPK3基因敲除。使用免疫泛素来分离特定的脑细胞群，我们发现 RIPK3在内皮细胞和免疫细胞中的表达水平最高。RIPK3 KO小鼠HMGB1降低 CCI后释放，减少脑组织和内皮细胞中IL-1β的处理，并维持 K48泛素化神经元蛋白和脑内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变的神经退变 疾病。我们假设内皮细胞和免疫细胞中的RIPK3信号诱导神经 脑挫裂伤后多条急性炎症通路的激活及其潜在的功能障碍 改善结果的治疗目标。为了验证这一假设，我们提出了三个具体目标：目标1， 确定CCI后特定脑细胞类型中RIPK3激活的上游调节机制 免疫泛素和流式细胞术分离特定脑细胞类型；目标2，定义细胞特定的功能角色 利用骨髓嵌合体和可诱导/条件性RIPK3在脑内与外周免疫细胞中的RIPK3 和目标3，以确定RIPK3下游可能有助于 颅脑损伤后慢性期的神经变性。

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.