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， 坏死和炎症。RIPKs通过RIPK 1-RIPK 3-MLKL坏死体介导程序性坏死 组装和通过涉及FLIP、半胱天冬酶-8、TRADD和RIPK 3等的核糖体的凋亡。 RIPK 1抑制剂目前正在用于几种全身性疾病的人体临床试验中。相比之下，RIPK 3 研究较少，部分原因是临床上可接受的RIPK 3抑制剂尚未上市。充分发挥两国 先前的研究显示，在TNF和Fas受体（上游）缺陷的小鼠中， RIPK 3的激活剂），我们使用遗传工具来询问RIPK 3在小鼠对照中的可能作用。 皮质撞击（CCI）模型。缺乏RIPK 3的小鼠在CCI后运动和认知功能得到改善， 在认知功能测试中比RIPK 1激酶死亡或MLKL敲除小鼠具有更大的保护作用，这表明 RIPK 3的独特作用-不依赖于坏死性凋亡，因为急性神经元细胞死亡或病变体积不是 通过RIPK 3敲除减少。使用免疫淘选分离特定的脑细胞群，我们发现 内皮细胞和免疫细胞中RIPK 3表达水平最高。RIPK 3 KO小鼠的HMGB 1 CCI后释放，减少脑组织和内皮细胞中的白细胞介素-1 β加工，并维持 神经元蛋白的K48泛素化和脑TBK 1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中的神经退行性变 疾病我们假设内皮细胞和免疫细胞中的RIPK 3信号传导诱导神经系统疾病， 通过激活多种急性炎症通路来治疗脑挫伤后的功能障碍， 治疗目标，以改善结果。为了验证这一假设，我们提出了三个具体目标：目标1， 在小鼠CCI后，鉴定调节特定脑细胞类型中RIPK 3活化的上游机制， 免疫淘选和流式细胞仪分离特定的脑细胞类型;目的2，确定细胞特异性功能作用， 使用骨髓嵌合体和诱导型/条件性RIPK 3的脑与外周免疫细胞中的RIPK 3 基因敲除小鼠;和目标3，以确定可能有助于RIPK 3下游的机制， TBI后慢性期的神经退行性变。

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.