Role of Cell Cycle Pathways in Traumatic Brain Injury

描述（由申请人提供）：创伤性脑损伤（TBI）导致神经元细胞死亡，并伴有星形胶质细胞增殖和与小胶质细胞激活相关的炎症。细胞周期蛋白的上调发生在CMS创伤后，似乎有助于有丝分裂后细胞（如神经元）的凋亡细胞死亡。它也可能导致创伤后神经胶质瘤和小胶质细胞活化。我们实验室最近的研究表明，啮齿动物TBI或脊髓损伤后，许多细胞周期蛋白的表达显著增加，这些蛋白在神经元中共表达，表现出caspase-3活化和凋亡的形态学特征。此外，在原代神经细胞培养中caspase-3依赖性凋亡的几个经典模型中，损伤与许多相同的细胞周期蛋白的上调有关。此外，初步研究表明，在体外和体内，抑制关键的细胞周期调节途径可减少损伤诱导的细胞死亡。因此，大鼠脑外伤后使用细胞周期抑制剂治疗可显著减少病变体积和周围胶质瘢痕；它还能显著改善脑损伤后的运动和认知功能。提出的研究旨在解决以下假设：(1)TBI上调神经元、星形胶质细胞和小胶质细胞中关键细胞周期成分的mRNA和蛋白水平；(2)这种上调促进了神经元的凋亡和星形胶质细胞的增殖；(3)细胞周期蛋白的上调有助于小胶质细胞的激活和随后相关炎症因子的释放；(4)细胞周期抑制剂治疗具有神经保护作用，其机制包括抑制神经元内固有的caspase通路，以及减少胶质细胞激活和减少小胶质细胞介导的炎症因子的释放。具体目的是证明：(1)a.脑外伤导致一些关键细胞周期相关基因/蛋白的表达增加，包括细胞周期蛋白D1、CDK4、CDK5和Rb在神经元和胶质细胞中；B.蛋白表达增加与神经元caspase依赖性凋亡、星形胶质细胞增殖、小胶质细胞活化和促进小胶质细胞相关炎症因子的释放有关；c. cyclin D1敲除小鼠表现出较弱的损伤诱导病理生物学，包括神经元凋亡、脑损伤、星形胶质瘢痕形成、小胶质相关炎症因子释放和创伤后神经功能缺损；(2) a.结构上不同的细胞周期抑制剂以剂量依赖的方式减少了大鼠和小鼠两种病理生物学上不同的TBI模型的病变体积，改善了认知和运动功能；b.细胞周期抑制剂降低脑外伤后的细胞周期激活，从而减少随后的神经元细胞死亡、反应性胶质瘤和小胶质细胞活化；C.延迟全身性给药细胞周期抑制剂，一个更临床相关的范例，是神经保护。

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) causes neuronal cell death combined with astroglial proliferation and inflammation associated with activation of microglia. Upregulation of cell cycle proteins occurs after CMS trauma, and appears to contribute to apoptotic cell death of post-mitotic cells such as neurons. It also likely contributes to posttraumatic gliosis and microglial activation. Recent studies in our laboratory have shown significantly increased expression of many cell cycle proteins after TBI or spinal cord injury in rodents, with the proteins co-expressed in neurons showing caspase-3 activation and morphological features of apoptosis. Moreover, in several classical models of caspase-3 dependent apoptosis in primary neuronal cell cultures, injury is associated with up-regulation of many of these same cell cycle proteins. In addition, pilot studies have indicated that inhibition of key cell cycle regulatory pathways reduces injury-induced cell death both in vitro and in vivo. Thus, treatment with a cell cycle inhibitor after TBI in rats markedly reduces lesion volumes and the surrounding glial scar; it also significantly improves motor and cognitive functions following brain injury. The proposed studies are intended to address the following hypotheses: (1) TBI up-regulates key cell cycle constituents at both the mRNA and protein levels in neurons, astrocytes, and microglia; (2) such an up-regulation promotes apoptosis in neurons and proliferation of astrocytes; (3) up-regulation of cell cycle proteins contributes to microglial activation and subsequent release of associated inflammatory factors; and (4) treatment with cell cycle inhibitors is neuroprotective, through mechanisms that include inhibition of the intrinsic caspase pathway in neurons, as well as reduced glial activation and diminished release of microglial mediated inflammatory factors. Specific aims are to demonstrate that: (1) a. TBI causes increased expression of a number of critical cell cycle related genes/proteins, including cyclin D1, CDK4, CDK5 and Rb in both neurons and glia; b. increased protein expression is associated with caspase-dependent apoptosis in neurons, proliferation of astroglia, activation of microglia and facilitated release of microglia-related inflammatory factors; c. cyclin D1 knockout mice show less intense injury-induced pathobiology including neuronal apoptosis, brain lesion, astroglial scar formation, release of microglial associated inflammatory factors, and post-traumatic neurological deficits; (2) a. structurally different cell cycle inhibitors in dose-dependent manner reduce lesion volumes and improve cognitive as well as motor function in two pathobiologically different TBI models in the rat and mouse; b. cell cycle inhibitors decrease cell cycle activation after TBI, thereby reducing subsequent neuronal cell death, reactive gliosis and microglial activation; c. delayed systemic administration of a cell cycle inhibitor, a more clinically relevant paradigm, is neuroprotective.