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，伴有与小胶质细胞活化相关的星形胶质细胞增殖和炎症。细胞周期蛋白的上调发生在CMS创伤后，并且似乎有助于有丝分裂后细胞如神经元的凋亡性细胞死亡。它也可能有助于创伤后神经胶质增生和小胶质细胞活化。本实验室最近的研究表明，啮齿类动物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激活和凋亡的形态学特征。此外，在原代神经元细胞培养物中caspase-3依赖性凋亡的几种经典模型中，损伤与许多这些相同细胞周期蛋白的上调相关。此外，初步研究表明，抑制关键的细胞周期调控途径减少损伤诱导的细胞死亡在体外和体内。因此，在大鼠TBI后用细胞周期抑制剂治疗显著减少了病变体积和周围的胶质瘢痕;它还显著改善了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在神经元、星形胶质细胞和小胶质细胞中在mRNA和蛋白水平上调关键细胞周期组分;（2）这种上调促进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活化和随后相关炎症因子的释放;和（4）用细胞周期抑制剂治疗是神经保护性的，其机制包括抑制神经元中的内在半胱天冬酶途径，以及减少神经胶质活化和减少小神经胶质介导的炎性因子的释放。具体目标是证明：（1）a。TBI引起神经元和神经胶质中的许多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、CDK 4、CDK 5和Rb; B.增加的蛋白质表达与神经元中的半胱天冬酶依赖性凋亡、星形胶质细胞的增殖、小胶质细胞的活化和小胶质细胞相关炎性因子的促进释放相关; c.细胞周期蛋白D1敲除小鼠显示出较低强度的损伤诱导的病理生物学，包括神经元凋亡、脑损伤、星形胶质细胞瘢痕形成、小胶质细胞相关炎性因子的释放和创伤后神经功能缺损;（2）a.在大鼠和小鼠的两种病理生物学上不同的TBI模型中，结构上不同的细胞周期抑制剂以剂量依赖性方式减少损伤体积并改善认知和运动功能; B.细胞周期抑制剂减少TBI后的细胞周期活化，从而减少随后的神经元细胞死亡、反应性神经胶质增生和小神经胶质活化; c.细胞周期抑制剂的延迟全身给药是一种更临床相关的范例，具有神经保护作用。

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.