Cell Cycle Pathways and Spinal Cord Injury

描述(由申请人提供)：脊髓损伤(SCI)导致神经细胞死亡，并伴有星形胶质细胞增殖和炎症，与小胶质细胞激活有关。细胞周期蛋白的上调发生在中枢神经系统(CMS)损伤后，并可能导致有丝分裂后细胞(如神经元和少突胶质细胞)的凋亡。它还可能导致创伤后胶质细胞增多症和小胶质细胞的激活。本实验室最近的研究表明，在啮齿动物脊髓损伤后，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的激活和细胞凋亡的形态特征。此外，在几种依赖caspase-3的原代神经元细胞培养的经典凋亡模型中，损伤与许多相同的细胞周期蛋白上调有关。此外，我们和其他人的研究表明，在体外和急性脑损伤后，抑制关键的细胞周期调节通路可以减少损伤诱导的细胞死亡。我们最近发现，在大鼠脊髓损伤后使用细胞周期抑制剂治疗显著减少了损伤体积和周围的神经胶质瘢痕；它还显著改善了损伤后的运动功能。本研究旨在解决以下假设：(1)SCI上调神经元、少突胶质细胞、星形胶质细胞和小胶质细胞中的关键细胞周期成分，并诱导细胞周期通路的激活；(2)这种上调和激活促进神经元和少突胶质细胞的凋亡；(3)细胞周期蛋白的上调和激活也有助于星形胶质细胞的增殖和小胶质细胞的激活以及随后相关炎症因子的释放；(4)细胞周期抑制剂的治疗具有神经保护作用，其机制包括抑制神经元和少突胶质细胞中caspase依赖的凋亡，减少神经胶质细胞的激活，减少小胶质细胞介导的炎症因子的释放，并减弱脊髓损伤诱导的免疫反应。其具体目的是证明：(1)脊髓损伤导致大鼠脊髓挫伤模型神经元和胶质细胞中关键的细胞周期相关基因/蛋白(包括c-myc、细胞周期蛋白D1、CDK4、Rb和E2F5)表达增加，细胞周期通路激活(CDKs激活和Rb磷酸化)；B.细胞周期蛋白表达/激活增加与caspase依赖的神经元和少突胶质细胞凋亡有关；C.细胞周期通路上调也与星形胶质细胞增殖有关；D.脊髓损伤后诱导细胞周期通路激活小胶质细胞并诱导相关炎症因子的释放；(2)中央(鞘内)给予两种结构不同的细胞周期抑制物(黄酮醇或罗索维汀)可减少脊髓损伤后细胞周期的激活，从而减少随后的神经元和少突胶质细胞死亡、反应性胶质增生和小胶质细胞的激活；b.中央给药(鞘内注射)可缩小损伤范围并促进脊髓损伤后的运动恢复；(3)全身给药对脊髓损伤后的功能结局和全身免疫反应有剂量依赖性的影响；b.全身给药减少脊髓损伤后细胞周期的激活，从而减少随后的神经元和少突胶质细胞死亡、反应性胶质增生和免疫激活；在治疗上，延迟全身给药可改善创伤后功能和组织学结果。

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) causes neuronal cell death combined with astroglial proliferation and inflammation associated with activation of microglia. Upregulation of cell cycle proteins occurs after central nervous system (CMS) trauma, and appears to contribute to apoptotic cell death of post-mitotic cells such as neurons and oligodendroglia. It also likely contributes to post-traumatic gliosis and microglial activation. Recent studies in our laboratory have shown significantly increased expression of many cell cycle proteins after SCI 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 upregulation of many of these same cell cycle proteins. In addition, studies by us and others indicate that inhibition of key cell cycle regulatory pathways reduces injury-induced cell death both in vitro and after acute brain injury. We recently found that treatment with a cell cycle inhibitor after SCI in rats markedly reduces lesion volumes and the surrounding glial scar; it also significantly improves motor functions following injury. The proposed studies are intended to address the following hypotheses: (1) SCI up-regulates key cell cycle constituents at both the mRNA and protein levels in neurons, oligodendroglia, astrocytes, and microglia and induces activation of the cell cycle pathways; (2) such upregulation and activation promotes apoptosis in neurons and oligodendroglia; (3) upregulation and activation of cell cycle proteins also contribute to proliferation of astrocytes as well as microglial activation and subsequent release of associated inflammatory factors; and (4) treatment with cell cycle inhibitors is neuroprotective, through mechanisms that include inhibition of caspase-dependent apoptosis in neurons and oligodendroglia, along with reduced glial activation, diminished release of microglial mediated inflammatory factors and attenuation of the SCI- induced immune response. The specific aims are to demonstrate that: (1) a. SCI causes increased expression of critical cell cycle related genes/proteins (including c-myc, cyclin D1, CDK4, Rb and E2F5) and activation of cell cycle pathways (activation of CDKs and phosphorylation of Rb) in neurons and glia in a rat spinal cord contusion model; b. increased cell cycle protein expression/activation is associated with caspase- dependent apoptosis in neurons and oligodendroglia; c. upregulation of cell cycle pathways is also associated with proliferation of astroglia; d. induction of cell cycle pathways after SCI activates microglia and induces the release of related inflammatory factors; (2) a. central (intrathecal) administration of two structurally different cell cycle inhibitors (Flavopiridol or roscovitine) decreases cell cycle activation after SCI, thereby reducing subsequent neuronal and oligodendroglial cell death, reactive gliosis and microglial activation; b. central administration of Flavopiridol or roscovitine reduces lesion size and improves motor recovery following SCI; (3) a. systemic administration of Flavopiridol has dose-dependent effects on functional outcome and the systemic immune response; b. systemic administration of Flavopiridol decreases cell cycle activation after SCI, thereby reducing subsequent neuronal and oligodendroglial cell death, reactive gliosis and immune activation; c. therapeutically relevant, delayed systemic administration of Flavopiridol improves post-traumatic function and histological outcome.