Cell Cycle Pathways and Spinal Cord Injury

脊髓损伤(SCI)引起神经细胞死亡和星形胶质细胞增殖 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 神经系统(CMS)创伤，并似乎有助于有丝分裂后细胞的凋亡死亡，如 神经元和少突胶质细胞。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。 我们实验室最近的研究表明，许多细胞周期蛋白的表达显著增加。 在啮齿动物脊髓损伤后，这些蛋白在神经元中共表达，显示caspase-3激活和 细胞凋亡的形态特征。此外，在caspase-3依赖的几个经典模型中， 原代培养神经细胞中的细胞凋亡，损伤与许多相同的细胞上调有关 循环蛋白。此外，我们和其他人的研究表明，对关键细胞周期调节的抑制 在体外和急性脑损伤后，通路均可减少损伤诱导的细胞死亡。我们最近发现， 大鼠脊髓损伤后应用细胞周期抑制剂治疗可显著减少损伤体积和周围组织 胶质疤痕；它还显著改善受伤后的运动功能。 拟议的研究旨在解决以下假设：(1)脊髓损伤上调关键细胞 神经元、少突胶质细胞、星形胶质细胞和小胶质细胞中mRNA和蛋白水平的周期成分 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 有助于星形胶质细胞的增殖以及小胶质细胞的激活和随后相关的释放 炎症因子；以及(4)细胞周期抑制剂的治疗是神经保护的，其机制是 包括抑制神经元和少突胶质细胞中依赖于caspase的凋亡，以及减少胶质细胞 小胶质细胞介导的炎症因子的激活、释放减少和脊髓损伤的减轻。 诱导免疫应答。 其具体目的是证明：(1)脊髓损伤导致关键细胞周期表达增加 相关基因/蛋白(包括c-myc、细胞周期蛋白D1、CDK4、Rb和E2F5)与细胞周期激活 大鼠脊髓神经元和神经胶质细胞中CDKs的激活和Rb的磷酸化 B.细胞周期蛋白表达/活化增加与caspase-1相关。 神经元和少突胶质细胞的依赖性凋亡；C.细胞周期通路的上调也是 与星形胶质细胞的增殖有关；D.脊髓损伤后细胞周期通路的诱导激活小胶质细胞和 诱导相关炎症因子的释放；(2)a.中央(鞘内)给药 结构上不同的细胞周期抑制剂(黄烷醇或罗索维汀)在以下情况下会降低细胞周期激活 脊髓损伤，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 激活；B.中心给药黄吡哆醇或罗索维汀可缩小病变面积并改善 脊髓损伤后的运动恢复；(3)a.全身用药具有剂量依赖效应 关于功能结局和全身免疫反应；B.全身给药 黄吡哆醇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、反应性胶质化和免疫激活；C.与治疗相关， 延迟全身给药可改善创伤后功能和组织学 结果。

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 immuneresponse. 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.