Cell Cycle Pathways and Spinal Cord Injury

脊髓损伤（SCI）导致神经元细胞死亡，伴有星形胶质细胞增殖， 与小胶质细胞活化相关的炎症。细胞周期蛋白的上调发生在中枢神经系统 神经系统（CMS）创伤，并似乎有助于有丝分裂后细胞的凋亡性细胞死亡， 神经元和少突胶质细胞。它也可能有助于创伤后神经胶质增生和小胶质细胞活化。 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 在啮齿类动物脊髓损伤后，神经元中共表达的蛋白质显示caspase-3激活， 凋亡的形态学特征。此外，在几种经典的caspase-3依赖性模型中， 在原代神经元细胞培养物中，损伤与许多这些相同细胞的上调有关。 循环蛋白此外，我们和其他人的研究表明，抑制关键的细胞周期调节 在体外和急性脑损伤后，这些信号通路减少损伤诱导的细胞死亡。我们最近发现， 在大鼠SCI后用细胞周期抑制剂治疗显著减少了损伤体积和周围环境， 神经胶质瘢痕;它还显著改善损伤后的运动功能。 本研究拟解决以下假设：（1）SCI上调了关键细胞的表达， 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 并诱导细胞周期途径的活化;（2）这种上调和活化促进 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 促进星形胶质细胞增殖以及小胶质细胞活化和随后释放相关的 炎性因子;和（4）用细胞周期抑制剂治疗是神经保护性的，通过机制， 包括抑制神经元和少突胶质细胞中半胱天冬酶依赖性凋亡，沿着减少的胶质细胞 激活，减少小胶质细胞介导的炎症因子的释放和脊髓损伤的衰减。 诱导免疫应答。 具体的目的是证明：（1）a. SCI导致关键细胞周期表达增加 相关基因/蛋白（包括c-myc、cyclin D1、CDK 4、Rb和E2 F5）和细胞周期激活 大鼠脊髓神经元和神经胶质细胞中的CDKs活化和Rb磷酸化途径 挫伤模型; B.增加的细胞周期蛋白表达/活化与半胱天冬酶相关， 神经元和少突胶质细胞中的依赖性凋亡; c.细胞周期途径的上调也是 与星形胶质细胞增殖相关; d. SCI后细胞周期途径的诱导激活了小胶质细胞， 诱导相关炎性因子的释放;（2）a.中枢（鞘内）给药 结构不同的细胞周期抑制剂（Flavopiridol或roscovitine）降低细胞周期激活后 SCI，从而减少随后的神经元和少突胶质细胞死亡，反应性神经胶质增生和小胶质细胞 激活; B. Flavopiridol或roscovitine的中央给药减小了病变大小并改善了 SCI后的运动恢复;（3）a. Flavopiridol全身给药具有剂量依赖性作用 对功能结果和全身免疫应答的影响; B.全身施用 Flavopiridol降低SCI后细胞周期活化，从而减少随后的神经元和 少突胶质细胞死亡、反应性神经胶质增生和免疫活化; c.治疗相关， Flavopiridol延迟全身给药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.