Role of Cell Cycle Pathways in Traumatic Brain Injury (TBI)

描述（由申请人提供）：尽管有许多积极的动物研究，创伤性脑损伤（TBI）后的临床神经保护试验在主要终点和一般人群方面一致失败。然而，大多数此类疗法都针对单一的损伤途径，并且针对非常早期的生化变化。我们之前的工作表明，细胞周期激活是脑外伤后继发性损伤的关键组成部分；根据我们和其他研究小组的研究，我们假设e2f1、2和3参与脑外伤后细胞周期相关神经元死亡（CRND）和小胶质细胞激活。在本项目中，我们建议使用高度特异性的E2F诱饵寡核苷酸（ODN）来证明阻断E2F的神经保护作用。我们将产生细胞特异性和可诱导的e2f1、2和3敲除和Prohibitin-1敲除，以证明神经元和小胶质细胞中的细胞周期激活代表了单独和附加的继发性损伤机制。我们还将使用视网膜母细胞瘤调节剂RRD-251来证实阻断E2Fs的神经保护作用。此外，我们建议将我们的研究重点扩展到急性损伤之外，并验证延迟细胞周期激活有助于TBI后数月的慢性神经变性和小胶质细胞激活的假设。具体目的是要表明：(1) tbi诱导的E2F转录因子的激活是导致急性神经元细胞死亡和小胶质细胞/星形胶质细胞反应性的关键事件，而用高度特异性的E2F诱导物寡核苷酸（ODN）序列或Rb调节剂RRD-251治疗可以减弱这些细胞变化，限制损伤诱导的病理生物学。(2)E2F 1-3是神经元细胞死亡和小胶质细胞激活的重要启动物，随着神经元中E2F活性的衰减，小胶质细胞表现出附加的神经保护作用；条件和细胞特异性三重敲除（E2F - 1-3）将通过E2F - 1-3 loxP小鼠与我们的诱导神经元和小胶质细胞特异性Cre小鼠交配产生；通过将Prohibitin-1 loxP小鼠与我们的可诱导的神经元和小胶质细胞特异性Cre小鼠配对，将产生条件和细胞特异性的Prohibitin-1敲入。(3)根据基于切片的细胞计数和荧光原位杂交（FISH）， TBI后的细胞周期激活持续超过急性期，并导致选择性易感脑区域某些神经元的多倍体逐渐增加；这些变化不会导致神经元的快速死亡，而是导致慢性进行性神经变性。(4)在创伤后1个月诱导条件性、神经元特异性或小胶质细胞特异性三重敲除（E2F -3），或使用泛cdk抑制剂CR8治疗，可减少进行性神经元高倍体，并减轻创伤后1年检测到的慢性进行性神经元丧失和相关神经损伤。

DESCRIPTION (provided by applicant): Despite numerous positive animal studies, clinical neuroprotection trials after traumatic brain injury (TBI) have uniformly failed with regard to primary end points and general populations. However, most such therapies have been directed toward a single proposed injury pathway and have targeted very early biochemical changes. Our prior work has shown that cell cycle activation is a key component of secondary injury following TBI; based upon our research and that from other groups, we hypothesize that E2F 1, 2, and 3 are involved in both cell cycle-related neuronal death (CRND) and microglial activation after brain trauma. In the present project we propose to use highly specific E2F decoy oligonucleotides (ODN) to demonstrate the neuroprotective effect of blocking the E2Fs. We will generate cell specific and inducible E2F 1, 2, and 3 knockouts and Prohibitin-1 knock-in to demonstrate that cell cycle activation in neurons and microglia represent separate and additive secondary injury mechanisms. We will also use the retinoblastoma modulator RRD-251 to confirm the neuroprotective effect of blocking the E2Fs. Moreover, we propose to expand our focus beyond the acute injury and test the hypothesis that delayed cell cycle activation contributes to chronic neurodegeneration and microglial activation months after TBI. Specific aims are to show that: (1) TBI-induced activation of E2F transcription factors is a critical event contributing to acute neuronal cell death and microglial/astrocyte reactivity, and that treatment with highly specific E2F decoy oligonucleotide (ODN) sequences or the Rb modulator RRD-251 attenuates these cellular changes and limits injury-induced pathobiology (2) E2F 1-3 are important contributing initiators of neuronal cell death and activation of microglia, with attenuation of E2F activity in neurons, and microglia showing additive neuroprotective effects; conditional and cell specific triple knockouts (E2F 1-3) will be generated by mating E2F 1-3 loxP mice with our inducible neuronal and microglia specific Cre mice; conditional and cell specific Prohibitin-1 knock-in will be generated by mating Prohibitin-1 loxP mice with our inducible neuronal and microglia specific Cre mice (3) Cell cycle activation after TBI persists beyond the acute period and leads to progressively increased polyploidy of certain neurons in selectively vulnerable brain regions as demonstrated by slide-based cytometry and fluorescence in- situ hybridization (FISH); these changes are not followed by rapid neuronal death but rather serve to predispose to chronic progressive neurodegeneration (4) Induction of conditional, neuron specific or microglial specific triple knockouts (E2F 1-3), or treatment with a the pan-CDK inhibitor CR8, at 1 month after trauma, reduce progressive neuronal hyperploidy, and attenuate chronic progressive neuronal loss and associated neurological impairment detected at 1 year after trauma.