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都失败了。然而，大多数这样的疗法已经针对单一的拟议损伤途径，并针对非常早期的生化变化。我们先前的工作表明，细胞周期激活是TBI后继发性损伤的关键组成部分;根据我们的研究和其他研究组的研究，我们假设E2 F 1、2和3参与脑创伤后细胞周期相关的神经元死亡（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E2 F诱饵寡核苷酸（ODN）来证明阻断E2 F的神经保护作用。我们将产生细胞特异性和可诱导的E2 F 1，2和3敲除和Prohibitin-1敲入，以证明神经元和小胶质细胞中的细胞周期激活代表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E2 Fs的神经保护作用。此外，我们建议扩大我们的重点超越急性损伤和测试的假设，延迟细胞周期激活有助于慢性神经变性和小胶质细胞激活后几个月的TBI。具体目标是表明：（1）TBI诱导的E2 F转录因子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，并且用高度特异性的E2 F诱饵寡核苷酸（ODN）序列或Rb调节剂RRD-251治疗减弱这些细胞变化并限制损伤诱导的病理生物学（2）E2 F 1- 3是神经元细胞死亡和小胶质细胞活化的重要促进引发剂，具有神经元中E2 F活性的减弱，并且小胶质细胞显示出附加的神经保护作用;条件性和细胞特异性三重敲除（E2 F 1-3）将通过使E2 F 1-3 loxP小鼠与我们的可诱导的神经元和小胶质细胞特异性Cre小鼠交配来产生;通过将Prohibitin-1 loxP小鼠与我们的诱导型神经元和小胶质细胞特异性Cre小鼠交配，将产生条件性和细胞特异性Prohibitin-1敲入。（3）TBI后的细胞周期激活持续超过急性期，并导致选择性脆弱脑区域中某些神经元的多倍性逐渐增加，如基于载玻片的细胞计数和荧光原位杂交（FISH）所证明的。（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（E2 F 1-3），或用泛CDK抑制剂CR 8治疗，减少进行性神经元超倍性，并减轻创伤后1年检测到的慢性进行性神经元丢失和相关的神经损伤。

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.