Mechanisms and Modulation of Cell Death in Traumatic Brain Injury

描述（由申请人提供）：创伤性脑损伤（TBI）或体外创伤性神经元损伤，部分通过激活半胱天冬酶引起神经元凋亡。在体内或体外创伤模型中抑制半胱天冬酶-3减少创伤后细胞凋亡，并改善临床相关TBI模型的功能结果。然而，这些研究中的一些表明，改善通常仅反映细胞死亡的延迟，其最终仍然发生而没有经典的凋亡表型。这表明，半胱天冬酶非依赖性途径可能在决定细胞的最终命运中发挥重要作用。最近的工作支持这一假设，证明胱天蛋白酶非依赖性细胞凋亡也有助于多种体外模型系统中的神经元细胞死亡，并且在急性脑缺血或TBI后发生与细胞凋亡形态学特征相关的凋亡诱导因子（AIF）从线粒体到细胞核的易位。此外，AIF易位可在低能量条件下发生，与聚ADP-核糖聚合酶I（PARP-1）的活化和烟酰胺腺嘌呤二核苷酸（NAD+）的还原相关。相比之下，半胱天冬酶激活通常与更保存的生物能量状态相关，并且需要腺苷5 '-三磷酸（ATP）。因此，在更严重的损伤后，或在细胞生物能量状态基本受损的病变部位的更中心区域内，半胱天冬酶非依赖性细胞凋亡可能比半胱天冬酶介导的细胞死亡发挥更大的作用。AIF介导的细胞凋亡可以通过负责内在半胱天冬酶激活的相同机制或通过PARP-1激活来启动。在前者中，AIF所起的作用只有在caspase激活被阻断时才可见。在后者中，AIF是主要的死亡诱发因素。PARP-1抑制或PARP敲除动物以及AIF载体蛋白亲环素A敲除动物显示AIF易位减少。我们建议利用一个完善的，控制皮质撞击（CCI）模型的TBI小鼠，以及在体外模型中选择，比较的机制，半胱天冬酶依赖性和半胱天冬酶非依赖性的程序性细胞死亡的神经元和它们的相对作用作为损伤严重程度和损伤定位的函数。具体假设包括：1）半胱天冬酶非依赖性途径和半胱天冬酶依赖性途径都有助于TBI后的创伤后细胞损失和相关的神经功能障碍，以及与DNA损伤相关的细胞培养模型中的凋亡性神经元细胞死亡; 2）在更严重的损伤后，或在扩大的损伤的更中心区域，与半胱天冬酶依赖性细胞死亡相比，半胱天冬酶非依赖性细胞凋亡被诱导到相对更大的程度，其中生物能量状态降低; 3）AIF促死亡结构域的细胞特异性、可诱导的“功能性”敲除，以及其中AIF易位被抑制的模型（PARP敲除，用PARP抑制剂处理，或亲环素A敲除），在TBI后或体外细胞损伤后显示减少的凋亡性细胞死亡，和; 4）抑制半胱天冬酶依赖性和半胱天冬酶非依赖性细胞死亡以相加或协同方式改善CCI后的恢复。我们提出了以下具体目标：1）比较轻、中或中-重度TBI后caspase依赖性和caspase非依赖性神经元细胞死亡的相对程度和位置; 2）通过比较两种可诱导的、神经元特异性的、促死亡结构域选择性的AIF转基因模型与它们的“非诱导”对照，研究AIF在TBI诱导的神经元死亡和行为恢复中的作用; 3）评估亲环素A敲除对TBI后和在选定的细胞培养模型中AIF易位、凋亡和行为结果的影响; 4）评估两种结构不同的PARP抑制剂或PARP-1敲除对TBI后和在所选细胞培养模型中的AIF易位、细胞凋亡和行为结果的影响，并确定这种作用对胱天蛋白酶抑制作用是加和的还是协同的。公共卫生相关性：创伤性脑损伤（TBI）是美国死亡和残疾的主要原因。更好地了解TBI的潜在机制将提供提高生存率和确保更完全康复的可能性。

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI), or traumatic neuronal injury in vitro, causes neuronal apoptosis, in part through activation of caspases. Inhibition of caspase-3, in both in vivo or in vitro trauma models, reduces post-traumatic apoptosis, and improves functional outcomes in clinically relevant TBI models. However, some of these studies indicate that improvements often reflect only a delay in cell death, which still occurs eventually without the classical apoptotic phenotype. This suggests that caspase-independent pathways might play an important role in determining the final fate of cells. Recent work supports this hypothesis, demonstrating that caspase- independent apoptosis also contributes to neuronal cell death in a variety of in vitro model systems, and that translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus, in association with apoptotic morphological features, occurs after acute brain ischemia or TBI. Moreover, AIF translocation can occur under low energetic conditions, in association with activation of poly-ADP-ribose polymerase I (PARP-1) and reduction of nicotinamide adenine dinucleotide (NAD+). In contrast, caspase activation is generally associated with a more preserved bioenergetic state and requires adenosine 5'-triphosphate (ATP). Thus caspase-independent apoptosis may play a greater role than caspase-mediated cell death after a more severe injury, or within more central regions of the evolving lesion - sites at which cellular bioenergetic state is substantially compromised. AIF-mediated apoptosis may be initiated either by the same mechanisms responsible for intrinsic caspase activation or through PARP-1 activation. In the former, the role played by AIF becomes visible only when caspase activation has been blocked. In the latter, AIF is the main death-inducing factor. PARP-1 inhibition or PARP knockout animals, as well as knockout of the AIF carrier protein cyclophilin A, show reduced AIF translocation. We propose to utilize a well-established, controlled cortical impact (CCI) model of TBI in mouse, as well as selected in vitro models, to compare mechanisms underlying both caspase- dependent and caspase-independent programmed cell death of neurons and their relative roles as a function of injury severity and injury localization. Specific hypotheses include: 1) both caspase-independent and caspase-dependent pathways contribute to post-traumatic cell loss and associated neurological dysfunction after TBI, as well as to apoptotic neuronal cell death in cell culture models associated with DNA damage; 2) caspase-independent apoptosis is induced to a relatively greater degree than caspase-dependent cell death after more severe insults, or at more central regions of the expanding lesion, where bioenergetic state is reduced; 3) cell specific, inducible 'functional" knockouts of AIF pro-death domains, as well as models in which AIF translocation is inhibited (PARP knockout, treatment with PARP inhibitors, or cyclophilin A knockout), show reduced apoptotic cell death after TBI or after cell injury in vitro, and; 4) inhibition of both caspase- dependent and caspase-independent cell death improves recovery after CCI in additive or synergistic fashion. We propose the following specific aims: 1) to compare the relative degree and location of caspase-dependent and caspase-independent neuronal cell death after mild, moderate or moderately-severe TBI; 2) to investigate the role of AIF in TBI-induced neuronal death and behavioral recovery by comparing two inducible, neuron-specific, pro-death domain selective AIF transgenic models versus their "non-induced" controls; 3) to evaluate the effects of cyclophilin A knockout on AIF translocation, apoptosis and behavioral outcome after TBI and in selected cell culture models and; 4) to evaluate the effects of two structurally-distinct PARP inhibitors or PARP-1 knockout on AIF translocation, apoptosis and behavioral outcome after TBI and in selected cell culture models, and determine whether such effects are additive or synergistic to that of caspase inhibition. PUBLIC HEALTH RELEVANCE: Traumatic brain injury (TBI) represents a major cause of death and disability in the United States. A better understanding of the mechanisms underlying TBI would offer the possibility of improving survival and insuring a more complete recovery.