Mechanisms and Modulation of Cell Death in Traumatic Brain Injury

描述(申请人提供)：创伤性脑损伤(TBI)，或体外创伤性神经元损伤，导致神经细胞凋亡，部分通过激活caspase。在体内或体外创伤模型中，抑制caspase-3可以减少创伤后的细胞凋亡，并改善临床相关的脑损伤模型的功能结果。然而，这些研究中的一些研究表明，改善通常只是反映了细胞死亡的延迟，这种情况最终仍然会发生，而不是经典的凋亡表型。这表明，caspase不依赖的通路可能在决定细胞最终命运方面发挥重要作用。最近的工作支持这一假说，表明在各种体外模型系统中，caspase非依赖性的细胞凋亡也有助于神经细胞的死亡，并且凋亡诱导因子(AIF)从线粒体到细胞核的移位与凋亡的形态特征有关，发生在急性脑缺血或脑损伤后。此外，AIF易位可以在低能量条件下发生，伴随着多ADP核糖聚合酶I(PARP-1)的激活和烟酰胺腺嘌呤二核苷酸(NAD+)的减少。相反，caspase的激活通常与更保存的生物能量状态有关，需要5‘-三磷酸腺苷(ATP)。因此，在更严重的损伤后，或者在不断演变的损伤的更中心区域-细胞生物能量状态实质上受到损害的位置上，caspase非依赖性的细胞凋亡可能发挥比caspase介导的细胞死亡更大的作用。AIF介导的细胞凋亡可能由与caspase激活相同的机制启动，也可能通过PARP-1激活启动。在前者中，只有当caspase激活被阻断时，AIF所起的作用才变得可见。在后者中，AIF是主要的致死因素。PARP-1抑制或PARP敲除动物，以及AIF载体蛋白亲环素A的敲除，都显示AIF易位减少。我们建议利用一种成熟的小鼠脑损伤受控皮质撞击(CCI)模型，以及选定的体外模型，比较caspase依赖和非caspase依赖的神经元程序性细胞死亡的机制，以及它们作为损伤严重程度和损伤定位的函数的相对作用。具体的假说包括：1)caspase非依赖途径和caspase依赖途径均导致脑创伤后细胞丢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神经细胞凋亡；2)在更严重的损伤后，或在扩大的损伤的更中心区域，诱导caspase非依赖的细胞死亡的程度相对更大，那里的生物能量状态降低；3)细胞特异性的、可诱导的AIF前死亡结构域的功能敲除，以及AIF易位被抑制的模型(PARP敲除、PARP抑制剂治疗或亲环素A敲除)显示，在体外脑损伤后或细胞损伤后，细胞凋亡减少；以及4)抑制caspase依赖和caspase非依赖的细胞死亡以相加或协同的方式促进CCI后的恢复。我们提出的具体目标如下：1)比较轻、中、中、重度脑损伤后caspase依赖和非caspase依赖的神经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、支持死亡结构域的AIF转基因模型与其“非诱导”对照，探讨AIF在脑创伤后神经元死亡和行为恢复中的作用；3)评价亲环素A基因敲除对脑损伤后和部分细胞培养模型中AIF易位、细胞凋亡和行为结局的影响；4)评价两种结构不同的PARP抑制剂或PARP-1基因敲除对脑创伤后AIF易位、细胞凋亡和行为结局的影响，并确定这些作用与抑制caspase的作用是相加的还是协同的。公共卫生相关性：在美国，创伤性脑损伤(TBI)是导致死亡和残疾的主要原因。更好地了解创伤性脑损伤的潜在机制将为提高存活率和确保更全面的康复提供可能性。

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.