Mechanisms and Treatment of CNS Edema

描述(申请人提供)：中风是美国第三大致死原因和最大致残原因，但目前还没有有效的治疗方法来治疗大多数病例。目前的治疗方案只是着眼于恢复血液流动，希望挽救处于危险状态的组织，但没有提供有针对性的神经保护。由于缺乏对缺血性卒中后继发性损伤的关键信号通路的了解，神经保护性治疗的发展一直受到阻碍。十多年来，半胱氨酸天冬氨酸氨基转移酶家族的死亡蛋白与脑缺血和神经变性有关。最近的证据表明，在缺血过程中，不同的caspase通路被激活。我们已经确定caspase-9/-6通路与脑缺血后神经元功能障碍和死亡有关。我们的数据显示，靶向caspase-9活性可以在缺血性损伤后提供实质性的神经保护。此外，我们发现caspase-9活性在脑缺血发病机制的两个方面是必需的：1)神经元变性和2)脑水肿的发展。水肿是由血管完整性的丧失引起的，而不是血管内皮细胞和周细胞死亡(BV)。这些细胞之间紧密连接的消除允许液体从小的颅内BV渗出。浮肿的形成是导致严重中风患者死亡和残疾的主要原因。内科治疗和外科减压术仅极小地改变了这一致病过程的自然历史。在我们的研究中，一种细胞意义上的caspase-9抑制剂Pen1-XBIR3降低了caspase-9的活性，并伴随着消除了水肿。这一发现开启了一个问题，即caspase-9活性是否是通过损害小脑BV的血管完整性而导致水肿的直接原因。我们的初步数据表明，激活的caspase-9通过减少基质金属蛋白酶9(MMP9)的表达来调节水肿。我们的数据还显示，成熟NGF前体proNGF的表达在卒中过程中增加。ProNGF是p75NTR的高亲和力配体，我们已经证明通过p75NTR信号激活caspase-9。P75NTR存在于脑内较小的BV中，卒中时p75NTR表达增加。我们的初步数据还表明，激活的caspase-9存在于BV中，抑制caspase-9可以阻止中风诱导的MMP-9的表达。我们现在提出的假设是，卒中水肿的发展是由原神经营养因子(ProNT)通过p75NTR传递的信号所介导的，它激活小BV中的caspase-9来裂解对血管完整性至关重要的底物。我们将利用体内和体外模型来检验这一假说，具体目的如下：目的1：确定ProNTs的诱导是否会在小BV中触发caspase-9的激活。目的2：确定p75NTR信号通路是否激活caspase-9并导致水肿。目的3：确定caspase-9裂解底物如何导致血管完整性丧失。

DESCRIPTION (provided by applicant): Stroke is the 3rd largest cause of death and the largest cause of disability in the U.S., yet there are no effective therapies for the vast majorityof cases. Present therapeutic options merely aim to restore blood flow in the hopes of salvaging at risk tissue, but offer no targeted neuroprotection. Development of neuroprotective therapies has been hindered by lack of knowledge of the signaling pathways critical in secondary injury following ischemic stroke. For more than a decade, the caspase family of death proteases has been implicated in cerebral ischemia and neurodegeneration. Recent evidence shows that distinct caspase pathways are activated during ischemia. We have identified the caspase-9/-6 pathway as responsible for neuronal dysfunction and death after ischemia. Our data show that targeting caspase-9 activity provides substantial neuroprotection following an ischemic insult. Moreover, we find that caspase-9 activity is required for two aspects of ischemic pathogenesis: 1) neuronal degeneration and 2) the development of cerebral edema. Edema is caused by a loss of vascular integrity, rather than death of endothelial cells and pericytes in the blood vessels (BV). The elimination of tight junctions between these cells allows extravasation of fluid from small intracranial BVs. Edema formation is a major contributor to death and disability in severe stroke. Medical therapies and surgical decompressive procedures have only minimally altered the natural history of this pathogenic process. In our studies, a cell permeant caspase-9 inhibitor, Pen1-XBIR3, reduces caspase-9 activity and concomitantly abolishes edema. This finding opens the question of whether caspase-9 activity is a direct cause of edema through the impairment of vascular integrity of small cerebral BVs. Our preliminary data suggest that active caspase-9 regulates edema by decreasing the expression of matrix metalloproteinase 9 (MMP-9). Our data also show that expression of the precursor of mature NGF, proNGF, increases during stroke. ProNGF is a high affinity ligand for p75NTR, and we have shown that signaling through p75NTR activates caspase-9. p75NTR is found in small BVs in the brain, and expression of p75NTR increases during stroke. Our preliminary data also show that activated caspase-9 is present in BVs, and that caspase-9 inhibition prevents the stroke-induced expression of MMP-9. We now propose the hypothesis that the development of edema in stroke is mediated by proneurotrophin (proNT) signaling through p75NTR, which activates caspase-9 in small BVs to cleave substrates vital to the integrity of the vessels. We will utilize in vivo an in vitro models to examine this hypothesis with the following Specific Aims: Aim 1: To determine if induction of proNTs triggers caspase-9 activation in small BVs. Aim 2: To determine if signaling via p75NTR activates caspase-9 and leads to edema. Aim 3: To determine how caspase-9 cleavage of substrates leads to loss of vascular integrity.