Zn2+, mitochondria and the induction of ischemic neurodegeneration

描述（由申请人提供）：缺血性脑损伤是老年人发病和死亡的主要原因，但目前的治疗方法很差，部分原因是我们对导致神经元丧失的致病机制的了解有限。损伤过程的一个关键触发因素似乎是急性能量损失，导致膜去极化，兴奋性神经递质谷氨酸的过度释放和神经元Ca2+积累。一个大的和持续的Ca2+上升（“Ca2+解除管制”）似乎是神经元死亡的指示。最近的证据暗示了另一种二价阳离子Zn2+的关键作用，它在大脑中含量丰富，通常受到严格调节。然而，缺血或长时间癫痫发作后，游离Zn2+在神经元中积累，观察到Zn2+螯合具有保护作用，暗示其在神经元死亡中起作用。培养研究表明，外源性Zn2+可以进入神经元并积聚在线粒体中，并严重破坏其功能。然而，内源性Zn2+在天然脑组织中积累所引起的损伤机制尚不清楚。因此，提出的项目旨在解决以下假设：Zn2+在海马锥体神经元中的积累对缺血性神经元损伤的启动起关键作用，部分是通过与线粒体的有害相互作用。初步研究表明，内源性Zn2+在受氧葡萄糖剥夺（OGD）的海马切片的锥体神经元中积累，在可检测到的Ca2+积累之前，Zn2+似乎进入线粒体，并有助于诱导Ca2+失调和细胞死亡。目的：我将应用荧光成像技术（使用单细胞和大容量负载指标）对急性海马切片进行检测，以检测OGD期间CA1神经元中Zn2+的积累，检测其与线粒体的相互作用，并确定其在急性OGD期间和随后的再灌注期间对Ca2+解除管制和细胞死亡的贡献。这一关键目标将寻求为上述假设提供第一个严格的检查，并检查一系列可能提供保护的干预措施，同时帮助阐明涉及损伤触发和表达阶段的事件顺序。Aim II将使用一系列方法来确定有害的Zn2+积累的来源和途径。这些“锌离子从何而来”的问题很复杂，但对制定最佳干预措施至关重要。Aim III将使用器官型切片培养模型来检查Zn2+在延迟性神经变性（OGD后3天）触发中的作用，以检查下游损伤过程并测试在缺血后提供保护的治疗干预措施。希望这些研究将为缺血性神经元损伤触发的事件序列提供新的见解，从而导致新的有效的神经保护策略。

DESCRIPTION (provided by applicant): Ischemic brain injuries are leading causes of morbidity and mortality to the aging population, but current therapy is poor in part because of our limited understanding of pathogenic mechanisms leading to neuronal loss. A critical trigger of the injury process seems to be acute energy loss, leading to membrane depolarization, excessive release of the excitatory neurotransmitter glutamate and neuronal Ca2+ accumulation. A large and persistent Ca2+ rise ("Ca2+ deregulation") seems to be indicative of neuronal death. Recent evidence implicates critical contributions of another divalent cation, Zn2+, which is abundant in the brain and is normally very tightly regulated. However after ischemia or prolonged seizures, free Zn2+ accumulates in neurons, and observations that Zn2+ chelation is protective implicates a role in neuronal death. Culture studies have revealed that exogenously applied Zn2+ can enter neurons and accumulate in mitochondria and powerfully disrupt their function. However, little is known about mechanisms of injury caused by the accumulation of endogenous Zn2+ in native brain tissues. The proposed project thus seeks to address the following hypothesis: Accumulation of Zn2+ in hippocampal pyramidal neurons contributes critically to the initiation of ischemic neuronal injury, in part via deleterious interactions with mitochondria. Preliminary studies indicate that endogenous Zn2+ accumulates in pyramidal neurons in hippocampal slices subjected to oxygen glucose deprivation (OGD), prior to detectable Ca2+ accumulation, and that the Zn2+ appears to enter mitochondria and contribute to the induction of Ca2+ deregulation and cell death. Aim I will apply fluorescent imaging techniques (using both single cell and bulk loaded indicators) to acute hippocampal slices to examine Zn2+ accumulation in CA1 neurons during OGD, examine its interactions with mitochondria and determine its contributions to Ca2+ deregulation and cell death during acute OGD, and the subsequent reperfusion period. This key aim will seek to provide the first rigorous examination of the above hypothesis, and examine a range of interventions that may offer protection while helping to elucidate the sequence of events involved in the triggering and expression stages of injury. Aim II will use a range of approaches to determine the sources and routes of the injurious Zn2+ accumulation. These issues of "where the Zn2+ comes from" are complex, yet crucial to development of optimal interventions. Aim III will use organotypic slice culture models to examine roles of Zn2+ in the triggering of delayed neurodegeneration (up to 3 days after the OGD), in order to examine downstream injury processes and test therapeutic interventions that may offer protection when delivered well after the ischemia. It is hoped that these studies will provide new insights as to the sequence of events involved in the triggering of ischemic neuronal injury which will lead to new and effective neuroprotective strategies. PUBLIC HEALTH RELEVANCE: Despite being a cause of tremendous morbidity to the aging population, treatment of stroke is presently poor in part because of limited understanding of the events set in motion by ischemia that culminate in loss of function and nerve cell death. In this study, nerve cells in slices of mouse brain will be examined during and after simulated ischemia to directly examine movements and effects of the metal ion, zinc, which seems to play critical yet presently poorly understood in the triggering of ischemic brain injury. It is hoped that these studies will provide new insights into critical early events in ischemia that will suggest new approaches for new and better treatments to decrease brain damage.