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群发病率和死亡率的主要原因，但目前的治疗效果不佳，部分原因是我们对导致神经元损失的致病机制的理解有限。损伤过程的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Ca 2+积累。大的和持续的Ca 2+升高（“Ca 2+失调”）似乎是神经元死亡的指示。最近的证据表明，另一种二价阳离子Zn 2+发挥了关键作用，它在大脑中含量丰富，通常受到非常严格的调节。然而，在缺血或长时间癫痫发作后，游离Zn 2+在神经元中积累，并且观察到Zn 2+螯合是保护性的，这暗示了在神经元死亡中的作用。培养研究表明，外源性应用的Zn 2+可以进入神经元并在线粒体中积累，并强烈破坏其功能。然而，很少有人知道的机制所造成的损伤内源性Zn 2+在天然脑组织中的积累。因此，拟议的项目旨在解决以下假设：海马锥体神经元中的Zn 2+的积累对缺血性神经元损伤的开始起关键作用，部分通过与线粒体的有害相互作用。初步研究表明，内源性Zn 2+积累在海马脑片的锥体神经元进行氧葡萄糖剥夺（OGD），检测到的Ca 2+积累之前，和Zn 2+似乎进入线粒体，并有助于诱导Ca 2+失调和细胞死亡。目的应用荧光成像技术（包括单细胞荧光成像和整体荧光成像）研究急性缺氧缺糖（OGD）时海马CA 1区神经元内Zn ~（2+）的蓄积，以及Zn ~（2+）与线粒体的相互作用，并确定Zn ~（2+）在急性缺氧缺糖（OGD）及随后的再灌注过程中对Ca ~（2+）失调和细胞死亡的作用。这一关键目标将寻求提供上述假设的第一个严格的检查，并检查一系列干预措施，可能提供保护，同时帮助阐明的触发和表达阶段的损伤所涉及的事件的顺序。目标II将使用一系列方法来确定有害的Zn 2+积累的来源和途径。这些问题的“锌2+来自哪里”是复杂的，但至关重要的最佳干预措施的发展。目的III将使用器官型切片培养模型来检查Zn 2+在触发延迟神经变性（OGD后长达3天）中的作用，以检查下游损伤过程并测试在缺血后良好递送时可能提供保护的治疗干预。我们希望这些研究将提供新的见解，涉及的事件触发缺血性神经元损伤，这将导致新的和有效的神经保护策略的顺序。

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.