Ion channel role-glutamate-independent neuronal injury

描述（由申请人提供）：卒中和全脑缺血是发病率和死亡率的主要原因。缺血导致进行性神经元死亡，即使在再灌注后。这种迟发性神经元死亡主要是由于过量的钙进入神经元。多年来，治疗策略集中在Ca 2+渗透性谷氨酸受体门控通道作为主要靶点。然而，通过治疗性使用谷氨酸受体拮抗剂来防止细胞死亡的努力令人失望。虽然谷氨酸受体拮抗剂的不耐受性和时间问题在很大程度上导致了临床试验的失败，但一个新兴的新概念也有利于其他Ca 2+进入来源在确定缺血性神经元死亡中可能同样重要的想法。我们现在有强有力的证据表明，氧化应激和氧自由基，产生于缺血，激活一种新的Ca 2 *-渗透性阳离子通道（自由基激活通道：FRAC）在皮层神经元和我们的初步数据表明，该通道可能是负责一个谷氨酸非依赖性的迟发性钙超载缺血性神经元死亡。我们的目标是充分表征离子特性，药理学概况和该通道的调节。我们将试图确定负责通道激活的自由基的具体形式和自由基如何激活FRAC的机制。将使用同步Ca 2+成像和膜片钳记录来量化通过FRAC的Ca 2+进入。我们还将确定自由基如何改变急性的兴奋性！分离的神经元和海马脑片的神经元。最后，使用体外和最终在体内缺血模型，我们将确定是否防止FRAC的激活保护神经元免于缺血性死亡。一般假设：由氧自由基（FRAC）激活的Ca 2 +-可渗透阳离子通道是负责的，至少部分地，在缺血性神经元死亡中谷氨酸非依赖性的，延迟的Ca 2+过载。FRAC的电生理学表征。（二）、FRA Cs的药理学特征和细胞内调节。（三）、FRAC激活诱导的Ca ~（2+）响应的荧光成像研究（四）、FRAC激活在谷氨酸非依赖性缺血性神经元损伤中的潜在作用我们的长期目标是确定除了谷氨酸受体之外的新靶点，以及保护脑细胞免受伴随中风的损伤的新策略。

DESCRIPTION (provided by applicant): Stroke and global brain ischemia are leading causes of morbidity and mortality. Ischemia causes progressive neuronal death, even after re-perfusion. This delayed neuronal death is largely due to excessive calcium entry into neurons. For many years, therapeutic strategies have focused on the Ca2+-permeable glutamate receptor gated channels as the main target. However, efforts to prevent cell death through the therapeutic use of glutamate receptor antagonists have been disappointing. Although intolerance of glutamate receptor antagonists and timing issues largely contribute to the failure of clinical trials, an emerging new concept also favors the idea that other sources of Ca2+ entry might be equally important in determining the ischemic neuronal death. We have now strong evidence that oxidative stress and oxygen free radicals, produced in ischemia, activate a novel Ca2*-permeable cation channel (the free radical activated channel: FRAC) in cortical neurons and our preliminary data demonstrated that this channel is likely responsible for a glutamate-independent delayed Ca2+ overload in ischemic neuronal death. Our objective is to fully characterize the ionic properties, pharmacology profile and the regulation of this channel. We will attempt to determine the specific form of free radicals responsible for the channel activation and the mechanism of how free radicals activate the FRAC. Simultaneous Ca2+imaging and patch-clamp recording will be used to quantify the Ca2+ entry through FRACs. We will also determine how free radicals alter the excitability of acute! dissociated neurons and the neurons in hippocampal slices. Finally, using in vitro and eventually in vivo ischemic models, we will determine if preventing the activation of FRACs protects neurons from ischemic death.General Hypothesis: Activation of a Ca2+-permeable cation channel by oxygen free radicals (the FRAC) is responsible, at least partially, for a glutamate-independent, delayed Ca2+ overload in ischemic neuronal death.Specific Aims:(1). Electrophysiological Characterization of FRACs.(2). Pharmacological Characterization and intracellular Regulation of FRA Cs.(3). Fluorescent imaging Study of Ca2+ Response Induced by the FRACActivation.(4). Potential Role of FRACActivation in Glutamate-independent Ischemic Neuronal InjuryOur long-term objective is to identify new targets, in addition to glutamate receptors, and novel strategies to protect brain cells from the damage that accompanies the stroke.