Elemental And Structural Organization Of Neurons And Glia

谷氨酸受体的NMDA亚型（NMDARs）在正常CNS功能中起着重要和不同的作用。然而，这些受体的过度激活导致过量的Ca 2+进入、线粒体钙（Ca 2+）过载和功能障碍，并且是兴奋性毒性损伤的关键早期事件。这建议将NMDAR作为抗兴奋性毒性疗法的靶点，但这种方法的患者结局普遍令人失望，这强烈表明NMDAR过度激活以外的因素在起作用。这一信息提示搜索其他重要的Ca 2+依赖性损伤途径。下面总结了最近的进展和正在进行的工作的具体目标。 目的#1：确定电压门控钙通道在钙依赖性神经变性中的作用。 虽然NMDAR在毒性Ca 2+负荷中明显起主导作用，但我们实验室和其他实验室的证据表明，Ca 2+进入的替代途径，例如通过电压门控钙通道（VGCC），可能会显著导致发育成熟神经元的毒性。因此，我们发现，虽然在一般情况下，在海马和皮层培养的神经元VGCC激活不促进显着的细胞死亡，VGCC激活引起更强的钙离子升高，在一个小的，但重要的神经元子集。这些神经元的特征在于VGCC的表达升高，其通过使人联想到经典兴奋性毒性途径的机制导致细胞死亡，即过度的Ca 2+负荷和神经元变性之前的线粒体功能障碍。结果表明谷氨酸毒性的一个辅助途径，其重要性可能在脑老化或与年龄相关的痴呆症中增加。 目的#2：确定锌在谷氨酸兴奋毒性和缺血性损伤中的作用。 短暂性脑缺血后细胞内锌（Zn ~（2+））浓度升高可导致神经元损伤，但其毒性机制尚不清楚。与Ca 2+类似，已提出Zn 2+通过线粒体功能障碍和/或ROS产生诱导毒性。最近在培养的海马神经元中的实验表明，Zn 2+可以通过VGCC和Ca 2+渗透性AMPA受体进入神经元，但是积累的Zn 2+仅在细胞外介质含有异常高（200 μ M）的外源性Zn 2+浓度时才是有毒的，如缺血后可能发生的。在有利于Zn 2+摄取的条件下，Ca 2+和Zn 2+都在线粒体内积累和共沉淀，但只有Ca 2+能够诱导线粒体肿胀、去极化和自由基产生，这是线粒体损伤的主要标志。这些数据支持工作假设，即兴奋性毒性线粒体功能障碍的经典机制必然是Ca 2+依赖性的，因此Zn 2+毒性的机制虽然仍然未知，但一定不同于Ca 2+的机制。

The NMDA subtype of the glutamate receptor (NMDARs) plays essential and diverse roles in normal CNS function. However over-activation of these receptors leads to excessive Ca2+ entry, mitochondrial calcium (Ca2+) overload and dysfunction, and is a crucial early event in excitotoxic injury. This recommends NMDARs as targets for anti-excitotoxic therapies, but the generally disappointing patient outcomes for such approaches strongly suggest that factors beyond the over-activation of NMDARs are at play. This information prompts searches for other important Ca2+-dependent injury pathways. Recent progress and the Specific Aims of ongoing work are summarized next. Aim #1: To define the role of voltage-gated calcium channels in calcium-dependent neurodegeneration. Although NMDARs clearly play the dominant role in toxic Ca2+ loading, evidence from our lab and others indicates that alternative routes of Ca2+ entry, for example, through voltage-gated calcium channels (VGCCs), can contribute significantly to toxicity in developmentally mature neurons. Thus, we find that while in general in hippocampal and cortical cultured neurons VGCC activation does not promote significant cell death, VGCC activation does evoke much stronger calcium elevations in a small but important subset of neurons. These neurons are characterized by elevated expression of VGCCs, which leads to cell death by mechanisms that are reminiscent of the classical excitotoxicity pathway, namely, excessive Ca2+ loading and mitochondrial dysfunction that precedes neuronal degeneration. The results demonstrate one ancillary pathway of glutamate toxicity, one whose significance is likely to increase during brain aging or in age-related dementia. Aim #2: To determine the role of zinc in glutamate excitotoxicity and ischemic injury. Elevation of intracellular zinc (Zn2+) following transient ischemia contributes to neuronal injury, but the mechanism(s) of Zn2+ toxicity remain unclear. In analogy to Ca2+, Zn2+ has been proposed to induce toxicity via mitochondrial dysfunction and/or ROS generation. Recent experiments in cultured hippocampal neurons reveal that Zn2+ can enter neurons through VGCCs and Ca2+-permeable AMPA receptors, but accumulated Zn2+ is only toxic when the extracellular medium contains unusually high (200 uM) exogenous Zn2+ concentrations, as might occur after ischemia. Under conditions that favor Zn2+ uptake, both Ca2+ and Zn2+ accumulate and co-precipitate within mitochondria, but only Ca2+ is capable of inducing mitochondrial swelling, depolarization, and free radical generation, which are the main hallmarks of mitochondrial damage. These data support the working hypothesis that classical mechanisms of excitotoxic mitochondrial dysfunction are necessarily Ca2+ dependent, so that mechanism(s) of Zn2+ toxicity, although still unknown, must be different from that of Ca2+.