Novel Mechanisms of Microglial Neurotoxicity at Physiological Oxygen

描述(由申请人提供)：神经炎在急性和慢性神经退行性疾病中都会发生，并导致神经损伤。小胶质细胞在脑内被激活。 炎症反应，释放细胞因子、一氧化氮(NO)和超氧化物歧化酶，以邻近神经元为靶标。绝大多数体外研究神经退行性疾病机制的研究都是在大气氧气中进行的，尽管这种pO2(21%，160 mm Hg)远远超过生理性脑pO2(~3%，23 mm Hg)。我们认为，PO2从根本上影响神经元损伤的机制，并影响治疗策略的成功。我们发现，在3%O2条件下，NO对皮层神经元呼吸的抑制作用是在大气O2条件下的10倍以上，而且与目前的学说不同，在没有抑制复合体IV的情况下发生，而发生在抑制复合体II的情况下。本研究将检验这一中心假设，即在脑生理性O2条件下，来自小胶质细胞诱导型一氧化氮合酶的NO通过S介导的琥珀酸脱氢酶在复合体III上游的失活而不是通过NO或过氧亚硝酸盐介导的抑制复合体IV而导致皮质神经元损伤。一种短链辅酶Q类似物被NQO1酶还原后，可以将电子从细胞质NAD(P)H直接传输到复合体III，将测试其绕过上游呼吸损伤，挽救ATP水平，并在3%或21%O2中改善神经元存活的能力。我们预测，诱导NRF2/抗氧化反应元件通路的药物将通过1)上调NQO1和2)增加内源性抗氧化剂谷胱甘肽(可以防止半胱氨酸S-亚硝硫醇修饰)而有效地与艾地苯酮协同作用。在目标1中，我们将确定PO2如何影响激活的小胶质细胞所产生的神经毒性的机制。在目标2中，我们将通过可逆的S亚硝基化介导的琥珀酸脱氢酶(复合体II)抑制来确定外源性NO是否会在生理(3%)O2条件下损害皮质神经元的线粒体功能。在目标3中，我们将确定艾地苯酮与Nrf2/抗氧化反应元件途径的诱导剂萝卜硫素联合使用时，是否可以绕过NO介导的呼吸抑制，防止小胶质细胞毒性。我们将首次使用海马XF24在生理3%O2下进行神经元呼吸测量，并将这项技术应用于检测由共培养的激活的小胶质细胞引起的神经元呼吸损伤。我们的长期目标是阐明神经损伤的线粒体机制，并确定治疗干预的新途径。在这里，我们将向这些目标迈出重要的一步，1)确定PO2如何影响神经元损伤，并有可能改变体外神经退行性研究的方式，2)提供关于绕过呼吸抑制是否是减少小胶质细胞神经毒性的可行策略的原则证明。

DESCRIPTION (provided by applicant): Neuroinflammation occurs in both acute and chronic neurodegenerative disorders and contributes to neural injury. Microglial cells are activated in the inflammatory response, releasing cytokines, nitric oxide (NO), and superoxide which target neighboring neurons. The overwhelming majority of studies investigating neurodegenerative disease mechanisms in vitro are conducted at atmospheric O2 despite the fact that this pO2 (21%, 160 mm Hg) far exceeds physiological brain pO2 (~3%, 23 mm Hg). We propose that pO2 fundamentally influences the mechanisms of neuronal injury and impacts the success of therapeutic strategies. We found that at 3% O2, NO-mediated inhibition of cortical neuron respiration is over 10-fold more potent than at atmospheric O2, and, in contrast to current dogma, occurs without inhibition of complex IV but with inhibition of complex II. This study will test the central hypothesis that at brain physiological O2, NO derived from microglial inducible nitric oxide synthase (iNOS) causes cortical neuron injury by S-nitrosylation-mediated inactivation of succinate dehydrogenase upstream of complex III rather than by NO or peroxynitrite-mediated inhibition of complex IV. Clinically safe idebenone, a short chain Coenzyme Q analogue which upon reduction by NQO1 enzyme can shuttle electrons from cytoplasmic NAD(P)H directly to complex III, will be tested for the ability to bypass an upstream respiratory impairment, rescue ATP levels, and improve neuronal survival at either 3% or 21% O2. We predict that drugs that induce the Nrf2/antioxidant response element pathway will potently synergize with idebenone by 1) upregulating NQO1 and by 2) increasing the pool of the endogenous antioxidant glutathione which can prevent cysteine S-nitrosothiol modifications. In aim 1 we will determine how pO2 influences the mechanism of neurotoxicity exerted by activated microglia. In aim 2 we will determine whether exogenous NO impairs mitochondrial function in cortical neurons at physiological (3%) O2 by reversible S-nitrosylation-mediated inhibition of succinate dehydrogenase (complex II). In aim 3 we will determine whether idebenone, when combined with an inducer of the Nrf2/antioxidant response element pathway, sulforaphane, can bypass NO-mediated respiratory inhibition and prevent microglial toxicity. We will for the first time perform neuronal respiration measurements at physiological 3% O2 using the Seahorse XF24 and also adapt this technology for examining neuronal respiratory impairments caused by co-cultured, activated microglia. Our long-term goals are to elucidate mitochondrial mechanisms of neural injury and identify new avenues for therapeutic intervention. Here, we will take important steps toward these goals by 1) determining how pO2 influences neuronal injury, with the potential for a paradigm-shifting change in the way in vitro neurodegenerative research is conducted, and by 2) providing proof-of-principle on whether bypass of respiratory inhibition is a viable strategy to reduce microglial neurotoxicity.