Novel Mechanisms of Microglial Neurotoxicity at Physiological Oxygen

生理氧下小胶质细胞神经毒性的新机制

基本信息

批准号：
8612571
负责人：
BRIAN M POLSTER
金额：
$ 33.58万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8612571
关键词：
Acute Antioxidants Binding Sites Brain Bypass Cell Culture Techniques Cell Death Cells Chronic Coculture Techniques Complex Cysteine Electron Transport Complex III Electrons Engineering Enzymes Esters Exposure to Genus Hippocampus Glutathione Goals Immune Impairment In Vitro Individual Inflammatory Response Injury Knock-out Link Measurement Measures Mediating Microglia Mitochondria Mitochondrial Proteins Modification NQO1 gene Nerve Degeneration Neurodegenerative Disorders Neuronal Injury Neurons Nitric Oxide Oxygen Pathway interactions Peptides Peroxonitrite Pharmaceutical Preparations Physiological Protein S Reaction Relative (related person)Research Resistance Respiration Response Elements Rest S-Nitrosothiols Succinate Dehydrogenase Sulfhydryl Compounds Sulforaphane Superoxides Technology Testing Therapeutic Therapeutic Intervention Time Toxic effect Tyrosine Ubiquinone analog attenuation brain cell complex IV cytokine design human NOS2A protein idebenone improved mitochondrial dysfunction mutant nerve injury neuroinflammation neuronal survival neurotoxicity nitration novel novel therapeutics overexpression planetary Atmosphere prevent public health relevance relating to nervous system research study respiratory success

项目摘要

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.

描述（由申请人提供）：神经炎症发生在急性和慢性神经退行性疾病中，并导致神经损伤。小胶质细胞在炎症反应，释放细胞因子，一氧化氮（NO）和靶向相邻神经元的超氧化物。尽管该PO2（21％，160 mm Hg）远远超过生理脑PO2（〜3％，23 mm Hg），但在大气O2上进行了大多数研究的大多数研究都在大气O2上进行了大气O2。我们建议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时，无介导的皮质神经元呼吸的抑制比大气O2高10倍以上，与当前的教条相反，与当前的教条相反，不受复杂IV的抑制，但抑制了复合物II。这项研究将检验一个中心假设：在脑生理O2上，无胶质诱导型一氧化氮合酶（INOS）衍生出来，从而导致S-硝基化介导的盐氨基甲基化脱氢酶上游的非活化III上游的脱氢酶的失活而导致皮质神经元损伤，而不是由NO或Peroxynitrite-Nitrite介导的复杂化合物的INBIND INBIND INBIND INBIND INBIND INBIND IV。临床上安全的iDebenone是一种短链辅酶Q类似物，在NQO1酶减少后，可以直接从细胞质NAD（P）H穿梭到复杂III的电子，以便能够绕过上游呼吸障碍，并在3％或21％O2上绕过上游呼吸障碍，营救ATP水平，并提高神经元水平。我们预测，诱导NRF2/抗氧化反应元件途径的药物将通过1）上调NQO1和2）增加内源性抗氧化剂谷胱甘肽的库有效地协同，从而可以预防半胱氨酸S-硝基硫醇修饰。在AIM 1中，我们将确定PO2如何影响活化的小胶质细胞施加的神经毒性机制。在AIM 2中，我们将通过可逆的S-亚硝基化介导的琥珀酸脱氢酶（复合物II）来确定在生理神经元（3％）O2处皮质神经元中的线粒体功能（3％）O2（复合物II）。在AIM 3中，我们将确定IDEBENONE与NRF2/抗氧化剂反应元件途径相结合时，Sulforaphane是否可以绕过NO介导的呼吸抑制并预防小胶质细胞毒性。我们将首次使用Seahorse XF24在生理3％O2上进行神经元呼吸测量，并适应该技术检查由共培养的，活化的小胶质细胞引起的神经元呼吸障碍。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，我们将通过1）确定PO2如何影响神经元损伤的重要步骤，并有可能进行体外神经退行性研究的方式变化范式变化，并通过2）为旁路旁路提供证明是否可以降低呼吸抑制作用是否有能力降低小型神经毒性。