The Role of Microglia and the Kynurenine Pathway in Huntington's Disease

描述(申请人提供)：亨廷顿病(HD)是一种致命的神经退行性疾病，由亨廷顿蛋白(HTT)中扩大的聚谷氨酰胺(PolyQ)途径引起。可悲的是，十年来对依赖多聚Q的神经退行性变机制的研究甚至没有产生一种治疗HD的有效方法。尽管已发现小分子在体外抑制突变的HTT片段的聚集，但许多小分子与其他蛋白质共享的二级结构结合，目前尚不清楚这些分子中是否有任何分子在更复杂的HD模型中有效和特异。我们最近在酿酒酵母中完成了单基因缺失菌株的功能丧失(LOF)基因组筛选，发现犬尿氨酸3-单加氧酶(KMO)是色氨酸降解KP中的一种酶，是突变的HTT毒性的有效抑制因子。在早期HD小鼠模型中，脑组织中两种神经毒性代谢物喹啉酸(Quin)和3-羟基犬尿氨酸(3-HK)的水平在纹状体和新皮质中升高；Quin和/或3-HK在三种HD小鼠模型中也有类似的升高。奎宁和3-HK长期以来一直被认为与HD的病理生理学有关。事实上，纹状体内注射奎宁和3-HK可引起纹状体损伤，这可能是N-甲基-D-天冬氨酸(NMDA)受体过度刺激(兴奋毒性)和自由基形成的组合所致。在我们的提案中，我们提供的数据显示，在使用HD小鼠模型的小型先导性研究中，高亲和力、口服生物可用KMO小分子抑制剂RO61-8048可以降低Quin、3-HK和突变的HTT在酵母中的毒性，并显著改善神经学指数、旋转臂性能、运动活动和步行距离。值得注意的是，我们发现KMO仅在小胶质细胞中表达。在早期HD患者的死后大脑和HD小鼠模型中，小胶质细胞的激活已被证明。然而，小胶质细胞在HD病理生理学中的作用知之甚少。我们发现，从HD小鼠分离的原代小胶质细胞显著增加了3-HK的水平。我们假设突变的HTT导致转录缺陷，激活小胶质细胞中的KP，通过药理学和遗传学方法抑制KP将改善HD小鼠模型的行为和病理结果。我们建议通过研究突变的HTT和KP在培养的小胶质细胞中的作用以及在小鼠HD模型中的作用来验证这些假设。这些实验将确定KMO抑制剂，如Ro 61-4048，在一项小型先导研究中显示了有希望的结果，是否值得进一步考虑作为HD疗法的临床前开发。更广泛地说，我们在小鼠身上的遗传学实验将确定KMO的药理抑制是否是治疗HD的真正治疗方法。在这个项目中，我们将使用遗传学和药理学方法来确定阻断与亨廷顿病有关的代谢途径(犬尿氨酸途径)是否对这种疾病的小鼠模型提供保护。犬尿氨酸途径主要在小胶质细胞中发现，小胶质细胞是大脑的巨噬细胞，在无症状的亨廷顿病患者中被异常激活。如果成功，我们的研究可能会导致对亨廷顿病患者进行该途径的小分子抑制剂的临床测试。

DESCRIPTION (provided by applicant): Huntington's Disease (HD) is a fatal neurodegenerative disorder caused by an expanded polyglutamine (polyQ) tract in the protein huntingtin (htt). Sadly, a decade of research into the mechanisms of polyQ- dependent neurodegeneration has failed to produce even a single effective therapy for HD. Although small molecules have been identified that inhibit the aggregation of a mutant htt fragment in vitro, many bind to secondary structures shared by other proteins, and it is not known if any of these molecules will be effective and specific in more complex models of HD. We recently completed a loss-of-function (LOF) genomic screen in S. cerevisiae with single gene deletion strains that identified kynurenine 3-monooxygenase (KMO), an enzyme in the KP of tryptophan degradation, as a potent suppressor of mutant htt toxicity. The brain levels of two neurotoxic metabolites in the KP, quinolinic acid (QUIN) and 3-hydroxykynurenine (3-HK), are increased in the striatum and neocortex in early grade HD; similar increases in QUIN and/or 3-HK are present in three mouse models of HD. QUIN and 3-HK have long been hypothetically linked to the pathophysiology of HD. Indeed, intrastriatal injection of QUIN together with 3-HK causes striatal lesions that may be mediated by the combination of N-methyl D-aspartate (NMDA) receptor over-stimulation (excitotoxicity) and free radical formation. In our proposal, we present data showing that Ro 61-8048, a high-affinity, orally bioavailable small molecule inhibitor of KMO, decreases QUIN, 3-HK and mutant htt toxicity in yeast, and significantly improved neurological index, rotarod performance, locomotor activity and ambulatory distance in a small pilot study using a mouse model of HD. Remarkably, we show KMO is expressed exclusively in microglia. Microglial activation has been documented in postmortem brains of early grade HD patients and in HD mouse models. However, little is known about the role of microglia in HD pathophysiology. We show that primary microglia isolated from HD mice have significantly increased levels of 3-HK. We hypothesize that mutant htt induces a transcriptional defect that activates the KP in microglia, and that inhibiting the KP via pharmacological and genetic approaches will improve behavioral and pathological outcome measures in HD mouse models. We propose to test these hypotheses by studying the role of mutant htt and the KP in cultured microglia and in vivo in mouse models of HD. These experiments will establish whether KMO inhibitors such as Ro 61-4048, which showed promising results in a small pilot study, deserves further consideration for pre-clinical development as a HD therapy. More broadly, our genetic experiments in mice will determine whether pharmacological inhibition of KMO is a bona fide therapeutic approach to treating HD. PUBLIC HEALTH RELEVANCE In this project we will use genetic and pharmacological approaches to determine if blocking a metabolic pathway implicated in Huntington's disease (the kynurenine pathway) confers protection in mouse models of this disorder. The kynurenine pathway is found predominantly in microglia, the macrophages of the brain, which are activated abnormally in pre-symptomatic Huntington's disease patients. If successful, our studies may lead to clinical tests of small molecule inhibitors of the pathway in patients with Huntington's disease.