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

描述（由申请人提供）：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）中的多聚谷氨酰胺（polyQ）道扩展引起。可悲的是，十年来对polyQ依赖性神经变性机制的研究未能产生针对HD的甚至单一有效疗法。尽管已经鉴定出小分子在体外抑制突变htt片段的聚集，但许多小分子结合到由其他蛋白质共享的二级结构，并且不知道这些分子中的任何一种是否在更复杂的HD模型中有效和特异。我们最近完成了一个功能丧失（LOF）的基因组筛选在S。用单基因缺失菌株鉴定了犬尿氨酸3-单加氧酶（KMO）（色氨酸降解KP中的一种酶）作为突变体htt毒性的有效抑制剂。KP中两种神经毒性代谢物喹啉酸（QUIN）和3-羟基犬尿氨酸（3-HK）的脑水平在早期HD的纹状体和新皮质中增加;在三种HD小鼠模型中，QUIN和/或3-HK的增加相似。长期以来，QUIN和3-HK一直被假设与HD的病理生理学相关。事实上，纹状体内注射QUIN和3-HK会引起纹状体病变，这可能是由N-甲基D-天冬氨酸（NMDA）受体过度刺激（兴奋性毒性）和自由基形成的组合介导的。在我们的提案中，我们提供的数据显示，Ro 61-8048，一种高亲和力，口服生物可利用的KMO小分子抑制剂，降低酵母中的QUIN，3-HK和突变htt毒性，并在使用HD小鼠模型的小型试点研究中显着改善神经指数，旋转棒性能，运动活性和步行距离。值得注意的是，我们发现KMO只在小胶质细胞中表达。在早期HD患者和HD小鼠模型的死后大脑中已经记录了小胶质细胞活化。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小鼠中分离的初级小胶质细胞具有显著增加的3-HK水平。我们假设突变htt诱导转录缺陷，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体htt和KP在培养的小胶质细胞和HD小鼠模型中的作用来测试这些假设。这些实验将确定KMO抑制剂如Ro 61-4048是否值得进一步考虑作为HD治疗的临床前开发，Ro 61 -4048在小型试点研究中显示出有希望的结果。更广泛地说，我们在小鼠中的遗传实验将确定KMO的药理学抑制是否是治疗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）是否能在这种疾病的小鼠模型中提供保护。犬尿氨酸途径主要存在于小胶质细胞、脑巨噬细胞中，其在症状前亨廷顿病患者中被异常激活。如果成功的话，我们的研究可能会导致亨廷顿病患者的小分子抑制剂的临床试验。

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.