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）中的聚谷氨酰胺（polyQ）通道扩大引起。遗憾的是，对多q依赖性神经退行性变机制的十年研究未能产生一种针对HD的有效治疗方法。尽管在体外已经发现了抑制突变htt片段聚集的小分子，但许多小分子与其他蛋白质共享的二级结构结合，并且尚不清楚这些小分子是否在更复杂的HD模型中有效和特异性。我们最近在酿酒酵母的单基因缺失菌株中完成了功能丧失（LOF）基因组筛选，鉴定出犬尿氨酸3-单加氧酶（KMO），一种色氨酸降解KP中的酶，是突变体htt毒性的有效抑制因子。早期HD患者纹状体和新皮层中两种KP神经毒性代谢物——喹啉酸（QUIN）和3-羟基犬尿氨酸（3-HK）水平升高；在三种HD小鼠模型中，QUIN和/或3-HK也出现类似的升高。长期以来，人们一直假设QUIN和3-HK与HD的病理生理有关。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（兴奋毒性）和自由基形成共同介导的。在我们的提议中，我们提供的数据显示，高亲和力，口服生物可利用的KMO小分子抑制剂Ro 61-8048，在酵母中降低QUIN， 3-HK和突变体htt毒性，并显着改善神经系统指数，旋转性能，运动活性和移动距离，使用HD小鼠模型进行小型先导研究。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患者死后的大脑和HD小鼠模型中有小胶质细胞激活的记录。然而，人们对小胶质细胞在HD病理生理中的作用知之甚少。我们发现从HD小鼠分离的初级小胶质细胞中3-HK的水平显著增加。我们假设突变的htt诱导了一个转录缺陷，激活了小胶质细胞中的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。我们拟通过研究突变体htt和KP在培养的小胶质细胞和体内HD小鼠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）是否值得进一步考虑作为HD治疗的临床前开发。Ro 61-4048在一项小型中试研究中显示出有希望的结果。更广泛地说，我们的小鼠遗传实验将确定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，我们将使用遗传和药理学方法来确定阻断与亨廷顿氏病有关的代谢途径（犬尿氨酸途径）是否能保护这种疾病的小鼠模型。犬尿氨酸途径主要存在于小胶质细胞中，即大脑的巨噬细胞，在症状前的亨廷顿舞蹈病患者中被异常激活。如果成功，我们的研究可能会导致在亨廷顿病患者中进行小分子抑制剂途径的临床试验。

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.