Clinical variability and Cell autonomy of Krabbe leukodystrophy

描述（由申请人提供）：本提案的目标有两个：1）了解为什么半乳糖神经氨酸酶（Galc）基因中的相同突变会导致克拉伯脑白质营养不良患者的临床变异性，以及2）了解GALC缺乏症是否直接靶向少突胶质细胞以外的脑细胞。这两个问题的答案对于准确诊断和设计更有效的疗法至关重要。这种疾病是由于溶酶体酶GALC中的常染色体隐性突变引起的，其导致严重的脱髓鞘和神经变性。超过85%的Krabbe患者在两岁时表现出发作性、进行性神经功能恶化和死亡，而其他人则有迟发性、不太严重的疾病。这种疾病通过测量GALC活性来诊断，并通过突变分析和临床特征来证实。然而，临床研究已经揭示，患者在GALC基因中携带的致病突变并不总是预测结果，并且这种变异性的原因尚不清楚。因此，很难预测 疾病的过程准确。不幸的是，唯一可用的治疗选择是在症状发生之前进行造血细胞移植（HCT），因此预后指标对于疾病管理至关重要。HCT仅部分改善存活率并减弱婴儿表型中的病程，推测是通过将GALC从供体细胞转移到患者的髓鞘形成细胞。Krabbe和其他脱髓鞘疾病的一个未回答的问题是，神经变性是否只是继发于脱髓鞘，或者神经元或其他脑细胞是否也是疾病过程的直接靶点。我假设：1） 除了突变之外，顺式多态性还影响GALC向溶酶体的运输，这对于GALC加工和活化是必不可少的，最终影响表型。2)除少突胶质细胞外的任何脑细胞都能产生毒性，最终损害神经元。两个具体的目标将测试这个想法：1）确定顺式多态性与Galc突变的组合对GALC在ER、高尔基体和溶酶体中的运输、加工和活性的影响，2）确定每种脑细胞类型中GALC缺失对髓鞘形成和神经变性的影响。对于第一个目标，我将测试是否有任何的顺式多态性，已报告共存的某些突变影响GALC的贩运。对于第二个目标，我将从Krabbe病的抽搐小鼠模型中纯化每种类型的脑细胞，并将它们以各种组合共培养，以阐明哪些细胞GALC功能丧失是重要的，哪些细胞是脱髓鞘神经变性中最重要的毒性靶点。这种方法是创新的，因为它第一次考虑了细胞自主性和克拉贝病中顺式多态性的影响。该项目将产生影响，因为它将使我们能够更好地了解哪些因素决定Krabbe患者的预后，并将提供设计合理有效治疗所需的信息。

DESCRIPTION (provided by applicant): The goal of this proposal is two-fold: 1) to understand why the same mutation in the galactosylceraminidase (Galc) gene results in ample clinical variability among Krabbe leukodystrophy patients, and 2) to understand if brain cells other than oligodendrocytes are directly targeted by GALC deficiency. Answers to these two questions are crucially important for accurate diagnosis and for the design of more effective therapies. This disease is due to autosomal recessive mutations in the lysosomal enzyme GALC, which cause severe demyelination and neurodegeneration. More than 85% of Krabbe patients show infantile-onset, progressive neurologic deterioration and death by two years of age, while others have a late onset, less severe disease. This disease is diagnosed by measuring GALC activity and confirmed by mutation analysis and clinical features. However clinical studies have revealed that the pathogenetic mutation that a patient carries in the GALC gene is not always predictive of outcome, and the reasons for such variability are unclear. Therefore, it is difficult to predict the disease course accurately. Unfortunately, the only available therapeutic option is hematopoietic cell transplantation (HCT) before symptoms occur, thus prognostic indicators are of utmost importance for disease management. HCT only partially improves survival and attenuates the disease course in the infantile phenotype, presumably by transfer of GALC from donor cells to myelinating cells of the patients. One unanswered question in Krabbe and other demyelinating disease is whether neurodegeneration is only secondary to demyelination, or if neurons or other brain cells are also direct targets of the disease process. I hypothesize that: 1) In addition to the mutation, cis-polymorphisms also affect the trafficking of GALC to the lysosome which is indispensable for GALC processing and activation, ultimately influencing the phenotype. 2) Any brain cell other than oligodendrocytes can produce toxicity which eventually damages neurons. Two specific aims will test this idea: 1) Identify the effect of the combination of cis-polymorphisms with Galc mutations on GALC trafficking, processing, and activity in the ER, Golgi, and lysosome, 2) Identify the effect of the absence of GALC in each brain cell type on myelination and neurodegeneration. For the first aim, I will test if any of the cis-polymorphisms that have been reported to coexist with certain mutations affects the trafficking of GALC. For the second aim, I will purify each type of brain cell from the twitcher mouse model of Krabbe disease and coculture them in various combinations to elucidate in which cells GALC loss-of-function is important and which cells are the most important target of toxicity in demyelinating neurodegeneration. This approach is innovative because for the first time it considers cell autonomy and the effect of cis- polymorphisms in Krabbe disease. The project will have impact because it will allow us to better understand which factors determine the prognosis of Krabbe patients, and will provide information necessary to design rational and effective therapies.