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酰胺酶（Galc）基因的相同突变会导致Krabbe白质营养不良患者的临床差异，2)了解Galc缺乏是否直接针对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治疗方法至关重要。这种疾病是由于溶酶体酶GALC的常染色体隐性突变，导致严重的脱髓鞘和神经退行性变。超过85%的Krabbe患者表现为婴儿发病，神经系统进行性恶化，到两岁时死亡，而其他患者则表现为发病较晚，病情较轻。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，临床研究表明，患者携带GALC基因的致病突变并不总是预测结果，而且这种变异性的原因尚不清楚。因此，很难预测

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.