Investigating the molecular basis of Canavan disease

研究卡纳万病的分子基础

• 批准号: 8638634
• 负责人: Maria Traka
• 金额: $ 23.7万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8638634
• 关键词: Acetates; Address; Amino Acids; Animals; Aspartic Acid; Aspartoacylase; Astrocytes; Brain; Canavan Disease; Catabolism; Coculture Techniques; Culture Media; Data; Defect; Development; Disease; Disease Progression; Ethylnitrosourea; Family; Gene Expression; Gene Proteins; Genes; Glutamates; Hydrolysis; Knowledge; Lead; Life; Link; Lipids; Mediating; Metabolism; Molecular; Mus; Mutant Strains Mice; Mutation; Myelin; N-acetylaspartate; Neurons; Nonsense Mutation; Oligodendroglia; Pathogenesis; Patients; Research; Retinal Ganglion Cells; Role; Spinal Cord; Stem cells; Structure; Symptoms; Testing; Urine; Wild Type Mouse; base; cell type; in vivo; leukodystrophy; member; mutant; myelin degeneration; myelination; postnatal; prevent; protein expression; public health relevance; repaired; solute; therapeutic target; white matter

DESCRIPTION (provided by applicant): Canavan disease (CD) is a rare autosomal recessive leukodystrophy that is caused by mutations of the aspartoacylase gene (ASPA). ASPA is highly expressed in mature oligodendrocytes (OLs), where it catalyzes the hydrolysis of the most abundant amino acid in the brain, N-acetyl-aspartate (NAA) to acetate and aspartic acid. The mechanism of CD pathogenesis, however, remains unknown. One hypothesis has been that the loss of ASPA results in reduced levels of acetate, a precursor for myelin lipid synthesis. We recently described the identification of the ENU-induced nonsense mutation, Q193X, in the mouse Aspa gene that results in the absence of detectable ASPA protein expression in Aspanur7 homozygous mutant mice, which display severe spongy degeneration (vacuolation) throughout the CNS, strikingly resembling CD. High levels of NAA are found in the CSF and urine of CD patients. Similarly, NAA is increased in the CNS of Aspanur7 mutants. Therefore, another hypothesis implicates elevated NAA levels as the leading cause of myelin degeneration observed in CD. This proposal aims to investigate the molecular mechanisms that are responsible for CD pathogenesis by taking advantage of the Aspanur7 mutant. Our previous studies and recent preliminary data on the Aspanur7 mouse indicate that myelin degeneration in CD is not primarily due to a limited supply of NAA- derived acetate for the myelin lipid synthesis. Thus, a role of ASPA outside myelination is also possible. Here, we favor the hypothesis that CD pathogenesis is caused by ASPA deficiency in mediating NAA clearance in the CNS and thereby protecting myelin and/or OLs from NAA damage. We plan to address this hypothesis by establishing cocultures of purified retinal ganglion neurons (RGCs) with oligodendrocyte progenitor cells (OPCs) that result in myelination (Specific Aim 1). By comparing myelination levels and structure between cocultures of OPCs derived from the ASPA-deficient mice and wild-type ones we will be able to determine whether ASPA is required or not for myelination. These cocultures will also be used to assess the possibility that ASPA has an NAA-scavenger activity in the CNS that protects myelin and/or OLs from NAA damage (Specific Aim 1). The proposed role of ASPA as NAA-scavenger in the CNS will be further investigated in vivo by generating ASPA-deficient animals that synthesize significantly reduced NAA levels due to the deficiency of the solute carrier family 25 member 12 (Slc25a12) gene (Specific Aim 2). We anticipate that the accumulation of NAA observed in the CNS of the Aspanur7 mutants will be significantly reduced in the Aspanur7/nur7;Slc25a12-/- animals, which might lead to amelioration of their CD symptoms as compared to the Aspanur7/nur7 mice, supporting that ASPA's activity in OLs protects OLs and/or myelin from the potential detrimental effects of NAA accumulation observed in CD. Overall, the results produced by the proposed Aims could promote our understanding on the mechanism of the CD pathogenesis and eventually help us develop therapeutic targets to prevent disease progression and thereby potentially enhance myelin repair in the CNS of the CD patients.

描述（由申请人提供）：Canavan病（CD）是由Aspartoyacylase基因（ASPA）突变引起的罕见常染色体隐性白细胞营养不良。 ASPA在成熟的少突胶质细胞（OLS）中高度表达，它催化了大脑中最丰富的氨基酸的水解，N-乙酰基天冬氨酸（NAA）至乙酸和天冬氨酸。但是，CD发病机理的机制仍然未知。一种假设是，ASPA的损失导致乙酸水平降低，乙酸水平是髓磷脂脂质合成的前体。我们最近描述了在小鼠ASPA基因中鉴定eNU诱导的胡说八道突变Q1193X，导致在均表现出严重的海胶变性（液化）的ASPANUR7纯合突变小鼠中没有可检测到的ASPA蛋白表达。 CD患者的CSF和尿液中发现了高水平的NAA。同样，在Aspanur7突变体的中枢神经系统中，NAA增加了。因此，另一个假设暗示NAA水平升高是CD中观察到的髓磷脂变性的主要原因。该建议旨在通过利用Aspanur7突变体来研究负责CD发病机理的分子机制。我们以前的研究和有关Aspanur7小鼠的最新初步数据表明，CD中的髓磷脂变性并不主要是由于髓磷脂脂质合成的NAA衍生乙酸供应有限。 因此，在髓鞘化之外的ASPA角色也是可能的。在这里，我们赞成以下假设：CD发病机理是由ASPA缺乏在中枢神经系统中介导NAA清除率的情况下引起的，从而保护髓磷脂和/或OLS免受NAA损伤。我们计划通过建立与少突胶质细胞祖细胞（OPC）建立纯化的视网膜神经元神经元（RGC）的共培养，从而解决这一假设（特定目标1）。通过比较源自ASPA缺陷小鼠和野生型的OPC的共培养水平和结构，我们将能够确定是否需要ASPA才能用于骨髓。这些共培养物还将用于评估ASPA在中枢神经系统中具有NAA范围活动的可能性，可保护髓磷脂和/或OLS免受NAA损害（特定AIM 1）。通过生成由于溶质载体家族25成员12（SLC25A12）基因的缺乏，将在体内进一步研究ASPA作为中枢神经系统中NAA范围的作用，从而在体内进一步研究。我们预计，在Aspanur7突变体中观察到的NAA的积累将大大减少，而Aspanur7/Nur7; Slc25a12 - / - 动物在Aspanur7/Nur7小鼠中的cd and和/aSPA的活性相比，可能会导致OLS和/或/或或/或或/或或/或sya的活动，这可能会导致其CD症状，这可能会导致其CD症状，从而使OLS和/或IM/OLS的活动受到影响。在CD中。总体而言，提出的目标产生的结果可以促进我们对CD发病机理机制的理解，并最终帮助我们开发治疗靶标，以防止疾病进展，从而可能增强CD患者中枢神经系统的髓磷脂修复。