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）：卡纳万病（CD）是一种罕见的常染色体隐性遗传性脑白质营养不良，由天冬氨酸酰化酶基因（ASPA）突变引起。 ASPA 在成熟的少突胶质细胞 (OL) 中高度表达，催化大脑中最丰富的氨基酸 N-乙酰天冬氨酸 (NAA) 水解为乙酸盐和天冬氨酸。然而，CD 的发病机制仍不清楚。一种假设是，ASPA 的缺失会导致乙酸盐水平降低，而乙酸盐是髓磷脂脂质合成的前体。我们最近描述了小鼠 Aspa 基因中 ENU 诱导的无义突变 Q193X 的鉴定，该突变导致 Aspanur7 纯合突变小鼠中缺乏可检测的 ASPA 蛋白表达，这些小鼠在整个中枢神经系统中表现出严重的海绵变性（空泡化），与光盘。 CD 患者的脑脊液和尿液中发现高浓度的 NAA。类似地，Aspanur7 突变体的 CNS 中 NAA 增加。因此，另一个假设表明 NAA 水平升高是 CD 中观察到的髓磷脂变性的主要原因。该提案旨在利用 Aspanur7 突变体研究 CD 发病机制的分子机制。我们之前的研究和最近对 Aspanur7 小鼠的初步数据表明，CD 中的髓磷脂变性并非主要是由于用于髓磷脂脂质合成的 NAA 衍生乙酸盐供应有限。 因此，ASPA 在髓鞘形成之外的作用也是可能的。在这里，我们支持这样的假设：CD 发病机制是由 ASPA 缺乏介导 CNS 中的 NAA 清除而引起的，从而保护髓磷脂和/或 OL 免受 NAA 损伤。我们计划通过建立纯化的视网膜神经节神经元 (RGC) 与少突胶质细胞祖细胞 (OPC) 的共培养来解决这一假设，从而导致髓鞘形成（具体目标 1）。通过比较来自 ASPA 缺陷小鼠和野生型小鼠的 OPC 共培养物之间的髓鞘形成水平和结构，我们将能够确定髓鞘形成是否需要 ASPA。这些共培养物还将用于评估 ASPA 在 CNS 中具有 NAA 清除剂活性的可能性，可保护髓磷脂和/或 OL 免受 NAA 损伤（具体目标 1）。拟议的 ASPA 作为 CNS 中 NAA 清除剂的作用将通过生成 ASPA 缺陷动物进行体内进一步研究，由于溶质载体家族 25 成员 12 (Slc25a12) 基因的缺陷，这些动物合成的 NAA 水平显着降低（具体目标 2） ）。我们预计 Aspanur7/nur7;Slc25a12-/- 动物中在 Aspanur7 突变体的 CNS 中观察到的 NAA 积累将显着减少，与 Aspanur7/nur7 小鼠相比，这可能会改善其 CD 症状，支持ASPA 在 OL 中的活性可以保护 OL 和/或髓磷脂免受 CD 中观察到的 NAA 积累的潜在有害影响。总体而言，所提出的目标产生的结果可以促进我们对 CD 发病机制的理解，并最终帮助我们开发治疗靶点以防止疾病进展，从而潜在地增强 CD 患者中枢神经系统的髓磷脂修复。