INTERVENTION STRATEGIES FOR NON-FOLATE RESPONSIVE NEURAL TUBE DEFECTS

 描述(申请人提供)：神经管缺陷(NTD)是人类最常见的出生缺陷之一。NTDS的原因是多因素的，包括遗传、环境和营养因素。母亲的叶酸(FA)状态是NTD易感性的最强联系之一。大量研究表明，在一些人群中，补充FA可以降低NTD患病率高达70%。尽管经过了40多年的密集努力，我们仍然不了解这些叶酸依赖过程背后的机制。我们已经开始利用一种新的小鼠NTD模型(Mthfd1l KO)来解决这一现有的数据差距，该模型紧密复制了人类NTD表型，并且不需要额外的营养干预来表达NTD表型。在这个新的小鼠模型中，一种特定的叶酸依赖酶(线粒体Mthfd1l)的丢失会导致NTDS。这是迄今与NTD易感性/病因学相关的最特异的代谢缺陷，表明FA为核苷酸和甲基团的生物合成提供了必要的一碳单位。这些生物合成途径在快速生长的胚胎中尤其活跃，在神经管关闭(NTC)期间它们支持细胞的增殖和死亡、迁移和分化。我们将使用这个小鼠模型检验下列特定的假设：(1)母体补充蛋氨酸、嘌呤、胸苷和S-腺苷蛋氨酸可以保护Mthfd1l KO(Mthfd1lz/z)零合子胚胎免受NTDS的影响，(2)Depakote(丙戊酸；VPA)是药物性NTDS的主要原因，它抑制线粒体1C代谢，因此甲酸盐可能可以预防由该致畸原引起的NTDS，以及(3)Mthfd1z/z胚胎的细胞增殖和凋亡、细胞迁移和分化程序被扰乱，导致神经管和口腔面部缺陷。我们证明，在母体中补充Mthfd1酶反应的产物甲酸盐，可以降低NTDS的发生率，并部分挽救缺乏功能性Mthfd1的胚胎的生长缺陷。在特定的目标1中，我们将确定Mthfd1l反应下游的哪些补充剂可以挽救NTD表型。这将在一些FA反应性和非反应性NTD突变株以及VPA敏感的小鼠株系中进行探索。我们将确定哪些细胞过程在Mthfd1lz/z胚胎和对VPA敏感的小鼠品系中存在失调，从而导致不适当的NTC。将进行代谢学和表观遗传学研究，以全面描述突变小鼠的特征。具体目标2将侧重于神经干细胞和神经脊干细胞对甲酸盐的需求，使用神经球生长和分化分析，以及其他表观遗传学研究。具体目标3将涉及对人类Mthfd1l基因进行DNA重新测序，并对脊柱裂队列中已识别的变异进行功能分析。这项研究计划通过阐明甲酸盐预防非FA反应性NTDS的潜在机制，为开发第一个针对非FA反应性NTDS的有效干预措施带来了希望。开发有益于非叶酸反应性NTDS的干预措施，对于预防这些可预防的出生缺陷至关重要。

 DESCRIPTION (provided by applicant): Neural tube defects (NTDs) are among the most common birth defects in humans. The causes of NTDs are multifactorial, including genetic, environmental, and nutritional factors. Maternal folic acid (FA) status is one of the strongest links to NTD susceptibility. Numerous studies have shown that supplemental FA can reduce NTD prevalence by as much as 70% in some populations. Despite more than 40 years of intensive effort, we still do not understand the mechanisms that underlie these folate-dependent processes. We have begun to address this existing data gap utilizing a new mouse NTD model (Mthfd1l KO) that closely replicates the human NTD phenotype, and does not require additional nutritional intervention to express the NTD phenotype. In this new mouse model, loss of a specific folate-dependent enzyme (mitochondrial MTHFD1L) leads to NTDs. This is the most specific metabolic defect yet associated with NTD susceptibility/etiology, and suggests that FA provides essential one-carbon units for nucleotide and methyl group biosynthesis. These biosynthetic pathways are especially active in the rapidly growing embryo, where they support cell proliferation and death, migration, and differentiation during neural tube closure (NTC). We will test the following specific hypotheses using this mouse model: (1) Maternal supplementation with methionine, purines, thymidylate and S-adenosylmethionine can protect against NTDs in nullizygous Mthfd1l KO (Mthfd1lz/z) embryos, (2) Depakote (Valproic Acid; VPA), the leading cause of pharmaceutical-induced NTDs, inhibits mitochondrial 1C metabolism, thus it is possible that formate can prevent NTDs caused by this teratogen, and (3) cell proliferation and apoptosis, cell migration, and differentiation programs are disrupted in Mthfd1lz/z embryos, leading to neural tube and orofacial defects. We demonstrated that maternal supplementation of MTHFD1L dams with formate, the product of the MTHFD1L enzymatic reaction, decreases the incidence of NTDs and partially rescues the growth deficit in embryos lacking a functional Mthfd1l. In Specific Aim 1 we will determine which supplements downstream of the MTHFD1L reaction can rescue the NTD phenotype. This will be explored in a number of FA responsive and non-responsive NTD mutant strains, and in VPA-sensitive mouse strains. We will identify which cellular processes are dysregulated in Mthfd1lz/z embryos and VPA-sensitive mouse strains, leading to improper NTC. Metabolomic and epigenetic studies will be pursued to fully characterize the mutant mice. Specific Aim 2 will focus on the requirement for formate in neural stem cells and neural crest stem cells using neurosphere growth and differentiation assays, as well as additional epigenetic investigations. Specific Aim 3 will involve DNA resequencing of the human MTHFD1L gene and functional analyses of identified variants in a spina bifida cohort. This research program offers hope for developing the first effective intervention for non-FA responsive NTDs by illuminating the underlying mechanisms by which formate prevents NTDs. Developing interventions that benefit non-folate responsive NTDs is crucial for preventing these preventable birth defects.