Defining disease mechanisms in SLC35A2 epilepsy

定义 SLC35A2 癫痫的疾病机制

基本信息

批准号：
10191063
负责人：
Peter B Crino
金额：
$ 68.96万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10191063
关键词：
3-Dimensional Adult Age of Onset Alleles Astrocytes Biological Assay Brain CRISPR/Cas technology Cell Fraction Cell Line Cell Nucleus Cells Clustered Regularly Interspaced Short Palindromic Repeats Complement Complementary DNA Conceptions Congenital disorders of glycosylation Cortical Dysplasia Cortical Malformation Coupled Data Defect Dendritic Spines Development Diagnostic radiologic examination Disease Electroencephalogram Electrophysiology (science)Electroporation Embryonic Development Epilepsy Event FDA approved Freezing Functional disorder Galactose Galactosyltransferases Genes Genotype Glutamates Glycoconjugates Glycosphingolipids Golgi Apparatus Guide RNA Human Immunoblotting Impairment Individual Induced pluripotent stem cell derived neurons Intractable Epilepsy Kinetics Knock-out Lead Length Link Lipids Liquid Chromatography Mass Spectrum Analysis Metabolic Diseases Microscopy Modeling Molecular Monitor Morphology Mosaicism Mus Mutation Neurites Neurons Oligodendroglia Organoids Parents Partial Epilepsies Pathogenicity Pathologic Pathology Pathway interactions Patients Phenotype Polysaccharides Population Process Production Protein Glycosylation Proteoglycan Refractory Resected Role Sampling Scanning Seizures Series Severities Severity of illness Somatic Mutation Specimen Supplementation Syndrome Therapeutic Time Translating Uridine Diphosphate Galactose Variant Work X Chromosome brain cell brain dysfunction brain tissue cell type clinical phenotype de novo mutation druggable target early onset effectiveness evaluation epileptic encephalopathies fetal genetic variant girls glycosylation hemimegalencephaly in utero induced pluripotent stem cell insertion/deletion mutation loss of function mRNA Expression migration mouse model multi-electrode arrays mutant neocortical nerve stem cell neural network neurodevelopment neurogenesis neuron development neuronal cell body novel offspring patch clamp precision medicine prevent protein transport relating to nervous system time use transcriptome sequencing

项目摘要

ABSTRACT Many epilepsy syndromes associated with severe, early-onset seizures result from de novo variants in genes involved in early brain development. Recent studies have also identified somatic variants in focal epilepsy associated with cortical malformations, including hemimegalencephaly and the more common focal cortical dysplasia (FCD) type 2. These post-zygotically acquired variants arise during neurogenesis and are therefore present in only a fraction of cells. Expanding on these early discoveries implicating somatic variants in epilepsy, we recently identified brain-specific somatic mutations in SLC35A2 in individuals with refractory neocortical epilepsy. Germline variants in SLC35A2 were previously implicated in a rare X-linked developmental and epileptic encephalopathy. Our data suggest that somatic variants in SLC35A2 may also be responsible for approximately 17% of intractable non-lesional focal epilepsy cases. The number of cells harboring a pathogenic SLC35A2 variant allele appears to correlate with disease severity, and several of the cases have FCD type 1a (FCD1a) pathology. Pathogenic variants in SLC35A2, both somatic and germline, prevent the Golgi-localized transporter from moving UDP-Galactose (UDP-Gal) into the Golgi apparatus for use in the formation of essential galactosylated glycans. There is theoretical, experimental, and observational data suggesting that Gal supplementation may be able to restore glycosylation to the cell to provide therapeutic benefit. In this study, we will define the functional consequences of SLC35A2 variants in epilepsy. Given that not all cells carry the variant allele in the individuals with a somatic SLC35A2 variant, in Aim 1 we seek to use resected human brain tissue specimens to identify the specific cell types in the brain harboring the variant alleles. This will allow us to determine which cell types contribute to SLC35A2 epilepsy and whether cell-type-specific burden dictates the pathological observations. In Aim 2 we will evaluate the effects of the variants on cell-type-specific glycosylation in human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells and mature glutamatergic neurons, a cell type that we have preliminary data to support involvement in the epileptogenic processes. Since nearly all patients with an SLC35A2 variant have seizures, and a significant fraction has FCD1a, in Aim 3 we will also characterize the effects of the variants on individual and neural network activity, neural migration, and neurodevelopment in both human (e.g., hiPSC-derived neurons, 3-D organoids) and mouse models (e.g., mouse neural progenitors and in utero electroporation). In Aims 2 and 3, we will assess the effectiveness of Gal to restore glycosylation and reverse effects on neuronal development or activity. Collectively, these studies will translate our exciting initial discovery implicating a novel pathway underlying a significant fraction of individuals suffering from intractable seizures into studies of the role of glycosylation defects underlying localized brain dysfunction in focal epilepsy. Given that the glycosylation pathway represents a potentially druggable target, this work may inform precision medicine approaches to the treatment of refractory epilepsy.

抽象的许多与严重、早发性癫痫发作相关的癫痫综合征是由基因的新生变异引起的参与早期大脑发育。最近的研究还发现了局灶性癫痫的体细胞变异与皮质畸形相关，包括半侧巨脑畸形和更常见的局灶性皮质畸形发育不良 (FCD) 2 型。这些合子后获得性变异在神经发生过程中出现，因此是仅存在于一小部分细胞中。扩展这些涉及癫痫体细胞变异的早期发现，我们最近在难治性新皮质个体中发现了 SLC35A2 的大脑特异性体细胞突变癫痫。 SLC35A2 的种系变异先前被认为与罕见的 X 连锁发育和癫痫性脑病。我们的数据表明 SLC35A2 的体细胞变异也可能是造成大约 17% 的顽固性非病变局灶性癫痫病例。携带病原体的细胞数量 SLC35A2 变异等位基因似乎与疾病严重程度相关，其中一些病例患有 FCD 1a 型 (FCD1a)病理学。 SLC35A2 的致病性变异（体细胞和种系）阻止高尔基体定位转运蛋白将 UDP-半乳糖 (UDP-Gal) 转移至高尔基体，用于形成必需的半乳糖基化聚糖。有理论、实验和观察数据表明 Gal 补充可能能够恢复细胞的糖基化以提供治疗益处。在这项研究中，我们将定义 SLC35A2 变异在癫痫中的功能后果。鉴于并非所有细胞都携带该变体具有体细胞 SLC35A2 变异的个体中的等位基因，在目标 1 中，我们寻求使用切除的人脑组织样本来识别大脑中含有变异等位基因的特定细胞类型。这将使我们能够确定哪些细胞类型导致 SLC35A2 癫痫，以及细胞类型特异性负担是否决定病理观察。在目标 2 中，我们将评估变体对细胞类型特异性糖基化的影响在人类诱导多能干细胞 (hiPSC) 衍生的神经祖细胞和成熟谷氨酸能细胞中神经元是一种细胞类型，我们有初步数据支持其参与癫痫发生过程。自从几乎所有携带 SLC35A2 变异的患者都会出现癫痫发作，并且很大一部分患有 FCD1a，在目标 3 中，我们将还描述了变异对个体和神经网络活动、神经迁移的影响，以及人类（例如 hiPSC 衍生的神经元、3-D 类器官）和小鼠模型（例如小鼠神经祖细胞和子宫内电穿孔）。在目标 2 和 3 中，我们将评估 Gal 的有效性：恢复糖基化并逆转对神经元发育或活动的影响。总的来说，这些研究将转化我们令人兴奋的初步发现，该发现暗示了很大一部分个体潜在的新途径患有顽固性癫痫发作，开始研究局部大脑糖基化缺陷的作用局灶性癫痫的功能障碍。鉴于糖基化途径代表了一个潜在的药物靶标，这这项工作可能会为难治性癫痫的治疗提供精准医学方法。