Identifying molecular and cellular deficits in a human model of myelin disease

 描述（由申请人提供）：Pelizaeus-Merzbacher病（PMD）是一种严重的儿科神经退行性髓鞘疾病，是称为脑白质营养不良的髓鞘疾病的原型。虽然是一种单基因的X连锁疾病，但在蛋白脂质蛋白1（PLP 1）中发现的基因突变的多样性以及与这些突变相关的广泛的临床表型混淆了先前在动物模型中研究PMD的尝试。在人类细胞中的直接研究也同样受到限制，这是由于初级少突胶质细胞的不可接近性和死后组织中结构的保存较差。随着诱导多能干细胞（iPSC）技术和细胞命运再造的出现，我们现在有了体外生成少突胶质细胞的方法，为PMD患者特异性细胞提供了前所未有的途径。我开发了一组独特的PMD患者样本，概括了在更大的患者人群中发现的基因型和表型异质性。我已经将每个细胞系重新编程为iPSCs，并验证了它们的多能性和PLP 1突变。基于我实验室在啮齿动物和其他实验室最近的人类实验方案中的工作，我已经使用发育线索快速而有力地产生PMD患者特异性和控制少突胶质细胞。初步体外结果显示PMD患者在PLP 1运输、内质网应激和少突胶质细胞形态学方面存在特定缺陷。当注射到小鼠体内时，来自对照人类胚胎干细胞和PMD点突变iPSC的少突胶质细胞能够移植，广泛迁移，并将髓鞘包裹在内源性轴突周围，但PMD系未能完全成熟，这表明该患者的疾病病因。本文提出的研究旨在对这一大组PMD衍生的少突胶质细胞进行体外和体内全面的分子和细胞分析，以确定ER应激、蛋白质转运和髓鞘超微结构中患者特异性的缺陷。这些缺陷的识别将直接搜索患者相关的 分子和基因治疗学。最终，我们希望小组的广度将使我们有机会通过疾病相关指标对PLP 1突变进行分类，使我们的结果在更广泛的PMD患者人群中推广。

 DESCRIPTION (provided by applicant): Pelizaeus-Merzbacher Disease (PMD) is a severe pediatric neurodegenerative myelin disorder and archetype for the class of myelin disorders known as leukodystrophies. Though a monogenic, X-linked disease, the diversity of genetic mutations found in proteolipid protein 1 (PLP1) and the wide spectrum of clinical phenotypes associated with these mutations have confounded prior attempts to study PMD in animal models. Direct studies in human cells have been similarly limited due to the inaccessibility of primary oligodendrocytes and poor preservation of structures in post mortem tissues. With the advent of induced pluripotent stem cell (iPSC) technologies and cell fate reengineering, we now have methods for oligodendrocyte generation in vitro, providing unprecedented access to PMD patient-specific cells. I have developed a panel of unique PMD patient samples that recapitulates the genotypic and phenotypic heterogeneity found across the greater patient population. I have reprogrammed each line into iPSCs and validated their pluripotent identity and PLP1 mutations. Building off my lab's work in rodents and other labs' recent human protocols, I have used developmental cues to quickly and robustly generate PMD patient-specific and control oligodendrocytes. Preliminary in vitro results reveal PMD patient-specific defects in PLP1 trafficking, endoplasmic reticulum stress, and oligodendrocyte morphology. When injected into mice, oligodendrocytes derived from both control human embryonic stem cells and PMD point mutation iPSCs were capable of engrafting, migrating extensively, and wrapping myelin around endogenous axons, but the PMD line failed to fully mature, suggesting a etiology of disease in this patient. The studies proposed here aim to perform comprehensive molecular and cellular analyses, in vitro and in vivo, across this large panel of PMD-derived oligodendrocytes in order to identify patient-specific deficits in ER stress, protein trafficking, nd myelin ultrastructure. Identification of these deficits will direct the search for patient-relevant molecular and genetic therapeutics. Ultimately, we hope that the breadth of the panel will grant us the first opportunity to classify PLP1 mutations by disease-relevant metrics, making our results generalizable across the wider PMD patient population.