Investigation of Pitt-Hopkins Syndrome pathophysiology using a human model

项目摘要 自闭症谱系障碍影响着全世界数百万人， 儿童、他们的家庭和卫生保健系统。Pitt-Hopkins综合征（PTHS）是一种ASD， TCF 4基因的新突变。PTHS的特征是严重的智力残疾， 发育和运动延迟，言语缺失，重复行为，特殊的面部完形，以及 胃肠道表现。虽然PTHS的遗传病因学已得到很好的确立，但细胞和神经系统的病理学改变可能与PTHS的发病有关。 人类患者的表型改变仍然没有完全了解，也不清楚TCF 4突变是如何发生的。 会导致这种异常。对PTHS的分子和细胞机制缺乏了解是一个 问题，因为直到这些信息变得可用，特定的改变途径不能治疗， 针对性地而且，没有神经病理学知识，是不可能治疗并最终治愈PTHS的 通过直接纠正基因组中的突变。 我们的长期目标是了解大脑中特定的遗传缺陷和改变的通路是如何导致 自闭症儿童表现出的衰弱的表型。本申请的目的是：（a）使用 体外神经发育的人类模型，以确定 PTHS中临床相关的TCF 4突变;和（B）提供概念验证， 策略可用于固定TCF 4表达，这种方法最终可用于基因治疗， PTHS。我们的中心假设是TCF 4突变导致特定细胞类型的异常表型。 神经系统，导致患者的神经症状。我们假设，患者来源的体外 PTHS模型比小鼠模型更能概括病理生理学，因为脑结构， 基因组结构和发育在啮齿动物和人类之间差异很大， 模型不能精确地模拟疾病的所有临床相关方面。在初步实验中，我们 获得了患者来源的脑类器官和体外培养的神经细胞类型，并将其用作人类 模型显示PTHS神经祖细胞表现出衰老和增殖降低， 伴随着Wnt信号传导和SOX 3表达的下调。此外，我们观察到PTHS 脑类器官不能发育出正常的解剖学组织的祖细胞结构， 显示出严重受损的燃烧性能。我们的预期成果/可交付成果包括： 操纵特定的改变的分子途径和神经细胞类型，并测试遗传学特性。 这种疾病的矫正策略，这可能会推动未来的药理学和基因研究 治疗PTHS。

PROJECT SUMMARY Autism-spectrum disorders impact millions of individuals worldwide, representing a heavy toll on affected children, their families, and the health care system. Pitt–Hopkins Syndrome (PTHS) is an ASD caused by de novo mutations in the TCF4 gene. PTHS is characterized by severe intellectual disability, pronounced developmental and motor delays, absence of speech, repetitive behaviors, peculiar facial gestalt, and gastrointestinal manifestations. While the genetic etiology of PTHS is well established, the cellular and neural phenotypic alterations in human patients are still not fully understood, nor is it clear how TCF4 mutations cause such abnormalities. Lack of understanding about PTHS's molecular and cellular mechanisms is a problem because, until this information becomes available, specific altered pathways cannot be therapeutically targeted. Moreover, without neuropathological knowledge, it is impossible to treat and eventually cure PTHS by directly correcting the mutation in the genome. Our long-term goal is to understand how specific genetic defects and altered pathways in the brain result in the debilitating phenotypes exhibited by autistic children. The objectives of this application are to: (a) use human models of neural development in vitro to define the cellular and neural pathological consequences of clinically relevant TCF4 mutations in PTHS; and (b) provide proof-of-concept that correctional molecular strategies can be used to fix TCF4 expression, an approach that could eventually be used as gene therapy for PTHS. Our central hypothesis is that TCF4 mutations cause aberrant phenotypes in specific cell types of the nervous system, leading to the patients' neurological symptoms. We postulated that patient-derived in vitro models of PTHS can better recapitulate the pathophysiology than mouse models, because brain structure, genome architecture and development vary greatly between rodents and humans, and current PTHS animal models do not closely mimic all the disease's clinically relevant aspects. In preliminary experiments, we obtained patient-derived brain organoids and cultured neural cell types in vitro and used them as human models to show that PTHS neural progenitor cells exhibit senescence and decreased proliferation, accompanied by downregulation of Wnt signaling and SOX3 expression. Moreover, we observed that PTHS brain organoids fail to develop normal anatomically organized progenitor structures and that PTHS neurons display severely impaired firing properties. Our anticipated results/deliverables include the identification and manipulation of specific altered molecular pathways and neural cell types and the testing of genetic correctional strategies for the disease, which could propel future research on pharmacological and gene therapy for PTHS.