Synaptic signaling in a human stem cell model of Angelman syndrome

描述(申请人提供)：染色体15q11-q13缺失的个体患有Angelman综合征(AS)，这是一种神经遗传性发育障碍，特征是智力残疾、运动性共济失调、言语缺失和癫痫发作。特定的基因就是 AS编码泛素蛋白连接酶UBE3A。在AS中，突触信号和可塑性的缺陷似乎在疾病表型中起着关键作用，但UBE3A及其相关下游靶点的确切功能作用尚不清楚。为了开发合适的AS治疗方法，有必要了解UBE3A缺失引起的病理生理变化。直到最近，还不可能检查受影响个体的脑神经元的功能特性。人类体细胞基因组重编程为诱导多能干细胞(IPSC)系的发现为模拟复杂遗传学的人类疾病提供了一种新的方法。我们最近成功地将AS患者和年龄匹配的对照组的皮肤成纤维细胞重新编程为ipscs，然后将这些细胞分化为功能神经元。 这保持了AS患者中UBE3A表达的印记表型。我们现在准备利用这些新的患者来源的细胞系来测试关于AS潜在生理缺陷的特定假设。第一个目的是使用电生理和免疫细胞化学方法来探索IPSC来源神经元的内在功能特性和突触连接的活性依赖性可塑性。抢救性实验将集中在即刻早期基因ARC和钙/钙调蛋白依赖的蛋白激酶CaMKII在AS相关突触信号缺陷中的作用。第二个目标将探索AS来源神经元中突触数量和树突密度的变化。救援实验将针对Ephexin-5的作用，Ephexin-5是UBE3A的底物，在发育过程中发挥调节兴奋性突触数量的作用。总体而言，这一方法可能被证明有助于确定药物开发的新靶点，并筛选旨在改善和/或治疗Angelman综合征的癫痫发作、运动障碍以及语言和认知障碍的潜在疗法。

DESCRIPTION (provided by applicant): Individuals with a deletion of chromosome 15q11-q13 suffer from Angelman syndrome (AS), a neurogenetic developmental disorder characterized by intellectual disability, motor ataxia, absent speech, and seizures. The specific gene that is responsible for AS encodes the ubiquitin protein ligase UBE3A. In AS, deficits in synaptic signaling and plasticity appear to play a critical role in the disease phenotype, but the exact functional role of UBE3A and its relevant downstream targets are unknown. In order to develop appropriate treatments for AS, it is necessary to understand the pathophysiological changes caused by UBE3A deletion. Until recently it has not been possible to examine the functional properties of brain neurons in affected individuals. The discovery of genomic reprogramming of human somatic cells into induced pluripotent stem cell (iPSC) lines provides a novel way to model human diseases with complex genetics. We have recently succeeded in reprogramming dermal fibroblasts from AS patients, as well as age-matched control subjects, into iPSCs, and then differentiated these cells into functional neurons that maintain the imprinting phenotype of UBE3A expression seen in AS patients. We are now poised to take advantage of these novel patient-derived cell lines to test specific hypotheses about the underlying physiological defects in AS. The first aim uses electrophysiological and immunocytochemical approaches to explore the intrinsic functional properties of iPSC-derived neurons and activity-dependent plasticity of synaptic connections. Rescue experiments will focus on the roles of the immediate early gene ARC and the calcium/calmodulin-dependent protein kinase CaMKII in AS-associated deficits in synaptic signaling. The second aim will explore changes in synapse number and dendritic spine density in AS-derived neurons. Rescue experiments will target the role of ephexin-5, a substrate of UBE3A that plays a role in regulating excitatory synapse number during development. Overall, this approach may prove useful for identifying novel targets for drug discovery and for screening potential therapeutics aimed at ameliorating and/or curing the seizures, movement disorders, and language and cognitive impairments in Angelman syndrome.