In vitro and in vivo functional analysis of Ikbkap in Familial Dysautonomia

Ikbkap 在家族性自主神经功能障碍中的体外和体内功能分析

• 批准号: 8007310
• 负责人: Marisa Zoe-Tatiana Jackson
• 金额: $ 3.35万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8007310
• 关键词: 5' Splice Site; Affect; Afferent Neurons; Autonomic nervous system; Biological Models; Cells; Congenital neurologic anomalies; Degenerative Disorder; Development; Disease; Dysautonomias; Exons; Experimental Models; Familial Dysautonomia; Genes; Hereditary Disease; Hereditary Sensory and Autonomic Neuropathies; Human; In Vitro; Introns; Knockout Mice; Lead; Mutation; Nerve Degeneration; Nervous system structure; Neurons; Pathogenesis; Play; Point Mutation; Proteins; RNA Interference; RNA Splicing; Role; Sensory; Sympathetic Nervous System; Testing; Tissues; Transcript; Translating; in vivo; migration; neoplastic; nerve supply; nervous system development; neuron development; premature; public health relevance

DESCRIPTION (provided by applicant): Sympathetic nervous system (SNS) abnormalities have been implicated in a wide variety of congenital, neurodegenerative and neoplastic diseases. However, few molecules have been specifically identified in the pathogenesis of human SNS-related diseases. Familial Dysautonomia (FD; Riley-Day Syndrome; hereditary sensory and autonomic neuropathy, type 3; HSAN3;OMIM 223900) is one of five well-recognized genetic disorders affecting the sensory and autonomic nervous systems. A highly conserved point mutation located in the donor splice site of intron 20 of the human IKBKAP gene has been identified in >99.5% of FD cases. The mutation weakens the intron-exon boundary definition between exon 20 and 21, leading to exon-20 deficient transcripts and premature truncation of the translated protein. The abnormal splicing preferentially occurs in the nervous system, but the cause and functional significance is not well understood. To better understand the function of IKAP, the protein encoded by the IKBKAP gene, in FD and specifically its role in sympathetic and sensory neuron development, I will 1) characterize the effects of IKAP knockdown by utilizing RNA interference (RNAi) in primary sympathetic and sensory neuron culture and 2) generate and characterized a conditional IKBKAP knockout (cKO) mouse to explore the cell autonomous function of IKAP in SNS development. These studies will test the hypothesis that loss of IKAP in developing neurons can lead to cell autonomous abnormalities involving neuron migration, differentiation, survival and/or target tissue innervation. PUBLIC HEALTH RELEVANCE: A highly conserved point mutation located in the donor splice site of intron 20 of the human IKBKAP gene has been found in >99.5% cases of Familial Dysautonomia (FD), a severe neuro-developmental and degenerative disease. This mutation leads to loss of IKAP protein, yet we understand very little about how it plays a role in disease. Here, I am proposing to investigate the role that IKAP has in developing sensory and sympathetic neurons, and I will develop and characterize a new conditional IKBKAP knockout mouse which will make it possible to study its mechanisms of action in an experimental model system.

描述（由申请人提供）：交感神经系统（SNS）异常与多种先天性、神经退行性和肿瘤性疾病有关。然而，在人类 SNS 相关疾病的发病机制中，很少有分子被具体鉴定。家族性自主神经功能障碍（FD；Riley-Day 综合征；遗传性感觉和自主神经病，3 型；HSAN3；OMIM 223900）是五种公认的影响感觉和自主神经系统的遗传性疾病之一。超过 99.5% 的 FD 病例中发现了位于人类 IKBKAP 基因内含子 20 供体剪接位点的高度保守点突变。该突变削弱了外显子 20 和 21 之间的内含子-外显子边界定义，导致外显子 20 转录本缺陷和翻译蛋白过早截短。异常剪接优先发生在神经系统中，但其原因和功能意义尚不清楚。为了更好地了解 IKAP（IKBKAP 基因编码的蛋白质）在 FD 中的功能，特别是其在交感神经和感觉神经元发育中的作用，我将 1) 通过利用 RNA 干扰 (RNAi) 来表征 IKAP 敲低对初级交感神经和感觉神经元发育的影响。感觉神经元培养；2) 生成并表征条件性 IKBKAP 敲除 (cKO) 小鼠，以探索 IKAP 在 SNS 发育中的细胞自主功能。这些研究将检验以下假设：发育中的神经元中 IKAP 的缺失会导致涉及神经元迁移、分化、存活和/或靶组织神经支配的细胞自主异常。 公共健康相关性：在超过 99.5% 的家族性自主神经功能障碍 (FD)（一种严重的神经发育和退行性疾病）病例中发现了位于人类 IKBKAP 基因内含子 20 供体剪接位点的高度保守点突变。这种突变导致 IKAP 蛋白丢失，但我们对其如何在疾病中发挥作用知之甚少。在这里，我提议研究 IKAP 在感觉和交感神经元发育中的作用，并且我将开发并表征一种新的条件性 IKBKAP 敲除小鼠，这将使在实验模型系统中研究其作用机制成为可能。