Investigating molecular mechanisms and treatments for CTNNB1 Syndrome using mouse and human models

CTNNB 1综合征是一种发育障碍，其特征为智力残疾、小头畸形、全面发育迟缓（运动、语言）和运动残疾（躯干肌张力减退、远端张力亢进伴痉挛性步态）。它是由CTNNB 1（β-连环蛋白）由于部分或完全缺失突变单倍不足引起的。CTNNB 1是智力残疾的重要危险基因。其他几种人类基因突变也会导致β-连环蛋白水平或功能降低以及类似的发育障碍。β-连环蛋白在两条对正常神经和神经肌肉发育和功能至关重要的途径中发挥关键作用-经典Wnt信号转导途径和基于钙粘蛋白的突触粘附复合物。由于对潜在的病理生理学机制的了解有限，CTNNB 1杂合子小鼠的研究有限，而且迄今还没有人类细胞模型，因此缺乏CTNNB 1综合征的治疗方法。我们建议在体内小鼠和体外人类细胞研究，以解决这些关键的差距。我们的CTNNB 1种系杂合子小鼠的初步研究显示，与对照组同窝小鼠相比，与这种疾病相关的表型，受损的联想和运动学习，以及降低的肌肉握力。我们的目标1研究将通过识别与CTNNB 1综合征特征相关的三种组织类型（前脑、脊髓和骨骼肌）的体内分子和功能变化，提供新的机制见解。我们将使用多学科的定量方法，质谱蛋白质组学，电生理记录，并进一步行为测试改变认知和运动能力。我们的目标2研究将利用体内小鼠模型来检验我们的假设，即使β-连环蛋白水平正常化的药物治疗将改善或补救由β-连环蛋白单倍不足引起的表型。我们将测试已被证明可以增加β-连环蛋白水平并改善CTNNB 1综合征表型相似的其他疾病小鼠模型的学习和运动缺陷的药物治疗。为了增加翻译相关性，Aim 3研究将在与CTNNB 1综合征特征相关的多种细胞类型中产生CTNNB 1杂合功能丧失的人细胞模型：皮质神经元、脊髓运动神经元和骨骼肌管。我们还将产生校正了突变等位基因的同基因回复突变体对照。我们将使用质谱蛋白质组学来定义由β-连环蛋白减少引起的分子变化。我们将测试用于校正β-连环蛋白和分子变化的药物治疗的功效。我们的研究结果将确定体外人类细胞和相应的体内小鼠组织类型之间的共享和独特的分子改变。共同的变化将确定核心病理生理机制。我们还可以在不同的组织中鉴定β-连环蛋白网络的新组分。我们的研究将在两个临床前模型中为药物治疗改善CTNNB 1综合征表型和改善受影响儿童生活质量的潜力提供关键的概念验证。

CTNNB1 Syndrome is a developmental disorder characterized by intellectual disabilities, microcephaly, global developmental delays (motor, language) and motor disabilities (truncal muscle hypotonia, distal hypertonia with spastic gait). It is caused by CTNNB1 (Beta-catenin) haploinsufficiency due to partial or complete deletion mutations. CTNNB1 is a significant risk gene for intellectual disabilities. Several other human gene mutations also cause reduced Beta-catenin levels or functions and similar developmental disorders. Beta-catenin plays key roles in two pathways critical for proper neural and neuromuscular development and function- the canonical Wnt signal transduction pathway and cadherin-based synaptic adhesion complexes. Treatments for CTNNB1 Syndrome are lacking due to limited knowledge of the underlying pathophysiological mechanisms, limited studies of CTNNB1 heterozygous mice and as yet no human cell models. We propose in vivo mouse and in vitro human cell studies to address these critical gaps. Preliminary studies of our CTNNB1 germline heterozygous mouse show phenotypes relevant to this disorder, impaired associative and motor learning, and reduced muscle grip strength, compared with control littermates. Our Aim 1 studies will provide novel mechanistic insights by identifying in vivo molecular and functional changes in three tissue types relevant to CTNNB1 syndrome features: forebrain, spinal cord and skeletal muscle. We will use multi-disciplinary quantitative approaches, mass spectrometry proteomics, electrophysiological recordings, and further behavioral testing for altered cognitive and motor capabilities. Our Aim 2 studies will utilize the in vivo mouse model to test our hypothesis that drug treatments that normalize Beta-catenin levels will improve or remedy the phenotypes caused by Beta-catenin haploinsufficiency. We will test drug treatments that have been shown to increase Beta-catenin levels and improve learning and motor deficits in mouse models of other disorders with similar phenotypes to CTNNB1 syndrome. To increase translational relevance, Aim 3 studies will generate human cell models of CTNNB1 heterozygous loss-of-function in multiple cell types relevant to CTNNB1 syndrome features: cortical glutamatergic neurons, spinal motoneurons and skeletal myotubes. We will also generate isogenic revertant controls with the mutated allele corrected. We will define molecular changes caused by reduced Beta-catenin, using mass spectrometry proteomics. We will test the efficacy of drug treatments for correction of Beta-catenin and the molecular changes. Our findings will identify both shared and unique molecular alterations between the in vitro human cells and the corresponding in vivo mouse tissue types. Shared changes will identify core pathophysiological mechanisms. We may also identify novel components of the Beta-catenin network in the different tissues. Our studies will provide critical proof-of-concept in two preclinical models for the potential of drug treatments to ameliorate phenotypes of CTNNB1 syndrome and improve quality of life for affected children.