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

CTNNB1综合征是一种以智力障碍、小头畸形、全身性发育迟缓(运动、语言)和运动障碍(躯干肌张力低下、远端高张力伴痉挛步态)为特征的发育障碍。它是由CTNNB1(Beta-Catenin)单倍体不足引起的，原因是部分或完全缺失突变。CTNNB1是智力残疾的重要风险基因。其他几个人类基因突变也会导致β-连环素水平或功能降低，以及类似的发育障碍。β-连环蛋白在两条对神经和神经肌肉的正常发育和功能至关重要的通路中发挥关键作用--典型的Wnt信号转导通路和基于钙粘附素的突触黏附复合体。CTNNB1综合征的治疗由于对潜在的病理生理机制的了解有限，对CTNNB1杂合子小鼠的研究有限，并且到目前为止还没有人类细胞模型。我们建议进行体内、小鼠和体外人类细胞研究，以解决这些关键的差距。对我们的CTNNB1生殖系杂合子小鼠的初步研究显示，与对照小鼠相比，与这种疾病相关的表型包括联想和运动学习受损，以及肌肉握力降低。我们的目标1研究将通过确定与CTNNB1综合征特征相关的三种组织类型的体内分子和功能变化来提供新的机制洞察：前脑、脊髓和骨骼肌。我们将使用多学科的定量方法、质谱学蛋白质组学、电生理记录，以及进一步的认知和运动能力改变的行为测试。我们的目标2研究将利用活体小鼠模型来验证我们的假设，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-连环蛋白单倍体不足引起的表型。我们将在与CTNNB1综合征表型相似的其他疾病小鼠模型中测试已被证明可提高β-连环素水平并改善学习和运动障碍的药物治疗。为了提高翻译相关性，Aim 3研究将建立与CTNNB1综合征特征相关的多种细胞类型的CTNNB1杂合性功能丧失的人类细胞模型：皮质谷氨酸能神经元、脊髓运动神经元和骨骼肌管。我们还将生成等基因回复对照，并纠正突变的等位基因。我们将使用质谱学蛋白质组学来定义由β-连环蛋白减少引起的分子变化。我们将测试药物治疗对纠正β-连环蛋白的疗效和分子变化。我们的发现将确定在体外人类细胞和相应的体内小鼠组织类型之间共享和独特的分子变化。共同的变化将确定核心的病理生理机制。我们还可以在不同的组织中鉴定出β-连环蛋白网络的新成分。我们的研究将在两个临床前模型中为药物治疗改善CTNNB1综合征表型和改善受影响儿童的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.