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MBNL loss of function in visceral smooth muscle as a model of myotonic dystrophy type 1

MBNL 内脏平滑肌功能丧失作为 1 型强直性肌营养不良模型

基本信息

批准号：
10605562
负责人：
Janel Ann Merkel Peterson
金额：
$ 4.77万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-15 至 2025-12-14
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10605562
关键词：
3&apos Untranslated Regions Adult Affect Affinity Alleles Alternative Splicing Anatomy Area Atrophic Binding Sites Biological Assay Blood Pressure Blood Vessels Brain Calcium Calcium Channel Cardiopulmonary Cell Line Clinical Research Cognitive Colorectal Complex Constipation Data Defect Deglutition Development Disease Embryo Esophagus Evaluation Event Exhibits Family Feces Fellowship Fetal Proteins Frequencies Functional disorder Gastrointestinal Motility Gastrointestinal Transit Gastrointestinal tract structure Genes Genetic Transcription Goals Heart High Prevalence Histologic Human Hyperplasia In Vitro Individual Intestines Investigation Knock-out Knockout Mice Large Intestine Life LoxP-flanked allele Maps Measurement Measures Messenger RNA Modeling Molecular Movement Mus Muscle Muscle Contraction Muscle Tonus Muscle Weakness Muscle function Muscular Dystrophies Myocardium Myotonia Myotonic Dystrophy Myotonic dystrophy type 1 Nutrient Obstruction Ontology Oral Pathogenesis Pathogenicity Pathologic Pathology Patients Pattern Periodicity Personal Satisfaction Phenotype Physiological Poly A Polyadenylation Protein Family Protein Isoforms Protein Kinase Proteins RNA RNA Interference RNA Processing RNA Splicing RNA-Binding Proteins Regulation Research Reverse Transcriptase Polymerase Chain Reaction Role RyR1 Severities Skeletal Muscle Smooth Muscle Smooth Muscle Myocytes Spliced Genes Structure Surveys Symptoms Tamoxifen Therapeutic Studies Thick Time Tissue Sample Tissue-Specific Gene Expression Tissues Trinucleotide Repeat Expansion Vascular Smooth Muscle Visceral Water affective disturbance autosome cell motility differential expression fluorescein isothiocyanate dextran gain of function gastrointestinal gastrointestinal system insight knock-down live cell imaging loss of function mRNA Stability motility disorder mouse model novel paralogous gene postnatal prevent release of sequestered calcium ion into cytoplasm skeletal muscle wasting spatiotemporal transcriptome sequencing transcriptomics uptake

项目摘要

ABSTRACT One in 8500 individuals are affected by myotonic dystrophy type 1 (DM1), the most common adult onset muscular dystrophy. Those affected suffer from multisystemic symptoms affecting the brain, heart, and skeletal muscle, which are active areas of investigation. DM1 patient surveys have identified gastrointestinal (GI) disturbances as a predominant patient complaint that affects daily life and well-being and include difficulty swallowing, pseudo-obstruction, and constipation. The cause of DM1 GI pathology is currently unknown and evidence supports a role for visceral smooth muscle dysfunction. The goal of this proposal is to determine the specific molecular mechanisms by which loss of MBNL function affects smooth muscle activity. DM1 is caused by a CTG trinucleotide repeat expansion, ranging between 50 to >4000 repeats, in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. DM1 pathology results from a toxic gain of function of the expanded DMPK RNA [(CUG)exp RNA]. (CUG)exp RNA sequesters and prevents activity of the muscleblind-like (MBNL) family of RNA binding proteins that regulate postnatal RNA processing networks and maintain adult RNA expression patterns. In DM1, loss of MBNL activity predominantly affects alternative splicing, leading to the expression of fetal protein isoforms in adult tissues, causing disease features. The role of two Mbnl paralogs in DM1 pathology is demonstrated by mouse Mbnl1 and/or Mbnl2 knockout (KO) that recapitulate DM1 phenotypes in brain, heart, and skeletal muscle. I am using tamoxifen inducible, conditional double KO of Mbnl1 and Mbnl2 in mice to determine the impact of MBNL loss of function in visceral smooth muscle. This will be the first investigation into the molecular mechanisms of DM1 GI features utilizing MBNL loss as previously established for other DM1 affected tissues. The sponsor’s lab generated homozygous floxed Mbnl1 and Mbnl2 alleles that were combined with a smooth muscle specific CreERT2 (smoCRE;dHOM mice). Five- week old smoCRE;dHOM mice exhibit delayed upper GI motility and known DM1-associated alternative splicing events after Mbnl knock out. Optimized dKO mice will be used to: (1) determine what GI phenotypes result from loss of MBNL, then (2) identify molecular mechanisms for smooth muscle dysfunction. In aim 1, lower GI motility will be assessed by bead expulsion assays and ex vivo assays will identify perturbations of muscle contractility and peristaltic patterning across intestinal segments. In aim 2, RNA sequencing data from both experimental mice and tissues from DM1 affected individuals will be used to identify conserved alternative splicing events, differentially expressed genes, and predominately affected gene ontologies. I anticipate identifying transcriptomic changes affecting calcium dynamics and will perform in vitro investigations of calcium handling in mouse primary smooth muscle cells and immortalized human smooth muscle cells. Together, these aims will address the myogenic basis for DM1 GI pathogenesis and drive DM1 smooth muscle research forward.

摘要每8500人中就有一人患有强直性肌营养不良1型（DM 1），这是最常见的成人发病肌肉萎缩症受影响的人患有影响大脑，心脏和骨骼的多系统症状肌肉，这是积极的调查领域。DM 1患者调查已确定胃肠道（GI）干扰是影响日常生活和健康的主要患者主诉，包括困难吞咽假性梗阻和便秘DM 1 GI病理的原因目前尚不清楚，证据支持内脏平滑肌功能障碍的作用。本提案的目的是确定 MBNL功能丧失影响平滑肌活性的特定分子机制。 DM 1是由CTG三核苷酸重复扩增引起的，在3'端，范围在50至>4000个重复之间。肌强直性营养不良症蛋白激酶（DMPK）基因的非翻译区。DM 1病理学结果来自于扩增的DMPK RNA [（CUG）exp RNA]的功能获得。（CUG）exp RNA螯合并阻止调节出生后RNA加工网络的肌盲样（MBNL）RNA结合蛋白家族并维持成人RNA表达模式。在DM 1中，MBNL活性的丧失主要影响替代治疗。剪接，导致胎儿蛋白异构体在成人组织中表达，引起疾病特征。的作用通过小鼠Mbnl 1和/或Mbnl 2敲除（KO）证实了两种Mbnl旁系同源物在DMl病理学中的作用，概括了脑、心脏和骨骼肌中的DM 1表型。我在使用他莫昔芬诱导型，条件性小鼠中Mbnl 1和Mbnl 2的双KO，以确定MBNL功能丧失对内脏平滑的影响。肌肉.这将是首次利用MBNL损失对DM 1 GI特征的分子机制进行研究如先前对其它DM 1受影响的组织所建立的。申办方实验室生成纯合floxed Mbnl 1 和与平滑肌特异性CreERT 2组合的Mbnl 2等位基因（smoCRE;dHOM小鼠）。五- 周龄的smoCRE;dHOM小鼠表现出延迟的上GI运动和已知的DM 1相关的选择性剪接 Mbnl敲除后的事件。优化的dKO小鼠将用于：（1）确定什么GI表型由以下引起： MBNL的损失，然后（2）确定平滑肌功能障碍的分子机制。在目标1中，降低GI运动性将通过珠粒排出试验进行评估，并且离体试验将鉴定肌肉收缩性的扰动和蠕动模式穿过肠段。在目标2中，将来自两个实验的RNA测序数据来自DM 1受影响个体的小鼠和组织将用于鉴定保守的可变剪接事件，差异表达的基因和主要受影响的基因本体。我希望能找出影响钙动力学的转录组学变化，并将在体外研究钙处理，小鼠原代平滑肌细胞和永生化人平滑肌细胞。总之，这些目标将解决DM 1 GI发病机制的肌源性基础，推动DM 1平滑肌研究向前发展。