Developing tools for calcium imaging in ITPR2-linked liver pathogenesis

开发 ITPR2 相关肝脏发病机制的钙成像工具

基本信息

批准号：
10727998
负责人：
Gyorgy Hajnoczky
金额：
$ 15.6万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10727998
关键词：
A kinase anchoring protein Address Affect Agonist Anhidrosis Area Binding Calcium Calcium Signaling Cell Line Cells Coupled Cytoplasm Dependovirus Diet Epithelial Cells Event Fructose Genetic Hepatocyte ITPR1 gene Image Inositol Japan Link Liver Liver Regeneration Lobule Measurement Measures Mediating Medical Metabolic Metabolic Diseases Metabolism Missense Mutation Mitochondria Modeling Monitor Mus Mutation Organ Outcome Outer Mitochondrial Membrane Pathogenesis Patients Pharmaceutical Preparations Photons Physiological Physiology Play Population Process Protein Isoforms Proteins Rare Diseases Rattus Reporter Reporting Research Role Satellite Viruses Signal Transduction Structure System Tacrolimus Binding Proteins Testing Tissues Viral Western Blotting bile formation cell type functional outcomes human disease in vivo inorganic phosphate intravenous injection intravital imaging liver cell proliferation liver function liver imaging mouse model multi-photon nano non-alcoholic fatty liver disease novel physiologic model receptor response scaffold sensor spatiotemporal tissue injury tool tool development tripolyphosphate vector

项目摘要

The liver is a vital organ, central to metabolic functions. Intracellular Ca2+ is a major regulator of cellular events affecting liver functions. The major Ca2+ release channels present in liver are the inositol 1, 4, 5-triphosphate (IP3) receptors (ITPR). Of the three ITPR isoforms, ITPR1 and ITPR2 are expressed in hepatocytes. ITPR1 expression corresponds to ~20% of total ITPR present making ITPR2 the principal Ca2+ release entity in liver. ITPR2 has been linked to key liver functions like bile formation, liver regeneration and hepatocyte proliferation. Reduced ITPR2 expression has been observed in a rat model of non-alcoholic fatty liver disease. The importance of ITPR2 as a Ca2+ release channel with downstream effects on liver functions has become even more interesting with the emergence of ER-mitochondria contacts (ERMC). We have previously shown that ITPR2 has been the most effective in local Ca2+ transfer at ERMC in cell lines. Recent studies indicate ERMC mediated local signaling and Ca2+ transfer to be associated with metabolic disorders involving liver. In summary, broad evidence indicates a fundamental role of ITPR2 in various liver functions, which are likely directly relevant for ITPR2-linked human diseases, but the underlying mechanisms remained difficult to study in physiological models. To solve this problem, we propose to create a novel toolkit that includes a genetic mouse model combined with viral delivery of fluorescent protein reporters for recording global and local cytoplasmic and mitochondrial Ca2+ signaling. We intend to introduce tools in the form of inducible and liver-specific ITPR2KO mouse model and subject hepatocytes to intracellular and intra-organellar Ca2+ measurements which will enable understanding the function of ITPR2 in Ca2+ signaling pertaining to liver physiology. We intend to deliver fluorescent cytoplasmic and mitochondrial Ca2+ probes with liver-targeted adeno-associated virus 8 (AAV8) that will allow intra-vital imaging to monitor spontaneous and agonist induced Ca2+ changes in live liver tissue. Furthermore, to study the ERMC local signaling we have developed drug-inducible synthetic ER-mitochondria linker constructs tagged with Ca2+ sensing fluorescent proteins. In order to understand the role of ITPR2 in ERMC structure and functions in the context of liver physiology, we plan to express the synthetic linkers in the inducible ITPR2KO mouse model. Thus, our project will yield a novel toolkit to study ITPR2 in the context of both global and local cytoplasmic and mitochondrial Ca2+ signaling that is expected to be broadly useful for determining the outcome of ITPR2 mutations and other ITPR2-linked conditions in physiological models.

肝脏是一个重要的器官，是代谢功能的核心。细胞内Ca2+是细胞内Ca2+的主要调节因子影响肝功能的细胞事件。肝脏中主要的 Ca2+ 释放通道是肌醇 1, 4, 5-三磷酸 (IP3) 受体 (ITPR)。在三种 ITPR 同工型中，ITPR1 和 ITPR2 在肝细胞中表达。 ITPR1 表达量相当于 ITPR 总量的约 20% 使 ITPR2 成为肝脏中主要的 Ca2+ 释放实体。 ITPR2已与关键肝脏相关具有胆汁形成、肝脏再生和肝细胞增殖等功能。减少ITPR2 已在非酒精性脂肪肝病大鼠模型中观察到表达。重要性 ITPR2 作为 Ca2+ 释放通道对肝功能具有下游影响的作用已变得更加均匀 ER-线粒体接触（ERMC）的出现更有趣。我们之前有过结果表明，ITPR2 在细胞系中 ERMC 局部 Ca2+ 转移中最有效。最近的研究表明 ERMC 介导的局部信号传导和 Ca2+ 转移与代谢相关涉及肝脏的疾病。总之，广泛的证据表明 ITPR2 的基本作用在各种肝功能中，这可能与 ITPR2 相关的人类直接相关疾病，但其潜在机制仍然难以在生理学中研究模型。为了解决这个问题，我们建议创建一个新的工具包，其中包括基因小鼠模型结合荧光蛋白报告基因的病毒传递进行记录全局和局部细胞质和线粒体 Ca2+ 信号传导。我们打算引入工具以诱导型和肝脏特异性 ITPR2KO 小鼠模型的形式，并对肝细胞进行细胞内和细胞器内 Ca2+ 测量，有助于了解其功能 ITPR2 在与肝脏生理学相关的 Ca2+ 信号传导中的作用。我们打算提供荧光使用肝靶向腺相关病毒 8 (AAV8) 进行细胞质和线粒体 Ca2+ 探针这将允许活体成像监测活体内自发的和激动剂诱导的 Ca2+ 变化肝组织。此外，为了研究 ERMC 局部信号传导，我们开发了药物诱导型用 Ca2+ 感应荧光蛋白标记的合成 ER-线粒体连接体构建体。在为了了解 ITPR2 在肝脏 ERMC 结构和功能中的作用生理学方面，我们计划在诱导型 ITPR2KO 小鼠模型中表达合成接头。因此，我们的项目将产生一个新颖的工具包，用于在全球和本地背景下研究 ITPR2 细胞质和线粒体 Ca2+ 信号传导有望广泛用于确定生理模型中 ITPR2 突变和其他 ITPR2 相关条件的结果。