Developing tools for calcium imaging in ITPR2-linked liver pathogenesis

肝脏是一个重要的器官，是代谢功能的中心。细胞内Ca 2+是一种主要的调节因子， 影响肝功能的细胞事件。肝脏中主要的钙释放通道是 肌醇1，4，5-三磷酸（IP 3）受体（ITPR）。在三种ITPR亚型中，ITPR 1和ITPR 2 在肝细胞中表达。ITPR 1表达相当于总ITPR的约20% 使ITPR 2成为肝脏中主要的Ca 2+释放实体。ITPR 2与关键肝脏有关 功能如胆汁形成、肝再生和肝细胞增殖。降低ITPR 2 在非酒精性脂肪肝疾病的大鼠模型中观察到表达。的重要性 ITPR 2作为Ca 2+释放通道对肝功能的下游作用已经变得均匀 随着ER-线粒体接触（ERMC）的出现，这一点更加有趣。我们先前已经 表明ITPR 2在细胞系ERMC的局部Ca 2+转移中是最有效的。最近 研究表明ERMC介导的局部信号传导和Ca 2+转移与代谢相关， 涉及肝脏的疾病总之，广泛的证据表明ITPR 2的基本作用 在各种肝功能中，这可能与ITPR 2相关的人直接相关。 疾病，但潜在的机制仍然难以在生理学上研究。 模型为了解决这个问题，我们建议创建一个新的工具包，其中包括一个基因， 用于记录的与病毒递送荧光蛋白报告基因组合的小鼠模型 整体和局部细胞质和线粒体Ca 2+信号传导。我们打算引进工具 以诱导型和肝脏特异性ITPR 2KO小鼠模型的形式， 细胞内和细胞器内的Ca 2+测量，这将有助于了解功能 ITPR 2在与肝脏生理学有关的Ca 2+信号传导中的作用。我们打算提供荧光 肝靶向腺相关病毒8（AAV 8）的细胞质和线粒体Ca 2+探针 这将允许活体内成像监测肝中自发和激动剂诱导的Ca 2+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的 用Ca 2+感应荧光蛋白标记的合成ER-线粒体接头构建体。在 为了了解ITPR 2在肝脏ERMC结构和功能中的作用， 为了进一步研究ITPR 2KO小鼠的生理学特性，我们计划在诱导型ITPR 2KO小鼠模型中表达合成接头。 因此，我们的项目将产生一个新的工具包，研究ITPR 2在全球和地方的背景下， 细胞质和线粒体Ca 2+信号传导，预期可广泛用于确定 ITPR 2突变和其他ITPR 2相关疾病在生理模型中的结果。

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.