Developing tools for calcium imaging in ITPR2-linked liver pathogenesis

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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.