An inducible expression system for the manipulation of autophagic flux in vivo

Much of our understanding of the intracellular regulation of macroautophagy/autophagy comes from in vitro studies. However, there remains a paucity of knowledge about how this process is regulated within different tissues during development, aging and disease in vivo. Because upregulation of autophagy is considered a promising therapeutic strategy for the treatment of diverse disorders, it is vital that we understand how this pathway functions in different tissues and this is best done by in vivo analysis. Similarly, to understand the role of autophagy in the pathogenesis of disease, it is important to study this process in the whole animal to investigate how tissue-specific changes in flux and cell-autonomous versus non-cell-autonomous effects alter disease progression. To this end, we have developed an inducible expression system to up- or downregulate autophagy in vivo, in zebrafish. We have used a modified version of the Gal4-UAS expression system to allow inducible expression of autophagy up- or downregulating transgenes by addition of tamoxifen. Using this inducible expression system, we have tested which transgenes robustly up- or downregulate autophagy and have validated these tools using Lc3-II blots and puncta analysis and disease rescue in a zebrafish model of neurodegeneration. These tools allow the temporal control of autophagy via the administration of tamoxifen and spatial control via tissue or cell-specific ERT2-Gal4 driver lines and will enable the investigation of how cell- or tissue-specific changes in autophagic flux affect processes such as aging, inflammation and neurodegeneration in vivo.

Automated summary

Much of our understanding of the regulation of macroautophagy/autophagy comes from in vitro studies, and there is still a lack of knowledge about its regulation in different tissues in vivo. Upregulation of autophagy is considered a promising therapeutic strategy, so it is important to understand its function in different tissues through in vivo analysis. To achieve this, an inducible expression system has been developed in zebrafish to up- or downregulate autophagy, allowing the investigation of how cell- or tissue-specific changes in autophagic flux affect processes such as aging, inflammation, and neurodegeneration.