Abstract
The process of controlled cellular death known as apoptosis has an important central role not only in normal homeostatic maintenance of tissues, but also in numerous diseases such as cancer, neurodegenerative, autoimmune, and cardiovascular diseases. As a result, new technologies with the capability to selectively detect apoptotic cells represent a central focus of research for the study of these conditions. We have developed a new biosensor for the detection of apoptotic cells, incorporating the targeted selectivity for apoptotic cells from Annexin V with the sensitivity of bioluminescence signal generation from a serum-stable mutant of Renilla luciferase (RLuc8). Our data presents a complete characterization of the structural and biochemical properties of this new Annexin-Renilla fusion protein (ArFP) construct, as well as a validation of its ability to detect apoptosis in vitro. Moreover, this work represents the first report of a bioluminescent Annexin V apoptosis sensor utilized in vivo. With this new construct, we examine apoptosis within disease-relevant animal models of surgery-induced ischemia/reperfusion, corneal injury, and retinal cell death as a model of age-related macular degeneration. In each of these experiments, we demonstrate successful application of the ArFP construct for detection and bioluminescence imaging of apoptosis within each disease or treatment model. ArFP represents an important new tool in the continuously growing kit of technologies for apoptosis detection, and our results from both in vitro and in vivo experiments suggest a diverse range of potential clinically relevant applications including cancer therapeutic screening and efficacy analysis, atherosclerosis and cardiovascular disease detection, and the monitoring of any number of other conditions in which apoptosis has a central role.
Highlights
The presence of controlled cell death has been identified in a diverse range of cell and tissue types as far back as the mid-nineteenth century.[1,2] it was not until the introduction of the unifying term ‘apoptosis’ in the 1970’s that the importance of this ubiquitous process was rigorously examined.[3]
This study represents the first description of a bioluminescent Annexin V apoptosis sensor successfully applied in vivo, providing new means to assess clinically relevant disease models involving apoptosis
We report a new bioluminescence-based Annexin V biosensor for the detection of apoptosis
Summary
The presence of controlled cell death has been identified in a diverse range of cell and tissue types as far back as the mid-nineteenth century.[1,2] it was not until the introduction of the unifying term ‘apoptosis’ in the 1970’s that the importance of this ubiquitous process was rigorously examined.[3]. Renilla luciferase (RLuc) is an enzyme natively expressed by the sea pansy Renilla reniformis that oxidatively decarboxylates its substrate coelenterazine to generate bioluminescence (Supplementary Figure S1).[25,26] Detection of this type of emission generally results in lower background and higher signal-to-noise ratios, providing a means of detection that is more sensitive than fluorescence, especially in the context of biological samples.[22,27] it has been shown that this wild-type RLuc is quickly rendered inactive in the presence of murine serum,[28] strictly limiting previous work with a Renilla luciferase-labeled Annexin V probe to applications in vitro.[29,30] Protein engineering of the wild-type RLuc protein has yield a luciferase variant (RLuc8) that exhibits a 200-fold increase in serum stability, as well as a 4-fold increase in light output.[28] This affords a unique opportunity to couple its use with relevant in vivo disease models to detect apoptosis, and analyze the role which apoptosis has within them We have utilized this RLuc[8] mutant to generate a chimeric bioluminescence-based Annexin V apoptosis detection construct. We demonstrate that, in addition to functioning as a specific sensor for apoptosis in vitro, this construct allows for the bioluminescence imaging of
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