Apoptosis Detection in Retinal Ganglion Cells Using Quantitative Changes in Multichannel Fluorescence Colocalization. Biosensors (Basel) 2022 Aug 28;12(9)
Date
09/24/2022Pubmed ID
36140078Pubmed Central ID
PMC9496076DOI
10.3390/bios12090693Scopus ID
2-s2.0-85138340626 (requires institutional sign-in at Scopus site)Abstract
KcapTR488 is a dual-fluorophore peptide sensor for the real-time reporting of programmed cell death by fluorescence imaging. KcapTR488 contains a nuclear localization sequence (NLS) conjugated with Texas Red, a caspase-cleavable sequence (DEVD), and a C-terminus conjugated to Alexa Fluor 488 (AF488). The synthesis and preliminary evaluation in cellulo of KcapTR488 for monitoring cell death by fluorescence imaging has been previously reported, but its utility in vivo has yet to be tested or validated. Herein, in vitro solution experiments verified the intramolecular fluorescence resonance energy transfer (FRET) between the two fluorophores and enabled a quantitative analysis of enzyme rates and selectivity. The sensor delivery kinetics in live rat models were quantified by ex vivo fluorescence microscopy. Studies in healthy control retinas demonstrated that KcapTR488 concentrated in the nucleus of retinal ganglion cells (RGC), with a strong colocalization of red and green fluorescence signals producing robust FRET signals, indicating an intact reporter. By contrast, using an acute but mild NMDA-induced retinal injury model, dual-color confocal ex vivo microscopy of cleaved KcapTR488 identified sensor activation as early as 2 h after injection. Quantitative changes in fluorescence colocalization were superior to changes in FRET for monitoring injury progression. Longitudinal monitoring revealed that the NLS-Texas Red fragment of the cleaved sensor moved out of the cell body, down the axon, and exited the retina, consistent with anterograde axonal transport. Thus, KcapTR488 may be a powerful tool to study RGC death pathways in live preclinical models of glaucoma.
Author List
Qiu X, Gammon ST, Johnson JR, Pisaneschi F, Millward SW, Barnett EM, Piwnica-Worms DAuthor
Edward M. Barnett MD, PhD Professor in the Ophthalmology and Visual Sciences department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsApoptosis
Caspases
Fluoresceins
Fluorescent Dyes
N-Methylaspartate
Peptides
Rats
Retinal Ganglion Cells
Sulfonic Acids
