Apoptosis Detection in Retinal Ganglion Cells Using Quantitative Changes in Multichannel Fluorescence Colocalization
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)...
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MDPI AG
2022-08-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/12/9/693 |
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| author | Xudong Qiu Seth T. Gammon James R. Johnson Federica Pisaneschi Steven W. Millward Edward M. Barnett David Piwnica-Worms |
| author_facet | Xudong Qiu Seth T. Gammon James R. Johnson Federica Pisaneschi Steven W. Millward Edward M. Barnett David Piwnica-Worms |
| author_sort | Xudong Qiu |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T00:35:21Z |
| format | Article |
| id | doaj.art-6393b174bd3545a69f429a9f8131334b |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-10T00:35:21Z |
| publishDate | 2022-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-6393b174bd3545a69f429a9f8131334b2023-11-23T15:17:33ZengMDPI AGBiosensors2079-63742022-08-0112969310.3390/bios12090693Apoptosis Detection in Retinal Ganglion Cells Using Quantitative Changes in Multichannel Fluorescence ColocalizationXudong Qiu0Seth T. Gammon1James R. Johnson2Federica Pisaneschi3Steven W. Millward4Edward M. Barnett5David Piwnica-Worms6Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USADepartment of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USAMallinckrodt Institute of Radiology, Washington University in St. Louis, St. Louis, MO 63130, USADepartment of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USADepartment of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USADepartment of Ophthalmology & Visual Sciences, Medical College of Wisconsin, Milwaukee, WI 53226, USADepartment of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USAKcapTR488 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.https://www.mdpi.com/2079-6374/12/9/693apoptosisretinal ganglion celldual-fluorophore peptide sensorfluorescence resonance energy transferNMDAanterograde axonal transport |
| spellingShingle | Xudong Qiu Seth T. Gammon James R. Johnson Federica Pisaneschi Steven W. Millward Edward M. Barnett David Piwnica-Worms Apoptosis Detection in Retinal Ganglion Cells Using Quantitative Changes in Multichannel Fluorescence Colocalization Biosensors apoptosis retinal ganglion cell dual-fluorophore peptide sensor fluorescence resonance energy transfer NMDA anterograde axonal transport |
| title | Apoptosis Detection in Retinal Ganglion Cells Using Quantitative Changes in Multichannel Fluorescence Colocalization |
| title_full | Apoptosis Detection in Retinal Ganglion Cells Using Quantitative Changes in Multichannel Fluorescence Colocalization |
| title_fullStr | Apoptosis Detection in Retinal Ganglion Cells Using Quantitative Changes in Multichannel Fluorescence Colocalization |
| title_full_unstemmed | Apoptosis Detection in Retinal Ganglion Cells Using Quantitative Changes in Multichannel Fluorescence Colocalization |
| title_short | Apoptosis Detection in Retinal Ganglion Cells Using Quantitative Changes in Multichannel Fluorescence Colocalization |
| title_sort | apoptosis detection in retinal ganglion cells using quantitative changes in multichannel fluorescence colocalization |
| topic | apoptosis retinal ganglion cell dual-fluorophore peptide sensor fluorescence resonance energy transfer NMDA anterograde axonal transport |
| url | https://www.mdpi.com/2079-6374/12/9/693 |
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