A Second Generation Activatable, Cell-Penetrating Peptide Probe for Imaging Effector Caspase Activity in an in vivo Model of RGC Degeneration

Abstract

Purpose: : Previously, we described a fluorescence probe based on an activatable strategy to detect apoptosis-associated caspase activity in vivo. This probe consisted of a cell-penetrating Tat peptide conjugated to an effector recognition sequence (DEVD) that was flanked by a fluorophore-quencher pair (Alexa Fluor 647 and QSY 21). Once exposed to effector caspases, the recognition sequence was cleaved, resulting in separation of the fluorophore-quencher pair and signal generation. We previously demonstrated the utility of this probe in detecting RGC apoptosis in an in vivo model. Recently, a second generation probe, KcapQ, with a modified cell-penetrating peptide sequence (KKKRKV), was developed and validated in vitro. This modification resulted in a probe that was more sensitive to effector caspase enzymes, displayed a higher quenching efficiency between the fluorophore-quencher pair, and was potentially less toxic to cells. The utility of KcapQ in imaging RGC apoptosis in an in vivo NMDA model was investigated.

Methods: : RGC apoptosis was induced in vivo in rats pharmacologically using intravitreal injection of NMDA (concentrations of 5 to 80 mM), followed by injection of KcapQ probe. Retinas were imaged as eyecups or flatmounts with a fluorescence microscope to detect activated probe. Retinal neurons with activated probe (primarily RGCs) were quantified in retinal flatmounts, assisted by retrograde labeling with FluoroGold in some animals. A second probe, Kcap₄₈₈, which lacks the fluorophore quencher, was co-injected with KcapQ₆₄₇ in several rats to distinguish uptake of probe from probe activation.

Results: : The number of RGCs displaying probe activation followed a dose-dependent pattern based on the concentration of NMDA used for intravitreal injection, as previously demonstrated for TcapQ. Examination of vertical retinal sections as well as retrograde labeling of RGCs with FluoroGold confirmed that probe activation was largely limited to RGCs. Co-injection of quenched and non-quenched versions of the Kcap probe with different fluorophores revealed diffuse uptake of probe by RGCs with selective activation.

Conclusions: : Our findings provide validation of this second generation activatable, cell-penetrating fluorescent probe for identifying RGC apoptosis in an in vivo model following intravitreal injection. This probe should be useful for in vivo imaging of RGC apoptosis in animal models of glaucoma and may eventually have applications in the diagnosis and management of human glaucoma

Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • apoptosis/cell death • imaging/image analysis: clinical