Caspase-Activated Cell-Penetrating Peptides Reveal Temporal Coupling Between Endosomal Release and Apoptosis in an RGC‑5 Cell Model

Caspase-activatable cell-penetrating peptide (CPP) probes, designed for efficient cell uptake and specificity via cleavable intramolecular quenched-fluorophore strategies, show promise for identifying and imaging retinal ganglion cell apoptosis <i>in vivo.</i> However, initial cell uptake and trafficking events cannot be visualized because the probes are designed to be optically quenched in the intact state. To visualize subcellular activation events in real-time during apoptosis, a new series of matched quenched and nonquenched CPP probes were synthesized. In both native and staurosporine-differentiated RGC-5 cells, probe uptake was time- and concentration-dependent through clathrine-, caveolin-, and pinocytosis-mediated endocytic mechanisms. During apoptosis, KcapTR488, a novel dual fluorophore CPP probe, revealed by multispectral imaging a temporal coupling of endosomal release and effector caspase activation in RGC-5 cells. The novel CPPs described herein provide new tools to study spatial and temporal regulation of endosomal permeability during apoptosis

Quantitative plot of probe activation from live images.

<p>(A) Plot of probe activation from images in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088855#pone-0088855-g003" target="_blank">Figure 3</a> B–F. (B) Probe activation normalized to maximum counts from an individual eye at each time point was averaged across all probe dose/NMDA concentration combinations. Probe activation increased significantly in the first 12 hours and generally decreased slowly thereafter. Data represent mean ± SEM.</p

Representative kinetics of TcapQ488 activation <i>in vivo</i>.

<p><i>In vivo</i> images were taken in a rat eye pretreated with 12.5 mM NMDA immediately before (A) and at 4 hours (B), 12 hours (C), 25 hours (D), 48 hours (E) and 72 hours (F) post intravitreal injection of 0.313 nmol TcapQ488. Evidence of initial probe activation was noted at 4 hours after TcapQ488 injection (B). Scale bar, 200 µm in all images.</p

Probe activation <i>in vivo</i> as a function of both NMDA concentration and probe dose.

<p>Retinal ganglion cell apoptosis was induced in rats by intravitreal injection of NMDA at various concentrations (0 (PBS only), 2.5, 12.5, 25 and 40 mM), followed by intravitreal injection of TcapQ488 (at 0 (PBS only), 0.097, 0.193, 0.313, 0.387 and 0.775 nmol) for a total of 27 NMDA-probe-dose combinations. Data reflect imaging at 12 hours post-probe injection. Probe signal increased as both NMDA concentration and probe dose increased. The plateau in TcapQ488 activation above 0.313 nmol indicates saturation of the dynamic range of probe dosing. n≥3 (5 to 8 eyes) at each combination. Data represent mean ± SEM.</p

Signal-to-noise ratio across NMDA concentrations and TcapQ488 probe doses.

<p>TcapQ488 activation data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088855#pone-0088855-g007" target="_blank">Figure 7</a> was normalized to PBS pre-treatment (i.e., NMDA = 0) for each probe dose to reflect relative “signal-to-noise.” For lower NMDA concentrations (2.5 and 12.5 mM), the ratio was highest at 0.097 nmol probe, while for higher NMDA concentrations (25 and 40 mM), the ratio was similar from probe doses 0.097 to 0.313 nmol. Data represent mean normalized labeled cells ± Error Propagation.</p

Dark-adapted B-wave amplitudes as a function of probe dose and time post-treatment.

<p>Treatment indicates PBS or probe only injections. (A–C) Comparison among pre-treatment, 1 week post-treatment, and 2 months post-treatment time points for each probe condition (PBS, 0.193 nmol TcapQ488, 0.387 nmol TcapQ488). (D–F) Comparison of each probe condition at each time point (pre-treatment, 1 week and 2 months post-treatment). There were no significant differences on dark-adapted B-wave amplitudes among probe conditions when tested at each time point. Data represent mean ± SD.</p

<i>In vivo</i> probe activation as a function of NMDA concentration.

<p>Rat eyes were pretreated with various NMDA concentrations (2.5, 12.5, 25, 40 mM) followed by 0.313 nmol TcapQ488. A control injection consisting of PBS was also performed to determine background probe activation levels in the absence of NMDA. All eyes were imaged at 4, 12, 24, 48 and 72 hours post-probe injection. Probe activation increased with increasing NMDA concentration at all time points examined. n≥3 (5 to 8 eyes) for each time point. Data represent mean ± SEM.</p

Correspondence of an <i>in vivo</i> fluorescent fundus image with an <i>ex vivo</i> retinal flat mount.

<p>(A) Fluorescent fundus image obtained <i>in vivo</i> using the CSLO (28 hours post-probe injection) from a rat eye pretreated with NMDA followed by TcapQ488. Strong, punctate fluorescent signals were detected in the retina ganglion cell (RGC) layer. (B) Higher magnification of the boxed area in A in which prominent fluorescent signals are highlighted. (C) <i>Ex vivo</i> flat mount of the same retina showed excellent correspondence with <i>in vivo</i> images in A and B, indicating that real time images reflect single cell resolution of probe activation. Scale bar: A, 200 µm; C, 100 µm.</p