Glaucoma is one of the leading causes of vision loss in the world. Glaucoma is an optic neuropathy characterized by the death of retinal ganglia cells (RGCs), axon loss, and an excavated appearance to the optic nerve head. Glaucoma is usually associated with elevated intraocular pressure (lOP), but a subset of "normal tension glaucoma" patients develops damage without ever manifesting high lOPs. This shows that lOP-independent mechanisms of RGC death are present in glaucoma. RGC death mechanisms in animal models of glaucoma and human glaucoma involve apoptosis. Regardless of the trigger, apoptotic cascade produces cell suicide by invoking a series of cellular events that has been conserved in RGCs. Apoptotic stimuli converge on the activation of a family of cytokine proteases known as capsizes (cysteine aspartases). Activation of members of the caspase family is necessary for programmed cell death in a number of biological processes. Particularly, evidence strongly points to caspase-3 as central to the execution pathway of apoptosis in many different cell types. While existing in a non-active pro-enzyme form in the cytosol of healthy cells, activated caspase-3 is a key "effector" protease in cells committed to apoptosis. Thus, to monitor the final commitment of target cells in death pathways, a strategy to directly quantify the cytosolic enzymatic activity of caspase-3 in vivo is needed. Our approach to selectively image apoptotic events both in vitro and in vivo involves the development of low molecular weight, peptide based optical imaging agents capable of entering living cells. These peptide based probes incorporate a fluorophore - quencher pair connected by a protease specific substrate recognition site, remaining optically silent. Upon cleavage by caspase-3 the fluorophore - quencher is disrupted allowing fluorescence detection of apoptotic cells. Originally our probes were based on the HIV-1 Tat transactivation protein sequence, and we have developed a new generation of peptide based probes optimized for optical imaging of caspase activity in vivo. Upon intravitreal (eye) administration, our peptide based probes selectively accumulate in retinal ganglion cells, the target cell injured in neurodegenerative diseases, including glaucoma. Thus, we plan to use this cell-type-specific property of our peptide probes in retinal pathology for molecular imaging applications, and combined diagnostic-therapeutic ("theragnostic") anti-apoptotic applications. In this way, we will generate noninvasive imaging agents for focal retention of fluorescence, an optical "hot spot", in target cells corresponding to enzymatically active caspase-3 as well as real-time imaging of therapeutic delivery to target cells in vitro and in vivo.
|Qiu, Xudong; Johnson, James R; Wilson, Bradley S et al. (2014) Single-cell resolution imaging of retinal ganglion cell apoptosis in vivo using a cell-penetrating caspase-activatable peptide probe. PLoS One 9:e88855|
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|Johnson, James R; Kocher, Brandon; Barnett, Edward M et al. (2012) Caspase-activated cell-penetrating peptides reveal temporal coupling between endosomal release and apoptosis in an RGC-5 cell model. Bioconjug Chem 23:1783-93|