There remains a strong need for imaging methods which will enable researchers and clinicians to assess biochemical activities within cells in vivo. The eye is an attractive model for this approach given its accessibility and the ease of optical imaging. One cellular process which is of considerable interest at this time is the sequence of intracellular events which leads to apoptosis. Activatable peptides containing enzyme specific cleavage sites which, when cleaved, result in a signal denoting the activity of the specified enzyme are a useful tool to assay the apoptotic cascade. Access of molecular imaging probes to the intracellular space has been greatly improved through the use of permeation peptides as a targeting moiety. We propose a strategy to enable the identification of retinal neurons undergoing apoptosis in vivo. Specifically, this project focuses on the detection and imaging of retinal ganglion cell (RGC) apoptosis in vivo in animal models of RGC degeneration. This novel approach will utilize modified Tat permeation peptides to facilitate uptake into RGCs of an activatable peptide designed for the detection of apoptotic protease (effector caspase) activity. The activatable permeation peptide is a small peptidomimetic consisting of an all D-amino acid modified Tat permeation peptide, an L-amino acid effector caspase recognition sequence, a far red quencher, and a far-red fluorophore. Upon cleavage of the caspase recognition sequence and subsequent loss of fluorescent quenching, fluorescence from the retained intracellular fluorophore will be detectable via fluorescence imaging. RGC apoptosis will be induced in rats either pharmacologically using intravitreal injection of staurosporine or through the induction of uniocular elevated intraocular pressure. Initially, the use of the activatable permeation peptide to identify effector caspases activity in RGCs will be validated using immunohistochemistry for effector caspases in histologic retinal preparations. Following this validation, our goal is to image and quantitate effector Caspase activity in these two models of RGC apoptosis using macroscopic fluorescence biomicroscopy in vivo. Utilizing the same two animal models of RGC apoptosis, fluorescence fundus imaging will be performed following intravitreal injection of the activatable permeation peptide. At present, there is no method available for clinicians to identify injured retinal nerve cells in patients with glaucoma, a common cause of blindness worldwide. The proposed imaging strategy should not only benefit the study of animal models of glaucoma, but may also ultimately improve our ability to diagnose and treat glaucoma in humans. The ability to image dying retinal neurons in living humans and animals will be an important advance, while the validation of this strategy in the eye will promote its application to a broad range of both tissues and diseases. ? ? ?
| Maxwell, Dustin; Chang, Qing; Zhang, Xu et al. (2009) An improved cell-penetrating, caspase-activatable, near-infrared fluorescent peptide for apoptosis imaging. Bioconjug Chem 20:702-9 |
| Barnett, Edward M; Zhang, Xu; Maxwell, Dustin et al. (2009) Single-cell imaging of retinal ganglion cell apoptosis with a cell-penetrating, activatable peptide probe in an in vivo glaucoma model. Proc Natl Acad Sci U S A 106:9391-6 |
