Abstract
Purpose :
An increasing number of eye disorders are associated with protein misfolding, oxidative stress, inflammation, and apoptosis. It is hypothesized that a cell-penetrable, protease-resistant amphiphilic mini chaperone peptide (CPPRAMC) having the core αA-crystallin chaperone region sequence can help treat such disorders. We have tested our hypothesis using cell culture and Caenorhabditis elegans (C. elegans) model systems.
Methods :
The CPPRAMC, having all D- amino acids, and the control peptides were synthesized (>95% pure) by GenScript. The anti-aggregation activity of the CPPRAMC was confirmed in vitro by testing the suppression of Aβ1-42 (25 µM) oligomerization and fibrillization under TEM. The suppression of mutant myocilin (DsRed-MYOC-Y437H) accumulation in TM cells was confirmed by fluorescence imaging. The ability of CPPRAMC to protect ARPE-19 cells from Aβ1-42-induced cytotoxicity was evaluated by performing an EarlyTox cell integrity assay on treated cells. The anti-oxidative property of CPPRAMC was demonstrated in ARPE-19 cells challenged with 7.5 mM sodium iodate for 24 hr. The cellular ROS activity was measured with 2′,7′-Dichloro-fluorescein diacetate. The therapeutic efficacy of the CPPRAMC was also evaluated in C. elegans wild-type (N2) worms subjected to stress and in transgenic (CL4176) worms expressing human Aβ1-42. The anti-inflammatory property of CPPRAMC was tested using HEK293 IL-6 reporter cells (Invivogen).
Results :
CPPRAMC treatment (5 µM) reduced the accumulation of DsRed-MYOC-Y437H protein in TM cells. Aβ peptide-treated (5 µM) ARPE-19 cells showed 25% apoptosis while cells treated with CPPRAMC (5 µM) + Aβ peptide showed 10% apoptotic cells. Doubling the molar concentration of CPPRAMC reduced the number of dead cells to 5%. CPPRAMC (5 µM) completely suppressed the NaIO3-induced ROS production. CPPRAMC ablates the LPS-induced release of IL-6 in mouse macrophages (Raw264.7) and blocks IL-6 signaling (50 pM) completely in HEK-Blue IL-6 reporter cells when used at 1 µM. CPPRAMC feeding augments the lifespan (46%) and increases the thermotolerance (51%) of wild-type C. elegans. CPPRAMC treatment delays β-amyloid induced paralysis and increases survival (18%) in transgenic CL4176 worms.
Conclusions :
The results suggest the beneficial effects of CPPRAMC in treating diseases associated with protein misfolding, oxidative stress, inflammation, and apoptosis that affect vision.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.