Citation: Sreekumar PG, Chothe P, Sharma KK, et al. Antiapoptotic properties of a-crystallin-derived peptide chaperones and characterization of their uptake transporters in human RPE cells. Invest Ophthalmol Vis Sci. 2013;54:278754: -279854: . DOI:10.1167 PURPOSE. The chaperone proteins, a-crystallins, also possess antiapoptotic properties. The purpose of the present study was to investigate whether 19 to 20-mer a-crystallin-derived mini-chaperone peptides (a-crystallin mini-chaperone) are antiapoptotic, and to identify their putative transporters in human fetal RPE (hfRPE) cells. METHODS.Cell death and caspase-3 activation induced by oxidative stress were quantified in early passage hfRPE cells in the presence of 19 to 20-mer aA-or aB-crystallin-derived or scrambled peptides. Cellular uptake of fluorescein-labeled, a-crystallin-derived mini-peptides and recombinant full-length aB-crystallin was determined in confluent hfRPE. The entry mechanism in hfRPE cells for a-crystallin mini-peptides was investigated. The protective role of polycaprolactone (PCL) nanoparticle encapsulated aB-crystallin mini-chaperone peptides from H 2 O 2 -induced cell death was studied.RESULTS. Primary hfRPE cells exposed to oxidative stress and either aA-or aB-crystallin minichaperones remained viable and showed marked inhibition of both cell death and activation of caspase-3. Uptake of full-length aB-crystallin was minimal while a time-dependent uptake of aB-crystallin-derived peptide was observed. The mini-peptides entered the hfRPE cells via the sodium-coupled oligopeptide transporters 1 and 2 (SOPT1, SOPT2). PCL nanoparticles containing aB-crystallin mini-chaperone were also taken up and protected hfRPE from H 2 O 2 -induced cell death at significantly lower concentrations than free aB-crystallin minichaperone peptide.CONCLUSIONS. aA-and aB-crystallin mini-chaperones offer protection to hfRPE cells and inhibit caspase-3 activation. The oligopeptide transporters SOPT1 and SOPT2 mediate the uptake of these peptides in RPE cells. Nanodelivery of aB-crystallin-derived mini-chaperone peptide offers an alternative approach for protection of hfRPE cells from oxidant injury.Keywords: a-crystallin, chaperone peptides, oxidative stress, RPE protection, oligopeptide transporters T he superfamily of small heat shock proteins (sHSPs) has attracted considerable attention in recent years because of its multifunctional cellular properties. The human genome encodes 10 members of the sHSP family, among which aAcrystallin and aB-crystallin are considered important members. 1 Both aA-and aB-crystallins have been studied extensively in the lens for their chaperone and related functions. However, recent studies have identified several novel functions for aA-and aBcrystallins in retina and other tissues in addition to their wellrecognized chaperone function. 2 Both a-crystallins are expressed in RPE cells and in the retina; higher expression of aBcrystallin was found in the RPE while aA-crystallin was found mostly in photoreceptors and astroglial and Mül...

Nuclear hormone receptors play a major role in many important biological processes. Most nuclear hormone receptors are ubiquitously expressed and regulate processes such as metabolism, circadian function, and development. They function in these processes to maintain homeostasis through modulation of transcriptional gene networks. In this study we evaluate the effectiveness of a nuclear hormone receptor gene to modulate retinal degeneration and restore the integrity of the retina. Currently, there are no effective treatment options for retinal degenerative diseases leading to progressive and irreversible blindness. In this study we demonstrate that the nuclear hormone receptor gene Nr1d1 (Rev-Erbα) rescues Nr2e3-associated retinal degeneration in the rd7 mouse, which lacks a functional Nr2e3 gene. Mutations in human NR2E3 are associated with several retinal degenerations including enhanced S cone syndrome and retinitis pigmentosa. The rd7 mouse, lacking Nr2e3, exhibits an increase in S cones and slow, progressive retinal degeneration. A traditional genetic mapping approach previously identified candidate modifier loci. Here, we demonstrate that in vivo delivery of the candidate modifier gene, Nr1d1 rescues Nr2e3 associated retinal degeneration. We observed clinical, histological, functional, and molecular restoration of the rd7 retina. Furthermore, we demonstrate that the mechanism of rescue at the molecular and functional level is through the re-regulation of key genes within the Nr2e3-directed transcriptional network. Together, these findings reveal the potency of nuclear receptors as modulators of disease and specifically of NR1D1 as a novel therapeutic for retinal degenerations.

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Hyaluronan (HA) is a naturally occurring glycosaminoglycan widely researched for its use as a biomaterial in tissue engineering, drug delivery, angiogenesis, and ophthalmic surgeries. The mechanical properties of this biomaterial can be altered to a required extent by chemically modifying the pendant reactive groups. However, derivatizing these polymers to a predetermined extent has been the Achilles heel for this process. In this study, we have investigated the factors controlling the derivatization of the carboxyl moieties of HA with amine containing thiol, cystamine dihydrochloride (Cys), via carbodiimide crosslinking chemistry. We used fractional factorial design to screen and identify the significant factor(s) affecting the reaction, and response surface methodology (RSM) to develop a model equation for predicting the degree of thiolation of HA. Also, we analyzed the reaction mechanism for potential side reactions. We observed that, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (mole ratio with repeat unit of HA) is the significant factor controlling the degree of amidation. The quadratic equations developed from RSM predict the formulation for a desired degree of amidation of HA and percentage of potential side product. Hence, derivatizing HA to a predetermined extent with minimal side product can be achieved using the statistical design of experiments.

BackgroundP23H rhodopsin, a mutant rhodopsin, is known to aggregate and cause retinal degeneration. However, its effects on retinal pigment epithelial (RPE) cells are unknown. The purpose of this study was to determine the effect of P23H rhodopsin in RPE cells and further assess whether LEDGF1-326, a protein devoid of heat shock elements of LEDGF, a cell survival factor, reduces P23H rhodopsin aggregates and any associated cellular damage.MethodsARPE-19 cells were transiently transfected/cotransfected with pLEDGF1-326 and/or pWT-Rho (wild type)/pP23H-Rho. Rhodopsin mediated cellular damage and rescue by LEDGF1-326 was assessed using cell viability, cell proliferation, and confocal microscopy assays. Rhodopsin monomers, oligomers, and their reduction in the presence of LEDGF1-326 were quantified by western blot analysis. P23H rhodopsin mRNA levels in the presence and absence of LEDGF1-326 was determined by real time quantitative PCR.Principal FindingsP23H rhodopsin reduced RPE cell viability and cell proliferation in a dose dependent manner, and disrupted the nuclear material. LEDGF1-326 did not alter P23H rhodopsin mRNA levels, reduced its oligomers, and significantly increased RPE cell viability as well as proliferation, while reducing nuclear damage. WT rhodopsin formed oligomers, although to a smaller extent than P23H rhodopsin. Further, LEDGF1-326 decreased WT rhodopsin aggregates.ConclusionsP23H rhodopsin as well as WT rhodopsin form aggregates in RPE cells and LEDGF1-326 decreases these aggregates. Further, LEDGF1-326 reduces the RPE cell damage caused by P23H rhodopsin. LEDGF1-326 might be useful in treating cellular damage associated with protein aggregation diseases such as retinitis pigmentosa.