Abstract
Purpose: :
Heat Shock Proteins (HSPs) function as molecular helper proteins (chaperones) to prevent protein aggregation and facilitate refolding of dysfunctional proteins, critical to the survival of all organisms. Akt, a serine-threonine protein kinase, is a key component of cell survival pathways. Here, we investigated the role of HSP70 and AKT on photoreceptor survival after experimental retinal detachment (RD) in rats.
Methods: :
Retinal detachment was induced in adult rats by subretinal injection of sodium hyaluronate. Expression levels of various HSP proteins was assessed by microarray analysis of the outer nuclear layer tissue (ONL) retrieved by Laser-Capture Microdissection in attached and detached retina 3 days after the initiation of RD. To study the role of HSP70, either an HSP70 inhibitor (Quercetin 100 mg/Kg), or vehicle (DMSO) was injected intraperitoneally 1hr before RD and animals were sacrificed 1, 3 or 7 days after RD. ONL thickness was measured and apoptotic cells were quantified using a TdT-dUTP terminal nick-end labeling (TUNEL) assay in cryo-sections. Expression of HSP70 levels and Akt activation was evaluated by western blots and immunohistochemistry.
Results: :
Microarray analysis showed that expression of HSP27, HSP70 and HSP90 were increased after 3 days of retinal detachment (3.3x, 4.1x, 3.8x [[Unsupported Character - Codename ­]]fold respectively p<0.05 for all of them). Phospho-Akt increased by 1.4 fold (p<0.05). There was increased co-immunoprecipitation of HSP70 with Akt after just 1 day of RD. Treatment with HSP70 inhibitor, Quercetin, decreased HSP70 expression levels by 64.8%(p<0.05), inhibited Akt phosphorylation by 18% ( p<0.05) on day 1 and by 33.1% (p<0.05) on day 3, and significantly increased TUNEL+ cells in the RD by 67.4% (p<0.05). However these changes did not correlate with a significant difference in the thickness of the ONL by day 7.
Conclusions: :
Heat shock proteins HSP27, HSP70 and HSP90 are increased in the retina following RD and may aid in photoreceptor cell survival by preventing dephosphorylation of pro-survival protein Akt. Inhibition of HSP70 did not alter ONL thickness following RD, perhaps due to redundant pathways.
Keywords: photoreceptors • apoptosis/cell death • cell survival