Abstract
In the present study, we tested the hypothesis that 17beta-estradiol (betaE2) is a neuroprotectant in the retina, using two experimental approaches: 1) hydrogen peroxide (H(2)O(2))-induced retinal neuron degeneration in vitro, and 2) light-induced photoreceptor degeneration in vivo. We demonstrated that both betaE2 and 17alpha-estradiol (alphaE2) significantly protected against H(2)O(2)-induced retinal neuron degeneration; however, progesterone had no effect. betaE2 transiently increased the phosphoinositide 3-kinase (PI3K) activity, when phosphoinositide 4,5-bisphosphate and [(32)gammaATP] were used as substrate. Phospho-Akt levels were also transiently increased by betaE2 treatment. Addition of the estrogen receptor antagonist tamoxifen did not reverse the protective effect of betaE2, whereas the PI3K inhibitor LY294002 inhibited the protective effect of betaE2, suggesting that betaE2 mediates its effect through some PI3K-dependent pathway, independent of the estrogen receptor. Pull-down experiments with glutathione S-transferase fused to the N-Src homology 2 domain of p85, the regulatory subunit of PI3K, indicated that betaE2 and alphaE2, but not progesterone, identified phosphorylated insulin receptor beta-subunit (IRbeta) as a binding partner. Pretreatment with insulin receptor inhibitor, HNMPA, inhibited IRbeta activation of PI3K. Systemic administration of betaE2 significantly protected the structure and function of rat retinas against light-induced photoreceptor cell degeneration and inhibited photoreceptor apoptosis. In addition, systemic administration of betaE2 activated retinal IRbeta, but not the insulin-like growth factor receptor-1, and produced a transient increase in PI3K activity and phosphorylation of Akt in rat retinas. The results show that estrogen has retinal neuroprotective properties in vivo and in vitro and suggest that the insulin receptor/PI3K/Akt signaling pathway is involved in estrogen-mediated retinal neuroprotection.
Highlights
Tive diseases [1, 2]
We tested the hypothesis that Oxidative stress-induced neuronal cell death has been impli17-estradiol (E2) is a neuroprotectant in the retina, cated in different neurological disorders and neurodegenerausing two experimental approaches: 1) hydrogen peroxide (H2O2)-induced retinal neuron degeneration in vitro, and 2) light-induced photoreceptor degeneration in vivo. We demonstrated that both E2 and 17␣-estradiol (␣E2) significantly protected against H2O2-induced retinal neuron degeneration; progesterone had no effect. E2 transiently increased the phosphoinositide 3-kinase (PI3K) activity, when phosphoinositide 4,5bisphosphate and [32␥ATP] were used as substrate
We presented preliminary data on this at the Retinal Degeneration meeting held at Burginstock, Switzerland [40] that 1) IR is expressed in our cultured rat retinal neurons; 2) 5 M E2 and 5 M ␣E2 activated IR; and 3) 5 M progesterone was ineffective
Summary
Primary Culture of Retinal Neurons—Timed pregnant Sprague-Dawley rats were ordered each week and the retinas of 10 –15 pups, 0 –2 days old, were removed with the aid of a dissecting microscope under sterile conditions in a tissue culture hood. The cells were incubated overnight with antirecoverin antibody (1:5,000) and anti-estrogen receptor-␣ antibody (1: 100) in TBS with 1% bovine serum albumin, 1% goat serum, and 0.01% Triton X-100. Three digitized images of similar total cell numbers were selected from each coverslip for counting and averaging and were considered as one independent experiment. Assays were performed directly on total cells in 50 l of the reaction mixture containing 0.2 mg/ml phosphoinositide 4,5-bisphosphate, 50 M ATP, 0.2 Ci of [␥-32P]ATP, 5 mM MgCl2, and 10 mM HEPES buffer, pH 7.5. Pull-down experiments were performed as described previously [38], using 5 g of GST fusion proteins that had been absorbed onto GST-Sepharose 4B matrix. Functional Rescue of Photoreceptor Cells Evaluated by Electroretinogram—Electroretinogram (ERG) recordings were performed as described previously [22].
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