Photoreceptor protection via blockade of BET epigenetic readers in a murine model of inherited retinal degeneration

Abstract

BackgroundThe bromodomain and extraterminal domain (BET) family proteins (BET2, BET3, and BET4) “read” (bind) histone acetylation marks via two distinct bromodomains (Brom1 and Brom2) facilitating transcriptional activation. These epigenetic “readers” play crucial roles in pathogenic processes such as inflammation. The role of BETs in influencing the degenerative process in the retina is however unknown.MethodsWe employed the rd10 mouse model (Pde6b rd10 mutation) of retinitis pigmentosa (RP) to examine the involvement of BET proteins in retinal neurodegeneration.ResultsInhibition of BET activity by intravitreal delivery of JQ1, a BET-specific inhibitor binding both Brom1 and Brom2, ameliorated photoreceptor degeneration and improved electroretinographic function. Rescue effects of JQ1 were related to the suppression of retinal microglial activation in vivo, as determined by decreased immunostaining of activation markers (IBA1, CD68, TSPO) and messenger RNA (mRNA) levels of inflammatory cytokines in microglia purified from rd10 retinas. JQ1 pre-treatment also suppressed microglial activation in vitro, decreasing microglial proliferation, migration, and mRNA expression of inflammatory cytokines (TNFα, MCP-1, IL-1β, IL-6, and RANTES). Expression of BET2, but not BET3 and BET4, was significantly elevated during photoreceptor degeneration at postnatal day (PN)24 in retinas of rd10 mice relative to age-matched wild-type controls. siRNA knockdown of BET2 but not BET4, and the inhibitor of Brom2 (RVX208) but not of Brom1 (Olinone), decreased microglial activation.ConclusionsThese findings indicate that BET inhibition rescues photoreceptor degeneration likely via the suppression of microglial activation and implicates BET interference as a potential therapeutic strategy for the treatment of degenerative retinal diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0775-4) contains supplementary material, which is available to authorized users.

Potential independent action of sigma receptor ligands through inhibition of the Kv2.1 channel

“…Mice were euthanized by CO 2 asphyxiation followed by cervical dislocation. Retinal cryosections were prepared according to our published methods [ 31 , 40 ]. Briefly, eyeballs were fixed overnight in 4% paraformaldehyde at 4 °C and then soaked in 30% sucrose (in PBS) overnight at 4 °C, and 10 μm sections were cut from the eyeballs frozen in the optimum cutting temperature (OCT) embedding medium.…”

“…The animal procedures were in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Details of ERG recording were described in our recent report [ 40 ]. Briefly, 40-day old σ1-R WT and σ1-R KO mice were anesthetized with ketamine/xylazine (80 and 10 mg/kg IP).…”

Partnership between epigenetic reader BRD4 and transcription factor CEBPD

“…Therefore, our data distinguished that BRD4 but not BRD2 or BRD3 was the determinant BET that controlled CEBPD expression. Furthermore, BD1 rather than BD2 was responsible for this BRD4 function; this result was unexpected given that in previous reports BD2 vs BD1 was shown to play a major role in non-SMC settings 5,21 .…”

“…First, BRD4 determined CEBPD expression levels; the role of BRD2 and BRD3 was minor (if any). We further distinguished that BD1 but not BD2 was critical for this BRD4 functiona finding worth more attention since BD1 and BD2 are highly similar and hence poorly differentiated in their biological functions 5,19,21 . Second, while TNFa as an extracellular stimulant upregulated CEBPD which prompted inflammatory SMC state transition (increased IL-1b, IL6, and MCP-1), using JQ1 to block BRD4 function abolished this TNFa stimulation.…”

Role of the sigma-1 receptor chaperone in rod and cone photoreceptor degenerations in a mouse model of retinitis pigmentosa

“…In this study, the majority of experiments were performed for a time course spanning from postnatal 3 weeks (3 W) to 6 W. Mice were euthanized by CO 2 asphyxiation followed by cervical dislocation at 3 W, 4 W, 5 W, and 6 W. Eyeballs were enucleated immediately and dissected. For morphometric and immunohistochemistry analyses, retinal cryosections were prepared according to our published methods [ 21 , 27 ]. Briefly, eyeballs were fixed in 4% paraformaldehyde for 7 h at 4 °C and then soaked in 30% sucrose in PBS for 14 h at 4 °C, and 10 μm sections were cut from the eyeballs frozen in optimum cutting temperature (OCT) embedding medium (Sakura Finetek USA, Inc., Torrance, CA).…”