Epigenetic maintenance of adult cone photoreceptors

Abstract

Purpose: After terminal differentiation most neurons persist and maintain their function throughout the lifetime. We were interested to study whether epigenetic factors such as microRNAs were involved in maintaining the function and genetic identity of terminally differentiated cone photoreceptors in adult mouse retinas.

Methods: We disrupted the microRNA machinery by depleting the epigenetic factor DiGeorge syndrome critical region gene 8 (DGCR8) specifically in differentiated mouse cone photoreceptors. The DGCR8 knockout in cones (C-Dgcr8-/-) was obtained by crossing a conditional Dgcr8 null allele to mice expressing Cre recombinase specifically in cone photoreceptors. MicroRNA and mRNA samples were analyzed by quantitative RT-PCR and deep sequencing. Gene arrays were used to profile gene expression. Cone protein samples served for Western Blot analysis. We studied the cone morphology by immunohistochemistry and their function by ex vivo electrophysiology and in vivo electroretinograms. Rescue experiments were performed be delivering wild-type Dgcr8 by conditional adeno-associated-viruses.

Results: Cre-expression started earliest at postnatal day 6 (P6) but mature microRNAs and DGCR8 proteins were detectable much longer in C-Dgcr8-/- samples. At P60, the ablation of DGCR8 was accomplished; mature microRNAs were missing whereas their precursors accumulated due to the lack of processing. Morphologically, the cones in C-Dgcr8-/- expressed significantly less cone opsins. The overall changes in the transcriptome compared to C-Dgcr8+/+ were little but members of the cone photoreceptor transduction cascade were significantly down-regulated. C-Dgcr8-/- mice were light-insensitive in the photopic range whereas their rod vision was normal. At P60, the number of light-insensitive cones was unchanged compared to wild-type numbers. Viral delivery of wild-type Dgcr8 to adult C-Dgcr8-/- could rescue cone opsin expression. Interestingly, the genetically, morphologically and functionally altered phenotype of C-Dgcr8-/- was not detectable in P30 animals.

Conclusions: In this new epigenetic state that presents the clinical picture of achromatopsia, cone photoreceptor numbers were unchanged and transcripts that are not specifically expressed in cones did not decrease. Our results thus suggest that DGCR8 dependent pathways are necessary for maintaining the adult epigenetic identity and function of a sensory neuron.

Keywords: 648 photoreceptors • 649 photoreceptors: visual performance