Reprogramming of adult rod photoreceptors prevents retinal degeneration

Abstract

A prime goal of regenerative medicine is to direct cell fates in a therapeutically useful manner. Retinitis pigmentosa is one of the most common degenerative diseases of the eye and is associated with early rod photoreceptor death followed by secondary cone degeneration. We hypothesized that converting adult rods into cones, via knockdown of the rod photoreceptor determinant Nrl, could make the cells resistant to the effects of mutations in rod-specific genes, thereby preventing secondary cone loss. To test this idea, we engineered a tamoxifen-inducible allele of Nrl to acutely inactivate the gene in adult rods. This manipulation resulted in reprogramming of rods into cells with a variety of cone-like molecular, histologic, and functional properties. Moreover, reprogramming of adult rods achieved cellular and functional rescue of retinal degeneration in a mouse model of retinitis pigmentosa. These findings suggest that elimination of Nrl in adult rods may represent a unique therapy for retinal degeneration.

Fig. 1.

Rod reprogramming therapy. (A) Hypothesis that the normal progression of photoreceptor degeneration in retinitis pigmentosa might be circumvented by reprogramming rods into cones via acute Nrl KO. (B) Mice homozygous for the mutant rd1 allele of rod-specific Pde6b have lost nearly all rod photoreceptors by P36. Germ-line deletion of Nrl on the Pde6b^rd1/rd1 background suppresses the degeneration phenotype. Note that both the Nrl^−/− and double mutants retinas display marked rosette formation in the ONL, a finding previously reported in the Nrl^−/− retina (17). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Scale bar, 50 μm.)

Fig. 2.

Engineering a conditional allele of Nrl. (A) Schematic of the conditional Nrl allele; the first coding exon (ex3) is flanked by loxP sites. (B) Nrl^f/f retinas are indistinguishable from WT retinas by H&E histology and ISH for rod (Nrl and Rho, rhodopsin) and cone (Opn1sw, short-wavelength cone opsin) transcripts. Germ-line recombination of the floxed Nrl alleles (Nrl^CKO/CKO) via Sox2-Cre recapitulates the Nrl^−/− phenotype. (Scale bar, 50 μm.)

Fig. 3.

Reprogramming of adult mouse rods into cone-like cells. (A) Timeline for the acute Nrl KO experiments. 4-OHT, 4-hydroxytamoxifen for Cre^ERTM induction. Unless otherwise specified, control is Nrl^+/f;Cre^ERTM + 4-OHT and acute Nrl KO is Nrl^f/f;Cre^ERTM + 4-OHT. (B) H&E and ISH staining for rod and cone transcripts. Gnb1, G protein β subunit 1 in rod transducin. Note that acute Nrl KO results in variable waviness of the ONL, but no frank rosette formation as observed in the germ-line KO. (C) Electron micrographs of photoreceptor nuclei in the ONL for control (Nrl^f/f;Cre^ERTM + vehicle) and acute Nrl KO (Nrl^f/f;Cre^ERTM + 4-OHT) mice. Sample control and reprogrammed nuclei are outlined in green; heterochromatin (h), euchromatin (e), and juxtanuclear mitochondria (m) are labeled. (Scale bar, 2 μm.) (D and E) Scotopic (D) and photopic (E) ERGs recorded from P63 control (n = 10) and acute Nrl KO mice (n = 8). P values for two-way ANOVA: **P < 0.01, ***P < 0.001. Error bars are SEM. (F) Families of responses to increasing light intensities for dark-adapted, isolated control, and acute Nrl KO retinas. Red traces, photoresponses to a light intensity of 392 photons/μm² (505 nm). (G) Responses to 505-nm test flashes recorded from isolated retinas, lacking RPE, under various conditions (n ≥ 3 animals per condition). Bleach, exposed to 520-nm light for 2 min followed by dark adaptation for 2.5 h; 9-cis-ROL, the 2.5-h dark adaptation was conducted in the presence of 130 µM 9-cis-retinol. Data were fitted with the Naka-Rushton hyperbolic function. Error bars are SEM.

Fig. 4.

Epigenetic barriers to reprogramming of rods into cones. Retinal DNA methylation status of the (A) Rho and (B) Opn1sw loci for four genotypes, C57BL/6 (WTe), Nrl^−/−, control (Nrl^+/f;Cre^ERT2 + 4-OHT), and acute Nrl KO (Nrl^f/f;Cre^ERT2 + 4-OHT). Each circle represents a single CpG (5′-CG-3′); shading indicates percentage methylation across ≥28 clones (from two biological replicates). Graph at right represents overall locus methylation; error bars are SD. Phastcons, vertebrate phylogenetic conservation track from UCSC Genome Browser.

Fig. 5.

Rod reprogramming prevents retinal degeneration. (A) Time course of photoreceptor degeneration in Rho^−/− retinas (Upper) and timeline for acute Nrl KO experiments on the Rho^−/− background (Lower). Control is Rho^−/−;Nrl^+/f;Cre^ERTM + 4-OHT and acute Nrl KO is Rho^−/−;Nrl^f/f;Cre^ERTM + 4-OHT. (B) H&E and antibody staining show increased preservation of the ONL and cone opsin expression in Rho^−/− animals treated with acute Nrl KO relative to controls. (Scale bar, 50 μm.) (C) Quantification of ONL cells in 300-μm segments of retina (n = 3 animals per genotype). ***P < 0.001 by unpaired t test. Error bars are SD. (D) Electron micrograph montages of the ONL, indicated by dashed lines. (Scale bar, 4 μm.) (E) Photopic ERGs recorded from control (n = 10) and acute Nrl KO mice (n = 10) on the Rho^−/− background. P values for two-way ANOVA: **P < 0.01, ***P < 0.001. Error bars are SEM.