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. 2017 Jun 13;114(24):6376-6381.
doi: 10.1073/pnas.1618757114. Epub 2017 May 30.

miR-183/96 plays a pivotal regulatory role in mouse photoreceptor maturation and maintenance

Lue Xiang  1   2 , Xue-Jiao Chen  1   2 , Kun-Chao Wu  1   2 , Chang-Jun Zhang  1   2 , Gao-Hui Zhou  1   2 , Ji-Neng Lv  1   2 , Lan-Fang Sun  1   2 , Fei-Fei Cheng  1   2 , Xue-Bi Cai  1   2 , Zi-Bing Jin  3   2
Affiliations

miR-183/96 plays a pivotal regulatory role in mouse photoreceptor maturation and maintenance

Lue Xiang et al. Proc Natl Acad Sci U S A. .

Abstract

MicroRNAs (miRNAs) are known to be essential for retinal maturation and functionality; however, the role of the most abundant miRNAs, the miR-183/96/182 cluster (miR-183 cluster), in photoreceptor cells remains unclear. Here we demonstrate that ablation of two components of the miR-183 cluster, miR-183 and miR-96, significantly affects photoreceptor maturation and maintenance in mice. Morphologically, early-onset dislocated cone nuclei, shortened outer segments and thinned outer nuclear layers are observed in the miR-183/96 double-knockout (DKO) mice. Abnormal photoreceptor responses, including abolished photopic electroretinography (ERG) responses and compromised scotopic ERG responses, reflect the functional changes in the degenerated retina. We further identify Slc6a6 as the cotarget of miR-183 and miR-96. The expression level of Slc6a6 is significantly higher in the DKO mice than in the wild-type mice. In contrast, Slc6a6 is down-regulated by adeno-associated virus-mediated overexpression of either miR-183 or miR-96 in wild-type mice. Remarkably, both silencing and overexpression of Slc6a6 in the retina are detrimental to the electrophysiological activity of the photoreceptors in response to dim light stimuli. We demonstrate that miR-183/96-mediated fine-tuning of Slc6a6 expression is indispensable for photoreceptor maturation and maintenance, thereby providing insight into the epigenetic regulation of photoreceptors in mice.

Keywords: degeneration; miR-183/96/182 cluster; photoreceptor; regulation; taurine transporter.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Generation of miR-183/96 DKO mice. (A) A conventional knockout strategy was used to generate the DKO mice. The neomycin resistance gene was preserved in the DKO mice. Neo, neomycin; tel, telomeric; cent, centromeric. (B) Genotyping strategy for the WT mice and DKO mutants. (C and D) RT-PCR (C) and qRT-PCR (D) analysis of miR-183/96/182 expression at P90. Relative expression levels of microRNAs are normalized to the level of U6. The normalized values represent mean ± SEM. n = 3. *P < 0.05, ***P < 0.001 between the DKO mice and their littermates, Student’s t test.
Fig. 2.
Fig. 2.
Depletion of miR-183/96 results in early-onset defects in nuclear polarization and shortened OSs of cones and abolished ERG in mice. (A) Cone arrestin immunostaining at P10, P30, and P180 in WT (Left) and DKO (Right) mouse retinas. White arrows point to cone nuclei located at the basal part of the ONL. (Scale bar: 20 µm.) (B) Flat retinal whole-mount immunostaining of PNA and cone arrestin at P10. (C) The number of cone nuclei on the outer, middle, and inner areas of the ONL shown in A at P10; n = 4. (D) Quantification of the PNA-positive OSs per mm2 as shown in B. n = 4. (E) Representative photopic ERG recordings of the DKO and WT mice at P30, P120, and P180. (F–G) Comparison of the b-wave amplitudes (F) and a-wave amplitudes (G) between the DKO mice and age-matched WTs. n = 4, 3, and 3 for photopic responses of mice at P30, P120, and P180, respectively. In C–G, the normalized values represent mean ± SEM. **P < 0.005, ***P < 0.001, Student’s t test.
Fig. 3.
Fig. 3.
Ablation of miR-183/96 causes progressive retinal degeneration. (A) Representative scotopic ERG recordings of DKO and WT mice. (B and C) b-wave (B) and a-wave (C) amplitudes for the DKO mice and age-matched controls. n = 4, 3, and 4 for scotopic responses in mice at P30, P120, and P180, respectively. The normalized values represent mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.001 between the DKO mice and age-matched controls, Student’s t test. (D) Quantification of the thickness of the ONL, OS/IS, and whole retina from SD-OCT images of mice at P30–P180. The red intensities on the curve of the DKO mice represent the thickness of the WT sample minus that in the DKO sample. Blue and red indicate the minimum and maximum thickness, respectively, in the WT and DKO samples at each point. n = 4 for each group.
Fig. 4.
Fig. 4.
Slc6a6 is a target of both miR-183 and miR-96 in the mouse retina. (A) Immunostaining reflects the endogenous expression of Slc6a6 in WT mouse retinas at P90. The red arrows point to cells with high expression. (Scale bar: 20 µm.) (B) Western blot verification of the expression of miR-183/96 target Slc6a6 in mouse tissues at P7 and P120. (C) Quantification of Slc6a6 protein level relative to Gapdh level at P7 shown in B. n = 3. (D) Schematic representation of the experiments. (E) AAV-based miR-183, miR-96, and control expression cassettes were driven by the pCMV-bGlobin promoter. (F) AAV-miR-183, AAV-miR-96, and their control vector were successfully expressed in retinas. (G and H) Slc6a6 was down-regulated in both miR-96 (G) and miR-183 (H) overexpressing WT retinas at P30. M1L, mouse 1 left eye; M1R, mouse 1 right eye, etc. (I) Quantification of Slc6a6 protein levels relative to GAPDH levels shown in G and H. n = 4. (J and K) The concentration of taurine was increased in the DKO heterozygous mice at P21 (J) and decreased in the WT mice overexpressing miR-183 or miR-96 at P44 (K). DKO Het, miR-183/96 DKO heterozygotes; conc, concentration. In C, and I–K, the normalized values represent mean ± SEM. Taurine concentrations are represented as median ± SEM. **P < 0.005, ***P < 0.001, Student’s t test.
Fig. 5.
Fig. 5.
Both silencing Slc6a6 and overexpressing Slc6a6 are detrimental to photoreceptors. (A) Slc6a6 RNAi sequences or spacer sequences were inserted downstream of mCherry into the AAV vectors. (B) qRT-PCR analysis of Slc6a6 expression in AAV-Slc6a6 RNAi-injected eyes compared with the control eyes. n = 3. (C and H) Retinas infected with AAV-Slc6a6 RNAi or AAV-Slc6a6 showed degenerative features in the photoreceptors. (D and I) Eyes injected with AAV-Slc6a6 RNAi or AAV-Slc6a6 displayed significantly attenuated scotopic ERG responses compared with the control eyes. n = 5 for AAV-Slc6a6 RNAi, n = 6 for AAV-Slc6a6 subretinal injection in mice. (E) Bar charts showing the general down-regulation of randomly-selected photoreceptor-specific genes. n = 4. (F) AAV-based Slc6a6 and control expression cassettes were driven by the pCMV-bGlobin promoter. (G) Slc6a6 expression in AAV-Slc6a6–injected retinas compared with the AAV-GFP–injected WT retinas assessed by qRT-PCR. n = 3. (J) Bar charts showing the general up-regulation of photoreceptor-specific genes. n = 4. In B–J, for the qRT-PCR bar plot, the normalized values represent mean ± SEM. For the ERG amplitude boxplot, the relative amplitudes are normalized to the mean amplitudes of control eyes; the normalized values represent median ± SEM. *P < 0.05; **P < 0.005; ***P < 0.001; n.s., not significant, Student’s t test.
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