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. 2024 May 21;14(1):64.
doi: 10.1186/s13578-024-01243-3.

Targeting miR-181a/b in retinitis pigmentosa: implications for disease progression and therapy

Bruna Lopes da Costa  1   2 , Peter M J Quinn  3 , Wen-Hsuan Wu  1   3 , Siyuan Liu  1   2 , Nicholas D Nolan  1   2 , Aykut Demirkol  1   3 , Yi-Ting Tsai  1   2 , Salvatore Marco Caruso  1   2 , Thiago Cabral  4   5 , Nan-Kai Wang  1   3 , Stephen H Tsang  6   7   8   9   10   11
Affiliations

Targeting miR-181a/b in retinitis pigmentosa: implications for disease progression and therapy

Bruna Lopes da Costa et al. Cell Biosci. .

Abstract

Background: Retinitis pigmentosa (RP) is a genetically heterogeneous group of degenerative disorders causing progressive vision loss due to photoreceptor death. RP affects other retinal cells, including the retinal pigment epithelium (RPE). MicroRNAs (miRs) are implicated in RP pathogenesis, and downregulating miR-181a/b has shown therapeutic benefit in RP mouse models by improving mitochondrial function. This study investigates the expression profile of miR-181a/b in RPE cells and the neural retina during RP disease progression. We also evaluate how miR-181a/b downregulation, by knocking out miR-181a/b-1 cluster in RPE cells, confers therapeutic efficacy in an RP mouse model and explore the mechanisms underlying this process.

Results: Our findings reveal distinct expression profiles, with downregulated miR-181a/b in RPE cells suggesting a protective response and upregulated miR-181a/b in the neural retina indicating a role in disease progression. We found that miR-181a/b-2, encoded in a separate genomic cluster, compensates for miR-181a/b-1 ablation in RPE cells at late time points. The transient downregulation of miR-181a/b in RPE cells at post-natal week 6 (PW6) led to improved RPE morphology, retarded photoreceptor degeneration and decreased RPE aerobic glycolysis.

Conclusions: Our study elucidates the underlying mechanisms associated with the therapeutic modulation of miR-181a/b, providing insights into the metabolic processes linked to its RPE-specific downregulation. Our data further highlights the impact of compensatory regulation between miR clusters with implications for the development of miR-based therapeutics.

Keywords: Aerobic glycolysis; Metabolic reprogramming; MicroRNAs; Retinal pigment epithelium; Retinitis pigmentosa.

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Conflict of interest statement

Stephen H Tsang receives financial support from Emendo and is on the scientific and clinical advisory board for Medical Excellence Capital and Nanoscope Therapeutics. The other authors have no conflicting interests.

Figures

Fig. 1
Fig. 1
Expression of miR-181a and miR-181b are dysregulated in the NR and RPE of Pde6βH620Q mice. A–B qPCR analysis of (A) miR-181a and B miR-181b expression in the NR of Pde6βWT and Pde6βH620Q mice at PW3, PW6, and PW10. N ≥ 4 mice. P-values are reported in black. Data are presented as mean ± SEM. Student’s t-test, unpaired. C–D qPCR analysis of (C) miR-181a and D miR-181b expression in the RPE cells of Pde6βWT and Pde6βH620Q mice at PW3, PW6, and PW10. N ≥ 4 mice. P-values are reported in black. Data are presented as mean ± SEM. Student’s t-test, unpaired. E Illustration of mouse model design. miR-181a/b are organized in two separate genomic clusters within mammalian cells: miR-181a/b-1 located on chromosome 1, and miR-181a/b-2 located on chromosome 2. Tamoxifen injection only mediates ablation of miR-181a/b-1
Fig. 2
Fig. 2
miR-181a/b shows dynamic expression upon tamoxifen injection in RPE cells of mutant mice. A Representative agarose gel electrophoresis result of genomic PCR to confirm miR-181a/b-1 ablation in the RPE cells of Pde6βH620Q mice. B–C qPCR analysis of miR-181a and miR-181b expression in the RPE cells of Pde6βH620QmiR-181a/b-1+/+ and Pde6βH620QmiR-181a/b-1–/− mice at (B) PW6, and C PW10. n ≥ 3 mice/genotype. P-values are reported in black. Data are presented as mean ± SEM. Student’s t-test, unpaired with Welch ‘s correction. D Representative agarose gel electrophoresis result of genomic PCR to confirm miR-181a/b-1 ablation in the RPE cells of Pde6βWT mice. E–F qPCR analysis of miR-181a and miR-181b expression in the RPE cells of Pde6βWTmiR-181a/b-1+/+ and Pde6βWTmiR-181a/b-1−/− mice at (E) PW6, and F PW10. n ≥ 4 mice/genotype. Data are presented as mean ± SEM. Student’s t-test, unpaired
Fig. 3
Fig. 3
miR-181a/b-2 compensates the lost of miR-181a/b-1 in RPE cells of mutant mice. A Illustration showing that lnc-Nr6a1 and miR-181a/b-2 share the same promoter. B–C qPCR analysis of (B) lnc-Nr6a1-1 and C lnc-Nr6a1-2 expression in the RPE cells of Pde6βH620QmiR-181a/b-1+/+ and Pde6βH620QmiR-181a/b-1−/− mice at PW6 and PW10. n ≥ 3 mice/genotype. Data are presented as mean ± SEM. Student’s t-test, unpaired. D–E qPCR analysis of miR-181a/b targets expression in the RPE cells of Pde6βH620QmiR-181a/b-1+/+ versus Pde6βH620QmiR-181a/b-1−/− mice at (D) PW6, and E PW10. n ≥ 4 mice/genotype. P-values are reported in black. Data are presented as mean ± SEM. Student’s t-test, unpaired with or without Welch ‘s correction
Fig. 4
Fig. 4
Transient miR-181a/b downregulation in the RPE cells of Pde6βH620Q mice at PW6 ameliorates disease phenotype. A–C Morphological analysis of Pde6βH620QmiR-181a/b-1+/+ and Pde6βH620QmiR-181a/b-1−/− retinas at PW6. A Representative histological sections. Scale bar 20 µm. The thickness profiles of (B) PRs ONL and C PS across the whole retina. N ≥ 3 mice/genotype. Each dot represents the average of the thicknesses at the specified point. P-values are reported in black. Data are presented as mean ± SEM. Student’s t-test, unpaired. D–G Immunofluorescence analysis of (D–E) C-Arrestin; and F–G Rhodopsin in Pde6βH620QmiR-181a/b-1+/+ and Pde6βH620QmiR-181a/b-1−/− retinas at PW6. Scale bar 20 µm in D–F. Number of cone pedicles quantified on the C-Arrestin slides are represented in (E). Fluorescence densitometry quantification of rhodopsin internalized into PRs nuclei layer is reported in (G). N = 3 mice/genotype. P-values are reported in black. Data are presented as mean SEM. Student’s t-test, unpaired. H–J Morphological analysis of Pde6βH620QmiR-181a/b-1+/+ and Pde6βH620QmiR-181a/b-1−/− retinas at PW10. H Representative histological sections. Scale bar 20 µm. The thickness profiles of (I) PRs ONL and J PS across the whole retina. N ≥ 3 mice/genotype. Each dot represents the average of the thicknesses at the specified point. Data are presented as mean ± SEM. Student’s t-test, unpaired
Fig. 5
Fig. 5
miR-181a/b downregulation ameliorated RPE morphology but did not increase mtDNA at PW6. A–D RPE morphometric analysis of the central area of RPE flat mounts using representative pictures of A Phalloidin staining from Pde6βH620QmiR-181a/b-1+/+ and Pde6βH620QmiR-181a/b-1−/− mice at PW6. N = 3 mice/genotype. Scale bar 20 µm. B Quantification of RPE cell area (in pixels) in the upper panel shows a heterogeneous distribution of RPE cell area in the Pde6βH620QmiR-181a/b-1+/+ in comparison to Pde6βH620QmiR-181a/b-1−/−. In the lower panel, we further confirmed the larger variability of the RPE cell area distribution in Pde6βH620QmiR-181a/b-1+/+ versus Pde6βH620QmiR-181a/b-1−/− mice. RPE cell area was quantified in six to eight areas of central RPE for each mice. N = 3 mice/genotype. In the upper panel data are presented as box plots showing the distribution of cell area for all RPE cells analyzed per group. In the lower panel, data are presented as mean ± SEM. Student’s t-test, unpaired. C Schematic showing how the health of RPE cells was scored. Scores 1 and 5 stand for the healthier and diseased morphology, respectively. D Qualitative scores attributed by two blinded observers to evaluate the RPE cells morphology of mice at PW6. N = 3 mice/genotype. Scores were attributed to four images of central RPE for each mice. Data are presented as mean of scores per mice ± SEM. Student’s t-test, unpaired. E mtDNA content measured by qPCR from Pde6βH620QmiR-181a/b-1+/+ and Pde6βH620QmiR-181a/b-1−/− eye cups at PW6. N = 4 mice/genotype. Data are presented as mean ± SEM. Student’s t-test, unpaired
Fig. 6
Fig. 6
LDHA expression decreases with downregulation of miR-181a/b in the RPE of Pde6βH620Q mice at PW6. A Illustration of the proposed hypothesis: miR-181a/b downregulation makes RPE cells healthier and decreases the aerobic glycolysis in those cells. B–C qPCR analysis of the expression of key regulators of aerobic glycolysis in the RPE cells of Pde6βH620QmiR-181a/b-1+/+ and Pde6βH620QmiR-181a/b-1−/− mice at (B) PW6, and C PW10. N ≥ 3 mice/genotype. Data are presented as mean ± SEM. Student’s t-test, unpaired. D–F Western Blotting analysis in (D) reveals decreased levels of LDHA protein in the RPE cells of Pde6βH620QmiR-181a/b-1−/− versus Pde6βH620QmiR-181a/b-1+/+. Data are normalized to β-actin. N = 3 mice/genotype. Please note that all compared bands are from the same blots. LDHA bands are quantified in (E) and PKM2 bands in (F). N = 6 mice/genotype. Data are presented as mean ± SEM. Student’s t-test, unpaired. G–K Analysis of the expression of key regulators of aerobic glycolysis at (G) mRNA and H–K protein levels in the NR of Pde6βH620QmiR-181a/b-1+/+ and Pde6βH620QmiR-181a/b-1−/− mice at PW6. Data are presented as mean ± SEM for N ≥ 3 mice/genotype and Student’s t-test, unpaired in (G). Please note that all compared bands are from the same blots in (H), LDHA bands are quantified in (I), PKM2 bands are quantified in (J), and pPKM2 (Tyr105) bands are quantified in (K). N = 3 mice/genotype. Data are presented as mean ± SEM. Student’s t-test, unpaired. (L–M) qPCR analysis reveals that miR-181a/b expression does not change in the NR of Pde6βH620QmiR-181a/b-1−/− in comparison to Pde6βH620QmiR-181a/b-1+/+ at (L) PW6, and M PW10. N = 4 mice/genotype. Data are presented as mean ± SEM. Student’s t-test, unpaired
Fig. 7
Fig. 7
miR-181a/b sustains aerobic glycolysis in RPE cells. Reduced expression of miR-181a/b at PW6 enhances the vitality of RPE cells. Consequently, these cells shift away from utilizing glucose via aerobic glycolysis for energy generation, favoring OXPHOS instead. This shift implies a potential increase in glucose availability for PR
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References

    1. Narayan DS, Wood JP, Chidlow G, Casson RJ. A review of the mechanisms of cone degeneration in retinitis pigmentosa. Acta Ophthalmol. 2016;94(8):748–754. doi: 10.1111/aos.13141. - DOI - PubMed
    1. Chrenek MA, Dalal N, Gardner C, Grossniklaus H, Jiang Y, Boatright JH, et al. Analysis of the RPE sheet in the rd10 retinal degeneration model. Adv Exp Med Biol. 2012;723:641–647. doi: 10.1007/978-1-4614-0631-0_81. - DOI - PMC - PubMed
    1. Wang W, Kini A, Wang Y, Liu T, Chen Y, Vukmanic E, et al. Metabolic deregulation of the blood-outer retinal barrier in retinitis pigmentosa. Cell Rep. 2019 doi: 10.1016/j.celrep.2019.06.093. - DOI - PMC - PubMed
    1. Napoli D, Biagioni M, Billeri F, Di Marco B, Orsini N, Novelli E, et al. Retinal pigment epithelium remodeling in mouse models of retinitis pigmentosa. Int J Mol Sci. 2021 doi: 10.3390/ijms22105381. - DOI - PMC - PubMed
    1. Kanow MA, Giarmarco MM, Jankowski CS, Tsantilas K, Engel AL, Du J, et al. Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye. Elife. 2017 doi: 10.7554/eLife.28899. - DOI - PMC - PubMed

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