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Dimitrios Stampoulis, Stephen E Moss; Analysis of retinal phenotype and complement homeostasis in aged Abca4-/- Rdh8-/- mice. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5986. doi: https://doi.org/.
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Reduction of all-trans-retinal to all-trans-retinol after its release from photoisomerized visual pigments, partly depends on its transfer by the ATP-binding cassette (ABC) transporter ABCA4, and on the dehydrogenase activity of retinol dehydrogenase 8 (RDH8). Mutations in the ABCA4 transporter gene cause recessive Stargardt macular degeneration (STGD1). Both STGD1 patients and Abca4-/- mice exhibit build-up of bisretinoid-containing lipofuscin pigments in the retinal pigment epithelium (RPE), increased oxidative stress, augmented complement activation and slow degeneration of photoreceptors. In mice deficient in both Abca4-/- and Rdh8-/-, bisretinoid levels in the RPE are further elevated when compared to the single knockout Abca4-/- mice. The purpose of this study was to investigate complement homeostasis in the retinas of aged Abca4-/- Rdh8-/- double knock out mice.
mRNA was extracted from the RPE-choroid of wild type (WT) and Abca4-/- Rdh8-/- mice at age 18-20 months. Gene expression was examined by qRT-PCR for CFH, C3, C5, C3aR, C5aR, CFI, CFB, CFD, CFP, CD59a, NLRP3 and CTSL. Fundus imaging was performed, and fundus autofluorescence images were acquired in order to assess the presence of lipofuscin in the RPE. Neuroretinal whole mounts were stained for collagen IV and GFAP. RPE flatmounts were stained for C3aR and C3 breakdown products C3b/iC3b/C3c. PFA-fixed sections of mouse retinas were stained for C3 and C3 breakdown products C3b/iC3b/C3c.
qRT-PCR showed down-regulation of the expression levels of CFH, CD59a and C5aR in the RPE-choroid in the Abca4-/- Rdh8-/- mice. Fundus imaging revealed accumulation of bisretinoids in the RPE. Retinal vasculature and microglia/astrocyte staining appeared normal in the mutant strain. Staining of RPE flatmounts for C3aR and C3b/iC3b/C3c showed striking relocalisation of these proteins in the RPE of the Abca4-/- Rdh8-/- mice.
Abca4-/- Rdh8-/- mice at 18-20 months showed lower expression levels in the RPE-choroid of complement genes when compared to age matched WTs. Collagen IV and GFAP staining of neuroretinal whole mounts appeared normal. Accumulation of bisretinoids in the RPE of Abca4-/- Rdh8-/- mice was observed, consistent with previous studies in Abca4-/- Rdh8-/- mice at an earlier age. However, subcellular relocalisation of C3aR and C3b/iC3b/C3c in the Abca4-/- Rdh8-/- mice indicates complement dysregulation in the RPE.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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