Purpose : A mouse model with a knockout of multifunctional protein 2 (MFP2), the central peroxisomal β-oxidation enzyme, shows retinal degeneration mimicking the pathology in MFP2 deficient patients. To understand the origins and mechanisms of this degeneration, specifically in the RPE, we used global and RPE-specific Mfp2 knockout mice.

Methods : Eyes were collected either in the morning (2-3h post light onset) or in the afternoon (7h post light onset) at ages between 3 weeks and 12 months. They were used for immunostaining, immunoblotting, LC-MS lipidomic analysis and TEM imaging.

Results : The first pathological signs in Mfp2^-/- RPE were lipid accumulations at 3w visualized by Perilipin-2 staining, which were not present at 2w, suggesting that peroxisomal β-oxidation is important for digestion of lipids coming from ingested POS, rich in VLC-PUFAs. The accumulation of neutral lipids and lipid species containing VLC-PUFAs was confirmed using lipidomics. TEM analysis at 3w showed accumulation of POS phagosomes, suggesting lysosomal dysfunction, confirmed with rhodopsin staining on RPE flat mounts. In addition, mitochondria were increased in number and were more circular in appearance. Immunostaining on flat mounts showed loss of the honeycomb structure and mislocalization of mitochondrial and peroxisomal markers. Mfp2^-/- RPE progressively dedifferentiated with mislocalization of ezrin, a reduction in visual cycle genes and increases in dedifferentiation markers PCNA and SMA. mTOR, previously shown to induce RPE dedifferentiation, was already activated at 3w. The distribution and expression of the principle lactate transporters MCT1 and 3 was altered, suggesting impairment of lactate transport in the retina of these mice. The RPE-specific MFP2 knockouts show similar RPE degeneration. Interestingly, this caused pathology in the neural retina, as evidenced by increased inflammation, loss of photoreceptor nuclei and visual function towards 10-12 months of age.

Conclusions : Our results strongly indicate a crucial role for peroxisomal β-oxidation in the digestion of POS, with defects leading to lysosomal and mitochondrial abnormalities in RPE cells and their dedifferentiation. The RPE-specific MFP2 knockouts showed that the RPE degeneration was cell autonomous, but had negative consequences for the underlying neural retina.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.