The murine retinal pigment epithelium requires peroxisomal ß-oxidation to maintain lysosomal function and prevent dedifferentiation.

Retinal pigment epithelium (RPE) cells have to phagocytose shed photoreceptor outer segments (POS) on a daily basis over the lifetime of an organism, but the mechanisms involved in the digestion and recycling of POS lipids are poorly understood. Although it was frequently assumed that peroxisomes may play an essential role, this was never investigated. Here, we show that global as well as RPE-selective loss of peroxisomal ß-oxidation in multifunctional protein 2 (MFP2) knockout mice impairs the digestive function of lysosomes in the RPE at a very early age, followed by RPE degeneration. This was accompanied by prolonged mammalian target of rapamycin activation, lipid deregulation, and mitochondrial structural anomalies without, however, causing oxidative stress or energy shortage. The RPE degeneration caused secondary photoreceptor death. Notably, the deterioration of the RPE did not occur in an Mfp2/rd1 mutant mouse line, characterized by absent POS shedding. Our findings prove that peroxisomal ß-oxidation in the RPE is essential for handling the polyunsaturated fatty acids present in ingested POS and shed light on retinopathy in patients with peroxisomal disorders. Our data also have implications for gene therapy development as they highlight the importance of targeting the RPE in addition to the photoreceptor cells.

Peroxisomal Disorders and Their Mouse Models Point to Essential Roles of Peroxisomes for Retinal Integrity.

Peroxisomes are multifunctional organelles, well known for their role in cellular lipid homeostasis. Their importance is highlighted by the life-threatening diseases caused by peroxisomal dysfunction. Importantly, most patients suffering from peroxisomal biogenesis disorders, even those with a milder disease course, present with a number of ocular symptoms, including retinopathy. Patients with a selective defect in either peroxisomal α- or ß-oxidation or ether lipid synthesis also suffer from vision problems. In this review, we thoroughly discuss the ophthalmological pathology in peroxisomal disorder patients and, where possible, the corresponding animal models, with a special emphasis on the retina. In addition, we attempt to link the observed retinal phenotype to the underlying biochemical alterations. It appears that the retinal pathology is highly variable and the lack of histopathological descriptions in patients hampers the translation of the findings in the mouse models. Furthermore, it becomes clear that there are still large gaps in the current knowledge on the contribution of the different metabolic disturbances to the retinopathy, but branched chain fatty acid accumulation and impaired retinal PUFA homeostasis are likely important factors.

Differential distribution of peroxisomal proteins points to specific roles of peroxisomes in the murine retina.

The retinal pathology in peroxisomal disorders suggests that peroxisomes are important to maintain retinal homeostasis and function. These ubiquitous cell organelles are mainly involved in lipid metabolism, which comprises α- and ß-oxidation and ether lipid synthesis. Although peroxisomes were extensively studied in liver, their role in the retina still remains to be elucidated. As a first step in gaining more insight into the role of peroxisomes in retinal physiology, we performed immunohistochemical stainings, immunoblotting and enzyme activity measurements to reveal the distribution of peroxisomes and peroxisomal lipid metabolizing enzymes in the murine retina. Whereas peroxisomes were detected in every retinal layer, we found a clear differential distribution of the peroxisomal lipid metabolizing enzymes in the neural retina compared to the retinal pigment epithelium. In particular, the ABC transporters that transfer lipid substrates into the organelle as well as several enzymes of the ß-oxidation pathway were enriched either in the neural retina or in the retinal pigment epithelium. In conclusion, our results strongly indicate that peroxisome function varies between different regions in the murine retina.

Peroxisomal Disorders and Retinal Degeneration.

Peroxisomal disorders are a group of inherited metabolic diseases, which can be incompatible with life in the postnatal period or allow survival into adulthood. Retinopathy is a recurrent feature in both the severely and mildly affected patients, which can be accompanied with other ophthalmological pathologies. Thanks to next-generation sequencing, patients originally identified with other inherited blinding diseases were reclassified as suffering from peroxisomal disorders. In addition, new peroxisomal gene defects or disease presentations exhibiting retinal degeneration were recently identified. The pathogenic mechanisms underlying retinopathy in peroxisomal disorders remain unresolved.

DHA Shortage Causes the Early Degeneration of Photoreceptors and RPE in Mice With Peroxisomal ß-Oxidation Deficiency.

Purpose: Patients deficient in peroxisomal ß-oxidation, which is essential for the synthesis of docosahexaenoic acid (DHA, C22:6n-3) and breakdown of very-long-chain polyunsaturated fatty acids (VLC-PUFAs), both important components of photoreceptor outer segments, develop retinopathy present with retinopathy. The representative mouse model lacking the central enzyme of this pathway, multifunctional protein 2 (Mfp2-/-), also show early-onset retinal decay and cell-autonomous retinal pigment epithelium (RPE) degeneration, accompanied by reduced plasma and retinal DHA levels. In this study, we investigated whether DHA supplementation can rescue the retinal degeneration of Mfp2-/- mice. Methods: Mfp2+/- breeding pairs and their offspring were fed a 0.12% DHA or control diet during gestation and lactation and until sacrifice. Offspring were analyzed for retinal function via electroretinograms and for lipid composition of neural retina and plasma with lipidome analysis and gas chromatography, respectively, and histologically using retinal sections and RPE flatmounts at the ages of 4, 8, and 16 weeks. Results: DHA supplementation to Mfp2-/- mice restored retinal DHA levels and prevented photoreceptor shortening, death, and impaired functioning until 8 weeks. In addition, rescue of retinal DHA levels temporarily improved the ability of the RPE to phagocytose outer segments and delayed the RPE dedifferentiation. However, despite the initial rescue of retinal integrity, DHA supplementation could not prevent retinal degeneration at 16 weeks. Conclusions: We reveal that the shortage of a systemic supply of DHA is pivotal for the early retinal degeneration in Mfp2-/- mice. Furthermore, we report that adequate retinal DHA levels are essential not only for photoreceptors but also for RPE homeostasis.

Cell Type-Selective Loss of Peroxisomal ß-Oxidation Impairs Bipolar Cell but Not Photoreceptor Survival in the Retina.

Retinal degeneration is a common feature in peroxisomal disorders leading to blindness. Peroxisomes are present in the different cell types of the retina; however, their precise contribution to retinal integrity is still unclear. We previously showed that mice lacking the central peroxisomal ß-oxidation enzyme, multifunctional protein 2 (MFP2), develop an early onset retinal decay including photoreceptor cell death. To decipher the function of peroxisomal ß-oxidation in photoreceptors, we generated cell type selective Mfp2 knockout mice, using the Crx promotor targeting photoreceptors and bipolar cells. Surprisingly, Crx-Mfp2-/- mice maintained photoreceptor length and number until the age of 1 year. A negative electroretinogram was indicative of preserved photoreceptor phototransduction, but impaired downstream bipolar cell signaling from the age of 6 months. The photoreceptor ribbon synapse was affected, containing free-floating ribbons and vesicles with altered size and density. The bipolar cell interneurons sprouted into the ONL and died. Whereas docosahexaenoic acid levels were normal in the neural retina, levels of lipids containing very long chain polyunsaturated fatty acids were highly increased. Crx-Pex5-/- mice, in which all peroxisomal functions are inactivated in photoreceptors and bipolar cells, developed the same phenotype as Crx-Mfp2-/- mice. In conclusion, the early photoreceptor death in global Mfp2-/- mice is not driven cell autonomously. However, peroxisomal ß-oxidation is essential for the integrity of photoreceptor ribbon synapses and of bipolar cells.

Peroxisomal Multifunctional Protein 2 Deficiency Perturbs Lipid Homeostasis in the Retina and Causes Visual Dysfunction in Mice.

Patients lacking multifunctional protein 2 (MFP2), the central enzyme of the peroxisomal ß-oxidation pathway, develop retinopathy. This pathway is involved in the metabolism of very long chain (VLCFAs) and polyunsaturated (PUFAs) fatty acids, which are enriched in the photoreceptor outer segments (POS). The molecular mechanisms underlying the retinopathy remain, however, elusive. Here, we report that mice with MFP2 inactivation display decreased retinal function already at the age of 3 weeks, which is accompanied by a profound shortening of the photoreceptor outer and inner segments, but with preserved photoreceptor ultrastructure. Furthermore, MFP2 deficient retinas exhibit severe changes in gene expression with downregulation of genes involved in the phototransduction pathway and upregulation of inflammation related genes. Lipid profiling of the mutant retinas revealed a profound reduction of DHA-containing phospholipids. This was likely due to a hampered systemic supply and retinal traffic of this PUFA, although we cannot exclude that the local defect of peroxisomal ß-oxidation contributes to this DHA decrease. Moreover, very long chain PUFAs were also reduced, with the exception of those containing ≥ 34 carbons that accumulated. The latter suggests that there is an uncontrollable elongation of retinal PUFAs. In conclusion, our data reveal that intact peroxisomal ß-oxidation is indispensable for retinal integrity, most likely by maintaining PUFA homeostasis.

A Simplified Geboes Score for Ulcerative Colitis.

BACKGROUND AND AIMS: The original Geboes Score [OGS] is the most commonly used histological score in ulcerative colitis [UC], but rather complicated to use in daily clinical practice. The aim of this study was to develop a Simplified Geboes Score [SGS] and to compare it with the OGS in patients newly diagnosed with UC. METHODS: All patients diagnosed with UC at a tertiary referral centre between 2005 and 2010, who had serial colonoscopies with biopsies, were retrospectively included. The 5-year endoscopic/histological evolution after diagnosis was recorded. Histological activity was scored by an experienced inflammatory bowel disease pathologist and three trained readers using the OGS and also the new SGS that only includes variables linked to active inflammatory disease. The correlation between endoscopic and histological activity and the histological inter-observer agreement were measured. RESULTS: A total of 528 slides from 339 colonoscopies of 103 UC patients were reviewed. Forty [12%] colonoscopies presented Mayo 0, 74 [22%] Mayo 1, 107 [31%] Mayo 2 and 118 [35%] Mayo 3. Active microscopic disease [≥ 3.1 in both scores] was described in 10/40 [25%] patients who were in complete endoscopic remission [Mayo 0], and 62/74 [84%] with mild endoscopic lesions [Mayo 1]. The correlation analysis between endoscopy and OGS/SGS did not show significant differences between the histological scores. The inter-observer agreement was moderate for all the grades of the SGS. CONCLUSIONS: The assessments of histological activity based on the OGS and the SGS were comparable in newly diagnosed active UC patients. Further prospective validation should now be done to replace the OGS with the SGS.