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.

Benefits and Caveats in the Use of Retinal Pigment Epithelium-Specific Cre Mice.

The retinal pigment epithelium (RPE) is an important monolayer of cells present in the outer retina, forming a major part of the blood-retina barrier (BRB). It performs many tasks essential for the maintenance of retinal integrity and function. With increasing knowledge of the retina, it is becoming clear that both common retinal disorders, like age-related macular degeneration, and rare genetic disorders originate in the RPE. This calls for a better understanding of the functions of various proteins within the RPE. In this regard, mice enabling an RPE-specific gene deletion are a powerful tool to study the role of a particular protein within the RPE cells in their native environment, simultaneously negating any potential influences of systemic changes. Moreover, since RPE cells interact closely with adjacent photoreceptors, these mice also provide an excellent avenue to study the importance of a particular gene function within the RPE to the retina as a whole. In this review, we outline and compare the features of various Cre mice created for this purpose, which allow for inducible or non-inducible RPE-specific knockout of a gene of interest. We summarize the various benefits and caveats involved in the use of such mouse lines, allowing researchers to make a well-informed decision on the choice of Cre mouse to use in relation to their research needs.

Mouse Models to Study Peroxisomal Functions and Disorders: Overview, Caveats, and Recommendations.

During the last three decades many mouse lines were created or identified that are deficient in one or more peroxisomal functions. Different methodologies were applied to obtain global, hypomorph, cell type selective, inducible, and knockin mice. Whereas some models closely mimic pathologies in patients, others strongly deviate or no human counterpart has been reported. Often, mice, apparently endowed with a stronger transcriptional adaptation, have to be challenged with dietary additions or restrictions in order to trigger phenotypic changes. Depending on the inactivated peroxisomal protein, several approaches can be taken to validate the loss-of-function. Here, an overview is given of the available mouse models and their most important characteristics.

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.

Integrated Omic Analysis Delineates Pathways Modulating Toxic TDP-43 Protein Aggregates in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a multi-systemic, incurable, amyloid disease affecting the motor neurons, resulting in the death of patients. The disease is either sporadic or familial with SOD1, C9orf72, FUS, and TDP-43 constituting the majority of familial ALS. Multi-omics studies on patients and model systems like mice and yeast have helped in understanding the association of various signaling and metabolic pathways with the disease. The yeast model system has played a pivotal role in elucidating the gene amyloid interactions. We carried out an integrated transcriptomic and metabolomic analysis of the TDP-43 expressing yeast model to elucidate deregulated pathways associated with the disease. The analysis shows the deregulation of the TCA cycle, single carbon metabolism, glutathione metabolism, and fatty acid metabolism. Transcriptomic analysis of GEO datasets of TDP-43 expressing motor neurons from mice models of ALS and ALS patients shows considerable overlap with experimental results. Furthermore, a yeast model was used to validate the obtained results using metabolite addition and gene knock-out experiments. Taken together, our result shows a potential role for the TCA cycle, cellular redox pathway, NAD metabolism, and fatty acid metabolism in disease. Supplementation of reduced glutathione, nicotinate, and the keto diet might help to manage the disease.

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.

Renal tubular peroxisomes are dispensable for normal kidney function.

Peroxisomes are specialized cellular organelles involved in a variety of metabolic processes. In humans, mutations leading to complete loss of peroxisomes cause multiorgan failure (Zellweger's spectrum disorders, ZSD), including renal impairment. However, the (patho)physiological role of peroxisomes in the kidney remains unknown. We addressed the role of peroxisomes in renal function in mice with conditional ablation of peroxisomal biogenesis in the renal tubule (cKO mice). Functional analyses did not reveal any overt kidney phenotype in cKO mice. However, infant male cKO mice had lower body and kidney weights, and adult male cKO mice exhibited substantial reductions in kidney weight and kidney weight/body weight ratio. Stereological analysis showed an increase in mitochondria density in proximal tubule cells of cKO mice. Integrated transcriptome and metabolome analyses revealed profound reprogramming of a number of metabolic pathways, including metabolism of glutathione and biosynthesis/biotransformation of several major classes of lipids. Although this analysis suggested compensated oxidative stress, challenge with high-fat feeding did not induce significant renal impairments in cKO mice. We demonstrate that renal tubular peroxisomes are dispensable for normal renal function. Our data also suggest that renal impairments in patients with ZSD are of extrarenal origin.

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.