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Peroxisomes are highly dynamic, oxidative organelles with key metabolic functions in cellular lipid metabolism, such as the β-oxidation of fatty acids and the synthesis of myelin sheath lipids, as well as the regulation of cellular redox balance. Loss of peroxisomal functions causes severe metabolic disorders in humans. Furthermore, peroxisomes also fulfil protective roles in pathogen and viral defence and immunity, highlighting their wider significance in human health and disease. This has sparked increasing interest in peroxisome biology and their physiological functions. This review presents an update and a continuation of three previous review articles addressing the unsolved mysteries of this remarkable organelle. We continue to highlight recent discoveries, advancements, and trends in peroxisome research, and address novel findings on the metabolic functions of peroxisomes, their biogenesis, protein import, membrane dynamics and division, as well as on peroxisome–organelle membrane contact sites and organelle cooperation. Furthermore, recent insights into peroxisome organisation through super-resolution microscopy are discussed. Finally, we address new roles for peroxisomes in immune and defence mechanisms and in human disorders, and for peroxisomal functions in different cell/tissue types, in particular their contribution to organ-specific pathologies.

Peroxisomes and mitochondria are ubiquitous, highly dynamic organelles with an oxidative type of metabolism in eukaryotic cells. Over the years, substantial evidence has been provided that peroxisomes and mitochondria exhibit a close functional interplay which impacts on human health and development. The so-called “peroxisome-mitochondria connection” includes metabolic cooperation in the degradation of fatty acids, a redox-sensitive relationship, an overlap in key components of the membrane fission machineries and cooperation in anti-viral signalling and defence. Furthermore, combined peroxisome-mitochondria disorders with defects in organelle division have been revealed. In this review, we present the latest progress in the emerging field of peroxisomal and mitochondrial interplay in mammals with a particular emphasis on cooperative fatty acid β-oxidation, redox interplay, organelle dynamics, cooperation in anti-viral signalling and the resulting implications for disease.

The absence or dysfunction of the peroxisomal membrane protein Acyl-CoA Binding Domain-Containing Protein 5 (ACBD5) is the cause of the most recently discovered peroxisomal disorder “Retinal Dystrophy with Leukodystrophy” (RDLKD). ACBD5 is a tail-anchored protein, anchored by its C-terminus into the peroxisomal membrane; hence, the bulk of its amino acid sequence faces the cytosol. With respect to ACBD5’s molecular functions, RDLKD is unique since it is not only an accessory protein for the import of very-long-chain fatty acids (VLCFAs) into peroxisomes but also the first identified peroxisomal tethering protein facilitating membrane contacts with the endoplasmic reticulum (ER). Consequently, RDLKD is neither a peroxisomal biogenesis disorder nor single enzyme deficiency, since a deficiency in ACBD5 likely affects several aspects of peroxisomal function including VLCFA degradation, ether lipid synthesis, docosahexaenoic acid synthesis but also the transfer of membrane lipids from the ER to peroxisomes. Hence, RDLKD appears to be a multifactorial disorder leading to a mosaic pathology, combining symptoms caused by the disruption of several pathways. In this review, we will highlight recent findings obtained from case reports of RDLKD patients as well as insights from ACBD5-deficient mouse models to better understand its complex retinal and brain pathology. Moreover, we will discuss the possible contribution of the different dysregulated metabolites in the neurological pathogenesis of this latest peroxisomal disorder.

Membrane-bound organelles such as mitochondria, peroxisomes, or the endoplasmic reticulum (ER) create distinct environments to promote specific cellular tasks such as ATP production, lipid breakdown, or protein export. During recent years, it has become evident that organelles are integrated into cellular networks regulating metabolism, intracellular signaling, cellular maintenance, cell fate decision, and pathogen defence. In order to facilitate such signaling events, specialized membrane regions between apposing organelles bear distinct sets of proteins to enable tethering and exchange of metabolites and signaling molecules. Such membrane associations between the mitochondria and a specialized site of the ER, the mitochondria associated-membrane (MAM), as well as between the ER and the plasma membrane (PAM) have been partially characterized at the molecular level. However, historical and recent observations imply that other organelles like peroxisomes, lysosomes, and lipid droplets might also be involved in the formation of such apposing membrane contact sites. Alternatively, reports on so-called mitochondria derived-vesicles (MDV) suggest alternative mechanisms of organelle interaction. Moreover, maintenance of cellular homeostasis requires the precise removal of aged organelles by autophagy-a process which involves the detection of ubiquitinated organelle proteins by the autophagosome membrane, representing another site of membrane associated-signaling. This review will summarize the available data on the existence and composition of organelle contact sites and the molecular specializations each site uses in order to provide a timely overview on the potential functions of organelle interaction.

Peroxisomes play an important role in the metabolism of a variety of biomolecules, including lipids and bile acids. Peroxisomal Membrane Protein 4 (PXMP4) is a ubiquitously expressed peroxisomal membrane protein that is transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα), but its function is still unknown. To investigate the physiological function of PXMP4, we generated a Pxmp4 knockout ( Pxmp4 −/− ) mouse model using CRISPR/Cas9-mediated gene editing. Peroxisome function was studied under standard chow-fed conditions and after stimulation of peroxisomal activity using the PPARα ligand fenofibrate or by using phytol, a metabolite of chlorophyll that undergoes peroxisomal oxidation. Pxmp4 −/− mice were viable, fertile, and displayed no changes in peroxisome numbers or morphology under standard conditions. Also, no differences were observed in the plasma levels of products from major peroxisomal pathways, including very long-chain fatty acids (VLCFAs), bile acids (BAs), and BA intermediates di- and trihydroxycholestanoic acid. Although elevated levels of the phytol metabolites phytanic and pristanic acid in Pxmp4 −/− mice pointed towards an impairment in peroxisomal α-oxidation capacity, treatment of Pxmp4 −/− mice with a phytol-enriched diet did not further increase phytanic/pristanic acid levels. Finally, lipidomic analysis revealed that loss of Pxmp4 decreased hepatic levels of the alkyldiacylglycerol class of neutral ether lipids, particularly those containing polyunsaturated fatty acids. Together, our data show that while PXMP4 is not critical for overall peroxisome function under the conditions tested, it may have a role in the metabolism of (ether)lipids.

Peroxisomes can be frequently found in proximity to other subcellular organelles such as the endoplasmic reticulum (ER), mitochondria or lysosomes. The tail-anchored protein ACBD5 was recently identified as part of a tethering complex at peroxisome-ER contact sites, interacting with the ER resident protein VAPB. Contact site disruption was found to significantly increase peroxisome motility, apparently interfering with intracellular positioning systems. Unlike other somatic cells, neurons have to distribute organelles across relatively long distances in order to maintain their extraordinary cellular polarity. Using confocal live imaging microscopy in cultured hippocampal neurons we observed that peroxisomes and mitochondria show a strikingly similar motility with approximately 10% performing microtubule-driven long range movements. In order to investigate if ER contacts influence overall peroxisome motility and cellular distribution patterns, hippocampal neurons were transfected with plasmids encoding ACBD5 to stimulate peroxisome-ER interactions. Overexpression of ACBD5 reduced peroxisomal long range movements in the neurites of the hippocampal cells by 70%, implying that ER attachment counteracts microtubule-driven peroxisome transport, while mitochondrial motility was unaffected. Moreover, the analyses of peroxisome distribution in fixed neurons unveiled a significant redistribution of peroxisomes towards the periphery of the perikaryon underneath the plasma membrane and into neurites, where peroxisomes are frequently found in close proximity to mitochondria. Surprisingly, further analysis of peroxisome and VAPB distribution upon ACBD5 expression did not reveal a substantial colocalization, implying this effect may be independent of VAPB. In line with these findings, expression of an ACBD5 variant unable to bind to VAPB still altered the localization of peroxisomes in the same way as the wild-type ACBD5. Thus, we conclude, that the VAPB-ACBD5 facilitated peroxisome-ER interaction is not responsible for the observed organelle redistribution in neurons. Rather, we suggest that additional ACBD5-binding proteins in neurons may tether peroxisomes to contact sites at or near the plasma membrane of neurons.

Peroxisomes are highly dynamic and responsive organelles, which can adjust their morphology, number, intracellular position, and metabolic functions according to cellular needs. Peroxisome multiplication in mammalian cells involves the concerted action of the membrane-shaping protein PEX11β and division proteins, such as the membrane adaptors FIS1 and MFF, which recruit the fission GTPase DRP1 to the peroxisomal membrane. The latter proteins are also involved in mitochondrial division. Patients with loss of DRP1, MFF or PEX11β function have been identified, showing abnormalities in peroxisomal (and, for the shared proteins, mitochondrial) dynamics as well as developmental and neurological defects, whereas the metabolic functions of the organelles are often unaffected. Here, we provide a timely update on peroxisomal membrane dynamics with a particular focus on peroxisome formation by membrane growth and division. We address the function of PEX11β in these processes, as well as the role of peroxisome–ER contacts in lipid transfer for peroxisomal membrane expansion. Furthermore, we summarize the clinical phenotypes and pathophysiology of patients with defects in the key division proteins DRP1, MFF, and PEX11β as well as in the peroxisome–ER tether ACBD5. Potential therapeutic strategies for these rare disorders with limited treatment options are discussed.

Patients deficient in the peroxisomal membrane protein ACBD5 regularly exhibit a dystrophy of the retina along with decline in visual acuity. Despite the prevalent retinal phenotype, information on the pathogenesis of the retinodystrophy is limited. To gain insight into the cellular, subcellular and molecular alterations occurring in the retina, we analyzed an ACBD5-deficient mouse model by immunofluorescence microscopy, electron microscopy, full-field electroretinography (ffERG) and as well as analytical and spatial mass spectrometry (MS)-based lipidomics techniques. Histological results implied that ACBD5-deficient mice exhibit a moderate degeneration of photoreceptor, bipolar, ganglion and retinal pigment epithelial cells accompanied, however, by a prominent activation of astroglia and microglia. Reduced a- and b-wave amplitudes from ffERG point to a severe functional dysregulation of retinal signal transduction with a focus at the level of the information-processing cell of the inner retina. At the lipidome level, very long-chain polyunsaturated fatty acids (VLC-PUFA) accumulated in phosphatidylcholines from retina homogenates, most likely disrupted by a decline in peroxisome functions. Remarkably, as revealed by MALDI MS imaging, these lipidome changes affected neither the whole retina nor the photoreceptor outer segments (POS), where VLC-PUFAs display the highest concentration in phospholipids of POS membrane discs. In contrast, VLC-PUFAs in ACBD5-deficient mice consistently accumulated in the inner retinal region from the outer (OPL) to inner plexiform layer (IPL). In line with VLC-PUFA-accumulations, photoreceptor ribbon synapses in the OPL showed morphological signs of degeneration on the ultrastructural level. Hence, peroxisomal dysfunction appears to affect cell type-specific lipid homeostasis, thereby disrupting local retinal membrane physiology leading to a severe neuroinflammation of the ACBD5-deficient mouse retina.

Ongoing technical and bioinformatics improvements in mass spectrometry (MS) allow for the identifying and quantifying of the enrichment of increasingly less-abundant proteins in individual fractions. Accordingly, this study reassessed the proteome of mouse liver peroxisomes by the parallel isolation of peroxisomes from a mitochondria- and a microsome-enriched prefraction, combining density-gradient centrifugation with a semi-quantitative SWATH-MS proteomics approach to unveil novel peroxisomal or peroxisome-associated proteins. In total, 1071 proteins were identified using MS and assessed in terms of their distribution in either high-density peroxisomal or low-density gradient fractions, containing the bulk of organelle material. Combining the data from both fractionation approaches allowed for the identification of specific protein profiles characteristic of mitochondria, the ER and peroxisomes. Among the proteins significantly enriched in the peroxisomal cluster were several novel peroxisomal candidates. Five of those were validated by colocalization in peroxisomes, using confocal microscopy. The peroxisomal import of HTATIP2 and PAFAH2, which contain a peroxisome-targeting sequence 1 (PTS1), could be confirmed by overexpression in HepG2 cells. The candidates SAR1B and PDCD6, which are known ER-exit-site proteins, did not directly colocalize with peroxisomes, but resided at ER sites, which frequently surrounded peroxisomes. Hence, both proteins might concentrate at presumably co-purified peroxisome-ER membrane contacts. Intriguingly, the fifth candidate, OCIA domain-containing protein 1, was previously described as decreasing mitochondrial network formation. In this work, we confirmed its peroxisomal localization and further observed a reduction in peroxisome numbers in response to OCIAD1 overexpression. Hence, OCIAD1 appears to be a novel protein, which has an impact on both mitochondrial and peroxisomal maintenance.