Peroxisome deficiency underlies failures in hepatic immune cell development and antigen presentation in a severe Zellweger disease model.

Peroxisome biogenesis disorders (PBDs) represent a group of metabolic conditions that cause severe developmental defects. Peroxisomes are essential metabolic organelles, present in virtually every eukaryotic cell and mediating key processes in immunometabolism. To date, the full spectrum of PBDs remains to be identified, and the impact PBDs have on immune function is unexplored. This study presents a characterization of the hepatic immune compartment of a neonatal PBD mouse model at single-cell resolution to establish the importance and function of peroxisomes in developmental hematopoiesis. We report that hematopoietic defects are a feature in a severe PBD murine model. Finally, we identify a role for peroxisomes in the regulation of the major histocompatibility class II expression and antigen presentation to CD4+ T cells in dendritic cells. This study adds to our understanding of the mechanisms of PBDs and expands our knowledge of the role of peroxisomes in immunometabolism.

Tissue-specific roles of peroxisomes revealed by expression meta-analysis.

Peroxisomes are primarily studied in the brain, kidney, and liver due to the conspicuous tissue-specific pathology of peroxisomal biogenesis disorders. In contrast, little is known about the role of peroxisomes in other tissues such as the heart. In this meta-analysis, we explore mitochondrial and peroxisomal gene expression on RNA and protein levels in the brain, heart, kidney, and liver, focusing on lipid metabolism. Further, we evaluate a potential developmental and heart region-dependent specificity of our gene set. We find marginal expression of the enzymes for peroxisomal fatty acid oxidation in cardiac tissue in comparison to the liver or cardiac mitochondrial ß-oxidation. However, the expression of peroxisome biogenesis proteins in the heart is similar to other tissues despite low levels of peroxisomal fatty acid oxidation. Strikingly, peroxisomal targeting signal type 2-containing factors and plasmalogen biosynthesis appear to play a fundamental role in explaining the essential protective and supporting functions of cardiac peroxisomes.

Multivariate analysis and model building for classifying patients in the peroxisomal disorders X-linked adrenoleukodystrophy and Zellweger syndrome in Chinese pediatric patients.

BACKGROUND: The peroxisome is a ubiquitous single membrane-enclosed organelle with an important metabolic role. Peroxisomal disorders represent a class of medical conditions caused by deficiencies in peroxisome function and are segmented into enzyme-and-transporter defects (defects in single peroxisomal proteins) and peroxisome biogenesis disorders (defects in the peroxin proteins, critical for normal peroxisome assembly and biogenesis). In this study, we employed multivariate supervised and non-supervised statistical methods and utilized mass spectrometry data of neurological patients, peroxisomal disorder patients (X-linked adrenoleukodystrophy and Zellweger syndrome), and healthy controls to analyze the role of common metabolites in peroxisomal disorders, to develop and refine a classification models of X-linked adrenoleukodystrophy and Zellweger syndrome, and to explore analytes with utility in rapid screening and diagnostics. RESULTS: T-SNE, PCA, and (sparse) PLS-DA, operated on mass spectrometry data of patients and healthy controls were utilized in this study. The performance of exploratory PLS-DA models was assessed to determine a suitable number of latent components and variables to retain for sparse PLS-DA models. Reduced-features (sparse) PLS-DA models achieved excellent classification performance of X-linked adrenoleukodystrophy and Zellweger syndrome patients. CONCLUSIONS: Our study demonstrated metabolic differences between healthy controls, neurological patients, and peroxisomal disorder (X-linked adrenoleukodystrophy and Zellweger syndrome) patients, refined classification models and showed the potential utility of hexacosanoylcarnitine (C26:0-carnitine) as a screening analyte for Chinese patients in the context of a multivariate discriminant model predictive of peroxisomal disorders.

A Cell-Free In Vitro Import System for Peroxisomal Proteins Containing a Type 2 Targeting Signal (PTS2).

Cell-free in vitro systems are invaluable tools to study the molecular mechanisms of protein translocation across biological membranes. We have been using such a strategy to dissect the mechanism of the mammalian peroxisomal matrix protein import machinery. Here, we provide a detailed protocol to import proteins containing a peroxisomal targeting signal type 2 (PTS2) into the organelle. The in vitro system consists of incubating a 35S-labeled reporter protein with a post-nuclear supernatant from rat/mouse liver. At the end of the incubation, the organelle suspensions are generally treated with an aggressive protease to degrade reporter proteins that did not enter peroxisomes, and the organelles are isolated by centrifugation and analyzed by SDS-PAGE and autoradiography. This in vitro system is particularly suited to characterize the functional consequences of PEX5 and PEX7 mutations found in patients affected with a peroxisomal biogenesis disorder.

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.

Comparison of human PEX knockout cell lines suggests a dual role of PEX1 in peroxisome biogenesis.

For the biogenesis and maintenance of peroxisomes several proteins, called peroxins, are essential. Malfunctions of these proteins lead to severe diseases summarized as peroxisome biogenesis disorders. The different genetic background of patient-derived cell lines and the residual expression of mutated PEX genes impede analysis of the whole spectrum of cellular functions of affected peroxins. To overcome these difficulties, we have generated a selected PEX knockout resource of HEK T-REx293 cells using the CRISPR/Cas9 technique. Comparative analyses of whole cell lysates revealed PEX-KO specific alterations in the steady-state level of peroxins and variations in the import efficacy of matrix proteins with a Type 2 peroxisomal targeting signal. One of the observed differences concerned PEX1 as in the complete absence of the protein, the number of peroxisomal ghosts is significantly increased. Upon expression of PEX1, import competence and abundance of peroxisomes was adjusted to the level of normal HEK cells. In contrast, expression of an alternatively spliced PEX1 isoform lacking 321 amino acids of the N-terminal region failed to rescue the peroxisomal import defects but reduced the number of peroxisomal vesicles. All in all, the data suggest a novel 'moonlighting' function of human PEX1 in the regulation of pre-peroxisomal vesicles.

Quantification of Very-Long-Chain and Branched-Chain Fatty Acids in Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

Peroxisomal disorders are a heterogeneous group of genetic disorders caused by impaired peroxisomal biogenesis or by defects in single peroxisomal proteins. The most common peroxisomal disorders are Zellweger spectrum disorders (ZSDs), due to pathogenic variants in one of the 13 PEX genes, and X-linked adrenoleukodystrophy/adrenomyeloneuropathy (X-ALD/AMN), due to pathogenic variants in ATP-binding cassette transporter type D1 (ABCD1) gene. Peroxisomes perform multiple essential cellular functions, including ß-oxidation of very-long-chain fatty acids (VLCFAs), pristanic acid and some bile acid intermediates, and α-oxidation of phytanic acid. In most patients, abnormal levels of VLCFAs and/or branched-chain fatty acids (BCFAs, e.g., phytanic and pristanic acids) are present; hence, measuring these analytes is critical when suspecting a peroxisomal disorder. This chapter describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify VLCFAs and BCFAs in plasma or serum for the diagnosis of peroxisomal disorders. The method consists of an acid hydrolysis step to release the fatty acids from their coenzyme A esters followed by derivatization using oxalyl chloride, dimethylaminoethanol, and then methyl iodide. The trimethyl-amino-ethyl (TMAE) iodide ester derivatives are analyzed using UPLC-MS/MS in positive electrospray ionization and multiple reaction-monitoring (MRM) mode. Quantitation is performed using a five-point calibration curve after normalizing with deuterated internal standards.

Hypomorphic mutation of PEX3 with peroxisomal mosaicism reveals the oscillating nature of peroxisome biogenesis coupled with differential metabolic activities.

Impaired peroxisome assembly caused by mutations in PEX genes results in a human congenital metabolic disease called Zellweger spectrum disorder (ZSD), which impacts the development and physiological function of multiple organs. In this study, we revealed a long-standing problem of heterogeneous peroxisome distribution among cell population, so called "peroxisomal mosaicism", which appears in patients with mild form of ZSD. We mutated PEX3 gene in HEK293 cells and obtained a mutant clone with peroxisomal mosaicism. We found that peroxisomal mosaicism can be reproducibly arise from a single cell, even if the cell has many or no peroxisomes. Using time-lapse imaging and a long-term culture experiment, we revealed that peroxisome biogenesis oscillates over a span of days; this was also confirmed in the patient's fibroblasts. During the oscillation, the metabolic activity of peroxisomes was maintained in the cells with many peroxisomes while depleted in the cells without peroxisomes. Our results indicate that ZSD patients with peroxisomal mosaicism have a cell population whose number and metabolic activities of peroxisomes can be recovered. This finding opens the way to develop novel treatment strategy for ZSD patients with peroxisomal mosaicism, who currently have very limited treatment options.

Beyond rare disorders: A new era for peroxisomal pathophysiology.

Metabolism is emerging as a central influencer of multiple disease states in humans. Peroxisomes are central metabolic organelles whose decreased function gives rise to severe peroxisomal diseases. Recently, it is becoming clear that, beyond such rare inborn errors, the deterioration of peroxisomal functions contributes to multiple and prevalent diseases such as cancer, viral infection, diabetes, and neurodegeneration. Despite the clear importance of peroxisomes in common pathophysiological processes, research on the mechanisms underlying their contributions is still sparse. Here, we highlight the timeliness of focusing on peroxisomes in current research on central, abundant, and society-impacting human pathologies. As peroxisomes are now coming into the spotlight, it is clear that intensive research into these important organelles will enable a better understanding of their contribution to human health, serving as the basis to develop new diagnostic and therapeutic approaches to prevent and treat human diseases.

Stop Codon Context-Specific Induction of Translational Readthrough.

Premature termination codon (PTC) mutations account for approximately 10% of pathogenic variants in monogenic diseases. Stimulation of translational readthrough, also known as stop codon suppression, using translational readthrough-inducing drugs (TRIDs) may serve as a possible therapeutic strategy for the treatment of genetic PTC diseases. One important parameter governing readthrough is the stop codon context (SCC)-the stop codon itself and the nucleotides in the vicinity of the stop codon on the mRNA. However, the quantitative influence of the SCC on treatment outcome and on appropriate drug concentrations are largely unknown. Here, we analyze the readthrough-stimulatory effect of various readthrough-inducing drugs on the SCCs of five common premature termination codon mutations of PEX5 in a sensitive dual reporter system. Mutations in PEX5, encoding the peroxisomal targeting signal 1 receptor, can cause peroxisomal biogenesis disorders of the Zellweger spectrum. We show that the stop context has a strong influence on the levels of readthrough stimulation and impacts the choice of the most effective drug and its concentration. These results highlight potential advantages and the personalized medicine nature of an SCC-based strategy in the therapy of rare diseases.

The biochemical basis of mitochondrial dysfunction in Zellweger Spectrum Disorder.

Peroxisomal biogenesis disorders (PBDs) are genetic disorders of peroxisome biogenesis and metabolism that are characterized by profound developmental and neurological phenotypes. The most severe class of PBDs-Zellweger spectrum disorder (ZSD)-is caused by mutations in peroxin genes that result in both non-functional peroxisomes and mitochondrial dysfunction. It is unclear, however, how defective peroxisomes contribute to mitochondrial impairment. In order to understand the molecular basis of this inter-organellar relationship, we investigated the fate of peroxisomal mRNAs and proteins in ZSD model systems. We found that peroxins were still expressed and a subset of them accumulated on the mitochondrial membrane, which resulted in gross mitochondrial abnormalities and impaired mitochondrial metabolic function. We showed that overexpression of ATAD1, a mitochondrial quality control factor, was sufficient to rescue several aspects of mitochondrial function in human ZSD fibroblasts. Together, these data suggest that aberrant peroxisomal protein localization is necessary and sufficient for the devastating mitochondrial morphological and metabolic phenotypes in ZSDs.

Mitochondria as emergency landing for abandoned peroxins.

Zellweger spectrum disorder (ZSD) is the most severe peroxisomal biogenesis disorder (PBD). Why ZSD patients not only loose functional peroxisomes but also present with severe mitochondrial dysfunction was a long-standing mystery. In this issue, Nuebel et al (2021) identified that loss of peroxisomes leads to re-routing of peroxisomal proteins to mitochondria, thereby impairing mitochondrial structure and function. The findings provide the first molecular understanding of the mitochondrial-peroxisomal link in ZSD.

Peroxisomes of the Brain: Distribution, Functions, and Associated Diseases.

Peroxisomes are versatile cell organelles that exhibit a repertoire of organism and cell-type dependent functions. The presence of oxidases and antioxidant enzymes is a characteristic feature of these organelles. The role of peroxisomes in various cell types in human health and disease is under investigation. Defects in the biogenesis of the organelle and its function lead to severe debilitating disorders. In this manuscript, we discuss the distribution and functions of peroxisomes in the nervous system and especially in the brain cells. The important peroxisomal functions in these cells and their role in the pathology of associated disorders such as neurodegeneration are highlighted in recent studies. Although the cause of the pathogenesis of these disorders is still not clearly understood, emerging evidence supports a crucial role of peroxisomes. In this review, we discuss research highlighting the role of peroxisomes in brain development and its function. We also provide an overview of the major findings in recent years that highlight the role of peroxisome dysfunction in various associated diseases.

A peroxisome deficiency-induced reductive cytosol state up-regulates the brain-derived neurotrophic factor pathway.

The peroxisome is a subcellular organelle that functions in essential metabolic pathways, including biosynthesis of plasmalogens, fatty acid ß-oxidation of very-long-chain fatty acids, and degradation of hydrogen peroxide. Peroxisome biogenesis disorders (PBDs) manifest as severe dysfunction in multiple organs, including the central nervous system (CNS), but the pathogenic mechanisms in PBDs are largely unknown. Because CNS integrity is coordinately established and maintained by neural cell interactions, we here investigated whether cell-cell communication is impaired and responsible for the neurological defects associated with PBDs. Results from a noncontact co-culture system consisting of primary hippocampal neurons with glial cells revealed that a peroxisome-deficient astrocytic cell line secretes increased levels of brain-derived neurotrophic factor (BDNF), resulting in axonal branching of the neurons. Of note, the BDNF expression in astrocytes was not affected by defects in plasmalogen biosynthesis and peroxisomal fatty acid ß-oxidation in the astrocytes. Instead, we found that cytosolic reductive states caused by a mislocalized catalase in the peroxisome-deficient cells induce the elevation in BDNF secretion. Our results suggest that peroxisome deficiency dysregulates neuronal axogenesis by causing a cytosolic reductive state in astrocytes. We conclude that astrocytic peroxisomes regulate BDNF expression and thereby support neuronal integrity and function.

7-Ketocholesterol- and 7ß-Hydroxycholesterol-Induced Peroxisomal Disorders in Glial, Microglial and Neuronal Cells: Potential Role in Neurodegeneration : 7-ketocholesterol and 7ß-hydroxycholesterol-Induced Peroxisomal Disorders and Neurodegeneration.

Peroxisomopathies are qualitative or quantitative deficiencies in peroxisomes which lead to increases in the level of very-long-chain fatty acids (VLCFA) and can be associated with more or less pronounced dysfunction of central nervous system cells: glial and microglial cells. Currently, in frequent neurodegenerative diseases, Alzheimer's disease (AD) and multiple sclerosis (MS), peroxisomal dysfunction is also suspected due to an increase in VLCFA, which can be associated with a decrease of plasmalogens, in these patients. Moreover, in patients suffering from peroxisomopathies, such as X-linked adrenoleukodystrophy (X-ALD), AD, or MS, the increase in oxidative stress observed leads to the formation of cytotoxic oxysterols: 7-ketocholesterol (7KC) and 7ß-hydroxycholesterol (7ß-OHC). These observations led to the demonstration that 7KC and 7ß-OHC alter the biogenesis and activity of peroxisomes in glial and microglial cells. In X-ALD, AD, and MS, it is suggested that 7KC and 7ß-OHC affecting the peroxisome, and which also induce mitochondrial dysfunctions, oxidative stress, and inflammation, could promote neurodegeneration. Consequently, the study of oxisome in peroxisomopathies, AD and MS, could help to better understand the pathophysiology of these diseases to identify therapeutic targets for effective treatments.

Peroxisome: Metabolic Functions and Biogenesis.

Peroxisome is an organelle conserved in almost all eukaryotic cells with a variety of functions in cellular metabolism, including fatty acid ß-oxidation, synthesis of ether glycerolipid plasmalogens, and redox homeostasis. Such metabolic functions and the exclusive importance of peroxisomes have been highlighted in fatal human genetic disease called peroxisomal biogenesis disorders (PBDs). Recent advances in this field have identified over 30 PEX genes encoding peroxins as essential factors for peroxisome biogenesis in various species from yeast to humans. Functional delineation of the peroxins has revealed that peroxisome biogenesis comprises the processes, involving peroxisomal membrane assembly, matrix protein import, division, and proliferation. Catalase, the most abundant peroxisomal enzyme, catalyzes decomposition of hydrogen peroxide. Peroxisome plays pivotal roles in the cellular redox homeostasis and the response to oxidative stresses, depending on intracellular localization of catalase.

Peroxisome Biogenesis Disorders.

Peroxisomes are presented in all eukaryotic cells and play essential roles in many of lipid metabolic pathways, including ß-oxidation of fatty acids and synthesis of ether-linked glycerophospholipids, such as plasmalogens. Impaired peroxisome biogenesis, including defects of membrane assembly, import of peroxisomal matrix proteins, and division of peroxisome, causes peroxisome biogenesis disorders (PBDs). Fourteen complementation groups of PBDs are found, and their complementing genes termed PEXs are isolated. Several new mutations in peroxins from patients with mild PBD phenotype or patients with phenotypes unrelated to the commonly observed impairments of PBD patients are found by next-generation sequencing. Exploring a dysfunctional step(s) caused by the mutation is important for unveiling the pathogenesis of novel mutation by means of cellular and biochemical analyses.

Fatty Acid Oxidation in Peroxisomes: Enzymology, Metabolic Crosstalk with Other Organelles and Peroxisomal Disorders.

Peroxisomes play a central role in metabolism as exemplified by the fact that many genetic disorders in humans have been identified through the years in which there is an impairment in one or more of these peroxisomal functions, in most cases associated with severe clinical signs and symptoms. One of the key functions of peroxisomes is the ß-oxidation of fatty acids which differs from the oxidation of fatty acids in mitochondria in many respects which includes the different substrate specificities of the two organelles. Whereas mitochondria are the main site of oxidation of medium-and long-chain fatty acids, peroxisomes catalyse the ß-oxidation of a distinct set of fatty acids, including very-long-chain fatty acids, pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid. Peroxisomes require the functional alliance with multiple subcellular organelles to fulfil their role in metabolism. Indeed, peroxisomes require the functional interaction with lysosomes, lipid droplets and the endoplasmic reticulum, since these organelles provide the substrates oxidized in peroxisomes. On the other hand, since peroxisomes lack a citric acid cycle as well as respiratory chain, oxidation of the end-products of peroxisomal fatty acid oxidation notably acetyl-CoA, and different medium-chain acyl-CoAs, to CO2 and H2O can only occur in mitochondria. The same is true for the reoxidation of NADH back to NAD+. There is increasing evidence that these interactions between organelles are mediated by tethering proteins which bring organelles together in order to allow effective exchange of metabolites. It is the purpose of this review to describe the current state of knowledge about the role of peroxisomes in fatty acid oxidation, the transport of metabolites across the peroxisomal membrane, its functional interaction with other subcellular organelles and the disorders of peroxisomal fatty acid ß-oxidation identified so far in humans.

Heimler Syndrome.

Heimler syndrome is a rare syndrome associating sensorineural hearing loss with retinal dystrophy and amelogenesis imperfecta due to PEX1 or PEX6 biallelic pathogenic variations. This syndrome is one of the less severe forms of peroxisome biogenesis disorders. In this chapter, we will review clinical, biological, and genetic knowledges about the Heimler syndrome.

A Mouse Model System to Study Peroxisomal Roles in Neurodegeneration of Peroxisome Biogenesis Disorders.

Fourteen PEX genes are currently identified as genes responsible for peroxisome biogenesis disorders (PBDs). Patients with PBDs manifest as neurodegenerative symptoms such as neuronal migration defect and malformation of the cerebellum. To address molecular mechanisms underlying the pathogenesis of PBDs, mouse models for the PBDs have been generated by targeted disruption of Pex genes. Pathological phenotypes and metabolic abnormalities in Pex-knockout mice well resemble those of the patients with PBDs. The mice with tissue- or cell type-specific inactivation of Pex genes have also been established by using a Cre-loxP system. The genetically modified mice reveal that pathological phenotypes of PBDs are mediated by interorgan and intercellular communications. Despite the illustrations of detailed pathological phenotypes in the mutant mice, mechanistic insights into pathogenesis of PBDs are still underway. In this chapter, we overview the phenotypes of Pex-inactivated mice and the current understanding of the pathogenesis underlying PBDs.