Alterations Of Peroxisomes Research Articles

Efficacy of HDAC Inhibitors in Driving Peroxisomal β-Oxidation and Immune Responses in Human Macrophages: Implications for Neuroinflammatory Disorders.

Elevated levels of saturated very long-chain fatty acids (VLCFAs) in cell membranes and secreted lipoparticles have been associated with neurotoxicity and, therefore, require tight regulation. Excessive VLCFAs are imported into peroxisomes for degradation by β-oxidation. Impaired VLCFA catabolism due to primary or secondary peroxisomal alterations is featured in neurodegenerative and neuroinflammatory disorders such as X-linked adrenoleukodystrophy and multiple sclerosis (MS). Here, we identified that healthy human macrophages upregulate the peroxisomal genes involved in β-oxidation during myelin phagocytosis and pro-inflammatory activation, and that this response is impaired in peripheral macrophages and phagocytes in brain white matter lesions in MS patients. The pharmacological targeting of VLCFA metabolism and peroxisomes in innate immune cells could be favorable in the context of neuroinflammation and neurodegeneration. We previously identified the epigenetic histone deacetylase (HDAC) inhibitors entinostat and vorinostat to enhance VLCFA degradation and pro-regenerative macrophage polarization. However, adverse side effects currently limit their use in chronic neuroinflammation. Here, we focused on tefinostat, a monocyte/macrophage-selective HDAC inhibitor that has shown reduced toxicity in clinical trials. By using a gene expression analysis, peroxisomal β-oxidation assay, and live imaging of primary human macrophages, we assessed the efficacy of tefinostat in modulating VLCFA metabolism, phagocytosis, chemotaxis, and immune function. Our results revealed the significant stimulation of VLCFA degradation with the upregulation of genes involved in peroxisomal β-oxidation and interference with immune cell recruitment; however, tefinostat was less potent than the class I HDAC-selective inhibitor entinostat in promoting a regenerative macrophage phenotype. Further research is needed to fully explore the potential of class I HDAC inhibition and downstream targets in the context of neuroinflammation.

Rise and fall of peroxisomes during Alzheimer´s disease: a pilot study in human brains

Peroxisomes are eukaryotic organelles that rapidly change in number depending on the metabolic requirement of distinct cell types and tissues. In the brain, these organelles are essential for neuronal migration and myelination during development and their dysfunction is associated with age-related neurodegenerative diseases. Except for one study analysing ABCD3-positive peroxisomes in neurons of the frontal neocortex of Alzheimer disease (AD) patients, no data on other brain regions or peroxisomal proteins are available. In the present morphometric study, we quantified peroxisomes labelled with PEX14, a metabolism-independent peroxisome marker, in 13 different brain areas of 8 patients each either with low, intermediate or high AD neuropathological changes compared to 10 control patients. Classification of patient samples was based on the official ABC score. During AD-stage progression, the peroxisome density decreased in the area entorhinalis, parietal/occipital neocortex and cerebellum, it increased and in later AD-stage patients decreased in the subiculum and hippocampal CA3 region, frontal neocortex and pontine gray and it remained unchanged in the gyrus dentatus, temporal neocortex, striatum and inferior olive. Moreover, we investigated the density of catalase-positive peroxisomes in a subset of patients (> 80 years), focussing on regions with significant alterations of PEX14-positive peroxisomes. In hippocampal neurons, only one third of all peroxisomes contained detectable levels of catalase exhibiting constant density at all AD stages. Whereas the density of all peroxisomes in neocortical neurons was only half of the one of the hippocampus, two thirds of them were catalase-positive exhibiting increased levels at higher ABC scores. In conclusion, we observed spatiotemporal differences in the response of peroxisomes to different stages of AD-associated pathologies.Graphical

Protective effects of milk thistle (Sylibum marianum) seed oil and α-tocopherol against 7β-hydroxycholesterol-induced peroxisomal alterations in murine C2C12 myoblasts: Nutritional insights associated with the concept of pexotherapy

Peroxisomes play an important role in regulating cell metabolism and RedOx homeostasis. Peroxisomal dysfunctions favor oxidative stress and cell death. The ability of 7β-hydroxycholesterol (7β-OHC; 50 μM, 24 h), known to be increased in patients with age-related diseases such as sarcopenia, to trigger oxidative stress, mitochondrial and peroxisomal dysfunction was studied in murine C2C12 myoblasts. The capacity of milk thistle seed oil (MTSO, 100 μg/mL) as well as α-tocopherol (400 µM; reference cytoprotective agent) to counteract the toxic effects of 7β-OHC, mainly at the peroxisomal level were evaluated. The impacts of 7β-OHC, in the presence or absence of MTSO or α-tocopherol, were studied with complementary methods: measurement of cell density and viability, quantification of reactive oxygen species (ROS) production and transmembrane mitochondrial potential (ΔΨm), evaluation of peroxisomal mass as well as topographic, morphologic and functional peroxisomal changes. Our results indicate that 7β-OHC induces a loss of cell viability and a decrease of cell adhesion associated with ROS overproduction, alterations of mitochondrial ultrastructure, a drop of ΔΨm, and several peroxisomal modifications. In the presence of 7β-OHC, comparatively to untreated cells, important quantitative and qualitative peroxisomal modifications were also identified: a) a reduced number of peroxisomes with abnormal sizes and shapes, mainly localized in cytoplasmic vacuoles, were observed; b) the peroxisomal mass was decreased as indicated by lower protein and mRNA levels of the peroxisomal ABCD3 transporter; c) lower mRNA level of Pex5 involved in peroxisomal biogenesis as well as higher mRNA levels of Pex13 and Pex14, involved in peroxisomal biogenesis and/or pexophagy, was found; d) lower levels of ACOX1 and MFP2 enzymes, implicated in peroxisomal β-oxidation, were detected; e) higher levels of very-long-chain fatty acids, which are substrates of peroxisomal β-oxidation, were found. These different cytotoxic effects were strongly attenuated by MTSO, in the same range of order as with α-tocopherol. These findings underline the interest of MTSO and α-tocopherol in the prevention of peroxisomal damages (pexotherapy).

Ultrastructure of cytopathological responses in Solanum tuberosum cv. Spounta infected with necrotic strain of potato virus Y

Ultrastructural responses of plant cell organelles in leaf tissues of potato cv. Spounta towards potato virus Y (PVY-necrotic and ordinary strains) infection were investigated using an electron microscope. Numerous cytopathological alterations in cell wall, middle lamella, nucleus, chloroplast, mitochondria, endoplasmic reticulum and peroxisomes have been observed compared to healthy tissues. Splitting, destroying, joining to neighboring protoplasts and necrosis in cell wall were observed. Cell nucleus was changed to lobe-like shape; damage in envelope and enlargement of pore complexes were observed. Mitochondria were increased in number, elongated and destroyed. Many changes in chloroplasts such as deformation, reduction in size and number, damage of envelope and grana thylakoids were observed. Virus particles and cytoplasmic inclusions were observed in ultra-thin sections. To our knowledge, obtained results showed that only peroxisomes could be responsible organelles for induction of necrotic symptoms, because they were increased in number and frequently existed only in plant cells infected with PVY-necrotic strain.

Prostate Cancer Proliferation Is Affected by the Subcellular Localization of MCT2 and Accompanied by Significant Peroxisomal Alterations.

Simple SummaryFatty acid β-oxidation is a dominant bioenergetic pathway in prostate cancer. It has recently been suggested that the specific targeting of monocarboxylate transporter 2 (MCT2) to peroxisomes contributed to an increase in β-oxidation rates and maintenance of the redox balance in prostate cancer cells. Here we provide evidence demonstrating that prostate cancer streamlines peroxisome metabolism by upregulating distinct pathways involved in lipid metabolism. Importantly, we show that the localization of MCT2 at peroxisomes is required for prostate cancer cell proliferation. Our results emphasize the importance of peroxisomes for prostate cancer development and highlight different cellular mechanisms that may be further explored as possible targets for prostate cancer therapy.Reprogramming of lipid metabolism directly contributes to malignant transformation and progression. The increased uptake of circulating lipids, the transfer of fatty acids from stromal adipocytes to cancer cells, the de novo fatty acid synthesis, and the fatty acid oxidation support the central role of lipids in many cancers, including prostate cancer (PCa). Fatty acid β-oxidation is the dominant bioenergetic pathway in PCa and recent evidence suggests that PCa takes advantage of the peroxisome transport machinery to target monocarboxylate transporter 2 (MCT2) to peroxisomes in order to increase β-oxidation rates and maintain the redox balance. Here we show evidence suggesting that PCa streamlines peroxisome metabolism by upregulating distinct pathways involved in lipid metabolism. Moreover, we show that MCT2 is required for PCa cell proliferation and, importantly, that its specific localization at the peroxisomal membranes is essential for this role. Our results highlight the importance of peroxisomes in PCa development and uncover different cellular mechanisms that may be further explored as possible targets for PCa therapy.

Mitochondrial fission factor (MFF) is a critical regulator of peroxisome maturation

Peroxisomes are highly dynamic subcellular compartments with important functions in lipid and ROS metabolism. Impaired peroxisomal function can lead to severe metabolic disorders with developmental defects and neurological abnormalities. Recently, a new group of disorders has been identified, characterised by defects in the membrane dynamics and division of peroxisomes rather than by loss of metabolic functions. However, the contribution of impaired peroxisome plasticity to the pathophysiology of those disorders is not well understood. Mitochondrial fission factor (MFF) is a key component of both the peroxisomal and mitochondrial division machinery. Patients with MFF deficiency present with developmental and neurological abnormalities. Peroxisomes (and mitochondria) in patient fibroblasts are highly elongated as a result of impaired organelle division. The majority of studies into MFF-deficiency have focused on mitochondrial dysfunction, but the contribution of peroxisomal alterations to the pathophysiology is largely unknown. Here, we show that MFF deficiency does not cause alterations to overall peroxisomal biochemical function. However, loss of MFF results in reduced import-competency of the peroxisomal compartment and leads to the accumulation of pre-peroxisomal membrane structures. We show that peroxisomes in MFF-deficient cells display alterations in peroxisomal redox state and intra-peroxisomal pH. Removal of elongated peroxisomes through induction of autophagic processes is not impaired. A mathematical model describing key processes involved in peroxisome dynamics sheds further light into the physical processes disturbed in MFF-deficient cells. The consequences of our findings for the pathophysiology of MFF-deficiency and related disorders with impaired peroxisome plasticity are discussed.

Dimethyl fumarate and monomethyl fumarate attenuate oxidative stress and mitochondrial alterations leading to oxiapoptophagy in 158N murine oligodendrocytes treated with 7β-hydroxycholesterol

Oxidative stress and mitochondrial dysfunction contribute to the pathogenesis of neurodegenerative diseases and favor lipid peroxidation, leading to increased levels of 7β-hydroxycholesterol (7β-OHC) which induces oxiapoptophagy (OXIdative stress, APOPTOsis, autoPHAGY). The cytoprotective effects of dimethylfumarate (DMF), used in the treatment of relapsing remitting multiple sclerosis and of monomethylfumarate (MMF), its main metabolite, were evaluated on murine oligodendrocytes 158 N exposed to 7β-OHC (50 μM, 24 h) with or without DMF or MMF (25 μM). The activity of 7β-OHC in the presence or absence DMF or MMF was evaluated on several parameters: cell adhesion; plasma membrane integrity measured with propidium iodide (PI), trypan blue and fluoresceine diacetate (FDA) assays; LDH activity; antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)); generation of lipid peroxidation products (malondialdehyde (MDA), conjugated dienes (CDs)) and protein oxidation products (carbonylated proteins (CPs)); reactive oxygen species (ROS) overproduction conducted with DHE and DHR123. The effect on mitochondria was determined with complementary criteria: measurement of succinate dehydrogenase activity, evaluation of mitochondrial potential (ΔΨm) and mitochondrial superoxide anions (O2●−) production using DiOC6(3) and MitoSOX, respectively; quantification of mitochondrial mass with Mitotracker Red, and of cardiolipins and organic acids. The effects on mitochondrial and peroxisomal ultrastructure were determined by transmission electron microscopy. Intracellular sterol and fatty acid profiles were determined. Apoptosis and autophagy were characterized by staining with Hoechst 33,342, Giemsa and acridine orange, and with antibodies raised against caspase-3 and LC3. DMF and MMF attenuate 7β-OHC-induced cytotoxicity: cell growth inhibition; decreased cell viability; mitochondrial dysfunction (decrease of succinate dehydrogenase activity, loss of ΔΨm, increase of mitochondrial O2●− production, alteration of the tricarboxilic acid (TCA) cycle, and cardiolipins content); oxidative stress induction (ROS overproduction, alteration of GPx, CAT, and SOD activities, increased levels of MDA, CDs, and CPs); changes in fatty acid and cholesterol metabolism; and cell death induction (caspase-3 cleavage, activation of LC3-I in LC3-II). Ultrastructural alterations of mitochondria and peroxisomes were prevented. These results demonstrate that DMF and MMF prevent major dysfunctions associated with neurodegenerative diseases: oxidative stress, mitochondrial dysfunction, apoptosis and autophagy.

Thermogenic flux induced by lignoceric acid in peroxisomes isolated from HepG2 cells and from X-adrenoleukodystrophy and control fibroblasts.

This work analyzes the thermogenic flux induced by the very long-chain fatty acid (VLCFA) lignoceric acid (C24:0) in isolated peroxisomes. Specific metabolic alterations of peroxisomes are related to a variety of disorders, the most frequent one being the neurodegenerative inherited disease X-linked adrenoleukodystrophy (X-ALD). A peroxisomal transport protein is mutated in this disorder. Due to reduced catabolism and enhanced fatty acid (FA) elongation, VLCFA accumulates in plasma and in all tissues, contributing to the clinical manifestations of this disorder. During peroxisomal metabolism, heat is produced but it is considered lost. Instead, it is a form of energy that could play a role in molecular mechanisms of this pathology and other neurodegenerative disorders. The thermogenic flux induced by lignoceric acid (C24:0) was estimated by isothermal titration calorimetry in peroxisomes isolated from HepG2 cells and from fibroblasts obtained from patients withX-ALD and healthy subjects. Heat flux induced by lignoceric acid in HepG2 peroxisomes was exothermic, indicating normal peroxisomal metabolism. In X-ALD peroxisomes the heat flux was endothermic, indicating the requirement of heat/energy, possibly for cellular metabolism. In fibroblasts from healthy subjects, the effect was less pronounced than in HepG2, a kind of cell known to have greater FA metabolism than fibroblasts. Our hypothesis is that heat is not lost but it could act as an activator, for example on the heat-sensitive pathway related to TRVP2 receptors. To investigate this hypothesis we focused on peroxisomal metabolism, considering that impaired heat generation could contribute to the development of peroxisomal neurodegenerative disorders.

Silencing of PPARαBb mRNA in brown trout primary hepatocytes: effects on molecular and morphological targets under the influence of an estrogen and a PPARα agonist.

The crosstalk between peroxisome proliferator-activated receptor α (PPARα) and estrogenic pathways are shared from fish to humans. Salmonid fish had an additional genome duplication, and two PPARα isoforms (PPARαBa and PPARαBb) were previously identified. Since a negative regulation between estrogen signaling and PPARα was described, a post-transcriptional gene silencing for PPARαBb was designed in primary brown trout hepatocytes. The aims of the study were to: (i) decipher the effects of PPARαBb knock-down on peroxisome morphology and on mRNA expression of potential target genes, and (ii) to assess the cross-interferences caused by an estrogenic compound (17α-ethinylestradiol - EE2) and a PPARα agonist (Wy-14,643 - Wy) using the established knock-down model. A knock-down efficiency of 70% was achieved for PPARαBb and its silencing significantly reduced the volume density of peroxisomes, but did not alter mRNA levels of the studied genes. Exposure to Wy did not change peroxisome morphology or mRNA expression, but under silencing conditions Wy rescued the volume density of peroxisomes to control levels, and increased acyl-coenzyme A oxidase 1-3l (Acox1-3l) mRNA. Exposure to EE2 caused a reduction of peroxisome volume density, but under silencing conditions this effect was abolished and ApoA1 mRNA level was diminished. The morphological alterations of peroxisomes by WY and EE2 demonstrated that obtained results are PPARαBb dependent, and suggest the regulation of unknown downstream targets of PPARαBb. In summary, PPARαBb is involved in the control of peroxisome size and/or number, which opens future opportunities to explore its regulation and molecular targets.

Induction of peroxisomal changes in oligodendrocytes treated with 7-ketocholesterol: Attenuation by α-tocopherol

The involvement of organelles in cell death is well established especially for endoplasmic reticulum, lysosomes and mitochondria. However, the role of the peroxisome is not well known, though peroxisomal dysfunction favors a rupture of redox equilibrium. To study the role of peroxisomes in cell death, 158 N murine oligodendrocytes were treated with 7-ketocholesterol (7 KC: 25–50 μM, 24 h). The highest concentration is known to induce oxiapoptophagy (OXIdative stress + APOPTOsis + autoPHAGY), whereas the lowest concentration does not induce cell death. In those conditions (with 7 KC: 50 μM) morphological, topographical and functional peroxisome alterations associated with modifications of the cytoplasmic distribution of mitochondria, with mitochondrial dysfunction (loss of transmembrane mitochondrial potential, decreased level of cardiolipins) and oxidative stress were observed: presence of peroxisomes with abnormal sizes and shapes similar to those observed in Zellweger fibroblasts, lower cellular level of ABCD3, used as a marker of peroxisomal mass, measured by flow cytometry, lower mRNA and protein levels (measured by RT-qPCR and western blotting) of ABCD1 and ABCD3 (two ATP-dependent peroxisomal transporters), and of ACOX1 and MFP2 enzymes, and lower mRNA level of DHAPAT, involved in peroxisomal β-oxidation and plasmalogen synthesis, respectively, and increased levels of very long chain fatty acids (VLCFA: C24:0, C24:1, C26:0 and C26:1, quantified by gas chromatography coupled with mass spectrometry) metabolized by peroxisomal β-oxidation. In the presence of 7 KC (25 μM), slight mitochondrial dysfunction and oxidative stress were found, and no induction of apoptosis was detected; however, modifications of the cytoplasmic distribution of mitochondria and clusters of mitochondria were detected. The peroxisomal alterations observed with 7 KC (25 μM) were similar to those with 7 KC (50 μM). In addition, data obtained by transmission electron microcopy and immunofluorescence microscopy by dual staining with antibodies raised against p62, involved in autophagy, and ABCD3, support that 7 KC (25–50 μM) induces pexophagy. 7 KC (25–50 μM)-induced side effects were attenuated by α-tocopherol but not by α-tocotrienol, whereas the anti-oxidant properties of these molecules determined with the FRAP assay were in the same range. These data provide evidences that 7 KC, at concentrations inducing or not cell death, triggers morphological, topographical and functional peroxisomal alterations associated with minor or major mitochondrial changes.

Peroxisome morphology in pathology.

Peroxisomes are remarkably dynamic and versatile organelles that are essential for human health and development. They respond to physiological changes in the cellular environment by adapting their morphology, number, enzyme content and metabolic functions accordingly. With the discovery of the first key peroxisomal morphology proteins, the investigation of peroxisomal shape, distribution and dynamics has become an exciting new field in cell biology and biomedical sciences because of its relation to organelle functionality and its impact on developmental and physiological processes. In this review, we summarize recent findings on peroxisome biology, dynamics and the modulation of peroxisome morphology, especially in mammals. Furthermore, we discuss the roles of peroxisome dynamics and morphology in cell pathology and present recent examples for alterations in peroxisome morphology under disease conditions. Besides defects in the peroxisomal morphology machinery, we also address peroxisome biogenesis disorders, alterations of peroxisome number during carcinogenesis and liver cirrhosis, and morphological alterations of peroxisomes during viral infection.

Effect of elevated [CO2] on cell structure and function in seed plants

Unprecedented increase in atmospheric carbon dioxide concentration [CO2] is regarded as one of the key factors responsible for global climate change. Evidence from palaeo-records indicates a close relationship between variation in atmospheric [CO2] and corresponding structural adaptations in plants. Exposure to elevated [CO2] alters plant structure by inducing changes in rate of cell division, cell expansion and cell cycling. Alterations in plant structure under elevated [CO2] at organ level is possibly a result of metabolic changes induced at cellular level. Chloroplast, the light-harvesting organelle, has been shown to be most seriously affected by high [CO2]. Prolonged exposure to elevated [CO2] induces accumulation of starch grains within chloroplasts leading to distortion of thylakoids. Higher rate of photosynthesis in plants grown at high [CO2] is accompanied by increased number of mitochondria to meet high cellular energy demand. These ultrastructural observations are corroborated by studies on nuclear genome that have revealed up as well as down-regulation of several genes involved in photosynthetic and respiratory pathways. In some species, exposure to elevated [CO2] results in accumulation of tannin-like vacuolar deposition, suggesting activation of phenylpropanoid pathway. Ultrastructural alterations in peroxisomes, endoplasmic reticulum, Golgi apparatus and cytoskeleton due to CO2 enrichment have received less attention. It is imperative to understand cellular changes occurring with elevated [CO2]so as to correlate these with the physiological alterations at plant level. A thorough insight into ultrastructural changes in response to increased carbon dioxide will provide a better, generalized understanding of plant response to rising atmospheric [CO2] in future. Disruption of cell organelle structure and function may also indicate a wider impact of climate change across plant (and animal) groups.

Organelle dynamics and dysfunction: A closer link between peroxisomes and mitochondria

Mitochondria and peroxisomes are ubiquitous subcellular organelles, which fulfil an indispensable role in the cellular metabolism of higher eukaryotes. Moreover, they are highly dynamic and display large plasticity. There is growing evidence now that both organelles exhibit a closer interrelationship than previously appreciated. This connection includes metabolic cooperations and cross-talk, a novel putative mitochondria-to-peroxisome vesicular trafficking pathway, as well as an overlap in key components of their fission machinery. Thus, peroxisomal alterations in metabolism, biogenesis, dynamics and proliferation can potentially influence mitochondrial functions, and vice versa. In this review, we present the latest progress in the emerging field of peroxisomal and mitochondrial interrelationship with a particular emphasis on organelle dynamics and its implication in diseases.

Psychosine-induced alterations in peroxisomes of twitcher mouse liver

Krabbe disease is a neuroinflammatory disorder in which galactosylsphingosine (psychosine) accumulates in nervous tissue. To gain insight into whether the psychosine-induced effects in nervous tissue extend to peripheral organs, we investigated the expression of cytokines and their effects on peroxisomal structure/functions in twitcher mouse liver (animal model of Krabbe disease). Immunofluorescence analysis demonstrated TNF-α and IL-6 expression, which was confirmed by mRNAs quantitation. Despite the presence of TNF-α, lipidomic analysis did not indicate a significant decrease in sphingomyelin or an increase in ceramide fractions. Ultrastructural analysis of catalase-dependent staining of liver sections showed reduced reactivity without significant changes in peroxisomal contents. This observation was confirmed by assaying catalase activity and quantitation of its mRNA, both of which were found significantly decreased in twitcher mouse liver. Western blot analysis demonstrated a generalized reduction of peroxisomal matrix and membrane proteins. These observations indicate that twitcher mouse pathobiology extends to the liver, where psychosine-induced TNF-α and IL-6 compromise peroxisomal structure and functions.

Fatty Acid Alterations in Liver Peroxisomes from n–3-Deficient Mice

A diet deficient in n–3 fatty acids dramatically reduces docosahexaenoic acid (4.8-fold) and 20:5n–3 content in murine total peroxisomal phospholipids, and conversely increases 22:5n–6 (17.1-fold) and also, to a lesser extent, 20:4n–6. This was also found in purified phosphatidylethanolamine and phosphatidylcholine. After changing the non-deficient diet (containing α-linolenic acid, ALA) to a deficient one (deficient in ALA), it took a very long time for docosahexaenoic acid concentration in peroxisomes to decline (>5 months). In contrast, after changing the deficient to a non-deficient diet, time to complete recovery was more rapid (3 weeks). Changes in 20:5n–3, 22:6n–3 and 20:4n–6 were generally stabilized within 2–4 weeks. Dietary n–3 fatty acids control the fatty acid composition of peroxisomal membranes, and thus possibly affect some of their functions.

Mitochondrial alterations of retinal pigment epithelium in age-related macular degeneration

Mitochondrial dysfunctions have been implicated in the pathophysiology of several age-related diseases including age-related macular degeneration (AMD), a progressive neurodegenerative disease affecting primarily the retinal pigment epithelium (RPE). The aims of our electron microscopic and morphometric studies were to reveal qualitative and quantitative alterations of mitochondria in human RPE from AMD and from age- and sex-matched controls. With increasing age a significant decrease in number and area of mitochondria, as well as loss of cristae and matrix density were found in both AMD and control specimens. These decreases were significantly greater in AMD than in normal aging. Alterations of mitochondria were accompanied by proliferation of peroxisomes and lipofuscin granules in both AMD and control specimens, although the difference between groups was significant only for peroxisomes. Unexpectedly, morphometric data showed that the RPE alterations seen in AMD may also develop in normal aging, 10–15 years after appearing in AMD patients. These findings suggest that (i) the severity of mitochondrial and peroxisomal alterations are different between AMD and normal aging, and (ii) the timing of damage to RPE may be critical for the development of AMD. We conclude that besides the well-documented age-related changes in mitochondrial DNA, alterations of mitochondrial membranes may also play a role in the pathogenesis of AMD. These membranes could be a new target for treatment of AMD and other age-related diseases.

A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPARα-dependent and -independent pathways

Branched-chain fatty acids (such as phytanic and pristanic acid) are ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the effects of these physiological compounds in vivo, wild-type and PPARalpha-deficient (PPARalpha-/-) mice were fed a phytol-enriched diet. This resulted in increased plasma and liver levels of the phytol metabolites phytanic and pristanic acid. In wild-type mice, plasma fatty acid levels decreased after phytol feeding, whereas in PPARalpha-/- mice, the already elevated fatty acid levels increased. In addition, PPARalpha-/- mice were found to be carnitine deficient in both plasma and liver. Dietary phytol increased liver free carnitine in wild-type animals but not in PPARalpha-/- mice. Investigation of carnitine biosynthesis revealed that PPARalpha is likely involved in the regulation of carnitine homeostasis. Furthermore, phytol feeding resulted in a PPARalpha-dependent induction of various peroxisomal and mitochondrial beta-oxidation enzymes. In addition, a PPARalpha-independent induction of catalase, phytanoyl-CoA hydroxylase, carnitine octanoyltransferase, peroxisomal 3-ketoacyl-CoA thiolase, and straight-chain acyl-CoA oxidase was observed. In conclusion, branched-chain fatty acids are physiologically relevant ligands of PPARalpha in mice. These findings are especially relevant for disorders in which branched-chain fatty acids accumulate, such as Refsum disease and peroxisome biogenesis disorders.

Using Laser Scanning Cytometry to Measure PPAR-Mediated Peroxisome Proliferation and β Oxidation

Laser scanning cytometry (LSC) is a new technology that combines the properties and advantages of flow cytometry (FC) and immunohistochemistry (IHC), thus providing qualitative and quantitative information on protein expression with the additional perspective provided by cell and tissue localization. Formalin-fixed, paraffin embedded liver sections from rats exposed to a Peroxisome Proliferator Activated Receptor (PPAR) agonist were stained with antibodies against peroxisomal targeting signal-1 (PTS-1) (a highly conserved tripeptide contained within all peroxisomal enzymes), Acyl CoA oxidase (AOX) (the rate limiting enzyme of peroxisomal beta oxidation), and catalase (an inducible peroxisomal antioxidant enzyme) to evaluate peroxisomal beta oxidation, oxidative stress, and peroxisome proliferation. The LSC showed increased AOX, catalase, and PTS-1 expression in centrilobular hepatocytes that correlated favorably with the microscopic observation of centrilobular hepatocellular hypertrophy and with the palmitoyl CoA biochemical assay for peroxisomal beta oxidation, and provided additional morphologic information about peroxisome proliferation and tissue patterns of activation. Therefore, the LSC provides qualitative and quantitative evaluation of peroxisome activity with similar sensitivity but higher throughput than the traditional biochemical methods. The additional benefits of the LSC include the direct correlation between histopathologic observations and peroxisomal alterations and the potential utilization of archived formalin-fixed tissues from a variety of organs and species.

Hepatic peroxisomes in isolated hyperpipecolic acidaemia: evidence supporting its classification as a single peroxisomal enzyme deficiency.

Hyperpipecolic acidaemia is still regarded as a peroxisomal assembly deficiency. The enzyme responsible for the accumulation of pipecolic acid is located in the peroxisomes in man. We studied the appearance and alterations of peroxisomes in liver biopsy material from three unrelated children suffering from isolated hyperpipecolic acidaemia, in which only the metabolism of pipecolic acid is disturbed, using light and electron microscopy after cytochemical staining for visualisation of peroxisomes. Morphometric results showed the presence of normal-sized to small peroxisomes, an increase in number and abnormally shaped organelles, suggesting enhancement of metabolic efficiency. In one case enlarged organelles were observed. Skin fibroblasts were studied in all patients: their peroxisomes appeared to be normal. The obvious presence of peroxisomes in isolated HPA indicates that this disorder should be classified as a single peroxisomal enzyme deficiency.

Endotoxin induces structure-function alterations of rat liver peroxisomes: Kupffer cells released factors as possible modulators.

We report that endotoxin treatment results in decreased amounts of peroxisomes as well as changes in structure and function of peroxisomal membranes. Peroxisomes isolated from the liver of control and treated animals showed a marked decrease in total protein, but no significant alteration in the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein profile. However, the Western blot study of the peroxisomal beta-oxidation enzymes and catalase showed an increase in those enzymes in the peroxisomal peak of normal density in endotoxin-treated rats. Disintegration of peroxisomal membranes by carbonate treatment from endotoxin-treated liver and change in the fluidity of peroxisomal membranes suggests alterations in peroxisomal membrane structure. No such alterations were found in mitochondrial or microsomal membranes of endotoxin-treated livers. The lipid analysis of these organelles showed that the only organelle affected was the peroxisome, with a significant decrease in the phospholipid and cholesterol concentrations. To understand the mechanism of endotoxin-mediated alterations in peroxisomes, we studied the possible role of Kupffer cell secreted soluble factors (tumor necrosis factor alpha [TNF-alpha]) on the peroxisomal structure/function. Inactivation/elimination of Kupffer cells by gadolinium chloride before endotoxin treatment did not normalize the overall peroxisomal protein amount and the lipid composition of isolated peroxisomes. However, the levels of individual protein amount in remaining peroxisomes were normalized. Endotoxin also decreased peroxisomal beta-oxidation, and this was partially restored with gadolinium treatment. These results clearly show that peroxisomes are severely affected by endotoxin treatment and suggest that the damage to this organelle may contribute, at least in part, to endotoxin-induced hepatic cytotoxicity.