Presence of thiamine pyrophosphate in mammalian peroxisomes

<p>Abstract</p> <p>Background</p> <p>Thiamine pyrophosphate (TPP) is a cofactor for 2-hydroxyacyl-CoA lyase 1 (HACL1), a peroxisomal enzyme essential for the α-oxidation of phytanic acid and 2-hydroxy straight chain fatty acids. So far, HACL1 is the only known peroxisomal TPP-dependent enzyme in mammals. Little is known about the transport of metabolites and cofactors across the peroxisomal membrane and no peroxisomal thiamine or TPP carrier has been identified in mammals yet. This study was undertaken to get a better insight into these issues and to shed light on the role of TPP in peroxisomal metabolism.</p> <p>Results</p> <p>Because of the crucial role of the cofactor TPP, we reanalyzed its subcellular localization in rat liver. In addition to the known mitochondrial and cytosolic pools, we demonstrated, for the first time, that peroxisomes contain TPP (177 ± 2 pmol/mg protein). Subsequently, we verified whether TPP could be synthesized from its precursor thiamine, <it>in situ</it>, by a peroxisomal thiamine pyrophosphokinase (TPK). However, TPK activity was exclusively recovered in the cytosol.</p> <p>Conclusion</p> <p>Our results clearly indicate that mammalian peroxisomes do contain TPP but that no pyrophosphorylation of thiamine occurs in these organelles, implying that thiamine must enter the peroxisome already pyrophosphorylated. Consequently, TPP entry may depend on a specific transport system or, in a bound form, on HACL1 translocation.</p

Peroxisomal 2-Hydroxyacyl-CoA Lyase Is Involved in Endogenous Biosynthesis of Heptadecanoic Acid.

Circulating heptadecanoic acid (C17:0) is reported to be a pathology risk/prognosis biomarker and a dietary biomarker. This pathology relationship has been shown to be reliably predictive even when independent of dietary contributions, suggesting that the endogenous biosynthesis of C17:0 is related to the pathological aetiology. Little is known about C17:0 biosynthesis, which tissues contribute to the circulating levels, and how C17:0 is related to pathology. Hacl1+/- mice were mated to obtain Hacl1-/- and Hacl1+/+ control mice. At 14 weeks, they were anesthetized for tissue collection and fatty acid analysis. Compared to Hacl1+/+, C15:0 was not significantly affected in any Hacl1-/- tissues. However, the Hacl1-/- plasma and liver C17:0 levels were significantly lower: ~26% and ~22%, respectively. No significant differences were seen in the different adipose tissues. To conclude, Hacl1 plays a significant role in the liver and plasma levels of C17:0, providing evidence it can be endogenously biosynthesized via alpha-oxidation. The strong inverse association of C17:0 with pathology raises the question whether there is a direct link between α-oxidation and these diseases. Currently, there is no clear evidence, warranting further research into the role of α-oxidation in relation to metabolic diseases

Glial β-Oxidation regulates drosophila energy metabolism

The brain's impotence to utilize long-chain fatty acids as fuel, one of the dogmas in neuroscience, is surprising, since the nervous system is the tissue most energy consuming and most vulnerable to a lack of energy. Challenging this view, we here show in vivo that loss of the Drosophila carnitine palmitoyltransferase 2 (CPT2), an enzyme required for mitochondrial β-oxidation of long-chain fatty acids as substrates for energy production, results in the accumulation of triacylglyceride-filled lipid droplets in adult Drosophila brain but not in obesity. CPT2 rescue in glial cells alone is sufficient to restore triacylglyceride homeostasis, and we suggest that this is mediated by the release of ketone bodies from the rescued glial cells. These results demonstrate that the adult brain is able to catabolize fatty acids for cellular energy production.This work was partially supported by the Flanders Fund for Scientific Research (FWO G 0.666.10N), NEUROBRAINNET IAP 7/16, Flemish Government Methusalem Grant, Spanish Ministry of Science (SAF2010-14906) and Innovation Ingenio-Consolider (CSD2010-00045) and Spanish Ministry of Economy and Competitiveness (SAF2013-45392).Peer Reviewe

Glial β-Oxidation regulates drosophila energy metabolism

The brain's impotence to utilize long-chain fatty acids as fuel, one of the dogmas in neuroscience, is surprising, since the nervous system is the tissue most energy consuming and most vulnerable to a lack of energy. Challenging this view, we here show in vivo that loss of the Drosophila carnitine palmitoyltransferase 2 (CPT2), an enzyme required for mitochondrial β-oxidation of long-chain fatty acids as substrates for energy production, results in the accumulation of triacylglyceride-filled lipid droplets in adult Drosophila brain but not in obesity. CPT2 rescue in glial cells alone is sufficient to restore triacylglyceride homeostasis, and we suggest that this is mediated by the release of ketone bodies from the rescued glial cells. These results demonstrate that the adult brain is able to catabolize fatty acids for cellular energy production.This work was partially supported by the Flanders Fund for Scientific Research (FWO G 0.666.10N), NEUROBRAINNET IAP 7/16, Flemish Government Methusalem Grant, Spanish Ministry of Science (SAF2010-14906) and Innovation Ingenio-Consolider (CSD2010-00045) and Spanish Ministry of Economy and Competitiveness (SAF2013-45392).Peer Reviewe

Peroxisome deficiency but not the defect in ether lipid synthesis causes activation of the innate immune system and axonal loss in the central nervous system

Background: Mice with peroxisome deficiency in neural cells (Nestin-Pex5 −/− ) develop a neurodegenerative phenotype leading to motor and cognitive disabilities and early death. Major pathologies at the end stage of disease include severe demyelination, axonal degeneration and neuroinflammation. We now investigated the onset and progression of these pathological processes, and their potential interrelationship. In addition, the putative role of oxidative stress, the impact of plasmalogen depletion on the neurodegenerative phenotype, and the consequences of peroxisome elimination in the postnatal period were studied. Methods: Immunohistochemistry in association with gene expression analysis was performed on Nestin-Pex5 −/− mice to document demyelination, axonal damage and neuroinflammation. Also Gnpat −/− mice, with selective plasmalogen deficiency and CMV-Tx-Pex5 −/− mice, with tamoxifen induced generalized loss of peroxisomes were analysed. Results: Activation of the innate immune system is a very early event in the pathological process in Nestin-Pex5 −/− mice which evolves in chronic neuroinflammation. The complement factor C1q, one of the earliest up regulated transcripts, was expressed on neurons and oligodendrocytes but not on microglia. Transcripts of other pro- and anti-inflammatory genes and markers of phagocytotic activity were already significantly induced before detecting pathologies with immunofluorescent staining. Demyelination, macrophage activity and axonal loss co-occurred throughout the brain. As in patients with mild peroxisome biogenesis disorders who develop regressive changes, demyelination in cerebellum and brain stem preceded major myelin loss in corpus callosum of both Nestin-Pex5 −/− and CMV-Tx-Pex5 −/− mice. These lesions were not accompanied by generalized oxidative stress throughout the brain. Although Gnpat −/− mice displayed dysmyelination and Purkinje cell axon damage in cerebellum, confirming previous observations, no signs of inflammation or demyelination aggravating with age were observed. Conclusions: Peroxisome inactivity triggers a fast neuroinflammatory reaction, which is not solely due to the depletion of plasmalogens. In association with myelin abnormalities this causes axon damage and loss

Lipid homeostasis and inflammatory activation are disturbed in classically activated macrophages with peroxisomal β-oxidation deficiency

Macrophage activation is characterized by pronounced metabolic adaptation. Classically activated macrophages show decreased rates of mitochondrial fatty acid oxidation and oxidative phosphorylation and acquire a glycolytic state together with their pro-inflammatory phenotype. In contrast, alternatively activated macrophages require oxidative phosphorylation and mitochondrial fatty acid oxidation for their anti-inflammatoryfunction. Although it is evident that mitochondrial metabolism is regulated during macrophage polarization and essential for macrophagefunction, little is known on the regulation and role of peroxisomal -oxidation during macrophage activation. In this study, we show that peroxisomal -oxidation is strongly decreased in classically activated bone-marrow-derived macrophages (BMDM) and mildly induced in alternatively activated BMDM. To examine the role of peroxisomal -oxidation in macrophages, we used Mfp2(-/-) BMDM lacking the key enzyme of this pathway. Impairment of peroxisomal -oxidation in Mfp2(-/-) BMDM did not cause lipid accumulation but rather an altered distribution of lipid species with very-long-chain fatty acids accumulating in the triglyceride and phospholipid fraction. These lipid alterations in Mfp2(-/-) macrophages led to decreased inflammatory activation of Mfp2(-/-) BMDM and peritoneal macrophages evidenced by impaired production of several inflammatory cytokines and chemokines, but did not affect anti-inflammatory polarization. The disturbed inflammatory responses of Mfp2(-/-) macrophages did not affect immune cell infiltration, as mice with selective elimination of MFP2 from myeloid cells showed normal monocyte and neutrophil influx upon challenge with zymosan. Together, these data demonstrate that peroxisomal -oxidation is involved in fine-tuning the phenotype of macrophages, probably by influencing the dynamic lipid profile during macrophage polarization

Synaptic Vesicle Docking: Sphingosine Regulates Syntaxin1 Interaction with Munc18

Consensus exists that lipids must play key functions in synaptic activity but precise mechanistic information is limited. Acid sphingomyelinase knockout mice (ASMko) are a suitable model to address the role of sphingolipids in synaptic regulation as they recapitulate a mental retardation syndrome, Niemann Pick disease type A (NPA), and their neurons have altered levels of sphingomyelin (SM) and its derivatives. Electrophysiological recordings showed that ASMko hippocampi have increased paired-pulse facilitation and post-tetanic potentiation. Consistently, electron microscopy revealed reduced number of docked vesicles. Biochemical analysis of ASMko synaptic membranes unveiled higher amounts of SM and sphingosine (Se) and enhanced interaction of the docking molecules Munc18 and syntaxin1. In vitro reconstitution assays demonstrated that Se changes syntaxin1 conformation enhancing its interaction with Munc18. Moreover, Se reduces vesicle docking in primary neurons and increases paired-pulse facilitation when added to wt hippocampal slices. These data provide with a novel mechanism for synaptic vesicle control by sphingolipids and could explain cognitive deficits of NPA patients