Characterization of cellular membranes under conditions of ether lipid deficiency

Etherlipide bilden eine Klasse von Lipiden, die sich durch ihre O-Alkyl-Bindung an der sn-1-Position ihres Glycerin-Rückgrats auszeichnen und deren erste Biosyntheseschritte im Peroxisom ablaufen. Plasmalogene, die wichtigsten Etherlipide, haben große physiologische Bedeutung, zum Beispiel als strukturgebende Moleküle in der Zellmembran. Ein Mangel an Etherlipiden führt im Menschen zu einer schweren Krankheit, rhizomelischer Chondrodysplasia punctata. Neueste Forschungsarbeiten zeigten, dass auch eine Untergruppe der GPI-Anker, einer posttranslationalen Modifikation, die Proteine an der Außenseite der Zellmembran verankert, eine Etherbindung beinhaltet und damit vom peroxisomalen Biosyntheseweg abhängig ist. GPI-verankerte Proteine werden innerhalb der Membran in kleine Subdomänen, „membrane rafts“, dirigiert. Mittels Nano-Elektrospray-Ionisierungs-Massenspektrometrie kann gezeigt werden, dass das Fehlen der Plasmalogene durch die erhöhte Synthese anderer Phospholipide, der Phosphatidylethanolamine, die die gleiche Kopfgruppe (Ethanolamin) wie die meisten Plasmalogene besitzen, kompensiert wird. In Zellkultur kann der Defekt in der peroxisomalen Etherlipid-Biosynthese umgangen werden, indem Vorläufermoleküle mit bereits vorhandener Etherbindung dem Medium beigemengt werden. Interessanterweise reagieren Fibroblasten auf die vermehrte Bildung von Plasmalogenen bei Verfügbarkeit einer solchen Substanz mit einer Reduktion von Phosphatidylethanolaminen. Das weist darauf hin, dass die insgesamte Menge an Ethanolamin-Phospholipiden konstant gehalten wird. Weiters wird bewiesen, dass eine O-Alkyl-Kette in GPI-Ankern nicht zwingend für deren Membranassoziation erforderlich ist. Hingegen zeigt sich, dass die Menge an einzelnen Raft-Proteinen, auch GPI-verankerten Proteinen, in Etherlipid-defizienten Zellen verändert ist. Die Rafts weisen zusätzlich ein verändertes Flotationsverhalten in einem Saccharose-Dichtegradienten auf. Als mögliche funktionelle Konsequenz dieser Veränderungen können leicht verkleinerte Acetylcholinrezeptor-Cluster bei der Enstehung der neuromuskulären Synapse in vitro an Hand von Myotuben aus Etherlipid-defizienten Mäusen, nachgewiesen werden.Ether lipids form a specialized class of lipids that are characterized by an O-alkyl bond at the sn-1 position of their glycerol backbone. The initial steps of their biosynthesis take place in the peroxisome. Among ether lipids, plasmalogens take on a dominant role. They perform important physiological functions, for example as constituents of cellular membranes. In humans, the lack of ether lipids causes a fatal disease, rhizomelic chondrodysplasia punctata. Research has also identified a subpopulation of glycosylphosphatidylinositol anchors, a posttranslational modification that targets proteins to the outer surface of the cell membrane, as an ether lipid. Proteins associated with the membrane by GPI anchorage are recruited to specialized domains called membrane rafts, which are also enriched in plasmalogens. These microdomains constitute important platforms for various cellular processes e.g. signal transduction. Nano-electrospray ionization mass spectrometric analysis of these primary cultured human fibroblasts shows that the cells compensate for the loss of plasmalogens by increasing the synthesis of other phospholipids, namely phosphatidylethanolamines, which share the head group (ethanolamine) with the majority of plasmalogens. Supplementation of ether lipid-deficient cultures with plasmalogen precursors that already contain an ether linkage circumvents the peroxisomal steps of ether lipid synthesis. Interestingly, the resulting rescue of plasmalogen levels causes a corresponding decline in phosphatidylethanolamines, so that the amount of total ethanolamine phospholipids is kept constant in every circumstance. In addition, the present study offers the proof that an O-alkyl chain is not necessarily required for the membrane anchoring of Thy-1, a GPI-anchored protein. However, sucrose density gradient centrifugation suggests a reorganization of membrane rafts upon depletion of ether lipids. Furthermore, some raft proteins, amongst others GPI-anchored proteins, exhibit changed amounts in western blot analysis and show an altered floating behavior in a density gradient. Additionally, preliminary experiments in murine myoblast cultures derived from ether lipid-deficient mice point to differences in the formation of the neuromuscular junction under conditions of ether lipid deficiency. Summarizing, in spite of compensatory mechanisms employed by human fibroblasts in response to ether lipid deficiency, a variety of changes, especially concerning cellular membranes, can be detected under these conditions

Peroxisomes in brain development and function

AbstractPeroxisomes contain numerous enzymatic activities that are important for mammalian physiology. Patients lacking either all peroxisomal functions or a single enzyme or transporter function typically develop severe neurological deficits, which originate from aberrant development of the brain, demyelination and loss of axonal integrity, neuroinflammation or other neurodegenerative processes. Whilst correlating peroxisomal properties with a compilation of pathologies observed in human patients and mouse models lacking all or individual peroxisomal functions, we discuss the importance of peroxisomal metabolites and tissue- and cell type-specific contributions to the observed brain pathologies. This enables us to deconstruct the local and systemic contribution of individual metabolic pathways to specific brain functions. We also review the recently discovered variability of pathological symptoms in cases with unexpectedly mild presentation of peroxisome biogenesis disorders. Finally, we explore the emerging evidence linking peroxisomes to more common neurological disorders such as Alzheimer's disease, autism and amyotrophic lateral sclerosis. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann

International Journal of Molecular Sciences / Ether Lipid Deficiency in Mice Produces a Complex Behavioral Phenotype Mimicking Aspects of Human Psychiatric Disorders

Ether lipids form a specialized subgroup of phospholipids that requires peroxisomes to be synthesized. We have previously detected that deficiency in these lipids leads to a severe disturbance of neurotransmitter homeostasis and release as well as behavioral abnormalities, such as hyperactivity, in a mouse model. Here, we focused on a more detailed examination of the behavioral phenotype of ether lipid-deficient mice (Gnpat KO) and describe a set of features related to human psychiatric disorders. Gnpat KO mice show strongly impaired social interaction as well as nestlet shredding and marble burying, indicating disturbed execution of inborn behavioral patterns. Also, compromised contextual and cued fear conditioning in these animals suggests a considerable memory deficit, thus potentially forming a connection to the previously determined ether lipid deficit in human patients with Alzheimers disease. Nesting behavior and the preference for social novelty proved normal in ether lipid-deficient mice. In addition, we detected task-specific alterations in paradigms assessing depression- and anxiety-related behavior. The reported behavioral changes may be used as easy readout for the success of novel treatment strategies against ether lipid deficiency in ameliorating nervous system-associated symptoms. Furthermore, our findings underline that ether lipids are paramount for brain function and demonstrate their relevance for cognitive, social, and emotional behavior. We hereby substantially extend previous observations suggesting a link between deficiency in ether lipids and human mental illnesses, particularly autism and attention-deficit hyperactivity disorder.(VLID)491278

Homeostasis of phospholipids — The level of phosphatidylethanolamine tightly adapts to changes in ethanolamine plasmalogens

AbstractEthanolamine plasmalogens constitute a group of ether glycerophospholipids that, due to their unique biophysical and biochemical properties, are essential components of mammalian cellular membranes. Their importance is emphasized by the consequences of defects in plasmalogen biosynthesis, which in humans cause the fatal disease rhizomelic chondrodysplasia punctata (RCDP). In the present lipidomic study, we used fibroblasts derived from RCDP patients, as well as brain tissue from plasmalogen-deficient mice, to examine the compensatory mechanisms of lipid homeostasis in response to plasmalogen deficiency. Our results show that phosphatidylethanolamine (PE), a diacyl glycerophospholipid, which like ethanolamine plasmalogens carries the head group ethanolamine, is the main player in the adaptation to plasmalogen insufficiency. PE levels were tightly adjusted to the amount of ethanolamine plasmalogens so that their combined levels were kept constant. Similarly, the total amount of polyunsaturated fatty acids (PUFAs) in ethanolamine phospholipids was maintained upon plasmalogen deficiency. However, we found an increased incorporation of arachidonic acid at the expense of docosahexaenoic acid in the PE fraction of plasmalogen-deficient tissues. These data show that under conditions of reduced plasmalogen levels, the amount of total ethanolamine phospholipids is precisely maintained by a rise in PE. At the same time, a shift in the ratio between ω-6 and ω-3 PUFAs occurs, which might have unfavorable, long-term biological consequences. Therefore, our findings are not only of interest for RCDP but may have more widespread implications also for other disease conditions, as for example Alzheimer's disease, that have been associated with a decline in plasmalogens

Ether lipid transfer across the blood-brain and placental barriers does not improve by inactivation of the most abundant ABC transporters

Phospholipid transport from the periphery to the brain is an understudied topic. When certain lipid species are deficient due to impaired synthesis, though, transfer across the blood-brain barrier is essential for replenishing lipids in the brain. For example, the deficiency in plasmalogens, the most abundant ether lipids in mammals, has detrimental effects on the brain, which is a major issue in inherited peroxisomal disorders but also contributes to more common disorders like Alzheimer's disease. Oral administration of alkylglycerols like batyl alcohol, which carry a pre-formed ether bond, enables replenishment of ether lipids in various peripheral tissues. However, plasmalogen deficiency in the brain cannot be overcome by this approach. Here, we tried to increase cerebral plasmalogen uptake by modulating the efflux transport across the blood-brain barrier. We hypothesized, based on previous literature, that at least some ether lipid species readily enter endothelial cells of the barrier through the transporter MFSD2A but are re-exported by ATP-binding cassette (ABC) transporters. By crossbreeding Mdr1a-/-/Mdr1b-/-/Bcrp-/- and ether lipid-deficient Gnpat-/- mice as well as pharmacological inhibition with MK-571 to inactivate the major ABC transporters at the blood-brain barrier, we evaluated the potential of combined ABC transporter inhibition and oral batyl alcohol administration for the treatment of plasmalogen deficiency. We found that even in the absence of the most abundant ABC transporters, batyl alcohol supplementation did not restore plasmalogen levels in the brain, despite the presence of a wide spectrum of ether lipid subspecies in the plasma as demonstrated by lipidomic analysis. Surprisingly, batyl alcohol treatment of pregnant Gnpat+/- dams had beneficial effects on the plasmalogen levels of Gnpat-/- offspring with defective ether lipid biosynthesis, independently of ABC transporter status at the placental barrier. Our results underline the autonomy of brain lipid homeostasis and indicate that peripheral supplementation of ether lipids is not sufficient to supply the brain with larger amounts of plasmalogens. Yet, the findings suggest that alkylglycerol treatment during pregnancy may pose a viable option to ameliorate some of the severe developmental defects of inborn ether lipid deficiency

Ether lipid transfer across the blood-brain and placental barriers does not improve by inactivation of the most abundant ABC transporters

Phospholipid transport from the periphery to the brain is an understudied topic. When certain lipid species are deficient due to impaired synthesis, though, transfer across the blood-brain barrier is essential for replenishing lipids in the brain. For example, the deficiency in plasmalogens, the most abundant ether lipids in mammals, has detrimental effects on the brain, which is a major issue in inherited peroxisomal disorders but also contributes to more common disorders like Alzheimer's disease. Oral administration of alkylglycerols like batyl alcohol, which carry a pre-formed ether bond, enables replenishment of ether lipids in various peripheral tissues. However, plasmalogen deficiency in the brain cannot be overcome by this approach. Here, we tried to increase cerebral plasmalogen uptake by modulating the efflux transport across the blood-brain barrier. We hypothesized, based on previous literature, that at least some ether lipid species readily enter endothelial cells of the barrier through the transporter MFSD2A but are re-exported by ATP-binding cassette (ABC) transporters. By crossbreeding Mdr1a-/-/Mdr1b-/-/Bcrp-/- and ether lipid-deficient Gnpat-/- mice as well as pharmacological inhibition with MK-571 to inactivate the major ABC transporters at the blood-brain barrier, we evaluated the potential of combined ABC transporter inhibition and oral batyl alcohol administration for the treatment of plasmalogen deficiency. We found that even in the absence of the most abundant ABC transporters, batyl alcohol supplementation did not restore plasmalogen levels in the brain, despite the presence of a wide spectrum of ether lipid subspecies in the plasma as demonstrated by lipidomic analysis. Surprisingly, batyl alcohol treatment of pregnant Gnpat+/- dams had beneficial effects on the plasmalogen levels of Gnpat-/- offspring with defective ether lipid biosynthesis, independently of ABC transporter status at the placental barrier. Our results underline the autonomy of brain lipid homeostasis and indicate that peripheral supplementation of ether lipids is not sufficient to supply the brain with larger amounts of plasmalogens. Yet, the findings suggest that alkylglycerol treatment during pregnancy may pose a viable option to ameliorate some of the severe developmental defects of inborn ether lipid deficiency

Reduced muscle strength in ether lipid-deficient mice is accompanied by altered development and function of the neuromuscular junction

Inherited deficiency in ether lipids, a subgroup of phospholipids whose biosynthesis needs peroxisomes, causes the fatal human disorder rhizomelic chondrodysplasia punctata. The exact roles of ether lipids in the mammalian organism and, therefore, the molecular mechanisms underlying the disease are still largely enigmatic. Here, we used glyceronephosphate O-acyltransferase knockout (Gnpat KO) mice to study the consequences of complete inactivation of ether lipid biosynthesis and documented substantial deficits in motor performance and muscle strength of these mice. We hypothesized that, probably in addition to previously described cerebellar abnormalities and myelination defects in the peripheral nervous system, an impairment of neuromuscular transmission contributes to the compromised motor abilities. Structurally, a morphologic examination of the neuromuscular junction (NMJ) in diaphragm muscle at different developmental stages revealed aberrant axonal branching and a strongly increased area of nerve innervation in Gnpat KO mice. Post-synaptically, acetylcholine receptor (AChR) clusters colocalized with nerve terminals within a widened endplate zone. In addition, we detected atypical AChR clustering, as indicated by decreased size and number of clusters following stimulation with agrin, in vitro. The turnover of AChRs was unaffected in ether lipid-deficient mice. Electrophysiological evaluation of the adult diaphragm indicated that although evoked potentials were unaltered in Gnpat KO mice, ether lipid deficiency leads to fewer spontaneous synaptic vesicle fusion events but, conversely, an increased post-synaptic response to spontaneous vesicle exocytosis. We conclude from our findings that ether lipids are essential for proper development and function of the NMJ and may, therefore, contribute to motor performance. Read the Editorial Highlight for this article on page 46