Overlapping and Distinct Features of Cardiac Pathology in Inherited Human and Murine Ether Lipid Deficiency.

Inherited deficiency in ether lipids, a subgroup of glycerophospholipids with unique biochemical and biophysical properties, evokes severe symptoms in humans resulting in a multi-organ syndrome. Mouse models with defects in ether lipid biosynthesis have widely been used to understand the pathophysiology of human disease and to study the roles of ether lipids in various cell types and tissues. However, little is known about the function of these lipids in cardiac tissue. Previous studies included case reports of cardiac defects in ether-lipid-deficient patients, but a systematic analysis of the impact of ether lipid deficiency on the mammalian heart is still missing. Here, we utilize a mouse model of complete ether lipid deficiency (Gnpat KO) to accomplish this task. Similar to a subgroup of human patients with rhizomelic chondrodysplasia punctata (RCDP), a fraction of Gnpat KO fetuses present with defects in ventricular septation, presumably evoked by a developmental delay. We did not detect any signs of cardiomyopathy but identified increased left ventricular end-systolic and end-diastolic pressure in middle-aged ether-lipid-deficient mice. By comprehensive electrocardiographic characterization, we consistently found reduced ventricular conduction velocity, as indicated by a prolonged QRS complex, as well as increased QRS and QT dispersion in the Gnpat KO group. Furthermore, a shift of the Wenckebach point to longer cycle lengths indicated depressed atrioventricular nodal function. To complement our findings in mice, we analyzed medical records and performed electrocardiography in ether-lipid-deficient human patients, which, in contrast to the murine phenotype, indicated a trend towards shortened QT intervals. Taken together, our findings demonstrate that the cardiac phenotype upon ether lipid deficiency is highly heterogeneous, and although the manifestations in the mouse model only partially match the abnormalities in human patients, the results add to our understanding of the physiological role of ether lipids and emphasize their importance for proper cardiac development and function.

Normal plasmalogen levels are maintained in tissues from mice with hepatocyte-specific deletion in peroxin 5.

On the basis of findings that cultured rat hepatocytes secrete lipoprotein with a high plasmalogen content and the occurrence of this lipid in human serum, it has been suggested that hepatocytes play a role in the supply of plasmalogens to tissues. We tested this hypothesis in a mouse with a hepatocyte-specific defect in peroxisomes, an organelle essentially required for plasmalogen biosynthesis. We analyzed plasmalogens in lipid extracts of forebrain, liver and five further tissues and in plasma by reaction with dansylhydrazine in hydrochloric acid, which cleaves the vinyl ether of plasmalogens and forms a fluorescent dansylhydrazone, which we quantified by reversed phase high performance liquid chromatography. Reaction with dansylhydrazine in acetic acid was used to quantify free aldehydes as a control. Our results show normal levels of plasmalogens in plasma and in all tissues examined, including forebrain and the liver, irrespective of the inactivation of hepatic peroxisomes. None of the selected ether lipids analyzed by mass spectrometry in plasma and liver was decreased in the mice deficient in liver peroxisomes. In contrast, we found three plasmenylcholine species which were even significantly increased in the livers of these animals. Quantification of mRNA expression of plasmalogen biosynthetic enzymes revealed particularly low expression of fatty acyl-CoA reductase, the key regulatory enzyme of plasmalogen biosynthesis, in liver, with and without hepatic peroxisome deficiency. Our results do not support the suggested role of hepatocytes in supplying plasmalogens to tissues.

Regulation of plasmalogen metabolism and traffic in mammals: The fog begins to lift.

Due to their unique chemical structure, plasmalogens do not only exhibit distinct biophysical and biochemical features, but require specialized pathways of biosynthesis and metabolization. Recently, major advances have been made in our understanding of these processes, for example by the attribution of the gene encoding the enzyme, which catalyzes the final desaturation step in plasmalogen biosynthesis, or by the identification of cytochrome C as plasmalogenase, which allows for the degradation of plasmalogens. Also, models have been presented that plausibly explain the maintenance of adequate cellular levels of plasmalogens. However, despite the progress, many aspects around the questions of how plasmalogen metabolism is regulated and how plasmalogens are distributed among organs and tissues in more complex organisms like mammals, remain unresolved. Here, we summarize and interpret current evidence on the regulation of the enzymes involved in plasmalogen biosynthesis and degradation as well as the turnover of plasmalogens. Finally, we focus on plasmalogen traffic across the mammalian body - a topic of major importance, when considering plasmalogen replacement therapies in human disorders, where deficiencies in these lipids have been reported. These involve not only inborn errors in plasmalogen metabolism, but also more common diseases including Alzheimer's disease and neurodevelopmental disorders.

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.

A Pex7 Deficient Mouse Series Correlates Biochemical and Neurobehavioral Markers to Genotype Severity-Implications for the Disease Spectrum of Rhizomelic Chondrodysplasia Punctata Type 1.

Rhizomelic chondrodysplasia punctata type 1 (RCDP1) is a peroxisome biogenesis disorder caused by defects in PEX7 leading to impairment in plasmalogen (Pls) biosynthesis and phytanic acid (PA) oxidation. Pls deficiency is the main pathogenic factor that determines the severity of RCDP. Severe (classic) RCDP patients have negligible Pls levels, congenital cataracts, skeletal dysplasia, growth and neurodevelopmental deficits, and cerebral hypomyelination and cerebellar atrophy on brain MRI. Individuals with milder or nonclassic RCDP have higher Pls levels, better growth and cognitive outcomes. To better understand the pathophysiology of RCDP disorders, we generated an allelic series of Pex7 mice either homozygous for the hypomorphic allele, compound heterozygous for the hypomorphic and null alleles or homozygous for the null allele. Pex7 transcript and protein were almost undetectable in the hypomorphic model, and negligible in the compound heterozygous and null mice. Pex7 deficient mice showed a graded reduction in Pls and increases in C26:0-LPC and PA in plasma and brain according to genotype. Neuropathological evaluation showed significant loss of cerebellar Purkinje cells over time and a decrease in brain myelin basic protein (MBP) content in Pex7 deficient models, with more severe effects correlating with Pex7 genotype. All Pex7 deficient mice exhibited a hyperactive behavior in the open field environment. Brain neurotransmitters analysis of Pex7 deficient mice showed a significant reduction in levels of dopamine, norepinephrine, serotonin and GABA. Also, a significant correlation was found between brain neurotransmitter levels, the hyperactivity phenotype, Pls level and the severity of Pex7 genotype. In conclusion, our study showed evidence of a genotype-phenotype correlation between the severity of Pex7 deficiency and several clinical and neurobiochemical phenotypes in RCDP1 mouse models. We propose that PA accumulation may underlie the cerebellar atrophy seen in older RCDP1 patients, as even relatively low tissue levels were strongly associated with Purkinje cells loss over time in the murine models. Also, our data demonstrate the interrelation between Pls, brain neurotransmitter deficiencies and the neurobehavioral phenotype, which could be further used as a valuable clinical endpoint for therapeutic interventions. Finally, these models show that incremental increases in Pex7 levels result in dramatic improvements in phenotype.

Plasmalogens, platelet-activating factor and beyond - Ether lipids in signaling and neurodegeneration.

Glycerol-based ether lipids including ether phospholipids form a specialized branch of lipids that in mammals require peroxisomes for their biosynthesis. They are major components of biological membranes and one particular subgroup, the plasmalogens, is widely regarded as a cellular antioxidant. Their vast potential to influence signal transduction pathways is less well known. Here, we summarize the literature showing associations with essential signaling cascades for a wide variety of ether lipids, including platelet-activating factor, alkylglycerols, ether-linked lysophosphatidic acid and plasmalogen-derived polyunsaturated fatty acids. The available experimental evidence demonstrates links to several common players like protein kinase C, peroxisome proliferator-activated receptors or mitogen-activated protein kinases. Furthermore, ether lipid levels have repeatedly been connected to some of the most abundant neurological diseases, particularly Alzheimer's disease and more recently also neurodevelopmental disorders like autism. Thus, we critically discuss the potential role of these compounds in the etiology and pathophysiology of these diseases with an emphasis on signaling processes. Finally, we review the emerging interest in plasmalogens as treatment target in neurological diseases, assessing available data and highlighting future perspectives. Although many aspects of ether lipid involvement in cellular signaling identified in vitro still have to be confirmed in vivo, the compiled data show many intriguing properties and contributions of these lipids to health and disease that will trigger further research.

Oral batyl alcohol supplementation rescues decreased cardiac conduction in ether phospholipid-deficient mice.

Plasmalogens (Pls) are a class of membrane phospholipids which serve a number of essential biological functions. Deficiency of Pls is associated with common disorders such as Alzheimer's disease or ischemic heart disease. A complete lack of Pls due to genetically determined defective biosynthesis gives rise to rhizomelic chondrodysplasia punctata (RCDP), characterized by a number of severe disabling pathologic features and death in early childhood. Frequent cardiac manifestations of RCDP include septal defects, mitral valve prolapse, and patent ductus arteriosus. In a mouse model of RCDP, reduced nerve conduction velocity was partially rescued by dietary oral supplementation of the Pls precursor batyl alcohol (BA). Here, we examine the impact of Pls deficiency on cardiac impulse conduction in a similar mouse model (Gnpat KO). In-vivo electrocardiographic recordings showed that the duration of the QRS complex was significantly longer in Gnpat KO mice than in age- and sex-matched wild-type animals, indicative of reduced cardiac conduction velocity. Oral supplementation of BA for 2 months resulted in normalization of cardiac Pls levels and of the QRS duration in Gnpat KO mice but not in untreated animals. BA treatment had no effect on the QRS duration in age-matched wild-type mice. These data suggest that Pls deficiency is associated with increased ventricular conduction time which can be rescued by oral BA supplementation.

Nestlet Shredding and Nest Building Tests to Assess Features of Psychiatric Disorders in Mice.

Mimicking the various facets of human psychiatric and neurodevelopmental disorders in animal models is a challenging task. Nevertheless, mice have emerged as a widely used model system to study pathophysiology and treatment strategies for these diseases. However, the corresponding behavioral tests are often elaborate and require extensive experience in behavioral testing. Here, we present protocols for two simple assays, nest building and nestlet shredding, that can serve as a starting point for the behavioral phenotyping of mouse models with (potential) features of psychiatric disorders. Both tests have been reported previously and we extend prior descriptions by including adaptations and refinements derived from our practical experience, like the use of the home cage instead of a fresh cage for nestlet shredding. Summarized, we provide ready-to-use protocols for two behavioral assays that allow the generation of robust data with minimal time and cost expenditure and enable an initial assessment of features of psychiatric or neurodevelopmental disorders in mouse models of these diseases.

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 Alzheimer's 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.

Impaired plasmalogen synthesis dysregulates liver X receptor-dependent transcription in cerebellum.

Synthesis of ethanolamine plasmalogen (PlsEtn) is regulated by modulating the stability of fatty acyl-CoA reductase 1 (Far1) on peroxisomal membrane, a rate-limiting enzyme in plasmalogen synthesis. Dysregulation of plasmalogen homeostasis impairs cholesterol biosynthesis in cultured cells by altering the stability of squalene epoxidase (SQLE). However, regulation of PlsEtn synthesis and physiological consequences of plasmalogen homeostasis in tissues remain unknown. In the present study, we found that the protein but not the transcription level of Far1 in the cerebellum of the Pex14 mutant mouse expressing Pex14p lacking its C-terminal region (Pex14ΔC/ΔC) is higher than that from wild-type mouse, suggesting that Far1 is stabilized by the lowered level of PlsEtn. The protein level of SQLE was increased, whereas the transcriptional activity of the liver X receptors (LXRs), ligand-activated transcription factors of the nuclear receptor superfamily, is lowered in the cerebellum of Pex14ΔC/ΔC and the mice deficient in dihydroxyacetonephosphate acyltransferase, the initial enzyme for the synthesis of PlsEtn. These results suggest that the reduction of plasmalogens in the cerebellum more likely compromises the cholesterol homeostasis, thereby reducing the transcriptional activities of LXRs, master regulators of cholesterol homeostasis.

Disturbed neurotransmitter homeostasis in ether lipid deficiency.

Plasmalogens, the most prominent ether (phospho)lipids in mammals, are structural components of most cellular membranes. Due to their physicochemical properties and abundance in the central nervous system, a role of plasmalogens in neurotransmission has been proposed, but conclusive data are lacking. Here, we targeted this issue in the glyceronephosphate O-acyltransferase (Gnpat) KO mouse, a model of complete deficiency in ether lipid biosynthesis. Throughout the study, focusing on adult male animals, we found reduced brain levels of various neurotransmitters. In the dopaminergic nigrostriatal tract, synaptic endings but not neuronal cell bodies were affected. Neurotransmitter turnover was altered in ether lipid-deficient murine as well as human post-mortem brain tissue. A generalized loss of synapses did not account for the neurotransmitter deficits, since the levels of several presynaptic proteins appeared unchanged. However, reduced amounts of vesicular monoamine transporter indicate a compromised vesicular uptake of neurotransmitters. As exemplified by norepinephrine, the release of neurotransmitters from Gnpat KO brain slices was diminished in response to strong electrical and chemical stimuli. Finally, addressing potential phenotypic correlates of the disturbed neurotransmitter homeostasis, we show that ether lipid deficiency manifests as hyperactivity and impaired social interaction. We propose that the lack of ether lipids alters the properties of synaptic vesicles leading to reduced amounts and release of neurotransmitters. These features likely contribute to the behavioral phenotype of Gnpat KO mice, potentially modeling some human neurodevelopmental disorders like autism or attention deficit hyperactivity disorder.

Alterations in the Plasma Levels of Specific Choline Phospholipids in Alzheimer's Disease Mimic Accelerated Aging.

Alzheimer's disease (AD) is the most common neurodegenerative disease and of continuously rising prevalence. The identification of easy-to-measure biomarkers capable to assist in the prediction and early diagnosis of AD is currently a main research goal. Lipid metabolites in peripheral blood of human patients have recently gained major attention in this respect. Here, we analyzed plasma of 174 participants (not demented at baseline; mean age: 75.70±0.44 years) of the Vienna Transdanube Aging (VITA) study, a longitudinal, population-based birth cohort study, at baseline and after 90 months or at diagnosis of probable AD. We determined the levels of specific choline phospholipids, some of which have been suggested as potential biomarkers for the prediction of AD. Our results show that during normal aging the levels of lysophosphatidylcholine, choline plasmalogen, and lyso-platelet activating factor increase significantly. Notably, we observed similar but more pronounced changes in the group that developed probable AD. Thus, our results imply that, in terms of choline-containing plasma phospholipids, the conversion to AD mimics an accelerated aging process. We conclude that age, even in the comparatively short time frame between 75 and 82.5 years, is a crucial factor in the quest for plasma lipid biomarkers for AD that must be carefully considered in future studies and trials.

ABCA8 Regulates Cholesterol Efflux and High-Density Lipoprotein Cholesterol Levels.

OBJECTIVE: High-density lipoproteins (HDL) are considered to protect against atherosclerosis in part by facilitating the removal of cholesterol from peripheral tissues. However, factors regulating lipid efflux are incompletely understood. We previously identified a variant in adenosine triphosphate-binding cassette transporter A8 (ABCA8) in an individual with low HDL cholesterol (HDLc). Here, we investigate the role of ABCA8 in cholesterol efflux and in regulating HDLc levels. APPROACH AND RESULTS: We sequenced ABCA8 in individuals with low and high HDLc and identified, exclusively in low HDLc probands, 3 predicted deleterious heterozygous ABCA8 mutations (p.Pro609Arg [P609R], IVS17-2 A>G and p.Thr741Stop [T741X]). HDLc levels were lower in heterozygous mutation carriers compared with first-degree family controls (0.86±0.34 versus 1.17±0.26 mmol/L; P=0.005). HDLc levels were significantly decreased by 29% (P=0.01) in Abca8b-/- mice on a high-cholesterol diet compared with wild-type mice, whereas hepatic overexpression of human ABCA8 in mice resulted in significant increases in plasma HDLc and the first steps of macrophage-to-feces reverse cholesterol transport. Overexpression of wild-type but not mutant ABCA8 resulted in a significant increase (1.8-fold; P=0.01) of cholesterol efflux to apolipoprotein AI in vitro. ABCA8 colocalizes and interacts with adenosine triphosphate-binding cassette transporter A1 and further potentiates adenosine triphosphate-binding cassette transporter A1-mediated cholesterol efflux. CONCLUSIONS: ABCA8 facilitates cholesterol efflux and modulates HDLc levels in humans and mice.

From peroxisomal disorders to common neurodegenerative diseases - the role of ether phospholipids in the nervous system.

The emerging diverse roles of ether (phospho)lipids in nervous system development and function in health and disease are currently attracting growing interest. Plasmalogens, a subgroup of ether lipids, are important membrane components involved in vesicle fusion and membrane raft composition. They store polyunsaturated fatty acids and may serve as antioxidants. Ether lipid metabolites act as precursors for the formation of glycosyl-phosphatidyl-inositol anchors; others, like platelet-activating factor, are implicated in signaling functions. Consolidating the available information, we attempt to provide molecular explanations for the dramatic neurological phenotype in ether lipid-deficient human patients and mice by linking individual functional properties of ether lipids with pathological features. Furthermore, recent publications have identified altered ether lipid levels in the context of many acquired neurological disorders including Alzheimer's disease (AD) and autism. Finally, current efforts to restore ether lipids in peroxisomal disorders as well as AD are critically reviewed.

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 463.

Peroxisomes in brain development and function.

Peroxisomes 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.

Ether lipid deficiency does not cause neutropenia or leukopenia in mice and men.

In this Letter, Dorninger et al. show that the deficiency in ether lipid biosynthesis alone is not sufficient to induce neutropenia or leukopenia, as has been suggested by a previous report using an inducible knockout mouse model of ether lipid deficiency.

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

Ethanolamine 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.