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.

Small GTPases in peroxisome dynamics.

In this review article, we summarize current knowledge on peroxisome biogenesis/functions and the role that small GTPases may play in these processes. Precise intracellular distribution of cell organelles requires their regulated association to microtubules and the actin cytoskeleton. In this respect, RhoGDP/RhoGTP favor binding of peroxisomes to microtubules and actin filaments. In its GTP-bound form, RhoA activates a regulatory cascade involving Rho kinaseII and non-muscle myosinIIA. Such interactions frequently depend on phosphoinositides (PIs) of which PI4P, PI(4,5)P2, and PI(3,5)P2 were found to be present in the peroxisomal membrane. PIs are pivotal determinants of intracellular signaling and known to regulate a wide range of cellular functions. In many of these functions, small GTPases are implicated. The small GTPase ADP-ribosylation factor 1 (Arf1), for example, is known to stimulate synthesis of PI4P and PI(4,5)P2 on the Golgi to regulate protein and lipid sorting. In vitro binding assays localized Arf1 and the COPI complex to peroxisomes. In light of the recent discussion of pre-peroxisomal vesicle generation at the ER, peroxisomal Arf1-COPI vesicles may serve retrograde transport of ER-resident components. A mass spectrometric screen localized various Rab proteins to peroxisomes. Overexpression of these proteins in combination with laser-scanning fluorescence microscopy co-localized Rab6, Rab8, Rab10, Rab14, and Rab18 with peroxisomal structures. By analogy to the role these proteins play in other organelle dynamics, we may envisage what the function of these proteins may be in relation to the peroxisomal compartment.

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.

Peripheral nervous system plasmalogens regulate Schwann cell differentiation and myelination.

Rhizomelic chondrodysplasia punctata (RCDP) is a developmental disorder characterized by hypotonia, cataracts, abnormal ossification, impaired motor development, and intellectual disability. The underlying etiology of RCDP is a deficiency in the biosynthesis of ether phospholipids, of which plasmalogens are the most abundant form in nervous tissue and myelin; however, the role of plasmalogens in the peripheral nervous system is poorly defined. Here, we used mouse models of RCDP and analyzed the consequence of plasmalogen deficiency in peripheral nerves. We determined that plasmalogens are crucial for Schwann cell development and differentiation and that plasmalogen defects impaired radial sorting, myelination, and myelin structure. Plasmalogen insufficiency resulted in defective protein kinase B (AKT) phosphorylation and subsequent signaling, causing overt activation of glycogen synthase kinase 3ß (GSK3ß) in nerves of mutant mice. Treatment with GSK3ß inhibitors, lithium, or 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) restored Schwann cell defects, effectively bypassing plasmalogen deficiency. Our results demonstrate the requirement of plasmalogens for the correct and timely differentiation of Schwann cells and for the process of myelination. In addition, these studies identify a mechanism by which the lack of a membrane phospholipid causes neuropathology, implicating plasmalogens as regulators of membrane and cell signaling.

Localization of Rab proteins to peroxisomes: a proteomics and immunofluorescence study.

A proteomics screen was initiated to identify Rab proteins regulating transport to and away from peroxisomes. Mass spectrometry-based protein correlation profiling of rat liver organelles and immunofluorescence analysis of the peroxisome candidate Rab proteins revealed Rab6, Rab10, Rab14 and Rab18 to associate with the peroxisomal membrane. While Rab14 localized to peroxisomes predominantly in its dominant-active form, other Rab proteins associated with peroxisomes in both their GTP- and GDP-bound state. In summary, our data suggest that Rab6, Rab10, Rab14 and Rab18 associate with the peroxisomal compartment and similar as previously shown for Rab8, Rab18 in its GDP-bound state favors peroxisome proliferation.

Impaired neurotransmission in ether lipid-deficient nerve terminals.

Isolated defects of ether lipid (EL) biosynthesis in humans cause rhizomelic chondrodysplasia punctata type 2 and type 3, serious peroxisomal disorders. Using a previously described mouse model [Rodemer, C., Thai, T.P., Brugger, B., Kaercher, T., Werner, H., Nave, K.A., Wieland, F., Gorgas, K., and Just, W.W. (2003) Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice. Hum. Mol. Genet., 12, 1881-1895], we investigated the effect of EL deficiency in isolated murine nerve terminals (synaptosomes) on the pre-synaptic release of the neurotransmitters (NTs) glutamate and acetylcholine. Both Ca(2+)-dependent exocytosis and Ca(2+)-independent efflux of the transmitters were affected. EL-deficient synaptosomes respire at a reduced rate and exhibit a lowered adenosin-5'-triphosphate/adenosine diphosphate (ATP/ADP) ratio. Consequently, ATP-driven processes, such as synaptic vesicle cycling and maintenance of Na(+), K(+) and Ca(2+) homeostasis, might be disturbed. Analyzing reactive oxygen species in EL-deficient neural and non-neural tissues revealed that plasmalogens (PLs), the most abundant EL species in mammalian central nervous system, considerably contribute to the generation of the lipid peroxidation product malondialdehyde. Although EL-deficient tissue contains less lipid peroxidation products, fibroblasts lacking ELs are more susceptible to induced oxidative stress. In summary, these results suggest that due to the reduced energy state of EL-deficient tissue, the Ca(2+)-independent efflux of NTs increases while the Ca(2+)-dependent release declines. Furthermore, lack of PLs is mainly compensated for by an increase in the concentration of phosphatidylethanolamine and results in a significantly lowered level of lipid peroxidation products in the brain cortex and cerebellum.

The phosphoinositide 3-kinase Vps34p is required for pexophagy in Saccharomyces cerevisiae.

PIds (phosphoinositides) are phosphorylated derivatives of the membrane phospholipid PtdIns that have emerged as key regulators of many aspects of cellular physiology. We have discovered a PtdIns3P-synthesizing activity in peroxisomes of Saccharomyces cerevisiae and have demonstrated that the lipid kinase Vps34p is already associated with peroxisomes during biogenesis. However, although Vps34 is required, it is not essential for optimal peroxisome biogenesis. The function of Vps34p-containing complex I as well as a subset of PtdIns3P-binding proteins proved to be mandatory for the regulated degradation of peroxisomes. This demonstrates that PtdIns3P-mediated signalling is required for pexophagy.

RhoA regulates peroxisome association to microtubules and the actin cytoskeleton.

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.

Disruption of blood-testis barrier dynamics in ether-lipid-deficient mice.

One of the major roles of Sertoli cells is to establish the blood-testis (Sertoli cell) barrier (BTB), which is permanently assembled and disassembled to accommodate the translocation of leptotene spermatocytes from the basal into the adluminal compartment of the seminiferous epithelium and to guarantee completion of meiosis and spermiogenesis. Recently, we have demonstrated spermatogenesis to be arrested before spermatid elongation in Gnpat-null mice with selective deficiency of ether lipids (ELs) whose functions are poorly understood. In this study, we have focused on the spatio-temporal expression of several BTB tight-junctional proteins in the first wave of spermatogenesis to obtain insights into the physiological role of ELs during BTB establishment and dynamics. Our data confirm the transient existence of Russell's intermediate or translocation compartment delineated by two separate claudin-3-positive luminal and basal tight junctions and reveal that EL deficiency blocks BTB remodeling. This block is associated with (1) downregulation and mistargeting of claudin-3 and (2) impaired BTB disassembly resulting in deficient sealing of the intermediate compartment as shown by increased BTB permeability to biotin. These results suggest that ELs are essential for cyclic BTB dynamics ensuring the sluice mechanism for leptotene translocation into the adluminal compartment.

Defects in myelination, paranode organization and Purkinje cell innervation in the ether lipid-deficient mouse cerebellum.

Ether lipids (ELs), particularly plasmalogens, are essential constituents of the mammalian central nervous system. The physiological role of ELs, in vivo, however is still enigmatic. In the present study, we characterized a mouse model carrying a targeted deletion of the peroxisomal dihydroxyacetonephosphate acyltransferase gene that results in the complete lack of ELs. Investigating the cerebellum of these mice, we observed: (i) defects in foliation patterning and delay in precursor granule cell migration, (ii) defects in myelination and concomitant reduction in the level of myelin basic protein, (iii) disturbances in paranode organization by extending the Caspr distribution and disrupting axo-glial septate-like junctions, (iv) impaired innervation of Purkinje cells by both parallel fibers and climbing fibers and (v) formation of axon swellings by the accumulation of inositol-tris-phosphate receptor 1 containing smooth ER-like tubuli. Functionally, conduction velocity of myelinated axons in the corpus callosum was significantly reduced. Most of these phenotypes were already apparent at P20 but still persisted in 1-year-old animals. In summary, these data show that EL deficiency results in severe developmental and lasting structural alterations at the cellular and network level of the cerebellum, and reveal an important role of ELs for proper brain function. Common molecular mechanisms that may underlie these phenotypes are discussed.

The ether lipid-deficient mouse: tracking down plasmalogen functions.

Chemical and physico-chemical properties as well as physiological functions of major mammalian ether-linked glycerolipids, including plasmalogens were reviewed. Their chemical structures were described and their effect on membrane fluidity and membrane fusion discussed. The recent generation of mouse models with ether lipid deficiency offered the possibility to study ether lipid and particularly plasmalogen functions in vivo. Ether lipid-deficient mice revealed severe phenotypic alterations, including arrest of spermatogenesis, development of cataract and defects in central nervous system myelination. In several cell culture systems lack of plasmalogens impaired intracellular cholesterol distribution affecting plasma membrane functions and structural changes of ER and Golgi cisternae. Based on these phenotypic anomalies that were accurately described conclusions were drawn on putative functions of plasmalogens. These functions were related to cell-cell or cell-extracellular matrix interactions, formation of lipid raft microdomains and intracellular cholesterol homeostasis. There are several human disorders, such as Zellweger syndrome, rhizomelic chondrodysplasia punctata, Alzheimer's disease, Down syndrome, and Niemann-Pick type C disease that are distinguished by altered tissue plasmalogen concentrations. The role plasmalogens might play in the pathology of these disorders is discussed.

Peroxisome biogenesis: where Arf and coatomer might be involved.

The present review summarizes recent observations on binding of Arf and COPI coat to isolated rat liver peroxisomes. The general structural and functional features of both Arf and coatomer were considered along with the requirements and dependencies of peroxisomal Arf and coatomer recruitment. Studies on the expression of mammalian Pex11 proteins, mainly Pex11alpha and Pex11beta, intimately related to the process of peroxisome proliferation, revealed a sequence of individual steps including organelle elongation/tubulation, formation of membrane and matrix protein patches segregating distinct proteins from each other, development of membrane constrictions and final membrane fission. Based on the similarities of the processes leading to cargo selection and concentration on Golgi membranes on the one hand and to the formation of peroxisomal protein patches on the other hand, an implication of Arf and COPI in distinct processes of peroxisomal proliferation is hypothesized. Alternatively, peroxisomal Arf/COPI might facilitate the formation of COPI-coated peroxisomal vesicles functioning in cargo transport and retrieval from peroxisomes to the ER. Recent observations suggesting transport of Pex3 and Pex19 during early steps of peroxisome biogenesis from the ER to peroxisomes inevitably propose such a retrieval mechanism, provided the ER to peroxisome pathway is based on transporting vesicles.

Phosphoinositide synthesis and degradation in isolated rat liver peroxisomes.

Analyzing peroxisomal phosphoinositide (PId(#)) synthesis in highly purified rat liver peroxisomes we found synthesis of phosphatidylinositol 4-phosphate (PtdIns4P), PtdIns(4,5)P(2) and PtdIns(3,5)P(2). PtdIns3P was hardly detected in vitro, however, was observed in vivo after [(32)P]-phosphate labeling of primary rat hepatocytes. In comparison with other subcellular organelles peroxisomes revealed a unique PId pattern suggesting peroxisomal specificity of the observed synthesis. Use of phosphatase inhibitors enhanced the amount of PtdIns4P. The results obtained provide evidence that isolated rat liver peroxisomes synthesize PIds and suggest the association of PId 4-kinase and PId 5-kinase and PId 4-phosphatase activities with the peroxisomal membrane.

Binding and functions of ADP-ribosylation factor on mammalian and yeast peroxisomes.

We have analyzed in vitro the binding characteristics of members of the ADP-ribosylation factor (ARF) family of proteins to a highly purified rat liver peroxisome preparation void of Golgi membranes and studied in vivo a role these proteins play in the proliferation of yeast peroxisomes. Although both ARF1 and ARF6 were found on peroxisomes, coatomer recruitment only depended on ARF1-GTP. Recruitment of ARF1 and coatomer to peroxisomes was significantly affected both by pretreating the animals with peroxisome proliferators and by ATP and a cytosolic fraction designated the intermediate pool fraction depleted of ARF and coatomer. In the presence of ATP, the concentrations of ARF1 and coatomer on peroxisomes were reduced, whereas intermediate pool fraction led to a concentration-dependent decrease in ARF and increase in coatomer. Brefeldin A, a fungal toxin that is known to reduce ARF1 binding to Golgi membranes, did not affect ARF1 binding to peroxisomes. In Saccharomyces cerevisiae, both ScARF1 and ScARF3, the yeast orthologs of mammalian ARF1 and ARF6, were implicated in the control of peroxisome proliferation. ScARF1 regulated this process in a positive manner, and ScARF3 regulated it in a negative manner.

Role of dihydroxyacetonephosphate acyltransferase in the biosynthesis of plasmalogens and nonether glycerolipids.

The variant CHO-K1 cell line, NRel-4, is unable to synthesize plasmalogens because of a severe reduction in dihydroxyacetonephosphate acyltransferase (DHAPAT) activity (Nagan, N., A. K. Hajra, L. K. Larkins, P. Lazarow, P. E. Purdue, W. B. Rizzo, and R. A. Zoeller. 1998. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol. Biochem. J. 332: 273-279). Northern analysis demonstrated that the loss of this activity was attributable to a severe reduction in mRNA levels for DHAPAT. Transfection of NRel-4 cells with a plasmid bearing the human DHAPAT cDNA recovered DHAPAT activity and plasmalogen biosynthesis. Examination of clonal isolates from the transfected population showed that recovery of as little as 10% of wild-type DHAPAT activity restored plasmalogen levels to 55% of normal, whereas in one isolate, NRel-4.15, which overexpressed DHAPAT activity by 6-fold over wild-type cells, plasmalogen levels were returned only to wild-type values. Although the rate of plasmenylethanolamine biosynthesis was restored in NRel-4.15, the biosynthesis of nonether glycerolipids was either decreased or unaffected, suggesting that peroxisomal DHAPAT does not normally contribute to nonether glycerolipid biosynthesis. These data demonstrate that a defect in the gene that codes for peroxisomal DHAPAT is the primary lesion in the NRel-4 cell line and that the peroxisomal DHAPAT is essential for the biosynthesis of plasmalogens in animal cells.

Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice.

Although known for almost 80 years, the physiological role of plasmalogens (PLs), the major mammalian ether lipids (ELs), is still enigmatic. Humans that lack ELs suffer from rhizomelic chondrodysplasia punctata (RCDP), a peroxisomal disorder usually resulting in death in early childhood. In order to learn more about the functions of ELs, we generated a mouse model for RCDP by a targeted disruption of the dihydroxyacetonephosphate acyltransferase gene. The mutant mice revealed multiple abnormalities, such as male infertility, defects in eye development, cataract and optic nerve hypoplasia, some of which were also observed in RCDP. Mass spectroscopic analysis demonstrated the presence of highly unsaturated fatty acids including docosahexaenoic acid (DHA) in brain PLs and the occurrence of PLs in lipid raft microdomains (LRMs) isolated from brain myelin. In mutants, PLs were completely absent and the concentration of brain DHA was reduced. The marker proteins flotillin-1 and F3/contactin were found in brain LRMs in reduced concentrations. In addition, the gap junctional protein connexin 43, known to be recruited to LRMs and essential for lens development and spermatogenesis, was down-regulated in embryonic fibroblasts of the EL-deficient mice. Free cholesterol, an important constituent of LRMs, was found in these fibroblasts to be accumulated in a perinuclear compartment. These data suggest that the EL-deficient mice allow the identification of new phenotypes not related so far to EL-deficiency (male sterility, defects in myelination and optic nerve hypoplasia) and indicate that PLs are required for the correct assembly and function of LRMs.