Phosphatidylserine in the Brain: Metabolism and Function
Hee-Yong Kim, Bill X. Huang, and Arthur A. Spector Laboratory of Molecular Signaling, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892-9410 United States
Prog Lipid Res. 2014 October ;
ABSTRACT
Phosphatidylserine (PS) is the major anionic phospholipid class particularly enriched in the inner leaflet of the plasma membrane in neural tissues. PS is synthesized from phosphatidylcholine or phosphatidylethanolamine by exchanging the base head group with serine in reactions are catalyzed by phosphatidylserine synthase 1 and phosphatidylserine synthase 2 located in the endoplasmic reticulum. Activation of Akt, Raf-1 and protein kinase C signaling, which supports neuronal survival and differentiation, requires interaction of these proteins with PS localized in the cytoplasmic leaflet of the plasma membrane. Furthermore, neurotransmitter release by exocytosis and a number of synaptic receptors and proteins are modulated by PS present in the neuronal membranes. Brain is highly enriched with docosahexaenoic acid (DHA), and brain PS has a high DHA content. By promoting PS synthesis, DHA can uniquely expand the PS pool in neuronal membranes and thereby influence PS-dependent signaling and protein function. Ethanol decreases DHA-promoted PS synthesis and accumulation in neurons, which may contribute to the deleterious effects of ethanol intake. Improvement of some memory functions has been observed in cognitively impaired subjects as a result of PS supplementation, but the mechanism is unclear.
1. Introduction
Phosphatidylserine (PS) is the major acidic phospholipid class that accounts for 13–15 % of the phospholipids in the human cerebral cortex [1]. In the plasma membrane, PS is localized exclusively in the cytoplasmic leaflet where it forms part of protein docking sites necessary for the activation of several key signaling pathways. These include the Akt, protein kinase C (PKC) and Raf-1 signaling that is known to stimulate neuronal survival, neurite growth and synaptogenesis [2–7]. Modulation of the PS level in the plasma membrane of neurons has significant impact on these signaling processes. The mechanism of PS-mediated activation of these neuronal signaling pathways is illustrated in Fig. 1.
In the synapses, PS plays an important role in exocytosis by influencing Ca2+-dependent membrane fusion between synaptic vesicles and the target plasma membrane, which is mediated by synaptotagmin and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex [8–11]. PS also modulates the AMPA glutamate receptor [12], interacts with synapsin I [13], and alters the conformation of the microtubule associated protein tau [14]. Furthermore, abnormal PS asymmetry in the synaptosomal membrane has been observed in mild cognitive impairment and Alzheimer’s disease [15]. The recent discovery of the critical role of PS in activating important signal transduction pathways and modulating neurotransmitter release and receptor function as well as implications in neuropathophysiology have renewed interest in PS in relation to brain function.
This review focuses on the metabolism and function of PS in the nervous system. Further details can be obtained from previous reviews dealing with PS function in the mammalian brain [16], cell and molecular biology involved in PS metabolism [17], the synthesis and intracellular transport of PS [18,19], the effects of docosahexaenoic acid (DHA) on neuronal PS function [5], the interrelationship between phosphatidylethanolamine (PE) and PS metabolism [20–22], and the effects of PS on membrane properties [23].
2. Phosphatidylserine synthesis in the brain
In mammalian tissues, PS is synthesized from either phosphatidylcholine (PC) or PE exclusively by Ca2+-dependent reactions where the head group of the substrate phospholipids is replaced by serine [20], as illustrated in Fig. 2. These base-exchange reactions are catalyzed by phosphatidylserine synthases (PSS) and so far two isoforms, PSS1 and PSS2 encoded by two separate genes, Pss1 and Pss2, respectively, have been identified.
PSS1 utilizes PC as its substrate, and PSS2 utilizes PE. These enzymes are localized in the endoplasmic reticulum, particularly enriched in the mitochondria associated membrane regions of the endoplasmic reticulum [24].
Together with testis and kidney, brain is one of the tissues that have high capacity to synthesize PS [25]. Also, the expression of PSS in the brain is among the highest. The serine base exchange enzymatic activities of rat cerebellar homogenates, cerebral cortical homogenates and cerebral cortical membranes were shown to be recovered in the insoluble floating fraction of TritonX-100 extracts, suggesting the localized presence of PS synthases in membrane lipid rafts [26,27]. Although intriguing, the possible contribution of microsomal contamination cannot be excluded. PS production is increased in cells of neuronal origin by compounds that trigger Ca2+ release, a finding consistent with the fact that PS synthesis is a Ca2+-dependent process [28].
The PC and PE substrates utilized for PS production can be de novo synthesized in microsomes by transfer of either phosphocholine or phosphoethanolamine from the respective cytidine diphosphate derivatives to 1,2-diacylglycerol [29]. PC synthesis is upregulated during neuronal differentiation. For example, substantial increases of PC wereobserved along with activation of two cytidine diphosphate-choline pathway enzymes, choline cytidylyltransferase and choline kinase when Neuro-2A or PC12 cells undergo differentiation and form neurites [30,31]. PC also can be synthesized by the phosphatidylethanolamine N-methyl transferase (PEMT) reaction through three sequential methylations of the ethanolamine head group of PE. Polyunsaturated PC species are synthesized largely by the PEMT pathway [32], suggesting that the PEMT reaction might be particularly important in regulating phospholipid molecular species composition where polyunsaturated species are abundant. Although PEMT was reported to be present in rat brain and bovine caudate nucleus, the activities detected were low [33,34]. Subsequent studies indicated that the PEMT pathway is negligible in neurons [35], and the only tissue where it is quantitatively significant is liver [36]. Nevertheless, the PEMT pathway appears to be important for normal hippocampal development, as indicated by the finding that Pemt knockout mice show more neuronal apoptosis and less hippocampal expression of calretinin, a marker of neuronal differentiation [37]. Considering the high level of polyunsaturated PS species in the brain, it is possible that the PC ultimately derived from the hepatic PEMT reaction may be an important substrate for neural PS synthesis catalyzed by PSS1.
2.1. Phosphatidyserine synthase 1 function
Although PSS1 expression is ubiquitous, studies with [3H]serine indicate that the brain has the highest specific activity for choline exchange which represents PSS1 activity. Purified human PSS1 can convert either PC or PE to PS in enzymatic assays in vitro, but PSS1 utilizes only PC in intact cells. An explanation for this difference may be selective phospholipid substrate availability in the membrane microdomains where PSS1 is localized. PC and PSS1 can provide sufficient PS to support neuronal differentiation, for the axon extension in cultured sympathetic neurons is not impaired by a PSS2 deficiency [38]. Furthermore, transactivation of the Pss1 promoter by the Sp and N-Myc transcription factors is high in neonatal brain, leading to higher PSS1 expression and activity as compared with other neonatal mouse tissues [39]. These findings indicate that PSS1 has an important role in PS synthesis in the developing brain. Primary cultures of cortical astrocytes have higher PSS1 activity than primary cortical neuron cultures, suggesting that astrocytes may be a major site of PS synthesis from PC in some brain regions [39].
The 1-stearoyl-2-docosahexanoyl (18:0, 22:6) PC molecular species is the preferred substrate for PS synthesis in cerebral cortical microsomes, and 18:0,22:6 is the most abundant PS species in the brain even though it is not an abundant brain PC species [5,6,40]. Although present in larger amounts, 1-palmitoyl-2-docosahexanoyl (16:0, 22:6) PC is not utilized efficiently by PSS1, and 16:0, 22:6 is a minor PS species in the brain. These results suggest that 18:0, 22:6-PC is particularly favored for the conversion to PS in the brain and provide additional evidence that PC is an important substrate for brain PS synthesis in vivo. Furthermore, MALDI-imaging mass spectrometry studies of mouse brain indicate that 18:0, 22:6-PC is selectively enriched in Purkinje neurons [41], suggesting that this substrate also may be an important source of PS in the cerebellum.
2.2 PSS2 function
Neurons obtained from neonatal mice contain relatively high levels of PSS2, indicating that PE also is an important source of neuronal PS during development. As opposed to PSS1, PSS2 utilizes only PE for PS synthesis under all conditions [42,43]. The tissue expression profile of PSS2 is different from that of PSS1. While PSS1 expression is ubiquitous, PSS2 is expressed highly in testis, brain and kidney [44], suggesting that PSS2 may have specialized roles in these tissues.
Recent studies with purified flag-tagged PSS2 demonstrate that the enzyme utilizes PE substrates containing either palmitate or stearate in the sn-1 position equally well. However, PSS2 prefers PE with DHA as opposed to either arachidonic acid or oleic acid (18:1n-9) in the sn-2 position. PSS2 isolated from a variety of cultured cell lines, as well as from microsomes of a Chinese hamster ovary cell mutant that lacks PSS1, also preferentially utilizes PE containing DHA in the sn-2 position. These findings, together with the higher expression and activity of PSS2 in the brain, suggest that PSS2 plays a key role in producing the high level of DHA-containing PS in the brain [44].
The serine base exchange activity has been observed in the Triton insoluble floating fractions from both rat cerebrocortex and cerebrocortical plasma membrane preparations [27]. The Triton insoluble fractions mainly converted PE to PS, indicating dominant presence of PSS2 activity in these membrane preparations. PKC also was present in the plasma membrane enriched fraction. Although this membrane fraction was enriched in
Na/K-ATPase, it also contained 10% as much NADPH cytochrome c reductase activity as in the microsomes. Therefore, it is possible that the PSS activity detected in this cerebrocortical plasma membrane fraction was due to microsomal contamination rather than the enzyme actually being present in the plasma membrane. If the PSS2 activity is indeed enriched in the plasma membrane preparation, local PS synthesis at the cerebrocortical plasma membrane may have a significant role in modulating PKC signaling where PS binding is required.
2.3. Deletion of phosphatidylserine synthase genes
Deletion of both Pss1 and Pss2 causes embryonic lethality in mice, indicating that PS synthesis is an essential metabolic function [45]. When only one of these genes is deleted, PS synthesis is reduced, but sufficient quantities still are produced for normal development and most physiological functions. Deletion of Pss1 does not produce a phenotype, and the mice are viable, fertile and have a normal life span. In these Pss1-deleted mice, the serine base exchange activity is decreased by 67% and 85% in liver homogenate and microsomes, respectively, and the liver PS content is reduced. However, the PS content in the brain is not altered and axonal extension is normal [45]. The Pss2 deletion does not cause a reduction in the PS content in liver, testis and brain, or a decrease in neuronal axon extension [38]. The total serine head group exchange activity is reduced over 90% in the testis, liver and brain [44], but unchanged in hepatocytes [38]. Elevation of PSS1 activity without any change in PSS1 mRNA expression has been observed in some studies of Pss2-deficient mice [38]. The fact that deletion of either Pss1 or Pss2 genes does not affect the PS level in the brain suggests possible compensatory mechanisms at the biochemical level; less PS metabolism through decarboxylation or phospholipase reactions, and/or less regulation by the remaining PSS in these mutants. The only abnormality resulting from deletion of Pss2 is infertility in some of the male mice [46].
3. Composition of brain phosphatidylserine
The PS content in human brain is maintained at the 13–14% level throughout the life [1]. As shown in Table 1 the synaptic plasma membrane, olfactory bulb and hippocampus of rats and mice contain markedly higher PS as compared to non-neuronal tissues such as liver and adrenal [38,47–50].
The fatty acid composition of PS, and for comparison that of PE and PC, contained in the gray and white matter of human brain, is shown in Table 2 [1, 51]. There are substantial differences in the fatty acid composition in gray and white matter PS. Gray matter PS contains considerably more DHA and less 18:1n-9 than white matter. Appreciable differences also occur in the PE fatty acid composition in gray and white matter, whereas comparatively small differences occur in the PC composition. DHA accounts for more than one-third of the total fatty acid and 80% of the polyunsaturated fatty acid in gray matter PS. A substantial amount of DHA is present in gray matter PE, but only a relatively small amount is present in PC. Gray matter PS and PE contain considerably more 18:0 and much less 16:0 than PC. Arachidonic acid is present primarily in PE, and there is little arachidonic acid in PS. According to one study [51], PE also contains the largest amount of docosapentaenoic acid (22:5n- 6), an arachidonic acid-derived product. Only trace amounts of linoleic acid (18:2n-6) are present in brain PS, PE and PC.
According to the positional distribution of the main fatty acids in bovine brain gray matter PS, 18:0 and DHA represent the most abundant fatty acids at the sn-1 and sn-2 positions, respectively [52]. Likewise, 18:0 and DHA are highly enriched at the sn-1 and sn-2 positions of PE, respectively. In contrast, the sn-2 position of PC contains only minor level of DHA, and 16:0 is more abundant than 18:0 at the sn-1 position. The molecular species analysis in the mouse brain by mass spectrometry also confirms the relatively high concentration of 18:0 and DHA in PS as shown in Table 3. The most abundant PS molecular species is 18:0,22:6-PS, which varies from 38% in the cerebellum to 59% in the cortex.
There is little 16:0,22:6-PS except in the olfactory bulb where it accounts for 22% of the PS. Three species that contain DHA are di-polyunsaturated, and together, they comprise 2 to 4% of the PS in these regions of the mouse brain.
The greater similarity in fatty acid composition and positional distribution between PS and PE than PS and PC in gray matter might indicate that PE is the more important glycerophosphatide substrate for PS synthesis in neurons. However, the metabolism of neuronal PS, PE and PC is interrelated as shown in Fig. 2, complicating any interpretations based on fatty acid compositional data. For example, the mitochondrial phosphatidylserine decarboxylase (PSD) reaction converts PS to PE [20], and a substantial amount of brain PE is synthesized by this reaction [53,54]. Therefore, one cannot discern whether the similarities in PS and PE fatty acid compositions are due primarily to PSS2 mediated conversion of PE to PS, or conversely, PSD mediated conversion of PS to PE. The fatty acyl composition in these phospholipids is further tailored by deacylation and reacylationof PS synthesis reactions [5]. Therefore, conclusions regarding PS biosynthesis based on fatty acid compositional similarities are highly tenuous.
3.1. Phosphatidylserine alkyl ethers and plasmalogens
Alkyl ether phosphoglycerides contain a 1-O-alkyl hydrocarbon chain, and plasmalogens are alkenyl ether phosphoglycerides that contain a 1-O-alkenyl hydrocarbon chain.
[U-14C]serine is incorporated into 1-O- alkyl, 2-acyl PS in cultured cerebral hemisphere
cells obtained from 16 day rat embryos, demonstrating that the developing brain can synthesize PS alkyl ethers [53]. In addition, small amounts of serine plasmalogens were detected in myelin from monkey, ox, mouse and human brain [51,55]. Previously, it has been reported that PS contained 13% 1-O-alkenyl hydrocarbon chains in the white matter whereas gray matter PS contained only 0.3% 1-O-alkenyl chains. By contrast, the PE in these fractions contained 47% and 21% 1-O-alkenyl hydrocarbon chains. In terms of the 1-O-alkenyl hydrocarbon chain composition, 18:0 accounts for more than half in gray matter, while18:1 was the most abundant 1-O-alkenyl hydrocarbon chain in white matter [51].
However, modern analytical techniques such as mass spectrometry detect 1-O-alkeny, 2-acyl species mostly in PE but rarely in PS from mammalian tissues.
PS alkyl ethers also have been detected in the lens of human eyes by mass spectrometry with collision-induced dissociation and ozone-induced dissociation [56]. These alkyl ethers contain saturated (16:0 and 18:0) and monounsaturated (18:1) hydrocarbon chains. Studies using liquid chromatography combined with tandem mass spectrometry also demonstrate the presence of serine plasmalogens in postmortem samples of human retina and optic nerve obtained from males and females between the ages of 72 and 94 years [57]. Polyunsaturated molecular species were present in the retinal serine plasmalogens. The function of the retinal PS alkyl ethers and serine plasmalogens is unknown, but the more highly unsaturated molecular species are thought to be involved in retinal signaling [57].
4. Sources of serine for the brain
Serine is required by the brain for the synthesis of proteins and three classes of lipids, PS, sphingolipids, and N-acylserines. As illustrated in Fig. 3, serine is obtained either by uptake from the cerebral circulation or by synthesis from glucose. The serine concentration in normal human plasma is 11.2 mg/L (107 μM), which accounts for 3% of the total plasma free amino acid content [58].Serine is transported across the blood brain barrier by three Na+-dependent neutral amino acid transporters present on the abluminal surface of the capillary endothelium [59]. The concentration of free amino acids in the brain extracellular fluid is estimated to be 10 % of the amount present in the plasma [59], so the serine concentration to which the neural cells are directly exposed is estimated to be about 10 μM.
Serine also is synthesized in astrocytes from glucose taken up by the brain. As opposed to most other cells, astrocytes do not convert all of the 3-phosphoglycerate intermediate formed by glycolysis to pyruvate. Instead, as shown in Fig. 3, these cells convert some 3-phosphoglycerate to serine in a pathway that requires three enzymatic steps; 3-phosphoglycerate dehydrogenase, 3-phosphohydroxypyruvate aminotransferase and 3-phosphoserine phosphatase [60]. The serine that is synthesized from glucose in astrocytesreleased into the extracellular fluid by the astrocyte Na+-dependent ASCT1 transporter, and serine is one of the neuronal trophic factors contained in astrocyte-conditioned medium.
Neurons cannot convert glucose to serine because they do not express the rate-limiting enzyme, 3-phosphoglycerate dehydrogenase. Therefore, they require a preformed source of serine, as indicated by studies showing that serine is an essential nutrient for survival and neuritogenesis of hippocampal and Purkinje neurons [60]. The Na+-dependent Asc1 transporter expressed in neurons facilitates the uptake of serine from the extracellular fluid, which is provided by either the astrocytes or the amino acid pool of the cerebral circulation [60].
A small amount of PS is present in plasma lipoproteins [61], and the blood brain barrier contains low density lipoprotein receptors and binding sites for high density lipoproteins [62–64]. It is possible that PS contained in lipoproteins might enter the brain and serve as an additional source of serine for the brain, although extensive studies with cholesterol indicated that plasma lipoproteins themselves are not transported into the brain [65].