Letter pubs.acs.org/chemneuro
Unique Lipid Chemistry of Synaptic Vesicle and Synaptosome Membrane Revealed Using Mass Spectrometry Kenneth T. Lewis,† Krishna R. Maddipati,‡ Akshata R. Naik,† and Bhanu P. Jena*,† †
Department of Physiology and ‡Department of Pathology, Lipidomics Core Facility, Wayne State University School of Medicine, Detroit, Michigan 48201, United States
ABSTRACT: Synaptic vesicles measuring 30−50 nm in diameter containing neurotransmitters either completely collapse at the presynaptic membrane or dock and transiently fuse at the base of specialized 15 nm cup-shaped lipoprotein structures called porosomes at the presynaptic membrane of synaptosomes to release neurotransmitters. Recent study reports the unique composition of major lipids associated with neuronal porosomes. Given that lipids greatly influence the association and functions of membrane proteins, differences in lipid composition of synaptic vesicle and the synaptosome membrane was hypothesized. To test this hypothesis, the lipidome of isolated synaptosome, synaptosome membrane, and synaptic vesicle preparation were determined by using mass spectrometry in the current study. Results from the study demonstrate the enriched presence of triacyl glycerols and sphingomyelins in synaptic vesicles, as opposed to the enriched presence of phospholipids in the synaptosome membrane fraction, reflecting on the tight regulation of nerve cells in compartmentalization of membrane lipids at the nerve terminal. KEYWORDS: Synaptosome, synaptic vesicle lipids, mass spectrometry
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conserved protein complex involved in membrane fusion and neurotransmission. In neurons, porosomes measuring approximately 15 nm are present at the presynaptic membrane and are composed of nearly 40 proteins, among them SNAREs, ion channels such as calcium channels, and the GTP-binding Gprotein Gαo.10 Studies using mass spectrometry on the isolated neuronal porosome complex demonstrate the presence of phosphatidylinositol phosphates (PIP’s) and phosphatidic acid (PA) among other lipids, and the enriched presence of ceramide (Cer), lysophosphatidylinositol phosphates (LPIP), and diacylglycerol (DAG), in comparison to synaptosomes.20 These results suggest the unique composition of lipids associated with the porosome complex, a domain of the presynaptic membrane of the synaptosome. Additionally, exposure of brain slices to exogenous PA, results in increase in glutamate release upon stimulation, demonstrating the involvement of PA in neurotransmitter release.20 Since different lipids occupy unique
n the past three decades, it has become increasingly clear that the role of lipids is as important as proteins, if not more, on the function of cellular membranes. Protein−lipid interactions govern cellular membrane functions from cell signaling and ion channel function, to cellular and subcellular compartmentalization, and membrane fission−fusion reactions.1−4 It is estimated that cellular membranes are composed of nearly 1000 different species of lipids.5 Understanding how lipids influence membrane function would require a clear understanding of their composition in various cellular organelles, and their specific interactions with resident proteins within those membranes. Cup-shaped lipoprotein structures called porosomes are present at the cell plasma membrane, where secretory vesicles transiently dock and fuse at its base to release intravesicular contents to the outside.6−17 Fusion of membrane-bound secretory vesicles at the porosome base is mediated by calcium and a specialized set of three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors called SNAREs.10,18,19 In neurons, for example, target membrane proteins SNAP-25 and syntaxin-1A called t-SNAREs present at the base of neuronal porosomes, and a synaptic vesicle-associated membrane protein (VAMP) or v-SNARE, are part of the © XXXX American Chemical Society
Received: January 20, 2017 Accepted: February 28, 2017 Published: February 28, 2017 A
DOI: 10.1021/acschemneuro.7b00030 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Letter
ACS Chemical Neuroscience
laboratory demonstrate a highly enriched SM and SV fraction for lipid analysis.20 Lipidomics performed utilizing mass spectrometry revealed key differences in the lipid composition of the three different sample groups: SS, SM, and SV. Isolated SS, besides containing SM and SV, has mitochondria and soluble lipids that are absent in the other fractions (SM, SV). When compared to synaptosome membrane alone, the SS fraction showed significant enrichment in 34 lipid species; including all significantly different ceramides, sphingosine, phosphatidic acids, phosphatidylinositol and sphingomyelins (Figure 2). The SM fraction contains the plasma membrane of the synaptosome along with all integral proteins, lipoprotein complexes such as the porosome, and some docked SV at the time of isolation (Figure 1). The SM fraction is found to be enriched in 24 lipid species, compared to the SS fraction; these include subtypes of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylserine (PS). As expected, the neurotransmitter-containing SV fraction is found to be simpler than the SM fraction, both in composition and function, hence not surprisingly just 5 lipid species are found to be enriched in SV, as opposed to 25 species in SM (Figures 2 and 3). Interestingly, 3 of the 5 enriched lipid species in SV are triacylglycerides, which did not have a single variant enriched in the SM fraction. The SV fraction is also enriched in both lysosphingomyelin and sphingomyelin as opposed to their absence in the SM fraction. Until recently, lipids were primarily seen as structural molecules that simply help compartmentalize the cell into distinct functional organelles; however, advances in lipid research have unraveled a plethora of vital cellular functions performed by lipids including signaling and regulation of membrane transport.4 Therefore, an understanding of the distribution of lipids in different cellular compartments will help determine their role in cell function. Consequently, the objective of the current study was to determine the lipid profiles of SV and SM, that would help better understand neurotransmission. The significant differences observed in their lipid profiles of SV and SM, reflects on little lipid mixing between the two compartments. The enriched presence of ceramides and sphingomyelins in isolated synaptosomes may primarily be due to the presence of mitochondria, since ceramides and sphingomyelins are concentrated in the mitochondria and cytosol while phospholipids are highly enriched in the plasma membrane and integral membrane fractions. In agreement, ceramides have a welldefined role in regulating mitochondrial-directed apoptosis and sphingolipids have been identified in mitochondrial signal transduction.21 Our results demonstrate that the composition of lipids in SM and SV differ greatly (Figure 4), and that phospholipids are enriched in the SM whereas triacylglycerides (TAGs) and sphingolipids are enriched in SV. It is therefore reasonable to propose that TAGs and sphingolipids present in SV may contribute to neurotransmitter release.10,22−25 Interestingly, it has been shown that increasing the relative ceramide concentration of cell membranes by depleting sphingomyelin decreases entry of the virus HCV, highlighting the importance of lipid composition on membrane fusion.26 Furthermore, in terms of evaluating the role of lipids in neurotransmission, it has been shown that presynaptic sphingolipids increase glutamate secretion in hippocampal neurons; demonstrating that the asymmetrical distribution of lipids we find in the SV and SM is indicative of their importance in neuronal function.27 Although little is known anout the effects of triacylglycerides on membrane
domains of the synaptosome membrane and influence neurotransmitter release, the composition of lipids in synaptic vesicle and that of the synaptosome membrane was hypothesized to be different, and to be tightly regulated. To test this hypothesis, and understand the native lipid−protein interactions involved in neurotransmission, the lipidome of isolated synaptosome membrane and isolated synaptic vesicles was determined in the current study. Results from the study confirm our hypothesis and demonstrate the unique lipid composition of synaptic vesicles, which differs from the synaptosome membrane.
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RESULTS AND DISCUSSION Lipid distribution in subfractions of synaptic nerve endings from rat brain was assessed using isolated presynaptic nerve terminals or synaptosomes (SS). The approximately 0.75−1.5 μm SS contain mitochondria, synaptic vesicles (SVs), and cytoskeletal elements, enveloped by plasma membrane. Hypo-osmotic lysis of purified SS enables centrifugal gradient separation of the synaptosomal membrane (SM) and SV for lipid distribution measurements. The enriched presence of isolated SS, SM, and SV is demonstrated from electron microscopy (EM), atomic force microscopy (AFM), and immunoblot analysis (Figure 1). Western blot analysis of isolated SV measuring approximately 30−50 nm demonstrates the enriched presence of the vesicle associated membrane protein VAMP, compared to total brain homogenate (BH) and the SM fraction (Figure 1). These results and the isolation of SS, SM, and SV routinely carried out in the
Figure 1. Morphology and immunochemistry of isolated synaptosome, synaptosomal membrane, and synaptic vesicle preparations from rat brain tissue. (A) Electron micrograph of isolated synaptosomes (bar = 100 nm). Note the 30−50 nm synaptic vesicles within synaptosomes. (B) Electron micrograph of isolated synaptosome membrane preparation (bar = 200 nm). Note some 30−50 nm synaptic vesicles (yellow arrowhead) adhering to the isolated synaptosome membranes (SM). (C) AFM micrograph of isolated synaptic vesicle (SV) preparation demonstrating the presence of synaptic vesicles (red arrowhead). (D) Western blot analysis of equal protein loads each of total brain homogenate (BH), SM, and SV fractions probed with antibody specific to VAMP-1/2/3, Gαo, Gαi3, and vimentin, shows the enriched presence in the SV fraction, the established SV-associated VAMP and Gαo proteins; and the porosome-associated SM proteins vimentin and Gαi3 in the SM fraction, demonstrating enriched isolation of the SM and SV fractions for lipid analysis. B
DOI: 10.1021/acschemneuro.7b00030 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Letter
ACS Chemical Neuroscience
fusion and secretion, their enriched presence in SV reflects a critical role in neurotransmission. The current study besides presenting asymmetrical lipid distribution in the synaptosome, highlights lipid species that may be functionally important in a specific domain at the presynaptic membrane. Results from the study further lend support for the transient or kiss-and-run mechanism of vesicle-plasma membrane fusion in addition to the traditional model of full vesicle collapse with the presynaptic membrane. The SV membrane completely merging and diffusing into the plasma membrane only to be separated from the plasma membrane just moments later to create new vesicles, would be highly energy-inefficient and complex, given the hundreds of different lipid species present in the two membrane compartments. In contrast, the transient kiss-and-run fusion events involving the synaptic vesicle establishing temporary continuity via a fusion pore with the presynaptic membrane, allowing fractional content release from SV’s during neurotransmission, would be highly efficient involving little lipid mixing between the two opposing membranes. This way, upon cessation of the secretory stimulus, the SV disengages from the plasma membrane, to undergo refilling with neurotransmitters via the neurotransmitter transporters present in the SV membrane. Since individual lipids can diffuse throughout the lipid membrane, a tight regulation of their distribution within and between membrane compartments is required, and hence, lipid mixing between compartments must be kept to a minimum. We therefore propose, that during neurotransmission, transient fusion is primarily utilized by the cell to prevent any major lipid and protein mixing between the opposing membranes, that would require unnecessary expenditure of energy to separate the two compartment membrane components, and consequently the repeated generation of new SV and SM. Additionally, the presence of neurotransmitter transporters at the SV membrane would be of little use, if the SV fully incorporates into the SM during neurotransmission. In agreement, many studies document the transient fusion of SV at the SM in neurons.23−25 Recent studies on the role of lysophosphatidic acid (LPA), which is a product of PA metabolism, in the regulation of neurotransmitter release by a mechanism involving Gi/o, were not discussed since LPA was not detected as a major synaptic vesicle (SV) and synaptosomal membrane (SM) lipid in the current study. Future studies, however, using labeled lipids will explore the relative distribution of LPA in both the SV and SM membrane compartments.28 Our observations demonstrate that even within the dynamic nerve terminal where SV rapidly dock and fuse to release neurotransmitters, the integrity of both the SV membrane lipids and the SM lipids is uncompromised. This comes as no surprise, since while minor changes in membrane lipids manifest major changes in the function of membrane proteins and protein complexes, membrane lipid composition varies between organelles and different microdomains at the plasma membrane of the cell.3 Moreover, the lipid composition is even known to differ between the inner and outer leaflet of the bilayer at a certain position of the cell plasma membrane.3 Given the estimated >1000 cellular membrane lipid species, it would be a major priority of the cell to prevent their mixing between cellular compartments.5 It will be critical therefore to determine the interactions that exist between SV proteins and lipids, and their influence on vesicle physiology and function. An understanding of how the various resident lipids in the SM and SV influence the activity of their respective resident proteins will be revelatory. Furthermore, these results provide a platform for a molecular
Figure 2. Heat map of lipid mass spectrometry data showing the lipid signal intensity in log scale, for four synaptosomal membrane (SM) and four synaptic vesicle (SV) fractions, relative to the mean signal of four separate synaptosome fractions prepared from rat brain tissue. Black colored tiles represent signals that were within 20% of the mean of the whole synaptosome signal, red tiles are those that were reduced compared to the synaptosome, and green tiles are those that were increased compared to the synaptosome. Note that triacylglycerides and sphingomyelins tend to be enriched in the SV compared to the SM fractions. C
DOI: 10.1021/acschemneuro.7b00030 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience
Figure 3. Principal component loadings of LC-MS lipidomic data for lipid species statistically significant (p < 0.05) when compared between (A) synaptosome (green) and synaptosome membrane (blue); (B) whole synaptosome and synaptic vesicle; and (C) synaptosome membrane and synaptic vesicle (orange). (D) Principal component plot of four samples from SS, SM, and SV when comparing lipids, significantly different between SM and SV. The clear separation of each membrane group suggests their lipid profile is similar within their own membrane compartment and different in other membrane compartments. (E,F) Multiple 3D views of the principle component plot from panel (D) including PC3 on the z-axis generated using the R3.2.2 scatter3d function. MO). The brain tissue was homogenized using 8 strokes at 900 rpm in a Teflon-glass homogenizer, and the total homogenate was diluted in HEPES-sucrose and centrifuged for 3 min at 2500g. The resulting supernatant fraction was further centrifuged for 13 min at 14 500g to obtain a pellet. The resultant pellet was resuspended in buffered sucrose solution and then loaded onto a 3−10−23% Percoll gradient. After centrifugation at 35 000g for 7 min, the fraction at the 10−23% Percoll gradient interface was collected and centrifuged at 4500g for 8 min to obtain a loose pellet containing the isolated synaptosome. Typically, synaptosomes were isolated at a concentration of 2.5 mg/mL, estimated using the Bradford protein assay.39 To isolate SM and SVs, the synaptosome preparation was diluted using 9 volumes of ice-cold water, resulting in the lysis of synaptosomes to release SVs, followed by 30 min
understanding of lipid−protein interactions at the nerve terminal, and their role in neurotransmission.29−38
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METHODS
Isolation of Synaptosome, Synaptosome Membrane, and Synaptic Vesicle. Synaptosomes (SS), synaptic vesicles (SV), and synaptosome membrane (SM) were prepared from rat brains according to published methods.20 For each experiment, Sprague−Dawley rats weighing 100−150 g were euthanized by CO2 inhalation, with all animal procedures preapproved by the Institution Animal Care & Use Committee (IACUC). Whole brains were isolated and placed in icecold buffered sucrose solution (5 mM Hepes, pH 7.4, 0.32 M sucrose) supplemented with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, D
DOI: 10.1021/acschemneuro.7b00030 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Letter
ACS Chemical Neuroscience
Figure 4. Schematic drawing of a synaptosome, depicting the major lipid species in the synaptosome (SM) membrane and in synaptic vesicles (SV). Note the enriched presence of TAGs and sphingomyelins in SV, compared to the SM membrane fraction. were washed three times in PBST and then incubated for 1 h at room temperature in horseradish peroxidase-conjugated secondary antibody at a dilution of 1:1000 in blocking buffer. The immunoblots were then washed three times in PBST, processed for enhanced chemiluminescence, and photographed. Estimation of Major Lipids in Isolated Synaptosomes, and Synaptic Vesicle Preparations. Lipid Extraction for Mass Spectrometry. Isolated synaptosome membrane and synaptic vesicles were extracted for lipids with methanol and methyl-tert-butyl ether (MTBE) according to published methods.25 Briefly, methanol (1.5 mL) containing 100 ng each of internal standards (diheptadecanoyl PC, diheptadecanoyl PE, diheptadecanoyl PS, diheptadecanoyl PA, diheptadecanoyl PG, diheptadecanoyl glycerol-d5,1,3- diheptadecanoyl-2-(10Z)heptdecenoyl glycerol-d5, 1-palmitoyl(d31)-2-oleoyl-sn-glycero-3-phosphoinositol, N-heptadecanoyl C-18 ceramide, N-heptadecanoyl C-18 sphingomyelin, and PAF-C16-d4) was added to a suspension of neuronal fractions (20 μL) followed by MTBE (5 mL) and then mixed well. The mixture was left for 1 h at room temperature with occasional mixing. Water (1.5 mL) was added to the mixture, mixed thoroughly, and centrifuged (1000g) for 5 min to assist the separation of phases. The upper organic phase was collected to a clean glass tube. The lower aqueous phase was extracted twice (2 mL each time) with MTBE saturated with methanol and water (10:3:2.5 v/v), and the extracts were combined. The MTBE extracts were evaporated to dryness under a gentle stream of nitrogen, and the residue was dissolved in LC-MS grade isopropanol/hexane/100 mM aqueous ammonium acetate (58:40:2 v/ v). The reconstituted lipid extract was analyzed for lipids by mass spectrometry. Mass Spectrometric Quantitation of Lipid Classes. Lipid extracts were directly infused into the TurboVion source via a syringe pump at 10 μL/min and analyzed by using a QTRAP5500 mass spectrometer (ABSCIEX). Multiple precursor ion and neutral loss scanning methods were used for information dependent acquisition of MS/MS data to detect and quantify the lipid classes as described earlier. Mass analyzer conditions used in the positive ion mode were as follows: ionization potential, 5500 V; declustering potential, 120 V; entrance potential, 9 V; collision cell exit potential, 9 V. Collision energy for the survey scan was 10 and 45 eV for enhanced product ion scans. In each scan, three ions with highest intensity were chosen for dependent product ion acquisition and the detected ions were excluded for the rest of the experiment after three occurrences. Data were analyzed for the identification of lipid species using LipidView software (ABSCIEX). Lipids were quantified against internal standards and normalized against protein values obtained by the Bradford assay.39
incubation on ice. The lysate was then centrifuged for 20 min at 30 000g, and the resultant pellet was resuspened to create the SM fraction, whereas the supernatant was centrifuged at 300 000g for 1 h to obtain enriched SVs. Electron and Atomic Force Microscopy of Isolated SS, SM, and SV Preparation. Synaptosomes (SS), synaptic vesicles (SV), and synaptosome membrane (SM) were resuspended in ice-cold PBS pH 7.4 containing 0.2% paraformaldehyde (PFA) and 0.2% glutaraldehyde (GA) for electron microscopy (EM) and atomic force microscopy (AFM). The subcellular fractions were washed in 10 volumes of PBS pH 7.4, and for AFM, SVs adhering to a mica surface were imaged. AFM was performed on SVs using a minor modification of our previously published procedure.15 A Nanoscope IIIa atomic force microscope from Digital Instruments (Santa Barbara, CA) was used to image the isolated SV. Images were obtained in the “tapping” mode, using silicon nitride tips with a spring constant of 0.38 N·m−1 and an imaging force of
