Sphingolipidomics investigation of the temporal dynamics after

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Sphingolipidomics investigation of the temporal dynamics after ischemic brain injury Hsi-Chun Chao, Tsung-Heng Lee, Chien-Sung Chiang, Sin-Yu Yang, Ching-Hua Kuo, and Sung-Chun Tang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00370 • Publication Date (Web): 16 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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Journal of Proteome Research

Sphingolipidomics investigation of the temporal dynamics after ischemic brain injury Hsi-Chun Chao†,‡, Tsung-Heng Lee†,‡, Chien-Sung Chiang§, Sin-Yu Yang§, Ching⊥ Hua Kuo†,‡, ,*, and Sung-Chun Tang§,* †School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan ‡ The Metabolomics Core Laboratory, Center of Genomic Medicine, National Taiwan University, Taipei, Taiwan § Stroke Center and Department of Neurology, National Taiwan University Hospital, Taipei, Taiwan

⊥Department of Pharmacy, National Taiwan University Hospital, Taipei, Taiwan

*Corresponding Author Ching-Hua Kuo Address: No.33, Linsen S. Rd., Taipei 100, Taiwan Tel.: +886.2.33668766 E-mail: [email protected] Sung-Chun Tang Address: No.7, Chung-Shan S.Rd., Taipei 100, Taiwan. Tel.: +886.2.23563279 E-mail:[email protected]

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Abstract Sphingolipids (SPL) have been proposed as a potential therapeutic target for strokes, but no reports have ever profiled the changes of the entire range of SPLs after stroke. This study applied sphingolipidomic methods to investigate the temporal and individual changes in the sphingolipidome including the effect of atorvastatin after ischemic brain injury. We conducted sphingolipidomic profiling of mouse brain tissue by liquid chromatography-electrospray ionization tandem mass spectrometry at 3 hour (hr) and 24 hr after 1 hr of middle cerebral artery occlusion (MCAO), and SPL levels were compared with those of the Sham control group. At 3 hr post MCAO, ceramides (Cers) exhibited an increase in levels of long-chain Cers but a decrease in very-long-chain Cers. Moreover, sphingosine, the precursor of sphingosine-1-phosphate (S1P) decreased and S1P increased at 3 hr after MCAO. In contrast to 3 hr, both long-chain and verylong-chain Cers showed an increased trend at 24 hr post MCAO. Most important, the administration of atorvastatin improved the neurological function of the mice and significantly reversed the SPL changes resulting from the ischemic injury. Furthermore, we used plasma samples from non-stroke control and stroke patients at time points of 72 hr after stroke, and found a similar trend of Cers as in the MCAO model. This study successfully elucidated the overall effect of ischemic injury on SPL metabolism with and without atorvastatin treatment. The network of SPL components that change upon ischemic damage may provide novel therapeutic targets for ischemic stroke. Keywords: Sphingolipidomics, ischemic stroke, neuron injury, long-chain ceramides, very-longchain ceramides, atorvastatin.


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Introduction Stroke is among the three leading causes of death worldwide and is the most frequent cause of permanent disability1-2. A complex of ischemic cascades involving a series of biochemical pathways in the ischemic brain has been proposed to respond to cell death and functional deficits. Sphingolipids (SPLs) are essential structural components of cellular membranes, playing prominent roles in the signal transduction that governs cell proliferation, differentiation and apoptosis3. SPLs are enriched in neuron cells, and few studies have indicated the significant changes in SPL levels after ischemic injury. Systemic study of the SPC changes after ischemic injury could help to delineate their roles in the pathophysiology of strokes. SPLs including sphinganine (dihydrosphingosine), sphingosine, ceramides (Cers), dihydroceramides, cerebrosides, sphingomyelin and their phosphate derivatives, have diverse biochemical properties. Significant changes of several SPLs levels have been observed after strokes. For example, sphingomyelin levels were found to decrease and ceramide levels were found to increase in a MCAO model4. Ceramides were shown to regulate a diverse range of cellstress responses5. However, previous studies have only focused on specific SPLs after ischemic injury4-7. Nevertheless, the biosynthesis and metabolic pathways of SPLs are closely connected, and their changes after ischemic injury may result in a new homeostatic condition of the sphingolipidome. Therefore, proﬁling the entire sphingolipidome can provide a better understanding of the complicated biological processes that occur after ischemic injury. Advancements in liquid chromatography-electrospray ionization tandem mass spectrometry (LCESI-MS/MS) have increased the power of sphingolipidomic technology and enabled the investigation of overall SPL changes after ischemic injury. Atorvastatin, a lipid lowering agent, reportedly has the potential to decrease stroke injury6-7. However, there have been no studies investigating the effect of atorvastatin on SPLs after ischemic injury. Since the SPL pathway has been demonstrated as potential therapeutic target to treat strokes, using sphingolipidomic technology to investigate time-dependent metabolic changes could help to understand the metabolic regulation of SPL pathways after ischemic injury and further identify potential therapeutic targets for stroke. This study used sphingolipidomic methods to study temporal dynamics of sphingolipids after ischemic brain injury using mice models receiving middle cerebral artery ischemic reperfusion (MCAO). Ceramide alteration was also studied using


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plasma samples from patients with ischemic stroke. We additionally used sphingolipidomic technology to investigate the effect of atorvastatin on SPL changes after ischemic injury to provide more mechanism insight for rational of use of statin in clinical therapy.

Materials and Methods Chemicals Lipid standards were all purchased from Avanti Polar Lipids (Alabaster, AL). MS-grade methanol and water were purchased from ScharlauChemie (Sentmenat, Barcelona, Spain), and LC-MS-grade isopropanol (IPA) was purchased from J.T. Baker (Phillipsburg, NJ). MS-grade formic acid solution (99%) and atorvastatin calcium were obtained from Sigma (St. Louis, MO), and MS-grade ammonium acetate and ethanol (EtOH) were purchased from Merck (Darmstadt, Germany). The atorvastatin calcium was prepared in PBS/EtOH (99.5:0.5) for a final concentration of 20 mg/mL. Analysis of sphingolipids The SPL pathway is shown in Figure 1A. All SPLs were analyzed using an Agilent 1290UHPLC system coupled with an Agilent 6460 triple quadrupole LC/MS (Agilent Technologies, Santa Clara, CA). An Agilent Eclipse Plus C18 (100 x 2.1 mm, 1.8 μm) column (Agilent Technologies, Santa Clara, CA) was employed to perform the separations. The mobile phase consisted of MeOH, water, and formic acid (60:40:0.2, v/v) with 10 mM ammonium acetate (solvent A) and MeOH, IPA, and formic acid (60:40:0.2, v/v) with 10 mM ammonium acetate (solvent B). The flow rate was 0.3 mL/min, and the gradient elution program was as follows: 0–1 min: linear gradient from 55 to 56.1% B, hold at 56.1% B for 2 min; 3–3.1 min: linear gradient from 56.1 to 80% B; 3.1–9.1 min: linear gradient from 80 to 90% B, hold at 90% B for 3 min; 12.1–15.1 min: linear gradient from 90 to 100% B, hold at 100% B for 3 min. The sample reservoir and column oven were maintained at 4 °C and 40 °C, respectively. The injection volume was 5 μL. Electrospray ionization in positive ion mode was employed to analyze sphingolipids with the following parameters: 325 °C dry gas temperature, 7 L/min dry gas flow rate, 35 psi nebulizer pressure, 325 °C sheath gas temperature, 11 L/min sheath gas flow rate, 4000 V capillary voltage, and 500 V nozzle voltage. SPLs were detected in precursor ion scan mode via monitoring the product ions of m/z 184, 264, and 266 for sphingomyelins, Cers/cerebrosides, and


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dihydroceramides, respectively, and multiple reaction monitoring mode was employed for further confirmation of sphingolipids and other single-chain SPLs. Quality control samples (QC) were prepared and was injected every 6 samples. Internal Standard of Cer(18:1/17:0) was added into every sample to check the reproducibility. MCAO procedure and atorvastatin administration Three-month-old male C57BL/6J mice were subjected to transient middle cerebral artery ischemic reperfusion (I/R) injury, as reported previously 8. Briefly, after a midline incision was made in the neck, the left external carotid and pterygopalatine arteries were isolated and ligated with 6-0 silk thread. The internal carotid artery was occluded at the peripheral site of the bifurcation with a small clip, and the common carotid artery was ligated with 6-0 silk thread. The external carotid artery was cut, and a 6-0 nylon monofilament with a tip that was blunted (0.20–0.22 mm) with a coagulator was inserted into the external carotid artery. After the clip on the internal carotid artery was removed, the nylon thread was advanced into the middle cerebral artery until light resistance was felt. The nylon thread and the common carotid artery ligature were removed after 1 hr of occlusion to initiate reperfusion. In the Sham group, the surgery proceeded only until the arteries were visualized. For atorvastatin administration, after the one hour MCAO treatment, a single dose of 20mg/kg atorvastatin or PBS vehicle was randomly assigned and intraperitoneally injected into the mice, followed by removal of the nylon thread. The Sham group received the same dose of PBS vehicle, and then the surgery was performed. At 3 or 24 hr of reperfusion, the mice were sacrificed with a lethal dose of isoflurane. The functional consequences of I/R injury were evaluated using a 5-point neurological deficit score described in our previous study (0, no deficit; 1, failure to extend right paw; 2, circling to the right; 3, falling to the right; and 4, inability to walk spontaneously)9. All of the experimental procedures described above were approved by the National Taiwan University Animal Care and Use Committees.

Primary neuronal cultures and oxygen-glucose deprivation model For primary neuron cells, dissociated cell neuron-enriched cultures of the cerebral cortex were established from 18-day-old Sprague–Dawley rat embryos, as described previously10. Experiments were performed in 7- to 9-day-old cultures. For oxygen and glucose deprivation (OGD), glucose-


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free Locke’s buffer (Locke without glucose) containing 154 mmol/L NaCl, 5.6 mmol/L KCl, 2.3 mmol/L CaCl2, 1 mmol/L MgCl2, 3.6 mmol/L NaHCO3, and 5 mmol/L HEPES (pH = 7.2) supplemented with gentamicin (5 mg/L) was used, and the culture dish was kept in an oxygen-free chamber containing a 95% N2 and 5% CO2 atmosphere for 3 hours. Then, the medium was replaced with Neurobasal medium (Gibco, Gaithersburg, MD, US), and the cells were incubated under usual culture conditions for either 0, 1, 3 or 24 hours. After that, the medium was removed, 400 μL of PBS was added into the dish, and the cells were scraped into Eppendorf tubes. After washing twice with PBS, the PBS was removed, and the cells were quenched in liquid nitrogen, followed by storage at -80°C. Sample preparation For the mouse brain studies, brain tissue samples were stored at -80 °C until use. Before extraction, the samples were homogenized by grinding in a mortar with liquid nitrogen. The extraction procedure was modified from Bligh and Dyer’s lipid extraction methods11. A volume of 500 μL of homogenization solvent (MeOH : Water = 4:1) was added to 10 mg of brain tissue. After homogenization, 200 μL of chloroform was added to the sample solution. The extraction was performed on a Geno/Grinder2010 (SPEX, Metuchen, NJ, US) for 3 min. To enhance the separation of the chloroform and water layers (liquid-liquid extraction), an additional 200 μL of chloroform and 200 μL of deionized water were both added to the sample solution, which was then subjected to Geno/Grinder2010 extraction for 3 min, followed by standing in an ice bath for 15 min. The resulting sample was then centrifuged at 15,000 rcf for 5 min at 4 °C. The lower layer of the supernatant was collected for SPL analysis. The extract was dried under a nitrogen stream and stored at -20 °C until use. Before LC-MS analysis, the dried residue was reconstituted with 200 μL of 100% methanol followed by sonication in a water bath at room temperature for 15 min, then centrifuged at 15,000 rcf for 5 min. An aliquot of the supernatant was filtered and subjected to LCMS analysis. For primary neuronal cultured cells studies, cells were washed with phosphate-buffered saline (PBS, pH = 7.4) twice, trypsinized from the dish for 2 min, and the medium was used to quench the trypsin activity. The cells were then centrifuged at 200 rcf for 15 min to remove the medium and was then washed twice with PBS. The residual PBS was removed by centrifugation at 200 rcf for 5 min. The extraction was similar to the above mentioned procedures for the mouse brain.


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Concentration normalization of the cell extracts were performed using precursor ion scan of 184 with LC-ESI-MS/MS before sphingolipidomic analysis12.

For the analysis of the clinical plasma samples, 50 µl of human plasma was added into 50 µ L of buffer containing 30 mM citric acid and 40 mM disodium hydrogen phosphate (pH 4.0). After vortexing, the mixture was extracted by 500 µ L of a 2:1 (v/v) mixture of CHCl3 : MeOH and vortexed at 1000 rpm for 5 min. Next, the sample was centrifuged at 15000 g for 5 min and the lower layer was transferred to a new tube. This procedure was repeated and the combined phases were dried by nitrogen stream and stored at -20 °C until use. Before LC-MS analysis, the dried residue was reconstituted with 50 μL of 100% methanol and then centrifuged at 15,000 rcf for 5 min. An aliquot of the supernatant was filtered and subjected to LC-MS analysis.

Collection of clinical plasma samples Clinical plasma samples, including samples from non-stroke controls and acute ischemic stroke patients were collected at the National Taiwan University Hospital and obtained with informed consent. The study was approved by the institutional review board of the National Taiwan University Hospital. Patients with acute ischemic stroke who were admitted within 24 hours were recruited per our established standard protocol 9. The diagnosis of acute ischemic stroke was confirmed by magnetic resonance imaging (diffusion-weighted image) or repeated computed tomographic exams 13. Blood samples were drawn at 72 hours after the onset of stroke. The blood samples were centrifuged at 300 g for 15 min, and the resultant plasma samples were stored at −80 °C until use.

Data interpretation and statistical analysis Integration of peak areas was accomplished using Agilent MassHunter software (Albuquerque, NM). The data obtained using the Agilent triple quadrupole instrument were converted into the comma-separated values format and processed using Microsoft Excel 2013. Statistical analyses were performed using the R programming software

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. The integrated peak area was further

normalized by the sample wet weight to normalize the potential difference between sample weights.


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The fold changes were shown as the mean ± SD, and Student’s t-test was used to test of significance. For total SLP subclass analysis in the atorvastatin administration study, the intensity of each SPL was tested by the Shapiro-Wilk test to confirm its distribution, then further standardized. The comparisons were further processed using Student’s t-test on the standardized results. For behavior scores in mice with the experimental stroke model, the results are expressed as mean ± SEM. These statistical test results were plotted using R and MetaboAnalyst 3.0 (www.metaboanalyst.ca)15. Significance was defined as p value < 0.05. Results Sphingolipidomics analysis of mouse brains by LC-MS This study used an LC-MS/MS platform to profile SPLs in mouse brain. An in-house library capable of identifying 87 different SPLs (supplementary table 1) was first established12. Although more than hundreds of SPL species have been identified in previous studies 16, we focused on SPLs which have higher contents and biological significance in mammals. Through narrowing down to those more important SPLs, analytical performance could be optimized so that ischemic injury caused SPL changes could be observed more clearly. The SPL extraction procedure was evaluated next, and the extraction recoveries of each SPL subclass were calculated using standard spiked mouse brain samples. The modified Bligh and Dyher method was adapted due to its’ better recovery (supplementary table 2). Figure 1(B) shows the overlaid LC-MS chromatogram of SPL extract from mouse brain tissue obtained by the optimized method. A total of 47 different SPLs including sphinganine (d18:0) (dihydrosphingosine), sphingosine(d18:1), sphingosine-1phosphate (d18:1) (S1P), 7 dihydroceramides (dhCers), 12 Cers, 14 sphingomyelins (SMs), and 12 cerebrosides (CBs) could be detected in mice brain tissues (Figure 1B and supplementary table 1).


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Figure 1. The metabolic pathways of sphingolipids (A) and the overlaid LC-MS chromatogram of SPL extract from mouse brain tissue (B).

Temporal dynamic changes of the SPL profile in MCAO mouse brains To elucidate SPL changes at different time periods after ischemic injury, we performed sphingolipidomic profiling of mouse brain tissue at time points 3 hr and 24 hr post MCAO, and the SPL profiles were compared with those of the Sham group. These SPLs were displayed in a heat map, which showed that MCAO caused significant changes in SPL profiles (Figure 2A). A decreased in SMs was observed at 3 hr post MCAO, and an increase in Cers was observed at 24 hr post MCAO. Using the signal intensities of the 48 SPLs, principal component analysis can separate three distinct groups (Figure 2B).


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Figure 2. The heat map (A) and the principal component analysis (PCA) score plot (B) of the SPL profiling results for mouse brains from different MCAO study groups. (MCAO study groups: Sham: without MCAO treatment; 3 hr: 3 hr post MCAO; 24 hr: 24 hr post MCAO)


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Figure 3 shows the individual SPL fold changes between different groups. Compared with the Sham group, Sphinganine (d18:1) (Figure 3(A)), the precursor of Cer in its de novo synthesis pathway, showed a significant decrease in concentration at both 3 hr and 24 hr post MCAO. dhCers and Cers showed distinct trends between species with long and very-long acyl chain (acyl chain length ≥ 22) at 3 hr post MCAO (Figure 3(B)(C)). The difference is especially obvious for Cers with significant decreases in very-long-chain Cers including Cer (18:1/23:0) and Cer (18:1/24:0) and a significant increase in the long chain Cer (18:1/16:0). On the other hand, most Cers significantly increased at 24 hr post MCAO. SMs showed significant decreases at both 3 hr and 24 hr post MCAO (Figure 3(D)). Compared to other SPL, the change was not significant for most of the CBs except CB (18:1/16:1) and CB (18:1/16:0), which showed significant decreases at 3 hr. CB (18:1/20:0) showed significant decreases at 24 hr (Figure 3(E)). S1P was the downstream metabolites of Cer. It significantly increased at 3 hr, and slightly decreased at 24 hr post MCAO (Figure 3(F)). Distinct trends between long-chain and very-long-chain Cers (acyl chain length ≥ 22) at 3 hr post MCAO were observed in the mouse brain. To elaborate their association with neuron injury, we further studied the expression of individual ceramide species on primary neuronal cultured cells in response to OGD treatment. Supplementary Figure 1 shows differential concentration changes could also be observed between long-chain and very-long-chain Cers on primary neuronal cultured cells at 0 hr post OGD.


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Figure 3. Bar charts for comparing the fold changes of (A) sphinganine (B) dhCers (C) Cers in different MCAO study groups. (MCAO study groups: Sham: without MCAO treatment; 3 hr: 3 hr post MCAO; 24 hr: 24 hr post MCAO.) *: p-value < 0.05; **: p-value < 0.01.


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Figure 3-continued. Bar charts for comparing the fold changes of (D) SMs (E) CBs (F) sphingosine and S1P in different MCAO study groups. (MCAO study groups: Sham: without MCAO treatment; 3 hr: 3 hr post MCAO; 24 hr: 24 hr post MCAO.) *: p-value < 0.05; **: p-value < 0.01.


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Long-chain and very-long-chain ceramides in ischemic stroke patients To further validate the findings of the different roles for long-chain and very-long-chain ceramides, we performed the Cer analysis using clinical samples. A total of 20 human plasma samples from 10 non-stroke controls and the same number of acute stroke patients, collected at 72hr after stroke were included for the study. Figure 4 shows the results of the human samples, opposite trends were observed for long-chain and very-long-chain ceramides after ischemic stroke. Long chain ceramides presented an increased trend with Cer, which showed significance in stroke patients. In contrast, very-long-chain ceramides decreased in ischemic stroke samples with Cer(d18:1/24:0), which showed significance. The results agree with our findings from the MCAO samples.

Figure 4. Bar charts for comparing the fold changes of ceramides in health control and ischemic stroke patients. *: p-value < 0.05; **: p-value < 0.01.

Effect of atorvastatin on the sphingolipid profile in MCAO mouse brains A single dose of 20 mg/kg atorvastatin was administered after an hour of occlusion, and the results were compared with those of mice that received PBS vehicle to investigate the effect of atorvastatin on the SPL pathway upon ischemic injury. A better improvement trend in neurological score was observed after single-dose atorvastatin administration (Supplementary Figure 2).


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To simplify the observations for the effect of atorvastatin on SPL concentration changes, SPLs in the same class were summarized. Because long-chain and very-long-chain Cers showed different responses to ischemic injury, they were examined separately for their concentration changes caused by MCAO and atorvastatin administration. Figure 5 shows the standardized results of different SPL levels with and without atorvastatin treatment. Briefly speaking, it revealed that atorvastatin could effectively reverse the SPL metabolism changes caused by MCAO injury. The increase in long-chain Cers at 3 hr and 24 hr post MCAO was not significant after atorvastatin administration compared with the Sham group. Additionally, the decrease and increase in verylong-chain Cer at 3 hr and 24 hr post MCAO, respectively, were not significant after atorvastatin treatment compared with the Sham group. Regarding the downstream metabolites sphingosine and S1P, a similar reversal could be observed after atorvastatin treatment.


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Figure 5. Box plots of the standardized abundance of each subclass of SPL obtained from different groups in the atorvastatin administration study. SPL subclass: sphinganine, sphingomyelin, longchain ceramides, very-long-chain ceramides, sphingosine, and sphingosine-1-phosphate. Atorvastatin administration study groups: Sham: no MCAO or atorvastatin treatment; 3 hr: 3 hr post MCAO; 24 hr: 24 hr post MCAO; +: with 20 mg/kg of atorvastatin treatment; -: without atorvastatin treatment. *: p-value < 0.05; **: p-value < 0.01.


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Discussion The de novo synthesis of Cers at 3 hr post MCAO Cers are the main products in the de novo synthesis of SPLs and are correlated with different signaling pathways including those of cell apoptosis17 and inflammation18. Because the levels of Cers of different acyl chain lengths were regulated by different Cer synthases, the concentration changes of different Cers at 3 hr and 24 hr post MCAO showed different patterns. In mammals, six distinct Cer synthases, abbreviated as CerS1-6, have been identified and are encoded by six distinct genes19-20; the proteins are responsible for synthesizing dhCer and Cers with different acyl chain lengths. To date, the functions of different acyl chain lengths on Cers remain poorly understood. With advancements in analytical instruments and SPL profiling technology, the different behavior of Cers with different chain lengths could be seen clearly. Our study identified an increase in long-chain Cers in the mouse brain at 3 hr post MCAO. Long-chain Cers, especially Cer (d18:1/18:0), are reported to be closely associated with JNK signaling, which relates to mitochondria dysfunction under ischemia21-22. Clinically, the elevation of Cer (d18:1/18:0) was observed in patients with poor outcome after stroke23. Other long-chain Cers are also reported to relate to different apoptotic pathways, such as TNF-related apoptosis2426

. In this study, we found that Cer (d18:1/16:0) and Cer (d18:1/18:0) significantly increased at

3 hr post MCAO. In contrast to the elevated concentrations of long-chain Cers, our study found a decrease in very-long-chain Cers soon after ischemic injury. F. D. Testai et al. have tested the changes of Cers in human cerebral endothelial cells (HCEC) under oxygen deprivation; they found a decreasing trend in very long chain Cers27. Interestingly, we also found that several very-longchain Cers, including Cer (d18:1/23:0) and Cer (d18:1/24:0), significantly decreased at 3 hr after I/R treatment, and other very-long-chain Cers have the same decreasing trends, which we observed similar decreased trend in the primary cultured neuron. The correlation between mouse brain model and primary cultured neurons in Cer concentration changes revealed that these changes are associated with the responses to neural injury.


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The differences in tendency and possibly function between the long-chain and very-longchain Cers have been mentioned in several studies20,28. In contrast to long-chain Cers, which are associated with cell apoptosis, very-long-chain Cers are reported to be related to cell proliferation and might play a protective role against apoptosis29-30. Vicent Menuz et al. also found similar protective role of very-long-chain Cers, reporting that HYL-2 may promote C. elegans survival by producing Cer(d18:1/20:0) and Cer(d18:1/22:0) under anoxia 31. SMs are the precursors of Cers through demyelination by sphingomyelinase, which is independent of de novo synthesis of Cers32. A general decreased trend in SMs was observed in our results. Previous studies have suggested that the demyelination of SMs, especially the neutral sphingomyelinase-related demyelination, might be related to neuron death under ischemic stress33. Shinji Soeda et al. suggested that the inhibition of demyelinase would help to prevent neuron death34. Another report also suggested that demyelination might be the key regulator of the production and function of Cers35. Combining our result of the decrease in SM with the increase in ceramide and the literature reports, we assumed that some of the Cer synthesis after MCAO was from the demyelination of SMs. Demyelination would form the loss of the head group from SM, which is phosphocholine. We further analyzed phosphocholine in OGD-treatment neuron cells, and the results showed a significant decrease of phosphocholine at both 3 and 24 hr post OGD (supplementary figure 3). As phosphocholine is also involved in the biosynthesis of other lipids such as phosphatidylcholines (PCs)

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, the decreased phosphocholine levels revealed that there

should be multiple lipid metabolic pathways involved after OGD-treatment.

S1P under I/R damage at 3 hr post MCAO S1P, a downstream metabolite of SPL metabolism, has been reported to be associated with cell growth and cell survival37-38. The activation of the S1P receptor by the agonist FTY720 has been reported to show a neuroprotective function in rats after ischemic stroke 39. In our study, we also found that S1P accumulated and that sphingosine, which is the precursor of S1P, decreased at 3 hr post MCAO. Therefore, sphingosine might be consumed for S1P synthesis. In addition, very long chain Cers were consumed at 3 hr, and S1P was the only downstream metabolite that increased at 3 hr. Therefore, one possible cell protective function of very-long-chain Cers might


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be to act as a precursor for increased synthesis of S1P. Further studies under such conditions are required to verify this phenomenon. SPL metabolism changes at 24 hr post MCAO SPL changes at 24 hr post MCAO were also investigated in this study. In contrast to 3 hr post MCAO, both long-chain and very-long-chain dhCers and Cers showed an increased trend at 24 hr post MCAO. It has been reported that serine palmitoyltransferase, which synthesizes sphinganine from serine and palmitoyl-CoA, is activated to enhance the de novo synthesis of Cers under hypoxia stress40. This phenomenon also has a lag effect because transcriptional up-regulation takes approximately 16 hr to increase the mRNA and protein levels of SPT41-42. Compared with 3 hr post MCAO, there are greater increases in both long-chain and very-long-chain Cers at 24 hr post MCAO, which indicates increased activation of whole de novo synthesis at this time point. The significant increase in long-chain Cers may suggest that further brain damage occurred at the reperfusion stage of MCAO43-44. Reperfusion-related injuries are highly associated with innate and adaptive immune responses in reperfused organs45. De novo synthesis of Cers could be activated by the activation of TNF-α signaling and cytokines, which would lead to Cer accumulation and further activate apoptosis signaling 46-47. Long-chain and very-long-chain ceramides in ischemic stroke patients Cer(d18:1/16:0) significantly increased and Cer(d18:1/24:0) significantly decreased in acute ischemic stroke patient plasma, comparing to non-stroke controls, which were similar in the MCAO and OGD neuron models. The results again support that different ceramide synthases involved in the ischemic injury result in different responses in long-chain Cers and very-longchain Cers. In the clinical perspective, future studies with more clinical samples are needed to clarify the potential diagnostic or prognostic roles of ceramide in ischemic stroke patients.

Effects of atorvastatin on SPL metabolism after ischemic injury Several recent studies have demonstrated the protective effect of atorvastatin on ischemic injury6-7,

48-50

interesting

. Compared with the MCAO-alone groups (vehicle groups), we found some

metabolic

differences

in

atorvastatin-treatment

groups.

After

atorvastatin


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administration, the decreased and increased trends of long-chain Cers and very-long-chain Cers, respectively, at 3 hr post MCAO were reversed to the level of the Sham group. In addition, the observed increase in S1P at 3 hr post MCAO was no longer significant compared with the Sham group. Although the influence of atorvastatin on sphingosine level was not as obvious as its effect on Cer or S1P, the level of sphingosine in the atorvastatin group was still closer to the level in the Sham group than to that of the MCAO-alone group. Since Cers and S1P are both involved in postischemic brain damage and protection, the reversal of changes by the atorvastatin intervention could provide additional confirmation of their roles in ischemic injury. It has been proposed that the pharmacological effect of statins may result from the inhibition of superoxide production and modulation of inflammation, decreasing lipid peroxidation, oxidative DNA damage, and microglial activation.51-53,54 In this study, we found that the de novo synthesis of Cers was altered after atorvastatin administration. However, whether this change is due to direct action on the SPL pathway or indirect effects from its protective function requires further study. In contrast to the changes in de novo synthesis, sphingomyelin levels in the atorvastatin treatment group did not show significant changes compared with the Sham group, which suggests that the concentration changes of Cers after atorvastatin administration could be mainly due to the alteration in de novo synthesis. At 24 hr post MCAO, the reversal of changes in Cer, sphingosine and S1P were not as significant as 3 hr post MCAO, but their levels were still closer to the Sham group than to the MCAO-alone group after atorvastatin administration. Conclusions In conclusion, this study was the first to use sphingolipidomic profiling techniques to investigate temporal dynamic changes in SPL concentrations after ischemic injury. Through the simultaneous measurement of metabolites in the SPL pathway, we were able to elucidate the overall effect of ischemic injury on SPL metabolism. In this study, we found that the de novo synthesis of Cers was significantly influenced and that Cers with different acyl chain lengths showed different responses to ischemic injury in MCAO mouse brains, OGD neuron cells, and stroke patient plasma. Through atorvastatin administration, we observed that the changes in de novo synthesis of SPLs were reversed, which provided additional confirmation of their roles in ischemic injury. The present study revealed the systemic changes of SPLs after ischemic injury,


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which may provide some directions for future studies to investigate novel therapeutic targets in the SPL pathway for ischemic stroke. Supporting Information: The following supporting information is available free of charge at ACS website. Supplementary protocols, the protocols for QC sample preparation and anlysis, reconstitution solvent preparation, and neurological score evaluation. Supplementary Figure 1, Bar Charts which compares the fold changes of individual ceramide on primary neuronal cultured cells in response to OGD treatment at 0, 3 and 24 hr. Supplementary Figure 2, The neurological scores evaluated at different time points after MCAO treatment and with or without atorvastatin treatment. Supplementary Figure 3, The bar charts for phosphocholine on primary neuronal cultured cells in response to OGD treatment at 3 and 24 hr. Supplementary Table 1, Molecule weight and retention time library for the sphingolipidomic analysis. Supplementary Table 2, Extraction recoveries of different sphingolipids.

Acknowledgement: The authors would like to thank the 3rd core facility at National Taiwan University Hospital for technical assistance and facility support.

Source of Funding: This work was supported by Grants (105-2113-M-002-013) and the National Taiwan University Hospital Grants (105-S3105).

DISCLOSURES All authors report no disclosures relevant to the manuscript


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Figures Legends Figure 1. The metabolic pathways of sphingolipids (A) and the overlaid LC-MS chromatogram of SPL extract from mouse brain tissue (B). Figure 2. The heat map (A) and the principal component analysis (PCA) score plot (B) of the SPL profiling results for mouse brains from different MCAO study groups. (MCAO study groups: Sham: without MCAO treatment; 3 hr: 3 hr post MCAO; 24 hr: 24 hr post MCAO) Figure 3. Bar charts for comparing the fold changes of (A) sphinganine (B) dhCers (C) Cers in different MCAO study groups. (MCAO study groups: Sham: without MCAO treatment; 3 hr: 3 hr post MCAO; 24 hr: 24 hr post MCAO.) *: p-value < 0.05; **: p-value < 0.01. Figure 4. Bar charts for comparing the fold changes of ceramides in health control and ischemic stroke patients. *: p-value < 0.05; **: p-value < 0.01. Figure 5. Box plots of the standardized abundance of each subclass of SPL obtained from different groups in the atorvastatin administration study. SPL subclass: sphinganine, sphingomyelin, longchain ceramides, very-long-chain ceramides, sphingosine, and sphingosine-1-phosphate. Atorvastatin administration study groups: Sham: no MCAO or atorvastatin treatment; 3 hr: 3 hr post MCAO; 24 hr: 24 hr post MCAO; +: with 20 mg/kg of atorvastatin treatment; -: without atorvastatin treatment. *: p-value < 0.05; **: p-value < 0.01.


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Table of graphic content (TOC) (For TOC only)


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Sphingolipidomics investigation of the temporal dynamics after

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