Reduced Cerebrospinal Fluid Levels of Lysophosphatidic Acid Docosahexaenoic Acid in Patients With Major Depressive Disorder and Schizophrenia.

BACKGROUND: Lysophosphatidic acid (LPA) is involved in numerous biological processes, including neurodevelopment, chronic inflammation, and immunologic response in the central nervous system. Autotaxin (ATX) is a secreted enzyme that produces LPA from lysophosphatidylcholine (LPC). Previous studies have demonstrated decreased protein levels of ATX in cerebrospinal fluid (CSF) of patients with major depressive disorder (MDD). Based on those studies, the current study investigated the levels of lysophospholipids species including LPA and related metabolic enzymes, in CSF of patients with MDD and schizophrenia (SCZ). METHODS: The levels of lysophospholipids species and related metabolic enzymes were measured with either liquid chromatography-tandem mass spectrometry or enzyme-linked immunosorbent assay. Japanese patients were diagnosed with DSM-IV-TR. CSF was obtained from age- and sex-matched healthy controls (n = 27) and patients with MDD (n = 26) and SCZ (n = 27). RESULTS: Of all lysophospholipids species, the levels of LPA 22:6 (LPA - docosahexaenoic acid) were significantly lower in patients with MDD and SCZ than in healthy controls. These levels were negatively correlated with several clinical symptomatic scores of MDD, but not those of SCZ. In addition, the levels of LPA 22:6 were significantly correlated with the levels of LPC 22:6 among all 3 groups. On the other hand, the levels of LPA 22:6 were not correlated with ATX activity in patients with MDD and SCZ. CONCLUSION: The lower levels of LPA 22:6 in patients with MDD and SCZ suggest an abnormality of LPA 22:6 metabolism. In addition, several depressive symptoms in patients with MDD were significantly associated with the lower levels of LPA 22:6, suggesting an involvement of LPA 22:6 in the pathophysiology of MDD.

Establishment of a Measurement System for Sphingolipids in the Cerebrospinal Fluid Based on Liquid Chromatography-Tandem Mass Spectrometry, and Its Application in the Diagnosis of Carcinomatous Meningitis.

OBJECTIVES: Sphingolipids have been demonstrated to be involved in many human diseases. However, measurement of sphingolipids, especially of sphingosine 1-phosphate (S1P) and dihydro-sphingosine 1-phosphate (dhS1P), in blood samples requires strict sampling, since blood cells easily secrete these substances during sampling and storage, making it difficult to introduce measurement of sphingolipids in clinical laboratory medicine. On the other hand, cerebrospinal fluid (CSF) contains few blood cells. Therefore, we attempted to establish a system based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the measurement of sphingolipids in the CSF, and applied it for the diagnosis of carcinomatous meningitis. METHODS: We developed and validated a LC-MS/MS-based measurement system for S1P and dhS1P and for ceramides and sphingosines, used this system to measure the levels of these sphingolipids in the CSF collected from the subjects with cancerous meningitis, and compared the levels with those in normal routine CSF samples. RESULTS: Both the measurement systems for S1P/dhS1P and for ceramides/sphingosines provided precision with the coefficient of variation below 20% for sphingolipids in the CSF samples. We also confirmed that the levels of S1P, as well as ceramides/sphingosines, in the CSF samples did not increase after the sampling. In the CSF samples collected from patients with cancerous meningitis, we observed that the ratio of S1P to ceramides/sphingosine and that of dhS1P to dihydro-sphingosine were higher than those in control samples. CONCLUSIONS: We established and validated a measurement system for sphingolipids in the CSF. The system offers promise for being introduced into clinical laboratory testing.

Alteration of the lysophosphatidic acid and its precursor lysophosphatidylcholine levels in spinal cord stenosis: A study using a rat cauda equina compression model.

Cauda equina compression (CEC) is a major cause of neurogenic claudication and progresses to neuropathic pain (NP). A lipid mediator, lysophosphatidic acid (LPA), is known to induce NP via the LPA1 receptor. To know a possible mechanism of LPA production in neurogenic claudication, we determined the levels of LPA, lysophosphatidylcholine (LPC) and LPA-producing enzyme autotaxin (ATX), in the cerebrospinal fluid (CSF) and spinal cord (SC) using a CEC as a possible model of neurogenic claudication. Using silicon blocks within the lumbar epidural space, we developed a CEC model in rats with motor dysfunction. LPC and LPA levels in the CSF were significantly increased from day 1. Importantly, specific LPA species (16:0, 18:2, 20:4) were upregulated, which have been shown to produce by ATX detected in the CSF, without changes on its level. In SC, the LPC and LPA levels did not change, but mass spectrometry imaging analysis revealed that LPC was present in a region where the silicon blocks were inserted. These results propose a model for LPA production in SC and CSF upon neurogenic claudication that LPC produced locally by tissue damages is converted to LPA by ATX, which then leak out into the CSF.

Evaluation of Lysophospholipid Measurement in Cerebrospinal Fluid Samples using Liquid Chromatography-Tandem Mass Spectrometry.

A quantification system for lysophospholipids (lysoPL) was developed, especially for blood samples, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, the lysoPL measurement in cerebrospinal fluid (CSF) has not been validated. Therefore, the present study aimed to validate the lysoPL measurement using CSF samples and to elucidate the possible clinical significance of the lysoPL measurement in CSF. For the validation, we observed a good precision and linearity in a sample with high lysoPL levels. The concentrations of lysoPL changed after incubation but the changes were smaller than those observed for serum samples. Moreover, we observed that the CSF levels of 16:0, 18:0 lysophosphatidylcholine, and 18:0, 18:1, and 20:4 lysophosphatidic acid were significantly higher in subjects with central nervous system invasion caused by hematological malignancies or carcinoma than in subjects with no abnormal CSF test results. In conclusion, an LC-MS/MS quantification system for lysoPL in CSF might be useful and could be applied to clinical laboratory testing.

Lysophosphatidic acids and their substrate lysophospholipids in cerebrospinal fluid as objective biomarkers for evaluating the severity of lumbar spinal stenosis.

Lysophospholipids (LPLs) are known to have potentially important roles in the initiation and maintenance of neuropathic pain in animal models. This study investigated the association between the clinical severity of lumbar spinal stenosis (LSS) and the cerebrospinal fluid (CSF) levels of LPLs, using human samples. We prospectively identified twenty-eight patients with LSS and fifteen controls with idiopathic scoliosis or bladder cancer without neurological symptoms. We quantified LPLs from CSF using liquid chromatography-tandem mass spectrometry. We assessed clinical outcome measures of LSS (Neuropathic Pain Symptom Inventory (NPSI) and Zurich Claudication Questionnaire (ZCQ)) and categorized patients into two groups according to their severity. Five species of lysophosphatidic acid (LPA), nine species of lysophosphatidylcholine (LPC), and one species of lysophosphatidylinositol (LPI) were detected. The CSF levels of all species of LPLs were significantly higher in LSS patients than controls. Patients in the severe NPSI group had significantly higher LPL levels (three species of LPA and nine species of LPC) than the mild group. Patients in the severe ZCQ group also had significantly higher LPL levels (four species of LPA and nine species of LPC). This investigation demonstrates a positive correlation between the CSF levels of LPLs and the clinical severity of LSS. LPLs are potential biomarkers for evaluating the severity of LSS.

Levels of lysophosphatidic acid in cerebrospinal fluid and plasma of patients with schizophrenia.

It is suggested that lysophosphatidic acid (LPA) plays a key role in the pathophysiology of schizophrenia. In this study, we measured LPA levels by enzyme-linked immunosorbent assay in cerebrospinal fluid (CSF) and plasma samples. The participants were 49 patients with schizophrenia and 49 normal healthy controls for CSF study, and 42 patients and 44 controls for plasma study. We found that LPA levels in the patients were not significantly different from those of controls in CSF (controls: 0.189 ±â€¯0.077 µM, patients: 0.175 ±â€¯0.067 µM; P = 0.318) and plasma samples (controls: 0.131 ±â€¯0.067 µM, patients: 0.120 ±â€¯0.075 µM; P = 0.465). On the other hand, CSF levels in medicated patients (0.162 ±â€¯0.061 µM) were significantly lower than those observed in unmedicated patients (0.224 ±â€¯0.067 µM, P = 0.038), suggesting that our findings could be masked by the influence of medication with antipsychotics. Interestingly, we detected significant negative correlation between PANSS scores and plasma LPA levels, especially in males and in unmedicated patients. Our result suggests that LPA levels in CSF and plasma samples would not serve as a diagnostic biomarker, but plasma levels could be used for symptomatic assessment of schizophrenia.

Lysophosphatidic acid is associated with neuropathic pain intensity in humans: An exploratory study.

The underlying mechanisms of neuropathic pain remain to be elucidated. Basic animal research has suggested that lysophosphatidic acids, which are bioactive lipids produced by autotaxin from lysophosphatidylcholine, may play key roles in the initiation and maintenance of neuropathic pain. Here, we investigated the clinical relevance of lysophosphatidic acids signaling on neuropathic pain in humans. Eighteen patients who had been diagnosed with neuropathic pain with varied etiologies participated in the study. Cerebrospinal fluid samples were obtained by lumbar puncture and the concentrations of 12 species of lysophosphatidic acids and lysophosphatidylcholine, autotaxin, and the phosphorylated neurofilament heavy subunit were measured. Pain symptoms were assessed using an 11-point numeric rating scale and the Neuropathic Pain Symptom Inventory regarding intensity and descriptive dimensions of neuropathic pain. The total lysophosphatidic acids were significantly associated with both pain intensity and symptoms. 18:1 and 20:4 lysophosphatidic acids in particular demonstrated the most correlations with dimensions of pain symptoms. Autotaxin and the phosphorylated neurofilament heavy subunit showed no association with pain symptoms. In conclusions, lysophosphatidic acids were significantly associated with pain symptoms in neuropathic pain patients. These results suggest that lysophosphatidic acids signaling might be a potential therapeutic target for neuropathic pain.

Elevated lysophosphatidic acid levels in the serum and cerebrospinal fluid in patients with multiple sclerosis: therapeutic response and clinical implication.

BACKGROUND: To date, although great effort has been made to identify biomarkers of multiple sclerosis (MS), it remains unclear whether lysophosphatidic acid (LPA) can be used as a biomarker for MS. METHODS: This study compared the LPA levels in the serum and cerebrospinal fluid (CSF) in patients with MS in relapse versus in remission and investigated the change in LPA levels in MS patients in relapse after treatment. Forty-one patients with relapsing-remitting MS (RRMS) (21 patients in relapse and 20 patients in remission) and 21 patients with non-inflammatory, non-vascular neurological diseases as controls were included in this study. MS patients in relapse received standard glucocorticoid treatment. LPA concentrations in serum and CSF were measured using an inorganic phosphate quantification assay. RESULTS: LPA levels in the serum and CSF were significantly higher in MS patients in relapse than in MS patients in remission and control patients (P < 0.05). The LPA level in MS patients in relapse was significantly reduced after treatment (P < 0.05). CONCLUSION: LPA concentrations in the serum and CSF may be used as biomarkers to monitor disease activity and therapeutic response in MS patients.

Increased levels of sphingosine-1-phosphate in cerebrospinal fluid of patients diagnosed with tick-borne encephalitis.

BACKGROUND: Tick-borne encephalitis (TBE) is a serious acute central nervous system infection that can result in death or long-term neurological dysfunctions. We hypothesize that changes in sphingosine-1-phosphate (S1P) concentration occur during TBE development. METHODS: S1P and interleukin-6 (IL-6) concentrations in blood plasma and cerebrospinal fluid (CSF) were measured using HPLC and ELISA, respectively. The effects of S1P on cytoskeletal structure and IL-6 production were assessed using rat astrocyte primary cultures with and without addition of plasma gelsolin and the S1P receptor antagonist fingolimod phosphate (FTY720P). RESULTS: We report that acute inflammation due to TBE virus infection is associated with elevated levels of S1P and IL-6 in the CSF of infected patients. This elevated concentration is observed even at the earliest neurologic stage of disease, and may be controlled by glucocorticosteroid anti-inflammatory treatment, administered to patients unresponsive to antipyretic drugs and who suffer from a fever above 39°C. In vitro, treatment of confluent rat astrocyte monolayers with a high concentration of S1P (5 µM) results in cytoskeletal actin remodeling that can be prevented by the addition of recombinant plasma gelsolin, FTY720P, or their combination. Additionally, gelsolin and FTY720P significantly decreased S1P-induced release of IL-6. CONCLUSIONS: TBE is associated with increased concentration of S1P and IL-6 in CSF, and this increase might promote development of inflammation. The consequences of increased extracellular S1P can be modulated by gelsolin and FTY720P. Therefore, blocking the inflammatory response at sites of infection by agents modulating S1P pathways might aid in developing new strategies for TBE treatment.

Vitamin D therapy in experimental allergic encephalomyelitis could be limited by opposing effects of sphingosine 1-phosphate and gelsolin dysregulation.

Several studies support a protective effect of vitamin D on multiple sclerosis and experimental allergic encephalomyelitis (EAE), but the mechanisms of these favorable effects are unclear. Our study demonstrates that sphingosine 1-phosphate (S1P) is upregulated in the serum and spinal cords of EAE rats, but that vitamin D reverses the upregulation to alleviate inflammation. Vitamin D, however, cannot prevent the disease process, suggesting that other factors may be involved. To identify additional factors that might limit vitamin D efficacy, we assessed the effects of vitamin D on plasma gelsolin (pGSN), a regulator of S1P that is downregulated in the CSF of MS patients. Our results show that pGSN is downregulated in the serum of EAE rats, whereas its cellular form, cytoplasmic gelsolin (cGSN), is upregulated in the spinal cord of EAE rats. Importantly, vitamin D causes a downregulation of both pGSN and cGSN, which may counteract the positive effects of S1P decrease. Furthermore, 48 and 42 kDa caspase-3 cleavage products of cGSN are detected in EAE spinal cords, suggesting enhanced apoptotic activity, but these cleaved products undergo a similar decrease upon vitamin D treatment. To directly test the role of cGSN in the apoptotic process, we performed RNA interference in PC-12, a rat sympathetic nerve cell line. Results verify that cGSN suppresses apoptosis induced by TNF-α. Collectively, these results support a therapeutic effect of vitamin D that is derived from its ability to reduce S1P, but is limited by its simultaneous effect in reducing pGSN and cGSN. Based on these observations, we postulate that combined therapy with recombinant human pGSN and vitamin D may produce more beneficial effect in treating multiple sclerosis.

Anti-lysophosphatidic acid antibodies improve traumatic brain injury outcomes.

BACKGROUND: Lysophosphatidic acid (LPA) is a bioactive phospholipid with a potentially causative role in neurotrauma. Blocking LPA signaling with the LPA-directed monoclonal antibody B3/Lpathomab is neuroprotective in the mouse spinal cord following injury. FINDINGS: Here we investigated the use of this agent in treatment of secondary brain damage consequent to traumatic brain injury (TBI). LPA was elevated in cerebrospinal fluid (CSF) of patients with TBI compared to controls. LPA levels were also elevated in a mouse controlled cortical impact (CCI) model of TBI and B3 significantly reduced lesion volume by both histological and MRI assessments. Diminished tissue damage coincided with lower brain IL-6 levels and improvement in functional outcomes. CONCLUSIONS: This study presents a novel therapeutic approach for the treatment of TBI by blocking extracellular LPA signaling to minimize secondary brain damage and neurological dysfunction.

Sphingosine lysolipids in the CNS: endogenous cannabinoid antagonists or a parallel pain modulatory system?

A significant number of patients experience chronic pain and the intractable side effects of currently prescribed pain medications. Recent evidence indicates important pain-modulatory roles for two classes of G-protein-coupled receptors that are activated by endogenous lipid ligands, the endocannabinoid (eCB) and sphingosine-1-phosphate (S1P) receptors, which are widely expressed in both the immune and nervous systems. In the central nervous system (CNS), CB1 cannabinoid and S1P1 receptors are most abundantly expressed and exhibit overlapping anatomical distributions and similar signaling mechanisms. The eCB system has emerged as a potential target for treatment of chronic pain, but comparatively little is known about the roles of S1P in pain regulation. Both eCB and S1P systems modulate pain perception via the central and peripheral nervous systems. In most paradigms studied, the eCB system mainly inhibits pain perception. In contrast, S1P acting peripherally at S1P1 and S1P3 receptors can enhance sensitivity to various pain stimuli or elicit spontaneous pain. However, S1P acting at S1P1 receptors and possibly other targets in the CNS can attenuate sensitivity to various pain stimuli. Interestingly, other endogenous sphingolipid derivatives might play a role in central pain sensitization. Moreover, these sphingolipids can also act as CB1 cannabinoid receptor antagonists, but the physiological relevance of this interaction is unknown. Overall, both eCB and sphingolipid systems offer promising targets for the treatment of chronic pain. This review compares and contrasts the eCB and S1P systems with a focus on their roles in pain modulation, and considers possible points of interaction between these systems.

Changes in the cerebrospinal fluid ceramide profile after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The purpose of this study was to investigate changes in the cerebrospinal fluid sphingolipid profile in patients with subarachnoid hemorrhage in relation to the occurrence of symptomatic vasospasm and outcome at hospital discharge. METHODS: The ceramide profile in the cerebrospinal fluid was determined by mass spectrometry in control subjects and patients with Fisher 3 grade subarachnoid hemorrhage within 48 hours of the bleed. Patients were prospectively followed and subcategorized based on the occurrence of symptomatic vasospasm and modified Rankin Scale at discharge. RESULTS: Compared to control subjects, patients with subarachnoid hemorrhage had higher cerebrospinal fluid levels of total ceramide (12.4±8.8 versus 54.6±49.3 pmol/mL; P<0.001). In the subgroup analysis, total ceramide levels in individuals with symptomatic vasospasm (104.2±57.0 pmol/mL) were higher than in those with asymptomatic vasospasm (32.4±25.7 pmol/mL; P=0.006) and no vasospasm (30.9±15.7 pmol/mL; P=0.003). In addition, compared to patients with a good outcome (modified Rankin Scale ≤3), individuals with poor outcome (modified Rankin Scale ≥4) had higher cerebrospinal fluid levels of total ceramide (79±25 versus 23±6 pmol/mL; P=0.008). When the relative contributions of the different ceramide species were calculated, a higher relative concentration of C(18:0) ceramide was observed in individuals with symptomatic vasospasm (P=0.018) and poor outcome (P=0.028). CONCLUSIONS: Ceramide profile changes occur in subarachnoid hemorrhage. In this small case-based series elevation of levels of this sphingolipid, particularly C(18:0), was associated with the occurrence of symptomatic vasospasm and poor neurological outcome after subarachnoid hemorrhage.

Plasma gelsolin modulates cellular response to sphingosine 1-phosphate.

Hypogelsolinemia is observed in patients with different states of acute or chronic inflammation such as sepsis, rheumatoid arthritis, and multiple sclerosis. In animal models of sepsis, repletion of plasma gelsolin reduces septic mortality. However, the functions of extracellular gelsolin and the mechanisms leading to its protective nature are poorly understood. Potential mechanisms involve gelsolin's extracellular actin scavenging function or its ability to bind bioactive lipids or proinflammatory mediators, which would limit inflammatory responses and prevent tissue damage. Here we report that human plasma gelsolin binds to sphingosine 1-phosphate (S1P), a pleiotropic cellular agonist involved in various immune responses, and to its synthetic structural analog FTY720P (Gilenya). The fluorescence intensity of a rhodamine B-labeled phosphatidylinositol 4,5-bisphosphate binding peptide derived from gelsolin and the optical density of recombinant human plasma gelsolin (rhpGSN) were found to decrease after the addition of S1P or FTY720P. Gelsolin's ability to depolymerize F-actin also decreased progressively with increasing addition of S1P. Transient increases in phosphorylation of extracellular signal-regulated kinase in bovine aortic endothelial cells (BAECs) after S1P treatment were inhibited by rhpGSN. The ability of S1P to increase F-actin content and the elastic modulus of primary astrocytes and BAECs was also prevented by rhpGSN. Evaluation of S1P and gelsolin levels in cerebrospinal fluid reveals a low concentration of gelsolin and a high concentration of S1P in samples obtained from patients suffering from lymphatic meningitis. These findings suggest that gelsolin-mediated regulation of S1P bioactivity may be important to maintain immunomodulatory balance at inflammatory sites.

Intrathecal increase of sphingosine 1-phosphate at early stage multiple sclerosis.

Sphingosine 1-phosphate (S1P) is a pleiotropic mediator that is critically involved in the development of an inflammatory response in various pathological conditions. We hypothesize that during the course of multiple sclerosis (MS) development, chronic inflammation will result in the alteration of S1P levels in blood and cerebrospinal fluid (CSF). We evaluated S1P concentrations in blood and CSF obtained from 66 subjects, including 40 patients diagnosed with MS and 26 subjects of a control group that included patients diagnosed with idiopathic cephalgia and idiopathic (Bell's) facial nerve palsy. HPLC techniques were used to determine S1P levels. We found that S1P concentrations in blood of the MS subject group (361.7+/-150.7 nM) did not differ from those of the control group (371.9+/-142.5 nM). However, S1P concentrations in CSF of the MS group were significantly higher (p<0.01) compared to the control group (2.2+/-2.7 versus 0.69+/-1.1 nM). The increase of S1P concentration in CSF of MS subjects suggests that this bioactive lipid is involved in chronic inflammation associated with MS and it may be useful to study S1P in a number of neurodegenerative diseases to provide better understanding of the mechanisms governing their development.

Role of lysophosphatidic acid in endothelin-1- and hematoma-induced alteration of cerebral microcirculation.

Cerebral hematoma increases cerebrospinal fluid (CSF) endothelin-1 (ET-1). Inhibitors of ET-1 synthesis prevent this increment and hematoma-induced modification of cerebral arteriolar reactivity. We hypothesized that intrathecal ET-1 injection could 1) modify pial arteriolar reactivity similarly to hematoma; 2) increase CSF lysophosphatidic acid (LPA), a potential contributor to altered cerebrovascular reactivity; and 3) reduce the level of adenosine 3',5'-cyclic monophosphate (cAMP) in the CSF. Either ET-1 (10(-7) M) or artificial CSF was injected over the left parietal cortex of newborn pigs. Four days later, cranial windows were implanted. CSF ET was increased from a basal level of 11 fmol/ml to 18 fmol/ml 4 days after ET-1 injection, whereas CSF cAMP was reduced from 2,700 to 950 fmol/ml. The mean diameter of pial arterioles was reduced 31%. In control animals, 10(-12) M ET caused dilation, and higher concentrations induced vasoconstriction. Four days after ET-1 injection topical ET-1 caused constriction instead of dilation at 10(-12) M, and constrictions at higher doses were enhanced. Norepinephrine-induced constrictions were potentiated in the ET-1-injected group. Dilations to cAMP-dependent (but not independent) vasodilators were attenuated after ET-1. The concentration of the vasoconstrictor lipid mediator LPA increased approximately fourfold. Thus intrathecal injection of ET-1 mimics hematoma-induced modification of cerebral vascular reactivity and increase in LPA production. The mechanism(s) of ET-1- and hematoma-induced modifications may involve LPA, which is known to contribute to the loss of dilator responses by inhibition of cAMP product on. The present study further suggests that ET-1 together with LPA could be causing changes in cerebrovascular reactivity following cerebral hemorrhage. ET-1 stimulates the release of LPA from brain parenchyma independent of serum so that LPA could serve as a secondary mediator.