Regulation of lipid synthesis in myelin modulates neural activity and is required for motor learning.

Brain function relies on both rapid electrical communication in neural circuitry and appropriate patterns or synchrony of neural activity. Rapid communication between neurons is facilitated by wrapping nerve axons with insulation by a myelin sheath composed largely of different lipids. Recent evidence has indicated that the extent of myelination of nerve axons can adapt based on neural activity levels and this adaptive myelination is associated with improved learning of motor tasks, suggesting such plasticity may enhance effective learning. In this study, we examined whether another aspect of myelin plasticity-changes in myelin lipid synthesis and composition-may also be associated with motor learning. We combined a motor learning task in mice with in vivo two-photon imaging of neural activity in the primary motor cortex (M1) to distinguish early and late stages of learning and then probed levels of some key myelin lipids using mass spectrometry analysis. Sphingomyelin levels were elevated in the early stage of motor learning while galactosylceramide levels were elevated in the middle and late stages of motor learning, and these changes were correlated across individual mice with both learning performance and neural activity changes. Targeted inhibition of oligodendrocyte-specific galactosyltransferase expression, the enzyme that synthesizes myelin galactosylceramide, impaired motor learning. Our results suggest regulation of myelin lipid composition could be a novel facet of myelin adaptations associated with learning.

Desorption ionization using through-hole alumina membrane offers higher reproducibility than 2,5-dihydroxybenzoic acid, a widely used matrix in Fourier transform ion cyclotron resonance mass spectrometry imaging analysis.

RATIONALE: DIUTHAME (desorption ionization using through-hole alumina membrane), a recently developed matrix-free ionization-assisting substrate, was examined for reproducibility in terms of mass accuracy and intensity using standard lipid and mouse brain sections. The impregnation property of DIUTHAME significantly improved the reproducibility of mass accuracy and intensity compared with 2,5-dihydroxybenzoic acid (DHB). METHODS: Frozen tissue sections were mounted on indium tin oxide-coated glass slides. DIUTHAME and DHB were applied to individual sections. Subsequently, a solution of a phosphatidylcholine standard, PC(18:2/18:2), was poured onto the DIUTHAME and matrix. Finally, the samples were subjected to laser desorption ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry. The reproducibility was tested by calculating the mean ± standard deviation values of mass errors and intensities of individual ion species. RESULTS: Analysis of the PC(18:2/18:2) standard showed significantly (p < 0.01) lower mass error for DIUTHAME-MS than for MALDI-MS. Endogenous PC(36:4) analysis in mouse brain section also showed significantly (p < 0.05) lower mass errors for DIUTHAME-MS. Furthermore, we investigated the mass error of some abundant lipid ions in brain sections and observed similar results. DIUTHAME-MS displayed lower signal intensity in standard PC analysis. Interestingly, it offered higher signal intensities for all the endogenous lipid ions. Lower fluctuations of both mass accuracies and signal intensities were observed in DIUTHAME-MS. CONCLUSIONS: Our results demonstrated that DIUTHAME-MS offers higher reproducibility for mass accuracies and intensities than MALDI-MS in both standard lipid and mouse brain tissue analyses. It can potentially be used instead of conventional MALDI-MS and mass spectrometry imaging analyses to achieve highly reproducible data for mass accuracy and intensity.

Change in Brain Plasmalogen Composition by Exposure to Prenatal Undernutrition Leads to Behavioral Impairment of Rats.

Epidemiological studies suggest that poor nutrition during pregnancy influences offspring predisposition to experience developmental and psychiatric disorders. Animal studies have shown that maternal undernutrition leads to behavioral impairment, which is linked to alterations in monoaminergic systems and inflammation in the brain. In this study, we focused on the ethanolamine plasmalogen of the brain as a possible contributor to behavioral disturbances observed in offspring exposed to maternal undernutrition. Maternal food or protein restriction between gestational day (GD) 5.5 and GD 10.5 resulted in hyperactivity of rat male adult offspring. Genes related to the phospholipid biosynthesis were found to be activated in the PFC, but not in the NAcc or striatum, in the offspring exposed to prenatal undernutrition. Corresponding to these gene activations, increased ethanolamine plasmalogen (18:0p-22:6) was observed in the PFC using mass spectrometry imaging. A high number of crossings and the long time spent in the center area were observed in the offspring exposed to prenatal undernutrition and were mimicked in adult rats via the intravenous injection of ethanolamine plasmalogen (18:0p-22:6) incorporated into the liposome. Additionally, plasmalogen (18:0p-22:6) increased only in the PFC, and not in the NAcc or striatum. These results suggest that brain plasmalogen is one of the key molecules to control behavior, and its injection using liposome is a potential therapeutic approach for cognitive impairment.SIGNIFICANCE STATEMENT Maternal undernutrition correlates to developmental and psychiatric disorders. Here, we found that maternal undernutrition in early pregnancy led to hyperactivity in rat male offspring and induced gene activation of phospholipid-synthesizing enzyme and elevation of ethanolamine plasmalogen (18:0p-22:6) level in the PFC. Intravenous injection of ethanolamine plasmalogen (18:0p-22:6) incorporated into the liposome maintained crossing activity and the activity was circumscribed to the center area for a long time period, as in prenatally undernourished offspring with aberrant behavior. Furthermore, the amount of ethanolamine plasmalogen (18:0p-22:6) increased in the PFC of the rat after injection. Our result suggests that brain plasmalogen is one of the key molecules to control behavior and that its injection using liposome is a potential therapeutic approach for cognitive impairment.

Sphingomyelin(d35:1) as a novel predictor for lung adenocarcinoma recurrence after a radical surgery: a case-control study.

BACKGROUND: To improve the postoperative prognosis of patients with lung cancer, predicting the recurrence high-risk patients is needed for the efficient application of adjuvant chemotherapy. However, predicting lung cancer recurrence after a radical surgery is difficult even with conventional histopathological prognostic factors, thereby a novel predictor should be identified. As lipid metabolism alterations are known to contribute to cancer progression, we hypothesized that lung adenocarcinomas with high recurrence risk contain candidate lipid predictors. This study aimed to identify candidate lipid predictors for the recurrence of lung adenocarcinoma after a radical surgery. METHODS: Frozen tissue samples of primary lung adenocarcinoma obtained from patients who underwent a radical surgery were retrospectively reviewed. Recurrent and non-recurrent cases were assigned to recurrent (n = 10) and non-recurrent (n = 10) groups, respectively. Extracted lipids from frozen tissue samples were subjected to liquid chromatography-tandem mass spectrometry analysis. The average total lipid levels of the non-recurrent and recurrent groups were compared. Candidate predictors were screened by comparing the folding change and P-value of t-test in each lipid species between the recurrent and non-recurrent groups. RESULTS: The average total lipid level of the recurrent group was 1.65 times higher than that of the non-recurrent group (P < 0.05). A total of 203 lipid species were increased (folding change, ≥2; P < 0.05) and 4 lipid species were decreased (folding change, ≤0.5; P < 0.05) in the recurrent group. Among these candidates, increased sphingomyelin (SM)(d35:1) in the recurrent group was the most prominent candidate predictor, showing high performance of recurrence prediction (AUC, 9.1; sensitivity, 1.0; specificity, 0.8; accuracy, 0.9). CONCLUSION: We propose SM(d35:1) as a novel candidate predictor for lung adenocarcinoma recurrence. Our finding can contribute to precise recurrence prediction and qualified postoperative therapeutic strategy for lung adenocarcinomas. TRIAL REGISTRATION: This retrospective study was registered at the UMIN Clinical Trial Registry ( UMIN000039202 ) on 21st January 2020.

Preferential Incorporation of Administered Eicosapentaenoic Acid Into Thin-Cap Atherosclerotic Plaques.

OBJECTIVE: n-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have beneficial effects on atherosclerosis. Although specific salutary actions have been reported, the detailed distribution of n-3 polyunsaturated fatty acids in plaque and their relevance in disease progression are unclear. Our aim was to assess the pharmacodynamics of EPA and DHA and their metabolites in atherosclerotic plaques. Approach and Results: Apolipoprotein E-deficient (Apoe-/-) mice were fed a Western diet supplemented with EPA (1%, w/w) or DHA (1%, w/w) for 3 weeks. Imaging mass spectrometry analyses were performed in the aortic root and arch of the Apoe-/- mice to evaluate the distribution of EPA, DHA, their metabolites and the lipids containing EPA or DHA in the plaques. Liquid chromatography-mass spectrometry and histological analysis were also performed. The intima-media thickness of atherosclerotic plaque decreased in plaques containing free EPA and EPAs attached with several lipids. EPA was distributed more densely in the thin-cap plaques than in the thick-cap plaques, while DHA was more evenly distributed. In the aortic root, the distribution of total EPA level and cholesteryl esters containing EPA followed a concentration gradient from the vascular endothelium to the media. In the aortic arch, free EPA and 12-hydroxy-EPA colocalized with M2 macrophage. CONCLUSIONS: Administered EPA tends to be incorporated from the vascular lumen side and preferentially taken into the thin-cap plaque.

Development of sheet-enhanced technique (Set) method for matrix-assisted laser desorption/ionization imaging mass spectrometry.

RATIONALE: The key to successful experiments in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is to apply the matrix uniformly to the sample. With the development of automated equipment, uniform matrix application has made great progress while the sample preparation required to acquire a better image becomes complicated. METHODS: The approach is to apply the matrix uniformly to tape and adhere it to the tissue section. We call this the sheet-enhanced technique (Set) method. RESULTS: The Set method promotes ionization of biomolecules as well as the spray method. This procedure does not require the preparation and application of a matrix solution for each experiment, dramatically reducing the time and effort of matrix deposition. CONCLUSIONS: In the present study, we have developed the Set method as a new matrix application method. The method promotes ionization of biomolecules as well as the spray method for MALDI-IMS.

Higher Accumulation of Docosahexaenoic Acid in the Vermilion of the Human Lip than in the Skin.

The vermilion of the human lip is a unique facial area because of certain distinguishing features from the adjacent tissues such as the white lip (skin) and oral mucosa. However, the distinction in terms of molecular distribution between the vermilion and skin has remained unexplored. Therefore, we aimed to map the human lip by mass spectrometry imaging to gain understanding of the free fatty acid distribution in the vermilion. The lip specimens trimmed off during cheiloplasty were analyzed using desorption electrospray ionization-mass spectrometry imaging. Distributions of two monounsaturated fatty acids and three polyunsaturated fatty acids were observed in the human lip tissue: palmitoleic acid (POA) and oleic acid (OA) and linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA), respectively. Although POA, OA, LA, and AA were differentially distributed across the vermilion and skin, DHA showed a higher accumulation in the epithelium of the vermilion compared to that in the skin. Our results clearly demonstrated the difference in fatty acid distributions between the vermilion and skin. The highly abundant DHA in the epithelium of the vermilion may have an antioxidant role and may thus protect the lip from aging. Our findings can provide a novel strategy for treating lip disorders.

Lipid-correlated alterations in the transcriptome are enriched in several specific pathways in the postmortem prefrontal cortex of Japanese patients with schizophrenia.

AIMS: Schizophrenia is a chronic relapsing psychiatric disorder that is characterized by many symptoms and has a high heritability. There were studies showing that the phospholipid abnormalities in subjects with schizophrenia (Front Biosci, S3, 2011, 153; Schizophr Bull, 48, 2022, 1125; Sci Rep, 7, 2017, 6; Anal Bioanal Chem, 400, 2011, 1933). Disturbances in prefrontal cortex phospholipid and fatty acid composition have been reported in subjects with schizophrenia (Sci Rep, 7, 2017, 6; Anal Bioanal Chem, 400, 2011, 1933; Schizophr Res, 215, 2020, 493; J Psychiatr Res, 47, 2013, 636; Int J Mol Sci, 22, 2021). For exploring the signaling pathways contributing to the lipid changes in previous study (Sci Rep, 7, 2017, 6), we performed two types of transcriptome analyses in subjects with schizophrenia: an unbiased transcriptome analysis solely based on RNA-seq data and a correlation analysis between levels of gene expression and lipids. METHODS: RNA-Seq analysis was performed in the postmortem prefrontal cortex from 10 subjects with schizophrenia and 5 controls. Correlation analysis between the transcriptome and lipidome from 9 subjects, which are the same samples in the previous lipidomics study (Sci Rep, 7, 2017, 6). RESULTS: Extraction of differentially expressed genes (DEGs) and further sequence and functional group analysis revealed changes in gene expression levels in phosphoinositide 3-kinase (PI3K)-Akt signaling and the complement system. In addition, a correlation analysis clarified alterations in ether lipid metabolism pathway, which is not found as DEGs in transcriptome analysis alone. CONCLUSIONS: This study provided results of the integrated analysis of the schizophrenia-associated transcriptome and lipidome within the PFC and revealed that lipid-correlated alterations in the transcriptome are enriched in specific pathways including ether lipid metabolism pathway.

Spatial distribution of the Shannon entropy for mass spectrometry imaging.

Mass spectrometry imaging (MSI) allows us to visualize the spatial distribution of molecular components in a sample. A large amount of mass spectrometry data comprehensively provides molecular distributions. In this study, we focus on the information in the obtained data and use the Shannon entropy as a quantity to analyze MSI data. By calculating the Shannon entropy at each pixel on a sample, the spatial distribution of the Shannon entropy is obtained from MSI data. We found that low-entropy pixels in entropy heat maps for kidneys of mice had different structures between two ages (3 months and 31 months). Such changes cannot be visualized by conventional imaging techniques. We further propose a method to find informative molecules. As a demonstration of the proposed scheme, we identified two molecules by setting a region of interest which contained low-entropy pixels and by exploring changes of peaks in the region.

Region-specific effects of Scrapper on the abundance of glutamate and gamma-aminobutyric acid in the mouse brain.

The brain consists of various areas with anatomical features. Neurons communicate with one another via excitatory or inhibitory synaptic transmission. Altered abundance of neurotransmitters, including glutamate and gamma-aminobutyric acid (GABA), in specific brain regions is closely involved in severe neurological diseases, such as schizophrenia and obsessive-compulsive disorder. SCRAPPER, a ubiquitin E3 ligase, regulates synaptic transmission. Scrapper gene deficiency results in defective neurotransmission due to excessive secretion of neurotransmitters. The present study employed matrix-assisted laser desorption/ionization imaging mass spectrometry to analyze the abundance of amino acid neurotransmitters in Scrapper knockout (SCR-KO) mice. SCR-KO mice exhibited significantly increased glutamate levels in the isocortex (CTX), corpus callosum (CC), thalamus (TH), midbrain (MB), cerebellar cortex (CBX), and caudoputamen (CP) and increased GABA levels in the CTX, CC, TH, MB, CBX and hypothalamus (HY) compared with wild-type mice. These findings indicate that Scrapper deficiency leads to upregulated glutamate and GABA levels in multiple regions. Our results show a differential, region-specific effect of Scrapper on the abundance of glutamate and GABA.

Mass spectrometry imaging reveals glycine distribution in the developing and adult mouse brain.

Glycine is an important amino acid in the central nervous system. The aberrant conditions of glycine concentrations cause sever neurological disorders, such as nonketotic-hyperglycinemia (NKH), also known as glycine encephalopathy. Therefore, a better understanding of its relative abundance and distribution in the developing and adult brains would provide insights into the pathogeneses of this kind of disorders. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging has been used for direct molecular-specific compound detection, distribution mapping, and identifying molecular species in tissue sections. Although a few reports have already shown the imaging of glycine using MALDI-MS in the adult mouse brain, they lack detailed neuroanatomical and developmental evaluations. We, thus, investigated the detailed distribution and abundance of glycine not only in the adult mouse brain but also in the developing mouse brain using this technique. In both brains, we detected derivatized glycine throughout the mouse brain. Interestingly, in both brains, derivatized glycine was abundantly detected in the brain stem. The other areas showed relatively lower signal intensities. As many model mice are used for glycine-related diseases, MALDI-MS is a suitable technique to analyze the pathogenesis of these diseases.

Discovery of lipid biomarkers correlated with disease progression in clear cell renal cell carcinoma using desorption electrospray ionization imaging mass spectrometry.

Clear cell renal cell carcinoma (ccRCC) often results in recurrence or metastasis, and there are only a few clinically effective biomarkers for early diagnosis and personalized therapy. Metabolic changes have been widely studied using mass spectrometry (MS) of tissue lysates to identify novel biomarkers. Our objective was to identify lipid biomarkers that can predict disease progression in ccRCC by a tissue-based approach. We retrospectively investigated lipid molecules in cancerous tissues and normal renal cortex tissues obtained from patients with ccRCC (n = 47) using desorption electrospray ionization imaging mass spectrometry (DESI-IMS). We selected eight candidate lipid biomarkers showing higher signal intensity in cancerous than in normal tissues, with a clear distinction of the tissue type based on the images. Of these candidates, low maximum intensity ratio (cancerous/normal) values of ions of oleic acid, m/z 389.2, and 391.3 significantly correlated with shorter progression-free survival compared with high maximum intensity ratio values (P = 0.011, P = 0.022, and P < 0.001, respectively). This study identified novel lipid molecules contributing to the prediction of disease progression in ccRCC using DESI-IMS. Our findings on lipid storage may provide a new diagnostic or therapeutic strategy for targeting cancer cell metabolism.

N-3 fatty acids modulate repeated stress-evoked pain chronicity.

N-3 fatty acids, including docosahexaenoic acid (DHA), have a beneficial effect in both pain and psychiatric disorders. In fact, we previously reported that stress-induced pain prolongation might be mediated through the suppression of the G-protein coupled-receptor 40/free fatty acid receptor 1 (GPR40/FFAR1), which is activated by DHA and long-chain fatty acids. However, the involvement of GPR40/FFAR1 ligands in the development of stress-induced chronic pain has not yet been described. In this study, we investigated the role of DHA in stress-evoked pain chronicity using diet-induced n-3 fatty acid deficient mice. The n-3 fatty acid deficient mice showed exacerbation of anxiety-like behavior after repeated exposure to social defeat stress. The intact n-3 fatty acid deficient mice showed a decrease in paw threshold values. On the other hand, paw withdrawal thresholds of defeated but not non-stressed, n-3 fatty acid deficient mice continued until day 49 after paw surgery. We evaluated changes in phosphatidylcholine composition in the brains of repeat stress-evoked chronic pain model mice which were not on n-3 fatty acid deficiency diets. On day 7 after paw surgery, phosphatidylcholines with DHA and other long-chain fatty acids were found to have decreased in the brains of stressed mice. Moreover, stress-induced persistent mechanical allodynia was improved by oral DHA supplementation. These results indicated that chronic stress may directly affect brain lipid composition; the related changes could be involved in chronic pain development. Our findings suggested that n-3 fatty acids, particularly DHA, are useful as a potential therapeutic target for stress-evoked chronic pain.

Dietary Intake of Green Nut Oil or DHA Ameliorates DHA Distribution in the Brain of a Mouse Model of Dementia Accompanied by Memory Recovery.

Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, has significant healthbenefits. Previous studies reported decreased levels of DHA and DHA-containing phosphatidylcholines inthe brain of animals suffering from Alzheimer's disease, the most common type of dementia; furthermore,DHA supplementation has been found to improve brain DHA levels and memory efficiency in dementia. Oilextracted from the seeds of Plukenetia volubilis (green nut oil; GNO) is also expected to have DHA like effectsas it contains approximately 50% α-linolenic acid, a precursor of DHA. Despite this, changes in the spatialdistribution of DHA in the brain of animals with dementia following GNO or DHA supplementation remainunexplored. In this study, desorption electrospray ionization imaging mass spectrometry (DESI-IMS) wasapplied to observe the effects of GNO or DHA supplementation upon the distribution of DHA in the brain ofmale senescence-accelerated mouse-prone 8 (SAMP8) mice, a mouse model of dementia. DESI-IMS revealedthat brain DHA distribution increased 1.85-fold and 3.67-fold in GNO-fed and DHA-fed SAMP8 mice,respectively, compared to corn oil-fed SAMP8 mice. Memory efficiency in SAMP8 mice was also improvedby GNO or DHA supplementation. In summary, this study suggests the possibility of GNO or DHAsupplementation for the prevention of dementia.

Analysis of potential anti-aging beverage Pru, a traditional Cuban refreshment, by desorption electrospray ionization-mass spectrometry and FTICR tandem mass spectrometry.

Aging has been established as a major risk factor for prevalent diseases and hence, the development of anti-aging medicines is of great importance. Recently, herbal fermented beverages have emerged as a promising source of potential anti-aging drug. Pru, a traditional Cuban refreshment produced by decoction and fermentation of multispecies plants with sugar, has been consumed for many years and is claimed to have multiple medicinal properties. Besides the traditional method, Pru is also manufactured industrially. The present study analyzed the major components of both traditional Pru (TP) and industrial Pru (IP) to reveal their potential application in promoting the health span. We performed desorption electrospray ionization-mass spectrometry (DESI-MS) and acquired mass spectra by scanning over the 50-1200 m/z range in both positive and negative ion modes. Fourier transform ion cyclotron resonance (FTICR) tandem mass spectrometry (MS/MS) was performed for validating the compound assignments. Three important compounds were identified by comparing the MS and MS/MS spectra with reported literature and the online database. One of the identified compounds, gluconic acid, was found to be the most abundant shared metabolite between TP and IP whereas the other two compounds, magnoflorine and levan were exclusively detected in TP. The present study is the first report of component profiling in Cuban traditional and industrial Pru using DESI-MS and FTICR MS/MS, and reveals the potential application of Pru as a health-promoting agent.

Lysophosphatidic acid precursor levels decrease and an arachidonic acid-containing phosphatidylcholine level increases in the dorsal root ganglion of mice after peripheral nerve injury.

In the current study, we aimed to analyze the lipid changes in the dorsal root ganglion (DRG) after sciatic nerve transection (SNT) using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). We found that the arachidonic acid-containing phosphatidylcholine (AA-PC), PC(16:0/20:4) largely increased, while PC(16:0/18:1), PC(18:0/18:1) and phosphatidic acid (PA)(36:2) levels largely decreased in the DRG following nerve injury. Previous studies show that the increase in PC(16:0/20:4) was associated with neuropathic pain and that decrease in PC(16:0/18:1), PC(18:0/18:1), and PA(36:2) were due to producing lysophosphatidic acid (LPA), an initiator for neuropathic pain. These results suggest that the lipid changes in DRG after SNT could be the result of changes for the cause of neuropathic pain. Thus, blocking of LPA could be potential for treatment of neuropathic pain.

A power law distribution of metabolite abundance levels in mice regardless of the time and spatial scale of analysis.

Biomolecule abundance levels change with the environment and enable a living system to adapt to the new conditions. Although, the living system maintains at least some characteristics, e.g. homeostasis. One of the characteristics maintained by a living system is a power law distribution of biomolecule abundance levels. Previous studies have pointed to a universal characteristic of biochemical reaction networks, with data obtained from lysates of multiple cells. As a result, the spatial scale of the data related to the power law distribution of biomolecule abundance levels is not clear. In this study, we researched the scaling law of metabolites in mouse tissue with a spatial scale of quantification that was changed stepwise between a whole-tissue section and a single-point analysis (25 µm). As a result, metabolites in mouse tissues were found to follow the power law distribution independently of the spatial scale of analysis. Additionally, we tested the temporal changes by comparing data from younger and older mice. Both followed similar power law distributions, indicating that metabolite composition is not diversified by aging to disrupt the power law distribution. The power law distribution of metabolite abundance is thus a robust characteristic of a living system regardless of time and space.