Long term administration of loquat leaves and their major component, ursolic acid, attenuated endogenous amyloid-ß burden and memory impairment.

Loquat (Eriobotrya japonica) leaves contain many bioactive components such as ursolic acid (UA) and amygdalin. We investigated the effects of loquat leaf powder and methanol extract in human neuroglioma H4 cells stably expressing the Swedish-type APP695 (APPNL-H4 cells) and C57BL/6 J mice. Surprisingly, the extract greatly enhanced cellular amyloid-beta peptide (Aß) 42 productions in APPNL-H4 cells. Administration of leaf powder increased Aß42 levels after 3 months and decreased levels after 12 months compared to control mice. Leaf powder had no effect on working memory after 3 months, but improved working memory after 12 months. Administration of UA decreased Aß42 and P-tau levels and improved working memory after 12 months, similar to the administration of leave powder for 12 months. Amygdalin enhanced cellular Aß42 production in APPNL-H4 cells, which was the same as the extract. Three-month administration of amygdalin increased Aß42 levels slightly but did not significantly increase them, which is similar to the trend observed with the administration of leaf powder for 3 months. UA was likely the main compound contained in loquat leaves responsible for the decrease in intracerebral Aß42 and P-tau levels. Also, amygdalin might be one of the compounds responsible for the transiently increased intracerebral Aß42 levels.

Microglial depletion exacerbates axonal damage and motor dysfunction in mice with cuprizone-induced demyelination.

The cuprizone (CPZ)-induced demyelination model, an animal model of Multiple sclerosis (MS), is characterized by demyelination and motor dysfunction due to microglial-mediated neuroinflammation. To determine the contribution of microglia to motor function during CPZ-induced demyelination, the microglia of mice in the CPZ-model were depleted using PLX3397 (PLX), an orally bioavailable selective colony stimulating factor 1 receptor inhibitor. PLX treatment aggravated motor dysfunction as shown by the pole, beam walk, ladder walk, and rotarod tests. PLX treatment removed microglia from the superior cerebellar peduncle (SCP), but not from the corpus callosum (CC). Although PLX treatment did not affect the degree of demyelination in both of CC and SCP, the expression of axonal damage marker APP (amyloid precursor protein) was increased. Increased TNF-α, IL-1ß, and iNOS expressions were observed in PLX-treated mice. These results suggest that microglial depletion exacerbates axonal damage and motor dysfunction in CPZ model mice. In this study, we found that microglia contribute to motor function and axon-protective effects in CPZ-induced demyelination.

GPR137 Inhibits Cell Proliferation and Promotes Neuronal Differentiation in the Neuro2a Cells.

The orphan receptor, G protein-coupled receptor 137 (GPR137), is an integral membrane protein involved in several types of cancer. GPR137 is expressed ubiquitously, including in the central nervous system (CNS). We established a GPR137 knockout (KO) neuro2A cell line to analyze GPR137 function in neuronal cells. KO cells were generated by genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and cultured as single cells by limited dilution. Rescue cells were then constructed to re-express GPR137 in GPR137 KO neuro2A cells using an expression vector with an EF1-alpha promoter. GPR137 KO cells increased cellular proliferation and decreased neurite outgrowth (i.e., a lower level of neuronal differentiation). Furthermore, GPR137 KO cells exhibited increased expression of a cell cycle regulator, cyclin D1, and decreased expression of a neuronal differentiation marker, NeuroD1. Additionally, GPR137 KO cells exhibited lower expression levels of the neurite outgrowth markers STAT3 and GAP43. These phenotypes were all abrogated in the rescue cells. In conclusion, GPR137 deletion increased cellular proliferation and decreased neuronal differentiation, suggesting that GPR137 promotes cell cycle exit and neuronal differentiation in neuro2A cells. Regulation of neuronal differentiation by GPR137 could be vital to constructing neuronal structure during brain development.

Suppressive Effect of Fruiting Bodies of Medicinal Mushrooms on Demyelination and Motor Dysfunction in a Cuprizone-Induced Multiple Sclerosis Mouse Model.

Epidemiologic studies have shown a high prevalence of multiple sclerosis (MS) in Europe and North America, and a low prevalence in East Asia. Mushrooms contain various biological response modifiers (BRMs) and are widely used in traditional Chinese medicine in East Asian countries. To investigate whether mushrooms have potential beneficial effects on MS, we administered mushrooms to cuprizone (bis-cyclohexanone-oxalyldihydrazone, CPZ)-induced MS model mice. This model is used to study the processes of demyelination in the CNS. The CPZ-induced demyelination is involved in the apoptotic death of mature oligodendrocytes, neuroinflammation, and motor dysfunction. Mice were fed a powdered diet containing 5% each mushroom and CPZ diet for 5 weeks, which coincides with peak demyelination. We measured the body weight of the mice, evaluated their motor function using a rotarod, and quantified the myelin levels using Black-Gold II staining. Ganoderma lucidum and Hericium erinaceus treatments showed recovery from weight loss. Pleurotus eryngii, G. lucidum, and Flammulina velutipes treatments significantly improved CPZ-induced motor dysfunction. P. eryngii, G. lucidum, F. velutipes, and H. erinaceus treatments effectively suppressed CPZ-induced demyelination. The four medicinal mushrooms may be promising BRMs for prevention and alleviation of the symptoms of MS.

Dietary oleic acid contributes to the regulation of food intake through the synthesis of intestinal oleoylethanolamide.

Introduction: Among the fatty acid ethanolamides (FAEs), oleoylethanolamide (OEA), linoleoylethanolamide (LEA), and palmitoylethanolamide (PEA) are reported to be involved in feeding regulation. In particular, OEA is well characterized as a satiety signal. Following food consumption, OEA is synthesized from oleic acid (OA) via an N-acyl phosphatidylethanolamine-specific phospholipase D-dependent pathway in the gastroenterocytes, and OEA induces satiety by recruiting sensory fibers. Thus, we hypothesized that dietary OA is an important satiety-inducing molecule. However, there has been no direct demonstration of the effect of dietary OA on satiety induction without the influence of the endogenous biosynthesis of OA from stearic acid (SA) or other FAEs. Methods: In this study, we used two experimental diets to test our hypothesis: (i) an OA diet (OAD; 38.4 mg of OA/g and 7.2 mg of SA/g) and (ii) a low OA diet (LOAD; 3.1 mg of OA/g and 42.4 mg of SA/g). Results: Relative to mice fed the OAD, mice fed the LOAD for two weeks exhibited reduced levels of jejunal OEA but not jejunal LEA and PEA. The LOAD-fed mice showed an increase in food intake and body weight gain. Moreover, LOAD-induced increase in food intake was immediately observed after the switch from the OAD, whereas these effects were diminished by the switch back to the OAD. Furthermore, treatment with OA and OEA diminished the effects of LOAD on food intake. Conclusion: Collectively, these results show that dietary OA is a key factor in the reduction of food intake and increase in satiety mediated by OEA signaling.

A peripheral lipid sensor GPR120 remotely contributes to suppression of PGD2-microglia-provoked neuroinflammation and neurodegeneration in the mouse hippocampus.

BACKGROUND: Neuroinflammation is a key pathological component of neurodegenerative disease and is characterized by microglial activation and the secretion of proinflammatory mediators. We previously reported that a surge in prostaglandin D2 (PGD2) production and PGD2-induced microglial activation could provoke neuroinflammation. We also reported that a lipid sensor GPR120 (free fatty acid receptor 4), which is expressed in intestine, could be activated by polyunsaturated fatty acids (PUFA), thereby mediating secretion of glucagon-like peptide-1 (GLP-1). Dysfunction of GPR120 results in obesity in both mice and humans. METHODS: To reveal the relationship between PGD2-microglia-provoked neuroinflammation and intestinal PUFA/GPR120 signaling, we investigated neuroinflammation and neuronal function with gene and protein expression, histological, and behavioral analysis in GPR120 knockout (KO) mice. RESULTS: In the current study, we discovered notable neuroinflammation (increased PGD2 production and microglial activation) and neurodegeneration (declines in neurogenesis, hippocampal volume, and cognitive function) in GPR120 KO mice. We also found that Hematopoietic-prostaglandin D synthase (H-PGDS) was expressed in microglia, microglia were activated by PGD2, H-PGDS expression was upregulated in GPR120 KO hippocampus, and inhibition of PGD2 production attenuated this neuroinflammation. GPR120 KO mice exhibited reduced intestinal, plasma, and intracerebral GLP-1 contents. Peripheral administration of a GLP-1 analogue, liraglutide, reduced PGD2-microglia-provoked neuroinflammation and further neurodegeneration in GPR120 KO mice. CONCLUSIONS: Our results suggest that neurological phenotypes in GPR120 KO mice are probably caused by dysfunction of intestinal GPR120. These observations raise the possibility that intestinal GLP-1 secretion, stimulated by intestinal GPR120, may remotely contributed to suppress PGD2-microglia-provoked neuroinflammation in the hippocampus.

2-Carba-lysophosphatidic acid is a novel ß-lysophosphatidic acid analogue with high potential for lysophosphatidic acid receptor activation and autotaxin inhibition.

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator that, along with its chemically stabilized analogue 2-carba-cyclic phosphatidic acid (2ccPA), induces various biological activities in vitro and in vivo. Although cPA is similar to lysophosphatidic acid (LPA) in structure and synthetic pathway, some of cPA biological functions apparently differ from those reported for LPA. We previously investigated the pharmacokinetic profile of 2ccPA, which was found to be rapidly degraded, especially in acidic conditions, yielding an unidentified compound. Thus, not only cPA but also its degradation compound may contribute to the biological activity of cPA, at least for 2ccPA. In this study, we determined the structure and examined the biological activities of 2-carba-lysophosphatidic acid (2carbaLPA) as a 2ccPA degradation compound, which is a type of ß-LPA analogue. Similar to LPA and cPA, 2carbaLPA induced the phosphorylation of the extracellular signal-regulated kinase and showed potent agonism for all known LPA receptors (LPA1-6) in the transforming growth factor-α (TGFα) shedding assay, in particular for LPA3 and LPA4. 2carbaLPA inhibited the lysophospholipase D activity of autotaxin (ATX) in vitro similar to other cPA analogues, such as 2ccPA, 3-carba-cPA, and 3-carba-LPA (α-LPA analogue). Our study shows that 2carbaLPA is a novel ß-LPA analogue with high potential for the activation of some LPA receptors and ATX inhibition.

Feeding regulation by oleoylethanolamide synthesized from dietary oleic acid.

Oleoylethanolamide (OEA), a well-known satiety factor, is produced during feeding in the proximal intestine. Enterocytes sense oleic acid in dietary fat via CD36 and convert it to OEA through NAPE-PLD dependent or independent pathways. The satiety function of OEA is known to involve peroxisome proliferator-activated receptor type-α (PPAR-α). OEA stimulates afferent sensory fibers (possibly those of the vagus nerve) and provoke the recruitment of feeding-controlling circuits in the brain that use oxytocin and histamine as neurotransmitters for regulating satiety. Dysfunction of OEA synthesis by high-fat feeding might contribute to increased weight and obesity. Here, we describe the roles played by OEA in the regulation of energy metabolism and food intake by introducing our preliminary data regarding this lipid mediator, and we briefly outline the biosynthesis and deactivation of OEA.

Ursolic acid treatment suppresses cuprizone-induced demyelination and motor dysfunction via upregulation of IGF-1.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system, characterized by apoptotic death of mature oligodendrocytes, neuroinflammation, and motor dysfunction. A pentacyclic triterpenoid compound, ursolic acid (UA), has various pharmacological activities, such as anti-inflammatory, anti-oxidative, and anti-apoptotic effects. In the present study, we investigated the effects of UA on cuprizone-induced demyelination, which is a model of MS. Oral administration of UA effectively suppressed cuprizone-induced demyelination and motor dysfunction via the enhancement of IGF-1 levels in the demyelinating lesions. Our results suggest that UA might be therapeutically useful for demyelination in MS.

Evaluation of the pharmacokinetics of 2-carba-cyclic phosphatidic acid by liquid chromatography-triple quadrupole mass spectrometry.

Cyclic phosphatidic acid (cPA) is a lysophospholipid mediator that suppresses cancer metastasis and osteoarthritis. It also has neuroprotective roles in diseases such as multiple sclerosis and delayed neuronal death following transient ischemia. In order to take advantage of the properties of cPA for the development of new therapeutic strategies, we have synthesized several cPA derivatives and discovered 2-carba-cPA (2ccPA) as a promising candidate. To develop 2ccPA as a therapeutic agent, we investigated the pharmacokinetic profile of 2ccPA by liquid chromatography-triple quadrupole mass spectrometry in this study. When 2ccPA was administered intraperitoneally to mice at a dose of 1.6 mg/kg, the half-life of 2ccPA in plasma was 16 min. The 2ccPA, dosed intraperitoneally to mice at 16 mg/kg, distributed to each organ including brain at 20 min after dosing. It was found that 2ccPA was stable in neutral or alkaline conditions (e.g., intestine) but unstable in acidic conditions (e.g., stomach). When 2ccPA was orally administrated to rats as a gastro-resistant form using an enterosoluble capsule, plasma 2ccPA levels peaked at 2 h, slowly declined thereafter and persistently detected even at 10 h after administration. Here, we present the findings on the effect of the continuous release of 2ccPA from the capsule to reduce the lysophospholipase D activity and also decrease plasma levels of lysophosphatidic acid in rat. These findings will be useful in further studies for evaluating the application of 2ccPA in several disorders.

Correction to: Protective and therapeutic role of 2-carbacyclic phosphatidic acid in demyelinating disease.

After publication of the article [1], it was brought to our attention that an acknowledgement was missing from the original version.

Quantitative determination of cyclic phosphatidic acid and its carba analog in mouse organs and plasma using LC-MS/MS.

Cyclic phosphatidic acid (cPA), an analog of lysophosphatidic acid, is involved in the regulation of many cellular processes. A sensitive and specific method to quantify the molecular species of cPA is important for studying the physiological and pathophysiological roles of cPA. Here, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantification method for the simultaneous detection of cPA species having various fatty acids (16:0, 18:0, 18:1, and 18:2) as well as 2-carba-cPA, a chemically synthesized analog of cPA. Chromatography was performed using a reversed-phase C18 column. cPA species were detected using a triple quadrupole mass spectrometer. cPA 17:0 was used as an internal standard. Intra- and interday precision values (CV%) were within 10%. The linear range of detection for each cPA species was 0.01 µg/mL to 5 µg/mL, with correlation coefficients of 0.998 or higher. The developed method was applied to the quantification of cPA species in mouse plasma and organs. The concentrations of cPA 16:0, 18:0, and 18:1 were revealed to be significantly reduced in the brains of cuprizone-treated mice, a model of multiple sclerosis, compared with control mice. These findings could be important for understanding the roles of cPA in the neurodegenerative processes associated with multiple sclerosis.

The flavonoid Baicalein attenuates cuprizone-induced demyelination via suppression of neuroinflammation.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system characterized by recurrent and progressive demyelination/remyelination cycles, neuroinflammation, oligodendrocyte loss, and axonal pathology. Baicalein isolated from the roots of Scutellaria baicalensis has been shown to exert anti-inflammatory and antioxidant effects. The cuprizone model is an established mouse model of MS and causes demyelination and motor dysfunction and induces neuroinflammation, such as glial activation and pro-inflammatory cytokine production. To determine whether Baicalein attenuates cuprizone-induced demyelination, we administrated Baicalein to cuprizone-exposed mice. Baicalein attenuated weight loss (P<0.05) and motor dysfunction (P<0.05) in the cuprizone model mice. Baicalein treatment effectively suppressed the demyelination (P<0.01) and gene expressions of CNP (P<0.05) and MBP (P<0.05). Baicalein treatment also inhibited the cuprizone-induced increase in Iba1-positive microglia (P<0.001), GFAP-positive astrocytes (P<0.001), and the gene expressions of CD11b (P<0.01), GFAP (P<0.05), TNFα (P<0.05), IL-1ß (P<0.05), and iNOS (p<0.01). We found that Baicalein treatment attenuated cuprizone-induced demyelination, glial activation, pro-inflammatory cytokine expression, and motor dysfunction. Our results suggest that Baicalein may be a useful therapeutic agent in demyelinating diseases to suppress neuroinflammation.

Protective and therapeutic role of 2-carba-cyclic phosphatidic acid in demyelinating disease.

BACKGROUND: Multiple sclerosis is a neuroinflammatory demyelinating and neurodegenerative disease of the central nervous system characterized by recurrent and progressive demyelination/remyelination cycles, neuroinflammation, oligodendrocyte loss, demyelination, and axonal degeneration. Cyclic phosphatidic acid (cPA) is a natural phospholipid mediator with a unique cyclic phosphate ring structure at the sn-2 and sn-3 positions of the glycerol backbone. We reported earlier that cPA elicits a neurotrophin-like action and protects hippocampal neurons from ischemia-induced delayed neuronal death. We designed, chemically synthesized, and metabolically stabilized derivatives of cPA: 2-carba-cPA (2ccPA), a synthesized compound in which one of the phosphate oxygen molecules is replaced with a methylene group at the sn-2 position. In the present study, we investigated whether 2ccPA exerts protective effects in oligodendrocytes and suppresses pathology in the two most common mouse models of multiple sclerosis. METHODS: To evaluate whether 2ccPA has potential beneficial effects on the pathology of multiple sclerosis, we investigated the effects of 2ccPA on oligodendrocyte cell death in vitro and administrated 2ccPA to mouse models of experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination. RESULTS: We demonstrated that 2ccPA suppressed the CoCl2-induced increase in the Bax/Bcl-2 protein expression ratio and phosphorylation levels of p38MAPK and JNK protein. 2ccPA treatment reduced cuprizone-induced demyelination, microglial activation, NLRP3 inflammasome, and motor dysfunction. Furthermore, 2ccPA treatment reduced autoreactive T cells and macrophages, spinal cord injury, and pathological scores in EAE, the autoimmune multiple sclerosis mouse model. CONCLUSIONS: We demonstrated that 2ccPA protected oligodendrocytes via suppression of the mitochondrial apoptosis pathway. Also, we found beneficial effects of 2ccPA in the multiperiod of cuprizone-induced demyelination and the pathology of EAE. These data indicate that 2ccPA may be a promising compound for the development of new drugs to treat demyelinating disease and ameliorate the symptoms of multiple sclerosis.

Excitotoxicity-induced prostaglandin D2 production induces sustained microglial activation and delayed neuronal death.

Excitotoxicity is the pivotal mechanism of neuronal death. Prostaglandins (PGs) produced during excitotoxicity play important roles in neurodegenerative conditions. Previously, we demonstrated that initial burst productions of PGD2, PGE2, and PGF2α are produced by cyclooxygenase-2 (COX-2) in the hippocampus following a single systemic kainic acid (KA) administration. In addition, we showed that blocking of all PG productions ameliorated hippocampal delayed neuronal death at 30 days after KA administration. To investigate the role of individual PGs in the delayed neuronal death, we performed intracerebroventricular injection of PGD2, PGE2, or PGF2α in rats whose hippocampal PG productions were entirely blocked by pretreatment of NS398, a COX-2 selective inhibitor. Administration of PGD2 and PGF2α had a latent contribution to the delayed neuronal death, sustained over 30 days after a single KA treatment. Furthermore, PGD2 enhanced microglial activation, which may be involved in the delayed neuronal death in the hippocampus. These findings suggest that excitotoxic delayed neuronal death is mediated through microglia activated by PGD2.