Increase in Cellular Lysophosphatidylserine Content Exacerbates Inflammatory Responses in LPS-Activated Microglia.

Mutations in alpha/beta-hydrolase domain containing (ABHD) 12 gene, which encodes lysophosphatidylserine (LysoPS) lipase, cause the neurodegenerative disease PHARC (Polyneuropathy, Hearing loss, Ataxia, Retinitis pigmentosa, Cataract). Since ABHD12 is expressed by microglia in the central nervous system and is localized to the endoplasmic reticulum, accumulation of intracellular LysoPS by ABHD12 mutations is assumed to be one of the pathological mechanisms associated with microglial activation in PHARC. However, the role of microglia in the PHARC brain and the relationship between microglial function and cellular LysoPS content remains unclear. Therefore, we explored the influence of cellular LysoPS content in microglial inflammatory responses. We evaluated the effects of inhibitors of cellular LysoPS metabolism, KC01 and DO-264, on inflammatory responses using a lipopolysaccharide (LPS)-stimulated mouse microglial cell line, BV-2 and primary microglia. Treatment of DO-264, an inhibitor of cellular LysoPS degradation, enhanced LPS-induced phagocytosis concomitant with the increase in cellular LysoPS content in BV-2 cells. On the other hand, treatment with KC01, an agent had been developed as an inhibitor of LysoPS synthase, reduced phagocytosis without affecting cellular LysoPS content. Such effects of both inhibitors on phagocytosis were also confirmed using primary microglia. KC01 treatment decreased nitric oxide (NO) production, accompanied by a reduction in inducible NO synthase expression in BV-2 microglia. KC01 also suppressed LPS-induced generation of intracellular reactive oxygen species and cytokines such as interleukin-6. Our results suggest that increase in cellular LysoPS levels can exacerbate microglial inflammatory responses. Treatment to prevent the increase in cellular LysoPS in microglia may have therapeutic potential for PHARC.

Zinc Potentiates Lipopolysaccharide-induced Nitric Oxide Production in Cultured Primary Rat Astrocytes.

Zn2+ plays a crucial role in the CNS where it accumulates in synaptic vesicles and is released during neurotransmission. Synaptically released Zn2+ is taken up by neurons and astrocytes. The majority of previous work has focused on neuronal damage caused by excess Zn2+. However, its effect on astrocyte function is not well understood. We examined the effect of extracellularly applied Zn2+ on nitric oxide (NO) production in primary cultured rat astrocytes, which were experimentally activated by lipopolysaccharide (LPS). Zn2+, at a concentration up to 125 µM, augmented LPS-induced NO production without affecting cell viability. LPS induced expression of both mRNA and protein of inducible NO synthase; this expression was enhanced by 125 µM Zn2+. Zn2+ also increased LPS-induced production of intracellular reactive oxygen species. Zn2+ enhanced the phosphorylation of p38-mitogen-activated protein kinase (MAPK) at 1-6 h after LPS treatment. The LPS-induced nuclear factor-kappaB (NFκB) activation was sustained for 6 h by Zn2+. Intracellular Zn2+ chelation with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) or inhibition of p38-MAPK diminished the Zn2+ enhancement of LPS-induced NO production. These findings suggest that activation of MAPK and NFκB is important for mediating Zn2+enhancement of LPS-induced NO production in astrocytes. Such changes may exacerbate glial and neuronal damage during neuroinflammation.

Microglia Endocytose Amyloid ß Through the Binding of Transglutaminase 2 and Milk Fat Globule EGF Factor 8 Protein.

Activation of glial cells has been observed in neurodegenerative diseases including Alzheimer's disease (AD). Aggregation of amyloid ß (Aß) is profusely observed as characteristic pathology in AD brain. In our previous study using microglial cell line BV-2, tissue-type transglutaminase (TG2) was found to be involved in phagocytosis (Kawabe et al., in Neuroimmunomodulation 22(4):243-249, 2015; Kawabe et al., Neurochem Res 2017). In the present study, we examined whether TG2 and milk fat globule EGF factor 8 protein (MFG-E8), an adaptor protein promotes macrophage to engulf apoptotic cells, were involved in Aß endocytosis. When the neuronal/glial mixed culture was stimulated freshly prepared Aß1-42 for 3 days, the incorporation of Aß was observed by immunofluorescence staining technique in Iba-1-positive microglia. Cystamine, a broad competitive inhibitor of TGs, suppressed it. When aggregated Aß was added to the mixed culture, the immunoreactivity of MFG-E8 surrounding Aß was observed, and then followed by microglial endocytosis. Using western blotting technique, MFG-E8 was detected in cell lysate of astrocyte culture, and was also detected in the medium. When microglia culture was incubated with astrocyte conditioned medium, MFG-E8 levels in microglia tended to increase. It is likely that microglia might utilize MFG-E8 released from astrocytes as well as that expressed in themselves in order to endocytose Aß aggregation. Furthermore, we confirmed that MFG-E8 could bind with TG2 in microglia culture by immunoprecipitate technique. These results suggest that microglia might uptake Aß as a complex of aggregated Aß/MFG-E8/TG2.

Inhibition of Gap Junction Elevates Glutamate Uptake in Cultured Astrocytes.

Glutamate uptake is a main function of astrocytes to keep extracellular glutamate levels low and protect neurons against glutamate-induced excitotoxicity. On the other hand, astrocyte networks formed by gap junctions, which are consisted with connexins and connecting neighboring cells, are reported to play a critical role in maintaining the homeostasis in the brain. In the present study, we examined the effects of gap junction inhibitors on the glutamate uptake activity in cultured rat cortical astrocytes. At first, we confirmed the effects of gap junction inhibitors, 1-octanol and carbenoxolone, on cell-cell communication by the scrape-loading assay using a fluorescent dye Lucifer yellow. Both of 1-octanol and carbenoxolone treatments for 20 min in cultured astrocytes significantly suppressed the cell-cell communication assessed as the distance of dye-spreading. 1-octanol and carbenoxolone increased the glutamate uptake by astrocytes and glutamate aspartate transporter (GLAST) expression on the cell membrane. These results suggest that gap junction inhibitors increase the glutamate uptake activity through the increase of GLAST proteins located on the cell membrane. The regulation of gap junction in astrocytes might protect neurons against glutamate-induced excitotoxicity.

Lauric Acid Alleviates Neuroinflammatory Responses by Activated Microglia: Involvement of the GPR40-Dependent Pathway.

In several neurodegenerative diseases such as Alzheimer's disease (AD), microglia are hyperactivated and release nitric oxide (NO) and proinflammatory cytokines, resulting its neuropathology. Mounting evidence indicates that dietary supplementation with coconut oil (CNO) reduces the cognitive deficits associated with AD; however, the precise mechanism(s) underlying the beneficial effect of CNO are unknown. In the present study, we examined the effects of lauric acid (LA), a major constituent of CNO, on microglia activated experimentally by lipopolysaccharide (LPS), using primary cultured rat microglia and the mouse microglial cell line, BV-2. LA attenuated LPS-stimulated NO production and the expression of inducible NO synthase protein without affecting cell viability. In addition, LA suppressed LPS-induced reactive oxygen species and proinflammatory cytokine production, as well as phosphorylation of p38-mitogen activated protein kinase and c-Jun N-terminal kinase. LA-induced suppression of NO production was partially but significantly reversed in the presence of GW1100, an antagonist of G protein-coupled receptor (GPR) 40, which is an LA receptor on the plasma membrane. LA also decreased LPS-induced phagocytosis, which was completely reversed by co-treatment with GW1100. Moreover, LA alleviated amyloid-ß-induced enhancement of phagocytosis. These results suggest that attenuation of microglial activation by LA may occur via the GPR40-dependent pathway. Such effects of LA may reduce glial activation and the subsequent neuronal damage in AD patients who consume CNO.

Transglutaminases Derived from Astrocytes Accelerate Amyloid ß Aggregation.

Activation of astrocytes has been observed in neurodegenerative diseases including Alzheimer's disease (AD). Transglutaminase (TG) is a crosslinking enzyme and contributes to cell adhesion, cytoskeleton construct, extracellular matrix formation, and so on. One of the isozymes, tissue-type TG (TG2) is reported to be activated in AD. Moreover, amyloid ß1-42 (Aß), which is aggregated and the aggregation is detected as characteristic pathology in AD brain, is known to be a substrate of TG2. However, contribution and derivation of TGs in brain for Aß aggregation remain to be clarified. In the present study, we examined the effects of cultured astrocytes prepared from rat embryonic brain cortex on Aß aggregation. When freshly prepared Aß was added to cultured astrocytes for 7 days, Aß monomer decreased and Aß oligomer unchanged. On the other hand, when Aß monomer was diluted with astrocytes conditioned medium, Aß oligomer increased time-dependently, and an inhibitor of TGs, cystamine, blocked it. Furthermore, when cultured astrocytes were stimulated with aggregated Aß, TG2 expression significantly increased. These results suggest that astrocytes could uptake Aß monomer to eliminate from brain; however, TGs derived from astrocytes might accelerate Aß aggregation and the aggregated Aß might enhance TG2 in astrocytes as a vicious cycle in pathological conditions. Adequate control of TGs expression and function in astrocytes would be an important factor in AD pathology.

Amphotericin B Increases Transglutaminase 2 Expression Associated with Upregulation of Endocytotic Activity in Mouse Microglial Cell Line BV-2.

Amphotericin B (AmB), a polyene antibiotic, is reported to cause the microglial activation to induce nitric oxide (NO) production and proinflammatory cytokines expression, and change neurotrophic factors expression in cultured microglia (Motoyoshi et al. in Neurochem Int 52:1290-1296, 2008). On the other hand, tissue-type transglutaminase (TG2) is involved in connection to phagocytes with apoptotic cells. Engulfment of neurons by activated microglia is thought to cause neurodegenerative diseases but detail is unclear, and involvement of TG2 in phagocytosis has been reported in our previous study using lipopolysaccharide-stimulated BV-2 cells (Kawabe et al. in Neuroimmunomodulation 22(4):243-249, 2015). In the present study, we examined the changes of TG2 expression, phagocytosis and pinocytosis in BV-2 cells stimulated by AmB. AmB stimulation increased TG2 expression and TG activity. Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were also up-regulated by AmB stimulation in BV-2 cells. Blockade of TG activity by cystamine, an inhibitor of TGs, suppressed AmB-enhanced TG2 expression, TG activity, NO production, phagocytosis and pinocytosis. Excessive NO production from microglia and/or facilitation of phagocytosis might be involved in neuronal death. To control TG activity might make possible to protect neurons and care for CNS diseases.

Mechanism for increased hepatic glycerol synthesis in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mouse: Urine glycerol and glycerol 3-phosphate as potential diagnostic markers of human citrin deficiency.

The mitochondrial aspartate-glutamate carrier isoform 2 (citrin) and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse has been a useful model of human citrin deficiency. One of the most prominent findings has been markedly increased hepatic glycerol 3-phosphate (G3P) following oral administration of a sucrose solution. We aimed to investigate whether this change is detectable outside of the liver, and to explore the mechanism underlying the increased hepatic G3P in these mice. We measured G3P and its metabolite glycerol in plasma and urine of the mice under various conditions. Glycerol synthesis from fructose was also studied using the liver perfusion system. The citrin/mGPD double-knockout mice showed increased urine G3P and glycerol under normal, fed conditions. We also found increased plasma glycerol under fasted conditions, while oral administration of different carbohydrates or ethanol led to substantially increased plasma glycerol. Fructose infusion to the perfused liver of the double-knockout mice augmented hepatic glycerol synthesis, and was accompanied by a concomitant increase in the lactate/pyruvate (L/P) ratio. Co-infusion of either pyruvate or phenazine methosulfate, a cytosolic oxidant, with fructose corrected the high L/P ratio, leading to reduced glycerol synthesis. Overall, these findings suggest that hepatic glycerol synthesis is cytosolic NADH/NAD(+) ratio-dependent and reveal a likely regulatory mechanism for hepatic glycerol synthesis following a high carbohydrate load in citrin-deficient patients. Therefore, urine G3P and glycerol may represent potential diagnostic markers for human citrin deficiency.

Acetate Attenuates Lipopolysaccharide-Induced Nitric Oxide Production Through an Anti-Oxidative Mechanism in Cultured Primary Rat Astrocytes.

The biomolecule acetate can be utilized for energy production, lipid synthesis, and several metabolic processes. Acetate supplementation reduces neuroglial activation in a model of neuroinflammation induced by intraventricular injection of lipopolysaccharide (LPS). To investigate the mechanisms underlying the anti-inflammatory effect of acetate on glial cells, we examined the effect of acetate on nitric oxide (NO) production, which was experimentally activated by LPS, in cultured primary rat astrocytes. Acetate attenuated the LPS-induced NO production in a dose-dependent manner, although cell viability was not affected. Acetate suppressed the phosphorylation of p38-mitogen-activated protein kinase 24 h after LPS treatment. Acetate decreased the LPS-induced production of intracellular reactive oxygen species (ROS) at 4-24 h concomitant with an increase in glutathione. Acetate rescued astrocytes from the hydrogen peroxide-induced cell death by reducing ROS levels. These findings suggest that attenuation of NO production by acetate may alleviate glial cell damage during neuroinflammation. Acetate may offer a glioprotective effect through an anti-oxidative mechanism.

Lipopolysaccharide-Stimulated Transglutaminase 2 Expression Enhances Endocytosis Activity in the Mouse Microglial Cell Line BV-2.

OBJECTIVES: In peripheral macrophages, tissue-type transglutaminase (TG2) is reported to be involved in phagocytosis of apoptotic cells. However, the contribution of TG2 to microglial phagocytosis has not been investigated. In this study, using a microglial cell line, BV-2, we examined the changes in TG2 expression, phagocytosis and pinocytosis in cells stimulated by lipopolysaccharide (LPS). METHODS: Cells of the mouse microglial cell line BV-2 were stimulated by LPS with or without cystamine, an inhibitor of TG enzyme activity, for 24 h. TG2 expression was measured by real-time RT-PCR and Western blotting. TG activity was evaluated using biotinylated pentylamine as a substrate. Pinocytosis was determined by uptake of 1-µm fluorescent microbeads. Phagocytosis was assessed by uptake of dead cells, human neuroblastoma SH-SY5Y cells, which were pretreated with H2O2 for 24 h. RESULTS: Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were up-regulated by LPS stimulation together with TG2 expression. Blockade of TG enzyme activity by cystamine suppressed TG2 expression, phagocytosis and pinocytosis. CONCLUSIONS: These results suggested that LPS-induced TG2 was involved in the mechanism of pinocytosis and phagocytosis in microglia.

Theophylline potentiates lipopolysaccharide-induced NO production in cultured astrocytes.

Elucidation of the functions of astrocytes is important for understanding of the pathogenic mechanism of various neurodegenerative diseases. Theophylline is a common drug for bronchial asthma and occasionally develops side-effects, such as acute encephalopathy; although the pathogenic mechanism of the side-effects is unknown. The lipopolysaccharide (LPS)-induced nitricoxide (NO) production is generally used for an index of the activation of astrocyte in vitro. In this study, in order to elucidate the effect of theophylline on the astrocytic functions, we examined the LPS-induced NO production and the expression of iNOS in cultured rat cortex astrocytes.Theophylline alone could not induce the NO production; however, NO production induced by LPS was enhanced by theophylline in a dose-dependent manner; and by isobutylmethylxanthine, a phosphodiesterase inhibitor. The theophylline enhancement of LPS-induced NO production was further increased by dibutyryl cyclic AMP, a membrane-permeable cAMP analog; and by forskolin, an adenylate cyclase activator. When the cells were preincubated with Rp-8-Br-cAMP, an inhibitor of protein kinase A, the theophylline enhancement of LPS-induced NO production was decreased. The extent of iNOS protein expression induced by LPS was also enhanced by theophylline.It is likely that phosphodiesterase inhibition is a major action mechanism for the theophylline enhancement of LPS-induced NO production in astrocytes. Theophylline-induced acute encephalopathy might be due to the hyper-activation of astrocytes via cAMP signaling to produce excess amount of NO.

Extracellular superoxide dismutase induced by dopamine in cultured astrocytes.

Under some pathological conditions in brain, a large amount of superoxide anion (O(2)(-)) is produced, causing various cellular damages. Among three isozymes of superoxide dismutase (SOD), extracellular (EC)-SOD should play a role to detoxify O(2)(-) in extracellular space; however, a little is known about EC-SOD in brain. Although dopamine (DA) stored in the synaptic vesicle is stable, the excess leaked DA is spontaneously oxidized to yield O(2)(-) and reactive DA quinones, causing damages of dopaminergic neurons. In the present study, we examined the effects of DA on SOD expression in cultured rat cortical astrocytes. By means of RT-PCR, all mRNA of three isozymes of SOD could be detected; however, only EC-SOD was increased by DA exposure for 24 h, dose-dependently. The expression of EC-SOD protein and the cell-surface SOD activity in astrocytes also increased with 100 µM DA exposure. The increase of EC-SOD mRNA by DA was inhibited by a DA transporter inhibitor, GBR12909, whereas it was not changed by DA receptor antagonists, SKF-83566 (D1) and haloperidol (D2). Furthermore, a monoamine oxidase inhibitor, pargyline, and antioxidants, N-acetyl-L-cysteine and glutathione, also did not affect the DA-induced expression of EC-SOD mRNA. On the other hand, an inhibitor of nuclear factor kappaB (NF-κB), ammonium pyrrolidine-1-carbodithioate, suppressed the DA-induced expression of EC-SOD mRNA. These results suggest that DA incorporated into the cells caused the induction of EC-SOD mRNA followed by the enhancements of EC-SOD protein level and the enzyme activity, and that NF-κB activation is involved in the mechanisms of the EC-SOD induction. The regulation of EC-SOD in astrocytes surrounding dopaminergic neurons may contribute to the defensive mechanism against oxidative stress in brain.

Extracellular superoxide dismutase in cultured astrocytes: decrease in cell-surface activity and increase in medium activity by lipopolysaccharide-stimulation.

Under pathological conditions such as ischemia/reperfusion, a large amount of superoxide anion (O(2) (-)) is produced and released in brain. Among three isozymes of superoxide dismutase (SOD), extracellular (EC)-SOD, known to be excreted outside cells and bound to extracellular matrix, should play a role to detoxify O(2) (-) in extracellular space; however, a little is known about EC-SOD in brain. In order to evaluate the SOD activity in extracellular space of CNS as direct as possible, we attempted to measure the cell-surface SOD activity on primary cultured rat brain cells by the inhibition of color development of a water-soluble tetrazolium due to O(2) (-) generation by xanthine oxidase/hypoxanthine added into extracellular medium of intact cells. The cell-surface SOD activity on cultured neuron and microglia was below the detection limit; however, that on cultured astrocyte was high enough to measure. By means of RT-PCR, all mRNA of three isozymes of SOD could be detected in the three types of the cells examined; however, the semi-quantitative analysis revealed that the level of EC-SOD mRNA in astrocytes was significantly higher than that in neurons and microglia. When astrocytes were stimulated with lipopolysaccharide (LPS) for 12-24 h, the cell-surface SOD activity decreased to a half, whereas the activity recovered after 36-48 h. The decrease in the activity was dependent on the LPS concentration. On the other hand, the SOD activity in the medium increased by the LPS-stimulation in a dose dependent manner; suggesting that the SOD protein localized on cell-surface, probably EC-SOD, was released into the medium. These results suggest that EC-SOD of astrocyte play a role for detoxification of extracellular O(2) (-) and the regulation of EC-SOD in astrocytes may contribute to the defensive mechanism against oxidative stress in brain.

Imipramine induces brain-derived neurotrophic factor mRNA expression in cultured astrocytes.

Depression is one of the most prevalent and livelihood-threatening forms of mental illnesses and the neural circuitry underlying depression remains incompletely understood. Recent studies suggest that the neuronal plasticity involved with brain-derived neurotrophic factor (BDNF) plays an important role in the recovery from depression. Some antidepressants are reported to induce BDNF expression in vivo; however, the mechanisms have been considered solely in neurons and not fully elucidated. In the present study, we evaluated the effects of imipramine, a classic tricyclic antidepressant drug, on BDNF expression in cultured rat brain astrocytes. Imipramine dose-dependently increased BDNF mRNA expression in astrocytes. The imipramine-induced BDNF increase was suppressed with inhibitors for protein kinase A (PKA) or MEK/ERK. Moreover, imipramine exposure activated transcription factor cAMP response element binding protein (CREB) in a dose-dependent manner. These results suggested that imipramine induced BDNF expression through CREB activation via PKA and/or ERK pathways. Imipramine treatment in depression might exert antidepressant action through BDNF production from astrocytes, and glial BDNF expression might be a target of developing novel antidepressants.

Acetate Suppresses Lipopolysaccharide-stimulated Nitric Oxide Production in Primary Rat Microglia but not in BV-2 Microglia Cells.

AIMS: To show that acetate attenuates neuroinflammatory responses in activated microglia. BACKGROUND: Dietary acetate supplementation alleviates neuroglial activation in a rat model of neuroinflammation induced by intraventricular administration of lipopolysaccharide (LPS). However, the precise mechanism(s) underlying the anti-inflammatory effect of acetate, is not fully understood. OBJECTIVE: To determine whether acetate has inhibitory effects on LPS-induced neuroinflammatory responses in microglia. METHODS: We examined LPS-stimulated nitric oxide (NO) production in primary rat microglia and BV-2 cells. Protein expression of inducible NO synthase (iNOS) was determined by western blot analysis. The intracellular generation of reactive oxygen species (ROS) and glutathione (GSH) were also evaluated. RESULTS: In primary microglia, acetate decreased LPS-stimulated NO production in a dose-dependent manner, reaching significance at greater than 10 mM, and cell viability was not affected. Acetate suppressed LPS-induced expression of iNOS protein concomitantly with the decrease in NO. The LPS-induced increase in intracellular ROS production was attenuated by acetate. In addition, acetate prevented LPS-induced reduction of GSH. Notably, such suppressive effects of acetate on NO and ROS production were not observed in BV-2 cells. CONCLUSION: These findings suggest that acetate may alleviate neuroinflammatory responses by attenuating NO and ROS production in primary microglia but not in BV-2 cells. Other: All animals received humane care, and the animal protocols used in this study were approved by the Ethics Committees for Animal Experimentation.

Extracellular Vesicles Derived From Canine Mesenchymal Stromal Cells in Serum Free Culture Medium Have Anti-inflammatory Effect on Microglial Cells.

Mesenchymal stem/stromal cells (MSCs) have been used as cell sources for treating dogs with naturally-occurring diseases. Extracellular vesicles (EVs) derived from MSCs are now recognized as pivotal to modulating the immune response and supporting tissue repair. Manufacture of MSC-EVs for clinical application mandates removal of the xeno-proteins, including fetal bovine serum. The objective of this study was to examine whether canine MSCs survived and secreted EVs in serum-free medium (SFM) conditions and to assess the immunomodulatory effect of EVs in vitro. Canine MSCs were found to survive and secrete EVs under SFM conditions. The surface markers of MSCs in the SFM were similar to MSCs in complete culture medium. Canine MSC-EVs had a diameter of ~300 nm and were positive for EV markers. MSC-derived EVs from the serum-free condition reduced the levels of IL-1ß by BV-2 cells in response to LPS stimulation. These results warrant further studies of the use of SFM for producing EVs derived from canine MSCs.

Lysophosphatidylinositol, an Endogenous Ligand for G Protein-Coupled Receptor 55, Has Anti-inflammatory Effects in Cultured Microglia.

Lysophosphatidylinositol (LysoPI), an endogenous ligand for G protein-coupled receptor (GPR) 55, has been known to show various functions in several tissues and cells; however, its roles in the central nervous system (CNS) are not well known. In particular, the detailed effects of LysoPI on microglial inflammatory responses remain unknown. Microglia is the immune cell that has important functions in maintaining immune homeostasis of the CNS. In this study, we explored the effects of LysoPI on inflammatory responses using the mouse microglial cell line BV-2, which was stimulated with lipopolysaccharide (LPS), and some results were confirmed also in rat primary microglia. LysoPI was found to reduce LPS-induced nitric oxide (NO) production and inducible NO synthase protein expression without affecting cell viability in BV-2 cells. LysoPI also suppressed intracellular generation of reactive oxygen species both in BV-2 cells and primary microglia and cytokine release in BV-2 cells. In addition, LysoPI treatment decreased phagocytic activity of LPS-stimulated BV-2 cells and primary microglia. The GPR55 antagonist CID16020046 completely inhibited LysoPI-induced downregulation of phagocytosis in BV-2 microglia, but did not affect the LysoPI-induced decrease in NO production. Our results suggest that LysoPI suppresses microglial phagocytosis via a GPR55-dependent pathway and NO production via a GPR55-independent pathway. LysoPI may contribute to neuroprotection in pathological conditions such as brain injury or neurodegenerative diseases, through its suppressive role in the microglial inflammatory response.

Deletion of SERP1/RAMP4, a component of the endoplasmic reticulum (ER) translocation sites, leads to ER stress.

Stress-associated endoplasmic reticulum (ER) protein 1 (SERP1), also known as ribosome-associated membrane protein 4 (RAMP4), is a Sec61-associated polypeptide that is induced by ER stress. SERP1-/- mice, made by targeted gene disruption, demonstrated growth retardation, increased mortality, and impaired glucose tolerance. Consistent with high levels of SERP1 expression in pancreas, pancreatic islets from SERP1-/- mice failed to rapidly synthesize proinsulin in response to a glucose load. In addition, reduced size and enhanced ER stress were observed in the anterior pituitary of SERP1-/- mice, and growth hormone production was slowed in SERP1-/- pituitary after insulin stimulation. Experiments using pancreatic microsomes revealed aberrant association of ribosomes and the Sec61 complex and enhanced ER stress in SERP1-/- pancreas. In basal conditions, the Sec61 complex in SERP1-/- microsomes was more cofractionated with ribosomes, compared with SERP1+/+ counterparts, in high-salt conditions. In contrast, after glucose stimulation, the complex showed less cofractionation at an early phase (45 min) but more at a later phase (120 min). Although intracellular insulin/proinsulin levels were not significantly changed in both genotypes, these results suggest that subtle changes in translocation efficiency play an important role in the regulation of ER stress and rapid polypeptide synthesis.

Pivotal role of inter-organ aspartate metabolism for treatment of mitochondrial aspartate-glutamate carrier 2 (citrin) deficiency, based on the mouse model.

Previous studies using citrin/mitochondrial glycerol-3-phosphate (G3P) dehydrogenase (mGPD) double-knockout mice have demonstrated that increased dietary protein reduces the extent of carbohydrate-induced hyperammonemia observed in these mice. This study aimed to further elucidate the mechanisms of this effect. Specific amino acids were initially found to decrease hepatic G3P, or increase aspartate or citrulline levels, in mGPD-knockout mice administered ethanol. Unexpectedly, oral glycine increased ammonia in addition to lowering G3P and increasing citrulline. Subsequently, simultaneous glycine-plus-sucrose (Gly + Suc) administration led to a more severe hyperammonemic state in double-KO mice compared to sucrose alone. Oral arginine, ornithine, aspartate, alanine, glutamate and medium-chain triglycerides all lowered blood ammonia following Gly + Suc administration, with combinations of ornithine-plus-aspartate (Orn + Asp) or ornithine-plus-alanine (Orn + Ala) suppressing levels similar to wild-type. Liver perfusion and portal vein-arterial amino acid differences suggest that oral aspartate, similar to alanine, likely activated ureagenesis from ammonia and lowered the cytosolic NADH/NAD+ ratio through conversion to alanine in the small intestine. In conclusion, Gly + Suc administration induces a more severe hyperammonemic state in double-KO mice that Orn + Asp or Orn + Ala both effectively suppress. Aspartate-to-alanine conversion in the small intestine allows for effective oral administration of either, demonstrating a pivotal role of inter-organ aspartate metabolism for the treatment of citrin deficiency.

Effects of Amphotericin B on the expression of neurotoxic and neurotrophic factors in cultured microglia.

Amphotericin B (AmB) is a polyene antibiotic and reported to have therapeutic effects on prion diseases, in which the microglial activation has been suggested to play important roles by proliferating and producing various factors such as nitric oxide, proinflammatory cytokines, and so on. However, the therapeutic mechanism of AmB on prion diseases remains elusive. In the present study, we investigated the effects of AmB on cellular functions of rat primary cultured microglia. We found that AmB, similarly as lipopolysaccharide (LPS), could activate microglia to produce nitric oxide via inducible nitric oxide synthase. Both AmB and LPS also induced mRNA expressions of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in microglia. AmB also changed the expression levels of neurotrophic factors mRNAs. AmB and LPS significantly down-regulated the level of ciliary neurotrophic factor mRNA. However, AmB, but not LPS, significantly up-regulated the level of glial cell-line derived neurotrophic factor mRNA in microglia. In addition, brain-derived neurotrophic factor mRNA expression level was tending upward by treatment with AmB, but not with LPS. Taken together, these results suggest that AmB regulates the microglial activation in different manner from LPS and that microglia may participate in the therapeutic effects of AmB on prion diseases by controlling the expression and production of such mediators.