Yoshiteru Kagawa

Inhibition of Fatty Acid Synthase Decreases Expression of Stemness Markers in Glioma Stem Cells

Cellular metabolic changes, especially to lipid metabolism, have recently been recognized as a hallmark of various cancer cells. However, little is known about the significance of cellular lipid metabolism in the regulation of biological activity of glioma stem cells (GSCs). In this study, we examined the expression and role of fatty acid synthase (FASN), a key lipogenic enzyme, in GSCs. In the de novo lipid synthesis assay, GSCs exhibited higher lipogenesis than differentiated non-GSCs. Western blot and immunocytochemical analyses revealed that FASN is strongly expressed in multiple lines of patient-derived GSCs (G144 and Y10), but its expression was markedly reduced upon differentiation. When GSCs were treated with 20 μM cerulenin, a pharmacological inhibitor of FASN, their proliferation and migration were significantly suppressed and de novo lipogenesis decreased. Furthermore, following cerulenin treatment, expression of the GSC markers nestin, Sox2 and fatty acid binding protein (FABP7), markers of GCSs, decreased while that of glial fibrillary acidic protein (GFAP) expression increased. Taken together, our results indicate that FASN plays a pivotal role in the maintenance of GSC stemness, and FASN-mediated de novo lipid biosynthesis is closely associated with tumor growth and invasion in glioblastoma.

Astrocyte-Expressed FABP7 Regulates Dendritic Morphology and Excitatory Synaptic Function of Cortical Neurons

Fatty acid binding protein 7 (FABP7) expressed by astrocytes in developing and mature brains is involved in uptake and transportation of fatty acids, signal transduction, and gene transcription. Fabp7 knockout (Fabp7 KO) mice show behavioral phenotypes reminiscent of human neuropsychiatric disorders such as schizophrenia. However, direct evidence showing how FABP7 deficiency in astrocytes leads to altered brain function is lacking. Here, we examined neuronal dendritic morphology and synaptic plasticity in medial prefrontal cortex (mPFC) of Fabp7 KO mice and in primary cortical neuronal cultures. Golgi staining of cortical pyramidal neurons in Fabp7 KO mice revealed aberrant dendritic morphology and decreased spine density compared with those in wild‐type (WT) mice. Aberrant dendritic morphology was also observed in primary cortical neurons co‐cultured with FABP7‐deficient astrocytes and neurons cultured in Fabp7 KO astrocyte‐conditioned medium. Excitatory synapse number was decreased in mPFC of Fabp7 KO mice and in neurons co‐cultured with Fabp7 KO astrocytes. Accordingly, whole‐cell voltage‐clamp recording in brain slices from pyramidal cells in the mPFC showed that both amplitude and frequency of action potential‐independent miniature excitatory postsynaptic currents (mEPSCs) were decreased in Fabp7 KO mice. Moreover, transplantation of WT astrocytes into the mPFC of Fabp7 KO mice partially attenuated behavioral impairments. Collectively, these results suggest that astrocytic FABP7 is important for dendritic arbor growth, neuronal excitatory synapse formation, and synaptic transmission, and provide new insights linking FABP7, lipid homeostasis, and neuropsychiatric disorders, leading to novel therapeutic interventions. GLIA 2016;64:48–62

Fatty acid-binding protein 7 regulates function of caveolae in astrocytes through expression of caveolin-1.

Fatty acid‐binding proteins (FABPs) bind and solubilize long‐chain fatty acids, controlling intracellular lipid dynamics. FABP7 is expressed by astrocytes in the developing brain, and suggested to be involved in the control of astrocyte lipid homeostasis. In this study, we sought to examine the role of FABP7 in astrocytes, focusing on plasma membrane lipid raft function, which is important for receptor‐mediated signal transduction in response to extracellular stimuli. In FABP7‐knockout (KO) astrocytes, the ligand‐dependent accumulation of Toll‐like receptor 4 (TLR4) and glial cell‐line‐derived neurotrophic factor receptor alpha 1 into lipid raft was decreased, and the activation of mitogen‐activated protein kinases and nuclear factor‐κB was impaired after lipopolysaccharide (LPS) stimulation when compared with wild‐type astrocytes. In addition, the expression of caveolin‐1, not cavin‐1, 2, 3, caveolin‐2, and flotillin‐1, was found to be decreased at the protein and transcriptional levels. FABP7 re‐expression in FABP7‐KO astrocytes rescued the decreased level of caveolin‐1. Furthermore, caveolin‐1‐transfection into FABP7‐KO astrocytes significantly increased TLR4 recruitment into lipid raft and tumor necrosis factor‐α production after LPS stimulation. Taken together, these data suggest that FABP7 controls lipid raft function through the regulation of caveolin‐1 expression and is involved in the response of astrocytes to the external stimuli. GLIA 2015;63:780–794

Cortical region-specific engraftment of embryonic stem cell-derived neural progenitor cells restores axonal sprouting to a subcortical target and achieves motor functional recovery in a mouse model of neonatal hypoxic-ischemic brain injury

Hypoxic-ischemic encephalopathy (HIE) at birth could cause cerebral palsy (CP), mental retardation, and epilepsy, which last throughout the individuals lifetime. However, few restorative treatments for ischemic tissue are currently available. Cell replacement therapy offers the potential to rescue brain damage caused by HI and to restore motor function. In the present study, we evaluated the ability of embryonic stem cell-derived neural progenitor cells (ES-NPCs) to become cortical deep layer neurons, to restore the neural network, and to repair brain damage in an HIE mouse model. ES cells stably expressing the reporter gene GFP are induced to a neural precursor state by stromal cell co-culture. Forty-hours after the induction of HIE, animals were grafted with ES-NPCs targeting the deep layer of the motor cortex in the ischemic brain. Motor function was evaluated 3 weeks after transplantation. Immunohistochemistry and neuroanatomical tracing with GFP were used to analyze neuronal differentiation and axonal sprouting. ES-NPCs could differentiate to cortical neurons with pyramidal morphology and expressed the deep layer-specific marker, Ctip2. The graft showed good survival and an appropriate innervation pattern via axonal sprouting from engrafted cells in the ischemic brain. The motor functions of the transplanted HIE mice also improved significantly compared to the sham-transplanted group. These findings suggest that cortical region specific engraftment of preconditioned cortical precursor cells could support motor functional recovery in the HIE model. It is not clear whether this is a direct effect of the engrafted cells or due to neurotrophic factors produced by these cells. These results suggest that cortical region-specific NPC engraftment is a promising therapeutic approach for brain repair.

Dorsal Forebrain-Specific Deficiency of Reelin-Dab1 Signal Causes Behavioral Abnormalities Related to Psychiatric Disorders

Reelin-Dab1 signaling is involved in brain development and neuronal functions. The abnormalities in the signaling through either reduction of Reelin and Dab1 gene expressions or the genomic mutations in the brain have been reported to be associated with psychiatric disorders. However, it has not been clear if the deficiency in Reelin-Dab1 signaling is responsible for symptoms of the disorders. Here, to examine the function of Reelin-Dab1 signaling in the forebrain, we generated dorsal forebrain-specific Dab1 conditional knockout mouse (Dab1 cKO) and performed a behavioral test battery on the Dab1 cKO mice. Although conventional Dab1 null mutant mice exhibit cerebellar atrophy and cerebellar ataxia, the Dab1 cKO mice had normal cerebellum and showed no motor dysfunction. Dab1 cKO mice exhibited behavioral abnormalities, including hyperactivity, decreased anxiety-like behavior, and impairment of working memory, which are reminiscent of symptoms observed in patients with psychiatric disorders such as schizophrenia and bipolar disorder. These results suggest that deficiency of Reelin-Dab1 signal in the dorsal forebrain is involved in the pathogenesis of some symptoms of human psychiatric disorders.

Fatty Acid Binding Protein 3 Is Involved in n–3 and n–6 PUFA Transport in Mouse Trophoblasts

BACKGROUND Low placental fatty acid (FA) transport during the embryonic period has been suggested to result in fetal developmental disorders and various adult metabolic diseases, but the molecular mechanism by which FAs are transported through the placental unit remains largely unknown. OBJECTIVE The aim of this study was to examine the distribution and functional relevance of FA binding protein (FABP), a cellular chaperone of FAs, in the mouse placenta. METHODS We clarified the localization of FABPs and sought to examine their function in placental FA transport through the phenotypic analysis of Fabp3-knockout mice. RESULTS Four FABPs (FABP3, FABP4, FABP5, and FABP7) were expressed with spatial heterogeneity in the placenta, and FABP3 was dominantly localized to the trophoblast cells. In placentas from the Fabp3-knockout mice (both sexes), the transport coefficients for linoleic acid (LA) were significantly reduced compared with those from wild-type mice by 25% and 44% at embryonic day (E) 15.5 and E18.5, respectively, whereas those for α-linolenic acid (ALA) were reduced by 19% and 17%, respectively. The accumulation of LA (18% and 27% at E15.5 and E18.5) and ALA (16% at E15.5) was also significantly less in the Fabp3-knockout fetuses than in wild-type fetuses. In contrast, transport and accumulation of palmitic acid (PA) were unaffected and glucose uptake significantly increased by 23% in the gene-ablated mice compared with wild-type mice at E18.5. Incorporation of LA (51% and 52% at 1 and 60 min, respectively) and ALA (23% at 60 min), but not PA, was significantly less in FABP3-knockdown BeWo cells than in controls, whereas glucose uptake was significantly upregulated by 51%, 50%, 31%, and 33% at 1, 20, 40, and 60 min, respectively. CONCLUSIONS Collectively FABP3 regulates n-3 (ω-3) and n-6 (ω-6) polyunsaturated FA transport in trophoblasts and plays a pivotal role in fetal development.

A novel trigger for cholesterol-dependent smooth muscle contraction mediated by the sphingosylphosphorylcholine-Rho-kinase pathway in the rat basilar artery: a mechanistic role for lipid rafts.

Hyperlipidemia is a risk factor for abnormal cerebrovascular events. Rafts are cholesterol-enriched membrane microdomains that influence signal transduction. We previously showed that Rho-kinase-mediated Ca2+ sensitization of vascular smooth muscle (VSM) induced by sphingosylphosphorylcholine (SPC) has a pivotal role in cerebral vasospasm. The goals of the study were to show SPC-Rho-kinase-mediated VSM contraction in vivo and to link this effect to cholesterol and rafts. The SPC-induced VSM contraction measured using a cranial window model was reversed by Y-27632, a Rho-kinase inhibitor, in rats fed a control diet. The extent of SPC-induced contraction correlated with serum total cholesterol. Total cholesterol levels in the internal carotid artery (ICA) were significantly higher in rats fed a cholesterol diet compared with a control diet or a β-cyclodextrin diet, which depletes VSM cholesterol. Western blotting and real-time PCR revealed increases in flotillin-1, a raft marker, and flotillin-1 mRNA in the ICA in rats fed a cholesterol diet, but not in rats fed the β-cyclodextrin diet. Depletion of cholesterol decreased rafts in VSM cells, and prevention of an increase in cholesterol by β-cyclodextrin inhibited SPC-induced contraction in a cranial window model. These results indicate that cholesterol potentiates SPC-Rho-kinase-mediated contractions of importance in cerebral vasospasm and are compatible with a role for rafts in this process.

Differential expression and regulatory roles of FABP5 and FABP7 in oligodendrocyte lineage cells.

Fatty-acid-binding proteins (FABPs) are key intracellular molecules involved in the uptake, transportation and storage of fatty acids and in the mediation of signal transduction and gene transcription. However, little is known regarding their expression and function in the oligodendrocyte lineage. We evaluate the in vivo and in vitro expression of FABP5 and FABP7 in oligodendrocyte lineage cells in the cortex and corpus callosum of adult mice, mixed cortical culture and oligosphere culture by immunofluorescent counter-staining with major oligodendrocyte lineage markers. In all settings, FABP7 expression was detected in NG2+/PDGFRα+ oligodendrocyte progenitor cells (OPCs) that did not express FABP5. FABP5 was detected in mature CC1+/MBP+ oligodendrocytes that did not express FABP7. Analysis of cultured OPCs showed a significant decrease in the population of FABP7-knockout (KO) OPCs and their BrdU uptake compared with wild-type (WT) OPCs. Upon incubation of OPCs in oligodendrocyte differentiation medium, a significantly lower percentage of FABP7-KO OPCs differentiated into O4+ oligodendrocytes. The percentage of mature MBP+ oligodendrocytes relative to whole O4+/MBP+ oligodendrocytes was significantly lower in FABP7-KO and FABP5-KO than in WT cell populations. The percentage of terminally mature oligodendrocytes with membrane sheet morphology was significantly lower in FABP5-KO compared with WT cell populations. Thus, FABP7 and FABP5 are differentially expressed in oligodendrocyte lineage cells and regulate their proliferation and/or differentiation. Our findings suggest the involvement of FABP7 and FABP5 in the pathophysiology of demyelinating disorders, neuropsychiatric disorder and glioma, conditions in which OPCs/oligodendrocytes play central roles.

Effects of intrathecal baclofen therapy on motor and cognitive functions in a rat model of cerebral palsy.

OBJECT Cerebral palsy (CP) arises in the early stages of brain development and manifests as spastic paresis that is often associated with cognitive dysfunction. Available CP treatments are aimed at the management of spasticity and include botulinum toxin administration, selective dorsal rhizotomy, and intrathecal baclofen (ITB). In this study, the authors investigated whether the management of spasticity with ITB therapy affected motor function and whether the release of spasticity was associated with an improvement in intellectual function. METHODS Newborn Sprague-Dawley rats were divided into the following groups: control, CP model, and CP model with ITB therapy. For the CP model, postnatal Day 7 (P7) rats were exposed to hypoxic conditions (8% O(2)) for 150 minutes after ligation of the right common carotid artery. In the groups receiving ITB therapy, a spinal catheter was connected to an osmotic pump filled with baclofen and placed in the spinal subarachnoid space on P21 in the early group and on P35 in the late group. A daily dose of 12 μg of baclofen was continuously administered until P49, resulting in 28 days of therapy in the early group and 14 days in the late group. Changes in spasticity in the CP and CP with ITB treatment groups were confirmed by assessing the motor evoked potential in the plantar muscle. RESULTS In the CP group, the time required to complete a beam-walking test on P49 was significantly longer than that in the control and ITB treatment groups (4.15 ± 0.60 vs 2.10 ± 0.18 and 2.22 ± 0.22 seconds, respectively). Results of the beam-walking test are expressed as the mean ± SD. Radial arm maze performance on P49 indicated that spatial reference memory had significantly deteriorated in the CP group compared with controls (2.33 ± 0.87 vs 0.86 ± 0.90 points); moreover, working memory was also negatively affected by CP (0.78 ± 1.09 vs 0.14 ± 0.38 points). Results of the memory tests are expressed as the mean ± SE. These memory functions did not recover after ITB treatment. CONCLUSIONS Management of spasticity with ITB therapy improved the walking ability in the rat CP model. Intrathecal baclofen therapy-which reduces harmful sensory and motor stimulations caused by spasticity to more optimal levels-contributed to motor function recovery; however, it had no effect on intellectual recovery as assessed by memory performance in the rat CP model.

Fatty acid-binding protein 4 (FABP4) and FABP5 modulate cytokine production in the mouse thymic epithelial cells.

Thymic stromal cells, including cortical thymic epithelial cells (cTEC) produce many humoral factors, such as cytokines and eicosanoids to modulate thymocyte homeostasis, thereby regulating the peripheral immune responses. In this study, we identified fatty acid-binding protein (FABP4), an intracellular fatty acid chaperone, in the mouse thymus, and examined its role in the control of cytokine production in comparison with FABP5. By immunofluorescent staining, FABP4+ cells enclosing the thymocytes were scattered throughout the thymic cortex with a spatial difference from the FABP5+ cell that were distributed widely throughout the cTEC. The FABP4+ cells were immunopositive for MHC class II, NLDC145 and cytokeratin 8, and were identified as part of cTEC. The FABP4+ cells were identified as thymic nurse cells (TNC), a subpopulation of cTEC, by their active phagocytosis of apoptotic thymocytes. Furthermore, FABP4 expression was confirmed in the isolated TNC at the gene and protein levels. To explore the function of FABP in TNC, TSt-4/DLL1 cells stably expressing either FABP4 or FABP5 were established and the gene expressions of various cytokines were examined. The gene expression of interleukin (IL)-7 and IL-18 was increased both in FABP4 and FABP5 over-expressing cells compared with controls, and moreover, the increase in their expressions by adding of stearic acids was significantly enhanced in the FABP4 over-expressing cells. These data suggest that both FABPs are involved in the maintenance of T lymphocyte homeostasis through the modulation of cytokine production, which is possibly regulated by cellular fatty acid-mediated signaling in TEC, including TNC.