Kazem Sharifi

FABP7 expression in normal and stab-injured brain cortex and its role in astrocyte proliferation

Reactive gliosis, in which astrocytes as well as other types of glial cells undergo massive proliferation, is a common hallmark of all brain pathologies. Brain-type fatty acid-binding protein (FABP7) is abundantly expressed in neural stem cells and astrocytes of developing brain, suggesting its role in differentiation and/or proliferation of glial cells through regulation of lipid metabolism and/or signaling. However, the role of FABP7 in proliferation of glial cells during reactive gliosis is unknown. In this study, we examined the expression of FABP7 in mouse cortical stab injury model and also the phenotype of FABP7-KO mice in glial cell proliferation. Western blotting showed that FABP7 expression was increased significantly in the injured cortex compared with the contralateral side. By immunohistochemistry, FABP7 was localized to GFAP+ astrocytes (21% of FABP7+ cells) and NG2+ oligodendrocyte progenitor cells (62%) in the normal cortex. In the injured cortex there was no change in the population of FABP7+/NG2+ cells, while there was a significant increase in FABP7+/GFAP+ cells. In the stab-injured cortex of FABP7-KO mice there was decrease in the total number of reactive astrocytes and in the number of BrdU+ astrocytes compared with wild-type mice. Primary cultured astrocytes from FABP7-KO mice also showed a significant decrease in proliferation and omega-3 fatty acid incorporation compared with wild-type astrocytes. Overall, these data suggest that FABP7 is involved in the proliferation of astrocytes by controlling cellular fatty acid homeostasis.

Fatty acid binding protein 7 as a marker of glioma stem cells.

Glioblastomas are the most aggressive brain tumors. Glioblastoma stem cells (GSCs) are thought to be responsible for the recurrence, chemoresistance, and poor prognosis of glioblastoma. Fatty acid binding protein 7 (FABP7), which is a cellular chaperone for a variety of omega‐3 fatty acids, is a known marker for neural stem cells. In this study, using a newly developed anti‐FABP7 antibody and patient‐derived GSC lines, we evaluated the expression of FABP7 in GSCs. Using immunocytochemistry, Western blotting, and qPCR analyses, FABP7 was found to be highly enriched in GSCs and its localization was found in cytosol and nuclei. FABP7 expression was significantly downregulated in differentiated GSCs induced by the addition of serum. In the glioma surgical specimens, FABP7 was highly expressed in the majority of glioblastoma. Double immunostaining for FABP7 and Sox2 showed that FABP7+Sox2+ tumor cells were significantly increased in glioblastoma (grade IV) compared with diffuse astrocytoma (grade II) and anaplastic astrocytoma (grade III). Our data introduces FABP7 as a marker for GSCs and further highlights its possible significance for glioma diagnosis and treatment.

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

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.

Identification of FABP7 in fibroblastic reticular cells of mouse lymph nodes

Fatty acids and their metabolites regulate immune cell function. The present study was undertaken to examine the detailed distribution of fatty acid binding proteins (FABPs), the cytosolic chaperones of fatty acids, in mouse peripheral immune organs. Using immunohistochemistry, FABP7 was localized to the alpha-smooth muscle actin (SMA)+ fibroblastic reticular cells, which construct the stromal reticula in the T cell areas of the peripheral lymph nodes and spleen. Immunoelectron microscopy showed that FABP7+ cells enclosed the collagen fibers, forming a conduit system, which transport lymph and associated low-molecular-mass proteins. In contrast, FABP5+ cells were distributed throughout the lymph node and contained well-developed lysosome and phagocytic materials within the cytoplasm. The mesenteric lymph nodes of FABP7 knockout mice showed normal histological features, but the percentage of CD4+ cells was significantly increased compared with that in wild-type mice. These data indicate that FABP7 may be involved in T cell homeostasis, possibly by modulating lipid metabolism in fibroblastic reticular cells within the peripheral lymph nodes.

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.

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.

Fatty Acid Binding Protein 7 Regulates Phagocytosis and Cytokine Production in Kupffer Cells during Liver Injury

Kupffer cells (KCs) are involved in the progression of liver diseases such as hepatitis and liver cancer. Several members of the fatty acid binding proteins (FABPs) are expressed by tissue macrophages, and FABP7 is localized only in KCs. To clarify the role of FABP7 in the regulation of KC function, we evaluated pathological changes of Fabp7 knockout mice during carbon tetrachloride-induced liver injury. During liver injury in Fabp7 knockout mice, serum liver enzymes were increased, cytokine expression (tumor necrosis factor-α, monocyte chemoattractant protein-1, and transforming growth factor-β) was decreased in the liver, and the number of KCs in the liver necrotic area was significantly decreased. Interestingly, in the FABP7-deficient KCs, phagocytosis of apoptotic cells was impaired, and expression of the scavenger receptor CD36 was markedly decreased. In chronic liver injury, Fabp7 knockout mice showed less fibrogenic response to carbon tetrachloride compared with wild-type mice. Taken together, FABP7 is involved in the liver injury process through its regulation of KC phagocytic activity and cytokine production. Such modulation of KC function by FABP7 may provide a novel therapeutic approach to the treatment of liver diseases.