Dermal macrophages set pain sensitivity by modulating the amount of tissue NGF through an SNX25-Nrf2 pathway.

Cross-talk between peripheral neurons and immune cells is important in pain sensation. We identified Snx25 as a pain-modulating gene in a transgenic mouse line with reduced pain sensitivity. Conditional deletion of Snx25 in monocytes and macrophages, but not in peripheral sensory neurons, in mice (Snx25cKO mice) reduced pain responses in both normal and neuropathic conditions. Bone marrow transplantation using Snx25cKO and wild-type mice indicated that macrophages modulated pain sensitivity. Expression of sorting nexin (SNX)25 in dermal macrophages enhanced expression of the neurotrophic factor NGF through the inhibition of ubiquitin-mediated degradation of Nrf2, a transcription factor that activates transcription of Ngf. As such, dermal macrophages set the threshold for pain sensitivity through the production and secretion of NGF into the dermis, and they may cooperate with dorsal root ganglion macrophages in pain perception.

Characterization of Glial Populations in the Aging and Remyelinating Mouse Corpus Callosum.

Cells in the white matter of the adult brain have a characteristic distribution pattern in which several cells are contiguously connected to each other, making a linear array (LA) resembling pearls-on-a-string parallel to the axon axis. We have been interested in how this pattern of cell distribution changes during aging and remyelination after demyelination. In the present study, with a multiplex staining method, semi-quantitative analysis of the localization of oligodendrocyte lineage cells (oligodendrocyte progenitors, premyelinating oligodendrocytes, and mature oligodendrocytes), astrocytes, and microglia in 8-week-old (young adult) and 32-week-old (aged) corpus callosum showed that young adult cells still include immature oligodendrocytes and that LAs contain a higher proportion of microglia than isolated cells. In aged mice, premyelinating oligodendrocytes were decreased, but microglia continued to be present in the LAs. These results suggest that the presence of microglia is important for the characteristic cell localization pattern of LAs. In a cuprizone-induced demyelination model, we observed re-formation of LAs after completion of cuprizone treatment, concurrent with remyelination. These re-formed LAs again contained more microglia than the isolated cells. This finding supports the hypothesis that microglia contribute to the formation and maintenance of LAs. In addition, regardless of the distribution of cells (LAs or isolated cells), astrocytes were found to be more abundant than in the normal corpus callosum at 24 weeks after cuprizone treatment when remyelination is completed. This suggests that astrocytes are involved in maintaining the functions of remyelinated white matter.

Large-scale electron microscopic volume imaging of interfascicular oligodendrocytes in the mouse corpus callosum.

Single oligodendrocytes produce myelin sheaths around multiple axons in the central nervous system. Interfascicular oligodendrocytes (IOs) facilitate nerve conduction, but their detailed morphologies remain largely unknown. In the present study, we three-dimensionally reconstructed IOs in the corpus callosum of adult mouse using serial block face scanning electron microscopy. The cell bodies of IOs were morphologically polarized and extended thick processes from the cytoplasm-rich part of the cell. Processes originating from the cell body of each IO can be classified into two types: one myelinates an axon without branching, while the other type branches and each branch myelinates a distinct axon. Myelin sheaths originating from a particular IO have biased thicknesses, wrapping axons of a limited range of diameters. Consistent with this finding, IOs transduced and visualized with a rabies viral vector expressing GFP showed statistically significant variation in their myelination patterns. We further reconstructed the sheath immediately adjacent to that derived from each of the analyzed IOs; the thicknesses of the pair of sheaths were significantly correlated despite emanating from different IOs. These results suggest that a single axon could regulate myelin sheath thicknesses, even if the sheaths are derived from distinct IOs. Collectively, our results indicate that the IOs have their own myelin profiles defined by myelin thickness and axonal diameter although axons may regulate thickness of myelin sheath.

Responses of perivascular macrophages to circulating lipopolysaccharides in the subfornical organ with special reference to endotoxin tolerance.

BACKGROUND: Circulating endotoxins including lipopolysaccharides (LPS) cause brain responses such as fever and decrease of food and water intake, while pre-injection of endotoxins attenuates these responses. This phenomenon is called endotoxin tolerance, but the mechanisms underlying it remain unclear. The subfornical organ (SFO) rapidly produces proinflammatory cytokines including interleukin-1ß (IL-1ß) in response to peripherally injected LPS, and repeated LPS injection attenuates IL-1ß production in the SFO, indicating that the SFO is involved in endotoxin tolerance. The purpose of this study is to investigate features of the IL-1ß source cells in the SFO of LPS-non-tolerant and LPS-tolerant mice. METHODS: We first established the endotoxin-tolerant mouse model by injecting LPS into adult male mice (C57BL/6J). Immunohistochemistry was performed to characterize IL-1ß-expressing cells, which were perivascular macrophages in the SFO. We depleted perivascular macrophages using clodronate liposomes to confirm the contribution of IL-1ß production. To assess the effect of LPS pre-injection on perivascular macrophages, we transferred bone marrow-derived cells obtained from male mice (C57BL/6-Tg (CAG-EGFP)) to male recipient mice (C57BL/6N). Finally, we examined the effect of a second LPS injection on IL-1ß expression in the SFO perivascular macrophages. RESULTS: We report that perivascular macrophages but not parenchymal microglia rapidly produced the proinflammatory cytokine IL-1ß in response to LPS. We found that peripherally injected LPS localized in the SFO perivascular space. Depletion of macrophages by injection of clodronate liposomes attenuated LPS-induced IL-1ß expression in the SFO. When tolerance developed to LPS-induced sickness behavior in mice, the SFO perivascular macrophages ceased producing IL-1ß, although bone marrow-derived perivascular macrophages increased in number in the SFO and peripherally injected LPS reached the SFO perivascular space. CONCLUSIONS: The current data indicate that perivascular macrophages enable the SFO to produce IL-1ß in response to circulating LPS and that its hyporesponsiveness may be the cause of endotoxin tolerance.

Kallikrein 6 secreted by oligodendrocytes regulates the progression of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), and experimental autoimmune encephalomyelitis (EAE) is a well-established animal model of the disease. Here, we examined the pathophysiological role of Kallikrein 6 (Klk6), a serine protease produced by oligodendrocytes (OLs), in EAE using Klk6 knockout (Klk6-/-) mice. Compared with Klk6+/+ (wild-type) mice, Klk6-/- mice showed milder EAE symptoms, including delayed onset and milder paralysis. Loss of Klk6 suppressed matrix metalloprotease-9 expression and diminished the infiltration of peripheral inflammatory cells into the CNS by decreasing blood-brain barrier (BBB) permeability and reducing expression levels of inflammatory cytokines, chemokines and their receptors. Scanning electron microscopic analysis revealed demyelination characterized by myelin detachment from the axons in the early phase of EAE progression (days 3-7) in Klk6+/+ mice but not in Klk6-/- mice. Interestingly, anti-MOG (myelin oligodendrocyte glycoprotein) autoantibody was also detected in the cerebrospinal fluid (CSF) and spinal cord on day 3 after MOG immunization. Furthermore, treatment of primary cultured OLs with anti-MOG autoantibody induced oligodendroglial morphological changes and increases in myelin basic protein and Klk6 expression. We also developed a novel enzyme-linked immunoabsorbent assay method for detecting activated KLK6 in human CSF. In human autopsy brain samples, expression of active KLK6 was detected in OLs using an antibody that specifically recognizes the protein's activated form. Taken together, our findings demonstrate that Klk6 secreted by OLs plays a critical role in the pathogenesis of EAE/MS and that it might serve as a potential therapeutic target for MS.

Microglia-derived neuregulin expression in psychiatric disorders.

Several studies have revealed that neuregulins (NRGs) are involved in brain function and psychiatric disorders. While NRGs have been regarded as neuron- or astrocyte-derived molecules, our research has revealed that microglia also express NRGs, levels of which are markedly increased in activated microglia. Previous studies have indicated that microglia are activated in the brains of individuals with autism spectrum disorder (ASD). Therefore, we investigated microglial NRG mRNA expression in multiple lines of mice considered models of ASD. Intriguingly, microglial NRG expression significantly increased in BTBR and socially-isolated mice, while maternal immune activation (MIA) mice exhibited identical NRG expression to controls. Furthermore, we observed a positive correlation between NRG expression in microglia and peripheral blood mononuclear cells (PBMCs) in mice, suggesting that NRG expression in human PBMCs may mirror microglia-derived NRG expression in the human brain. To translate these findings for application in clinical psychiatry, we measured levels of NRG1 splice-variant expression in clinically available PBMCs of patients with ASD. Levels of NRG1 type III expression in PBMCs were positively correlated with impairments in social interaction in children with ASD (as assessed using the Autistic Diagnostic Interview-Revised test: ADI-R). These findings suggest that immune cell-derived NRGs may be implicated in the pathobiology of psychiatric disorders such as ASD.

Yokukansan Reduces Cuprizone-Induced Demyelination in the Corpus Callosum Through Anti-inflammatory Effects on Microglia.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). The release of inflammatory cytokines and pro-oxidant molecules from microglia has been shown to play a key role in the pathology of MS. Thus, suppression of microglial cell activation is an attractive therapeutic option. Yokukansan, a traditional Japanese herbal medicine, has been shown to suppress microglial activity in the CNS. However, whether or not yokukansan reduces demyelination observed in the CNS during MS remains unknown. In this study, female C57BL/6 mice were fed a diet containing 0.2% cuprizone (bis-cyclohexanone oxaldihydrazone) to induce demyelination in the corpus callosum. We investigated whether or not yokukansan reduces cuprizone-induced demyelination using immunohistochemical analyses. Furthermore, we examined the in vitro anti-inflammatory effects of yokukansan on LPS-stimulated BV2 cells, a murine microglial cell line. Luxol fast blue staining and immunostaining for myelin basic protein demonstrated that yokukansan reduces demyelination of the corpora callosa of cuprizone-fed mice. In addition, yokukansan significantly decreased the number of activated microglial cells in the corpora callosa of cuprizone-fed mice. Furthermore, treatment with 500 µg/ml yokukansan suppressed the expression of interleukin-1ß and inducible nitric-oxide synthase mRNA and protein in LPS-stimulated BV2 cells. These findings suggest that yokukansan reduces demyelination owing to anti-inflammatory effects on microglia. As yokukansan has few adverse effects, yokukansan has the potential to be a novel option to treat MS.

Interferon regulatory factor 7 participates in the M1-like microglial polarization switch.

Microglia are generally considered the immune cells of the central nervous system. Recent studies have demonstrated that under specific polarization conditions, microglia develop into two different phenotypes, termed M1-like and M2-like microglia. However, the phenotypic characteristics of M1-like- and M2-like-polarized microglia and the mechanisms that regulate polarization are largely unknown. In this study, we characterized lipopolysaccharide-treated M1-like and IL-4-treated M2-like microglia and investigated the mechanisms that regulate phenotypic switching. The addition of M2-like microglial conditioned medium (CM) to primary neurons resulted in an increase in neurite length when compared with neurons treated with M1-like microglial CM, possibly because of the enhanced secretion of neurotrophic factors by M2-like microglia. M1-like microglia were morphologically characterized by larger soma, whereas M2-like microglia were characterized by long processes. M2-like microglia exhibited greater phagocytic capacity than M1-like microglia. These features switched in response to polarization cues. We found that expression of interferon regulatory factor 7 (IRF7) increased during the M2-like to M1-like switch in microglia in vitro and in vivo. Knockdown of IRF7 using siRNA suppressed the expression of M1 marker mRNA and reduced phosphorylation of STAT1. Our findings suggest that IRF7 signaling may play an important role in microglial polarization switching.

Minocycline reduces remyelination by suppressing ciliary neurotrophic factor expression after cuprizone-induced demyelination.

Remyelination is disrupted in demyelinating diseases such as multiple sclerosis, but the underlying pathogenetic mechanisms are unclear. In this study, we employed the murine cuprizone model of demyelination, in which remyelination occurs after removal of the toxin from the diet, to examine the cellular and molecular changes during demyelination and remyelination. Microglia accumulated in the corpus callosum during weeks 2-4 of the cuprizone diet, and these cells remained activated 2 weeks after the change to the normal diet. To examine the role of microglia in remyelination, mice were treated with minocycline to inactivate these cells after cuprizone-induced demyelination. Minocycline treatment reduced the number of CC1-positive oligodendrocytes, as well as levels of myelin basic protein (MBP) and CNPase in the remyelination phase. The expression of CNTF mRNA in the corpus callosum increased after 4 weeks on the cuprizone diet and remained high 2 weeks after the change to the normal diet. Minocycline suppressed CNTF expression during the remyelination phase on the normal diet. Primary culture experiments showed that CNTF was produced by microglia in addition to astrocytes. In vitro, CNTF directly affected the differentiation of oligodendrocytic cells. These findings suggest that minocycline reduces remyelination by suppressing CNTF expression by microglia after cuprizone-induced demyelination.

Amino acid transporter Asc-1 (SLC7A10) expression is altered in basal ganglia in experimental Parkinsonism and L-dopa-induced dyskinesia model mice.

In Parkinson's disease (PD), a decrease in dopamine levels in the striatum causes abnormal circuit activity in the basal ganglia, resulting in increased output via the substantia nigra pars reticulata (SNr). A characteristic feature of glutamatergic synaptic transmission in the basal ganglia circuitry under conditions of dopamine depletion is enhanced synaptic activity of NMDA receptors. However, the cause of this NMDA receptor hyperactivity is not fully understood. We focused on Asc-1 (SLC7A10), an alanine-serine-cysteine transporter, as one of the factors that regulate NMDA receptor activity by modulating D-serine and glycine concentration in synaptic clefts. We generated PD model mice by injection of 6-hydroxydopamine into the unilateral medial forebrain bundle and analyzed the expression level of Asc-1 mRNA in the nuclei of basal ganglia (the external segment of the globus pallidus (GPe), subthalamic nucleus (STN), and SNr) compared to control mice. Each nucleus was dissected using laser microdissection, and RNA was extracted and quantified by quantitative PCR. Asc-1 mRNA expression was significantly higher in the GPe and lower in the SNr under the PD state than that in control naïve mice. The STN showed no change in Asc-1 mRNA expression. We further modeled L-dopa-induced dyskinesia by administering L-dopa continuously for 14 days to the PD model mice and found that Asc-1 mRNA expression in the GPe and SNr became close to that of control mice, regardless of the presence of abnormal involuntary movements. The present study revealed that Asc-1 mRNA expression is differentially regulated in the basal ganglionic nuclei in response to striatal dopamine concentration (depleted or replenished) and suggests that Asc-1 can be a therapeutic target for the amelioration of motor symptoms of PD.

Juvenile social isolation affects the structure of the tanycyte-vascular interface in the hypophyseal portal system of the adult mice.

Accumulating evidence indicates that social stress in the juvenile period affects hypothalamic-pituitary-adrenal (HPA) axis activity in adulthood. The biological mechanisms underlying this phenomenon remain unclear. We aimed to elucidate them by comparing adult mice that had experienced social isolation from postnatal day 21-35 (juvenile social isolation (JSI) group) with those reared normally (control group). JSI group mice showed an attenuated HPA response to acute swim stress, while the control group had a normal response to this stress. Activity levels of the paraventricular nucleus in both groups were comparable, as shown by c-Fos immunoreactivities and mRNA expression of c-Fos, Corticotropin-releasing factor (CRF), Glucocorticoid receptor, and Mineralocorticoid receptor. We found greater vascular coverage by tanycytic endfeet in the median eminence of the JSI group mice than in that of the control group mice under basal condition and after acute swim stress. Moreover, CRF content after acute swim stress was greater in the median eminence of the JSI group mice than in that of the control group mice. The attenuated HPA response to acute swim stress was specific to JSI group mice, but not to control group mice. Although a direct link awaits further experiments, tanycyte morphological changes in the median eminence could be related to the HPA response.

SNX25 regulates proinflammatory cytokine expression via the NF-κB signal in macrophages.

Innate immunity is the first line of defense against bacterial infection and is initiated by macrophages. Sorting nexin 25 (SNX25) is an SNX family member and is reported to negatively regulate TGF-ß signaling by enhancing TGF receptor degradation. However, few studies have focused on the relationship between SNX25 and the immune system. We knocked down SNX25 expression in macrophages and examined inflammatory cytokine expression, a hallmark of innate immunity, after lipopolysaccharide stimulation. SNX25 knockdown increased proinflammatory cytokine expression in RAW 264.7 cells. In addition, SNX25 knockdown activated the NF-κB signal by promoting ubiquitination of IκBα. These results suggest that SNX25 inhibits the NF-κB signal and thereby regulates proinflammatory cytokine expression in macrophages.

Olig2-astrocytes express neutral amino acid transporter SLC7A10 (Asc-1) in the adult brain.

We have reported that the transcription factor Olig2 labels a subpopulation of astrocytes (Olig2-astrocytes), which show distribution patterns different from those of GFAP-expressing astrocytes (GFAP-astrocytes) in the adult brain. Here, to uncover the specific functions of Olig2-astrocytes, we first analyzed public single-cell RNA-seq databases of adult mouse brains. Unbiased classification of gene expression profiles and subsequent gene ontology analyses revealed that the majority of Olig2-astrocytes belonged to an astrocytic cluster that is enriched for transporter-related genes. SLC7A10 (also known as ASC-1) was one of the representative neutral amino acid transporter genes in the cluster. To complement the in silico data analyses, we differentially isolated Olig2- and GFAP-astrocytes from the same frozen section of the lateral globus pallidus using laser microdissection and compared their gene expression by quantitative reverse transcription PCR. We confirmed that Olig2 and GFAP mRNAs were preferentially expressed in the Olig2- and GFAP-astrocytes, respectively, indicating that the laser microdissection method yielded minimal cross-contamination between two types of cells. The Olig2-astrocytes expressed significantly higher levels of SLC7A10 mRNA than the GFAP-astrocytes, corroborating the in silico data. We next localized SLC7A10 protein by immunohistochemistry in the lateral globus pallidus, which was also genetically labeled for Olig2. SLC7A10 co-localized with Olig2-genetic labeling, especially on the fine processes of Olig2-astrocytes. These results are consistent with the recent discovery that SLC7A10 is expressed not only in neurons but also in a subset of astrocytes. Taken together, our findings suggest that SLC7A10 exerts specific functions in Olig2-astrocytes of the adult brain.

Gene Expression Profiles of Human Cerebral Organoids Identify PPAR Pathway and PKM2 as Key Markers for Oxygen-Glucose Deprivation and Reoxygenation.

Ischemic stroke is one of the most common neurological diseases. However, the impact of ischemic stroke on human cerebral tissue remains largely unknown due to a lack of ischemic human brain samples. In this study, we applied cerebral organoids derived from human induced pluripotent stem cells to evaluate the effect of oxygen-glucose deprivation/reoxygenation (OGD/R). Pathway analysis showed the relationships between vitamin digestion and absorption, fat digestion and absorption, peroxisome proliferator-activated receptor (PPAR) signaling pathway, and complement and coagulation cascades. Combinational verification with transcriptome and gene expression analysis of different cell types revealed fatty acids-related PPAR signaling pathway and pyruvate kinase isoform M2 (PKM2) as key markers of neuronal cells in response to OGD/R. These findings suggest that, although there remain some limitations to be improved, our ischemic stroke model using human cerebral organoids would be a potentially useful tool when combined with other conventional two-dimensional (2D) mono-culture systems.

Gly192 at hinge 2 site in the chaperonin GroEL plays a pivotal role in the dynamic apical domain movement that leads to GroES binding and efficient encapsulation of substrate proteins.

The subunit structure of chaperonin GroEL is divided into three domains; the apical domain, the intermediate domain, and the equatorial domain. Each domain has a specific role in the chaperonin mechanism. The 'hinge 2' site of GroEL contains three glycine residues, Gly192, Gly374, and Gly375, connecting the apical domain and the intermediate domain. In this study, to understand the importance of the hinge 2 amino acid residues in chaperonin function, we substituted each of these three glycine residues to tryptophan. The GroEL mutants G374W and G375W were functionally similar to wild-type GroEL. However, GroEL G192W showed a significant decrease in the ability to assist the refolding of stringent substrate proteins. Interestingly, from biochemical assays and characterization using surface plasmon resonance analysis, we found that GroEL G192W was capable of binding GroES even in the absence of ATP to form a very stable GroEL-GroES complex, which could not be dissociated even upon addition of ATP. Electron micrographs showed that GroEL G192W intrinsically formed an asymmetric double ring structure with one ring locked in the 'open' conformation, and it is postulated that GroES binds to this open ring in the absence of ATP. Trans-binding of both substrate protein and GroES was observed for this binary complex, but simultaneous binding of both substrate and GroES (a mechanism that ensures substrate encapsulation) was impaired. We postulate that alteration of Gly192 severely compromises an essential movement that allows efficient encapsulation of unfolded protein intermediates.

Neural expression of sorting nexin 25 and its regulation of tyrosine receptor kinase B trafficking.

Sorting nexin 25 (SNX25) belongs to the sorting nexin superfamily, whose members are responsible for membrane attachment to organelles of the endocytic system. Recent reports point to critical roles for SNX25 as a negative regulator of transforming growth factor ß signaling, but the expression patterns of SNX25 in the central nervous system (CNS) remain almost uncharacterized. Here, we show widespread neuronal expression of SNX25 protein and Snx25 mRNA using immunohistochemistry and in situ hybridization. As an exception, SNX25 was present in the Bergmann glia of the cerebellum. SNX25 immunoreactivity was found in cholinergic and catecholaminergic neurons. Moreover, SNX25 colocalized with tropomyosin receptor kinase B (TrkB) in the neurons of the cortex and hippocampus. In vitro, SNX25 can interact with full-length TrkB, but not with its C-terminal-truncated isoform. Overexpression of SNX25 accelerated degradation of full-lengh TrkB, indicating that SNX25 promotes the trafficking of TrkB for lysosomal degradation. These findings suggest that SNX25 is a new actor in endocytic signaling, perhaps contributing to the regulation of BDNF-TrkB signaling in the CNS.

Circadian rhythms of sorting nexin 25 in the mouse suprachiasmatic nucleus.

Entrainment of mammalian circadian rhythms requires receptor-mediated signaling in the hypothalamic suprachiasmatic nucleus (SCN), the site of the master circadian pacemaker. Receptor-mediated signaling is regulated by endocytosis, indicating that endocytosis-related proteins contribute to SCN pacemaking. Sorting nexin 25 (SNX25) belongs to the sorting nexin superfamily, whose members are responsible for membrane attachment to organelles of the endocytic system. In this study, we showed that Snx25 mRNA and SNX25 protein are highly expressed and exhibit remarkable circadian rhythms in the SCN of adult mice. Expression was maximal at about zeitgeber time (ZT) 16 in the subjective night and minimal at ZT8 in the subjective day. Prominent SNX25 immunoreactivity was found in the arginine vasopressin-positive neurons of the SCN. These findings suggest that SNX25 is a new actor in endocytic signaling, perhaps contributing to the circadian pacemaking system.

Biosynthesis, processing, and secretion of glial cell line-derived neurotrophic factor in astroglial cells.

Glial cell line-derived neurotrophic factor (GDNF) is synthesized as a precursor, proGDNF. However, the molecular mechanisms for the processing and secretion of GDNF are not fully characterized, since the amount of its biosynthesis and secretion in glial cells are below the detection limit of western blotting. We established stably GDNF-overexpressing C6 cells, and this enabled us to monitor its spontaneous secretion, as well as its processed forms in the cells. GDNF secretion was augmented by stimulation with high potassium, while it was inhibited by treatment with either tunicamycin, an inhibitor of protein glycosylation, or brefeldin A, a disturbing factor of ER-Golgi transport. Wild-type GDNF transfected cells secreted three forms of processed GDNF. After deglycosylation, the highest molecular weight of secreted GDNF showed the same mobility on electrophoresis as recombinant human GDNF without a whole pro-domain. Mutations in the pro-domain and two cysteines at the C-terminal of GDNF markedly diminished the secretion of resultant proteins into the culture medium. GDNF proteins having mutations in the putative furin-consensus sequence were secreted partly as unprocessed forms, and forms with lower molecular weights than a mature form were secreted from the C6 cells. Taking these observations together, we conclude that GDNF is likely secreted both with and without processing by furin-like proteases, and that the pro-domain and C-terminal cysteines of GDNF play important roles in its processing and secretion in cultured astrocytes and C6 cells.

The Role of the Wnt Signaling Pathway in Upper Jaw Development of Chick Embryo.

Cleft lip with or without cleft palate (CLP) usually results from a failure of the medial nasal prominences to fuse with the lateral and maxillary prominences. This failure inhibits facial morphogenesis regulated by several major morphogenetic signaling pathways. We hypothesized that CLP results from the failure of the Wnt signaling pathway. To examine whether Wnt signaling can influences upper jaw development, we applied beads soaked with Dickkopf-1 (Dkk-1), Alsterpaullone (AL) or Wnt3a to the right side of the maxillary prominence of the chick embryo. The embryo showed a defect of the maxilla on the treated side, and skeletal staining revealed hypoplasia of the premaxilla and palatine bone as a result of Dkk-1-soaked bead implantation. 5-bromo-2'-deoxyuridine (BrdU)-positive cell numbers in the treated maxillary prominence were significantly lower at both 24 and 48 hr after implantation. Down-regulation of the expression of Bmp4, Tbx22, Sox9, and Barx1 was confirmed in the maxillary prominence treated with Dkk-1, which indicated that the deformity of the maxillary bone was controlled by gene targets of the Wnt signaling pathway. Expression of N-cadherin was seen immunohistochemically in the maxillary prominences of embryos at 6 hr and increased at 24 hr after AL treatment. Wnt signaling enhanced by AL or Wnt3a up-regulated the expression levels of Msx1, Bmp4, Tbx22, Sox9, and Barx1. Our data suggest that the Wnt signaling pathway regulates maxillary morphogenesis and growth through Bmp4, Tbx22, Sox9, and Barx1. Wnt signaling might regulate N-cadherin expression via Msx1, resulting in cell aggregation for osteochondrogenesis.

Identification of a novel GDNF mRNA induced by LPS in immune cell lines.

Glial cell line-derived neurotrophic factor (GDNF) is a survival factor for many neuronal cell types and has wide ranging effects within the central nervous system. To investigate the expression of the GDNF gene in immune cell lines under inflammatory conditions, we pharmacologically estimated the induction of GDNF mRNA in RAW264.7 cells. RT-PCR analysis revealed that LPS-induced GDNF mRNA in RAW264.7 cells does not include exon 3, which encodes the translational start site of this gene. A novel type of GDNF mRNA cloned by 5'-RACE consisted of the previous exon 4 and its flanking 5' upstream region, akin to a single exon gene. A similar type of human GDNF mRNA was also detected in a human neuroblastoma cell line, SH-SY5Y, without any stimulation. This novel (Ex4) GDNF mRNA was also upregulated by LPS in primary cultured rat macrophages, microglia and astrocytes and was found to exist in mouse brain. Ex4 GDNF protein produced by transfected HEK293 cells was mainly detected in cell lysates, but in conditioned medium only after PMA stimulation. Ex4 GDNF protein was found to exist as an unglycosylated form in both the transfected cells and the conditioned medium while full-type GDNF protein is glycosylated. PMA-stimulated augmentation of unglycosylated Ex4 GDNF protein was demonstrably regulated at the post-translational level.