Yoko Tohyama

Neurotrophin secretion from cultured microglia.

Because microglia have been suggested to produce neurotrophins, we tested this ability in vitro. Rat primary microglia were found to constitutively secrete a limited amount of brain‐derived neurotrophic factor (BDNF), but nerve growth factor (NGF) and neurotrophin‐3 (NT‐3) were undetectable in the conditioned medium. Stimulation of the cells with lipopolysaccharide (LPS) increased BDNF secretion, and induced NGF secretion. As a first step to examine this regulation system, the association of protein kinase C (PKC) was pharmacologically analyzed. A PKC activator, phorbol‐12‐myristate‐13‐acetate, enhanced the secretion of BDNF. Pre‐treatment of microglia with a PKC inhibitor, bisindolylmaleimide, suppressed LPS‐stimulated BDNF secretion as well as the constitutive one. These results suggest that the PKC signaling cascade is closely associated with BDNF secretion. Among PKC isoforms, PKCα probably plays a role in BDNF secretion, based on the results of experiments using a specific PKC activator, 1‐oleoyl‐2‐acetyl‐sn‐glycerol, and a specific PKC inhibitor, Gö 6976, and by immunoblotting. Taken together, these findings suggest that the secretion of BDNF from microglia is regulated through PKCα‐associated signal transduction mechanism. J. Neurosci. Res. 65:322–331, 2001.

Ability of rat microglia to uptake extracellular glutamate

Since it has been suggested that microglia in vivo act as glutamate scavengers, this possibility was investigated in primary cultured microglia. The microglia showed specific abilities to uptake (14)C-glutamate depending on incubation time and numbers of cells used. The activity was suppressed by a specific inhibitor for a glial cell-type transporter, glutamate transporter-1 (GLT-1) (EAAT2). However, that of cultured astrocytes was not affected. These results suggest that microglia uptake glutamate by means of GLT-1. Supporting these results, immunoblotting revealed the presence of GLT-1 in the microglia, while only glutamate-aspartate transporter (GLAST) (EAAT1: another glial cell-type transporter) was detected in the astrocytes. All together, these results indicate that microglia can act as glutamate scavengers in vivo by expressing the glutamate transporter GLT-1.

Ceramide activates microglia to enhance the production/secretion of brain‐derived neurotrophic factor (BDNF) without induction of deleterious factors in vitro

In analyzing the regulation of neurotrophin production/secretion from microglia, C8‐ceramide (d‐erythro‐sphingosine, N‐octanoyl‐) was found to induce secretion of brain‐derived neurotrophic factor (BDNF) from microglia in vitro. In the present study, the action of C8‐ceramide in secreting neurotrophic and harmful factors was investigated and compared with the effects of lipopolysaccharide (LPS). C8‐ceramide as well as LPS enhanced the production/secretion of BDNF but, different from LPS, did not induce tumor necrosis factor α, interleukin‐1β, or nitric oxide. The C8‐ceramide‐induced BDNF release was significantly suppressed by protein kinase C (PKC) inhibitor, bisindolylmaleimide, which targets PKC isoforms, α, β, γ, δ and ε. However, it was not suppressed by a specific inhibitor of PKCα. Furthermore, PKCβ and γ were undetected in the microglia. Therefore, PKCδ and/or ε appear to be functioning PKC isoforms. In contrast, none of the mitogen‐activated protein kinases (MAPKs) and none of the transcription factors, including the cAMP response element‐binding transcription factor (CREB) and nuclear factor κB (NFκB) were activated in the microglia in response to C8‐ceramide. These results indicate that ceramide‐induced BDNF release in microglia is mediated by a signaling pathway associated with PKCδ and/or ε, but not with activation of MAPKs, CREB and NFκB.

Phylogenetic relation of lungfish indicated by the amino acid sequence of myelin DM20

The cDNA of lungfish Protopterus annectens myelin DM20 was cloned, and the complete amino acid sequence of Protopterusannectens DM20 was deduced. When five possible phylogenetic trees were tested for the DM20 sequences, the maximum likelihood method supported tree 1 [((tetrapods, lungfish), coelacanth), zebrafish, shark] or tree 5 [(tetrapods, lungfish), (coelacanth, zebrafish), shark]. Both tree 1 and tree 5 indicate that lungfish is phylogenetically the closest to tetrapods among the living fishes.

Neuronal regulation by which microglia enhance the production of neurotrophic factors for GABAergic, catecholaminergic, and cholinergic neurons.

A phenomenon-in which microglia are activated in axotomized rat facial nucleus suggests that a certain neuronal stimulus triggers the activation of microglia. However, how the microglial characteristics are regulated by this neuronal stimulus has not previously been determined. In this study, therefore, the regulation of microglial properties by neurons was characterized in vitro from a neurotrophic perspective. To evaluate the neurotrophic effects of microglia stimulated with neurons, the effects of conditioned medium (CM) of microglia stimulated with neuronal CM (NCM) were assessed in neuronal cultures. The amounts of tyrosine hydroxylase (TH) in neuronal culture exposed to CM of microglia stimulated with NCM was much more than those in neurons exposed to CM of control microglia, suggesting that neuronal stimulus enhances the production of neurotrophic factors for catecholaminergic neurons in microglia. Therefore, the neurotrophic effects of CM of microglia stimulated with NCM were analyzed in detail. The immunocytochemical and biochemical experiments revealed that the CM of microglia stimulated with NCM enhances the survival/maturation of GABAergic and catecholaminergic neurons. The levels of choline acetyltransferase specific to cholinergic neurons also significantly increased in response to stimulation with the same microglial CM. These results allowed us to investigate the production of neurotrophic factors in the CM of microglia stimulated with NCM. The results indicated that NCM induces nerve growth factor (NGF), and enhances neurotrophin-4/5 (NT-4/5), transforming growth factor beta1 (TGFbeta1), glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), interleukin-3 (IL-3), and IL-10 in microglia. The promoted neurotrophic effects of CM of microglia stimulated with NCM were significantly abrogated by deprivation of neurotrophic factors by means of an immunoprecipitation method. Taken together, neuronal stimulus was found to activate microglia to produce more neurotrophic factors as above, thereby changing microglia into more neurotrophic cells.

Protein kinase Cα requirement in the activation of p38 mitogen-activated protein kinase, which is linked to the induction of tumor necrosis factor α in lipopolysaccharide-stimulated microglia

Abstract Activated microglia have been suggested to produce a cytotoxic cytokine, tumor necrosis factor α (TNFα), in many pathological brains. Thus, determining the molecular mechanism of this induction and suppression has been the focus of a great deal of research. Using lipopolysaccharide (LPS) as an experimental inducer of TNFα, we investigated the regulatory mechanism by which TNFα is induced or suppressed in microglia. We found that LPS-induced TNFα is suppressed by pretreatment with the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580. Similar suppression was achieved by pretreatment with specific protein kinase C (PKC) inhibitors, Go6976, myristoylated pseudosubstrate (20–28), and bisindolylmaleimide. These results suggest that PKCα activity as well as p38MAPK activity is associated with TNFα induction in LPS-stimulated microglia. The requirement of PKCα in LPS-dependent TNFα induction was verified in PKCα-downregulated microglia which could be induced by phorbol-12-myristate-13-acetate pretreatment. Simultaneously, PKCα was found to be requisite for the activation of p38MAPK in LPS-stimulated microglia. In addition, the PKCα levels in the LPS-stimulated microglia were observed to decrease in response to the p38MAPK inhibitor, indicating that the PKCα levels are regulated by the p38MAPK activity. We therefore concluded that PKCα and p38MAPK are interactively linked to the signaling cascade inducing TNFα in LPS-stimulated microglia, and that in this cascade, PKCα is requisite for the activation of p38MAPK, leading to the induction of TNFα.

Nonparticipation of nuclear factor kappa B (NFκB) in the signaling cascade of c-Jun N-terminal kinase (JNK)- and p38 mitogen-activated protein kinase (p38MAPK)-dependent tumor necrosis factor alpha (TNFα) induction in lipopolysaccharide (LPS)-stimulated microglia

The molecular mechanism of cytotoxic cytokine tumor necrosis factor alpha (TNFalpha) induction in microglia remains to be clarified. We have previously reported that p38 mitogen-activated protein kinase (p38MAPK) is an important signaling molecule for the induction of TNFalpha in lipopolysaccharide (LPS)-stimulated microglia. Recently, we have shown that c-Jun N-terminal kinase (JNK) is associated with the induction of TNFalpha. Furthermore, using an NFkappaB inhibitor (SN50), we discovered that activation of nuclear factor kappaB (NFkappaB) may also be linked to TNFalpha induction. We therefore examined the relationship between NFkappaB and the two MAPKs (p38MAPK and JNK) in the signaling cascade of TNFalpha induction in LPS-stimulated microglia. NFkappaB inhibitor SN50 decreased the induction of TNFalpha under the suppressed NFkappaB activation. However, SN50 was found to prevent the activation of MKK3/6-p38MAPK and MKK4-JNK pathways. On the other hand, the other NFkappaB inhibitor ammonium pyrrolidine dithiocarbamate (APDC) neither prevented the activation of p38MAPK and JNK nor inhibited TNFalpha induction in LPS-stimulated microglia, although it was confirmed to serve as an NFkappaB inhibitor. These results suggest that both MKK3/6-p38MAPK and MKK4-JNK pathways are important signaling cascades leading to the induction of TNFalpha in LPS-stimulated microglia, but that NFkappaB itself is not required for this induction.

Differential suppression of endotoxin-inducible inflammatory cytokines by nuclear factor kappa B (NFκB) inhibitor in rat microglia

The molecular mechanism by which the deleterious cytokines interleukin 1 beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) are induced in endotoxin-stimulated microglia was investigated from the viewpoint of signal transduction. Neither cytokine is produced in nonstimulated rat microglia, but both are remarkably induced by stimulation with endotoxin lipopolysaccharide (LPS). LPS-inducible IL-1beta was significantly suppressed by pretreatment with the nuclear factor kappa B (NFkappaB) inhibitor ammonium pyrrolidine dithiocarbamate (APDC), but TNFalpha was not. APDC was actually confirmed to suppress the degradation of IkappaBalpha and IkappaBbeta in microglia, indicating a role for the inhibitor of NFkappaB activation. Taken together, these results suggest that the induction of IL-1beta and TNFalpha in endotoxin-stimulated microglia is differentially regulated at the level of NFkappaB activation.

Activation of microglia by endotoxin suppresses the secretion of glial cell line–derived neurotrophic factor (GDNF) through the action of protein kinase Cα (PKCα) and mitogen-activated protein kinases (MAPKS)

The ability of microglia to produce/secrete glial cell line–derived neurotrophic factor (GDNF) in vitro was examined. Immunoblotting analysis revealed that nonstimulated microglia release limited amounts of GDNF with molecular sizes of 14 and 17 kDa. However, the secreted amounts significantly decreased when the microglia were activated with the endotoxin lipopolysaccharide (LPS). Comparison of the amounts of GDNF in the cells and the conditioned medium between the nonstimulated microglia and LPS‐stimulated microglia clarified that the secretion of GDNF, but not its production, is strongly suppressed when the microglia are activated with LPS. The inhibitor experiments suggested that the GDNF secretion is depressed by a signaling cascade associated with protein kinase C alpha (PKCα) and/or mitogen‐activated protein kinases (MAPKs). As expected from the above results, a PKC activator suppressed the secretion of GDNF in nonstimulated microglia. Taken together, these results demonstrated that microglia have the ability to produce and secrete GDNF in vitro, and that the secretion is suppressed by stimulation with endotoxin, probably due to a signaling mechanism involving PKCα and/or MAPKs.

Gene Structure and Amino Acid Sequence of Latimeria chalumnae (Coelacanth) Myelin DM20: Phylogenetic Relation of the Fish

The structure of Latimeria chalumnae (coelacanth) proteolipid protein/DM20 gene excluding exon 1 was determined, and the amino acid sequence of Latimeria DM20 corresponding to exons 2–7 was deduced. The nucleotide sequence of exon 3 suggests that only DM20 isoform is expressed in Latimeria. The structure of proteolipid protein/DM20 gene is well preserved among human, dog, mouse, and Latimeria. Southern blot analysis indicates that Latimeria DM20 gene is a single-copy gene. When the amino acid sequences of DM20 were compared among various species, Latimeria was more similar to tetrapods than other fishes including lungfish, confirming the previous finding by immunoreactivity (Waehneldt and Malotka 1989 J. Neurochem. 52:1941–1943). However, when phylogenetic trees were constructed from the DM20 sequences, lungfish was clearly the closest to tetrapods. Latimeria was situated outside of lungfish by the maximum likelihood method. The apparent similarity of Latimeria DM20 to tetrapod proteolipid protein/DM20 is explained by the slow amino acid substitution rate of Latimeria DM20.