Language：English   Publishing type：Research paper (scientific journal)  

A number of clinical and experimental studies have revealed a strong association between periodontitis and accelerated cognitive decline in Alzheimer's disease (AD); however, the mechanism of the association is unknown. In the present study, we tested the hypothesis that cathepsin (Cat) B plays a critical role in the initiation of neuroinflammation and neural dysfunction following chronic systemic exposure to lipopolysaccharide from Porphyromonas gingivalis (PgLPS) in mice (1 mg/kg, daily, intraperitoneally). Young (2 months old) and middle-aged (12 months old) wild-type (WT; C57BL/6N) or CatB-deficient (CatB^{− / −}) mice were exposed to PgLPS daily for 5 consecutive weeks. The learning and memory function were assessed using the passive avoidance test, and the expression of amyloid precursor protein (APP), CatB, TLR2 and IL-1β was analyzed in brain tissues by immunohistochemistry and Western blotting. We found that chronic systemic exposure to PgLPS for five consecutive weeks induced learning and memory deficits with the intracellular accumulation of Aβ in neurons in the middle-aged WT mice, but not in young WT or middle-aged CatB^{− / −} mice. PgLPS significantly increased the expression of CatB in both microglia and neurons in middle-aged WT mice, while increased expression of mature IL-1β and TLR2 was restricted to microglia in the hippocampus of middle-aged WT mice, but not in that of the middle-aged CatB^{− / −} ones. In in vitro studies, PgLPS (1 µg/ml) stimulation upregulated the mean mRNA expression of IL-1β, TLR2 and downregulated the protein levels of IκBα in the cultured MG6 microglia as well as in the primary microglia from WT mice, which were significantly inhibited by the CatB-specific inhibitor CA-074Me as well as by the primary microglia from CatB^{− / −} mice. Furthermore, the mean mRNA expression of APP and CatB were significantly increased in the primary cultured hippocampal neurons after treatment with conditioned medium from PgLPS-treated WT primary microglia, but not after treatment with conditioned medium neutralized with anti-IL-1beta, and not after treatment with conditioned medium from PgLPS-treated CatB^{− / −} primary microglia or with PgLPS directly. Taken together, these findings indicate that chronic systemic exposure to PgLPS induces AD-like phenotypes, including microglia-mediated neuroinflammation, intracellular Aβ accumulation in neurons and impairment of the learning and memory functions in the middle-aged mice in a CatB-dependent manner. We propose that CatB may be a therapeutic target for preventing periodontitis-associated cognitive decline in AD.

DOI： 10.1016/j.bbi.2017.06.002

Language：English   Publishing type：Research paper (scientific journal)  

The expression of cathepsin S (CatS), a microglia-specific lysosomal cysteine protease in the brain, is regulated by the intrinsic microglial circadian clock. We herein report that the diurnal variation of evoked synaptic responses of cortical neurons disappeared in cathepsin S-deficient (CatS^−/−) mice. The dendritic spine density of the cortical neurons was significantly reduced by incubation with a recombinant CatS. Furthermore, CatS^−/− mice exhibited impaired social interaction and social novelty recognition in the three-chamber test. These findings indicate that the circadian clock-regulated secretion of CatS from microglia is involved in the diurnal variation of synaptic responses and dendritic spine density through the proteolytic modification of perisynaptic ECM molecules. Therefore, a dysfunction of the diurnal synaptic responses due to CatS deficiency may lead to social behavior abnormalities.

DOI： 10.1016/j.bbrc.2017.06.061

Language：English   Publishing type：Research paper (scientific journal)  

The expression of cathepsin S (CatS), a microglia-specific lysosomal cysteine protease in the brain, is regulated by the intrinsic microglial circadian clock. We herein report that the diurnal variation of evoked synaptic responses of cortical neurons disappeared in cathepsin S-deficient (CatS^−/−) mice. The dendritic spine density of the cortical neurons was significantly reduced by incubation with a recombinant CatS. Furthermore, CatS^−/− mice exhibited impaired social interaction and social novelty recognition in the three-chamber test. These findings indicate that the circadian clock-regulated secretion of CatS from microglia is involved in the diurnal variation of synaptic responses and dendritic spine density through the proteolytic modification of perisynaptic ECM molecules. Therefore, a dysfunction of the diurnal synaptic responses due to CatS deficiency may lead to social behavior abnormalities.

DOI： 10.1016/j.bbrc.2017.06.061

Language：English   Publishing type：Research paper (scientific journal)  

Cathepsin E (CatE), an aspartic protease, has a limited distribution in certain cell types such as gastric cells. CatE is not detectable in the normal brain, whereas it is increasingly expressed in damaged neurons and activated microglia of the pathological brain. Neurons expressing high levels of CatE showed apparent morphological changes, including a marked shrinkage of the cytoplasmic region and beading of neurites, suggesting neuronal damage. The intracellular level of CatE in neurons is strictly regulated at both transcriptional and translational levels. Although the up-regulation of CatE may cause pathological changes in neurons, little information is available about the precise outcome of the increased expression of CatE in neurons. In this study, we have attempted to clarify the outcome of up-regulated CatE gene expression in neurons using the P19 cell neuronal differentiation after the overexpression of CatE. We unexpectedly found that the overexpression of CatE interfered with neuronal differentiation of P19 cells through an impairment of cell aggregate formation. Pepstatin A, an aspartic protease inhibitor, restored the impaired cell aggregation of P19/CatE cells. The small number of P19 cells differentiated into neurons had abnormal morphology characterized by their fusiform cell bodies with short processes. Furthermore, CatE proteolytically cleaved the extracellular domain of N-cadherin. These observations suggest that the overexpression of CatE interferes with neuronal differentiation of P19 cells through an impairment of cell aggregate formation, possibly through proteolytic degradation of N-cadherin.

DOI： 10.1007/s10571-016-0376-x

Language：English   Publishing type：Research paper (scientific journal)  

Cathepsin E (CatE), an aspartic protease, has a limited distribution in certain cell types such as gastric cells. CatE is not detectable in the normal brain, whereas it is increasingly expressed in damaged neurons and activated microglia of the pathological brain. Neurons expressing high levels of CatE showed apparent morphological changes, including a marked shrinkage of the cytoplasmic region and beading of neurites, suggesting neuronal damage. The intracellular level of CatE in neurons is strictly regulated at both transcriptional and translational levels. Although the up-regulation of CatE may cause pathological changes in neurons, little information is available about the precise outcome of the increased expression of CatE in neurons. In this study, we have attempted to clarify the outcome of up-regulated CatE gene expression in neurons using the P19 cell neuronal differentiation after the overexpression of CatE. We unexpectedly found that the overexpression of CatE interfered with neuronal differentiation of P19 cells through an impairment of cell aggregate formation. Pepstatin A, an aspartic protease inhibitor, restored the impaired cell aggregation of P19/CatE cells. The small number of P19 cells differentiated into neurons had abnormal morphology characterized by their fusiform cell bodies with short processes. Furthermore, CatE proteolytically cleaved the extracellular domain of N-cadherin. These observations suggest that the overexpression of CatE interferes with neuronal differentiation of P19 cells through an impairment of cell aggregate formation, possibly through proteolytic degradation of N-cadherin.

DOI： 10.1007/s10571-016-0376-x

Language：English   Publishing type：Research paper (scientific journal)  

It has long been believed that microglia morphologically transform into the activated state by retracting their long processes and consuming pathogens when bacteria infect into the brain parenchyma. In the present study, however, we showed for the first time that murine cortical microglia extend their processes towards focally injected Porphyromonas gingivalis. This P. gingivalis-induced microglial process extension was significantly increased during the light (sleeping) phase than the dark (waking) phase. In contrast, focally injected ATP-induced microglial process extension was significantly increased during the dark phase than the light phase. Furthermore, in contrast to the P2Y 12 receptor-mediated mechanism of ATP-induced microglial process extension, the P. gingivalis-mediated microglial process extension was mediated by P2Y 6 receptors. The infection of bacteria such as P. gingivalis to the brain parenchyma may induce the secretion of UDP from microglia at the site of infection, which in turn induces the process extension of the neighboring microglia.

DOI： 10.1038/srep30006

Language：English   Publishing type：Research paper (scientific journal)  

It has long been believed that microglia morphologically transform into the activated state by retracting their long processes and consuming pathogens when bacteria infect into the brain parenchyma. In the present study, however, we showed for the first time that murine cortical microglia extend their processes towards focally injected Porphyromonas gingivalis. This P. gingivalis-induced microglial process extension was significantly increased during the light (sleeping) phase than the dark (waking) phase. In contrast, focally injected ATP-induced microglial process extension was significantly increased during the dark phase than the light phase. Furthermore, in contrast to the P2Y 12 receptor-mediated mechanism of ATP-induced microglial process extension, the P. gingivalis-mediated microglial process extension was mediated by P2Y 6 receptors. The infection of bacteria such as P. gingivalis to the brain parenchyma may induce the secretion of UDP from microglia at the site of infection, which in turn induces the process extension of the neighboring microglia.

DOI： 10.1038/srep30006

Language：English   Publishing type：Research paper (scientific journal)  

Hypoxia has been recently proposed as a neuroinflammatogen, which drives microglia to produce proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α; (TNF-α;), and IL-6. Considering the fact that propolis has hepatoprotective, antitumor, antioxidative, and anti-inflammatory effects, propolis may have protective effects against the hypoxia-induced neuroinflammatory responses. In this study, propolis (50 g/mL) was found to significantly inhibit the hypoxia-induced cytotoxicity and the release of proinflammatory cytokines, including IL-1β, TNF-α;, and IL-6, by MG6 microglia following hypoxic exposure (1% O ₂, 24 h). Furthermore, propolis significantly inhibited the hypoxia-induced generation of reactive oxygen species (ROS) from mitochondria and the activation of nuclear factor-B (NF-B) in microglia. Moreover, systemic treatment with propolis (8.33 mg/kg, 2 times/day, i.p.) for 7 days significantly suppressed the microglial expression of IL-1β, TNF-α;, IL-6, and 8-oxo-deoxyguanosine, a biomarker for oxidative damaged DNA, in the somatosensory cortex of mice subjected to hypoxia exposure (10% O₂, 4 h). These observations indicate that propolis suppresses the hypoxia-induced neuroinflammatory responses through inhibition of the NF-B activation in microglia. Furthermore, increased generation of ROS from the mitochondria is responsible for the NF-B activation. Therefore, propolis may be beneficial in preventing hypoxia-induced neuroinflammation.

DOI： 10.1155/2013/906726

Language：English   Publishing type：Research paper (scientific journal)  

Hypoxia has been recently proposed as a neuroinflammatogen, which drives microglia to produce proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α; (TNF-α;), and IL-6. Considering the fact that propolis has hepatoprotective, antitumor, antioxidative, and anti-inflammatory effects, propolis may have protective effects against the hypoxia-induced neuroinflammatory responses. In this study, propolis (50 g/mL) was found to significantly inhibit the hypoxia-induced cytotoxicity and the release of proinflammatory cytokines, including IL-1β, TNF-α;, and IL-6, by MG6 microglia following hypoxic exposure (1% O ₂, 24 h). Furthermore, propolis significantly inhibited the hypoxia-induced generation of reactive oxygen species (ROS) from mitochondria and the activation of nuclear factor-B (NF-B) in microglia. Moreover, systemic treatment with propolis (8.33 mg/kg, 2 times/day, i.p.) for 7 days significantly suppressed the microglial expression of IL-1β, TNF-α;, IL-6, and 8-oxo-deoxyguanosine, a biomarker for oxidative damaged DNA, in the somatosensory cortex of mice subjected to hypoxia exposure (10% O₂, 4 h). These observations indicate that propolis suppresses the hypoxia-induced neuroinflammatory responses through inhibition of the NF-B activation in microglia. Furthermore, increased generation of ROS from the mitochondria is responsible for the NF-B activation. Therefore, propolis may be beneficial in preventing hypoxia-induced neuroinflammation.

DOI： 10.1155/2013/906726

Language：English   Publishing type：Research paper (scientific journal)  

Liposomes containing phosphatidylserine (PSL) produce PGE₂ after being phagocytosed by microglia, but the precise underlying mechanism behind it still remains unclear. Here, we showed that liposomes consisting of phosphatidylserine and lysophosphatidylcholine, a lipolysis product of phosphatidylcholine by PLA₂, were phagocytosed by microglia, but failed to induce secretion of PGE₂. Furthermore, PSL-induced PGE₂ secretion was significantly inhibited by MJ33, an aiPLA₂ inhibitor, but not by AACOCF₃, a cPLA₂ inhibitor. PSL also produced PGD₂ and 15d-PGJ₂ in microglia. We thus hypothesize that free arachidonic acid is supplied through aiPLA₂-mediated lipolysis of phagocytosed phosphatidylcholine, leading to the production of PGH₂ and its downstream metabolites.

DOI： 10.1016/j.jneuroim.2013.06.011

Language：English   Publishing type：Research paper (scientific journal)  

Liposomes containing phosphatidylserine (PSL) produce PGE₂ after being phagocytosed by microglia, but the precise underlying mechanism behind it still remains unclear. Here, we showed that liposomes consisting of phosphatidylserine and lysophosphatidylcholine, a lipolysis product of phosphatidylcholine by PLA₂, were phagocytosed by microglia, but failed to induce secretion of PGE₂. Furthermore, PSL-induced PGE₂ secretion was significantly inhibited by MJ33, an aiPLA₂ inhibitor, but not by AACOCF₃, a cPLA₂ inhibitor. PSL also produced PGD₂ and 15d-PGJ₂ in microglia. We thus hypothesize that free arachidonic acid is supplied through aiPLA₂-mediated lipolysis of phagocytosed phosphatidylcholine, leading to the production of PGH₂ and its downstream metabolites.

DOI： 10.1016/j.jneuroim.2013.06.011

Event date： 2018.10

Language：Japanese  

Venue：福岡県歯科医師会館   Country：Japan  

低ホスファターゼ症（HPP）は、組織非特異的アルカリホスファターゼ（ALP）遺伝子異常による先天性代謝性骨疾患であり、骨石灰化の低下等に加え、乳歯の早期脱落が特徴の１つである。歯の異常と骨疾患は関連が深く、医科との連携が求められる。今回我々は、下顎乳前歯の早期脱落を主訴に当科を来院し、小児科での精査の結果HPPと診断された１例を経験した。その治療経過について報告する。