Decreases in the number of microglia and neural circuit dysfunction elicited by developmental exposure to neonicotinoid pesticides in mice.

Neonicotinoids are insecticides widely used in the world. Although neonicotinoids are believed to be toxic only to insects, their developmental neurotoxicity in mammals is a concern. Therefore, we examined the effects of developmental exposure to neonicotinoids on immune system in the brain and post-developmental behaviors in this study. Imidacloprid or clothianidin was orally administered to dams at a dosage of 0.1 mg/kg/day from embryonic day 11 to postnatal day 21. Imidacloprid decreased sociability, and both imidacloprid and clothianidin decreased locomotor activity and induced anxiety, depression and abnormal repetitive behaviors after the developmental period. There was no change in the number of neurons in the hippocampus of mice exposed to imidacloprid. However, the number and activity of microglia during development were significantly decreased by imidacloprid exposure. Imidacloprid also induced neural circuit dysfunction in the CA1 and CA3 regions of the hippocampus during the early postnatal period. Exposure to imidacloprid suppressed the expression of csf1r during development. Collectively, these results suggest that developmental exposure to imidacloprid decreases the number and activity of microglia, which can cause neural circuit dysfunction and abnormal behaviors after the developmental period. Care must be taken to avoid exposure to neonicotinoids, especially during development.

A simple air-liquid interface exposure system for exposing cultured human 3D epidermis and cornea to PM2.5 collected through cyclonic separation.

Particulate matter (PM) is among the major air pollutants suspended in the atmosphere. PM2.5 has a particle size of 2.5 µm; it is known to cause inflammation, especially in the respiratory tract and skin. Since the skin acts a primary barrier against harmful environmental substances that may enter the body, it is highly exposed to PM2.5 present in the environment. However, the adverse health effects of PM2.5 exposure on human skin have not been accurately examined due to the lack of a system that exposes human epidermal tissue to the actual environmental concentration of PM2.5. In this study, we developed an air-liquid interface exposure system for exposing cultured human 3D epidermis and cornea to PM2.5 collected through cyclonic separation. PM2.5 suspension was nebulized in an acrylic chamber, and the resulting mist was pumped through a diffusion dryer into a glass exposure chamber. A particle counter was connected to the exposure chamber to continuously measure the spatial mass concentration of PM. Human 3D epidermis was cultured in the exposure chamber. Exposure of the human 3D epidermis to PM aerosol increased interleukin-8 release into the media around 50 µg/m3. Mass concentrations above 100 µg/m3 caused cell death. Furthermore, a human corneal model showed similar responses against PM2.5 exposure as 3D epidermis. The air-liquid interface exposure system developed in this study is considered useful for evaluating the health effects induced by environmental PM2.5 and can be used as an alternative to experiments involving actual human or animals.

Maternal DHA intake in mice increased DHA metabolites in the pup brain and ameliorated MeHg-induced behavioral disorder.

Although pregnant women's fish consumption is beneficial for the brain development of the fetus due to the DHA in fish, seafood also contains methylmercury (MeHg), which adversely affects fetal brain development. Epidemiological studies suggest that high DHA levels in pregnant women's sera may protect the fetal brain from MeHg-induced neurotoxicity, but the underlying mechanism is unknown. Our earlier study revealed that DHA and its metabolite 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP) produced by cytochrome P450s (P450s) and soluble epoxide hydrolase (sEH) can suppress MeHg-induced cytotoxicity in mouse primary neuronal cells. In the present study, DHA supplementation to pregnant mice suppressed MeHg-induced impairments of pups' body weight, grip strength, motor function, and short-term memory. DHA supplementation also suppressed MeHg-induced oxidative stress and the decrease in the number of subplate neurons in the cerebral cortex of the pups. DHA supplementation to dams significantly increased the DHA metabolites 19,20-epoxydocosapentaenoic acid (19,20-EDP) and 19,20-DHDP as well as DHA itself in the fetal and infant brains, although the expression levels of P450s and sEH were low in the fetal brain and liver. DHA metabolites were detected in the mouse breast milk and in human umbilical cord blood, indicating the active transfer of DHA metabolites from dams to pups. These results demonstrate that DHA supplementation increased DHA and its metabolites in the mouse pup brain and alleviated the effects of MeHg on fetal brain development. Pregnant women's intake of fish containing high levels of DHA (or DHA supplementation) may help prevent MeHg-induced neurotoxicity in the fetus.

Suppressive Effects of Docosahexaenoic Acid Intake on Increased Seizure Susceptibility after Growth Due to Febrile Seizures in Infancy.

Febrile seizures are seizures accompanied by a fever and frequently occur in children six months to five years of age. Febrile seizures are classified as simple or complex, and complex febrile seizures increase the risk of temporal lobe epilepsy after growth. Therefore, it is important to interfere with epileptogenesis after febrile seizures to prevent post-growth epilepsy. The present study challenged nutritional intervention using docosahexaenoic acid (DHA). Febrile seizures were induced in mice at the age of 10 d using a heat chamber, and seizure sensitivity was examined using pentylenetetrazol (PTZ) administration after growth. PTZ increased the seizure score and shortened the latency in the complex febrile seizure group compared to the control, hyperthermia and simple febrile seizure groups. Mice in the complex febrile seizure group showed abnormal electroencephalograms pre- and post-PTZ administration. Therefore, seizure susceptibility increases the episodes of complex febrile seizures. DHA supplementation after febrile seizures clearly suppressed the increased seizure susceptibility due to complex febrile seizures experienced in infancy. DHA also attenuated microglial activation after complex febrile seizures. Taken together, DHA suppressed microglial activation following complex febrile seizures, which may contribute to protecting the brain from post-growth seizures. The intake of DHA in infancy may protect children from high fever-induced developmental abnormalities.

Regulation of IDO2 by the Aryl Hydrocarbon Receptor (AhR) in Breast Cancer.

Indoleamine 2,3-dioxygenase 2 (IDO2) is a tryptophan-catabolizing enzyme and a homolog of IDO1 with a distinct expression pattern compared with IDO1. In dendritic cells (DCs), IDO activity and the resulting changes in tryptophan level regulate T-cell differentiation and promote immune tolerance. Recent studies indicate that IDO2 exerts an additional, non-enzymatic function and pro-inflammatory activity, which may play an important role in diseases such as autoimmunity and cancer. Here, we investigated the impact of aryl hydrocarbon receptor (AhR) activation by endogenous compounds and environmental pollutants on the expression of IDO2. Treatment with AhR ligands induced IDO2 in MCF-7 wildtype cells but not in CRISPR-cas9 AhR-knockout MCF-7 cells. Promoter analysis with IDO2 reporter constructs revealed that the AhR-dependent induction of IDO2 involves a short-tandem repeat containing four core sequences of a xenobiotic response element (XRE) upstream of the start site of the human ido2 gene. The analysis of breast cancer datasets revealed that IDO2 expression increased in breast cancer compared with normal samples. Our findings suggest that the AhR-mediated expression of IDO2 in breast cancer could contribute to a pro-tumorigenic microenvironment in breast cancer.

The effect of mask fit test on the association between the concentration of metals in biological samples and the results of time-weighted average personal exposure: A study on Japanese male welders.

OBJECTIVES: The mask fit test confirms whether the wearing condition of the wearer's face and the facepiece of the respirators are used appropriately. This study aimed to examine whether the results of the mask fit test affect the association between the concentration of metals related to welding fumes in biological samples and the results of time-weighted average (TWA) personal exposures. METHODS: A total of 94 male welders were recruited. Blood and urine samples were obtained from all participants to measure the metal exposure levels. Using personal exposure measurements, the 8-h TWA (8 h-TWA) of respirable dust, TWA of respirable Mn, and 8-h TWA of respirable Mn were calculated. The mask fit test was performed using the quantitative method specified in the Japanese Industrial Standard T8150:2021. RESULTS: Fifty-four participants (57%) passed the mask fit test. Only in the Fail group of the mask fit test, it was observed that blood Mn concentrations be positively associated with the results of TWA personal exposure after adjusting for multivariate factors (8-h TWA of respirable dust; coefficient, 0.066; standard error (SE), 0.028; P = 0.018, TWA of respirable Mn: coefficient, 0.048; SE, 0.020; P = 0.019, 8 h-TWA of respirable Mn: coefficient, 0.041; SE, 0.020; P = 0.041). CONCLUSIONS: The results clarify that welders with high concentrations of welding fumes in their breathing air zone are exposed to dust and Mn if there is leaking air owing to the lack of fitness between respirators and the wearer's face when using human samples in Japan.

Complementary spectroscopy studies and potential activities of levan-type fructan produced by Bacillus paralicheniformis ND2.

This study aimed at the production of marine bacterial exopolysaccharides (EPS) as biodegradable and nontoxic biopolymers, competing the synthetic derivatives, with detailed structural and conformational analyses using spectroscopy techniques. Twelve marine bacterial bacilli were isolated from the seawater of Mediterranean Sea, Egypt, then screened for EPS production. The most potent isolate was identified genetically as Bacillus paralicheniformis ND2 by16S rRNA gene sequence of ~99 % similarity. Plackett-Burman (PB) design identified the optimization conditions of EPS production, which yielded the maximum EPS (14.57 g L-1) with 1.26-fold increase when compared to the basal conditions. Two purified EPSs namely NRF1 and NRF2 with average molecular weights (Mw¯) of 15.98 and 9.70 kDa, respectively, were obtained and subjected for subsequent analyses. FTIR and UV-Vis reflected their purity and high carbohydrate contents while EDX emphasized their neutral type. NMR identified the EPSs as levan-type fructan composed of ß-(2-6)-glycosidic linkage as a main backbone, and HPLC explained that the EPSs composed of fructose. Circular dichroism (CD) suggested that NRF1 and NRF2 had identical structuration with a little variation from the EPS-NR. The EPS-NR showed antibacterial activity with the maximum inhibition against S. aureus ATCC 25923. Furthermore, all the EPSs revealed a proinflammatory action through dose-dependent increment of expression of proinflammatory cytokine mRNAs, IL-6, IL-1ß and TNFα.

Microglial inflammatory reactions regulated by oxidative stress.

Microglia are immune cells in the brain that can respond to endogenous and exogenous substrates to elicit inflammatory reactions. The transcription factor nuclear factor kappa-light-chain-enhancer of activated B induces proinflammatory gene expression in response to foreign matter via pattern recognition receptors; thus, nuclear factor kappa-light-chain-enhancer of activated B is a master regulator of inflammation. During the inflammatory process, very large amounts of reactive oxygen species are generated and promote the onset and progression of inflammation. Interestingly, nuclear factor kappa-light-chain-enhancer of activated B drives the transcription of superoxide dismutase 2 in many types of cells, including microglia. Superoxide dismutase 2 is an antioxidative enzyme that catalyzes the dismutation of superoxide anions into molecular oxygen and hydrogen peroxide. Of note, nuclear factor kappa-light-chain-enhancer of activated B can initiate inflammation to elicit proinflammatory gene expression, while its transcription product superoxide dismutase 2 can suppress inflammation. In this review, we use recent knowledge to describe the interaction between oxidative stress and nuclear factor kappa-light-chain-enhancer of activated B and discuss the complicated role of microglial superoxide dismutase 2 in inflammation.

Associations between welding fume exposure and neurological function in Japanese male welders and non-welders.

OBJECTIVES: There are some studies reporting the association between (manganese [Mn]) exposure to welding fume and neurological dysfunction. This study examined the relationship between Mn exposure and neurological behavior in Japanese male welders and non-welders using biological samples, which to date has not been assessed in Japan. METHODS: A total of 94 male welders and 95 male non-welders who worked in the same factories were recruited. The blood and urine samples were obtained from all the participants to measure Mn exposure levels. Neurological function tests were also conducted with all participants. The sampling of the breathing air zone using a personal sampler was measured for welders only. RESULTS: The odds ratios (ORs) for the Working Memory Index (WMI) scores were significantly higher among all participants in the low blood Mn concentration group than those in the high blood Mn concentration group (OR, 2.77; 95% confidence interval [CI], 1.24, 6.19; P = .013). The association of WMI scores and blood Mn levels in welders had the highest OR (OR, 3.73; 95% CI, 1.04, 13.38; P = .043). Although not statistically significant, a mild relationship between WMI scores and blood Mn levels was observed in non-welders (OR, 2.09; 95% CI, 0.63, 6.94; P = .227). CONCLUSIONS: The results revealed a significant positive relationship between blood Mn and neurological dysfunction in welders. Furthermore, non-welders at the same factories may be secondarily exposed to welding fumes. Further research is needed to clarify this possibility.

Polycyclic aromatic hydrocarbons in urban particle matter exacerbate movement disorder after ischemic stroke via potentiation of neuroinflammation.

BACKGROUND: A recent epidemiological study showed that air pollution is closely involved in the prognosis of ischemic stroke. We and others have reported that microglial activation in ischemic stroke plays an important role in neuronal damage. In this study, we investigated the effects of urban aerosol exposure on neuroinflammation and the prognosis of ischemic stroke using a mouse photothrombotic model. RESULTS: When mice were intranasally exposed to CRM28, urban aerosols collected in Beijing, China, for 7 days, microglial activation was observed in the olfactory bulb and cerebral cortex. Mice exposed to CRM28 showed increased microglial activity and exacerbation of movement disorder after ischemic stroke induction. Administration of core particles stripped of attached chemicals from CRM28 by washing showed less microglial activation and suppression of movement disorder compared with CRM28-treated groups. CRM28 exposure did not affect the prognosis of ischemic stroke in null mice for aryl hydrocarbon receptor, a polycyclic aromatic hydrocarbon (PAH) receptor. Exposure to PM2.5 collected at Yokohama, Japan also exacerbated movement disorder after ischemic stroke. CONCLUSION: Particle matter in the air is involved in neuroinflammation and aggravation of the prognosis of ischemic stroke; furthermore, PAHs in the particle matter could be responsible for the prognosis exacerbation.

Chemokine expression in human 3-dimensional cultured epidermis exposed to PM2.5 collected by cyclonic separation.

Fine particulate matter (PM2.5) exposure has a risk of inducing several health problems, especially in the respiratory tract. The skin is the largest organ of the human body and is therefore the primary target of PM2.5. In this study, we examined the effects of PM2.5 on the skin using a human 3-dimensional cultured epidermis model. PM2.5 was collected by cyclonic separation in Yokohama, Japan. Global analysis of 34 proteins released from the epidermis revealed that the chemokines, chemokine C-X-C motif ligand 1 (CXCL1) and interleukin 8 (IL-8), were significantly increased in response to PM2.5 exposure. These chemokines stimulated neutrophil chemotaxis in a C-X-C motif chemokine receptor 2-dependent manner. The oxidative stress and signal transducer and activator of transcription 3 pathways may be involved in the increased expression of CXCL1 and IL-8 in the human epidermis model. Interestingly, in the HaCaT human keratinocyte cell line, PM2.5 did not affect chemokine expression but did induce IL-6 expression, suggesting a different effect of PM2.5 between the epidermis model and HaCaT cells. Overall, PM2.5 could induce the epidermis to release chemokines, followed by neutrophil activation, which might cause an unregulated inflammatory reaction in the skin.

CaMK phosphatase (CaMKP/POPX2/PPM1F) inhibitors suppress the migration of human breast cancer MDA-MB-231 cells with loss of polarized morphology.

CaMK phosphatase (CaMKP/POPX2/PPM1F) is a Ser/Thr protein phosphatase that belongs to the PPM family. Accumulating evidence suggests that CaMKP is involved in the pathogenesis of various diseases, including cancer. To clarify the relationship between CaMKP activity and human breast cancer cell motility, we examined the phosphatase activity of CaMKP in cell extracts. CaMKP activity assays of the immunoprecipitates prepared from the cell extract revealed that cells exhibiting higher motility had higher CaMKP activity, with no significant differences in the specific activity being observed. Two CaMKP-specific inhibitors, 1-amino-8-naphthol-4-sulfonic acid (ANS) and 1-amino-8-naphthol-2,4-disulfonic acid (ANDS), inhibited the migration of highly invasive MDA-MB-231 breast cancer cells without significant cytotoxicity, while an inactive analog, naphthionic acid, did not. Furthermore, the cells lost their elongated morphology and assumed a rounded shape following treatment with ANS, whereas they retained their elongated morphology following treatment with naphthionic acid. Consistent with these findings, ANS and ANDS significantly enhanced the phosphorylation level of CaMKI, a cellular substrate of CaMKP, while naphthionic acid did not. The present data suggest that CaMKP could be a novel therapeutic target for cancer metastasis.

Isolation of Fluorescent Benzoxazines with Neuroprotective Activity from the Olive Weevil Pimelocerus perforatus.

The weevil Pimelocerus perforatus poses a serious pest problem for olive cultivation in Japan. Two new racemic fluorescent benzoxazines, designated as pimeforazine A ((±)-1) and pimeforazine B ((±)-2), were successfully isolated from P. perforatus. Their structures, including the absolute configurations of their resolved enantiomers, were determined using spectroscopic methods, single-crystal X-ray diffraction, and electronic circular dichroism calculations. The neuroprotective activity of the isolated compounds was evaluated against hydrogen peroxide-induced cellular damage in SH-SY5Y human neuroblastoma cells. Compounds (±)-1 and (±)-2 exhibited neuroprotective effects.

Levetiracetam Suppresses the Infiltration of Neutrophils and Monocytes and Downregulates Many Inflammatory Cytokines during Epileptogenesis in Pilocarpine-Induced Status Epilepticus Mice.

Acute brain inflammation after status epilepticus (SE) is involved in blood-brain barrier (BBB) dysfunction and brain edema, which cause the development of post-SE symptomatic epilepsy. Using pilocarpine-induced SE mice, we previously reported that treatment with levetiracetam (LEV) after SE suppresses increased expression levels of proinflammatory mediators during epileptogenesis and prevents the development of spontaneous recurrent seizures. However, it remains unclear how LEV suppresses neuroinflammation after SE. In this study, we demonstrated that LEV suppressed the infiltration of CD11b+CD45high cells into the brain after SE. CD11b+CD45high cells appeared in the hippocampus between 1 and 4 days after SE and contained Ly6G+Ly6C+ and Ly6G-Ly6C+ cells. Ly6G+Ly6C+ cells expressed higher levels of proinflammatory cytokines such as IL-1ß and TNFα suggesting that these cells were inflammatory neutrophils. Depletion of peripheral Ly6G+Ly6C+ cells prior to SE by anti-Ly6G antibody (NIMP-R14) treatment completely suppressed the infiltration of Ly6G+Ly6C+ cells into the brain. Proteome analysis revealed the downregulation of a variety of inflammatory cytokines, which exhibited increased expression in the post-SE hippocampus. These results suggest that Ly6G+Ly6C+ neutrophils are involved in the induction of acute brain inflammation after SE. The proteome expression profile of the hippocampus treated with LEV after SE was similar to that after NIMP-R14 treatment. Therefore, LEV may prevent acute brain inflammation after SE by suppressing inflammatory neutrophil infiltration.

A CCR5 antagonist, maraviroc, alleviates neural circuit dysfunction and behavioral disorders induced by prenatal valproate exposure.

BACKGROUND: Valproic acid (VPA) is a clinically used antiepileptic drug, but it is associated with a significant risk of a low verbal intelligence quotient (IQ) score, attention-deficit hyperactivity disorder and autism spectrum disorder in children when it is administered during pregnancy. Prenatal VPA exposure has been reported to affect neurogenesis and neuronal migration and differentiation. In addition, growing evidence has shown that microglia and brain immune cells are activated by VPA treatment. However, the role of VPA-activated microglia remains unclear. METHODS: Pregnant female mice received sodium valproate on E11.5. A microglial activation inhibitor, minocycline or a CCR5 antagonist, maraviroc was dissolved in drinking water and administered to dams from P1 to P21. Measurement of microglial activity, evaluation of neural circuit function and expression analysis were performed on P10. Behavioral tests were performed in the order of open field test, Y-maze test, social affiliation test and marble burying test from the age of 6 weeks. RESULTS: Prenatal exposure of mice to VPA induced microglial activation and neural circuit dysfunction in the CA1 region of the hippocampus during the early postnatal periods and post-developmental defects in working memory and social interaction and repetitive behaviors. Minocycline, a microglial activation inhibitor, clearly suppressed the above effects, suggesting that microglia elicit neural dysfunction and behavioral disorders. Next-generation sequencing analysis revealed that the expression of a chemokine, C-C motif chemokine ligand 3 (CCL3), was upregulated in the hippocampi of VPA-treated mice. CCL3 expression increased in microglia during the early postnatal periods via an epigenetic mechanism. The CCR5 antagonist maraviroc significantly suppressed neural circuit dysfunction and post-developmental behavioral disorders induced by prenatal VPA exposure. CONCLUSION: These findings suggest that microglial CCL3 might act during development to contribute to VPA-induced post-developmental behavioral abnormalities. CCR5-targeting compounds such as maraviroc might alleviate behavioral disorders when administered early.

Regulation of Inflammation-Related Genes through Fosl1 Suppression in a Levetiracetam-Treated Pilocarpine-Induced Status Epilepticus Mouse Model.

Levetiracetam (LEV) suppresses the upregulation of proinflammatory molecules that occurs during epileptogenesis after status epilepticus (SE). Based on previous studies, LEV likely helps prevent the onset of epilepsy after insults to the brain, unlike other conventional anti-epileptic drugs. Recently, we discovered that the increase in Fosl1 expression that occurs after lipopolysaccharide (LPS) stimulation is suppressed by LEV and that Fosl1 inhibition suppresses inflammation in BV-2 microglial cells. These data indicate that Fosl1 is an important target of LEV and a key factor in preventing epilepsy onset. In this study, we examined the effects of LEV on Fosl1 expression and neuroinflammation in vivo. During epileptogenesis, the post-SE upregulation of hippocampal levels of Fosl1 and many inflammatory factors were suppressed by LEV. Fosl1 expression showed a characteristic pattern different from that of the expression of Fos, an immediate-early gene belonging to the same Fos family. At 2 days after SE, Fosl1 was predominantly expressed in astrocytes but was rarely detected in microglia, whereas Fos expression was distributed in various brain cell types. The expression of A2 astrocyte markers was similar to that of Fosl1 and was significantly suppressed by LEV. These results suggest that LEV may regulate astrocyte reactivity through regulation of Fosl1.

Involvement of polycyclic aromatic hydrocarbons and endotoxin in macrophage expression of interleukin-33 induced by exposure to particulate matter.

Air pollutants are important factors that contribute to the development and/or exacerbation of allergic inflammation accompanied by asthma, but experimental evidence still needs to be collected. Interleukin 33 (IL-33) is closely involved in the onset and progression of asthma. In this study, we examined the effects of particulate matter (PM) on IL-33 expression in macrophages. PM2.5 collected in Yokohama, Japan by the cyclone device significantly induced IL-33 expression in human THP-1 macrophages, and the induction was clearly suppressed by pretreatment with the aryl hydrocarbon receptor (AhR) antagonist CH-223191 or the Toll-like receptor 4 (TLR4) antagonist TAK-242. PM2.5-induced IL-33 expression was significantly attenuated in AhR-knockout or TLR4-mutated macrophages, suggesting an important role of polycyclic aromatic hydrocarbons (PAHs) and endotoxin in IL-33 stimulation. PM samples derived from tunnel dust slightly but significantly induced IL-33 expression, while road dust PM did not affect IL-33 expression. The PAH concentration in tunnel dust was higher than that in road dust. Tunnel dust or road dust PM contained less endotoxin than PM2.5 collected in Yokohama. These data suggest that the potency of IL-33 induction could depend on the concentration of PAHs as well as endotoxin in PMs. Caution regarding PAHs and endotoxin levels in air pollutants should be taken to prevent IL-33-induced allergic inflammation.

Kiss1-dependent and independent release of luteinizing hormone and testosterone in perinatal male rats.

Prenatal and postnatal biphasic increases in plasma testosterone levels derived from perinatal testes are considered critical for defeminizing/masculinizing the brain mechanism that regulates sexual behavior in male rats. Hypothalamic kisspeptin neurons are indispensable for stimulating GnRH and downstream gonadotropin, as well as the consequent testicular testosterone production/release in adult male rats. However, it is unclear whether kisspeptin is responsible for the increase in plasma testosterone levels in perinatal male rats. The present study aimed to investigate the role of Kiss1/kisspeptin in generating perinatal plasma LH and the consequent testosterone increase in male rats by comparing the plasma testosterone and LH profiles of wild-type (Kiss1+/+) and Kiss1 knockout (Kiss1-/-) male rats. A biphasic pattern of plasma testosterone levels, with peaks in the prenatal and postnatal periods, was found in both Kiss1+/+ and Kiss1-/- male rats. Postnatal plasma testosterone and LH levels were significantly lower in Kiss1-/- male rats than in Kiss1+/+ male rats, whereas the levels in the prenatal embryonic period were comparable between the genotypes. Exogenous kisspeptin challenge significantly increased plasma testosterone and LH levels and the number of c-Fos-immunoreactive GnRH neurons in neonatal Kiss1-/- and Kiss1+/+ male rats. Kiss1 and Gpr54 (kisspeptin receptor gene) were found in the testes of neonatal rats, but kisspeptin treatment failed to stimulate testosterone release in the cultured testes of both genotypes. These findings suggest that postnatal, but not prenatal, testosterone increase in male rats is mainly induced by central kisspeptin-dependent stimulation of GnRH and consequent LH release.

FosL1 Is a Novel Target of Levetiracetam for Suppressing the Microglial Inflammatory Reaction.

We previously showed that the antiepileptic drug levetiracetam (LEV) inhibits microglial activation, but the mechanism remains unclear. The purpose of this study was to identify the target of LEV in microglial activity suppression. The mouse microglial BV-2 cell line, cultured in a ramified form, was pretreated with LEV and then treated with lipopolysaccharide (LPS). A comprehensive analysis of LEV targets was performed by cap analysis gene expression sequencing using BV-2 cells, indicating the transcription factors BATF, Nrf-2, FosL1 (Fra1), MAFF, and Spic as candidates. LPS increased AP-1 and Spic transcriptional activity, and LEV only suppressed AP-1 activity. FosL1, MAFF, and Spic mRNA levels were increased by LPS, and LEV only attenuated FosL1 mRNA expression, suggesting FosL1 as an LEV target. FosL1 protein levels were increased by LPS treatment and decreased by LEV pretreatment, similar to FosL1 mRNA levels. The FosL1 siRNA clearly suppressed the expression of TNFα and IL-1ß. Pilocarpine-induced status epilepticus increased hippocampus FosL1 expression, along with inflammation. LEV treatment significantly suppressed FosL1 expression. Together, LEV reduces FosL1 expression and AP-1 activity in activated microglia, thereby suppressing neuroinflammation. LEV might be a candidate for the treatment of several neurological diseases involving microglial activation.

Janohigenins: Long-chain anacardic acid derivatives with neuroprotective activity from Ophiopogon japonicus seeds.

Eight hitherto undescribed long-chain anacardic acid derivatives, janohigenins, were isolated from the endosperm of Ophiopogon japonicus seed, and their structures were elucidated employing spectroscopic and chemical methods. The neuroprotective activity of the isolated compounds was evaluated against rotenone-induced cellular damage in SH-SY5Y human neuroblastoma cells. Janohigenins exhibited noticeable neuroprotection at 1 µM.