Sodium Valproate Use in Japanese Patients with Schizophrenia and Coronavirus Disease Is Associated with an Increased Risk of Pneumonia.

Schizophrenia is a known risk factor for coronavirus disease (COVID-19) infection and severity, and certain psychotropic drugs have been linked to increased mortality in infected patients with schizophrenia. However, little evidence exists regarding this risk. We retrospectively examined the association between mood stabilizers and the risk of pneumonia in patients with schizophrenia. This study included 99 patients with schizophrenia or schizoaffective disorder who were infected with COVID-19 in 2022 and met the inclusion criteria. After conducting propensity score matching to align patient backgrounds and concomitant medications, we assessed the impact of mood stabilizers, specifically sodium valproate, on the risk of pneumonia development. Univariate analysis revealed that patients with schizophrenia and COVID-19 who developed pneumonia were more likely to be older (64.5 [14.2] vs. 57.4 [11.5] years, p = 0.008) and using sodium valproate (44.4% vs. 16.7%, p = 0.004). Even after propensity score matching, patients who developed pneumonia were still more likely to be receiving sodium valproate than not (58.8% vs. 20.0%, p = 0.003). Sodium valproate use may be a risk factor for the development of pneumonia in patients with chronic schizophrenia who are infected with COVID-19 during long-term hospitalization.

Analysis of the effect of brexpiprazole on sleep architecture in patients with schizophrenia: A preliminary study.

BACKGROUND: Brexpiprazole is an atypical antipsychotic drug widely used in Japan for the treatment of schizophrenia. Previous studies have investigated the therapeutic effects of some antipsychotics on sleep variables; however, to our knowledge, the effects of brexpiprazole on sleep architecture have not been examined in patients with schizophrenia. Therefore, we aimed to exploratorily investigate the effect of brexpiprazole on sleep variables measured by polysomnography in patients with schizophrenia. METHODS: This study included 10 patients with schizophrenia who were originally treated with haloperidol alone. Sleep variables of the participants were measured using polysomnography. After excluding those who did not meet the study criteria, seven patients (five men and two women; mean age [SD], 59.0 [10.0] years) were eligible for further analysis. Polysomnography was repeated at 4 weeks after the participants were prescribed brexpiprazole in addition to haloperidol. We compared the sleep architecture of the participants, measured using polysomnography, before and after taking brexpiprazole. RESULTS: Add-on brexpiprazole significantly prolonged rapid eye movement latency, increased the duration and percentage of stage N2 and stage N3 sleep (min, %), and decreased the duration and percentage of stage rapid eye movement sleep (min, %) at a significance level of nominal p < 0.05. CONCLUSION: Although not significant after correcting for multiple comparisons, the present results showed that add-on brexpiprazole could alter the sleep architecture of patients with schizophrenia. Future studies are warranted to replicate these findings and to further investigate the beneficial influence of brexpiprazole on sleep.

Whole-cell observation of ZIO-stained Golgi apparatus in rat hepatocytes with serial block-face scanning electron microscope, SBF-SEM.

The Golgi apparatus, which plays a role in various biosynthetic pathways, is usually identified in electron microscopy by the morphological criteria of lamellae. A 3-dimensional analyses with serial block-face scanning electron microscope (SBF-SEM), a volume-SEM proficient in obtaining large volumes of data at the whole-cell level, could be a promising technique for understanding the precise distribution and complex ultrastructure of Golgi apparatus, although optimal methods for such analyses remain unclear since the observation can be hampered with sample charging and low image contrast, and manual segmentation often requires significant manpower. The present study attempted the whole-cell observation and semi-automatic classification and segmentation of the Golgi apparatus in rat hepatocytes for the first time by SBF-SEM via ZIO staining, a classical osmium impregnation. The staining electron-densely visualized individual Golgi lamellae, and their ultrastructure could stably be observed without any noticeable charging. The simple thresholding of the serial images enabled the efficient reconstruction of the labeled Golgi apparatus, which revealed plural Golgi apparatus in one hepatocyte. The combination of the heavy metal-based histochemistry of zinc, iodine and osmium (ZIO) staining and SBF-SEM was useful in the 3-dimensional observation of the Golgi apparatus at the whole-cell level because of two technical advantages: (i) visualization of the Golgi apparatus without any heavy metal staining and efficient acquisition of the block-face images without additional conductive staining or any devices for eliminating charging; (ii) easy identification of the staining and hassle-free, semi-automatic classification and segmentation by simple thresholding of the images. This novel approach could elucidate the topographic characteristics of the Golgi apparatus in hepatocytes.

Expression of high mobility group B1 and toll-like receptor-nuclear factor κB signaling pathway in chronic subdural hematomas.

Chronic subdural hematoma (CSDH) is an angiogenic and inflammatory disease. Toll-like receptors (TLRs) transduce intracellular signals, resulting in the activation of nuclear factor κB (NF-κB), which leads to the production of inflammatory cytokines. High-mobility group box 1 (HMGB1) functions as a mediator of inflammatory responses through TLRs. In this study, we examined the expression of HMGB1 and components of the Toll-like receptor and NF-κB signaling pathways in the outer membrane of CSDH. Eight patients whose outer membrane was successfully obtained during trepanation surgery were included in this study. The expression of TLR4, myeloid differentiation factor 88 (MyD88), interleukin-1 receptor-associated kinase 4 (IRAK4), TNF receptor-associated factor 6 (TRAF6), TGFß-activated kinase 1 (Tak1), interferon regulatory factors 3 (IRF3), IκB kinase ß (IKKß), IKKγ, IκBε, IκBα, NF-κB/p65 and ß-actin was examined by Western blot analysis. The expression of TLR4, NF-κB/p65 and interleukin-6 (IL-6) was also examined by immunohistochemistry. The concentrations of HMGB1 and IL-6 in CSDH fluids were measured using ELISA kits. Above-mentioned molecules were detected in all cases. In addition, TLR4, NF-κB/p65 and IL-6 were localized in the endothelial cells of vessels within CSDH outer membranes. The concentrations of HMGB1 and IL-6 in CSDH fluids were significantly higher than that in the CSF and serum. There existed a correlation between the concentrations of HMGB1 and IL-6 in CSDH fluids. Our data suggest that HMGB1 in CSDH fluids produces the inflammatory cytokine IL-6 in endothelial cells through the Toll-like receptor and NF-κB signaling pathways. Anti-HMGB1 therapy might be a useful method to treat the growth of CSDH.

Expression of Autophagy Signaling Molecules in the Outer Membranes of Chronic Subdural Hematomas.

Chronic subdural hematoma (CSDH) is fundamentally treatable, although it sometimes recurs. We observed, however, several cases of spontaneous resolution of CSDH outer membranes, even in a trabecular type of CSDH, after a trepanation surgical procedure. In this study, we examined the expression of molecules of the autophagy signaling pathway in CSDH outer membranes. Eight patients whose outer membranes were obtained successfully during trepanation were included in this study. By Western blot analysis, we examined the expression of mammalian target of rapamycin (mTOR); GßL; UNC-51-like kinase-1 (ULK1); Beclin-1; autophagy-related genes (Atg) 3, 5, 7, 12, 13, and 16L1ß,α; the autophagy marker Light Chain3A/B (LC3A/B); and ß-actin, which constitute the autophagy signaling pathway. The expression levels of Beclin-1, Atg12, and LC3A/B were also examined by immunohistochemistry. Almost all of these molecules could be detected in all samples. Beclin-1, Atg12, and LC3A/B were found to be localized in the endothelial cells of vessels and fibroblasts in CSDH. We detected molecules of the autophagy signaling pathway in CSDH outer membranes. Autophagy contributes to the tissue homeostatic process, maintaining cellular integrity by clearing debris. Our data suggest that autophagy might play an important role in the spontaneous resolution of CSDH. Therefore, these molecules may be novel therapeutic targets for the treatment of those with CSDH.

Subarachnoid hemorrhage induces neuronal nitric oxide synthase phosphorylation at Ser1412 in the dentate gyrus of the rat brain.

INTRODUCTION: We previously demonstrated that cyclic AMP-dependent protein kinase (PKA) phosphorylates neuronal nitric oxide synthase (nNOS) at Ser1412 in the hippocampal dentate gyrus after forebrain ischemia; this phosphorylation event activates NOS activity and might contribute to depression after cerebral ischemia. In this study, we revealed chronological and topographical changes in the phosphorylation of nNOS at Ser1412 immediately after subarachnoid hemorrhage (SAH). METHODS: In a rat single-hemorrhage model of SAH, the hippocampus and adjacent cortex were collected up to 24 h after SAH. Samples from rats that were not injected with autologous blood were used as controls. NOS was partially purified from crude samples via an ADP-agarose gel. Levels of nNOS, nNOS phosphorylated at Ser1412 (p-nNOS), PKA, and p-PKA at Thr197 were studied in the rat hippocampus and cortex using Western blot analyses and immunohistochemistry. RESULTS: According to the Western blot analysis, levels of p-nNOS at Ser1412 were significantly increased in the hippocampus, but not in the cortex, between 1 and 3 h after SAH. Immunohistochemistry revealed the phosphorylation of nNOS at Ser1412 and PKA at Thr197 in the dentate gyrus, but not in the CA1 area, 1 h after SAH. An injection of saline instead of blood also significantly increased levels of p-nNOS at Ser1412 in the hippocampus 1 h after the injection. CONCLUSIONS: An immediate increase in intracranial pressure (ICP) might induce transient cerebral ischemia and promote the PKA-mediated phosphorylation of nNOS at Ser1412 in the dentate gyrus. This signal transduction pathway induces the excessive production of nitric oxide (NO) and might be involved in cognitive dysfunction after SAH.

Immature morphological properties in subcellular-scale structures in the dentate gyrus of Schnurri-2 knockout mice: a model for schizophrenia and intellectual disability.

Accumulating evidence suggests that subcellular-scale structures such as dendritic spine and mitochondria may be involved in the pathogenesis/pathophysiology of schizophrenia and intellectual disability. Previously, we proposed mice lacking Schnurri-2 (Shn2; also called major histocompatibility complex [MHC]-binding protein 2 [MBP-2], or human immunodeficiency virus type I enhancer binding protein 2 [HIVEP2]) as a schizophrenia and intellectual disability model with mild chronic inflammation. In the mutants' brains, there are increases in C4b and C1q genes, which are considered to mediate synapse elimination during postnatal development. However, morphological properties of subcellular-scale structures such as dendritic spine in Shn2 knockout (KO) mice remain unknown. In this study, we conducted three-dimensional morphological analyses in subcellular-scale structures in dentate gyrus granule cells of Shn2 KO mice by serial block-face scanning electron microscopy. Shn2 KO mice showed immature dendritic spine morphology characterized by increases in spine length and decreases in spine diameter. There was a non-significant tendency toward decrease in spine density of Shn2 KO mice over wild-type mice, and spine volume was indistinguishable between genotypes. Shn2 KO mice exhibited a significant reduction in GluR1 expression and a nominally significant decrease in SV2 expression, while PSD95 expression had a non-significant tendency to decrease in Shn2 KO mice. There were significant decreases in dendrite diameter, nuclear volume, and the number of constricted mitochondria in the mutants. Additionally, neuronal density was elevated in Shn2 KO mice. These results suggest that Shn2 KO mice serve as a unique tool for investigating morphological abnormalities of subcellular-scale structures in schizophrenia, intellectual disability, and its related disorders.

Expression of Caspase Signaling Components in the Outer Membranes of Chronic Subdural Hematomas.

Chronic subdural hematoma (CSDH) is fundamentally treatable through surgery, although CSDH recurs in some cases. We have observed several cases of spontaneous resolution of CSDH outer membranes, including in trabecular CSDH, after trepanation surgery. In this study, we examined the expression of molecules involved in caspase signaling in CSDH outer membranes. Eight patients whose outer membranes were obtained successfully during trepanation surgery were included in this study. The expression of Fas; Fas-associated death domain (FADD); tumor necrosis factor receptor type 1-associated death domain (TRADD); receptor-interacting protein (RIP); caspases 3, 7, 8, and 9; poly-(ADP-ribose) polymerase (PARP); DNA fragmentation factor 45 (DFF45) and ß-actin was examined by Western blot analysis. The expression levels of PARP, caspase-3, and cleaved caspase-3 were also examined by immunohistochemistry. Fas; FADD; TRADD; RIP; caspases 3, 7, 8, and 9; PARP, and DFF45 were detected in nearly all samples. Caspase-3 and PARP were localized in the endothelial cells of vessels and in fibroblasts in CSDH outer membranes. In addition, cleaved caspase-3 was detected in fibroblasts. We detected molecules of the caspase signaling pathway in CSDH outer membranes. In particular, cleaved caspase-3 was detected, which suggests that apoptosis may occur within these membranes. Thus, during the growth of CSDH outer membranes, the caspase signaling pathway may be restrained. Once the pathway is activated, gradual resolution of CSDH outer membranes may occur. Therefore, these molecules may be novel therapeutic targets for intractable CSDH.

Activation of Nuclear Factor-kappa B in Endothelial Cells of Chronic Subdural Hematoma Outer Membranes.

BACKGROUND: Chronic subdural hematoma (CSDH) is considered an angiogenic and inflammatory disease. Nuclear factor-kappa B (NF-κB) induces the production of inflammatory cytokines and adhesion molecules, which play an essential role in angiogenesis and inflammation. Recently, the double-stranded RNA-activated protein kinase (PKR) was shown to directly interact with NF-κB subunits to influence its transcriptional activity. OBJECTIVE: To examine the expression of NF-κB signaling pathway components and PKR in CSDH outer membranes. METHODS: Eight patients whose outer membranes were successfully obtained during trepanation surgery were included in this study. The IκBα, IKKß, IKKγ, NF-κB, phosphorylated ( p )-NF-κB, and PKR expression levels were examined using western blotting analysis. NF-κB expression was also examined using immunohistochemistry. We investigated whether CSDH fluid could activate NF-κB in cultured endothelial cells in Vitro. RESULTS: The IκBα, IKKß, IKKγ, and NF-κB levels were approximately the same. Additionally, p -NF-κB and PKR were detected at similar levels. Immunostaining showed that NF-κB was expressed in the vascular endothelium. p -NF-κB expression in endothelial cells was significantly induced immediately after treatment with CSDH fluid. Furthermore, NF-κB activation was significantly inhibited by treatment with antibodies directed against vascular endothelial growth factor. CONCLUSION: PKR might activate NF-κB through vascular endothelial growth factor in endothelial cells, which might be associated with endothelial cell proliferation in the CSDH outer membrane. Thus, the NF-κB signaling pathway could play a critical role in CSDH growth.

Inhibitory Mechanism of the Outer Membrane Growth of Chronic Subdural Hematomas.

We previously demonstrated that the inflammatory cytokine interleukin-6 (IL-6) activates the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway in fibroblasts within the outer membranes of chronic subdural hematomas (CSDHs), and the activation of this pathway may induce CSDH outer membrane growth. The inhibitory system for this signal transduction pathway is unknown. CSDH fluids were obtained from 10 patients during trepanation surgery as the case group, and cerebrospinal fluid (CSF) samples were obtained from seven patients suffering from subarachnoid hemorrhage (SAH) on Day 1 as the control group. The concentrations of IL-6, soluble IL-6 receptor (sIL-6R), and soluble gp130 (sgp130) in CSDH fluid and CSF were measured using enzyme immunoassay kits. The co-localization of IL-6 and sgp130 in CSDH fluid was examined by immunoprecipitation. The expression levels of STAT3, JAK2, suppressor of cytokine signaling 3 (SOCS3), and protein inhibitor of activated Stat3 (PIAS3) in the outer membranes of CSDHs were examined by immunostaining. Soluble IL-6R and sgp130 concentrations in CSDH fluid were significantly higher than those in CSF after SAH. Sgp130 and IL-6 were co-immunoprecipitated from CSDH fluid. Immunostaining revealed STAT3, JAK2, SOCS3, and PIAS3 expression in fibroblasts located in the outer membranes of CSDHs. Soluble gp130 binds to IL-6/sIL-6R and acts as an antagonist of the JAK/STAT signaling pathway. SOCS3 also binds to JAK and inhibits its signaling pathway. In addition, PIAS3 regulates STAT3 activation. These factors might down-regulate the IL-6/JAK/STAT signaling pathway in fibroblasts within CSDH outer membranes. Therefore, these molecules may be novel therapeutic targets for the inhibition of CSDH growth.

Activation of Signal Transducer and Activator of Transcription 3 in Endothelial Cells of Chronic Subdural Hematoma Outer Membranes.

OBJECTIVE: Chronic subdural hematoma (CSDH) is considered an angiogenic and inflammatory disease. Interleukin-6, a well-known inflammatory cytokine, activates the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. We previously reported that the JAK/STAT pathway is activated in fibroblasts in the outer membrane of CSDH. More recently, signal transducer and activator of transcription 3 (STAT3) has been shown to have a role in angiogenesis. We examined the expression of STAT3 in endothelial cells in the outer membrane of CSDH. METHODS: This study included 7 patients whose outer membranes were successfully obtained during trephination surgery. The expression of STAT3 and phosphorylated STAT3 was examined by Western blot analysis and immunohistochemistry. We also examined whether CSDH fluid could activate STAT3 in cultured endothelial cells in vitro. RESULTS: STAT3 and phosphorylated STAT3 were detected in all cases. Immunostaining showed that STAT3 and phosphorylated STAT3 were expressed not only in fibroblasts but also in vascular endothelium. Expression of phosphorylated STAT3 in endothelial cells was significantly induced immediately after treatment of CSDH fluid in vitro. The activation of STAT3 was significantly inhibited by treatment with antibodies that were directed against interleukin-6; however, this was suppressed by antibodies that were directed against vascular endothelial growth factor, but not significantly. CONCLUSIONS: Interleukin-6 might dominantly activate STAT3 in endothelial cells, which might have a central role in endothelial cell proliferation and angiogenesis of CSDH outer membranes.

Increased ICP promotes CaMKII-mediated phosphorylation of neuronal NOS at Ser847 in the hippocampus immediately after subarachnoid hemorrhage.

Early brain injury has recently been identified as an indicator of poor prognosis after subarachnoid hemorrhage (SAH). Calmodulin-dependent protein kinase IIα (CaMKIIα) has been shown to phosphorylate neuronal NOS (nNOS) at Ser(847), resulting in a reduction in nNOS activity. In this study, we revealed chronological changes in the phosphorylation of nNOS at Ser(847) in the hippocampus and cortex immediately after SAH. In a rat single-hemorrhage model of SAH, the hippocampus and adjacent cortex were collected up to 24h after SAH. Samples from rats that were not injected with blood were used as controls. NOS was partially purified from the crude samples using ADP-agarose affinity chromatography. Western blot analysis revealed that nNOS phosphorylated (p-nNOS) at Ser(847) was significantly increased in the hippocampus, but not in the cortex, at 1h after SAH compared with that resulting from the control treatment. Immunoreactivity of p-nNOS at Ser(847) was observed in interneurons of the hippocampus at 1h after SAH. Injection of saline instead of blood also significantly induced p-nNOS at Ser(847) levels in the hippocampus at 1h after injection. The colocalization of CaMKIIα and nNOS was transiently increased in the hippocampus at 0.5h after SAH. Our data suggest that immediately after SAH, an increase in intracranial pressure might induce transient cerebral ischemia, potentially promoting the phosphorylation of nNOS at Ser(847) by CaMKIIα in the hippocampus. The activation of p-nNOS at Ser(847) in the hippocampus may alleviate ischemic insults immediately after SAH to exert a neuroprotective effect against early brain injury.

Expression of Mitogen-Activated Protein Kinases in Chronic Subdural Hematoma Outer Membranes.

Growth factors and inflammatory cytokines activate the mitogen-activated protein kinase (MAPK) cascade. Previous studies have shown that chronic subdural hematoma (CSDH) fluid contains these factors and cytokines. In this study, expression of three major MAPK cascade transmitters in the outer membrane of CSDH was assessed. Eleven patients whose outer membrane and CSDH fluid were successfully obtained during trepanation surgery were included in this study. Expression of extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK, p38, p-p38, c-Jun N-terminal kinase (JNK), p-JNK, and actin was examined by Western blot and immunostaining. We examined whether CSDH fluid could activate MAPKs in cultured endothelial cells (ECs) or fibroblasts in vitro. Western blot analysis showed that p-ERK was present in all samples, whereas p-p38 and p-JNK were detected, but not in all cases. Immunostaining showed that all three p-MAPKs were expressed in vascular endothelium. However, only p-ERK was expressed in fibroblasts. Expression of p-extracellular signal-regulated kinase kinase (MEK) and p-ERK in ECs and fibroblasts was significantly induced immediately after treatment with CSDH fluid, whereas p-p38 and p-JNK expression was significantly induced in ECs 60 min after treatment, but not in fibroblasts. Activation of MEK was significantly inhibited by treatment with antibodies directed against interleukin-6 and vascular endothelial growth factor in ECs, but not in fibroblasts. Inflammatory cytokines and growth factors in CSDH fluids might activate major MAPKs in ECs, which might be associated with neovascularization in the outer membrane of CSDH. These MAPK pathways could become novel targets for treatment of CSDHs.

The first case in Asia of 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency (HSD10 disease) with atypical presentation.

2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (2M3HBD) deficiency (HSD10 disease) is a rare inborn error of metabolism, and <30 cases have been reported worldwide. This disorder is typically characterized by progressive neurodegenerative disease from 6 to 18 months of age. Here, we report the first patient with this disorder in Asia, with atypical clinical presentation. A 6-year-old boy, who had been well, presented with severe ketoacidosis following a 5-day history of gastroenteritis. Urinary organic acid analysis showed elevated excretion of 2-methyl-3-hydroxybutyrate and tiglylglycine. He was tentatively diagnosed with ß-ketothiolase (T2) deficiency. However, repeated enzyme assays using lymphocytes showed normal T2 activity and no T2 mutation was found. Instead, a hemizygous c.460G>A (p.A154T) mutation was identified in the HSD17B10 gene. This mutation was not found in 258 alleles from Japanese subjects (controls). A normal level of the HSD17B10 protein was found by immunoblot analysis but no 2M3HBD enzyme activity was detected in enzyme assays using the patient's fibroblasts. These data confirmed that this patient was affected with HSD10 disease. He has had no neurological regression until now. His fibroblasts showed punctate and fragmented mitochondrial organization by MitoTracker staining and had relatively low respiratory chain complex IV activity to those of other complexes.

Eotaxin-3 activates the Smad pathway through the transforming growth factor beta 1 in chronic subdural hematoma outer membranes.

Chronic subdural hematoma (CSDH) is considered to be an inflammatory disease. Eosinophils are frequently expressed in the outer membrane of CSDH and are major sources of transforming growth factor beta (TGF-ß). The mothers against decapentaplegic (Smad)-signaling pathway, which is activated by TGF-ß, has been shown to be involved with fibrosis. In the present study, we compared the concentrations of eotaxin-3, eosinophil-specific chemoattractant, and TGF-ß between CSDH fluid and cerebrospinal fluid (CSF) from control patients. We also explored the expression of the Smad-signaling pathway in the outer membrane of CSDH. Eight patients whose outer membrane and 12 whose CSDH fluid were successfully obtained during trepanation surgery were included in the study. Concentrations of eotaxin-3 and TGF-ß were measured by enzyme immunoassay kits. Expression levels of Smad2, phosphorylated Smad3, Smad3, Smad4, and actin were examined by Western blot analysis. In addition, expression of Smad3 was also examined by immunohistochemistry. Concentrations of eotaxin-3 and TGF-ß in CSDH fluid were significantly higher than those in CSF. Smad2, Smad3, phosphorylated Smad3, and Smad4 were detected in all cases. Smad3 was shown to be present in fibroblasts. These findings indicate that eotaxin-3 is expressed in CSDH fluid, inducing eosinophils into the outer membrane and resulting in elevation of TGF-ß with the Smad pathway activated by TGF-ß. These data suggest a potential mechanism for CSDH formation and growth.

Expression of the JAK/STAT3/SOCS3 signaling pathway in herniated lumbar discs.

The inflammatory cytokine interleukin-6 (IL-6) plays an important role in causing symptoms of lumbar disk herniation. The present study clarifies the expression of the signaling pathway of IL-6 in herniated discs. Homogenates prepared from lumbar herniated discs from 10 patients were assessed. The expression of janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), phosphorylated (p)-STAT3 at Tyr(705), suppressor of cytokine signaling 3 (SOCS3) and actin was examined by Western blot analysis. The expression of JAK1, STAT3, and p-STAT3 at Tyr(705) was also examined by immunostaining. JAK1, STAT3, p-STAT3 at Tyr(705) and SOCS3 were detected in almost all cases. Immunoreactivity against JAK1 and STAT3 was observed mainly in chondrocytes, whereas immunoreactivity against p-STAT3 at Tyr(705) was observed in the nuclei of chondrocytes. The JAK/STAT signaling pathway might be activated by IL-6 and transmit messages from the cell surface to the nucleus, and the pathway is negatively regulated by SOCS3. These JAK1, STAT3 and SOCS3 molecules might tightly regulate and play a role in the degeneration of chondrocytes within herniated discs.

Different modifications of phosphorylated Smad3C and Smad3L through TGF-ß after spinal cord injury in mice.

Transforming growth factor-ß (TGF-ß) is an anti-inflammatory cytokine and is expressed in the injured spinal cord. TGF-ß signals through receptors to activate Smad proteins, which translocate into the nucleus. In the present study, we investigated the chronological alterations and cellular locations of the TGF-ß/Smad signaling pathway following spinal cord injury (SCI) in mice. ELISA analysis showed that the concentration of interleukin-6 (IL-6) in injured spinal cords significantly increases immediately after SCI, while the concentration of TGF-ß gradually increased after SCI, peaked at 2 days, and then gradually decreased. Immunohistochemical studies revealed that Smad3 was mainly expressed in neurons of the spinal cord. Phosphorylated Smad3 at the C-terminus (p-Smad3C) was stained within the motor neurons in the anterior horn, while phosphorylated Smad3 at the linker regions (p-Smad3L) was expressed in astrocytes within gray matter. These findings suggest that SCI induces gradual increases in TGF-ß and induces different activation of p-Smad3C and p-Smad3L. Phosphorylated Smad3C might be involved in neuronal degeneration after SCI, and p-Smad3L may play a role in glial scar formation by astrocytes.

Phosphorylation of neuronal nitric oxide synthase at Ser1412 in the dentate gyrus of rat brain after transient forebrain ischemia.

We previously demonstrated that calmodulin-dependent protein kinase IIα (CaM-KIIα) phosphorylates nNOS at Ser(847) in the hippocampus after forebrain ischemia; this phosphorylation attenuates NOS activity and might contribute to resistance to post-ischemic damage. We also revealed that cyclic AMP-dependent protein kinase (PKA) could phosphorylate nNOS at Ser(1412)in vitro. In this study, we focused on chronological and topographical changes in the phosphorylation of nNOS at Ser(1412) after rat forebrain ischemia. The hippocampus and adjacent cortex were collected at different times, up to 24h, after 15min of forebrain ischemia. NOS was partially purified from crude samples using ADP agarose gel. Neuronal NOS, phosphorylated (p)-nNOS at Ser(1412), PKA, and p-PKA at Thr(197) were studied in the rat hippocampus and cortex using Western blot analysis and immunohistochemistry. Western blot analysis revealed that p-nNOS at Ser(1412) significantly increased between 1 and 6h after reperfusion in the hippocampus, but not in the cortex. PKA was cosedimented with nNOS by ADP agarose gel. Immunohistochemistry revealed that phosphorylation of nNOS at Ser(1412) and PKA at Thr(197) occurred in the subgranular layer of the dentate gyrus. Forebrain ischemia might thereby induce temporary activation of PKA at Thr(197), which then phosphorylates nNOS at Ser(1412) in the subgranular layer of the dentate gyrus.

Deficiency of schnurri-2, an MHC enhancer binding protein, induces mild chronic inflammation in the brain and confers molecular, neuronal, and behavioral phenotypes related to schizophrenia.

Schnurri-2 (Shn-2), an nuclear factor-κB site-binding protein, tightly binds to the enhancers of major histocompatibility complex class I genes and inflammatory cytokines, which have been shown to harbor common variant single-nucleotide polymorphisms associated with schizophrenia. Although genes related to immunity are implicated in schizophrenia, there has been no study showing that their mutation or knockout (KO) results in schizophrenia. Here, we show that Shn-2 KO mice have behavioral abnormalities that resemble those of schizophrenics. The mutant brain demonstrated multiple schizophrenia-related phenotypes, including transcriptome/proteome changes similar to those of postmortem schizophrenia patients, decreased parvalbumin and GAD67 levels, increased theta power on electroencephalograms, and a thinner cortex. Dentate gyrus granule cells failed to mature in mutants, a previously proposed endophenotype of schizophrenia. Shn-2 KO mice also exhibited mild chronic inflammation of the brain, as evidenced by increased inflammation markers (including GFAP and NADH/NADPH oxidase p22 phox), and genome-wide gene expression patterns similar to various inflammatory conditions. Chronic administration of anti-inflammatory drugs reduced hippocampal GFAP expression, and reversed deficits in working memory and nest-building behaviors in Shn-2 KO mice. These results suggest that genetically induced changes in immune system can be a predisposing factor in schizophrenia.