Long-lasting increases in GABAB receptor subunit levels in hippocampal dentate gyrus of mice with a single systemic injection of trimethyltin.

We have recently shown delayed increases in GABAB receptor (GABABR) subunit protein levels in the hippocampal dentate gyrus (DG), but not in the pyramidal CA1 and CA3 regions, at 15-30 days after the systemic single administration of trimethyltin (TMT) in mice. An attempt was thus made to determine whether the delayed increases return to the control levels found in naive mice afterward. In the DG on hippocampal slices obtained at 90 days after the administration, however, marked increases were still seen in protein levels of both GABABR1 and GABABR2 subunits without significant changes in calbindin and glial fibrillary acidic protein (GFAP) levels on immunoblotting analysis. Fluoro-Jade B staining clearly revealed the absence of degenerated neurons from the DG at 90 days after the administration. Although co-localization was invariably detected between GABABR2 subunit and GFAP in the DG at 30 days on immunohistochemical analysis, GABABR2-positive cells did not merge well with GFAP-positive cells in the DG at 90 days. These results suggest that both GABABR1 and GABABR2 subunits would be tardily and sustainably up-regulated by cells other than neurons and astrocytes in the murine DG at 90 days after a systemic single injection of TMT.

Pharmacokinetics of L-theanine and the effect on amino acid composition in mice administered with L-theanine.

L-theanine, an amino acid component of the tea leaves of Camellia sinensis, is sold in Japan as a supplement for good sleep. Although several studies in humans and mice have reported the effects of L-theanine on brain function, only a few reports have comprehensively clarified the disposition of theanine administered to mice and its effects on concentrations of other blood amino acids. In this study, we aimed to determine the changes in the blood levels of L-theanine administered to mice and amino acid composition of the serum. L-theanine were administered to four-week-old Std-ddY male mice orally or via tail vein injection. L-theanine and other amino acids in serum prepared from blood collected at different time points post-dose were labeled with phenylisothiocyanate and quantified. The serum concentration of orally administered L-theanine peaked 15 min after administration. The area under the curve for tail vein injection revealed the bioavailability of L- theanine to be approximately 70%. L-theanine administration did not affect any amino acid levels in the serum, but a significant increase in the peak area overlapping the Glycine (Gly) peak was observed 30 min after administration. L-theanine administered to mice was rapidly absorbed and eliminated, suggesting that taking L-theanine as a supplement is safe without affecting its own levels or serum levels of other amino acids. However, considering that Gly, similar to L-theanine, is used as a dietary supplement for its anxiolytic effects and to improve sleep, determining the effects of L-theanine administration on Gly is important and needs further research.

Post-translational modifications of the apelin receptor regulate its functional expression.

Post-translational modifications (PTMs) are protein modifications that occur after protein biosynthesis, playing a crucial role in regulating protein function. They are involved in the functional expression of G-protein-coupled receptors (GPCRs), as well as intracellular and secretory protein signaling. Here, we aimed to investigate the PTMs of the apelin receptor (APLNR), a GPCR and their potential influence on the receptor's function. In an in vitro experiment using HEK cells, we only observed glycosylation as a PTM of the APLNR and ineffective receptor signaling by the agonist, (Pyr1)-apelin-13. In contrast, when analyzing mouse spinal cord, we detected glycosylation and other PTMs, excluding isopeptidation. This suggests that additional PTMs are involved in the functional expression of the APLNR in vitro. In summary, these findings suggest that the APLNR in vivo requires multiple PTMs for functional expression. To comprehensively understand the pharmacological effects of the APLNR, it is essential to establish an in vitro system that adequately replicates the receptor's PTM profile. Nonetheless, it is crucial to overcome the challenge of heat-sensitive proteolysis in APLNR studies. By elucidating the regulation of PTMs, further research has the potential to advance the analysis and pharmacological studies of both the apelin/APLNR system and GPCR signal modulation.

Delayed Expression of Both GABABR1 and GABABR2 Subunits in Murine Hippocampal Dentate Gyrus After a Single Systemic Injection of Trimethyltin.

Trimethyltin (TMT) has been used as a cytotoxin to neurons rather than glial cells in the mammalian hippocampus. The systemic administration of TMT led to declined fluorescence of ZnAF-2 DA staining as a marker of intact mossy fibers and increased fluorescence of Fluoro-Jade B staining as a marker of degenerated neurons during the initial 2 to 5 days after the administration with later ameliorations within 30 days in the hippocampal dentate gyrus (DG) and CA3 region in mice. On immunoblotting analysis, both GABABR1 and GABABR2 subunit levels increased during 15 to 30 days after TMT along with significant decreases in glutamatergic GluA1 and GluA2/3 receptor subunit levels during 2 to 7 days in the DG, but not in other hippocampal regions such as CA1 and CA3 regions. Immunohistochemical analysis revealed the constitutive and inducible expression of GABABR2 subunit in cells immunoreactive to an astrocytic marker as well as neuronal markers in the DG with the absence of neither GABABR1a nor GABABR1b subunit from cells positive to an astrocytic marker. These results suggest that both GABABR1 and GABABR2 subunits may be up-regulated in cells other than neurons and astroglia in the DG at a late stage of TMT intoxication in mice.

Apelin/Apelin Receptor System: Molecular Characteristics, Physiological Roles, and Prospects as a Target for Disease Prevention and Pharmacotherapy.

Among the various orphan G protein-coupled receptors, apelin receptor (APJ), originally identified in the human genome as an orphan G-protein-coupled receptor, was deorphanised in 1998 with the discovery of its endogenous ligand, apelin. Apelin and APJ mRNA are widely expressed in peripheral tissues and the central nervous system in mammals. In this review, we discuss the characteristics, pharmacology, physiology, and pathology of the apelin/APJ system in mammals. Several physiological roles of the apelin/APJ system have been reported, including in homeostasis, cardiovascular maintenance, angiogenesis, and neuroprotection. In cellular signaling, apelin has been shown to drive the PI3K/Akt, MAPK, and PKA signaling pathways, leading to cell proliferation and protection from excitotoxicity. Apelin is also found in breast milk; therefore, apelin is believed to contribute to the establishment of the infant immune system. Furthermore, activation of the apelin/APJ system is reported to restore muscular weakness associated with aging. Thus, the apelin/APJ system represents a novel target for the prevention of several important cardiovascular and neurodegenerative diseases and the maintenance of health during old age.

Regulatory effects associated with changes in intracellular potassium level in susceptibility to mitochondrial depolarization and excitotoxicity.

Excitotoxicity has been believed to be one of the causes of neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. So far, much research has been done to suppress the neuronal excessive excitations, however, we still have not achieved full control, which may be due to the lack of some factors. As a matter of course, there is an urgent need to clarify all mechanisms that inhibit the onset and progression of neurodegenerative diseases. We found that potassium ion level regulation may be important in the sense that it suppresses mitochondrial depolarization rather than hyperpolarization of cell membrane potential. Minoxidil, an opener of ATP-activated potassium (KATP) channels decreased injury with middle cerebral artery occlusion in vivo experiment using TTC staining. In the primary cortical neurons, N-methyl-D-aspartate (NMDA)-induced mitochondrial depolarization was suppressed by minoxidil treatment. Minoxidil inhibited the increase in levels of cleaved caspase 3 and the release of cytochrome c into the cytosol, further reducing potassium ion levels. It was observed decreased potassium levels in neurons by the treatment of minoxidil. Those effects of minoxidil were blocked by glibenclamide. Therefore, it was suggested that minoxidil, via opening of KATP channels, reduced intracellular potassium ion level that contribute to mitochondrial depolarization, and suppressed subsequent NMDA-induced mitochondrial depolarization. Our findings suggest that the control of ion levels in neurons could dominate the onset and progression of neurodegenerative diseases.

Alleviation by GABAB Receptors of Neurotoxicity Mediated by Mitochondrial Permeability Transition Pore in Cultured Murine Cortical Neurons Exposed to N-Methyl-D-aspartate.

Mitochondrial permeability transition pore (PTP) is supposed to at least in part participate in molecular mechanisms underlying the neurotoxicity seen after overactivation of N-methyl-D-aspartate (NMDA) receptor (NMDAR) in neurons. In this study, we have evaluated whether activation of GABAB receptor (GABABR), which is linked to membrane G protein-coupled inwardly-rectifying K+ ion channels (GIRKs), leads to protection of the NMDA-induced neurotoxicity in a manner relevant to mitochondrial membrane depolarization in cultured embryonic mouse cortical neurons. The cationic fluorescent dye 3,3'-dipropylthiacarbocyanine was used for determination of mitochondrial membrane potential. The PTP opener salicylic acid induced a fluorescence increase with a vitality decrease in a manner sensitive to the PTP inhibitor ciclosporin, while ciclosporin alone was effective in significantly preventing both fluorescence increase and viability decrease by NMDA as seen with an NMDAR antagonist. The NMDA-induced fluorescence increase and viability decrease were similarly prevented by pretreatment with the GABABR agonist baclofen, but not by the GABAAR agonist muscimol, in a fashion sensitive to a GABABR antagonist. Moreover, the GIRK inhibitor tertiapin canceled the inhibition by baclofen of the NMDA-induced fluorescence increase. These results suggest that GABABR rather than GABAAR is protective against the NMDA-induced neurotoxicity mediated by mitochondrial PTP through a mechanism relevant to opening of membrane GIRKs in neurons.

Endoscopic hematoma evacuation for acute subdural hematoma in a young patient: a case report.

Case: The standard treatment for acute subdural hematoma (ASDH) is large craniotomy; decompressive craniectomy may also be carried out, if needed, to prevent secondary brain damage. Recently, an endoscopic procedure for elderly patients with ASDH was carried out and reported; its safety and effectiveness were emphasized because of minimal invasiveness. We report a young case and discuss its difficulties and tips.A 31-year-old man was found to be in a state of general convulsion. At the time of admission, we observed severe consciousness disturbance, anisocoria, and left hemiparesis. Computed tomography showed a massive subdural hematoma with marked midline shift. Outcome: Osmotherapy and emergency trepanation improved anisocoria. An endoscopic procedure under local anesthesia was sequentially selected. After surgery, the patient's symptoms clearly improved. Conclusion: Although the standard treatment for ASDH is craniotomy, endoscopic surgery may be useful in some cases.

Regulation of allergic airway inflammation by adoptive transfer of CD4+ T cells preferentially producing IL-10.

Anti-inflammatory pharmacotherapy for asthma has mainly depended on the inhalation of glucocorticoids, which non-specifically suppress immune responses. If the anti-inflammatory cytokine interleukin (IL)-10 can be induced by a specific antigen, asthmatic airway inflammation could be suppressed when individuals are exposed to the antigen. The purpose of this study was to develop cellular immunotherapeutics for atopic diseases using IL-10-producing CD4+ T cells. Spleen cells isolated from ovalbumin (OVA)-sensitized mice were cultured with the antigen, OVA and growth factors, IL-21, IL-27 and TGF-ß for 7 days. After the 7-day culture, the CD4+ T cells were purified using a murine CD4 magnetic beads system. When the induced CD4+ T cells were stimulated by OVA in the presence of antigen-presenting cells, IL-10 was preferentially produced in vitro. When CD4+ T cells were adoptively transferred to OVA-sensitized mice followed by intratracheal OVA challenges, IL-10 was preferentially produced in the serum and bronchoalveolar lavage fluid in vivo. IL-10 production coincided with the inhibition of eosinophilic airway inflammation and epithelial mucus plugging. Most of the IL-10-producing CD4+ T cells were negative for Foxp3 and GATA-3, transcription factors of naturally occurring regulatory T cells and Th2 cells, respectively, but double positive for LAG-3 and CD49b, surface markers of inducible regulatory T cells, Tr1 cells. Collectively, most of the induced IL-10-producing CD4+ T cells could be Tr1 cells, which respond to the antigen to produce IL-10, and effectively suppressed allergic airway inflammation. The induced Tr1 cells may be useful for antigen-specific cellular immunotherapy for atopic diseases.

Apelin deficiency accelerates the progression of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons. Recent studies have implicated that chronic hypoxia and insufficient vascular endothelial growth factor (VEGF)-dependent neuroprotection may lead to the degeneration of motor neurons in ALS. Expression of apelin, an endogenous ligand for the G protein-coupled receptor APJ, is regulated by hypoxia. In addition, recent reports suggest that apelin protects neurons against glutamate-induced excitotoxicity. Here, we examined whether apelin is an endogenous neuroprotective factor using SOD1(G93A) mouse model of ALS. In mouse CNS tissues, the highest expressions of both apelin and APJ mRNAs were detected in spinal cord. APJ immunoreactivity was observed in neuronal cell bodies located in gray matter of spinal cord. Although apelin mRNA expression in the spinal cord of wild-type mice was not changed from 4 to 18 weeks age, that of SOD1(G93A) mice was reduced along with the paralytic phenotype. In addition, double mutant apelin-deficient and SOD1(G93A) displayed the disease phenotypes earlier than SOD1(G93A) littermates. Immunohistochemical observation revealed that the number of motor neurons was decreased and microglia were activated in the spinal cord of the double mutant mice, indicating that apelin deficiency pathologically accelerated the progression of ALS. Furthermore, we showed that apelin enhanced the protective effect of VEGF on H(2)O(2)-induced neuronal death in primary neurons. These results suggest that apelin/APJ system in the spinal cord has a neuroprotective effect against the pathogenesis of ALS.

[Observation of de novo formation and growth of an intracranial aneurysm: a case report].

We observed a de novo formation and growth of an aneurysm in a 43-year-old woman who was followed up after treatment of a subarachnoid hemorrhage (SAH). In 2002, the patient, whose mother had a history of SAH, presented with SAH at the age of 36. Three-dimensional computed tomography angiography (3D-CTA) and digital subtraction angiography showed an aneurysm in the right internal carotid-posterior communicating artery. The aneurysm was clipped and postoperative course was uneventful without neurological deficit. The patient was followed up by 3D-CTA and magnetic resonance angiography every 6 months, because of an untreated small aneurysm, 3 mm in diameter, in the left middle cerebral artery (MCA). The MCA aneurysm remained unchanged but a de novo aneurysm, 1.5 mm in diameter, developed in the right anterior cerebral artery (ACA) 6 years after the first surgery. The ACA aneurysm grew to 4 mm in diameter during the following 10 months but the MCA aneurysm remained unchanged. Both aneurysms were clipped in one session. The MCA aneurysm had a smooth wall but the ACA aneurysm had an irregular and thin wall. The postoperative course was uneventful. Young female patients who have developed SAH with familial history, like this case, should receive long-term follow up to check whether a de novo aneurysm has developed.

Apelin is a crucial factor for hypoxia-induced retinal angiogenesis.

OBJECTIVE: To investigate the role of endogenous apelin in pathological retinal angiogenesis. METHODS AND RESULTS: The progression of ischemic retinal diseases, such as diabetic retinopathy, is closely associated with pathological retinal angiogenesis, mainly induced by vascular endothelial growth factor (VEGF) and erythropoietin. Although antiangiogenic therapies using anti-VEGF drugs are effective in treating retinal neovascularization, they show a transient efficacy and cause general adverse effects. New therapeutic target molecules are needed to resolve these issues. It was recently demonstrated that the apelin/APJ system, a newly deorphanized G protein-coupled receptor system, is involved in physiological retinal vascularization. Retinal angiography and mRNA expression were examined during hypoxia-induced retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Compared with age-matched control mice, retinal apelin expression was dramatically increased during the hypoxic phase in oxygen-induced retinopathy model mice. APJ was colocalized in proliferative cells, which were probably endothelial cells of the ectopic vessels in the vitreous body. Apelin deficiency hardly induced hypoxia-induced retinal angiogenesis despite the upregulation of VEGF and erythropoietin mRNA in oxygen-induced retinopathy model mice. Apelin small and interfering RNA suppressed the proliferation of endothelial cells independent of the VEGF/VEGF receptor 2 signaling pathway. CONCLUSIONS: These results suggest that apelin is a prerequisite factor for hypoxia-induced retinal angiogenesis.

[A successfully treated case of cerebrospinal fluid fistula caused by fracture of the sella turcica].

Fracture of the sella turcica is rare and is associated with many complications. We successfully treated a cerebrospinal fluid fistula caused by a fracture of the sella turcica. A 66-year-old male in a motor vehicle accident was admitted to an outside hospital with disturbance of consciousness. A computed tomography (CT) scan of the head revealed a subarachnoid hemorrhage and pneumocephalus. Cerebrospinal fluid rhinorrhea developed after admission. Repair of the fistula was attempted without success, and the patient was transferred to our hospital for further examination and treatment. A fracture of the sella turcica was clearly visualized on coronal and sagittal head CT and on a three-dimensional reconstructed CT (3D-CT) image. The source of the CSF fistula was thought to be the anterior wall of the sella turcica. Through a bifrontal interhemispheric approach, the cerebrospinal fluid fistula was repaired microscopically with the assistance of endoscopy. Postoperatively, the fistula stopped completely. Coronal and sagittal head CT and 3D-CT images are useful for making a diagnosis of CSF leakage. Endoscopic images can assist in observation of the dead angle of the microscope.