New Dihydropyridine Derivative Attenuates NF-κB Activation via Suppression of Calcium Influx in a Mouse BV-2 Microglial Cell Line.

Activated microglia contribute to many neuroinflammatory diseases in the central nervous system. In this study, we attempted to identify an anti-inflammatory compound that could suppress microglial activation. We performed high-throughput screening with a chemical library developed at our institute. We performed a luciferase assay of nuclear factor-kappa B (NF-κB) reporter stable HT22 cells and identified a compound that was confirmed to inhibit the anti-inflammatory response in BV2 microglial cells. The selected dihydropyridine derivative can suppress the expression response of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor (TNF), as well as NF-κB phosphorylation and nuclear translocation, and reduce the intracellular calcium level. Thus, our identified compound has a potential role in suppressing microglial activation and may contribute to the development of a new therapeutic molecule against neuroinflammatory diseases.

The GPR84 molecule is a mediator of a subpopulation of retinal microglia that promote TNF/IL-1α expression via the rho-ROCK pathway after optic nerve injury.

Resident microglia are important to maintain homeostasis in the central nervous system, which includes the retina. The retinal microglia become activated in numerous pathological conditions, but the molecular signatures of these changes are poorly understood. Here, using an approach based on FACS and RNA-seq, we show that microglial gene expression patterns gradually change during RGC degeneration induced by optic nerve injury. Most importantly, we found that the microglial cells strongly expressed Tnf and Il1α, both of which are known to induce neurotoxic reactive astrocytes, and were characterized by Gpr84high -expressing cells in a particular subpopulation. Moreover, ripasudil, a Rho kinase inhibitor, significantly blunted Gpr84 expression and cytokine induction in vitro and in vivo. Finally, GPR84-deficient mice prevented RGC loss in optic nerve-injured retina. These results reveal that Rho kinase-mediated GPR84 alteration strongly contribute to microglial activation and promote neurotoxicity, suggesting that Rho-ROCK and GPR84 signaling may be potential therapeutic targets to prevent the neurotoxic microglial phenotype induced by optic nerve damage, such as occurs in traumatic optic neuropathy and glaucoma.

Anti-angiogenic effects of valproic acid in a mouse model of oxygen-induced retinopathy.

Pathological retinal angiogenesis contributes to the pathogenesis of several ocular diseases. Valproic acid, a widely used antiepileptic drug, exerts anti-angiogenic effects by inhibiting histone deacetylase (HDAC). Herein, we investigated the effects of valproic acid and vorinostat, a HDAC inhibitor, on pathological retinal angiogenesis in mice with oxygen-induced retinopathy (OIR). OIR was induced in neonatal mice by exposure to 80% oxygen from postnatal day (P) 7 to P10 and to atmospheric oxygen from P10 to P15. Mice were subcutaneously injected with valproic acid, vorinostat, or vehicle once a day from P10 to P14. At P15, retinal neovascular tufts and vascular growth in the central avascular zone were observed in mice with OIR. Additionally, immunoreactivity for phosphorylated ribosomal protein S6 (pS6), an indicator of mammalian target of rapamycin (mTOR) activity, was detected in the neovascular tufts. Both valproic acid and vorinostat reduced the formation of retinal neovascular tuft without affecting vascular growth in the central avascular zone. Valproic acid reduced the pS6 immunoreactivity in neovascular tufts. Given that vascular endothelial growth factor (VEGF) activates mTOR-dependent pathways in proliferating endothelial cells of the neonatal mouse retina, these results suggest that valproic acid suppresses pathological retinal angiogenesis by interrupting VEGF-mTOR pathways.

Active Sites of Spinoxin, a Potassium Channel Scorpion Toxin, Elucidated by Systematic Alanine Scanning.

Peptide toxins from scorpion venoms constitute the largest group of toxins that target the voltage-gated potassium channel (Kv). Spinoxin (SPX) isolated from the venom of scorpion Heterometrus spinifer is a 34-residue peptide neurotoxin cross-linked by four disulfide bridges. SPX is a potent inhibitor of Kv1.3 potassium channels (IC50 = 63 nM), which are considered to be valid molecular targets in the diagnostics and therapy of various autoimmune disorders and cancers. Here we synthesized 25 analogues of SPX and analyzed the role of each amino acid in SPX using alanine scanning to study its structure-function relationships. All synthetic analogues showed similar disulfide bond pairings and secondary structures as native SPX. Alanine replacements at Lys(23), Asn(26), and Lys(30) resulted in loss of activity against Kv1.3 potassium channels, whereas replacements at Arg(7), Met(14), Lys(27), and Tyr(32) also largely reduced inhibitory activity. These results suggest that the side chains of these amino acids in SPX play an important role in its interaction with Kv1.3 channels. In particular, Lys(23) appears to be a key residue that underpins Kv1.3 channel inhibition. Of these seven amino acid residues, four are basic amino acids, suggesting that the positive electrostatic potential on the surface of SPX is likely required for high affinity interaction with Kv1.3 channels. This study provides insight into the structure-function relationships of SPX with implications for the rational design of new lead compounds targeting potassium channels with high potency.

SH-SY5Y human neuronal cells with mutations of the CDKN2B-AS1 gene are vulnerable under cultured conditions.

Glaucoma is a common cause of blindness worldwide. Genetic effects are believed to contribute to the onset and progress of glaucoma, but the underlying pathological mechanisms are not fully understood. Here, we set out to introduce mutations into the CDKN2B-AS1 gene, which is known as being the closely associated with glaucoma, in a human neuronal cell line in vitro. We introduced gene mutations with CRISPR/Cas9 into exons and introns into the CDKN2B-AS1 gene. Both mutations strongly promoted neuronal cell death in normal culture conditions. RNA sequencing and pathway analysis revealed that the transcriptional factor Fos is a target molecule regulating CDKN2B-AS1 overexpression. We demonstrated that gene mutation of CDKN2B-AS1 is directly associated with neuronal cell vulnerability in vitro. Additionally, Fos, which is a downstream signaling molecule of CDKN2B-AS1, may be a potential source of new therapeutic targets for neuronal degeneration in diseases such as glaucoma.

Mass spectrometry of short peptides reveals common features of metazoan peptidergic neurons.

The evolutionary origins of neurons remain unknown. Although recent genome data of extant early-branching animals have shown that neural genes existed in the common ancestor of animals, the physiological and genetic properties of neurons in the early evolutionary phase are still unclear. Here, we performed a mass spectrometry-based comprehensive survey of short peptides from early-branching lineages Cnidaria, Porifera and Ctenophora. We identified a number of mature ctenophore neuropeptides that are expressed in neurons associated with sensory, muscular and digestive systems. The ctenophore peptides are stored in vesicles in cell bodies and neurites, suggesting volume transmission similar to that of cnidarian and bilaterian peptidergic systems. A comparison of genetic characteristics revealed that the peptide-expressing cells of Cnidaria and Ctenophora express the vast majority of genes that have pivotal roles in maturation, secretion and degradation of neuropeptides in Bilateria. Functional analysis of neuropeptides and prediction of receptors with machine learning demonstrated peptide regulation of a wide range of target effector cells, including cells of muscular systems. The striking parallels between the peptidergic neuronal properties of Cnidaria and Bilateria and those of Ctenophora, the most basal neuron-bearing animals, suggest a common evolutionary origin of metazoan peptidergic nervous systems.

Dose conversion in opioid rotation from continuous intravenous infusion of morphine hydrochloride injection to fentanyl patch in the management of cancer pain.

Opioid rotation has been proposed for management of cancer pain. No studies directly investigating dose equivalence between morphine injection (continuous IV administration) and the transdermal fentanyl patch have been reported. Therefore, we examined dose conversion ratios in patients undergoing opioid rotation from morphine injection to fentanyl patches. The subjects consisted of 45 patients admitted to Kitasato University East Hospital. Medical records were consulted to determine the "basic dose of morphine injection immediately prior to rotation" and the "basic dose of fentanyl patch after rotation". Equivalent doses and conversion ratios obtained with the expression of (daily dose of morphine injection (mg)/daily delivered dose of fentanyl patch (mg)) were determined from the relationship between the data by regression analysis. The regression equation obtained was Y=50.882X-13.96, r²=0.8922, where X and Y are daily doses of morphine injection and fentanyl patch, respectively. Equivalent doses and conversion ratios for daily dose of morphine injection (mg): daily delivered dose of fentanyl patch (mg) (patch dose mg/3 days) were 16.6 mg: 0.6 mg (2.5 mg)=28:1, 47.1 mg: 1.2 mg (5 mg) = 39:1 and 169.2 mg: 3.6 mg (15 mg)=47:1. In other reports, the ratio of morphine vs. fentanyl at 50:1 had no relation to the dose. While the present study suggested that in opioid rotation from low dose, 50:1 is not enough for the fentanyl patch. The dose conversion ratio of morphine injection to fentanyl patch was different at the low doses and high doses of morphine.

[Liver Injury Risk Factors in Amyotrophic Lateral Sclerosis Patients Treated with Riluzole].

Riluzole, a drug used in the management of amyotrophic lateral sclerosis (ALS), is associated with a high incidence of liver failure. It is imperative to determine risk factors and severity of liver injury in patients taking riluzole to devise an appropriate treatment regimen. We, therefore, studied risk factors for liver injury in ALS patients who were prescribed riluzole at Kitasato University East Hospital from 1999 to 2015. Of the 222 patients enrolled in this study, 113 and 109 patients were diagnosed with mild to moderate (grade 1 or 2) and without (grade 0) liver injury, respectively. Prediction of risk factors was determined using binary logistical regression analyses. The results showed that 50.9% (n=113) of ALS patients developed mild to moderate liver injury; 71.7% and 53.1% of patients were concurrently using CYP1A2 inhibitors (p=0.005) and diclofenac (p=0.032), respectively; 55.8% of patients with liver injury had a history of smoking (p=0.011). Multivariate analyses revealed that the concurrent use of CYP1A2 inhibitors [odds ratio (OR) 2.152, 95% confidence interval (CI) 1.225-3.780, p=0.008] and history of smoking (OR 1.938, 95% CI 1.125-3.340, p=0.017) were independent risk factors for liver injury in patients receiving riluzole. In conclusion, treatment of ALS patients with riluzole, smoking habits, and concurrent use of CYP1A2 inhibitors are independent liver injury risk factors. Further studies on liver injury are warranted in ALS patients treated with riluzole to comprehensively understand the underlying mechanisms of riluzole-associated liver toxicity.

A Chemometrics-driven Strategy for the Bioactivity Evaluation of Complex Multicomponent Systems and the Effective Selection of Bioactivity-predictive Chemical Combinations.

Although understanding their chemical composition is vital for accurately predicting the bioactivity of multicomponent drugs, nutraceuticals, and foods, no analytical approach exists to easily predict the bioactivity of multicomponent systems from complex behaviors of multiple coexisting factors. We herein represent a metabolic profiling (MP) strategy for evaluating bioactivity in systems containing various small molecules. Composition profiles of diverse bioactive herbal samples from 21 green tea extract (GTE) panels were obtained by a high-throughput, non-targeted analytical procedure. This employed the matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) technique, using 1,5-diaminonaphthalene (1,5-DAN) as the optical matrix for detecting GTE-derived components. Multivariate statistical analyses revealed differences among the GTEs in their antioxidant activity, oxygen radical absorbance capacity (ORAC). A reliable bioactivity-prediction model was constructed to predict the ORAC of diverse GTEs from their compositional balance. This chemometric procedure allowed the evaluation of GTE bioactivity by multicomponent rather than single-component information. The bioactivity could be easily evaluated by calculating the summed abundance of a few selected components that contributed most to constructing the prediction model. 1,5-DAN-MALDI-MS-MP, using diverse bioactive sample panels, represents a promising strategy for screening bioactivity-predictive multicomponent factors and selecting effective bioactivity-predictive chemical combinations for crude multicomponent systems.