Cytoprotective effects of Hangekobokuto against corticosterone-induced cell death in HT22 cells.

The hypothalamic-pituitary-adrenal (HPA) system plays an important role in stress response. Chronic stress is thought to induce neuronal damage and contribute to the pathogenesis of psychiatric disorders by causing dysfunction of the HPA system and promoting the production and release of glucocorticoids, including corticosterone and cortisol. Several clinical studies have demonstrated the efficacy of herbal medicines in treating psychiatric disorders; however, their effects on corticosterone-induced neuronal cell death remain unclear. Here, we used HT22 cells to evaluate the neuroprotective potential of herbal medicines used in neuropsychiatry against corticosterone-induced hippocampal neuronal cell death. Cell death was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reduction and Live/Dead assays. Hangekobokuto, Kamikihito, Saikokaryukotsuboreito, Kamishoyosan, and Yokukansan were supplied in the form of water-extracted dried powders. Exposure of HT22 cells to ≥ 100 µM corticosterone decreased MTT values. Exposure to 500 µM corticosterone alone reduced MTT values to 18%, while exposure to 10 µM Mifepristone (RU486)-a glucocorticoid receptor antagonist-restored values to 36%. Corticosterone-induced cell death was partially suppressed by treatment with RU486. At 100 µg/mL, Hangekobokuto significantly suppressed the decrease in MTT values (15-32%) and increase in the percentage of ethidium homodimer-1-positive dead cells caused by corticosterone exposure (78-36%), indicating an inhibitory effect on cell death. By contrast, Kamikihito, Saikokaryukotsuboreito, Kamishoyosan, and Yokukansan did not affect corticosterone-induced cell death. Therefore, our results suggest that Hangekobokuto may ameliorate the onset and progression of psychiatric disorders by suppressing neurological disorders associated with increased levels of glucocorticoids.

2(3H)-Dihydrofranolactone metabolites from Pleosporales sp. NUH322 as anti-amyotrophic lateral sclerosis drugs.

Amyotrophic lateral sclerosis (ALS) is a devastating motor disease with limited treatment options. A domestic fungal extract library was screened using three assays related to the pathophysiology of ALS with the aim of developing a novel ALS drug. 2(3H)-dihydrofuranolactones 1 and 2, and five known compounds 3-7 were isolated from Pleosporales sp. NUH322 culture media, and their protective activity against the excitotoxicity of ß-N-oxalyl-L-α,ß-diaminopropionic acid (ODAP), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamatergic agonist, was evaluated under low mitochondrial glutathione levels induced by ethacrynic acid (EA) and low sulfur amino acids using our developed ODAP-EA assay. Additional assays evaluated the recovery from cytotoxicity caused by transfected SOD1-G93A, an ALS-causal gene, and the inhibitory effect against reactive oxygen species (ROS) elevation. The structures of 1 and 2 were elucidated using various spectroscopic methods. We synthesized 1 from D-ribose, and confirmed the absolute structure. Isolated and synthesized 1 displayed higher ODAP-EA activities than the extract and represented its activity. Furthermore, 1 exhibited protective activity against SOD1-G93A-induced toxicity. An ALS mouse model, SOD1-G93A, of both sexes, was treated orally with 1 at pre- and post-symptomatic stages. The latter treatment significantly extended their lifespan (p = 0.03) and delayed motor deterioration (p = 0.001-0.01). Our result suggests that 1 is a promising lead compound for the development of ALS drugs with a new spectrum of action targeting both SOD1-G93A proteopathy and excitotoxicity through its action on the AMPA-type glutamatergic receptor.

Utility of a Novel Micro-Spraying Device for Intranasal Administration of Drug Solutions to Mice.

Intranasal administration has attracted attention as a means of delivering drugs because it bypasses the blood-brain barrier. However, conventional intranasal administration of drug solutions to mice using the micropipette method (MP method) is complicated and time-consuming because it requires small doses to be administered under inhalation anesthesia. This study evaluated the effectiveness of a novel intranasal administration method using Micro FPS™, a novel micro-spraying device (the MSD method). The MSD method allowed more reliable administration of the solution to the nasal mucosa than the MP method did. The transfer of inulin, a model water-soluble macromolecule compound, to the olfactory bulb and brain (cerebrum, cerebellum, brainstem, and striatum) was similar with the two methods. It also allowed the drug to be administered in a shorter time. These results suggest that the MSD method is simpler and more rapid than the MP method for intranasal administration of drugs to mice and achieves comparable delivery of inulin to the olfactory bulb and brain. Therefore, the Micro FPS™ device is a potentially useful tool for intranasal drug administration to rodents and could facilitate the development of intranasal formulations, contributing to drug development for central nervous system diseases.

Anti-Inflammatory Effects of Miyako Bidens pilosa in a Mouse Model of Amyotrophic Lateral Sclerosis and Lipopolysaccharide-Stimulated BV-2 Microglia.

Neuroinflammation is a fundamental feature in the pathogenesis of amyotrophic lateral sclerosis (ALS) and arises from the activation of astrocytes and microglial cells. Previously, we reported that Miyako Bidens pilosa extract (MBP) inhibited microglial activation and prolonged the life span in a human ALS-linked mutant superoxide dismutase-1 (SOD1G93A) transgenic mouse model of ALS (G93A mice). Herein, we evaluated the effect of MBP on microglial activation in the spinal cord of G93A mice and lipopolysaccharide-stimulated BV-2 microglial cells. The administration of MBP inhibited the upregulation of the M1-microglia/macrophage marker (interferon-γ receptor (IFN-γR)) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6) in G93A mice. However, MBP did not affect the increase in the M2-microglia/macrophage marker (IL-13R) and anti-inflammatory cytokines (transforming growth factor (TGF)-ß and IL-10) in G93A mice. BV-2 cell exposure to MBP resulted in a decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction activity and bromodeoxyuridine incorporation, without an increase in the number of ethidium homodimer-1-stained dead cells. Moreover, MBP suppressed the production of lipopolysaccharide-induced pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in BV-2 cells. These results suggest that the selective suppression of M1-related pro-inflammatory cytokines is involved in the therapeutic potential of MBP in ALS model mice.

An Increase in Peroxiredoxin 6 Expression Induces Neurotoxic A1 Astrocytes in the Lumbar Spinal Cord of Amyotrophic Lateral Sclerosis Mice Model.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease with selective degeneration of motor neurons. It has been reported that an increase in the levels of inflammatory cytokines and glial cells such as reactive astrocytes is closely involved in the pathological progression of ALS. Recently, the levels of neuropathic cytotoxic (A1) astrocytes among reactive astrocytes have reportedly increased in the central nervous system of ALS mice, which induce motor neuron degeneration through the production of inflammatory cytokines and secretion of neuropathic factors. Hence, elucidating the induction mechanism of A1 astrocytes in ALS is important to understand the mechanism of disease progression in ALS. In this study, we observed that the expression of peroxiredoxin 6 (PRDX6), a member of the peroxiredoxin family, was markedly upregulated in astrocytes of the lumbar spinal cord of SOD1G93A mice model for ALS. Additionally, when PRDX6 was transiently transfected into the mouse astrocyte cell line C8-D1A and human astrocytoma cell line U-251 MG, the mRNA expression of complement C3 (a marker for A1 astrocyte phenotype) and inflammatory cytokines was increased. Furthermore, the mRNA expression of C3 and inflammatory cytokine was increased in C8-D1A and U-251 MG cells stably expressing PRDX6, and the increased mRNA expression was significantly suppressed by MJ33 (lithium[1-hexadecoxy-3-(2,2,2-trifluoroethoxy) propan-2-yl] methyl phosphate), an inhibitor of the phospholipase A2 activity of PRDX6. Our results suggest that the expression of PRDX6 in astrocytes plays an important role in the induction of A1 astrocytes and expression of inflammatory cytokines in the ALS mice model.

Schwann cell derived-peroxiredoxin protects motor neurons against hydrogen peroxide-induced cell death in mouse motor neuron cell line NSC-34.

Schwann cells and oligodendrocytes secrete proteins that promote neuron survival, but their role in amyotrophic lateral sclerosis (ALS) is unclear. To address this question, we evaluated the effect of molecules secreted by Schwann cells on reactive oxygen species (ROS)-induced motor neuronal cell death. We observed that in motor neuron cell line NSC-34 cultures, the conditioned medium (CM) from Schwann cell line YST-1 (YST-1 CM) cultures had a protective effect against hydrogen peroxide-induced cell death. However, this protective effect of YST-1 CM was abolished by removing peroxiredoxin 1-4 (PRDX1-4) from the CM. We found that the expression of PRDX1 mRNA was markedly downregulated in the lumbar spinal cord of the superoxide dismutase 1 (SOD1)G93A mouse model of ALS. We also found that transient transfection of YST-1 cells with G93A SOD1 resulted in reduced PRDX1 mRNA expression. Additionally, in the mutant transfected cells, YST-1 CM showed decreased neuroprotective effect against hydrogen peroxide-induced NSC-34 cell death compared to those transfected with WT SOD1. Our results suggest that Schwann cells protect motor neurons from oxidative stress by secreting PRDX1 and that the reduction of PRDX secreted from Schwann cells contributes to increased ROS and associated motor neuronal death in ALS.

Update on the pathological roles of prostaglandin E2 in neurodegeneration in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective degeneration of upper and lower motor neurons. The pathogenesis of ALS remains largely unknown; however, inflammation of the spinal cord is a focus of ALS research and an important pathogenic process in ALS. Prostaglandin E2 (PGE2) is a major lipid mediator generated by the arachidonic-acid cascade and is abundant at inflammatory sites. PGE2 levels are increased in the postmortem spinal cords of ALS patients and in ALS model mice. Beneficial therapeutic effects have been obtained in ALS model mice using cyclooxygenase-2 inhibitors to inhibit the biosynthesis of PGE2, but the usefulness of this inhibitor has not yet been proven in clinical trials. In this review, we present current evidence on the involvement of PGE2 in the progression of ALS and discuss the potential of microsomal prostaglandin E synthase (mPGES) and the prostaglandin receptor E-prostanoid (EP) 2 as therapeutic targets for ALS. Signaling pathways involving prostaglandin receptors mediate toxic effects in the central nervous system. In some situations, however, the receptors mediate neuroprotective effects. Our recent studies demonstrated that levels of mPGES-1, which catalyzes the final step of PGE2 biosynthesis, are increased at the early-symptomatic stage in the spinal cords of transgenic ALS model mice carrying the G93A variant of superoxide dismutase-1. In addition, in an experimental motor-neuron model used in studies of ALS, PGE2 induces the production of reactive oxygen species and subsequent caspase-3-dependent cytotoxicity through activation of the EP2 receptor. Moreover, this PGE2-induced EP2 up-regulation in motor neurons plays a role in the death of motor neurons in ALS model mice. Further understanding of the pathophysiological role of PGE2 in neurodegeneration may provide new insights to guide the development of novel therapies for ALS.

Photothermal therapeutic ability of copper open-shell nanostructures that are effective in the second biological transparency window based on symmetry breaking-induced plasmonic properties.

In this study, a photothermal therapy agent that works efficiently in the second biological transparency window was developed based on the localized surface plasmon (LSP) resonance of symmetry-broken open-shell nanostructures of low-cost Cu (CuOSNs). The strong LSP resonance and superior photothermal conversion ability in the second biological transparency window were achieved by generating the dipolar bonding mode due to the plasmon hybridization between the nanoshell dipole and the nanohole dipole at the opening edge in CuOSNs derived from the symmetry breaking of a Cu nanoshell. Oxidative dissolution of CuOSNs in water was significantly suppressed by successive coating with the self-assembled monolayer of 16-mercaptohexadecanoic acid and a thin silica layer. Furthermore, the stability in phosphate buffered saline, which models the biological environment, was attained by further coating the nanoparticles with polyethylene glycol. It was demonstrated from in vitro cell tests using HeLa cells that the cytotoxicity of CuOSNs was effectively suppressed by the surface protection. The viability of HeLa cells incubated with CuOSNs was decreased under the irradiation of low intensity 1060 nm laser with increasing number of CuOSNs. These results demonstrate that low-cost symmetry-broken Cu-based nanostructures can act as an excellent photothermal therapy agent in the second biological transparency window.

Intranasal Administration of N-acetyl-L-cysteine Combined with Cell-Penetrating Peptide-Modified Polymer Nanomicelles as a Potential Therapeutic Approach for Amyotrophic Lateral Sclerosis.

Intranasal administration is a promising route for direct drug delivery to the brain; its combination with nanocarriers enhances delivery. We have previously shown that intranasal administration combined with PEG-PCL-Tat (a nanocarrier) efficiently delivers drugs to the brain and exhibits excellent therapeutic efficacy against brain diseases. We aimed to clarify whether intranasal administration combined with PEG-PCL-Tat represents a useful drug delivery system (DDS) for amyotrophic lateral sclerosis (ALS) pharmacotherapy. We used N-acetyl-L-cysteine (NAC) as a model drug with low transferability to the spinal cord and determined the physicochemical properties of NAC/PEG-PCL-Tat. After intranasal administration of NAC/PEG-PCL-Tat, we measured the survival duration of superoxide dismutase-1 G93A mutant transgenic mice (G93A mice), widely used in ALS studies, and quantitatively analyzed the tissue distribution of NAC/PEG-PCL-Tat in ddY mice. The mean particle size and zeta potential of NAC/PEG-PCL-Tat were 294 nm and + 9.29 mV, respectively. Treatment with repeated intranasal administration of NAC/PEG-PCL-Tat considerably prolonged the median survival of G93A mice by 11.5 days compared with that of untreated G93A mice. Moreover, the highest distribution after a single administration of NAC/PEG-PCL-Tat was measured in the spinal cord. These results suggest that intranasal administration combined with PEG-PCL-Tat might represent a useful DDS for ALS therapeutics.

Association Between Inappropriate Use of Over-The-Counter Drugs for Allergic Rhinitis and Side Effects on the Central Nervous system-a Cross-Sectional Survey.

Purpose: Antihistamine over-the-counter (OTC) drugs for allergic rhinitis are widely used and cause central nervous system side effects. Most available data on anti-allergic drugs are on controlled usage. It is necessary to investigate the occurrence of side effects in the context of self-medication to avoid inappropriate use. We aimed to clarify the association between the usage of OTC anti-allergic drugs and central nervous system side effects. Patients and Methods: An online, anonymous, cross-sectional study was conducted using a structured questionnaire. People who had used OTC anti-allergic drugs in the year prior to the study were recruited. To assess the association between inappropriate drug use and the occurrence of side effects, a binary logistics regression analysis was performed according to three dosage forms (oral only, nasal only, and oral and nasal combined use). Results: Somnolence was experienced by 59.1% of the participants using the OTC drug for allergic rhinitis. Using logistic regression analysis, "drug use exceeding the upper limit" was seen to be associated with side effects in only oral (Somnolence: OR = 1.41, 95% CI = 1.17-1.70; Dull head: OR=1.41, 95% CI = 1.16-1.70; Loss of concentration: OR = 1.25, 95% CI = 1.04-1.49) and oral and nasal combined use groups (Somnolence: OR = 1.33, 95% CI = 1.04-1.71; Dull head: OR = 1.47, 95% CI = 1.15-1.89; Loss of concentration: OR = 1.51, 95% CI = 1.19-1.91). Furthermore, "expired drug use" was associated with side effects in the nasal spray-only group (Somnolence: OR = 1.31, 95% CI = 1.07-1.60; Dull head: OR =1.25, 95% CI = 1.02-1.53; Loss of concentration: OR = 1.24, 95% CI = 1.00-1.54). Conclusion: Inappropriate use was common among users of OTC allergic rhinitis drugs. Differences in side effects depending on the dosage form used were observed.

Modulation Technique of Localized Surface Plasmon Resonance of Palladium Nanospheres by Coating with Titanium Dioxide Shell for Application to Photothermal Therapy Agent.

Although plasmonic palladium (Pd) nanospheres are thermodynamically stable and have high photothermal conversion due to the free and bound electron coupling associated with the intrinsic high interband transition, they have not attracted attention as a photothermal conversion material for next-generation photothermal cancer therapy. This is because the Pd nanospheres generate the localized surface plasmon resonance (LSPR) intrinsically in the ultraviolet region, which is far away from the biological transparent window (750-900 nm). In this study, we controlled the LSP wavelength of Pd nanospheres by coating with high refractive index TiO2 shells taking advantage of the Pd LSPR which is highly sensitive to changes in the local refractive index around the nanospheres. Our calculations indicated that the absorption cross section at 808 nm (corresponding to the wavelength used for photothermal treatment) was increased by 4.5 times by redshifting the LSPR and increasing the extinction intensity associated with the coating with TiO2 shell. Experiments confirmed the theoretical prediction in that the LSPR of the synthesized Pd nanospheres with a diameter of 81 nm was significantly redshifted by coating with amorphous TiO2 shell, resulting in significant large extinction intensity at 808 nm. The photothermal conversion efficiency was estimated to be 50%. In vitro cell tests, HeLa cells incubated with 100-300 µg/mL TiO2-coated Pd nanospheres were efficiently killed by irradiating 808 nm laser (1.8 W) even though the nanospheres with the same concentrations showed little cytotoxicity. These results indicate that the Pd nanospheres coated with high refractive index shells can be promising as a photothermal therapy agent.

Nose-to-brain/spinal cord delivery kinetics of liposomes with different surface properties.

The administration of liposomes via nose-to-brain delivery is expected to become a strategy for efficient drug delivery to the central nervous system. Efficient nose-to-brain delivery and the kinetics of drugs administered in this manner depend on the properties of liposomes. However, there is a lack of basic knowledge of which liposomes are suitable for this purpose. Here, a qualitative study of intranasally administered liposomes (positively charged, neutral, and negatively charged, with or without polyethylene glycol [PEG] modification; particle size <100 nm) was performed to elucidate their dynamics in the brain and spinal cord. Additionally, a quantitative investigation was performed to ascertain their distribution in each part of the brain and spinal cord. The effects of liposome surface charge and PEG modification on the kinetics and distribution post intranasal administration were investigated via two experiments. Qualitative evaluation was performed via ex vivo observation after intranasal administration of fluorescently labeled liposomes. Neutral PEG-modified liposomes were distributed throughout the brain and spinal cord 60 min after administration, and the fluorescence intensity increased with time. By contrast, non-PEG-modified neutral liposomes showed particularly strong fluorescence in the olfactory bulb, and the fluorescence was localized in the anterior part of the brain. Positively charged liposomes showed low fluorescence around the lateral part of the brain and lumbar spinal cord 60 min after administration. Low fluorescence was observed in the whole brain and spinal cord, with strong fluorescence being observed in the olfactory bulb after 120 min of administration. Negatively charged liposomes showed no fluorescence at 60 min after administration, but low fluorescence was observed throughout the brain and spinal cord 120 min after administration. We quantified the radioactivity in the brain and spinal cord after intranasal administration of radioisotope-labeled liposomes. Neutral liposomes showed the highest distribution by area under the drug concentration-time curve (AUC60-120) in the brain and spinal cord compared to other liposomes. Compared with negatively charged liposomes, positively charged liposomes had a higher distribution in the olfactory bulb and forebrain, while negatively charged liposomes had a higher distribution in the hindbrain and bulbospinal tract cord. In addition, the distribution of PEG-modified neutral liposomes in the brain and spinal cord was significantly enhanced compared to that of non-PEG-modified neutral liposomes after 90 min of intranasal administration. These results indicate that surface charge and PEG modification strongly affect the efficiency of nose-to-brain delivery kinetics, and that PEG-modified neutral liposomes are excellent carriers for drug delivery to a wide area of the brain and spinal cord.

Possible role of transcriptional regulation of 5-HT1A receptor in the midbrain on unadaptation to stress in mice.

The ability to adapt to stress is an essential defensive function of a living body, and disturbance of this ability in the brain may contribute to the development of affective illness. Previously, we reported that mice exposed to unadaptable restraint stress show emotional abnormality. Moreover, this emotional abnormality was alleviated by chronic treatment with flesinoxan, a serotonin (5-HT)1A receptor agonist. 5-HT1A receptor expression is regulated by several transcription factors such as nuclear deformed epidermal autoregulatory factor (NUDR/Deaf-1) and five prime repressors under dual repression binding protein 1 (Freud-1). The present study was designed to investigate the expression levels of 5-HT1A receptor and its transcription factors in the midbrain and hippocampus of stress-adaptive and -unadaptive mice. Mice were exposed to 14 days of repeated adaptable (1 h/day) or repeated unadaptable (4 h/day) restraint stress, or were left in their home cage (non-stressed groups). In a western blot analysis, a significant increase in the expression levels of 5HT1A receptor protein were observed in the hippocampal membrane fraction in stress-adaptive mice. In contrast, the expression levels of 5-HT1A receptor protein in stress-unadaptive mice were significantly increased in both cytoplasmic and membrane fraction of the midbrain. Furthermore, real-time PCR analysis revealed that, in the midbrain of stress-unadaptive mice, the expression levels of 5-HT1A receptor mRNA and Freud-1 or NUDR mRNA were significantly increased and decreased, respectively. These results suggest that increased expression of 5-HT1A receptor due to decrease in the expression of Freud-1 and NUDR in the midbrain may play a pivotal role in the emotional abnormality of stress-unadaptive mice.

Preventive effect of rebamipide on NSAID-induced lower gastrointestinal tract injury using FAERS and JADER.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for their antipyretic, analgesic, and anti-inflammatory properties. However, various aspects of NSAID-induced lower gastrointestinal tract injury remain unclear, and effective prophylaxis has not been established. Based on its pharmacological effect and clinical trials, rebamipide may prevent lower gastrointestinal tract injury, although this evidence is limited by the small scale of trials. The present study used the FDA Adverse Event Reporting System (FAERS) and the Japanese Adverse Event Reporting Database (JADER) to assess the efficacy of rebamipide in combination with loxoprofen and diclofenac in preventing NSAID-induced lower gastrointestinal tract injury. The calculated reporting odds ratio and 95% confidence interval (CI) for rebamipide in combination with loxoprofen and diclofenac were 1.15 (95% CI 0.88-1.51) and 1.28 (95% CI 0.82-2.01) for FAERS, and 0.50 (95% CI 0.35-0.71) and 0.43 (95% CI 0.27-0.67) for JADER, respectively. No signal was detected when combining drugs. These results suggest a prophylactic effect of rebamipide on NSAID-induced lower gastrointestinal tract injury.

Present State and Future Perspectives of Prostaglandins as a Differentiation Factor in Motor Neurons.

Spinal motor neurons have the longest axons that innervate the skeletal muscles of the central nervous system. Motor neuron diseases caused by spinal motor neuron cell death are incurable due to the unique and irreplaceable nature of their neural circuits. Understanding the mechanisms of neurogenesis, neuritogenesis, and synaptogenesis in motor neurons will allow investigators to develop new in vitro models and regenerative therapies for motor neuron diseases. In particular, small molecules can directly reprogram and convert into neural stem cells and neurons, and promote neuron-like cell differentiation. Prostaglandins are known to have a role in the differentiation and tissue regeneration of several cell types and organs. However, the involvement of prostaglandins in the differentiation of motor neurons from neural stem cells is poorly understood. The general cell line used in research on motor neuron diseases is the mouse neuroblastoma and spinal motor neuron fusion cell line NSC-34. Recently, our laboratory reported that prostaglandin E2 and prostaglandin D2 enhanced the conversion of NSC-34 cells into motor neuron-like cells with neurite outgrowth. Moreover, we found that prostaglandin E2-differentiated NSC-34 cells had physiological and electrophysiological properties of mature motor neurons. In this review article, we provide contemporary evidence on the effects of prostaglandins, particularly prostaglandin E2 and prostaglandin D2, on differentiation and neural conversion. We also discuss the potential of prostaglandins as candidates for the development of new therapeutic drugs for motor neuron diseases.

A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells.

Superoxide dismutase 1 (SOD1) is a metalloenzyme with high structural stability, but a lack of Cu and Zn ions decreases its stability and enhances the likelihood of misfolding, which is a pathological hallmark of amyotrophic lateral sclerosis (ALS). A growing body of evidence has demonstrated that misfolded SOD1 has prion-like properties such as transmissibility between cells and intracellular propagation of misfolding of natively folded SOD1. Recently, we found that SOD1 is misfolded in the cerebrospinal fluid of sporadic ALS patients, providing a route by which misfolded SOD1 spreads via the extracellular environment of the central nervous system. Unlike intracellular misfolded SOD1, it is unknown which extracellular misfolded species is most relevant to prion-like properties. Here, we determined a conformational feature of extracellular misfolded SOD1 that is linked to prion-like properties. Using culture media from motor neuron-like cells, NSC-34, extracellular misfolded wild-type, and four ALS-causing SOD1 mutants were characterized as a metal-free, disulfide oxidized form of SOD1 (apo-SOD1S-S). Extracellular misfolded apo-SOD1S-S exhibited cell-to-cell transmission from the culture medium to recipient cells as well as intracellular propagation of SOD1 misfolding in recipient cells. Furthermore, culture medium containing misfolded apo-SOD1S-S exerted cytotoxicity to motor neuron-like cells, which was blocked by removal of misfolded apo-SOD1S-S from the medium. We conclude that misfolded apo-SOD1S-S is a primary extracellular species that is linked to prion-like properties.

Quantitative analysis of inulin distribution in the brain focused on nose-to-brain route via olfactory epithelium by reverse esophageal cannulation.

This study aimed to determine the effect of intranasal dosing speed and administrating volume of nose-to-brain delivery on candidates for peptide drugs (molecular weight ca. 1-10 kDa). Using inulin as the model molecule of a peptide drug, intranasal administration by cannulation from the airway side through the esophagus was tested in mice. This was done to determine the quantitative distribution levels of the drug in the brain and cerebral spinal fluid (CSF). Distribution levels were increased with slower and constant speed (5 µL/min), with higher dosing volume equivalent to nasal volume per body weight in mice (25 µL), and were recorded 0.27% injected dose per gram of tissue (ID/g) in the brain, and 0.24% injected dose per milliliter (ID/mL) in the CSF at 60 min. Then, brain distribution resulting from reverse cannulation was two times more than that of the typical intranasal administration method using a micropipette. In addition, the percentage of inulin estimated to reach the brain via direct transport (%DTP) during reverse cannulation was estimated to be 93%, suggesting that ~95% of the total dose was transferred directly to the brain via the olfactory mucosa. These results show that distribution of the peptide drug in the brain was increased through constant administration at a slow and constant speed.

Highly Efficient Conversion of Motor Neuron-Like NSC-34 Cells into Functional Motor Neurons by Prostaglandin E2.

Motor neuron diseases are a group of progressive neurological disorders that degenerate motor neurons. The neuroblastoma × spinal cord hybrid cell line NSC-34 is widely used as an experimental model in studies of motor neuron diseases. However, the differentiation efficiency of NSC-34 cells to neurons is not always sufficient. We have found that prostaglandin E2 (PGE2) induces morphological differentiation in NSC-34 cells. The present study investigated the functional properties of PGE2-differentiated NSC-34 cells. Retinoic acid (RA), a widely-used agent inducing cell differentiation, facilitated neuritogenesis, which peaked on day 7, whereas PGE2-induced neuritogenesis took only 2 days to reach the same level. Whole-cell patch-clamp recordings showed that the current threshold of PGE2-treated cell action potentials was lower than that of RA-treated cells. PGE2 and RA increased the protein expression levels of neuronal differentiation markers, microtubule-associated protein 2c and synaptophysin, and to the same extent, motor neuron-specific markers HB9 and Islet-1. On the other hand, protein levels of choline acetyltransferase and basal release of acetylcholine in PGE2-treated cells were higher than in RA-treated cells. These results suggest that PGE2 is a rapid and efficient differentiation-inducing factor for the preparation of functionally mature motor neurons from NSC-34 cells.

Decaturenol A and known oxalicine related meroterpenoids isolated from Penicillium decaturense RO050, and their new biological activities.

Decaturenol A (1), a new oxalicine related meroterpenoid, has been isolated from Penicillium decaturense RO050 along with seven known compounds (2-8). The structure of 1 was elucidated by spectroscopic data. The effects of isolated compounds (1-8) on endoplasmic reticulum (ER) stress-induced cell death in HT22 hippocampal nerve cells and on the interleukin 10 (IL-10)-induced expression of CD163, a M2 phenotype marker, in human monocyte-derived macrophages were evaluated. While decaturenol A (1) exhibited a protective effect on ER stress-induced cell death in HT22 cells at 10 µM, on the other hand oxalicine A (7) showed cytotoxic activity (IC50 = 5.9 µM). Additionally, decaturenol A (1), decaturins D (2), E (3), and B (4) inhibited the IL-10-induced expression of CD163 each at a concentration of 20 µg/mL.

Generation of Cellular Reactive Oxygen Species by Activation of the EP2 Receptor Contributes to Prostaglandin E2-Induced Cytotoxicity in Motor Neuron-Like NSC-34 Cells.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease characterized by progressive degeneration of motor neurons in the central nervous system. Prostaglandin E2 (PGE2) plays a pivotal role in the degeneration of motor neurons in human and transgenic models of ALS. We have shown previously that PGE2 directly induces neuronal death through activation of the E-prostanoid (EP) 2 receptor in differentiated NSC-34 cells, a motor neuron-like cell line. In the present study, to clarify the mechanisms underlying PGE2-induced neurotoxicity, we focused on generation of intracellular reactive oxygen species (ROS) and examined the effects of N-acetylcysteine (NAC), a cell-permeable antioxidant, on PGE2-induced cell death in differentiated NSC-34 cells. Dichlorofluorescein (DCF) fluorescence analysis of PGE2-treated cells showed that intracellular ROS levels increased markedly with time, and that this effect was antagonized by a selective EP2 antagonist (PF-04418948) but not a selective EP3 antagonist (L-798,106). Although an EP2-selective agonist, butaprost, mimicked the effect of PGE2, an EP1/EP3 agonist, sulprostone, transiently but significantly decreased the level of intracellular ROS in these cells. MTT reduction assay and lactate dehydrogenase release assay revealed that PGE2- and butaprost-induced cell death were each suppressed by pretreatment with NAC in a concentration-dependent manner. Western blot analysis revealed that the active form of caspase-3 was markedly increased in the PGE2- and butaprost-treated cells. These increases in caspase-3 protein expression were suppressed by pretreatment with NAC. Moreover, dibutyryl-cAMP treatment of differentiated NSC-34 cells caused intracellular ROS generation and cell death. Our data reveal the existence of a PGE2-EP2 signaling-dependent intracellular ROS generation pathway, with subsequent activation of the caspase-3 cascade, in differentiated NSC-34 cells, suggesting that PGE2 is likely a key molecule linking inflammation to oxidative stress in motor neuron-like NSC-34 cells.