Photosensitizer-singlet oxygen sensor conjugated silica nanoparticles for photodynamic therapy and bioimaging.

Intracellular singlet oxygen (1O2) generation and detection help optimize the outcome of photodynamic therapy (PDT). Theranostics programmed for on-demand phototriggered 1O2 release and bioimaging have great potential to transform PDT. We demonstrate an ultrasensitive fluorescence turn-on sensor-sensitizer-RGD peptide-silica nanoarchitecture and its 1O2 generation-releasing-storing-sensing properties at the single-particle level or in living cells. The sensor and sensitizer in the nanoarchitecture are an aminomethyl anthracene (AMA)-coumarin dyad and a porphyrin or CdSe/ZnS quantum dots (QDs), respectively. The AMA in the dyad quantitatively quenches the fluorescence of coumarin by intramolecular electron transfer, the porphyrin or QD moiety generates 1O2, and the RGD peptide facilitates intracellular delivery. The small size, below 200 nm, as verified by scanning electron microscopy and differential light scattering measurements, of the architecture within the 1O2 diffusion length enables fast and efficient intracellular fluorescence switching by the tandem ultraviolet (UV)-visible or visible-near-infrared (NIR) photo-triggering. While the red emission and 1O2 generation by the porphyrin are continually turned on, the blue emission of coumarin is uncaged into 230-fold intensity enhancement by on-demand photo-triggering. The 1O2 production and release by the nanoarchitecture enable spectro-temporally controlled cell imaging and apoptotic cell death; the latter is verified from cytotoxic data under dark and phototriggering conditions. Furthermore, the bioimaging potential of the TCPP-based nanoarchitecture is examined in vivo in B6 mice.

Distribution of Signal Peptides in Microvesicles from Activated Macrophage Cells.

Extracellular vesicles, such as microvesicles (LEV) and exosomes (SEV), play an important role in intercellular signaling by encapsulating functional molecules and delivering them to specific cells. Recent studies showed that signal peptides (SPs), which are derived from sequences at the N-terminal of newly synthesized proteins, exhibited biological activity in the extracellular fluid. We previously reported that SPs were secreted into the extracellular fluid via SEV; however, it remains unclear whether the release of SPs occurs via LEV. In the present study, we demonstrated that SP fragments from human placental secreted alkaline phosphatase (SEAP) were present in LEV as well as SEV released from RAW-Blue cells, which stably express an NF-κB-inducible SEAP reporter. When RAW-Blue cells were treated with LPS at 0-10,000 ng/mL, SEAP SP fragments per particle were more abundant in LEV than in SEV, with fragments in LEV and SEV reaching a maximum at 1000 and 100 ng/mL, respectively. The content of SEAP SP fragments in LEV from IFNγ-stimulated RAW-Blue cells was higher than those from TNFα-stimulated cells, whereas that in SEV from TNFα-stimulated RAW-Blue cells was higher than those from IFNγ-stimulated cells. Moreover, the content of SEAP SP fragments in LEV and SEV decreased in the presence of W13, a calmodulin inhibitor. Collectively, these results indicate that the transportation of SP fragments to extracellular vesicles was changed by cellular activation, and calmodulin was involved in their transportation to LEV and SEV.

Signal Sequence-Dependent Orientation of Signal Peptide Fragments to Exosomes.

Signal peptides (SPs) not only mediate targeting to the endoplasmic reticulum (ER) but also play important roles as biomarkers and substances with physiological activity in extracellular fluids including blood. SPs are thought to be degraded intracellularly, making it unclear how they are transported from the ER to the extracellular fluid. In a recent study, we showed that a C-terminal fragment of the SP of a type I membrane protein, amyloid precursor protein (APP), was secreted into the extracellular fluid via exosomes using transformed HEK293 cells expressing APP SP flanking a reporter protein. In the present study, we demonstrate that a N-terminal fragment of the SP from a type II membrane protein, human placental secreted alkaline phosphatase (SEAP), is contained in exosomes and secreted into the extracellular fluid using HEK-Blue hTLR3 cells, which express both a human toll-like receptor 3 gene and an inducible SEAP reporter gene. When HEK-Blue hTLR3 cells were stimulated with a TLR3 ligand, a N-terminal fragment of SEAP SP in exosomes was increased in parallel with SEAP secretion in a concentration-dependent manner. These results indicated that SP fragments are exosomal components. In addition, migrating SP fragments were determined by characteristics of the signal-anchor sequence of membrane proteins. Furthermore, we found that SP fragments could bind to calmodulin (CALM), which is a cytosolic protein and also a component of exosomes, suggesting its involvement in the transportation of SP fragments from the endoplasmic reticulum to exosomes.

Calmodulin as a Key Regulator of Exosomal Signal Peptides.

Signal peptides (SPs) and their fragments play important roles as biomarkers and substances with physiological functions in extracellular fluid. We previously reported that SP fragments were released into extracellular fluid via exosomes and bound to calmodulin (CaM), an exosomal component, in a cell-free system. However, it currently remains unclear whether CaM intracellularly interacts with SP fragments or is involved in the trafficking of these fragments to exosomes. Therefore, the present study examined the binding of CaM to SP fragments in T-REx AspALP cells, transformed HEK293 cells expressing amyloid precursor protein (APP) SP flanking a reporter protein, and their exosomes. APP SP fragments were detected in exosomes from T-REx AspALP cells in the absence of W13, a CaM inhibitor, but were present in lower amounts in exosomes from W13-treated cells. Cargo proteins, such as Alix, CD63, and CD81, were increased in W13-treated T-REx AspALP cells but were decreased in their exosomes. Furthermore, CaM interacted with heat shock protein 70 and CD81 in T-REx AspALP cells and this increased in the presence of W13. APP SP fragments were detected in intracellular CaM complexes in the absence of W13, but not in its presence. These results indicate that CaM functions as a key regulator of the transport of SP fragments into exosomes and plays novel roles in the sorting of contents during exosomal biogenesis.

Effectiveness of Long-Term Physiotherapy in Parkinson's Disease: A Systematic Review and Meta-Analysis.

BACKGROUND: Long-term physiotherapy is acknowledged to be crucial to manage motor symptoms for Parkinson's disease (PD) patients, but its effectiveness is not well understood. OBJECTIVE: This systematic review and meta-analysis aimed to assess the evidence regarding the effectiveness of long-term physiotherapy to improve motor symptoms and reduce antiparkinsonian medication dose in PD patients. METHODS: Pubmed, Cochrane, PEDro, and CINAHL were searched for randomized controlled trials before August 31, 2020 that investigated the effectiveness of physiotherapy for 6 months or longer on motor symptoms and levodopa-equivalent dose (LED) in PD patients with Hoehn and Yahr stage 1- 3. We performed random effects meta-analyses for long-term physiotherapy versus no/control intervention and estimated standard mean differences with 95% confidence intervals (CIs). Levels of evidence were rated by the Grading of Recommendation Assessment, Development and Evaluation approach. RESULTS: From 2,940 studies, 10 studies involving 663 PD patients were assessed. Long-term physiotherapy had favorable effects on motor symptoms in off medication state [- 0.65, 95% CI - 1.04 to - 0.26, p = 0.001] and LED [- 0.49, 95% CI - 0.89to - 0.09, p = 0.02]. Subgroup analyses demonstrated favorable effects on motor symptoms in off medication state by aerobic exercise [- 0.42, 95% CI - 0.64 to - 0.20, p < 0.001] and LED by multidisciplinary rehabilitation of primarily physiotherapy [- 1.00, 95% CI - 1.44 to - 0.56, p < 0.001]. Quality of evidence for aerobic exercise and multidisciplinary rehabilitation were low and very low. CONCLUSION: This review provided evidence that long-term physiotherapy has beneficial impact on motor symptoms and antiparkinsonian medication dose in PD patients and could motivate implementation of long-term physiotherapy.

Size and surface modification of silica nanoparticles affect the severity of lung toxicity by modulating endosomal ROS generation in macrophages.

BACKGROUND: As the application of silica nanomaterials continues to expand, increasing chances of its exposure to the human body and potential harm are anticipated. Although the toxicity of silica nanomaterials is assumed to be affected by their physio-chemical properties, including size and surface functionalization, its molecular mechanisms remain unclear. We hypothesized that analysis of intracellular localization of the particles and subsequent intracellular signaling could reveal a novel determinant of inflammatory response against silica particles with different physico-chemical properties. RESULTS: We employed a murine intratracheal instillation model of amorphous silica nanoparticles (NPs) exposure to compare their in vivo toxicities in the respiratory system. Pristine silica-NPs of 50 nm diameters (50 nm-plain) induced airway-centered lung injury with marked neutrophilic infiltration. By contrast, instillation of pristine silica particles of a larger diameter (3 µm; 3 µm-plain) significantly reduced the severity of lung injury and neutrophilic infiltration, possibly through attenuated induction of neutrophil chemotactic chemokines including MIP2. Ex vivo analysis of alveolar macrophages as well as in vitro assessment using RAW264.7 cells revealed a remarkably lower cellular uptake of 3 µm-plain particles compared with 50 nm-plain, which is assumed to be the underlying mechanism of attenuated immune response. The severity of lung injury and neutrophilic infiltration was also significantly reduced after intratracheal instillation of silica NPs with an amine surface modification (50 nm-NH2) when compared with 50 nm-plain. Despite unchanged efficacy in cellular uptake, treatment with 50 nm-NH2 induced a significantly attenuated immune response in RAW264.7 cells. Assessment of intracellular redox signaling revealed increased reactive oxygen species (ROS) in endosomal compartments of RAW264.7 cells treated with 50 nm-plain when compared with vehicle-treated control. In contrast, augmentation of endosomal ROS signals in cells treated with 50 nm-NH2 was significantly lower. Moreover, selective inhibition of NADPH oxidase 2 (NOX2) was sufficient to inhibit endosomal ROS bursts and induction of chemokine expressions in cells treated with silica NPs, suggesting the central role of endosomal ROS generated by NOX2 in the regulation of the inflammatory response in macrophages that endocytosed silica NPs. CONCLUSIONS: Our murine model suggested that the pulmonary toxicity of silica NPs depended on their physico-chemical properties through distinct mechanisms. Cellular uptake of larger particles by macrophages decreased, while surface amine modification modulated endosomal ROS signaling via NOX2, both of which are assumed to be involved in mitigating immune response in macrophages and resulting lung injury.

Secretion of signal peptides via extracellular vesicles.

Signal peptides (SPs) consist of short peptide sequences present at the N-terminal of newly synthesizing proteins and act as a zip code for the translocation of the proteins to the endoplasmic reticulum (ER). It was thought that the SPs are intracellularly degraded after translocation to the ER; however, recent studies showed cleaved SPs have diverse roles for controlling cell functions in auto- and/or intercellular manners. In addition, it still remains obscure how SP fragments translocate away from the site where they are produced. Extracellular vesicles (EV) are important for intercellular communication and can transport functional molecules to specific cells. In this study, we show that SPs are involved in EV from T-REx AspALP cells that were transfected with a human APP SP-inducible expression vector. There was no difference in the average particle size or particle concentration of EV collected from T-REx AspALP cells and T-REx Mock cells. When the SP content in the EV was examined by mass spectrometry, the C-terminal fragment of APP SP was identified in the exosomes (SEV) of T-REx AspALP cells. In our preparation of SEV fractions, no ER-specific proteins were detected; therefore, SPs may be included in SEV but not in the debris of degraded ER. This is the first indication that SPs are secreted from cells via EV.

The sodium-glucose cotransporter-2 inhibitor Tofogliflozin prevents the progression of nonalcoholic steatohepatitis-associated liver tumors in a novel murine model.

BACKGROUND: Diabetes and obesity contribute to the pathogenesis of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). However, how diabetes and obesity accelerate liver tumorigenesis remains to be fully understood. Moreover, to verify the therapeutic potential of anti-diabetic drugs, there exists a strong need for appropriate animal models that recapitulate human pathophysiology of NASH and HCC. METHODS: We established a novel murine model of NASH-associated liver tumors using genetically obese melanocortin 4 receptor-deficient mice fed on Western diet in combination with a chemical procarcinogen, and verified the validity of our model in evaluating drug efficacy. FINDINGS: Our model developed multiple liver tumors together with obesity, diabetes, and NASH within a relatively short period (approximately 3 months). In this model, sodium glucose cotransporter 2 inhibitor Tofogliflozin prevented the development of NASH-like liver phenotypes and the progression of liver tumors. Tofogliflozin attenuated p21 expression of hepatocytes in non-tumorous lesions in the liver. INTERPRETATION: Tofogliflozin treatment attenuates cellular senescence of hepatocytes under obese and diabetic conditions. This study provides a unique animal model of NASH-associated liver tumors, which is applicable for assessing drug efficacy to prevent or treat NASH-associated HCC.

Detection of Alzheimer's disease-related neuroinflammation by a PET ligand selective for glial versus vascular translocator protein.

A substantial and constitutive expression of translocator protein (TSPO) in cerebral blood vessels hampers the sensitive detection of neuroinflammation characterized by greatly induced TSPO expression in activated glia. Here, we conducted in vivo positron emission tomography (PET) and in vitro autoradiographic imaging of normal and TSPO-deficient mouse brains to compare the binding properties of 18F-FEBMP, a relatively novel TSPO radioligand developed for human studies based on its insensitivity to a common polymorphism, with 11C-PK11195, as well as other commonly used TSPO radioligands including 11C-PBR28, 11C-Ac5216 and 18F-FEDAA1106. TSPO in cerebral vessels of normal mice was found to provide a major binding site for 11C-PK11195, 11C-PBR28 and 18F-FEDAA1106, in contrast to no overt specific binding of 18F-FEBMP and 11C-Ac5216 to this vascular component. In addition, 18F-FEBMP yielded PET images of microglial TSPO with a higher contrast than 11C-PK11195 in a tau transgenic mouse modeling Alzheimer's disease (AD) and allied neurodegenerative tauopathies. Moreover, TSPO expression examined by immunoblotting was significantly increased in AD brains compared with healthy controls, and was well correlated with the autoradiographic binding of 18F-FEBMP but not 11C-PK11195. Our findings support the potential advantage of comparatively glial TSPO-selective radioligands such as 18F-FEBMP for PET imaging of inflammatory glial cells.

Peripheral benzodiazepine receptor/18 kDa translocator protein positron emission tomography imaging in a rat model of acute brain injury.

OBJECTIVE: The activation of microglia in various brain pathologies is accompanied by an increase in the expression of peripheral benzodiazepine receptor/18 kDa translocator protein (PBR/TSPO). However, whether activated microglia have a neuroprotective or neurotoxic effect on neurons in the brain is yet to be determined. In this study, we investigated the ability of the novel PBR/TSPO ligand FEPPA to detect activated microglia in an animal model of primary neurotoxic microglia activation. METHODS: [18F] FEPPA positron emission tomography (PET) imaging was performed before and after intraperitoneal administration of lipopolysaccharide (LPS) (LPS group) or saline (control group) in a unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of Parkinson's disease. Images were compared between these groups. After imaging, the brains were collected, and the activated microglia at the disease sites were analyzed by the expression of inflammatory cytokines and immunohistochemistry staining. These results were then comparatively examined with those obtained by PET imaging. RESULTS: In the unilateral 6-OHDA lesion rat model, the PBR/TSPO PET signal was significantly increased in the LPS group compared with the saline group. As the increased signal was observed 4 h after the injection, we considered it an acute response to brain injury. In the post-imaging pathological examination, activated microglia were found to be abundant at the site where strong signals were detected, and the expression of the inflammatory cytokines TNF-α and IL-1ß was increased. Intraperitoneal LPS administration further increased the expression of inflammatory cytokines, and the PBR/TSPO PET signal increased concurrently. The increase in inflammatory cytokine expression correlated with enhanced signal intensity. CONCLUSIONS: PET signal enhancement by PBR/TSPO at the site of brain injury correlated with the activation of microglia and production of inflammatory cytokines. Furthermore, because FEPPA enables the detection of neurotoxic microglia on PET images, we successfully constructed a novel PET detection system that can monitor neurodegenerative diseases.

Inhibition of cellular inflammatory mediator production and amelioration of learning deficit in flies by deep sea Aspergillus-derived cyclopenin.

In the course of screening lipopolysaccharide (LPS)-induced nitric oxide (NO) production inhibitors, two related benzodiazepine derivatives, cyclopenol and cyclopenin, were isolated from the extract of a deep marine-derived fungal strain, Aspergillus sp. SCSIOW2. Cyclopenol and cyclopenin inhibited the LPS-induced formation of NO and secretion of IL-6 in RAW264.7 cells at nontoxic concentrations. In terms of the mechanism underlying these effects, cyclopenol and cyclopenin were found to inhibit the upstream signal of NF-κB activation. These compounds also inhibited the expression of IL-1ß, IL-6, and inducible nitric oxide synthase (iNOS) in mouse microglia cells, macrophages in the brain. In relation to the cause of Alzheimer's disease, amyloid-ß-peptide is known to induce inflammation in the brain. Therefore, the present study investigated the ameliorative effects of these inhibitors on an in vivo Alzheimer's model using flies. Learning deficits were induced by the overexpression of amyloid-ß42 in flies, and cyclopenin but not cyclopenol was found to rescue learning impairment. Therefore, novel anti-inflammatory activities of cyclopenin were identified, which may be useful as a candidate of anti-inflammatory agents for neurodegenerative diseases.

Dipeptidyl peptidase-4 inhibition prevents nonalcoholic steatohepatitis-associated liver fibrosis and tumor development in mice independently of its anti-diabetic effects.

Nonalcoholic steatohepatitis (NASH) is a hepatic phenotype of the metabolic syndrome, and increases the risk of cirrhosis and hepatocellular carcinoma (HCC). Although increasing evidence points to the therapeutic implications of certain types of anti-diabetic agents in NASH, it remains to be elucidated whether their effects on NASH are independent of their effects on diabetes. Genetically obese melanocortin 4 receptor-deficient (MC4R-KO) mice fed Western diet are a murine model that sequentially develops hepatic steatosis, NASH, and HCC in the presence of obesity and insulin resistance. In this study, we investigated the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor anagliptin on NASH and HCC development in MC4R-KO mice. Anagliptin treatment effectively prevented inflammation, fibrosis, and carcinogenesis in the liver of MC4R-KO mice. Interestingly, anagliptin only marginally affected body weight, systemic glucose and lipid metabolism, and hepatic steatosis. Histological data and gene expression analysis suggest that anagliptin treatment targets macrophage activation in the liver during the progression from simple steatosis to NASH. As a molecular mechanism underlying anagliptin action, we showed that glucagon-like peptide-1 suppressed proinflammatory and profibrotic phenotypes of macrophages in vitro. This study highlights the glucose metabolism-independent effects of anagliptin on NASH and HCC development.

Novel Oxindole-Curcumin Hybrid Compound for Antioxidative Stress and Neuroprotection.

Oxidative stress plays an important role in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. The oxindole compound GIF-2165X-G1 is a hybrid molecule composed of the oxindole skeleton of the neuroprotective compound GIF-0726-r and the polyphenolic skeleton of the antioxidant curcumin. We previously reported that novel oxindole derivatives such as GIF-0726-r and GIF-2165X-G1 prevent endogenous oxidative stress-induced cell death in mouse hippocampal HT22 cells. In this study, we present a detailed investigation of the effect of GIF-2165X-G1 on endogenous oxidative stress in HT22 cells in comparison with GIF-0726-r and curcumin. GIF-2165X-G1 exhibited more potent neuroprotective activity than GIF-0726-r or curcumin and had less cytotoxicity than that observed with curcumin. Both GIF-0726-r and GIF-2165X-G1 were found to have ferrous ion chelating activity similar to that exhibited by curcumin. GIF-2165 X-G1 and curcumin induced comparable antioxidant response element transcriptional activity. Although the induction of heme oxygenase-1, an antioxidant response element-regulated gene product, was much stronger in curcumin-treated cells than in GIF-2165X-G1-treated cells, it turned out that the induction of heme oxygenase-1 is dispensable for neuroprotection. These results demonstrate that the introduction of the polyphenol skeleton of curcumin to the oxindole GIF-0726-r improves neuroprotective features. Furthermore, intrastriatal injection of GIF-2165X-G1 alleviated apomorphine-induced rotation and prevented dopaminergic neuronal loss in a 6-hydroxydopamine mouse model of Parkinson's diseases. Collectively, our novel findings indicate that the novel oxindole compound GIF-2165X-G1 serves to delay the progression of Parkinson's disease by suppressing oxidative stress.

Clinical features of freezing of gait in Parkinson's disease patients.

OBJECTIVE: To clarify the clinical features of freezing of gait (FOG) in Parkinson's disease (PD) patients by classification into two groups: Clinically observed FOG (CFOG) and self-reported FOG (SFOG). METHODS: Two hundred twenty-nine PD patients were medically examined in an examination room as well as subjected to a New Freezing of Gait Questionnaire (NFOG-Q) and analysis of nonmotor symptoms including sleep, cognition, depression, and fatigue. RESULTS: The prevalence of CFOG was 17.9%, while 53.7% of the patients without CFOG reported the presence of FOG via the NFOG-Q. Univariate analysis revealed that CFOG was associated with longer disease duration, motor dysfunction, sleepiness, fatigue, and cognitive dysfunction. These symptoms, excluding akinesia, apathy, rapid eye movement (REM) sleep Behavior Disorder, and cognitive dysfunction, were also associated with SFOG. Multivariate analysis revealed that long PD duration, postural instability, and gait difficulty (PIGD), along with fatigue, were independent factors for SFOG. CONCLUSIONS: SFOG and CFOG have many common clinical features. Although the clinical relevance of SFOG remains unclear, careful attention should be paid to related features in clinical practice.

Anti-Claudin Antibodies as a Concept for Development of Claudin-Directed Drugs.

Claudin (CLDN) proteins, a tetra-transmembrane family containing over 20 members, have been identified as key structural and functional components of intercellular seals, tight junctions (TJs). CLDNs are involved in the barrier and fence functions of TJs. Loosening the TJ barrier is one strategy for increasing drug absorption and delivery to the brain. Due to aberrant CLDN expression, the TJ fence function is frequently dysregulated in carcinogenesis. In addition, CLDN-1 is a co-receptor for the hepatitis C virus. Together these characteristics indicate CLDNs as promising targets for drug development, and CLDN binders are potential candidates for delivering drugs, treating cancer, and preventing viral infection. Before 2008, a receptor-binding fragment of Clostridium perfringens enterotoxin was the only CLDN binder available. Since then, several challenges regarding the generation of monoclonal antibodies against CLDNs have been surmounted, leading to breakthroughs in CLDN-targeted drug development. Here, we provide an overview of the recent progress in technology using created CLDN binders-anti-CLDN monoclonal antibodies.

Effectiveness of Home-Based Exercises Without Supervision by Physical Therapists for Patients With Early-Stage Amyotrophic Lateral Sclerosis: A Pilot Study.

OBJECTIVE: To verify the effects of structured home-based exercises without supervision by a physical therapist in patients with early-stage amyotrophic lateral sclerosis (ALS). DESIGN: A historical controlled study that is part of a multicenter collaborative study. SETTING: Rehabilitation departments at general hospitals and outpatient clinics with a neurology department. PARTICIPANTS: Patients (N=21) with ALS were enrolled and designated as the home-based exercise (Home-EX) group, and they performed unsupervised home-based exercises. As a control group, 84 patients with ALS who underwent supervised exercise with a physical therapist for 6 months were extracted from a database of patients with ALS and matched with the Home-EX group in terms of their basic attributes and clinical features. INTERVENTION: The Home-EX group was instructed to perform structured home-based exercises without supervision by a physical therapist that consisted of muscle stretching, muscle training, and functional training for 6 months. MAIN OUTCOME MEASURES: The primary outcome was the score on the ALS Functional Rating Scale-Revised (ALSFRS-R), which is composed of 3 domains: bulbar function, limb function, and respiratory function. The score ranges from 0 to 48 points, with a higher score indicating better function. RESULTS: In the Home-EX group, 15 patients completed the home-based exercises for 6 months, and 6 patients dropped out because of medical reasons or disease progression. No adverse events were reported. The Home-EX group was found to have a significantly higher respiratory function subscore and total score on the ALSFRS-R than the control group at follow-up (P<.001 and P<.05, respectively). CONCLUSIONS: Structured home-based exercises without supervision by a physical therapist could be used to alleviate functional deterioration in patients with early-stage ALS.

Novel oxindole derivatives prevent oxidative stress-induced cell death in mouse hippocampal HT22 cells.

The current medical and surgical therapies for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease offer symptomatic relief but do not provide a cure. Thus, small synthetic compounds that protect neuronal cells from degeneration are critically needed to prevent and treat these. Oxidative stress has been implicated in various pathophysiological conditions, including neurodegenerative diseases. In a search for neuroprotective agents against oxidative stress using the murine hippocampal HT22 cell line, we found a novel oxindole compound, GIF-0726-r, which prevented oxidative stress-induced cell death, including glutamate-induced oxytosis and erastin-induced ferroptosis. This compound also exerted a protective effect on tunicamycin-induced ER stress to a lesser extent but had no effect on campthothecin-, etoposide- or staurosporine-induced apoptosis. In addition, GIF-0726-r was also found to be effective after the occurrence of oxidative stress. GIF-0726-r was capable of inhibiting reactive oxygen species accumulation and Ca2+ influx, a presumed executor in cell death, and was capable of activating the antioxidant response element, which is a cis-acting regulatory element in promoter regions of several genes encoding phase II detoxification enzymes and antioxidant proteins. These results suggest that GIF-0726-r is a low-molecular-weight compound that prevents neuronal cell death through attenuation of oxidative stress. Among the more than 200 derivatives of the GIF-0726-r synthesized, we identified the 11 most potent activators of the antioxidant response element and characterized their neuroprotective activity in HT22 cells.

Inducible nitric oxide synthase during the late phase of sepsis is associated with hypothermia and immune cell migration.

Hypothermia is a significant sign of sepsis, which is associated with poor prognosis, but few mechanisms underlying the regulation of hypothermia are known. Inducible nitric oxide synthase (iNOS) is a key inflammatory mediator of sepsis. However, the therapeutic benefit of iNOS inhibition in sepsis is still controversial, and requires elucidation in an accurate model system. In this study, wild-type (WT) mice showed temperature drops in a biphasic manner at the early and late phase of sepsis, and all mice died within 48 h of sepsis. In contrast, iNOS-knockout (KO) mice never showed the second temperature drop and exhibited improved mortality. Plasma nitric oxide (NO) levels of WT mice increased in the late phase of sepsis and correlated to hypothermia. The results indicate that iNOS-derived NO during the late phase of sepsis caused vasodilation-induced hypothermia and a lethal hypodynamic state. The expression of the iNOS mRNA was high in the lung of WT mice with sepsis, which reflects the pathology of acute respiratory distress syndrome (ARDS). We obtained the results in a modified keyhole-type cecal ligation and puncture model of septic shock induced by minimally invasive surgery. In this accurate and reproducible model system, we transplanted the bone marrow cells of GFP transgenic mice into WT and iNOS-KO mice, and evaluated the role of increased pulmonary iNOS expression in cell migration during the late phase of sepsis. We also investigated the quantity and type of bone marrow-derived cells (BMDCs) in the lung. The number of BMDCs in the lung of iNOS-KO mice was less than that in the lung of WT mice. The major BMDCs populations were CD11b-positive, iNOS-negative cells in WT mice, and Gr-1-positive cells in iNOS-KO mice that expressed iNOS. These results suggest that sustained hypothermia may be a beneficial guide for future iNOS-targeted therapy of sepsis, and that iNOS modulated the migratory efficiency and cell type of BMDCs in septic ARDS.

Visualization of Arc promoter-driven neuronal activity by magnetic resonance imaging.

Visualization of direct neuronal activity to understand brain function is one of the most important challenges in neuroscience. We have previously demonstrated that in vivo and in vitro gene expression of the ferritin reporter system could be detected by magnetic resonance imaging (MRI). In addition, increased neuronal activity induces Arc, an immediate early gene, and insertion of a destabilized fluorescent reporter dVenus under Arc promoter control has been used for monitoring neuronal activities in the brain by optical imaging. In this study, to visualize Arc promoter-driven neuronal activities directly, we generated transgenic mice and cell lines that express a destabilized fusion reporter ferritin-mKate2 under Arc promoter control. When transgenic mice and cell lines were treated with pilocarpine, a non-selective muscarinic agonist, an increase in T2-weighted image signal was successfully found in neuronal cells. There was a difference in peak time between MRI and fluorescence imaging, which might result from the binding process of iron with ferritin. Visualization of Arc promoter-driven neuronal activity is essential to understand neural mechanisms underlying cognitive processes and complex behaviors, and could be a useful tool for therapeutic approaches in the brain by MRI.