Cell-cell interaction determines cell fate of mesoderm-derived cell in tongue development through Hh signaling.

Dysfunction of primary cilia leads to genetic disorder, ciliopathies, which shows various malformations in many vital organs such as brain. Multiple tongue deformities including cleft, hamartoma, and ankyloglossia are also seen in ciliopathies, which yield difficulties in fundamental functions such as mastication and vocalization. Here, we found these tongue anomalies in mice with mutation of ciliary protein. Abnormal cranial neural crest-derived cells (CNCC) failed to evoke Hh signal for differentiation of mesoderm-derived cells into myoblasts, which resulted in abnormal differentiation of mesoderm-derived cells into adipocytes. The ectopic adipose subsequently arrested tongue swelling formation. Ankyloglossia was caused by aberrant cell migration due to lack of non-canonical Wnt signaling. In addition to ciliopathies, these tongue anomalies are often observed as non-familial condition in human. We found that these tongue deformities could be reproduced in wild-type mice by simple mechanical manipulations to disturb cellular processes which were disrupted in mutant mice. Our results provide hints for possible future treatment in ciliopathies.

Ghost roads and the destruction of Asia-Pacific tropical forests.

Roads are expanding at the fastest pace in human history. This is the case especially in biodiversity-rich tropical nations, where roads can result in forest loss and fragmentation, wildfires, illicit land invasions and negative societal effects1-5. Many roads are being constructed illegally or informally and do not appear on any existing road map6-10; the toll of such 'ghost roads' on ecosystems is poorly understood. Here we use around 7,000 h of effort by trained volunteers to map ghost roads across the tropical Asia-Pacific region, sampling 1.42 million plots, each 1 km2 in area. Our intensive sampling revealed a total of 1.37 million km of roads in our plots-from 3.0 to 6.6 times more roads than were found in leading datasets of roads globally. Across our study area, road building almost always preceded local forest loss, and road density was by far the strongest correlate11 of deforestation out of 38 potential biophysical and socioeconomic covariates. The relationship between road density and forest loss was nonlinear, with deforestation peaking soon after roads penetrate a landscape and then declining as roads multiply and remaining accessible forests largely disappear. Notably, after controlling for lower road density inside protected areas, we found that protected areas had only modest additional effects on preventing forest loss, implying that their most vital conservation function is limiting roads and road-related environmental disruption. Collectively, our findings suggest that burgeoning, poorly studied ghost roads are among the gravest of all direct threats to tropical forests.

RGMa collapses the neuronal actin barrier against disease-implicated protein and exacerbates ALS.

Repulsive guidance molecule A (RGMa) was originally identified as a neuronal growth cone-collapsing factor. Previous reports have demonstrated the multifunctional roles of RGMa mediated by neogenin1. However, the pathogenic involvement of RGMa in amyotrophic lateral sclerosis (ALS) remains unclear. Here, we demonstrated that RGMa concentration was elevated in the cerebrospinal fluid of both patients with ALS and transgenic mice overexpressing the mutant human superoxide dismutase1 (mSOD1 mice). Treatment with humanized anti-RGMa monoclonal antibody ameliorated the clinical symptoms in mSOD1 mice. Histochemical analysis revealed that the anti-RGMa antibody significantly decreased mutant SOD1 protein accumulation in the motor neurons of mSOD1 mice via inhibition of actin depolymerization. In vitro analysis revealed that the anti-RGMa antibody inhibited the cellular uptake of the mutant SOD1 protein, presumably by reinforcing the neuronal actin barrier. Collectively, these data suggest that RGMa leads to the collapse of the neuronal actin barrier and promotes aberrant protein deposition, resulting in exacerbation of the ALS pathology.

Specific AAV2/PHP.eB-mediated gene transduction of CA2 pyramidal cells via injection into the lateral ventricle.

Given its limited accessibility, the CA2 area has been less investigated compared to other subregions of the hippocampus. While the development of transgenic mice expressing Cre recombinase in the CA2 has revealed unique features of this area, the use of mouse lines has several limitations, such as lack of specificity. Therefore, a specific gene delivery system is required. Here, we confirmed that the AAV-PHP.eB capsid preferably infected CA2 pyramidal cells following retro-orbital injection and demonstrated that the specificity was substantially higher after injection into the lateral ventricle. In addition, a tropism for the CA2 area was observed in organotypic slice cultures. Combined injection into the lateral ventricle and stereotaxic injection into the CA2 area specifically introduced the transgene into CA2 pyramidal cells, enabling us to perform targeted patch-clamp recordings and optogenetic manipulation. These results suggest that AAV-PHP.eB is a versatile tool for specific gene transduction in CA2 pyramidal cells.

Preferential arborization of dendrites and axons of parvalbumin- and somatostatin-positive GABAergic neurons within subregions of the mouse claustrum.

The claustrum coordinates the activities of individual cortical areas through abundant reciprocal connections with the cerebral cortex. Although these excitatory connections have been extensively investigated in three subregions of the claustrum-core region and dorsal and ventral shell regions-the contribution of GABAergic neurons to the circuitry in each subregion remains unclear. Here, we examined the distribution of GABAergic neurons and their dendritic and axonal arborizations in each subregion. Combining in situ hybridization with immunofluorescence histochemistry showed that approximately 10% of neuronal nuclei-positive cells expressed glutamic acid decarboxylase 67 mRNA across the claustral subregions. Approximately 20%, 30%, and 10% of GABAergic neurons were immunoreactive for parvalbumin (PV), somatostatin (SOM), and vasoactive intestinal polypeptide, respectively, in each subregion, and these neurochemical markers showed little overlap with each other. We then reconstructed PV and SOM neurons labeled with adeno-associated virus vectors. The dendrites and axons of PV and SOM neurons were preferentially localized to their respective subregions where their cell bodies were located. Furthermore, the axons were preferentially extended in a rostrocaudal direction, whereas the dendrites were relatively isotropic. The present findings suggest that claustral PV and SOM neurons might execute information processing separately within the core and shell regions.

Ift88 regulates enamel formation via involving Shh signaling.

OBJECTIVES: The ciliopathies are a wide spectrum of human diseases, which are caused by perturbations in the function of primary cilia. Tooth enamel anomalies are often seen in ciliopathy patients; however, the role of primary cilia in enamel formation remains unclear. MATERIALS AND METHODS: We examined mice with epithelial conditional deletion of the ciliary protein, Ift88, (Ift88fl / fl ;K14Cre). RESULTS: Ift88fl / fl ;K14Cre mice showed premature abrasion in molars. A pattern of enamel rods which is determined at secretory stage, was disorganized in Ift88 mutant molars. Many amelogenesis-related molecules expressing at the secretory stage, including amelogenin and ameloblastin, enamelin, showed significant downregulation in Ift88 mutant molar tooth germs. Shh signaling is essential for amelogenesis, which was found to be downregulated in Ift88 mutant molar at the secretory stage. Application of Shh signaling agonist at the secretory stage partially rescued enamel anomalies in Ift88 mutant mice. CONCLUSION: Findings in the present study indicate that the function of the primary cilia via Ift88 is critical for the secretory stage of amelogenesis through involving Shh signaling.

Effects of CEACAM1 in oral keratinocytes on HO-1 expression induced by Candida ß-glucan particles.

OBJECTIVE: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a member of the carcinoembryonic antigen family. Although its expression has been found in chronic oral inflammatory epithelium, this study aimed to know whether CEACAM1 in oral keratinocytes participates in host immune response against Candida albicans . METHODOLOGY: We investigated CEACAM1 expression in oral keratinocytes induced by C. albicans as well as by Candida cell wall component ß-glucan particles (ß-GPs). Furthermore, the effects of CEACAM1 on ß-GPs-induced heme oxygenase-1 (HO-1) expression and its related signals were examined. RESULTS: Fluorescence staining showed CEACAM1 expression in oral keratinocytes (RT7) cells, whereas quantitative reverse transcription (RT)-PCR indicated that both live and heat-killed C. albicans increased CEACAM1 mRNA expression in RT7 cells. Examinations using quantitative RT-PCR and western blotting indicated that CEACAM1 expression was also increased by ß-GPs derived from C. albicans . Specific siRNA for CEACAM1 decreased HO-1 expression induced by ß-GPs from C. albicans as well as the budding yeast microorganism Saccharomyces cerevisiae . Moreover, knockdown of CEACAM1 decreased ß-GPs-induced ROS activity in the early phase and translocation of Nrf2 into the nucleus. CONCLUSION: CEACAM1 in oral keratinocytes may have a critical role in regulation of HO-1 for host immune defense during Candida infection.

Fluorochromized tyramide-glucose oxidase as a multiplex fluorescent tyramide signal amplification system for histochemical analysis.

Tyramide signal amplification (TSA) is a highly sensitive method for histochemical analysis. Previously, we reported a TSA system, biotinyl tyramine-glucose oxidase (BT-GO), for bright-filed imaging. Here, we develop fluorochromized tyramide-glucose oxidase (FT-GO) as a multiplex fluorescent TSA system. FT-GO involves peroxidase-catalyzed deposition of fluorochromized tyramide (FT) with hydrogen peroxide produced by enzymatic reaction between glucose and glucose oxidase. We showed that FT-GO enhanced immunofluorescence signals while maintaining low background signals. Compared with indirect immunofluorescence detections, FT-GO demonstrated a more widespread distribution of monoaminergic projection systems in mouse and marmoset brains. For multiplex labeling with FT-GO, we quenched antibody-conjugated peroxidase using sodium azide. We applied FT-GO to multiplex fluorescent in situ hybridization, and succeeded in labeling neocortical interneuron subtypes by coupling with immunofluorescence. FT-GO immunofluorescence further increased the detectability of an adeno-associated virus tracer. Given its simplicity and a staining with a high signal-to-noise ratio, FT-GO would provide a versatile platform for histochemical analysis.

Multi-scale light microscopy/electron microscopy neuronal imaging from brain to synapse with a tissue clearing method, ScaleSF.

The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscopy/electron microscopy (LM/EM) imaging with a glutaraldehyde-resistant tissue clearing method, ScaleSF. Our multi-scale neuronal imaging incorporates (1) brain-wide macroscopic observation, (2) mesoscopic circuit mapping, (3) microscopic subcellular imaging, and (4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied this technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects.

Immune response to cytosolic DNA via intercellular receptor modulation in oral keratinocytes and fibroblasts.

OBJECTIVE: Double-strand (ds) DNA-enveloped viruses can cause oral infection. Our aim is to investigate whether oral mucosal cells participate in immune response against cytosolic dsDNA invasion. METHODS: We examined the response to transfected herpes simplex virus (HSV) dsDNA via intracellular receptors in oral keratinocytes (RT7) and fibroblasts (GT1), and the effect of TNF-α on those responses. RESULTS: Transfected dsDNA increased CXCL10 expression via NF-κB activation in both cell types, while those responses were inhibited by knockdown of RIG-I, an RNA sensor. Although IFI16, a DNA sensor, was expressed in the nuclei of both types, its knockdown decreased transfected dsDNA-induced CXCL10 expression in GT1 but not RT7 cells. IFI16 in GT1 cells was translocated into cytoplasm from nuclei, which was attributed to immune response to cytosolic dsDNA. TNF-α enhanced transfected dsDNA-induced CXCL10, and knockdown of IFI16 decreased TNF-α and dsDNA-driven CXCL10 expression in both RT7 and GT1 cells. Finally, the combination of TNF-α and transfected dsDNA resulted in translocation of IFI16 from nuclei to cytoplasm in RT7 cells. CONCLUSION: RIG-I and IFI16 in oral mucosal cells may play important roles in host immune response against DNA viral infection, while TNF-α contributes to development of an antiviral system via those intracellular receptors.

Effects of CEACAM1 in oral keratinocytes on HO-1 expression induced by Candida β-glucan particles

Abstract Objective Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a member of the carcinoembryonic antigen family. Although its expression has been found in chronic oral inflammatory epithelium, this study aimed to know whether CEACAM1 in oral keratinocytes participates in host immune response against Candida albicans . Methodology We investigated CEACAM1 expression in oral keratinocytes induced by C. albicans as well as by Candida cell wall component β-glucan particles (β-GPs). Furthermore, the effects of CEACAM1 on β-GPs-induced heme oxygenase-1 (HO-1) expression and its related signals were examined. Results Fluorescence staining showed CEACAM1 expression in oral keratinocytes (RT7) cells, whereas quantitative reverse transcription (RT)-PCR indicated that both live and heat-killed C. albicans increased CEACAM1 mRNA expression in RT7 cells. Examinations using quantitative RT-PCR and western blotting indicated that CEACAM1 expression was also increased by β-GPs derived from C. albicans . Specific siRNA for CEACAM1 decreased HO-1 expression induced by β-GPs from C. albicans as well as the budding yeast microorganism Saccharomyces cerevisiae . Moreover, knockdown of CEACAM1 decreased β-GPs-induced ROS activity in the early phase and translocation of Nrf2 into the nucleus. Conclusion CEACAM1 in oral keratinocytes may have a critical role in regulation of HO-1 for host immune defense during Candida infection.

Stability of tropical forest tree carbon-water relations in a rainfall exclusion treatment through shifts in effective water uptake depth.

Increasing severity and frequency of drought is predicted for large portions of the terrestrial biosphere, with major impacts already documented in wet tropical forests. Using a 4-year rainfall exclusion experiment in the Daintree Rainforest in northeast Australia, we examined canopy tree responses to reduced precipitation and soil water availability by quantifying seasonal changes in plant hydraulic and carbon traits for 11 tree species between control and drought treatments. Even with reduced soil volumetric water content in the upper 1 m of soil in the drought treatment, we found no significant difference between treatments for predawn and midday leaf water potential, photosynthesis, stomatal conductance, foliar stable carbon isotope composition, leaf mass per area, turgor loss point, xylem vessel anatomy, or leaf and stem nonstructural carbohydrates. While empirical measurements of aboveground traits revealed homeostatic maintenance of plant water status and traits in response to reduced soil moisture, modeled belowground dynamics revealed that trees in the drought treatment shifted the depth from which water was acquired to deeper soil layers. These findings reveal that belowground acclimation of tree water uptake depth may buffer tropical rainforests from more severe droughts that may arise in future with climate change.

High-fat diet-induced activation of SGK1 promotes Alzheimer's disease-associated tau pathology.

Type 2 diabetes mellitus (T2DM) has long been considered a risk factor for Alzheimer's disease (AD). However, the molecular links between T2DM and AD remain obscure. Here, we reported that serum-/glucocorticoid-regulated kinase 1 (SGK1) is activated by administering a chronic high-fat diet (HFD), which increases the risk of T2DM, and thus promotes Tau pathology via the phosphorylation of tau at Ser214 and the activation of a key tau kinase, namely, GSK-3ß, forming SGK1-GSK-3ß-tau complex. SGK1 was activated under conditions of elevated glucocorticoid and hyperglycemia associated with HFD, but not of fatty acid-mediated insulin resistance. Elevated expression of SGK1 in the mouse hippocampus led to neurodegeneration and impairments in learning and memory. Upregulation and activation of SGK1, SGK1-GSK-3ß-tau complex were also observed in the hippocampi of AD cases. Our results suggest that SGK1 is a key modifier of tau pathology in AD, linking AD to corticosteroid effects and T2DM.

MicroRNAs regulate distal region of mandibular development through Hh signaling.

Mandibular anomalies are often seen in various congenital diseases, indicating that mandibular development is under strict molecular control. Therefore, it is crucial to understand the molecular mechanisms involved in mandibular development. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating the level of gene expression. We found that the mesenchymal conditional deletion of miRNAs arising from a lack of Dicer (an essential molecule for miRNA processing, Dicerfl/fl ;Wnt1Cre), led to an abnormal groove formation at the distal end of developing mandibles. At E10.5, when the region forms, inhibitors of Hh signaling, Ptch1 and Hhip1 showed increased expression at the region in Dicer mutant mandibles, while Gli1 (a major mediator of Hh signaling) was significantly downregulated in mutant mandibles. These suggest that Hh signaling was downregulated at the distal end of Dicer mutant mandibles by increased inhibitors. To understand whether the abnormal groove formation inDicer mutant mandibles was caused by the downregulation of Hh signaling, mice with a mesenchymal deletion of Hh signaling activity arising from a lack of Smo (an essential molecule for Hh signaling activation, Smofl/fl ;Wnt1Cre) were examined. Smofl/fl ;Wnt1Cre mice showed a similar phenotype in the distal region of their mandibles to those in Dicerfl/fl ;Wnt1Cre mice. We also found that approximately 400 miRNAs were expressed in wild-type mandibular mesenchymes at E10.5, and six microRNAs were identified as miRNAs with binding potential against both Ptch1 and Hhip1. Their expressions at the distal end of the mandible were confirmed by in situ hybridization. This indicates that microRNAs regulate the distal part of mandibular formation at an early stage of development by involving Hh signaling activity through controlling its inhibitor expression level.

Exclusive labeling of direct and indirect pathway neurons in the mouse neostriatum by an adeno-associated virus vector with Cre/lox system.

We developed an adeno-associated virus (AAV) vector-based technique to label mouse neostriatal neurons comprising direct and indirect pathways with different fluorescent proteins and analyze their axonal projections. The AAV vector expresses GFP or RFP in the presence or absence of Cre recombinase and should be useful for labeling two cell populations exclusively dependent on its expression. Here, we describe the AAV vector design, stereotaxic injection of the AAV vector, and a highly sensitive immunoperoxidase method for axon visualization. For complete details on the use and execution of this protocol, please refer to Okamoto et al. (2020).

Overlapping Projections of Neighboring Direct and Indirect Pathway Neostriatal Neurons to Globus Pallidus External Segment.

Indirect pathway medium-sized spiny neurons (iMSNs) in the neostriatum are well known to project to the external segment of the globus pallidus (GPe). Although direct MSNs (dMSNs) also send axon collaterals to the GPe, it remains unclear how dMSNs and iMSNs converge within the GPe. Here, we selectively labeled neighboring dMSNs and iMSNs with green and red fluorescent proteins using an adeno-associated virus vector and examined axonal projections of dMSNs and iMSNs to the GPe in mice. Both dMSNs and iMSNs formed two axonal arborizations displaying topographical projections in the dorsoventral and mediolateral planes. iMSNs displayed a wider and denser axon distribution, which included that of dMSNs. Density peaks of dMSN and iMSN axons almost overlapped, revealing convergence of dMSN axons in the center of iMSN projection fields. These overlapping projections suggest that dMSNs and iMSNs may work cooperatively via interactions within the GPe.

Visualizing and Modulating Mitophagy for Therapeutic Studies of Neurodegeneration.

Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control.

Association Between Overtime-Working Environment and Psychological Distress Among Japanese Workers: A Multilevel Analysis.

OBJECTIVE: The study aims to examine the association between overtime-working environment (OWE) and individual psychological distress among Japanese workers. METHODS: Data of 7786 workers from 101 companies in Japan were analyzed. Psychological distress was assessed through a 29-item questionnaire. The OWE was evaluated by calculating the proportion of workers whose monthly overtime was 45 hours or more in a workplace. Multilevel logistic regression was used. RESULTS: As 10% increase in the OWE was associated with a 16% higher risk of individual psychological distress after adjustment of individual covariates, including overtime working hours. Cross-level interaction showed that the risk was varied depending on individual overtime working hours. CONCLUSIONS: OWE was associated with the psychological distress of workers. It is necessary to create a non-OWE at workplaces to prevent psychological distress for workers.

Disease Control Status and Safety of Telemedicine in Patients With Lifestyle Diseases　- A Multicenter Prospective Observational Study in Japan.

Background: The Japanese Ministry of Health, Labour and Welfare has tried to promote telemedicine since 2018, but faces difficulties in increasing the use of telemedicine partly due to a lack of clinical evidence. This study investigated the disease control status and safety of telemedicine, which, in Japan, is provided under the National Health Insurance system, for the treatment of lifestyle diseases under the present legal restraints. Methods and Results: This multicenter prospective observational study started in April 2018 and enrolled 34 patients with lifestyle diseases, including hypertension, dyslipidemia, and diabetes. Primary and secondary outcome measures included control status, serial changes in clinical indices, and the safety of telemedicine 6 months after implementation. Control status was assessed by the attending physician, and differences in blood pressure (BP), low density lipoprotein cholesterol (LDL-C), or HbA1c levels were evaluated. Of the 34 patients, 29 were successfully introduced to telemedicine and followed-up for 6 months. Median patient age was 77 years, 14 (48.3%) were men, 24 (82.8%) had hypertension, 17 (58.6%) had dyslipidemia, and 9 (31.0%) had diabetes. At the 6-month follow-up, no patients had experienced exacerbation of underlying diseases, with no significant changes in BP, LDL-C, or HbA1c. Moreover, no telemedicine-associated adverse events were observed. Conclusions: Telemedicine can be a safe and feasible option for managing lifestyle diseases under the present legal restraints.