The synaptic protein α-synuclein has been identified as a major component of Lewy bodies, a pathological hallmark of Parkinson's disease (PD). Prior to the formation of Lewy bodies, mislocalization and aggregation of the α-synuclein in brain tissue is frequently observed in various neurodegenerative diseases. Aberrant accumulation and localization of α-synuclein are also observed in the aging human brain, for which reason aging is regarded as a risk factor for neurodegenerative disease. To investigate changes in α-synuclein properties in the aging brain, we compared α-synuclein immunoreactivity in brain tissue of young (2-years-old) and middle-aged (6-years-old) common marmoset (Callithrix jacchus). Our analyses revealed marked changes in α-synuclein immunoreactivity in the olfactory bulb of common marmosets of these age cohorts. Perikaryal α-synuclein aggregations were formed in the olfactory bulb in middle-aged animals. We also observed signals of α-synuclein accumulation in hippocampus in this cohort; however, unlike in the olfactory bulb, hippocampal α-synuclein signals were localized in the synaptic terminals. We did not observe either of these features in younger marmosets, which suggest that aging may play a role in these phenomena. Our results using common marmoset brain corresponded with the observation that the α-synuclein aggregations were first occurred from olfactory bulb in human normal aged and PD brain. Therefore, common marmoset is expected as useful model for α-synuclein pathology.

Strictly controlled dendrite patterning underlies precise neural connection. Dendrite self-avoidance is a crucial system preventing self-crossing and clumping of dendrites. Although many cell-surface molecules that regulate self-avoidance have been identified, the signaling pathway that orchestrates it remains poorly understood, particularly in mammals. Here, we demonstrate that the LKB1-SIK kinase pathway plays a pivotal role in the self-avoidance of Purkinje cell (PC) dendrites by ensuring dendritic localization of Robo2, a regulator of self-avoidance. LKB1 is activated in developing PCs, and PC-specific deletion of LKB1 severely disrupts the self-avoidance of PC dendrites without affecting gross morphology. SIK1 and SIK2, downstream kinases of LKB1, mediate LKB1-dependent dendrite self-avoidance. Furthermore, loss of LKB1 leads to significantly decreased Robo2 levels in the dendrite but not in the cell body. Finally, restoration of dendritic Robo2 level via overexpression largely rescues the self-avoidance defect in LKB1-deficient PCs. These findings reveal an LKB1-pathway-mediated developmental program that establishes dendrite self-avoidance.

Objective: To assess the safety and efficacy of edoxaban for the treatment of venous thromboembolism (VTE) secondary to active malignancy. Materials and Methods: We enrolled 48 patients with newly diagnosed VTE secondary to active malignancy that was treated with oral edoxaban for 1 year between September 2014 and August 2015. We retrospectively examined the presence or absence of recurrent symptomatic VTE, VTE-related mortality, and bleeding events. Results: No recurrent symptomatic VTE or VTE-related deaths were recorded, enabling efficient assessment. Treatment safety was determined based on the reports of bleeding. Bleeding was reported in two patients, with serious bleeding in one of them. Conclusion: Edoxaban is safe and effective for the treatment of VTE secondary to active malignancy.

To investigate the effect of global cerebral ischemia on brain cell proliferation in young adult macaques, we infused 5-bromo-2'-deoxyuridine (BrdU), a DNA replication indicator, into monkeys subjected to ischemia or sham-operated. Subsequent quantification by BrdU immunohistochemistry revealed a significant postischemic increase in the number of BrdU-labeled cells in the hippocampal dentate gyrus, subventricular zone of the temporal horn of the lateral ventricle, and temporal neocortex. In all animals, 20-40% of the newly generated cells in the dentate gyrus and subventricular zone expressed the neural progenitor cell markers Musashi1 or Nestin. A few BrdU-positive cells in postischemic monkeys were double-stained for markers of neuronal progenitors (class III beta-tubulin, TUC4, doublecortin, or Hu), neurons (NeuN), or glia (S100beta or GFAP). Our results suggest that ischemia activates endogenous neuronal and glial precursors residing in diverse locations of the adult primate central nervous system.

Mutations in the Vacuolar protein sorting 35 (VPS35) gene have been linked to familial Parkinson's disease (PD), PARK17. VPS35 is a key component of the retromer complex, which plays a central role in endosomal trafficking. However, whether and how VPS35 deficiency or mutation contributes to PD pathogenesis remain unclear. Here, we analyzed human induced pluripotent stem cell (iPSC)-derived neurons from PD patients with the VPS35 D620N mutation and addressed relevant disease mechanisms. In the disease group, dopaminergic (DA) neurons underwent extensive apoptotic cell death. The movement of Rab5a- or Rab7a-positive endosomes was slower, and the endosome fission and fusion frequencies were lower in the PD group than in the healthy control group. Interestingly, vesicles positive for cation-independent mannose 6-phosphate receptor transported by retromers were abnormally localized in glial cells derived from patient iPSCs. Furthermore, we found α-synuclein accumulation in TH positive DA neurons. Our results demonstrate the induction of cell death, endosomal dysfunction and α -synuclein accumulation in neural cells of the PD group. PARK17 patient-derived iPSCs provide an excellent experimental tool for understanding the pathophysiology underlying PD.

Dravet syndrome (DS) is an infantile-onset developmental and epileptic encephalopathy characterized by an age-dependent evolution of drug-resistant seizures and poor developmental outcomes. Functional impairment of gamma-aminobutyric acid (GABA)ergic interneurons due to loss-of-function mutation of SCN1A is currently considered the main pathogenesis. In this study, to better understand the age-dependent changes in the pathogenesis of DS, we characterized the activity of different brain regions in Scn1a knockout rats at each developmental stage.

Precisely regulated spatiotemporal gene expression is essential for the establishment of neural circuits. In contrast to the increasing evidence for transcriptional regulation of axon guidance cues and receptors, the role of posttranscriptional regulation in axon guidance, especially in vivo, remains poorly characterized. Here, we demonstrate that the expression of Slit receptor Robo3/Rig-1, which plays crucial roles in axonal midline crossing, is regulated by a neural RNA-binding protein Musashi1 (Msi1). Msi1 binds to Robo3 mRNA through RNA recognition motifs and increases the protein level of Robo3 without affecting its mRNA level. In Msi1-deficient precerebellar neurons, Robo3 protein, but not its mRNA, is dramatically reduced. Moreover, similar to defects in Robo3-deficient mice, axonal midline crossing and neuronal migration of precerebellar neurons are severely impaired in Msi1-deficient mice. Together, these findings indicate that Msi1-mediated posttranscriptional regulation of Robo3 controls midline crossing of precerebellar neurons.

The 14-3-3 proteins participate in many aspects of plant physiology by interacting with phosphorylated proteins and thereby regulating target protein functions. In Arabidopsis plant, the ubiquitin ligase ATL31 controls 14-3-3 stability via both direct interaction and ubiquitination, and this consequently regulates post-germinative growth in response to carbon and nitrogen nutrient availability. Since 14-3-3 proteins regulate the activities of many key enzymes related to nutrient metabolism, one would anticipate that they should play an essential role not only in vegetative but also in reproductive tissue. Because fruit yield largely depends on carbon and nitrogen availability and their utilization, the function of 14-3-3 proteins was analyzed in tomato fruit tissue. Here, we isolated and characterized an ubiquitin ligase SlATL31 (Solyc03g112340) from tomato and demonstrated that SlATL31 has ubiquitin ligase activity as well as interaction with tomato 14-3-3 proteins, suggesting the possibility that the SlATL31 functions as an ubiquitin ligase for 14-3-3 similarly to its Arabidopsis ortholog. Furthermore, we performed proteomic analysis of 14-3-3 interacting proteins and identified 106 proteins as putative 14-3-3 targets including key enzymes for carbon metabolism and photosynthesis. This 14-3-3 interactome result and available transcriptome profile suggest a considerable yet complex role of 14-3-3 proteins in tomato fruit tissue.

Neuronal Elav-like (nElavl or neuronal Hu) proteins are RNA-binding proteins that regulate RNA stability and alternative splicing, which are associated with axonal and synaptic structures. nElavl proteins promote the differentiation and maturation of neurons via their regulation of RNA. The functions of nElavl in mature neurons are not fully understood, although Elavl3 is highly expressed in the adult brain. Furthermore, possible associations between nElavl genes and several neurodegenerative diseases have been reported. We investigated the relationship between nElavl functions and neuronal degeneration using Elavl3-/- mice. Elavl3-/- mice exhibited slowly progressive motor deficits leading to severe cerebellar ataxia, and axons of Elavl3-/- Purkinje cells were swollen (spheroid formation), followed by the disruption of synaptic formation of axonal terminals. Deficit in axonal transport and abnormalities in neuronal polarity was observed in Elavl3-/- Purkinje cells. These results suggest that nElavl proteins are crucial for the maintenance of axonal homeostasis in mature neurons. Moreover, Elavl3-/- mice are unique animal models that constantly develop slowly progressive axonal degeneration. Therefore, studies of Elavl3-/- mice will provide new insight regarding axonal degenerative processes.

Systemic steroid injections are used to treat idiopathic sudden-onset sensorineural hearing loss (ISSHL) and some inner ear disorders. Recent studies show that transtympanic (TT) steroid injections are effective for treating ISSHL. As in vivo monitoring of drug delivery dynamics for inner ear is lacking, its time course and dispersion of drugs is unknown. Here, we used a new in vivo imaging system to monitor drug delivery in live mice and to compare drug concentrations over time after TT and systemic injections.

The present study aimed to investigate the involvement of the NMDA receptor (NMDAR) and/or nitric oxide (NO) pathway in ketamine-induced behavioral sensitization. Mice received repeated subcutaneous administration of ketamine (25mg/kg), once daily or once weekly for a total of five doses. Even three administrations of ketamine, daily or weekly, induced a rapid increase in locomotor activity in wild-type (WT), but not in GluN2D knockout (GluN2D-KO) mice. Furthermore, for WT mice receiving daily ketamine, elevated locomotor activity was maintained after a 1-month withdrawal period; however, this was not the case when ketamine was administered weekly. The effect of acute ketamine on nNOS activities was estimated with nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) histochemistry. Ketamine rapidly increased the number of NADPH-d activated cells and strongly stained dendrites in the dorsal striatum and prefrontal cortex of WT mice, but not GluN2D-KO mice. These results suggest that ketamine-induced locomotor sensitization and nNOS activation in the frontal cortex-striatum neuronal circuit are positively correlated and that the NMDAR GluN2D subunit plays an important role in the acquisition and maintenance of ketamine-induced behavioral sensitization.

Disasters, including terrorism and earthquakes, are significant threats to people and may lead to many people requiring rescue. The longer the rescue takes, the higher the chances of an individual contracting acute compartment syndrome (ACS). ACS is fatal if diagnosed too late, and early diagnosis and treatment are essential.

Focused-ion beam-scanning electron microscopic (FIB-SEM) tomography enables easier acquisition of a series of ultrastructural, sectional images directly from resin-embedded biological samples. In this study, to clarify the three-dimensional (3D) architecture of glomerular endothelial cells (GEnCs) in adult rats, we manually extracted GEnCs from serial FIB-SEM images and reconstructed them on an Amira reconstruction software. The luminal and basal surface structures were clearly visualized in the reconstructed GEnCs, although only the luminal surface structures could be observed by conventional SEM. The luminal surface visualized via the reconstructed GEnCs was quite similar to that observed through conventional SEM, indicating that 3D reconstruction could be performed with high accuracy. Thus, we successfully described the 3D architecture of normal GEnCs in adult rats more clearly and precisely than ever before. The GEnCs were found to consist of three major subcellular compartments, namely, the cell body, cytoplasmic ridges, and sieve plates, in addition to two associated subcellular compartments, namely, the globular protrusions and reticular porous structures. Furthermore, most individual GEnCs made up a "seamless" tubular shape, and some of them formed an autocellular junction to make up a tubular shape. FIB-SEM tomography with reconstruction is a powerful approach to better understand the 3D architecture of GEnCs. Moreover, the morphological information revealed in this study will be valuable for the 3D pathologic evaluation of GEnCs in animal and human glomerular diseases and the structural analysis of developmental processes in the glomerular capillary system.

The DJ-1 gene, a causative gene for familial Parkinson's disease (PD), has been reported to have various functions, including transcriptional regulation, antioxidant response, and chaperone and protease functions; however, the molecular mechanism associated with the pathogenesis of PD remains elusive. To further explore the molecular function of DJ-1 in the pathogenesis of PD, we compared protein expression profiles in brain tissues from wild-type and DJ-1-deficient mice. Two-dimensional difference gel electrophoresis analysis and subsequent analysis using data mining methods revealed alterations in the expression of molecules associated with energy production. We demonstrated that DJ-1 deletion inhibited S-nitrosylation of endogenous Parkin as well as overexpressed Parkin in neuroblastoma cells and mouse brain tissues. Thus, we used genome editing to generate neuroblastoma cells with DJ-1 deletion or S-nitrosylated cysteine mutation in Parkin and demonstrated that these cells exhibited similar phenotypes characterized by enhancement of cell death under mitochondrial depolarization and dysfunction of mitochondria. Our data indicate that DJ-1 is required for the S-nitrosylation of Parkin, which positively affects mitochondrial function, and suggest that the denitrosylation of Parkin via DJ-1 inactivation might contribute to PD pathogenesis and act as a therapeutic target.

Caffeine is a psychoactive substance that not only improves wakefulness, but also slows the cognitive decline caused by aging. However, at present, there are no reports about the effects of caffeine withdrawal, including headaches and changes in brain functional networks (nerve activity). Headache may occur approximately 24 h after discontinuing caffeine intake in chronic caffeine drinkers. The current study aimed to examine the brain functional activity via resting-state functional magnetic resonance imaging in chronically caffeinated and decaffeinated groups to investigate changes in brain activity caused by caffeine. C57BL/6J mice were included in the analysis, and they underwent 9.4-T ultrahigh-field magnetic resonance imaging. The mice were classified into the control, chronic caffeinated, and caffeine withdrawal grsoups. Mice were divided into three groups: 1) not exposed to caffeine (control); 2) treated with caffeine at a concentration of 0.3 mg/mL for 4 weeks (chronic caffeinated); and 3) treated as before with caffeine and withdrawn from caffeine for 24 h. After the three groups were examined, functional connectivity matrices were calculated using brain imaging analysis tools, and independent component analysis was performed. The results showed that caffeine administration activated neural activity areas in the stress response system. Furthermore, 24h after caffeine withdrawal, the results showed an increase in pain-related neural activity. In addition, caffeine administration was shown to activate the dentate gyrus, one of the hippocampal regions, and to decrease the neural activity in the olfactory bulb and anterior cingulate cortex. In the current research, the neural activity of specific brain regions changed after chronic caffeine administration and withdrawal.

GPR56, a member of the adhesion G protein-coupled receptor family, is abundantly expressed in cells of the developing cerebral cortex, including neural progenitor cells and developing neurons. The human GPR56 gene has multiple presumptive promoters that drive the expression of the GPR56 protein in distinct patterns. Similar to coding mutations of the human GPR56 gene that may cause GPR56 dysfunction, a 15-bp homozygous deletion in the cis-regulatory element upstream of the noncoding exon 1 of GPR56 (e1m) leads to the cerebral cortex malformation and epilepsy. To clarify the expression profile of the e1m promoter-driven GPR56 in primate brain, we generated a transgenic marmoset line in which EGFP is expressed under the control of the human minimal e1m promoter. In contrast to the endogenous GPR56 protein, which is highly enriched in the ventricular zone of the cerebral cortex, EGFP is mostly expressed in developing neurons in the transgenic fetal brain. Furthermore, EGFP is predominantly expressed in GABAergic neurons, whereas the total GPR56 protein is evenly expressed in both GABAergic and glutamatergic neurons, suggesting the GABAergic neuron-preferential activity of the minimal e1m promoter. These results indicate a possible pathogenic role for GABAergic neuron in the cerebral cortex of patients with GPR56 mutations.

Plants are able to sense and mediate the balance between carbon (C) and nitrogen (N) nutrient availability to optimize metabolism and growth, described as the C/N response. To clarify the C/N signalling mechanism, C/N-insensitive plants were obtained from an Arabidopsis FOX hunting population, which over-expresses full-length cDNAs for individuals. The resulting cni2-D (carbon/nitrogen insensitive 2-dominant) plant was found to overcome the post-germination growth checkpoint and to expand green cotyledons in disrupted high C/low N stress conditions. The CNI2 gene encodes ABI1, a phosphatase type 2C protein, which negatively regulates abscisic acid (ABA) signal transduction. Over-expressors of ABI1 were found to be insensitive to disrupted C/N stress, whereas the loss-of function mutant abi1-2 was hypersensitive, suggesting that ABI1 plays an essential role in the plant C/N response. By contrast, the C/N-dependent growth phenotype observed in wild-type plants was not associated with endogenous ABA content. Accordingly, the ABA-insensitive mutant abi1-1, which could not bind to the ABA-ABA receptor complex, was not insensitive and restored normal sensitivity to high C/low N stress. The canonical ABA signalling mutants abi4 and abi5 were also sensitive to disrupted C/N stress. Further gene expression analysis demonstrated that several genes in the SnRK2s and SnRK1s pathways are transcriptionally affected by high C/low N stress in wild-type plants regardless of the lack of increased endogenous ABA contents, whereas the expression of these genes were significantly suppressed in ABI1 over-expressors. Taken together, these results suggest direct cross-talk between C/N and non-canonical ABA signalling pathways, regulated by ABI1, in plants.

Ubiquitin ligases play important roles in regulating various cellular processes by modulating the protein function of specific ubiquitination targets. The Arabidopsis Tóxicos en Levadura (ATL) family is a group of plant-specific RING-type ubiquitin ligases that localize to membranes via their N-terminal transmembrane-like domains. To date, 91 ATL isoforms have been identified in the Arabidopsis genome, with several ATLs reported to be involved in regulating plant responses to environmental stresses. However, the functions of most ATLs remain unknown. This study, involving transcriptome database analysis, identifies ATL15 as a sugar responsive ATL gene in Arabidopsis. ATL15 expression was rapidly down-regulated in the presence of sugar. The ATL15 protein showed ubiquitin ligase activity in vitro and localized to plasma membrane and endomembrane compartments. Further genetic analyses demonstrated that the atl15 knockout mutants are insensitive to high glucose concentrations, whereas ATL15 overexpression depresses plant growth. In addition, endogenous glucose and starch amounts were reciprocally affected in the atl15 knockout mutants and the ATL15 overexpressors. These results suggest that ATL15 protein plays a significant role as a membrane-localized ubiquitin ligase that regulates sugar-responsive plant growth in Arabidopsis.

Nutrient availability, in particular the availability of sugar [carbon (C)] and nitrogen (N), is important for the regulation of plant metabolism and development. In addition to independent utilization of C and N nutrients, plants sense and respond to the balance of C and N nutrients (C/N-nutrient) available to them. High C/low N-nutrient stress has been shown to arrest early post-germinative growth while promoting progression to senescence in Arabidopsis. Although several signaling components of the C/N-nutrient response have been identified, the inclusive molecular basis of plant C/N-nutrient response remains unclear. This proteome analysis evaluated phosphorylation dynamics in response to high C/low N-nutrient stress. Phosphoproteomics under conditions of C/N-nutrient stress showed a global change in the phosphorylation status of proteins, including plasma membrane H+-ATPase, carbon and nitrogen metabolic enzymes and signaling proteins such as protein kinases and transcription factors. Further analyses suggested that SNF1-related protein kinase 1 (SnRK1) is involved in primary C/N-nutrient signal mediation via the transcriptional regulation of C/N-regulatory kinases. We also identified a leucine-rich repeat receptor-like kinase with extracellular malectin-like domain, named as LMK1, which was shown to possess cell death induction activity in plant leaves. These results provide important insight into the C/N-nutrient signaling pathways connecting nutrition stress to various cellular and physiological processes in plants.

In-resin CLEM (Correlative Light and Electron Microscopy) of Epon-embedded cells involves correlating fluorescence microscopy with electron microscopy in the same Epon-embedded ultrathin section. This method offers the advantage of high positional accuracy compared to standard CLEM. However, it requires the expression of recombinant proteins. In order to detect the localization of endogenous target(s) and their localized ultrastructures of Epon-embedded samples using in-resin CLEM, we investigated whether immunological and affinity-labeling using fluorescent dyes applied to in-resin CLEM of Epon-embedded cells. The orange fluorescent (λem ∼550 nm) and far-red (λem ∼650 nm) fluorescent dyes examined maintained a sufficient level of fluorescent intensity after staining with osmium tetroxide and subsequent dehydration treatment with ethanol. Immunological in-resin CLEM of mitochondria and the Golgi apparatus was achieved using anti-TOM20, anti-GM130 antibodies, and fluorescent dyes. Two-color in-resin CLEM revealed that wheat germ agglutinin-puncta showed the ultrastructures of multivesicular body-like structures. Finally, taking the advantage of high positional accuracy, volume in-resin CLEM of mitochondria in the semi-thin section (2 μm thick) of Epon-embedded cells was performed by focused ion beam scanning electron microscopy. These results suggested that the application of immunological reaction and affinity-labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells is suitable for analyzing the localization of endogenous targets and their ultrastructures by scanning and transmission electron microscopy.