Biology subjects

Watanabe, M.

Publications and source records attributed to Watanabe, M..

At least 91 records · Page 5Linked to original sources

bioRxiv2020

Neurexins play a crucial role in cerebellar granule cell survival by organizing autocrine machinery for neurotrophins

Neurexins (NRXNs) are among the key presynaptic cell adhesion molecules that regulate synapse function and formation via trans-synaptic interaction with postsynaptic ligands. Here, we generated cerebellar granule cell (CGC)-specific Nrxn triple-knockout (TKO) mice to allow the deletion of all NRXNs. Unexpectedly, most CGCs died in these mice. The requirement of NRNXs for cell survival was reproduced in cultured CGCs. We showed that the axons of cultured Nrxn TKO CGCs that were not in contact with the postsynaptic structure had defects in the formation of presynaptic protein cluster and action potential-induced Ca2+ influx. Additionally, these cells were impaired in the secretion from axons of depolarization-induced fluorescence-tagged brain-derived neurotrophic factor (BDNF), and the cell-survival defect was rescued by the application of BDNF. Our results suggest that CGC survival is maintained by autocrine neurotrophic factors, and that NRXNs organize the presynaptic protein clusters and the autocrine neurotrophic factor secretory machinery independent of contact with postsynaptic ligands.

neuroscience↗

bioRxiv2020

A Specific Neuroligin3-αNeurexin1 Code Regulates GABAergic Synaptic Function in Mouse Hippocampus

Synapse formation and regulation require interactions between pre- and postsynaptic proteins, notably cell adhesion molecules (CAMs). It has been proposed that the functions of neuroligins (Nlgns), postsynaptic CAMs, rely on the formation of trans-synaptic complexes with neurexins (Nrxns), presynaptic CAMs. Nlgn3 is a unique Nlgn isoform that localizes at both excitatory and inhibitory synapses. However, Nlgn3 function mediated via Nrxn interactions is unknown. Here, we demonstrate that Nlgn3 localizes at postsynaptic sites apposing vesicular glutamate transporter 3-expressing (VGT3+) inhibitory terminals and regulates VGT3+ inhibitory interneuron-mediated synaptic transmission in mouse organotypic slice cultures. Gene expression analysis of interneurons revealed that the Nrxn1+AS4 splice isoform is highly expressed in VGT3+ interneurons as compared with other interneurons. Most importantly, postsynaptic Nlgn3 requires presynaptic Nrxn1+AS4 expressed in VGT3+ interneurons to regulate inhibitory synaptic transmission. Our results indicate that specific Nlgn-Nrxn interactions generate distinct functional properties at synapses.

neuroscience↗

bioRxiv2020

Marker-assisted selection of trees with MALE STERALITY 1 in Cryptomeria japonica D. Don

Practical use of marker-assisted selection (MAS) is limited in conifers because of the difficulty with developing markers due to a rapid decrease in linkage disequilibrium, the limited genomic information available, and the diverse genetic backgrounds among breeding material collections. First, in this study, two families were produced by artificial crossing between two male-sterile trees, Shindai11 and Shindai12, and a plus tree, Suzu-2 (Ms1/ms1) (S11-S and S12-S families, respectively). The segregation ratio between male-sterile and male-fertile trees did not deviate significantly from the expected 1:1 ratio in either family. These results clearly suggested that the male-sterile gene of Shindai11 and Shindai12 is MALE STERALITY 1 (MS1). Because some markers reported previously have not been linkage mapped, we constructed a partial linkage map of the region encompassing MS1 using the S11-S and S12-S families. For the S11-S and S12-S families, 19 and 18 markers were mapped onto the partial linkage maps of MS1 region, respectively. There was collinearity (conserved gene order) between the two partial linkage maps. Two markers (CJt020762_ms1-1 and reCj19250_2335) were mapped to the same position as the MS1 locus on both maps. Of these markers, we used CJt020762 for MAS in this study. According to the MAS results for 650 trees from six prefectures of Japan (603 trees from breeding materials and 47 trees from the Ishinomaki natural population), five trees in Niigata Prefecture and one tree in Yamagata Prefecture had heterozygous ms1-1, and three trees in Miyagi Prefecture had heterozygous ms1-2. The results obtained in this study suggested that there may be geographical hotspots for the ms1-1 and ms1-2 alleles. Because MAS can be used effectively to reduce the labor and time required for selection of trees with a male-sterile gene, the number of breeding materials should increase in the future.

genetics↗

bioRxiv2020

Molecular detection of pathogens in ticks and fleas collected from companion dogs and cats in East and Southeast Asia

Ticks and fleas are considered amongst the most important arthropod vectors of medical and veterinary concern due to their ability to transmit pathogens to a range of animal species including dogs, cats and humans. By sharing a common environment with humans, companion animal-associated parasitic arthropods may potentially transmit zoonotic vector-borne pathogens (VBPs). This study aimed to molecularly detect pathogens from ticks (n = 392) and fleas (n = 248) collected from companion dogs and cats in East and Southeast Asia. Of the 392 ticks tested, 37 (9.4%) scored positive for at least one pathogen with Hepatozoon canis being the most prevalent (5.4%), followed by Ehrlichia canis (1.8%), Babesia vogeli (1%), Anaplasma platys (0.8%) and Rickettsia spp. (1%) [including Rickettsia sp. (0.5%), Rickettsia asembonensis (0.3%), Rickettsia felis (0.3%)]. Out of 248 fleas tested, 106 (42.7%) were harboring at least one pathogen with R. felis being the most common (19.4%), followed by Bartonella spp. (16.5%), Rickettsia asembonensis (10.9%) and Candidatus Rickettsia senegalensis (0.4%). Furthermore, 35 Rhipicephalus sanguineus sensu lato ticks were subjected to phylogenetic analysis, of which 34 ticks belonged to the tropical and only one belonged to the temperate lineage (Rh. sanguineus sensu stricto). Our data reveals the circulation of different VBPs in ticks and fleas of dogs and cats from Asia, including zoonotic agents, which may represent a potential risk to animal and human health. Author summaryTicks and fleas are among the most important vectors of pathogens infesting many animal species including humans worldwide. Although a number of vector-borne pathogens have been detected in dogs and cats from East and Southeast Asia, investigation in ticks and fleas collected from them are scant. In order to provide an overview of the pathogens circulating in ticks and fleas from companion dogs and cats in Asia, ticks (n = 392) and fleas (n = 248) were collected from privately-owned dogs and cats from China, Taiwan, Indonesia, Malaysia, Singapore, Thailand, Philippines and Vietnam and molecularly screened for the presence of pathogens. Overall, multiple pathogens were found in ticks (i.e., Babesia vogeli, Hepatozoon canis, Ehrlichia canis, Anaplasma platys and Rickettsia spp.) and fleas (i.e., Rickettsia spp. and Bartonella spp.) from the sampling areas. Of the ticks tested, 9.4% scored positive for at least one pathogen and of fleas 42.7% harbored at least one pathogen with Rickettsia felis being the most common (19.4%). Overall, of the detected pathogens, R. felis stood out as the most important due to its zoonotic potential. The result of this study should increase awareness among pet owners and veterinary practitioners regarding the importance of ticks and fleas, and their transmitted pathogens.

molecular biology↗

bioRxiv2020

Human iPSC-derived hypertrophic chondrocytes reveal a mutation-specific unfolded protein response in chondrodysplasias

Chondrodysplasias are hereditary diseases caused by mutations in the components of growth cartilage. Although the unfolded protein response (UPR) has been identified as a key disease mechanism in mouse models, no suitable in vitro system has been reported to analyze the pathology in humans. Here, utilizing human chondrodysplasia-specific iPSCs, we examined the UPR caused by mutations in MATN3 or COL10A1. In growth plate-like structures formed from iPSC-derived sclerotome in vivo, the hypertrophic zone was disrupted, and induced hypertrophic chondrocytes in vitro showed varying levels of ER stress depending on the mutation. Autophagy inducers and chemical chaperones succeeded in reducing ER stress only in some mutants, while transcriptome analysis revealed many mutation-specific changes in genes involved in apoptosis, metabolism, and protein trafficking. In this way, our system has allowed the precise evaluation of the UPR caused by each mutation, opening up new avenues for treatment of individual chondrodysplasia patients.

cell biology↗

bioRxiv2020

Enhancer Remodeling Promotes Tumor-Initiating Activity in NRF2-Activated Non-Small Cell Lung Cancers

Transcriptional dysregulation, which can be caused by genetic and epigenetic alterations, is a fundamental feature of many cancers. A key cytoprotective transcriptional activator, NRF2, is often aberrantly activated in non-small cell lung cancers (NSCLCs) and supports both aggressive tumorigenesis and therapeutic resistance. Herein, we found that persistently activated NRF2 in NSCLCs generates enhancers at gene loci that are not normally regulated by transiently activated NRF2 under physiological conditions. Elevated accumulation of CEBPB in NRF2-activated NSCLCs was found to be one of the prerequisites for establishment of the unique NRF2-dependent enhancers, among which NOTCH3 enhancer was shown to be critical for the promotion of tumor-initiating activity. Enhancer remodeling mediated by NRF2-CEBPB cooperativity promotes tumor-initiating activity and drives malignancy of NRF2-activated NSCLCs via establishment of the NRF2-NOTCH3 regulatory axis.

cancer biology↗

bioRxiv2020

Subepidermal blisters are healed by wedge-shaped keratinocytes of hair follicle origin

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that tissue development can be sacrificed for the sake of wound regeneration and suggest that tissue repair does not coincide with the reactivation of developmental programs in all regenerative contexts. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases.

cell biology↗

bioRxiv2020

A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

Neuronal synapses undergo structural and functional changes throughout life, essential for nervous system physiology. However, these changes may also perturb the excitatory/inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable. Here, we report the design and characterization of CPTX, a synthetic synaptic organizer combining structural elements from cerebellin-1 and neuronal pentraxin-1 to interact with presynaptic neurexins and postsynaptic AMPA-type ionotropic glutamate receptors. CPTX induced the formation of excitatory synapses in vitro and in vivo and restored synaptic functions, motor coordination, spatial and contextual memories, and locomotion in mouse models for cerebellar ataxia, Alzheimers disease and spinal cord injury, respectively. Thus, CPTX represents a prototype for novel structure-guided biologics that can efficiently repair or remodel neuronal circuits. One Sentence SummaryStructural biology information was used to design CPTX, a synthetic protein that induces functional excitatory synapses and restores normal behaviors in mouse models of neurological diseases.

neuroscience↗

bioRxiv2020

Neuroligin3 Splice Isoforms Shape Mouse Hippocampal Inhibitory Synaptic Function

Synapse formation is a dynamic process essential for neuronal circuit development and maturation. At the synaptic cleft, trans-synaptic protein-protein interactions constitute major biological determinants of proper synapse efficacy. The balance of excitatory and inhibitory synaptic transmission (E-I balance) stabilizes synaptic activity. Dysregulation of the E-I balance has been implicated in neurodevelopmental disorders including autism spectrum disorders. However, the molecular mechanisms underlying E-I balance remain to be elucidated. Here, we investigate Neuroligin (Nlgn) genes that encode a family of postsynaptic adhesion molecules known to shape excitatory and inhibitory synaptic function. We demonstrate that Nlgn3 protein differentially regulates inhibitory synaptic transmission in a splice isoform-dependent manner at hippocampal CA1 synapses. Distinct subcellular localization patterns of Nlgn3 isoforms contribute to the functional differences observed among splice variants. Finally, single-cell sequencing analysis reveals that Nlgn1 and Nlgn3 are the major Nlgn genes and that expression of Nlgn splice isoforms are highly diverse in CA1 pyramidal neurons.

neuroscience↗

bioRxiv2019

TGFβ superfamily signaling regulates the state of human stem cell pluripotency and competency to create telencephalic organoids

Telencephalic organoids generated from human pluripotent stem cells (hPSCs) are emerging as an effective system to study the distinct features of the developing human brain and the underlying causes of many neurological disorders. While progress in organoid technology has been steadily advancing, many challenges remain including rampant batch-to-batch and cell line-to-cell line variability and irreproducibility. Here, we demonstrate that a major contributor to successful cortical organoid production is the manner in which hPSCs are maintained prior to differentiation. Optimal results were achieved using fibroblast-feeder-supported hPSCs compared to feeder-independent cells, related to differences in their transcriptomic states. Feeder-supported hPSCs display elevated activation of diverse TGF{beta} superfamily signaling pathways and increased expression of genes associated with naive pluripotency. We further identify combinations of TGF{beta}-related growth factors that are necessary and together sufficient to impart broad telencephalic organoid competency to feeder-free hPSCs and enable reproducible formation of brain structures suitable for disease modeling. HIGHLIGHTSO_LIhPSC maintenance conditions influence outcomes in cortical organoid formation C_LIO_LIIdentification of an intermediate pluripotency state optimal for cortical organoids C_LIO_LIFeeder support involves activation of diverse TGF{beta} signaling pathways C_LIO_LIThe organoid-promoting effects of feeders can be mimicked by a TGF{beta} factor mixture C_LI

developmental biology↗

bioRxiv2019

Role of microRNA-21 in hypertrophic cardiac remodeling

Hypertension is a major public health problem among with aging population worldwide. It causes cardiac remodeling, including hypertrophy and interstitial fibrosis, which leads to development of hypertensive heart disease (HHD). Although microRNA-21 (miR-21) is associated with fibrogenesis in multiple organs, its impact on hypertrophic cardiac remodeling in hypertension is not known. Circulating miR-21 level was higher in patients with HHD than that in the control subjects. It also positively correlated with serum myocardial fibrotic markers. MiR-21 expression levels were significantly upregulated in the mice hearts after angiotensin II (Ang II) infusion or transverse aortic constriction (TAC) compared with control mice. Expression level of programmed cell death 4 (PDCD4), a main target of miR-21, was significantly decreased in Ang II infused mice and TAC mice compared with control mice. Expression levels of transcriptional activator protein 1 (AP-1) and transforming growth factor-{beta}1 (TGF-{beta}1), which were downstream targets of PDCD4, were increased in Ang II infused mice and TAC mice compared with control mice. In vitro, mirVana-miR-21-specific inhibitor attenuated Ang II-induced PDCD4 downregulation and contributed to subsequent deactivation of AP-1/TGF-{beta}1 signaling pathway in neonatal rat cardiomyocytes. Thus, suppression of miR-21 prevents hypertrophic cardiac remodeling by regulating PDCD4, AP-1, and TGF-{beta}1 signaling pathway.

molecular biology↗

bioRxiv2019

Enhanced emotional memory retrieval by chemogenetic activation of locus coeruleus norepinephrine neurons

The ability to retrieve memory store in response to the environment is essential for animal behavioral adaptation. Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation. However, the role of the central NE system in memory retrieval remains unclear. In this study, we developed a neural chemogenetic activation strategy using insect olfactory Ionotropic Receptors (IRs), and used it for selective stimulation of NE neurons in the locus coeruleus (LC) in transgenic mice. Ligand-induced activation of LC NE neurons resulted in enhancement of the retrieval process of conditioned taste aversion, which was mediated through at least partly adrenergic receptors in the amygdala. Pharmacological blockade of LC activity confirmed the facilitative role of these neurons in memory retrieval. Our findings indicate that the LC-amygdalar pathway is required and sufficient for enhancing the recall of taste associative memory.

neuroscience↗

bioRxiv2019

GluD1 knockout mice with a pure C57BL/6N background show impaired fear memory, social interaction, and enhanced depressive-like behavior

The GluD1 gene is associated with susceptibility for schizophrenia, autism, depression, and bipolar disorder. However, the function of GluD1 and how it is involved in these conditions remain elusive. In this study, we generated a GluD1-knockout (GluD1-KO) mouse line with a pure C57BL/6N genetic background and performed several behavioral analyses. Compared to a control group, GluD1-KO mice showed no significant anxiety-related behavioral differences, evaluated using behavior in an open field, elevated plus maze, a light-dark transition test, the resident-intruder test of aggression and sensorimotor gating evaluated by the prepulse inhibition test. However, GluD1-KO mice showed (1) hyper locomotor activity in the open field, (2) decreased sociability and social novelty preference in the three-chambered social interaction test, (3) impaired memory in contextual, but not cued fear conditioning tests, and (4) enhanced depressive-like behavior in a forced swim test. Pharmacological studies revealed that enhanced depressive-like behavior in GluD1-KO mice was restored by the serotonin reuptake inhibitors imipramine and fluoxetine, but not the norepinephrine transporter inhibitor desipramine. In addition, biochemical analysis revealed no significant difference in protein expression levels, such as other glutamate receptors in the synaptosome and postsynaptic densities prepared from the frontal cortex and the hippocampus. These results suggest that GluD1 plays critical roles in fear memory, sociability, and depressive-like behavior.

neuroscience↗

bioRxiv2019

Identification of neural oscillations and epileptiform changes in human brain organoids

Brain organoids represent a powerful tool for the study of human neurological diseases, particularly those impacting brain growth and structure. However, many diseases manifest with clear evidence of physiological and network abnormality in the absence of anatomical changes. This raises the question of whether organoids possess sufficient neural network complexity to model these conditions. Here, we explore the network level functions of brain organoids using calcium sensor imaging and extracellular recording approaches that together reveal the existence of complex network behaviors reminiscent of intact brain preparations. We demonstrate highly abnormal and epileptiform-like activity in organoids derived from MECP2 mutant patients compared to isogenic controls accompanied by modest transcriptomic differences revealed by single cell analyses. We also rescue key physiological activities with an unconventional neuromodulatory drug, Pifithrin-. Together, these findings provide an essential foundation for the utilization of brain organoids to study intact and disordered human brain network formation and illustrate their utility in therapeutic discovery.

neuroscience↗

bioRxiv2019

Calretinin positive neurons form an excitatory amplifier network in the spinal cord dorsal horn

The passage of nociceptive information is relayed through the spinal cord dorsal horn, a critical area in sensory processing. The neuronal circuits in this region that underpin sensory perception must be clarified to better understand how dysfunction can lead to pathological pain. This study used an optogenetic approach to selectively activate neurons that contain the calcium-binding protein calretinin (CR). We show that CR+ interneurons form an interconnected network that can initiate and sustain enhanced excitatory signaling, and directly relays signals to lamina I projection neurons. In vivo photoactivation of CR+ interneurons resulted in a significant nocifensive behavior that was morphine sensitive and cause a conditioned place aversion. Furthermore, halorhodopsin-mediated inhibition of CR+ interneurons elevated sensory thresholds. These results suggest that neuronal circuits in the superficial dorsal horn that involve excitatory CR+ neurons are important for the generation and amplification of pain, and identify these interneurons as a future analgesic target.

neuroscience↗

bioRxiv2019

Cortisol levels adter cold exposure are independent of adrenocorticotropic hormone stimulation

We previously showed that postmortem serum levels of adrenocorticotropic hormone (ACTH) were significantly higher in cases of hypothermia (cold exposure) than other causes of death. This study examined how the human hypothalamic-pituitary-adrenal axis, and specifically cortisol, responds to hypothermia.\n\nHuman samplesAutopsies on 205 subjects (147 men and 58 women; age 15-98 years, median 60 years) were performed within 3 days of death. Cause of death was classified as either hypothermia (cold exposure, n=14) or non-cold exposure (controls; n=191). Cortisol levels were determined in blood samples obtained from the left and right cardiac chambers and common iliac veins using a chemiluminescent enzyme immunoassay. Adrenal gland tissue samples were stained for cortisol using a rabbit anti-human polyclonal antibi.\n\nCell cultureAtT20, a mouse ACTH secretory cell line, and Y-1, a corticosterone secretory cell line derived from a mouse adrenal tumor, were analyzed in mono-and co-culture, and times courses of ACTH (in AtT20) and corticosterone (in Y-1) secretion were assessed after low temperature exposure mimicking hypothermia and compared with data for samples collected postmortem for other causes of death. However, no correlation between ACTH concentration and cortisol levels was observed in hypothermia cases. Immunohistologic analyses of samples from hypothermia cases showed that cortisol staining was localized primarily to the nucleus rather than the cytoplasm of cells in the zona fasciculata of the adrenal gland. During both mono-culture and co-culture, AtT20 cells secreted high levels of ACTH after 10-15 minutes of cold exposure, whereas corticosterone secretion by Y-1 cells increased slowly during the first 15-20 minutes of cold exposure. Similar to autopsy results, no correlation was detected between ACTH levels and corticosterone secretion, either in mono-culture or co-culture experiments. These results suggested that ACTH-independent cortisol secretion may function as a stress response during cold exposure.

biochemistry↗

bioRxiv2018

mitoNEET Regulates Mitochondrial Iron Homeostasis Interacting with Transferrin Receptor

Iron is an essential trace element for regulation of redox and mitochondrial function, and then mitochondrial iron content is tightly regulated in mammals. We focused on a novel protein localized at the outer mitochondrial membrane. Immunoelectron microscopy revealed transferrin receptor (TfR) displayed an intimate relationship with the mitochondria, and mass spectrometry analysis also revealed mitoNEET interacted with TfR in vitro. Moreover, mitoNEET was endogenously coprecipitated with TfR in the heart, which indicates that mitoNEET also interacts with TfR in vivo. We generated mice with cardiac-specific deletion of mitoNEET (mitoNEET-knockout). Iron contents in isolated mitochondria were significantly increased in mitoNEET-knockout mice compared to control mice. Mitochondrial reactive oxygen species (ROS) were higher, and mitochondrial maximal capacity and reserve capacity were significantly decreased in mitoNEET-knockout mice, which was consistent with cardiac dysfunction evaluated by echocardiography. The complex formation of mitoNEET with TfR may regulate mitochondrial iron contents via an influx of iron. A disruption of mitoNEET could thus be involved in mitochondrial ROS production by iron overload in the heart.

cell biology↗

bioRxiv2018

Conditional upregulation of KCC2 selectively enhances neuronal inhibition during seizures

Abstract/SummaryEfficacious neuronal inhibition is sustained by the neuronal K+Cl- co-transporter KCC2, and loss of KCC2 function through injury or mutation is associated with altered GABAergic signalling and neuronal seizures. Here we report a transgenic mouse with conditional KCC2 overexpression that results in increased membrane transport function. Increased KCC2 has little impact on behavioural and in vitro assays of neuronal excitability and GABAA receptor responses under resting conditions. In contrast, increased KCC2 imparts resistance to seizure-like neuronal activity in hippocampal slices and prevents the progression of mice into behavioural status epilepticus following multiple kainic acid doses. Our results demonstrate a transgenic mouse to facilitate investigations into the role of KCC2 in brain function, and provide a proof of principle that targeting KCC2 may be an effective way to selectively enhance neuronal inhibition to mitigate against diseases that involve an imbalance between excitation and inhibition.

neuroscience↗