Biology subjects

Kobayashi, K.

Publications and source records attributed to Kobayashi, K..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

The role of desmoplakin for neuronal function in the dentate gyrus and anxiety-related behavior

Desmoplakin (Dsp) is a component of desmosomal cell-cell junctions that interacts with the cadherin complex and cytoskeletal intermediate filaments. In addition to its function as an adhesion component, Dsp is involved in various biological processes, such as gene expression, differentiation, and migration. Dsp is specifically expressed in the hippocampal dentate gyrus (DG) in the central nervous system. However, it is unclear how Dsp impacts hippocampal function and its related behaviors. Using an adeno-associated virus knockdown system in mice, we provide evidence that Dsp in the DG maintains hippocampal functions, including neuronal activity and adult neurogenesis, and contributes to anxiolytic-like effects. Dsp protein is mostly localized in mature granule cells in the adult DG. Dsp knockdown in the DG resulted in a lowered expression of an activity-dependent transcription factor FosB, and an increased expression of mature neuronal markers, such as calbindin. In addition, the suppression of Dsp decreases serotonin responsiveness at the DG output mossy fiber synapses and alters adult neurogenic processes in the subgranular zone of the DG. Moreover, DG- specific Dsp knockdown mice showed an increase in anxiety-like behaviors. Taken together, this research uncovers an unexplored function for Dsp in the central nervous system and suggests that Dsp in the DG may function as a regulator to maintain proper neuronal activation and adult neurogenesis, and contribute to the adaptation of emotion-related behavior.

neuroscience↗

bioRxiv2023

Functional and structural analysis of a cyclization domain in a cyclic beta-1,2-glucan synthase

Cyclic {beta}-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic {beta}-1,2-glucans (C{beta}Gs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that {beta}-glucosidase-resistant compounds are produced by TiCGSCy with linear {beta}-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are C{beta}Gs. Next, action pattern analyses using {beta}-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide as the minimum length of the substrate. Nevertheless, longer substrate {beta}-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce C{beta}Gs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of {beta}-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear difference in the reaction pathways between these enzymes. Therefore, we propose a novel reaction mechanism of TiCGSCy. Thus, we propose that the present group of CGSs should be defined as a new glycoside hydrolase family, GHxxx. Statements and DeclarationsCompeting Interests: The authors declare no competing interests. Key PointsIt was clearly evidenced that cyclization domain alone produces cyclic {beta}-1,2-glucans. The domain exclusively catalyzes transglycosylation without hydrolysis. The present catalytic domain should be defined as a new glycoside hydrolase family.

biochemistry↗

bioRxiv2023

Acquisition of Auditory Discrimination Mediated by Different Processes through Two Distinct Circuits Linked to the Lateral Striatum

The striatum, the central hub of cortico-basal ganglia loops, contains functionally heterogeneous subregions distinguished by the topographic patterns of structural connectivity. These subregions mediate various processes of procedural learning. However, it remains unclear when and how striatal subregions engage in the acquisition of sensory stimulus-based decision-making. A neuroimaging of regional brain activity shows that the anterior dorsolateral striatum (aDLS) and posterior ventrolateral striatum (pVLS) in rats are activated in a different temporal pattern during the acquisition phase of auditory discrimination. Chronic and transient pharmacologic manipulations show that the aDLS promotes the behavioral strategy driven by the stimulus- response association while suppressing that by the response-outcome association, and that the pVLS contributes to forming and maintaining the stimulus-response strategy. Electrophysiological recording indicates that subpopulations of aDLS neurons predominantly represent the outcome of specific behaviors at the initial period of discrimination learning, and that pVLS subpopulations encode the beginning and ending of each behavior according to the progress of learning. In addition, other subpopulations of striatal neurons indicate sustained activation after obtaining reward with distinct patterns reflecting the stimulus-response associations. Our findings demonstrate that aDLS and pVLS neurons integrate the new learning of auditory discrimination in spatiotemporally and functionally different manners.

animal behavior and cognition↗

bioRxiv2023

Single-cell resolution functional networks during sleep aresegregated into spatially intermixed modules

The common neural mechanisms underlying the reduction of consciousness during sleep and anesthesia remain unclear. Previous studies have examined changes in network structure only using recordings with limited spatial resolution, which has hindered the investigation of the critical spatial scales from the micro (single neurons) to the meso (groups of neurons) for the reduction of consciousness. To address this issue, by leveraging fast, single-cell resolution, and wide-field two-photon microscopy, we recorded calcium signals from approximately 10,000 neurons across multiple cortical regions in awake, sleeping, and anesthetized mice. This single-cell resolution data enabled us to investigate the scales at which changes in network structure compared to an awake state are commonly observed during sleep and anesthesia. We found that at the single-cell scale, both sleep and anesthesia exhibited higher network modularity, indicating a segregated network, compared to an awake state. Despite this segregation, modules were spatially intermixed in all three states. In contrast, at the mesoscale, there were no consistent differences in modularity between states, and modules were spatially localized. Our multi-scale analysis provides novel insights into the cellular-scale organization of functional networks commonly associated with the reduction of consciousness and highlights a scale-dependent organization of network structures.

neuroscience↗

bioRxiv2023

Dynamics of motor direction representation in the primate premotor and primary motor cortices during sensorimotor learning

Sensorimotor learning requires reorganization of neuronal activity in the premotor cortex (PM) and primary motor cortex (M1). However, how PM- and M1-specific reorganization occurs in primates remains unclear. We conducted calcium imaging of these areas in common marmosets while they learned a two-target reaching (pull/push) task. Throughout learning, the dorsorostral PM (PMdr) showed peak activity earlier than the dorsocaudal PM (PMdc) and M1. PMdr showed decreased representation of newly introduced (push) movement, whereas PMdc and M1 maintained high representation. Many task-related neurons in PMdc and M1 exhibited a strong preference to either movement direction. PMdc neurons dynamically switched their preferred direction, whereas M1 neurons stably retained their preferred direction. Differences in preferred direction between adjacent neurons in PMdc increased during learning. These results suggest that in primate sensorimotor learning, dynamic motor representation in PMdc converts the cognitive sensorimotor signals of PMdr to stable and specific motor representation of M1.

neuroscience↗

bioRxiv2023

Chemogenetic activation of target neurons expressing insect Ionotropic Receptors in the mammalian central nervous system by systemic administration of ligand precursors

The IR-mediated neuronal activation (IRNA) technology allows stimulation of neurons in the brain that heterologously-express members of the insect chemosensory IR repertoire in response to their cognate ligands. In the current protocol, a ligand against the complex consisting of IR84a and IR8a subunits, phenylacetic acid (PhAc), is locally injected into a brain region, because of a low efficiency of PhAc for the delivery into the brain across the blood-brain barrier. To circumvent this invasive injection, here we developed a strategy for activation of target neurons in the brain through peripheral administration with a precursor of PhAc, methyl ester of PhAc (PhAcM), which is efficiently transferred into the brain and converted to the mature ligand by endogenous esterase activities. Peripheral administration of with PhAcM activated IR84a/IR8a-expressing neurons in the locus coeruleus of mice and increased the release of neurotransmitters in their nerve terminal regions. (S)-2-phenylpropionic acid ((S)-PhPr) was newly identified as a ligand for IR84a/IR8a, and peripheral administration with the methyl ester of PhPr with the S-configuration [(S)-PhPrM] caused similar effects on the target neurons. In addition, cell-type specific expression of IR84a/IR8a complex in the striatum of rats was unilaterally induced with a viral vector based on the Cre-loxP system. Peripheral administration with PhAcM or (S)-PhPrM stimulated the neurotransmitter release in the ipsilateral terminal regions of the vector-injected striatum, and PhAcM administration resulted in rotational behavior. Finally, we demonstrated that the metabolites of the peripherally administered-radiolabeled (S)-PhPrM accumulated in the IR84a/IR8a-expressing region in the striatum of the vector-injected rats. These results demonstrate that the systemic IRNA technique provides a powerful strategy for remote manipulation of diverse types of target neurons in the mammalian central nervous system.

neuroscience↗

bioRxiv2023

Immune Response of Transplanted Kidney Tissues Assembled from Organoid Building Blocks

The increasing scarcity of organs and the significant morbidity linked to dialysis requires the development of engineered kidney tissues from human-induced pluripotent stem cells. To accomplish this, integrative approaches that synergize scalable kidney organoid differentiation, tissue biomanufacturing, and comprehensive assessment of their immune response and host integration are essential. Here, we create engineered human kidney tissues composed of kidney organoid building blocks (OBBs) and transplant them into mice reconstituted with allogeneic human immune cells. We assess their host vascular integration, in vivo maturation, and their ability to trigger human immune responses. Tissue-infiltrating human immune cells are composed of effector T cells and innate cells. This immune infiltration leads to kidney tissue injury characterized by reduced microvasculature, enhanced kidney cell apoptosis, and a unique inflammatory gene signature comparable to kidney organ transplant rejection in humans. Upon treatment with the immunosuppressive agent Rapamycin, the induced immune response is greatly suppressed. Our model serves as a translational platform to study engineered kidney tissue immunogenicity and develop novel therapeutic targets for kidney rejection.

bioengineering↗

bioRxiv2023

A type VII secretion system toxin functioning as a biofilm-specific intercellular signal in Bacillus subtilis

Biofilms are cooperative bacterial communities, in which diffusible signal-mediated quorum sensing coordinates the activities of member cells. However, limited diffusion in extracellular polymeric substance (EPS)-filled biofilms suggests existence of another intercellular signaling mechanism that is more adapted to the biofilm environment. Here, I demonstrate that the yfjA operon of Bacillus subtilis encodes one such signaling mechanism. The yfjA operon was induced by the two-component system DegSU, which controls biofilm development. Deleting the yfjA operon resulted in increased expression of DegSU-regulated genes under nutrient-rich conditions, hastening the onset of sporulation in biofilms. The yfjA operon encoded the type VII secretion system effector toxin YFJ in addition to its related proteins. YFJ toxin-mediated intercellular competition required EPS components which promote close cell-cell association, suggesting that the contact-dependent YFJ toxin system functions specifically in biofilms. The YFJ toxin also functioned as an intercellular signal that controlled the activity of DegSU. I propose that the YFJ toxin system is a biofilm-specific intercellular signaling mechanism that controls cell fate in biofilms in response to nutrient conditions and population size.

microbiology↗

bioRxiv2023

Opioidergic activation of descending pain inhibitory system underlies placebo analgesia

Placebo analgesia is caused by inactive treatment, implicating endogenous brain function involvement. However, the underlying neurobiological mechanisms remain unclear. We found that -opioid signals in the medial prefrontal cortex (mPFC) activate the descending pain inhibitory system to initiate placebo analgesia in neuropathic pain rats. Chemogenetic manipulation demonstrated that specific activation of -opioid receptor-positive (MOR+) neurons in the mPFC or suppression of the mPFC-ventrolateral periaqueductal gray (vlPAG) circuit inhibited placebo analgesia in rats. MOR+ neurons in the mPFC are monosynaptically connected and directly inhibit L5 pyramidal neurons that project to the vlPAG via GABAA receptors. Thus, intrinsic opioid signaling in the mPFC disinhibits excitatory outflow to the vlPAG by suppressing MOR+ neurons, leading to descending pain inhibitory system activation that initiates placebo analgesia. One Sentence SummarySugar pills relieve pain by activating the intrinsic pain inhibitory system via opioidergic signals in the prefrontal cortex.

neuroscience↗

bioRxiv2023

Emotional association enhances perceptual memory through amygdalo-cortical inputs during NREM sleep

Emotional arousal is thought to enhance the consolidation of associated memories by activating the basolateral amygdala (BLA) and its projections to memory-storing regions1-4. Although the importance of both rapid eye movement (REM) and non-REM (NREM) sleep-state specific BLA activity for emotional memory processing has been proposed5-9, how and when the BLA interacts with other brain regions to enhance memory consolidation remains unclear10. Here, by adding emotional information to a perceptual recognition task that relies on top-down inputs from frontal to sensory cortices, we demonstrated that the BLA not only associates emotional information with perceptual information, but also enhances the retention of associated perceptual memory through BLA-frontal projections. Electrophysiological recordings revealed that emotional association increases the reactivation of coordinated activity across the BLA-frontal-sensory region during NREM sleep, but not during REM sleep. Notably, this inter-regional coordinated reactivation during NREM sleep was entrained to the BLA high-frequency oscillations in the emotional condition, suggesting that the BLA triggers inter-regional interaction. Optogenetic silencing of BLA terminals in the frontal cortex during NREM sleep, but not REM sleep, disrupted the enhanced retention of the perceptual memory, but not the association itself or the emotional component of associative memory. Our results indicate that the inter-regional coordination through the BLA-cortical inputs during NREM sleep is causally required for memory enhancement by emotional arousal.

neuroscience↗

bioRxiv2023

Plastid anionic lipids are required for membrane development and protochlorophyllide synthesis in etioplasts

Dark-germinated angiosperms develop the chloroplast precursors called etioplasts in cotyledon cells. Etioplasts develop lattice membrane structures called prolamellar bodies (PLBs), where the chlorophyll intermediate protochlorophyllide (Pchlide) forms a ternary complex with NADPH and light-dependent NADPH-Pchlide oxidoreductase (LPOR). The lipid bilayers of etioplast membranes are mainly composed of galactolipids, which play important roles in membrane-associated processes in etioplasts. Although etioplast membranes also contain two anionic lipids, phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG), the roles of these anionic lipids are unknown. To reveal the importance of PG and SQDG for the development of etioplasts, we characterized etiolated Arabidopsis mutants deficient in the biosynthesis of PG and SQDG. A partial deficiency in PG biosynthesis loosened the lattice structure of PLBs and impaired the insertion of Mg2+ into protoporphyrin IX, leading to a significant decrease in Pchlide content. Although a complete lack of SQDG biosynthesis did not notably affect both PLB formation and Pchlide biosynthesis, the lack of SQDG in addition to the partial deficiency of PG caused strong impairments of these processes. The results suggested that PG is required for PLB formation and Pchlide biosynthesis, whereas SQDG plays an auxiliary role in these processes. Notably, the PG deficiency and the lack of SQDG oppositely affected the dynamics of LPOR complexes after photoconversion, suggesting different involvements of PG and SQDG in the organization of LPOR complexes. Our data demonstrate pleiotropic roles of anionic lipids in etioplast development.

plant biology↗

bioRxiv2023

State-dependent role of interhemispheric pathway for motor recovery in primates

Whether and how the interhemispheric pathway is involved in post-injury motor recovery is controversial. Unidirectional chemogenetic blockade of the interhemispheric pathway from the contralesional to ipsilesional premotor cortex impaired dexterous hand movements during the early recovery stage after lesioning the lateral corticospinal tract in macaques. Furthermore, electrocorticographical recording showed that the low frequency band activity of the ipsilesional premotor cortex around the movement onset was decreased by the blockade during the early recovery stage, while it was increased by blockade during the intact state and the late recovery stage. These results demonstrate that the action of the interhemispheric pathway changed from inhibition to facilitation, leading to the involvement of the ipsilesional sensorimotor cortex in hand movements during the early recovery stage. The present study offers new insights into the state-dependent role of the interhemispheric pathway and a therapeutic target in the early recovery stage after lesioning of the corticospinal tract.

neuroscience↗

bioRxiv2023

Universal method for generating knockout mice in multiple genetic backgrounds using zygote electroporation

Genetically engineered mouse models are essential tools for understanding mammalian gene functions and disease pathogenesis. Genome editing allows for the generation of these models in multiple inbred strains of mice without backcrossing. Zygote electroporation dramatically removed the barrier for introducing the CRISPR-Cas9 complex in terms of cost and labour. However, the editing conditions and protocols to produce knockout lines have been optimised for a limited number of strains or stocks. Here, we demonstrate a novel and universal approach for generating knockout mice in multiple inbred strains. By combining in vitro fertilisation and electroporation, we obtained founders for knockout alleles in 8 common inbred strains. Long-read sequencing analysis detected not only intended mutant alleles but also differences in read frequency of intended and unintended alleles among strains. Successful germline transmission of knockout alleles demonstrated that our novel approach can establish mutant mice targeting the same locus in multiple inbred strains for phenotyping analysis, contributing to reverse genetics and human disease research. Summary statementUniversal method for zygote genome editing in multiple inbred mouse strains allows for generation of novel mutant mice for understanding mammalian gene function and human disease pathogenesis.

genetics↗

bioRxiv2023

Novel glycoside hydrolase family enzymes from Escherichia coli are associated with osmo-regulated periplasmic glucan synthesis

Most Gram-negative bacteria synthesize osmo-regulated periplasmic glucans (OPG) in the periplasm or extracellular space. Many pathogens lose their pathogenicity by knocking out opgG, an OPG-related gene indispensable for OPG synthesis. However, the biochemical functions of OpgG and OpgD, a paralog of OpgG, have not been elucidated. In this report, structural and functional analyses of OpgG and OpgD from Escherichia coli revealed that these proteins are {beta}-1,2-glucanases with remarkably different activity, establishing a new glycoside hydrolase family. Furthermore, a reaction mechanism with an unprecedentedly long proton transfer pathway is proposed for OpgD. The conformation of the region that forms the reaction pathway differs noticeably between OpgG and OpgD, which explains the observed low activity of OpgG. The findings enhance our understanding of OPG biosynthesis and provide insights into functional diversity for this novel enzyme family.

biochemistry↗

bioRxiv2023

Global disinhibition and corticospinal plasticity for drastic recovery after spinal cord injury

The induction of large-scale plasticity in the adult brain should be key for recovery from severe damage of the central nervous system. Here, drastic motor recovery was observed after subhemisection spinal cord injury in macaques that received intensive training and cortical electrical stimulation. During recovery, movement-related activity increased in ipsilesional sensorimotor areas and functional connectivity from ipsilesional to contralesional areas was strengthened. Electrical stimulation applied widely across bilateral sensorimotor areas induced muscle twitches in affected and intact forelimbs. The interhemispheric inhibition observed before injury was switched to facilitation. Furthermore, massive re-routing occurred in corticospinal axons from the contralesional motor cortex. Such global disinhibition and massive plasticity would open the workspace for the reorganization of motor networks to recruit novel areas for recovery. One Sentence SummaryGlobal disinhibition and corticospinal plasticity for drastic recovery after spinal cord injury in macaque monkeys.

neuroscience↗

bioRxiv2023

Structural basis for lysophosphatidylserine recognition by GPR34

GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-Gi complex bound with either the LysoPS analogue S3E-LysoPS, which contains an ethoxy group at the sn-1 position, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. In both structures, the ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster, and the aromatic fatty acid surrogate of M1 forms stable hydrophobic interactions with the cavity, thus acting as a superagonist. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34, suggesting its short signal duration. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34.

biochemistry↗

bioRxiv2023

ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

Pregnane X receptor (PXR) is one of the key regulators of drug metabolism, gluconeogenesis, and lipid synthesis in the human liver. Activation of PXR by drugs such as rifampicin, simvastatin, and efavirenz causes adverse reactions such as drug{square}drug interaction, hyperglycemia, and dyslipidemia. The inhibition of PXR activation has merit in preventing such adverse events. Here, we demonstrated that bromodomain containing protein 9 (BRD9), a component of non-canonical brahma-related gene 1-associated factor (ncBAF), one of the chromatin remodelers, interacts with PXR. Rifampicin-mediated induction of CYP3A4 expression was attenuated by iBRD9, an inhibitor of BRD9, in human primary hepatocytes and CYP3A/PXR-humanized mice, indicating that BRD9 enhances the transcriptional activation of PXR in vitro and in vivo. Chromatin immunoprecipitation assay reveled that iBRD9 treatment resulted in attenuation of the rifampicin-mediated binding of PXR to the CYP3A4 promoter region, suggesting that ncBAF functions to facilitate the binding of PXR to its response elements. Efavirenz-induced hepatic lipid accumulation was attenuated by iBRD9 in C57BL/6J mice, suggesting that the inhibition of BRD9 would be useful to reduce the risk of efavirenz-induced hepatic steatosis. Collectively, we found that inhibitors of BRD9, a component of ncBAF that plays a role in assisting transactivation by PXR, would be useful to reduce the risk of PXR-mediated adverse reactions.

pharmacology and toxicology↗

bioRxiv2022

A dark matter in sake brewing: Origin of microbes producing a Kimoto-style fermentation starter

In Kimoto-style fermentation, a fermentation starter is produced before the primary brewing process to stabilize fermentation. Nitrate-reducing bacteria, mainly derived from brewing water, produce nitrite, and lactic acid bacteria such as Leuconostoc can proliferate because of their tolerance towards low temperature and their low nutritional requirements. Later, Lactobacillus becomes the dominant genus, leading to weakly acidic conditions that contribute to control yeasts and undesired bacterial contaminants. However, the sources of these microorganisms that play a pivotal role in Sake brewing have not yet been revealed. Thus, comprehensive elucidation of the microbiome is necessary. In this study, we performed 16S rRNA amplicon sequencing analysis after sampling from floor, equipment surfaces, and raw materials for making fermentation starters, including koji, and water in Tsuchida Sake brewery, Gunma, Japan. Amplicon sequence variants (ASVs) between the external environments and the fermentation starter were compared, and it was verified that the microorganisms in the external environments, such as built environments, equipment surfaces, and raw materials in the sake brewery, were introduced into the fermentation starter. Furthermore, various adventitious microbes present in the fermentation starter of early days and from the external environments were detected in a nonnegligible proportion in the starter, which may impact the taste and flavor. These findings illuminate the uncharacterized microbial dark matter of sake brewing, the sources of microbes in Kimoto-style fermentation.

microbiology↗