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Kato, S.

Publications and source records attributed to Kato, S..

At least 55 records · Page 3Linked to original sources
bioRxiv2023

Cell specificity of adeno-associated virus (AAV) serotypes in human cortical organoids

Human-derived cortical organoids (hCOs) recapitulate cell diversity and 3D structure found in the human brain and offer a promising model for discovery of new gene therapies targeting neurological disorders. Adeno-associated viruses (AAVs) are the most promising vehicles for non-invasive gene delivery to the central nervous system (CNS), but reliable and reproducible in vitro models to assess their clinical potential are lacking. hCOs can take on these issues as they are a physiologically relevant model to assess AAV transduction efficiency, cellular tropism, and biodistribution within the tissue parenchyma, all of which could significantly modulate therapeutic efficacy. Here, we examine a variety of naturally occurring AAV serotypes and measure their ability to transduce neurons and glia in hCOs from multiple donors. We demonstrate cell tropism driven by AAV serotype and hCO donor and quantify fractions of neurons and astrocytes transduced with GFP as well as overall hCO health.

neuroscience↗
bioRxiv2023

Neuroimmune cortical organoids overexpressing C4A exhibit multiple schizophrenia endophenotypes

Elevated expression of the complement component 4A (C4A) protein has been linked to an increased risk of schizophrenia (SCZ). However, there are few human models available to study the mechanisms by which C4A contributes to the development of SCZ. In this study, we established a C4A overexpressing neuroimmune cortical organoid (NICO) model, which includes mature neuronal cells, astrocytes, and functional microglia. The C4A NICO model recapitulated several neuroimmune endophenotypes observed in SCZ patients, including modulation of inflammatory genes and increased cytokine secretion. C4A expression also increased microglia-mediated synaptic uptake in the NICO model, supporting the hypothesis that synapse and brain volume loss in SCZ patients may be due to excessive microglial pruning. Our results highlight the role of C4A in the immunogenetic risk factors for SCZ and provide a human model for phenotypic discovery and validation of immunomodulating therapies.

neuroscience↗
bioRxiv2022

Dissection of insular cortex layer 5 reveals sublayers with opposing modulatory roles in appetitive behavior

The insular cortex (insula) is known to play a modulatory role in motivated behaviors including feeding and drinking. Previous studies have revealed that the anterior and posterior subregions of the insula have differential subcortical efferents and roles, yet the anatomical and functional heterogeneity among the cortical layers remains poorly understood. Here, we show that layer 5 of the mouse dysgranular insula has two distinct neuronal subpopulations along the entire anterior-posterior axis: the upper layer (L5a) population, expressing NECAB1, projects bilaterally to the lateral and capsular divisions of the central amygdala, and the deeper layer (L5b) population, expressing CTIP2, projects ipsilaterally to the parasubthalamic nucleus and the medial division of the central amygdala. Optogenetically activating L5a and L5b neuronal populations in thirsty mice led to suppressed and facilitated water spout licking, respectively, in a single-spout test. However, the opto-activation induced no avoidance against or preference for the spout paired with the opto-stimulation in a two-spout choice test, indicating no induction of emotional valences by the opto-activation per se. Our results suggest sublayer-specific bidirectional modulatory roles of insula layer 5 in the motivational aspect of appetitive behavior.

neuroscience↗
bioRxiv2022

Catecholaminergic cell type-specific expression of Cre recombinase in knock-in transgenic rats generated by the Combi-CRISPR technology

BackgroundCell groups containing catecholamines provide a useful model to study the molecular and cellular mechanisms underlying the morphogenesis, physiology, and pathology of the central nervous system. For this purpose, it is necessary to establish a system to induce catecholaminergic group-specific expression of Cre recombinase. Recently, we introduced a gene cassette encoding 2A peptide fused to Cre recombinase into the site between the C-terminus and translational termination codons of the rat tyrosine hydroxylase (TH) open reading frame by the Combi-CRISPR technology, which is a genomic editing method to enable an efficient knock-in (KI) of long DNA sequence into a target site. However, the expression patterns of the transgene and its function as well as the effect of the mutation on the biochemical and behavioral phenotypes in the KI strains have not been characterized yet. New MethodWe aimed to evaluate the usefulness of TH-Cre KI rats as an experimental model for investigating the structure and function of catecholaminergic neurons in the brain. ResultsWe detected cell type-specific expression of Cre recombinase and site-specific recombination activity in the representative catecholaminergic groups in the TH-Cre KI rat strains. In addition, we measured TH expression level and catecholamine accumulation in the brain regions, and spontaneous locomotion, indicating that catecholamine metabolism and general behavior are apparently normal in these KI rats. ConclusionsTH-Cre KI rat strains produced by the Combi-CRISPR system offer a beneficial model to study the molecular and cellular mechanics for the morphogenesis, physiology, and pathology of catecholamine-containing neurons in the brain.

neuroscience↗
bioRxiv2022

Highly selective transgene expression through double-floxed inverted orientation system by using a unilateral spacer sequence

The double-floxed inverted orientation (DIO) system with adeno-associated viral (AAV) vector provides a beneficial approach to express transgenes in specific cell populations having Cre recombinase. A significant issue with this system is the protection against non-specific expression of transgenes in tissues after vector injection. We here show that Cre-independent recombination in AAV genome carrying the DIO sequence occurs during the production of viral vectors in packaging cells, which results in transgene expression in off-target populations. Introduction of a relatively longer nucleotide sequence between two recognition sites at the unilateral side of the transgene cassette, termed a unilateral spacer sequence (USS), is useful to suppress recombination during the vector production, leading to the protection of non-specific transgene expression with enhanced gene expression selectivity. Our DIO/USS system offers a powerful strategy for highly specific Cre-dependent transgene expression, aiming at various applications for structural and functional analyses of target cell populations.

neuroscience↗
bioRxiv2022

Reinstating olfactory bulb derived limbic gamma oscillations alleviates depression

Although the etiology of major depressive disorder remains poorly understood, impairment of gamma oscillations recently emerged as a potential biomarker for major depression. The olfactory bulb (OB) is a major source of brain wide gamma oscillations and bulbectomy is an animal model for depression. Here we demonstrate that chemogenetic suppression of OB neuronal activity or temporally suppressing the OB to pyriform cortex synaptic transmission decreased gamma oscillation power in multiple brain areas associated with depression-like behaviors. To assess the hypothesized link between depression and diffuse depression of gamma oscillations, we employed gamma phase-dependent closed loop neuromodulation of cortical areas, paced by the native OB output. This procedure alleviated depressive-like behaviors in animals and suggests that restoring gamma oscillations may improve depression in humans. One Sentence SummaryRole of limbic gamma oscillations in depression

neuroscience↗
bioRxiv2022

Information theory rules out the reflex-chain model of C. elegans locomotion

Despite decades of research, whether the C. elegans traveling-wave sinusoidal body pattern during locomotion is produced (a) by the undulations of the head followed by wave propagation down the body, or (b) via centrally coordinated posture control along the body, is still under debate. By studying relationships between the time series of postural angles along the body extracted from videos of moving worms, we find that the reflex-chain model can be refuted, in both forward and backward locomotion as well as during swimming and crawling behaviors. We show that information theory applied to animal behavior can yield insights into the neural control of behavior.

neuroscience↗
bioRxiv2022

A novel genomic DNA library preparation method with low GC bias

The amount of input DNA available to prepare next-generation sequencing (NGS) libraries is often limited, which can lead to GC content bias and enrichment of specific genomic regions with currently available protocols. In this study, we used breath capture technology to incorporate sequencing adapters into DNA to develop a novel cost-effective protocol for the preparation of genomic DNA libraries. We performed a benchmarking experiment comparing our protocol with common commercially available kits for genomic DNA library preparation with input DNA amount in the range of 1 to 50 ng. Our protocol can generate high-quality genomic sequence data with a marked improvement in coverage breadth and low GC bias, in contrast to standard protocols. Further, our protocol reduces sample handling time and reagent costs, and requires comparatively fewer enzymatic steps relative to other protocols, making it suitable for a range of genomics applications.

genomics↗
bioRxiv2021

Recording and neural circuit-based manipulation of the maternal oxytocin pulses in mice

Pulsatile release of the hormone oxytocin (OT) mediates uterine contraction during parturition and milk ejection during lactation1-3. These pulses are generated by unique activity patterns of the central neuroendocrine OT neurons located in the paraventricular and supraoptic hypothalamus. Classical studies have characterized putative OT neurons by in vivo extracellular recording techniques in rats and rabbits under anesthesia1, 4-7 or awake8-10. Due to technical limitations, however, the identity of OT neurons in these previous studies was speculative based on their electrophysiological characteristics and axonal projection to the posterior pituitary, not on OT gene expression. To pinpoint OT neural activities among other hypothalamic neurons that project to the pituitary11, 12 and make better use of cell-type-specific neuroscience toolkits13, a mouse model needs to be developed for studies of parturition and lactation. We herein introduce viral genetic approaches in mice to characterize the maternal activities of OT neurons by fiber photometry. During lactation, a sharp photometric peak of OT neurons appeared at approximately 520 s following simultaneous suckling stimuli from three pups. The amplitude of the peaks increased as the mother mice experienced lactation, irrespective of the age of the pups, suggesting the intrinsic plasticity of maternal OT neurons. Based on a mono-synaptic input map to OT neurons, we pharmacogenetically activated the inhibitory neurons in the bed nucleus of the stria terminalis and found suppression of the activities of OT neurons. Collectively, our study illuminates temporal dynamics in the maternal neural activities of OT neurons and identifies one of its modulatory circuits. Highlights- Pulsatile activities of genetically-defined OT neurons in mother mice were recorded in vivo. - The maternal experience-dependent plasticity of the OT neural activities was found. - Input-mapping of OT neurons in mother mice was performed by rabies-mediated trans-synaptic tracing. - Photometric peaks of OT neurons were suppressed by the activation of BST inhibitory neurons.

neuroscience↗
bioRxiv2021

Demuxalot: scaled up genetic demultiplexing for single-cell sequencing

Demultiplexing methods have facilitated the widespread use of single-cell RNA sequencing (scRNAseq) experiments by lowering costs and reducing technical variations. Here, we present demuxalot: a method for probabilistic genotype inference from aligned reads, with no assumptions about allele ratios and efficient incorporation of prior genotype information from historical experiments in a multi-batch setting. Our method efficiently incorporates additional information across reads originating from the same transcript, enabling up to 3x more calls per read relative to naive approaches. We also propose a novel and highly performant tradeoff between methods that rely on reference genotypes and methods that learn variants from the data, by selecting a small number of highly informative variants that maximize the marginal information with respect to reference single nucleotide variants (SNVs). Our resulting improved SNV-based demultiplex method is up to 3x faster, 3x more data efficient, and achieves significantly more accurate doublet discrimination than previously published methods. This approach renders scRNAseq feasible for the kind of large multi-batch, multi-donor studies that are required to prosecute diseases with heterogeneous genetic backgrounds.

bioinformatics↗
bioRxiv2021

Hierarchical confounder discovery in the experiment-machine learning cycle

The promise of using machine learning (ML) to extract scientific insights from high dimensional datasets is tempered by the frequent presence of confounding variables, and it behooves scientists to determine whether or not a model has extracted the desired information or instead may have fallen prey to bias. Due both to features of many natural phenomena and to practical constraints of experimental design, complex bioscience datasets tend to be organized in nested hierarchies which can obfuscate the origin of a confounding effect and obviate traditional methods of confounder amelioration. We propose a simple non-parametric statistical method called the Rank-to-Group (RTG) score that can identify hierarchical confounder effects in raw data and ML-derived data embeddings. We show that RTG scores correctly assign the effects of hierarchical confounders in cases where linear methods such as regression fail. In a large public biomedical image dataset, we discover unreported effects of experimental design. We then use RTG scores to discover cross-modal correlated variability in a complex multi-phenotypic biological dataset. This approach should be of general use in experiment-analysis cycles and to ensure confounder robustness in ML models.

bioinformatics↗
bioRxiv2021

Ultra-small cells and DPANN genome unveiled inside an extinct vent chimney

Chemosynthetic organisms flourish around deep-sea hydrothermal vents where energy-rich fluids are emitted from metal sulfide chimneys. In contrast to actively venting chimneys, the nature of microbial life in extinct chimneys without fluid venting remains largely unknown. Here, the occurrence of ultra-small cells in silica-filled grain boundaries inside an extinct chimney is demonstrated by high-resolution bio-signature mapping. The ultra-small cells are associated with extracellularly precipitated Cu2O nanocrystals. Single-gene analysis shows that the chimney interior is dominated by a member of Pacearchaeota known as a major phylum of DPANN. Genome-resolved metagenomic analysis reveals that the chimney Pacearchaeota member is equipped with a nearly full set of genes for fermentation-based energy generation from nucleic acids, in contrast to previously characterized Pacearchaeota members lacking many genes for nucleic acid fermentation. We infer that the ultra-small cells associated with silica and extracellular Cu2O nanocrystals in the grain boundaries are Pacearchaeota, on the basis of the experimentally demonstrated capability of silica to concentrate nucleic acids from seawater and the presence of Cu-exporting genes in a reconstructed Pacearchaeota genome. Given the existence of ~3-billion-year-old submarine hydrothermally deposited silica, proliferation of microbial life using silica-bound nucleic acids might be relevant to the primitive vent biosphere.

microbiology↗
bioRxiv2020

Sleep is required for odor exposure to consolidate memory and remodel olfactory synapses.

Animals with complex nervous systems demand sleep for memory consolidation and synaptic remodeling. Here we show that though the Caenorhabditis elegans nervous system has a limited number of neurons, sleep is necessary for both processes. In addition, it is unclear in any system if sleep collaborates with experience to alter synapses between specific neurons and whether this ultimately affects behavior. C. elegans neurons have defined connections and well-described contributions to behavior. We show that spaced odor-training and post-training sleep induce long-term memory. Memory consolidation, but not acquisition, requires a pair of interneurons, the AIYs, which play a role in odor-seeking behavior. In worms that consolidate memory, both sleep and odor conditioning are required to diminish inhibitory synaptic connections between the AWC chemosensory neurons and the AIYs. Thus, we demonstrate in a living organism that sleep is required for events immediately after training that drive memory consolidation and alter synaptic structures.

neuroscience↗
bioRxiv2020

Optimization and scaling of patient-derived brain organoids uncovers deep phenotypes of disease

Cerebral organoids provide unparalleled access to human brain development in vitro. However, variability induced by current culture methodologies precludes using organoids as robust disease models. To address this, we developed an automated Organoid Culture and Assay (ORCA) system to support longitudinal unbiased phenotyping of organoids at scale across multiple patient lines. We then characterized organoid variability using novel machine learning methods and found that the contribution of donor, clone, and batch is significant and remarkably consistent over gene expression, morphology, and cell-type composition. Next, we performed multi-factorial protocol optimization, producing a directed forebrain protocol compatible with 96-well culture that exhibits low variability while preserving tissue complexity. Finally, we used ORCA to study tuberous sclerosis, a disease with known genetics but poorly representative animal models. For the first time, we report highly reproducible early morphological and molecular signatures of disease in heterozygous TSC+/- forebrain organoids, demonstrating the benefit of a scaled organoid system for phenotype discovery in human disease models.

neuroscience↗
bioRxiv2020

Resolving the data asynchronicity in high-speed atomic force microscopy measurement via the Kalman Smoother

High-speed atomic force microscopy (HS-AFM) is a scanning probe microscopy that can capture structural dynamics of biomolecules in real time at single molecule level near physiological condition. Albeit much improvement of the instruments, while scanning one frame of HS-AFM movies, biomolecules often change their conformations largely. Thus, the obtained frame images can be hampered by the time-difference, the asynchronicity, in the data acquisition. Here, to resolve this data asynchronicity in the HS-AFM movie, we developed Kalman filter and smoother methods, some of the sequential Bayesian filtering approaches. The Kalman filter/smoother methods use alternative steps of a short time-propagation by a linear dynamical system and a correction by the likelihood of AFM data acquired pixel by pixel. We first tested the method using a toy model of a diffusing cone, showing that the Kalman smoother method outperforms to reproduce the ground-truth movie, compared to that mimics the raw AFM movie, and the Kalman filter result. We then applied the Kalman smoother to a synthetic movie for conformational change dynamics of a motor protein, i.e., dynein, confirming the superiority of the Kalman smoother. Finally, we applied the Kalman smoother to two real HS-AFM movies, FlhAc and centralspindlin, reducing distortion and noise in the AFM movies. The method is general and can be applied to any HS-AFM movies.

biophysics↗
bioRxiv2020

Timing of fruiting rather than topography determines the direction of vertical seed dispersal by mammals and birds

Vertical seed dispersal toward higher or lower altitudes has been recognized as one of the critical processes for plants to escape from climate change. Studies exploring vertical seed dispersal are scarce, preventing the prediction of future vegetation dynamics. In the present study, we show that the timing of fruiting, rather than topography, determines the direction of vertical seed dispersal by mammals and birds across mountains in central Japan. We found strong uphill seed dispersal of summer fruiting cherry and weak downhill seed dispersal of summer-to-autumn fruiting cherry, irrespective of mountains and animals. The ascent or descent of animals, following the altitudinal gradients in food plant phenology in the temperate zone, was considered to be a driver of the biased seed dispersal. We found that megafauna (i.e., bears) intensively dispersed seeds vertically. The results suggest that the timing of fruiting and megafauna strongly affect whether animal-dispersed temperate plants can maintain their populations under climate change.

ecology↗
bioRxiv2020

Comprehensive analyses of the cysteine thiol oxidation of PKM2 reveals the effects of multiple oxidation on cellular oxidative stress response

Redox regulation of proteins via cysteine residue oxidation is known to be involved in the control of various cellular signal pathways. Pyruvate kinase M2 (PKM2), a rate-limiting enzyme in glycolysis, is critical for the metabolic shift from glycolysis to the pentose phosphate pathway under oxidative stress in cancer cell growth. The PKM2 tetramer acts as pyruvate kinase (PK), whereas the PKM2 dimer, which is induced by Cys358 oxidation, has reduced PK activity. Here, we identified four oxidation-sensitive cysteine residues (Cys152, Cys358, Cys423, and Cys424) responsible for three different oxidation forms. Possibly due to obstruction of the dimer-dimer interface, sulfenylation (-SOH) at Cys424 inhibited tetramer formation and PK activity. Cys423 is responsible for intermolecular disulfide bonds with heterologous proteins. In addition, intramolecular polysulfide linkage (-Sn-, n[greater double equals]3) possible between Cys152 and Cys358 also is induced. We found that cells expressing the oxidation-resistant, constitutive-tetramer PKM2 (PKM2C358,424A) show a higher intracellular reactive oxygen species (ROS) and greater sensitivity to ROS-generating reagents and ROS-inducible anti-cancer drugs. These results highlight the possibility that PKM2 inhibition via Cys358 and Cys424 oxidation contributes to the elimination of excess ROS and oxidative stress.

molecular biology↗