Biology subjects

Okano, H.

Publications and source records attributed to Okano, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Shared GABA transmission pathology in dopamine agonist- and antagonist-induced dyskinesia

Dyskinesia is involuntary movement caused by long-term medication with dopamine-related agents: the dopamine agonist, L-DOPA, to treat Parkinsons disease (L-DOPA-induced dyskinesia [LID]) or dopamine antagonists to treat schizophrenia (tardive dyskinesia [TD]). However, it remains unknown why distinct types of medications for distinct neuropsychiatric disorders induce similar involuntary movements. Here, we searched for a shared structural footprint using magnetic resonance imaging-based macroscopic screening and super-resolution microscopy-based microscopic identification. We identified the enlarged axon terminals of striatal medium spiny neurons in both LID and TD model mice. The striatal overexpression of vesicular gamma-aminobutyric acid transporter (VGAT) was necessary and sufficient for modeling these structural changes; VGAT levels gated the functional and behavioral alterations in dyskinesia models. Our findings indicate that lowered type 2 dopamine receptor signaling with repetitive dopamine fluctuations is a common cause of VGAT overexpression and late-onset dyskinesia formation, and that reducing dopamine fluctuation rescues dyskinesia pathology via VGAT downregulation. HighlightsO_LIEnhancement of GABAergic transmission is a shared mechanism between LID and TD. C_LIO_LIVGAT levels in MSNs govern the structure and function of MSN presynaptic terminals. C_LIO_LIGain and loss of VGAT function in MSNs exacerbates and ameliorates dyskinesia. C_LIO_LILowered D2 signaling with repetitive DA fluctuations causes VGAT overexpression. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=188 HEIGHT=200 SRC="FIGDIR/small/550763v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@15041b4org.highwire.dtl.DTLVardef@9bf41org.highwire.dtl.DTLVardef@1eb7ee4org.highwire.dtl.DTLVardef@1d7dae8_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Proteomic characterization of aging-driven changes in the mouse brain by co-expression network analysis

Brain aging causes a progressive decline in functional capacity and is a strong risk factor for dementias such as Alzheimers disease. To characterize age-related proteomic changes in the brain, we used quantitative proteomics to examine brain tissues, cortex and hippocampus, of mice at three age points (3, 15, and 24 months old), and quantified more than 7,000 proteins in total with high reproducibility. We found that many of the proteins upregulated with age were extracellular proteins, such as extracellular matrix proteins and secreted proteins, associated with glial cells. On the other hand, many of the significantly downregulated proteins were associated with synapses, particularly postsynaptic density, specifically in the cortex but not in the hippocampus. Our datasets will be helpful as resources for understanding the molecular basis of brain aging.

molecular biology↗

bioRxiv2023

Infant attachment behaviors reflect the parenting style of individual caregiver in common marmosets

Childrens physical, cognitive, and emotional maturation require adequate interactions and secure attachment with their primary caregivers. However, the causal relationships between parenting components and infant attachment behaviors remain unclear. New World monkey common marmosets provide an ideal model of human parent-infant relations because, like humans, infant attachment is shared among family caregivers, including parents and older siblings using intricate vocal communications. Combining the natural variations in parenting styles and subsecond-scale microanalyses of dyadic vocal and physical interactions, we demonstrate that marmoset infants signal their need by context-dependent call use and selectively maintain proximity with familiar caregivers. The infant attachment behaviors are individually tuned according to the caregivers parenting style; infants keep producing isolation calls when they are carried by rejecting caregivers and exhibit avoidance toward neglectful and rejecting caregivers. Family-deprived infants failed to develop such adaptive uses of attachment behaviors or age-appropriate autonomy and exhibited disorganized attachment behaviors. Thus, marmoset infants shape their ability to modulate their intricate attachment system depending on the caregivers attitude through early social interactions with their family caregivers. Demonstrating the significant similarity of the infant attachment system between marmosets and humans, these findings will pave a path to elucidating the neural mechanisms of the infant attachment system.

animal behavior and cognition↗

bioRxiv2023

An efficient induction method for human spinal lower motor neurons and high-throughput image analysis at the single cell level

This study presents a newly developed method to rapidly and efficiently induce human spinal lower motor neurons (LMNs) from induced pluripotent stem cells (iPSCs) for elucidation of the amyotrophic lateral sclerosis (ALS) pathomechanism and drug screening. Previous methods had several limitations such as poor efficiency and low purity of LMN induction and labor intensiveness of the induction and evaluation procedures. Our new protocol achieved nearly 80% induction efficiency in only 2 weeks by combining a small molecule-based approach and transduction of transcription factors. To overcome cellular heterogeneity, we analyzed morphology and viability of iPSC-derived LMNs on a cell-by-cell basis using time-lapse microscopy and machine learning, thus establishing a highly accurate pathophysiological evaluation system. Our rapid, efficient, and simplified protocol and single cell-based evaluation method allow the conduct of large-scale analysis and drug screening using iPSC-derived motor neurons.

neuroscience↗

bioRxiv2023

Modeling the marmoset brain using embryonic stem cell-derived cerebral assembloids

Studying the non-human primate (NHP) brain is required for the translation of rodent research to humans, but remains a challenge for molecular, cellular, and circuit-level analyses in the NHP brain due to the lack of in vitro NHP brain system. Here, we report an in vitro NHP cerebral model using marmoset (Callithrix jacchus) embryonic stem cell-derived cerebral assembloids (CAs) that recapitulate inhibitory neuron migration and cortical network activity. Cortical organoids (COs) and ganglionic eminence organoids (GEOs) were induced from cjESCs and fused to generate CAs. GEO cells expressing the inhibitory neuron marker LHX6 migrated toward the cortical side of CAs. COs developed their neural activity from a synchronized pattern to an unsynchronized pattern as COs matured. CAs showed mature neural activity with an unsynchronized pattern. The marmoset assembloid system will provide an in vitro platform for the NHP neurobiology and facilitate translation into humans in neuroscience research, regenerative medicine, and drug discovery.

neuroscience↗

bioRxiv2023

Highly efficient induction of functionally mature excitatory neurons from feeder-free human ES/iPS cells

Cortical excitatory neurons (Cx neurons) are the most dominant neuronal cell type in the cerebral cortex, which play a central role in cognition, perception, intellectual behavior and emotional processing. Robust in vitro induction of Cx neurons may facilitate as a tool for the elucidation of brain development and pathomechanism of the intractable neurodevelopmental and neurodegenerative disorders including Alzheimers disease, and thus potentially contribute to drug development. Here, we report a defined method for efficient induction of Cx neurons from the feeder-free-conditioned human embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). By using this method, human ES/iPS cells could be differentiated into ~99% MAP2-positive neurons by three weeks, and these induced neurons, within five weeks, presented various characteristics of mature excitatory neurons such as strong expression of glutamatergic neuron-specific markers (subunits of AMPA and NDMA receptors and CAMKII), highly synchronized spontaneous firing and excitatory postsynaptic current (EPSC). Moreover, the Cx neurons showed susceptibility to the toxicity of A{beta}42 oligomers and excitotoxicity of excessive glutamates, which is another advantage in terms of toxicity test and searching for the therapeutic agents. Taken together, this study provides a novel research platform for the study of neural development and degeneration based on the feeder-free human ES/iPS cell system.

neuroscience↗

bioRxiv2022

Functional Decomposition of Metabolism allows a system-level quantification of fluxes and protein allocation towards specific metabolic functions

Quantifying the contribution of individual molecular components to complex cellular processes is a grand challenge in systems biology. Here we establish a general theoretical framework (Functional Decomposition of Metabolism, FDM) to quantify the contribution of every metabolic reaction to metabolic functions, e.g. the biosynthesis of metabolic building blocks such as amino acids. This allows us to obtain a plethora of results for E. coli growing in different conditions. A detailed quantification of energetic costs for biosynthesis and biomass growth on glucose shows that ATP generated during de novo biosynthesis of building blocks almost balances the ATP costs of peptide chain polymerization, the single largest energy expenditure for growing cells. This leaves the bulk of energy generated by fermentation and respiration (consuming 1/3 of the glucose intake) during aerobic growth unaccounted for. FDM also enabled the quantification of protein allocated towards each metabolic function, unveiling linear enzyme-flux relations for biosynthesis. These results led us to derive a function-based coarse-grained model to capture global protein allocation and overflow metabolism, without relying on curated pathway annotation or clustering of gene expression data.

systems biology↗

bioRxiv2022

Multi-modal brain magnetic resonance imaging database covering marmosets with a wide age range

Magnetic resonance imaging (MRI) is a noninvasive neuroimaging method beneficial for the identification of normal developmental and aging processes and data sharing. Marmosets have a relatively shorter life expectancy (approximately 10 years) than other primates, including, humans because they grow and age faster. Hence, the common marmoset model is effective in aging research. The current study investigated the aging process of the marmoset brain and provided an open MRI database on marmosets with a wide age range. The Brain/MINDS Marmoset Brain MRI Dataset contains brain MRI information on 216 marmosets aged between 1 and 10 years. During its release date, it is the largest public dataset worldwide. Further, it comprises multi contrast MRI images. In addition, 91 of 216 animals have corresponding ex vivo high-resolution MRI datasets. Our MRI database, which is available at the Brain/MINDS Data portal might help understand the effects of different factors, such as age, sex, body size, and fixation, on the brain. Moreover, it can contribute to and accelerate brain science studies worldwide.

neuroscience↗

bioRxiv2022

A Metabolic Sum Rule Dictates Bacterial Response to Short-Chain Fatty Acid Stress

Short-chain fatty acids (SCFAs) such as acetate accumulate in fermentative environments, inhibiting many types of bacteria. While it is known that cells accumulate SCFAs to high concentrations internally, the cause of SCFA toxicity is not understood. By forcing Escherichia coli cells to accumulate a variety of "useless metabolites", we establish via extensive omic analysis a metabolic sum rule, by which the accumulation of exogenous metabolites such as acetate forces the depletion of endogenous metabolites. The latter leads to bottlenecks in biosynthesis, manifested as a simple linear relation between useless metabolite accumulation and growth reduction. Guided by quantitative models, we show that acetate-stressed cells optimize growth by partially acidifying their own cytoplasm, which reduces acetate accumulation, restoring the endogenous metabolites as allowed by the sum rule.

microbiology↗

bioRxiv2022

Strategy for selective acquisition of transgenic marmosets using the piggyBac transposon system

Transgenic (Tg) nonhuman primates (NHPs) are excellent potential models due to their physiological similarities with humans. In NHPs, the lentiviral vector is the only available transgenesis method, but transgene sizes are limited. The piggyBac (PB) transposon system is a DNA element sandwiched between two terminal inverted repeats (TRs) that allows transposases to introduce larger genes than lentiviral vectors. However, embryos with integrated transgenes are difficult to distinguish using this system. To address this issue, we placed the Enhanced Green Fluorescent Protein (EGFP) gene inside the TRs and the humanized kusabira orange 1 (hKO1) fluorescent protein gene with the proteolysis-promoting sequence d2PEST outside the TRs to create an indicator for gene integration using fluorescent protein expression. In HEK293T cells, hKO1 and EGFP co-expression after transfection gradually changed to decay of hKO1 expression and enhancement of EGFP expression by transposase within five days. In mouse and marmoset embryos, hKO1 expression was attenuated, while only EGFP expression was observed at the blastocyst stage. All offspring obtained through embryo transfer of EGFP-expressing embryos were Tg; thus, we established a new PB system capable of determining Tg embryos and successfully produced Tg marmosets, for the first time, in NHPs.

molecular biology↗

bioRxiv2022

The Brain/MINDS Marmoset Connectivity Atlas: Exploring bidirectional tracing and tractography in the same stereotaxic space

We report on the implementation and features of the Brain/MINDS Marmoset Connectivity Atlas, BMCA, a new resource that provides access to anterograde neuronal tracer data in the prefrontal cortex of a marmoset brain. Neuronal tracers combined with fluorescence microscopy are a key technology for the systematic mapping of structural brain connectivity. We selected the prefrontal cortex for mapping due to its important role in higher brain functions. This work introduces the BMCA standard image preprocessing pipeline and tools for exploring and reviewing the data. We developed the BMCA-Explorer, which is an online image viewer designed for data exploration. Unlike other existing image explorers, it visualizes the data of different individuals in a common reference space at an unprecedented high resolution, facilitating comparative studies. To foster the integration with other marmoset brain image databases and cross-species comparisons, we added fiber tractography data from diffusion MRI, retrograde neural tracer data from the Marmoset Brain Connectivity Atlas project, and tools to map image data between marmoset and the human brain image space. This version of BMCA allows direct comparison between the results of 52 anterograde and 164 retrograde tracer injections in the cortex of the marmoset.

neuroscience↗

bioRxiv2022

LRRK2 Phosphorylates Neuronal Elav RNA-Binding Proteins to RegulatePhenotypes Relevant to Parkinson's Disease

Parkinsons disease (PD) is characterized by accumulation of -synuclein and the loss of dopaminergic neurons. Mutations which cause an increase in the kinase activity of Leucine-Rich-Repeat Kinase-2 (LRRK2) are a major inherited cause of PD. Research continues to determine which targets LRRK2 phosphorylates to cause disease. Polymorphisms in the locus of ELAVL4, an RNA-binding protein are a risk-factor for Parkinsons disease and an ELAV family member was identified in Drosophila as required for pathology instigated by human mutant LRRK2. We discovered that three neuronal ELAVs including ELAVL4 (also known as HuD) are phosphorylated by LRRK2. This controls binding of neuronal ELAVs to mRNA and their post- transcriptional regulation of mRNA abundance and splicing in neuronal cell lines and the mouse midbrain. LRRK2 G2019S functionally inhibits neuronal ELAVs effects on mRNA abundance, while enhancing their effects on mRNA splicing. The combination of LRRK2 G2019S and ELAVL4-/- causes accumulation of LRRK2 and -synuclein, loss of dopaminergic neurons and motor deficits. Targets of neuronal ELAVs are also selectively misregulated in cells and tissues of PD patients. Together, this suggests that misregulation of neuronal ELAVs, triggered by LRRK2 mutations may contribute to the characteristic pathology of Parkinsons disease. Brief SummaryLRRK2, a kinase linked to Parkinsons disease, phosphorylates the neuronal ELAV RNA-binding proteins to aggravate key hallmarks of Parkinsons disease including accumulation of -synuclein and motor deficits in mice.

cell biology↗

bioRxiv2022

A Genome-Scale Metabolic Model of Marine Heterotroph Vibrio splendidus sp. 1A01

While the Vibrio splendidus species is best known as an opportunistic pathogen in oysters, the Vibrio splendidus sp. 1A01 strain was first identified as an early colonizer of synthetic chitin particles incubated in seawater. To gain a better understanding of its metabolism, a genome-scale metabolic model (GSMM) of V. splendidus sp. 1A01 was reconstructed. GSMMs enable us to simulate all metabolic reactions in a bacterial cell using Flux Balance Analysis. A draft model was built using an automated pipeline from BioCyc. Manual curation was then performed based on experimental data, in part by gap-filling metabolic pathways and tailoring the models biomass reaction to V. splendidus sp. 1A01. The challenges of building a metabolic model for a marine microorganism like V. splendidus sp. 1A01 are described.

microbiology↗

bioRxiv2022

Connectional architecture of the prefrontal cortex in the marmoset brain

Prefrontal cortex (PFC) has dramatically expanded in primates, but its organization and interactions with other brain regions are only partially understood. We performed high-resolution connectomic mapping of marmoset PFC and found two contrasting corticocortical and corticostriatal projection patterns: "patchy" projections that formed many columns of submillimeter scale in nearby and distant regions and "diffuse" projections that spread widely across the cortex and striatum. Parcellation-free analyses revealed representations of PFC gradients in these projections local and global distribution patterns. We also demonstrated column-scale precision of reciprocal cortico-cortical connectivity, suggesting that PFC contains a mosaic of discrete columns. Diffuse projections showed considerable diversity in the laminar patterns of axonal spread. In mice, columnar projections were much less conspicuous, underscoring the importance of the primate model. Altogether, these fine-grained analyses reveal important principles of local and long-distance PFC circuits in marmosets and provide insights into the functional organization of the primate brain.

neuroscience↗

bioRxiv2021

biPACT: a method for three-dimensional visualization of mouse spinal cord circuits of long segments with high resolution

BackgroundThe spatial complexity of neuronal circuits in the central nervous system is a hurdle in understanding and treating brain and spinal cord injuries. Although several methods have recently been developed to render the spinal cord transparent and label specific neural circuits, three-dimensional visualization of long segments of spinal cord with high resolution remains challenging. New MethodWe present a method that combines tissue staining of neuronal tracts traced with biotinylated dextran amine (BDA) and a modified passive clarity clearing protocol to describe individual fibers in long segments of mouse spinal cord. ResultsCorticospinal tract was traced with BDA with a mouse model of thoracic spinal cord injury. The spinal cord was stained and cleared in two weeks with four solutions: staining solution, hydrogel solution, clearing solution, and observation solution. The samples were observed with a light-sheet microscope, and three-dimensional reconstruction was performed with ImageJ software. High resolution-images comparable with tissue sections were obtained continuously and circumferentially. By tiling, it was possible to obtain high-resolution images of long segments of the spinal cord. The tissue could be easily re-stained in case of fading, Comparison with Existing MethodsThe present method does not require special equipment, can label specific circuits without genetic technology, and re-staining rounds can be easily implemented. It enables to visualize individual neural fiber of specific neural circuit in long spinal cord segments. ConclusionsBy using simple neural staining, and clearing methods, it was possible to acquire a wide range of high-resolution three-dimensional images of the spinal cord. HighlightsO_LINo special devices or genetic tracers are required for a new clearing method C_LIO_LINeuronal fibers are individually depicted in long segments of mouse spinal cord. C_LIO_LIRe-staining of neuronal fiber is possible. C_LIO_LIStereotaxic observation is achieved by 3-D reconstruction with open-source software. C_LI

neuroscience↗

bioRxiv2021

Simple and efficient differentiation of human iPSCs into contractible skeletal muscles for muscular disease modeling

Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patients pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) can potentially recapitulate disease pathology more accurately than existing disease models when differentiated into affected cell types. Thus, successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection procedures, and clonal variations must be overcome. Moreover, their functionality to exhibit muscular contraction has never been reported. Here, we demonstrated that bulk MYOD1- hiPSCs established with puromycin selection, but not with G418 selection, showed high differentiation efficiency, generating more than 80% Myogenin (MyoG)+ and Myosin heavy chain (MHC)+ muscle cells within seven days. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties compared with those of clonally established MYOD1- hiPSCs, suggesting that the bulk method may minimize the effects of clonal variations. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical pulse stimulation, indicating their functionality. Together, the findings indicate that our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and facilitates the generation of muscular disease models. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/468571v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@12c091eorg.highwire.dtl.DTLVardef@ad1ccorg.highwire.dtl.DTLVardef@53ba90org.highwire.dtl.DTLVardef@fd65f5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Novel System to Monitor In Vivo Neural Graft Activity After Spinal Cord Injury

Expectations for neural stem/progenitor cell (NS/PC) transplantation as a treatment for spinal cord injury (SCI) are increasing. However, whether and how grafted cells are incorporated into the host neural circuit and contribute to motor function recovery remain unknown. The aim of this project was to establish a novel non-invasive in vivo imaging system to visualize the activity of neural grafts by which we can simultaneously demonstrate the circuit-level integration between the graft and host, and the contribution of graft neuronal activity to host behaviour. We introduced Akaluc, a newly engineered luciferase, under control of a potent neuronal activity-dependent synthetic promoter, E-SARE, into NS/PCs and engrafted the cells into SCI model mice. Through the use of this system, we reveal that the activity of grafted cells was integrated with host behaviour and driven by host neural circuit inputs. This non-invasive system is expected to help elucidate the therapeutic mechanism of cell transplantation treatment for SCI and determine better therapy techniques that maximize the function of cells in the host circuit.

neuroscience↗

bioRxiv2021

A role of cryptochrome for magnetic field-dependent improvement of sleep quality, lifespan and motor function in Drosophila

Understanding the molecular genetic basis of animal magnet reception has been one of the big challenges in biology. Recently it was discovered that the magnetic sense of Drosophila melanogaster is mediated by the ultraviolet (UV)-A/blue light photoreceptor cryptochrome (Cry). Here, using the fruit fly as a magnet receptive model organism, we show that exposure to a specific AC magnetic field during night time affected the health of the fly. AC magnetic field exposure showed lifespan extension under starvation, sleep improvement and prevention of decreased motor function. In contrast, all the health improvement effect was not observed in cryptochrome mutant flies (cryb). We showed that AC magnetic field exposure prevented motor dysfunction in Gauchers disease model Drosophila. The data suggests that magnetic field-dependent improvement of sleep quality, lifespan and motor function is mediated through a cry-dependent pathway in animals.

animal behavior and cognition↗