Biology subjects

Yoshimura, H.

Publications and source records attributed to Yoshimura, H..

7 recordsLinked to original sources

bioRxiv2025

Oligo DNA-based quantum dot (QD) single-particle tracking for multicolor single-molecule imaging

Quantum dot single-particle tracking (QD-SPT), a powerful tool for analyzing membrane domains that are critical to various cellular processes, is widely used in membrane molecular dynamics research. QDs, which possess both a broad excitation range and a narrow emission bandwidth, are inherently suited for multicolor imaging at various wavelengths. However, applying multicolor QD-SPT with multiple biomolecular targets has been challenging due to the limited methods for specifically conjugating QDs to biomolecules. Here, we propose a DNA hybridization-based QD labeling method that generates several specific interactions based on sequence. QD fused with 20-mer oligo DNA specifically labeled membrane lipid 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) covalently bound to complementary oligo DNA, forming a stable label that is suitable for SPT. The combination of polyA-polyT sequence as the linker oligo caused more QDs to fuse to DPPE compared with a random sequence linker. Oligo DNA-based QD-SPT accurately reflected the diffusion dynamics of DPPE measured using the single-fluorescence tracking technique and was compatible with conventional QD-SPT utilizing secondary antibody Fab. Using different pairs of oligo DNA sequences, we successfully achieved multicolor QD-SPT that distinguishes the lateral diffusion of DPPE and a membrane protein, GABAA receptor (GABAAR), within the same cell. The oligo DNA-based QD labeling method developed in this study is anticipated to substantially advance simultaneous multicolor QD-SPT of different living cell membrane molecules specific to DNA sequences.

biophysics↗

bioRxiv2025

A subtype of ultrasonic vocalizations during highly palatable food consumption in rats identified by machine learning-assisted classification

Identifying behavioral and physiological responses to rewarding stimuli is essential for understanding positive emotional states in animals and for investigating their neural basis. Ultrasonic vocalizations (USVs) in rats are modulated by various behavioral contexts and are thought to represent affective-like states. However, their diversity during palatable food consumption remains underexplored. In this study, we investigated acoustic features of USVs in male rats consuming chocolate, a highly palatable food. Using a machine-learning-assisted logistic regression model trained on spectrogram features, we identified a distinct USV subtype--the 40-kHz inverted-U type--that was selectively emitted during chocolate consumption. The emission of this subtype was tightly time-locked to chocolate feeding behavior. Systemic administration of naloxone, an opioid receptor antagonist, significantly reduced the emission of the 40-kHz inverted-U USVs during chocolate intake. These findings suggest that these USVs may reflect internal states associated with palatable feeding and are under modulation by the endogenous opioid system. Moreover, our data demonstrate the utility of machine learning for high-throughput, objective classification of USV subtypes. This framework provides a promising approach for decoding emotion-related vocal expressions in rats and highlights the potential of specific USVs as behavioral readouts for studying the neurobiology of positive emotions. Significance StatementUltrasonic vocalizations (USVs) are high-frequency calls produced by rats known to correlated with emotional and motivational states, yet their diversity and functional relevance remain underexplored. In this study, we identified a novel 40-kHz inverted-U USV subtype associated with chocolate consumption. These calls were closely tied to feeding behavior and were selectively suppressed by an opioid receptor antagonist, suggesting modulation by the brains reward system. Using machine learning-assisted classification, we established a robust method to detect these vocalizations. Our findings highlight the potential of specific USVs as non-invasive, real-time behavioral indicators of internal states, offering new tools for studying affect, reward, and communication in animal models.

neuroscience↗

bioRxiv2024

Spiking network model of the cerebellum as a reinforcement learning machine

The cerebellum has been considered to perform error-based supervised learning via long-term depression (LTD) at synapses between parallel fibers and Purkinje cells (PCs). Since the discovery of multiple synaptic plasticity other than LTD, recent studies have suggested that synergistic plasticity mechanisms could enhance the learning capability of the cerebellum. Indeed, we have proposed a concept of cerebellar learning as a reinforcement learning (RL) machine. However, there is still a gap between the conceptual algorithm and its detailed implementation. To close this gap, in this research, we implemented a cerebellar spiking network as an RL model in continuous time and space, based on known anatomical properties of the cerebellum. We confirmed that our model successfully learned a state value and solved the mountain car task, a simple RL benchmark. Furthermore, our model demonstrated the ability to solve the delay eyeblink conditioning task using biologically plausible internal dynamics. Our research provides a solid foundation for cerebellar RL theory that challenges the classical view of the cerebellum as primarily a supervised learning machine.

neuroscience↗

bioRxiv2024

Discovery and characterization of stereodefined PMO-gapmers targeting tau

Antisense oligonucleotides (ASOs) are an important class of therapeutics to treat genetic diseases, and expansion of this modality to neurodegenerative disorders has been an active area of research. To realize chronic administration of ASO therapeutics to treat neurogenerative diseases, new chemical modifications improving activity and safety profile are still needed. Furthermore, it is highly desirable to develop a single stereopure ASO with defined activity and safety profile to avoid any efficacy and safety concerns due to the batch-to-batch variation in the composition of diastereomers. Herein, a stereopure PMO-gapmer was developed as a new construct to improve safety and stability by installing charge-neutral PMOs at the wing region and by fully controlling phosphorus stereochemistries. The developed stereopure PMO-gapmer construct was applied to the discovery of ASO candidates for the reduction of microtubule-associated protein tau (MAPT, tau). Sequence screening targeting MAPT followed by screening of optimal phosphorus stereochemistry identified stereopure development candidates. While evaluating the stereopure PMO-gapmers, we observed a dramatic difference in safety profile among stereoisomers in which only one phosphorus stereochemistry differs. These results further highlight the benefits of developing stereopure ASOs as safe and well-characterized candidates for clinical studies.

neuroscience↗

bioRxiv2024

Induction of the aggresome and insoluble tau aggregation using an optogenetic tool

Tauopathy is a spectrum of diseases characterized by fibrillary tau aggregate formation in neurons and glial cells in the brain. Tau aggregation originates in the brainstem and entorhinal cortex and then spreads throughout the brain in Alzheimers disease (AD), which is the most prevalent type of tauopathy. Understanding the mechanism by which locally developed tau pathology propagates throughout the brain is crucial for comprehending AD pathogenesis. Therefore, a novel model of tau pathology that artificially induces tau aggregation in targeted cells at specific times is essential. This study describes a novel optogenetic module, OptoTau, which is a human tau with the P301L mutation fused with a photosensitive protein CRY2olig, inducing various forms of tau according to the temporal pattern of blue light illumination pattern. Continuous blue light illumination for 12 h to Neuro2a cells that stably express OptoTau (OptoTauKI cells) formed clusters along microtubules, many of which eventually accumulated in aggresomes. Conversely, methanol-resistant tau aggregation was formed when alternating light exposure and darkness in 30-min cycles for 8 sets per day were repeated over 8 days. Methanol-resistant tau was induced more rapidly by repeating 5-min illumination followed by 25-min darkness over 24 h. These results indicate that OptoTau induced various tau aggregation stages based on the temporal pattern of blue light exposure. Thus, this technique exhibits potential as a novel approach to developing specific tau aggregation in targeted cells at desired time points. SignificanceThis study developed an approach to manipulate tau aggregation in a blue light-dependent manner using cells that stably express OptoTau, which is an optogenetic tool based on the CRY2olig module. Tau accumulation in aggresomes or stable tau aggregation were selectively induced by blue light illumination conditions. These results are crucial as they provide a new technological basis for establishing a singular point of tau aggregation in specific targeted cells at a particular time.

biophysics↗

bioRxiv2023

Intracellular Tau Fragment Droplets Serve as Seeds for Tau Fibrils

Intracellular tau aggregation requires a local protein concentration increase, referred to as "droplets". However, the cellular mechanism for droplet formation is poorly understood. Here, we expressed OptoTau, a P301L mutant tau fused with CRY2olig, a light-sensitive protein that can form homo-oligomers. Under blue light exposure, OptoTau increased tau phosphorylation and was sequestered in aggresomes. Suppressing aggresome formation by nocodazole formed tau granular clusters in the cytoplasm. The granular clusters disappeared by discontinuing blue light exposure or 1,6-hexanediol treatment suggesting that intracellular tau droplet formation requires microtubule collapse. Expressing OptoTau-{Delta}N, a species of N-terminal cleaved tau observed in the Alzheimers disease brain, formed 1,6-hexanediol and detergent-resistant tau clusters in the cytoplasm with blue light stimulation. This intracellular stable tau clusters acted as a seed for tau fibrils in vitro. These results suggest that tau droplet formation and N-terminal cleavage are necessary for neurofibrillary tangles formation in neurodegenerative diseases.

cell biology↗

bioRxiv2021

Cellular adhesion is a controlling factor in neutrophil extracellular trap formation induced by antineutrophil cytoplasmic antibodies

Anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) is a life-threatening condition characterized by improper activation of neutrophils and release of neutrophil extracellular traps (NETs) in small vessels. This study aimed to explain the role of NETs in AAV pathogenesis by investigating a link between neutrophil adhesion and NET release. We leveraged an imaging flow cytometry-based assay and three-dimensional culture to demonstrate that neutrophil adhesion is essential for ANCA induced NET formation. We confirmed this requirement for cell adhesion using standard microscopy on ultra-low attachment hydrogel surfaces and demonstrate that this depends on the focal adhesion kinase pathway as determined using inhibitors for multiple targets in this process. ANCA increased expression of {beta}2 integrins on neutrophils, and we confirmed that these integrins were required for NET formation using blocking antibodies. Finally, inhibitors for oxidative burst prevented NET formation, and this oxidative burst was mediated by the focal adhesion pathway. Overall, our findings reveal a central role for neutrophil attachment in NET formation in response to ANCA, helping to explain the restricted localization pattern of vessel damage, and suggesting that targeting neutrophil adhesion factors may be beneficial in preventing pathological damage from NETs during AAV.

immunology↗