Biology subjects

Kiyonaka, S.

Publications and source records attributed to Kiyonaka, S..

8 recordsLinked to original sources

bioRxiv2026

Off-target-free chemogenetic platform that decodes physiological roles of target GPCRs

G-protein-coupled receptors (GPCRs) are essential mediators of cellular processes, and their dysfunction is implicated in various diseases. Although pharmacological approaches are powerful for elucidating GPCR functions, careful consideration of potential off-target effects remains crucial. Here, we present an off-target-free chemogenetic platform to dissect the physiological roles of target GPCRs. As a proof-of-concept, we focused on the adenosine A2A receptor (A2AR), a prototypical GPCR involved in neurodegenerative diseases. Leveraging the ligand recognition mechanisms of A2AR, we designed a prominent chemogenetic pair comprising a clinical A2AR antagonist and an engineered A2AR mutant that is insensitive to the antagonist but retains responsiveness to adenosine. Using this pair, we uncovered the critical role of A2AR in neurite outgrowth under hypoxic conditions in neuron-like cells, while avoiding potential off-target effects. Furthermore, we demonstrate the generalizability of this chemogenetic approach to other class A GPCRs, offering a versatile platform for receptor-specific dissection of GPCR signaling.

biochemistry↗

bioRxiv2026

Native receptor-targeted chemogenetics enables cell-type-specific inhibition of endogenous receptors in freely moving mice

Understanding brain function requires tools that allow precise manipulation of receptor signaling in defined cell types within intact neural circuits. Optogenetics and conventional chemogenetic approaches primarily enable cell-type-specific control of neuronal excitability using engineered receptors or ion channels. However, direct and reversible inhibition of endogenous neurotransmitter receptors in defined cell types has remained technically inaccessible. Here, we introduce native receptor-targeted chemogenetics (NARCH), a chemogenetic strategy that integrates structure-guided receptor engineering with allosteric ligand design to achieve reversible and temporally precise inhibition of endogenous receptor signaling with cell-type specificity in vivo. By applying NARCH to metabotropic glutamate receptor 1 (mGlu1), we demonstrate that mGlu1 signaling in cerebellar Purkinje cells is required for stabilization of motor learning across training sessions in freely moving mice. NARCH thus establishes a receptor-level chemogenetic framework for causal analysis of neural circuits and behavior.

neuroscience↗

bioRxiv2024

A fluorophore-ligand conjugate for the rapid and reversible staining of native AMPA receptors in living neurons

The subcellular localizations of neurotransmitter receptors are strictly regulated in neurons. Changes in the trafficking of -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-type glutamate receptors (AMPARs) play an essential role in synaptic plasticity, which is the cellular basis of learning and memory. To explore receptor trafficking, genetically encoded approaches (e.g., the fusion of fluorescent proteins to receptors) are often used. However, concerns remain that genetic approaches cannot fully reproduce the receptor function that is inherent to neurons. Herein, we report a chemical probe, PFQX1(AF488), for the visualization of cell-surface AMPARs without any genetic manipulation to neurons. The rapid and reversible staining features of this probe allowed the visualization of AMPAR accumulation in dendritic spines during synaptic plasticity in living hippocampal neurons. Moreover, the mechanisms of this synaptic accumulation, for which genetically encoded approaches have given controversial results, were revealed by integrating two chemical methods: PFQX1(AF488) and covalent chemical labeling.

neuroscience↗

bioRxiv2024

Nanoscale organization is changed in native, surface AMPARs by mouse brain region and tauopathy

The distribution of synaptic and extra-synaptic AMPA receptors (AMPARs) on neuronal plasma membranes is correlated with learning and memory. Although AMPAR organization has been extensively studied in neuronal cultures, its native cell-surface distribution in intact adult brain tissue across distinct brain regions and in neurodegenerative pathology remains poorly understood. Here, we combine a selective small-molecule labeling strategy with two-color 3D super-resolution dSTORM imaging to map native surface AMPAR organization at the nanoscale in 30 micron thick mouse brain slices. We find that wild-type mice exhibit marked regional differences in AMPAR organization, with the CA1 hippocampus containing a substantially larger extrasynaptic AMPAR pool than the nearby motor and somatosensory cortex. In the PS19 tauopathy mouse model, at an age preceding overt neurodegeneration, AMPAR organization is selectively disrupted in the hippocampus but largely preserved in the cortex. Specifically, we observe depletion of the extrasynaptic receptor pool together with reduced synaptic nanodomain organization, revealing early molecular-scale synaptic remodeling associated with tau pathology. These findings provide direct structural insight into region- and disease-dependent AMPAR organization in intact adult brain tissue and establish a broadly applicable framework for nanoscale investigation of synaptic receptor architecture in health and neurological disease.

neuroscience↗

bioRxiv2023

Bioorthogonal chemical labelling of endogenous neurotransmitter receptors in living mouse brains

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent chemical labelling of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labelling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labelling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-colour receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

biochemistry↗

bioRxiv2021

Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution dynamics

Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/extrusion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein we report a new method termed "Fixation-driven chemical crosslinking of exogenous ligands (FixEL)" which traps and images exogenously administered molecules-of-interest (MOI) in complex tissues. This method relies on proteins-MOI interactions, and chemical crosslinking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules and their dynamics, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOI such as PET tracer derivatives or drug-like small molecules. Clear imaging of a nanobody distributed in the whole brain was also achieved with high spatial resolution using 2D/3D mode.

neuroscience↗

bioRxiv2021

Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue

Direct activation of cell-surface receptors is highly desirable for elucidating the physiological roles of receptors. However, subtype-selective ligands are very limited because of the high homology among receptor subtypes. A potential approach for selective activation of a receptor subtype is chemogenetics, in which both point mutagenesis of the receptors and designed ligands are used. However, ligand-binding properties are affected in most current methods. Here, we developed a chemogenetic method for direct activation of metabotropic glutamate receptor 1 (mGlu1), which plays essential roles in cerebellar functions in the brain. Our screening identified a mGlu1 mutant, mGlu1(N264H), that was directly activated by palladium complexes. Notably, a palladium complex showing low cytotoxicity successfully activated mGlu1 in mGlu1(N264H) knock-in mice, revealing that activation of endogenous mGlu1 is sufficient to evoke the critical cellular mechanism of synaptic plasticity, a basis of motor learning in the cerebellum.

neuroscience↗

bioRxiv2020

Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking

The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for AMPA-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a new method for the rapid and selective labeling of chemical probes to AMPARs by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological conditions. This method allowed us to quantify AMPAR distribution and trafficking, which revealed some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method was also successfully expanded to selectively label NMDA-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons.

biochemistry↗