Biology subjects

Sakimura, K.

Publications and source records attributed to Sakimura, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Asymmetric turnover dynamics of Nav1.6 voltage-gated sodium channels at the axon initial segment

Nav1.6 voltage-gated sodium channels are critical for shaping action potentials, and their precise localization at the axon initial segment (AIS) is essential for neuronal excitability. However, the turnover mechanisms that maintain this spatial pattern remain unclear. Here, we present a genetically engineered mouse in which endogenous Nav1.6 carries a Cre-switchable fluorescent tag, enabling us to simultaneously trace the turnover of pre-existing and newly synthesized Nav1.6 without perturbing AIS structure. Using this system, we determine the physiological lifetimes of Nav1.6 at the AIS in vivo and in vitro, and further uncover the spatially asymmetric turnover dynamics, with the proximal and distal AIS organizing a source-to-sink gradient. Based on the quantified parameters, computational modeling clarifies that this asymmetry promotes efficient clearance of older molecules, thereby supporting AIS quality control. Collectively, these findings provide a technical and conceptual foundation for understanding how molecular turnover contributes to AIS homeostasis.

neuroscience↗

bioRxiv2026

Stressor-specific dynamic patterns of noradrenaline release in the paraventricular nucleus of the hypothalamus in freely moving mice

An organism is constantly challenged with various stressors. These stress signals ultimately converge on the hypothalamic paraventricular nucleus (PVN), where they are integrated by corticotropin-releasing hormone (CRH)-producing neurons that are primarily involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Noradrenaline (NA), among others, is recognized as the major transmitter that regulates the PVN-CRH neurons, and thereby is involved in the regulation of the HPA axis as well as the autonomic outflow. Previous studies have demonstrated that stress increases NA release within the PVN1 and that NA activates CRH neurons2. However, NA release patterns from the axon terminals in the PVN upon stress exposure have not yet been studied, because continuous monitoring of transmitter release became possible only recently. In the present study, we aimed to monitor stressor-dependent NA release patterns in the PVN. To continuously monitor NA release in freely moving mice, a fluorescent NA sensor (GRABNA)3,4 was expressed in the PVN, and the emitted signals were recorded via fibre photometry. Following stress exposure, NA release took place over distinct timescales ranging from seconds to hours. For example, a pulsatile NA release was observed in seconds in response to an acute nociceptive stressor. In contrast, sustained physical stressors, such as tail suspension and restraint, induced much more prolonged NA release that persists throughout the stress exposure period over tens of minutes. Intraperitoneal administration of lipopolysaccharide produced a gradual increase in NA release that persisted for several hours. These findings demonstrate that NA is released into the PVN in a stress-specific manner. In addition, elevated NA release was associated with increased behavioural activity, characteristic of each stressor. Together, these findings provide a framework for understanding how the temporal dynamics of NA release in the PVN encode diverse stress signals, and regulate neuroendocrine outputs.

neuroscience↗

bioRxiv2026

High basal autophagic activity in the brain revealed by systemic quantitative analysis using GFP-LC3-RFP mice

Autophagy is a fundamental intracellular degradation pathway with vital physiological functions. Although it is well known that autophagy is activated during starvation, the extent of basal autophagy remains unclear owing to challenges in measuring autophagic flux in vivo. In this study, we developed autophagy reporter (GFP-LC3-RFP) mice and quantified basal autophagic flux across tissues by comparing normal and autophagy-deficient conditions. Comparative analyses revealed uniformly low basal autophagic flux during embryogenesis, but significant tissue-specific variation in adult mice. In contrast to previous assumptions that basal autophagy in the brain is low, the brain, along with the liver and kidney, exhibited higher basal autophagic flux than the heart, skeletal muscle, and intestine. These data serve as foundational information on basal autophagic flux in mammals and provide a plausible explanation for the severe neurological phenotypes linked to autophagy gene mutations in mice and humans.

cell biology↗

bioRxiv2025

Differential Effects of Inducible Cerebellar Granule Cell and Purkinje Cell Ablation on Motor Coordination and Motor Learning in Adult Mice

The cerebellum regulates motor coordination and motor learning through highly organized circuits composed mainly of granule cells (GCs) and Purkinje cells (PCs). To investigate their distinct roles, we generated two lines of inducible transgenic mice in which either GCs or PCs could be selectively ablated in adulthood by administration of the progesterone receptor antagonist RU-486. This system combined a Cre recombinase-progesterone receptor fusion, in which Cre activity is induced in an RU-486-dependent manner, with a Cre-dependent diphtheria toxin A expression to achieve cell-type-specific ablation. High-dose RU-486 induced nearly complete loss of either GCs or PCs and resulted in severe ataxia. When partial ablation was induced by low-dose RU-486, different phenotypes emerged. Mice retaining about 20% of PCs were still able to improve motor coordination in the rotarod test and maintained performance in the balance beam test comparable to that of controls. In contrast, mice with about 30% of GCs remaining showed marked deficits, failing to improve across rotarod trials and exhibiting reduced latency to fall in the balance beam test. These results suggest that while both GCs and PCs are indispensable for motor coordination, a sufficient number of GCs is required for both motor coordination and motor learning. This inducible ablation model highlights the differential contributions of cerebellar neurons and provides a valuable tool to dissect circuit-specific functions in the adult brain.

neuroscience↗

bioRxiv2025

Visualization of stochastic expression of clustered protocadherin β isoforms in vivo

The stochastic expression of clustered protocadherin (cPcdh) establishes a single-cell identity fundamental to cellular self/non-self-discrimination. However, it has been challenging to reveal the spatiotemporal patterning of the stochastic cPcdh expression in vivo. We developed XFP (tdTomato or GFP) knock-in mice using a new strategy to enhance XFP expression, which allows us to visualize cPcdh{beta}3 or 19-positive cells throughout the brain. These mouse lines demonstrate the cell-type selectivity, spatial biases, inter-individual differences, left-right asymmetry, developmental regulation, alteration in pathological or aging brain, and monoallelic expression of stochastic cPcdh{beta} expression in vivo. Our findings further demonstrate that the cPcdh{beta}3 expression undergoes significant changes in the mature brain over time. These results demonstrate the potential of these reporter mice to advance our understanding of cPcdh cellular barcode in vivo.\n\nOne Sentence SummaryNovel established cPcdh{beta} reporter mouse lines reveal stochastic expression of cPcdh{beta} isoforms in vivo.

neuroscience↗

bioRxiv2025

Neuropeptide CRH prevents premature differentiation of OPCs following CNS injury and in early postnatal development

The role of neuropeptides and their receptors in oligodendrocyte progenitor cells (OPCs) has largely been overlooked so far. Here, we describe a new subpopulation of corticotropin-releasing hormone (CRH)-expressing OPCs that aggregate around acute brain injuries and exhibit an elevated capacity to differentiate into myelinating oligodendrocytes (OLs). We found that CRH expression in OPCs is rapidly induced de novo as a transient response within the first 72 hours after injury. As target cells, we identified CRH receptor type 1 (CRHR1)-expressing OPCs which show a decreased differentiation velocity. We demonstrate that CRH/CRHR1 system inactivation increases the speed of OL generation compromising the long-term survival of OLs after acute injury. Furthermore, we prove that a CRH/CRHR1 system deficiency under non-injury conditions leads to increased early postnatal oligodendrogenesis and alterations in adult myelination. Altogether, we show that OPC-derived CRH not only actively influences the injury environment through the interaction with CRHR1-expressing OPCs, but also identify the G-protein coupled receptor CRHR1 as a critical modulator of oligodendrogenesis at early postnatal stages with lasting effects on adult myelination.

neuroscience↗

bioRxiv2024

AUTS2, a causative gene for microcephaly, regulates division of intermediate progenitor cells and production of upper-layer neurons

AUTS2 mutations often exhibit neurodevelopmental disorders and microcephaly. However, how AUTS2 regulates neuron production and affects brain size remains unclear. Here, we show that AUTS2 cooperates with the Polycomb complex PRC2 to regulate gene expression and cortical neurogenesis. Auts2 mutant mice exhibit reduced division of intermediate progenitor cells (IPCs), leading to decreased neurons and thickness in the upper-layer cortex. Expression of Robo1 is increased in the mutants, which in turn suppresses IPC division. Transcriptome and chromatin profiling experiments show that, in IPCs, AUTS2 primarily represses transcription of genes, including Robo1. Promoter region of AUTS2 target genes is enriched with H3K27me3, a repressive histone modification, but its levels are reduced in Auts2 mutants. AUTS2 interacts with PRC2, and together, they promote IPC division. These suggest that AUTS2 collaborates with PRC2 to repress gene transcription through H3K27 trimethylation, promoting neuron production. This sheds light on AUTS2 pathophysiological mechanisms in neurogenesis and microcephaly.

neuroscience↗

bioRxiv2023

NMDAR Phosphoproteome Controls Synaptic Growth and Learning

In the mammalian brain, NMDA receptors (NMDARs) activation triggers a calcium-dependent signal transduction cascade resulting in postsynaptic remodeling and behavioral learning. However, the phosphoprotein signal flow through this transduction network is poorly understood. Here, we show that NMDAR-dependent phosphorylation drives the assembly of protein signaling complexes that regulate synaptic morphology and behavior. We performed large-scale phosphoproteomic analyses of protein kinase target proteins in successive layers of the signaling network in mouse striatal/accumbal slices. NMDARs activation resulted in the phosphorylation of 194 proteins, including Rho GTPase regulators. CaMKII-mediated phosphorylation of ARHGEF2 increased its RhoGEF activity, thereby activating the RhoA-Rho-kinase pathway. Subsequent phosphoproteomics of Rho-kinase revealed 221 protein targets, including SHANK3. Experimental validation revealed a pathway from NMDAR-dependent calcium influx through CaMKII, ARHGEF2, Rho-kinase, and SHANK3 to coordinate assembly of an actin-tethered postsynaptic complex of SHANK3/NMDAR/PSD95/DLGAP3 for spine growth and aversive learning. These findings show that NMDARs initiate metabolic phosphorylation for learning.

neuroscience↗

bioRxiv2023

Brn3a controls the soma localization and axonal extension patterns of developing spinal dorsal horn neurons

The spinal dorsal horn comprises heterogeneous neuronal populations, that interconnect with one another to form neural circuits modulating various types of sensory information. Decades of evidence has revealed that transcription factors expressed in each neuronal progenitor subclass play pivotal roles in the cell fate specification of spinal dorsal horn neurons. However, the development of subtypes of these neurons is not fully understood in more detail as yet and warrants the investigation of additional transcription factors. In the present study, we examined the involvement of the POU domain-containing transcription factor Brn3a in the development of spinal dorsal horn neurons. Analyses of Brn3a expression in the developing spinal dorsal horn neurons in mice demonstrated that Brn3a was downregulated in majority of the Brn3a-lineage neurons during embryonic stages (Brn3a-transient neurons), whereas a limited population continued to express Brn3a after E18.5 (Brn3a-persistent neurons). Brn3a knockout disrupted the localization pattern of Brn3a-persistent neurons, indicating a critical role of this transcription factor in the development of these neurons. In contrast, Brn3a overexpression in Brn3a-transient neurons directed their localization in a manner similar to that in Brn3a-persistent neurons. Moreover, Brn3a-overexpressing neurons exhibited increased axonal extension to the ventral and ventrolateral funiculi, where the axonal tracts of Brn3a-persistent neurons reside. These results suggest that Brn3a controls the soma localization and axonal extension patterns of Brn3a-persistent spinal dorsal horn neurons.

neuroscience↗

bioRxiv2022

Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of phosphorylation remain unclear. Herein, we identify ULK1 as a kinase responsible for phosphorylation of p62. ULK1 co-localizes with p62 bodies, and directly interacts with p62. This phosphorylation allows KEAP1 to be retained within p62 bodies, activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351 corresponding to human Ser349 is replaced by Glu. These mice, but not phosphodefective p62S351A/S351A mice, exhibit NRF2 hyperactivation and growth retardation, the latter caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis. p62S351E/+ mice are a phenocopy of systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insight into the role of phase separation in this process.

cell biology↗

bioRxiv2021

Neurexins in serotonergic neurons regulate serotonin transmission and complex mouse behaviors

Extensive serotonin (5-HT) innervation throughout the brain corroborates 5-HTs modulatory role in numerous cognitive activities. Volume transmission is the major mode for 5-HT transmission but mechanisms underlying 5-HT signaling are still largely unknown. Abnormal brain 5-HT levels and function have been implicated in autism spectrum disorder (ASD). Neurexin (Nrxn) genes encode presynaptic cell adhesion molecules important for the regulation of synaptic neurotransmitter release, notably glutamatergic and GABAergic transmission. Mutations in Nrxn genes are associated with neurodevelopmental disorders including ASD. However, the role of Nrxn genes in the 5-HT system is poorly understood. Here, we generated a mouse model with all three Nrxn genes disrupted specifically in 5-HT neurons to study how Nrxns affect 5-HT transmission. Loss of Nrxns in 5-HT neurons reduced the number of serotonin neurons in the early postnatal stage, impaired 5-HT release, and decreased 5-HT release sites and serotonin transporter expression. Furthermore, 5-HT neuron-specific Nrxn knockout reduced sociability and increased depressive-like behavior. Our results highlight functional roles for Nrxns in 5-HT neurotransmission, 5-HT neuron survival, and the execution of complex behaviors.

neuroscience↗

bioRxiv2021

Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum

Experience-dependent plasticity is a key feature of brain synapses for which neuronal N-Methyl-D-Aspartate receptors (NMDARs) play a major role, from developmental circuit refinement to learning and memory. Astrocytes also express NMDARs although their exact function has remained controversial. Here we identify a circuit function for GluN2C NMDAR, a subtype highly expressed in astrocytes, in layer-specific tuning of synaptic strengths in mouse hippocampal CA1 pyramidal neurons. Interfering with astrocyte NMDAR or GluN2C NMDAR activity reduces the range of presynaptic strength distribution specifically in the stratum radiatum inputs without an appreciable change in the mean presynaptic strength. Mathematical modeling shows that narrowing of the width of presynaptic release probability distribution compromises the expression of long-term synaptic plasticity. Our findings suggest a novel feedback signaling system that uses astrocyte GluN2C NMDARs to adjust basal synaptic weight distribution of Schaffer collateral inputs, which in turn impacts computations performed by the CA1 pyramidal neuron.

neuroscience↗

bioRxiv2021

Activation of extrasynaptic kainate receptors drives hilar mossy cell activity

Mossy cells (MCs) of the dentate gyrus (DG) are key components of an excitatory associative circuit established by reciprocal connections with dentate granule cells (GCs). MCs are implicated in place field encoding, pattern separation and novelty detection, as well as in brain disorders such as temporal lobe epilepsy and depression. Despite their functional relevance, little is known about the determinants that control MC activity. Here, we examined whether MCs express functional kainate receptors (KARs), a subtype of glutamate receptors involved in neuronal development, synaptic transmission and epilepsy. Using mouse hippocampal slices, we found that bath application of submicromolar and micromolar concentrations of the KAR agonist kainic acid induced inward currents and robust MC firing. These effects were abolished in GluK2 KO mice, indicating the presence of functional GluK2-containing KARs in MCs. In contrast to CA3 pyramidal cells, which are structurally and functionally similar to MCs, and express synaptic KARs at mossy fiber (MF) inputs (i.e., GC axons), we found no evidence for KAR-mediated transmission at MF-MC synapses, indicating that most KARs at MCs are extrasynaptic. Immunofluorescence and immunoelectron microscopy analyses confirmed the extrasynaptic localization of GluK2-containing KARs in MCs. Finally, blocking glutamate transporters, a manipulation that increases extracellular levels of endogenous glutamate, was sufficient to induce KAR-mediated inward currents in MCs, suggesting that MC-KARs can be activated by increases in ambient glutamate. Our findings provide the first direct evidence of functional extrasynaptic KARs at a critical excitatory neuron of the hippocampus. Significance StatementHilar mossy cells (MCs) are an understudied population of hippocampal neurons that form an excitatory loop with dentate granule cells. MCs have been implicated in pattern separation, spatial navigation, and epilepsy. Despite their importance in hippocampal function and disease, little is known about how MC activity is recruited. Here, we show for the first time that MCs express extrasynaptic kainate receptors (KARs), a subtype of glutamate receptors critically involved in neuronal function and epilepsy. While we found no evidence for synaptic KARs in MCs, KAR activation induced strong action potential firing of MCs, raising the possibility that extracellular KARs regulate MC excitability in vivo and may also promote dentate gyrus hyperexcitability and epileptogenesis.

neuroscience↗

bioRxiv2020

Neurexins play a crucial role in cerebellar granule cell survival by organizing autocrine machinery for neurotrophins

Neurexins (NRXNs) are among the key presynaptic cell adhesion molecules that regulate synapse function and formation via trans-synaptic interaction with postsynaptic ligands. Here, we generated cerebellar granule cell (CGC)-specific Nrxn triple-knockout (TKO) mice to allow the deletion of all NRXNs. Unexpectedly, most CGCs died in these mice. The requirement of NRNXs for cell survival was reproduced in cultured CGCs. We showed that the axons of cultured Nrxn TKO CGCs that were not in contact with the postsynaptic structure had defects in the formation of presynaptic protein cluster and action potential-induced Ca2+ influx. Additionally, these cells were impaired in the secretion from axons of depolarization-induced fluorescence-tagged brain-derived neurotrophic factor (BDNF), and the cell-survival defect was rescued by the application of BDNF. Our results suggest that CGC survival is maintained by autocrine neurotrophic factors, and that NRXNs organize the presynaptic protein clusters and the autocrine neurotrophic factor secretory machinery independent of contact with postsynaptic ligands.

neuroscience↗

bioRxiv2020

Common synaptic phenotypes arising from diverse mutations in the human NMDA receptor subunit GluN2A

Dominant mutations in the human gene GRIN2A, encoding NMDA receptor (NMDAR) subunit GluN2A, make a significant and growing contribution to the catalogue of published single-gene epilepsies. Understanding the disease mechanism in these epilepsy patients is complicated by the surprising diversity of effects that the mutations have on NMDARs. We have examined the cell-autonomous effect of five GluN2A mutations, 3 loss-of-function and 2 gain-of-function, on evoked NMDAR-mediated synaptic currents (NMDA-EPSCs) in CA1 pyramidal neurons in cultured hippocampal slices. Despite the mutants differing in their functional incorporation at synapses, prolonged NMDA-EPSC current decays (with only marginal changes in charge transfer) were a common effect for both gain- and loss-of-function mutants. Modelling NMDA-EPSCs with mutant properties in a CA1 neuron revealed that the effect of GRIN2A mutations can lead to abnormal temporal integration and spine calcium dynamics during trains of concerted synaptic activity. Investigations beyond establishing the molecular defects of GluN2A mutants are much needed to understand their impact on synaptic transmission.

neuroscience↗

bioRxiv2020

A Specific Neuroligin3-αNeurexin1 Code Regulates GABAergic Synaptic Function in Mouse Hippocampus

Synapse formation and regulation require interactions between pre- and postsynaptic proteins, notably cell adhesion molecules (CAMs). It has been proposed that the functions of neuroligins (Nlgns), postsynaptic CAMs, rely on the formation of trans-synaptic complexes with neurexins (Nrxns), presynaptic CAMs. Nlgn3 is a unique Nlgn isoform that localizes at both excitatory and inhibitory synapses. However, Nlgn3 function mediated via Nrxn interactions is unknown. Here, we demonstrate that Nlgn3 localizes at postsynaptic sites apposing vesicular glutamate transporter 3-expressing (VGT3+) inhibitory terminals and regulates VGT3+ inhibitory interneuron-mediated synaptic transmission in mouse organotypic slice cultures. Gene expression analysis of interneurons revealed that the Nrxn1+AS4 splice isoform is highly expressed in VGT3+ interneurons as compared with other interneurons. Most importantly, postsynaptic Nlgn3 requires presynaptic Nrxn1+AS4 expressed in VGT3+ interneurons to regulate inhibitory synaptic transmission. Our results indicate that specific Nlgn-Nrxn interactions generate distinct functional properties at synapses.

neuroscience↗

bioRxiv2020

Gene Deletion Studies Reveal A Critical Role For Prostamide/Prostaglandin F2α Synthase In Prostamide F2α Formation

Prostamide/prostaglandin F synthase (PM/PGFS) is an enzyme with very narrow substrate specificity and is dedicated to the biosynthesis of prostamide and prostaglandin F2. The importance of this enzyme, relative to the aldoketoreductase (AKR) series, in providing functional tissue prostamide F2 levels was determined by creating a line of PM/PGFS gene deleted mice. Deletion of the gene encoding PM/PGFS (fam213b) was accomplished by a two exon disruption. Prostamide F2 levels in WT and PM/PGFS KO mice were determined by LC/MS/MS. Intraocular pressure was measured by tonometry and outflow facility was measured by the iPerfusion method in enucleated mouse eyes. Deletion of fam213b had no observed effect on behavior, appetite, or fertility. Intraocular pressure was significantly elevated by approximately 4 mmHg in PM/PGFS KO mice compared to littermate WT mice. No effect on pressure dependent outflow facility occurred, which is consistent with the typically reported prostanoid effect of increasing outflow through the unconventional pathway. The elevation of intraocular pressure caused by deletion of the gene encoding the PM/PGFS enzyme likely results from a diversion of the endoperoxide precursor pathway to provide increased levels of those prostanoids that raise intraocular pressure, namely prostaglandin D2 (PGD2), thromboxane A2 (TxA2). It follows that PM/PGFS may serve an important regulatory role in the eye by providing PGF2 and prostamide F2 to constrain the influence of those prostanoids that raise intraocular pressure.

pharmacology and toxicology↗

bioRxiv2020

AUTS2 Governs Cerebellar Development, Purkinje Cell Maturation, Motor Function and Social Communication

Autism susceptibility candidate 2 (AUTS2), a risk gene for autism spectrum disorders (ASDs), is implicated in telencephalon development. Because AUTS2 is also expressed in the cerebellum where defects have been linked to ASDs, we investigated AUTS2 functions in the cerebellum. AUTS2 is specifically localized in Purkinje cells (PCs) and Golgi cells during postnatal development. Auts2 conditional knockout (cKO) mice exhibited smaller and deformed cerebella containing immature-shaped PCs with reduced expression of Cacna1a. Auts2 cKO and knock-down experiments implicated AUTS2 participation in elimination and translocation of climbing fiber synapses, and restriction of parallel fiber synapse numbers. Auts2 cKO mice exhibited behavioral impairments in motor learning and vocal communications. Because Cacna1a is known to regulate synapse development in PCs, it suggests that AUTS2 is required for PC maturation to elicit normal development of PC synapses and thus the impairment of AUTS2 may cause cerebellar dysfunction related to psychiatric illnesses such as ASDs.

neuroscience↗