Biology subjects

Kaibuchi, K.

Publications and source records attributed to Kaibuchi, K..

12 recordsLinked to original sources

bioRxiv2026

KCNQ2/3 Regulates the Synaptic Remodeling Associated with Drug Seeking in Addiction

Drug-seeking behavior during withdrawal represents a critical obstacle to addiction treatment. In the nucleus accumbens, hyperactive dopamine D1 receptor-expressing medium spiny neurons (D1R-MSNs) promote cocaine-seeking through aberrant synaptic remodeling, including synapse formation and calcium-permeable AMPA receptor (CP-AMPAR) insertion. However, the intermediate molecular control mechanism remains unclear. We identified KCNQ2/3 potassium channels as key regulators of synaptic pathology during withdrawal. Cocaine-conditioned mice showed increased spine density, enhanced surface CP-AMPAR, and elevated neuronal activity 14 days after withdrawal. These phenotypes were reversed by repeated administration of KCNQ2/3 openers or D1R-MSN-specific expression of constitutively active KCNQ2. Functional restoration of KCNQ2/3 suppressed cocaine-seeking behavior and normalized D1R-MSN excitability. These findings suggest that sustained KCNQ2/3 deactivation drives synaptic remodeling during withdrawal, while channel activation offers a potential therapeutic strategy. Furthermore, the results position KCNQ2/3 as a master regulator of drug-seeking behavior and channel activation in D1R-MSNs as a logical target for relapse prevention in addiction.

neuroscience↗

bioRxiv2026

Comprehensive transcriptomic and proteomic profiling of mTOR-related epilepsy

Malformations of cortical development (MCDs) are a major cause of drug-resistant epilepsy (DRE) in children. A subset of MCDs, including focal cortical dysplasia type II, tuberous sclerosis complex, and hemimegalencephaly, shares characteristic histopathological features and somatic mutations in genes within the PI3K-AKT-mTOR signaling cascade, and is termed mTORopathies. However, the molecular mechanisms that drive epileptogenesis and consequent uncontrollable seizures in these disorders remain poorly understood. Here, we performed integrated genomic, transcriptomic, and proteomic analyses of 60 surgical brain specimens from 42 patients with MCDs and 18 with mesial temporal lobe epilepsy. Targeted genomic sequencing identified 19 somatic mutations in PI3K-AKT-mTOR pathway genes, including novel variants in AKT3 (L51H) and MTOR (A512T). RNA sequencing comparing mTORopathy brains with controls, along with cross-validation using an independent public dataset, identified 287 consistently differentially expressed genes that were consistently altered. Downregulated genes were enriched in oxidative phosphorylation (OXPHOS) and mitochondrial metabolic pathways, whereas upregulated genes were associated with gliogenesis and cellular senescence. Proteomic profiling identified 44 differentially expressed proteins, including significant reductions in OXPHOS-related proteins (COX5B, NDUFS4, GOT2, RAB27B, and DKK3). Our study provides a patient-matched, multi-omics dataset of human mTOR-related epilepsies from a single-institution cohort. Integrated analyses across transcriptomic and proteomic layers highlight impairment of OXPHOS as a molecular hallmark of mTORopathies, Integrated transcriptomic and proteomic analyses identified impaired OXPHOS as a molecular hallmark, potentially contributing to focal hypometabolism in mTORopathy. These findings offer mechanistic insights into epileptogenesis and provide a valuable resource for understanding molecular pathologies and developing mechanism-based therapies for mTOR-related epilepsy.

neuroscience↗

bioRxiv2026

Single-cell-resolved spatial multi-omics identifies mTOR-driven neuronal and astroglial pathogenicity underlying epileptogenic focal cortical dysplasia

Focal cortical dysplasia type II (FCDII) is a malformation of cortical development caused by somatic mutations in the mTOR signaling pathway. Two hallmark pathological cell types in FCDII, dysmorphic neurons (DNs) and balloon cells (BCs), arise as a result of somatic mutations in the mTOR signaling pathway and are implicated in the pathophysiology of drug-resistant epilepsy. However, how these somatic mutations reshape cell states within the human cortex remains poorly understood. Here, we integrate imaging-based spatial transcriptomics (iST), single-nucleus RNA sequencing, and proteomics of surgically resected FCDIIb tissue to define the transcriptional and proteomic profiles of DNs and BCs. Spatial mapping of iST data resolved transcriptional signatures in histologically validated DNs and BCs within FCDIIb sections. Integrative omics analysis further revealed that DNs show upregulation of PI3K-AKT-mTOR and p53-CROT metabolic programs accompanied by suppression of synaptic signaling, whereas BCs exhibit transcriptional signatures of reactive astrocytes with increased phagocytic and immune-like activity. These data delineate cell-type-specific consequences of somatic mTOR pathway mutations at single-cell resolution and reveal previously unrecognized metabolic and immunoregulatory mechanisms contributing to epileptogenesis in drug-resistant epilepsy. Our study establishes a spatial multi-omics framework for dissecting human cortical malformations and highlights potential therapeutic targets for drug-resistant epilepsy.

neuroscience↗

bioRxiv2025

Distinct effects of nonselective Rho-kinase inhibitor fasudil and selective Rho-kinase 2 inhibitor KD025 on serotonin and dopamine release in the nucleus accumbens of mice

Recent studies have indicated that the Rho GTPase family and Rho-kinases are associated with psychiatric diseases, such as schizophrenia. Rho-kinases have two subtypes, Rho-kinases 1 and 2 that regulate actin dynamics and mediate neurite outgrowth, spine morphology in neurons, and neurotransmitter release in vitro and ex vivo. However, the precise role of Rho-kinases in neurotransmitter release in vivo remains unclear. To clarify the role of Rho-kinases 1 and 2 in serotonin and dopamine release in the nucleus accumbens (NAc) of mice in vivo, we investigated the effect of a nonselective Rho-kinase inhibitor, fasudil, and a selective Rho-kinase 2 inhibitor, KD025, using an in vivo microdialysis technique. Fasudil perfusion (1-20 M) into the NAc increased the basal extracellular serotonin level but did not affect dopamine levels, whereas KD025 (10-20 M) had little effect on basal serotonin and dopamine levels. Notably, fasudil perfusion into the NAc suppressed depolarization-induced serotonin and dopamine release in a dose-dependent manner, whereas KD025 selectively suppressed depolarization-induced serotonin release. Our results suggested that Rho-kinases 1 and 2 are associated with dopamine and serotonin release, respectively, and that both may have significant but distinct roles in the regulation of serotonin and dopamine release in the NAc.

neuroscience↗

bioRxiv2025

Twinfilin-1 phosphorylation in reelin signaling regulates actin dynamics and spine development

Reelin is an extracellular glycoprotein essential for neuronal migration, spine development, and synaptic plasticity. Impaired reelin signaling is linked to neurological disorders, including schizophrenia and autism. While reelin mutant (reeler) mice exhibit behavioral deficits associated with impaired spine formation, the underlying molecular mechanisms remain unclear. We identified Twinfilin-1 (Twf1) as a downstream effector of reelin signaling via phosphoproteomic analysis, based on its reduced tyrosine phosphorylation in reeler mice. We found that Src regulated Twf1 phosphorylation at tyrosine 309, and reelin stimulation increased Twf1 phosphorylation in neurons, an effect blocked by the Src inhibitor PP2. A phospho-resistant Twf1 mutant (Twf1 Y309F) showed reduced capping protein binding and a lower F/G-actin ratio. Twf1Y309F mice exhibited cognitive deficits, reduced spine density, smaller spine head size, and a decreased F/G-actin ratio in synaptosomes. These findings highlight Twf1 phosphorylation as a key component of reelin signaling involved in actin remodeling and spine development.

neuroscience↗

bioRxiv2024

Antipsychotic-like effects of the selective Rho-kinase 2 inhibitor KD025 in genetic and pharmacological mouse models of schizophrenia

1Copy number variations in the ARHGAP10 gene encoding Rho GTPase-activating protein 10 are significantly associated with schizophrenia. ARHGAP10 negatively regulates RhoA/Rho-kinase (ROCK) signaling. We previously demonstrated that fasudil, a non-selective ROCK inhibitor, exhibited antipsychotic-like effects in several mouse models of schizophrenia. ROCK has two subtypes, ROCK1 and ROCK2. ROCK1 is mainly expressed in the thymus and blood, while ROCK2 is predominantly expressed in the brain. Therefore, it is expected that like fasudil, selective ROCK2 inhibitors will exhibit antipsychotic-like effects, accompanied by a lower incidence of adverse effects due to ROCK1 inhibition. Here, we used genetic and pharmacological models of schizophrenia to investigate whether the selective ROCK2 inhibitor KD025 would show antipsychotic-like effects with a favorable adverse effect profile. Oral administration of KD025 suppressed the abnormal increase in the phosphorylation level of myosin phosphatase-targeting subunit 1, a substrate of ROCK, and ameliorated the decreased spine density of layer 2/3 pyramidal neurons in the medial prefrontal cortex of Arhgap10 S490P/NHEJ mice. Furthermore, KD025 mitigated the methamphetamine-induced impairment of visual discrimination (VD) in Arhgap10 S490P/NHEJ and wild-type mice. KD025 also reduced MK-801-induced impairments of VD, novel object recognition, and hyperlocomotion. Regarding side effects that are commonly seen with typical antipsychotics, KD025 did not affect systolic blood pressure and did not induce extrapyramidal symptoms, hyperprolactinemia, or hyperglycemia at the effective dosage in naive wild-type mice. Taken together, KD025 shows antipsychotic-like effects with a favorable adverse effect profile in genetic and pharmacological mouse models of schizophrenia.

pharmacology and toxicology↗

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↗

bioRxiv2022

Homeodomain-less MEIS1 suppresses CUL3-dependent ATOH1 degradation to regulate cerebellar granule cell development

The development of the complex nervous system is strictly controlled by diverse isoforms produced from individual genes, but the underlying machinery remains unclear. Our long-read cDNA sequencing identifies more than 700 genes with high isoform diversity in cerebellar granule cell progenitors (GCPs). One such gene, Meis1, produces MEIS1-FL and MEIS1-HdL isoforms, which include and lack the homeodomain, respectively. Our previous study showed that MEIS1-FL localizes to nuclei and promotes ATOH1 protein degradation through transcriptional regulation, thereby promoting GCP differentiation. In contrast, our in vivo electroporation experiment in this study shows that MEIS1-HdL inhibits GCP differentiation. MEIS1-HdL localizes in the cytoplasm and inhibits the degradation of ATOH1 mediated by CUL3, which is a newly identified E3 ligase for ATOH1. MEIS1-HdL enhances the binding of the COP9 signalosome to CUL3, which suppresses ATOH1 polyubiquitination. This study demonstrates that functionally antagonistic isoforms derived from a single gene cleverly control neural progenitor differentiation.

cell biology↗

bioRxiv2022

LIS1 RNA-binding orchestrates the mechanosensitive properties of embryonic stem cells in AGO2-dependent and independent ways

Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the activity of the molecular motor cytoplasmic dynein. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it regulates the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the expression of extracellular matrix (ECM) and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post- transcriptional regulation underlying development and mechanosensitive processes.

cell biology↗

bioRxiv2021

Stress-impaired reward pathway promotes distinct feeding behavior patterns

Psychosocial stress can impact feeding behavior outcomes. Although many studies have examined alterations to food intake, little is known about how stress affects feeding behavior patterns. To determine the impact of psychological stress on feeding behavior patterns, mice were subjected to various psychosocial stressors (social isolation, intermittent high-fat-diet, or physical restraint) prior to timed observations in a feeding arena that incorporated multiple bait loci. In addition, in vivo microdialysis was used to assess the effects of stressors on the reward system by measuring dopamine levels in the nucleus accumbens (NAcc) shell. Impaired feeding behavior patterns characterized by significant deviations in bait selection (i.e. fixated feeding) and prolonged periods of eating (i.e. protracted feeding) were observed in stressed mice relative to non-stressed controls. In addition to clear behavioral effects, the stressors also negatively impacted dopamine levels at the nucleus accumbens shell. Normalization of dopamine reversed the fixated feeding behavior, whereas specifically inhibiting neuronal activity in the dopaminergic neurons of the ventral tegmental area that project to the nucleus accumbens shell caused similar impairments in feeding. Given that the deviations were not consistently accompanied by changes in the amount of bait consumed, body weight, or metabolic factors, the qualitative effects of psychosocial stressors on feeding behavior likely reflect perturbations to a critical pathway in the mesolimbic dopamine system. These findings provide compelling evidence that aberrations in feeding behavior patterns can be developed as sensitive biomarkers of psychosocial stress and possibly a prodromal state of neuropsychiatric diseases. Significance StatementFeeding behavior can be affected by neuropsychiatric disorders including psychosocial stressors, and the evaluation of eating behavior was mainly based on food intake. However, it is speculated that not only food intake but also feeding behavior patterns can be affected in such disorders. The biological processes underlying the feeding behavior patterns have not been clarified yet. We found that aberrant feeding behaviors in mice characterized by fixated feeding were provoked by psychosocial stressors. The qualitative effects of psychosocial stressors on feeding behavior reflect perturbations in the mesolimbic dopamine system. These findings provide compelling evidence that aberrations in feeding behavior patterns can be developed as sensitive biomarkers of psychosocial stress and possibly a prodromal state of neuropsychiatric diseases.

neuroscience↗

bioRxiv2020

CyclinD1 controls development of cerebellar granule cell progenitors through phosphorylation and stabilization of ATOH1

Here we report that CyclinD1 (CCND1) directly regulates both the proliferative and immature states of cerebellar granule cell progenitors (GCPs). CCND1 not only accelerates cell cycle but also upregulates ATOH1 protein, an essential transcription factor that maintains GCPs in an immature state. In cooperation with CDK4, CCND1 directly phosphorylates Ser309 of ATOH1, which inhibits additional phosphorylation at S328, consequently preventing Ser328 phosphorylation-dependent ATOH1 degradation. PROX1 downregulates Ccnd1 expression by histone-deacetylation of Ccnd1 promoter in GCPs, leading to cell cycle exit and differentiation. WNT signaling upregulates PROX1 expression in GCPs. These findings suggest that WNT-PROX1-CCND1-ATOH1 signaling cascade cooperatively controls proliferation and immaturity of GCPs. We revealed that the expression and phosphorylation levels of these molecules dynamically change during cerebellar development, which was suggested to determine appropriate differentiation rates from GCPs to GCs at distinct developmental stages. This study contributes to understanding the regulatory mechanism of GCPs as well as neural progenitors.

developmental biology↗

bioRxiv2019

Tau-tubulin kinase 1 and amyloid-β peptide induce phosphorylation of collapsin response mediator protein-2 and enhance neurite degeneration in Alzheimer disease mouse models

The accumulation of phosphorylated tau protein (pTau) in the entorhinal cortex (EC) is the earliest tau pathology in Alzheimers disease (AD). Tau tubulin kinase-1 (TTBK1) is a neuron-specific tau kinase and expressed in the EC and hippocampal regions in both human and mouse brains. Here we report that collapsin response mediator protein-2 (CRMP2), a critical mediator of growth cone collapse, is a new downstream target of TTBK1 and is accumulated in the EC region of early stage AD brains. TTBK1 transgenic mice show severe axonal degeneration in the perforant path, which is exacerbated by crossing with Tg2576 mice expressing Swedish familial AD mutant of amyloid precursor protein (APP). TTBK1 mice show accumulation of phosphorylated CRMP2 (pCRMP2), in the EC at 10 months of age, whereas age-matched APP/TTBK1 bigenic mice show pCRMP2 accumulation in both the EC and hippocampal regions. Amyloid-{beta} peptide (A{beta}) and TTBK1 suppresses the kinetics of microtubule polymerization and TTBK1 reduces the neurite length of primary cultured neurons in Rho kinase-dependent manner in vitro. Silencing of TTBK1 or expression of dominant-negative Rho kinase demonstrates that A{beta} induces CRMP2 phosphorylation at threonine 514 in a TTBK1-dependent manner, and TTBK1 enhances A{beta}-induced CRMP2 phosphorylation in Rho kinase-dependent manner in vitro. Furthermore, TTBK1 expression induces pCRMP2 complex formation with pTau in vitro, which is enhanced upon A{beta} stimulation in vitro. Finally, pCRMP2 forms a complex with pTau in the EC tissue of TTBK1 mice in vivo, which is exacerbated in both the EC and hippocampal tissues in APP/TTBK1 mice. These results suggest that TTBK1 and A{beta} synergistically induce phosphorylation of CRMP2, which may be causative for the neurite degeneration and somal accumulation of pTau in the EC neurons, indicating critical involvement of TTBK1 and pCRMP2 in the early AD pathology.

neuroscience↗