Biology subjects

Yamasaki, T.

Publications and source records attributed to Yamasaki, T..

14 recordsLinked to original sources

bioRxiv2026

Disruption of CaMKII biomolecular condensation induces attention-deficit/hyperactivity disorder-like phenotypes

CaMKII is a multifunctional kinase essential for synaptic plasticity and memory formation. While its canonical role involves enzymatic phosphorylation, recent evidence suggests CaMKII also functions through liquid-liquid phase separation (LLPS) with substrate proteins, including GluN2B-containing NMDA receptors. However, the physiological significance remains unclear. Here, we generated CaMKII subtype knock-in (KI) mice carrying a point mutation (I205K) in the hydrophobic pocket, a key interface required for LLPS. These mice exhibited a complete loss of structural long-term potentiation (sLTP) despite normal spine morphology, marked hyperactivity and profound deficits in aversive memory formation. Atomoxetine, an approved attention-deficit/hyperactivity disorder (ADHD) treatment, ameliorated the hyperactive phenotype. Notably, we identified a patient carrying the I205N variant presenting with ADHD and mild intellectual disability, mirroring the behavioral features observed in I205K KI mice. Additional neurodevelopmental disorder-associated variants within the same hydrophobic pocket similarly disrupted LLPS in vitro. Molecular dynamics simulations revealed these variants destabilize the CaMKII-GluN2B interaction through distinct mechanisms that perturb the dynamic stability of the binding interface. These findings establish CaMKII-mediated phase separation as critical for linking synaptic molecular assembly to cognitive function and provide a unifying molecular basis for synaptic disorganization, hyperactivity, and memory deficits associated with neurodevelopmental disorders. SignificanceCaMKII is essential for synaptic plasticity and memory, yet the contribution of its non-catalytic functions, including liquid-liquid phase separation (LLPS), to brain function remains unclear. Here, we show that disrupting CaMKII LLPS with its synaptic partners impairs synaptic localization, abolishes LTP, and causes profound memory deficits in vivo. A knock-in mouse carrying the I205K mutation showed hyperactivity and impaired learning. This recapitulate the clinical phenotypes of a human patients with the I205N variant, which is associated with ADHD and intellectual disability. Together, these findings establish LLPS as a critical mechanism underlying CaMKII function in the brain and provide a disease-relevant framework for understanding synaptic dysfunction in neurodevelopmental disorders.

neuroscience↗

bioRxiv2026

SCD1 inhibition synergizes with TMZ to improve IDH1 MUT glioma survival of mice via lipotoxic stress

Mutations in the isocitrate dehydrogenase enzyme (IDH1) are prevalent in low-grade gliomas, such as oligodendrogliomas. Other than surgery, radiation, and chemotherapy, few options for treatment exist. The standard of care for patients currently consists of maximal safe resection, as well as the potential use of radiation and chemotherapy, typically followed by radiographic surveillance. Although significant advances have been achieved, these have not translated into meaningful improvements in overall survival, warranting the development of novel therapies. Herein, we demonstrate that the inhibition of SCD1 in vitro leads to decreased colony formation and is more specific to IDH1MUT glioma than the IDH1WT cells, We further identified MF-438 as an active inhibitor of this enzyme and showed that this inhibitor is linked with iron transport and ferroptosis. MF438 depleted oleic (C18:1) and palmitoleic acid (C16:1) levels, driving saturated phosphatidylcholine (PC) accumulation and ER stress (INSIG1, SEL1L upregulation). TS603 exhibited selective polyunsaturated phosphatidylcholine reduction with downregulation of GPX4, FTH1, and KEAP1, and upregulation of NCOA4, SLC11A2, ALDH7A1, and DPP4, consistent with ferroptosis priming via ferritinophagy-driven expansion of the labile iron pool, as confirmed by FerroOrange flow cytometry. Neutral lipid metabolism genes (LPIN1, LDLR, PNPLA3, ACSL1), intracellular lipid transport genes (TMEM41B, OSBP, STARD4), and lipid droplet organization genes (SQLE, CHKA, AUP1) were coordinately upregulated in TS603 after treatment with MF-438. MF438+TMZ activated the integrated stress response (ATF3, DDIT3, IRF1, CDKN1A, GADD45B) and synergistically suppressed TS603 neurosphere growth (p=0.0326). In vivo combination between MF-438 and TMZ showed improved survival versus the TMZ-alone group, in an IDH1-mutant oligodendroglioma model. Tissue analyses showed significant reduced Ki67 expression, a marker of cellular proliferation, in the combination treatment. Collectively, these findings suggest that targeting lipid metabolism may enhance the efficacy of standard-of-care therapy in IDH1-mutant oligodendroglioma.

Cancer Biology↗

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

Global identification of AGO3-RNA interactions in Chlamydomonas reveals the small RNA-mediated regulation of nuclear and chloroplast gene expression

MicroRNAs (miRNAs) form complexes with Argonaute (AGO) proteins and bind to mRNAs with complementary sequences to repress their expression. Organisms typically possess several hundred miRNAs regulating diverse biological processes. Although the roles of miRNAs have been well elucidated in multicellular organisms, they remain largely unexplored in unicellular organisms. In the green alga Chlamydomonas reinhardtii, the first unicellular organism in which miRNAs were discovered, identifying miRNA target genes remains challenging. Here, we report the high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) to generate global AGO3-RNA interaction maps, identifying 131 mRNAs derived from nuclear genes. We also detected mRNAs from two chloroplast genes, suggesting that AGO3 may also influence chloroplast gene expression. Our findings indicate that Chlamydomonas small RNAs repress nuclear and chloroplast gene expression. Furthermore, HITS-CLIP analyses are now feasible for any RNA-binding protein in Chlamydomonas, offering new opportunities to uncover the functions of RNA-binding proteins of interest.

plant biology↗

bioRxiv2024

A novel VCP modulator, KUS121, attenuates atherosclerosis progression by maintaining intracellular ATP and mitigating ER stress in endothelial cells

BackgroundEndoplasmic reticulum (ER) stress signaling pathways have pivotal roles in atherosclerosis progression. Recently, we have developed Kyoto University Substance (KUS) 121, which selectively inhibits ATPase activities of valosin-containing protein (VCP) and consequently saves intracellular ATP consumption and mitigates ER stress. Methods and ResultsWe assessed the efficacy of KUS121 against atherosclerosis by its daily injection into Apoe-/- mice fed with Western Diet (WD) for 8 weeks. Consequently, KUS121 treatment reduced atherosclerosis progression by approximately 40% in atherosclerotic plaques. Interestingly, we found that C/EBP homologous protein (CHOP), an established ER stress marker, was mainly expressed in plaque endothelium. Therefore, we assessed the action of KUS121 in endothelial cells using the human endothelial cell line (EA.hy926 cells). As a result, KUS121 prevented ER stress-induced apoptosis and downregulated the IRE1 (Inositol-requiring enzyme) -associated inflammatory pathways. Consistent with these in vitro findings, KUS121 treatment also significantly reduced endothelial apoptosis assessed by TUNEL staining and inflammation examined by immunostaining of Nuclear factor kappa B (NF-{kappa}B) and Intercellular adhesion molecule (ICAM) 1 at plaque endothelium. We also demonstrated that KUS121 maintained ATP levels in EA.hy926 cells and atherosclerotic plaque lesions using the single-wavelength or the FRET-based fluorescent ATP sensor. Supplementation of intracellular ATP by Methyl pyruvate (MePyr) attenuated ER stress-induced apoptotic and inflammatory pathways in endothelial cells, which could be the main mechanism how KUS121 reduces ER stress. ConclusionsKUS121 can be a new therapeutic option for atherosclerotic diseases by maintaining intracellular ATP levels and attenuating ER stress-induced apoptosis and inflammation in plaque endothelium.

pathology↗

bioRxiv2024

DMT1 contributes to MF- 438 - mediated inhibition of glioma cells

Elevated SCD1 expression has been associated with enhanced cancer cell survival, proliferation, and resistance to therapy in many cancer types including gliomas. Hereby, we investigate the impact of MF-438 on SCD1-mediated lipid metabolism and its consequences on glioma growth and survival. Our data reveals an IDHmut-specific inhibitory effect of MF438 on gliomas. Also, we delineate a dual mechanism of action: while SCD1-mediated lipid metabolism is hindered by MF-438 treatment, MF-438 also exerts an SCD1-independent inhibition on DMT1 expression. Supporting data from the DMT1 blocker underscores its significance in MF-438s anti-glioma efficacy.

cancer biology↗

bioRxiv2024

Targeting the sphingolipid rheostat in IDH1mut glioma alters cholesterol homeostasis and triggers apoptosis via membrane degradation

The cross-regulation of metabolism and trafficking is not well understood for the vital sphingolipids and cholesterol constituents of cellular compartments. While reports are starting to surface on how sphingolipids like sphingomyelin (SM) dysregulate cholesterol levels in different cellular compartments (Jiang et al., 2022), limited research is available on the mechanisms driving the relationship between sphingolipids and cholesterol homeostasis, or its biological implications. Previously, we have identified sphingolipid metabolism as a unique vulnerability for IDH1mut gliomas via a rational drug design. Herein, we show how modulating sphingolipid levels affects cholesterol homeostasis in brain tumors. However, we unexpectedly discovered for the first time that C17 sphingosine and NDMS addition to cancer cells alters cholesterol homeostasis by impacting its cellular synthesis, uptake, and efflux leading to a net decrease in cholesterol levels and inducing apoptosis. Our results reflect a reverse correlation between the levels of sphingosines, NDMS, and unesterified, free cholesterol in the cells. We show that increasing sphingosine and NDMS (a sphingosine analog) levels alter not only the trafficking of cholesterol between membranes but also the efflux and synthesis of cholesterol. We also demonstrate that despite the effort to remove free cholesterol by ABCA1-mediated efflux or by suppressing machinery for the influx (LDLR) and biosynthetic pathway (HMGCR), apoptosis is inevitable for IDH1mut glioma cells. This is the first study that shows how altering sphingosine levels directly affects cholesterol homeostasis in cancer cells and can be used to manipulate this relationship to induce apoptosis in IDH1mut gliomas.

cancer biology↗

bioRxiv2023

In vivo visualization of intracellular pH gradient in brain using PET imaging

Intracellular pH (pHi) is a valuable index for predicting hypoxic brain damage. However, no positron emission tomography (PET) probe is currently available for monitoring pHi in vivo. In this study, we developed a new approach for visualizing monoacylglycerol lipase (MAGL) activity in the brain. This approach used PET with a new probe [11C]HC-A, an azetidine carbamate inhibitor, whose uptake and residence depended on the pHi gradient was evaluated with in silico, in vitro, and in vivo assessments. Molecular dynamics simulations predicted that complex (complex-A) between HC-A and MAGL would be difficult to hydrolyze under acidic conditions. In vitro assessment using rat brain homogenate showed that [11C]HC-A reacted with MAGL to yield [11C]complex-A, which was rapidly hydrolyzed to liberate 11CO2. The 11CO2 liberation rate was slower at lower pH. In PET with [11C]HC-A using ischemic rats, the radioactivity clearance rate, which reflects the production rate of 11CO2 in the brain, was lower in a remarkably hypoxic area than in the contralateral region. In conclusion, we successfully visualized the pHi gradient in the brain using PET imaging.

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↗

bioRxiv2021

Radiosynthesis and preclinical evaluation of a carbon-11 labeled PDE7 inhibitor for PET neuroimaging

BackgroundDysfunction of cyclic nucleotide phosphodiesterase 7 (PDE7) has been associated with excess intracellular cAMP concentrations, fueling pathogenic processes that are implicated in neurodegenerative disorders. The aim of this study was to develop a suitable PDE7-targeted positron emission tomography (PET) probe that allows non-invasive mapping of PDE7 in the mammalian brain. MethodsBased on a spiro cyclohexane-1,4-quinazolinone scaffold with known inhibitory properties towards PDE7, we designed and synthesized a methoxy analog that was suitable for carbon-11 labeling. Radiosynthesis was conducted with the respective desmethyl precursor using [11C]MeI. The resulting PET probe, codenamed [11C]26, was evaluated by cell uptake studies, ex vivo biodistribution and radiometabolite studies, as well as in vivo PET experiments in rodents and nonhuman primates (NHP). ResultsTarget compound 26 and the corresponding phenolic precursor were synthesized in 2-3 steps with overall yields of 49.5% and 12.4%, respectively. An inhibitory constant (IC50) of 31 nM towards PDE7 was obtained and no significant interaction with other PDE isoforms were observed. [11C]26 was synthesized in high molar activities (170 - 220 GBq/{micro}mol) with radiochemical yields of 34{+/-}7%. In vitro cell uptake of [11C]26 was 6-7 folds higher in PDE7 overexpressing cells, as compared to the controls, whereas an in vitro specificity of up to 90% was measured. Ex vivo metabolite studies revealed a high fraction of intact parent in the rat brain (98% at 5 min and 75% at 30 min post injection). Considerable brain penetration was further corroborated by ex vivo biodistribution and PET imaging studies - the latter showing heterogenic brain uptake. While marginal specific binding was observed by PET studies in rodents, a moderate, but dose-dependent, blockade was observed in the NHP brain following pretreatment with non-radioactive 26. ConclusionIn this work, we report on the preclinical evaluation of [11C]26 (codename [11C]P7-2104), a PDE7-targeted PET ligand that is based on a spiroquinazolinone scaffold. [11C]26 displayed promising in vitro performance characteristics, a moderate degree of specific binding in PET studies with NHP. Accordingly, [11C]26 will serve as a valuable lead compound for the development of a new arsenal of PDE7-targeted probes with potentially improved in vivo specificity.

neuroscience↗

bioRxiv2021

Structural basis of LhcbM5-mediated state transitions in green algae

In green algae and plants, state transitions serve as a short-term light acclimation process to regulate light harvesting capacity of photosystems I and II (PSI and PSII). During the process, a portion of the light-harvesting complexes II (LHCII) are phosphorylated, dissociate from PSII and bind PSI to form PSI-LHCI-LHCII supercomplex. Here we report high-resolution structures of PSI-LHCI-LHCII supercomplex from Chlamydomonas reinhardtii, revealing the mechanism of assembly between PSI-LHCI complex and two phosphorylated LHCII trimers containing all four types of LhcbM proteins. Two specific LhcbM isoforms, namely LhcbM1 and LhcbM5, directly interact with the PSI core through their phosphorylated amino-terminal regions. Furthermore, biochemical and functional studies on mutant strains lacking either LhcbM1 or LhcbM5 indicate that only LhcbM5 is indispensable in the supercomplex formation. The results unraveled the specific interactions and potential excitation energy transfer routes between green algal PSI and two phosphorylated LHCIIs.

plant biology↗

bioRxiv2020

UV-A radiation rapidly activates photoprotective mechanisms in Chlamydomonas reinhardtii

Conversion of light energy into chemical energy through photosynthesis in the chloroplasts of photosynthetic organisms is essential for photoautotrophic growth. However, the conversion of excess light energy into thermal energy by non-photochemical quenching (NPQ) is important for avoiding the generation of reactive oxygen species and maintaining efficient photosynthesis. In the unicellular green alga Chlamydomonas reinhardtii, NPQ is activated as a photoprotective mechanism through wavelength-specific light signaling pathways mediated by the phototropin (blue light) and UVR8 (ultra-violet light, UV) photoreceptors. NPQ-dependent photoprotection improves cell survival under high-light conditions; however, the biological significance of photoprotection being activated by light with different qualities remains poorly understood. Here, we demonstrate that NPQ-dependent photoprotection is activated more rapidly by UV than by visible light. We found that induction of gene expression and protein accumulation related to photoprotection was significantly faster and greater in magnitude under UV treatment compared to that under blue- or red-light treatment. Furthermore, the action spectrum of UV-dependent induction of photoprotective factors implied that Chlamydomonas sense relatively long-wavelength UV (including UV-A/B), whereas the model dicot plant Arabidopsis thaliana preferentially senses relatively short-wavelength UV (mainly UV-B/C) for induction of photoprotective responses. Therefore, we hypothesize that Chlamydomonas developed a UV response distinct from that of land plants. One-sentence summaryIn contrast to land plants, which sense short-wave UV light, the unicellular green alga Chlamydomonas senses long-wavelength UV light for photoprotective responses.

plant biology↗

bioRxiv2020

Activation of prodynorphin neurons in the dorsomedial hypothalamus inhibits food intake and promotes positive valence

The regulation of food intake is one of the major research areas in the study of metabolic syndromes such as obesity. Gene targeting studies have clarified the roles of hypothalamic neurons in feeding behaviour. However, our understanding of neural function under physiological conditions is still limited. Immediate early genes, such as activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), are useful markers of neuronal activity. Here, we investigated the role of Arc/Arg3.1 gene-expressing neurons in the hypothalamus after refeeding using the targeted recombination in active populations method. We identified refeeding-responsive prodynorphin/cholecystokinin neurons in the dorsomedial hypothalamus that project to the paraventricular hypothalamic nucleus. Chemogenetic activation of these neurons decreased food intake and promoted positive valence. Our findings provide insight into the role of newly identified hedonic neurons in the process of feeding-induced satiety.

physiology↗

bioRxiv2020

IDH1 Mutations Induce Organelle Defects Via Dysregulated Phospholipids

Cytosolic IDH1 enzyme plays a key, but currently unexplored, role in lipid biosynthesis. Using Raman imaging microscopy, we identified heterogeneous lipid profiles in cellular organelles attributed uniquely to IDH1 mutations. Via organelle lipidomics, we found an increase in saturated and monounsaturated fatty acids in the endoplasmic reticulum of IDH1mut cells compared with IDHWT glioma. We showed that these fatty acids incorporate into phospholipids and induce organelle dysfunctions, with prominent dilation of Golgi apparatus, which can be restored by transient knockdown of stearyl-CoA desaturase or inhibition of D-2-hydroxyglutarate (D-2HG) formation. We validated these findings using tissue from patients with glioma. Oleic acid addition led to increased sensitivity to apoptosis of IDH1mut cells compared with IDHWT. Addition of D-2HG to U251WT cells lead in increased ER and Golgi apparatus dilation. Collectively, these studies provide clinically relevant insights into the functional link between IDH1mut-induced lipid alterations and organelle dysfunction, with therapeutic implications. SignificanceGliomas are devastating tumors, with the most aggressive form--glioblastoma multiforme-- correlated with a mean patient survival of 14.5 months. No curative treatment exists to date. Low-grade glioma (LGG) with the isocitrate dehydrogenase 1 (IDH1) mutation, R132H, provides a survival benefit to patients. Understanding the unique metabolic profile of IDH1mut could provide clues regarding its association with longer survival and information about therapeutic targets. Herein, we identified lipid imbalances in organelles, generated by IDHmut in cells and patient tissue, that were responsible for Golgi dilation and that correlated with increased survival. Addition of oleic acid, which tilted the balance towards elevated levels of monounsaturated fatty acids produced IDH1mut-specific cellular apoptosis. HighlightsO_LISingle-organelle omics revealed unique alterations in lipid metabolism due to IDH1-mutations. C_LIO_LIIDH mutation leads to organelle-wide structural defects. C_LIO_LIIDH1 mutation leads to increased monounsaturated fatty acids levels in glioma cells and oligodendroglioma patient samples. C_LIO_LILipid alterations affect the membrane integrity of the Golgi apparatus. C_LIO_LIIncreased D-2HG induced SCD expression and elevated monounsaturated fatty acids C_LIO_LITilting the balance toward more-abundant monounsaturated fatty acids leads to specific IDH1mut glioma apoptosis. C_LI

cancer biology↗