Biology subjects

Takahashi, M.

Publications and source records attributed to Takahashi, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Glucosylation of Endogenous Haustorium-inducing factors Underpins Kin Avoidance in Parasitic Plants

Parasitic plants rarely attack themselves, suggesting the activity of a kin-avoidance mechanism. In the root parasitic plant Phtheirospermum japonicum, prehaustorium formation is triggered by host-secreted haustorium-inducing factors (HIFs), but it is unresponsive to its own root exudates. Here we report the identification of the spontaneous prehaustorium 1 (spoh1) mutant, which forms prehaustoria without external HIFs. spoh1 harbors a point mutation in the gene encoding UDP-glycosyltransferase GT72B1, an enzyme that glucosylates and thereby inactivates phenolic HIFs. Notably, PjGT72B1 possesses a different substrate specificity from its ortholog of the host Arabidopsis. Introduction of PjGT72B1 in Arabidopsis reduced HIF activity, indicating that HIF glucosylation regulates haustorium induction by hosts. Our findings suggest that parasitic plants have evolved kin-avoidance mechanisms through the glucosylation of endogenous HIFs.

plant biology↗

bioRxiv2025

Real-time imaging of rotation during synthesis by the replisome

During chromosome replication, unwinding by the helicase and synthesis by the polymerases can lead to overwinding and supercoiling of DNA. The mechanical consequences of these events and resulting local dynamics at the replication fork are not well understood. To address these issues, we developed a transverse DNA flow-stretching approach to spatially resolve the parental, leading and lagging strands in real-time. Using bacteriophage T7 as a model system, this approach revealed bursts of high-speed replisome rotation that support continuous DNA synthesis. Surprisingly, excessive rotation does not reduce replisome speed, but increases pausing, reduces processivity, and increases the number of polymerases. Taken together, our observations reveal intrinsic pathways to overcome challenges posed by unfavorable DNA topologies during DNA replication.

biophysics↗

bioRxiv2025

Human TIMELESS, a potential circadian clock regulator, plays an essential role in survival and reawakening of metastasis-initiating cells in bone

Bone metastasis is becoming increasingly common globally. In such cases, cancer cells disseminate from the primary site to bone, subsequently entering a dormant state. Following a certain incubation period, the cells reawaken and grow, forming a metastatic mass. These dormant cells are called metastasis-initiating cells (MICs), but their survival and reawakening are poorly understood. Here, we established an in vivo MIC-reawakening mouse model in bone. Microarray analysis demonstrated enrichment for mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid synthesis in isolated MICs. In addition, transcriptional regulator TIMELESS was identified as an independent poor prognostic factor by using clinical transcriptomic datasets, which was validated by immunostaining on 209 breast cancer cases. TIMELESS-deficient breast, prostate, and bladder cancer cell lines exhibited reduced viability and MIC reawakening in bone. Single-cell analysis on the epigenetic landscape of MICs revealed that motif activities of CLOCK and BMAL1/ARNTL were enhanced in a cluster with increased TIMELESS accessibility scores. As TIMELESS regulates stemness through the OXPHOS metabolic state, an inhibitor targeting the metabolic pathway, MP-A08, was identified to suppress MIC reawakening and growth in bone more efficiently than cisplatin. These results suggest that TIMELESS regulates survival and reawakening in MICs and MP-A08 could contribute to adjuvant therapeutic strategies.

cancer biology↗

bioRxiv2025

SNARE Protein Ykt6 Drives AMPAR Insertion at Synaptic Terminals During LTP

Long-Term Potentiation (LTP), a crucial form of synaptic plasticity essential for memory and learning, depends on protein synthesis and the upregulation of GluA1 at postsynaptic terminals. While extensive research has focused on the role of endosomal trafficking in GluA1 regulation, the contribution of endoplasmic reticulum (ER) trafficking pathways remains largely unexplored. A key opportunity to investigate this emerged from Ykt6, an evolutionarily conserved SNARE protein and a master regulator of vesicular fusion along ER-trafficking pathways. Here, we demonstrate that Ykt6 is highly expressed in the mammalian hippocampus, where it localizes to synaptic spines and regulates GluA1 surface expression in an LTP-dependent manner. Furthermore, we found that Ykt6 modulates synaptic vesicle pool dynamics as well as the amplitude and frequency of miniature excitatory postsynaptic currents. Ykt6 loss of function has been linked to -synuclein pathology, a hallmark of Lewy Body Dementias (LBDs), where -synuclein misfolding in the hippocampus disrupts LTP. Taken together, our findings establish Ykt6 as a critical SNARE protein in hippocampal function during LTP, with significant implications for neurodegenerative disorders such as LBDs.

neuroscience↗

bioRxiv2025

Cleavage of MALAT1 RNA by 14-nt sgRNA-guided tRNase ZL

We have been developing a gene suppression technology, tRNase ZL-utilizing efficacious (TRUE) gene silencing, in which artificially designed small guide RNA (sgRNA) guides tRNase ZL to cleave cellular target RNA. In this study, we examined 14-nt linear-type sgRNAs, which are fully 2'-O-methylated and have full phosphorothioate linkages, for their ability to suppress a level of a nuclear-localized long non-coding RNA, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1). The MALAT1 RNA is implied to be involved in stress responses and diseases including cancers. Specifically, we designed six 14-nt linear-type sgRNAs, sgRM1-sgRM6 that target the human MALAT1 RNA. sgRM1, sgRM2 and sgRM6 suppressed the MALAT1 RNA level, while the other sgRNAs showed little effect. In order to demonstrate that the suppression effect of sgRM1, sgRM2 and sgRM6 on the MALAT1 RNA level is caused by TRUE gene silencing, we performed in vitro tRNase ZL cleavage assay, microscopic analysis for nuclear existence of sgRNA, and tRNase ZL knockdown experiment. For the in vitro tRNase ZL cleavage assay, three 30-nt MALAT1 RNA fragments, TM1, TM2 and TM6 were prepared, which were RNA targets for sgRM1, sgRM2 and sgRM6, respectively. All of the sgRNAs guided recombinant tRNase ZL in vitro to cleave their own targets, although the cleavage efficiency changed depending on target/sgRNA pairs. By fluorescence microscopy, a 14-nt 5'-Alexa568-labeled sgRNA released from liposome was observed to be distributed ubiquitously in A549 cells with higher density in the nucleus, where both the target MALAT1 RNA and tRNase ZL exist. Knockdown of tRNase ZL by siRNA attenuated the suppression effect of sgRM1, sgRM2 and sgRM6 on the MALAT1 RNA level. We also demonstrated that the effective sgRNAs sgRM1, sgRM2 and sgRM6 reduce A549 cell viability.

molecular biology↗

bioRxiv2025

A bright synthetic near-infrared luciferin enhances the capabilities of deep-tissue bioluminescence imaging using firefly luciferases

Synthetic bioluminescence reactions exhibiting near-infrared (NIR)-shifted spectra have been explored to improve deep-tissue imaging through the design of firefly luciferin analogues. Although the NIR bioluminescence reactions improve the tissue penetration of bioluminescence signals from deep tissues, their photon output is markedly lower compared to the natural reaction with D-luciferin and firefly luciferase (Fluc), often by an order of magnitude or more. Consequently, in most instances, the sensitivity of NIR bioluminescence imaging (NIR-BLI) has not yet substantially surpassed that of BLI with the natural firefly reaction. Here, we present a synthetic firefly luciferin, named AkaSuke, that generates intense NIR bioluminescence ({lambda}max = 680 nm) in reaction with Fluc, greatly improving the detection sensitivity beyond that of the D-luciferin/Fluc reaction for targeting deep tissue. AkaSuke enables sensitive visualizations of ectopic hematogenesis through entire tissues of mice over time following transplantation of bone marrow stem cells labeled with Fluc. We additionally identify a Japanese firefly luciferase, DkumLuc1, that displays higher catalytic activities for bioluminescence emission of AkaSuke compared to typical Fluc, resulting in detection sensitivity comparable to that of AkaLumine/Akaluc reaction, one of the most sensitive bioluminescence systems for deep tissue imaging. We further propose the potential of the AkaSuke/DkumLuc1 reaction as an orthogonal pair with the AkaLumine/Akaluc for sensitive dual-target tracking in mice. Overall results suggest that AkaSuke enhances the capabilities of deep-tissue bioluminescence imaging using Fluc and its variant, and could serve as an emerging benchmark for the molecular design of NIR luciferin analogues.

cancer biology↗

bioRxiv2024

Single-Cell Temporal Atlas of Myeloid Cells in the Live Haemorrhagic Brain

Innate immune cells contribute to both secondary brain injury and repair following intracerebral hemorrhage (ICH). However, the specific signaling pathways that govern initial inflammatory and subsequent reparative myeloid programs in living patients remain poorly understood. To better characterize mononuclear phagocyte cell changes over time, we generated a single-cell transcriptomic dataset of paired hematoma clot evacuates and peripheral blood samples from 10 patients following ICH (5 - 290 hours). We identified distinct populations of activated and TNF-low microglia, as well as a unique, highly activated population of CD14+ monocytes in the hematoma. Perturbation analysis identified TNF signaling as the primary driver of hematoma monocyte activation. Custom temporal trajectory analysis using single-cell foundation model embeddings found that this TNF response in monocytes was transient, peaking early after hemorrhage and decreasing over the following 48 hours as monocytes shifted to reparative transcriptional programs. Transiently activated microglia emerged as the likely acute source of TNF, signaling through monocyte TNFR2. Surprisingly, acute TNF signaling in CD14+ monocytes was also associated with better severity-adjusted neurological outcomes both in our cohort and an independent validation cohort. These findings suggest acute TNF signaling between activated microglia and hematoma-associated monocytes, particularly through TNFR2, may contribute to recovery following ICH. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/630187v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@ff4fa0org.highwire.dtl.DTLVardef@1c4cc5borg.highwire.dtl.DTLVardef@1449761org.highwire.dtl.DTLVardef@2e7ea6_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Data-independent acquisition coupled with electron-activated dissociation for in-depth structure elucidation of fatty acid ester of hydroxy fatty acids

Fatty acid esters of hydroxy fatty acid (FAHFAs) are a biologically important class of lipids known for their anti-inflammatory and anti-diabetic effects in animals. The physiological activity of FAHFAs varies depending on the length of the carbon chain, number and position of double bonds (DBs), and the position of the hydroxyl (OH) group. Moreover, gut bacteria produce FAHFAs with more diverse structures than those produced by the host, which necessitates a FAHFA-lipidomics approach grasping their diverse structures to fully understand the physiological and metabolic significance of FAHFAs. In this study, we developed a methodology for in-depth structural elucidation of FAHFAs. First, FAHFAs were enriched using a solid-phase extraction (SPE) system coated with titanium and zirconium dioxide, which separated these analytes from neutral lipids and phospholipids. The fractionated metabolites were then derivatized using N,N-dimethylethylenediamine (DMED) to facilitate FAHFA detection in the positive ion mode of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. A data-independent acquisition technique known as sequential window acquisition of all theoretical mass spectra (SWATH-DIA) was used to collect sequential MS/MS spectra of the DMED-derivatized fatty acid metabolites. Structural elucidation was based on the fragment ions generated by electron-activated dissociation (EAD). DMED-FAHFAs were annotated using the newly updated MS-DIAL program, and FAHFA isomers were quantified using the MRMPROBS program, which quantifies lipids based on SWATH-MS/MS chromatograms. This procedure was applied to profile the FAHFAs present in mouse fecal samples, characterizing seven structures at the molecular species level, 63 structures at the OH position-resolved level, and 15 structures at both the DB and OH position-resolved levels using the MS-DIAL program. In the MRMPROBS analysis, 2OH and 3OH hydroxy fatty acids with more than 20 carbon atoms were predominantly expressed, while 5OH-13OH hydroxy fatty acids with 16 or 18 carbon atoms were the major components, abundant at positions 5, 7, 9, and 10. Furthermore, age-related changes in FAHFA isomers were also observed, where FAHFA 4:0/2O(FA 26:0) and FAHFA 16:0/10O(FA 16:0) significantly increased with age. In conclusion, our study offers a novel LC-SWATH-EAD-MS/MS technique with the updates of computational MS to facilitate in-depth structural lipidomics of FAHFAs. TOC graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/627939v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1d33c09org.highwire.dtl.DTLVardef@10a97b5org.highwire.dtl.DTLVardef@6e2c35org.highwire.dtl.DTLVardef@1186da5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Self-organization of spermatogenic wave coordinates sustained sperm production in the mouse testis

Spermatogenesis takes place in the testis, relying on the ordered turnover of differentiating cells supplied from stem cells. Classic histological analyses have revealed that this process shows hierarchical spatiotemporal patterning known as the spermatogenic cycle, wave, and descent of segmental order, indicative of currently underexplored mechanisms of tissue- and organ-scale homeostasis. Here, using mice, we conducted high-resolution, wide-field, and ultra long-term live imaging studies in vivo and ex vivo, combined with whole-organ mapping of differentiation stages. Such trans-scale measures demonstrate how stereotypic local cell turnover is coordinated into characteristic phase waves propagating along the seminiferous tubules, further organized into organ-scale patterning over the tubule loops. Minimal mathematical modeling shows that such higher-order dynamics can emerge from the local coupling of autonomous oscillators, which are rooted in delayed feedback interplay between stem and differentiating cells via retinoic acid signaling. These findings highlight a self-organization mechanism underpinning organ-scale homeostasis and constant sperm production.

developmental biology↗

bioRxiv2024

Cholesterol sulfate prevents maternal-fetal conflict by locally modulating immune reactivity

Although placental mammals are at high risk of maternal-fetal immune conflict, it is unclear how the semi-allogeneic fetus avoids rejection. Cholesterol sulfate (CS) is a bioactive metabolite that inhibits leukocyte migration and activation. This study investigated the roles and cells associated with CS in pregnant mice and humans. CS was sequentially produced by maternal-derived endometrial cells and fetal-derived placental trophoblasts before and after placentation. When mated with allogeneic males, CS-deficient mice showed increased fetal resorption rates under induced placental inflammation. This phenotype disappeared when the activity of the CS-producing enzyme was restored in the placenta. Placental CS levels were reduced in patients with "villitis of unknown etiology." Thus, we uncovered the spatiotemporal control of CS production and its relevance to local immunosuppression during pregnancy.

immunology↗

bioRxiv2024

Dnmt1 determines bone length by regulating energy metabolism of growth plate chondrocytes

Chondrocytes differentiated from mesenchymal stem cells play a role in determining skeletal patterns by ossification. However, the mechanism by which maintenance DNA methylation in chondrocytes regulates differentiation and skeletal formation is unclear. In the Musculoskeletal Knowledge Portal, Dnmt1 was significantly associated with "Height". Long bones in the limbs of Dnmt1-deficient (Dnmt1{Delta}Prx1) mice are significantly shortened due to decreased chondrocyte proliferation and accelerated differentiation. Integrated analysis of RNA-Seq and MBD-Seq revealed that in Dnmt1{Delta}Prx1 chondrocytes reduced DNA methylation resulted in increased expression of genes related to energy metabolism and to ossification. Metabolomic analyses confirmed that levels of nearly all energy metabolites were increased in Dnmt1{Delta}Prx1 chondrocytes. These results indicate that Dnmt1-mediated maintenance DNA methylation governs chondrocyte differentiation by regulating energy metabolism through both gene expression and modulation of metabolite supplies. Taken together, this study suggests that appropriate DNA methylation status in chondrocytes can orchestrate growth plate mineralization and subsequently determine bone length.

developmental biology↗

bioRxiv2024

Systematic Targeting of Protein Complexes with Molecular COUPLrs

Molecular glues that engage protein complexes have transformed the study of cell biology and have had a direct impact on clinical oncology. However, the identification of new glue classes and their corresponding protein complexes has remained largely serendipitous. To overcome this challenge, we report the development of molecular COUPLrs, elaborated small molecules flanked by two cysteine-reactive warheads, as well as CONNECT, an integrated chemical proteomic platform for target deconvolution. By profiling a library of molecular COUPLrs across 13 cancer cell lines, we uncovered hundreds of proteins that can be coupled together, including in some cases in mutant selective fashions. We develop an advanced COUPLr for the oncogene EML4-ALK, which engages the fusion outside of its kinase domain, restricts protein dynamics, and disrupts EML4-ALK signaling. Collectively, molecular COUPLrs substantially expand the scope of proteins that can be chemically connected, providing an unbiased approach to identify small molecules that target protein complexes.

systems biology↗

bioRxiv2024

LAG-3 blockade reactivates the CD8+ T cell expansion program to re-expand contracted clones in the tumor

Effective cancer immunotherapy relies on the clonal proliferation and expansion of CD8+ T cells in the tumor1,2. However, our insights into clonal expansions are limited, owing to an inability to track the same clones in tumors over time. Here, we developed a multi-tumor mouse model system to track hundreds of expanding and contracting CD8+ T cell clones over multiple timepoints in tumors of the same individual. Through coupling of clonal expansion dynamics and single-cell RNA/TCR-seq data, we identified a transcriptomic signature in PD-1+Ly108+ precursor exhausted cells3,4 that strongly predicts rates of intratumoral clone expansion in mice and humans. We found that expression of the signature successfully stratifies melanoma patient outcomes to PD-1/PD-L1 blockade5,6. Downregulation of the signature precedes clone contraction - a phase in which clones contract but maintain revivable precursor exhausted cells in the tumor. LAG-3 blockade - an FDA-approved therapy whose effects on CD8+ T cell responses are currently unclear7, re-activates the expansion signature, re-expanding pre-existing clones, including previously contracted clones. These findings reveal how the study of clonal expansion dynamics provide a powerful pan-immunotherapy signature for monitoring immunotherapies, including PD-1/PD-L1 and LAG-3 blockade, with implications for their future development.

immunology↗

bioRxiv2024

Polyphyletic domestication and inter-lineage hybridization magnified genetic diversity of cultivated melon, Cucumis melo L.

A total of 212 melon accessions with diverse geographical origins were classified into large and small seed-types by length of seed at the boundary of 9 mm, and into five populations based on polymorphisms in the nuclear genome. They were further divided into three maternal lineages, Ia, Ib, and Ic, by polymorphisms in the chloroplast genome. By combining these three classifications, the Europe/US subsp. melo and the East Asian subsp. agrestis were characterized as [large seed, Ib, PopA1 or A2] and [small seed, Ia, PopB1 or B2], respectively, indicating nearly perfect divergence in both nuclear and cytoplasm genomes. In contrast, in South and Southeast Asia, in addition to the Europe/US and East Asian types, recombinant types were also frequently found, indicating unclear genetic differentiation in South and Southeast Asia. Such an intermixed structure of genetic variation supported the Indian origin of Ia and Ib types of melon. Seed length was intermediate, between the large and small seed-types, and chloroplast type was a mixture of Ia and Ib in Momordica, suggesting its origin from the recombinant type. In Africa, three lineages of melon were distributed allopatrically and showed distinct divergence. Subsp. agrestis of the Ic type proved to be endemic to Africa, indicating its African origin.

evolutionary biology↗

bioRxiv2024

Structural robustness and temporal vulnerability of the starvation-responsive metabolic network in liver of healthy and obese mice

Adaptation to starvation is a multi-molecular and temporally ordered process, that could be impaired in obesity. To elucidate how the healthy liver regulates various molecules in a temporally ordered manner during starvation and how obesity disrupts this process, we measured time course multiomic data in the liver of wild-type (WT) and leptin-deficient obese (ob/ob) mice during starvation. Using the measured data, we constructed a starvation-responsive metabolic network, that is a transomic network including responsive molecules and their regulatory relationships during starvation, and analyzed the structure of the network. In WT mice, ATP and AMP, the energy indicators, regulated various metabolic reactions in the network as the hub molecules, both of which were not responsive in ob/ob mice. However, the structural properties of the network were maintained in ob/ob mice. In WT mice, the molecules in the network were temporally ordered through metabolic process coordinated by the hub molecules including ATP and AMP and were positively or negatively co-regulated. By contrast, both temporal order and co-regulation were disrupted in ob/ob mice. Taken together, the starvation-responsive metabolic network is structurally robust, but temporally vulnerable by the loss of responsiveness of the hub molecules in obesity. In addition, we proposed a potential therapeutic target to treat the negative effects of obesity on intermittent fasting to extend lifespan. One Sentence SummaryHub molecules activate or inhibit various molecules in a temporally ordered manner in healthy liver, and the regulatory network is structurally robust but temporally vulnerable to obesity.

systems biology↗

bioRxiv2024

Co-utilization of microalgae and heterotrophic microorganisms improves wastewater treatment efficiency

Wastewater treatment using co-culture systems of microalgae and heterotrophic microorganisms is expected to be useful under atmospheric dilute carbon dioxide conditions. In this study, we investigated the combination of microalgae and heterotrophic microorganisms to improve the efficiency of wastewater treatment. Furthermore, to elucidate the cause of the changes in wastewater treatment efficiency in the co-culture system, changes in gene expression were revealed through transcriptome analysis. Three types of microalgae and five heterotrophic microorganisms were used in combination for wastewater treatment. The combination of Chlamydomonas reinhardtii NIES-2238 and Saccharomyces cerevisiae SH-4 showed the highest wastewater treatment efficiency. Using this combination for artificial wastewater treatment, the removal rates of TOC (Total organic carbon), PO43-, and NH4+ reached 80%, 93%, and 63%, respectively, after 18 h of treatment. Transcriptome analysis revealed that the combined wastewater treatment altered the expression of 1371 and 692 genes in C. reinhardtii and S. cerevisiae, respectively. The genes upregulated in C. reinhardtii included those related to molecular and ion transport. Genes upregulated in S. cerevisiae included those related to cell protection from various types of damage and stress. To the best of our knowledge, this is the first study to show that a combination of green algae and yeast improves the efficiency of wastewater treatment. As both the green alga C. reinhardtii and the yeast S. cerevisiae are highly safe microorganisms, the establishment of their effective combination for wastewater treatment is highly significant.

bioengineering↗

bioRxiv2024

Asymmetric Engagement of Dimeric CRL3KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes

UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal1, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition2,3. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.

molecular biology↗

bioRxiv2024

Visualization of intracellular ATP dynamics in the whole kidney under pathophysiological conditions using the kidney slice culture system

ATP depletion plays a central role in the pathogenesis of kidney diseases. Recently, we reported spatiotemporal intracellular ATP dynamics during ischemia reperfusion (IR) using GO-ATeam2 mice systemically expressing an ATP biosensor. However, observation from the kidney surface did not allow visualization of deeper nephrons or accurate evaluation of ATP synthesis pathways. Here we established a novel ATP imaging system using slice culture of GO-ATeam2 mouse kidneys, evaluated ATP synthesis pathway, and analyzed intracellular ATP dynamics using ex vivo IR and cisplatin nephropathy models. We demonstrated that proximal tubules (PTs) were strongly dependent on oxidative phosphorylation (OXPHOS), whereas podocytes relied on both OXPHOS and glycolysis using ATP synthesis inhibitors. We also confirmed that the ex vivo IR model could recapitulate ATP dynamics in vivo; ATP recovery in PTs after reperfusion varied depending on ischemia length, whereas ATP in distal tubules (DTs) recovered well even after long ischemia. After cisplatin administration, ATP levels in PTs decreased first, followed by a decrease in DTs. An organic cation transporter 2 inhibitor suppressed cisplatin uptake in kidney slices, leading to better ATP recovery in PTs, but not in DTs. Finally, we confirmed that a mitochondria protection reagent delayed cisplatin-induced ATP decrease in PTs. This novel system may provide new insights into the energy dynamics and pathogenesis of kidney disease.

molecular biology↗