Biology subjects

Hirano, M.

Publications and source records attributed to Hirano, M..

32 records · Page 2Linked to original sources

bioRxiv2022

Chronic Glucocorticoid Stress Reveals Increased Energy Expenditure and Accelerated Aging as Cellular Features of Allostatic Load

Stress triggers anticipatory physiological responses that promote survival, a phenomenon termed allostasis. However, the chronic activation of energy-dependent allostatic responses results in allostatic load, a dysregulated state that predicts functional decline, accelerates aging, and increases mortality in humans. The energetic cost and cellular basis for the damaging effects of allostatic load have not been defined. Here, by longitudinally profiling three unrelated primary human fibroblast lines across their lifespan, we find that chronic glucocorticoid exposure increases cellular energy expenditure by [~]60%, along with a metabolic shift from glycolysis to mitochondrial oxidative phosphorylation (OxPhos). This state of stress-induced hypermetabolism is linked to mtDNA instability, non-linearly affects age-related cytokines secretion, and accelerates cellular aging based on DNA methylation clocks, telomere shortening rate, and reduced lifespan. Pharmacologically normalizing OxPhos activity while further increasing energy expenditure exacerbates the accelerated aging phenotype, pointing to total energy expenditure as a potential driver of aging dynamics. Together, our findings define bioenergetic and multi-omic recalibrations of stress adaptation, underscoring increased energy expenditure and accelerated cellular aging as interrelated features of cellular allostatic load.

cell biology↗

bioRxiv2022

Active haptic training improves somatosensory-motor feedback control in the motor skills of experts

We perceive the external world through both externally generated and self-generated sensory stimuli (i.e., passive and active perception). While the performance in passive perception is improved by training or repetition through the functional and structural reorganization of the central nervous system (i.e., perceptual learning), the mechanisms by which perceptual learning occurs in active perception remain unclear. Here, we sought to explore the mechanisms underlying the improvement of active somatosensory perception and sensorimotor skills through active perceptual learning. Because we previously found that active perceptual learning depends on the expertise of the motor task to be performed, this study focused primarily on trained individuals. To this end, active haptic training (AHT) that targets the active somatosensory perception during the piano keystroke was used as a means of inducing active perceptual learning. We found four main results. First, participants actively modulated the muscle coordination patterns to optimize the movements required by the task through AHT without any explicit instruction on it by the experimenter, suggesting the involvement of active exploration in active perceptual learning. Second, AHT increased the relative reliance on afferent sensory information relative to the predicted one during the piano keystroke. Third, perceptual sensitivity of externally generated keystroke motions remained unchanged through AHT. Finally, AHT improved feedback control of repetitive keystroke movements in expert pianists. These results suggest that active perceptual learning involves changes in both the predictive integration process and active exploration and that the improved feedback control of fine movements benefits from the improvement in active perception. Significant statementOur ability to perceive both externally generated and self-generated sensory stimuli can be enhanced through training, known as passive and active perceptual learning. Here, we sought to explore the mechanisms underlying active perceptual learning by using active haptic training (AHT), which has been demonstrated to enhance the somatosensory perception of a finger in a trained motor skill. First, AHT reorganized the muscular coordination during the piano keystroke. Second, AHT increased the relative reliance on afferent sensory information relative to predicted one, in contrast to no increment of overall perceptual sensitivity. Finally, AHT improved feedback movement control of keystrokes. These results suggest that active perceptual learning involves active exploration and adaptation of predictive sensory integration, which underlies the co-enhancement of active perception and feedback control of movements.

neuroscience↗

bioRxiv2022

The RNA helicase DDX39B activates FOXP3 RNA splicing to control T regulatory cell fate

Genes associated with increased susceptibility to multiple sclerosis (MS) have been identified, but their functions are incompletely understood. One of these genes codes for the RNA helicase DExD/H-Box Polypeptide 39B (DDX39B), which shows genetic and functional epistasis with interleukin-7 receptor- gene (IL7R) in MS-risk. Based on evolutionary and functional arguments we postulated that DDX39B enhances immune tolerance decreasing MS risk. Consistent with such a role we show that DDX39B controls the expression of many MS susceptibility genes and important immune-related genes. Among these we identified ForkheadBox P3 (FOXP3), which codes for the master transcriptional factor in CD4+/CD25+ T regulatory cells. DDX39B knockdown led to loss of immune-regulatory and gain of immune-effector expression signatures. Splicing of FOXP3 introns, which belong to a previously unrecognized subclass of introns with C-rich polypyrimidine tracts, was exquisitely sensitive to DDX39B levels. Given the importance of FOXP3 in autoimmunity, this work cements DDX39B as an important guardian of immune tolerance.

molecular biology↗

bioRxiv2021

Leukocyte cytokine responses in adult patients with mitochondrial DNA defects

Patients with oxidative phosphorylation (OxPhos) defects causing mitochondrial diseases appear particularly vulnerable to infections. Although OxPhos defects modulate cytokine production in vitro and in animal models, little is known about how circulating leukocytes of patients with inherited mitochondrial DNA (mtDNA) defects respond to acute immune challenges. In a small cohort of healthy controls (n=21) and patients (n=12) with either the m.3243A>G mutation or single, large-scale mtDNA deletions, we examined: i) cytokine responses (IL-6, TNF-, IL-1{beta}) in response to acute lipopolysaccharide (LPS) exposure, and ii) sensitivity to the immunosuppressive effects of glucocorticoid signaling (dexamethasone) on cytokine production. In dose-response experiments to determine the half-maximal effective LPS concentration (EC50), relative to controls, leukocytes from patients with mtDNA deletions showed 74 - 79% lower responses for IL-6 and IL-1{beta} (pIL-6=0.031, pIL-1{beta}=0.009). Moreover, IL-6 response to LPS in presence of GC was also blunted in cells from patients with mtDNA deletions (pIL-6=0.006), but not in leukocytes from patients with the m.3243A>G mutation. Overall, these ex vivo data provide preliminary evidence that some systemic OxPhos defects may compromise immune cytokine responses and glucocorticoid sensitivity. Further work in larger cohorts is needed to define the nature of immune dysregulation in patients with mitochondrial disease, and their potential implications for disease phenotypes.

immunology↗

bioRxiv2021

OxPhos Dysfunction Causes Hypermetabolism and Reduces Lifespan in Cells and in Patients with Mitochondrial Diseases

Patients with primary mitochondrial diseases present with fatigue and multi-system disease, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. Integrating data from 17 cohorts of patients with mitochondrial diseases (n=690), we find that clinical mitochondrial disorders increase resting energy expenditure, a state termed hypermetabolism. In a longitudinal cellular model of primary patient-derived fibroblasts from multiple donors, we show that genetic and pharmacological disruptions of oxidative phosphorylation (OxPhos) similarly trigger increased energy consumption in a cell-autonomous manner, despite near-normal OxPhos coupling efficiency. Hypermetabolism is associated with mtDNA instability, activation of the integrated stress response, increased extracellular secretion of age-related cytokines and metabokines including GDF15, as well as an accelerated rate of telomere erosion and epigenetic aging, and a reduced Hayflick limit. Together with these dynamic measures, we have generated a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations in response to OxPhos dysfunction. The increased energetic cost of living, or hypermetabolism, in cells and organisms with OxPhos defects has important biological and clinical implications.

cell biology↗

bioRxiv2021

A Multi-Omics and Bioenergetics Longitudinal Aging Dataset in Primary Human Fibroblasts with Mitochondrial Perturbations

Aging is a process of progressive change. In order to develop biological models of aging, longitudinal datasets with high temporal resolution are needed. Here we report a multi-omic longitudinal dataset for cultured primary human fibroblasts measured across their replicative lifespans. Fibroblasts were sourced from both healthy donors (n=6) and individuals with lifespan-shortening mitochondrial disease (n=3). The dataset includes cytological, bioenergetic, DNA methylation, gene expression, secreted proteins, mitochondrial DNA copy number and mutations, cell-free DNA, telomere length, and whole-genome sequencing data. This dataset enables the bridging of mechanistic processes of aging as outlined by the "hallmarks of aging", with the descriptive characterization of aging such as epigenetic age clocks. Here we focus on bridging the gap for the hallmark mitochondrial metabolism. Our dataset includes measurement of healthy cells, and cells subjected to over a dozen experimental manipulations targeting oxidative phosphorylation (OxPhos), glycolysis, and glucocorticoid signaling, among others. These experiments provide opportunities to test how cellular energetics affect the biology of cellular aging. All data are publicly available at our webtool: https://columbia-picard.shinyapps.io/shinyapp-Lifespan_Study/

cell biology↗

bioRxiv2021

A comprehensive analysis of gene expression changes in a high replicate and open-source dataset of differentiating hiPSC-derived cardiomyocytes

We performed a comprehensive analysis of the transcriptional changes within and across cell populations during human induced pluripotent stem cell (hiPSC) differentiation to cardiomyocytes. Using the single cell RNA-seq combinatorial barcoding method SPLiT-seq, we sequenced >20,000 single cells from 55 independent samples representing two differentiation protocols and multiple hiPSC lines. Samples included experimental replicates ranging from undifferentiated hiPSCs to mixed populations of cells at D90 post-differentiation. As expected, differentiated cell populations clustered by time point, with differential expression analysis revealing markers of cardiomyocyte differentiation and maturation changing from D12 to D90. We next performed a complementary cluster-independent sparse regression analysis to identify and rank genes that best assigned cells to differentiation time points. The two highest ranked genes between D12 and D24 (MYH7 and MYH6) resulted in an accuracy of 0.84, and the three highest ranked genes between D24 and D90 (A2M, H19, IGF2) resulted in an accuracy of 0.94, revealing that low dimensional gene features can identify differentiation or maturation stages in differentiating cardiomyocytes. Expression levels of select genes were validated using RNA FISH. Finally, we interrogated differences in differentiation population composition and cardiac gene expression resulting from two differentiation protocols, experimental replicates, and three hiPSC lines in the WTC-11 background to identify sources of variation across these experimental variables.

cell biology↗

bioRxiv2021

Early Dynamics of Chronic Myeloid Leukemia on Nilotinib Predicts Deep Molecular Response

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by the BCR-ABL1 tyrosine kinase1,2. ABL1-selective tyrosine kinase inhibitors (TKIs) including nilotinib have dramatically improved the prognosis of patients with CML3-7. The ultimate goal of CML treatment is likely to be TKI-free maintenance of deep molecular response (DMR), which is defined by quantitative measurement of BCR-ABL1 transcripts on the international scale (IS)8, and durable DMR is a prerequisite to reach this goal9. Thus, an algorithm to enable the early prediction of DMR achievement on TKI therapy is quite valuable. Here, we show that our mathematical framework based on a clinical trial dataset10 can accurately predict the response to frontline nilotinib. We found that our simple dynamical model can predict nilotinib response by using two common laboratory findings (observation values): IS11,12 and white blood cell (WBC) count. Furthermore, our proposed method identified patients who failed to achieve DMR with high fidelity according to the data collected only at three initial time points during nilotinib therapy. Since our model relies on the general properties of TKI response, our framework would be applicable to CML patients who receive frontline nilotinib or other TKIs in clinical practice. Significance StatementChronic myeloid leukemia (CML) is a myeloproliferative disorder caused by the BCR-ABL1 tyrosine kinase. The goal of this treatment is the sequential achievement of deep molecular response (DMR). Tyrosine kinase inhibitors (TKIs) are effective in the reduction because they inhibit CML cell proliferation. However, because of individual differences in the TKI efficacy, some patients are unable to achieve DMR, so that early prediction of DMR reachability is necessary for personalized medicine. By combining time series analysis and mathematical modeling, we developed a mathematical method that accurately predicts patients who do not achieve DMR in the early stages of TKI administration. Our prediction method gives a basis of effective personalized treatments for CML patients.

systems biology↗

bioRxiv2020

Synergistic effect of deoxynucleosides and AAV gene therapy for thymidine kinase 2 deficiency

Autosomal recessive thymidine kinase 2 (TK2) mutations causes TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants and children. Treatment with deoxycytidine and thymidine ameliorates mitochondrial defects and extends lifespan of Tk2 knock-in mouse (TK2-/-); however, efficacy is limited by age- and tissue-dependent expression of the cytosolic enzymes Tk1 and Dck. Thus, therapies aimed at systemic restoration of TK2 activity are needed. Here, we demonstrate that delivery of human TK2 cDNA to Tk2-/- mice using AAV9 efficiently rescued Tk2 activity in all the tissues tested except kidney, delayed disease onset, and increased lifespan. Sequential treatment of Tk2-/- mice with AAV9 first followed by AAV2 at different ages allowed us to reduce the viral dose while further prolonging the lifespan. Furthermore, addition of deoxycytidine and deoxythymidine supplementation to AAV9 + AAV2 treated Tk2-/- mice dramatically improved mtDNA copy numbers in liver and kidney, animal growth, and lifespan. These data indicate that combined pharmacological and gene therapies may be highly efficacious for human TK2 deficiency.

genetics↗

bioRxiv2020

Spatial and single-cell transcriptional landscape of human cerebellar development

ABSTRACTCerebellar development and function require precise regulation of molecular and cellular programs to coordinate motor functions and integrate network signals required for cognition and emotional regulation. However, molecular understanding of human cerebellar development is limited. Here, we combined spatially resolved and single-cell transcriptomics to systematically map the molecular, cellular, and spatial composition of early and mid-gestational human cerebellum. This enabled us to transcriptionally profile major cell types and examine the dynamics of gene expression within cell types and lineages across development. The resulting ‘Developmental Cell Atlas of the Human Cerebellum’ demonstrates that the molecular organization of the cerebellar anlage reflects cytoarchitecturally distinct regions and developmentally transient cell types that are insufficiently captured in bulk transcriptional profiles. By mapping disease genes onto cell types, we implicate the dysregulation of specific cerebellar cell types, especially Purkinje cells, in pediatric and adult neurological disorders. These data provide a critical resource for understanding human cerebellar development with implications for the cellular basis of cerebellar diseases.Competing Interest StatementA.B.R, C.R., and G.Se. are shareholders of Split Bioscience.View Full Text

developmental biology↗

bioRxiv2020

Cell states beyond transcriptomics: integrating structural organization and gene expression in hiPSC-derived cardiomyocytes

We present a quantitative co-analysis of RNA abundance and sarcomere organization in single cells and an integrated framework to predict subcellular organization states from gene expression. We used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes expressing mEGFP-tagged alpha-actinin-2 to develop quantitative image analysis tools for systematic and automated classification of subcellular organization. This captured a wide range of sarcomeric organization states within cell populations that were previously difficult to quantify. We performed RNA FISH targeting genes identified by single cell RNA sequencing to simultaneously assess the relationship between transcript abundance and structural states in single cells. Co-analysis of gene expression and sarcomeric patterns in the same cells revealed biologically meaningful correlations that could be used to predict organizational states. This study establishes a framework for multi-dimensional analysis of single cells to study the relationships between gene expression and subcellular organization and to develop a more nuanced description of cell states. Graphical AbstractTranscriptional profiling and structural classification was performed on human induced pluripotent stem cell-derived cardiomyocytes to characterize the relationship between transcript abundance and subcellular organization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/081083v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@8f89c4org.highwire.dtl.DTLVardef@19dc813org.highwire.dtl.DTLVardef@1ba7c20org.highwire.dtl.DTLVardef@2b3daa_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Topoisomerase III-beta is required for efficient replication of positive-sense RNA viruses

Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-{beta} (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.

microbiology↗

bioRxiv2019

Microbial single-cell RNA sequencing by split-pool barcoding

Single-cell RNA-sequencing (scRNA-seq) has become an essential tool for characterizing multi-celled eukaryotic systems but current methods are not compatible with bacteria. Here, we introduce microSPLiT, a low cost and high-throughput scRNA-seq method that works for gram-negative and gram-positive bacteria and can resolve transcriptional states that remain hidden at a population level. We applied microSPLiT to >25,000 Bacillus subtilis cells sampled from different growth stages, creating a detailed atlas of changes in metabolism and lifestyle. We not only retrieve detailed gene expression profiles associated with known but rare states such as competence and PBSX prophage induction, but also identify novel and unexpected gene expression states including heterogeneous activation of a niche metabolic pathway in a subpopulation of cells. microSPLiT empowers high-throughput analysis of gene expression in complex bacterial communities.

bioengineering↗

bioRxiv2019

Human Aging DNA Methylation Signatures are Conserved but Accelerated in Cultured Fibroblasts

Aging is associated with progressive and site-specific changes in DNA methylation (DNAm). These global changes are captured by DNAm clocks that accurately predict chronological age in humans but relatively little is known about how clocks perform in vitro. Here we culture primary human fibroblasts across the cellular lifespan ([~]6 months) and use four different DNAm clocks to show that age-related DNAm signatures are conserved and accelerated in vitro. The Skin & Blood clock shows the best linear correlation with chronological time (r=0.90), including during replicative senescence. Although similar in nature, the rate of epigenetic aging is approximately 62x times faster in cultured cells than in the human body. Consistent with in vivo data, cells aged under hyperglycemic conditions exhibit an approximately three years elevation in baseline DNAm age. Moreover, candidate gene-based analyses further corroborate the conserved but accelerated biological aging process in cultured fibroblasts. Fibroblasts mirror the established DNAm topology of the age-related ELOVL2 gene in human blood and the rapid hypermethylation of its promoter cg16867657, which correlates with a linear decrease in ELOVL2 mRNA levels across the lifespan. Using generalized additive modeling on twelve timepoints across the lifespan, we also show how single CpGs exhibit loci-specific, linear and nonlinear trajectories that reach rates up to -47% (hypomethylation) to +23% (hypermethylation) per month. Together, these high temporal resolution global, gene-specific, and single CpG data highlight the conserved and accelerated nature of epigenetic aging in cultured fibroblasts, which may constitute a system to evaluate age-modifying interventions across the lifespan.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC=\"FIGDIR/small/605295_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (60K):\norg.highwire.dtl.DTLVardef@101e4f2org.highwire.dtl.DTLVardef@15df049org.highwire.dtl.DTLVardef@4a704dorg.highwire.dtl.DTLVardef@112f970_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract\n\nC_FIG

cell biology↗