Biology subjects

Nakamura, K.

Publications and source records attributed to Nakamura, K..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Mitochondrial protein FKBP8 captures PDZD8 to form mitochondria-ER contacts

Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identified the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-Electron Microscopy (Cryo-EM) tomography, and correlative light-EM (CLEM). Single molecule tracking revealed highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and capture at MERCS. Overexpression of FKBP8 was sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrated their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.

cell biology↗

bioRxiv2023

Identification of Core Yeast Species and Microbe-Microbe Interactions Impacting Larval Growth of Drosophila in the Wild

Microbiota consisting of various fungi and bacteria have a significant impact on the physiological functions of the host. However, it is unclear which species are essential to this impact and how they affect the host. This study analyzed and isolated microbes from natural food sources of Drosophila larvae, and investigated their functions. Hanseniaspora uvarum is the predominant yeast responsible for larval growth in the earlier stage of fermentation. As fermentation progresses, Acetobacter orientalis emerges as the key bacterium responsible for larval growth, although yeasts and lactic acid bacteria must coexist along with the bacterium to stabilize this host-bacterial association. By providing nutrients to the larvae in an accessible form, the microbiota contributes to the upregulation of various genes that function in larval cell growth and metabolism. Thus, this study elucidates the key microbial species that support animal growth under microbial transition.

developmental biology↗

bioRxiv2023

The Neonatal Gyrencephalic Cortex Maintains Regionally Distinct Streams of Neuroblasts

Neurodevelopmental mechanisms have evolved to support the formation of diverse brain structures, such as in humans, during the perinatal period. Here, we demonstrate that neonatal gyrencephalic brains harbor an expanded subventricular zone, termed the Arc, defined by tiered arrangement of doublecortin (DCX)-expressing neuroblasts and vascular enrichment at the ventricular wall. The Arc is the origin of dorsal and ventral populations of migratory neuroblasts that target multiple regions involved in higher cognitive functions. Arc-derived migratory streams, primarily from the caudal ganglionic eminence, are composed of diverse neuronal subtypes with distinct spatial and migratory-receptor profiles. Our findings indicate the Arc is a structure present in phylogenetically divergent species that supports the expansion of postnatal neuronal migration, contributing to a protracted formation of cortical circuits in gyrencephalic brains. One-Sentence SummaryThe ventricular cytoarchitecture of gyrencephalic brains supports an ongoing supply of migratory neurons to the neonatal cortex.

neuroscience↗

bioRxiv2023

NEMO reshapes the protein aggregate interface and promotes aggrephagy by co-condensation with p62

NEMO is a ubiquitin-binding protein which regulates canonical NF-{kappa}B pathway activation in innate immune signaling, cell death regulation and host-pathogen interactions. Here we identified an NF-{kappa}B-independent function of NEMO in proteostasis regulation by promoting autophagosomal clearance of protein aggregates. NEMO-deficient cells accumulate misfolded proteins upon proteotoxic stress and are vulnerable to proteostasis challenges. Moreover, a patient with a mutation in the NEMO gene resulting in defective binding of NEMO to linear ubiquitin chains, developed a widespread mixed brain proteinopathy, including -synuclein, tau and TDP-43 pathology. NEMO amplifies linear ubiquitylation at -synuclein aggregates and promotes the local concentration of p62 into foci. In vitro, NEMO lowers the threshold concentrations required for ubiquitin-dependent phase transition of p62. In summary, NEMO reshapes the aggregate surface for efficient autophagosomal clearance by providing a mobile phase at the aggregate interphase favoring co-condensation with p62.

cell biology↗

bioRxiv2023

Infant attachment behaviors reflect the parenting style of individual caregiver in common marmosets

Childrens physical, cognitive, and emotional maturation require adequate interactions and secure attachment with their primary caregivers. However, the causal relationships between parenting components and infant attachment behaviors remain unclear. New World monkey common marmosets provide an ideal model of human parent-infant relations because, like humans, infant attachment is shared among family caregivers, including parents and older siblings using intricate vocal communications. Combining the natural variations in parenting styles and subsecond-scale microanalyses of dyadic vocal and physical interactions, we demonstrate that marmoset infants signal their need by context-dependent call use and selectively maintain proximity with familiar caregivers. The infant attachment behaviors are individually tuned according to the caregivers parenting style; infants keep producing isolation calls when they are carried by rejecting caregivers and exhibit avoidance toward neglectful and rejecting caregivers. Family-deprived infants failed to develop such adaptive uses of attachment behaviors or age-appropriate autonomy and exhibited disorganized attachment behaviors. Thus, marmoset infants shape their ability to modulate their intricate attachment system depending on the caregivers attitude through early social interactions with their family caregivers. Demonstrating the significant similarity of the infant attachment system between marmosets and humans, these findings will pave a path to elucidating the neural mechanisms of the infant attachment system.

animal behavior and cognition↗

bioRxiv2023

Two Ascending Thermosensory Pathways from the Lateral Parabrachial Nucleus That Mediate Behavioral and Autonomous Thermoregulation

Thermoregulatory behavior in homeothermic animals is an innate behavior to defend body core temperature from environmental thermal challenges in coordination with autonomous thermoregulatory responses. In contrast to the progress in understanding the central mechanisms of autonomous thermoregulation, those of behavioral thermoregulation remain poorly understood. We have previously shown that the lateral parabrachial nucleus (LPB) mediates cutaneous thermosensory afferent signaling for thermoregulation. To understand the thermosensory neural network for behavioral thermoregulation, in the present study, we investigated the roles of ascending thermosensory pathways from the LPB in avoidance behavior from innocuous heat and cold in rats. Neuronal tracing revealed two segregated groups of LPB neurons projecting to the median preoptic nucleus (MnPO), a thermoregulatory center (LPB[->]MnPO neurons), and those projecting to the central amygdaloid nucleus (CeA), a limbic emotion center (LPB[->]CeA neurons). While LPB[->]MnPO neurons include separate subgroups activated by heat or cold exposure of rats, LPB[->]CeA neurons were only activated by cold exposure. By selectively inhibiting LPB[->]MnPO or LPB[->]CeA neurons using tetanus toxin light chain or chemogenetic or optogenetic techniques, we found that LPB[->]MnPO transmission mediates heat avoidance, whereas LPB[->]CeA transmission contributes to cold avoidance. In vivo electrophysiological experiments showed that skin cooling-evoked thermogenesis in brown adipose tissue requires not only LPB[->]MnPO neurons but also LPB[->]CeA neurons, providing a novel insight into the central mechanism of autonomous thermoregulation. Our findings reveal an important framework of central thermosensory afferent pathways to coordinate behavioral and autonomous thermoregulation and to generate the emotions of thermal comfort and discomfort that drive thermoregulatory behavior.

neuroscience↗

bioRxiv2023

Oncogenic drivers dictate immune control of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a genetically heterogeneous, aggressive hematological malignancy induced by distinct oncogenic driver mutations. The effect of specific AML oncogenes on immune activation or suppression has not been investigated. Here, we examine immune responses in genetically distinct models of AML and demonstrate that specific AML oncogenes dictate immunogenicity, the quality of immune response and immune escape through immunoediting. Specifically, expression of NrasG12D alone is sufficient to drive a potent anti-leukemia response through increased MHC Class II expression that can be overcome with increased expression of Myc. These data have important implications for the design and implementation of personalized immunotherapies for patients with AML. Statement of SignificanceThe endogenous immune response against acute myeloid leukemia (AML) is determined by leukemia-specific oncogenic driver mutations. Mutant Nras drives immunological selection of AML.

cancer biology↗

bioRxiv2022

Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

Old animals display significant alterations in sleep-wake patterns such as increases in sleep fragmentation and sleep propensity. Here we demonstrated that dorsomedial hypothalamus-specific PR-domain containing protein 13-knockout (DMH-Prdm13-KO) mice recapitulated age-associated sleep alterations such as sleep fragmentation and increased sleep attempts during sleep deprivation (SD). These phenotypes were further exacerbated during aging, with increased adiposity and decreased physical activity, resulting in shortened lifespan. Dietary restriction (DR), a well-known anti-aging intervention in diverse organisms, ameliorated age-associated sleep alterations, whereas these effects of DR were abrogated in DMH-Prdm13-KO mice. Moreover, overexpression of Prdm13 in the DMH ameliorated sleep fragmentation and excessive sleepiness during SD in old mice. Therefore, maintaining Prdm13 signaling in the DMH might play an important role to control sleep-wake patterns during aging.

physiology↗

bioRxiv2022

Use of the particle agglutination/particle agglutination-inhibition test for antigenic analysis of SARS-CoV-2

The antigenicity of SARS-CoV-2 is a critical issue for the effectiveness of the vaccine, and thus it should be phenotypically evaluated by serological assays as new field isolates emerge. The hemagglutination/hemagglutination-inhibition (HA/HI) tests are well-known as a representative method for antigenic analysis of influenza viruses, but SARS-CoV-2 is unlikely to agglutinate to human or guinea pig red blood cells. Therefore, the antigenic analysis requires complicated enzyme-linked immunosorbent assay (ELISA) or cell-based assays such as the microneutralization assay. In this study, we developed the particle agglutination/particle agglutination-inhibition (PA/PAI) test to easily and rapidly quantify the virus and antibody using human angiotensin-converting enzyme 2 (hACE2)-bound latex beads. The PA titer was positively correlated with the plaque-forming units. The PAI titer using post-infection Syrian hamster antisera clearly revealed the antigenic difference between the omicron and previous variants. The results show the PAI test is useful for easy and rapid antigenic analysis of SARS-CoV-2.

microbiology↗

bioRxiv2022

The SARS-CoV-2 Omicron BA.1 spike G446S potentiates HLA-A*24:02-restricted T cell immunity

Although the Omicron variant of the SARS-CoV-2 virus is resistant to neutralizing antibodies, it retains susceptibility to cellular immunity. Here, we characterized vaccine-induced T cells specific for various SARS-CoV-2 variants and identified HLA-A*24:02-restricted CD8+ T cells that strongly suppressed Omicron BA.1 replication. Mutagenesis analyses revealed that a G446S mutation, located just outside the N-terminus of the cognate epitope, augmented TCR recognition of this variant. In contrast, no enhanced suppression of replication was observed against cells infected with the prototype, Omicron BA.2, and Delta variants that express G446. The enhancing effect of the G446S mutation was lost when target cells were treated with inhibitors of tripeptidyl peptidase II, a protein that mediates antigen processing. These results demonstrate that the G446S mutation in the Omicron BA.1 variant affects antigen processing/presentation and potentiates antiviral activity by vaccine-induced T cells, leading to enhanced T cell immunity towards emerging variants.

immunology↗

bioRxiv2022

Prostaglandin EP3 receptor-expressing preoptic neurons bidirectionally control body temperature via tonic GABAergic signaling

The circuit mechanism of the thermoregulatory center in the preoptic area (POA) is unknown. Using rats, here we show prostaglandin EP3 receptor-expressing POA neurons (POAEP3R neurons) as a pivotal bidirectional controller in the central thermoregulatory mechanism. POAEP3R neurons are activated in response to elevated ambient temperature, but inhibited by prostaglandin E2, a pyrogenic mediator. Chemogenetic stimulation of POAEP3R neurons at room temperature reduces body temperature by enhancing heat dissipation, whereas inhibition of them elicits hyperthermia involving brown fat thermogenesis, mimicking fever. POAEP3R neurons innervate sympathoexcitatory neurons in the dorsomedial hypothalamus (DMH) via tonic inhibitory signaling. Although many POAEP3R neuronal cell bodies express a glutamatergic mRNA marker, paradoxically, their axons in the DMH predominantly contain terminals with GABAergic presynaptic proteins, which are increased by chronic heat exposure. These findings indicate that tonic GABAergic inhibitory signaling from POAEP3R neurons is a fundamental determinant of body temperature for thermal homeostasis and fever.

physiology↗

bioRxiv2021

In vivo CRISPRi screen reveals the differential requirement for mitochondrial respiratory chain function between in vivo and in vitro tumor growth

The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and significant processes such as tumor dissemination and metastases remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells downregulate glycolytic mechanisms. These studies demonstrate that discrete perturbations of mitochondrial metabolism impact in vivo tumor growth in a context-specific manner and provides systems-level evidence that respiratory function modulates tumor growth in vivo, suggesting that ATP limits growth and metastatic potential.

cancer biology↗

bioRxiv2021

An interactive deep learning-based approach reveals mitochondrial cristae topologies

Outer and inner mitochondrial membranes are highly specialized structures with distinct functional properties. Reconstructing complex 3D ultrastructural features of mitochondrial membranes at the nanoscale requires analysis of large volumes of serial scanning electron tomography data. While deep-learning-based methods improved in sophistication recently, time-consuming human intervention processes remain major roadblocks for efficient and accurate analysis of organelle ultrastructure. In order to overcome this limitation, we developed a deep-learning image analysis platform called Python-based Human-In-the-LOop Workflows (PHILOW). Our implementation of an iterative segmentation algorithm and Three-Axis-Prediction method not only improved segmentation speed, but also provided unprecedented ultrastructural detail of whole mitochondria and cristae. Using PHILOW, we found that 42% of cristae surface exhibits tubular structures that are not recognizable in light microscopy and 2D electron microscopy. Furthermore, we unraveled a fundamental new regulatory function for the dynamin-related GTPase Optic Atrophy 1 (OPA1) in controlling the balance between lamellar versus tubular cristae subdomains.

cell biology↗

bioRxiv2021

Strategy for detecting off-target sites in genome-edited rice

Genome-editing using the CRISPR-Cas9 system can substantially accelerate crop breeding. Because off-target editing is the main problem with this system, a reliable method for comprehensively detecting off-target sites is required for the editing of food crop genomes. However, a method that accurately predicts off-target sites has not been established. In this study, we performed a SITE-Seq analysis to predict potential off-target sites. SITE-Seq is an unbiased method applicable for the in vitro detection of double-strand breaks (DSBs). To analyze SITE-Seq data, we developed a novel Galaxy system, which can perform simple and reproducible analyses without a command line operation. We conducted a SITE-Seq analysis of a rice genome modified by OsFH15 gRNA-Cas9, and identified 41 DSB sites in the annotated regions. Amplicon-sequencing revealed mutations at one off-target site in the genome-edited rice. The presence of an uncommon protospacer adjacent motif (NTG PAM) likely makes this off-target site difficult to identify using in silico methods. Of the six tested programs, only CRISPRdirect predicted this off-target site, but it also predicted 6,080 off-target sites in total. These results suggest the SITE-Seq method presented herein can efficiently predict off-target sites and is useful for assessing the safety of genome-edited food.

bioinformatics↗

bioRxiv2021

Mediodorsal thalamus is critical for updating during extra-dimensional shifts but not reversals in the attentional set-shifting task

Cognitive flexibility, attributed to frontal cortex, is vital for navigating the complexities of everyday life. The mediodorsal thalamus (MD), interconnected to frontal cortex, may influence cognitive flexibility. Here male rats performed an attentional set-shifting task measuring intra-dimensional and extra-dimensional shifts in sensory discriminations. MD lesion rats needed more trials to learn the rewarded sensory dimension. However, once the choice response strategy was established, learning further two-choice discriminations in the same sensory dimension, and reversals of the reward contingencies in the same dimension, were unimpaired. Critically though, MD lesion rats were impaired during the extra-dimensional shift, when they must rapidly update the optimal choice response strategy. Behavioral analyses showed MD lesion rats had significantly reduced correct within trial second choice responses. This evidence shows transfer of information via the MD is critical when monitoring and rapid within trial updates in established choice response strategies are required after there is a rule change. Significance statementWe demonstrate for the first time that rodent mediodorsal (MD) thalamus is a critical node when choice response strategies need to change rapidly after a within session rule change but not after reversals of reward contingencies during reward guided learning. MD interactions with orbitofrontal cortex are critical for value based learning, while MD interactions with medial prefrontal cortex are critical for rapid within trial updating of optimal choice response rules. MD interactions with the orbitofrontal cortex are not always necessary for reversal learning.

animal behavior and cognition↗

bioRxiv2021

Pharmacologic Therapy for Engraftment Arrhythmia Induced by Transplantation of Human Cardiomyocytes

BackgroundEngraftment arrhythmias (EAs) are observed in large animal studies of intramyocardial transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) for myocardial infarction. Although transient, the risk posed by EA presents a barrier to clinical translation. ObjectivesWe hypothesized that clinically approved antiarrhythmic drugs can prevent EA-related mortality as well as suppress tachycardia and arrhythmia burden. MethodshPSC-CM were transplanted into the infarcted porcine heart by surgical or percutaneous delivery to induce EA. Following a screen of antiarrhythmic agents, a prospective study was conducted to determine the effectiveness of amiodarone plus ivabradine in preventing cardiac death and suppressing EA. ResultsEA was observed in all subjects, and amiodarone-ivabradine treatment was well-tolerated. None of the treated subjects experienced the primary endpoint of cardiac death, unstable EA or heart failure compared to 5/8 (62.5%) in the control cohort (hazard ratio 0.00; 95% confidence interval, 0-0.297; p = 0.002). Overall survival including two deaths in the treated cohort from immunosuppression-related infection showed borderline improvement with treatment (hazard ratio 0.21; 95% confidence interval, 0.03-1.01; p = 0.05). Without treatment, peak heart rate averaged 305 {+/-} 29 beats per min (bpm), whereas in treated subjects peak daily heart rate was significantly restricted to 185{+/-}9 bpm (p = 0.006). Similarly, treatment reduced peak daily EA burden from 96.8 {+/-} 2.9% to 76.5 {+/-} 7.9% (p = 0.003). Antiarrhythmic treatment was safely discontinued after approximately one-month of treatment without recrudescence of arrhythmia. ConclusionsThe risk of engraftment arrhythmia following hPSC-CM transplantation can be reduced significantly by combined amiodarone and ivabradine drug therapy. Condensed AbstractEngraftment arrhythmia (EA) is a transient but serious complication of cardiac remuscularization therapy. Using a porcine model of cardiac remuscularization and EA, ivabradine and amiodarone were independently effective in suppressing tachycardia and arrhythmia, respectively. Baseline amiodarone combined with adjunctive ivabradine successfully prevented cardiac death, unstable EA and heart failure (hazard ratio 0.00; 95% confidence interval, 0-0.297; p = 0.002) and significantly suppressed peak daily heart rate and arrhythmia burden (p=0.006 and 0.003, respectively). Antiarrhythmic treatment was successfully discontinued after one-month without recrudescence of arrhythmia. We conclude that EA can be suppressed by combined amiodarone and ivabradine drug therapy.

pharmacology and toxicology↗

bioRxiv2021

Metabolic flexibility via mitochondrial BCAA carrier SLC25A44 is required for optimal fever

Importing necessary metabolites into the mitochondrial matrix is a crucial step of fuel choice during stress adaptation. Branched chain-amino acids (BCAA, Valine, Leucine, and Isoleucine) are essential for anabolic processes like protein synthesis, but they are also imported into the mitochondria for catabolic reactions. What controls the distinct subcellular BCAA utilization during stress adaptation is insufficiently understood. The present study reports the role of SLC25A44, a recently identified mitochondrial BCAA carrier (MBC), in the regulation of mitochondrial BCAA catabolism and adaptive response to fever. We found that mitochondrial BCAA oxidation in brown adipose tissue (BAT) is significantly enhanced during fever in response to the pyrogenic mediator prostaglandin E2 (PGE2) and psychological stress. Genetic deletion of MBC in a BAT-specific manner blunts mitochondrial BCAA oxidation and non-shivering thermogenesis following intracerebroventricular PGE2 administration. At a cellular level, MBC is required for mitochondrial BCAA deamination as well as the synthesis of mitochondrial amino acids and TCA intermediates. Together, these results illuminate the role of MBC as a determinant of metabolic flexibility to mitochondrial BCAA catabolism and optimal febrile responses. This study also offers an opportunity to control fever by rewiring the subcellular BCAA fate.

cell biology↗

bioRxiv2020

A novel, ataxic mouse model of Ataxia Telangiectasia caused by a clinically relevant nonsense mutation

Ataxia Telangiectasia (A-T) and Ataxia with Ocular Apraxia Type 1 (AOA1) are devastating neurological disorders caused by null mutations in the genome stability genes, A-T mutated (ATM) and Aprataxin (APTX), respectively. Our mechanistic understanding and therapeutic repertoire for treating these disorders is severely lacking, in large part due to the failure of prior animal models with similar null mutations to recapitulate the characteristic loss of motor coordination (i.e., ataxia) and associated cerebellar defects. By increasing genotoxic stress through the insertion of null mutations in both the Atm (nonsense) and Aptx (knockout) genes in the same animal, we have generated a novel mouse model that for the first time develops a progressively severe ataxic phenotype associated with atrophy of the cerebellar molecular layer. We find biophysical properties of cerebellar Purkinje neurons are significantly perturbed (e.g., reduced membrane capacitance, lower action potential thresholds, etc.), while properties of synaptic inputs remain largely unchanged. These perturbations significantly alter Purkinje neuron neural activity, including a progressive reduction in spontaneous action potential firing frequency that correlates with both cerebellar atrophy and ataxia over the animals first year of life. Double mutant mice also exhibit a high predisposition to developing cancer (thymomas) and immune abnormalities (impaired early thymocyte development and T-cell maturation), symptoms characteristic of A-T. Lastly, by inserting a clinically relevant nonsense-type null mutation in Atm, we demonstrate that Small Molecule Read-Through (SMRT) compounds can restore ATM production, indicating their potential as a future A-T therapeutic.

neuroscience↗