Biology subjects

Nakamura, K.

Publications and source records attributed to Nakamura, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Overexpression of alpha synuclein in Midbrain Dopamine Neurons Reduces Dopamine Release Without Cell Loss and Drives Mild Motor Deficits in Mice

It has proven challenging to faithfully recapitulate the key pathological, physiological, and behavioral features of Parkinsons Disease (PD) in animals. Here we used adeno-associated virus (AAV) vectors to achieve cell type-specific overexpression of wild-type human -synuclein (syn) and a fluorophore (mCherry) in midbrain dopamine neurons to model PD in mice. We found that AAVs drove selective expression of both syn and mCherry in midbrain dopamine neurons. In conjunction with approximately 2-fold overexpression of syn, we found several histopathological markers of PD-like pathology, including progressive accumulation of phosphorylated and aggregated syn, ubiquitin, and a reduction in the expression of tyrosine hydroxylase, without overt cell loss. In parallel, syn overexpression drove a profound loss of evoked dopamine release, without a substantive change in the intrinsic properties of dopamine neurons, nor in striatal dopamine content. Finally, syn overexpression led to mild locomotor deficits. Together, these findings suggest that moderate syn overexpression can mimic some aspects of premotor and early symptomatic phases of PD, including markers of Lewy Body-like pathology and functional loss of evoked dopamine release. This model may be useful for investigating cellular and circuit mechanisms related to PD pathogenesis and progression.

neuroscience↗

bioRxiv2025

Human pluripotent stem cell-derived intestinal epithelial cells maintain small intestine-specific functions over time, even with repeated cell division

The human colon cancer-derived cell line Caco-2 is widely used in drug discovery due to its barrier function and transporter activity. However, Caco-2 cells have extremely low drug metabolic capacity, resulting in discrepancies with human physiology. In this study, we conducted experiments on human intestinal epithelial cells generated from pluripotent stem cell-derived organoids. We assessed cell morphology, gene expression, barrier and transporter functions, drug metabolic capacity, and cytotoxicity in relation to cell growth and the effects of cellular aging. The results indicate that organoid-derived intestinal epithelial cells may be helpful as a new model cell for drug discovery. Understanding the advantages of drug metabolic capacity and cytotoxicity among cryopreserved human enterocytes, the human colon cancer-derived cell line Caco-2, and human pluripotent stem cell-derived intestinal epithelial cells within microphysiological systems and organ-on-chip technologies is essential for the development of an appropriate model system for the small intestine.

cell biology↗

bioRxiv2025

Phage defense and genome editing using novel retrons sourced from isolated environmental bacteria

Retrons are bacterial immune systems that protect a bacterial population against phages by killing infected hosts. Retrons typically comprise a reverse transcriptase, a template noncoding RNA that is partially reverse transcribed into RT-DNA, and a toxic effector. The reverse transcriptase, noncoding RNA, and RT-DNA complex sequester the toxic effector until triggered by phage infection, at which point the toxin is released to induce cell death. Due to their ability to produce single-stranded DNA in vivo, retrons have also been engineered to produce donor templates for genome editing in both prokaryotes and eukaryotes. However, the current repertoire of experimentally characterized retrons is limited, with most retrons sourced from clinical and laboratory strains of bacteria. To better understand retron biology and natural diversity, and to expand the current toolbox of retron-based genome editors, we developed a pipeline to isolate retrons and their bacterial hosts from a variety of environmental samples. Here, we present six of these novel retrons, each isolated from a different host bacterium. We characterize the full operon of these retrons and test their ability to defend against a panel of E. coli phages. For two of these retrons, we further unravel their mechanism of defense by identifying the phage genes responsible for triggering abortive infection. Finally, we engineer these retrons for genome editing in E. coli, demonstrating their potential use in a biotechnological application.

microbiology↗

bioRxiv2024

Improvement of D-lactic acid production from methanol by metabolically engineered Komagataella phaffii via ultra-violet mutagenesis

Methanol has attracted attention as an alternative carbon source to petroleum. Komagataella phaffii, a methanol-assimilating yeast, is a useful host for the chemical production from methanol. A previous study successfully constructed a metabolically engineered K. phaffii GS115/S8/Z3 strain capable of producing D-lactic acid from methanol. In this study, we aimed to develop a strain with improved D-lactic acid production by applying ultra-violet mutagenesis to the D-lactic acid-producing strain, GS115/S8/Z3. The resulting mutant strain DLac_Mut2_221 produced 5.38 g/L of D-lactic acid from methanol, a 1.52-fold increase compared to the parent strain GS115/S8/Z3. Transcriptome analysis revealed 167 differentially expressed genes in DLac_Mut2_221, comprising 104 upregulated and 63 downregulated genes. These results suggest that the improvement in D-lactic acid production from methanol in the mutant strain involves three important mechanisms: (1) avoiding excessive formaldehyde accumulation, (2) activating the glyoxylate pathway, and (3) reducing the expression of O-glycosylated proteins required for cell wall stability. Metabolic engineering strategies for K. phaffii based on the knowledge gained from this study will contribute to improving the productivity of various useful chemicals from methanol.

bioengineering↗

bioRxiv2024

Cell position-based evaluation of mechanical features of cells in multicellular systems

Measurement of mechanical forces of cell-cell interactions is important for studying the emergence of diverse three-dimensional morphologies of multicellular organisms. We previously reported an image-based statistical method for inferring effective pairwise forces of cell-cell interactions (i.e., attractive/repulsive forces), where a cell particle model was fitted to cell tracking data acquired by live imaging. However, because the particle model is a coarse-grained model, it remains unclear how the pairwise forces relates to sub-cellular mechanical components including cell-cell adhesive forces. Here we applied our inference method to cell tracking data generated by vertex models that assumed sub-cellular components. Through this approach, we investigated the relationship between the effective pairwise forces and various sub-cellular components: cell-cell adhesion forces, cell surface tensions, cell-extracellular matrix (ECM) adhesion, traction forces between cells and ECM, cell growth, etc. We found that the cell-cell adhesion forces were attractive, and both the cell surface tensions and cell-ECM adhesive forces were repulsive, etc. These results indicate that sub-cellular mechanical components can contribute to the effective attractive/repulsive forces of cell-cell interactions. This comprehensive analysis provides theoretical bases for linking the pairwise forces to the sub-cellular mechanical components: this showcase is useful for speculating the sub-cellular mechanical components from the information of cell positions, and for interpreting simulation results based on particle models.

biophysics↗

bioRxiv2024

CHCHD2 mutant mice display mitochondrial protein accumulation and disrupted energy metabolism

Mutations in the mitochondrial cristae protein CHCHD2 lead to a late-onset autosomal dominant form of Parkinsons disease (PD) which closely resembles idiopathic PD, providing the opportunity to gain new insights into the mechanisms of mitochondrial dysfunction contributing to PD. To begin to address this, we used CRISPR genome-editing to generate CHCHD2 T61I point mutant mice. CHCHD2 T61I mice had normal viability, and had only subtle motor deficits with no signs of premature dopaminergic (DA) neuron degeneration. Nonetheless, CHCHD2 T61I mice exhibited robust molecular changes in the brain including increased CHCHD2 insolubility, accumulation of CHCHD2 protein preferentially in the substantia nigra (SN), and elevated levels of -synuclein. Metabolic analyses revealed an increase in glucose metabolism through glycolysis relative to the TCA cycle with increased respiratory exchange ratio, and immune-electron microscopy revelated disrupted mitochondria in DA neurons. Moreover, spatial genomics revealed decreased expression of mitochondrial complex I and III respiratory chain proteins, while proteomics revealed increased respiratory chain and other mitochondrial protein-protein interactions. As such, the CHCHD2 T61I point-mutation mice exhibit robust mitochondrial disruption and a consequent metabolic shift towards glycolysis. These findings thus establish CHCHD2 T61I mice as a new model for mitochondrial-based PD, and implicate disrupted respiratory chain function as a likely causative driver.

neuroscience↗

bioRxiv2024

Comparative analysis of trans-chromosomic rodent models reveals improved somatic hypermutation and class-switch recombination in rats

Humanized rodent models, especially humanization of genetic/genomic components involved in immunity have significantly advanced our understanding of human immune system. Here, we utilized trans-chromosomic (Tc) technology to generate a TC-mAb rat model that stably harbors a mouse artificial chromosome carrying full-length human immunoglobulin (Ig) heavy and kappa light chain genes (IGHK-NAC) in a rat Ig knockout background. In contrast with TC-mAb mice, serum human IgG concentration was found higher than IgM. Number of lymphocytes was recovered, and B cell population in the spleen was normal. Remarkably, repertoire analysis revealed similarities between the model and human PBMCs; somatic hypermutation and class-switch recombination also more closely resembled humans. Furthermore, immunization resulted in generation of antigen-specific human antibodies. Collectively, our strategy to generate both rat and mouse models through introduction of the identical IGHK-NAC offers unprecedented opportunities to comprehensively evaluate genomic regulation and its outcomes associated with genomic sequences and host-derived protein factors.

bioengineering↗

bioRxiv2024

Melanocortin system activates carotid body arterial chemoreceptors in hypertension

BackgroundThe bodys internal milieu is controlled by a system of interoceptors coupled to motor outflows that drive compensatory adaptive responses. These include the arterial chemoreceptors, best known for sensing arterial oxygen. In cardiometabolic diseases, such as essential hypertension, the carotid bodies (CB) exhibit heightened reflex sensitivity and tonic activity without an apparent stimulus. The mechanisms behind CB sensitization in these conditions are not well understood. MethodsGuided by functional genomics, a range of functional assays is used to interrogate downstream intracellular and interorgan signalling pathways involved in arterial chemosensory function. ResultsHere, we report the presence of the Melanocortin 4 receptor (MC4R) in the mammalian CB and show its elevated expression in experimental hypertension. We demonstrate that melanocortin agonists activate arterial chemosensory cells, modulating CB chemosensory afferent drive to influence both resting and chemoreflex-evoked sympathetic and ventilatory activity. Transcriptional analysis of hypertensive CB implicates the activation of the Mash1 (Ascl1) regulatory network in driving elevated Mc4r expression. ConclusionsCollectively, our data indicate a primarily pathophysiological role of melanocortin signalling in arterial chemosensation, contributing to excess sympathetic activity in cardiometabolic disease.

neuroscience↗

bioRxiv2024

Development of a DSM test battery to determine depression-like marmoset

Depression is a serious and high-incidence mental disorder that can lead to suicide. Despite much depression research using humans and rodents to date, it has been difficult to completely overcome depression and elucidate its etiology. Marmoset monkeys have been utilized to study neuropsychiatric disorders in recent years. Since the clinical diagnostic criteria for psychiatric disorders are mainly based on multiple behavioral changes, test battery systems, a multifaceted and comprehensive strategy, are applied to depression research. Here, we devised a test battery with six tests to investigate depression-like symptoms corresponding to clinical criteria and set criteria for a test indicator-based judgment. We did a usability confirmation experiment using a drug and judged three of nine marmosets as depression-like. This rate is comparable with the incidence rate of human patients; i.e., 10-30% of patients who received reserpine treatment suffer from depression. Thus, we propose the test battery we constructed in this study will contribute to the study of depression.

neuroscience↗

bioRxiv2024

Distinct roles of two thalamostriatal systems in learning processes of visual discrimination in common marmosets

The thalamostriatal projections arising from the intralaminar thalamic nuclei (ILN) constitute the principal source of input information to specified subregions of the striatum, a key structure of the cortico-basal ganglia circuitry. However, the roles of primate ILN in cortico-basal ganglia circuit functions remain unclear. Here, we performed immunotoxin-induced selective targeting of two representative structures of the ILN, the parafascicular nucleus (Pf) and centre median nucleus (CM) projecting to the caudate nucleus (Cd) and putamen (Pu), respectively, in common marmosets. Elimination of Pf-Cd neurons resulted in impaired reversal learning of a two-choice visual discrimination task, whereas removal of CM-Pu neurons disturbed the task acquisition. No marked impact of such manipulations was observed on either motor skill learning or spontaneous locomotor activity. Our findings reveal that the two thalamostriatal systems play distinct roles in the learning processes of external cue-dependent decision-making in nonhuman primates.

neuroscience↗

bioRxiv2024

Effect of phage variation on Shiga toxin 2 (Stx2) production and the virulence of Stx-producing Escherichia coli

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes serious gastrointestinal illness, including hemorrhagic colitis and hemolytic uremic syndrome. Although all known Stxs (Stx1 and Stx2) are encoded by bacteriophages (Stx phages), the production of Stx2 is known to be a major risk factor for severe STEC infections. The production of Stx2, but not Stx1, is tightly coupled with the induction of Stx phages, and Stx2 production levels vary between STEC strains, even within the same serotype. Here, we analyzed the genomic diversity of all Stx phages in 71 strains representing the entire O145:H28 lineage, one of the major STECs. Our analysis revealed the highly dynamic nature of the Stx phages in O145:H28, including the independent acquisition of similar Stx phages by different sublineages and the frequent changes in Stx phages in the same sublineages due to the gain and loss of Stx phages. Analyses of Stx2 production levels in O145:H28 strains and K-12 lysogens of Stx2 phages of specific groups and types, which were defined by their early region sequences and CI repressors, respectively, revealed that short-tailed Stx2a phages (S-Stx2a phages) confer significantly greater Stx2 production to host strains than long-tailed Stx2a phages (L-Stx2a phages). However, L-Stx2a phages that encode a specific type of CI repressor promoted Stx2 production, comparable to the level of production among S-Stx2a phages, as well as promoted virulence to host strains, exceeding the level among other L-Stx2a phages. We also showed a clear link between the phage induction efficiency, which was primarily determined by the early region of each phage, and the level of Stx2 production by host strains. These results provide important insights into the diversification and dynamism of Stx phages and the relationship between the variations in Stx2 phages and the amount of Stx2 production by their host strains. Author summaryShiga toxin (Stx)-producing Escherichia coli (STEC) is an important human intestinal pathogen that causes severe illnesses. These bacteria produce Stx1, Stx2 or both toxins, but the production of Stx2 is an important measure of the virulence of STEC strains. While both types of Stx are encoded by bacteriophages (Stx phages), Stx2 production is tightly coupled with phage induction, and variations in Stx2 phages have been associated with variations in Stx2 production levels by their host O157:H7 STEC strains. However, in non-O157 STEC strains, the variation in Stx phages and its association with host strain production of Stx2 have not yet been fully analyzed. This systematic study of Stx phages in O145:H28 STEC reveals not only the marked genomic diversity and dynamism of Stx phages in this STEC lineage but also that short-tailed Stx2 phages and a specific group of long-tailed Stx2 phages induce high levels of Stx2 production by host strains, and this increased production is linked to the efficient induction of phages.

microbiology↗

bioRxiv2024

Thermostability and binding properties of single-chained Fv fragments derived from therapeutic antibodies

Small antibody fragments have recently been used as alternatives to full-length monoclonal antibodies in therapeutic applications. One of the most popular fragment antibodies is single-chain fragment variables (scFvs), consisting of variable heavy (VH) and variable light (VL) domains linked by a flexible peptide linker. scFvs have small molecular sizes, which enables good tissue penetration and low immunogenicity. Despite these advantages, the use of scFvs, especially for therapeutic purpose, is still limited because of the difficulty to regulate the binding activity and conformational stability. In this study, we constructed and analyzed 10 scFv fragments derived from 10 representatives of FDA-approved mAbs to evaluate their physicochemical properties. Differential scanning calorimetry analysis showed that scFvs exhibited relatively high but varied thermostability, from 50 to 70 {degrees}C of melting temperatures, and different unfolding cooperativity. Surface plasmon resonance analysis revealed that scFvs fragments that exhibit high stability and cooperative unfolding likely tend to maintain antigen binding. This study demonstrated the comprehensive physicochemical properties of scFvs derived from FDA-approved antibodies, providing insights into antibody design and development.

bioengineering↗

bioRxiv2024

Differential and temporally dynamic involvement of primate amygdala nuclei in face animacy and reward information processing

Decision-making is influenced by both expected reward and social factors, such as who offered the outcomes. Thus, although a reward might originally be independent from social factors, the two elements are closely related. However, whether and how they are processed separately or conjointly remains unclear. Here, we show that neurons in distinct sub-nuclei of the amygdala encode expected reward and face animacy, which is a vital aspect of face perception. Although these encoding processes are distinct, they rely on partially shared neuronal circuits with characteristic temporal dynamics. Two male macaque monkeys made saccades under different social and reward contexts, created by presenting facial images with independent attributes: animacy (a monkey or cartoon face) and associated reward (large or small). The stimulus image was presented twice per trial: during the initial stimulus encoding (S1) and before saccades were made (S2). A longer gaze duration for eye region of the monkey versus cartoon images indicated more robust social engagement for realistic faces. During S1, a similar number of lateral nucleus neurons encoded either animacy only with a monkey-image preference, reward only with a large-reward preference, or both. Conversely, neurons in the basal and central nuclei primarily encoded reward, preferring large-versus small-reward associated face images. The reward-dependent modulation was continuous after S1, but was more conspicuous during S1 in the basal nucleus and during both S1 and S2 in the central nucleus. This anatomically- and temporally-specific encoding in the amygdala may underlie the computation and integration of face animacy and reward information. Significance StatementReward and social information are closely related but originally independent, as both influence our decision-making. The amygdala has been associated with both reward and social information coding. However, whether and how they are processed separately or conjointly by individual neurons in the amygdala remains unclear. We found that neurons in the lateral and basal nuclei encoded face animacy, which is an important aspect of social information, and reward, respectively, during sensory processing. Neurons in the central nucleus encoded reward information during the execution phase. This provides new clarity regarding the mechanisms of separate or integrated social and reward information processing within the amygdala.

neuroscience↗

bioRxiv2023

Paradoxical mTORC1-Dependent microRNA-mediated Translation Repression in the Nucleus Accumbens of Mice Consuming Alcohol Attenuates Glycolysis

mTORC1 promotes protein translation, learning and memory, and neuroadaptations that underlie alcohol use and abuse. We report that activation of mTORC1 in the nucleus accumbens (NAc) of mice consuming alcohol promotes the translation of microRNA (miR) machinery components and the upregulation of microRNAs (miRs) expression including miR-34a-5p. In parallel, we detected a paradoxical mTORC1-dependent repression of translation of transcripts including Aldolase A, an essential glycolytic enzyme. We found that miR-34a-5p in the NAc targets Aldolase A for translation repression and promotes alcohol intake. Our data further suggest that glycolysis is inhibited in the NAc manifesting in an mTORC1-dependent attenuation of L-lactate, the end product of glycolysis. Finally, we show that systemic administration of L-lactate attenuates mouse excessive alcohol intake. Our data suggest that alcohol promotes paradoxical actions of mTORC1 on translation and glycolysis which in turn drive excessive alcohol use.

neuroscience↗

bioRxiv2023

Systems-level analyses dissociate genetic regulators of reactive oxygen species and energy production

Respiratory chain dysfunction can decrease ATP and increase reactive oxygen species (ROS) levels. Despite the importance of these metabolic parameters to a wide range of cellular functions and disease, we lack an integrated understanding of how they are differentially regulated. To address this question, we adapted a CRISPRi- and FACS-based platform to compare the effects of respiratory gene knockdown on ROS to their effects on ATP. Focusing on genes whose knockdown is known to decrease mitochondria-derived ATP, we showed that knockdown of genes in specific respiratory chain complexes (I, III and CoQ10 biosynthesis) increased ROS, whereas knockdown of other low ATP hits either had no impact (mitochondrial ribosomal proteins) or actually decreased ROS (complex IV). Moreover, although shifting metabolic conditions profoundly altered mitochondria-derived ATP levels, it had little impact on mitochondrial or cytosolic ROS. In addition, knockdown of a subset of complex I subunits--including NDUFA8, NDUFB4, and NDUFS8--decreased complex I activity, mitochondria-derived ATP and supercomplex level, but knockdown of these genes had differential effects on ROS. Conversely, we found an essential role for ether lipids in the dynamic regulation of mitochondrial ROS levels independent of ATP. Thus, our results identify specific metabolic regulators of cellular ATP and ROS balance that may help dissect the roles of these processes in disease and identify therapeutic strategies to independently target energy failure and oxidative stress. SignificanceMitochondrial respiration generates both energy (ATP) and reactive oxygen species (ROS). Insufficient energy and increased ROS from respiratory chain dysfunction may be central to the pathophysiology of neurodegenerative diseases and aging. We established a screening platform using CRISPR and fluorescent-cell sorting to compare the impact of decreasing respiratory chain proteins on ROS and ATP levels. The results provide the first systems-level analysis of how ROS and ATP are differentially regulated, and identify genes and respiratory chain complexes that can manipulate each independently. These findings advance our understanding of the relative contributions of ATP and ROS to disease pathophysiology, and guide the development of therapies to preserve energy while minimizing ROS.

systems biology↗

bioRxiv2023

Rapid and dynamic evolution of a giant Y chromosome in Silene latifolia

To test hypotheses about the evolution of massive sex-linked regions in plants, we sequenced the genome of Silene latifolia, whose giant heteromorphic sex chromosomes were first discovered in 1923. It has long been known that the Y consists mainly of a male-specific region which does not recombine with the X in male meiosis, and that this region carries the primary sex-determining genes, and other genes contributing to male functioning. However, only with a whole Y chromosome assembly can the candidates be validated experimentally, as we describe. Our new results also illuminate the genomic changes as the ancestral chromosome evolved into the current XY pair, testing ideas about why large regions of sex-linkage evolve, and the mechanisms creating the present recombination pattern. One sentence summaryBased on the whole genome sequences of Silene latifolia, a model species for plant sex chromosome evolution, we describe discovery of genes underlying male-female flower differences, and relate the results to ideas about the evolution of the vast non-recombining regions of the Y chromosome.

genetics↗

bioRxiv2023

Representation of Protein Dynamics Disentangled by Time-structure-based Prior

Representation learning (RL) is a universal technique for deriving low-dimensional disentangled representations from high-dimensional observations, aiding a multitude of downstream tasks. RL has been extensively applied to various data types, including images and natural language. Here, we analyze molecular dynamics (MD) simulation data of biomolecules in terms of RL to obtain disentangled representations related to their conformational transitions. Currently, state-of-the-art RL techniques, which are mainly motivated by the variational principle, try to capture slow motions in the representation (latent) space. Here, we propose two methods based on alternative perspective on the disentanglement in the representation space. The methods introduce a simple prior that imposes temporal constraints in the representation space, serving as a regularization term to facilitate capturing disentangled representations of dynamics. The introduction of this simple prior aids in characterizing the conformational transitions of proteins. Indeed, comparison with other methods via the analysis of MD simulation trajectories for alanine dipeptide and chignolin validates that the proposed methods construct Markov state models (MSMs) whose implied time scales are comparable to state-of-the-art methods. By coarse-graining MSMs, we further show the methods aid to detect physically important interactions for conformational transitions. Overall, our methods provide good representations of complex biomolecular dynamics for downstream tasks, allowing for better interpretations of conformational transitions.

biophysics↗

bioRxiv2023

Effective mechanical potential of cell-cell interaction in tissues harboring cavity and in cell sheet toward morphogenesis

Measuring mechanical forces of cell-cell interactions is important for studying morphogenesis in multicellular organisms. We previously reported an image-based statistical method for inferring effective mechanical potentials of pairwise cell-cell interactions by fitting cell tracking data with a theoretical model. However, whether this method is applicable to tissues with non-cellular components such as cavities remains elusive. Here we evaluated the applicability of the method to cavity-harboring tissues. Using synthetic data generated by simulations, we found that the effect of expanding cavities was added to the pregiven potentials used in the simulations, resulting in the inferred effective potentials having an additional repulsive component derived from the expanding cavities. Interestingly, simulations by using the effective potentials reproduced the cavity-harboring structures. Then, we applied our method to the mouse blastocysts, and found that the inferred effective potentials can reproduce the cavity-harboring structures. Pairwise potentials with additional repulsive components were also detected in two-dimensional cell sheets, by which curved sheets including tubes and cups were simulated. We conclude that our inference method is applicable to tissues harboring cavities and cell sheets, and the resultant effective potentials are useful to simulate the morphologies.

biophysics↗