Biology subjects

Kamiya, A.

Publications and source records attributed to Kamiya, A..

12 recordsLinked to original sources

bioRxiv2025

DISC1-PML protein interaction for congenital CMV infection-induced cortical neural progenitor deficit: perturbance of host signaling via viral IE1

Congenital CMV infection is the most common perinatal infection, affecting up to 0.5% of infants. This elicits long-term disabilities that include neuropsychiatric manifestations, such as intellectual disability, microcephaly. Despite its high prevalence, the underlying mechanism of how congenitally acquired CMV infection causes brain pathology remains unknown. Here we discovered the molecular interplay of key host (DISC1 and PML) and viral (IE1) proteins within the neural progenitor cells, which underlay an attenuated neural progenitor proliferation. Abolishing the viral IE1 protein by delivering IE1-targeting CRISPR/Cas9 to fetal brain rescued this progenitor cell deficit, a key pathology in congenital CMV infection. A selective targeting to a viral-specific protein by the CRISPR/Cas9 system is minimal in off-target effects. Therefore, we believe that a pivotal role of IE1 in an attenuated neural progenitor proliferation in the developing cortex through its interfering with interaction between host DISC1 and PML proteins.

developmental biology↗

bioRxiv2023

Random walk and cell morphology dynamics in Naegleria gruberi

Amoeboid cell movement and migration are wide-spread across various cell types and species. Microscopy-based analysis of the model systems Dictyostelium and neutrophils over the years have uncovered generality in their overall cell movement pattern. Under no directional cues, the centroid movement can be quantitatively characterized by their persistence to move in a straight line and the frequency of re-orientation. Mathematically, the cells essentially behave as a persistent random walker with memory of two characteristic time-scale. Such quantitative characterization is important from a cellular-level ethology point of view as it has direct connotation to their exploratory and foraging strategies. Interestingly, outside the amoebozoa and metazoa, there are largely uncharacterized species in the excavate taxon Heterolobosea including amoeboflagellate Naegleria. While classical works have shown that these cells indeed show typical amoeboid locomotion on an attached surface, their quantitative features are so far unexplored. Here, we analyzed the cell movement of Naegleria gruberi by employing long-time phase contrast imaging to automatically track individual cells. We show that the cells move as a persistent random walker with two time-scales that are close to those known in Dictyostelium and neutrophils. Similarities were also found in the shape dynamics which is characterized by the appearance, splitting and annihilation of the curvature waves along the cell edge. Our analysis based on the Fourier descriptor and a neural network classifier point to importance of morphology features unique to Naegleria including complex protrusions and the transient bipolar dumbbell morphologies.

cell biology↗

bioRxiv2023

Microglial cannabinoid receptor type1 mediates social memory deficits produced by adolescent THC exposure and 16p11.2 duplication

Adolescent cannabis use increases the risk for cognitive impairments and psychiatric disorders. Cannabinoid receptor type 1 (Cnr1) is expressed not only in neurons and astrocytes, but also in microglia, which shape synaptic connections during adolescence. Nonetheless, until now, the role of microglia in mediating the adverse cognitive effects of delta-9-tetrahydrocannabinol (THC), the principal psychoactive constituent of cannabis, has been unexplored. Here, we report that adolescent THC exposure produces microglial apoptosis in the medial prefrontal cortex (mPFC), which was exacerbated in the mouse model of 16p11.2 duplication, a representative copy number variation (CNV) risk factor for psychiatric disorders. These effects are mediated by microglial Cnr1, leading to reduction in the excitability of mPFC pyramidal-tract neurons and deficits in social memory in adulthood. Our findings highlight the importance of microglial Cnr1 to produce the adverse effect of cannabis exposure in genetically vulnerable individuals.

neuroscience↗

bioRxiv2023

R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

Here, we construct genome-scale maps for R-loops, three-stranded nucleic acid structures comprised of a DNA/RNA hybrid and a displaced single strand of DNA, in the proliferative and differentiated zones of the human prenatal brain. We show that R-loops are abundant in the progenitor-rich germinal matrix, with preferential formation at promoters slated for upregulated expression at later stages of differentiation, including numerous neurodevelopmental risk genes. RNase H1-mediated contraction of the genomic R-loop space in neural progenitors shifted differentiation toward the neuronal lineage and was associated with transcriptomic alterations and defective functional and structural neuronal connectivity in vivo and in vitro. Therefore, R- loops are important for fine-tuning differentiation-sensitive gene expression programs of neural progenitor cells.

genomics↗

bioRxiv2023

Drug metabolic activity as a selection factor for pluripotent stem cell-derived hepatic progenitor cells

As a metabolic organ, the liver plays a variety of roles, including detoxification. It has been difficult to obtain stable supplies of hepatocytes for transplantation and for accurate hepatotoxicity determination in drug discovery research. Human pluripotent stem cells, capable of unlimited self-renewal, may be a promising source of hepatocytes. In order to develop a stable supply of embryonic stem cell (ESC)-derived hepatocytes, we have purified human ESC-derived hepatic progenitor cells with exposure to cytocidal puromycin by using their ability to metabolize drugs. Hepatic progenitor cells stably proliferated at least 2^20-fold over 120 days, maintaining hepatic progenitor cell-like properties. High drug-metabolizing hepatic progenitor cells can be matured into liver cells by suppressing hepatic proliferative signals. The method we developed enables the isolation and proliferation of functional hepatic progenitors from human ESCs, thereby providing a stable supply of high-quality cell resources at high efficiency. Cells produced by this method may facilitate cell therapy for hepatic diseases and reliable drug discovery research.

cell biology↗

bioRxiv2022

Immune-related molecular changes in the olfactory epithelium, structural alteration in the olfactory bulb, and psychosis

Smell deficits and neurobiological changes in the olfactory bulb (OB) and olfactory epithelium (OE) have been observed in schizophrenia and related disorders. The OE is the most peripheral olfactory system located outside the cranium, and is connected with the brain via direct neuronal projections to the OB. Nevertheless, it is unknown whether and how a disturbance of the OE affects the OB in schizophrenia and related disorders. Addressing this gap would be the first step in studying the impact of OE pathology in the disease pathophysiology in the brain. In this cross-species study, we observed that chronic, local OE inflammation with a set of upregulated genes in an inducible olfactory inflammation (IOI) mouse model led to a volume reduction, layer structure changes, and alterations of neuron functionality in the OB. Furthermore, IOI model also displayed behavioral deficits relevant to negative symptoms (avolition) in parallel to smell deficits. In first episode psychosis (FEP) patients, we observed a significant alteration in immune/inflammation-related molecular signatures in olfactory neuronal cells (ONCs) enriched from biopsied OE and a significant reduction in the OB volume, compared with those of healthy controls (HC). The increased expression of immune/inflammation-related molecules in ONCs was significantly correlated to the OB volume reduction in FEP patients, but no correlation was found in HCs. Moreover, the increased expression of human orthologues of the IOI genes in ONCs was significantly correlated with the OB volume reduction in FEP, but not in HCs. Together, our study implies a potential mechanism of the OE-OB pathology in patients with psychotic disorders (schizophrenia and related disorders). We hope that this mechanism may have a cross-disease implication, including COVID-19-elicited mental conditions that include smell deficits.

molecular biology↗

bioRxiv2021

Direct neuronal conversion of microglia/macrophages reinstates neurological function after stroke

Ischaemic brain injury causes permanent neuronal loss, which often results in persistent severe neurological dysfunctions. Although generating new neurons in the injured brain would be an ideal approach to replenish the lost neurons for repairing the damage, the adult mammalian brain retains only limited neurogenic capability. Here, we show that direct conversion of microglia/macrophages into neurons in the brain has great potential as a therapeutic strategy for ischaemic brain injury. After transient middle cerebral artery occlusion in adult mice, microglia/macrophages converge at the lesion core of the striatum, where neuronal loss is prominent. Targeted expression of a neurogenic transcription factor, NeuroD1, in microglia/macrophages in the injured striatum enables their conversion into induced neuronal cells that functionally integrate into the existing neuronal circuits. Furthermore, NeuroD1-mediated induced neuronal cell generation significantly improves neurological function in the mouse stroke model, and ablation of these cells abolishes the gained functional recovery. Our findings thus demonstrate that neuronal conversion contributes directly to functional recovery after stroke and shed further light on the development of therapies for ischaemic brain injury by in situ neuronal conversion technology.

neuroscience↗

bioRxiv2021

Drug metabolism activity is a Critical Intrinsic Selector for Hepatocytes to elongate cell lifespan by using a cell-killing antibiotic

Hepatocytes can be used to study the pathogenesis of liver diseases and drug discovery research. Human hepatocytes are, however, hardly expandable in vitro making it difficult to secure large numbers of cells from one donor. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of human hepatocytes to ensure a constant supply. We purified human hepatocytes by selection with cytocidal puromycin, and cultured them for more than 60 population doublings over a span of more than 350 days. These results show that this simple culture system with usage of the cytocidal antibiotics enables efficient hepatocyte proliferation and is an effective method for generating a stable supply of hepatocytes for drug discovery research at a significant cost reduction.

cell biology↗

bioRxiv2020

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

Zone I and zone III hepatocytes metabolize ammonia through urea cycle and drug by cytochrome P450, respectively. Ammonia has a cytotoxic effect, and can therefore be used as a selection agent for enrichment of hepatocytes. Besides, isolated hepatocytes from livers can be propagated ex vivo under appropriate condition. However, it has not been investigated so far whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed the ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human pluripotent stem cells (PSCs) that are embryonic stem cells (ESCs) and induced pluripotent stem cells. Hepatocyte-like cells after exposure to ammonia highly expressed the CPS1 gene that metabolizes ammonia to carbamoyl phosphate. The resistance to cytotoxicity or cell death by ammonia is probably attributed to the metabolic activity of ammonia in the cells. In addition to the ammonia metabolism-related genes, ammonia-selected PSC-derived hepatocytes increased expression of the CYP3A4 gene, one of the cytochrome P450 genes, that is mainly expressed in zone III hepatocytes. Ammonia-selected hepatocyte-like cells derived from both ESCs and iPSCs can be propagated in vitro up to 30 population doublings for more than 190 days without affecting expression of the liver-associated genes, implying that the ammonia-selected cells have immortality or equivalent life span on the appropriate feeder cells like ESCs and iPSCs. The long-term cultivation of ammonia-selected hepatocyte-like cells resulted in the increased expression of hepatocyte-associated genes such as the CPS1 and CYP3A4 genes. The ammonia selection method to enrich a hepatocyte population was also applicable to immortalized cells from the liver. Ammonia treatment in combination with in vitro propagation will be used to obtain large amounts of hepatocytes or hepatocyte-like cells for pharmacology, toxicology and regenerative medicine.

cell biology↗

bioRxiv2019

Brain-synthesized estrogens regulate cortical migration in a sexually divergent manner

Estrogens play an important role in the sexual dimorphisms that occur during brain development, including the neural circuitry that underlies sex-typical and socio-aggressive behaviors. Aromatase, the enzyme responsible for the conversion of androgens to estrogens, is expressed at high levels during early development in both male and female cortices, suggesting a role for brain-synthesized estrogens during corticogenesis. This study investigated how the local synthesis of estrogens affects neurodevelopment of the cerebral cortex, and how this differs in males and females by knockdown expression of the Cyp19a1 gene, which encodes aromatase, between embryonic day 14.5 and postnatal day 0 (P0). The effects of Cyp19a1 knockdown on neural migration was then assessed. Aromatase was expressed in the developing cortex of both sexes, but at significantly higher levels in male than female mice. Under basal conditions, no obvious differences in cortical migration between male and female mice were observed. However, knockdown of Cyp19a1 increased the number GFP-positive cells in the cortical plate, with a concurrent decrease in the subventricular zone/ventricular zone in P0 male mice. The opposite effect was observed in females, with a significantly reduced number of GFP-positive cells migrating to the cortical plate. These findings have important implications for our understanding of the role of fetal steroids for neuronal migration during cerebral cortex development. Moreover, these data indicate that brain-synthesized estrogens regulate radial migration through distinct mechanisms in males and females.

neuroscience↗

bioRxiv2018

In vivo epigenetic editing of sema6a promoter reverses impaired transcallosal connectivity caused by C11orf46/ARL14EP neurodevelopmental risk gene

Many neuropsychiatric risk genes contribute to epigenetic regulation of gene expression but very little is known about specific chromatin-associated mechanisms governing the formation and maintenance of neuronal connectivity. Here we show that transcallosal connectivity is critically dependent on C11orf46 (also known as ARL14EP), a small nuclear protein encoded in the chromosome 11p13 Wilms Tumor, Aniridia, Genitourinary Abnormalities, intellectual disability (formerly referred to as Mental Retardation) (WAGR) risk locus. C11orf46 haploinsufficiency in WAGR microdeletion cases was associated with severe hypoplasia of the corpus callosum. In utero short hairpin RNA-mediated C11orf46 knockdown disrupted transcallosal projections of cortical pyramidal neurons, a phenotype that was rescued by wild type C11orf46 but not the C11orf46R236H mutant associated with autosomal recessive intellectual disability. Multiple genes encoding key regulators of axonal growth and differentiation, including Sema6A, were hyperexpressed in C11orf46-knockdown neurons. Importantly, RNA-guided epigenetic editing of neuronal Sema6a gene promoters via a dCas9 protein-conjugated SunTag scaffold with multimeric (10x) C11orf46 binding during early developmental periods, resulted in normalization of expression and rescue of transcallosal dysconnectivity via repressive chromatin remodeling, including up-regulated histone H3K9 methylation by the KAP1-SETDB1 repressor complex. Our study demonstrates that interhemispheric communication is highly sensitive to locus-specific remodeling of neuronal chromatin, revealing the therapeutic potential for shaping the brains connectome via gene-targeted designer activators and repressor proteins.

developmental biology↗

bioRxiv2018

Glutathione shapes glutamatergic transmission

Glutamate is the most abundant excitatory neurotransmitter, present at the bulk of cortical synapses, and participating in many physiologic and pathologic processes ranging from learning and memory to stroke. The tripeptide, glutathione, is one third glutamate and present at up to low millimolar intracellular concentrations in brain, mediating antioxidant defenses and drug detoxification. Because of the substantial amounts of brain glutathione and its rapid turnover under homeostatic control, we hypothesized that glutathione is a relevant reservoir of glutamate, and could influence synaptic excitability. We find that drugs which inhibit generation of glutamate by the glutathione cycle elicit decreases in cytosolic glutamate and decreased miniature excitatory post synaptic potential (mEPSC) frequency. In contrast, pharmacologically decreasing the biosynthesis of glutathione leads to increases in cytosolic glutamate and enhanced mEPSC frequency. The glutathione cycle can compensate for decreased excitatory neurotransmission when the glutamate-glutamine shuttle is inhibited. Glutathione may be a physiologic reservoir of glutamate neurotransmitter.\n\nSignificanceGlutathione is the principal antioxidant and redox regulator in cells. In addition to its essential roles in redox homeostasis it functions as cofactors for a multitude of enzymes. We show here that glutathione is a reservoir for synaptic glutamate, the excitatory neurotransmitter in the central nervous system. Deficits in glutathione have been linked to multiple neurodegenerative and neuropsychiatric disorders. Accordingly, agents that restore glutathione-glutamate homeostasis may afford therapeutic benefit.

neuroscience↗