Biology subjects

Chun, H.

Publications and source records attributed to Chun, H..

14 recordsLinked to original sources

bioRxiv2025

A comprehensive view of somatic mosaicism by single-cell DNA analysis

Single-cell DNA sequencing offers a powerful means of studying somatic mosaicism but requires careful analysis to mitigate DNA amplification-related artifacts. We performed primary template-directed amplification (PTA) and sequencing of 102 nuclei from postmortem lung and colon tissues of a 74-year-old male. Single-cell mutation burdens and spectra were validated by duplex sequencing and revealed heterogeneity across organs and cells, including signatures of APOBEC activity and tobacco exposure. Cells from both tissues exhibited chromosomal aneuploidies, loss of chromosome Y, and chromosomal rearrangements including rearrangements of the T-cell receptor loci indicative of T-cells. Shared embryonic mutations between cells enabled reconstruction of cellular ancestries from the zygote, which were validated by bulk sequencing. Collectively, we demonstrate a comprehensive approach for single-cell genomics that yields an expansive view of diverse somatic mutation types from development through aging across diverse tissues--insights that are obscured in bulk sequencing and only partially captured by other single-cell methods.

genomics↗

bioRxiv2025

Update on the reproduction and interpretation of DIANA fMRI

Three years ago, our group reported direct imaging of neuronal activity (DIANA) with high spatiotemporal resolution, but its reproducibility and signal origin remain controversial. Here, we report the results of our reproduction experiments of DIANA fMRI performed to date using forelimb electrical stimulation at various magnetic field strengths in anesthetized mice, along with the characteristics of DIANA signal, called the pseudo-steady state (PSS). Theoretical analysis and Bloch simulations demonstrated that the spatial location and temporal phase of PSS oscillations are primarily determined by frequency-offset, and that their spatiotemporal superposition can generate peak signals in specific regions at specific timing that closely resemble DIANA signals. These findings suggest that if PSS oscillations are the primary source of DIANA signals, it may be premature to interpret them as neuronal responses to sensory stimulation. Further studies are needed to clarify the relationship between DIANA signals and brain activation.

neuroscience↗

bioRxiv2025

Potential beneficial effects of PD-1/PD-L1 blockade in Alzheimer's disease: A systematic review and meta-analysis of preclinical and clinical studies

Programmed cell death-1 (PD-1) and its ligand (PD-L1) play key roles in cancer immune evasion and in modulating neuroinflammation. This systematic review and meta-analysis investigated the effect of PD-1/PD-L1 blockade on pathology and cognitive function in Alzheimers disease (AD) in preclinical and clinical studies. Relevant studies were systematically identified using the MEDLINE, Embase, CENTRAL, and Web of Science databases from their inception until April 10, 2024. In total, 33 studies were included in this meta-analysis, conducted using R software. Preclinical studies revealed that blockade of PD-1 signaling reduces amyloid-beta plaque burden, tau phosphorylation, and astrocyte reactivity in AD mouse models, accompanied by improvements in cognitive function in behavioral tests. Furthermore, clinical studies demonstrated the beneficial effect of PD-1 signaling inhibitors on cognitive function in cancer patients. This study highlights the necessity for additional research to clarify the exact mechanisms by which PD-1/PD-L1 inhibition impacts AD pathology and cognitive function, opening avenues for potential therapeutic strategies targeting this pathway in AD.

neuroscience↗

bioRxiv2024

Direct imaging of neural activity reveals neural circuits via spatiotemporal activation mapping

Two years ago, our group reported direct imaging of neuronal activity (DIANA), a functional magnetic resonance imaging (fMRI) technique that directly detects neuronal activity at high spatiotemporal resolution. In this study, we successfully reproduced the DIANA response in medetomidine-anesthetized mice using forelimb electrical stimulation at 11.7 T. More importantly, we showed that multiple neural circuits can be effectively revealed by DIANA fMRI through spatiotemporal activation mapping. The spatiotemporal activation mapping proposed here utilizes the temporal information of the DIANA response, that is, the time when the DIANA response reaches its peak, which is a unique feature that distinguishes it from the activation mapping method used in existing fMRI. Based on DIANA activation areas, we identified several neural circuits involved in forelimb sensory processing in the somatosensory network, which includes multiple brain regions: ventral posterolateral nucleus of the thalamus (VPL), posteromedial thalamic nucleus (POm), forelimb primary somatosensory cortex (S1FL), secondary somatosensory cortex (S2), primary motor cortex (M1), and secondary motor cortex (M2). Additionally, we also identified a pain-related neural circuit involving brain regions of the anterior cingulate cortex (ACC) and mediodorsal nucleus (MD). Interestingly, the spatiotemporal activation mapping also allowed us to identify subregions with different DIANA response times within the same functional region (e.g., VPL, POm, S1FL, and S2). Our study highlights the potential of DIANA fMRI to advance our understanding of sensory information processing throughout the brain and to provide insight into the spatiotemporal dynamics of brain networks at the level of neural circuits.

neuroscience↗

bioRxiv2024

Epidermal Growth Factor Receptor Regulates Beclin-1 in Hyperoxia

While delivery of supplemental oxygen is a life-saving therapy, exposure to high levels of oxygen, called hyperoxia, is associated with increased mortality in the intensive care unit (ICU). Hyperoxia leads to oxidant-mediated acute lung injury (ALI) and pulmonary cell death, called hyperoxic acute lung injury (HALI). Elucidation of molecular mechanisms in HALI could identify therapeutic targets in ALI. In the current study, we examined in vivo effects of HALI on Beclin-1 (BCN1), a molecule that regulates autophagy and cell death. Effects of HALI on BCN1 and autophagy markers were examined in wildtype mice. Analysis of BCN1 and autophagy was completed via Western blot, RT-qPCR, and immunohistochemistry. In wildtype mice, HALI led to increased BCN1 in the lung and in the alveolar epithelium. HALI resulted in significant alterations in markers of autophagy in the lung, including reduced microtubule-associated protein 1B-light chain (LC3B)-II/-I ratios, suggesting reduced autophagic flux. HALI caused increased LDH release in human alveolar type-II cells derived from induced pluripotent stem cells (AT2siPSC), as well as reduced LC3B-II/-I ratios. We previously showed that inhibition of the tyrosine kinase receptor epidermal growth factor receptor (EGFR) is protective in HALI. EGFRWa5/+ mice, which have genetically reduced EGFR activity and improved survival in HALI, showed increased total BCN1, reduced phosphorylated-(p-)/total BCN1 ratios, and decreased LC3B-II/-I ratios in the lung in HALI compared with wildtype. Administration of wortmannin, a phosphatidylinositol-3 kinase (PI3K) inhibitor which decreases BCN1-mediated autophagy, led to increased mortality in HALI in wildtype mice. These data support that regulation of BCN1 and autophagy by EGFR is a protective mechanism in HALI, a pathway which warrants further study for its therapeutic potential. KEY MESSAGESHALI is associated with increased mortality in the ICU and causes alveolar epithelial cell death, but the role of BCN1 in HALI is not well defined. This study shows that BCN1, a molecule involved in autophagy and cell death, is regulated by EGFR in HALI in vivo. These results are significant because regulation by BCN1 and autophagy by EGFR is a novel pathway in HALI with therapeutic potential that warrants further study.

cell biology↗

bioRxiv2024

Astrocytic hemoglobin is an H2O2-decomposing peroxidase and therapeutic target for Alzheimer's disease

Summary paragraphHemoglobin (Hb) is well-known for transporting oxygen in red blood cells within blood vessels1. Although Hb is also present in the brain2, its role remains poorly understood. Here, we show that Hb, found in astrocytes of neurodegenerative animal models and patients, displays significant antioxidant effects through its H2O2-decomposing peroxidase activity, and a small molecule enhancer boosts this activity, reducing aberrant H2O2 and mitigating H2O2-induced neurodegeneration. To counteract the harmful effects of aberrant H2O2-production in Alzheimers disease (AD), we developed KDS12025, a blood-brain barrier (BBB)-permeable small molecule that effectively enhances the peroxidase activity of Hb by a hundredfold, especially at a low level of Hb. KDS12025 and its analogs achieve this enhancement through its electron-donating amine group. KDS12025 reduces H2O2 levels in astrocytes, exhibits neuroprotective effects, and reverses memory impairment in AD models. Gene-silencing of Hb{beta} abrogates KDS12025s impact in both culture and animal models of AD. Moreover, KDS12025 prevented the death of dopaminergic neurons in a Parkinsons disease (PD) model without altering the oxygen-transporting function of Hb. KDS12025 extended survival and improved motor function even in the severe amyotrophic lateral sclerosis (ALS) mouse model. Our findings propose Hb as a new therapeutic target for neurodegenerative diseases, with KDS12025 emerging as a first-in-class drug candidate that enhances Hbs peroxidase activity to reduce H2O2. Boosting Hbs peroxidase activity with KDS12025 mitigates oxidative stress and alleviates neurodegeneration in AD, PD, and ALS with broad applicability for numerous oxidative-stress-driven diseases.

neuroscience↗

bioRxiv2023

Myospreader improves gene editing in skeletal muscle by myonuclear propagation

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader", a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.

cell biology↗

bioRxiv2023

Astrocytic connection to engram neurons increased after learning

Astrocytes directly participate in learning and memory. However, the structural association between astrocytes and memory-encoding engram neurons after learning remains to be elucidated. We developed astrocyte-enhanced green fluorescent protein reconstitution across synaptic partners (eGRASP) to examine tripartite synapses between astrocytes and engram neurons. Using astrocyte-eGRASP, we found that astrocytes had increased connections to engram neurons after learning. Dendritic spines with astrocytic contacts showed enhanced morphology. Live-cell imaging of astrocyte-eGRASP revealed that astrocytic connections are stabilized by neuronal activity. These results indicate that astrocytes distinguish contact between engram neurons and generate engram-specific contact patterns during learning.

neuroscience↗

bioRxiv2022

Impaired ERK MAPK activation in mature osteoblasts enhances bone formation via the mTOR pathway

Emerging evidence supports that osteogenic differentiation of skeletal stem cells (SSCs) is a key determinant of overall bone formation and bone mass. Despite extensive studies showing mitogen-activated protein kinase (MAPK) function in osteoblast differentiation, none of these studies properly show in vivo evidence of impacting post-lineage commitment and subsequent maturation. Here, we describe how the extracellular signal-regulated kinase (ERK) pathway in osteoblasts controls bone formation by suppressing the mechanistic target of rapamycin (mTOR) pathway. We also show that, while ERK inhibition blocks the differentiation of osteogenic precursors when initiated at an early stage, ERK inhibition surprisingly promotes the later stages of osteoblast differentiation. Accordingly, inhibition of the ERK pathway using a small compound inhibitor or conditional deletion of the MAP2Ks Mek1 and Mek2, in mature osteoblasts and osteocytes (Mek1/2Dmp1), markedly increased bone formation due to augmented osteoblast differentiation. Mice with inducible deletion of the ERK pathway in mature osteoblasts (Mek1/2Ocn-Ert) also displayed similar phenotypes, demonstrating that this phenotype reflects continuous postnatal inhibition of late-stage osteoblast maturation. Mechanistically, ERK inhibition increases mitochondrial function and SGK1 phosphorylation via mTOR2 activation, which leads to osteoblast differentiation and production of angiogenic and osteogenic factors to promote bone formation. This phenotype was partly reversed by inhibiting mTOR. Our study uncovers a surprising dichotomy of ERK pathway functions in osteoblasts, whereby ERK activation promotes the early differentiation of osteoblast precursors, but inhibits the subsequent differentiation of committed osteoblasts via mTOR-mediated regulation of mitochondrial function and SGK1.

molecular biology↗

bioRxiv2021

Competition between hematopoietic stem and progenitor cells controls hematopoietic stem cell compartment size.

Cellular competition for limiting hematopoietic factors is a physiologically regulated but poorly understood process. Here, we studied this phenomenon by hampering hematopoietic progenitor access to Leptin receptor+ mesenchymal stem/progenitor cells (MSPCs) and endothelial cells (ECs). We show that HSC numbers increased by 2-fold when multipotent and lineage-restricted progenitors failed to respond to CXCL12 produced by MSPCs and ECs. HSCs were qualitatively normal, and HSC expansion only occurred when early hematopoietic progenitors but not differentiated hematopoietic cells lacked CXCR4. Furthermore, the MSPC and EC transcriptomic heterogeneity was remarkably stable, suggesting that it is impervious to dramatic changes in hematopoietic progenitor interactions. Instead, HSC expansion was caused by increased availability of membrane-bound stem cell factor (mSCF) on MSPCs and ECs due to reduced consumption by cKit-expressing hematopoietic progenitors. These studies revealed an intricate homeostatic balance between HSCs and proximal hematopoietic progenitors regulated by cell competition for limited amounts of mSCF.

immunology↗

bioRxiv2021

Intravital imaging of cerebral microinfarct reveals an astrocyte reaction led to glial scar

Cerebral microinfarct increases the risk of dementia. But how microscopic cerebrovascular disruption affects the brain tissue in cellular-level are mostly unknown. Herein, with a longitudinal intravital imaging, we serially visualized in vivo dynamic cellular-level changes in astrocyte, pericyte and neuron as well as microvascular integrity after the induction of cerebral microinfarction for 1 month in mice. At day 2-3, it revealed a localized edema with acute astrocyte loss, neuronal death, impaired pericyte-vessel coverage and extravascular leakage indicating blood-brain barrier (BBB) dysfunction. At day 5, edema disappeared with recovery of pericyte-vessel coverage and BBB integrity. But brain tissue continued to shrink with persisted loss of astrocyte and neuron in microinfarct until 30 days, resulting in a collagen-rich fibrous scar surrounding the microinfarct. Notably, reactive astrocytes appeared at the peri-infarct area early at day 2 and thereafter accumulated in the peri-infarct. Oral administration of a reversible monoamine oxidase B inhibitor significantly decreased the astrocyte reactivity and fibrous scar formation. Our result suggests that astrocyte reactivity may be a key target to alleviate the impact of microinfarction.

neuroscience↗

bioRxiv2021

Astrocytes render memory flexible

Cognitive flexibility is an essential ability to adapt to changing environment and circumstances. NMDAR has long been implicated in cognitive flexibility, but the precise molecular and cellular mechanism is not well understood. Here, we report that astrocytes regulate NMDAR tone through Best1-mediated glutamate and D-serine release, which is critical for cognitive flexibility. Co-release of D-serine and glutamate is required for not only homosynaptic LTD but also heterosynaptic LTD, which is induced at unstimulated synapses upon release of norepinephrine and activation of astrocytic 1-AR during homosynaptic LTP. Remarkably, heterosynaptic LTD at unstimulated synapses during memory acquisition is required for later repotentiation LTP during reversal learning, laying a foundation for flexible memory and cognitive flexibility. Our study sheds light on the pivotal role of astrocytes in orchestrating multiple synapses during memory formation and determining the fate of consolidated memory to be retained as a flexible memory. HighlightsO_LIAstrocytes regulate NMDAR tone via Best1-mediated glutamate and D-serine release C_LIO_LIActivation of astrocytic 1-AR induces heterosynaptic LTD via NMDAR tone C_LIO_LIHeterosynaptic LTD is required for repotentiation LTP and spatial reversal learning C_LIO_LIAstrocytic regulation of NMDAR tone is critical for metaplasticity and flexible memory C_LI

neuroscience↗

bioRxiv2021

Optical regulation of endogenous RhoA reveals switching of cellular responses by signal amplitude

How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, the small GTPase RhoA regulates both focal adhesion (FA) growth or disassembly, but whether RhoA serves as a switch selecting between cellular outcomes, or if outcomes are simply modulated by additional factors in the cell, is not clear. Here, we develop a photoswitchable RhoA guanine exchange factor, psRhoGEF, to precisely control endogenous RhoA activity. We also develop a FRET-based biosensor to allow visualization of RhoA activity together with psRhoGEF control. Using these new optical tools, we discover that low levels of RhoA activation preferentially induce FA disassembly in a Src-dependent manner, while high levels induce both FA growth and disassembly in a ROCK-dependent manner. Thus, rheostatic control of RhoA activation with photoswitchable RhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.

bioengineering↗

bioRxiv2020

Photon-directed Multiplexed Enzymatic DNA Synthesis for Molecular Digital Data Storage

New storage technologies are needed to keep up with the global demands of data generation. DNA is an ideal storage medium due to its stability, information density and ease of readout with advanced sequencing techniques. However, progress in writing DNA is stifled by the continued reliance on chemical synthesis methods. The enzymatic synthesis of DNA is a promising alternative, but thus far has not been well demonstrated in a highly parallelized manner. Here, we report a novel multiplexed enzymatic DNA synthesis method using maskless photolithography. Rapid uncaging of Co2+ ions by patterned UV light activates Terminal deoxynucleotidyl Transferase (TdT) for spatially-selective synthesis on an array surface. Spontaneous quenching of reactions by the diffusion of excess caging molecules confines synthesis to light patterns and controls the extension length. We show that our multiplexed synthesis method can be used to store digital data by encoding 12 unique DNA oligonucleotide sequences with music from the 1985 Nintendo video game Super Mario Brothers, which is equivalent to 84 trits or 110 bits of data.

synthetic biology↗