Biology subjects

Won, J.

Publications and source records attributed to Won, J..

15 recordsLinked to original sources

bioRxiv2026

Reduced cortical VPS26B levels are associated with altered glutamate receptor expression and synaptic protein loss in the primary motor cortex of a Parkinsonian mouse model

Parkinsons disease (PD) is associated with motor impairment and cortical synaptic dysfunction, which involve altered glutamate receptor trafficking, yet the underlying mechanisms remain incompletely understood. VPS26B, a component of the retromer complex, regulates GluA1 recycling in the trans-entorhinal cortex region. However, its role in the primary motor cortex (M1) under Parkinsonian conditions has not been explored. Here, we show that VPS26B levels are reduced in the M1 of an MPTP-induced PD mouse model, accompanied by decreased surface GluA1 and synaptic protein levels. VPS26B overexpression partially attenuated these alterations. In the accelerating rotarod test, VPS26B-deficient mice exhibited unstable motor performance following MPTP administration, whereas VPS26B overexpression was associated with improved performance in both wild-type and knockout mice. These findings suggest that cortical VPS26B may contribute to maintaining glutamate receptor surface expression and synaptic protein levels, especially under Parkinsonian conditions, with potential implications for motor learning.

neuroscience↗

bioRxiv2025

Precision at Every Scale: Efficiency in AI-Driven De Novo Antibody Design

The precise de novo design of antibodies remains a therapeutic challenge. The AI platform, GaluxDesign, was evaluated in a high-efficiency Precision-Scale Workflow by synthesizing and testing only 50 full-length IgG candidates per epitope across eight distinct epitopes from six therapeutic targets. This campaign yielded a 10.5% binder rate (estimated EC50 < 100 nM), identifying target-specific binders for seven of eight epitopes, with multiple candidates exhibiting sub-nanomolar to single-digit nanomolar dissociation constants (Kd). We further assessed the same workflow on nine shared benchmark targets selected for external comparison, where GaluxDesign identified target-specific binders for eight of nine targets, demonstrating strong target-level performance relative to previously reported de novo antibody design approaches. Together, these results establish a high-efficiency, precision-scale workflow for generating novel, high-affinity therapeutic antibodies.

bioengineering↗

bioRxiv2025

Optimizing twin prime editing components for scalable genome editing and therapy in spinocerebellar ataxia type 3

Recent advances in prime editing technologies using CRISPR modules fused with reverse transcriptase (RT) have enabled efficient and precise reprogramming of target genomic sequences. Twin prime editing using two coordinated prime editor complexes is a promising strategy for inducing extensive genomic modifications via reverse transcribed complementary templates. However, current twin prime editing systems still require improvements in editing efficiency, accuracy, and intended edit predictability. Here, efficiency and precision of twin prime editing were enhanced via engineering and optimizing conventional SpCas9(H840A)-RT-based prime editor components. A La domain-fused prime editor (La-SpCas9(H840A)-RT) and optimized pegRNAs were developed, achieving a 1.75 {+/-} 0.21-fold increase in gene editing efficiency at multiple genomic loci in human-derived cell lines without increasing off-target activity. La-SpCas9(H840A)-RT facilitated efficient [~]2.8 kb GFP transgene knock-in at target loci and eliminated the expanded polyQ tract in ATXN3 in patient-derived mutant cell lines modeling spinocerebellar ataxia type 3. The advanced twin prime editing platform expands genome engineering capabilities beyond existing CRISPR-based systems and holds great promise for diverse biotechnological and therapeutic applications.

bioengineering↗

bioRxiv2025

Precise, Specific, and Sensitive De Novo Antibody Design Across Multiple Cases

The precision design of antibodies, which naturally recognize diverse molecules through six variable loops, remains a critical challenge in therapeutic molecule discovery. In this study, we demonstrate that precise, sensitive, and specific antibody design can be achieved without prior antibody information across eight distinct target proteins. For each target, binders were identified from a yeast display scFv library of approximately 106 sequences, constructed by combining 102 designed light chain sequences with 104 designed heavy chain sequences. Binders with varying binding strengths were identified for all eight targets, including a case where no experimentally resolved target protein structure was available, demonstrating the highest level of precision compared to previous de novo antibody design reports. To further validate the designed antibodies, they were characterized in the IgG format for five distinct targets. The antibodies exhibited favorable developability properties, positioning them as promising hit or lead candidates. Notably, for one target in particular, the IgG-formatted antibodies exhibited affinity, activity, and developability, comparable to a commercial antibody, highlighting the sensitivity of the design method. Furthermore, binders capable of distinguishing closely related protein subtypes or mutants were identified, demonstrating that the method can achieve high molecular specificity. Cryo-EM analysis experimentally validated the designs accuracy by confirming that the key binding interactions were precisely as designed. These findings underscore the effectiveness of precision molecular design based on atomic-accuracy structure prediction. This study establishes computational antibody design as a viable approach for generating therapeutic molecules with tailored properties, with promising potential for achieving the efficacy and safety required for successful therapeutics.

bioengineering↗

bioRxiv2025

Astrocytic MAOB-GABA axis as a molecular brake on repair following spinal cord injury

Neuroregeneration and remyelination rarely occur in the adult mammalian brain and spinal cord following central nervous system (CNS) injury. The glial scar has been proposed as a major contributor to this failure in the regenerative process. However, its underlying molecular and cellular mechanisms remain unclear. Here, we report that monoamine oxidase B (MAOB)-dependent excessive GABA release from reactive astrocytes suppresses CNS repair system by reducing BDNF and TrkB expression in severe spinal cord injury (SCI) animal models. Genetic deletion of MAOB in a mouse SCI model promotes both functional and tissue recovery. Notably, the selective MAOB inhibitor, KDS2010, facilitates recovery and regeneration by disinhibiting the BDNF-TrkB axis in a rat SCI model. Its dose-dependent effects were further validated in a monkey SCI model. Moreover, KDS2010 demonstrates a tolerable safety profile and dose-proportional pharmacokinetics in healthy humans during a phase 1 clinical trial. Our findings identify the astrocytic MAOB-GABA axis as a crucial molecular and cellular brake on CNS repair system following SCI and highlight translational potential of KDS2010 as a promising therapeutic candidate for SCI treatment.

neuroscience↗

bioRxiv2025

Glutamatergic heterogeneity in the neuropeptide projections from the lateral hypothalamus to the mouse olfactory bulb

The direct pathway from the lateral hypothalamus to the mouse olfactory bulb (OB) includes neurons that express the neuropeptide orexin-A, and others that do not. The OB-projecting neurons that do not express orexin-A are present in an area of the lateral hypothalamus known to contain neurons that express the neuropeptide melanin-concentrating hormone (MCH). We used virally mediated anterograde tract tracing and immunohistochemistry for orexin-A and MCH to demonstrate that the OB is broadly innervated by axon projections from both populations of neurons. Orexin-A and MCH were expressed in each OB layer across its anterior to posterior axis. Both orexin-A and MCH neurons are genetically heterogeneous, with subsets that co-express an isoform of vesicular glutamate transporter (VGLUT). We used high-resolution confocal imaging to test whether the projections from orexin-A and MCH neurons to the OB reflect this glutamatergic heterogeneity. The majority ([~]57%) of putative orexin-A axon terminals overlapped with VGLUT2, with smaller proportions that co-expressed VGLUT1, or that did not overlap with either VGLUT1 or VGLUT2. In contrast, only [~]26% of putative MCH axon terminals overlapped with VGLUT2, with the majority not overlapping with either VGLUT. Therefore, the projections from the lateral hypothalamus to the OB are genetically heterogeneous and include neurons that can release two different neuropeptides. The projections from both populations are themselves genetically heterogeneous with distinct ratios of glutamatergic and non-glutamatergic axon terminals. Three Key PointsO_LIA direct anterograde projection from orexin-A-expressing neurons in the hypothalamus to the mouse olfactory bulb (OB) was confirmed using virally mediated tract tracing. C_LIO_LIOrexin-A and melanin-concentrating hormone (MCH), two neuropeptides primarily produced in the lateral hypothalamus are broadly expressed throughout the mouse OB. C_LIO_LIApproximately 64% of putative orexin-A axon terminals overlapped with vesicular glutamate transporter type 1 (VGLUT1) or type 2 (VGLUT2). In contrast, only 25% of putative MCH axon terminals overlapped with VGLUT2. C_LI

neuroscience↗

bioRxiv2024

Accurate antibody loop structure prediction enables zero-shot design of target-specific antibodies

Protein loops, characterized by their versatile structures with varying sizes and shapes, can recognize a wide range of targets with high specificity and affinity. The variable loops of the antibody complementarity-determining region (CDR) are particularly crucial for immune responses and therapeutic applications due to their effective target recognition capabilities. Accurate structure prediction of these antibody loops is essential for the efficient in silico design of target-binding antibodies for therapeutic or industrial use. However, predicting antibody loop structures is challenging due to the lack of evolutionary information from related proteins. Thus, a successful ab initio structure prediction method, which operates without structural templates or related sequences, is crucial for the effective design of antibody loop-mediated interactions. This study demonstrates that highly accurate antibody loop structure prediction enables the effective zero-shot design of target-binding antibody loops. The performance of loop design has been shown to depend on the accuracy of ab initio loop structure prediction, as tested with two versions of our design model. The high affinity, diversity, novelty, and specificity of the antibody loops designed with these new methods were validated experimentally on four target proteins.

bioengineering↗

bioRxiv2023

Hypothalamic neuronal activation in primates drives naturalistic goal-directed eating behavior

Eating addiction is the primary cause of modern obesity. Although the causal role of the lateral hypothalamic area (LHA) for eating is demonstrated in rodents, there is no evidence in primates regarding naturalistic eating behaviors. We investigated the role of LHA GABAergic (LHAGABA) neurons in eating by chemogenetics in three macaques. LHAGABA neuron activation significantly increased naturalistic goal-directed behaviors and food motivation, which was specific for palatable food. PET and MRS validated the chemogenetic activation. Rs-fMRI result revealed that functional connectivity (FC) between the LHA and frontal areas was increased, while the FC between the frontal cortices was decreased after the LHAGABA neuron activation. Thus, our study elucidates the role of LHAGABA neurons in eating and obesity therapeutics for primates and humans.

neuroscience↗

bioRxiv2023

Host genetic variation guides hepacivirus clearance, chronicity, and liver fibrosis in mice

Background & AimsHuman genetic variation is thought to guide the outcome of hepatitis C virus (HCV) infection but model systems within which to dissect these host genetic mechanisms are limited. Norway rat hepacivirus (NrHV), closely related to HCV, causes chronic liver infection in rats but causes acute self-limiting hepatitis in typical strains of laboratory mice, which resolves in two weeks. The Collaborative Cross (CC) is a robust mouse genetics resource comprised of a panel of recombinant inbred strains, which model the complexity of the human genome and provide a system within which to understand diseases driven by complex allelic variation. Approach & ResultsWe infected a panel of CC strains with NrHV and identified several that failed to clear virus after 4 weeks. Strains displayed an array of virologic phenotypes ranging from delayed clearance (CC046) to chronicity (CC071, CC080) with viremia for at least 10 months. Body weight loss, hepatocyte infection frequency, viral evolution, T-cell recruitment to the liver, liver inflammation and the capacity to develop liver fibrosis varied among infected CC strains. ConclusionsThese models recapitulate many aspects of HCV infection in humans and demonstrate that host genetic variation affects a multitude of virus and host phenotypes. These models can be used to better understand the molecular mechanisms that drive hepacivirus clearance and chronicity, the virus and host interactions that promote chronic disease manifestations like liver fibrosis, therapeutic and vaccine performance, and how these factors are affected by host genetic variation.

microbiology↗

bioRxiv2023

CRISPR-Cas-Docker: Web-based in silico docking and machine learning-based classification of crRNAs with Cas proteins

MotivationCRISPR-Cas-Docker is a web server for in silico docking experiments with CRISPR RNAs (crRNAs) and Cas proteins. This web server aims at providing experimentalists with the optimal crRNA-Cas pair predicted computationally when prokaryotic genomes have multiple CRISPR arrays and Cas systems, as frequently observed in metagenomic data. CRISPR-Cas-Docker provides two methods to predict the optimal Cas protein given a particular crRN sequence: a structure-based method (in silico docking) and a sequence-based method (machine learning classification). For the structure-based method, users can either provide experimentally determined 3D structures of these macromolecules or use an integrated pipeline to generate 3D-predicted structures for in silico docking experiments. ResultsCRISPR-Cas-Docker is an optimized and integrated platform that provides users with 1) 3D-predicted crRNA structures and AlphaFold-predicted Cas protein structures, 2) the top-10 docking models for a particular crRNA-Cas protein pair, and 3) machine learning-based classification of crRNA into its Cas system type. Availability and implementationCRISPR-Cas-Docker is available as an open-source tool under the GNU General Public License v3.0 on GitHub. It is also available as a web server.

bioinformatics↗

bioRxiv2022

Characterization of TMEM43 as a novel ion channel

The TMEM43 gene has been reported to play supportive but critical roles in human diseases including cancer, arrhythmogenic right ventricular cardiomyopathy (ARVC), and auditory neuropathy spectrum disorder (ANSD). However, direct characterization of the TMEM43 protein itself and its role in the brain remain unexplored. In this study, we demonstrated that TMEM43 confers ion channel activities via the lipid bilayer reconstitution of purified TMEM43 protein and further characterized TMEM43 as a pH-sensing cation channel in the heterologous expression system. TMEM43 was shown to conduct transjunctional potentials between adjacent cells, further facilitating electrical couplings of the gap junctions. In the hippocampus of TMEM43 knockout (KO) mice, we observed a decrease in astrocytic dye diffusion and potassium buffering, an increase in neuronal excitability, and alterations in AMPA/NMDA ratio and LTP. The electrophysiological changes in the KO mice led to a disturbance in memory retrieval which was rescued with TMEM43 overexpression. These results indicate that TMEM43 actively participates in gap junction networks of the hippocampus to prevent neurons from hyperexcitability, which is critical for memory retrieval. Together, our study elucidates the molecular and functional identities of TMEM43 and underscores its role in memory retrieval.

molecular biology↗

bioRxiv2020

Platycodin D prevents both lysosome- and TMPRSS2-driven SARS-CoV-2 infection in vitro by hindering membrane fusion

An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to the global public health. Herbal medicines and their derived natural products have drawn much attention to treat COVID-19, but there has been no natural product showing inhibitory activity against SARS-CoV-2 infection with detailed mechanism. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection-routes via lysosome- and transmembrane protease, serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host-entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by a pharmacological inhibition or gene-silencing of NPC1, which is mutated in Niemann-Pick type C (NPC) patients displaying disrupted membrane cholesterol. Finally, readily available local foods or herbal medicines containing PG root show the similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that a brief disruption of membrane cholesterol can be a novel therapeutic approach against SARS-CoV-2 infection.

pharmacology and toxicology↗

bioRxiv2020

Nasal commensal, Staphylococcus epidermidis shapes the mucosal environment to prevent influenza virus invasion through Serpine1 induction

Our recent study presented evidence that Staphylococcus epidermidis (S. epidermidis) was the most frequently encountered microbiome component in healthy human nasal mucus and that S. epidermidis could induce interferon (IFN)-dependent innate immunity to control acute viral lung infection. The serine protease inhibitor Serpine1 was identified to inhibit influenza A virus (IAV) spread by inhibiting glycoprotein cleavage, and the current study supports an additional mechanism of Serpine1 induction in the nasal mucosa, which can be regulated through S. epidermidis and IFN signaling. The exposure of in vivo mice to human S. epidermidis increased IFN-{lambda} secretion in nasal mucosa and prevented an increase in the burden of IAV in the lung. S. epidermidis-inoculated mice exhibited the significant induction of Serpine1 in vivo in the nasal mucosa, and by targeting airway protease, S. epidermidis-induced Serpine1 inhibited the intracellular invasion of IAV to the nasal epithelium and led to restriction of IAV spreading to the lung. Furthermore, IFN-{lambda} secretion was involved in the regulation of Serpine1 in S. epidermidis-inoculated nasal epithelial cells and in vivo nasal mucosa, and this was biologically relevant for the role of Serpine1 as an interferon-stimulated gene in the upper airway. Together, our findings reveal that human nasal commensal S. epidermidis manipulates the suppression of serine protease in in vivo nasal mucosa through Serpine1 induction and protects the nasal mucosa from IAV invasion through IFN-{lambda} signaling. IMPORTANCEPreviously, we proved that nasal microbiome could enhance IFN-related innate immune responses to protect the respiratory tract against influenza virus infection. The present study shows a great understanding of the intimate association of S. epidermidis-regulated IFN-lambda induction and serine protease inhibitor in nasal mucosa. Our data demonstrate that S. epidermidis-regulated Serpine1 suppresses the invasion of influenza virus through suppression of airway serine protease at the level of nasal mucosa and impedes IAV spread to the respiratory tract. Thus, human nasal commensal S. epidermidis represents a therapeutic potential for treating respiratory viral infections via the change of cellular environment in respiratory tract. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

immunology↗

bioRxiv2020

Impaired hand dexterity function in a non-human primate model with chronic Parkinson's disease

Symptoms of Parkinsons disease (PD) caused by loss of dopaminergic neurons are accompanied by movement disorders, including tremors, rigidity, bradykinesia, and akinesia. Non-human primate (NHP) models with PD play an essential role in the analysis of PD pathophysiology and behavior symptoms. As impairments of hand dexterity function can affect activities of daily living in patients with PD, research on hand dexterity function in NHP models with chronic PD is essential. Traditional rating scales previously used in the evaluation of animal spontaneous behavior were insufficient due to factors related to subjectivity and passivity. Thus, experimentally designed applications for an appropriate apparatus are necessary. In this study, we aimed to longitudinally assess hand dexterity function using hand dexterity task (HDT) in NHP-PD models. To validate this assessment, we analyzed an alteration in Parkinsonian tremor symptoms, loss of dopaminergic neuron, and positron emission tomography (PET) imaging of dopamine transporters (DAT) in these models. HDT latency significantly increased in NHP-PD models. In addition, a significant inverse correlation between HDT and DAT was identified, but no local bias was found. The correlation with intention tremor symptoms was lower than the resting tremor. In conclusion, the evaluation of HDT may reflect behavioral symptoms of NHP-PD models. Furthermore, HDT was effectively used to experimentally distinguish intention tremors from other tremors.

neuroscience↗

bioRxiv2020

Gait analysis of healthy rhesus monkeys using a pressure-sensing walkway system

Gait analysis in non-human primate models has been performed to elucidate the neural systems involved in controlling quadrupedal locomotor behavior. This study aimed to use a pressure-sensing walkway to identify characteristics of species-specific quadrupedal locomotion in rhesus monkeys. A total of nine healthy adult female rhesus monkeys (Macaca mulatta) were used for gait analysis. We measured the temporo-spatial and kinetic parameters of quadrupedal gait using a custom-built pressure-sensing walkway and compared the left- and right-side parameters to assess the symmetry of the gait pattern. All temporo-spatial and kinetic parameter values showed no significant differences among the nine rhesus monkeys for both the forelimbs and hindlimbs. However, significant differences were observed between forelimb and hindlimb kinetic parameters such as peak vertical force, vertical impulse, and the percentage of body weight distribution. All kinetic parameter values were higher for the forelimbs than for the hindlimbs. These data indicated that the center of gravity in healthy rhesus monkeys is located at the forelimbs rather than at the hindlimbs while walking. Furthermore, the symmetry indices considered for symmetric gait pattern showed a low variability. Most median symmetry index values were nearly zero, indicating no difference between the right and left sides. This study described valid methods for assessing gait parameters and demonstrated rhesus-specific characteristics of quadrupedal locomotion, providing a basis for the assessment of gait normality in rhesus monkeys.

animal behavior and cognition↗