Biology subjects

Ha, C.

Publications and source records attributed to Ha, C..

14 recordsLinked to original sources

bioRxiv2026

Chronic NLRP3 inflammasome activation drives neutrophil brain entry and interactions with microglia

NOD-like receptor family pyrin domain-containing 3 (NLRP3) is a cytosolic regulator of an inflammasome-mediated innate immune response. In the central nervous system (CNS), NLRP3 inflammasome activation has been implicated in multiple neurodegenerative diseases, yet the mechanisms by which it contributes to disease remain unclear. Here, we investigated the CNS effects of chronic NLRP3 activation using a humanized NLRP3 gain-of-function mouse model (hNLRP3D305N). Bulk brain analyses confirmed constitutive inflammasome activation, widespread cytokine induction, and the increased presence of blood-associated proteins suggestive of dysfunction at CNS border sites and the blood-brain barrier (BBB). Furthermore, cerebrospinal fluid (CSF) neurofilament light chain levels were elevated, indicating neuronal damage. Single-cell RNA-sequencing of CD45+ immune cells in the brain demonstrated that microglia adopt distinct reactive states and that peripheral immune cells infiltrate the CNS, with neutrophils emerging as the predominant infiltrating immune cell type. This finding was confirmed by untargeted bulk brain and CSF proteomics that also suggest neutrophil reactivity. Immunohistochemistry further revealed regional neutrophil entry into the brain parenchyma, concurrent with reactive microglia and engulfment of neutrophils, suggesting functional microglia-neutrophil interactions. Collectively, these findings establish a direct pathogenic role for the NLRP3 inflammasome in the CNS independent of other neurodegeneration-related disease pathologies.

neuroscience↗

bioRxiv2025

CNS-penetrant NLRP3 inhibitor achieves durable weight loss and reverses hypothalamic inflammation in diet-induced obesity

The NLRP3 inflammasome is a key mediator of innate immunity that integrates inflammatory and metabolic stress signals. Increased and/or chronic activation of this critical pathway has been implicated in obesity, with hypothalamic neuroinflammation linked to dysregulation of energy balance. TN-783 is an investigational, CNS-penetrant, small-molecule NLRP3 inhibitor that potently suppressed inflammasome activation across multiple in vitro assays. In diet-induced obese (DIO) mice, only TN-783, and not the peripherally restricted NLRP3 inhibitor TN-101, produced progressive and sustained weight loss, underscoring the requirement for central target engagement. Weight loss was driven by a persistent reduction in food intake across both acute and chronic phases, without altering energy expenditure. This effect was further characterized by selective reduction of fat mass, with minimal impact on lean tissues. Mechanistically, NLRP3 inhibition attenuated DIO-induced hypothalamic neuroinflammation and partially reversed obesity-associated molecular changes based on transcriptomic and proteomic profiling of the hypothalamus. Beyond monotherapy, TN-783 enhanced the effects of the GLP-1 receptor agonist semaglutide by amplifying weight loss, reinitiating weight loss after semaglutide effect had plateaued, and maintaining the weight loss benefit after semaglutide withdrawal. Discontinuation of TN-783 resulted in reversal of both weight and feeding effects, indicating that its therapeutic activity requires ongoing target engagement rather than permanent remodeling of metabolic pathways. Collectively, these observations support central NLRP3 inhibition as a distinct and promising approach for obesity treatment, offering robust induction and sustained maintenance of weight loss while preserving reversibility.

neuroscience↗

bioRxiv2025

Anti-Aβ immunotherapy-mediated amyloid clearance attenuates microglial activation without inducing exhaustion at residual plaques

Anti-amyloid {beta}-peptide (A{beta}) immunotherapy was developed to reduce amyloid plaque pathology and slow cognitive decline during progression of Alzheimers disease. Efficient amyloid plaque clearance has been proven in clinical trials testing anti-A{beta} antibodies, with the impact on cognitive endpoints correlating with the extent of plaque removal. However, treatment is associated with adverse side-effects, such as oedema and haemorrhages, which are potentially linked to the induced immune response. To improve the safety profile of these molecules, it is imperative to understand the consequences of anti-A{beta} antibody treatment on immune cell function. Here, we investigated the effects of long-term chronic anti-A{beta} treatment on amyloid plaque pathology and microglial response in the APP-SAA triple knock-in mouse model. Mice were treated weekly with anti-A{beta} antibody from 4-8 months of age. Long-term treatment with anti-A{beta} results in a robust and dose-dependent removal of amyloid plaque pathology, with a higher efficiency for removing diffuse over dense-core plaques. Analysis of the CSF proteome indicates a reduction of markers for neurodegeneration including Tau and -Synuclein, as well as immune cell related proteins. Bulk RNA-seq revealed a dose-dependent decrease in brain-wide disease-associated microglial (DAM) and glycolytic gene expression, which is supported by a parallel decrease of glucose uptake and protein levels of Triggering receptor of myeloid cells 2 (Trem2) protein, a major immune receptor involved in DAM activation of microglia. In contrast, DAM activation around remaining plaques remains high regardless of treatment dose. In addition, microglia surrounding remaining plaques display a dose-dependent increase in microglial clustering and a selective increase in antigen presenting and immune signalling proteins. These findings demonstrate that long-term chronic anti-A{beta} mediated removal of A{beta} leads to a dose dependent decrease in brain-wide microglial DAM activation and neurodegeneration, while microglia at residual plaques display a combined DAM and antigen presenting phenotype that suggests a continued treatment response. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/645950v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@6d1a49org.highwire.dtl.DTLVardef@d8c14dorg.highwire.dtl.DTLVardef@7ae330org.highwire.dtl.DTLVardef@1d96cc3_HPS_FORMAT_FIGEXP M_FIG Graphical abstract: Schematic overview of the effects of chronic long-term anti-A treatment in APP-SAA mice Schematic was created with BioRender.com C_FIG

neuroscience↗

bioRxiv2025

GPR34 regulates microglia state and loss-of-function rescues TREM2 metabolic dysfunction

Microglia are implicated in modifying neurodegenerative disease risk in the central nervous system (CNS). GPR34 is a microglia-enriched G-protein coupled receptor that detects cytotoxic lipids upregulated in Alzheimers Disease (AD). Since dysregulated lipid metabolism occurs in disease, we hypothesized GPR34 could act with other lipid sensors, such as TREM2, to regulate microglial function. Here, we report that GPR34 knockout (KO) rescues dysregulated cholesterol metabolism in TREM2 KO iPSC-derived microglia (iMG) and alone promotes fatty acid catabolism without the proton leak observed in TREM2 KO. Loss of GPR34 downregulated ERK signaling, while its agonism promoted interaction with and activation of ERK. In healthy and amyloid mouse models, Gpr34 KO accelerated the conversion of homeostatic microglia to disease-associated microglia (DAM) states. Additionally, in Gpr34 KO amyloid mouse brain, the frequency of large plaques was increased compared to WT, indicating that Gpr34 KO microglia may promote amyloid aggregation. Overall, our data suggest GPR34 as a therapeutic target for modulating microglial function to slow AD progression.

neuroscience↗

bioRxiv2024

Lysosomes cell autonomously regulate myeloid cell states and immune responses

Myeloid cells maintain tissue homeostasis via the recognition, engulfment, and lysosomal clearance of dying cells and cellular debris, which is often accompanied by changes from homeostatic to reactive states. While a role for phagocytic receptors in gating these transitions has been described1,2, less is known about if and how lysosomes can contribute to transcriptional and functional plasticity. To determine how lysosomal health impacts myeloid cell states, we evaluated microglia and macrophages deficient for progranulin (encoded by Grn), a lysosomal protein with pleiotropic functions whose loss is associated with several neurodegenerative diseases3-8. Single-cell RNA-sequencing of the aged mouse brain identified a Grn knockout (KO)-specific microglial subpopulation marked by high GPNMB expression that displays hallmarks of lysosomal dysfunction, including lipofuscin accumulation. Epigenetic analysis of aged microglia revealed MITF/TFE transcription factors as key mediators of the transcriptional states associated with Grn deficiency. In addition to identifying a core myeloid cell transcriptional response to diverse lysosomal stressors, targeted perturbations of various lysosomal properties in vitro uncovered a cell autonomous, TREM2- independent, response to lysosomal deacidification (via v-ATPase or VPS34 loss of function) that overlaps with Grn KO microglia phenotypes, including the induction of a lysosomal gene program, increased proliferation, and secretion of pro-inflammatory cytokines. Compound loss-of-function approaches established GPNMB upregulation upon lysosomal stress is required for the compensatory response to enhance lysosomal function via promoting acidification. Finally, pharmacological endolysosomal reacidification through sodium/proton exchanger inhibition partially rescued Grn KO microglia phenotypes. Overall, these data establish a fundamental link between lysosomal health and myeloid cell epigenetic, transcriptional, and functional states observed in neurodegeneration models.

neuroscience↗

bioRxiv2024

Identification of immunogenic and cross-reactive chikungunya virus-specific CD4+ T cell epitopes in chronic chikungunya viral arthritic disease in humans

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes acute febrile illness that can progress into chronic chikungunya virus disease (CHIKVD) marked by persistent debilitating arthralgia. At present, the exact cause of chronic CHIKVD is not understood, and in humans, the targets of CD4+ T cells in CHIKV are currently unknown. Here, by stimulating peripheral blood mononuclear cells (PBMCs) collected from patients suffering from chronic CHIKVD with peptides spanning the entire CHIKV genome, we provide a comprehensive landscape of CHIKV CD4+ T cell epitopes. We identified 123 novel CD4+ T cell epitopes and three immunodominant regions in E1, nsP1 and CP proteins. The immunodominance of these E1, nsP1 and CP regions was mapped to optimal epitopes, characterized by the capacity to bind to many common HLA class II allelic variants. In addition, we designed and validated a new CHIKV-specific CD4+ T cell epitope megapool, spanning both structural and non-structural proteins, which can be a useful tool to study CHIKV-specific T cell responses in small blood volumes, typically available in pediatric or clinical samples. Finally, by in silico assessment of the conservation of the CHIKV proteome in a diverse set of alphaviruses, we defined CHIKV epitopes conserved across arthritogenic and encephalitic viruses. Overall, our work is the first to identify CD4+ T cell targets of CHIKV in humans, expanding our capacity to study the role of T cells in CHIKV pathogenesis and mapping targets of alphaviruses for vaccine design.

immunology↗

bioRxiv2024

Fc-engineered large molecules targeting blood-brain barrier transferrin receptor and CD98hc have distinct central nervous system and peripheral biodistribution compared to standard antibodies

The blood-brain barrier (BBB) poses a significant challenge drug delivery to the brain. BBB-crossing molecules are emerging as a new class of therapeutics with significant potential for central nervous system (CNS) indications. In particular, transferrin receptor (TfR)- and CD98 heavy chain (CD98hc)-targeting molecules have been demonstrated to cross the BBB for enhanced brain delivery. Previously, we reported TfR and CD98hc antibody transport vehicles (ATVTfR and ATVCD98hc) that utilize these BBB receptors to improve CNS drug delivery1,2. Here, we provide a comprehensive and unbiased biodistribution characterization of ATVTfR and ATVCD98hc compared to a standard IgG at a multiscale level, ranging from whole-body to brain region- and cell type-targeting specificity. Mouse whole-body tissue clearing revealed distinct organ localization for each molecule. In the CNS, ATVTfR and ATVCD98hc not only achieves enhanced brain delivery but importantly, much broader parenchymal distribution in contrast to the severely limited distribution observed with a standard antibody that was not able to be improved even at very high dose levels. Using cell sorting and single-cell RNA sequencing of mouse brain, we revealed that standard IgG predominantly localizes to perivascular and leptomeningeal cells and reaches the CNS by entering the CSF, rather than crossing the BBB. In contrast, ATVTfR and ATVCD98hc enables broad parenchymal cell-specific distribution via transcytosis through brain endothelial cells (BECs) along the neurovasculature. Finally, we extended the translational relevance of our findings by revealing enhanced and broad brain and spinal cord biodistribution of ATVTfR compared to standard IgG in cynomolgus monkey. Taken together, this multiscale analysis reveals in-depth biodistribution differences between ATVTfR, ATVCD98hc, and standard IgG. These results may better inform platform selection for specific therapeutic targets of interest, optimally matching platforms to desired CNS target engagement, peripheral organ exposures, and predict or potentially reduce off-target effects.

neuroscience↗

bioRxiv2023

Chikungunya-virus-specific CD4+ T cells are associated with chronic chikungunya viral arthritic disease in humans

Chikungunya virus (CHIKV) is a mosquito-borne re-emerging viral infection that can cause chronic chikungunya viral arthritic disease (CHIKVD), which is characterized by incapacitating arthralgia and inflammation that can last for months to years following infection. Despite CHIKV outbreaks occurring worldwide and several vaccines currently in development, immune responses to CHIKV in humans remains largely understudied, and targets of T cell response are currently unknown. In this study, we tested peripheral blood mononuclear cells (PBMCs) collected from patients diagnosed with CHIKV infection during the 2014-2015 outbreak in Colombia against pools of overlapping peptides sequentially spanning each CHIKV protein. Using high-resolution flow cytometry, we detected robust CHIKV-specific CD4+, but not CD8+ T cell responses, in these patients. Patients still experiencing disease symptoms six years after infection displayed significantly stronger CHIKV- specific CD4+ T cell responses against nsP1, nsP2 and E2 proteins, compared to patients that resolved the infection. CHIKV-specific CD4+ T cells in symptomatic patients displayed a significantly lower Th1 CD4+ helper T cell responses and were enriched within the Th17 CD4+ helper subset, identifying tumor necrosis factor-alpha (TNF) as the predominantly produced cytokine. In conclusion, this study comprehensively characterizes the T cell response against CHIKV in humans during the chronic phase and provides insights into the role of T cells and possible treatment of CHIKVD.

immunology↗

bioRxiv2023

DNL343 is an investigational CNS penetrant eIF2B activator that prevents and reverses the effects of neurodegeneration caused by the Integrated Stress Response

The integrated stress response (ISR) is a conserved pathway in eukaryotic cells that is activated in response to multiple sources of cellular stress. Although acute activation of this pathway restores cellular homeostasis, intense or prolonged ISR activation perturbs cell function and may contribute to neurodegeneration. DNL343 is an investigational CNS-penetrant small molecule ISR inhibitor designed to activate the eukaryotic initiation factor 2B (eIF2B) and suppress aberrant ISR activation. DNL343 reduced CNS ISR activity and neurodegeneration in a dose-dependent manner in two established in vivo models - the optic nerve crush injury and an eIF2B loss of function (LOF) mutant - demonstrating neuroprotection in both and preventing motor dysfunction in the LOF mutant mouse. Treatment with DNL343 at a late stage of disease in the LOF model reversed elevation in plasma biomarkers of neuroinflammation and neurodegeneration and prevented premature mortality. Several proteins and metabolites that are dysregulated in the LOF mouse brains were normalized by DNL343 treatment, and this response is detectable in human biofluids. Several of these biomarkers show differential levels in CSF and plasma from patients with vanishing white matter disease (VWMD), a neurodegenerative disease that is driven by eIF2B LOF and chronic ISR activation, supporting their potential translational relevance. This study demonstrates that DNL343 is a brain penetrant ISR inhibitor capable of attenuating neurodegeneration in mouse models and identifies several biomarker candidates that may be used to assess treatment responses in the clinic.

neuroscience↗

bioRxiv2023

Targeting Transferrin Receptor to Transport Antisense Oligonucleotides Across the Blood-Brain Barrier

Antisense oligonucleotides (ASO) are promising therapies for neurological disorders, though they are unable to cross the blood-brain barrier (BBB) and must be delivered directly to the central nervous system (CNS). Here, we use a human transferrin receptor (TfR)-binding molecule to transport ASO across the BBB in mice and non-human primates, termed oligonucleotide transport vehicle (OTV). Systemically delivered OTV drives significant, cumulative, and sustained knockdown of the ASO target across multiple CNS regions and all major cell types. Further, systemic OTV delivery enables more uniform ASO biodistribution and knockdown compared to two other clinically relevant ASO delivery routes: a standard, high affinity TfR antibody, or direct ASO delivery to the CSF. Together, our data support systemically delivered OTV as a potential therapeutic platform for neurological disorders. One-Sentence SummarySystemically dosed OTV delivered via TfR1 targeting shows widespread and cumulative target knockdown in the mouse and NHP CNS.

neuroscience↗

bioRxiv2022

Mild SARS-CoV-2 infection results in long-lasting microbiota instability

Viruses targeting mammalian cells can indirectly alter the gut microbiota, potentially compounding their phenotypic effects. Multiple studies have observed a disrupted gut microbiota in severe cases of SARS-CoV-2 infection that require hospitalization. Yet, despite demographic shifts in disease severity resulting in a large and continuing burden of non-hospitalized infections, we still know very little about the impact of mild SARS-CoV-2 infection on the gut microbiota in the outpatient setting. To address this knowledge gap, we longitudinally sampled 14 SARS-CoV-2 positive subjects who remained outpatient and 4 household controls. SARS-CoV-2 cases exhibited a significantly less stable gut microbiota relative to controls, as long as 154 days after their positive test. These results were confirmed and extended in the K18-hACE2 mouse model, which is susceptible to SARS-CoV-2 infection. All of the tested SARS-CoV-2 variants significantly disrupted the mouse gut microbiota, including USA-WA1/2020 (the original variant detected in the United States), Delta, and Omicron. Surprisingly, despite the fact that the Omicron variant caused the least severe symptoms in mice, it destabilized the gut microbiota and led to a significant depletion in Akkermansia muciniphila. Furthermore, exposure of wild-type C57BL/6J mice to SARS-CoV-2 disrupted the gut microbiota in the absence of severe lung pathology. IMPORTANCETaken together, our results demonstrate that even mild cases of SARS-CoV-2 can disrupt gut microbial ecology. Our findings in non-hospitalized individuals are consistent with studies of hospitalized patients, in that reproducible shifts in gut microbial taxonomic abundance in response to SARS-CoV-2 have been difficult to identify. Instead, we report a long-lasting instability in the gut microbiota. Surprisingly, our mouse experiments revealed an impact of the Omicron variant, despite producing the least severe symptoms in genetically susceptible mice, suggesting that despite the continued evolution of SARS-CoV-2 it has retained its ability to perturb the intestinal mucosa. These results will hopefully renew efforts to study the mechanisms through which Omicron and future SARS-CoV-2 variants alter gastrointestinal physiology, while also considering the potentially broad consequences of SARS-CoV-2-induced microbiota instability for host health and disease.

microbiology↗

bioRxiv2022

Influence of residual microbial nucleic acid in instruments for hip joint arthroplasty on false-positive results of metagenomic sequencing

The purpose of this study was to analyze the effect of residual microbial nucleic acid in instruments for hip joint arthroplasty on false-positive sequencing results. Samples were taken from 3 different acetabular reamer for hip arthroplasty in 7 different hospitals. The whole process was strictly aseptic, metagenomic next-generation sequencing (mNGS) was performed according to standard operating procedures. The sterility of instruments was confirmed by culture method. The sequencing results of specimens from different hospitals were compared to analyze the difference of background bacteria. Bioinformatics analysis and visualization were presented through R language. A total of 26 samples were processed by mNGS, including 24 instrument swab samples, 1 blank swab control, and 1 blank water control. 254,314,707 reads were sequenced in all samples. The results showed that 1.13% of Clean Reads can be matched to pathogenic microorganism genomes, of which bacterial sequences account for 87.48%, fungal sequences account for 11.18%, parasite sequences account for 1.26%, and virus sequences account for 0.06%. The results of PCA (Principal Component Analysis) demonstrated that the distribution of bacteria on the surface of instruments was significantly different between medical institutions. Through the Venn diagram, it was found that 465 species of bacteria in all region hospitals, Liaocheng Peoples Hospital had a maximum of 340 species of bacteria, followed by Guanxian County Peoples Hospital with 169 species. The clustering heat map illustrated that the distribution of bacterial groups in three different instrument samples in the same hospital was basically the same, and the bacterial genera varied significantly among hospitals. The residual microbial nucleic acid fragments are mainly bacterial DNA and represent differences in different medical institutions, The establishment of independent background bacterial libraries in different medical institutions can effectively improve the accuracy of mNGS diagnosis and help to exclude background microorganisms interference.

genomics↗

bioRxiv2021

Fibrillar Aβ causes profound microglial metabolic perturbations in a novel APP knock-in mouse model

Microglial dysfunction is believed to play a pathogenic role in Alzheimers disease (AD). Here, we characterize the amyloid-{beta} related pathology and microglial responses in an engineered APP knock-in mouse model of familial AD. This model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered glial responses and neurodegeneration. Leveraging multi-omics approaches, we found lipid accumulation and an exacerbated disease-associated transcriptomic response in methoxy-X04-positive, phagocytic microglia. Together, these findings highlight the potential of this novel, open-access mouse model to investigate AD pathogenesis and demonstrate that fibrillar A{beta} triggers lipid dysregulation and immuno-metabolic perturbations in phagocytic microglia. HighlightsO_LINovel open-access APP KI mouse model shows salient AD pathological features C_LIO_LIDeep phenotyping of sorted microglia reveals profound lipidomic perturbations in line with Alois Alzheimers original descriptions of glial adipose inclusions C_LIO_LIImmunometabolic perturbations are exacerbated in microglia accumulating fibrillar A{beta} C_LI

neuroscience↗

bioRxiv2019

Defining the relative and combined contribution of CTCF and CTCFL to genomic regulation

BackgroundUbiquitously expressed CTCF is involved in numerous cellular functions, such as organizing chromatin into TAD structures. In contrast, its paralog, CTCFL is normally only present in testis. However, it is also aberrantly expressed in many cancers. While it is known that shared and unique zinc finger sequences in CTCF and CTCFL enable CTCFL to bind competitively to a subset of CTCF binding sites as well as its own unique locations, the impact of CTCFL on chromosome organization and gene expression has not been comprehensively analyzed in the context of CTCF function. Using an inducible complementation system, we analyze the impact of expressing CTCFL and CTCF-CTCFL chimeric proteins in the presence or absence of endogenous CTCF to clarify the relative and combined contribution of CTCF and CTCFL to chromosome organization and transcription. ResultsWe demonstrate that the N terminus of CTCF interacts with cohesin which explains the requirement for convergent CTCF binding sites in loop formation. By analyzing CTCF and CTCFL binding in tandem we identify phenotypically distinct sites with respect to motifs, targeting to promoter/intronic intergenic regions and chromatin folding. Finally, we reveal that the N, C and zinc finger terminal domains play unique roles in targeting each paralog to distinct binding sites, to regulate transcription, chromatin looping and insulation. ConclusionThis study clarifies the unique and combined contribution of CTCF and CTCFL to chromosome organization and transcription, with direct implications for understanding how their co-expression deregulates transcription in cancer.

genomics↗