Biology subjects

Jagodic, M.

Publications and source records attributed to Jagodic, M..

16 recordsLinked to original sources

bioRxiv2026

Advancing Neurological Liquid Biopsy via Six-Base Sequencing of Cerebrospinal Fluid Cell-Free DNA

Real-time molecular insights into the human brain are crucial for understanding neurological conditions and development of soluble biomarkers but challenged by the limited access to target tissue. Here we utilize 6-base sequencing to profile DNA modifications of cell-free DNA released from neural cells into cerebrospinal fluid. This strategy provides molecular, quantitative and qualitative insights into the ongoing neurodegenerative biology, which paves the way for liquid biopsy approaches to study neurological conditions.

neuroscience↗

bioRxiv2026

Single-cell analysis suggests coordinated immune-cell redistribution between blood and cerebrospinal fluid in early multiple sclerosis

Despite immune-cell infiltration being a hallmark of multiple sclerosis (MS), the interplay between the periphery and central nervous system immune responses is still incompletely characterized. We performed single-cell transcriptomic and V(D)J sequencing of paired blood and cerebrospinal fluid (CSF) immune cells from treatment-naive relapsing-remitting women with MS and compared them to age- and sex-matched healthy controls. Across major immune lineages, we identified coordinated, compartment-specific immune alterations, with enrichment of activated and memory lymphocyte populations in the CSF and concomitant depletion of related populations in peripheral blood, suggesting their recruitment from blood to CSF. Clonally expanded CD4 memory T-cells, together with activated, expanded IgM-positive B-cells, accumulated predominantly in the CSF of people with MS compared to healthy subjects. Interestingly, tissue-primed cytotoxic populations and CXCR3-associated memory populations were depleted from the CSF of MS, implying their recruitment to the target tissue in early disease. These findings reveal coordinated, compartment-specific immune changes in early MS and provide a systems-level view of immune-cell trafficking between peripheral and central nervous system compartments.

immunology↗

bioRxiv2026

Epigenomic profiling of cerebrospinal fluid cells identifies immune regulatory alterations and implicates protocadherins in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), where DNA methylation may play a role by connecting genetic and environmental risk factors. We performed whole-genome DNA methylation profiling of cerebrospinal fluid (CSF) cells from relapsing-remitting MS patients and matched controls, identifying 2,710 differentially methylated positions (DMPs) and 4,330 regions (DMRs). These changes were enriched in immune signaling, adhesion and migration processes, and were accompanied by corresponding RNA expression changes. MS-associated methylation changes enriched in the cohesin chromatin regulation pathway mapped to enhancers of T helper 17 (Th17) cells, whereas in other T cell types they were mapping to bivalent enhancers and repressed chromatin. Notably, this pathway comprised multiple Protocadherin (PCDH) genes, typically expressed in neuronal cells, that displayed consistent methylation and expression changes in CSF cells. Expression of shared intracellular domain of PCDH{gamma} cluster proteins was confirmed in peripheral blood T cells by flow cytometry as well as expression of PCDH{gamma} cluster genes in memory CD4+ T cell subsets. Moreover, co-expression analysis suggests a role of PCDH genes in aryl hydrocarbon receptor (AHR) signaling. In summary, DNA methylation changes in CSF resident cells reflect dysregulated T cell activation and migration in MS and suggest a novel role of protocadherin molecules in MS pathogenesis.

immunology↗

bioRxiv2026

Targeted DNA methylation editing in vivo

The number of epigenome-wide association studies linking CpG DNA methylation with disease, traits and exposures, continues to rise. Despite the rapid development of epigenome editing tools, establishing causation remains challenging, particularly in vivo. In this study, we developed and characterized three Cre-dependent CRISPR-based mouse lines that enable locus-specific DNA methylation deposition by either constitutive or inducible dCas9-DNMT3A expression. We demonstrate robust highly locus-specific DNA methylation deposition at MHC class II (H2-Ab1) and interleukin 6 (Il6) genes in bone marrow-derived myeloid cells ex vivo. Moreover, neuron-specific methylation targeting resulted in reduced cannabinoid receptor 1 (Cnr1) expression in striatal neurons in vivo. Notably, we demonstrate that the causal effect of DNA methylation on gene expression is locus-dependent, reinforcing the necessity of such editing tools for detailed understanding of the role of DNA methylation and for addressing the causality of disease-associated CpGs.

molecular biology↗

bioRxiv2026

Whole-genome profiling of native 5-hydroxymethylation in human neurons with long-read sequencing

The 5-hydroxymethylcytosine (5hmC) modification of DNA is particularly prevalent in neurons and thereby a hallmark of the brains epigenetic landscape. While 5mC DNA methylation is a well-known player in genome stability and transcriptional regulation, the role of 5hmC remains largely unknown. Here, we used long-read Oxford Nanopore Technology (ONT) to profile whole-genome, native 5mC and 5hmC levels in sorted neuronal nuclei samples from human post-mortem brain tissue. We applied different models for DNA modification calling and compared with array-based 5mC and 5hmC levels derived from the same samples, demonstrating high sample-wise correlations. Annotation across genomic and regulatory features, as well as chromatin states, generated by the International Human Epigenome Consortium, revealed high levels of 5hmC in introns, actively transcribed genes and (distal) enhancers. Pathway analysis of genes with high levels of 5hmC (> 60%) were enriched in neuron-related terms, with functional variety when stratifying across chromatin states. Analysis of transcription factor motifs in highly methylated regions, demonstrated 5hmC- and 5mC-specific enrichment affecting downstream regulatory networks. Altogether, our study demonstrates the potential of ONT to characterize whole-genome, native 5hmC and 5mC DNA modifications in human neurons, specifically highlighting the enrichment of 5hmC in actively transcribed regions and enhancers in the human brain.

neuroscience↗

bioRxiv2025

Anoctamin-2-specific T Cells Link Epstein-Barr Virus to Multiple Sclerosis

Multiple sclerosis (MS) occurs when the central nervous system (CNS) is damaged by misguided adaptive immune responses, likely caused by a combination of environmental factors in genetically susceptible individuals. A known prerequisite for disease is Epstein-Barr virus (EBV) infection, and previous studies have demonstrated elevated Epstein-Barr virus nuclear antigen 1 (EBNA1) antibodies which cross-react with the calcium-activated chloride channel anoctamin-2 (ANO2) in persons with MS (pwMS). ANO2-reactive antibodies have been associated with greater neuroaxonal damage in MS, although their exact effector function is still uncertain. Here, we demonstrate that ANO2 is also the target of IFN{gamma}-producing CD4+ T cells, which are more frequent in untreated and natalizumab-treated pwMS compared to control individuals. Immunisation of SJL/J mice with either ANO2 or EBNA1 elicited cross-reactive CD4+ T cell and antibody responses in vivo. Pre-immunisation of young mice with ANO2 worsened proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), which in older mice included atypical clinical phenotype, immune infiltration into the brain and reduced survival. EAE exacerbation was recapitulated with the adoptive co-transfer of ANO2 and myelin antigen-specific CD4+ T cells, and ANO2-specific T cells alone could induce the cell death of ANO2-expressing glial cells in vitro. T cell clones with cross-reactivity to both EBNA1 and ANO2 antigens could be isolated from natalizumab-treated pwMS. Single cell sequencing of EBNA1 and ANO2-specific T cell receptors (TCR) from four pwMS revealed a significant overlap between their antigen-specific expanded TCR repertoires within donors and transcriptomic analysis showed cross-reactive T cells to have predominantly activated and cytotoxic phenotypes. In summary, we report the first mechanistic evidence that EBNA1 CD4+ T cells can target the MS-associated autoantigen ANO2, thereby establishing a link between EBV infection and development of autoimmune neuroinflammatory disease.

immunology↗

bioRxiv2025

Oligodendroglia as functional effectors of Multiple Sclerosis risk variants

Multiple sclerosis (MS) is a neuroinflammatory disease for which a large number of non-coding single nucleotide polymorphisms (SNPs) have been associated with disease risk/susceptibility. Immune cells have been suggested as the principal functional effector cell types of these common variants. Here, we identify 76 MS-associated SNPs whose loci present accessible chromatin in homeostatic and diseased oligodendroglia (OLG), including both oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes (MOLs). By applying high-throughput functional genomics, we found that a subset of these SNPs led to variant- and cell-specific regulatory effects in human induced pluripotent stem cell-derived oligodendroglia. Phenotypic profiling of these variants indicated that rs483180:PHGDH interfered with human OPC proliferation via long-range chromatin interactions with the S100A6 locus, while variants at rs2248137:CYP24A1 impaired oligodendrocyte differentiation by regulating BCAS1 expression. In addition, variants at rs1415069:DIPK1A enhanced secretion of the cytokine CCL2 by oligodendroglia, suggesting that these variants might be implicated in OLG-driven immune cell recruitment in MS. These findings position oligodendroglia as important drivers of MS pathogenesis through modulation of both their cell-intrinsic oligodendroglial function and intercellular communication by non-coding MS risk variants.

cell biology↗

bioRxiv2025

Human placental stem cells induce a novel multiple myeloid cell-driven immunosuppressive program that ameliorates proinflammatory CNS pathology

Despite a growing interest in Amniotic Epithelial Cell (AEC)-based therapies, the immune responses triggered by AEC transplantation in vivo remain poorly characterized. In particular, how direct exposure to AECs within the central nervous system (CNS) shapes the local immune environment is currently unknown. Herein we describe a novel CNS- specific immunoregulatory pathway induced by intracisternal delivery of human AECs. Local immune responses induced by AECs in the brain led to recruitment of immunosuppressive Arginase 1+ (ARG1+) macrophages and a novel population of myeloid-derived suppressor cells with eosinophilic characteristics, which we term Eo- MDSCs. We further demonstrate that Eo-MDSCs produce Maresin 2 (MaR2), a specialized pro-resolving mediator (SPM) involved in the resolution of inflammation. In a mouse model of Multiple Sclerosis (MS), treatment of established disease with AECs induced immunological responses that resulted in reduced numbers of pathogenic macrophages and T helper (TH)17 cells, increased anti-inflammatory T cell subsets, and enhanced myelin phagocytosis, all of which led to functional recovery. These findings suggest that AEC therapy has the potential to target CNS-intrinsic inflammatory processes in MS, providing a strong rationale for translation into the clinic.

neuroscience↗

bioRxiv2025

Systematic comparison of dCas9-based DNA methylation epimodifiers over time indicates efficient on-target and widespread off-target effects

CRISPR/dCas9-based epigenome editing systems, including DNA methylation epimodifiers, have greatly advanced molecular functional studies revolutionizing their precision and applicability. Despite their promise, challenges such as the magnitude and stability of the on-target editing and unwanted off-target effects underscore the need for improved tool characterization and design. We systematically compared specific targeting of the BACH2 gene promoter and genome-wide off-target effects of available and novel dCas9-based DNA methylation editing tools over time. We demonstrate that multimerization of the catalytic domain of DNA methyltransferase 3A enhances editing potency but also induces widespread, early methylation deposition at low-to-medium methylated promoter-related regions with specific gRNAs and, interestingly, also with non-targeting gRNAs. A small fraction of the methylation changes associated with transcriptional dysregulation and mapped predominantly to bivalent chromatin associating both with transcriptional repression and activation. Additionally, specific non-targeting control gRNA caused pervasive and long-lasting methylation-independent transcriptional alterations particularly in genes linked to RNA and energy metabolism. CRISPRoff emerged as the most efficient tool for stable targeting of the BACH2 promoter, with fewer and less stable off-target effects compared to other epimodifiers but with persistent transcriptome alterations. Our findings highlight the delicate balance between potency and specificity of epigenome editing and provide critical insights into the design and application of future tools to improve their precision and minimize unintended consequences.

molecular biology↗

bioRxiv2024

GeneSetCluster 2.0: a comprehensive toolset for summarizing and integrating gene-sets analysis

BackgroundGene-Set Analysis (GSA) is commonly used to analyze high-throughput experiments. However, GSA cannot readily disentangle clusters or pathways due to redundancies in upstream knowledge bases, which hinders comprehensive exploration and interpretation of biological findings. To address this challenge, we developed GeneSetCluster, an R package designed to summarize and integrate GSA results. Over time, we and users as well identified limitations in the original version, such as difficulties in managing redundancies across multiple gene-sets, large computational times, and its lack of accessibility for users without programming expertise. ResultsWe present GeneSetCluster 2.0, a comprehensive upgrade that delivers methodological, computational, interpretative, and user-experience enhancements. Methodologically, GeneSetCluster 2.0 introduces a novel approach to address duplicated gene-sets and implements a seriation-based clustering algorithm that reorders results, aiding pattern identification. Computationally, the package is optimized for parallel processing, significantly reducing execution time. GeneSetCluster 2.0 enhances cluster annotations by associating clusters with relevant tissues and biological processes to improve biological interpretation, particularly for human and mouse data. To broaden accessibility, we have developed a user-friendly web application enabling non-programmers to use it. This version also ensures seamless integration between the R package, catering to users with programming expertise, and the web application for broader audiences. We evaluated the updates in a single-cell RNA public dataset. ConclusionGeneSetCluster 2.0 offers substantial improvements over its predecessor. Furthermore, by bridging the gap between bioinformaticians and clinicians in multidisciplinary teams, GeneSetCluster 2.0 facilitates collaborative research. The R package and web application, along with detailed installation and usage guides, are available on GitHub (https://github.com/TranslationalBioinformaticsUnit/GeneSetCluster2.0), and the web application can be accessed at https://translationalbio.shinyapps.io/genesetcluster/.

bioinformatics↗

bioRxiv2024

Single-nuclei histone modification profiling of the adult human central nervous system unveils epigenetic memory of developmental programs

The adult human central nervous system (CNS) is remarkably complex, with neural cells displaying extensive transcriptional heterogeneity. However, how different layers of epigenetic regulation underpin this heterogeneity is poorly understood. Here, we profile the adult human CNS from distinct regions, for chromatin accessibility at the single-nuclei level. In addition, we simultaneously co-profiled the histone modifications H3K27me3 and H3K27ac at the single nuclei-level, providing their first map in all major human CNS cell types. We unveil primed chromatin signatures at HOX loci in spinal cord-derived human oligodendroglia (OLG) but not microglia. These signatures were reminiscent of developmental OLG but were decoupled from robust gene expression. Moreover, using high-resolution Micro-C, we show that induced pluripotent stem cell (iPS) derived human OLGs exhibit a HOX chromatin architecture compatible with the primed chromatin in adult OLGs, and bears a strong resemblance not only to OLG developmental architecture, but also high-grade pontine gliomas. Thus, adult OLG retain epigenetic memory from developmental states, which might enable them to promptly transcribe Hox genes, in contexts of regeneration, but also make them susceptible to gliomagenesis.

neuroscience↗

bioRxiv2020

STATegra: Multi-omics data integration - A conceptual scheme with a bioinformatics pipeline

Technologies for profiling samples using different omics platforms have been at the forefront since the human genome project. Large-scale multi-omics data hold the promise of deciphering different regulatory layers. Yet, while there is a myriad of bioinformatics tools, each multi-omics analysis appears to start from scratch with an arbitrary decision over which tools to use and how to combine them. It is therefore an unmet need to conceptualize how to integrate such data and to implement and validate pipelines in different cases. We have designed a conceptual framework (STATegra), aiming it to be as generic as possible for multi-omics analysis, combining machine learning component analysis, non-parametric data combination and a multi-omics exploratory analysis in a step-wise manner. While in several studies we have previously combined those integrative tools, here we provide a systematic description of the STATegra framework and its validation using two TCGA case studies. For both, the Glioblastoma and the Skin Cutaneous Melanoma cases, we demonstrate an enhanced capacity to identify features in comparison to single-omics analysis. Such an integrative multi-omics analysis framework for the identification of features and components facilitates the discovery of new biology. Finally, we provide several options for applying the STATegra framework when parametric assumptions are fulfilled, and for the case when not all the samples are profiled for all omics. The STATegra framework is built using several tools, which are being integrated step-by-step as OpenSource in the STATegRa Bioconductor package https://bioconductor.org/packages/release/bioc/html/STATegra.html.

bioinformatics↗

bioRxiv2020

Systematic GWAS-assessment of disease modules reveals a multi-omic MS module strongly associated with risk factors

BackgroundThere are few (if any) practical guidelines for predictive and falsifiable multi-omics data integration that systematically integrate existing knowledge. Disease modules are popular concepts for interpreting genome-wide studies in medicine but have so far not been systematically evaluated and may lead to corroborating multi-omic modules. MethodsWe assessed eight module identification methods in 57 previously published expression and methylation studies of 19 diseases using GWAS enrichment analysis. Next, we applied the same strategy for multi-omics integration of 19 datasets of multiple sclerosis (MS), and further validated the resulting module using both GWAS and risk-factor associated genes from several independent cohorts. ResultsOur benchmark of modules showed that in immune-associated diseases modules inferred from clique-based methods were the most enriched for GWAS-genes. The multi-omics case study using MS revealed the robust identification of a module of 220 genes. Strikingly, most genes of the module was differentially methylated upon the action of one or several environmental risk factors in MS (n = 217, P = 10-47) and were also independently validated for association with five different risk factors of MS, which further stressed the high genetic and epigenetic relevance of the module for MS. ConclusionWe believe our analysis provides a workflow for selecting modules and our benchmark study may help further improvement of disease module methods. Moreover, we also stress that our methodology is generally applicable for combining and assessing the performance of multi-omics approaches for complex diseases.

bioinformatics↗

bioRxiv2020

Gsta4 controls apoptosis of differentiating adult oligodendrocytes during homeostasis and remyelination via the mitochondria-associated Fas/Casp8/Bid-axis

Arrest of oligodendrocyte (OL) differentiation and remyelination following myelin damage in Multiple Sclerosis (MS) are associated with disease progression but the underlying mechanism are elusive. We show that Glutathione S-transferase 4 (Gsta4) is highly expressed during adult OL differentiation and that its loss prevents differentiation into myelinating OLs. Also, Gsta4 appeared to be a novel target for Clemastine, in clinical trial for MS. Over-expression of Gsta4 reduced the expression of Fas and activity along the mitochondria-associated Casp8/Bid-axis in adult pre-OLs from the corpus callosum, together promoting enhanced pre-OL survival during differentiation. The Gsta4-mediated input on apoptosis during adult OL differentiation was further verified in the LPC and EAE model, where Casp8 were reduced in pre-OLs with high Gsta4 expression in an immune response-independent fashion. Our results place Gsta4 as a key regulator of intrinsic mechanisms beneficial for OL differentiation and remyelination, and as a possible target for future MS therapies.

neuroscience↗

bioRxiv2020

Profiling of small non-coding RNAs across cellular and biofluid compartments: implications for multiple sclerosis immunopathology

Multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), is associated with dysregulation of microRNAs (miRNA). We here analyzed all classes of small non-coding RNAs (sncRNAs) in matching peripheral blood mononuclear cells (PBMCs), plasma, cerebrospinal fluid (CSF) cells and cell-free CSF from relapsing-remitting (RRMS, n=12 in relapse, n=11 in remission), secondary progressive (SPMS, n=6) MS patients and non-inflammatory and inflammatory neurological disease controls (NINDC, n=11; INDC, n=5). We show widespread changes in small nuclear, nucleolar, transfer RNAs and miRNAs. In CSF cells, 133/133 and 115/117 differentially expressed sncRNAs are increased in RRMS relapse compared to remission and RRMS compared to NINDC, respectively. In contrast, 65/67 differentially expressed PBMC sncRNAs are decreased in RRMS compared to NINDC. The striking contrast between periphery and CNS suggests that sncRNA-mediated mechanisms, including alternative splicing, RNA degradation and mRNA translation, regulate the transcriptome of pathogenic cells primarily in the target organ.

molecular biology↗

bioRxiv2019

On the prediction of protein abundance from RNA

BackgroundProfiling of mRNA expression is an important method to identify biomarkers but complicated by limited correlations between mRNA expression and protein abundance. We hypothesised that these correlations could be improved by mathematical models based on measuring splice variants and time delay in protein translation. MethodsWe characterised time-series of primary human naive CD4+ T cells during early T-helper type 1 differentiation with RNA-sequencing and mass-spectrometry proteomics. We then performed computational time-series analysis in this system and in two other key human and murine immune cell types. Linear mathematical mixed time-delayed splice variant models were used to predict protein abundances, and the models were validated using out-of-sample predictions. Lastly, we re-analysed RNA-Seq datasets to evaluate biomarker discovery in five T-cell associated diseases, validating the findings for multiple sclerosis (MS) and asthma. ResultsThe new models demonstrated median correlations of mRNA-to-protein abundance of 0.79-0.94, significantly out-performing models not including the usage of multiple splice variants and time-delays, as shown in cross-validation tests. Our mathematical models provided more differentially expressed proteins between patients and controls in all five diseases. Moreover, analysis of these proteins in asthma and MS supported their relevance. One marker, sCD27, was clinically validated in MS using two independent cohorts, for treatment response and prognosis. ConclusionOur splice variant and time-delay models substantially improved the prediction of protein abundance from mRNA data in three immune cell-types. The models provided valuable biomarker candidates, which were validated in clinical studies of MS and asthma. We propose that our strategy is generally applicable for biomarker discovery.

bioinformatics↗