Biology subjects

Uhlen, P.

Publications and source records attributed to Uhlen, P..

13 recordsLinked to original sources

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

A neoantigen-microbead platform for personalized T cell cancer vaccination

Colorectal cancer (CRC) is a leading cause of cancer mortality and is characterized by a high tumor mutational burden, making it responsive to immunotherapy. We developed a bioinformatic and manufacturing pipeline for personalized cancer vaccines and evaluated it in the MC38 colon adenocarcinoma mouse model. Thirty-six high-scoring neoantigens (NAGs) were identified by exome and transcriptome analysis, produced as six purified polypeptides, and coupled to paramagnetic beads. Intralymphatic vaccination of C57BL/6 mice with NAG beads induced robust NAG-specific T cell and antibody responses, resulting in significant inhibition of MC38 tumor growth. Treated tumors displayed increased necrosis and CD8+ T cell infiltration. Compared with soluble peptides, bead-coupled antigens elicited superior protection. Studies in T cell-deficient and antibody-depleted mice confirmed that both CD4+ and CD8+ T cells mediated the antitumor effect. These findings highlight the potential of NAG bead vaccination as an effective immunotherapy for CRC.

cancer biology↗

bioRxiv2025

Decoding calcium oscillation frequency in transcriptional regulation

Cells continuously experience fluctuating intracellular calcium (Ca{superscript 2}) signals that orchestrate diverse processes such as transcription, proliferation, and apoptosis. Temporal features of Ca{superscript 2} dynamics, including oscillation frequency, are hypothesized to encode information, allowing cells to discriminate between relevant and stochastic signals. However, the mechanisms of frequency decoding and their transcriptional consequences remain incompletely understood. To address this, we investigated how defined Ca{superscript 2} oscillation frequencies are translated into signaling cascades and gene expression programs in human non-excitable cells. Using optogenetic control of melanopsin-mediated Ca{superscript 2} influx, we induced slow (8 mHz) or fast (15 mHz) oscillations with identical single-pulse kinetics to isolate the effect of frequency. We found that TNF and IL8 transcription via NF-{kappa}B displayed sigmoidal frequency dependence, strictly requiring regular periodic stimulation, while random or low-frequency inputs with equal cumulative Ca{superscript 2} exposure were ineffective. Bulk RNA sequencing revealed a MYC-centered transcriptional response, with 116 of 215 differentially expressed genes predicted as MYC targets, despite unchanged MYC mRNA levels. Label-free phosphoproteomics identified PRKDC, CHEK2 and ATM as the top upstream kinases, forming a network linking Ca{superscript 2} oscillations to cell cycle and stress signaling. These findings demonstrate that cells can decode Ca{superscript 2} oscillation frequency through a multi-kinase network that tunes transcription via NF-{kappa}B and MYC, providing mechanistic insight into how temporal dynamics of second messengers shape cellular decision-making.

cell biology↗

bioRxiv2025

Development of a protocol utilizing single-cell analysis for the differentiation of human iPSCs into SOX6+ midbrain dopaminergic neurons.

Parkinsons disease (PD) is characterized by the progressive loss of midbrain dopaminergic (mDA) neurons, leading to severe debilitating motor impairment. Recent single-cell analyses have revealed that specific mDA subpopulations, such as SOX6 AGTR1 neurons, are most vulnerable to degeneration. Current human pluripotent stem cell differentiation protocols fail to selectively generate these subtypes. Here, we describe a multiomic-guided strategy that enriches SOX6+ mDA neurons by combining enhancing Sonic Hedgehog agonism with prolonged Wnt activation. This approach accelerates floor plate specification, increases expression of mDA developmental markers, and substantially increases the yield of SOX6 mature mDA neurons compared to prior attempts. Following intrastriatal transplantation into hemiparkinsonian mice, these cells restored motor function in approximately 4 months and generated grafts containing SOX6 A9-like neurons. Our work thus establishes a reproducible differentiation platform for generating the PD-susceptible mDA subtype, providing a foundation for precision disease modelling and subtype-targeted cell replacement therapies.

cell biology↗

bioRxiv2025

Glioma-induced DNMT3A-dependent reduction of DNA methylation in microglia promotes a transient anti-tumoral phenotype.

Glioblastoma, aggressive primary brain tumors with a dismal prognosis, promote the recruitment of microglia, brain resident innate immune cells, and ultimately their activation toward a tumor-supportive phenotype that increases gliomal proliferation and invasion capability. Here, we report that upon stimulation by glioma cells, microglia transit via a reactive state holding anti-tumoral properties coupled to reduced DNMT3A chromatin occupancy and DNA demethylation that promote microglial pro-inflammatory gene expressions. We find that upon repression of Dnmt3a expression in microglia, those cells maintain anti-tumoral attributes in vitro and in vivo. In a syngeneic immunocompetent glioblastoma mouse model, brain delivery of antisense oligonucleotide targeting Dnmt3a expression led to reduced tumor growth. Taken together, our results reveal the involvement of DNA demethylation in the control of glioma cells-induced microglia activation and indicate that microglial DNMT3A is a potentially therapeutic target to treat brain neoplasms such as glioblastoma that include a microglial component.

immunology↗

bioRxiv2024

Spatial dynamics of mammalian brain development and neuroinflammation by multimodal tri-omics mapping

The ability to spatially map multiple layers of the omics information over different time points allows for exploring the mechanisms driving brain development, differentiation, arealization, and alterations in disease. Herein we developed and applied spatial tri-omic sequencing technologies, DBiT ARP-seq (spatial ATAC-RNA-Protein-seq) and DBiT CTRP-seq (spatial CUT&Tag- RNA-Protein-seq) together with multiplexed immunofluorescence imaging (CODEX) to map spatial dynamic remodeling in brain development and neuroinflammation. A spatiotemporal tri-omic atlas of the mouse brain was obtained at different stages from postnatal day P0 to P21, and compared to the regions of interest in the human developing brains. Specifically, in the cortical area, we discovered temporal persistence and spatial spreading of chromatin accessibility for the layer-defining transcription factors. In corpus callosum, we observed dynamic chromatin priming of myelin genes across the subregions. Together, it suggests a role for layer specific projection neurons to coordinate axonogenesis and myelination. We further mapped the brain of a lysolecithin (LPC) neuroinflammation mouse model and observed common molecular programs in development and neuroinflammation. Microglia, exhibiting both conserved and distinct programs for inflammation and resolution, are transiently activated not only at the core of the LPC lesion, but also at distal locations presumably through neuronal circuitry. Thus, this work unveiled common and differential mechanisms in brain development and neuroinflammation, resulting in a valuable data resource to investigate brain development, function and disease.

neuroscience↗

bioRxiv2024

Single-Cell Transcriptomics Reveals the Molecular Logic Underlying Ca2+ Signaling Diversity in Human and Mouse Brain

The calcium ion (Ca2+) is a ubiquitous intracellular signaling molecule that plays a critical role in the adult and developing brain. However, the principles governing the specificity of Ca2+ signaling remain unresolved. In this work, we comprehensively analyzed the Ca2+ signaling transcriptome in the adult mouse brain and developing human brain. We found that neurons form non-stochastic Ca2+-states that are reflective of their cell types and functionality, with evidence suggesting that the diversity is driven by lineage-specific developmental changes. Focusing on the neocortical development, we reveal that an unprecedented number of Ca2+ genes are tightly regulated and evolutionarily conserved, capturing functionally driven differences within radial glia and neuronal progenitors. In summary, our study provides an in-depth understanding of the cellular and temporal diversity of Ca2+ signaling and suggests that Ca2+ signaling is dynamically tailored to specific cell states. One Sentence SummaryThe expression of Ca2+ signaling genes is finely tuned to cellular states, reflecting a spectrum of differences that range from lineage specificity to subtle functional distinctions within cortical radial glia.

neuroscience↗

bioRxiv2023

Defined human PSC culture conditions robustly maintain human PSC pluripotency through Ca2+ signaling.

Human pluripotent stem cells (hPSCs) have significant potential for disease modeling and cell therapies. However, their clinical applicability is limited due to the need for undefined conditions for PSC cultivation, which increase the risk of immunogenicity, result in batch-to-batch variability and finite scalability. These limitations may be circumvented by xeno-free, defined culture conditions. However, biological processes that preserve robust, homogenous PSCs in defined conditions remain to be characterized. Here, we compared gene expression data from over 100 hPSC cell lines cultivated in undefined and defined culture conditions. Defined culture conditions significantly reduced inter-PSC line variability, highlighting the importance of standardization to minimize PSC biases. This variability is concurrent with decreased germ layer differentiation and increased expression of Ca2+-binding proteins. The significance of tightly controlled Ca2+ signaling in hPSC pluripotency in defined culture conditions was also confirmed. A deeper understanding of these processes may aid in standardizing defined hPSC culture conditions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/552440v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@1e5729aorg.highwire.dtl.DTLVardef@c6fa28org.highwire.dtl.DTLVardef@1515b91org.highwire.dtl.DTLVardef@52c6b6_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefEidhof et al. compared gene expression array data obtained from more than 100 hPSC cell lines to study the biological differences between PSCs cultivated under defined and undefined culture conditions. HighlightsO_LIDefined culture conditions reduce inter-hPSC line variability. C_LIO_LIDefined conditions decrease the expression of germ layer differentiation markers. C_LIO_LICa2+ signaling associated genes capture specific hPSC states in defined conditions. C_LIO_LIHigh Ca2+ activity drives pluripotency, low Ca2+ activity drives differentiation. C_LI

cell biology↗

bioRxiv2022

Whole-Brain Three-Dimensional Imaging of RNAs at Single-Cell Resolution

Whole-brain three-dimensional (3D) imaging is desirable to obtain a comprehensive and unbiased view of architecture and neural circuitry. However, current spatial analytic methods for brain RNAs are limited to thin sections or small samples. Here, we combined multiple new techniques to develop TRIC-DISCO, a new pipeline that allows imaging of RNA spatial distributions in whole adult mouse brains. First, we developed Tris-mediated retention of in situ hybridization signal during clearing (TRIC), which produces highly transparent tissue while maintaining the RNA signal intensities. We then combined TRIC with DISCO clearing (TRIC-DISCO) by controlling temperature during the in situ hybridization chain reaction (isHCR) to ensure uniform whole-brain staining. This pipeline eliminates the requirements for both strict RNase-free environments and workflow-compatible RNase inhibitors. Our TRIC-DISCO pipeline enables simple and robust, single-cell, whole-brain, 3D imaging of transcriptional signatures, cell-identity markers, and noncoding RNAs across the entire brain.

neuroscience↗

bioRxiv2022

Anatomy of the complete mouse eye vasculature indevelopment and pathology explored by light-sheet fluorescence microscopy

Eye development and function rely on precise establishment, regression and maintenance of its many sub-vasculatures. These crucial vascular properties have been extensively investigated in eye development and disease utilizing genetic and experimental mouse models. However, due to technical limitations, individual studies have often restricted their focus to one specific sub-vasculature. Here, we apply a workflow that allows for visualisation of complete vasculatures of mouse eyes of various developmental stages. Through tissue depigmentation, immunostaining, clearing and light-sheet fluorescence microscopy (LSFM) entire vasculatures of the retina, vitreous (hyaloids) and uvea were simultaneously imaged at high resolution. In silico dissection provided detailed information on their 3D architecture and interconnections. By this method we describe remodelling of the postnatal iris vasculature following its disconnection to the feeding hyaloid vasculature. In addition, we demonstrate examples of conventional and LSFM-mediated analysis of choroidal neovascularisation after laser-induced wounding, showing added value of the presented workflow in analysis of modelled eye disease. These advancements in visualisation and analysis of the respective eye vasculatures in development and complex eye disease open for novel observations of their functional interplay at a whole-organ level.

developmental biology↗

bioRxiv2021

Arg1+ microglia are critical for shaping cognition in female mice

Diversity within microglia, the resident brain immune cells, is reported. Whether microglial subsets constitute different subtypes with intrinsic properties and unique functions has not been fully elucidated. Here, we describe a microglial subtype characterized by the expression of the enzyme Arginase-1, i.e. Arg1 +microglia, which is found predominantly in the cholinergic neuron-rich forebrain region during early postnatal development. Arg1+ microglia are frequently observed in close apposition to neurons and exhibit a distinctive molecular signature reflecting a reactive profile. Arg1 deficiency in microglia results in impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, and cognitive behavioural deficiencies in female mice. Our results expand on microglia diversity and provide insights into distinctive spatiotemporal functions exerted by microglial subtypes.

neuroscience↗

bioRxiv2019

Live detection of neural stem and glioblastoma cells by an oligothiophene derivative

Here we report a luminescent conjugated oligothiophene (LCO), named p-HTMI, for non-invasive and non-amplified real-time detection of live human patient-derived glioblastoma (GBM) cells and embryonic neural stem cells (NSCs). While p-HTMI stained only a small fraction of other cell types investigated, the mere addition of p-HTMI to the cell culture resulted in efficient detection of NSCs or GBM cells from rodents and humans within minutes. p-HTMI is functionalized with a methylated imidazole moiety resembling the side chain of histidine/histamine, and non-methylated analogues were not functional. p-HTMI were readily able to detect a subpopulation of human cells in vivo in mouse brain sections with tumors developed from orthotopically transplanted patient-derived GBM cells. Cell sorting experiments of human GBM cells demonstrated that p-HTMI labeled the same population as CD271, a proposed marker for stem cell-like cells and rapidly migrating cells in glioblastoma. Our results suggest that the LCO p-HTMI is a versatile tool for immediate and selective detection of subpopulations of neural stem and glioma cells.

bioengineering↗

bioRxiv2019

Recombinant spider silk protein matrices facilitate multi-analysis of calcium-signaling in neural stem cell-derived AMPA-responsive neurons

Neural progenitors or stem cells (NSCs) show great promise in drug discovery and clinical application. Yet few efforts have been made to optimize biocompatible materials for such cells to be expanded and used in clinical conditions. We have previously demonstrated that NSCs are readily cultured on substrates of certain recombinant spider silk protein without addition of animal- or human-derived components. The question remains however whether this material allows differentiation into functional neurons and glia, and whether such differentiation can take place also when the NSCs are cultured within the material in a pseudo-3D context. Here we demonstrate that \"foam\"-like structures generated from recombinant spider silk protein (4RepCT) provided excellent matrices for the generation and multicellular analysis of functional excitatory neurons from NSCs without addition of animal- or human-derived components. NSCs isolated from the cerebral cortices of rat embryos were cultured on either 4RepCT matrices shaped as foam-like structures without coating, or on conventional polystyrene plates coated with poly-L-ornithine and fibronectin. Upon treatment with recombinant proteins including the growth factor BMP4 or a combination of BMP4 and the signaling factor Wnt3a, the cortical NSCs cultured in 4RepCT foam-like structures differentiated efficiently into neurons that responded to glutamate receptor agonists, such as AMPA, to at least the same extent as control cultures. Matrices derived from recombinant spider silk proteins thus provide a functional microenvironment for neural stem cells without any animal- or human-derived components, and can be employed in the development of new strategies in stem cell research and tissue engineering.

bioengineering↗