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See detailSingle-cell transcriptomics of human iPSC differentiation dynamics reveal a core molecular network of Parkinson’s disease
Novak, Gabriela; Kyriakis, Dimitrios UL; Grzyb, Kamil UL et al

in Communications Biology (2022), 5(1), 1--19

Parkinson’s disease (PD) is the second-most prevalent neurodegenerative disorder, characterized by the loss of dopaminergic neurons (mDA) in the midbrain. The underlying mechanisms are only partly ... [more ▼]

Parkinson’s disease (PD) is the second-most prevalent neurodegenerative disorder, characterized by the loss of dopaminergic neurons (mDA) in the midbrain. The underlying mechanisms are only partly understood and there is no treatment to reverse PD progression. Here, we investigated the disease mechanism using mDA neurons differentiated from human induced pluripotent stem cells (hiPSCs) carrying the ILE368ASN mutation within the PINK1 gene, which is strongly associated with PD. Single-cell RNA sequencing (RNAseq) and gene expression analysis of a PINK1-ILE368ASN and a control cell line identified genes differentially expressed during mDA neuron differentiation. Network analysis revealed that these genes form a core network, members of which interact with all known 19 protein-coding Parkinson’s disease-associated genes. This core network encompasses key PD-associated pathways, including ubiquitination, mitochondrial function, protein processing, RNA metabolism, and vesicular transport. Proteomics analysis showed a consistent alteration in proteins of dopamine metabolism, indicating a defect of dopaminergic metabolism in PINK1-ILE368ASN neurons. Our findings suggest the existence of a network onto which pathways associated with PD pathology converge, and offers an inclusive interpretation of the phenotypic heterogeneity of PD. [less ▲]

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See detailSingle-cell transcriptional profiling and gene regulatory network modeling in Tg2576 mice reveal gender-dependent molecular features preceding Alzheimer-like pathologies
Ali, Muhammad UL; Huarte, Oihane; Heurtaux, Tony UL et al

in Molecular Neurobiology (2022), in press (doi:10.1007/s12035-022-02985-2)(in press),

Alzheimer’s disease (AD) onset and progression is influenced by a complex interplay of several environmental and genetic factors, one of them gender. Pronounced gender differences have been observed both ... [more ▼]

Alzheimer’s disease (AD) onset and progression is influenced by a complex interplay of several environmental and genetic factors, one of them gender. Pronounced gender differences have been observed both in the relative risk of developing AD and in clinical disease manifestations. A molecular level understanding of these gender disparities is still missing, but could provide important clues on cellular mechanisms modulating the disease and reveal new targets for gender-oriented disease-modifying precision therapies. We therefore present here a comprehensive single-cell analysis of disease-associated molecular gender differences in transcriptomics data from the neocortex, one of the brain regions most susceptible to AD, in one of the most widely used AD mouse models, the Tg2576 model. Cortical areas are also most commonly used in studies of post-mortem AD brains. To identify disease-linked molecular processes that occur before the onset of detectable neuropathology, we focused our analyses on an age with no detectable plaques and microgliosis. Cell-type specific alterations were investigated at the level of individual genes, pathways, and gene regulatory networks. The number of differentially expressed genes (DEGs) was not large enough to build context-specific gene regulatory networks for each individual cell type, and thus, we focused on the study of cell types with dominant changes and included analyses of changes across the combination of cell types. We observed significant disease-associated gender differences in cellular processes related to synapse organization and axonogenesis, and identified a limited set of transcription factors, including Egr1 and Klf6, as key regulators of many of the disease-associated and gender-dependent gene expression changes in the model. Overall, our analyses revealed significant celltype-specific gene expression changes in individual genes, pathways and subnetworks, including gender-specific and gender-dimorphic changes in both upstream transcription factors and their downstream targets, in the Tg2576 AD model before the onset of overt disease. This opens a window into molecular events that could determine gender-susceptibility to AD, and uncovers tractable target candidates for potential gender-specific precision medicine for AD. [less ▲]

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See detailStress hormone signalling inhibits Th1 polarization in a CD4 T-cell-intrinsic manner via mTORC1 and the circadian gene PER1
Capelle, Christophe M.; Chen, Anna; Zeng, Ni et al

in Immunology (2022), 165(4), 428--444

Stress hormones are believed to skew the CD4 T-cell differentiation towards a Th2 response via a T-cell-extrinsic mechanism. Using isolated primary human naïve and memory CD4 T cells, here we show that ... [more ▼]

Stress hormones are believed to skew the CD4 T-cell differentiation towards a Th2 response via a T-cell-extrinsic mechanism. Using isolated primary human naïve and memory CD4 T cells, here we show that both adrenergic- and glucocorticoid-mediated stress signalling pathways play a CD4 naïve T-cell-intrinsic role in regulating the Th1/Th2 differentiation balance. Both stress hormones reduced the Th1 programme and cytokine production by inhibiting mTORC1 signalling via two parallel mechanisms. Stress hormone signalling inhibited mTORC1 in naïve CD4 T cells (1) by affecting the PI3K/AKT pathway and (2) by regulating the expression of the circadian rhythm gene, period circadian regulator 1 (PER1). Both stress hormones induced the expression of PER1, which inhibited mTORC1 signalling, thus reducing Th1 differentiation. This previously unrecognized cell-autonomous mechanism connects stress hormone signalling with CD4 T-cell differentiation via mTORC1 and a specific circadian clock gene, namely PER1. [less ▲]

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See detailCombinatorial analysis reveals highly coordinated early-stage immune reactions that predict later antiviral immunity in mild COVID-19 patients
Capelle, Christophe M.; Ciré, Séverine; Domingues, Olivia et al

in Cell Reports Medicine (2022), 3(4), 100600

While immunopathology has been widely studied in patients with severe COVID-19, immune responses in non-hospitalized patients have remained largely elusive. We systematically analyze 484 peripheral ... [more ▼]

While immunopathology has been widely studied in patients with severe COVID-19, immune responses in non-hospitalized patients have remained largely elusive. We systematically analyze 484 peripheral cellular or soluble immune features in a longitudinal cohort of 63 mild and 15 hospitalized patients versus 14 asymptomatic and 26 household controls. We observe a transient increase of IP10/CXCL10 and interferon-β levels, coordinated responses of dominant SARS-CoV-2-specific CD4 and fewer CD8 T cells, and various antigen-presenting and antibody-secreting cells in mild patients within 3 days of PCR diagnosis. The frequency of key innate immune cells and their functional marker expression are impaired in hospitalized patients at day 1 of inclusion. T cell and dendritic cell responses at day 1 are highly predictive for SARS-CoV-2-specific antibody responses after 3 weeks in mild but not hospitalized patients. Our systematic analysis reveals a combinatorial picture and trajectory of various arms of the highly coordinated early-stage immune responses in mild COVID-19 patients. [less ▲]

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See detailA new brain organoid model to study Parkinson’s Disease
Bolognin, Silvia UL; Smits, Lisa UL; Nickels, Sarah Louise UL et al

in Biomedical Science and Engineering (2021)

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See detailSingle‑nuclei chromatin profiling of ventral midbrain reveals cell identity transcription factors and cell‑type‑specific gene regulatory variation
Gui, Yujuan; Grzyb, Kamil UL; Thomas, Melanie UL et al

in Epigenetics and Chromatin (2021)

Background: Cell types in ventral midbrain are involved in diseases with variable genetic susceptibility, such as Parkinson’s disease and schizophrenia. Many genetic variants affect regulatory regions and ... [more ▼]

Background: Cell types in ventral midbrain are involved in diseases with variable genetic susceptibility, such as Parkinson’s disease and schizophrenia. Many genetic variants affect regulatory regions and alter gene expression in a cell-type-specific manner depending on the chromatin structure and accessibility. Results: We report 20,658 single-nuclei chromatin accessibility profiles of ventral midbrain from two genetically and phenotypically distinct mouse strains. We distinguish ten cell types based on chromatin profiles and analysis of accessible regions controlling cell identity genes highlights cell-type-specific key transcription factors. Regulatory variation segregating the mouse strains manifests more on transcriptome than chromatin level. However, cell-type-level data reveals changes not captured at tissue level. To discover the scope and cell-type specificity of cis-acting variation in midbrain gene expression, we identify putative regulatory variants and show them to be enriched at differentially expressed loci. Finally, we find TCF7L2 to mediate trans-acting variation selectively in midbrain neurons. Conclusions: Our data set provides an extensive resource to study gene regulation in mesencephalon and provides insights into control of cell identity in the midbrain and identifies cell-type-specific regulatory variation possibly underlying phenotypic and behavioural differences between mouse strains. [less ▲]

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See detailThe Parkinson’s-disease-associated mutation LRRK2-G2019S alters dopaminergic differentiation dynamics via NR2F1
Walter, Jonas; Bolognin, Silvia UL; Poovathingal, Suresh et al

in Cell Reports (2021)

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See detailPatient-derived organoids and orthotopic xenografts of primary and recurrent gliomas represent relevant patient avatars for precision oncology.
Golebiewska, Anna UL; Hau, Ann-Christin; Oudin, Anaïs et al

in Acta Neuropathologica (2020)

Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique ... [more ▼]

Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1, and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1, ATRX, TP53, MDM2/4, amplification of EGFR, PDGFRA, MET, CDK4/6, MDM2/4, and deletion of CDKN2A/B, PTCH, and PTEN. Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology. [less ▲]

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See detailPrimary and recurrent glioma patient-derived orthotopic xenografts (PDOX) represent relevant patient avatars for precision medicine
Golebiewska, Anna UL; Hau, Ann-Christin; Oudin, Anais et al

E-print/Working paper (2020)

Patient-derived cancer models are essential tools for studying tumor biology and preclinical interventions. Here, we show that glioma patient-derived orthotopic xenografts (PDOXs) enable long-term ... [more ▼]

Patient-derived cancer models are essential tools for studying tumor biology and preclinical interventions. Here, we show that glioma patient-derived orthotopic xenografts (PDOXs) enable long-term propagation of patient tumors and represent clinically relevant patient avatars. We created a large collection of PDOXs from primary and recurrent gliomas with and without mutations in IDH1, which retained histopathological, genetic, epigenetic and transcriptomic features of patient tumors with no mouse-specific clonal evolution. Longitudinal PDOX models recapitulate the limited genetic evolution of gliomas observed in patient tumors following treatment. PDOX-derived standardized tumor organoid cultures enabled assessment of drug responses, which were validated in mice. PDOXs showed clinically relevant responses to Temozolomide and to targeted treatments such as EGFR and CDK4/6 inhibitors in (epi)genetically defined groups, according to MGMT promoter and EGFR/CDK status respectively. Dianhydrogalactitol, a bifunctional alkylating agent, showed promising potential against glioblastoma. Our study underlines the clinical relevance of glioma PDOX models for translational research and personalized treatment studies. [less ▲]

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See detailSingle-cell transcriptomics reveals multiple neuronal cell types in human midbrain-specific organoids
Smits, Lisa UL; Magni, Stefano UL; Kinugawa, Kaoru et al

in Cell and Tissue Research (2020)

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See detailNew insights into the early mechanisms of epileptogenesis in a zebrafish model of Dravet syndrome
Tiraboschi, Ettore; Martina, Silvia UL; van der Ent, Wietske et al

in Epilepsia (2020)

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See detailThe Effect of ATP INDUCED CALCIUM DYNAMICS ON EPITHELIAL TO MESENCHYMAL TRANSITIONS
Grzyb, Kamil UL

Doctoral thesis (2019)

Cells respond to a multitude of external triggers by a limited number of signaling pathways activated by receptors on plasma membrane, such as receptor tyrosine kinases (RTKs) or G protein-coupled ... [more ▼]

Cells respond to a multitude of external triggers by a limited number of signaling pathways activated by receptors on plasma membrane, such as receptor tyrosine kinases (RTKs) or G protein-coupled receptors (GPCRs). These pathways do not simply convey the downstream signal, but instead the signal is very often processed by encoding and integrated with the current state of the cell. A traditional transcriptional analysis tends to provide an averaged output measured in a population, what often masks the behavior of individual cells. However, with recent single cell techniques developments, it is possible to investigate transcription in individual living cells. This contributed tremendously to the understanding of development and progression of many diseases including cancer. The more we understand about this high complexity of signaling mechanisms and multitude of cellular safety countermeasures, the more we see cancer as a microevolution state of “rebellious cells” (cells entering the fate opposite to the one intended) following a patch through a discreet system. This thesis specifically focused on the temporal aspect of signaling in the context of the epithelia-to-mensenchymal transition (EMT) by combining single cell experiments and bioinformatics analysis. We investigated cellular signaling changes in response to different dynamical profiles of the stimuli. In particular, we used the HMLER cell line, which is a metastatic breast cancer model for the epithelial to mesenchymal transition. By applying stochastic or oscillatory pulses of extracellular ATP-induced Ca2+ signals with different interspike intervals, we were investigating different transcription states from those evoked by constant ATP-induced Ca2+ dose responses. In order to precisely apply those stimulation profiles, we have developed and established a perfusion system. This device allows to treat population of cells simultaneously with the exact same dynamical profiles. Cells treated by these well controlled signals were subsequently processed by the single cell RNA-seq technique Drop-seq for transcriptional analysis. The resulting high dimensional digital gene expression matrices were analyzed by a developed high-throughput computational analysis pipeline. This analysis includes the identification of differentially expressed genes and cellular clusters (states) by dimensionality reduction methods (PCA, t-SNE) and pathway analysis. We evaluated changes and trends of genes from difference dynamical profiles by investigating their involvement in stress, stemness and regulation of motility. First, we confirmed that oscillatory stimulation with extracellular ATP (eATP) tends to lower the burden of cellular stress and apoptosis related pathways while maintaining its other effector functions compared to constant eATP stimulation. Interestingly, stochastic spiking of extracellular ATP in our setup led to a massive (~80%) increase in overall differential gene expression compared to deterministic oscillatory stimulation with the same period. Consequentially, stochastic signaling seems to activate a much wider range of biological pathways, which indicates the much higher complexity in information processing capability of producing rebellious cells during cancer progression and metastasis. On the other hand, our findings suggests that oscillatory eATP stimulation could contribute to EMT by lowering ID3 expression compared to stochastic stimulation where we observed a stronger upregulation of IRS2. Finally, we integrated the DEGs into biological processes involved in each conditions and put these new insights into the context of the eATP-induced Ca2+ induced epithelial to mesenchymal transition. Overall, this thesis has applied recent single cell technologies to characterize underlying principles of cellular heterogeneity induced by cell signaling and specifically investigated the complex mechanisms of cell fate in the context of EMT [less ▲]

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See detailSingle-cell transcriptomics reveals multiple neuronal cell types in human midbrain-specific organoids
Smits, Lisa UL; Magni, Stefano UL; Grzyb, Kamil UL et al

E-print/Working paper (2019)

Human stem cell-derived organoids have great potential for modelling physiological and pathological processes. They recapitulate in vitro the organisation and function of a respective organ or part of an ... [more ▼]

Human stem cell-derived organoids have great potential for modelling physiological and pathological processes. They recapitulate in vitro the organisation and function of a respective organ or part of an organ. Human midbrain organoids (hMOs) have been described to contain midbrain-specific dopaminergic neurons that release the neurotransmitter dopamine. However, the human midbrain contains also additional neuronal cell types, which are functionally interacting with each other. Here, we analysed hMOs at high-resolution by means of single-cell RNA-sequencing (scRNA-seq), imaging and electrophysiology to unravel cell heterogeneity. Our findings demonstrate that hMOs show essential neuronal functional properties as spontaneous electrophysiological activity of different neuronal subtypes, including dopaminergic, GABAergic, and glutamatergic neurons. Recapitulating these in vivo features makes hMOs an excellent tool for in vitro disease phenotyping and drug discovery. [less ▲]

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See detailCaSiAn: a Calcium Signaling Analyzer tool
Moein, Mahsa UL; Grzyb, Kamil UL; Gonçalves Martins, Teresa et al

in Bioinformatics (2018), 1

Ca2þ is a central second messenger in eukaryotic cells that regulates many cellular proc- esses. Recently, we have indicated that typical Ca2þ signals are not purely oscillatory as widely assumed, but ... [more ▼]

Ca2þ is a central second messenger in eukaryotic cells that regulates many cellular proc- esses. Recently, we have indicated that typical Ca2þ signals are not purely oscillatory as widely assumed, but exhibit stochastic spiking with cell type and pathway specific characteristics. Here, we present the Calcium Signaling Analyzer (CaSiAn), an open source software tool that allows for quantifying these signal characteristics including individual spike properties and time course statis- tics in a semi-automated manner. CaSiAn provides an intuitive graphical user interface allowing experimentalists to easily process a large amount of Ca2þ signals, interactively tune peak detection, revise statistical measures and access the quantified signal properties as excel or text files. [less ▲]

Detailed reference viewed: 152 (16 UL)