Single-cell transcriptomics of human iPSC differentiation dynamics reveal a core molecular network of Parkinson’s disease

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 ▲]

Stress hormone signalling inhibits Th1 polarization in a CD4 T-cell-intrinsic manner via mTORC1 and the circadian gene PER1

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 ▲]

A new brain organoid model to study Parkinson’s Disease

in Biomedical Science and Engineering (2021)

Single-Cell Transcriptomics and In Situ Morphological Analyses Reveal Microglia Heterogeneity Across the Nigrostriatal Pathway

in Frontiers in Immunology (2021)

Patient-derived organoids and orthotopic xenografts of primary and recurrent gliomas represent relevant patient avatars for precision oncology.

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 ▲]

Single-cell transcriptomics reveals multiple neuronal cell types in human midbrain-specific organoids

in Cell and Tissue Research (2020)

The Effect of ATP INDUCED CALCIUM DYNAMICS ON EPITHELIAL TO MESENCHYMAL TRANSITIONS

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 ▲]

CaSiAn: a Calcium Signaling Analyzer tool

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 ▲]