Publications by authors named "Alexander Skupin"

46 Publications

The Contribution of Microglia to Neuroinflammation in Parkinson's Disease.

Int J Mol Sci 2021 Apr 28;22(9). Epub 2021 Apr 28.

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4367 Esch-sur-Alzette, Luxembourg.

With the world's population ageing, the incidence of Parkinson's disease (PD) is on the rise. In recent years, inflammatory processes have emerged as prominent contributors to the pathology of PD. There is great evidence that microglia have a significant neuroprotective role, and that impaired and over activated microglial phenotypes are present in brains of PD patients. Thereby, PD progression is potentially driven by a vicious cycle between dying neurons and microglia through the instigation of oxidative stress, mitophagy and autophagy dysfunctions, a-synuclein accumulation, and pro-inflammatory cytokine release. Hence, investigating the involvement of microglia is of great importance for future research and treatment of PD. The purpose of this review is to highlight recent findings concerning the microglia-neuronal interplay in PD with a focus on human postmortem immunohistochemistry and single-cell studies, their relation to animal and iPSC-derived models, newly emerging technologies, and the resulting potential of new anti-inflammatory therapies for PD.
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http://dx.doi.org/10.3390/ijms22094676DOI Listing
April 2021

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

Front Immunol 2021 29;12:639613. Epub 2021 Mar 29.

Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg.

Microglia are the resident immune effector cells of the central nervous system (CNS) rapidly reacting to various pathological stimuli to maintain CNS homeostasis. However, microglial reactions in the CNS may also worsen neurological disorders. Hence, the phenotypic analysis of microglia in healthy tissue may identify specific poised subsets ultimately supporting or harming the neuronal network. This is all the more important for the understanding of CNS disorders exhibiting regional-specific and cellular pathological hallmarks, such as many neurodegenerative disorders, including Parkinson's disease (PD). In this context, we aimed to address the heterogeneity of microglial cells in susceptible brain regions for PD, such as the nigrostriatal pathway. Here, we combined single-cell RNA-sequencing with immunofluorescence analyses of the murine nigrostriatal pathway, the most affected brain region in PD. We uncovered a microglia subset, mainly present in the midbrain, displaying an intrinsic transcriptional immune alerted signature sharing features of inflammation-induced microglia. Further, an morphological screening of inferred cellular diversity showed a decreased microglia complexity in the midbrain when compared to striatum. Our study provides a resource for the identification of specific microglia phenotypes within the nigrostriatal pathway, which may be relevant in PD.
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http://dx.doi.org/10.3389/fimmu.2021.639613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8039119PMC
March 2021

l-Isoaspartyl Methyltransferase Deficiency in Zebrafish Leads to Impaired Calcium Signaling in the Brain.

Front Genet 2020 21;11:612343. Epub 2021 Jan 21.

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.

Isomerization of l-aspartyl and l-asparaginyl residues to l-isoaspartyl residues is one type of protein damage that can occur under physiological conditions and leads to conformational changes, loss of function, and enhanced protein degradation. Protein l-isoaspartyl methyltransferase (PCMT) is a repair enzyme whose action initiates the reconversion of abnormal l-isoaspartyl residues to normal l-aspartyl residues in proteins. Many lines of evidence support a crucial role for PCMT in the brain, but the mechanisms involved remain poorly understood. Here, we investigated PCMT activity and function in zebrafish, a vertebrate model that is particularly well-suited to analyze brain function using a variety of techniques. We characterized the expression products of the zebrafish homologous genes and . Both zebrafish proteins showed a robust l-isoaspartyl methyltransferase activity and highest mRNA transcript levels were found in brain and testes. Zebrafish morphant larvae with a knockdown in both the and genes showed pronounced morphological abnormalities, decreased survival, and increased isoaspartyl levels. Interestingly, we identified a profound perturbation of brain calcium homeostasis in these morphants. An abnormal calcium response upon ATP stimulation was also observed in mouse hippocampal HT22 cells knocked out for . This work shows that zebrafish is a promising model to unravel further facets of PCMT function and demonstrates, for the first time , that PCMT plays a pivotal role in the regulation of calcium fluxes.
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http://dx.doi.org/10.3389/fgene.2020.612343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859441PMC
January 2021

Roles of bacteriophages, plasmids and CRISPR immunity in microbial community dynamics revealed using time-series integrated meta-omics.

Nat Microbiol 2021 01 2;6(1):123-135. Epub 2020 Nov 2.

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.

Viruses and plasmids (invasive mobile genetic elements (iMGEs)) have important roles in shaping microbial communities, but their dynamic interactions with CRISPR-based immunity remain unresolved. We analysed generation-resolved iMGE-host dynamics spanning one and a half years in a microbial consortium from a biological wastewater treatment plant using integrated meta-omics. We identified 31 bacterial metagenome-assembled genomes encoding complete CRISPR-Cas systems and their corresponding iMGEs. CRISPR-targeted plasmids outnumbered their bacteriophage counterparts by at least fivefold, highlighting the importance of CRISPR-mediated defence against plasmids. Linear modelling of our time-series data revealed that the variation in plasmid abundance over time explained more of the observed community dynamics than phages. Community-scale CRISPR-based plasmid-host and phage-host interaction networks revealed an increase in CRISPR-mediated interactions coinciding with a decrease in the dominant 'Candidatus Microthrix parvicella' population. Protospacers were enriched in sequences targeting genes involved in the transmission of iMGEs. Understanding the factors shaping the fitness of specific populations is necessary to devise control strategies for undesirable species and to predict or explain community-wide phenotypes.
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http://dx.doi.org/10.1038/s41564-020-00794-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7752763PMC
January 2021

ROS networks: designs, aging, Parkinson's disease and precision therapies.

NPJ Syst Biol Appl 2020 10 26;6(1):34. Epub 2020 Oct 26.

Infrastructure for Systems Biology Europe (ISBE.NL), Amsterdam, The Netherlands.

How the network around ROS protects against oxidative stress and Parkinson's disease (PD), and how processes at the minutes timescale cause disease and aging after decades, remains enigmatic. Challenging whether the ROS network is as complex as it seems, we built a fairly comprehensive version thereof which we disentangled into a hierarchy of only five simpler subnetworks each delivering one type of robustness. The comprehensive dynamic model described in vitro data sets from two independent laboratories. Notwithstanding its five-fold robustness, it exhibited a relatively sudden breakdown, after some 80 years of virtually steady performance: it predicted aging. PD-related conditions such as lack of DJ-1 protein or increased α-synuclein accelerated the collapse, while antioxidants or caffeine retarded it. Introducing a new concept (aging-time-control coefficient), we found that as many as 25 out of 57 molecular processes controlled aging. We identified new targets for "life-extending interventions": mitochondrial synthesis, KEAP1 degradation, and p62 metabolism.
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http://dx.doi.org/10.1038/s41540-020-00150-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589522PMC
October 2020

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

Acta Neuropathol 2020 12 3;140(6):919-949. Epub 2020 Oct 3.

Department of Neurosurgery, Henry Ford Health System, Detroit, MI, 48202, USA.

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.
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http://dx.doi.org/10.1007/s00401-020-02226-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666297PMC
December 2020

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

Cell Tissue Res 2020 Dec 31;382(3):463-476. Epub 2020 Jul 31.

Luxembourg Centre for Systems Biomedicine (LCSB), Developmental and Cellular Biology, University of Luxembourg, Belvaux, Luxembourg.

Human stem cell-derived organoids have great potential for modelling physiological and pathological processes. They recapitulate in vitro the organization 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, glutamatergic and serotonergic neurons. Recapitulating these in vivo features makes hMOs an excellent tool for in vitro disease phenotyping and drug discovery.
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http://dx.doi.org/10.1007/s00441-020-03249-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7683480PMC
December 2020

New insights into the early mechanisms of epileptogenesis in a zebrafish model of Dravet syndrome.

Epilepsia 2020 03 24;61(3):549-560. Epub 2020 Feb 24.

Chemical Neuroscience Group, Center for Molecular Medicine Norway, University of Oslo, Oslo, Norway.

Objective: To pinpoint the earliest cellular defects underlying seizure onset (epileptogenic period) during perinatal brain development in a new zebrafish model of Dravet syndrome (DS) and to investigate potential disease-modifying activity of the 5HT receptor agonist fenfluramine.

Methods: We used CRISPR/Cas9 mutagenesis to introduce a missense mutation, designed to perturb ion transport function in all channel isoforms, into scn1lab, the zebrafish orthologue of SCN1A (encoding voltage-gated sodium channel alpha subunit 1). We performed behavioral analysis and electroencephalographic recordings to measure convulsions and epileptiform discharges, followed by single-cell RNA-Seq, morphometric analysis of transgenic reporter-labeled γ-aminobutyric acidergic (GABAergic) neurons, and pharmacological profiling of mutant larvae.

Results: Homozygous mutant (scn1lab ) larvae displayed spontaneous seizures with interictal, preictal, and ictal discharges (mean = 7.5 per 20-minute recording; P < .0001; one-way analysis of variance). Drop-Seq analysis revealed a 2:1 shift in the ratio of glutamatergic to GABAergic neurons in scn1lab larval brains versus wild type (WT), with dynamic changes in neuronal, glial, and progenitor cell populations. To explore disease pathophysiology further, we quantified dendritic arborization in GABAergic neurons and observed a 40% reduction in arbor number compared to WT (P < .001; n = 15 mutant, n = 16 WT). We postulate that the significant reduction in inhibitory arbors causes an inhibitory to excitatory neurotransmitter imbalance that contributes to seizures and enhanced electrical brain activity in scn1lab larvae (high-frequency range), with subsequent GABAergic neuronal loss and astrogliosis. Chronic fenfluramine administration completely restored dendritic arbor numbers to normal in scn1lab larvae, whereas similar treatment with the benzodiazepine diazepam attenuated seizures, but was ineffective in restoring neuronal cytoarchitecture. BrdU labeling revealed cell overproliferation in scn1lab larval brains that were rescued by fenfluramine but not diazepam.

Significance: Our findings provide novel insights into early mechanisms of DS pathogenesis, describe dynamic cell population changes in the scn1lab brain, and present first-time evidence for potential disease modification by fenfluramine.
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http://dx.doi.org/10.1111/epi.16456DOI Listing
March 2020

Data-driven multiscale modeling reveals the role of metabolic coupling for the spatio-temporal growth dynamics of yeast colonies.

BMC Mol Cell Biol 2019 Dec 19;20(1):59. Epub 2019 Dec 19.

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, 2, avenue de l'Université, Esch-sur-Alzette, L-4365, Luxembourg.

Background: Multicellular entities like mammalian tissues or microbial biofilms typically exhibit complex spatial arrangements that are adapted to their specific functions or environments. These structures result from intercellular signaling as well as from the interaction with the environment that allow cells of the same genotype to differentiate into well-organized communities of diversified cells. Despite its importance, our understanding how this cell-cell and metabolic coupling lead to functionally optimized structures is still limited.

Results: Here, we present a data-driven spatial framework to computationally investigate the development of yeast colonies as such a multicellular structure in dependence on metabolic capacity. For this purpose, we first developed and parameterized a dynamic cell state and growth model for yeast based on on experimental data from homogeneous liquid media conditions. The inferred model is subsequently used in a spatially coarse-grained model for colony development to investigate the effect of metabolic coupling by calibrating spatial parameters from experimental time-course data of colony growth using state-of-the-art statistical techniques for model uncertainty and parameter estimations. The model is finally validated by independent experimental data of an alternative yeast strain with distinct metabolic characteristics and illustrates the impact of metabolic coupling for structure formation.

Conclusions: We introduce a novel model for yeast colony formation, present a statistical methodology for model calibration in a data-driven manner, and demonstrate how the established model can be used to generate predictions across scales by validation against independent measurements of genetically distinct yeast strains.
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http://dx.doi.org/10.1186/s12860-019-0234-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923950PMC
December 2019

Mitochondrial morphology provides a mechanism for energy buffering at synapses.

Sci Rep 2019 12 4;9(1):18306. Epub 2019 Dec 4.

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Belvaux, L-4367, Luxembourg.

Mitochondria as the main energy suppliers of eukaryotic cells are highly dynamic organelles that fuse, divide and are transported along the cytoskeleton to ensure cellular energy homeostasis. While these processes are well established, substantial evidence indicates that the internal structure is also highly variable in dependence on metabolic conditions. However, a quantitative mechanistic understanding of how mitochondrial morphology affects energetic states is still elusive. To address this question, we here present an agent-based multiscale model that integrates three-dimensional morphologies from electron microscopy tomography with the molecular dynamics of the main ATP producing components. We apply our modeling approach to mitochondria at the synapse which is the largest energy consumer within the brain. Interestingly, comparing the spatiotemporal simulations with a corresponding space-independent approach, we find minor spatial effects when the system relaxes toward equilibrium but a qualitative difference in fluctuating environments. These results suggest that internal mitochondrial morphology is not only optimized for ATP production but also provides a mechanism for energy buffering and may represent a mechanism for cellular robustness.
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http://dx.doi.org/10.1038/s41598-019-54159-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893035PMC
December 2019

A Statistical View on Calcium Oscillations.

Adv Exp Med Biol 2020 ;1131:799-826

Centre for Mathematical Medicine and Biology, School of Mathematical Sciences, University of Nottingham, Nottingham, UK.

Transient rises and falls of the intracellular calcium concentration have been observed in numerous cell types and under a plethora of conditions. There is now a growing body of evidence that these whole-cell calcium oscillations are stochastic, which poses a significant challenge for modelling. In this review, we take a closer look at recently developed statistical approaches to calcium oscillations. These models describe the timing of whole-cell calcium spikes, yet their parametrisations reflect subcellular processes. We show how non-stationary calcium spike sequences, which e.g. occur during slow depletion of intracellular calcium stores or in the presence of time-dependent stimulation, can be analysed with the help of so-called intensity functions. By utilising Bayesian concepts, we demonstrate how values of key parameters of the statistical model can be inferred from single cell calcium spike sequences and illustrate what information whole-cell statistical models can provide about the subcellular mechanistic processes that drive calcium oscillations. In particular, we find that the interspike interval distribution of HEK293 cells under constant stimulation is captured by a Gamma distribution.
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http://dx.doi.org/10.1007/978-3-030-12457-1_32DOI Listing
October 2019

Mutations in Disrupt Endoplasmic Reticulum-Mitochondria Contact Sites Interfering with Calcium Homeostasis and Mitochondrial Dynamics in Parkinson's Disease.

Antioxid Redox Signal 2019 12 21;31(16):1213-1234. Epub 2019 Aug 21.

Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg.

The outer mitochondrial membrane protein Miro1 is a crucial player in mitochondrial dynamics and calcium homeostasis. Recent evidence indicated that Miro1 mediates calcium-induced mitochondrial shape transition, which is a prerequisite for the initiation of mitophagy. Moreover, altered Miro1 protein levels have emerged as a shared feature of monogenic and sporadic Parkinson's disease (PD), but, so far, no disease-associated variants in have been identified. Here, we aim to explore the genetic and functional contribution of mutations to PD in patient-derived cellular models. For the first time, we describe heterozygous mutations in two PD patients (het c.815G>A; het c.1348C>T) and identified mitochondrial phenotypes with reduced mitochondrial mass in patient fibroblasts. Both mutations led to decreased endoplasmic reticulum-mitochondrial contact sites and calcium dyshomeostasis. As a consequence, energy metabolism was impaired, which in turn caused increased mitophagy. Our study provides functional evidence that is a genetic risk factor for PD, further implicating Miro1 in calcium homeostasis and mitochondrial quality control.
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http://dx.doi.org/10.1089/ars.2018.7718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798875PMC
December 2019

Stem cell-associated heterogeneity in Glioblastoma results from intrinsic tumor plasticity shaped by the microenvironment.

Nat Commun 2019 04 16;10(1):1787. Epub 2019 Apr 16.

NorLux Neuro-Oncology Laboratory, Department of Oncology, Luxembourg Institute of Health, L-1526, Luxembourg, Luxembourg.

The identity and unique capacity of cancer stem cells (CSC) to drive tumor growth and resistance have been challenged in brain tumors. Here we report that cells expressing CSC-associated cell membrane markers in Glioblastoma (GBM) do not represent a clonal entity defined by distinct functional properties and transcriptomic profiles, but rather a plastic state that most cancer cells can adopt. We show that phenotypic heterogeneity arises from non-hierarchical, reversible state transitions, instructed by the microenvironment and is predictable by mathematical modeling. Although functional stem cell properties were similar in vitro, accelerated reconstitution of heterogeneity provides a growth advantage in vivo, suggesting that tumorigenic potential is linked to intrinsic plasticity rather than CSC multipotency. The capacity of any given cancer cell to reconstitute tumor heterogeneity cautions against therapies targeting CSC-associated membrane epitopes. Instead inherent cancer cell plasticity emerges as a novel relevant target for treatment.
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http://dx.doi.org/10.1038/s41467-019-09853-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467886PMC
April 2019

MIC-MAC: An automated pipeline for high-throughput characterization and classification of three-dimensional microglia morphologies in mouse and human postmortem brain samples.

Glia 2019 08 14;67(8):1496-1509. Epub 2019 Apr 14.

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Belval, Luxembourg.

The phenotypic changes of microglia in brain diseases are particularly diverse and their role in disease progression, beneficial, or detrimental, is still elusive. High-throughput molecular approaches such as single-cell RNA-sequencing can now resolve the high heterogeneity in microglia population for a specific physiological condition, however, the relation between the different microglial signatures and their surrounding brain microenvironment is barely understood. Thus, better tools to characterize the phenotypic variations of microglia in situ are needed, particularly for human brain postmortem samples analysis. To address this challenge, we developed MIC-MAC, a Microglia and Immune Cells Morphologies Analyser and Classifier pipeline that semiautomatically segments, extracts, and classifies all microglia and immune cells labeled in large three-dimensional (3D) confocal image stacks of mouse and human brain samples. Our imaging-based approach enables automatic 3D-morphology characterization and classification of thousands of individual microglia in situ and revealed species- and disease-specific morphological phenotypes in mouse aging, human Alzheimer's disease, and dementia with Lewy Bodie's samples. MIC-MAC is a precision diagnostic tool that allows a rapid, unbiased, and large-scale analysis of microglia morphological states in mouse models and patient brain samples.
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http://dx.doi.org/10.1002/glia.23623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6617786PMC
August 2019

Neural Stem Cells of Parkinson's Disease Patients Exhibit Aberrant Mitochondrial Morphology and Functionality.

Stem Cell Reports 2019 05 11;12(5):878-889. Epub 2019 Apr 11.

Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, 4362 Belvaux, Luxembourg. Electronic address:

Emerging evidence suggests that Parkinson's disease (PD), besides being an age-associated disorder, might also have a neurodevelopment component. Disruption of mitochondrial homeostasis has been highlighted as a crucial cofactor in its etiology. Here, we show that PD patient-specific human neuroepithelial stem cells (NESCs), carrying the LRRK2-G2019S mutation, recapitulate key mitochondrial defects previously described only in differentiated dopaminergic neurons. By combining high-content imaging approaches, 3D image analysis, and functional mitochondrial readouts we show that LRRK2-G2019S mutation causes aberrations in mitochondrial morphology and functionality compared with isogenic controls. LRRK2-G2019S NESCs display an increased number of mitochondria compared with isogenic control lines. However, these mitochondria are more fragmented and exhibit decreased membrane potential. Functional alterations in LRRK2-G2019S cultures are also accompanied by a reduced mitophagic clearance via lysosomes. These findings support the hypothesis that preceding mitochondrial developmental defects contribute to the manifestation of the PD pathology later in life.
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http://dx.doi.org/10.1016/j.stemcr.2019.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522948PMC
May 2019

Single-cell transcriptomics reveals distinct inflammation-induced microglia signatures.

EMBO Rep 2018 11 11;19(11). Epub 2018 Sep 11.

NORLUX Neuro-Oncology Laboratory, Department of Oncology, Luxembourg Institute of Health, Luxembourg, Luxembourg

Microglia are specialized parenchymal-resident phagocytes of the central nervous system (CNS) that actively support, defend and modulate the neural environment. Dysfunctional microglial responses are thought to worsen CNS diseases; nevertheless, their impact during neuroinflammatory processes remains largely obscure. Here, using a combination of single-cell RNA sequencing and multicolour flow cytometry, we comprehensively profile microglia in the brain of lipopolysaccharide (LPS)-injected mice. By excluding the contribution of other immune CNS-resident and peripheral cells, we show that microglia isolated from LPS-injected mice display a global downregulation of their homeostatic signature together with an upregulation of inflammatory genes. Notably, we identify distinct microglial activated profiles under inflammatory conditions, which greatly differ from neurodegenerative disease-associated profiles. These results provide insights into microglial heterogeneity and establish a resource for the identification of specific phenotypes in CNS disorders, such as neuroinflammatory and neurodegenerative diseases.
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http://dx.doi.org/10.15252/embr.201846171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6216255PMC
November 2018

Loss of inter-cellular cooperation by complete epithelial-mesenchymal transition supports favorable outcomes in basal breast cancer patients.

Oncotarget 2018 Apr 13;9(28):20018-20033. Epub 2018 Apr 13.

Institute for Systems Biology, Seattle, WA, USA.

According to the sequential metastasis model, aggressive mesenchymal (M) metastasis-initiating cells (MICs) are generated by an epithelial-mesenchymal transition (EMT) which eventually is reversed by a mesenchymal-epithelial transition (MET) and outgrowth of life-threatening epithelial (E) macrometastases. Paradoxically, in breast cancer M signatures are linked with more favorable outcomes than E signatures, and M cells are often dispensable for metastasis in mouse models. Here we present evidence at the cellular and patient level for the cooperation metastasis model, according to which E cells are MICs, while M cells merely support E cell persistence through cooperation. We tracked the fates of co-cultured E and M clones and of fluorescent CDH1-promoter-driven cell lines reporting the E state derived from basal breast cancer HMLER cells. Cells were placed in suspension state and allowed to reattach and select an EMT cell fate. Flow cytometry, single cell and bulk gene expression analyses revealed that only pre-existing E cells generated E cells, mixed E/M populations, or stem-like hybrid E/M cells after suspension and that complete EMT manifest in M clones and CDH1-negative reporter cells resulted in loss of cell plasticity, suggesting full transdifferentiation. Mechanistically, E-M coculture experiments supported the persistence of pre-existing E cells where M cells inhibited EMT of E cells in a mutual cooperation via direct cell-cell contact. Consistently, M signatures were associated with more favorable patient outcomes compared to E signatures in breast cancer, specifically in basal breast cancer patients. These findings suggest a potential benefit of complete EMT for basal breast cancer patients.
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http://dx.doi.org/10.18632/oncotarget.25034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5929443PMC
April 2018

Data-driven dynamical model indicates that the heat shock response in is tailored to handle natural temperature variation.

J R Soc Interface 2018 05;15(142)

Institute of Quantitative and Theoretical Biology, Heinrich Heine University, Düsseldorf, Germany

Global warming exposes plants to severe heat stress, with consequent crop yield reduction. Organisms exposed to high temperature stresses typically protect themselves with a heat shock response (HSR), where accumulation of unfolded proteins initiates the synthesis of heat shock proteins through the heat shock transcription factor HSF1. While the molecular mechanisms are qualitatively well characterized, our quantitative understanding of the underlying dynamics is still very limited. Here, we study the dynamics of HSR in the photosynthetic model organism with a data-driven mathematical model of HSR. We based our dynamical model mostly on mass action kinetics, with a few nonlinear terms. The model was parametrized and validated by several independent datasets obtained from the literature. We demonstrate that HSR quantitatively and significantly differs if an increase in temperature of the same magnitude occurs abruptly, as often applied under laboratory conditions, or gradually, which would rather be expected under natural conditions. In contrast to rapid temperature increases, under gradual changes only negligible amounts of misfolded proteins accumulate, indicating that the HSR of efficiently avoids the accumulation of misfolded proteins under conditions most likely to prevail in nature. The mathematical model we developed is a flexible tool to simulate the HSR to different conditions and complements the current experimental approaches.
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http://dx.doi.org/10.1098/rsif.2017.0965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6000179PMC
May 2018

CaSiAn: a Calcium Signaling Analyzer tool.

Bioinformatics 2018 09;34(17):3052-3054

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Belvaux L-4367, Luxembourg.

Summary: Ca2+ is a central second messenger in eukaryotic cells that regulates many cellular processes. Recently, we have indicated that typical Ca2+ 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 statistics in a semi-automated manner. CaSiAn provides an intuitive graphical user interface allowing experimentalists to easily process a large amount of Ca2+ signals, interactively tune peak detection, revise statistical measures and access the quantified signal properties as excel or text files.

Availability And Implementation: CaSiAn is implemented in Java and available on Github (https://github.com/mmahsa/CaSiAn) as well as on the project page (http://r3lab.uni.lu/web/casa).

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6129310PMC
September 2018

On the phase space structure of IP induced Ca signalling and concepts for predictive modeling.

Chaos 2018 Apr;28(4):045115

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, 7, Rue de Swing, Belval L-4367, Luxembourg.

The correspondence between mathematical structures and experimental systems is the basis of the generalizability of results found with specific systems and is the basis of the predictive power of theoretical physics. While physicists have confidence in this correspondence, it is less recognized in cellular biophysics. On the one hand, the complex organization of cellular dynamics involving a plethora of interacting molecules and the basic observation of cell variability seem to question its possibility. The practical difficulties of deriving the equations describing cellular behaviour from first principles support these doubts. On the other hand, ignoring such a correspondence would severely limit the possibility of predictive quantitative theory in biophysics. Additionally, the existence of functional modules (like pathways) across cell types suggests also the existence of mathematical structures with comparable universality. Only a few cellular systems have been sufficiently investigated in a variety of cell types to follow up these basic questions. IP induced Casignalling is one of them, and the mathematical structure corresponding to it is subject of ongoing discussion. We review the system's general properties observed in a variety of cell types. They are captured by a reaction diffusion system. We discuss the phase space structure of its local dynamics. The spiking regime corresponds to noisy excitability. Models focussing on different aspects can be derived starting from this phase space structure. We discuss how the initial assumptions on the set of stochastic variables and phase space structure shape the predictions of parameter dependencies of the mathematical models resulting from the derivation.
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http://dx.doi.org/10.1063/1.5021073DOI Listing
April 2018

Cell Fate Decision as High-Dimensional Critical State Transition.

PLoS Biol 2016 Dec 27;14(12):e2000640. Epub 2016 Dec 27.

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.

Cell fate choice and commitment of multipotent progenitor cells to a differentiated lineage requires broad changes of their gene expression profile. But how progenitor cells overcome the stability of their gene expression configuration (attractor) to exit the attractor in one direction remains elusive. Here we show that commitment of blood progenitor cells to the erythroid or myeloid lineage is preceded by the destabilization of their high-dimensional attractor state, such that differentiating cells undergo a critical state transition. Single-cell resolution analysis of gene expression in populations of differentiating cells affords a new quantitative index for predicting critical transitions in a high-dimensional state space based on decrease of correlation between cells and concomitant increase of correlation between genes as cells approach a tipping point. The detection of "rebellious cells" that enter the fate opposite to the one intended corroborates the model of preceding destabilization of a progenitor attractor. Thus, early warning signals associated with critical transitions can be detected in statistical ensembles of high-dimensional systems, offering a formal theory-based approach for analyzing single-cell molecular profiles that goes beyond current computational pattern recognition, does not require knowledge of specific pathways, and could be used to predict impending major shifts in development and disease.
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http://dx.doi.org/10.1371/journal.pbio.2000640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5189937PMC
December 2016

Analysis of mitochondrial metabolism in situ: Combining stable isotope labeling with selective permeabilization.

Metab Eng 2017 09 15;43(Pt B):147-155. Epub 2016 Dec 15.

Technische Universiät Braunschweig, Department of Bioinfomatics and Biochemistry and Braunschweig Integrated Center of Systems Biology (BRICS), Rebenring 56, 38106 Braunschweig, Germany; Luxemburg Centre for Systems Biomedicine, University of Luxemburg, House of Biomedicine II, L-4367 Belvaux, Luxemburg; Computational Biology of Infection Research, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. Electronic address:

To date, it is well-established that mitochondrial dysfunction does not only play a vital role in cancer but also in other pathological conditions such as neurodegenerative diseases and inflammation. An important tool for the analysis of cellular metabolism is the application of stable isotope labeled substrates, which allow for the tracing of atoms throughout metabolic networks. While such analyses yield very detailed information about intracellular fluxes, the determination of compartment specific fluxes is far more challenging. Most approaches for the deconvolution of compartmented metabolism use computational models whereas experimental methods are rare. Here, we developed an experimental setup based on selective permeabilization of the cytosolic membrane that allows for the administration of stable isotope labeled substrates directly to mitochondria. We demonstrate how this approach can be used to infer metabolic changes in mitochondria induced by either chemical or genetic perturbations and give an outlook on its potential applications.
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http://dx.doi.org/10.1016/j.ymben.2016.12.005DOI Listing
September 2017

Inhibition of the mitochondrial calcium uniporter rescues dopaminergic neurons in pink1 zebrafish.

Eur J Neurosci 2017 02 28;45(4):528-535. Epub 2016 Dec 28.

Bateson Centre, University of Sheffield, Sheffield, UK.

Mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of early onset Parkinson's disease (PD). Loss of PINK1 function causes dysregulation of mitochondrial calcium homeostasis, resulting in mitochondrial dysfunction and neuronal cell death. We report that both genetic and pharmacological inactivation of the mitochondrial calcium uniporter (MCU), located in the inner mitochondrial membrane, prevents dopaminergic neuronal cell loss in pink1 * mutant zebrafish (Danio rerio) via rescue of mitochondrial respiratory chain function. In contrast, genetic inactivation of the voltage dependent anion channel 1 (VDAC1), located in the outer mitochondrial membrane, did not rescue dopaminergic neurons in PINK1 deficient D. rerio. Subsequent gene expression studies revealed specific upregulation of the mcu regulator micu1 in pink1 * mutant zebrafish larvae and inactivation of micu1 also results in rescue of dopaminergic neurons. The functional consequences of PINK1 deficiency and modified MCU activity were confirmed using a dynamic in silico model of Ca triggered mitochondrial activity. Our data suggest modulation of MCU-mediated mitochondrial calcium homeostasis as a possible neuroprotective strategy in PINK1 mutant PD.
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http://dx.doi.org/10.1111/ejn.13473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5324670PMC
February 2017

Pro-inflammatory Macrophages Sustain Pyruvate Oxidation through Pyruvate Dehydrogenase for the Synthesis of Itaconate and to Enable Cytokine Expression.

J Biol Chem 2016 Feb 17;291(8):3932-46. Epub 2015 Dec 17.

From the Luxembourg Centre for Systems Biomedicine, University of Luxembourg, 6 Avenue de Swing, L-4367 Belvaux, Luxembourg

Upon stimulation with Th1 cytokines or bacterial lipopolysaccharides, resting macrophages shift their phenotype toward a pro-inflammatory state as part of the innate immune response. LPS-activated macrophages undergo profound metabolic changes to adapt to these new physiological requirements. One key step to mediate this metabolic adaptation is the stabilization of HIF1α, which leads to increased glycolysis and lactate release, as well as decreased oxygen consumption. HIF1 abundance can result in the induction of the gene encoding pyruvate dehydrogenase kinase 1 (PDK1), which inhibits pyruvate dehydrogenase (PDH) via phosphorylation. Therefore, it has been speculated that pyruvate oxidation through PDH is decreased in pro-inflammatory macrophages. However, to answer this open question, an in-depth analysis of this metabolic branching point was so far lacking. In this work, we applied stable isotope-assisted metabolomics techniques and demonstrate that pyruvate oxidation is maintained in mature pro-inflammatory macrophages. Glucose-derived pyruvate is oxidized via PDH to generate citrate in the mitochondria. Citrate is used for the synthesis of the antimicrobial metabolite itaconate and for lipogenesis. An increased demand for these metabolites decreases citrate oxidation through the tricarboxylic acid cycle, whereas increased glutamine uptake serves to replenish the TCA cycle. Furthermore, we found that the PDH flux is maintained by unchanged PDK1 abundance, despite the presence of HIF1. By pharmacological intervention, we demonstrate that the PDH flux is an important node for M(LPS) macrophage activation. Therefore, PDH represents a metabolic intervention point that might become a research target for translational medicine to treat chronic inflammatory diseases.
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http://dx.doi.org/10.1074/jbc.M115.676817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4759172PMC
February 2016

Neurological Diseases from a Systems Medicine Point of View.

Methods Mol Biol 2016 ;1386:221-50

Luxembourg Centre for Systems Biomedicine (LCSB), Université du Luxembourg, 7, Avenue des Hauts-Fourneaux, L-4362, Esch-Belval, Luxembourg.

The difficulty to understand, diagnose, and treat neurological disorders stems from the great complexity of the central nervous system on different levels of physiological granularity. The individual components, their interactions, and dynamics involved in brain development and function can be represented as molecular, cellular, or functional networks, where diseases are perturbations of networks. These networks can become a useful research tool in investigating neurological disorders if they are properly tailored to reflect corresponding mechanisms. Here, we review approaches to construct networks specific for neurological disorders describing disease-related pathology on different scales: the molecular, cellular, and brain level. We also briefly discuss cross-scale network analysis as a necessary integrator of these scales.
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http://dx.doi.org/10.1007/978-1-4939-3283-2_11DOI Listing
October 2016

Multiscale Modeling Indicates That Temperature Dependent [Ca2+]i Spiking in Astrocytes Is Quantitatively Consistent with Modulated SERCA Activity.

Neural Plast 2015 4;2015:683490. Epub 2015 Aug 4.

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, 7 Avenue des Hauts-Fourneaux, 4362 Esch-sur-Alzette, Luxembourg ; National Centre for Microscopy and Imaging Research, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0608, USA.

Changes in the cytosolic Ca(2+) concentration ([Ca(2+)]i) are the most predominant active signaling mechanism in astrocytes that can modulate neuronal activity and is assumed to influence neuronal plasticity. Although Ca(2+) signaling in astrocytes has been intensively studied in the past, our understanding of the signaling mechanism and its impact on tissue level is still incomplete. Here we revisit our previously published data on the strong temperature dependence of Ca(2+) signals in both cultured primary astrocytes and astrocytes in acute brain slices of mice. We apply multiscale modeling to test the hypothesis that the temperature dependent [Ca(2+)]i spiking is mainly caused by the increased activity of the sarcoendoplasmic reticulum ATPases (SERCAs) that remove Ca(2+) from the cytosol into the endoplasmic reticulum. Quantitative comparison of experimental data with multiscale simulations supports the SERCA activity hypothesis. Further analysis of multiscale modeling and traditional rate equations indicates that the experimental observations are a spatial phenomenon where increasing pump strength leads to a decoupling of Ca(2+) release sites and subsequently to vanishing [Ca(2+)]i spikes.
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http://dx.doi.org/10.1155/2015/683490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539483PMC
April 2016

Stemness of the hybrid Epithelial/Mesenchymal State in Breast Cancer and Its Association with Poor Survival.

PLoS One 2015 28;10(5):e0126522. Epub 2015 May 28.

Institute for Systems Biology, Seattle, WA 98109, United States of America.

Breast cancer stem cells (CSCs) are thought to drive recurrence and metastasis. Their identity has been linked to the epithelial to mesenchymal transition (EMT) but remains highly controversial since--depending on the cell-line studied--either epithelial (E) or mesenchymal (M) markers, alone or together have been associated with stemness. Using distinct transcript expression signatures characterizing the three different E, M and hybrid E/M cell-types, our data support a novel model that links a mixed EM signature with stemness in 1) individual cells, 2) luminal and basal cell lines, 3) in vivo xenograft mouse models, and 4) in all breast cancer subtypes. In particular, we found that co-expression of E and M signatures was associated with poorest outcome in luminal and basal breast cancer patients as well as with enrichment for stem-like cells in both E and M breast cell-lines. This link between a mixed EM expression signature and stemness was explained by two findings: first, mixed cultures of E and M cells showed increased cooperation in mammosphere formation (indicative of stemness) compared to the more differentiated E and M cell-types. Second, single-cell qPCR analysis revealed that E and M genes could be co-expressed in the same cell. These hybrid E/M cells were generated by both E or M cells and had a combination of several stem-like traits since they displayed increased plasticity, self-renewal, mammosphere formation, and produced ALDH1+ progenies, while more differentiated M cells showed less plasticity and E cells showed less self-renewal. Thus, the hybrid E/M state reflecting stemness and its promotion by E-M cooperation offers a dual biological rationale for the robust association of the mixed EM signature with poor prognosis, independent of cellular origin. Together, our model explains previous paradoxical findings that breast CSCs appear to be M in luminal cell-lines but E in basal breast cancer cell-lines. Our results suggest that targeting E/M heterogeneity by eliminating hybrid E/M cells and cooperation between E and M cell-types could improve breast cancer patient survival independent of breast cancer-subtype.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126522PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447403PMC
April 2016

Protocols and programs for high-throughput growth and aging phenotyping in yeast.

PLoS One 2015 30;10(3):e0119807. Epub 2015 Mar 30.

Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting "Colony Forming Units". To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119807PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4379057PMC
April 2016

Critical transitions in chronic disease: transferring concepts from ecology to systems medicine.

Curr Opin Biotechnol 2015 Aug 10;34:48-55. Epub 2014 Dec 10.

Experimental Neurobiology Group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Campus Belval, 7 Avenue des Hauts-Fourneaux, L-4362 Esch-sur-Alzette, Luxembourg. Electronic address:

Ecosystems and biological systems are known to be inherently complex and to exhibit nonlinear dynamics. Diseases such as microbiome dysregulation or depression can be seen as complex systems as well and were shown to exhibit patterns of nonlinearity in their response to perturbations. These nonlinearities can be revealed by a sudden shift in system states, for instance from health to disease. The identification and characterization of early warning signals which could predict upcoming critical transitions is of primordial interest as prevention of disease onset is a major aim in health care. In this review, we focus on recent evidence for critical transitions in diseases and discuss the potential of such studies for therapeutic applications.
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http://dx.doi.org/10.1016/j.copbio.2014.11.020DOI Listing
August 2015

Reliable encoding of stimulus intensities within random sequences of intracellular Ca2+ spikes.

Sci Signal 2014 Jun 24;7(331):ra59. Epub 2014 Jun 24.

Mathematical Cell Physiology, Max Delbrück Center for Molecular Medicine, Robert Rössle Straße 10, Berlin 13125, Germany. Department of Physics, Humboldt University Berlin, Newtonstraße 15, Berlin 12489, Germany.

Ca(2+) is a ubiquitous intracellular messenger that regulates diverse cellular activities. Extracellular stimuli often evoke sequences of intracellular Ca(2+) spikes, and spike frequency may encode stimulus intensity. However, the timing of spikes within a cell is random because each interspike interval has a large stochastic component. In human embryonic kidney (HEK) 293 cells and rat primary hepatocytes, we found that the average interspike interval also varied between individual cells. To evaluate how individual cells reliably encoded stimuli when Ca(2+) spikes exhibited such unpredictability, we combined Ca(2+) imaging of single cells with mathematical analyses of the Ca(2+) spikes evoked by receptors that stimulate formation of inositol 1,4,5-trisphosphate (IP3). This analysis revealed that signal-to-noise ratios were improved by slow recovery from feedback inhibition of Ca(2+) spiking operating at the whole-cell level and that they were robust against perturbations of the signaling pathway. Despite variability in the frequency of Ca(2+) spikes between cells, steps in stimulus intensity caused the stochastic period of the interspike interval to change by the same factor in all cells. These fold changes reliably encoded changes in stimulus intensity, and they resulted in an exponential dependence of average interspike interval on stimulation strength. We conclude that Ca(2+) spikes enable reliable signaling in a cell population despite randomness and cell-to-cell variability, because global feedback reduces noise, and changes in stimulus intensity are represented by fold changes in the stochastic period of the interspike interval.
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http://dx.doi.org/10.1126/scisignal.2005237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092318PMC
June 2014