How nuclear envelope dynamics can direct laminopathy phenotypes

DATA AVAILABILITY : No data was used for the research described in the article.The nuclear envelope separates the genome from the cytoplasmic environment. However, the nuclear envelope is also physically associated with the genome and exerts influence on gene expression and genome modification. The nucleus is dynamic, changing shape and responding to cell movement, disassembling and assembling during cell division, and undergoing rupture and repair. These dynamics can be impacted by genetic disease, leading to a family of diseases called laminopathies. Their disparate phenotypes suggest that multiple processes are affected. We highlight three such processes here, which we believe can be used to classify most of the laminopathies. While much still needs to be learned, some commonalities between these processes, such as proteins involved in nuclear envelope formation and rupture repair, may drive a variety of laminopathies. Here we review the latest information regarding nuclear dynamics and its role in laminopathies related to mutations in the nuclear lamina and linker of nucleoskeleton and cytoskeleton complex (LINC) proteins.https://www.sciencedirect.com/journal/current-opinion-in-cell-biologyhj2024ImmunologyPhysiologySDG-03:Good heatlh and well-bein

Prediction of Biochemical Recurrence Based on Molecular Detection of Lymph Node Metastasis After Radical Prostatectomy.

Background Molecular detection of lymph node (LN) micrometastases by analyzing mRNA expression of epithelial markers in prostate cancer (PC) patients provides higher sensitivity than histopathological examination. Objective To investigate which type of marker to use and whether molecular detection of micrometastases in LNs was predictive of biochemical recurrence. Design setting and participants LN samples from PC patients undergoing radical prostatectomy with extended LN dissection between 2009 and 2011 were examined for the presence of micrometastases by both routine histopathology and molecular analyses. Outcome measurements and statistical analysis The mRNA expression of a panel of markers of prostate epithelial cells, prostate stem cell-like cells, epithelial-to-mesenchymal transition, and stromal activation, was performed by quantitative real-time polymerase chain reaction. The expression levels of these markers in LN metastases from three PC patients were compared with the expression levels in LN from five control patients without PC in order to identify the panel of markers best suited for the molecular detection of LN metastases. The predictive value of the molecular detection of micrometastases for biochemical recurrence was assessed after a follow-up of 10 yr. Results and limitations Prostate epithelial markers are better suited for the detection of occult LN metastases than molecular markers of stemness, epithelial-to-mesenchymal transition, or reactive stroma. An analysis of 1023 LNs from 60 PC patients for the expression of prostate epithelial cell markers has revealed different expression levels and patterns between patients and between LNs of the same patient. The positive predictive value of molecular detection of occult LN metastasis for biochemical recurrence is 66.7% and the negative predictive value is 62.5%. Limitations are sample size and the hypothesis-driven selection of markers. Conclusions Molecular detection of epithelial cell markers increases the number of positive LNs and predicts tumor recurrence already at surgery. Patient summary We show that a panel of epithelial prostate markers rather than single genes is preferred for the molecular detection of lymph node micrometastases not visible at histopathological examination

Developmental neurotoxicity in a human model system

There is an urgent scientific and regulatory need for development and implementation of new approach methods (NAM), such as cell culture test methods. NAM can give infor-mation about mechanisms of toxicity and about pathways disturbed after test compound exposure. Such knowledge plays an increasingly larger role in modern toxicology. Partic-ularly, in the field of developmental neurotoxicity (DNT), in vitro test methods can model different stages of neurodevelopment and thereby inform on stage-specific toxicities. However, there is not yet a suitable test system available for every stage of neurodevel-opment. This thesis addresses this gap in two different ways: first, by investigating mech-anisms of neurotoxicity at various stages of neurodevelopment, and second, by establish-ing new test methods for stages not covered so far. In the first part of this thesis, human pluripotent stem cells (hPSC) were differentiated into neuroepithelial precursors (NEP) to investigate transcriptome changes triggered by the well-known teratogen valproic acid and other histone deacetylase inhibitors (HDACi) during very early neurodevelopment. Short exposures (up to 24 h) altered histone acetylation and the transcriptome only tran-siently. Transcriptome changes after drug exposure both reflected the mode of action (MoA) of a compound and described an altered differentiation state. Altered differentia-tion was observed after longer HDACi exposures (at least three days) accompanied by altered histone methylation. This study showed that epigenetic modifications can have neuroteratogenic effects by changing the chromatin state. In the second part of this work, it was examined in how far neurotoxicology of complex toxins could be evaluated with human neuronal cultures. To explore the neurotoxicity hazard of microcystins (MC) in humans, two mature neuronal cell types (LUHMES and peripheral neurons) were exposed to MC-LF. Clear neurotoxic effects were identified at low μM concentrations. In this study, the hazard data obtained in vitro were combined with literature exposure data for a gen-eral risk assessment. In the final part of this thesis, a novel test system was developed to close an obvious gap in the available neurotoxicity test battery: identification of toxicants interfering with ionotropic glutamate receptors. An hPSC-based differentiation protocol was established yielding a mixed cortical culture (MCC) with about 50% N-methyl-D-as-partate receptor (NMDA-R) expressing neurons. Toxicants known to interfere with gluta-mate receptors, like ibotenic acid and domoic acid, triggered changes in intracellular free calcium ion concentration which could be inhibited by antagonists like ketamine and phencyclidine. Hence, MCC contribute to the currently available test systems for investi-gation of compounds affecting important neuronal receptors. The studies presented in this thesis led to the development of novel in vitro test systems that allow the study of mode of action (MoA) of toxicants during different stages of neurodevelopment and the screening of unknown compounds of potential neurotoxicity.publishe

CaFFEE : A program for evaluating time courses of Ca2+ dependent signal changes of complex cells loaded with fluorescent indicator dyes

Quantification of changes in intracellular free Ca2+ concentrations [Ca2+]i is fundamental to the understanding of the physiology of single cells in response to both environmental and endogenous stimuli. Here we present easy to use freeware that allows especially the evaluation of [Ca2+]i signals in complex and mixed cultures. The program CaFFEE (Calcium Fluorescent Flash Evaluating Engine) enables the user to evaluate the response of hundreds of cells to treat-ments that influence [Ca2+]i. CaFFEE processes large quantities of image data, automatically identifies individual cells in mixed, heterogeneous populations, and evaluates their fluorescence signal. All data are exported in spreadsheet format, and data on thousands of cells can be batch-processed. Moreover, the program optimizes the visual representation of time-lapse image data for user-guided data exploration (setting of parameters for semi-automated data processing). The freeware allows the standardized and transparent processing of imaging data independent of the platform used to generate the data.publishe

Examination of microcystin neurotoxicity using central and peripheral human neurons

Microcystins (MC) are a group of cyanobacterial toxins that comprises MC-LF and other cyclic heptapeptides, best known as potent hepatotoxicants. Cell culture and epidemiological studies suggest that MC might also affect the nervous system, when there is systemic exposure e.g. via drinking water or food. We asked whether in vitro studies with human neurons could provide estimates on the neurotoxicity hazard of MC-LF. First, we used LUHMES neurons, a well-established test system for neurotoxicants and neuropathological processes. These central nervous system cells expressed OATP1A2, a presumed carrier of MC-LF, and we observed selective neurite toxicity in the µM range (EC20 = 3.3 µM ≈ 3.3 µg/ml). Toxicity paralleled transcriptome changes pointed towards attenuated cell maintenance and biosynthetic processes. Prolonged exposure for up to four days did not increase toxicity. As a second model, we used human dorsal root ganglia-like neurons. These peripheral nervous system cells represent parts of the nervous system not protected by the blood brain barrier in humans. Toxicity was observed in a similar concentration range (EC20 = 7.4 µM). We conclude that MC-LF poses a potential neurotoxic hazard in humans. The adverse effect concentrations observed here were orders of magnitude higher than those presumed to be encountered after normal nutritional or environmental exposure. However, the low µM concentrations found to be toxic are close to levels that may be reached after very excessive algae supplement intake.publishe

A human stem cell-derived test system for agents modifying neuronal N-methyl-D-aspartate-type glutamate receptor Ca²⁺-signalling

Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neuroepithelial stem cell (NESC) population, we generate "mixed cortical cultures" (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profiling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-D-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell-derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca2+ signalling. Changes of the intracellular free calcium ion concentration ([Ca2+]i) were measured by fluorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range. MCC responded to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and a subpopulation of 50% had functional NMDA-R. The receptor was modulated by Mg2+, Zn2+ and Pb2+ in the expected ways, and various toxicologically relevant agonists (quinolinic acid, ibotenic acid, domoic acid) triggered [Ca2+]i responses in MCC. Antagonists, such as phencyclidine, ketamine and dextromethorphan, were also readily identified. Thus, the MCC developed here may fill an important gap in the panel of test systems available to characterize the effects of chemicals on neurotransmitter receptors.publishe

A human stem cell-derived test system for agents modifying neuronal N-methyl-d-aspartate-type glutamate receptor Ca2+-signalling

Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neuroepithelial stem cell (NESC) population, we generate "mixed cortical cultures" (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profiling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-D-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell-derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca2+ signalling. Changes of the intracellular free calcium ion concentration ([Ca2+]i) were measured by fluorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range. MCC responded to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and a subpopulation of 50% had functional NMDA-R. The receptor was modulated by Mg2+, Zn2+ and Pb2+ in the expected ways, and various toxicologically relevant agonists (quinolinic acid, ibotenic acid, domoic acid) triggered [Ca2+]i responses in MCC. Antagonists, such as phencyclidine, ketamine and dextromethorphan, were also readily identified. Thus, the MCC developed here may fill an important gap in the panel of test systems available to characterize the effects of chemicals on neurotransmitter receptors.publishe

Human neuronal signaling and communication assays to assess functional neurotoxicity

Prediction of drug toxicity on the human nervous system still relies mainly on animal experiments. Here, we developed an alternative system allowing assessment of complex signaling in both individual human neurons and on the network level. The LUHMES cultures used for our approach can be cultured in 384-well plates with high reproducibility. We established here high-throughput quantification of free intracellular Ca2+ concentrations [Ca2+]i as broadly applicable surrogate of neuronal activity and verified the main processes by patch clamp recordings. Initially, we characterized the expression pattern of many neuronal signaling components and selected the purinergic receptors to demonstrate the applicability of the [Ca2+]i signals for quantitative characterization of agonist and antagonist responses on classical ionotropic neurotransmitter receptors. This included receptor sub-typing and the characterization of the anti-parasitic drug suramin as modulator of the cellular response to ATP. To exemplify potential studies on ion channels, we characterized voltage-gated sodium channels and their inhibition by tetrodotoxin, saxitoxin and lidocaine, as well as their opening by the plant alkaloid veratridine and the food-relevant marine biotoxin ciguatoxin. Even broader applicability of [Ca2+]i quantification as an end point was demonstrated by measurements of dopamine transporter activity based on the membrane potential-changing activity of this neurotransmitter carrier. The substrates dopamine or amphetamine triggered [Ca2+]i oscillations that were synchronized over the entire culture dish. We identified compounds that modified these oscillations by interfering with various ion channels. Thus, this new test system allows multiple types of neuronal signaling, within and between cells, to be assessed, quantified and characterized for their potential disturbance.publishe

Time and space-resolved quantification of plasma membrane sialylation for measurements of cell function and neurotoxicity

While there are many methods to quantify the synthesis, localization, and pool sizes of proteins and DNA during physiological responses and toxicological stress, only few approaches allow following the fate of carbohydrates. One of them is metabolic glycoengineering (MGE), which makes use of chemically modified sugars (CMS) that enter the cellular biosynthesis pathways leading to glycoproteins and glycolipids. The CMS can subsequently be coupled (via bio-orthogonal chemical reactions) to tags that are quantifiable by microscopic imaging. We asked here, whether MGE can be used in a quantitative and time-resolved way to study neuronal glycoprotein synthesis and its impairment. We focused on the detection of sialic acid (Sia), by feeding human neurons the biosynthetic precursor N-acetyl-mannosamine, modified by an azide tag. Using this system, we identified non-toxic conditions that allowed live cell labeling with high spatial and temporal resolution, as well as the quantification of cell surface Sia. Using combinations of immunostaining, chromatography, and western blotting, we quantified the percentage of cellular label incorporation and effects on glycoproteins such as polysialylated neural cell adhesion molecule. A specific imaging algorithm was used to quantify Sia incorporation into neuronal projections, as potential measure of complex cell function in toxicological studies. When various toxicants were studied, we identified a subgroup (mitochondrial respiration inhibitors) that affected neurite glycan levels several hours before any other viability parameter was affected. The MGE-based neurotoxicity assay, thus allowed the identification of subtle impairments of neurochemical function with very high sensitivity.publishe

Stem Cell-Derived Immature Human Dorsal Root Ganglia Neurons to Identify Peripheral Neurotoxicants

Safety sciences and the identification of chemical hazards have been seen as one of the most immediate practical applications of human pluripotent stem cell technology. Protocols for the generation of many desirable human cell types have been developed, but optimization of neuronal models for toxicological use has been astonishingly slow, and the wide, clinically important field of peripheral neurotoxicity is still largely unexplored. A two-step protocol to generate large lots of identical peripheral human neuronal precursors was characterized and adapted to the measurement of peripheral neurotoxicity. High content imaging allowed an unbiased assessment of cell morphology and viability. The computational quantification of neurite growth as a functional parameter highly sensitive to disturbances by toxicants was used as an endpoint reflecting specific neurotoxicity. The differentiation of cells toward dorsal root ganglia neurons was tracked in relation to a large background data set based on gene expression microarrays. On this basis, a peripheral neurotoxicity (PeriTox) test was developed as a first toxicological assay that harnesses the potential of human pluripotent stem cells to generate cell types/tissues that are not otherwise available for the prediction of human systemic organ toxicity. Testing of more than 30 chemicals showed that human neurotoxicants and neurite growth enhancers were correctly identified. Various classes of chemotherapeutic agents causing human peripheral neuropathies were identified, and they were missed when tested on human central neurons. The PeriTox test we established shows the potential of human stem cells for clinically relevant safety testing of drugs in use and of new emerging candidates