Current treatments against organophosphate poisoning (OPP) do not directly address effects mediated by the overstimulation of nicotinic acetylcholine receptors (nAChR). Non-oxime bispyridinium compounds (BPC) promote acetylcholine esterase-independent recovery of organophosphate-induced paralysis. Here, we test the hypothesis that they act by positive modulatory action on nAChRs. Using two-electrode voltage clamp analysis in combination with mutagenesis and molecular docking analysis, the potency and molecular mode of action of a series of nine BPCs was investigated on human α7 and muscle-type nAChRs expressed in Xenopus laevis oocytes. The investigated BPCs inhibited α7 and/or muscle-type nAChRs with IC50 values in the high nanomolar to high micromolar range. Further analysis of the most potent analogues revealed a noncompetitive, voltage-dependent inhibition. Co-application with the α7-selective positive allosteric modulator PNU120596 and generation of α7/5HT3 receptor chimeras excluded direct interaction with the PNU120596 binding site and binding to the extracellular domain of the α7 nAChR, suggesting that they act as open channel blockers (OCBs). Molecular docking supported by mutagenesis localized the BPC binding area in the outer channel vestibule between the extracellular and transmembrane domains. Analysis of BPC action on other cation-selective channels suggests a rather nonspecific inhibition of pentameric cation channels. BPCs have been shown to ameliorate organophosphate-induced paralysis in vitro and in vivo. Our data support molecular action as OCBs at α7 and muscle-type nAChRs and suggest that their positive physiological effects are more complex than anticipated and require further investigation.

Neonicotinoids (neonics) are amongst the most commonly used class of pesticides globally. In the United States, imidacloprid (IMI) is extensively used for agriculture and in other common applications such as house-hold pest control. Regular exposure to IMI, and several of its known metabolites including IMI-olefin and desnitroimidacloprid (DN-IMI), has been shown to be harmful to many organisms including mammals, birds, and fish. Studies show that neonics bind human nicotinicacetylcholine receptors (nAChRs) and cause cellular toxicity. In the dopaminergic Lund human mesencephalic (LUHMES) cell line, IMI and other neonics (10-100 mu M) have been recently shown to activate intracellular calcium signaling through nAChRs. Thus, we examined proteomic responses of LUHMES cells to a 48-h treatment with 50 mu M IMI, IMI-olefin, or DN-IMI. Our findings show differential effects of these neonics on cellular protein expression. Bioinformatic analysis of significantly altered proteins indicates an effect of IMI, IMI-olefin, and DN-IMI on protein synthesis and ribosomal function. These findings suggest a role for protein synthesis and transcriptional regulation in neonic-mediated dopaminergic neurotoxicity.

AbstractIn vitro models of the peripheral nervous system would benefit from further refinements to better support studies on neuropathies. In particular, the assessment of pain-related signals is still difficult in human cell cultures. Here, we harnessed induced pluripotent stem cells (iPSCs) to generate peripheral sensory neurons enriched in nociceptors. The objective was to generate a culture system with signaling endpoints suitable for pharmacological and toxicological studies. Neurons generated by conventional differentiation protocols expressed moderate levels of P2X3 purinergic receptors and only low levels of TRPV1 capsaicin receptors, when maturation time was kept to the upper practically useful limit of 6 weeks. As alternative approach, we generated cells with an inducible NGN1 transgene. Ectopic expression of this transcription factor during a defined time window of differentiation resulted in highly enriched nociceptor cultures, as determined by functional (P2X3 and TRPV1 receptors) and immunocytochemical phenotyping, complemented by extensive transcriptome profiling. Single cell recordings of Ca2+-indicator fluorescence from >9000 cells were used to establish the “fraction of reactive cells” in a stimulated population as experimental endpoint, that appeared robust, transparent and quantifiable. To provide an example of application to biomedical studies, functional consequences of prolonged exposure to the chemotherapeutic drug oxaliplatin were examined at non-cytotoxic concentrations. We found (i) neuronal (allodynia-like) hypersensitivity to otherwise non-activating mechanical stimulation that could be blocked by modulators of voltage-gated sodium channels; (ii) hyper-responsiveness to TRPV1 receptor stimulation. These findings and several other measured functional alterations indicate that the model is suitable for pharmacological and toxicological studies related to peripheral neuropathies.

Aspidasept (Pep19-2.5) and its derivative Pep19-4LF ("Aspidasept II") are anti-infective and anti-inflammatory synthetic polypeptides currently in development for application against a variety of moderate to severe bacterial infections that could lead to systemic inflammation, as in the case of severe sepsis and septic shock, as well as application to non-systemic diseases in the case of skin and soft tissue infections (SSTI). In the present study, Aspidasept and Aspidasept II and their part structures were analysed with respect to their toxic behavior in different established models against a variety of relevant cells, and in electrophysiological experiments targeting the hERG channel according to ICH S7B. Furthermore, the effects in mouse models of neurobiological behavior and the local lymph node according to OECD test guideline 429 were investigated, as well as a rat model of repeated dose toxicology according to ICH M3. The data provide conclusive information about potential toxic effects, thus specifying a therapeutic window for the application of the peptides. Therefore, these data allow us to define Aspidasept concentrations for their use in clinical studies as parenteral application.

Human cell-based neural organoids are increasingly being used for investigations of neurotoxicity and to study the pathophysiology of neurodegenerative diseases. Here, we present a fast and robust method to generate 3D cultured human dopaminergic neurons (LUHMES) for toxicity testing and long-term culture. Moreover, a plating step was introduced to allow generation of neurite networks with a defined 2D orientation and several mm length while all cell bodies (somata) remained in a 3D, dome-like structure. These cultures, named 2.5D (for 2.5 dimensional), offer new approaches to quantify toxicant effects on organoids by standard technology and in high throughput. For instance, the system reacted to the parkinsonian model toxicants MPP+, rotenone and MG-132, and to the ferroptosis-inducer erastin. Moreover, stable incorporation of human stem cell-derived astrocytes or microglia was possible. Addition of astrocytes stabilized the post mitotic state of the LUHMES neurons and thereby allowed formation of a stable microphysiological system. Neuroprotection against the proteasome inhibitor MG-132 and the ferroptosis-inducer erastin was mediated by such glia, exemplifying the crucial protective role of astrocytes in neurodegeneration. The modularity of the system was further employed to incorporate microglia together with astrocytes into the organoids. Such ratio-defined, three cell type-based organoids will allow new approaches to study human pathophysiology and toxicology of the nervous system.

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.

Event Abstract Back to Event Micro electrode arrays to investigate neuron-glia crosstalk in neuropathic pain in-vitro models Francesca Izzi1, 2*, Dominik Loser2, 3, 4 and Paolo Cesare1, 2 1 Natural and Medical Sciences Institute, Germany 2 Natural and Medical Sciences Institute, Germany 3 NMI Technologie Transfer GmbH, Germany 4 Hochschule Albstadt-Sigmaringen, Life Sciences Faculty, Germany Motivation Chronic Neuropathic Pain (NP) is frequently associated with peripheral nerve injury or disease. It affects 6-8% of the population, and current treatments are inadequate, since they have limited efficacy. Interest in understanding the mechanisms that underpin neuropathic pain has been growing in the last years, but the exact mechanisms haven’t been clarified yet. Experimental models of neuropathic pain showed that non-neuronal cells, in particular Schwann cells and satellite glia cells (SGCs), play a very active role in the development of sensory abnormalities. What is less clear is exactly how these non-neuronal cells induce the hyper-excitable state in pain signaling neurons associated with neuropathic pain condition. Materials and methods Mice between p13 and p20 were euthanized with CO2, and the vertebral column was removed. Each column was cut along its main axis, and for each half dorsal root ganglia (DRG) were removed. DRG were dissociated enzymatically, and dead cells and debris were removed by gradient centrifugation. Pure sensory neurons were obtained using the Neuron Isolation Kit (Miltenyi Biotec). Co-cultures of neurons-glia and cultures of pure sensory neurons were plated on micro electrode arrays (MEA) and glass coverslips coated with polyethyleneimine (PEI) 0.075 g/ml, and laminin 20 µg/ml, supplied with modified Neurobasal-A medium and kept at 37°C with 5% CO2. After 2-4 days in vitro, the electrical activity of each MEA was recorded using MEA2100 system (Multichannel System), and calcium imaging was performed. Results By using a high-density MEA system, we were able to record the electrical activity of both pure sensory neurons and co-cultures of neurons-glia derived from murine dorsal root ganglia (DRG). To analyse the differential excitability of co-cultures of neurons-glia and pure neurons, capsaicin was applied to every cell culture. Co-cultures showed an increase in electrical excitability in response to capsaicin already after 1 day in vitro. Cultures of pure sensory neurons showed electrical activity in response to capsaicin after 1 day in vitro, although lower if compared to that of the co-cultures. Surprisingly, this activity was completely lost after 3 days in vitro. To further investigate this effect, calcium imaging experiments were performed on these cultures. Application of capsaicin could evoke in both cases an increase in intracellular calcium concentration and no significant difference was noticed between purified and non-purified cultures. This demonstrates that purified sensory neurons do actually express the TRPV1 receptor although they are somehow unable to generate action potentials in response to capsaicin application. Discussion and conclusion It has been demonstrated that sensory neurons possess electrical activity in response to different excitatory stimuli already after 1 day in vitro; however, in the case of purified neurons, this activity disappears after 2 days in vitro. This cannot be explained by a lack in the capsaicin receptor TRPV1, whose presence has been proved by calcium-imaging experiment. The ability of purified sensory neurons to maintain their electrical activity shortly after being plated suggests the presence of a “memory effect”, that allows them to behave as in vivo, effect that disappears as the culture timeline increases, as noticed from the MEA recording data. All these data combined lead to the hypothesis that non-neuronal cells, such as Schwann cells and satellite glial cells, play an active role in the activity of sensory neurons, and their absence lead to a loss of functionality in these neurons. Future studies, which include perforated patch-clamp experiments, will be required to understand the loss of electrical activity in purified sensory neurons, identify proteins that are dysregulated in purified sensory neurons, and their relationship with non-neuronal cells. Figure legends: Figure1: Co-culture of sensory neurons and glia on MEA. Figure 2: Culture of purified sensory neurons on MEA. Figure 1 Figure 2 Acknowledgements This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 116072. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA. Keywords: neuropathic pain, dorsal root ganglia, sensory neurons, Neuroglia, MEA Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Poster Presentation Topic: Microphysiological systems Citation: Izzi F, Loser D and Cesare P (2019). Micro electrode arrays to investigate neuron-glia crosstalk in neuropathic pain in-vitro models. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00029 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Mrs. Francesca Izzi, Natural and Medical Sciences Institute, Reutlingen, 72770, Germany, Francesca.Izzi@nmi.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Francesca Izzi Dominik Loser Paolo Cesare Google Francesca Izzi Dominik Loser Paolo Cesare Google Scholar Francesca Izzi Dominik Loser Paolo Cesare PubMed Francesca Izzi Dominik Loser Paolo Cesare Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Event Abstract Back to Event Development of a microelectrode array (MEA) based neurotoxicity assay for detecting the seizurogenic activity of novel drug candidates Dominik Loser1, 2, 3*, Timm Danker2, Clemens Möller1, Anita Niedworok3 and Udo Kraushaar3 1 Hochschule Albstadt-Sigmaringen, Life Sciences Faculty, Germany 2 NMI Technologie Transfer GmbH, Germany 3 Natural and Medical Sciences Institute, Germany During the early phase of drug development, the identification of possible side effects of novel drug candidates on the neuronal activity is very important. Until now most of the neurotoxicity assays are based on in vivo or in vitro animal models, which are problematic in terms of ethical issues and predictivity for humans. Over the last few years the continuous improvement of human iPSC derived neurons has increased their importance in this field, since they offer a great opportunity for the investigation of compound effects directly on a complex in vitro system of human origin. By combining these cells with the microelectrode array (MEA) technique we can investigate the functional neurotoxicity of novel drug candidates on a neuronal network and thereby detect their seizurogenic activity. In order to achieve this goal, we examined the effects of different coating and plating conditions on the development of the electrical activity of the human iPSC derived neurons (GlutaNeurons, Cellular Dynamics International CDI, US) that were cultured on 24-well glass Multiwell-MEAs (Multi Channel Systems MCS, GER). We tested three different coatings: 0.1% PEI, 0.07% PEI, which was diluted in a ready-to-use borate buffer, and the 0.07% PEI coating in combination with a pre-dotting with Laminin (10 µg/ml). The cells were dotted on the electrode fields of the MEAs in medium with a Laminin concentration of 10 µg/ml. The results showed no major differences between the tested coatings. Therefore, and because the handling of the 0.07% PEI coating is easier compared to the other tested coatings, we continued the study with the 0.07% PEI coating and explored the effects of different plating conditions. We examined the influences of a lower and a higher Laminin concentration in the dotting medium (10 µg/ml and 100 µg/ml) and in the medium (1 µg/ml and 33.3 µg/ml) that was used to fill-up the wells after the cells attached to the bottom of the wells. The cells that were plated in the higher Laminin concentration showed an earlier outgrowth of neurites as well as an earlier increase of the electrical activity and the occurrence of synchronous bursting compared to the cells plated in the lower Laminin concentration. The results suggest that there is a difference in the development of the cells in the tested plating conditions. The higher Laminin concentration led to an earlier outgrowth of neurites which could have favored an earlier formation of a neuronal network and thereby an earlier occurrence of synchronous burst activity. These findings have to be further investigated to establish a reliable neurotoxicity assay for detecting the seizurogenic activity of novel drug candidates in the future. Acknowledgements The authors would like to thank Dr. Sabine Lange (CDI) for providing the cells and useful information as well as Multi Channel Systems for providing the Glass Multiwell MEAs. This work was partially funded by the Baden-Württemberg Ministry of Science, Research and Art. Keywords: Assay development, human iPSC derived neurons, microelectrode array, seizurogenic compounds, Neurotoxicity Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Oral Presentation Topic: Assay development Citation: Loser D, Danker T, Möller C, Niedworok A and Kraushaar U (2019). Development of a microelectrode array (MEA) based neurotoxicity assay for detecting the seizurogenic activity of novel drug candidates. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00016 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Mr. Dominik Loser, Hochschule Albstadt-Sigmaringen, Life Sciences Faculty, Albstadt, Germany, dominik.loser@nmi.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Dominik Loser Timm Danker Clemens Möller Anita Niedworok Udo Kraushaar Google Dominik Loser Timm Danker Clemens Möller Anita Niedworok Udo Kraushaar Google Scholar Dominik Loser Timm Danker Clemens Möller Anita Niedworok Udo Kraushaar PubMed Dominik Loser Timm Danker Clemens Möller Anita Niedworok Udo Kraushaar Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

In vitro models of the peripheral nervous system would benefit from further refinements to better support studies on neuropathies. In particular, the assessment of pain-related signals is still difficult in human cell cultures. Here, we harnessed induced pluripotent stem cells (iPSCs) to generate peripheral sensory neurons enriched in nociceptors. The objective was to generate a culture system with signaling endpoints suitable for pharmacological and toxicological studies. Neurons generated by conventional differentiation protocols expressed moderate levels of P2X3 purinergic receptors and only low levels of TRPV1 capsaicin receptors, when maturation time was kept to the upper practically-useful limit of 6 weeks. As alternative approach, we generated cells with an inducible NGN1 transgene. Ectopic expression of this transcription factor during a defined time window of differentiation resulted in highly-enriched nociceptor cultures, as determined by functional (P2X3 and TRPV1 receptors) and immunocytochemical phenotyping, complemented by extensive transcriptome profiling. Single cell recordings of Ca 2+ -indicator fluorescence from >9,000 cells were used to establish the “fraction of reactive cells” in a stimulated population as experimental endpoint, that appeared robust, transparent and quantifiable. To provide an example of application to biomedical studies, functional consequences of prolonged exposure to the chemotherapeutic drug oxaliplatin were examined at non-cytotoxic concentrations. We found (i) neuronal (allodynia-like) hypersensitivity to otherwise non-activating mechanical stimulation that could be blocked by modulators of voltage-gated sodium channels; (ii) hyper-responsiveness to TRPV1 receptor stimulation. These findings and several other measured functional alterations indicate that the model is suitable for pharmacological and toxicological studies related to peripheral neuropathies.