Miroslav Cik

Vesicular Glutamate Transporter VGLUT2 Expression Levels Control Quantal Size and Neuropathic Pain

Uptake of l-glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). Three transporters (VGLUT1–VGLUT3) are expressed in the mammalian CNS, with partial overlapping expression patterns, and VGLUT2 is the most abundantly expressed paralog in the thalamus, midbrain, and brainstem. Previous studies have shown that VGLUT1 is necessary for glutamatergic transmission in the hippocampus, but the role of VGLUT2 in excitatory transmission is unexplored in glutamatergic neurons and in vivo. We examined the electrophysiological and behavioral consequences of loss of either one or both alleles of VGLUT2. We show that targeted deletion of VGLUT2 in mice causes perinatal lethality and a 95% reduction in evoked glutamatergic responses in thalamic neurons, although hippocampal synapses function normally. Behavioral analysis of heterozygous VGLUT2 mice showed unchanged motor function, learning and memory, acute nociception, and inflammatory pain, but acquisition of neuropathic pain, maintenance of conditioned taste aversion, and defensive marble burying were all impaired. Reduction or loss of VGLUT2 in heterozygous and homozygous VGLUT2 knock-outs led to a graded reduction in the amplitude of the postsynaptic response to single-vesicle fusion in thalamic neurons, indicating that the vesicular VGLUT content is critically important for quantal size and demonstrating that VGLUT2-mediated reduction of excitatory drive affects specific forms of sensory processing.

Identifying Modulators of hERG Channel Activity Using the PatchXpress® Planar Patch Clamp

The authors used the PatchXpress® 7000A system to measure compound activity at the hERG channel using procedures that mimicked the “gold-standard” conventional whole-cell patch clamp. A set of 70 compounds, including hERG antagonists with potencies spanning 3 orders of magnitude, were tested on hERG302-HEK cells using protocols aimed at either identifying compound activity at a single concentration or obtaining compound potency from a cumulative concentration dependence paradigm. After exposure to compounds and subsequent washout of the wells to determine reversibility of the block, blockade by a reference compound served as a quality control. Electrical parameters and voltage dependence were similar to those obtained using a conventional whole-cell patch clamp. Rank order of compound potency was also comparable to that determined by conventional methods. One exception was flunarizine, a particularly lipophilic compound. The PatchXpress® accurately identified the activity of 29 moderately potent antagonists, which only weakly displace radiolabeled astemizole and are false negatives in the binding assay. Finally, no false hits were observed from a collection of relatively inactive compounds. High-quality data acquisition by PatchXpress® should help accelerate secondary screening for ion channel modulators and the drug discovery process

Translation of a Tumor Microenvironment Mimicking 3D Tumor Growth Co-culture Assay Platform to High-Content Screening

For drug discovery, cell-based assays are becoming increasingly complex to mimic more realistically the nature of biological processes and their diversifications in diseases. Multicellular co-cultures embedded in a three-dimensional (3D) matrix have been explored in oncology to more closely approximate the physiology of the human tumor microenvironment. High-content analysis is the ideal technology to characterize these complex biological systems, although running such complex assays at higher throughput is a major endeavor. Here, we report on adapting a 3D tumor co-culture growth assay to automated microscopy, and we compare various imaging platforms (confocal vs. nonconfocal) with correlating automated image analysis solutions to identify optimal conditions and settings for future larger scaled screening campaigns. The optimized protocol has been validated in repeated runs where established anticancer drugs have been evaluated for performance in this innovative assay.

Neurotrophic and Antileukemic Daphnane Diterpenoids from Synaptolepis kirkii

Biological assay guided fractionation of a dichloromethane extract of Synaptolepis kirkii led to the isolation of four new and five known daphnane-type diterpene orthoesters, whose structure was established by spectroscopic data. Full spectroscopic data of the new and known natural products are reported here for the first time. Pronounced neurotrophic and substantial antileukaemia activities of these compounds were found in in vitro assays.

Sustained synchronized neuronal network activity in a human astrocyte co-culture system

Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer’s disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal)function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases.

Phaedra, a Protocol-Driven System for Analysis and Validation of High-Content Imaging and Flow Cytometry

High-content screening has brought new dimensions to cellular assays by generating rich data sets that characterize cell populations in great detail and detect subtle phenotypes. To derive relevant, reliable conclusions from these complex data, it is crucial to have informatics tools supporting quality control, data reduction, and data mining. These tools must reconcile the complexity of advanced analysis methods with the user-friendliness demanded by the user community. After review of existing applications, we realized the possibility of adding innovative new analysis options. Phaedra was developed to support workflows for drug screening and target discovery, interact with several laboratory information management systems, and process data generated by a range of techniques including high-content imaging, multicolor flow cytometry, and traditional high-throughput screening assays. The application is modular and flexible, with an interface that can be tuned to specific user roles. It offers user-friendly data visualization and reduction tools for HCS but also integrates Matlab for custom image analysis and the Konstanz Information Miner (KNIME) framework for data mining. Phaedra features efficient JPEG2000 compression and full drill-down functionality from dose-response curves down to individual cells, with exclusion and annotation options, cell classification, statistical quality controls, and reporting.

Using Human iPSC-Derived Neurons to Model TAU Aggregation

Alzheimer’s disease and frontotemporal dementia are amongst the most common forms of dementia characterized by the formation and deposition of abnormal TAU in the brain. In order to develop a translational human TAU aggregation model suitable for screening, we transduced TAU harboring the pro-aggregating P301L mutation into control hiPSC-derived neural progenitor cells followed by differentiation into cortical neurons. TAU aggregation and phosphorylation was quantified using AlphaLISA technology. Although no spontaneous aggregation was observed upon expressing TAU-P301L in neurons, seeding with preformed aggregates consisting of the TAU-microtubule binding repeat domain triggered robust TAU aggregation and hyperphosphorylation already after 2 weeks, without affecting general cell health. To validate our model, activity of two autophagy inducers was tested. Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model, highly suitable to screen for compounds that modulate TAU aggregation.

Investigation of signalling cascades induced by neurotrophic synaptolepis factor K7 reveals a critical role for novel PKCε.

This study elucidates signalling cascades involved in the neurotrophic effects induced by an active compound of Synaptolepis kirkii, a plant that is used against snakebites and for treatment of epilepsy. The active compound of this plant, synaptolepis factor K7 (K7), is suggested to exert anti-tumoral and neurotrophic actions via modulation of PKC. In SH-SY5Y cells synthesis of the neuronal marker growth-associated protein 43 was increased upon 48h treatment with K7. Immunofluorescent staining of neurites revealed an increased neurite formation by synaptolepis factor K7. Short-term signal transduction events were followed at the level of extracellular-regulated kinase phosphorylation. Extracellular-regulated kinase (ERK) phosphorylation was transiently increased upon stimulation with synaptolepis factor K7 (300nM) with a maximal effect at 30min. Use of the general PKC inhibitor bisindolylmaleimide I blocked the K7-induced ERK phosphorylation suggesting involvement of PKC. Conversely, inhibition of conventional PKCs, α, β and γ by treatment with Go6976 did not inhibit ERK phosphorylation up to 1μM. Use of a specific-PKCε translocation inhibitor peptide or RNAi-mediated knockdown of PKC-epsilon (ε) abolished the K7-induced ERK phosphorylation implicating PKCε in K7 function. This was confirmed by the observed increase in PKCε translocation and autophosphorylation induced by the compound. These data show that synaptolepis factor K7 induces neuronal differentiation of SH-SY5Y cells concomitant with a transient increase in ERK phosphorylation that is mediated by activation of PKCε.