Electrophysiological Effect of uPA Inhibition on Synaptic Plasticity Through Modulation of the Extracellular Matrix

Event Abstract Back to Event Electrophysiological Effect of uPA Inhibition on Synaptic Plasticity Through Modulation of the Extracellular Matrix Sebastiaan Van De Vijver1*, Stephan Missault2, Jeroen Van Soom3, Pieter Van Der Veken3, Jurgen Joossens3, Stefanie Dedeurwaerdere2 and Michele Giugliano1 1 University of Antwerp, Theoretical Neurobiology and Neuroengineering Lab, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Belgium 2 University of Antwerp, Experimental Laboratory of Translational Neuroscience and Otolaryngology, Faculty of Medicine and Health Sciences, Belgium 3 University of Antwerp, Laboratory of Medicinal Chemistry, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Belgium Motivation The extracellular matrix (ECM) is a critical regulator of synaptic plasticity1. Consequently, proteases hydrolysing the ECM's proteins (matrix metalloproteinases2,3; MMP) are expected to influence plasticity by remodelling the ECM. Indeed, MMPs have been implicated in, for example, axon extension and guidance during development4 and are linked to the structural effects of long term potentiation on synapses5,6. By degrading proteins with a role in stability, ECM remodelling allows new synaptic connections to be formed. Here, we investigate the effect of a key regulator of this process, urokinase plasminogen activator (uPA). uPA activates MMPs through its upstream action, permitting synaptic changes to occur5. Recent findings have indeed highlighted the role of the plasminogen activator system in shaping plastic changes in the hippocampal mossy fiber pathway7. We aim to investigate the role of uPA in neuronal plasticity through targeted inhibition. Our hypothesis is that by blocking uPA control, the ECM will remain in a 'stiffened' state that prevents plastic changes of the synapses. Due to the dynamic nature of synaptic connections and the interplay between activity and connectivity, we postulate that reducing plasticity will drive the network to a different electrophysiological state. Material and Methods Primary cortical cultures of mammalian neurons were prepared as in 8, according to guidelines on animal welfare. 60-electrode polyethyleneimine-coated MEAs (60MEA200/30iR-ITO-gr, MultiChannel Systems) were employed. The uPA inhibitor9, dissolved in DMSO, was added to cultures in two concentration steps. MEAs were recorded at DIV27-30 for three one-hour intervals: before, after adding uPA inhibitor (100 nM), and after raising the concentration (10 µM). Between phases, ten-minute pauses were incorporated. Control cultures were treated equally, except that the solution contained only DMSO. Analysis was performed using custom-written Matlab scripts (The MathWorks, USA) and QSpikeTools10. Results After spike detection, four features were extracted (Fig. 1). To check for toxicity, the number of active electrodes was compared, no significant effect of the uPA inhibitor could be found (p > 0.05, Mann-Whitney test). The effect on the network activity was assessed using the average firing rate and burst features, specifically burst duration and average bursting rate. At the highest concentration, the network bursting rate decreased by about 60% compared to control (p < 0.05, Mann-Whitney test). Discussion The results reveal that the uPA inhibitor indeed affects the neuronal network's characteristics. The underlying cause for the decrease in bursting frequency is currently under investigation. Impairment of synaptic changes by the ECM could, for example, lead to a change in functional connectivity or to an imbalance of excitation and inhibition, explaining the altered bursting frequency. Additional research is needed to correlate the effect to its interaction with uPA, since the compound also has an affinity for both kallikrein (KLK) 4 and 8 (IC50 range of 5 - 20 nM). The KLKs also function as proteases and are linked to synaptic plasticity11, potentially causing them to act in conjunction with uPA. Conclusion The extracellular matrix has been revealed to be a key player in the regulation of synaptic plasticity. How the different molecular entities that shape the ECM are involved in this regulation is largely unknown. Here, we investigated the effect of one of these regulators, uPA, on the ECM by studying changes in the electrophysiological features of cortical networks, as measured by MEAs. We found that uPA inhibitor indeed affects the network, however follow-up research is required to fully elucidate uPA's electrophysiological impact and larger role in regulating the ECM. References 1. Mukhina, I. V., Korotchenko, S. A. & Dityatev, A. E. Extracellular matrix molecules, their receptors, and extracellular proteases as synaptic plasticity modulators. Neurochem. J. 6, 89–99 (2012). 2. Kähäri, V. M. & Saarialho-Kere, U. Matrix metalloproteinases in skin. Exp. Dermatol. 6, 199–213 (1997). 3. Stamenkovic, I. Extracellular matrix remodelling: the role of matrix metalloproteinases. J. Pathol. 200, 448–464 (2003). 4. Galko, M. J. & Tessier-Lavigne, M. Function of an axonal chemoattractant modulated by metalloprotease activity. Science 289, 1365–1367 (2000). 5. Yoshida, S. & Shiosaka, S. Plasticity-related serine proteases in the brain. Int. J. Mol. Med. 3, 405–409 (1999). 6. Wright, J. W. & Harding, J. W. Contributions of matrix metalloproteinases to neural plasticity, habituation, associative learning and drug addiction. Neural Plast. (2009). 7. Wiera, G. & Mozrzymas, J. W. Extracellular proteolysis in structural and functional plasticity of mossy fiber synapses in hippocampus. Front. Cell. Neurosci. 9, 1–21 (2015). 8. Reinartz, S., Biro, I., Gal, A., Giugliano, M. & Marom, S. Synaptic dynamics contribute to long-term single neuron response fluctuations. Front. Neural Circuits 8, 71 (2014). 9. Joossens, J. et al. Small, potent, and selective diaryl phosphonate inhibitors for urokinase-type plasminogen activator with in vivo antimetastatic properties. J. Med. Chem. 50, 6638–6646 (2007). 10. Mahmud, M., Pulizzi, R., Vasilaki, E. & Giugliano, M. QSpike tools: a generic framework for parallel batch preprocessing of extracellular neuronal signals recorded by substrate microelectrode arrays. Front. Neuroinform. 8, 26 (2014). 11. Yousef, G. M., Kishi, T. & Diamandis, E. P. Role of kallikrein enzymes in the central nervous system. Clin. Chim. Acta 329, 1–8 (2003). Figure Legend Figure 1. Network characteristics of uPA inhibitor treated cultures (blue bars, n = 8) compared to control (grey bars, n = 3) at three distinct timepoints: before addition, at 100 nM and at 10 µM. Data is shown as mean and SEM. Figure 1 Acknowledgements We are grateful to Mr. D. Van Dyck and Mr. M. Wijnants for excellent technical assistance. Financial support from the 7th Framework Programme of the European Commission (“NEUROACT”, contracts no. 264872 and 286403), University of Antwerp (BOF2015), FWO (fellowship 11K3716N and research project GA00913N) and the BELSPO Interuniversity Attraction Poles Program (IUAP) is kindly acknowledged. Keywords: Extracellular Matrix, synaptic plasticity, cortical culture, urokinase plasminogen activator Conference: MEA Meeting 2016 | 10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016. Presentation Type: Poster Presentation Topic: MEA Meeting 2016 Citation: Van De Vijver S, Missault S, Van Soom J, Van Der Veken P, Joossens J, Dedeurwaerdere S and Giugliano M (2016). Electrophysiological Effect of uPA Inhibition on Synaptic Plasticity Through Modulation of the Extracellular Matrix. Front. Neurosci. Conference Abstract: MEA Meeting 2016 | 10th International Meeting on Substrate-Integrated Electrode Arrays. doi: 10.3389/conf.fnins.2016.93.00064 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: 22 Jun 2016; Published Online: 24 Jun 2016. * Correspondence: Dr. Sebastiaan Van De Vijver, University of Antwerp, Theoretical Neurobiology and Neuroengineering Lab, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Antwerp, Belgium, sebastiaan.vandevijver@uantwerpen.be 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 Sebastiaan Van De Vijver Stephan Missault Jeroen Van Soom Pieter Van Der Veken Jurgen Joossens Stefanie Dedeurwaerdere Michele Giugliano Google Sebastiaan Van De Vijver Stephan Missault Jeroen Van Soom Pieter Van Der Veken Jurgen Joossens Stefanie Dedeurwaerdere Michele Giugliano Google Scholar Sebastiaan Van De Vijver Stephan Missault Jeroen Van Soom Pieter Van Der Veken Jurgen Joossens Stefanie Dedeurwaerdere Michele Giugliano PubMed Sebastiaan Van De Vijver Stephan Missault Jeroen Van Soom Pieter Van Der Veken Jurgen Joossens Stefanie Dedeurwaerdere Michele Giugliano 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.