amyloid beta peptides exhibit functional ...

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During the course of this work E. Kirazov was recipient of an Alexander von Humboldt .... [7] Butterfield D. A., S. M. Yatin, S. Varadarajan, T. Koppal. Meth. Enzy-.
Доклади на Българската академия на науките Comptes rendus de l’Acad´emie bulgare des Sciences Tome 61, No 7, 2008

BIOLOGIE Neurobiologie

AMYLOID BETA PEPTIDES EXHIBIT FUNCTIONAL NEUROTOXICITY TO CORTICAL NETWORK CULTURES Evgeni Kirazov, Ludmil Kirazov, Olaf Schroeder ∗ , Alexandra Gramowski∗∗ , Evgenija Vassileva, Cyrill Naydenov∗∗∗ , Dieter Weiss∗∗ (Submitted by Corresponding Member R. Radomirov on April 16, 2008)

Abstract Amyloid beta (Aβ) peptides have been suggested to play an essential role in the generation of Alzheimer’s disease (AD). In spite of the abundance of findings the exact correlation of Aβ and AD remains unclear. Aβ could influence cognition by impairment of basic neurotransmission mechanisms without causing directly neuronal death. This view is supported by the hypothesis of Wang et al. [1] that Aβ may produce dementia by disrupting neuronal signalling prior to cell death. Although there are many reports on neurotoxic effects of amyloid beta peptides, some questions remain still open. In this paper we describe the acute effects of two amyloid beta peptides (1– 40 and 1–42) and the biologically active fragment 25–35 on the electrical activity of cortical network cultures on microelectrode arrays (MEA-neurochips). Key words: amyloid beta peptides, neurotoxicity

Aβ has been shown to bind to a number of neuronal membrane proteins and receptors. On neurons these are the N-methyl-D-aspartate receptor, integrins, α7 nicotinic acetylcholine receptor, α4 nicotinic receptor, P75 neurotrophin receptor and the receptor for advanced glycation end products (see e.g. [ 2–4]). Aβ also binds to a number of receptors expressed in glial membranes (for recent review, see [ 5]). Therefore it is not surprising that Aβ can affect synaptic function and plasticity. Researchers have only recently begun to address the mechanisms by which During the course of this work E. Kirazov was recipient of an Alexander von Humboldt Foundation grant.

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Aβ peptides impair synaptic function. Such studies are focused on identification of the cell surface receptors and the signalling pathways mediating Aβ-induced synaptotoxicity. We studied the acute effects of two amyloid beta peptides (1–40 and 1–42) and the biologically active fragment 25–35 on the electrical activity of cortical network cultures on MEA-neurochips characterized by 200 quantitative parameters. With this approach a precise assessment of the influence of the amyloid beta peptides is possible. Our data showed a more pronounced activity decline in response to the application of fragments 25–35 and 1–42, with a significant decrease at 25 nM as the lowest concentration tested. However, 1–40 reveals a stronger inhibition and biphasic neuroactive effect at low concentrations with an EC50 at 15 µM. All effects were observable within 3–4 min after application and were reversible upon medium change. Material and methods. Experiments were carried out on MEAs from CNNS, Denton, TX, USA. For descriptions of the techniques used to produce microelectrode arrays and for obtaining long term cell neuronal networks, see [ 6]. MEAs are glass substrates (5 × 5 cm) with thin indium tin oxide conductors and a central 0.6 × 0.6 mm2 large 8 × 8 matrix of 64 electrodes. Data recording and spike sorting were performed with Multichannel Acquisition Processor Systems (Plexon Inc., Dallas, TX, USA). The Multichannel Acquisition Processor System receives the preamplified neuronal signals from 64 electrodes and provides gain, filtering and digitisation (40 kHz). The Digital Signal Processing System allowed the collection of waveforms, spike signal detection (maximum of 32 recording channels), and separation of up to four different units (neurons) per channel. Frontal cortex cells from embryonic day 14–16 NMRI mice were seeded onto MEA surfaces. The networks show spontaneous activity which stabilizes in terms of coordinated spike and burst patterns after four weeks. Such networks remain spontaneously active and pharmacologically responsive for more than 12 months. The age of the cultures used here ranged from days in vitro (div) 28 to div 68. For each cell culture, native activity and several episodes with increasing concentrations of the test substances were obtained for spike train analysis. All Aβ peptides were applied to the networks after recording a stable phase of native activity for at least 20 minutes. Preparing the solutions of Aβ we took special precautions to preserve its ability to cause oxidative stress [ 7] and prevent the formation of Aβ fibrils, keeping the peptide in its monomeric form [8]. NeuroProof (NeuroProof GmbH, Rostock, Germany) has optimized the technology to study electrical activity patterns of neuronal networks on MEA-neurochips as functional screening. Despite their complexity, the neurophysiological action profiles of neuroactive compounds are sensitive and selective as well as robust and stable, allowing a precise pharmacological “fingerprinting” and the 906

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establishment of a database derived from data on well-characterized neuroactive substances. The complex description of the changes caused by treatment of a neuronal network co-cultured with glia allows a sophisticated approach to quantify the complex effects of neuroactive agents, of unknown compounds, and of complex mixtures. With the technology framework Pattern Expert, an advanced data analysing tool for pattern recognition, the influence of compounds on electrical activity patterns can be studied and interpreted. Furthermore, it was shown that every compound has its own fingerprint of changing the network activity pattern in a specific way [9–11]. For each of the stable episodes we computed more than 200 parameter values which describe the general spike train activity, the activity in bursts, the burst shape, the regularity of oscillation and the network synchronicity. With feature selection and score methods from pattern recognition theory we selected the best suited parameters. For those four general categories, we found the following parameters with the highest relevance: • Burst shape: the average duration of plateaus in bursts (burst plateau); • Synchronisation: the number of coordinated spikes within 1 ms (simplex); • General activity in bursts: mean burst amplitude (burst amplitude); • General spiking activity: mean spike rate (spike rate). Results and discussion. Aβ1-40, Aβ1-42 and Aβ25-35 decrease the spike rate with increased concentration. We see an EC50 with about 15 µM for all three substances. Although we have a significant difference for the Hill Coefficient nH. The course of the concentration response curve differs from an ideal sigmoidal curve. The effects of all Aβ peptides were readily reversible on removal of Aβ by consecutive medium changes. Employing the data analysing tool for pattern recognition – Pattern Expert, we compared the influence of four “fingerprinted” compounds to the effects of Aβ peptides. The compounds are as follows: baclofen (a direct agonist of GABA B receptors), levetiracetam (opposes the activity of negative modulators of GABAand glycine-gated currents and partially inhibits N-type calcium currents in neuronal cells), fentanyl (a strong agonist at µ- and kappa-opiate receptors), and diazepam (a GABAA receptor agonist). The effects of the first three compounds on cultured neuronal networks are shown in Fig. 1. Pattern recognition and classification experiments indicate a similarity of the effects of Aβ peptides with GABAA receptor agonists such as diazepam, clonazepam and propofol (Fig. 2). We observe a course of cessation, which is known for example from GABAA agonists. This could lead to the hypothesis of the involvement of at least one ion-channel receptor mechanism, as it is controversially discussed in literature. These results lead us to the hypothesis that the rapid neurotoxic effect of 6

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Fig. 1. Selected spike train parameters for baclofen, levetiracetam and fentanyl. Native activity = 100%; n = 14 for all compounds

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Fig. 2. Selected spike train parameters for diazepam, Aβ1–40 and Aβ1–42. Native activity = 100%; for diazepam n = 14, for Aβ1–40 n = 10, for Aβ1–42 n = 12

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increased amyloid beta peptide concentrations on the synaptic function could cause the early memory loss in Alzheimer’s disease, preceding the synaptic loss and neuron degeneration. By hypothesis this mechanism seems to be a similar mechanism as it is described in literature for amnesia caused by benzodiazepine treatment. It is not clear if the concentration-response curve has a biphasic course, indicating the effects of several mechanisms of actions of the applied substance; this has to be studied in a future work.

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NeuroProof GmbH Rostock, Germany



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Department of Chemistry and Biochemistry Medical University of Sofia 1431 Sofia, Bulgaria

E. Kirazov, L. Kirazov, O. Schroeder et al.