DP-b99 Modulates Matrix Metalloproteinase Activity and Neuronal Plasticity

et al. (2014) DP-b99 Modulates Matrix Metalloproteinase Activity and Neuronal Plasticity. PLoS ONE 9(6): e99789. doi:10.1371/journal.pone.0099789 DP-b99 Modulates Matrix Metalloproteinase Activity and Neuronal Plasticity Marine Yeghiazaryan 0 Izabela Rutkowska-Wlodarczyk 0 Anna Konopka 0 Grzegorz M. Wilczyn ski 0 Armenuhi Melikyan 0 Eduard Korkotian 0 Leszek Kaczmarek 0 Izabela Figiel 0 Effie C. Tsilibary, National Center for Scientific Research Demokritos, Greece 0 1 Department of Neurophysiology, The Nencki Institute of Experimental Biology, Warsaw, Poland, 2 Department of Molecular and Cellular Neurobiology, The Nencki Institute of Experimental Biology, Warsaw, Poland, 3 D-Pharm Ltd , Kiryat Weizmann Science Park, Rehovot , Israel , 4 Department of Neurobiology, Weizmann Institute , Rehovot , Israel DP-b99 is a membrane-activated chelator of zinc and calcium ions, recently proposed as a therapeutic agent. Matrix metalloproteinases (MMPs) are zinc-dependent extracellularly operating proteases that might contribute to synaptic plasticity, learning and memory under physiological conditions. In excessive amounts these enzymes contribute to a number of neuronal pathologies ranging from the stroke to neurodegeneration and epileptogenesis. In the present study, we report that DP-b99 delays onset and severity of PTZ-induced seizures in mice, as well as displays neuroprotective effect on kainate excitotoxicity in hippocampal organotypic slices and furthermore blocks morphological reorganization of the dendritic spines evoked by a major neuronal MMP, MMP-9. Taken together, our findings suggest that DP-b99 may inhibit neuronal plasticity driven by MMPs, in particular MMP-9, and thus may be considered as a therapeutic agent under conditions of aberrant plasticity, such as those subserving epileptogenesis. - Funding: This work has been supported by the 7FP EU grant Plasticise, ERANET NEURON grant TBI Epi, and grant no. N N301 665140 from National Science Centre Poland (IF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors declare that Armenuh Melikyan is an employee of D-Pharm and that D-Pharm provided the test compounds, free of charge, under a material transfer agreement, to the Kaczmarek group in Poland. D-Pharm has been, and is currently developing DP-b99 for acute ischemic stroke and acute pancreatitis, respectively. D-Pharm is currently sponsoring a small Phase 2 clinical trial in acute pancreatitis. D-Pharm and the Kaczmarek group were previously part of the FP7 funded Plasticise consortium, under whose auspices the collaboration originally started. This does not alter the authors adherence to PLOS ONE policies on sharing data and materials. DP-b99[1,2-Bis(2-aminophenoxy)ethane-N,N,NN-tetraaceticacid, N,N-di(octyloxyethyl ester), N,N-disodium salt, CAS Number: 222315-66-4] is a lipophilic, cell permeable diester, derivative of BAPTA that is able to selectively chelate transition metals such as zinc, copper, and iron within membranes [1]. DPb99 was initially developed by D-Pharm Ltd. as a neuroprotectant for acute ischemic stroke [2] and preclinical studies with radiolabeled DP-b99 in rats indicate that DP-b99 penetrates the rodent brain. This promising compound was undergoing evaluation in phase III clinical trials [3], but recent studies revealed lack of neuroprotective capacity for DP-b99 under those conditions [4]. However, despite the phase III MACSI trial failure, the drug may yet be efficacious in other indications. DP-b99 was previously shown to prevent zinc-induced neuronal cell death [5]. Zinc is considered as a key mediator and modulator of the neuronal death associated with transient global ischemia and sustained seizures [6], [7]. It was demonstrated that DP-b99 effectively attenuates microglial activation and diminishes the activity of zinc-dependent matrix metalloproteinase 9 (MMP-9) and tumor necrosis factor-a (TNF-a) - converting enzyme in vitro and in vivo [1], [8], [9]. Thus, it seems to be important to evaluate efficacy of DP-b99 in blocking MMP-mediated processes in the brain. Matrix metalloproteinases (MMPs) are a family of secreted and transmembrane endopeptidases that exert their function through cleavage of proteins of the extracellular matrix. All MMPs are active at neutral pH, require calcium for activity and contain a zinc atom within the catalytic domain [10]. MMPs are involved in numerous physiological phenomena, such as development, cell migration, learning and memory [11], [12]. They are also implicated in various pathophysiological processes and subsequent regenerative attempts, including cancer, epilepsy, stroke, inflammation, neurodegeneration and wound healing. Notably, one type of MMP, MMP-9, has recently been recognized as contributing to learning and memory, as well as synaptic plasticity, at the physiological and morphological levels [13], [14], [15], [16], [17].The association of MMP-9 with neuroplasticity has also been supported by studies involving MMP-9 knockout mice and transgenic rats overexpressing the enzyme. For instance, MMP-9 knockout mice showed decreased sensitivity to chemical kindling [18], [19], whereas transgenic rats overexpressing MMP-9 developed enhanced seizure progression [18]. Considering all of the above, we therefore postulate that the pharmacological inhibition of MMPs might be beneficial by suppressing seizure progression in response to chemical kindling. Kindling (repeated chemical stimulation of the brain, which inhibits GABAergic transmission in CNS) is widely accepted as a functional model of neuronal network activity, in which the altered Materials and Methods Animals A total of 14 wild-type male C57Bl/6 mice (3 months old at the start of the experiment), 2530 g, were used in the present study. All animals were housed in the same facility with a 12:12 h lightdark cycle in the animal house of the Nencki Institute of Experimental Biology. The experimental protocols were approved by the Local Ethical Committee on Animal Experiments of the Nencki Institute (Permit Number: 174/2011) and all efforts were made to minimize animal suffering and to decrease the number of animal used. PTZ Kindling Animals received a dose of 35 mg/kg PTZ (Sigma-Aldrich) intraperitoneally (i.p.), once every 48 hours till mice were kindled and sacrificed. After each PTZ injection, animals were placed into chambers and behavioral seizure activity was recorded within 30 min after administration. Seizure activity was classified according to the following scale (adapted from [21]: 0, no convulsive behavior; 1, facial movements; 2, rhythmic head movements, head nodding; 3, unilateral forelimb clonus; 4, bilateral forelimb clonus and rearing; 5, falling and clonic convulsions; 6, death. Mice were considered kindled when seizures at score 4 and 5 occurred after each PTZ injection for three consecutive days. Animals (...truncated)