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1

MMP-9 inhibitors as a new drug that blocks the development of epilepsy

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Rejmak-Kozicka E., Khomiak D., Kaczmarek L.
Acta Neurobiologiae Experimentalis
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2017
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tom 77
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nr Suppl.1
INTRODUCTION: Matrix metalloproteinase-9 (MMP-9) is a member of matrix metalloproteinase family that remodels the extracellular matrix. Recently, cumulative evidence indicates that MMP-9 is upregulated in experimental epilepsy models. Increased MMP-9 is also implicated in clinical epilepsy studies. AIM(S): Thus, we hypothesize that MMP-9 may be a novel therapeutic target for epilepsy and some agents, such as OAT1 or OAT2, may be potential antiepileptogenic drugs. METHOD(S): First we estimated IC50 of selected inhibitors using recombinant MMP‑9 and DQ gelatin, fluorescent substrate of MMP-9. Next, we estimated the level of cleavage of MMP-9 substrate – Nectin-3. To determine whether Nectin-3 cleavage might be blocked by MMP-9 inhibitors, we added to hippocampal neurons different concentrations of inhibitors upon 50 μM glutamate stimulation. Extracts from whole cell lysates were analyzed on Western blots. We also adapted gel zymography, method for estimating the level of MMP-9. Due to the MMP-9 low brain expression level and its secretion on the synapse upon neuronal stimulation we decided to inject mice with PTZ (50 mg/kg, 15 min). Next we added inhibitors of MMP-9 directly to developing buffer. RESULTS: IC50 value for OAT1 is 0,5 nM and for OAT2 is 13 nM. Glutamate-dependent stimulation of Nectin-3 cleavage was abolished only in the presence of OAT1 in 5 μM concentration in culture. Further, the gel zymography analysis showed inhibition of MMP-9 activity in the gel with OAT1 in the buffer. The inhibitor’s specificity towards MMP-9 was supported by the absence of MMP-2 activity in this probe, which is another abundant in the brain metalloproteinase showing gelatinase activity. CONCLUSIONS: This results strongly confirm that OAT1 is specific towards MMP‑9 and could be used in animal models of epileptogenesis. FINANCIAL SUPPORT: PBS3/A8/36/2015 from The National Centre for Research and Development.
2

Regulatoin of Nectin-3 processing in the hippocampus mediared by MMP-9 under the chronic stress conditions

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Rejmak-Kozicka E., Kalita K., Kaczmarek L.
Acta Neurobiologiae Experimentalis
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2015
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tom 75
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nr Supl.
BACKGROUND AND AIMS: Nectin-3 is Ca2+-independent immunoglobulin (Ig)-like cell-cell adhesion molecule (CAMs) that is involved in the organization of various types of intercellular junctions, including interneuronal synapses. How cleavage of nectin-3 is regulated in neuronal cells is poorly understood. Emerging evidence suggest a role for CAMs and extracellular matrix remodeling in mechanisms that underlie the behavioral effects of stress. We tested the hypothesis that nectin-3 is involved in hippocampal region-specific effects induced by chronic stress. METHODS: Adult male Spraque-Dawley rats were restrained 6 h/ day for 21 days in wire mesh restrainers in their home cages. In the experiments that involved Western blot and gel zymography analyses, the rats were sacrificed by decapitation and CA1 or CA3 of the hippocampal formation were collected for synaptoneurosomes preparation. RESULTS: The results show an increase in the nectin-3 cleavage in synaptoneurosomal fraction obtained from the hippocampal CA1 field stimulated with glutamate in the group of rats subjected to chronic stress procedure as compared to the control group. Proteolytic cleavage of the nectin-3 results in the appearance of a 20 kDa nectin-3 derived fragment. The cleavage was inhibited by the MMP-9 inhibitor (inhibitor I). Moreover, we shown that the increased nectin-3 proteolysis under chronic stress conditions correlates with the elevated MMP-9 activity in the rat hippocampal CA1 fragment. In addition, taking into account the possibility of non-specific activity of the inhibitor we used we demonstrated experimentally its specific activity towards MMP-9. To test the connection between the nectin-3 shedding and activity of the MMP-9 in vitro experiments were performed on the hippocampal cultures. CONCLUSION: Obtained results clearly confirm that this protease causes fragmentation of the nectin-3. Furthermore, it was shown that this process depends on the activation of NMDA receptor and the presence of Ca2+/calmodulin.
3

MMP-9 activity in animal model of traumatic brain injury

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Pijet B., Stefaniuk M., Rejmak-Kozicka E., Kaczmarek L.
Acta Neurobiologiae Experimentalis
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2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: Epilepsy in 20% of cases, develops as an effect of traumatic brain injury (TBI). Recent evidences indicate important role of extracellular matrix metalloproteinase-9 (MMP-9) in neuronal circuitry remodeling and synaptic plasticity. AIM(S): The aim of the present study was to evaluate the MMP-9 activity changes, dendritic spines density after brain injury and the influence of MMP‑9 expression level on spontaneous seizures appearance after TBI. METHOD(S): We used Controlled Cortical Impact (CCI) as a model of TBI in animals with altered MMP-9 levels (lacking: MMP-9 KO; overexpressing: MMP-9 OE) and their WT siblings. 12 weeks after CCI animals were subjected to continuous video-EEG monitoring. To verify MMP-9 changes after TBI: gel zymography and in situ hybridization were used. For dendritic spine alterations staining using liophilic dye DiI were performed. RESULTS: TBI resulted in progressive cortex (Cx) degeneration during 30 days after TBI. This effect was MMP-9 dependent. In MMP-9 KO animals degeneration volume degree was significantly smaller compared to wildtype siblings and MMP-9 OE mice. Gel zymography analysis showed time-associated elevation of MMP-9 activity in ipsilateral Cx and Hp, also in the thalamus samples during 3 days after CCI. Moreover, in situ hybridization showed increase of MMP-9 mRNA expression which reached the peak 6 hours post-CCI. Density of the dendritic spines measured 7 and 14 d after CCI was significantly decreased in ipsilateral Cx and CA1. EEG recordings with threshold test showed decreased seizure latency in MMP-9 OE mice while increased in MMP-9 KO. Interestingly total seizure number was the highest in MMP-9 OE animals. CONCLUSIONS: We described the correlation between TBI and MMP-9 activity and action post trauma. We indicated that MMP-9 might be an important factor for major dendritic spine reshaping, observed after brain injury, which in consequence may lead to altered sensibility of neuronal circuits to trigger seizures. FINANCIAL SUPPORT: This work was supported by PBS Program founded by The National Centre of Research and Development.
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