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3 Acta Neurobiologiae Experimentalis

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3’ untranslated region polymorphisms of matrix metalloproteinase 9 and their role in schizophrenia: the role in the local translation

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Lepeta K. , Dziembowska M. , Kaczmarek L.

nr Suppl.1

Recent studies have implicated MMP-9 in schizophrenia, in particular, Domenici et al reported highly elevated plasma levels of MMP-9 in schizophrenic patients and Rybakowski et al. demonstrated an association of MMP-9 5’UTR polymorphism -1562C/T with schizophrenia. Furthermore, Dziembowska et al. have shown that MMP-9 is locally translated in neurons in response to synaptic stimulation. Since 3’UTR plays essential role in mRNA transport to the dendrites and in its local translation, MMP-9 3’UTR polymorphisms may affect synaptic availability of the enzyme. In order to verify if SNPs affect local translation of MMP-9 or its mRNA transport we have made two types of vectors with human MMP-9 containing two 3’UTR variants as well as the inactive form of MMP-9 under human synapsin I promoter. The gene constructs enable MMP-9 protein visualization by its fusion to Venus fluorescent protein and additionally contain myristoylation sequence, which is responsible for the cell membrane docking. In result, locally translated MMP-9 at the synapse could be observed. Currently, we investigate if the polymorphism influences efficiency of MMP-9 mRNA transport and local translation under basal conditions or after stimulation. To enable MMP-9 mRNA tracking in the dendrites we will use MS2 system on living neurons under basal conditions and after stimulation of the two studied 3’UTR variants.

MMP-9 activation after neuronal stimulation is a polyadenylation dependent process

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Dziembowska M. , Janusz A. , Romanowska E. , Kaczmarek M.

nr 3

Recent studies indicate that MMP-9 (gelatinase B that regulates pericellular environment through the cleavage of protein components of the extracellular matrix) plays a role in synaptic plasticity. Szklarc zyk et al. (2002), Konopacki et al. (2008) and Wilczynski et al. (2008) have demonstrated the presence of mRNA and protein for MMP-9 at postsynaptic sides of rat hippocampal neurons. It was also shown by Michaluk et al. (2007) that gelatinolytic activity of MMP9 increases after stimulation of rat neuronal cultures with either glutamate or bicuculine. We observed the presence of MMP-9 protein and mRNA in synaptoneurosomes, the synaptic fraction isolated from hippocampus. To detect the activity of MMP-9 we measured the cleavage of its substrate, β-dystroglycan. By the use of this readout we showed that MMP-9 is activated 5 to 10 mintes after neuronal stimulation. We postulate here that MMP-9 is translated from dendritically-localized mRNA and the protein is produced in response to synaptic stimulation. Its rapid and local translation and secretion is a polyadenylation- and local translation-dependent process.

Abnormal AMPA receptor trafficking in a mouse model of fragile X syndrome

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Chojnacka M. , Beroun A. , Kuzniewska B. , Kaczmarek L. , Dziembowska M.

nr Suppl.1

INTRODUCTION: Fragile X syndrome (FXS) a common inherited form of mental retardation and autism is caused by lack of expression of fragile X mental retardation protein (FMRP). FMRP is RNA-binding protein that regulates local translation of many synaptic proteins, including AMPA-type glutamate receptors subunits. Accumulated evidence indicate that proper rates of exocytosis and endocytosis of glutamate receptors play a key role in synaptic plasticity. However, current state of knowledge of AMPA receptor trafficking in FXS models is incomplete. AIM(S): The aim of this study was to analyze AMPA receptor trafficking in a mouse model of fragile X syndrome. METHOD(S): We used synaptoneurosomes (SN) isolated from Fmr1 KO and wild-type (WT) mice and stimulated them in vitro with NMDA/glutamate. To determine levels of surface and intracellular GluR1, GluR2 and GluR3 we used crosslinking of SN with BS3 reagent followed by western blot analysis. To confirm our biochemical results we investigated the synaptic calcium-permeable AMPA receptors using whole-cell patch-clamp recordings. RESULTS: We found that SN stimulation produced an increase in the surface glutamate receptor subunits only in WT mice. We also found that surface GluR2 protein level was significantly higher in Fmr1 KO SN in basal conditions, when compared to WT. The electrophysiological experiments confirmed higher abundance of GluR2‑containing AMPA receptors in the hippocampus of Fmr1 KO mice. CONCLUSIONS: Our results indicate that Fmr1 KO mice exhibit abnormal AMPA receptor trafficking and it is demonstrated by elevated amount GluR2.