Wyniki wyszukiwania

1

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

100%

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

Acta Neurobiologiae Experimentalis

|

2017

|

tom 77

|

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.

2

SRF regulates the expression of genes that may control epilepsy

100%

Kalita K., Kuzniewska B., Nader K., Dabrowski M., Kaczmarek L.

Acta Neurobiologiae Experimentalis

|

2015

|

tom 75

|

nr Supl.

BACKGROUND AND AIMS: Although the transcription factor serum response factor (SRF) has been suggested to play a role in activity-dependent gene expression and mediate plasticity-associated structural changes in the hippocampus, no solid evidence has been provided for its role in brain pathology. A genome-wide program of activity-induced genes that are regulated by SRF also remains unknown. RESULTS: In the present study, we showed that the inducible-conditional deletion of SRF in the adult mouse hippocampus increased the epileptic phenotype in the kainic acid model of epilepsy, reflected by increase in the susceptibility to spontaneous seizure development and more severe seizures. Moreover, we observed a robust decrease in activity-induced gene transcription in SRF knockout mice at 6 hours after kainic acid injections. We characterized the genetic program controlled by SRF in neurons and found that SRF target genes are associated with synaptic plasticity and epilepsy. Several of these SRF targets function as regulators of inhibitory/excitatory balance and the structural plasticity of neurons. We also identified novel direct SRF targets in neurons: Npas4, Gadd45g, and Zfp36. CONCLUSIONS: Altogether, our data indicate that proteins that are highly upregulated by neuronal stimulation, identified in the present study as SRF targets, function as endogenous protectors against overactivation by increasing the level of inhibition or modulating dendritic spine number and morphology. Thus, the lack of these effector proteins in SRF knockout animals may lead to uncontrolled excitation and eventually epilepsy.

3

Increased vulnerability to oxidative stress in lymphocytes from SAD and FAD patients

100%

Bialopiotrowicz, Kuzniewska B., Kuchamakova N., Kuznicki J., Wojda U.

Acta Neurobiologiae Experimentalis

|

2009

|

tom 69

|

nr 3

Alzheimer’s disease (AD) is the most common neurodegenerative disorder in which certain molecular changes are observed not only in neurons but also in peripheral cells. Growing evidence suggests that AD post-mitotic neurons exhibit increased apoptotic response to oxidative stress, mitochondria dysfunction and calcium dyshomeostasis. We hypothesized that some of these alterations could be observed in peripheral lymphocytes and studied for potential diagnostic purposes. We analyzed apoptotic response to the redox stress evoked by 2-deoxy-Dribose in immortalized lymphocytes from 18 patients with sporadic AD (SAD), from 2 familial AD (FAD) patients with novel mutations in presenilin 1: P117R and I213F, and from 20 agematched healthy individuals. Using two independent fl ow cytometry methods for quantifi cation of apoptosis, we found that SAD and FAD lymphocytes show enhanced apoptotic response to the redox stress. This apoptotic response was accompanied by decline in mitochondrial membrane potential measured with JC-1 as well as by increased activities of caspase 9 and caspase 3. However, no changes in the expression of two calcium-binding proteins: calmyrin 1 and calreticulin were observed. This study emphasizes that increased susceptibility to redox stress and associated upregulation of mitochondrial apoptotic pathway is characteristic not only for AD neurons, but also for AD lymphocytes. Thus, human lymphocytes could be used in further studies on AD pathogenesis.

4

Fragile X mental retardation protein regulates synaptic translation of neuroligins

100%

Podsiadlowska J. J., Kuzniewska B., Milek J., Urbanska K., Dziembowska M.

Acta Neurobiologiae Experimentalis

|

2017

|

tom 77

|

nr Suppl.1

INTRODUCTION: Neuroligins (NLGNs) are postsynaptic cell adhesion proteins which bind to their presynaptic partners neurexins across the synaptic cleft. Thus, NLGNs are crucial for the formation, maturation and maintenance of synapses. In rodents, neuroligins are encoded by four genes: Nlgn1, Nlgn2, Nlgn3 and Nlgn4. The mutations in Nlgn3 and Nlgn4 genes are associated with autistic phenotype. Another cause of autistic behaviors, fragile X syndrome, results from the lack of fragile X mental retardation protein (FMRP). FMRP binds to neuronal mRNAs and regulate local translation of transcripts that play an important role in synaptic signaling and plasticity. AIM(S): We aimed to determine if synaptic translation of Nlgn1, Nlgn2 and Nlgn3 mRNAs is regulated by FMRP. METHOD(S): We used Fmr1 knock-out mice (Fmr1 KO) and their wild type (WT) littermates to isolate synaptoneurosomes, which were stimulated in vitro to induce local protein synthesis. We performed FMRP IP on synaptoneurosomes and FISH combined with FMRP immunostaining on cultured neurons to investigate Nlgns mRNAs interaction with FMRP. The polyribosome fractionation was used to elucidate if FMRP regulates Nlgns mRNAs local translation. To study the surface versus intracellular NLGNs distribution at WT and Fmr1 KO synapses we have chosen chemical crosslinking and biotinylation assays, followed by Western blotting. RESULTS: We show that mRNAs for three studied neuroligins interact directly with FMRP in synaptoneurosomes and Nlgn1, Nlgn2, Nlgn3 mRNAs colocalize with FMRP in dendritic granules of cultured hippocampal neurons. The Nlgn1, Nlgn2 and Nlgn3 mRNAs associate with translating polyribosomes in response to synaptic stimulation and Fmr1 KO mice exhibit upregulated local translation due to the lack of FMRP. Finally, the excessive local synthesis of NLGN proteins at Fmr1 KO synapses leads to their elevated level on the postsynaptic membrane. CONCLUSIONS: Nlgn1, Nlgn2 and Nlgn3 mRNAs are locally translated at the synapse and FMRP regulates this process. FINANCIAL SUPPORT: Supported by NCN grant Sonata Bis 2014/14/E/NZ3/00375.

5

Serum response factor (SRF) regulates matrix metalloproteinase-9 (MMP-9) transcription in neurons

86%

Kuzniewska B., Blazejczyk M., Malik A., Jaworski J., Kaczmarek L., Kalita-Bykowska K.

Acta Neurobiologiae Experimentalis

|

2011

|

tom 71

|

nr S

MMP-9 is an endopeptidase playing important role in neuronal plasticity. Although multiple factors regulating MMP-9 expression have been described in different cell types, the molecular mechanism directly controlling its transcription in neurons remains poorly understood. The aim of the current study was to determine, whether SRF/c-Fos pathway is involved in the transcriptional regulation of MMP-9 in neurons. Real-Time PCR analysis revealed strong upregulation of MMP-9 mRNA levels after stimulation of rat primary cortical neurons with BDNF. Additionally, elevated MMP-9 gelatinolytic activity was observed. To investigate mechanism of MMP-9 promoter regulation, we used luciferase gene reporter assay system in which luciferase gene is controlled by MMP-9 promoter fragment (-1369/+35). Treatment of neurons with BDNF led to MMP-9 promoter activation, that was dependent on ERK1/2 actvity, as demonstrated using selective inhibitor or overexpressing constitutively active MKK1. As in MMP-9 promoter there are two AP-1 binding sites, we investigated whether AP-1 contributes to the BDNF-mediated MMP-9 transcription in neurons. MMP-9 reporter construct was induced upon overexpression of different AP-1 dimers in neurons, the most potent being those containing c-Fos. Moreover, BDNFinduced activation of the MMP-9 reporter construct was reduced if proximal, but not distal, AP-1 binding site was mutated. Furthermore knocking-down c-Fos expression in neurons by shRNA decreased MMP-9 gene activation in response to BDNF. As c-fos gene is a known target of SRF, we tested whether SRF can contribute to MMP-9 transcription. Inhibition of SRF by the overexpression of dominant-negative mutant of SRF or using shRNA targeting SRF, abolished BDNF-induced activation of MMP-9 promoter. Our data indicate that MMP-9 expression in neurons can be induced by BDNF. The signal propagation could involve ERK1/2 pathway and SRF-mediated transcription of c-fos gene resulting in activation of MMP-9 promoter.

6

Cell cycle malfunctions in lymphocytes from sporadic Alzheimer’s disease patients

86%

Bialopiotrowicz E., Kuzniewska B., Kochamakova-Trojanowska N. M., Barcikowska M., Kuznicki J., Wojda U.

Acta Neurobiologiae Experimentalis

|

2011

|

tom 71

|

nr 1

Alzheimer’s disease (AD) constitutes one of the leading causes of disability with enormous socioeconomic costs. Most of AD cases occur sporadically and have unknown etiology (SAD). The Abeta peptide deposition in neuritic plaques is one of AD major hallmarks. Nevertheless, the role of Abeta as a primary driver in AD progression arouses controversy. Mounting evidence suggests that neurodegeneration results from the cell cycle reentry occurring in AD neurons. Recent data indicate that some of the cell cycle changes can be also observed in peripheral cells. Thus, our aim was to investigate whether any cell cycle abnormalities occur in transcriptome or proteome of lymphocytes from SAD patients. This study was performed in immortalized lymphocytes from 18 SAD patients and 26 healthy age-matched individuals. PCR arrays experiments showed that 43% of the 90 investigated cell cycle genes were down-regulated in SAD, whereas 4% were up-regulated comparing to control lymphocytes. Since most significant changes referred to the genes engaged in G1/S control, we assessed the levels of key proteins involved in G1/S transition with immunobloting. The most striking difference occurred in p21 protein level, which was significantly elevated for SAD in respect to controls. Furthermore, we measured distribution of cells in G1, S and G2/M cell cycle phases using flow cytometry. Our results showed increased % of cells in G1 phase with adequate decrease in % of cells in S phase for SAD lymphocytes. However, estimation of proliferation rate for SAD and control lymphocytes revealed no significant differences. We therefore used the following methods to assess the lengths of G1 and S phases: flow cytometry analysis of PI-labeled cells after nocodazole treatment and BrdU pulse chase labeling. SAD lymphocytes indicated significant prolongation of G1 phase and simultaneous shortening of S phase. In addition, treating the cells with gamma-secretase inhibitor L685,458 did not affect the observed cell cycle dysregulations, which highlighted that the impairments of cell cycle in SAD lymphocytes are not linked to gamma-secretase activity. Taken together, this study emphasizes that disturbances in the cell cycle control are common for AD neurons as well as SAD lymphocytes. Thus, human lymphocytes could be used in further studies on AD pathogenesis and diagnostics

7

MiR-132 regulates MMP-9 protein levels in vivo by targeting its 3’UTR region

86%

Kuzniewska B., Gewartowska O., Jasinska M., Gruchota J., Rejmak K., Dziembowski A., Dziembowska M.

Acta Neurobiologiae Experimentalis

|

2017

|

tom 77

|

nr Suppl.1

INTRODUCTION: MicroRNAs (miRNAs) are small noncoding RNAs that bind to target sites in mRNAs, leading to translational repression. MiRNAs are present in dendrites and synapses where they are believed to fine‑tune the local expression of synaptic proteins. MiR-132 is a neuronal activity-regulated microRNA that controls the morphology of dendritic spines and synaptic transmission. Similar activities have recently been attributed to matrix metalloproteinase-9 (MMP-9), an extrasynaptic protease. Our previous studies show that miR-132 can directly regulate Mmp-9 mRNA by targeting its 3’UTR in cultured primary neurons. AIM(S): In the current study, we aimed at verification whether miR-132 regulates the expression of Mmp-9 in vivo in the mouse brain. METHOD(S): To determine whether miR-132 binds to the 3’UTR of Mmp-9 mRNA, the luciferase reporter assay using the coding sequence of firefly luciferase fused with the 3’UTR of Mmp-9 mRNA. Next, CRISP-Cas9 technology was used, in order to introduce mutations in putative binding site for miR-132 in 3’UTR of Mmp-9 locus in mice. Subsequently, gelatin zymography was used to evaluate the levels of MMP‑9 protein in different brain regions of mutant and control mice. RESULTS: Overexpression of miR-132 in cortical neurons significantly reduced the luciferase activity of MMP‑9 3’UTR reporter. Importantly, miR-132 failed to regulate the mutated MMP‑9 3’UTR luciferase reporter, confirming the functionality of the predicted sequence within the 3’UTR of MMP-9. Mutation in 3’UTR region of MMP-9 targeted by miR-132 in mice, resulted in higher MMP-9 protein levels in different brain regions of mutant mice as compared to controls. CONCLUSIONS: We show, that miR-132 binds to the 3’UTR of Mmp-9 mRNA in primary cortical neurons. Moreover, we developed a new mouse model to study miR-132 – Mmp-9 interaction in vivo. Our data suggest, that miR-132 targets 3’UTR of Mmp-9 mRNA in vivo and can regulate MMP-9 protein in mouse brain. FINANCIAL SUPPORT: Supported by NCN OPUS 2014/15/B/NZ3/01054.

Ograniczanie wyników

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

SRF regulates the expression of genes that may control epilepsy

Increased vulnerability to oxidative stress in lymphocytes from SAD and FAD patients

Fragile X mental retardation protein regulates synaptic translation of neuroligins

Serum response factor (SRF) regulates matrix metalloproteinase-9 (MMP-9) transcription in neurons

Cell cycle malfunctions in lymphocytes from sporadic Alzheimer’s disease patients

MiR-132 regulates MMP-9 protein levels in vivo by targeting its 3’UTR region