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1

Matrix metalloproteinase-9-a culprit in epilepsy?

100%
Wilczynski G. M.
Acta Neurobiologiae Experimentalis
|
2009
|
tom 69
|
nr 1
Temporal lobe epilepsy (TLE) is a chronic devastating disease in which aberrant synaptic plasticity plays a major role. Recently, MMP-9, a matrix metalloproteinase, has been implicated synaptic plasticity, long-term potentiation and learning and memory formation. Therefore, MMP-9 might play a pathogenic role in epileptogenesis. Indeed, the recent study revealed MMP-9 as a novel synaptic enzyme, and a key pathogenic factor in two distinct animal models of TLE: kainate-evoked-epilepsy and pentylenetetrazole (PTZ) kindling-induced epilepsy. In particular, sensitivity to PTZ-induced epileptogenesis is decreased in MMP-9 knockout (KO) mice, whereas it is increased in MMP-9-overexpressing rats. Moreover, confocal- and immunoelectron-microscopic analyses demonstrated that MMP-9 associates with hippocampal dendritic spines bearing asymmetric (excitatory) synapses. In addition, both MMP-9 protein levels as well as its enzymatic activity become strongly increased upon seizures. Furthermore, MMP-9-defi ciency diminishes seizure-evoked pruning of dendritic spines and, most importantly (with regards to epileptogenesis), it decreases aberrant synapse formation following mossyfi bers sprouting. Taken together, the aforementioned results suggest that the synaptic pool of MMP-9 is critically involved in the sequence of events that underlie epileptogenesis in two commonly used models of TLE.
2

Synaptic metalloproteinases in plasticity and epileptogenesis

75%
Wilczynski G. M.
Acta Neurobiologiae Experimentalis
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2007
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tom 67
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nr 3
3

The high resolution morphological study of MMP-9 and its mRNA in the rat hippocampus

51%
Konopacki F. A., Wilczynski G. M., Lasiecka Z., Kaczmarek L.
Acta Neurobiologiae Experimentalis
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2005
|
tom 65
|
nr 3
4

Activity-dependent rearrangement of the neuronal cell nucleus

51%
Szczepankiewicz A. A., Walczak A., Parobczak K., Wilczynski G. M.
Acta Neurobiologiae Experimentalis
|
2013
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tom 73
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nr Suppl.1
It is now firmly established that long-lasting synaptic plasticity involves dramatic changes in gene expression occurring under the influence of specific signaling pathways and transcription factors. Numerous studies have shown that DNA and histone epigenetic modifications play key roles in neuronal plasticity. Recent studies in non-neuronal cells, indicated the existence of epigenetic mechanism of yet another class, related to the nuclei structural remodeling and very poorly understood in neurons. Therefore, we decided to study the ultrastructure of the cell nuclei in the hippocampal dentate gyrus granule neurons upon seizures induced by kainic acid, an analog of glutamate. Under these conditions the granular neurons instead of degradation, undergo an intensive plasticity phenomena. We found that seizures led to rapid and dramatic enlargement and striking reorganization of internal component-structures of interchromatin granule clusters (IGCs) in granular cell’s nucleus. Moreover, unlike IGCs of control animals, the reorganized IGCs contained activated RNA polymerase II CTD phosphoepitopes. These observations may suggest involvement of IGC in activity-dependent transcription events in neurons.
5

Cre/lox transgenic mice as a tool to study CD44 function in the brain

51%
Kruk P., Olech-Kochanzyk G., Wilczynski G. M., Dzwonek J.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: The role of CD44 protein in astrocytes in physiological and pathological conditions in the brain remains largely unknown. To study its function the transgenic animal models were used. The CD44 knock-out mice are commercially available, however the compensatory effect of ICAM‑1 molecule forthe CD44 deficiency has been previously described. For that reason, we decided to create conditional knockout mice where we can control the time of gene silencing during animal development by tamoxifen (TAM) administration. Moreover, we generated a conditional overexpression mouse line in which the transgene overexpression is also initiated by TAM. By using CreERT2 fusion protein driven by GFAP (glial fibrillary acidic protein) promoter, we can achieve inducible astrocyte‑specific CD44 knock‑out/overexpression line in which CD44 gene becomes altered in astrocytes of the adult brain upon the tamoxifen-driven activation of Cre recombinase, at the chosen time point. This gives us an ability to change the CD44 expression after the mice reach adulthood. AIM(S): The aim of our work is to validate two conditional double transgenic mouse lines created with the use of Cre/lox system to study the function of CD44 protein in astrocytes. METHOD(S): For the comparison of Cre/lox activation efficacy, mice were injected with TAM either every 12 hours (10 mg/ml) or every 24 hours (20 mg/ml) for the duration of 5 constitutive days. Then, the effect of the transgene activation was validated using western blot and immunohistochemistry techniques. RESULTS: Validation studies confirm CD44 overexpression model works. CD44 overexpression can be seen in all hippocampus, cerebral cortex and cerebellum. Immunohistochemistry staining shows increased level of CD44 in astrocytes of cerebral cortex and hippocampus, especially in the molecular layer of dentate gyrus. CONCLUSIONS: Described inducible CD44 transgenic mouse lines are the first animal models that can help scientists study the yet undiscovered function of CD44 protein in astrocytes.
6

Semispecific TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) displays cytotoxic activity by induction of apoptosis, autophagy and protein aggregation in U937 cells

51%
Bialy L. P., Fayet J., Wilczynski G. M., Mlynarczuk-Bialy I.
Folia Histochemica et Cytobiologica
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2018
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tom 56
|
nr 4
7

Coiled bodies in the nuclei of undifferentiated crypt base cells in the human small intestine

51%
Wilczynski G. M., Rowinski J., Sulikowska-Rowinska A.
Folia Histochemica et Cytobiologica
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1999
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tom 37
|
nr 2
8

Extracellular matrix metalloproteinase inhibitor S24994 aboiishes LTP in CA1 field of rat's hippocampus

45%
Okulski P., Konopacki F., Balcerzyk M., Wilczynski G. M., Kaczmarek L.
Acta Neurobiologiae Experimentalis
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2005
|
tom 65
|
nr 3
9

Role of posttranslational modifications of tubulin in neuronal function

45%
Kasprzak A. A., Kliszcz B., Szkop M., Wloga D., Wilczynski G. M.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
The axon is a slender projection of a neuron that conducts electrical impulses to synapses in muscles or other neurons. A number of cellular components such as mitochondria and vesicles are transported along the axon by microtubule-based molecular motors (kinesins and dyneins). The most important factors controlling the axonal transport are the structure and function of the motors involved and the condition of the rails (the microtubule, MT). In the last decade, a compelling evidence has been provided that MTs contain marks (posttranslational modifications, PTMs) that indicate what kind of activity should take place in a given MT segment. These cues are read and interpreted by molecular motors, MAPS and other proteins. Kinesin-1, the major motor that transports cargoes along MTs is a homodimer with a pair of MT-binding sites on each end of the molecule. In some conditions, kinesin-1, besides interacting with MT using its N-terminal motor domains, can also bind another MT by its tail site producing sliding of one MT relative to another. This type of pair sliding can be used to sort MTs in the same way it occurs in the mitotic spindle and also act as an efficient way to move large amounts of tubulin, in the form of short MTs. Both activities have been observed in Drosophila cultured neurons. The effects of PTMs on the interaction of the motor domain with MT are to some extent characterized, but the effects of PTMs in the cargo-MT on the MT pair sliding have never been examined. Currently, we are exploring the impact of two well-known PTMs (detyrosination and polyglutamylation) and one recently reported (polyamination) on MT-MT sliding. To that end, we have developed an assay in which the sliding is observed in vitro and quantified by kymographic analysis. Surprisingly, the velocity of the sliding was highly variable along the track indicating that its efficiency may be sensitive to many cellular and developmental mechanisms. FINANCIAL SUPPORT: Supported in part by NCN Grant 2014/13/B/NZ1/03995.
10

Imaging method of neuronal nuclei in the mouse brain

45%
Czaban L., Broszkiewicz M., Magalska A., Ruszczycki B., Wilczynski G. M.
Acta Neurobiologiae Experimentalis
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2013
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tom 73
|
nr Suppl.1
Little is known about the relationship between neuronal gene expression and the architecture of the neuronal cell nucleus. We wanted to study the cell nuclei in neurons of the mouse cerebral cortex, using confocal microscopic immunocytochemistry. We used a segmentation algorithm, based on continuous boundary tracing, able to reconstruct the nucleus surface and to separate adjacent nuclei (Walczak et al. 2013). The algorithm did not use a rigid threshold what made it robust against variations in image intensity and poor contrast. However, when we analyzed mouse, but not rat, neuronal nuclei there have occurred a considerable problem with an appropriate segmentation. This problem was related to the presence of the discrete chromocenters, which are much more prominent in the mouse that in other species. Therefore, in order to assure the proper segmentation, we used sections co-immunostained for the lamin protein. Our refined program is an efficient segmentation tool for crowded and overlapping objects in 3D space, regardless of the particular species. It allows us to study quantitatively the architecture of the neuronal nucleus using confocal-microscopic approach.
11

The role of adhesion protein CD44 in the shape changes of astrocytes

45%
Konopka A., Swiech L., Jaworski J., Wilczynski G. M., Dzwonek J.
Acta Neurobiologiae Experimentalis
|
2013
|
tom 73
|
nr Suppl.1
CD44 is a widely distributed type I transmembrane glycoprotein and functions as the major hyaluronan receptor on most cell types. CD44 through interaction with actin cytoskeleton affects the transmission of signals from the outside to the inside of the cell in many tissues and organs. Primary cultures of astrocyte are diverse in their morphology and many factors can influence on it. In vivo astrocyte also are able to change their shape in response to various stimuli. The appearance of reactive astrocytes in vivo with thicker and longer processes and increased cellular content of glial fibrillary acidic protein (GFAP) has been observed in the CNS after various types of injury caused by physical, chemical, and pathological trauma. Furthermore, it has been showed that CD44 expression increases after brain injury. In our study we investigated the influence of knock down of CD44 by specific shRNA and CD44 overexpression on the astrocytes shape changes. Our results indicate that knock down of CD44 in astrocytes results in more regular and flat shape. In contrast the overexpression of CD44 promotes more irregular, radial-like shape of astrocyte. Our data support the hypothesis that CD44 plays role in morphological changes of astrocyte and give the opportunity to investigate its role in pathological processes such as brain injury.
12

The role of hippocampal PML in transgenic mouse circadian rhythm

45%
Olech-Kochanczyk G., Kiryk-Jaskiewicz A., Domanskyi A., Konopka W., Wilczynski G. M.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: Circadian clock is an evolutionarily well-conserved mechanisms in higher organisms. PML protein implicated in many important biological processes (response to DNA damage, cell division control) influences also circadian rhythm by regulating nuclear localization of Per2, a significant positive regulator of the clock transcriptional mechanisms. AIM(S): Our aim is to explain how overexpression or knock‑out of PML affect the oscillations of levels of proteins involved in circadian rhythm in hippocampus. METHOD(S): We generated transgenic mouse models with overexpression or knock-out of PML gene that are induced in specific time and location in brain. The project consists of two tasks: 1) generation of transgenic animals with overexpression of PML gene using AAV vectors encoding PML and mCherry reporter gene, and 2) generation of transgenic animals with knock-out of PML gene using AAV vectors encoding PML gene-targeting gRNAs and Cas9 (CRISPR/Cas9 system). RESULTS: We generated AAV vectors with PML-mCherry and microinjected them into the hippocampus of mice that were subject to behavioral tests (in the IntelliCage system). AAV PML-mCherry mice showed impairment in circadian activity 45 days after surgery and displayed proper spatial learning of cage corners with appetitive reinforcement and slower re-learning process. Next, we designed gRNAs for PML exon I and II and generated plasmid vectors containing gRNAs for the PML gene and tdTomato reporter. Using both these vectors and vectors containing Cas9 endonuclease fused with GFP reporter we transfected NIH 3T3 cells to induce PML gene knock-out in vitro. Next, using T7E1 endonuclease we confirmed that knock-out of PML works. Afterwards, we generated AAV vectors encoding PML gene-targeting gRNAs and Cas9 endonuclease. Both vectors were injected into the hippocampus. CONCLUSIONS: Thus far research on PML function in the circadian rhythm was usually performed in vitro. Our tools enable us to study the role of PML in mammalian molecular clock mechanisms in vivo.
13

The role of CD44 protein in synapse phagocytosis by astrocytes in vitro

39%
Chwedorowicz A., Kaza B., Zareba-Koziol M., Konopka A., Wilczynski G. M., Dzwonek J.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: CD44 adhesion molecule is highly expressed in astrocytes but its role in these cells is unknown. Recently a novel function of astrocytes in the brain in synapse phagocytosis has been described but the molecular mechanisms underlying these processes remain largely unknown. Phagocytic receptors MEGF10 and MERTK have been shown to be involved in this phenomenon. However, CD44 was shown to act as phagocytic receptor in macrophages. We hypothesized that CD44 can regulate phagocytosis in astrocytes and can be involved in synapse elimination. To investigate the role of CD44 in astrocyte-mediated synapse pruning, we used an in vitro engulfment assay, where astrocytes were cultured in the presence of synaptoneurosomes. We investigated whether altered CD44 expression level in astrocytes influence the efficiency of engulfement of synaptoneurosomes conjugated with a pH‑sensitive fluorescent dye. AIM(S): The aim of our work was to determine whether CD44 protein regulates synapse phagocytosis by astrocytes in vitro. METHOD(S): To check if CD44 regulates synapse phagocytosis by astrocytes we used primary astrocytes cultures in vitro that were transfected with specific CD44shRNA to knockdown CD44 expression or controls (empty pSuper). Astrocytes were cultured in the presence of synaptoneurosomes conjugated with pHrodo fluorescent dye. The amount of synaptoneurosomes engulfed by astrocytes were monitored in living cells with the use of confocal microscopy. Three-dimentional reconstructions of astrocytes and analysis of engulfed puncta were performed using Imaris software. Live imaging of transfected cells before and after incubation with fluorescently labaled synaptoneurosomes were performed. RESULTS: We observed significantly higher number of engulfed puncta inside CD44 depleted cells comparing to controls. CONCLUSIONS: Our results suggest that CD44 inhibits phagocytosis of synaptoneurosomes by astrocytes. FINANCIAL SUPPORT: The work was supported by the National Science Center grant No 2015/17/B/ NZ4/02540.
14

Arc functional neighbourhood in the neuronal cell nucleus

39%
Parobczak K., Rutkowska-Wlodarczyk L., Szczepankiewicz A. A., Khanema-Jakobsen T., Schubert M., Bramham C. R., Wilczynski G. M.
Acta Neurobiologiae Experimentalis
|
2013
|
tom 73
|
nr Suppl.1
Arc protein is a versatile factor connecting memory formation, plasticity changes and neuronal activity. Through regulation of actin polymerization, Arc contributes to synapse expansion and may furthermore influence synapse strength via management of AMPA receptor turnover. The function of Arc in the neuronal cell nucleus is poorly understood. In this work we performed structural, functional and biochemical analysis to identify Arc`s nuclear interactome. Using confocal microscopy we investigated Arc`s functional neighborhood and found that it occupied internal parts of the nucleus, closely to hnRNPs. This observation were confirmed with electron microscopy, which demonstrated that Arc localizes mainly at the peripheral areas of chromatin. Furthermore, pull-down-based biochemical experiments suggested that Arc interacts with splicing machinery. Collectively, our data suggest that nuclear Arc is involved in the gene expression phenomena.
15

Are early immediate genes activated in learninginduced plasticity?

39%
Radwanska A., Filipkowski R. K., Konopacki F. A., Wilczynski G. M., Zakrzewska R., Kossut M.
Acta Neurobiologiae Experimentalis
|
2007
|
tom 67
|
nr 3
16

Irregular nuclear shapes of neural stem cells in adult murine dentate gyrus

39%
Grymanowska A. W., Martin A., Olszynski K. H., Wilczynski G. M., Magalska A., Filipkowski R. K.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: In adult hippocampal neurogenesis, stem cells and their derivative progenitors are generated. They differentiate into neurons as they migrate from the subgranular zone to the granule cell layer of the dentate gyrus, maintaining homeostatic tissue regeneration and supporting brain plasticity. Depending on the stage of the neurogenesis, different subpopulations of cells of the neurogenic lineage can be distinguished, i.e. neural stem cells (NSC, type 1), intermediate progenitor cells (type 2a and type 2b), neuroblasts (type 3) and granule neurons (type 4). Little is known about the architecture of nuclei in these cells, while the cell nucleus is known to be highly organized with numerous functional and structural domains as well as dynamic organization of chromatin governed by epigenetic mechanisms which were shown to respond to external signals. AIM(S): We aimed to distinguish type 1 through 4 cells and investigate their nuclear shape. METHOD(S): Transgenic Nestin-GFP mice were used. Cell types were identified with immunohistochemistry and morphological features: type 1 (GFP+, one long neural process), type 2a (GFP+), type 2b (GFP+/DCX+), type 3 (DCX+), type 4 (NeuN+). Confocal microscopy was used to collect z‑stack files of the nuclei of different cell types. RESULTS: We observed irregularity in shape of the nuclei in type 1 cells (NSC) with the presence of nuclear envelope invaginations. When selected layers were analyzed, NSC nuclei turned out to have reduced circularity, roundness and solidity when compared with other cell types. CONCLUSIONS: The irregularity observed and nuclear envelope invaginations seem to be characteristics of the “stemness” as the shape of the nucleus becomes more regular with successive stages of neurogenesis. The biological significance of the observed phenomenon is not yet clear and further studies are necessary to better understand the process of adult neurogenesis at the nuclear level. FINANCIAL SUPPORT: The work was supported by National Science Centre, Poland, grant no. 2014/14/M/NZ4/00561.
17

Transcription factor Yin Yang 1 regulates in vivo MMP-9 expression in the rat hippocampusRylski

39%
Rylski M., Bielinska B., Saganek R., Konopacki F. A., Wilczynski G. M., Kaczmarek L.
Acta Neurobiologiae Experimentalis
|
2005
|
tom 65
|
nr 3
18

Beta-dystroglycan as a target for MMP-9, in response to enhanced neuronal activity

39%
Michaluk P., Kolodziej L., Mioduszewska B., Wilczynski G. M., Dzwonek J., Kaczmarek L.
Acta Neurobiologiae Experimentalis
|
2007
|
tom 67
|
nr 3
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