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

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Kruk P., Olech-Kochanzyk G., Wilczynski G. M., Dzwonek J.

Acta Neurobiologiae Experimentalis

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2017

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tom 77

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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.

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CD44 adhesion molecule interacts with vesicular transport regulators in neurons

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Skupien A., Zareba-Koziol M., Dzwonek J.

Acta Neurobiologiae Experimentalis

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2017

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tom 77

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nr Suppl.1

INTRODUCTION: The role of extracellular matrix and its cellular receptors in acquisition of proper dendrite morphology in neurons has remained enigmatic. Previously we have shown that CD44 adhesion molecule, the main hyaluronan receptor, plays a crucial inhibitory role in dendritic tree arborization. Additionally, our results clearly demonstrate that CD44 defines the structure of Golgi apparatus, suggesting that CD44 may regulate dendritic arbor development by modulating the Golgi apparatus morphology and positioning AIM(S): The aim of our work is to find molecular partners of CD44 in neurons. METHOD(S): Immunoprecipitation and subsequent mass spectrometry analysis were used to unravel new CD44-interacting proteins in cortical neurons cultured in vitro. Obtained results were validated by Western Blot analysis. RESULTS: Mass spectrometry analysis pointed out several potential CD44-interacting partners in neurons. In the group of identified proteins we have distinguished many molecules involved in cellular vesicles transport. One of them is ERC2, the protein that belongs to Rab3-interacting molecule (RIM)‑binding protein family. We confirmed the results obtained by mass spectrometry by immunoprecipitation and Western Blot methods. ERC2 protein was co-immunoprecipitated with anti-CD44 antibody, but not with the IgG control antibody. CONCLUSIONS: We have shown for the first time, that CD44 interacts with ERC2 in neurons. These results suggest that the cellular mechanism underlying CD44-dependent modulation of dendritic tree development involves the regulation of cellular vesicular transport in neuronal cells. FINANCIAL SUPPORT: 2014/15/N/NZ4/01912.

3

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

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Konopka A., Swiech L., Jaworski J., Wilczynski G. M., Dzwonek J.

Acta Neurobiologiae Experimentalis

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2013

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tom 73

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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.

4

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

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Chwedorowicz A., Kaza B., Zareba-Koziol M., Konopka A., Wilczynski G. M., Dzwonek J.

Acta Neurobiologiae Experimentalis

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2017

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tom 77

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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.

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The role of adhesion molecule CD44 in dendritic tree arborization

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Dzwonek J., Gorlewicz A., Konopka A., Swiech L., Jaworski J., Wilczynski G.

Acta Neurobiologiae Experimentalis

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2011

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tom 71

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nr S

Dendritic arborization patterns define neuronal subtypes, and have important functional implications, determining how signals coming from individual synapses are integrated. Developing dendrites of neurons are responsive to extrinsic signals. Although several secreted proteins, cell surface receptors and adhesion molecules have been recently shown to be involved in dendrite morphogenesis, the role of extracellular matrix (ECM) components and molecular mechanisms of signal transduction from ECM to the neuronal cells involved in these processes are still poorly understood. The main component of the ECM in the brain is hyaluronan (HA). The major receptor for hyaluronan is CD44 adhesion molecule which mediates the response of cells to their extracellular microenvironment. The aim of this study was to investigate the role of CD44 in regulation of dendritic tree arborization. First, we examined the expression pattern of CD44 at the protein and RNA level in the rat brain by immunohistochemical, in situ hybridization and in situ PCR assays. All our experimental approaches clearly point to the neuronal localization of CD44, in addition to widely accepted presence in glia. Next, we investigated the role of CD44 in the hippocampal neurons cultured in vitro using the shRNA technology and anti-CD44 function-blocking antibody. The morphometric analysis show that cells with diminished expression of CD44 have more complex dendritic tree then control cells. Moreover, we have shown that treatment of neuronal cells with CD44 blocking antibody caused activation of small Rho GTPases (Cdc42, Rac1 and RhoA), which were previously shown to regulate dendritic tree arborization. These observations indicate that the members of small Rho GTPase family can be downstream effectors of CD44 in neuronal cells. The results of our experiments point to the importance of CD44 protein for the development of dendritic tree.

6

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

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Michaluk P., Kolodziej L., Mioduszewska B., Wilczynski G. M., Dzwonek J., Kaczmarek L.

Acta Neurobiologiae Experimentalis

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2007

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tom 67

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nr 3

7

Expression and function of extracellular matrix metalloproteinase-9 in human epilepsy

64%

Konopka A., Grajkowska W., Ziemianska K., Roszkowski M., Rysz A., Koperski L., Kaczmarek L., Wilczynski G., Dzwonek J.

Acta Neurobiologiae Experimentalis

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2011

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tom 71

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nr S

Focal cortical dysplasia (FCD) is a developmental brain disorder characterized by abnormalities of cytoarchitecture and neuronal morphology. FCD is associated with pharmacologically intractable forms of epilepsy in both children and adults. The mechanisms that underlie FCD-associated seizures are unclear. It is believed that a pathological plasticity, including abnormality of synaptic connections, plays the crucial role in this disease. Recent studies indicate the role of interactions between nerve cells and the extracellular matrix in the processes of plasticity. Matrix metalloproteinases are enzymes, which are able to degrade the extracellular matrix components, so they can play an important role in these interactions. Results of experiments using rodent models showed that extracellular matrix metalloproteinase-9 (MMP-9) can play an important role in epileptogenesis. There is no data demonstrating that MMP-9 is involved in the development of epilepsy in human. The aim of this study was to determine whether MMP-9 might play a role in FCD - related epilepsy. Expression of MMP-9 was investigated in human brain tissue derived from people suffering from epilepsy. The immunohistochemistry and antibody microarray methods were used. The control group consisted of the autopsy brain samples. The results indicate that the expression of MMP-9 in the human brain tissue with FCD is increased. The highest immunoreactivity occurs in cytoplasm of abnormal neurons. Moreover, among the 7 tested matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13), MMP-9 is present in greatest concentration in the FCD tissue homogenates. The results support the hypothesis of the possible role of MMP-9 in the development of human epilepsy and give an opportunity to develop new treatments.

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Synaptic remodeling depends on signaling between serotonin receptors and the extracellular matrix

64%

Wlodarczyk J., Bijata M., Labus J., Guseva D., Michaluk P., Rusakov D. A., Dityatev A., Dzwonek J., Ponimaskin E.

Acta Neurobiologiae Experimentalis

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2017

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tom 77

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nr Suppl.1

The rewiring of synaptic circuitry pertinent to memory formation in the brain has often been associated with morphological changes in dendritic spines and extracellular matrix (ECM) remodeling. Here, we linked these processes by uncovering the signaling pathway involving the serotonin 5-HT7 receptors (5 HT7R) the matrix metalloproteinase-9 (MMP-9), the hyaluronan receptor CD44, and the small GTPase Cdc42. We highlight a physical interaction between 5‑HT7R and CD44 (identified as a novel MMP 9 substrate in neurons) on the nanoscale, and find that 5-HT7R stimulation increases local MMP 9 activity triggering dendritic spines remodeling, synaptic pruning and impairment of long-term potentiation (LTP). The underlying molecular machinery involves 5-HT7R-mediated activation of MMP-9, which leads to CD44 cleavage followed by Cdc42 activation. Pharmacological/genetic suppression of this pathway rescues the 5-HT7R-induced synaptic changes and the deficit in LTP. Our results thus reveal causal interactions in a previously unknown molecular mechanism regulating neuronal plasticity. FINANCIAL SUPPORT: The work was supported by the National Science Centre (grant no. DEC-2012/06/M/ NZ3/00163), TANGO1/269352/NCBR/2015, Deutsche Forschungsgemeinschaft (grant no. PO732, excellence cluster REBIRTH), and ERA-NET Neuron/BMBF funding for the TargetECM project to E.P and A.D.

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Udzial regulacji ekspresji genow w procesach integracji informacji w komorkach nerwowych.

46%

Kaczmarek L., Kaminska-Kaczmarek B., Savonenko A., Lukasiuk K., Jaworski J., Biedermann I., Zielinski K., Werka T., Nikolaev E., Szklarczyk A., Dzwonek J., Kowalczyk A., Konopka D., Nowicka D., Rydz M., Filipkowski R., Figiel-Ozog I.

Działalność Naukowa PAN. Wybrane Zagadnienia

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2000

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tom 09

46-48

Ograniczanie wyników

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

CD44 adhesion molecule interacts with vesicular transport regulators in neurons

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

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

The role of adhesion molecule CD44 in dendritic tree arborization

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

Expression and function of extracellular matrix metalloproteinase-9 in human epilepsy

Synaptic remodeling depends on signaling between serotonin receptors and the extracellular matrix

Udzial regulacji ekspresji genow w procesach integracji informacji w komorkach nerwowych.