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1

The effect of tetrahydrocarbozoles on the ER calcium release and SOCE in medium spiny neurons from transgenic YAC128 mice, a model of Huntington's disease

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Maciag F., Czeredys M., Kuznicki J.
Acta Neurobiologiae Experimentalis
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2015
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tom 75
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nr Supl.
BACKGROUND AND AIMS: Huntington’s disease (HD) is a genetic neurodegenerative disorder caused by an extended polyglutamine tail in the huntingtin (HTT) protein and manifesting itself by neurodegeneration of medium spiny neurons (MSN) in the striatum. An early event in the pathology of HD is deregulated Ca2+ homeostasis (Giacomello et al. 2013). One of the mechanisms that regulate Ca2+ homeostasis is store-operated Ca2+ entry (SOCE), which was shown to be enhanced in HD (Wu et al. 2011). However, the mechanism by which mutated HTT affects SOCE is still unknown and there is no effective treatment of HD. RESULTS: Therefore, we assessed the alterations of the Ca2+ signalosome in the striatum of transgenic YAC128 mice, a model of HD (Czeredys et al. 2013). In MSN isolated from these mice we detected an about 10% increase in the basal Ca2+ level. We found that the activity of SOCE was enhanced about 30%. Since the deregulation of Ca2+ homeostasis and signaling is considered to be the primary event in HD, the aim of this work was to investigate the effect of compounds called tetrahydrocarbazoles on the ER Ca2+ release induced by mGluR1/5 receptor agonist, DHPG (3,5-dihydroxyglycine), as well as their influence on SOCE in MSN from YAC128 mice. It was previously shown that tetrahydrocarbazoles stabilize the ER Ca2+ release induced by carbachole in HEK293 cells overexpressing mutated presenilin 1, a cellular Alzheimer’s disease model (Honarnejad et al. 2014). We have confirmed by immunostaining that in our in vitro model of HD, MSN culture from YAC128 mice, mGluR1/5 receptors are specifically expressed. The MSN cells were treated with chosen tetrahydrocarbazoles and Ca2+ was imaged with Fura-2 AM. CONCLUSIONS: Our preliminary data suggest that some tested compounds have a stabilizing effect on Ca2+ level in HD cellular model. Tetrahydrocarbazoles could be potentially used as drugs stabilizing the disturbed Ca2+ homeostasis in Huntington’s disease.
2

Studies of CacyBP/SIP protein in beta-catenin dysregulation using m128 HD mice and the CacyBP zebrafish knockout

100%
Czeredys M., Romaszko A., Kuznicki J.
Acta Neurobiologiae Experimentalis
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2017
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tom 77
|
nr Suppl.1
INTRODUCTION: Mutated huntingtin has been shown to affect gene expression in brains of Huntington’s disease (HD) mice models. One of these genes is CacyBP/SIP encoding calcyclin-binding protein (CacyBP/SIP), which is 2-fold overexpressed in the striatum of YAC128 mice, a model of HD. A higher increase in the CacyBP/SIP dimer than in the monomer was found in the striatum of 3-month-old HD mice. Moreover, we detected a decrease in total protein ubiquitination, while the level of β‑catenin was higher in the striatum of HD transgenic mice as compared to wild-type mice. AIM(S): To determine the effect of increased dimerization of CacyBP/SIP protein in YAC128 model and the effect of decreased level of Cacybp in zebrafish on β‑catenin signaling. METHOD(S): Medium Spiny Neurons (MSNs) and glial cultures isolated from the striatum of YAC128 and wild‑type mice were used to analyze the level of β‑catenin and protein ubiquitination by western blotting. Proximity Ligation Assay (PLA) was used to study CacyBP/ SIP dimerization in these cultures. Zebrafish lines with knockout of cacybp were generated using CRISPR/Cas9 technology. RESULTS: We did not detect any changes in β‑catenin or total protein ubiquitination in MSN or glial cultures. We observed the presence of CacyBP/SIP dimers using PLA. Currently, we are studying if the increased level of CacyBP/SIP dimers affects β‑catenin and its ubiquitination in YAC128 MSNs. Preliminary data from the cacybp zebrafish knockout shows disturbances in β‑catenin protein level in total protein extracts. CONCLUSIONS: In MSNs and glial cells from YAC128 mice we did not find any changes in β‑catenin and protein ubiquitination, which were observed in the striatum of adult HD mice. Increased dimerization of CacyBP might disturb degradation of β‑catenin during aging in HD. The cacybp zebrafish knockouts will allow us to find out if Cacybp is involved in β‑catenin signaling and what are its other potential functions. FINANCIAL SUPPORT: This study was supported by The National Science Centre in Poland. Grant no. 2014/15/D/ NZ3/05181 for MCZ.
3

Dysfunction of store-operated calcium entry as an early event in the pathogenesis of neurodegenerative diseases

88%
Czeredys M., Gruszczynska-Biegala J., Schacht T., Methner A., Kuznicki J.
Acta Neurobiologiae Experimentalis
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2013
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tom 73
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nr Suppl.1
Store-operated calcium entry (SOCE) is a mechanism that regulates calcium influx from the extracellular space which affects calcium signalling in the cell and has been implicated with neuronal cell death. We hypothesized that SOCE might be altered at the early stages of Alzheimer’s (AD) and Huntington’s (HD) disease. We used PC12 cells with an inducible expression of mutated fulllength huntingtin as a cellular model of Huntington’s disease. Calcium measurements were performed by single cell imaging with the Fura-2. We found SOCE parameters were changed as a result of the expression of mutant huntingtin. We next investigated if these differences were caused by changes in the mRNA expression of genes involved in SOCE. Similar analyses are currently being conducted using mouse models of HD (YAC128) and AD (APP V717I) using custom-made TaqMan Low Density Arrays containing probes for genes involved in calcium homeostasis and signalling. Our preliminary results suggest that the expression of mutant proteins such as huntingtin or amyloid precursor protein affect the expression of selected components of calcium homeostasis and signalling pathways.
4

CacyBP/SIP and Beta-catenin homeostasis in the YAC128 mice model of Huntington’s disease

88%
Czeredys M., Latoszek E., Ludwiczak J., Dunin-Horkawicz S., Kuznicki J.
Acta Neurobiologiae Experimentalis
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2019
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tom 79
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nr Suppl.1
INTRODUCTION: We previously detected a 2-fold increase of CacyBP/SIP gene expression encoding calcyclin‑binding protein (CacyBP/SIP) and increased CacyBP/SIP dimerization in the striatum of YAC128 mice, a model of Huntington’s disease (HD). In these mice, mutated huntingtin is overexpressed in neurons. In agreement with the suggested role of CacyBP/ SIP in β‑catenin degradation, we observed a decrease in total protein ubiquitination and a higher level of β‑catenin in the striatum of HD mice as compared to wild‑type animals. AIM(S): To check if there is a relationship between the level of CacyBP/SIP dimerization in the YAC128 model and β‑catenin signaling and neuronal neurodegeneration. METHOD(S): To impair or increase the ability of CacyBP/SIP to dimerize, mutations in its coiled‑coil dimerization domain were computationally designed using the crystal structure as a template and the Rosetta energy function and molecular dynamics methods. Native gel electrophoresis was used to confirm the effects of mutations in dimer stabilization and destabilization. In cultures of medium spiny neurons (MSNs) isolated from the striatum of YAC128 mice, CacyBP/SIP mutants were overexpressed by lentiviruses and used to analyze the level of β‑catenin and its ubiquitination by immunoprecipitation and western blotting. RESULTS: In HD MSNs in which wild‑type CacyBP/SIP was overexpressed, the level of β‑catenin and its ubiquitination was unchanged relative to the control vector. The effects of mutations leading to increased (K21W and a double mutant T30R, S33E) or decreased ability of CacyBP/SIP to dimerize (D11A, E14A, and L18A) on β‑catenin homeostasis are being analyzed. CONCLUSIONS: CacyBP/SIP protein mutants with different abilities to dimerize allow us to establish the role of dimerization on the β‑catenin level, which might be related to HD pathology. FINANCIAL SUPPORT: Supported by National Science Centre in Poland. Grant no. 2014/15/D/NZ3/05181 to MC.
5

Role of Huntingtin-associated protein 1 in the regulation of SOCE in medium spiny neurons from transgenic YAC128 mice, a model of Huntington's disease

76%
Czeredys M., Maciag F., Vigont V., Kaznacheyeva E., Methner A., Kuznicki J.
Acta Neurobiologiae Experimentalis
|
2015
|
tom 75
|
nr Supl.
BACKGROUND AND AIMS: Huntington’s disease (HD) is a hereditary neurodegenerative disease caused by the expansion of a polyglutamine stretch in the huntingtin (HTT) protein and characterized by deregulated Ca2+ homeostasis (Giacomello et al. 2013). One of the mechanisms that regulate Ca2+ homeostasis is store-operated Ca2+ entry (SOCE) (Majewski and Kuźnicki 2015), which is enhanced in HD (Wu et al. 2011). The mechanism by which mutated HTT affects SOCE is unknown. The changes in Ca2+ homeostasis could be explained by increased expression of huntingtin-associated protein 1 (Hap1) mRNA (3-fold) and HAP1 protein (about 2-fold) in the striatum of YAC128 mice, which we detected (Czeredys et al. 2013). If HAP1 influences ER Ca2+ release mediated by IP3R in MSN from YAC128 mice, which was shown in other HD models by Tang et al. (2003, 2004), its increased level may explain changes in SOCE described by Wu et al. (2011). The aim of this study is to determine the role of HAP1 protein in the regulation of SOCE in MSN from transgenic mice YAC128, an HD model. METHODS: Using prepared lentiviral constructs we overexpressed HAP1a or HAP1b in YAC128 neurons, and in HEK293 cells overexpressing mutant HTT. We imaged cells with Fura-2AM, a selective fluorescent Ca2+ probe. SOCE was measured after depletion of intracellular Ca2+ by mGluR1/5 receptor agonist, DHPG (3,5- dihydroxyglycine) and subsequent incubation of cells in 2 mM Ca2+ media. RESULTS: In HD MSN we detected about 10% increase in basal Ca2+ level and found that the activity of SOCE was enhanced about 30%, thereby confirming results of Wu et al. (2011). The preliminary results showed that overexpressed Hap1 protein is involved in SOCE pathway in studied cells. Using electrophysiology we will also determine changes in the SOC currents in MSN and SK-N-SH cells, both transduced with HAP1 and mutant HTT. CONCLUSION: The investigation of the role of HAP1 protein in deregulated.
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