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1

Proximity ligation assay visualizes in cultured neurons the interaction between endogenous ORAI1 and STIM proteins

100%
Gruszczynska-Biegala J., Kuznicki J.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
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nr S
Calcium sensors STIM1 and STIM2, located in ER, and calcium channel forming protein ORAI1 are involved in the store-operated calcium entry (SOCE). The process relies on extracellular calcium influx through the plasma membrane channels. In non-excitable cells STIM interaction with ORAI1 is a crucial element of SOCE, but in neurons its mechanism remains unclear. We showed earlier that STIM1 is likely involved in thapsigargin induced SOCE, while STIM2 is mostly active after EGTA-driven depletion of extracellular Ca2+ (Gruszczynska-Biegala et al. 2011). Depletion of calcium from ER increased number of puncta-like colocalization of YFP-STIM1 and ORAI1, but not of YFP-STIM2 and ORAI1. In contrast, reduction of extracellular calcium level triggered puncta formation for both YFP-STIM1/ORAI1 and YFP-STIM2/ORAI1. In this work we aimed to determine whether after SOCE induction it is possible to detect complexes containing endogenous STIMs and ORAI1. We showed that in cultured rat cortical neurons STIM1 and STIM2 can interact with ORAI1 what can be observed by proximity ligation assay (PLA). Using PLA we were able to visualize fluorescent dots, which represent the site where two antibodies are bound: one against ORAI1 and another one against either STIM1 or STIM2. These dots identify likely the complexes between STIMs and ORAI1. The interaction between them was quantified and found to correlate well with the number of exogenous complexes formed under the same conditions. To confirm that observed PLA dots represent authentic STIMORAI1 complexes we use different pairs of anti-STIM and antiORAI1 antibodies. The positive findings will allow us to confirm that the interaction between endogenous STIMs and ORAI1 occurs in neurons during SOCE in vivo and to demonstrate the feasibility of the PLA technique with antibodies of low specificity.
2

Endogenous STIM2 and endogenous ORAI1 form a calcium-sensitive and thapsigargin-insensitive complex in cortical neurons

100%
Gruszczynska-Biegala J., Kuznicki J.
Acta Neurobiologiae Experimentalis
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2013
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tom 73
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nr 1
ER calcium sensors (STIM1, STIM2) and calcium channel-ORAI1 interaction is crucial for store-operated calcium entry (SOCE) in non-excitable cells, but in neurons their localization and dynamics are not clear. We showed earlier that in neurons STIM1 is involved in thapsigargin induced SOCE, while STIM2 is active after EGTA-driven depletion of extracellular Ca2+ (Klejman et al. 2009, Gruszczynska-Biegala et al. 2011). To confirm that this is not due to the overexpression of exogenous proteins we used Proximity Ligation Assay to analyze activities of endogenous proteins. Cortical neurons were cultured in 2 mM CaCl2, 2 mM EGTA or 2 µM thapsigargin, fixed and incubated with primary antibodies anti-STIM2 and anti-ORAI1. The pairs of appropriate secondary antibodies with conjugated oligonucleotides were then added and Duolink II was performed to create the fluorescent products. We detected in situ the endogenous STIM2/ORAI1 complexes in somata and quantified in single neurons the number of hetero- and homo-complexes. The amount of hetero-complexes increased up to 10-fold in response to calcium depletion by EGTA. The number of STIM2/ORAI1 endogenous complexes correlated well with the number of overexpressed YFP-STIM2/ORAI1 complexes formed under the same conditions (Gruszczynska-Biegala et al. 2011). By co-immunoprecipitation we confirmed the in situ interaction between endogenous STIM2 and ORAI1 and that the interaction is increased after Ca2+ depletion in the medium. In conclusion, the present study provides a novel finding that endogenous STIM2 can physically interact and form hetero-complexes with endogenous ORAI1 in TG-insensitive manner, suggesting that the proteins are key molecules that underlie the regulation of basal calcium levels in neurons and constitutive calcium entry. This work was supported by funds from a Polish Ministerial grant (1900/B/P01/2010/39, JK) and from National Science Centre (2011/01/D/NZ3/02051, JGB)
3

Sensivity of store-operated calcium entry to antagonists of monotropic receptors

81%
Gruszczynska-Biegala J., Sladowska 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: In non-excitable cells Ca2+ enters through Store-Operated Ca2+ Entry (SOCE) pathway, involving STIM1, STIM2 and Orai1 proteins. Upon activation of neurons [Ca2+] is increased in the cytoplasm as a result of Ca2+ influx from the extracellular environment mainly through voltage-operated Ca2+ channels and ionotropic receptor-operated Ca2+ channels (IR). Recently, another possibility has been shown into neurons – Ca2+ influx via SOCE. Our earlier data indicated that both STIMs are involved in Ca2+ homeostasis in neurons, form complexes with endogenous ORAI1 (Gruszczynska-Biegala and Kuznicki 2013, J Neurochem), but played a distinct role in SOCE (Gruszczynska-Biegala et al. 2011, PLoS ONE). The aim of this study is to determine, which receptors react with STIM proteins and are involved in SOCE. The potential STIM partnersin plasma membrane, belong to 3 types of IR (NMDAR, AMPAR and kainate receptors – KR). METHODS: In cultured cortical neurons we recorded single-cell Ca2+ levels using the ratiometric Ca2+ indicator Fura-2AM. SOCE was measured after depletion of intracellular Ca2+ stores by thapsigargin (TG) and subsequent incubation of cells in 2 mM Ca2+ media. To investigate the involvement of IR in TG-induced Ca2+ entry, we applied antagonists of these receptors such as NS-102 (KR), CNQX (AMPAR/KR), NBQX (AMPAR), MK-801 (NMDAR), memantine (NMDAR), and D-AP5 (NMDAR). RESULTS: We found that SOCE was decreased by CNQX, NBQX, D-AP5, memantine but insignificant changes were observed in the presence of MK801 and NS-102. The results showed that NMDA and AMPA receptors are involved in SOCE pathway. The interaction between endogenous STIM1/STIM2 with IR will be checked by co-immunoprecipitation. CONCLUSIONS: The identification of new partners of STIMs will allow us to better understand the mechanisms of SOCE in healthy neurons and during Alzheimer’s disease degeneration. Supported by funds from a National Science Centre (2011/01/D/NZ3/02051, JGB).
4

Cross-talk between stim proteins and ionotropic receptors in neurons

81%
Gruszczynska-Biegala J., Sladowska M., Kuznicki J.
Acta Neurobiologiae Experimentalis
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2017
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tom 77
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nr Suppl.1
INTRODUCTION: Depletion of Ca2+ in endoplasmic reticulum (ER) is sensed by STIM proteins, which then activate influx of these ions via Orai channels from the extracellular environment. This process is called the Store-Operated Calcium Entry (SOCE), and its role in non-excitatory cells is to refill ER with Ca2+. In neurons, however, SOCE is also used for signaling and seems to involve several types of channels. AIM(S): The aim of this study is to determine which ionotropic receptors (IR) react with STIM proteins and are involved in calcium influx during SOCE. METHOD(S): In cultured cortical neurons we recorded single-cell Ca2+ levels using Fura-2AM. SOCE was measured after depletion of intracellular Ca2+ stores by thapsigargin and subsequent incubation of cells in 2 mM Ca2+ media. To investigate the involvement of IR in SOCE, we applied antagonists of these receptors such as CNQX and NBQX (AMPAR), MK‑801, memantine and D-AP5 (NMDAR). To determine the effects of SOCE on IR agonist-induced Ca2+ entry we applied SOCE inhibitors (ML9, SKF96365). The co-immunoprecipitation assay was used to detect the interaction between STIMs and IR. Electrophysiology experiments are also being performed. RESULTS: We report that ML-9 and SKF reduced AMPAand NMDA‑induced Ca2+ influx. In addition, SOCE was decreased by CNQX, NBQX, D‑AP5, memantine, but no significant effect was observed in the presence of MK801. Physical association of endogenous STIM proteins with endogenous GluA1 or GluA2 subunits of AMPAR and N2B subunit of NMDAR we detected by immunoprecipitation. Application of SOCE inhibitor, SKF96365, had no effect on the amplitude of AMPA-mediated miniature excitatory postsynaptic currents (mEPSCs). CONCLUSIONS: Ca2+ measurements using specific inhibitors and immunoprecipitation experiments indicate that STIM proteins might participate in neuronal signaling by the interaction with ionotropic receptors such as AMPA and NMDA. FINANCIAL SUPPORT: Supported by funds from a National Science Centre (2011/01/D/NZ3/02051, JGB).
5

STIM1 and STIM2 behave differently in neurons during store operated calcium entry

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Gruszczynska-Biegala J., Pomorski P., Kuznicki J.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
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nr 1
Store Operated Calcium Entry (SOCE) is a common phenomenon in non-excitable cells. The process relies on extracellular calcium influx through the plasma membrane (PM) channels, tightly regulated by endoplasmic reticulum (ER) calcium concentration. This influx allows refilling of the ER after Ca2+ release to the cytoplasm. The proteins involved in this process are calcium sensors STIM1 and STIM2 (located in ER), and calcium channel forming protein called ORAI1 (located in PM). Complexes of the STIM proteins with ORAI1 were identified in the fluorescent microscopy and called \”puncta\”. In neurons the molecular mechanism of SOCE is unclear. Our previous research led to the identification and characterization of STIM1 in the brain and neurons (Acta Neurobiol. Exp. 2009, 69:413-28; Neurochem. Int. 2009, 54:49-55). In this study we found that also STIM2 is expressed in neurons and aimed our work to compare the function of STIM proteins. In cultured cortical neurons, overexpressing YFP-STIM1/YFP-STIM2 and ORAI1, we observed changes of the fluorescence distribution from dispersed before to aggregated complexes of STIM1-ORAI1 and STIM2-ORAI1 after treatment with thapsigargin (TG). We also found that depletion of calcium from ER increased the number of STIM1-ORAI1 puncta much more than of STIM2-ORAI1 puncta. Then we analyzed the effects of STIM1/ORAI1 or STIM2/ORAI1 expression on intracellular calcium level during SOCE using Ca2+ imaging in two types of experiments. The first one was an analysis of SOCE after depletion of intracellular Ca2+ stores by TG and subsequent incubation of cells in 2 mM Ca2+ media. These measurements were performed also in the presence of SOCE inhibitors (ML-9 or 2-APB). SOCE was enhanced in neurons transfected with STIM1/ORAI1, but not with STIM2/ORAI1. Moreover, both inhibitors reduced calcium influx by about 70% in neurons expressing STIM1/ORAI1, while produced no significant change in neurons transfected with STIM2/ ORAI1. In the second type of experiments a removal of extracellular Ca2+ caused a sustained decrease in intracellular calcium in all experimental setups, however the highest decrease was observed in neurons transfected with STIM2/ORAI1. In store-repleted cells, an increase in constitutive Ca2+ entry was observed with STIM1/ ORAI1 and STIM2/ORAI1 expression, but not with STIM expression alone. STIM2/ORAI1-mediated constitutive Ca2+ level was raised by 50 µM 2-APB, but not in case of STIM1/ORAI1 transfectants. Based on these observations we suggest that in neurons STIM1 and STIM2 proteins have distinct role in SOCE.
6

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

71%
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
|
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.
7

Immunolocalization of STIM1 in the mouse brain

71%
Skibinska-Kijek A., Wisniewska M. B., Gruszczynska-Biegala J., Methner A., Kuznicki J.
Acta Neurobiologiae Experimentalis
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2009
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tom 69
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nr 4
Capacitative Calcium Entry (CCE) in neurons seems to depend, as in non-excitatory cells, on endoplasmic reticulum calcium sensors STIM1 or STIM2. We show localization of STIM1 in the mouse brain by immunohistochemistry with a specific antibody. STIM1 immunoreactivity has wide, but not uniform, distribution throughout the brain and is observed in neuropil and cells. The most intensive immunoreactivity is observed in Purkinje neurons of cerebellum. High/moderate levels of immunostaining are found in hippocampus, cerebral cortex and in cortico-medial amygdala, low in thalamus and basolateral amygdala. Co-staining with anti-NeuN antibody identify STIM1 immunopositive cells as neurons. Real time PCR demonstrates that Stim2 expression is 7-fold higher than that of Stim1 in hippocampus and 3-fold in other regions. Immunoblotting confirms that levels of STIMs vary in different brain regions. The data show that STIM1 and STIM2 are present in the brain, thus both can be involved in CCE, depending on neuronal type.
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