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The maintenance of proper cytosolic Ca 2+ level is crucial for neuronal survival, and dysregulation of Ca 2+ homeostasis is found in a variety of neurological disorders, including Alzheimer's disease. According to the "Ca 2+ hypothesis of aging", Ca 2+ disturbances precede the onset of AD symptoms and lead to neurodegeneration. STIM and ORAI proteins are involved in neuronal physiological and pathological processes as essential components of the store-operated Ca 2+ entry. Our previous data suggested that overexpression of STIM2 and ORAI1 might increase basal neuronal cytosolic Ca 2+ level. We generated double transgenic mice overexpressing these two genes in neurons, expecting that the increased basal Ca 2+ concentration will lead to premature neurodegeneration. We observed changes in Ca 2+ homeostasis and electrophysiological properties in acute brain slices of STIM2/ORAI1 neurons. However, we did not observe any augmentation of neurodegenerative processes, as tested by Fluoro-Jade ® C staining and assessment of amyloidogenesis. The battery of behavioral tests did not show any signs of accelerated aging. We conclude that changes of calcium homeostasis induced by overexpression of STIM2 and ORAI1 had no substantial adverse effects on neurons and did not lead to early neurodegeneration.

Neuronal Store-Operated Ca 2+ Entry (nSOCE) plays an essential role in refilling endoplasmic reticulum Ca 2+ stores and is critical for Ca 2+ -dependent neuronal processes. SOCE sensors, STIM1 and STIM2, can activate Orai, TRP channels and AMPA receptors, and inhibit voltage-gated channels in the plasma membrane. However, the link between STIM, SOCE, and NMDA receptors, another key cellular entry point for Ca 2+ contributing to synaptic plasticity and excitotoxicity, remains unclear. Using Ca 2+ imaging, we demonstrated that thapsigargin-induced nSOCE was inhibited in rat cortical neurons following NMDAR inhibitors. Blocking nSOCE by its inhibitor SKF96365 enhanced NMDA-driven [Ca 2+ ] i . Modulating STIM protein level through overexpression or shRNA inhibited or activated NMDA-evoked [Ca 2+ ] i , respectively. Using proximity ligation assays, immunofluorescence, and co-immunoprecipitation methods, we discovered that thapsigargin-dependent effects required interactions between STIMs and the NMDAR2 subunits. Since STIMs modulate NMDAR-mediated Ca 2+ levels, we propose targeting this mechanism as a novel therapeutic strategy against neuropathological conditions that feature NMDA-induced Ca 2+ overload as a diagnostic criterion.

Orai proteins form highly selective Ca 2+ release-activated channels (CRACs). They play a critical role in store-operated Ca 2+ entry (SOCE; i.e., the influx of external Ca 2+ that is induced by the depletion of endoplasmic reticulum Ca 2+ stores). Of the three Orai homologs that are present in mammals (Orai1-3), the physiological function of Orai1 is the best described. CRACs are formed by both homomeric assemblies and heteromultimers of Orais. Orai1 and Orai2 can form heteromeric channels that differ in conductivity during SOCE, depending on their Orai1-to-Orai2 ratio. The present study explored the potential consequences of ORAI1 overexpression in neurons where the dominant isoform is Orai2. We established the Tg(ORAI1)Ibd transgenic mouse line that overexpresses ORAI1 in brain neurons. We observed seizure-like symptoms in aged (≥15-month-old) female mice but not in males of the same age. The application of kainic acid and bicuculline to slices that were isolated from 8-month-old (±1 month) female Tg(ORAI1)Ibd mice revealed a significantly lower frequency of interictal bursts compared with samples that were isolated from wildtype mice. No differences were observed in male mice of a similar age. A battery of behavioral tests showed that context recognition decreased only in female transgenic mice. The phenotype that was observed in female mice suggests that ORAI1 overexpression may affect neuronal activity in a sex-dependent manner. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Store-operated calcium entry (SOCE) is the flow of calcium ions (Ca 2+ ) into cells in response to the depletion of intracellular Ca 2+ stores that reside predominantly in the endoplasmic reticulum (ER). The role of SOCE has been relatively well understood for non-excitable cells. It is mediated mostly by the ER Ca 2+ sensor STIM1 and plasma membrane Ca 2+ channel Orai1 and serves to sustain Ca 2+ signaling and refill ER Ca 2+ stores. In contrast, because of the complexity of Ca 2+ influx mechanisms that are present in excitable cells, our knowledge about the function of neuronal SOCE (nSOCE) is still nascent. This review summarizes the available data on the molecular components of nSOCE and their relevance to neuronal signaling. We also present evidence of disturbances of nSOCE in neurodegenerative diseases (namely Alzheimer's disease, Huntington's disease, and Parkinson's disease) and traumatic brain injury. The emerging important role of nSOCE in neuronal physiology and pathology makes it a possible clinical target., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Huntington's disease (HD) is a hereditary neurodegenerative disease caused by a polyglutamine expansion within the huntingtin (HTT) gene. One of the cellular functions that is dysregulated in HD is store-operated calcium entry (SOCE), a process in which the depletion of Ca 2+ from the endoplasmic reticulum (ER) induces Ca 2+ influx from the extracellular space. We detected an enhanced activity of SOC channels in medium spiny neurons (MSNs) from YAC128 mice, a transgenic model of HD, and investigated whether this could be reverted by tetrahydrocarbazoles. The compound 6-bromo-N-(2-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride was indeed able to restore the disturbed Ca 2+ homeostasis and stabilize SOCE in YAC128 MSN cultures. We also detected a beneficial effect of this compound on the mitochondrial membrane potential. Since dysregulated Ca 2+ homeostasis is believed to be one of the pathological hallmarks of HD, this compound might be a lead structure for HD treatment., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The mechanism of lithium's therapeutic action remains obscure, hindering the discovery of safer treatments for bipolar disorder. Lithium can act as an inhibitor of the kinase GSK3α/β, which in turn negatively regulates β-catenin, a co-activator of LEF1/TCF transcription factors. However, unclear is whether therapeutic levels of lithium activate β-catenin in the brain, and whether this activation could have a therapeutic significance. To address this issue we chronically treated mice with lithium. Although the level of non-phospho-β-catenin increased in all of the brain areas examined, β-catenin translocated into cellular nuclei only in the thalamus. Similar results were obtained when thalamic and cortical neurons were treated with a therapeutically relevant concentration of lithium in vitro. We tested if TCF7L2, a member of LEF1/TCF family that is highly expressed in the thalamus, facilitated the activation of β-catenin. Silencing of Tcf7l2 in thalamic neurons prevented β-catenin from entering the nucleus, even when the cells were treated with lithium. Conversely, when Tcf7l2 was ectopically expressed in cortical neurons, β-catenin shifted to the nucleus, and lithium augmented this process. Lastly, we silenced tcf7l2 in zebrafish and exposed them to lithium for 3 days, to evaluate whether TCF7L2 is involved in the behavioral response. Lithium decreased the dark-induced activity of control zebrafish, whereas the activity of zebrafish with tcf7l2 knockdown was unaltered. We conclude that therapeutic levels of lithium activate β-catenin selectively in thalamic neurons. This effect is determined by the presence of TCF7L2, and potentially contributes to the therapeutic response., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In this review we describe the present knowledge about store operated Ca²⁺ entry (SOCE) in neurons and the proteins involved in this process: STIM, as well as Orai and TRP channels. We address the issue of whether SOCE is used only to refill Ca²⁺ in the ER or whether Ca²⁺ that enters the neuronal cell during SOCE also performs signaling functions. We collected the data indicating that SOCE and its components participate in the important processes in neurons. This has implications for identifying new drug targets for the treatment of brain diseases. Evidence indicates that in neurodegenerative diseases Ca²⁺ homeostasis and SOCE components become dysregulated. Thus, different targets and strategies might be identified for the potential treatment of these diseases. This article is part of a Special Issue entitled: 13th European symposium on calcium., (Copyright © 2015. Published by Elsevier B.V.)

In non-excitatory cells, stromal interaction molecule 1 (STIM1) and STIM2 mediate store-operated calcium entry via an interaction with ORAI1 calcium channels. However, in neurons, STIM2 over-expression appears to play a role in calcium homeostasis that is different from STIM1 over-expression. The aim of this study was to establish the role and localization of native STIM2 in the neuronal cell. Co-immunoprecipitation experiments revealed that the interaction between endogenous STIM2 and ORAI1 was greater in a low-calcium medium than in a high-calcium medium. Using a Proximity Ligation Assay (PLA), the number of apparent complexes of endogenous STIM2 with ORAI1 was quantified. No change in the number of PLA signals was observed in the presence of thapsigargin, which depletes calcium from the endoplasmic reticulum (ER). However, the number of apparent STIM2-ORAI1 complexes increased when intracellular and subsequently ER calcium concentrations were decreased by BAPTA-AM or a low-calcium medium. Both Fura-2 acetoxymethyl ester calcium imaging and PLA in the same neuronal cell indicated that the calcium responses correlated strongly with the number of endogenous STIM2-ORAI1 complexes. The small drop in calcium levels in the ER caused by decreased intracellular calcium levels appeared to initiate the calcium-sensitive and thapsigargin-insensitive interaction between STIM2 and ORAI1. We show in neuronal somata the formation of endogenous complexes of stromal interaction molecule 2 (STIM2) with ORAI1 calcium channels. Their number increased when intracellular Ca²⁺ concentrations were decreased by the Ca²⁺ chelator BAPTA-AM or a low-calcium medium (EGTA), but did not in the presence of thapsigargin (TG). We conclude that the small drop of Ca²⁺ level in endoplasmic reticulum, due to the decreased level of intracellular Ca²⁺, is sufficient to trigger STIM2-ORAI1 complex formation in a thapsigargin-insensitive manner., (© 2013 International Society for Neurochemistry.)

Background: LEF1/TCF transcription factors and their activator β-catenin are effectors of the canonical Wnt pathway. Although Wnt/β-catenin signaling has been implicated in neurodegenerative and psychiatric disorders, its possible role in the adult brain remains enigmatic. To address this issue, we sought to identify the genetic program activated by β-catenin in neurons. We recently showed that β-catenin accumulates specifically in thalamic neurons where it activates Cacna1g gene expression. In the present study, we combined bioinformatics and experimental approaches to find new β-catenin targets in the adult thalamus., Results: We first selected the genes with at least two conserved LEF/TCF motifs within the regulatory elements. The resulting list of 428 putative LEF1/TCF targets was significantly enriched in known Wnt targets, validating our approach. Functional annotation of the presumed targets also revealed a group of 41 genes, heretofore not associated with Wnt pathway activity, that encode proteins involved in neuronal signal transmission. Using custom polymerase chain reaction arrays, we profiled the expression of these genes in the rat forebrain. We found that nine of the analyzed genes were highly expressed in the thalamus compared with the cortex and hippocampus. Removal of nuclear β-catenin from thalamic neurons in vitro by introducing its negative regulator Axin2 reduced the expression of six of the nine genes. Immunoprecipitation of chromatin from the brain tissues confirmed the interaction between β-catenin and some of the predicted LEF1/TCF motifs. The results of these experiments validated four genes as authentic and direct targets of β-catenin: Gabra3 for the receptor of GABA neurotransmitter, Calb2 for the Ca(2+)-binding protein calretinin, and the Cacna1g and Kcna6 genes for voltage-gated ion channels. Two other genes from the latter cluster, Cacna2d2 and Kcnh8, appeared to be regulated by β-catenin, although the binding of β-catenin to the regulatory sequences of these genes could not be confirmed., Conclusions: In the thalamus, β-catenin regulates the expression of a novel group of genes that encode proteins involved in neuronal excitation. This implies that the transcriptional activity of β-catenin is necessary for the proper excitability of thalamic neurons, may influence activity in the thalamocortical circuit, and may contribute to thalamic pathologies.

Store-operated Ca(2+) entry (SOCE) over the plasma membrane is activated by depletion of intracellular Ca(2+) stores and has only recently been shown to play a role in CNS processes like synaptic plasticity. However, the direct effect of SOCE on the excitability of neuronal networks in vitro and in vivo has never been determined. We confirmed the presence of SOCE and the expression of the calcium sensors STIM1 and STIM2, which convey information about the calcium load of the stores to channel proteins at the plasma membrane, in neurons and astrocytes. Inhibition of SOCE by pharmacological agents 2-APB and ML-9 reduced the steady-state neuronal Ca(2+) concentration, reduced network activity, and increased synchrony of primary neuronal cultures grown on multi-electrode arrays, which prompted us to elucidate the relative expression of STIM proteins in conditions of pathologic excitability. Both proteins were increased in brains of chronic epileptic rodents and strongly expressed in hippocampal specimens from medial temporal lobe epilepsy patients. Pharmacologic inhibition of SOCE in chronic epileptic hippocampal slices suppressed interictal spikes and rhythmized epileptic burst activity. Our results indicate that SOCE modulates the activity of neuronal networks in vitro and in vivo and delineates SOCE as a potential drug target., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Nuclear localization of β-catenin is a hallmark of canonical Wnt signaling, a pathway that plays a crucial role in brain development and the neurogenesis of the adult brain. We recently showed that β-catenin accumulates specifically in mature thalamic neurons, where it regulates the expression of the Ca(v)3.1 voltage-gated calcium channel gene. Here, we investigated the mechanisms underlying β-catenin accumulation in thalamic neurons. We report that a lack of soluble factors produced either by glia or cortical neurons does not impair nuclear β-catenin accumulation in thalamic neurons. We next found that the number of thalamic neurons with β-catenin nuclear localization did not change when the Wnt/Dishevelled signaling pathway was inhibited by Dickkopf1 or a dominant negative mutant of Dishevelled3. These results suggest a WNT-independent cell-autonomous mechanism. We found that the protein levels of APC, AXIN1, and GSK3β, components of the β-catenin degradation complex, were lower in the thalamus than in the cortex of the adult rat brain. Reduced levels of these proteins were also observed in cultured thalamic neurons compared with cortical cultures. Finally, pulse-chase experiments confirmed that cytoplasmic β-catenin turnover was slower in thalamic neurons than in cortical neurons. Altogether, our data indicate that the nuclear localization of β-catenin in thalamic neurons is their cell-intrinsic feature, which was WNT-independent but associated with low levels of proteins involved in β-catenin labeling for ubiquitination and subsequent degradation.

The interaction between Ca(2+) sensors STIM1 and STIM2 and Ca(2+) channel-forming protein ORAI1 is a crucial element of store-operated calcium entry (SOCE) in non-excitable cells. However, the molecular mechanism of SOCE in neurons remains unclear. We addressed this issue by establishing the presence and function of STIM proteins. Real-time polymerase chain reaction from cortical neurons showed that these cells contain significant amounts of Stim1 and Stim2 mRNA. Thapsigargin (TG) treatment increased the amount of both endogenous STIM proteins in neuronal membrane fractions. The number of YFP-STIM1/ORAI1 and YFP-STIM2/ORAI1 complexes was also enhanced by such treatment. The differences observed in the number of STIM1 and STIM2 complexes under SOCE conditions and the differential sensitivity to SOCE inhibitors suggest their distinct roles. Endoplasmic reticulum (ER) store depletion by TG enhanced intracellular Ca(2+) levels in loaded with Fura-2 neurons transfected with YFP-STIM1 and ORAI1, but not with YFP-STIM2 and ORAI1, which correlated well with the number of complexes formed. Moreover, the SOCE inhibitors ML-9 and 2-APB reduced Ca(2+) influx in neurons expressing YFP-STIM1/ORAI1 but produced no effect in cells transfected with YFP-STIM2/ORAI1. Moreover, in neurons transfected with YFP-STIM2/ORAI1, the increase in constitutive calcium entry was greater than with YFP-STIM1/ORAI1. Our data indicate that both STIM proteins are involved in calcium homeostasis in neurons. STIM1 mainly activates SOCE, whereas STIM2 regulates resting Ca(2+) levels in the ER and Ca(2+) leakage with the additional involvement of STIM1.

beta-Catenin, together with LEF1/TCF transcription factors, activates genes involved in the proliferation and differentiation of neuronal precursor cells. In mature neurons, beta-catenin participates in dendritogenesis and synaptic function as a component of the cadherin cell adhesion complex. However, the transcriptional activity of beta-catenin in these cells remains elusive. In the present study, we found that in the adult mouse brain, beta-catenin and LEF1 accumulate in the nuclei of neurons specifically in the thalamus. The particular electrophysiological properties of thalamic neurons depend on T-type calcium channels. Cav3.1 is the predominant T-type channel subunit in the thalamus, and we hypothesized that the Cacna1g gene encoding Cav3.1 is a target of the LEF1/beta-catenin complex. We demonstrated that the expression of Cacna1g is high in the thalamus and is further increased in thalamic neurons treated in vitro with LiCl or WNT3A, activators of beta-catenin. Luciferase reporter assays confirmed that the Cacna1G promoter is activated by LEF1 and beta-catenin, and footprinting analysis revealed four LEF1 binding sites in the proximal region of this promoter. Chromatin immunoprecipitation demonstrated that the Cacna1g proximal promoter is occupied by beta-catenin in vivo in the thalamus, but not in the hippocampus. Moreover, WNT3A stimulation enhanced T-type current in cultured thalamic neurons. Together, our data indicate that the LEF1/beta-catenin complex regulates transcription of Cacna1g and uncover a novel function for beta-catenin in mature neurons. We propose that beta-catenin contributes to neuronal excitability not only by a local action at the synapse but also by activating gene expression in thalamic neurons.

Recent findings indicate that Store Operated Ca(2+) Entry (SOCE) in non-excitable cells is based on the interaction of ER calcium sensor STIM1 with the plasma membrane Ca(2+) channel protein ORAI1. However, despite physiological evidence for functional SOCE in neurons, its mechanism is not known. Using PCR, immunoblotting and immunohistochemical methods we show that STIM1 protein is present in the mouse brain. The protein and mRNA levels of STIM1 are similar in the thalamus, the hippocampus, the cortex and the amygdala and the higher level is observed in the cerebellum. Immunohistochemistry of the cortex and the hippocampus of brain sections shows that STIM1 is present in cell bodies and dendrites of pyramidal neurons. In the cerebellum STIM1 is present in Purkinje and granule cells. The same immunostaining pattern is observed in cultured hippocampal and cortical neurons. Localization of YFP-STIM1 and ORAI1 changes from a dispersed pattern in untreated cortical neurons to puncta-like pattern in cells with a Ca(2+) store depleted by thapsigargin treatment. The YFP-STIM1(D76A) dominant positive mutant, which is active regardless of the Ca(2+) level in ER, concentrates as puncta even without depletion of the neuronal Ca(2+) store. Also, this mutant forces ORAI1 redistribution to form puncta-like staining. We suggest that in neurons, just as in non-excitable cells, the STIM1 and ORAI1 proteins are involved in SOCE.

Neuronal Ca(2+) homeostasis and Ca(2+) signaling regulate multiple neuronal functions, including synaptic transmission, plasticity, and cell survival. Therefore disturbances in Ca(2+) homeostasis can affect the well-being of the neuron in different ways and to various degrees. Ca(2+) homeostasis undergoes subtle dysregulation in the physiological ageing. Products of energy metabolism accumulating with age together with oxidative stress gradually impair Ca(2+) homeostasis, making neurons more vulnerable to additional stress which, in turn, can lead to neuronal degeneration. Neurodegenerative diseases related to aging, such as Alzheimer's disease, Parkinson's disease, or Huntington's disease, develop slowly and are characterized by the positive feedback between Ca(2+) dyshomeostasis and the aggregation of disease-related proteins such as amyloid beta, alfa-synuclein, or huntingtin. Ca(2+) dyshomeostasis escalates with time eventually leading to neuronal loss. Ca(2+) dyshomeostasis in these chronic pathologies comprises mitochondrial and endoplasmic reticulum dysfunction, Ca(2+) buffering impairment, glutamate excitotoxicity and alterations in Ca(2+) entry routes into neurons. Similar changes have been described in a group of multifactorial diseases not related to ageing, such as epilepsy, schizophrenia, amyotrophic lateral sclerosis, or glaucoma. Dysregulation of Ca(2+) homeostasis caused by HIV infection or by sudden accidents, such as brain stroke or traumatic brain injury, leads to rapid neuronal death. The differences between the distinct types of Ca(2+) dyshomeostasis underlying neuronal degeneration in various types of pathologies are not clear. Questions that should be addressed concern the sequence of pathogenic events in an affected neuron and the pattern of progressive degeneration in the brain itself. Moreover, elucidation of the selective vulnerability of various types of neurons affected in the diseases described here will require identification of differences in the types of Ca(2+) homeostasis and signaling among these neurons. This information will be required for improved targeting of Ca(2+) homeostasis and signaling components in future therapeutic strategies, since no effective treatment is currently available to prevent neuronal degeneration in any of the pathologies described here., (Copyright 2008 IUBMB)

Calretinin (CR) is an EF-hand calcium binding protein expressed at high level in neurons. To identify regulatory elements in CR gene promoter, cultured rat cortical cells were transfected with constructs containing its 5'-end deletion mutants and the luciferase reporter gene. A fragment ending at -115 bp upstream of the transcription start site had high promoter activity and was able to induce expression of luciferase specifically in neuronal cells of cortical cultures. The wild type sequence of -115/+54 CR promoter fragment preferentially drove the expression of green fluorescent protein analog in cells of neuronal phenotype differentiated from multipotent human cell line DEV. Electrophoretic mobility shift assays (EMSA) revealed that the -115/-71 CR gene promoter region contains a binding site for a factor present in brain nuclear extract. Among oligonucleotides containing consensus binding sites for transcription factors within this region, the one representing AP2 binding site was able to compete formation of a protein complex. Mutations of this site prevented the binding between brain protein(s) and the -115/+54 CR gene promoter region and abolished the preferential expression of reporter gene in neuronal cells of DEV line. Thus, the AP2-like element seems to be essential for the neuron-specific activity of the CR gene promoter.

The calcyclin-binding protein (CacyBP) binds calcyclin (S100A6) at physiological levels of [Ca(2+)] and is highly expressed in brain neurons. Subcellular localization of CacyBP was examined in neurons and neuroblastoma NB-2a cells at different [Ca(2+)](i). Immunostaining indicates that CacyBP is present in the cytoplasm of unstimulated cultured neurons in which resting [Ca(2+)](i) is known to be approximately 50 nm. When [Ca(2+)](i) was increased to above 300 nm by KCl treatment, the immunostaining was mainly apparent as a ring around the nucleus. Such perinuclear localization of CacyBP was observed in untreated neuroblastoma NB-2a cells in which [Ca(2+)](i) is approximately 120 nm. An additional increase in [Ca(2+)](i) to above 300 nm by thapsigargin treatment did not change CacyBP localization. However, when [Ca(2+)](i) in NB-2a cells dropped to 70 nm, because of BAPTA/AM treatment, perinuclear localization was diminished. Ca(2+)-induced translocation of CacyBP was confirmed by immunogold electron microscopy and by fluorescence of NB-2a cells transfected with an EGFP-CacyBP vector. Recombinant CacyBP can be phosphorylated by protein kinase C in vitro. In untreated neuroblastoma NB-2a cells, CacyBP is phosphorylated on a serine residue(s), but exists in the dephosphorylated form in BAPTA/AM-treated cells. Thus, phosphorylation of CacyBP occurs in the same [Ca(2+)](i) range that leads to its perinuclear translocation.