Dmitry Lim

Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients.

Background Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca2+ entry (SOCE), which is activated by a depletion of the intracellular Ca2+ pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca2+-sensor, Stim1, and the plasmalemmal Ca2+ channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca2+ influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients. Methodology/Principal Findings The present study employed Ca2+ imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs) as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La3+ and Gd3+. Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca2+ release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca2+ buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs. Conclusions SOCE is remodelled in EPCs from RCC patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis.

Amyloid Beta Deregulates Astroglial mGluR5- Mediated Calcium Signaling via Calcineurin and NF-kB

The amyloid hypothesis of Alzheimers disease (AD) suggests that soluble amyloid β (Aβ) is an initiator of a cascade of events eventually leading to neurodegeneration. Recently, we reported that Aβ deranged Ca2+ homeostasis specifically in hippocampal astrocytes by targeting key elements of Ca2+ signaling, such as mGluR5 and IP3R1. In the present study, we dissect a cascade of signaling events by which Aβ deregulates glial Ca2+: (i) 100 nM Aβ leads to an increase in cytosolic calcium after 4–6 h of treatment; (ii) mGluR5 is increased after 24 h of treatment; (iii) this increase is blocked by inhibitors of calcineurin (CaN) and NF‐kB. Furthermore, we show that Aβ treatment of glial cells leads to de‐phosphorylation of Bcl10 and an increased CaN‐Bcl10 interaction. Last, mGluR5 staining is augmented in hippocampal astrocytes of AD patients in proximity of Aβ plaques and co‐localizes with nuclear accumulation of the p65 NF‐kB subunit and increased staining of CaNAα. Taken together our data suggest that nanomolar [Aβ] deregulates Ca2+ homeostasis via CaN and its downstream target NF‐kB, possibly via the cross‐talk of Bcl10 in hippocampal astrocytes.

Aβ leads to Ca2+ signaling alterations and transcriptional changes in glial cells

The pathogenesis of Alzheimers disease includes accumulation of toxic amyloid beta (Aβ) peptides. A recently developed cell-permeable peptide, termed Tat-Pro, disrupts the complex between synapse-associated protein 97 (SAP97) and the α-secretase a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), thereby leading to an alteration of the trafficking of the enzyme, which is important for nonamyloidogenic processing of amyloid precursor protein (APP). We report that Tat-Pro treatment, as well as the treatment with exogenous Aβ, deregulates Ca(2+) homeostasis specifically in astrocytes through increased expression of key components of Ca(2+) signaling, metabotropic glutamate receptor-5 and inositol 1,4,5-trisphosphate receptor-1. This is accompanied by potentiation of (S)-3,5-dihydroxyphenylglycine-induced Ca(2+) transients. Calcineurin inhibition reverts all these effects. Furthermore, our data demonstrate that astrocytes express all the components for the amyloidogenic and nonamyloidogenic processing of APP including APP itself, beta-site APP-cleaving enzyme 1 (BACE1), ADAM10, γ-secretase, and SAP97. Indeed, treatment with Tat-Pro for 48 hours significantly increased the amount of Aβ(1-42) in the medium of cultured astrocytes. Taken together, our results suggest that astroglia might be active players in Aβ production and indicate that the calcium hypothesis of Alzheimers disease may recognize glial cells as important intermediates.

A novel Ca2+-mediated cross-talk between endoplasmic reticulum and acidic organelles: Implications for NAADP-dependent Ca2+ signalling

Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as the ideal trigger of spatio-temporally complex intracellular Ca(2+) signals. However, the identity of the intracellular Ca(2+) store(s) recruited by NAADP, which may include either the endolysosomal (EL) or the endoplasmic reticulum (ER) Ca(2+) pools, is still elusive. Here, we show that the Ca(2+) response to NAADP was suppressed by interfering with either EL or ER Ca(2+) sequestration. The measurement of EL and ER Ca(2+) levels by using selectively targeted aequorin unveiled that the preventing ER Ca(2+) storage also affected ER Ca(2+) loading and vice versa. This indicates that a functional Ca(2+)-mediated cross-talk exists at the EL-ER interface and exerts profound implications for the study of NAADP-induced Ca(2+) signals. Extreme caution is warranted when dissecting NAADP targets by pharmacologically inhibiting EL and/or the ER Ca(2+) pools. Moreover, Ca(2+) transfer between these compartments might be essential to regulate vital Ca(2+)-dependent processes in both organelles.

Glial calcium signalling in Alzheimer's disease.

The most accredited (and fashionable) hypothesis of the pathogenesis of Alzheimer Disease (AD) sees accumulation of β-amyloid protein in the brain (in both soluble and insoluble forms) as a leading mechanism of neurotoxicity. How β-amyloid triggers the neurodegenerative disorder is at present unclear, but growing evidence suggests that a deregulation of Ca(2+) homeostasis and deficient Ca(2+) signalling may represent a fundamental pathogenic factor. Given that symptoms of AD are most likely linked to synaptic dysfunction (at the early stages) followed by neuronal loss (at later and terminal phases of the disease), the effects of β-amyloid have been mainly studied in neurones. Yet, it must be acknowledged that neuroglial cells, including astrocytes, contribute to pathological progression of most (if not all) neurological diseases. Here, we review the literature pertaining to changes in Ca(2+) signalling in astrocytes exposed to exogenous β-amyloid or in astrocytes from transgenic Alzheimer disease animals models, characterized by endogenous β-amyloidosis. Accumulated experimental data indicate deregulation of Ca(2+) homeostasis and signalling in astrocytes in AD, which should be given full pathogenetic consideration. Further studies are warranted to comprehend the role of deficient astroglial Ca(2+) signalling in the disease progression.

Differential deregulation of astrocytic calcium signalling by amyloid-β, TNFα, IL-1β and LPS

In Alzheimers disease (AD), astrocytes undergo complex morphological and functional changes that include early atrophy, reactive activation and Ca(2+) deregulation. Recently, we proposed a mechanism by which nanomolar Aβ42 deregulates mGluR5 and InsP3 receptors, the key elements of astrocytic Ca(2+) signalling toolkit. To evaluate the specificity of these changes, we have now investigated whether the effects of Aβ42 on Ca(2+) signalling machinery can be reproduced by pro-inflammatory agents (TNFα, IL-1β, LPS). Here we report that Aβ42 (100nM, 72h) significantly increased mRNA levels of mGluR5, InsP3R1 and InsP3R2, whereas pro-inflammatory agents reduced expression of these specific mRNAs. Furthermore, DHPG-induced Ca(2+) signals and store operated Ca(2+) entry (SOCE) were augmented in Aβ42-treated cells due to up-regulation of a set of Ca(2+) signalling-related genes including TRPC1 and TRPC4. Opposite changes were observed when astrocytes were treated with TNFα, IL-1β and LPS. Last, the effects observed on SOCE by treating wild-type astrocytes with Aβ42 were also identified in untreated astrocytes from 3×Tg-AD animals, suggesting a link to the AD pathology. Our results demonstrate that effects of Aβ42 on astrocytic Ca(2+) signalling differ from and may contrast to the effects of pro-inflammatory agents.

Activation of TRPV4 channels reduces migration of immortalized neuroendocrine cells.

J. Neurochem. (2011) 116, 606–615.

Tropisetron attenuates amyloid-beta-induced inflammatory and apoptotic responses in rats.

Alzheimers disease (AD) is a neurodegenerative disorder featured by deposition of beta‐amyloid (Aβ) plaques in the hippocampus and associated cortices and progressive cognitive decline. Tropisetron, a selective 5‐HT3 receptor antagonist, is conventionally used to counteract chemotherapy‐induced emesis. Recent investigations describe antiphlogistic properties for tropisetron. It has been shown that tropisetron protects against rat embolic stroke. We investigated protective properties of tropisetron in a beta‐amyloid (Aβ) rat model of AD and possible involvement of 5‐HT3 receptors.

Endoplasmic Reticulum Ca2+ Handling and Apoptotic Resistance in Tumor‐Derived Endothelial Colony Forming Cells

Truly endothelial progenitor cells (EPCs) can be mobilized from bone marrow to support the vascular network of growing tumors, thereby sustaining the metastatic switch. Endothelial colony forming cells (ECFCs) are the only EPC subtype belonging to the endothelial phenotype and capable of incorporating within neovessels. The intracellular Ca2+ machinery plays a key role in ECFC activation and is remodeled in renal cellular carcinoma‐derived ECFCs (RCC‐ECFCs). Particularly, RCC‐ECFCs seems to undergo a drop in endoplasmic reticulum (ER) Ca2+ concentration ([Ca2+]ER). This feature is remarkable when considering that inositol‐1,4,5‐trisphosphate (InsP3)‐dependent ER‐to‐mitochondria Ca2+ transfer regulates the intrinsic apoptosis pathway. Herein, we sought to assess whether: (1) the [Ca2+]ER and the InsP3‐induced ER‐mitochondria Ca2+ shuttle are reduced in RCC‐ECFCs; and (2) the dysregulation of ER Ca2+ handling leads to apoptosis resistance in tumor‐derived cells. RCC‐ECFCs displayed a reduction both in [Ca2+]ER and in the InsP3‐dependent mitochondrial Ca2+ uptake, while they expressed normal levels of Bcl‐2 and Bak. The decrease in [Ca2+]ER was associated to a remarkable ER expansion in RCC‐ECFCs, which is a hallmark of ER stress, and did not depend on the remodeling of the Ca2+‐transporting and the ER Ca2+‐storing systems. As expected, RCC‐ECFCs were less sensitive to rapamycin‐ and thapsigargin‐induced apoptosis; however, buffering intracellular Ca2+ levels with BAPTA dampened apoptosis in both cell types. Finally, store‐operated Ca2+ entry was seemingly uncoupled from the apoptotic machinery in RCC‐ECFCs. Thus, the chronic underfilling of the ER Ca2+ pool could confer a survival advantage to RCC‐ECFCs and underpin RCC resistance to pharmacological treatment. J. Cell. Biochem. 117: 2260–2271, 2016.

Calcium Signalling Toolkits in Astrocytes and Spatio-Temporal Progression of Alzheimer's Disease

Pathological remodelling of astroglia represents an important component of the pathogenesis of Alzheimers disease (AD). In AD astrocytes undergo both atrophy and reactivity; which may be specific for different stages of the disease evolution. Astroglial reactivity represents the generic defensive mechanism, and inhibition of astrogliotic response exacerbates b-amyloid pathology associated with AD. In animal models of AD astroglial reactivity is different in different brain regions, and the deficits of reactive response observed in entorhinal and prefrontal cortices may be linked to their vulnerability to AD progression. Reactive astrogliosis is linked to astroglial Ca(2+) signalling, this latter being widely regarded as a mechanism of astroglial excitability. The AD pathology evolving in animal models as well as acute or chronic exposure to β-amyloid induce pathological remodelling of Ca(2+) signalling toolkit in astrocytes. This remodelling modifies astroglial Ca(2+) signalling and may be linked to cellular mechanisms of AD pathogenesis.