Heimdall, an alternative protein issued from a ncRNA related to kappa light chain variable region of immunoglobulins from astrocytes: a new player in neural proteome.

The dogma "One gene, one protein" is clearly obsolete since cells use alternative splicing and generate multiple transcripts which are translated into protein isoforms, but also use alternative translation initiation sites (TISs) and termination sites on a given transcript. Alternative open reading frames for individual transcripts give proteins originate from the 5'- and 3'-UTR mRNA regions, frameshifts of mRNA ORFs or from non-coding RNAs. Longtime considered as non-coding, recent in-silico translation prediction methods enriched the protein databases allowing the identification of new target structures that have not been identified previously. To gain insight into the role of these newly identified alternative proteins in the regulation of cellular functions, it is crucial to assess their dynamic modulation within a framework of altered physiological modifications such as experimental spinal cord injury (SCI). Here, we carried out a longitudinal proteomic study on rat SCI from 12 h to 10 days. Based on the alternative protein predictions, it was possible to identify a plethora of newly predicted protein hits. Among these proteins, some presented a special interest due to high homology with variable chain regions of immunoglobulins. We focus our interest on the one related to Kappa variable light chains which is similarly highly produced by B cells in the Bence jones disease, but here expressed in astrocytes. This protein, name Heimdall is an Intrinsically disordered protein which is secreted under inflammatory conditions. Immunoprecipitation experiments showed that the Heimdall interactome contained proteins related to astrocyte fate keepers such as "NOTCH1, EPHA3, IPO13" as well as membrane receptor protein including "CHRNA9; TGFBR, EPHB6, and TRAM". However, when Heimdall protein was neutralized utilizing a specific antibody or its gene knocked out by CRISPR-Cas9, sprouting elongations were observed in the corresponding astrocytes. Interestingly, depolarization assays and intracellular calcium measurements in Heimdall KO, established a depolarization effect on astrocyte membranes KO cells were more likely that the one found in neuroprogenitors. Proteomic analyses performed under injury conditions or under lipopolysaccharides (LPS) stimulation, revealed the expression of neuronal factors, stem cell proteins, proliferation, and neurogenesis of astrocyte convertor factors such as EPHA4, NOTCH2, SLIT3, SEMA3F, suggesting a role of Heimdall could regulate astrocytic fate. Taken together, Heimdall could be a novel member of the gatekeeping astrocyte-to-neuroprogenitor conversion factors.

Astrocytes express aberrant immunoglobulins as putative gatekeeper of astrocytes to neuronal progenitor conversion.

Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and they resemble aberrant immunoglobulins observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In addition, the constant heavy chain is also present in adult rat astrocytes, whereas in primary astrocytes from human fetus we identified constant and variable kappa chains as well as the substitution lambda chains known to be involved in pre-B cells. To gather insights into the function of these neural IgGs, CRISPR-Cas9 of IgG2B constant heavy chain encoding gene (Igh6), IgG2B overexpression, proximal labeling of rat astrocytes IgG2B and targets identification through 2D gels were performed. In Igh6 KO astrocytes, overrepresentation of factors involved in hematopoietic cells, neural stem cells, and the regulation of neuritogenesis have been identified. Moreover, overexpression of IgG2B in astrocytes induces the CRTC1-CREB-BDNF signaling pathway known to be involved in gliogenesis, whereas Igh6 KO triggers the BMP/YAP1/TEAD3 pathway activated in astrocytes dedifferentiation into neural progenitors. Proximal labeling experiments revealed that IgG2B is N-glycosylated by the OST complex, addressed to vesicle membranes containing the ATPase complex, and behaves partially like CD98hc through its association with LAT1. These experiments also suggest that proximal IgG2B-LAT1 interaction occurs concomitantly with MACO-1 and C2CD2L, at the heart of a potentially novel cell signaling platform. Finally, we demonstrated that these chains are synthesized individually and associated to recognize specific targets. Indeed, intermediate filaments Eif4a2 and Pdia6 involved in astrocyte fate constitute targets for these neural IgGs. Taken together, we hypothese that neural aberrant IgG chains may act as gatekeepers of astrocytes' fate.

The Antibody Dependant Neurite Outgrowth Modulation Response Involvement in Spinal Cord Injury.

Spinal cord injury (SCI) represents a major medical challenge. At present, there is still no cure to treat it efficiently and enable functional recovery below the injury site. Previously, we demonstrated that inflammation determines the fate of the physiopathology. To decipher the molecular mechanisms involved in this process, we performed a meta-analysis of our spatio-temporal proteomic studies in the time course of SCI. This highlighted the presence of IgG isotypes in both spinal cord explants and their secretomes. These IgGs were detected in the spinal cord even if no SCI occurred. However, during the time course following SCI, abundance of IgG1 and IgG2 subclasses (a, b, c) varied according to the spatial repartition. IgG1 was clearly mostly abundant at 12 h, and a switch to IgG2a was observed after 24 h. This IgG stayed predominant 3, 7, and 10 days after SCI. A protein related to IgM as well as a variable heavy chain were only detected 12 h after lesion. Interestingly, treatment with RhoA inhibitor influenced the abundance of the various IgG isotypes and a preferential switch to IgG2c was observed. By data reuse of rat dorsal root ganglion (DRG) neurons RNAseq datasets and RT-PCR experiments performed on cDNA from DRG sensory neurons ND7/23 and N27 dopaminergic neural cell lines, we confirmed expression of immunoglobulin heavy and light chains (constant and variable) encoding genes in neurons. We then identified CD16 and CD32b as their specific receptors in sensory neuron cell line ND7/23 and their activation regulated neurites outgrowth. These results suggest that during SCI, neuronal IgG isotypes are released to modulate neurites outgrowth. Therefore, we propose a new view of the SCI response involving an antibody dependent neurite outgrowth modulation (ADNM) which could be a precursor to the neuroinflammatory response in pathological conditions.

Impact of SOCE Abolition by ORAI1 Knockout on the Proliferation, Adhesion, and Migration of HEK-293 Cells.

Store-operated calcium entry (SOCE) provided through channels formed by ORAI proteins is a major regulator of several cellular processes. In immune cells, it controls fundamental processes such as proliferation, cell adhesion, and migration, while in cancer, SOCE and ORAI1 gene expression are dysregulated and lead to abnormal migration and/or cell proliferation. In the present study, we used the CRISPR/Cas9 technique to delete the ORAI1 gene and to identify its role in proliferative and migrative properties of the model cell line HEK-293. We showed that ORAI1 deletion greatly reduced SOCE. Thereby, we found that this decrease and the absence of ORAI1 protein did not affect HEK-293 proliferation. In addition, we determined that ORAI1 suppression did not affect adhesive properties but had a limited impact on HEK-293 migration. Overall, we showed that ORAI1 and SOCE are largely dispensable for cellular proliferation, migration, and cellular adhesion of HEK-293 cells. Thus, despite its importance in providing Ca2+ entry in non-excitable cells, our results indicate that the lack of SOCE does not deeply impact HEK-293 cells. This finding suggests the existence of compensatory mechanism enabling the maintenance of their physiological function.

ORAI3 silencing alters cell proliferation and promotes mitotic catastrophe and apoptosis in pancreatic adenocarcinoma.

Changes in cytosolic free Ca2+ concentration play a central role in many fundamental cellular processes including muscle contraction, neurotransmission, cell proliferation, differentiation, gene transcription and cell death. Many of these processes are known to be regulated by store-operated calcium channels (SOCs), among which ORAI1 is the most studied in cancer cells, leaving the role of other ORAI channels yet inadequately addressed. Here we demonstrate that ORAI3 channels are expressed in both normal (HPDE) and pancreatic ductal adenocarcinoma (PDAC) cell lines, where they form functional channels, their knockdown affecting store operated calcium entry (SOCE). More specifically, ORAI3 silencing increased SOCE in PDAC cell lines, while decreasing SOCE in normal pancreatic cell line. We also show the role of ORAI3 in proliferation, cell cycle, viability, mitotic catastrophe and cell death. Finally, we demonstrate that ORAI3 silencing impairs pancreatic tumor growth and induces cell death in vivo, suggesting that ORAI3 could represent a potential therapeutic target in PDAC treatment.

Co-targeting Mitochondrial Ca2+ Homeostasis and Autophagy Enhances Cancer Cells' Chemosensitivity.

Mitochondria are important cell death checkpoints, and mitochondrial Ca2+ overload is considered as a potent apoptotic intrinsic pathway inducer. Here, we report that this Ca2+ apoptosis link is largely ineffective in inducing cell-death just by itself and required a concomitant inhibition of autophagy to counteract its pro-survival action. In such condition, an acute mitochondrial stress revealed by a DRP1-mediated mitochondrial dynamic remodeling is observed concomitantly with mitochondrial depolarization, release of cytochrome c, and efficient apoptosis induction. We also uncover that mitochondrial Ca2+ status modulates the function of autophagy as a sensitizer for chemotherapies. This priming mediated by mitochondrial Ca2+ overload and inhibition of autophagy sensitizes many cancer cells types to different chemotherapies with independent mechanisms of action. Collectively, our results redefine an important cell signaling pathway, uncovering new combined therapies for the treatment of diseases associated with mitochondrial Ca2+ homeostasis disorders such as cancer.

Control of cell death/survival balance by the MET dependence receptor.

Control of cell death/survival balance is an important feature to maintain tissue homeostasis. Dependence receptors are able to induce either survival or cell death in presence or absence of their ligand, respectively. However, their precise mechanism of action and their physiological importance are still elusive for most of them including the MET receptor. We evidence that pro-apoptotic fragment generated by caspase cleavage of MET localizes to the mitochondria-associated membrane region. This fragment triggers a calcium transfer from endoplasmic reticulum to mitochondria, which is instrumental for the apoptotic action of the receptor. Knock-in mice bearing a mutation of MET caspase cleavage site highlighted that p40MET production is important for FAS-driven hepatocyte apoptosis, and demonstrate that MET acts as a dependence receptor in vivo. Our data shed light on new signaling mechanisms for dependence receptors' control of cell survival/death balance, which may offer new clues for the pathophysiology of epithelial structures.

TRPV1 variants impair intracellular Ca2+ signaling and may confer susceptibility to malignant hyperthermia.

PURPOSE: Malignant hyperthermia (MH) is a pharmacogenetic disorder arising from uncontrolled muscle calcium release due to an abnormality in the sarcoplasmic reticulum (SR) calcium-release mechanism triggered by halogenated inhalational anesthetics. However, the molecular mechanisms involved are still incomplete. METHODS: We aimed to identify transient receptor potential vanilloid 1 (TRPV1) variants within the entire coding sequence in patients who developed sensitivity to MH of unknown etiology. In vitro and in vivo functional studies were performed in heterologous expression system, trpv1-/- mice, and a murine model of human MH. RESULTS: We identified TRPV1 variants in two patients and their heterologous expression in muscles of trpv1-/- mice strongly enhanced calcium release from SR upon halogenated anesthetic stimulation, suggesting they could be responsible for the MH phenotype. We confirmed the in vivo significance by using mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation associated with MH in humans. We showed that the TRPV1 antagonist capsazepine slows the heat-induced hypermetabolic response in this model. CONCLUSION: We propose that TRPV1 contributes to MH and could represent an actionable therapeutic target for prevention of the pathology and also be responsible for MH sensitivity when mutated.

Paclitaxel Treatment and Proprotein Convertase 1/3 (PC1/3) Knockdown in Macrophages is a Promising Antiglioma Strategy as Revealed by Proteomics and Cytotoxicity Studies.

High grade gliomas are the most common brain tumors in adult. These tumors are characterized by a high infiltration in microglial cells and macrophages. The immunosuppressive tumor environment is known to orient immune cells toward a pro-tumoral and anti-inflammatory phenotype. Therefore, the current challenge for cancer therapy is to find a way to reorient macrophages toward an antitumoral phenotype. Previously, we demonstrated that macrophages secreted antitumoral factors when they were invalidated for the proprotein converstase 1/3 (PC1/3) and treated with LPS. However, achieving an activation of macrophages via LPS/TLR4/Myd88-dependent pathway appears yet unfeasible in cancer patients. On the contrary, the antitumor drug Paclitaxel is also known to activate the TLR4 MyD88-dependent signaling pathway and mimics LPS action. Therefore, we evaluated if PC1/3 knock-down (KD) macrophages could be activated by Paclitaxel and efficient against glioma. We report here that such a treatment of PC1/3 KD macrophages drove to the overexpression of proteins mainly involved in cytoskeleton rearrangement. In support of this finding, we found that these cells exhibited a Ca2+ increase after Paclitaxel treatment. This is indicative of a possible depolymerization of microtubules and may therefore reflect an activation of inflammatory pathways in macrophages. In such a way, we found that PC1/3 KD macrophages displayed a repression of the anti-inflammatory pathway STAT3 and secreted more pro-inflammatory cytokines. Extracellular vesicles isolated from these PC1/3 KD cells inhibited glioma growth. Finally, the supernatant collected from the coculture between glioma cells and PC1/3 KD macrophages contained more antitumoral factors. These findings unravel the potential value of a new therapeutic strategy combining Paclitaxel and PC1/3 inhibition to switch macrophages toward an antitumoral immunophenotype.

Ferroquine, the next generation antimalarial drug, has antitumor activity.

Despite the tremendous progress in medicine, cancer remains one of the most serious global health problems awaiting new effective therapies. Here we present ferroquine (FQ), the next generation antimalarial drug, as a promising candidate for repositioning as cancer therapeutics. We report that FQ potently inhibits autophagy, perturbs lysosomal function and impairs prostate tumor growth in vivo. We demonstrate that FQ negatively regulates Akt kinase and hypoxia-inducible factor-1α (HIF-1α) and is particularly effective in starved and hypoxic conditions frequently observed in advanced solid cancers. FQ enhances the anticancer activity of several chemotherapeutics suggesting its potential application as an adjuvant to existing anticancer therapy. Alike its parent compound chloroquine (CQ), FQ accumulates within and deacidifies lysosomes. Further, FQ induces lysosomal membrane permeabilization, mitochondrial depolarization and caspase-independent cancer cell death. Overall, our work identifies ferroquine as a promising new drug with a potent anticancer activity.

Targeting of short TRPM8 isoforms induces 4TM-TRPM8-dependent apoptosis in prostate cancer cells.

Since its cloning a decade ago, TRPM8 channel has emerged as a promising prognostic marker and a putative therapeutic target in prostate cancer (PCa). However, recent studies have brought to light the complexity of TRPM8 isoforms in PCa. Consequently, the respective role of each TRPM8 isoform needs to be deciphered prior to considering TRPM8 as an attractive therapeutic target. Full-length (6 transmembrane (TM)-domain) TRPM8 channel is overexpressed in early PCa and repressed in advanced prostate tumors whereas the localization of the truncated, 4TM-TRPM8 channel (4 transmembrane (TM)-domain), in the membranes of endoplasmic reticulum (ER) is independent of the pathogenic status of epithelial cells. In the same line, expression of non-channel cytoplasmic small TRPM8 isoforms (namely sM8) is conserved in cancer cells. In this study, we identify sM8s as putative regulator of PCa cell death. Indeed, suppression of sM8 isoforms was found to induce concomitantly ER stress, oxidative stress, p21 expression and apoptosis in human epithelial prostate cancer cells. We furthermore demonstrate that induction of such mechanisms required the activity of 4TM-TRPM8 channels at the ER-mitochondria junction. Our study thus suggests that targeting sM8 could be an appropriate strategy to fight prostate cancer.

The calcium-signaling toolkit: Updates needed.

Here, we review the role of Ca(2+) in apoptosis, namely that ER Ca(2+) depletion or a sustained elevation of cytosolic or mitochondrial Ca(2+) concentration are sufficient to trigger apoptosis. These concepts have emerged by the use of ER stressor agents that decrease the ER Ca(2+) pool by inhibiting SERCA pumps. However, aside from their well-known actions on Ca(2+) homeostasis disruption leading to apoptosis, new evidence show that some ER Ca(2+) modulators have significant implications in other Ca(2+)-mediated or Ca(2+)-independent pathways determining cell fate suggesting a more complex regulation of apoptosis by intracellular Ca(2+). Here, we discuss the crucial interplay between Ca(2+) mediated apoptosis, the Unfold Protein Response and autophagy determining cell fate, and the molecular compounds that have been used to depict these pathways. This review of the literature clearly shows the need for new inhibitors that do not interfere concomitantly with autophagy and Ca(2+) signaling. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.

Molecular Consequences of Proprotein Convertase 1/3 (PC1/3) Inhibition in Macrophages for Application to Cancer Immunotherapy: A Proteomic Study.

Macrophages provide the first line of host immune defense. Their activation triggers the secretion of pro-inflammatory cytokines and chemokines recruiting other immune cells. In cancer, macrophages present an M2 anti-inflammatory phenotype promoting tumor growth. In this way, strategies need to be develop to reactivate macrophages. Previously thought to be expressed only in cells with a neural/neuroendocrine phenotype, the proprotein convertase 1/3 has been shown to also be expressed in macrophages and regulated as a function of the Toll-like receptor immune response. Here, we investigated the intracellular impact of the down-regulation of the proprotein convertase 1/3 in NR8383 macrophages and confirmed the results on macrophages from PC1/3 deficient mice. A complete proteomic study of secretomes and intracellular proteins was undertaken and revealed that inhibition of proprotein convertase 1/3 orient macrophages toward an M1 activated phenotype. This phenotype is characterized by filopodial extensions, Toll-like receptor 4 MyD88-dependent signaling, calcium entry augmentation and the secretion of pro-inflammatory factors. In response to endotoxin/lipopolysaccharide, these intracellular modifications increased, and the secreted factors attracted naïve T helper lymphocytes to promote the cytotoxic response. Importantly, the application of these factors onto breast and ovarian cancer cells resulted in a decrease viability or resistance. Under inhibitory conditions using interleukin 10, PC1/3-knockdown macrophages continued to secrete inflammatory factors. These data indicate that targeted inhibition of proprotein convertase 1/3 could represent a novel type of immune therapy to reactivate intra-tumoral macrophages.

Disrupting the dynamic equilibrium of ORAI channels determines the phenotype of malignant cells.

We recently unraveled a finely tuned oncogenic mechanism in which genetic and tumor microenvironment alterations act together on a crucial calcium signaling pathway. This pathway involves an interconnected equilibrium of calcium channels functioning like a binary star system in which ORAI1 homomers and ORAI1/3 heteromers are two companion stars under the influence of each other that orbit around the cancer hallmarks of apoptosis resistance and enhanced proliferation.

TRPV6 calcium channel translocates to the plasma membrane via Orai1-mediated mechanism and controls cancer cell survival.

Transient receptor potential vanilloid subfamily member 6 (TRPV6) is a highly selective calcium channel that has been considered as a part of store-operated calcium entry (SOCE). Despite its first discovery in the early 2000s, the role of this channel in prostate cancer (PCa) remained, until now, obscure. Here we show that TRPV6 mediates calcium entry, which is highly increased in PCa due to the remodeling mechanism involving the translocation of the TRPV6 channel to the plasma membrane via the Orai1/TRPC1-mediated Ca(2+)/Annexin I/S100A11 pathway, partially contributing to SOCE. The TRPV6 calcium channel is expressed de novo by the PCa cell to increase its survival by enhancing proliferation and conferring apoptosis resistance. Xenografts in nude mice and bone metastasis models confirmed the remarkable aggressiveness of TRPV6-overexpressing tumors. Immunohistochemical analysis of these demonstrated the increased expression of clinical markers such as Ki-67, prostate specific antigen, synaptophysin, CD31, and CD56, which are strongly associated with a poor prognosis. Thus, the TRPV6 channel acquires its oncogenic potential in PCa due to the remodeling mechanism via the Orai1-mediated Ca(2+)/Annexin I/S100A11 pathway.

Remodeling of channel-forming ORAI proteins determines an oncogenic switch in prostate cancer.

ORAI family channels have emerged as important players in malignant transformation, yet the way in which they reprogram cancer cells remains elusive. Here we show that the relative expression levels of ORAI proteins in prostate cancer are different from that in noncancerous tissue. By mimicking ORAI protein remodeling observed in primary tumors, we demonstrate in in vitro models that enhanced ORAI3 expression favors heteromerization with ORAI1 to form a novel channel. These channels support store-independent Ca(2+) entry, thereby promoting cell proliferation and a smaller number of functional homomeric ORAI1-based store-operated channels, which are important in supporting susceptibility to apoptosis. Thus, our findings highlight disrupted dynamic equilibrium of channel-forming proteins as an oncogenic mechanism.

Complex modulation of the cold receptor TRPM8 by volatile anaesthetics and its role in complications of general anaesthesia.

The mechanisms by which volatile general anaesthetics (VAs) produce a depression of central nervous system are beginning to be better understood, but little is known about a number of side effects. Here, we show that the cold receptor transient receptor potential melastatin 8 (TRPM8) undergoes a complex modulation by clinical concentrations of VAs in dorsal root ganglion neurons and HEK-293 cells heterologously expressing TRPM8. VAs produced a transient enhancement of TRPM8 through a depolarizing shift of its activation towards physiological membrane potentials, followed by a sustained TRPM8 inhibition. The stimulatory action of VAs engaged molecular determinants distinct from those used by the TRPM8 agonist. Transient TRPM8 activation by VAs could explain side effects such as inhibition of respiratory drive, shivering and the cooling sensation during the beginning of anaesthesia, whereas the second phase of VA action, that associated with sustained TRPM8 inhibition, might be responsible for hypothermia. Consistent with this, both hypothermia and the inhibition of respiratory drive induced by VAs are partially abolished in Trpm8-knockout animals. Thus, we propose TRPM8 as a new clinical target for diminishing common and serious complications of general anaesthesia.

Differential effects of thapsigargin analogues on apoptosis of prostate cancer cells: complex regulation by intracellular calcium.

The inhibition of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) by thapsigargin (Tg) and Tg-type analogues is considered to trigger cell death by activation of apoptotic pathways. Some of these analogues may be useful as antineoplastic agents after appropriate targeting as peptide conjugated prodrugs to cancer cells. With this in mind, this study evaluates the effect on LNCaP androgen-sensitive cancer cells of thapsigargin substituted with 12-aminododecanoyl linkers and Leu (Leu-8ADT), aspartate (Asp-8ADT) or Boc-8ADT. Our results show that both Leu-8ADT and Asp-8ADT result in rapid ER calcium depletion and an influx of calcium across the plasma membrane by activation of store-operated calcium entry. By contrast, ER Ca(2+) depletion by Boc-8ADT is a very slow process that does not perceptibly increase cytosolic Ca(2+) and activate store-operated calcium entry, because the inhibition of SERCA with this compound is very slow. Nevertheless, we find that Boc-8ADT is a more efficient inducer of apoptosis than both Tg and Leu-8ADT. Compared with Tg and the other analogues, apoptosis induced by Asp-8ADT is very modest, although this compound also activates store-operated calcium entry and at high concentrations (1 µm) causes severe morphological changes, reflecting decreased cell viability. We conclude that many factors need to be considered for optimization of these compounds in antineoplastic drug design. Among these ER stress induced by Ca(2+) endoplasmic reticulum mobilization seems particularly important, whereas the early cytosolic increase of Ca(2+) concentration preceding the executive phase of apoptosis appears to be of no, or little, consequence for a subsequent apoptotic effect.

TRPV2 mediates adrenomedullin stimulation of prostate and urothelial cancer cell adhesion, migration and invasion.

Adrenomedullin (AM) is a 52-amino acid peptide initially isolated from human pheochromocytoma. AM is expressed in a variety of malignant tissues and cancer cell lines and was shown to be a mitogenic factor capable of stimulating growth of several cancer cell types. In addition, AM is a survival factor for certain cancer cells. Some data suggest that AM might be involved in the progression cancer metastasis via angiogenesis and cell migration and invasion control. The Transient Receptor Potential channel TRPV2 is known to promote in prostate cancer cell migration and invasive phenotype and is correlated with the stage and grade of bladder cancer. In this work we show that AM induces prostate and urothelial cancer cell migration and invasion through TRPV2 translocation to plasma membrane and the subsequent increase in resting calcium level.