Lysosomal TRPML1 triggers global Ca2+ signals and nitric oxide release in human cerebrovascular endothelial cells.

Lysosomal Ca2+ signaling is emerging as a crucial regulator of endothelial Ca2+ dynamics. Ca2+ release from the acidic vesicles in response to extracellular stimulation is usually promoted via Two Pore Channels (TPCs) and is amplified by endoplasmic reticulum (ER)-embedded inositol-1,3,4-trisphosphate (InsP3) receptors and ryanodine receptors. Emerging evidence suggests that sub-cellular Ca2+ signals in vascular endothelial cells can also be generated by the Transient Receptor Potential Mucolipin 1 channel (TRPML1) channel, which controls vesicle trafficking, autophagy and gene expression. Herein, we adopted a multidisciplinary approach, including live cell imaging, pharmacological manipulation, and gene targeting, revealing that TRPML1 protein is expressed and triggers global Ca2+ signals in the human brain microvascular endothelial cell line, hCMEC/D3. The direct stimulation of TRPML1 with both the synthetic agonist, ML-SA1, and the endogenous ligand phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) induced a significant increase in [Ca2+]i, that was reduced by pharmacological blockade and genetic silencing of TRPML1. In addition, TRPML1-mediated lysosomal Ca2+ release was sustained both by lysosomal Ca2+ release and ER Ca2+- release through inositol-1,4,5-trisphophate receptors and store-operated Ca2+ entry. Notably, interfering with TRPML1-mediated lysosomal Ca2+ mobilization led to a decrease in the free ER Ca2+ concentration. Imaging of DAF-FM fluorescence revealed that TRPML1 stimulation could also induce a significant Ca2+-dependent increase in nitric oxide concentration. Finally, the pharmacological and genetic blockade of TRPML1 impaired ATP-induced intracellular Ca2+ release and NO production. These findings, therefore, shed novel light on the mechanisms whereby the lysosomal Ca2+ store can shape endothelial Ca2+ signaling and Ca2+-dependent functions in vascular endothelial cells.

The ß3-AR agonist BRL37344 ameliorates the main symptoms of X-linked nephrogenic diabetes insipidus in the mouse model of the disease.

X-linked nephrogenic diabetes insipidus (X-NDI) is a rare congenital disease caused by inactivating mutations of the vasopressin type-2 receptor (AVPR2), characterized by impaired renal concentrating ability, dramatic polyuria, polydipsia and risk of dehydration. The disease, which still lacks a cure, could benefit from the pharmacologic stimulation of other GPCRs, activating the cAMP-intracellular pathway in the kidney cells expressing the AVPR2. On the basis of our previous studies, we here hypothesized that the ß3-adrenergic receptor could be such an ideal candidate. We evaluated the effect of continuous 24 h stimulation of the ß3-AR with the agonist BRL37344 and assessed the effects on urine output, urine osmolarity, water intake and the abundance and activation of the key renal water and electrolyte transporters, in the mouse model of X-NDI. Here we demonstrate that the ß3-AR agonism exhibits a potent antidiuretic effect. The strong improvement in symptoms of X-NDI produced by a single i.p. injection of BRL37344 (1 mg/kg) was limited to 3 h but repeated administrations in the 24 h, mimicking the effect of a slow-release preparation, promoted a sustained antidiuretic effect, reducing the 24 h urine output by 27%, increasing urine osmolarity by 25% and reducing the water intake by 20%. At the molecular level, we show that BRL37344 acted by increasing the phosphorylation of NKCC2, NCC and AQP2 in the renal cell membrane, thereby increasing electrolytes and water reabsorption in the kidney tubule of X-NDI mice. Taken together, these data suggest that human ß3-AR agonists might represent an effective possible treatment strategy for X-NDI.

ß3-Adrenoceptor as a new player in the sympathetic regulation of the renal acid-base homeostasis.

Efferent sympathetic nerve fibers regulate several renal functions activating norepinephrine receptors on tubular epithelial cells. Of the beta-adrenoceptors (ß-ARs), we previously demonstrated the renal expression of ß3-AR in the thick ascending limb (TAL), the distal convoluted tubule (DCT), and the collecting duct (CD), where it participates in salt and water reabsorption. Here, for the first time, we reported ß3-AR expression in the CD intercalated cells (ICCs), where it regulates acid-base homeostasis. Co-localization of ß3-AR with either proton pump H+-ATPase or Cl-/HCO3 - exchanger pendrin revealed ß3-AR expression in type A, type B, non-A, and non-B ICCs in the mouse kidney. We aimed to unveil the possible regulatory role of ß3-AR in renal acid-base homeostasis, in particular in modulating the expression, subcellular localization, and activity of the renal H+-ATPase, a key player in this process. The abundance of H+-ATPase was significantly decreased in the kidneys of ß3-AR-/- compared with those of ß3-AR+/+ mice. In particular, H+-ATPase reduction was observed not only in the CD but also in the TAL and DCT, which contribute to acid-base transport in the kidney. Interestingly, we found that in in vivo, the absence of ß3-AR reduced the kidneys' ability to excrete excess proton in the urine during an acid challenge. Using ex vivo stimulation of mouse kidney slices, we proved that the ß3-AR activation promoted H+-ATPase apical expression in the epithelial cells of ß3-AR-expressing nephron segments, and this was prevented by ß3-AR antagonism or PKA inhibition. Moreover, we assessed the effect of ß3-AR stimulation on H+-ATPase activity by measuring the intracellular pH recovery after an acid load in ß3-AR-expressing mouse renal cells. Importantly, ß3-AR agonism induced a 2.5-fold increase in H+-ATPase activity, and this effect was effectively prevented by ß3-AR antagonism or by inhibiting either H+-ATPase or PKA. Of note, in urine samples from patients treated with a ß3-AR agonist, we found that ß3-AR stimulation increased the urinary excretion of H+-ATPase, likely indicating its apical accumulation in tubular cells. These findings demonstrate that ß3-AR activity positively regulates the expression, plasma membrane localization, and activity of H+-ATPase, elucidating a novel physiological role of ß3-AR in the sympathetic control of renal acid-base homeostasis.

Intracellular Ca2+ signalling: unexpected new roles for the usual suspect.

Cytosolic Ca2+ signals are organized in complex spatial and temporal patterns that underlie their unique ability to regulate multiple cellular functions. Changes in intracellular Ca2+ concentration ([Ca2+]i) are finely tuned by the concerted interaction of membrane receptors and ion channels that introduce Ca2+ into the cytosol, Ca2+-dependent sensors and effectors that translate the elevation in [Ca2+]i into a biological output, and Ca2+-clearing mechanisms that return the [Ca2+]i to pre-stimulation levels and prevent cytotoxic Ca2+ overload. The assortment of the Ca2+ handling machinery varies among different cell types to generate intracellular Ca2+ signals that are selectively tailored to subserve specific functions. The advent of novel high-speed, 2D and 3D time-lapse imaging techniques, single-wavelength and genetic Ca2+ indicators, as well as the development of novel genetic engineering tools to manipulate single cells and whole animals, has shed novel light on the regulation of cellular activity by the Ca2+ handling machinery. A symposium organized within the framework of the 72nd Annual Meeting of the Italian Society of Physiology, held in Bari on 14-16th September 2022, has recently addressed many of the unexpected mechanisms whereby intracellular Ca2+ signalling regulates cellular fate in healthy and disease states. Herein, we present a report of this symposium, in which the following emerging topics were discussed: 1) Regulation of water reabsorption in the kidney by lysosomal Ca2+ release through Transient Receptor Potential Mucolipin 1 (TRPML1); 2) Endoplasmic reticulum-to-mitochondria Ca2+ transfer in Alzheimer's disease-related astroglial dysfunction; 3) The non-canonical role of TRP Melastatin 8 (TRPM8) as a Rap1A inhibitor in the definition of some cancer hallmarks; and 4) Non-genetic optical stimulation of Ca2+ signals in the cardiovascular system.

Transient receptor potential ankyrin 1 (TRPA1) mediates reactive oxygen species-induced Ca2+ entry, mitochondrial dysfunction, and caspase-3/7 activation in primary cultures of metastatic colorectal carcinoma cells.

Colorectal carcinoma (CRC) represents the fourth most common cancer worldwide and is the third most common cause of malignancy-associated mortality. Distant metastases to the liver and lungs are the main drivers of CRC-dependent death. Pro-oxidant therapies, which halt disease progression by exacerbating oxidative stress, represent an antitumour strategy that is currently exploited by chemotherapy and ionizing radiation. A more selective strategy to therapeutically exploit reactive oxygen species (ROS) signaling would consist in targeting a redox sensor that is up-regulated in metastatic cells and is tightly coupled to the stimulation of cancer cell death programs. The non-selective cation channel, Transient Receptor Potential Ankyrin 1 (TRPA1), serves as a sensor of the cellular redox state, being activated to promote extracellular Ca2+ entry by an increase in oxidative stress. Recent work demonstrated that TRPA1 channel protein is up-regulated in several cancer types and that TRPA1-mediated Ca2+ signals can either engage an antiapoptotic pro-survival signaling pathway or to promote mitochondrial Ca2+ dysfunction and apoptosis. Herein, we sought to assess for the first time the outcome of TRPA1 activation by ROS on primary cultures of metastatic colorectal carcinoma (mCRC cells). We found that TRPA1 channel protein is up-regulated and mediates enhanced hydrogen peroxide (H2O2)-induced Ca2+ entry in mCRC cells as compared to non-neoplastic control cells. The lipid peroxidation product 4-hydroxynonenal (4-HNE) is the main ROS responsible for TRPA1 activation upon mCRC cell exposure to oxidative stress. TRPA1-mediated Ca2+ entry in response to H2O2 and 4-HNE results in mitochondrial Ca2+ overload, followed by mitochondrial depolarization and caspase-3/7 activation. Therefore, targeting TRPA1 could represent an alternative strategy to eradicate metastatic CRC by enhancing its sensitivity to oxidative stress.

Targeting unfolded protein response reverts ER stress and ER Ca2+ homeostasis in cardiomyocytes expressing the pathogenic variant of Lamin A/C R321X.

BACKGROUND: We previously demonstrated that an Italian family affected by a severe dilated cardiomyopathy (DCM) with history of sudden deaths at young age, carried a mutation in the Lmna gene encoding for a truncated variant of the Lamin A/C protein (LMNA), R321X. When expressed in heterologous systems, such variant accumulates into the endoplasmic reticulum (ER), inducing the activation of the PERK-CHOP pathway of the unfolded protein response (UPR), ER dysfunction and increased rate of apoptosis. The aim of this work was to analyze whether targeting the UPR can be used to revert the ER dysfunction associated with LMNA R321X expression in HL-1 cardiac cells. METHODS: HL-1 cardiomyocytes stably expressing LMNA R321X were used to assess the ability of 3 different drugs targeting the UPR, salubrinal, guanabenz and empagliflozin to rescue ER stress and dysfunction. In these cells, the state of activation of both the UPR and the pro-apoptotic pathway were analyzed monitoring the expression levels of phospho-PERK, phospho-eIF2α, ATF4, CHOP and PARP-CL. In addition, we measured ER-dependent intracellular Ca2+ dynamics as indicator of proper ER functionality. RESULTS: We found that salubrinal and guanabenz increased the expression levels of phospho-eIF2α and downregulated the apoptosis markers CHOP and PARP-CL in LMNA R321X-cardiomyocytes, maintaining the so-called adaptive UPR. These drugs also restored ER ability to handle Ca2+ in these cardiomyocytes. Interestingly, we found that empagliflozin downregulated the apoptosis markers CHOP and PARP-CL shutting down the UPR itself through the inhibition of PERK phosphorylation in LMNA R321X-cardiomyocytes. Furthermore, upon empagliflozin treatment, ER homeostasis, in terms of ER ability to store and release intracellular Ca2+ was also restored in these cardiomyocytes. CONCLUSIONS: We provided evidence that the different drugs, although interfering with different steps of the UPR, were able to counteract pro-apoptotic processes and to preserve the ER homeostasis in R321X LMNA-cardiomyocytes. Of note, two of the tested drugs, guanabenz and empagliflozin, are already used in the clinical practice, thus providing preclinical evidence for ready-to-use therapies in patients affected by the LMNA R321X associated cardiomyocytes.

TRPML1-Induced Lysosomal Ca2+ Signals Activate AQP2 Translocation and Water Flux in Renal Collecting Duct Cells.

Lysosomes are acidic Ca2+ storage organelles that actively generate local Ca2+ signaling events to regulate a plethora of cell functions. Here, we characterized lysosomal Ca2+ signals in mouse renal collecting duct (CD) cells and we assessed their putative role in aquaporin 2 (AQP2)-dependent water reabsorption. Bafilomycin A1 and ML-SA1 triggered similar Ca2+ oscillations, in the absence of extracellular Ca2+, by alkalizing the acidic lysosomal pH or activating the lysosomal cation channel mucolipin 1 (TRPML1), respectively. TRPML1-dependent Ca2+ signals were blocked either pharmacologically or by lysosomes' osmotic permeabilization, thus indicating these organelles as primary sources of Ca2+ release. Lysosome-induced Ca2+ oscillations were sustained by endoplasmic reticulum (ER) Ca2+ content, while bafilomycin A1 and ML-SA1 did not directly interfere with ER Ca2+ homeostasis per se. TRPML1 activation strongly increased AQP2 apical expression and depolymerized the actin cytoskeleton, thereby boosting water flux in response to an hypoosmotic stimulus. These effects were strictly dependent on the activation of the Ca2+/calcineurin pathway. Conversely, bafilomycin A1 led to perinuclear accumulation of AQP2 vesicles without affecting water permeability. Overall, lysosomal Ca2+ signaling events can be differently decoded to modulate Ca2+-dependent cellular functions related to the dock/fusion of AQP2-transporting vesicles in principal cells of the CD.

ß3 Adrenergic Receptor Agonist Mirabegron Increases AQP2 and NKCC2 Urinary Excretion in OAB Patients: A Pleiotropic Effect of Interest for Patients with X-Linked Nephrogenic Diabetes Insipidus.

We previously reported the novel finding that ß3-AR is functionally expressed in the renal tubule and shares its cellular localization with the vasopressin receptor AVPR2, whose physiological stimulation triggers antidiuresis by increasing the plasma membrane expression of the water channel AQP2 and the NKCC2 symporter in renal cells. We also showed that pharmacologic stimulation of ß3-AR is capable of triggering antidiuresis and correcting polyuria, in the knockout mice for the AVPR2 receptor, the animal model of human X-linked nephrogenic diabetes insipidus (XNDI), a rare genetic disease still missing a cure. Here, to demonstrate that the same response can be evoked in humans, we evaluated the effect of treatment with the ß3-AR agonist mirabegron on AQP2 and NKCC2 trafficking, by evaluating their urinary excretion in a cohort of patients with overactive bladder syndrome, for the treatment of which the drug is already approved. Compared to baseline, treatment with mirabegron significantly increased AQP2 and NKCC2 excretion for the 12 weeks of treatment. This data is a step forward in corroborating the hypothesis that in patients with XNDI, treatment with mirabegron could bypass the inactivation of AVPR2, trigger antidiuresis and correct the dramatic polyuria which is the main hallmark of this disease.

Neuroprotective Effect of Antiapoptotic URG7 Protein on Human Neuroblastoma Cell Line SH-SY5Y.

Up-regulated Gene clone 7 (URG7) is a protein localized in the endoplasmic reticulum (ER) and overexpressed in liver cells upon hepatitis B virus (HBV) infection. Its activity has been related to the attenuation of ER stress resulting from HBV infection, promoting protein folding and ubiquitination and reducing cell apoptosis overall. While the antiapoptotic activity of URG7 in HBV-infected cells may have negative implications, this effect could be exploited positively in the field of proteinopathies, such as neurodegenerative diseases. In this work, we aimed to verify the possible contribution of URG7 as a reliever of cellular proteostasis alterations in a neuronal in vitro system. Following tunicamycin-induced ER stress, URG7 was shown to modulate different markers of the unfolded protein response (UPR) in favor of cell survival, mitigating ER stress and activating autophagy. Furthermore, URG7 promoted ubiquitination, and determined a reduction in protein aggregation, calcium release from the ER and intracellular ROS content, confirming its pro-survival activity. Therefore, in light of the results reported in this work, we hypothesize that URG7 offers activity as an ER stress reliever in a neuronal in vitro model, and we paved the way for a new approach in the treatment of neurodegenerative diseases.

Evidence of the different effect of mercury and cadmium on the hIAPP aggregation process.

hIAPP is a hormone consisting of 37 aminoacids that shows a strong tendency to self-assemble into ß-sheet-rich aggregates, which evolve to form insoluble aggregates that seem to be associated with ß-cell degeneration in Type 2 Diabetes Mellitus. Numerous factors, intrinsic and extrinsic to the peptide molecule, appear to influence the hIAPP aggregation process. Different metal ions are able to interact with the hIAPP molecule, modulating its secondary structure and subsequently the peptide's capacity to aggregate. In this study, the effect of Hg2+ and Cd2+ on the hIAPP aggregation process was evaluated using direct and indirect methods. The kinetics and morphology of amyloid aggregate formation were respectively evaluated with Thioflavin T assays and electron microscopy, while the ability of the peptide to incorporate into POPC PLMs and form ion channels was monitored by single-channel current measurements. Hg2+ and Cd2+ each seem to modulate the peptide's ability to aggregate in a different way, suggesting a different mechanism of hIAPP toxicity.

Role of Nuclear Lamin A/C in the Regulation of Nav1.5 Channel and Microtubules: Lesson From the Pathogenic Lamin A/C Variant Q517X.

In this work, we studied an lmna nonsense mutation encoding for the C-terminally truncated Lamin A/C (LMNA) variant Q517X, which was described in patients affected by a severe arrhythmogenic cardiomyopathy with history of sudden death. We found that LMNA Q517X stably expressed in HL-1 cardiomyocytes abnormally aggregates at the nuclear envelope and within the nucleoplasm. Whole-cell patch clamp experiments showed that LMNA Q517X-expressing cardiomyocytes generated action potentials with reduced amplitude, overshoot, upstroke velocity and diastolic potential compared with LMNA WT-expressing cardiomyocytes. Moreover, the unique features of these cardiomyocytes were 1) hyper-polymerized tubulin network, 2) upregulated acetylated α-tubulin, and 3) cell surface Nav1.5 downregulation. These findings pointed the light on the role of tubulin and Nav1.5 channel in the abnormal electrical properties of LMNA Q517X-expressing cardiomyocytes. When expressed in HEK293 with Nav1.5 and its ß1 subunit, LMNA Q517X reduced the peak Na+ current (INa) up to 63% with a shift toward positive potentials in the activation curve of the channel. Of note, both AP properties in cardiomyocytes and Nav1.5 kinetics in HEK293 cells were rescued in LMNA Q517X-expressing cells upon treatment with colchicine, an FDA-approved inhibitor of tubulin assembly. In conclusion, LMNA Q517X expression is associated with hyper-polymerization and hyper-acetylation of tubulin network with concomitant downregulation of Nav1.5 cell expression and activity, thus revealing 1) new mechanisms by which LMNA may regulate channels at the cell surface in cardiomyocytes and 2) new pathomechanisms and therapeutic targets in cardiac laminopathies.

The Non-Gastric H+/K+ ATPase (ATP12A) Is Expressed in Mammalian Spermatozoa.

H+/K+ ATPase Type 2 is an heteromeric membrane protein involved in cation transmembrane transport and consists of two subunits: a specific α subunit (ATP12A) and a non-specific ß subunit. The aim of this study was to demonstrate the presence and establish the localization of ATP12A in spermatozoa from Bubalus bubalis, Bos taurus and Ovis aries. Immunoblotting revealed, in all three species, a major band (100 kDa) corresponding to the expected molecular mass. The ATP12A immunolocalization pattern showed, consistently in the three species, a strong signal at the acrosome. These results, described here for the first time in spermatozoa, are consistent with those observed for the ß1 subunit of Na+/K+ ATPase, suggesting that the latter may assemble with the α subunit to produce a functional ATP12A dimer in sperm cells. The above scenario appeared to be nicely supported by 3D comparative modeling and interaction energy calculations. The expression of ATP12A during different stages of bovine sperm maturation progressively increased, moving from epididymis to deferent ducts. Based on overall results, we hypothesize that ATP12A may play a role in acrosome reactions. Further studies will be required in order to address the functional role of this target protein in sperm physiology.

Pro-inflammatory cytokines as emerging molecular determinants in cardiolaminopathies.

Mutations in Lamin A/C gene (lmna) cause a wide spectrum of cardiolaminopathies strictly associated with significant deterioration of the electrical and contractile function of the heart. Despite the continuous flow of biomedical evidence, linking cardiac inflammation to heart remodelling in patients harbouring lmna mutations is puzzling. Therefore, we profiled 30 serum cytokines/chemokines in patients belonging to four different families carrying pathogenic lmna mutations segregating with cardiac phenotypes at different stages of severity (n = 19) and in healthy subjects (n = 11). Regardless lmna mutation subtype, high levels of circulating granulocyte colony-stimulating factor (G-CSF) and interleukin 6 (IL-6) were found in all affected patients' sera. In addition, elevated levels of Interleukins (IL) IL-1Ra, IL-1ß IL-4, IL-5 and IL-8 and the granulocyte-macrophage colony-stimulating factor (GM-CSF) were measured in a large subset of patients associated with more aggressive clinical manifestations. Finally, the expression of the pro-inflammatory 70 kDa heat shock protein (Hsp70) was significantly increased in serum exosomes of patients harbouring the lmna mutation associated with the more severe phenotype. Overall, the identification of patient subsets with overactive or dysregulated myocardial inflammatory responses could represent an innovative diagnostic, prognostic and therapeutic tool against Lamin A/C cardiomyopathies.

Aquaporin-1 Facilitates Transmesothelial Water Permeability: In Vitro and Ex Vivo Evidence and Possible Implications in Peritoneal Dialysis.

We previously showed that mesothelial cells in human peritoneum express the water channel aquaporin 1 (AQP1) at the plasma membrane, suggesting that, although in a non-physiological context, it may facilitate osmotic water exchange during peritoneal dialysis (PD). According to the three-pore model that predicts the transport of water during PD, the endothelium of peritoneal capillaries is the major limiting barrier to water transport across peritoneum, assuming the functional role of the mesothelium, as a semipermeable barrier, to be negligible. We hypothesized that an intact mesothelial layer is poorly permeable to water unless AQP1 is expressed at the plasma membrane. To demonstrate that, we characterized an immortalized cell line of human mesothelium (HMC) and measured the osmotically-driven transmesothelial water flux in the absence or in the presence of AQP1. The presence of tight junctions between HMC was investigated by immunofluorescence. Bioelectrical parameters of HMC monolayers were studied by Ussing Chambers and transepithelial water transport was investigated by an electrophysiological approach based on measurements of TEA+ dilution in the apical bathing solution, through TEA+-sensitive microelectrodes. HMCs express Zo-1 and occludin at the tight junctions and a transepithelial vectorial Na+ transport. Real-time transmesothelial water flux, in response to an increase of osmolarity in the apical solution, indicated that, in the presence of AQP1, the rate of TEA+ dilution was up to four-fold higher than in its absence. Of note, we confirmed our data in isolated mouse mesentery patches, where we measured an AQP1-dependent transmesothelial osmotic water transport. These results suggest that the mesothelium may represent an additional selective barrier regulating water transport in PD through functional expression of the water channel AQP1.

Activation of the Thiazide-Sensitive Sodium-Chloride Cotransporter by Beta3-Adrenoreceptor in the Distal Convoluted Tubule.

We previously showed that the beta-3 adrenergic receptor (BAR3) is expressed in most segments of the nephron where its agonism promotes a potent antidiuretic effect. We localized BAR3 in distal convoluted tubule (DCT) cells expressing the thiazide-sensitive sodium-chloride cotransporter (NCC). Aim of this study is to investigate the possible functional role of BAR3 on NCC modulation in DCT cells. Here, we found that, in mice, the knockout of BAR3 was paralleled by a significant attenuation of NCC phosphorylation, paralleled by reduced expression and activation of STE-20/SPS1-related proline-alanine-rich kinase (SPAK) and WNKs the main kinases involved in NCC activation. Conversely, in BAR1/2 knockout mice, we found reduced NCC abundance with no changes in the phosphorylation state of NCC. Moreover, selective BAR3 agonism promotes both SPAK and NCC activation in wild-type mouse kidney slices. In conclusion, our findings suggest a novel role for BAR3 in the regulation of NCC in DCT.

N-acetylaspartate release by glutaminolytic ovarian cancer cells sustains protumoral macrophages.

Glutaminolysis is known to correlate with ovarian cancer aggressiveness and invasion. However, how this affects the tumor microenvironment is elusive. Here, we show that ovarian cancer cells become addicted to extracellular glutamine when silenced for glutamine synthetase (GS), similar to naturally occurring GS-low, glutaminolysis-high ovarian cancer cells. Glutamine addiction elicits a crosstalk mechanism whereby cancer cells release N-acetylaspartate (NAA) which, through the inhibition of the NMDA receptor, and synergistically with IL-10, enforces GS expression in macrophages. In turn, GS-high macrophages acquire M2-like, tumorigenic features. Supporting this in␣vitro model, in silico data and the analysis of ascitic fluid isolated from ovarian cancer patients prove that an M2-like macrophage phenotype, IL-10 release, and NAA levels positively correlate with disease stage. Our study uncovers the unprecedented role of glutamine metabolism in modulating macrophage polarization in highly invasive ovarian cancer and highlights the anti-inflammatory, protumoral function of NAA.

Functional Characterization of Two Novel Mutations in SCN5A Associated with Brugada Syndrome Identified in Italian Patients.

BACKGROUND: Brugada syndrome (BrS) is an autosomal dominantly inherited cardiac disease characterized by "coved type" ST-segment elevation in the right precordial leads, high susceptibility to ventricular arrhythmia and a family history of sudden cardiac death. The SCN5A gene, encoding for the cardiac voltage-gated sodium channel Nav1.5, accounts for ~20-30% of BrS cases and is considered clinically relevant. METHODS: Here, we describe the clinical findings of two Italian families affected by BrS and provide the functional characterization of two novel SCN5A mutations, the missense variant Pro1310Leu and the in-frame insertion Gly1687_Ile1688insGlyArg. RESULTS: Despite being clinically different, both patients have a family history of sudden cardiac death and had history of arrhythmic events. The Pro1310Leu mutation significantly reduced peak sodium current density without affecting channel membrane localization. Changes in the gating properties of expressed Pro1310Leu channel likely account for the loss-of-function phenotype. On the other hand, Gly1687_Ile1688insGlyArg channel, identified in a female patient, yielded a nearly undetectable sodium current. Following mexiletine incubation, the Gly1687_Ile1688insGlyArg channel showed detectable, albeit very small, currents and biophysical properties similar to those of the Nav1.5 wild-type channel. CONCLUSIONS: Overall, our results suggest that the degree of loss-of-function shown by the two Nav1.5 mutant channels correlates with the aggressive clinical phenotype of the two probands. This genotype-phenotype correlation is fundamental to set out appropriate therapeutical intervention.

PKA compartmentalization links cAMP signaling and autophagy.

Autophagy is a highly regulated degradative process crucial for maintaining cell homeostasis. This important catabolic mechanism can be nonspecific, but usually occurs with fine spatial selectivity (compartmentalization), engaging only specific subcellular sites. While the molecular machines driving autophagy are well understood, the involvement of localized signaling events in this process is not well defined. Among the pathways that regulate autophagy, the cyclic AMP (cAMP)/protein kinase A (PKA) cascade can be compartmentalized in distinct functional units called microdomains. However, while it is well established that, depending on the cell type, cAMP can inhibit or promote autophagy, the role of cAMP/PKA microdomains has not been tested. Here we show not only that the effects on autophagy of the same cAMP elevation differ in different cell types, but that they depend on a highly complex sub-compartmentalization of the signaling cascade. We show in addition that, in HT-29 cells, in which autophagy is modulated by cAMP rising treatments, PKA activity is strictly regulated in space and time by phosphatases, which largely prevent the phosphorylation of soluble substrates, while membrane-bound targets are less sensitive to the action of these enzymes. Interestingly, we also found that the subcellular distribution of PKA type-II regulatory PKA subunits hinders the effect of PKA on autophagy, while displacement of type-I regulatory PKA subunits has no effect. Our data demonstrate that local PKA activity can occur independently of local cAMP concentrations and provide strong evidence for a link between localized PKA signaling events and autophagy.