Endosomal Acid-Base Homeostasis in Neurodegenerative Diseases.

Neurodegenerative disorders are debilitating and largely untreatable conditions that pose a significant burden to affected individuals and caregivers. Overwhelming evidence supports a crucial preclinical role for endosomal dysfunction as an upstream pathogenic hub and driver in Alzheimer's disease (AD) and related neurodegenerative disorders. We present recent advances on the role of endosomal acid-base homeostasis in neurodegeneration and discuss evidence for converging mechanisms. The strongest genetic risk factor in sporadic AD is the ε4 allele of Apolipoprotein E (ApoE4), which potentiates pre-symptomatic endosomal dysfunction and prominent amyloid beta (Aß) pathology, although how these pathways are linked mechanistically has remained unclear. There is emerging evidence that the Christianson syndrome protein NHE6 is a prominent ApoE4 effector linking endosomal function to Aß pathologies. By functioning as a dominant leak pathway for protons, the Na+/H+ exchanger activity of NHE6 limits endosomal acidification and regulates ß-secretase (BACE)-mediated Aß production and LRP1 receptor-mediated Aß clearance. Pathological endosomal acidification may impact both Aß generation and clearance mechanisms and emerges as a promising therapeutic target in AD. We also offer our perspective on the complex role of endosomal acid-base homeostasis in the pathogenesis of neurodegeneration and its therapeutic implications for neuronal rescue and repair strategies.

Store-independent activation of Orai1 by SPCA2 in mammary tumors.

Ca(2+) is an essential and ubiquitous second messenger. Changes in cytosolic Ca(2+) trigger events critical for tumorigenesis, such as cellular motility, proliferation, and apoptosis. We show that an isoform of Secretory Pathway Ca(2+)-ATPase, SPCA2, is upregulated in breast cancer-derived cells and human breast tumors, and suppression of SPCA2 attenuates basal Ca(2+) levels and tumorigenicity. Contrary to its conventional role in Golgi Ca(2+) sequestration, expression of SPCA2 increased Ca(2+) influx by a mechanism dependent on the store-operated Ca(2+) channel Orai1. Unexpectedly, SPCA2-Orai1 signaling was independent of ER Ca(2+) stores or STIM1 and STIM2 sensors and uncoupled from Ca(2+)-ATPase activity of SPCA2. Binding of the SPCA2 amino terminus to Orai1 enabled access of its carboxyl terminus to Orai1 and activation of Ca(2+) influx. Our findings reveal a signaling pathway in which the Orai1-SPCA2 complex elicits constitutive store-independent Ca(2+) signaling that promotes tumorigenesis.

Emerging links between endosomal pH and cancer.

Extracellular acidification is a well-known driver of tumorigenesis that has been extensively studied. In contrast, the role of endosomal pH is novel and relatively unexplored. There is emerging evidence from a growing number of studies showing that the pH of endosomal compartments controls proliferation, migration, stemness, and sensitivity to chemoradiation therapy in a variety of tumors. Endosomes are a crucial hub, mediating cellular communication with the external environment. By finely regulating the sorting and trafficking of vesicular cargo for degradation or recycling, endosomal pH determines the fate of plasma membrane proteins, lipids, and extracellular signals including growth factor receptors and their ligands. Several critical regulators of endosomal pH have been identified, including multiple isoforms of the family of electroneutral Na+/H+ exchangers (NHE) such as NHE6 and NHE9. Recent studies have shed light on molecular mechanisms linking endosomal pH to cancer malignancy. Manipulating endosomal pH by epigenetic reprogramming, small molecules, or nanoparticles may offer promising new options in cancer therapy. In this review, we summarize evidence linking endosomal pH to cancer, with a focus on the role of endosomal Na+/H+ exchangers and how they affect the prognosis of cancer patients, and also suggest how regulation of endosomal pH may be exploited to develop new cancer therapies.

Amyloid clearance defect in ApoE4 astrocytes is reversed by epigenetic correction of endosomal pH.

Endosomes have emerged as a central hub and pathogenic driver of Alzheimer's disease (AD). The earliest brain cytopathology in neurodegeneration, occurring decades before amyloid plaques and cognitive decline, is an expansion in the size and number of endosomal compartments. The strongest genetic risk factor for sporadic AD is the ε4 allele of Apolipoprotein E (ApoE4). Previous studies have shown that ApoE4 potentiates presymptomatic endosomal dysfunction and defective endocytic clearance of amyloid beta (Aß), although how these two pathways are linked at a cellular and mechanistic level has been unclear. Here, we show that aberrant endosomal acidification in ApoE4 astrocytes traps the low-density lipoprotein receptor-related protein (LRP1) within intracellular compartments, leading to loss of surface expression and Aß clearance. Pathological endosome acidification is caused by ε4 risk allele-selective down-regulation of the Na+/H+ exchanger isoform NHE6, which functions as a critical leak pathway for endosomal protons. In vivo, the NHE6 knockout (NHE6KO) mouse model showed elevated Aß in the brain, consistent with a causal effect. Increased nuclear translocation of histone deacetylase 4 (HDAC4) in ApoE4 astrocytes, compared with the nonpathogenic ApoE3 allele, suggested a mechanistic basis for transcriptional down-regulation of NHE6. HDAC inhibitors that restored NHE6 expression normalized ApoE4-specific defects in endosomal pH, LRP1 trafficking, and amyloid clearance. Thus, NHE6 is a downstream effector of ApoE4 and emerges as a promising therapeutic target in AD. These observations have prognostic implications for patients who have Christianson syndrome with loss of function mutations in NHE6 and exhibit prominent glial pathology and progressive hallmarks of neurodegeneration.

NHA2 promotes cyst development in an in vitro model of polycystic kidney disease.

KEY POINTS: Significant and selective up-regulation of the Na+ /H+ exchanger NHA2 (SLC9B2) was observed in cysts of patients with autosomal dominant polycystic kidney disease. Using the MDCK cell model of cystogenesis, it was found that NHA2 increases cyst size. Silencing or pharmacological inhibition of NHA2 inhibits cyst formation in vitro. Polycystin-1 represses NHA2 expression via Ca2+ /NFAT signalling whereas the dominant negative membrane-anchored C-terminal fragment (PC1-MAT) increased NHA2 levels. Drugs (caffeine, theophylline) and hormones (vasopressin, aldosterone) known to exacerbate cysts elicit NHA2 expression. Taken together, the findings reveal NHA2 as a potential new player in salt and water homeostasis in the kidney and in the pathogenesis of polycystic kidney disease. ABSTRACT: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2 encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The molecular pathways linking polycystins to cyst development in ADPKD are still unclear. Intracystic fluid secretion via ion transporters and channels plays a crucial role in cyst expansion in ADPKD. Unexpectedly, we observed significant and selective up-regulation of NHA2, a member of the SLC9B family of Na+ /H+ exchangers, that correlated with cyst size and disease severity in ADPKD patients. Using three-dimensional cultures of MDCK cells to model cystogenesis in vitro, we showed that ectopic expression of NHA2 is causal to increased cyst size. Induction of PC1 in MDCK cells inhibited NHA2 expression with concordant inhibition of Ca2+ influx through store-dependent and -independent pathways, whereas reciprocal activation of Ca2+ influx by the dominant negative membrane-anchored C-terminal tail fragment of PC1 elevated NHA2. We showed that NHA2 is a target of Ca2+ /NFAT signalling and is transcriptionally induced by methylxanthine drugs such as caffeine and theophylline, which are contraindicated in ADPKD patients. Finally, we observed robust induction of NHA2 by vasopressin, which is physiologically consistent with increased levels of circulating vasopressin and up-regulation of vasopressin V2 receptors in ADPKD. Our findings have mechanistic implications on the emerging use of vasopressin V2 receptor antagonists such as tolvaptan as safe and effective therapy for polycystic kidney disease and reveal a potential new regulator of transepithelial salt and water transport in the kidney.

Histone deacetylase-mediated regulation of endolysosomal pH.

The pH of the endolysosomal system is tightly regulated by a balance of proton pump and leak mechanisms that are critical for storage, recycling, turnover, and signaling functions in the cell. Dysregulation of endolysosomal pH has been linked to aging, amyloidogenesis, synaptic dysfunction, and various neurodegenerative disorders, including Alzheimer's disease. Therefore, understanding the mechanisms that regulate luminal pH may be key to identifying new targets for managing these disorders. Meta-analysis of yeast microarray databases revealed that nutrient-limiting conditions inhibited the histone deacetylase (HDAC) Rpd3 and thereby up-regulated transcription of the endosomal Na+/H+ exchanger Nhx1, resulting in vacuolar alkalinization. Consistent with these findings, Rpd3 inhibition by the HDAC inhibitor and antifungal drug trichostatin A induced Nhx1 expression and vacuolar alkalinization. Bioinformatics analysis of Drosophila and mouse databases revealed that caloric control of the Nhx1 orthologs DmNHE3 and NHE6, respectively, is also mediated by HDACs. We show that NHE6 is a target of the transcription factor cAMP-response element-binding protein (CREB), a known regulator of cellular responses to low-nutrient conditions, providing a molecular mechanism for nutrient- and HDAC-dependent regulation of endosomal pH. Of note, pharmacological targeting of the CREB pathway to increase NHE6 expression helped regulate endosomal pH and correct defective clearance of amyloid Aß in an apoE4 astrocyte model of Alzheimer's disease. These observations from yeast, fly, mouse, and cell culture models point to an evolutionarily conserved mechanism for HDAC-mediated regulation of endosomal NHE expression. Our insights offer new therapeutic strategies for modulation of endolysosomal pH in fungal infection and human disease.

Direct Comparison of Manganese Detoxification/Efflux Proteins and Molecular Characterization of ZnT10 Protein as a Manganese Transporter.

Manganese homeostasis involves coordinated regulation of specific proteins involved in manganese influx and efflux. However, the proteins that are involved in detoxification/efflux have not been completely resolved nor has the basis by which they select their metal substrate. Here, we compared six proteins, which were reported to be involved in manganese detoxification/efflux, by evaluating their ability to reduce manganese toxicity in chicken DT40 cells, finding that human ZnT10 (hZnT10) was the most significant contributor. A domain swapping and substitution analysis between hZnT10 and the zinc-specific transporter hZnT1 showed that residue Asn(43), which corresponds to the His residue constituting the potential intramembranous zinc coordination site in other ZnT transporters, is necessary to impart hZnT10's unique manganese mobilization activity; residues Cys(52) and Leu(242) in transmembrane domains II and V play a subtler role in controlling the metal specificity of hZnT10. Interestingly, the His → Asn reversion mutant in hZnT1 conferred manganese transport activity and loss of zinc transport activity. These results provide important information about manganese detoxification/efflux mechanisms in vertebrate cells as well as the molecular characterization of hZnT10 as a manganese transporter.

Calcium-ATPases: Gene disorders and dysregulation in cancer.

Ca(2+)-ATPases belonging to the superfamily of P-type pumps play an important role in maintaining low, nanomolar cytoplasmic Ca(2+) levels at rest and priming organellar stores, including the endoplasmic reticulum, Golgi, and secretory vesicles with high levels of Ca(2+) for a wide range of signaling functions. In this review, we introduce the distinct subtypes of Ca(2+)-ATPases and their isoforms and splice variants and provide an overview of their specific cellular roles as they relate to genetic disorders and cancer, with a particular emphasis on recent findings on the secretory pathway Ca(2+)-ATPases (SPCA). Mutations in human ATP2A2, ATP2C1 genes, encoding housekeeping isoforms of the endoplasmic reticulum (SERCA2) and secretory pathway (SPCA1) pumps, respectively, confer autosomal dominant disorders of the skin, whereas mutations in other isoforms underlie various muscular, neurological, or developmental disorders. Emerging evidence points to an important function of dysregulated Ca(2+)-ATPase expression in cancers of the colon, lung, and breast where they may serve as markers of differentiation or novel targets for therapeutic intervention. We review the mechanisms underlying the link between calcium homeostasis and cancer and discuss the potential clinical relevance of these observations. 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.

Secretory pathway Ca2+ -ATPases promote in vitro microcalcifications in breast cancer cells.

Calcification of the breast is often an outward manifestation of underlying molecular changes that drive carcinogenesis. Up to 50% of all non-palpable breast tumors and 90% of ductal carcinoma in situ present with radiographically dense mineralization in mammographic scans. However, surprisingly little is known about the molecular pathways that lead to microcalcifications in the breast. Here, we report on a rapid and quantitative in vitro assay to monitor microcalcifications in breast cancer cell lines, including MCF7, MDA-MB-231, and Hs578T. We show that the Secretory Pathway Ca2+ -ATPases SPCA1 and SPCA2 are strongly induced under osteogenic conditions that elicit microcalcifications. SPCA gene expression is significantly elevated in breast cancer subtypes that are associated with microcalcifications. Ectopic expression of SPCA genes drives microcalcifications and is dependent on pumping activity. Conversely, knockdown of SPCA expression significantly attenuates formation of microcalcifications. We propose that high levels of SPCA pumps may initiate mineralization in the secretory pathway by elevating luminal Ca2+ . Our new findings offer mechanistic insight and functional implications on a widely observed, yet poorly understood radiographic signature of breast cancer.

The Na+/H+ exchanger NHE6 modulates endosomal pH to control processing of amyloid precursor protein in a cell culture model of Alzheimer disease.

Early intervention may be key to safe and effective therapies in patients with Alzheimer disease. Endosomal dysfunction is an early step in neurodegeneration. Endosomes are a major site of production of Aß peptide from the processing of amyloid precursor protein (APP) by clipping enzymes (ß- and γ-secretases). The ß-secretase enzyme BACE1 requires acidic lumen pH for optimum function, and acid pH promotes Aß aggregation. The Na(+)/H(+) exchanger NHE6 provides a leak pathway for protons, limiting luminal acidification by proton pumps. Like APP, NHE6 expression was induced upon differentiation of SH-SY5Y neuroblastoma cells and localized to an endosomal compartment. Therefore, we investigated whether NHE6 expression altered APP localization and processing in a stably transfected cell culture model of human APP expression. We show that co-expression with NHE6 or treatment with the Na(+)/H(+) ionophore monensin shifted APP away from the trans-Golgi network into early and recycling endosomes in HEK293 cells. NHE6 alkalinized the endosomal lumen, similar to monensin, and significantly attenuated APP processing and Aß secretion. In contrast, Aß production was elevated upon NHE6 knockdown. We show that NHE6 transcript and protein levels are lowered in Alzheimer brains relative to control. These findings, taken together with emerging genetic evidence linking endosomal Na(+)/H(+) exchangers with Alzheimer disease, suggest that proton leak pathways may regulate Aß generation and contribute to disease etiology.

Cellular calcium dynamics in lactation and breast cancer: from physiology to pathology.

Breast cancer is the second leading cause of cancer mortality in women, estimated at nearly 40,000 deaths and more than 230,000 new cases diagnosed in the U.S. this year alone. One of the defining characteristics of breast cancer is the radiographic presence of microcalcifications. These palpable mineral precipitates are commonly found in the breast after formation of a tumor. Since free Ca(2+) plays a crucial role as a second messenger inside cells, we hypothesize that these chelated precipitates may be a result of dysregulated Ca(2+) secretion associated with tumorigenesis. Transient and sustained elevations of intracellular Ca(2+) regulate cell proliferation, apoptosis and cell migration, and offer numerous therapeutic possibilities in controlling tumor growth and metastasis. During lactation, a developmentally determined program of gene expression controls the massive transcellular mobilization of Ca(2+) from the blood into milk by the coordinated action of calcium transporters, including pumps, channels, sensors and buffers, in a functional module that we term CALTRANS. Here we assess the evidence implicating genes that regulate free and buffered Ca(2+) in normal breast epithelium and cancer cells and discuss mechanisms that are likely to contribute to the pathological characteristics of breast cancer.

Essential role for vacuolar acidification in Candida albicans virulence.

Fungal infections are on the rise, with mortality above 30% in patients with septic Candida infections. Mutants lacking V-ATPase activity are avirulent and fail to acidify endomembrane compartments, exhibiting pleiotropic defects in secretory, endosomal, and vacuolar pathways. However, the individual contribution of organellar acidification to virulence and its associated traits is not known. To dissect their separate roles in Candida albicans pathogenicity we generated knock-out strains for the V0 subunit a genes VPH1 and STV1, which target the vacuole and secretory pathway, respectively. While the two subunits were redundant in many vma phenotypes, such as alkaline pH sensitivity, calcium homeostasis, respiratory defects, and cell wall integrity, we observed a unique contribution of VPH1. Specifically, vph1Δ was defective in acidification of the vacuole and its dependent functions, such as metal ion sequestration as evidenced by hypersensitivity to Zn(2+) toxicity, whereas stv1Δ resembled wild type. In growth conditions that elicit morphogenic switching, vph1Δ was defective in forming hyphae whereas stv1Δ was normal or only modestly impaired. Host cell interactions were evaluated in vitro using the Caco-2 model of intestinal epithelial cells, and murine macrophages. Like wild type, stv1Δ was able to inflict cellular damage in Caco-2 and macrophage cells, as assayed by LDH release, and escape by filamentation. In contrast, vph1Δ resembled a vma7Δ mutant, with significant attenuation in host cell damage. Finally, we show that VPH1 is required for fungal virulence in a murine model of systemic infection. Our results suggest that vacuolar acidification has an essential function in the ability of C. albicans to form hyphae and establish infection.

Unconventional chemiosmotic coupling of NHA2, a mammalian Na+/H+ antiporter, to a plasma membrane H+ gradient.

Human NHA2, a newly discovered cation proton antiporter, is implicated in essential hypertension by gene linkage analysis. We show that NHA2 mediates phloretin-sensitive Na(+)-Li(+) counter-transport (SLC) activity, an established marker for hypertension. In contrast to bacteria and fungi where H(+) gradients drive uptake of metabolites, secondary transport at the plasma membrane of mammalian cells is coupled to the Na(+) electrochemical gradient. Our findings challenge this paradigm by showing coupling of NHA2 and V-type H(+)-ATPase at the plasma membrane of kidney-derived MDCK cells, resulting in a virtual Na(+) efflux pump. Thus, NHA2 functionally recapitulates an ancient shared evolutionary origin with bacterial NhaA. Although plasma membrane H(+) gradients have been observed in some specialized mammalian cells, the ubiquitous tissue distribution of NHA2 suggests that H(+)-coupled transport is more widespread. The coexistence of Na(+) and H(+)-driven chemiosmotic circuits has implications for salt and pH regulation in the kidney.

A DNA nanodevice for mapping sodium at single-organelle resolution.

Cellular sodium ion (Na+) homeostasis is integral to organism physiology. Our current understanding of Na+ homeostasis is largely limited to Na+ transport at the plasma membrane. Organelles may also contribute to Na+ homeostasis; however, the direction of Na+ flow across organelle membranes is unknown because organellar Na+ cannot be imaged. Here we report a pH-independent, organelle-targetable, ratiometric probe that reports lumenal Na+. It is a DNA nanodevice containing a Na+-sensitive fluorophore, a reference dye and an organelle-targeting domain. By measuring Na+ at single endosome resolution in mammalian cells and Caenorhabditis elegans, we discovered that lumenal Na+ levels in each stage of the endolysosomal pathway exceed cytosolic levels and decrease as endosomes mature. Further, we find that lysosomal Na+ levels in nematodes are modulated by the Na+/H+ exchanger NHX-5 in response to salt stress. The ability to image subcellular Na+ will unveil mechanisms of Na+ homeostasis at an increased level of cellular detail.

Plant-specific cation/H+ exchanger 17 and its homologs are endomembrane K+ transporters with roles in protein sorting.

The complexity of intracellular compartments in eukaryotic cells evolved to provide distinct environments to regulate processes necessary for cell proliferation and survival. A large family of predicted cation/proton exchangers (CHX), represented by 28 genes in Arabidopsis thaliana, are associated with diverse endomembrane compartments and tissues in plants, although their roles are poorly understood. We expressed a phylogenetically related cluster of CHX genes, encoded by CHX15-CHX20, in yeast and bacterial cells engineered to lack multiple cation-handling mechanisms. Of these, CHX16-CHX20 were implicated in pH homeostasis because their expression rescued the alkaline pH-sensitive growth phenotype of the host yeast strain. A smaller subset, CHX17-CHX19, also conferred tolerance to hygromycin B. Further differences were observed in K(+)- and low pH-dependent growth phenotypes. Although CHX17 did not alter cytoplasmic or vacuolar pH in yeast, CHX20 elicited acidification and alkalization of the cytosol and vacuole, respectively. Using heterologous expression in Escherichia coli strains lacking K(+) uptake systems, we provide evidence for K(+) ((86)Rb) transport mediated by CHX17 and CHX20. Finally, we show that CHX17 and CHX20 affected protein sorting as measured by carboxypeptidase Y secretion in yeast mutants grown at alkaline pH. In plant cells, CHX20-RFP co-localized with an endoplasmic reticulum marker, whereas RFP-tagged CHX17-CHX19 co-localized with prevacuolar compartment and endosome markers. Together, these results suggest that in response to environmental cues, multiple CHX transporters differentially modulate K(+) and pH homeostasis of distinct intracellular compartments, which alter membrane trafficking events likely to be critical for adaptation and survival.

Endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44 functions independently and downstream of multivesicular body formation.

The multivesicular body (MVB) is an endosomal intermediate containing intralumenal vesicles destined for membrane protein degradation in the lysosome. In Saccharomyces cerevisiae, the MVB pathway is composed of 17 evolutionarily conserved ESCRT (endosomal sorting complex required for transport) genes grouped by their vacuole protein sorting Class E mutant phenotypes. Only one integral membrane protein, the endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44, has been assigned to this class, but its role in the MVB pathway has not been directly tested. Herein, we first evaluated the link between Nhx1 and the ESCRT proteins and then used an unbiased phenomics approach to probe the cellular role of Nhx1. Select ESCRT mutants (vps36Δ, vps20Δ, snf7Δ, and bro1Δ) with defects in cargo packaging and intralumenal vesicle formation shared multiple growth phenotypes with nhx1Δ. However, analysis of cellular trafficking and ultrastructural examination by electron microscopy revealed that nhx1Δ cells retain the ability to sort cargo into intralumenal vesicles. In addition, we excluded a role for Nhx1 in Snf7/Bro1-mediated cargo deubiquitylation and Rim101 response to pH stress. Genetic epistasis experiments provided evidence that NHX1 and ESCRT genes function in parallel. A genome-wide screen for single gene deletion mutants that phenocopy nhx1Δ yielded a limited gene set enriched for endosome fusion function, including Rab signaling and actin cytoskeleton reorganization. In light of these findings and the absence of the so-called Class E compartment in nhx1Δ, we eliminated a requirement for Nhx1 in MVB formation and suggest an alternative post-ESCRT role in endosomal membrane fusion.

Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs.

Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H(+)-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma(-) phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca(2+) and H(+) surges triggered by the antimicrobial agent amiodarone, and impaired Ca(2+) sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.

Secretory pathway Ca2+-ATPase SPCA2 regulates mitochondrial respiration and DNA damage response through store-independent calcium entry.

A complex interplay between the extracellular space, cytoplasm and individual organelles modulates Ca2+ signaling to impact all aspects of cell fate and function. In recent years, the molecular machinery linking endoplasmic reticulum stores to plasma membrane Ca2+ entry has been defined. However, the mechanism and pathophysiological relevance of store-independent modes of Ca2+ entry remain poorly understood. Here, we describe how the secretory pathway Ca2+-ATPase SPCA2 promotes cell cycle progression and survival by activating store-independent Ca2+ entry through plasma membrane Orai1 channels in mammary epithelial cells. Silencing SPCA2 expression or briefly removing extracellular Ca2+ increased mitochondrial ROS production, DNA damage and activation of the ATM/ATR-p53 axis leading to G0/G1 phase cell cycle arrest and apoptosis. Consistent with these findings, SPCA2 knockdown confers redox stress and chemosensitivity to DNA damaging agents. Unexpectedly, SPCA2-mediated Ca2+ entry into mitochondria is required for optimal cellular respiration and the generation of mitochondrial membrane potential. In hormone receptor positive (ER+/PR+) breast cancer subtypes, SPCA2 levels are high and correlate with poor survival prognosis. We suggest that elevated SPCA2 expression could drive pro-survival and chemotherapy resistance in cancer cells, and drugs that target store-independent Ca2+ entry pathways may have therapeutic potential in treating cancer.

The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma.

A small population of self-renewing stem cells initiate tumors and maintain therapeutic resistance in glioblastoma (GBM). Given the limited treatment options and dismal prognosis for this disease, there is urgent need to identify drivers of stem cells that could be druggable targets. Previous work showed that the endosomal pH regulator NHE9 is upregulated in GBM and correlates with worse survival prognosis. Here, we probed for aberrant signaling pathways in patient-derived GBM cells and found that NHE9 increases cell surface expression and phosphorylation of multiple receptor tyrosine kinases (RTKs) by promoting their escape from lysosomal degradation. Downstream of NHE9-mediated receptor activation, oncogenic signaling pathways converged on the JAK2-STAT3 transduction axis to induce pluripotency genes Oct4 and Nanog and suppress markers of glial differentiation. We used both genetic and chemical approaches to query the role of endosomal pH in GBM phenotypes. Loss-of-function mutations in NHE9 that failed to alkalinize endosomal lumen did not increase self-renewal capacity of gliomaspheres in vitro. However, monensin, a chemical mimetic of Na+/H+ exchanger activity, and the H+ pump inhibitor bafilomycin bypassed NHE9 to directly alkalinize the endosomal lumen resulting in stabilization of RTKs and induction of Oct4 and Nanog. Using orthotopic models of primary GBM cells we found that NHE9 increased tumor initiation in vivo. We propose that NHE9 initiates inside-out signaling from the endosomal lumen, distinct from the established effects of cytosolic and extracellular pH on tumorigenesis. Endosomal pH may be an attractive therapeutic target that diminishes stemness in GBM, agnostic of specific receptor subtype.

Vesicular distribution of Secretory Pathway Ca²+-ATPase isoform 1 and a role in manganese detoxification in liver-derived polarized cells.

Manganese is a trace element that is an essential co-factor in many enzymes critical to diverse biological pathways. However, excess Mn(2+) leads to neurotoxicity, with psychiatric and motor dysfunction resembling parkinsonism. The liver is the main organ for Mn(2+) detoxification by excretion into bile. Although many pathways of cellular Mn(2+) uptake have been established, efflux mechanisms remain essentially undefined. In this study, we evaluated a potential role in Mn(2+) detoxification by the Secretory Pathway Ca(2+), Mn(2+)-ATPase in rat liver and a liver-derived cell model WIF-B that polarizes to distinct bile canalicular and sinusoidal domains in culture. Of two known isoforms, only secretory pathway Ca(2+)-ATPase isoform 1 (SPCA1) was expressed in liver and WIF-B cells. As previously observed in non-polarized cells, SPCA1 showed overlapping distribution with TGN38, consistent with Golgi/TGN localization. However, a prominent novel localization of SPCA1 to an endosomal population close to, but not on the basolateral membrane was also observed. This was confirmed by fractionation of rat liver homogenates which revealed dual distribution of SPCA1 to the Golgi/TGN and a fraction that included the early endosomal marker, EEA1. We suggest that this novel pool of endosomes may serve to sequester Mn(2+) as it enters from the sinusoidal/basolateral domains. Isoform-specific partial knockdown of SPCA1 delayed cell growth and formation of canalicular domain by about 30% and diminished viability upon exposure to Mn(2+). Conversely, overexpression of SPCA1 in HEK 293T cells conferred tolerance to Mn(2+) toxicity. Taken together, our findings suggest a role for SPCA1 in Mn(2+) detoxification in liver.