InteractiveFly: GeneBrief

Sarco/endoplasmic reticulum Ca(2+)-ATPase: Biological Overview | References

Gene name - Sarco/endoplasmic reticulum Ca(2+)-ATPase

Synonyms -

Cytological map position - 60A11-60A12

Function - endoplasmic reticulum calcium pump

Keywords - mutants disrupt Wingless signaling by sequestering Armadillo/beta-catenin away from the signaling pool - Seipin interacts with SERCA, whose activity is reduced in dSeipin mutants, leading to reduced ER calcium levels - THADA binds SERCA and acts on it as an uncoupler - tracheal budding

Symbol - SERCA

FlyBase ID: FBgn0263006

Genetic map position - chr2R:23,926,911-23,935,894

NCBI classification - P-type_ATPase_SERCA

Cellular location - ER transmembrane

NCBI links: EntrezGene, Nucleotide, Protein

Calcium homeostasis in the lumen of the endoplasmic reticulum is required for correct processing and trafficking of transmembrane proteins, and defects in protein trafficking can impinge on cell signaling pathways. This study shows that mutations in the endoplasmic reticulum calcium pump SERCA disrupt Wingless signaling by sequestering Armadillo/beta-catenin away from the signaling pool. Armadillo remains bound to E-cadherin, which is retained in the endoplasmic reticulum when calcium levels there are reduced. Using hypomorphic and null SERCA alleles in combination with the loss of the plasma membrane calcium channel Orai allowed definition of three distinct thresholds of endoplasmic reticulum calcium. Wingless signaling is sensitive to even a small reduction, while Notch and Hippo signaling are disrupted at intermediate levels, and elimination of SERCA function results in apoptosis. These differential and opposing effects on three oncogenic signaling pathways may complicate the use of SERCA inhibitors as cancer therapeutics (Suisse, 2019).

Transmembrane proteins must pass through the secretory pathway to reach the cell surface, where they can interact with other cells and respond to signaling cues. Disrupting the environment in the first secretory compartment, the endoplasmic reticulum (ER), causes misfolding of transmembrane and secreted proteins and elicits a stress response that can either restore proteostasis or trigger apoptosis. The ER acts as a store of intracellular calcium (Ca2+) that can be rapidly released into the cytoplasm to trigger a variety of cellular responses. The sarcoplasmic-ER ATPase (SERCA) actively pumps Ca2+ into the ER, increasing its concentration to 1,000-fold higher than in the cytosol. Depletion of Ca2+ from the ER is sensed by Stromal interaction molecule (Stim), which encodes an endoplasmic reticulum-membrane protein that is an essential component of the store-operated calcium entry mechanism, which in neurons regulates flight. Stim, which accumulates at ER-plasma membrane junctions and activates Orai, a Ca2+ channel in the plasma membrane that mediates store-operated calcium entry (SOCE). SERCA colocalizes with Stim-Orai complexes, allowing entering Ca2+ to be pumped directly into the ER. SOCE maintains Ca2+ homeostasis in the ER so that Ca2+-binding proteins can fold correctly. In the absence of SERCA, the cell-surface receptor Notch, which has extracellular EGF and Lin-12/Notch repeats that interact with Ca2+, fails to mature (Suisse, 2019).

Wnt signaling relies on the bifunctional β-catenin protein, which acts as an essential linker between E-cadherin (E-Cad) and α-catenin at adherens junctions (AJs), but also enters the nucleus and regulates target gene expression in cells that receive a Wnt signal. In the absence of Wnt, cytoplasmic β-catenin is phosphorylated within a destruction complex, leading to its ubiquitination and degradation. Junctional β-catenin is distinct from the pool available for Wnt signaling, and excess E-Cad can remove β-catenin from the signaling pool. The extracellular domain of E-Cad binds Ca2+ ions at the junctions between cadherin domains, giving it a rigid structure. The cadherin family also includes the large protocadherins Fat and Dachsous, which restrict growth by activating the Hippo signaling pathway and regulate planar cell polarity. The precise conformation of these molecules depends on Ca2+ binding by only a subset of their cadherin domain linkers (Suisse, 2019).

There has been significant interest in using SERCA inhibitors such as thapsigargin as cancer therapeutics due to their ability to induce ER stress and apoptosis. Their general toxicity means that they would need to be targeted to specific cancer cell types. However, activating mutations in Notch that are found in certain types of leukemia may make this receptor especially sensitive to reduced SERCA function. This study, shows that a hypomorphic mutation in Drosophila SERCA preferentially affects signaling by the Wnt Wingless (Wg), because E-Cad is retained in the ER and sequesters bound Armadillo (Arm)/β-catenin. Complete loss of SERCA function leads to apoptosis, but an intermediate reduction in ER Ca2+ induced by mutating orai in the hypomorphic SERCA background disrupts Hippo signaling, leading to overgrowth and Notch signaling. These results imply that Wnt-driven cancers may be the most sensitive to SERCA inhibition but highlight the risk that inhibitors may activate cell proliferation through the Hippo pathway (Suisse, 2019).

Transmembrane proteins must pass through the secretory pathway to reach the cell surface, where they can interact with other cells and respond to signaling cues. Disrupting the environment in the first secretory compartment, the endoplasmic reticulum (ER), causes misfolding of transmembrane and secreted proteins and elicits a stress response that can either restore proteostasis or trigger apoptosis. The ER acts as a store of intracellular calcium (Ca2+) that can be rapidly released into the cytoplasm to trigger a variety of cellular responses. The sarcoplasmic-ER ATPase (SERCA) actively pumps Ca2+ into the ER, increasing its concentration to 1,000-fold higher than in the cytosol. Depletion of Ca2+ from the ER is sensed by Stim, which accumulates at ER-plasma membrane junctions and activates Orai, a Ca2+ channel in the plasma membrane that mediates store-operated calcium entry (SOCE). SERCA colocalizes with Stim-Orai complexes, allowing entering Ca2+ to be pumped directly into the ER (Alonso, 2012). SOCE maintains Ca2+ homeostasis in the ER so that Ca2+-binding proteins can fold correctly. In the absence of SERCA, the cell-surface receptor Notch, which has extracellular EGF and Lin-12/Notch repeats that interact with Ca2+, fails to mature (Periz, 1999, Roti, 2013; Suisse, 2019 and references therein).

Wnt signaling relies on the bifunctional β-catenin protein, which acts as an essential linker between E-cadherin (E-Cad) and α-catenin at adherens junctions (AJs), but also enters the nucleus and regulates target gene expression in cells that receive a Wnt signal. In the absence of Wnt, cytoplasmic β-catenin is phosphorylated within a destruction complex, leading to its ubiquitination and degradation. Junctional β-catenin is distinct from the pool available for Wnt signaling, and excess E-Cad can remove β-catenin from the signaling pool. The extracellular domain of E-Cad binds Ca2+ ions at the junctions between cadherin domains, giving it a rigid structure. The cadherin family also includes the large protocadherins Fat and Dachsous, which restrict growth by activating the Hippo signaling pathway and regulate planar cell polarity. The precise conformation of these molecules depends on Ca2+ binding by only a subset of their cadherin domain linkers (Suisse, 2019).

There has been significant interest in using SERCA inhibitors such as thapsigargin as cancer therapeutics due to their ability to induce ER stress and apoptosis. Their general toxicity means that they would need to be targeted to specific cancer cell types. However, activating mutations in Notch that are found in certain types of leukemia may make this receptor especially sensitive to reduced SERCA function (Roti, 2013). This study shows that a hypomorphic mutation in Drosophila SERCA preferentially affects signaling by the Wnt Wingless (Wg), because E-Cad is retained in the ER and sequesters bound Armadillo (Arm)/β-catenin. Complete loss of SERCA function leads to apoptosis, but an intermediate reduction in ER Ca2+ induced by mutating orai in the hypomorphic SERCA background disrupts Hippo signaling, leading to overgrowth and Notch signaling. These results imply that Wnt-driven cancers may be the most sensitive to SERCA inhibition but highlight the risk that inhibitors may activate cell proliferation through the Hippo pathway (Suisse, 2019).

Characterization of a hypomorphic SERCA mutant allele revealed that E-Cad trafficking is especially sensitive to reduced ER Ca2+ levels and that retention of E-Cad in the ER under these mild stress conditions sequesters Arm away from the pool available for Wg signaling. A similar ER retention of E-Cad and desmosomal cadherins, leading to the loss of cell adhesion, has been demonstrated in human keratinocytes in Darier disease, which results from a mutation in SERCA2. In addition, ER stress promotes the differentiation of mouse intestinal stem cells, suggesting that this may be a physiological mechanism to reduce the Wnt signaling that is required for stem cell maintenance. Ca2+ is essential for the homophilic binding of cadherin extracellular domains that mediates cell adhesion. Cadherin monomers contain multiple cadherin domains separated by hinge regions that can each bind three Ca2+ ions, stabilizing the molecule to form a rod-like structure that is resistant to protease cleavage. In larger cadherins, some of the linker regions are Ca2+ free and remain flexible. Cadherin folding into the correct conformation may thus be very sensitive to Ca2+ levels in the ER. In mammalian cells, Tg-induced ER stress leads to O-GlcNAc glycosylation of the E-Cad cytoplasmic domain, blocking its exit from the ER. However, this modification depends on caspase induction by ER stress-induced apoptosis, which does not occur in SERCAdsm mutant clones. It is also possible that E-Cad is not affected by ER Ca2+ levels directly, but is especially sensitive to the general reduction in secretion caused by the loss of SERCA (Suisse, 2019).

Arm that is bound to E-Cad at the ER membrane appears to be unavailable for Wg signaling. In mammalian cells, β-catenin forms a complex with E-Cad during co-translation in the ER and helps to transport E-Cad from the ER to the Golgi. Depleting ER Ca2+ levels may enhance the binding of Arm to E-Cad at the ER, as low extracellular Ca2+ induces rapid Arm recruitment to E-Cad at the plasma membrane. Because E-Cad competes with adenomatous polyposis coli and Axin to bind to the Arm domains, a stronger Arm-E-Cad interaction could both protect Arm from degradation and prevent it from translocating into the nuclei of Wg-receiving cells. The mechanism by which β-catenin enters the nucleus is poorly understood, and it is possible that mislocalization at the ER membrane would exclude it from docking with the partner proteins required for nuclear import (Suisse, 2019).

Using two SERCA alleles and a SERCA orai mutant combination, this study produced three distinct levels of ER Ca2+ that revealed the differential sensitivities of three oncogenic pathways. Wg signaling is the most sensitive, as it is disturbed by the weak allele SERCAdsm; while Notch trafficking is also abnormal in this mutant background, Notch target genes can still be activated. A further reduction in ER Ca2+ produced by disrupting SOCE prevents Notch and Hippo signaling, probably through effects on the trafficking of Notch and the large protocadherin Fat, but only complete loss of SERCA induces apoptosis. These findings have important implications for the use of SERCA inhibitors such as Tg as cancer therapeutics, even when targeted to specific cell types. Although it may be possible to selectively block Wnt-driven cancers with low doses of such inhibitors, the level of inhibition needed to prevent Notch signaling is likely to actually enhance tumor invasiveness by downregulating FAT family members and thus disrupting Hippo signaling (Suisse, 2019).

Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism

Seipin, the gene that causes Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), is important for adipocyte differentiation and lipid homeostasis. Previous studies in Drosophila revealed that Seipin promotes ER calcium homeostasis through the Ca(2+)-ATPase SERCA, but little is known about the events downstream of perturbed ER calcium homeostasis that lead to decreased lipid storage in Drosophila dSeipin mutants. This study shows that glycolytic metabolites accumulate and the downstream mitochondrial TCA cycle is impaired in dSeipin mutants. The impaired TCA cycle further leads to a decreased level of citrate, a critical component of lipogenesis. Mechanistically, Seipin/SERCA-mediated ER calcium homeostasis is important for maintaining mitochondrial calcium homeostasis. Reduced mitochondrial calcium in dSeipin mutants affects the TCA cycle and mitochondrial function. The lipid storage defects in dSeipin mutant fat cells can be rescued by replenishing mitochondrial calcium or by restoring the level of citrate through genetic manipulations or supplementation with exogenous metabolites. Together, these results reveal that Seipin promotes adipose tissue lipid storage via calcium-dependent mitochondrial metabolism (Ding, 2018).

Impaired lipid metabolism is associated with an imbalance in energy homeostasis and many other disorders. Excessive lipid storage results in obesity, while a lack of adipose tissue leads to lipodystrophy. Clinical investigations reveal that obesity and lipodystrophy share some common secondary effects, especially non-alcoholic fatty liver disease and severe insulin resistance. Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2/CGL2) is one of the most severe lipodystrophy diseases. Patients with BSCL2 manifest almost total loss of adipose tissue as well as fatty liver, insulin resistance, and myohypertrophy. BSCL2 results from mutation of the Seipin gene, which is highly conserved from yeast to human (Ding, 2018).

To study the function of Seipin, genetic models were established in different organisms, including yeast, fly, and mouse, and in human cells. As a transmembrane protein residing in the endoplasmic reticulum (ER) and in the vicinity of lipid droplet (LD) budding sites, Seipin has been shown to be involved in LD formation, phospholipid metabolism, lipolysis, and ER calcium homeostasis. As a result of the functional studies in these models, several factors that interact with Seipin protein were identified, such as the phosphatidic acid phosphatase lipin, 14-3-3β, and glycerol-3-phosphate acyltransferase (GPAT). Drosophila Seipin (dSeipin) functions tissue autonomously in preventing ectopic lipid accumulation in salivary gland (a non-adipose tissue) and in promoting lipid storage in fat tissue. The non-adipose tissue phenotype is likely attributed to the increased level of phosphatidic acid (PA) generated by elevated GPAT activity. In adipose tissue Seipin interacts with the ER Ca2+-ATPase SERCA, whose activity is reduced in dSeipin mutants, leading to reduced ER calcium levels. Further genetic analysis suggested that the perturbed level of intracellular calcium contributes to the lipodystrophy. However, it is not known how the depleted ER calcium pool causes decreased lipid storage (Ding, 2018).

Besides the ER, mitochondria are another important intracellular calcium reservoir. Mitochondrial calcium is mainly derived from the ER through the IP3R channel. IP3R not only releases calcium from the ER into the cytosol, but also provides sufficient Ca2+ at mitochondrion-associated ER membranes (MAMs) for activation of the mitochondrial calcium uniporter. The mitochondrial Ca2+ level varies greatly in different cell types and can be modulated by influx and efflux channel proteins, such as MCU and NCLX, a mitochondrial Na+/Ca2+ exchanger. A proper mitochondrial Ca2+ level is implicated in mitochondrial integrity and function. Mitochondrial calcium is needed to support the activity of the mitochondrial matrix dehydrogenases in the TCA cycle. TCA cycle intermediates are used for the synthesis of important compounds, including glucose, amino acids, and fatty acids. Acetyl-CoA, as the basic building block of fatty acids, is generally derived from glycolysis, the TCA cycle, and fatty acid β-oxidation. In mammalian adipocytes, acetyl-CoA derived from the TCA cycle intermediate citrate is crucial for de novo lipid biosynthesis, which contributes significantly to lipid storage (Ding, 2018 and references therein).

This study used multiple comparative omics to analyze the proteomic, transcriptomic, and metabolic differences between larval fat cells of dSeipin mutants and wild type. The results reveal an impairment in channeling glycolytic metabolites to mitochondrial metabolism in dSeipin mutant fat cells, and scarcity of mitochondrial Ca2+, are the causative factors of this metabolic dysregulation. Evidence is provided showing that dSeipin lipodystrophy is rescued by restoring mitochondrial calcium or replenishing citrate. It is proposed that the low ER Ca2+ level in dSeipin mutants cannot maintain a sufficiently high mitochondrial Ca2+ concentration to support the TCA reactions. This in turn leads to reduced lipogenesis in dSeipin mutants (Ding, 2018).

Seipin promotes fat tissue lipid storage via calcium-dependent mitochondrial metabolism. Defective ER calcium homeostasis in dSeipin mutants is associated with reduced mitochondrial calcium and impaired mitochondrial function, such as low production of TCA cycle metabolites. Restoring mitochondrial calcium levels or replenishing citrate, a key TCA cycle product and also an important precursor of lipogenesis, rescues the lipid storage defects in dSeipin mutant fat cells (Ding, 2018).

This study investigated the underlying causes of Seipin-dependent lipodystrophy by integrating multiple omic analyses, including RNA-seq, quantitative proteomics, and metabolomic analysis. Compared to previous studies based on genetics and traditional cellular phenotypic analysis, these combinatory omic approaches provide an unprecedented spectrum of molecular phenotypes, which not only add new information but also pinpoint logical directions for further investigations (Ding, 2018).

Omics analyses, in particular lipidomic analysis, have been utilized to investigate the underlying mechanisms in several previous Seipin studies and led to the finding that PA is elevated in several Seipin mutant models. In this study, based on genetic rescuing assays and quantitative proteomics analysis, it was initially proposed that downregulated glycolysis is the cause of lipodystrophy. However, both the RNA-seq results and metabolomic data argue against this possibility and suggest a new mechanism. Despite reduced levels of glycolytic enzymes, transcription of the corresponding genes is not affected, and glycolytic metabolites, in particular pyruvate, are increased in dSeipin mutants compared to wild type. Metabolomic data further show that citrate and isocitrate, which are the products of the first two steps of the mitochondrial TCA cycle, are dramatically decreased in dSeipin mutants, suggesting a defective metabolic flow downstream of pyruvate. These results lead to a new possibility that the lipid storage defects in dSeipin mutants are caused by a defective TCA cycle and this is indeed supported by the metabolic flux analysis. These findings further suggest the involvement of mitochondria. In line with this, the previous discovery that fatty acid β-oxidation is elevated in dSeipin mutant fat cells may reflect compensation for the reduced TCA cycle and lipogenesis. This possibility is supported by the results of genetic and citrate-supplement rescue experiments and by citrate measurements (Ding, 2018).

It is known that glycolytic enzymes and metabolites are regulated by a metabolic feedback loop, which may complicate the explanation of genetic interactions. The current findings highlight that although genetic analysis and rescue results provide important clues, multiple lines of evidence are critical for unraveling complex intracellular pathways. In this case, the combination of omic results and genetic analysis led to the finding that mitochondrial metabolism is important in Seipin-associated lipodystrophy (Ding, 2018).

Mitochondria are hubs in key cellular metabolic processes, including the TCA cycle, ATP production, and amino acid catabolism. Mitochondria also play a central role in lipid homeostasis by controlling two seemingly opposite metabolic pathways, lipid biosynthesis, and fatty acid breakdown. Therefore, impairment of mitochondrial function in different tissues may lead to different, even opposite, phenotypes in lipid storage. In tissues where lipid biosynthesis is the major pathway, defective mitochondria might result in reduced lipid storage, whereas in tissues where fatty acid oxidation prevails, the same defect might lead to increased lipid storage. Reduced lipid storage in dSeipin mutants suggests the former case. The reduced level of citrate and other TCA cycle products in dSeipin mutants suggests an impairment of mitochondrial function. The reduction of OCR and ATP production, the decreased Rhod-2 staining, and the aberrant enrichment of mitochondria within autophagosomes all further support this notion. Interestingly, in mouse brown adipose tissue, Seipin mutation increases mitochondrial respiration along with normal MitoTracker labeling. The discrepancies suggest that Seipin may have cell type-specific functions. Unlike white adipose tissue, which favors lipid storage/biosynthesis, brown adipose tissue is prone to fatty acid breakdown (Ding, 2018).

The link between mitochondria and Seipin was concealed in several previous studies. GPATs, which are recently reported Seipin-interacting proteins, participate in many mitochondrial processes. For example, mitochondria from brown adipocytes that are deficient in GPAT4 exhibit high oxidative levels, and mitochondrial GPAT is required for mitochondrial dynamics. PA, which is elevated in Seipin mutants, is required for mitochondrial morphology and function. Similarly, mitochondrial impairments were also observed in various lipodystrophic conditions. Downregulation of mitochondrial transcription and altered mitochondrial function were indicated in type III congenital generalized lipodystrophy. Multiple mitochondrial metabolic processes are altered in mice with lipodystrophy caused by Zmpste24 mutation. HIV patients treated with anti-retroviral therapy manifest partial lipodystrophy and impaired mitochondria in adipocytes. Moreover, mitochondrial dysfunction in adipose tissue triggers lipodystrophy and systemic disorders in mice. Therefore, the contribution of mitochondrial dysfunction to the cause or development of lipodystrophic conditions warrants further examination (Ding, 2018).

It has been previously reported that dSeipin/SERCA-mediated ER calcium homeostasis is critical for lipid storage (Bi, 2014). Consistent with this, transcripts encoding calcium signaling factors are enriched in the genes that are differentially expressed between dSeipin mutants and wild type. Mitochondrial calcium is transported from the ER through the ER-resident channel IP3R. The reduction of mitochondrial calcium in dSeipin mutant fat cells suggests that the decreased ER calcium leads to an insufficient level of mitochondrial calcium. Importantly, RNAi of a putative Drosophila mitochondrial calcium efflux channel (NCLX/CG18660) not only restores the mitochondrial calcium level but also rescues the lipid storage defects in dSeipin mutants, indicating that mitochondrial calcium is key for dSeipin-mediated lipid storage. This explains the previous finding that the lipid storage defects in dSeipin mutants are rescued by RNAi of RyR, which is not required for ER-mitochondrion calcium transport, but not by RNAi of IP3R (Ding, 2018).

Cellular calcium has been linked to lipid storage and related diseases in recent studies. Comprehensive genetic screening in Drosophila showed that ER calcium-related proteins are key regulators of lipid storage. In particular, SERCA, as the sole ER calcium influx channel and an interacting partner of Seipin, has been repeatedly implicated in lipid metabolism. Dysfunctional lipid metabolism can disrupt ER calcium homeostasis by inhibiting SERCA and further disturbing systemic glucose homeostasis. Increased SERCA expression was shown to have dramatic anti-diabetic benefits in mouse models. In a genomewide association study, SERCA was been found to be associated with obesity. In addition, cellular calcium influx is important for transcriptional programming of lipid metabolism, including lipolysis in mice. The current study further elucidates that ER calcium and mitochondrial calcium are important for cellular lipid homeostasis. It also provides a new insight into the pathogenic mechanism of congenital lipodystrophy (Ding, 2018).

Since Seipin mutations lead to opposite effects on lipid storage in adipose tissue (lipodystrophy) and non-adipose tissues (ectopic lipid storage), numerous studies have been carried out to understand the underlying mechanisms. In Seipin mutants, elevated GPAT activity leads to an increased level of PA. This may cause the formation of supersized lipid droplets in non-adipose cells because of the fusogenic property of PA in lipid leaflets, and may also lead to adipogenesis defects due to the potential role of PA as an inhibitor of preadipocyte differentiation. The Seipin-mediated lipid storage phenotype is further complicated by the role of Seipin in lipid droplet formation, which is mainly studied in unicellular eukaryotic yeast or in cultured cells from multicellular eukaryotic organisms. Seipin has been found in the ER-LD contact sites, which are considered as essential subcellular foci for LD formation/maturation. Moreover, in mammalian adipose tissue, the role of Seipin in lipogenesis or lipolysis may also be masked by the defect in early adipogenesis (Ding, 2018).

How can previous findings in different model organisms and different cell types be reconciled? Seipin has been characterized as a tissue-autonomous lipid modulator. It is likely that Seipin participates in lipid metabolism via distinct mechanisms in different tissues. Alternatively, the metabolic processes that involve Seipin may have different outcomes in different tissues. For example, mitochondria have a different impact on lipid metabolism in different tissues: In non-fat cells, mitochondria mainly direct energy mobilization, whereas in fat cells, mitochondria mainly lead anabolism. The molecular role of Seipin and the phenotypic outcomes in Seipin mutants may rely on specific cellular and developmental contexts (Ding, 2018).

THADA regulates the organismal balance between energy storage and heat production

Human susceptibility to obesity is mainly genetic, yet the underlying evolutionary drivers causing variation from person to person are not clear. One theory rationalizes that populations that have adapted to warmer climates have reduced their metabolic rates, thereby increasing their propensity to store energy. This study uncovered the function of a gene that supports this theory. THADA is one of the genes most strongly selected during evolution as humans settled in different climates. THADA knockout flies are obese, hyperphagic, have reduced energy production, and are sensitive to the cold. THADA binds the sarco/ER Ca2+ ATPase (SERCA)> and acts on it as an uncoupler. Reducing SERCA activity in THADA mutant flies rescues their obesity, pinpointing SERCA as a key effector of THADA function. In sum, this identifies THADA as a regulator of the balance between energy consumption and energy storage, which was selected during human evolution (Moraru, 2017).

Obesity has reached pandemic proportions, with 13% of adults worldwide being obese. Although the modern diet triggers this phenotype, 60%-70% of an individual's susceptibility to obesity is genetic. The underlying evolutionary drivers that cause susceptibility vary from person to person and are not clear. Since obesity is most prevalent in populations that have adapted to warm climates, an emerging theory proposes that populations in warm climates evolved low metabolic rates to reduce heat production, making them prone to obesity. In contrast, populations in cold climates evolved high energy consumption for thermogenesis, making them more resistant to obesity. This theory predicts the existence of genes that have been selected in the human population by climate adaptation which regulate the balance between heat production and energy storage (Moraru, 2017).

The gene Thyroid Adenoma Associated (THADA) has played an important role in human evolution. Comparison of the Neanderthal genome with the genomes of current humans reveals that SNPs in THADA were the most strongly positively selected SNPs genome-wide in the evolution of modern humans. Furthermore, as hominins left Africa circa 70,000 years ago, they adapted to colder climates. Genome-wide association studies (GWAS) identified THADA as one of the top genes that was evolutionarily selected in response to cold adaptation, suggesting a link between THADA and energy metabolism. THADA was also identified as one of the top risk loci for type 2 diabetes by GWAS. Although follow-up studies could not confirm an association between THADA SNPs and various aspects of insulin release or insulin sensitivity, some studies did find an association between THADA and pancreatic β-cell response or marginal evidence for an association with body mass index. In sum, THADA has been connected to both metabolism and adaptation to climate. Nonetheless, nothing is known about the function of THADA in animal biology, at the physiological or the molecular level. Animals lacking THADA function have not yet been described. An analysis of the amino acid sequence of THADA provides little or no hints regarding its molecular function (Moraru, 2017).

To study the function of THADA, THADA knockout flies were generated. THADA knockout animals are obese and produce less heat than controls, making them sensitive to the cold. THADA binds the sarco/ER Ca2+ ATPase (SERCA) and regulates organismal metabolism via calcium signaling. In addition to unveiling the physiological role and molecular function of this medically relevant gene, the results also show that one gene that has been strongly selected during human evolution in response to environmental temperature plays a functional role in regulating the balance between heat production and energy storage, affecting the propensity to become obese (Moraru, 2017).

This study reports the physiological and molecular function of THADA in animals. THADA mutants were found to be obese, sensitive to the cold, and have reduced heat production compared with controls. THADA interacts physically with SERCA and modulates its activity. The combination of improved calcium pumping and cold sensitivity of THADA mutants indicates that THADA acts as an SERCA uncoupler, similar to sarcolipin. This interaction between THADA and SERCA appears to be an important part of THADA function, since the obesity phenotype of THADA mutants can be rescued by mild SERCA knockdown (Moraru, 2017).

Calcium signaling is increasingly coming into the spotlight as an important regulator of organismal metabolism. In a genome-wide in vivo RNAi screen in Drosophila to search for genes regulating energy homeostasis, calcium signaling was the most enriched gene ontology category among obesity-regulating genes (Baumbach, 2014). Cytosolic calcium levels can alter organismal adiposity by more than 10-fold (from 15% to 250% of control levels) (Baumbach, 2014), indicating that it is an important regulator of organismal metabolism. In line with these numbers, THADAKO flies have 250% the triglyceride levels of control flies. The phenotypes observed for other regulators of calcium signaling all point in the same general direction that high ER calcium leads to hyperphagia and obesity. Likewise, mice heterozygous for a mutation in IP3R are susceptible to developing glucose intolerance on a high-fat diet (Moraru, 2017).

The molecular mechanisms by which ER calcium regulates organismal metabolism are not yet fully understood, but this important question will surely be the subject of intense research in the near future. Calcium levels are known to regulate activity of tricarboxylic acid cycle enzymes such as α-ketoglutarate dehydrogenase, isocitrate dehydrogenase, and pyruvate dehydrogenase, which could explain part of the effect of calcium on metabolism (Moraru, 2017).

THADA mutation leads to obesity due to roles of THADA both in the fat body and in neurons. This has also been observed for IP3R mutants. Calcium signaling regulates lipid homeostasis directly and cell-autonomously in the fat body, as observed in seipin mutants (Bi, 2014) or when Stim expression was modulated specifically in fat tissue. In addition, it regulates feeding via the CNS. Interestingly, while THADA mutant females have elevated glycogen levels, THADA mutant males do not. It is not known why this is the case: it could be due to the higher energetic demand in females compared with males, leading to stronger metabolic phenotypes in females, or THADA might regulate glycogen metabolism differently in the two sexes (Moraru, 2017).

GWAS identified THADA as one of the top risk loci for type 2 diabetes. The data presented in this study indicates that THADA regulates lipid metabolism and feeding, suggesting that the association between THADA and diabetes may be causal in nature. THADA mutant flies develop obesity, but have normal circulating sugar levels under standard laboratory food conditions. Interestingly, mouse mutants for IP3R likewise do not become insulin resistant under a regular diet, but do become insulin resistant on a high-fat diet. Combined, these data suggest that the primary effect of altered THADA activity and calcium signaling is on lipid metabolism, and that a combination with high-fat feeding may be required to lead to type 2 diabetes over time. This could potentially explain why follow-up association studies did not find links between THADA and insulin sensitivity but did find links between THADA and adiposity (Moraru, 2017 and references therein).

Insects are ectotherms, meaning that their internal physiological sources of heat are not sufficient to control their body temperature. Nonetheless they do produce heat, and the main sources of heat are either of muscular origin due to movement or shivering, or of biochemical origin from futile cycles that consume ATP with no net work. For instance, bumblebees preheat their flight muscles by simultaneously activating phosphofructokinase and fructose 1,6-bisphosphatase, which catalyze opposing enzymatic reactions, leading to the futile hydrolysis of ATP and release of heat. Drosophila also have mitochondrial uncoupling proteins, which potentially generate a futile metabolic cycle by dissipating the mitochondrial membrane potential. It is proposed in this stduy that uncoupled hydrolysis of ATP by SERCA could constitute one additional example of such a futile cycle that produces heat. It cannot be excluded, however, that THADA knockout flies might also have changes in their evaporative heat loss contributing to their reduced thermogenesis. The thermogenic phenotypes in THADA knockout flies are relatively mild, perhaps reflecting the ectothermic nature of flies. Hence it will be of interest to study in the future the metabolic parameters of THADA knockout mice (Moraru, 2017).

The combination of cold sensitivity and obesity in THADA mutant animals is interesting in terms of the evolutionary origins of the current obesity pandemic. The prevalence of obesity is highest in populations that have adapted to warmer climates, suggesting that people in warm climates evolved reduced metabolic rates to prevent overheating, and in combination with a modern diet this reduced metabolic rate leads to obesity. Interestingly, THADA is a gene that provides support for this theory. SNPs in THADA are among the SNPs genome-wide that have been most strongly selected as humans adapted to climates of different temperatures). Indeed, comparison of the Neanderthal genome with the genomes of current humans reveals that SNPs in THADA were the most strongly positively selected SNPs genome-wide in the evolution of modern humans. The data presented in this study show that THADA simultaneously affects sensitivity to cold and obesity. Uncoupled SERCA ATPase activity is a major contributor to non-shivering thermogenesis. Similar to animals mutant for another SERCA uncoupling protein, sarcolipin, this study found that THADA mutants are sensitive to the cold. This provides a possible explanation for why evolution selected for SNPs in THADA. In addition, THADA, via SERCA, also regulates lipid homeostasis. THADA thereby provides a genetic and molecular link between climate adaptation and obesity (Moraru, 2017).

Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca2+ signaling in the Drosophila midgut

Precise regulation of stem cell activity is crucial for tissue homeostasis and necessary to prevent overproliferation. In the Drosophila adult gut, high levels of reactive oxygen species (ROS) has been detected with different types of tissue damage, and oxidative stress has been shown to be both necessary and sufficient to trigger intestinal stem cell (ISC) proliferation. However, the connection between oxidative stress and mitogenic signals remains obscure. In a screen for genes required for ISC proliferation in response to oxidative stress, this study identified two regulators of cytosolic Ca2+ levels, transient receptor potential A1 (TRPA1) and ryanodine receptor (RyR). Characterization of TRPA1 and RyR demonstrates that Ca2+ signaling is required for oxidative stress-induced activation of the Ras/MAPK pathway, which in turns drives ISC proliferation. These findings provide a link between redox regulation and Ca2+ signaling and reveal a novel mechanism by which ISCs detect stress signals (Xu, 2017).

This study found that the two cation channels TRPA1 and RyR are critical for cytosolic Ca2+ signaling and ISC proliferation. Under homeostatic conditions, the basal activities of TRPA1 and RyR are required for maintaining cytosolic Ca2+ in ISCs to ensure their self-renewal activities and normal tissue turnover. Agonists, including but not limited to low levels of ROS, could be responsible for the basal activities of TRPA1 and RyR. Under tissue damage conditions, increased ROS stimulates the channel activities of TRPA1 to mediate increases in cytosolic Ca2+ in ISCs. As for RyR, besides its potential to directly sense ROS, it is known to act synergistically with TRPA1 in a positive feedback mechanism to release more Ca2+ from the ER into the cytosol upon sensing the initial Ca2+ influx through TRPA1 (Xu, 2017).

Previously, Deng (2015) identified L-glutamate as a signal that can activate metabotropic glutamate receptor (mGluR) in ISCs, which in turn modulates the cytosolic Ca2+ oscillation pattern via phospholipase C (PLC) and inositol-1,4,5-trisphosphate (InsP3). Interestingly, L-glutamate and mGluR RNAi mainly affected the frequency of Ca2+ oscillation in ISCs, while their influence on cytosolic Ca2+ concentration was very weak. Strikingly, the number of mitotic cells induced by L-glutamate (i.e. an increase from a basal level of ~5 per midgut to ~10 per midgut) is far less than what has been observed in tissue damage conditions (depending on the severity of damage, the number varies from ~20 to more than 100 per midgut following damage). Consistent with this, in a screen for regulators of ISC proliferation in response to tissue damage, this study tested three RNAi lines targeting mGluR (BL25938, BL32872, and BL41668, which was used by Deng, 2015), and none blocked the damage response in ISCs, suggesting that L-glutamate and mGluR do not play a major role in damage repair of the gut epithelium (Xu, 2017).

This study found that ROS can trigger Ca2+ increases through the redox- sensitive cation channels TRPA1 and RyR under damage conditions. In particular, it was demonstrated using voltage-clamp experiments that the TRPA1-D isoform, which is expressed in the midgut, is sensitive to the oxidant agent paraquat. In addition, the results of previous studies have demonstrated the direct response of RyR to oxidants via single channel recording and showed that RyR could amplify TRPA1-mediated Ca2+ signaling through the Ca2+-induced Ca2+ release (CICR) mechanism. Interestingly, expression of oxidant- insensitive TRPA1-C isoform in the ISCs also exhibits a tendency to induce ISC proliferation, indicating that ROS may not be the only stimuli for TRPA1 and RyR under physiological conditions. Possible other activators in the midgut may be irritant chemicals, noxious thermal/mechanical stimuli, or G-protein-coupled receptors (Xu, 2017).

Altogether, the concentration of cytosolic Ca2+ in ISCs appears to be regulated by a number of mechanisms/inputs including mGluR and the ion channels TRPA1 and RyR. Although mGluR might make a moderate contribution to cytosolic Ca2+ in ISCs, TRPA1 and RyR have a much stronger influence on ISC Ca2+ levels. Thus, it appears that the extent to which different inputs affect cytosolic Ca2+ concentration correlates with the extent of ISC proliferation (Xu, 2017).

Although, as a universal intracellular signal, cytosolic Ca2+ controls a plethora of cellular processes, this study demonstrated that cytosolic Ca2+ levels regulate Ras/MAPK activity in ISCs. Specifically, trpA1 RNAi or RyR RNAi were found to block Ras/MAPK activation in stem cells, and forced cytosolic Ca2+ influx by SERCA RNAi induces Ras/MAPK activity. Moreover, Ras/MAPK activation is an early event following increases in cytosolic Ca2+, since increased dpErk signal was observed in stem cells expressing SERCA RNAi before they undergo massive expansion, and when Yki RNAi was co-expressed to block proliferation. It should be noted that a more variable pattern of pErk activation was observed with prolonged increases of cytosolic Ca2+, suggesting complicated regulations via negative feedback, cross-activation, and cell communication at late stages of Ca2+ signaling. This might explain why Deng failed to detect pErk activation after 4 days induction of Ca2+ signaling (Deng, 2015). Previously, Ras/MAPK activity was reported to increase in ISCs, regulating proliferation rather than differentiation, in regenerating midguts, which is consistent with the findings about TRPA1 and RyR (Xu, 2017).

The Calcineurin A1/CREB-regulated transcription coactivator/CrebB pathway previously proposed to act downstream of mGluR-calcium signaling (Deng, 2015) is not likely to play a major role in high Ca2+-induced ISC proliferation, as multiple RNAi lines targeting CanA1 or CrebB were tested and none of them suppressed SERCA RNAi-induced ISC proliferation. In support of this model, it was also found that the active forms of CanA1/ CRTC/ CrebB cannot stimulate mitosis in ISCs when their cytosolic Ca2+ levels are restricted by trpA1 RNAi, whereas mitosis induced by the active forms of Ras or Raf is not suppressed by trpA1 RNAi (Xu, 2017).

Prior to this study, it has been shown that paracrine ligands such as Vn from the visceral muscle, and autocrine ligands such as Spi and Pvf ligands from the stem cells, can stimulate ISC proliferation via RTK-Ras/MAPK signaling. It study found that multiple RTK ligands in the midgut are down-regulated by trpA1 RNAi expression in the ISCs, including spi and pvf1 that can be induced by SERCA RNAi. Further, it was demonstrated that high Ca2+ fails to induce ISC proliferation in the absence of EGFR. As spi is induced by EGFR-Ras/MAPK signaling in Drosophila cells, and DNA binding mapping (DamID) analyses indicate that spi might be a direct target of transcriptional factors downstream of EGFR-Ras/MAPK in the ISCs, the autocrine ligand Spi might therefore act as a positive feedback mechanism for EGFR-Ras/MAPK signaling in ISCs (Xu, 2017).

In summary, this study identifies a mechanism by which ISCs sense microenvironment stress signals. The cation channels TRPA1 and RyR detect oxidative stress associated with tissue damage and mediate increases in cytosolic Ca2+ in ISCs to amplify and activate EGFR-Ras/MAPK signaling. In vertebrates, a number of cation channels, including TRPA1 and RyR, have been associated with tumor malignancy. The current findings, unraveling the relationship between redox-sensing, cytosolic Ca2+, and pro-mitosis Ras/MAPK activity in ISCs, could potentially help understand the roles of cation channels in stem cells and cancers, and inspire novel pharmacological interventions to improve stem cell activity for regeneration purposes and suppress tumorigenic growth of stem cells (Xu, 2017).

SERCA is critical to control the Bowditch effect in the heart

The Bowditch effect or staircase phenomenon is the increment or reduction of contractile force when heart rate increases, defined as either a positive or negative staircase. The healthy and failing human heart both show positive or negative staircase, respectively, but the causes of these distinct cardiac responses are unclear. Different experimental approaches indicate that while the level of Ca(2+) in the sarcoplasmic reticulum is critical, the molecular mechanisms are unclear. This study demonstrates that Drosophila melanogaster shows a negative staircase which is associated to a slight but significant frequency-dependent acceleration of relaxation (FDAR) at the highest stimulation frequencies tested. It was further shown that the type of staircase is oppositely modified by two distinct SERCA mutations. The dominant conditional mutation SERCA(A617T) induced positive staircase and arrhythmia, while SERCA(E442K) accentuated the negative staircase of wild type. At the stimulation frequencies tested, no significant FDAR could be appreciated in mutant flies. The present results provide evidence that two individual mutations directly modify the type of staircase occurring within the heart and suggest an important role of SERCA in regulating the Bowditch effect (Balcazar, 2018).

Ma2/d promotes myonuclear positioning and association with the sarcoplasmic reticulum

The cytoplasm of striated myofibers contains a large number of membrane organelles, including sarcoplasmic reticulum (SR), T-tubules and the nuclear membrane. These organelles maintain a characteristic juxtaposition that appears to be essential for efficient inter-membranous exchange of RNA, proteins and ions. This study found that the membrane-associated Muscle-specific alpha2/delta (Ma2/d) subunit of the Ca(2+) channel complex localizes to the SR and T-tubules, and accumulates at the myonuclear surfaces. Furthermore, Ma2/d mutant larval muscles exhibit nuclear positioning defects, disruption of the nuclear-SR juxtapositioning, as well as impaired larval locomotion. Ma2/d localization at the nuclear membrane depends on the proper function of the nesprin ortholog Msp300 and the BAR domain protein Amphiphysin (Amph). Importantly, live imaging of muscle contraction in intact Drosophila larvae indicated altered distribution of Sarco/Endoplamic Reticulum Ca(2+)-ATPase (SERCA) around the myonuclei of Ma2/d mutant larvae. Co-immunoprecipitation analysis supports association between Ma2/d and Amph, and indirectly with Msp300. It is therefore suggested that Ma2/d, in association with Msp300 and Amph, mediates interactions between the SR and the nuclear membrane (Reuveny, 2018).

SERCA directs cell migration and branching across species and germ layers

Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. This study shows that the sarco-endoplasmic reticulum Ca(2+) reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca(2+) levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca(2+) differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca(2+) in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca(2+) signaling, established by SERCA, in iterative budding (Truong, 2017).

SERCA performs diverse regulatory functions, ranging from roles in periodic contractility in muscle to ER stress and protein folding. The current findings reveal a new function for SERCA, as a conserved controller of iterative budding. The initiation of new buds and encoding of the timing of formation of these buds has been proposed to be controlled by growth factor morphogens. Specifically, FGF10 acting on airway epithelial FGFR2b (in mammals) or Branchless acting on Breathless (in Drosophila) are required for proper branching of mammalian lungs or Drosophila trachea, respectively. Unidentified morphogens have also been proposed to act as a 'branching clock' that work with FGF signaling to coordinate the branching program. In contrast to this hypothesis that unidentified growth factor morphogens serve as the 'clock' to direct the timing of branching, this study showsthat SERCA is a central organizer that directs the onset and rate of budding. Morphogens must operate upstream of SERCA, because SERCA blockade stalls the branching program, while supply of exogenous morphogens (e.g. FGFs) is insufficient to overcome this blockade. Thus, it is proposed that SERCA must integrate inputs from morphogens like FGF and establishes a differential in Ca2+ levels at branching tips to indicate the timing for directed cell migration and branch formation (Truong, 2017).

This novel role of SERCA as a central organizer of branching seems highly conserved, as branching in both invertebrates and vertebrates, as well as tissues from all germ layers, requires SERCA. In all these systems, branch iteration rate is determined by the level of SERCA function; these effects are mediated by controlling cell migration. SERCA's effects are not mediated by altering cell shape, and do not require alterations in proliferation. Live Ca2+ imaging in Drosophila reveals that SERCA directs cell migration at branch points by establishing a local Ca2+ differential, where the Ca2+ level is higher in the leading cell that migrates to form a new branch. The cells trailing behind it maintain comparatively lower Ca2+ levels. Loss of this local Ca2+ differential halts migration and branching. Reinstatement of this local Ca2+ differential, whether by lifting of SERCA blockade or by PKC activation, restores cell migration and branching (Truong, 2017).

Beyond the Ca2+ differential revealed by light-sheet imaging of Drosophila embryos, episodic Ca2+ impulses were observed to propagate through the tracheal epithelium as the cells migrate and fuse to form their branched network. These propagating Ca2+ waves have been predicted by computational modeling, yet they do not appear to be important for branch iteration, raising the question as to their function. A recent publication on tracheal tube anastomosis did not implicate these whole-cell Ca2+ impulses in membrane fusion. The increase in frequency of these impulses upon fusion of cells from adjacent segments suggests they may be a response to cell-cell contact, which could in turn modulate cell membrane machinery. Similar Ca2+ impulses have been described in other cell types, such as in fungi following contact with a pathogen. The remarkable similarity of these Ca2+ impulses from animals to fungi suggests that they are highly conserved and may have been adapted by evolution to suit each specific cellular environment. The function of these impulses may, alternatively, relate to maintenance or elongation of the branched network that has formed. Indeed, in mammalian lung, periodic Ca2+ waves course through airway smooth muscle, inducing waves of contractility. These waves are thought to mechano-regulate branching morphogenesis, whereby abolishing the Ca2+ waves impairs airway growth and elongation (Truong, 2017).

The results consistently demonstrate that SERCA instructs budding across germ layers, tissue types, and species, suggesting that the role of SERCA may be more broadly generalizable. A conserved regulator simplifies understanding of how a vast array of branched tissues could arise from one platform, and specialize based on local morphogen inputs. Thus, the current findings may unite disparate observations of Ca2+ signaling involvement in other types of branching, such as axonal pathfinding, plant gravitotropism, angiogenesis, and endothelial wound healing. A centralized control of branching also holds implications for understanding a range of disease mechanisms. Regarding the lung, the significance of reduced epithelial SERCA function has been highlighted in human and animal studies of asthma as well as in other burgeoning diseases such as cystic fibrosis, lung fibrosis and lung cancer. This study suggests that these oft intractable pulmonary challenges may feature SERCA-related lesions of cell migration. Examples include airway remodeling in asthma, alveolar remodeling in fibrosis, or lung cancer invasiveness. More generally, altered SERCA expression or function has been associated with numerous cancers, and changes in SERCA expression have been reported during cell lineage differentiation. Therefore, a wider opportunity may lie in determining how SERCA-mediated Ca2+ switching helps cells find not just their route, but also their fate (Truong, 2017).

Probing the fractal pattern of heartbeats in Drosophila pupae by visible optical recording system

Judiciously tuning heart rates is critical for regular cardiovascular function. The fractal pattern of heartbeats - a multiscale regulation in instantaneous fluctuations - is well known for vertebrates. The most primitive heart system of the Drosophila provides a useful model to understand the evolutional origin of such a fractal pattern as well as the alterations of fractal pattern during diseased statuses. A non-invasive visible optical heart rate recording system especially suitable for long-term recording was developed by using principal component analysis (PCA) instead of fluorescence recording system to avoid the confounding effect from intense light irradiation. To deplete intracellular Ca(2+) levels, the expression of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) was tissue-specifically knocked down. The SERCA group shows longer heart beat intervals as compared to the control group. The multiscale correlation of SERCA group, on the other hand, is weaker than that of the control Drosophila. It is concluded that fractal correlations were presented in control group but were disrupted by the heart specific SERCA depletion (Lin, 2016).

Bafilomycin A disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion

Autophagosome-lysosome fusion and autolysosome acidification constitute late steps in the autophagic process necessary to maintain functional autophagic flux and cellular homeostasis. Both of these steps are disrupted by the V-ATPase inhibitor bafilomycin A1, but the mechanisms potentially linking them are unclear. The role of lysosomal acidification in autophagosome-lysosome fusion was recently revisited, using an in vivo approach in Drosophila. Surprisingly, vesicle fusion remained active in V-ATPase-depleted cells, indicating that autophagosome-lysosome fusion and autolysosome acidification are 2 separable processes. In contrast, bafilomycin A1 inhibited both acidification and fusion, consistent with its effects in mammalian cells. Together, these results imply that this drug inhibits fusion independently of its effect on V-ATPase-mediated acidification. The ER-calcium ATPase Ca-P60A/dSERCA was identified as a novel target of bafilomycin A1. Autophagosome-lysosome fusion was defective in Ca-P60A/dSERCA-depleted cells, and bafilomycin A1 induced a significant increase in cytosolic calcium concentration and disrupted Ca-P60A/SERCA-mediated fusion. Thus, bafilomycin A1 disrupts autophagic flux by independently inhibiting V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion (Mauvezin, 2015).

Complementary genomic screens identify SERCA as a therapeutic target in NOTCH1 mutated cancer

Notch1 is a rational therapeutic target in several human cancers, but as a transcriptional regulator, it poses a drug discovery challenge. To identify Notch1 modulators, two cell-based, high-throughput screens were performed for small-molecule inhibitors and cDNA enhancers of a NOTCH1 allele bearing a leukemia-associated mutation. Sarco/endoplasmic reticulum calcium ATPase (SERCA) channels emerged at the intersection of these complementary screens. SERCA inhibition preferentially impairs the maturation and activity of mutated Notch1 receptors and induces a G0/G1 arrest in NOTCH1-mutated human leukemia cells. A small-molecule SERCA inhibitor has on-target activity in two mouse models of human leukemia and interferes with Notch signaling in Drosophila. These studies "credential" SERCA as a therapeutic target in cancers associated with NOTCH1 mutations (Roti, 2013).

Seipin promotes adipose tissue fat storage through the ER Ca(2)(+)-ATPase SERCA

Adipose tissue is central to the regulation of lipid metabolism. Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), one of the most severe lipodystrophy diseases, is caused by mutation of the Seipin gene. Seipin plays an important role in adipocyte differentiation and lipid homeostasis, but its exact molecular functions are still unknown. This study shows that Seipin physically interacts with the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) in both Drosophila and man. SERCA, an endoplasmic reticulum (ER) calcium pump, is solely responsible for transporting cytosolic calcium into the ER lumen. Like dSeipin, dSERCA cell-autonomously promotes lipid storage in Drosophila fat cells. dSeipin affects dSERCA activity and modulates intracellular calcium homeostasis. Adipose tissue-specific knockdown of the ER-to-cytosol calcium release channel ryanodine receptor (RyR) partially restores fat storage in dSeipin mutants. These results reveal that Seipin promotes adipose tissue fat storage by regulating intracellular calcium homeostasis (Bi, 2014).

Lipids are major cellular energy sources, membrane components, and signal molecules. Adipose tissue is the main storage site for neutral lipids. Proper lipid storage by adipose tissue is important for human health. Excess or impaired lipid storage in adipose tissue leads respectively to obesity and lipodystrophy, which are tightly associated with numerous metabolic syndromes such as diabetes, dyslipidemia, hypertriglyceridemia, and hepatic steatosis (Bi, 2014).

Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), one of the most severe lipodystrophy diseases in man, is characterized by a near-total loss of adipose tissue from birth or early infancy, severe insulin resistance, fatty liver, and muscular hypertrophy. BSCL2 is caused by mutation of the Seipin gene. Seipin encodes a homo-oligomeric protein that is integral to the endoplasmic reticulum (ER) membrane. Studies in yeast, flies, mice, and various cell lines have shown that loss of Seipin function leads to severe lipodystrophy, suppressed adipocyte differentiation, aberrant lipid droplet formation, and ectopic lipid accumulation. However, the exact molecular functions of Seipin remain unknown (Bi, 2014).

Calcium is an important intracellular signal responsible for regulating multiple cellular processes. The ER is the main intracellular calcium storage site and plays a key role in the maintenance of intracellular calcium homeostasis. The ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP3R) are two calcium channels that release calcium from the ER to the cytosol in response to cellular stimuli. The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps cytosolic calcium into the ER lumen and maintains a high calcium concentration difference between the ER lumen and the cytosol at rest. Several studies indicate that ER calcium homeostasis is critical for adipogenesis and lipid storage. For instance, the SERCA inhibitor thapsigargin inhibits the early stages of adipogenesis in cultured cells. The ER calcium sensor STIM1 was found to negatively regulate the differentiation of 3T3-L1 preadipocytes. Drosophila IP3R mutants exhibit excessive food intake and obesity (Bi, 2014).

This study identified that the ER Ca2+-ATPase SERCA is a Seipin binding protein in both Drosophila and man. It was further shown that dSERCA is required cell autonomously for lipid storage in Drosophila fat cells, and Seipin affects SERCA calcium pump activity and regulates intracellular calcium homeostasis in Drosophila. Reducing ER-to-cytosol calcium release restores the lipid storage function of fat cells in dSeipin mutants. These findings may provide an effective therapy for BSCL2 (Bi, 2014).

SERCA pumps are critical for maintaining ER calcium homeostasis and the activity of SERCA is tightly regulated in different cell types. SERCA has been linked to lipodystrophy; the level of SERCA2 is reduced in cells lacking lipin-1 or its activator protein NEP1-R1. Several lines of evidence favor a specific connection between Seipin and SERCA. First, unlike in lipin-1 mutant cells, the protein level of dSERCA is unchanged in dSeipin mutants. Second, overexpression of dSERCA in wild-type does not cause an obvious fat storage phenotype; however, it partially, but significantly, rescues the fat storage defect in dSeipin mutants. In addition, on its own, IP3R RNAi causes a much stronger phenotype than RyR RNAi, but it cannot rescue the dSeipin mutant phenotype, while RyR RNAi rescues strongly. Third, endogenous hSeipin can be coprecipitated with hSERCA2. Purified hSeipin and hSERCA can interact, but a small deletion of hSeipin totally abolishes the hSeipin/hSERCA interaction without affecting the ER localization of hSeipin. Phospholamban (PLN) and sarcolipin (SLN), the best-studied regulators of SERCA in muscle, inhibit SERCA pumps by reducing their affinity for cytosolic calcium. Neither PLN nor SLN is conserved in fly, suggesting that other factors regulate SERCA activity. This study suggested that Seipin may act as a conserved positive regulator of SERCA in adipose tissue, although the mechanisms for modulation of SERCA activity by Seipin are still unknown (Bi, 2014).

Quantitative proteomic mass spectrometry analysis suggests that the lipodystrophy in dSeipin mutants may be due to impaired lipogenesis and elevated fatty acid β-oxidation. This is supported by a previous finding that elevated lipogenesis, induced by overexpressing SREBP, can rescue the fat storage defect of dSeipin mutants. How calcium homeostasis affects lipogenesis and fatty acid β-oxidation is largely unknown. It was reported that mitochondrial fatty acid oxidation can be activated by calcium in isolated rat liver mitochondria. Moreover, SREBP-2 was found to be activated by the SERCA inhibitor thapsigargin through depletion of Insig-1 in cultured CHO cells. This is in contrast to the current findings and cannot at present help to explain why dSERCA RNAi or dSeipin mutants have reduced lipogenesis in fat cells (Bi, 2014).

The ER is the major intracellular calcium storage site as well as being the organelle responsible for lipid biosynthesis and protein folding. The accumulation of misfolded or unfolded proteins results in ER stress. Similarly, loss of ER lumenal calcium triggers ER stress. Interestingly, ER stress leads to elevated lipid storage in yeast and hepatic cells, but the underlying mechanisms are not fully understood. Moreover, hepatic overexpression of SERCA in obese mice reduced chronic ER stress and improved hepatic steatosis. This study has shown that impaired SERCA activity leads to reduced fat storage in adipose tissue. These seemingly opposite effects of impaired ER calcium homeostasis on fat storage in hepatocytes and adipocytes may reflect the tissue-specific manner in which lipid metabolism is regulated. Similar tissue-specific effects on fat storage have also been observed when autophagy is impaired. Hepatocyte-specific knockout of ATG7 or ATG5 by RNAi results in elevated fat storage, while knockdown of ATG5 or ATG7 in the preadipocyte cell line 3T3-L1 leads to decreased TAG accumulation. In addition, mice with adipocyte-specific knockout of ATG7 are lean and have greatly reduced white adipocyte mass (Bi, 2014).

ER calcium homeostasis and ER stress have previously been linked to transcriptional regulation, chaperone activity, and protein folding. At present, it is unknown whether the effect of ER calcium homeostasis on fat storage, such as lipogenesis and fatty acid β-oxidation, is mediated at the gene transcriptional level, the protein activity level, or both. The possibility cannot be ruled out that the elevated cytosol calcium level, as well as the reduced ER calcium level, may contribute to the lipodystrophy. Indeed, it has been reported that in lymphocytes the activity of DGAT is inhibited by an increase in intracellular calcium induced by the ionophore A23187. In addition, previous work suggests that lipolysis is strongly activated in differentiating adipocytes in Seipin mutant mice. However, the increase in intracellular calcium has antilipolytic effects mainly by decreasing phosphorylation of hormone-sensitive lipase (HSL). Therefore, it remains to be determined whether altered intracellular calcium homeostasis in the fat cells of dSeipin mutants has an effect on lipolysis (Bi, 2014).

These findings may be important in man for the following reasons. First, both Seipin and SERCA are highly conserved from fly to human, and endogenous hSeipin and hSERCA2b physically interact. Second, the ER calcium homeostasis regulators STIM1 and SERCA have previously been reported to affect the early stages of adipocyte differentiation, which is consistent with the role of Seipin in adipocyte differentiation. Third, BSCL2 patients are at much higher risk of hypertrophic cardiomyopathy and mild mental retardation, which may also be caused by disruption of intracellular calcium signaling. The finding that modulating ER calcium homeostasis through RyR significantly rescues the dSeipin mutant phenotype suggests a potential therapeutic approach for treating BSCL2 disease and possibly other lipodystrophies (Bi, 2014).

Drosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves

Calcium signalling is a highly versatile cellular communication system that modulates basic functions such as cell contractility, essential steps of animal development such as fertilization and higher-order processes such as memory. This study probed the function of calcium signalling in Drosophila wing imaginal discs through a combination of ex vivo and in vivo imaging and genetic analysis. Wing discs were found display slow, long-range intercellular calcium waves (ICWs) when mechanically stressed in vivo or cultured ex vivo. These slow imaginal disc intercellular calcium waves (SIDICs) are mediated by the inositol-3-phosphate receptor, the endoplasmic reticulum (ER) calcium pump SERCA and the key gap junction component Inx2. The knockdown of genes required for SIDIC formation and propagation negatively affects wing disc recovery after mechanical injury. These results reveal a role for ICWs in wing disc homoeostasis and highlight the utility of the wing disc as a model for calcium signalling studies (Picchio, 2013).

This study describes the occurrence of slow, long-range ICWs in imaginal discs (SIDICs), in vivo and ex vivo. The InsP3R, SERCA and Inx2 were identified as necessary components for SIDICs generation and propagation. Finally, it was found that the SIDICs constitute a response to mechanical stress, probably supporting the wound healing and regeneration (Picchio, 2013).

In comparison with previously reported calcium signals, such as the calcium flashes observed in the embryo or the calcium transients in the pupal notum, the SIDICs are different in several aspects: the propagation speed, the duration of the phenomenon and the latency between the source of stress and the generation of the wave. The dissimilarities in the type of calcium signal observed in wing discs and the embryo and pupal notum could reflect inherent differences in the composition of the tissues studied. Alternatively, the SIDICs and the calcium flashes may be encoding different types of information that fulfill different purposes during wound healing and regeneration. In the embryo, the calcium flashes recruit hemocytes, whereas in the pupal notum they coordinate cell contraction. To further study the function of the SIDICs in vivo, a more sophisticated in vivo imaging setup will need to be developed. The ex vivo setup, however, proves to be a valuable substitute (Picchio, 2013).

The wing disc has been an exquisite model for developmental genetics. This work expands the utility of this model by revealing that it can be employed for the study of calcium signalling and ICWs. Wound healing and regeneration probably require a complex interplay between developmental pathways and the ability to coordinate cells during morphogenetic movements. Interestingly, calcium signalling has been proposed to be at the nexus of many signaling pathways and this nexus function could be essential for its role during regeneration. It will be interesting to see whether the SIDICs link, and perhaps help to orchestrate, different signalling pathways and morphogenetic movements during wound healing and regeneration of Drosophila imaginal wing disc (Picchio, 2013).

Novel Drosophila model of myotonic dystrophy type 1: phenotypic characterization and genome-wide view of altered gene expression

Myotonic dystrophy type 1 (DM1) is a multisystemic RNA-dominant disorder characterized by myotonia and muscle degeneration. In DM1 patients, the mutant DMPK transcripts containing expanded CUG repeats form nuclear foci and sequester the Muscleblind-like 1 splicing factor, resulting in mis-splicing of its targets. However, several pathological defects observed in DM1 and their link with disease progression remain poorly understood. In an attempt to fill this gap, this study generated inducible transgenic Drosophila lines with increasing number of CTG repeats. Targeting the expression of these repeats to the larval muscles recapitulates in a repeat-size-dependent manner the major DM1 symptoms such as muscle hypercontraction, splitting of muscle fibers, reduced fiber size or myoblast fusion defects. Comparative transcriptional profiling performed on the generated DM1 lines and on the muscleblind (mbl)-RNAi line revealed that nuclear accumulation of toxic CUG repeats can affect gene expression independently of splicing or Mbl sequestration. Also, in mblRNAi contexts, the largest portion of deregulated genes corresponds to single-transcript genes, revealing an unexpected impact of the indirect influence of mbl on gene expression. Among the single-transcript Mbl targets is Muscle protein 20 involved in myoblast fusion and causing the reduced number of nuclei in muscles of mblRNAi larvae. Finally, by combining in silico prediction of Mbl targets with mblRNAi microarray data, the calcium pump dSERCA was found to be a Mbl splice target and it was shown that the membrane dSERCA isoform is sufficient to rescue a DM1-induced hypercontraction phenotype in a Drosophila model (Picchio, 2013).

Drosophila has already proved to be a powerful tool for conducting genetic screening and global analyses on the effect of CTG repeats in DM1. So far, an inducible line expressing 480 interrupted CTG repeats has been used at the adult stage and shows age-dependent muscle degeneration. This study generated a set of three inducible site-specific lines expressing 240, 600 or 960 interrupted CTG repeats. In some cases of DM1, the existence of variant repeats interrupting the pure CTG expansions have been observed. Interestingly, interruptions allow either repeat stabilization or repeats contraction. If in one peculiar DM1 family CCG and CGG variants are associated with Charcot-Marie-Tooth symptoms, the role of interruptions remains unclear in other patients. The interrupted CTG repeats have already been used to generate different animal models of DM1. One could consider a possibility of additive toxic effect in all these models. However, the CTCGA interruption motif commonly used for these models is different from already described variants and its toxicity as well as unstability have not been not reported so far (Picchio, 2013).

This study used CTG size variation to compensate for age effect in third instar larvae. The study assessed larval muscles instead of adult muscle for three reasons: (1) segmentally repeated larval musculature is organized in a stereotyped network of muscle fibers and is easy to analyze at morphological and functional levels, (2) establishing and characterizing larval DM1 model appears attractive for future genetic rescue approaches and molecular screening applications and (3) adult lethality of the Mef>mblRNAi line prevents comparative analyses with DM1 lines in adult flies (Picchio, 2013). 

As observed in patients, it was found that expressing an increasing number of CTG repeats in larval somatic muscles leadd to the formation of nuclear foci and that these foci co-localize with Drosophila MBNL1 ortholog, Mbl. As the number of repeats positively influences the number of foci per nucleus and worsen muscle phenotypes, the new Drosophila model of DM1 presented here could be of interest for simulating disease progression and (or) severity (Picchio, 2013). 

Global analysis of muscle pattern in this model reveals a histopathological defect called ‘splitting fibers’ already observed in mbnl1 knockout mice as well as in DM1 patients. Here, splitting occurs during larval stages characterized by rapid muscle growth. As observed in dorsal oblique fibers, it is initiated at muscle endings at the level of interaction with tendon cells. This suggests that splitting results from affected muscle attachment to tendon cells and (or) abnormal sarcomeric organization that weaken the integrity of myofibrils. This latter hypothesis is supported by decreased expression of two sarcomere components (Mhc, up) in the DM1960 line. Surprisingly, the Mef>240CTG line which doesn not exhibit visible foci within muscle nuclei displays altered motility associated with muscle splitting but no fiber defects. This observation suggests that splitting is sufficient to alter motility (Picchio, 2013). 

Also, SBM and VL3 fiber examination shows reduced muscle size. So far, it has been shown in primary cell culture of myoblasts from DM1 patients that the ability of DM1-derived myoblasts to fuse is affected, consequently reducing myotube length. This study reports that expressing non-coding CTG repeats affects in vivo myoblast fusion. Interestingly, microarray data and RT–qPCR performed at embryonic and larval stages on mutant lines have shown decreased expression of Mp20 encoding an actin-binding protein involved in Drosophila myoblast fusion. Mp20 appears as an attractive candidate gene for myoblast fusion defects in DM1, since by overexpressing Mp20 during myogenesis, the number of nuclei per fiber was rescued in DM1 lines and in the mbl attenuated line. Surprisingly, Mp20 does not appear to undergo alternative splicing (single-transcript gene according to Flybase), suggesting that its Mbl-dependent down-regulation could occur through an indirect effect of Mbl. It is also noteworthy that one human counterpart of Mp20, the Calponin 3 gene, has been found to be involved in myoblast fusion in vitro. Thus, genes of the Mp20/Calponin family appear as attractive candidates to be tested for their role in DM1 muscle defects in humans (Picchio, 2013). 

Finally, this study reports that mutant larvae and in particular those from DM1960 and mbl attenuated lines display altered motility with affected complex movements. Interestingly, by measuring the contractility index and sarcomere size, it was found that both the lines exhibit hypercontraction, a phenotype related to myotonia. It has been previously shown that mbnl1 disruption in the mouse also leads to myotonia. In the Drosophila DM1 model, the effect of CTG repeat size on the severity of myotonia was observed, so that the DM1600 line exhibits intermediate hypercontraction phenotypes when compared with DM1240 and DM1960. As not only hypercontraction but also affected myoblast fusion account for a reduced muscle size in pathological lines, this study assume that both parameters need to be repaired to fully rescue muscle length (Picchio, 2013).

It was also found that the severity of several phenotypes is positively correlated with the size of the CTG repeats. This was followed by comparative transcriptional profiling on DM1600 and DM1960 lines. First, during validation of selected candidate genes from microarray analyses, repeat-size-dependent deregulation of genes involved in carbohydrate and nitrogen metabolism was observed. A more systematic classification of candidates deregulated in a repeat-size-dependent manner and having human orthologs was then performed based on the ratio of their fold-change between the two conditions and on their function. Data reveals that genes encoding transporter proteins are significantly enriched among gene categories down-regulated in larvae carrying high repeat numbers (Picchio, 2013). 

Among these, repeat-size-dependent deregulation of smvt, whose human orthologs (SLC5A3, SLC5A5, SLC5A8 and SLC5A12) encode myo-inositol transporters, and CG17597/SCP-2, involved in phosphatidylinositol transfer and signaling, was validated. It is known that phosphatidylinositol is a derivative of myo-inositol, suggesting that both transporters may work in the same pathway. However, how the alterations of transporters influence the accumulation of the inositol forms and how this is connected to muscle defects remain to be investigated. It was also found that two genes involved in the sarcomere structure Mhc and up were both down-regulated specifically in the DM1960 context. In DM1 patients, it has been shown that Mhc ortholog MYH14 and up orthologs TNNT2 and TNNT3 are mis-spliced. Besides, another report provides evidence that in a Drosophila mbl null mutant, up transcripts are mis-spliced as well. However, the link between the Mhc and up gene deregulations and DM1 muscle phenotypes and their impact on DM1 pathogenesis have not yet been investigated. This study speculate that down-regulation of Mhc and up might be involved in splitting fiber phenotype observed in DM1 larvae (Picchio, 2013). 

Comparative genomic analyses shows that a high percentage of genes with misregulated expression (∼70%) do not undergo alternative splicing but are sorted out in the Mef>mblRNAi context. As Mbl binds specifically to double-stranded RNA structures, the study hypothesized that it may influence transcript stability of this class of genes as already observed with MBNL1 in C2C12 cells. Alternatively, Mbl might play an indirect role on single-transcript genes via mis-splicing of transcription factors that regulate their expression. In order to gain insights into the second hypothesis, potential common regulators of Mbl-deregulated single-transcript genes were identified using the bioinformatics i-cisTarget approach. Interestingly, several transcription factors known to act in musclessuch as dMef2 and GATA factor Panier (Pnr) were found as potential transcriptional regulators of candidate genes. More importantly, the same transcription factors were found deregulated in transcriptional profiling experiments under all pathological conditions and most of them (including dMef2 and Pnr) were also predicted in silico to be targets of Mbl. Thus, these data reveal an important contribution of single-transcript gene deregulation in the Drosophila DM1 model and point to an indirect role of Mbl in the regulation of gene expression via mis-splicing of key myogenic factors. As a matter of fact, this mechanism may play a role in the regulation of Mp20 expression, one of dMef2 targets. Interestingly, both qPCR and microarray experiments showed that Mp20 expression is down-regulated in pathological contexts leading to myoblast fusion defects. Consequently, the study suggests an indirect role of Mbl in Mp20 expression through misregulation of dMef2 alternative splicing (Picchio, 2013). 

In DM1960 larvae, an Mbl-dependent muscle hypercontraction phenotype related to myotonia was observed. By associating microarray data with in silico prediction of Mbl direct targets, the dSERCA gene was identified as a putative candidate for Mbl-driven mis-splicing and hypercontraction phenotype. By RT–qPCR, it was confirmed that the isoforms B-H of dSERCA containing exons 8 or 11 encoding the transmembrane domain show decreased expression in mbl attenuated and in DM1960 lines. This indicates that in the Mbl-deficient context, the exons 8 or 11 of dSERCA are spliced out, leading to the production of dSERCA isoforms devoid of the transmembrane domain. This switch in dSERCA isoforms is consistent with the immunostaining of DM1 larval muscles, in which the membrane-associated dSERCA protein is barely detectable at muscle surface or even in sarcomere for the Mef>mblRNAi line, whereas the level of free dSERCA in nuclei appears to be enhanced in DM1 lines. It has been previously shown that in DM1 patient muscles, as a result of MBNL1 sequestration, SERCA1 exon 22 in the 3' part of the transcript is excluded leading to the formation of a neonatal isoform of SERCA1. This isoform is expected to cause muscle degeneration but so far, no functional analysis has been performed to confirm this hypothesis. However, patients with Brody's disease, which is caused by different mutations in the SERCA1a gene, manifest impairment of skeletal muscle relaxation among other symptoms. In addition, it has been shown that dSERCA plays a key role in muscle contraction and heartbeat frequency and rythmicity in flies, suggesting that it might be involved in muscle hypercontraction phenotypes and myotonia in DM1 muscles (Picchio, 2013). 

To date, the only gene functionally implicated in myotonia in DM1 is the CIC-1 encoding a muscle-specific chloride channel. CIC-1 transcripts have been found to undergo MBNL1- and CUGBP1-dependent splice modifications causing muscle delayed relaxation and pathogenic muscle defects. However, analyses performed on HSA(LR) myotonic mice reveals that ClC-1 channels account for muscle hyperexcitability in young but not in old DM1 animals, suggesting alteration of conductance other than chloride currents. Consequently, this study tested if the loss of dSERCA function and in particular depletion in its isoforms carrying the transmembrane domain could indeed affect muscle contractility. By using a pharmacological tool, CPA, a highly specific inhibitor of SERCA, which binds to the entry channel, it was found that the contractility of CPA-treated larval muscles is severely affected. Next, by performing rescue experiments by overexpressing the transmembrane isoform of dSERCA in DM1 lines with hypercontracted phenotypes, it was found that the contractility index is significantly improved. Thus, these data provide the first evidence in an animal model of DM1 that SERCA mis-splicing is involved in muscle hypercontraction (Picchio, 2013).

Parasitoid wasp venom SERCA regulates Drosophila calcium levels and inhibits cellular immunity

Because parasite virulence factors target host immune responses, identification and functional characterization of these factors can provide insight into poorly understood host immune mechanisms. The fruit fly Drosophila melanogaster is a model system for understanding humoral innate immunity, but Drosophila cellular innate immune responses remain incompletely characterized. Fruit flies are regularly infected by parasitoid wasps in nature and, following infection, flies mount a cellular immune response culminating in the cellular encapsulation of the wasp egg. The mechanistic basis of this response is largely unknown, but wasps use a mixture of virulence proteins derived from the venom gland to suppress cellular encapsulation. To gain insight into the mechanisms underlying wasp virulence and fly cellular immunity, a joint transcriptomic/proteomic approach was used to identify venom genes from Ganaspis sp.1 (G1), a previously uncharacterized Drosophila parasitoid species; G1 venom was found to contain a highly abundant sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. Accordingly, it was found that fly immune cells termed plasmatocytes normally undergo a cytoplasmic calcium burst following infection, and that this calcium burst is required for activation of the cellular immune response. It was further found that the plasmatocyte calcium burst is suppressed by G1 venom in a SERCA-dependent manner, leading to the failure of plasmatocytes to become activated and migrate toward G1 eggs. Finally, by genetically manipulating plasmatocyte calcium levels, fly immune success against G1 and other parasitoid species was altered. This characterization of parasitoid wasp venom proteins led to the identification of plasmatocyte cytoplasmic calcium bursts as an important aspect of fly cellular immunity (Mortimer, 2013).

A new genetic model of activity-induced Ras signaling dependent pre-synaptic plasticity in Drosophila

Techniques to induce activity-dependent neuronal plasticity in vivo allow the underlying signaling pathways to be studied in their biological context. This study demonstrates activity-induced plasticity at neuromuscular synapses of Drosophila double mutant for comatose (an NSF mutant) and Kum (Calcium ATPase at 60A: a SERCA mutant), and presents an analysis of the underlying signaling pathways. comt; Kum (CK) double mutants exhibit increased locomotor activity under normal culture conditions, concomitant with a larger neuromuscular junction synapse and stably elevated evoked transmitter release. The observed enhancements of synaptic size and transmitter release in CK mutants are completely abrogated by: a) reduced activity of motor neurons; b) attenuation of the Ras/ERK signaling cascade; or c) inhibition of the transcription factors Fos and CREB. All of which restrict synaptic properties to near wild type levels. Together, these results document neural activity-dependent plasticity of motor synapses in CK animals that requires Ras/ERK signaling and normal transcriptional activity of Fos and CREB. Further, novel in vivo reporters of neuronal Ras activation and Fos transcription also confirm increased signaling through a Ras/AP-1 pathway in motor neurons of CK animals, consistent with results from the genetic experiments. Thus, this study: a) provides a robust system in which to study activity-induced synaptic plasticity in vivo; b) establishes a causal link between neural activity, Ras signaling, transcriptional regulation and pre-synaptic plasticity in glutamatergic motor neurons of Drosophila larvae; and c) presents novel, genetically encoded reporters for Ras and AP-1 dependent signaling pathways in Drosophila (Freeman, 2010).

This study describes a new model for activity-dependent pre-synaptic plasticity in Drosophila. In the double mutant combination of comt and Kum, sustained elevation of neural activity (potentially including seizure-like motor neuron firing under normal rearing conditions) results in the expansion of motor synapses with a concomitant increase in transmitter release. These synaptic changes are mediated by the Ras/ERK signaling cascade and the activity of at least two key transcription factors, CREB and Fos. In vivo reporter assays also directly demonstrate Ras activation and enhanced transcription of Fos in the nervous system. CK is the only genetic model of synaptic plasticity in Drosophila in which pre-synaptic plasticity has been correlated with the Ras/ERK signaling cascade. This result is especially relevant given the wide conservation of the Ras/ERK signaling cascade in plasticity and recent demonstrations of the involvement of this signaling cascade in learning behavior in flies. Significant insights into Ras mediated regulation of both synapse growth and transmitter release are also presented (Freeman, 2010).

Non-invasive methods to manipulate neural activity in select neurons continue to be an important experimental target in plasticity research. In Drosophila, combinations of the eag and Shaker potassium channel mutants have long been used to chronically alter neural activity and study downstream cellular events. In recent years, transgenic expression of modified Shaker channels has also been generated and used to alter excitability in both neurons and muscles. However, the CK model of activity-dependent plasticity was developed since in synaptic changes in CK were consistently more robust than eag Sh and core plasticity-related signaling components were activated in a predictable manner in CK mutants. Another advantage with CK is the option of acutely inducing seizures as has been used to identify activity-regulated genes. CK thus combines advantages of both eag Sh and seizure mutants, and as is shown in this study, leads to an activity-dependent increase in synaptic size and transmitter release. It is believed that this model will prove highly beneficial to the large community of researchers who investigate synaptic plasticity in Drosophila. The utility of more recent techniques (such as the ChannelRhodopsin or the newly reported temperature sensitive TrpA1 channel transgenes) to induce neural activity-dependent synaptic plasticity at Drosophila motor synapses has not been tested yet and it will be interesting to see if these afford greater experimental flexibility in the future (Freeman, 2010).

Signal transduction through the Ras cascade has been shown to affect both dendritic and pre-synaptic plasticity in invertebrate and vertebrate model systems. In mammalian neurons, Ras signaling has been linked to hippocampal slice LTP, changes in dendritic spine architecture and plasticity of cultured neurons. In this context, Ras signaling has been shown to impinge on downstream MAP kinase signaling, thus implicating a canonical signaling module already established as a mediator of long-term plasticity in vertebrates. In Drosophila, expression of a mutant constitutively active Ras that is predicted to selectively target ERK leads to synapse expansion and increased localized phosphorylation of ERK at pre-synaptic terminals. In light of these observations, tests were performed to see if Ras signaling os necessary and sufficient for synaptic plasticity in CK. The results suggest that synaptic changes in CK are driven by stimulated Ras/ERK signaling in Drosophila motor neurons, and these can be replicated by directly enhancing Ras signaling in these cells. Furthermore Ras activation was found to be sufficient to cause stable elevation in pre-synaptic transmitter release. Finally, evidence is provided to show that synaptic effects of Ras activation require the function of both Fos and CREB in motor neurons. The consistency of signaling events in CK with those observed in mammalian preparations makes this a more useful and generally applicable genetic model of synaptic plasticity (Freeman, 2010).

In vivo reporters of neural activity have been difficult to design but offer better experimental resolution and flexibility over standard immuno-histochemical or RNA in situ methods to detect changes in gene expression in the brain. Thus, a good reporter permits increased temporal and spatial resolution, the option of live imaging (for fluorescent reporters) and in the case of transcriptional reporters, better understanding of cis-regulatory elements that control activity-dependent gene expression. This paper describes two genetically encoded reporters with utility clearly beyond the current study; a Raf based reporter to detect Ras activation in neurons and an enhancer based reporter to detect transcription of Fos (Freeman, 2010).

The Ras binding domain of Raf has been used previously to detect Ras expression in yeast, mammalian cell lines, and recently in hippocampal neuron dendrites. This study used a similar strategy to model the reporter using the conserved Ras binding domain and the cysteine-rich domain (RBD + CRD) from Drosophila Raf, under the reasonable assumption that this would provide sensitive reporter activity in neurons. This is the first time that a Ras reporter has been utilized in an intact metazoan organism to measure changes in endogenous Ras activity. In addition to confirming Ras activation in CK brains, it is expected that this reporter will find widespread use in tracing Ras activation in multiple tissues through development and in response to signaling changes in the entire organism. Since the reporter is based on the GAL4-UAS system, it can be expressed in tissues of choice, limiting reporter activity to regions of interest. Indeed, the experiments with the eye-antennal imaginal disc illustrate the utility of this reporter in identifying regions of activated Ras signaling during eye development (Freeman, 2010).

The Fos transcriptional reporter is one of the very few activity-regulated reporters in existence in Drosophila and it should find broad acceptance as a tool to map neural circuits in the fly brain that show activity-dependent plasticity. The reporter believed to be reasonably accurate since it is expressed in expected tissue domains (embryonic leading edge cells, for instance), and also co-localizes extensively with anti-Fos staining in the larval brain. There are several recognizable transcription factor binding motifs that can be detected in this 5 kb region of DNA (including binding sites for CREB, Fos, Mef2 and c/EBP). Which of these transcription factors regulate activity-dependent Fos expression from this enhancer is currently unknown. However, future experiments that dissect functional elements in this large enhancer region are expected to refine and identify these regulatory elements. Such studies are likely to lead the way in the development of a new generation of neural activity reporters in the brain (Freeman, 2010).

Functions of SERCA orthologs in other species

A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle

Muscle contraction depends on release of Ca(2+) from the sarcoplasmic reticulum (SR) and reuptake by the Ca(2+)adenosine triphosphatase SERCA. A putative muscle-specific long noncoding RNA has been discovered that encodes a peptide of 34 amino acids and that was named dwarf open reading frame (DWORF). DWORF localizes to the SR membrane, where it enhances SERCA activity by displacing the SERCA inhibitors, phospholamban, sarcolipin, and myoregulin. In mice, overexpression of DWORF in cardiomyocytes increases peak Ca(2+) transient amplitude and SR Ca(2+) load while reducing the time constant of cytosolic Ca(2+) decay during each cycle of contraction-relaxation. Conversely, slow skeletal muscle lacking DWORF exhibits delayed Ca(2+) clearance and relaxation and reduced SERCA activity. DWORF is the only endogenous peptide known to activate the SERCA pump by physical interaction and provides a means for enhancing muscle contractility (Nelson, 2016).

Small molecular allosteric activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) attenuates diabetes and metabolic disorders

Dysregulation of endoplasmic reticulum (ER) Ca(2+) homeostasis triggers ER stress leading to the development of insulin resistance in obesity and diabetes. Impaired function of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) has emerged as a major contributor to ER stress. This study pharmacologically activated SERCA2b in a genetic model of insulin resistance and type 2 diabetes (ob/ob mice) with a novel allosteric activator, CDN1163, which markedly lowered fasting blood glucose, improved glucose tolerance, and ameliorated hepatosteatosis but did not alter glucose levels or body weight in lean controls. Importantly, CDN1163-treated ob/ob mice maintained euglycemia comparable with that of lean mice for >6 weeks after cessation of CDN1163 administration. CDN1163-treated ob/ob mice showed a significant reduction in adipose tissue weight with no change in lean mass, assessed by magnetic resonance imaging. They also showed an increase in energy expenditure using indirect calorimetry, which was accompanied by increased expression of uncoupling protein 1 (UCP1) and UCP3 in brown adipose tissue. CDN1163 treatment significantly reduced the hepatic expression of genes involved in gluconeogenesis and lipogenesis, attenuated ER stress response and ER stress-induced apoptosis, and improved mitochondrial biogenesis, possibly through SERCA2-mediated activation of AMP-activated protein kinase pathway. The findings suggest that SERCA2b activation may hold promise as an effective therapy for type-2 diabetes and metabolic dysfunction (Kang, 2016).

Sarcolipin and phospholamban inhibit the calcium pump by populating a similar metal ion-free intermediate state

Microsecond molecular dynamics (MD) simulations and protein pKa calculations were performed of the muscle calcium pump (sarcoplasmic reticulum Ca(2+)-ATPase, SERCA) in complex with sarcolipin (SLN) to determine the mechanism by which SLN inhibits SERCA. SLN and its close analog phospholamban (PLN) are membrane proteins that regulate SERCA by inhibiting Ca(2+) transport in skeletal and cardiac muscle. Although SLN and PLB binding to SERCA have different functional outcomes on the coupling efficiency of SERCA, both proteins decrease the apparent Ca(2+) affinity of the pump, suggesting that SLN and PLB inhibit SERCA by using a similar mechanism. Recently, MD simulations showed that PLB inhibits SERCA by populating a metal ion-free, partially-protonated E1 state of the pump, E1. X-ray crystallography studies at 40-80 mM Mg(2+) have proposed that SLN-bound SERCA populates E1.Mg(2+), an intermediate with Mg(2+) bound near transport site I. To test this proposed mode of SLN regulation, a 0.5-m&mi;s MD simulation of E1.Mg(2+)-SLN was performed in a solution containing 100 mM K(+) and 3 mM Mg(2+), with calculation of domain dynamics in the cytosolic headpiece and side-chain ionization and occupancy in the transport sites. SLN was found to increases the distance between residues E771 and D800, thereby rendering E1.Mg(2+) incapable of producing a competent Ca(2+) transport site I. Following removal of Mg(2+,) a 2-m&mi;s MD simulation of Mg(2+)-free SERCA-SLN showed that Mg(2+) does not re-bind to the transport sites, indicating that SERCA-SLN does not populate E1.Mg(2+) at physiological conditions. Instead, protein pKa calculations indicate that SLN stabilizes a metal ion-free SERCA state protonated at residue E771, but ionized at E309 and D800. It is concludes that both SLN and PLB inhibit SERCA by populating a similar metal ion-free intermediate state. It is proposed that (1) this partially-protonated intermediate serves as the consensus mechanism for SERCA inhibition by other members of the SERCA regulatory subunit family including myoregulin and sarcolamban, and (2) this consensus mechanism is utilized to regulate Ca(2+) transport in skeletal and cardiac muscle, with important implications for therapeutic approaches to muscle dystrophy and heart failure (Espinoza-Fonseca, 2015).

SERCA2 dysfunction in Darier disease causes endoplasmic reticulum stress and impaired cell-to-cell adhesion strength: rescue by Miglustat

Darier disease (DD) is a severe dominant genetic skin disorder characterized by the loss of cell-to-cell adhesion and abnormal keratinization. The defective gene, ATP2A2, encodes sarco/endoplasmic reticulum (ER) Ca2+ -ATPase isoform 2 (SERCA2), a Ca2+ -ATPase pump of the ER. This study shows that Darier keratinocytes (DKs) display biochemical and morphological hallmarks of constitutive ER stress with increased sensitivity to ER stressors. Desmosome and adherens junctions (AJs) displayed features of immature adhesion complexes: expression of desmosomal cadherins (desmoglein 3 (Dsg3) and desmocollin 3 (Dsc3)) and desmoplakin was impaired at the plasma membrane, as well as E-cadherin, beta-, alpha-, and p120-catenin staining. Dsg3, Dsc3, and E-cadherin showed perinuclear staining and co-immunostaining with ER markers, indicative of ER retention. Consistent with these abnormalities, intercellular adhesion strength was reduced as shown by a dispase mechanical dissociation assay. Exposure of normal keratinocytes to the SERCA2 inhibitor thapsigargin recapitulated these abnormalities, supporting the role of loss of SERCA2 function in impaired desmosome and AJ formation. Remarkably, treatment of DKs with the orphan drug Miglustat, a pharmacological chaperone, restored mature AJ and desmosome formation, and improved adhesion strength. These results point to an important contribution of ER stress in DD pathogenesis and provide the basis for future clinical evaluation of Miglustat in Darier patients (Savignac, 2014).

Privileged coupling between Ca(2+) entry through plasma membrane store-operated Ca(2+) channels and the endoplasmic reticulum Ca(2+) pump

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) is the third element of capacitative calcium entry. It colocalizes with STIM1 and Orai1 at puncta, where it couples plasma membrane store-operated Ca(2+) channels (SOC) to Ca(2+) pumping into the ER. The efficiency of this calcium entry-calcium refilling (CECR) coupling is comparable to the classic excitation-response transduction mechanisms. This allows efficient filling of the endoplasmic reticulum (ER) with the Ca(2+) entering through SOC channels with little progression of the Ca(2+) wave towards the cell core. CECR coupling is very sensitive to changes in stoichiometry among STIM, Orai and SERCA, with excess Orai antagonizing ER refilling. ER takes up most of the calcium load that enters through SOC, whereas mitochondria take up a very small fraction. This difference is due to the spatial positioning with regard to SOC, the amplitude of the high Ca(2+) microdomains, and the differences in the Ca(2+) affinity of the uptake mechanisms (Alonso, 2012).


Search PubMed for articles about Drosophila SERCA

Alonso, M. T., Manjarres, I. M. and Garcia-Sancho, J. (2012). Privileged coupling between Ca(2+) entry through plasma membrane store-operated Ca(2+) channels and the endoplasmic reticulum Ca(2+) pump. Mol Cell Endocrinol 353(1-2): 37-44. PubMed ID: 21878366

Balcazar, D., Regge, V., Santalla, M., Meyer, H., Paululat, A., Mattiazzi, A. and Ferrero, P. (2018). SERCA is critical to control the Bowditch effect in the heart. Sci Rep 8(1): 12447. PubMed ID: 30127403

Baumbach, J., Hummel, P., Bickmeyer, I., Kowalczyk, K. M., Frank, M., Knorr, K., Hildebrandt, A., Riedel, D., Jackle, H. and Kuhnlein, R. P. (2014). A Drosophila in vivo screen identifies store-operated calcium entry as a key regulator of adiposity. Cell Metab 19(2): 331-343. PubMed ID: 24506874

Bi, J., Wang, W., Liu, Z., Huang, X., Jiang, Q., Liu, G., Wang, Y. and Huang, X. (2014). Seipin promotes adipose tissue fat storage through the ER Ca(2)(+)-ATPase SERCA. Cell Metab 19(5): 861-871. PubMed ID: 24807223

Deng, H., Gerencser, A. A. and Jasper, H. (2015). Signal integration by Ca(2+) regulates intestinal stem-cell activity. Nature 528(7581): 212-217. PubMed ID: 26633624

Ding, L., Yang, X., Tian, H., Liang, J., Zhang, F., Wang, G., Wang, Y., Ding, M., Shui, G. and Huang, X. (2018). Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism. Embo J 37(17). PubMed ID: 30049710

Espinoza-Fonseca, L. M., Autry, J. M. and Thomas, D. D. (2015). Sarcolipin and phospholamban inhibit the calcium pump by populating a similar metal ion-free intermediate state. Biochem Biophys Res Commun 463(1-2): 37-41. PubMed ID: 25983321

Freeman, A., et al. (2010). A new genetic model of activity-induced Ras signaling dependent pre-synaptic plasticity in Drosophila. Brain Res. 1326: 15-29. PubMed Citation: 20193670

Kang, S., Dahl, R., Hsieh, W., Shin, A., Zsebo, K. M., Buettner, C., Hajjar, R. J. and Lebeche, D. (2016). Small molecular allosteric activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) attenuates diabetes and metabolic disorders. J Biol Chem 291(10): 5185-5198. PubMed ID: 26702054

Lin, C., Chang, Y. C., Cheng, Y. C., Lai, P. J., Yeh, C. H., Hsieh, W. H., Hu, K., Wu, J. T., Lee, H. H., Lo, M. T. and Ho, Y. L. (2016). Probing the fractal pattern of heartbeats in Drosophila pupae by visible optical recording system. Sci Rep 6: 31950. PubMed ID: 27535299

Mauvezin, C. and Neufeld, T. P. (2015). Bafilomycin A disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion. Autophagy 11(8):1437-8. PubMed ID: 26156798

Moraru, A., Cakan-Akdogan, G., Strassburger, K., Males, M., Mueller, S., Jabs, M., Muelleder, M., Frejno, M., Braeckman, B. P., Ralser, M. and Teleman, A. A. (2017). THADA regulates the organismal balance between energy storage and heat production. Dev Cell 41(1): 72-81. PubMed ID: 28399403

Mortimer, N. T., Goecks, J., Kacsoh, B. Z., Mobley, J. A., Bowersock, G. J., Taylor, J. and Schlenke, T. A. (2013). Parasitoid wasp venom SERCA regulates Drosophila calcium levels and inhibits cellular immunity. Proc Natl Acad Sci U S A 110(23): 9427-9432. PubMed ID: 23690612

Nelson, B. R., Makarewich, C. A., Anderson, D. M., Winders, B. R., Troupes, C. D., Wu, F., Reese, A. L., McAnally, J. R., Chen, X., Kavalali, E. T., Cannon, S. C., Houser, S. R., Bassel-Duby, R. and Olson, E. N. (2016). A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle. Science 351(6270): 271-275. PubMed ID: 26816378

Periz, G. and Fortini, M. E. (1999). Ca(2+)-ATPase function is required for intracellular trafficking of the Notch receptor in Drosophila. EMBO J 18(21): 5983-5993. PubMed ID: 10545110

Picchio, L., Plantie, E., Renaud, Y., Poovthumkadavil, P. and Jagla, K. (2013). Novel Drosophila model of myotonic dystrophy type 1: phenotypic characterization and genome-wide view of altered gene expression. Hum Mol Genet 22: 2795-2810. PubMed ID: 23525904

Reuveny, A., Shnayder, M., Lorber, D., Wang, S. and Volk, T. (2018). Ma2/d promotes myonuclear positioning and association with the sarcoplasmic reticulum. Development 145(17). PubMed ID: 30093550

Roti, G., Carlton, A., Ross, K. N., Markstein, M., Pajcini, K., Su, A. H., Perrimon, N., Pear, W. S., Kung, A. L., Blacklow, S. C., Aster, J. C. and Stegmaier, K. (2013). Complementary genomic screens identify SERCA as a therapeutic target in NOTCH1 mutated cancer. Cancer Cell 23(3): 390-405. PubMed ID: 23434461

Savignac, M., Simon, M., Edir, A., Guibbal, L. and Hovnanian, A. (2014). SERCA2 dysfunction in Darier disease causes endoplasmic reticulum stress and impaired cell-to-cell adhesion strength: rescue by Miglustat. J Invest Dermatol 134(7): 1961-1970. PubMed ID: 24390139

Suisse, A. and Treisman, J. E. (2019). Reduced SERCA function preferentially affects Wnt signaling by retaining E-Cadherin in the endoplasmic reticulum. Cell Rep 26(2): 322-329. PubMed ID: 30625314

Truong, T. V., Bower, D. J., Featherstone, N. C., Connell, M. G., Al Alam, D., Frey, M. R., Trinh, L. A., Fernandez, G. E., Warburton, D., Fraser, S. E., Bennett, D. and Jesudason, E. C. (2017). SERCA directs cell migration and branching across species and germ layers. Biol Open 6(10): 1458-1471. PubMed ID: 28821490

Xu, C., Luo, J., He, L., Montell, C. and Perrimon, N. (2017). Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca2+ signaling in the Drosophila midgut. Elife 6. PubMed ID: 28561738

Zhou, Y., Huang, S., Shen, H., Ma, M., Zhu, B. and Zhang, D. (2017). Detection of glutathione in oral squamous cell carcinoma cells With a fluorescent probe during the course of oxidative stress and apoptosis. J Oral Maxillofac Surg 75(1): 223 e221-223 e210. PubMed ID: 27637779

Biological Overview

date revised: 20 February 2019

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