Work from our laboratory and others provide evidence that calreticulin (CRT), a prominent Ca2+-binding/buffering protein in the endoplasmic reticulum (ER) of eukaryotic cells, may be involved in pollen formation and function. Here, we show for the first time the expression pattern of the PhCRT1 gene and CRT accumulation in relation to exchangeable Ca2+ in Petunia hybrida developing anther, and discuss probable roles for this protein in the male gametophyte development.Pollen development in the anther in angiosperms depends on complicated cellular interactions associated with the expression of gametophytic and sporophytic genes which control fundamental processes during microsporo/gametogenesis, such as exo/endocytosis, intracellular transport, cell signaling, chromatin remodeling, and cell division. Most if not all of these cellular processes depend of local concentration of calcium ions  Ca2+ during the successive stages of microsporo/gametogenesis. We confirmed a permanent presence of both CRT and exchangeable Ca2+ in the germ line and tapetal cells, where these factors preferentially localized to the ER which is known to be the most effective intracellular Ca2+ store in eukaryotic cells. In addition, our immunoblots revealed a gradual increase in CRT level from the microsporocyte stage through the meiosis and the highest CRT level at the microspore stage, when both microspores and tapetal cells show extremely high secretory activity correlated with the biogenesis of the sporoderm.Using northern hybridization, western blot analysis, fluorescent in situ hybridization (FISH), immunocytochemistry, and potassium antimonate precipitation, we report that 2+ homeostasis during pollen grains formation. This Ca2+-buffering chaperone seems to be essential for pollen development and maturation since a high rate of protein synthesis and protein folding within the ER as well as intracellular Ca2+ homeostasis are strictly required during the multi-step process of pollen development.Our present data provide support for a key role of CRT in developing anther of angiosperms – regulation of CaThe online version contains supplementary material available at 10.1186/s12870-021-03409-4. Petunia) shed their pollen in a bicellular state . During microsporogenesis Fig. a-f, diplate Fig. g and the2+ [Differences between the successive stages of pollen development are reflected in their transcriptomic profiles. For example, in different plant species more genes are expressed in microspores and bicellular pollen but their number progressively declines in mature pollen . Althoug2+ –6.2+-binding/buffering protein in the ER of eukaryotic cells [Ginkgo [Liriodendron [Nicotiana [Haemanthus [Arabidopsis [Petunia [Nicotiana anther [CRT genes (CRT1/2 but not CRT3) was confirmed in Arabidopsis developing and mature anthers [Brassica [2+ storage/mobilization and protein folding [2+ homeostasis are strictly required during the multi-step processes of microsporo/gametogenesis.Work from our laboratory and others provide evidence that CRT, a prominent Caic cells  may be iic cells –10. CRT  [Ginkgo , Liriodeodendron , Nicotiaicotiana , Haemantemanthus , Arabidobidopsis  and Petu[Petunia –20. But a anther . Second, anthers . Third, Brassica . In plan folding , 9. BothPhCRT gene (belonging to the subgroup CRT1/2) and showed its elevated expression during the key reproductive events in the pistil, when the level of exchangeable Ca2+ changes dynamically [. We have also demonstrated that Petunia pollen tubes accumulate of both PhCRT1/2 mRNA and CRT protein in the ER-rich cytoplasmic regions [PhCRT1/2 expression impairs pollen tube elongation [PhCRT1 gene and CRT accumulation in relation to exchangeable Ca2+ in Petunia developing anther, and discuss probable roles for this protein in the male gametophyte development.Recently, we have cloned and characterized the full-length cDNA of the amically , 18. We  regions . Moreoveongation . Thus, wPhCRT1 in relation to subsequent stages of pollen development, we first investigated PhCRT1 mRNA transcript levels at particular phases of microsporo/gametogenesis  and in dry pollen collected from mature anther at the anther dehiscence. Northern blot analysis of total Petunia anther RNA revealed that the highest level of PhCRT1 expression was at the microsporocyte stage before meiosis  were detected throughout anther development in Nicotiana, while CRT expression was much lower in dry or hydrated pollen. This result indicates that the transcriptional activity of PhCRT1 or the stabilization of PhCRT1 mRNA decreases with progressive anther development and pollen maturation.To analyze the expression pattern of sis Fig. a. ExpresPetunia anthers and dry pollen. Using the polyclonal antibody against CRT (CRT PAb) that we have previously demonstrated to be specific for CRT in Petunia [Nicotiana anther which showed a particularly dense CRT immunolabeling in developing pollen and in the active tapetum through tetrads to pollen grains, and much lower signal in dry and hydrated pollen [PhCRT1 gene occurs at the beginning of microsporogenesis, however, the whole pool of transcripts do not undergo translation at once, and do not contribute to increase in CRT level at the microsporocyte stage. Activation of translation  starts with a slight delay reaching its highest level at the microspore stage what is correlated with biogenesis of the sporoderm.Next, we performed western blot analysis of total proteins isolated from whole  Petunia , we deted pollen . MoreovePhCRT1 mRNAs in developing pollen and the tapetal cells, samples of anther dissected from flowers buds or open flowers were fixed, embedded, sectioned, processed for FISH and visualized by fluorescence microscopy. Several anther developmental stages were selected for FISH using standard methylene blue staining of semi-thin sections: diploid microsporocytes .To determine distribution of tes Fig. a, dyads/tes Fig. e, early tes Fig. i, late mtes Fig. m, and mates Fig. q. Cross lls Fig. b. A complls Fig. f. Howevells Fig. j, n. Somlls Fig. f, j, n. lls Fig. r. At thiges Fig. n. A veryges Fig. r. Also vges Fig. f were nePhCRT1 mRNA transcripts in developing anther correlates with the microsporocyte stage  corresponding to exchangeable Ca2+ were observed in the same localizations where CRT was found; there were plasmodesmata  cytosolic free Ca2+ acts as a second messenger in multiple signal transduction pathways; and (3) loosely bound (exchangeable) Ca2+ is associated with fixed and mobile anions and is in dynamic equilibrium with the free Ca2+. Exchangeable Ca2+ is sequestered in different organelles or cell compartments, such as ER and the cell wall, where it can be associated with specific proteins that buffer and release Ca2+ to control its local concentration. This mechanism suggests an important role of Ca2+-binding/buffering proteins in stabilization of Ca2+ homeostasis in living cells. A good candidate for providing this function is CRT.The angiosperm anther is a male reproductive organ whose development is strictly regulated. To date, our understanding of the biological role of Caunctions : (1) ins2+ have similar distribution patterns during pollen development in Petunia anther. This is the first study to fully characterize the sites of localization of these two factors during the complicated intercellular communication that occurs between the developing male gametophyte and the sporophytic cells in the anther. First, we confirmed a permanent presence of CRT and Ca2+ ppts in the cytoplasm of the germ line and tapetal cells during the successive stages of microsporo/gametogenesis. The importance of exchangeable Ca2+ in the regulation of anther development has often been suggested for years. For example, Tian et al. [2+ ppts accumulate within tapetum and locules of rice anthers, but only few in developing microspores. Dynamic localization patterns of exchangeable Ca2+ were also observed in anthers of tabacco [2+ has been suggested to control the formation of vacuoles from mitochondria to regulate microspore development [2+ ppts compared to fertile anthers in male sterile plants [2+ in specific times and positions during pollen development, and that its abnormal distribution is related to pollen abortion. Second, our immunogold and cytochemical experiments revealed that both CRT and Ca2+ ppts preferentially localizes to the ER of these cells. The ER is known to be the most effective intracellular Ca2+ store in eukaryotic cells [2+-binding/buffering protein typically residing in the ER lumen [2+ with high affinity and capacity, it can serve as a mobile intracellular store of easily releasable Ca2+ and control its local concentration within the cytoplasm. Third, we detected the epitopes binding CRT PAb and Ca2+ ppts at plasmodesmata connecting adjacent microsporocytes or adjacent tapetal cells as well as connecting microsporocytes with the tapetal cells. It has long been recognized that plasmodesmata of higher plants contain a central strand of tightly compressed ER that creates the cytosolic sleeve providing continuity of cytoplasm between adjacent cells [2+ at plasmodesmata connecting adjacent cells in Petunia pollen chambers most likely corresponds with the ER position.Here we have demonstrated that both CRT and exchangeable Can et al.  found th tabacco , lettuce tabacco  and oil  tabacco . In wheaelopment . In addie plants , 29. Theic cells , whereasER lumen . Becausent cells . Thus, tPhCRT1 gene, highly expressed in Petunia anther, encodes a CRT1 isoform belonging to the CRT1/2 subgroup. These isoforms work as primary proteins within a general ER chaperone framework related to regulation of Ca2+ homeostasis [2+-binding ability [CRT1/2 genes were highly expressed in Arabidopsis developing/mature anther, whereas the expression of CRT3 gene was limited to mature anther/pollen. We previously revealed that the PhCRT gene expressed in Petunia pistil also belongs to the CRT1/2 subclass [PhCRT1/2 during pistil transmitting tract maturation, pollen-pistil interactions, double fertilization, and early embryogenesis [2+ changed dynamically [Petunia pollen tubes growing in vitro accumulated of both PhCRT1/2 mRNA and CRT protein in the ER-rich regions [2+ gradient in elongated pollen tubes [PhCRT1/2 expression strongly impairs pollen tube growth [2+ homeostasis during pollen development in Petunia anther. This idea seems to be supported by results obtained by Yu et al. [2+-binding protein, OsDEX1, which is expressed in tapetal cells and microspores during early anther development in rice. In osdex1 mutant anther, tapetal cell degeneration is delayed and degradation of the callosic cell wall surrounding the microspores is compromised, leading to aborted pollen formation and complete male sterility. Recombinant OsDEX1 is able to bind Ca2+ and regulate Ca2+ homeostasis in vitro, and osdex1 exhibited disturbed Ca2+ homeostasis in tapetal cells. These findings confirm a fundamental role of Ca2+-binding proteins in the development of tapetal cells and pollen formation, possibly via modulating the Ca2+ homeostasis during pollen formation.A key piece of data in support of our idea is our finding that the eostasis –10. Inst ability  suggesti ability  who reposubclass . We showogenesis  when theamically . We also regions , where Cen tubes  Finally,e growth . Therefou et al. . These a2+ in the same compartment, strongly suggesting that this pool of Ca2+ is bound by CRT. Since CRT has typical ER targeting and retention signals, it resides primarily in the ER lumen [Lupinus and Arabidopsis. We have previously demonstrated that both CRT and exchangeable Ca2+ were localized to the extracellular peripheries of highly specialized plant cells, such as pollen tubes and synergids [2+ ppts were found in extracellular peripheries of elongated pollen tubes. Our present results have revealed the same localization patterns for both CRT and exchangeable Ca2+ in the callosic cell wall surrounding dyads and tetrads during microsporogenesis in Petunia anther. Moreover, our double labeling experiments using the CRT PAb and monoclonal antibody against (1 → 3)-β-glucan  have confirmed that CRT co-localized with callose in this specific cell wall. Based on our previous and present data, we cannot exclude the possibility that this external Ca2+ store is equally important during the male gametophyte development as the main internal store, which is the ER. It should be noted that the neutral polymer of (1 → 3)-β-glucan does not bind Ca2+. Thus, we postulate that excess of exchangeable Ca2+ is translocated from the cell cytosol not only to the ER but also to the callosic cell wall, and bound by CRT present there in order to maintaining Ca2+ homeostasis in meiocytes during microsporogenesis. We proposed this idea previously when we observed co-localization of CRT, callose and Ca2+ ppts in the internal cell wall of growing pollen tube [One of our most interesting observations was that CRT was concentrated in the callosic cell wall surrounding dyads and tetrads. Accumulation of the CRT PAb in the callose deposition begins with the first meiotic division, peaking in the tetrad stage, and then disappears as the callosic cell wall is lysed after meiosis. This localization pattern of CRT corresponds well with the high level of loosely bound CaER lumen . HoweverER lumen . AlthougER lumen  providedynergids , 18, 22.len tube , 16, 22.2+-binding/buffering lectin-like molecular chaperone involved in proper folding, quality control, and maturation of newly synthesized glycoproteins within the secretory pathway [Petunia anther has the most common type of tapetum in angiosperms – the secretory (glandular) type. During microsporogenesis, the specialized tapetal cells remains around the anther locule. They exhibit highest secretory activity from the tetrade stage until the late microspore stage, and then they start breaking down just before the anther dehiscence. Tapetal cells play a vital role by secreting proteins, signals, and pollen wall material to ensure microspore/pollen development. In general, secretory cells are characterized by very high rate of protein synthesis and numerous ER cisternae and dictyosomes in close association with the plasma membrane. We have observed similar ultrastructure in the Petunia tapetal cells, in which CRT was highly abundant. Our immunogold experiments further revealed that CRT preferentially localizes to the ER, which is prominent in the cytoplasm of the tapetal cells during microsporogenesis. Thus, we suggest that in addition to regulating Ca2+ homeostasis, the high level of CRT expression in tapetum reflects a role for CRT as a chaperone to achieve high secretory activity of this highly specialized tissue to ensure pollen development. In fact, our immunoblots revealed a gradual increase in CRT level from the microsporocyte stage through the meiosis; the highest CRT level was at the microspore stage, when both microspores and tapetal cells show extremely high secretory activity correlated with the biogenesis of the sporoderm. By contrast, the highest level of PhCRT1 mRNA was observed at the microsporocyte stage; expression then decreased gradually through the subsequent stages of pollen development and dropped significantly in dry pollen. Together, these results indicate that PhCRT1 transcription in Petunia anther starts before meiosis, whereas CRT translation is postponed and reaches maximum level at the microspore stage, when the highest rates of protein synthesis and protein folding in the ER may be strictly required. These results are partially consistent with the previous studies by Nardi et al. [Nicotiana anther who reported the expression of CRT mRNA throughout microsporogenesis until the mature pollen stage, when the level of CRT mRNA significantly decreased. However, these authors detected the strongest immunohistochemical signal using the anti-spinach CRT antiserum in both the germ line and tapetal cells at the tetrad stage, when the sporoderm biogenesis starts. Regardless of these slight differences, the obtained results support the hypothesis that CRT’s chaperone activity may facilitate the high rate of protein synthesis required for pollen development.It is commonly accepted that CRT acts as a Ca pathway . The Peti et al.  in NicotBrassica anther [2+ ppts were found in the same localizations, including the ER of degenerating tapetum and the residual tapetum. Both CRT and Ca2+ ppts were also found in the apertures of maturing pollen grains. Therefore, further investigations are needed to explain CRT’s role at the late stage of pollen development within the anther.Interestingly, CRT was also detected in the lipid-rich tapetosomes that are abundant organelles in secretory tapetum of a anther . At an ea anther . SubsequPetunia developing anther, such as microsporocytes, dyads/tetrads, microspores, pollen grains, and the tapetal cells. The immunogold research have shown highly selective CRT distribution in specific nuclear sub-domains, especially in the nuclei of the tapetal cells with dispersed chromatin and large nucleoli. In these cells, we have detected CRT within chromatin, including the perichromatin areas and the nucleolus-associated chromatin. Several theories could explain the functional importance of CRT within the nucleus. Perichromatin fibrils are the sites of transcription and co-transcriptional processing of pre-mRNA and maturation of pre-RNA/mRNA [Petunia anther corresponded with sites of exchangeable Ca2+. This could indicate that CRT modulates nuclear Ca2+ homeostasis by regulating the local Ca2+ concentration. The lumen of the nuclear envelope is contiguous with the lumen of the rough ER and can form a branching intra-nuclear network which could serve as a mobile store of Ca2+ [2+ dynamics (free Ca2+ ions) in in vivo rapeseed microspores as well as in those reprogrammed to in vitro embryogenesis, establishing a link between changes in the cytosolic Ca2+ level and its nuclear distribution in the male gametophyte. Such dynamic changes in the Ca2+ levels between the nucleus and the cytosol require a very efficient and mobile Ca2+ store. CRT seems to be an excellent candidate to fulfill this role.Finally, we report the presence of CRT in the nuclei of several gametophytic and sporophytic cells in RNA/mRNA . Therefo of Ca2+ . Recentl of Ca2+  revealed2+ during pollen development in the anther. Based on the obtained results, we strongly believe that CRT1 plays crucial role in regulation of Ca2+ homeostasis during formation of the male gametophyte in angiosperms, and its further functioning as a pollen tube. Currently, a role of diverse ER chaperones, including CRT isoforms, in pollen development and the tube formation is under heated debate and opposing arguments are put forward by different research groups. We have recently proved that post-transcriptional silencing of the PhCRT1/2 expression strongly impairs pollen tube growth [Arabidopsis triple mutant of CRT1/2/3 grows and develops almost normally, while calnexin (CNX1) expression is crucial for normal pollen development, pollen germination and the tube elongation. Nevertheless, these authors suggest that both CNX and CRT proteins play essential and overlapping roles during vegetative growth and the male gametophyte development, and their complex relationship is strictly required for normal pollen formation, pollen germination and the tube elongation. It has to be noted, however, that the recent work published by Wakasa et al. [CNX genes does not have any impact for obtaining viable progeny in rice. Therefore, future functional studies are needed to help us sort out these possibilities.Our data for the first time clearly show an interplay between CRT1 and exchangeable Cae growth . In conte growth  indicatea et al.  has showPetunia hybrida were grown at room temperature in the Department of Cellular and Molecular Biology, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University in Toruń, Poland. Fresh anthers of Petunia were dissected from flowers at different developmental stages: microsporocytes, dyads/tetrads, microspores, and pollen grains. To study callose metabolism during microsporogenesis, germ line cells were isolated mechanically from one of five anthers of the same flower, stained with 0.1% aniline blue according to a standard protocol, and observed under a fluorescence microscope. This staining was helpful in determining particular developmental stages of microsporogenesis. For northern hybridization and western blot analysis, dissected anthers and dry pollen collected from flowers at the anther dehiscence were frozen in a liquid nitrogen and stored at − 80 °C until they were used. For light and electron microscopy, dissected anthers were fixed, dehydrated in ethanol, and embedded according to the protocols described below. To analyze the histological features of the anther at each of the developmental stages, semi-thin sections of the embedded anthers were stained with 0.1% methylene blue and observed by light microscopy. All experiments described below were repeated at least three times during several growing seasons with similar results.Commercial cultivars of PhCRT1 antisense probe and the Ph18S probe, were prepared as described previously [PhCRT1 insert was cut with Xba I and purified on CHROMA SPIN™ + TE − 100 columns. A 1-μg aliquot served as template to generate the DIG-labeled molecular probe using the DIG RNA Labeling Kit (SP6/T7) according to the manufacturer’s instructions (Roche). To obtain DIG-labeled 18S probe, plasmid containing the internal part of the Ph18S rRNA sequence was used as template. The probe was generated by PCR according to the manufacturer’s instructions .Two digoxigenin (DIG)-labelled molecular probes, the full-length eviously . In brie2O, supplemented with two volumes of denaturing buffer containing 50% formamide, 6.1% formaldehyde, 1 × MOPS, 1 × loading dye buffer, and 40 mg/dm− 3 ethidium bromide, and heat denatured. RNA samples were resolved on a 1.2% formaldehyde-agarose gel, capillary transferred onto Immobilon-NY+ membrane (Millipore), rinsed with H2O mQ, and UV crosslinked. The membrane was prehybridized for 40 min in DIG Easy Hyb buffer (Roche) at 65 °C, and then hybridized overnight to the DIG-labeled PhCRT11 probe at a final concentration 150 ng/ml. Chemiluminescence detection was performed according to the manufacturer’s instruction . To reprobe the membrane with Ph18S DIG-labeled probe, the blot was stripped twice at 80 °C in 50% formamide, 5% SDS, 50 mM Tris–HCl, pH 7.5, for 60 min, and then rinsed with 2 × SSC. Rehybridization was performed in the same conditions as hybridization described above. Northern hybridization was performed a minimum of three times, and representative blots were shown. Quantification of signals was done with Image Gauge 3.4 software (Science Lab99). All data obtained from the northern blot experiments were subjected to one-way ANOVA test.Northern hybridization was performed according to the protocol described previously . In briewas homogenized in liquid nitrogen, and soluble proteins were extracted in 50 mM Tris–HCl (pH 7.5), 1 mM EGTA, 2 mM DTT plus 1 mM PMSF and cOmplete Protease Inhibitor Cocktail (Roche) according to the manufacturer’s recommendation. The homogenates were centrifuged at 16,000 g for 30 min at 4 °C. Protein concentrations of the supernatants were measured, equal amounts of proteins were separated by electrophoresis on a 12.0% SDS–PAGE gel, and then the proteins were semi-dry transferred to Amersham PVDF Hybond-P membrane . Blocked blot was probed with a rabbit polyclonal antibody CRT PAb produced by Napier et al. [Immunoblotting was performed according to the protocol described previously . In brier et al. , washed,v/v) formaldehyde and 0.25% (v/v) glutaraldehyde in phosphate-buffered saline  for 1 h at room temperature (slight vacuum infiltration) followed by overnight fixation at 4 °C. The fixed samples were washed in PBS, dehydrated through a graded series of ethanol, embedded in LR Gold resin (Fluka), sectioned into semi-thin sections (cross sections through the anther), and transferred onto microscope slides covered with Biobond . The DIG-labeled antisense PhCRT1 molecular probe (generated above) was used at a final concentration of 0.5 μg/μl. Prehybridization and hybridization were carried out in 50% formamide, 4 × SSC, 5 × Denhardt’s, and 50 mM sodium phosphate buffer for 1 h at 42 °C and overnight at 37 °C, respectively. Signals were detected using primary mouse anti-DIG (Roche) and secondary goat-anti-mouse IgG-Alexa Fluor 488 (Life Technologies) antibodies in PBS. For negative control, the molecular probe was omitted. In the final step, DNA was stained with 1 mg/ml Hoechst 33342 (Molecular Probes) and the stained sections were mounted in ProLong Gold antifade reagent (Life Technologies). The sections were analyzed using a Nicon Eclipse 80i microscope. FISH was performed a minimum of three times, and representative data were shown.FISH was performed according to the protocol described previously  with sliw/v) bovine serum albumin (BSA) in PBS buffer, pH 7.2, the sections were incubated in 1:20 dilution of a primary CRT PAb in PBS supplemented with 0.3% (w/v) BSA and then incubated with: (i) a goat anti-rabbit IgG Cy3® secondary antibody (Sigma-Aldrich) for immunofluorescence labeling; (ii) a goat anti-rabbit IgG 1 nm gold-conjugated  secondary antibody for immunogold-silver staining technique; (iii) a goat anti-rabbit IgG 20 nm gold-conjugated  secondary antibody for immunogold labeling. For CRT and callose immunolocalizations, a double labeling technique was performed as previously described [Immunocytochemical localization of CRT was performed according to the protocols described previously , 18, 22 escribed , 22. In 2+ was performed according to the protocol described previously [w/v) potassium antimonate, 2% (v/v) glutaraldehyde, and 2% (v/v) formaldehyde in 0.1 M phosphate buffer  for 4 h at room temperature, and then subsequently postfixed with 1% (v/v) osmium tetroxide (OsO4) in the same buffer-antimonate solution for 12 h at 4 °C. The fixed samples were dehydrated in graduated ethanol concentrations and embedded in Spurr resin (Merck) according to the standard protocol. Ultra-thin sections (cross sections through the anther) were collected on copper grids, stained with 2.5% (w/v) uranyl acetate and 0.4% (w/v) lead citrate solutions, and examined by transmission electron microscopy (Jeol EM 1010) at 80 kV. The presence of Ca2+ in the Ca2+-antimonate precipitates (Ca2+ ppts) was confirmed previously using energy-dispersive X-ray microanalysis [Localization of exchangeable Caeviously , 22. In analysis . For negAdditional file 1: Figure S1. Northern blot analysis (a and b) and western blot analysis (c and d) in whole Petunia anthers during subsequent stages of pollen development  and in dry pollen (Pd). aPhCRT1 mRNA, bPh18S rRNA, cPhCRT, dPhGAPDH (boxed). Numbers above each blot point out separate lines; M protein marker . The top and bottom blot edges are marked with solid lines.


As a ubiquitous second messenger, calcium (Ca2+) can interact with numerous cellular proteins to regulate multiple physiological processes and participate in a variety of diseases, including hepatitis B virus (HBV) infection, which is a major cause of hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. In recent years, several studies have demonstrated that depends on the distinct Ca2+ channels on the plasma membrane, endoplasmic reticulum, as well as mitochondria, HBV can elevate cytosolic Ca2+ levels. Moreover, within HBV-infected cells, the activation of intracellular Ca2+ signaling contributes to viral replication via multiple molecular mechanisms. Besides, the available evidence indicates that targeting Ca2+ signaling by suitable pharmaceuticals is a potent approach for the treatment of HBV infection. In the present review, we summarized the molecular mechanisms related to the elevation of Ca2+ signaling induced by HBV to modulate viral propagation and the recent advances in Ca2+ signaling as a potential therapeutic target for HBV infection.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-021-00762-7. The chronic infection of the hepatitis B virus (HBV) is a public health concern and could cause different forms of liver diseases, including hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) around the world . DespiteThe regulation of cytosolic Ca2+ signaling mainly depends on the distinct ion channels located in the PM, ER, as well as mitochondria . For exaIn hepatocytes, the increase of cytosolic Ca2+ is mainly from the extracellular space, ER, and mitochondria. Among HBV-encoded proteins, HBX is found to be responsible for inducing extracellular Ca2+ influx , and theAs the largest intracellular Ca2+ storage, ER plays a very important role in regulating cytosolic Ca2+ levels . CurrentWithin the cells, reduced ER Ca2+ storage could activate Ca2+ sensors or SECAR channels to uptake Ca2+ , 25. On Mitochondria is a vital cellular organelle with the function of participating in the regulation of cellular Ca2+ signaling to promote the tricarboxylic acid cycle and ATP synthesis . The groAs a partially double-stranded DNA virus, HBV contains 4 overlapping open reading frames (ORFs): P, S, C, and X. The P ORF encodes Pol protein. S ORF encodes S, preS1, and preS2 domains to construct the LS (large surface), MS (middle surface), and S  proteins. Two genes locate in C ORF and are responsible for the expression of HBc and precore proteins. The X region is the least ORF, and it is capable of encoding HBX protein. After the virus interacts with the receptor sodium taurocholate cotransporting polypeptide (NTCP), it enters into the cells and uncoated. Sequentially, the genome of the virus was transferred into the nucleus and converted into covalently closed circular DNA (cccDNA). Next, the cccDNA forms a minichromosome and transcripts to 4 lengths of viral mRNA, including 3.5 kb preC mRNA and pregenomic RNA (pgRNA), 2.4 and 2.1 kb envelope mRNAs, and 0.7 kb X mRNA. The preC mRNA encodes precore protein, which is further cleaved and secrets as HBe. pgRNA translates HBc and Pol proteins as well as acts as a template for viral replication. The 2.4 and 2.1 kb envelope mRNAs encode LS, MS, and S proteins. Additionally, the X mRNA translates HBX protein. After these viral RNAs translate into HBV proteins, the pgRNA is encapsulated into core particles, and further reversely transcribes into viral DNA. Then, the viral particles containing HBV DNA are enveloped and secret from the cells , 32.In the past years, several works have been focused on understanding the role of Ca2+ signaling mediated by HBV in accelerating its replication. As a virus non-structural protein, HBX plays a critical role in HBV replication , 32. A rThe innate immune response is the first line of host defense against HBV infection. Binding to the interferon-α/β receptor, the innate immune molecule interferon (IFN) could initiate Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways to induce the expression of IFN-stimulated genes (ISGs) to further eliminate the virus . HoweverTo date, the current studies also demonstrated that increased Ca2+ mediated by HBX enhanced HBV core assembly  in hepatocytes  Because of the importance of Ca2+ signaling in HBV infection, the increasing evidence has demonstrated that controlling intracellular Ca2+ or Ca2+ channels by suitable pharmaceuticals is a potential strategy to control HBV replication Fig. . For insAs mentioned, HBV core assembly is promoted by Ca2+ signaling . The worThe evidence presenting here indicates that HBV can utilize multiple mechanisms to elevate the levels of intracellular Ca2+ . In turn, the increased cytosolic Ca2+ is capable of facilitating viral replication in several manners. Moreover, the available evidence suggests that the use of different kinds of pharmaceuticals that targeting Ca2+ signaling is a potent strategy for HBV. Until now, significant progress has been made in identifying the potential targets, including Ca2+ channels and Ca2+ dependent proteins, for pharmacological intervention . An in-dSpecifically, HBX has been demonstrated to participate in viral replication , 32, altBesides these, our knowledge on the role of Ca2+ signaling mediated by HBX on viral replication was mainly from the in vitro cellular models. The effect of Ca2+ signaling on HBX-dependent HBV replication in animal models as well as patients with HBV infection is unknown. Furthermore, although Ca2+ signaling facilitates HBV replication, the influence of cytosolic Ca2+ signaling on the development of HBV-associated diseases, including hepatitis, cirrhosis, and HCC, is not well clarified. Therefore, future studies are needed to provide more insights into the biological processes associated with the alteration of Ca2+ signaling mediated by HBV.


This problem is exacerbated by the close connections and crosstalk between cGMP-signalling and calcium (Ca2+)-signalling in photoreceptors. In this review, we summarize key aspects of cGMP-signalling and Ca2+-signalling relevant for hereditary photoreceptor degeneration. The topics covered include cGMP-signalling targets, the role of Ca2+ permeable channels, relation to energy metabolism, calpain-type proteases, and how the related metabolic processes may trigger and execute photoreceptor cell death. A focus is then put on cGMP-dependent mechanisms and how exceedingly high photoreceptor cGMP levels set in motion cascades of Ca2+-dependent and independent processes that eventually bring about photoreceptor cell death. Finally, an outlook is given into mutation-independent therapeutic approaches that exploit specific features of cGMP-signalling. Such approaches might be combined with suitable drug delivery systems for translation into clinical applications.The second messengers, cGMP and Ca The retina is a neuronal tissue devoted to the conversion of light-stimuli into electrochemical signals that can be interpreted by the central nervous system. The key conversion step is performed by photoreceptors, which are compartmentalized cells with an outer segment (OS) harboring the components of the phototransduction cascade, an inner segment (IS) containing mitochondria, a cell body with nucleus and organelles, and a synaptic region providing for connectivity with second-order neurons. Two main types of photoreceptors can be distinguished: rod photoreceptors (rods) and cone photoreceptors (cones). Rods respond to dim light and enable vision at night, whereas cones respond to bright daylight. In humans, cones are essential for high-resolution color vision .2+. In the dark, high levels of cGMP in photoreceptor OSs activate the cyclic nucleotide–gated channel (CNGC) to allow for an influx of Na2+ (≈80% of ion flux) and Ca2+ ions (≈20%) [2+ inhibit guanylyl cyclase–activating protein (GCAP), restricting GC activity [The phototransduction cascade relies to a large extent on the interplay of two essential signalling molecules: cyclic-Guanosine-Mono-Phosphate (cGMP) and Cas (≈20%) . cGMP iss (≈20%) . High leactivity , and proactivity , 34, 79.activity  Fig. 1)2+. In th+ and Ca2+ in the dark needs to be compensated for by other ion channels and transporters. Ca2+ ions are exported from the OS by the Na+/Ca2+/K+ exchanger (NCKX), which uses high extra-cellular to intra-cellular electrochemical gradients of Na+ and K+ ions to drive out Ca2+ [+ ions flowing into the OS through both CNGC and NCKX diffuse to the IS where they are exported by the ATP-driven Na+/K+ exchanger (NKX)  affect retinal photoreceptors, and in most cases these diseases are untreatable , 77. Wit+-levels , 65. Morent role , 77. Phoho genes , 69, 81.ho genes . Overall2+-signalling relevant for hereditary photoreceptor degeneration. Indeed, it appears that both too low and too high cGMP and/or Ca2+ signalling may be lethal for photoreceptors, when present during extended periods of time [2+ signalling for phototransduction and normal photoreceptor physiology. We then detail the potential roles that Ca2+ permeable channels and downstream Ca2+-dependent processes may play in degenerative events. Eventually, we highlight recent evidence indicating how exceedingly high photoreceptor cGMP levels and cGMP-dependent mechanisms set in motion cascades of Ca2+-dependent and Ca2+-independent processes that bring about photoreceptor cell death.In this review, we summarize key aspects of cGMP-signalling and Ca of time ). We lay+ and Ca2+ influx. Rod photoreceptor CNGC is composed of CNGA1 and CNGB1a subunits expressed in a 3:1 ratio [2+ influx in photoreceptors. Because Ca2+ has been suggested as a major disease driver for photoreceptor degeneration [CNGCs are heterotetrameric ion channels expressed in the OS membrane of rods and cones, where they mediate Na:1 ratio . Cone ph:1 ratio . In bothneration , CNGC maneration .2+ influx in photoreceptor OS mediates inhibition of cGMP synthesis in a regulatory feedback loop that normally ensures that both cGMP and Ca2+ levels in the OS are kept within physiological ranges. However, when this feedback loop is broken by mutations in RD-genes, the result can be pathological changes in the intracellular concentrations of either cGMP or Ca2+ or both at once. For instance, loss-of-function mutations in PDE6 genes cause an elevation of photoreceptor cGMP levels, overactivate CNGC, increase Ca2+ influx, and cause photoreceptor cell death [2+ depletion, disinhibition of GC, exceedingly high cGMP production, and again photoreceptor cell death [2+ levels, high in case of PDE6 mutations and low in CNGC mutations. Both types of genetic defects invariably result in photoreceptor death, with the common point being high photoreceptor cGMP. On the other hand, it is important to consider that Ca2+ channels and exchangers are tightly coupled in a cellular system, therefore reduced function of CNGC may be compensated in a cell by decreased activity of exchangers. At any rate, high levels of cGMP will overactivate cGMP-dependent protein kinase G (PKG) [As shown in Fig. ll death , 57. Conll death , 38, 112 G (PKG) , 112, th G (PKG) .PRKG1 gene gives rise to the alternatively spliced α and β isoforms of PKG1, while the PRKG2 gene encodes for PKG2 [The prototypic cellular target for cGMP-signalling is PKG . PKG is for PKG2 . To be afor PKG2 , 75.Prkg1 gene [Cngb1−/− x Prkg1−/− double mutant mice, in which both CNGC and PKG are dysfunctional, photoreceptor degeneration was markedly delayed when compared to Cngb1−/− single mutants [Although cell death in retinal degeneration is often believed to be driven by apoptosis, several studies hint at the involvement of a non-apoptotic cGMP/PKG-dependent cell death mechanism triggered by accumulation of cGMP in the degenerating photoreceptors , 77. In kg1 gene , yet it kg1 gene . In Cngb mutants .Taken together, PKG activation by high levels of photoreceptor cGMP is very likely to play an important role in triggering photoreceptor cell death. As far as the mechanisms triggered by high cGMP and PKG are concerned, two important questions remain: (1) At present it is not entirely clear which PKG isoform is responsible for photoreceptor cell death, a question that may be further addressed with conditional PKG knockout studies. (2) As a kinase, PKG has at least several hundred potential phosphorylation targets. While for instance the ryanodine receptor-2  or CNGC 2+ signalling via PDEs. There are 11 structurally related but functionally distinct mammalian PDE gene families (PDEs 1–11). PDEs 5, 6, and 9 are specific for cGMP; PDEs 4, 7, and 8 hydrolyse cAMP only; while PDEs 1, 2, 3, 10, and 11 can degrade both cGMP and cAMP [To terminate cGMP-signalling, the cGMP molecule is broken down by enzymes belonging to the phosphodiesterase (PDE) group, which specifically hydrolyse the 3′5′ cyclic phosphate bond. Interestingly, there is a potential for crosstalk between cGMP-, cyclic Adenosine-Mono-Phosphate (cAMP)-, and Caand cAMP . Among aand cAMP , 51. To 2+ is via adenylyl cyclase (AC), which is stimulated by Ca2+ to produce cAMP [2+ influx could conceivably raise photoreceptor cAMP levels. Moreover, cGMP activates the cAMP-specific PDE2, while it inhibits cAMP-specific PDE3. Thus, depending on the PDE expression pattern in a given cell type, cGMP can either increase or decrease cAMP-signalling [rd2 mouse retina; however, it is unclear whether this increase was specific to photoreceptors or to other cell types of the retina [rd1 mouse, elevation of retinal cAMP content was shown to be restricted to the inner retina [One possible connection between cyclic nucleotide signalling and Cauce cAMP . cAMP-siuce cAMP  and cGMPgnalling . With ree retina . In the r retina . Since n2+-dependent cysteine proteases. Even though their discovery dates back to 1964 [2+, respectively [2+ concentrations are thought to reach micromolar levels at most, calpain-1 is considered to be the most active isoform in vivo. Curiously, calpain-1 deficient mice present an apparently normal phenotype [Calpains are a group of Ca to 1964 , earlier to 1964 . The mosectively . Since thenotype , while chenotype , suggest2+-influx due to accumulation of cGMP may contribute to calpain protease activity, which was found to be associated to photoreceptor cell death [2+ concentrations, thus suggesting that when calpain-2 activation occurs, the cell may have entered a degenerative stage at which it can no longer maintain the normal intracellular Ca2+ homeostasis (approx. 500 nm). Thus, calpain-2 activation may happen relatively late during the final stages of cell death.Calpain-1 and calpain-2 appear to have opposing functions in the brain . While cll death , 50, 72.ll death , 67. Mosll death . RemarkaN-methyl-N-nitrosourea (MNU) [rd1 mouse model for RD [rd1 mouse [2+-activated calpain-type proteases are likely contributors to photoreceptor cell death.The inhibition of calpains with SNJ-1945 decreased cell death in a rat model of photoreceptor degeneration induced by ea (MNU) , while tl for RD . The tred1 mouse . Overall2+ prevents processing and release of AIF [The activity of calpain may contribute to retinal degeneration via activation of apoptosis-inducing factor (AIF) . The AIFe of AIF . In the e of AIF . All the+ to add poly(ADP-ribose) polymers to target proteins in response to a variety of cellular stresses [2+, indicating that both may take part in a common cell death pathway [N-methyl-d-aspartic acid (NMDA) toxicity model in rat primary cortical neurons, PARP and calpain were found to be linked via PARP-1 induced alterations in mitochondrial Ca2+ homeostasis [. One possible connection could be the excessive consumption of NAD+ by overactivated PARP-1. Since the intracellular pools of NAD+ and ATP are linked, the depletion of NAD+ could lead to a breakdown of photoreceptor energy metabolism (see below). Future studies may reveal the nature of relationship between calpains and PARP in retinal degeneration.Enzymes belonging to the 18-member poly(ADP-ribose) polymerase (PARP) family are involved in the repair of DNA damage but have also been linked to a variety of disease conditions , 89. Thestresses . Overact pathway , 117. In pathway . Yet, ca pathway , includi pathway . In addi pathway . Similareostasis . In a moeostasis . Hence, 2+ may influence photoreceptor metabolism and energy consumption.To maintain their OS dark current and to achieve single photon light sensitivity , photore+ and K+ ion gradient. The Na+ gradient is maintained by the activity of the ATP-driven NKX in the IS [+ and Ca2+ increases further, and the photoreceptor membrane potential becomes more positive than it would otherwise be in darkness. This requires extra NKX activity and likely puts additional strain on photoreceptor energy metabolism.The highest energy demanding function in a photoreceptor is the active transport of ions against their concentration and electrical gradients . As mentn the IS . Remarkan the IS , and conn the IS . On the n the IS . This, h2+ channels (VGCC) in the IS, cell body, and synapse [2+ influx is counterbalanced by the ATP-driven plasma membrane Ca2+-ATPase (PMCA) [2+-signalling impact photoreceptor energy metabolism in ways that are likely to have a bearing for retinal disease pathogenesis.Moreover, the continuous depolarization triggers a sustained activation of voltage-gated Ca synapse , 105. Ine (PMCA) . Hence, 2+ levels, alternative hypotheses that may be referred to as “high Ca2+” and “low Ca2+” hypotheses, respectively [2+ signalling might be targeted to delay RD progression. Pharmacological inhibition of Ca2+ channels has been suggested as a means to slow down degeneration in rd1 [Cngb1−/− x rd1 [Cngb1−/− x rd10 [2+ permeable channels might be of lesser importance for photoreceptor cell death [2+ signalling in cellular physiology, any targeting of Ca2+ permeable channels must be very specific. In case of RP, only rod-specific channels should be inhibited to avoid interference with cone-mediated vision. While specific pharmacological inhibition of channels may be feasible in some cases [2+ may also be detrimental to photoreceptors [2+ permeable channels really are a suitable target for pharmacological inhibition intervention.Paradoxically, photoreceptor degeneration has been reported to be linked to both increased and decreased intracellular Caectively , 26. Thun in rd1 , 96, 102/− x rd1  and Cngb− x rd10  double-mll death , 88. Becme cases , geneticme cases  may alloeceptors , raisingrd1 and rd10 murine models, reducing photoreceptor cGMP levels and inhibiting cGMP-dependent cytotoxicity [Since the imbalance of cGMP levels seems to be one of the initial events in photoreceptor cell death, neuroprotective strategies targeting upstream events may prevent or slow down the course of the disease. One way to target cGMP-signalling is to reduce the intracellular pool of guanine nucleotides . For instoxicity .rd1, rd2, and rd10 mice in vivo [Effectors downstream of the cGMP-signalling cascade, such as CNGCs or PKG, may also be considered possible targets . In part in vivo  suggestiDelivery of therapeutic compounds or antisense oligonucleotides to photoreceptors remains a complicated task primarily due to the protected environment that these cells require. Drugs delivered by systemic administration methods need to cross either the inner blood-retinal barrier (iBRB) or outer blood-retinal barrier (oBRB) . The iBRl-lactide)-based particles have proved promising for retinal drug delivery. One system provided retention of small hydrophilic drugs in the retina for up to 3 months after IVT injection, while the drug solution administered without the delivery system could not be detected after 1 month. [Local administration methods, such as intravitreal (IVT) injection, could circumvent parts of the BRB to make drug delivery more effective. Yet, rapid clearance of drug from the vitreous environment and the inner limiting membrane (ILM) at the vitreoretinal interphase still limits drug uptake by photoreceptors. To address the first issue, injectable drug-loaded implants have been developed based on hydrogel, providing sustained drug release for up to 6 months . Non-bio1 month. . Unfortu1 month. . Hence, 1 month. , which c1 month. . However1 month. . While t1 month.  and PEG-1 month.  have bee2+ levels [2+ levels, often by targeting Ca2+-permeable channels [2+ was in fact not as important as thought. In addition, in recent years, it has become increasingly clear that apoptosis—while involved in retinal development—is unlikely to be related to cell death caused by RD-gene mutations [In the past 25 years, attempts at treatment development for retinal degeneration mirrored those pursued for neurodegenerative brain diseases. These attempts were based on two main premises, namely that (1) photoreceptor degeneration was governed by apoptosis as the causative cell death mechanism , 76, and+ levels . Accordichannels , 102. Unutations , 77.2+) with CNGC mutations (high cGMP but low Ca2+). While Ca2+ levels are elevated in only one of the two situations, cGMP is elevated in both, strongly suggesting that photoreceptor cell death was driven by cGMP-dependent processes. Nevertheless, the added strain on energy metabolism that may be induced by increased Ca2+ influx may aggravate the situation further, so that, when high cGMP and high Ca2+ come together, cell death is precipitated. Therefore, in certain genetic constellations targeting Ca2+-signalling may be considered for adjuvant treatment, to maximize the effectiveness of cGMP-targeting approaches.In many genetically distinct types of RD, the rise in intracellular cGMP can be directly linked to photoreceptor degeneration , 77. Whi2+-signalling. Indeed, recent studies have shown that interference with cGMP signalling, when combined with a suitable drug delivery vehicle, holds great promise for RD treatment development. Future pre-clinical studies shall attempt to validate these findings and hopefully subsequent clinical trials will translate them into mutation-independent therapy.In summary, the evidence available today suggests that therapeutic approaches in RD should focus on targeting cGMP-signalling rather than Ca


CD157/BST-1 (a member of the ADP-ribosyl cyclase family) is expressed at variable levels in 97% of patients with acute myeloid leukemia (AML), and is currently under investigation as a target for antibody-based immunotherapy. We used peripheral blood and bone marrow samples from patients with AML to analyse the impact of CD157-directed antibodies in AML survival and in response to cytarabine (AraC) ex vivo. The study was extended to the U937, THP1 and OCI-AML3 AML cell lines of which we engineered CD157-low versions by shRNA knockdown. CD157-targeting antibodies enhanced survival, decreased apoptosis and reduced AraC toxicity in AML blasts and cell lines. CD157 signaling activated the PI3K/AKT/mTOR and MAPK/ERK pathways and increased expression of Mcl-1 and Bcl-XL anti-apoptotic proteins, while decreasing expression of Bax pro-apoptotic protein, thus preventing Caspase-3 activation. The primary CD157-mediated anti-apoptotic mechanism was Bak sequestration by Mcl-1. Indeed, the Mcl-1-specific inhibitor S63845 restored apoptosis by disrupting the interaction of Mcl-1 with Bim and Bak and significantly increased AraC toxicity in CD157-high but not in CD157-low AML cells. This study provides a new role for CD157 in AML cell survival, and indicates a potential role of CD157 as a predictive marker of response to therapies exploiting Mcl-1 pharmacological inhibition. The dismal prognosis of AML is largely due to the acquisition of resistance to chemotherapy and leukemia relapse, which often overlap with deregulation of the apoptotic machinery3. However, growing understanding of the molecular basis of AML have led to the introduction of new treatment options that exploit novel mechanisms of action4. New drugs are rapidly being developed to expand the AML treatment landscape, with the promise of ultimately improving patient outcomes, especially in elderly patients and relapsed/refractory AML, the areas of greatest unmet need5.Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous malignant disease characterized by abnormal proliferation of undifferentiated myeloid progenitors in the bone marrow (BM) and peripheral blood, impaired hematopoiesis and an aggressive clinical course. Intensive remission-inducing chemotherapy, followed by consolidation chemotherapy or allogeneic hematopoietic stem cell transplantation, in eligible patients with a high risk of relapse, still remains the standard therapeutic paradigm. However, mortality remains high, and most patients succumb to their disease in less than 5 years6. The basic idea is to kill leukemic cells by targeting the pro-survival members of the Bcl-2 family proteins with specific small molecule inhibitors that mimic the function of the BH3-only proteins, hence named BH3-mimetics. Currently, six BH3-mimetic drugs have entered clinical trials8 whereas the Bcl-2 inhibitor, ABT-199/venetoclax has been approved for therapy10. Although highly active, venetoclax monotherapy has shown limited efficacy in AML patients, and it proved to be more effective when used in combination with either hypomethylating agents or low-dose AraC for treatment-naive elderly patients11. However, a non-negligible minority of patients are refractory12 while the majority of patients who achieved remission, ultimately relapse13. In a clinical trial on relapsed and refractory AML, sensitivity to venetoclax increased with increased Bcl-2 expression; in contrast, sensitivity decreased with increased expression of myeloid cell leukemia-1 (Mcl-1)14, a critical anti-apoptotic member of the Bcl-2 family and target of various inhibitors now being used in clinical trials, such as S63845. Therefore, on the one hand, AML treatment needs the rational design of effective drug combination strategies, on the other, we need to be able to identify patients most likely to benefit from therapies encompassing specific BH3-mimetic drugs, for example through the discovery of predictive biomarkers.In AML, suppression of apoptosis by expression of anti-apoptotic members of the Bcl-2 family is one of the principal mechanisms underlying disease pathogenesis, resistance and relapse, making the anti-apoptotic members of the family attractive therapeutic targets15 and subsequently identified by RF3 monoclonal antibody as a cell membrane protein expressed in bone marrow stromal cells where it supports the growth of hematopoietic B cell progenitors16, hence the name bone marrow stromal cell antigen 1 (BST-1). CD157 is expressed by normal granulocytes, monocytes and more immature myeloid precursors. CD157 interaction with the heparin binding domain of selected extracellular matrix (ECM) proteins  promotes intracellular signaling in leukocytes, and regulate their adhesion17 and transmigration18. As a GPI-anchored protein, CD157 must associate with β1 and β2-integrins for signaling to occur. CD157-directed agonistic antibodies are effective in mimicking the signaling effects of the myeloid cells/ECM interaction21.CD157/BST-1 glycoprotein was originally discovered as Mo5 myeloid antigen22 and mesothelioma23, CD157 overexpression correlated with tumor invasiveness, aggressiveness, and decreased sensitivity to platinum-based chemotherapy24, suggesting that CD157 is more than a bystander marker. In AML, CD157 is expressed both at diagnosis and relapse, with highest expression in myelomonocytic and monocytic AML subtypes . In addition, CD157 is also expressed in CD34+CD38− leukemia-initiating cells that contribute to AML relapse25. Moreover, CD157 is currently under investigation as a target for antibody-based immunotherapy in AML (NCT02353143)26. However, the functional role of CD157 in the biology of AML has never been explored. To address this issue, we used CD157-directed antibodies to reveal that CD157 activates pro-survival signals in AML and in response to treatment. The mechanistic basis of this pro-survival effect was investigated in detail using AML cell lines expressing high and low levels of CD157 that confirmed the correlation between high CD157 expression, apoptosis inhibition and reduced sensitivity to chemotherapy. High CD157-expressing cell lines showed increased activation of the PI3K/AKT/mTOR and MAPK/ERK signaling pathways, converging on Mcl-1. Our study reveals a functional role of CD157 in pro-survival signaling in AML cells and provides a rationale for the use of CD157 as a predictive marker of response to therapies exploiting Mcl-1 pharmacological inhibition.We previously showed that in two non-hematological malignancies, ovarian carcinomal-glutamine and 100-unit penicillin, 100 μg/ml streptomycin. According to the number of cells obtained from each sample, AML cells were used immediately for ex vivo experiments, or frozen in FCS with 20% DMSO.Peripheral blood (n = 11) or bone marrow (n = 8) samples from patients with newly diagnosed AML without any prior treatment, were obtained for ex vivo studies from the A.O.U Città della Salute e della Scienza  and from the A.O.S. Croce e Carle  hospitals. The average age of patients was 57 years (range 25–86), with five males and 14 females. Supplementary Table l-glutamine. Morphology of cell lines was monitored routinely, and mycoplasma contamination was excluded using a PCR-based assay. DNA fingerprinting analysis using 16 different, highly polymorphic short tandem repeat loci  confirmed cell authenticity.U937 and THP1 AML cell lines  were grown in RPMI-1640 medium supplemented with 10% FCS; OCI-AML3 cells  were cultured in DMEM medium supplemented with 20% FCS. Culture media were supplemented with 100-unit penicillin, 100 μg/ml streptomycin and 2 mM The SY11B5 anti-CD157 (IgG1) mouse monoclonal antibody (mAb) was produced in-house and affinity-purified on protein G. RF3 anti-CD157 (IgG1) mAb produced in mouse was purchased from MBL . The isotype control IgG1 produced in mouse  was purchased from Biolegend . Purified murine IgG Fc fragments were from Jackson ImmunoResearch . Antibodies to phospho-mTOR Ser-2448 , phospho-p70S6K Thr-389 , phospho-AKT Ser-473 , phosphor-GSK-3β Ser-9 , phosho-ERK1/2 Thr-202/Tyr-204 , phosho-S6 ribosomal protein Ser-235/236 , phosho-4E-BP1 Thr-37/46 , mTOR , AKT , GSX3β  PARP-1 , Caspase-3 , Caspase-9 , Bcl-XL , Bcl-2 , Bim , Bax , Bak , Mcl-1 , HRP-conjugated anti-mouse IgG1 (#7076) and HRP-conjugated anti-Rabbit IgG1 (#7074) were obtained from Cell Signaling Technology . Antibodies to β-actin (MAB-24008-HRP) and β-tubulin (MAB-80142-HRP) were purchased from Immunological Sciences . AraC was purchased from Sigma-Aldrich, S63845 and ABT-199/venetoclax inhibitors from Selleck Chemicals . Both ABT-199 and S63845 were dissolved in DMSO. Fibronectin from human plasma, Fibrinogen and Collagen type I were from Sigma-Aldrich .5/sample) with a PE-labeled mAb against CD157  for 20 min at 4 °C. Leukemic blasts were analyzed by multiparametric flow cytometry and gated based on CD45 expression  and side scatter (CD45dim and SSClow). To further characterize blast subpopulations the following antibodies were used: CD33-FITC, CD34-PE/Cy7, CD64-PE, CD117-PE, CD123-PE/Cy7, HLA-DR-FITC (Biolegend). Specifically, this back-gating allowed us to identify leukemic myeloid blasts  and leukemic monocytic populations . Data were analyzed using FlowJo software Version 10.6 (BD Biosciences). CD157 relative Mean Fluorescence Intensity (MFI) was normalized to the MFI of lymphocytes, which are CD157 negative. CD157 MFI ratio was calculated according to the formula: (MFI of CD157 in blasts − MFI of CD157 in lymphocytes)/MFI of CD157 in lymphocytes.CD157 surface expression on AML samples and cell lines was assessed by flow cytometry  by staining cells  encoding the BST-1 short-harpin RNA (shRNA) (target sequences: 5′-GAGTCAGACTGCTTGTATA-3′ (shCD157#1) and 5′-CCTGAGCGATGTTCTGTAT-3′ (shCD157#2) or a scrambled negative control (scrambled sequence: 5′-TTCTCCGAACGTGTCACGTT-3′), as previously described24. PCR products were analyzed by electrophoresis on 1% agarose gel stained with Midori Green Advance DNA Stain .Total RNA was extracted from transduced AML cell lines using TRI reagent (Sigma-Aldrich), and quality and quantity checked with NanoDrop . 2 µg of RNA were reverse transcribed with the M-MLV Reverse Transcriptase (Invitrogen) and Oligo-dT primers (Sigma-Aldrich), as previously described5/ml) or cell lines (4 × 105/ml) were treated for 24 h (unless otherwise indicated) at 37 °C in standard culture medium or in serum-free conditions, as indicated, with affinity purified SY11B5 anti-CD157 mAb (10 µg/ml), or with an isotype-matched mIgG (10 µg/ml), as control. Then, cells were washed once in RPMI 1640 medium and processed according to the indicated assay.In selected experiments, primary AML cells   for 20 min at room temperature in the dark. Annexin V fluorescence was determined with a BD FACS Canto (BD Biosciences) flow cytometer and the membrane integrity of cells was simultaneously assessed by PI exclusion method. Flow cytometry analysis was performed by FlowJo software Version 10.6 (BD Bioscience). At least three independent experiments were performed with AML cell lines. Patient samples were chosen on the basis of availability of adequate cell number and experiments were performed once.4/well) or AML cell lines (5 × 104/well) were seeded in 96-well plates in 100 µl of culture medium supplemented with FCS and treated as indicated. Drugs at the indicated dilutions were added to each well. At each time point, 10 µl/well of PrestoBlue reagent (Thermo Fisher Scientific) was added for 1 h at 37 °C, then fluorescence was measured by using the Infinite M200 Pro Microplate Reader  with excitation/emission wavelengths set at 560/590 nm. Cell viability was determined by comparing the fluorescence values before and after treatment and normalizing the data to controls. Half-maximal effective concentration (EC50) values were calculated by non-linear regression algorithms and a four-parameter logistic model with log-transformed data using GraphPad Prism 7 software.Primary AML cells . Equal amounts of proteins were separated by an Any-kD Critereon TGX Stain-free PreCast gel (BioRad) and transferred to nitrocellulose membranes . After blocking with 2% BSA for 1 h, membranes were cut to minimize antibody use and each strip was separately probed with the indicated primary antibody, followed by the appropriate HRP-conjugated secondary antibody. Immunoreactive proteins were detected using enhanced chemiluminescence, images were captured with a ChemiDoc XRS + System. Band densitometry measurements were performed with Image Lab Software (BioRad).Cells were washed and harvested in ice-cold phosphate-buffered saline and subsequently lysed in extraction buffer  supplemented with 1 mM Na3VO4, 1 mM DTT and protease inhibitor cocktail. An equivalent of 1 mg/sample of protein extracts was incubated overnight at 4 °C with anti-Mcl-1  or anti-Bim  antibodies , and subjected to immunoprecipitation with 20 µl of anti-rabbit IgG-coated Sepharose beads  for 3 h at 4 °C. Proteins were eluted by boiling samples for 5 min at 95 °C with 3× SDS reducing sample buffer and then analyzed by Western blotting. Equal amounts (20 µg) of lysate was prepared as described above, and used as whole-cell lysate control.For immunoprecipitation, after the indicated treatments, cell extracts were obtained using lysis buffer  supplemented with 5 mM EDTA, 10 mM NaF, 1 mM Nat-test. When the data were not normally distributed, the comparison of variables was performed with a Wilcoxon signed rank test for paired data or with a Mann–Whitney test for unpaired data. All statistical analyses were performed using GraphPad Prism 7.0 . For all analyses, p values < 0.05 were considered statistically significant .Unless otherwise indicated, results of in vitro experiments are expressed as means ± SEM of at least three independent experiments. Comparisons among multiple groups were performed with both one-way and two-way ANOVA models. Differences between two groups were analyzed using unpaired two-tailed Student’s 28. To explore the potential role of CD157 signaling in AML cell survival in vitro, we used BM and peripheral blood samples from 19 newly-diagnosed AML patients . Then, cell apoptosis was quantified by Annexin V/PI double staining and flow cytometry analysis. After 24 h of ex vivo culture, AML cell apoptosis ranged from 15.6 to 92.5%. However, for every sample tested, the percentage of apoptotic cells  decreased after CD157 mAb treatment with respect to untreated controls Fig. A. Five ATo determine whether the pro-survival effect mediated by CD157 was specific to leukemic blasts, flow cytometry analysis was carried out on cell subsets identified through forward scatter (FSC) and side scatter (SSC) parameters at baseline (T0) and following 24 h ex vivo culture in the presence or absence of anti-CD157 mAb (or control mIgG) /extracellular signal kinase (ERK) signal transduction pathways, known to be activated through CD157 targeting in monocytes31, and GSK-3β was previously found to be associated with poor survival outcome in AML patients32. Next, to gather evidence that CD157 stimulation could modulate apoptosis, AML cells were treated with anti-CD157 mAb or mIgG as before, and analysed by Western blot for expression of proteins belonging to the Bcl-2-family. Although Bcl-2 expression was marginally affected, the anti-apoptotic proteins Mcl-1 and Bcl-XL were strongly upregulated while the pro-apoptotic protein Bax was clearly downregulated following CD157 antibody-binding. Moreover, CD157 stimulation reduced the proteolytic cleavage of Caspase-3 and its substrate PARP-1, considered to be hallmarks of apoptosis  mTOR and its downstream substrates p70S6K, pS6 ribosomal protein and 4E-BP1; (2) ERK and (3) AKT at its Ser-473, leading to (4) Ser-9 inactivating phosphorylation of Glycogen Synthase Kinase 3β (GSK-3β)  and OCI-AML3 (AML FAB M4 subtype) human AML cell lines which express variable levels of CD157 mRNA and protein  targeting CD157 or a scramble control sequence that conserved endogenous expression of CD157 (CD157high cells). CD157 expression in the genetically engineered cell lines was assessed by RT-PCR . After overnight starvation, cells were maintained in one of the following conditions: (1) standard culture, (2) without FCS, (3) with 10% FCS for 10 min and (4) with 10% FCS for 2 h. Cell extracts were then analysed by Western blot. In standard culture conditions, both CD157-high THP1 and U937 expressed higher Mcl-1 protein levels compared to their respective CD157-low counterparts. In the presence of FCS, CD157-high THP1 and U937 showed more robust phosphorylation of AKT at Ser-493, GSK-3β kinase at Ser-9 and pS6 ribosomal protein at Ser-235/236 and increased the expression of Mcl-1  with respect to their CD157-low versions  of AraC decreased from 4.8 µM in U937/CD157-high cells to 2.9 µM in U937/CD157-low cells (p = 0.0093). Moreover, following treatment with 10 µM AraC, the percentage of apoptotic U937/CD157-low cells was significantly higher than that of U937/CD157-high cells  , regardless of CD157 expression levels  and effectors of apoptosis (Bax and Bak)These data indicate that CD157 promotes survival of leukemic blasts through Mcl-1 upregulation, and suggest that sequestration of Bak by Mcl-1 might represent the primary CD157-mediated anti-apoptotic mechanism in AML cells.43, arousing considerable interest toward Mcl-1 as a therapeutic target in AML. Since Mcl-1 inhibitors are most likely to be effective in AML therapy when used in combination with a “priming” agent44, we compared the efficacy of S63845 in combination with AraC in U937 cells with high or low CD157. Cells were treated for 24 h with increasing concentrations of AraC either as single agent or in combination with increasing concentrations of S63845, maintaining a constant ratio between the two drugs (S63845:AraC = 1:100). Cell viability was then determined by PrestoBlue assays, which measures metabolic activity. The results showed that in CD157-high, but not in CD157-low U937 cells, the addition of S63845 significantly enhanced AraC-induced cell death at all doses considered, with respect to AraC alone  surface proteome in which CD157 was identified as one of a restricted set of markers with significantly higher expression in BCP-ALL cells with respect to normal bone marrow B cell populations.In this study, we identified the biological function of CD157 in AML showing that CD157 favors survival and confers protection from stress or drug-induced apoptosis in primary AML blasts and AML cell lines. Two different experimental approaches support this notion. Firstly, antibody-directed binding of CD157 supported survival and reduced AraC-induced apoptosis in primary AML blasts ex vivo. Secondly, knockdown of CD157 increased apoptosis in AML cell lines exposed to nutrient deprivation conditions, and increased their sensitivity to AraC toxicity. These findings are in line with previous studies from our laboratory demonstrating that high levels of CD157 expression can promote tumor cell survival and induce drug resistance in non-hematological tumors, namely epithelial ovarian cancer47 was reported in a small cohort of patients with AML25. Although is a preliminary observation, this report supports the idea that CD157 expression is associated with aggressive disease progression in vivo.The correlation between high CD157 expression levels and the group of patients with “adverse prognosis” according to the European Leukemia Net (ELN) classification-201733, our data showed that CD157 exerts its pro-survival effects in AML cells by transducing signals from external cues. Indeed, in primary AML cells, CD157 stimulation by antibody binding activated the PI3K/AKT/mTOR and MAPK/ERK pathways, while inactivating GSK3-β kinase thus leading to extended ex vivo survival and reduced sensitivity to AraC treatment. In addition, stronger phosphorylation of AKT, GSK-3β, mTOR and its downstream effectors was found in CD157-high versus CD157-low AML cell lines exposed to FCS (as source of ECM proteins) or to purified ECM proteins that bind CD15717. PI3K/AKT/mTOR and MAPK/ERK intracellular signaling pathways, as well as GSK3-β, regulate the expression and activity of apoptotic proteins ultimately ruling the balance between survival versus death of cells48. In leukemic cells, aberrant activation of these intracellular signals converge towards apoptosis escape, favoring tumor growth and eventually leading to resistance to chemotherapy49. The results of this study unveiled that CD157 takes part to the cross-talk between the PI3K/AKT/mTOR pathway and apoptosis, contributing to leukemic cell survival. In particular, CD157-mediated intracellular signals enhanced both Mcl-1 and Bcl-XL anti-apoptotic proteins; in contrast, they decreased Bax pro-apoptotic protein and prevented Caspase-3 activation, thus mitigating the apoptotic response in primary AML cells. Accordingly, knockdown of CD157 in U937 and THP1 cells reduced Mcl-1 and resulted in increased sensitivity to AraC toxicity in U937 cells. In U937 cells, the pro-apoptotic protein Bim was primarily bound to Mcl-1, while binding of Bim to Bcl-2 was barely detectable. In addition, in CD157-high U937 cells, Mcl-1 sequestered the pro-apoptotic effector protein Bak precluding the activation of the Bax/Bak-dependent mitochondrial apoptosis50. Consistently, treatment with S63845, which binds with high affinity to the BH3-binding groove of Mcl-151, displaced Bim and Bak bound to Mcl-1 resulting in activation of the Bax/Bak-mediated apoptosis52  to leukemic cells could be to conjugate them to target-directing antibodies. It is conceivable to envision that CD157-specific antibodies might be suitable candidates for this purpose.Mcl-1 expression is essential for survival of human stem and progenitor cells and cardiac myocyte functioning, hinting to a relevant risk of severe on-target side effects in patients treated with Mcl-1 inhibitors. Clinical data gathered from ongoing phase I clinical trials  will provide better insight both into the therapeutic efficacy and side effects of Mcl-1 inhibitorsSupplementary Information 1.Supplementary Information 2.


D-serine, ATP). To sense diverse input, astrocytes express a plethora of G-protein coupled receptors, which couple, via Gi/o and Gq, to the intracellular Ca2+ release channel IP3-receptor (IP3R). Indeed, manipulating astrocytic IP3R-Ca2+ signaling is highly consequential at the network and behavioral level: Depleting IP3R subtype 2 (IP3R2) results in reduced GPCR-Ca2+ signaling and impaired synaptic plasticity; enhancing IP3R-Ca2+ signaling affects cognitive functions such as learning and memory, sleep, and mood. However, as a result of discrepancies in the literature, the role of GPCR-IP3R-Ca2+ signaling, especially under physiological conditions, remains inconclusive. One primary reason for this could be that IP3R2 has been used to represent all astrocytic IP3Rs, including IP3R1 and IP3R3. Indeed, IP3R1 and IP3R3 are unique Ca2+ channels in their own right; they have unique biophysical properties, often display distinct distribution, and are differentially regulated. As a result, they mediate different physiological roles to IP3R2. Thus, these additional channels promise to enrich the diversity of spatiotemporal Ca2+ dynamics and provide unique opportunities for integrating neuronal input and modulating astrocyte–neuron communication. The current review weighs evidence supporting the existence of multiple astrocytic-IP3R isoforms, summarizes distinct sub-type specific properties that shape spatiotemporal Ca2+ dynamics. We also discuss existing experimental tools and future refinements to better recapitulate the endogenous activities of each IP3R isoform.Astrocytes are sensitive to ongoing neuronal/network activities and, accordingly, regulate neuronal functions  by the context-dependent release of several gliotransmitters (e.g., glutamate, glycine,  Indeed, astrocytic Ca2+ signaling was shown to regulate synaptic transmission, synaptic plasticity, and to influence behavior (3Rs) mediated Ca2+ signaling (IP3R-Ca2+) is regarded a primary generator of astrocytic Ca2+ signaling. Upon activation of Gq-GPCRs, the main input pathway of astrocytes, phospholipase C breaks down PIP2 into DAG and IP3, activating IP3R predominantly located on the membrane of endoplasmic reticulum (ER) resulting in Ca2+ release  such as glutamate, D-serine, and ATP, which have distinct influences over neuronal activity. Initially, IP3R subtype 2 (IP3R2) was the only recognized Ca2+ channel in astrocytes; However, advanced Ca2+ imaging techniques have since identified novel Ca2+ sources, including mitochondria  entirely appears to need remedying. This section considers the historical data from which the dogmatic view of astrocytic-IP3R2 has flowed and reviews the evidence for other IP3R subtypes.There are three mammalian IP., IP3R1 , IP3R2 , triggered Ca2+ events in the astrocytic processes of IP3R2KO mice  mice  mice  are wideffer IP3 , or an Itabolism . To studor IP3R1  and IP3Rtrocytes .3R-Ca2+ signaling in astrocytes. The most used DREADDs are the excitatory Gq or inhibitory Gi-coupled receptors, hM3Dq and hM4Di, respectively (derived from human M3/M4 muscarinic receptor). Both receptors are activated by a pharmacologically inert but bioavailable ligand clozapine-N-oxide (CNO) while being non-responsive to endogenous GPCR ligands  enable the selective activation of GPCR-IP ligands . hM3Dq h ligands , fear re ligands , and mem ligands . While tysiology ; Secondlysiology , temporarocesses .3R-Ca2+ signaling cascade using light. Light activation has been achieved by introducing to astrocytes either a mammalian light-sensitive Gq/Gi/o-protein-coupled photopigment, Melanopsin  is not reproducible (3R response (3Rs from cellular compartments distinct from those of the endogenous receptors, limiting their ability to recreate physiologically relevant Ca2+ profiles. To address this, next-generation activation tools are being engineered to mimic the subcellular distribution of endogenous receptors (While having great potential for controlling astrocytic activation, a central question is to what extent do the chimeras mimic the signaling of wild-type receptors. GPCRs can have multiple signaling axis, e.g., multiple G-protein axes, β-arrestins, wnt-frizzled, or the hedgehog-smoothened axes , and theoducible . While mresponse . Anothereceptors .2+ signaling in astrocytes. Understanding the subtleties of these signals will require detailed knowledge of the astrocytic Ca2+ signaling toolbox along with the generation and characterization of more sophisticated tools to control and accurately recapitulate the physiologically relevant Ca2+ signals.In the last decade, substantial progress has been made revealing diverse spatio-temporal CaMS and MA performed the literature survey, wrote the manuscript, and prepared the figures and tables. MS, MA, AP, KM, and SHRO reviewed, finalized, and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To counterbalance potentially detrimental consequences of Ca2+, neurons are equipped with sophisticated mechanisms that function to keep its concentration in a tightly regulated range. Calcium pumps belonging to the P-type family of ATPases: plasma membrane Ca2+-ATPase (PMCA), sarco/endoplasmic Ca2+-ATPase (SERCA) and secretory pathway Ca2+-ATPase (SPCA) are considered efficient line of defense against abnormal Ca2+ rises. However, their role is not limited only to Ca2+ transport, as they present tissue-specific functionality and unique sensitive to the regulation by the main calcium signal decoding protein—calmodulin (CaM). Based on the available literature, in this review we analyze the contribution of these three types of Ca2+-ATPases to neuropathology, with a special emphasis on mental diseases.Calcium in mammalian neurons is essential for developmental processes, neurotransmitter release, apoptosis, and signal transduction. Incorrectly processed Ca Among three main calcium pumps, the plasma membrane Ca2+-ATPase (PMCA) and sarco/endoplasmic Ca2+-ATPase (SERCA) are known for decades while the secretory pathway Ca2+-ATPase has been discovered in 2000s by two independent laboratories that described novel mutations leading to Hailey-Hailey disease [2+ and function to restore cytosolic Ca2+ concentration [Ca2+]c to the resting, nanomolar level following neuronal stimulations. They belong to the superfamily of mammalian P-type ATPases and are characterized by formation of a phosphorylated enzyme intermediate during catalytic cycle c to the basal level following neuronal excitation c. On the other hand, proteins such as CaM (CALM1 and CALM2), CaMKII  and CaM-like proteins were found to be downregulated in the brain or secretion fluids  and shows two-fold higher velocity of Ca2+ transport compared to ubiquitous SERCA2b [2+ uptake can be ascribed to SERCA3 [Despite high structural homology, all isoforms of SERCA possess different affinity for calcium and unique kinetic properties. For instance, SERCA2a has a two-fold lower affinity for [Ca SERCA2b . This tro SERCA3 . This feo SERCA3 ,150,151.2+ homeostasis can lead, among others, to ER stress and accumulation of unfolded or misfolded proteins, which are detected for most neurodegenerative diseases, including AD, PD, HD, and ALS [Disturbances in ER Ca and ALS ,153,154. and ALS . Further and ALS .An interesting observation was identification of several ATP2A2 mutations in autosomal dominant skin disorder—Darier’s disease (DD), a disorder frequently associated with several mental diseases  ,160,161.2+, thus attenuating transport rate by ~50% [2+-CaMKII at Thr17 causes PLN dissociation from SERCA, thereby removes its inhibitory effect, and increases Ca2+ uptake to the SR [Although the direct regulation by CaM has been shown only for PMCA, a growing body of evidence indicates that CaM can significantly, but indirectly, participate in modulation of SERCA activity. The well-recognized mechanism exists in cardiac and skeletal muscles and is based on regulation of SERCA activity by endogenous molecule—phospholamban (PLN) ,163. Thi by ~50% . It was  by ~50% . Phosphoo the SR . The deco the SR . Althoug2+ depletion from ER, finally leading to the cell death.Astrocytes, the important elements of glia, are an integral part of the CNS that couple the activity of neurons and blood-brain barrier (BBB). The proper function of BBB requires cooperation between endothelium, astrocytes, neurons, and extracellular matrix. An important role in a progression of neurological diseases is a disruption of the BBB integrity ,169. Deg2+ affinity, SPCA also represents strong preference for Mn2+ ions. This Ca2+/Mn2+ transporter possesses unique structural elements in the N-terminus and transmembrane (TM) region determining orientation and selectivity of the ion transport during phosphoryl-transfer reactions. Particularly, SPCA pump is crucial for maintaining the sufficient supply of Mn2+ for glycosyltransferases and sulfotransferases in the Golgi lumen [2+ and Mn2+, while the overload of these ions may trigger neurotoxicity resulting in several neurological disorders [SPCA pump, as a member of the P2A subfamily, shares some common structural and mechanistic properties of SERCA , yet in gi lumen . On the isorders .In human, two SPCA isoforms, SPCA1 and SPCA2 are encoded by ATP2C1 and ATP2C2 genes, respectively. These isoforms share nearly 60% of sequence identity and they exhibit distinct expression pattern and tissue distributions in the human body . Althoug2+ transport by SPCA pump [2+ affinity while SPCA1a displays significantly lower apparent Ca2+ affinity than SPCA2. Remarkably, SPCA1a displays a two-fold higher maximal ATPase activity in the presence of Ca2+ as compared to Mn2+ conditions, whereas SPCA2 shows similar maximal turnover rates for both ions. These differences in biochemical properties of SPCA isoforms seem to be determined, at least in part, by EF hand-like motif that is present in SPCA1a N-terminus, but absent in SPCA2. It is believed that such motif influences lower affinity and higher Ca2+ capacity of this pump promoting stronger Ca2+-dependent autophosphorylation. This feature may have physiological implications in cells with a high calcium load and/or fulfilling secretory functions [SPCA pump is presumed to be composed of ten membrane-spanning helices (M1-M10) and cytosolic headpiece containing conservative motifs, which are critical for transport functions in a SERCA-like manner, including the Thr–Gly–Glu loop in A site, the phosphate-accepting Asp residue, the ATP- and FITC-binding region, and the Asp–Pro–Pro–Arg loop between the N and P sites. Unlike SERCA, SPCA pump lacks long cytoplasmic tail and elongated luminal loops linking some of the transmembrane domains, and importantly, only one calcium-binding region in SPCA protein overlaps with site II in the transmembrane domains of SERCA . The resPCA pump . Human Sunctions .2+ can be released mainly by IP3 receptors following extracellular stimuli. It is now widely accepted that GA, together with ER and mitochondria, plays an important role in the regulation of cytosolic Ca2+. It has been shown that the maintenance of the Ca2+ content provided by SPCA1 appears essential for the correct structure of the entire GA and for important functions of the secretory pathway.The GA is associated with post-translational processing of proteins destined for secretion as well as their incorporation into the ER, lysosomes or plasma membranes. In the GA, calcium can reach ~300 μM, due to the action of SERCA and SPCA localized in this organelle ,192. StoEarly study on SPCA1 indicated its vital role during brain development and closure of neural tube ,193. AddA unique SPCA1 expression pattern has been described during ischemic/reperfusion brain injury (IRI), which was affected by pre-ischemic challenge. Whereas IRI induced the depression of SPCA-mediated calcium transport in hippocampus, ischemic preconditioning (IPC) had a partial protective effect on SPCA activity . Similar2+ transport, SPCA also controls manganese homeostasis, which is important for brain development. Moreover, it represents the only known system for cellular Mn2+ detoxification. In rat brain, SPCA1 was upregulated following Mn2+ exposure, but impaired regulation of Mn2+ transport may trigger neuronal pathology. Although SPCA2 shows more restricted distribution, its relatively high level in brain could be more important taking into account the correlations between manganese neurotoxicity and PD [2+ concentration exerted the toxic, inhibitory effect on SPCA activity in cultured mice neurons and glia [2+ and Ca2+ can occupy the same ion-transport site, Mn2+ toxicity may also affect Ca2+ homeostasis. At elevated levels, Mn2+ can also compete with magnesium-binding sites in many functional proteins, enhance oxidative stress or lead to accumulation of intracellular toxic metabolites [2+ poisoning than neurons, prolonged dyshomeostasis may result in fragmentation of the GA. The role of Mn2+ in the etiology of neurodegenerative diseases has been widely documented. Excess Mn2+ was shown to induce a neurological disorder with symptoms similar to PD [2+ contributed to AD associated with impaired cognitive function and cognitive decline [Besides Cay and PD ,203. Higand glia . In addiabolites . Finallyar to PD . Moreove decline .2+ overload, which can lead to additional injury. These processes are also associated with specific brain areas, as well as with BBB dysfunction. Mechanisms underlying the neuropathological changes are observed in different cell types and frequently result in improper communication between brain structures, including cooperation between neurons and glial cells [2+ circulation. Furthermore, astrocytes are an integral part of the BBB [2+ concentration are under feedback control of Ca2+/CaM complex [2+/CaM complex, which binds to nitric oxide synthase (NOS), and actives the enzyme producing NO from L-arginine. The most important target for NO is guanylate cyclase (GC). High level of NO can contribute to excitotoxicity following a stroke and neurodegenerative diseases. In addition, NOS generates superoxide, which is involved in both cell injury and signaling [Changes in the GA morphology observed in a number of neuronal diseases such as AD, ALS, and stroke include GA fragmentation and intracellular Caal cells . Astrocyal cells . The act the BBB . Interes complex ,212,213.ignaling .2+ released after the GA fragmentation caused by pathological conditions triggers overactivation of eNOS in a Ca2+/CaM-dependent way. Moreover, increased Mn2+ levels in astrocytes elevated the expression of iNOS and activated soluble guanylate cyclase (sGC), which is suggested to be a causative factor for PD [2+/CaM comprises the activation of CaMKII, the enzyme controlling multiple signaling pathways in the brain. It should be noted that the effects of CaMKII may be reversed by antagonistic action of protein phosphatases, particularly PP2B, which is also a Ca2+/CaM-dependent enzyme. Phosphorylation of nNOS at Ser847 by CaMKII was shown to decrease NO generation, but increase superoxide generation [2+ into the cytoplasm. Nitrosative/oxidative stress may also induce BBB rupture and impair metabolic network of astrocytes [It has been demonstrated that Car for PD . sGC expr for PD . The secneration . Subsequtrocytes .2+ in the ER and the GA is mainly released through the inositol-1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RYR) channels, which are also regulated by Ca2+/CaM complex [3Rs has been shown in ataxia and neurodegenerative diseases such as AD and HD [Ca complex ,220. TheD and HD . Recent D and HD .2+ and/or Mn2+ concentration. Participation of several ion transporting systems and cooperation of intracellular organelles is required for adaptation to stress conditions that allows protection against neurotoxicity. The importance of cooperation between GA, ER, and mitochondria in maintenance of calcium homeostasis has been suggested in many studies. Under pathological conditions improper control of Ca2+ circulation, its excessive accumulation and disturbed mitochondrial energy production preclude the formation of physiological Ca2+ signaling networks leading to neurotoxicity and cell death.The functionality of GA appears to be more complex, especially in its ability to counterbalance excessive Ca2+ signaling is frequently observed in majority of mental and neurodegenerative diseases. Because one of the most distinguishable features of Ca2+ is ambivalence, the dysfunctions of neuronal Ca2+-ATPases have recently acquired growing awareness and prominence. So far, no massive and uncontrolled neuronal death due to pumps dysfunctions have been identified. Instead, they are usually linked to less severe phenotypic changes that possibly originate from mild alterations in Ca2+ concentration in subtle cellular compartments or domains. It should be underlined that at different steps of subsequent Ca2+-induced processes, the Ca2+/CaM complex plays a decisive control function. Up to now, the studies revealed only certain human diseases, mostly with genetic defects in Ca2+ pumps. These phenotypes have gained considerable attention, however an intensified research on animal models is soon expected to describe the global effects of nongenetic pump alterations as well. Undoubtedly, knowing the consequences of Ca2+ pumps dysfunctions will help to understand the importance of Ca2+ signaling in development of neuropathologies. In view of that, future work should reveal the relationship between PMCA, SERCA and SPCA and their integration into dynamic processes of neuronal death, adaptation, and repair. The special attention should be given to CaM-regulatory mechanisms as primarily responsible for neuronal signaling in physiological and pathological states. This may significantly contribute to the identification of therapeutic strategies centered on neuronal calcium pumps.There is now substantial evidence that defective Ca