InteractiveFly: GeneBrief

Calcineurin A1 : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Calcineurin is a Ca2+/calmodulin-activated, Ser-Thr protein phosphatase that is essential for the translation of Ca²⁺ signals into changes in cell function and development. A dominant modifier screen was carried out in the Drosophila eye using an activated form of Calcineurin A1 (FlyBase name: Protein phosphatase 2B at 14D), the catalytic subunit, to identify new targets, regulators, and functions of calcineurin. An examination of 70,000 mutagenized flies yielded nine specific complementation groups, four that enhanced and five that suppressed the activated calcineurin phenotype. The gene canB2, which encodes the essential regulatory subunit of calcineurin, was identified as a suppressor group, demonstrating that the screen was capable of identifying genes relevant to calcineurin function. A second suppressor group was sprouty, a negative regulator of receptor tyrosine kinase signaling. Wing and eye phenotypes of ectopic activated calcineurin and genetic interactions with components of signaling pathways have suggested a role for calcineurin in repressing Egf receptor/Ras signal transduction. On the basis of these results, it is proposed that calcineurin, upon activation by Ca²⁺-calmodulin, cooperates with other factors to negatively regulate Egf receptor signaling at the level of Sprouty and the GTPase-activating protein Gap1 (Sullivan, 2002).

Calcineurin, the only protein phosphatase regulated by both Ca²⁺ and calmodulin, is a key player in Ca²⁺ signal transduction from yeast to humans and has been implicated in a wide array of processes, from disease progression to development (for a general review, see Rusnak, 2000). In the mammalian immune system, calcineurin is essential for T-cell activation and is the target of immunosuppressant drugs such as cyclosporin (Liu, 1991; Clipstone, 1992). An abundant neuronal protein, calcineurin has been implicated in various forms of synaptic plasticity (reviewed in Yakel, 1997). In addition, calcineurin is involved both in the development of cardiac valves (Ranger, 1998) and in hypertrophy of cardiac muscle (Molkentin, 1998) following disease or injury (Sullivan, 2002 and references therein).

The enzyme consists of an ~60-kD catalytic subunit, calcineurin A (canA), bound to the regulatory subunit, calcineurin B (see Drosophila Calcineurin B), a 19-kD EF-hand Ca²⁺-binding protein (reviewed in Klee, 1998). CanB is essential for phosphatase activity and can be dissociated from canA only by denaturants. CanA has two variable regions at the N and C termini, a highly conserved catalytic domain, and a regulatory region. The regulatory region consists of a binding site for Ca²⁺-calmodulin and a short autoinhibitory domain that blocks substrate access to the active site in the absence of Ca²⁺-calmodulin (Sullivan, 2002).

A Ca²⁺-calmodulin-independent, constitutively active phosphatase is made by deleting the canA regulatory region (O'Keefe, 1992). Studies from a number of different organisms indicate that, aside from a small degree of Ca²⁺ sensitivity mediated by canB, activated calcineurin functions identically to the full-length, Ca²⁺-calmodulin-activated form (Mendoza, 1996; Shibasaki, 1996; Winder, 1998; Sullivan, 2002 and references therein).

Calcineurin is activated by a sustained increase in intracellular Ca²⁺ levels that can result from the opening of intracellular Ca²⁺ channels in response to phosphoinositide (PI) signaling (reviewed in Berridge 1993). PI signaling is initiated by the activation of a phosphatidylinositol-specific phospholipase C, either PLCß by G-protein-coupled receptors (GPCR) or PLCgamma by receptor tyrosine kinases (RTK). PI-PLCs cleave phosphatidylinositol 4,5-bisphosphate (PIP₂) to yield inositol 1,4,5-trisphosphate (InsP₃), which then activates the InsP₃ receptor Ca²⁺ channel (Sullivan, 2002).

GPCRs and RTKs activate an integrated signaling network that includes the Ras/mitogen-activated protein (MAP) kinase cascade, PI3-kinase, and the small GTPase Rho. Depending upon the cellular context, these pathways can either antagonize or cooperate with each other and with PI signaling. For example, T-cell activation (Crabtree, 1999) requires the activation of both NFAT, which is transduced to the nucleus upon dephosphorylation by calcineurin, and AP1, which acts downstream of Ras and MAP kinase (Sullivan, 2002).

Conversely, PI signaling has been found to antagonize the Ras pathway in Drosophila. The Egf receptor and Ras/MAP kinase cascade are essential for formation of wing veins and photoreceptor (R) cells in the eye. Mutations in the single phospholipase Cgamma gene, small wing (sl), cause the formation of extra R7 cells and wing vein material and also genetically interact with Egf-receptor-signaling components. A recently proposed model for sl-mediated repression of Egf receptor signaling was based on the identification of the GTPase-activating protein Gap1 as an InsP₄ receptor. PLCgamma-generated InsP₃ is converted to InsP₄, which then activates Gap. Gap converts the active form of Ras, Ras-GTP, to the inactive form, Ras-GDP (Sullivan, 2002 and references therein).

The Drosophila genome contains three canA genes and two canB genes that are 75% and 88% similar to the vertebrate genes, respectively. To date, no mutants have been described for any of the five genes. To study calcineurin function in Drosophila, a constitutively active form of CanA1 was expressed during imaginal development and the resulting phenotypes were examined. The activated calcineurin rough eye phenotype was used to perform a genetic modifier screen. Specific enhancers and suppressors were successfully isolated and characterized and two suppressors were identified as canB2 and sprouty. The activated calcineurin rough eye was also tested extensively for genetic interactions with an array of signaling cascades. Taken together, the genetic evidence is consistent with calcineurin functioning as a negative regulator of Egf receptor/Ras signaling during imaginal development, possibly in the same pathway as PLCgamma (Sullivan, 2002).

ThecanA gene Pp2B-14D was used for these experiments because it is expressed throughout development, including in eye discs (Brown, 1994). The adjacent canA gene at 14F1 (gene designation CG9819) encodes a protein that is 83% identical to Pp2B-14D; however, canA-14F is not represented in the expressed sequence tag collection and the expression pattern has not been characterized. The Calcineurin A1 gene at 100B4, which was incorrectly localized to 21B, is undetectable by Northern analysis (Guernini, 1992) and appears to have a highly restricted expression pattern (Sullivan, 2002).

An activated form of Pp2B-14D, canA^act, was made by deleting the autoinhibitory and calmodulin-binding domains (O'Keefe, 1992; Mendoza, 1996). The canA^act construct was expressed in Drosophila under the control of glass response elements, which induce transcription uniformly in cells posterior to the morphogenetic furrow in the eye imaginal disc (Sullivan, 2002).

Flies carrying one copy of the canA^act.gl transgene have mild rough eyes compared to wild type, and the eyes of flies carrying two copies exhibit a stronger phenotype. Consistent with observations in other systems, neither full-length CanA nor activated canA without a functional CanB-binding domain causes any detectable phenotypes when expressed throughout development (Sullivan, 2002).

Removing one copy of glass by introducing the null allele gl^60J strongly suppresses the canA^act.gl rough eye phenotype. This demonstrates that the canA^act.gl rough eye phenotype is dependent on glass and is not caused by the insertion site of the transgene or by some other factor (Sullivan, 2002).

Reducing the dosage of canB-4F or canB2 by introducing deficiencies that uncover the 4F or 43E genomic region results in suppression of the canA^act.gl rough eye. Western blots confirmed that CanB protein is present in the eye disc; however, it is not known whether the protein is derived from one or both canB genes. Consistent with the effect of reduced CanB levels, canB4F^gl, which alone has no phenotype, increases the severity of the canA^act.gl rough eye (Sullivan, 2002).

Because expression occurs throughout the later stages of eye development, glass-dependent transgenes can affect many different processes. On the basis of these observations, activated calcineurin may have multiple effects on the differentiation and morphology of photoreceptor and other cell types. However, no effect of canA^act.gl on cell proliferation or cell death was observed (Sullivan, 2002).

Several lines of evidence demonstrate that phosphatase activity is required for ectopic canA^act phenotypes in Drosophila. Genetically raising or lowering the level of CanB, which is essential for activity, respectively enhances or suppresses the phenotype of canA^act. Transgenic flies expressing a form of canA^act that lacks an intact CanB-binding site are indistinguishable from wild-type flies. Finally, the full-length phosphatase, which is inactive in the absence of Ca²⁺-calmodulin, does not have a detectable phenotype when overexpressed throughout development (Sullivan, 2002).

Activated calcineurin has been used reliably to identify physiologically relevant functions of calcineurin in a number of systems (Fruman, 1995; Mendoza, 1996; Molkentin, 1998; Winder, 1998). However, it remains formally possible that calcineurin does not normally function in the cells or at the stage of development in which canA^act has a detectable phenotype, even though Pp2B-14D appears to be ubiquitously expressed. The role of calcineurin must be confirmed by mutational analysis of the canA and canB genes. Despite this caveat, the genetic screen presented in this study can still be used to identify physiological targets of calcineurin in Drosophila, as well as to provide insight into the roles calcineurin may play in development (Sullivan, 2002).

The canA^act.gl screen yielded 11 complementation groups, 9 of which failed to modify rough eyes caused by other glass-induced transgenes. This demonstrates that the majority of the modifier groups do not act through the glass enhancer. The nine specific modifiers were then divided into class I genes, which act downstream of calcineurin, and class II genes, which act at the level of CanB (Sullivan, 2002).

Consistent with this classification, the class II group CS2-1 is CanB2. The allele CS2-1⁸⁷ has an inversion that breaks within 400 bp of the CanB2 start of transcription. CS2-1⁸⁷ and CS2-1¹⁸⁰ have decreased protein levels compared to similarly staged controls. Finally, the lethality of CS2-1¹⁸⁰ is partially rescued by CanB-4F. The ability of CanB-4F to rescue the CanB2 lesion suggests that the CanB-4F protein can at least partially substitute for CanB2. More importantly, isolation of the calcineurin regulatory subunit in the canA^act.gl modifier screen demonstrates that the screen is capable of identifying genes that are required for calcineurin function (Sullivan, 2002).

The class I modifier group CS3-3 failed to complement the hypomorphic sprouty alleles sty^Delta5 and sty^Delta64; both sty^Delta5 and sty^Delta64 also suppressed canA^act.gl, and the sty gene from CS3-3⁵¹⁸ harbored a nonsense mutation (Q250Stop). Therefore, it is concluded that the CS3-3 complementation group is sprouty. The fact that sty falls into the class II group suggests that sprouty functions downstream of calcineurin and/or in a parallel pathway (Sullivan, 2002).

Two lines of evidence suggest that calcineurin is a negative regulator of Egf receptor/Ras signaling. First, a negative regulator of RTK signaling, sprouty, was isolated as a suppressor of the canA^act.gl rough eye phenotype in the dominant modifier screen. Both sprouty and canA^act suppress wing vein formation and reduce the number of photoreceptor cells per ommatidium. Egf receptor/Ras signaling is essential for both wing vein and R-cell formation (Sullivan, 2002).

A thorough examination of genetic interactions between canA^act and components of RTK and other signaling pathways has confirmed that canA^act specifically represses the Egf receptor/Ras pathway and that it acts upstream in the pathway. The lack of convincing genetic interactions with other signaling pathways in the imaginal eye disc does not rule out a role for calcineurin in these pathways in other developmental contexts. With the exception of pnt, activated calcineurin was not modified by components downstream of Ras and was modified only by a subset of genes that act between the Egf receptor and Ras. While Gap1 and sty alleles modify the effects of activated calcineurin, drk and cbl do not. Thus calcineurin may act downstream of, or parallel to, drk and cbl. The more downstream components of the Ras/MAP kinase pathway may not interact with activated calcineurin because they are too far removed from the point(s) of intersection between calcineurin and the pathway. Alternatively, these components may not be limiting, so that reduction of gene dose, which is the basis of a dominant modifier screen, would have no appreciable effect (Sullivan, 2002).

The hypermorphic allele Egfr^E1 inhibits Ras signaling; thus it might be expected to enhance the effects of activated calcineurin. However, low levels of inappropriate Egf receptor activity in eye development are thought to increase secretion of the Egf receptor antagonist Argos. The Argos protein inhibits subsequent Egf receptor signaling that is required for photoreceptor determination. Thus, suppression of the Egfr^E1 rough eye by canA^act.gl may be the result of activated calcineurin inhibiting inappropriate Egf receptor signaling (Sullivan, 2002).

Consistent with these findings, PLCgamma is a negative regulator of Egf receptor/Ras signaling in eye and wing development. However, PLCgamma was identified in this study as a strong suppressor of activated calcineurin, although biochemically PLCgamma has been placed upstream of calcineurin in the PI signaling pathway. One explanation is that PLCgamma acts on one of the other canA genes. Another possibility is that the signaling pathways activated by PLCgamma parallel to calcineurin are required for calcineurin function (Sullivan, 2002).

In a recent model, PLCgamma has been proposed to inhibit Egf receptor/Ras signaling via the activation of Gap1 by InsP₄. The results presented in this study suggest that PLCgamma is also acting through calcineurin. The genetic evidence presented indicates that calcineurin intersects with the Ras pathway at roughly the same point that PLCgamma does, and thus a modified model is proposed for the function of PI signaling in Drosophila development. Additionally, the fact that calcineurin can be activated by any sustained Ca²⁺ flux suggests a mechanism by which other signaling pathways, such as GPCRs acting via PLCß, can modulate Egf receptor signaling (Sullivan, 2002).

A simplified schematic is presented that illustrates upstream Egf receptor signaling components in an eye disc cell. PLCgamma is activated by the Egf receptor and cleaves PIP₂ to yield InsP₃. PLCgamma is proposed to negatively regulate Egf receptor signaling through InsP₄, which is generated from InsP₃ by an InsP₃-3 kinase. Gap1 is then activated by InsP₄, which results in the inhibition of Ras. Sprouty, which may be linked to the Egf receptor by the adaptor protein Drk, may facilitate the inactivation of Ras by Gap. In this model, it is proposed that PLCgamma also acts via Ca²⁺ and calcineurin. Genetic evidence suggests that calcineurin acts at the level of sty and Gap1, although it should be noted that calcineurin may act further upstream, e.g., at the level of InsP₄. In addition, it is possible that calcineurin is activated by other Ca²⁺ signaling pathways (Sullivan, 2002).

In conclusion, it has been demonstrated that a dominant modifier screen can be used successfully to isolate mutations in genes involved in calcineurin function. The mutations in the calcineurin B gene that was isolated in the screen will help determine the roles of calcineurin in Drosophila development. In addition, compelling genetic evidence was presented that calcineurin negatively regulates the Egf receptor/Ras signaling pathway at the level of Gap1 and sprouty. Calcineurin may act directly by dephosphorylating one or more signaling components, or it may target a transcription factor and act indirectly through changes in gene expression. More work will be needed to elucidate the molecular mechanism, and the modifiers isolated in the canA^act.gl screen should prove valuable in this endeavor. Furthermore, given the conservation of signal transduction between fruit flies and vertebrates, it is likely that the signaling network that was identified is employed in other organisms (Sullivan, 2002).

Drosophila Calcineurin B2 function is required for myofilament formation and troponin I isoform transition in Drosophila indirect flight muscle

Mutations in calcineurin B2 gene cause the collapse of indirect flight muscles during mid stages of pupal development. Examination of cell fate-specific markers indicates that unlike mutations in genes such as vestigial, calcineurin B2 does not cause a shift in cell fate from indirect flight muscle (IFM) to direct flight muscle (DFM). Genetic and molecular analyses indicate a severe reduction of myosin heavy chain gene expression in calcineurin B2 mutants, which accounts at least in part for the muscle collapse. Myofibrils in calcineurin B2 mutants display a variety of phenotypes, ranging from normal to a lack of sarcomeric structure. Calcineurin B2 also plays a role in the transition to an adult-specific isoform of troponin I during the late pupal stages, although the incompleteness of this transition in calcineurin B2 mutants does not contribute to the phenotype of muscle collapse. Together, these findings suggest a molecular basis for the indirect flight muscle hypercontractility phenotype observed in flies mutant for Drosophila calcineurin B2 (Gajewski, 2005).

This report further characterizes the IFM collapse phenotype of the canB2[EP(2)0774] mutation. Studies of mutations in other loci that produce IFM collapse revealed two major causes for the phenotype: change of cell fate in the adepithelial cells of the 3rd Instar lava, or hypercontraction of the IFM muscle fibers. In mutants that cause a change in cell fate, such as vg[null], a change in muscle cell fate can be clearly demonstrated by the loss of IFM-specific markers, and the ectopic expression of DFM-specific markers. No such changes are observed in canB2 mutant IFM. Unlike vg[null] mutants, the 88Factin-GFP reporter is expressed strongly in canB2 mutants, even after collapse of the IFM. A DFM-specific marker, gD1142.1-lacZ, expressed in a subset of DFM, also showed no alteration of expression pattern in canB2 mutants. The expression of these reporters in the expected places indicates proper fate determination for the precursor cells that form the DFM and IFM (Gajewski, 2005).

Disruption of the myofibrillar structure by mutation of the fli I locus, encoding a member of the gelsolin protein family, involved in the capping, severing, and bundling of actin filaments, partially suppresses the IFM collapse phenotype, pointing to hypercontraction rather than a shift in cell fate as a cause. Addition of two doses of the fli I[3] allele to a canB2 mutant background significantly increases the numbers of uncollapsed DLM. However, the suppression is not complete, and this may be due to the relatively mild effect of the fli I[3] allele. Null alleles of fli I cause lethality in the early embryonic stages. The fli I[3] allele is a less severe mutation, caused by a change of a highly conserved glycine to serine. It is possible that even with the disruptions of the sarcomeric structure, fli I[3] does not completely inhibit IFM contraction (Gajewski, 2005).

A reduction of calcineurin function has a profound effect on the expression of the mhc gene in the IFM. One copy of canB2[EP(2)0774] enhances the severity of IFM defects in flies heterozygous for the antimorphic Mhc[5] allele. mhc transcripts are barely detectable in the IFM of canB2 mutant flies, and many of the mutant myofibrils have greatly reduced or completely absent thick filaments. However, there is no interference with the tissue-specific splicing of the five versions of Mhc exon 11. The reduction of mhc expression is not due to a nonspecific reduction in transcription; the levels of GFP transcript from a reporter driven by mhc upstream sequences (mhc-GFP) are also reduced in a mutant background, but expression of actin88F is unaffected. The simplest explanation is that transcription of mhc is greatly reduced in the absence/reduction of calcineurin function, but further studies will be needed to confirm it (Gajewski, 2005).

The function of calcineurin in transcriptional activation is well documented, for example, its role in regulating transcription factors such as NFAT and Mef2. There are multiple Dmef2 binding sites upstream of the mhc gene, as well as a binding site for the zinc-finger transcription factor CF2. Work in other systems has established that calcineurin can activate Mef2 both directly and indirectly; it is likely that this will also hold true for Drosophila. Whether calcineurin can affect CF2 activity is not yet known, but the phosphorylation state of CF2 has been demonstrated to play a role in its regulation via the EGFR pathway in Drosophila ovaries. Phosphorylated CF2 is found predominantly in the cytoplasm of the anterodorsal follicle cells, where it is fated to be degraded. It is speculated that removal of the phosphate group allows entry in the nucleus CF2 is expressed in all three muscle types of the Drosophila embryo (Bagni, 2002), but it is not yet known if this protein is required for IFM development. It will be of interest to investigate whether CF2 is expressed in the developing IFM, and what effects calcineurin function (or lack thereof) would possibly have on its subcellular localization (Gajewski, 2005).

It is also interesting to note that the lack/reduction of calcineurin has a much more drastic effect on mhc transcript levels in the IFM than it does on the various muscle types of the abdomen. The amount of total mhc transcripts are readily detectable in the mutant abdominal musculature, but not in the mutant IFM under the same PCR conditions. The transcript is not completely missing in the mutant IFM; if extra PCR cycles are done, or extra fly equivalents of cDNA are added for the mutants, a mhc band can be amplified. The reason for greater IFM sensitivity to lack of calcineurin function is unknown, and warrants further investigation. It may be that calcineurin is part of a system to promote maximum expression of mhc. The IFM are the largest muscles in the fly, and their tightly packed hexagonal arrangement of thick and thin filaments (unique in the fly musculature) could require increased expression of myosin and other structural proteins. There are numerous examples of mutations in muscle structural protein genes that result in a flightless phenotype, but do not impair the functions of other types of muscles (Gajewski, 2005).

The myofilament structure of the canB2 mutants reflects the reduction in mhc transcripts. While about 20% of the adult mutant IFM tissue examined resembled wild type, the majority of samples exhibited some degree of defect. Some myofibrils had patches of organized filament structure at the periphery, but have no recognizable structures in focus at the center region. This is likely the result of hypercontraction, which can lead to random myofilament orientation. In the most severely affected myofibrils, no organized structures of any sort could be detected. Examination of longitudinal sections confirmed this range of phenotypes. Some samples resembled the wild type sarcomeric pattern. Mildly affected mutant tissue had broken Z-bands, partial or missing M-lines (indicative of reduced or missing thick filaments), and shorter sarcomeres (indicative of hypercontraction). The most severely affected mutant muscles lacked any Z-bands or M-lines. The mutant pupal samples tended to display the most severe myofibrillar phenotypes. It is likely that using adults for examination selects against the most severe phenotypes; the animals examined in the pupal stage are likely to represent those that would not have successfully eclosed, and a small sample of canB2 mutant pupae could easily display a propensity for the strongest defects. The canB2[EP(2)0774] mutation is semi-lethal; life cycle analysis reveals that many of the animals that die do so in the pupal stages. It may be that the most severe canB2 phenotypes render the animals unable to eclose, although the role, if any, of the IFM is this process is yet to be confirmed. It is also possible that the most severe canB2 phenotypes could impair other muscles (Gajewski, 2005).

The effect of the canB2 mutation on Tn I expression represents a possible novel role for calcineurin, that being in different isoform formation. Although no direct role for calcineurin in the control of RNA splicing has yet been demonstrated, it is interesting to note that phosphorylation status of SR proteins plays a role in their localization within the nucleus, and assembly, disassembly, and activity of the spliceosome may by influenced by a cycle of protein phosphorylation. The degree of phosphorylation is believed to effect protein-protein and protein-RNA interactions in the spliceosomal complexes. The splicing of at least one variant exon of the mouse CD44 gene is coupled to signal transduction via the protein kinase C/ras signaling pathway. Therefore, it is possible that calcineurin helps regulate a system responsible for transition in the pupal IFM from the smaller Tn I isoform to the larger version, by control of the phosphorylation states of one or more proteins in the spliceosome complex that are required for inclusion of the third exon (Gajewski, 2005).

It should be noted that the effects of the canB2 mutation on the relative levels of the two Tn I isoforms in the adult IFM are highly variable. In some PCR experiments, the smaller, exon 3 lacking transcript is predominant, but in others, both forms can be clearly seen. However, the results for the wild type adults are consistent: the larger transcript is clearly present, with little or no smaller form detected, in multiple repeats of the experiment. Thus, it must be considered that the differential formation of the Tn I isoforms may not be a direct result of altered calcineurin control of splicing in the IFM, but an indirect consequence of the physiological status of mutant versus normal muscle. That is, the switch to the larger exon 3 containing isoform may normally occur in a wild type genetic background due to some signal (or muscle state) perceived and transmitted within IFM that is of a proper developmental age and competency. In canB2 mutants, an abnormal cellular environment may exist in some or all IFM that prevents the normal sensing of this signal and subsequent isoform switch. Thus, the variability observed in the relative ratio of the two Tn I mRNA forms may simply reflect a nonequivalent status of collapsed muscles as to their competency to sense and execute this developmental molecular switch (Gajewski, 2005).

Taken together, these results have provided mechanistic insights into the cause of IFM collapse in canB2 mutants. Cell fate changes can be ruled out, as can problems with mhc isoform production. In canB2 mutants, the transition to the adult Tn I splice variant is incomplete at best, but this change occurs after the time when the muscles collapse, so an altered stoichiometry of troponin isoforms cannot contribute to this phenotype. Reduction of calcineurin function in the IFM leads to lower levels of mhc transcripts and a variable reduction in the numbers of thick filaments. This reduction in mhc expression is likely a major contributing factor in the collapse of the canB2 mutant IFM. Heterozygotes of Mhc[1], which is a null allele, have reduced numbers of thick filaments and partial hypercontraction of the IFM. However, there is a striking difference in the collapse phenotypes of canB2 and various mhc mutations. In canB2 mutants, without fail, the collapse of the IFMs is directed towards the posterior of the thorax. In a number of different mhc mutant alleles, the IFM can bunch to either. The most severe myofibrillar phenotypes also suggest problems with more than just mhc. The strongest canB2 phenotypes had no Z-bands or any semblance of sarcomeric structure, an effect seen in some mutations that cause defects in the thin filaments. In animals homozygous for the Tn I allele heldup[3] (hdp[3]), which is functionally a null in the IFM, pupal myofibrils showed diffuse Z-bands at 42 h APF, and no sarcomeric structures by 46-48 h APF. Since no Z-bands in the most severely affected canB2 mutant pupae, it is possible that Z-bands could form and break down in a manner similar to hdp[3] mutants. Therefore, it is quite likely that expression and/or processing of other muscle structural proteins are regulated by calcineurin activity, and these warrant future investigation (Gajewski, 2005).

REGULATION

A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT

Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1-4) is essential for vertebrate development and function. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca²⁺ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). This study used a genome-wide RNA interference (RNAi) screen in Drosophila to identify additional regulators of the signalling pathway leading from Ca²⁺-calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca²⁺ influx, Ca²⁺-calmodulin-calcineurin signalling and NFAT kinases) are conserved across species, even though Ca²⁺-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) has been identified as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates (Gwack, 2006).

To validate the use of genome-wide RNAi screening in Drosophila to identify regulators of the Ca²⁺-calcineurin-NFAT signalling pathway, an NFAT-GFP (green fluorescent protein) fusion protein containing the entire regulatory domain of NFAT1 was used. This domain bears >14 phosphorylated serines, 13 of which are dephosphorylated by calcineurin. Five of the thirteen serines are located in the SRR-1 motif, which controls exposure of the nuclear localization sequence (NLS) and is a target for phosphorylation by CK1; three are located in the SP-2 motif, which can be phosphorylated by GSK3 after a priming phosphorylation by protein kinase A (PKA); and four are located in the SP-3 motif, for which a relevant kinase had yet to be identified at the time this study was initiated. The SP-2 and SP-3 motifs do not directly regulate the subcellular localization of NFAT1, but their dephosphorylation increases both the probability of NLS exposure and the affinity of NFAT for DNA. NFAT-GFP was correctly regulated in Drosophila S2R⁺ cells: it was phosphorylated and localized to the cytoplasm under resting conditions; it became dephosphorylated and translocated to the nucleus with appropriate kinetics in response to Ca²⁺ store depletion with the sarcoplasmic/endoplasmic reticulum ATPase (SERCA) inhibitor thapsigargin; and its dephosphorylation and nuclear translocation were both sensitive to the calcineurin inhibitor cyclosporin A (CsA). S2R⁺ cells treated with limiting amounts of thapsigargin displayed intermediate phosphorylated forms of NFAT-GFP, most likely reflecting progressive dephosphorylation of serines within individual conserved motifs of the regulatory domain. Depletion of the primary NFAT regulator, calcineurin, by RNAi in S2R⁺ cells inhibited thapsigargin-dependent dephosphorylation and nuclear import of NFAT-GFP. Thus, the major pathways regulating NFAT phosphorylation and subcellular localization (store-operated Ca²⁺ influx, calcineurin activation and NFAT phosphorylation/dephosphorylation) are conserved in Drosophila and appropriately regulate vertebrate NFAT (Gwack, 2006).

A genome-wide RNAi screen on unstimulated S2R⁺ cells was performed, and aberrant nuclear localization of NFAT-GFP was scored. Positive candidates obtained in the screen include (1) Na⁺/Ca²⁺ exchangers and SERCA Ca²⁺ ATPases, the knockdown of which would be expected to increase basal levels of intracellular free Ca²⁺ ([Ca²⁺]_i); (2) the scaffold protein Homer, which has been linked to Ca²⁺ influx and Ca²⁺ homeostasis; (3) stromal interaction molecule (STIM), a recently identified regulator of store-operated Ca²⁺ influx, and (4) several protein kinases that control NFAT function either directly via phosphorylation or indirectly via basal [Ca²⁺]_i levels, calcineurin activity, or other kinases. To identify kinases that directly phosphorylate the NFAT regulatory domain, Flag-tagged human homologues of selected Drosophila kinases were expressed in HEK293 cells, and anti-Flag immunoprecipitates were tested in an in vitro kinase assay for their ability to phosphorylate a GST-NFAT1(1-415) fusion protein. Three kinases -- protein kinase cGMP-dependent (PRKG1), DYRK2 and interleukin (IL)-1 receptor-associated kinase 4 (IRAK4) -- showed strong activity in this assay. In cells, only DYRK2 countered the dephosphorylation of NFAT-GFP by calcineurin, even though both PRKG1 and DYRK2 were expressed at high levels. CD4⁺ T_H1 cells isolated from Irak4^-/- mice showed normal NFAT1 dephosphorylation, re-phosphorylation and nuclear transport compared to control T_H1 cells. Therefore focus was placed on DYRK-family kinases as potential direct regulators of NFAT (Gwack, 2006).

DYRKs constitute an evolutionarily conserved family of proline- or arginine-directed protein kinases belonging to the CMGC family of cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), GSK and CDK-like kinases (CLKs). The DYRK family has multiple members that can be predominantly nuclear (DYRK1A and DYRK1B) or cytoplasmic (DYRK2-4 and homeodomain interacting protein kinase 3 (HIPK3)/DYRK6). RT-PCR and western blotting suggested that DYRK1A and DYRK2 were major representatives of nuclear and cytoplasmic DYRKs, respectively, in Jurkat T cells, a conclusion supported by several additional observations. (1) Overexpression of DYRK2 prevented dephosphorylation of NFAT-GFP after ionomycin treatment; overexpression of wild-type DYRK2, but not a kinase-dead mutant of DYRK2, prevented NFAT nuclear localization in thapsigargin-treated cells. The slower-migrating form of NFAT might indicate that DYRK2, a proline-directed kinase, acts in part by phosphorylating the SPRIEITP docking sequence on NFAT1 and thereby blocking the calcineurin-NFAT interaction. However, DYRK was still effective when the potential DYRK phosphorylation sites in the docking sequence were eliminated by substituting HPVIVITGP for SPRIEITPS. (2) Both wild-type and kinase-dead DYRK co-immunoprecipitated with NFAT1. (3) Depletion of the DYRK-family candidate CG40478 in S2R⁺ cells did not affect (and DYRK2 overexpression in Jurkat T cells only slightly diminished) Ca²⁺ mobilization in response to thapsigargin. (4) Most importantly, depletion of endogenous DYRK1A with DYRK1A-specific short interfering RNAs (siRNA) in HeLa cells stably expressing NFAT-GFP increased the rate and extent of NFAT1 dephosphorylation and nuclear import while slowing re-phosphorylation and nuclear export. These results show that DYRK1A and DYRK2 are physiological negative regulators of NFAT activation in cells. The absence of basal NFAT dephosphorylation in DYRK1A-depleted cells may reflect both the expression of other DYRK family members in human cells and the predominantly nuclear localization of DYRK1A (Gwack, 2006).

DYRKs are direct NFAT1 kinases that selectively phosphorylate the SP-3 motif, but nevertheless control the overall phosphorylation of NFAT1. Flag-tagged DYRK2 expressed in HEK cells, as well as bacterially expressed recombinant DYRK1A and DYRK2, phosphorylated peptides corresponding to the SP-3 motif of the NFAT1 regulatory domain in vitro, but did not phosphorylate SRR-1 or SP-2 peptides or an SP-3 peptide with serine to alanine substitutions in the known phosphoserine residues. Two serine residues (underlined) in the SP-3 motif (SPQRSRSPSPQPSPHVAPQDD) fit the known sequence preference of DYRK kinases [R(x)xx(S/T)(P/V)], and both are known to be phosphorylated in cells. DYRK is reported to prime for GSK3-mediated phosphorylation of eukaryotic initiation factor 2B-epsilon (eIF2B-epsilon) and the microtubule-associated protein tau, as well as for GSK3- and CK1-mediated phosphorylation of OMA-1. Therefore whether DYRK kinases could also prime for GSK3- and CK1-mediated phosphorylation of NFAT1 was investigated. Pre-phosphorylation of the NFAT1 regulatory domain by DYRK2 led to robust phosphorylation by GSK3 and induced the mobility shift characteristic of phosphorylation of the SP-2 and SP-3 motifs; this shift was not observed after pre-phosphorylation by PKA. Furthermore, pre-phosphorylation by DYRK2 accelerated CK1-mediated phosphorylation of GST-NFAT1(1-415) by at least twofold. In contrast, DYRK2 did not prime phosphorylation of the SP-2 peptide by GSK3 nor the SRR-1 peptide by CK1, consistent with the fact that neither motif is a substrate for DYRK. This 'discontiguous' priming mechanism is distinct from conventional priming, which requires phosphorylation at +4 and -3 for GSK3 and CK1, respectively. A less likely interpretation is that the conventional priming sites for CK1 and GSK3 are efficiently phosphorylated by DYRK in the context of the GST-NFAT1(1-415) protein, although they are not phosphorylated in the peptide context (Gwack, 2006).

The kinase-dead mutant of DYRK2 shows that DYRK regulates the transcriptional activity of NFAT. Wild-type DYRK2 strongly diminished NFAT-dependent activity, whereas the kinase-dead mutant increased NFAT-dependent luciferase activity of the IL2 promoter, of an NFAT-activating protein 1 (AP-1) reporter, or of an AP-1-independent promoter (the kappa3 site of the tumour-necrosis factor-alpha (TNF-alpha) promoter). Similarly, wild-type DYRK2 diminished production of endogenous IL-2 by stimulated Jurkat T cells, whereas kinase-dead DYRK2 had the opposite effect (Gwack, 2006).

These data indicate that DYRK is a key kinase that regulates NFAT1 phosphorylation. DYRK, GSK3 and CK1 target completely distinct motifs of the NFAT1 regulatory domain, but DYRK-mediated phosphorylation of the SP-3 motif primes for further phosphorylation of the distinct SRR-1 and SP-2 motifs by CK1 and GSK3, respectively, thus facilitating complete phosphorylation and deactivation of NFAT1. This mechanism, which has been termed 'discontiguous priming', is reminiscent of that recently proposed for C. elegans oocyte maturation protein 1 (OMA-1), in which phosphorylation of Thr 239 by the DYRK-family kinase minibrain kinase 2 (MBK-2) potentiates GSK3-mediated phosphorylation of Thr 339. It is likely that DYRK2, DYRK3 and DYRK4, which are localized to the cytoplasm, function primarily as 'maintenance' kinases that sustain the phosphorylation status of cytoplasmic NFAT in resting cells, whereas DYRK1A and DYRK1B, which are localized to the nucleus, re-phosphorylate nuclear NFAT and promote its nuclear export. Notably, NFAT dephosphorylation may also proceed through a sequential mechanism, with dephosphorylation of the SRR-1 motif promoting dephosphorylation of the SP-2 and SP-3 motifs by increasing their accessibility to calcineurin. DYRK1A and the endogenous calcineurin regulator DSCR1/RCN/calcipressin-1 are both localized to the Down's syndrome critical region on human chromosome 21; thus, overexpression of these negative regulators of NFAT could contribute (by inhibiting NFAT activation) to the neurological and immunological developmental anomalies observed in individuals with chromosome 21 trisomy (Gwack, 2006).

Genome-wide RNAi screening in Drosophila is a valid and powerful strategy for exploring novel aspects of signal transduction in mammalian cells, provided that key members of the signalling pathway are evolutionarily conserved and represented in the Drosophila genome. This study used the method to identify conserved regulators of the purely vertebrate transcription factor NFAT; this is the first example of a genome-wide RNAi screen that crosses evolutionary boundaries in this manner. It is likely that conserved aspects of the regulation of other mammalian processes will also be successfully defined by developing assays in Drosophila cells (Gwack, 2006).

Drosophila Calcineurin promotes induction of innate immune responses

The sophisticated adaptive immune system of vertebrates overlies an ancient set of innate immune-response pathways, which have been genetically dissected in Drosophila. Although conserved regulatory pathways have been defined, calcineurin, a Ca2+-dependent phosphatase, has not been previously implicated in Drosophila immunity. Calcineurin activates mammalian immune responses by activating the nuclear translocation of the vertebrate-specific transcription factors NFAT1-4. In Drosophila, infection with gram-negative bacteria promotes the activation of the Relish transcription factor through the Imd pathway. The activity of this pathway in the larva is modulated by nitric oxide (NO). This study shows that the input by NO is mediated by calcineurin. Pharmacological inhibition of calcineurin suppressed the Relish-dependent gene expression that occurs in response to gram-negative bacteria or NO. One of the three calcineurin genes in Drosophila, CanA1, mediates NO-induced nuclear translocation of Relish in a cell-culture assay. A CanA1 RNA interference (RNAi) transgene suppressed immune induction in larvae upon infection or upon treatment with NO donors, whereas a gain-of-function CanA1 transgene activated immune responses in untreated larvae. Interestingly, CanA1 RNAi in hemocytes but not the fat body was sufficient to block immune induction in the fat body. Thus, CanA1 provides an additional input into Relish-promoted immune responses and functions in hemocytes to promote a tissue-to-tissue signaling cascade required for robust immune response (Dijkers, 2007).

Altogether, these findings show that calcineurin contributes to innate immune responses and conveys an NO signal that activates AMP production in the Drosophila larva. The bacteria (Ecc15) fed to the larvae remain confined to the gut but nonetheless induce responses in the fat body. Because infection induces NOS in the gut, NO produced in the gut might signal to hemocytes, which then induce responses in the fat body. This proposal is supported by previous demonstrations that NOS contributes to immune induction and that Domino mutant larvae, which have a severe reduction of hemocytes (among other defects), fail to induce Dipt in response to NO or to natural infection. Furthermore, psidin gene function in hemocytes promotes fat-body expression of AMPs (Brennan, 2007). The demonstration that CanA1 is required in hemocytes for the immune response in the fat body provides further support of this proposal. In this model, the response of the hemocyte to NO is independent of Imd, like the response of S2 cells, whereas robust induction of AMPs in downstream tissues requires Imd. Consequently, Imd acts downstream of NO to induce AMPs in larvae. These findings argue that tissue-to-tissue signaling plays a role in a natural infection model in larvae and that CanA1 participates in this signaling (Dijkers, 2007).

Nebula/DSCR1 upregulation delays neurodegeneration and protects against APP-induced axonal transport defects by restoring calcineurin and GSK-3beta signaling

Post-mortem brains from Down syndrome (DS) and Alzheimer's disease (AD) patients show an upregulation of the Down syndrome critical region 1 protein (DSCR1), but its contribution to AD is not known. To gain insights into the role of DSCR1 in AD, this study explored the functional interaction between DSCR1 and the amyloid precursor protein (APP), which is known to cause AD when duplicated or upregulated in DS. The Drosophila homolog of DSCR1, Nebula, was found to delay neurodegeneration and ameliorates axonal transport defects caused by APP overexpression. Live-imaging reveals that Nebula facilitates the transport of synaptic proteins and mitochondria affected by APP upregulation. Furthermore, Nebula upregulation was shown to protect against axonal transport defects by restoring calcineurin and GSK-3beta signaling altered by APP overexpression, thereby preserving cargo-motor interactions. As impaired transport of essential organelles caused by APP perturbation is thought to be an underlying cause of synaptic failure and neurodegeneration in AD, these findings imply that correcting calcineurin and GSK-3beta signaling can prevent APP-induced pathologies. The data further suggest that upregulation of Nebula/DSCR1 is neuroprotective in the presence of APP upregulation and provides evidence for calcineurin inhibition as a novel target for therapeutic intervention in preventing axonal transport impairments associated with AD (Shaw, 2013).

Although upregulation of APP had been shown to negatively influence axonal transport in mouse and fly models, mechanisms by which APP upregulation induces transport defects are poorly understood. Several hypotheses have been proposed, including titration of motor/adaptor by APP, impairments in mitochondrial bioenergetics, altered microtubule tracks, or aberrant activation of signaling pathways. The motor/adaptor titration theory suggests that excessive APP-cargos titrates the available motors away from other organelles, thus resulting in defective transport of pre-synaptic vesicles. The finding that Nebula co-upregulation enhanced the movement and delivery of both synaptotagmin and APP to the synaptic terminal argues against this hypothesis. In addition, earlier findings suggest that Nebula upregulation alone impaired mitochondrial function and elevated ROS level, thus implying that Nebula is not likely to rescue APP-dependent phenotypes by selectively restoring mitochondrial bioenergetics. Furthermore, consistent with a recent report showing normal microtubule integrity in flies overexpressing either APP-YFP or activated GSK-3βM (Weaver, 2013), the data revealed normal gross microtubule structure in flies with APP overexpression. Together, these results suggest that changes in gross microtubule structure and stability is not a likely cause of APP-induced transport defects (Shaw, 2013).

Instead, the current results support the idea that Nebula facilitates axonal transport defects by correcting APP-mediated changes in phosphatase and kinase signaling pathways. First, APP upregulation was found to elevate intracellular calcium level and calcineurin activity, and restoring calcineurin activity to normal suppresses the synaptotagmin aggregate accumulation in axons. The observed increase in calcium and calcineurin activity is consistent with reports of calcium dyshomeostasis and elevated calcineurin phosphatase activity found in AD brains, as well as reports demonstrating elevated neuronal calcium level due to APP overexpression and increased calcineurin activation in Tg2576 transgenic mice carrying the APP^swe mutant allele. Second, APP upregulation resulted in calcineurin dependent dephosphorylation of GSK-3β at Ser9 site, a process thought to activate GSK-3β kinase. APP upregulation also triggered calcineurin-independent phosphorylation at Tyr216 site, which has been shown to enhance GSK-3β activity. The kinase(s) that phosphorylates APP at Tyr216 is currently not well understood, it will be important to study how APP leads to Tyr216 phosphorylation in the future. Based on the current results, it is envisioned that APP overexpression ultimately leads to excessive calcineurin and GSK-3β activity, whereas nebula overexpression inhibits calcineurin to prevent activation of GSK-3β. The findings that nebula co-overexpression prevents GSK-3β activation and enhances the transport of APP-YFP vesicles are consistent with a recent report by Weaver (2013), in which it was found decreasing GSK-3β in fly increases the speed of APP-YFP movement. Furthermore, consistent with the current result that APP upregulation triggers GSK-3β enhancement and severe axonal transport defect, Weaver did not detect changes in GFP-synaptotagmin movement in the absence of APP upregulation (Shaw, 2013).

Active GSK-3β has been shown to influence the transport of mitochondria and synaptic proteins including APP, although the exact mechanism may differ between different cargos and motors. One mechanism proposed for GSK-3β-mediated regulation of axonal transport is through phosphorylation of KLC1, thereby disrupting axonal transport by decreasing the association of the anterograde molecular motor with its cargos. Accordingly, this study found that APP reduces KLC-synaptotagmin interaction while Nebula upregulation preserves it. Synaptotagmin transport in both the anterograde and retrograde directions are affected, consistent with previous reports showing that altering either the anterograde kinesin or retrograde dynein is sufficient affected transport in both directions. The results also support work suggesting that synaptotagmin can be transported by the kinesin 1 motor complex in addition to the kinesin 3/imac motor. As kinesin 1 is known to mediate the movement of both APP and mitochondria and phosphorylation of KLC had been shown to inhibit mitochondrial transport, detachment of cargo-motor caused by GSK-3β mediated phosphorylation of KLC may lead to general axonal transport problems as reported in this study. However, GSK-3β activation may also perturb general axonal transport by influencing motor activity or binding of motors to the microtubule tract. Interestingly, increased levels of active GSK-3β and phosphorylated KLC and dynein intermediate chain (DIC), a component of the dynein retrograde complex, have been observed in the frontal complex of AD patients. Genetic variability for KLC1 is thought to be a risk factor for early-onset of Alzheimer's disease. There is also increasing evidence implicating GSK-3β in regulating transport by modulating kinesin activity and exacerbating neurodegeneration in AD through tau hyperphosphorylation. It will be interesting to investigate if Nebula also modulates these processes in the future (Shaw, 2013).

SAlthough calcineurin had been shown to regulate many important cellular pathways, the link between altered calcineurin and axonal transport, especially in the context of AD, had not been established before. This study shows that calcineurin can regulate axonal transport through both GSK-3β independent and dependent pathways. This is supported by observation that the severity of the aggregate phenotype was worse for flies expressing APP and active calcineurin than it was for flies expressing APP and active GSK-3β. These findings point to a role for calcineurin in influencing axonal transport directly, perhaps through dephosphorylation of motor or adaptor proteins. The data also indicate that calcineurin in part modulates axonal transport through dephosphorylation of GSK-3β as discussed above; however, upregulation of APP is necessary for the induction of severe axonal transport problems, mainly by causing additional enhancement of GSK-3β signaling. GSK3 inhibition is widely discussed as a potential therapeutic intervention for AD; results suggest that perhaps calcineurin is a more effective target for delaying degeneration by preserving axonal transport (Shaw, 2013).

DSCR1 and APP are both located on chromosome 21 and upregulated in DS. Overexpression of DSCR1 alone had been contradictorily implicated in both conferring resistance to oxidative stress and in promoting apoptosis. Upregulation of Nebula/DSCR1 had also been shown to negatively impact learning and memory in fly and mouse models through altered calcineurin pathways. How could upregulation of DSCR1 be beneficial? It is proposed that DSCR1 upregulation in the presence of APP upregulation compensates for the altered calcineurin and GSK-3β signaling, shifting the delicate balance of kinase/phosphatase signaling pathways close to normal, therefore preserving axonal transport and delaying neurodegeneration. It is also proposed that axonal transport defects and synapse dysfunction caused by APP upregulation in the Drosophila model system occur prior to accumulation of amyloid plaques and severe neurodegeneration, similar to that described for a mouse model (Shaw, 2013).

DS is characterized by the presence of AD neuropathologies early in life, but most DS individuals do not exhibit signs of dementia until decades later, indicating that there is a delayed progression of cognitive decline. The upregulation of DSCR1 may in fact activate compensatory cell signaling mechanisms that provide protection against APP-mediated oxidative stress, aberrant calcium, and altered calcineurin and GSK3-β activity (Shaw, 2013).

A TRPV channel in Drosophila motor neurons regulates presynaptic resting Ca(2+) levels, synapse growth, and synaptic transmission

Presynaptic resting Ca²⁺ influences synaptic vesicle (SV) release probability. This study reports that a TRPV channel, Inactive (Iav), maintains presynaptic resting [Ca²⁺] by promoting Ca²⁺ release from the endoplasmic reticulum in Drosophila motor neurons, and is required for both synapse development and neurotransmission. Iav activates the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin, which is essential for presynaptic microtubule stabilization at the neuromuscular junction. Thus, loss of Iav induces destabilization of presynaptic microtubules, resulting in diminished synaptic growth. Interestingly, expression of human TRPV1 in Iav-deficient motor neurons rescues these defects. The absence of Iav causes lower SV release probability and diminished synaptic transmission, whereas Iav overexpression elevates these synaptic parameters. Together, these findings indicate that Iav acts as a key regulator of synaptic development and function by influencing presynaptic resting [²⁺] (Sullivan, 2014).

Protein Interactions

Requirement of the calcineurin subunit gene canB2 for indirect flight muscle formation in Drosophila

Calcineurin is a calcium-activated protein phosphatase involved in multiple aspects of cardiac and skeletal muscle development and disease. Genes encoding calcineurin subunit proteins are highly conserved among animal species. Toward the goal of identifying new calcineurin-interacting loci that function in myogenic processes, an activated form of mouse calcineurin A was expressed in Drosophila and suppressors of the phosphatase-induced lethality were screened for. A mutation in the canB2 gene, which encodes a regulatory subunit of Drosophila calcineurin, can suppress a pupal developmental arrest phenotype to adult viability. Since canB2 is an essential gene and rare homozygous escapers are flightless, canB2 expression and function was further analyzed in pupae and adults. The gene is expressed in the forming indirect flight muscles and central nervous system during pupal development. A canA gene is comparably expressed in these tissues. Consistent with the observed muscle expression, canB2 mutants exhibit severe defects in the organization of their indirect flight muscles, a phenotype that is likely caused by muscle hypercontractility. Together, these findings demonstrate a vital role for the phosphatase in this specific facet of Drosophila myogenesis and show conserved fly and vertebrate calcineurin genes contribute prominently to fundamental processes of muscle formation and function (Gajewski, 2003).

Drosophila Calcineurin B2 function is required for myofilament formation and troponin I isoform transition in Drosophila indirect flight muscle

Mutations in calcineurin B2 gene cause the collapse of indirect flight muscles during mid stages of pupal development. Examination of cell fate-specific markers indicates that unlike mutations in genes such as vestigial, calcineurin B2 does not cause a shift in cell fate from indirect flight muscle (IFM) to direct flight muscle (DFM). Genetic and molecular analyses indicate a severe reduction of myosin heavy chain gene expression in calcineurin B2 mutants, which accounts at least in part for the muscle collapse. Myofibrils in calcineurin B2 mutants display a variety of phenotypes, ranging from normal to a lack of sarcomeric structure. Calcineurin B2 also plays a role in the transition to an adult-specific isoform of troponin I during the late pupal stages, although the incompleteness of this transition in calcineurin B2 mutants does not contribute to the phenotype of muscle collapse. Together, these findings suggest a molecular basis for the indirect flight muscle hypercontractility phenotype observed in flies mutant for Drosophila calcineurin B2 (Gajewski, 2005).

This report further characterizes the IFM collapse phenotype of the canB2[EP(2)0774] mutation. Studies of mutations in other loci that produce IFM collapse revealed two major causes for the phenotype: change of cell fate in the adepithelial cells of the 3rd Instar lava, or hypercontraction of the IFM muscle fibers. In mutants that cause a change in cell fate, such as vg[null], a change in muscle cell fate can be clearly demonstrated by the loss of IFM-specific markers, and the ectopic expression of DFM-specific markers. No such changes are observed in canB2 mutant IFM. Unlike vg[null] mutants, the 88Factin-GFP reporter is expressed strongly in canB2 mutants, even after collapse of the IFM. A DFM-specific marker, gD1142.1-lacZ, expressed in a subset of DFM, also showed no alteration of expression pattern in canB2 mutants. The expression of these reporters in the expected places indicates proper fate determination for the precursor cells that form the DFM and IFM (Gajewski, 2005).

Disruption of the myofibrillar structure by mutation of the fli I locus, encoding a member of the gelsolin protein family, involved in the capping, severing, and bundling of actin filaments, partially suppresses the IFM collapse phenotype, pointing to hypercontraction rather than a shift in cell fate as a cause. Addition of two doses of the fli I[3] allele to a canB2 mutant background significantly increases the numbers of uncollapsed DLM. However, the suppression is not complete, and this may be due to the relatively mild effect of the fli I[3] allele. Null alleles of fli I cause lethality in the early embryonic stages. The fli I[3] allele is a less severe mutation, caused by a change of a highly conserved glycine to serine. It is possible that even with the disruptions of the sarcomeric structure, fli I[3] does not completely inhibit IFM contraction (Gajewski, 2005).

A reduction of calcineurin function has a profound effect on the expression of the mhc gene in the IFM. One copy of canB2[EP(2)0774] enhances the severity of IFM defects in flies heterozygous for the antimorphic Mhc[5] allele. mhc transcripts are barely detectable in the IFM of canB2 mutant flies, and many of the mutant myofibrils have greatly reduced or completely absent thick filaments. However, there is no interference with the tissue-specific splicing of the five versions of Mhc exon 11. The reduction of mhc expression is not due to a nonspecific reduction in transcription; the levels of GFP transcript from a reporter driven by mhc upstream sequences (mhc-GFP) are also reduced in a mutant background, but expression of actin88F is unaffected. The simplest explanation is that transcription of mhc is greatly reduced in the absence/reduction of calcineurin function, but further studies will be needed to confirm it (Gajewski, 2005).

The function of calcineurin in transcriptional activation is well documented, for example, its role in regulating transcription factors such as NFAT and Mef2. There are multiple Dmef2 binding sites upstream of the mhc gene, as well as a binding site for the zinc-finger transcription factor CF2. Work in other systems has established that calcineurin can activate Mef2 both directly and indirectly; it is likely that this will also hold true for Drosophila. Whether calcineurin can affect CF2 activity is not yet known, but the phosphorylation state of CF2 has been demonstrated to play a role in its regulation via the EGFR pathway in Drosophila ovaries. Phosphorylated CF2 is found predominantly in the cytoplasm of the anterodorsal follicle cells, where it is fated to be degraded. It is speculated that removal of the phosphate group allows entry in the nucleus CF2 is expressed in all three muscle types of the Drosophila embryo (Bagni, 2002), but it is not yet known if this protein is required for IFM development. It will be of interest to investigate whether CF2 is expressed in the developing IFM, and what effects calcineurin function (or lack thereof) would possibly have on its subcellular localization (Gajewski, 2005).

It is also interesting to note that the lack/reduction of calcineurin has a much more drastic effect on mhc transcript levels in the IFM than it does on the various muscle types of the abdomen. The amount of total mhc transcripts are readily detectable in the mutant abdominal musculature, but not in the mutant IFM under the same PCR conditions. The transcript is not completely missing in the mutant IFM; if extra PCR cycles are done, or extra fly equivalents of cDNA are added for the mutants, a mhc band can be amplified. The reason for greater IFM sensitivity to lack of calcineurin function is unknown, and warrants further investigation. It may be that calcineurin is part of a system to promote maximum expression of mhc. The IFM are the largest muscles in the fly, and their tightly packed hexagonal arrangement of thick and thin filaments (unique in the fly musculature) could require increased expression of myosin and other structural proteins. There are numerous examples of mutations in muscle structural protein genes that result in a flightless phenotype, but do not impair the functions of other types of muscles (Gajewski, 2005).

The myofilament structure of the canB2 mutants reflects the reduction in mhc transcripts. While about 20% of the adult mutant IFM tissue examined resembled wild type, the majority of samples exhibited some degree of defect. Some myofibrils had patches of organized filament structure at the periphery, but have no recognizable structures in focus at the center region. This is likely the result of hypercontraction, which can lead to random myofilament orientation. In the most severely affected myofibrils, no organized structures of any sort could be detected. Examination of longitudinal sections confirmed this range of phenotypes. Some samples resembled the wild type sarcomeric pattern. Mildly affected mutant tissue had broken Z-bands, partial or missing M-lines (indicative of reduced or missing thick filaments), and shorter sarcomeres (indicative of hypercontraction). The most severely affected mutant muscles lacked any Z-bands or M-lines. The mutant pupal samples tended to display the most severe myofibrillar phenotypes. It is likely that using adults for examination selects against the most severe phenotypes; the animals examined in the pupal stage are likely to represent those that would not have successfully eclosed, and a small sample of canB2 mutant pupae could easily display a propensity for the strongest defects. The canB2[EP(2)0774] mutation is semi-lethal; life cycle analysis reveals that many of the animals that die do so in the pupal stages. It may be that the most severe canB2 phenotypes render the animals unable to eclose, although the role, if any, of the IFM is this process is yet to be confirmed. It is also possible that the most severe canB2 phenotypes could impair other muscles (Gajewski, 2005).

The effect of the canB2 mutation on Tn I expression represents a possible novel role for calcineurin, that being in different isoform formation. Although no direct role for calcineurin in the control of RNA splicing has yet been demonstrated, it is interesting to note that phosphorylation status of SR proteins plays a role in their localization within the nucleus, and assembly, disassembly, and activity of the spliceosome may by influenced by a cycle of protein phosphorylation. The degree of phosphorylation is believed to effect protein-protein and protein-RNA interactions in the spliceosomal complexes. The splicing of at least one variant exon of the mouse CD44 gene is coupled to signal transduction via the protein kinase C/ras signaling pathway. Therefore, it is possible that calcineurin helps regulate a system responsible for transition in the pupal IFM from the smaller Tn I isoform to the larger version, by control of the phosphorylation states of one or more proteins in the spliceosome complex that are required for inclusion of the third exon (Gajewski, 2005).

It should be noted that the effects of the canB2 mutation on the relative levels of the two Tn I isoforms in the adult IFM are highly variable. In some PCR experiments, the smaller, exon 3 lacking transcript is predominant, but in others, both forms can be clearly seen. However, the results for the wild type adults are consistent: the larger transcript is clearly present, with little or no smaller form detected, in multiple repeats of the experiment. Thus, it must be considered that the differential formation of the Tn I isoforms may not be a direct result of altered calcineurin control of splicing in the IFM, but an indirect consequence of the physiological status of mutant versus normal muscle. That is, the switch to the larger exon 3 containing isoform may normally occur in a wild type genetic background due to some signal (or muscle state) perceived and transmitted within IFM that is of a proper developmental age and competency. In canB2 mutants, an abnormal cellular environment may exist in some or all IFM that prevents the normal sensing of this signal and subsequent isoform switch. Thus, the variability observed in the relative ratio of the two Tn I mRNA forms may simply reflect a nonequivalent status of collapsed muscles as to their competency to sense and execute this developmental molecular switch (Gajewski, 2005).

Taken together, these results have provided mechanistic insights into the cause of IFM collapse in canB2 mutants. Cell fate changes can be ruled out, as can problems with mhc isoform production. In canB2 mutants, the transition to the adult Tn I splice variant is incomplete at best, but this change occurs after the time when the muscles collapse, so an altered stoichiometry of troponin isoforms cannot contribute to this phenotype. Reduction of calcineurin function in the IFM leads to lower levels of mhc transcripts and a variable reduction in the numbers of thick filaments. This reduction in mhc expression is likely a major contributing factor in the collapse of the canB2 mutant IFM. Heterozygotes of Mhc[1], which is a null allele, have reduced numbers of thick filaments and partial hypercontraction of the IFM. However, there is a striking difference in the collapse phenotypes of canB2 and various mhc mutations. In canB2 mutants, without fail, the collapse of the IFMs is directed towards the posterior of the thorax. In a number of different mhc mutant alleles, the IFM can bunch to either. The most severe myofibrillar phenotypes also suggest problems with more than just mhc. The strongest canB2 phenotypes had no Z-bands or any semblance of sarcomeric structure, an effect seen in some mutations that cause defects in the thin filaments. In animals homozygous for the Tn I allele heldup[3] (hdp[3]), which is functionally a null in the IFM, pupal myofibrils showed diffuse Z-bands at 42 h APF, and no sarcomeric structures by 46-48 h APF. Since no Z-bands in the most severely affected canB2 mutant pupae, it is possible that Z-bands could form and break down in a manner similar to hdp[3] mutants. Therefore, it is quite likely that expression and/or processing of other muscle structural proteins are regulated by calcineurin activity, and these warrant future investigation (Gajewski, 2005).

The calcineurin regulator Sra plays an essential role in female meiosis in Drosophila

The Drosophila modulatory calcineurin-interacting protein (MCIP) sarah (sra) is essential for meiotic progression in oocytes. Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract; it is unknown whether calcium signaling is involved. MCIPs [also termed regulators of calcineurin (RCNs), calcipressins, or DSCR1 (Down's syndrome critical region 1)] are highly conserved regulators of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase 1 and 2. Although overexpression experiments in several organisms have revealed that MCIPs inhibit calcineurin activity, their in vivo functions remain unclear. Eggs from sra null mothers are arrested at anaphase of meiosis I. This phenotype was due to loss of function of sra specifically in the female germline. Sra is physically associated with the catalytic subunit of calcineurin, and its overexpression suppresses the phenotypes caused by constitutively activated calcineurin, such as rough eye or loss of wing veins. Hyperactivation of calcineurin signaling in the germline cells resulted in a meiotic-arrest phenotype, which can also be suppressed by overexpression of Sra. All these results support the hypothesis that Sra regulates female meiosis by controlling calcineurin activity in the germline. This is the first unambiguous demonstration that the regulation of calcineurin signaling by MCIPs plays a critical role in a defined biological process (Takeo, 2006; Horner, 2006).

sarah mutation disrupts several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. The conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation. Despite its independence from a sperm trigger, egg activation in Drosophila involves calcium-mediated pathways that are likely to be analogous to those in other animals. It is intriguing that among these downstream events is the acquisition of the egg's competence to remodel the sperm nucleus into the male pronucleus (Horner, 2006).

To explore the in vivo function of the Drosophila MCIP Sra, a null mutation in this locus was created by gene targeting. Homozygotes of the null allele sra^KO are semilethal during larval or pupal stages. In addition, sra^KO females were sterile, and their ovulation is abnormal (Ejima, 2004). These phenotypes were rescued by either sarah transgenes. Taken together, these results unambiguously demonstrate that sra is responsible for the phenotypes associated with sra^KO, which include developmental defects in both sexes and ovulation and sterility in females (Takeo, 2006).

There is no apparent morphological abnormality in ovarian development in sra^KO females, but eggs from sra^KO mothers failed to hatch. Wild-type eggs at 2 hr after deposition already have completed meiosis and undergo synchronous mitotic nuclear division. In contrast, eggs—hereafter referred to as sra eggs—from sra^KO mothers had a localized DAPI-stained signal in the cortical region near the anterior pole, indicating that sra eggs are arrested during meiosis. To analyze this phenotype in detail, the pattern of chromosome segregation and the spindle shape of sra eggs were examined. Spindle microtubules were visualized with tubulin antibody staining. In wild-type females, mature oocytes are arrested at metaphase of meiosis I, during which the chromosomes are seen as a large mass of chromatin. After the release of meiotic arrest during ovulation, individual chromosome arms become visible and migrate toward the poles; the chromosomes subsequently undergo meiosis II, after which nuclear fusion and the mitotic divisions of the zygote take place. In sra eggs, the meiotic chromosomes were seen in between the metaphase plate and the poles. It was confirmed that the oocytes taken from sra mutants including sra^GS3080 and sra^GS3168 are arrested at metaphase I as in the wild-type. Therefore, sra eggs are arrested at anaphase I shortly after the meiotic resumption from the metaphase I arrest (Takeo, 2006).

To determine whether the meiotic-arrest phenotype of sra eggs is caused by loss of function in the germline or somatic cells of sra mothers, mutant germline clones were generated by the flippase-dominant female sterile (FLP-DFS) technique. All eggs laid by wild-type females completed meiotic divisions, whereas most eggs (98%) from sra mothers arrested at anaphase of meiosis I. sra germline clones were also arrested at anaphase I, reproducing the phenotype of sra^KO. A few sra eggs, including germline clones, were arrested at meiosis II. In these eggs, the two spindles were perpendicular to each other, rather than in tandem as in the wild-type. These results demonstrate that the meiotic defects in sra eggs are specifically attributable to the loss of function of sra in the female germline. Consistent with this conclusion, sra is highly expressed in the female germline during oogenesis (Ejima, 2004) and in early embryos. Furthermore, the meiotic-arrest phenotype caused by sra mutations was almost fully rescued by nos-GAL4/UASp-sra transgenes. These results establish that sra is required in the germline for meiotic progression in Drosophila females (Takeo, 2006).

Sra is a Drosophila member of the modulatory calcineurin-interacting protein (MCIP) family of proteins, which are known to function as endogenous regulators of calcineurin. Calcineurin consists of two subunits, CnA and CnB. The Drosophila genome contains three genes encoding CnA subunits (CanA1, Pp2B-14D, and CanA-14F) and two genes encoding CnB subunits (CanB and CanB2). The functions of calcineurin have been poorly analyzed in Drosophila. It is hypothesized that sra functions as an endogenous regulator of calcineurin in Drosophila. Analyses of the expression pattern of Drosophila calcineurin genes by RT-PCR revealed that all three CnA and both CnB genes were expressed in larvae and adult females, but among these, only Pp2B-14D, CanA-14F, and CanB2 are expressed in early embryos and ovaries. Therefore, these three calcineurin subunits are candidates for interacting with Sra in the female germline (Takeo, 2006).

A constitutively active form of calcineurin can be created by truncating the C-terminal part of CnA. Misexpression of the active form of Pp2B-14D (Pp2B-14D^act) causes morphological abnormalities in eyes and wings. To determine the effects of sra on calcineurin signaling, whether activated calcineurin-dependent phenotypes can be modified by coexpression of sra was tested. Overexpression of sra alone in developing eyes by using GMR-GAL4 did not induce any phenotypic change. Flies misexpressing Pp2B-14D^act showed a mild rough-eye phenotype, which was completely suppressed by coexpression of sra. Similarly, misexpression of Pp2B-14D^act in the posterior compartment of developing imaginal discs by using en-GAL4 resulted in loss of wing veins and reduction of wing size. These wing phenotypes were also completely suppressed by coexpression of sra, whereas overexpression of sra alone had no effect on wing morphology. Furthermore, overexpression of sra rescued the lethality induced by the muscle-specific expression of Pp2B-14D^act by using 24B-GAL4. All these results clearly show that sra has an inhibitory effect on calcineurin signaling (Takeo, 2006).

If Sra acts as an inhibitor of calcineurin in vivo, it was speculated that calcineurin signaling is hyperactivated in sra mutants; that is, hyperactivation of calcineurin signaling might also affect the meiotic phenotype as in sra mutants. It was found that females carrying nos-GAL4 and UASp-Pp2B-14D^act had fully developed ovaries, but were sterile or semisterile, depending on the transgenic lines. The sterility or semisterility caused by nos>Pp2B-14D^act was effectively rescued by co-overexpression of sra, demonstrating that sra counteracts activated calcineurin (Takeo, 2006).

To characterize the meiotic phenotype caused by Pp2B-14D^act, eggs were stained from semisterile females expressing nos>Pp2B-14D^act (Ejima, 2004) with DAPI and tubulin antibody to visualize chromosomes and spindles, respectively. A total of 63 eggs were examined. Of these, 10% (6/63) developed normally, whereas 14% (9/63) had neither DAPI signaling nor tubulin antibody staining. The remaining 76% showed complex abnormalities that could be classified into three types: (1) dispersed chromatins with no obvious spindle (33%); (2) apparently normal chromosomes with an abnormal spindle (38%), and (3) a mass of chromatin with an apparently normal spindle (5%). Also the nuclei of mature oocytes taken from nos>Pp2B-14D^act (Ejima, 2004) females were observed to see whether meiotic arrest at metaphase I is normal. It was found that the majority had abnormal nuclei containing dispersed chromatins, and that the remaining were arrested at metaphase I or anaphase I. These results suggest that calcineurin signaling was activated to a greater extent in the germline of females constructed in this way than in sra mutants. Taken together, these results demonstrate that the regulation of calcineurin signaling is critical for female meiosis, and its regulator Sra/MCIP is essential for meiotic progression at the time of egg activation in the Drosophila female (Takeo, 2006).

In vertebrates whose meiotic arrest occurs at metaphase II, arrest is released at the time of fertilization. The mechanisms of meiotic arrest and resumption have been extensively studied in mice and frogs, and several key components have been identified, including Cdc2/Cyclin B (MPF) and MAP kinase. In addition, the so-called “Ca²⁺ transient” mediated by IP₃ signaling has been linked to egg activation; this transient promotes completion of meiosis, ion-channel opening, and cortical granule exocytosis. Recent studies have revealed that Ca²⁺/calmoduin-dependent protein kinase II (CaMKII) is physiologically activated in mouse oocytes in response to fertilizing sperm. CaMKII is implicated in the regulation of the timing of re-entry into mitosis through the phosphorylation of Cdc25C, a phosphatase mediating G1/M transition by dephosphorylating MPF in Xenopus. More recently, CaMKII was shown to phosphorylate an anaphase-promoting complex/cyclosome (APC/C) inhibitor, Emi1-related protein (Erp1), resulting in its degradation and thereby releasing the brakes on the cell cycle from metaphase II in Xenopus eggs (Takeo, 2006).

Less is known about the mechanism of egg activation in Drosophila. Genetic screens for female-sterile mutations have identified several genes involved in female meiosis. For example, twine, a homolog of Drosophila cdc25, is required for arrest at metaphase I in mature oocytes. cortex (cort) and grauzone (grau) mutant eggs exhibit meiotic arrest at meiosis II with defects in cytoplasmic polyadenylation and translation of maternal bicoid mRNAs. cort encodes an APC/C activator protein Cdc20, suggesting that APC/C-Cdc20^Cort-mediated inactivation of MPF is required for the translational control of poly(A)-dependent maternal mRNA. grau encodes a member of the C2H2-type zinc-finger protein family and activates transcription of cort to induce the completion of female meiosis. In addition, a recent study reported that a small cell-cycle regulator, Cks30A, plays an essential role in meiotic progression by associating with Cdk1 (Cdc2)/cyclin complexes and mediating Cyclin A degradation in the female germline. Therefore, cell-cycle regulators involved in meiotic progression are likely to be conserved between vertebrates and Drosophila (Takeo, 2006).

The involvement of calcineurin signaling in female meiosis has not previously been described in any organism. These studies on sra are the first to demonstrate that regulation of a Ca²⁺-dependent phosphatase is critical for the progression of female meiosis. Analyses of mutations in calcineurin genes and identification of the substrates in germline cells should facilitate further understanding of the role of calcineurin signaling in female meiosis (Takeo, 2006).

Calcineurin isoforms are involved in Drosophila Toll immune signaling

Because excessive or inadequate responses can be detrimental, immune responses to infection require appropriate regulation. Networks of signaling pathways establish versatility of immune responses. Drosophila melanogaster is a powerful model organism for dissecting conserved innate immune responses to infection. For example, the Toll pathway, which promotes activation of NF-kappaB transcription factors Dorsal/Dorsal-related immune factor (Dif), was first identified in Drosophila. Together with the IMD pathway, acting upstream of NF-kappaB transcription factor calcineurin A1, acts on Relish during infection. However, it is not known whether there is a role for calcineurin in Dorsal/Dif immune signaling. This article demonstrates involvement of specific calcineurin isoforms, protein phosphatase at 14D (Pp2B-14D)/calcineurin A at 14F (CanA-14F), in Toll-mediated immune signaling. These isoforms do not affect IMD signaling. In cell culture, pharmacological inhibition of calcineurin or RNA interference against homologous calcineurin isoforms Pp2B-14D/CanA-14F, but not against isoform calcineurin A1, decreased Toll-dependent Dorsal/Dif activity. A Pp2B-14D gain-of-function transgene promoted Dorsal nuclear translocation and Dorsal/Dif activity. In vivo, Pp2B-14D/CanA-14F RNA interference attenuated the Dorsal/Dif-dependent response to infection without affecting the Relish-dependent response. Altogether, these data identify a novel input, calcineurin, in Toll immune signaling and demonstrate involvement of specific calcineurin isoforms in Drosophila NF-kappaB signaling (Li, 2014).

Bidirectional regulation of Amyloid precursor protein-induced memory defects by Nebula/DSCR1: A protein upregulated in Alzheimer's disease and Down syndrome

Aging individuals with Down syndrome (DS) have an increased risk of developing Alzheimer's disease (AD), a neurodegenerative disorder characterized by impaired memory. Memory problems in both DS and AD individuals usually develop slowly and progressively get worse with age, but the cause of this age-dependent memory impairment is not well understood. This study examines the functional interactions between Down syndrome critical region 1 (DSCR1) and Amyloid-precursor protein (APP), proteins upregulated in both DS and AD, in regulating memory. Using Drosophila as a model, this study found that overexpression of nebula (fly homolog of DSCR1) initially protects against APP-induced memory defects by correcting calcineurin and cAMP signaling pathways but accelerates the rate of memory loss and exacerbates mitochondrial dysfunction in older animals. Transient upregulation of Nebula/DSCR1 or acute pharmacological inhibition of calcineurin in aged flies protected against APP-induced memory loss. These data suggest that calcineurin dyshomeostasis underlies age-dependent memory impairments and further imply that chronic Nebula/DSCR1 upregulation may contribute to age-dependent memory impairments in AD in DS (Shaw, 2015).

Down syndrome (DS), due to full or partial triplication of chromosome 21, greatly increases the risk of Alzheimer's disease (AD). By age 65, ~75% of DS individuals will develop dementia as compared to 13% of age-matched controls. Despite an early presence of the neurochemical changes seen in AD brains, dementia is delayed in most DS individuals until after mid-life, suggesting both a genetic risk for dementia and the existence of a neuroprotective period before the onset of memory impairments. The mechanism underlying this age-dependent memory decline is poorly understood, but the well known connection between DS and AD provides a unique opportunity to identify common genetic factors contributing to AD and age-associated dementia (Shaw, 2015).

To uncover mechanisms underlying age-dependent memory decline in AD and DS, this study examined the functional interactions between two genes encoded by chromosome 21 and upregulated in both DS and AD. The amyloid precursor protein (App), encoded by chromosome 21, is a known risk gene for AD because either mutations or duplication of App is associated with familial AD. Studies have shown that overexpression of the wild-type human APP in both mouse and Drosophila causes cognitive deficits before β-amyloid accumulation, suggesting that APP perturbation could contribute to dementia independent of β-amyloid plaques. Another gene encoded by chromosome 21 that is likely to play a crucial role in AD is the Down syndrome critical region 1 (Dscr1; also known as Rcan-1) gene. Postmortem brains from both DS and AD patients show an upregulation of DSCR1 mRNA and protein levels. Oxidative stress, APP upregulation, and β-amyloid exposure have also been shown to induce DSCR1 upregulation. DSCR1 encodes an evolutionarily conserved inhibitor of calcineurin, a serine/threonine calcium/calmodulin phosphatase important for numerous physiological pathways, including memory, cell death, and immunity. Studies have shown that altering levels of DSCR1 in mouse and Nebula (fly homolog of DSCR1) in Drosophila severely impaired memory. However, upregulation of Nebula/DSCR1 has been shown to both promote and inhibit cell survival after oxidative stress, as well as protect against APP-induced neurodegeneration and axonal transport defects. Thus, it remains unknown how Nebula/DSCR1 upregulation will affect APP-induced memory defects (Shaw, 2015).

Drosophila and humans share conserved cell signaling components and pathways essential for learning and memory formation, thus providing an effective model system for studying mechanisms contributing to age-dependent memory impairments and neurological disorders. Drosophila has also been used successfully as a model system to investigate mechanisms underlying various neurological disorders. Using Drosophila, this study shows that overexpression of nebula rescued memory impairments induced by APP upregulation through inhibition of calcineurin. These protective effects did not persist during aging, and Nebula co-upregulation instead accelerated age-dependent memory impairments, increased reactive oxygen species (ROS), and enhanced mitochondrial dysfunctions in aged flies. Furthermore, transient upregulation of Nebula or acute pharmacological inhibition of calcineurin in aged flies was sufficient to restore APP-induced memory loss. These findings suggest that Nebula/DSCR1 upregulation may contribute to progressive dementia by initially rescuing APP-induced memory loss but accelerating the rate of memory impairment in older animals (Shaw, 2015).

These findings reveal a complex and novel role for Nebula/DSCR1 upregulation in regulating APP-induced memory loss during aging. First, it was shown that upregulation of Nebula initially protects against APP-induced memory impairments by restoring calcineurin-mediated signaling in young flies. Second, persistent upregulation of Nebula was found to contribute to the poor memory performance of APP and Nebula flies during aging. Third, aging is accompanied by elevations in calcineurin activity, and acute inhibition of calcineurin can improve the memory performance of older control and APP overexpressing flies. Together, these results suggest that Nebula/DSCR1 upregulation may delay the onset of memory loss but contribute to progressive dementia in older individuals with DS. Therefore, this study has wide implications for memory loss during natural aging and in AD and DS and shines light on restoring calcineurin or regulating Nebula/DSCR1 levels as potential therapeutic strategies for age-dependent memory loss (Shaw, 2015).

Nebula/DSCR1 is a multifunctional protein that inhibits calcineurin and modulates mitochondria function and oxidative stress response. Previous reports have indicated that upregulation of either Nebula/DSCR1 or APP alone impaired memory. Therefore, it is unexpected that co-upregulation of Nebula and APP restored both STM and LTM of young flies. Such results were confirmed using two different mushroom body drivers: C739-Gal4 and MB-GeneSwitch-Gal4. The use of mushroom body drivers is advantageous because it circumvents the problem of locomotor defects associated with pan-neuronal APP overexpression, and the Drosophila mushroom bodies has been shown to be structures important for memory retrieval, a process disrupted in AD-related memory loss. The current biochemical and behavioral data indicate that Nebula rescues memory loss by correcting APP-induced calcineurin hyperactivation, as well as deficits in PKA activity and CREB phosphorylation. These results are consistent with the finding that a fine balance in calcineurin and PKA signaling are crucial for normal memory. However, genetics and behavioral data indicate that restoring GSK-3β hyperactivation in APP overexpressing flies, shown previously to rescue axonal transport defects, is not sufficient to rescue the memory deficits. Furthermore, because APP and Nebula overexpressing flies restored STM despite the presence of mitochondrial dysfunction, the data highlight that correcting calcineurin disturbances in younger flies is more beneficial for memory than restoring mitochondrial dysfunction (Shaw, 2015).

Age-associated memory impairment occurs in many species ranging from Drosophila to humans; understanding mechanisms contributing to this process may provide useful insights into changes responsible for dementia in age-related neurological disorders such as AD. The current data provide two important revelations concerning cellular changes contributing to age-dependent memory decline. First, the finding highlight that elevation in calcineurin activity is a previously unidentified mechanism contributing to memory decline during natural aging in Drosophila. This is supported by biochemical data showing increases in calcineurin activity during aging, as well as behavioral data illustrating that transient pharmacological inhibition of calcineurin can significantly improve the memory performance of old wild-type flies. Second, chronic upregulation of Nebula also triggers severe mitochondrial dysfunction that can override the protective effect of calcineurin inhibition by Nebula in flies overexpressing APP, implying that long-term Nebula upregulation may contribute to memory loss in APP overexpressing flies during aging. By measuring ATP content and ROS levels within the fly brain, this study showed that chronic Nebula overexpression both on its own or in the presence of APP significantly exacerbated mitochondrial dysfunction and elevated ROS. Conversely, short-term upregulation of APP and Nebula in aged flies or transient pharmacological inhibition of calcineurin in older flies with chronic APP overexpression both resulted in normal STM performance compared with age-matched control. These results support the notion that chronic Nebula upregulation during aging enhances age-dependent memory impairments in flies with APP overexpression and further suggest that proper mitochondrial function plays an important role in memory preservation in older flies. This interpretation is supported by a report that STM of older flies is particularly sensitive to mutations that elevate ROS, whereas the STM of younger flies is not affected by ROS elevation (Shaw, 2015).

It is proposed that Nebula/DSCR1 upregulation plays a two-pronged role in regulating APP-induced phenotypes in DS. Nebula/DSCR1 upregulation initially protects against APP-induced memory loss by correcting calcineurin-mediated signaling, but chronic Nebula/DSCR1 overexpression triggers severe mitochondrial dysfunction and ROS elevation that potentially leads to rapid decline in memory during aging in DS. Interestingly, β-amyloid has been shown to trigger upregulation of DSCR1, and DSCR1 upregulation is also associated with tau hyperphosphorylation. It will be particularly interesting in the future to study the effects of Nebula/DSCR1 in modifying β-amyloid and tau-associated memory impairments and to test whether preventing mitochondrial dysfunction and ROS elevations in older animals while correcting calcineurin signaling could alleviate memory problems associated with Nebula/DSCR1 and APP overexpression as seen in some cases of DS and AD (Shaw, 2015).

DEVELOPMENTAL BIOLOGY

Galactokinase is a novel modifier of calcineurin-induced Cardiomyopathy in Drosophila

Activated/uninhibited calcineurin is both necessary and sufficient to induce cardiac hypertrophy, a condition that often leads to dilated cardiomyopathy, heart failure, and sudden cardiac death. Constitutively active calcineurin was expressed in the adult heart of Drosophila melanogaster and enlarged cardiac chamber dimensions and reduced cardiac contractility were identified. In addition, expressing constitutively active calcineurin in the fly heart using the Gal4/UAS system induced an increase in heart wall thickness. A targeted genetic screen was performed for modifiers of calcineurin-induced cardiac enlargement based on previous calcineurin studies in the fly and galactokinase was identified as a novel modifier of calcineurin-induced cardiomyopathy. Genomic deficiencies spanning the galactokinase locus, transposable elements that disrupt galactokinase, and cardiac-specific RNAi knockdown of galactokinase suppressed constitutively active calcineurin-induced cardiomyopathy. In addition, in flies expressing constitutively active calcineurin using the Gal4/UAS system, a transposable element in galactokinase suppressed the increase in heart wall thickness. Lastly, genetic disruption of galactokinase suppressed calcineurin-induced wing vein abnormalities. Collectively, this study has generated a model for discovering novel modifiers of calcineurin-induced cardiac enlargement in the fly and has identified galactokinase as a previously unknown regulator of calcineurin-induced cardiomyopathy in adult Drosophila (Lee, 2014).

EFFECTS OF MUTATION

A dominant modifier screen in the Drosophila eye was performed using an activated form of the calcineurin catalytic subunit to identify new targets, regulators, and functions of calcineurin. The canA^act.gl rough eye phenotype is modifiable; i.e., it is sensitive to transgene dose and is specifically modified by CanB, which is essential for CanA function. An isogenic wild-type stock was prepared and canA^act.gl was inserted onto the chromosome 3 balancer TM3, which carries the dominant visible marker Sb. 70,000 progeny of TM3-canA^act.gl (TCAG) females and EMS- or X-ray-treated males were screened. Each individual F₁ with an enhanced or suppressed TCAG rough eye was backcrossed to TCAG to confirm the modification and to determine the chromosomal location. About 21% of the modifiers initially isolated bred true, and stable lines of the confirmed modifiers were established over either TCAG or the chromosome 2 balancer CCAG (CyO-canA^act.gl). Because chromosome 2 balancers, including CyO, harbor one or more suppressors of the TCAG phenotype, chromosome 2 suppressors were difficult to balance and are thus underrepresented in the final tally. Modifiers on chromosome 1 were also underrepresented, in part because the balancers carry a dominant eye mutation, Bar, that significantly interfered with scoring TCAG modification. A total of 5 viable and 123 lethal modifiers were isolated in the screen (Sullivan, 2002).

Modifier groups that act directly on the glass enhancer do not specifically modify canA^act.gl. Nonspecific groups were identified by testing whether any of the complementation groups modified rough eyes caused by unrelated, glass-dependent transgenes. Two of the enhancer groups, CE3-1 and CE2-1, modified rough eye phenotypes caused by other glass-dependent transgenes, such as sina^gl, a Ras pathway component, and Rho1^gl. All complementation groups were additionally tested with Ras^v12, phyllopod, yan^act, and reaper glass-dependent transgenes, but only CE3-1 and CE2-1 modified the rough eye phenotypes caused by these transgenes (Sullivan, 2002).

The specific modifier groups were further separated into two classes by determining whether they modified the canA^act.gl rough eye phenotype caused by TCAGB (TM3-canA^act.gl,canB^gl). Class I genes, such as Ca²⁺ signaling components or dephosphorylation targets, act downstream of calcineurin and will modify the rough eye phenotype of TCAG and TCAGB. However, class II groups, which act at the level of canB, such as canB or factors that regulate its expression, will modify TCAG but not TCAGB. Only two groups, CE3-3 and CS2-1, failed to modify TCAGB. Class I and class II modifier groups were mapped by meiotic recombination and by failure to complement deficiencies. The results from both methods were used to estimate the cytological map position of each group (Sullivan, 2002).

Meiotic mapping localized the class II group CS2-1 to 44A;50B, and deficiencies refined the region to 42B3;43E18. Polytene chromosome analysis revealed a large deficiency in CS2-1¹²⁸ that uncovered 43E6;44B1. CS2-1⁸⁷ was an inversion with breakpoints at 43A1-2 and 43E13-18. The left breakpoint of CS2-1⁸⁷ fails to complement two independent alleles of prickle (pk), a gene in 43A1 that is required for tissue polarity in the wing, haltere, and notum. However, the other CS2-1 alleles complement pk alleles, the pk mutation in CS2-1⁸⁷ is viable, and pk does not modify TCAG. The right inversion breakpoint, 43E13-18, is lethal when uncovered by deficiencies in the 43E region, and these deficiencies also fail to complement other CS2-1 alleles. Thus, the right breakpoint of the CS2-1⁸⁷ inversion corresponds to the CS2-1 TCAG suppressor (Sullivan, 2002).

One of the canB genes, CanB2, maps to 43E16 and was a strong candidate for CS2-1. The CS2-1¹²⁸ deficiency uncovered canB2, since CS2-1¹²⁸/CyO,cn flies were cn, indicating that the deficiency breaks to the left of cn. PCR on genomic DNA from homozygous CS2-1⁸⁷ flies revealed that the right breakpoint of the insertion occurred between base pair positions -452 and +60, relative to the canB2 start of transcription. The gene on the left side of the breakpoint, cn, was not disrupted in CS2-1⁸⁷, because CS2-1⁸⁷/CyO,cn flies are cn⁺. Additionally, Western blots of homozygous CS2-1⁸⁷, CS2-1¹²⁸, and CS2-1¹⁸⁰ larvae that were probed with canB antibodies revealed that, compared to similarly staged controls, total canB protein levels are reduced in CS2-1 alleles (Sullivan, 2002).

Rescue was carried out using the UAS-GAL4 system on CS2-1¹⁸⁰, which has no detectable lesions aside from canB2 and has a late larval/pupal lethal stage. CS2-1¹⁸⁰/CyO;GAL4^hs flies were crossed to CS2-1¹⁸⁰/CyO;canB-4F^UAS, and the GAL4^hs/canB-4F^UAS progeny were screened for CS2-1¹⁸⁰ (i.e., Cy⁺) animals. Heat shock was not used because basal GAL4^hs activity at 25° induces UAS transgenes at a low level. In three independent crosses, the percentage of CS2-1¹⁸⁰ adults was increased from <0.2% to an average of 11%. Thus, ectopic canB-4F successfully rescues the lethality associated with CS2-1¹⁸⁰ (Sullivan, 2002).

Deficiency mapping localized CS3-3 to 63C6;63E, and the X-ray allele CS3-3¹⁵⁴ had a deletion spanning 63C2-5;63E1-4. The EMS allele CS3-3⁵¹⁸ failed to complement sty^Delta5 and sty^Delta64, which are hypomorphic alleles of sprouty, a gene in 63D1. In addition, the sty alleles were able to suppress TCAG. Sequencing the sty ORF from CS3-3⁵¹⁸ revealed that the codon corresponding to glutamine residue 250 was mutated into a stop codon. This mutation removes one of the two sty homology domains and the cysteine-rich region and is predicted to be nonfunctional. Sprouty is a negative regulator of RTK signaling in Drosophila, including Fgf receptor and Egf receptor signaling. Sprouty protein can bind to the E3 ligase Cbl, the adaptor protein Drk, and Gap1 and has been proposed to facilitate Gap1 inactivation of Ras. A single gene in flies, sprouty, has at least five homologs in mammalian genomes (Sullivan, 2002).

Increased signaling through the Egf receptor, caused by either the presence of ectopic Egfr/Ras signaling components or hypomorphic mutations in negative regulators, results in the development of extra photoreceptors and wing vein material. Conversely, a decrease in Egf receptor signaling reduces both wing vein formation and the number of photoreceptor cells. Ectopic canA^act causes defects in eye and wing vein development consistent with repression of Egfr/Ras signaling. Misexpression of canA^act in the posterior compartment of the developing wing by using GAL4^en results in a truncation of wing veins and a decrease in compartment size. GAL4^sev drives expression in the presumptive R3, R4, and R7 photoreceptor cells, as well as in cone cells. Sections of GAL4^sev/canA^act.UAS eyes reveal a decrease in the number of photoreceptor cells per ommatidium. Usually the missing cell is R7, but occasionally R3 or R4 are also absent. In addition, TCAG and TCAGB discs were examined for a decrease in active MAP kinase levels by using an antibody specific for the diphosphorylated, active form of MAP kinase (Sigma, M8159). However, posterior to the furrow the levels of active MAP kinase were too low to reliably detect any effect of activated calcineurin on signaling output from the Ras pathway (Sullivan, 2002).

To examine the interaction between calcineurin and individual components of the Egfr pathway, the ability of mutations in these components to modify the activated calcineurin phenotype was tested. Hypomorphic mutations in Egfr, Ras, pnt, sty, Gap1, and small wing modified activated calcineurin, although this was not the case for most downstream components of the Egfr pathway. TCAGB is enhanced by removing one copy of Egfr, Ras, or pnt and was suppressed by Gap1 and small wing. Both TCAGB and TCAG suppress the rough eye caused by hypermorphic Egfr alleles: flies that have one copy of Egfr^E1 and TCAGB have a rough eye that closely resembles that of TCAGB alone. TCAG is not detectably modified by hypomorphic Egfr, Ras, or pnt alleles. Aside from CS3-3, none of the modifier groups corresponded to Egf receptor/Ras signaling components that genetically interact with TCAG. However, it is possible that these genes are present among the 61 single hits, which have not been characterized (Sullivan, 2002).

Pan-neuronal knockdown of calcineurin reduces sleep in the fruit fly, Drosophila melanogaster

Sleep is a unique physiological state, which is behaviorally defined, and is broadly conserved across species from mammals to invertebrates such as insects. Because of the experimental accessibility provided by various novel animal models including the fruit fly there have been significant advances in the understanding of sleep. Although the physiological functions of sleep have not been fully elucidated, accumulating evidence indicates that sleep is necessary to maintain the plasticity of neuronal circuits and, hence, is essential in learning and memory. Calcineurin (Cn) is a heterodimeric phosphatase composed of CnA and CnB subunits and known to function in memory consolidation in the mammalian brain, but its neurological functions in the fruit fly are largely unknown. This study shows that Cn is an important regulator of sleep in Drosophila. A pan-neuronal RNA interference-mediated knockdown of Cn expression resulted in sleep loss, whereas misexpression of the constitutively active form of a CnA protein led to increased sleep. Furthermore, CnA knockdown also impaired the retention of aversive olfactory memory. These results indicate a role for Cn and calcium-dependent signal transduction in sleep and memory regulation and may bring insight into the relationship between them (Tomita, 2011; full text of article).

EVOLUTIONARY HOMOLOGS

Calcineurin function in yeast and slime mold

The PMC1 gene in S. cerevisiae encodes a vacuolar Ca2+ ATPase required for growth in high-Ca²⁺ conditions. Previous work has shown that Ca²⁺ tolerance can be restored to pmc1 mutants by inactivation of calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase sensitive to the immunosuppressive drug FK506. Calcineurin decreases Ca²⁺ tolerance of pmc1 mutants by inhibiting the function of VCX1, which encodes a vacuolar H+/Ca²⁺ exchanger related to vertebrate Na+/Ca²⁺ exchangers. The contribution of VCX1 in Ca²⁺ tolerance is low in strains with a functional calcineurin and is high in strains which lack calcineurin activity. In contrast, the contribution of PMC1 to Ca²⁺ tolerance is augmented by calcineurin activation. Consistent with these positive and negative roles of calcineurin, expression of a vcx1::lacZ reporter is slightly diminished and a pmc1::lacZ reporter is induced up to 500-fold by processes dependent on calcineurin, calmodulin, and Ca²⁺. It is likely that calcineurin inhibits VCX1 function mainly by posttranslational mechanisms. Activities of VCX1 and PMC1 help to control cytosolic free Ca²⁺ concentrations because their function can decrease pmc1::lacZ induction by calcineurin. Additional studies with reporter genes and mutants indicate that PMR1 and PMR2A, encoding P-type ion pumps required for Mn2+ and Na+ tolerance, may also be induced physiologically in response to high-Mn2+ and -Na+ conditions through calcineurin-dependent mechanisms. In these situations, inhibition of VCX1 function may be important for the production of Ca²⁺ signals. It is proposed that elevated cytosolic free Ca²⁺ concentrations, calmodulin, and calcineurin regulate at least four ion transporters in S. cerevisiae in response to several environmental conditions (Cunningham, 1996).

S. cerevisiae mutants that exhibit phenotypes (calcium resistance and vanadate sensitivity) similar to those of calcineurin-deficient mutants were classified into four complementation groups (crv1,2,3 and 4). Crv1 is allelic to cnb1, a mutation in the regulatory subunit of calcineurin. The nucleotide sequences of mutant crv2 are identical to those of BCK1/SLK1/SKC1/SSP31 and the sequences of mutant crv3 match those of MPK1/SLT2; both genes are involved in the MAP kinase cascade. A calcineurin-deletion mutation (delta cnb1), which by itself has no detectable effect on growth and morphology, enhances some phenotypes (slow growth and morphological abnormality) of crv2 and crv3 mutants. The phenotypes of crv2 and crv3 mutants are partially suppressed by Ca²⁺ or by overproduction of the calcineurin subunits (Cmp2 and Cnb1). Like the calcineurin-deficient mutant, crv2 and crv3 mutants are defective in recovery from alpha-factor-induced growth arrest. The defect in recovery of the delta cnb1 mutant is suppressed by overexpression of MPK1. These results indicate that the calcineurin-mediated and the Mpk1- (Bck1-) mediated signaling pathways act in parallel to regulate functionally redundant cellular events important for growth (Nakamura, 1996).

The catalytic subunit of Ca²⁺/calmodulin(CaM)-dependent protein phosphatase (calcineurin A, protein phosphatase 2B) was isolated from Dictyostelium discoideum. A complete cDNA of 2146 bp predicts a protein of 623 amino acids with homology to calcineurin A from other organisms and a similar molecular architecture. However, the Dictyostelium protein contains N-terminal and C-terminal extra domains causing a significantly higher molecular mass than found in any of its known counterparts. Recombinant Dictyostelium calcineurin A was purified from Escherichia coli cells and shows similar enzymatic properties as does the enzyme from other sources. A band of approximately 80 kDa migrates on gels and possesses an endogenous CaM-binding activity. Both the mRNA for calcineurin A and the protein are expressed during the growth phase. During early development the abundance of the protein is reduced and then increases to peak after 10 h of starvation, when tight aggregates have formed (Dammann, 1996).

Calcineurin is a Ca2+/calmodulin-regulated protein phosphatase required for Saccharomyces cerevisiae to respond to a variety of environmental stresses. Calcineurin promotes cell survival during stress by dephosphorylating and activating the Zn-finger transcription factor Crz1p/Tcn1p. Using a high-throughput assay, 119 yeast kinases were screened for their ability to phosphorylate Crz1p in vitro; the casein kinase I homolog Hrr25p was identifed. Hrr25p negatively regulates Crz1p activity and nuclear localization in vivo. Hrr25p binds to and phosphorylates Crz1p in vitro and in vivo. Overexpression of Hrr25p decreases Crz1p-dependent transcription and antagonizes its Ca2+-induced nuclear accumulation. In the absence of Hrr25p, activation of Crz1p by Ca2+/calcineurin is potentiated. These findings represent the first identification of a negative regulator for Crz1p, and establish a novel physiological role for Hrr25p in antagonizing calcineurin signaling (Kafadar, 2003).

Cryptococcus neoformans is a fungal pathogen that causes meningitis in immunocompromised patients. Its growth is sensitive to the immunosuppressants FK506 and cyclosporin, which inhibit the Ca²⁺- calmodulin-activated protein phosphatase calcineurin. Calcineurin is required for growth at 37°C and the virulence of C. neoformans. Calcineurin is also required for mating. FK506 blocks mating of C. neoformans via FKBP12-dependent inhibition of calcineurin, and mutants lacking calcineurin are bilaterally sterile. Calcineurin is not essential for the initial fusion event, but is required for hyphal elongation and survival of the heterokaryon produced by cell fusion. It is also required for hyphal elongation in diploid strains and during asexual haploid fruiting of MATalpha cells in response to nitrogen limitation. Because mating and haploid fruiting produce infectious basidiospores, these studies suggest a second link between calcineurin and the virulence of C. neoformans. Calcineurin regulates filamentation and 37°C growth via distinct pathways. Together with studies revealing that calcineurin mediates neurite extension and neutrophil migration in mammals, these findings indicate that calcineurin plays a conserved role in the control of cell morphology (Cruz, 2001).

The Caenorhabditis elegans homologue of Down's syndrome critical region 1, RCN-1, inhibits multiple functions of the phosphatase calcineurin

A conserved family of calcineurin-regulating proteins whose members have been implicated in several disease models such as Down's syndrome, Alzheimer's disease, and cardiac hypertrophy has been identified in several organisms including yeast, mice, and humans. Caenorhabditis elegans rcn-1 belongs to this family of calcineurin regulators, and shows approximately 40% identity with the human homologue DSCR-1. rcn-1 is expressed in hypodermal cells, nerve cords and various neurons, vulva epithelial and muscle cells, marginal cells of the pharynx, and structures of the male tail. rcn-1 expression is upregulated by calcineurin activity. RCN-1 binds to calcineurin A from C.elegans lysate in a calcium-dependent manner, and inhibits bovine calcineurin phosphatase activity dose-dependently. In addition, overexpression of RCN-1 results in calcineurin-deficient phenotypes such as small body size, cuticle defects, fertility defects, slow growth, and serotonin-resistant egg-laying defects. Moreover, phenotypes observed in gain-of-function calcineurin mutant animals were restored to normal by RCN-1 overexpression. These results demonstrate an effective and specific inhibition of calcineurin in vitro as well as in vivo by RCN-1 (Lee, 2003).

Calcineurin regulates coordinated outgrowth of zebrafish regenerating fins

Vertebrates develop organs and appendages in a proportionally coordinated manner, and animals that regenerate them do so to the same dimensions as the original structures. Coordinated proportional growth involves controlled regulation between allometric and isometric growth programs, but it is unclear what executes this control. Calcineurin inhibition results in continued allometric outgrowth of regenerating fins beyond their original dimensions. Calcineurin inhibition also maintains allometric growth of juvenile fins and induces it in adult fins. Furthermore, calcineurin activity is low when the regeneration rate is highest, and its activity increases as the rate decreases. Growth measurements and morphometric analysis of proximodistal asymmetry indicate that calcineurin inhibition shifts fin regeneration from a distal growth program to a proximal program. This shift is associated with the promotion of retinoic acid signaling. Thus, this study has identified a calcineurin-mediated mechanism that operates as a molecular switch between position-associated isometric and allometric growth programs (Kujawski, 2014).

Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB

Activating AMPK or inactivating calcineurin slows ageing in C. elegans and both have been implicated as therapeutic targets for age-related pathology in mammals. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs; see Drosophila Crtc) are a family of cofactors involved in diverse physiological processes including energy homeostasis, cancer and endoplasmic reticulum stress. This study shows that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. CRTC-1 is a direct AMPK target (See Drosophila SNF1A/AMP-activated protein kinase), and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity (Mair, 2011).

These data indicate that CRTC-1 is the critical direct longevity target of both AMPK and calcineurin in C. elegans and identify a new role for CRTCs and CREB in modulating longevity. They also represent the first analysis of the transcriptional profiles of long-lived activated AMPK and deactivated calcineurin organisms and suggest the primary longevity-associated role of these perturbations is the modulation of CRTC-1 and CRH-1 transcriptional activity. Notably, both the FOXO transcription factor daf-16 and genes involved in autophagy have also been implicated in AMPK and calcineurin longevity, respectively. Further work to determine precisely where the AMPK-calcineurin-CRTC-1 pathway converges with FOXO and autophagy will be enlightening. It will also be interesting to determine if CRTC-1 mediates downstream effects of kinases other than AMPK. In mammals, CRTCs are regulated by multiple CAMKL kinase family members, and additive effects are seen of AMPK and related kinases on the localization of CRTC-1, in particular the MAP/microtubule affinity-regulating kinase (MARK) par-1, indicating that this kinase may also regulate CRTC-1 in vivo. At present, however, AMPK is the only CAMKL kinase shown to be a positive regulator of longevity (Mair, 2011).

Collectively, these data identify CRTC-1 as a central node linking the upstream lifespan modifiers AMPK and calcineurin to CREB activity via a shared signal-transduction pathway, and demonstrate that post-translational modification of CRTC-1 is required for their effects on longevity. Complementing the pro-longevity effects of inhibiting CRTC function in C. elegans, reducing components of the CRTC/CREB pathway has recently been shown to confer health benefits to mice. Given the evolutionary conservation of this pathway from C. elegans to mammals it will be fascinating to determine the role of CRTCs both as mammalian ageing modulators and as potential drug targets for patients with metabolic disorders and cancer (Mair, 2011).

Calcineurin protein interactions

The protein phosphatase activity of calcineurin (CaN) is activated through calcium binding to both calmodulin and the B subunit of CaN. The purpose of this study was to determine which domain(s) in the CaN B subunit is required for either binding to the CaN A subunit or for transducing the effects of B subunit Ca²⁺ binding to the stimulation of the CaN A subunit phosphatase activity. Interaction of CaN B regulatory subunit with the CaN A catalytic subunit requires hydrophobic residues within the CaN A sequence 328-390. Selected hydrophobic residues within the B subunit were mutated to Glu to Gln. CaN B subunit mutants BE-1 (Val115/Leu116 to Glu), BE-2 (Val156/157/168/169 to Glu), and BQ-2 (Val156/157/168/169 to Gln) were expressed and purified. The three mutant B subunits bind 45Ca²⁺ normally. Mutants BE-2 and BQ-2 interact with a GST fusion protein containing the B subunit binding domain of the CaN A subunit (residues 328-390); they stimulate the phosphatase activity of the CaN A subunit. Mutant BE-1 has a 3-fold reduced affinity for binding CaN A, and this mutant, even at saturating concentrations, gives very little stimulation of CaN A phosphatase activity. It is concluded that residues Val115/Leu116 in the B subunit participate in high-affinity binding to the A subunit and are required for transducing the effects of B subunit Ca²⁺ binding in stimulation of CaN A phosphatase activity (Watanabe, 1996).

Using a genetic screen in yeast, a new family of proteins conserved in fungi and animals has been identified that inhibits calcineurin function when overexpressed. Overexpression of the yeast protein Rcn1p or the human homologs DSCR1 or ZAKI-4 inhibits two independent functions of calcineurin in yeast -- the activation of the transcription factor Tcn1p and the inhibition of the H+/Ca²⁺ exchanger Vcx1p. Purified recombinant Rcn1p and DSCR1 binds calcineurin in vitro and inhibits its protein phosphatase activity. Signaling via calmodulin, calcineurin, and Tcn1p induces Rcn1p expression, suggesting that Rcn1p operates as an endogenous feedback inhibitor of calcineurin. Surprisingly, rcn1 null mutants exhibit phenotypes similar to those of Rcn1p-overexpressing cells. This effect may be due to lower expression of calcineurin in rcn1 mutants during signaling conditions. Thus, Rcn1p levels may fine-tune calcineurin signaling in yeast. The structural and functional conservation between Rcn1p and DSCR1 suggests that the mammalian Rcn1p-related proteins, termed calcipressins, will modulate calcineurin signaling in humans and potentially contribute to disorders such as Down Syndrome (Kingsbury, 2000).

Negative regulation of calcineurin signaling

Calcineurin plays a pivotal role in the T cell receptor (TCR)-mediated signal transduction pathway and serves as a common target for the immunosuppressants FK506 and cyclosporin A. A novel endogenous calcineurin binding protein named Cabin 1 has been identified that inhibits calcineurin-mediated signal transduction. The interaction between Cabin 1 and calcineurin is dependent on PKC activation. Overexpression of Cabin 1 or its N-terminal truncation mutants inhibits the transcriptional activation of calcineurin-responsive elements in the interleukin-2 promoter and blocks dephosphorylation of NF-AT upon T cell activation. These results suggest a negative regulatory role for Cabin 1 in calcineurin signaling and provide a possible mechanism of feedback inhibition of TCR signaling through cross-talk between protein kinases and calcineurin (Sun, 1998).

Structure of mammalian calcipressin/DSCR1 and interaction with calcineurin

Calcipressin 1 is an endogenous inhibitor of calcineurin, which is a serine/threonine phosphatase under the control of Ca(2+) and calmodulin. Calcipressin 1 is encoded by DSCR1, a gene on human chromosome 21 with seven exons, exons 1-4 are alternative first exons (isoforms 1-4). Calcipressin 1 isoform 1 has an N-terminal coding region longer than that previously described, and this generates a new polypeptide of 252 amino acids. This polypeptide is able to interact with calcineurin A and to inhibit NF-AT-mediated transcriptional activation. Endogenous calcipressin 1 exists as a complex together with the calcineurin A and B heterodimer. Calcipressin 1 is a phosphoprotein that increases its capacity to inhibit calcineurin when phosphorylated at the FLISPP motif, and this phosphorylation also controls the half-life of calcipressin 1 by accelerating its degradation. Additionally, further phosphorylation sites have been detected outside the FLISPP motif and these contribute to the complex phosphorylation pattern of calcipressin 1. Taking these results into consideration it is suggested that phosphorylation of calcipressin 1 is involved in the regulation of the phosphatase activity of calcineurin and can therefore act as a modulator of calcineurin-dependent cellular pathways (Genesca, 2003).

Down's syndrome is one of the major causes of mental retardation and congenital heart malformations. Other common clinical features of Down's syndrome include gastrointestinal anomalies, immune system defects and Alzheimer's disease pathological and neurochemical changes. The most likely consequence of the presence of three copies of chromosome 21 is the overexpression of its resident genes, a fact which must underlie the pathogenesis of the abnormalities that occur in Down's syndrome. DSCR1, the product of a chromosome 21 gene highly expressed in brain, heart and skeletal muscle, is overexpressed in the brain of Down's syndrome fetuses, and interacts physically and functionally with calcineurin A, the catalytic subunit of the Ca(2+)/calmodulin-dependent protein phosphatase PP2B. The DSCR1 binding region in calcineurin A is located in the linker region between the calcineurin A catalytic domain and the calcineurin B binding domain, outside of other functional domains previously defined in calcineurin A. DSCR1 belongs to a family of evolutionarily conserved proteins with three members in humans: DSCR1, ZAKI-4 and DSCR1L2. Overexpression of DSCR1 and ZAKI-4 inhibits calcineurin-dependent gene transcription through the inhibition of NF-AT translocation to the nucleus. Together, these results suggest that members of this newly described family of human proteins are endogenous regulators of calcineurin-mediated signaling pathways and as such, they may be involved in many physiological processes (Fuentes, 2000).

Calcineurin phosphatase activity regulates the nuclear localization of the nuclear factor of activated T cells (NFAT) family of transcription factors during immune challenge. Calcineurin inhibitors, such as the cyclosporin A-cyclophilin A and FK506-FKBP12 complexes, regulate this enzymatic activity noncompetitively by binding at a site distinct from the enzyme active site. A family of endogenous protein inhibitors of calcineurin was recently identified and shown to block calcineurin-mediated NFAT nuclear localization and transcriptional activation. One such inhibitor, Down's Syndrome Critical Region 1 (DSCR1), functions in T cell activation, cardiac hypertrophy, and angiogenesis. A small region of DSCR1, the C-terminal 57 residues encoded by exon 7, has been identified is a potent inhibitor of calcineurin activity in vitro and in vivo (Chan, 2005).

Inhibition of the calcineurin-NFAT signalling pathway is one of the main challenges for immunosuppression therapy to avoid the severe side effects of the current anticalcineurinic drugs, cyclosporin A and FK506. The members of the calcipressin family are endogenous inhibitors of calcineurin. Two independent motifs within human calcipressin 1, the ELHA and the PxIxxT motifs, interact with calcineurin in an independent functional manner. The main finding here is that the ELHA-containing calcineurin-inhibitor CALP1 (CIC) motif is the responsible for the in vivo inhibition of calcineurin-mediated NFAT-dependent cytokine gene expression in human T cells. The identification of the CIC motif could be used as a starting point for the development of new immunosuppressive drugs for use in transplantation and autoimmune diseases (Aubereda, 2006).

Calcineurin and NFAT

A new facet of calcium signaling involves the nuclear import of the NF-AT transcription factors from their dormant position in the cytoplasm. The protein phosphatase calcineurin appears to play an essential role in activating NF-AT nuclear import, as the calcineurin inhibitors cyclosporin A and FK506 block dephosphorylation and nuclear import of NF-AT. Calcium signaling induces an association between NF-AT4 and calcineurin; these molecules are transported, as a complex, to the nucleus, where calcineurin continues to dephosphorylate NF-AT4. It is proposed that a nuclear complex of NF-AT4 and calcineurin maintains calcium signaling by counteracting a vigorous nuclear NF-AT kinase (Shibasaki, 1996).

Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response. Two new isoforms of the transcription factor NFAT1 are the predominant isoforms expressed in murine and human T cells. When expressed in Jurkat T cells, recombinant NFAT1 is regulated, as expected, by the calmodulin-dependent phosphatase calcineurin, and its function is inhibited by the immunosuppressive agent cyclosporin A (CsA). Transactivation by recombinant NFAT1 in Jurkat T cells requires dual stimulation with ionomycin and phorbol ester; this activity is potentiated by coexpression of constitutively active calcineurin and is inhibited by CsA. Immunocytochemical analysis indicates that recombinant NFAT1 localizes in the cytoplasm of transiently transfected T cells and translocates into the nucleus in a CsA-sensitive manner following ionomycin stimulation. When expressed in COS cells, however, NFAT1 is capable of transactivation, but it is not regulated correctly: its subcellular localization and transcriptional function are not affected by stimulation of the COS cells with ionomycin and phorbol. Recombinant NFAT1 can mediate transcription of the interleukin-2, interleukin-4, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor promoters in T cells, suggesting that NFAT1 contributes to the CsA-sensitive transcription of these genes during the immune response (Luo, 1996).

It is thought that inositol-1,4,5-trisphosphate-Ca²⁺ signaling has a function in dorsoventral axis formation in Xenopus embryos; however, the immediate target of free Ca²⁺ is unclear. The secreted Wnt protein family comprises two functional groups, the canonical Wnt and Wnt/Ca²⁺ pathways. The Wnt/Ca²⁺ pathway interferes with the canonical Wnt pathway, but the underlying molecular mechanism is poorly understood. The complementary DNA coding for the Xenopus homolog of nuclear factor of activated T cells (XNF-AT) was cloned. A gain-of-function, calcineurin-independent active XNF-AT mutation (CA XNF-AT) inhibits anterior development of the primary axis, as well as Xwnt-8-induced ectopic dorsal axis development in embryos. A loss-of-function, dominant negative XNF-AT mutation (DN XNF-AT) induces ectopic dorsal axis formation and expression of the canonical Wnt signaling target molecules siamois and Xnr3. Xwnt-5A induces translocation of XNF-AT from the cytosol to the nucleus. These data indicate that XNF-AT functions as a downstream target of the Wnt/Ca²⁺ and Ins(1,4,5)P³-Ca²⁺ pathways, and has an essential role in mediating ventral signals in the Xenopus embryo through suppression of the canonical Wnt pathway (Saneyoshi, 2002).

Intracellular calcium is one of the important signals that initiates the myogenic program. The calcium-activated phosphatase calcineurin is necessary for the nuclear import of the nuclear factor of activated T cell (NFAT) family members, which interact with zinc finger GATA transcription factors. Whereas GATA-6 plays a role in the maintenance of the differentiated phenotype in vascular smooth muscle cells (VSMCs), it is unknown whether the calcineurin pathway is associated with GATA-6 and plays a role in the differentiation of VSMCs. The smooth muscle-myosin heavy chain (Sm-MHC) gene is a downstream target of GATA-6, and provides a highly specific marker for differentiated VSMCs. Using immunoprecipitation Western blotting, it has been shown that NFATc1 interacts with GATA-6. Consistent with this, NFATc1 further potentiates GATA-6-activated Sm-MHC transcription. Induction of VSMCs to the quiescent phenotype causes nuclear translocation of NFATc1. In differentiated VSMCs, blockage of calcineurin down-regulates the amount of GATA-6-DNA binding as well as the expression of Sm-MHC and its transcriptional activity. These findings demonstrate that the calcineurin pathway is associated with GATA-6 and is required for the maintenance of the differentiated phenotype in VSMCs (Wada, 2002).

The Notch and Calcineurin/NFAT pathways have both been implicated in control of keratinocyte differentiation. Induction of the p21^WAF1/Cip1 gene by Notch 1 activation in differentiating keratinocytes is associated with direct targeting of the RBP-Jκ protein to the p21 promoter. Notch 1 activation functions also through a second Calcineurin-dependent mechanism acting on the p21 TATA box-proximal region. Increased Calcineurin/NFAT activity by Notch signaling involves downregulation of Calcipressin (see Drosophila Sarah), an endogenous Calcineurin inhibitor, through a HES-1-dependent mechanism. Besides control of the p21 gene, Calcineurin contributes significantly to the transcriptional response of keratinocytes to Notch 1 activation, both in vitro and in vivo. In fact, deletion of the Calcineurin B1 gene in the skin results in a cyclic alopecia phenotype, associated with altered expression of Notch-responsive genes involved in hair follicle structure and/or adhesion to the surrounding mesenchyme. Thus, an important interconnection exists between Notch 1 and Calcineurin-NFAT pathways in keratinocyte growth/differentiation control (Mammucari, 2005).

Levels of extra- and intra-cellular calcium play a major role in keratinocyte growth/differentiation control, and the calcium/Calmodulin-dependent phosphatase Calcineurin has been implicated in this process. Calcineurin is the only known serine/threonine phosphatase under calcium/calmodulin control. Among the proteins that are dephosphorylated as a consequence of Calcineurin activation are the nuclear factors of activated T cells (NFATs). Increased Calcineurin activity promotes the localization of NFATs to the nucleus, and its effect is counteracted by the phosphorylation of these factors by a number of both constitutive and inducible kinases such as GSK3, CK1, p38, and JNK1. Such a complexity of regulation is reflected by the fact that induction of NFAT-dependent transcription by Calcineurin activation is not immediately associated with increases in intracellular calcium levels, but requires a prolonged stimulus consistent with an oscillatory and accumulative mechanism of NFAT dephosphorylation and nuclear translocation (Mammucari, 2005).

Studies on the biological function of Calcineurin have been greatly facilitated by the use of the inhibitory drugs Cyclosporin A (CsA) and FK506. Several endogenous Calcineurin inhibitors have also been reported. Among these is Calcipressin (CALP1), also known as the DSCR1 gene product, located in the Down Syndrome Critical Region of human chromosome 21 and mouse chromosome 16. This protein binds directly to the CnA subunit and inhibits its activity. Importantly, Calcipressin gene expression is under direct positive control of Calcineurin/NFAT activity, so that this protein is thought to function as a feedback inhibitor of Calcineurin signaling, with an impact on T cell activation as well as the response to different stress stimuli in cardiac hypertrophy (Mammucari, 2005).

The function of Calcineurin has been elucidated in great detail in T cells, but has also been studied in the hematopoietic, neuronal, myogenic, and vascular systems. Calcineurin/NFAT activity has also been directly implicated in keratinocyte growth/differentiation control and, in vivo, in control of the hair cycle. Molecular analysis of the role of this pathway in keratinocytes has focused on control of p21 gene transcription. Induction of p21^(WAF1/Cip1) is one of the earliest regulatory events associated with keratinocyte differentiation, contributing to withdrawal from the cell cycle. In mouse primary keratinocytes, p21 expression is induced by increased extracellular calcium, and the responsive region of the p21 promoter maps to a 78 bp GC-rich region close to the TATA box, containing six Sp1/Sp3 binding sites. Calcineurin induces activation of this promoter through the Calcineurin-dependent association of NFAT with the transcription factors Sp1/Sp3 (Mammucari, 2005).

Notch 1 activation induces p21 transcription not only through direct binding of the RBP-Jκ protein to the p21 promoter, but also through the calcium/Calcineurin-responsive TATA box-proximal region. Underlying this effect, induction of Calcineurin/NFAT activity by Notch signaling involves downregulation of Calcipressin, in opposition to positive control of this gene by Calcineurin/NFAT itself. Besides control of p21 expression, Calcineurin signaling plays a significantly broader role in the transcriptional response of keratinocytes to Notch 1 activation. In particular, inducible deletion of the CnB1 gene in the skin causes a cyclic alopecia phenotype that is linked to altered expression of several Notch-responsive genes involved in hair follicle structure and adhesion to the surrounding mesenchyme (Mammucari, 2005).

Spatial and temporal regulation of coronary vessel formation by calcineurin-NFAT signaling

Formation of the coronary vasculature requires reciprocal signaling between endothelial, epicardially derived smooth muscle and underlying myocardial cells. These studies show that calcineurin-NFAT signaling functions in endothelial cells within specific time windows to regulate coronary vessel development. Mouse embryos exposed to cyclosporin A (CsA), which inhibits calcineurin phosphatase activity, failed to develop normal coronary vasculature. To determine the cellular site at which calcineurin functions for coronary angiogenesis, calcineurin was deleted in endothelial, epicardial and myocardial cells. Disruption of calcineurin-NFAT signaling in endothelial cells resulted in the failure of coronary angiogenesis, recapitulating the coronary phenotype observed in CsA-treated embryos. By contrast, deletion of calcineurin in either epicardial or myocardial cells had no effect on coronary vasculature during early embryogenesis. To define the temporal requirement for NFAT signaling, developing embryos were treated with CsA at overlapping windows from E9.5 to E12.5 and coronary development was examined at E12.5. These experiments demonstrated that calcineurin-NFAT signaling functions between E10.5 and E11.5 to regulate coronary angiogenesis. Consistent with these in vivo observations, endothelial cells exposed to CsA within specific time windows in tissue culture were unable to form tubular structures and their cellular responses to VEGF-A were blunted. Thus, these studies demonstrate specific temporal and spatial requirements of NFAT signaling for coronary vessel angiogenesis. These requirements are distinct from the roles of NFAT signaling in the angiogenesis of peripheral somatic vessels, providing an example of the environmental influence of different vascular beds on the in vivo endothelial responses to angiogenic stimuli (Zeini, 2009).

Calcineurin signaling regulates neural induction through antagonizing the BMP pathway

Development of the nervous system begins with neural induction, which is controlled by complex signaling networks functioning in concert with one another. Fine-tuning of the bone morphogenetic protein (BMP) pathway is essential for neural induction in the developing mammalian embryos. However, the molecular mechanisms by which cells integrate the signaling pathways that contribute to neural induction have remained unclear. This study found that neural induction is dependent on the Ca(2+)-activated phosphatase calcineurin (CaN). Fibroblast growth factor (FGF)-regulated Ca(2+) entry activates CaN, which directly and specifically dephosphorylates BMP-regulated Smad1/5 proteins. Genetic and biochemical analyses revealed that CaN adjusts the strength and transcriptional output of BMP signaling and that a reduction of CaN activity leads to an increase of Smad1/5-regulated transcription. As a result, FGF-activated CaN signaling opposes BMP signaling during gastrulation, thereby promoting neural induction and the development of anterior structures (Cho, 2014).

Calcineurin channels and receptors

Chemoattractants stimulate neutrophil migration by activating signaling pathways including repeated transient increases in intracellular free calcium, [Ca²⁺]i. A motile neutrophil sends out many pseudopods, some of which adhere to the substrate; to continue moving forward, the cell must release these attachments. Adhesion can be actively regulated, and neutrophils in which [Ca²⁺]i transients are inhibited become stuck on fibronectin or vitronectin, extracellular matrix proteins that neutrophils encounter in vivo. Function-blocking antibodies to beta 3 integrins or the alpha v beta 3 heterodimer restore motility on vitronectin to [Ca²⁺]i-buffered cells, indicating that an alpha v beta 3-like integrin is responsible for the [Ca²⁺]i-sensitive adhesion. The density of alpha v beta 3 integrins in the adherent membrane of neutrophils migrating on vitronectin is much higher at the leading edge than at the rear, but [Ca²⁺]i buffering or inhibition of Ca²⁺-calmodulin-activated protein phosphatase 2B (calcineurin) leads to accumulation of alpha v beta 3 on the adherent surface at the rear of the cell. The polarized distribution of alpha v beta 3 integrins in migrating neutrophils is maintained by [Ca²⁺]i-dependent release of adhesion followed by endocytosis of these integrins and recycling to the leading edge (Lawson, 1995).

Rat brain sodium channels are phosphorylated at multiple serine residues by cAMP-dependent protein kinase. Soluble rat brain phosphatases have been identified that dephosphorylate purified sodium channels. Five separable forms of sodium channel phosphatase activity have been observed. Three forms (two, approximately 234 kDa and one, 192 kDa) are identical or related to phosphatase 2A. The two major peaks of phosphatase 2A-like activity, A1 and B1, are enriched in either B' or B alpha. The remaining two activities (approximately 100 kDa each) probably represent calcineurin. Each is relatively insensitive to okadaic acid, is active only in the presence of CaCl2 and calmodulin, and contains a 19-kDa polypeptide recognized by a monoclonal antibody raised against the B subunit of calcineurin. Treatment of synaptosomes with okadaic acid to inhibit phosphatase 2A, or with cyclosporin A to inhibit calcineurin, increases apparent phosphorylation of sodium channels at cAMP-dependent phosphorylation sites. These results indicate that both phosphatase 2A and calcineurin dephosphorylate sodium channels in rat brain, and thus may counteract the effect of cAMP-dependent phosphorylation on sodium channel activity (Chen, 1995).

The M current regulates neuronal excitability, with its amplitude resulting from high open probability modal M channel behavior. The M current is affected by changes in intracellular calcium levels. It is proposed that internal calcium acts by regulating M channel modal gating. Intracellular application of a preactivated form of the calcium-dependent phosphatase calcineurin (CaN420) inhibits the macroscopic M current, while its application to excised inside-out patches reduces high open probability M channel activity. Addition of ATP reverses the action of CaN420 on excised patches. The change in M channel gating induced by CaN420 is different from the effect of muscarine. A kinetic model supports the proposition that shifts in channel gating induced by calcium-dependent phosphorylation and dephosphorylation control M current amplitude (Marrion, 1996).

Whole-cell recording in the superficial layers of the developing superior colliculus (sSC) reveals a large drop in NMDA receptor (NMDAR) current decay time synchronized across all neurons and occurring consistently between post-natal (P) day 10 and P11. Blocking the Ca²⁺/calmodulin-dependent phosphatase calcineurin (CaN) in the postsynaptic neuron can abolish this drop. The regulation is induced prematurely by 1-2 hr of electrical stimulation in P10 collicular slices only if CaN and NMDAR currents can be activated in the neuron. These data suggest that a long-lasting, CaN-mediated control of NMDAR kinetics is rapidly initiated by heightened activity of the NMDAR itself and demonstrate a novel developmental and tonic function of CaN that can play an important role in modulating the plasticity of the developing CNS (Shi, 2000).

It is likely that this developmental regulation of the NMDAR serves functions that are qualitatively different from desensitization in more mature brain. For example, the CaN effect on NMDARCs described here is large. During the synaptogenic period from P6-P21, NMDARC decay time decreases by ~18 ms. Close to half of this decrease is caused by the sudden appearance of CaN activity at P11. By the end of the following week, the slower incorporation of the NR2A subunit into sSC synaptic NMDARs could be responsible for the additional shortening of the NMDARC decay time. Rat eyes open on P13-P14, and the sudden increase in visual activity resulting from pattern visual could readily damage collicular neurons if the mechanism of CaN-dependent NMDARC downregulation did not exist. In addition, the rapid downregulation of NMDARC decay time on P11 probably reduces much of the amplification of synaptic function caused by NMDAR activity. This loss of amplification may account for the sustained decreases in spontaneous spiking activity reported in the sSC after P10 by other investigators, and it may also serve to abruptly limit ongoing synapticplasticity in the sSC. In short, this developmental function of CaN appears to be an exceptionally rapid and potent homeostatic mechanism that uses an increase in Ca²⁺ through the NMDAR channel to tonically decrease the potency of the NMDAR posttranslationally. This could maintain a nontoxic level of intracellular free Ca²⁺ in the face of a sudden increase in the activity arriving at collicular neurons. It is likely that in the visual system the increase in CaN activity results from the first powerful activation of the central visual pathway by light. It is also possible that similar sudden increases in activity occur in other regions of the nervous system. This activity-dependent, tonic, CaN-mediated control system may be broadly distributed within the vertebrate CNS (Shi, 2000).

A final significant property of the developmental change in CaN activity reported here is that it is not associated with changes in total CaN protein levels. The only event necessary to initiate phosphatase activity may therefore be the activation exerted by the NMDAR itself. Nevertheless, CaN is an elaborately regulated enzyme. Thus, changes in the activity or the synaptic localization of AKAP proteins, FKBP or cyclophorin family members, DARPP-32, CHP, or Cabin 1 may be mediating an additional level of control. Alternatively, or in addition, a prolonged depression of kinase activity could contribute to both the onset and stability of the CaN effect in sSC NMDARCs. The prolonged CaN activation at sSC synapses may also arise from a fundamentally different mechanism, such as protection of the phosphatases Fe-Zn active center from oxidation by superoxide dismutase. Regardless of the precise mechanism through which the prolongation of synaptic CaN activity is exerted, these developmental data suggest an interaction that may be retained in the mature brain. Prolonged activation of synaptic CaN could protect neurons from excitotoxicity in the face of seizure, ischemia, trauma, or disease-induced tonic increases in NMDAR activity. Thus, interventions that amplify or maintain this response may prove useful in the clinical treatment of a variety of neurological dysfunctions (Shi, 2000).

Small conductance Ca2+-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. In addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels (Bildl, 2004).

Ca(2+)/calcineurin-dependent inactivation of neuronal L-type Ca(2+) channels requires priming by AKAP-anchored protein kinase A

Within neurons, Ca(2+)-dependent inactivation (CDI) of voltage-gated L-type Ca(2+) channels shapes cytoplasmic Ca(2+) signals. CDI is initiated by Ca(2+) binding to channel-associated calmodulin and subsequent Ca(2+)/calmodulin activation of the Ca(2+)-dependent phosphatase, calcineurin (CaN), which is targeted to L channels by the A-kinase-anchoring protein AKAP79/150. This study reports that CDI of neuronal L channels is abolished by inhibition of PKA activity or PKA anchoring to AKAP79/150 and that CDI is also suppressed by stimulation of PKA activity. Although CDI was reduced by positive or negative manipulation of PKA, interference with PKA anchoring or activity lowered Ca(2+) current density whereas stimulation of PKA activity elevated it. In contrast, inhibition of CaN reduced CDI but had no effect on current density. These results suggest a model wherein PKA-dependent phosphorylation enhances neuronal L current, thereby priming channels to undergo CDI, and Ca(2+)/calmodulin-activated CaN actuates CDI by reversing PKA-mediated enhancement of channel activity (Dittmer, 2014).

γCaMKII shuttles Ca(2+)/CaM to the nucleus to trigger CREB phosphorylation and gene expression

Activity-dependent CREB (see Drosophila CrebB) phosphorylation and gene expression are critical for long-term neuronal plasticity. Local signaling at voltage gated CaV1 channels triggers these events, but how information is relayed onward to the nucleus remains unclear. This study reports a mechanism that mediates long-distance communication within cells: a shuttle that transports Ca(2+)/calmodulin (see Drosophila Calmodulin) from the surface membrane to the nucleus. This study shows that the shuttle protein is γCaMKII (see Drosophila CaMKII), its phosphorylation at Thr287 by βCaMKII protects the Ca(2+)/CaM signal, and CaN triggers its nuclear translocation. Both betaCaMKII and CaN act in close proximity to CaV1 channels, supporting their dominance, whereas γCaMKII operates as a carrier, not as a kinase. Upon arrival within the nucleus, Ca(2+)/CaM activates CaMKK and its substrate CaMKIV, the CREB kinase. This mechanism resolves long-standing puzzles about CaM/CaMK-dependent signaling to the nucleus. The significance of the mechanism is emphasized by dysregulation of CaV1, γCaMKII, βCaMKII, and CaN in multiple neuropsychiatric disorders (Ma, 2014).

Calcineurin and muscle development and function

Slow- and fast-twitch myofibers of adult skeletal muscles express unique sets of muscle-specific genes, and these distinctive programs of gene expression are controlled by variations in motor neuron activity. It is well established that, as a consequence of more frequent neural stimulation, slow fibers maintain higher levels of intracellular free calcium than fast fibers, but the mechanisms by which calcium may function as a messenger linking nerve activity to changes in gene expression in skeletal muscle have been unknown. Here, fiber-type-specific gene expression in skeletal muscles is shown to be controlled by a signaling pathway that involves calcineurin, a cyclosporin-sensitive, calcium-regulated serine/threonine phosphatase. Activation of calcineurin in skeletal myocytes selectively up-regulates slow-fiber-specific gene promoters (Chin, 1998).

The myoglobin (Mb) and troponin I slow (TnIs) genes are expressed selectively in slow, oxidative skeletal muscle fibers, whereas the muscle creatine kinase (MCK) gene is expressed most abundantly in the fast, glycolytic myofiber subtype. To test whether these genes might respond differently to a calcineurin-stimulated signaling pathway, skeletal myogenic cells were transfected with reporter genes linked to well-characterized control regions from these genes, along with an expression vector encoding a constitutively active (calcium-insensitive) form of calcineurin that retains sensitivity to inhibition by cyclosporin A. Transcriptional activity of the slow-fiber-specific myoglobin and TnIs promoters is stimulated in cultured skeletal myotubes (C2C12) by active calcineurin, as measured by expression of luciferasein cotransfection assays. In contrast, activity of the fast-fiber-specific MCK promoter, or of other strong (CMV) or weak (minimal TATA element) promoters, is unaffected by activated calcineurin. The induction of the myoglobin promoter in the presence of the calcineurin expression plasmid is inhibited by cyclosporin A. This result indicates the specificity of the response, since the effect of cyclosporin A is to bind cyclophilin and form a protein complex that binds calcineurin and inhibits its protein phosphatase activity. The same relative potency of calcineurin-dependent transactivation (myoglobin and TnIs is much more potent than MCK, CMV, or TATA promoters) is observed in Sol8 myotubes, a different myogenic cell line. In contrast, forced expression of activated calcineurin had no effect on promoter activity in undifferentiated myoblasts or in 3T3 fibroblasts, demonstrating a requirement for muscle-specific factors in the calcineurin-stimulated pathway for transcriptional control of the myoglobin and TnIs promoters. Inhibition of calcineurin activity by administration of cyclosporin A to intact animals promotes slow-to-fast fiber transformation (Chin, 1998).

Transcriptional activation of slow-fiber-specific transcription appears to be mediated by a combinatorial mechanism involving proteins of the NFAT and MEF2 families. The finding that the myoglobin and TnIs promoters can be transcriptionally regulated by a calcineurin-dependent mechanism suggests the participation of NFAT transcription factors in the signaling cascade. Examination of the complete nucleotide sequences of these functionally defined transcriptional control regions (2.0 and 4.2 kb, respectively) reveals two 8-bp elements within each that match the consensus-binding sequence for NFAT transcription factors. The response to activated calcineurin of the native promoter sequences was compared to that of mutated promoters in which these putative NFAT recognition elements were ablated by site-directed mutagenesis. Disruption of putative NFAT recognition elements within both the myoglobin and TnIs promoters diminishes the response to activated calcineurin, indicating that the transactivation mechanism is likely to involve DNA binding of NFAT proteins. Transduction of the calcineurin-directed signal to the native myoglobin and TnIs promoters exhibits a saturable dose-response relationship with respect to the activated calcineurin expression plasmid; diminished reporter gene activation was evident across the entire dose range examined. Some degree of calcineurin-dependent transactivation persists after ablation of identifiable NFAT binding sites within these transcriptional control regions. Thus, either cryptic binding sites for NFAT proteins that cannot be recognized by inspection of the DNA sequence are present, or calcineurin-dependent signaling to these promoters can be driven without direct DNA binding of NFAT proteins. Nuclear localization of NFAT proteins in skeletal myocytes is under the control of calcineurin. These results identify a molecular mechanism by which different patterns of motor nerve activity promotes selective changes in gene expression to establish the specialized characteristics of slow and fast myofibers (Chin, 1998).

Calcineurin-dependent pathways have been implicated in the hypertrophic response of skeletal muscle to functional overload (OV). Skeletal muscles overexpressing an activated form of calcineurin (CnA*) exhibit a phenotype indistinguishable from wild-type counterparts under normal weightbearing conditions and respond to OV with a similar doubling in cell size and slow fiber number. These adaptations occur despite the fact that CnA* muscles display threefold higher calcineurin activity and enhance dephosphorylation of the calcineurin targets NFATc1, MEF2A, and MEF2D. Moreover, when calcineurin signaling is compromised with cyclosporin A, muscles from OV wild-type mice display a lower molecular weight form of CnA, originally detected in failing hearts, whereas CnA* muscles are spared this manifestation. OV-induced growth and type transformations are prevented in muscle fibers of transgenic mice overexpressing a peptide that inhibits calmodulin from signaling to target enzymes. Taken together, these findings provide evidence that both calcineurin and its activity-linked upstream signaling elements are crucial for muscle adaptations to OV and that, unless significantly compromised, endogenous levels of this enzyme can accommodate large fluctuations in upstream calcium-dependent signaling events (Dunn, 2000).

Regarding the potential identity of contractile activity-dependent signal transduction events, there is mounting evidence that calcineurin must interact with parallel calcium-sensitive signaling pathways in order to fully activate downstream target genes. For instance, calcineurin synergizes with phorbol ester-dependent pathways to stimulate the IL-2 promoter in T lymphocytes and the expression of atrial natriuretic factor in cardiomyocytes. Similarly, calcineurin acts in conjunction with CaM-dependent kinase IV to fully activate the myoglobin promoter in cultured skeletal myocytes and the Nur77 promoter in T lymphocytes. Moreover, retroviral-mediated gene transfer of CnA* induces skeletal myogenesis in vitro only in the presence of extracellular Ca²⁺. Additionally, there is evidence that MAP kinase pathways are activated in response to increased contractile activity and play a role in regulation of the slow fiber phenotype. In this context, MEF2 is an enticing candidate as an integrator of calcineurin and other activation-linked signal transduction pathways, since this transcription factor is both dephosphorylated by calcineurin and phosphorylated by various CaM kinases, ERK5, p38, and PKC (Dunn, 2000 and references therein).

An alternative possibility is that calcineurin signaling may converge with other activity-linked pathways via the association of GATA with NFAT. Indeed, activation of calcineurin promotes the association of these two transcription factors via the dephosphorylation of NFATc1 and increased expression of GATA-2 under conditions of skeletal myocyte growth. Consistent with findings from hypertrophic myocytes, this protein is upregulated in the plantaris in response to muscle overload, but not lowered by CsA treatment, suggesting that this transcription factor may be important for growth but not necessarily a gene target of calcineurin. The fact that GATA is also known to associate with MEF2, and that fiber hypertrophy is observed only when NFATc1 and MEF2 are dephosphorylated and GATA-2 increases, leads to the idea that NFAT, MEF2, and GATA proteins act in synergy to transactivate target genes that lead to fiber growth in response to OV. Future studies should help identify the particular permutations of these transcription factors involved in the activation of slow fiber-specific genes versus those modulating adult fiber size (Dunn, 2000 and references therein).

Myf5 is a member of the muscle regulatory factor family of transcription factors and plays an important role in the determination, development, and differentiation of skeletal muscle. However, factors that regulate the expression and activity of Myf5 itself are not well understood. Recently, a role for the calcium-dependent phosphatase calcineurin was suggested in three distinct pathways in skeletal muscle: differentiation, hypertrophy, and fiber-type determination. It is proposed that one downstream target of calcineurin and the calcineurin substrate NFAT in skeletal muscle is regulation of Myf5 gene expression. Myotube cultures were used that contain both multinucleated myotubes and quiescent, mononucleated cells termed 'reserve' cells, which share many characteristics with satellite cells. Treatment of such myotube cultures with the calcium ionophore ionomycin results in an approximately 4-fold increase in Myf5 mRNA levels, but similar effects are not observed in proliferating myoblast cultures, indicating that Myf5 is regulated by different pathways in different cell populations. The increase in Myf5 mRNA levels in myotube cultures requires the activity of calcineurin and NFAT, and can be specifically enhanced by overexpressing the NFATc isoform. Immunohistochemical analyses and fractionation of the cell populations were used to demonstrate that the calcium regulated expression of Myf5 occurs in the mononucleated reserve cells. It is concluded that Myf5 gene expression is regulated by a calcineurin- and NFAT-dependent pathway in the reserve cell population of myotube cultures. These results may provide important insights into the molecular mechanisms responsible for satellite cell activation and/or the renewal of the satellite cell pool following activation and proliferation (Friday, 2001).

Multiple intracellular signaling pathways have been shown to regulate the hypertrophic growth of cardiac myocytes including mitogen-activated protein kinase (MAPK) and calcineurin-nuclear factor of activated T-cells. However, it is uncertain if individual regulatory pathways operate in isolation or if interconnectivity between unrelated pathways is required for the orchestration of the entire hypertrophic response. To this end, the interconnectivity between calcineurin-mediated cardiac myocyte hypertrophy and p38 MAPK signaling was investigated in vitro and in vivo. Calcineurin promotes down-regulation of p38 MAPK activity and enhances expression of the dual specificity phosphatase MAPK phosphatase-1 (MKP-1). Transgenic mice expressing activated calcineurin in the heart are characterized by inactivation of p38 and increased MKP-1 expression during early postnatal development, before the onset of cardiac hypertrophy. In vitro, cultured neonatal cardiomyocytes infected with a calcineurin-expressing adenovirus and stimulated with phenylephrine demonstrate reduced p38 phosphorylation and increased MKP-1 protein levels. Activation of endogenous calcineurin with the calcium ionophore decreases p38 phosphorylation and increases MKP-1 protein levels. Inhibition of endogenous calcineurin with cyclosporin A decreases MKP-1 protein levels and increases p38 activation in response to agonist stimulation. To further investigate potential cross-talk between calcineurin and p38 through alteration in MKP-1 expression, the MKP-1 promoter was characterized and determined to be calcineurin-responsive. These data suggest that calcineurin enhances MKP-1 expression in cardiac myocytes; this expression is associated with p38 inactivation (Lim, 2001).

Increases in the expression of endothelin-1 (ET-1) in cardiac myocytes play a critical role in the development of heart failure in vivo. Whereas norepinephrine (NE) is a potent inducer of ET-1 expression in cardiac myocytes, the signaling pathways that link NE to inducible cardiac ET-1 expression are unknown. Adrenergic stimulation results in an increase in intracellular calcium levels, which in turn activates calcineurin. Stimulation with NE markedly increases the expression of the ET-1 gene in primary cardiac myocytes from neonatal rats. This increase is severely attenuated by a beta-adrenergic antagonist, metoprolol, but not by an alpha-adrenergic antagonist, prazosin. Consistent with these data, the beta-adrenergic agonist isoproterenol (ISO) activates the rat ET-1 promoter activity to an extent that is similar to NE. The ISO-stimulated increase in promoter activity is significantly inhibited by a Ca²⁺-antagonist, nifedipine, and an immunosuppressant, cyclosporin A, which blocks calcineurin. Mutation analysis indicated that the GATA4 binding site is required for ISO-responsive ET-1 transcription. Stimulation with ISO enhances the interaction between NFATc and GATA4 in cardiac myocytes. Consistent with this interaction, overexpression of GATA4 and NFATc synergistically activates the ET-1 promoter. These findings demonstrate that NE-stimulated ET-1 expression in cardiac myocytes is mediated predominantly via a beta-adrenergic pathway, and that calcium-activated calcineurin-GATA4 plays a role in this process (Morimoto, 2001).

Signaling events controlled by calcineurin promote cardiac hypertrophy, but the degree to which such pathways are required to transduce the effects of various hypertrophic stimuli remains uncertain. In particular, the administration of immunosuppressive drugs that inhibit calcineurin has inconsistent effects in blocking cardiac hypertrophy in various animal models. As an alternative approach to inhibiting calcineurin in the hearts of intact animals, transgenic mice were engineered to overexpress a human cDNA encoding the calcineurin-binding protein, myocyte-enriched calcineurin-interacting protein-1 (hMCIP1) under control of the cardiac-specific, alpha-myosin heavy chain promoter (alpha-MHC). In unstressed mice, forced expression of hMCIP1 results in a 5%-10% decline in cardiac mass relative to wild-type littermates, but otherwise produced no apparent structural or functional abnormalities. However, cardiac-specific expression of hMCIP1 inhibits cardiac hypertrophy, reinduction of fetal gene expression, and progression to dilated cardiomyopathy, all of which otherwise results from expression of a constitutively active form of calcineurin. Expression of the hMCIP1 transgene also inhibits hypertrophic responses to beta-adrenergic receptor stimulation or exercise training. These results demonstrate that levels of hMCIP1 producing no apparent deleterious effects in cells of the normal heart are sufficient to inhibit several forms of cardiac hypertrophy, and suggest an important role for calcineurin signaling in diverse forms of cardiac hypertrophy. The future development of measures to increase expression or activity of MCIP proteins selectively within the heart may have clinical value for prevention of heart failure (Rothermel, 2001).

Nerve activity can induce long-lasting, transcription-dependent changes in skeletal muscle fibers and thus affect muscle growth and fiber-type specificity. Calcineurin signaling has been implicated in the transcriptional regulation of slow muscle fiber genes in culture, but the functional role of calcineurin in vivo has not been unambiguously demonstrated. The up-regulation of slow myosin heavy chain (MyHC) and a MyHC-slow promoter induced by slow motor neurons in regenerating rat soleus muscle is prevented by the calcineurin inhibitors cyclosporin A (CsA), FK506, and the calcineurin inhibitory protein domain from cain/cabin-1. In contrast, calcineurin inhibitors do not block the increase in fiber size induced by nerve activity in regenerating muscle. The activation of MyHC-slow induced by direct electrostimulation of denervated regenerating muscle with a continuous low frequency impulse pattern is blocked by CsA, showing that calcineurin function in muscle fibers and not in motor neurons is responsible for nerve-dependent specification of slow muscle fibers. Calcineurin is also involved in the maintenance of the slow muscle fiber gene program because in the adult soleus muscle, cain causes a switch from MyHC-slow to fast-type MyHC-2X and MyHC-2B gene expression, and the activity of the MyHC-slow promoter is inhibited by CsA and FK506 (Serrano, 2001).

A new role for the calcineurin/NFAT pathway in neonatal myosin heavy chain expression via the NFATc2/MyoD complex during mouse myogenesis

The calcineurin/NFAT (nuclear factor of activated T-cells, see Drosophila NFAT homolog) signaling pathway is involved in the modulation of the adult muscle fiber type, but its role in the establishment of the muscle phenotype remains elusive. This study shows that the NFAT member NFATc2 cooperates with the basic helix-loop-helix transcription factor MyoD to induce the expression of a specific myosin heavy chain (MHC) isoform, the neonatal one, during embryogenesis. This cooperation is crucial, as Myod/Nfatc2 double-null mice die at birth, with a dramatic reduction of the major neonatal MHC isoform normally expressed at birth in skeletal muscles, such as limb and intercostal muscles, whereas its expression is unaffected in myofibers mutated for either factor alone. Using gel shift and chromatin immunoprecipitation assays, NFATc2 was found bound to the neonatal Mhc gene, whereas NFATc1 and NFATc3 would preferentially bind the embryonic Mhc gene. Evidence is provided that MyoD synergistically cooperates with NFATc2 at the neonatal Mhc promoter. Altogether, these findings demonstrate that the calcineurin/NFAT pathway plays a new role in establishing the early muscle fiber type in immature myofibers during embryogenesis (Daou, 2013)

Calcineurin and T cell function

The activation of calcineurin, a calcium- and calmodulin-dependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4+CD8+ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FK506 treatment blocks the generation of mature TCRhighCD4+CD8- and TCRhighCD4-CD8+ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (RAG-1) transcription and the downregulation of CD4 and CD8 expression are inhibited by FK506 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation does not appear to be required for negative selection based on the observations that negative selection of TCR alpha beta T cells in the H-2b male thymus (a negative selecting environment) is not inhibited by in vivo treatment with FK506 and that there is no rescue of the endogenous superantigen-mediated clonal deletion of V beta 6 and V beta 11 thymocytes in FK506-treated CBA/J mice. Different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes (Wang, 1995).

Embryonic stem (ES) cells and mice lacking the predominant isoform (alpha) of the calcineurin A subunit (CNA alpha) were prepared to study the role of this serine/threonine phosphatase in the immune system. T and B cell maturation appears to be normal in CNA alpha -/- mice. CNA alpha -/- T cells respond normally to mitogenic stimulation (i.e., PMA plus ionomycin, concanavalin A, and anti-CD3 epsilon antibody). However, CNA alpha -/- mice generated defective antigen-specific T cell responses in vivo. Mice produced from CNA alpha -/- ES cells injected into RAG-2-deficient blastocysts have a similar defective T cell response, indicating that CNA alpha is required for T cell function per se, rather than for an activity of other cell types involved in the immune response. CNA alpha -/- T cells remain sensitive to both cyclosporin A and FK506, suggesting that CNA beta or another CNA-like molecule can mediate the action of these immunosuppressive drugs. Thus CNA alpha -/- mice provide an animal model for dissecting the physiologic functions of calcineurin as well as the effects of FK506 and CsA (Zhang, 1996).

Calcineurin and vascular development

Vascular development requires an orderly exchange of signals between growing vessels and their supporting tissues, but little is known of the intracellular signaling pathways underlying this communication. Mice with disruptions of both NFATc4 and the related NFATc3 genes die around E11 with generalized defects in vessel assembly as well as excessive and disorganized growth of vessels into the neural tube and somites. Since calcineurin is thought to control nuclear localization of NFATc proteins, a mutation was introduced into the calcineurin B gene that prevents phosphatase activation by Ca²⁺ signals. These CnB mutant mice exhibit vascular developmental abnormalities similar to the NFATc3/c4 null mice. Calcineurin function is transiently required between E7.5 and E8.5. Hence, early calcineurin/NFAT signaling initiates the later cross-talk between vessels and surrounding tissues that pattern the vasculature (Graef, 2001).

Calcineurin signaling plays a role in development and synaptic organization of cerebellar granule cells during the postnatal period

Primary culture of postnatal cerebellar granule cells provides a model system that recapitulates many molecular events of developing granule cells in vivo. Depolarization of cultured granule cells increases intracellular Ca(2+) and activates Ca(2+)/calmodulin-dependent calcineurin (CaN) phosphatase. This Ca(2+) signaling mimics some of the signaling events for proliferation, migration, and differentiation of granule cells in vivo. This study investigated the genome-wide expression profiles of depolarization- and CaN-regulated genes in cultured mouse granule cells and addressed their relevance to gene regulation in developing granule cells in vivo. Granule cells were cultured under a nondepolarization condition (5 mM KCl) and a depolarization condition (25 mM KCl) with and without the CaN inhibitor FK506. Gene expression profiles between depolarization and nondepolarization and between FK506 treatment and untreatment were analyzed by microarray techniques. Both depolarization and FK506 treatment influence expression levels of a large number of genes, most of which are overlapping, however, are conversely regulated by these two treatments. Importantly, many of the FK506-responsive genes are up- or down-regulated in parallel with gene expression in postnatal granule cells in vivo. The FK506-down-regulated genes are highly expressed in proliferating/premigratory granule cells and many of these genes encode cellular components involved in cell proliferation, migration, and differentiation. In contrast, the FK506-up-regulated genes are predominantly expressed in postmigratory granule cells, including many functional molecules implicated in synaptic transmission and modulation. This investigation demonstrates that the CaN signaling plays a pivotal role in development and synaptic organization of granule cells during the postnatal period (Sato, 2005).

Calcineurin is universally involved in vesicle endocytosis at neuronal and nonneuronal secretory cells

Calcium influx triggers and accelerates endocytosis in nerve terminals and nonneuronal secretory cells. Whether calcium/calmodulin-activated calcineurin, which dephosphorylates endocytic proteins, mediates this process is highly controversial for different cell types, developmental stages, and endocytic forms. Using three preparations that previously produced discrepant results (i.e., large calyx-type synapses, conventional cerebellar synapses, and neuroendocrine chromaffin cells containing large dense-core vesicles), this study found that calcineurin gene knockout consistently slowed down endocytosis, regardless of cell type, developmental stage, or endocytic form (rapid or slow). In contrast, calcineurin and calmodulin blockers slowed down endocytosis at a relatively small calcium influx, but did not inhibit endocytosis at a large calcium influx, resulting in false-negative results. These results suggest that calcineurin is universally involved in endocytosis. They may also help explain the discrepancies among previous pharmacological studies. It is therefore suggested that calcineurin should be included as a key player in mediating calcium-triggered and -accelerated vesicle endocytosis (Wu, 2014).

AKAP-anchored PKA maintains neuronal L-type calcium channel activity and NFAT transcriptional signaling

L-type voltage-gated Ca²⁺ channels (LTCC) couple neuronal excitation to gene transcription. LTCC activity is elevated by the cyclic AMP (cAMP)-dependent protein kinase (PKA) and depressed by the Ca²⁺-dependent phosphatase calcineurin (CaN), and both enzymes are localized to the channel by A-kinase anchoring protein 79/150 (AKAP79/150). AKAP79/150 anchoring of CaN also promotes LTCC activation of transcription through dephosphorylation of the nuclear factor of activated T cells (NFAT). This study reports that the basal activity of AKAP79/150-anchored PKA maintains neuronal LTCC coupling to CaN-NFAT signaling by preserving LTCC phosphorylation in opposition to anchored CaN. Genetic disruption of AKAP-PKA anchoring promoted redistribution of the kinase out of postsynaptic dendritic spines, profound decreases in LTCC phosphorylation and Ca²⁺ influx, and impaired NFAT movement to the nucleus and activation of transcription. Thus, LTCC-NFAT transcriptional signaling in neurons requires precise organization and balancing of PKA and CaN activities in the channel nanoenvironment, which is only made possible by AKAP79/150 scaffolding (Murphy, 2014).

Calcineurin and neurite extension

Growth cones generate spontaneous transient elevations of intracellular Ca²⁺ that regulate the rate of neurite outgrowth. These Ca²⁺ waves inhibit neurite extension via the Ca²⁺-dependent phosphatase calcineurin (CN) in Xenopus spinal neurons. Pharmacological blockers of CN (cyclosporin A and deltamethrin) and peptide inhibitors of CN [the Xenopus CN (xCN) autoinhibitory domain and African swine fever virus protein A238L] block the Ca²⁺-dependent reduction of neurite outgrowth in cultured neurons. Time-lapse microscopy of growing neurites demonstrates directly that the reduction in the rate of outgrowth by Ca²⁺ transients is blocked by cyclosporin A. In contrast, expression of a constitutively active form of xCN in the absence of waves results in shorter neurite lengths similar to those seen in the presence of waves. The developmental expression pattern of xCN transcripts in vivo coincides temporally with axonal pathfinding by spinal neurons, supporting a role of CN in regulating Ca²⁺-dependent neurite extension in the spinal cord. Ca²⁺ wave frequency and Ca²⁺-dependent expression of GABA are not affected by inhibition or activation of CN. However, phosphorylation of the cytoskeletal element GAP-43, which promotes actin polymerization, is reduced by Ca²⁺ waves and enhanced by suppression of CN activity. CN ultimately acts on the growth cone actin cytoskeleton, because disrupting actin microfilaments with cytochalasin D or stabilizing them with jasplakinolide negates the effects of suppressing or activating CN. Destabilization or stabilization of microtubules with colcemide or taxol results in Ca²⁺-independent inhibition of neurite outgrowth. The results identify components of the cascade by which Ca²⁺ waves act to regulate neurite extension (Lautermilch, 2000).

Axon outgrowth is the first step in the formation of neuronal connections, but the pathways that regulate axon extension are still poorly understood. NFAT proteins belong to the Rel/Dorsal family of transcription factors. Mice deficient in calcineurin-NFAT signaling have dramatic defects in axonal outgrowth, yet have little or no defect in neuronal differentiation or survival. In vitro, sensory and commissural neurons lacking calcineurin function or NFATc2, c3, and c4 are unable to respond to neurotrophins or netrin-1 with efficient axonal outgrowth. Neurotrophins and netrins stimulate calcineurin-dependent nuclear localization of NFATc4 and activation of NFAT-mediated gene transcription in cultured primary neurons. These data indicate that the ability of these embryonic axons to respond to growth factors with rapid outgrowth requires activation of calcineurin/NFAT signaling by these factors. The precise parsing of signals for elongation, turning and survival could allow independent control of these processes during development (Graef, 2003).

NFAT transcription complexes are appealing candidates for regulating aspects of neuronal morphogenesis because they integrate extracellular signals. Cell membrane signaling results in the assembly of NFAT transcription complexes in the nucleus and the activation of genes that are dependent on the cell type in which the signal is received. A rise in intracellular Ca2+ activates the serine/threonine phosphatase calcineurin and rapidly dephosphorylates the four cytoplasmic subunits NFATc1-4. Dephosphorylation of serines in the amino-termini of NFATc proteins by calcineurin exposes nuclear localization sequences leading to their rapid nuclear import. NFATc cytoplasmic subunits require other transcription factors for DNA binding, including AP-1, MEF2, GATA4, and additional factors generically referred to as nuclear partners (NFATn). The nuclear components of NFAT transcription complexes are often regulated by the PKC and Ras/MAPK pathways. Hence, the assembly of NFAT transcription complexes requires that Ca2+/calcineurin signaling be coincident with other signals. Nuclear import of NFATc family members is opposed by rapid export induced by rephosphorylation mediated by the sequential actions of PKA and GSK3. The rapid export of NFATc proteins from the nucleus can make NFAT signaling responsive to receptor occupancy and/or Ca2+ channel dynamics (Graef, 2003 and references therein).

Evidence is provided for an unexpected role for calcineurin and NFATc family members in controlling the outgrowth of embryonic axons. The results suggest that calcineurin/NFAT signaling is required specifically for axon outgrowth stimulated by growth factors like neurotrophins and netrins and provides a potential regulatory site for controlling axonal elongation independent of neuronal survival (Graef, 2003).

Axon pathfinding depends on attractive and repulsive turning of growth cones to extracellular cues. Localized cytosolic Ca2+ signals are known to mediate the bidirectional responses, but downstream mechanisms remain elusive. Calcium-calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) phosphatase have been shown to provide a switch-like mechanism to control the direction of Ca(2+)-dependent growth cone turning. A relatively large local Ca2+ elevation preferentially activates CaMKII to induce attraction, while a modest local Ca2+ signal predominantly acts through CaN and phosphatase-1 (PP1) to produce repulsion. The resting level of intracellular Ca2+ concentrations also affects CaMKII/CaN operation: a normal baseline allows distinct turning responses to different local Ca2+ signals, while a low baseline favors CaN-PP1 activation for repulsion. Moreover, the cAMP pathway negatively regulates CaN-PP1 signaling to inhibit repulsion. Finally, CaMKII/CaN-PP1 also mediates netrin-1 guidance. Together, these findings establish a complex Ca2+ mechanism that targets the balance of CaMKII/CaN-PP1 activation to control distinct growth cone responses (Wen, 2004).

Axon guidance by a number of guidance molecules has been shown to depend on localized Ca2+ signals in the growth cone. Using direct focal photoactivated release of caged Ca2+ in the growth cone, it has been found that a localized Ca2+ signal in the growth cone is sufficient to induce growth cone attraction as well as repulsion, depending on the resting level of intracellular Ca2+ concentrations ([Ca2+]i) at the growth cone. Similarly, studies on netrin-1 guidance have indicated that different Ca2+ signals might underlie distinct turning responses induced by netrin-1 gradients. These results not only demonstrate a crucial role for Ca2+ signals in growth cone guidance but also indicate that complex Ca2+ mechanisms may operate to control distinct growth cone responses to a wide spectrum of external molecules (Wen, 2004 and references therein).

How different turning responses are generated by distinct Ca2+ signals, however, remains unknown. It is conceivable that different local Ca2+ signals, integrated with the baseline level of [Ca2+]i in the growth cone, activate distinct pathways to mediate attraction and repulsion, respectively. The complexity of Ca2+ signaling in axon guidance is further increased by a series of recent studies demonstrating that Ca2+-dependent growth cone responses can be further modulated by the cAMP pathway: elevation of cAMP leads to switching of repulsion to attraction and vice versa. Does the cAMP pathway act upstream to modify the characteristics of Ca2+ signals (local and/or global) or target the downstream effectors of Ca2+ for the switching? It was recently shown that cAMP/cGMP can affect L-type Ca2+ channels to alter intracellular Ca2+ signals induced by netrin-1, thus placing cAMP/cGMP upstream of Ca2+ in mediating bidirectional turning responses. However, whether cAMP also plays a role downstream of Ca2+ signaling in guidance remains to be evaluated. Most importantly, the question of what downstream targets mediate distinct Ca2+-dependent turning behaviors is still unanswered. Do attraction and repulsion involve the same or separate downstream signaling cascades? In this study, a direct local Ca2+ elevation approach was used to study downstream mechanisms of Ca2+-dependent bidirectional turning responses of nerve growth cones. The use of direct local elevation of intracellular Ca2+ concentrations through photoactivated release of caged Ca2+ bypasses membrane receptor activation and can largely avoid crosstalk among different signaling pathways, thus allowing a focus on intracellular Ca2+ and its downstream events during distinct turning responses. Evidence suggests that Ca2+-calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN)-phosphatase-1 (PP1) mediate attraction and repulsion, respectively. Significantly, CaMKII/CaN-PP1 acts as a bimodal switch to control the direction of growth cone turning in response to different Ca2+ signals (local and global) by preferentially activating one component over the other. It is further shown that the cAMP pathway negatively regulates the CaN-PP1 side of the switch to modulate growth cone responses. Finally, evidence is presented that the CaMKII/CaN-PP1 mechanism also mediates netrin-1 guidance. These findings thus provide significant insights toward the downstream mechanisms underlying various turning behaviors induced by complex Ca2+ signals (Wen, 2004).

Neural activity regulates synaptic properties and dendritic structure in vivo through calcineurin/NFAT signaling

The calcium-regulated protein phosphatase Calcineurin (CaN) participates in synaptic plasticity and the regulation of transcription factors, including Nuclear Factor of Activated T cells (NFAT). To understand how CaN contributes to neuronal circuit development, whole-cell mEPSC recordings and multiphoton imaging were performed in the visual system of living Xenopus laevis tadpoles electroporated to express either a CaN phosphatase inhibitor or N-VIVIT, a nuclear localization sequence-tagged VIVIT peptide that blocks the binding of CaN to select substrates including NFAT. Both strategies increased mEPSC frequency and dendritic arbor complexity in tectal neurons over 3 days. Expression of either of two constitutively active Xenopus NFATs (CA-NFATs) restored normal synaptic properties in neurons expressing N-VIVIT. However, the morphological phenotype was only rescued by a CA-NFAT bearing an intact regulatory domain, implying that transcriptional control of morphological and electrophysiological properties of neurons is mediated by distinct NFAT interactions (Schwartz, 2009).

Calcineurin, LTP and memory

The threshold for hippocampal-dependent synaptic plasticity and memory storage is thought to be determined by the balance between protein phosphorylation and dephosphorylation mediated by the kinase PKA and the phosphatase calcineurin. To establish whether endogenous calcineurin acts as an inhibitory constraint in this balance, the effect of genetically inhibiting calcineurin on plasticity and memory was examined. Using the doxycycline-dependent rtTA system to express a calcineurin inhibitor reversibly in the mouse brain, it has been found that the transient reduction of calcineurin activity facilitates LTP in vitro and in vivo. This facilitation is PKA dependent and persists over several days in vivo. It is accompanied by enhanced learning and strengthened short- and long-term memory in several hippocampal-dependent spatial and nonspatial tasks. The LTP and memory improvements are reversed fully by suppression of transgene expression. These results demonstrate that endogenous calcineurin constrains LTP and memory (Malleret, 2001).

The main finding of this study is that the regulated inhibition of the phosphatase CN leads to enhanced LTP both in vitro and in vivo, and to improved learning and memory storage. The parallel in time course of the increased persistence of LTP in awake animals and of the memory improvement strongly suggest a correlation between the duration of LTP and memory storage. Improved cognitive performance was observed both on spatial and nonspatial hippocampal-dependent tasks, consistent with the multipurpose role of the hippocampus in human declarative memory. Moreover, with different tasks, different temporal components of memory were improved. Thus, the complex object recognition task, involving brief training sessions, multiple objects, spatial transfer, and object change, elicit a weak form of memory that is strengthened at early and intermediate time points by the CN inhibitor, but does not persist longer in mutants than in controls. By contrast, a more robust form of memory elicited by a more intense training is maintained and persists for over a week longer in mutants expressing the CN inhibitor when compared to controls (Malleret, 2001).

Facilitated learning and memory was also observed in the Morris water maze and was evident not only with traditional measurements of spatial performance, such as escape latency, but also with more specific aspects of performance such as the precision of navigation. These measures suggest that mutant mice expressing the CN inhibitor retain spatial information more efficiently than controls. The persistent memory for the first platform position associated with the efficient learning of a second platform position suggests an overall enhanced capacity for memory storage with the CN inhibitor. Further, the rapid adaptation to spatial changes observed in mutants expressing the CN inhibitor on both the Morris water maze and the object exploration task suggest increased cognitive flexibility, a process that depends on the hippocampus (Malleret, 2001).

One of the molecular mechanisms allowing transmitted signals to persist or decay is thought to be the balance between phosphatase and kinase activity. Much evidence suggests that PKA and CN specifically regulate this balance and thereby serve as a gate for LTP. In the current study, evidence in support of this model is provided by demonstrating that shifting the endogenous balance away from calcineurin activity positively modulates synaptic plasticity in a PKA-dependent manner. Further, the data indicate that altering CN activity transiently in the adult brain is sufficient to positively or negatively control synaptic plasticity and memory storage. The effects observed suggest that CN is essential both for early events of plasticity and memory and for downstream pathways that contribute to persistent changes in plasticity and memory storage (Malleret, 2001).

Mechanistically, early and transient forms of plasticity and memory are known to rely on the covalent modification of pre-existing proteins while long-term forms require activation of transcription factors such as CREB, and protein synthesis. One possible mechanism for the facilitatory effect of the CN inhibitor may be a decrease in the activity of PP1, a protein phosphatase positively regulated by CN through dephosphorylation of inhibitor-1 (I-1). PP1 inhibition has been shown to promote the induction of LTP, whereas increased PP1 activity, produced by genetic suppression of I-1, has been shown to affect certain forms of LTP in some hippocampal regions. Since PP1 is effective in modulating CaMKII, a kinase critical for the transmission of postsynaptic signals required for the induction of LTP, it is possible that increased CaMKII activity mediated by lower PP1 activity facilitates the induction of LTP. Raising the signal for the induction of LTP, through genetic upregulation of NMDA-R function, has been shown to enhance LTP, learning, and memory. The findings suggest that LTP and memory enhancements can be similarly achieved by relieving a constraint downstream of the NMDA-R and that this constraint is exercised by CN (Malleret, 2001).

The effect of the CN inhibitor on long-lasting changes in plasticity and memory may be mediated by modulation of transcriptional control. Thus, the prolonged maintenance of LTP and of memory may arise from augmented CREB transcriptional activity via reduced CREB dephosphorylation by PP1. In this context, it is important to note that unlike the phenotypes observed in Drosophila mutants expressing active CREB, the CN inhibitor does not convert labile memory into long-lasting memory. It is, however, able to strengthen or prolong different phases of memory, suggesting that CN inhibition modulates rather than mediates memory processes (Malleret, 2001).

The PKA and CN pathways may also interact antagonistically at sites other than CREB. For example, CN can inhibit specific isoforms of adenylyl cyclase required for PKA activation. Similarly, PKA and CN can regulate, in opposite ways, phosphorylation sites on key proteins in synaptic transmission, such as the NMDA-R or AMPA receptor. The effect of inhibiting CN may also occur through processes additional to or independent of the cAMP pathway. For instance, CN inhibitor may modulate Ca²⁺-dependent kinases such as CaMKII or PKC through control of intracellular Ca²⁺ mobilization by regulation of inositol 1,4,5-triphosphate receptors. Finally, the PKA/CN gate most likely represents only one of several activator/suppressor mechanisms regulating plasticity and memory (Malleret, 2001).

Several genetic approaches have been used to study the molecular mechanisms of hippocampal functions such as memory. Standard genetic techniques, however, have suffered from the limitation that the genetic modification is permanent. Here, the usefulness of the rtTA system for such studies has been demonstrated by showing that inducible and reversible transgene expression allows temporary improvement of complex cognitive functions and of brain plasticity. The ability to achieve such reversible improvements in the adult animal demonstrates that no permanent changes in neuronal circuits are involved and that the effects result specifically from molecular and biochemical changes elicited by a reduction in CN activity. In this context, the rtTA system could be further exploited to assess the timing of the requirement of CN in such processes, for instance in various stages of memory storage such as memory consolidation or retrieval since CN has been suggested to be involved in both processes. Transgene expression was found in several brain structures and, therefore, the contribution of other structures, in addition to the hippocampus, to the enhancement of long-term memory, cannot be excluded. Overall, however, these results may provide a clear target for potential treatment of learning and memory disorders (Malleret, 2001).

The calcium-dependent phosphatase calcineurin and its downstream transcriptional effector nuclear factor of activated T cells (NFAT) are important regulators of inducible gene expression in multiple cell types. In T cells, calcineurin-NFAT signaling represents a critical event for mediating cellular activation and the immune response. The widely used immunosuppressant agents cyclosporin and FK506 are thought to antagonize the immune response by directly inhibiting calcineurin-NFAT signal transduction in lymphocytes. To unequivocally establish the importance of calcineurin signaling as a mediator of the immune response, the gene encoding the predominant calcineurin isoform expressed in lymphocytes, calcineurin Abeta (CnAbeta), was deleted. CnAbeta^-/- mice are viable as adults, but display defective T cell development characterized by fewer total CD3 cells and reduced CD4 and CD8 single positive cells. Total peripheral T cell numbers are significantly reduced in CnAbeta^-/- mice and are defective in proliferative capacity and IL-2 production in response to PMA/ionomycin and T cell receptor cross-linking. CnAbeta^-/- mice also are permissive to allogeneic tumor-cell transplantation in vivo, similar to cyclosporin-treated wild-type mice. A mechanism for the compromised immune response is suggested by the observation that CnAbeta^-/- T cells are defective in stimulation-induced NFATc1, NFATc2, and NFATc3 activation. These results establish a critical role for CnAbeta signaling in regulating T cell development and activation in vivo (Bueno, 2002).

Metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) was investigated in hippocampal CA1 pyramidal neurons of 6- to 8-d-old [postnatal days 6-8 (P6-P8)] and 21- to 25-d-old (P21-P25) rats. In P6-P8 rats, induction of LTD depends on the activity of group II mGluRs. In P21-P25 rats, however, this LTD disappears, and instead, NMDA receptor (NMDAR)-dependent LTD appears. A bath containing a specific calcineurin (CaN) inhibitor restores the group II mGluR-dependent LTD in the neurons of the P21-P25 rats. Although postsynaptic injection of CaN inhibitors suppresses NMDAR-dependent LTD, it does not affect induction of group II mGluR-dependent LTD. These results demonstrate that CaN plays different roles in the induction of two forms of LTD: presynaptic CaN inhibits group II mGluR-dependent LTD, whereas postsynaptic CaN facilitates NMDAR-dependent LTD. These findings are the first demonstration in vitro of group II mGluR-dependent LTD that is negatively regulated by CaN via an age-dependent mechanism (Li, 2002).

Calcineurin and sensory signal transduction

Animals sense and adapt to variable environments by regulating appropriate sensory signal transduction pathways. Calcineurin plays a key role in regulating the gain of sensory neuron responsiveness across multiple modalities. C. elegans animals bearing a loss-of-function mutation in TAX-6, a calcineurin A subunit, exhibit pleiotropic abnormalities, including many aberrant sensory behaviors. The tax-6 mutant defect in thermosensation is consistent with hyperactivation of the AFD thermosensory neurons. Conversely, constitutive activation of TAX-6 causes a behavioral phenotype consistent with inactivation of AFD neurons. In olfactory neurons, the impaired olfactory response of tax-6 mutants to an AWC-sensed odorant is caused by hyperadaptation, which is suppressible by a mutation causing defective olfactory adaptation. Taken together, these results suggest that stimulus-evoked calcium entry activates calcineurin, which in turn negatively regulates multiple aspects of sensory signaling (Kuhara, 2002).

Two types of cation channels, TAX-4/TAX-2 and OSM-9, are expressed in the AWC olfactory neurons. Genetic analyses suggest that odor sensing activates primary olfactory transduction through the TAX-4/TAX-2 channel, which allows calcium entry to activate AWC neurons, whereas odor-provoked calcium influx through the OSM-9 channel only affects olfactory adaptation. tax-6 animals are hyperadaptable to AWC-sensed isoamyl alcohol. Exposure to isoamyl alcohol for only 10 min is sufficient for tax-6 animals to adapt. This hyperadaptable phenotype and the partially defective olfactory response of tax-6 mutants to isoamyl alcohol are both completely suppressed by an osm-9 mutation. These results suggest that TAX-6 represses OSM-9-dependent olfactory adaptation in AWC. Taken together, two possible models are proposed for the role of TAX-6 in AWC signaling. TAX-6 could be activated by calcium entry through the primary signal transduction channel TAX-4/TAX-2 upon activation of the odorant (IAA) receptor, and the activated TAX-6 could inhibit the adaptation machinery. Alternatively, TAX-6 could be activated by the odorant (IAA)-evoked calcium influx through the OSM-9 channel, and the activated TAX-6 could negatively regulate opening of the OSM-9 channel that is required for isoamyl alcohol adaptation (Kuhara, 2002).

These models on the role of TAX-6 as a negative regulator for OSM-9-dependent olfactory adaptation in AWC might paradoxically imply that TAX-6 could be a positive regulator for TAX-4/TAX-2-dependent primary sensory signaling. If TAX-6 is a direct positive regulator of AWC primary transduction, at least partially defective olfactory responses to isoamyl alcohol could be expected in osm-9 tax-6 double mutants. It was found, however, that osm-9 tax-6 mutants show completely normal olfactory response to isoamyl alcohol. This result argues against a direct positive role of TAX-6 in AWC primary signaling. The results on osm-9 tax-6 mutants are also inconsistent with a direct negative regulatory role of TAX-6 in AWC primary signaling. If that were true, hyperattractive olfactory responses to isoamyl alcohol would be seen in osm-9 tax-6 mutants (Kuhara, 2002).

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