InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

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 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-arrestphenotype, 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 (Gorlach, 2000; Kingsbury, 2000; Lee, 2003; Fuentes, 2000). 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 (Sullivan, 2002; Gajewski, 2003). Misexpression of the active form of Pp2B-14D (Pp2B-14D^act) causes morphological abnormalities in eyes and wings (Sullivan, 2002). 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).

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).

Phospho-regulation pathways during egg activation in Drosophila melanogaster

Egg activation is the series of events that transition a mature oocyte to an egg capable of supporting embryogenesis. Increasing evidence points toward phosphorylation as a critical regulator of these events. This study used Drosophila melanogaster to investigate the relationship between known egg activation genes and phosphorylation changes that occur upon egg activation. Using the phosphorylation states of four proteins-Giant Nuclei, Young Arrest, Spindly, and Vap-33-1-as molecular markers, this study showed that the egg activation genes sarah, CanB2, and cortex are required for the phospho-regulation of multiple proteins. This study showed that an additional egg activation gene, prage, regulates the phosphorylation state of a subset of these proteins. Finally, this study shows that Sarah and calcineurin are required for the Anaphase Promoting Complex/Cyclosome (APC/C)-dependent degradation of Cortex following egg activation. From these data, a model is presented in which Sarah, through the activation of calcineurin, positively regulates the APC/C at the time of egg activation, which leads to a change in phosphorylation state of numerous downstream proteins (Krauchunas, 2013).

REGULATION

Protein Interactions

Sra Forms a Complex with CnA and CnB Subunits

MCIPs have been shown to inhibit calcineurin activity by binding directly to the CnA subunit (Fuentes, 2000). Whether Sra interacts with Drosophila calcineurin subunits, which were expressed in the female germline, was examined. In this experiment, Drosophila S2 cultured cells were first transfected with sra and Pp2B-14D-myc expression constructs and then immunoprecipitated them with Sra antibody. Immunoblotting with Myc antibody revealed that Sra indeed binds to Pp2B-14D. Thus, it is clear that Sra is capable of forming a complex with Pp2B-14D when coexpressed in S2 cells. It has been shown that calcineurin subunits are more stable when they form a complex. When CanB2 plasmid was cotransfected, an increased (2.7 times) amount of Pp2B-14D protein was coimmunoprecipitated together with CanB2. Western analysis of cell lysates suggested that Pp2B-14D, CanB2, and Sra are more stable when they were coexpressed. In addition to Pp2B-14D, it was also found that CanA-14F, another CnA subunit expressed in the germline, could bind to Sra, implying at least that it can form a complex with Pp2B-14D and CanB2 or CanA-14F and CanB2 (Takeo, 2006).

The WAVE regulatory complex links diverse receptors to the actin cytoskeleton

The WAVE regulatory complex (WRC) controls actin cytoskeletal dynamics throughout the cell by stimulating the actin-nucleating activity of the Arp2/3 complex at distinct membrane sites. However, the factors that recruit the WRC to specific locations remain poorly understood. This study has identified a large family of potential WRC ligands, consisting of approximately 120 diverse membrane proteins, including protocadherins, ROBOs, netrin receptors, neuroligins, GPCRs, and channels. Structural, biochemical, and cellular studies reveal that a sequence motif that defines these ligands binds to a highly conserved interaction surface of the WRC formed by the Sarah and Abi subunits. Mutating this binding surface in flies resulted in defects in actin cytoskeletal organization and egg morphology during oogenesis, leading to female sterility. These findings directly link diverse membrane proteins to the WRC and actin cytoskeleton and have broad physiological and pathological ramifications in metazoans (Chen, 2014).

A consensus peptide motif, WIRS, specifically binds to a unique surface formed by the Sra and Abi subunits of the WRC. Strict conservation of the binding surface suggests that this interaction is broadly important to metazoans. The WIRS motif defines a novel class of WRC ligands that contains ~120 diverse membrane proteins. Genetic data further show that mutating the WIRS binding site of the WRC in Drosophila disrupts actin cytoskeleton organization and egg morphology during oogenesis, leading to female sterility, and also disrupts development of the visual system. In summary, these data characterize a widespread and conserved interaction that may link numerous membrane proteins to the WRC and the actin cytoskeleton (Chen, 2014).

The WIRS binding surface is contributed by both the Sra and Abi subunits of the WRC and therefore is only present in the fully assembled complex. Consequently, the WIRS interaction is unique to the intact WRC and cannot occur with individual subunits or subcomplexes. This may have important functional implications because, in cells, individual WRC subunits may form complexes with other proteins. For example, Sra1 binds the fragile-X mental retardation syndrome protein FMRP, along with the translation initiation factor eIF4E, using an interaction surface that is normally buried within the WRC. Moreover, Abi has been shown to interact with other proteins independent of its assembly into the WRC, including another member of the Wiskott-Aldrich syndrome protein WASP and the Diaphanous-related formin. Finally, the Nap1 ortholog Hem1 was suggested to exist in large complexes distinct from the WRC. These various complexes likely have distinct cellular functions. For example, the Sra1-FMRPeIF4E complex regulates mRNA localization and protein translation, and the Abi complexes were shown to regulate the actin cytoskeleton in processes distinct from those regulated by the WRC. Therefore, the multisubunit nature of the WIRS binding site may provide a mechanism to specifically regulate the intact WRC (Chen, 2014).

WIRS proteins can directly recruit the WRC to membranes, likely in cooperation with the other classes of WRC ligands. WIRS proteins may also have additional effects on the biochemical activity of the WRC. For example, this study has demonstrated that, although the minimal WIRS motif does not activate the WRC, sequences flanking the motif can potentiate (as in PCDH10) or inhibit (as in PCDH17) activity of the WRC in vitro. Therefore, WIRS proteins may exert different effects on the activity of the assembly, again likely in cooperation with other WRC ligands such as Rac1 or kinases. Alternatively, WIRS proteins could act as a scaffold and modulate WRC activity by coordinately recruiting the complex and other ligands. For example, the cytoplasmic tail of the NMDA receptor subunit NR2B could potentially corecruit cyclin-dependent kinase 5 (Cdk5) and the WRC to facilitate phosphorylation and consequent activation of WAVE. In fact, many WIRS-containing proteins are thought to function as scaffolds, including APC, Ankyrin, WTX/Amer1, Shroom, and Shank. Finally, many WIRS proteins are cell-cell adhesion receptors, which are often densely clustered at the plasma membrane. Such clustering would locally concentrate the WRC, a process known to increase the activity of WASP proteins toward the Arp2/3 complex (Chen, 2014).

Finally, WIRS/WRC interactions themselves are likely regulated. In fact, the data suggest that the WIRS motif (F-x-T/S-FX- X) could be modulated by phosphorylation. High-affinity binding requires Thr or Ser at the third position of the WIRS motif. No other residues examined were tolerated. Thus, it is likely that Thr/Ser phosphorylation at this position would block binding as well. Indeed, phosphorylation of various WIRS sites has been identified not only in global phosphoproteome studies but also by site-specific mutagenesi. Together, these various mechanisms could bring a large range of regulatory dynamics to locally tune WRC activity and consequent actin assembly in vivo (Chen, 2014).

The conservation of the WIRS binding surface in virtually all metazoans suggests that the WIRS/WRC interaction is broadly important and unique to animals because it is absent from other eukaryotes, including protists, fungi, and plants. It is notable that the WIRS binding surface is found even in nonmetazoan choanoflagellates, suggesting that WIRS/WRC interactions appeared more than 700 million years ago in an early ancestor that predates metazoans. Choanoflagellates are considered to be the closest living relatives to metazoans because they encode many metazoan-specific protein domains, including various cell adhesion molecules and proteins enriched in the nervous system. Although choanoflagellates are generally considered unicellular organisms, they can form simple colonies, leading to the possibility that the WIRS interaction arose to maintain multicellularity. However, this interaction may not be strictly necessary for multicellularity, as the WIRS binding surface is not found in the placozoan T. adhaerens, a primitive, amoeboid- like metazoan that lacks tissues or organs but is made up of distinct cell types. Moreover, a significant number of nonadhesion proteins also contain WIRS motifs, indicating that the WIRS interaction likely developed additional functions (Chen, 2014).

In this study, the search was limited to proteins whose WIRS motifs were conserved in four of seven representative species. Among the ~120 WIRS proteins, some display high conservation of their WIRS motifs. These include netrin receptors and ROBO proteins, whose WIRS motifs are conserved from human to C. elegans, despite a significant divergence in the overall sequences of their cytoplasmic tails. The WIRS motifs of many other proteins, including protocadherins and neuroligins, are conserved in all vertebrates examined (from human to zebrafish). It is noted that, by using conservation as a criterion in the search, other bona fide WIRS ligands that are less conserved might have been missed (Chen, 2014).

This study has demonstrated biological functions of WIRS/WRC interactions in animals by using Drosophila oogenesis as a model system. Defects observed by disrupting the WIRS binding surface, which resulted in defective egg morphology, disrupted actin cytoskeleton, and female sterility, resemble defects that arise from knocking out the WRC, suggesting that the WIRS interaction plays a major role in regulating WRC function during oogenesis in flies. Additionally, it was observed that the WIRS binding site is also important to the WRC in its non-cell-autonomous function of regulating photoreceptor axonal targeting in developing optic lobes. It is believed that many more WIRS-mediated regulatory functions are yet to be discovered. In support of this assertion, in C. elegans, WIRS-mediated interaction of the neuronal adhesion receptor SYG-1 with the WRC has been shown to regulate actin assembly at presynaptic sites in the neuromuscular junction of the egglaying motor neuron HSN and consequently is critical in initiating both synaptogenesis and axonal branching. It has been proposed that WIRS/WRC interactions are of general and diverse importance to animals throughout development (Chen, 2014).

Future studies are needed to reveal which specific WIRScontaining ligands are important to particular processes. Prior data in the literature suggest candidate WIRS proteins during oogenesis. Two membrane-associated proteins, P08630 (Tec29 tyrosine kinase) and Q9VCX1 (locomotion defects protein, Loco), both contain WIRS motifs and have been shown to regulate nurse cell dumping. Loco was also found to regulate the cortical actin cytoskeleton in glia. Phenotypic analysis also reveals an opposite oogenesis defect, which is similar to those observed in kugelei mutants deficient for dFAT2, another WIRS-containing protein. It remains to be determined whether these proteins or others are directly linked to the WRC during this process (Chen, 2014).

A variety of evidence also exists in the literature, suggesting functional roles of the WIRS interaction in other biological processes. In addition to PCDH10 and PCDH19, the WIRS proteins DCC and ROBO and the epithelial sodium channel ENaC (γ subunit) have been genetically linked to the WRC. DCC and ROBO differentially regulate the abundance and subcellular localization of the WRC to control the actin cytoskeleton in C. elegans embryonic epidermis. The WRC and Rac1 were found to be essential in regulating the activity of ENaC. The current data suggest that these genetic interactions may be due to direct physical interactions of WIRS motifs with the WRC. The functions of many other WIRS proteins, only a few of which have been previously linked to the actin cytoskeleton (e.g., glutamate receptor NR2B and the postsynaptic cell adhesion molecule Neuroligin1), may also depend on an interaction with the WRC. As a notable example, a 21 amino acid sequence of the Neuroligin1 cytoplasmic tail harboring a WIRS motif (PGIQPLHTFNTFTGGQNNTLP, WIRS bold is required for presynaptic terminal maturation (Chen, 2014).

Although it is still very premature to link WIRS/WRC interactions to any disease, several cases are suggestive. For example, seven cases of epilepsy and mental retardation in females (EFMR) were reported to arise from truncations of the cytoplasmic tail of PCDH19, all resulting in the loss of its WIRS motif. Additionally, partial truncation of the DCC cytoplasmic tail, along with its WIRS motif, caused congenital mirror movement in four affected members of a three generation Italian family. Finally, a point mutation (S1359C) that disrupts the WIRS site (LDSFES, S1359) in the adenomatous polyposis coli (APC) protein was associated with three unrelated cases of hepatoblastoma (Chen, 2014).

In summary, this study has defined and characterized a large family of potential WRC ligands unique to metazoans. A large and diverse set of membrane proteins comprises this class, many with important biological functions. These findings provide a mechanistic framework to understand how these proteins signal downstream to the actin cytoskeleton via direct interaction with the WRC and how their mutations may ultimately lead to disease (Chen, 2014).

DEVELOPMENTAL BIOLOGY

RNA in situ hybridization showed that sra mRNA is present in the central nervous system of the third instar larvae, with a relatively intense signal in the brain and weak signals in the ventral ganglion. A relatively low, but ubiquitous expression level was observed in leg and wing imaginal discs, and no signal was detected in the eye-antennal discs. A strong signal was detected in all neurons of the adult brain. There is no specific localization of mRNA within the brain. The gene was also expressed at high levels in the nurse cells and oocytes. These results support the idea that sra functions in neurons during oogenesis (Ejima, 2004).

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/Sarah, 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 declin. 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).

EFFECTS OF MUTATION

Mutations in the sarah gene cause female sterility

Independent sarah (sra) alleles were found in genetic screens for sterile Drosophila females whose embryos arrest development very soon after fertilization. sarah corresponds to CG6072, which encodes calcipressin, a highly conserved inhibitor of the calcium- and calmodulin-dependent phosphatase, calcineurin (Ejima, 2004; Kingsbury, 2000). These sra mutations are at a minimum strong hypomorphs; the accompanying article by Takeo (2006) describes an authentic sra null allele whose phenotypic effects are very similar to those described in this study (Horner, 2006).

Mutations of sarah affect neither the fertility of males nor the viability of adults of either sex. However, sarah mutations have previously been shown to influence learning and memory (Chang, 2003), as well as ovulation and courtship behaviors (Ejima, 2004). Although sarah mutant females lay eggs, no embryos hatch (Ejima, 2004). To determine the underlying defect, these eggs and their precursors were examined in more detail. Oocytes within the ovaries of sra mutant females appear normal, as determined by staining for DNA and tubulin. In the wild-type, mature oocytes are arrested in metaphase I because the meiotic spindle pulls homologous chromosomes to opposite spindle poles while the homologs are held together by chiasmata, generating tension. This metaphase I arrest also occurred in all observed sra oocytes, as established by a single mass of chromatin at the midpoint of a single spindle. Thus, sra mutations do not affect bivalent formation or metaphase arrest prior to ovulation (Horner, 2006).

To characterize the developmental defect, heteroallelic sarah females and heterozygous control females were mated to wild-type males and allowed to lay eggs for 2 hr. Laid eggs were fixed and stained with propidium iodide to visualize the chromosomes and with α-tubulin antibodies to reveal the spindles. Eggs laid by control females developed as expected for 0–2 hr embryos; most had completed meiosis and were undergoing cleavage divisions and gastrulation (Horner, 2006).

In contrast, none of the eggs laid by sarah heteroallelic females progressed beyond meiosis, even though these females mated avidly with males and produced fertilized eggs. The characteristic phenotype was arrest during anaphase of the first meiotic division. A few eggs from mothers of certain presumably weaker genotypes showed other meiotic aberrations, probably due to low residual sra activity. Some anaphase I figures were abnormal, with chromosomes scattered throughout the spindle. A few eggs appeared to be in meiosis II because two spindles were observed. However, these two spindles were generally parallel to one another instead of end to end as in wild-type meiosis II, and the two spindles were usually asynchronous, with one in metaphase and the other in anaphase. In a few eggs, some pycnotic chromosomes appeared to have separated from the main spindle and nucleated secondary spindles. Apparently normal polar bodies formed in a single egg, so meiosis may rarely complete in eggs from mothers with the weakest sra alleles (Horner, 2006).

To verify that sra eggs usually arrest in anaphase I, they were examined by high-resolution Feulgen and Giemsa staining. In every arrested anaphase, three large chromosomes are directed toward the two poles of the spindle; occasionally, the tiny fourth chromosome is seen to lead each of the two groups of chromosomes. Sister chromatids must therefore remain attached (at least at their centromeres) during the anaphase movements. The accumulation of “middle pole” material that ordinarily forms late in anaphase I and eventually demarcates the boundary between the two metaphase II spindles was never seen, indicating that the arrest occurs early in anaphase I. Consistent with the fact that egg activation in Drosophila is independent of fertilization, the eggs laid by virgin sra mutant females also arrest at anaphase I (Horner, 2006).

Mutant germline clones were created via the FLP-dominant female sterile technique. The eggs produced by mutant germlines were double stained with Feulgen and Giemsa for high-resolution cytometry of meiotic figures. Anaphase I arrest was observed in all cases. The genotype of the germline therefore determines the sra mutant phenotype in eggs. Transcripts of sra accumulate to high levels in germline nurse cells and oocytes during oogenesis (Ejima, 2004).

Bicoid mRNA is neither fully polyadenylated nor translated in sarah mutant eggs

The translation of Bicoid (Bcd) upon activation organizes anterior development in embryos. During oogenesis, bcd mRNA is synthesized in nurse cells and transported to oocytes, where it remains untranslated. Upon activation, bcd mRNA is rapidly polyadenylated and translated. To test for effects of sarah on bcd mRNA polyadenylation, the length of the poly(A) tails on bcd mRNAs was measured by using the PCR poly(A) test (PAT). In the wild-type, more than 120 A's are added to these RNAs within 1 hr of egg laying and activation. In contrast, eggs laid by sra mutant females add only about 64 nucleotides of poly(A) to bcd mRNA upon egg laying, similar to the 80 base extension that occurs in cortex mutants (Horner, 2006).

The failure of sra mutant eggs to fully polyadenylate bcd transcripts predicts these eggs will be compromised in their ability to translate Bcd protein. To test this assumption, lysates from fertilized, laid eggs of sra mutant females were probed for Bcd on Western blots. As controls, ovaries from wild-type females as well as fertilized and unfertilized laid eggs from wild-type and siblings heterozygous for the same sra alleles were also examined. In wild-type and heterozygous controls, Bcd is not observed in mature, unactivated oocytes but accumulates to high levels upon activation, as expected. However, no Bcd translation was observed in eggs laid by any sra mutants (Horner, 2006).

Vitelline membranes are cross-linked in sarah mutant eggs

To ascertain whether VM (vitelline membrane) cross-linking occurs in sra eggs, mated females were allowed to lay eggs for 2 hr timed intervals, and these in vivo activated eggs were treated with 50% bleach for two minutes. Non-cross-linked eggs lysed within the 2 min incubation, but if VM cross-linking and subsequent eggshell hardening occurred, eggs became resistant to 50% bleach. Eggs laid by sra females undergo VM cross-linking to the same extent as those from heterozygous controls. In contrast, all mature stage-14 ovarian oocytes of all genotypes are lysed by bleach. Although VM cross-linking occurs in sra eggs activated in vivo by egg laying, results from in vitro activation suggest that the cross-linking may be slower or less efficient than in wild-type eggs (Horner, 2006).

The male pronucleus does not mature in sarah mutant eggs

Mutations in sra do not prevent the fertilization of eggs by sperm; using an antibody that recognizes the sperm tail, the sperm tail was observed in the majority of the eggs produced by mated sra mothers. By examining the sperm nucleus in fertilized sra eggs, it was possible to investigate the interaction between egg activation and maturation of the sperm nucleus into the male pronucleus. In fertilized sra eggs, the sperm nucleus is round, but its diameter is only 1.7 ± 0.04 μm, much smaller than that of fully decondensed male pronuclei seen just prior to pronuclear fusion (Horner, 2006).

To determine if histone exchange occurs in the wild-type sperm nucleus of sra mutant eggs, fertilized laid eggs were stained with propidium iodide for DNA and an antibody that recognizes histone H1, which is not present in mature sperm before fertilization. The sperm nucleus in these eggs is consistently associated with histone H1. Thus, paternal chromosomes associate with histone H1 from maternal stores in sra mutant eggs; it is presumed that H1 targeting requires previous replacement of sperm protamines with the core histones on paternal chromatin (Horner, 2006).

The very first steps of sperm nuclear envelope breakdown, chromosome decondensation, and the replacement of protamines with histones therefore occur in sarah mutant eggs. Sperm produced by males homozygous for the male-sterile gene sneaky cannot undergo these steps even in wild-type eggs. As a result, the DNA of sneaky sperm nuclei in eggs cannot be stained by the membrane-impermeable dye propidium iodide and retains an elongated needle-shaped configuration. In contrast, the sperm nucleus in sarah eggs can be stained by propidium iodide and assumes a rounded shape. Mutations in the sesame gene, which encodes the maternal factor Hira, a histone chaperone required for nucleosome assembly, result in a failure of core histones to incorporate into the sperm nucleus. Sarah-mediated activation events thus appear to be required for neither sneaky nor sesame function; nor do they appear to be required for protamine/histone exchange in general (Horner, 2006).

However, further development of the sperm nucleus does not occur in sra eggs. To determine if the sperm nucleus replicates its genome, laid, fertilized sra mutant eggs were stained for proliferating cell nuclear antigen (PCNA). PCNA functions as a processivity factor of DNA polymerase δ during DNA replication; in early dividing Drosophila embryos, it colocalizes with nuclei during interphase and occasionally in late anaphase or telophase, but never in metaphase. PCNA was never detected on either the sperm nucleus or the anaphase-arrested female nucleus. In wild-type controls, staining was observed on the male and female apposed pronuclei 70% of the time. Sperm nuclei prior to apposition did not stain with PCNA in the wild-type controls (Horner, 2006).

Fertilized eggs for the mitotic marker phosphohistone H3 (Serine 10), which is normally deposited on chromosomes during prophase. Maternally derived chromosomes stained intensely, as expected given their cell-cycle arrest, but the majority of sperm nuclei (n = 8/9) in the same embryos did not stain for phosphohistone H3. Together, these findings indicate that the sperm nuclei are arrested in a pre-S phase, premitotic state in the absence of maternal sra function. Although it may appear paradoxical that the male and female genomes in these eggs are arrested at very different stages of the cell cycle, precedents in sea urchin zygotes indicate that the cell cycles of male and female pronuclei are independently regulated, probably by the differential accumulation of active MPF in the respective nuclei (Horner, 2006).

Fertilized sra eggs were stained for tubulin as well as for DNA to determine whether the sperm nucleus could elaborate the prominent sperm aster along which the female pronucleus migrates to allow pronuclear apposition. The sperm aster is first visible in wild-type zygotes when the female genome is undergoing anaphase II and then grows enormously during telophase II. However, no sperm aster, nor indeed any organized microtubule system associated with sperm nuclei, was observed in sra eggs (Horner, 2006).

Sperm nuclei in sra mutant eggs thus become arrested prior to DNA replication, prophase of the first mitotic division, formation of the sperm aster, and nuclear apposition. The results taken together indicate that sperm nuclear-envelope breakdown, initial chromatin decondensation, and histone exchange are dependent on male-supplied factors such as sneaky and maternal factors including sesame, but these early steps are independent of sarah-mediated egg activation. Later steps in sperm activation do require Sarah and concomitant egg activation (Horner, 2006).

Cyclin B is elevated in sra mutant eggs

Because injection of nondegradable cyclin B causes early anaphase arrest of syncytial blastoderm mitoses, cyclin B elevation might explain the anaphase I arrest seen in sra mutant embryos. Thus lysates from fertilized, laid eggs of sra mutant females were probed for cyclin B on Western blots. As controls, eggs from wild-type females (fertilized and unfertilized) and sra heterozygotes were also tested, as were activated eggs from cortex mutant mothers that were terminally arrested in metaphase II. Eggs from sra mothers showed an elevation of cyclin B when they were compared to those from sra heterozyogotes and wild-type fertilized eggs. It is presumed that the low levels of cyclin B in the latter embryos result from asynchronous development of the population during the 2 hr collection, so that only a small proportion of embryos are in mitosis. Wild-type unfertilized eggs and cortex eggs showed elevated cyclin B levels similar to those seen in sra mutants, consistent with meiotic arrest. These results likely reflect a role for Sarah as a regulator of M phase-promoting factor (MPF) during egg activation (Horner, 2006).

A model for the role of sarah in egg activation

In vertebrates and marine invertebrates, fertilization triggers a transient rise in free Ca²⁺, and this rise is responsible for subsequent activation events such as modification of the eggshell, prevention of polyspermy, and cell-cycle resumption. This study shows that a protein involved in a calcium signal-transduction pathway is necessary for several egg-activation events in Drosophila. The sarah gene product is a calcipressin; the human calcipressin DSCR1 can directly bind to and inhibit calcineurin, the only known phosphatase that is dependent on both calcium and calmodulin. The accompanying paper by Takeo (2006) verifies that the fly Sarah protein shares these biochemical properties (Horner, 2006).

The present understanding of egg activation in Xenopus and the activities of calcipressin make strong predictions for the role of sra in meiotic reactivation. Upon fertilization of frog eggs, Ca²⁺ activates calmodulin-dependent protein kinase II (CaMKII), which in turn directs the inactivation of Anaphase Promoting Complex (APC) inhibitors such as Erp1/Emi2. The APC in turn inactivates MPF through the destruction of cyclin, relieving the meiotic block and initiating other egg-activation events. The current results suggest that essentially the same pathway operates during the activation of Drosophila eggs. It is hypothesized that Sarah acts early in the pathway by mediating the antagonistic relationship between calcineurin and CaMKII; that is, CaMKII activity upon egg activation requires the inhibition of calcineurin by Sarah (Horner, 2006).

Consistent with this model is the similarity of the sarah phenotype to that associated with mutations in another Drosophila gene called cortex. The Cortex protein is a member of the Cdc20 protein family, whose members serve as specificity factors and activators for the APC. Meiosis arrests normally at metaphase I in cortex oocytes and resumes when the eggs are laid but then soon arrests again at metaphase II. The variation in the phase of the meiotic arrest (metaphase II versus anaphase I) being an exception, other aspects of egg activation are similarly affected by mutations in cortex and sra (Horner, 2006).

The meiotic arrest in both sra and cortex eggs is most simply explained by the failure of the APC to target certain molecules for degradation upon egg laying. In cortex eggs, cyclin A fails to be degraded. The cyclin B component of MPF remains undegraded in sra eggs. These results are consistent with the mitotic-arrest phenotypes seen in early Drosophila embryos expressing nondegradable cyclin A (metaphase arrest) or nondegradable cyclin B (early anaphase arrest). Different APC targets could remain undegraded in sra and cortex eggs because there are at least two Cdc20-like proteins (Cortex and Fzy) in Drosophila eggs. APC^Cortex and APC^Fzy may have different substrate specificities and may be differently regulated by APC inhibitors downstream of CaMKII (Horner, 2006).

The failure of the eggs laid by sra mutant mothers to translate the maternal bcd mRNA can also be understood in the same theoretical framework. The translation of bcd requires the polyadenylation of its mRNA; the enzyme poly(A) polymerase that catalyzes polyadenylation is phosphorylated and thus negatively regulated by MPF. Failure of APC activation in sra mutant eggs would therefore prevent both MPF inactivation and bcd mRNA translation (Horner, 2006).

Although meiosis and bcd translation are both disrupted in sra eggs, vitelline membrane (VM) cross-linking is apparently normal. VM cross-linking also occurs in other mutants that block female meiotic progression; such mutants include cortex, grauzone, prage, and wispy. In fact, VM reorganization can occur independently of every other known egg-activation event, including bcd mRNA translation, the degradation of other maternal mRNAs, and microtubule depolymerization. If there is only a single trigger for egg activation, it must therefore be able to activate at least two autonomous downstream pathways (one for VM cross-linking and a second for other events). Because in vitro activated eggs are defective in several aspects of embryonic development, it is difficult to interpret the finding of delayed VM modification in sra eggs upon in vitro activation. Although sra function is not formally required for VM organization, sra-dependent processes might nonetheless impinge on its efficiency (Horner, 2006).

In summary, results indicate that 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).

Expression level of sarah, a homolog of DSCR1, is critical for ovulation and female courtship behavior in Drosophila

To better understand the genetic bases of postmating responses in Drosophila melanogaster females, a collection of P{GS} insertion lines were screened and two insertions in were identified sarah (sra), whose misexpression in the nervous system induced high levels of ovulation in virgins. The gene sra encodes a protein similar to human Down syndrome critical region 1 (DSCR1). The ovulation phenotype was reproduced in transgenic virgins expressing UAS-sra in the nervous system. The flies also extruded the ovipositor toward courting males as seen in wild-type mated females, supporting the notion that ovulation and behavioral patterns are physiologically coupled. The sra insertions were found to be hypomorphic alleles with reduced expression levels. Females homozygous for these alleles show: (1) spontaneous ovulation in virgins, (2) sterility with impaired meiotic progression, and (3) compromised postmating responses with lower ovulation level, higher remating rate, and shorter period for restoration of receptivity. No obvious defects were observed in the homozygous males. The gene sra is predominantly expressed in oocytes, nurse cells, and the nervous system. Taken together, these results indicate that the expression level of sra is critical for ovulation and female courtship behavior, including their postmating changes (Ejima, 2004; full text of paper).

The Drosophila homolog of Down syndrome critical region 1 gene regulates learning: implications for mental retardation

Mental retardation is the most common phenotypic abnormality seen in Down syndrome (DS) patients, yet the underlying mechanism remains mysterious. DS critical region 1 (DSCR1), located on chromosome 21, is overexpressed in the brain of DS fetus and encodes an inhibitor of calcineurin, but its physiological significance is unknown. To study its functional importance and role in mental retardation in DS, Drosophila mutants of nebula, an ortholog of human DSCR1, were generated. Both nebula loss-of-function and overexpression mutants exhibit severe learning defects that are attributed by biochemical perturbations rather than maldevelopment of the brain. These results, combined with data showing that the same biochemical signaling pathway is altered in human DS fetal brain tissue overexpressing DSCR1, suggest that alteration of DSCR1 expression could contribute to mental retardation in DS (Chang, 2003; full text of article).

To investigate the role of nebula in learning and memory, a Pavlovian olfactory associative learning and memory test was used. Significant defect in learning was observed after a single trial of training for homozygous nla¹ and nla² mutants, as compared to nla¹ heterozygotes, precise excision line (nla^PJ), and CS flies. The learning defects are not attributed by abnormal sensorimotor responses, because both odor and shock avoidances were indistinguishable among the fly lines tested. Nevertheless, the possibility that some of the performance defects seen in the nebula mutants could be caused by subtle alteration in sensitivity to stimuli cannot be ruled out (Chang, 2003).

Short-term memory is thought to last 60 min after training in Drosophila. Thus short-term memory was investigated in different nebula alleles by examining the performance at different time points after training. The same rate of normal memory decay seen for the different fly lines suggests that short-term memory is likely intact and that the low performance value obtained right after training may be a reflection of defective learning, albeit the possibility cannot be ruled out that nebula is required for immediate short-term memory (during the 3 min in between training and the first possible test time point). To further examine the role of nebula in the memory pathway, long-term memory was tested in the nebula mutants. Long-term memory in Drosophila is associated with phosphorylated cAMP-responsive element binding protein (pCREB) and protein synthesis and is evident 1 day after 10 trials of spaced training with 15-min rest intervals between trainings. Strikingly, it was found that homozygous nebula mutants showed a virtual absence of long-term memory, whereas control flies displayed ~40% memory retention 24 h after training. The observed defect in long-term memory is not caused by the initial deficiency in acquisition, because the performance of nla¹ and nla² homozygotes immediately after spaced training did not differ significantly from CS flies. Together, these results indicate that increasing the number of training sessions can improve the learning performance of nebula mutants, and more importantly, nebula is required for effective learning and long-term memory (Chang, 2003).

Despite the overall normal brain structure, it is possible that the learning and memory deficits seen for the nebula mutants are caused by structural defects specifically in the mushroom bodies, the center for learning and memory in Drosophila. To examine the integrity of the mushroom bodies, the α, β, and γ lobes were visualized with Fasciclin II antibody. Of all 60 nla¹ homozygous mutant flies examined, no visible structural defect in the mushroom bodies was detected, and the α, β, and γ lobes were similar to the control flies. This finding implies that the nebula mutation does not cause maldevelopment of the mushroom bodies and that defects in learning and memory are not caused by gross structural defect (Chang, 2003).

To understand whether the mutation in nebula significantly alters the biochemical activity predicted for the gene, calcineurin activity level was examined in the nebula mutant flies. Calcineurin activity detected in the homogenate of nla¹ homozygotes was ~40% higher than CS control and nla¹ heterozygotes, indicating that nebula functions as an endogeneous inhibitor of calcineurin as other members in the calcipressin family. The signaling pathways downstream from calcineurin was examined and it was found that homozygous nla¹ mutants showed a substantial decrease in cAMP-dependent protein kinase (PKA) activity, the amount of pCREB, and the level of d-jun transcripts. Given that PKA and pCREB are important for normal learning and long-term memory, the observed biochemical perturbations in the nebula mutant may contribute to the observed deficiency in learning and memory (Chang, 2003).

By using Drosophila as a simple model organism, this study demonstrated that nebula mediates learning and long-term memory. Observations that both nebula loss-of-function and overexpression mutants displayed learning defects suggest that precise regulation of nebula-mediated calcineurin signaling is necessary to maintain optimum learning. Furthermore, these results, along with data that the same biochemical pathway is disrupted in human trisomy 21 fetal brain tissue, strongly implicate the involvement of DSCR1 in mental retardation in DS (Chang, 2003).

This study focused on the function of DSCR1 in learning and memory to address its role in mental retardation associated with DS. However, DSCR1 is also expressed in other tissues such as the heart and skeletal muscle and may thus regulate diverse calcineurin-dependent processes. Furthermore, calcineurin has been shown to regulate the transcription of DSCR1, suggesting the existence of a complex negative feedback regulation circuit. It will be interesting to investigate whether such regulatory mechanism also controls nebula expression in flies. Combined with its amenability to genetic manipulations and behavioral assays, Drosophila can be used to identify some of the genotype-phenotype relations in DS. In addition, the finding that defective learning in nebula overexpressing flies is likely caused by functional defects rather than developmental defects raises the exciting possibility of pharmacological intervention to ameliorate at least some of the cognitive deficits in DS patients. Drosophila may be used as a tool to rapidly screen for drugs that treat learning and memory deficits by restoring the balance of kinases and phosphatases. Genetic screens that identify suppressors of nebula in the learning and memory pathway will also provide insights into the underlying mechanism of mental retardation in DS (Chang, 2003).

Drosophila melanogaster homolog of Down syndrome critical region 1 is critical for mitochondrial function

Mitochondrial dysfunction has emerged as a common theme that underlies numerous neurological disorders, including Down syndrome. Down syndrome cultures and tissues show mitochondrial damage such as impaired mitochondrial enzyme activities, defective mitochondrial DNA repairs and accumulation of toxic free radicals, but the cause of mitochondrial dysfunction remains elusive. This study demonstrated that the Drosophila melanogaster homolog of human Down syndrome critical region gene 1 (DSCR1), nebula (also known as sarah, sra), has a crucial role in the maintenance of mitochondrial function and integrity. nebula protein is located in the mitochondria. An alteration in the abundance of nebula affects mitochondrialenzyme activities, mitochondrial DNA content, and the number and size of mitochondria. Furthermore, nebula interacts with the ADP/ATP translocator and influences its activity. These results identify nebula/DSCR1 as a regulator of mitochondrial function and integrity and further suggest that an increased level of DSCR1 may contribute to the mitochondrial dysfunction seen in Down syndrome (Chang, 2005).

EVOLUTIONARY HOMOLOGS

Identification of calcipressin genes

Calcineurin is the conserved target of the immunosuppressants cyclosporin A and FK506. Using the yeast two-hybrid system, a novel calcineurin binding protein, CBP1, as been identified from the pathogenic fungus Cryptococcus neoformans. CBP1 binds to calcineurin in vitro and in vivo, and FKBP12-FK506 inhibits CBP1 binding to calcineurin. Cryptococcus neoformans cbp1 mutant strains exhibit modest defects in growth under stress conditions and virulence, similar to but less severe than the phenotypes of calcineurin mutants. Saccharomyces cerevisiae mutants lacking the CBP1 homolog RCN1 are, like calcineurin mutants, sensitive to lithium cation stress. CBP1 shares a central peptide sequence motif, SPPxSPP, with related proteins in S. cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster, Caenorhabditis elegans and humans, and peptides containing this motif altered calcineurin activity in vitro. Interestingly, the human CBP1 homolog DSCR1 is encoded by the Down syndrome candidate region interval on chromosome 21, is highly expressed in the heart and central nervous system, and may play a role in calcineurin functions in heart development, neurite extension and memory (Gorlach, 2000).

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).

A recently recognized gene family, conserved from yeast to humans, includes Down syndrome candidate region 1 gene (DSCR1), Adapt78 (recognized as the hamster ortholog of the DSCR1 isoform 4), ZAKI-4 (renamed DSCR1-like 1, DSCR1L1) and DSCR1L2 (a novel gene on human chromosome 1), along with yeast and C. elegans single members. The proposed family identifies are a putative single-strand nucleic acid binding domain similar to the RNA recognition motif, and a unique, highly-conserved serine-proline motif. A bioinformatics-driven molecular biology approach was used to characterize the murine members of DSCR1-like gene family. Systematic EST database search and RT-PCR allowed identification of the murine DSCR1, DSCR1L1 and DSCR1L2. The sequences of the respective protein products are of 198, 197 and 241 amino acids, respectively, and are very similar to the corresponding human proteins. The very broad expression pattern of the murine DSCR1 genes is similar to that of the human genes. Using a radiation hybrid panel, the murine DSCR1-like family members were mapped. The murine DSCR1 ortholog is located on the chromosome 16, in a region corresponding to that on human chromosome 21 just upstream of the Down syndrome candidate region. DSCR1L1 and DSCR1L2 murine genes are also located in chromosomal segments of chromosome 17 and 4, respectively, exactly corresponding to those containing the respective human homologs on chromosomes 6 and 1. Description of the mouse orthologs for DSCR1-like genes will allow knockout mice to be obtained for specific family members (Strippoli, 2000).

The Caenorhabditis elegans homologue of Down 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 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).

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 syndrome is one of the major causes of mental retardation and congenital heart malformations. Other common clinical features of Down 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 syndrome. DSCR1, the product of a chromosome 21 gene highly expressed in brain, heart and skeletal muscle, is overexpressed in the brain of Down 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 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).

Transcriptional regulation of mammalian calcipressin

The transcriptional regulation of Down syndrome critical region isoform 4 (DSCR1.4) is mediated by the calcineurin/nuclear factor of activated T cells (NFAT) pathway in neural cells. Stimuli that elicit an increase in the intracellular concentrations of calcium, such as membrane depolarization, induced de novo transcription of DSCR1.4, with mRNA expression peaking after 4 h and then declining. Action via the physiologically relevant L-type calcium channel was confirmed by blockade with nifedipine and verapamil. This calcium-dependent transcription of DSCR1.4 is inhibited by the calcineurin inhibitors cyclosporin A and FK506. Deletional analysis showed that the calcium- and calcineurin-dependent activation is mediated by the promoter region between nucleotides -350 and -166, a region that contains putative NFAT-binding motifs. Exogenous NFATc2 potently augmented the DSCR1.4 promoter transcriptional activity, and the involvement of endogenous NFAT signaling pathway in DSCR1.4 transcription was confirmed by the suppression of depolarization-inducible promoter activity with the NFAT inhibitor peptide VIVIT. Exogenous overexpression of DSCR1 protein (calcipressin 1) resulted in the inhibition of the transcription of DSCR1.4 and NFAT-dependent signaling. These findings suggest that calcineurin-dependent induction of DSCR1.4 product may represent an important auto-regulatory mechanism for the homeostatic control of NFAT signaling in neural cells (Cano, 2005).

Expression of DSCR1/Calcipressin

DSCR1 acts as a negative regulator of calcineurin-mediated signaling and its transcript is overexpressed in the Down syndrome (DS) fetal brain. To evaluate the possible involvement of DSCR1 in DS, the mouse gene was cloned and its expression was analyzed in the central nervous system (CNS). Early expression of Dscr1 is detected mainly in the heart tube and in the CNS in rhombomere 4 and the pretectum. From embryonic day 14.5 onwards, Dscr1 is widely distributed in the CNS but becomes more restricted as the brain matures. Its neuronal expression pattern in the adult, preferentially in Purkinje and pyramidal cells, was confirmed by double labeling with glial fibrillary acidic protein. Although Dscr1 is present in trisomy in the Ts65Dn mouse, the adult brain expression pattern is not significantly altered. This expression pattern indicated that Dscr1 is a developmentally regulated gene involved in neurogenesis and cardiogenesis and suggests that it may contribute to the alterations observed in these organ systems in DS patients (Casas, 2001).

Calcipressin mutation

Calcineurin links calcium signaling to transcriptional responses in the immune, nervous and cardiovascular systems. To determine the function of the calcipressins, a family of putative calcineurin inhibitors, the calcineurin-dependent process of T cell activation was assessed in mice engineered to lack the gene encoding calcipressin 1 (Csp1). Csp1 regulates calcineurin in vivo, and genes triggered in an immune response have unique transactivation thresholds for T cell receptor stimulation. In the absence of Csp1, the apparent transactivation thresholds for all these genes are shifted because of enhanced calcineurin activity. This unbridled calcineurin activity drives Fas ligand expression, which normally requires high T cell receptor stimulation and results in the premature death of T helper type 1 cells. Thus, calcipressins modulate the pattern of calcineurin-dependent transcription, and may influence calcineurin activity beyond calcium to integrate a broad array of signals into the cellular response (Ryeom, 2003).

Calcipressin protects against acute calcium-mediated stress damage, including transient oxidative stress

Although DSCR1 (Adapt78) has been associated with successful adaptation to oxidative stress and calcium stress and with devastating diseases such as Alzheimer's and Down syndrome, no rationale for these apparently contradictory findings has been tested. In fact, DSCR1 (Adapt78) has not yet been proved to provide protection against acute oxidative stress or calcium stress. This question was addressed using cross-adaptation to H2O2 and the calcium ionophore A23187, stable DSCR1 (Adapt78) transfection and overexpression in hamster HA-1 cells, 'tet-off' regulated DSCR1 (Adapt78) isoform 1 transgene expression in human PC-12 cells, and DSCR1 (Adapt78) antisense oligonucleotides to test the ability of the DSCR1 (Adapt78) protein product calcipressin 1 (a calcineurin inhibitor) to protect against oxidative stress and calcium stress. Under all conditions, resistance to oxidative stress and calcium stress increased as a function of DSCR1 (Adapt78)/calcipressin 1 expression and decreased as gene/protein expression diminished. It is concluded that cells may transiently use increased expression of the DSCR1 (Adapt78) gene product calcipressin 1 to provide short-term protection against acute oxidative stress and other calcium-mediated stresses, whereas chronic overexpression may be associated with Alzheimer's disease progression (Ermak, 2002).

RCAN1 (DSCR1 or Adapt78) stimulates expression of GSK-3beta

The RCAN1 protein (previously called calcipressin 1 or MCIP1) binds to calcineurin, a serine/threonine phosphatase (PP2B), and inhibits its activity. Regulated overexpression of an RCAN1 transgene (this gene was previously called DSCR1 or Adapt78) also stimulates expression of the GSK-3beta kinase, which can antagonize the action of calcineurin. GSK-3beta is regulated by RCAN1 at a post-transcriptional level. In humans, high RCAN1 expression is found in the brain, where at least two mRNA isoforms have been reported. Therefore, expression of the various RCAN1 isoforms, resulting from differential splicing and alternative promotors in human brain, was further examined. At least three distinct RCAN1s were detected: RCAN1-1 Short at 31 kDa (RCAN1-1S), RCAN1-1 Long at 38 kDa (RCAN1-1 L), and RCAN1-4. Furthermore, the levels of RCAN1-1S, but not RCAN1-1 L or RCAN1-4 correlated with the levels of GSK-3beta. This suggests that RCAN1-1S might induce production of GSK-3beta in vivo. While RCAN1s can regulate calcineurin and GSK-3beta, it has also been shown that calcineurin and GSK-3beta can regulate RCAN1s. This study proposes a new model (incorporating all these findings) in which cells maintain an equilibrium between RCAN1s, calcineurin, and GSK-3beta (Ermak, 2006).

Integration of Notch 1 and calcineurin/NFAT signaling pathways in keratinocyte growth and differentiation control; Increased Calcineurin/NFAT activity by Notch signaling involves downregulation of Calcipressin

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, 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).

DSCR1, a downstream target of VEGF, participates in endothelial cell migration and angiogenesis

Vascular endothelial growth factor (VEGF) is a principal stimulator of angiogenesis. However, the downstream targets of VEGF in endothelial cells (ECs) are not entirely clarified. Survey of downstream targets of VEGF in human ECs identified a number of genes, including Down syndrome candidate region 1 (DSCR1). This study confirmed the inducible expression of DSCR1 in ECs by Northern and Western blottings. Moreover, VEGF-stimulated induction of DSCR1 is blocked by anti-VEGF receptor-2 monoclonal antibody (mAb), or the specific calcineurin inhibitors cyclosporin A and FK506. The expression of DSCR1 in ECs of neovessels was shown by immunohistochemical analysis. Whether DSCR1 plays any roles in angiogenesis was examined. The specific downregulation of DSCR1 expression by antisense oligonucleotide (AS-ODN) inhibits VEGF-stimulated migration of ECs as well as angiogenesis in vivo. AS-ODN inhibits the spreading of ECs on vitronectin, as well as on the immobilized anti-alphavbeta3 mAb, but not on anti-alphavbeta5 mAb. Moreover, AS-ODN inhibits tyrosine phosphorylation of focal adhesion kinase when ECs are plated on a vitronectin-coated dish. Immunoprecipitation followed by Western blotting showed the coimmunoprecipitation of DSCR1 and integrin alphavbeta3. These results suggest that DSCR1 is involved in angiogenesis by regulating adhesion and migration of ECs via the interaction with integrin alphavbeta3 (Iizuka, 2004).

Down syndrome candidate region 1 isoform 1 mediates angiogenesis through the calcineurin-NFAT pathway

Down syndrome candidate region 1 (DSCR1) is one of more than 50 genes located in a region of chromosome 21 that has been implicated in Down syndrome. DSCR1 can be expressed as four isoforms, one of which, isoform 4 (DSCR1-4), has recently been found to be strongly induced by vascular endothelial growth factor A [VEGF-A(165)] and to provide a negative feedback loop that inhibits VEGF-A(165)-induced endothelial cell proliferation in vitro and angiogenesis in vivo. Another DSCR1 isoform, DSCR1-1L, is also up-regulated by VEGF-A(165) in cultured endothelial cells and is strongly expressed in several types of pathologic angiogenesis in vivo. In contrast to DSCR1-4, the overexpression of DSCR1-1L induced the proliferation and activation of the transcription factor NFAT in cultured endothelial cells and promoted angiogenesis in Matrigel assays in vivo, even in the absence of VEGF-A. Similarly, small interfering RNAs specific for DSCR1-1L and DSCR1-4 had opposing inhibitory and stimulatory effects, respectively, on these same functions. DSCR1-4 is thought to inhibit angiogenesis by inactivating calcineurin, thereby preventing activation and nuclear translocation of NFAT, a key transcription factor. In contrast, DSCR1-1L, regulated by a different promoter than DSCR1-4, activates NFAT and its proangiogenic activity is inhibited by cyclosporin, an inhibitor of calcineurin. In sum, DSCR1-1L, unlike DSCR1-4, potently activates angiogenesis and could be an attractive target for antiangiogenesis therapy (Qin, 2006).

Chronic overexpression of the calcineurin inhibitory gene DSCR1 is associated with Alzheimer's disease

It was hypothesized that DSCR1 (Adapt78) might also be involved in the development of Alzheimer's disease. To address this question DSCR1 was examined in multiple human tissues and significant expression was found in brain, spinal cord, kidney, liver, mammary gland, skeletal muscle, and heart. Within the brain DSCR1 is predominantly expressed in neurons within the cerebral cortex, hippocampus, substantia nigra, thalamus, and medulla oblongata. When DSCR1 mRNA expression in post-mortem brain samples from Alzheimer's disease patients was compared with expression in individuals who had died with no Alzheimer's diagnosis, it was found that DSCR1 mRNA levels were about twice as high in age-matched Alzheimer's patients as in controls. DSCR1 mRNA levels were actually three times higher in patients with extensive neurofibrillary tangles (a hallmark of Alzheimer's disease) than in controls. In comparison, post-mortem brain samples from Down syndrome patients (who suffer Alzheimer's symptoms) also exhibited DSCR1 mRNA levels two to three times higher than controls. Using a cell culture model it was discovered that the amyloid beta(1-42) peptide, which is a major component of senile plaques in Alzheimer's, can directly induce increased expression of DSCR1. These findings associate DSCR1 with such major hallmarks of Alzheimer's disease as amyloid protein, senile plaques, and neurofibrillary tangles (Ermak, 2001).

Increased dosage of DYRK1A and DSCR1 delays neuronal differentiation in neocortical progenitor cells

Down's syndrome (DS), a major genetic cause of mental retardation, arises from triplication of genes on human chromosome 21. This study shows that DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A; Drosophila homolog - Minibrain) and DSCR1 (DS critical region 1; Drosophila homolog - Nebula/Sarah), two genes lying within human chromosome 21 and encoding for a serine/threonine kinase and calcineurin regulator, respectively, are expressed in neural progenitors in the mouse developing neocortex. Increasing the dosage of both proteins in neural progenitors leads to a delay in neuronal differentiation, resulting ultimately in alteration of their laminar fate. This defect is mediated by the cooperative actions of DYRK1A and DSCR1 in suppressing the activity of the transcription factor NFATc. In Ts1Cje mice, a DS mouse model, dysregulation of NFATc in conjunction with increased levels of DYRK1A and DSCR1 were observed. Furthermore, counteracting the dysregulated pathway ameliorates the delayed neuronal differentiation observed in Ts1Cje mice. In sum, these findings suggest that dosage of DYRK1A and DSCR1 is critical for proper neurogenesis through NFATc and provide a potential mechanism to explain the neurodevelopmental defects in DS (Kurabayashi, 2013).

DSCR1 and Down syndrome

The Down syndrome critical region 1 (DSCR1) gene is present in the region of human chromosome 21 and the syntenic region of mouse chromosome 16, trisomy of which is associated with congenital heart defects observed in Down syndrome. DSCR1 encodes a regulatory protein in the calcineurin/NFAT signal transduction pathway. During valvuloseptal development in the heart, DSCR1 is expressed in the endocardium of the developing atrioventricular and semilunar valves, the muscular interventricular septum, and the ventricular myocardium. Human DSCR1 contains an NFAT-rich calcineurin-responsive element adjacent to exon 4. Transgenic mice generated with a homologous regulatory region of the mouse DSCR1 gene linked to lacZ (DSCR1(e4)/lacZ) show gene activation in the endocardium of the developing valves and aorticopulmonary septum of the heart, recapitulating a specific subdomain of endogenous DSCR1 cardiac expression. DSCR1(e4)/lacZ expression in the developing valve endocardium colocalizes with NFATc1 and, endocardial DSCR1(e4)/lacZ, is notably reduced or absent in NFATc1^−/− embryos. Furthermore, expression of the endogenous DSCR1(e4) isoform is decreased in the outflow tract of NFATc1^−/− hearts, and the DSCR1(e4) intragenic element is trans-activated by NFATc1 in cell culture. In trisomy 16 (Ts16) mice, expression of endogenous DSCR1 and DSCR1(e4)/lacZ colocalizes with anomalous valvuloseptal development, and transgenic Ts16 hearts have increased β-galactosidase activity. DSCR1 and DSCR1(e4)/lacZ also are expressed in other organ systems affected by trisomy 16 in mice or trisomy 21 in humans including the brain, eye, ear, face, and limbs. Together, these results show that DSCR1(e4) expression in the developing valve endocardium is dependent on NFATc1 and support a role for DSCR1 in normal cardiac valvuloseptal formation as well as the abnormal development of several organ systems affected in individuals with Down syndrome (Lange, 2004).

Trisomy 21 results in Down syndrome, but little is known about how a 1.5-fold increase in gene dosage produces the pleiotropic phenotypes of Down syndrome. Two genes, DSCR1 and DYRK1A, lie within the critical region of human chromosome 21 and act synergistically to prevent nuclear occupancy of NFATc transcription factors, which are regulators of vertebrate development. Mathematical modelling was used to predict that autoregulation within the pathway accentuates the effects of trisomy of DSCR1 and DYRK1A, leading to failure to activate NFATc target genes under specific conditions. Observations of calcineurin-and Nfatc-deficient mice, Dscr1- and Dyrk1a-overexpressing mice, mouse models of Down syndrome and human trisomy 21 are consistent with these predictions. It is suggested that the 1.5-fold increase in dosage of DSCR1 and DYRK1A cooperatively destabilizes a regulatory circuit, leading to reduced NFATc activity and many of the features of Down syndrome. More generally, these observations suggest that the destabilization of regulatory circuits can underlie human disease (Arron, 2006).

REFERENCES

Search PubMed for articles about Drosophila sarah

Arron, J. R., et al. (2006). NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21. Nature 441(7093): 595-600. Medline abstract: 16554754

Aubareda, A., Mulero, M. C. and Perez-Riba, M. (2006). Functional characterization of the calcipressin 1 motif that suppresses calcineurin-mediated NFAT-dependent cytokine gene expression in human T cells. Cell Signal 18(9): 1430-8. Medline abstract: 16406492

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Ermak, G., Morgan, T. E. and Davies, K. J. (2001). Chronic overexpression of the calcineurin inhibitory gene DSCR1 (Adapt78) is associated with Alzheimer's disease. J. Biol. Chem. 276(42): 38787-94. Medline abstract: 11483593

Ermak, G., Harris, C. D. and Davies, K. J. (2002). The DSCR1 (Adapt78) isoform 1 protein calcipressin 1 inhibits calcineurin and protects against acute calcium-mediated stress damage, including transient oxidative stress. FASEB J. 16(8): 814-24. Medline abstract: 12039863

Ermak, G., Harris, C. D., Battocchio, D. and Davies, K. J. (2006). RCAN1 (DSCR1 or Adapt78) stimulates expression of GSK-3beta. FEBS J. 273(10): 2100-9. Medline abstract: 16649988

Fuentes, J. J., et al. (2000). DSCR1, overexpressed in Down syndrome, is an inhibitor of calcineurin-mediated signaling pathways. Hum. Mol. Genet. 9: 1681-1690. Medline abstract: 10861295

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Kurabayashi, N. and Sanada, K. (2013). Increased dosage of DYRK1A and DSCR1 delays neuronal differentiation in neocortical progenitor cells. Genes Dev 27: 2708-2721. PubMed ID: 24352425

Lange, A. W., Molkentin, J. D. and Yutzey, K. E. (2004). DSCR1 gene expression is dependent on NFATc1 during cardiac valve formation and colocalizes with anomalous organ development in trisomy 16 mice. Dev. Biol. 266(2): 346-60. Medline abstract: 14738882

Lee, J. I., et al. (2003). The Caenorhabditis elegans homologue of down syndrome critical region 1, RCN-1, inhibits multiple functions of the phosphatase calcineurin. J. Mol. Biol. 328: 147-156. Medline abstract: 12684004

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date revised: 23 June 2023

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