Over-Expression of DSCR1 Protects against Post-Ischemic Neuronal Injury

Background and Purpose

The Down syndrome candidate region 1 (DSCR1) gene is located on human chromosome 21 and its protein is over-expressed in brains of Down syndrome individuals. DSCR1 can modulate the activity of calcineurin, a phosphatase abundant in the brain, but its influence on stroke outcome is not clear. We compared stroke outcome in wildtype (WT) and transgenic (DSCR1-TG) mice which over-express isoform 1 of human DSCR1.

Methods

Transient cerebral ischemia was produced by occlusion of the middle cerebral artery for 0.5 h. After 23.5 h reperfusion, we assessed neurological impairment, brain infarct and edema volume, leukocyte infiltration and markers of inflammation. Intrinsic resistance to apoptosis following glucose deprivation was also assessed in primary cultures of WT and DSCR1-TG neurons.

Results

In contrast to WT, DSCR1-TG mice had an improved neurological deficit score, greater grip strength, attenuated infarct volume and brain swelling, and lacked hippocampal lesions after stroke. Expression of mouse DSCR1-1, but not DSCR1-4, mRNA and protein was increased by ischemia in both WT and DSCR1-TG. Brain calcineurin activity was increased to a similar degree after ischemia in each genotype. DSCR1-TG mice had fewer infiltrating neutrophils and activated microglia compared with WT, in association with an attenuated upregulation of several pro-inflammatory genes. Neurons from DSCR1-TG mice were more resistant than WT neurons to apoptotic cell death following 24 h of glucose deprivation.

Conclusions

Over-expression of DSCR1 in mice improves outcome following stroke. Mechanisms underlying this protection may involve calcineurin-independent, anti-inflammatory and anti-apoptotic effects mediated by DSCR1 in neurons.     

Down syndrome candidate region-1 protein interacts with Tollip and positively modulates interleukin-1 receptor-mediated signaling

Background

The Down syndrome candidate region-1 gene (DSCR1, also known as RCAN1) is situated close to the Down Syndrome Critical Region (DSCR), which contains genes responsible for many features of Down syndrome. DSCR1 modulates calcineurin phosphatase activity, though its functional role is incompletely understood.

Methods

Here we investigated the role of DSCR1-1S isoform in IL-1 receptor (IL-1R)-mediated signaling by analyzing interaction between DSCR1-1S and the IL-1R pathway components Tollip, IRAK-1, and TRAF6.

Results

Co-immunoprecipitation analyses of HEK293 cells revealed that DSCR1-1S interacted with Tollip, an IRAK-1 inhibitor, leading to the dissociation of IRAK-1 from Tollip. Similarly, both DSCR1-1S and Tollip interacted with TRAF6, with DSCR1 reducing interaction between Tollip and TRAF6. DSCR1-1S also stimulated IL-1R-mediated signaling pathways, TAK1 activation, NF-κB transactivation, and IL-8 production, all downstream consequences of IL-1R activation.

General significance

Together, these results suggest that DSCR1-1S isoform positively modulates IL-1R-mediated signaling pathways by regulating Tollip/IRAK-1/TRAF6 complex formation.     

Overexpression of the human <Emphasis Type="Italic">MNB/DYRK1A</Emphasis> gene induces formation of multinucleate cells through overduplication of the centrosome

Background  

Previously we cloned the human MNB/DYRK1A gene from the "Down syndrome critical region" on chromosome 21. This gene encodes a dual specificity protein kinase that catalyzes its autophosphorylation on serine/threonine and tyrosine residues. But, the functions of the MNB/DYRK1A gene in cellular processes are unknown.     

Induction of Apoptosis and Autophagy Using Ectopic DSCR1 Expression in Breast Cancer Cells

Down syndrome critical region 1 gene (DSCR1) is an anti-angiogenesis gene that inhibits the growth of tumor cells. In this study, the role of autophagy and apoptosis in DSCR1-induced cytotoxicity were investigated in MDA-MB-468 breast cancer cells. Lentivirus vector harboring DSCR1 (LV-DSCR1⁺) was constructed in HEK 293 cells and the optimal dosage of lentivirus vector for infection was determined by the MTT assay. After infection of cells using LV-DSCR1⁺, acridine orange and ethidium bromide staining was performed to investigation of apoptosis and autophagy. Expression of DSCR1 and marker genes for angiogenesis (VEGF), apoptosis (Bax and Bcl2) and autophagy (LC3 and Beclin) were determined by Real time PCR. The cellular morphological changes related to apoptosis and autophagy was happened after 48 hours of viral infection. Fragmented bright orange nucleuses and vacuoles were observed due to the cell apoptosis and autophagy after acridine orange and ethidium bromide staining. Upregulation of Bax, Lc3, DSCR1 and Beclin1 and downregulation of Bcl2 and VEGF was detected due to treatment with LV-DSCR1⁺. These results demonstrated that LV-DSCR1⁺ can induce apoptosis and autophagy, therefore suggesting that it may serves as an efficient tool to breast cancer treatment.     

Protein levels of genes encoded on chromosome 21 in fetal Down Syndrome brain (Part V): Overexpression of phosphatidyl-inositol-glycan class P protein (DSCR5)

Summary. Down Syndrome (DS, trisomy 21) is the most common genetic cause of mental retardation. The completed sequencing of genes encoded on chromosome 21 provides excellent basic information, however the molecular mechanisms leading to the phenotype of DS remain to be elucidated. Although overexpression of chromosome 21 encoded genes has been documented information at the protein expression level is mandatory as it is the proteins that carry out function. We therefore decided to evaluated expression level of seven proteins whose genes are encoded on chromosome 21: DSCR4, DSCR5, DSCR6; KIR4.2, GIRK2, KCNE1 and KCNE2 in fetal cortex brain of DS and controls at the early second trimester of pregnancy by Western blotting. -actin and neuron specific enolase (NSE) were used to normalise cell loss and neuronal loss. DSCR5 (PIG-P), a component of glycosylphosphatidylinositol-N-acetylglucosaminyltransferase (GPI-GnT), was overexpressed about twofold, even when levels were normalised with NSE. DSCR6 was overexpressed in addition but when normalised versus NSE, levels were comparable to controls. DSCR4 was not detectable in fetal brain. Potassium channels KIR4.2 and GIRK2 were comparable between DS and controls, whereas KCNE1 and KCNE2 were not detectable. Quantification of these proteins encoded on chromosome 21 revealed that not all gene products of the DS critical region are overexpressed in DS brain early in life, indicating that the DS phenotype cannot be simply explained by the gene dosage effect hypothesis. Overexpression of PIG-P (DSCR5) may lead to or represent impaired glycosylphosphatidylinositol-N-acetylglucosaminyltransferase mediated posttranslational modifications and subsequent anchoring of proteins to the plasma membrane.     

NF-kappaB-inducing kinase phosphorylates and blocks the degradation of Down syndrome candidate region 1

Down syndrome, the most frequent genetic disorder, is characterized by an extra copy of all or part of chromosome 21. Down syndrome candidate region 1 (DSCR1) gene, which is located on chromosome 21, is highly expressed in the brain of Down syndrome patients. Although its cellular function remains unknown, DSCR1 expression is linked to inflammation, angiogenesis, and cardiac development. To explore the functional role of DSCR1 and the regulation of its expression, we searched for novel DSCR1-interacting proteins using a yeast two-hybrid assay. Using a human fetal brain library, we found that DSCR1 interacts with NF-kappaB-inducing kinase (NIK). Furthermore, we demonstrate that NIK specifically interacts with and phosphorylates the C-terminal region of DSCR1 in immortalized hippocampal cells as well as in primary cortical neurons. This NIK-mediated phosphorylation of DSCR1 increases its protein stability and blocks its proteasomal degradation, the effects of which lead to an increase in soluble and insoluble DSCR1 levels. We show that an increase in insoluble DSCR1 levels results in the formation of cytosolic aggregates. Interestingly, we found that whereas the formation of these inclusions does not significantly alter the viability of neuronal cells, the overexpression of DSCR1 without the formation of aggregates is cytotoxic.     

Molecular and cellular characterization of the Down syndrome critical region protein 2

Down syndrome (DS) is caused by trisomy for human chromosome 21 and is the most common genetic cause of mental retardation. The distal 10 Mb region of the long arm of the chromosome has been proposed to be associated with many of the abnormalities seen in DS. This region is often referred to as the Down syndrome critical region (DSCR). We report here the results of our analyses of the DSCR protein 2 (DSCR2). Results from transiently transfected COS-1 and HEK293 cells suggest that DSCR2 is synthesized as a 43 kDa precursor protein, from which the N-terminus is cleaved resulting in a polypeptide of 41 kDa. The polypeptide is modified by still uncharacterized co- or post-translational modifications increasing the predicted molecular weight of 32.8 kDa by about 10 kDa. Analyses of the only putative N-glycosylation site by in vitro mutagenesis excluded the possibility of the contribution of N-glycosylation to this increase in molecular weight. Further, the results of intracellular localization studies and membrane fractionation assays indicate that DSCR2 is targeted to a cytoplasmic compartment as a soluble form.     

Epigenetic Modulations Induction Using DSCR1 Ectopic Expression in Breast Cancer Cells

Today, prognosis, diagnosis and treatment of cancers are progressing with non-invasive methods, including investigation and modification of the DNA methylation profile in cancer cells. One of the effective factors in regulating gene expression in mammals is DNA methylation. Methylation alterations of genes by external factors can change the expression of genes and inhibit the cancer. In the present study, we investigated the effect of Down syndrome critical region 1 gene (DSCR1) ectopic expression on the methylation status of the BCL-XL, ITGA6, TCF3, RASSF1A, DOK7, VIM and CXCR4 genes in breast cancer cell lines. The effect of DSCR1 ectopic expression on cell viability in MCF7, MDA-MB-468, MDA-MB-231 and MCF10A cell lines was evaluated using MTT assay after the cells treated by lentivirus vectors harboring DSCR1 for 72 hours. Methylation status of BCL-XL, ITGA6, TCF3, RASSF1A, DOK7, VIM and CXCR4 genes in breast cancer cell lines was assessed by Restriction Enzyme PCR (REP) method. Also, methylation changes of these genes in breast cancer cell lines after treatment by lentivirus vectors harboring DSCR1 for 7 days were analyzed by REP method. To confirm the effect of DSCR1 on methylation of genes, Real-time PCR was performed. The MTT assay results indicated that DSCR1 ectopic expression reduced cell viability in all three human breast cancer cell lines. Our results showed that DSCR1 ectopic expression after 6 days reversed the hypomethylation status of the BCL-XL, ITGA6, TCF3, VIM and CXCR4 genes and hypermethylation of RASSF1A and DOK7 genes. The expression levels of BCL-XL, ITGA6, TCF3, VIM and CXCR4 mRNA significantly reduced (P<0.05) and the expression levels of RASSF1A and DOK7 mRNA significantly increased (P<0.05). Our findings reveal for the first time the impact of DSCR1 ectopic expression on the methylation status of breast cancer cells and identify a novel agent for epigenetic therapy.     

Nebula/DSCR1 Upregulation Delays Neurodegeneration and Protects against APP-Induced Axonal Transport Defects by Restoring Calcineurin and GSK-3β 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, we explored the functional interaction between DSCR1 and the amyloid precursor protein (APP), which is known to cause AD when duplicated or upregulated in DS. We find that the Drosophila homolog of DSCR1, Nebula, delays 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, we show that Nebula upregulation protects against axonal transport defects by restoring calcineurin and GSK-3β 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, our findings imply that correcting calcineurin and GSK-3β signaling can prevent APP-induced pathologies. Our 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.     

Effects of <Emphasis Type="Italic">sarah/nebula</Emphasis> knockdown on Aβ42-induced phenotypes during <Emphasis Type="Italic">Drosophila</Emphasis> development

The Down syndrome critical region 1 (DSCR1), a Down syndrome-associated protein, is an endogenous inhibitor of the Ca²⁺-dependent phosphatase calcineurin. It has been also suggested to be associated with Alzheimer’s disease (AD) but the role of DSCR1 in the pathogenesis of AD still remains controversial. In this paper, we investigated the effects of knockdown of sarah (sra), a Drosophila DSCR1 ortholog, on the Aβ42-induced developmental phenotypes of Drosophila. Knockdown of sra showed detrimental effects on the rough eye phenotype and survival of Aβ42-expressing flies without altering the Aβ42 accumulation. Furthermore, the knockdown of sra increased glial cell numbers in the larval brains and its susceptibility to oxidative stress. Overexpression of an active form of calcineurin produced similar results to sra knockdown as they both exacerbated the Aβ42-induced rough eye phenotype. However, sra knockdown did not alter apoptosis or c-Jun N-terminal kinase activation in Aβ42-expressing flies. In conclusion, our results suggest that sra does play an important role in Aβ42-induced developmental defects in Drosophila without affecting its stress responses.     

Division of labor in the growth cone by DSCR1

Local protein synthesis directs growth cone turning of nascent axons, but mechanisms governing this process within compact, largely autonomous microenvironments remain poorly understood. In this issue, Wang et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201510107) demonstrate that the calcineurin regulator Down syndrome critical region 1 protein modulates both basal neurite outgrowth and growth cone turning.Connectivity and function of a mature nervous system requires precise wiring between neurons and with target cells throughout embryonic development. Severe to mild errors in connectivity lead to neurodevelopmental disorders ranging from profound intellectual deficits to subtle behavioral abnormalities (Van Battum et al., 2015). For example, evidence from many groups has shown defective neuronal connections in several autism spectrum disorders, such as Fragile X syndrome and tuberous sclerosis complex, as well as in motor deficits and degenerative muscle diseases (Nie et al., 2010; Doers et al., 2014; Bakos et al., 2015). However, most disease etiologies cannot be unilaterally attributed to abnormal axon guidance, as many of the relevant proteins are functional in other important neurological processes, such as dendritic spine maturation and function (Hoeffer and Klann, 2010; Holt and Schuman, 2013).One key player involved in morphogenesis and innervation of developing neurons is the nerve growth cone, the dynamic and motile sensory tip of growing axons and dendrites. Growth cones function with a large degree of autonomy from the cell soma, as they transduce contacted soluble and substratum-bound ligands into signals that coordinate cytoskeletal changes to regulate the rate and direction of axon outgrowth (Lowery and Van Vactor, 2009). Both growth-promoting and -inhibiting molecules are expressed along the pathways of developing axons and local discontinuities (e.g., gradients and borders) of extracellular cues are amplified into local biochemical changes within growth cones. Although many groups have given us insight into these processes over the past few decades, crucial mechanisms underlying guidance are still poorly understood (Goodhill, 2016). In this issue, Wang et al. demonstrate that Down syndrome critical region 1 protein (DSCR1) has two distinct roles in growth cones to control neurite outgrowth and guidance.Importantly, biochemical changes within growth cones have been shown to both directly modulate the cytoskeleton and indirectly affect motility by regulating local synthesis of new proteins (Holt and Schuman, 2013). Despite significant advances, it is still unclear why and how growth cones use protein synthesis–independent and –dependent mechanisms to regulate motility. Precise spatiotemporal control of translation likely provides additional levels of cellular regulation. For example, local protein synthesis is controlled by numerous mRNA binding and trafficking proteins, which may be regulated by classic second messengers (Akiyama and Kamiguchi, 2015). Many proteins synthesized at the growth cone are ubiquitinated at a higher rate than those trafficked from the cell body, and protein degradation is regulated by axon guidance cues (Deglincerti et al., 2015). Distinct pathways could also be activated by newly synthesized proteins via their relative lack of posttranslational modifications. Finally, new protein synthesis may sensitize growth cones to different types and concentrations of ligands. Interestingly, in vitro experiments have shown that basal axon outgrowth is independent of local protein synthesis, whereas local protein synthesis is necessary for guidance. For example, Nie et al. (2010) found that in a mouse model of tuberous sclerosis complex, which displays a defect in the regulation of the mTOR complex (a translational hub in the growth cone), Ephrin A–dependent local protein synthesis was required for proper retinogeniculate mapping, but no defects in retinal axon growth were observed (Nie et al., 2010). It is also interesting to note that several inherited autism spectrum disorders exhibit misregulation of protein synthesis (Van Battum et al., 2015), suggesting these mechanisms have important roles in human central nervous system assembly.Down syndrome, or trisomy 21, affects human development and is caused, in part, by elevated expression of genes encoded by chromosome 21, resulting in intellectual disabilities. One particular protein implicated is DSCR1 (Fuentes et al., 2000), also known as regulator of calcineurin (RCAN1). One established function of DSCR1 is to inhibit calcineurin (CaN), which is a calcium- and calmodulin-dependent serine/threonine protein phosphatase. DSCR1 binds and inhibits CaN, whereas phosphorylation of DSCR1 releases CaN, which may actively or passively lead to CaN activation. DSCR1 and CaN are highly expressed in developing neurons (Fuentes et al., 2000), where they may cooperate to control morphological differentiation. DSCR1 also interacts with Fragile X mental retardation protein (FMRP), which is lost in Fragile X syndrome (Verkerk et al., 1991; Wang et al., 2012). FMRP is an mRNA binding protein that regulates local protein synthesis in dendritic spines and neuronal growth cones (Ashley et al., 1993; Sidorov et al., 2013). Moreover, previous results from Chang et al. (2013) suggest that DSCR1 and FMRP1 may participate in common biological pathways leading to intellectual disability, including the maturation of dendritic spines.In their most recent work, Wang et al. (2016) demonstrate that DSCR1 serves a dual function in growth cones to regulate both neurite outgrowth and guidance. Gain and loss of function of DSCR1 leads to increased and decreased axon extension in developing mouse hippocampal neurons, respectively. Consistent with the role of DSCR1 as a CaN inhibitor, DSCR1^−/− growth cones have elevated CaN activity as indicated by reduced phosphorylated cofilin (nonphosphorylated cofilin is the active form of this actin depolymerizing factor), which leads to loss of F-actin and short axons. In contrast, DSCR1 transgenic neurons, which express 1.5-fold excess DSCR1 compared with wild-type neurons, display elevated phosphorylated cofilin (the inactive form) and increased F-actin in their growth cones, increasing neurite extension.In chemotropic turning assays, DSCR1^−/− neurons fail to orient toward brain-derived neurotrophic factor (BDNF), whereas transgenic DSCR1 neurons exhibit enhanced turning. Interestingly, Wang et al. (2016) find that although activation of CaN and cofilin caused by loss of DSCR1 function reduces axon extension, misregulation of CaN is not responsible for defective chemotropic turning toward BDNF by DSCR1^−/− neurons. This result suggests that a different DSCR1-dependent target regulates axon turning. Here, Wang et al. (2016) find that local protein synthesis in response to BDNF depends on DSCR1 and Fmr1. They show that enhanced protein synthesis in growth cones and axon turning in DSCR1 transgenic neurons is abrogated in Fmr1 knockdown neurons. Together these results support a model where DSCR1 functions as an important regulatory switch to direct distinct aspects of axon growth and guidance machinery.Wang et al. (2016) illustrate for the first time divergent activities of DSCR1 in the regulation of axon outgrowth and guidance, but many open questions remain. For example, there appears to be an important difference between the regulation of DSCR1 in axon guidance versus dendritic spine morphogenesis. Previous work by Wang et al. (2012) showed that Fmr1 is dephosphorylated by CaN in response to BDNF, which promotes protein translation. However, in their current work, inhibition of CaN does not prevent chemotropic turning toward a BDNF, which depends on DSCR1, Fmr1 and protein synthesis. Therefore, the role of CaN-mediated dephosphorylation of Fmr1 downstream of BDNF and DSCR1 is unclear in growth cone turning. It is also interesting to note that Wang et al. (2016) observe changes in total cofilin levels in growth cones from DSCR1 transgenic and knockout neurons, as well as after inhibition of CaN, suggesting that there may be homeostatic aspects of regulation of these pathways yet to be explored. Given the complexities of cofilin regulation in actin polymerization and evidence that cofilin can be locally translated in axons (Piper et al., 2006), it is clear that additional work is necessary to understand these pathways more completely.Finally, how DSCR1 dysfunction contributes specifically to neural developmental disorders associated with Down syndrome is not known. DSCR1 gain-of-function experiments may most closely match chromosomal triplication conditions in developing trisomy 21 neurons. Under these conditions, Wang et al. (2016) find increased axon extension, as well as enhanced turning toward BDNF by mouse hippocampal neurons. However, it is not clear how elevated DSCR1 expression will affect CaN-cofilin signaling and protein synthesis downstream of the wide range of growth promoting and inhibiting guidance cues that use these signals (Gomez and Letourneau, 2014). It is also not clear how DSCR1 overexpression will affect human neurons under similar conditions. To address this question, specific classes of excitatory and inhibitory neurons differentiated from human embryonic stem cells and induced pluripotent stem cells (hiPSCs) should be tested. For example, in these in vitro systems, Crispr-Cas–mediated correction of specific genes of hiPSCs from Down syndrome patients or selected triplication of chromosome 21 target genes of unaffected hiPSCs could be used to test the necessity and sufficiency of specific genes on cellular phenotypes observed in vitro. If specific requirements for DSCR1 or other candidate genes can be identified by assaying neuronal morphogenesis of human cells in vitro, these findings would provide an excellent platform for therapeutic drug screening of treatments that rescue these cellular phenotypes.     

Direct biomechanical induction of endogenous calcineurin inhibitor Down Syndrome Critical Region-1 in cardiac myocytes

Signaling through the protein phosphatase calcineurin may play a critical role in cardiac hypertrophy. The gene for Down Syndrome Critical Region-1 (DSCR1) encodes a protein that is an endogenous calcineurin inhibitor. This study was designed to test the hypothesis that DSCR1 is directly induced by biomechanical stimuli. Neonatal rat cardiac myocytes were exposed to biaxial cyclic mechanical strain; mechanical strain upregulated DSCR1 mRNA expression in a time- and amplitude-dependent manner (3.4 +/- 0.2-fold at 8% strain for 6 h, n = 11, P < 0.01), and this induction was angiotensin II and endothelin I independent. Biomechanical induction of DSCR1 mRNA was partially blocked by calcineurin inhibition with cyclosporine A (30 +/- 5%, n = 3, P < 0.01). DSCR1 promoter-reporter experiments showed that mechanical strain induced DSCR1 promoter activity by 2.3-fold and that this induction was completely inhibited by cyclosporin A. Furthermore, DSCR1 gene expression was increased in the left ventricles of mice with pressure-overload hypertrophy induced by transverse aortic banding. These data demonstrate that biomechanical strain directly induces gene expression for the calcineurin inhibitor DSCR1 in cardiac myocytes, indicating that mechanically induced DSCR1 may regulate the hypertrophic response to mechanical overload.     

DSCR1 gene expression is dependent on NFATc1 during cardiac valve formation and colocalizes with anomalous organ development in trisomy 16 mice

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 beta-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.     

Raf-1 is a binding partner of DSCR1

Down syndrome critical region 1 (DSCR1) is recognized as an endogenous calcineurin inhibitor. DSCR1 is induced in endothelial cells and may play an important role in inflammation and angiogenesis. To address a novel function of DSCR1, we searched interacting partners of DSCR1. We performed pull-down analysis using DSCR1 as a bait and identified Raf-1 as a binding partner. The association of Raf-1 was confirmed by co-immunoprecipitation in GM7373 cells expressing green fluorescence protein tagged DSCR1. We determined two Raf-1 binding regions in DSCR1; one in the N-terminus and the other in the C-terminus regions. We further demonstrated that calpain cleaved DSCR1 and generated fragments with different binding affinity to Raf-1 or calcineurin. These results constitute the first demonstration of Raf-1 as a binding partner of DSCR1, and suggest a novel role of DSCR1.     

Aggregate formation and synaptic abnormality induced by DSCR1

Aggregation of conformation-abnormal peptides probably plays a key role in the pathogenesis of many neurodegenerative diseases. DSCR1 Down syndrome (DS) critical region 1, was identified from a chromosomal region (21q22.1-q22.2) for the clinical manifestations of DS when an extra-copy is present. We report that expression of DSCR1 in several cell types, including primary neurons, causes microtubule-dependent aggresome-like inclusion body formation. Disease-associated huntingtin (Q148) and ataxin-3 (Q84) co-localize with DSCR1 aggregates. Neurons bearing DSCR1 aggregates show reduced synaptophysin staining in processes. DSCR1 residues 31-90 constitute an aggregation-prone domain that is predicted to form a hydrophobic patch on the protein surface when residues 1-30 are removed. This study identifies a novel function of DSCR1 that may underlie DS neuropathology.