Kasper Kjaer-Sorensen

Severe Preeclampsia-Related Changes in Gene Expression at the Maternal-Fetal Interface Include Sialic Acid-Binding Immunoglobulin-Like Lectin-6 and Pappalysin-2

Preeclampsia (PE), which affects 4-8% of human pregnancies, causes significant maternal and neonatal morbidity and mortality. Within the basal plate, placental cytotrophoblasts (CTBs) of fetal origin invade the uterus and extensively remodel the maternal vasculature. In PE, CTB invasion is often shallow, and vascular remodeling is rudimentary. To better understand possible causes, we conducted a global analysis of gene expression at the maternal-fetal interface in placental samples from women with PE (n = 12; 24-36 wk) vs. samples from women who delivered due to preterm labor with no evidence of infection (n = 11; 24-36 wk), a condition that our previous work showed is associated with normal CTB invasion. Using the HG-U133A&B Affymetrix GeneChip platform, and statistical significance set at log odds-ratio of B >0, 55 genes were differentially expressed in PE. They encoded proteins previously associated with PE [e.g. Flt-1 (vascular endothelial growth factor receptor-1), leptin, CRH, and inhibin] and novel molecules [e.g. sialic acid binding Ig-like lectin 6 (Siglec-6), a potential leptin receptor, and pappalysin-2 (PAPP-A2), a protease that cleaves IGF-binding proteins]. We used quantitative PCR to validate the expression patterns of a subset of the genes. At the protein level, we confirmed PE-related changes in the expression of Siglec-6 and PAPP-A2, which localized to invasive CTBs and syncytiotrophoblasts. Notably, Siglec-6 placental expression is uniquely human, as is spontaneous PE. The functional significance of these novel observations may provide new insights into the pathogenesis of PE, and assaying the circulating levels of these proteins could have clinical utility for predicting and/or diagnosing PE.

A substrate specificity-determining unit of three Lin12-Notch repeat modules is formed in trans within the pappalysin-1 dimer and requires a sequence stretch C-terminal to the third module

Members of the pappalysin family of metzincin metalloproteinases, pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) and PAPP-A2 (pappalysin-2), regulate the bioavailability of insulin-like growth factors (IGFs) by specific proteolytic inactivation of IGF-binding proteins (IGFBPs). PAPP-A cleaves IGFBP-4 and IGFBP-5, whereas PAPP-A2 cleaves only IGFBP-5. The pappalysins contain three Lin12-Notch repeat (LNR1–3) modules, previously considered unique to the Notch receptor family in which they function to regulate receptor cleavage. In contrast to the Notch receptor where three LNR modules are tandemly arranged, LNR3 is separated by more than 1000 residues from LNR1–2 in the pappalysin sequence. Each of the three LNR modules of PAPP-A is required for proteolysis of IGFBP-4, but not IGFBP-5. However, we here find that a C-terminal truncated variant of PAPP-A, which lacks LNR3 and therefore activity against IGFBP-4, cleaves IGFBP-4 when co-expressed with a PAPP-A variant, which is mutated in the active site. This suggests that LNR3 from the inactive subunit interacts in trans with LNR1–2 of the truncated PAPP-A subunit to form a functional trimeric LNR unit. We also show that formation of such a functional LNR unit depends on dimerization, as dissociation of a mutated non-covalent PAPP-A dimer results in reduced activity against IGFBP-4, but not IGFBP-5. Using PAPP-A/PAPP-A2 chimeras, we demonstrate that PAPP-A2 LNR1–2, but not LNR3, are functionally conserved with respect to IGFBP proteolysis. Additionally, we find that a sequence stretch C-terminal to LNR3 and single residues (Asp1521, Arg1529, and Asp1530) within this are required for LNR functionality.

Molecular Cloning and Characterization of Porcine Na+/K+-ATPase Isoforms α1, α2, α3 and the ATP1A3 Promoter

Na+/K+-ATPase maintains electrochemical gradients of Na+ and K+ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na+/K+-ATPase exists in four different isoforms (α1–α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na+/K+-ATPase mutations, we have determined the porcine coding sequences of the α1–α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na+ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na+ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by ATP1A3 mutations.

Papp-a2 modulates development of cranial cartilage and angiogenesis in zebrafish embryos

ABSTRACT Pregnancy-associated plasma protein A2 (PAPP-A2, also known as pappalysin-2) is a large metalloproteinase that is known to be required for normal postnatal growth and bone development in mice. We here report the detection of zebrafish papp-a2 mRNA in the chordamesoderm, notochord and lower jaw of zebrafish (Danio rerio) embryos, and that papp-a2-knockdown embryos display broadened axial mesoderm, notochord bends and severely reduced cranial cartilages. Genetic data link these phenotypes to insulin-like growth factor (Igf)-binding protein-3 (Igfbp-3) and bone morphogenetic protein (Bmp) signaling, and biochemical analysis show specific Igfbp-3 proteolysis by Papp-a2, implicating Papp-a2 in the modulation of Bmp signaling by Igfbp-3 proteolysis. Knockdown of papp-a2 additionally resulted in angiogenesis defects, strikingly similar to previous observations in embryos with mutations in components of the Notch system. Accordingly, we find that Notch signaling is modulated by Papp-a2 in vivo, and, furthermore, that human PAPP-A2 is capable of modulating Notch signaling independently of its proteolytic activity in cell culture. Based on these results, we conclude that Papp-a2 modulates Bmp and Notch signaling by independent mechanisms in zebrafish embryos. In conclusion, these data link pappalysin function in zebrafish to two different signaling pathways outside the IGF system.

Calmodulin mutations causing catecholaminergic polymorphic ventricular tachycardia confer opposing functional and biophysical molecular changes

Calmodulin (CaM) is the central mediator of intracellular Ca2+ signalling in cardiomyocytes, where it conveys the intricate Ca2+ transients to the proteins controlling cardiac contraction. We recently linked two separate mutations in CaM (N53I and N97S) to dominantly inherited catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmic disorder in which exercise or acute emotion can lead to syncope and sudden cardiac death. Given the ubiquitous presence of CaM in all eukaryote cells, it is particular intriguing that carriers of either mutation show no additional symptoms. Here, we investigated the effects of the CaM CPVT mutations in a zebrafish animal model. Three‐day‐old embryos injected with either CaM mRNA showed no detectable pathologies or developmental abnormalities. However, embryos injected with CPVT CaM mRNA displayed increased heart rate compared to wild‐type CaM mRNA under β‐adrenergic stimulation, demonstrating a conserved dominant cardiac specific effect between zebrafish and human carriers of these mutations. Motivated by the highly similar physiological phenotypes, we compared the effects of the N53I and N97S mutations on the biophysical and functional properties of CaM. Surprisingly, the mutations have opposing effects on CaM C‐lobe Ca2+ binding affinity and kinetics, and changes to the CaM N‐lobe Ca2+ binding are minor and specific to the N53I mutation. Furthermore, both mutations induce differential perturbations to structure and stability towards unfolding. Our results suggest different molecular disease mechanisms for the CPVT (N53I and N97S mutations) and strongly support that cardiac contraction is the physiological process most sensitive to CaM integrity.

The α2Na+/K+-ATPase is critical for skeletal and heart muscle function in zebrafish.

Summary The Na+/K+-ATPase generates ion gradients across the plasma membrane, essential for multiple cellular functions. In mammals, four different Na+/K+-ATPase &agr;-subunit isoforms are associated with characteristic cell-type expression profiles and kinetics. We found the zebrafish &agr;2Na+/K+-ATPase associated with striated muscles and that knockdown causes a significant depolarization of the resting membrane potential in slow-twitch fibers of skeletal muscles. Abrupt mechanosensory responses were observed in &agr;2Na+/K+-ATPase-deficient embryos, possibly linked to a postsynaptic defect. The &agr;2Na+/K+-ATPase deficiency reduced the heart rate and caused a loss of left-right asymmetry in the heart tube. Similar phenotypes from knockdown of the Na+/Ca2+ exchanger indicated a role for the interplay between these two proteins in the observed phenotypes. Furthermore, proteomics identified up- and downregulation of specific phenotype-related proteins, such as parvalbumin, CaM, GFAP and multiple kinases, thus highlighting a potential proteome change associated with the dynamics of &agr;2Na+/K+-ATPase. Taken together, our findings show that zebrafish &agr;2Na+/K+-ATPase is important for skeletal and heart muscle functions.

Pregnancy-associated plasma protein A (PAPP-A) modulates the early developmental rate in zebrafish independently of its proteolytic activity

Background: Pregnancy-associated plasma protein-A (PAPP-A) is known to regulate insulin-like growth factor (IGF) bioavailability by specific IGF binding protein proteolysis. Results: Early developmental delay resulting from zebrafish papp-a knockdown can be rescued by wild-type or proteolytically inactive zebrafish Papp-a. Conclusion: Papp-a has functionality independent of its proteolytic activity. Significance: This is the first report of non-proteolytic PAPP-A function with important implications for understanding the biology of PAPP-A. Pregnancy-associated plasma protein-A (PAPP-A) is a large metalloproteinase specifically cleaving insulin-like growth factor (IGF) binding proteins, causing increased IGF bioavailability and, hence, local regulation of IGF receptor activation. We have identified two highly conserved zebrafish homologs of the human PAPP-A gene. Expression of zebrafish Papp-a, one of the two paralogs, begins during gastrulation and persists throughout the first week of development, and analyses demonstrate highly conserved patterns of expression between adult zebrafish, humans, and mice. We show that the specific knockdown of zebrafish papp-a limits the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside of the Igf system. Biochemical analysis of recombinant zebrafish Papp-a demonstrates conservation of proteolytic activity, specificity, and the intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficiently as wild-type Papp-a. Thus, the developmental phenotype of papp-a knockdown is not a consequence of lacking Papp-a proteolytic activity. We conclude that Papp-a possesses biological functions independent of its proteolytic activity. Our data represent the first evidence for a non-proteolytic function of PAPP-A.

Loss-of-activity-mutation in the cardiac chloride-bicarbonate exchanger AE3 causes short QT syndrome

Patients with short QT syndrome (SQTS) may present with syncope, ventricular fibrillation or sudden cardiac death. Six SQTS susceptibility genes, encoding cation channels, explain <25% of SQTS cases. Here we identify a missense mutation in the anion exchanger (AE3)-encoding SLC4A3 gene in two unrelated families with SQTS. The mutation causes reduced surface expression of AE3 and reduced membrane bicarbonate transport. Slc4a3 knockdown in zebrafish causes increased cardiac pHi, short QTc, and reduced systolic duration, which is rescued by wildtype but not mutated SLC4A3. Mechanistic analyses suggest that an increase in pHi and decrease in [Cl−]i shortened the action potential duration. However, other mechanisms may also play a role. Altered anion transport represents a mechanism for development of arrhythmia and may provide new therapeutic possibilities.Mutations in potassium and calcium channel genes have been associated with cardiac arrhythmias. Here, Jensen et al. show that an anion transporter chloride-bicarbonate exchanger AE3 is also responsible for the genetically-induced mechanism of cardiac arrhythmia, suggesting new therapeutic targets for this disease

Female versus male biological identities of nanoparticles determine the interaction with immune cells in fish

Biomolecule decoration of nanoparticles provides a corona that modulates how the nanoparticles interact with biological milieus. The corona composition has proved to reflect the differences in the repertoire of proteins to which the nanoparticles are exposed, and as a result the same nanoparticles can acquire a differential biological identity. Here we examined whether a unique biological identity acquired from sex-specific protein repertoires could alter the degree of nanoparticle uptake by cognate immune cells. We chose zebrafish as a model species of which blood plasma is sexually contrasted by the unique presence/absence of the egg yolk precursor protein vitellogenin. Sex-specific protein coronas were thus formed around 70 nm SiO2 nanoparticles using female/male blood plasma from zebrafish or fetal bovine serum as a non-native reference. In contrast to protein coronas formed of male blood plasma, a “female” biological identity of the nanoparticles was represented by prevailing contribution of vitellogenins to the corona proteome. We then exposed zebrafish blood cells to the three types of pre-formed nanoparticle–protein complexes and compared nanoparticle uptake using flow cytometry. Lymphoid and myeloid populations of the blood cells preferentially accumulated the nanoparticles with a female biological identity, irrespective of the sex of the fish from which the cells were obtained. The concept of repertoire differences in the corona proteome therefore deserves further attention, as various factors such as sex-specific biological conditions exemplified in this study could alter the nanoparticle–cell interactions.

Ephrin-A1-EphA4 signaling negatively regulates myelination in the central nervous system

During development of the central nervous system not all axons are myelinated, and axons may have distinct myelination patterns. Furthermore, the number of myelin sheaths formed by each oligodendrocyte is highly variable. However, our current knowledge about the axo‐glia communication that regulates the formation of myelin sheaths spatially and temporally is limited. By using axon‐mimicking microfibers and a zebrafish model system, we show that axonal ephrin‐A1 inhibits myelination. Ephrin‐A1 interacts with EphA4 to activate the ephexin1‐RhoA‐Rock‐myosin 2 signaling cascade and causes inhibition of oligodendrocyte process extension. Both in myelinating co‐cultures and in zebrafish larvae, activation of EphA4 decreases myelination, whereas myelination is increased by inhibition of EphA4 signaling at different levels of the pathway, or by receptor knockdown. Mechanistically, the enhanced myelination is a result of a higher number of myelin sheaths formed by each oligodendrocyte, not an increased number of mature cells. Thus, we have identified EphA4 and ephrin‐A1 as novel negative regulators of myelination. Our data suggest that activation of an EphA4‐RhoA pathway in oligodendrocytes by axonal ephrin‐A1 inhibits stable axo‐glia interaction required for generating a myelin sheath.