Fraternal twins: Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1, two homologous EF-hand containing calcium binding adaptor proteins with distinct functions

Changes in the intracellular calcium concentration govern cytoskeletal rearrangement, mitosis, apoptosis, transcriptional regulation or synaptic transmission, thereby, regulating cellular effector and organ functions. Calcium binding proteins respond to changes in the intracellular calcium concentration with structural changes, triggering enzymatic activation and association with downstream proteins. One type of calcium binding proteins are EF-hand super family proteins. Here, we describe two recently discovered homologous EF-hand containing adaptor proteins, Swiprosin-1/EF-hand domain containing 2 (EFhd2) and Swiprosin-2/EF-hand domain containing 1 (EFhd1), which are related to allograft inflammatory factor-1 (AIF-1). For reasons of simplicity and concision we propose to name Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1 from now on EFhd2 and EFhd1, according to their respective gene symbols. AIF-1 and Swiprosin-1/EFhd2 are already present in Bilateria, for instance in Drosophila melanogaster and Caenhorhabditis elegans. Swiprosin-2/EFhd1 arose later from gene duplication in the tetrapodal lineage. Secondary structure prediction of AIF-1 reveals disordered regions and one functional EF-hand. Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1 exhibit a disordered region at the N-terminus, followed by two EF-hands and a coiled-coil domain. Whereas both proteins are similar in their predicted overall structure they differ in a non-homologous stretch of 60 amino acids just in front of the EF-hands. AIF-1 controls calcium-dependent cytoskeletal rearrangement in innate immune cells by means of its functional EF-hand. We propose that Swiprosin-1/EFhd2 as well is a cytoskeleton associated adaptor protein involved in immune and brain cell function. Pro-inflammatory conditions are likely to modulate expression and function of Swiprosin-1/EFhd2. Swiprosin-2/EFhd1, on the other hand, modulates apoptosis and differentiation of neuronal and muscle precursor cells, probably through an association with mitochondria. We suggest furthermore that Swiprosin-2/EFhd1 is part of a cellular response to oxidative stress, which could explain its pro-survival activity in neuronal, muscle and perhaps some malignant tissues

Chronic Activation of Hepatic Nrf2 Has No Major Effect on Fatty Acid and Glucose Metabolism in Adult Mice

The transcription factor NF-E2-related factor 2 (Nrf2) induces cytoprotective genes, but has also been linked to the regulation of hepatic energy metabolism. In order to assess the pharmacological potential of hepatic Nrf2 activation in metabolic disease, Nrf2 was activated over 7 weeks in mice on Western diet using two different siRNAs against kelch-like ECH-associated protein 1 (Keap1), the inhibitory protein of Nrf2. Whole genome expression analysis followed by pathway analysis demonstrated successful knock-down of Keap1 expression and induction of Nrf2-dependent genes involved in anti- oxidative stress defense and biotransformation, proving the activation of Nrf2 by the siRNAs against Keap1. Neither the expression of fatty acid- nor carbohydrate-handling proteins was regulated by Keap1 knock-down. Metabolic profiling of the animals did also not show effects on plasma and hepatic lipids, energy expenditure or glucose tolerance. The data indicate that hepatic Keap1/Nrf2 is not a major regulator of glucose or lipid metabolism in mice

Impact of Swiprosin-1/Efhd2 on Adult Hippocampal Neurogenesis

Swiprosin-1/Efhd2 (Efhd2) is highly expressed in the CNS during development and in the adult. EFHD2 is regulated by Ca2+ binding, stabilizes F-actin, and promotes neurite extension. Previous studies indicated a dysregulation of EFHD2 in human Alzheimer's disease brains. We hypothesized a detrimental effect of genetic ablation of Efhd2 on hippocampal integrity and specifically investigated adult hippocampal neurogenesis. Efhd2 was expressed throughout adult neuronal development and in mature neurons. We observed a severe reduction of the survival of adult newborn neurons in Efhd2 knockouts, starting at the early neuroblast stage. Spine formation and dendrite growth of newborn neurons were compromised in full Efhd2 knockouts, but not upon cell- autonomous Efhd2 deletion. Together with our finding of severe hippocampal tauopathy in Efhd2 knockout mice, these data connect Efhd2 to impaired synaptic plasticity as present in Alzheimer's disease and identify a role of Efhd2 in neuronal survival and synaptic integration in the adult hippocampus

Distinct housing conditions reveal a major impact of adaptive immunity on the course of obesity-induced type 2 diabetes

Obesity is associated with adipose tissue inflammation, insulin resistance, and the development of type 2 diabetes (T2D). However, our knowledge is mostly based on conventional murine models and promising preclinical studies rarely translated into successful therapies. There is a growing awareness of the limitations of studies in laboratory mice, housed in abnormally hygienic specific pathogen-free (SPF) conditions, as relevant aspects of the human immune system remain unappreciated. Here, we assessed the impact of housing conditions on adaptive immunity and metabolic disease processes during high-fat diet (HFD). We therefore compared diet-induced obesity in SPF mice with those housed in non-SPF, so-called "antigen exposed" (AE) conditions. Surprisingly, AE mice fed a HFD maintained increased insulin levels to compensate for insulin resistance, which was reflected in islet hyperplasia and improved glucose tolerance compared to SPF mice. By contrast, we observed higher proportions of effector/memory T cell subsets in blood and liver of HFD AE mice accompanied by the development of nonalcoholic steatohepatitis-like liver pathology. Thus, our data demonstrate the impact of housing conditions on metabolic alterations. Studies in AE mice, in which physiological microbial exposure was restored, could provide a tool for revealing therapeutic targets for immune-based interventions for T2D patients

Regulation of body weight and energy homeostasis by neuronal cell adhesion molecule 1

Susceptibility to obesity is linked to genes regulating neurotransmission, pancreatic beta-cell function and energy homeostasis. Genome-wide association studies have identified associations between body mass index and two loci near cell adhesion molecule 1 (CADM1) and cell adhesion molecule 2 (CADM2), which encode membrane proteins that mediate synaptic assembly. We found that these respective risk variants associate with increased CADM1 and CADM2 expression in the hypothalamus of human subjects. Expression of both genes was elevated in obese mice, and induction of Cadm1 in excitatory neurons facilitated weight gain while exacerbating energy expenditure. Loss of Cadm1 protected mice from obesity, and tract-tracing analysis revealed Cadm1-positive innervation of POMC neurons via afferent projections originating from beyond the arcuate nucleus. Reducing Cadm1 expression in the hypothalamus and hippocampus promoted a negative energy balance and weight loss. These data identify essential roles for Cadm1-mediated neuronal input in weight regulation and provide insight into the central pathways contributing to human obesity.</p

Analyses of the function of the murine protein EFhd2/Swiprosin-1 in B cell development and immune response in vivo

Das Kalzium-bindende EFhd2 Protein wird in allen B Zellstadien exprimiert. Unsere Arbeiten zeigten, dass es B Zellrezeptorsignale in WEHI231 B Zellen verstärkt. Außerdem ist EFhd2 Bestandteil der konservierten transkriptionellen T und B Gedächtnissignatur. EFhd2 könnte daher eine Funktion in der B Zellentwicklung sowie bei der antigenabhängigen B Zellaktivierung ausüben. Das Ziel dieser Arbeit war es, die Funktion des EFhd2 Proteins in vivo in B Zellen zu entschlüsseln. Zu diesem Zweck wurde eine konstitutive EFhd2 defiziente c57Bl/6 Mauslinie aus erworbenen embryonalen Stammzellen etabliert und charakterisiert. Während die B und T Zelldifferenzierung EFhd2 defizienter Mäuse bis auf signifikant reduzierte, peritoneale CD4+ T Zellen ohne Defizite normal verläuft, wurden deutliche Unterschiede in der antigenabhängigen B Zelldifferenzierung festgestellt. So traten in einjährigen Mäusen spontan mehr gegen doppelsträngige DNA gerichtete Autoantikörper auf; dies nivellierte sich nach eineinhalb Jahren jedoch wieder. Die Typ I T Zell-unabhängige IgM Immunantwort nach TNP-LPS Immunisierung war an Tag 12, nicht jedoch bei späten Zeitpunkten, signifikant verbessert. Bei der T Zell-abhängigen Immunantwort gegen NP-KLH ergaben sich weder nach primärer Immunisierung noch nach Reimmunisierung quantitative Unterschiede. Allerdings bildeten wiederholt immunisierte EFhd2 defiziente Mäuse deutlich höher affine NP-spezifische IgG1 Antikörper als Wildtyp Mäuse. Um die Hypothese zu überprüfen, dass die höher affinen IgG1 Antikörper eine Folge verstärkter primärer Keimzentrumsreaktionen sind, wurden Keimzentrums B Zellen nach Immunisierung mit Schaferythrozyten analysiert. Während sich die Oberflächenexpression von IgM, MHCII und CD40 nicht zwischen Wildtyp und EFhd2 defizienten Mäusen unterschied, waren am Tag 7, nicht jedoch an Tag 12, mehr Keimzentrums B Zellen und größere Keimzentren in EFhd2 defizienten Mäusen vorhanden. Dies führt vermutlich zu einem erhöhten Selektionsdruck in Keimzentren von EFhd2 defizienten Mäusen. Die mit dieser Mauslinie erhobenen Daten lassen die Schlussfolgerung zu, dass EFhd2 ein transienter, negativer Regulator der B Zellaktivierung und möglicherweise auch der peripheren Immuntoleranz ist. Für weitergehende Untersuchungen, die sich auch auf den Menschen ausdehnen könnten, wurden im Rahmen dieser Arbeit monoklonale EFhd2-spezifische Antikörper generiert, die in zytoplasmatischen Färbungen in Maus und Mensch einsetzbar sind.The calcium-binding protein EFhd2 is expressed in all B cell stages. Our previous work has shown that EFhd2 amplifies B cell receptor signals in WEHI231 B cells. Moreover, EFhd2 is part of a conserved transcriptional, memory B and T cells signature. Thus, EFhd2 could play a role in B cell differentiation as well as in antigen-dependent B cell activation. The aim of this work was to elucidate the in vivo function of EFhd2 in B cells. For this purpose a constitutive EFhd2 deficient c57Bl/6 mouse line was established from commercial available embryonic stem cells and characterized. While B and T cell differentiation in EFhd2 deficient mice is normal, aside from significantly reduced peritoneal CD4+ T cells, there are major differences in B cell activation. For instance, one-year-old mice developed spontaneously more anti double-stranded DNA autoantibodies, but this effect was evened out after one and a half year of age. Type I T cell independent IgM immune response after TNP-LPS immunization was transient, but not at late time points significantly enhanced. T cell dependent immune responses against NP-KLH revealed no quantitative differences, neither in response to primary immunization nor after booster immunization. However, repeatedly immunized EFhd2 deficient mice exhibited clearly higher affine NP-specific IgG1 antibodies compared to wild type mice. To test the hypothesis that the higher affine IgG1 antibodies are a consequence of enhanced primary germinal center reactions, germinal center B cells were analyzed after SRBC immunization. Whereas the surface expression of IgM, MHCII and CD40 did not differ between wild type and EFhd2 deficient B cells, EFhd2 deficient mice developed significantly more germinal center B cells and larger germinal centers on day 7 but not on day 12. Thus, the selection pressure in germinal centers of EFhd2 deficient mice is most likely enhanced favoring selection of high affinity B cells. The data suggest that EFhd2 is a transient, negative regulator of B cell activation and potentially peripheral tolerance. Further analysis that could possibly involve the human system will be facilitated by monoclonal EFhd2-specific antibodies, which have been established here for use in cytoplasmic stainings

The Adaptor Protein Swiprosin-1/EFhd2 Is Dispensable for Platelet Function in Mice

Background Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity, but may also cause pathological vessel occlusion. Reorganizations of the platelet cytoskeleton and agonist-induced intracellular Ca2+-mobilization are crucial for platelet hemostatic function. EF-hand domain containing 2 (EFhd2, Swiprosin-1) is a Ca2+-binding cytoskeletal adaptor protein involved in actin remodeling in different cell types, but its function in platelets is unknown. Objective Based on the described functions of EFhd2 in immune cells, we tested the hypothesis that EFhd2 is a crucial adaptor protein for platelet function acting as a regulator of Ca2+-mobilization and cytoskeletal rearrangements. Methods and Results We generated EFhd2-deficient mice and analyzed their platelets in vitro and in vivo. Efhd2-/- mice displayed normal platelet count and size, exhibited an unaltered in vivo life span and showed normal Ca2+-mobilization and activation/aggregation responses to classic agonists. Interestingly, upon stimulation of the immunoreceptor tyrosine-based activation motif-coupled receptor glycoprotein (GP) VI, Efhd2-/- platelets showed a slightly increased coagulant activity. Furthermore, absence of EFhd2 had no significant impact on integrin-mediated clot retraction, actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo EFhd2-deficiency resulted in unaltered hemostatic function and unaffected arterial thrombus formation. Conclusion These results show that EFhd2 is not essential for platelet function in mice indicating that other cytoskeletal adaptors may functionally compensate its loss

The adaptor protein Swiprosin-1/EFhd2 is dispensable for platelet function in mice.

BACKGROUND: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity, but may also cause pathological vessel occlusion. Reorganizations of the platelet cytoskeleton and agonist-induced intracellular Ca2+-mobilization are crucial for platelet hemostatic function. EF-hand domain containing 2 (EFhd2, Swiprosin-1) is a Ca2+-binding cytoskeletal adaptor protein involved in actin remodeling in different cell types, but its function in platelets is unknown. OBJECTIVE: Based on the described functions of EFhd2 in immune cells, we tested the hypothesis that EFhd2 is a crucial adaptor protein for platelet function acting as a regulator of Ca2+-mobilization and cytoskeletal rearrangements. METHODS AND RESULTS: We generated EFhd2-deficient mice and analyzed their platelets in vitro and in vivo. Efhd2-/- mice displayed normal platelet count and size, exhibited an unaltered in vivo life span and showed normal Ca2+-mobilization and activation/aggregation responses to classic agonists. Interestingly, upon stimulation of the immunoreceptor tyrosine-based activation motif-coupled receptor glycoprotein (GP) VI, Efhd2-/- platelets showed a slightly increased coagulant activity. Furthermore, absence of EFhd2 had no significant impact on integrin-mediated clot retraction, actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo EFhd2-deficiency resulted in unaltered hemostatic function and unaffected arterial thrombus formation. CONCLUSION: These results show that EFhd2 is not essential for platelet function in mice indicating that other cytoskeletal adaptors may functionally compensate its loss