Impaired myocellular Ca2+ cycling in protein phosphatase PP2A-B56α KO mice is normalized by ß-adrenergic stimulation.

The activity of protein phosphatase 2A (PP2A) is determined by the expression and localization of the regulatory B-subunits. PP2A-B56α is the dominant isoform of the B'-family in the heart. Its role in regulating the cardiac response to ß-adrenergic stimulation is not yet fully understood. We therefore generated mice deficient in B56α to test the functional cardiac effects in response to catecholamine administration versus corresponding WT mice. We found the decrease in basal PP2A activity in hearts of KO mice was accompanied by a counter-regulatory increase in the expression of B' subunits (ß and γ) and higher phosphorylation of sarcoplasmic reticulum Ca2+ regulatory and myofilament proteins. The higher phosphorylation levels were associated with enhanced intraventricular pressure and relaxation in catheterized KO mice. In contrast, at the cellular level, we detected depressed Ca2+ transient and sarcomere shortening parameters in KO mice at basal conditions. Consistently, the peak amplitude of the L-type Ca2+ current was reduced and the inactivation kinetics of ICaL were prolonged in KO cardiomyocytes. However, we show ß-adrenergic stimulation resulted in a comparable peak amplitude of Ca2+ transients and myocellular contraction between KO and WT cardiomyocytes. Therefore, we propose higher isoprenaline-induced Ca2+ spark frequencies might facilitate the normalized Ca2+ signaling in KO cardiomyocytes. In addition, the application of isoprenaline was associated with unchanged L-type Ca2+ current parameters between both groups. Our data suggest an important influence of PP2A-B56α on the regulation of Ca2+ signaling and contractility in response to ß-adrenergic stimulation in the myocardium.

A Carboxy-terminal Smarcb1 Point Mutation Induces Hydrocephalus Formation and Affects AP-1 and Neuronal Signalling Pathways in Mice.

The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal differentiation. Mutations of its core subunit SMARCB1 result in a broad spectrum of pathologies, including aggressive rhabdoid tumours or neurodevelopmental disorders. Other mouse models have addressed the influence of a homo- or heterozygous loss of Smarcb1, yet the impact of specific non-truncating mutations remains poorly understood. Here, we have established a new mouse model for the carboxy-terminal Smarcb1 c.1148del point mutation, which leads to the synthesis of elongated SMARCB1 proteins. We have investigated its impact on brain development in mice using magnetic resonance imaging, histology, and single-cell RNA sequencing. During adolescence, Smarcb11148del/1148del mice demonstrated rather slow weight gain and frequently developed hydrocephalus including enlarged lateral ventricles. In embryonic and neonatal stages, mutant brains did not differ anatomically and histologically from wild-type controls. Single-cell RNA sequencing of brains from newborn mutant mice revealed that a complete brain including all cell types of a physiologic mouse brain is formed despite the SMARCB1 mutation. However, neuronal signalling appeared disturbed in newborn mice, since genes of the AP-1 transcription factor family and neurite outgrowth-related transcripts were downregulated. These findings support the important role of SMARCB1 in neurodevelopment and extend the knowledge of different Smarcb1 mutations and their associated phenotypes.

DNA barcoding reveals that injected transgenes are predominantly processed by homologous recombination in mouse zygote.

Mechanisms that ensure repair of double-strand DNA breaks (DSBs) are instrumental in the integration of foreign DNA into the genome of transgenic organisms. After pronuclear microinjection, exogenous DNA is usually found as a concatemer comprising multiple co-integrated transgene copies. Here, we investigated the contribution of various DSB repair pathways to the concatemer formation. We injected mouse zygotes with a pool of linear DNA molecules carrying unique barcodes at both ends and obtained 10 transgenic embryos with 1-300 transgene copies. Sequencing the barcodes allowed us to assign relative positions to the copies in concatemers and detect recombination events that occurred during integration. Cumulative analysis of approximately 1,000 integrated copies reveals that over 80% of them underwent recombination when their linear ends were processed by synthesis-dependent strand annealing (SDSA) or double-strand break repair (DSBR). We also observed evidence of double Holliday junction (dHJ) formation and crossing over during the concatemer formations. Sequencing indels at the junctions between copies shows that at least 10% of DNA molecules introduced into the zygotes are ligated by non-homologous end joining (NHEJ). Our barcoding approach, verified with Pacific Biosciences Single Molecule Real-Time (SMRT) long-range sequencing, documents high activity of homologous recombination after DNA microinjection.

TrkC Is Essential for Nephron Function and Trans-Activates Igf1R Signaling.

BACKGROUND: Injury to kidney podocytes often results in chronic glomerular disease and consecutive nephron malfunction. For most glomerular diseases, targeted therapies are lacking. Thus, it is important to identify novel signaling pathways contributing to glomerular disease. Neurotrophic tyrosine kinase receptor 3 (TrkC) is expressed in podocytes and the protein transmits signals to the podocyte actin cytoskeleton. METHODS: Nephron-specific TrkC knockout (TrkC-KO) and nephron-specific TrkC-overexpressing (TrkC-OE) mice were generated to dissect the role of TrkC in nephron development and maintenance. RESULTS: Both TrkC-KO and TrkC-OE mice exhibited enlarged glomeruli, mesangial proliferation, basement membrane thickening, albuminuria, podocyte loss, and aspects of FSGS during aging. Igf1 receptor (Igf1R)-associated gene expression was dysregulated in TrkC-KO mouse glomeruli. Phosphoproteins associated with insulin, erb-b2 receptor tyrosine kinase (Erbb), and Toll-like receptor signaling were enriched in lysates of podocytes treated with the TrkC ligand neurotrophin-3 (Nt-3). Activation of TrkC by Nt-3 resulted in phosphorylation of the Igf1R on activating tyrosine residues in podocytes. Igf1R phosphorylation was increased in TrkC-OE mouse kidneys while it was decreased in TrkC-KO kidneys. Furthermore, TrkC expression was elevated in glomerular tissue of patients with diabetic kidney disease compared with control glomerular tissue. CONCLUSIONS: Our results show that TrkC is essential for maintaining glomerular integrity. Furthermore, TrkC modulates Igf-related signaling in podocytes.

Reference Genes across Nine Brain Areas of Wild Type and Prader-Willi Syndrome Mice: Assessing Differences in Igfbp7, Pcsk1, Nhlh2 and Nlgn3 Expression.

Prader−Willi syndrome (PWS) is a complex neurodevelopmental disorder caused by the deletion or inactivation of paternally expressed imprinted genes at the chromosomal region 15q11−q13. The PWS-critical region (PWScr) harbors tandemly repeated non-protein coding IPW-A exons hosting the intronic SNORD116 snoRNA gene array that is predominantly expressed in brain. Paternal deletion of PWScr is associated with key PWS symptoms in humans and growth retardation in mice (PWScr model). Dysregulation of the hypothalamic−pituitary axis (HPA) is thought to be causally involved in the PWS phenotype. Here we performed a comprehensive reverse transcription quantitative PCR (RT-qPCR) analysis across nine different brain regions of wild-type (WT) and PWScr mice to identify stably expressed reference genes. Four methods (Delta Ct, BestKeeper, Normfinder and Genorm) were applied to rank 11 selected reference gene candidates according to their expression stability. The resulting panel consists of the top three most stably expressed genes suitable for gene-expression profiling and comparative transcriptome analysis of WT and/or PWScr mouse brain regions. Using these reference genes, we revealed significant differences in the expression patterns of Igfbp7, Nlgn3 and three HPA associated genes: Pcsk1, Pcsk2 and Nhlh2 across investigated brain regions of wild-type and PWScr mice. Our results raise a reasonable doubt on the involvement of the Snord116 in posttranscriptional regulation of Nlgn3 and Nhlh2 genes. We provide a valuable tool for expression analysis of specific genes across different areas of the mouse brain and for comparative investigation of PWScr mouse models to discover and verify different regulatory pathways affecting this complex disorder.

DHHC7-mediated palmitoylation of the accessory protein barttin critically regulates the functions of ClC-K chloride channels.

Barttin is the accessory subunit of the human ClC-K chloride channels, which are expressed in both the kidney and inner ear. Barttin promotes trafficking of the complex it forms with ClC-K to the plasma membrane and is involved in activating this channel. Barttin undergoes post-translational palmitoylation that is essential for its functions, but the enzyme(s) catalyzing this post-translational modification is unknown. Here, we identified zinc finger DHHC-type containing 7 (DHHC7) protein as an important barttin palmitoyl acyltransferase, whose depletion affected barttin palmitoylation and ClC-K-barttin channel activation. We investigated the functional role of barttin palmitoylation in vivo in Zdhhc7-/- mice. Although palmitoylation of barttin in kidneys of Zdhhc7-/- animals was significantly decreased, it did not pathologically alter kidney structure and functions under physiological conditions. However, when Zdhhc7-/- mice were fed a low-salt diet, they developed hyponatremia and mild metabolic alkalosis, symptoms characteristic of human Bartter syndrome (BS) type IV. Of note, we also observed decreased palmitoylation of the disease-causing R8L barttin variant associated with human BS type IV. Our results indicate that dysregulated DHHC7-mediated barttin palmitoylation appears to play an important role in chloride channel dysfunction in certain BS variants, suggesting that targeting DHHC7 activity may offer a potential therapeutic strategy for reducing hypertension.

DGK and DZHK position paper on genome editing: basic science applications and future perspective.

For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems.

SNORD116 and growth hormone therapy impact IGFBP7 in Prader-Willi syndrome.

PURPOSE: Prader-Willi syndrome (PWS) is a neurodevelopmental disorder with hypothalamic dysfunction due to deficiency of imprinted genes located on the 15q11-q13 chromosome. Among them, the SNORD116 gene appears critical for the expression of the PWS phenotype. We aimed to clarify the role of SNORD116 in cellular and animal models with regard to growth hormone therapy (GHT), the main approved treatment for PWS. METHODS: We collected serum and induced pluripotent stem cells (iPSCs) from GH-treated PWS patients to differentiate into dopaminergic neurons, and in parallel used a Snord116 knockout mouse model. We analyzed the expression of factors potentially linked to GH responsiveness. RESULTS: We found elevated levels of circulating IGFBP7 in naive PWS patients, with IGFBP7 levels normalizing under GHT. We found elevated IGFBP7 levels in the brains of Snord116 knockout mice and in iPSC-derived neurons from a SNORD116-deleted PWS patient. High circulating levels of IGFBP7 in PWS patients may result from both increased IGFBP7 expression and decreased IGFBP7 cleavage, by downregulation of the proconvertase PC1. CONCLUSION: SNORD116 deletion affects IGFBP7 levels, while IGFBP7 decreases under GHT in PWS patients. Modulation of the IGFBP7 level, which interacts with IGF1, has implications in the pathophysiology and management of PWS under GHT.

A Comprehensive Review of Genetically Engineered Mouse Models for Prader-Willi Syndrome Research.

Prader-Willi syndrome (PWS) is a neurogenetic multifactorial disorder caused by the deletion or inactivation of paternally imprinted genes on human chromosome 15q11-q13. The affected homologous locus is on mouse chromosome 7C. The positional conservation and organization of genes including the imprinting pattern between mice and men implies similar physiological functions of this locus. Therefore, considerable efforts to recreate the pathogenesis of PWS have been accomplished in mouse models. We provide a summary of different mouse models that were generated for the analysis of PWS and discuss their impact on our current understanding of corresponding genes, their putative functions and the pathogenesis of PWS. Murine models of PWS unveiled the contribution of each affected gene to this multi-facetted disease, and also enabled the establishment of the minimal critical genomic region (PWScr) responsible for core symptoms, highlighting the importance of non-protein coding genes in the PWS locus. Although the underlying disease-causing mechanisms of PWS remain widely unresolved and existing mouse models do not fully capture the entire spectrum of the human PWS disorder, continuous improvements of genetically engineered mouse models have proven to be very powerful and valuable tools in PWS research.

A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart.

Whereas myosin 18B (Myo18B) is known to be a critical sarcomeric protein, the function of myosin 18A (Myo18A) is unclear, although it has been implicated in cell motility and Golgi shape. Here, we show that homozygous deletion (homozygous tm1a, tm1b, or tm1d alleles) of Myo18a in mouse is embryonic lethal. Reminiscent of Myo18b, Myo18a was highly expressed in the embryo heart, and cardiac-restricted Myo18a deletion in mice was embryonic lethal. Surprisingly, using Western blot analysis, we were unable to detect the known isoforms of Myo18A, Myo18Aα and Myo18Aß, in mouse heart using a custom C-terminal antibody. However, alternative anti-Myo18A antibodies detected a larger than expected protein, and RNA-Seq analysis indicated that a novel Myo18A transcript is expressed in mouse ventricular myocytes (and human heart). Cloning and sequencing revealed that this cardiac isoform, denoted Myo18Aγ, lacks the PDZ-containing N terminus of Myo18Aα but includes an alternative N-terminal extension and a long serine-rich C terminus. EGFP-tagged Myo18Aγ expressed in ventricular myocytes localized to the level of A-bands in sarcomeres, and Myo18a knockout embryos at day 10.5 exhibited disorganized sarcomeres with wavy thick filaments. We additionally generated myeloid-restricted Myo18a knockout mice to investigate the role of Myo18A in nonmuscle cells, exemplified by macrophages, which express more Myo18Aß than Myo18Aα, but no defects in cell shape, motility, or Golgi shape were detected. In summary, we have identified a previously unrecognized sarcomere component, a large novel isoform (denoted Myo18Aγ) of Myo18A. Thus, both members of class XVIII myosins are critical components of cardiac sarcomeres.

Endothelial C-Type Natriuretic Peptide Acts on Pericytes to Regulate Microcirculatory Flow and Blood Pressure.

BACKGROUND: Peripheral vascular resistance has a major impact on arterial blood pressure levels. Endothelial C-type natriuretic peptide (CNP) participates in the local regulation of vascular tone, but the target cells remain controversial. The cGMP-producing guanylyl cyclase-B (GC-B) receptor for CNP is expressed in vascular smooth muscle cells (SMCs). However, whereas endothelial cell-specific CNP knockout mice are hypertensive, mice with deletion of GC-B in vascular SMCs have unaltered blood pressure. METHODS: We analyzed whether the vasodilating response to CNP changes along the vascular tree, ie, whether the GC-B receptor is expressed in microvascular types of cells. Mice with a floxed GC-B ( Npr2) gene were interbred with Tie2-Cre or PDGF-Rß-Cre ERT2 lines to develop mice lacking GC-B in endothelial cells or in precapillary arteriolar SMCs and capillary pericytes. Intravital microscopy, invasive and noninvasive hemodynamics, fluorescence energy transfer studies of pericyte cAMP levels in situ, and renal physiology were combined to dissect whether and how CNP/GC-B/cGMP signaling modulates microcirculatory tone and blood pressure. RESULTS: Intravital microscopy studies revealed that the vasodilatatory effect of CNP increases toward small-diameter arterioles and capillaries. CNP consistently did not prevent endothelin-1-induced acute constrictions of proximal arterioles, but fully reversed endothelin effects in precapillary arterioles and capillaries. Here, the GC-B receptor is expressed both in endothelial and mural cells, ie, in pericytes. It is notable that the vasodilatatory effects of CNP were preserved in mice with endothelial GC-B deletion, but abolished in mice lacking GC-B in microcirculatory SMCs and pericytes. CNP, via GC-B/cGMP signaling, modulates 2 signaling cascades in pericytes: it activates cGMP-dependent protein kinase I to phosphorylate downstream targets such as the cytoskeleton-associated vasodilator-activated phosphoprotein, and it inhibits phosphodiesterase 3A, thereby enhancing pericyte cAMP levels. These pathways ultimately prevent endothelin-induced increases of pericyte calcium levels and pericyte contraction. Mice with deletion of GC-B in microcirculatory SMCs and pericytes have elevated peripheral resistance and chronic arterial hypertension without a change in renal function. CONCLUSIONS: Our studies indicate that endothelial CNP regulates distal arteriolar and capillary blood flow. CNP-induced GC-B/cGMP signaling in microvascular SMCs and pericytes is essential for the maintenance of normal microvascular resistance and blood pressure.

Nephron-specific knockin of the PIKfyve-binding-deficient Vac14L156R mutant results in albuminuria and mesangial expansion.

Intracellular trafficking processes play a key role for the establishment and maintenance of membrane surfaces in renal epithelia. Therefore, dysfunctions of these trafficking processes could be key events and important determinants in the onset and progression of diseases. The presence of cellular vacuoles-observed in many histologic analyses of renal diseases-is a macroscopic hint for disturbed intracellular trafficking processes. However, how vacuoles develop and which intracellular pathways are directly affected remain largely unknown. Previous studies showed that in some cases, vacuolization is linked to malfunction of the Vac14 complex. This complex, including the scaffold protein Vac14, the lipid kinase PIKfyve, and its counteracting lipid phosphatase Fig4, regulates intracellular phosphatidylinositol phosphate levels, which in turn, control the maturation of early-into-late endosomes, as well as the processing of autophagosomes into autophagolysosomes. Here, we analyzed the role of Vac14 in mice and observed that the nephron-specific knockin of the PIKfyve-binding-deficient Vac14L156R mutant led to albuminuria, accompanied by mesangial expansion, increased glomerular size, and an elevated expression of several kidney injury markers. Overexpression of this Vac14 variant in podocytes did not reveal a strong in vivo phenotype, indicating that Vac14-dependent trafficking processes are more important for tubular than for glomerular processes in the kidney. In vitro overexpression of Vac14L156R in Madin-Darby canine kidney cells had no impact on apico-basal polarity defects but resulted in a faster reassembly of junctional structures after Ca2+ depletion and delayed endo- and transcytosis rates. Taken together, our data suggest that increased albuminuria of Vac14L156R-overexpressing mice is a consequence of a lowered endo- and transcytosis of albumin in renal tubules.

Differential effects of anoctamins on intracellular calcium signals.

The Ca2+-activated Cl- channel TMEM16A [anoctamin (ANO)1] is homologous to yeast Ist2 and has been shown to tether the cortical endoplasmic reticulum (ER) to the plasma membrane. We therefore examined whether ANO1 and other members of the ANO family affect intracellular Ca2+ ([Ca2+]i) signals. It is shown that expression of ANO1 augments Ca2+ store release upon stimulation of GPCRs, whereas knockdown of ANO1, or lack of Ano1 expression in Ano1-/- animals, as shown in an earlier report, inhibits Ca2+ release. ANO6, and -10 show similar effects, whereas expression of ANO4, -8, and -9 attenuate filling of the ER store. The impact of ANO1 and -4 were examined in more detail. ANO1 colocalized and interacted with IP3R, whereas ANO4 colocalized with SERCA Ca2+ pumps and interacted with ORAI-1 channels, respectively. ANO1 Cl currents were rapidly activated exclusively through Ca2+ store release, and remained untouched by influx of extracellular Ca2+ In contrast expression of ANO4 caused a delayed activation of membrane-localized ANO6 channels, solely through store-operated Ca2+ entry via ORAI. Ca2+ signals were inhibited by knocking down expression of endogenous ANO1, -5, -6, and -10 and were also reduced in epithelial cells from Ano10-/- mice. The data suggest that ANOs affect compartmentalized [Ca2+]i signals, which may explain some of the cellular defects related to ANO mutations.-Cabrita, I., Benedetto, R., Fonseca, A., Wanitchakool, P., Sirianant, L., Skryabin, B. V., Schenk, L. K., Pavenstädt, H., Schreiber, R., Kunzelmann, K. Differential effects of anoctamins on intracellular calcium signals.

Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2.

Autism spectrum disorders comprise a range of neurodevelopmental disorders characterized by deficits in social interaction and communication, and by repetitive behaviour. Mutations in synaptic proteins such as neuroligins, neurexins, GKAPs/SAPAPs and ProSAPs/Shanks were identified in patients with autism spectrum disorder, but the causative mechanisms remain largely unknown. ProSAPs/Shanks build large homo- and heteromeric protein complexes at excitatory synapses and organize the complex protein machinery of the postsynaptic density in a laminar fashion. Here we demonstrate that genetic deletion of ProSAP1/Shank2 results in an early, brain-region-specific upregulation of ionotropic glutamate receptors at the synapse and increased levels of ProSAP2/Shank3. Moreover, ProSAP1/Shank2(-/-) mutants exhibit fewer dendritic spines and show reduced basal synaptic transmission, a reduced frequency of miniature excitatory postsynaptic currents and enhanced N-methyl-d-aspartate receptor-mediated excitatory currents at the physiological level. Mutants are extremely hyperactive and display profound autistic-like behavioural alterations including repetitive grooming as well as abnormalities in vocal and social behaviours. By comparing the data on ProSAP1/Shank2(-/-) mutants with ProSAP2/Shank3αß(-/-) mice, we show that different abnormalities in synaptic glutamate receptor expression can cause alterations in social interactions and communication. Accordingly, we propose that appropriate therapies for autism spectrum disorders are to be carefully matched to the underlying synaptopathic phenotype.

Regulation and Function of TMEM16F in Renal Podocytes.

The Ca2+-activated phospholipid scramblase and ion channel TMEM16F is expressed in podocytes of renal glomeruli. Podocytes are specialized cells that form interdigitating foot processes as an essential component of the glomerular filter. These cells, which participate in generation of the primary urine, are often affected during primary glomerular diseases, such as glomerulonephritis and secondary hypertensive or diabetic nephropathy, which always leads to proteinuria. Because the function of podocytes is known to be controlled by intracellular Ca2+ signaling, it is important to know about the role of Ca2+-activated TMEM16F in these cells. To that end, we generated an inducible TMEM16F knockdown in the podocyte cell line AB8, and produced a conditional mouse model with knockout of TMEM16F in podocytes and renal epithelial cells of the nephron. We found that knockdown of TMEM16F did not produce proteinuria or any obvious phenotypic changes. Knockdown of TMEM16F affected cell death of tubular epithelial cells but not of glomerular podocytes when analyzed in TUNEL assays. Surprisingly, and in contrast to other cell types, TMEM16F did not control intracellular Ca2+ signaling and was not responsible for Ca2+-activated whole cell currents in podocytes. TMEM16F levels in podocytes were enhanced after inhibition of the endolysosomal pathway and after treatment with angiotensin II. Renal knockout of TMEM16F did not compromise renal morphology and serum electrolytes. Taken together, in contrast to other cell types, such as platelets, bone cells, and immune cells, TMEM16F shows little effect on basal properties of podocytes and does not appear to be essential for renal function.

Lmbrd1 expression is essential for the initiation of gastrulation.

The rare inborn cblF defect of cobalamin metabolism is caused by mutations in the limb region 1 (LMBR1) domain containing 1 gene (LMBRD1). This defect is characterized by massive accumulation of free cobalamin in lysosomes and loss of mitochondrial succinyl-CoA synthesis and cytosolic methionine synthesis. Affected children suffer from heart defects, developmental delay and megaloblastic anemia. LMBRD1 encodes for LMBD1, a predicted lysosomal cobalamin transport protein. In this study, we determine the physiological function of LMBRD1 during embryogenesis by generating Lmbrd1 deficient mice using the Cre/LoxP system. Complete loss of Lmbrd1 function is accompanied by early embryonic death in mice. Whole mount in situ hybridization studies against bone morphogenetic protein 4 and Nodal show that initial formation of the proximal-distal axis is unaffected in early embryonic stages whereas the initiation of gastrulation is disturbed shown by the expression pattern of even skipped homeotic gene 1 and fibroblast growth factor 8 in Lmbrd1 deficient mice. We conclude that intact function of LMBD1 is essential for the initiation of gastrulation.

Anoctamins support calcium-dependent chloride secretion by facilitating calcium signaling in adult mouse intestine.

Intestinal epithelial electrolyte secretion is activated by increase in intracellular cAMP or Ca(2+) and opening of apical Cl(-) channels. In infants and young animals, but not in adults, Ca(2+)-activated chloride channels may cause secretory diarrhea during rotavirus infection. While detailed knowledge exists concerning the contribution of cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) channels, analysis of the role of Ca(2+)-dependent Cl(-) channels became possible through identification of the anoctamin (TMEM16) family of proteins. We demonstrate expression of several anoctamin paralogues in mouse small and large intestines. Using intestinal-specific mouse knockout models for anoctamin 1 (Ano1) and anoctamin 10 (Ano10) and a conventional knockout model for anoctamin 6 (Ano6), we demonstrate the role of anoctamins for Ca(2+)-dependent Cl(-) secretion induced by the muscarinic agonist carbachol (CCH). Ano1 is preferentially expressed in the ileum and large intestine, where it supports Ca(2+)-activated Cl(-) secretion. In contrast, Ano10 is essential for Ca(2+)-dependent Cl(-) secretion in jejunum, where expression of Ano1 was not detected. Although broadly expressed, Ano6 has no role in intestinal cholinergic Cl(-) secretion. Ano1 is located in a basolateral compartment/membrane rather than in the apical membrane, where it supports CCH-induced Ca(2+) increase, while the essential and possibly only apical Cl(-) channel is CFTR. These results define a new role of Ano1 for intestinal Ca(2+)-dependent Cl(-) secretion and demonstrate for the first time a contribution of Ano10 to intestinal transport.

Evolutionary and molecular facts link the WWC protein family to Hippo signaling.

The scaffolding protein KIBRA (also called WWC1) is involved in the regulation of important intracellular transport processes and the establishment of cell polarity. Furthermore, KIBRA/WWC1 is an upstream regulator of the Hippo signaling pathway that controls cell proliferation and organ size in animals. KIBRA/WWC1 represents only one member of the WWC protein family that also includes the highly similar proteins WWC2 and WWC3. Although the function of KIBRA/WWC1 was studied intensively in cells and animal models, the importance of WWC2 and WWC3 was not yet elucidated. Here, we describe evolutionary, molecular, and functional aspects of the WWC family. We show that the WWC genes arose in the ancestor of bilateral animals (clades such as insects and vertebrates) from a single founder gene most similar to the present KIBRA/WWC1-like sequence of Drosophila. This situation was still maintained until the common ancestor of lancelet and vertebrates. In fish, a progenitor-like sequence of mammalian KIBRA/WWC1 and WWC2 is expressed together with WWC3. Finally, in all tetrapods, the three family members, KIBRA/WWC1, WWC2, and WWC3, are found, except for a large genomic deletion including WWC3 in Mus musculus. At the molecular level, the highly conserved WWC proteins share a similar primary structure, the ability to form homo- and heterodimers and the interaction with a common set of binding proteins. Furthermore, all WWC proteins negatively regulate cell proliferation and organ growth due to a suppression of the transcriptional activity of YAP, the major effector of the Hippo pathway.

FHL2 regulates the resolution of tissue damage in chronic inflammatory arthritis.

OBJECTIVE: We analysed the role of the adaptor molecule four-and-a-half Lin11, Isl-1 & Mec-3 (LIM) domain protein 2 (FHL2) in the activation of fibroblast-like synoviocytes in human rheumatoid arthritis (RA) and tumour necrosis factor α (TNFα)-dependent animal models of the disease. METHODS: Synovial tissues of patients with RA and osteoarthritis (OA) as well as hind paw sections from arthritic human TNFα transgenic (hTNFtg) mice and synovial fibroblasts from these were analysed. The effects of cytokines on the expression of FHL2 and disease-relevant matrixmetalloproteases (MMPs) were determined. Analyses of human tissue specimens from patients treated with anti-TNFα as well as anti-TNFα treatment of hTNFtg mice were performed to substantiate the TNFα effects on FHL2 levels. FHL2(-/-) mice and hTNFtg mice (with constitutive or inducible transgene expression) were crossbred to generate TNFα overexpressing FHL2-deficient animals. Signalling pathways were analysed in cells from these mice and in human cells after knock down of FHL2 by western blot. RESULTS: FHL2 levels were higher in RA than in OA and in hTNFtg than in wild-type mice. Surprisingly, while transforming growth factor (TGF)ß-induced FHL2 expression, TNFα suppressed FHL2. In vivo, anti-TNFα treatment led to higher FHL2 levels both in RA patients and hTNFtg mice. The loss of FHL2 increased joint destruction in hTNFtg mice, which was accompanied by elevated MMP-13. In vitro, TNFα-mediated MMP-13 was significantly higher in FHL2(-/-) cells and after knock down of FHL2, which was caused by prolonged p38 MAPK activation. CONCLUSIONS: These data suggest that FHL2 serves as a protective factor and that, rather than promoting the pathology, the upregulation of FHL2 in RA occurs in frame of a regenerative attempt.

KIBRA (KIdney/BRAin protein) regulates learning and memory and stabilizes Protein kinase Mζ.

The WWC1 gene has been genetically associated with human episodic memory performance, and its product KIdney/BRAin protein (KIBRA) has been shown to interact with the atypical protein kinase protein kinase M ζ (PKMζ). Although recently challenged, PKMζ remains a candidate postsynaptic regulator of memory maintenance. Here, we show that PKMζ is subject to rapid proteasomal degradation and that KIBRA is both necessary and sufficient to counteract this process, thus stabilizing the kinase and maintaining its function for a prolonged time. We define the binding sequence on KIBRA, a short amino acid motif near the C-terminus. Both hippocampal knock-down of KIBRA in rats and KIBRA knock-out in mice result in decreased learning and memory performance in spatial memory tasks supporting the notion that KIBRA is a player in episodic memory. Interestingly, decreased memory performance is accompanied by decreased PKMζ protein levels. We speculate that the stabilization of synaptic PKMζ protein levels by KIBRA may be one mechanism by which KIBRA acts in memory maintenance. KIBRA/WWC1 has been genetically associated with human episodic memory. KIBRA has been shown to be post-synaptically localized, but its function remained obscure. Here, we show that KIBRA shields PKMζ, a kinase previously linked to memory maintenance, from proteasomal degradation via direct interaction. KIBRA levels in the rodent hippocampus correlate closely both to spatial memory performance in rodents and to PKMζ levels. Our findings support a role for KIBRA in memory, and unveil a novel function for this protein.