SANE,a novel LEM domain protein,regulates bone morphogenetic protein signaling through interaction with Smad1

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily that play important roles in bone formation, embryonic patterning, and epidermal-neural cell fate decisions. BMPs signal through pathway specific mediators such as Smads1 and 5, but the upstream regulation of BMP-specific Smads has not been fully characterized. Here we report the identification of SANE (Smad1 Antagonistic Effector), a novel protein with significant sequence similarity to nuclear envelop proteins such as MAN1. SANE binds to Smad1/5 and to BMP type I receptors and regulates BMP signaling. SANE specifically blocks BMP-dependent signaling in Xenopus embryos and in a mammalian model of bone formation but does not inhibit the TGF-beta/Smad2 pathway. Inhibition of BMP signaling by SANE requires interaction between SANE and Smad1, because a SANE mutant that does not bind Smad1 does not inhibit BMP signaling. Furthermore, inhibition appears to be mediated by inhibition of BMP-induced Smad1 phosphorylation, blocking ligand-dependent nuclear translocation of Smad1. These studies define a new mode of regulation for intracellular BMP/Smad1 signaling.     

Correlation between butyrylcholinesterase variants and sensitivity to soman toxicity

Butyrylcholinesterase (BChE) is synthesized in the liver and found in high concentrations in blood plasma, liver, heart, pancreas, vascular endothelium, skin, brain white matter, smooth muscle cells and adipocytes. BChE is a non specific enzyme that hydrolyzes different choline esters (succinylcholine, mivacurium) and many other drugs such as aspirin, cocaine and procaine. The enzyme is also considered as a bioscavenger due to its ability to neutralize the toxic effects of organophosphorus compounds (nervous system fs agents) such as soman. BChE displays several polymorphisms that influence its serum activity; therefore they could determine the individual sensitivity to chemical nerve agents. In this study, we investigated the correlation between BChE variants and the degree of enzyme inhibition and reactivation after soman application on blood samples of 726 individuals. The blood samples of individuals expressing abnormal variants, were more sensitive to soman compared to variants of homozygotes and heterozygotes for U-allele. We found significant differences in the degree of enzyme reactivation between different variants (with and without U-presence).     

SDF‐1/CXCR4 signalling is involved in blood vessel growth and remodelling by intussusception

The precise mechanisms of SDF‐1 (CXCL12) in angiogenesis are not fully elucidated. Recently, we showed that Notch inhibition induces extensive intussusceptive angiogenesis by recruitment of mononuclear cells and it was associated with increased levels of SDF‐1 and CXCR4. In the current study, we demonstrated SDF‐1 expression in liver sinusoidal vessels of Notch1 knockout mice with regenerative hyperplasia by means of intussusception, but we did not detect any SDF‐1 expression in wild‐type mice with normal liver vessel structure. In addition, pharmacological inhibition of SDF‐1/CXCR4 signalling by AMD3100 perturbs intussusceptive vascular growth and abolishes mononuclear cell recruitment in the chicken area vasculosa. In contrast, treatment with recombinant SDF‐1 protein increased microvascular density by 34% through augmentation of pillar number compared to controls. The number of extravasating mononuclear cells was four times higher after SDF‐1 application and two times less after blocking this pathway. Bone marrow‐derived mononuclear cells (BMDC) were recruited to vessels in response to elevated expression of SDF‐1 in endothelial cells. They participated in formation and stabilization of pillars. The current study is the first report to implicate SDF‐1/CXCR4 signalling in intussusceptive angiogenesis and further highlights the stabilizing role of BMDC in the formation of pillars during vascular remodelling.     

Dynamics of the Developing Chick Chorioallantoic Membrane Assessed by Stereology,Allometry, Immunohistochemistry and Molecular Analysis

The chick chorioallantoic membrane (CAM) is a widely used model for the study of angiogenesis, tumour growth, as well as drug efficacy. In spite of this, little is known about the developmental alteration from its appearance to the time of hatching. In the current study the CAM has been studied by classical stereology and allometry. Expression levels of selected angiogenesis-related molecules were estimated by RT-PCR and cell dynamics assessed by proliferation and apoptosis assays. Absolute CAM volume increased from a low of 0.47 ± 0.11 cm³ at embryonic day 8 (E8) to a high of 2.05 ± 0.27 cm³ at E18, and then decreased to 1.6 ± 0.47 cm³ at E20. On allometric analysis, three growth phases were identifiable. Between E8-13 (phase I), the CAM grew fastest; moderately in phase II (E13-18) but was regressing in phase III (E18-20). The chorion, the mesenchyme and the allantoic layers grew fastest in phase I, but moderately in phase II. The mesenchyme grew slowly in phase III while the chorion and allantois were regressing. Chorionic cell volume increased fastest in phase I and was regressing in phase III. Chorionic capillaries grew steadily in phase I and II but regressed in phase III. Both the chorion and the allantois grew by intrinsic cell proliferation as well as recruitment of cells from the mesenchyme. Cell proliferation was prominent in the allantois and chorion early during development, declined after E17 and apoptosis started mainly in the chorion from E14. VEGFR2 expression peaked at E11 and declined steadily towards E20, VEGF peaked at E13 and E20 while HIF 1α had a peak at E11 and E20. Studies targeting CAM growth and angiogenesis need to take these growth phases into consideration     

Contribution of HIF-P4H isoenzyme inhibition to metabolism indicates major beneficial effects being conveyed by HIF-P4H-2 antagonism

Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases (HIF-P4Hs 1–3) are druggable targets in renal anemia, where pan-HIF-P4H inhibitors induce an erythropoietic response. Preclinical data suggest that HIF-P4Hs could also be therapeutic targets for treating metabolic dysfunction, although the contributions of HIF-P4H isoenzymes in various tissues to the metabolic phenotype are inadequately understood. Here, we used mouse lines that were gene-deficient for HIF-P4Hs 1 to 3 and two preclinical pan-HIF-P4H inhibitors to study the contributions of these isoenzymes to the anthropometric and metabolic outcome and HIF response. We show both inhibitors induced a HIF response in wildtype white adipose tissue (WAT), liver, and skeletal muscle and alleviated metabolic dysfunction during a 6-week treatment period, but they did not alter healthy metabolism. Our data indicate that HIF-P4H-1 contributed especially to skeletal muscle and WAT metabolism and that its loss lowered body weight and serum cholesterol levels upon aging. In addition, we found HIF-P4H-3 had effects on the liver and WAT and its loss increased body weight, adiposity, liver weight and triglyceride levels, WAT inflammation, and cholesterol levels and resulted in hyperglycemia and insulin resistance, especially during aging. Finally, we demonstrate HIF-P4H-2 affected all tissues studied; its inhibition lowered body and liver weight and serum cholesterol levels and improved glucose tolerance. We found very few HIF target metabolic mRNAs were regulated by the inhibition of three isoenzymes, thus suggesting a potential for selective therapeutic tractability. Altogether, these data provide specifications for the future development of HIF-P4H inhibitors for the treatment of metabolic diseases.     

Forebrain-Specific Loss of BMPRII in Mice Reduces Anxiety and Increases Object Exploration

To investigate the role of Bone Morphogenic Protein Receptor Type II (BMPRII) in learning, memory, and exploratory behavior in mice, a tissue-specific knockout of BMPRII in the post-natal hippocampus and forebrain was generated. We found that BMPRII mutant mice had normal spatial learning and memory in the Morris water maze, but showed significantly reduced swimming speeds with increased floating behavior. Further analysis using the Porsolt Swim Test to investigate behavioral despair did not reveal any differences in immobility between mutants and controls. In the Elevated Plus Maze, BMPRII mutants and Smad4 mutants showed reduced anxiety, while in exploratory tests, BMPRII mutants showed more interest in object exploration. These results suggest that loss of BMPRII in the mouse hippocampus and forebrain does not disrupt spatial learning and memory encoding, but instead impacts exploratory and anxiety-related behaviors.     

Characterization of the Munc13-calmodulin interaction by photoaffinity labeling

Sensing of and response to transient increases in the residual presynaptic Ca2+ levels are important adaptive mechanisms that define the short-term plasticity characteristics of neurons. Due to their essential function in synaptic vesicle priming and in the modulation of synaptic strength, Munc13 proteins have emerged as key regulators of these adaptive mechanisms. Indeed, Munc13-1 and ubMunc13-2 contain a conserved calmodulin (CaM) binding site and the Ca2+ -dependent interaction of these Munc13 isoforms with CaM constitutes a molecular mechanism that transduces residual Ca2+ signaling to the synaptic exocytotic machinery. Here, we used Munc13-derived model peptides in photoaffinity labeling (PAL) experiments to demonstrate the stoichiometric and Ca2+ -dependent CaM binding of the other members of the Munc13 family, bMunc13-2 and Munc13-3, via structurally distinct non-conserved binding sites. A PAL-based Ca2+ titration assay revealed that all Munc13 isoforms can form a complex with CaM already at low Ca2+ concentrations just above resting levels, underscoring the Ca2+ sensor/effector function of this interaction in short-term synaptic plasticity phenomena.     

Withaferin A Targets Intermediate Filaments Glial Fibrillary Acidic Protein and Vimentin in a Model of Retinal Gliosis

Gliosis is a biological process that occurs during injury repair in the central nervous system and is characterized by the overexpression of the intermediate filaments (IFs) glial fibrillary acidic protein (GFAP) and vimentin. A common thread in many retinal diseases is reactive Müller cell gliosis, an untreatable condition that leads to tissue scarring and even blindness. Here, we demonstrate that the vimentin-targeting small molecule withaferin A (WFA) is a novel chemical probe of GFAP. Using molecular modeling studies that build on the x-ray crystal structure of tetrameric vimentin rod 2B domain we reveal that the WFA binding site is conserved in the corresponding domain of tetrameric GFAP. Consequently, we demonstrate that WFA covalently binds soluble recombinant tetrameric human GFAP at cysteine 294. In cultured primary astrocytes, WFA binds to and down-regulates soluble vimentin and GFAP expression to cause cell cycle G₀/G₁ arrest. Exploiting a chemical injury model that overexpresses vimentin and GFAP in retinal Müller glia, we demonstrate that systemic delivery of WFA down-regulates soluble vimentin and GFAP expression in mouse retinas. This pharmacological knockdown of soluble IFs results in the impairment of GFAP filament assembly and inhibition of cell proliferative response in Müller glia. We further show that a more severe GFAP filament assembly deficit manifests in vimentin-deficient mice, which is partly rescued by WFA. These findings illustrate WFA as a chemical probe of type III IFs and illuminate this class of withanolide as a potential treatment for diverse gliosis-dependent central nervous system traumatic injury conditions and diseases, and for orphan IF-dependent pathologies.