X-linked microtubule-associated protein, Mid1, regulates axon development

Opitz syndrome (OS) is a genetic neurological disorder. The gene responsible for the X-linked form of OS, Midline-1 (MID1), encodes an E3 ubiquitin ligase that regulates the degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). However, how Mid1 functions during neural development is largely unknown. In this study, we provide data from in vitro and in vivo experiments suggesting that silencing Mid1 in developing neurons promotes axon growth and branch formation, resulting in a disruption of callosal axon projections in the contralateral cortex. In addition, a similar phenotype of axonal development was observed in the Mid1 knockout mouse. This defect was largely due to the accumulation of PP2Ac in Mid1-depleted cells as further down-regulation of PP2Ac rescued the axonal phenotype. Together, these data demonstrate that Mid1-dependent PP2Ac turnover is important for normal axonal development and that dysregulation of this process may contribute to the underlying cause of OS

The Tripartite Motif Protein MADD-2 Functions with the Receptor UNC-40 (DCC) in Netrin-Mediated Axon Attraction and Branching

Neurons innervate multiple targets by sprouting axon branches from a primary axon shaft. We show here that the ventral guidance factor unc-6 (Netrin), its receptor unc-40 (DCC), and the gene madd-2 stimulate ventral axon branching in C. elegans chemosensory and mechanosensory neurons. madd-2 also promotes attractive axon guidance to UNC-6 and assists unc-6- and unc-40-dependent ventral recruitment of the actin regulator MIG-10 in nascent axons. MADD-2 is a tripartite motif protein related to MID-1, the causative gene for the human developmental disorder Opitz syndrome. MADD-2 and UNC-40 proteins preferentially localize to a ventral axon branch that requires their function; genetic results indicate that MADD-2 potentiates UNC-40 activity. Our results identify MADD-2 as an UNC-40 cofactor in axon attraction and branching, paralleling the role of UNC-5 in repulsion, and provide evidence that targeting of a guidance factor to specific axonal branches can confer differential responsiveness to guidance cues.National Institutes of Health (U.S.) (Grant number GM0680678

Regulation of the MID1 protein function is fine-tuned by a complex pattern of alternative splicing

Clinical features of Opitz BBB/G syndrome are confined to defects of the developing ventral midline, whereas the causative gene, MID1, is ubiquitously expressed. Therefore, a non-redundant physiological function of the MID1 product appears to be developmentally restricted. Here, we report the identification of several alternative MID1 exons in human, mouse and fugu. We show that splice variants of the MID1 gene that are comparable in terms of function occur in the three organisms, suggesting an important role in the regulation of the MID1 protein function. Accordingly, we observed differential MID1 transcript patterns in a tissue-specific manner by Northern blot and RT-PCR. The identified splice variants cause loss-of-function effects via several mechanisms. Some introduce a stop codon followed by a novel poly(A+) tail, leading to the formation of C-terminally truncated proteins. Dominant negative effects through altered binding to the MID1-interacting protein agr4 in vitro could be demonstrated in a couple of cases. Others carry premature termination codons without poly(A+) tails. These are degraded by nonsense mediated mRNA decay (NMD). Our data reveal a mechanism conserved in human, mouse and fugu that regulates developmentally restricted MID1 activity and suggest NMD to be critical in the translational regulation of a ubiquitously transcribed mRNA

Anti-Inflammatory Extract from Soil Algae Chromochloris zofingiensis Targeting TNFR/NF-κB Signaling at Different Levels

Inflammatory skin diseases, including atopic dermatitis (AD) and psoriasis, are increasing in populations worldwide. The treatment of patients with AD and other forms of skin inflammation is mainly based on the use of topical corticosteroids or calcineurin inhibitors, which can cause significant side effects with long-term use. Therefore, there is a great need for the development of more effective and less toxic anti-inflammatory agents suitable for the treatment of chronic skin lesions. Here, we screened a number of strains from the ASIB 505 terrestrial algae collection and identified a green algae Chromochloris zofingiensis with pronounced anti-inflammatory properties. We found that a crude nonpolar extract of C. zofingiensis (ID name NAE_2022C), grown upon nitrogen deprivation, acts as a bioactive substance by inhibiting TNFR/NF-κB responses in human skin keratinocyte HaCaT cells. We also found that NAE_2022C suppressed the secretion of pro-inflammatory cytokine tumor necrosis factor α (TNFα) and several Th1- and Th2-related chemokines in a reconstituted human epidermis. The TNFR/NF-κB pathway analysis showed multiple inhibitory effects at different levels and disclosed a direct targeting of IKKβ by the extract. Bioassay-guided fractionation followed by high-resolution mass spectrometry detected diacylglyceryl-trimethylhomoserine (DGTS), Lyso-DGTS (LDGTS), 5-phenylvaleric acid, theophylline and oleamide as leading metabolites in the active fraction of NAE_2022C. Further analysis identified betaine lipid DGTS (32:0) as one of the active compounds responsible for the NAE_2022C-mediated NF-κB suppression. Overall, this study presents an approach for the isolation, screening, and identification of anti-inflammatory secondary metabolites produced by soil algae

The Caenorhabditis elegans homolog of the Opitz syndrome gene, madd-2/Mid1, regulates anchor cell invasion during vulval development

Mutations in the human Mid1 gene cause Opitz G/BBB syndrome, which is characterized by various midline closure defects. The Caenorhabditis elegans homolog of Mid1, madd-2, positively regulates signaling by the unc-40 Netrin receptor during the extension of muscle arms to the midline and in axon guidance and branching. During uterine development, a specialized cell called anchor cell (AC) breaches the basal laminae separating the uterus from the epidermis and invades the underlying vulval tissue. AC invasion is guided by an UNC-6 Netrin signal from the ventral nerve cord and an unknown guidance signal from the vulval cells. Using genetic epistasis analysis, we show that madd-2 regulates AC invasion downstream of or in parallel with the Netrin signaling pathway. Measurements of AC shape, polarity and dynamics indicate that MADD-2 prevents the formation of ectopic AC protrusions in the absence of guidance signals. We propose that MADD-2 represses the intrinsic invasive capacity of the AC, while the Netrin and vulval guidance cues locally overcome this inhibitory activity of MADD-2 to guide the AC ventrally into the vulval tissue. Therefore, developmental cell invasion depends on a precise balance between pro- and anti-invasive factors

5-Methoxyleoligin, a Lignan from Edelweiss, Stimulates CYP26B1-Dependent Angiogenesis In Vitro and Induces Arteriogenesis in Infarcted Rat Hearts In Vivo

<div><p>Background</p><p>Insufficient angiogenesis and arteriogenesis in cardiac tissue after myocardial infarction (MI) is a significant factor hampering the functional recovery of the heart. To overcome this problem we screened for compounds capable of stimulating angiogenesis, and herein investigate the most active molecule, 5-Methoxyleoligin (5ML), in detail.</p> <p>Methods and Results</p><p>5ML potently stimulated endothelial tube formation, angiogenic sprouting, and angiogenesis in a chicken chorioallantoic membrane assay. Further, microarray- and knock down- based analyses revealed that 5ML induces angiogenesis by upregulation of CYP26B1. In an <i>in vivo</i> rat MI model 5ML potently increased the number of arterioles in the peri-infarction and infarction area, reduced myocardial muscle loss, and led to a significant increase in LV function (plus 21% 28 days after MI).</p> <p>Conclusion</p><p>The present study shows that 5ML induces CYP26B1-dependent angiogenesis <i>in vitro</i>, and arteriogenesis <i>in vivo</i>. Whether or not CYP26B1 is relevant for <i>in vivo</i> arteriogenesis is not clear at the moment. Importantly, 5ML-induced arteriogenesis <i>in vivo</i> makes the compound even more interesting for a post MI therapy. 5ML may constitute the first low molecular weight compound leading to an improvement of myocardial function after MI.</p> </div

5ML alters the transcription of a small set of genes; CYP26B1 is the causal mediator of 5ML -induced angiogenesis.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058342#pone-0058342-g003" target="_blank">Figures 3A and 3B</a> show median values +/− S.D. of microarray-based analyses of gene-regulation by 5ML in relation to controls 6 and 24 hours after the addition of 5ML (10 µM). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058342#pone-0058342-g003" target="_blank">Figure 3C</a> shows that despite the fact that the knockdown of CYP1A1 had a significant inhibitory effect on spontaneous angiogenesis in HUVECs; CYP1A1 knockdown had no effect on angiogenesis increased by 5ML. Similarly, CYP26B1 knockdown potently repressed spontaneous angiogenesis in HUVECS and in addition completely abrogated 5ML-induced tube formation. Shown are mean values of two independent experiments performed in triplicates +/− SD. * p<0.05. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058342#pone-0058342-g003" target="_blank">Figure 3D</a> shows Western blot analyses of effects of the CYP1A1- and the CYP26B1-specific knockdown on protein expression of the corresponding proteins. Asterisks indicate significant differences (* p<0.05) compared to the corresponding controls.</p