Surface expression and limited proteolysis of ADAM10 are increased by a dominant negative inhibitor of dynamin

<p>Abstract</p> <p>Background</p> <p>The amyloid precursor protein (APP) is cleaved by β- and γ-secretases to generate toxic amyloid β (Aβ) peptides. Alternatively, <it>α</it>-secretases cleave APP within the Aβ domain, precluding Aβ formation and releasing the soluble ectodomain, sAPPα. We previously showed that inhibition of the GTPase dynamin reduced APP internalization and increased release of sAPPα, apparently by prolonging the interaction between APP and α-secretases at the plasma membrane. This was accompanied by a reduction in Aβ generation. In the present study, we investigated whether surface expression of the α-secretase ADAM (a disintegrin and metalloprotease)10 is also regulated by dynamin-dependent endocytosis.</p> <p>Results</p> <p>Transfection of human embryonic kidney (HEK) cells stably expressing M3 muscarinic receptors with a dominant negative dynamin I mutant (dyn I K44A), increased surface expression of both immature, and mature, catalytically active forms of co-expressed ADAM10. Surface levels of ADAM10 were unaffected by activation of protein kinase C (PKC) or M3 receptors, indicating that receptor-coupled shedding of the ADAM substrate APP is unlikely to be mediated by inhibition of ADAM10 endocytosis in this cell line. Dyn I K44A strongly increased the formation of a C-terminal fragment of ADAM10, consistent with earlier reports that the ADAM10 ectodomain is itself a target for sheddases. The abundance of this fragment was increased in the presence of a γ-secretase inhibitor, but was not affected by M3 receptor activation. The dynamin mutant did not affect the distribution of ADAM10 and its C-terminal fragment between raft and non-raft membrane compartments.</p> <p>Conclusions</p> <p>Surface expression and limited proteolysis of ADAM10 are regulated by dynamin-dependent endocytosis, but are unaffected by activation of signaling pathways that upregulate shedding of ADAM substrates such as APP. Modulation of ADAM10 internalization could affect cellular behavior in two ways: by altering the putative signaling activity of the ADAM10 C-terminal fragment, and by regulating the biological function of ADAM10 substrates such as APP and N-cadherin.</p

Genome-Wide Discovery of Somatic Regulatory Variants in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3′ UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.&nbsp

F-Spondin/spon1b Expression Patterns in Developing and Adult Zebrafish

F-spondin, an extracellular matrix protein, is an important player in embryonic morphogenesis and CNS development, but its presence and role later in life remains largely unknown. We generated a transgenic zebrafish in which GFP is expressed under the control of the F-spondin (spon1b) promoter, and used it in combination with complementary techniques to undertake a detailed characterization of the expression patterns of F-spondin in developing and adult brain and periphery. We found that F-spondin is often associated with structures forming long neuronal tracts, including retinal ganglion cells, the olfactory bulb, the habenula, and the nucleus of the medial longitudinal fasciculus (nMLF). F-spondin expression coincides with zones of adult neurogenesis and is abundant in CSF-contacting secretory neurons, especially those in the hypothalamus. Use of this new transgenic model also revealed F-spondin expression patterns in the peripheral CNS, notably in enteric neurons, and in peripheral tissues involved in active patterning or proliferation in adults, including the endoskeleton of zebrafish fins and the continuously regenerating pharyngeal teeth. Moreover, patterning of the regenerating caudal fin following fin amputation in adult zebrafish was associated with F-spondin expression in the blastema, a proliferative region critical for tissue reconstitution. Together, these findings suggest major roles for F-spondin in the CNS and periphery of the developing and adult vertebrate

Calcium Dependent CAMTA1 in Adult Stem Cell Commitment to a Myocardial Lineage

The phenotype of somatic cells has recently been found to be reversible. Direct reprogramming of one cell type into another has been achieved with transduction and over expression of exogenous defined transcription factors emphasizing their role in specifying cell fate. To discover early and novel endogenous transcription factors that may have a role in adult-derived stem cell acquisition of a cardiomyocyte phenotype, mesenchymal stem cells from human and mouse bone marrow and rat liver were co-cultured with neonatal cardiomyocytes as an in vitro cardiogenic microenvironment. Cell-cell communications develop between the two cell types as early as 24 hrs in co-culture and are required for elaboration of a myocardial phenotype in the stem cells 8-16 days later. These intercellular communications are associated with novel Ca(2+) oscillations in the stem cells that are synchronous with the Ca(2+) transients in adjacent cardiomyocytes and are detected in the stem cells as early as 24-48 hrs in co-culture. Early and significant up-regulation of Ca(2+)-dependent effectors, CAMTA1 and RCAN1 ensues before a myocardial program is activated. CAMTA1 loss-of-function minimizes the activation of the cardiac gene program in the stem cells. While the expression of RCAN1 suggests involvement of the well-characterized calcineurin-NFAT pathway as a response to a Ca(2+) signal, the CAMTA1 up-regulated expression as a response to such a signal in the stem cells was unknown. Cell-cell communications between the stem cells and adjacent cardiomyocytes induce Ca(2+) signals that activate a myocardial gene program in the stem cells via a novel and early Ca(2+)-dependent intermediate, up-regulation of CAMTA1