Mutations in human presenilin (PS) genes cause aggressive forms of familial Alzheimer's disease. Presenilins are polytopic proteins that harbour the catalytic site of the c-secretase complex and cleave many type I transmembrane proteins including b-amyloid precursor protein (APP), Notch and syndecan 3. Contradictory results have been published concerning whether PS mutations cause 'abnormal' gain or (partial) loss of function of c-secretase. To avoid the possibility that wild-type PS confounds the interpretation of the results, we used presenilin-deficient cells to analyse the effects of different clinical mutations on APP, Notch, syndecan 3 and N-cadherin substrate processing, and on c-secretase complex formation. A loss in APP and Notch substrate processing at e and S3 cleavage sites was observed with all presenilin mutants, whereas APP processing at the c site was affected in variable ways. PS1-D9 and PS1-L166P mutations caused a reduction in b-amyloid peptide (Ab) 40 production whereas PS1-G384A mutant significantly increased Ab 42 . Interestingly PS2, a close homologue of PS1, appeared to be a less efficient producer of Ab than PS1. Finally, subtle differences in c-secretase complex assembly were observed. Overall, our results indicate that the different mutations in PS affect c-secretase structure or function in multiple ways.

Nicastrin and presenilin are two major components of the γ-secretase complex, which executes the intramembrane proteolysis of type I integral membrane proteins such as the amyloid precursor protein (APP) and Notch. Nicastrin is synthesized in fibroblasts and neurons as an endoglycosidase-H-sensitive glycosylated precursor protein (immature nicastrin) and is then modified by complex glycosylation in the Golgi apparatus and by sialylation in the trans-Golgi network (mature nicastrin). These modifications are not observed with exogenously overexpressed nicastrin. Under normal cell culture conditions, only mature nicastrin is expressed at the cell surface and binds to the presenilin heterodimers. Mature nicastrin has a half-life of more than 24 hours. In the absence of presenilin 1 and 2,nicastrin remains entirely endoglycosidase H sensitive, is retained in the endoplasmic reticulum and is slowly degraded. Single presenilin 1 or presenilin 2 deficiency affects glycosylation of nicastrin to a lesser extent than the combined presenilin deficiencies, suggesting a correlation between either the transport of nicastrin out of the endoplasmic reticulum or the concomitant complex glycosylation of nicastrin, and γ-secretase activity. However, when complex glycosylation of nicastrin was inhibited using mannosidase I inhibitors, γ-secretase cleavage of APP or Notch was not inhibited and the immature nicastrin still associates with presenilin and appears at the cell surface. Complex glycosylation of nicastrin is therefore not needed for γ-secretase activity. Because the trafficking of nicastrin to the Golgi apparatus is dependent on presenilins, our data point to a central role of presenilin in nicastrin maturation/localization, which could help to partially resolve the `spatial paradox'.

Vascular endothelial growth factor (VEGF) and β‐catenin both act broadly in embryogenesis and adulthood, including in the skeletal and vascular systems. Increased or deregulated activity of these molecules has been linked to cancer and bone‐related pathologies. By using novel mouse models to locally increase VEGF levels in the skeleton, we found that embryonic VEGF over‐expression in osteo‐chondroprogenitors and their progeny largely pheno‐copied constitutive β‐catenin activation. Adult induction of VEGF in these cell populations dramatically increased bone mass, associated with aberrant vascularization, bone marrow fibrosis and haematological anomalies. Genetic and pharmacological interventions showed that VEGF increased bone mass through a VEGF receptor 2‐ and phosphatidyl inositol 3‐kinase‐mediated pathway inducing β‐catenin transcriptional activity in endothelial and osteoblastic cells, likely through modulation of glycogen synthase kinase 3‐β phosphorylation. These insights into the actions of VEGF in the bone and marrow environment underscore its power as pleiotropic bone anabolic agent but also warn for caution in its therapeutic use. Moreover, the finding that VEGF can modulate β‐catenin activity may have widespread physiological and clinical ramifications.

ADAM10 is involved in the proteolytic processing and shedding of proteins such as the amyloid precursor protein (APP), cadherins, and the Notch receptors, thereby initiating the regulated intramembrane proteolysis (RIP) of these proteins. Here, we demonstrate that the sheddase ADAM10 is also subject to RIP. We identify ADAM9 and -15 as the proteases responsible for releasing the ADAM10 ectodomain, and Presenilin/␥-Secretase as the protease responsible for the release of the ADAM10 intracellular domain (ICD). This domain then translocates to the nucleus and localizes to nuclear speckles, thought to be involved in gene regulation. Thus, ADAM10 performs a dual role in cells, as a metalloprotease when it is membrane-bound, and as a potential signaling protein once cleaved by ADAM9/15 and the ␥-Secretase. ADAMs8 (A disintegrin and metalloprotease) are type 1 transmembrane proteins related to snake venom integrin ligands and metalloproteases. All 38 different family members feature a common modular ectodomain structure (1-4) (Fig. 1A). In addition to the membrane-bound, full-length prototype, soluble ADAM variants have also been identified, consisting of only the ectodomain or fragments thereof that are released into the intercellular space. Such variants are generated by partial gene duplication (ADAM9) (5), alternative splicing (ADAM12) (6, 7), or proteolysis (ADAMs 8, 13, and 19) (8 -10). ADAMs can be grouped either by their tissue distribution and/or functional properties. One major group (ADAMs 2, 3, 5, 6, 16, 18, 20, 21, 24, 25, 26, 29, and 30) is expressed exclusively in the male gonad, with an emerging role in sperm maturation. A second group (ADAMs 2,7,11,18,22,23,and 29) is characterized by an inactive protease domain, and they seem to be mainly important for cell adhesion and fusion. A large third group of ADAMs displays a broad expression profile and has demonstrated (ADAMs 8,9,10,12,17,19, and 28) or predicted (ADAMs 15,20,21,30, and 33) proteolytic activity. These proteases play a major role in the ectodomain shedding of proteins involved in paracrine signaling, cell adhesion, and intracellular signaling (reviewed in Refs. 11 and 12). The site specificity of the cleavage of these substrates is rather relaxed, and apparently different family members can mutually compensate for each other. This has been illustrated particularly well for the amyloid precursor protein (APP) (13-17).ADAM10 is one of the proteolytically active ADAM members (15, 18 -21). The list of ADAM10 substrates is still growing, confirming the central role of ADAM10 in many important biological processes, such as cell migration and axonal navigation (robo receptors and ephrins (22, 23), cell adhesion (cadherins (19, 21), CD44 and L1 (24)), and regulation of immune reactions, and control of apoptosis (FasL) (25). Importantly, genetic ablation of ADAM10 in vertebrates (15) and invertebrates (26 -29) mainly results in loss of Notch phenotypes, indicating the crucial role for this protease in the Notch signaling pathway (30,31). Finally, AD...

The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3–4 weeks by using Gateway® cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.

The Presenilins are part of the ␥-secretase complex that is involved in the regulated intramembrane proteolysis of amyloid precursor protein and other type I integral membrane proteins. Nicastrin, Pen-2, and Aph1 are the other proteins of this complex. The Presenilins probably contribute the catalytic activity to the protease complex. However, several investigators reported normal A␤-peptide generation in cells expressing Presenilins mutated at the putative catalytic site residue Asp-257, contradicting this hypothesis. Because endogenously expressed wild type Presenilin could contribute to residual ␥-secretase activity in these experiments, we have reinvestigated the problem by expressing mutated Presenilins in a Presenilin-negative cell line. We confirm that Presenilins with mutated Asp residues are catalytically inactive. Unexpectedly, these mutated Presenilins are still partially processed into amino-and carboxyl-terminal fragments by a "Presenilinase"-like activity. They are also able to rescue Pen-2 expression and Nicastrin glycosylation in Presenilin-negative cells and become incorporated into large ϳ440-kDa complexes as assessed by blue native gel electrophoresis. Our study demonstrates that the catalytic activity of Presenilin and its other functions in the generation, stabilization, and transport of the ␥-secretase complex can be separated and extends the concept that Presenilins are multifunctional proteins.

Islet and hepatocyte transplantation are associated with tissue factor-dependent activation of coagulation which elicits instant blood mediated inflammatory reaction, thereby contributing to a low rate of engraftment. The aim of this study was i) to evaluate the procoagulant activity of human adult liver-derived mesenchymal progenitor cells (hALPCs), ii) to compare it to other mesenchymal cells of extra-hepatic (bone marrow mesenchymal stem cells and skin fibroblasts) or liver origin (liver myofibroblasts), and iii) to determine the ways this activity could be modulated. Using a whole blood coagulation test (thromboelastometry), we demonstrated that all analyzed cell types exhibit procoagulant activity. The hALPCs pronounced procoagulant activity was associated with an increased tissue factor and a decreased tissue factor pathway inhibitor expression as compared with hepatocytes. At therapeutic doses, the procoagulant effect of hALPCs was inhibited by neither antithrombin activators nor direct factor Xa inhibitor or direct thrombin inhibitors individually. However, concomitant administration of an antithrombin activator or direct factor Xa inhibitor and direct thrombin inhibitor proved to be a particularly effective combination for controlling the procoagulant effects of hALPCs both in vitro and in vivo. The results suggest that this dual antithrombotic therapy should also improve the efficacy of cell transplantation in humans.

The conditional Cre/loxP system and/or the doxycycline (Dox) inducible Tet-on/off system are widely used in mouse transgenesis but often require time consuming, inefficient cloning/screening steps and extensive mouse breeding strategies. We have therefore developed a highly efficient Gateway- and recombinase-mediated cassette exchange (RMCE)-compatible system to target conditional and/or inducible constructs to the ROSA26 locus of F1 hybrid Bl6/129 ESCs, called G4 ROSALUC ESCs. By combining the Cre/loxP system with or without the inducible Tet-on system using Gateway cloning, we can rapidly generate spatial and/or temporal controllable gain-of-function constructs that can be targeted to the RMCE-compatible ROSA26 locus of the G4 ROSALUC ESCs with efficiencies close to 100 %. These novel ESC-based technologies allow for the creation of multiple gain-of-function conditional and/or inducible transgenic ESC clones and mouse lines in a highly efficient and locus specific manner. Importantly, incorporating insulator sequences into the Dox-inducible vector system resulted in robust, stable transgene expression in undifferentiated ESCs but could not fully overcome transgene mosaicism in the differentiated state.