The metalloproteinase and major amyloid precursor protein (APP) ␣-secretase candidate ADAM10 is responsible for the shedding of proteins important for brain development, such as cadherins, ephrins, and Notch receptors. Adam10 ؊/؊ mice die at embryonic day 9.5, due to major defects in development of somites and vasculogenesis. To investigate the function of ADAM10 in brain, we generated Adam10 conditional knock-out (cKO) mice using a Nestin-Cre promotor, limiting ADAM10 inactivation to neural progenitor cells (NPCs) and NPC-derived neurons and glial cells. The cKO mice die perinatally with a disrupted neocortex and a severely reduced ganglionic eminence, due to precocious neuronal differentiation resulting in an early depletion of progenitor cells. Premature neuronal differentiation is associated with aberrant neuronal migration and a disorganized laminar architecture in the neocortex. Neurospheres derived from Adam10 cKO mice have a disrupted sphere organization and segregated more neurons at the expense of astrocytes. We found that Notch-1 processing was affected, leading to downregulation of several Notch-regulated genes in Adam10 cKO brains, in accordance with the central role of ADAM10 in this signaling pathway and explaining the neurogenic phenotype. Finally, we found that ␣-secretasemediated processing of APP was largely reduced in these neurons, demonstrating that ADAM10 represents the most important APP ␣-secretase in brain. Our study reveals that ADAM10 plays a central role in the developing brain by controlling mainly Notch-dependent pathways but likely also by reducing surface shedding of other neuronal membrane proteins including APP.

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...

SUMMARYThe disintegrin and metalloproteinase Adam10 has been implicated in the regulation of key signaling pathways that determine skin morphogenesis and homeostasis. To address the in vivo relevance of Adam10 in the epidermis, we have selectively disrupted Adam10 during skin morphogenesis and in adult skin. K14-Cre driven epidermal Adam10 deletion leads to perinatal lethality, barrier impairment and absence of sebaceous glands. A reduction of spinous layers, not associated with differences in either proliferation or apoptosis, indicates that loss of Adam10 triggers a premature differentiation of spinous keratinocytes. The few surviving K14-Adam10-deleted mice and mice in which Adam10 was deleted postnatally showed loss of hair, malformed vibrissae, epidermal hyperproliferation, cyst formation, thymic atrophy and upregulation of the cytokine thymic stromal lymphopoetin (TSLP), thus indicating non cell-autonomous multi-organ disease resulting from a compromised barrier. Together, these phenotypes closely resemble skin specific Notch pathway loss-of-function phenotypes. Notch processing is indeed strongly reduced resulting in decreased levels of Notch intracellular domain fragment and functional Notch signaling. The data identify Adam10 as the major Site-2 processing enzyme for Notch in the epidermis in vivo, and thus as a central regulator of skin development and maintenance.

␥-Secretase is the protease responsible for amyloid ␤ peptide release and is needed for Notch, N-Cadherin, and possibly other signaling pathways. The protease complex consists of at least four subunits, i.e., Presenilin, Aph1, Pen2, and Nicastrin. Two different genes encode Aph1A and Aph1B in man. A duplication of Aph1B in rodents has given rise to a third gene, Aph1C. Different mixes of ␥-secretase subunits assemble in at least four human and six rodent complexes but it is not known whether they have different activities in vivo. We report here the inactivation of the three Aph1 genes in mice. Aph1A ؊/؊ embryos show a lethal phenotype characterized by angiogenesis defects in the yolk sac, neuronal tube malformations, and mild somitogenesis defects. Aph1B ؊/؊ or C ؊/؊ or the combined Aph1BC ؊/؊ mice (which can be considered as a model for total Aph1B loss in human) survive into adulthood. However, Aph1BC ؊/؊ deficiency causes a mild but significant reduction in amyloid ␤ percursor protein processing in selective regions of the adult brain. We conclude that the biochemical and physiological repercussions of genetically reducing ␥-secretase activity via the different Aph1 components are quite divergent and tissue specific. Our work provides in vivo evidence for the concept that different ␥-secretase complexes may exert different biological functions. In the context of Alzheimer's disease therapy, this implies the theoretical possibility that targeting specific ␥-secretase subunit combinations could yield less toxic drugs than the currently available general inhibitors of ␥-secretase activity.Alzheimer ͉ intramembrane cleavage ͉ Presenilin ͉ knockout T he multimolecular complex ␥-secretase cleaves proteins in their transmembrane domain. The complex consists of at least four subunits called Presenilin (Psen), Nicastrin (Nct), Pen2, and Aph1 (1-3). The Psens provide the catalytic subunits of the complex (4), although the precise functional contribution of the other subunits remains to be clarified. Mutations in the genes encoding presenilin 1 (PSEN1) or its homologue presenilin 2 (PSEN2) cause familial Alzheimer's disease (5, 6). Besides amyloid ␤ (A␤) precursor protein (APP), ␥-secretase cleaves an increasing list of type I transmembrane proteins including Notch (7) and N-Cadherin (8) (for a full review, see ref. 9).Until now, ␥-secretase has largely been considered as a homogenous activity, but especially in mammals the situation is probably more complicated (10). Two different Psen genes and two (human) or three (rodent) Aph1 genes that can be alternatively spliced have been identified. Aph1A or Aph1B and Psen1 or Psen2 are incorporated in a mutually exclusive way into different complexes as demonstrated recently, providing formal proof that at least four different complexes in man (and six in mouse) can be generated (11,12). The question remains however whether those different complexes have also different physiological functions. Because ␥-secretase is considered a potential drug target in Alzheimer's disease, a...