The disintegrin/metalloproteinase Adam10 is essential for epidermal integrity and Notch-mediated signaling.

The 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.

The disintegrin/metalloproteinase ADAM10 is essential for the establishment of the brain cortex.

The metalloproteinase and major amyloid precursor protein (APP) alpha-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 alpha-secretase-mediated processing of APP was largely reduced in these neurons, demonstrating that ADAM10 represents the most important APP alpha-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.

Lack of a-disintegrin-and-metalloproteinase ADAM10 leads to intracellular accumulation and loss of shedding of the cellular prion protein in vivo.

BACKGROUND: The cellular prion protein (PrPC) fulfils several yet not completely understood physiological functions. Apart from these functions, it has the ability to misfold into a pathogenic scrapie form (PrPSc) leading to fatal transmissible spongiform encephalopathies. Proteolytic processing of PrPC generates N- and C-terminal fragments which play crucial roles both in the pathophysiology of prion diseases and in transducing physiological functions of PrPC. A-disintegrin-and-metalloproteinase 10 (ADAM10) has been proposed by cell culture experiments to be responsible for both shedding of PrPC and its α-cleavage. Here, we analyzed the role of ADAM10 in the proteolytic processing of PrPC in vivo. RESULTS: Using neuron-specific Adam10 knockout mice, we show that ADAM10 is the sheddase of PrPC and that its absence in vivo leads to increased amounts and accumulation of PrPC in the early secretory pathway by affecting its posttranslational processing. Elevated PrPC levels do not induce apoptotic signalling via p53. Furthermore, we show that ADAM10 is not responsible for the α-cleavage of PrPC. CONCLUSION: Our study elucidates the proteolytic processing of PrPC and proves a role of ADAM10 in shedding of PrPC in vivo. We suggest that ADAM10 is a mediator of PrPC homeostasis at the plasma membrane and, thus, might be a regulator of the multiple functions discussed for PrPC. Furthermore, identification of ADAM10 as the sheddase of PrPC opens the avenue to devising novel approaches for therapeutic interventions against prion diseases.

Gamma-secretase and the intramembrane proteolysis of Notch.

Gamma-secretase is the crucial proteolytic activity that releases the Notch intracellular domain and is therefore a central player in the canonical Notch-signaling transduction pathway. We discuss here briefly the discovery of gamma-secretase and what is known on its structure and function. Recent work also indicates that the assembly and activity of gamma-secretase might be regulated by novel cell biological mechanisms. Finally we explore the recent insight that there are several gamma-secretase complexes in mammalian and discuss possibilities to use gamma-secretase as a drug target in Alzheimer's disease and cancer.

ADAM10, the rate-limiting protease of regulated intramembrane proteolysis of Notch and other proteins, is processed by ADAMS-9, ADAMS-15, and the gamma-secretase.

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/gamma-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 gamma-Secretase.

(Make) stick and cut loose--disintegrin metalloproteases in development and disease.

"A disintegrin and metalloprotease" (ADAM) proteases form a still growing family of about 40 type 1 transmembrane proteins. They are defined by a common modular ectodomain architecture that combines cell deadhesion/adhesion and fusion motifs (disintegrin and cysteine-rich domains), with a Zn-protease domain capped by a large prodomain. Their ectodomain thus strikingly resembles snake venom disintegrin proteases, which by combined integrin blocking and extracellular proteolysis, can cause extensive tissue damage after snake bites. A surprisingly large proportion (13 ADAMs) is exclusively expressed in the male gonads, and only a minority can be found throughout all tissues. As predicted by their amino acid sequence, a major proportion of this family has not maintained a functional protease domain, most probably rendering them into pure adhesion and/or fusion proteins. For most ADAMs, the respective key function has remained elusive. Despite their overall conserved ectodomain structure, ADAMs appear to be subdivided into those with a predominant role in direct adhesion (e.g., ADAMs 1, 2, and 3) and those mainly acting as proteases (e.g., ADAMs 10 and 17). Only for a few of them are functions of more than one domain documented (e.g., ADAM9 in cell fusion and proteolysis). Several ADAMs exist in both membrane-resident and secreted isoforms; the functional significance of this dichotomy is in most cases still unclear. Knockout phenotypes have been informative only in a few cases (ADAMs 1, 2, 10, 12, 15, 17, and 19) and are mainly related to their protease function. A common denominator of ADAM-mediated proteolysis is the ectodomain shedding of a broad spectrum of substrates, including paracrine growth factors like epidermal growth factor receptor (EGFR) ligands, cell adhesion molecules like CD44 or cadherins, and the initiation of regulated intramembrane proteolysis (RIP), whereby the transmembrane fragment of the respective substrate is further cleaved by an intramembrane cleaving protease to release an intracellular domain acting as a nuclear transcription regulator. Most ADAMs feature a significant overlap of substrate specificities, explaining why an inactivation of individual ADAMs only rarely causes major phenotypes.

Differential contribution of the three Aph1 genes to gamma-secretase activity in vivo.

Gamma-secretase is the protease responsible for amyloid beta 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 gamma-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 beta percursor protein processing in selective regions of the adult brain. We conclude that the biochemical and physiological repercussions of genetically reducing gamma-secretase activity via the different Aph1 components are quite divergent and tissue specific. Our work provides in vivo evidence for the concept that different gamma-secretase complexes may exert different biological functions. In the context of Alzheimer's disease therapy, this implies the theoretical possibility that targeting specific gamma-secretase subunit combinations could yield less toxic drugs than the currently available general inhibitors of gamma-secretase activity.