A large body of literature is available on wound healing in humans. Nonetheless, a standardized ex vivo wound model without disruption of the dermal compartment has not been put forward with compelling justification. Here, we present a novel wound model based on application of negative pressure and its effects for epidermal regeneration and immune cell behaviour. Importantly, the basement membrane remained intact after blister roof removal and keratinocytes were absent in the wounded area. Upon six days of culture, the wound was covered with one to three-cell thick K14+Ki67+ keratinocyte layers, indicating that proliferation and migration were involved in wound closure. After eight to twelve days, a multi-layered epidermis was formed expressing epidermal differentiation markers (K10, filaggrin, DSG-1, CDSN). Investigations about immune cell-specific manners revealed more T cells in the blister roof epidermis compared to normal epidermis. We identified several cell populations in blister roof epidermis and suction blister fluid that are absent in normal epidermis which correlated with their decrease in the dermis, indicating a dermal efflux upon negative pressure. Together, our model recapitulates the main features of epithelial wound regeneration, and can be applied for testing wound healing therapies and investigating underlying mechanisms.

/pdf/re-epithelialization-and-immune-cell-behaviour-in-an-ex-vivo-3lsvtwo6xm.pdf

Re-epithelialization and immune cell behaviour in an ex vivo human skin model.

Rapid progress in deciphering the biological mechanism of Alzheimer's disease (AD) has arisen from the application of molecular and cell biology to this complex disorder of the limbic and association cortices. In turn, new insights into fundamental aspects of protein biology have resulted from research on the disease. This beneficial interplay between basic and applied cell biology is well illustrated by advances in understanding the genotype-to-phenotype relationships of familial Alzheimer's disease. All four genes definitively linked to inherited forms of the disease to date have been shown to increase the production and/or deposition of amyloid β-protein in the brain. In particular, evidence that the presenilin proteins, mutations in which cause the most aggressive form of inherited AD, lead to altered intramembranous cleavage of the β-amyloid precursor protein by the protease called γ-secretase has spurred progress toward novel therapeutics. The finding that presenilin itself may be the long-sought γ-...

Alzheimer's Disease: Genes, Proteins, and Therapy

Notch signaling defines an evolutionarily ancient cell interaction mechanism, which plays a fundamental role in metazoan development. Signals exchanged between neighboring cells through the Notch receptor can amplify and consolidate molecular differences, which eventually dictate cell fates. Thus, Notch signals control how cells respond to intrinsic or extrinsic developmental cues that are necessary to unfold specific developmental programs. Notch activity affects the implementation of differentiation, proliferation, and apoptotic programs, providing a general developmental tool to influence organ formation and morphogenesis.

http://science.sciencemag.org/content/sci/284/5415/770.full.pdf

Notch Signaling: Cell Fate Control and Signal Integration in Development

The distinct protein aggregates that are found in Alzheimer's, Parkinson's, Huntington's and prion diseases seem to cause these disorders. Small intermediates - soluble oligomers - in the aggregation process can confer synaptic dysfunction, whereas large, insoluble deposits might function as reservoirs of the bioactive oligomers. These emerging concepts are exemplified by Alzheimer's disease, in which amyloid beta-protein oligomers adversely affect synaptic structure and plasticity. Findings in other neurodegenerative diseases indicate that a broadly similar process of neuronal dysfunction is induced by diffusible oligomers of misfolded proteins.

Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.

Cerebral deposition of amyloid beta peptide (Abeta) is an early and critical feature of Alzheimer's disease. Abeta generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: beta-secretase and gamma-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of beta-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increased the amount of beta-secretase cleavage products, and these were cleaved exactly and only at known beta-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreased the amount of beta-secretase cleavage products, and purified BACE protein cleaved APP-derived substrates with the same sequence specificity as beta-secretase. Finally, the expression pattern and subcellular localization of BACE were consistent with that expected for beta-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease.

/pdf/beta-secretase-cleavage-of-alzheimer-s-amyloid-precursor-7frdw77i59.pdf

Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE.

The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism

Signalling through the receptor protein Notch, which is involved in crucial cell-fate decisions during development, requires ligand-induced cleavage of Notch. This cleavage occurs within the predicted transmembrane domain, releasing the Notch intracellular domain (NICD), and is reminiscent of gamma-secretase-mediated cleavage of beta-amyloid precursor protein (APP), a critical event in the pathogenesis of Alzheimer's disease. A deficiency in presenilin-1 (PS1) inhibits processing of APP by gamma-secretase in mammalian cells, and genetic interactions between Notch and PS1 homologues in Caenorhabditis elegans indicate that the presenilins may modulate the Notch signalling pathway. Here we report that, in mammalian cells, PS1 deficiency also reduces the proteolytic release of NICD from a truncated Notch construct, thus identifying the specific biochemical step of the Notch signalling pathway that is affected by PS1. Moreover, several gamma-secretase inhibitors block this same step in Notch processing, indicating that related protease activities are responsible for cleavage within the predicted transmembrane domains of Notch and APP. Thus the targeting of gamma-secretase for the treatment of Alzheimer's disease may risk toxicity caused by reduced Notch signalling.

A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain.

Notch proteins are ligand-activated transmembrane receptors involved in cell-fate selection throughout development1,2,3. No known enzymatic activity is contained within Notch and the molecular mechanism by which it transduces signals across the cell membrane is poorly understood. In many instances, Notch activation results in transcriptional changes in the nucleus through an association with members of the CSL family of DNA-binding proteins (where CSL stands for CBF1, Su(H), Lag-1)1,2,3,4. As Notch is located in the plasma membrane and CSL is a nuclear protein, two models have been proposed to explain how they interact (Fig. 1) . The first suggests that the two interact transiently at the membrane1,5,6,7. The second postulates that Notch is cleaved by a protease, enabling the cleaved fragment to enter the nucleus6,8,9,10,11,12,13,14. Here we show that signalling by a constitutively active membrane-bound Notch-1 protein requires the proteolytic release of the Notch intracellular domain (NICD), which interacts preferentially with CSL. Very small amounts of NICD are active, explaining why it is hard to detect in the nucleus in vivo. We also show that it is ligand binding that induces release of NICD.

Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain.

Notch belongs to a family of transmembrane proteins that are widely conserved from flies to vertebrates and are thought to be involved in cell-fate decisions. In Drosophila, the Suppressor of hairless (Su(H)) gene and genes of the Enhancer of split (E(Spl)) complex, which encode proteins of the basic helix-loop-helix type have been implicated in the Notch signalling pathway. Mammalian homologues of E(Spl), such as the mouse Hairy enhancer of split (HES-1), have been isolated. Both HES-1 and the intracellular domain of murine Notch (mNotch) are able to block MyoD-induced myogenesis. Here we show that activated forms of mNotch associate with the human analogue of Su(H), KBF2/RBP-J kappa (refs 8,9) and act as transcriptional activators through the KBF2-binding sites of the HES-1 promoter.

Raphael Kopan

Papers

The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism

A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain.

Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain.

Signalling downstream of activated mammalian Notch.

Notch Signaling: From the Outside In