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Autophagy has been implicated in the ageing process, but whether autophagy activation extends lifespan in mammals is unknown. Here we show that ubiquitous overexpression of Atg5, a protein essential for autophagosome formation, extends median lifespan of mice by 17.2%. We demonstrate that moderate overexpression of Atg5 in mice enhances autophagy, and that Atg5 transgenic mice showed anti-ageing phenotypes, including leanness, increased insulin sensitivity and improved motor function. Furthermore, mouse embryonic fibroblasts cultured from Atg5 transgenic mice are more tolerant to oxidative damage and cell death induced by oxidative stress, and this tolerance was reversible by treatment with an autophagy inhibitor. Our observations suggest that the leanness and lifespan extension in Atg5 transgenic mice may be the result of increased autophagic activity.

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Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell-cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD 416 G and DSPD 748 G) in vitro, and point mutations substituting the Asp of DVPD 416 G and DSPD 748 G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD 416 G generated a 74-kDa fragment, which was in turn cleaved at DSPD 748 G, yielding 47-and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas-paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix. INTRODUCTIONApoptotic cell death is a fundamental biological process crucial for proper organism development and for maintenance of tissue homeostasis (Raff, 1992). Apoptosis causes characteristic morphological changes that include membrane blebbing, decreased adhesion and intercellular contacts, chromatin condensation, nuclear fragmentation, and the packing of the nuclear fragments into membrane-enclosed apoptotic bodies (Wyllie et al., 1980;Darzynkiewicz et al., 1994). Caspases are a family of highly conserved aspartate-specific cysteine-proteases related to mammalian interleukin-1␤-converting enzyme, which are involved in the final execution phase of apoptosis (Nicholson, 1996;Nicholson and Thornberry, 1997). The substrates for caspases are now known to include a growing number of molecules involved in cytoskeletal regulation and signaling, including ␣-fodrin, gelsolin, growth arrest-specific gene (Gas 2), MEKK-1, and PKC (Brancolini et al., 1995;Emoto et al., 1995;Martin et al., 1995;Ghayur et al., 1996;Cardone et al., 1997;Kothakota et al., 1997;Janicke et al., 1998). Recent studies have shown that attachment to the extracellular matrix (ECM) actively regulates cell survival, and that cells undergoing apoptosis lose their connections to neighboring cells and to the ECM. For example, epithelial and endothelial cells undergo apoptosis ("anoikis") when displaced from the ECM (Frisch and Francis, 1994;Re et al., 1994), whereas cell adhesion to ECM suppresses apoptosis in mammary epithelial cells (Frisch and Francis, 1994;Boudreau et al., 1995;Aharoni et al., 1996).Cell attachment to the ECM i...

Cain͞cabin1 is an endogenous inhibitor of calcineurin (Cn), a calcium-dependent serine͞threonine phosphatase involved in various cellular functions including apoptosis. We show here that during apoptosis cain͞cabin1 is cleaved by calpain at the carboxyl terminus to generate a cleavage product with a molecular mass of 32 kDa as a necessary step leading to Cn-mediated cell death.