Autophagy, an evolutionarily conserved lysosomal degradation pathway, has a key role in tissue homeostasis, health and disease4 .
In 2003, we showed that the Caenorhabditis elegans autophagy gene bec-1 (orthologue of yeast ATG6, mammalian beclin 1) was required for lifespan extension in nematodes with a loss of function in the insulin signalling pathway5 .
Subsequently, numerous loss-of-function studies in C. elegans and Drosophila have confirmed an essential role for the autophagy machinery in longevity1,2 , and tissue-specific deletion of core autophagy genes have shown that autophagy delays age-related changes in mouse tissues, including kidney and heart6,7 .
Moreover, physiological inducers (such as caloric restriction) as well as pharmacological inducers (such as spermidine) of autophagy increase lifespan in mice1,8,9 .
Despite these clues that autophagy may be a longevity pathway in mammals, definitive evidence that increased basal autophagy extends mammalian healthspan and lifespan is lacking.
An earlier study（10） demonstrated an increase in lifespan of mice that transgenically overexpress ATG5.
However, it is unclear how overexpression of ATG5, a protein necessary for autophagy but not directly involved in the regulation of autophagy levels, results in increased autophagy.
Moreover, ATG5 has other key functions, such as the regulation of inflammation11, and these roles are not shared by other genes in the autophagy pathway.
Therefore, it is imperative to use a more direct and specific genetic approach to assess the effects of enhanced basal autophagy on mammalian lifespan and healthspan.
To do so, we focused on the mammalian autophagy protein, beclin 1 (encoded by Becn1) 12, which is part of an autophagy-specific class III phosphatidylinositol-3-OH kinase (PI3K) complex13 that has a key role in the regulation of the initiation of autophagosome formation14.
Beclin1=autophagy
We recently reported the construction of mice with a Phe-to-Ala knock-in substitution mutation in the BH3 domain of beclin 1 (F121A; corresponding to F123A in human beclin 1)15 that decreases the binding of two negative regulators of autophagy (BCL2 and BCL-XL) to beclin 1 in vitro16,17. Using these mice, we performed co-immunoprecipitation of endogenous beclin 1 with BCL2 in muscle, heart, kidney and liver of two-month-old wild-type and homozygous knock-in mice. We observed a marked reduction in beclin 1 co-immunoprecipitation with BCL2 in the tissues of the knock-in mice (Fig. 1a, b). In parallel, we analysed autophagic flux by crossing wild-type or knock-in mice with animals that transgenically express green fluorescent protein (GFP)-tagged LC318, a fluorescent marker of autophagosomes. In skeletal muscle, heart, renal glomeruli, proximal convoluted tubules and liver, knock-in mice had significantly increased numbers of GFP–LC3 puncta compared to wild-type control littermates (Fig. 1c, d; Extended Data Fig. 1). In all tissues except for the liver, there was a further increase in GFP–LC3 puncta after treatment with chloroquine, an inhibitor of lysosomal acidification and autophagosome–lysosomal fusion, indicating that the increased numbers of GFP–LC3 puncta in knock-in mice represents a true increase in basal autophagic flux, rather than a block in autophagosomal maturation. We further confirmed that knock-in mice had increased autophagic flux by western blot analyses. Both hearts and kidneys had increased conversion of LC3-I to LC3-II (the lipidated, autophagosome-associated form of LC3), decreased levels of total LC3 and decreased levels of the autophagy substrate p6219 (Fig. 1e, f). Similar findings were also observed in the hearts and kidneys of six- to eight-month-old mice (Extended Data Fig. 2), indicating that the effects of the knock-in mutation are sustained over time in adulthood.
Beclin 1-BCL2自噬调节复合物的破坏促进小鼠的寿命
碱基突变解除自噬的抑制后，自噬增强，寿命增加。
参考文献：Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.
