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Autophagy proteins LC3B, ATG5 and ATG12 participate in quality control after mitochondrial damage and influence lifespan.

Autophagy (2011-12-16)
Sören Mai, Britta Muster, Jürgen Bereiter-Hahn, Marina Jendrach
ABSTRACT

Mitochondrial health is maintained by the quality control mechanisms of mitochondrial dynamics (fission and fusion) and mitophagy. Decline of these processes is thought to contribute to aging and neurodegenerative diseases. To investigate the role of mitochondrial quality control in aging on the cellular level, human umbilical vein endothelial cells (HUVEC) were subjected to mitochondria-targeted damage by combining staining of mitochondria and irradiation. This treatment induced a short boost of reactive oxygen species, which resulted in transient fragmentation of mitochondria followed by mitophagy, while mitochondrial dynamics were impaired. Furthermore, targeted mitochondrial damage upregulated autophagy factors LC3B, ATG5 and ATG12. Consequently these proteins were overexpressed in HUVEC as an in vitro aging model, which significantly enhanced the replicative life span up to 150% and the number of population doublings up to 200%, whereas overexpression of LAMP-1 did not alter the life span. Overexpression of LC3B, ATG5 and ATG12 resulted in an improved mitochondrial membrane potential, enhanced ATP production and generated anti-apoptotic effects, while ROS levels remained unchanged and the amount of oxidized proteins increased. Taken together, these data relate LC3B, ATG5 and ATG12 to mitochondrial quality control after oxidative damage, and to cellular longevity.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
OxyBlot Protein Oxidation Detection Kit, The OxyBlot Protein Oxidation Detection Kit provides the reagents to perform the immunoblot detection of carbonyl groups introduced into proteins by oxidative reactions with ozone or oxides of nitrogen or by metal catalyzed oxidation.
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Anti-ATG5 (N-terminal) antibody produced in rabbit, affinity isolated antibody, PBS solution
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Anti-LC3 antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
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Senescence Cells Histochemical Staining Kit, sufficient for 100 tests
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Acid phosphatase Assay Kit,  kit sufficient for 1,000 assays (multiwell plates),  kit sufficient for 100 assays (tubes)