Cardiolipin Regulates Mitophagy Through The Pkc Pathway

Cardiolipin (CL), the signature phospholipid of mitochondrial membranes, is important for cardiovascular health. Perturbation of CL metabolism is implicated in cardiovascular disease (CVD). The link between CL and CVD may be explained by the physiological roles of CL in pathways that are cardioprotective, such as autophagy/mitophagy and the mitogen-activated protein kinase (MAPK) pathways. My dissertation work focuses on elucidating how CL influences mitophagy and MAPK pathways. crd1Δ was synthetically lethal/sick with the general autophagy mutants atg8Δ, atg18Δ and mitophagy mutant atg32Δ, suggesting that autophagy/mitophagy may be deficient in cells lacking CL. Microscopic examination of mitophagy revealed decreased translocation of GFP-tagged mitochondrial proteins into the vacuole of crd1Δ cells. This was confirmed by a decreased level of free GFP generated by cleavage of GFP-tagged mitochondrial protein after delivery into the vacuole by mitophagy. These findings indicated that mitophagy is decreased in CL-deficient cells. Expression of ATG8 was increased in crd1Δ cells at 37˚C, suggesting that nonselective autophagy was upregulated to compensate for decreased mitophagy. The PKC and HOG MAPK pathways are known to be required for mitophagy. crd1Δ growth defects are exacerbated by deletion of HOG pathway genes SHO1, SSK1, STE50 and HOG1, and rescued by stimulating the HOG pathway and upregulating the PKC pathway. These findings suggested the possibility that MAPK pathways are defective in crd1Δ cells. Phosphorylation of Slt2p and Hog1p in response to stimulants was decreased in crd1Δ, consistent with defective activation of these MAPK pathways. Interestingly, upregulating PKC by transforming the cell with a vector expressing a constitutively activated Pkc1p rescued defective mitophagy in crd1Δ. These results suggest that the mechanism underlying defective mitophagy caused by loss of CL is a defective PKC pathway

The Role of Cardiolipin in Cardiovascular Health

Cardiolipin (CL), the signature phospholipid of mitochondrial membranes, is crucial for both mitochondrial function and cellular processes outside of the mitochondria. The importance of CL in cardiovascular health is underscored by the life-threatening genetic disorder Barth syndrome (BTHS), which manifests clinically as cardiomyopathy, skeletal myopathy, neutropenia, and growth retardation. BTHS is caused by mutations in the gene encoding tafazzin, the transacylase that carries out the second CL remodeling step. In addition to BTHS, CL is linked to other cardiovascular diseases (CVDs), including cardiomyopathy, atherosclerosis, myocardial ischemia-reperfusion injury, heart failure, and Tangier disease. The link between CL and CVD may possibly be explained by the physiological roles of CL in pathways that are cardioprotective, including mitochondrial bioenergetics, autophagy/mitophagy, and mitogen activated protein kinase (MAPK) pathways. In this review, we focus on the role of CL in the pathogenesis of CVD as well as the molecular mechanisms that may link CL functions to cardiovascular health

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field