William A. Dunn

A unified nomenclature for yeast autophagy-related genes

The authors would like to thank Drs. Jan A.K.W. Kiel, Ida J. van der Klei, Beth Levine, Fulvio Reggiori, and Takahiro Shintani for helpful comments on the manuscript, and the many researchers in the yeast field who have agreed to changes in the standard names of various genes.

Human Atherosclerotic Plaque Contains Viable Invasive Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis

To the Editor: Because epidemiological evidence supports an association between cardiovascular and periodontal disease, we assessed whether periodontal pathogens were present in atherosclerotic lesions. To detect invasive bacteria, the natural tropism of the bacteria toward human tissues was exploited. Further, bacterial presence was demonstrated using quantitative polymerase chain reaction (Q-PCR). This confirms the presence of periodontal pathogens in atherosclerotic lesions, whereby the bacteria could contribute to the vascular pathology either directly through their cytotoxicity or indirectly by inducing or exacerbating inflammation. Cardiovascular disease (CVD) is the leading cause of death in the in the United States.1 According to the American Heart Association’s statistics from 2003, there were no previous symptoms in 50% of men and 63% of women who died suddenly from CHD. In a 10-year follow-up study, ≈25% of coronary deaths in males and 15% in females occurred in persons in the lowest two quintiles of the multivariate Framingham Heart Study risk scores.2 This and other data have led to an emerging paradigm shift from coronary heart disease having a purely hereditary/nutritional causation to possibly having an infectious component.3 Many epidemiological studies strongly suggest that periodontitis may be a risk factor for coronary heart disease (CHD).4 Serologically, edentulousness and serum IgG-antibodies to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in 1163 men were recently shown to be associated with CHD.5 In a larger prospective study of 6950 subjects, the same authors provide serological evidence that an infection caused by major periodontal pathogens is associated with future stroke.6 Previous studies have identified 16S rRNA of oral microbial pathogens, including P gingivalis and A actinomycetemcomitans , …

Porphyromonas gingivalis Traffics to Autophagosomes in Human Coronary Artery Endothelial Cells

ABSTRACT Porphyromonas gingivalis is a periodontal pathogen that also localizes to atherosclerotic plaques. Our previous studies demonstrated that P. gingivalis is capable of invading endothelial cells and that intracellular bacteria are contained in vacuoles that resemble autophagosomes. In this study, we have examined the trafficking of P. gingivalis 381 to the autophagic pathway. P. gingivalis 381 internalized by human coronary artery endothelial (HCAE) cells is located within vacuoles morphologically identical to autophagosomes. The progression ofP. gingivalis 381 through intracellular vacuoles was analyzed by immunofluorescence microscopy. Vacuoles containingP. gingivalis colocalize with Rab5 and HsGsa7p early after internalization. At later times, P. gingivaliscolocalizes with BiP and then progresses to a vacuole that contains BiP and lysosomal glycoprotein 120. Late endosomal markers and the lysosomal cathepsin L do not colocalize with P. gingivalis 381. The intracellular survival of P. gingivalis 381 decreases over 8 h in HCAE cells pretreated with the autophagy inhibitors 3-methyladenine and wortmannin. In addition, the vacuole containing P. gingivalis 381 lacks BiP but contains cathepsin L in the presence of wortmannin. These results suggest that P. gingivalis 381 evades the endocytic pathway to lysosomes and instead traffics to the autophagosome.

Autophagy and amino acid homeostasis are required for chronological longevity in Saccharomyces cerevisiae

Following cessation of growth, yeast cells remain viable in a nondividing state for a period of time known as the chronological lifespan (CLS). Autophagy is a degradative process responsible for amino acid recycling in response to nitrogen starvation and amino acid limitation. We have investigated the role of autophagy during chronological aging of yeast grown in glucose minimal media containing different supplemental essential and nonessential amino acids. Deletion of ATG1 or ATG7, both of which are required for autophagy, reduced CLS, whereas deletion of ATG11, which is required for selective targeting of cellular components to the vacuole for degradation, did not reduce CLS. The nonessential amino acids isoleucine and valine, and the essential amino acid leucine, extended CLS in autophagy‐deficient as well as autophagy‐competent yeast. This extension was suppressed by constitutive expression of GCN4, which encodes a transcriptional regulator of general amino acid control (GAAC). Consistent with this, GCN4 expression was reduced by isoleucine and valine. Furthermore, elimination of the leucine requirement extended CLS and prevented the effects of constitutive expression of GCN4. Interestingly, deletion of LEU3, a GAAC target gene encoding a transcriptional regulator of branched side chain amino acid synthesis, dramatically increased CLS in the absence of amino acid supplements. In general, this indicates that activation of GAAC reduces CLS whereas suppression of GAAC extends CLS in minimal medium. These findings demonstrate important roles for autophagy and amino acid homeostasis in determining CLS in yeast.

Bacterial interactions with the autophagic pathway.

Bacteria have evolved a variety of mechanisms to invade eukaryotic cells and survive intracellularly. Once inside, bacterial pathogens often modulate their phagosome to establish an intracellular niche for survival and replication. A subset of intracellular pathogens, including Brucella abortus, Legionella pneumophila and Porphyromonas gingivalis, are diverted from the endosomal pathway to the auto‐phagic pathway. Once within the autophagosome, each in some way presumably modifies this compartment to establish an environment necessary for its survival. Transit into autophagosomes represents an avenue by which to escape host defences. In this review, we examine the biochemical and morphological evidence for the survival of some bacterial pathogens by replicating within an autophagosome‐like compartment.

Impaired Autophagy: A Mechanism of Mitochondrial Dysfunction in Anoxic Rat Hepatocytes

Autophagy selectively removes abnormal or damaged organelles such as dysfunctional mitochondria. The mitochondrial permeability transition (MPT) is a marker of impaired mitochondrial function that is evident in hepatic ischemia/reperfusion (I/R) injury. However, the relationship between mitochondrial dysfunction and autophagy in I/R injury is unknown. Cultured rat hepatocytes and mouse livers were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. Expression of autophagy‐related protein 7 (Atg7), Beclin‐1, and Atg12, autophagy regulatory proteins, was analyzed by western blots. Some hepatocytes were incubated with calpain 2 inhibitors or infected with adenoviruses encoding green fluorescent protein (control), Atg7, and Beclin‐1 to augment autophagy. To induce nutrient depletion, a condition stimulating autophagy, hepatocytes were incubated in an amino acid–free and serum‐free medium for 3 hours prior to onset of anoxia. For confocal imaging, hepatocytes were coloaded with calcein and tetramethylrhodamine methyl ester to visualize onset of the MPT and mitochondrial depolarization, respectively. To further examine autophagy, hepatocytes were infected with an adenovirus expressing green fluorescent protein–microtubule‐associated protein light chain 3 (GFP‐LC3) and subjected to A/R. Calpain activity was fluorometrically determined with succinyl‐Leu‐Leu‐Val‐Tyr‐7‐amino‐4‐methylcoumarin. A/R markedly decreased Atg7 and Beclin‐1 concomitantly with a progressive increase in calpain activity. I/R of livers also decreased both proteins. However, inhibition of calpain isoform 2, adenoviral overexpression, and nutrient depletion all substantially suppressed A/R‐induced loss of autophagy proteins, prevented onset of the MPT, and decreased cell death after reoxygenation. Confocal imaging of GFP‐LC3 confirmed A/R‐induced depletion of autophagosomes, which was reversed by nutrient depletion and adenoviral overexpression. Conclusion: Calpain 2–mediated degradation of Atg7 and Beclin‐1 impairs mitochondrial autophagy, and this subsequently leads to MPT‐dependent hepatocyte death after A/R. (HEPATOLOGY 2008.)

Autophagy is required for extension of yeast chronological life span by rapamycin

Rapamycin is an antibiotic that stimulates autophagy in a wide variety of eukaryotes, including the budding yeast Saccharomyces cerevisiae. Low concentrations of rapamycin extend yeast chronological life span (CLS). We have recently shown that autophagy is required for chronological longevity in yeast, which is attributable in part to a role for autophagy in amino acid homeostasis. We report herein that low concentrations of rapamycin stimulate macroautophagy during chronological aging and extend CLS.

How shall i eat thee

If you work in the field of autophagy we do not really need to tell you that this research area has grown tremendously. Along with that growth has developed a need for some unification of the nomenclature. In 2003, researchers working with the yeast model system proposed the use of the acronym ATG to denote AuTophaGy-related genes,1 and this designation has also been adopted for most of the genes involved in autophagy in higher eukaryotes. Similarly, a common nomenclature for isoforms of lysosome associated protein type 2 (LAMP-2) was recently proposed, hopefully reducing some of the confusion resulting from the use of multiple names.2 At this time we thought it worthwhile to consider the terms being used to describe different types of lysosomal or vacuolar degradative pathways. Many names are being introduced, and this is reasonable to the extent that these various processes have distinct features; each unique process needs a specific name to avoid confusion, and to eliminate the need for a lengthy description. It would be helpful, however, if the community agreed on their use. Finally, the addition or use of a name that implies a unique process must be backed up by data that justify the nomenclature. Thus, researchers should verify that a process is specific before using a name that implies specificity. For example, to demonstrate selectivity in organelle degradation it is incumbent upon the researcher to show that the organelle in question, and not other organelles, is sequestered and/or degraded with kinetics that distinguish it from a bulk, nonspecific process. There are many types of autophagy. To our knowledge, the term “autophagy” (from the Greek “auto” for “self ” and “phagein” meaning “to eat”) was first used in a 1963 review article by Christian de Duve.3 The first reference we have found in a research paper is in regard to a possible role of autophagy in lung cancer;4 however, as this work was published in an Italian journal, we are not able to comment on this in any authoritative manner. The following year, de Duve published a highly referenced review,5 and by this time the authors unquestionably refer to the process of macroautophagy, although the actual term was introduced later.6 Perhaps the most distinguishing feature of macroautophagy for the purposes of this discussion is that it involves the generally nonspecific (see ref. 7 for an exception) sequestration of cytoplasm within a non-lysosomal/vacuolar compartment, usually delimited by a double or multiple membrane; this compartment is typically referred to as an autophagosome. Another long-standing term that has not seen tremendous usage of late, but that is experiencing renewed interest, is “crinophagy” that is derived from the Greek “crin” meaning, “to secrete”. As far as we can tell this name also derives from de Duve.8 “Crinophagy” was originally used to describe the direct fusion of secretory vesicles with lysosomes (e.g., see refs. 9–11), resulting in the formation of a “crinosome.”12 This topic has attracted recent attention because of possible connections with diabetes, as crinophagy appears to be used for the regulated degradation of vesicle-stored insulin.13 It is not known whether insulin degradation incorporates any aspects of macroautophagy, but that possibility has not been ruled out. We suggest that we retain the use of the term “crinophagy” as it was originally described; if it turns out that the degradation of insulin does involve a macroautophagic process, we think we will need to introduce another name. We note that “insulinophagy” should be avoided because the target of degradation would presumably be the vesicles that contain insulin rather than the hormone itself. One possibility would be “secrephagy” to note that the target is secretory vesicles, or alternatively “macrocrinophagy.” There is probably no controversy about the use or meaning of the name “chaperonemediated autophagy” (CMA),14 which is a process involving the direct translocation of

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.

Rapamycin Activates Autophagy and Improves Myelination in Explant Cultures from Neuropathic Mice

Misexpression and cytosolic retention of peripheral myelin protein 22 (PMP22) within Schwann cells (SCs) is associated with a genetically heterogeneous group of demyelinating peripheral neuropathies. PMP22 overproducer C22 and spontaneous mutant Trembler J (TrJ) mice display neuropathic phenotypes and affected nerves contain abnormally localized PMP22. Nutrient deprivation-induced autophagy is able to suppress the formation of PMP22 aggregates in a toxin-induced cellular model, and improve locomotor performance and myelination in TrJ mice. As a step toward therapies, we assessed whether pharmacological activation of autophagy by rapamycin (RM) could facilitate the processing of PMP22 within neuropathic SCs and enhance their capacity to myelinate peripheral axons. Exposure of mouse SCs to RM induced autophagy in a dose- and time-dependent manner and decreased the accumulation of poly-ubiquitinated substrates. The treatment of myelinating dorsal root ganglion (DRG) explant cultures from neuropathic mice with RM (25 nm) improved the processing of PMP22 and increased the abundance and length of myelin internodes, as well as the expression of myelin proteins. Notably, RM is similarly effective in both the C22 and TrJ model, signifying that the benefit overlaps among distinct genetic models of PMP22 neuropathies. Furthermore, lentivirus-mediated shRNA knockdown of the autophagy-related gene 12 (Atg12) abolished the activation of autophagy and the increase in myelin proteins, demonstrating that autophagy is critical for the observed improvement. Together, these results support the potential use of RM and other autophagy-enhancing compounds as therapeutic agents for PMP22-associated demyelinating neuropathies.