Kim D. Finley

Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila

Autophagy is involved with the turnover of intracellular components and the management of stress responses. Genetic studies in mice have shown that suppression of neuronal autophagy can lead to the accumulation of protein aggregates and neurodegeneration. However, no study has shown that increasing autophagic gene expression can be beneficial to an aging nervous system. Here we demonstrate that expression of several autophagy genes is reduced in Drosophila neural tissues as a normal part of aging. The age-dependent suppression of autophagy occurs concomitantly with the accumulation of insoluble ubiquitinated proteins (IUP), a marker of neuronal aging and degeneration. Mutations in the Atg8a gene (autophagy-related 8a) result in reduced lifespan, IUP accumulation and increased sensitivity to oxidative stress. In contrast, enhanced Atg8a expression in older fly brains extends the average adult lifespan by 56% and promotes resistance to oxidative stress and the accumulation of ubiquitinated and oxidized proteins. These data indicate that genetic or age-dependent suppression of autophagy is closely associated with the buildup of cellular damage in neurons and a reduced lifespan, while maintaining the expression of a rate-limiting autophagy gene prevents the age-dependent accumulation of damage in neurons and promotes longevity.

The Selective Macroautophagic Degradation of Aggregated Proteins Requires the PI3P-Binding Protein Alfy

There is growing evidence that macroautophagic cargo is not limited to bulk cytosol in response to starvation and can occur selectively for substrates, including aggregated proteins. It remains unclear, however, whether starvation-induced and selective macroautophagy share identical adaptor molecules to capture their cargo. Here, we report that Alfy, a phosphatidylinositol 3-phosphate-binding protein, is central to the selective elimination of aggregated proteins. We report that the loss of Alfy inhibits the clearance of inclusions, with little to no effect on the starvation response. Alfy is recruited to intracellular inclusions and scaffolds a complex between p62(SQSTM1)-positive proteins and the autophagic effectors Atg5, Atg12, Atg16L, and LC3. Alfy overexpression leads to elimination of aggregates in an Atg5-dependent manner and, likewise, to protection in a neuronal and Drosophila model of polyglutamine toxicity. We propose that Alfy plays a key role in selective macroautophagy by bridging cargo to the molecular machinery that builds autophagosomes.

Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain

p62 has been proposed to mark ubiquitinated protein bodies for autophagic degradation. We report that the Drosophila melanogaster p62 orthologue, Ref(2)P, is a regulator of protein aggregation in the adult brain. We demonstrate that Ref(2)P localizes to age-induced protein aggregates as well as to aggregates caused by reduced autophagic or proteasomal activity. A similar localization to protein aggregates is also observed in D. melanogaster models of human neurodegenerative diseases. Although atg8a autophagy mutant flies show accumulation of ubiquitin- and Ref(2)P-positive protein aggregates, this is abrogated in atg8a/ref(2)P double mutants. Both the multimerization and ubiquitin binding domains of Ref(2)P are required for aggregate formation in vivo. Our findings reveal a major role for Ref(2)P in the formation of ubiquitin-positive protein aggregates both under physiological conditions and when normal protein turnover is inhibited.

p62/SQSTM1 and ALFY interact to facilitate the formation of p62 bodies/ALIS and their degradation by autophagy

Accumulation of ubiquitinated proteins in cytoplasmic and/or nuclear inclusions is a hallmark of several diseases associated with premature cell death. SQSTM1/p62 is known to bind ubiquitinated substrates and aid their aggregation and degradation by macro-autophagy. We show here that p62 is required to recruit the large phosphoinositide-binding protein ALFY to cytoplasmic p62 bodies generated upon amino acid starvation or puromycin-treatment. ALFY, as well as p62, is required for formation and autophagic degradation of cytoplasmic ubiquitin-positive inclusions. Moreover, both p62 and ALFY localize to nuclear promyleocytic leukemia (PML) bodies. The Drosophila p62 homologue Ref(2)P accumulates in ubiquitinated inclusions in the brain of flies carrying mutations in the ALFY homologue Blue cheese, demonstrating that ALFY is required for autophagic degradation of p62-associated ubiquitinated proteins in vivo. We conclude that p62 and ALFY interact to organize misfolded, ubiquitinated proteins into protein bodies that become degraded by autophagy.

p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.

dissatisfaction Encodes a Tailless-like Nuclear Receptor Expressed in a Subset of CNS Neurons Controlling Drosophila Sexual Behavior

The dissatisfaction (dsf) gene is necessary for appropriate sexual behavior and sex-specific neural development in both sexes. dsf males are bisexual and mate poorly, while mutant females resist male courtship and fail to lay eggs. Males and females have sex-specific neural abnormalities. We have cloned dsf and rescued both behavioral and neural phenotypes. dsf encodes a nuclear receptor closely related to the vertebrate Tailless proteins and is expressed in both sexes in an extremely limited set of neurons in regions of the brain potentially involved in sexual behavior. Expression of a female transformer cDNA under the control of a dsf enhancer in males leads to dsf-like bisexual behavior.

Cardioprotection requires taking out the trash.

Autophagy is a critical cellular housekeeping process that is essential for removal of damaged or unwanted organelles and protein aggregates. Under conditions of starvation, it is also a mechanism to break down proteins to generate amino acids for synthesis of new and more urgently needed proteins. In the heart, autophagy is upregulated by starvation, reactive oxygen species, hypoxia, exercise, and ischemic preconditioning, the latter a well-known potent cardioprotective phenomenon. The observation that upregulation of autophagy confers protection against ischemia/reperfusion injury and inhibition of autophagy is associated with a loss of cardioprotection conferred by pharmacological conditioning suggests that the pathway plays a key role in enhancing the heart’s tolerance to ischemia. While many of the antecedent signaling pathways of preconditioning are well-defined, the mechanisms by which preconditioning and autophagy converge to protect the heart are unknown. In this review we discuss mechanisms that potentially underlie the linkage between cardioprotection and autophagy in the heart.

Autophagy: More Than a Nonselective Pathway

Autophagy is a catabolic pathway conserved among eukaryotes that allows cells to rapidly eliminate large unwanted structures such as aberrant protein aggregates, superfluous or damaged organelles, and invading pathogens. The hallmark of this transport pathway is the sequestration of the cargoes that have to be degraded in the lysosomes by double-membrane vesicles called autophagosomes. The key actors mediating the biogenesis of these carriers are the autophagy-related genes (ATGs). For a long time, it was assumed that autophagy is a bulk process. Recent studies, however, have highlighted the capacity of this pathway to exclusively eliminate specific structures and thus better fulfil the catabolic necessities of the cell. We are just starting to unveil the regulation and mechanism of these selective types of autophagy, but what it is already clearly emerging is that structures targeted to destruction are accurately enwrapped by autophagosomes through the action of specific receptors and adaptors. In this paper, we will briefly discuss the impact that the selective types of autophagy have had on our understanding of autophagy.

Genetic Modifiers of the Drosophila Blue Cheese Gene Link Defects in Lysosomal Transport With Decreased Life Span and Altered Ubiquitinated-Protein Profiles

Defects in lysosomal trafficking pathways lead to decreased cell viability and are associated with progressive disorders in humans. Previously we have found that loss-of-function (LOF) mutations in the Drosophila gene blue cheese (bchs) lead to reduced adult life span, increased neuronal death, and widespread CNS degeneration that is associated with the formation of ubiquitinated-protein aggregates. To identify potential genes that participate in the bchs functional pathway, we conducted a genetic modifier screen based on alterations of an eye phenotype that arises from high-level overexpression of Bchs. We found that mutations in select autophagic and endocytic trafficking genes, defects in cytoskeletal and motor proteins, as well as mutations in the SUMO and ubiquitin signaling pathways behave as modifiers of the Bchs gain-of-function (GOF) eye phenotype. Individual mutant alleles that produced viable adults were further examined for bchs-like phenotypes. Mutations in several lysosomal trafficking genes resulted in significantly decreased adult life spans and several mutants showed changes in ubiquitinated protein profiles as young adults. This work represents a novel approach to examine the role that lysosomal transport and function have on adult viability. The genes characterized in this study have direct human homologs, suggesting that similar defects in lysosomal transport may play a role in human health and age-related processes.

Selective Types of Autophagy

The focus of this special issue of the International Journal of Cell Biology is to underscore the recent developments in the field of macroautophagy and how this degradative pathway intersects with cellular metabolism, complex physiological functions, and human diseases. During the last decade, autophagy has become an expanding field in biomedical life sciences due to its involvement with numerous intracellular processes. Autophagy also plays a role in pathology, and it has the therapeutic potential to be the target for the treatment of specific human diseases. Early studies suggested that autophagy was a nonselective process in which cytoplasmic structures were randomly sequestered into autophagosomes before being delivered to the mammalian lysosome or the plant and yeast vacuole for degradation. Now there is growing evidence that unwanted cellular structures can be selectively recognized and exclusively eliminated within cells (F. Reggiori et al., “Selective types of autophagy”). This is achieved through the action of specific autophagy receptors, as reviewed by C. Behrends and S. Fulda in “Receptor proteins in selective autophagy”) and studied by K. Marchbank et al. “MAP1B interaction with the FW domain of the autophagic receptor Nbr1 facilitates its association to the microtubule network”. Thus excess or damaged organelles including mitochondria (A. May et al., “The many faces of mitochondrial autophagy: making sense of contrasting observations in recent research”; Y. Hirota et al., “The physiological role of mitophagy: new insights into phosphorylation events”), peroxisomes (A. Till et al., “Pexophagy: the selective degradation of peroxisomes”), lipid droplets (R. Singh and A. Cuervo, “Lipophagy: connecting autophagy and lipid metabolism”), endoplasmic reticulum and ribosomes (E. Cebollero et al., “Reticulophagy and ribophagy: regulated degradation of protein production factories”) can be specifically sequestered by autophagosomes and targeted to the lysosome for degradation. Importantly, there is growing evidence that selective autophagy subtypes also have a wide range of physiological functions. In yeast, the cytosol-to-vacuole (Cvt) pathway transports hydrolases into the vacuole, which is reviewed by M. Umekawa and D. Klionsky in “The cytoplasm-to-vacuole targeting pathway: a historical perspective”. In eukaryotes, autophagy plays a central role in both innate and acquired immunity. Further sequestration and elimination of invading pathogens such as Salmonella and Staphylococcus aureus have been exploited to study autophagosome biogenesis (T. Noda et al., “Three-axis model for Atg recruitment in autophagy against Salmonella”; M. Mauthe et al., “WIPI-1 positive autophagosome-like vesicles entrap pathogenic Staphylococcus aureus for lysosomal degradation”). In pancreas cells, autophagy has recently been shown to specifically turn over secretory granules, as described by M. Vaccaro in “Zymophagy: selective autophagy of secretory granules”. Dysregulation of autophagic function has been implicated in a growing list of disease processes and has underscored the selective or substrate-specific versions of the pathway. Examples in this special issue include the clearance of aggregates associated with neurological diseases, as reviewed by T. Lamark and T. Johansen in “Aggrephagy: selective disposal of protein aggregates by macroautophagy” and by I. Nezis in “Selective autophagy in Drosophila”. In terms of cancer biology, autophagy has been viewed as having dual roles in both tumor suppression and progression. K. Hughson et al. in “Implications of therapy-induced selective autophagy on tumor metabolism and survival” review how activation of autophagy selective forms can be used as a potential therapeutic approach for the treatment of specific cancers. Adding to the complexity of autophagic function and regulation, the article by K. Juenemann and E. Reits “Alternative macroautophagic pathways” explores alternative macroautophagic pathways that are independent of key core autophagy components such as Beclin-1 or Atg5. We expect future research on the mechanism and regulation of selective autophagy, and the physiological importance of this pathway in human disease will be very exciting and expand on the findings highlighted in this issue of IJCB. Fulvio Reggiori Maasaki Komatsu Kim Finley Anne Simonsen