Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

BMP pathway regulation of insulin signaling components promotes lipid storage in Caenorhabditis elegans.

A small number of peptide growth factor ligands are used repeatedly in development and homeostasis to drive programs of cell differentiation and function. Cells and tissues must integrate inputs from these diverse signals correctly, while failure to do so leads to pathology, reduced fitness, or death. Previous work using the nematode C. elegans identified an interaction between the bone morphogenetic protein (BMP) and insulin/IGF-1-like signaling (IIS) pathways in the regulation of lipid homeostasis. The molecular components required for this interaction, however, were not fully understood. Here we report that INS-4, one of 40 insulin-like peptides (ILPs), is regulated by BMP signaling to modulate fat accumulation. Furthermore, we find that the IIS transcription factor DAF-16/FoxO, but not SKN-1/Nrf, acts downstream of BMP signaling in lipid homeostasis. Interestingly, BMP activity alters sensitivity of these two transcription factors to IIS-promoted cytoplasmic retention in opposite ways. Finally, we probe the extent of BMP and IIS interactions by testing additional IIS functions including dauer formation, aging, and autophagy induction. Coupled with our previous work and that of other groups, we conclude that BMP and IIS pathways have at least three modes of interaction: independent, epistatic, and antagonistic. The molecular interactions we identify provide new insight into mechanisms of signaling crosstalk and potential therapeutic targets for IIS-related pathologies such as diabetes and metabolic syndrome.

Molecular definitions of autophagy and related processes.

Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research.

Autophagy in C. elegans development.

Autophagy involves the sequestration of cytoplasmic contents in a double-membrane structure referred to as the autophagosome and the degradation of its contents upon delivery to lysosomes. Autophagy activity has a role in multiple biological processes during the development of the nematode Caenorhabditis elegans. Basal levels of autophagy are required to remove aggregate prone proteins, paternal mitochondria, and spermatid-specific membranous organelles. During larval development, autophagy is required for the remodeling that occurs during dauer development, and autophagy can selectively degrade components of the miRNA-induced silencing complex, and modulate miRNA-mediated silencing. Basal levels of autophagy are important in synapse formation and in the germ line, to promote the proliferation of proliferating stem cells. Autophagy activity is also required for the efficient removal of apoptotic cell corpses by promoting phagosome maturation. Finally, autophagy is also involved in lipid homeostasis and in the aging process. In this review, we first describe the molecular complexes involved in the process of autophagy, its regulation, and mechanisms for cargo recognition. In the second section, we discuss the developmental contexts where autophagy has been shown to be important. Studies in C. elegans provide valuable insights into the physiological relevance of this process during metazoan development.

C. elegans ADAMTS ADT-2 regulates body size by modulating TGFß signaling and cuticle collagen organization.

Organismal growth and body size are influenced by both genetic and environmental factors. We have utilized the strong molecular genetic techniques available in the nematode Caenorhabditis elegans to identify genetic determinants of body size. In C. elegans, DBL-1, a member of the conserved family of secreted growth factors known as the Transforming Growth Factor ß superfamily, is known to play a major role in growth control. The mechanisms by which other determinants of body size function, however, is less well understood. To identify additional genes involved in body size regulation, a genetic screen for small mutants was previously performed. One of the genes identified in that screen was sma-21. We now demonstrate that sma-21 encodes ADT-2, a member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted metalloproteases. ADAMTS proteins are believed to remodel the extracellular matrix and may modulate the activity of extracellular signals. Genetic interactions suggest that ADT-2 acts in parallel with or in multiple size regulatory pathways. We demonstrate that ADT-2 is required for normal levels of expression of a DBL-1-responsive transcriptional reporter. We further demonstrate that adt-2 regulatory sequences drive expression in glial-like and vulval cells, and that ADT-2 activity is required for normal cuticle collagen fibril organization. We therefore propose that ADT-2 regulates body size both by modulating TGFß signaling activity and by maintaining normal cuticle structure.

Methods to Determine the Role of Autophagy Proteins in C. elegans Aging.

This chapter describes methods for the analysis of autophagy proteins in C. elegans aging. We discuss the strains to be considered, the methods for the delivery of double-stranded RNA, and the methods to measure autophagy levels, autophagic flux, and degradation by autophagy.

A Non-Cell-Autonomous Role of BEC-1/BECN1/Beclin1 in Coordinating Cell-Cycle Progression and Stem Cell Proliferation during Germline Development.

The decision of stem cells to proliferate and differentiate is finely controlled. The Caenorhabditis elegans germline provides a tractable system for studying the mechanisms that control stem cell proliferation and homeostasis [1-4]. Autophagy is a conserved cellular recycling process crucial for cellular homeostasis in many different contexts [5], but its function in germline stem cell proliferation remains poorly understood. Here, we describe a function for autophagy in germline stem cell proliferation. We found that autophagy genes such as bec-1/BECN1/Beclin1, atg-16.2/ATG16L, atg-18/WIPI1/2, and atg-7/ATG7 are required for the late larval expansion of germline stem cell progenitors in the C. elegans gonad. We further show that BEC-1/BECN1/Beclin1 acts independently of the GLP-1/Notch or DAF-7/TGF-ß pathways but together with the DAF-2/insulin IGF-1 receptor (IIR) signaling pathway to promote germline stem cell proliferation. Similar to DAF-2/IIR, BEC-1/BECN1/Beclin1, ATG-18/WIPI1/2, and ATG-16.2/ATG16L all promote cell-cycle progression and are negatively regulated by the phosphatase and tensin homolog DAF-18/PTEN. However, whereas BEC-1/BECN1/Beclin1 acts through the transcriptional regulator SKN-1/Nrf1, ATG-18/WIPI1/2 and ATG-16.2/ATG16L exert their function through the DAF-16/FOXO transcription factor. In contrast, ATG-7 functions in concert with the DAF-7/TGF-ß pathway to promote germline proliferation and is not required for cell-cycle progression. Finally, we report that BEC-1/BECN1/Beclin1 functions non-cell-autonomously to facilitate cell-cycle progression and stem cell proliferation. Our findings demonstrate a novel non-autonomous role for BEC-1/BECN1/Beclin1 in the control of stem cell proliferation and cell-cycle progression, which may have implications for the understanding and development of therapies against malignant cell growth in the future.

Detection of Autophagy in Caenorhabditis elegans by Western Blotting Analysis of LGG-1.

A common way to measure the induction of autophagy in yeast and mammalian cells is to compare the amount of Atg8/LC3-I with that of Atg8-PE/LC3-II by using western blot analysis. This is because changes in the amount of LC3-II correlate closely with changes in the number of autophagosomes present in cells. Atg8/LC3 is initially synthesized as an unprocessed form, which is proteolytically processed to form Atg8/LC3-I, and then this is modified into the phosphatidylethanolamine (PE)-conjugated Atg8-PE/LC3-II form. Atg8/LC3-II is membrane bound, whereas Atg8-PE/LC3-I is cytosolic. By associating with both the inner and outer membranes of the autophagosome, Atg8-PE/LC3-II is the only autophagy reporter that is reliably associated with completed autophagosomes. In the nematode Caenorhabditis elegans, the ortholog of Atg8/LC3 is LGG-1. Here, we discuss how changes in the levels of LGG-1-II (and the paralog LGG-2) protein can, with appropriate controls, be used to monitor autophagy activity in nematodes and present a protocol for monitoring changes in the protein levels of different forms of LGG-1 by western blotting.

RNAi-Mediated Inactivation of Autophagy Genes in Caenorhabditis elegans.

RNA interference (RNAi) is a process that results in the sequence-specific silencing of endogenous mRNA through the introduction of double-stranded RNA (dsRNA). In the nematode Caenorhabditis elegans, RNA inactivation can be used at any specific developmental stage or during adulthood to inhibit a given target gene. Investigators can take advantage of the fact that, in C. elegans, RNAi is unusual in that it is systemic, meaning that dsRNA can spread throughout the animal and can affect virtually all tissues except neurons. Here, we describe a protocol for the most common method to achieve RNAi in C. elegans, which is to feed them bacteria that express dsRNA complementary to a specific target gene. This method has various advantages, including the availability of libraries that essentially cover the whole genome, the ability to treat animals at any developmental stage, and that it is relatively cost effective. We also discuss how RNAi specific to autophagy genes has proven to be an excellent method to study the role of these genes in autophagy, as well as other cellular and developmental processes, while also highlighting the caveats that must be applied.

Detection of Autophagy in Caenorhabditis elegans.

Autophagy is a dynamic and catabolic process that results in the breakdown and recycling of cellular components through the autophagosomal-lysosomal pathway. Many autophagy genes identified in yeasts and mammals have orthologs in the nematode Caenorhabditis elegans. In recent years, gene inactivation by RNA interference (RNAi) and chromosomal mutations has been useful to probe the functions of autophagy in C. elegans, and a role for autophagy has been shown to contribute to multiple processes, such as the adaptation to stress, longevity, cell death, cell growth control, clearance of aggregation-prone proteins, degradation of P granules during embryogenesis, and apoptotic cell clearance. Here, we discuss some of these roles and describe methods that can be used to study autophagy in C. elegans. Specifically, we summarize how to visualize autophagy in embryos, larva, or adults, how to detect the lipidation of the ubiquitin-like modifier LGG-1 by western blot, and how to inactivate autophagy genes by RNAi.

Detection of Autophagy in Caenorhabditis elegans Using GFP::LGG-1 as an Autophagy Marker.

In yeast and mammalian cells, the autophagy protein Atg8/LC3 (microtubule-associated proteins 1A/1B light chain 3B encoded by MAP1LC3B) has been the marker of choice to detect double-membraned autophagosomes that are produced during the process of autophagy. A lipid-conjugated form of Atg8/LC3B is localized to the inner and outer membrane of the early-forming structure known as the phagophore. During maturation of autophagosomes, Atg8/LC3 bound to the inner autophagosome membrane remains in situ as the autophagosomes fuse with lysosomes. The nematode Caenorhabditis elegans is thought to conduct a similar process, meaning that tagging the nematode ortholog of Atg8/LC3-known as LGG-1-with a fluorophore has become a widely accepted method to visualize autophagosomes. Under normal growth conditions, GFP-modified LGG-1 displays a diffuse expression pattern throughout a variety of tissues, whereas, when under conditions that induce autophagy, the GFP::LGG-1 tag labels positive punctate structures, and its overall level of expression increases. Here, we present a protocol for using fluorescent reporters of LGG-1 coupled to GFP to monitor autophagosomes in vivo. We also discuss the use of alternative fluorescent markers and the possible utility of the LGG-1 paralog LGG-2.

Detecting Autophagy in Caenorhabditis elegans Embryos Using Markers of P Granule Degradation.

Autophagy plays an active role during the early stages of embryogenesis in the nematode Caenorhabditis elegans. Although their exact function is unknown, P granules are ribonucleoprotein particles that play a role in germ cell specification. The localization of P granules is restricted to the germline precursor cells in wild-type embryos, as a result of their degradation in the somatic cell lineage. Autophagy is known to be required for the degradation of P granules, as mutations in various autophagy genes, including those encoding the adaptor SEPA-1 and the p62-like adaptor SQST-1, result in the accumulation of the P granule components PGL-1 and PGL-3 (termed PGL granules) in the somatic cells of C. elegans embryos. In this protocol, we present a methodology for using fusion reporters of SEPA-1, SQST-1, and PGL-1 that have aided in the identification of new genes for normal autophagy activity by screening for mutant animals that lack the degradation of these autophagy substrates.

Guidelines for monitoring autophagy in Caenorhabditis elegans.

The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.

C. elegans CEP-1/p53 and BEC-1 are involved in DNA repair.

p53 is a transcription factor that regulates the response to cellular stress. Mammalian p53 functions as a tumor suppressor. The C. elegans p53, cep-1, regulates DNA-damage induced germline cell death by activating the transcription of egl-1 and ced-13. We used the C. elegans model to investigate how, in the whole animal, different forms of DNA damage can induce p53-dependent versus p53-independent cell death and DNA repair. DNA damage was induced by ultraviolet type C (UVC) radiation, or 10-decarbamoyl mitomycin C (DMC, an agent known to induce mammalian p53-independent cell death). Wild-type or cep-1 loss-of-function mutant animals were assayed for germline cell death and DNA lesions. Wild-type animals displayed greater removal of UVC-lesions over time, whereas cep-1 mutant animals displayed increased UVC-lesion retention. The cep-1 mutation increased UVC-lesion retention directly correlated with a reduction of progeny viability. Consistent with DMC inducing p53-independent cell death in mammalian cells DMC induced a C. elegans p53-independent germline cell death pathway. To examine the influence of wild-type CEP-1 and DNA damage on C. elegans tumors we used glp-1(ar202gf)/Notch germline tumor mutants. UVC treatment of glp-1 mutant animals activated the CEP-1 target gene egl-1 and reduced tumor size. In cep-1(gk138);glp-1(ar202gf) animals, UVC treatment resulted in increased susceptibility to lesions and larger tumorous germlines. Interestingly, the partial knockdown of bec-1 in adults resulted in a CEP-1-dependent increase in germline cell death and an increase in DNA damage. These results strongly support cross-talk between BEC-1 and CEP-1 to protect the C. elegans genome.

MiR-34 modulates Caenorhabditis elegans lifespan via repressing the autophagy gene atg9.

Evidence for a regulatory role of the miR-34 family in senescence is growing. However, the exact role of miR-34 in aging in vivo remains unclear. Here, we report that a mir-34 loss-of-function mutation in Caenorhabditis elegans markedly delays the age-related physiological decline, extends lifespan, and increases resistance to heat and oxidative stress. We also found that RNAi against autophagy-related genes, atg4, bec-1, or atg9, significantly reversed the lifespan-extending effect of the mir-34 mutants. Furthermore, miR-34a inhibits Atg9A expression at the post-transcriptional level in vitro, and the miR-34a binding sequences in the 3'-UTR of Atg9A contributes to the modulation of Atg9A expression by miR-34a. Our results demonstrate that the C. elegans mir-34 mutation extends lifespan by enhancing autophagic flux in C. elegans, and that miR-34 represses autophagy by directly inhibiting the expression of the autophagy-related proteins Atg9 in mammalian cells.

Autophagy genes are required for normal lipid levels in C. elegans.

Autophagy is a cellular catabolic process in which various cytosolic components are degraded. For example, autophagy can mediate lipolysis of neutral lipid droplets. In contrast, we here report that autophagy is required to facilitate normal levels of neutral lipids in C. elegans. Specifically, by using multiple methods to detect lipid droplets including CARS microscopy, we observed that mutants in the gene bec- 1 (VPS30/ATG6/BECN1), a key regulator of autophagy, failed to store substantial neutral lipids in their intestines during development. Moreover, loss of bec-1 resulted in a decline in lipid levels in daf-2 [insulin/IGF-1 receptor (IIR) ortholog] mutants and in germline-less glp-1/Notch animals, both previously recognized to accumulate neutral lipids and have increased autophagy levels. Similarly, inhibition of additional autophagy genes, including unc-51/ULK1/ATG1 and lgg-1/ATG8/MAP1LC3A/LC3 during development, led to a reduction in lipid content. Importantly, the decrease in fat accumulation observed in animals with reduced autophagy did not appear to be due to a change in food uptake or defecation. Taken together, these observations suggest a broader role for autophagy in lipid remodeling in C. elegans.

Autophagy links lipid metabolism to longevity in C. elegans.

The cellular recycling process of autophagy is emerging as a central player in many of the conserved longevity pathways in C. elegans, but the underlying mechanisms that link autophagy and life span remain unclear. In a recent study, we provided evidence to suggest that autophagy modulates aging through an effect on lipid homeostasis. Specifically, we identified a role for autophagy in a longevity model in which germline removal in C. elegans extends life span. Life-span extension in these animals is achieved, at least in part, through increased expression of the lipase LIPL-4. We found that autophagy and LIPL-4-dependent lipolysis are both upregulated in germline-less animals and work interdependently to prolong life span. While these genetic results lend further support to a growing link between autophagy and lipid metabolism, our findings are the first to suggest a possible molecular mechanism by which autophagy modulates organismal aging.

Autophagy and lipid metabolism coordinately modulate life span in germline-less C. elegans.

BACKGROUND: The cellular recycling process of autophagy is emerging as a key player in several longevity pathways in Caenorhabditis elegans. Here, we identify a role for autophagy in long-lived animals lacking a germline and show that autophagy and lipid metabolism work interdependently to modulate aging in this longevity model. RESULTS: Germline removal extends life span in C. elegans via genes such as the lipase LIPL-4; however, less is known of the cellular basis for this life-span extension. Here, we show that germline loss induces autophagy gene expression via the forkhead box A (FOXA) transcription factor PHA-4 and that autophagy is required to extend longevity. We identify a novel link between autophagy and LIPL-4, because autophagy is required to maintain high lipase activity in germline-deficient animals. Reciprocally, lipl-4 is required for autophagy induction. Coordination between autophagy and lipolysis is further supported by the finding that inhibition of TOR (target of rapamycin), a major negative regulator of autophagy, induces lipl-4 expression, and TOR levels are reduced in germline-less animals. TOR may therefore function as a common upstream regulator of both autophagy and lipl-4 expression in germline-less animals. Importantly, we find that the link between autophagy and LIPL-4 is relevant to longevity, because autophagy is induced in animals overexpressing LIPL-4 and autophagy is required for their long life span, recapitulating observations in germline-less animals. CONCLUSIONS: Collectively, our data offer a novel mechanism by which autophagy and the lipase LIPL-4 interdependently modulate aging in germline-deficient C. elegans by maintaining lipid homeostasis to prolong life span.

The Atg6/Vps30/Beclin 1 ortholog BEC-1 mediates endocytic retrograde transport in addition to autophagy in C. elegans.

Autophagy and endocytosis are dynamic and tightly regulated processes that contribute to many fundamental aspects of biology including survival, longevity, and development. However, the molecular links between autophagy and endocytosis are not well understood. Here, we report that BEC-1, the C. elegans ortholog of Atg6/Vps30/Beclin1, a key regulator of the autophagic machinery, also contributes to endosome function. In particular we identify a defect in retrograde transport from endosomes to the Golgi in bec-1 mutants. MIG-14/Wntless is normally recycled from endosomes to the Golgi through the action of the retromer complex and its associated factor RME-8. Lack of retromer or RME-8 activity results in the aberrant transport of MIG-14/Wntless to the lysosome where it is degraded. Similarly, we find that lack of bec-1 also results in mislocalization and degradation of MIG-14::GFP, reduced levels of RME-8 on endosomal membranes, and the accumulation of morphologically abnormal endosomes. A similar phenotype was observed in animals treated with dsRNA against vps-34. We further identify a requirement for BEC-1 in the clearance of apoptotic corpses in the hermaphrodite gonad, suggesting a role for BEC-1 in phagosome maturation, a process that appears to depend upon retrograde transport. In addition, autophagy genes may also be required for cell corpse clearance, as we find that RNAi against atg-18 or unc-51 also results in a lack of cell corpse clearance.