MARINE

Marine Environmental Research 62 (2006) S420–S425

www.elsevier.com/locate/marenvrev

ENVIRONMENTAL

RESEARCH

Short communication

Autophagy: Role in surviving environmental stress

Michael N. Moore *, J. Icarus Allen, Paul J. Somerfield

Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1 3DH, UK

Abstract

This conceptual paper addresses the role of lysosomal autophagy in cellular defence against oxi-dative stress. A hypothesis is proposed that autophagic removal of oxidatively damaged organellesand proteins provides a second tier of defence against oxidative stress. Age pigment or lipofuscin is aproduct of oxidative attack on proteins and lipids and can accumulate in lysosomes, where it cangenerate reactive oxygen species (ROS) and inhibit lysosomal function, resulting in autophagic fail-ure. It is further hypothesised that repeated triggering of augmented autophagy can protectively min-imise lipofuscin generation; and that animals living in fluctuating environments, where autophagy isrepeatedly stimulated by natural stressors, will be generically more tolerant of pollutant stress. Datafor resistance to pollutant stress is presented, together with evidence for a correlation between lyso-somal stability and macrobenthic diversity. Finally, we speculate that organisms making up func-tional ecological assemblages in fluctuating environments, where up-regulation of autophagyshould provide a selective advantage, may be pre-selected to be tolerant of pollutant-induced oxida-tive stress.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Autophagy; Caloric restriction; Functional ecological assemblages; Lipofuscin; Lysosomes; Lyso-somal stability; Macrofaunal diversity; Mussels; Oxidative stress; Pollutants; Selection; Tolerance

Autophagy is the degradation of cellular components in lysosomes and is implicated inmany disease processes, cell death and adaptive responses (Cuervo, 2004; Levine, 2005).This group of at least three related processes (i.e., macro- and micro-autophagy and chap-eronin-mediated autophagy) is essential for maintenance of cellular homeostasis (Cuervo,2004), and morphological evidence has long indicated that lysosomal autophagy is a

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doi:10.1016/j.marenvres.2006.04.055

* Corresponding author. Tel.: +44 1752 633120; fax: 44 1752 633101.E-mail address: mnm@pml.ac.uk (M.N. Moore).

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highly conserved mechanism evolutionarily (Cuervo, 2004). While the genes for autophagywere initially discovered in yeast, analogues of those genes are now being found in diversegroups of animals including nematode worms, fruit-flies and mammals (Cuervo, 2004).Damaged cellular constituents and redundant products are removed by lysosomal autoph-agy, but it is also critically involved, along with other proteolytic systems (e.g. protea-somes), in the continuous turnover of intracellular components. Autophagy isup-regulated in times of stress or physiological change: by breaking down longer-livedproteins and organelles, and recycling the products into protein-synthesis and energy-pro-duction pathways, the process allows cells to be temporarily self-sustaining during periodswhen nutrients are restricted (Bergamini et al., 2003; Cuervo, 2004; Levine, 2005). In thispaper we consider the role of lysosomal autophagy as a protective system against the con-sequences of oxidative stress, and discuss its significance in the development of tolerance inaquatic ecosystems.

Autophagy is often considered to be primarily a survival strategy in multicellularorganisms, which either is initiated by stressors (e.g., restricted nutrients, hyperthermia,hypoxia and salinity increase; Moore et al., 2006a). However, recent evidence indicatesthat autophagy is much more than just a survival process and is, in fact, intimatelyinvolved in cell physiology (Cuervo, 2004; Lockshin and Zakeri, 2004; Moore, 1988,2004; Moore et al., 2006a). Augmented autophagy is apparently controlled by switchingoff the mTOR (mammalian target of rapamycin) kinase: mTOR signalling is involved inmany aspects of cell growth-regulation and has also been implicated in some cancers(Fig. 1; Levine, 2005). mTOR is evolutionarily conserved in eukaryotes and has been var-iously described in yeast, nematodes, molluscs, insects, crustaceans, birds and mammals(Cammalleri et al., 2003; Beaumont et al., 2001; Levine, 2005; Klionsky and Emr,2000). This signalling system is classically switched off by nutrient deprivation andhypoxia, with resultant up-regulation of autophagy which has been described in musselsand snails (Bayne et al., 1978; Moore and Halton, 1973; Moore et al., 1979, 1985).

In eukaryotic cells, the first tier of defence against oxidative damage is provided byxenobiotic transporters, biotransformation enzymes and anti-oxidant protection enzymes,like superoxide dismutase and catalase (Livingstone, 2001; Moore et al., 2004). Lysosomalautophagy provides a second line of defence, by removing oxidatively damaged proteins,and impaired organelles and even portions of the nucleus and DNA (Bergamini et al.,2003; Cuervo, 2004; Fig. 1). Consequently, when the first line of defensive systems is over-come, autophagy protects the cell against the harmful effects of damaged and malfunction-ing proteins, which can form aggregates that will accumulate irreversibly in cells (Gruneet al., 2004; Moore, 2004; Figs. 1 and 2A). Autophagy may also serve as a third tier ofdefence: when autophagic capabilities are compromised, autophagic processes can contrib-ute to all three types of cell death (i.e., Type 1 – apoptotic; Type 2 – autophagic; and Type3 – necrotic; Lockshin and Zakeri, 2004) to remove irreversibly damaged cells in order tomaintain organ integrity (Pipe and Moore, 1986a; Fig. 2). There also appear to be molec-ular cross-talk with crossover points in the pathways leading to all three forms of celldeath (Lockshin and Zakeri, 2004).

Pollutant exposure often causes oxidative attack on the protein machinery and organ-elles of the cell (Livingstone, 2001; Fig. 1). An effective ability to up-regulate the autopha-gic process will obviously be advantageous to organisms exposed to pollutants, since thiswill facilitate the removal of damaged cellular constituents and conserve cell function(Cuervo, 2004; Kirchin et al., 1992; Lockshin and Zakeri, 2004; Figs. 1 and 2A); and also

Fig. 1. The role autophagy in normal protein turnover and in degradation of damaged proteins and organelles.Reactive oxygen species (ROS) contribute to the formation of lipofuscin, a type of protein aggregate, which iscurrently believed to produce further ROS and inhibit enzymic breakdown of autophagocytosed cellularconstituents. It is proposed that repeated stimulation of enhanced autophagy by various environmental stimuli,including certain stressors, will result in a more effective recycling of cellular materials, consequently reducinglipofuscin formation and protecting the cell from oxidative attack. Adapted from Brunk and Terman (2002) andMoore et al. (2006a); SOD – superoxide dismutase.

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reduce the amount of age-pigment (lipofuscin) produced (Grune et al., 2004; Fig. 1). Lip-ofuscin accumulates in lysosomes as a result of peroxidation of autophagocytosed proteinsassociated with protein aggregates and oxidatively damaged organelles (Fig. 1), and waspreviously considered to be just cellular junk (Grune et al., 2004). However, new evidenceindicates that lipofuscin binds iron, which generates reactive oxygen species (ROS), result-ing in exacerbation of oxidative damage (Brunk and Terman, 2002). Brunk and Terman(2002) also hypothesise that lipofuscin binds lysosomal hydrolases, thereby inhibiting pro-tein degradation. The outcome is ‘‘failed autophagy’’ with autophagic accumulation ofessentially undegradable damaged organelles, proteins, phospholipids and lipids contrib-uting to further lipofuscin formation (Brunk and Terman, 2002; Krishnakumar et al.,1994; Moore, 1988; Moore et al., 2006a; Fig. 1).

Failed autophagy has been observed in molluscs, fish and mammals exposed to pollu-tants (Allen and Moore, 2004; Moore, 1988; Moore et al., 2004, 2006a). Reports on otheranimals tend to be largely descriptive, so systematic observations of other phyletic groupswould be beneficial to the development of a generic understanding of the role of autoph-agy in ameliorating the harmful effects of pollutants.

Since autophagy is one of the most highly evolutionarily conserved molecular processesin the eukaryotes (Cuervo, 2004; Klionsky and Emr, 2000), it is hypothesised that up-reg-

Fig. 2. (A) Relationship between lysosomal stability and cellular health status, showing how autophagicdegradation acts as a second tier of defence against oxidative stress; and possibly as a third tier when autophagiccapabilities are compromised and the autophagic system may trigger cell death (i.e., apoptotic – type I,autophagic – type II or necrotic – type III) for removal of irreversibly damaged cells. Lysosomal stability is usedhere as an integrated indicator of cellular wellbeing, that can be used as a generic yard-stick for interpreting theresponses of many biomarkers (Allen and Moore, 2004; Moore et al., 2004, 2006a). (B) Macrofaunal diversityand lysosomal stability (mean ± 95% CL, n = 10) in mussel hepatopancreatic digestive cells from 4 sites (1–4),sampled along a pollutant gradient in Norway (indicated by RPAHs in mussels) shows that mussels from the mostcontaminated site 4 are more similar to the cleanest mussels at site 1 than are mussels from sites 2 and 3.Macrofaunal diversity H 0 (mean ± 95% CL, n = 4) was measured at adjacent sites. (C) Lysosomal stability isdirectly correlated with macrofaunal diversity. Diversity data is derived from Gray et al. (1988) and Moore(1988).

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ulation of this system may play a significant role in adaptation to environments where oxi-dative stress is a major selection pressure, and where the facility to rapidly delete andrecycle oxidatively damaged proteins and organelles will provide an evolutionary advan-tage. Estuarine and coastal environments, where fluctuating salinity, nutrient, temperatureand oxygen regimes are all important selective drivers and also inducers of autophagy, arelikely to be areas where it would be an adaptive advantage to be able to minimise theeffects of oxidative stress. Furthermore, due to the fact that nearshore and estuarine envi-ronments are those most likely to be subjected to anthropogenic stress; we have a scenariowhere effective anti-oxidant protection may provide an evolutionary advantage. Conse-quently, we would expect to observe functional ecological groupings of organisms incoastal and estuarine environments that have generic and highly effective first- and sec-ond-tier defensive systems for dealing with oxidative stress. The corollary of this is thatan ability for rapid up-regulation of autophagy is a probable necessity for acquisitionof tolerance in high stress environmental scenarios. Many molluscs have such a capabilityfor augmented autophagy, including mussels and marine and terrestrial snails (Bayneet al., 1978; Lowe et al., 1981; Marigomez et al., 2005; Moore, 2004; Moore and Halton,1973; Moore et al., 1985, 1986, 2006a; Pipe and Moore, 1986b). Augmented autophagyinduced by short term starvation has a protective effect against copper and phenanthrenemediated oxidative stress in mussels (Moore, 2004; Moore et al., 2006b). Further supportcomes from a study of blue mussels from a highly contaminated site in Norway whichshowed evidence of tolerance, in comparison with less contaminated mussels (Moore,1988; Fig. 2B). Samples from four sites along a pollution gradient showed that musselsfrom site 4 were the most contaminated, although lysosomal stability in these mussels indi-cated that they were more similar to the cleanest site 1, than were animals from sites 2 and3 (Fig. 2B). Although these mussels had relatively high lysosomal stability, they showedclear evidence of autophagy, placing them in Tier 2, whereas mussels from sites 2 and 3with low stability were clearly in Tier 3 (Fig. 2A and B; Moore, 1988). The inference hereis that up-regulated autophagy may have been selected in this population (Moore, 1988).Lysosomal stability was also strongly correlated with macrobenthic diversity (H 0) in spa-tially adjacent sites (Fig. 2C; R = 0.946, P < 0.01); and although these are just correla-tions, they may be a reflection of the universality of autophagic reactions to stress.

In addition to studies of single populations and species, many studies have examinedchanges in the structure of communities along marine pollution gradients (Fig. 2B).Indeed, this is a standard component of most pollution monitoring schemes. One commonfinding is that responses are often manifest at taxonomic levels higher than species, com-monly as high as phyla (Olsgard et al., 1998). Organisms within different phyla are consid-ered to have different sensitivities to stress (Warwick and Clarke, 1993), raising theinteresting possibility that this reflects long-established evolutionary differences in thephysiology of organisms within different phyla. While autophagic mechanisms are highlyconserved among eukaryotes, we hypothesise that ability to up-regulate this process variesamong phyla, and this underlies differences in their ability to colonise parts of the coastaland estuarine environment, and also contributes to their differential sensitivities to anthro-pogenic stress. We propose that representative organisms from a range of phyla making upfunctional ecological assemblages are assessed to determine the extent to which their rel-ative capabilities for up-regulation of autophagy and the degradative (retrograde) compo-nent of protein turnover contribute to their ability to survive natural and anthropogenicstress.

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In conclusion, lysosomal autophagy appears to provide a second line of defence againstoxidative stress, and the capability to effectively up-regulate this process is probably a sig-nificant factor contributing to the ability of some organisms to tolerate stressful and pol-luted environments.

Acknowledgement

The work forms part of the PREDICT 2 and AMBLE Projects supported by theDepartment for Environment, Food and Rural Affairs (Defra, UK), Contract Nos.AE1136 and ME3109.

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