The Key Role of Autophagy and its Relationship with Apoptosis in Lepidopteran Larval Midgut Remodeling

C H A P T E R

22 The Key Role of Autophagy and its Relationship with Apoptosis in Lepidopteran Larval Midgut Remodeling Eleonora Franzetti, Davide Romanelli, and Gianluca Tettamanti O U T L I N E Remodeling of Midgut in Silkworm: Apoptosis is Activated Later The Role of Autophagy and Apoptosis in the Larval Midgut Regulation of Cell Death by Hormones Another Part of the Story: BmAtg1 and Starvation-Induced Autophagy

Introduction 334 Autophagy and Apoptosis in Lepidoptera 335 Autophagy in Larval Organs 335 The Relationship Between Autophagy and Apoptosis 338 The Midgut of Lepidoptera 339 The Larval Midgut of Lepidoptera as a Model to Study Cell Death Processes 339 Remodeling of Midgut in Silkworm: Autophagy Comes First 340

M.A. Hayat (ed): Autophagy, Volume 3 DOI: http://dx.doi.org/10.1016/B978-0-12-405529-2.00022-6

Conclusions and Future Perspectives

342 343 344 346 347

Acknowledgments 347 References 347

333

© 2014 Elsevier Inc. All rights reserved.

334

22.  THE KEY ROLE OF AUTOPHAGY

Abstract

Cell death is a primary cellular response essential for the development, differentiation, homeostasis, and survival of organisms. This highly heterogeneous process, which includes apoptotic and autophagy-based cell death, can be activated by distinct biochemical cascades, and can display different morphological features. For this reason, a precise characterization of the numerous cell death modalities described so far in eukaryotes, and of their relationships, constitutes a major challenge for current research. Cell death-associated phenomena occur extensively in the larvae of holometabolous insects (i.e. Lepidoptera) during development and metamorphosis to eliminate tissues and organs that the adult does not need. Several larval organs of lepidopteran larvae have been used over the years to study autophagy and apoptosis; however, the current literature is basically fragmentary and confusing. The completion of genome sequencing in Bombyx mori and the development of molecular tools to manipulate the expression of autophagic and apoptotic genes that have now been identified in the silkworm opened up new perspectives and made it possible to analyze in-depth the cell death processes that occur in these insects. This chapter summarizes current knowledge about autophagy research in Lepidoptera. The use of the larval midgut is discussed as a model for studying the roles and regulation of autophagy, and for gaining insight as to how autophagy and apoptosis cooperate in cell death events in lepidopteran larval organs.

INTRODUCTION Various mechanisms have evolved in eukaryotic cells to accomplish cell death. The four most important types of cell death processes, defined by the Nomenclature Committee on Cell Death, are apoptosis, autophagic cell death, cornification, and programmed necrosis (Kroemer et  al., 2009). However, while cornification and programmed necrosis have been identified in specific biological settings or are associated almost exclusively with pathological conditions, apoptosis and autophagy have been widely described to occur in various developmental contexts. In holometabolous insects, cell death phenomena occur extensively during development and metamorphosis, and they are necessary to eliminate tissues and organs typical of the embryonic or larval life (Tettamanti et al., 2008b). In Drosophila, several examples have been described and well characterized over the years, such as the death of larval midgut and salivary gland. In contrast, the picture is more complex in Lepidoptera. In fact, there is no general agreement about the cell death processes that occur in the larval organs of these insects. Indeed, this fragmentation of data is likely due more to the different experimental approaches and markers that have been used in those studies than to a truly different behavior of the organs. Among insects, butterflies and moths have been widely used to study processes related to metamorphosis because the larva is amenable to performing endocrinological, electrophysiological, and developmental biology studies. Several Lepidoptera species have been used in the past to analyze the destruction of body tissues and organs through autophagy and apoptosis. Moreover, the wide repertoire of new molecular tools that have been established for several species belonging to this taxon makes Lepidoptera an excellent model system for tackling a broad range of questions concerning autophagy and apoptosis.

IV.  AUTOPHAGY AND APOPTOSIS

Autophagy and Apoptosis in Lepidoptera

335

AUTOPHAGY AND APOPTOSIS IN LEPIDOPTERA Autophagy in Larval Organs Up to the early 1980s, studies of autophagy in Lepidoptera were mainly based on morphological analyses. A few years after the term “autophagy” appeared (de Duve and Wattiaux, 1966), Locke and Collins described the isolation of cell organelles within paired membranes derived from Golgi in the fat body of Calpodes ethlius larvae (Locke and Collins, 1968). They reported the complete sequence of autophagosome formation and suggested that the isolated compartments could constitute regions of massive lysis. This finding was confirmed by using a specific staining reaction for acid phosphatase, a lysosomal enzyme whose activity was detected in these “storage granules” (Larsen, 1976). This massive cellular autolysis in the larva caused a deep rearrangement of this tissue before the adult emerged. In subsequent years, autophagic compartments and increases in lysosome numbers have reportedly been detected in other larval/pupal tissues and organs beyond the fat body, including midgut, wing epithelium, silk gland, and intersegmental muscles (see Tettamanti et al. (2011) for a comprehensive review on the topic) (Figure 22.1). Along with the identification of the two main players in the autophagic process, the autophagosome and the lysosome, several research groups analyzed the signals and the signal transduction pathway that regulate autophagy in butterflies and moths in the 1970s and 1980s. These studies demonstrated that 20-hydroxyecdysone (20E) can trigger the onset of autophagy. Accordingly, injection of this hormone in the body cavity of the larva induces an increase in the numbers of secondary lysosomes and mitophagy in midgut (Radford and Misch, 1971) and fat body (de Priester et  al., 1979) cells. Additional information was derived from experiments performed ex vivo. In fact, by administering 20E to the fat body isolated from fifth-instar larvae before the programmed occurrence of autophagy (critical period), self-digestion of the cells could be set in motion (Dean, 1978), while a fat body taken soon after the critical period continues with the autophagic sequence in hormone-free medium (Dean, 1978). These results confirmed that autophagy is induced by ecdysone, and also demonstrated that once the cells are committed to eliciting an autophagic process, the autophagic program does not require the persistent presence of the hormone for completion. The beginning of the twenty-first century witnessed the birth of a second age for the study of autophagy in Lepidoptera. The initiation of several expressed sequence tag (EST) projects in various Lepidoptera species, the completion of genome sequencing in the silkworm, Bombyx mori, the development of RNAi, stable germline transformation, and viral vectors for transient gene expression, offered new opportunities for analyzing in-depth the autophagic processes that occur in these insects and for manipulating the expression of autophagic genes that are progressively identified in silkworm (Figure 22.1). Bioinformatics analysis performed by Zhang and colleagues (Zhang et al., 2009) revealed that homologues of most of the autophagy-related gene or protein (ATG) genes originally identified in yeast and subsequently in higher eukaryotes are present in the B. mori genome. Along with 11 ATG genes, genes involved in the phosphatidylinositol 3-kinase (PI3K) I and PI3K III signal transduction pathway and in the formation of autophagosomes were found. In particular, most of these genes are involved in the two ubiquitin-like conjugation

IV.  AUTOPHAGY AND APOPTOSIS

336

22.  THE KEY ROLE OF AUTOPHAGY

Autophagy Molecular/ Biochemical evidence (gene expression, protein expression)

Morphological/cell biology evidence (TEM, caspase activation, DNA fragmentation)

Molecular/ Biochemical evidence (gene expression, protein expression)

Misch, 1965 Redford and Misch, 1971 Tettamanti et al., 2007 Vilaplana et al., 2008 Goncu and Parlak, 2011

Khoa and Takeda, 2012b

Uwo et al., 2002 Tettamanti et al., 2007 Tettamanti et al., 2007 Shinoara et al., 2008 Vilaplana et al., 2008 Goncu and Parlak, 2011 Khoa et al., 2012 Khoa and Takeda, 2012a Xu et al., 2012

Parthasarathy & Palli, 2007 Vilaplana et al., 2008 Khoa et., 2012 Khoa and Takeda, 2012a

Locke and Collins, 1965 Locke and Collins, 1978 Larsen, 1970 Larsen, 1976 Sass and Kovacs, 1977 Komuves et al., 1985 Muller et al., 2004 Sumithra et al., 2010 Tial et al., in press

Tian et al., in press

Muller et al., 2004 Sumithra et al., 2010 Kaneko et al, 2011 Tian et al., 2012

Kaneko et al, 2011 Tian et al., 2012

Matsuura et al., 1976 Tashiro et al., 1976 Goncu and Parlak, 2009 Li et al., 2010 Goncu and Parlak, 2011

Li et al., 2010 Li et al., 2011 Khoa and Takeda, 2012b

Li et al., 2010 Goncu and Parlak, 2011 Khoa and Takeda, 2012a

Zhang et al., 2009 Li et al., 2010 Khoa and Takeda, 2012a

Morphological/cell biology evidence (TEM, acid phosphatase, Lysotracker)

Midgut

Fat body

Silk gland

Ovary Other organs

Apoptosis

Mpakou et al., 2006 Mpakou et al., 2008

Mpakou et al., 2006 Mpakou et al., 2008 Beaulaton & Lockshin, 1977 Lockshin & Beaulaton, 1979

Facey and Lockshin, 2010

Dai and Gilbert, 1999 Hoffman & Weeks, 2001 Kinch et al. 2003

FIGURE 22.1  Current literature on autophagy and apoptosis in lepidopteran larval organs.

systems, Atg8–PE and Atg12–Atg5–Atg16. Two recent studies have expanded the number of ATG genes that are now available for the silkworm (Casati et al., 2012; Tian et al., 2013). In both studies, considerable attention was devoted to BmATG1, in which Drosophila has a pivotal regulatory role in activating both developmental-programmed autophagy and starvation-induced autophagy. In particular, Tian and coworkers (Tian et al., 2013) demonstrated that five BmATG genes (BmATG1, BmATG2, BmATG8, BmATG9, and BmATG18) are 20E-primary-reponsive genes, and an ecdysone-responsive element was identified in the

IV.  AUTOPHAGY AND APOPTOSIS

Autophagy and Apoptosis in Lepidoptera

337

BmATG1 promoter region. The number of genes regulating autophagy has been recently extended through the identification of two paralogous targets of rapamycin (Tor) genes, BmTOR1 and BmTOR2 (Zhou et  al., 2010), encoding for factors that lie on the regulatory pathway of the autophagic process. Both BmTOR genes are upregulated by two autophagypromoting signals, starvation and 20E, although with differing sensitivity (Zhou et al., 2010). Because in higher eukaryotes the pathways through which hormones and nutrients regulate autophagy are different, but both converge on Tor, it is reasonable to hypothesize that the silkworm Tor has a key role in the signaling pathway that regulates autophagy. In general, several of the genes that have been identified in silkworm are actively transcribed in different tissues during development and metamorphosis, or are upregulated by starvation; thus, most of them will surely be good candidates for future investigations. A growing interest in ATG genes led to the derivation of the crystal structure of BmAtg8, a clear-cut marker of the autophagic process (Hu et al., 2010). In fact, Atg8 is a ubiquitous protein among eukaryotes, and after its recruitment to the phagophore, is involved in the membrane-expansion step. BmAtg8 has several residues and a ubiquitin-fold domain at the C-terminus conserved in different species, thus implicating a central role in the autophagic pathway. Although with small differences, such as the absence of an identifiable BmATG10, the identification, expression, and structural characterization of the 24 autophagy-related genes in the silkworm confirm the existence of a well-organized autophagy pathway in this insect (Zhang et al., 2009). However, the exact mechanisms of action of the autophagy pathway remain to be elucidated. An Atg8 homolog has also been identified in Galleria mellonella. GmAtg8 belongs to the Atg8 family of ubiquitin-like proteins, and shares the highest sequence identity with B. mori BmAtg8 (Khoa and Takeda, 2012b). The analysis of its expression profile showed that the GmATG8 transcript and its protein product are present in different organs during the development of the larva, such as midgut, fat body, silk gland, ovary, and Malpighian tubules. Moreover, increased levels of mRNA and protein were found in larvae undergoing starvation (Khoa and Takeda, 2012b). Some recent studies performed on different silkworm larval tissues attempted to characterize the sequential gene activation triggered by 20E that leads to cell death in B. mori tissues. In insects, the effects of 20E are mediated by a heterodimeric nuclear receptor formed by the ecdysone receptor (EcR) and ultraspiracle (USP). Among the three isoforms of the ecdysone receptor, EcR-B1 has been shown to actively take part in the onset of death processes in the silk gland. Its protein levels reach a maximum just before the larval to pupal transformation, when autophagosomes appear (Goncu and Parlak, 2009). Although the main early and late genes involved in the cis-regulation downstream of EcR in B. mori are similar to those of Drosophila, gene recruitment is different in the two insects. This difference in behavior is particularly evident when the regulation of autophagic and apoptotic processes within the same tissue is dissected. Accordingly, Li et al. (2010) demonstrated that in the silk gland, the expression of BmEcR, BmE74A, BmE75C, and BmBR-C peaks at the onset of the autophagic process, while BmBFTZ-F1, BmHR39, and BmE75B are more likely involved in apoptosis initiation. Based on the results obtained in silkworm fat body and previous reports in Drosophila, Tian et al. (2013) propose a model for the 20E signal in inducing autophagy: 20E blocks Torc1 activity to induce autophagosome initiation by phosphorylation (to break the gate) and upregulates ATG genes (to provide the flow) by transcriptional regulation, thus inducing autophagy.

IV.  AUTOPHAGY AND APOPTOSIS

338

22.  THE KEY ROLE OF AUTOPHAGY

The Relationship Between Autophagy and Apoptosis Autophagic and apoptotic features coexist in many organs that die during metamorphosis, a phenomenon that has been widely described in the silk gland, fat body, midgut, and other tissues of Lepidoptera in which autophagy has been shown to play a key role in degradation (we refer the reader to Tettamanti et  al. (2011) for a review on this topic). DNA fragmentation, the appearance of nuclei with condensed chromatin, caspase activation, and the presence of unusual apoptotic bodies and identifiable phagocytes that cleanse the tissue by removing cell debris in certain organs, all represent features that are not typically seen in autophagy-mediated cell death. The issue of caspase activation during the cell death of these organs deserves particular attention because it does not seem to be a feature exclusively linked to apoptosis. In fact, in two lepidopteran series, the larval midgut at the pupal stage exhibits positive immunostaining with an antibody specific for cleaved caspase-3 (Tettamanti et al., 2007; Vilaplana et al., 2007), and ovarian nurse cells that degenerate during oogenesis can be labeled by a specific in situ assay for activated caspases (Mpakou et  al., 2006). In addition, the expression of BmCaspase-C has been assessed in B. mori silk gland (Li et al., 2010), and the administration of a specific caspase inhibitor to dying motoneurons impairs the late phase of cell death, which is autophagy dependent (Hoffman and Weeks, 2001). In contrast, the opposite situation has been described in Manduca sexta fat body, where no evidence of executioner caspase activity, such as caspase-3 and caspase-7, was found (Muller et al., 2004). In G. mellonella, where autophagy and apoptosis collaborate to remodel larval midgut during metamorphosis, the expression of caspase-1, an effector caspase in Lepidoptera, is strictly correlated with the occurrence of the apoptotic process (Khoa et al., 2012). According to the current literature, at least two settings linking autophagy and apoptosis in Lepidoptera can be outlined: (1) Autophagy-associated programmed cell death (PCD), where autophagy is the driving force that promotes cell death, and although the autophagic machinery is functional, it does not involve precocious cytochrome c release, apoptosome formation, and caspase recruitment. This hypothesis is based on evidence collected in Manduca sexta labial glands (Facey and Lockshin, 2010). Although no caspase activity was detected during metamorphosis, the authors demonstrated an increase in lysosomal proteolytic activity when the gland disintegrated. In this scenario, cathepsin B may play the major proteolytic role similar to the apoptotic cascade in mammals. (2) Caspase-dependent autophagic cell death that involves activation of effector caspases after loss of mitochondrial function (Hoffman and Weeks, 2001). In addition to these two scenarios, it must be emphasized that a series of situations exist in which the borders are not clear because autophagy can be accompanied by DNA fragmentation or nuclear condensation; the executioner caspases, however, are not activated (Muller et al., 2004). The overlap between autophagy and apoptosis, and the evidence that some morphological, biochemical, and molecular features are not exclusive to either autophagy or apoptosis (Berry and Baehrecke, 2007; Nezis et  al., 2009) have prompted a search for possible mediators common to these two processes. In this context, attention has been focused on Inhibitor of Apoptosis Protein (IAP) and on specific autophagic genes. In fact, IAP expression is modified during midgut remodeling in Lepidoptera (Vilaplana et  al., 2007; Khoa and Takeda, 2012a). The IAP protein Bruce in Drosophila is fundamental for the autophagic

IV.  AUTOPHAGY AND APOPTOSIS

The Midgut of Lepidoptera

339

processes (Hou et al., 2008), thus suggesting a mechanistic link between autophagy and cell death (Nezis et al., 2010). Concerning the autophagic genes, it has been shown in mammals that Atg5 plays a role in autophagosome formation and is also involved in a pro-apoptotic signaling pathway through cytochrome c release and caspase activation (Yousefi et al., 2006). Given that in silkworm BmATG5 shows a peculiar expression pattern during the development of the larval midgut and silk gland (Li et  al., 2010; Franzetti et  al., 2012), it could be a good candidate as mediator of the connection between the autophagic and the apoptotic pathway. A concerted action between autophagy and apoptosis seems to occur in the removal of larval tissues in Lepidoptera, raising the question of what might explain this synergy. EisenbergLerner et  al. (2009) analyzed this issue and concluded that the co-occurrence of autophagy and apoptosis within the same tissue does not represent mere redundancy, and postulated at least three possible scenarios: (1) A collaboration between autophagy and apoptosis to lead to cell death. In this setting autophagy could work as a back-up system to ensure cell death if the apoptotic process failed, but it could also establish a partnership with apoptosis to maximize the death process. (2) Apoptosis and autophagy could have different goals, the latter acting as a prosurvival mechanism that helps cells maintain homeostasis until a point of no return is reached, after which apoptosis is activated and the cell dies. (3) Autophagy may enable apoptosis by participating in the regulation of some molecular mechanisms of the apoptotic machinery. Although all three hypotheses can be envisaged for Lepidoptera, we cannot yet disentangle this puzzling issue owing to the current lack of detailed information about the molecular mechanisms underpinning the connection between apoptosis and autophagy as well as to the complexity of the phenotypic features demonstrated in the dying tissues in the larvae. In this context, the larval midgut of Lepidoptera, where both processes have been observed, could provide an interesting experimental model to analyze such an overlap among different cell death modalities and address this issue.

THE MIDGUT OF LEPIDOPTERA The Larval Midgut of Lepidoptera as a Model to Study Cell Death Processes The larval midgut of Lepidoptera is extensively remodeled during metamorphosis. While previous work has shown that apoptosis occurs in this organ, robust biochemical and molecular evidence for the involvement of autophagy is still lacking; furthermore, the role of autophagy, and the relationship between these two processes in the larval midgut epithelium undergoing cell death, is still cryptic. Thus, the current literature is basically fragmentary and confusing. To overcome this knowledge fragmentation, we began several years ago to characterize the morphofunctional features of the larval midgut remodeling process that occurs in these insects during metamorphosis. In particular, we analyzed the cell death mechanisms that are involved in the demise of larval midgut cells, focusing our attention on autophagy and apoptosis. Because there is much confusion regarding acceptable methods for monitoring autophagy in higher eukaryotes and cell death mechanisms taking place in organs and

IV.  AUTOPHAGY AND APOPTOSIS

340

22.  THE KEY ROLE OF AUTOPHAGY

tissues of Lepidoptera appear to have somewhat peculiar features, it was essential to broadly characterize the morphological, biochemical, and molecular features of autophagy and apoptosis in this organ in order to establish a solid starting platform for accomplishing the functional manipulations that will be necessary to assess the role of these two cell deathrelated processes. For the most part, our experiments were performed in the silkworm, B. mori, although other lepidopteran models, such as the tobacco budworm, Heliothis virescens, and the gypsy moth, Lymantria dispar (Tettamanti et al., 2007, 2008a,b; Franzetti et al., 2012), have been used to investigate specific features. For silkworm we analyzed animals during the fifth larval instar, which is the last larval instar, the spinning phase and the pupal phase. During the spinning phase, the larva stops feeding and starts wandering (wandering stage); it takes two days to produce the silk cocoon (SD1 and SD2), and then it becomes a prepupa (Franzetti et al., 2012). The midgut represents the central and largest region of the alimentary canal of the larva. It consists of an epithelium formed by columnar and goblet cells. Stem cells are localized at the base of the epithelium, and they can divide and differentiate into mature midgut cells. At the wandering stage, stem cells proliferate and start to form a new pupal epithelium that will become the midgut epithelium in the adult. A concurrent degeneration of larval midgut cells can be observed from the spinning stage onwards. In fact, the midgut epithelium is pushed toward the lumen, and at later stages, columnar and goblet cells give rise to a compact mass of cells in the lumen, called the yellow body, which is progressively degraded. While forming the yellow body, cells modify their shape, contacts among them are lost, and the number of cells decreases progressively, thus suggesting the intervention of cell death processes (Figure 22.2A–D).

Remodeling of Midgut in Silkworm: Autophagy Comes First Preliminary TEM analysis revealed that several autophagic compartments can be observed in the midgut cells from the spinning stage. These were characterized by a double-limiting membrane, which is a hallmark of autophagosomes. Autolysosomes were surrounded by lysosomes, and contained digested cellular material and organelles at different stages of degeneration. All these structures were highly represented until the early pupal stages. Morphometric analysis confirmed the TEM observations and demonstrated a significant increase in autophagic compartments in midgut cells at the spinning stage (Franzetti et al., 2012). We evaluated the expression of three autophagy-related genes, BmATG5, BmATG6, and BmATG8, that encode proteins involved in the early stages of autophagosome biogenesis, thus being good markers to assess the onset of the autophagic program. All three genes were highly expressed at the wandering stage, before the morphological features of autophagy appeared (Franzetti et al., 2012). For one of these genes, BmATG8, we also analyzed the expression and processing of the encoded protein. In fact, this protein is considered an undisputed and specific marker for autophagy, as discussed above. A band corresponding to the phosphatidylethanolamine-conjugated form of Atg8 (Atg8–PE), which is associated with the autophagosome membrane and thus an indicator suggesting the presence of mature autophagosomes, could be detected from the wandering stage up to the early pupal stage (Figure 22.2E).

IV.  AUTOPHAGY AND APOPTOSIS

The Midgut of Lepidoptera

341

FIGURE 22.2  Degeneration of larval midgut in B. mori and cell death features. (A–D) Cross-sections of silkworm midgut. Larval midgut consists of a highly folded monolayered epithelium (e) formed by columnar, goblet, and stem cells (A). From the wandering stage (B), stem cells (arrowheads) start to form the pupal midgut (p), while the old larval midgut epithelium (yb) is sloughed into the lumen (l) and the cells die (C, D). (E) Autophagy. Occurrence of autophagy in larval midgut cells is demonstrated by the expression pattern of BmAtg8 and its processing. D6: Fifth larval instar Day 6; SD2: Spinning stage Day 2; PD3: Pupal stage Day 3. (F–H) Apoptosis. Nuclear fragmentation (n) is confirmed by TUNEL assay (G, H): only degenerating cells within the yellow body (yb) show DNA undergoing fragmentation (brown staining, G), while the new pupal epithelium (p) is negative. (H) negative control.

IV.  AUTOPHAGY AND APOPTOSIS

342

22.  THE KEY ROLE OF AUTOPHAGY

Immunohistochemical analysis showed that Atg8 protein was localized in larval midgut cells and in the yellow body at later stages, while it was absent in the new pupal midgut epithelium. These data confirmed that autophagosomes are specifically formed in larval midgut cells. Because the formation of autolysosomes requires the lysosome recruitment and fusion to autophagosomes, lysosomal activity was evaluated by monitoring acid phosphatase. The activity of this enzyme increased considerably during the spinning period, when high autophagic activity is detected. As demonstrated by a specific histochemical staining for acid phosphatase, this high lysosomal activity detected at the spinning stage was restricted to the cells of the larval midgut that were forming the yellow body. By using TEM analysis it was possible to appreciate the punctate localization of the enzyme acid phosphatase activity in the cytoplasm of degenerating cells. In particular, in some of these dots, acid phosphatase staining and degenerated cellular structures coexisted, thus confirming these compartments to be autolysosomes (Franzetti et al., 2012). When cells are deprived of nutrients, they set autophagy in motion to generate molecules and energy to cope with starvation, as demonstrated in several eukaryotic models. To assess whether autophagy has a prosurvival role in the midgut during metamorphosis, we evaluated three parameters that are related to the metabolic activity of the cell: protein content, ATP production, and mitochondrial activity. We analyzed midgut samples from late fifth larval instar up to early pupae because autophagy peaks in this window. Initiation of wandering resulted in a reduction of protein concentration, which continued during the entire spinning period. Interestingly, ATP levels increased 30-fold immediately thereafter, at the SD1 stage, and they rose heavily until the prepupal phase. In accordance with this massive increase in ATP levels, a specific histochemical staining showed that high mitochondrial activity was present in the midgut epithelium at the beginning of the spinning phase. Thus, we concluded from these experiments that the sudden decrease in protein content followed by a massive increase in the amount of ATP and mitochondrial activity in the time frame of the maximum occurrence of autophagy suggests that autophagy determines the degradation of long-lived proteins in the larval midgut to provide amino acids for ATP production by central carbon metabolism (Franzetti et al., 2012).

Remodeling of Midgut in Silkworm: Apoptosis is Activated Later Morphological features typical for the apoptotic process, such as nuclear condensation and nuclear fragmentation (Figure 22.2F), were visible in larval midgut cells from the prepupal phase. It is very simple to identify the nuclei undergoing apoptosis. They differ greatly from nuclei of midgut cells of larvae at the feeding stage, which show a completely different chromatin organization and distribution. All these morphological features of apoptosis could be detected in midgut cells until late pupal stages (Franzetti et al., 2012). DNA fragmentation in these apoptotic cells started at the prepupal phase and then became significant in pupae. TUNEL assay helped us to evaluate where the nuclei undergoing DNA fragmentation were localized in midgut tissues. Some positive cells became visible in the larval midgut from the late spinning phase, then their number increased significantly within the yellow body of midguts at the pupal stage (Figures 22.2G,H).

IV.  AUTOPHAGY AND APOPTOSIS

The Midgut of Lepidoptera

343

To determine the timing of apoptosis-related gene expression, we quantified mRNA levels of ICE2 and IAP by using real-time polymerase chain reaction (PCR). While ICE2 was highly expressed at the end of the fifth larval instar and then its expression decreased, IAP expression was maintained at high levels until the wandering stage. This increased transcription of the prodeath factor ICE2 at the end of last larval instar, followed by a decrease in the expression of the antideath factor IAP at early spinning, marks the onset of the apoptotic signaling pathway (Franzetti et al., 2012). In fact, in accordance with these data on gene expression, the presence of activated effector caspases could be detected in midgut tissues immediately thereafter: a strong 15-kDa band corresponding to activated caspase-3 was visible from the late spinning phase onward, and the protein expression was also retained at later stages. By using a specific substrate for caspases, we could precisely quantify the activity of this enzyme and the results confirmed the trend seen on western blots. In fact, the activity peaked at late spinning stage, and after dropping, was progressively resumed from PD1 onwards. In this case, too, cells positive for activated caspase-3 were always localized in the larval midgut or in the yellow body, but not in the new pupal epithelium (Tettamanti et al., 2007; Franzetti et al., 2012). This pattern was similar to that described above for Atg8 staining and acid phosphatase activity. At this point we wanted to know what happens to these cells at later stages, once the apoptotic program is activated. We focused on the cell membrane because the loss of plasma membrane integrity is considered a point of no return during cell death and this parameter is used to evaluate cell viability. As demonstrated by our experiments based on Ho33258-PI double staining, the plasma membrane of some, but not all, cells within the yellow body was permeable to both probes. These were damaged cells that had lost membrane integrity, as confirmed by TEM images. In contrast, cells with a normal nucleus, and thus still viable, were not stained by PI. These experiments demonstrated that membrane integrity is lost progressively in groups of cells, and not suddenly in the whole yellow body, and suggested that larval midgut cells inside the yellow body are degraded and disappear gradually (Franzetti et  al., 2012). We did not find any evidence of phagocytes that can remove apoptotic bodies in the midgut tissues. However, this is not unusual because this organ is in direct contact with the external surface of the animal, and is therefore considered as outer environment. Therefore, we hypothesize that this membrane damage at late stages of degeneration might indicate the occurrence of secondary necrosis. Secondary necrosis represents the natural outcome of apoptosis when scavengers are not available to remove apoptotic bodies. Although the loss of membrane integrity in cells that belong to the yellow body, and the high lysosomal activity in midgut tissues at late pupal stages are good indications of secondary necrosis in these cells, some experiments are currently being performed in our laboratory to analyze the distribution of caspases in the midgut. Clearly, the detection of activated caspase-3 released from these cells would provide undisputable evidence to substantiate such a hypothesis (Silva, 2010).

The Role of Autophagy and Apoptosis in the Larval Midgut What might the overall role of midgut remodeling be? Given that no water or food is taken up by the animal after the wandering stage, we think that the self-digestion of larval midgut cells by autophagy, and the recycling of the breakdown products by the pupal

IV.  AUTOPHAGY AND APOPTOSIS

344

22.  THE KEY ROLE OF AUTOPHAGY

midgut, may provide a means of obtaining materials and energy to construct pupal and adult structures. This hypothesis is supported by two pieces of evidence. Firstly, the delayed, gradual, and asynchronous death of yellow body cells shown by Ho33258-PI double-staining fits with a gradual release and supply of nutrients and molecules from the larval to the pupal epithelium. Secondly, we demonstrated in a previous paper that the new pupal midgut has morphofunctional features typical of an absorbing epithelium, such as the presence of membrane transporters Glut2, Glut5, and Na+/K+-ATPase, and shows marked activity for some hydrolytic enzymes involved in the final digestion of sugars, such as trehalase and sucrase, or in the intermediate digestion of proteins, such as aminopeptidase N (Tettamanti et al., 2007). In addition, both a well-developed apical brush-border and strong mitochondrial activity detected in the pupal and adult midgut, detected by NADH staining, are consistent with a functional absorbing epithelium with high metabolic activity. This evidence has been recently confirmed by experiments performed on purified brush-border membrane vesicles (BBMVs). BBMVs represent a useful tool to study transport mechanisms, as the transport phenomenon can be separated from the metabolic event, and due to the complete control of the composition of the internal and external compartments, transport processes can be better understood. The characterization of L-leucine transport in BBMVs prepared from the imago midgut shows that apical membranes of this epithelium are able to transport this amino acid (Casartelli M., personal communication). All these data demonstrate how the new pupal epithelium is able to recycle nutrients and materials derived from the degradation of the yellow body. Based on these considerations and data, we can conceive a working model as follows (Figure 22.3). At the wandering/early spinning stage, stem cells start to proliferate. Autophagy is activated in the larval midgut epithelium, and cells digest their content to produce ATP because at this stage the larva does not feed (Figure 22.3A). Later on, apoptosis intervenes and leads to the progressive demise of groups of cells within the yellow body (Figure 22.3B). Because no phagocytes are available, apoptosis ends up in a secondary necrosis process and membrane integrity is lost. Once these cells are dead, they release molecules from their cytoplasm, and these molecules can be absorbed and used to nurture and maintain the new pupal epithelium (Figure 22.3C).

Regulation of Cell Death by Hormones What about the signals that trigger cell death in the larval midgut? We previously demonstrated that ecdysone and juvenile hormone can regulate cell death processes in the midgut of Lepidoptera (Tettamanti et al., 2008a). In fact, administering juvenile hormone before commitment and 20E after commitment delays and fosters, respectively, the growth and differentiation of stem cells, and the occurrence of cell death in the yellow body. Moreover, by injecting 20E into developmentally arrested and 20E-deficient host last-instar larvae that have been parasitized by the wasp Toxoneuron nigriceps, which show a failure of 20E surge, the midgut remodeling process can be rescued: regular midgut development is immediately triggered after the injection of 20E in these larvae, and the final result is comparable to that of control animals. These hormone-based experiments suggest that endocrine signals are involved in the regulation of the midgut replacement process.

IV.  AUTOPHAGY AND APOPTOSIS

The Midgut of Lepidoptera

345

FIGURE 22.3  Sequence of activation of autophagy, apoptosis, and secondary necrosis in the larval midgut of silkworm during metamorphosis.

IV.  AUTOPHAGY AND APOPTOSIS

346

22.  THE KEY ROLE OF AUTOPHAGY

We are now investigating how autophagy and apoptosis are specifically regulated by 20E and other signals. One possibility is that a 20E pulse at the end of the larval stage (commitment peak) triggers the onset of the autophagic response detected at the wandering/spinning stage, while a second, more consistent, 20E pulse at early pupal stages (molting peak) is related to the massive apoptotic burst in pupae, which drives cell demise. Autophagy might also be activated by nutritional signals in accordance with its prosurvival role that has been ubiquitously characterized in eukaryotic cells, while the late, subsequent recruitment of apoptosis is regulated by ecdysone signals. However, we are still at the beginning of this work, and further experiments are required to confirm this hypothesis and to better address this issue.

Another Part of the Story: BmAtg1 and Starvation-Induced Autophagy The larval organs of Lepidoptera offer, in addition, an opportunity to study the relationship between programmed autophagy and starvation-induced autophagy. Some years ago, Rusten and colleagues showed in Drosophila how developmental autophagy is mediated by downregulation of PI3K signaling, a pathway also involved in starvation-induced autophagy in the fat body (Rusten et  al., 2004). In this pathway, Atg1 is a pivotal factor and activates autophagy in a TOR-dependent manner when the cell is subjected to metabolic stress. To verify a possible relationship between programmed autophagy and starvation-induced autophagy in the midgut of Lepidoptera, we recently cloned the full-length coding sequence of ATG1 in silkworm, performed an in silico analysis of the protein, and analyzed the expression of this gene in larvae subjected to prolonged starvation (Casati et al., 2012). We isolated two full-length cDNAs of 2,175 (transcript variant A) and 2,271 (transcript variant B) bases representing ATG1 in the silkworm. Phylogenetic analysis indicated that BmATG1 was closely related to orthologues of other insects. The encoded BmAtg1 proteins shared extensive homology with orthologues from yeast to mammals, showing high conservation at the N-terminal region where the catalytic domain and ATP- and Mg-binding sites are located. Unfortunately, real-time PCR analysis showed that starvation had only a slight effect on BmATG1 expression in the midgut, while it significantly influenced the BmATG1 mRNA copy number in the fat body, inducing an upregulation of the transcripts 24 h after food withdrawal and thus confirming this organ to be a highly responsive tissue following nutrient deprivation. Interestingly, at the end of the fifth day of starvation, which corresponded to the first day of the spinning phase (SD1), the expression of the BmATG1 gene in the midgut of the starved larvae increased significantly in comparison to that of control larvae (Casati et al., 2012). Notwithstanding the lack of a transcriptional upregulation of this gene following starvation in the midgut, additional investigations are necessary to assess the putative regulation of BmAtg1 at the post-translational level in the midgut. In fact, several sites of phosphorylation, SUMOylation, and glycosylation have been identified on the protein by in silico analysis. Thus, it will be important to assess the role of post-translational modifications at these specific residues. In addition, a detailed analysis of the autophagic response in starved larvae and specific functional experiments are necessary to delve deeper into the story. This additional work might also shed light on the different response to starvation of the larval midgut and fat body, two organs that, although characterized by different metabolic functions, have been frequently considered to be responsive to similar regulatory signals.

IV.  AUTOPHAGY AND APOPTOSIS

REFERENCES

347

CONCLUSIONS AND FUTURE PERSPECTIVES Although some larval organs of Lepidoptera represent interesting experimental models to analyze different cell death processes operating within the same developmental context, and to investigate their relationships, many questions are yet to be answered, especially concerning autophagy. The three most intriguing questions are the following: 1. What is the true role of developmental autophagy in larval tissues of lepidopterans? Autophagy might be necessary to achieve cell death in organs and tissues not readily accessible to phagocytes, such as in the midgut. Alternatively, autophagy may help the apoptotic process by enhancing its efficacy or perhaps by offsetting its inefficiencies, but in any case it would be necessary to achieve large-scale histolysis. A third possibility is that autophagy could intervene as a prosurvival process that helps the larva to cope with starvation as soon as it approaches metamorphosis, and as we showed in the midgut, it could help the animal to exploit molecules that are present in a larval tissue that would become obsolete in the imago with a consequent loss of these molecules. Only an accurate, comparative analysis of the autophagic process in different larval organs can unveil the true role of autophagy in each of them. 2. Does starvation have a role in developmental autophagy? Programmed autophagy in insect larval tissues is switched on by ecdysteroids. However, given that this self-digestion process occurs during the food starvation period that the larva experiences during metamorphosis, can nutrient deprivation contribute at least to maintain the autophagic process as active once it is started by the hormone signal? In other words, do hormone signals or starvation play the most important and direct role in the autophagic process in larval tissues and organs? 3. In biological systems where autophagy coexists or cooperates with apoptosis, which molecular signals are specific for initiating autophagy and apoptosis rather than being mediators that regulate this cross-talk? Present and future work in Lepidoptera is, and will be, focused on the search for genes and proteins that initiate and regulate autophagy, and aim to identify the complex interactions that link autophagy and apoptosis. This will surely help us to understand what roles this self-digesting process plays in different larval tissues both during the development of the animal and under physiological versus stress conditions.

Acknowledgments The authors wish to thank all the colleagues who have collaborated in the past few years to unravel the midgut story. This work was in part financed by a grant from the Italian Ministry of University and Research (PRIN 2008, protocol 2008SMMCJY) and by FAR 2012 (University of Insubria) to Gianluca Tettamanti.

References Berry, D.L., Baehrecke, E.H., 2007. Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila. Cell 131, 1137–1148. Casati, B., Terova, G., Cattaneo, A.G., et  al., 2012. Molecular cloning, characterization and expression analysis of ATG1 in the silkworm, Bombyx mori. Gene 511, 326–337.

IV.  AUTOPHAGY AND APOPTOSIS

348

22.  THE KEY ROLE OF AUTOPHAGY

de Duve, C., Wattiaux, R., 1966. Functions of lysosomes. Annu. Rev. Physiol. 28, 435–492. de Priester, W., van Pelt-Verkuil, E., de Leeuw, G., 1979. Demonstration of acid phosphatase activity induced by 20-hydroxyecdysone in the fat body of Calliphora. Cell Tissue Res. 200, 435–442. Dean, R.L., 1978. The induction of autophagy in isolated insect fat body by beta-ecdysone. J. Insect Physiol. 24, 439–447. Eisenberg-Lerner, A., Bialik, S., Simon, H.U., et  al., 2009. Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ. 16, 966–975. Facey, C.O.B., Lockshin, R.A., 2010. The execution phase of autophagy associated PCD during insect metamorphosis. Apoptosis 15, 639–652. Franzetti, E., Huang, Z.J., Shi, Y.X., et al., 2012. Autophagy precedes apoptosis during the remodeling of silkworm larval midgut. Apoptosis 17, 305–324. Goncu, E., Parlak, O., 2009. Morphological changes and patterns of ecdysone receptor B1 immunolocalization in the anterior silk gland undergoing programmed cell death in the silkworm, Bombyx mori. Acta Histochem. 111, 25–34. Hoffman, K.L., Weeks, J.C., 2001. Role of caspases and mitochondria in the steroid-induced programmed cell death of a motoneuron during metamorphosis. Dev. Biol. 229, 517–536. Hou, Y.C.C., Chittaranjan, S., Barbosa, S.G., et al., 2008. Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis. J. Cell Biol. 182, 1127–1139. Hu, C., Zhang, X.A., Teng, Y.B., et al., 2010. Structure of autophagy-related protein Atg8 from the silkworm Bombyx mori. Acta Crystallogr. F66, 787–790. Khoa, D.B., Takeda, M., 2012a. Expression analysis of inhibitor of apoptosis and related caspases in the midgut and silk gland of the greater wax moth, Galleria mellonella, during metamorphosis and under starvation. Gene 510, 133–141. Khoa, D.B., Takeda, M., 2012b. Expression of autophagy 8 (Atg8) and its role in the midgut and other organs of the greater wax moth, Galleria mellonella, during metamorphic remodelling and under starvation. Insect Mol. Biol. 21, 473–487. Khoa, D.B., Trang, L.T., Takeda, M., 2012. Expression analyses of caspase-1 and related activities in the midgut of Galleria mellonella during metamorphosis. Insect Mol. Biol. 21, 247–256. Kroemer, G., Galluzzi, L., Vandenabeele, P., et al., 2009. Classification of cell death: recommendations of the nomenclature committee on cell death 2009. Cell Death Differ. 16, 3–11. Larsen, W.J., 1976. Cell remodeling in the fat body of an insect. Tissue Cell 8, 73–92. Li, Q.R., Deng, X.J., Yang, W.Y., et al., 2010. Autophagy, apoptosis, and ecdysis-related gene expression in the silk gland of the silkworm (Bombyx mori) during metamorphosis. Can. J. Zool. 88, 1169–1178. Locke, M., Collins, J.V., 1968. Protein uptake into multivesicular bodies and storage granules in the fat body of an insect. J. Cell Biol. 36, 453–483. Mpakou, V.E., Nezis, I.P., Stravopodis, D.J., et  al., 2006. Programmed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx mori. Dev. Growth Differ. 48, 419–428. Muller, F., Adori, C., Sass, M., 2004. Autophagic and apoptotic features during programmed cell death in the fat body of the tobacco hornworm. Eur. J. Cell Biol. 83, 67–78. Nezis, I.P., Lamark, T., Velentzas, A.D., et  al., 2009. Cell death during Drosophila melanogaster early oogenesis is mediated through autophagy. Autophagy 5, 298–302. Nezis, I.P., Shravage, B.V., Sagona, A.P., et al., 2010. Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis. J. Cell Biol. 190, 523–531. Radford, S.V., Misch, D.W., 1971. The cytological effect of ecdysterone on the midgut cells of the flesh-fly Sarcophaga bullata. J. Cell Biol. 49, 702–711. Rusten, T.E., Lindmo, K., Juhasz, G., et al., 2004. Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway. Dev. Cell 7, 179–192. Silva, M.T., 2010. Secondary necrosis: the natural outcome of the complete apoptotic program. FEBS Lett. 584, 4491–4499. Tettamanti, G., Grimaldi, A., Casartelli, M., et  al., 2007. Programmed cell death and stem cell differentiation are responsible for midgut replacement in Heliothis virescens during prepupal instar. Cell Tissue Res. 330, 345–359. Tettamanti, G., Grimaldi, A., Pennacchio, F., et al., 2008a. Toxoneuron nigriceps parasitization delays midgut replacement in fifth-instar Heliothis virescens larvae. Cell Tissue Res. 332, 371–379.

IV.  AUTOPHAGY AND APOPTOSIS

REFERENCES

349

Tettamanti, G., Salo, E., Gonzalez-Estevez, C., et al., 2008b. Autophagy in invertebrates: insights into development, regeneration and body remodeling. Curr. Pharm. Des. 14, 116–125. Tettamanti, G., Cao, Y., Feng, C., et al., 2011. Autophagy in Lepidoptera: more than old wine in new bottle. Invert. Surv. J. 8, 5–14. Tian, L., Ma, L., Guo, E., et al., 20-hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body. Autophagy 9, 1172–1187. Vilaplana, L., Pascual, N., Perera, N., et  al., 2007. Molecular characterization of an inhibitor of apoptosis in the Egyptian armyworm, Spodoptera littoralis, and midgut cell death during metamorphosis. Insect Biochem. Mol. Biol. 37, 1241–1248. Yousefi, S., Perozzo, R., Schmid, I., et al., 2006. Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat. Cell Biol. 8, 1124–1146. Zhang, X., Hu, Z.Y., Li, W.F., et al., 2009. Systematic cloning and analysis of autophagy-related genes from the silkworm Bombyx mori. BMC Mol. Biol. 10, 50–58. Zhou, S., Zhou, Q., Liu, Y., et al., 2010. Two Tor genes in the silkworm Bombyx mori. Insect Mol. Biol. 19, 727–735.

IV.  AUTOPHAGY AND APOPTOSIS