Detection of autophagy in tissue by standard immunohistochemistry: possibilities and limitations

Transmission electron microscopy (TEM) is currently the standard method to monitor autophagy in tissue. Because TEM is labor intensive, we recently questioned whether marker proteins could be found for unambiguous detection of autophagy in tissue using standard immunohistochemical techniques. Our findings indicated that the identification of autophagy-specific biomarkers for tissue is highly compromised due to lack of differential gene expression. In this respect, TEM remains an indispensable technique for evaluation of autophagy in situ. Nevertheless, immunohistochemical staining of microtubule-associated protein 1 light chain 3 (LC3) appeared to be a valuable technique to detect autophagosome formation in tissue but only when this protein is overexpressed, e.g., in GFP-LC3 transgenic animals. Furthermore, demonstration of granular cytoplasmic ubiquitin inclusions by immunohistochemistry may be an attractive technique to measure autophagic cell degeneration in some human pathologies such as neurodegenerative diseases, heart failure and atherosclerosis.     

Immunohistochemical analysis of macroautophagy

Transmission electron microscopy (TEM) is an indispensable standard method to monitor macroautophagy in tissue samples. Because TEM is time consuming and not suitable for daily routine, many groups try to identify macroautophagy in tissue by conventional immunohistochemistry. The aim of the present study was to evaluate whether immunohistochemical assessment of macroautophagy-related marker proteins such as LC3, ATG5, CTSD/cathepsin D, BECN1/Beclin 1 or SQSTM1/p62 is feasible and autophagy-specific. For this purpose, livers from starved mice were used as a model because hepatocytes are highly sensitive to autophagy induction. ATG7-deficient mouse livers served as negative control. Our findings indicate that unambiguous immunodetection of LC3 in paraffin-embedded tissue specimens was hampered due to low in situ levels of this protein. Maximum sensitivity could only be obtained using high-quality, isoform-specific antibodies, such as antibody 5F10, in combination with Envision+ signal amplification. Moreover, LC3 stains were optimal in neutral-buffered formalin-fixed tissue, immersed in citrate buffer during antigen retrieval. However, even when using this methodology, LC3 monitoring required overexpression of the protein, e.g., in GFP-LC3 transgenic mice. This was not only the case for the liver but also for other organs including heart, skeletal muscle, kidney and gut. Immunohistochemical detection of the autophagy-related proteins ATG5, CTSD or BECN1 is not recommendable for monitoring autophagy, due to lack of differential gene expression or doubtful specificity. SQSTM1 accumulated in autophagy-deficient liver, thus it is not a useful marker for tissue with autophagic activity. We conclude that TEM remains an indispensable technique for in situ evaluation of macroautophagy, particularly in clinical samples for which genetic manipulation or other in vitro techniques are not feasible.     

Why Sick Cells Produce Tumors: The Protective Role of Autophagy

Cells exploit autophagy for survival to metabolic stress in vitro as well as in tumors where it localizes to regions of metabolic stress suggesting its role as a survival pathway. Consistent with this survival function, deficiency in autophagy impairs cell survival, but also promotes tumor growth, creating a paradox that the loss of a survival pathway leads to tumorigenesis. There is evidence that autophagy is a homeostatic process functioning to limit the accumulation of poly-ubiquitinated proteins and mutant protein aggregates associated with neuronal degeneration. Interestingly, we found that deficiency in autophagy caused by monoallelic loss of beclin1 or deletion of atg5 leads to accelerated DNA damage and chromosomal instability demonstrating a mutator phenotype. These cells also exhibit enhanced chromosomal gains or losses suggesting that autophagy functions as a tumor suppressor by limiting chromosomal instability. Thus the impairment of survival to metabolic stress due to deficiency in autophagy may be compensated by an enhanced mutation rate thereby promoting tumorigenesis. The protective role of autophagy may be exploited in developing novel autophagy modulators as rational chemotherapeutic as well as chemopreventive agents.

Addendum to:

Autophagy Supresses Tumor Progression by Limiting Chromosomal Instability

R. Mathew, S. Kongara, B. Beaudoin, C.M. Karp, K. Bray, K. Degenhardt, G. Chen, S. Jin and E. White

Genes Dev 2007; 21:1367-81     

Autophagy and Insulin/TOR Signaling in Caenorhabditis elegans pcm-1 Protein Repair Mutants

Biological responses due to nutrient deprivation in the nematode Caenorhabditis elegans, including L1 diapause and autophagy during dauer formation, can be mediated through the linked DAF-2/insulin/IGF receptor and target-of-rapamycin (TOR) kinase pathways. Here we discuss how altered insulin/TOR signaling may underlie the previously reported phenotypes of worms with a null mutation in the pcm-1 gene that results in reduced autophagy during dauer formation and decreased L1 arrest survival. PCM-1 encodes a protein repair methyltransferase and mutants of the encoding pcm-1 gene are incapable of converting spontaneously damaged l-isoaspartyl residues in cellular proteins to normal forms by this pathway. We speculate that PCM-1 may function either directly or indirectly as an inhibitor of insulin/TOR signaling, perhaps in a role to balance autophagy with alternative protein degradation pathways that may be more specific for recognizing age-damaged proteins.

Addendum to:

The L-Isoaspartyl-O-Methyltransferase in Caenorhabditis elegans Larval Longevity and Autophagy

T.A. Gomez, K.L. Banfield, D.M. Trogler and S.G. Clarke

Developmental Biol 2007; 303:493-500     

The Inositol Trisphosphate Receptor in the Control of Autophagy

The second messenger myo-inositol-1,4,5-trisphosphate (IP₃) acts on the IP₃ receptor (IP₃R), an IP3-activated Ca²⁺ channel of the endoplasmic reticulum (ER). The IP₃R agonist IP3 inhibits starvation-induced autophagy. The IP₃R antagonist xestospongin B induces autophagy in human cells through a pathway that requires the obligate contribution of Beclin-1, Atg5, Atg10, Atg12 and hVps34, yet is inhibited by ER-targeted Bcl-2 or Bcl-X_L, two proteins that physically interact with IP₃R. Autophagy can also be induced by depletion of the IP₃R by small interfering RNAs. Autophagy induction by IP3R blockade cannot be explained by changes in steady state levels of Ca²⁺ in the endoplasmic reticulum (ER) and the cytosol. Autophagy induction by IP₃R blockade is effective in cells lacking the obligate mediator of ER stress IRE1. In contrast, IRE1 is required for autophagy induced by ER stress-inducing agents such a tunicamycin or thapsigargin. These findings suggest that there are several distinct pathways through which autophagy can be initiated at the level of the ER.

Addendum to:

Regulation of Autophagy by the Inositol Trisphosphate Receptor

A. Criollo, M.C. Maiuri, E. Tasdemir, I. Vitale, A.A. Fiebig, D. Andrews, J. Molgo, J. Diaz, S. Lavandero, F. Harper, G. Pierron, D. di Stefano, R. Rizzuto, G. Szabadkai and G. Kroemer

Cell Death Differ 2007; In press     

The role of Beclin 1 in IR-induced crosstalk between autophagy and G2/M cell cycle arrest

ObjectivesBeclin 1 is a well-established core mammalian autophagy protein. Autophagy has been demonstrated to play roles in cellular responses to DNA damage, such as cell cycle regulation and apoptosis. In the present study, we investigated the exact mechanism by which Beclin 1 acts as a bridge between autophagy and cell cycle, when cells are exposed to ionizing radiation (IR).Materials and methodsWestern blotting and coimmunoprecipitation were performed to investigate protein expression levels and interactions. Immunofluorescence was used to monitor the localization and distribution of the indicated proteins. The levels of apoptosis and cell cycle changes were evaluated by flow cytometry. Double thymidine deoxyribonucleoside (TdR) blocking was conducted to differentiate G2 from mitotic delay. In vitro kinase assays using ATM kinase were performed to elucidate the specific phosphorylation site in Beclin 1.ResultsIn this study, we show that Beclin 1 knockdown reduces IR-induced autophagy. IR enhanced Beclin 1/PIK3CIII complex activity as demonstrated by the results of coimmunoprecipitation and immunofluorescence assays. An investigation to assess the possible relationship between autophagy and G2/M arrest showed that, similar to the autophagy inhibitor 3MA, Beclin 1 knockdown delayed IR-induced G2/M arrest. Furthermore, the interactions between Beclin 1 and several G2/M checkpoint-related proteins, namely, PLK1 and CDC25C, were observed to increase. In addition, we observed that both 3MA and Beclin 1 inhibition decreased IR-induced apoptosis. Regarding the potential mechanism associated with this phenomenon, we showed that IR induced the interaction between Beclin 1 and Tip60 as well as their redistribution. Furthermore, we demonstrated that Beclin 1 T57 may be a targeted phosphorylation site for ATM.ConclusionsIn the present study, we demonstrate the crucial and intricate roles of Beclin 1 in IR-induced autophagy, G2/M cell cycle arrest, and apoptosis. Additionally, Tip60 and ATM were identified as important molecular regulators of Beclin 1. Our findings show the precise mechanism of crosstalk between IR-induced autophagy and G2/M cell cycle arrest.     

AMP-Activated Protein Kinase: A Universal Regulator of Autophagy?

Autophagy is a lysosomal pathway involved in the turnover of cellular macromolecules and organelles. Starvation and various other stresses increase autophagic activity above the low basal levels observed in unstressed cells, where it is kept down by mammalian target of rapamycin complex 1 (mTORC1). In starved cells, LKB1 activates AMP-activated protein kinase (AMPK) that inhibits mTORC1 activity via a pathway involving tuberous sclerosis complex 1 and 2 (TSC1/2) and its substrate Rheb. The present study suggests that AMPK inhibits mTORC1 and autophagy also in non-starved cells. Various Ca²⁺ mobilizing agents (vitamin D compounds, thapsigargin, ATP and ionomycin) activate AMPK via activation of Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β), and this pathway is required for Ca²⁺-induced mTORC1 inhibition and autophagy. Thus, we propose that an increase in free cytosolic Ca²⁺ ([Ca²⁺]c) induces autophagy via the CaMKK/β-AMPK-TSC1/2-Rheb-mTORC1 signaling pathway and that AMPK is a more general regulator of autophagy than previously expected.

Addendum to:

Control of Macroautophagy by Calcium, Calmodulin-Dependent Kinase Kinase-β and Bcl-2

M. Høyer-Hansen, L. Bastholm, P. Szyniarowski, M. Campanella, G. Szabadkai, T. Farkas, K. Bianchi, N. Fehrenbacher, F. Elling, R. Rizzuto, I.S. Mathiasen and M. Jäättelä

Mol Cell 2007; 25:193-205     

Autophagy Can Be a Killer Even in Apoptosis-Competent Cells

Despite abundant evidence for autophagic cell death as a morphological type, the notion that autophagy can actually contribute mechanistically to the cell's death is controversial. In cells capable of apoptosis, autophagic cell death has been dismissed by some authors as a morphologically unusual form of apoptosis. But strong recent evidence for autophagy-mediated death of cells rendered incapable of apoptosis has been criticized on the grounds that this cell death is too artificial to be relevant to normal cells. We here argue from our own and other recent evidence that autophagy can mediate the death even of apoptosis-competent cells.

Addendum to:

Role of Phosphoinositide 3-Kinase in the Autophagic Death of Serum-Deprived PC12 Cells.

A. Guillon-Munos, M.X.P. van Bemmelen and P.G.H. Clarke

Apoptosis 2005; 10:1031-41.     

Pathways that Regulate Autophagy and their Role in Mediating Tumor Response to Treatment

Addenda to:

Rapamycin-Sensitive Pathway Regulates Mitochondrial Membrane Potential, Autophagy and Survival in Irradiated MCF-7 Cells

Paglin S, Lee N-Y, Nakar C, Fitzgerald M, Plotkin J, Deuel B, Hackett N, McMahill M, Sphicas E, Lampen N and Yahalom J.

Cancer Res 2005; 65:11061-70.

In addition to their role in cellular homeostasis, pathways that regulate autophagy affect both tumorigenesis and tumor response to treatment. Therefore, understanding regulation of autophagy in treated cancer cells is relevant to discovery of molecular targets for development of anti-cancer drugs. Our recent report points to radiation-induced inactivation of mTOR pathway as an underlying mechanism of radiation-induced autophagy in the human breast cancer cell line MCF-7. Most importantly, radiation-induced inactivation of this pathway was detrimental to cell survival and was associated with reversal of mitochondrial ATPase activity and mitochondrial hyperpolarization, decreased level of eukaryotic initiation factor 4G (eIF4G) and increased phosphorylation of p53. Future analysis of the interrelationship among these events and the role each of them plays in cell survival following radiation will increase our ability to employ the mTOR pathway in anti-cancer therapy.     

Initiation of Apoptosis and Autophagy by Photodynamic Therapy

This study was designed to examine modes of cell death after photodynamic therapy (PDT). Murine leukemia L1210 cells and human prostate Bax-deficient DU-145 cells were examined after PDT-induced photodamage to the endoplasmic reticulum (ER). Previous studies indicated that this resulted in a substantial loss of Bcl-2 function. Both apoptosis and autophagy occurred in L1210 after ER photodamage with the latter predominating after 24 hr. These processes were characterized by altered cellular morphology, chromatin condensation, loss of mitochondrial membrane potential, and formation vacuoles containing cytosolic components. Western blots demonstrated processing of LC3-I to LC3-II, a marker for autophagy. Inhibitors of apoptosis and/or autophagy were then used to delineate the contributions of the two pathways to the effects of PDT. In DU145 cells, PDT initiated only autophagy. PI3-kinase inhibitors suppressed autophagy in both cell lines as indicated by inhibition of vacuolization and LC3 processing. Autophagy may play a role in the ability of photodynamic therapy to stimulate immunologic recognition of target cells.

Addendum to

Initiation of Apoptosis and Autophagy by Photodynamic Therapy

D. Kessel, M.G.H. Vicente and J.J. Reiners Jr.

Lasers Surg Med 2006; In press     

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.     

Inositol and IP3 Levels Regulate Autophagy—Biology and Therapeutic Speculations

We recently showed that lithium induces autophagy via inositol monophosphatase (IMPase) inhibition, leading to free inositol depletion and reduced myo-inositol-1,4,5-triphosphate (IP₃) levels. This represents a novel way of regulating mammalian autophagy, independent of the mammalian target of rapamycin (mTOR). Induction of autophagy by lithium led to enhanced clearance of autophagy substrates, like mutant huntingtin fragments and mutant a-synucleins, associated with Huntington’s disease (HD) and some autosomal dominant forms of Parkinson’s disease (PD), respectively. Similar effects were observed with a specific IMPase inhibitor and mood-stabilizing drugs that decrease inositol levels. This may represent a new therapeutic strategy for upregulating autophagy in the treatment of neurodegenerative disorders, where the mutant protein is an autophagy substrate. In this Addendum, we review these findings, and some of the speculative possibilities they raise.

Addendum to:

Lithium induces autophagy by inhibiting inositol monophosphatase

S. Sarkar, R.A. Floto, Z. Berger, S. Imarisio, A. Cordenier, M. Pasco, L.J. Cook, D.C. Rubinsztein

J Cell Biol 2005; 170:1101-11.     

A Novel Role for Autophagy in Neurodevelopment

We recently showed that Ambra1, a WD40-containing ~130 KDa protein, is a novel activating molecule in Beclin 1-regulated autophagy and plays a role in the development of the nervous system. Ambra1 binds to Beclin 1 and favors Beclin 1/Vps34 interaction. At variance with these factors, Ambra1 is highly conserved among vertebrates only, and its expression is mostly confined to the neuroepithelium during early neurogenesis. Ambra1 functional inactivation in mouse led to lethality in utero (starting from embryonic day 14.5), characterized by severe neural tube defects associated with autophagy impairment, unbalanced cell proliferation, accumulation of ubiquitinated proteins, and excessive apoptosis. We also demonstrated that hyperproliferation was the earliest detectable abnormality in the developing neuroepithelium, followed by a wave of caspase-dependent cell death. These findings provided in vivo evidence supporting the existence of a complex interplay between autophagy, cell proliferation and cell death during neural development in mammals. In this Addendum, we review our findings in the contexts of autophagy and neurodevelopment and consider some of the issues raised.

Addendum to:

Ambra1 Regulates Autophagy and the Development of the Nervous System

G.M. Fimia, A. Stoykova, A. Romagnoli, L. Giunta, S. Di Bartolomeo, R. Nardacci, M. Corazzari, C. Fuoco, A. Ucar, P. Schwartz, P. Gruss, M. Piacentini, K. Chowdhury and F. Cecconi

Nature 2007; In press     

Signal-Dependent Control of Autophagy and Cell Death in Colorectal Cancer Cell: The Role of the p38 Pathway

Autophagy is a vacuolar process leading to the degradation of long-lived proteins and cytoplasmic organelles in eukaryotes. This process has an important role in normal and cancer cells during adaptation to changing environmental conditions, cellular and tissue remodeling, and cell death.

To date, several signaling cascades have been described to regulate autophagy in a cell type-specific and signal-dependent manner.

We found that pharmacological blockade of the p38 pathway in colorectal cancer cells, either by the inhibitor SB202190 or by genetic ablation of p38α kinase, causes cell cycle arrest and autophagic cell death. In these cells, a complex network of intracellular kinase cascades controls autophagy and survival since the effect of p38α blockade is differentially affected by the pharmacological inhibition of MEK1, PI3K class I and III, and mTOR or by the differentiation status.

Collectively, our results suggest an opportunity for exploiting the pharmacological manipulation of the p38α pathway in the treatment of colorectal cancer. Given the number of drugs, currently available or under development, that target the p38 pathway, it stands to reason that elucidating the molecular mechanisms that link p38 and autophagy might have an impact on the clinical translation of these drugs.

Addendum to:

A Novel Cell Type-Specific Role of p38α in the Control of Autophagy and Cell Death in Colorectal Cancer Cells

F. Comes, A. Matrone, P. Lastella, B. Nico, F.C. Susca, R. Bagnulo, G. Ingravallo, S. Modica, G. Lo Sasso, A. Moschetta, G. Guanti and C. Simone

Cell Death Differ 2007; 14: 693-702     

Shaping Cellular Form and Function by Autophagy

In addition to its familiar role in non-selective bulk degradation of cellular material, autophagy can also bring about specific changes in the structure and function of cells. Autophagy has been proposed to operate in a substrate-selective mode to carry out this function, although evidence to demonstrate selectivity has been lacking. A recent study of synapse formation in the nervous system of the nematode Caenorhabditis elegans now provides experimental evidence for substrate-selective autophagy. Synapses form when presynaptic cells contact their postsynaptic partners during development. This contact induces the assembly of synaptically-localized protein complexes in the postsynaptic cell that contain scaffolding proteins and neurotransmitter receptors. When presynaptic contact was blocked, autophagy in the postsynaptic cell was induced. Substrate selectivity was evident in this system: the g-aminobutyric acid type A receptor (GABA_A receptor), an integral-membrane neurotransmitter receptor, trafficked from the cell surface to autophagosomes. By contrast, the acetylcholine receptor, a structurally-similar neurotransmitter receptor, remained on the cell surface. This result provides experimental support for the idea that autophagy can bring about changes in cell structure and behavior by degrading specific cellular proteins, particularly cell surface receptors that are often important for regulating cell growth, differentiation and function.

Addendum to:

Presynaptic Terminals Independently Regulate Synaptic Clustering and Autophagy of GABA_A Receptors in Caenorhabditis elegans

.A.M. Rowland, J.E. Richmond, J.G. Olsen, D.H. Hall and B. A. Bamber

J Neurosci 2006; 26:1711-20     

BH3-Only Proteins and BH3 Mimetics Induce Autophagy by Competitively Disrupting the Interaction between Beclin 1 and Bcl-2/Bcl-XL

Beclin 1 has recently been identified as novel BH3-only protein, meaning that it carries one Bcl-2-homology-3 (BH3) domain. As other BH3-only proteins, Beclin 1 interacts with anti-apoptotic multidomain proteins of the Bcl-2 family (in particular Bcl-2 and its homologue Bcl-X_L) by virtue of its BH3 domain, an amphipathic α-helix that binds to the hydrophobic cleft of Bcl-2/Bcl-X_L. The BH3 domains of other BH3-only proteins such as Bad, as well as BH3-mimetic compounds such as ABT737, competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-X_L. This causes autophagy of mitochondria (mitophagy) but not of the endoplasmic reticulum (ER-phagy). Only ER-targeted (not mitochondrion-targeted) Bcl-2/Bcl-X_L can inhibit autophagy induced by Beclin 1, and only Beclin 1-Bcl-2/Bcl-X_L complexes present in the ER (but not those present on heavy membrane fractions enriched in mitochondria) are disrupted by ABT737. These findings suggest that the Beclin 1-Bcl-2/Bcl-X_L complexes that normally inhibit autophagy are specifically located in the ER and point to an organelle-specific regulation of autophagy. Furthermore, these data suggest a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrion-permeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-X_L at the level of the endoplasmic reticulum.

Addendum to:

Functional and Physical Interaction Between Bcl-X_L and a BH3-Like Domain in Beclin-1

M.C. Maiuri, G. Le Toumelin, A. Criollo, J.-C. Rain, F. Gautier, P. Juin, E. Tasdemir, G. Pierron, K. Troulinaki, N. Tavernarakis, J.A. Hickman, O. Geneste and G. Kroemer

EMBO J 2007; In press