Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy 2

Autophagy has been investigated intensively in Aspergilli, and in particular in the genetically amenable species Aspergillus nidulans, which is well suited to investigate intracellular traffic.¹²⁵⁴ In A. oryzae, autophagy has been monitored by the rapamycin-induced and Atg8-dependent delivery of DsRed2, which is normally cytosolic, to the vacuoles.²⁵⁸ In A. nidulans, autophagy has been monitored by the more “canonical” GFP-Atg8 proteolysis assays, by monitoring the delivery of GFP-Atg8 to the vacuole (by time-lapse microscopy), and by directly following the biogenesis of GFP-Atg8-labeled phagophores and autophagosomes, which can be tracked in large numbers using kymographs traced across the hyphal axis. In these kymographs, the autophagosome cycle starting from a PAS “draws” a cone whose apex and base correspond to the “parental” PAS punctum and to the diameter of the “final” autophagosome, respectively.¹²⁵⁵ Genetic analyses revealed that autophagosomes normally fuse with the vacuole in a Rab7-dependent manner. However, should Rab7 fusogenic activity be mutationally inactivated, autophagosomes can traffic to the endosomes in a RabB/Rab5- and CORVET-dependent manner.¹²⁵⁵ An important finding was that RabO/Rab1 plays a key role in A. nidulans autophagy (and actually can be observed on the phagophore membranes). This finding agrees with previous work in S. cerevisiae demonstrating that Ypt1 (the homolog of RAB1) is activated by the Trs85-containing version of TRAPP, TRAPPIII, for autophagy.^1256,1257 This crucial involvement of RabO/Ypt1 points at the ER as one source of membrane for autophagosomes. The suitability of A. nidulans for in vivo microscopy has been exploited to demonstrate that nascent phagophores are cradled by ER-associated structures resembling mammalian omegasomes.¹²⁵⁵ The macroautophagic degradation of whole nuclei that has been observed in A. oryzae⁶⁹² might be considered as a specialized version of reticulophagy. Finally, autophagosome biogenesis has also been observed using a PtdIns3P-binding GFP-tagged FYVE domain probe in mutant cells lacking RabB/Rab5. Under these genetic conditions Vps34 cannot be recruited to endosomes and is entirely at the disposition of autophagy,¹²⁵⁵ such that PtdIns3P is only present in autophagic membranes.

Mitophagy has been studied in M. oryzae, by detecting the endogenous level of porin (a mitochondrial outer membrane protein) by western blot, and by microscopy observation of vacuolar accumulation of mito-GFP.⁶⁸⁰ Mitophagy is involved in regulating the dynamics of mitochondrial morphology and/or mitochondrial quality control, during asexual development and invasive growth in M. oryzae. Pexophagy has also been studied in rice-blast fungus and it serves no obvious biological function, but is naturally induced during appressorial development, likely for clearance of excessive peroxisomes prior to cell death.¹²⁵⁸ Methods to monitor pexophagy in M. oryzae include microscopy observation of the vacuolar accumulation of GFP-SRL (peroxisome-localized GFP), and detection of the endogenous thiolase,¹²⁵⁸ or Pex14 levels.

7. 
   Food biotechnology. Required for yeast cell survival under a variety of stress conditions, autophagy has the potential to contribute to the outcome of many food fermentation processes. For example, autophagy induction is observed during the primary fermentation of synthetic grape must¹²⁵⁹ and during sparkling wine production (secondary fermentation).¹²⁶⁰ A number of genome-wide studies have identified vacuolar functions and autophagy as relevant processes during primary wine fermentation or for ethanol tolerance, based on gene expression data or cell viability of knockout yeast strains.^{1259,1261-1265} However, determining the relevance of autophagy to yeast-driven food fermentation processes requires experimentation using some of the methods available for S. cerevisiae as described in these guidelines.
   

In the food/health interface, the effect of some food bioactive compounds on autophagy in different human cell types has already attracted some attention.^1267,1268 Interpreting the results of this type of research, however, warrants 2 cautionary notes.¹²⁶⁹ First, the relationship between health status and autophagic activity is obviously far from being direct. Second, experimental design in this field must take into account the actual levels of these molecules in the target organs after ingestion, as well as exposure time and their transformations in the human body. In addition, attention must be paid to the fact that several mechanisms might contribute to the observed biological effects. Thus, relevant conclusions about the actual involvement of autophagy on the health-related effect of food bioactive compounds would only be possible by assaying the correct molecules in the appropriate concentrations.

8. 
   Honeybee. The reproductive system of bees, or insects whose ovaries exhibit a meroistic polytrophic developmental cycle can be a useful tool to analyze and monitor physiological autophagy. Both queen and worker ovaries of Africanized A. mellifera display time-regulated features of cell death that are, however, linked to external stimuli.¹²⁷⁰ Features of apoptosis and autophagy are frequently associated with the degeneration process in bee organs, but only more recently has the role of autophagy been highlighted in degenerating bee tissues. The primary method currently being used to monitor autophagy is following the formation of autophagosomes and autolysosomes by TEM. This technique can be combined with cytochemical and immunohistochemical detection of acid phosphatase as a marker for autolysosomes.^1271,1272 Acidotropic dyes can also be used to follow autophagy in bee organs, as long as the cautions noted in this article are followed. The honeybee genome has been sequenced, and differential gene expression has been used to monitor Atg18 in bees parasitized by Varroa destructor.¹²⁷³
   
9. 
   Human. Considering that much of the research conducted today is directed at understanding the functioning of the human body, in both normal and disease states, it is pertinent to include humans and primary human tissues and cells as important models for the investigation of autophagy. Although clinical studies are not readily amenable to these types of analyses, it should be kept in mind that the MTORC1 inhibitor rapamycin, the lysosomal inhibitors chloroquine and hydroxychloroquine, and the microtubule depolymerizing agent colchicine are all available as clinically approved drugs. However, these drugs have serious side effects, which often impede their clinical use to study autophagy (e.g., severe immunesuppressive effects of rapamycin; gastrointestinal complaints, bone marrow depression, neuropathy and acute renal failure induced by colchicine; gastrointestinal complaints, neuropathy and convulsions, retinopathy induced by [hydroxyl]chloroquine). Theses side effects may in part be exacerbated by potential inhibition of macroautophagy in itself by these drugs.¹²⁷⁴ In cancer treatetment, for example, autophagy inhibiting drugs are used in combination with other anticancer drugs to increase their potency. Conversely, normal tissues such as kidney induce macroautophagy in response to anticancer drugs to resist against their toxicity;¹²⁷⁵ additional blockade of autophagy could worsen normal tissue toxicity and cause serious side effects. Therefore, the potential for serious adverse effects and toxicity of these drugs warrants caution, especially when studying a role of autophagy in high-risk patients, such as the critically ill. Furthermore, fresh biopsies of some human tissues are possible to obtain. Blood, in particular, as well as samples of adipose and muscle tissues, can be obtained from needle biopsies or from elective surgery. For example, in a large study, adipocytes were isolated from pieces of adipose tissue (obtained during surgery) and examined for INS/insulin signaling and autophagy. It was demonstrated that autophagy was strongly upregulated (based on LC3 flux, EM, and lipofuscin degradation) in adipocytes obtained from obese patients with type 2 diabetes compared with nondiabetic subjects.²⁷⁷
   

A major caveat of the work concerning autophagy on human tissue is the problem of postmortem times, agonal state, premortem clinical history (medication, diet, etc.) and tissue fixation. Time to fixation is typically longer in autopsy material than when biopsies are obtained. For tumors, careful sampling to avoid necrosis, hemorrhagic areas and non-neoplastic tissue is required. The problem of fixation is that it can diminish the antibody binding capability; in addition, especially in autopsies, material is not obtained immediately after death.^1277,1278 The possibilities of postmortem autolysis and fixation artifacts must always be taken into consideration when interpreting changes attributed to autophagy.¹²⁷⁹ Analyses of these types of samples require not only special antigen retrieval techniques, but also histopathological experience to interpret autophagy studies by IHC, immunofluorescence or TEM. Nonetheless, at least one recent study demonstrated that LC3 and SQSTM1 accumulation can be readily detected in autopsy-derived cardiac tissue from patients with chloroquine- and hydroxychloroquine-induced autophagic vacuolar cardiomyopathy.⁹²⁰ Despite significant postmortem intervals, sections of a few millimeters thickness cut from fresh autopsy brain and fixed in appropriate glutaraldehyde-formalin fixative for EM, can yield TEM images of sufficient ultrastructural morphology to discriminate different autophagic vacuole subtypes and their relative regional abundance in some cases (R. Nixon, personal communication).

The situation is even worse with TEM, where postmortem delays can cause vacuolization. Researchers experienced in the analysis of TEM images corresponding to autophagy should be able to identify these potential artifacts because autophagic vacuoles should contain cytoplasm. While brain biopsies may be usable for high quality TEM (Fig. 29, 30), this depends upon proper handling at the intraoperative consultation stage, and such biopsies are performed infrequently except for brain tumor diagnostic studies. Conversely, biopsies of organs such as the digestive tract, the liver, muscle and the skin are routinely performed and thus nearly always yield high-quality TEM images. When possible, nonsurgical biopsies are preferable since surgery is usually performed in anesthetized and fasting patients, 2 conditions possibly affecting autophagy. Moreover, certain surgical procedures require tissue ischemia-reperfusion strategies that can also affect autophagy level.¹²⁸⁰ An analysis that examined liver and skeletal muscle from critically ill patients utilized tissue biopsies that were taken within 30 ± 20 min after death and were flash-frozen in liquid nitrogen followed by storage at -80°C.¹⁰¹⁰ Samples could subsequently be used for EM and western blot analysis.

A major limitation of studying patient biopsies is that only static measurements can be performed. This limitation does not apply, however, for dynamic experiments on tissue biopsies or cells derived from biopsies, as described above.²⁷⁷ Multiple measurements over time, especially when deep (vital) organs are involved, are impossible and ethically not justifiable. Hence, quantitative flux measurements are virtually impossible in patients. To overcome these problems to the extent possible and to gain a more robust picture of the autophagic status, observational studies need to include 2 different aspects. First, a static marker for phagophore or autophagosome formation needs to be measured. This can be done by assessing ultrastructural changes with TEM and/or on the molecular level by measuring LC3-II protein levels. Second, accumulation of autophagy substrates, such as SQSTM1 and (poly)ubiquitinated proteins, can provide information on the overall efficacy of the pathway and can be a surrogate marker of the consequences of altered autophagic flux, especially when autophagy is insufficient, although these changes can also be affected by the ubiquitin-proteasome system as mentioned above. In addition, and even more so when problems with specific pathways are suspected (e.g., mitophagy), specific substrates of these pathways should be determined. Again, none of these measurements on its own provides enough information on (the efficacy of) autophagy, because other processes may confound every single parameter. However, the combination of multiple analyses should be informative. Of note, there has been recent interest in assessing markers of autophagy and autophagic flux in right atrial biopsy samples obtained from patients undergoing cardiac surgery.^1281,1282 Evidence to date suggests that cardiac surgery may be associated with an increase in autophagic flux, and that this response may protect the heart from perioperative cardiac ischemia-reperfusion injury.¹²⁸¹ Although still in its infancy with regard to autophagy, it is worth pointing out that mathematical modeling has the power to bridge whole body in vivo data with in vitro data from tissues and cells. The usefulness of so-called hierarchical or multilevel modeling has thus been demonstrated when examining the relevance of INS/insulin signaling to glucose uptake in primary human adipocytes compared with whole-body glucose homeostasis.¹²⁸³

Lipophagy is an important pathway of lipid droplet clearance in hepatocytes, and the extent of lipophagy modulates the lipid content in these cells. Hepatocytes break down lipid droplets through lipophagy as a pathway of endogenous lipid clearance in response to hormones or daily rhythms of nutrient supply.¹⁰¹¹ LC3-II colocalizes with lipid droplets, indicating a role for autophagy in the mobilization of free fatty acids.⁷⁸² Little is known regarding the changes of lipophagy under pathological conditions, such as drug toxicity, alcoholic steatohepatitis or nonalcoholic steatohepatitis (NASH). The accumulation of lipid droplets in hepatocytes activates ATG5 in the droplets, and initiates a lipophagy process; in addition, increased influx of fatty acids in hepatocytes results in oxidant stress, ER stress and autophagy,^1284,1285 as indicated by the fact that there is enhanced staining of LC3-II in NASH tissue.^1284,1286 However, autophagy flux is impaired in liver specimens of NASH patients as indicated by increased levels of SQSTM1.¹²⁸⁷ Therefore, the value of using LC3-II staining in tissue as an indication of autophagy or lipophagy is in question.

A stepwise process can be proposed for linking changes in the autophagic pathway to changes in disease outcome. First, in an observational study, the changes in the autophagic pathway (see above) should be quantified and linked to changes in disease outcome. To prove causality, a subsequent autophagy-modifying intervention should be tested in a randomized study. Before an intervention study is performed in human patients, the phenotype of (in)active autophagy contributing to poor outcome should be established in a validated animal model of the disease. For the validation of the hypothesis in an animal model, a similar two-step process is suggested, with the assessment of the phenotype in a first stage, followed by a proof-of-concept intervention study (see Large animals).

10. 
    Hydra. Hydra is a freshwater cnidarian animal that provides a unique model system to test autophagy. The process can be analyzed either in the context of nutrient deprivation, as these animals easily survive several weeks of starvation,^1288,1289 or in the context of regeneration, because in the absence of protease inhibitors, bisection of the animals leads to an uncontrolled wave of autophagy. In the latter case, an excess of autophagy in the regenerating tip immediately after amputation is deleterious.^1290-1292 Most components of the autophagy and MTOR pathways are evolutionarily conserved in Hydra.¹²⁸⁹ For steady-state measurements, autophagy can be monitored by western blot for ATG8/LC3, by immunofluorescence (using antibodies to ATG8/LC3, lysobisphosphatidic acid or RPS6KA/RSK), or with dyes such as MitoFluor Red 589 and LysoTracker Red. Flux measurements can be made by following ATG8/LC3 turnover using lysosomal protease inhibitors (leupeptin and pepstatin A) or in vivo labeling using LysoTracker Red. It is also possible to monitor MTOR activity with phosphospecific antibodies to RPS6KB and EIF4EBP1 or to examine gene expression by semiquantitative RT-PCR, using primers that are designed for Hydra. Autophagy can be induced by RNAi-mediated knockdown of Kazal1,^1290,1291 or with rapamycin treatment, and can be inhibited with wortmannin or bafilomycin A₁.^1288,1289
    
11. 
    Large animals. This section refers in particular to mammals other than humans. Assessment of autophagy (and, in particular, autophagic flux) in clinically relevant large animal models is critical in establishing its (patho)physiological role in multiple disease states. For example, evidence obtained in swine suggests that upregulation of autophagy may protect the heart against damage caused by acute myocardial infarction/heart attack.¹²⁹³ Ovine models of placental insufficiency leading to intrauterine growth restriction have shown that there is no change in the expression of markers of autophagy in the fetus in late gestation¹²⁹⁴ or in the lamb at 21 days after birth.¹²⁹⁵ Furthermore, there is an increase in markers of autophagy in the placenta of human intrauterine growth restriction pregnancies.¹²⁹⁶ Studies in rabbits suggest a protective role of upregulated autophagy against critical illness-induced multiple organ failure and muscle weakness,^1297,1298 which is corroborated by human studies.^1009,1010 Conversely, autophagy may contribute to the pathogenesis of some types of tissue injury, at least in the lung.^1299,1300
    

In contrast with cell culture experiments, precise monitoring of autophagic flux is practically impossible in vivo in large animals. Theoretically, repetitive analyses of small tissue biopsies should be performed to study ultrastructural and molecular alterations over time in the presence or absence of an autophagy inhibitor (e.g., chloroquine). However, several practical problems impede applicability of this approach. First, repetitive sampling of small needle biopsies in the same animal (a major challenge by itself) could be assumed to induce artifacts following repetitive tissue destruction, especially when deep (vital) organs are involved. In addition, chemical inhibitors of autophagy have considerable side effects and toxicity, hampering their usage. Also, the general physical condition of an animal may confound results obtained with administration of a certain compound, for instance altered uptake of the compound when perfusion is worse.

Therefore, in contrast to cells, where it is more practical to accurately document autophagic flux, we suggest the use of a stepwise approach in animal models to provide a proof of concept with an initial evaluation of sequellae of (in)active autophagy and the relation to the outcome of interest.

First, prior to an intervention, the static ultrastructural and molecular changes in the autophagic pathway should be documented and linked to the outcome of interest (organ function, muscle mass or strength, survival, etc.). These changes can be evaluated by light microscopy, EM and/or by molecular markers such as LC3-II. In addition, the cellular content of specific substrates normally cleared by autophagy should be quantified, as, despite its static nature, such measurement could provide a clue about the results of altered autophagic flux in vivo. These autophagic substrates can include SQSTM1 and (poly)ubiquitinated substrates or aggregates, but also specific substrates such as damaged mitochondria. As noted above, measurement of these autophagic substrates is mainly informative when autophagic flux is prohibited/insufficient, and, individually, all have specific limitations for interpretation. As mentioned several times in these guidelines, no single measurement provides enough information on its own to reliably assess autophagy, and all measurements should be interpreted in view of the whole picture. In every case, both static measurements reflecting the number of autophagosomes (ultrastructural and/or molecular) and measurements of autophagic substrates as surrogate markers of autophagic flux need to be combined. Depending on the study hypothesis, essential molecular markers can further be studied to pinpoint at which stage of the process autophagy may be disrupted.

Second, after having identified a potential role of autophagy in mediating an outcome in a clinically relevant large animal model, an autophagy-modifying intervention should be tested. For this purpose, an adequately designed, randomized controlled study of sufficient size on the effect of a certain intervention on the phenotype and outcome can be performed in a large animal model. Alternatively, the effect of a genetic intervention can be studied in a small animal model with clinical relevance to the studied disease.

As mentioned above, exact assessment of autophagic flux requires multiple time points, which cannot be done in the same animal. Alternatively, different animals can be studied for different periods of time. Due to the high variability between animals, however, it is important to include an appropriate control group and a sufficiently high number of animals per time point as corroborated by statistical power analyses. This requirement limits feasibility and the number of time points that can be investigated. The right approach to studying autophagy in large animals likely differs depending on the question that is being addressed. Several shortcomings regarding the methodology, inherent to working with large animals, can be overcome by an adequate study design. As for every study question, the use of an appropriate control group with a sufficient number of animals is crucial in this regard.

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