Department of Biochemistry, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 695011, IndiaComprehensive Care Centre for Movement Disorders, Department of Neurology, Sree Chitra Tirunal Institute for Medical Sciences and Technology,hiruvananthapuram 695011, India
r t i c l e i n f o
rticle history:eceived 12 February 2014eceived in revised form 7 March 2014ccepted 12 March 2014
hemical compounds studied in this article:-Methyladenine (PubChem CID: 1673)afilomycin A1 (PubChem CID: 6436223)ortmannin (PubChem CID: 312145)
The strategy for interpreting the role of autophagy on the basis of evidence obtained through autophagicinhibition sounds logical, but is beset with practical constraints. The knock down of autophagy-related(ATG) gene(s) or blockage of class III PI3-Kinase are the most common approaches for inhibitingautophagy. However, during stressful conditions, autophagy may operate in synchrony with other pro-cesses such as apoptosis; autophagy-related genes, unlike what their name implies, exert their regulationon apoptosis as well. Knocking down such genes not only blocks autophagy but also renders apoptosisdefective, making the interpretation of autophagic roles unreliable. Similarly, class III PI3-Kinase aids ininitiating autophagy but it is not a quintessential autophagic regulator. Class III PI3-Kinase also has a rolein regulating almost all membrane transport in cells. Blocking it not only inhibits autophagy, but alsohampers all the membrane trades, including endosomal transport. The pharmacological inhibitors usedto block autophagy by blocking class III PI3-Kinase further compound these limitations with their off-target effects. Knowing the limitations involved in blocking a target or using an autophagy-blocking toolis a prerequisite for designing the experiments meant for analyzing autophagic functions. This review
eywords:utophagypoptosis-Methyladeninetg5
attempts to provide a detailed overview about the practical constraints involved in using autophagicinhibition as a strategy to understand autophagy.
Fig. 1. Articles, published after 2007, describing the role of autophagy were selectedand classified based on the strategies mentioned in it for analyzing autophagy (A).The articles that explain role of autophagy based on the evidences obtained throughits inhibition were categorized again based on the strategies utilized for blockingautophagy. The percentage of articles in each category was calculated (B). The fre-quency at which different autophagic inhibitor used or gene targets utilized for theinhibition of autophagy in experiments mentioned in the articles has been calculated
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. Introduction
It is a truism that some processes, by their very nature, areialectical. This is particularly true of autophagy, a cellular process,hich in some cases protects cells from death but in other, seems to
ause it. During autophagy, organelles and proteins are sequesteredn a double membrane vesicle called the autophagosome, whoseontents are digested in the lysosomes. Though autophagy is gen-rally considered as a housekeeping process, which is meant toegrade and recycle the damaged proteins and organelles in theell, it remains unpredictable whether or not it retains the sameunction during a condition of cellular stress. Under stress, inducedutophagy can switch over from its housekeeping mode to either arogrammed cell death mechanism or, depending upon the modend intensity of the stress, a process delaying/enhancing apopto-is [1,2]. Otherwise, autophagy may choose to remain in rescueode, where it digests any of the cell’s proteins and organelles
hat were damaged during this stress event [3]. As if to upend thexisting notion regarding autophagy, there are contrasting reportsbout its role in different cellular contexts induced by the sameode of stress [4,5]. Researchers with a profound authority on
he subject of autophagy have critically evaluated the reasons forhe widely-reported discrepancies regarding its role [6,7]. Accord-ng to these experts, such contradictions are most likely due toactors such as misinterpretation of the experimental observa-ions, technical difficulties in assaying autophagy and a lack ofonvincing strategies to disrupt autophagy without altering otherrucial cellular processes. Though recent reports in the litera-ure exhibit appreciable improvements in using advanced toolsor assaying autophagy and in exercising caution in interpretingutophagic experiments, the problems regarding the inhibition ofutophagy are not yet fully sorted. On the contrary, the imper-ections of inhibition tools become exposed with advancementsn the knowledge regarding the complex mechanisms regulatingutophagy.
Current strategies to inhibit autophagy mainly involve block-ng of class III PI3-Kinase (an enzyme complex having a role inssembling autophagosomes) using pharmacological inhibitors,isrupting lysosomal functions with lysosomotropic agents, orsing lysosomal enzyme inhibitors, thus preventing digestion ofutophagic cargo, and blocking the expression of proteins reg-lating autophagy using genetic approaches. It is obvious thaturrent understanding regarding the role of autophagy was builtp after various studies on the inhibition of autophagy. Hence,utophagic inhibitors are, in other words, autophagic interpretersnd elucidating the role of autophagy will depend much on theccuracy of these inhibitors or inhibition strategies. Thus, a discus-ion on the shortcomings of inhibition tools and the limitationsnvolved in utilizing molecular targets for blocking autophagyould also be a reflection of our impediments in understandingutophagy.
. A bird’s eye – view of autophagic inhibition studies
A PubMed search for articles published from 2007 to June 2013ith ‘autophagy’ as a keyword, yielded about 5500 entries, 1193
f which were articles that described the role of autophagy. About3% of these 1193 articles explain the autophagic role based onhe experimental evidence obtained through autophagic inhibition,hereas 15% explain it on the basis of evidence obtained by both
ctivation and inhibition of autophagy (Fig. 1A). These articles were
urther categorized using the citation manager Zotero, and repre-ented based on the strategy used for blocking autophagy (Fig. 1B).oreover, the relative frequency at which different pharmacologi-
al inhibitors or gene-targets were utilized in the experiments (for
and expressed in percentage (C).
the purpose of blocking autophagy) was also calculated (Fig. 1C).The information obtained from the survey on these selected arti-cles is concluded as follows: (i) class III PI3-Kinase blockage andATG gene – knockdowns are the most preferred routes for blockingautophagy. About 27% of articles explain the autophagic role on thebasis of class III PI3-Kinase mediated inhibition of autophagy whileanother 27% attribute the role to ATG gene knockdowns. Evidencesobtained from both ATG gene knock-downs and class III PI3-Kinasemediated autophagic blockage was used in 17% of articles to explainthe autophagic role. (ii) 3-Methyladenine (3-MA), a pharmacolog-ical blocker of class III PI3-Kinase, was the most frequently usedtool for blocking autophagy. (iii) ATG5 and BECLIN1 were the mosttargeted genes when genetic methods were employed for blockingautophagy. (iv) Lysosomal inhibition was the least used methodto block autophagy, but when used, Bafilomycin A1, a lysosomalV-ATPase blocker, was the most preferred autophagic inhibitor.From these facts, it is safe to assume that the reliability of infer-ences about the autophagic role is compromised by the limitationsinvolved in using 3-MA, the most used inhibitor, or targeting ATG5and BECLIN1, the most targeted genes, for blocking autophagy. The
priority given to the topics discussed below are based on the factsrevealed from the above mentioned article survey.
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. Issues involved in inhibiting autophagy through class IIII3-Kinase blockage
As mentioned above, most of the information about the rolef autophagy was obtained from its inhibition through blockinglass III PI3-Kinase, an enzyme complex required for the forma-ion of the autophagosome. Pharmacological inhibitors, especially-MA and Wortmannin, were traditionally used for this purpose.owever, all known PI3-Kinases have their ATP binding sites at
he kinase domain in common, for they are evolutionarily related;he inhibitors (3-MA and Wortmannin) that prevent autophagy bylocking such sites in class III PI3-Kinase were also found to inter-ere with the activity of related kinases, especially that of class II3-Kinase [8]. A known limitation in the above mentioned strategyf autophagic inhibition is that any blockage in class I PI3-Kinaseignaling can interrupt the activity of its downstream target PKB, ainase known for phosphorylating and activating a wide range ofroteins involved in growth, survival and proliferation in responseo growth factor signals [9]. Some tumor cell lines routinely used forxperimental purposes are dependent upon PKB signaling (Onco-enic addiction), while others, upon inhibition become increasinglyensitive to several apoptosis-inducing stresses [10,11]. Thus, as aesult of the traditional autophagic inhibitors acting on class I PI3-inases, the validity of assumptions about their role in autophagy
s compromised. Even if molecules tailor-made for inhibiting classII PI3-Kinase were developed, it is highly unlikely that the speci-city problem regarding autophagy inhibition would have beenolved because the class III PI3-Kinase itself is not quintessen-ially evolved for autophagic regulation. Instead, it regulatesellular membrane trafficking processes including endocytosis, ret-ograde transport of vesicle components between different cellularrganelles, and transport across the nuclear membrane [12,13].egulating autophagy is, indeed, just one role among its differ-nt membrane trades. In certain cases, such as in non-canonicalutophagy, class III PI3-Kinase is not required for the activationr maintenance of autophagy and blockage of its activity neveruarantees autophagic blockage [14]. This makes it incumbent tonow the nature of induced autophagy before choosing the PI3-inase route for inhibiting it. All these complications make class IIII3-Kinase a sloppy experimental target for inhibiting autophagy.evertheless, class III PI3-Kinase deserves recognition, since muchf the current knowledge about autophagy has come about as aesult of studies with this target.
The annals of 3-MA as an autophagic inhibitor deserve some spe-ial attention in this context. The autophagy-inhibiting propertyf 3-MA was demonstrated in 1982 by Seglen et al. [15] How-ver, it took about eighteen years to identify class III PI3-Kinases its target [16]. All the general criticisms pertaining to the speci-city of an autophagic inhibitor that mediates its action throughI3-Kinase can also be leveled at 3-MA, but for more than twoecades it enjoyed its status as a classic autophagic inhibitor. Only
ater, when a report authored by Wu et al. demonstrated the dualature of 3-MA in regulating autophagy, were the credentials of-MA as an autophagic inhibitor questioned [17]. According tohis report, 3-MA could inhibit autophagy in starved cells, butn cells cultured with sufficient nutrients, it exerted only a tran-ient effect on class III PI3-Kinase. In these cases, it could induce,nstead of inhibit, autophagy by disrupting the anti-autophagicctivity of mTOR. Given that 3-MA is the most frequently usedool for inhibiting autophagy in research, the delay in exposingts autophagy-inducing nature has been quite surprising. There arether known concerns regarding the use of 3-MA as an autophagic
nhibitor. 3-MA can inhibit glycolytic enzymes and promote glyco-en breakdown-independent of its autophagy-regulating abilities18]. Independent of its autophagic inhibition property, the induc-ion of cell death by 3-MA through apoptosis has been reported
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recently [19,20]. All these factors point out the need to be cautiousin interpreting the role of autophagy, based on autophagic blockageby 3-MA, without other experimental support.
The efforts to overcome the above-described limitations in usingPI3-Kinase inhibitors for blocking autophagy are worth mention-ing. Autophagic inhibitors derived from 3-MA exhibit enhancedqualities in comparison to 3-MA; these include higher solubil-ity, greater effectiveness at lower concentrations, and enhancedinhibitory effects on class III PI3-Kinase without blockage of classI kinase [21]. Screening of a kinase inhibitor library helps recog-nize the autophagic inhibition potential of KU55933, a known ATMKinase inhibitor [22], which can inhibit autophagy by blocking classIII PI-Kinase without influencing class I PI3-Kinase activity or AKTsignaling [23]. A recent screening identified Spautin-1 as a promis-ing autophagic inhibitor that can block autophagy by inhibitingUPS10 and UPS13, two ubiquitin-specific peptidases capable of reg-ulating the deubiquitination of Beclin1, a component in the class IIIPI3-Kinase complex [24]. The inhibitors described here may not beexpected to solve all the existing limitations involved in autophagicinhibition, but some of these pharmacological agents may becomeeffective replacements for traditional inhibitors in the near future.
4. Issues involved in targeting autophagy related genes –Who put ‘A’ in ATG?
With advancements in knowledge about the mechanism ofautophagy, new vistas for inhibiting autophagy, in the form of genetargets, have opened up for researchers. The most frequently usedgenetic approaches to inhibit autophagy involve transient knockdown of autophagy related genes with siRNA [25], permanent dele-tion of specific autophagic genes as in the case of ATG−/− mouseembryonic fibroblasts [26], and knock down of ATG genes in mousemodels (including Cre-lox based “conditional” knock down) [27].As mentioned before, ATG5, BECLIN1 (ATG6) and ATG7 are the mostpopular targets utilized by autophagy researchers for inhibitingautophagy. The products of these genes are more or less involvedin the proper development of autophagosomes as well as the pos-itive regulation of autophagy. Beclin1 can induce autophagy byfunctioning as a scaffold for assembling a multiprotein complex(with class III PI3-Kinase, UVRAG and Ambra1 as its major compo-nents) that can positively regulate autophagosome developmentand autophagy [28]. Atg7 and Atg5 are proteins involved in theubiquitin-like conjugation system required for the developmentof autophagosomes [29]. In terms of flexibility and accuracy, thegenetic blockage of these autophagy-related genes seems to haverelative advantages over pharmacological autophagic inhibitors,albeit with its own issues.
For instance, consider Beclin1. Genetic inhibition of Beclin1 is,indeed, a robust strategy to block autophagy, but the caveats asso-ciated with it should not be overlooked. First of all, Beclin1 is nota quintessential pro-autophagic regulator but more or less a pro-tein placed at the hub of a complex apoptosis – autophagy signalingaxis. Caspases activated during apoptosis are capable of altering thepro-autophagic function of Beclin1 by cleaving it. Such cleaved frag-ments of Beclin1 function in an apoptosis amplification loop ratherthan in autophagic machinery, by entering into the mitochondriaand releasing the pro-apoptotic factors, thereby enhancing theongoing apoptosis [30]. This may be one of the reasons for thereduction of apoptosis in Beclin1 knock-down cells in response tostress, even though this is interpreted as the ability of autophagyto curb apoptosis. Another potential interconnection of Beclin1
with apoptosis is demonstrated in the form of its interaction withBcl2 – a known anti-apoptotic protein. Such an interaction nega-tively regulates autophagy [31]. An additional matter of concernin using Beclin1 as an autophagic inhibition target is its occasional
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nvolvement in negative autophagic regulation through its inter-ction with a protein called Rubicon. Through such interaction,eclin1 can down-regulate autophagy by slowing down the fusionf autophagosomes and lysosomes [32,33]. Similarly, the inhibitionf Beclin1 never guarantees autophagic inhibition in all experimen-al contexts, as seen in the case of non-canonical autophagy, where
ost autophagy-related proteins including Beclin1 were not foundo be involved [14].
Akin to the case of Beclin1, Atg5 also exhibits apoptosis connec-ion. When cleaved by calpain (released under apoptosis-inducedtress), Atg5 can switch over from being an autophagic regulator toeing an apoptosis initiator [34]. Atg5 can also activate caspases byinding to FADD, a component of an extrinsic apoptotic pathway35]. There are other complications involved in using Atg5 as anutophagy inhibition target. For instance, Atg5, depending upon itshosphorylation status (regulated by p38 signaling)—can change
ts function from a pro-autophagic factor to a negative regulator ofutophagy [36]. Similarly, non-canonical forms of autophagy with-ut Atg5 involvement were widely reported [37]. Another practicalonstraint often encountered during an experimental situation ishe need to inhibit atg5 expression completely for efficient block-ng of autophagy because only low levels of Atg5 are required forutophagic activity and failure to completely inhibit it may possiblyllow autophagy to continue unabated [38].
Atg7 also may have alternative substrates and roles beyondhe autophagic pathway. The role of Atg7 in modulating p53 andhereby regulating the cell cycle during the period of metabolictress has been reported [39]. Positive involvement of Atg7 in apo-tosis during lysosome dysfunctions is also known [40]. The Atg12rotein, one of the most utilized targets for experimental block-ge of autophagy, is also known to be involved in apoptosis. Itan deactivate anti – apoptotic Bcl2 family protein and initiateitochondria-mediated apoptosis. In mice, the knockout of genes
lone cannot be relied upon to ascertain the role of autophagyecause inhibition of different autophagic genes causes differ-nt phenotypic effects. For instance, Beclin1 knockout mice werembryonically lethal, whereas Atg7 and Atg5 knockdown mice sur-ived even after birth [41–43].
“Who put the ‘A’ in Atg12: Autophagy or Apoptosis” is a curi-us question raised by Letai et al. while reviewing the involvementf Atg12 in apoptosis [44]. Considering the involvement of othernown autophagy proteins in apoptosis, we strongly feel the ques-ion needs to be modified as “Who put ‘A’ in all the Atg proteins:utophagy or Apoptosis”. Anyhow, the involvement of Atg proteins
n apoptosis is considered the most concerning factor in study-ng the role of autophagy in cell death. Another concern is thenvolvement of Atg proteins in a wider range of cellular processeshan previously anticipated. Two excellent reviews on autophagyelated proteins, recently published in EMBO reports, collate allnown extra-autophagic roles of Atg, ranging from host defenseo cytoskeleton remodeling [45,46]. The frequency at which noveleports appear on the ‘moonlighting’ of Atg proteins, indirectlyespeaks the possibly erroneous interpretations of autophagic roleased on the inhibition of Atg proteins.
. Inhibiting autophagy through the lysosomal route: Is itnducing ‘autophagic frustrations’ along with autophagiclocking?
Inhibiting autophagy by blocking lysosomal activity also has itsair share of issues. Given that the lysosomes are the organelles
n which the contents of autophagosomes are finally loadednd digested, blocking lysosomal activity with pharmacologicalnhibitors stalls autophagy at its final step [47]. In a strict sense,his condition does not indicate a complete absence of autophagy,
l Research 82 (2014) 1–8
but the presence of a defective form of it. Instead of abolishing theentire autophagy process, the lysosomal route of autophagic inhibi-tion leaves the autophagosomes burdened with undigested cargoand probably ushers additional stresses, other than the effects ofautophagic blockage, to cells in experimental conditions. This con-dition, according to Gottlieb and Mentzer, is ‘frustrated autophagy’,which can often lead to the release of autophagosomes by exocyto-sis and cause inflammation in surrounding cells [48]. Moreover,blocking lysosomes to inhibit autophagy also indirectly blocksrelated process such as endocytosis [49]. All these facts under-score the limitations in using the lysosomal route of autophagicinhibition for analyzing the role of autophagy.
Available pharmacological tools for inhibiting autophagythrough lysosomal blockage include bafilomycin A1 (an antibioticcapable of altering lysosomal acidic environment, thereby changingprotease activity by blocking the lysosomal proton pump), Chloro-quine, Ammonium chloride, Monensin (lysosomotropic agents thatdirectly alter the acidic pH of lysosomes), and E64d and Pepstatin(lysosomal enzyme blockers). Criticism about the non-specificity ofautophagic inhibitors in general can also be leveled against lysoso-mal inhibitors. Bafilomycin A1 as an autophagic inhibitor is specificonly for a short time period, beyond which it has undesirableeffects such as proteosomal inhibition and blockage of endosometransport [50]. The targets of bafilomycin are not specifically con-fined to lysosomes, but are widely distributed in endosomes andplasma membrane and have crucial roles in the cell survival. Forinstance, the V-ATPase present in the plasma membrane of tumorcells helps to maintain a slightly alkaline pH in the cytosol, whichis essential to prevent acid-induced apoptosis and promote antitu-mor drug resistance [51]. The extended use of E64d and PepstatinA in experimental conditions may probably induce rather thanblock autophagy because E64d blocks calpain, which in turn caninduce autophagy [52]. Lysosomotropic agents such as chloroquine,NH4Cl or Monensin also have drawbacks as they can block all acidicorganelles (other than lysosomes) in the cells and cause unpre-dictable side effects. Monensin, other than inhibiting autophagy,inflicts damage on mitochondria and the Golgi complex; thereforeit may not be a popular experimental autophagic inhibitor [53]. Allthese show that the strategy of gaining information on autophagyby inhibiting it through the lysosomal route with the above men-tioned pharmacological agents lacks precision unless supported byevidence obtained from inhibition through genetic approaches.
6. Inhibiting autophagy to study its role – is it tooone–dimensional to analyze some complex characteristicsautophagy is supposed to have?
How is it that autophagy in some cases is protective and incertain other cases destructive to cells? Researchers often try tosolve this paradox, at least theoretically, by assuming that the rela-tionship between autophagy and cell death is quantitative; in otherwords, a moderate level of autophagy is protective for the cell,whereas a higher level is detrimental [54,55]. If such an assumptionholds true, autophagy induced during a stress, switches from pro-survival to a pro-death form after a certain threshold level. Thisthreshold may vary depending upon the nature or intensity of thestress. Such a limit also determines whether protective or destruc-tive autophagy predominates during a stress (Fig. 2). The switchingof autophagic role during a stress period has already been reportedin the literature [56]. Studying the role of autophagy on the basis ofits inhibition could only detect which type of autophagy predom-
inates during the stress, but would not help analyze the possibleinversion of autophagic functions in the midst of the stress. Thelimitations in studying qualitative changes of autophagy on thebasis of its inhibition are one of the probable reasons that the above
V. Vinod et al. / Pharmacological Research 82 (2014) 1–8 5
Fig. 2. It is believed that moderate autophagy during a stress is protective where as too much of autophagy is destructive to cells. If such an argument holds true, thenautophagy induced during a stress, after a certain threshold level, switchover its role from pro-survival to a pro-death form. This possible qualitative change in the role ofautophagy during a stress period is not easy to analyze by inhibiting autophagy. For example, in case-1, protective autophagy dominates and only after reaching a higherquantitative level, autophagy switches its role to death. Inhibition of autophagy in this condition increases cell death and autophagic role is interpreted as protective. Thethreshold level at which autophagy switches its role may vary depending upon the mode and intensity of a stress. In case-2, autophagy induced during the stress, even at al s cell di mids( the th
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ower level turned to be destructive. Inhibition of autophagy in this condition reducen both cases fails to address the qualitative changes occurring in autophagy in thefast or slow depending upon the nature or intensity of the stress) that determines
entioned assumptions remains as a theoretical rhetoric ratherhan an experimentally proven fact. Arriving at probable solutionso such limitations will require a paradigm shift in the approach tohe problem. Instead of directly analyzing qualitative changes inutophagy that is marred with limitations, it is advisable to seekhe signal(s) that regulate such a change during stress. The logicalossibility of the existence of a ‘switch signal’ capable of switchinghe role of autophagy (whose timing of appearance after thetress may indirectly set the above mentioned threshold level ofutophagy for its switch) cannot be overlooked. Such a ‘switch sig-al’, which helps to convert the pro-survival form of autophagy to
ts pro-death form is not entirely a hypothetical construct but existsn reality in Dictyostelium, a haploid eukaryotic organism. Perhapsuch a signal exists in mammalian cells too, but it may remainidden in the complex autophagy signaling circuit or exposed forll to see, only for its significance to go unrecognized [55].
In late stages of cell death, both autophagy and apoptosis cane considered as two facets of the same process. Apoptosis is the
rime mechanism of cell death, which can systematically dismantle
cell, whereas autophagy can act as a process and help apopto-is by digesting organelles and proteins in the lysosomes. Thesewo processes working together can help the cell to die efficiently,
eath and autophagy role is interpreted as pro-death. Anyhow autophagic inhibitiont of a stress period. The possibility of a second signal, whose timing of appearancereshold level of the autophagy for its functional switch could not be overlooked.
avoid necrosis, and reduce the complications of inflammation tothe surrounding cells. If autophagy is blocked, dying cells may havepossible mechanisms to upregulate apoptosis and thus probablyavoid delays or inefficiencies in cell death due to the lack of anautophagic backup. The blocked autophagic components may evenbe directed to enhance apoptosis. In The ability of Atg5 and Beclin1to take part in apoptosis is an indication of such a logical pos-sibility. However, enhanced apoptosis as observed in autophagyblocked cells is often interpreted as the ability of autophagy to delayapoptosis (Fig. 3). Considering the complex connections betweenapoptosis and autophagy, we strongly feel that it is unwise toexclude the alternative possibility of apoptosis enhancement as acompensation for the loss of autophagic assistance in cell death.The failure to address these possibilities is indeed another limita-tion involved in inhibition studies meant to analyze autophagic rolein stress induced cell death.
7. Some general rules observed by researchers to solve the
inhibition puzzles
An inquisitive reader of autophagy-related articles will not findit difficult to notice that the researchers, in order to interpret the
6 V. Vinod et al. / Pharmacological Research 82 (2014) 1–8
Fig. 3. Figure explains two theoretical possibilities for the enhancement of apoptosis when autophagy was inhibited. Apoptosis enhancement in autophagy inhibited cellsis conventionally interpreted as the ability of autophagy to delay apoptosis. Considering the complex cross talk between apoptosis and autophagy, an alternate possibilityof apoptosis enhancement as compensation for lack of autophagic assistance in executing cell death cannot be overlooked. The ability of autophagic proteins to involve ina
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poptosis during cell death is pointing toward such a possibility.
ole of autophagy, follow some basic rules to tackle the limitationsnvolved in blocking autophagy. Inhibiting autophagy with multi-le strategies is the most widely used approach. Such a strategyill ensure that a particular phenotype observed is due to the lack
f autophagy and not due to the off – target effects of the com-onents used for blocking autophagy. Nowadays, reviewers may
nsist that the researchers not only derive conclusions by knockingown multiple ATG genes to inhibit autophagy but also substanti-te such conclusions with evidence obtained from pharmacologicalnhibition of autophagy. The use of precaution in designing thexperiment to overcome the limitations of inhibition strategy by isnother useful approach followed by researchers. For instance,hile using 3-MA, researchers used to take extra effort to show that
utophagy was actually inhibited by it at a given experimental con-ition [57]. Since autophagy independent of ATG genes is reportedrequently, researchers often ensure that autophagy is actually
eing inhibited by targeting related genes such as ATG7, ATG5nd BECLIN1 [58]. When using lysosomal inhibitors for blockingutophagy, researchers have to restrict the experimental durationo short time periods, since lysosomal inhibitors exert effects other
than autophagic inhibition when treated in cells for extended timeperiods (in most cases such a reduction in the experimental dura-tion is not practically possible and that is a probable reason forthe relatively low frequency of the use of lysosomal inhibitorsfor blocking autophagy). Another appreciable approach is the useof advanced genetic approaches for blocking autophagy. The use ofdominant-negative mutant ATGs for blocking autophagy is oneexample. Such a type of inhibition provides a higher level of speci-ficity, as compared to the conventional siRNA-mediated inhibitionof ATG genes. Inhibiting autophagy using over expressing mutantproteins does not block the expression of its non-mutated coun-terparts, but only blocks its autophagy-specific function and leavesthe non-mutated proteins available for its other non-autophagicfunctions. For instance, over expression of the mutant form of Atg5(Atg5K130R) could selectively block the interaction of Atg5 withAtg12 and thus interfere in the formation of Atg5–Atg12, a protein
conjugate required for proper autophagosome assembly [26]. Suchmutant Atg5 expression is not expected to block the availability ofnormal Atg5 proteins for its apoptotic functions, unlike in the caseof siRNA-mediated blockage of ATG5.
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. Conclusion
From an autophagist’s point of view, all of the availableutophagic inhibitors and gene target have their own diabolicalide effects in the form of off -target activities and pleiotropic effectsespectively. Considering the complexity of autophagy and its reg-lation, designing precise tools and finding apt targets for blockingutophagy is a daunting task. Fulfilling such a task is like finding a
fitting shoe for Cinderella’ [59]. But with no magic around to help,ow it will be found is the actual puzzle that demands a solution.
ppendix A. Supplementary data
Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.phrs.2014.03.005.
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