1521-0111/90/3/371–384$25.00 http://dx.doi.org/10.1124/mol.116.105171MOLECULAR PHARMACOLOGY Mol Pharmacol 90:371–384, September 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

MINIREVIEW—A LATIN AMERICAN PERSPECTIVE ON ION CHANNELS

Modulation of Autophagy by Calcium Signalosome inHuman Disease

Eduardo Cremonese Filippi-Chiela, Michelle S. Viegas, Marcos Paulo Thomé,Andreia Buffon, Marcia R. Wink, and Guido LenzGraduate Program in Hepatology and Gastroenterology, Faculty of Medicine (E.C.F.-C.), and Gene Therapy Center (M.S.V.), Hospitalde Clínicas de Porto Alegre; Department of Biophysics and Center of Biotechnology (M.P.T., G.L.) and Laboratory of Biochemical andCytological Analysis, Faculty of Pharmacy (M.R.W.), Federal University of Rio Grande do Sul (UFRGS); and Department of HealthSciences and Cell Biology Laboratory, Federal University of Health Sciences of Porto Alegre (A.B.), Porto Allegre, Brazil

Received May 4, 2016; accepted July 18, 2016

ABSTRACTAutophagy is a catabolic process that is largely regulated byextracellular and intracellular signaling pathways that are centralto cellular metabolism and growth. Mounting evidence hasshown that ion channels and transporters are important forbasal autophagy functioning and influence autophagy to handlestressful situations. Besides its role in cell proliferation andapoptosis, intracellular Ca21 is widely recognized as a keyregulator of autophagy, acting through the modulation ofpathways such as the mechanistic target of rapamycin com-plex 1, calcium/calmodulin-dependent protein kinase kinase 2,and protein kinase C. Proper spatiotemporal Ca21 availability,coupled with a controlled ionic flow among the extracellular

milieu, storage compartments, and the cytosol, is critical indetermining the role played by Ca21 on autophagy and on cellfate. The crosstalk between Ca21 and autophagy has a centralrole in cellular homeostasis and survival during several physio-logic and pathologic conditions. Here we review the main findingsconcerning the mechanisms and roles of Ca21-modulated auto-phagy, focusing on human disorders ranging from cancer toneurologic diseases and immunity. By identifying mechanisms,players, and pathways that either induce or suppress autophagy,new promising approaches for preventing and treating humandisorders emerge, including those based on the modulation ofCa21-mediated autophagy.

IntroductionBasic Mechanisms of Autophagy Modulation

Autophagy is an evolutionary conserved catabolic process bywhich cells degrade and recycle self-components to maintaincellular homeostasis. By targeting intracellular substrates, this

lysosomal degradative pathway generates a pool of essentialmolecules required for several cellular functions (Mizushimaet al., 2008). Although nutritional stress is considered theclassic trigger of autophagy (Lum et al., 2005; Onodera andOhsumi, 2005), growth factor availability, hypoxia, aggregatedproteins, injured organelles, DNA damage, and infection canalso initiate an autophagic response (Choi et al., 2013; Filippi-Chiela et al., 2015; Galluzzi et al., 2015). Because autophagyworks as a protective and quality control mechanism, itsdysfunction has been implicated in apoptotic cell death and inthe onset of several human pathologies (Jiang and Mizushima,2014). Additionally, excessive autophagy was suggested todirectly drive cell killing if other cell death mechanisms areimpaired (Liu and Levine, 2015).

This work was supported by Conselho Nacional de DesenvolvimentoCientífico e Tecnológico (CNPq), Universal (Grants 475882/2012-1 and458139/2014-9) and Novas Terapias Portadoras de Futuro (Grant 457394/2013-7); PROBITEC-CAPES (Grant 004/2012) and ICGEB BRA11/01.E.C.F.C., M.S.V., and M.P.T. are or were recipients of CNPq fellowships, andE.C.F.C. is a recipient of CAPES fellowship. M.R.W. and G.L. are recipients ofresearch fellowships from CNPq.

E.C.F.C. and M.S.V. contributed equally to this work.dx.doi.org/10.1124/mol.116.105171.

ABBREVIATIONS: AMPK, 59 AMP-activated protein kinase; ATG, autophagy-related; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-tetra-aceticacid; BECN1, beclin 1 protein; [Ca21]c, cytosolic Ca21 concentration; [Ca21]ER, cytosolic Ca21 concentration; CaMKK2, calcium/calmodulin-dependent protein kinase kinase 2, b; CaMK4, calcium/calmodulin-dependent protein kinase IV; CRAC, Ca21-release-activated Ca21 channel; ER,endoplasmic reticulum; IP3-R, inositol 1,4,5-triphosphate receptor; JNK, c-Jun N-terminal kinases; LPS, lipopolysaccharide; LRRK2, leucine-richrepeat kinase-2; MAP1LC3A or LC3, icrotubule-associated protein 1A/1B-light chain 3; MCUR1, mitochondrial calcium uniporter regulator 1;MICU1, mitochondrial Ca21 uptake 1; mTORC1, mechanistic target of rapamycin complex 1; NAADP, nicotinic acid adenine dinucleotidephosphate; PD, Parkinson’s disease; RAPA, rapamycin; SQSTM1/p62, sequestosome 1 protein; STIM1, sensor stromal interaction molecule 1;TKO, triple knockout; TPC, two-pore channels; TRP, transient receptor potential; TRPM2/3, transient receptor potential calcium channel melastatin2 and 3; TRPML1/3, mucolipin 1 and 3; VDAC1, voltage-dependent anion channel 1; VGCC, voltage-gated calcium channels.

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Autophagy is classified as macroautophagy, microautophagy,and chaperone-mediated autophagy according to the differentways that cytosolic contents are delivered to lysosomes. Inmacroautophagy (hereafter referred to as autophagy), the cyto-solic cargo is captured and transported to lysosomes through adouble-membrane organelle called autophagosome (Fig. 1).In the other types, lysosomal membrane receptors directlymediate cargo internalization (Boya et al., 2013).Extracellular and intracellular signals that modulate auto-

phagy act on the activity of mechanistic target of rapamycincomplex 1 (mTORC1, a suppressor of autophagy) or AMP-activated protein kinase (AMPK, an activator of autophagy),as summarized in Fig. 1, boxes 1 and 3. Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), which isactivated by the Ca21-calmodulin complex, is themain proteininvolved in linking Ca21 to energetic balance and glucosehomeostasis. The most important target of CaMKK2 formetabolism modulation is the AMPK protein. Once activated,AMPK directly alters cell metabolism to replenish cellularATP levels, acting on proteins involved in fatty acid oxidationand autophagy. One of the most important targets of AMPKis mTORC1 (Hurley et al., 2005; Green et al., 2011). Thispathway, which involves several members of the autophagy-related (ATG) family of proteins, modulates the machinerythat executes autophagosome formation. These proteins areactivated in a coordinated fashion through post-translationalmodifications and the formation of complexes (Feng et al.,2014). Curiously, additional nonautophagic functions havebeen attributed to ATG proteins, including phagosome matu-ration, modulation of intracellular transport, apoptosis, andexocytosis (Subramani and Malhotra, 2013). The proteincomplex that initiates autophagy involves the ATG1 protein(also called as ULK1), which is modulated by mTORC1 andAMPK with opposite effects (Egan et al., 2011; Kim et al.,2011; Loffler et al., 2011). Notably, AMPK not only directlyphosphorylates ULK1, but also suppresses mTORC1 (Kimet al., 2011; Mao and Klionsky, 2011). Activated ULK1complex, together with Vps34 complex, induces the phago-phore isolation. Autophagosome completion is further con-trolled by the Atg12-Atg5-Atg16L complex, which drives themembrane expansion, and the LC3 protein conjugated tophosphatidylethanolamine, forming the LC3-II, which is in-volved in cargo targeting, membrane closure, and autophago-some maturation (Fig. 1, box 3) (Hara et al., 2008; Itakuraet al., 2008; He and Klionsky, 2009; Galluzzi et al., 2015).Autophagy selectivity is dictated by the ubiquitination ofdistinct targets, which are recognized by autophagy adapterslike SQSTM1/p62 and Nbr1, which mediate cargo binding toLC3-II attached to autophagosome membranes (Russell et al.,2014). Selective autophagy for organelles degradation, suchas mitophagy (mitochondria) or reticulophagy (ER), is criti-cal for maintaining proper cellular function and for prevent-ing the accumulation of dysfunctional or excessive cellularcomponents. Indeed, cells that are defective to autophagyor that are induced to suppress autophagy can have theirhomeostasis disturbed and be sensitized to apoptosis (Fimiaet al., 2013).A single signal can induce both autophagy and apoptosis in

the same cell. Generally, autophagy suppresses apoptosis thusinfluencing the response to infection and the recognition ofdead cells, the capacity of tumor cells to adapt to metabolicstress and respond to therapy, the sensitivity of neurons to

hypoxia, and the toxicity of intracellular protein aggregates.The best described mechanism in the autophagy-apoptosiscrosstalk involves the interaction between the autophagicprotein Beclin1 (BECN1) and antiapoptotic proteins from theBH3 family, including Bcl-2, Mcl-1, and Bcl-XL. This interac-tion blocks the role of BECN1 in autophagy but does not alterthe function of the antiapoptotic proteins (Pattingre et al.,2005; Kang et al., 2011). Similarly, ATG12 interacts with Mcl-1 and Bcl-2, leading to the suppression of the antiapoptoticactivity of Mcl-1 and Bcl-2, promoting apoptosis (Rubinsteinet al., 2011). Another mechanism underlying this crosstalkinvolves the cleavage of the autophagic protein ATG5 bycalpain, leading to reduced autophagy and increased apopto-sis by targeting the truncated ATG5 protein to the mitochon-dria (Yousefi et al., 2006). Finally, active caspases cleaveseveral autophagic proteins, such as p62, ATG3, ATG5 andBECN1, thus reducing cytoprotective autophagy and favoringapoptosis (Marino et al., 2014). The C-terminal fragment ofBECN1 translocates to the mitochondria and contributes totriggering apoptosis similarly to the truncated fragment ofATG5 (Wirawan et al., 2010).Autophagy is intimately tied to the main signaling path-

ways controlling the balance of anabolic and catabolic cellularprocesses. Pathways that positively control cell growth andproliferation (e.g., PI3k/AKT and MAPK) usually activatemTORC1, thus suppressing autophagy. In contrast, stress-activated pathways (e.g., AMPK and p53) are related tomTORC1 inhibition and autophagy activation (Jung et al.,2010). Besides these autophagy modulators, mounting evi-dence has shown that ion channels and transporters also havethe ability to control autophagy. Although different ions areimplicated in the regulation of autophagy, calcium (Ca21) is byfar the most important.The concentration of Ca21 is precisely controlled in terms of

signal amplitude and spatiotemporal distribution. This tightregulation is essential for the communication of the extracel-lular milieu with different cellular compartments involved inCa21 homeostasis during processes such as cell cycle, pro-liferation, apoptosis, migration, and defense. In the plasmamembrane, Ca21 channels, including the voltage-gated Ca21

channel (VGCC) and the transient receptor potential (TRP)family of channels, allow the movement of Ca21 along itsconcentration gradient (Catterall, 2011). Intracellular Ca21

indirectly maintains autophagy at low levels in healthy cellsas a result of its central role in ATP production by mitochon-dria. Disturbances in Ca21 transfer from the ER to themitochondria promote an energetic imbalance that triggersautophagy. Furthermore, Ca21 appears to be fundamental forthe maintenance of acidic pH in lysosomes, which is crucial tothe proper autophagic flux and the degradative propertiesof the autophagolysosome (Fig. 1, box 4). In addition to basalroles in cell homeostasis, cytosolic Ca21 plays a complex partin autophagy induced by different stimuli. Moreover, depend-ing on its levels, the duration of the waves and the subcellulardistribution, Ca21 can have a dual impact on autophagy, asdiscussed in the next section (Decuypere et al., 2015).Ca21-related components (hereafter called Ca21 signal-

osome, which includes Ca21 channels from the ER, mitochon-dria and lysosomes, Ca21 channels in the plasma membrane,Ca21 buffering proteins, and Ca21-dependent proteins) medi-ate cellular homeostasis and survival through autophagicinduction in many physiologic contexts, which has been well

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Fig. 1. The mechanism of autophagy. Autophagy is modulated by several intracellular and extracellular signals. Insulin growth factor (IGF), insulin,and growth factors activate their tyrosine kinase (TK) receptors and trigger intracellular pathways that suppress autophagy. The activation ofmetabotropic receptors in the plasmamembrane can increase intracellular levels of inositol triphosphate (IP3) and trigger the release of Ca2+ by the ER,leading to autophagy through the CaMKK2-AMPK pathway. Damaged intracellular components or energetic imbalance activate autophagy through thep53 andAMPKpathway, respectively. mTORC1 is central in integrating all these signals tomodulate autophagy (box 1, arrows do not necessarilymean adirect link between the signals and mTORC1). Autophagy can also be triggered by ULK1 activation by AMPK (box 2). After the activation of autophagy,ATG proteins mediate the isolation of the precursor membrane and its expansion around cytosolic components. Some adaptors like the SQSTM1/p62protein can participate on this step. This process continues with the autophagosome formation, which involves the LC3 protein (box 3) and the ATG5-ATG12-ATG16L1 complex (represented by the pink circle in the main scheme). Finally, the autophagosome fuses with lysosomes (box 4) to form theautolysosome, where cellular components are degraded and recycled. In box 5, we summarize key information about the distribution of Ca2+ insubcellular components and the main receptors, channels, and other proteins involved in the control of Ca2+ concentration in each compartment.

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described elsewhere (Decuypere et al., 2011, 2015; Parys et al.,2012; Kondratskyi et al., 2013). Here we discuss the complexlink between Ca21 signalosome and autophagy, focusing onits impact on pathologic human conditions, including tumorformation, neurologic diseases, and infection.

Complex Link between Ca21 Signalosome and Autophagy

Intracellular Ca21 is storedmainly in the ER (∼0.4–0.8mM)but also in mitochondria (∼200 nM) and lysosomes (0.4–0.6 mM) (Christensen et al., 2002; Shigetomi et al., 2016). Itsflow from these compartments to the cytosol and vice versa istightly but dynamically controlled by key players of the Ca21

signalosome: the inositol 1,4,5-triphosphate receptor (IP3-R,also called ITPR) in the ER membrane, which is the mostimportant intracellular Ca21 release channel (Berridge,2009); the voltage-dependent anion channel 1 (VDAC1) andmitochondrial calcium uniporter regulator 1 (MCUR1) in theouter and inner mitochondria membranes, respectively(Williams et al., 2013a); and the receptors mucolipin 1/3(TRPML1/3) and transient receptor potential calcium channelmelastatin 2 and 3 (TRPM2/3) in the lysosome (Christensenet al., 2002). In addition, plasma membrane channels controlthe influx of Ca21 from the extracellular environment (Fig. 1,box 5). Through these molecular components, cells alterthe quantity and the activity of key components of Ca21

-dependent pathways to maintain cellular homeostasis inresponse to environmental or cellular alterations.The influence of Ca21 in both basal and induced autophagy

occurs through several mechanisms (Fig. 2) (Decuypere et al.,2011, 2015; Parys et al., 2012; Kondratskyi et al., 2013). TheIP3-R is the main effector of Ca21 signalosome that linksenvironmental signals to autophagy since its ligand, IP3, isincreased on exposure of cells to ATP, hypoxia, hormones,antibodies, growth factors, and neurotransmitters (Fig. 1)(Berridge, 2009). For instance, hypoxia induces an increase incytosolic Ca21 concentration ([Ca21]c) through phospholipaseC activation, IP3 increase, and Ca21 release through IP3-Rfrom the ER, followed by the activation of the CaMKK2-AMPK-mTOR pathway and autophagy. Inhibition of phos-pholipase C reduces the adaptive capacity of cells to survive tooxygen deprivation (Jin et al., 2016). Downstream of theCaMKK2-AMPK pathway, both canonical (which involvesthe core machinery of Atg proteins, as shown in Fig. 1) andnoncanonical (autophagy that occurs in the absence of somekey ATG proteins) autophagy pathways can be stimulated. Ofnote, deficiency of both BECN1 or ATG7 only partiallysuppressed autophagy induced by the increase of [Ca21]c(Høyer-Hansen et al., 2007). Supporting this, theMCF7 breastcancer cell line, which expresses undetectable levels of BECN1(Liang et al., 1999), triggers autophagy in response to theincrease of [Ca21]c through the activation of CaMKK2-AMPKindependent of BECN1, which suggests an alternative path-way downstream of AMPK (Høyer-Hansen et al., 2007) .The modulation of autophagy by Ca21 is involved in the

response of cells to several stressful conditions, such as inneurons during hypoxia (next section), as well as in tumor cellsduring the metabolic adaptation along the carcinogenesis andin immune cells responding to infections. However, thisinfluence varies depending on the context of autophagy (Fig.2). Ca21 can induce autophagy (Fig. 2, left, green box) asevidenced by the ability of xestospongin B, a specific inhibitor

of IP3-R, to block autophagy induced by starvation (Decuypereet al., 2011). Similarly, the suppression of Ca21 signalingwhen autophagy is activated leads to a reduction of autophagicflux, as observed after the treatment with rapamycin (RAPA),hypoxia, and proteasome inhibition (Williams et al., 2013b),suggesting Ca21 as an important second messenger involvedin autophagy induction. Indeed, under homeostatic condi-tions, autophagy is maintained at low levels, and the disrup-tion of certain cell mechanism, including alterations in thehomeostasis of Ca21, may trigger autophagy as an adaptiveresponse. On the other hand, Ca21 can inhibit autophagy (Fig.2, right, red box), as suggested by the increase in basalautophagy induced by xestospongin B (Decuypere et al.,2011), similar to the knockdown of IP3-R or the suppressionof IP3 formation in some cell models, including SK-N-SHhuman neural precursor cells, HeLa cells, DT40 B-cell lym-phoma chicken cells, and rat1 murine fibroblasts (Sarkaret al., 2005; Criollo et al., 2007; Vicencio et al., 2009; Cárdenaset al., 2010). This could be attributed to the impairment ofCa21 efflux from the ER to themitochondria and a subsequentreduction of ATP levels, which triggers autophagy through theAMPK-ULK1 pathway, in a mTOR-independent way. Thus,we can infer that the dual role played by Ca21 on autophagydepends on whether autophagy is at basal levels (in whichsuppression of Ca21 signaling increases autophagy) or in-duced (in which suppression of Ca21 signaling suppressesautophagy) (Decuypere et al., 2015) (Fig. 2).The control of Ca21 transfer and availability varies in

subcellular compartments, and directly interferes with auto-phagy (Gordon et al., 1993). The most important processrelated to this is the local transfer of Ca21 from the ER tothe mitochondria, which is finely controlled by local proteinsand is crucial for cell fate (Fig. 3, box 1). VDAC1, a Ca21

channel located at the mitochondrial outer membrane, allowsthe transfer of Ca21 from the ER to the intermembrane spacethrough a direct interaction with IP3-R. Subsequently, mito-chondrial Ca21 uptake 1 (MICU1) and MCUR1 transportCa21 from the intermembrane space to the lumen of mito-chondria (Mallilankaraman et al., 2012a; Williams et al.,2013a). Inside the organelle, Ca21 regulates the activity ofthree key dehydrogenases from the Krebs cycle, thus allowingnormal ATP production. It also regulates other mitochondrialprocesses, such as fatty-acid oxidation, amino-acid catabolism,F-ATPase activity, manganese superoxide dismutase, aspar-tate and glutamate carriers, and the adenine-nucleotidetranslocase (McCormack et al., 1990; Jouaville et al., 1999;Shoshan-Barmatz et al., 2010). Thus, compromising Ca21

transfer from the ER to the mitochondria leads to mitochon-drial malfunctioning, decreased ATP levels, AMPK activation,and autophagy. In addition, other alterations inmitochondrialCa21 signaling can lead to mitophagy (Cárdenas and Foskett,2012; Rimessi et al., 2013; Williams et al., 2013a), and Ca21

overload can induce cell death through the mitochondrialpathway (Qian et al., 1999) (Fig. 3, box 1, bottom). Together,these observations show that control of both the quantity ofCa21 in each subcellular components and the spatiotemporalflow of Ca21 through these compartments is fundamental todefine cell fate. An imbalance in these mechanisms, similarlyto disturbances in autophagy, may induce cell death. Indeed,cells have evolved mechanisms to avoid cell death caused byCa21 disturbances. Overexpression of cell death suppressorTMBIM6/BI-1 (Bax inhibitor 1, which is a Ca21 channel), for

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instance, in conditions of low [Ca21]ER, fundamentally con-tributes to Ca21 transfer from the ER to the mitochondria,acting as an ER Ca21-leak channel and a sensitizer of IP3-R(Kiviluoto et al., 2012; Bultynck et al., 2014). TMBIM6 alsoinduces autophagy to contribute to themetabolic adaptation ofthose cells with low [Ca21]ER and low ATP availability (Sanoet al., 2012).Another fundamental link between Ca21 and autophagy is

based on lysosomes, which have emerged as a novel Ca21

storage compartment that functionally crosstalks with theER in the spatiotemporal control of Ca21 (Lopez-Sanjurjoet al., 2013; Morgan et al., 2013). Lysosomes have NAADP-dependent two-pore channels (TPC), which allow the releaseof Ca21 in a NAADP-dependent way. This Ca21 can stimulateIP3-R, in a Ca21-induced Ca21 release. Another consequenceof TPC-mediated signaling is the alkalinization of lysosomes,which impairs the autophagosome-lysosome fusion and ham-pers the autophagic flux (Kilpatrick et al., 2013; Lu et al.,2013). Thus, disturbances in Ca21 efflux from IP3-R tolysosomes may also block the autophagic flux. Finally, Ca21

present in lysosomes is important not only to maintain thebasal autophagic flux but also to be altered by cells in contexts

of induced autophagy. During starvation, there is an increasein Ca21 release from the lysosomes, activating calcineurinthat leads to the nuclear accumulation of the transcriptionfactor TFEB. TFEB coordinates the transcription of genesinvolved in lysosomal biogenesis and autophagy, thus in-creasing the autophagic potential of starved cells (Medinaet al., 2015).During starvation, cells trigger a set of alterations, probably

to maintain Ca21 homeostasis and cell functioning. To avoid amassive release of Ca21 in response to the increase of IP3, cellsincrease the concentration of Ca21 buffering proteins in theER (Decuypere et al., 2011, 2015). Cells also activate c-JunN-terminal kinases (JNK), which phosphorylates Bcl-2 andreleases BECN1 to induce autophagy (Wei et al., 2008) (seeFig. 3, box 2, for more details). Similarly, RAPA increases[Ca21]c as a result of increased Ca21 efflux through the IP3-R,despite the decrease of Ca21 leakage from the ER; however,whether all the above-mentioned alterations are guided byautophagy, as well as the influence of autophagy on Ca21, isunclear (Decuypere et al., 2013). ATG7-deficient cells expandtheir ER Ca21 stores and increase [Ca21]ER, probably tocompensate the reduction of autophagy and restore the

Fig. 2. Crosstalk between Ca2+ signal-osome and autophagy. Ca2+ influencesautophagy induced by several contexts,including rapamycin treatment, hypoxia,extracellular ATP and starvation (greenbox). On the other hand, Ca2+ signalosomecomponents are involved in the mainte-nance of basal autophagy at low levelsthrough mechanisms that involve thecontrol of Ca2+ efflux from the ER (throughthe IP3-R channel), the role of Ca2+ inmitochondria and lysosomes, and the for-mation of the IP3-R/Bcl-2/BECN1 complex(red box). Letters indicate the experimen-tal evidences for each situation, as depictedon the bottom.

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autophagic flux (Jia et al., 2011). Instead, the knockdown ofATG5, which fully suppresses autophagy, does not inducethese changes, nor does it alter the increase of [Ca21]c inducedby extracellular ATP or ionomycin (Decuypere et al., 2013).Therefore, the role played by autophagy on the Ca21 signal-osome remains obscure, and additional data are necessary toallow any conclusion about this connection.

Autophagy, Ca21, and the Central Nervous System

The most dominant phenotypes of autophagy gene deletionare related to the nervous system (Komatsu et al., 2006), asexemplified by the development of progressive deficits inmotor function and accumulation of cytoplasmic inclusion

bodies in neurons fromATG5KOmice (Hara et al., 2006). It hasbeen proposed fromyeast studies that asymmetric divisions canconcentrate faulty organelles in one daughter cell destined todie, therefore constantly cleaning these organelles from cells ina proliferative population (Mogk and Bukau, 2014). Postmitoticcells, such as neurons, cannot rely on this mechanism andtherefore are much more dependent on autophagy for theircleanup. This is the basis for the role of autophagy in severalneurologic diseases (Ghavami et al., 2014). Particularly inage-related diseases, both macroautophagy and chaperone-mediated autophagy become less efficient with time, con-tributing to the gradual decline in cognitive performance(Martinez-Vicente, 2015). Additionally, alterations in pro-teins that target mitochondria to autophagic degradation,

Fig. 3. Molecular pathways linking auto-phagy and components of the Ca2+ sig-nalosome in cancer, neurology, andimmunity. In the light blue backgroundare shown the main pathways connectingextracellular and intracellular signalsthat modulate ion channels and/or ionflow through cellular components in can-cer. Alterations in [Ca2+]c can be inducedthrough the increase of ion entry from theextracellular environment or through therelease of Ca2+ from the ER. The increasein [Ca2+]c can activate the CaMKK2-AMPK pathway, leading to autophagy ina mTOR-dependent or independent way.The unfolded protein response (UPR) canalso trigger the release of Ca2+ and activa-tion of autophagy, including reticulophagy(REphagy). The light red backgroundshows the main pathways linking Ca2+

from synaptic NMDA receptors andVGCCs to signaling pathways that mod-ulate autophagy and themain functions ofautophagy in ischemia and neurodegen-eration. PC, preconditioning. In the yel-low background (bottom) are shown themain findings linking ions and ion chan-nels to autophagy in immunity. Box 1:Mechanism and channels involved in Ca2+

transfer from the ER to the mitochondria;at the bottom, the consequences of normaland altered Ca2+ transfer on cell fate areshown. Box 2: Mechanisms of IP3-R/Bcl-2/BECN1 complex functioning andmodulation.

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such as PINK and PARKIN, suggest the importance ofautophagy in keeping neurons healthy (Koyano et al.,2014).The most important signaling pathways that link Ca21 to

autophagy are the Ca21-CaMKK2-AMPK pathway and themTORC1 pathway. Synaptic, but not nonsynaptic, glutama-tergic receptors signal to mTORC1 through Ca21 entry viaVGCC (Lenz and Avruch, 2005) (Fig. 3). Accordingly, inhibi-tors of VGCC induce autophagy in PC12 pheochromocytomacells (Williams et al., 2008). Another pathway that links Ca21

to autophagy with a strong relevance in neurons involves theprotease calpain, which cleaves ATG5; the truncated ATG5translocates from the cytosol to the mitochondria, inhibitingautophagy and promoting apoptosis (Yousefi et al., 2006).Calpain inhibitors induce autophagy in PC12 cells and lead toA53T a-synuclein clearance, reducing the accumulation ofEGFP-HDQ71 aggregates in a zebrafish model of Huntingtondisease (Williams et al., 2008).a-Synuclein forms intracellular aggregates in neurons,

which is the pathologic hallmark of Parkinson’s disease(PD). The accumulation of a-synuclein likely occurs as a resultof the resistance of protein aggregates to autophagy (Cuervoet al., 2004); in a vicious cycle, mutated a-synuclein and post-translational modifications of the wild-type protein furtherimpair the autophagic pathway (Winslow et al., 2010). In aprocess that appears to be directly involved with the patho-genesis of PD, this leads to neuronal death. Thus, thecombined modulation of Ca21 signaling and autophagyemerges as a promising target to control the progression ofPD. In accordance with this hypothesis, recent findings haveshown that Ca21 homeostasis is altered in PD (Rivero-Rioset al., 2014; Schöndorf et al., 2014). Molecular effectors linkingCa21 and autophagy in PD have been increasingly described.Mutations in the leucine-rich repeat kinase-2 (LRRK2) genecause late-onset PD, whereas mutations in two genes classi-cally involved in mitophagy, PINK1 and Parkin, are linked tothe early onset form of PD (Klein and Westenberger, 2012;Grenier et al., 2013; Ashrafi et al., 2014). LRRK2 localizes tolysosomes and controls Ca21 release through a mechanismthat involves TPC andNAADP-dependent Ca21 channels. Thelatter is a receptor for NAADP, a potent Ca21 mobilizingsignal. The release of Ca21 from the lysosome then causesrelease of Ca21 from the ER to amplify cytosolic Ca21 signals,leading to autophagy (Gomez-Suaga et al., 2012).Mutations inLRRK2 in mouse cortical neurons lead to neurite shortening,reduced capacity of Ca21 buffering, mitochondria depolariza-tion, and Ca21 imbalance, causing mitophagy. Importantly,the inhibition of L-type Ca21 channels suppresses mitophagyand dendritic shortening (Cherra et al., 2013). Accordingly,mitochondrial dysfunction has been closely related to PDpathogenesis (Ryan et al., 2015). In addition to its role inautophagy, PINK1 decreases Ca21 uptake by mitochondria,leading to energetic imbalance and potentially to autophagythrough both the increase of AMP/ATP ratio and the increaseof reactive oxygen species. Since alterations in mitochondrialCa21 can trigger autophagy (Rimessi et al., 2013), PINK1-mutated neurons are more vulnerable to Ca21-induced celldeath, another potential cause of PD pathogenesis (Gandhiet al., 2009).In ischemia and preconditioning, the usual “good and bad”

status of autophagy applies clearly. The level and durationof autophagy seem to determine whether it plays a positive

or a negative role in neuronal survival after ischemia/re-perfusion (Sheng and Qin, 2015). In central nervous systemischemia, autophagy is crucial for the protective effects ofpreconditioning and is also protective in reperfusion (Yanet al., 2013; Zhang et al., 2013). Accordingly, high expressionof TSC1, reductions in mTORC1 activity and higher auto-phagy levels are responsible for the resistance of theneurons from the cornus ammonis area 3 of the hippocampusin relation to the cornus ammonis area 1 (Papadakis et al.,2013), further supporting the protective effects that con-trolled levels of autophagy have on neurons. On the otherhand, during severe ischemia, the deletion of ATG genes orthe pharmacologic blockage of autophagy is protective,indicating that excessive autophagy is involved in neuronaldeath (Sheng and Qin, 2015). Although most cells die withsigns of autophagy, in several situations, autophagy can bepart of the mechanism of death, and the drastic meta-bolic imbalance produced by ischemia seems to be one suchsituation.One potential mechanism for the different intensity and

duration of autophagy comes, indirectly, from studyingAMPK. In neonatal ischemia, only the initial increase inAMPK activity is independent of CaMKK2, whereas theprolonged CaMKK2-dependent activity of AMPK was delete-rious to neurons. Interestingly, the synaptotoxic effects of Aboligomer is also mediated by Ca21increase and the activationof CaMKK2 and AMPK. Unfortunately, despite the activationof AMPK being a well established activator of autophagy inmost cells, including neurons (Di Nardo et al., 2014), auto-phagy was not assessed in these studies (Mairet-Coello et al.,2013), and therefore its involvement can only be implied.These studies suggest that Ca21-mediated long-lasting acti-vation of CaMKK2, AMPK, and, probably, autophagy isneurotoxic, whereas short and less intense activation ofautophagy is neuroprotective.Taken together, these data position Ca21 increase as an

important modulator of autophagy in neurons. This ion seemsto play a role in the clearance of components to avoid or toretard neurodegenerative disease. Mutation in subunits of theVGCC increases LC3-II and SQSTM1/p62, indicating that areduction in the autophagy flux in mice and that mutations inthese genes are among the ones that lead to neurodegenera-tion in Drosophila (Tian et al., 2015). Thus, mild activationof autophagy represents a potential strategy to curb theseprogressive diseases (Ravikumar et al., 2004; Schaeffer et al.,2012).It is surprising to see that several studies thoroughly

evaluated signaling pathways such as mTOR and AMPK incontexts involving energy restriction or aging without evalu-ating the role of autophagy. It would be important to definethese fundamental questions: What is the proportion ofpathophysiologic alterations mentioned that affects auto-phagy? What is the importance of autophagy in the responseof cells, tissues, and organisms in these injuries and pathol-ogies? This will be fundamental for the understanding of therole autophagy plays in the different stages of neuropathicdiseases and to better design interventions to target auto-phagy to mitigate the progression of these diseases; but, giventhe risk of high and long-lasting autophagy to induce neuronalcell death, modulation of autophagy will have to be strictlyfine-tuned to make its modulation applicable to the treatmentof neurologic conditions.

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Autophagy, Ca21, and Cancer

The role of autophagy in cancer depends on the step of thecarcinogenesis. During cancer initiation, autophagy acts as achemopreventive mechanism, contributing to the mainte-nance of genome integrity and to the elimination of procarci-nogens. Animals lacking key ATG genes present an increasedincidence of spontaneous tumors, including hepatocellularcarcinoma, lung adenocarcinoma, and B cell lymphoma (Yueet al., 2003) (Zhi and Zhong, 2015). Corroborating this, theectopic expression of BECN1 in breast cancer cells lackingendogenous BECN1 gene restored the autophagic capacity ofthese cells and suppressed their tumorigenesis in vivo (Lianget al., 1999). In addition, autophagy also plays a role in theprogression of premalignant to malignant lesions, includingvery aggressive tumors like pancreatic cancer (Yang et al.,2011), breast cancer (Kim et al., 2011) and colorectal cancer(Burada et al., 2015). Loss of autophagy may favor both tumorinitiation and the transition to a metastatic and therapy-insensitive state. However, the autophagic capacity seems tobe restored by tumors after the acquisition of the malignantphenotype, then contributing to tumor progression (Galluzziet al., 2015). During tumor progression and resistance, auto-phagy acts predominantly as a tumor-supporting mechanism.It provides energetic substrates for metabolic adaptation,favoring tumor resistance to hypoxia and starvation, twocontexts in which Ca21-mediated autophagy is important todefine cell fate. Considering the response to therapy, auto-phagy has also been suggested as a key mechanism for tumorresistance; so the rational inhibition of autophagy emerges asan alternative to sensitize cancer cells to chemotherapeutics(Sui et al., 2013; Filippi-Chiela et al., 2015). Indeed, 28 clinicaltrials using the strategy of combining chemotherapeutics withinhibitors of autophagic flux, mainly cloroquine and hydroxy-cloroquine, are in progress for more than 15 tumor types(clinicaltrials.gov as of July 2016). Under specific conditions,however, autophagy can act as an oncosuppressive mecha-nism, contributing to the anticancer immunosurveillance andto the degradation of potentially oncogenic proteins (Galluzziet al., 2015). Therefore, it is fundamental to fully understandthe mechanisms underlying its modulation. In addition to theclassic pathways, Ca21 has been increasingly established asa key modulator of autophagy in tumor cells, which arefrequently exposed to nutrient deprivation, ER stress, hyp-oxia, metabolic stress, and environmental alterations. Allthese conditions induce autophagy that is at least partiallymediated by Ca21 (Monteith et al., 2007, 2012; Kondratskyiet al., 2013), as depicted in Fig. 3 and discussed in this section.Signaling that links the increase of [Ca21]c to autophagy

induction in cancer involves several pathways. In HeLa cells,both the Ca21 chelator BAPTA-AM and the IP3-R inhibitorxestopongin B suppress starvation-induced autophagy (Fig. 3)(Decuypere et al., 2011). Molecularly, this response involvesthe increase of Ca21-binding proteins concomitant with adecrease in ER Ca21-leak rate (Decuypere et al., 2011). Thesealterations occur through modulation of the IP3-R/Bcl-2/BECN1 complex (see Fig. 3, box 2), which controls the ERCa21 stores and autophagy (Vicencio et al., 2009). The bindingof Bcl-2 to IP3-R hampers the efflux of Ca21 from the ER(Pattingre et al., 2005; Høyer-Hansen et al., 2007), and theoverexpression of ER-targeted Bcl-2, but not Bcl-2 targetedto the mitochondria, stabilizes the complex and inhibits

autophagy that depends on Ca21 from the ER (Criollo et al.,2007). Corroborating this, the phosphorylation of Bcl-2 byJNK during starvation and after treatment with IP3-Rantagonists releases BECN1 and allows the initiation ofautophagy (Wang et al., 2008; Vicencio et al., 2009). Theknockdown of IP3-R also leads to an accumulation of auto-phagosomes in HeLa cells. In this case, the effect is not due tothe release of BECN1 from the complex with IP3-R since cellsdeficient in TGM2 (a regulator of IP3-R that inhibits IP3R-mediated Ca21 release and IP3R-mediated autophagy)showed increased IP3-R-mediated Ca21 signaling and in-creased autophagosome formation (Hamada et al., 2014).Finally, BECN1 is recruited by IP3-R during starvation andsensitizes IP3-R to low levels of IP3, allowing the release ofCa21 from the ER (Decuypere et al., 2011). Thus, we can inferthat, during starvation (and probably other stressful condi-tions), the release of Ca21 from the ER may contribute to agreater extent to the induction of autophagy than theformation of the BECN1 complex (see Fig. 1). Since autophagyis involved in tumor resistance, the modulation of Ca21

release from the ER has a potential to be tested as a targetmechanism to suppress autophagy and sensitize tumor cells totherapy.IP3-R is also involved in autophagy induced by extracellular

ATP, which binds to P2 purinergic receptors and triggers theformation of IP3 in breast cancer cells. Binding of IP3 to IP3-Rleads to Ca21 release from the ER, subsequent activation ofCaMKK2-AMPK, and autophagy, which is totally suppressedby BAPTA-AM (Høyer-Hansen et al., 2007). In cervix cancercells, in turn, the toxicity of extracellular ATP is mediatedmainly by the uptake of extracellular adenosine and AMPKactivation. In this model, autophagy plays a cytotoxic role andthe cotreatment with Ca21 chelator EGTA does not suppressATP-induced cell death (Mello et al., 2015). Together, thesedata suggest that the role played by Ca21 in the response toextracellular ATP may depend on the model of study. Themechanisms underlying the role played by Ca21 in the linkbetween extracellular ATP and autophagy require furtherinvestigation.The role of autophagy induced by the increase of [Ca21]c in

cancer relies on the genetic and epigenetic profiles of eachcancer type (Table 1). In breast cancer, the release of Ca21

from the ER triggers autophagy, which positively correlateswith the toxicity of ER stressors, such as ATP and thapsigar-gin. In these cells, autophagy is part of the toxicity induced bythese treatments (Høyer-Hansen et al., 2005, 2007). In colonand prostate cancer cells, autophagy triggered by the sameaforementionedER stressors or by theCa21 ionophoreA23187plays a protective role (Ding et al., 2007). This was indirectlycorroborated in HepG2 hepatocarcinoma cells, where theactivation of autophagy using RAPA protects cells from ERstress-induced apoptosis (Kapuy et al., 2014). In fibroblastsand in normal colon cells, however, autophagy induced by thesame ER stressors contributed to cell death (Ding et al., 2007).Finally, in renal carcinoma, the influx of Ca21 and zincthrough the TRPM3 channel in the plasma membrane, re-spectively, activates the CaMKK2-AMPK pathway and sup-presses the increase of miR-241, a microRNA that targetsLC3, thus stimulating autophagy. In this model, autophagycontributes to tumor growth (Hall et al., 2014). These variedoutcomes may be attributed to: 1) the presence or absence andthe levels of key proteins involved in the connection between

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TABLE

1Roleof

Ca2

+-m

odulated

autoph

agyin

canc

erThetablesu

mmarizes

themainfindings

connectingau

toph

agyan

dCa2

+in

cancer.

Themod

elof

study

,thetrea

tmen

tthat

causesCa2

+alteration

san

dau

toph

agy,

themod

ulation

ofCa2

+av

ailability,an

ditsconsequ

encesin

autoph

agyan

dthemod

ulation

ofau

toph

agyusedto

assess

therole

ofau

toph

agyaresh

own.

Cellan

dCan

cerTyp

eIn

ducerof

Ca2

+Im

balance

andAutoph

agy

Ca2

+Mod

ulation

andCon

sequ

encesto

Autoph

agy

Autoph

agyMod

ulation

aRolein

Autoph

agy

Referen

ce

MCF7brea

stcancer

Extracellular

ATP

(relea

seof

Ca2

+

from

theER

throug

hIP

3-R)

BAPTA/AM:redu

cesthe%

ofGFP-LC3+

cellsfrom

40%

to5%

3-MA

(35%

to,

5%)

Not

assessed

Høy

er-H

ansen

etal.,20

07BECN1KD

(35%

to5%

)ATG7KD

(35%

to12

%)

MCF7brea

stcanc

erIono

mycin

(Ca2

+iono

phore)

BAPTA/AM:redu

cesthe%

ofGFP-LC3+

cellsfrom

40%

to10

%3-MA

(35%

to,

5%)

Not

assessed

Høy

er-H

ansen

etal.,20

07BECN1KD

(35%

to15

%)

ATG7KD

(35%

to12

%)

MCF7brea

stcanc

erTha

psigargin(TG;inhibitor

ofER

Ca2

+-A

TPas

ewhich

maintains

high

leve

lsof

Ca2

+in

thecytosol)

BAPTA/AM:redu

cesthe%

ofGFP-LC3+

cellsfrom

52%

to5%

3-MA

(45%

to18

%)

Noas

sessed

Høy

er-H

ansen

etal.,20

07BECN1KD

(45%

to30

%)

ATG7KD

(45%

to28

%)

MCF7brea

stcanc

erVitam

inD

analog

EB10

89(increa

ses

cytosolicCa2

+,b

utthemecha

nism

isnot

fullykn

own)

Not

assessed

3-MA

(80%

to20

%)-de

crea

sedcell

death

Cytotox

icHøy

er-H

ansen

etal.,20

05Ove

rexp

ressionof

BECN1increa

sed

cellde

ath

Helacervix

aden

ocarcino

ma

Starvationusing

HBSS(increa

ses

cytosolicCa2

+from

theER

byIP

3-R;also

disrup

tsthe

IP3-R/Bcl-2/BECN1complex

)

BAPTA/AM

andxe

stospo

ngin

(IP3-R

inhibitor):redu

cesthe%

ofGFP-LC3-po

sitive

cellsan

dthe

LC3IIleve

lsto

controlleve

ls

Not

assessed

Not

assessed

Decuy

pere,20

11

Helacervix

aden

ocarcino

ma

Starvationusing

HBSS(increa

ses

cytosolicCa2

+from

theER

byIP

3-R;also

disrup

tsthe

IP3-R/Bcl-2/BECN1complex

)

Ove

rexp

ressionof

Bcl2:

redu

ced

arou

ndaha

lfthepe

rcen

tage

ofGFP-LC3-po

sitive

cells

Not

assessed

Not

assessed

bVicen

cio,

2009

HCT11

6coloncancer

ATP;

Not

assessed

ATG6KD

andATG8KD

-increa

sed

casp

aseactiva

tion

andap

optotic

cellde

athfrom

∼30

%to

47%

and

62%

resp

ective

ly;

Cytop

rotective

Ding,

2007

TG-indu

cedER

stress

3MA

also

increa

sedcellde

ath,

but

data

arenot

show

nA23

187-indu

cedER

stress

(Ca2

+ionop

hore)

DU14

5pr

ostate

cancer

ATP

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assessed

ATG6KD,ATG8KD

and3-MA

indu

cedcellde

ath,

butda

taareno

tsh

own.

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rotective

Ding,

2007

TG-ind

uced

ER

stress;

Obs

:theefficien

cyis

notsh

own

A23

187-indu

cedER

stress

(Ca2

+

ionop

hore)

Murineem

bryo

nic

fibrob

lasts

TG-ind

uced

ER

stress

Not

assessed

ATG5KD

decrea

sedcellde

ath

Cytotox

icDing,

2007

A23

187-indu

cedER

stress

CCD-18C

onormal

colon

TG-indu

cedER

stress

Not

assessed

3-MA

decrea

sedcellde

ath

Cytotox

icDing,

2007

A23

187-indu

cedER

stress

Hep

G2Hep

atocarcinom

aTG-ind

ucedER

stress

Not

assessed

Rap

amycin

andmetyrap

one(m

TOR

inhibitor):increa

sedau

toph

agy

andcellviab

ility

Cytop

rotective

(ind

irect)

Kap

uy,

2014

DT40

chicke

nlymph

oma

IP3-R

triple

knocko

ut(TKO)cells

Restoration

ofIP

3-R

expr

ession

decrea

sedba

salau

toph

agy

Rap

amycin:increa

sedau

toph

agyin

WT

butnot

inDT40

TKO

cells

Not

assessed

bKhan

and

Joseph

,20

10

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ndop

lasm

icreticu

lum;I

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sitol1,4,5-tripho

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KD,kn

ockd

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G,thap

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-MA,3-methy

lade

nin.

aThepe

rcen

tage

sin

thepa

rentheses

indicate

thepe

rcen

tage

ofau

toph

agy-po

sitive

cellsbe

fore

(first

value)

andafter(secon

dva

lue)

autoph

agyinhibition

.bDespite

notbe

ingas

sessed

,in

thesecontexts,

autoph

agyis

know

nto

becytopr

otective

(Lum

etal.,20

05;O

nod

eraan

dOhsu

mi,20

05).

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Ca21 and autophagy in each cellular context; 2) the differentsensitivity of distinct cells to modulators of Ca21 signaling;and 3) the multiple alterations (both related to the amountand function) of key proteins required for different cell fates incancer cells, such as autophagy and cell death. Indeed, severalcomponents of Ca21 pathways are central to determine thefate of cancer cells after different stimuli, depending on the celltype, the extent of the cell damage, and the injured cellularcomponent (Bernardi and Rasola, 2007; Harr and Distelhorst,2010). These data are clinically relevant since, at least in thesemodels, the modulation of Ca21-mediated autophagy sensi-tizes cancer cells to death. Importantly, the role played byCa21-mediated autophagy in cancer cells in comparison withtheir normal counterpart, described for colon tissue, suggeststhat the modulation of this mechanism may sensitize cancercells to die without affecting normal cells.Increase of [Ca21]c Can also Suppress Autophagy.

Amino acids induce a rise in intracellular Ca21 levels, whichtriggers the activation of mTORC1 and hVps34 through thedirect binding of Ca21/calmodulin to hVps34, which sup-presses autophagy (Gulati et al., 2008). The increase in[Ca21]c can also suppress autophagy through the activationof calpains and ATG5 cleavage (Yousefi et al., 2006). Calpainsare associated to cellular migration, cell survival, and apopto-sis resistance, thus making the Ca21-calpain system animportant oncogenic signal related to tumor progression(Storr et al., 2011). Finally, cells with nonfunctional IP3-R[DT40 triple-knockout (TKO) cells, from a chicken lymphoma]show lower basal mTORC1 activity and higher basal auto-phagic levels than DT40 cells in which IP3-R WT expressionwas restored exogenously. DT40 TKO cells also present adelayed apoptotic response, which could be due to increasedbasal autophagy (Cárdenas et al., 2010; Khan and Joseph,2010). The absence of IP3-R hampers the transfer of Ca21 fromthe ER to mitochondria; as a consequence, DT40 TKO cellshave 60% lower basal O2 consumption rate than WT cells anduse autophagy as a metabolic adaptation (Cárdenas et al.,2010). Corroborating this, the knockdown of MCUR1 reducedO2 consumption rate, activated AMPK, and induced auto-phagy (Mallilankaraman et al., 2012b); however, a keyquestion remains unsolved in this model. Considering thatCa21 may be fundamental to the maintenance of acidic pH inlysosomes, how cells with nonfunctional IP3-R maintain highenough levels of Ca21 in the lysosome to guarantee an intensebasal autophagic flux?Actually, some other questions related to the link between

Ca21 and autophagy in cancer remain unanswered, such as: 1)What is the role of Ca21-mediated autophagy in the responseof cancer cells to therapy? 2) Why does Ca21-mediated auto-phagy contribute to cell survival in some conditions and tocell death in others? 3) Are there noncanonical autophagypathways mediating the activation of autophagy by Ca21

in cancer? 4) Does the modulation of any component ofCa21 signalosome have a potential to modulate autophagyclinically?Two points deserve attention in these questions. The first is

related to the role of Ca21 for normal mitochondria function-ing and cell metabolism. Recent data suggest that cancer-resistant cells usually present both increased autophagy(Sui et al., 2013) and oxidative phosphorylation (Viale et al.,2014; Vellinga et al., 2015). Disturbances in mitochondrialCa21 may sensitize cells through energetic imbalance and

autophagy, therefore triggering even more autophagy. Thus,the modulation of both Ca21 dynamics in mitochondria (forinstance, suppressing VDAC1 or MCU1) in combination withautophagy inhibitors could be of therapeutic value in cancertherapy. In this sense, the modulation of Ca21 may alsointerfere with the autophagic flux. Thus, the manipulation ofCa21 availability for lysosomes and mitochondria, for in-stance, may directly affect the activity of these organellesand the autophagic flux, sensitizing some cancer cells to die.Indeed, several clinical trials in cancer have tested thecombination of chemotherapeutics with compounds that sup-press the fusion of autophagosome to lysosome to disrupt theautophagic flux.In conclusion, Ca21-mediated autophagy emerges as a

potential target to be modulated to sensitize tumor cells andcontrol tumor progression. Ca21 is involved in cytoprotectiveautophagy induced by starvation, hypoxia, ER stress, andincreased extracellular ATP, all features commonly present ingrowing solid tumors but not in healthy homeostatic tissues.Furthermore, cancer cells reprogram their metabolism, in-cluding changes in autophagy and oxidative phosporylation,both modulated by Ca21 (Ward and Thompson, 2012). Thesealterations seem to be involved in tumor progression andresistance so that the modulation of metabolism has beenproposed as a therapeutic target. In this sense, the inhibitionof Ca21 transfer from the ER to mitochondria in combinationwith autophagy inhibition may cause an energetic collapse,leading cancer cells to die, including those cells that takeadvantage of oxidative phosphorylation to resist to therapy(Viale et al., 2014; Vellinga et al., 2015). Thus, the modulationof Ca21 signaling in combination with chemotherapy couldspecifically suppress autophagy. Ultimately, the developmentof modulators of specific Ca21 signalosome components is apossibility that deserves to be further investigated.

Crosstalk between Ca21 and Autophagy in Infection andInflammation

Autophagy is known to control key aspects of innate andadaptive immunity in multicellular organisms. Apart from itsparticipation in the capture and elimination of pathogens,autophagy also takes part in the process of inflammatory andimmune responses. Notably, autophagy has been shown toinfluence the antigenic profile and the immunogenic release ofsignals in antigen-presenting cells; therefore, it impacts cellsurvival, proliferation, plasticity, and function of dendriticcells and T-lymphocytes. The main findings concerning theregulation of autophagy by Ca21 in the immune context areshown in Fig. 3 and detailed in the following paragraphs.A number of infection agents subvert host defenses to

survive and proliferate intracellularly. The selective removalof suchmicroorganisms by autophagy, also termed xenophagy,is thought to act downstream of the pattern recognitionreceptors, thereby facilitating effector responses that lead topathogen destruction. Xenophagy also modulates the functionof a range of inflammatory mediators at various levels,including cytokine production (Puleston and Simon, 2014).During urinary tract infection, caused by uropathogenic

Escherichia coli, autophagy determines the role of TRPcation channels. After uropathogenic E. coli strains get ac-cess to the host cytoplasm, they are targeted by LC3-II- andSQSTM1/p62-positive autophagosomes; however, they avoid

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degradation by neutralizing the autophagolysosomal pH,which severely compromises organelle function by disturbingbactericidal properties and ion homeostasis. The TRPML3channel on the lysosomal membrane seems to respond to pHchanges, mediating Ca21 efflux to the cytosol. The stimulationof TRPML3 spontaneously induces lysosome exocytosis, thusexpelling the pathogen from the cell in a nonlytic manner.Remarkably, knockdown of ATG5 or BECN1 from bladderepithelial cells significantly suppressed bacterial expulsion,which was also observed by pretreatment of infected cells withBAPTA-AM, suggesting a role for Ca21 in this mechanism. Onthe contrary, cells exposed toML-SI1, a TRPML antagonist, orknockdown for TRPML3, markedly increased intracellularbacterial load (Miao et al., 2015).Viral spreading can also be controlled by autophagy. The

detection of vesicular stomatitis virus, for instance, results intype 1 interferon production due to the ability of autophagy todeliver viral ligands to TLR7 in dendritic cells (Lee et al., 2007).Notably, an impressive strategy to manipulate autophagy isobserved during rotavirus infection, linked to severe gastroen-teritis. Basically, the viral ER-anchored glycoprotein NSP4, apore-forming viroporin, elicits the leakage of Ca21 from the ERto the cytosol. This ion mobilization, in turn, activates auto-phagy through the CaMKK2-AMPK pathway; CaMKK2inhibition by STO-609 abrogated LC3-II detection. The disrup-tion of ER-Ca21 homeostasis is only the primary function ofNSP4. Further, it potentiates the activation of the ER Ca21

sensor stromal interaction molecule 1 (STIM1), embedded inthe ER membrane. The STIM1 oligomer conformation contrib-utes to [Ca21]c increase by allowing Ca21 influx across theplasmamembrane through activation of Orai1 channels, a typeof Ca21-release-activated Ca21 (CRAC) channel (Hyser et al.,2013). Later, the rotavirus also interferes with autophagymembrane trafficking to transport viral ER-associated proteinsto sites of genome replication and particle assembly since lowerexpression of ATG3 and ATG5 highly reduce virus yield.Furthermore, the virus blocks autophagosomematuration onceNSP4/LC3-II structures were not shown to progress to auto-phagolysosomes (Crawford et al., 2012). The typical role ofmTOR as an autophagic inhibitor is challenged in response toLPS and cellular septic insult, where activation of mTOR isrequired for autophagy induction. In this context, regulation ofautophagy also involves CaMK1 and CaMK4, two malfunc-tional members of the CaMK family of proteins. In macro-phages, lipopolysaccharide (LPS) induces ER stress, resultingin Ca21 mobilization and CaMK1 activation. This signalingstimulates autophagy in a CaMKK2-AMPK-dependent path-way through mechanisms unrelated to mTORC1, establishingthat autophagy and mTORC1 activity are required simulta-neously in specific cellular contexts (Guo et al., 2013). Later, amore detailed characterization of the regulatory role of CaMKsduring autophagy was described, focusing in CaMK4. Isolatedmacrophages isolated from CaMK42/2 mice exhibit reducedlevels of ATG5/12, ATG7, and LC3B in response to LPS. Theproposedmechanism relies on the inhibition of GSK-3b activityand FBXW7 recruitment by CaMK4, which prevents theproteasomal degradation of mTOR, preserving mTORC1 activ-ity, thereby increasing autophagy (Zhang et al., 2014). Understimulation, CaMK42/2 and WT macrophages express similarlevels of mTOR mRNA, suggesting that mTOR regulation byCaMK4 occurs at a posttranscriptional level. Moreover,CaMK4-mTOR dependent autophagy is essential to IL-6

production by macrophages and adaptive responses to cyto-protection in renal tubular cells during inflammatory state.OnTCR stimulation, in T-lymphocytes, theCa21 response is

initiated by efflux of Ca21 from ER stores, ultimately activat-ing CRAC channels on the plasmamembrane, which promotesextracellular Ca21 influx. In ATG7-deficient T-cells, however,the ER compartment is abnormally expanded and intracellu-lar Ca21 stores are increased, probably owing to impaired ERCa21 depletion and failed redistribution of STIM-1 in theER-plasma membrane junctions. Treatment with thapsigar-gin rescues the defective Ca21 influx in autophagy-deficientT cells. These results clearly demonstrate that autophagyregulates both the ER homeostasis and the calcium mobiliza-tion, which are interrelated events (Jia et al., 2011).More than an infection-reacting mechanism, inflammation

also responds to the loss of cellular homeostasis caused bytrauma, ischemia, or chemical injury. All these autophagicstress-inducers regulate signaling pathways that are involvedin cell metabolism, tissue remodeling, and repair. This raisesthe question of whether Ca21-modulated autophagy is alsoinvolved in these responses. In this scenario, a more detailedstudy relating Ca21 signalosome, phagocyte recruitment, andcytokine production and secretion would be of great interestfor therapeutic improvements. Another aspect that remainsblurred is the crosstalk between autophagy and immunity inthe context of other human diseases, including neurodegen-erative disorders and cancer.

DiscussionCa21 plays a key role in the maintenance of basal auto-

phagy, as well as in induced autophagy. Ca21-mediatedautophagy is involved in the pathogenesis and progression ofhuman diseases, and the modulation of Ca21 signalosomecomponents present a great potential for use in therapeuticinterventions for both therapy and prophylaxis.In neurodegenerative diseases, some potential targets in-

clude the modulation of calpain signaling and the reestablish-ment of Ca21 homeostasis, including the control of L-typechannels and Ca21 release from lysosomes. This increases thedegradation of a-synuclein in Huntington and PD patientsand controls neuronal cell death. Also, the suppression ofautophagy during the more severe phase of ischemia maycontribute to reduce the neuronal damage. In cancer, thesuppression of Ca21 release from the ER, as well as thesuppression of the influx of Ca21 to the mitochondria, maycontribute to sensitize cancer cells to therapy, especially in themetabolic adaptation that follows chemotherapy. In addition,the modulation of lysosome Ca21 signaling emerges as analternative to block the autophagic flux to sensitize cancercells to die. In immunity, the ability to pharmacologicallymodulate members of the Ca12 signalosome, which werestrategically manipulated by pathogens along host-parasitecoevolution, represents a promising therapeutic target. Theuse of CaMKK2 modulators, such as STO-609 or receptoragonists, in specific points of the infection cycle might be analternative to be used in combination with broadly usedmicrobicidal agents.Although some aspects of the modulation of autophagy by

Ca21 remain unknown, it is clear that themodulation dependson the spatiotemporal distribution of Ca21, as well as on theautophagy inducer and on the context in which Ca21 and

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autophagy are modulated; however, the signaling pathwaysconnecting them and probably the role played by key compo-nents that interfere in cell outcome after Ca21-mediated auto-phagy need to be further investigated. This may includeproteins that interact with components of Ca21 signalosome,such as IP3-R in the ER, VDAC1 in the mitochondria, andTRPML1/3 in the lysosome, as well as proteins that modulatethe CaMKK2-AMPK-mTOR-ULK1 pathway and the activity ofCa21 channels in the plasma membrane. It is expected that, inthe coming years, some of these mechanisms will be revealed;and, as hypothesized by Kondratskyi et al. (2013), the modu-lation of some components of Ca21 signalosome that influenceautophagy will be therapeutically tested in different humanpathologies. Notwithstanding, it is important to share acautionary note. Some methods used to access the role ofCa21 in autophagy such as Ca21 ionophores, thapsigargin-induced depletion of ER Ca21 stores, and the Ca21 chelatorBAPTA may induce cell stress at high concentrations, and theautophagy that follows may be activated by this stress ratherthan by a direct signaling involving Ca21 (Decuypere et al.,2011, 2015; Cárdenas and Foskett, 2012). Thus, some conclu-sions based on in vitro studiesmust be interpretedwith caution.In conclusion, the study of the link between Ca21 and

autophagy, two finely controlled and biologic relevant sys-tems, has a great potential to contribute to our understandingof the cause of neural or inflammatory diseases as well ascancer. In addition, Ca21-modulated autophagy represents anopportunity for the development of new or adjuvant thera-peutic strategies to control, prevent, or treat illnesses associ-ated to these systems.

Acknowledgments

The authors thankAlexandraVigna and Pitia F. Ledur for criticallyreading the manuscript. ASPET thanks Dr. Katie Strong for copy-editing of this article.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Filippi-Chiela, Viegas, Thomé, Buffon, Wink, Lenz.

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