TITLE PAGE Modulation of Autophagy by Calcium Signalosome in Human

Molecular Pharmacology Fast Forward. Published on July 19, 2016 as DOI: 10.1124/mol.116.105171 This article has not been copyedited and formatted. The final version may differ from this version. MOL #105171

TITLE PAGE

Modulation of Autophagy by Calcium Signalosome in Human Disease

Authors:

Eduardo Cremonese Filippi-Chiela, Michelle S. Viegas, Marcos Paulo Thomé, Andreia Buffon, Marcia R. Wink, Guido Lenz

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ECFC: Graduate Program in Hepathology and Gastroenterology, Faculty of Medicine, Hospital de Clínicas de Porto Alegre, RS, Brazil;

MSV: Gene Therapy Center, Hospital de Clínicas de Porto Alegre, RS, Brazil; molpharm.aspetjournals.org MPT: Department of Biophysics and Center of Biotechnology, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil;

MW: Laboratory of Biochemical and Cytological Analysis, Faculty of Pharmacy, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil;

AB: Department of Health Sciences and Cell Biology Laboratory, Federal University of

Health Sciences of Porto Alegre; at ASPET Journals on September 27, 2021

GL: Department of Biophysics and Center of Biotechnology, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.

RUNNING TITLE PAGE

Running Title:

Autophagy and Ca2+ in human disease

Corresponding Author: Name: Guido Lenz Address: Universidade Federal do Rio Grande do Sul, Instituto de Biociências, Departamento de Biofísica. Av. Bento Gonçalves, 9500, Bairro Agronomia, CEP: 91501-970, Porto Alegre, RS, Brasil. Downloaded from Telephone: (51) 33087613 Fax: (51) 33087003 E-mail: [email protected]

molpharm.aspetjournals.org Document Statistics: Number of text pages: 43 Number of tables: 1 Number of figures: 3 Number of references: 108 Number of words in the Abstract: 191 Number of words in the Introduction: 1196 Number of words in the Discussion: 550 at ASPET Journals on September 27, 2021

Abbreviation List:

2+ 2+ [Ca ]c, Cytosolic Ca concentration 2+ 2+ [Ca ]ER, Cytosolic Ca concentration AMPK, 5' AMP-activated Protein Kinase BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-tetraacetic acid BECN1, Beclin 1 protein CAMKK2, Calcium/calmodulin-Dependent Protein Kinase Kinase 2, beta CAMK4, Calcium/calmodulin-Dependent Protein Kinase IV CRAC, Ca2+-release-activated Ca2+ channel ER, Endoplasmic Reticulum IP3-R, Inositol 1,4,5-triphosphate Receptor LPS, Lipopolysaccharide MAP1LC3A or LC3, Microtubule-Associated Protein 1A/1B-Light Chain 3 MCUR1, Mitochondrial Calcium Uniporter Regulator 1 MICU1, Mitochondrial Ca2+ uptake 1 NAADP, Nicotinic Acid Adenine Dinucleotide Phosphate RAPA, Rapamycin SQSTM1/p62, Sequestosome 1 protein TKO, Triple knockout TPC, Two Pore Channels TRPM2/3, Transient Receptor Potential Calcium Channel Melastatin 2 and 3 TRPML1/3, Mucolipin 1 and 3 VGCC, Voltage-Gated Calcium Channels

ABSTRACT

Autophagy is a catabolic process largely regulated by extra- and intracellular signaling pathways that are central to cellular metabolism and growth.

Mounting evidence has shown that ion channels and transporters are important for basal autophagy functioning and influence autophagy as a means to handle stressful

2+ situations. Besides its role in cell proliferation and apoptosis, intracellular Ca is widely Downloaded from recognized as a key regulator of autophagy, acting through the modulation of pathways such as mTORC1, CaMKK2 and Protein Kinase C. Proper spatio-temporal

Ca2+ availability coupled with a controlled ionic flow among the extracellular milieu, molpharm.aspetjournals.org storage compartments and the cytosol are critical to determine the role played by Ca2+ on autophagy and on cell fate. The crosstalk between Ca2+ and autophagy has a central role in cellular homeostasis and survival during several physiological and pathological conditions. Here we review the main findings concerning the mechanisms at ASPET Journals on September 27, 2021 and roles of Ca2+-modulated autophagy, focusing on human disorders ranging from cancer to neurological diseases and immunity. By identifying mechanisms, players and pathways that either induce or suppress autophagy, new promising approaches for preventing and treating human disorders emerge, including those based on the modulation of Ca2+-mediated autophagy.

INTRODUCTION

Basic Mechanisms of Autophagy Modulation

Autophagy is an evolutionary conserved catabolic process by which cells degrade and recycle self-components to maintain cellular homeostasis. By targeting intracellular substrates, this lysosomal degradative pathway generates a pool of essential molecules required for several cellular functions (Mizushima et al., 2008). Downloaded from Although nutritional stress is considered the classic trigger of autophagy (Lum et al.,

2005; Onodera and Ohsumi, 2005), growth factor availability, hypoxia, aggregated proteins, injured organelles, DNA damage and infection can also initiate an autophagic molpharm.aspetjournals.org response (Choi et al., 2013; Filippi-Chiela et al., 2015; Galluzzi et al., 2015). Because autophagy works as a protective and quality control mechanism, its dysfunction has been implicated in apoptotic cell death and in the onset of several human pathologies (Jiang and Mizushima, 2014). Additionally, excessive autophagy was at ASPET Journals on September 27, 2021 suggested to directly drive cell killing if other cell death mechanisms are impaired (Liu and Levine, 2015).

Autophagy is classified as macroautophagy, microautophagy and chaperone- mediated autophagy according to the different ways that cytosolic contents are delivered to lysosomes. In macroautophagy (hereafter referred to as autophagy), the cytosolic cargo is captured and transported to lysosomes through a double membrane organelle called autophagosome (Fig. 1). In the other types, lysosomal membrane receptors directly mediate cargo internalization (Boya et al., 2013).

Extra- and intracellular signals that modulate autophagy act on the activity of mTOR Complex 1 (mTORC1; a suppressor of autophagy) or AMP-Activated Protein

Kinase (AMPK; an activator of autophagy), as summarized in Fig. 1 - Box 1 and 3.

CAMKK2, which is activated by the Ca2+-calmodulin complex, is the main protein involved in linking Ca2+ to energetic balance and glucose homeostasis. The most

important target of CAMKK2 for metabolism modulation is the AMPK protein. Once activated, AMPK directly alters cell metabolism to replenish cellular ATP levels, acting on proteins involved in fatty acid oxidation and autophagy. One of the most important targets of AMPK is mTORC1 (Green et al., 2011; Hurley et al., 2005). This pathway, which involves several members of the autophagy-related (ATG) family of proteins, modulates the machinery that executes autophagosome formation. These proteins are activated in a coordinated fashion through post-translational modifications and the Downloaded from formation of complexes (Feng et al., 2014). Curiously, additional non-autophagic functions have been attributed to ATG proteins, including phagosome maturation, modulation of intracellular transport, apoptosis and exocytosis (Subramani and molpharm.aspetjournals.org Malhotra, 2013). The protein complex that initiates autophagy involves the ATG1 protein (also called as ULK1), which is modulated by mTORC1 and AMPK with opposite effects (Egan et al., 2011; Kim et al., 2011; Loffler et al., 2011). Noteworthy,

AMPK not only directly phosphorylates ULK1, but also suppresses mTORC1 (Kim et al., 2011; Mao and Klionsky, 2011). Activated ULK1 complex together with Vps34 at ASPET Journals on September 27, 2021 complex induce the phagophore isolation. Autophagosome completion is further controlled by the Atg12-Atg5-Atg16L complex, which drives the membrane expansion, and the LC3 protein conjugated to phosphatidylethanolamine, forming the

LC3-II, which is involved in cargo targeting, membrane closure and autophagosome maturation (Fig. 1, Box 3) (Galluzzi et al., 2015; Hara et al., 2008; He and Klionsky,

2009; Itakura et al., 2008). Autophagy selectivity is dictated by the ubiquitination of distinct targets, which are recognized by autophagy adapters like SQSTM1/p62 and

Nbr1, which mediate cargo binding to LC3-II attached to autophagosome membranes (Russell et al., 2014). Selective autophagy for organelles degradation, such as mitophagy (mitochondria) or reticulophagy (ER), is critical for maintaining proper cellular function and for preventing the accumulation of dysfunctional or excessive cellular components. Indeed, cells that are defective to

autophagy or that are induced to suppress autophagy can have their homeostasis disturbed and be sensitized to apoptosis (Fimia et al., 2013).

A single signal can induce both autophagy and apoptosis in the same cell.

Generally, autophagy suppresses apoptosis by influencing the response to infection and the recognition of dead cells, the capacity of tumor cells to adapt to metabolic stress and respond to therapy, the sensitivity of neurons to hypoxia, and the toxicity of intracellular protein aggregates. The best described mechanism in the autophagy- Downloaded from apoptosis crosstalk involves the interaction between the autophagic protein Beclin1

(BECN1) and anti-apoptotic proteins from the BH3 family, including Bcl-2, Mcl-1 and

Bcl-XL. This interaction blocks the role of BECN1 in autophagy but does not alter the molpharm.aspetjournals.org function of the anti-apoptotic proteins (Kang et al., 2011; Pattingre et al., 2005).

Similarly, ATG12 interacts with Mcl-1 and Bcl-2, leading to the suppression of the antiapoptotic activity of Mcl-1 and Bcl-2, promoting apoptosis (Rubinstein et al., 2011).

Another mechanism underlying this crosstalk involves the cleavage of the autophagic protein ATG5 by calpain, leading to reduced autophagy and increased apoptosis by at ASPET Journals on September 27, 2021 targeting the truncated ATG5 protein to the mitochondria (Yousefi et al., 2006). Finally, active caspases cleave several autophagic proteins, such as p62, ATG3, ATG5 and

BECN1, thus reducing cytoprotective autophagy and favoring apoptosis (Marino et al.,

2014). The C-terminal fragment of BECN1 translocates to the mitochondria and contributes to triggering apoptosis similarly to the truncated fragment of ATG5

(Wirawan et al., 2010).

Autophagy is intimately tied to the main signaling pathways controlling the balance of anabolic and catabolic cellular processes. Pathways that positively control cell growth and proliferation (e.g. PI3k/AKT and MAPK) usually activate mTORC1, thus suppressing autophagy. In contrast, stress-activated pathways (e.g. AMPK and p53) are related to mTORC1 inhibition and autophagy activation (Jung et al., 2010). Besides these autophagy modulators, mounting evidence has shown that ion channels and

transporters also have the ability to control autophagy. Although different ions are implicated in the regulation of autophagy, calcium (Ca2+) is by far the most important.

The concentration of Ca2+ is precisely controlled in terms of signal amplitude and temporal-spatial requirements. This tight regulation is essential for the communication of the extracellular milieu with different cellular compartments involved in Ca2+ homeostasis during processes such as cell cycle, proliferation, apoptosis, migration and defense. In the plasma membrane, Ca2+ channels including the voltage-gated Downloaded from Ca2+channel (VGCC) and Transient Receptor Potential (TRP) family of channels, allow the movement of Ca2+ along its concentration gradient (Catterall, 2011). Intracellular

Ca2+ indirectly maintains autophagy at low levels in healthy cells, due to its central role molpharm.aspetjournals.org in ATP production by mitochondria. Disturbances in Ca2+ transfer from the ER to the mitochondria promote an energetic imbalance that triggers autophagy. Furthermore,

Ca2+ appears to be fundamental for the maintenance of acidic pH in lysosomes, which is crucial to the proper autophagic flux and the degradative properties of the autophagolysosome (Fig. 1 - Box 4). In addition to basal roles in cell homeostasis, at ASPET Journals on September 27, 2021 cytosolic Ca2+ plays a complex part in autophagy induced by different stimuli.

Moreover, depending on its levels, the duration of the waves and the subcellular distribution, Ca2+ can have a dual impact on autophagy, as discussed in the next section (Decuypere et al., 2015).

Ca2+-related components (hereafter called Ca2+ signalosome, which includes

Ca2+ channels from the ER, mitochondria and lysosomes, Ca2+ channels in the plasma membrane, Ca2+ buffering proteins and Ca2+-dependent proteins) mediate cellular homeostasis and survival through autophagic induction in many physiological contexts, which has been well-described elsewhere (Decuypere et al., 2015; Decuypere et al.,

2011; Kondratskyi et al., 2013; Parys et al., 2012). Here we discuss the complex link between Ca2+ signalosome and autophagy, focusing on its impact on pathological human conditions, including tumor formation, neurological diseases and infection.

The complex link between Ca2+ signalosome and autophagy

Intracellular Ca2+ is stored mainly in the ER (~0.4-0.8 mM), but also in mitochondria (~200 nM) and lysosomes (0.4-0.6 mM) (Christensen et al., 2002;

Shigetomi et al., 2016). Its flow from these compartments to the cytosol and vice versa is tightly but dynamically controlled by key players of the Ca2+ signalosome: the Inositol

1,4,5-triphosphate Receptor (IP3-R, also called as ITPR) in the ER membrane, which is the most important intracellular Ca2+ release channel (Berridge, 2009); the Voltage- Downloaded from Dependent Anion Channel 1 (VDAC1) and Mitochondrial Calcium Uniporter Regulator

1 (MCUR1) in the outer and inner mitochondria membranes, respectively (Williams et al., 2013a); and the receptors Mucolipin 1/3 (TRPML1/3) and Transient Receptor molpharm.aspetjournals.org

Potential Calcium Channel Melastatin 2 and 3 (TRPM2) in the lysosome (Christensen et al., 2002). In addition, plasma membrane channels control the influx of Ca2+ from the extracellular environment (Fig. 1 - Box 5). Through these molecular components, cells alter the quantity and the activity of key components of Ca2+-dependent pathways to at ASPET Journals on September 27, 2021 maintain cellular homeostasis in response to environmental or cellular alterations.

The influence of Ca2+ in both basal and induced autophagy occurs through several mechanisms (Fig. 2) (Decuypere et al., 2015; Decuypere et al., 2011;

Kondratskyi et al., 2013; Parys et al., 2012). The IP3-R is the main effector of Ca2+ signalosome that links environmental signals to autophagy since its ligand, IP3, is increased upon exposure of cells to ATP, hypoxia, hormones, antibodies, growth factors and neurotransmitters (Fig. 1) (Berridge, 2009). For instance, hypoxia induces

2+ 2+ an increase in cytosoic Ca concentration ([Ca ]c) through phospholipase C activation, IP3 increase and Ca2+ release through IP3-R from the ER, followed by the activation of the CAMKK2-AMPK-mTOR pathway and autophagy. Inhibition of phospholipase C reduces the adaptive capacity of cells to survive to oxygen deprivation (Jin et al., 2016). Downstream to the CAMKK2-AMPK pathway, both canonical (which involves the core machinary of Atg proteins, as shown in Fig. 1) and

non-canonical (autophagy that occurs in the absence of some key ATG proteins) pathways can be stimulated. Of note, deficiency of both BECN1 or ATG7 only partially

2+ suppressed autophagy induced by the increase of [Ca ]c (Hoyer-Hansen et al., 2007).

Supporting this, MCF7 breast cancer cell line, which express undetectable levels of

2+ BECN1 (Liang et al., 1999), triggered autophagy in response to the increase of [Ca ]c through the activation of CAMKK2-AMPK independent of BECN1, which suggests an alternative pathway downstream of AMPK (Hoyer-Hansen et al., 2007) . Downloaded from The modulation of autophagy by Ca2+ is involved in the response of cells to several stressful conditions, such as in neurons during hypoxia (next section), as well as in tumor cells during the metabolic adaptation along the carcinogenesis and in molpharm.aspetjournals.org immune cells responding to infections. However, this influence varies depending on the context of autophagy (Fig. 2). Ca2+ can induce autophagy (Fig. 2 – left, green box) as evidenced by the ability of Xestospongin B, a specific inhibitor of IP3-R, to block autophagy induced by starvation (Decuypere et al., 2011). Similarly, the suppression of at ASPET Journals on September 27, 2021 Ca2+ signaling when autophagy is activated leads to a reduction of autophagic flux, such as is observed after the treatment with rapamycin (RAPA), hypoxia and proteasome inhibition (Williams et al., 2013b), suggesting Ca2+ as an important second messenger involved in autophagy induction. Indeed, under homeostatic conditions, autophagy is maintained at low levels and the disruption of certain cell mechanism, including Ca2+ alterations in the homeostasis of Ca2+, may trigger autophagy as an adaptive response. On the other hand, Ca2+ can inhibit autophagy (Fig. 2 – right, red box), as suggested by the increase in autophagy induced by Xestospongin B

(Decuypere et al., 2011), similar to the knockdown of IP3-R or the suppression of IP3 formation in some cell models, including SK-N-SH human neural precursor cells, HeLa cells, DT40 B-cell lymphoma chicken cells and Rat1 murine fibroblasts (Cardenas et al., 2010; Criollo et al., 2007; Sarkar et al., 2005; Vicencio et al., 2009). This could be attributed to the impairment of Ca2+ efflux from the ER to the mitochondria and a

subsequent reduction of ATP levels, which triggers autophagy through the AMPK-

ULK1 pathway, in a mTOR-independent way. Thus, we can infer that the dual role played by Ca2+ on autophagy depends on whether autophagy is at basal levels (in which suppression of Ca2+ signaling increases autophagy) or induced (in which suppression of Ca2+ signaling supresses autophagy) (Decuypere et al., 2015) (Fig. 2).

The control of Ca2+ transfer and availability varies in subcellular compartments, and directly interferes with autophagy. (Gordon et al., 1993). The most important Downloaded from process related to this is the local transfer of Ca2+ from the ER to the mitochondria, which is finely controlled by local proteins and crucial for cell fate (Fig. 3 - Box 1).

VDAC1, a Ca2+ channel located at the mitochondrial outer membrane, allows the molpharm.aspetjournals.org transfer of Ca2+ from the ER to the intermembrane space through a direct interaction with IP3-R. Subsequently, Mitochondrial Ca2+ uptake 1 (MICU1) and MCUR1 transport

Ca2+ from the intermembrane space to the lumen of mitochondria (Mallilankaraman et al., 2013; Williams et al., 2013a). Inside the organelle, Ca2+ regulates the activity of at ASPET Journals on September 27, 2021 three key dehydrogenases from the Krebs cycle, thus allowing normal ATP production.

It also regulates other mitochondrial processes, such as fatty-acid oxidation, amino- acid catabolism, F-ATPase activity, manganese superoxide dismutase, aspartate and glutamate carriers and the adenine-nucleotide translocase (Jouaville et al., 1999;

McCormack et al., 1990; Shoshan-Barmatz et al., 2010). Thus, compromising Ca2+ transfer from the ER to the mitochondria leads to mitochondrial malfunctioning, decreased ATP levels, AMPK activation and autophagy. In addition, other alterations in mitochondrial Ca2+ signaling can lead to mitophagy (Cardenas and Foskett, 2012;

Rimessi et al., 2013; Williams et al., 2013a), while Ca2+ overload can induce cell death through the mitochondrial pathway (Qian et al., 1999) (Fig 3 - Box 1, bottom).

Altogether these observations show that the control of both the quantity of Ca2+ in each subcellular components and the spatio-temporal flow of Ca2+ through these compartments is fundamental to define cell fate. An imbalance in these mechanisms,

similarly to disturbances in autophagy, may induce cell death. Indeed, cells have evolved mechanisms to avoid cell death caused by Ca2+ disturbances. The overexpression of cell death suppressor TMBIM6/BI-1 (Bax Inhibitor 1, which is a Ca2+

2+ 2+ channel), for instance, in conditions of low [Ca ]ER, fundamentally contributes to Ca transfer from the ER to the mitochondria, acting as an ER Ca2+-leak channel and a sensitizer of IP3-R (Bultynck et al., 2014; Kiviluoto et al., 2012). TMBIM6 also induces

2+ autophagy to contribute to the metabolic adaptation of those cells with low [Ca ]ER and Downloaded from low ATP availability (Sano et al., 2012).

Another fundamental link between Ca2+ and autophagy is based on lysosomes, which have emerged as a novel Ca2+ storage compartment that functionally crosstalks molpharm.aspetjournals.org with the ER in the spatio-temporal control of Ca2+ (Lopez-Sanjurjo et al., 2013; Morgan et al., 2013). Lysosomes have NAADP-dependent two pore channels (TPCNs), which allow the release of Ca2+ in a NAADP-dependent way. This Ca2+ can stimulate IP3-R, in a Ca2+-induced Ca2+ release. Another consequence of TPNC-mediated signaling is at ASPET Journals on September 27, 2021 the alkalinization of lysosomes, which impairs the autophagosome-lysosome fusion and hampers the autophagic flux (Kilpatrick et al., 2013; Lu et al., 2013). Thus, disturbances in Ca2+ efflux from IP3-R to lysosomes may also block the autophagic flux. Finally, Ca2+ present in lysosomes is important not only to maintain the basal autophagic flux, but also to be altered by cells in contexts of induced autophagy. During starvation there is an increase of Ca2+ release from the lysosomes, activating calcineurin that leads to the nuclear accumulation of the transcription factor TFEB.

TFEB coordinates the transcription of genes involved in lysosomal biogenesis and autophagy, thus increasing the autophagic potential of starved cells (Medina et al.,

2015).

During starvation, cells trigger a set of alterations probably to maintain Ca2+ homeostasis and cell functioning. In order to avoid a massive release of Ca2+ in response to the increase of IP3, cells increase the concentration of Ca2+ buffering

proteins in the ER (Decuypere et al., 2015; Decuypere et al., 2011). Cells also activate

JNK, which phosphorylates Bcl-2 and releases BECN1 to induce autophagy (Wei et al.,

2+ 2008) (see Fig. 3 – Box 2 for more details). Similarly, RAPA increases [Ca ]c due to increased Ca2+ efflux through the IP3-R, despite the decrease of Ca2+ leakage from the

ER. However, whether all above-mentioned alterations are guided by autophagy as well as the influence of autophagy on Ca2+ are not clear (Decuypere et al., 2013).

2+ 2+ ATG7-deficient cells expand their ER Ca stores and increase [Ca ]ER, probably to Downloaded from compensate the reduction of autophagy and restore the autophagic flux (Jia et al.,

2011). Instead, the knockdown of ATG5, which fully suppresses autophagy, does not

2+ induce these changes nor does it alter the increase of [Ca ]c induced by extracellular molpharm.aspetjournals.org ATP or ionomycin (Decuypere et al., 2013). Therefore, the role played by autophagy on the Ca2+ signalosome remains obscure, and additional data is necessary to allow any conclusion about this connection.

at ASPET Journals on September 27, 2021 Autophagy, Ca2+ and the Central Nervous System

The most dominant phenotypes of autophagy gene deletion are related to the nervous system (Komatsu et al., 2006), as exemplified by the development of progressive deficits in motor function and accumulation of cytoplasmic inclusion bodies in neurons from ATG5 KO mice (Hara et al., 2006). It has been proposed from yeast studies that asymmetric divisions can concentrate faulty organelles in one daughter cell destined to die, therefore constantly cleaning these organelles from cells in a proliferative population (Mogk and Bukau, 2014). Post-mitotic cells, such as neurons cannot rely on this mechanism and therefore are much more dependent on autophagy for their cleanup. This is the basis for the role of autophagy in several neurological diseases (Ghavami et al., 2014). Particularly in age related diseases, both macroautophagy and chaperone-mediated autophagy become less efficient with time, contributing to the gradual decline in cognitive performance (Martinez-Vicente, 2015).

Additionally, alterations in proteins that target mitochondria to autophagic degradation such as PINK and PARKIN suggest the importance of autophagy in keeping neurons healthy (Koyano et al., 2014).

The most important signaling pathways that link Ca2+ to autophagy are the

Ca2+-CAMKK2-AMPK pathway and the mTORC1 pathway. Synaptic, but not non- synaptic, glutamatergic receptors signal to mTORC1 through Ca2+ entry via VGCC

(Lenz and Avruch, 2005) (Fig. 3). Accordingly, inhibitors of VGCC induce autophagy in Downloaded from PC12 pheochromocytoma cells (Williams et al., 2008). Another pathway that links Ca2+ to autophagy with a strong relevance in neurons involves the protease calpain, which cleaves ATG5; the truncated ATG5 translocates from the cytosol to the mitochondria molpharm.aspetjournals.org inhibiting autophagy and promoting apoptosis (Yousefi et al., 2006). Calpain inhibitors induce autophagy in PC12 cells and lead to A53T α-synuclein clearance, reducing the accumulation of EGFP-HDQ71 aggregates in a zebrafish model of Huntington’s

Disease (Williams et al., 2008).

Alpha-synuclein forms intracellular aggregates in neurons, which is the at ASPET Journals on September 27, 2021 pathological hallmark of Parkinson's Disease (PD). The accumulation of α-synuclein likely occurs due to the resistance of protein aggregates to autophagy (Cuervo et al.,

2004); in a vicious cycle, mutated α-synuclein and post-translational modifications of the wild-type protein further impair the autophagic pathway (Winslow et al., 2010). In a process that appears to be directly involved with the pathogenesis of PD, this leads to neuronal death. Thus, the combined modulation of Ca2+ signaling and autophagy emerges as a promising target to control the progression of PD. In accordance with this hypothesis, recent findings have shown that Ca2+ homeostasis is also altered in PD

(Rivero-Rios et al., 2014; Schondorf et al., 2014). Molecular effectors linking Ca2+ and autophagy in PD have been increasingly described. Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause late-onset PD, whereas mutations in two genes classically involved in mitophagy, PINK1 and Parkin, are linked to the early onset form of PD (Ashrafi et al., 2014; Grenier et al., 2013; Klein and Westenberger, 2012).

LRRK2 localizes to lysosomes and controls Ca2+ release through a mechanism that involves two pore channels (TPC) and NAADP-dependent Ca2+ channels. The latter is a receptor for NAADP, a potent Ca2+ mobilizing signal. The release of Ca2+ from the lysosome then causes release of Ca2+ from the ER to amplify cytosolic Ca2+ signals, leading to autophagy (Gomez-Suaga et al., 2012). Mutations in LRRK2 in mouse cortical neurons lead to neurite shortening, reduced capacity of Ca2+ buffering, mitochondria depolarization and Ca2+ imbalance that cause mitophagy. Importantly, the Downloaded from inhibition of L-type Ca2+ channels suppresses mitophagy and dendritic shortening

(Cherra et al., 2013). Accordingly, mitochondrial dysfunction has been closely related to PD pathogenesis (Ryan et al., 2015). In addition to its role in autophagy, PINK1 molpharm.aspetjournals.org decreases Ca2+ uptake by mitochondria, leading to energetic imbalance and potentially to autophagy through both the increase of AMP/ATP ratio and the increase of reactive oxygen species. Since alterations in mitochondrial Ca2+ can trigger autophagy (Rimessi et al., 2013), PINK1-mutated neurons are more vulnerable to Ca2+-induced cell death, another potential etiology of the PD pathogenesis (Gandhi et al., 2009). at ASPET Journals on September 27, 2021

In ischemia and preconditioning, the usual “good and bad” status of autophagy applies very clearly. The level and duration of autophagy seem to determine whether it plays a positive or a negative role in neuronal survival after ischemia/reperfusion

(Sheng and Qin, 2015). In central nervous system ischemia, autophagy is crucial for the protective effects of pre-conditioning and is also protective in reperfusion (Yan et al., 2013; Zhang et al., 2013). Accordingly, high expression of TSC1, reduction in mTORC1 activity and higher autophagy levels are responsible for the resistance of the neurons from the cornus ammonis area 3 (CA3) of the hippocampus in relation to the cornus ammonis area 1 (CA1) (Papadakis et al., 2013), further supporting the protective effects that controlled levels of autophagy have on neurons. On the other hand, during severe ischemia, the deletion of ATG genes or the pharmacological blockage of autophagy is protective, indicating that excessive autophagy is involved in neuronal death (Sheng and Qin, 2015). Although most cells die with signs of

autophagy, in several situations autophagy can be part of the mechanism of death, and the drastic metabolic imbalance produced by ischemia seems to be one such situations.

One potential mechanism for the different intensity and duration of autophagy comes, indirectly, from studying AMPK. In neonatal ischemia, only the initial increase in

AMPK activity is independent of CAMKK2, whereas the prolonged CAMKK2-dependent activity of AMPK was deleterious to neurons. Interestingly, the synaptotoxic effects of Downloaded from Aβ oligomer is also mediated by Ca2+increase and the activation of CAMKK2 and

AMPK. Unfortunately, despite the activation of AMPK being a well-established activator of autophagy in most cells, including neurons (Di Nardo et al., 2014), autophagy was molpharm.aspetjournals.org not assessed in these studies (Mairet-Coello et al., 2013) and therefore its involvement can only be implied. These studies suggest that Ca2+-mediated long-lasting activation of CAMKK2, AMPK, and (probably) autophagy is neurotoxic, while short and less intense activation of autophagy is neuroprotective.

Taken together, these data position Ca2+ increase as an important modulator of at ASPET Journals on September 27, 2021 autophagy in neurons. This ion seems to play a role in the clearance of components, for avoiding or retarding neurodegenerative disease. Mutation in subunits of the

VGCC increases LC3-II and SQSTM1/p62, indicating a reduction in the autophagy flux in mice and that mutations in these genes are among the ones that lead to neurodegeneration in Drosophila (Tian et al., 2015). Thus, mild activation of autophagy represent a potential strategy to curb these progressive 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 in contexts involving energy restriction or aging without evaluating the role of autophagy. It would be very important to define these fundamental questions: what is the proportion of pathophysiological alterations mentioned that affects autophagy? What is the importance of autophagy in the response of cells, tissues and organisms in these injuries and pathologies? This will be

fundamental for the understanding of the role autophagy plays in the different stages of neuropathic diseases and to better design interventions to target autophagy to mitigate the progression of these diseases. But, given the risk of high and long lasting autophagy to induce neuronal cell death, modulation of autophagy will have to be strictly fine-tuned in order to make its modulation applicable to the treatment of neurological conditions.

Downloaded from Autophagy, Ca2+ and Cancer

The role of autophagy in cancer depends on the step of the carcinogenesis.

During cancer initiation, autophagy acts as a chemopreventive mechanism, molpharm.aspetjournals.org contributing to the maintenance of genome integrity and to the elimination of pro- carcinogens. Animals lacking key ATG genes present an increased incidence of spontaneous tumors, including hepatocellular carcinoma, lung adenocarcinoma and B cell lymphoma (Yue et al., 2003) (Zhi and Zhong, 2015). Corroborating this, the ectopic at ASPET Journals on September 27, 2021 expression of BECN1 in breast cancer cells lacking endogenous BECN1 gene restored the autophagic capacity of these cells and suppressed their tumorigenesis in vivo

(Liang et al., 1999). In addition, autophagy also plays a role in the progression of pre- malignant to malignant lesions, including very 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 tumor initiation and the transition to a metastatic and therapy-insensitive state. However, the autophagic capacity seems to be restored by tumors after the acquisition of the malignant phenotype, then contributing to tumor progression (Galluzzi et al., 2015). During tumor progression and resistance, autophagy acts predominantly as a tumor supporting mechanism. It provides energetic substrates for metabolic adaptation, favoring tumor resistance to hypoxia and starvation, two contexts in which Ca2+-mediated autophagy is important to define cell fate. Considering the response to therapy, autophagy has also been

suggested as a key mechanism for tumor resistance, so the rational inhibition of autophagy emerges as an alternative to sensitize cancer cells to chemotherapeutics

(Filippi-Chiela et al., 2015; Sui et al., 2013). Indeed, 28 clinical trials that use the strategy of combining chemotherapeutics with inhibitors of autophagic flux, mainly cloroquine and hydroxycloroquine, are in progress, for more than 15 tumor types

(clinicaltrials.gov as of July 2016). Under specific conditions, however, autophagy can act as an oncosupressive mechanism, contributing to the anticancer immuno- Downloaded from surveillance and to the degradation of potentially oncogenic proteins (Galluzzi et al.,

2015). For this, it is fundamental to fully understand the mechanisms underlying its modulation. In addition to the classic pathways, Ca2+ has been increasingly established molpharm.aspetjournals.org as a key modulator of autophagy in tumor cells, which are frequently exposed to nutrient deprivation, ER stress, hypoxia, metabolic stress and environmental alterations. All these conditions induce autophagy that is at least partially mediated by

Ca2+ (Kondratskyi et al., 2013; Monteith et al., 2012; Monteith et al., 2007), as depicted at ASPET Journals on September 27, 2021 in Fig. 3 and discussed in this section.

2+ The signaling that links the increase of [Ca ]c to autophagy induction in cancer involves several pathways. In HeLa cells, both the Ca2+ chelator BAPTA-AM and the IP3-R inhibitor xestopongin B suppress starvation-induced autophagy (Fig. 3)

(Decuypere et al., 2011). Molecularly, this response involves the increase of Ca2+- binding proteins (CaBP) concomitant with a decrease of ER Ca2+-leak rate (Decuypere et al., 2011). These alterations occur through modulation of the IP3-R/Bcl-2/BECN1 complex (see Fig. 3 - box 2), which controls the ER Ca2+ stores and autophagy

(Vicencio et al., 2009). The binding of Bcl-2 to IP3-R hampers the efflux of Ca2+ from the ER (Hoyer-Hansen et al., 2007; Pattingre et al., 2005), and the overexpression of

ER-targeted Bcl-2, but not Bcl-2 targeted to the mitochondria, stabilizes the complex and inhibits autophagy that depends on the Ca2+ from ER (Criollo et al., 2007).

Corroborating this, the phosphorylation of Bcl-2 by JNK during starvation and after the

treatment with IP3-R antagonists releases BECN1 and allows the initiation of autophagy (Vicencio et al., 2009; Wang et al., 2008). The knockdown of IP3-R also leads to an accumulation of autophagosomes in HeLa cells. In this case, the effect is not due to the release of BECN1 from the complex with IP3-R, since cells deficient in

TGM2 (a regulator of IP3-R that inhibits IP3R-Mediated Ca2+ release and IP3R- mediated autophagy) showed increased IP3-R-mediated Ca2+ signaling and increased autophagosome formation (Hamada et al., 2014). Finally, BECN1 is recruited by IP3-R Downloaded from during starvation, and sensitizes IP3-R to low levels of IP3, allowing the release of Ca2+ from the ER (Decuypere et al., 2011). Thus, we can infer that, during starvation (and probably other stressful conditions), the release of Ca2+ from the ER may contribute to molpharm.aspetjournals.org a greater extent to the induction of autophagy than the formation of the BECN1 complex (see Fig. 1). Since autophagy is involved in tumor resistance, the modulation of Ca2+ release from the ER has a potential to be tested as a target mechanism to suppress autophagy and sensitize tumor cells to therapy. at ASPET Journals on September 27, 2021 IP3-R is also involved in autophagy induced by extracellular ATP, which binds to P2 purinergic receptors and triggers the formation of IP3 in breast cancer cells.

Binding of IP3 to IP3-R leads to Ca2+ release from the ER, subsequent activation of

CAMKK2-AMPK and autophagy, which is totally suppressed by BAPTA-AM (Hoyer-

Hansen et al., 2007). In cervix cancer cells, in turn, the toxicity of extracellular ATP is mediated mainly by the uptake of extracellular adenosine and AMPK activation. In this model, autophagy plays a cytotoxic role and the co-treatment with Ca2+ chelator EGTA does not suppress ATP-induced cell death (Mello et al., 2014). Altogether, these data suggest that the role played by Ca2+ in the response to extracellular ATP may depend on the model of study. The mechanisms underlying the role played by Ca2+ in the link between extracellular ATP and autophagy require further investigation.

2+ The role of autophagy induced by the increase of [Ca ]c in cancer relies on the genetic and epigenetic profiles of each cancer type (Table 1). In breast cancer the

release of Ca2+ from the ER triggers autophagy, which positively correlates with the toxicity of the treatment. In these cells, autophagy is part of the toxicity induced by ER stressors, such as ATP and thapsigargin (Hoyer-Hansen et al., 2005; Hoyer-Hansen et al., 2007). In colon and prostate cancer cells, autophagy triggered by the same above- mentioned ER stressors or by the Ca2+ ionophore A23187 plays a protective role (Ding et al., 2007). This was indirectly corroborated in HepG2 hepatocarcinoma cells, where the activation of autophagy using RAPA protects cells from ER stress-induced Downloaded from apoptosis (Kapuy et al., 2014). In fibroblasts and in normal colon cells, however, autophagy induced by the same ER stressors contributed to cell death (Ding et al.,

2007). Finally, in renal carcinoma the influx of Ca2+ and zinc through the TRPM3 molpharm.aspetjournals.org channel in the plasma membrane, respectively, activates the CAMKK2-AMPK pathway and suppresses the increase of miR-241, a microRNA that targets LC3, thus stimulating autophagy. In this model, autophagy contributes to tumor growth (Hall et al.,

2014). These varied outcomes may be attributed to: (i) the presence/absence and the levels of key proteins involved in the connection between Ca2+ and autophagy in each at ASPET Journals on September 27, 2021 cellular context; (ii) the different sensitivity of distinct cells to modulators of Ca2+ signaling; and (iii) the multiple alterations (both related to the amount and function) of key proteins required for different cell fates in cancer cells, such as autophagy and cell death. Indeed, several components of Ca2+ pathways are central to determine the fate of cancer cells after different stimuli, depending on the cell type, the extent of the cell damage and the injured cellular component (Bernardi and Rasola, 2007; Harr and

Distelhorst, 2010). These data are clinically relevant since, at least in these models, the modulation of Ca2+-mediated autophagy sensitizes cancer cells to death. Importantly, the role played by Ca2+-mediated autophagy in cancer cells in comparison to their normal counterpart, described for colon tissue, suggests that the modulation of this mechanism may sensitize cancer cells to die without affecting normal cells.

2+ The increase of [Ca ]c, however, can also suppress autophagy. Amino acids induce a rise in intracellular Ca2+ levels, which triggers the activation of mTORC1 and hVps34 through the direct binding of Ca2+/calmodulin to hVps34, which suppresses

2+ autophagy (Gulati et al., 2008). The increase of [Ca ]c can also suppress autophagy through the activation of calpains and ATG5 cleavage (Yousefi et al., 2006). Calpains are associated to cellular migration, cell survival and apoptosis resistance, thus making the Ca2+-calpain system an important oncogenic signal related to tumor progression Downloaded from (Storr et al., 2011). Finally, cells with nonfunctional IP3-R (DT40 TKO cells, from a chicken lymphoma) show lower basal mTORC1 activity and higher basal autophagic levels than DT40 cells in which IP3-R WT expression was restored exogenously. DT40 molpharm.aspetjournals.org TKO cells also present a delayed apoptotic response, which could be due to increased basal autophagy (Cardenas et al., 2010; Khan and Joseph, 2010). The absence of IP3-

R hampers the transfer of Ca2+ from the ER to mitochondria; as a consequence, DT40

TKO cells have 60% lower basal O2 consumption rate than WT cells and use autophagy as a metabolic adaptation (Cardenas et al., 2010). Corroborating this, the at ASPET Journals on September 27, 2021 knockdown of MCUR1 reduced O2 consumption rate, activated AMPK and induced autophagy (Mallilankaraman et al., 2012). However, a key question remains unsolved in this model. Considering that Ca2+ may be fundamental to the maintenance of acidic pH in lysosomes, how cells with nonfunctional IP3-R maintain high enough levels of

Ca2+ in the lysosome to guarantee an intense basal autophagic flux?

Actually, some other questions related to the link between Ca2+ and autophagy in cancer remain unanswered, such as: (a) What is the role of Ca2+-mediated autophagy in the response of cancer cells to therapy? (b) Why does Ca2+-mediated autophagy contributes to cell survival in some conditions and to cell death in others?

(c) Are there non-canonical autophagy pathways mediating the activation of autophagy by Ca2+ in cancer? (d) Does the modulation of any component of Ca2+ signalosome have a potential to modulate autophagy clinically?

Two points deserve attention in these questions. The first is related to the role of Ca2+ for normal mitochondria functioning and cell metabolism. Recent data suggest that cancer resistant cells usually present both increased autophagy (Sui et al., 2013) and oxidative phosphorylation (Vellinga et al., 2015; Viale et al., 2014). Disturbances in mitochondrial Ca2+ may sensitize cells due to energetic imbalance and autophagy, therefore triggering even more autophagy. Thus, the modulation of both Ca2+ dynamics in mitochondria (for instance suppressing VDAC1 or MCU1) in combination with Downloaded from autophagy inhibitors could be of therapeutic value in cancer therapy. In this sense, the modulation of Ca2+ may also interfere with the autophagic flux. Thus, the manipulation of Ca2+ availability for lysosomes and mitochondria, for instance, may directly affect the molpharm.aspetjournals.org activity of these organelles and the autophagic flux, sensitizing some cancer cells to die. Indeed, several clinical trials in cancer have tested the combination of chemotherapeutics with compounds that suppress the fusion of autophagosome to lysosome, since the disruption of the autophagic flux. at ASPET Journals on September 27, 2021 In conclusion, Ca2+-mediated autophagy emerges as a potential target to be modulated to sensitize tumor cells and control tumor progression. Ca2+ is involved in cytoprotective autophagy induced by starvation, hypoxia, ER stress and increased extracellular ATP, all features commonly present in growing solid tumors but not in healthy homeostatic tissues. Furthermore, cancer cells reprogram their metabolism, including changes in autophagy and oxidative phosporylation, both modulated by Ca2+

(Ward and Thompson, 2012). These alterations seem to be involved in tumor progression and resistance, so that the modulation of metabolism has being proposed as a therapeutic target. In this sense, the inhibition of Ca2+ transfer from the ER to mitochondria in combination with autophagy inhibition may cause an energetic collapse, leading cancer cells to die, including those cells that take advantage of oxidative phosphorylation to resist to therapy (Vellinga et al., 2015; Viale et al., 2014).

Thus, the modulation of Ca2+ signaling in combination with chemotherapy could

specifically suppress autophagy. Ultimately, the development of modulators of specific

Ca2+ signalosome components is a possibility that deserves to be further investigated.

The crosstalk between Ca2+ and autophagy in infection and inflammation

Autophagy is known to control key aspects of innate and adaptive immunity in multicellular organisms. Apart from its participation in the capture and elimination of pathogens, autophagy also takes part in the process of inflammatory and immune responses. Notably, autophagy has been shown to influence the antigenic profile and Downloaded from the immunogenic release of signals in antigen-presenting cells, therefore it impacts cell survival, proliferation, plasticity and function of dendritic cells and T-lymphocytes. The

2+ main findings concerning the regulation of autophagy by Ca in the immune context molpharm.aspetjournals.org are shown in Fig. 3 and detailed in the following paragraphs.

A number of infection agents subvert host defenses to survive and proliferate intracellularly. The selective removal of such microorganisms by autophagy, also

termed xenophagy, is thought to act downstream of the pattern recognition receptors at ASPET Journals on September 27, 2021 thereby facilitating effector responses that lead to pathogen destruction. Xenophagy also modulates the function of a range of inflammatory mediators at various levels, including cytokine production (Puleston and Simon, 2014).

During urinary tract infection, caused by uropathogenic E.coli (UPEC), autophagy determines the role of TRP cation channels. After UPEC strains get access to the host cytoplasm, they are targeted by LC3-II- and SQSTM1/p62-positive autophagosomes. However, they avoid degradation by neutralizing the autophagolysosomal pH, which severely compromises organelle function by disturbing bactericidal properties and ion homeostasis. The TRPML3 channel on the lysosomal membrane seems to respond to pH changes, mediating Ca2+ efflux to the cytosol. The stimulation of TRPML3 spontaneously induces lysosome exocytosis, thus expelling the pathogen from the cell in a non-lytic manner. Remarkably, knockdown of ATG5 or

BECN1 from bladder epithelial cells significantly suppressed bacterial expulsion, which

was also observed by pretreatment of infected cells with BAPTA-AM, suggesting a role for Ca2+ in this mechanism. On the contrary, cells exposed to ML-SI1, a TRPML antagonist, or knockdown for TRPML3, markedly increased intracellular bacterial load

(Miao et al., 2015).

Viral spreading can also be controlled by autophagy. The detection of vesicular stomatitis virus, for instance, results in type I interferon production due to the ability of autophagy to deliver viral ligands to TLR7 in dendritic cells (Lee et al., 2007). Downloaded from Noteworthy, an impressive strategy to manipulate autophagy is observed during rotavirus infection, linked to severe gastroenteritis. Basically, the viral ER-anchored glycoprotein NSP4, a pore-forming viroporin, elicits the leakage of Ca2+ from the ER to molpharm.aspetjournals.org the cytosol. This ion mobilization, in turn, activates autophagy through the CAMKK2-

AMPK pathway; CAMKK2 inhibition by STO-609 abrogated LC3-II detection. The disruption of ER-Ca2+ homeostasis is only the primary function of NSP4. Further, it potentiates the activation of the ER Ca2+ sensor stromal interaction molecule1 (STIM1), at ASPET Journals on September 27, 2021 embedded in the ER membrane. The STIM1 oligomer conformation contributes to

2+ 2+ [Ca ]c increase by allowing Ca influx across the plasma membrane, through activation of Orai1 channels, a type of Ca2+-release-activated Ca2+ (CRAC) channel

(Hyser et al., 2013). Later, the rotavirus also interferes with autophagy membrane trafficking to transport viral ER-associated proteins to sites of genome replication and particle assembly, since lower expression of ATG3 and ATG5 highly reduce virus yield.

Furthermore, the virus blocks autophagosome maturation, once NSP4/LC3-II structures were not shown to progress to autophagolysosomes (Crawford et al., 2012).

The typical role of mTOR as an autophagic inhibitor is challenged in response to LPS and cellular septic insult, where activation of mTOR is required for autophagy induction.

In this context, regulation of autophagy also involves CAMK1 and CAMK4, two malfunctional members of the CAMK family of proteins. In macrophages, LPS induces

ER stress, resulting in Ca2+ mobilization and CAMK1 activation. This signaling

stimulates autophagy in a CAMKK2AMPK-dependent pathway through mechanisms unrelated to mTORC1, establishing that autophagy and mTORC1 activity are required simultaneously in specific cellular contexts (Guo et al., 2013). Latter, a more detailed characterization of the regulatory role of CAMKs during autophagy was described, focusing in CAMK4. Isolated macrophages isolated from CAMK4-/- mice exhibit reduced levels of ATG5/12, ATG7 and LC3B in response to LPS. The proposed mechanism relies on the inhibition of GSK-3β activity and FBXW7 recruitment by Downloaded from CAMK4, which prevents the proteasomal degradation of mTOR, preserving mTORC1 activity, thereby increasing autophagy (Zhang et al., 2014). Under stimulation,

CAMK4-/- and WT macrophages express similar levels of mTOR mRNA, suggesting molpharm.aspetjournals.org that mTOR regulation by CAMK4 occurs at a posttranscriptional level. Moreover,

CAMK4-mTOR dependent autophagy is essential to IL-6 production by macrophages and adaptive responses to cytoprotection in renal tubular cells during inflammatory state. at ASPET Journals on September 27, 2021 Upon TCR stimulation, in T-lymphocytes, the Ca2+ response is initiated by efflux of Ca2+ from ER stores, ultimately activating CRAC channels on the plasma membrane, which promotes extracellular Ca2+ influx. However, in ATG7-deficient T-cells the ER compartment is abnormally expanded, while intracellular Ca2+ stores are increased, probably due to impaired ER Ca2+ depletion and failed redistribution of STIM-1 in the

ER-plasma membrane junctions. Treatment with Thapsigargin rescues the defective

Ca2+ influx in autophagy-deficient T-cells. These results clearly demonstrate that autophagy regulates both the ER homeostasis and the calcium mobilization, which are interrelated events (Jia et al., 2011).

More than an infection-reacting mechanism, inflammation also responds to the loss of cellular homeostasis caused by trauma, ischemia or chemical injury. All these autophagic stress-inducers regulate signaling pathways that are involved in cell metabolism, tissue remodeling and repair. This raises the question of whether Ca2+-

modulated autophagy is also involved in these responses. In this scenario, a more detailed study relating Ca2+ signalosome, phagocyte recruitment and cytokine production/secretion would be of great interest for therapeutic improvements. Another aspect that remains blurred is the crosstalk between autophagy and immunity in the context of other human diseases, including neurodegenerative disorders and cancer.

Discussion Downloaded from

Ca2+ plays a key role in the maintenance of basal autophagy as well as in induced autophagy. Ca2+-mediated autophagy is involved in the pathogenesis and progression of human diseases, and the modulation of Ca2+ signalosome components molpharm.aspetjournals.org present a great potential to be used in therapeutic interventions for both therapy and prophylaxis.

In neurodegenerative diseases, some potential targets include the modulation of calpain signaling and the reestablishment of Ca2+ homeostasis, including the control at ASPET Journals on September 27, 2021 of L-type channels and Ca2+ release from lysosomes. This increases the degradation of

α-synuclein in Huntington and Parkinson patients and controls neuronal cell death.

Also, the suppression of autophagy during the more severe phase of ischemia may contribute to reduce the neuronal damage. In cancer, the suppression of Ca2+ release from the ER as well as the suppression of the influx of Ca2+ to the mitochondria may contribute to sensitize cancer cells to therapy, especially in the metabolic adaptation that follows chemotherapy. In addition, the modulation of lysosome Ca2+ signaling emerges as an alternative to block the autophagic flux to sensitize cancer cells to die.

In immunity, the ability to pharmacologically modulate members of the Ca+2 signalosome, which were strategically manipulated by pathogens along host-parasite co-evolution, represents a promising therapeutic target. The use of CAMKK2 modulators, such as STO-609 or receptor agonists, in specific points of the infection

cycle might be an alternative to be used in combination with broadly used microbicidal agents.

Although some aspects of the modulation of autophagy by Ca2+ remain unknown, it is clear that the modulation depends on the spatio-temporal distribution of

Ca2+ as well as on the autophagy inducer and on the context in which Ca2+ and autophagy are modulated. However, the signaling pathways connecting them and probably the role played by key components that interfere in cell outcome after Ca2+- Downloaded from mediated autophagy need to be further investigated. This may include proteins that interact with components of Ca2+ signalosome, such as IP3-R in the ER, VDAC1 in the mitochondria and TRPML1/3 in the lysosome, as well as proteins that modulate the molpharm.aspetjournals.org

CAMKK2-AMPK-mTOR-ULK1 pathway and the activity of Ca2+ channels in the plasma membrane. It is expected that, in the coming years, some of these mechanisms will be revealed and, as hypothesized by Kondratskyi et al, the modulation of some components of Ca2+ signalosome that influence autophagy will be therapeutically at ASPET Journals on September 27, 2021 tested in different human pathologies (Kondratskyi et al., 2013). Notwithstanding, it is important to share a cautionary note with other authors. Some methods used to access the role of Ca2+ in autophagy such as Ca2+ ionophores, thapsigargin-induced depletion of ER Ca2+ stores and the Ca2+ chelator BAPTA may induce cell stress at high concentrations, and the autophagy that follows may be activated by this stress rather than by a direct signaling involving Ca2+ (Cardenas and Foskett, 2012; Decuypere et al., 2015; Decuypere et al., 2011). Thus, some conclusions based on in vitro studies must be interpreted with caution.

In conclusion, as two finely controlled and biological relevant systems, the study of the link between Ca2+ and autophagy has a great potential to contribute to our understanding of the etiology of neural or inflammatory diseases as well as cancer. In addition, Ca2+-modulated autophagy represents an opportunity for the development of

new or adjuvant therapeutic strategies to control, prevent or treat illnesses associated to these systems.

ACKNOWLEDGEMENT

We thank Alexandra Vigna and Pitia F. Ledur for critically reading the manuscript.

Downloaded from AUTHORSHIP CONTRIBUTIONS

Wrote of the manuscript: Filippi-Chiela, Viegas, and Lenz. molpharm.aspetjournals.org Contributed to the writing of the manuscript: Thomé, Buffon, and Wink.

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest to disclose.

at ASPET Journals on September 27, 2021

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FOOTNOTES

* ECFC and MSV contributed equally to this work.

This work was supported by Conselho Nacional de Desenvolvimento Científico e

Tecnológico (CNPq), Universal (475882/2012-1 and 458139/2014-9) and Novas

Terapias Portadoras de Futuro (457394/2013-7); PROBITEC-CAPES (004/2012) and

ICGEB BRA11/01. ECFC, MSV and MPT are or were recipient of CNPq fellowships and ECFC is recipient of CAPES fellowship. MRW and GL are recipients of research Downloaded from fellowship from CNPq.

FIGURE LEGENDS molpharm.aspetjournals.org

Fig. 1 - The mechanism of autophagy. Autophagy is modulated by several intra- and extracellular signals. Insulin Growth Factor (IGF), insulin and growth factors activate their receptors (TK - tyrosine kinase) and trigger intracellular pathways that suppress at ASPET Journals on September 27, 2021 autophagy. The activation of metabotropic receptors in the plasma membrane 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 the p53 and AMPK pathway, respectively. The mTOR Complex 1 (mTORC1) is central in integrating all these signals to modulate autophagy (Box 1 - arrows do not necessarily means a direct 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/p62 protein 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 the autolysosome, where cellular components are degraded and recycled. In box 5 we summarize key information about the distribution of Ca2+ in subcellular components and the main receptors, channels and other proteins involved in the control of Ca2+ concentration in each compartment.

Fig. 2 - The crosstalk between Ca2+ signalosome and autophagy. Ca2+ influences autophagy induced by several contexts, including Rapamycin treatment, hypoxia, Downloaded from extracellular ATP and starvation (green box). On the other hand, Ca2+ signalosome components are involved in the maintenance of basal autophagy at low levels, through mechanisms that involves the control of Ca2+ efflux from the ER (through the IP3-R molpharm.aspetjournals.org channel), the role of Ca2+ in mitochondria and lysosomes and the formation of the IP3-

R/Bcl-2/BECN1 complex (red box). Letters indicate the experimental evidences for each situation, as depicted on the bottom. at ASPET Journals on September 27, 2021

Fig. 3 - Molecular pathways linking autophagy and components of the Ca2+ signalosome in cancer, neurology and immunity. In the light blue background are shown the main pathways connecting extracellular and intracellular signals that modulate ion channels and/or ion flow through cellular components in cancer.

2+ Alterations in [Ca ]c can be induced through the increase of ion entry from the extracellular environment or through the release of Ca2+ from the ER. The increase in

2+ [Ca ]c can activate the CAMKK2-AMPK pathway, leading to autophagy in a mTOR- dependent or independent way. The Unfolded Protein Response (UPR) can also trigger the release of Ca2+ and activation of autophagy, including reticulophagy

(REphagy). The light red background shows the main pathways linking Ca2+ from synaptic NMDA receptors and VGCCs to signaling pathways that modulate autophagy and the main functions of autophagy in ischemia and neurodegeneration. PC –

preconditioning. In the yellow background (bottom) the main findings linking ions and ion channels 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 normal and altered Ca2+ transfer on cell fate are shown. Box 2: mechanisms of IP3-

R/Bcl-2/BECN1 complex functioning and modulation.

TABLE LEGENDS Downloaded from

TABLE 1 - The role of Ca2+-modulated autophagy in cancer. The table summarizes the main findings connecting autophagy and Ca2+ in cancer. The model of study, the molpharm.aspetjournals.org treatment that causes Ca2+ alterations and autophagy, the modulation of Ca2+ availability and its consequences in autophagy and the modulation of autophagy used to assess the role of autophagy are shown.

at ASPET Journals on September 27, 2021

MOL #105171

TABLE 1 Ca2+ Modulation and consequences to Cell and cancer type Inducer of Ca2+ imbalance and autophagy Autophagy modulation a Role in autophagy Ref autophagy 3-MA (35% to <5%) Extracellular ATP (release of Ca2+ from the ER BAPTA/AM - reduces the % of GFP-LC3+ BECN1 KD (35% to 5%) Molecular PharmacologyFastForward.PublishedonJuly19,2016asDOI:10.1124/mol.116.105171

MCF7 breast cancer Not assessed Høyer-Hansen, 2007 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. through IP3-R) cells from 40% to 5% ATG7 KD (35% to 12%)

3-MA (35% to <5%) BAPTA/AM - reduces the % of GFP-LC3+ BECN1 KD (35% to 15%) MCF7 breast cancer Ionomycin (Ca2+ ionophore) Not assessed Høyer-Hansen, 2007 cells from 40% to 10% ATG7 KD (35% to 12%)

3-MA (45% to 18%) Thapsigargin (TG; inhibitor of ER Ca2+- ATPase BAPTA/AM - reduces the % of GFP-LC3+ BECN1 KD (45% to 30%) MCF7 breast cancer No assessed Høyer-Hansen, 2007 which maintains high levels of Ca2+ in the cytosol) cells from 52% to 5% ATG7 KD (45% to 28%)

3-MA (80% to 20%) - decreased cell death Vitamin D analogue EB1089 (increases cytosolic MCF7 breast cancer Not assessed Overexpression of BECN1 increased Cytotoxic Høyer-Hansen, 2005 Ca2+, but the mechanism is not fully known) cell death

BAPTA/AM and xestospongin (IP3-R Starvation using HBSS (increases cytosolic Ca2+ inhibitor) - reduces the % of GFP-LC3- Hela cervix from the ER by IP3-R; also disrupts the IP3-R/Bcl- positive cells and the LC3II levels to Not assessed Not assessed Decuypere, 2011 adenocarcinoma 2/BECN1 complex). control levels

Starvation using HBSS (increases cytosolic Ca2+ Overexpression of Bcl2 - reduced around Hela cervix b from the ER by IP3-R; also disrupts the IP3-R/Bcl- a half the percentage of GFP-LC3- Not assessed Not assessed Vivencio 2009 adenocarcinoma 2/BECN1 complex). positive cells

ATG6 KD and ATG8 KD - increased caspase activation and apoptotic cell ATP; death from ~30% to 47% and 62% TG-induced ER stress; Not assessed Cytoprotective Ding, 2007 HCT116 Colon cancer respectively; A23187-induced ER stress (Ca2+ ionophore) 3MA also increased cell death, but data are not shown

ATP; ATG6 KD, ATG8 KD and 3-MA induced DU145 Prostate Cancer TG-induced ER stress; Not assessed cell death, but data are not shown. Cytoprotective Ding, 2007 A23187-induced ER stress (Ca2+ ionophore) Obs: the efficiency is not shown TG-induced ER stress; Murine embryonic A23187-induced ER stress; Not assessed ATG5 KD decreased cell death Cytotoxic Ding, 2007 fibroblasts

TG-induced ER stress; CCD-18Co normal colon A23187-induced ER stress; Not assessed 3-MA decreased cell death Cytotoxic Ding, 2007

Rapamycin and metyrapone (mTOR inhibitor) - increased autophagy and Cytoprotective HepG2 Hepatocarcinoma TG-induced ER stress Not assessed Kapuy, 2014 cell viability (indirect)

Restoration of IP3-R expression Rapamycin - increased autophagy in DT40 chicken lymphoma IP3-R triple knock out (TKO) cells Not assessed b Khan and Joseph, 2010 decreased basal autophagy WT but not in DT40 TKO cells a. The percentages in the parenthesis indicate the percentage of autophagy-positive cells before (first value) and after (second value) autophagy inhibition. b. Despite not assessed, in these contexts autophagy is known to be cytoprotective (Lum et al., 2005; Onodera and Ohsumi, 2005) . Abbreviations: 3-MA, 3-methyladenin; ER, Endoplasmic Reticulum; KD, knockdown; TG, Thapsigargin; IP3-R, Inositol 1,4,5-triphosphate Receptor.

Downloaded from from Downloaded molpharm.aspetjournals.org at ASPET Journals on September 27, 2021 27, September on Journals ASPET at FILIPPI-CHIELA & VIEGAS, 2016 FIGURE 1

IGF Insulin Ionotropic Growth Metabotropic ATP Molecular PharmacologyFactor Fast Forward.Receptor Published on July 19, 2016 as DOI: 10.1124/mol.116.105171Hormones This article has not been copyedited and formatted. The final version may differ from this version. Receptor Neurotransmitters Extracell

INSULIN α Intracell IGF-R1 TK α PIP2 DAG RECEPTOR G-protein RECEPTOR Phospholipase C

PI3k/Akt Endoplasmic MAPK TP53 TSC1/2 IP3 Reticulum Rheb 2+ 2+ AMPK CAMKK2 Ca mTORC1 Box 5 Box 1 2+ Downloaded from LKB1 Box 2 Free Ca Calpain IP3-R LC3 Box 4 Ca2+ leak molpharm.aspetjournals.org LC3-I ATG13 ULK1 FIP200 L LC3-II at ASPET Journals on September 27, 2021 Vps34 Box 3 BECN1 Autolysosome p150 ATG14 Phagophore Autophagosome ATG5-ATG12 ATG16L1 + Lysosome

Box 1 Box 2 2+ Box 5 CAMKK2 Ca Extracellular mTOR Complex 1 (mTORC1) AMP/ATP Glucose Environment Starvation Infectious Agents Oxygen (hypoxia) AMPK TSC2 1-2mM Oncogenes Cytokines NMDA TRPC VDCC Insulin ATP Ca2+ channels Glucose Aminoacids Rheb P P IP3-R and IP3 mTOR mLST8 ULK1 P ATG13 mTORC1 IP3-R / BECN1 / Bcl2 complex AMP/ATP Raptor FIP200 TGM2 Starvation Tumor supressors Endoplasmic Rapamycin Cytosol Cell Stress AUTOPHAGY 100nM Reticulum 0.4-0.8mM Box 3 Box 4 Lysosome + + ? 2+ LC3-II + Na /H Pump Exchange H Ca

+ Mitochondria Lysosome H Lysosomal Permease PE Ca2+ 200nM ? 0.4-0.6mM + + + + H Na VDAC1 H H + TRPML3 / TRPML1 TRPML1 and 3 + H MICU1 and MCU TRPM2 Adaptor H Lysosomal Acid Hidrolases BI-1 TFEB Phagophore Cargo FILIPPI-CHIELA & VIEGAS, 2016 FIGURE 2

2+ Molecular Pharmacology Fast Forward. Published on July 19,2+ 2016 as DOI: 10.1124/mol.116.105171 Ca signalosomeThis article hasis notpositively been copyedited involved and formatted. TheCa final version signalosome may differ from this inhibits version. autophagy or in induced autophagy maintains basal autophagy at low levels d 2+ Starvation Ca released 2+ from lysosomes Ca in IP3-R / IP3 mitochondria b (MCOLN1) JNK Signaling f MG132 Normal ATP (proteas. inhibitor) Calcineurin a b levels c Extracel. ATP c TFEB d a AMPK BECN1 Hypoxia a

Bcl2 Downloaded from a Availability IP3-R Rapamycin a Autophagy of BECN1 2+ 2+ e L-type Ca channels molpharm.aspetjournals.org Experimental (plasma membrane) Evidence Experimental Ca 2+ signals Evidence at ASPET Journals on September 27, 2021 Involved in the 2+ induction of Autophagy Ca signals Inhibit or maintain BAPTA suppresses Xestospongin B basal autophagy at low leves autophagy induced a b suppresses autophagy by RAPA, hypoxia induced by starvation a Knockdown of IP3-R Inhibition of IP3-R and extracellular ATP (Ex.: Xestospongin B) b KD of TGM2 Depletion of IP3 BAPTA blocks the BAPTA blocks the c d c d activation of TFEB (a regulator of IP3-R) (lithium cloride) induced by MG132 induced by starvation Antagonist of plasma e Diturbances in membrane L-type mitochondrial Ca2+ f Ca2+ channels

Increase basal autophagy FILIPPI-CHIELA & VIEGAS, 2016 FIGURE 3

2+ ‘Intracellular’ Ca ‘Extracellular’ Ca2+ 2+ Thapsigargin Ca ionophores Molecular Pharmacology Fast Forward. Published on July 19, 2016 as DOI: 10.1124/mol.116.105171 CANCER This article Vhasitamin not been D andcopyedited andIonomycin formatted. The final version may differ from thisNEURO version. Synaptic A23187 NMDA receptors analogues TRPM3 (e.g. EB1089)

Phospholipase C VGCC ATP P2y DAG α PIP2 G-protein 2+ (Gi, Gq/11 or Gs) [Ca ]c

STARVATION IP3 2+ CAMKK2 AMPK mTORC1 [Ca ]c JNK Calpain UPR AUTOPHAGY Box2 ER P IP3-R Stress Toxic during Ischemia AUTOPHAGY Bcl2 REphagy

versus Downloaded from P Protective at PC and AMPK ATP BECN1 reperfusion Box1 Functional Removal of aggregated Mitochondria proteins molpharm.aspetjournals.org

lysosome Nucleus pH4-5 STIM1 Autophagy and lysosome Orai1 gene transcription NSP4

2+ 2+ at ASPET Journals on September 27, 2021 Ca 2+ Ca TFEB LC3-II [Ca ] pH7 Calcineurin c pTFEB Viroplast pH7 2+ [Ca ]c FBXW7 CAMKK2 CAMK4 mTORC1 P AMPK P GSK-3β mTORC1 AUTOPHAGY 2+ Ca IMMUNITY TLR? IL6 Box1 Sepsis / LPS ER Cytosol Box2 A Cytosol B P Cytosol ER JNK ER NAF-1 NAF-1 BH3 BH3 mimetics P domain BCL2 IP3R BCL2 IP3R P DAPK P VDAC1 IP3R MCUR1 HSPA9 BECN1 2+ BECN1 Normal Ca transfer: normal mitochondria functioning

2+ ATP AMPK Autophagy Altered Ca Cell Survival Mitophagy Autophagy Autophagy transfer Cell Death