title page modulation of autophagy by calcium signalosome in...

MOL #105171

1

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

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;

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;

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

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

at ASPE

T Journals on A

ugust 30, 2020m

olpharm.aspetjournals.org

Dow

nloaded from

http://molpharm.aspetjournals.org/

MOL #105171

2

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. Telephone: (51) 33087613 Fax: (51) 33087003 E-mail: [email protected] 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

Abbreviation List:

[Ca2+]c, Cytosolic Ca2+ concentration [Ca2+]ER, Cytosolic Ca2+ 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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

3

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

situations. Besides its role in cell proliferation and apoptosis, intracellular Ca2+ is widely

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,

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

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.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

4

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).

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

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

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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

5

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

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

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

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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

6

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-

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

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 anti-

apoptotic 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

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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

7

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

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

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,

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.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

8

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-

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

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

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

an increase in cytosoic Ca2+ concentration ([Ca2+]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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

9

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

suppressed autophagy induced by the increase of [Ca2+]c (Hoyer-Hansen et al., 2007).

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

BECN1 (Liang et al., 1999), triggered autophagy in response to the increase of [Ca2+]c

through the activation of CAMKK2-AMPK independent of BECN1, which suggests an

alternative pathway downstream of AMPK (Hoyer-Hansen et al., 2007) .

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

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

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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

10

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

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

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

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,


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

11

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+

channel), for instance, in conditions of low [Ca2+]ER, fundamentally contributes to Ca2+

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

autophagy to contribute to the metabolic adaptation of those cells with low [Ca2+]ER and

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

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

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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

12

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.,

2008) (see Fig. 3 – Box 2 for more details). Similarly, RAPA increases [Ca2+]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).

ATG7-deficient cells expand their ER Ca2+ stores and increase [Ca2+]ER, probably to

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

induce these changes nor does it alter the increase of [Ca2+]c induced by extracellular

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.

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).


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

13

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

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

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

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).


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

14

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

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

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).

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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

15

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

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

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

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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

16

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.

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,

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

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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

17

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-

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

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

in Fig. 3 and discussed in this section.

The signaling that links the increase of [Ca2+]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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

18

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

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

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.

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.

The role of autophagy induced by the increase of [Ca2+]c in cancer relies on

the genetic and epigenetic profiles of each cancer type (Table 1). In breast cancer the


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

19

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

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

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

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.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

20

The increase of [Ca2+]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

autophagy (Gulati et al., 2008). The increase of [Ca2+]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

(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

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

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?


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

21

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

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

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.

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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

22

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

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

main findings concerning the regulation of autophagy by Ca2+ in the immune context

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

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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

23

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).

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

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),

embedded in the ER membrane. The STIM1 oligomer conformation contributes to

[Ca2+]c increase by allowing Ca2+ 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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

24

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

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

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.

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+-


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

25

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

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

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

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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

26

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+-

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

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

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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

27

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.

AUTHORSHIP CONTRIBUTIONS

Wrote of the manuscript: Filippi-Chiela, Viegas, and Lenz.

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 ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

28

REFERENCES

Ashrafi G, Schlehe JS, LaVoie MJ and Schwarz TL (2014) Mitophagy of damaged mitochondria

occurs locally in distal neuronal axons and requires PINK1 and Parkin. J Cell Biol 206(5):655-

670.

Bernardi P and Rasola A (2007) Calcium and cell death: the mitochondrial connection. Subcell

Biochem 45:481-506.

Berridge MJ (2009) Inositol trisphosphate and calcium signalling mechanisms. Biochim Biophys

Acta 1793(6):933-940.

Bultynck G, Kiviluoto S and Methner A (2014) Bax inhibitor-1 is likely a pH-sensitive calcium

leak channel, not a H+/Ca2+ exchanger. Sci Signal 7(343):pe22.

Burada F, Nicoli ER, Ciurea ME, Uscatu DC, Ioana M and Gheonea DI (2015) Autophagy in

colorectal cancer: An important switch from physiology to pathology. World J Gastrointest

Oncol 7(11):271-284.

Cardenas C and Foskett JK (2012) Mitochondrial Ca(2+) signals in autophagy. Cell Calcium

52(1):44-51.

Cardenas C, Miller RA, Smith I, Bui T, Molgo J, Muller M, Vais H, Cheung KH, Yang J, Parker I,

Thompson CB, Birnbaum MJ, Hallows KR and Foskett JK (2010) Essential regulation of cell

bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria. Cell 142(2):270-

283.

Catterall WA (2011) Voltage-gated calcium channels. Cold Spring Harb Perspect Biol

3(8):a003947.

Cherra SJ, 3rd, Steer E, Gusdon AM, Kiselyov K and Chu CT (2013) Mutant LRRK2 elicits calcium

imbalance and depletion of dendritic mitochondria in neurons. Am J Pathol 182(2):474-484.

Choi AM, Ryter SW and Levine B (2013) Autophagy in human health and disease. N Engl J Med

368(19):1845-1846.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

29

Christensen KA, Myers JT and Swanson JA (2002) pH-dependent regulation of lysosomal

calcium in macrophages. J Cell Sci 115(Pt 3):599-607.

Crawford SE, Hyser JM, Utama B and Estes MK (2012) Autophagy hijacked through viroporin-

activated calcium/calmodulin-dependent kinase kinase-beta signaling is required for rotavirus

replication. Proc Natl Acad Sci U S A 109(50):E3405-3413.

Criollo A, Maiuri MC, Tasdemir E, Vitale I, Fiebig AA, Andrews D, Molgo J, Diaz J, Lavandero S,

Harper F, Pierron G, di Stefano D, Rizzuto R, Szabadkai G and Kroemer G (2007) Regulation of

autophagy by the inositol trisphosphate receptor. Cell Death Differ 14(5):1029-1039.

Cuervo AM, Stefanis L, Fredenburg R, Lansbury PT and Sulzer D (2004) Impaired degradation of

mutant alpha-synuclein by chaperone-mediated autophagy. Science 305(5688):1292-1295.

Czech MP (2000) PIP2 and PIP3: complex roles at the cell surface. Cell 100(6):603-606.

Decuypere JP, Kindt D, Luyten T, Welkenhuyzen K, Missiaen L, De Smedt H, Bultynck G and

Parys JB (2013) mTOR-Controlled Autophagy Requires Intracellular Ca(2+) Signaling. PLoS One

8(4):e61020.

Decuypere JP, Parys JB and Bultynck G (2015) ITPRs/inositol 1,4,5-trisphosphate receptors in

autophagy: From enemy to ally. Autophagy 11(10):1944-1948.

Decuypere JP, Welkenhuyzen K, Luyten T, Ponsaerts R, Dewaele M, Molgo J, Agostinis P,

Missiaen L, De Smedt H, Parys JB and Bultynck G (2011) Ins(1,4,5)P3 receptor-mediated Ca2+

signaling and autophagy induction are interrelated. Autophagy 7(12):1472-1489.

Di Nardo A, Wertz MH, Kwiatkowski E, Tsai PT, Leech JD, Greene-Colozzi E, Goto J, Dilsiz P,

Talos DM, Clish CB, Kwiatkowski DJ and Sahin M (2014) Neuronal Tsc1/2 complex controls

autophagy through AMPK-dependent regulation of ULK1. Hum Mol Genet 23(14):3865-3874.

Ding WX, Ni HM, Gao W, Hou YF, Melan MA, Chen X, Stolz DB, Shao ZM and Yin XM (2007)

Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival. J Biol

Chem 282(7):4702-4710.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

30

Feng Y, He D, Yao Z and Klionsky DJ (2014) The machinery of macroautophagy. Cell Res

24(1):24-41.

Filippi-Chiela EC, Bueno e Silva MM, Thome MP and Lenz G (2015) Single-cell analysis

challenges the connection between autophagy and senescence induced by DNA damage.

Autophagy 11(7):1099-1113.

Fimia GM, Kroemer G and Piacentini M (2013) Molecular mechanisms of selective autophagy.

Cell Death Differ 20(1):1-2.

Galluzzi L, Pietrocola F, Bravo-San Pedro JM, Amaravadi RK, Baehrecke EH, Cecconi F, Codogno

P, Debnath J, Gewirtz DA, Karantza V, Kimmelman A, Kumar S, Levine B, Maiuri MC, Martin SJ,

Penninger J, Piacentini M, Rubinsztein DC, Simon HU, Simonsen A, Thorburn AM, Velasco G,

Ryan KM and Kroemer G (2015) Autophagy in malignant transformation and cancer

progression. Embo J 34(7):856-880.

Gandhi S, Wood-Kaczmar A, Yao Z, Plun-Favreau H, Deas E, Klupsch K, Downward J, Latchman

DS, Tabrizi SJ, Wood NW, Duchen MR and Abramov AY (2009) PINK1-associated Parkinson's

disease is caused by neuronal vulnerability to calcium-induced cell death. Mol Cell 33(5):627-

638.

Ghavami S, Shojaei S, Yeganeh B, Ande SR, Jangamreddy JR, Mehrpour M, Christoffersson J,

Chaabane W, Moghadam AR, Kashani HH, Hashemi M, Owji AA and Los MJ (2014) Autophagy

and apoptosis dysfunction in neurodegenerative disorders. Prog Neurobiol 112:24-49.

Gomez-Suaga P, Luzon-Toro B, Churamani D, Zhang L, Bloor-Young D, Patel S, Woodman PG,

Churchill GC and Hilfiker S (2012) Leucine-rich repeat kinase 2 regulates autophagy through a

calcium-dependent pathway involving NAADP. Hum Mol Genet 21(3):511-525.

Gordon PB, Holen I, Fosse M, Rotnes JS and Seglen PO (1993) Dependence of hepatocytic

autophagy on intracellularly sequestered calcium. J Biol Chem 268(35):26107-26112.

Green MF, Anderson KA and Means AR (2011) Characterization of the CaMKKbeta-AMPK

signaling complex. Cell Signal 23(12):2005-2012.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

31

Grenier K, McLelland GL and Fon EA (2013) Parkin- and PINK1-Dependent Mitophagy in

Neurons: Will the Real Pathway Please Stand Up? Front Neurol 4:100.

Gulati P, Gaspers LD, Dann SG, Joaquin M, Nobukuni T, Natt F, Kozma SC, Thomas AP and

Thomas G (2008) Amino acids activate mTOR complex 1 via Ca2+/CaM signaling to hVps34. Cell

Metab 7(5):456-465.

Guo L, Stripay JL, Zhang X, Collage RD, Hulver M, Carchman EH, Howell GM, Zuckerbraun BS,

Lee JS and Rosengart MR (2013) CaMKIalpha regulates AMP kinase-dependent, TORC-1-

independent autophagy during lipopolysaccharide-induced acute lung neutrophilic

inflammation. J Immunol 190(7):3620-3628.

Hall DP, Cost NG, Hegde S, Kellner E, Mikhaylova O, Stratton Y, Ehmer B, Abplanalp WA,

Pandey R, Biesiada J, Harteneck C, Plas DR, Meller J and Czyzyk-Krzeska MF (2014) TRPM3 and

miR-204 establish a regulatory circuit that controls oncogenic autophagy in clear cell renal cell

carcinoma. Cancer Cell 26(5):738-753.

Hamada K, Terauchi A, Nakamura K, Higo T, Nukina N, Matsumoto N, Hisatsune C, Nakamura T

and Mikoshiba K (2014) Aberrant calcium signaling by transglutaminase-mediated

posttranslational modification of inositol 1,4,5-trisphosphate receptors. Proc Natl Acad Sci U S

A 111(38):E3966-3975.

Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, Yokoyama M,

Mishima K, Saito I, Okano H and Mizushima N (2006) Suppression of basal autophagy in neural

cells causes neurodegenerative disease in mice. Nature 441(7095):885-889.

Harr MW and Distelhorst CW (2010) Apoptosis and autophagy: decoding calcium signals that

mediate life or death. Cold Spring Harb Perspect Biol 2(10):a005579.

Hoyer-Hansen M, Bastholm L, Mathiasen IS, Elling F and Jaattela M (2005) Vitamin D analog

EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death. Cell

Death Differ 12(10):1297-1309.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

32

Hoyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, Bianchi K,

Fehrenbacher N, Elling F, Rizzuto R, Mathiasen IS and Jaattela M (2007) Control of

macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell

25(2):193-205.

Hurley RL, Anderson KA, Franzone JM, Kemp BE, Means AR and Witters LA (2005) The

Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases.

J Biol Chem 280(32):29060-29066.

Hyser JM, Utama B, Crawford SE, Broughman JR and Estes MK (2013) Activation of the

endoplasmic reticulum calcium sensor STIM1 and store-operated calcium entry by rotavirus

requires NSP4 viroporin activity. J Virol 87(24):13579-13588.

Jia W, Pua HH, Li QJ and He YW (2011) Autophagy regulates endoplasmic reticulum

homeostasis and calcium mobilization in T lymphocytes. J Immunol 186(3):1564-1574.

Jin Y, Bai Y, Ni H, Qiang L, Ye L, Shan Y and Zhou M (2016) Activation of autophagy through

calcium-dependent AMPK/mTOR and PKCtheta pathway causes activation of rat hepatic

stellate cells under hypoxic stress. FEBS Lett 590(5):672-682.

Jouaville LS, Pinton P, Bastianutto C, Rutter GA and Rizzuto R (1999) Regulation of

mitochondrial ATP synthesis by calcium: evidence for a long-term metabolic priming. Proc Natl

Acad Sci U S A 96(24):13807-13812.

Jung CH, Ro SH, Cao J, Otto NM and Kim DH (2010) mTOR regulation of autophagy. FEBS Lett

584(7):1287-1295.

Kang R, Zeh HJ, Lotze MT and Tang D (2011) The Beclin 1 network regulates autophagy and

apoptosis. Cell Death Differ 18(4):571-580.

Kapuy O, Vinod PK and Banhegyi G (2014) mTOR inhibition increases cell viability via

autophagy induction during endoplasmic reticulum stress - An experimental and modeling

study. FEBS Open Bio 4:704-713.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

33

Khan MT and Joseph SK (2010) Role of inositol trisphosphate receptors in autophagy in DT40

cells. J Biol Chem 285(22):16912-16920.

Kilpatrick BS, Eden ER, Schapira AH, Futter CE and Patel S (2013) Direct mobilisation of

lysosomal Ca2+ triggers complex Ca2+ signals. J Cell Sci 126(Pt 1):60-66.

Kim MJ, Woo SJ, Yoon CH, Lee JS, An S, Choi YH, Hwang SG, Yoon G and Lee SJ (2011)

Involvement of autophagy in oncogenic K-Ras-induced malignant cell transformation. J Biol

Chem 286(15):12924-12932.

Kiviluoto S, Schneider L, Luyten T, Vervliet T, Missiaen L, De Smedt H, Parys JB, Methner A and

Bultynck G (2012) Bax inhibitor-1 is a novel IP(3) receptor-interacting and -sensitizing protein.

Cell Death Dis 3:e367.

Klein C and Westenberger A (2012) Genetics of Parkinson's disease. Cold Spring Harb Perspect

Med 2(1):a008888.

Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, Ueno T, Koike M, Uchiyama Y,

Kominami E and Tanaka K (2006) Loss of autophagy in the central nervous system causes

neurodegeneration in mice. Nature 441(7095):880-884.

Kondratskyi A, Yassine M, Kondratska K, Skryma R, Slomianny C and Prevarskaya N (2013)

Calcium-permeable ion channels in control of autophagy and cancer. Front Physiol 4:272.

Koyano F, Okatsu K, Kosako H, Tamura Y, Go E, Kimura M, Kimura Y, Tsuchiya H, Yoshihara H,

Hirokawa T, Endo T, Fon EA, Trempe JF, Saeki Y, Tanaka K and Matsuda N (2014) Ubiquitin is

phosphorylated by PINK1 to activate parkin. Nature 510(7503):162-166.

Lee HK, Lund JM, Ramanathan B, Mizushima N and Iwasaki A (2007) Autophagy-dependent

viral recognition by plasmacytoid dendritic cells. Science 315(5817):1398-1401.

Lenz G and Avruch J (2005) Glutamatergic regulation of the p70S6 kinase in primary mouse

neurons. J Biol Chem 280(46):38121-38124.

Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H and Levine B (1999)

Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402(6762):672-676.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

34

Lu Y, Hao BX, Graeff R, Wong CW, Wu WT and Yue J (2013) Two pore channel 2 (TPC2) inhibits

autophagosomal-lysosomal fusion by alkalinizing lysosomal pH. J Biol Chem 288(33):24247-

24263.

Mairet-Coello G, Courchet J, Pieraut S, Courchet V, Maximov A and Polleux F (2013) The

CAMKK2-AMPK kinase pathway mediates the synaptotoxic effects of Abeta oligomers through

Tau phosphorylation. Neuron 78(1):94-108.

Mallilankaraman K, Cardenas C, Doonan PJ, Chandramoorthy HC, Irrinki KM, Golenar T,

Csordas G, Madireddi P, Yang J, Muller M, Miller R, Kolesar JE, Molgo J, Kaufman B, Hajnoczky

G, Foskett JK and Madesh M (2013) MCUR1 is an essential component of mitochondrial Ca2+

uptake that regulates cellular metabolism. Nat Cell Biol 14(12):1336-1343.

Mallilankaraman K, Doonan P, Cardenas C, Chandramoorthy HC, Muller M, Miller R, Hoffman

NE, Gandhirajan RK, Molgo J, Birnbaum MJ, Rothberg BS, Mak DO, Foskett JK and Madesh M

(2012) MICU1 is an essential gatekeeper for MCU-mediated mitochondrial Ca(2+) uptake that

regulates cell survival. Cell 151(3):630-644.

Marino G, Niso-Santano M, Baehrecke EH and Kroemer G (2014) Self-consumption: the

interplay of autophagy and apoptosis. Nat Rev Mol Cell Biol 15(2):81-94.

Martinez-Vicente M (2015) Autophagy in neurodegenerative diseases: From pathogenic

dysfunction to therapeutic modulation. Semin Cell Dev Biol 40:115-126.

McCormack JG, Halestrap AP and Denton RM (1990) Role of calcium ions in regulation of

mammalian intramitochondrial metabolism. Physiol Rev 70(2):391-425.

Medina DL, Di Paola S, Peluso I, Armani A, De Stefani D, Venditti R, Montefusco S, Scotto-

Rosato A, Prezioso C, Forrester A, Settembre C, Wang W, Gao Q, Xu H, Sandri M, Rizzuto R, De

Matteis MA and Ballabio A (2015) Lysosomal calcium signalling regulates autophagy through

calcineurin and TFEB. Nat Cell Biol 17(3):288-299.

Miao Y, Li G, Zhang X, Xu H and Abraham SN (2015) A TRP Channel Senses Lysosome

Neutralization by Pathogens to Trigger Their Expulsion. Cell 161(6):1306-1319.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

35

Mogk A and Bukau B (2014) Mitochondria tether protein trash to rejuvenate cellular

environments. Cell 159(3):471-472.

Monteith GR, Davis FM and Roberts-Thomson SJ (2012) Calcium channels and pumps in

cancer: changes and consequences. J Biol Chem 287(38):31666-31673.

Monteith GR, McAndrew D, Faddy HM and Roberts-Thomson SJ (2007) Calcium and cancer:

targeting Ca2+ transport. Nat Rev Cancer 7(7):519-530.

Papadakis M, Hadley G, Xilouri M, Hoyte LC, Nagel S, McMenamin MM, Tsaknakis G, Watt SM,

Drakesmith CW, Chen R, Wood MJ, Zhao Z, Kessler B, Vekrellis K and Buchan AM (2013) Tsc1

(hamartin) confers neuroprotection against ischemia by inducing autophagy. Nat Med

19(3):351-357.

Parys JB, Decuypere JP and Bultynck G (2012) Role of the inositol 1,4,5-trisphosphate

receptor/Ca2+-release channel in autophagy. Cell Commun Signal 10(1):17.

Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD and

Levine B (2005) Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell

122(6):927-939.

Puleston DJ and Simon AK (2014) Autophagy in the immune system. Immunology 141(1):1-8.

Qian T, Herman B and Lemasters JJ (1999) The mitochondrial permeability transition mediates

both necrotic and apoptotic death of hepatocytes exposed to Br-A23187. Toxicol Appl

Pharmacol 154(2):117-125.

Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R,

O'Kane CJ and Rubinsztein DC (2004) Inhibition of mTOR induces autophagy and reduces

toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat

Genet 36(6):585-595.

Rimessi A, Bonora M, Marchi S, Patergnani S, Marobbio CM, Lasorsa FM and Pinton P (2013)

Perturbed mitochondrial Ca2+ signals as causes or consequences of mitophagy induction.

Autophagy 9(11):1677-1686.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

36

Rivero-Rios P, Gomez-Suaga P, Fdez E and Hilfiker S (2014) Upstream deregulation of calcium

signaling in Parkinson's disease. Front Mol Neurosci 7:53.

Rubinstein AD, Eisenstein M, Ber Y, Bialik S and Kimchi A (2011) The autophagy protein Atg12

associates with antiapoptotic Bcl-2 family members to promote mitochondrial apoptosis. Mol

Cell 44(5):698-709.

Ryan BJ, Hoek S, Fon EA and Wade-Martins R (2015) Mitochondrial dysfunction and mitophagy

in Parkinson's: from familial to sporadic disease. Trends Biochem Sci 40(4):200-210.

Sano R, Hou YC, Hedvat M, Correa RG, Shu CW, Krajewska M, Diaz PW, Tamble CM, Quarato G,

Gottlieb RA, Yamaguchi M, Nizet V, Dahl R, Thomas DD, Tait SW, Green DR, Fisher PB,

Matsuzawa S and Reed JC (2012) Endoplasmic reticulum protein BI-1 regulates Ca(2)(+)-

mediated bioenergetics to promote autophagy. Genes Dev 26(10):1041-1054.

Sarkar S, Floto RA, Berger Z, Imarisio S, Cordenier A, Pasco M, Cook LJ and Rubinsztein DC

(2005) Lithium induces autophagy by inhibiting inositol monophosphatase. J Cell Biol

170(7):1101-1111.

Schaeffer V, Lavenir I, Ozcelik S, Tolnay M, Winkler DT and Goedert M (2012) Stimulation of

autophagy reduces neurodegeneration in a mouse model of human tauopathy. Brain 135(Pt

7):2169-2177.

Schondorf DC, Aureli M, McAllister FE, Hindley CJ, Mayer F, Schmid B, Sardi SP, Valsecchi M,

Hoffmann S, Schwarz LK, Hedrich U, Berg D, Shihabuddin LS, Hu J, Pruszak J, Gygi SP, Sonnino S,

Gasser T and Deleidi M (2014) iPSC-derived neurons from GBA1-associated Parkinson's disease

patients show autophagic defects and impaired calcium homeostasis. Nat Commun 5:4028.

Sheng R and Qin ZH (2015) The divergent roles of autophagy in ischemia and preconditioning.

Acta Pharmacol Sin 36(4):411-420.

Shigetomi E, Patel S and Khakh BS (2016) Probing the Complexities of Astrocyte Calcium

Signaling. Trends Cell Biol 26(4):300-312.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

37

Shoshan-Barmatz V, De Pinto V, Zweckstetter M, Raviv Z, Keinan N and Arbel N (2010) VDAC, a

multi-functional mitochondrial protein regulating cell life and death. Mol Aspects Med

31(3):227-285.

Storr SJ, Carragher NO, Frame MC, Parr T and Martin SG (2011) The calpain system and cancer.

Nat Rev Cancer 11(5):364-374.

Subramani S and Malhotra V (2013) Non-autophagic roles of autophagy-related proteins.

EMBO Rep 14(2):143-151.

Sui X, Chen R, Wang Z, Huang Z, Kong N, Zhang M, Han W, Lou F, Yang J, Zhang Q, Wang X, He C

and Pan H (2013) Autophagy and chemotherapy resistance: a promising therapeutic target for

cancer treatment. Cell Death Dis 4:e838.

Tian X, Gala U, Zhang Y, Shang W, Nagarkar Jaiswal S, di Ronza A, Jaiswal M, Yamamoto S,

Sandoval H, Duraine L, Sardiello M, Sillitoe RV, Venkatachalam K, Fan H, Bellen HJ and Tong C

(2015) A voltage-gated calcium channel regulates lysosomal fusion with endosomes and

autophagosomes and is required for neuronal homeostasis. PLoS Biol 13(3):e1002103.

Vellinga TT, Borovski T, de Boer VC, Fatrai S, van Schelven S, Trumpi K, Verheem A, Snoeren N,

Emmink BL, Koster J, Rinkes IH and Kranenburg O (2015) SIRT1/PGC1alpha-Dependent Increase

in Oxidative Phosphorylation Supports Chemotherapy Resistance of Colon Cancer. Clin Cancer

Res 21(12):2870-2879.

Viale A, Pettazzoni P, Lyssiotis CA, Ying H, Sanchez N, Marchesini M, Carugo A, Green T, Seth S,

Giuliani V, Kost-Alimova M, Muller F, Colla S, Nezi L, Genovese G, Deem AK, Kapoor A, Yao W,

Brunetto E, Kang Y, Yuan M, Asara JM, Wang YA, Heffernan TP, Kimmelman AC, Wang H,

Fleming JB, Cantley LC, DePinho RA and Draetta GF (2014) Oncogene ablation-resistant

pancreatic cancer cells depend on mitochondrial function. Nature 514(7524):628-632.

Vicencio JM, Ortiz C, Criollo A, Jones AW, Kepp O, Galluzzi L, Joza N, Vitale I, Morselli E, Tailler

M, Castedo M, Maiuri MC, Molgo J, Szabadkai G, Lavandero S and Kroemer G (2009) The


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

38

inositol 1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1.

Cell Death Differ 16(7):1006-1017.

Wang SH, Shih YL, Ko WC, Wei YH and Shih CM (2008) Cadmium-induced autophagy and

apoptosis are mediated by a calcium signaling pathway. Cell Mol Life Sci 65(22):3640-3652.

Ward PS and Thompson CB (2012) Metabolic reprogramming: a cancer hallmark even warburg

did not anticipate. Cancer Cell 21(3):297-308.

Wei Y, Pattingre S, Sinha S, Bassik M and Levine B (2008) JNK1-mediated phosphorylation of

Bcl-2 regulates starvation-induced autophagy. Mol Cell 30(6):678-688.

Williams A, Sarkar S, Cuddon P, Ttofi EK, Saiki S, Siddiqi FH, Jahreiss L, Fleming A, Pask D,

Goldsmith P, O'Kane CJ, Floto RA and Rubinsztein DC (2008) Novel targets for Huntington's

disease in an mTOR-independent autophagy pathway. Nat Chem Biol 4(5):295-305.

Williams GS, Boyman L, Chikando AC, Khairallah RJ and Lederer WJ (2013a) Mitochondrial

calcium uptake. Proc Natl Acad Sci U S A 110(26):10479-10486.

Williams JA, Hou Y, Ni HM and Ding WX (2013b) Role of intracellular calcium in proteasome

inhibitor-induced endoplasmic reticulum stress, autophagy, and cell death. Pharm Res

30(9):2279-2289.

Winslow AR, Chen CW, Corrochano S, Acevedo-Arozena A, Gordon DE, Peden AA, Lichtenberg

M, Menzies FM, Ravikumar B, Imarisio S, Brown S, O'Kane CJ and Rubinsztein DC (2010) alpha-

Synuclein impairs macroautophagy: implications for Parkinson's disease. J Cell Biol

190(6):1023-1037.

Wirawan E, Vande Walle L, Kersse K, Cornelis S, Claerhout S, Vanoverberghe I, Roelandt R, De

Rycke R, Verspurten J, Declercq W, Agostinis P, Vanden Berghe T, Lippens S and Vandenabeele

P (2010) Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and

enhances apoptosis by promoting the release of proapoptotic factors from mitochondria. Cell

Death Dis 1:e18.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

39

Yan WJ, Dong HL and Xiong LZ (2013) The protective roles of autophagy in ischemic

preconditioning. Acta Pharmacol Sin 34(5):636-643.

Yang S, Wang X, Contino G, Liesa M, Sahin E, Ying H, Bause A, Li Y, Stommel JM, Dell'antonio G,

Mautner J, Tonon G, Haigis M, Shirihai OS, Doglioni C, Bardeesy N and Kimmelman AC (2011)

Pancreatic cancers require autophagy for tumor growth. Genes Dev 25(7):717-729.

Yousefi S, Perozzo R, Schmid I, Ziemiecki A, Schaffner T, Scapozza L, Brunner T and Simon HU

(2006) Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol

8(10):1124-1132.

Yue Z, Jin S, Yang C, Levine AJ and Heintz N (2003) Beclin 1, an autophagy gene essential for

early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci U S A

100(25):15077-15082.

Zhang X, Howell GM, Guo L, Collage RD, Loughran PA, Zuckerbraun BS and Rosengart MR

(2014) CaMKIV-dependent preservation of mTOR expression is required for autophagy during

lipopolysaccharide-induced inflammation and acute kidney injury. J Immunol 193(5):2405-

2415.

Zhang X, Yan H, Yuan Y, Gao J, Shen Z, Cheng Y, Shen Y, Wang RR, Wang X, Hu WW, Wang G

and Chen Z (2013) Cerebral ischemia-reperfusion-induced autophagy protects against neuronal

injury by mitochondrial clearance. Autophagy 9(9):1321-1333.

Zhi X and Zhong Q (2015) Autophagy in cancer. F1000Prime Rep 7:18.


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

40

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

fellowship from CNPq.

FIGURE LEGENDS

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

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


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

41

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,

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

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.

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.

Alterations in [Ca2+]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

[Ca2+]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 –


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

42

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

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

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 ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


MOL #105171

43

TABLE 1 Cell and cancer type Inducer of Ca2+ imbalance and autophagy Ca2+ Modulation and consequences to

autophagy Autophagy modulation a Role in autophagy Ref

MCF7 breast cancer Extracellular ATP (release of Ca2+ from the ER through IP3-R)

BAPTA/AM - reduces the % of GFP-LC3+ cells from 40% to 5%

3-MA (35% to <5%) BECN1 KD (35% to 5%) ATG7 KD (35% to 12%)

Not assessed Høyer-Hansen, 2007

MCF7 breast cancer Ionomycin (Ca2+ ionophore) BAPTA/AM - reduces the % of GFP-LC3+ cells from 40% to 10%

3-MA (35% to <5%) BECN1 KD (35% to 15%) ATG7 KD (35% to 12%)

Not assessed Høyer-Hansen, 2007

MCF7 breast cancer Thapsigargin (TG; inhibitor of ER Ca2+- ATPase which maintains high levels of Ca2+ in the cytosol)

BAPTA/AM - reduces the % of GFP-LC3+ cells from 52% to 5%

3-MA (45% to 18%) BECN1 KD (45% to 30%) ATG7 KD (45% to 28%)

No assessed Høyer-Hansen, 2007

MCF7 breast cancer Vitamin D analogue EB1089 (increases cytosolic Ca2+, but the mechanism is not fully known)

Not assessed

3-MA (80% to 20%) - decreased cell death

Overexpression of BECN1 increased cell death

Cytotoxic Høyer-Hansen, 2005

Hela cervix adenocarcinoma

Starvation using HBSS (increases cytosolic Ca2+ from the ER by IP3-R; also disrupts the IP3-R/Bcl-

2/BECN1 complex).

BAPTA/AM and xestospongin (IP3-R inhibitor) - reduces the % of GFP-LC3-

positive cells and the LC3II levels to control levels

Not assessed Not assessed Decuypere, 2011

Hela cervix adenocarcinoma

Starvation using HBSS (increases cytosolic Ca2+ from the ER by IP3-R; also disrupts the IP3-R/Bcl-

2/BECN1 complex).

Overexpression of Bcl2 - reduced around a half the percentage of GFP-LC3-

positive cells Not assessed Not assessed b Vivencio 2009

HCT116 Colon cancer

ATP; TG-induced ER stress;

A23187-induced ER stress (Ca2+ ionophore) Not assessed

ATG6 KD and ATG8 KD - increased caspase activation and apoptotic cell death from ~30% to 47% and 62%

respectively; 3MA also increased cell death, but data

are not shown

Cytoprotective Ding, 2007

DU145 Prostate Cancer ATP;

TG-induced ER stress; A23187-induced ER stress (Ca2+ ionophore)

Not assessed ATG6 KD, ATG8 KD and 3-MA induced

cell death, but data are not shown. Obs: the efficiency is not shown

Cytoprotective Ding, 2007

Murine embryonic fibroblasts

TG-induced ER stress; A23187-induced ER stress;

Not assessed ATG5 KD decreased cell death Cytotoxic Ding, 2007

CCD-18Co normal colon TG-induced ER stress;

A23187-induced ER stress;

Not assessed 3-MA decreased cell death Cytotoxic Ding, 2007

HepG2 Hepatocarcinoma TG-induced ER stress Not assessed

Rapamycin and metyrapone (mTOR inhibitor) - increased autophagy and

cell viability

Cytoprotective (indirect) Kapuy, 2014

DT40 chicken lymphoma IP3-R triple knock out (TKO) cells Restoration of IP3-R expression decreased basal autophagy

Rapamycin - increased autophagy in WT but not in DT40 TKO cells Not assessed b Khan and Joseph, 2010

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.

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

Molecular Pharm

acology Fast Forward. Published on July 19, 2016 as D

OI: 10.1124/m

ol.116.105171 at ASPET Journals on August 30, 2020 molpharm.aspetjournals.org Downloaded from


L

Extracell

Autophagosome+ Lysosome

AutolysosomePhagophore

Endoplasmic Reticulum

CaAMPKmTORC1

MAPKTP53

PI3k/Akt

ATG5-ATG12ATG16L1

Calpain

LC3

LC3-II

LC3-I

LC3-II

PE

AdaptorCargo

TKRECEPTOR

Insulin

INSULIN RECEPTOR

Growth Factor

TSC1/2Rheb

IGF-R1

IGF

LysosomeH+

Na+Ca

H+ H+H+

H+

H+

H+

2+

Na /H Pump Exchange

TRPML3 / TRPML1

Lysosomal Permease

+ +

Lysosomal Acid Hidrolases

FIGURE 1

Intracell

mTOR Complex 1 (mTORC1)

mTORRaptor

mLST8

Cell Stress

Aminoacids

Oxygen (hypoxia)

GlucoseInsulin

CytokinesOncogenes

Tumor supressors

Infectious Agents

ATP

AMP/ATP

StarvationRapamycin

IP3-R

IP3

Metabotropic Receptor

G-protein PIP2 DAGα α

Phospholipase C

ULK1FIP200 ATG13

AMPK

mTORC1

TSC2

ULK1FIP200ATG13

BECN1p150

Vps34

P

AUTOPHAGY

ATG14

Box 3 Box 4

Box 2Box 1

Box 3

Box 4

Box 2Box 1

AMP/ATP GlucoseStarvation

Phagophore

Rheb

CAMKK2

LKB1

2+

Ca2+

P

P

CAMKK2

IonotropicReceptor

ATPHormonesNeurotransmitters

EndoplasmicReticulum0.4-0.8mM

Mitochondria200nM

Ca2+

Lysosome0.4-0.6mM

IP3-R and IP3IP3-R / BECN1 / Bcl2 complex

TGM2

VDAC1MICU1 and MCU

TRPML1 and 3TRPM2

TFEB

?

?

Free Ca

2+

2+

Box 5

Box 5

Extracellular Environment

1-2mMVDCCNMDA TRPC

BI-1

Ca2+ channels

Cytosol100nM

Ca leak2+

FILIPPI-CHIELA & VIEGAS, 2016


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


2+

Involved in the induction of Autophagy

Inhibition of IP3-R(Ex.: Xestospongin B)

Inhibit or maintain basal autophagy at low leves

Depletion of IP3(lithium cloride)

Knockdown of IP3-R

Xestospongin Bsuppresses autophagy

induced by starvation

BAPTA suppressesautophagy induced

by RAPA, hypoxia and extracellular ATP

FILIPPI-CHIELA & VIEGAS, 2016 FIGURE 2

Antagonist of plasma membrane L-type

Ca channels

KD of TGM2 (a regulator of IP3-R)BAPTA blocks the

induced by MG132Diturbances in

mitochondrial Ca2+2+

Ca signals

ExperimentalEvidence

AutophagyRapamycin

Extracel. ATP

IP3-RBcl2

BECN1

Availability of BECN1

Ca inmitochondria

2+

Normal ATP levels

AMPK

Starvation Ca released from lysosomes

(MCOLN1)

2+

TFEB

Ca signalosome is positively involved in induced autophagy

Ca signalosome inhibits autophagy or maintains basal autophagy at low levels

2+2+

IP3-R / IP3 Signaling

a

c

ba

c

b

e f

a

b

e

f

L-type Ca channels(plasma membrane)

2+2+

dBAPTA blocks the activation of TFEB

induced by starvation

a

cb

da

2+Ca signals

Calcineurin

d

c

MG132 (proteas. inhibitor)

Hypoxiaa

a

JNK

Increase basal autophagy

d

ExperimentalEvidence


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


Nucleus

UPR

CAMKK2

AUTOPHAGY

ATPAMPK

[Ca ]2+c

AMPK

AUTOPHAGY

mTORC1

ATPP2y

JNK

STARVATIO

N

IonomycinA23187

PBcl2

BECN1

IP3

FIGURE 3

PIP2DAG

Phospholipase C

P

CANCER

Sepsis / LPS

[Ca ]2+c

CAMK4

AUTOPHAGY

FBXW7

GSK-3

IL6

PP

mTORC1

TLR?

STIM1

Ca2+NSP4

[Ca ]2+c

CAMKK2

AMPK

mTORC1

Orai1

Viroplast

LC3-II

pH4-5

pH7

pH7

Ca2+

lysosome

Vitamin D and analogues(e.g. EB1089)

IP3-R

Thapsigargin Ca ionophores2+

ERStress

‘Extracellular’ Ca2+‘Intracellular’ Ca2+

β

IMMUNITYCa

2+

G-protein(Gi, Gq/11 or Gs)

α

[Ca ]2+c

Toxic during Ischemia

Protective at PC and reperfusion

Removal of aggregatedproteins

VGCC

SynapticNMDA receptors

Calpain

NEURO

versusREphagy

Functional Mitochondria

VDAC1

ERCytosol

HSPA9IP3R MCUR1

ERCytosol

BH3 domain

BECN1

BCL2 IP3R

NAF-1

ERCytosol

BECN1

BCL2 IP3R

NAF-1JNK

P

DAPK P

BH3 mimetics

P

P

Autophagy Autophagy

Box1

Box2

Box2

Box1

A B

Normal Ca transfer: normal mitochondria functioning

Altered Ca transfer

ATP AMPK AutophagyMitophagyCell Death

2+

2+ Cell Survival

TFEB

pTFEB

Calcineurin

Autophagy and lysosomegene transcription

TRPM3

FILIPPI-CHIELA & VIEGAS, 2016


at ASPE

T Journals on A

ugust 30, 2020m


Dow

nloaded from


title page modulation of autophagy by calcium signalosome in...

Documents