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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.
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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
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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.
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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
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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
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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
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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.
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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
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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
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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,
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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
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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).
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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).
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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
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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
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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
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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
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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
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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.
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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?
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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
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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
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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
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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+-
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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
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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
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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.
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FOOTNOTES
* ECFC and MSV contributed equally to this work.
This work was supported by Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), Universal (475882/2012-1 and 458139/2014-9) and Novas
Terapias Portadoras de Futuro (457394/2013-7); PROBITEC-CAPES (004/2012) and
ICGEB BRA11/01. ECFC, MSV and MPT are or were recipient of CNPq fellowships
and ECFC is recipient of CAPES fellowship. MRW and GL are recipients of research
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
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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 –
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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.
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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.
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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
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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
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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
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