IP(3) Receptors, Mitochondria, and Ca Signaling: Implications for Aging.

Abstract

The tight interplay between endoplasmic-reticulum-(ER-) and mitochondria-mediated Ca(2+) signaling is a key determinant of cellular health and cellular fate through the control of apoptosis and autophagy. Proteins that prevent or promote apoptosis and autophagy can affect intracellular Ca(2+) dynamics and homeostasis through binding and modulation of the intracellular Ca(2+)-release and Ca(2+)-uptake mechanisms. During aging, oxidative stress becomes an additional factor that affects ER and mitochondrial function and thus their role in Ca(2+) signaling. Importantly, mitochondrial dysfunction and sustained mitochondrial damage are likely to underlie part of the aging process. In this paper, we will discuss the different mechanisms that control intracellular Ca(2+) signaling with respect to apoptosis and autophagy and review how these processes are affected during aging through accumulation of reactive oxygen species.

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SAGE-Hindawi Access to Research
Journal of Aging Research
Volume 2011, Article ID 920178, 20 pages
doi:10.4061/2011/920178
Review Article
IP3Receptors, Mitochondria, andCa2+Signaling:
ImplicationsforAging
Jean-PaulDecuypere,Giovanni Monaco,LudwigMissiaen, HumbertDe Smedt,
JanB. Parys, and GeertBultynck
Laboratory of Molecular and Cellular Signaling, Department of Molecular and Cellular Biology, K.U.Leuven,
Campus Gasthuisberg O/N-1, Herestraat 49, Bus 802, 3000 Leuven, Belgium
Correspondence should be addressed to Geert Bultynck, geert.bultynck@med.kuleuven.be
Received 15 October 2010; Revised 23 December 2010; Accepted 5 January 2011
Academic Editor: Christiaan Leeuwenburgh
Copyright © 2011 Jean-Paul Decuypere et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
The tight interplay between endoplasmic-reticulum-(ER-) and mitochondria-mediated Ca2+signaling is a key determinant of
cellular health and cellular fate through the control of apoptosis and autophagy. Proteins that prevent or promote apoptosis
and autophagy can affect intracellular Ca2+dynamics and homeostasis through binding and modulation of the intracellular
Ca2+-release and Ca2+-uptake mechanisms. During aging, oxidative stress becomes an additional factor that affects ER and
mitochondrialfunctionandthustheirroleinCa2+signaling.Importantly,mitochondrialdysfunctionandsustainedmitochondrial
damage are likely to underlie part of the aging process. In this paper, we will discuss the different mechanisms that control
intracellular Ca2+signaling with respect to apoptosis and autophagy and review how these processes are affected during aging
through accumulation of reactive oxygen species.
1.IntracellularCa2+Signaling
Intracellular Ca2+signaling is important in the regula-
tion of multiple cellular processes, including development,
proliferation, secretion, gene activation, and cell death.
The formation of these Ca2+signals is dependent on
many cellular Ca2+-binding and Ca2+-transporting proteins,
present in the various cell compartments of which the en-
doplasmic reticulum (ER) forms the main intracellular Ca2+
store [1]. The resting cytosolic [Ca2+] remains very low
(∼100nM), through active extrusion of Ca2+by pumps in
the plasma membrane or in intracellular organelles, like the
sarco/endoplasmic reticulum Ca2+ATPase (SERCA) pump
in the ER. Due to SERCA activity and intraluminal Ca2+-
binding proteins, the ER can accumulate Ca2+in more
than thousandfold excess compared to the cytosol [1, 2].
In the ER lumen, Ca2+functions as an important cofactor
for ER chaperones, thereby aiding in the proper folding
of newly synthesized proteins [3]. Reciprocally, the Ca2+-
binding chaperones affect the Ca2+capacity of the ER by
buffering Ca2+[2]. In addition, two tetrameric ER Ca2+-
release channels exist that, upon stimulation, release Ca2+
into the cytosol, thereby provoking Ca2+signaling: the
inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and the
ryanodine receptor (RyR). They are similar in function and
structure but differ in regulation, conductance, and expres-
sion profile [4, 5]. The rise in cytosolic [Ca2+] following
its release from the ER results in various Ca2+-dependent
intracellular events. The exact cellular outcome depends
on the spatiotemporal characteristics of the generated Ca2+
signal [6]. Since close contact sites between the ER and
the mitochondria, involving direct molecular links with the
IP3R, exist (Figure 1), it is clear that ER-originating Ca2+
signals critically affect the mitochondrial function.
During aging, ER Ca2+homeostasis alters and becomes
dysregulated [7]. Most observations support a decline in
ER [Ca2+] and in ER Ca2+release (due to lower activity
of SERCA, IP3R, and RyR), but contradictory findings have
been published, possibly related to the cell type under
investigation (Figure 2). In addition, ER Ca2+release and

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2Journal of Aging Research
RyR
RyR
RyR
CaBC
CaBC
CaBC
CaBC
Extracellular space
Agonist
7TM-receptor
PIP2
EGF ligand
EGF receptor
EGF receptor
PLC-γ
PLC-β
IP3
ER
SERCA
SERCA
SERCA
CaBC
Cyt C
Cyt C
Fhit
Cyt C
Bcl-2/Xl
IP3R
IP3R
IP3R
IP3R
Grp75
Grp75
Grp75
VDAC
VDAC
VDAC
MCU
MCU
Sig-1R
MFN2MFN1/2
IP3R
Sirtuins
Sirtuins
ATP
production/
metabolism
PML
PP2a
P
P
ROS
AnkB
BAX
BAK
BAX
BAK
p66Shc
Mitochondrion
Bcl-2/Xl
Bcl-2/Xl
Bcl-2/Xl
Akt
Akt
Figure 1: In a healthy cell, ER Ca2+-handling components tightly regulate mitochondrial function and bioenergetics, representing the
different key players involved in intracellular Ca2+signalling with particular emphasis on the ER-mitochondria connections. The ER-Ca2+
content is regulated by channels and pumps (IP3Rs, RyRs, SERCAs) and by Ca2+-binding chaperones (CaBCs). IP3stimulates ER Ca2+
release and consequently the transfer of Ca2+(red dots) from ER to mitochondria. Mitochondrial Ca2+, transported via VDAC, is directly
or indirectly involved in cellular energy metabolism and in the secondary production of reactive oxygen species (ROS). It is clear that IP3R-
mediated Ca2+release ought to be tightly regulated to sustain mitochondrial activity and function. As a consequence, Ca2+-flux properties
of IP3Rs are tightly and dynamically regulated by accessory proteins involved in cell death and survival, like Bcl-2, Bcl-Xl, PKB/Akt, Sigma-1
receptor (Sig-1R)/Ankyrin B (AnkB), and the recently identified PML. It is important to note that different regulatory mechanisms occur
at the IP3R, which may help cell survival (like Bcl-2, Bcl-Xl, PKB/Akt) or help to promote cell death (like PML). The latter is essential to
prevent the survival of altered, damaged, or oncogenic cells. Thus, a tight balance between both outcomes is a requisite for cellular health
and homeostasis, and a dynamic switch from prosurvival to prodeath is likely essential. In this paradigm, the production of ROS might
contribute to the survival of cells by efficient detection of damaged/altered mitochondria and their removal by autophagy, while preventing
excessive apoptosis. In addition, controlled apoptosisis likelyto be important to eliminatecells, in which the removal of altered mitocondria
by autophagy is not sufficient, thereby avoiding tumor genesis. In this process, the recently identified tumor suppressor PML may play a
crucial role as it promotes IP3R-mediated Ca2+transfer from the ER into the mitochondria by dephosphorylating and suppressing PKB/Akt
activity through PP2A. While PKB/Akt is known to suppress IP3R-channel activity by phosphorylation of the IP3R, the recruitment of
PP2AviaPMLattheinterorganellarER/mitochondrialcomplexdephosphorylatesandinactivatesPKB/Akt.ThissuppressesPKB-dependent
phosphorylationofIP3RandthuspromotesCa2+releasethroughthischannelandCa2+transferintothemitochondria.Atthemitochondrial
level, the tumor suppressor Fhit has been shown to increase the affinity for the mitochondrial Ca2+uniporter (MCU), thereby enhancing
the uptake of mitochondrial Ca2+at low and physiologically relevant levels of agonist-induced Ca2+signals. Green arrows: stimulation; red
lines: inhibition; black arrows: Ca2+flux.

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Journal of Aging Research3
Ca2+signaling
Suppressed
Normal
Exaggerated
Most agec d cells
Reduced IP3R/RyR levels
Reduced SERCA levels/activity
Reduced ER Ca2+buffering
Decreased ER Ca2+content
Dysfunctional IP3Rs
Neurodegenerative disease
Chronic heart failure
Increased IP3R and RyR
levels/activity
Increased RyR activity
Aged neurons
Cardiac hypertrophy
Aged rat hearts
Enhanced
Normal cells
Huntington’s disease
Attenuated
RyR upregulation
Enhanced IP3signaling
Increased IP3R levels
Figure 2: Altered Ca2+signalingduring aging and in age-related diseases. The Ca2+dyshomeostasisduring age is dependent on the cell type
and the context. Most aged cells display decreased ER Ca2+content and release, due to declined IP3R or RyR levels, reduced SERCA activity,
and decreased Ca2+buffering by intraluminal Ca2+-binding chaperones. However, in neurons and rat hearts, an enhanced Ca2+signaling is
found, caused by increasing IP3R or RyR activity. Age-related diseases (neurodegeneration, cardiac hypertrophy, and chronic heart failure)
are also characterized by enhanced Ca2+signaling. However, this property may be disease dependent, since a mouse model for Huntington’s
disease displayed attenuated IP3R1 activity due to impaired binding of Grp78 to IP3R1. Hence, caution should be taken with general claims.
subsequent Ca2+uptake by mitochondria regulate reactive
oxygen species (ROS)production, autophagy, and cell death,
processes implicated in aging.
In apreviousreview[8],we havefocusedon mechanisms
regulating the Ca2+content in the ER and its relevance for
the development of physiological versus pathophysiological
Ca2+signalling. In the present review, we will focus on the
subsequent step which is the mechanisms responsible for
controlling Ca2+transfer from the ER to the mitochondria.
The Ca2+level in the mitochondrial matrix plays an impor-
tant role in the progression of apoptosis and autophagy
[9, 10]. Here,we will especially analyze how theCa2+transfer
to the mitochondria as well as apoptosis and autophagy
are affected by the aging process in general and by reactive
oxygen species in particular.
2.Mitochondrial Ca2+Handling
In contrast with the role of the ER, the role of the mitochon-
dria in physiological Ca2+handling was underestimated or
even ignored for a long time, but due to the seminal work
of Rizzuto and his colleagues [11], this role is now generally
accepted.
The electrochemical gradient (Δψm = −180mV) be-
tween the inside and outside of energized mitochondria
forms the driving force for the Ca2+uptake in the mitochon-
drial matrix, which implies the transfer of Ca2+ions over
both the outer mitochondrial membrane (OMM) and the
inner mitochondrial membrane (IMM).
The Ca2+ions taken up into the mitochondrial matrix
stimulate the mitochondrial ATP production by regulating
the activities of isocitrate dehydrogenase, α-ketoglutarate
dehydrogenase, and pyruvate dehydrogenase, three dehydro-
genases of the Krebs cycle [12, 13]. Also other mitochondrial
processes as fatty acid oxidation, amino acid catabolism,
aspartate and glutamate carriers, the adenine-nucleotide
translocase, Mn-superoxide dismutase,and F1-ATPase activ-
ity, are regulated by mitochondrial Ca2+[12, 14, 15].
The ATP produced by the mitochondria is subsequently
transferred to the cytoplasm; it will so especially regulate
the activity of ATP-sensitive proteins localized in the close
vicinity of the mitochondria. Two major proteins involved in
Ca2+transport, the SERCA, responsible for loading the ER,
and the IP3Rs, responsible for Ca2+release from the ER, are
stimulated by ATP. The bidirectional relation between Ca2+
release and ATP production allows for a positive feedback
regulation between ER and mitochondria during increased
energetic demand [16].
The uptake of Ca2+in the mitochondria will also affect
Ca2+signaling. The local Ca2+concentration near the mito-
chondria will depend on both the amount of Ca2+released
by the IP3R and that taken up by the mitochondria. This
will in turn depend on the efficiency of the coupling between
both. Since both the SERCA pumps and the IP3Rs are also
regulatedbyCa2+,thelocalCa2+concentrationinthevicinity
of the mitochondria will determine the refilling of the
ER and eventually the spatiotemporal characteristics of the
subsequent Ca2+signals. The way in which the Ca2+signals
are affected depends on the exact subcellular localization of
the mitochondria, the production of ROS, the local Ca2+
concentration, the IP3R isoform expressed, and may as well
involve stimulation as inhibition of the signals [16–19].
Furthermore, the connection between mitochondria and the
ER can be highly dynamic as the local Ca2+concentration
can also affect mitochondrial motility and ER-mitochondria
associations in various ways [20].

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4Journal of Aging Research
3.TransportProteinsInvolved in
the TransferofCa2+betweenERand
Mitochondria
3.1. IP3Rs. The first key player is the IP3R, the main Ca2+-
releasechannelintheERofmostcelltypes.TheIP3Rconsists
of 4 subunits of about 310kDa each (i.e., about 2700 a.a.).
In mammals, three different IP3R isoforms are expressed
(IP3R1, IP3R2, and IP3R3) while diversity is increased by
splicing and the formation of both homo- and heteromeric
channels [4, 21, 22]. All IP3R isoforms are activated by IP3,
though with varying affinity [23]. Low Ca2+concentrations
stimulate but high Ca2+concentrations inhibit the IP3Rs
[24–27]. Further modulation of the IP3Rs is performed
by ATP, phosphorylation, and protein-protein interactions
[4, 28–30].
ForefficientCa2+transferbetweenERandmitochondria,
it is important that IP3Rs are localized very close to the
mitochondrial Ca2+-uptake sites. As different IP3R isoforms
exist, an important point is whether interaction with the
mitochondria is isoform specific [31]. In CHO cells, IP3R3
is the least expressed isoform, but it demonstrated the
highest degree of colocalization with the mitochondria and
consequently its silencing had the most profound effects on
mitochondrial Ca2+signals [32]. However, this does not rep-
resent a general rule as, for example, in astrocytes IP3R2 was
found to preferentially colocalize within the mitochondria
[33]. These differences in intracellular localization of the
IP3Risoformsmaybeduetodifferencesinrelativeexpression
levels of the various IP3R isoforms and in subcellular
localization among different cell types [34]. Moreover, the
physiological setting [35] and the differentiation status [36]
determine the subcellular localization of the various IP3R
isoforms in a given cell type.
3.2. Voltage-Dependent Anion Channels: The Main Ca2+-
Transport System across the OMM. The Ca2+fluxes through
the OMM are mainly determined by voltage-dependent
anion channels (VDAC). Of the 3 existing VDAC isoforms,
VDAC1 is the most abundant in most cell types [37]. It was
demonstrated that the transient overexpression of VDAC in
various cell types led to an increased Ca2+concentration
in the mitochondria, leading to a higher susceptibility for
ceramide-induced cell death [38].
VDAC, however, allows also the transport of other ions
and metabolites, including ATP. It has therefore multiple
functions in the cell and is a central player in the crosstalk
between the cytoplasm and mitochondria. In this manner,
VDAC is also implicated in the induction of apoptosis by
various stimuli [15].
The permeabilization of the OMM is a crucial step in
apoptosis, but how this is exactly performed is not yet clear.
Proteins belonging to the B-cell CLL/lymphoma-2 (Bcl-2)-
protein family appear anyway to be necessary [39, 40].
Several Bcl-2-family members can affect the permeability of
the OMM, for example, by binding to VDAC and regulating
its properties or by forming multimeric channel complexes.
Independently of the mechanism by which the increase in
permeability of the OMM is achieved, it allows the release
of the apoptogenic factors present in the intermembrane
space to the cytoplasm and the progression of apoptosis
[15, 40–42].
3.3. Ca2+-Transport Systems across the IMM. In contrast to
the Ca2+-transport system across the OMM, that of the IMM
is not yet well characterized. For a long time, the main
IMM Ca2+-transport system was named the mitochondrial
Ca2+uniporter. Additionally, a so-called rapid mode of
mitochondrial Ca2+uptake was described, but the nature of
neither was known [43].
Three different highly Ca2+-selective channels that may
contribute to this process were meanwhile characterized,
that is, MiCa [44], mCa1, and mCa2 [45]. Two of these
channels, MiCa and mCa1, have properties compatible with
the former uniporter and may represent species- and/or cell-
type-dependent variability [43]. At the molecular level, the
mitochondrial Ca2+-uptake channels are not yet identified,
but evidence for a role of a number of proteins has been
presented [46, 47]. Recently, a Ca2+-binding protein, named
MICU1, which appears essential for mitochondrial Ca2+
uptake,was described [48]. It is, however, not known wheth-
er it actually forms (part of) a Ca2+channel or functions as
Ca2+bufferor Ca2+sensor. Interestingly, the tumor suppres-
sor protein Fhit (fragile histidine triad) seems to promote
mitochondrial Ca2+uptake by increasing the affinity of
the mitochondrial Ca2+uniporter at the ER/mitochondrial
microdomain [49].
Finally, the permeabilization transition pore (PTP) is
another channel of still unknown nature [50]. It is voltage
and Ca2+dependent and is sensitive to cyclosporine A. It
is not selective for Ca2+as the open conformation of the
PTP has a high conductance for all ions, including Ca2+, and
for molecules up to 1500Da [51]. Its long-time activation
leads to the demise of the cell, either by apoptosis or else
by necrosis, depending on whether PTP opening occurs in
only a small part of the mitochondria or in all of them,
respectively [51, 52].
In addition, Ca2+/Na+and Ca2+/H+exchangers are also
present in the IMM. Their main function is probably to
export Ca2+from the matrix, but they may also contribute
to Ca2+uptake under certain conditions [43].
4.Structuraland RegulatoryProteins
Involved inthe ControlofCa2+
TransferbetweenERand Mitochondria
Mitochondria-associated ERmembranes(MAMs)wereorig-
inally described as sites for lipid synthesis and lipid transfer
between ER and mitochondria [53]. These MAMs are, how-
ever, also ideally suited for Ca2+exchange [14]. Several pro-
teinsmay participate in thestabilization ofthose MAMsand,
through this stabilization, affect Ca2+transfer between ER
and mitochondria. Other proteins may be directly involved
in regulating the Ca2+-transport proteins described above.

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Journal of Aging Research5
4.1. Glucose-Regulated Protein 75. Glucose-regulated protein
75 (Grp75) belongs to the Hsp70 family of chaperones but
is not inducible by heat shock [54, 55]. Importantly, it can
couple the IP3R to VDAC1 and allows for a better transfer of
the Ca2+ions from the ER to the mitochondrial matrix [56].
The increased Ca2+signals in the mitochondria were not due
to an increased ER-mitochondria contact area. These results
indicate that Grp75 is probably not the main determinant
for the ER-mitochondrial linkage but regulates the Ca2+flux
between ER and mitochondria by controlling the interaction
between the IP3R and VDAC1.
4.2. Sigma-1 Receptor. The ER chaperone proteins known as
sigma receptors are targets for certain neurosteroids. Based
on their biochemical and pharmacological properties, two
subclasses, sigma-1 and sigma-2 receptors, are distinguished
but only the sigma-1 receptor was cloned and properly char-
acterized [57, 58]. The sigma-1 receptor is involved in many
physiological functions as well as in several pathological
conditions [58].
Sigma-1 receptors are especially enriched at the MAMs
[59]. A specific interaction between the Ca2+-binding chap-
erone BiP and the sigma-1 receptor was described [59].
This interaction depends on the ER Ca2+concentration: a
decreaseinERCa2+concentrationleadstotheirdissociation,
whereby both proteins become active chaperones.
The sigma-1 receptor regulates several ion channels,
includingtheIP3Rs[58].Agonistsofsigma-1receptorscould
so potentiate agonist-induced Ca2+release in NG108 cells
[60]. Hereby, an interaction between the sigma-1 receptor,
cytoskeletal ankyrin B, and IP3R3 was demonstrated [61]. In
CHO cells, the sigma-1 receptor also interacted with IP3R3,
but here ankyrin was not observed in the complex. Finally, a
specific role was found for the sigma-1 receptor stabilizing
the IP3R3 present at the MAMs, and so regulating Ca2+
transfer between ER and mitochondria [59].
4.3. Mitofusins. Mitofusin 1 and 2 are two dynamin-related
GTPases acting on mitochondria. Mitofusin 2 is enriched
at MAMs. The absence of mitofusin 2 not only affected ER
andmitochondrialmorphologybutalsoreducedthenumber
of contact points between ER and mitochondria by about
40% [62]. Mitofusin 2 on the ER appeared necessary for
connecting the two organelles by directly interacting with
either mitofusin 1 or mitofusin 2 on the OMM. Moreover,
the diminished interaction observed in the absence of
mitofusin 2 affected Ca2+transfer between the ER and the
mitochondria. A too strong ER-mitochondria interaction
may also be detrimental as overexpression of mitofusin 2 led
to apoptosis [63].
4.4. Bcl-2-Family Members. Bcl-2 is the prototype of a large
family containing both anti- and proapoptotic proteins. The
antiapoptotic members of this family, including Bcl-2 itself,
are characterized by the presence of 4 Bcl-2-homology (BH)
domains (BH1 to 4). The proapoptotic members either
have 3 BH domains (BH1, BH2, and BH3) as, for example,
Bax and Bak, or only a single BH3 domain, as for example,
Bim, Bid, and Bad (the so-called BH3-only proteins) [39].
The BH1, BH2, and BH3 domains of the antiapoptotic
proteins, as Bcl-2 and Bcl-Xl, form together a hydrophobic
cleft that can bind the amphipathic α-helical BH3 domain
of proapoptotic proteins. In this manner, the antiapoptotic
Bcl-2family membersantagonize apoptosisatthelevelofthe
mitochondria by binding and neutralizing proapoptotic Bax
and Bak [39, 64]. In addition to this mitochondrial function,
antiapoptotic Bcl-2 family members also act on the ER Ca2+
homeostasis [65, 66]. The exact mechanism is, however, not
yet clarified, and effects on several Ca2+-binding or Ca2+-
transporting proteins were described, including on the IP3R
[67–69].
Although there is an agreement that the antiapoptotic
proteinsas Bcl-2bindto theIP3R,there isamong the various
studies a discrepancy with respect to the exact binding site
and to the functional consequences. The results obtained are
summarized here below.
Firstly, cells lacking Bax/Bak displayed a decreased ER
Ca2+-store content, which was associated with an increased
(i) amount of Bcl-2 bound to the IP3R, (ii) protein-kinase-
A-(PKA-) dependent phosphorylation of the IP3R, and (iii)
Ca2+leak rate from the ER. Hence, increasing the ratio
of antiapoptotic over proapoptotic Bcl-2-family members
seemed to decrease the ER Ca2+-store content by promoting
the Ca2+leak via hyperphosphorylation and hyperactivation
of the IP3R [70].
Secondly, IP3Rs were described to be activated by Bcl-Xl.
Bcl-Xl bound to all three IP3R isoforms, thereby sensitizing
them to low IP3 concentrations [71, 72]. The interaction
site was demonstrated to be the C-terminal part of IP3R1
[71]. The binding of Bcl-Xl to the IP3Rs is important for
the protection of cells against apoptotic stimuli, since the
overexpression of Bcl-Xl in IP3R triple-knockout (TKO)
cells did not provoke resistance against apoptotic stimuli.
By ectopically overexpressing the different IP3R isoforms in
the TKO cells, it was found that all IP3R isoforms were
sensitized by Bcl-Xl and so conferred resistance against
apoptoticstimuli. However,a declinein steady-state ERCa2+
levels was only found in TKO cells ectopically expressing
IP3R3 [72], suggesting that decreased ER Ca2+levels are
not a requisite for cellular protection against apoptosis. The
antiapoptotic action may therefore be due to the enhanced
Ca2+-spiking activity resulting from the sensitization of the
IP3Rs, and be mediated either by increased mitochondrial
bioenergetics or by modulation of transcriptional activity
and gene expression [71, 72]. A similar mechanism was
recently proposed for Bcl-2 and Mcl-1 [73].
Thirdly, an inhibition of the IP3-induced Ca2+release
by Bcl-2 was also demonstrated [74]. In contrast to the
work discussed above, the interaction site was mapped to the
regulatory domain of IP3R1; moreover, the interaction was
mediated through the BH4domain ofBcl-2,a domain which
is not involved in the interaction with the C-terminus of the
IP3R [73, 75]. A peptide corresponding to the Bcl-2-binding
site on IP3R1 specifically disrupted this interaction and in
this way counteracted the functional effects of Bcl-2 on the
IP3R [75, 76].

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6 Journal of Aging Research
4.5. PKB/Akt and Promyelocytic Leukemia Protein. Another
regulatory mechanism of the Ca2+-flux properties of the
IP3R is its phosphorylation via PKB/Akt [29, 77, 78].
Upon prosurvival stimulation of cells, the prosurvival kinase
PKB/Akt binds and phosphorylates the IP3R, thereby reduc-
ing its Ca2+-release activity. This mechanism underpins the
increased resistance of cells towards apoptotic stimuli by
inhibiting the Ca2+flux into the mitochondria and may be
perused by tumor cells, yielding a survival advantage. The
latter has been shown to occur in glioblastoma cells that
displayhyperactivePKB/Akt,leadingtoIP3Rhyperphospho-
rylation and suppression of IP3R-channel activity [77].
Very recently, extranuclear promyelocytic leukemia pro-
tein (PML) has been shown to be present at the ER and
mitochondrial-associated membranes, thereby promoting
ER Ca2+release. At these microdomains, PML controls the
Ca2+-flux properties of the IP3R by recruiting PP2A, which
dephosphorylates PKB/Akt. The latter suppresses its kinase
activity and thus the PKB/Akt-mediated phosphorylation of
the IP3R, resulting in increased IP3R-mediated Ca2+transfer
into the mitochondria and thus OMM permeabilization
[79, 80]. This mechanism supplements the other known
functions of PML in the nucleus of higher eukaryotes. PML
nuclear bodies seem to contribute to its tumor suppressive
action byinhibiting cellcycleprogression and promotingcell
death [81].
5.The TransferofCa2+betweenthe IP3Rand
Mitochondria inApoptosisand Autophagy
FromthepreviousitisclearthatCa2+transferfromtheERto
the mitochondrial matrix is crucial for regulating mitochon-
drial functions, including bioenergetics. The mitochondrial
Ca2+signal can,however, also controlthechoicebetween cell
survival and cell death, as it can participate in the induction
and progression of apoptosis and autophagy [9, 10].
5.1. IP3Rs and Mitochondrial Ca2+in Apoptosis and Necrosis.
Different studies have placed the IP3R as central player in
the transfer of Ca2+into the mitochondria. Many cell types
display the propagation of agonist-induced Ca2+signals into
the interior of the mitochondria [11, 82].
Ca2+uptake in the mitochondria is crucial for multiple
important cellular functions, but the risk of mitochondrial
Ca2+overload exists, which may result in the induction
of cell death. At a high concentration, mitochondrial Ca2+
supports opening of the PTP in the IMM [51, 83]. This
opening leads to the release of ions (including Ca2+) and
molecules (including ATP), mitochondrial depolarization,
ROS production, cessation of oxidative phosphorylation
followed by ATP hydrolysis, matrix swelling by osmotic
forces, remodeling of the IMM, and eventually rupture of
the OMM [52]. Subsequently various apoptogenic factors,
including cytochrome C (CytC), apoptosis-inducing factor,
Smac/Diablo, HtrA2/Omi, and endonuclease G, are released
from the mitochondria [40]. These apoptogenic factors
will activate effector caspases, as caspase-3 and caspase-7,
and lead the cell into the execution phase of apoptosis.
Permeabilization of the OMM is therefore considered as
the decisive event in the development of cell death [84].
Given the proximity of IP3Rs to the mitochondrial Ca2+-
entry sites, IP3-induced Ca2+spikes appear ideally suited for
the stimulation of apoptosis [85], while the knockdown of
the IP3R by siRNA led to thesuppression of the Ca2+transfer
to the mitochondria.
In addition to this canonical pathway, the group of
Mikoshiba recently showed that not only excessive IP3R-
mediated Ca2+release and the concomitant mitochondrial
Ca2+overload but also the loss of IP3R function may lead to
apoptosis by lowering the mitochondrial membrane poten-
tial[86].Inthisstudy,itwasshown thatERstressinneuronal
cell leads to attenuation of IP3R function by impairing the
positive regulation of IP3R1 by the ER chaperone Grp78,
which acts as a major regulator of the unfolded protein
response and thus prevents ER stress. The loss of Grp78
bindingtotheluminaldomainoftheIP3R1leadstoimpaired
subunit assembly and thus dysfunctional channels. This
property seems selective for IP3R1, since Grp78 knockdown
attenuated IP3R1-mediated Ca2+release but did not affect
IP3R2- or IP3R3-mediated Ca2+release. Hence, it is interest-
ing to note that Ca2+transfer from the ER to mitochondria
requires a fine-tuned regulation, in which both suppressed
and excessive Ca2+transfer leads to apoptosis.
While a severe impairment of IP3R1 function and
attenuatedCa2+releaseleadtomitochondrialapoptosis,low-
level Ca2+signaling from ER to mitochondria or enhancing
ER-originating Ca2+oscillations elicits a prosurvival action
by stimulating the mitochondrial energy production or by
inducing transcription of specific genes [9, 31, 67, 69, 87].
In this paradigm, Bcl-Xl has been proposed to promote cell
survival through its direct action on the IP3R by enhancing
prosurvival Ca2+signaling, increasing mitochondrial bio-
energetics and activation of signaling via nuclear factor of
activated T cells [71, 72].
Mitochondrial Ca2+is a central factor in several neu-
rodegenerative diseases as Alzheimer’s disease, Parkinson’s
disease, and Huntington’s disease [88]. The inhibition of
cell death by preventing mitochondrial Ca2+overload or
by preventing the collapse of the mitochondrial membrane
potential is likely therapeutically relevant for the treatment
of these diseases. In contrast, enhancement of mitochon-
drial Ca2+overload can lead to inhibition of tumor cell
growth. Stimulation of the Ca2+transfer between ER and
mitochondria could lead to increased apoptosis and in this
way inhibit uncontrolled cellular proliferation [89]. In this
concept, it is not surprising that many tumor suppressor
proteins emerge as regulators of the transfer of Ca2+from
the ER to the mitochondria, like Fhit and PML. Fhit acts
at the mitochondrial level by increasing the affinity of the
mitochondrial Ca2+uniporter, thereby promoting mito-
chondrial Ca2+elevations at low levels of agonist-induced
Ca2+signaling [49]. PML acts at the level of the ER, where
it is recruited by the IP3R via a phosphorylation-dependent
process involving Akt and PP2A, thereby promoting Ca2+
transfer between the ER and the mitochondria and inducing
celldeath[79,80].Mutationsorablationofproteins,likeFhit

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Journal of Aging Research7
and PML, which may involve attenuated ER/mitochondrial
Ca2+transfers, has been associated with the development of
tumors.
5.2. IP3Rs and Mitochondrial Ca2+in Autophagy. Autophagy
is a delivery pathway used for the lysosomal degradation of
long-lived proteins, protein aggregates, damaged organelles,
and foreign pathogens. In stress situations (e.g., nutrient
starvation),thisprocessoffersthecellafresh poolofbuilding
blocks and has thus a prosurvival function [90]. Cells in
those conditions have to make the decision between survival
(autophagy) and death (apoptosis). Important crosstalks
exist between these two pathways [91, 92]. Interestingly,
Ca2+and IP3Rs have been implicated in both apoptosis
and autophagy, although the role of Ca2+in autophagy
only recently emerged [9, 10, 93]. Nonetheless, Ca2+/IP3Rs
may represent key players in the apoptosis-autophagy deci-
sion.
The first results on Ca2+in autophagy even appeared
contradictory. On the one hand, autophagy was activated by
an increase of the cytosolic Ca2+concentration [94–96]. On
the other hand, autophagy was also activated by conditions
that all would lead to a decrease of the IP3R activity and/or
cytosolic Ca2+concentration and therefore potentially of
the mitochondrial Ca2+concentration [97–100]. In a recent
report, itwasshown thatIP3Ractivity isnecessary toprovide
for a basal Ca2+signal to the mitochondria, in order to
control mitochondrial bioenergetics. IP3R knockdown or
inhibition will blunt these Ca2+signals, thereby compromis-
ing mitochondrial ATP production. The resulting increase
in AMP/ATP ratio will subsequently activate autophagy via
AMP-activated protein kinase (AMPK) [87].
Other results indicate that IP3Rscould inhibit autophagy
through a scaffold function, via binding of both Bcl-
2 and Beclin-1 (an essential autophagy protein), thereby
promoting the anti-autophagic interaction between these
twoproteins. Treatment ofHeLa cellswith the IP3Rinhibitor
xestospongin B promoted the release of Beclin-1 from the
IP3R-Bcl-2 complex, leading to autophagy activation [101].
So far, the data on Ca2+-stimulated autophagy concern
the Ca2+in the cytosol [94–96] or ER [102, 103]. It is
not yet clear whether the IP3R is hereby involved, although
treatment with an IP3R inhibitor did blunt cadmium-
induced autophagy stimulation [95]. The exact mechanism
by which Ca2+promotes autophagy is also still under debate.
AMPK-dependent [94], AMPK-independent [96], or ERK-
dependent pathways [95] are all possible.
Taken together, these data indicate that a specific, low-
intensity Ca2+transfer from ER to mitochondria is necessary
to inhibit autophagy, while an increase of the cytosolic Ca2+
concentration would activate autophagy.
6.Implicationsof Ca2+Signaling inAging
6.1. Aging: A Process of Disorganization. All biological pro-
cesses involved in the transformation of a fertilized egg into
a mature individual capable of reproduction are driven by
a purposeful genetic program. Through evolution, natural
selection has favored individuals that are reproductively
successful [104, 105]. Biological systems, like everything else
in the universe, change as a result of entropic changes.
Entropy is the tendency for concentrated energy to disperse
when unhindered. Natural selection has resulted in sufficient
relative strengths of the chemical bonds in our molecules
to prevent entropic changes and also installed repair and
replacement mechanisms. Evolution has therefore kept the
biomolecules in a functional state until reproductive matu-
ration.
After sexual maturation, there is no longer a species-
survival benefit for indefinitely maintaining these energy
states and, hence, the fidelity in most molecules. As we
grow older, stochastic or random events not driven by a
genetic program cause energy loss resulting in biologically
inactive or malfunctioning molecules. Aging is therefore
characterized by increasing entropy. The intrinsic thermo-
dynamic instability of the molecules whose precise three-
dimensional structures are no longer maintained leads to
covalent modifications such as glycation, conformational
changes, aggregation and precipitation, amyloid formation,
altered protein degradation, synthesis rates, and nuclear and
mitochondrial DNA damage and alterations. When the loss
of structure and, hence, function ultimately exceeds repair
and turnover capacity, vulnerability to pathology and age-
associated diseases increases. Because of the randomness
of the molecular disorder underlying aging, the loss of
molecular fidelity varies within the body. The weakest links
in this system will be the first that lead to disease, like in
the vascular system and in cells with a high tendency for
cancer development. The very heterogeneous aging process
contrasts with the virtually identical stages of development
until adulthood [106]. In this respect, we will here focus
on the age-related disorganization in the Ca2+signaling
machinery, ROS production, and autophagy.
6.2. Mechanism Involved in Aging: ROS, Mitochondria, and
Autophagy. The role of ROS accumulation and subsequent
macromolecular damage in age-related degeneration has
been supported by a plethora of cellular and biological
data from various model systems and organisms [107].
Antioxidants act as ROS scavengers and protect against
the detrimental effects of cellular ROS exposure. Genet-
ically, genes that extend lifespan were clustered in the
IGF-1/insulin-like signaling pathway in a variety of model
systems [108]. Nongenetic mechanisms to extend lifespan
in different organisms are achieved by caloric restriction
and/or by physical activity [109–113]. The composition of
the diet during caloric restriction is important; addition of
antioxidants (like vitamins, flavonoids), minerals (like Zn
and Se), and other compounds such as caffeine, omega 3,
and fatty acids has been shown to enhance lifespan [114]. It
shouldbenoted,however,thatmoststudiesconcerningthese
mechanisms wereperformedinyeastandanimalmodels,but
not yet in humans [115].
Here, we will discuss the molecular mechanisms of
ROS underlying aging. First, we will discuss the remodeling
of Ca2+signaling during aging. This is important since

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8Journal of Aging Research
Dysfunctional
mitochondrion
Extracellular space
Agonist
7TM-receptor
PIP2
HPAs
EGF ligand
EGF receptor
EGF receptor
PLC-γ
PLC-β
IP3
ER
SERCA
RyR
CaBC
ROS
ROS
ROS
ROS
ROS
ROS
Apoptosis
Cyt C
Cyt C
Cyt C
Bcl-2/Xl
IP3R
BAX
BAK
Grp75
VDAC
VDAC
MCU
MCU
AnkB
MFN2MFN1/2
CaBC
IP3R
BAX
BAK
Sig-1R
Bcl-2/Xl
PTP opening
OMM permeabilization
p66Shc
Figure 3: Ca2+signalling and key events involved in aging. Aging cells display decreased function or expression of ER proteins (IP3Rs,
RyRs, SERCAs, Ca2+-binding chaperones (CaBC)), increased cytosolic [Ca2+], suppressed agonist-mediated signaling, and accumulation
of damaged mitochondria due to declined autophagic activity. The simultaneous increase in disorganization and dysfunction of the Ca2+-
handling proteins and the decline in autophagy will result in the exaggerated production and excessive accumulation of ROS. These events
may lead to both ER stress and mitochondrial dysfunction, like PTP opening and OMM permeabilization with the consequent release of
apoptogenic factors and cell death. p66Shcand sirtuins take part in this scenario. P66Shctranslocates to mitochondria upon oxidative-stress-
induced PKCβ phosphorylation and peptidylprolyl isomerization by Pin1, thereby supporting ROS production. Sirtuins are downregulated
and unable to exert its antiaging effect. It is important to note that while p66Shcablation leads to lifespan extension, high levels of p66Shc
have been observed in centenarians. While in normal cells, ROS help to detect and remove altered mitochondria through autophagy,
thereby maintaining cellular health, the excessive release of ROS in combination with the decline in autophagy observed during aging
may underpin the age-related cell-death processes. In this respect, the recently identified inhibitors of EGF-receptor signaling, the high-
performanceadvanced agephenotype proteins (HPA-1 andHPA-2), whoseknockdownpromoteslocomotoryhealth span ofC. elegans, may
point towards an important role of proper agonist-induced Ca2+signaling via the IP3R axis. The relevance of these ligands or of attenuated
agonist-induced signalingin humans needs to be established. However, recent evidence indicates that dysfunction of IP3Rs during ER stress
promotes cell death and underlies a neurodegenerative disease, like Huntington’s disease. Given the central role of proper IP3R function for
mitochondrial bioenergetics and ATP production, the decline of IP3R activity observed during ER stress or attenuated upstream signaling
linkedto IP3maybe very relevantforage-related apoptosisbut require further investigation.Green arrows:stimulation;red lines:inhibition;
black arrows: Ca2+flux; dashed-green arrow: stimulation/damage.
the OMM permeabilization is critically controlled by the
elevation of the mitochondrial Ca2+concentration, thereby
serving as a coincidence detector with ROS [116]. Next, we
will focus on the signaling cascade involving sirtuins, p66Shc,
and autophagy in the regulation of mitochondrial function.
Aschematicoverviewoftheinteractionbetweenthedifferent
molecular key players in aging is provided in Figure 3.
6.2.1. Ca2+Signaling in Aging. Altered intracellular Ca2+sig-
naling is a hallmark of neurodegeneration, like in Alzheim-
er’s and Huntington’s disease [117–120]. Different models
have been proposed for familial Alzheimer’s-disease-linked
presenilin mutations, including the function of presenilins
as Ca2+-leak channels [121], an increase in the expression
level of IP3Rs [122], or the direct activation of IP3Rs or

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Journal of Aging Research9
RyRs [123–125]. In any case, it is clear that exaggerated Ca2+
signaling is an upstream event in the pathophysiology of
Alzheimer’s disease and contributes to the ROS-mediated
cell toxicity [126]. However, the changes in Ca2+signaling
that occur in neurodegenerative diseases may be dependent
on the type of disease. For instance, a mouse model
for Huntington’s disease revealed dysfunctional IP3R Ca2+-
release channel activity in the cerebrum and striatum, which
was caused by a prominent decline in the association of
Grp78, a positive regulator of the IP3R1-channel formation,
with the IP3R1 [86].
Other age-related diseases also display altered Ca2+sig-
naling. Cardiac hypertrophy, forexample, is characterized by
enhanced IP3signaling, leading to spontaneous Ca2+-release
events that underlie arrhythmias [127]. Also chronic heart
failure can be a consequence of excessive phosphorylation of
RyR, leading to an increased Ca2+leak [128] (Figure 2).
However, the role and mechanism of ER Ca2+signaling
in aging is less clear [129], although most studies suggest
altered Ca2+signaling during aging (Figure 2). In most cell
types, ER Ca2+dyshomeostasis was caused by a decreased
ER Ca2+content and a decreased Ca2+release from the
ER, while the cytosolic [Ca2+] was increased. These effects
were the result of a decline in SERCA and/or IP3R and/or
RyR activity, caused by changes in mRNA or protein levels,
phosphorylation events, or oxidative damage to SERCA [7].
In addition, intraluminal Ca2+-buffering protein levels often
decline during age, in part also through oxidative damage
[130] (Figure 2). Also VDAC undergoes posttranslational
modificationsinagedcells,possiblythroughoxidativebreak-
upoftryptophanresidues,therebyincreasing thesusceptibil-
ity to apoptosis [131]. This is in line with evidence showing
that superoxide can lead to mitochondrial permeabilization
in a VDAC-dependent manner [132]. In yeast, this phe-
nomenon can be protected by Cu/Zn-superoxide dismutase,
a protein known for its protective role against aging [133].
Some cell types, however, display Ca2+dyshomeostasis
in a different way (Figure 2). Studies in aged rat hearts,
for example, showed increased IP3R levels [134]. Also aged
neuronal cells displayed reduced sensitivity towards caffeine,
which may be caused by a decline in the steady-state ER
Ca2+levels [135–137]. The latter may be due to a decreased
SERCA Ca2+-pump activity, a limited supply of ATP or
an increased Ca2+leak from the ER. Other studies pointed
to a prolonged Ca2+-induced Ca2+release, resulting in an
inhibition of synaptic strength and long-term potentiation
[138, 139].
Interestingly, IP3R characteristics also appear to be
altered in aged brain tissues [140], as IP3R density and IP3
binding to the IP3R were decreased in aged rat cerebellum.
The same observation of decreased IP3 binding was made
in aged mice cerebellum [141]. However, the cellular IP3
content increased with age [142]. These findings suggest a
role for the phosphoinositide/Ca2+signaling in the impaired
neuronal responsiveness during aging. In this respect, more
recent work revealed that stimulation of IP3Rs in old astro-
cytes increased protection against ROS and subsequently
neuroprotection [143].
Moreover, in aged MII-stage eggs, it was found that
the IP3R1 was proteolytically cleaved by caspase-3, resulting
in a leaky 95-kDa C-terminal IP3R1 fragment containing
the channel pore [144, 145]. In contrast, when the C-
terminalchanneldomainwasrecombinantlyexpressedinthe
mouse oocytes, the sperm-factor-induced Ca2+oscillations
were abolished and the eggs displayed an apoptotic and
fragmented phenotype. Previously, we had shown that
caspase-3-dependent cleavage of the IP3R augmented the
late phase of apoptosis by providing a prolonged ER Ca2+
leak [146]. However, in healthy cells, the Ca2+leak through
a recombinantly expressed C-terminal channel domain was
very small. Hence, the caspase-3-dependent cleavage of the
IP3R may participate in cellular Ca2+overload via a second-
hitmechanism. Inthecaseofagedoocytes,accumulatedROS
may be the second hit. Currently, it is not clear whether IP3R
cleavage contributes to the aging process by overloading the
mitochondria with Ca2+and sensitizing them towards ROS
accumulation. In addition, ROS may also directly regulate
IP3R activity, since it is known that oxidizing agents like
thimerosal sensitize IP3Rs by stimulating intramolecular
interactions between the suppressor and ligand-binding
domain [147]. Taken together, IP3R/Ca2+signaling appears
to be affected in aged cells. Abnormal Ca2+signals may
then affect many processes (ROS production/protection,
autophagy, apoptosis, synaptic transmission, etc.) that are
altered during aging (summarized in Figure 5).Nevertheless,
the overall changes in ER Ca2+handling observed during
aging seem relatively small compared to the changes found
in Alzheimer’s disease [129].
Recently, an elegant study on Caenorhabditis elegans re-
enforced the paradigm that the activation of IP3R pathways
may be considered in therapeutic applications for treating
age-related decline in skeletal muscle function (sarcopenia)
[148]. Indeed, using an RNAi screen, the authors identified
two critical factors that delayed the age-associated decline in
locomotory health span of C. elegans in a high-performance
advanced age phenotype (HPA-1 and HPA-2). The concept
underpinning this study was that locomotory decline in
humans contributes to frailty and loss of independence.
Although the exact mechanism is not yet known, it is
clear that HPA-1 and HPA-2 attenuate epidermal-growth-
factor-(EGF-) dependent signaling via the EGF receptor
[148]. When HPA-1 and HPA-2 are disrupted, EGF signaling
via the EGF receptor will increase. The activation of the
EGF-signaling pathway normally leads to cell proliferation,
survival, integrity, and differentiation. Importantly, phos-
pholipase C-γ (PLC-γ) and IP3Rs were demonstrated to act
downstream ofEGF-receptorsignaling, therebycontributing
to prolonged health span in these animals. This is the
very first report considering the role of EGF signaling
in aging. Therefore, the exact mechanism of how these
signaling pathways affect human aging remains to be further
clarified, but restoring the attenuated IP3R-mediated Ca2+
signaling and reestablishing normal mitochondrial func-
tion may be an attractive hypothesis in combination with
chemical induction of autophagy (Figure 4). Nevertheless, a
decline in G-protein-coupled receptor-dependent signaling
hasbeenobservedin theskeletalmuscleandintestine ofaged

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10 Journal of Aging Research
Rapamycin/spermidine
Dysfunctional
mitochondrion
Extracellular space
Agonist
7TM-receptor
PIP2
HPAs
EGF ligand
EGF receptor
EGF receptor
PLC-γ
PLC-β
IP3
IP3
IP3
IP3
ER
SERCA
RyR
CaBC
ROS
Bcl-2/Xl
IP3R
BAX
BAK
BAX
BAK
Grp75
VDAC
VDAC
MCU
MCU
Sig-1R AnkB
MFN2MFN1/2
CaBC
IP3R
Cyt C
Sirtuins
Sirtuins
Bcl-2/Xl
Autophagy
mitophagy
p66Shc
Figure 4: A speculative antiaging strategy based on restoring IP3R-mediated Ca2+signaling and chemical induction of autophagy. Provided
the concept thataging cells are characterized by suppressed IP3signalingor attenuated IP3R, Ca2+-release activity is relevant in humans,and
elevating IP3levels may compensate for the decline in the IP3/IP3R-signaling axis. This may contribute to a decline in the p66Shc-mediated
ROS production, an activation of sirtuin-dependent mitochondrial biogenesis, and the lowering of ROS production. The final step of this
compensatory response consistsin the autophagic removal of the damaged mitochondria. Hence, chemical induction of autophagy (e.g., by
rapamycinor spermidine) is likelycritical for successful and healthy agingin humanbeings. It is importantto note thatthis concept is based
on a recent report on C. elegans, in which ablations of inhibitors of EGF signaling enhance IP3R signaling and promote healthy lifespan
extension. Green arrows: stimulation; red lines: inhibition; black arrows: Ca2+flux.
rats [149]. The underlying mechanism involved a prominent
decrease in the levels of Gq/11and Giprotein levels.
6.2.2. Sirtuins. Sirtuins are a conserved family of proteins
that are linked to longevity and stress tolerance in Saccha-
romycescerevisiae [150].Sirtuinshavebeenidentifiedasanti-
aging genes,since increasing theiractivity prolongedlifespan
not only in yeast, but also in C. elegans and Drosophila
melanogaster and is thought to act similarly in mammals
[151–153]. In this respect, age is often associated with
reduced sirtuin levels. In aged mouse embryonic fibroblasts,
progressive loss of the sirtuin-1 protein, but not mRNA,
was observed [154]. However, other studies show that this
is at least tissue specific; sirtuin-1 activity was reduced in
rat hearts, but not in adipose tissue [155], and reduced
sirtuin-1 expression was found only in distinctive parts of
the mouse brain [156]. Sirtuins, which retard aging as a
function of their gene dosage, display unique biochemical
activities, that is, NAD-dependent protein deacetylase [157,
158]. The subsequent deacetylation of sirtuin substrates
alters their activity (activation or inhibition). In mammals,
sirtuin-1 deacetylates a variety of key transcription factors
and cofactors, like p53 [159], FOXO proteins [160, 161],
peroxisome proliferation activating receptor (PPAR)-γ co-
activator-1α (PGC-1α) [162], and nuclear factor-κB [163].
The effects of sirtuin-1 on these factors elicit stress tolerance
and metabolic changes reminiscent of caloric restriction,
while caloric restriction upregulates sirtuin-1 levels, and
mice lacking sirtuin-1 did not display phenotypic responses
upon caloric restriction [160, 164–166]. Since sirtuins are
regulated by NAD+, their activity will be influenced by the
NAD+/NADHratioandthusbythemetabolicstateofthecell
[167]. Hence, sirtuins may be influenced not only by caloric
restriction but also by physical activity, both associated with
longevity and increased insulin sensitivity [168, 169].
Importantly, sirtuin-1 also regulates mitochondrial biol-
ogy[150,167],anotherkeyaspect inaging, sincethenumber

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Journal of Aging Research11
Aging
Mitochondrial
function
Autophagy
(mitophagy)
Apoptosis
Ca2+
ROS
(a)
Aging
Mitochondrial
function
Autophagy
(mitophagy)
Apoptosis
Physical activity
caloric restriction
ROS
Pin1
Sirtuins
PKCβ
p66Shc
(b)
Figure 5: Network of interactions between sirtuins, p66Shc, Ca2+, and ROS, which affect mitochondrial function, autophagy, and apoptosis,
thereby controlling aging-dependent processes. (a) Ca2+signals may increase or prevent aging. Ca2+signals are characterized by different
spatiotemporal characteristics and subsequently different outcomes on mitochondrial function, autophagy, and apoptosis. For example, a
constitutive Ca2+transfer from ER to mitochondria would stimulate mitochondrial function and inhibit autophagy and apoptosis, while
a mitochondrial Ca2+overload would be proapoptotic. The interplay between mitochondrial Ca2+elevations and ROS production is a
critical determinant in the apoptotic outcome at the level of the mitochondria, which function as co-incidence detectors. Therefore, high
mitochondrial Ca2+concentrations andROS act as a double-hit mechanism, triggering mitochondrial-dependent apoptosis. (b) Sirtuins are
mainly antiaging genes via the promotion of mitochondrial function and autophagy and inhibition of apoptosis. They also act inhibitingly
on ROS. Sirtuin function may be enhanced by restricting caloric intake or increasing physical activity, thereby extending lifespan. Increased
ROSactivatethePin1-p66Shccomplex,which,inturn,promotestheproduction ofROSandsubsequentlymitochondrialdamage.Therefore,
p66Shcmay help to target damaged mitochondria and activate cellular processes that deal with dysfunctional mitochondria and oxidative
stress. The outcome, however, can be dual: aging may be enhanced via a complete removal of the cell through apoptosis, while the selective
removal of the damaged mitochondria through mitophagy, leaving the cell with predominantly healthy mitochondria, may slow down the
aging process. Green arrows: stimulation; red lines: inhibition; black arrows: stimulation or inhibition.
offunctionalmitochondriaisknowntodeclineduringaging.
This has been proposed to underlie aging in diseases like
type-2 diabetes [170, 171]. In contrast, increasing mito-
chondrial activity will increase the metabolic rate, enhance
glucose metabolism, and improve insulin sensitivity. Even
without an increase in the metabolic rate, caloric restriction
might be beneficial by inducing mitochondrial biogenesis
via sirtuin-1 [165, 172, 173]. Activation of sirtuin-1 has
been shown to be involved in mitochondrial biogenesis and
improved mitochondrial function by deacetylation of PGC-
1α, thereby lowering ROS production [162].
Sirtuin-1 also suppressed stress-induced apoptosis, while
the lack of sirtuin-1 inhibited autophagy in vivo [174]. In
addition, the extension of lifespan upon caloric restriction
wasproposedtobedependentontheinductionofautophagy
by sirtuin-1 [175]. The underlying mechanism probably
involves the deacetylation of certain autophagy proteins,
such as Atg5, Atg7, and Atg8 [174, 175]. A schematic over-
view of the role of sirtuins in aging is depicted in Figure 5.
6.2.3. p66Shc. Recent research revealed the role of p66Shc,
the 66kDa isoform of the Shc (Src homolog and collagen
homolog) family [176]. Although p66Shcforms stable com-
plexes with Grb2, an adaptor protein for the Ras-exchange
factor SOS, it has little effect on Ras-mediated signaling
[177].
Nevertheless, p66Shcis activated by oxidative stress via
phosphorylation on Ser36, and this mechanism is indispens-
ableforp66Shc’slifespan regulation[178,179].Micein which
p66Shchas been deleted displayed a prolonged lifespan with a
decreased mitochondrial metabolism and ROS production,
while lacking pathophysiological characteristics or effects
on body size. MEF cells from p66Shc−/−animals displayed
resistance towards oxidative-stress-induced apoptosis in a
p53-dependent manner [176].
ROS arise from the mitochondrial electron-transfer
chain or from exogenous sources, like UV and ionizing radi-
ations. p66Shcis involved in mitochondrial ROS production.
In basal conditions, about one fifth of p66Shcis localized
to the intermembrane space of the mitochondria, while
oxidative stress dramatically increases the mitochondria-
associatedp66Shcduetoitsmitochondrialtranslocationfrom
the cytosol [180]. In the mitochondria, p66Shcinteracts
with CytC, promoting the shuttling of electrons from CytC
to molecular oxygen [181]. The latter may underlie the
increased ROS production upon p66Shcoverexpression and
the decreased ROS production in p66Shcknockout cells. In
addition, p66Shcknockoutcellsdisplayed decreasedoxidative
capacity, thereby redirecting metabolic energy conversion
from oxidative toward glycolyticpathways. Therefore, p66Shc
may provide a molecular switch to oxidative-stress-induced
apoptosis by controlling mitochondrial ROS production.

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12Journal of Aging Research
It should be noted, however, that studies in yeast correlated
higher respiration rates combined with decreased oxidative
stress and increased lifespan [182]. This suggests that the
respiration rate per se is not the important factor for ROS
production, but more likely the electron transmit time and
the availability of oxygen [183].
In normal cells, oxidative stress leads to compromised
mitochondrial Ca2+homeostasis, which is an early event
of mitochondrial damage [107, 176]. This is observed as a
decreased mitochondrial Ca2+signal upon agonist stimula-
tion in cells challenged with H2O2despite a normal cytosolic
Ca2+signal. Importantly, cells lacking p66Shcseemed to
be protected against oxidative challenge, since their mito-
chondrial Ca2+signaling upon agonist stimulation was not
impaired in the presence of H2O2[176]. Similar results were
found in MEF cells lacking Pin-1, a peptidylprolyl isomerase
catalyzing cis/trans isomerization of phosphorylated Ser-
Pro bonds, where the reduction of agonist-induced Ca2+
signals in mitochondria upon oxidative stress was signifi-
cantly smaller. These findings suggest a phosphorylation-
dependent conformational change in Pin-1 targets, like
p66Shc.
Recent work provided important mechanistic insights
into the role of p66Shcin the early mitochondrial response
to oxidative stress [178, 179]. ROS are known to activate a
variety of kinases, including protein kinase C (PKC) β. The
activation of PKCβ will cause the phosphorylation of p66Shc
on Ser36, although other kinases may also participate in this
process. Indeed, the mitochondrial fraction of p66Shcduring
oxidative challenge was severely reduced after treatment
with PKCβ inhibitors. As a result, Ser36-phosphorylated
p66Shcwill interact with Pin-1. The catalytic activity of
Pin-1 may result in cis/trans isomerization of Ser36-Pro37,
thereby triggering the exposure of a mitochondrial targeting
sequence or an interaction with mtHsp70, a mitochondrial
heat-shock protein. This process may underlie selective
targeting of p66Shcto mitochondria undergoing oxidative
challenge. The mitochondrial targeting of p66Shcinvolves its
protein-phosphatase-(PP-) 2A-mediated dephosphorylation
and dissociation from mtHsp70, although the mechanism
of their contribution is not fully elucidated. In the inter-
membrane space, p66Shcwill interact with reduced CytCand
enhance intramitochondrial H2O2 production. The latter
and its more damaging reaction products, the hydroxyl
radicals, have been shown to trigger the opening of the
PTP [184]. This will perturb mitochondrial structure and
function, resulting in mitochondrial permeabilization, CytC
release, and apoptosis induction, and subsequently lead to
a coordinated cell-death response and the removal of the
cellcontainingdamagedmitochondria. However,inaddition
to apoptosis, autophagy may be involved in removing the
subpopulationofcompromisedmitochondriasufferingfrom
oxidative challenge. Interestingly, this autophagy-mediated
removal of damaged mitochondria can be triggered through
PTP opening [185]. This will result in the removal of
the organelles that are damaged by the oxidative stress (a
process termed mitophagy), while maintaining the healthy
mitochondria. Accordingto these findings, it isinteresting to
note that aging has been associated with declined autophagy
activity [186], while autophagy activity is a requisite for
lifespan extension in C. elegans [187]. In this way, p66Shc
may be important for mitochondrial quality control through
the autophagy-mediated removal of damaged mitochondria.
However, during aging, the number of mitochondria suffer-
ing from oxidativestress may increase, while theircleanupby
the autophagic system may become limiting, leading to the
accumulationofunprocessedoxidation-damagedmitochon-
dria. Importantly, in mouse models for aging, the levels of
p66Shcseemed to decline, while its phosphorylation at Ser36
was enhanced [188]. This correlated with higher free-radical
production and accumulation of damage caused by ROS.
Strikingly, fibroblasts obtained from centenarians dis-
played elevated levels of p66Shc[189], indicating that basal
mitochondrial p66Shcplays an important role in normal
cell-damage management of stress and in damage repair.
Indeed, the selective removal of damaged mitochondria
may contribute to lifespan extension. In addition, it is
interesting to note that increased physical activity has been
associated with lifespan extension and lower mortality,
althoughthisisassociatedwithincreasedmitochondrialROS
production due to an increased metabolic rate. Therefore, it
is conceivable that exercise may promote adaptation to ROS
by upregulating ROS scavengers, causing a natural resistance
against ROS or against cellular damage in general [167].
Hence, it may be worth investigating whether p66Shclevels
are affected by exercise and whether this may contribute to
increased cleanup of damaged mitochondria or resistance
against ROS. A schematic overview of the role of p66Shcin
aging is depicted in Figure 5.
6.2.4. Autophagy. It has become increasingly clear that
autophagy plays a central role in the aging process, in
which it is involved in the removal of damaged organelles
or of protein aggregates by engulfment in autophagosomes
followed by lysosomal degradation. First of all, autophagy
was demonstrated to decrease with increasing life time
[186]. Caloric restriction slowed down the age-related
impairment of autophagy in skeletal muscle of rats [190].
In addition, chemical induction of autophagy by spermi-
dine or by rapamycin prolonged lifespan [191, 192]. In
contrast, animals with compromised capacity to perform
autophagywereshortlivinganddisplayedneurodegenerative
phenotypes, probablydueto the accumulationofdeleterious
accumulation of protein aggregates [193–195]. Moreover, it
is clear that damaged mitochondria ought to be removed,
while harboring the healthy mitochondria, which are needed
for cell survival. In any case, the accumulation of damaged
mitochondria and their impaired removal is a hallmark of
aging and will contribute to decreased cell viability. There-
fore, mitochondrial qualitycontrol is essential forpropercell
survival.
The “selective” recognition of damaged mitochondria
by autophagosomes without affecting healthy mitochondria
remains very poorly understood. However, the first compo-
nentsessential for“selective” mitophagyhavebeen identified
in yeast: Uth1, an OMM protein, and Aup1, a mitochon-
drial phosphatase [196–198]. Additional components of

Page 13

Journal of Aging Research13
organelle-specific autophagy have been revealed in a system-
atic screen, including Atg11, Atg20, Atg24, Atg32, and Atg33
[199, 200]. Atg32 is proposed as the receptor for mitophagy
via the local recruitment of Atg8, an essential component
of the autophagosome formation. NIX/BNIP3L [201, 202],
BNIP3 [203], PARKIN [204], and PINK-1 [205–210] were
proposed to be involved in mitochondrial degradation in
mammalian cells. PARKIN is selectively recruited by dys-
functional mitochondria, thereby mediating the engulfment
of these mitochondria by the autophagosomes [204]. A
recent study provided clear insights into the underlying
mechanism, which required the accumulation of the kinase
PINK-1 on damaged mitochondria. In healthy mitochon-
dria, PINK-1 is maintained at a low level by voltage-
dependent proteolysis [210]. In mitochondria with sus-
tained damage, PINK-1 levels rapidly accumulated. The
latter was required and sufficient to recruit PARKIN to
the mitochondria providing a mechanism for the selective
removal of damaged mitochondria by autophagy. Impor-
tantly, mutations in PINK-1 or PARKIN associated with
Parkinson’s disease abolished the recruitment of PARKIN
by PINK-1 to the mitochondria, allowing the accumulation
of damaged mitochondria. Another recent study revealed
the mitochondrial protein NIX as the selective mitophagy
receptor for the removal of damaged mitochondria by
binding and recruiting LC3/GABARAP proteins [211]. The
latterareubiquitin-likemodifiersrequiredfortheelongation
of autophagosomal membranes.
Besides these mitophagy receptors, mitochondrial pro-
teases and chaperones were needed to prevent the accumu-
lation of misfolded and aggregated proteins within the mito-
chondria [167].
Finally, various studies point towards a role of ROS
upstream of autophagy [212]. Accumulation of ROS directly
affects different key players essential for the induction of
autophagy, including the activation of the protein kinases
AMPK and JNK, the inhibition of other kinases (Akt and
TOR), and the inhibition of LC3 delipidation. These pro-
cesseswill stimulateautophagy,therebyalleviatingtheoxida-
tive stress by removing the ROS-generating mitochondria.
7.Conclusions
Upstream Ca2+and ROS signaling tightly control cellular
homeostasis by regulating fundamental cell-death and cell-
survival processes like apoptosis and autophagy. It is clear
that many proteins that mediate apoptosis and autophagy
directly affect Ca2+signaling through interaction with the
ER and mitochondrial Ca2+-release and/or Ca2+-uptake
mechanisms. Furthermore, these Ca2+-signaling proteins
contribute to the functional and physical linking between
ER and mitochondria. Importantly, the interplay between
ER and mitochondrial Ca2+signaling and ROS signaling
mediates the detection, the efficient targeting, and removal
of mitochondria with sustained damage. This is the key for
cellular homeostasis as well as for homeostasis at the level of
thewhole organism. In thisrespect, theefficientandselective
removal of damaged mitochondria by autophagy is a crucial
element in the maintenance of cellular health, whereby
the poisonous accumulation of ROS from dysfunctional
mitochondria and eventual cell death via apoptosis are
avoided. Recent studies point towards a central role for
impaired autophagy and inadequate removal of damaged
mitochondria during aging. At the level of the organism,
apoptosis will be the ultimate resort to remove seriously
damaged cells. This will particularly affect the lifespan of
nondividing cells, like neurons, thereby affecting the lifespan
of the whole organism.
Acknowledgments
Work performed in the laboratory of the authors in this area
was supported by the Research Council of the K.U.Leuven
(Concerted Action GOA 04/07 and 09/012 and OT-START
research funding STRT1/10/044) and by the Research
FoundationFlanders(FWO-Vlaanderen) (GrantsG.0604.07,
G073109N, and G072409N). J. P. Decuypere and G. Monaco
are, respectively, recipients of a Ph.D. fellowship from the
AgencyforInnovationbyScienceandTechnology(IWT)and
the Research Foundation Flanders (FWO-Vlaanderen).
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