835

Journal of Alzheimers Disease 37 (2013) 835848

DOI 10.3233/JAD-131001
IOS Press

Elevated [Ca2+]i Levels Occur with

Decreased Calpain Activity in Aged
Fibroblasts and Their Reversal by
Energy-Rich Compounds: New Paradigm for
Alzheimers Disease Prevention
Huey T. Nguyena , Darrell R. Sawmillera , Olga Markova and Ming Chena,b,
a Aging

Research Laboratory, Bay Pines VA Healthcare System, Bay Pines, FL, USA
of Molecular Pharmacology and Physiology, University of South Florida College of Medicine,
Tampa, FL, USA

b Department

Accepted 8 June 2013

Abstract. Elevated intracellular Ca2+ levels in the aging brain are widely thought to hyperactivate Ca2+ signaling and Ca2+ dependent enzymes, leading to neuronal death through an excitatory mechanism in Alzheimers disease (AD). This Ca2+
overload hypothesis has been questioned by our theoretical analyses. To better understand the relationship between the level
and functionality of Ca2+ in aging, in this study we simultaneously measured intracellular Ca2+ transients and calpain activity in
cultured human fibroblasts. We found that Ca2+ transitions elicited by bradykinin were indeed overstayed or elevated in levels
in old cells but, remarkably, calpain activity was decreased compared to young cells. Also, treating young cells with the energy
inhibitor rotenone or with H2 O2 recapitulated the Ca2+ overstay and calpain inactivation found in old cells. More importantly,
treating old cells with high-energy compounds such as phosphoenol pyruvate or phosphocreatine, which boosted cellular ATP
content, reduced the Ca2+ overstay and re-activated calpain. Moreover, Ca2+ levels and calpain activity were dramatically raised
in the dying cells killed by detergent. Finally, Ca2+ oscillations induced by low dose of bradykinin in old cells exhibited lower
spike frequency, but higher overall levels. Collectively, these results suggest that (a) Ca2+ overload in old cells arises from an
inefficient Ca2+ handling system compromised by age-related energy depletion and oxidative stress; and (b) despite elevated
levels, the functionality of Ca2+ signaling has diminished in old cells. Thus, the study reinforces the concept that tonic promotion
of bioenergetics and Ca2+ signaling function is a reasonable and new paradigm to protect the aging brain cells to prevent AD.
Keywords: Alzheimers disease, calcium, calpain, energy

INTRODUCTION
Healthy cell maintains a 10,000-fold Ca2+ concentration gradient across the membrane and this steep
gradient is essential for the cell to quickly respond
to physiological stimuli. Ca2+ signaling regulates
Correspondence to: Ming Chen, Ph.D., Medical R&D/151, Bay
Pines VA Healthcare System and University of South Florida, 10,000
Bay Pines Blvd., Bay Pines, FL 33744, USA. Tel.: +1 727 398 6661;
Ex. 4049; E-mail: ming.chen@va.gov.

neurotransmission and brain function and this role

dictates the need for precise maintenance of Ca2+
homeostasis in the cell and any disturbance, however
subtle, could lead to profound consequences in neuronal function. The knowledge leads to a generally
accepted concept that a Ca2+ dysfunction is the primary underpinning in brain aging and development
of Alzheimers disease (AD), but the mechanism of
how the dysfunction occurs and its consequences have
remained elusive and controversial [1, 2].

ISSN 1387-2877/13/$27.50 2013 IOS Press and the authors. All rights reserved

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H.T. Nguyen et al. / Higher Ca2+ Levels Cause Lower Signaling

Many studies have found that [Ca2+ ]i levels are

persistently elevated in aged cells as compared to
young cells [35]. These and other findings have been
taken to support a widely-held calcium overload
hypothesis positing that the Ca2+ overload in neuronal cells is the final common pathway that causes
cognitive impairments through a mechanism of hyperactivating Ca2+ -dependent enzymes such as calpain,
leading eventually to cell death in AD [6]. According
to the hypothesis, a number of clinical trials aiming to
improve mental conditions by Ca2+ antagonists in the
elderly have been carried out, but have not yielded the
expected results up to now [7, 8]. But unexpectedly,
other clinical studies have found that Ca2+ agonists
benefit the aging brain [9, 10], thus raising questions
about the hypothesis.
Moreover, a number of studies over the years have
found that despite elevated [Ca2+ ]i levels, Ca2+ dependent enzymes/proteins such as calpain, protein
kinase C, calcineurin, calmodulin, Ca2+ /calmodulindependent kinases and calbindin in various cell types
have decreased their functional activities during aging
[1119]. These studies have revealed an apparent
disparity between the measured [Ca2+ ]i levels and
physiological function of Ca2+ in aged cells. Furthermore, we have reasoned that because Ca2+ signaling
is a highly energy-dependent process [1, 2] and calpain plays a critical role in life and cognition [2022],
it seems more likely that their physiological function should undergo down-regulation in the aging
brain where energy metabolisms and cognitive ability
are declining [23, 24]. These and other considerations [2527] have thus raised critical questions about
the scientific soundness of the current hypothesis.
It appears that the mechanisms of age-related Ca2+
alterations can be much more complex than currently
thought and thus further studies are necessary to
resolve the disparities and related issues.
In an attempt to address the issues especially
the relationship between the measured levels and
physiological function of Ca2+ , in this study we simultaneously measured Ca2+ transients and the in situ
activity of calpain in cultured human fibroblasts. Our
data showed that elevated [Ca2+ ]i levels in aged
cells occurred with a concomitant decrease of calpain
activity and, importantly, these impairments can be
attenuated by several high-energy compounds. These
compounds contain energy-rich bond of phosphoryl, acyl, or thioesterol group, liberating at least 7
kilocalories of free energy (
G ) per mole under
standard conditions. The best known high-energy compounds are ATP, GTP, phosphoenol pyruvate (PEP),

phosphocreatine (PCr), and acetyl coenzyme A

(ACoA) [28, 29].
MATERIALS AND METHODS
Materials
Human skin fibroblast cell lines from healthy
young and aged donors were purchased from ATCC
(Gaithersburg, MD). Fluo-4 AM and pluronic acid
were from Invitrogen (Grand Island, NY). Calpain substrate IV and BAPTA-AM were from EMD-Millipore
(Temecula, CA). Cell culture medium, MTT test kit,
bradykinin, rotenone, H2 O2 , ATP, PEP, PCr, ACoA,
protease inhibitor cocktail, and other chemicals were
from Sigma-Aldrich (St. Louis, MO).
Methods
Cell culture
Fibroblasts were routinely maintained in DMEM
supplemented with 10% fetal bovine serum and
100 g/ml streptomycin and 100 U/ml penicillin at
37 C with 5% CO2 . The cells within 10 passages were
used in the experiments and no significant differences
in [Ca2+ ]i or calpain activity were found in these cells
among the passages by preliminary experiments.
Confocal [Ca2+ ]i imaging
Cells were cultured on 25 mm glass-bottomed Petri
dishes designed for fluorimetry (MatTek, MA). For
experiment, the cells at approximately 50% confluence were incubated in Tyrodes buffer (140 mM NaCl,
4 mM KCl, 1 mM MgCl2 , 2 mM CaCl2 , 10 mM glucose, 10 mM, pH 7.4) and loaded with Fluo-4 AM
(3 M) and pluronic acid (0.07%) for 30 min. The
testing agents were added to the cells after recording baseline for 3 min. [Ca2+ ]i imaging was performed
using a non-ratiometric dye Fluo-4 AM in an Olympus Fluoview 1000 laser scanning confocal microscope
at wavelength 488 nm for excitation and 516 nm for
emission. Images were collected by 256 128 pixel
images at recording rate of 1 frame per 2 seconds. Ca2+
transients were expressed as fluorescence intensity or
relative [Ca2+ ]i levels computed using software from
GraphPad (San Diego, CA).
Cell viability assay
This was conducted using an MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] test

H.T. Nguyen et al. / Higher Ca2+ Levels Cause Lower Signaling

kit (Sigma, IL) according to the manufacturers

instructions.
Measurement of intracellular calpain activity
The procedure used a cell-permeable fluorogenic
calpain-specific substrate (Calpain substrate IV, EMDMillipore, CA) as previously described [30]. For
fluorescence imaging, cultured fibroblasts were incubated in a buffer (140 mM NaCl, 10 mM Glucose,
4 mM KCl, 1 mM MgCl2 , 2 mM CaCl2 , 2 mM
-mercaptoethanol, and 10 mM HEPES, pH 7.2) containing 20 M calpain substrate and the fluorescence
was determined in a confocal microscope using a
wavelength 360/480 nm filter. For kinetic studies, the
fibroblasts subcultured in a 96-well cell culture plate
(Corning, NY) to confluence were incubated in the
same buffer containing 10 M calpain substrate. The
time course of the reaction was determined first and the
subsequent measurements were conducted after incubation of the reaction mixtures for 15 min at 37 C.
The fluorescence generated was measured in a BioTek
FLX800 plate reader using wavelength 360 nm for
emission and 480 nm for excitation. All samples were
measured in triplicate and the values were normalized
to the cellular protein concentrations determined with
a Protein assay kit (Bio-Rad, CA).
Measurement of cellular ATP content
The procedure was carried out using a luciferasebased ATP assay kit (Molecular Probes, OR) according
to the manufacturers protocol. Young and old fibroblasts subcultured to confluence in a 12-well plate were
incubated with vehicle or luciferin-luciferase reaction
agents for 10 min at 37 C. At the end of incubation,
the cells were removed from the plate using a rubber
cell scraper, lysed, and processed for measurements.
Bioluminescent signals generated were determined at
570 nm for emission in a BioTek Flx800 plate reader.
Equal sampling of the cells was confirmed by protein
assay. All samples were measured in triplicate and the
average values were presented as relative cellular ATP
content.
Calpain protein levels in the cells
Cultured young and old fibroblasts were harvested,
washed, and lysed in a buffer (145 mM NaCl, 2 mM
MgCl2 , 5 mM EGTA, 1x protease inhibitor cocktail,
20 mM Tris-HCl, pH 7.2, and 1% Triton X-100). The
protein contents were normalized and analyzed by
western blotting probed with a monoclonal antibody

837

to human -calpain (dilution 1 : 1000) (CalBiochem,

CA) or a polyclonal antibody to human m-calpain
(dilution 1 : 500) (Triple Point Biologics, WA) as
described previously [31].
Statistical analysis
Unpaired student t-tests were used for comparisons
between the sample groups, Pearson product-moment
correlations were performed, and the intra- and interassay coefficients of variation were calculated using
software from GraphPad and SigmaPlot 11 (San Jose,
CA). p values <0.05 were considered significant.
RESULTS
[Ca2+ ]i transient overstay in old broblasts
We first measured [Ca2+ ]i transients in cultured
fibroblasts from a young (age 22) and an old (age
96) healthy donor labeled with calcium dye Fluo-4
AM monitored by confocal microscope. When cells
were stimulated by 25 nM bradykinin (BK), an inositol
1,4,5-trisphosphate receptor-activating Ca2+ agonist,
bright fluorescence was elicited in approximately 90%
of the cells (Fig. 1). No major difference was seen in the
fluorescence intensity between the young and old cells
in the first 50 seconds. But afterwards, the signal in the
young cells decayed rapidly and disappeared by 100
seconds, whereas in the old cells it stayed substantially
longer and typically did not disappear until after 200
seconds (Fig. 1). The effects by BK were no longer seen
when cells were pretreated with 10 nM BAPTA-AM,
an intracellular Ca2+ chelator (data not shown).
Measurements of the fluorescence changes as a function of time showed that the ascending phases of the
Ca2+ transients in the young and old cells were quite
similar when they were put together for comparison
(Fig. 2a). But, while the transient in the young cells
was sharp and near symmetrical, the transient in the
old cells exhibited a slightly higher amplitude and a
remarkably prolonged tail upon decaying to the resting level (Fig. 2a). This transient overstay, or excessive
[Ca2+ ]i level, in old cells was similar to those reported
by previous studies [35].
To address why Ca2+ transients were overstayed,
we reasoned that since old cells commonly suffer from
energy depletion and oxidative stress, these insults
may contribute to the observed [Ca2+ ]i overstay. If so,
then the [Ca2+ ]i overstay might be mimicked in the
young cells by inhibiting energy or by inducing reactive oxygen species. We pretreated the young cells with

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H.T. Nguyen et al. / Higher Ca2+ Levels Cause Lower Signaling

Fig. 1. Old fibroblasts retained the Ca2+ transient longer than young cells. Cultured human fibroblast cells labeled with fluo-4 AM were treated
with bradykinin (25 nM) and time-lapsed microphotographs were taken at the time intervals indicated (magnification 400). Small squares
denote the intracellular areas selected for confocal measurements (see Fig. 2a). BK, bradykinin; Rest, resting cells; Y, young; O, old cells.

Fig. 2. Representative [Ca2+ ]i transients evoked by BK in old and young fibroblasts and the effects of Rot, H2 O2 , PEP, or PCr. a) Young and
old cells in response to BK alone (25 nM). The areas inside the cells measured were shown in Fig. 1 (small square). b) Young cells pretreated
with Rot (1 M) or H2 O2 (1 mM) for 10 min followed by BK. c) Old cells pretreated with PEP or PCr (1 mM each) for 10 min followed by
BK. d) Quantitation of the transient curves (in ac) as [Ca2+ ]i levels under various concentrations of the agents. Values are means SEM from
three separation experiments using different pairs of young and old cells. *p < 0.01; FI, fluorescence intensity; AUC, area under the curve; AU,
arbitrary unit; pyru, pyruvate.

H.T. Nguyen et al. / Higher Ca2+ Levels Cause Lower Signaling

mitochondrial complex I inhibitor rotenone (1.0 M)

or with H2 O2 (1 mM) for 10 min prior to the addition of BK and found that both treatments caused an
apparent [Ca2+ ]i overstay, so that the [Ca2+ ]i curves
in the young cells now looked much like that in old
cells (Fig. 2b, compared to 2a). Reducing the doses of
rotenone (to 0.5 M) or H2 O2 (to 0.6 mM) caused less
severe [Ca2+ ]i overstay (not shown).
Very recently, we have found that promoting cellular
bioenergetics by a group of high-energy compounds
can substantially overcome the impairments caused
by energy depletion and oxidative stress and promote cell survival [26]. Since Ca2+ signaling is a
highly energy-dependent process, it was possible that
these compounds may also restore Ca2+ balance in
old cells, so we pretreated the old cells for 10 min
with PEP or PCr, two high-energy compounds [28,
29]. It was found that they remarkably reduced the
BK-evoked Ca2+ overstay (Fig. 2c), but pyruvate and
creatine, their cognate counterparts without energyrich bond, did not show the same effects (not shown,
see below). This suggested that the presence of such
bond in the compounds was necessary for the observed
effects.
Quantitative conversion of the Ca2+ transient curves
obtained from three different young and old cell lines
into [Ca2+ ]i levels (as area under the curve) revealed
that the transient alterations correlated well with the
changes of [Ca2+ ]i levels under various experimental conditions (Fig. 2d). For example, the BK-evoked
[Ca2+ ]i levels in old cells increased by 67.2% relative to the young cell level (Fig. 2d, left panel).
Meanwhile, rotenone or H2 O2 raised [Ca2+ ]i levels, respectively, in a dose-related manner in young
cells (middle panel). In old cells, rotenone further
increased [Ca2+ ]i to a dangerous level (middle panel,
O+Rot), and an MTT test showed that such cells
were near death (not shown). In contrast, treating the
old cells with PEP and PCr at increasing concentrations gradually decreased the excessive [Ca2+ ]i levels
(right panel). Additionally, three other high-energy
compounds, namely Acetyl-CoA (ACoA), acetyl
L-carnitine (ALC) and S-adenosylmethionine (SAM)
[29, 32, 33], also decreased [Ca2+ ]i levels in old cells
(not shown). In young cells, PEP exhibited a similar
effect (right panel).
Calpain activity in old cells
To see how altered [Ca2+ ]i levels in old cells affect
the efficacy of Ca2+ signaling, we measured the in situ
enzymatic activity of calpain in cultured fibroblasts

839

using a cell-permeable fluorogenic calpain substrate.

This substrate efficiently entered the cells and generated blue fluorescence when monitored by microscope
at wavelength 360/480 mm (Fig. 3a). The fluorescence
intensity in an old cell line (age 85) was visibly lower
than in a young line (age 22), but it increased considerably by the addition of 1 mM PEP, an effect that was
abolished by the presence of 10 M calpeptin, a cellpermeable calpain inhibitor (Fig. 3a). Kinetic study
of the reaction further revealed that while the fluorescence generation in both young and old cells was
time-dependent and displayed saturable rate kinetics,
the fluorescence intensity in the old cells was lower
than in the young cells at resting state, but was robustly
increased by 1 mM PEP and the effect was abolished
by calpeptin (Fig. 3b).
Quantitative measurements in five different young
(age 1729) and old (age 8196) cell lines showed that
the average calpain activity in the old cells decreased
to 51.7% of the young cell level (Fig. 4a). Meanwhile,
treating young cells with increasing doses of rotenone
(0.5 and 1.0 M) or H2 O2 (0.5 and 1.0 mM) gradually decreased calpain activity (Fig. 4b and Table 1).
Rotenone also decreased the activity in old cells
(Fig. 4b). In contrast, treating old cells with increasing
concentrations of PEP or PCr (0.5 and 1.0 mM) gradually re-activated calpain (Fig. 4c and Table 1), effects
that were completely abrogated by 1 M rotenone
(Fig. 4c, shown only its effect on 1 mM PEP). Again,
pyruvate or creatine (1 mM) did not enhance calpain
activity significantly (shown only the effect of pyruvate). Meanwhile, PEP in the young cells also robustly
boosted calpain activity to 3.4-fold and the effect was
blocked by rotenone (Fig. 4d and Table 1).
Interestingly, it was notable that calpain activities
measured in these cells were inversely correlated to
the [Ca2+ ]i levels under various experimental conditions (compare Fig. 4 to Fig. 2d). ACoA, ALC, and
SAM also exhibited strong calpain-activating effects
(Table 1).
High-energy compounds boosted cellular energy
level
These results suggested that the high-energy compounds enhanced Ca2+ signals and calpain activity by
affecting the energy states of the cell. To confirm this
possibility, we determined the effects of these compounds on cellular ATP content. Results showed that
the average ATP content in the five old fibroblast lines
at resting state was lowered to 55.2% relative to that
in the five young cell lines (Fig. 5a), indicating an

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Fig. 3. Fluorescence generated by PEP in fibroblasts incubated with a fluorogenic calpain substrate. a) Typical fluorescence intensity in a young
(age 26) and an old (age 85) fibroblast lines captured at wavelength of 360/480 mm after incubating with 20 M calpain substrate (magnification
400). O + PEP, 5 min after the addition of PEP (1 mM) in old cells; O + CPT + PEP, old cells pretreated with calpeptin (CPT, 20 M) for 10 min
before the addition of PEP. b) Representative time course showing the fluorescence generated in young and old cells and the effects of PEP
alone or in the presence of calpeptin. Blank, calpain substrate only.

Fig. 4. Calpain activity in young and old fibroblasts and the effects of Rot and energy-rich compounds. a) Average calpain activity in five young
and five old cell lines in the resting state. b) Young cells pretreated with rotenone (Rot) or H2 O2 at increasing concentrations. c) Old cells
treated with PEP or PCr at increasing concentrations and the effects of Rot and pyruvate (Pyru). d) Young cells in response to PEP and Rot for
comparison. All values are means SEM from five different young and old cell lines by three independent assays (n = 3). *p < 0.01; **p < 0.001,
versus their respective controls.

H.T. Nguyen et al. / Higher Ca2+ Levels Cause Lower Signaling

841

Fig. 5. Relative ATP content in young and old fibroblasts and the effects of various agents. a) Average ATP levels in five young and five old
fibroblast lines in the resting state. b) Young cells treated with increasing doses of rotenone. c) Old cells treated with PEP or Pcr at increasing
concentrations. d) Young cells treated with PEP for comparison. The cells were pretreated with various agents for 10 min and ATP levels were
determined using a bioluminescent ATP assay kit (see Methods). All values are means SEM from five young and five old cell lines by three
independent measurements (n = 3). *p < 0.01; **p < 0.001, verses their respective controls.
Table 1
[Ca2+ ]i levels, calpain activity and ATP content in young and old fibroblasts in response to rotenone and energy-rich compoundsa,b
Reagent
Basal
Rot 0.5 m
1.0 m
PEP 0.5 mM
1.0 mM
PEP 1.0 mM + Rot 1 M
Pyruvate 1 mM
PCr 0.5 mM
1.0 mM
PCr 1 mM + Rot 1 M
Creatine 1 mM
ACoA 1 mM
ALC 1 mM
SAM 1 mM

[Ca2+ ]i (AU)
Young
Old
10.0 0.9
14.2 1
19.5 2
7.3 1

7.6 1

16.7 2
22.3 3b
13.1 2
9.7 1
15.2 2
16.5 2
10.6 1
8.9 1
15.9 2
16.1 2
13.3 2
11.7 1
12.4 1

Calpain (%)
Young
Old
100 9
57.3 5
45.9 4
210 15
82.5 5
127 10
215 19
81.3 9
121 10

51.7 4
42.5 4b
91.4 8
128 11
50.3 4
55.1 5
87.2 8
132 14
54.3 5
56.7 5

ATP (%)
Young

Old

100 10
71.1 5
53.2 5

55.2 5

290 25
80.3 8
118 9
278 21
76.7 7
123 11

85.2 7
104 9
96.3 8

43.1 4b
81.2 7
128 12
56.4 5
59.2 5
80.4 7
130 12
58.6 5
61.4 5
87.5 8
117 10
109 10

are means SEM from three [Ca2+ ]i or five (calpain and ATP) different young and old fibroblast cell lines.
in near-death condition.

a values
b cells

inefficient energetic state in the old cells as previous studies reported [34]. Treating young cells with
rotenone (0.5 and 1 M) diminished ATP levels to
62.1% and 53.2%, respectively. But treating old cells
with increasing concentrations of PEP or PCr (0.5 and
1 mM) progressively boosted ATP levels (Fig. 5b and
Table 1). Again, the effects were abolished by rotenone
(shown only its effect on PEP). ACoA, ALC, and SAM
also exhibited ATP-enhancing effects, but pyruvate and
creatine did not (Fig. 5b, shown only the effect of pyruvate; and Table 1). Moreover, PEP (1 mM) strongly
promoted ATP levels by 2.9-fold in the young cells,

an effect that was blocked by rotenone (Fig. 5d and

Table 1).
Duration of calpain activation and calpain protein
levels in old cells
To see how long the high-energy compoundsstimulated calpain activation could last, we extended
the duration of the measurements of the effects and
found that the fluorescence intensity stimulated by PEP
or PCr (1 mM each) in old fibroblasts did not change
after 1 h, though gradually decreased afterwards

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Fig. 6. Duration of the calpain activity stimulated by PEP or PCr. a) Time-lapsed microphotographs showing the progressive changes of
fluorescence after stimulation by PEP or PCr (1 mM each) in old fibroblast cells preincubated with calpain substrate for 10 min. b) Time-course
of the dynamic calpain activity changes after the addition of PEP or PCr. Control, resting cells. Values are means SEM from five old cell lines
by three independent measurements.

(Fig. 6a). Calpain activity assay revealed that after

6 h, there were still 61.6% and 53.2% of the activity
remained from the peak levels by PEP and PCr stimulation, respectively (Fig. 6b). Thus, calpain activation
by these compounds was lasting for a significant length
of time.
To corroborate the finding of calpain inactivation in
old cells (Fig. 4a), we determined the protein levels of
both - and m-calpains in the young and old cells by
western blotting using two specific antibodies, which
clearly distinguish the two calpain isoforms as we previously reported [31]. It was found that the average
protein levels of -calpain and m-calpain in the five old
cells were reduced by 29.5% and 37.3%, respectively,
relative to the five young cell levels, whereas -actin
levels remained unchanged in the cells (Fig. 7a, b).
[Ca2+ ]i and calpain activity in the dying cells
Given the altered [Ca2+ ]i and calpain activity in
aged cells, it was of importance to see how they
change in the cell death process, the proximate cause

Fig. 7. Protein levels of - and m-calpains in cultured fibroblasts

from young and old fibroblasts. a) Cells from five young or five
old donors (ages indicated) were analyzed by western blotting
probed separately with antibodies specific to human -calpain or
to m-calpain. b) Mean values computed from the density of the
immunoreactive bands in (a). CAPN, calpain.

H.T. Nguyen et al. / Higher Ca2+ Levels Cause Lower Signaling

843

Fig. 8. [Ca2+ ]i transients and calpain activity in the dying cells. a) [Ca2+ ]i curves recorded in the young or old cells killed by 0.05% Triton
X-100 and cell death was confirmed by MTT assay. b) Calpain activity measured in the same cells killed by Triton or by overdose rotenone.
Data from the living young or old cells are presented for comparison. Values are mean SEM from three cell lines, p < 0.05 (n = 3). Tx, Triton
X-100; Rot, overdose of rotenone (1 mM).

of AD and an intensive study area today. We measured both [Ca2+ ]i levels and calpain activity in the
cells killed by 0.05% Triton X-100. Ca2+ transients
in the dying young or old cells surged dramatically
and stayed for about 5 minutes before sharply sinking to below the resting level and flattening out as a
straight line (Fig. 8a). No significant differences were
found between the young and old cells, but calpain
activity in the dead cells was spectacularly raised to
as high as proximately 2-fold or 4-fold in the young
and old cells, respectively, over their levels in the living state (Fig. 8b). Also, killing the cells by overdose
rotenone (1 mM) resulted in similar [Ca2+ ]i raises (not
shown) and calpain overactivation (Fig. 8b). Overdose
H2 O2 (10 mM) caused similar calpain activation (not
shown). No significant difference in calpain activity
was observed between the young and old cells, or
among the ways by which the cells were killed. Cell
death by the treatments was confirmed by MTT test
(not shown). Thus, while [Ca2+ ]i level elevation in old
cells continued its way into the cell death process, calpain activity was altered in opposite direction in old
vs. dying cells.

cells, typical spikes of the oscillation in a young (age

17) cell looked fairly sharp and regular, whereas the
spikes in an old (age 92) cell were visibly blunted with
lower number of the spikes within the same period of
time (Fig. 9a). Computing these parameters as spike
frequency or overall Ca2+ level (total area under the
curves) revealed that the average spike frequency in

Ca2+ oscillations in young and old cells

Agonist-elicited [Ca2+ ]i changes in the cell can
manifest either as a prominent single transient or
as repetitive oscillations by different agonist concentrations [35] and it was curious to see how such
oscillations were altered in old cells. In our hands,
repetitive Ca2+ oscillations were induced by 30 pM
BK in about 8% of the fibroblasts. Of these reactive

Fig. 9. Representative Ca2+ oscillations in young or old fibroblasts.

a) Ca2+ oscillations evoked by 30 pM BK in a young (age 17) or an
old (age 92) cell. b) The spike frequency of the Ca2+ oscillations in
the young or old cells. c) Quantitation of the oscillations as overall
Ca2+ levels (total area under the curve, AUC). Means SEM from
three independent measurements. *p < 0.01.

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H.T. Nguyen et al. / Higher Ca2+ Levels Cause Lower Signaling

three old cells was lowered by 28.1% (Fig. 9b), but their
overall Ca2+ levels were increased by 10.2% (Fig. 9c)
compared to young cells. Thus, an inverse relationship
may exist between the spike frequency and the overall Ca2+ levels in these cells under the experimental
conditions.
DISCUSSION
Inefcient Ca2+ handling system in aged cells
Ca2+ signaling in vivo is controlled by a
highly sophisticated Ca2+ maintenance system, which
involves many channels regulated by cholinergic,
glutamatergic, PI3 -, ryanodine-receptors, Na+ /Ca2+
exchangers, Ca2+ -ATPase pumps, and many other elements [1, 2]. Given the complexity of the system and
the difficulty to directly measure the resting Ca2+
activity in the cell or dynamic Ca2+ signals in the
brain, the state of the Ca2+ handling system in the aging
brain is often inferred from the measurements of the
Ca2+ transients evoked by agonists in cultured cells or
tissues [35]. Human fibroblast has been widely considered a useful model to evaluate AD-related changes
for designing intervention strategy [3, 36, 37].
In this study, we demonstrated that the BK-evoked
[Ca2+ ]i transients in old fibroblasts were indeed overstayed, or excessive in levels, as previously reported
[35] (Figs. 1 and 2a). Since the change did not occur
significantly on the Ca2+ entry phase (though slightly
higher amplitude in some cells), it appears that the elevated levels are not resulting from severe alterations
in the Ca2+ channels or receptor density in old cells.
Instead we found that the change occurred mainly as
slowed extrusion and this change can be recapitulated
in young cells by rotenone or H2 O2 (Fig. 2b) and substantially reversed by PEP and PCr in old cells (Fig. 2c).
The data suggest that the BK-evoked [Ca2+ ]i overload
in old cells arises from an inefficient Ca2+ handling
system, i.e., inefficient extrusion pumps compromised
by energy depletion and oxidative stress. Indeed, it
has been shown that Ca2+ -ATPase pumps are inactivated during aging [38, 39]. While other mechanisms
such as altered Na+ /Ca2+ exchangers or Ca2+ buffering proteins [1, 2] may also be involved, the restored
Ca2+ transients by high-energy compounds in old cells
strongly suggest that energy deficiency plays a major
role in the inefficient Ca2+ handling system.
The inefficient Ca2+ handling system, by whatever mechanisms, would be expected to exhibit lower,
rather than higher, functional activity of Ca2+ signals and Ca2+ -dependent enzymes. This reasoning is

now substantiated by our finding that calpain activity

was decreased in old cells (Figs. 4 and 6). Though
contrasting with current views [7, 8], this finding
agrees with a number of previous studies showing that
many Ca2+ -dependent enzymes/proteins are inactivated during aging, including calpain, protein kinase C,
calcineurin, calmodulin, Ca2+ /calmodulin-dependent
kinases and calbindin [1119]. These studies together
indicate, beyond reasonable doubt, that the functionality of the Ca2+ signaling system is diminished in
old cells, despite Ca2+ overload. Thus, caution should
be taken when interpreting data from measurements
of intracellular Ca2+ levels in old cells to assess its
functional changes, a key modification target for AD
prevention. The low activity of calpain in old cells may
also be partly attributed to the lowered protein levels
(Fig. 7) and oxidative stress (Fig. 4b), to which the
thiol group of its active center is known to be particularly sensitive [40]. However, the positive correlation
of calpain activity to energy levels (Figs. 4 and 5) and
its negative correlation to Ca2+ levels (Fig. 2b) argue
that insufficient energy and Ca2+ signaling potency
are perhaps the major contributors to the inactivated
calpain in old cells.
Calpain controversy
Contrasting to the calpain inactivation in old cells,
we also found that the enzyme was dramatically activated in the dying cells (Fig. 8), indicating that calpain
activity undergoes a bi-phasic activity changes, or
in opposite directions in aging vs. pathological conditions. This finding may help explain a previous report
showing that the enzyme is drastically activated in the
brains of AD patients (where massive cell death has
occurred) [41]. This report has prompted many studies
to attenuate cell death in AD by calcium antagonists
(7, 8). However, a broad consensus today is that AD
intervention should start in the pre-symptomatic, or
normal aging, stage [42, 43]. Thus, activating Ca2+
signaling during aging would be a more reasonable
strategy for preventing the disease [24]. But controversies remain, as it has been reported that inhibiting
calpain can improve cognition in the animal models
of AD [44, 45] or that [Ca2+ ]i is not altered in the
fibroblasts from aged individuals in the absence of AD
[36]. Thus, further studies are required to resolve the
controversy and to better understand the changes of calpain in the aging brain where it can also be influenced
by many other factors such as calpastatin, phospholipids, and phosphorylation of calpain substrates [46,
47]. It must be noted, however, that Ca2+ agonists, not

H.T. Nguyen et al. / Higher Ca2+ Levels Cause Lower Signaling

antagonists, have been shown to benefit aging brains

in clinical trials [9, 10].
Unanswered questions
Our study also raises interesting and critical questions. For example, Ca2+ rises have been widely
thought to cause cell death [68], but we have described
three distinct types of Ca2+ rises in the cell: lifesupporting, age-related, and death-triggered. They can
be clearly distinguished since the first is above the
basal line in young cells (Fig. 2a, Young); the second
is excessive over the young cell level and occurs in old
cells (Fig. 2a, Old) where the severity of the excess
is inversely correlated with calpain activity (Figs. 2d
and 4); and the third is the Ca2+ gradient collapse in
cell death (Fig. 7a). These three fundamentally distinct
types of Ca2+ rises result from three energy states in
the cell: high, low, and none. Such conceptual distinctions must be unambiguously made before any rational
AD intervention strategies can be designed.
Higher levels of metals usually correlate to greater
biological activity, but why do the higher Ca2+ levels
in old cells, by whatever reason, down-regulate calpain activity? Studies have established that unlike other
metals, Ca2+ acts as rapid waves in vivo [35] and that
Ca2+ -dependent enzymes including calpain in vivo can
be regulated by wave frequency [4850]. In light of this
concept, we found that the BK-elicited Ca2+ spikes in
old cells displayed lower frequency but slightly higher
overall Ca2+ levels (Fig. 9). If such Ca2+ oscillations
more closely reflect the physiological Ca2+ waves,
then their frequency changes may further explain why
lower Ca2+ signaling efficacy in old cells can manifest
itself as higher global Ca2+ levels.
If calpain activity is regulated by waves and energy,
then how can it be sharply activated in the dying cells
by flooding Ca2+ but no wave and energy? This paradox remains unexplained at present and awaits further
studies. It should be pointed out, however, that this
non-physiological phenomenon, perhaps being part
of the self-destructive autolysis in cell death, should
not overly complicate our studies because only the
age-related, physiological changes are the basis for
understanding of and designing intervention strategy
for AD [24].
Bioenergetics and Ca2+ signaling
Despite the remaining questions and controversies,
the most important finding of this study is that energyrich compounds such as PEP, PCr, ACoA, ALC and

845

SAM can substantially restore the energy balance and

the vigor of Ca2+ signaling in aged cells (Figs. 2, 4
and Table 1). Although their precise modes of action
are unclear at the present time, these compounds have
quite different molecular structures (enol or guanido
phosphate, and thioester) and act as key intermediates
in the aerobic citric acid cycle, the primary source of
ATP synthesis in the brain [28]. PCr can be taken up by
the cell through a sodium-dependent transporter [51].
ACoA, ALC, and SAM can directly enter mitochondria
and affect oxidative phosphorylation [52, 53]. Despite
their diverse roles in the body, our data suggest that
the cellular ATP content they boosted (Fig. 5) is the
common driving force for the observed effects [54].
Furthermore, the intimate relationship between bioenergetics and Ca2+ signaling may help explain why
Ca2+ and calpain both play vital roles in cognition
[21, 22], a highly energy-dependent process [54, 55].
Although the mechanism of origins of AD remains
highly controversial, an emerging consensus today is
that energy/mitochondrial dysfunction can be the earliest modifiable defect in the aging brain [5658],
which has been shown to cause A and tau deposition in the animal brain [59, 60] and undermine
animal learning and memory [61, 62]. In this context,
our findings may reinforce the model by suggesting
that the inefficient Ca2+ signaling is a key mediator through which energy deficits can mechanistically
impair neurotransmission/cognition, a highly Ca2+ dependent process [24]. Of particular interest is that
the inactivated calpain may reasonably explain the agerelated accumulation of its physiological substrates,
such as tau and perhaps also APP as truncated protein
remnants in the brain [30, 63].
We summarize the main findings of this study in a
model (Fig. 10) proposing that energy, Ca2+ signal,
and calpain activity decline hand-in-hand during normal aging. But, if these three pathways continue to
decline to zero in some elderlydue to their older age
or faster declining rate as influenced by inherited or
acquired risk factorscell death and sporadic AD will
result. This simple scheme, however, has been complicated by the dramatic rises of Ca2+ levels and calpain
activity at cell death, making them two tantalizing
intervention targets under the questionable NIH/NIA
definition of senile dementia as a discrete disease
[24]. We, however, emphasize that it is the modifiable
energy and Ca2+ signal deficiencies during the natural aging stage that should serve as rational targets
for AD prevention. It is also our view that energy and
Ca2+ deficiencies result from the inefficiency of the
entire energy and Ca2+ handling systems, rather than

846

H.T. Nguyen et al. / Higher Ca2+ Levels Cause Lower Signaling

Fig. 10. A schematic summary of the findings in this study. It suggests that energy decline during aging causes inefficient Ca2+ signal function
and decreased calpain activity, underlying cognitive decline. In the normally aged elderly, these three pathways decline to a certain extent but
aging cells survive. Whereas in the elderly with AD, the three pathways will continue to decline to zerodue to their older age or faster decline
rate as influenced by risk factorsleading to brain cell death within the human lifespan. This simple mechanism, however, is complicated by
the dramatically rises of Ca2+ levels and calpain activity at cell death, making them two seemingly attractive but false intervention targets. We
emphasize that it is the modifiable energy and Ca2+ signal deficiencies during natural aging that should serve as rational targets for prevention
of sporadic AD.

from any single error (a holistic rather than reductive

view) [24].
In this context, the energy and Ca2+ -signal activating effects of the energy-rich compounds, together
with their neuroprotective roles reported by us [29]
and by other investigators [32, 33, 54, 64], raise the
possibility that these compounds, and many other asyet-unidentified bioenergy-boosting substances, may
strengthen and protect the aging brain cells. This strategy, if combined with other neuroprotective agents
(such as antioxidants and metabolic stimulators), especially enhanced by targeting the modifiable risk factors
in late life, will extend the lifespan of the aging brain
cells to catch up with the demographic changes, the ultimate cause for rapid increase of sporadic AD today.
In this unprecedented endeavor in medical history, the
simple but reliable assays used in this study may prove to
be key tools that are critically needed for screening and
developing more efficient medications in future studies.
ACKNOWLEDGMENTS
This work is supported by the Merit Review program
of the U.S. Department of Veterans Affairs and also by
The CWS Foundation.
Authors disclosures available online (http://www.jalz.com/disclosures/view.php?id=1829).

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