High-energy compounds mobilize intracellular Ca2+ and activate

calpain in cultured SH-SY5Y cells

Huey T. Nguyena and Ming Chena,b,

a
Aging Research Laboratory, Bay Pines VA Healthcare System, Bay Pines, FL 33744, USA.
b
Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa,
Florida.

Running title: Energy drives Ca2+ signals

Key words: Alzheimers, energy, calcium, calpain, amyloid

To whom correspondence should be addressed. E-mail: ming.chen@va.gov

Abstract

Deficiency in energy metabolisms is perhaps the earliest modifiable defect in brain aging and
sporadic Alzheimers disease (sAD). Several high-energy compounds (HECs) such as ATP,
phosphoenolpyruvate, phosphocreatine and acetyl coenzyme A have been shown to exhibit
neuroprotective effects. To understand their mechanism of actions, we tested the effects of
these HECs on intracellular Ca2+, a central regulator in brain function. Our data showed that
the HECs robustly and dose-dependently mobilized intracellular Ca2+ in cultured SH-SY5Y
cells, and the actions were sensitive to intracellular Ca2+ chelator BAPTA-AM or energy
metabolism blocker rotenone. The Ca2+ influx triggered by the HECs was from both
extracellular medium and intracellular stores and the HECs also induced repetitive Ca2+
oscillations. As these actions were similar to those of classical Ca2+ agonists, the HECs may
be viewed as a new group of physiological Ca2+ agonists. We also found that the HECs
promoted the intracellular activity of calpain, a Ca2+-dependent protease, and the enzyme
activity fluctuated in concert with cellular energy levels, suggesting that calpain activity may
also be energy-driven or energy-dependent. These findings may add to current knowledge for
the regulatory mechanisms of Ca2+ and calpain. Since Ca2+ and calpain undergo critical
dysfunction in brain aging but the underlying mechanisms remain elusive, our work may
provide a new perspective for understanding some of the critical issues. More importantly, the
HECs, as key intermediates in glucose catabolism, the primary source of energy supply in the
brain, may be used as potential drugs for rational prevention of sAD.

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INTRODUCTION

The mechanism of origins of Alzheimers disease (AD) has remains disputable today, but an
emerging consensus in the research field is that energy/mitochondrial dysfunction is perhaps
the earliest modifiable defect in brain aging, thus targeting this defect could be an ideal point
of entry for AD prevention [1, 2]. In consistent with this view we have recently found that
several high-energy compounds (HECs), namely ATP, phosphoenolpyruvate (PEP),
phosphocreatine (PCr) and acetyl coenzyme A (ACoA), promote the physiological processing
of -amyloid precursor protein (APP) and enhance the survival of neurons from energy
depletion- or oxidative stress-induced cell death [3]. These and other findings [4-6] are
suggestive that HECs may serve as potential medications for preventing AD. HECs are
intermediates in the critic acid cycle and they contain distinct energy-rich bonds that liberate
at least 7 kilocalories of free energy (-Go) per mole under standard conditions [7].

But, what is the mechanism whereby the HECs promote cell survival? We speculated that
among many metabolic pathways, mobilization of intracellular Ca2+ may be of particular
importance, because ATP is a well-known Ca2+ agonist [8], and Ca2+ signaling is vital for cell
function, maintenance and survival [9]. Ca2+ signaling is made possible by rapid cross-
membrane movements of Ca2+ ions against a steep, 10,000-fold gradient driven by powerful
Ca2+-ATPase pumps. So, Ca2+ signaling is a highly energy-dependent process [9, 10].

So, in this study we tested the effects of several HECs on intracellular Ca2+ and found that
they potently mobilized Ca2+ transients and activated the enzymatic activity of calpain, a Ca2+-
dependent protease [11]. Since both Ca2+ signaling and calpain activity undergo critical
changes during aging, underlying many old-age diseases such as AD and Parkinsons but the
mechanism of the alterations involved has yet to be fully understood [12-14], our study may
help explain some of the key issues from a new perspective.

1. Materials and Methods

1.1. Materials

SH-SY5Y neuroblastoma cells were purchased from ATCC (Gaithersburg, MD). Intracellular
Ca2+ dye Fluo-4 AM was from Invitrogen (Grand Island, NY) and calpain substrate IV and
BAPTA-AM were from EMD-Millipore (Temecula, CA). ATP, PEP, PCr, ACoA, GTP, PP,
rotenone, calpeptin and other chemicals were from Sigma-Aldrich (St. Louis, MO).

1.2. Methods

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2.2.1. Confocal Ca2+ imaging. Cells cultured to approximately 50% confluence on 25 mm
glass-bottomed Petri dishes designed for fluorimetry (MatTek, MA) were loaded with non-
ratiometric Ca2+ dye Fluo-4 AM (3 M) and pluronic acid (0.07%) for 30 min. Ca 2+ imaging
was performed in Olympus Fluoview 1000 laser scanning confocal microscope at the
wavelength of 488/516 nm. Images were collected by 256 128 pixel images at recording
rate of 1 frame per 2 seconds. Ca2+ transients were expressed as fluorescence intensity (FI)
or relative [Ca2+]i levels computed the FI using software from GraphPad (San Diego, CA).

2.2.2. Intracellular calpain activity assay. The procedure used a fluorescence resonance
energy transfer (FRET)-based fluorogenic and cell-permeable calpain substrate (Calpain
substrate IV, EMD-Millipore, CA) as previously described [15]. For kinetic studies, the cells
cultured to confluence in a 96-well cell culture plate (Corning, NY) were incubated in the same
buffer containing 10 M calpain substrate and the measurements were conducted after
incubation for 20 min at 37oC. The emitted fluorescence was determined in a BioTek FLX800
plate reader using wavelength of 360/480 nm.

2.2.3. Measurement of cellular ATP content. The procedure used a luciferase-based

bioluminescent ATP assay kit (Molecular Probes, OR) according to the manufacturers
protocol. The cells subcultured to confluence in a 12-well plate were incubated with vehicle or
reconstituted luciferin-luciferase agents for 20 min at 37oC. The cells were then removed from
the plate, lysed and processed for measurements. Bioluminescent signals were measured in
a BioTek Flx800 plate reader at 570 nm. Equal sampling of the cells was confirmed using a
protein assay kit (Invitrogen, Grand Island, NY) .

2.2.4. Statistical analysis. Unpaired student t-tests were used for comparisons between the
sample groups, and the intra- and inter-assay variations were calculated using SigmaPlot
software (San Jose, CA). P values <0.05 were considered significant.

2. RESULSTS

2.1. HECs mobilize intracellular Ca2+

To study the mechanism of the HECs in promoting cell survival [3], we reasoned that the
protective actions of the HECs may involve Ca2+ mobilization, because Ca2+ is a lifeline for
cell viability and function. Also, because ATP is a potent Ca2+ agonist [8], it was reasonable to
assume that other HECs may also mobilize intracellular Ca2+. To validate this hypothesis, we
first determined the effect of PEP, which possesses perhaps the highest energy-rich bond
found in living organisms (-Go = 14.8 kcal/mol versus 7.3 kcal/mol for ATP) [7].

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In cultured SH-SY5Y neuroblastoma cells labeled with calcium dye Fura-4, PEP was found to
evoke green fluorescence in a concentration-dependent manner (Fig. 1a). As shown, 0.1 mM
PEP lit up only a few cells with moderate fluorescent intensity. But at 0.5 mM it sharply
increased both the signal intensity and the number of responsive cells. Further increase of the
PEP concentration to 1 mM activated almost all cells with even stronger signals (Fig. 1a;
representative cells from a total of more than 150 cells imaged). This effect of PEP was
completely abolished by pretreating the cells with 10 nM BAPTA-AM, an intracellular Ca2+
chelator, or with 1 M rotenone, a respiratory chain complex 1 inhibitor (Fig. 1a, two images at
right). The dynamic Ca2+ transient traces corresponding to the fluorescence intensity in Fig.
1a were recorded as a function of time (Fig. 1b). Three curves from each image were
obtained by monitoring three intracellular spots (small squares shown in Fig. 1a, the 4 images
at left).

The Ca2+ transient elicited by PEP was markedly reduced either in the calcium-free medium
or in the standard medium with the presence of thaspigagin (TG, 10 M), a
sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor (Fig. 1c, compare -Ca2+ and
+TG to standard medium, Std; the effect of TG only without PEP was also shown for
comparison). Moreover, in the calcium-free and thaspigagin-containing medium (-Ca2++TG),
PEP did not elicit significant Ca2+ transient. Similar results were also obtained with two other
HECs, PCr and ACoA (-Go = 10.3 and 7.7 kcal/mol, respectively) [7] (data not shown).
Thus, Ca2+ influx elicited by the HECs entered the cytosol from both extracellular medium and
intracellular stores (through calcium release-activated calcium channels), similar to the
mechanisms of nicotine and glutamate, two commonly used Ca2+ agonists [16, 17].

We also found that PEP, PCr and ACoA mobilized Ca2+ in a dose-dependent manner (Fig.
1d). Under the identical concentration (1 mM each), their potencies were comparable to ATP,
but considerably greater than glutamate. The effects of the HECs were blocked by BAPTA-
AM or by rotenone. However, cogent counterparts of these HECs without energy-rich bond,
namely pyruvate, creatine and CoA, did not exhibit significant effects (Fig. 1d, right). Thus,
the presence of high-energy bond was essential for the compounds to exhibit the Ca2+-
mobilizing effects.

2.2. HECs activated calpain in SH-SY5Y cells

To see whether the HECs boosted the functional activity of Ca2+-dependent enzymes, we
determined their effects on intracellular activity of calpain. ATP has been reported to activate
calpain [18]. The choice of calpain over other Ca2+-dependent enzymes was also because it is
involved in the pathogenesis of Alzheimers and other diseases but the underlying mechanism
has remained elusive [13].

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Using a FRET-based fluorogenic and cell-permeable calpain substrate and monitored under
microscope at the wavelength of 360/480 nm, we found that PEP elicited blue fluorescence in
SH-SY5Y cells (Fig. 2a). The signal intensity was gradually enhanced by increasing
concentrations of PEP and the effects were inhibited by rotenone (1 M) or calpeptin (20 M),
a cell-permeable calpain inhibitor (Fig. 2a). Time-course studies revealed that the PEP-
induced calpain activity exerted saturable rate kinetics under various experimental conditions
(Fig. 2b). PCr and ACoA exhibited similar effects (not shown). Quantitative measurements
showed that PEP, PCr and ACoA boosted calpain activity in a concentration-dependent
manner (Fig. 2c). Kinetic studies further revealed that PEP had an EC50 = 0.37 mM (Fig. 2d).
Similar experiments found EC50 = 0.39 mM and 0.42 mM for PCr and ACoA, respectively.

Comparison of the calpain-activating potencies of PEP, PCr, ACoA and two other HECs,
guanosine triphosphate (GTP) and pyrophosphate (PP), showed that they all promoted
calpain activity with potencies comparable to that of ATP, but significantly greater than that of
glutamate when tested at the same concentration (1 mM each) (Fig. 2e). Again, the effects of
the HECs were abolished by rotenone or calpeptin (Fig. 2e, the effects of rotenone and
calpeptin on GTP and PP not shown). Pyruvate, creatine and CoA did not display the
significant effects (Fig. 2e, right).

3.3. HECs boosted cellular ATP content

These experiments suggested that the HECs activated Ca2+ by promoting energy levels of the
cell. To confirm this mechanism, we determined the effects of the HECs on cellular ATP
content. Using a luciferase-based ATP assay, we found that PEP, PCr, ACoA, GTP and PP
all robustly boosted ATP levels to various degrees in SH-SY5Y cells, and the effects were
concentration-related and sensitive to rotenone (Fig. 3). Interestingly, nicotine, glutamate also
enhanced ATP levels, but their efficacies were much less than those of HECs. Again,
pyruvate, creatine and CoA were ineffective, suggesting that the increased ATP levels in the
cells by the HECs were attributable to the presence of the high-energy bonds in the
compounds.

3.4. HECs evoked Ca2+ oscillations

Ca2+ agonists such as ATP and nicotine in cultured cells at different concentrations can
induce either a single Ca2+ transient or repetitive Ca2+ oscillations [16, 19], and it was curious
to see whether HECs could induce Ca2+ oscillations in SH-SY5Y cell. In our hands, low
concentration of PEP, PCr and ACoA (10 M each) induced repetitive Ca2+ oscillations in
10.6% (15/141, active over total cells imaged), 9.6% (12/125) and 5.4% (5/93) of the cells,
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respectively (Fig. 4). At the concentrations lower than 10 M, the HECs did not evoke any
detectable signals over baseline. At higher concentrations (above 0.1 mM), they each induced
a strong single Ca2+ transient (see Fig. 1b). The reasons for the distinct responses of the
HECs at different concentrations were unclear at the present time.

4. DISCUSSION

4.1. The link between energy and Ca2+

This study shows that several energy-rich intermediates generated in the citric cycles of
mitochondria can directly mobilize intracellular Ca2+ (Fig. 1), thus affecting the spatiotemporal
aspects of Ca2+ signals in SH-SY5Y cells. Mitochondria are known to affect Ca2+ signals by
changing the concentrations of ATP, NAD(P)H and reactive oxygen species via both feed-
back and feed-forward mechanisms [9, 10].

While the precise mechanism of Ca2+ activating effects of the HECs is unclear at the present
time, it has been reported that PEP is a mitochondrial permeability transition pore (mPTP)
inducer and it enhances mitochondrial swelling and Ca2+-dependent permeability in
mitochondria (20, 21). PCr can be taken up into cell by a sodium-dependent transporter and
serve as a high-efficient energy reserve system in the body (22). ACoA can directly enter
mitochondria and affects the oxidative phosphorylation process (23). Despite distinct initial
pathways involved, we have found that HECs robustly boost energy levels in the cell, but their
cogent counterparts without energy-rich bond do not. It is thus reasonable to assume that the
free energy they released or enhanced by accelerating glucose catabolism in the
mitochondria is a common effector that drives Ca2+ mobilization. Interestingly, nicotine and
glutamate have also been reported to enhance energy levels (Fig. 3) [24], suggesting that
activation of energy metabolisms is an integral part of the Ca2+ mobilization process and, thus,
boosting energy levels will activate Ca2+ signals in the cell.

HECs promoted Ca2+ entry from both extracellular space and intracellular stores (Fig. 1c) and,
at low concentrations, they also induced repetitive Ca2+ oscillations in the cell (Fig. 4). As
these actions are similar to those of classical Ca2+ agonists, and HECs also promote Ca2+
signals in human fibroblasts [15], it thus appears that the HECs can be a new group of
physiological Ca2+ agonists acting in both neuronal and peripheral cell types.

Because PEP, PCr and ACoA have distinct high-energy bonds (phosphoric ester of enol,
guanido phosphate or acyl thioester, respectively), but do not have a purine ring [7], they
should be expected to act through different pathways than purinergic receptors used by ATP
[8]. These findings may add to current knowledge of the regulatory mechanisms of Ca2+

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signaling, though it remains to determine what the precise mode of actions of the HECs are in
the cell and whether or how they affect the G protein-related signal cascades.

Importantly, the intimate relationship between energy and Ca2+ signaling is also highlighted by
considering several Ca2+-dependent physiological processes altogether: neurotransmission,
fertilization, cell growth and muscle contraction [25]. A common feature is that each of them
represents an active work of the body and, apparently, such work will not occur unless the
body has already acquired sufficient amounts of free energy. Thus, these Ca2+-dependent
physiological processes are also energy-dependent or energy-consuming processes, so
promoting energy metabolisms will enhance their functionalities in the body.

4.2. Calpain activity and bioenergetics

We have also found that the HECs activate calpain and the calpain activity fluctuates in
concert with cellular energy levels (Figs. 2 and Fig. 3). Such an intimate relationship between
energy and calpain implies that calpain may also be viewed as an energy-driven or energy-
dependent protease, a unique feature that, in fact, can be directly inferred from the energy-
dependent nature of Ca2+ signaling per se.

However, this feature at first glance may seem to confront the knowledge that proteolysis in
the body generally occurs spontaneously and it is protein synthesis that is energy-dependent.
So, how can calpain be energy-dependent?

This intriguing question prompts us to take a closer look at the unique properties of calpain.
Unlike many other proteases that act spontaneously and randomly, calpain in vivo only makes
specific and limited cleavages on designated substrates and this converts them into active
enzymes/proteins in the Ca2+ signaling cascades [26]. Conceivably, such actions will not
occur spontaneously or randomly, but must be controlled by physiological demands. But, how
can calpain execute such a controlled action given it does not exhibit a high sequence
specificity on its substrates [27]? Our findings may suggest a clue: as an energy-driven
protease, calpain activation will occur only when physiological demands arise and sufficient
energy is available, such as during neurotransmission, fertilization, cell growth and muscle
contraction. Indeed, calpain plays vital roles in each of these Ca2+- and energy-dual
dependent processes [28-31].

4.3. Ca2+ and calpain changes in brain aging

Ca2+ signaling and calpain activity undergo critical dysfunction during aging, underlying many
age-related diseases such as Alzheimers, Parkinsons, muscular dystrophies, osteoporosis
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and cataracts [13], but the mechanism involved and intervention approaches have remained
poorly understood [12-14]. As the static Ca2+ levels have been found to be elevated in old
cells, it is widely believed that Ca2+ and calpain are activated during aging. We, however,
have recently found that the functional activities of Ca2+ signaling and calpain are decreased
in aged human fibroblasts. This suggests that their mechanism of age-related alterations may
be much more complex than currently thought [15].

Now, the present study may provide a new perspective for clarifying controversy. As energy
metabolisms will inevitably decline during aging, the functional decline of energy-dependent
Ca2+ signaling would be expected to decline, most likely in the forms of reduced frequency
and amplitude of the Ca2+ waves [15]. This in turn would result in inefficient
neurotransmission/memory in the aging brain. At the same time, the activity of energy-
dependent calpain will concomitantly decline, and this may account for the age-related
depositions of its physiological substrates such as tau and perhaps A as well (32).

Based on the findings, we propose an overall model for the natural history of sAD (Fig. 5).
Energy deficiency is the earliest and common underpinning for many age-related
conditions (e.g., bone and hearing loss). It also causes Ca2+ signaling deficits underling
memory deficiency and plaque and tangle formation in all elderly, though only some of
them will develop sAD (for reasons, see ref. 14). Therefore, energy and Ca signaling
deficiencies would emerge as ideal points of entry for early intervention of sAD. In this
context, the HECs, key intermediates in glucose catabolism, the primary source of energy
supply in the brain, may prove to be a new category of drugs for sAD and other age-
related disorders upon further studies.

Acknowledgement

This material is the result of work supported with resources and the use of facilities at the Bay
Pines VA Healthcare System. The contents of this paper do not represent the views of the
Department of Veterans Affairs or the United States Government. The work is also supported
in part by the CWS Foundation. We thank Drs. Jessica Chang and Valentina Echeverria for
kindly sharing research reagents with us.

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Figure legends:

Fig. 1. HECs mobilized intracellular Ca2+ in SH-SY5Y cells. (a) PEP at various
concentrations evoked fluorescent signals in sequential stimulations. In testing the effects
of BAPTA-AM (Bap, 10 nM) and rotenone (Rot, 1 M), cells were preincubated with the
inhibitors for 10 min before the addition of PEP. Small squares denote the subcellular
areas selected for confocal measurements. (b) Fluorescence traces corresponding to the
signal intensities of various conditions in (a) were measured as a function of time on the
selected intracellular areas shown in (a). FI, fluorescence intensity. (c) Effects of PEP (0.5
mM) under various conditions: in the standard medium (Std); in the calcium-free medium (-
Ca2+); in the standard medium with the presence of thaspigagin (Std+TG, 0.2 M; the
effect of TG only also shown for comparison); or in the calcium-free medium with the
presence of TG (-Ca2++TG). (d) Dose-dependent Ca2+-mobilizing actions of HECs in
comparison with glutamate (Glu) and ATP. Fluorescent signals evoked by the agents
were converted to Ca2+ concentrations as area under the curve and expressed in
arbitrary unit (AU). BAPTA-AM and rotenone were used as described in (a). The effects
of pyruvate (Pyru), creatine (Cr) and coenzyme A (CoA) were also shown. All values are
means + SEM from three independent experiments. *, p < 0.01; **, p < 0.001, versus basal
level.

Fig. 2. HECs promoted calpain activity in SH-SY5Y cells. (a) PEP at the indicated
concentrations evoked fluorescent signals in the cells incubated with calpain substrate.
Rot (1 M) and calpeptin (CPT, 20 M) were added 10 min prior to the addition of PEP.
(b) Representative time-course data showing calpain activity enhanced by PEP at
different concentrations. CPT+PEP, cells preincubated with CPT followed by the addition
of PEP. Blank, calpain substrate only. (c) Three HECs activated calpain in a dose-

11
dependent manner. (d) Determination of the EC50 for PEP. (e) Relative efficacies of HECs
in promoting calpain activity in comparison of glutamate (Glu). CPT and Rot were used as
described in (a). All agents were tested at 1 mM each (except for Rot and CPT) using the
assay described in (b) after incubation for 20 min. Values are means + SEM from five
independent assays. *, p < 0.01; **, p < 0.001, versus basal level.

Fig. 3. HECs boosted cellular ATP content in dose-dependent manner and in comparison
with nicotine (Nic) and glutamate (Glu). The agents were incubated with SH-SY5Y cells for 20
min and ATP levels were measured using a luciferase-based ATP assay kit (see Methods).
In testing the effects of rotenone on HECs, rotenone was preincubate with the cell for 10 min
before the addition of the HECs. Values are means + SEM from three independent
experiments. *, p < 0.01; **, p < 0.001, verses basal level.

Fig. 4. HECs at low concentrations induced Ca2+ oscillations in cultured SH-SY5Y cells.
The cells were incubated with PEP, PCr or ACoA (10 M each) and the Ca2+ signals were
recorded as a function of time by confocal Ca2+ imaging. FI, fluorescent intensity.

Fig. 5. Our overall model for the natural history of sAD. Energy deficiency is perhaps
the earliest modifiable defect in aging and the common underpinning for many age-related
conditions (bone and hearing loss, etc.). It also causes Ca2+ signaling dysfunction,
underling memory (Memo) deficits and the depositions of plaques and tangles in all
elderly, though only some of them develop sAD (for reasons, see Chen et al., 2011). Our
model emphasizes that energy and Ca2+ signaling deficiencies, but not their downstream
lesions (e.g., plaques and tangles), are the best targets for pharmaceutical prevention of
sAD. , functional decline.

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