European Journal of Pharmaceutical Sciences 46 (2012) 17–25

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

Neuroprotective effect of undecylenic acid extracted from Ricinus communis L.

through inhibition of l-calpain
Eunyoung Lee a, Ji-Eun Eom a, Hye-Lin Kim a, Da-Hye Kang a, Kyu-Yeon Jun a, Duk Sang Jung b,
Youngjoo Kwon a,⇑
a
College of Pharmacy, Division of Life & Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea
b
Department of Chemistry, Cheju National University, 1 Ara 1-dong, Jeju 690-756, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: The key neuropathological features of Alzheimer’s disease are abnormal deposition of Ab plaques and
Received 30 November 2011 insoluble Ab peptides in extracellular brain and intracellular neurofibril tangles induced by abnormal
Accepted 31 January 2012 tau hyperphosphorylation. l-Calpain is one of the factors that bridge these Ab- and hyperphosphorylated
Available online 8 February 2012
tau-mediated pathological pathways. Undecylenic acid (UDA), a naturally occurring unsaturated fatty
acid, was discovered as a l-calpain inhibitor by screening a chemical library using a substrate specific
Keywords: l-calpain assay method. UDA inhibited Ab oligomerization and Ab fibrillation and reversed Ab-induced
l-Calpain inhibitor
neuronal cell death. In addition, UDA scavenged ROS and reversed the levels of proapoptotic proteins
Undecylenic acid
Extraction of Ricinus communis L.
induced by ROS in SH-SY5Y cells. UDA inhibited l-calpain activity with better potency than the known
p25 Generation peptide-like l-calpain inhibitor, MDL28170, in SH-SY5Y and HEK293T cells transfected with the catalytic
Tau hyperphosphorylation subunit of l-calpain. These results suggest that UDA is a novel non-peptide-like l-calpain inhibitor with
good cell permeability and potent neuroprotective effect.
Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Calpains, cytoplasmic cysteine proteases, are activated by

Alzheimer’s disease (AD) is a major neurodegenerative disorder
of the aging brain (Conway et al., 2003). The characteristics of AD
are neurofibrillary tangles (NFTs) comprised of hyperphosphoryl-
ated tau and senile plaques made up of aggregated amyloid beta
protein (Ab) (Selkoe, 1991). Calpain may be one of the factors that
bridge the two pathways (Camins et al., 2006). The insoluble Ab
peptide is caused by proteolytic cleavage of amyloid precursor pro-
tein (APP), a type-1 transmembrane protein (Kang et al., 1987). The
N-terminal of APP cleaved by b-secretase (also called BACE1/Asp2/
memapsin2) becomes the soluble APP-b and successive cleavage
by c-secretase generates insoluble Ab. Through the secretion
pathway, sorted insoluble Ab is accumulated in outside the cell
(Buxbaum et al., 1998). Depending on where c-secretase cuts in
C-terminal APP, Ab1–40 and Ab1–42 are mainly formed (Younkin,
1998). In particular, the accumulation of Ab1–42 results in Ab
oligomers and fibrils which result in the forming of extracellular
amyloid plaques (Demattos et al., 2001). In AD, chronic toxicity
of aggregated Ab1–42 could lead to sustained intracellular calcium
elevations in susceptible central neurons and thus calpain activa-
tion (Saito et al., 1993). Eventually, the Ab peptide causes synapse
and neuronal cell loss (Smith et al., 2006).
⇑ Corresponding author. Tel.: +82 2 3277 4653; fax: +82 2 3277 3051.
E-mail address: ykwon@ewha.ac.kr (Y. Kwon).
increased intracellular calcium and are essential for normal physi-
ologic neuronal function. However, alteration in calcium
homeostasis generated by insoluble Ab leads to persistent hyperac-
tivation of calpains. The two most widely distributed isoforms are
l-calpain (calpain1) and m-calpain (calpain2). l-Calpain requires
micromolar concentrations of calcium for full activity and m-cal-
pain requires millimolar concentrations of calcium in vitro. l-Cal-
pain is the major form in neuronal cells and many blood
elements (Mellgren, 1990; Saido et al., 1992; Salamino et al.,
1993). Hyperactivated l-calpain cleaves a number of neuronal
substrates including p35, which negatively affects neuronal struc-
ture and function, leading to inhibition of essential neuronal sur-
vival mechanisms. CDK5, a protein kinase, is involved in terminal
differentiation of neurons and muscle cells. l-Calpain-mediated
p35 cleavage into p25 results in the increase in CDK5 activity be-
cause p25/CDK5 has a more stable conformation than p35/CDK5.
The prolonged activation of CDK5 by complexing with p25 induces
tau to be hyperphosphorylated, leading to the formation of neuro-
fibrillary tangles (NFTs) and neurodegeneration in AD (Nikolic
et al., 1996; Patrick et al., 1999; Lee et al., 2000; Hashiguchi
et al., 2002; Cruz et al., 2003; Noble et al., 2003). Conclusively, l-
calpain plays an important role in Ab-mediated NFT formation
and the development of a l-calpain inhibitor may be a good strat-
egy for drug developers targeting AD. In particular, targeting the
p35 cleavage activity of l-calpain among its proteolytic activities

0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2012.01.015
18 E. Lee et al. / European Journal of Pharmaceutical Sciences 46 (2012) 17–25
with other diverse substrates is of great interest in order to develop
l-calpain specific inhibitors in consideration of several reasons as
follows. First, the accumulation of p25 itself was observed in the
brain of early stage AD patients but not in the terminal stage AD
patients. Second, p25/CDK5 causes cytoskeletal disruption and
apoptosis as well as tau hyperphosphorylation in neurons (Nikolic
et al., 1996; Patrick et al., 1999). Third, there are neither introns in
the open reading frame of p35, a neuronal-specific CDK5 regulator,
nor internal methionine near the beginning of the p25 sequence,
suggesting that p25 is only generated by proteolytic cleavage of
calpain (Lee et al., 2000; Chae et al., 1997).
In order to develop l-calpain inhibitors with selectivity against
other enzymes belonging to the cysteine protease family and
against other endogenous substrates of l-calpain, we previously
developed an efficient high throughput screening system using
fluorescence probes (Kang et al., 2009). In this study, we were pro-
vided with a profiled small molecular chemical library containing
6480 compounds from the Korean Chemical Bank to discover novel
calpain inhibitors as potent drug-like compounds for AD. Several
potent compounds were selected from the chemical library.
Among the 6480 compounds, undecylenic acid (UDA) extracted
from Ricinus communis L. was chosen for further study due to its
low toxicity and potent calpain inhibitory activity.
UDA, an 11-carbon monounsaturated fatty acid (C11H2O2),
found naturally in the body (occurring in sweat), is produced com-
mercially by vacuum distillation of castor oil, via the pyrolysis of
ricinoleic acid. It is an economical antifungal agent and the active
ingredient in many topical over-the-counter antifungal prepara-
tions (Shapiro and Rothaman, 1945). We, in the current study,
found UDA to have a neuroprotective effect against neurotoxin-
induced apoptosis in SH-SY5Y, a human neuroblastoma cell, for
the first time. The mechanism underlying the neuroprotective
effect of UDA was studied and it functioned as a l-calpain inhibi-
tor, leading to reduction of p25 formation, CDK5 activation, and tau
phosphorylation. UDA may represent a potential adjuvant strategy
for AD treatment.
2. Materials and methods
2.1. Cell cultures
SH-SY5Y, a human neuroblastoma cell line (ATCC CRL-2266),
and HEK293T, a human kidney epithelial cell line (ATCC CRL-
11268), were purchased from the Korean Cell Line Bank (Seoul,
Korea). SH-SY5Y and HEK293T cells were grown in Minimum
Essential Medium (MEM, Welgene, Korea) and Dulbecco’s modified
Eagle’s medium (DMEM, Welgene, Korea), respectively, in a
humidified incubator maintained at 37 °C under 5% CO2. Each med-
ium contained 10% fetal bovine serum (FBS, Hyclone laboratories
Inc. USA) and 1% penicillin/streptomycin (Sigma Chemical Co.,
USA).
2.2. Fluorometric assay for l-calpain, cathepsin B and cathepsin L
A fluorometric assay was performed in 96-well plates and fluo-
rescence intensity in each well was measured using a Microplate
Fluorescence Reader (SpectraMAX GEMINI EM, Molecular De-
vices); the IC50 values were obtained using the data graphing soft-
ware, TableCurve 2D (Systat software Inc.). Fluorescence intensity
was expressed as relative fluorescence units (RFU). RFU was calcu-
lated by subtracting the RFU of the no-enzyme control from all
other values. To determine percent inhibition, the percent change
in RFU between the activity of the enzyme in the presence and ab-
sence of compounds was calculated. The RFU value calculated in
the absence of compound represents 100% enzyme activity.
The fluorometric l-calpain assay was performed as described
previously (Kang et al., 1009). l-Calpain (human erythrocyte) was
obtained from Calbiochem (Darmstadt, Germany). Pep1 and pep2
were substrates of l-calpain and synthesized by the Peptron Corp.
(Daejeon, Korea). Pep1 was derived from p35 cleavage site ((2-
Abz)-Ser-Thr-Phe-Ala-Gln-Pro-(3-nitrotyrosine)-NH2) and pep2
originated from the calpain cleavage site in a-spectrin ((2-Abz)-
Glu-Val-Tyr-Gly-Met-Met-(3-nitrotyrosine)-NH2). MDL28170 and
E64d (Sigma, USA) were used as positive controls for l-calpain inhi-
bition. The assay was performed in a final volume of 100 lL. Stock
solution of pep1, pep2 and compounds were prepared in dimethyl-
sulfoxide (DMSO) and stored at 20 °C until needed. l-Calpain inhi-
bition was assayed in reaction buffer (50 mM Tris–HCl, 50 mM NaCl,
1 mM EDTA, 1 mM EGTA and 5 mM b-mercaptoethanol, pH 7.5)
with 100 lM pep1 or pep2, 2.5 mM CaCl2 and 5.25 U/mL l-calpain.
The reaction was initiated by adding in the following order: sub-
strate, l-calpain, test compound and CaCl2 solution. Thereafter,
the mixture was incubated with shaking at room temperature for
30 min. Fluorescence intensities were determined by using wave-
lengths of 320 nm for excitation and 420 nm for emission.
Cathepsin B and L inhibitory activities were assayed in the reac-
tion buffer (50 mM NaOAc–HCl, 2 mM dithiothreitor (DTT), 2 mM
EDTA, pH 5.5 for cathepsin B and 0.1 M NaOAc–HCl, 1 mM EDTA,
0.1% b-mercaptoethanol, pH 5.5 for cathepsin L) with 20 lM sub-
strate and 1.5 nM cathepsin B or 4 nM cathepsin L. Cathepsin B
and L were obtained from Calbiochem (Darmstadt, Germany).
The substrates used were RR-AMC (Sigma, USA) for cathepsin B
and Z-FR-AMC (Sigma, USA) for cathepsin L. The cathepsins were
reductively activated by preincubation in assay buffer at 37 °C for
30 min prior to initiating the reaction with substrate and test com-
pounds. Afterwards, the reaction mixture was incubated at room
temperature for 30 min. Fluorescence intensities were determined
by using wavelengths of 360 nm for excitation and 450 nm for
emission. CA-074, Z-FF-FMK and nimodipine were used as a
cathepsin B inhibitor, cathepsin L inhibitor and calcium channel
blocker, respectively, and all were purchased from Sigma Chemical
Co. (USA).
2.3. Cell viability test
SH-SY5Y cells were seeded in 96-well microplates (105 cells per
each well in 100 lL medium) for 24 h. Then, cells were rinsed twice
with phosphate-buffered saline (PBS). After washing, the cells were
incubated in FBS free media with 10 mM glutamate combined with
10 mM glycine, 2 mM H2O2 and 25 lM Ab1–42 (Bachem, Switzer-
land), or neurotoxin with compound. Cells were treated with glu-
tamate and H2O2 for 24 h. Ab1–42 was treated for 72 h. Prior to
Ab1–42 treatment, Ab1–42 needed oligomerization since it has been
reported that the Ab1–42 oligomer was more toxic than Ab1–42 fibril
(Dahlgren et al., 2002). The oligomerised Ab1–42 was obtained as
previously reported with minor modification (Dahlgren et al.,
2002). Lyophilized, HPLC-purified Ab1–42 was reconstituted in
hexafluoroisopropanol (HFIP, Sigma, USA) at 20–23 °C using amber
microcentrifuge tubes for 30 min and then evaporated in a speed
vacuum to remove residual HFIP. The dried Ab1–42 was dissolved
in DMSO with sonication at room temperature in a water bath
and then was aliquoted. The Ab1–42 aliquot was kept at 20 °C be-
fore use. Each aliquot was diluted with cold PBS, vortexed for 30 s
and then incubated for 16 h at 4 °C for oligomerization before
treatment of cells. During the experiments, pre-oligomerized Ab
aliquots were added to a final concentration of 25 lM directly to
the cell media. Finally, after treating with neurotoxin only or
cotreating with neurotoxin and compound, 5 lL of cell counting
kit-8 solution (Dojindo, Japan) was added to each well. The micro-
plate was incubated at 37 °C for an additional 1 h and the
 E. Lee et al. / European Journal of Pharmaceutical Sciences 46 (2012) 17–25
absorbance of each well was assessed at 450 nm using an ELISA
Microplate Reader (VersaMax, Molecular Devices).
2.4. Evaluation of the effect of compound on amyloidal plaque
formation
SH-SY5Y cells were exposed to 25 lM of pre-oligomerized Ab1–
42 in the absence or presence of each compound which included
CA074, Z-FF-FMK, pepstatin A, nimodipine, MDL28170 and UDA
(all 20 lM) for 72 h at 37 °C in a humidified incubator with 5%
CO2. The amyloidal plaque was observed and imaged with a fluo-
rescence inverted microscope with magnification of 20 for objec-
tive lens and 10 for ocular lens (Axiovert 200, Germany).
2.5. Measurement of intracellular reactive oxygen species formation
Free radical production was measured by incubating the cells
with the fluorescent probe 20 ,70 -dichlorodihydrofluorescein diace-
tate (DCFH2-DA, Molecular Probe, USA). DCFH2-DA, which is
oxidized to fluorescent 20 ,70 -dichlorofluorescin (DCF) by hydroper-
oxides, was used to measure the relative levels of cellular perox-
ides (Fu et al., 1998). SH-SY5Y cells were incubated in a 24-well
culture dish (4  105 cells per well in 400 lL of medium) for
24 h. Subsequently, cells were rinsed with PBS. After washing, cells
were treated with various compounds in FBS free medium and
then incubated at 37 °C for 24 h. After incubation, cells were trea-
ted with 400 lM H2O2 for different durations (5, 10, 15 and
20 min). Each well was rinsed with standard medium (138 mM
NaCl, 2.7 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 10 mM PBS and
10 mM glucose, pH 7.4). Next, cells were treated with 5 lM of
DCFH2-DA for 30 min at 37 °C in the standard medium. Subse-
quently, cells were washed once with standard medium and the
fluorescence was measured using excitation and emission wave-
lengths of 485 nm and 535 nm, respectively in a microplate fluo-
rescence reader (SpectraMAX GEMINI EM, Molecular Devices).
2.6. Thioflavin T fluorescence assay
The thioflavin T (ThT) fluorescence assay was performed with
synthetic Ab1–42 (Bachem, Switzerland) as described previously
(Kim et al., 2006). The Ab1–42 solution at a final concentration of
25 lM was incubated at 37 °C for 48 h in PBS (50 mM NaH2PO4,
100 mM NaCl, 0.02% NaN3) to get aggregation in the presence or
absence of test compounds. After incubation, 15 lM ThT was
added to each well containing the aggregated Ab fibril, followed
by additional incubation for 15 min at 37 °C. ThT fluorescence
was measured at 450 nm emission/490 nm excitation wavelengths
in a microplate fluorescence reader (SpectraMAX GEMINI EM,
Molecular Devices). Readings were normalized to the DMSO-only
negative control and expressed as a percentage of relative fluores-
cence to control. Tannic acid, shown previously to inhibit Ab aggre-
gation, was used as a positive control (Ono et al., 2004a,b).
2.7. Atomic force microscopy (AFM) measurements
The Ab1–42 solution in a final concentration of 12.5 lM was
freshly prepared in PBS and aliquoted. The Ab1–42 solution exists
in monomer when freshly prepared. Oligomerized Ab1–42 was pre-
pared according to the method described in the cell viability assay.
The fibril form of Ab1–42 was arranged by incubation at 37 °C for
48 h. During the oligomerization process, each compound was
co-incubated to a final concentration of 20 lM to check the ability
of compounds to block Ab1–42 oligomerization. Twenty microliters
of each aliquot of monomer, oligomer, fibril Ab1–42 sample solution
19
and Ab1–42 oligomerized with co-incubation of each compound
was placed on the mica and dried. Then the sample was scanned
by AFM (XE-70, Park Systems, Korea), and analyzed by XEI (Park
Systems, Korea). The reading scale of AFM micrograph was a
4 lm  4 lm square, composed of 256  256 pixels. Each micro-
graph was measured with 256 maximum heights from each line
per pixel and average maximum height was obtained. The inhibi-
tion percentage of oligomerization was calculated from average
maximum heights of compound cotreated versus oligomerized
Ab1–42 without compound.
2.8. Western blotting analysis
The cells were lysed in lysis buffer solution containing 50 mM
Tris–HCl, 300 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM
MgCl2, 1 mM CaCl2, 1 mM PMSF and 1% protease inhibitor cocktail.
Protein concentrations of soluble extracts in the lysate were deter-
mined by BCA™ Protein Assay Kit (Pierce, USA). A total of 40 lg of
protein was loaded per well. Samples were run on a 12% polyacryl-
amide gel and transferred to a polyvinylidene fluoride membrane
(Millipore Corporation, USA). Membranes were blocked for 1 h at
room temperature in TBST with 5% skim milk. Membranes were
incubated with each antibody of a-tubulin (1:1000 dilution, load-
ing control), caspase-3 (1:1000), p35/25 (1:1000), tau and p-tau
(1:1000) in TBST with 5% skim milk overnight at 4 °C. Secondary
antibodies used were anti-mouse IgG horseradish peroxidase for
tau and p-tau, and anti-rabbit IgG horseradish peroxidase for a-
tubulin and p35/25 antibodies. All primary and secondary antibod-
ies used were purchased from Cell Signaling Technology Inc (USA).
After incubation with each antibody, membranes were washed
three times with TBST and then were detected with the ECL plus
Western blotting detection reagent (Ab frontier, Korea). Western
blot images were analyzed using Multi-Gauge Software (Fuji photo
film Co., Ltd., Japan).
2.9. l-Calpain assay in cells
In order to measure l-calpain inhibitory activity of compounds
in live SH-SY5Y and HEK293T cells, the human CAPN1 gene which
encodes the l-calpain catalytic subunit responsible for proteolytic
activity was synthesized (Bioneer, Korea) (Camins et al., 2006).
The plasmid of CAPN1-encoding pcDNA3.1/His A was subcloned
using the restriction sites EcoR V and Xho I and transfected into
SH-SY5Y and HEK293T cells, then incubated for various times using
WelFect QTM Plus (Welgene, Korea). The proper transfection time
was set up by monitoring the extent of protein expression with
Western blotting analysis using l-calpain antibody (Cell Signaling
Tech., USA). After transfection, cells were incubated with each com-
pound for 210 min in calpain reaction buffer additionally containing
0.1% Triton X-100. Cleavage product of pep1 by l-calpain was mea-
sured with a kinetic mode on the microplate fluorescence reader
(SpectraMAX GEMINI EM, Molecular Devices) at 37 °C. Fluorescence
was measured at 320 nm excitation/420 nm emission wavelengths.
2.10. Statistical analysis
In all experiments, data are expressed as mean ± standard devi-
ation, with at least three replicates in each experimental group.
Comparison of differences was conducted by using an unpaired,
two-tailed Student t-test. The difference was considered statisti-
cally significant when the p value was 60.05.
20 E. Lee et al. / European Journal of Pharmaceutical Sciences 46 (2012) 17–25
3. Results
3.1. Discovery of a novel l-calpain inhibitor by screening a small
molecular chemical library
A small molecular chemical library provided by Korean Chemi-
cal Bank was screened to discover novel calpain inhibitors using
the fluorometric l-calpain assay method established in our labora-
tory. The library was composed of 6480 compounds which have di-
verse chemical cores. Several potent compounds were selected
from the chemical library (Kang et al., 2009). UDA showed a mild
l-calpain inhibitory activity compared to MDL28170 but was more
potent than E64d in cell-free assay (Table 1). MDL28170 has been
known as one of l-calpain inhibitors categorized into peptidyl
aldehyde derivatives while E64d belongs to the peptidyl epoxide
derivative family (Shirasaki et al., 2006; Wang and Yuen, 1997).
Most of the known l-calpain inhibitors including peptidyl alde-
hyde, peptidyl epoxide, diketopiperazine and a-ketoamide deriva-
tives possess peptide characteristics and highly reactive aldehyde
or epoxide moieties which cause inadequate selectivity for
l-calpain to other cysteine proteases as well as poor metabolic
stability or/and cell permeability. MDL28170 inhibited cathepsin
B and cathepsin L to almost the same extent as l-calpain inhibition
and showed more cathepsin L inhibitory activity over l-calpain
(Table 1). UDA has a totally different structure compared to known
l-calpain inhibitors and originates from R. communis L. Among
several potent compounds selected from the chemical library
screening, UDA was of interest and selected for further study.
3.2. Neuroprotective effect of UDA against neurotoxin-induced SH-
SY5Y cell death
Glutamate, H2O2, and Ab1–42 are all well known neurotoxins
which have been previously shown to induce apoptosis in neuronal
cell types including SH-SY5Y (Smith et al., 2006; Choi and Roth-
man, 1990; Hoyt et al., 1997). Cell viability of SH-SY5Y was mark-
edly decreased after exposure to these neurotoxins, including
glutamate and H2O2 (Fig. 1A). Oligomerized Ab1–42 caused apopto-
sis in SH-SY5Y cells to a lesser extent compared to treatment of
glutamate and H2O2, respectively (Fig. 1B and C). Therefore, the
morphological changes of SH-SY5Y cells were monitored after
treatment with oligomerized Ab1–42 in the absence or presence of
compounds (Fig. 2A). Some synthetic compounds showed good
l-calpain inhibitory activity from the library, however unlike
UDA, they were not studied further due to their high toxicity, (data
not shown). The 72 h treatment of oligomerized Ab1–42 decreased
cell viability to 84.5 ± 2.8% versus controls treated with 0.1%
Table 1
The inhibitory activity of compounds against l-calpain, cathepsin B, and cathepsin L.
Compound Inhibitory activity as IC50 (lM)a of
l-Calpain
b c
On pep1 On pep2
MDL28170 0.0990 ± 0.0002 0.0552 ± 0.0001
CA074 >100 >100
Z-FF-FMK 0.3971 ± 0.0004 1.1100 ± 0.010
E64d 62.26 ± 7.73 77.60 ± 1.43
UDA 5.37 ± 0.71 >100
a
Each data point represents mean ± SD from three different experiments performed in triplicate.
b
Calpain inhibitory activity of chemicals on pep1 substrate was evaluated in reaction buffer (50 mM Tris–HCl, 50 mM NaCl, 1 mM EDTA, 5 mM b-mercaptoethanol, pH 7.5)
with 100 lM pep1, 2.5 mM CaCl2, and 5.25 U/ml l-calpain. Thereafter, reaction mixture was incubated with shaking at RT for 30 min. Fluorescence intensities were measured
using 360 nm excitation and 420 nm emission wavelengths. The substrate of pep1 possess the l-calpain-cleavage site in p35 which is [2-Abz]-Ser-Thr-Phe-Ala-Gln-Pro-[3-
nitrotyrosine]-NH2.
c
Calpain inhibitory activity of chemicals on pep2 substrate was assayed as following the method used for that on pep1. The pep2 substrate ([2-Abz]-Glu-Val-Tyr-Gly-Met-
Met-[3-nitrotyrosine]-NH2) was originated from a-spectrin.
DMSO. The decreased cell viability mediated by oligomerized
Ab1–42 was attenuated by co-treatment with CA074, Z-FF-FMK,
pepstatin A, MDL28170 and UDA (Fig. 1A). Among the compounds,
Z-FF-FMK, which is known as a specific inhibitor of cathepsin L,
inhibited both cathepsin B and L to a similar extent (Table 1) and
recovered the cell viability up to 98.7 ± 0.4%. UDA co-treatment
also enhanced viability of Ab1–42-treated cells to 91.3 ± 0.7% with
a statistical significance, even though its effect was much less than
that of Z-FF-FMK (Fig. 1A). The cell viability was decreased to
45.6 ± 9.5% in glutamate-treated cells and was recovered by co-
treatment with compounds to a very small extent. The viability
of glutamate-treated cells was recovered up to 63.6 ± 9.7% by
UDA (Fig. 1B). The treatment of hydrogen peroxide decreased cell
viability to 56.4 ± 13.5% compared to control. Only UDA co-treat-
ment, compared to other compounds, recovered the viability of
H2O2-treated cells significantly and up to 80.0 ± 5.4% (Fig. 1C). In
summary, UDA showed neuroprotective effect against glutamate,
H2O2 and Ab1–42 induced cell death. Among these, the neuroprotec-
tive effect of UDA against H2O2 was most significant.
3.3. Effect of UDA against Ab-induced cell aggregation, Ab
oligomerization and fibrillation in vitro
Exposure to Ab1–42 led to significant Ab aggregates in SH-SY5Y
cells called amyloidal plaques. The amyloidal plaques are one of
the major pathological events in brains of AD patients (Conway
et al., 2003; Selkoe, 1991). Changes in Ab-induced cell aggregation
in SH-SY5Y cells treated with 25 lM Ab1–42 in the absence and
presence of test compounds (20 lM) were monitored and imaged
with a fluorescence inverted microscope (Fig. 2A). Cells treated
with only aggregated Ab1–42 showed significant morphological
changes. But there were little or no aggregated forms in cells co-
treated with Z-FF-FMK, MDL28170 and UDA, while cells co-treated
with CA074, pepstatin A and nimodipine showed severe aggrega-
tion. We further tested the effect of UDA on aggregation of
Ab1–42 treatment. This test was performed with formation of amy-
loid fibrils, which can be readily assayed by binding to ThT (Reinke
et al., 2009). Synthetic Ab1–42 (25 lM) was incubated in the
absence or presence of either MDL28170, UDA and tannic acid at
final concentrations of 20 or 50 lM at 37 °C for 48 h. UDA inhibited
Ab fibril formation by 46.1 ± 5.3%, although UDA was less potent
than tannic acid which was used as positive control (Ono et al.,
2004a,b) (Fig. 2B). Ab1–42 has been reported to aggregate to form
oligomers and fibrils en route to deposition of amyloid plagues
associated with AD. Since oligomers are more toxic to mouse neu-
rons than fibrils, we evaluated whether UDA could inhibit Ab olig-
omerization using AFM micrographs which capture the differences
Cathepsin B Cathepsin L
0.0958 ± 0.0045 0.0031 ± 0.0003
0.0037 ± 0.0003 >100
0.0656 ± 0.0029 0.0837 ± 0.0008
14.39 ± 0.18 2.41 ± 0.09
>100 2.85 ± 0.26
 E. Lee et al. / European Journal of Pharmaceutical Sciences 46 (2012) 17–25 21

Fig. 1. UDA prevented neurotoxin-induced viability loss and apoptosis of SH-SY5Y cells. SH-SY5Y cells were incubated with each neurotoxin including 25 lM pre-
oligomerized Ab1–42 for 72 h (A), 10 mM glutamate with 10 mM glycine for 24 h (B) and 2 mM H2O2 for 24 h (C) in the absence and presence of 20 lM of each compound at
37 °C. Bars represent the mean ± SD from three different experiments performed in triplicate. ⁄p < 0.05, ⁄⁄p < 0.01, significantly different from each neurotoxin-only-treated
group.

between monomers, oligomers and fibrils of Ab1–42 (Ahmed et al.,
2010). AFM micrographs of the fibril form of Ab1–42 were distinctly
different from those of monomers and oligomers as shown in the
upper panel of Fig. 2C. When either UDA or MDL28170 was co-
incubated during the Ab1–42 oligomerization process, as shown in
bottom panel of Fig. 2C, both compounds inhibited the formation
of Ab1–42 oligomers. MDL28170 and UDA each inhibited Ab1–42 oli-
gomer formation by 75.31 ± 4.28% and 64.65 ± 3.07%, respectively,
which were calculated by using average maximum height obtained
from XEI program (Fig. 2D). These results suggested that UDA
might be able to inhibit Ab oligomerization as well as fibrillation.
3.4. Scavenging effect of UDA on oxidative stress-induced free radical
formation
UDA attenuated H2O2-induced apoptosis effectively in SH-SY5Y
cells (Fig. 1C). Therefore, the ability of UDA to directly scavenge
H2O2-induced reactive oxygen species (ROS) formation was evalu-
ated to determine a potential mechanism underlying the neuro-
protective effect of UDA. ROS formation was investigated within
SH-SY5Y cells by monitoring a conversion of DCFH2-DA to DCF.
We first observed H2O2-producing free radicals in a rapid and
dose-dependent manner in cells (data not shown). Pre-incubation
with 20 lM MDL28170 or UDA for 24 h prior to treatment with
400 lM H2O2 prevented the increase in fluorescence generated
by H2O2 (Fig. 3). UDA reduced the levels of free radicals generated
by H2O2 in cells with the maximum effect within 10 min after
treatment and with a better potency than MDL28170.
3.5. Effect of UDA on tau hyperphosphorylation and apoptosis induced
by GOx in SH-SY5Y cells
We observed UDA attenuated H2O2-induced apoptosis through
scavenging of free radicals in SH-SY5Y cells. These results necessi-
tated the evaluation of UDA effects on expression of proteins re-
lated to l-calpain activity. When l-calpain is over-activated in
cells, it results in p25 generation and tau hyperphosphorylation,
ultimately leading to neuronal cell death. SH-SY5Y cells were trea-
ted with glucose oxidase (GOx) which catalyses the oxidation of
glucose to D-glucono-d-lactone with the production of H2O2. GOx
induced apoptosis by continual release of H2O2 (Markesbery,
1997). GOx at 30 mU/mL elevated l-calpain hyperactivation and
apoptosis related proteins such as p25, hyperphosphorylated tau,
activated caspase and cleaved poly(ADP-ribose)polymerase
22 E. Lee et al. / European Journal of Pharmaceutical Sciences 46 (2012) 17–25

(A) (B)

(C) (D)

Fig. 2. UDA reduced the formation of amyloidal plaques in SH-SY5Y cells induced by 25 lM pre-oligomerized Ab1–42 (A) through preventing Ab fibrillation (B) and
oligomerization in vitro (C and D). (A) SH-SY5Y cells were incubated for 72 h with 25 lM pre-oligomerized Ab1–42 in the absence or presence of 20 lM of test compounds. The
amyloidal plaque formed was observed and imaged with a fluorescence inverted microscope at 200 magnification (Axiovert 200, Germany). (B) Ab1–42 aggregation was
induced in the absence or presence of compounds through incubation at 37 °C for 48 h. Then, ThT was added at a final concentration of 10 lM followed by incubation for
15 min at 37 °C. The fluorescence measured at 450 nm (ex) and 490 nm (em) was directly proportional to the amount of Ab1–42 fibril formed. Bars represent means ± SD from
six independent experiments. ⁄p < 0.05, ⁄⁄p < 0.01 vs control group treated with only Ab1–42. (C) AFM micrographs showed the difference between Ab1–42 monomers,
oligomers and fibrils. Ab1–42 was incubated at 20 °C (monomers), at 4 °C for 16 h (oligomers), and at 37 °C for 48 h (fibrils) in PBS. UDA or MDL28170 was co-incubated
during the oligomerization process. Twenty microliters of each aliquot of monomer, oligomer, fibril Ab1–42 sample solution and Ab1–42 oligomerized with co-incubation of
each compound was placed on the mica and dried. Then each sample was scanned by AFM. (D) The inhibition % of oligomerization was calculated as indicated in Methods.
Bars represent means ± SD from three independent experiments. ⁄⁄⁄p < 0.001 vs Ab1–42 oligomer without compounds treated.

(PARP). Caspase-3 is one of the main executioner caspases in the
apoptotic process. Caspase-3 exists within the cell as an inactive
proform called procaspase-3 which undergoes proteolytic cleavage
to be an active protein. The caspase-3 activation can be also veri-
fied by the proteolytic cleavage of PARP, an endogenous caspase-
3 substrate (Tenn and Wang, 2007). The treatment of 30 mU/mL
GOx resulted in an increase in levels of p25, phospho-tau, cleaved
PARP, and cleaved caspase-3 compared to control values (second
lane in Fig. 4), but UDA treatment (20 lM) remarkably decreased
these levels as shown in Fig. 4. On the other hand, the treatment
of 20 lM MDL28170 did not decrease these levels as much as UDA.
3.6. l-Calpain inhibitory activity of UDA in cells
UDA inhibited l-calpain activity with much less potency than
MDL28170 in a cell free in vitro experiment (Table 1). However,
UDA reduced l-calpain-mediated p35 cleavage to p25,
hyperphosphorylation of tau, caspase activation and PARP activa-
tion with better or equivalent potency in SH-SY5Y cells than
MDL28170. These results led us to develop an assay method to
evaluate l-calpain inhibitory activity of test compound in cells
which might predict directly l-calpain-mediated biological activ-
ity. We synthesized the CAPN1 gene encoding the large subunit
of l-calpain, which is responsible for catalytic proteolysis. Then,
it was transfected into HEK293T and SH-SY5Y cells. A transfection
time of 8 h showed the maximum degree of l-calpain protein
expression (Fig. 5A) in both cell types. Cells were treated with
the substrate, 10 lM pep1, together with 1 lM ionomycin in the
absence or presence of MDL28170 and UDA after the 8 h transfec-
tion. Ionomycin, a calcium ionophore, enhances calcium influx into
the cell (Morgan and Jacob, 1994), which then activates l-calpain.
The cells treated with pep1 and co-treated with 1 lM ionomycin
showed higher fluorescence intensity compared to pep1 treated
cells. This was due to increases in pep1 cleavage induced by
 E. Lee et al. / European Journal of Pharmaceutical Sciences 46 (2012) 17–25
Fig. 3. UDA prevented oxidative stress-induced free radical formation in SH-SY5Y
cells. SH-SY5Y cells were loaded with 5 lM DCFH2-DA for 30 min at 37 °C and then
exposed to 400 lM of H2O2 for 30 min. Before H2O2 treatment, cells were pre-
incubated for 24 h with 20 lM MDL28170 and UDA. Values are the means ± SD of
three independent experiments.
Fig. 4. UDA has a neuroprotective effect against a continuous oxidative stress. UDA
down regulated the conversion of p35 to p25 leading to a decrease in tau
phosphorylation. The elevated levels of both PARP and caspase-3 by GOx treatment
in SH-SY5Y cells showed decreases in the active forms when UDA was treated. SH-
SY5Y cells were treated with GOx (30 mU/mL) in the absence or presence of 20 lM
MDL28170 and UDA for 4 h at 37 °C in a humidified atmosphere with 5% CO2. The
cells were lysed and the soluble protein was analyzed by Western blot.
l-calpain activation when calcium influx occurred. The saturation
on fluorescence intensity was observed after about 80 min when
signals were measured in kinetic mode (Fig. 5B). The effect of
MDL28170 and UDA on l-calpain activity in HEK293T (Fig. 5C)
and SH-SY5Y cells (Fig. 5D) is shown using the value at 80 min.
UDA inhibited l-calpain with better potency than MDL28170 in
HEK293T cells and with equivalent potency in SH-SY5Y cells.
4. Discussion
We have screened a chemical library made up of 6480 com-
pounds which consisted of both synthetic and natural products
possessing diverse pharmacophores. Using the screening method
developed previously in our laboratory (Kang et al., 2009), UDA
was found to be a substrate-specific l-calpain inhibitor. The struc-
23
ture of UDA is much different from known calpain inhibitors and
the function of UDA regarding neuroprotective effect has not yet
been reported so far (Kang et al., 2009; Shirasaki et al., 2006; Wang
and Yuen, 1997; Cuerrier et al., 2007). Pep1 substrate is derived
from p35, a CDK5 activity regulator and pep2 originates from spec-
trin. The IC50 of UDA on l-calpain against pep1 was 5.37 ± 0.71 lM,
while that against pep2 was more than 100 lM. Further, other
known l-calpain inhibitors, MDL28170 and E64d, showed no
substrate specificity (Table 1). Although UDA has much less
activity than MDL28170 (0.0990 ± 0.0002 lM for pep1 and
0.0552 ± 0.0001 lM for pep2), we suggest UDA could be a novel
potent l-calpain inhibitor with influential activity in vivo due to
following reasons. Although E64d in vitro activity against calpains
and cathepsins is poorer than other known inhibitors including
MDL28170, E64 and E64c (Lampi et al., 1992; Azuma et al., 1992;
Tsubokawa et al., 2006), E64d has been extensively utilized in
in vivo experiments because of its enhanced cell permeability,
leading to good in vivo efficacy. In our in vitro experiments, UDA
showed much better l-calpain inhibitory activity than E64d
(62.26 ± 7.73 lM for pep1 and 77.60 ± 1.43 lM for pep2). Addi-
tionally, UDA is much less toxic; the LD50 of UDA after oral treat-
ment for mice is 8150 mg/kg (Newell et al., 1949). UDA also
showed competent neuroprotective effects against glutamate-,
H2O2- and Ab1–42-induced cell death in SH-SY5Y cells (Fig. 1).
Therefore, UDA was further evaluated in this study for its potential
use as an AD therapeutic. The key neuropathological features of AD
are the abnormal deposition of Ab1–42-mediated plaques and re-
lated insoluble peptides in extracellular spaces in brain. The neuro-
toxicity exerted by aggregated Ab may be mediated by several
mechanisms, such as the generation of ROS, escalation of intracel-
lular Ca2+ concentrations and induction of apoptosis (Selkoe, 1991;
Camins et al., 2006; Esteban, 2004). Another distinct biomarker of
AD is intracellular neurofibril tangles which are induced by abnor-
mal tau hyperphosphorylation. Alteration in calcium homeostasis
generated by insoluble Ab leads to persistent hyperactivation of
calpains. Hyperactivated l-calpain cleaves a number of neuronal
substrates including p35. The cleavage of p35 to p25 produces a
more stable CDK5-p25 complex. This leads to prolonged CDK5 acti-
vation and mislocation of CDK5, which results in tau hyperphosph-
orylation, neurofibril tangles and ultimately neuronal cell death
(Saito et al., 1993; Patrick et al., 1999). UDA attenuated Ab-medi-
ated cell death in SH-SY5Y cells with equipotency compared to
MDL28170 (Fig. 1A and Fig. 2A). Barry et al. demonstrated that
Z-FF-FMK, known as a cathepsin L inhibitor, could block
Ab-mediated induction of the apoptotic cascade (Bloland and
Campbell, 2004). The inhibitory activity of UDA against cathepsin
L (IC50 value of 2.85 ± 0.26 lM) was much weaker than that of
Z-FF-FMK (0.0837 ± 0.0008 lM) in vitro, while UDA protected
Ab-mediated cell death with almost equipotency to Z-FF-FMK. In
addition, UDA could inhibit Ab fibril formation by 46.1 ± 5.3%
which was determined in the ThT assay (Fig. 2B). Since the ThT as-
say cannot differentiate Ab oligomers from Ab fibrils, we estimated
the effect of UDA on the formation of oligomers using AFM analy-
sis. AFM micrographs of UDA treatment during Ab oligomerization
(oligomer vs UDA micrographs in Fig. 2C) showed no fibril shape
and the size of oligomers was lessened. It can be suggested that
UDA blocked the oligomerization process. It was recently reported
that the Ab oligomer was more toxic than Ab fibrils and Ab fibril
formation was reported as a self-detoxification mechanism (Sakon-
o and Zako, 2010; Walsh et al., 2000; Wang et al., 2008). In this
sense, it is worthy to note that UDA is able to inhibit Ab oligomer-
ization. It has been known that insoluble Ab1–42 generates ROS
and induces cell membrane damage, which leads to calcium
influx (Selkoe, 1991; Camins et al., 2006; Walsh et al., 2000).
Therefore, the ability of UDA to alleviate Ab oligomerization and
fibrillation might affect intracellular Ca2+ levels. Among the tested
24 E. Lee et al. / European Journal of Pharmaceutical Sciences 46 (2012) 17–25

(A) (B)

(C) (D)

Fig. 5. UDA reduced l-calpain activity in cells with better potency than MDL28170. (A) HEK293T and SH-SY5Y cells were transfected with 3 lg of CAPN1 encoded with
pcDNA3.1/His A for diverse time periods and then the extent of l-calpain expression was determined using Western blot. (B) Cells transfected with pcDNA 3.1/His A_CAPN1
for 8 h were harvested and resuspended in calpain reaction buffer containing 0.1% triton X-100. The cells were incubated with pep1 and ionomycin in the absence or presence
of either MDL28170 or UDA. The relative change in fluorescence induced by l-calpain-mediated pep1 cleavage was measured in kinetic mode using a microplate fluorescence
reader (SpectraMAX GEMINI EM, Molecular Devices) at 37 °C. Fluorescence was measured at 320 nm (ex) and 420 nm (em). (C),(D) Fluorescence obtained at an endpoint of
80 min from (B) were depicted in bar graphs in HEK293T and SH-SY5Y cells, respectively. Values are the means ± SD of three independent experiments performed in
triplicate. ns, not significant; ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001 vs ionomycin-treated group only.

compounds, UDA also showed the most potent neuroprotective ef-
fect against H2O2-mediated toxicity in SH-SY5Y cells (Fig. 1C).
These results can be explained by a ROS scavenging effect of
UDA. UDA inhibited H2O2-generated ROS by measuring the
DCFH2-DA conversion to DCF with a better potency than
MDL28170 (Fig. 3). From the Western blots of SH-SY5Y cells, it
was confirmed that l-calpain inhibitory activity of UDA reduced
the cleavage of p35 to p25, decreased tau hyperphosphorylation,
and ultimately inhibited the activation of PARP and caspase-3 in
GOx-treated SH-SY5Y cell (Fig. 4).
Most of the peptide-like l-calpain inhibitors showed no speci-
ficity to l-calpain over other cysteine proteases. Other problems
of these l-calpain inhibitors are poor membrane permeability
and poor metabolic stability (Kang et al., 2009; Shirasaki et al.,
2006; Wang and Yuen, 1997; Cuerrier et al., 2007). l-Calpain
(Calpain 1) exists as a heterodimer consisting of a large 80 kDa cat-
alytic subunit encoded by CAPN1 and a common 29 kDa regulatory
subunit encoded by CAPN4 (Aoki et al., 1986; Ohno et al., 1986).
Upon exposure to Ca2+, l-calpain is activated by subunit autolysis
by separation from the regulatory subunit (Goll et al., 1992). UDA
reduced l-calpain activity with better potency than MDL28170 in
SH-SY5Y and HEK293T cells when the catalytic subunit of l-cal-
pain was overexpressed (Fig. 5). UDA could be a distinctive potent
l-calpain inhibitor possessing good cell permeability, non-toxicity
and structural stability.
5. Conclusion
A multitude of neurodegenerative cellular events induced by l-
calpain hyperactivation leads to caspase-3 protease activity that
controls programmed cell death (Tenn and Wang, 2007; Eliasson
et al., 1997). Thus, UDA is likely able to slow the progression of
AD by inhibiting l-calpain and the many pathways such as insol-
uble Ab oligomerization and fibrillation, Ab-mediated ROS forma-
tion, neurofibril tangles formation and cell death. The treatment
of combination of Docosahexaenoic acid (a type of omega-3 fatty
acid), uridine, and choline to aged gerbils showed significantly bet-
ter performance on learning and memory tests than untreated ger-
bil group (Holguin et al., 2008). Although further in vivo studies are
necessary to demonstrate the potential of UDA as an adjuvant ther-
apeutic for AD, the current study may pave the way to develop
non-peptide-like novel l-calpain inhibitors which can reverse
the levels of insoluble Ab- and hyperphosphorylated tau-induced
apoptotic proteins.
Acknowledgements
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by
the Ministry of Education, Science and Technology (2011-0011007)
 E. Lee et al. / European Journal of Pharmaceutical Sciences 46 (2012) 17–25
and by the Ewha Global Top5 Grant 2011 of Ewha Womans
University. Jun K.Y. was partially supported by Ewha Womans
University.
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