Protective effects of β-sheet breaker α/β-hybrid peptide against amyloid β-induced neuronal apoptosis in vitro

Article abstract of DOI:10.1111/cbdd.12912

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid-β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid-β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β-sheet breaker α/β-hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀-induced neurotoxicity in human neuroblastoma SH-SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀-induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro-apoptotic protein Bax and up-regulating antiapoptotic protein Bcl-2. These results suggest that α/β-hybrid peptide has neuroprotective effects against Aβ₄₀-induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.

Full text of DOI:10.1111/cbdd.12912

Received: 19 February 2016
DOI: 10.1111/cbdd.12912
Revised: 13 September 2016
Accepted: 8 November 2016
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R E S E A R C H A R T I C L E
Protective effects of β-sheet breaker α/β-hybrid peptide against
amyloid β-induced neuronal apoptosis in vitro
Sourav Kumar1
Ashim Paul2
Sourav Kalita2
Anup Kumar Ghosh3
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Bhubaneswar Mandal2
Amal Chandra Mondal1,4
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1Neuroscience Research Unit, Department of
Physiology, Raja Peary Mohan College, Uttarpara,
Hooghly, West Bengal, India
2Laboratory of Peptide and Amyloid
Research, Department of Chemistry, Indian
Institute of Technology Guwahati (IITG), North
Guwahati, Assam, India
3Department of Instrumentation Science, Jadavpur
University, Kolkata, West Bengal, India
4School of Life Sciences, Jawaharlal Nehru
University, New Delhi, India
Alzheimer’s disease is most common neurodegenerative disorder and is character-
ized by increased production of soluble amyloid-β oligomers, the main toxic species
predominantly formed from aggregation of monomeric amyloid-β (Aβ). Increased
production of Aβ invokes a cascade of oxidative damages to neurons and eventually
leads to neuronal death. This study was aimed to investigate the neuroprotective ef-
fects of a β-sheet breaker α/β-hybrid peptide (BSBHp) and the underlying mecha-
nisms against Aβ40-induced neurotoxicity in human neuroblastoma SH-SY5Y cells.
Cells were pretreated with the peptide Aβ40 to induce neurotoxicity. Assays for cell
viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen
species (ROS), intracellular free Ca2+, and key apoptotic protein levels were per-
formed in vitro. Our results showed that pretreatment with BSBHp significantly at-
tenuates Aβ40-induced toxicity by retaining cell viability, suppressing generation of
ROS, Ca2+ levels, and effectively protects neuronal apoptosis by suppressing pro-
apoptotic protein Bax and up-regulating antiapoptotic protein Bcl-2. These results
suggest that α/β-hybrid peptide has neuroprotective effects against Aβ40-induced oxi-
dative stress, which might be a potential therapeutic agent for treating or preventing
neurodegenerative diseases.
Correspondence
Amal Chandra Mondal, School of Life Sciences,
Jawaharlal Nehru University (JNU), New Delhi,
India.
Email: acmondal@mail.jnu.ac.in
and
Bhubaneswar Mandal, Laboratory of Peptide and
Amyloid Research, Department of Chemistry,
Indian Institute of Technology Guwahati (IITG),
North Guwahati, Assam, India.
Email: bmandal@iitg.ernet.in
Funding information
Science and Engineering Research Board, Grant/
Award Number: PDF/2016/000892; RNP-UGC;
SAP DRS I-UGC; DBT NER, Govt of India,
Grant/Award Number: DBT (NER-BPMC)
[BT/347/NE/TBP/2012].
K E Y W O R D S
Alzheimer’s disease, amyloid-β peptide, apoptosis, human cerebrospinal fluid, neuroprotection,
SH-SY5Y cell, β-sheet breaker α/β-hybrid peptide
which impair hippocampal synaptic plasticity and cause cog-
nitive dysfunction.[2] Extensive accumulation of Aβ peptide
in brain parenchyma and cerebral vessel walls, called cerebral
1
INTRODUCTION
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Alzheimer’s disease (AD), a protein-misfolding neurodegen-
erative disease, is characterized by progressive loss of mem-
ory and cognitive ability in elderly population.[1] The central
event in AD is the formation of soluble oligomers of Aβ from
dysregulated proteolytic cleavage of amyloid precursor pro-
tein (APP) by two proteases β- and γ-secretases, respectively,
amyloid angiopathy (CAA), and senile plaques (SPs), plays
a key role in Alzheimer’s disease.[3] Neuropathological hall-
marks in AD brain are aggregation and fibrillization of Aβ
leading to extracellular deposition of neuritic plaques and
hyperphosphorylated microtubule-associated Tau protein,
called neurofibrillary tangles (NFT).[4] In addition, synaptic
dysfunction, alteration of neuronal metabolism, loss of neu-
rotransmission, and ultimately loss of neuronal cell occur.[5,6]
Aβ is a 4.3-kDa peptide composed of 39–42 amino acids.
Aggregation and misfolding of Aβ is the reversible trans-
formation of nonpathogenic soluble native peptide first into
Abbreviations: AD, Alzheimer’s disease; AβO, amyloid-β oligomers; Ant,
anthranilic acid; APP, amyloid precursor protein; BSBp, β-sheet breaker
peptide; BSBHp, β-sheet breaker α/β-hybrid peptide; CAA, cerebral amy-
loid angiopathy; h-CSF, human cerebrospinal fluid; NFT, neurofibrillary
tangles; ROS, reactive oxygen species; SPs, senile plaques.
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Chem Biol Drug Des 2016; 1–13
wileyonlinelibrary.com/journal/cbdd
© 2016 John Wiley & Sons A/S
1

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KUMAR et Al.
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soluble toxic oligomers and further to insoluble amyloi-
dogenic fibrils composed of β-sheet structures.[7] Several
studies reported that soluble oligomers are more toxic to
neurons in cultures than insoluble amyloidogenic peptide
and are responsible for neuronal cell death by synapse loss,
membrane disruption, altered mitochondrial metabolism,
impaired Ca2+ homeostasis, DNA damage, and inflammatory
processes.[8–10]
Aβ-induced neurotoxicity and its underlying mechanism
is complex and remains obscure, but several pieces of evi-
dence imply that enhancement of oxidative stress provoked
by Aβ is associated with AD.[11] It has been reported that
Aβ can fragmentize and thus generate free radicals with
potent lipid peroxidation. In addition, Aβ-induced toxicity in
cell culture supports that oxidative stress induced by ROS
plays a vital role in Aβ-mediated neuronal degeneration. The
excessive production of ROS can cause cellular damage and
subsequent cell death; thus, ROS is a sensitive marker of
oxidative injury. Therefore, pharmacological intake of ROS
modifying β-sheet breakers peptide can be considered as one
of the therapeutic strategies to treat Aβ-induced toxicity in
AD and could protect neurons from Aβ-induced oxidative
stress.
mature neuron-like structure, resembles with morphology
and biochemistry of human neurons. It reveals expression of
several neurospecific markers including synaptic protein sv-
2, nuclear marker NeuN, as well as the presence of synapses.
Reports from other laboratories have shown that these cells
also express mature tau isoforms and tau protein. So, this cel-
lular in vitro model is useful for primary screening of drugs
particularly in AD.
In this study, we have investigated the effects of the
BSBHp on Aβ40-induced neurotoxicity on human neuro-
blastoma SH-SY5Y cells. We evaluated the neuroprotective
effects and the underlying mechanism of BSBHp against Aβ-
mediated neuronal cell death, generation of ROS, intracel-
lular Ca2+ release, through mitochondrial apoptotic pathway
(Bax, Bcl-2, caspase 9, caspase 3, cytoplasmic and mitochon-
drial cytochrome-C) in vitro. We reported here that BSBHp
exerted the neuroprotective effects against Aβ not only by
regulating the expressions of pro-apoptotic proteins, but also
through restoring the intracellular levels of Ca2+ and ROS
generation. We also demonstrated the effect of BSBHp for
disruption of Aβ aggregates present in h-CSF of Alzheimer’s
patients of late stage.
A class of synthetic peptide called β-sheet breaker peptide
(BSBp) can inhibit the formation of β-sheet-rich pathologi-
cal conformers of Aβ peptide and reverse the misfolded con-
formation of pathogenic Aβ peptide. Previously, Soto et al.
introduced a series of BSBps specially iAβ5 that is comprised
of a recognition motif homologous to pathogenic Aβ42 and
also contains a proline-rich β-breaker element.[12] Most of the
previously reported BSBps contain either α-amino acids or
α-amino acid derivatives. They are degradable by proteolysis.
Because β-amino acids are nonproteinogenic, β-amino acid-
containing peptides are better choice for drug design for their
better stability against proteolytic degradation.[13]
2
MATERIALS AND METHODS
Reagents and solvents
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2.1
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Rink amide MBHA resin (loading 0.7 mmol/g), Fmoc amino
acids, coupling reagents (BOP and PyBOP), and human
Alzheimer’s β-amyloid (Aβ40) were purchased from GL
Biochem (Shanghai). DIPEA, thioflavin T, Congo red, and
5(6)-carboxyfluorescein were purchased from Sigma. All the
lipids were purchased from Avanti Polar Lipid, Inc. DMF,
DCM, and acetonitrile of HPLC grade were obtained from
Merck (India). Human cerebrospinal fluid (h-CSF) sam-
ples (late stage) were obtained from Guwahati Neurological
Research Center (GNRC) and Hospital, Guwahati, India,
for performing biophysical studies acceding to the bioethics
policy of the hospital.
We have demonstrated the design and synthesis of a new
class of β-sheet breaker peptide (e.g., Ac–Leu–Ant–Phe–
Phe–Asp–NH2) that contains an anthranilic acid (Ant) as a
β-sheet breaker unit. Ant is a noncoded aromatic β-amino
acid, which is nonproteinogenic, stable against proteolytic
degradation, and also a precursor for the biosynthesis of
tryptophan. Unlike proteinogenic amino acids, Ant and its
homologs are conformationally rigid and strongly favor helix
or turn conformation when formed hybrid peptide with the
α-amino acids.[14–16] Collectively, we named them as β-sheet
breaker α/β-hybrid peptide (BSBHp). Such BSBHps have
been identified as non-amyloidogenic and exhibited inhib-
itory and disruptive efficacy against Aβ aggregation.[17]
Therefore, Ant-containing α/β-hybrid peptides can be a
benign candidate for drug design against AD and various
protein-misfolding diseases.
2.2
Peptide synthesis
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The described peptides were synthesized by standard
Fmoc/tBu-based solid-phase peptide synthesis method on
MBHA-Rink amide resin (loading 0.7 mmol/g). For each
amino acid attachment, 2 equiv of Fmoc amino acids,
2.5 equiv of coupling reagent (BOP), and 5 equiv of base
(DIPEA) were used for incomplete reaction, and coupling
cycles were repeated, followed by capping with acetic
anhydride (2 equiv) and N-methyl imidazole (3 equiv).
Fmoc deprotection was performed with 20% piperidine in
DMF. The final peptide was cleaved from the resin using a
cleavage cocktail (90% TFA, 5% DCM, and 5% H2O) for
Herein, we have used human neuroblastoma SH-SY5Y
cells as in vitro cellular model of AD as it differentiates into

KUMAR et Al.
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5 hr. The crude peptide was precipitated by cold diethyl
ether followed by centrifugation to achieve crude solid
peptide.
of aliquot (20 μM) was added over the dark side of carbon-
coated copper grid and allowed to float for 1 min. Then, 2%
uranyl acetate solution (10 μl) was added onto it and allowed
to float for another 1 min. Then, the excess solution was
removed using a blotting paper. The sample was dried at
room temperature and kept in desiccators before taking TEM
analysis on JEOL (Model: JEM 2100) instrument at 200 kV.
In case of amyloidogenic study for the peptide solution,
the samples were prepared in a similar fashion as described
above.
2.3
Liquid chromatography and mass
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spectrometry
Crude peptides were purified by RP-HPLC (Waters 600E) using
a C18-μ Bondapak column at a flow rate of 5 ml/min. Binary
solvent systems were used, solvent A (0.1% TFA in H2O) and
solvent B (0.1% TFA in CH3CN). A Waters 2,489 UV detec-
tor was used with dual detection at 214 and 254 nm. A total
run time of 20 min was used and gradient used for purification
was 5%–100% CH3CN for 18 min followed by 100% CH3CN
till 20 min. Purity of the peptides was confirmed from Waters
UPLC-MS system (ESI +ve mode), and Micromass Q-TOF
equipped with Masslynx software was used. Mobile phase com-
prised solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA
in CH3CN) on C18 column with a flow rate of 0.25 ml/min.
Dual wavelengths were selected at 214 nm and 254 nm. Linear
gradient of 5%–100% A was used in a total run time of 8 min.
2.6
Peptide preparation and treatment
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Commercially available Aβ40 was dissolved in small vol-
ume (20 μl) of TFA to obtain disaggregated Aβ40. TFA was
evaporated using nitrogen gas. To remove TFA completely,
HFIP was added and evaporated using nitrogen gas. This pro-
cess was repeated twice and the disaggregated Aβ40 was kept
at −20°C before biophysical studies. For cell-based studies,
commercially available Aβ40 peptide was dissolved in ster-
ile deionized water at a concentration of 50 mM, incubated
in a capped vial at 37°C for 2 days to induce the generation
of toxic species.[19] Monomeric Aβ40 peptide was also co-
incubated with different concentration of BSBHp for 2 days
and added to SH-SY5Y cells. All assays were performed
after 24-hr co-incubation with or without Aβ40 and BSBHp.
2.4
Carboxyfluorescein dye-loaded large
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unilamellar vesicles (LUVs) leakage study
The LUVs were prepared using three different lipids, DMPC,
cholesterol, and GM1 with 68:30:2 molar ratios in 50 mM
HEPES buffer of pH 7.4.[20,21] The formation of LUVs was
confirmed by transmission electron microscope (TEM). For
the leakage study, the peptide and lipid were taken in 1:20
molar ratios, and the dye (carboxyfluorescein) release was
monitored using Fluoromax-4, Horiba instrument. The exci-
tation was at 485 nm and emission was monitored at 516 nm
with 3 nm of bandwidth. At the end of the experiment, 10 μl
of Triton-X-100 was added to obtain complete dye release
from the vesicle and the final fluorescence was measured.
The % of leakage (dye release) was calculated as
2.7
Cell culture
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The human neuroblastoma SH-SY5Y cells were obtained
from American Type Culture Collection (ATCC, CRL-2266)
and grown in DMEM/F12 medium (Cat. No. Gibco/BRL,
Life technologies, Grand Island, NY, USA) supplemented
with 10% (v/v) fetal bovine serum (FBS) (Cat. No. Gibco/
BRL, Life Technologies, NY, USA), 100 U/ml penicillin,
and 100 μg/ml streptomycin at 37°C and 5% CO2.[18] Cells
were differentiated with 10 μM of all-trans retinoic acid (RA)
(Sigma-Aldrich, St. Louis, MO, USA) for 5 days. Cells
were seeded in a FBS-free media for 12 hr prior to the treat-
ment with Aβ40 and BSBHp peptides. Aβ40 was dissolved
in deionized distilled water at a concentration of 10 μM. The
stock solution was diluted to desired concentrations imme-
diately before use. The control cells were added with the
same medium without Aβ40. Confluent cells were used in all
assays, and each experiment was performed in triplicate.
(observed fluorescence−initial fluorescence)
% Leakage=
×100%
(total fluorescence−initial fluorescence)
From the instrument, the text file was taken and graph
was plotted using oRigin pRo 8 software. For each data point,
three different sets of replicate solutions were scanned sep-
arately and the average was taken with observed standard
deviation.
2.8
MTT reduction assay for cell viability
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2.5
(TEM)
Transmission electron microscopy
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Cell viability was assessed using conventional MTT reduc-
tion assay. SH-SY5Y cells were seeded into a 96-well plate
at a density of 1.5 × 104 cells/well and quantified mito-
chondrial activity of living cells by colorimetric 3-(4,5-dim
ethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
We first prepared a stock solution of large unilamellar vesi-
cles (LUVs) of concentration of 2 M. The prepared stock LUV
solution (2 M) was diluted to 20 μM for TEM analysis. 10 μl

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KUMAR et Al.
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assay.[22] Cells were preincubated with different concentra-
tions (0.5, 1, 3, 5, and 10 μM) of BSBHp for 24 hr to study
the self-toxicity. After that, Aβ40 (10 μM) was co-incubated
with or without BSBHp for 24 hr at 37°C. MTT (Cat. No.
M5655; Sigma, St. Louis, MO, USA) was prepared at a
concentration of 5 mg/ml in PBS and a total of 100 μl MTT
solution was added to each well. The cells were incubated
at 37°C and 5% CO2 for 24 hr. After incubation, the MTT
solution was aspirated and 200 μl of dimethylsulfoxide
(DMSO, Cat. No. GRM 5856; Himedia, India) was added
to each well to sufficiently dissolve the formazan precipi-
tate. The amount of formazan was evaluated by measuring
absorbance in a microplate reader (Bio-Rad, CA, USA) at
570 nm, and reference filter was used at 690 nm. All data
were normalized to control cells from three independent
experiments.
pattern. The protocol was followed as described previously
with some modifications.[24] Briefly, Hoechst 33258 (Cat.
No. 861405 Sigma, St. Louis, MO, USA) stain was added
to the culture medium at a concentration of 10 μg/ml for
10 min at 37°C. After that, cells were washed twice with
phosphate-buffered saline (PBS) containing 1% (V/W) par-
aformaldehyde. Cells were mounted on a clean glass slide
with mounting solution (0.1 M citrate, 0.25 M NaH2PO4,
glycerol) and the slide kept it in a dark place in 4°C for
20 min. Nuclear morphology was observed under fluores-
cence microscope (Olympus ×51, Japan) with excitation
filter of 350 nm and emission of 460 nm. The number of
cells with condensed or fragmented nucleus was counted
for the indication of apoptosis.
2.11
Measurement of generation of
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intracellular reactive oxygen species (ROS)
2.9
Lactate dehydrogenase (LDH) release
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The intracellular ROS generation was measured by dichlo-
rodihydrofluorescein diacetate (DCFH-DA) as described
previously[25] with some modifications. In brief, follow-
ing the drug treatment, the medium was aspirated and cells
were incubated with DCFH-DA (Cat. No. D6883; Sigma, St.
Louis, MO, USA) at a concentration of 10 μM/L for 40 min
at 37°C. The nonfluorescent molecule DCFH-DA is cleaved
intracellularly by nonspecific esterases and turns to highly
fluorescent 2′,7′-dichlorofluorescin (DCF) upon oxidation
by ROS. Cells were then washed twice with PBS and cel-
lular DCF was determined by a fluorescent microplate reader
(FLX 800, Biotek, USA), and the fluorescence intensity was
measured by using an excitation wavelength at 490 nm and
emission wavelength at 520 nm.
assay for cell membrane damage
LDH is a soluble cytosolic enzyme present in most eukary-
otic cells, and its release into the culture medium is a key
marker for cell death due to the damage of plasma mem-
brane. Cell death was assessed by measuring the release
of LDH into the medium.[23] The increase in the LDH
activity in the culture supernatant is proportional to the
number of lysed cells. SH-SY5Y cells were seeded into
FBS-free medium for 12 hr and then Aβ40 alone or with
different concentrations of BSBHp (0.5, 1, 3, 5, and 10 μM)
co-incubated with 10 μM of Aβ40 for 24 hr. After 24-hr
treatment, 50 μl culture supernatants were collected from
each well. Extracellular LDH release was evaluated by
using colorimetric assay kit (Cat. No. ab102526 Abcam,
Cambridge, MA, USA) as per manufacturer’s protocol.
Total LDH release was determined by adding 1% (V/V)
Triton-X-100 into the cell for 1 hr. This represents maxi-
mal LDH release as the positive control with 100% cyto-
toxicity. The absorbance was measured at test wavelength
of 490 nm with 660 nm as a reference filter. LDH leak-
age was expressed as the percentage (%) of the total LDH
activity (LDH in the supernatant + LDH in the cell lysate),
according to the equation:
2.12
Measurement of intracellular calcium
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(Ca2+)
The intracellular free Ca2+ levels in suspension of SH-
SY5Y cells were measured by Ca2+-sensitive dye Fura-
2AM by previously defined method.[26] Shortly, after
treatment with respective peptides, 5 μm of Fura-2AM
(Cat. No. 14591 Cayman, USA) was added to the culture
medium at 37°C for 45 min. After that, each plate (1 × 106
cells) was washed twice with Krebs-Ringer–HEPES
(KRH) buffer (135 mM NaCl, 5 mM KCl, 1 mM MgSO4,
0.4 mM KH2PO4, 1 mM CaCl2, 5.5 mM glucose, 20 mM
HEPES, pH 7.4). Fluorescence intensity was measured
by using fluorescent microplate reader (FLX 800, Biotek,
USA) with excitation and emission wavelengths of 488
and 525 nm respectively. The results are the mean SEM
of quadruplicate measurements from three separate exper-
iments and are expressed as the percentage increase in
relative fluorescence units (RFU) relative to the reading
at baseline.
%LDH released=(LDH activity in the medium∕total LDH activity)×100.
2.10
Identification of apoptotic cells
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The morphologies of nuclear chromatin were assessed by
Hoechst 33258 staining after co-incubation of SH-SY5Y
cells with Aβ40 and BSBHp or Aβ40 alone. Hoechst 33258
is a DNA-binding dye. Apoptotic cells were identified by
their characteristics such as condensed chromatin, fragmen-
tation of nucleus, and bright staining, whereas nuclei from
normal cells demonstrated a normal uniform chromatin

KUMAR et Al.
5
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secondary antibody at room temperature for 2 hr and washed
it three times in the TBST buffer. After stripping the mem-
brane with stripping solution, anti-β-actin rabbit polyclonal
antibody (Cat. No. ab8227; Abcam, Cambridge, MA, USA;
1:5,000 dilutions in 3% BSA) was used as a control to reduce
internal blot variability. Immunoreactive bands were visual-
ized by using the ECL substrate solution (Cat. No. 32106;
Thermo Scientific, Rockford, USA). The electrophoresis
image analysis system (Smartview 2001, S/N: SV-0002202,
Japan) was used for analysis of the optical density (OD)
value of each band and normalized against corresponding
β-actin band.
2.13
Measurement of cytochrome-C
|
Cytochrome-C (cyt-C) expression was measured in both the
cytosolic and mitochondrial fractions. Cytosolic and mito-
chondrial fractions were prepared using mitochondrial/cyto-
sol fractionation kit as per manufacturer’s instruction (Cat.
No. K256-25 Biovision, USA). Briefly, treated cells were
centrifuged at 700 g for 5 min at 4°C. Cells were washed
in PBS and centrifuged, and the supernatant was discarded.
Then, 0.5 ml of cytosolic extraction buffer was added that
contains DTT and protease inhibitor. After incubation for
10 min, cells were homogenized and centrifuged at 700 g
for 10 min. Supernatant was collected as cytosolic fraction
of protein. The pellet was resuspended with 120 μl of mito-
chondrial fractionation buffer with DTT and protease inhibi-
tor. The mixture was vortexed for 30 s and marked as mito-
chondrial protein fraction. The protein expression from each
fraction was analyzed by Western blot technique described
below.
2.15
Thioflavin T fluorescence assay of h-
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CSF sample from AD patients
A stock solution of thioflavin T (ThT) of 50 μM concentra-
tion in PBS (50 mM, pH 7.4) was prepared and stored at 4°C
with proper protection to prevent degradation from light. For
disaggregation of Aβ40 present in human cerebrospinal fluid
(CSF), CSF sample alone and with 10-fold molar excess of
breaker peptides in PBS (pH 7.4) was incubated at 37°C in a
water bath. For fluorescence assay, 40 μl of aliquot from the
stock was mixed with 200 μl of ThT solution (50 μM) and
total volume was made up to 400 μl using PBS. Fluorescence
emission was measured at 485 nm and excitation at 440 nm,
using a slit of 5 nm on a Fluoromax-4, Horiba instrument.
2.14
Western blot analysis
|
Western blot analysis was performed for Bax, Bcl-2, caspase-
9, caspase-3, cytochrome-C (cyto and mito) protein expres-
sion. After treatment with BSBHp and Aβ40 peptides with
indicated dosage for 24 hr, about 5 × 106 cells were washed
twice with cold PBS and homogenized in ice-cold lysis
buffer (50 mM Tris, pH-8.0 150 mM NaCl, 1% NP-40, 5 mM
EDTA, pH 8.0, 1% deoxycholic acid) containing protease
cocktail inhibitor (Cat. No. P8340; Sigma, St. Louis, MO,
USA). Respective protein samples were electrophoresed on
15% (W/V) SDS–polyacrylamide gel (Mini-PROTEAN®
tetra cell with mini-trans Blot®, Bio-Rad, USA) and subse-
quently transferred from gel onto a nitrocellulose membrane
(Millipore, USA). The membrane was incubated with fresh
blocking buffer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl,
0.05% Tween-20 containing 5% nonfat-dried milk) for 1 hr at
room temperature and then probed with respective monoclo-
nal primary mouse anti-Bcl-2 (Cat. No. SC-7382; Santa Cruz
Biotechnology, Inc., USA; 1:800 dilution in 3% BSA), mouse
anti-Bax (Cat. No. SC-7480; Santa Cruz Biotechnology, Inc.,
USA; 1:800 dilution in 3% BSA), polyclonal rabbit anticas-
pase-3 (Cat. No. ab13847; Abcam, Cambridge, MA, USA;
1:700 dilution in 3% BSA), monoclonal rabbit anticaspase-9
(Cat. No. ab32539; Abcam, Cambridge, MA, USA; 1:1,000
dilution in 3% BSA), polyclonal rabbit anticytochrome-C
(Cat. No. ab90529; Abcam, Cambridge, MA, USA; 1:1,000
dilution in 3% BSA) at 4°C overnight. Nitrocellulose mem-
branes were washed three times in TBST and incubated in
the respective HRP-conjugated goat anti-mouse (Cat. No.
Sc-2005; Santa Cruz Biotechnology, Inc., USA; 1:5,000
dilution in 3% BSA), goat anti-rabbit (Cat. No. ab97051;
Abcam, Cambridge, MA, USA; 1:5,000 dilution in 3% BSA)
2.16
Congo red-stained birefringence
|
A 20 μl aliquot from the stock was placed over a glass slide
followed by 20 μl of the saturated Congo red solution. The
excess solution was removed using a blotting paper, dried
at room temperature, and analyzed under a Leica ICC50 HD
polarizable microscope.
2.17
Statistical analysis
|
All data were represented as mean standard error of mean
(SEM) for three independent experiments. Data were statis-
tically analyzed with ANOVA followed by Student’s t test
using gRAphpAd pRisM 6.0 Software (GraphPad Software, Inc.,
San Diego, CA, USA). Mean values were considered to be
statistically significant at p < .05.
3
RESULTS
|
We have synthesized a β-sheet breaker α/β-hybrid peptide
(BSBHp, Ac–Leu–Ant–Phe–Phe–Asp–NH2) and a known
control breaker peptide (CBp, Ac–Leu–Pro–Phe–Phe–Asp–
NH2)[12] via solid-phase peptide synthesis (SPPS) follow-
ing Fmoc/tBu orthogonal protection strategy. The synthe-
sized peptides were characterized by LC-MS (Figure 1). The

6
KUMAR et Al.
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FIGURE 1 LC-MS picture of the purified BSBHp: Calculated mass for C37H45N6O8 is 701.32 [M + H]+, while observed 701.31 [M + H]+ (a, b). LC-
MS picture of the purified CBp: Calculated mass for C35H47N6O8 is 679.34 [M + H]+, while observed 679.44 [M + H]+ (c, d) [Colour figure can be viewed
at wileyonlinelibrary.com]
BSBHp was found non-amyloidogenic and efficiently disrupts
the amyloid aggregates of Aβ40 in vitro.[17] As the soluble
oligomers are more toxic than the mature amyloid,[27] it was
important to check whether the BSBHp disrupts the amyloid
aggregates of Aβ into toxic soluble oligomers or to nontoxic
species. To address this problem, we have carried out a dye-
loaded large unilamellar vesicle (LUV) leakage assay in vitro
before conducting cell-based neurotoxicity studies. LUV
leakage study is a proof of presence of toxic oligomeric spe-
cies[27] in a solution. Amount of dye leakage is proportional to
the amount of the toxic soluble oligomeric species as toxicity
is originated by pore formation in cell membrane or vesicle.
LUVs were prepared as described in Materials and Methods
section and characterized by TEM (Figure 2a). For the LUV
leakage study, we prepared five different sets of solutions with
two replicas of those solutions. We added the breaker peptides
(BSBHp and CBp) to the preaggregated (3 days old) Aβ solu-
tion and incubated them for a total of 10 days (3 + 7 days),
before LUV leakage study. Five sets of solutions were used for
the vesicle leakage studies, namely untreated (a) and untreated
LUVs, (b) Aβ40 (incubated for 12 hr), (c) Aβ40 (incubated for
10 days), (d) Aβ40 and BSBHp (1:10, added after 3 days), and
(e) Aβ40 and CBp (1:10, added after 3 days).
in comparison with 12-hour-old Aβ fibril. The results con-
firmed that soluble oligomers caused more toxic effect on
LUVs by pore formation and dye leakage in comparison
with the mature fibrils. We observed lesser extent of dye
leakage when BSBHp (blue, Figure 2b, c) was present with
Aβ40 and that leakage was almost comparable with that
from the untreated LUVs. Therefore, it was concluded that
presence of BSBHps disaggregates the amyloid fibrils of
Aβ into nontoxic species.
To determine the toxic effect of soluble oligomeric mis-
folded Aβ peptide (Aβ40) for 24 hr in SH-SY5Y cells, MTT
reduction assays were used to determine the cell viability. As
shown in Figure 3a, BSBHp at each of these concentrations
(0.5–10 μM) alone did not produce any cytotoxicity in SH-
SY5Y cells. Cells were treated with 10 μM Aβ40 with or with-
out different concentrations of BSBHp (0.5–10 μM). After
24-hr exposure to Aβ40 (10 μM) alone, cell viability was sig-
nificantly (p < .01) decreased by 36.5 3.1% as compared
to control (no treatment) cells. However, this toxic effect
was significantly (p < .01) ameliorated when co-incubated
with BSBHp at a concentration of 3 μM (79 4.5%), 5 μM
(84 3.05%), and 10 μM (87 2.2%) on cell viability
assessed by MTT reduction assay (Figure 3b).
The pore formation on LUVs followed by dye
release was monitored by time-dependent fluorescence
assay (Figure 2a (i) and (ii), b, c). We observed that the
12-hour-old Aβ40 (magenta, Figure 2b, c) caused more dye
leakage (~53% in 72 hr), which confirmed the presence of
toxic oligomeric species (in solution a). Again, 10-day-old
Aβ40 sample (cyan, Figure 2b, c) caused less LUV leakage
Aggregated Aβ40 induces neuronal cell death by disrupt-
ing the cell membrane. To assess the membrane damage,
LDH release assay was performed. Lactate dehydrogenase is
a cytosolic enzyme; after treating with 10 μM Aβ40 alone in
SH-SY5Y cells for 24 hr, 45 2.8% increase in LDH release
was found into the medium, which is an indicator of cytotoxic
damage of cells. However, when cells were incubated with

KUMAR et Al.
7
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(a)
(b)
(c)
(i)
FIGURE 2 (a) TEM images of
synthesized LUVs and both the images
represent same information. (b) % of dye
leakage from LUVs over 0–100 min and at
0–72 hr (v), by untreated LUVs (black), LUVs
treated with 12-hour-old Aβ (magenta), LUVs
treated with 10-day-old Aβ (cyan), LUVs
treated with Aβ in the presence of BSBHp
(blue), and LUVs treated with Aβ in the
presence of CBp (red) [Colour figure can be
viewed at wileyonlinelibrary.com]
(ii)
FIGURE 3 The characterization of BSBHp on the viability of SH-SY5Y cells. (a) To study the self-toxicity of synthesized BSBHp on cell viability
of SH-SY5Y cells. Cells were incubated with different concentrations (0.5–10 μM) of BSBHp for 24 hr, which indicates that BSBHp has no toxic effect and
up to 85% cells were alive. (b) Aβ40 monomer (10 μM) was co-incubated with 0.5, 1, 3, 5, and 10 μM or without BSBHp for 48 hr at 37°C. After 24 hr of
treatment, cell viability was measured by MTT assay. Values are represented as mean SEM of three separate experiments. **p < .01, compared to control
group and ##p < .01 compared to Aβ40-treated group, one-way ANOVA
respective concentrations of BSBHp, only 3 μM (25 1.7%),
5 μM (21 2%), and 10 μM (19 2.9%) significantly (p < .01)
reduce LDH release as compared to Aβ40-treated (45 2.8%)
cells(Figure 4a). ThisresultsuggeststhatBSBHphasneuropro-
tective effect on Aβ40-induced cytotoxicity in SH-SY5Y cells.
Therefore, 3 μM dose of BSBHp was used for all experiments.
Cellular morphology was also observed in phase-contrast
microscope after 24-hr co-incubation with Aβ40 (10 μM) plus
BSBHp (3 μM) and Aβ40 (10 μM) alone (Figure 4b).
Aggregated Aβ40 induces apoptosis and apoptotic cells
are characterized by condensed or fragmented nuclei. To
study the cytoprotective effect of BSBHp on apoptosis
induced by Aβ40, cells were stained with DNA-binding dye
Hoechst 33258. 10 μM of Aβ40 was incubated with or with-
out 3 μM of BSBHp for 24 hr. Fluorescence microscopic
images showed that Aβ40-treated (52 2.3) cells exhibit
significant (p < .01) nuclear fragmentation and increased
number of apoptotic cells as compared to untreated con-
trol cells (12 1.4). However, cells treated combined
with Aβ40 and BSBHp had intact nuclei and exhibit signif-
icant (p < .05) reduction in the number of apoptotic cells
(29 3.0) (Figure 5a, b).
To determine the effect of BSBHp on oxidative
stress induced by aggregated Aβ40 in SH-SY5Y cells,
intracellular ROS generation was measured by fluores-
cent dye DCFH-DA. It was found that cells treated with
10 μM of Aβ40 for 24 hr significantly (p < .01) exhibit
increase (208 4.3) in the intensity of DCF fluores-
cence as compared to untreated cells. Incubation of cells
with Aβ40 and BSBHp significantly (p < .05) inhibited
(156 4.2) Aβ40-induced ROS production in SH-SY5Y
cells (Figure 6).
Impeding of Ca2+ homeostasis leads to impairment of
neuronal activity, neurogenesis, and neurotransmitter release
that causes the progression of pathogenesis of AD and also
induces mitochondrial apoptotic cascade.[28–30] We measured
intracellular free Ca2+ by fluorescent Ca2+ indicator, Fura-
2AM after incubation of cells with Aβ40 (10 μM) alone or
co-incubated with BSBHp (3 μM). Our result showed that
Aβ40 significantly (p < .01) elevates intracellular Ca2+ level
(158 2.7%) in SH-SY5Y cells as compared to untreated
cells (64 4.4%). Moreover, after incubation with Aβ40 and
BSBHp, elevated Ca2+ levels significantly (p < .01) reduced
(96.5 4.6) as compared to Aβ40-treated cells as shown in

8
KUMAR et Al.
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FIGURE 4 Effect of BSBHp on Aβ40-induced cytotoxicity in respect to LDH release on SH-SY5Y cells. Aβ40 (10 μM) was co-incubated with (0.5–
10 μM) or without BSBHp for 48 hr at 37°C and then added to culture medium. (a) After 24 hr of treatment, cytotoxicity was measured by % of LDH release
from the damaged cell membrane. (b) Phase-contrast micrograph image (40X) shows cellular morphology of SH-SY5Y cells after treatment with 3 μM of
BSBHp alone or with Aβ40 for 24 hr at 37°C. All values are represented as mean SEM of three separate experiments. **p < .01 compared to control group
and ##p < .01 compared to Aβ40-treated group, one-way ANOVA
FIGURE 5 BSBHp prevents apoptosis of SH-SY5Y cells from toxic oligomers of aggregated Aβ40. (a) Shown cells were exposed to Aβ40 (10 μM)
alone or BSBHp (3 μM) co-incubated with Aβ40 (10 μM) for 24 hr. Cells were stained with Hoechst 33258 and viewed under fluorescence microscope. Arrow
indicates condensed and fragmented nuclei. (b) Apoptotic cells with altered nuclear structure were quantified after Aβ40 treatment with the presence or
absence of BSBHp. Results are shown from three separate experiments. Data are mean SEM of three independent experiments. **p < .01 compared to
control group and ##p < .05 compared to Aβ40-treated group [Colour figure can be viewed at wileyonlinelibrary.com]

KUMAR et Al.
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FIGURE 6 Effect of BSBHp on Aβ40 (10 μM)-induced reactive
oxygen species (ROS) generation in SH-SY5Y cells after 24-hr incubation.
Cells were treated with Aβ40 (10 μM) or BSBHp (3 μM) alone and/
or co-incubated with both the peptides. The amount of intracellular
ROS formation was determined by oxidation of DCFH-DA to DCF by
fluorescent microplate reader. Values are mean SEM of three separate
experiments. **p < .01 compared to control group and ##p < .05 compared
to Aβ40-treated group
FIGURE 8 Effect of BSBHp on Aβ40 (10 μM)-induced Bcl-2 and
Bax expression in SH-SY5Y cells. Aβ40 (10 μM) was co-incubated with
or without BSBHp (3 μM) for 24 hr. (a) Shown representative Western
blot analysis of Bax and Bcl-2 expression. (b) And (c) shown Bax and
Bcl-2 expression (changes fold to control), respectively. The protein bands
were quantified using Smartview image analysis software, and values are
expressed as mean SEM (N = 3 animals per group) of three independent
experiments. **p < .05 compared to control group and ##p < .05 compared
to Aβ40-treated group
approximately 2.8-fold as compared with the untreated con-
trol cells. However, the expression of Bax protein was down-
regulated by reducing the Bax/Bcl-2 ratio after co-incubation
of Aβ40 with BSBHp-treated SH-SY5Y cells (Figure 8).
As caspases are the important proteases that are activated
by diverse stimuli by the process of apoptosis, and Western
blot analysis was performed to investigate the expression
profile of caspase-9 and caspase-3, following the respective
treatments. Our results indicate that treatment with Aβ40
significantly increases (p < .05) the expression of caspase-9
and caspase-3 protein level as compared to control cells.
Consequently, BSBHp significantly decreases (p < .05) the
caspase-9 and caspase-3 levels after 24-hr co-incubation with
Aβ40 (Figure 9).
FIGURE 7 Effect of BSBHp (3 μM) on Aβ40 (10 μM)-induced
elevation of intracellular calcium (Ca2+) in SH-SY5Y cells. After respective
treatment, cells were incubated with Fura-2AM and relative fluorescent
intensity was measured by fluorescence microplate reader. Results are
shown as mean SEM of three separate experiments. **p < .01 compared
to control group and ##p < .01 compared to Aβ40-treated group
Figure 7. This result suggests that BSBHp has a protective
role in Ca2+ homeostasis by blocking the aggregation of
Aβ40.
To examine the release of cytochrome-C from both the
cytosolic and mitochondrial fractions, Western blot analysis
was performed after respective treatment in SH-SY5Y cells.
Results showed that Aβ40 treatment significantly (p < .05)
increases the release of cytochrome-C from mitochondria to
cytosol, as compared to control. However, co-incubation with
BSBHp significantly reverses (p < .05) the Aβ40-induced
release of cytochrome-C from mitochondria to cytosol
(Figure 10). The above results demonstrated that BSBHp has
antiapoptotic effect against aggregated Aβ40-induced neu-
ronal apoptosis mediated through mitochondrial apoptotic
pathway by blocking Aβ40 aggregation.
Following the treatment with Aβ40 and BSBHp on SH-
SY5Y cells, Western blot analysis was performed to assess
the mechanistic role of proteins associated with mitochondrial
apoptotic cascades. Mitochondria-mediated apoptosis was
regulated by Bcl-2 family of proteins including pro-apoptotic
regulator Bax and antiapoptotic factor Bcl-2. Under stressed
condition, Bax regulates the release of cytochrome-C from
mitochondria followed by the activation of caspase-9 and
caspase-3 for the induction of apoptosis.[31] Our results showed
that Aβ40-treated cells up-regulated Bax expression, showed
a slight change in Bcl-2 protein level, and thereby a signifi-
cant increase (p < .05) in the ratio of Bax/Bcl-2 expression
We also performed the effect of BSBHp on the reduction
in amyloid formation in human cerebrospinal fluid (h-CSF).

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KUMAR et Al.
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FIGURE 9 Effect of BSBHp on Aβ40 (10 μM)-induced caspase-9 and caspase-3 expression in SH-SY5Y cells. Aβ40 (10 μM) was co-incubated with or
without BSBHp (3 μM) for 24 hr). (a) Shown representative Western blot analysis of caspase 9 and caspase 3 expression. (b) and (c) shown caspase 9 and
caspase 3 expression (changes fold to control), respectively. The protein bands were quantified using Smartview image analysis, and values are expressed as
mean SEM from three independent experiments. **p < .05 compared to control group and ##p < .05 compared to Aβ40-treated group
FIGURE 10 Effect of BSBHp on Aβ40 (10 μM)-induced cytochrome-C release in SH-SY5Y cells. Aβ40 (10 μM) was co-incubated with or without
BSBHp (3 μM) for 24 hr. (a) Shown representative Western blot analysis of cytosolic and mitochondrial fractions of cytochrome-C expression. (b) and (c)
shown cytosolic and mitochondrial cytochrome-C protein (changes fold to control), respectively. The protein bands were quantified using Smartview image
analysis, and values are expressed as mean SEM from three independent experiments. **p < .05, compared to control group and ##p < .05 compared to
Aβ40-treated group
As Aβ aggregates as plaques in the extracellular spaces of the
neuronal cells that are in direct contact with h-CSF, presence
of Aβ aggregates in h-CSF can be identified by histopatho-
logical investigations.[32] For the present study, h-CSF sam-
ples were obtained from the AD patients and the presence of
amyloid was confirmed by thioflavin T (ThT) fluorescence
study (Figure 11a, b). A 50 μM solution of Aβ40 was prepared
in PBS of pH 7.4 and incubated that at 37°C for 10 days.
After 10 days, ThT fluorescence was measured with differ-
ent concentrations (20, 50, and 100 μl) of Aβ40 (Figure 11a)
and fixed concentration of ThT. From the ThT fluorescence,
we noticed that with increasing amount of Aβ40, fluorescence
intensity increased, which indicated that amyloid was pres-
ent. Similarly, we performed ThT fluorescence study of the
h-CSF sample from the patients with AD and observed that
the fluorescence intensity increased with increasing concen-
tration of h-CSF (Figure 11b). Therefore, the presence of
amyloid aggregates in h-CSF was confirmed, and from the
normalized spectra, the concentration of Aβ in a CSF sample
was found to be ~23 μM; 10-fold molar excess of BSBHp was
added with the h-CSF sample, and the time-dependent kinet-
ics of disaggregation of amyloid aggregates was monitored
(Figure 11c). In the ThT assay, we observed that fluorescence
intensity of CSF was suppressed excessively in the presence
of BSBHp (blue, Figure 11c), while h-CSF alone (black,
Figure 11c) showed increased fluorescence. Again, h-CSF
alone showed clear fiber in TEM (Figure 11d(i)) and green
gold birefringence under cross-polarized light when stained

KUMAR et Al.
11
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(a)
(b)
(c)
(d)
(i)
(e)
(i)
(ii)
(iii)
(ii)
(iii)
FIGURE 11 Fluorescence spectra of ThT (25 μM) in the absence (black) and presence of 20 μl (red), 50 μl (blue), and 100 μl (magenta) of Aβ (a) and
human cerebrospinal fluid (h-CSF) (b). (c) Time-dependent ThT assay for the disruption of the amyloid present in h-CSF in the absence (black) and presence
BSBHp (blue) and CBp (red), respectively. (d) TEM images of h-CSF in the absence (i) and presence of BSBHp (ii) and CBp (iii). (e) Congo red-stained
birefringence images of h-CSF in the absence (i) and presence of BSBHp (ii) and CBp (iii). All the images were taken after 10 days of incubation at 37°C.
Scale bars indicated as 200 nm and 20 μm. Experiments were carried out in PBS (50 mM) at pH 7.4 and 37°C. Total cuvette volume was 400 μl (200 μl ThT
(50 μM) + sample (20, 50, 100 μl) + remaining volume of PBS) [Colour figure can be viewed at wileyonlinelibrary.com]
with Congo red [(Figure 11e (i)]. No such fiber [(Figure 11d
(ii)] or birefringence [Figure 11e (ii)] was observed in the
presence of BSBHp, indicating the absence of amyloid.
Our study demonstrated that BSBHp potentially disrupts and
redissolves the Aβ40 fibrils in vitro that generate nontoxic
species. Thus, inhibitory effects of BSBHp on underlying
mechanisms of Aβ40-induced apoptotic neurodegeneration
were studied on in vitro human neuroblastoma SH-SY5Y
cells. To the best of our knowledge, this type of β-amino
acid-containing breaker peptide was not used before for the
inhibition of Aβ40-induced neuronal apoptosis.
It is well documented that toxic oligomers or protofibrils of
Aβ produce maximum toxicity than mature fibrils due to their
ability of perforation in cell membrane and the induction of
cytotoxicity of neuronal cell.[8] We first examined it in large
unilamellar vesicles (LUVs) that are considered to mimic cell
membrane. Extent of dye-release from dye-entrapped LUVs
clearly indicated immature fibrils to be more efficient for pore
formation in the LUVs than mature fibrils. Also, it was found
that application of BSBHp demolishes such pore formation.
Our findings revealed that SH-SY5Y cells treated with 10 μM
of Aβ40 exhibit a decrease in MTT reduction and a maximum
release of LDH into the medium. The novelty is that co-
incubation with BSBHp protects SH-SY5Y cells from Aβ40-
induced toxicity studied by MTT and LDH release assay.
Results from LDH assay confirmed that BSBHp significantly
prevents the Aβ40-induced membrane damage, which is cor-
roborated with the findings of in vitro dye leakage assay
4
DISCUSSION
|
There is abundant evidence suggesting that accumulation
of amyloid-β in the brain is responsible for the formation of
senile plaque resulting in the neuronal degeneration, apop-
tosis, cognitive and memory impairments.[33] Amyloid-β
precursor protein (AβPP)-derived monomeric amyloid beta
is self-assembled into highly toxic soluble Aβ oligomers
(AβO) and high molecular weight Aβ oligomers and protofi-
brils are the predominant Aβ species in the native soluble
protein fraction of the AD brain.[34] Thus, we consider that
the anthranilic acid-containing β-sheet breaker α/β-hybrid
peptide (BSBHp), which can inhibit self-aggregation, even
reverse the aggregation of the pathogenic Aβ40 can be a
potential therapeutic strategy for AD.
BSBHp is a novel class of conformationally restricted
β-sheet breaker peptide, which has a recognition motif of the
target pathogenic peptide Aβ40. The β-breaker element con-
tains anthranilic acid (Ant, an aromatic β-amino acid) unit
and is relatively more stable against proteolytic degradation.

12
KUMAR et Al.
|
using carboxyfluorescein-entrapped LUVs. The neuroprotec-
tive effect of BSBHp was also confirmed by Hoechst stain-
ing assay, analysis of nuclear morphology, cellular apoptosis,
and nuclear fragmentation. So, these findings support that
BSBHp has potential role to inhibit self-aggregation and for-
mation of toxic oligomeric species of Aβ40. Furthermore, we
investigated the protective role of BSBHp on Aβ40-induced
mitochondrial apoptotic pathway and ROS production into
the neuronal cells.
apoptosis in SH-SY5Y cells by inhibiting Aβ40 aggregation.
As far as we know, this is the first report to demonstrate that
BSBHp has the neuroprotective effects against Aβ-induced
neurotoxicity in SH-SY5Y cells. The protection effects of
BSBHp against neuronal atrophy may help to provide the
pharmacological basis of its clinical use in the treatment for
neurodegeneration in AD.
ACKNOWLEDGEMENTS
Several lines of evidence showed that extensive oxida-
tive stress is generated in AD brain during disease progres-
sion, which may cause neuronal apoptosis leading to cell
death.[35,36] It was reported that soluble oligomers of Aβ40
can penetrate phospholipids bilayer of cell membrane, and
induction of neurodegenerative events like lipid peroxida-
tion, synaptic alteration, dysregulation of Ca2+ homeostasis,
and also activation of mitochondrial apoptotic pathway ulti-
mately leads to cell death.[37,38] This study demonstrated that
BSBHp significantly reduces Aβ40-induced ROS generation
by both the reversion of aggregation and formation of toxic
oligomeric Aβ40. It has been pointed out that accumulation of
oligomeric Aβ40 altered the Ca2+ homeostasis that disrupts
synaptic transmission, membrane integrity and initiates apop-
totic neurodegeneration.[39,40] We found that BSBHp effec-
tively disrupted protofibrils and maintained Aβ40-induced
Ca2+ homeostasis in SH-SY5Y cells.
We highly acknowledge the grants provided by DBT (NER-
BPMC) [BT/347/NE/TBP/2012] to BM and ACM. DST
PURSE-II, UPE-II, RNP, SAP DRS-I (UGC grants) to ACM
are duly acknowledged. We thank CIF, IIT Guwahati, for
LC-MS and TEM studies. We express our sincere grati-
tude to CIF, School of Life Sciences, JNU, New Delhi, and
thanks to RPM College, Uttarpara, for their kind support and
co-operations.
AUTHOR’S CONTRIBUTION
B.M. and A.P. designed the β-sheet breaker α/β-hybrid pep-
tide (BSBHp) and its biophysical application in cell-free
systems. A.C.M., S. Kumar, and A.K.G. designed the cell-
based framework of research. AP., S. Kumar, and S. Kalita
performed all the experiments. B.M., A.C.M, A.P., and S.
Kumar analyzed the results and wrote the manuscript.
Neuronal apoptosis caused by aggregated Aβ40 is consid-
ered to be a major pathological hallmark in AD. Aβ-induced
neuronal apoptosis was mediated through the generation of
intracellular ROS and mitochondrial impairment on neuronal
cells.[41] It is also reported that ROS are involved in the reg-
ulation of mitochondrial cascade and activation of apoptotic
proteins.[42] Bcl-2 family member proteins such as Bax and
Bcl-2 are involved in the Aβ-induced mitochondrial path-
way.[43] Under pathological condition, elevation of Bax trans-
located into the mitochondrial membrane, can homodimerize
there by activating several caspases, and alter the mitochon-
drial activity by promoting apoptotic factors into the cyto-
plasm.[44,45] Thus, it is found that BSBHp alleviates Bax/Bcl-2
ratio and also Aβ40-induced Bax expression. Another import-
ant factor is that release of cytochrome-C from mitochondria
to cytosol triggers the activation of caspase-9 followed by
caspase-3 and switching on the downstream signaling path-
way.[46] Our results clearly demonstrated that BSBHp effec-
tively suppresses the Aβ40-induced caspase-9 and caspase-3
protein expression, thereby maintaining neuronal cell death
by inhibiting Aβ40 aggregation and disruption of fibrillar
form of Aβ40. We also demonstrated the effect of BSBHp on
aggregated Aβ in the h-CSF sample of the patients with AD
in vitro. The BSBHp expressively disaggregated the fibrillar
assembly present in the h-CSF sample.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
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How to cite this article: Kumar S, Paul A, Kalita S, Ghosh AK,
Mandal B, and Mondal AC. (2016), Protective effects of β-sheet
breaker α/β-hybrid peptide against amyloid β-induced neuronal
apoptosis in vitro. Chem Biol Drug Des. 2016;00:1–13.
doi:10.1111/cbdd.12912.