1. Dose-dependentincrease ofResveratrol’sNeuroprotective effects in a SIRT1-
Independent Alzheimer’s disease model
Gbolahan Olarewaju
0587370
Monday April 4, 2011
Dr. M. Bakovic

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SECTION A
Introduction
Among the many neurodegenerative diseases that occur in the elderly, Alzheimer’s
disease (AD) has been one of the most researched. This disease marked with progressive
cognitive decline is the leading from of dementia in the elderly. It currently affects 1 in 11
Canadians over the age of 65 [1]. Since its discovery, the disease has been investigated at length
to provide an explanation of the observed signs in living AD patients which include memory
loss, decline in cognition and physical coordination [1]. The overall effect of the disease on the
brain is neuronal death leading to cortical and hippocampal shrinkage. The actual cause of cell
death in AD is not fully known but according to the National Institute of Communicative
Diseases and Stroke and the Alzheimer's disease and Related Disorders Association (NINCDS-
ADRDA) the criteria for AD diagnosis are the presence of neurofibrillary tangles (NFT) and
most especially dense neuritic plaques [2, 3].
Neurofibrillary tangles (NFT) are dense bundles of un-branched filaments composed of a
microtubule-associated protein Tau [3]. In AD Tau becomes hyperphosphorylated and self-
assembles into the filaments known as NFT. NFTs block axonal transport mechanisms and could
be an explanation for the neuronal death that comes with AD. However, NFT are associated with
other neurodegenerative diseases and their origin in AD is not fully understood [3]. Neuritic
plaques on the other hand have been labeled as criteria for the diagnosis of definite AD. The
plaques are abnormal extracellular accumulations of various proteins, the most prominent of
which is the Beta-amyloid peptide (AB). Beta-amyloid is a 39-43 amino acid peptide that is a
product of regular proteolytic cleavage of the Amyloid Precursor Protein (APP) [2]. APP can be
cleaved by α and γ-secretase enzymes to release p3 peptides or by Beta-site APP Cleaving
Enzyme 1 (BACE-1 or β-secretase) and γ -secretase to release AB [2]. The latter of the cleavage
pathways is referred to as the amyloidogenic pathway. Neither the complete composition of the
plaques nor how they are assembled is known. In relation to AD pathology however, they have
been implicated to cause neuronal cell death by blocking cell to cell neurotransmission at
synapses, causing local oxidative stress and triggering and sustaining inflammatory responses
[4].
Current pharmacotherapy for AD has focused on two areas; the cholinergic-deficit
hypothesis and the amyloid hypothesis. Post-mortem studies revealed that choline uptake and
acetylcholine release are decreased in the brains of AD patients [5]. The cholinergic-deficit
hypothesis explains this imbalance in acetylcholine (Ach) causes many of the AD symptoms [6].
As such researchers have tried to restore Ach balance by inhibiting cholinesterases which
breakdown Ach. The results of their research were drugs like Tacrine, Donepezil and
Galantamine [4]. These drugs have been moderate in their alleviation of AD symptoms and have
side effects like hepatotoxicity, nausea and diarrhea [7]. As such, there is a current search for
better solutions that target the root cause of AD. Such therapies would target the mechanisms
behind AB production and modulate its pathological effects [8]. For example inhibition of β-
secretase (BACE-1) and stimulating α-secretase are targets for therapies as they would lead to a
decrease in the amyloid plaque load [8]. An alternative is introducing small molecules that
disrupt the aggregation of AB [8]. AB immunotherapy is also being investigated; specifically the
use of vaccines that mimic parts of the AB sequence as their antigenic component is being

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investigated [9]. These are just examples of how future therapies could target AB production and
plaque formation mechanisms.
More recently, researchers are looking at potential therapies for treating the cytotoxic
effects of AB accumulation which are oxidative stress and inflammation. Among such therapies
is the use of natural polyphenolic compounds such as those belonging to the stilbenoid group
especially Resveratrol and its derivatives. Resveratrol is present in a variety of plants including
vegetables and fruits. Grapes such as the wine grape (Vitis vinifera, L) are the most important
dietary source of Resveratrol [10]. Resveratrol has been shown to have neuroprotective abilities
in AD models especially in relation to oxidative stress and inflammation. A study by Jang and
Surh showed that pretreatment of PC12 rat pheochromocytoma cells with Resveratrol stopped
AB induced intracellular reactive oxygen species (ROS) accumulation [11]. Other studies have
shown that Resveratrol up-regulates cellular antioxidants such as glutathione [12]. More
recently, attention has been paid to Resveratrol as an anti-inflammatory agent due to its status as
a SIRT1 activity inducer. SIRT1 is an NF-κB de-acetylase and a member of the Sirtuin family.
NF-κB is a pro-inflammatory transcription factor that enhances the expression of cytokine genes.
This indirect inhibition of NF-κBby SIRT1 promotion has further implicated Resveratrol as a
potential therapy in AD pathology treatment [13]. There has been some discrepancy regarding
the mechanism of protection that Resveratrol uses; whether it is solely via its interaction with
SIRT1 or by other means [14]. My research will focus on further understanding the relationship
between Resveratrol and AD pathology.
Rationale
As has already been stated, Alzheimer’s disease is the leading form of dementia in the
elderly and at this time the available treatments for the disease are focused on treating the
observed symptoms. There have not been any successful prophylactic therapies. Current
treatments for AD such as NMDA receptor stimulators and cholinesterase inhibitors have side
effects and are modest in their benefits. NSAIDs seems to be protective against AD development
but do not work for pre-existing cases [4]. The ideal therapy should be effective and relatively
free of undesirable side effects. Potential prophylactics should be safe, inexpensive and readily
available. Resveratrol seems to fulfill all these criteria as no side effects have been found and it is
can be obtained from natural sources. It is very promising in both prophylaxis and treatment
after the disease has developed. Many epidemiological studies have shown a decreased incidence
of AD with wine consumption [15]. Resveratrol’s effectiveness in preventing AD development
probably lies in its ability to decrease AB production and aggregation into plaques. Along with
preventing development, there is a wealth of evidence supporting its role as an anti-oxidant and
anti-inflammatory agent. What is currently lacking in the literature, however, is whether
Resveratrol requires SIRT1 to accomplish detectable neuroprotection [14]. Across all studies, a
recurring limitation is that Resveratrol has a very low oral bioavailability [14]. Studies that have
administered Resveratrol orally have determined that its oral bioavailability is considerably less
than 1% [16]. Thus, there is a need for further investigation to tackle this limitation. To that
effect, the objective of my research is 2-fold; (a) to improve the efficacy of Resveratrol in vivo
by increasing its bioavailability, (b) to elucidate the in vitro neuroprotective effects of
Resveratrol independent of SIRT-1. Keeping with these objectives, my hypothesis is:
Independent of SIRT1and in a dose-dependent manner, Resveratrol will induce substantial

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neuroprotective effects against AB-induced cytotoxicity in Alzheimer’s disease provided its
availability at the target tissue is improved.
SECTION B: Improving the efficacy of Resveratrol in vivo by increasing its bioavailability
Introduction
It has already been mentioned that the major limitation regarding Resveratrol and its in
vivo application is its extremely low bioavailability. This percentage is representative of the fact
that Resveratrol is heavily metabolized in the gut and the liver which leaves less that 1% of the
original dose in the blood getting to the site of action. When Resveratrol is taken orally, it
undergoes extensive Phase II metabolism in the liver which is either glucuronidation or sulfation
[16]. Studies that have administered oral doses and performed Liquid Chromatography/Mass
Spectrometry on the plasma have revealed the most abundant metabolites of Resveratrol first-
pass metabolism to be the glucuronides [16]. Current research shows that flavanoids can inhibit
the glucuronidation of Resveratrol in the liver [17]. This would improve its systemic
bioavailability and as such is not AD specific. A common theory regarding the Resveratrol
conjugates is that they serve as a pool from which active Resveratrol can be liberated at the
target tissue by β-glucuronidases [16]. I will employ this theory in my procedure to by inducing
production of these enzymes at the target site for AD (the brain) while administering Resveratrol
by 2 different routes. The dose-dependent pharmacokinetics of Resveratrol will also be observed
using a range of doses that will incorporate the amounts used in various studies [18]. This phase
of my study will be performed in vivo.
Experimental Procedures
This procedure will involve the use of 2 populations (4 each) of nude mice. All mice will
be administered Resveratrol either intravenously or orally. The first population of mice will be
modified by CNS grafting of embryonic Human Neural Progenitor Cells (HNPC) which have
been modified to promote β-glucuronidase (β-glu) production as can be seen from a study by
Buchet et al [19]. The second population will be regular nude mice, serving as controls. The
embryonic HNPC will be proliferated in vitro in the presence of basic fibroblast growth factor
[20] and co-transduced with a recombinant lentiviral vector encoding for human β-glu [19]. The
HNPC will then be grafted into the striatum of the nude mice where they will be allowed to be
integrated for 1 week [19].
The 8 mice will be split into four (4) groups as follows; (a) Intravenous Resveratrol Nude
mice, (b) Oral Resveratrol Nude mice, (c) IV Resveratrol HNPC mice, and (d) Oral Resveratrol
HNPC mice. Each group will receive the same escalating sequential doses of Resveratrol. Eight
doses will be used on separate days; 25, 50, 100, 250, 500mg, 1, 2.5 and 5g. After each dose,
blood samples will be collected at 4-hourly intervals until 24 hours after administration (4,
8…24). The blood samples will be centrifuged for 15 minutes to separate the plasma. Resveratrol
and its metabolites will be separated and quantified using UV- High Performance Liquid
Chromatography. The mean peak plasma concentrations of Resveratrol and its glucuronic
conjugate will be compared between mice strains as well as routes of administration.

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Expected Outcomes: It is expected that in the mice receiving IV Resveratrol, the amounts of
active Resveratrol in circulation will be greater than the Oral group (Fig. 2 Appendix). It is also
expected that in the HNPC mice, the mean peak plasma concentrations of Resveratrol will be
higher than in the regular mice (Fig. 2 Appendix). This will be evidence of a reversal of
Resveratrol glucuronidation in the brain. All concentrations are expected to increase in a dose-
dependent fashion (Fig. 1 Appendix) as elimination of Resveratrol is by first-order [21].
Anticipated Problems and Solutions: Potential problems with this study lie in both the design and
the applicability. There is a chance that the HNP cells may not integrate properly, in which case I
would artificially induce β-glu translation at the target site. This may be achieved by
administering Androgen and Resveratrol concomitantly [22]. The other problem is that
intravenous administration is not an attractive route for supplying Resveratrol. It is my hope that
the dosage increases in my study will reveal concentrations that come close to the expected
concentrations for IV administration. This would be beneficial to the possibility of Resveratrol as
a supplement.
SECTION C: Elucidating the in vitro neuroprotective effects of Resveratrol independent of
SIRT-1
Introduction
It is a well known fact that Resveratrol is a SIRT1 inducer. SIRT1 belongs to the Sirtuin
family and has many molecular targets. Of importance in AD pathology are key features of
neurodegeneration such as oxidative stress and inflammation. Resveratrol belongs to a class of
molecules that increase SIRT1 catalytic activity [14]. Resveratrol does this by binding to the
SIRT1 N-terminal. In AD pathology, Resveratrol has been shown to counteract AB toxicity in
vitro and this has been attributed to either its natural anti-oxidant or its SIRT1 induction ability.
AB-induced cytotoxicity is mediated by reactive oxygen intermediates (ROI). Resveratrol has
been shown to attenuate this effect through NF-κBdown-regulation. This interaction with NF-κB
has a two-fold effect, the second being anti-inflammatory. NF-κB is a pro-inflammatory
transcription factor as it enhances cytokine genes which play a role in AB toxicity in AD [13]. It
is not clear whether Resveratrol will exert its effect in this regard independent of SIRT1 because
SIRT1 also de-acetlyates the RelA/p65 subunit of NF-κB which regulates the NF-κB pathway.
Some studies have shown that Resveratrol also increased endogenous free-radical scavengers
[14] which would contribute to its anti-oxidant role but SIRT1 is still a confounding variable.
The positive effects of Resveratrol in AD pathology are not restricted to oxidative stress and
inflammation but this will be the focus of this phase of my research. This phase will be an in
vitro study, in which I will observe the effects of Resveratrol in an SIRT1-inhibited environment.
Another issue with Resveratrol is the idea that it may exhibit hormetic characteristics [23].
Hormesis refers to the possibility that some of these substances may produce stimulating or
favorable effects at low doses and toxic effects at higher doses. This has not been confirmed in
Resveratrol as there have been a wide range of effects seen in different studies using
concentrations [18]. To investigate this issue, I will use a range of concentrations that includes
those of the majority in the literature.

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Experimental Procedures:
Trans-Resveratrol (3,5,4'-trihydroxy-trans-stilbene; about 99% purity) will be used in this
phase of my study. Cell cultures used will be Rat pheochromocytoma PC12 cells. There will be 3
study groups; (a) Resveratrol, (b) SIRT1-inhibited Resveratrol, and (c) Control group. The
control group will not be administered Resveratrol. As PC12 cells already express SIRT1 in their
cytoplasm [24], it will not be induced in any of the groups. The SIRT1 inhibitor indole (2-
Chloro-5,6,7,8,9,10-hexahydro-cyclohepta[b]indole-6-carboxylic Acid Amide) will be used in
group (b) [25]. Beta-amyloid (AB1-42) will be used to induce AD-like cytotoxicity. MTT 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] will be used in culture analysis.
All groups of PC12 cells will be treated with AB1-42 for 24 hours. Group (a) will receive
AB in the presence of Resveratrol alone while Group (b) will receive it in the presence of
Resveratrol and the indole. Group (c) will only receive AB treatment. After treatment, the
cultures will be fractioned for analysis. An MTT reduction assay will be performed on all groups
to give an idea of the redox state of the cells. Mitochondrial membrane potential analysis via the
cationic probe JC-1 [11] will be run to observe the dissipation of membrane potential. Net
intracellular ROI accumulation will be measured using DCF-DA (a fluorescent probe). Another
fraction will undergo Western blotting (SDS-polyacrylamide gel electrophoresis) to detect pro-
apoptotic Bax and anti-apoptotic Bcl-XL proteins. These proteins will be quantified by
densitometric analysis and their ratio is a measure of cell survival/death. Finally the activation of
NF-κB will be assessed by an Electrophoretic Mobility Shift Assay (EMSA) using an
oligonucleotide containing a consensus NF-κB binding element.
The above procedure will be repeated and adjusted accordingly for Resveratrol concentrations of
25, 50 and 100µM.
Expected Outcomes: In the MTT assay, I predict that Resveratrol will exert cytoprotective
effects (Fig. 4 Appendix). In terms of suppression of AB induced membrane potential
dissipation, Resveratrol will have a positive effect. It is also expected that ROI accumulation as
well as the Bax:Bcl-XL ratio will be decreased in the presence of Resveratrol (Fig. 5 Appendix).
Finally DNA-binding activity of NF-κBis expected to be inhibited by Resveratrol (Fig. 6
Appendix). It is expected that these combined cytoprotective effects of Resveratrol in the indole
intervention group will be less than in the Resveratrol-only group. However, I hypothesize that
these effects will be significant compared to the control group and will increase in a dose-
dependent manner.
Anticipated Problems and Solutions: The potential problems with this study lie in the in vivo
application of the findings. Much like this study, most cell culture or animal studies have been
conducted short term [4]. This is not very effective because in clinical trials, patients already
suffer from extensive neuronal loss and damage. Moreover, there haven’t been any convincing
studies in humans yet [16]. More long term studies are needed to show benefits of Resveratrol in
slowly developing AD and to aid the transition from in vitro to in vivo. It is my hope that the
findings of this study will provide more understanding about Resveratrol for such studies.

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CONCLUSION
This proposed study is biphasic. The in vivo phase will compare intravenous and oral
Resveratrol bioavailabilities and explore the possibility of reversing Resveratrol metabolism at
the required site for Alzheimer’s disease. The in vitro phase will elucidate the SIRT1-
independent neuroprotective mechanisms of Resveratrol in AB-induced stress; a model that
mimics AD pathology. The findings of this study will not only show that Resveratrol can act
independent of SIRT1 in a dose-dependent manner but will set the pace for future research into
administration of Resveratrol as a supplement.
References
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14. Albani D. et al (2010) Neuroprotective properties of resveratrolin different
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APPENDIX
Fig 1: Proposed Mean peak plasma
resveratrol concentrations (Cmax)
in mice who received single oral doses of 25
to 5000 mg. IV dose is expected to have a
steeper gradient. Extracted from Walle
Fig 2: LC/radioactivity tracings with fraction collection of
a 0–12-h urine extract after a 25-mg oral dose of
Resveratrol M1–M5 are glucuronides and sulfate
conjugates. RV is resveratrol [Walle].
Proposed IV doses are expected to a higher RV peak and
lower M1-5 peaks. The same is expected for HNPC rats.
HPLC in this study will be performed on plasma and doses
will not be radioactive
Fig 3: Proposed cytoprotection effect of
increased Resveratrol concentrations on PC12
cells treated with AB. AB 1-42 will be used
in this study. Graphic extracted from Jang
Fig 4: Proposed cytoprotection effect of
Resveratrol on PC12 cells treated with AB 1-42.
Bottom curve represents absence of Resveratrol
and upper curve represents Resveratrol
intervention. Viability will be determined by MTT
reduction assay. Graphic extracted from Jang

10. Resveratrol and Alzheimer’s Disease Olarewaju2011
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APPENDIX
Fig 5: Proposed effect of resveratrol on the
levels of Bcl-XL (upper panel) and Bax (lower
panel) after AB induced stress.
Graphic extracted from Jang
Fig. 6: Proposed effect of increased
resveratrol on AB-induced DNA binding
activity of NF-kB. Nuclear extracts will be
subject to EMSA.
Lane 1: Probe only
Lane 2: Control
Lane 3: AB1-42 only
Lane 4: AB1-42 + Resveratrol (5mM)
Lane 5: AB1-42 + Resveratrol (25mM)
Graphic extracted from Jang