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Pomegranate Seed oil nanoemulsions for the prevention and treatment of neurodegenerative diseases: The case of genetic CJD

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Pomegranate seed oil nanoemulsions for the prevention and treatment of
neurodegenerative diseases: the case of genetic CJD
Michal Mizrahi, Bsc
a,1
, Yael Friedman-Levi, PhD
a,1
, Liraz Larush, PhD
b
, Kati Frid, Bsc
a
,
Orli Binyamin, MSc
a
, Dvir Dori, MD
a
, Nina Fainstein, PhD
a
, Haim Ovadia, PhD
a
,
Tamir Ben-Hur, Md, PhD
a
, Shlomo Magdassi, PhD
b
, Ruth Gabizon, PhD
a,
a
Department of Neurology, The Agnes Ginges Center for Human Neurogenetics, Hadassah University Hospital, Jerusalem, Israel
b
Casali Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel
Received 8 November 2013; accepted 24 March 2014
Abstract
Neurodegenerative diseases generate the accumulation of specific misfolded proteins, such as PrP
Sc
prions or A-beta in Alzheimer's diseases,
and share common pathological features, like neuronal death and oxidative damage. To test whether reduced oxidation alters disease manifestation,
we treated TgMHu2ME199K mice,modeling for genetic prion disease, with Nano-PSO, a nanodroplet formulation of pomegranate seed oil (PSO).
PSO comprises large concentrations of a unique polyunsaturated fatty acid, Punicic acid, among the strongest natural antioxidants. Nano-PSO
significantly delayed disease presentation when administered to asymptomatic TgMHu2ME199K mice and postponed disease aggravation
in already sick mice. Analysis of brain samples revealed that Nano-PSO treatment did not decrease PrP
Sc
accumulation, but rather reduced lipid
oxidation and neuronal loss, indicating a strong neuroprotective effect. We propose that Nano-PSO and alike formulations may be both beneficial
and safe enough to be administered for long years to subjects at risk or to those already affected by neurodegenerative conditions.
© 2014 Elsevier Inc. All rights reserved.
Key words: Neurodegeneration; Oxidation; Prion; PSO; Nanoparticles
Background
Neurodegenerative diseases are late onset fatal disorders that
affect large numbers of individuals in our society.
1
Since clinical
signs typically present after considerable irreversible loss of brain
cells have occurred, therefore there is a clear unmet need not
only for delaying disease progression in diagnosed patients but also
for preventing disease manifestation in subjects at risk. Ongoing
efforts in the search of disease modifying agents are mainly
focused on screening for molecules that can dismantle or inhibit the
formation of misfolded keydisease proteins aggregates,
2,3
which individually characterize each of these conditions, such as
Amyloid βin Alzheimer's disease (AD), αsynuclein in Parkinson's
disease (PD) and PrP
Sc
in prion diseases such as Creutzfeldt Jacob
disease (CJD),
4,5
considered the hallmark of neurodegeneration.
6,7
A complementary concept would be to look for therapeutic
targets common to all neurodegenerative diseases, as is the case
for sensitivity to oxidative stress.
8
In fact, most of the aggregated
key disease proteins mentioned above are oxidized,
9,10
and in
prion diseases oxidation of Met residues in PrP helix 3 precedes
the acquisition of protease resistance by this protein.
11
Also brain
lipids are oxidized in these and other brain diseases,
12,13
suggesting lipid oxidation may play an important role in the
pathogenesis of neurodegenerative diseases.
14-16
Indeed, oxidized
phospholipids generate compounds such as 4-oxo-2-nonenal and
acrolein, which are predominantly toxic to braincells.
17
Consistent
with this, we propose to investigate whether safe α-oxidant
reagents could be beneficial for individuals at risk of developing
neurodegenerative conditions, constituting today a large fraction of
the world population.
1,18-22
To this effect, we tested whether administration of pomegranate
seed oil (PSO), either in its natural form added to food or as a water
Nanomedicine: Nanotechnology, Biology, and Medicine
xx (2014) xxxxxx
nanomedjournal.com
Funding: This project was funded by a grant from the Israel Science
Foundation (ISF) and by the Agnes Ginges Center. At present time, a
commercial entity (Granalix) is being formed based on the presented results
Corresponding author. Department of Neurology, Hadassah University
Hospital, Jerusalem, Israel.
E-mail address: gabizonr@hadassah.org.il (R. Gabizon).
1
Equal contribution.
Please cite this article as: Mizrahi M., et al., Pomegranate seed oil nanoemulsions for the prevention and treatment of neurodegenerative diseases: the case
of genetic CJD. Nanomedicine: NBM 2014;xx:1-11, http://dx.doi.org/10.1016/j.nano.2014.03.015
http://dx.doi.org/10.1016/j.nano.2014.03.015
1549-9634/© 2014 Elsevier Inc. All rights reserved.
soluble Nano-emulsion, can delay the clinical advance and
ameliorate prion and neurodegeneration pathological features in
TgMHu2ME199K mice,
23
which model for genetic CJD (gCJD)
linked to the E200K PrP mutation.
24
As of today, therapeutic
intervention in all forms of human prion diseases had failed.
25-28
PSO comprises a unique component, punicic acid (PA), a
conjugated polyunsaturated fatty acid considered as one of the
strongest natural antioxidants.
29
Unsaturated fatty acids such
as linoleic acid, similar to PA, were shown to readily cross the blood
brain barrier (BBB).
30-32
PA is present only in PSO (60-80%) and
in Trichosanthes kirilowii (40%),
33
and was effective in protecting
tissue lipid profiles in inflammatory disease models.
34
PSO
lack of toxicity and partial bioavailability was already established
in humans.
33
An additional antioxidant, β-sitosterol, which
was demonstrated to accumulate in the plasma membrane of
brain cells,
35
is present in PSO at significantly higher concentra-
tions as compared to oils from other plants,
36
indicating PSO may
constitute a natural compound with stronger antioxidant activities
than its individual components.
To increase the bioavailability and activity of PSO, we
generated water soluble nanoemulsions hereby denominated
Nano-PSO.
37
This approach, as is the case for delivery systems
such as phospholipid micelles or nanoparticles,
38-40
may change
the target and distribution of the oil components between different
organs, thereby enabling a longer circulation which may increase
the levels of PA available to pass the BBB.
TgMHu2ME199K mice
23
express human-mouse chimeric
E199K PrP on a null (for homozygous) or a wt PrP (for
heterozygous) background. Mice from both lines suffer from
progressive neurological symptoms as early as 5-6 months of age
and deteriorate to a terminal condition several months thereafter,
concomitant with the accumulation of a truncated form of PK
resistant.
23,41
TgMHu2ME199K mice exhibit typical pathological
features of human CJD and of general neurodegeneration.
21
This
model therefore represents the most stringent challenge for candidate
therapies in neurodegenerative diseases.
We show here that administration of PSO significantly
delayed disease onset in TgMHu2ME199K mice, constituting a
proof of concept that a natural antioxidant may fight neurode-
generation. Nano-PSO delayed disease onset and progression in
a considerably faster mode and lower dose than natural PSO.
Most important, it was only Nano-PSO and not PSO that could
prevent further advance of disease when administrated to already
sick TgMHu2ME199K mice. No aberrant side effect was observed
in the time frame (months) and PSO doses used in these experiments.
Analysis of brain samples revealed that while accumulation of PK
resistant PrP was not affected by PSO formulations, brains from
treated mice exhibited a strong neuroprotection effect, as seen by
decreased lipid oxidation and neuronal loss, as well as increased
synapthophysin expression and neurogenesis.
Methods
Animal experiments
All animal experiments were conducted under the guidelines
and supervision of the Hebrew University Ethical Committee,
which approved of the methods employed in this project
(Permit Number: MD-11746-5).
Treatment of TgMHu2ME199K mice
PSO and Nano-PSO were administrated to TgMHu2-
ME199K mice modeling for E200K CJD expressing human-
mouse chimeric E199K PrP on a null (for homozygous) or a wt
PrP (for heterozygous) background,
23
as described in Table 2.
In the PSO experiments,
1-3
mousepelletedfeedtowhichPSO
was added as described below was unlimitedly administered to
young and older mice as applicable for as long as described in
the table. Nano-PSO was administered either by gavage 5 times
aweekasinexperiment4(150μl/day), or by adding Nano-PSO
to the mice drinking water (experiment 5). At the end of the
experiments, mice were sacrificed and their brains processed
for pathological and biochemical experiments.
Mice scoring for disease signs
TgMHu2ME199K mice were followed twice a week for the
appearance of spontaneous neurological disease. Mice were
scored for disease severity and progression according to the scale
described here and in Table 1. No clinical signs: 0; Initial hind limbs
weakness = 1; Partial hind limbs weakness = 1.5; Significant hind
limb/s weakness or paralysis = 2; Significant hind limb/s weakness
or paralysis with significant legs clasping = 2.5; Full paralysis in one
limb = 3; Full paralysis in one limb, and significant weakness at the
other hind foot = 3.5; Full paralysis in both limbs = 4; Death = 5.
Mice were sacrificed at designated time points when required,
according to the ethical requirements of the Hebrew University
Animal Authorities when they were too sick or paralyzed to reach
food and water, or after losing 20% body weight.
Preparation of PSO-enriched food
1 kg of mouse pelleted food (Harlan, Taklad) was dissolved in
water and subsequently supplemented with 25 ml of PSO (Flavex,
Germany). Mixture was next reassembled and dehydrated as pellets.
Preparation of O/W pomegranate oil nanoemulsion by sonication
A nanoemulsion with 10.8% oil fraction was prepared as
follows: 1.56 g of Pomegranate oil, 0.65 g of Tween 80, and
0.39 g of glyceryl monooleate were mixed by a magnetic stirrer
for 20 min. 2.5 g from the above mixture was mixed by a
magnetic stirrer with 0.277 g of glycerol, for 15 min. 2 g of the
glycerol mixture was then added drop wise to 8 g of deionized
Table 1
Score of disease signs.
Score Disease signs
1 Initial hind limb weakness
1.5 Partial hind limb weakness
2 Significant hind limb weakness/paralysis
2.5 +Legs clasping
3 +Full paralysis in one limb
3.5 +Significant weakness at the other hind foot
4 Full paralysis in both limbs
5 Death
2M. Mizrahi et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2014) xxxxxx
water. A crude white emulsion was obtained. At the second stage
this crude emulsion was sonicated using a horn sonicator
(model Vibra-Cell, Sonics & Materials Inc., USA) for 10 min at
750 W. The samples were cooled in an ice water bath during the
sonication process. A bluish nano emulsion was obtained.
Dynamic light scattering
Droplets size measurements were performed with a Zetasizer
Nano-S (Malvern Instruments Ltd., Worcestershire, UK). Size
measurements were performed in triplicates after dilution of the
emulsion in water. The coarse white emulsion droplets size was in
the range of few microns. The average droplets size of the O/W
nano emulsion used in these experiments was 135 ± 12 nm.
Statistical analysis
The survival curves were compared using the KaplanMeier
analysis with log rank test calculating Χsquares on one degree of
freedom, Pvalues and medians were calculated and are stated
through the manuscript.
The clinical score severity curves in mice were compared
between control and experimental groups using MannWhitney
test (two tailed) and performed on the means + SEM (number of
mice in each experimental group are detailed in Table 2).
Western blot analysis
Brains from TgMHu2ME199K mice at the designated end point
of the experiments were homogenized at 10% (W/V) in 10 mM
TrisHCl, pH 7.4 and 0.3 M sucrose. For Proteinase K digestions,
30 μl of 10% brain homogenates extracted with 2% sarkosyl on ice
was incubated with 40 mg/ml Proteinase K for 30 min at 37 °C.
Samples were boiled in the presence of SDS, subjected to SDS
PAGE and immunoblotted with α-PrP pAb RTC.
11
Immunocytochemistry
Four μm thick sections of formalin fixed, paraffin
embedded brains of treated and untreated TgMHu2ME199K
mice were evaluated for the levels of oxidized phospholipids
with EO6 mAb (Avanti) and for the levels of neuronal synapses
with an α-Synaptophysin pAb (Novus).
Neurogenesis
For identifying proliferating brain cells (neurogenesis),
10 months old wt, as well as Nano-PSO treated and untreated
TgMHu2ME199K mice were injected intraperitoneally with
Bromodeoxyuridine (BrdU, Sigma-Aldrich, 50 μg/1 g body
weight) for 7 consecutive days. Subsequently, mice were
anesthetized with a lethal dose of pentobarbital and brains were
perfused via the ascending aorta with ice-cold PBS followed
by cold 4% paraformaldehyde. Tissues were deep frozen in liquid
nitrogen, and next serial 10 μM coronal sections were immuno-
stained for BrdU (rat α-BrdU, Serotec) as previously described.
42
Results
Delay of disease onset following administration of PSO in food
to young and asymptomatic TgMHu2ME199K mice
Groups of asymptomatic TgMHu2ME199K mice
(TgMHu2ME199K/wt and TgMHu2ME199K/KO),
23
as well
as 7 months old TgMHu2ME199K/KO mice already presenting
significant neurological signs (see Table 1 for description of
disease scores, and Table 2 for details about the experimental
groups) were fed either with regular rodent food or with food
enriched with pomegranate seed oil (PSO) at a concentration
of 25 ml oil/kg. Since mice consume about 3-4 g of food/day,
we may assume treated mice received about 100 μlofPSOper
day or 700 μl of PSO/week. Disease progression in each
TgMHu2ME199K mouse was evaluated by frequent scoring of
clinical signs, and by calculating twice a week the average of
group scores in the treated animals as compared to untreated
littermate groups generated from the same male and several of
its sibling females. Administration of PSO in food to young
TgMHu2ME199K mice was initiated just before they reached
3 months of age, the first time point in which PK resistant PrP can
be easily detected in the still asymptomatic TgMHu2ME199K
mice.
23,41
Controls and treated mice from the same experiment
were sacrificed simultaneously (at 270 days of age) and thereafter
their brains processed for biochemical and pathological analysis. In
the older mice, treatment commenced at 200 days (average score
in the group = 2) and continued for 9 weeks before termination of
the experiment. No adverse effects were observed in any of
the mice following the long term administration with the oil
enriched food.
Figure 1 shows the effect of PSO administration on the clinical
presentation and advance of the disease in TgMHu2ME199K
mice. Results were similar for treatment of heterozygous and
homozygous TgMHu2ME199K mice (panels AI and BI),
consistent with our recent results indicating that wt PrP does not
participate in disease presentation in the genetic mice,
41
and show
a significant delay in disease presentation in the treated mice
(Pb0.02 for both panels Aand B, see methods for description of
statistical analysis). To better understand the meaning of this result
for medical practice, we evaluated the progression of disease for
individual mice at two clinical time points, as represented by the
percentage of mice under score 2 (Aand Bpanels II), or under
score 2.5 (Aand Bpanels III). We found that PSO administration
in food could confer a beneficial effect to most treated mice when
data was estimated for the more advance stage, as depicted by the
2.5 score (difference in medians between treated and untreated
groups were 60 and 80 days (panels Aand B)(P=0.04,χ
2
=3.9;
P=0.02,χ
2
= 4.1)). At score 2, less than 50% of the mice reacted
to the treatment, as indicated by a median difference of only 10
or 20 days respectably (PN0.05). These results indicate that the
beneficial effect of PSO on disease presentation (for most mice)
Table 2
Groups in in vivo experiments.
Treatment Exp # Genetic
background
Gender Number of mice
experiment/control
Days of
treatment
PSO 1 Tg/wt Male 5/4 86-270
PSO 2 Tg/ko Male 8/7 76-270
PSO 3 Tg/ko Female 6/7 190-270
Nano-PSO 4 Tg/ko Female 6/6 70-270
Nano-PSO 5 Tg/ko Male 11/10 236-300
3M. Mizrahi et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2014) xxxxxx
can be observed only after long term consumption of the oil, and
in many cases manifest when disease is already well advanced.
As for the treatment of older and sick mice, Figure 1,Cshows no
difference in the average group scores or on the progression of
disease for individual mice between PSO treated and untreated
mice 200 days old sick mice (panels Iand II).
Overall, these findings constitute the proof of concept
that PSO may serve as an anti-prion/neuroprotective compound,
but also present the limitations of such treatment; it requires
long term administration of the anti-oxidant from the subclinical
stage, while results may be apparent only when subjects are
already affected.
Increased delay of disease onset and of disease progression
following the administration of Nano-PSO to young and old
TgMHu2ME199K mice
To generate a more effective and bioavailable formulation,
we emulsified PSO to form oil nanodroplets with an average
diameter of approximately 150 nm (see methods), hereby
denominated Nano-PSO. As stated in the introduction, such
formulations may allow for a longer circulation time of the now
dispersed drug and thereby larger activity.
43
Figure 2,Apresents
the results of an experiment comparable to those depicted in
Figure 1,Aand Bfor PSO enriched food. In this case, Nano-
PSO was administrated to TgMHu2ME199K/KO mice from
70 to 270 days by gavage (15 μlPSO/day;5daysaweekor
75 μl/week). As in the previous experiment, treated and untreated
mice were sacrificed at the same time point, to compare
pathological parameters. The dose of PSO in the Nano-PSO
formulation used in this experiment constitutes approximately
10% of the PSO administered as enriched food in the experiments
described in Figure 1.PanelAI of Figure 2 shows a significant
difference between the average group score of disease as related
to the time of Nano-PSO administration (Pb0.001). When
comparing effect on individual mice, panel A(II and III) indicate
that administration of Nano-PSO resulted in a more rapid response
to treatment as compared to TgMHu2ME199K mice treated with
PSO enriched food. Already at score 1 (panel II), which is a very
mild and early diagnosed disease condition (see Table 1) there is a
40 days difference between the median of treated and untreated
mice (P=0.002,χ
2
= 9.55). In addition, the graph in panel III
shows that while the median difference between the treated
and untreated groups at score 2 was only 65 days (P=0.0082,
χ
2
= 6.98), 50% of the treated mice never reached score 2 during
the duration of the experiment. In fact, none of the mice treated
Figure 1. Natural PSO delays disease onset in TgMHu2ME199K mice. Young TgMHu2ME199K/wt and TgMHu2ME199K/ko mice or 190 days old
TgMHu2ME199K/KO mice were treated with regular or with PSO enriched food for the designated time course (Table 2). Mice were scored for disease signs as
described in Table 1. Figures (I) in panels (A),(B) and (C): Average group score as related to age of mice. Figures (II) in panels (A) and (B): % of mice under
score 2 as related to age of mice. Figures (III) in panels (A) and (B): Percentage of mice under score 2.5 as related to age of mice. Figure (II) in panel (C):%of
mice aggravated by 0.5 score.
4M. Mizrahi et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2014) xxxxxx
with Nano-PSO reached score 2.5 in the frame of the experiment
(see Figure 2,AI), as opposed to 50 to 80% of the mice treated with
PSO food. These results indicate that Nano-PSO can delay both the
onset and the progression of disease at a much shorter time
frame and at a much lower dose than natural PSO, indicating
this and alike formulations may be suitable and effective for
treatment of humans at risk, as is the case for mutation carriers
of pathogenic PrP mutations.
44
Next, we administered Nano-PSO to TgMHu2ME199K mice
already suffering from severe disease (individual scores of mice
were between 2.5 and 3 at beginning of treatment), and then scored
the mice 2-3 times a week to establish further advance of clinical
signs. In this experiment, Nano-PSO was added to the mice
drinking water for 5 days each week (9 weeks total). The calculated
dose of PSO for each mouse was 30 μl/day or 150 μl/week.
Figure 2,Bdepicts the results of these experiments. Panel I
demonstrated a significant difference (Pb0.05) between the
average rate of disease advance between treated and untreated
mice. Indeed, Nano-PSO was able to detent clinical deterioration
for the duration of the treatment (63 days) and even to maintain
a difference in scores between treated and untreated mice after
treatment was terminated.
Panel BII presents the individual deterioration by half a score
ratio of treated and untreated mice. It shows that Nano-PSO
administration delayed the advance of disease for each treated
mouse and that the difference in the median of both curves was
again significant (42 days, P= 0.01, χ
2
= 6.635). We therefore
conclude that while natural PSO exerts a statistically significant
anti-prion activity at some conditions, it is Nano-PSO that is
suitable as a suitable therapeutic agent. A direct comparison of
the beneficial activity of PSO versus Nano-PSO at different
scores of disease is presented in Table 3.
Figure 2. Administration of Nano-PSO to TgMHu2ME199K mice significantly delays prion disease onset and progression. Young and old TgMHu2ME199K/KO
mice were treated by gavage with Nano-PSO (young, panel (A)) or by addition of the novel formulation to the drinking water (older mice, panel (B)). Mice were
scored for disease signs as described in Table 1.Figure (I) in panel (A): Average group score asrelated to age of mice.Figure (II) in panel (A): % of mice under score
1 as related to age of mice. Figure (III) in panel (A): % of mice under score 2 as related to age of mice. Figure (I) in panel (B): Average group score as related to time
of days of treatment. Figure (II) in panel (B): % of mice aggravated by 0.5 score as related to time of treatment.
5M. Mizrahi et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2014) xxxxxx
No reduction in disease related PrP in PSO/Nano-PSO treated
TgMHu2ME199K mice
Disease related PrP forms, mostly resistant to digestion by
proteinase K (PK), are the main markers of prion diseases, and
are also considered the major/only components of the infectious
prion agents.
4
In our TgMHu2ME199K model, mutant PrP can
be detected as a PK resistant and truncated form (PrP
ST
) already
at 3 months of age, when mice are still asymptomatic for the
disease. PK resistant PrP levels increase with age, in parallel with
disease progression.
41
Figure 3 shows individual brain extracts
of TgMHu2ME199K mice treated with PSO or Nano-PSO
(from experiments 2 and 4, 270 days at end point) when
immunoblotted with αPrP pAb RTC.
11
No difference in the
total PrP levels was observed in the corresponding groups before
PK digestion, indicating that administration of PSO in both forms
(panel Aas PSO, panel Bas Nano-PSO) had no effect on PrP
expression. Most important, no reduction in PK resistant PrP was
observed in the treated brains in spite of the profound difference in
disease scorebetweentreated and untreated mice (Figures 1 and 2).
Actually, the signal for PK resistant PrP even increased slightly
in some of the treated mice, as was the case for scrapie infected
mice treated with Simvastatin.
45
This implies that PSO does not
interfere with the pathological process or aberrant metabolic
pathway which leads to the accumulation of disease related PrP in
TgMHu2ME199K mice, but may rather induce neuroprotective
pathways that manifest as reduced cell death. Such a mechanism
may even explain an apparent increase in prion protein accumulation
in the treated brains.
PSO formulations protect neurons from death
Next, we immunostained paraffin embedded brain slices from
wt mice as well as from treated and untreated TgMHu2ME199K
mice with an α-synapthophysin antibody. The samples used were
from experiments 2 (PSO) and 4 (Nano-PSO), as in the
experiments described in Figure 3. Synapthophysin is an integral
membrane protein located in presynaptic vesicles.
46,47
Decreased
levels of this and other synaptic proteins are considered a general
marker of neurodegeneration
48-50
and were observed in the brains
of CJD patients, bovine spongiform encephalopathy (BSE)
infected cattle and scrapie infected mice.
51-54
Figure 4,Ashows that while synapthophysin levels were
largely reduced in the untreated TgMHu2ME199K mice, as seen
by the reduce number of brown dots, as compared to age matched
wt mice, such immunostaining was significantly restored in the
treated mice from the parallel groups for both experiments. This
indicates that early treatment with PSO formulations may prevent
not only the advance of clinical signs but also the presentation of
general neurodegenerative features of prion and other neurode-
generative diseases, as is the case of synapthophysin expression.
Next, and to further establish whether PSO formulations
can inhibit neuronal death, we counted in age matched wt,
as well as in treated and untreated TgMHu2ME199K mice frozen
sections the number of cells in the CA1 and CA3 regions of the
hippocampus, as manifested by DAPI staining. Figure 4,Bshows
the results of such an experiments for sick TgMHu2ME199K
mice treated with and without Nano-PSO (experiment 5).
Indeed, while the brains from the sick TgMHu2ME199K mice
presented significant neuronal loss as compared to those of wt mice
(see thinner blue sections and numbers in the graph bellow), such
reduction was partially prevented by the Nano-PSO treatment.
Indeed, differences between cell numbers in treated and untreated
sections, as well as between non-treated to wt mice were statistically
significant (Pb0.01 or Pb0007, respectively). Death of neurons
in the hippocampus was shown to correlate with dementia,
55
suggesting both that our TgMHu2ME199K mice model is a good
model of neurodegeneration and that Nano-PSO exerts a strong
neuroprotective affect in the most severe conditions, as represented
by treatment of already sick TgMHu2ME199K mice.
Nano-PSO may restore hippocampal neurogenesis in sick
TgMHu2ME199K mice
Numerous new studies demonstrate that during normal aging,
certain areas of the brain retain pluripotent precursors with the
capacity of self-renewal.
56,57
This feature, also known as adult
neurogenesis, may be impaired in neurodegenerative diseases
such as CJD and AD.
58,59
To evaluate levels of neurogenesis
in treated and untreated TgMHu2ME199K mice, we injected
Bromodeoxyuridine (BrdU), a synthetic analog of thymidine
commonly used in the detection of proliferating cells in living
tissues
60
to wt and TgMHu2ME199K mice of several ages as well
as to Nano-PSO treated TgMHu2ME199K mice (experiment 5).
Mice were sacrificed a week after the first injection and new
neurons in the granular zone of the dentate gyrus were detected in
frozen sections with an α-BrdU antibody. Figure 4,Cshows that at
4 months of age, when the TgMHu2ME199K mice were still
asymptomatic, there was no significant difference in the number of
new cells between their brains and those of wt mice. Contrarily, in
Table 3
Comparing the clinical effect of the effect of PSO to Nano-PSO.
Experimental group Parameter measured PSO Nano-PSO
Young, asymptomatic Δ= The difference in medians between
treated and untreated groups
Score 1 = no difference Score 1 = 40 days
Pb0.002
Score 2 = 10 days At score 2 65days
Pb0.082
Score 2.5 = 80 days
Pb0.04
No score 2.5 intreated mice
Old, symptomatic Δ= The difference in deterioration
by half a score ratio
No difference 42 days
Pb0.01
Statistically significant
6M. Mizrahi et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2014) xxxxxx
older mice already presenting signs of disease (6 and 10 months of
age) the number of new cells in TgMHu2ME199K brains was
significantly lower that the corresponding new cells in age matched
wt mice (Pb0.002), indicating reduced neurogenesis, at least
at this age, is a feature of neurodegeneration more than a property
of aging. While only marginally significant (Pb0.12), the number
of new cells in Nano-PSO treated mice was higher than in the non-
treated mice, indicating a possible additional neuroprotective
property of Nano-PSO treatment.
PSO protects cell lipids from oxidative insults
As reviewed above, lipid and protein oxidation are important
features of all neurodegenerative diseases.
13,12,61
To establish
whether Nano-PSO can reduce lipid oxidation, we immuno-
stained paraffin embedded brain slices brain from wt and form
control and treated TgMHu2ME199K mice with EO6, an anti-
oxidized phospholipids mAb
62
which was shown to detect
oxidized lipids in MS plaques.
13
In Figure 5, we show that while
we could not detect any α-EO6 immunostaining in wt mice of
different ages (no dispersed brown color or specific features),
TgMHu2ME199K mice brain sections from experiment 4
presented strong EO6 immunostaining in all cerebellar layers
(dispersed brown in general and in cerebellum's Purkinje cells).
These results are consistent with our previous findings, indicating
a large sensitivity of TgMHu2ME199K mice Purkinje cells to
disease aggravation.
63
Our results also show that Nano-PSO
treated sections present considerable lower levels of EO6 staining,
both general and cell specific, as compared to the parallel untreated
brains. In samples from experiment 5, mice treated when already
sick, we show that EO6 may also recognize star like plaques in the
brains of the untreated, but not in the treated TgMHu2ME199K
mice. These results demonstrate that lipid oxidation may be
an important feature of prion diseases and that Nano-PSO
formulations can reduce/prevent such oxidation, concomitantly
with inhibition of cell death and disease aggravation.
Discussion
A significant number of natural anti-oxidants are ubiquitously
present in a healthy human diet. Many of them, such as
Sulforaphane from Broccoli, Curcumin and EGCG from green
tea were recognized for their neuroprotective properties in cells
and tested in appropriate animal models.
64-66
However, their in
vivo activity was limited by the sub-pharmacological doses
presented in food, their poor bioavailability to humans, rapid
chemical degradation and reduced distribution to different
organs in the body, in particular the CNS. In this work, and
after PSO by itself was found to be clinically active in its natural
oil form, we made an effort to overcome such limitations by
tailoring a more active formulation in the form of Nano-PSO.
We show here that administration of PSO in food may delay the
onset of spontaneous genetic prion disease in the TgMHu2ME199K
mouse line, constituting a proof of concept that natural antioxidants
Figure 3. No reduction in disease related PrP in PSO/Nano-PSO treated Tg mice: Brain extracts of mice sacrificed at the end point of experiments 2 and 4 were
digested in the presence and absence of PK, as described in the methods, and immunoblotted with αPrP pAb RTC.
11
Panel (A): PSO treated mice, last 2 lanes
represent scrapie infected brains (RML, and wt mice). Panel (B): Nano-PSO treated mice, last 2 lanes represent scrapie infected brains (RML, and wt mice).
7M. Mizrahi et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2014) xxxxxx
may exert a beneficial neuroprotective effect. However, it was only
Nano-PSO, a novel formulation in which natural PSO was converted
into soluble nanodroplets, which generated an impressive clinical
effect at a much lower PSO dose for both prevention and treatment of
progressive prion disease in the TgMHu2ME199K mice. Our results
also show that the mechanism of activity of PSO formulations was
not prion specific, or at least not PrP specific, since the long term
administration of the oil did not affect the expression of mutant
PrP or its accumulation as a PK resistant PrP form. Rather than a
specific anti-prion effect, these PSO formulations, concomitant with
an impressive clinical outcome, presented a wide neuroprotective
effect, in the form of reduced neuronal death and lipid oxidation
and increased neurogenesis, which may be valuable also for the
treatment of an array of neurodegenerative conditions. Indeed the
combination of an unsaturated fatty acid that can most probably
cross the BBB,
30
and the increased bioavailability conferred by the
nanoparticle formulation,
37
may explain the impressive neuropro-
tective activity of Nano-PSO.
The search for disease modifiers for the treatment of
neurodegenerative diseases is focused mostly on screening for
reagents such as antibodies or small molecules that may dismantle
or inhibit the aggregation of misfolded specific key proteins, as is
the case for PrP
Sc
in prion diseases,
67
A-beta in AD and synuclein
in Parkinson's disease,
68-71
or reduce the expression of the normal
key proteins, in the form of siRNAs
72
or other molecules.
73
While
these pathways may well lead to effective drugs, we propose an
alternative approach in the form of brain protection from common
neurodegeneration features, as is the case for neuroinflammation
and oxidative stress. Indeed, Nano-PSO generated a strong clinical
effect with an excellent safety profile which may allow its
prolonged administration to individuals at risk. The formulation
described here, oil nanodroplets of a specific size, may be further
improved by optimizing particle size, Nanoemulsion ingredients,
stability features and physicochemical principles of preparation.
Pharmacokinetics experiments in progress in our laboratory may
help to fine-tune the most active formulations, before we engage in
clinical trials.
Indeed, since Nano-PSO can most probably be classified as a
safereagent, even in the levels of safety of a food supplement,
such trials may come about soon enough. A comprehensive clinical
Figure 4. Neuroprotection of PSO formulations (A) PSO and Nano-PSO restore synapthophysin expression in sick mice: Paraffin embedded brain slices of wt,
as well as PSO and Nano-PSO treated and untreated TgMHu2ME199K/KO mice (experiments 2 and 4), were immunostained with an α-synapthophysin
antibody. Magnification in all pictures is × 20. (B) Nano-PSO protects hippocampal cells from death: Frozen sections the number of cells in the CA1 and CA3
regions of the hippocampus, as manifested by DAPI staining. Statistical analysis of average from 4 mice in each group was performed by T-test.
(C) Neurogenesis in treated and untreated TgMHu2ME199K mice: Slides of mice treated with BrdU and subsequently stained with an α-BrdU antibody were
counted for reacting cells. Average number of cells in slides of 4 mice was plotted in the graph. Statistical significance was evaluated by T-test.
8M. Mizrahi et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2014) xxxxxx
trial for CJD patients was recently described.
28
While the results for
doxycycline were negative, the experimental design may be useful in
our and other cases. In addition, we speculate that once sophisticated
disease specific reagents become available, they may well be
combined with general neuroprotective agents such as Nano-PSO
for the best possible outcome in patients and at risk individuals.
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... ROT is reported to categorically produce dyskinesias and dopaminergic neuronal degeneration in the midbrain of rodents [3,25] by uncoupling complex I from the mitochondrial respiratory chain [26,27]. In contrast, Nano-PSO is said to categorically neuro-protect or neuro-restore neurological functional morphology in mouse models of Creutzfeldt-Jakob disease (n = 4 TgMHu2ME199K mice [28]; n = 6-10 TgMHu2ME199K mice [29]), multiple sclerosis (n = 6-8 female C57BL/6 mice), or Alzheimer's disease (n = 5-18 5xFAD × C57BL/6 mice [24]) [24,[28][29][30][31]. It is also claimed that Nano-PSO categorically reduces cognitive decline in patients with multiple sclerosis (Group A: n = 12; Group B: n = 10) [31]. ...
... ROT is reported to categorically produce dyskinesias and dopaminergic neuronal degeneration in the midbrain of rodents [3,25] by uncoupling complex I from the mitochondrial respiratory chain [26,27]. In contrast, Nano-PSO is said to categorically neuro-protect or neuro-restore neurological functional morphology in mouse models of Creutzfeldt-Jakob disease (n = 4 TgMHu2ME199K mice [28]; n = 6-10 TgMHu2ME199K mice [29]), multiple sclerosis (n = 6-8 female C57BL/6 mice), or Alzheimer's disease (n = 5-18 5xFAD × C57BL/6 mice [24]) [24,[28][29][30][31]. It is also claimed that Nano-PSO categorically reduces cognitive decline in patients with multiple sclerosis (Group A: n = 12; Group B: n = 10) [31]. ...
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