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Modulating PP2A Methylation using Natural Extracts
Kristen Huber1, Michael Voronkov1, Jose Fernandez1, Karl Rouzard1, Eduardo Perez1,
Maxwell Stock1, Jeffry Stock2
1 Signum Nutralogix, 11 Deer Park Drive Suite 202, Monmouth Junction, New Jersey, U.S.A.
2 Molecular Biology Department, Princeton University, Princeton, New Jersey, U.S.A.
Table 1: Fruit extracts
Fruit Extract
PPMT (+) CH3 scaffold (A) subunit !
(-) CH3 regulatory (B) subunit PP2A
holoenzyme AC core dimer Catalytic (C) subunit PME Avocado
Blueberry
Cranberry
Grapefruit (Oil)
Juniper Berry
Maqui Berry
Mulberry
Pomegranate (Juice)
Schisandra
Strawberry
Estimated IC50
(µg/mL)
>25
>50
>30
25
5
>10
>10
>27
6
>5
Table 2: Seed extracts
Seed Extract
!
Avocado
Black Raspberry
Blueberry
Celery
Coffee
Cranberry
Fennel
Grape
Guarana
Red Raspberry
Estimated IC50
(µg/mL)
6
6
8
7
6
10
1
0.4
1.5
1
Table 3: Root, bark & leaf extracts
Table 4: Other natural extracts
Natural Extract
Estimated IC50
Natural Extract
Estimated IC50
(µg/mL)
(µg/mL)
Maca Root
3
Almond
>5
Goldenseal Root
4
Hazelnut
>10
Magnolia Bark
2
Cocoa Butter
12.5
Pygeum Bark
3
Cocoa Powder
6.25
Red Raspberry Leaf
2
Echinacea Angustifolia
4
!
Echinacea
12
!
Natural Extracts Affect
on Phosphatase Activity
Interestingly, a reduced risk of neurodegeneration has been
highlighted in epidemiological studies simply by consumption of
certain common foods, such as coffee. Therefore, we sought to
determine if natural botanical extracts from common sources
are able to modulate the methylation state and thus activity of
PP2A. From this study, we have not only identified a variety of
natural extracts that are able to modulate PP2A methylation but
were able to isolate one of the components in coffee that is
responsible for its neuroprotective effects. We demonstrate that
eicosanoyl-5-hydoxytryptamide, or EHT™, conserves PP2A
methylation and thus reduces Tau and α-synuclein hyperphosphorylation, hallmarks of Alzheimer’s and Parkinson’s
diseased brains. Additionally, EHT™ was able to restore
cognitive and motor deficits in Parkinson’s and Alzheimer’s
disease animal models. EHT™ also has the added benefit of
antioxidant and anti-inflammatory properties. Modulation of
PP2A’s methylation status by natural extracts shows the value
of natural dietary components for our everyday wellbeing in
addition to expanding the view of PP2A methylation as a target
mechanism for brain health.
Specific Activity (umol/min/mg)
Figure 1: PP2A assembly and methylation.
Epidemiological evidence suggests a
O
reduced risk of neurodegerative disease HO
N
H (CH2)18CH3
with higher coffee consumption.
N
Therefore, coffee was selected for further
H
investigation. Multiple rounds of isolation
Figure 3: Structure of
eicosanoyl-5-hydroxylead to the discovery of EHT™. Not only
tryptamide (EHT™).
does EHT™ enhance PP2A methylation, a
reduction in the hyper-phosphorylated form of Tau and αsynuclein, hallmarks of Alzheimer’s and Parkinson’s disease,
was also observed by western blot analysis.
A
Control
1.0
1.0
EHT™
(0.01%)
C
WT+
Control
EHT™
(0.1%)
Demethylated
PP2A
0.8
0.8
Total
PP2A-C
0.5
*
*
3
2
0
Syn Tg +
120mg/Kg/day
EHT™
0.4
0.4
0.3
0.01
0.1
Tau
EHT™
-0-
1
0.1
10
1
100
10
100
+
+-0
+
++
10
+
+
-
+
+
+
Wt Tg Wt Tg Wt Tg
Ctrl EHT™ EHT™
12 mpk 120 mpk
80
70
60
50
40
30
20
10
0
**
GFP GFP/ I2
I2/
EHT™
EHT™
1000
1000
Tau 4R
MARK4
D
10 µM EHT
Control
EHT™
(0.01%)
D
EHT™
(0.1%)
pSer129
α-Syn
- phospho-Tau
α-Syn
HMW
- total Tau
EHT™ Exhibits Antioxidant
& Anti-inflammatory Properties
B
150
C
10
0
Figure 2: Natural extracts affect on PP2A-AC phosphatase activity.
Figure 5: EHT™’s antioxidant and anti-inflammatory properties. A) EHT™ antioxidant capacity
measured by ABTS colorometric radical scavenging. B-C) Primary microglia cultures were
treated with LPS and measured for B) iNOS expression via western blot analysis or C) nitric
oxide production via Griess Reagent System in the presence of EHT™.
EHT™ was further characterized for its antioxidant and antiinflammatory properties. EHT™ exhibited slightly weaker activity
than ascorbic acid. Furthermore, EHT™ was able to reduce
iNOS expression and nitric oxide production in in microglia.
E
*
*
50
GFP
GFP
GFP-Sig N/C
GFP/EHT I2
*
50
100
0
Figure 4: EHT™ preserves PP2A methylation
Syn I
and reduces Tau and α-syn phosphorylation
A) Dose-response curve of EHT™ using
purified PP2A AC dimer that was methylated
monomer
using LCMT1 and S-[methyl-3H]-adenosyl-Lmethionine B) Western blots of cortical tissue lysates from wild-type mice probed with
antibodies to demethylated PP2A (deMe-PP2A) and total PP2A-C subunit C) Western blot of
transfected brain slices probed with pS262 and total Tau antibodies D) Western blots of lysates
from α-Syn Tg mice probed with phospho-S129-α-Syn antibody and total α-Syn antibody (Syn I).
A
**
1
0.6
0.6
Total
Tau
30
C
*
4
Syn Tg +
Control
0.7
pS262
Tau
40
B
5
Log [EHT™] (µM)
50
As a secondary screen, select natural extracts at a
concentration of 5 µg/mL were further analyzed to ensure they
have little to no affect on PP2A’s phosphatase activity as judged
by the chromogenic substrate, p-nitrophenyl phosphate. Our
results show that while these natural extracts prevent
demethylation of PP2A, they do not have an affect on pNPP
phosphatase activity, with the exception of magnolia bark.
Okadaic acid was used as a positive control.
A
0.9
0.001
60
20
B
1.1
We then sought to determine if EHT™ would improve cognitive
and motor function in animal disease models. Male mouse pups
transgenic for human α-synuclein and brains of new born rat
pups expressing PP2A inhibitor I2 N- and C-terminal fragments
via viral transduction, were used as Parkinson's and Alzheimer’s
disease models respectively. Under these conditions, EHT™
was able to recover cognitive and motor deficits in addition to
restoring PP2A activity.
Discrimination Index (%)
A small library of approximately 30 natural extracts was created
using isopropanol based extraction methods at 65°C. These
extracts were assayed for their ability to preserve the
methylation status of PP2A AC dimer in the presence of protein
phosphatase methylesterase-1, or PME-1, using a radioactive
filter binding assay. Data were collected in triplicate and the IC50
value was determined via the four parameter logistic curve fit
using SigmaPlot graphical software. Results indicate a
distinction between extract categories where the seed and root,
bark and leaf categories consistently prevented PP2A
demethylation with an IC50 better than 5 µg/mL.
EHT™ Improves Cognitive and Motor Deficit
In Alzheimer’s and Parkinson’s Disease Models
PP2Ac
(demethylated/total)
Protein phosphatase 2A (PP2A), a major serine/threonine
phosphatase, has been implicated in a broad range of cellular
functions anywhere from development to disease. Consisting of
a scaffolding (A), regulatory (B) and catalytic (C) subunit, this
trimeric holoenzyme is highly regulated through structural
assembly, post-translational modifications and small molecule
interactions. Methylation of PP2A’s carboxy-terminal tail has not
only been implicated in modulating its activity and specificity but
it has also been shown to be of particular importance in
neurodegenerative diseases such as Alzheimer’s and
Parkinson’s disease. For this reason, modulators of PP2A’s
methylation state would be of particular importance.
Reduces Tau and α-synuclein Phosphorylation
Relative PP2A Demethylation
Abstract
EHT™ Maintains PP2A Methylation &
Nesting Score
Natural Extracts Affect
on PP2A Methylation
PP2A activity
% of control
!
N/C/Sig
I2/EHT
40
30
20
10
0
GFP
GFP/Sig
N/C
GFP GFP/EHT I2
N/C/Sig
I2/EHT
Figure 6: EHT™ enhances cognitive and motor function. A-B) Nesting behavior of wild-type
and α-synuclein mice, a Parkinson’s disease model, at 9 months of age in the presence of
EHT™ C) EHT™ prevents the cognitive impairment in rats in an I2 induced cognitive
impairment model for Alzheimer's as judged by an object location task. D) PP2A activity from
I2-N/C rats treated with EHT™ E) PP2A methylation from I2-N/C rats treated with EHT™.
Conclusion
•  Natural extracts made from seeds, roots, bark, and leaves
alter PP2A methylation status in the presence of PME-1.
•  Isolated coffee component, EHT™, exhibits preservation of
PP2A methylation, reduced Tau and α-synuclein hyperphosphorylation and improved cognitive and motor function in
Alzheimer’s and Parkinson’s disease models.
•  PP2A methylation system poses as a promising target for
neurodegenerative disease.
•  Natural dietary components exhibit value for use in brain
health.
References
1. 
2. 
3. 
Basurot-Islas et. al. Therapeutic benefits of a component of coffee in a rat model of
Alzheimer disease. Neurobiology of Aging. In Press
Lee et. al. Neuroprotective and anti-inflammatory properties of a coffee component in the
MPTP model of Parkinson’s disease. Neuropathics Jan 2013. 10(1): 143-153.
Lee et. al. Enhanced phosphatase activity attenuates α-synucleinopathy in a mouse model.
J NeuroSci, May 2011. 31(19):6963-6971.

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