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Identification of Folate-sensitive signaling pathway in the Central Nervous System
Shani Blumenreich𝟏, Vered Vayman, Shani Martsiano, Yaarit Biala, Nathalie Weizman,
Merav Reizman, Nur abu Ahmad and Aron Troen
The Robert H. Smith Faculty of Agriculture, Food & Environment,
The Hebrew University of Jerusalem, Rehovot, Israel. Amirim project 2015
References:
Background:
Folic acid or Vitamin B9 is well known for its important role in the nervous system development and correct
closure of the neural tube. Folate is essential for numerous functions due to its role as one carbon group donor,
Including: nucleotide synthesis, role in DNA methylation and other biological methylations[1].
Folate deficiency and elevated plasma homocysteine (which can be caused due to low levels of Folate) are
associated with increased risk of age-related cognitive decline, cerebrovascular and neurodegenerative
disease. Troen et al. previously found that methionine, a product of folate metabolism, can mitigate cognitive
and neurochemical effects of folate-deficiency in young rats, independently of homocysteine
(Troen et al, J Nutr 2008)[2] . However, as yet, these experiments have not been replicated in older rats.
Furthermore, the molecular underpinnings of this response and their downstream effects on cellular function are
unclear.
Our working hypothesis was that folate deficiency would increase the severity of brain aging, and this would be moderated by methionine.
Aim:
To identify folate- and methionine-responsive signaling pathways in brain that could mediate neurocognitive risk, and to determine their interaction with age.
• Key pathways in the brain, which are involved in cognitive decline, were affected by folate. In some cases these genes were responsive to
methionine availability, independently of homocysteine. These affects were mild (10%-20% mean fold change, relative to young control) but
significant, and may affect long-term brain health.
• We identified folate-responsive regulatory genes including members of the VEGF and Wnt signal transduction pathways, which are nececssary
for maintaining brain vascular perfusion[3].
• Young rats are more affected by diet than old rats (in terms of significant changes). To the extent this is true for humans it suggests the
importance of early intervention for the prevention of dementia.
Discussion:
Experimental design:
1. Sezgin et al. Alzheimer's disease and epigenetic diet. Neurochemistry International, October 2014; 78: p. 105-116.
2. Troen AM et al. Cognitive impairment in folate-deficient rats corresponds to depleted brain phosphatidylcholine and is prevented
by dietary methionine without lowering plasma homocysteine. The Journal of Nutrition, December 2008; 138: p. 2502-2509. 3. Zerlin M et al. Wnt/Frizzled signaling in angiogenesis. Angiogenesis, 2008; 11: p.63-69.
Vascular regulatory genes:
Angiogenesis:
Fig. 4: Decreased VCAM1 expression due to Folate Deficiency was prevented by Methionine (independent of Hcy).
Wnt signaling pathway:
Heat map for all tested genes:
Overall effect:
Results: Plasma Homocysteine:
Fig. 1: Folate deficiency elevated plasma total homocysteine and was not corrected by methionine. No difference was seen by age.
N Interaction Diet Age
19 V V V
10 - V V
29 - - V
6 - V -
37 - - -
101 19 35 58 ∑N:
Overall:
Affect of Diet:
N FDM FD C
4 a b a Folate responsive, correction
by Methionine
0 b b a
Folate responsive, without
Methionine sensitivity (possibly
Homocysteine sensitive)
9 b a a Methionine responsive
14 b a ab
Trend of change by Folate
and trend direction change
by Methionine
6 b ab a
Methionine responsive
depending on Folate
availability
1 ab b a Folate responsive, partial
correction by Methionine
34 ∑N:
Fig. 5: Decreased VEGFR2 expression due to Folate Deficiency was prevented by Methionine (independent of Hcy).
Fig. 6: Increased APC2 expression due to Folate Deficiency was prevented by Methionine (independent of Hcy).
*Groups with different letters (a,b) differ significantly (P≤0.05).
5 Methyl
Tetrahydrofolate
Methionine
Homocysteine
Cysteine
SAM
SAH
X
X-CH3
Tetrahydrofolate
5, 10 Methyl
Tetrahydrofolate
Methionine
synthase
(MS)
Folate cycle
MTHFR
MAT
Methionine metabolism
Old (18 months)
male Sprague
Dawley rats
Control Diet
(C)
Folate
deficient Diet
(FD)
Folate deficient
with
supplemental
methionine Diet
(FDM)
10 weeks
(RT Real time PCR)
nCounter® Gene
Expression,
Nanostring
technologies.
(Western blot)
Young (3 months)
male Sprague
Dawley rats
Metabolic Response:
Fig. 3: Increased MAT2a expression due to Folate Deficiency was mitigated by Methionine (independent of Hcy).
nCounter® Gene expression assay- Nanostring technologies:
Quantitative and sensitive method for measuring mRNA abundance, linearly from 1 copy to more than 300,000 copies.
0
10
20
30
40
50
Young Old
Mea
n t
Hcy
[μ
M]
Plasma Homocysteine± SE
a a
b b
b b
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Young Old
Rel
ativ
e to
Yo
un
g C
on
tro
l
Methionine synthase
Control
FD
FDM
ab a
b
Age: P.value<0.05 Diet: P.value<0.05 Age*Diet: P.value<0.05
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Young Old
Rel
ativ
e to
Yo
un
g C
on
tro
l
Methionine adenosyltransferase 2a
Control
FD
FDM
Age: P.value<0.05 Diet: P.value<0.05 Age*Diet: P.value<0.05
a
b ab
Fig. 2: Decreased MS expression due to Folate Deficiency was prevented by Methionine (independent of Hcy).
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Young OldRel
ativ
e to
Yo
un
g C
on
tro
l
VCAM1
Control
FD
FDM
Age: P.value<0.05 Diet: P.value<0.05 Age*Diet: P.value<0.05
ab a
b
0
0.2
0.4
0.6
0.8
1
1.2
Control FD FDM
Rel
ativ
e to
Co
ntr
ol
VEGFR2
a a
b
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Control FD FDM
Rel
ativ
e to
Co
ntr
ol
APC2 Mean expression ± SE
ab
a
b
*Heat map is relative to Young control.
*Genes with expression less than 100 counts were excluded.
Old control
Old FD
Old FDM
Young FD
Young FDM
Legend (Fig 1-3): Mean± SE in Old and Young Rats (n=8
per group). Different letters (a,b) differ significantly
(P≤0.05). C – Control FD – Folate Deficient FDM– Folate deficient + Methionine
Legend (Fig 4): Mean± SE in Old and Young
Rats (n=8 per group). Legend (Fig 5-6): Mean± SE in Old and
Young Rats (n=16 per group).
Different letters (a,b) differ significantly
(P≤0.05). C – Control FD – Folate Deficient FDM– Folate deficient + Methionine