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