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Identification of Folate-sensitive signaling pathway in the Central Nervous System Shani Blumenreich , 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-CH 3 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 Mean tHcy [μM] 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 Relative to Young Control 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 Relative to Young Control 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 Old Relative to Young Control 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 Relative to Control VEGFR2 a a b 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Control FD FDM Relative to Control 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
