S-adenosylmethionine/homocysteine cycle alterations modify DNA methylation status with consequent deregulation of PS1 and BACE and beta-amyloid production

Abstract

Few diseases are characterized by high homocysteine (HCY) and low folate and vitamin B12 blood levels. Alzheimer disease (AD) is among these. It has already been shown that DNA methylation is involved in amyloid precursor protein (APP) processing and β-amyloid (Aβ) production through the regulation of Presenilin1 (PS1) expression and that exogenous S-adenosylmethionine (SAM) can silence the gene reducing Aβ production. Here we demonstrate that BACE (β-secretase), as well as PS1, is regulated by methylation and that the reduction of folate and vitamin B12 in culture medium can cause a reduction of SAM levels with consequent increase in presenilin1 and BACE levels and with increase in Aβ production. The simultaneous administration of SAM to the deficient medium can restore the normal gene expression, thus reducing the Aβ levels. The use of deprived medium was intended to mimic a mild nutritional deficit involved in the onset of AD.

Introduction

Elevated HCY plasma level is a serious risk factor for the onset of AD (Seshadri et al., 2002), as well as for cardiovascular pathologies. Moreover, SAM levels decrease in AD patients and, generally, with aging (Beyer et al., 2003, Bottiglieri and Hyland, 1994, Mizrahi et al., 2003, Morris, 2003, Morrison et al., 1996). These data lead us to hypothesize that alterations in SAM/HCY cycle (producing HCY accumulation) are responsible for decreased SAM levels and, in turn, for reduced DNA methylation (Medina et al., 2001, Miner et al., 1997). HCY accumulation causes the accumulation of S-adenosylhomocysteine (SAH) because of the reversibility of the reaction converting SAH to HCY and adenosine (Ado); the equilibrium dynamics favors SAH synthesis. The reaction proceeds in the hydrolytic direction only if HCY and Ado are efficiently removed (Fig. 1). SAH is a strong DNA methyltransferases inhibitor, which reinforces DNA hypomethylation (Chiang et al., 1996, Clarke et al., 1998, Lu, 2000, Weir and Scott, 1999).

The great relevance of Aβ production in AD is largely documented and accepted (Chaney et al., 2003, De Strooper and Konig, 1999, Selkoe, 2000) as well as the importance of reducing this protein in the therapy (Dodart et al., 2002, Haass, 2002). On the other hand, it is also well understood that a complete elimination is not possible because of the physiological activity of Aβ and APP (Atwood et al., 2003, Kontush et al., 2001, Munch and Robinson, 2002). Particular attention has been dedicated to the regulation of γ- and β-secretases, in order to reduce the amyloidogenic cutting of APP (Ohno et al., 2004, Selkoe, 2001). APP could be processed by γ- (PS1 and PS2) and α-secretases (ADAM10 and TACE) producing non-amyloidogenic peptides, or by γ- and β-secretases (BACE) producing the Aβ fragments (Evin et al., 2003, Marlow et al., 2003, Nunan and Small, 2000). The balance between the different secretase activities could be very important in the maintenance of the physiologic Aβ levels (De Strooper, 2000, De Strooper et al., 1998, Haass and De Strooper, 1999, Li et al., 2000, Plant et al., 2003, Wolfe et al., 1999). Secretase activities also could not be completely eliminated since they are implicated in several cellular functions (like Notch1 processing) besides Aβ processing (Cai et al., 2001, Hardy and Selkoe, 2002, Shen et al., 1997, Sinha et al., 1999, Wong et al., 1997).

In order to verify the correlation between the SAM/HCY cycle, DNA methylation and AD, we prepared a medium deprived of folate and vitamin B12 to study DNA methylation, gene expression and protein synthesis on SK-N-SH and SK-N-BE neuroblastoma cells. Folate and vitamin B12 are essential for the transformation of HCY to SAM; the failure of this step leads to decreased SAM synthesis, accumulation of HCY and methyltransferases inhibition. As previously shown, exogenous SAM can inhibit the demethylation of a gene promoter and modulate PS1 expression (Fuso et al., 2001, Scarpa et al., 2003); in this case, we administrate SAM to revert the hypothesized demethylating effect of medium deprivation.