Pathological conditions associated with abnormal folate status range from genetic to acquired disorders, highlighting the importance of this vitamin in key physiological processes in the CNS (Djukic, 2007
; Obeid et al., 2007b
). Because regulation of folate metabolism is highly complex, CNS folate deficiency or impaired availability can occur in the settings of normal or decreased systemic folate levels. Both cause altered methyltransferase-catalyzed reactions, leading to defects in amino acid metabolism, phospholipid and neurotransmitter biosynthesis, DNA repair and gene expression. In cultured cells, folate deficiency inhibits phosphatase activity (Chan et al., 2008
), and folate antagonists induce PP2A demethylation (Yoon et al., 2007
). Methylation differentially modulates the affinity of PP2A core enzyme for specific regulatory subunits, and is essential for ABαC formation (Janssens et al., 2008
). Yet, the regulatory mechanisms underlying the interplay between LCMT-1, PME-1 and PP2A, and their physiological significance for neuronal homeostasis remain essentially unknown. Using cultured neuroblastoma cells, we show that the major pathway by which folate deficiency induces tau phosphorylation and cell death involves downregulation of LCMT-1 and subsequent loss of ABαC. Our experiments indicate that folate deficiency does not demethylate pre-existing PP2A holoenzymes, in agreement with earlier in vitro
studies suggesting that binding of B subunits to the methylated core enzyme prevents demethylation by PME-1 (Tolstykh et al., 2000
). Rather, folate deprivation induced the de novo
accumulation of PP2A enzymes in an unmethylated state. This is in line with earlier studies of PP2A biogenesis proposing that ABαC holoenzyme assembly requires pre-activation of inactive PP2A by PP2A phosphatase activator (PTPA) and sequential methylation by LCMT-1 (Fellner et al., 2003
; Hombauer et al., 2007
). Our data suggest that folate starvation precludes the methylation of newly-synthesized PP2A enzymes by: 1) Inhibiting LCMT-1 activity towards PP2A as a result of decreased SAM/SAH ratio; and 2) Inducing LCMT-1 downregulation through mechanisms that remain to be elucidated. As dwindling pools of methylated C became available for Bα binding, Bα subunits that are unstable as monomers become targeted for degradation (Janssens et al., 2008
). Accordingly, the sizeable accumulation of demethylated C was associated with Bα downregulation in cellular and brain tissue extracts. Ultimately, Bα downregulation led to N2a cell death, as reported in HeLa cells (Lee and Pallas, 2007
). Knockdown of LCMT-1 in N2a cells substantially reduced PP2A methylation, leading to Bα downregulation and cell death, as reported in Hela and COS7 cells (Longin et al., 2007
; Lee and Pallas, 2007
). Conversely, methylation likely promotes ABαC stabilization (Tolstykh et al., 2000
), and methylated C and Bα expression levels increased in N2a-LCMT1 cells. Downregulation of demethylated C pools was observed in both N2a-LCMT1 and N2a-Bα cells. Thus, shifting intracellular PP2A composition towards preferential enrichment in methylated ABαC isoforms protects PP2A against demethylation.
Remarkably, overexpression of either LCMT-1 or Bα was sufficient to protect cells against folate deficiency-mediated tau phosphorylation and cell death. In this context, it is noteworthy that in yeast, overexpression of the Bα homolog, cdc55
, reverses at least one phenotype induced by deletion of the LCMT-1 homolog, PPM1
(Wu et al., 2000
). It is well established that LCMT-1-mediated methylation regulates ABαC formation. Our data provide surprising new evidence for the influence of Bα bioavailability on LCMT-1 regulation. Bα overexpression prevented the loss of LCMT-1 induced by folate starvation, and Bα knockdown promoted LCMT-1 downregulation. Thus, ABαC holoenzymes likely form a close complex with, or stabilize LCMT-1 enzymes. Indeed, while demethylated C was enriched with PME-1 in the nucleus as previously observed in Hela cells (Longin et al., 2008
), methylated ABαC and LCMT-1 co-localized in the cytosol and other subcellular structures of N2a cells (not shown). The differential spatial regulation of PME-1/demethylated and LCMT-1/methylated PP2A pools may explain why silencing PME-1 is not equivalent to overexpressing LCMT-1 in our cellular assays.
Significantly, overexpression of either LCMT-1 or Bα induced the downregulation of demethylated C and PME-1 in N2a cells cultured in NF medium, an effect that was further accentuated in FD medium. Conversely, PME-1 accumulated in folate-starved cells following LCMT-1 or Bα knockdown. PME-1 induces PP2A inactivation and demethylation (Xing et al., 2008
). It forms a stable complex with and sequesters inactive PP2A (Ogris et al., 1999
; Longin et al., 2004
; Hombauer et al., 2007
). Our data further point to a role for PME-1 in regulating PP2A turnover and protecting demethylated C pools against degradation. They also suggest that endogenous PME-1 expression levels become readjusted in response to detrimental fluctuations in demethylated C concentrations.
There is increasing evidence linking alterations in one-carbon metabolism with PP2A deregulation and tau phosphorylation. Downregulation of LCMT-1 and PP2A methylation correlate with enhanced tau phosphorylation in hyperhomocysteinemic mice (Sontag et al., 2007
). Injection of Hcy into rat cerebral ventricles induces C subunit downregulation and tau phosphorylation (Luo et al., 2007
). Vena caudalis Hcy injection for 2 weeks inactivates PP2A, and increases PME-1 expression and tau phosphorylation in the hippocampus without affecting LCMT-1 expression (Zhang et al., 2007
). In cultured cells, folate deficiency promotes tau phosphorylation by increasing Hcy levels and inhibiting Ser/Thr phosphatase activity (Chan et al., 2008
). We observed that folate deficiency-mediated tau phosphorylation and toxicity were associated with LCMT-1 and Bα downregulation, but these effects could not be rescued by decreasing intracellular PME-1 levels. Although we cannot exclude the possibility that PME-1 expression and activity are regulated by Hcy, our results strongly suggest that folate deficiency regulates PP2A methylation and tau phosphorylation via a pathway primarily involving SAH-induced LCMT-1 inhibition and/or downregulation. Since Hcy likely becomes rapidly oxidized into neurotoxic homocysteic acid upon injection, additional mechanisms like oxidative stress may contribute to the deregulation of PME-1, PP2A and tau in the studies by Zhang et al. (2007)
Tau phosphorylation increases in mice fed a diet combining folate and vitamin E deficiency with iron supplementation (Chan and Shea, 2006
), but the contribution of vitamin deprivation and oxidative stress in tau changes cannot be distinguished. Here, we demonstrate that dietary folate deficiency alone affects the regulation of methylation metabolites, PP2A and tau in mouse brain, in a region-specific manner. Under our experimental conditions, the extent of PP2A demethylation in response to dietary changes was the most significant in the cortex and cerebellum. It was associated with a significant decrease of LCMT-1 and Bα expression. In agreement with the observation that ABαC is a predominant tau phosphatase (Sontag et al., 1996
), enhanced tau phosphorylation only occurred under conditions where Bα expression levels decreased below a certain threshold, relative to basal levels. Compared to other regions examined, folate deprivation did not significantly affect the SAM/SAH ratio and PP2A methylation in the striatum. The high demand for methylation reactions by catecholamine-O-methyltransferase in dopaminergic terminals probably differentially affects striatal SAM metabolism (Zhu, 2002
). Compared to other brain areas, the striatum is also highly enriched in Bα mRNA and proteins (Strack et al., 1998
), which may protect neurons against folate starvation-induced PP2A demethylation. While LF and FD diets decreased the SAM/SAH ratio and PP2A methylation levels in the mid brain, these effects were relatively modest compared to the cortical and cerebellar regions. In response to the FD diet, there was no marked loss of LCMT-1 and Bα, and little changes in tau phosphorylation in the mid brain. Basal methylated PP2A levels were lower in the mid brain than in other regions, which may render PP2A pools in this region less sensitive to SAH fluctuations. It is likely that compensatory or other mechanisms, e.g. increase in LCMT-1 and/or Bα expression, and activation of signaling pathways leading to inhibition of tau kinases, could be triggered in response to folate deficiency and protect selective neurons against the loss of ABαC and/or tau phosphorylation. For instance, tau phosphorylation is controlled by the balance between PP2A and glycogen-synthase kinase 3β activities (Meske et al., 2008
). These protective mechanisms could operate in a time-dependent manner, and specific brain regions and neuronal cell populations may be able to cope better than others with low folate-induced toxicity.
In conclusion, our studies suggest that LCMT-1 is a critical intermediate of folate’s role in the CNS. Notably, the extent of the downregulation of LCMT-1 and Bα correlates with the severity of phosphorylated tau pathology in AD (Sontag et al., 2004a
). Our cellular and mouse data reinforce the strong connection between LCMT-1-dependent ABαC holoenzyme formation/stabilization and tau regulation. Thus, offsetting the neuronal loss of LCMT-1 or Bα could be a valuable therapeutic approach for tauopathies and folate-dependent CNS disorders.