Overexposure to Mn is considered as a risk factor of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, characterizing as motor and cognitive performance . Methionine, one of sulfur-contained essential amino acids, functions as a precursor for protein synthesis, methyl donator. No effective intervention has been applied to treat neurodegenerative impairment relative to Mn exposure. In the present study, we evaluated the molecular basic and potential therapeutic of regulation PP2Ac demethylation on Mn-induced neurotoxicity in vivo and vitro. We found methionine supplementary ameliorates the cognitive deficits in rats and N2a cells cytotoxicity induced by Mn. The protection of methionine, which scavenges ROS and donates methyl contributes to down regulate PP2Ac demethylation, thus decrease tau hyperphosphorylation. Furthermore, ABL127 administration reverses Mn cytotoxicity with down-regulation PP2Ac methylation and tau hyperphosphorylation accumulation. To our knowledge, our study firstly hightlights that PP2Ac is likely a prospective therapeutic target in Mn neurotoxicity.
Accumulating evidences demonstrated that Mn overexposure induced school-age students cognitive deficit as well as cells lesion [23, 24]. Methionine level was declined in animal brain after manganese treated . Thus, methionine is involved in neurotoxicity induced by Mn. In present study, methionine improved learning and memory impairment in animal and protected N2a cells from apoptosis caused by Mn in vivo. However, methionine protecting cytotoxicity induced by Mn has not been fully elucidated. Considering methionine and its sequence metabolisms, it is not surprising that methionine exerts oxidative defense, protein structure and cellular regulation. Our investigation indicated that PP2Ac are likely a prospective therapeutic target for neurotoxicity induced by Mn over-exposure, whose methylation is critical for regulating tau hyerphosphorylation. It seemed that oxidative stress instead of one-carbon cycle governed the methylation of PP2Ac in N2a exposure to Mn. Both methionine and ABL127 reverted demethylation of PP2Ac through their antioxidant properties (Fig. 9). Though methionine plays a crucial role in cell metabolism, no dose-response relationship was detected in cell viability after supplemented different level methionine, with optimal protective level at 10 mg/L,which is similar to the level in mediate. Methionine has also been demonstrated to protect cecal tonsils cellar apoptosis, the conflict evidence from animal and clinical trials showed that excessive methionine intake is likely contribute to Alzheimer’s-like neurodegeneration . The narrow adequate level of methionine in cellular was maintained by its metabolism process, which ubiquitously occurs in mammalian. In its metabolism procedure, a methyl was generated then transferred into DNA, protein methylation modification and methionine was recycled. The intermediate productions of methionine metabolism like SAM, Hcys and SAH have been demonstrated to involve in various diseases .
As a hallmark of tau pathology, tau hyperphosporylation can caused abnormally neurofibrillary tangle contributable to destruction of the neuronal cytoskeleton and axonal transport . Many neurotoxins can induce tau hyperphosphorylation, including aluminum, MPTP, BMAA, etc [28–32]. More than 30 tau phosphorylation sites were tested so far in neuronal diseases, with limited sites phosphorylation related to neurodegenerative diseases . The present study indicated that in our animal or cell model, the manganese exposure level we set caused tested sites at Ser199, Ser202 sites, Ser396 and Ser404 phosphorylation, with Ser199, Ser396 more sentivity. Other literatures have discovered that other heavy metals, including cadmium, lead caused tau hyperphosphorylation, but showing different phosphorylated sites [33–36]. Even exposed to Mn, the sites of phosphorylation of tau were not consistent in different models. Methionine supplement attenuates Tau phosphorylation, which is consistent with some literatures. Conflicting evidences was reported by Tapia and his colleagues . The balance of tau phosphorylation is supervised by numerous protein kinases and phosphatases. Various protein kinases are related to neurodegenerative diseases, including casein kinase, calcium calmodulin-dependent kinase and glycogen synthase kinase-3β (GSK-3β) ,which specifically was identified to get involved in tau phosphorylation in PC12 cells exposed to Mn . How Mn stimulated tau phosphorylation has not been fully understood yet. Nonetheless, the impact of phosphatases on tau hyperphosphorylation induced by Mn has not been fully elucidated yet.
PP2A, a major member of phosphoprotein phosphatases, its posttranslational modifications are responsible for dephosphorization in tau in neurodegenerative diseases. PP2A is composed of three subunits; those are a scaffolding unit (A), a regulatory unit (B) and a catalytic unit(C). Specifically, the methylation status of catalytic unit is crucial for PP2A activity. It have been demonstrated that the methylation of catalytic unit enhanced PP2A activity and decreased tau hyper phosphorylation. Conversely, demethylation of PP2Ac is contributed to dephosphorylate of hyperphosphorylated tau in AD brain [10, 37–40]. In present study, Mn caused demethylated PP2Ac increasing, paralleled with methylated PP2Ac decrease, which implicated inactivation of PP2A (Fig. 9). However, other investigation proved that lower level Mn2+ activated PP2A . Of note, the Mn concentration applied in Zhang et al. was 0.1-1% percent of ours.
Both one-carbon-cycle and oxidative were assumed to regulate PP2Ac methylation modification. Methionine and its intermediate, S-adenosylmethionine (SAM) were demonstrated to offer a methyl to promote PP2Ac methylation, thus dephosphorylate tau . It is known little about the impact of Mn on methionine metabolism. We found methionine supplement consistently increase SAM and decreased SAH level whatever with or without manganese, suggesting that decreases in methionine cycle activity was associated with tau hyperphosphorylation caused by Mn. Moreover, previous study indicated that oxidative stress can promptly and continuously demethylation PP2Ac, independent of other signal pathway . It is well accepted that oxidative stress can directly interact with cysteine residue then impact spatial structure and biological effects of protein. Sequence analysis pointed out that a pairs of conserved cysteine residues C266/269 close to the active site of PP2Ac , which is likely a potential target of oxidative stress. This hypothesis was proved that disulfide bond reducing agent seized the increasing ROS, restoring PP2A activity as well . Our present results demonstrated that Mn exposure enhanced ROS and declined GSH, which might partly explain the mechanism of PP2Ac demethylation after Mn exposure (Fig. 9).
The reversible methylation and demethylation of Leu309 at carboxyl end of PP2Ac is catalyzed by leucine carboxyl methyltransferase-1 (LCMT-1) and protein phosphatase methylesterase-1 (PME-1) . The levels of LCMT-1 and methylated PP2Ac are decreased, accordant with hyperphosphorylation of tau in AD brains . Deregulation of PP2Ac methylation also disrupts the interaction between PP2A and tau, and alters tau distribution . These previous studies have indicated that methylation of PP2Ac is essential for it to dephosphorylate tau. It has been discovered that Mn, being required for PP2A phosphatase activity, is evicted while the interaction between PME-1 and PP2A , but how does Mn disrupt the level of LCMT-1 and PME-1 hasn’t been explained yet. There was evidence show that ROS can cover the active site of PP2A to LCMT-1, thus deregulate PP2A activity [43, 49].