In agreement with a recent report showing that up-regulation of PP2A catalytic subunit promotes axonal formation and elongation while PP2A inhibition prevents differentiation (Zhu et al. 2010
), we found that neurite-like process outgrowth is facilitated in N2a-WT C cells. However, instead of looking at the effect of modulating PP2A activity, we chose here to focus our attention on assessing the potential role of PP2A methylation in N2a cell differentiation, which have been widely used to study formation of neurite-like processes. In the context of these studies, it is important to note that C subunit methylation likely modulates PP2A substrate specificity (Reviewed in Janssens et al. 2008
) through its role in PP2A biogenesis (Hombauer et al. 2007
). We and others have shown that the methylation-site L309Δ mutant cannot bind to regulatory B subunits, but can still associate with other regulatory subunits from the B′ and B″ families(Nunbhakdi-Craig et al. 2007
; Longin et al. 2007
). In addition, down-regulation of LCMT1 and methylated PP2A expression levels lead to a loss of Bα and compensatory changes inintracellular PP2A subunit composition that can affect its substrate specificity and targeting (Schild et al. 2006a
; Gentry et al. 2005
; Lee and Pallas 2007
; Longin et al. 2007
; Sontag et al. 2008
). Our data provide the first evidence that modulating PP2A methylation can dramatically affect the differentiation process in N2a cells. We have also obtained similar results in preliminary studies performed in human SH-SY5Y neuroblastoma and rat PC12 cells (E. Sontag, unpublished results). Notably, enhancing LCMT1 expression was able to trigger serum-independent differentiation. Data from the double N2a-LCMT1 + L309Δ clones indicated that the bability of LCMT1 to drive the initiation and extension of neurite-like processes was primarily mediated by PP2A. Most importantly, the extent of both process formation and elongation appeared to be highly correlated with the total levels of methylated PP2A enzymes present in our single and double clones. Interestingly, protein methylation is involved in neurite outgrowth in PC12 cells (Cimato et al. 1997
) and general inhibition of methyltransferase activity interferes with neuronal differentiation of P19 embryonal cells (Hong et al. 2008
). Accordingly, the abilityof LCMT1 to promote neuritogenesis was impeded by incubating N2a cells with 100μM SAH, a universal inhibitor of cellular methylation reactions. In agreement with the role of this metabolite in inhibiting LCMT1 methyltransferase activity (Leulliot et al. 2004
), we have previously reported that supplementing N2a cell culture medium with 100 μM SAH induces a 40–50% decrease in endogenous methylated C levels (Sontag et al. 2007
). Thus, together with our findings that the L309Δ mutant blocks neuritogenesis in N2a and N2a-LCMT1 cells, these observations strongly support the hypothesis that PP2A is one of the methylated proteins critically required for commencement of the neuronal differentiation process.
Results from PME-1 overexpression studies in N2a and N2a-LCMT1 cells also indicate that increased PP2A demethylation has an adverse effect on the initiation of neurite-like process formation. This may be in part because PME-1 overexpression induces a loss of methylated PP2A and subsequent loss of Bα in N2a cells (Sontag et al. 2008
), and the resulting down-regulation of Bα prevents differentiation (). Bα-containing PP2A holoenzymes are very abundant in neurons where they regulate microtubule stability (Nunbhakdi-Craig et al. 2007
) and the phosphorylation state of tau (Sontag et al. 2008
; Xu et al. 2008
), amyloid precursor protein (APP) (Sontag et al. 2007
) and neurofilament proteins (Strack et al. 1997
). It is well recognized that the microtubule cytoskeleton (Fukushima et al. 2009
), tau (Stoothoff and Johnson 2005
), APP (Stokin and Goldstein 2006
) and neurofilament proteins (Szaro and Strong 2010
) all play a critical role in axonal growth. In addition, many signaling pathways, including the MAP kinase (ERK) pathway (Van Kanegan et al. 2005
), are regulated by Bα-containing PP2A holoenzymes. Based on these observations, it is difficult to pinpoint the precise mechanisms that directly or indirectly contribute to Bα-dependent effects on N2a cell differentiation in our study.
It has been reported that differentiation of SH-SY5Y cells or PC12 cells does not affect Bα mRNA or protein expression levels. In contrast, the mRNA and/or protein levels of Bβ, Bγ, and Bδ, which also belong to the PP2A regulatory B subunit family, but are much less abundant than Bα, become either up- or down-regulated during the differentiation process in those cells. As a result, it had been proposed that those are more likely to be specifically required for early neurite formation (Strack 2002
; Schild et al. 2006b
). Indeed, Bγ plays a key role in MAP kinase-dependent PC6-3 cell differentiation (Strack 2002
). Recently, PP2A holoenzymes containing the B′β and B′δ subunits have also been implicated in nerve growth factor-mediated PC12 cell differentiation (Van Kanegan and Strack 2009
), and dendritic branching (Brandt et al. 2008
). While Bα protein expression levels do not fluctuate during the differentiation process, our results indicate that, nevertheless, these basal amounts are critically required for proper neuritogenesis. Indeed, Bα knockdown, which also leads to a subsequent loss of LCMT1 expression levels in N2a cells (Sontag et al. 2008
), inhibited N2a cell differentiation (), thereby mimicking the effects of LCMT1 knockdown (). On the other hand, overexpression of Bα, which is associated with down-regulation of demethylated C levels (Sontag et al. 2008
), promoted process elongation in serum-starved N2a cells (). Thus, there is a very close interrelationship between LCMT1-dependent PP2A methylation, Bα expression levels and process formation. Yet, we cannot exclude the possibility that variations in intracellular Bα amounts also indirectly influence neuritogenesis by inducing a compensatory increase or decrease in the levels of other regulatory “B” subunit-containing PP2A holoenzymes that play a role in the differentiation process. While many PP2A isoforms have the potential to regulate neuronal differentiation, it is likely that they are recruited in response to distinct extracellular or intracellular stimuli, are functioning in well-defined signaling pathways, and have a differential cellular and subcellular distribution. Further studies will be needed to elucidate their respective contribution to each step of the differentiation process by identifying their specific substrates. However, the lack of suitable B-specific antibodies, and the complex regulation and widespread cellular effects exerted by various PP2A isoforms make this a quite difficult prospect.
Interestingly, tau- and microtubule-containing straight projections reminiscent of neuronal axons were the hallmark of differentiated N2a-LCMT1 cells (). Secondary neuritic branching was observed in differentiated N2a, N2a-Wt C and N2a-LCMT1 + Wt C, but not in N2a-LCMT1 cells. These discrepancies reinforce the central dogma that PP2A activity and methylation differentially modulate PP2A function, and have divergent effects on the growth of primary and secondary cellular processes. Significantly, we observed that the organization of F-actin was altered in N2a-LCMT1 cells relative to controls. Together with microtubules, the actin cytoskeleton plays a critical role in neuritic development (Dehmelt and Halpain 2004
). Axon formation, elongation and branching are highly sensitive to local perturbation of microtubule and actin dynamics (Luo 2002
). It is thus tempting to speculate that local changes in actin stability could underlie the observed differentiated phenotype of N2a-LCMT1 cells. However, in-depth studies are required to fully understand the mechanisms by which PP2A methylation affects neuronal morphogenesis.
As observed with LCMT1 overexpression, PME-1 knockdown led to the formation of elongated processes. However, they appear somehow anomalous, being quite thin compared to the standard neurites of differentiated N2a cells. We have previously reported that PME-1 silencing in N2a cells does not significantly affect Bα and LCMT1 expression levels (Sontag et al. 2008
). While enhanced PME-1 expression inhibits N2a cell differentiation, the aberrant process formation induced by artificially decreasing intracellular PME-1 levels suggests that threshold levels of this PP2A methylesterase need to be maintained for ensuring normal neurite-like process development. Indeed, while LCMT1 and PME-1 selectively regulate PP2A methylation state, combined data from our differentiation experiments and earlier studies (Sontag et al. 2008
) indicate that they do not simply exert opposite actions on PP2A regulation. When examined altogether, our results suggest that a subtle equilibrium between LCMT1 and PME-1 expression levels and activity, and their subsequent modulation of the methylation/demethylation state of specific PP2A isoforms, is required for proper coordinated development of the neuritic network.