There is rising interest in the role of α-Syn aggregation in neurodegeneration. One theory states that α-Syn aggregation impairs neuronal activity (Cookson and van der Brug, 2008
) and others suggest that protofibrillar oligomers of α-Syn are toxic (Goldberg and Lansbury, 2000
, Lashuel and Lansbury, 2006
). We have found that in vivo α-Syn aggregation is associated with hyperphosphorylation of the key PP2A substrate, tyrosine hydroxylase, in dopaminergic neurons (Alerte et al., 2008
). In vitro studies have demonstrated that recombinant α-Syn also readily polymerizes into amyloidogenic fibrils that are structurally similar to those found in human brain (Conway et al., 1998
, Hashimoto et al., 1998
, Giasson et al., 1999
, Narhi et al., 1999
). Taken together the data suggest that α-Syn aggregation has the potential to impair PP2A activity, although that possibility had not been previously explored. In the present study we assessed PP2A activity as a function of α-Syn aggregation in various model systems. Our findings with recombinant proteins demonstrated a direct effect of α-Syn aggregation on PP2A activity impairment. Using DLB brain and α-Syn triplication brain we noted a loss of PP2A activity in association with α-Syn aggregation, supporting the hypothesis that PP2A activity appears to be attenuated in response to α-Syn aggregation, a process that can be accelerated by α-Syn Ser129 phosphorylation.
Hansen and colleagues long ago demonstrated that DLB frontal cortex contains abundant Lewy body pathology (Hansen et al., 1993
) and our data () concur with their findings. More recent work has shown that, as previously anticipated (Spillantini et al., 1997
) α-Syn is the major protein component of LBs (Anderson et al., 2006
) indicating that evaluation of frontal cortex for changes associated with α-Syn aggregation should be instructive.
We measured PP2A activity in frontal cortex of control and DLB brains using established methods with specificity controls (Cohen et al., 1989
, Sontag et al., 2004
, Lou et al., 2010
) and found that DLB brains, which contain significantly more aggregated α-Syn () also had significantly less PP2A activity than controls. Because α-Syn is a normal activator of PP2A, it is therefore likely that as α-Syn becomes sequestered in Lewy bodies; levels of soluble functional α-Syn become diminished, causing PP2A to be less active in brain regions harboring synucleinopathy. This is also supported by our findings from α-Syn triplication frontal cortex where there was a huge loss of PP2A activity compared to the soluble fraction of the identical tissue (), even though α-Syn protein content was high in both soluble and insoluble fractions. Taken together, the data imply that a reduction in soluble α-Syn and/or an increase in α-Syn PSer129 levels is sufficient to impair PP2A activity in brains with synucleinopathy, and in DLB brains it appears that it may be the combination of α-Syn aggregation and elevated PSer129 levels that act to diminish PP2A activity. These findings have implications for hyperphosphorylation of PP2A substrates, such as tau protein, which is consistently hyperphosphorylated in AD brain as well as in some cases of PD and DLB (Arima et al., 1999
) where abundant synucleinopathy is also found. There is also a possibility that as α-Syn becomes aggregated, PP2A may remain bound to it and become sequestered in Lewy bodies, reducing the soluble pool of active PP2A in the brain. Data from the triplication brain hint at this possibility (, compare PP2A in S and P fractions) a possibility that we are further exploring.
It is noteworthy that the substrate KRpTIRR used in our assays can be dephosphorylated by other serine/threonine phosphatases besides PP2A, including PP4 and PP5. Furthermore, recent studies have shown that PP5 is implicated in Alzheimer’s disease (Liu et al., 2005; Sanchez-Ortiz et al., 2009). Nonetheless, there are at least three lines of evidence that strongly support the role for PP2A as proposed herein: (first) our prior work measured the activity of PP2A that was immunoprecipitated with a PP2A-specific antibody, which allowed us to demonstrate that PP2A activity is enhanced by α-Syn (Peng et al., 2005
); (second) our recombinant PP2A cell free assays with soluble α-Syn or aggregated α-Syn () gave results similar to our findings in control and DLB brains (); (third) inhibition of phosphatase activity with fostriecin paralleled the loss of PP2A activity noted in DLB brains (). As previously stated, we cannot rule out a potential effect on PP4 as well, a possibility that should be further explored.
While α-Syn and the catalytic subunit of PP2A are known to interact (Peng et al., 2005
), no one knows if α-Syn might also bind B subunits of PP2A to in some manner contribute to holoenzyme assembly or localization. Both α-Syn and PP2A are localized to mitochondria in neuronal cells (Perez et al., 2002
, Wang et al., 2009
) and B subunits of PP2A have also been identified on mitochondria including subunits B56 (Ruvolo et al., 2002
) and Bβ2 (Dagda et al., 2003
We have begun exploring the mechanisms underlying α-Syn-mediated regulation of PP2A and the changes that occur with α-Syn aggregation. Preliminary data suggest that aggregated α-Syn may undergo a structural change that alters its ability to bind the PP2A catalytic subunit, which may then shift the phosphatase to a less active conformational state. In sum, this study reveals that α-Syn overexpression stimulates PP2A activity and α-Syn aggregation impairs PP2A activity, and may affect PP4 as well; implying that a gain or loss of normal α-Syn function in brains with synucleinopathy may induce phosphatase dysregulation. Importantly, novel therapies to modulate PP2A activity are being explored (Lu et al., 2009
, Corcoran et al., 2010
) and if successful, such treatments could benefit conditions in which levels of functional soluble α-Syn are found to vary.