Interestingly, Lobbestael et al. previously reported that protein phosphatase 1 (PP1) interacts with and dephosphorylates LRRK2 in vitro (Lobbestael et al.
2013). However, we did not identify PP1 as a modulator of LRRK2-induced toxicity in our
Drosophila-based phosphatase screen. Instead, we have identified the three components of PP2A, i.e., scaffolding (PP2A-29B), regulatory (wdb) and catalytic (mts) subunits, which are required to form a functional holoenzyme, as genetic modulators of LRRK2 function. Our finding is consistent with a recent report by Athanasopoulos and colleagues who found that LRRK2 is able to interact with all the three subunits of PP2A in cultured cells, and that silencing the catalytic subunit of PP2A by shRNA aggravated cellular degeneration induced by the pathogenic LRRK2 R1441C mutant (Athanasopoulos et al.
2016). Here, we have independently verified the finding by means of a different approach using an in vivo model system. Importantly, we have demonstrated the relevance of PP2A in DA neurons expressing the LRRK2 G2019S transgene. Moreover, we showed that treatment of LRRK2-mutant flies with fingolimod, a drug that is currently being used to treat multiple sclerosis, ameliorates their disease-associated phenotypes but otherwise has no apparent effects on the climbing performance of control flies. All the compounds tested also have no overt effects on other DA neuronal cluster examined except for PPL1, which they exert a positive effect. In the presence of PP2A overexpression, we found that LRRK2 phosphorylation at Ser-1292 is reduced, which suggests a mechanism for how PP2A may regulate LRRK2 activity. This is consistent with a recent report demonstrating that Ser1292 dephosphorylation is mediated by phosphatases that are sensitive to calyculin A (PP1) and okadaic acid (PP2) (Reynolds et al.
2014). At the same time, we also found that S6K, a reported PP2A substrate (Bielinski and Mumby
2007; Hahn et al.
2010), functionally interacts with LRRK2. In the presence of LRRK2, S6K phosphorylation is enhanced, although we do not know if their relationship is direct or indirect. Notwithstanding this, given that LRRK2-mediated enhancement of protein translation is thought to underlie its neurotoxicity (Imai et al.
2008; Martin et al.
2014a), it is perhaps not surprising to note that the modulation of S6K activity, which normally promotes protein synthesis via the phosphorylation of ribosomal S6, can influence LRRK2 actions. Notably, the ribosomal protein s15 was recently identified as a key pathogenic substrate of LRRK2 (Martin et al.
2014b). Whether PP2A can reverse the phosphorylation of s15 by LRRK2 is unclear but would undoubtedly be an important question to address. Another area worth investigating is the potential relationship between LRRK2-induced enhancement in protein synthesis and mitochondrial dysfunction, particularly in view of our finding that S6K inhibition can rescue the abnormally enlarged mitochondrial size in DA neurons of LRRK2 mutant flies. It is tempting to speculate that the TOR pathway, which coordinates protein synthesis and mitochondrial activity, may help to connect the dots. Indeed, rapamycin-mediated inhibition of mTOR was reported to rescue the pathological phenotype of LRRK2 flies (Tain et al.
2009). Moreover, Penny and colleagues recently showed that the TOR pathway is involved in the regulation of synaptic homeostasis by LRRK2 in
Drosophila neuromuscular junction (Penney et al.
2016). Interestingly, the authors further found that LRRK2 collaborates with S6K to promote synaptic enhancement of neurotransmitter release at the neuromuscular junction. It is noteworthy to mention that mTOR-mediated phosphorylation of S6K at Thr-389 is pivotal for its activation (Burnett et al.
1998). This is the same site that we have observed to exhibit enhanced phosphorylation (for p70 S6K) in the presence of LRRK2. Clearly, future studies should be conducted to unravel how the various components linked to LRRK2 pathways converge on impairing protein synthesis and mitochondrial function to bring about pathogenic outcomes.