. Ribosomal protein s15 phosphorylation mediates LRRK2 neurodegeneration in Parkinson's disease. Cell. 2014 Apr 10;157(2):472-85. PubMed.

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  1. The paper highlights the ribosomal protein S15 as a novel pathogenic LRRK2 substrate. Their hypothesis is supported by both in vivo and in vitro models of LRRK2 pathogenesis.

    The hypothesis that G2019S LRRK2 toxicity leads to bulk increase in protein synthesis is interesting because it adds to the growing body of evidence linking LRRK2 and other Parkinson’s disease-related proteins to changes in protein homeostasis in the cell. More specifically, it adds to the increasing lines of evidence linking neurodegeneration to translation misregulation.

    S15’s role in G2019S LRRK2-induced degeneration doesn’t extend to all pathogenic LRRK2 mutations, as demonstrated by their work on R1441C. It will be interesting to see if S15’s role is limited to the G2019S mutation, or has broader implications for LRRK2-induced Parkinson’s disease.   

     

  2. The most important finding of the paper, in my view, is the biochemical and genetic demonstration that aberrant regulation of protein synthesis contributes to LRRK2-G2019S pathogenesis. This resonates with earlier studies in Drosophila Parkinson's disease (PD) models that link altered mRNA metabolism and translation to PD pathogenesis.

    The authors argue that the ribosomal protein s15 is a physiological substrate of LRRK2. The strongest evidence is that s15 phosphorylation is reduced by ~50 percent in the Drosophila LRRK2 null mutant. Clearly other unidentified kinase(s) can also phosphorylate s15. What is missing from the paper is whether s15 phosphorylation is reduced when LRRK2 is knocked out or knocked down in mammalian cells, and whether wild-type, phospho-deficient, and phospho-mimetic s15 exhibit differential activities in rescuing the cellular phenotypes observed in LRRK2 knockout animals, such as apoptosis of kidney cells. This is an important point, because the current controversy regarding whether 4E-BP is a genuine substrate of LRRK2 was caused by the inability  to confirm in mammalian cells the findings derived from fly models.

    However, I don't think that the authors have completely convinced me that LRRK2-G2019S phosphorylation of s15 triggers neuronal injury by ramping up translation. One key piece of data presented was that the phospho-mimetic s15, called TD, is toxic to cultured mammalian neurons when overexpressed. However, the authors show no data that TD s15 promotes bulk translation in these neurons, or that the toxicity of TD s15 can be blocked by partial inhibition of global translation. More importantly, they did not show whether TD s15 promotes translation and is toxic to dopaminergic neurons in vivo in transgenic animals.

    The reliance on overexpression in cells throughout this study could be a cause of concern. Human neurons derived from LRRK2-G2019S patient iPSCs are now available. It would be interesting to test whether altered s15 phosphorylation is responsible for the disease-relevant phenotypes observed in these neurons. Neurons derived from LRRK2-GS knock-in animals can also be used for this purpose. With regard to the levels of LRRK2 autophosphorylation being far higher than phosphorylation of s15, I don't think that observation alone can be used to argue against s15 being a bona fide LRRK2 substrate. Phosphorylation of 4E-BP by LRRK2 in vitro is also weaker than LRRK2 autophosphorylation. It could be that the in-vitro kinase assay condition favors LRRK2 autophosphorylation, which may not be the case in vivo, or that LRRK2 intrinsically has higher autophosphorylation activity compared to other kinases

    As we have discussed in our previous publications, increased translation could lead to neuronal injury through a number of mechanisms. First, given that protein synthesis is a highly energy-demanding process, stimulation of protein translation by pathogenic LRRK2 could perturb cellular energy and redox homoeostasis. This could be especially detrimental in aged cells or stressed post-mitotic cells such as dopaminergic neurons where energy reserve is already low. Second, increased protein synthesis could lead to the accumulation of misfolded or aberrant proteins, overwhelming the already-compromised ubiquitin proteasome and molecular chaperone systems in aged or stressed cells. Third, altered translation by pathogenic LRRK2 kinase may compromise synapse structure and function, which is known to involve regulated local protein synthesis. Deregulation of this process could lead to synaptic dysfunction and eventual neurodegeneration.

    The authors have tried to distinguish their study from our previous studies on LRRK2 and eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP), in which we made the first link between altered translation and LRRK2 pathogenesis (see Imai et al., 2008). Unfortunately, their data on LRRK2 and 4E-BP are all embedded in supplementary information and many details of their experiments are not available.

    First, the source of their p-4E-BP and 4E-BP antibodies is not clear. The p-4E-BP western blots look very different from those in published works, which generally show multiple bands of 4E-BP because 4E-BP is phosphorylated in many sites. The p-4E-BP blots in this paper show only a single band.

    Secondly, the result showing lack of 4E-BP phosphorylation by LRRK2-GS in flies is subjected to alternative explanation. The authors used a cell-type specific Ddc-Gal4 driver to express LRRK2-GS in dopaminergic neurons, but used whole fly head extracts to detect p-4E-BP. Assuming their antibody is specific to p-4E-BP, since LRRK2-GS is only expressed in ~200 dopaminergic neurons in an adult fly head composed of millions of neurons and non-neuron cells that all express endogenous LRRK2 and 4E-BP, it is unlikely that any stimulating effect of LRRK2-GS on 4E-BP phosphorylation can be observed. A ubiquitous Gal4 driver should be used to drive LRRK2-GS expression.

    Thirdly, the reporter experiment showing that the microRNA pathway does not mediate LRRK2-GS effects on mRNA translation is misleading. The effect of LRRK2 on the micro-RNA pathway is specific to particular microRNAs and mRNA targets (Gehrke et al., 2010). Based on the limited information described for their bicistronic reporter, it appears to have no miRNA binding sites. If so, one cannot expect manipulating the microRNA pathway to have any effect on reporter expression.

    Fourthly, the genetic experiment showing that heterozygosity of eIF4E has no effect on LRRK2-GS toxicity by no means disproves the model that 4E-BP phosphorylation is relevant to LRRK2-GS toxicity. The hypomorphic eIF4E alleles only show partial reduction of eIF4E mRNA expression. No information is available as to whether this affects eIF4E protein expression or cap-dependent translation. A better way to  test the model would be to express WT and phospho-mutant forms of 4E-BP in LRRK2-GS background to see if there is genetic interaction, as we did  previously (Imai et al., 2008). 

    Finally, we carried out our previous studies on LRRK2 phosphorylation of 4E-BP using cell lines or transgenic flies expressing LRRK2-I2020T, whereas this study and the other cited mammalian studies all used LRRK2-GS. Although it is generally assumed that the T2020T and G2019S mutations all boost LRRK2 kinase activity, it may not be correct to assume that they act through the same pathogenic mechanism. Moreover, the phosphorylation sites in 4E-BP acted on by LRRK2 are also known targets for mTORC1, which are hyperactive in cultured mammalian cells. In order to observe 4E-BP phosphorylation by LRRK2 in mammalian cells, we used serum-starvation treatment to inhibit Ins/PI3K/mTORC1 signaling. Based on our reading of this study and the other published studies trying to duplicate our past work, it appears that this point was overlooked.

    Thus, until the right experiments using the appropriate reagents are performed in mammalian cells, it is premature to claim that the LRRK2-4E-BP connection is dead. In fact, the more scientific way to consider this study and previous work is that LRRK2 may act on more than one substrate (4E-BP, s15, etc) to regulate translation. Future studies exploring the relationship and relative contributions of 4E-BP and s15 phosphorylation to LRRK2 toxicity will test this hypothesis.

    References:

    . Phosphorylation of 4E-BP by LRRK2 affects the maintenance of dopaminergic neurons in Drosophila. EMBO J. 2008 Sep 17;27(18):2432-43. PubMed.

    . Pathogenic LRRK2 negatively regulates microRNA-mediated translational repression. Nature. 2010 Jul 29;466(7306):637-41. PubMed.

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