Synuclein as a LRRK2 substrate

The relationship between LRRK2 and alpha-synuclein, two genes linked to familial PD in an autosomal dominant manner, is still largely unknown. Although studies focussing on the relationship between these two genes are scant, current knowledge points more often than not to a diffuse relationship between LRRK2 and alpha-synuclein with both genes acting at separate levels in a complex pathogenic PD process  The recent report by Qing et al. proposing that LRRK2 directly phosphorylates alpha-synuclein at serine 129 provides the intriguing possibility that the PD pathogenic processes may not be as complicated as it is generally believed to be.

However, the report raises some questions. Many LRRK2 researchers have been confronted with the difficulty of preparing active recombinant enzyme. Qing et al used transfection of eukaryotic expression plasmids in HEK293T cells as expression system, a good choice as this system has previously been shown to yield active LRRK2 enzymes. However, the authors used crude cell extracts as a source of enzyme. The conclusion that LRRK2 directly phosphorylates alpha-synuclein in this system is therefore tenuous as the reaction mix presumably contains multiple alternative kinases endogenously present in these cells. What is the level of purity of the enzyme used in this study? Also, does the method of protein preparation as applied in the study yield active protein? Interestingly, the G2019S mutant which has consistently been shown to possess higher phosphorylation activity than WT yields higher phosphorylation levels of alpha-synuclein while cell extracts of normal cells yields no phosphorylation. Is it then possible that LRRK2 WT or G2019S induces or activates another kinase in HEK293T cells which then effectively phosphorylates alpha-synuclein? Also, the study proposed end point readouts at 6 hours of incubation. Does this mean that the proposed alpha-synuclein phosphorylation is a very slow process? What are the kinetics of the proposed alpha-synuclein phosphorylation? As suggested in previous comments on this issue, the inclusion of a kinase dead negative control would be a very useful addition to the study.

The validation of this finding will require first a thorough confirmation at the in vitro level to discriminate between LRRK2 phosphorylation and phosphorylation by other cellular kinases. The use of proteins with a high level of purity and confirmed activity should help to address this issue. If the phosphorylation of alpha-synuclein by LRRK2 is true and physiological, then we have an extremely exciting development on our hands.