Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are intimately linked to both familial and sporadic Parkinson's disease. LRRK2 is a large protein kinase able to bind and hydrolyse GTP. A wealth of in vitro studies have established that the distinct pathogenic LRRK2 mutants differentially affect those enzymatic activities, either causing an increase in kinase activity without altering GTP binding/GTP hydrolysis, or displaying no change in kinase activity but increased GTP binding/decreased GTP hydrolysis. Importantly, recent studies have shown that all pathogenic LRRK2 mutants display increased kinase activity towards select kinase substrates when analysed in intact cells. To understand those apparently discrepant results, better insight into the cellular role(s) of normal and pathogenic LRRK2 is crucial. Various studies indicate that LRRK2 regulates numerous intracellular vesicular trafficking pathways, but the mechanism(s) by which the distinct pathogenic mutants may equally interfere with such pathways has largely remained elusive. Here, we summarize the known alterations in the catalytic activities of the distinct pathogenic LRRK2 mutants and propose a testable working hypothesis by which the various mutants may affect membrane trafficking events in identical ways by culminating in increased phosphorylation of select substrate proteins known to be crucial for membrane trafficking between specific cellular compartments.
Parkinson's disease (PD) is a common neurodegenerative disorder of largely unknown aetiology, with ageing as a major risk factor . Over a decade ago, mutations in the Leucine-Rich Repeat kinase 2 (LRRK2) gene were reported to cause autosomal-dominant PD [2,3]. Subsequent reports revealed that mutations in LRRK2 comprise the most common cause for familial PD and that several LRRK2 variants either positively or negatively correlate with PD risk [4,5], highlighting the importance of LRRK2 in disease pathogenesis.
The LRRK2 protein comprises various domains implicated in protein–protein interactions and a central region consisting of a Ras-of-complex (ROC) GTPase domain and a kinase domain connected via a C-terminal of ROC (COR) domain (Figure 1). All currently identified pathogenic mutations cluster in this central region, suggesting that targeting those activities may allow for the development of disease-modifying therapies. Towards this end, highly selective, potent and brain-permeable LRRK2 kinase inhibitors have been developed , and our understanding of the alterations in the catalytic activities of the various pathogenic LRRK2 mutants has greatly increased over the past years. However, there remain big gaps in our understanding of the cellular role(s) of LRRK2 in neuronal as well as non-neuronal cells, even though such understanding is critical for successfully bringing LRRK2-related drugs into the clinic.
Schematic representation of the domain structure of LRRK2, including armadillo repeats (ARM), ankyrin repeats (ANK), leucine-rich repeats (LRRs), a ROC domain, a COR domain, a kinase domain and a C-terminal WD40 domain.
LRRK2 is expressed in many tissues, suggesting that it regulates events common to various distinct cell types. Indeed, pathogenic LRRK2 has been reported to have an impact upon several conserved vesicular trafficking steps related to endocytic uptake, endosome–lysosome and autophagosome–lysosome trafficking as well as retromer-mediated trafficking to and from the Golgi complex . Mechanistically, this may involve direct and/or indirect regulation of several distinct Rab proteins [7–14]. Rab proteins comprise a family of over 60 small GTPases which function as molecular switches, alternating between a GTP-bound active form and a GDP-bound inactive form. They are reversibly localized to distinct intracellular membranes where they interact with coat components, motor proteins and SNARE proteins to control vesicle budding, microtubule (MT)-dependent vesicle motility and vesicle fusion . Interestingly, a subset of Rab proteins have recently been found to serve as LRRK2 kinase substrates, with phosphorylation thought to inactivate Rab protein function and thus the specific vesicular transport steps which these Rab proteins are regulating . Since LRRK2 has also been described to bind to MT and may regulate their dynamics , alterations in MT stability may result in (additional) downstream effects on a vast array of vesicular trafficking routes. Here, we review current knowledge and propose a testable working model by which the distinct altered catalytic activities of the pathogenic LRRK2 mutants may have an impact upon multiple MT-mediated vesicular trafficking steps through a common mechanism.
The LRRK2 kinase and GTPase activities
A set of pathogenic LRRK2 mutations have been described, including the R1441C/G/H and N1437H mutations in the ROC domain, the Y1699C mutation in the COR domain, and the G2019S and I2020T mutations in the kinase domain (Figure 1). The most prominent G2019S mutation in the kinase domain has been consistently shown to increase LRRK2 kinase activity as assessed using in vitro phosphorylation assays [14,17,18]. Thus, the G2019S mutant may act in a gain-of-function manner, with enhanced kinase activity towards defined substrates detrimental to cell survival. Indeed, the enhanced kinase activity of the G2019S mutant has been shown to correlate with neurotoxicity which can be reverted by either pharmacological or genetic kinase inhibition [19–21]. However, none of the other pathogenic mutations in the ROC or COR domain seem associated with increased inherent LRRK2 kinase activity in vitro [14,17,18].
LRRK2 is also able to bind GTP and displays inherent GTPase activity. Pathogenic mutations in the ROC and COR domain (R1441C/G, Y1699C) have been found to display increased GTP binding and decreased GTPase activity [22–24], with no changes observed with the G2019S mutation [23,25–27]. Interestingly, a R1398H polymorphism in the ROC domain of LRRK2 associated with decreased PD risk  has recently been reported to display decreased GTP binding and increased GTP hydrolysis, opposite to that reported for pathogenic mutants in the ROC and COR domain , and similar results have been described for an artificial R1398L mutation [25–27]. These data suggest that pathogenic mutations in the ROC and COR domain increase the amount of GTP-bound LRRK2, and that such a GTP-bound form of LRRK2 may be pathogenic. In support of this view, decreasing the GTP-bound state of pathogenic LRRK2 seems beneficial to cell survival in in vitro and animal models , indicating that specific and brain-permeable LRRK2 GTP-binding inhibitors may provide an alternative therapeutic strategy for at least some PD cases due to mutations in the ROC and the COR domain.
Altogether, a picture has emerged whereby LRRK2-mediated pathogenicity results either from mutations which increase kinase activity without altering the GTP-bound state of LRRK2, or from mutations which increase the GTP-bound state without altering kinase activity. However, a recent seminal study found that all pathogenic LRRK2 mutants analysed (R1441C/G/H, Y1699C, G2019S, I2020T) increase the phosphorylation of a set of bona fide LRRK2 kinase substrate proteins in intact cells, whilst at the same time corroborating that only the G2019S mutant increases substrate phosphorylation when measured in in vitro phosphorylation assays using purified components . Whilst awaiting independent validation by other laboratories, this is a crucially important finding, as it suggests that the distinct pathogenic LRRK2 mutants may all act through a common pathogenic mechanism by increasing the phosphorylation of LRRK2 kinase substrates in intact cells, and thus that LRRK2 kinase inhibitors may be beneficial to the entire LRRK2-linked PD spectrum.
The LRRK2 MT connection
How may pathogenic LRRK2 mutants with unaltered inherent kinase activity in vitro cause increased substrate phosphorylation in a cellular context? A wide variety of scenarios are possible. For example, the distinct pathogenic LRRK2 mutants may differentially regulate other kinases and/or phosphatases which also impinge upon the Rab substrates or their respective regulatory proteins  (Figure 2A,B). Indeed, various kinases and phosphatases have been reported to modulate the phosphorylation status of some Rab proteins. Whilst this is most prominently observed during mitosis , non-mitotic alterations in Rab phosphorylation at sites distinct from or identical to those predicted and/or shown to be phosphorylated by LRRK2 have been reported as well [32–34]. However, there is currently no evidence that all pathogenic LRRK2 mutants except for the G2019S mutant regulate the activities of other kinases and/or phosphatases which have an impact upon the phosphorylation status of the select Rab proteins which are LRRK2 kinase substrates.
Possible models for how all pathogenic LRRK2 mutants may cause increased Rab protein phosphorylation in intact cells.
Along similar lines, the distinct pathogenic LRRK2 mutants may differentially interact with Rab kinases and/or phosphatases, altering their activities and/or substrate specificities towards the Rab substrates, thereby causing changes in the overall phosphorylation status of these proteins in intact cells (Figure 2C). Indeed, select pathogenic LRRK2 mutants have been shown to display altered interactions with various protein kinases and phosphatases [33–38], but a careful side-by-side analysis of all pathogenic LRRK2 mutants with respect to those interactions, as well as the identification of the precise kinases and/or phosphatase(s) responsible for further modulating the phosphorylation status of the Rab proteins which serve as LRRK2 kinase substrates is currently missing.
Another scenario depends on differences in the subcellular localization of the distinct pathogenic LRRK2 mutant proteins. If all pathogenic LRRK2 mutants except the G2019S mutant display altered intracellular localization overlapping with that of the identified LRRK2 kinase substrate(s), such increased molecular proximity may lead to enhanced substrate protein phosphorylation similar to that mediated by the kinase-activating G2019S mutation, even though not associated with an increase in in vitro kinase activity per se (Figure 2D). Such alterations in the subcellular localization of pathogenic mutant LRRK2 proteins may further be due to GTP-binding-mediated differences in protein–protein interactions. Indeed, the large number of reported LRRK2 interactors [39,40] suggests an important role for protein interactors in regulating the cellular biology of LRRK2, even though there is currently no evidence for interactions which are selectively enhanced by all pathogenic LRRK2 mutants except for G2019S.
Since MT are required for many intracellular cargo transport processes, the reported interactions of LRRK2 with MTs warrant particular attention. Endogenous LRRK2 has been consistently shown to physically interact and co-localize with MTs [41–44]. Such interactions may alter MT dynamics, with expected downstream effects on many distinct MT-mediated vesicular transport events . Alternatively, MT binding may increase the amount of LRRK2 in molecular proximity to various transport vesicles which carry distinct Rab proteins, thereby locally increasing their phosphorylation status. Thus, an enhanced MT association of select pathogenic LRRK2 mutants may result in increased Rab substrate protein phosphorylation in the absence of increased inherent kinase activity, with downstream effects on vesicular trafficking steps governed by those Rab proteins.
This model predicts that all pathogenic mutants with the exception of G2019S display increased MT association, and this indeed has been described for several LRRK2 mutants when compared with G2019S , consistent with observations from our laboratory (unpublished). The dynamics of MT are modulated by post-translational tubulin modifications which seem to be recognized by different molecular motor proteins, thereby further contributing to the establishment and maintenance of polarized vesicular trafficking. Given that many vesicular transport events occur preferentially along stable MT tracks [16,46–48], the model further predicts a preferential association of pathogenic LRRK2 with a subpopulation of stable MTs, which has been reported at least for the pathogenic R1441C and Y1699C mutants . In this manner, just a few strategically placed pathogenic LRRK2 molecules, with equal inherent kinase activity when compared with the wild-type molecule, may be able to phosphorylate significant amounts of Rab proteins bound to various transport vesicles which move up and down MT tracks to their respective destinations. Interestingly, R1441C/G, Y1699C, G2019S and I2020T mutants have all been shown to enhance trans-Golgi network clustering when co-expressed with Rab7L1 , and primary fibroblasts from R1441C, Y1699C and G2019S LRRK2 PD patients all display the same autophagic alterations . These observations suggest that all pathogenic LRRK2 mutants may converge onto the same functional outputs, even though the precise links between differential Rab phosphorylation and MT binding underlying the cellular phenotypes currently remain unknown.
Corroborating the proposed working model will require careful and correlative side-by-side analysis of all pathogenic LRRK2 mutants with respect to MT interactions, kinase activity and GTP binding. A testable prediction of this model is that the subcellular localization of all pathogenic LRRK2 mutants except for G2019S should be altered when decreasing LRRK2 GTP binding either by introducing the protective R1398H variant or by pharmacologically interfering with LRRK2 GTP binding, and decreasing GTP binding should result in decreased phosphorylation of the Rab substrate proteins mediated by all pathogenic LRRK2 mutants except for the G2019S mutant. The model further predicts that any LRRK2-mediated Rab protein phosphorylation causes deficits in vesicular trafficking, and future studies are required to determine whether LRRK2-mediated phosphorylation causes alterations in the GTP-bound (active) state of the Rab proteins, or changes in their interactions with select motor proteins, either one of which would interfere with vesicle mobility .
Over the past decade, significant progress has been made in our understanding of how distinct pathogenic LRRK2 mutations alter the catalytic activities of this enzyme. A picture has emerged whereby either aberrant kinase activity or an increase in the amount of GTP-bound LRRK2 may confer pathogenicity, even though the underlying mechanism(s) have remained elusive. The recent identification of Rab proteins as LRRK2 interactors and/or kinase substrates, together with the well-established role(s) of LRRK2 in MT-related processes allows for the proposal of a simple and testable working model by which all pathogenic LRRK2 mutants may culminate in identical changes in Rab-related membrane trafficking events relevant to disease pathogenesis and in a manner dependent on kinase activity. Whilst much work remains to be done to understand the cell biology underlying LRRK2-related PD, studies of this type will greatly aid in translating LRRK2 kinase inhibitors into the clinic, with a positive impact upon the life of millions of people currently suffering from this debilitating neurodegenerative disease.
Work in the laboratory is supported by FEDER, grants from the Spanish Ministry of Economy and Competitiveness [grant number SAF2014-58653-R], the BBVA Foundation and the Michael J. Fox Foundation (MJFF).
The Authors declare that there are no competing interests associated with the manuscript.