Leucine Rich Repeat Kinase 2: beyond Parkinson’s and beyond kinase inhibitors

KEYWORDS:

• LRRK2
• Parkinson’s disease
• kinase
• GTPase
• ROCO
• drug discovery

1. Introduction

Leucine Rich Repeat Kinase 2 (LRRK2) is a multidomain enzyme with dual kinase and GTPase activities. Following the identification of mutations in the LRRK2 gene linked to familial Parkinson’s disease (PD) [1,2], it was subsequently realized that these represent probably the single most common genetic cause of Parkinson’s [3]. Coupled to data from a number of research groups suggesting that the kinase activity of LRRK2 is required for the pathological changes leading to PD in these cases [4,5], these insights have afforded LRRK2 status as a priority putative drug target for this disorder [6]. In the 13 years since LRRK2 rose to prominence, discoveries relating to a number of aspects of LRRK2 biology, both in terms of protein function and the role it plays in diseases other than PD, have broadened our understanding of LRRK2 and expanded the potential importance of this protein to therapeutic discovery efforts.

2. LRRK2 and human disease

The interest and research activity directed toward understanding LRRK2 has been driven by the involvement of this protein in a host of human disorders, most notably Parkinson’s. Intriguingly, this has been through a variety of mechanisms – including coding variants causing disease in an apparently autosomal dominant fashion, coding variants acting as disease risk factors, and genome-wide association (GWA) with disease risk for common noncoding, genomic variants [7,8].

2.1. LRRK2 in Parkinson’s

In 2004, two back-to-back papers reported that mutations in the gene encoding LRRK2 segregate with autosomal dominant forms of PD [1,2]. There are at least three strong arguments that explain why LRRK2 research has been so prominent since the identification of mutations linked to Parkinson’s. First, mutations in LRRK2 represent the most common genetic cause of familial disease, and the gene sits within an important risk factor locus for sporadic PD [9]. Second, the clinical and pathological phenotype of LRRK2-linked PD is almost indistinguishable from idiopathic PD cases – one intriguing difference being the neuropathology, with a small but significant minority of cases presenting with Parkinson’s in the absence of Lewy bodies [10,11]. Third, LRRK2 possesses a biochemical activity that is both well characterized and extensively exploited in terms of pharmacological manipulation: a kinase activity [12]. In addition, this activity is increased by some pathological mutations [13]. For all these reasons, inhibiting LRRK2 holds much promise as a PD therapeutic approach, an area where there is an urgent need for disease-modifying drugs [14]. Whether this is feasible, and beneficial for patients, remains to be tested in clinical trials.

2.2. The kinase activity of LRRK2

LRRK2 kinase activity has been implicated in a number of important cellular roles, via multiple cell-signaling pathways [15]. The relevance of this domain in disease is highlighted by the three-fold increase of kinase activity in vitro in the presence of the G2019S mutation [12]. This was recently reinforced by the discovery that a subset of Rab GTPases act as physiological LRRK2 substrates, and that these are hyperphosphorylated by the majority of disease-segregating mutants in a cellular context [13]. In order to explore the contribution of LRRK2 kinase activity in the biology of the cell and in disease, a host of cellular and animal models expressing the hyperactive G2019S LRRK2 mutant have been generated and characterized. In addition, the availability of multiple highly potent and selective LRRK2 kinase inhibitors has allowed researchers to dissect the contribution of kinase activity to cellular phenotypes under endogenous conditions. The emerging picture is one where LRRK2 kinase activity is important for a number of neuronal and non-neuronal processes related to membrane remodeling, including autophagy, vesicular trafficking, and cytoskeletal dynamics [15], although the precise relationship of kinase activity to the etiology of PD is unclear.

2.3. Beyond PD

One of the most fascinating aspects of LRRK2 genetics is that it is a pleomorphic locus, with involvement in a number of different disorders (Figure 1). Specifically, the LRRK2 locus has been implicated in GWA for multibacillary leprosy and inflammatory bowel disease (IBD), as well as coding variants being linked to cancer [16–19]. As for the GWA with PD, the precise mechanism linking the association with the etiology of leprosy and IBD is not completely clear and, in contrast to PD, relatively under explored. One possible common thread is a role for LRRK2 in innate immunity [20]. Equally, the molecular mechanisms connecting LRRK2 and cancer are not well understood. Most importantly, the potential utility of compounds and approaches targeting LRRK2 in a clinical setting for these disorders has not been addressed.

Figure 1. LRRK2 as a pleomorphic locus in human disease. The LRRK2 gene has been implicated in a range of disparate human diseases, with the clearest and best understood links between this protein and Parkinson’s disease. LRRK2 has also been linked to inflammatory bowel disease, risk of infection with multibacillary leprosy and cancer. Structural model for dimeric LRRK2 courtesy of Dr Johannes Gloeckner, and derived from reference [23].

2.4. Beyond the kinase

The sheer number of studies documenting the pathobiology of LRRK2 bears witness to how complex the biology of LRRK2 is, likely reflecting the complexity of its multi-domain organization. In addition to being a kinase, LRRK2 is also a GTPase (one of only three proteins in the human proteome to have such a dual activity) and possesses several protein/protein interaction modules. There is accumulating evidence that the Roc/GTPase domain, likely in its guanine-nucleotide bound state, is important not only as an intramolecular regulator of LRRK2 kinase activity but also as a platform for interaction with heterologous partners, including PKA, Sec16A, PAK6, Rab proteins, and tubulins [15]. Accordingly, pathogenic mutations around this domain have been reported to slow GTP hydrolysis, thus maintaining the protein in its active state and, as consequence, prolonging the downstream effects of LRRK2 signaling. There is also increasing insight into the identity and regulation of phosphorylation sites in the LRRK2 open reading frame, including the cluster of serine residues at the N-terminus (Ser910/935/955/973), and Ser 1292 and Ser1444 within the Roc. Phosphorylation of these residues is important for 14-3-3 protein binding to LRRK2 and GTPase activity, and consequent subcellular localization and interaction with cellular targets [21,22]. All these extra-kinase domains increase the level of complexity of LRRK2 biology, but also expand the opportunities to target its cellular function beyond kinase activity. Recent advances in our understanding of the three-dimensional organization of LRRK2 represent a major step toward being able to target LRRK2 beyond the kinase domain [23]; however, it is important to note that we do not yet have atomic resolution structures for full length human LRRK2.

3. Expert opinion

LRRK2 is a challenging target to engage, due partly to the complexity of its domain organization and the relative dearth of information relating to the physiological role that it performs/validated readouts for function. Preclinical experiments in nonhuman primates using LRRK2 kinase inhibitors have highlighted that there may be on-target effects associated with directly targeting the kinase activity of LRRK2 [24]. Despite this, there are a number of active drug discovery programs targeting LRRK2 [25]. Moving beyond pre-clinical studies, the next step for testing LRRK2 inhibitors will be the move into phase I trials to examine the safety of this approach in humans. If inhibitors pass key safety tests, the existence of a cohort of LRRK2 mutation carriers in the PD population, as well as presymptomatic carriers, provide a route to testing the hypothesis that inhibiting LRRK2 can slow the progression of Parkinson’s, or alternatively delay the onset of symptoms and clinical diagnosis. If these phase II/III trials are successful as a proof of concept, further trials will then be required to examine whether this approach can be applied to the wider population of Parkinson’s patients without genetically defined disease.

Beyond kinase activity, it is critical that a better mechanistic understanding of the other activities of LRRK2, and impact of mutations on these, is arrived at. This is especially important given that there is not yet a consensus as to whether alterations in kinase activity are the central event in PD pathogenesis linked to LRRK2. Equally, although there is evidence that LRRK2 kinase activity is important to neuronal cell death, it is not firmly established that inhibiting LRRK2 kinase activity will correct the underlying etiological insult that leads to nigral cell death – even in cases with the G2019S mutation. As such, a deeper understanding of the other activities of LRRK2 is an absolute requirement and may provide alternative routes to targeting LRRK2 in human disease if LRRK2 kinase inhibitors do not prove efficacious in clinical trials. The GTPase activity of LRRK2 in particular offers itself as a potential alternative target, especially in the light of advances in our ability to target GTPases [26].

Looking beyond the protein activities of LRRK2, much progress remains to be made in dissecting the links between this gene and human disease – both in PD (especially in terms of the GWA) and in disorders such as IBD and leprosy. Deciphering exactly how variation at the LRRK2 locus contributes to disease risk for these disorders has the potential to open new avenues of investigation for modulating LRRK2 activity, with insight into expression and splicing events linked to LRRK2 being of particular interest [7].

In the decade since LRRK2 came to prominence as a putative drug target for human disease, a huge amount of progress has been made toward understanding how this protein functions within the cell and how it contributes to disease. There remain, however, significant hurdles to overcome in order to successfully engage LRRK2, even in the narrow context of kinase activity and Parkinson’s, and it will be important over the coming years to consider this protein in a holistic fashion – exploring the full range of LRRK2 activities, and the full extent of its involvement in disease. This, in turn, will provide the best chance of success in converting this protein from a putative drug target into a validated one.

Declaration of interest

P. A. Lewis serves as a member of the BBSRC pool of experts, and is a member of the Parkinson’s UK Grant Assessment Panel 1, the Scientific Advisory Panel for Ataxia UK and the Research Strategy Committee of the Multiple Sclerosis Society. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Additional information

Funding

E.Greggio has received support from the Michael J. Fox Foundation for Parkinson’s Research. S. Cogo is supported by a studentship from the Fondazione CARIPARO and by a fellowship from the Fondazione Aldo Gini. P. A. Lewis has received support from the Medical Research Council [grants MR/N026004/1 and MR/L010933/1], Parkinson’s UK [fellowship F1002], the Biotechnology and Biological Sciences Research Council [CASE studentship BB/M017222/1] and Michael J. Fox Foundation for Parkinson’s Research.