LRRK2 signaling pathways: the key to unlocking neurodegeneration?

Opinion

LRRK2 signaling pathways: the key to unlocking neurodegeneration? Daniel C. Berwick and Kirsten Harvey Department of Pharmacology, School of Pharmacy, University of London, 29–39 Brunswick Square, London, WC1N 1AX, UK

Mutations in PARK8, encoding leucine-rich repeat kinase 2 (LRRK2), are a major cause of Parkinson’s disease. We contrast data suggesting that changes in LRRK2 activity cause alterations in mitogen-activated protein kinase, translational control, tumor necrosis factor a/Fas ligand and Wnt signaling pathways with the cell biological functions of LRRK2 such as vesicle trafficking. Despite scarce in vivo data on cell signaling, involvement in diverse cell biological functions suggests a role for LRRK2 as an upstream regulator in events leading to neurodegeneration. To stimulate discussion and give direction for future research, we further suggest that despite the importance of the catalytic activity for cytotoxicity, the main cellular function of LRRK2 is linked to assembly of signaling complexes. Parkinson’s disease Parkinson’s disease (PD) is the second most common neurodegenerative disorder, estimated to afflict 6 million people worldwide [1,2]. Typically, PD is a disease of the elderly, with an age of onset between 60 and 80 years [3]. The classic symptoms of this disorder are resting tremor and bradykinesia [4], but cognitive and psychiatric symptoms such as depression and dementia are also common [2]. The typical hallmarks of PD in post-mortem brain tissue are loss of dopaminergic neurons and the deposition of cytoplasmic eosinophilic inclusions (Lewy bodies) enriched in the protein a-synuclein in the substantia nigra [2]. The cell biology of parkinsonian neurodegeneration remains poorly understood, but a protracted and progressive loss of neuronal cell function that ultimately culminates in cell death can be assumed. Impairments in a number of cellular systems have been suggested to underlie idiopathic PD (Figure 1). These include mitochondrial defects, increased oxidative stress, failed proteolytic pathways, impairments in autophagy, synaptic dysfunction, and disruption to axonal transport mechanisms [1,3,5,6]. However, although it is very likely that these cell biological defects all contribute during the process of neurodegeneration, the initial molecular events that trigger PD remain elusive. Genetics of PD The identification of mutations in PARK genes in families with hereditary forms of the disease has revolutionized the study of PD [7]. Understanding the function of the proteins encoded by PARK genes will hopefully further our underCorresponding author: Harvey, K. ([email protected]).

standing of the mechanisms leading to inherited and idiopathic forms of PD. In particular, modulating defects caused by mutated PARK genes could lead to new disease treatments that do not simply replace lost dopamine, but arrest neurodegeneration. For example, the PARK1/4 gene encoding the main component of Lewy bodies, asynuclein, is undoubtedly relevant to idiopathic PD. The expanding number of PARK genes has been reviewed in detail elsewhere [3,7]. Mutations in PARK8, encoding leucine-rich repeat kinase 2 (LRRK2), cause autosomal dominant PD and are by far the most common cause of familial PD, accounting for as much as 40% of all cases in some populations [8] and up to 5% of apparently sporadic cases [8]. In addition, they seem to be of particular relevance to idiopathic PD, because patients with PARK8 mutations almost invariably present features of typical idiopathic PD, such as late-onset, typical motor deficits and brain pathology [9]. Therefore, we suggest the involvement of LRRK2 in biological processes that are central to triggering idiopathic PD. LRRK2 is a signal transduction protein LRRK2 belongs to the ROCO family of proteins, characterized by the unique combination of a Ras in complex proteins (Roc) domain with intrinsic GTPase activity, and a Cterminal of Roc (COR) domain [10]. Throughout nature, Roc and COR domains are always expressed together in the same molecule, thus suggesting a combined function for these domains. The kinase domain of LRRK2 is situated C-terminal of the COR domain of LRRK2 and is most similar to that of a paralog, LRRK1, but also showing sequence identity to receptor-interacting protein kinases (RIPKs) and, more distantly, certain mitogen-activated protein kinase kinase kinases (MAPKKKs) [11]. The presence of GTPase and protein kinase domains in the molecule has inevitably led to the suggestion that LRRK2 is a cell signaling protein. Initial biochemical studies favored a mechanism whereby LRRK2 GTPase activity controlled kinase activity, and the ‘output’ of LRRK2 was the phosphorylation of substrate proteins [12]; however, recent advances suggest a more complex relationship between LRRK2 kinase and GTPase activities [13]. In particular, the LRRK2 Roc domain can be phosphorylated by the kinase domain [14–16], and mutation of a Roc domain autophosphorylation site to a phosphothreonine-mimicking glutamate reduces GTP binding [15]. In addition, further protein–protein interaction domains in LRRK2, such as the leucine-rich repeat (LRR) and WD40 propeller

0962-8924/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2011.01.001 Trends in Cell Biology, May 2011, Vol. 21, No. 5

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Key: Microtubules

Damaged mitochondrion

Dynein complex

Secretory vesicle

Signalling activity Endosome

(i)

Neurotransmitter Multi-vesicular body

Transmembrane receptor

(ii)

Autophagosome

Ligand

(iii)

Lysosome

Ligand-bound receptor Mis-folded/aggregated protein

(iv) Terminal bouton

(vi)

(v) Retrograde axonal transport

(vii)

Nu

Soma

cle u

s

(viii)

TRENDS in Cell Biology

Figure 1. Cell biological processes linked to LRRK2 and neurodegeneration. A schematic neuron divided into two sections: the soma and axonal terminal bouton. For simplicity, postsynaptic/dendritic events are not included, although these include many of the same processes described in axons. (i) PARK8 mutations have been suggested to reduce neurotransmitter release, weakening synaptic strength. (ii) Old or damaged mitochondria become leaky, poisoning the cell with reactive oxygen species. Mitochondrial impairments have long been linked to PD, and LRRK2 is suggested to localize to these organelles. (iii) Altered cell signaling pathways, which are activated by the binding of an extracellular ligand to a transmembrane receptor, are linked to PD. Activated receptors are rapidly subjected to endocytosis, another process linked to LRRK2. (iv) Neurons are acutely dependent on the long-distance, microtubule-based, transport of cargo between the soma and axons and dendrites. This includes the retrograde transport (e.g. to the soma) of damaged mitochondria and endocytic vesicles that contain activated transmembrane receptors (‘signalosomes’). LRRK2 interacts with microtubules, suggesting that these trafficking events might be impaired by PARK8 mutations. (v, vi) In the soma, endocytic vesicles are internalized into multivesicular bodies (MVBs), thus terminating signaling, and damaged mitochondria are engulfed by autophagosomes. LRRK2 affects autophagy, and this process is frequently deregulated in neurodegeneration. (vii) Aggregates of misfolded protein, which might be related to a dysfunction of proteolytic pathways as proposed in PD, are also targeted for autophagy. Protein aggregates are a hallmark of neurodegenerative disease, not least the a-synuclein-rich Lewy bodies seen in PD. (viii) The processes of endocytosis and autophagy both terminate at lysosomes. Here, both MVBs and autophagosomes release their contents, which are destroyed by the acidic intralysosomal environment. It is important to note the interconnectedness of these processes. Therefore, any subtle impairment in one system might eventually affect many other systems.

motifs, create scope for a third model, in which LRRK2 functions as a scaffolding protein. These are particularly well described in MAPK signaling (e.g. kinase suppressor of Ras and the c-Jun N-terminal kinase (JNK)-interacting proteins). MAPK scaffolding proteins have been shown to nucleate preformed signaling complexes, often at precise subcellular locations, thus ensuring that pathways are only activated by appropriate upstream signals [17]. Therefore, three possible models for the function of LRRK2 as a signaling protein are suggested (Figure 2). These roles of LRRK2 as kinase, GTPase and scaffolding protein are not mutually exclusive. The cell biological functions of LRRK2 have been subject to intense investigation. In particular, studies in cultured cells have established that overexpression of LRRK2 causes apoptosis, and that this is enhanced by pathogenic PARK8 mutations [12]. Furthermore, both the kinase and GTPase activities of LRRK2 are required for inducing cell death [18,19]. These results are certainly encouraging, because they provide a rationale for how pathogenic mutations in the GTPase and kinase domain might cause neurodegeneration. However, cell death represents the final 258

stage of neurodegeneration, and it remains to be seen whether these studies will shed light on the crucial early stages of PD. Cell biological studies using overexpression and knockdown in cell lines, and studies on knockout and transgenic Caenorhabditis elegans, Drosophila and mouse model organisms, have also implicated LRRK2 in decreased neurite outgrowth, mitochondrial dysfunction, increased protein translation, altered endocytosis and autophagy – events that precede cell death [20,21]. Although there remains a lack of corroborating in vivo data to establish the cell signaling events leading to these cell biological effects, the evidence that LRRK2 function impinges on neurite outgrowth, protein translation, vesicle trafficking and autophagy is fairly convincing. The important question is: how is LRRK2 affecting these processes? Signal transduction mechanisms control a variety of cell biological processes. Furthermore, diseases caused by pathologic mutations affecting the activity of GTPases and kinases are not without precedence. For example, gain-of-function mutations in Ha-Ras and B-Raf [22,23] or the loss-of-function mutation in Akt2 [24] are important in oncogenesis and diabetes, respectively. Thus, if patho-

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LRRK2 Mutations, possible modes of action

(a) Common mutations R1441C/G/H Y1669C G2019S I2020T

LRR

Roc

COR

Kinase

WD40

Kinase

WD40

(b) Possible mechanisms of action Kinase

Autophosphorylation LRR

Roc

COR

Substrates

GTPase

GDP LRR

Roc

COR

Kinase

WD40

COR

Kinase

WD40

Kinase

WD40

X GTP LRR

Roc

Effector

Scaffold LRR

Components A A,B and C of signalling cascade

Roc

B

COR

C TRENDS in Cell Biology

Figure 2. LRRK2 domains, mutations and modes of action. (a) A graphical representation of the domain structure of LRRK2, with locations of known missense mutations depicted. There are five major domains in LRRK2: LRR, Roc, COR, a kinase domain (kinase) and a WD40 domain (WD40). The LRR and WD40 domains are believed to be important for protein–protein interactions, whereas the Roc and COR domains [10] influence LRRK2 GTPase activity. The sequence of the kinase domain has the highest sequence similarity to receptor-interacting serine/ threonine protein kinase (RIPK) and MAPKKK kinases [11]. Many amino acid substitutions resulting from genetic variation have been found throughout LRRK2; however, the vast majority do not appear to segregate with PD (red circles). By contrast, the labeled mutations within the Roc, COR and kinase domains have been proved to be pathogenic. (b) Possible modes of action. (i) LRRK2 could function as a conventional kinase, phosphorylating and thus activating or inhibiting substrates. In addition, self-regulation by autophosphorylation is probable. (ii) LRRK2 could function like a small GTPase, under the control of GTP or GDP binding. When GDP-bound it is inactive, and unable to bind effector proteins (upper panel). However, when it is GTP-bound, LRRK2 can elicit downstream signaling (lower panel). (iii) LRRK2 might function as a scaffolding protein, serving to pre-assemble signaling complexes at specific subcellular locations. Note that these three possibilities are not mutually exclusive.

logic PARK8 mutations alter the function of LRRK2 in signaling, this could explain not only the apparent ‘upstream’ role of LRRK2 in the etiology of PD, but also the range of cell biological defects linked to parkinsonian neurodegeneration. Interestingly, although single-nucleotide polymorphisms have been found throughout PARK8, the only changes that have been proven to cause PD are located within exons encoding the Roc, COR and kinase domains [8]. This suggests that pathogenic PARK8 muta-

tions compromise the signaling functions of LRRK2. Thus, we suggest a role for LRRK2 as a signaling protein in healthy cells, and that a disruption of these pathways leads to aberrant cell biological functions and ultimately neurodegeneration. In this review, we appraise the experimental evidence that LRRK2 might function as a signal transduction protein. In particular, we examine data linking LRRK2 to MAPK cascades, but also cover translational control via the mammalian target of rapamycin complex-1 (mTORC1), tumor necrosis factor-a (TNFa)/Fas ligand (FasL) cascades, and Wnt signaling pathways. This is still an emerging field of research, and therefore we are cautious in our interpretations. Nonetheless, we suggest that the evidence indicates that LRRK2 can affect the activity of a variety of signaling cascades, and suggest that this points towards a role for LRRK2 in processes that influence many signaling pathways (e.g. endocytosis or axonal transport) (Figure 1). LRRK2 in MAPK signaling pathways MAPK pathways are undoubtedly the best-known cell signaling cascades, and have been linked to the regulation of a plethora of cell biological phenomena. In mammalian cells, there are four MAPK pathways [17] (Box 1, Figure I). In principle at least, the deregulation of any of these cascades could lead to neurodegeneration, and altered expression of components of most MAPK pathways have been observed in post-mortem tissue from patients with PD (Box 1). This alone is sufficient to explain why researchers interested in linking LRRK2 to signal transduction have focused on MAPK pathways. However, the observation of sequence similarity between the kinase domain of LRRK2 and those of certain MAPKKKs stimulated interest further. Most of the work investigating roles for LRRK2 in MAPK pathways has taken the form of overexpression studies in cultured cell lines. In most studies, MAPK activation was assessed in cells transiently expressing wild-type or mutant LRRK2, using western blotting with phosphospecific antibodies [12,25–29]. One study included the use of cell lines stably expressing exogenous LRRK2 [25]. Frequently investigated pathogenic LRRK2 mutants include R1441C, Y1699C and G2019S, which fall within the Roc, COR and kinase domains, respectively. However, artificial mutants have also been designed to abrogate the kinase activity of LRRK2 (‘kinase-dead’ mutations) or to prevent the Roc domain from binding guanine nucleotides (‘GTP-non-binding’ mutations). Studies into the effects of LRRK2 on extracellular signal-regulated kinase 1/2 (ERK1/2) activity are conflicting (summarized in Table 1). Although we believe that emerging trends support a role of wild-type and mutant LRRK2 in activating this signaling pathway [12,25,26], no conclusions can be made regarding the modulatory effects of LRRK2 mutants on ERK1/2 activation in comparison to wild-type LRRK2 [12,25–27]. The only study that found a decrease in ERK1/2 phosphorylation upon wild-type and mutant LRRK2 expression also found that wild-type LRRK2 protected cells from H2O2-induced cell death. This protective effect was abrogated by ERK1/2 pathway inhibitors, suggesting an activation of ERK1/2 signaling upon 259

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Box 1. MAPK cascades and their links to Parkinson’s disease MAPK cascades are well-defined signaling pathways that obey an ordered structure, consisting of a MAPKKK upstream of a MAPKK, which is in turn upstream of a MAPK. The MAPK is usually considered the effector kinase because, when activated, it phosphorylates many downstream targets. However, there is growing evidence of an additional tier of MAPK signaling, the MAPK-activated protein kinases (MAPKAPs) [17,49]. In many systems, MAPKKKs are activated by small GTPases such as Ras and Rac, and by other kinases that are inevitably categorized as MAPKKKKs. Note also that there can be positive crosstalk at the level of MAPKKK signaling to MAPKKs, whereas MAPK activation can lead to negative feedback and suppression of other pathways [49]. In mammals, all four major MAPK pathways – ERK1/2, p38 MAPK, JNK, and ERK5 [17] – have been linked to PD. In particular, postmortem studies show ERK1/2, p38 MAPK and JNK to be activated in PD brains [50]. These observations are supported by the observations that ERK1/2 and JNK are activated by 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [51–53], compounds commonly used to induce neurodegeneration in animal PD models. p38 MAPK is also activated by MPTP [54].

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Activation of the generally pro-apoptotic p38 MAPK and JNK cascades in PD brains is not unexpected [49]. ERK1/2 activation appears counterintuitive, as this pathway is usually pro-survival and essential for normal neuronal function [55,56]. Nevertheless, ERK1/2 activity is central to cell death elicited by dopamine treatment [57], and a growing number of in vitro experiments indicate that there are neurotoxic effects of increased ERK1/2 activity [55]. These include work in dopaminergic neurons showing that overexpressed ERK1, p38a or JNK1 are sufficient to elicit apoptosis. Intriguingly, in all cases this can be rescued by co-transfection of Parkin, encoded by PARK2, which is disrupted in recessive forms of early onset PD [58]. ERK5 activity has not been investigated in PD brains. However, a growing number of studies in a variety of cell types, including dopaminergic neurons, demonstrate a pro-survival role for ERK5, and it has also been suggested that this pathway can elicit cell death under certain circumstances [59,60]. Thus, there is particularly strong evidence linking ERK1/2 and JNK signaling to PD, and roles for p38 MAPK and ERK5 cannot be excluded. Because these pathways control a multitude of cellular processes, MAPK pathways make interesting candidates for mediating the effects of pathogenic LRRK2 mutations (Figure I).

MAPK pathways

Stimulus

GTPase

MAPKKKK

Mitogens, synaptic activity

Inflammation, death receptors, stress Rac

Ras

Mitogens, stress

PAK1/2

MAPKKK

A/B/C-Raf

MLK3, TAK1

MEKK1/4, MLK3, ASK1

MEKK2/3

MAPKK

MEK1/2

MKK3/6

MKK4/7

MEK5

ERK1/2

p38α/β/γ/δ

JNK1/2/3

ERK5

MAPK

Substrates e.g. MAPKAPs, transcription factors TRENDS in Cell Biology

Figure I. Key signaling components of the four major MAPK pathways.

LRRK2 overexpression under conditions of oxidative stress [27]. To date, the only study looking into ERK5 activation found no effect of wild-type or mutant LRRK2 on ERK1/2 or ERK5 signaling in one expression system, but reported an increase in ERK5 activity when wild-type or mutant LRRK2 was overexpressed in another [12]. Despite the fact that results on the effect of LRRK2 on ERK1/2 and ERK5 signaling are mixed, it is interesting to note that the majority of studies favor ERK1/2 activation upon LRRK2 overexpression. We acknowledge that this trend requires further confirmation, and suggest that the investigation of the effect of LRRK2 on ERK1/2 and ERK5 signaling should include studies performed under conditions of pathway activation and inhibition, as well as the effects of LRRK2 knockdown on ERK1/2 and ERK5 signal260

ing. After establishing these conditions, an investigation of the relevance of LRRK2 kinase, GTP binding, GTPase activity and PD mutants for pathway activation will be easier to interpret. At present, there is no clear evidence linking LRRK2 catalytic activity to the activation of ERK1/ 2 and ERK5 pathways, suggesting to us that LRRK2 is more likely to function as a scaffold in these pathways. In this model, the role of LRRK2 would be to ensure the correct subcellular localization of cell signaling components required for pathway activation. Investigations of the roles for LRRK2 in the ‘stressactivated’ MAPK cascades, p38 MAPK and JNK, showed convincing evidence of interactions between LRRK2 and signaling components [28–30], but no clear effect on the activity of these pathways [12,25,27–29]. Biochemical

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Table 1. Summary of reports on the effects of LRRK2 on MAPK pathway activation System a Overexpression (R1441C/G, Y1699C, G2019S, I2020T)

Cell Type HEK293

SH-SY5Y PARK8 mutation carriers with or without PD (G2019S) Overexpression (G2019S versus non-transfected cells and wild-type kinase dead mutants) Overexpression (Y1699C)

Overexpression Overexpression (G2019S) Overexpression (transient and inducible stable) (R1441C, G2019S)

Leukocytes from blood samples SH-SY5Y

HEK293

HEK293 HEK293 HEK293

Effect " p-ERK1/2, " p-ERK5 (independent of GTP-binding; all wild-type and mutant constructs stimulated ERK1/2/5 phosphorylation) p-p38 and p-JNK, no change " p-JNK (Independent of GTP-binding) p-ERK1/2, p-p38 and p-ERK5, no change # p-JNK (but paralleled by decreased total JNK protein); p-p38 and p-ERK, no statistically significant changes " p-ERK1/2

Kinase activity required b No (K1906M)

Reference [12]

N/A (K1906M) No (K1906M) N/A (K1906M) N/A

[32]

No (K1906M)

[26]

#p-ERK1/2 (effect further enhanced by Y1699C mutant compared with wild-type, which also suppressed H2O2-mediated ERK1/2 activation) p-JNK, no change p-p38 and p-JNK, no change p-p38 and p-JNK, no change

No (kinase domain deletion)

[27]

N/A N/A N/A

[28] [29]

" p-ERK1/2 (Partially dependent on kinase activity, G2019S less effective at inducing increased p-ERK1/2 than wild-type; all wild-type and mutant constructs stimulated ERK1/2 phosphorylation) p-p38 and p-JNK, no change

No (K1906N)

[25]

N/A (K1906N)

a

All studies except one [26] included LRRK2 wild-type and some used pathogenic LRRK2 mutants as indicated.

b

Absolute requirement of kinase activity for MAPK pathway activation. Artificial kinase-dead mutants are indicated. N/A, not applicable.

experiments suggested that LRRK2 binds to three MAPKKs (MKK3, MKK6, and MKK7) [28,29] and four MAPK scaffolding proteins, the JNK-interacting proteins (JIPs) 1–4 [30]. MKK3 and MKK6 phosphorylate and activate p38 MAPKs, and MKK7 plays an identical role in JNK signaling. Similarly, JIPs 1–3 are involved in the JNK pathway, and JIP4 is involved in p38 MAPK signaling. Further experiments suggested that the interactions between LRRK2 and stress-activated MAPK signaling components might be of biological relevance. Overexpression of MKK6 in HEK293 cells caused the redistribution of LRRK2 to membranes, and co-transfection of LRRK2 with JIP1 increased JIP1 steadystate protein levels [29,30]. These interactions create a strong link between LRRK2 and both the p38 MAPK and JNK pathways. However, there is a remarkable lack of evidence for any effect of LRRK2 on the activity of these cascades. No fewer than four overexpression studies in HEK293 cells reported no change in p38 MAPK phosphorylation [12,16,25,29], and five failed to find any effect on JNK activity [12,16,25,27,29]. The only study to date in SH-SY5Y cells found that LRRK2 overexpression activates JNK signaling in this system, although, in accord with studies in HEK293 cells, no effects on p38 MAPK activity were observed [12]. These studies have not yet provided a definitive link between LRRK2 and stress-activated MAPK pathways. Nonetheless, changes in the localization of LRRK2 upon coexpression with MKK6 are intriguing, and fuel the need for further investigations of interplay between MAPK cascades and LRRK2 [31]. Research linking LRRK2 to MAPK pathways is clearly still at a preliminary stage, with a lack of consensus on

many observations. However, we would contend that there is sufficient evidence supporting a link between LRRK2 and MAPK cascades to encourage focused experiments on well-defined questions (Box 4). In particular, we wish to draw attention to studies showing ERK1/2 activation by LRRK2 [12,25,26], and interactions between LRRK2 and JNK/p38 signaling components [28–30]. In general, experimental methods could be improved by the use of positive controls for MAPK pathway activation, such as constitutively active mutants of established upstream MAPK components. This would allow greater certainty in the description of results obtained upon overexpression of LRRK2. Changes in MAPK phosphorylation should be quantified in optimized assays with sufficient sensitivity whenever possible, to obtain statistically significant data. We also encourage loss-of-function studies, because it is possible that the effect of LRRK2 knockdown will have a clearer effect than overexpression on MAPK pathway activation. In addition, only one study showed no effect of LRRK2 on phospho-c-Jun levels under activating conditions following treatment with anisomycin [12], but no experiments have been performed to date under conditions of MAPK pathway activation using upstream activators (e.g. growth factors). In our opinion, this is particularly important, because LRRK2 might not have any activating effect under basal conditions, but when a pathway is already active, LRRK2 might be a potent positive or negative modulator. Indeed, to date, the possibility that LRRK2 might inhibit MAPK pathways does not appear to have been investigated at all. Experiments under activating or inhibiting conditions might also help 261

Opinion to explain differences observed in the published data. Experimental conditions in the investigations conducted to date might have had different effects on MAPK activation because culture conditions influence the activity of MAPK pathways (e.g. the amounts and treatment of serum in culture media affect the amounts of active growth factors). Importantly, there is also a lack of in vivo data to support these studies. The observation of altered MAPK signaling in LRRK2 transgenic mice or post-mortem brain tissue would inspire greater confidence. To date, the sole in vivo investigation of MAPK pathway activity was a pilot study performed in leukocytes from patients with PD carrying the G2019S mutation [32]. The data suggested trends that might be in agreement with some of the in vitro observations described above, but such in vivo work needs further investigation. LRRK2 in other signal transduction pathways A number of studies have investigated LRRK2 function in three other signaling paradigms: translational control, tumor necrosis factor-a (TNF-a)/Fas Ligand (FasL) pathways and Wnt cascades (how deregulation of these pathways might cause PD is outlined in Box 2). LRRK2 was first implicated in increased protein synthesis by genetic studies in Drosophila melanogaster [33,34]. Flies lacking dLRRK, the Drosophila ortholog of LRRK1 and LRRK2, showed decreased phosphorylation of the translational control protein, initiation factor-4E binding protein-1 (4E-BP1). 4E-BP1 phosphorylation correlates positively with the activity of the protein synthesis machinery [35]. In agreement with this finding, LRRK2 overexpression in mammalian HEK293 cells was shown to increase 4E-BP1 phosphorylation [33]. Nonetheless, in vitro kinase assays suggest that the phosphorylation of 4E-BP1 by LRRK2 is not direct [36]. An additional link of dLRRK2 to increased protein synthesis was made by the observation that LRRK2 can repress certain microRNAs that are able to inhibit translation [37]. The genetic aspect of this story is certainly strong, although studies in Drosophila come with the obvious caveats that dLRRK is also the homolog of LRRK1, and that establishing LRRK2 as

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‘upstream’ of 4E-BP1 gives no clue as to how far upstream it is. Nevertheless, this finding warrants further investigation in mammalian systems. TNF-a/FasL pathways A single study has linked LRRK2 to the extrinsic apoptotic TNF-a and FasL pathways [38]. Overexpressed LRRK2 was found to bind the Fas-associated protein with death domain (FADD). Interestingly, this interaction was strengthened by four pathogenic PARK8 mutations, but brought back to the interaction strength of the wild-type LRRK2 FADD interaction by the introduction of an artificial kinase-dead mutation [38]. This observation is in accord with previous studies demonstrating the importance of kinase activity for the cytotoxic effects of LRRK2 in cell systems [19]. Furthermore, the cytotoxicity caused by LRRK2 overexpression could be blocked by inhibiting FADD or downstream signaling [38], providing a potential alternative therapeutic target. These data are undoubtedly exciting, because they suggest a plausible mechanism for the apoptosis elicited by overexpressed LRRK2. However, although this story provides an elegant explanation for the cell death caused by PARK8 mutations, it remains difficult to extend this mechanism to cover early events in neurodegeneration preceding cell death, such as synaptic dysfunction or axonal degeneration. Wnt signaling pathways LRRK2 has been linked to Wnt signaling by three observations. Firstly, LRRK2 has been reported to interact with disheveled (DVL) proteins 1–3 [39] and glycogen synthase kinase-3 (GSK-3) [40], both of which are central components of Wnt signaling cascades [41]. Secondly, pathogenic PARK8 mutations affecting residues in the Roc and COR domains were found to modulate the interaction with DVL proteins [39], whereas the G2019S mutation affected the interaction with GSK-3 [40]. Thirdly, microarray studies examining the effect of knocking down LRRK2 reported altered expression of a number of Wnt signaling proteins [42]. We acknowledge that the link between LRRK2 and Wnt cascades is at an early stage, but the possibility that

Box 2. Links between parkinsonian neurodegeneration and translational control, TNF-a/FasL and Wnt signaling Translational control, TNF-a/FasL and Wnt signaling mechanisms have been linked to LRRK2, and deregulation of these pathways has the potential to affect neurodegeneration. Correctly regulated protein synthesis is undoubtedly a requirement for most cellular processes, and a subtle defect in translation could lead to impairments in any number of cell biological systems, which might be expected to elicit a protracted loss of cellular function. In neurons, perturbed translation is particularly likely to cause defects, because certain types of long-term potentiation are acutely dependent on protein synthesis [61]. It is therefore not surprising that a number of studies linked deregulated protein translation to neurodegeneration [62], and that rapamycin, an inhibitor of the masterregulator of translational control mTORC1 [35], is neuroprotective in animal models of PD [63]. Thus, in principle, a role for LRRK2 in translational control is consistent with an upstream defect caused by PARK8 mutations leading to neurodegeneration. Signaling elicited by FasL and TNFa is strongly linked to apoptosis and neurodegeneration [64]. These death receptor ligands activate an extrinsic apoptotic pathway, initiating the recruitment of caspase 8 to plasma membrane complexes via an interaction with FADD [65]. This

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process results in the activation of a caspase cascade, resulting in the proteolytic cleavage of a variety of proteins ultimately leading to controlled cell death. Extrinsic cell death pathways are undoubtedly central to PD, and it is fascinating that they should have been linked to LRRK2. Nevertheless, it is generally accepted that cell death is a late event in neurodegenerative processes, and we would suggest that these pathways are unlikely to be involved in the initial cellular impairments through which PARK8 mutations trigger PD. Wnt cascades have also been implicated in PD. For example, Parkin, the product of the PARK2 gene, has been reported to inhibit Wnt signaling [66], and the central Wnt component GSK-3 and the Wnt signaling-activated transcription factor Nurr1 have been linked to PD in genetic screens [67,68]. Deregulated Wnt signaling represents a feasible initiating event in neurodegeneration. Not only are Wnt signals important in the acute regulation of synaptic function, but deregulation in the longer term would also lead to altered gene expression and disrupted vesicle trafficking pathways [69]. Tantalizingly, a unifying hypothesis for the etiology of Alzheimer’s disease has already been constructed around deregulated Wnt signaling [43].

Opinion LRRK2 might influence these pathways is fascinating, considering that deregulation of Wnt signaling has been implicated in the earliest stages of Alzheimer’s disease, the most common neurodegenerative disorder [43]. Can signaling data predict function? The evidence supporting a role for LRRK2 in signaling pathways is largely preliminary, and the precise function of LRRK2 remains to be defined. However, it is remarkable that this protein has already been linked to such a variety of signal transduction pathways. In our opinion, LRRK2 does not appear to be a genuine MAPKKK. This is supported by the observation that many of the reported effects of LRRK2 on MAPKs appear to be independent of LRRK2 kinase activity (Table 1). In addition, LRRK2 does not appear to phosphorylate MKK3, MKK6 or MKK7 in vivo [28,29], despite the apparently robust interactions of LRRK2 with these MAPKKs. Indeed, a growing body of evidence supports the idea that LRRK2 might not phosphorylate heterogeneous substrates at all, and that the output of LRRK2 might be via the GTPase domain (Box 3). The number of different signaling pathways suggested to be under the control of LRRK2 is fascinating, and suggests an upstream role for LRRK2 in cell signaling, or alternatively a function that can influence multiple pathways. One intriguing possibility is that LRRK2 has a role in endocytosis, a process crucially important in all cell signaling paradigms that involve the activation of a plasma membrane receptor by an extracellular ligand [44]. In response to ligand binding, signaling pathways are activated at the plasma membrane. Activated receptors are rapidly internalized and trafficked further into the cell via the endosomal system, until they ultimately reach the lysosome and are degraded [44,45]. Importantly, endocytosed receptors continue to signal during their transit through the endosomal system [44,45]. Indeed, activated receptors have been reported to activate different path-

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ways at different subcellular locations during their journey into the cell [44,45]. It is only once receptors reach the lysosome that signaling is switched off entirely [44,45]. Evidence to support a role for LRRK2 at the interface of signal transduction and endocytosis comes from studies showing a direct link between LRRK2 and endocytosis [46], suggestions of increased kinase activity of LRRK2 at membranes [31], and effects of LRRK2 overexpression on autophagy [26], a process requiring lysosomes. As we outline above, lysosomes are the final stage in endocytosis, and altered endocytosis would be predicted to affect the availability of lysosomes to the autophagic machinery. In addition, endocytosis and exocytosis of synaptic vesicles is fundamentally important for synaptic function, another process affected during neurodegeneration. Interestingly, a number of papers have also implicated LRRK2 in microtubule function [40,47,48]. Endocytosis is largely dependent on the cytoskeleton, especially in neurons, where axonal transport requires the trafficking of vesicles over long tracts of stabilized microtubules [45]. In essence, deregulation of endocytosis, microtubule stability and cell signaling could happen long before any more obvious neurodegenerative changes can be detected. This would be consistent with the progressive loss of cell function that is characteristic of neurodegeneration (Figure 1). Concluding remarks In conclusion, we believe that the published data are more supportive of an upstream role for LRRK2 in functions influencing a variety of signal transduction cascades rather than any one pathway. We suggest therefore that LRRK2 might function as a scaffold at the interface of signaling, endocytosis and microtubule function. We believe this to be the best interpretation of the current data that allows for an inclusive explanation of how PARK8 mutations cause early defects in neuronal function, leading to the progressive neurodegeneration observed in PD.

Box 3. LRRK2 or LRR GTPase 2? Three principal mechanisms for the participation of LRRK2 in signaling pathways are suggested by the LRRK2 domain structure: (i) as a kinase phosphorylating downstream substrates; (ii) as a GTPase; and/or (iii) as a scaffolding protein. Thus, the idea that LRRK2 kinase activity might not be required for ERK1/2 activation (Table 1) and the recent suggestion that LRRK2 GTPase activity might be the more likely output of this protein [13] are intriguing. This idea is supported by the observation that a major site of LRRK2 autophosphorylation is the Roc domain, suggesting that GTPase activity is downstream of kinase activity [14–16]. Furthermore, it is curious that despite extensive substrate identification studies in a number of laboratories, including a powerful kinase substrate tracking and elucidation (KESTREL) screen [70], only a handful of LRRK2 substrates have been proposed. These include moesin and 4E-BP1 [33,70]. However, LRRK2 does not phosphorylate moesin in cells [71], and in vitro, 4E-BP1 is poorly phosphorylated by LRRK2 [36], suggesting that neither of these proteins are genuine LRRK2 substrates. Other candidate substrates, such as a-synuclein, b-tubulin and Foxo1, have been suggested, but remain to be independently confirmed [48,72,73]. Remarkably, therefore, the only reproducible substrate of LRRK2 is itself.

Nevertheless, LRRK2 kinase activity is important. Cytotoxicity induced by LRRK2 overexpression, which is potentiated by pathogenic PARK8 mutations, is greatly reduced by kinase inactivation, whereas the G2019S mutation is widely accepted to increase kinase activity [19]. So how can the lack of substrates be reconciled with the importance of kinase activity? The most obvious answer is that some effects of LRRK2 do not involve phosphorylation, and for those that do, the substrates remain unidentified. Although we cannot exclude this explanation, it requires two distinct modes of action for LRRK2. We would therefore suggest an alternative, more parsimonious explanation. We suggest that the GTPase domain of LRRK2 might be the only in vivo target for LRRK2 kinase activity. In this model, autophosphorylation would be required for GTPase domain function (perhaps more as a maturation step than as regulation per se). Kinasedead LRRK2, with no autophosphorylation, would simply represent LRRK2 with decreased output, and hence less cytotoxicity. Moreover, should autophosphorylation prove to decrease GTPase domain activity, it would be expected that the G2019S mutant, which has higher autophosphorylation levels, would lower this activity, thereby eliciting an identical effect to the Roc and COR mutations R1441C and Y1699C. In contrast to this hypothesis, one recent study showed no effect of the G2019S mutant on GTPase activity [18].

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Box 4. Unanswered questions Research into the function of LRRK2 is clearly at an early stage, and thus there are many important experiments that need to be performed. Below, we outline the questions that we believe are most pertinent for making sense of the cell signaling data published to date, and also those best suited to advancing the field. 1. The effect of LRRK2 overexpression on JNK and p38 MAPK signaling under conditions of stress. The evidence that LRRK2 might play an upstream role in stress-activated MAPK signaling is fairly strong, but the absence of any effect on downstream activity is unexpected. It is possible that LRRK2 might inhibit these pathways, and thus it is logical to examine these pathways under the conditions in which they are active, for example, following treatment with TNF-a or lipopolysaccharide. 2. The effect of LRRK2 overexpression on ERK5 activity. This pathway has been overlooked and needs attention, particularly because ERK5 pathways are inhibited by the same compounds as ERK1/2 signaling. 3. The effect of LRRK2 loss-of-function. Almost all current data have come from overexpression studies. The need for complementary loss-of-function (e.g. small interfering RNA knockdown) experiments is becoming ever greater.

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4. Requirement for LRRK2 GTPase activity. A general shift of focus away from the kinase activity of LRRK2 towards interacting proteins and the function of the RocCOR tandem domain is required. In the context of MAPK signaling, the need to examine the role of LRRK2 GTPase domain activity is particularly strong. 5. In vivo studies. With one exception, investigations into the effects of LRRK2 on signal transduction pathways have focused on cellculture models, because in vitro studies are more amenable to teasing apart the detailed mechanisms of signaling cascades. Nevertheless, investigations into the effect of LRRK2 mutations in vivo (in particular using rodent or zebrafish models) will allow for the development of hypotheses based on more physiologically relevant data. Finally, we would add a general need for quantitative studies. Although most work will require western blotting, which is a poorly quantitative method, more certainty can be achieved with statistical analysis to show that observed changes are significant. In addition, the use of positive controls (e.g. parallel transfection of established activators of MAPK pathways) would improve the interpretation of results.

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