The development of inhibitors of leucine-rich repeat kinase 2 (LRRK2) as a therapeutic strategy for Parkinson's disease: the current state of play.

Current therapeutic approaches for Parkinson's disease (PD) are based around treatments that alleviate symptoms but do not slow or prevent disease progression. As such, alternative strategies are needed. A promising approach is the use of molecules that reduce the function of leucine-rich repeat kinase (LRRK2). Gain-of-function mutations in LRRK2 account for a notable proportion of familial Parkinson's disease cases, and significantly, elevated LRRK2 kinase activity is reported in idiopathic Parkinson's disease. Here, we describe progress in finding therapeutically effective LRRK2 inhibitors, summarising studies that range from in vitro experiments to clinical trials. LRRK2 is a complex protein with two enzymatic activities and a myriad of functions. This creates opportunities for a rich variety of strategies and also increases the risk of unintended consequences. We comment on the strength and limitations of the different approaches and conclude that with two molecules under clinical trial and a diversity of alternative options in the pipeline, there is cause for optimism. LINKED ARTICLES: This article is part of a themed issue on Building Bridges in Neuropharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.8/issuetoc.

LRRK2 Biology from structure to dysfunction: research progresses, but the themes remain the same.

Since the discovery of leucine-rich repeat kinase 2 (LRRK2) as a protein that is likely central to the aetiology of Parkinson's disease, a considerable amount of work has gone into uncovering its basic cellular function. This effort has led to the implication of LRRK2 in a bewildering range of cell biological processes and pathways, and probable roles in a number of seemingly unrelated medical conditions. In this review we summarise current knowledge of the basic biochemistry and cellular function of LRRK2. Topics covered include the identification of phosphorylation substrates of LRRK2 kinase activity, in particular Rab proteins, and advances in understanding the activation of LRRK2 kinase activity via dimerisation and association with membranes, especially via interaction with Rab29. We also discuss biochemical studies that shed light on the complex LRRK2 GTPase activity, evidence of roles for LRRK2 in a range of cell signalling pathways that are likely cell type specific, and studies linking LRRK2 to the cell biology of organelles. The latter includes the involvement of LRRK2 in autophagy, endocytosis, and processes at the trans-Golgi network, the endoplasmic reticulum and also key microtubule-based cellular structures. We further propose a mechanism linking LRRK2 dimerisation, GTPase function and membrane recruitment with LRRK2 kinase activation by Rab29. Together these data paint a picture of a research field that in many ways is moving forward with great momentum, but in other ways has not changed fundamentally. Many key advances have been made, but very often they seem to lead back to the same places.

Downregulated Wnt/ß-catenin signalling in the Down syndrome hippocampus.

Pathological mechanisms underlying Down syndrome (DS)/Trisomy 21, including dysregulation of essential signalling processes remain poorly understood. Combining bioinformatics with RNA and protein analysis, we identified downregulation of the Wnt/ß-catenin pathway in the hippocampus of adult DS individuals with Alzheimer's disease and the 'Tc1' DS mouse model. Providing a potential underlying molecular pathway, we demonstrate that the chromosome 21 kinase DYRK1A regulates Wnt signalling via a novel bimodal mechanism. Under basal conditions, DYRK1A is a negative regulator of Wnt/ß-catenin. Following pathway activation, however, DYRK1A exerts the opposite effect, increasing signalling activity. In summary, we identified downregulation of hippocampal Wnt/ß-catenin signalling in DS, possibly mediated by a dose dependent effect of the chromosome 21-encoded kinase DYRK1A. Overall, we propose that dosage imbalance of the Hsa21 gene DYRK1A affects downstream Wnt target genes. Therefore, modulation of Wnt signalling may open unexplored avenues for DS and Alzheimer's disease treatment.

Pathogenic LRRK2 variants are gain-of-function mutations that enhance LRRK2-mediated repression of ß-catenin signaling.

BACKGROUND: LRRK2 mutations and risk variants increase susceptibility to inherited and idiopathic Parkinson's disease, while recent studies have identified potential protective variants. This, and the fact that LRRK2 mutation carriers develop symptoms and brain pathology almost indistinguishable from idiopathic Parkinson's disease, has led to enormous interest in this protein. LRRK2 has been implicated in a range of cellular events, but key among them is canonical Wnt signalling, which results in increased levels of transcriptionally active ß-catenin. This pathway is critical for the development and survival of the midbrain dopaminergic neurones typically lost in Parkinson's disease. METHODS: Here we use Lrrk2 knockout mice and fibroblasts to investigate the effect of loss of Lrrk2 on canonical Wnt signalling in vitro and in vivo. Micro-computed tomography was used to study predicted tibial strength, while pulldown assays were employed to measure brain ß-catenin levels. A combination of luciferase assays, immunofluorescence and co-immunoprecipitation were performed to measure canonical Wnt activity and investigate the relationship between LRRK2 and ß-catenin. TOPflash assays are also used to study the effects of LRRK2 kinase inhibition and pathogenic and protective LRRK2 mutations on Wnt signalling. Data were tested by Analysis of Variance. RESULTS: Loss of Lrrk2 causes a dose-dependent increase in the levels of transcriptionally active ß-catenin in the brain, and alters tibial bone architecture, decreasing the predicted risk of fracture. Lrrk2 knockout cells display increased TOPflash and Axin2 promoter activities, both basally and following Wnt activation. Consistently, over-expressed LRRK2 was found to bind ß-catenin and repress TOPflash activation. Some pathogenic LRRK2 mutations and risk variants further suppressed TOPflash, whereas the protective R1398H variant increased Wnt signalling activity. LRRK2 kinase inhibitors affected canonical Wnt signalling differently due to off-targeting; however, specific LRRK2 inhibition reduced canonical Wnt signalling similarly to pathogenic mutations. CONCLUSIONS: Loss of LRRK2 causes increased canonical Wnt activity in vitro and in vivo. In agreement, over-expressed LRRK2 binds and represses ß-catenin, suggesting LRRK2 may act as part of the ß-catenin destruction complex. Since some pathogenic LRRK2 mutations enhance this effect while the protective R1398H variant relieves it, our data strengthen the notion that decreased canonical Wnt activity is central to Parkinson's disease pathogenesis.

L'RRK de Triomphe: a solution for LRRK2 GTPase activity?

Leucine-rich repeat kinase 2 (LRRK2) is a central protein in the pathogenesis of Parkinson's disease (PD), yet its normal function has proved stubbornly hard to elucidate. Even though it remains unclear how pathogenic mutations affect LRRK2 to cause PD, recent findings provide increasing cause for optimism. We summarise here the developing consensus over the effect of pathogenic mutations in the Ras of complex proteins and C-terminal of Roc domains on LRRK2 GTPase activity. This body of work has been greatly reinforced by our own study of the protective R1398H variant contained within the LRRK2 GTPase domain. Collectively, data point towards the pathogenicity of GTP-bound LRRK2 and strengthen a working model for LRRK2 GTPase function as a GTPase activated by dimerisation. Together with the identification of the protective R1398H variant as a valuable control for pathogenic mutations, we have no doubt that these triumphs for the LRRK2 field will accelerate research towards resolving LRRK2 function and towards new treatments for PD.

Protective LRRK2 R1398H Variant Enhances GTPase and Wnt Signaling Activity.

Mutations in LRRK2 are a common cause of familial and idiopathic Parkinson's disease (PD). Recently, the LRRK2 GTPase domain R1398H variant was suggested in genetic studies to confer protection against PD but mechanistic data supporting this is lacking. Here, we present evidence that R1398H affects GTPase function, axon outgrowth, and Wnt signaling in a manner opposite to pathogenic LRRK2 mutations. LRRK2 R1398H GTPase domain dimerization and GTP hydrolysis were increased whereas GTP binding was reduced, leading to a decrease in active GTP-bound LRRK2. This protective variant also increased axon length of primary cortical neurones in comparison to wild-type LRRK2, whereas the R1441G LRRK2 pathogenic mutant decreased axon outgrowth. Importantly, R1398H enhanced the stimulatory effect of LRRK2 on canonical Wnt signaling whereas the G2385R risk variant, in accordance with all previously tested pathogenic LRRK2 mutants, had the opposite effect. Molecular modeling placed R1398H in close proximity to PD-causing mutations suggesting that this protective LRRK2 variant, like familial mutations, affects intramolecular RocCOR domain interactions. Thus, our data suggest that R1398H LRRK2 is a bona fide protective variant. The opposite effects of protective versus PD associated LRRK2 variants on GTPase function and canonical Wnt signaling activity also suggests that regulation of these two basic signaling mechanisms is important for neuronal function. We conclude that LRRK2 mediated Wnt signaling and GTPase function are fundamental in conferring disease susceptibility and have clear implications for therapeutic target identification.

A direct interaction between leucine-rich repeat kinase 2 and specific ß-tubulin isoforms regulates tubulin acetylation.

Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and ß-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three ß-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of ß-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.

The regulation and deregulation of Wnt signaling by PARK genes in health and disease.

Wingless/Int (Wnt) signaling pathways are signal transduction mechanisms that have been widely studied in the field of embryogenesis. Recent work has established a critical role for these pathways in brain development, especially of midbrain dopaminergic neurones. However, the fundamental importance of Wnt signaling for the normal function of mature neurones in the adult central nervous system has also lately been demonstrated by an increasing number of studies. Parkinson's disease (PD) is the second most prevalent neurodegenerative disease worldwide and is currently incurable. This debilitating disease is characterized by the progressive loss of a subset of midbrain dopaminergic neurones in the substantia nigra leading to typical extrapyramidal motor symptoms. The aetiology of PD is poorly understood but work performed over the last two decades has identified a growing number of genetic defects that underlie this condition. Here we review a growing body of data connecting genes implicated in PD--most notably the PARK genes--with Wnt signaling. These observations provide clues to the normal function of these proteins in healthy neurones and suggest that deregulated Wnt signaling might be a frequent pathomechanism leading to PD. These observations have implications for the pathogenesis and treatment of neurodegenerative diseases in general.

LRRK2: an éminence grise of Wnt-mediated neurogenesis?

The importance of leucine-rich repeat kinase 2 (LRRK2) to mature neurons is well-established, since mutations in PARK8, the gene encoding LRRK2, are the most common known cause of Parkinson's disease. Nonetheless, despite the LRRK2 knockout mouse having no overt neurodevelopmental defect, numerous lines of in vitro data point toward a central role for this protein in neurogenesis. Roles for LRRK2 have been described in many key processes, including neurite outgrowth and the regulation of microtubule dynamics. Moreover, LRRK2 has been implicated in cell cycle control, suggesting additional roles in neurogenesis that precede terminal differentiation. However, we contend that the suggested function of LRRK2 as a scaffolding protein at the heart of numerous Wnt signaling cascades provides the most tantalizing link to neurogenesis in the developing brain. Numerous lines of evidence show a critical requirement for multiple Wnt pathways in the development of certain brain regions, not least the dopaminergic neurons of the ventral mid-brain. In conclusion, these observations indicate a function of LRRK2 as a subtle yet critical mediator of the action of Wnt ligands on developing neurons. We suggest that LRRK2 loss- or gain-of-function are likely modifiers of developmental phenotypes seen in animal models of Wnt signaling deregulation, a hypothesis that can be tested by cross-breeding relevant genetically modified experimental strains.

The importance of Wnt signalling for neurodegeneration in Parkinson's disease.

PD (Parkinson's disease) is a devastating progressive motor disorder with no available cure. Over the last two decades, an increasing number of genetic defects have been found that cause familial and idiopathic forms of PD. In parallel, the importance of Wnt signalling pathways for the healthy functioning of the adult brain and the dysregulation of these pathways in neurodegenerative disease has become apparent. Cell biological functions disrupted in PD are partially controlled by Wnt signalling pathways and proteins encoded by PARK genes have been shown to modify Wnt signalling. This suggests the prospect of targeting Wnt signalling pathways to modify PD progression.

LRRK2 functions as a Wnt signaling scaffold, bridging cytosolic proteins and membrane-localized LRP6.

Mutations in PARK8, encoding leucine-rich repeat kinase 2 (LRRK2), are a frequent cause of Parkinson's disease (PD). Nonetheless, the physiological role of LRRK2 remains unclear. Here, we demonstrate that LRRK2 participates in canonical Wnt signaling as a scaffold. LRRK2 interacts with key Wnt signaling proteins of the ß-catenin destruction complex and dishevelled proteins in vivo and is recruited to membranes following Wnt stimulation, where it binds to the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) in cellular models. LRRK2, therefore, bridges membrane and cytosolic components of Wnt signaling. Changes in LRRK2 expression affects pathway activity, while pathogenic LRRK2 mutants reduce both signal strength and the LRRK2-LRP6 interaction. Thus, decreased LRRK2-mediated Wnt signaling caused by reduced binding to LRP6 may underlie the neurodegeneration observed in PD. Finally, a newly developed LRRK2 kinase inhibitor disrupted Wnt signaling to a similar extent as pathogenic LRRK2 mutations. The use of LRRK2 kinase inhibition to treat PD may therefore need reconsideration.

LRRK2 signaling pathways: the key to unlocking neurodegeneration?

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 α/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.

A simple technique for the prediction of interacting proteins reveals a direct Brn-3a-androgen receptor interaction.

The formation of multiprotein complexes constitutes a key step in determining the function of any translated gene product. Thus, the elucidation of interacting partners for a protein of interest is of fundamental importance to cell biology. Here we describe a simple methodology for the prediction of novel interactors. We have applied this to the developmental transcription factor Brn-3a to predict and verify a novel interaction between Brn-3a and the androgen receptor (AR). We demonstrate that these transcription factors form complexes within the nucleus of ND7 neuroblastoma cells, while in vitro pull-down assays show direct association. As a functional consequence of the Brn-3a-AR interaction, the factors bind cooperatively to multiple elements within the promoter of the voltage-gated sodium channel, Nav1.7, leading to a synergistic increase in its expression. Thus, these data define AR as a direct Brn-3a interactor and verify a simple interacting protein prediction methodology that is likely to be useful for many other proteins.

Regulation of Brn-3a N-terminal transcriptional activity by MEK1/2-ERK1/2 signalling in neural differentiation.

The POU family transcription factor Brn-3a is required for the differentiation and survival of sensory neurones, and is phosphorylated in neuroblastoma cells following treatment with all-trans retinoic acid (RA). Mutation of serines-121 and -122 of Brn-3a to alanine blocks its phosphorylation and impairs RA-mediated neurite outgrowth. Here we show that this deficit in differentiation is mimicked by a single mutation at serine-122, and demonstrate a similar requirement for a second residue, threonine-39. Like Brn-3a, the neuropeptide Galanin has been implicated in the development of sensory neurones. We show that Brn-3a over-expression acts synergistically with RA treatment to up-regulate Galanin promoter activity; that the activity of the N-terminal transcriptional activation domain of Brn-3a is increased following RA treatment; and that both these effects require threonine-39 and serine-122. In addition, we demonstrate that the RA-mediated activation of Galanin promoter activity and Brn-3a N-terminal transcriptional activity are both blocked by pan-MEK inhibitors, and show that the expression of a constitutively-active mutant of MEK1, but not MEK5, is sufficient to increase Brn-3a activity. These results reveal an important role for the ERK1/2 pathway in Brn-3a regulation during RA-mediated neuronal differentiation and define the neuropeptide Galanin as a novel target of this transcription factor.

Protein kinase B phosphorylation of PIKfyve regulates the trafficking of GLUT4 vesicles.

Insulin-stimulated glucose uptake involves the recruitment of the glucose transporter 4 isoform (GLUT4) from an intracellular location to the plasma membrane of fat and muscle cells. Although the activation of the PI3-kinase/protein kinase B (PKB) pathway is central to this effect of insulin, the key substrates for PKB that are involved require identification. Here we report that serine318 on the FYVE domain-containing PtdIns3P 5-kinase (PIKfyve) is a novel substrate for PKB, and show that phosphorylation stimulates the PtdIns3P 5-kinase activity of the enzyme. We also demonstrate that PIKfyve is phosphorylated on serine318 in intact cells in response to insulin, in a PI3-kinase-dependent manner, and that PIKfyve colocalises with a highly motile subpopulation of insulin-regulated aminopeptidase (IRAP)/GLUT4 vesicles. Finally, we demonstrate that overexpression of a PIKfyve[S318A] mutant in 3T3-L1 adipocytes enhances insulin-stimulated IRAP/GLUT4 vesicle translocation to the plasma membrane suggesting a role for PKB-dependent phosphorylation of PIKfyve in insulin-regulated IRAP/GLUT4 trafficking. The phosphorylation and activation of PIKfyve by PKB provides a novel signalling paradigm that may link plasma membrane-localised PtdIns(3,4,5)P3 signals via a protein kinase cascade to regulated PtdIns(3,5)P2 production, and thereby to the control of trafficking of other membrane cargos.

The identification of ATP-citrate lyase as a protein kinase B (Akt) substrate in primary adipocytes.

Protein kinase B (Akt) plays a central role in cellular regulation, although many of the physiologically relevant substrates for the kinase remain to be identified. In this study, we have isolated a protein from primary epididymal adipocytes with an apparent molecular weight of 125,000. This protein exhibited immunoreactivity, in an insulin-dependent manner, with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) as well as a phosphospecific antibody that recognizes serine 21/9 of GSK-3alpha/beta. MALDI-TOF mass spectrometry revealed the protein to be ATP-citrate lyase, suggesting that the two phosphospecific antibodies recognize phosphoserine 454, a previously reported insulin- and isoproterenol-stimulated ATP-citrate lyase phosphorylation site. Indeed, both insulin and isoproterenol stimulated the phosphorylation of this protein on the site recognized by the phosphospecific antibodies in a wortmannin-sensitive and -insensitive manner, respectively. In addition, transient expression of a constitutively active protein kinase B in primary adipocytes mimicked the effect of insulin on ATP-citrate lyase phosphorylation. Furthermore, ATP-citrate lyase was phosphorylated in vitro by recombinant protein kinase B on the same site. Taken together, these results demonstrate that serine 454 of ATP-citrate lyase is a novel and major in vivo substrate for protein kinase B.