Leucine-rich repeat kinase 2 mutations and Parkinson’s disease: three questions

Mutations in the gene encoding LRRK2 (leucine-rich repeat kinase 2) were first identified in 2004 and have since been shown to be the single most common cause of inherited Parkinson’s disease. The protein is a large GTP-regulated serine/threonine kinase that additionally contains several protein–protein interaction domains. In the present review, we discuss three important, but unresolved, questions concerning LRRK2. We first ask: what is the normal function of LRRK2? Related to this, we discuss the evidence of LRRK2 activity as a GTPase and as a kinase and the available data on protein–protein interactions. Next we raise the question of how mutations affect LRRK2 function, focusing on some slightly controversial results related to the kinase activity of the protein in a variety of in vitro systems. Finally, we discuss what the possible mechanisms are for LRRK2-mediated neurotoxicity, in the context of known activities of the protein.     

Leucine-rich repeat kinase 2 disturbs mitochondrial dynamics via Dynamin-like protein

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are the leading causes of genetically inherited Parkinson's disease (PD) identified so far. The underlying mechanism whereby missense alterations in LRRK2 initiate neurodegeneration remains largely unclear. Mitochondrial dysfunction has been recognized to contribute to the pathogenesis of both sporadic and familial PD. The pathogenic gain-of-function mutant form of LRRK2, LRRK2 G2019S, is associated with elevated kinase activity and PD. Here we show that LRRK2 G2019S can cause defects in the morphology and dynamics of mitochondria in cortical neurons. In neurons, endogenous LRRK2 and the mitochondrial fission factor Dynamin like protein 1 (DLP1) interact with and partially co-localize with each other. DLP1 plays an essential role in LRRK2-induced mitochondrial fission. In support of this, expression of LRRK2 leads to the translocation of DLP1 from the cytosol to the mitochondria and knockdown of DLP1 expression inhibits LRRK2-induced mitochondrial fission. In addition, co-expression of LRRK2 and DLP1 induces mitochondrial clearance. Furthermore, we have found that expression of LRRK2 leads to increased reactive oxygen species levels in cells. Taken together, our results provide insights into the pathobiology of LRRK2 and suggest that LRRK2 G2019S may induce neuronal dysfunction or cell death by disturbing normal mitochondrial fission/fusion dynamics and function.     

Leucine‐rich repeat kinase 2 and Parkinson's disease

Leucine‐rich repeat kinase 2 (LRRK2) is a large multidomain protein that is expressed in many tissues and participates in numerous biological pathways. Mutations in LRRK2 are recognized as genetic risk factors for familial Parkinson's disease (PD) and may also represent causal factors in the more common sporadic form of PD. The structure of LRRK2 comprises a combination of GTPase, kinase, and scaffolding domains. This functional diversity, combined with a potentially central role in genetic and idiopathic PD motivates significant effort to further credential LRRK2 as a therapeutic target. Here, we review the current understanding for LRRK2 function in normal physiology and PD, with emphasis on insight gained from proteomic approaches.     

Leucine-rich repeat kinase 2 (LRRK2)/PARK8 possesses GTPase activity that is altered in familial Parkinson's disease R1441C/G mutants

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are linked to the most common familial forms and some sporadic forms of Parkinson's disease (PD). The LRRK2 protein contains two well-known functional domains, MAPKKK-like kinase and Rab-like GTPase domains. Emerging evidence shows that LRRK2 contains kinase activity which is enhanced in several PD-associated mutants of LRRK2. However, the GTPase activity of LRRK2 has yet to be formally demonstrated. Here, we produced and purified the epitope-tagged LRRK2 protein from transgenic mouse brain, and showed that purified brain LRRK2 possesses both kinase and GTPase activity as assayed by GTP binding and hydrolysis. The brain LRRK2 is associated with elevated kinase activity in comparison to that from transgenic lung or transfected cultured cells. In transfected cell cultures, we detected GTP hydrolysis activity in full-length as well as in GTPase domain of LRRK2. This result indicates that LRRK2 GTPase can be active independent of LRRK2 kinase activity (while LRRK2 kinase activity requires the presence of LRRK2 GTPase as previously shown). We further found that PD mutation R1441C/G in the GTPase domain causes reduced GTP hydrolysis activity, consistent with the altered enzymatic activity in the mutant LRRK2 carrying PD familial mutations. Therefore, our study shows the biochemical characteristics of brain-specific LRRK2 which is associated with robust kinase and GTPase activity. The distinctive levels of kinase/GTPase activity in brain LRRK2 may help explain LRRK2-associated neuronal functions or dysfunctions in the pathogenesis of PD.     

The role of leucine-rich repeat kinase 2 (LRRK2) in Parkinson's disease

Parkinson's disease, like many common age-related conditions, is now recognized to have a substantial genetic component. Here, I discuss how mutations in a large complex gene--leucine-rich repeat kinase 2 (LRRK2)--affect protein function, and I review recent evidence that LRRK2 mutations affect pathways that involve other proteins that have been implicated in Parkinson's disease, specifically α-synuclein and tau. These concepts can be used to understand disease processes and to develop therapeutic opportunities for the treatment of Parkinson's disease.     

Leucine-rich repeat kinase 2 modulates retinoic acid-induced neuronal differentiation of murine embryonic stem cells

Background

Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson''s disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment.

Methodology/Principal Findings

In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/− cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture.

Conclusion/Significance

Parkinson''s disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.     

Leucine-rich repeat kinase 2 mutants I2020T and G2019S exhibit altered kinase inhibitor sensitivity

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a frequent cause of late-onset autosomal dominant Parkinson’s disease (PD). Some disease-associated mutations directly affect LRRK2 kinase activity and inhibition of LRRK2 is viewed as a potential therapeutic treatment for PD. We demonstrate by both binding and enzymatic assays that alterations in the kinase activity of the PD-associated mutants I2020T and G2019S are due in part to altered ATP affinity. In binding assays, G2019S and I2020T have approximately 2-fold lower and 6-fold higher ATP affinity, respectively, than wild-type LRRK2. Furthermore, using an in vitro kinase activity assay, we demonstrate that at ATP concentrations close to cellular levels (1 mM) I2020T is approximately 10-fold more resistant to ATP-competitive kinase inhibitors than wild-type whereas G2019S is 1.6-fold more sensitive. These results predict that LRRK2 status may impact kinase inhibitor potencies in vivo or in cellular models.     

Phenylethanolamine N-methyltransferase has beta-carboline 2N-methyltransferase activity: hypothetical relevance to Parkinson's disease

Mammalian brain has a β-carboline 2N-methyltransferase activity that converts β-carbolines, such as norharman and harman, into 2N-methylated β-carbolinium cations, which are structural and functional analogs of the Parkinsonian-inducing toxin 1-methyl-4-phenylpyridinium cation (MPP⁺). The identity and physiological function of this β-carboline 2N-methylation activity was previously unknown. We report pharmacological and biochemical evidence that phenylethanolamine N-methyltransferase (EC 2.1.1.28) has β-carboline 2N-methyltransferase activity. Specifically, purified phenylethanolamine N-methyltransferase (PNMT) catalyzes the 2N-methylation (21.1 pmol/h per unit PNMT) of 9-methylnorharman, but not the 9N-methylation of 2-methylnorharmanium cation. LY134046, a selective inhibitor of phenylethanolamine N-methyltransferase, inhibits (IC₅₀ 1.9 μM) the 2N-methylation of 9-methylnorharman, a substrate for β-carboline 2N-methyltransferase. Substrates of phenylethanolamine N-methyltransferase also inhibit β-carboline 2N-methyltransferase activity in a concentration-dependent manner. β-Carboline 2N-methyltransferase activity (43.7 pmol/h/mg protein) is present in human adrenal medulla, a tissue with high phenylethanolamine N-methyltransferase activity.

We are investigating the potential role of N-methylated β-carbolinium cations in the pathogenesis of idiopathic Parkinson’s disease. Presuming that phenylethanolamine N-methyltransferase activity forms toxic 2N-methylated β-carbolinium cations, we propose a novel hypothesis regarding Parkinson’s disease—a hypothesis that includes a role for phenylethanolamine N-methyltransferase-catalyzed formation of MPP⁺-like 2N-methylated β-carbolinium cations.     

Leucine-rich repeat region of decorin binds to filamin-A

Decorin is a member of the family of small leucine-rich proteoglycans found in the extracellular matrix and has an important role in promoting fiber formation and in controlling cell proliferation. Here, we have investigated whether the leucine-rich repeat (LRR) region of decorin interacts with proteins from human lung fibroblasts by using a yeast two-hybrid assay. We report that the LRR region of decorin interacts with the cytoskeletal protein, filamin-A (ABP-280), a peripheral cytoplasmic protein. This interaction is dependent on the 288 carboxyl-terminal amino acids of filamin-A, which correspond to repeats 22-24 of its conserved beta-sheet structure. We also show that the recombinant LRR region of decorin binds to filamin-A in vitro, and that the deglycosylated core protein of decorin coprecipitates with filamin-A, whereas intact decorin does not. Together, these results suggest that proteins containing the LRR motif that interact with filamin-A may be present in the cytoplasm or at the plasma membrane.     

Mitochondria and ubiquitin-proteasomal system interplay: relevance to Parkinson's disease

The cellular mechanisms that may underlie the death of dopaminergic neurons in Parkinson's disease are ubiquitin-proteasomal system (UPS) impairment, mitochondrial dysfunction, and oxidative stress. The goal of this work was to elucidate the correlation between mitochondrial dysfunction and UPS impairment, focusing on the role of oxidative stress. Our data revealed that mitochondria-DNA-depleted cells (rho0) are compromised at the mitochondrial and UPS levels and also show an alteration of the oxidative status. In parental cells (rho+), MPP(+) induced a clear inhibition of complex I activity, as well as an increase in ubiquitinylated protein levels, which was not observed in cells treated with lactacystin. Moreover, MPP(+) induced a decreased in the 20S chymotrypsin-like and peptidyl-glutamyl peptide hydrolytic-like proteolytic activities after 24 h of exposure. ROS production was increased in rho+ cells treated with MPP(+) or lactacystin, at early treatment periods. MPP(+) induced an increase in carbonyl group formation in rho+ cells. The results suggest that a mitochondrial alteration leads to an imbalance in the cellular oxidative status, inducing a proteasomal deregulation, which may exacerbate protein aggregation, and consequently degenerative events.     

Identification of chemicals to inhibit the kinase activity of leucine-rich repeat kinase 2 (LRRK2), a Parkinson's disease-associated protein

Parkinson’s disease (PD) is a late-onset neurodegenerative disease which occurs at more than 1% in populations aging 65-years and over. Recently, leucine-rich repeat kinase 2 (LRRK2) has been identified as a causative gene for autosomal dominantly inherited familial PD cases. LRRK2 G2019S which is a prevalent mutant found in familial PD patients with LRRK2 mutations, exhibited kinase activity stronger than that of the wild type, suggesting the LRRK2 kinase inhibitor as a potential PD therapeutics. To develop such therapeutics, we initially screened a small chemical library and selected compound 1, whose IC₅₀ is about 13.2 μM. To develop better inhibitors, we tested five of the compound 1 derivatives and found a slightly better inhibitor, compound 4, whose IC₅₀ is 4.1 μM. The cell-based assay showed that these two chemicals inhibited oxidative stress-induced neurotoxicity caused by over-expression of a PD-specific LRRK2 mutant, G2019S. In addition, the structural analysis of compound 4 suggested hydrogen bond interactions between compound 4 and Ala 1950 residue in the backbone of the ATP binding pocket of LRRK2 kinas domain. Therefore, compound 4 may be a promising lead compound to further develop a PD therapeutics based on LRRK2 kinase inhibition.     

The I2020T Leucine-rich repeat kinase 2 transgenic mouse exhibits impaired locomotive ability accompanied by dopaminergic neuron abnormalities

ABSTRACT: BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) is the gene responsible for autosomal-dominant Parkinson's disease (PD), PARK8, but the mechanism by which LRRK2 mutations cause neuronal dysfunction remains unknown. In the present study, we investigated for the first time a transgenic (TG) mouse strain expressing human LRRK2 with an I2020T mutation in the kinase domain, which had been detected in the patients of the original PARK8 family. RESULTS: The TG mouse expressed I2020T LRRK2 in dopaminergic (DA) neurons of the substantia nigra, ventral tegmental area, and olfactory bulb. In both the beam test and rotarod test, the TG mice exhibited impaired locomotive ability in comparison with their non-transgenic (NTG) littermates. Although there was no obvious loss of DA neurons in either the substantia nigra or striatum, the TG brain showed several neurological abnormalities such as a reduced striatal dopamine content, fragmentation of the Golgi apparatus in DA neurons, and an increased degree of microtubule polymerization. Furthermore, the tyrosine hydroxylasepositive primary neurons derived from the TG mouse showed an increased frequency of apoptosis and had neurites with fewer branches and decreased outgrowth in comparison with those derived from the NTG controls. CONCLUSIONS: The I2020T LRRK2 TG mouse exhibited impaired locomotive ability accompanied by several dopaminergic neuron abnormalities. The TG mouse should provide valuable clues to the etiology of PD caused by the LRRK2 mutation.     

The G2385R variant of leucine-rich repeat kinase 2 associated with Parkinson's disease is a partial loss-of-function mutation

Autosomal-dominant missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a common genetic cause of PD (Parkinson's disease). LRRK2 is a multidomain protein with kinase and GTPase activities. Dominant mutations are found in the domains that have these two enzyme activities, including the common G2019S mutation that increases kinase activity 2-3-fold. However, there is also a genetic variant in some populations, G2385R, that lies in a C-terminal WD40 domain of LRRK2 and acts as a risk factor for PD. In the present study we show that the G2385R mutation causes a partial loss of the kinase function of LRRK2 and deletion of the C-terminus completely abolishes kinase activity. This effect is strong enough to overcome the kinase-activating effects of the G2019S mutation in the kinase domain. Hsp90 (heat-shock protein of 90 kDa) has an increased affinity for the G2385R variant compared with WT (wild-type) LRRK2, and inhibition of the chaperone binding combined with proteasome inhibition leads to association of mutant LRRK2 with high molecular mass native fractions that probably represent proteasome degradation pathways. The loss-of-function of G2385R correlates with several cellular phenotypes that have been proposed to be kinase-dependent. These results suggest that the C-terminus of LRRK2 plays an important role in maintaining enzymatic function of the protein and that G2385R may be associated with PD in a way that is different from kinase-activating mutations. These results may be important in understanding the differing mechanism(s) by which mutations in LRRK2 act and may also have implications for therapeutic strategies for PD.     

Expression and localization of Parkinson's disease-associated leucine-rich repeat kinase 2 in the mouse brain

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) have been identified as the cause of familial Parkinson's disease (PD) at the PARK8 locus. To begin to understand the physiological role of LRRK2 and its involvement in PD, we have investigated the distribution of LRRK2 mRNA and protein in the adult mouse brain. In situ hybridization studies indicate sites of mRNA expression throughout the mouse brain, with highest levels of expression detected in forebrain regions, including the cerebral cortex and striatum, intermediate levels observed in the hippocampus and cerebellum, and low levels in the thalamus, hypothalamus and substantia nigra. Immunohistochemical studies demonstrate localization of LRRK2 protein to neurones in the cerebral cortex and striatum, and to a variety of interneuronal subtypes in these regions. Furthermore, expression of LRRK2 mRNA in the striatum of VMAT2-deficient mice is unaltered relative to wild-type littermate controls despite extensive dopamine depletion in this mouse model of parkinsonism. Collectively, our results demonstrate that LRRK2 is present in anatomical brain regions of direct relevance to the pathogenesis of PD, including the nigrostriatal dopaminergic pathway, in addition to other regions unrelated to PD pathology, and is likely to play an important role in the normal function of telencephalic forebrain neurones and other neuronal populations.     

Leucine-rich repeat receptor-like kinase1 is a key membrane-bound regulator of abscisic acid early signaling in Arabidopsis

Abscisic acid (ABA) is important in seed maturation, seed dormancy, stomatal closure, and stress response. Many genes that function in ABA signal transduction pathways have been identified. However, most important signaling molecules involved in the perception of the ABA signal or with ABA receptors have not been identified yet. Receptor-like kinase1 (RPK1), a Leu-rich repeat (LRR) receptor kinase in the plasma membrane, is upregulated by ABA in Arabidopsis thaliana. Here, we show the phenotypes of T-DNA insertion mutants and RPK1-antisense plants. Repression of RPK1 expression in Arabidopsis decreased sensitivity to ABA during germination, growth, and stomatal closure; microarray and RNA gel analysis showed that many ABA-inducible genes are downregulated in these plants. Furthermore, overexpression of the RPK1 LRR domain alone or fused with the Brassinosteroid-insensitive1 kinase domain in plants resulted in phenotypes indicating ABA sensitivity. RPK1 is involved in the main ABA signaling pathway and in early ABA perception in Arabidopsis.     

Matrix metalloproteinase-3 is activated by HtrA2/Omi in dopaminergic cells: relevance to Parkinson's disease

Dopaminergic neurons in the substantia nigra are particularly vulnerable, and their degeneration leads to Parkinson's disease. We have previously reported that matrix metalloproteinase-3 (MMP-3) activity is involved in dopaminergic neurodegeneration by multiple mechanisms and that this requires activation of MMP-3 from proMMP-3 by an intracellular serine protease. HtrA2/Omi is a mitochondrial serine protease that has been shown in non-dopaminergic cells to translocate into the cytosol where it triggers apoptosis. In the present study we sought to determine whether HtrA2/Omi might cause activation of MMP-3 in dopaminergic neuronal cells using CATH.a cell line. Mitochondrial stress induced by rotenone led to MMP-3 activation and HtrA2/Omi translocation into the cytosol. The MMP-3 activation involved HtrA2/Omi, because both pharmacological inhibition and siRNA-induced knockdown of HtrA2/Omi attenuated the activation induced by rotenone or MPP+. Overexpression of mature HtrA2/Omi, but not mutant HtrA2/Omi, resulted in MMP-3 activity increase and cell death. Addition of recombinant and catalytically active HtrA2/Omi to lysate of untreated cells led to activation of the endogenous MMP-3, and incubation of the HtrA2/Omi with recombinant proMMP-3 caused cleavage of proMMP-3 to a 48kD protein, corresponding to the active form, which was accompanied by an increase in MMP-3 activity. Taken together, the data indicate that HtrA2/Omi, which normally exists in the mitochondria, can cause MMP-3 activation in the cytosol under a cell stress condition, which can ultimately lead to demise of dopaminergic neuronal cells.     

The Parkinson's disease-associated protein, leucine-rich repeat kinase 2 (LRRK2), is an authentic GTPase that stimulates kinase activity

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the leading cause of autosomal dominant Parkinson's disease (PD). LRRK2, a member of the ROCO protein family, contains both Ras GTPase-like (Roc) and kinase (MAPKKK) domains, as well as other functional motifs. Here, we have identified LRRK2 as the first mammalian ROCO protein that is an authentic and functional GTPase, defined by the ability to bind GTP and undergo intrinsic GTP hydrolysis. Furthermore, the Roc domain is sufficient for this native GTPase activity and binds and hydrolyzes GTP indistinguishably from the Ras-related small GTPase, Rac1. The PD-associated mutation, R1441C, located within the Roc domain, leads to an increase in LRRK2 kinase activity and a decrease in the rate of GTP hydrolysis, compared to the wild-type protein, in an in vitro assay. This finding suggests that the R1441C mutation may help stabilize an activated state of LRRK2. Additionally, LRRK2-mediated phosphorylation is stimulated upon binding of non-hydrolyzable GTP analogs, suggesting that LRRK2 is an MAPKKK-activated intramolecularly by its own GTPase. Since GTPases and MAPKKKs are upstream regulators of multiple signal transduction cascades, LRRK2 may play a central role in integrating pathways involved in neuronal cell signaling and the pathogenesis of PD.