Ancient Origin of the Parkinson Disease Gene LRRK2

Dominant mutations in the LRRK2 gene, a member of the Roco family, cause both familial and sporadic Parkinson disease. LRRK genes had so far been detected only in bilaterian animals. In deuterostomes, including humans, two LRRK genes (LRRK1 and LRRK2) exist, while in protostomes a single LRRK gene has been found. In this study, I combine structural and phylogenetic analyses to show that the cnidarian Nematostella vectensis has four LRRK genes. One of them is a bona fide orthologue of the human LRRK2 gene, demonstrating that this gene has an ancient origin. Two others are, respectively, orthologues of the deuterostome LRRK1 and the protostome LRRK genes. The fourth gene is probably cnidarian-specific. This precise characterization of the early evolution of LRRK genes in animals has important implications, because it indicates that the Drosophila and Caenorhabditis LRRK genes, which are studied to gain an understanding of LRRK2 function, are not true orthologues of the human Parkinson disease gene. Novel functional insights are also gained by comparison of the structures of LRRK2 genes in distantly related species.     

Pathogenic LRRK2 mutations do not alter gene expression in cell model systems or human brain tissue

Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances.     

Sequence conservation between porcine and human LRRK2

Leucine-rich repeat kinase 2 (LRRK2) is a member of the ROCO protein superfamily (Ras of complex proteins (Roc) with a C-terminal Roc domain). Mutations in the LRRK2 gene lead to autosomal dominant Parkinsonism. We have cloned the porcine LRRK2 cDNA in an attempt to characterize conserved and therefore likely functional domains. The LRRK2 cDNA contains an open reading frame of 7,578 bp. The predicted LRRK2 protein consists of 2,526 amino acids of 86–93% identity with its mammalian couterparts. The deduced amino acid sequence of encoded porcine LRRK2 protein displays extensive homology with its human counterpart, with greatest similarities in those regions that contain the kinase domain, the Roc domain and the COR motif. Expression of porcine LRRK2 mRNA in various organs and tissues is similar to its human counterpart and not limited to the brain. The obtained data show that the LRRK2 sequence and expression patterns are conserved across species. The porcine LRRK2 gene was mapped to chromosome 5q25. The results obtained suggest that the LRRK2 gene might be of particular interest in our attempt to generate a transgenic porcine model for Parkinson’s disease. The sequence of the porcine LRRK2 cDNA, encoding the LRRK2/dardarin protein, and the genomic sequence of LRRK2 have been submitted to DDBJ/EMBL/GenBank under the Accessions Numbers EU019992, and EU019994, respectively.     

The Role of LRRK2 in Intracellular Organelle Dynamics

Pathogenic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene hyperactivate LRRK2 kinase activity and lead to the development of Parkinson’s disease (PD). Membrane recruitment of LRRK2 and the identification of RAB GTPases as bona fide LRRK2 substrates strongly indicate that LRRK2 regulates intracellular trafficking. This review highlights the current literature on the role of LRRK2 in intracellular organelle dynamics. With a focus on the effects of LRRK2 on microtubule function, mitochondrial dynamics, the autophagy-lysosomal pathway, and synaptic vesicle trafficking, it summarizes our current understanding of how intracellular dynamics are altered upon pathogenic LRRK2 hyperactivation.     

The Parkinson disease gene LRRK2: evolutionary and structural insights

Mutations in the human leucine-rich repeat kinase 2 (LRRK2) gene are associated with both familial and sporadic Parkinson disease (PD). LRRK2 belongs to a gene family known as Roco. Roco genes encode for large proteins with several protein domains. Particularly, all Roco proteins have a characteristic GTPase domain, named Roc, plus a domain of unknown function called COR. In addition, LRRK2 and several other Roco proteins also contain a protein kinase domain. In this study, I use a combination of phylogenetic and structural analyses of the COR, Roc, and kinase domains present in Roco proteins to describe the origin and evolutionary history of LRRK2. Phylogenetic analyses using these domains demonstrate that LRRK2 emerged from a duplication that occurred after the protostome-deuterostome split. The duplication was followed by the acquisition by LRRK2 proteins of a specific type of N-terminal repeat, described here for the first time. This repeat is absent in the proteins encoded by the paralogs of LRRK2, called LRRK1 or in protostome LRRK proteins. These results suggest that Drosophila or Caenorhabditis LRRK genes may not be good models to understand human LRRK2 function. Genes in the slime mold Dictyostelium discoideum with structures very similar to those found in animal LRRK genes, including the protein kinase domain, have been described. However, phylogenetic analyses suggest that this structural similarity is due to independent acquisitions of distantly related protein kinase domains. Finally, I confirm in an extensive sequence analysis that the Roc GTPase domain is related but still substantially different from small GTPases, such as Rab, Ras, or Rho. Modeling based on known kinase structures suggests that mutations in LRRK2 that cause familiar PD may alter the local 3-dimensional folding of the LRRK2 protein without affecting its overall structure.     

Temporal expression of mutant LRRK2 in adult rats impairs dopamine reuptake

Parkinson's disease (PD) results from progressive degeneration of dopaminergic neurons. Most PD cases are sporadic, but some have pathogenic mutation in the individual genes. Mutation of the leucine-rich repeat kinase-2 (LRRK2) gene is associated with familial and sporadic PD, as exemplified by G2019S substitution. While constitutive expression of mutant LRRK2 in transgenic mice fails to induce neuron death, transient expression of the disease gene by viral delivery causes a substantial loss of dopaminergic neurons in mice. To further assess LRRK2 pathogenesis, we created inducible transgenic rats expressing human LRRK2 with G2019S substitution. Temporal overexpression of LRRK2(G2019S) in adult rats impaired dopamine reuptake by dopamine transporter (DAT) and thus enhanced locomotor activity, the phenotypes that were not observed in transgenic rats constitutively expressing the gene throughout life time. Reduced DAT binding activity is an early sign of dopaminergic dysfunction in asymptomatic subjects carrying pathogenic mutation in LRRK2. Our transgenic rats recapitulated the initiation process of dopaminergic dysfunction caused by pathogenic mutation in LRRK2. Inducible transgenic approach uncovered phenotypes that may be obscured by developmental compensation in constitutive transgenic rats. Finding in inducible LRRK2 transgenic rats would guide developing effective strategy in transgenic studies: Inducible expression of transgene may induce greater phenotypes than constitutive gene expression, particularly in rodents with short life time.     

GTPase Activity Plays a Key Role in the Pathobiology of LRRK2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with late-onset, autosomal-dominant, familial Parkinson''s disease (PD) and also contribute to sporadic disease. The LRRK2 gene encodes a large protein with multiple domains, including functional Roc GTPase and protein kinase domains. Mutations in LRRK2 most likely cause disease through a toxic gain-of-function mechanism. The expression of human LRRK2 variants in cultured primary neurons induces toxicity that is dependent on intact GTP binding or kinase activities. However, the mechanism(s) underlying LRRK2-induced neuronal toxicity is poorly understood, and the contribution of GTPase and/or kinase activity to LRRK2 pathobiology is not well defined. To explore the pathobiology of LRRK2, we have developed a model of LRRK2 cytotoxicity in the baker''s yeast Saccharomyces cerevisiae. Protein domain analysis in this model reveals that expression of GTPase domain-containing fragments of human LRRK2 are toxic. LRRK2 toxicity in yeast can be modulated by altering GTPase activity and is closely associated with defects in endocytic vesicular trafficking and autophagy. These truncated LRRK2 variants induce similar toxicity in both yeast and primary neuronal models and cause similar vesicular defects in yeast as full-length LRRK2 causes in primary neurons. The toxicity induced by truncated LRRK2 variants in yeast acts through a mechanism distinct from toxicity induced by human α-synuclein. A genome-wide genetic screen identified modifiers of LRRK2-induced toxicity in yeast including components of vesicular trafficking pathways, which can also modulate the trafficking defects caused by expression of truncated LRRK2 variants. Our results provide insight into the basic pathobiology of LRRK2 and suggest that the GTPase domain may contribute to the toxicity of LRRK2. These findings may guide future therapeutic strategies aimed at attenuating LRRK2-mediated neurodegeneration.     

Mutations in LRRK2/dardarin associated with Parkinson disease are more toxic than equivalent mutations in the homologous kinase LRRK1

Several mutations have been found in the leucine-rich repeat kinase 2 gene (LRRK2), encoding the protein dardarin, which are associated with autosomal dominant Parkinson disease. We have previously shown that mutant LRRK2/dardarin is toxic to neurons and neuron-like cell lines in culture and that some mutations are also associated with an inclusion-body phenotype. There is a homologous kinase, LRRK1, which has a similar domain structure but is not known to carry mutations causing Parkinson disease. In the current study, we introduced mutations at equivalent residues in both LRRK2 and LRRK1 to determine their effects in cells. We show that mutations in dardarin are more prone to form inclusion bodies in transfected cells and are more toxic than equivalent mutations in LRRK1. This work suggests that dardarin/LRRK2 is inherently more damaging than LRRK1.     

Re-examination of the dimerization state of leucine-rich repeat kinase 2: predominance of the monomeric form

Mutations in the LRRK2 (leucine-rich repeat kinase 2) gene have been identified in PARK8, a major form of autosomal-dominantly inherited familial Parkinson's disease, although the biochemical properties of LRRK2 are not fully understood. It has been proposed that LRRK2 predominantly exists as a homodimer on the basis of the observation that LRRK2, with a theoretical molecular mass of 280 kDa, migrates at 600 kDa (p600 LRRK2) on native polyacrylamide gels. In the present study, we biochemically re-examined the nature of p600 LRRK2 and found that p600 LRRK2 was fractionated with a single peak at ~272 kDa by ultracentrifugation on a glycerol gradient. In addition, p600 LRRK2 behaved similarly to monomeric proteins upon two-dimensional electrophoretic separation. These results suggested a monomeric composition of p600 LRRK2 within cells. The p600 LRRK2 exhibited kinase activity as well as GTP-binding activity, and forced dimerization of LRRK2 neither upregulated its kinase activity nor altered its subcellular localization. Collectively, we conclude that the monomer form of LRRK2 is predominant within cells, and that dimerization is dispensable for its enzymatic activity.     

Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent known cause of autosomal dominant Parkinson's disease. The LRRK2 gene encodes a Roco protein featuring a Ras of complex proteins (ROC) GTPase and a kinase domain linked by the C-terminal of ROC (COR) domain. Here, we explored the effects of the Y1699C pathogenic LRRK2 mutation in the COR domain on GTPase activity and interactions within the catalytic core of LRRK2. We observed a decrease in GTPase activity for LRRK2 Y1699C comparable to the decrease observed for the R1441C pathogenic mutant and the T1348N dysfunctional mutant. To study the underlying mechanism, we explored the dimerization in the catalytic core of LRRK2. ROC-COR dimerization was significantly weakened by the Y1699C or R1441C/G mutation. Using a competition assay, we demonstrated that the intra-molecular ROC : COR interaction is favoured over ROC : ROC dimerization. Interestingly, the intra-molecular ROC : COR interaction was strengthened by the Y1699C mutation. This is supported by a 3D homology model of the ROC-COR tandem of LRRK2, showing that Y1699 is positioned at the intra-molecular ROC : COR interface. In conclusion, our data provides mechanistic insight into the mode of action of the Y1699C LRRK2 mutant: the Y1699C substitution, situated at the intra-molecular ROC : COR interface, strengthens the intra-molecular ROC : COR interaction, thereby locally weakening the dimerization of LRRK2 at the ROC-COR tandem domain resulting in decreased GTPase activity.     

Leucine-Rich Repeat Kinase 2 (LRRK2)-Deficient Rats Exhibit Renal Tubule Injury and Perturbations in Metabolic and Immunological Homeostasis

Genetic evidence links mutations in the LRRK2 gene with an increased risk of Parkinson’s disease, for which no neuroprotective or neurorestorative therapies currently exist. While the role of LRRK2 in normal cellular function has yet to be fully described, evidence suggests involvement with immune and kidney functions. A comparative study of LRRK2-deficient and wild type rats investigated the influence that this gene has on the phenotype of these rats. Significant weight gain in the LRRK2 null rats was observed and was accompanied by significant increases in insulin and insulin-like growth factors. Additionally, LRRK2-deficient rats displayed kidney morphological and histopathological alterations in the renal tubule epithelial cells of all animals assessed. These perturbations in renal morphology were accompanied by significant decreases of lipocalin-2, in both the urine and plasma of knockout animals. Significant alterations in the cellular composition of the spleen between LRRK2 knockout and wild type animals were identified by immunophenotyping and were associated with subtle differences in response to dual infection with rat-adapted influenza virus (RAIV) and Streptococcus pneumoniae. Ontological pathway analysis of LRRK2 across metabolic and kidney processes and pathological categories suggested that the thioredoxin network may play a role in perturbing these organ systems. The phenotype of the LRRK2 null rat is suggestive of a complex biology influencing metabolism, immune function and kidney homeostasis. These data need to be extended to better understand the role of the kinase domain or other biological functions of the gene to better inform the development of pharmacological inhibitors.     

Prevention of intracellular degradation of I2020T mutant LRRK2 restores its protectivity against apoptosis

Leucine-rich repeat kinase 2 (LRRK2) is the causal gene for autosomal dominant familial Parkinson’s disease. We have previously reported a novel molecular feature characteristic to I2020T mutant LRRK2: higher susceptibility to post-translational degradation than the wild-type LRRK2. In the present study, we demonstrated that the protective effect of I2020T LRRK2 against hydrogen peroxide-induced apoptosis was impaired in comparison with the wild-type molecule. When the intracellular level of the protein had been allowed to recover by treatment with proteolysis inhibitors, the protective effect of I2020T LRRK2 against apoptosis was increased. We further confirmed that a decrease in the intracellular protein level of WT LRRK2 by knocking down resulted in a reduction of protectivity against apoptosis. These results suggest that higher susceptibility of I2020T mutant LRRK2 to intracellular degradation than the wild-type molecule may be one of the mechanisms involved in the neurodegeneration associated with this LRRK2 mutation.     

Dependence of Leucine-rich Repeat Kinase 2 (LRRK2) Kinase Activity on Dimerization

Dominant missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson disease. LRRK2 encodes a serine/threonine protein kinase, and pathogenic mutations may increase kinase activity. Intrinsic GTP binding in the GTPase domain may govern kinase activity through an internal signal transduction cascade. As with many protein kinases, LRRK2 self-interacts through mechanisms that may regulate enzymatic activity. We find that the disruption of either GTPase or kinase activity enhances the formation of high molecular weight oligomers and prevents the formation of LRRK2 dimer structures. In addition, brief application of the broad spectrum kinase inhibitor staurosporine ablates LRRK2 dimers and promotes LRRK2 high molecular weight oligomers. LRRK2 interactions with other proteins in cell lines are kinase-independent and include chaperones and cell cytoskeleton components, suggesting that LRRK2 self-assembly principally dictates complex size. To further explore the mechanics of kinase activation, we separate soluble LRRK2 protein that encodes the pathogenic G2019S mutation into high molecular weight oligomers, dimers, and monomers and find that kinase activity resides with dimeric LRRK2. Some PD-associated mutations that increase kinase activity in vitro significantly increase the proportion of dimer structures relative to total LRRK2 protein, providing additional insight into how pathogenic mutations may alter normal enzymatic regulation. Targeting and tracking LRRK2 dimerization may provide a clear way to observe LRRK2 kinase activity in living cells, and disruption of dimeric LRRK2 through kinase inhibition or other means may attenuate pathogenic increases in LRRK2 enzymatic output.     

Regulation of LRRK2 Stability by the E3 Ubiquitin Ligase CHIP

Dominantly inherited mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common cause of familial Parkinson''s disease (PD) and have also been identified in individuals with sporadic PD. Although the exact cellular function of LRRK2 remains unknown, most PD-linked mutations appear to be toxic to cells in culture via mechanisms that depend on the kinase activity of LRRK2 or on the formation of cytoplasmic inclusions. Here we show that the E3 ubiquitin ligase CHIP physically associates with LRRK2 and regulates the cellular abundance of LRRK2. We further show that LRRK2 forms a complex with overexpressed and endogenous CHIP and Hsp90. Our data indicates that the destabilization of LRRK2 by CHIP is due to ubiquitination and proteasome-dependent degradation. Hsp90 can attenuate CHIP-mediated degradation and this can be blocked by the Hsp90 inhibitor geldanamycin. These findings provide important insight into the cellular regulation of LRRK2 stability and may lead to the development of therapeutics to treat PD based on controlling LRRK2 stability.     

GTPase activity and neuronal toxicity of Parkinson's disease-associated LRRK2 is regulated by ArfGAP1

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 encodes a large multi-domain protein with GTPase and kinase activity. Initial data indicates that an intact functional GTPase domain is critically required for LRRK2 kinase activity. PD-associated mutations in LRRK2, including the most common G2019S variant, have variable effects on enzymatic activity but commonly alter neuronal process morphology. The mechanisms underlying the intrinsic and extrinsic regulation of LRRK2 GTPase and kinase activity, and the pathogenic effects of familial mutations, are incompletely understood. Here, we identify a novel functional interaction between LRRK2 and ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1). LRRK2 and ArfGAP1 interact in vitro in mammalian cells and in vivo in brain, and co-localize in the cytoplasm and at Golgi membranes. PD-associated and functional mutations that alter the GTPase activity of LRRK2 modulate the interaction with ArfGAP1. The GTP hydrolysis activity of LRRK2 is markedly enhanced by ArfGAP1 supporting a role for ArfGAP1 as a GTPase-activating protein for LRRK2. Unexpectedly, ArfGAP1 promotes the kinase activity of LRRK2 suggesting a potential role for GTP hydrolysis in kinase activation. Furthermore, LRRK2 robustly and directly phosphorylates ArfGAP1 in vitro. Silencing of ArfGAP1 expression in primary cortical neurons rescues the neurite shortening phenotype induced by G2019S LRRK2 overexpression, whereas the co-expression of ArfGAP1 and LRRK2 synergistically promotes neurite shortening in a manner dependent upon LRRK2 GTPase activity. Neurite shortening induced by ArfGAP1 overexpression is also attenuated by silencing of LRRK2. Our data reveal a novel role for ArfGAP1 in regulating the GTPase activity and neuronal toxicity of LRRK2; reciprocally, LRRK2 phosphorylates ArfGAP1 and is required for ArfGAP1 neuronal toxicity. ArfGAP1 may represent a promising target for interfering with LRRK2-dependent neurodegeneration in familial and sporadic PD.     

GTP binding is essential to the protein kinase activity of LRRK2, a causative gene product for familial Parkinson's disease

Leucine-rich repeat kinase 2 (LRRK2), a product of a causative gene for the autosomal-dominant form of familial Parkinson's disease (PARK8), harbors a Ras-like small GTP binding protein-like (ROC) domain besides the kinase domain, although the relationship between these two functional domains remains elusive. Here we show by thin-layer chromatographic analysis that LRRK2 stably binds GTP but lacks a GTPase activity in HEK293 and Neuro-2a cells. A ROC domain mutation that converts LRRK2 to a guanine nucleotide-free form (T1348N) abolishes the kinase activity of LRRK2 as well as its phosphate incorporation upon metabolic labeling. The phosphorylation of LRRK2 was inhibited by potential inhibitors for cyclic AMP-dependent protein kinase. These data suggest that binding of GTP to the ROC domain regulates the kinase activity of LRRK2 as well as its phosphorylation by other kinase(s).     

Reevaluation of Phosphorylation Sites in the Parkinson Disease-associated Leucine-rich Repeat Kinase 2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an important cause of late-onset, autosomal dominant familial Parkinson disease and contribute to sporadic Parkinson disease. LRRK2 is a large complex protein with multiple functional domains, including a Roc-GTPase, protein kinase, and multiple protein-protein interaction domains. Previous studies have suggested an important role for kinase activity in LRRK2-induced neuronal toxicity and inclusion body formation. Disease-associated mutations in LRRK2 also tend to increase kinase activity. Thus, enhanced kinase activity may therefore underlie LRRK2-linked disease. Similar to the closely related mixed-lineage kinases, LRRK2 can undergo autophosphorylation in vitro. Three putative autophosphorylation sites (Thr-2031, Ser-2032, and Thr-2035) have been identified within the activation segment of the LRRK2 kinase domain based on sequence homology to mixed-lineage kinases. Phosphorylation at one or more of these sites is critical for the kinase activity of LRRK2. Sensitive phopho-specific antibodies to each of these three sites have been developed and validated by ELISA, dot-blot, and Western blot analysis. Using these antibodies, we have found that all three putative sites are phosphorylated in LRRK2, and Ser-2032 and Thr-2035 are the two important sites that regulate LRRK2 kinase activity.