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.     

A continuous and direct assay to monitor leucine-rich repeat kinase 2 activity

Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain enzyme displaying activities of GTP hydrolase and protein threonine/serine kinase in separate domains. Mutations in both catalytic domains have been linked to the onset of Parkinson’s disease, which triggered high interest in this enzyme as a potential target for drug development, particularly focusing on inhibition of the kinase activity. However, available activity assays are discontinuous, involving either radioactivity detection or coupling with antibodies. Here we describe a continuous and direct assay for LRRK2 kinase activity, combining a reported peptide sequence optimized for LRRK2 binding and an established strategy for fluorescence emission on magnesium ion chelation by phosphorylated peptides carrying an artificial amino acid. The assay was employed to evaluate apparent steady-state parameters for the wild type and two mutant forms of LRRK2 associated with Parkinson’s disease as well as to probe the effects of GTP, GDP, and autophosphorylation on the kinase activity of the enzyme. Staurosporine was evaluated as an inhibitor of the wild-type enzyme. It is expected that this assay will aid in mechanistic investigations of LRRK2.     

Identification of compounds that inhibit the kinase activity of leucine-rich repeat kinase 2

Mutations in leucine-repeat rich kinase 2 (LRRK2) are the most common known cause of late-onset Parkinson’s disease. In this study, a novel system to purify active recombinant LRRK2 expressed in mammalian cells was generated. This recombinant enzyme was used to characterize the specificity of LRRK2 and identify small compounds that can inhibit the kinase activity. Recombinant LRRK2 was shown to autophosphorylate and phosphorylate MBP and a peptide (LRRKtide) corresponding to the T688 site in moesin. A series of well-characterized kinase peptide substrates was not modified by LRRK2 demonstrating remarkable specificity. G2019S, the most common disease-causing mutation in LRRK2, increased kinase activity more dramatically than previously appreciated (∼10-fold). Several small molecules sharing a basic indolocarbazole structure (Gö6976, K-252a, and staurosporine) where identified as potent inhibitors of LRRK2 kinase activity. These findings provide important insights and tools to study the mechanisms of LRRK2 pathobiology, and could lead to therapeutic applications.     

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 Parkinson’s disease kinase LRRK2 autophosphorylates its GTPase domain at multiple sites

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited Parkinson’s disease (PD). The protein is large and complex, but pathogenic mutations cluster in a region containing GTPase and kinase domains. LRRK2 can autophosphorylate in vitro within a dimer pair, although the significance of this reaction is unclear. Here, we mapped the sites of autophosphorylation within LRRK2 and found several potential phosphorylation sites within the GTPase domain. Using mass spectrometry, we found that Thr1343 is phosphorylated and, using kinase dead versions of LRRK2, show that this is an autophosphorylation site. However, we also find evidence for additional sites in the GTPase domain and in other regions of the protein suggesting that there may be multiple autophosphorylation sites within LRRK2. These data suggest that the kinase and GTPase activities of LRRK2 may exhibit complex autoregulatory interdependence.     

Ethyl pyruvate has a neuroprotective effect through activation of extracellular signal-regulated kinase in Parkinson’s disease model

Ethyl pyruvate (EP), a simple derivative of endogenous pyruvate, has an anti-inflammatory function. Recently, the protective neurological effects of EP have been reported in cell culture and animal models of neurological diseases. The present study investigates the protective effects of EP on dopaminergic cell death in Parkinson’s disease models. The selective death of dopaminergic neurons in substantia nigra was prevented by EP in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse models. EP also suppressed the 1-methyl-4-pyridinium-induced cell death of SH-SY5Y cells and restored the phosphorylation of extracellular signal-regulated kinase. Thus, EP has neuroprotective effects of EP in Parkinson’s disease and its related signaling pathways.     

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.     

Autophosphorylation in the leucine-rich repeat kinase 2 (LRRK2) GTPase domain modifies kinase and GTP-binding activities

The leucine-rich repeat kinase 2 (LRRK2) protein has both guanosine triphosphatase (GTPase) and kinase activities, and mutation in either enzymatic domain can cause late-onset Parkinson disease. Nucleotide binding in the GTPase domain may be required for kinase activity, and residues in the GTPase domain are potential sites for autophosphorylation, suggesting a complex mechanism of intrinsic regulation. To further define the effects of LRRK2 autophosphorylation, we applied a technique optimal for detection of protein phosphorylation, electron transfer dissociation, and identified autophosphorylation events exclusively nearby the nucleotide binding pocket in the GTPase domain. Parkinson-disease-linked mutations alter kinase activity but did not alter autophosphorylation site specificity or sites of phosphorylation in a robust in vitro substrate myelin basic protein. Amino acid substitutions in the GTPase domain have large effects on kinase activity, as insertion of the GTPase-associated R1441C pathogenic mutation together with the G2019S kinase domain mutation resulted in a multiplicative increase (∼ 7-fold) in activity. Removal of a conserved autophosphorylation site (T1503) by mutation to an alanine residue resulted in greatly decreased GTP-binding and kinase activities. While autophosphorylation likely serves to potentiate kinase activity, we find that oligomerization and loss of the active dimer species occur in an ATP- and autophosphorylation-independent manner. LRRK2 autophosphorylation sites are overall robustly protected from dephosphorylation in vitro, suggesting tight control over activity in vivo. We developed highly specific antibodies targeting pT1503 but failed to detect endogenous autophosphorylation in protein derived from transgenic mice and cell lines. LRRK2 activity in vivo is unlikely to be constitutive but rather refined to specific responses.     

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.     

Controlling aggregation propensity in A53T mutant of alpha-synuclein causing Parkinson’s disease

Understanding α-synuclein in terms of fibrillization, aggregation, solubility and stability is fundamental in Parkinson’s disease (PD). The three familial mutations, namely, A30P, E46K and A53T cause PD because the hydrophobic regions in α-synuclein acquire β-sheet configuration, and have a propensity to fibrillize and form amyloids that cause cytotoxicity and neurodegeneration. On simulating the native form and mutants (A30P, E46K and A53T) of α-synuclein in water solvent, clear deviations are observed in comparison to the all-helical 1XQ8 PDB structure. We have identified two crucial residues, ⁴⁰Val and ⁷⁴Val, which play key roles in β-sheet aggregation in the hydrophobic regions 36-41 and 68-78, respectively, leading to fibrillization and amyloidosis in familial (A53T) PD. We have also identified V40D_V74D, a double mutant of A53T (the most amyloidogenic mutant). The simultaneous introduction of these two mutations in A53T nearly ends its aggregation propensity, increases its solubility and positively enhances its thermodynamic stability.     

Pathogenic Parkinson’s disease mutations across the functional domains of LRRK2 alter the autophagic/lysosomal response to starvation

LRRK2 is one of the most important genetic contributors to Parkinson’s disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consistently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data highlight the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations.     

Assessing the prevalence of PINK1 genetic variants in South African patients diagnosed with early- and late-onset Parkinson’s disease

Mutations in the PINK1 gene are the second most common cause after parkin of autosomal recessive early-onset Parkinson’s disease (PD). PINK1 is a protein kinase that is localized to the mitochondrion and is ubiquitously expressed in the human brain. Recent studies aimed at elucidating the function of PINK1, have found that it has neuroprotective properties against mitochondrial dysfunction and proteasomally-induced apoptosis. In the present study, we aimed to investigate the prevalence of PINK1 genetic variants in 154 South African PD patients from all ethnic groups. Mutation screening was performed using the High-Resolution Melt technique and direct sequencing. A total of 16 sequence variants were identified: one known homozygous mutation (Y258X), two heterozygous missense variants (P305A and E476K), and 13 polymorphisms of which five were novel. No homozygous exonic deletions were detected. The novel P305A variant was found in a female patient of Black Xhosa ethnicity who has a positive family history of the disease and an age at onset of 30 years. This variant has the potential to modulate enzymatic activity due to its location in the kinase domain. This is the first report on mutation screening of PINK1 in the South African population. Results from the present study showed that point mutations and homozygous exonic deletions in PINK1 are not a common cause of PD in the South African population.     

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.     

AMP-activated protein kinase is activated in Parkinson’s disease models mediated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

The selective loss of dopaminergic neurons in the substantia nigra pars compacta is a feature of Parkinson’s disease (PD). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity is the most common experimental model used to investigate the pathogenesis of PD. Administration of MPTP in mice produces neuropathological defects as observed in PD and 1-methyl-4-pyridinium (MPP⁺) induces cell death when neuronal cell cultures are used. AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. In the present study, we demonstrated that AMPK is activated by MPTP in mice and MPP⁺ in SH-SY5Y cells. The inhibition of AMPK by compound C resulted in an increase in MPP⁺-induced cell death. We further showed that overexpression of AMPK increased cell viability after exposure to MPP⁺ in SH-SY5Y cells. Based on these results, we suggest that activation of AMPK might prevent neuronal cell death and play a role as a survival factor in PD.     

Targeting phosphatases as the next generation of disease modifying therapeutics for Parkinson’s disease

Phosphorylation is a key post-translational modification for cellular signaling, and abnormalities in this process are observed in several neurodegenerative disorders. Among these disorders, Parkinson’s disease (PD) is particularly intriguing as there are both genetic causes of disease that involve phosphorylation, and pathological hallmarks of disease composed of a hyperphosphorylated protein. Two of the major genes linked to PD are themselves kinases – leucine rich repeat kinase 2 (LRRK2) and phosphatase and tensin induced homolog kinase 1 (PINK1). Mutations in LRRK2 lead to its increased kinase activity and dominantly inherited PD, while mutations in PINK1 lead to loss of function and recessive PD. A third genetic linkage to disease is α-synuclein, a protein that is heavily phosphorylated in Lewy bodies and Lewy neurites, the pathological hallmarks of PD. The phosphorylation of α-synuclein at various residues influences its aggregation, either positively or negatively, thereby impacting its central role in disease pathogenesis. Given these associations of phosphorylation with PD, modulation of this modification is an attractive therapeutic strategy. The kinases that act in these disease relevant pathways have been the primary target for such approaches. But, the development of kinase inhibitors has been complicated by the necessary specificity to retain safety, the redundancy of kinases leading to lack of efficacy, and the difficulties in overcoming the blood–brain barrier. The field of modulating phosphatases has the potential to overcome some of these issues and provide the next generation of therapeutic targets for PD. In this review, we address the phosphorylation pathways involved in PD, the kinases and issues related to their inhibition, and the evolving field of the phosphatases relevant in PD and how they may be targeted pharmacologically.     

The effect of the partially restricted sit-to-stand task on biomechanical variables in subjects with and without Parkinson’s disease

The aim of this study was to explore the electromyographic, kinetic and kinematic patterns during a partially restricted sit-to-stand task in subjects with and without Parkinson’s disease (PD). If the trunk is partially restricted, different behavior of torques and muscle activities could be found and it can serve as a reference of the deterioration in the motor performance of subjects with PD. Fifteen subjects participated in this study and electromyography (EMG) activity of the tibialis anterior (TA), soleus (SO), vastus medialis oblique (VMO), biceps femoris (BF) and erector spinae (ES) were recorded and biomechanical variables were calculated during four phases of the movement. Subjects with PD showed more flexion at the ankle, knee and hip joints and increased knee and hip joint torques in comparison to healthy subjects in the final position. However, these joint torques can be explained by the differences in kinematic data. Also, the hip, knee and ankle joint torques were not different in the acceleration phase of movement. The use of a partially restricted sit-to-stand task in PD subjects with moderate involvement leads to the generation of joint torques similar to healthy subjects. This may have important implications for rehabilitation training in PD subjects.     

Role of genomics in translational research for Parkinson’s disease

Research on Parkinson’s disease (PD) has made remarkable progress in recent decades, due largely to new genomic technologies, such as high throughput sequencing and microarray analyses. Since the discovery of a linkage of a missense mutation of the α-synuclein (αS) gene to a rare familial dominant form of PD in 1996, positional cloning and characterization of a number of familial PD risk factors have established a hypothesis that aggregation of αS may play a major role in the pathogenesis of PD. Furthermore, dozens of sensitizing alleles related to the disease have been identified by genome wide association studies (GWAS) and meta-GWAS, contributing to a better understanding of the pathological mechanisms of sporadic PD. Thus, the knowledge obtained from the association studies will be valuable for “the personal genome” of PD. Besides summarizing such progress, this paper focuses on the role of microRNAs in the field of PD research, since microRNAs might be promising as a biomarker and as a therapeutic reagent for PD. We further refer to a recent view that neurodegenerative diseases, including PD, coexist with metabolic disorders and are stimulated by type II diabetes, the most common disease among elderly populations. The development of genomic approaches may potentially contribute to therapeutic intervention for PD.     

Indolinone based LRRK2 kinase inhibitors with a key hydrogen bond

The most prevalent leucine-rich repeat kinase 2 (LRRK2) mutation G2019S is associated with Parkinson’s disease (PD). It enhances kinase activity and has been identified in both familial and sporadic cases. Kinase activity was reported to be required for LRRK2 mutants to exert their toxic effects. Hence LRRK2 kinase inhibition may be a promising therapeutic target for PD. Here we report on the discovery and characterization of indolinone based LRRK2 inhibitors. Indolinone 15b, the most potent and selective inhibitor of the present series, is characterized by an IC₅₀ of 15 nM against wild-type LRRK2 and 10 nM against the LRRK2 G2019S mutant, respectively. Compound 15b was further evaluated in a kinase panel including 46 human protein kinases and in a zebrafish embryo phenotype assay, which enabled toxicity determination in whole organisms.