Mechanisms in dominant parkinsonism: The toxic triangle of LRRK2, α‐synuclein,and tau

Parkinson's disease (PD) is generally sporadic but a number of genetic diseases have parkinsonism as a clinical feature. Two dominant genes, α‐synuclein (SNCA) and leucine‐rich repeat kinase 2 (LRRK2), are important for understanding inherited and sporadic PD. SNCA is a major component of pathologic inclusions termed Lewy bodies found in PD. LRRK2 is found in a significant proportion of PD cases. These two proteins may be linked as most LRRK2 PD cases have SNCA‐positive Lewy bodies. Mutations in both proteins are associated with toxic effects in model systems although mechanisms are unclear. LRRK2 is an intracellular signaling protein possessing both GTPase and kinase activities that may contribute to pathogenicity. A third protein, tau, is implicated as a risk factor for PD. We discuss the potential relationship between these genes and suggest a model for PD pathogenesis where LRRK2 is upstream of pathogenic effects through SNCA, tau, or both proteins.     

LRRK2 directing ER‐to‐Golgi transport? (ER)yES!

Mutations in leucine‐rich repeat kinase 2 (LRRK2) represent the most frequent genetic lesions associated with Parkinson's disease (PD). LRRK2 function and the pathogenic mechanisms of LRRK2 genetic mutations remain poorly understood. Cho et al report in this issue of The EMBO Journal a new interaction between LRRK2 and the ER exit site (ERES) protein Sec16A, important for anterograde transport of secretory proteins. Moreover, a disease‐associated mutation disrupts LRRK2–Sec16A interaction and ERES function.     

Molecular docking studies of bacoside from Bacopa monnieri with LRRK2 receptor

Bacosides, constituents of Bacopa monnieri (Linn.), are reported to be potential therapeutic saponins in the cure of Parkinson’s disease (PD). However, detailed mechanism for control of PD by bacosides is not well documented. PD has been reported to be caused by genetic mutations in leucine-rich repeat kinase 2 (LRRK2) leading to higher kinase activity that has been identified as a major cause of familial PD. The LRRK2 was thus proposed as an important marker in the pathogenesis of PD. This suggests that inhibition of LRRK2 holds promise as a potential treatment for PD. Our study focuses on the possible application of bacoside A constituents as potential inhibitors of LRRK2. In this work, we have carried out the in silico molecular docking studies of bacoside A constituents with LRRK2, proposing their role as an inhibitor in PD. The study has revealed the significant interactions between bacosaponin and LRRK2 having ten H-bonds at receptor-ligand site with binding affinity ?7.5 kcal/mol. Hence, amongst the studied triglycosidic saponins, bacosaponin was analyzed to be a better ligand, proposing it to be a major constituent in inhibiting enzymatic activities of mutated LRRK2.     

<Emphasis Type="Italic">LRRK2</Emphasis> Low-penetrance Mutations (Gly2019Ser) and Risk Alleles (Gly2385Arg)—Linking Familial and Sporadic Parkinson’s Disease

The identification of mutations in the leucine-rich repeat kinase 2 (LRRK2) gene as a cause of autosomal dominant Parkinson’s disease (PD) was a major step forward in the genetic dissection of this disorder. However, what makes LRRK2 unique among the known PD-causing genes is that a low-penetrance mutation, Gly2019Ser, is a frequent determinant not only of familial, but also of sporadic PD in several populations from South Europe, North Africa and Middle East. Moreover, a different polymorphic variant, Gly2385Arg, is a frequent risk factor for PD among Chinese and Japanese populations. Currently, the Gly2019Ser and Gly2385Arg variants represent the most relevant PD-causing mutation and risk allele, respectively, linking the etiology of the familial and the sporadic forms of this disease. Understanding how the dysfunction of LRRK2 protein leads to neurodegeneration might provide crucial insights for unraveling the molecular mechanisms of PD and for developing disease-modifying therapies. Special issue dedicated to Dr. Moussa Youdim.     

The WD40 Domain Is Required for LRRK2 Neurotoxicity

Background

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson disease (PD). LRRK2 contains an “enzymatic core” composed of GTPase and kinase domains that is flanked by leucine-rich repeat (LRR) and WD40 protein-protein interaction domains. While kinase activity and GTP-binding have both been implicated in LRRK2 neurotoxicity, the potential role of other LRRK2 domains has not been as extensively explored.

Principal Findings

We demonstrate that LRRK2 normally exists in a dimeric complex, and that removing the WD40 domain prevents complex formation and autophosphorylation. Moreover, loss of the WD40 domain completely blocks the neurotoxicity of multiple LRRK2 PD mutations.

Conclusion

These findings suggest that LRRK2 dimerization and autophosphorylation may be required for the neurotoxicity of LRRK2 PD mutations and highlight a potential role for the WD40 domain in the mechanism of LRRK2-mediated cell death.     

LRRK2 mutations and neurotoxicant susceptibility

Interactions between genetic and environmental factors are thought to contribute to the pathogenesis of the majority of Parkinson’s disease (PD) cases. However, our understanding of these interactions is at an early stage. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of hereditary PD. Penetrance of LRRK2 mutations is incomplete and variable, suggesting that other environmental or genetic factors may contribute to the development of the disorder. Recently, using animal models, several attempts have been made to understand if LRRK2 may mediate sensitivity to environmental neurotoxicants. Here, we critically review the most current data on how LRRK2 mutations influence neurotoxicity in PD models.     

LRRK2 mutations impair depolarization-induced mitophagy through inhibition of mitochondrial accumulation of RAB10

ABSTRACT

Parkinson disease (PD) is a disabling, incurable disorder with increasing prevalence in the western world. In rare cases PD is caused by mutations in the genes for PINK1 (PTEN induced kinase 1) or PRKN (parkin RBR E3 ubiquitin protein ligase), which impair the selective autophagic elimination of damaged mitochondria (mitophagy). Mutations in the gene encoding LRRK2 (leucine rich repeat kinase 2) are the most common monogenic cause of PD. Here, we report that the LRRK2 kinase substrate RAB10 accumulates on depolarized mitochondria in a PINK1- and PRKN-dependent manner. RAB10 binds the autophagy receptor OPTN (optineurin), promotes OPTN accumulation on depolarized mitochondria and facilitates mitophagy. In PD patients with the two most common LRRK2 mutations (G2019S and R1441C), RAB10 phosphorylation at threonine 73 is enhanced, while RAB10 interaction with OPTN, accumulation of RAB10 and OPTN on depolarized mitochondria, depolarization-induced mitophagy and mitochondrial function are all impaired. These defects in LRRK2 mutant patient cells are rescued by LRRK2 knockdown and LRRK2 kinase inhibition. A phosphomimetic RAB10 mutant showed less OPTN interaction and less translocation to depolarized mitochondria than wild-type RAB10, and failed to rescue mitophagy in LRRK2 mutant cells. These data connect LRRK2 with PINK1- and PRKN-mediated mitophagy via its substrate RAB10, and indicate that the pathogenic effects of mutations in LRRK2, PINK1 and PRKN may converge on a common pathway.

Abbreviations : ACTB: actin beta; ATP5F1B: ATP synthase F1 subunit beta; CALCOCO2: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide m-chlorophenylhydrazone; Co-IP: co-immunoprecipitation; EBSS: Earle’s balanced salt solution; GFP: green fluorescent protein; HSPD1: heat shock protein family D (Hsp60) member 1; LAMP1: lysosomal associated membrane protein 1; LRRK2: leucine rich repeat kinase 2; IF: immunofluorescence; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; OMM: outer mitochondrial membrane; OPTN: optineurin; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RHOT1: ras homolog family member T1; ROS: reactive oxygen species; TBK1: TANK binding kinase 1; WB: western blot.     

Leucine-rich repeat kinase 2 induces α-synuclein expression via the extracellular signal-regulated kinase pathway

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of autosomal-dominant Parkinson's disease (PD). The second known autosomal-dominant PD gene (SNCA) encodes α-synuclein, which is deposited in Lewy bodies, the neuropathological hallmark of PD. LRRK2 contains a kinase domain with homology to mitogen-activated protein kinase kinase kinases (MAPKKKs) and its activity has been suggested to be a key factor in LRRK2-associated PD. Here we investigated the role of LRRK2 in signal transduction pathways to identify putative PD-relevant downstream targets. Over-expression of wild-type [wt]LRRK2 in human embryonic kidney HEK293 cells selectively activated the extracellular signal-regulated kinase (ERK) module. PD-associated mutants G2019S and R1441C, but not kinase-dead LRRK2, induced ERK phosphorylation to the same extent as [wt]LRRK2, indicating that this effect is kinase-dependent. However, ERK activation by mutant R1441C and G2019S was significantly slower than that for [wt]LRRK2, despite similar levels of expression. Furthermore, induction of the ERK module by LRRK2 was associated to a small but significant induction of SNCA, which was suppressed by treatment with the selective MAPK/ERK kinase inhibitor U0126. This pathway linking the two dominant PD genes LRRK2 and SNCA may offer an interesting target for drug therapy in both familial and sporadic disease.     

On the road to leucine-rich repeat kinase 2 signalling: evidence from cellular and in vivo studies

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Although PD has long been considered a purely sporadic disorder, genetic research has revealed an underlying genetic cause in at least 10% of all PD cases. To date, mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial PD. Moreover, given the strong clinical and neuropathological similarities between LRRK2 PD and the sporadic forms of the disease, the notion is supported that the unravelling of the molecular pathways underlying LRRK2 PD will greatly contribute to our general understanding of PD. Therefore, intense research efforts have been focused on the understanding of the physiological function of LRRK2 and its relation to PD. To date, progress has been made in these fields based on the study of LRRK2 cell culture models, the identification of LRRK2 interaction partners and kinase substrates and the generation of LRRK2 animal models. In this review, the current insights into the cellular role of LRRK2 are discussed. The overview reveals a potential involvement of LRRK2 in major cell signalling pathways including apoptosis, cytoskeleton dynamics, protein translation, mitogen-activated protein kinase signalling and specific dopaminergic functions, consistent with its proposed role as a signal transduction protein.     

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.     

The role of the LRRK2 gene in Parkinsonism

Parkinson’s disease (PD), like many common age-related conditions, has been recognized to have a substantial genetic component. Multiple lines of evidence suggest that Leucine-rich repeat kinase 2 (LRRK2) is a crucial factor to understanding the etiology of PD. LRRK2 is a large, widely expressed, multi-domain and multifunctional protein. LRRK2 mutations are the major cause to inherited and sporadic PD. In this review, we discuss the pathology and clinical features which show diversity and variability of LRRK2-associated PD. In addition, we do a thorough literature review and provide theoretical data for gene counseling. Further, we present the evidence linking LRRK2 to various possible pathogenic mechanism of PD such as α-synuclein, tau, inflammatory response, oxidative stress, mitochondrial dysfunction, synaptic dysfunction as well as autophagy-lysosomal system. Based on the above work, we investigate activities both within GTPase and outside enzymatic regions in order to obtain a potential therapeutic approach to solve the LRRK2 problem.     

Dysregulated LRRK2 signaling in response to endoplasmic reticulum stress leads to dopaminergic neuron degeneration in C. elegans

Mutation of leucine-rich repeat kinase 2 (LRRK2) is the leading genetic cause of Parkinson's Disease (PD), manifested as age-dependent dopaminergic neurodegeneration, but the underlying molecular mechanisms remain unclear. Multiple roles of LRRK2 may contribute to dopaminergic neurodegeneration. Endoplasmic reticulum (ER) stress has also been linked to PD pathogenesis, but its interactive mechanism with PD genetic factors is largely unknown. Here, we used C. elegans, human neuroblastoma cells and murine cortical neurons to determine the role of LRRK2 in maintaining dopaminergic neuron viability. We found that LRRK2 acts to protect neuroblastoma cells and C. elegans dopaminergic neurons from the toxicity of 6-hydroxydopamine and/or human α-synuclein, possibly through the p38 pathway, by supporting upregulation of GRP78, a key cell survival molecule during ER stress. A pathogenic LRRK2 mutant (G2019S), however, caused chronic p38 activation that led to death of murine neurons and age-related dopaminergic-specific neurodegeneration in nematodes. These observations establish a critical functional link between LRRK2 and ER stress.     

The role of posttranslational modifications of α-synuclein and LRRK2 in Parkinson's disease: Potential contributions of environmental factors

Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease (AD), and the most prevalent movement disorder. PD is characterized by dopaminergic neurodegeneration in the substantia nigra, but its etiology has yet to be established. Among several genetic variants contributing to PD pathogenesis, α-synuclein and leucine-rich repeat kinase (LRRK2) are widely associated with neuropathological phenotypes in familial and sporadic PD. α-Synuclein and LRRK2 found in Lewy bodies, a pathogenic hallmark of PD, are often posttranslationally modified. As posttranslational modifications (PTMs) are key processes in regulating the stability, localization, and function of proteins, PTMs have emerged as important modulators of α-synuclein and LRRK2 pathology. Aberrant PTMs altering phosphorylation, ubiquitination, nitration and truncation of these proteins promote PD pathogenesis, while other PTMs such as sumoylation may be protective. Although the causes of many aberrant PTMs are unknown, environmental risk factors may contribute to their aberrancy. Environmental toxicants such as rotenone and paraquat have been shown to interact with these proteins and promote their abnormal PTMs. Notably, manganese (Mn) exposure leads to a PD-like neurological disorder referred to as manganism—and induces pathogenic PTMs of α-synuclein and LRRK2. In this review, we highlight the role of PTMs of α-synuclein and LRRK2 in PD pathogenesis and discuss the impact of environmental risk factors on their aberrancy.     

Molecular basis of Parkinsons’s disease linked to LRRK2 mutations

Parkinson’s disease (PD) is a common neurodegenerative disorder whose symptoms are consistent with death of dopaminergic neurons in the substantia nigra of the brain. The pathogenesis of PD involves several factors, such as α-synuclein aggregation, oxidative stress, mitochondrial dysfunction, and activation of apoptosis, but the exact molecular mechanism of neurodegeneration remains obscure. PD is usually sporadic, while rare monogenic forms have been identified and described in the past 15 years. Familial Parkinson’s disease is most commonly associated with mutations of the leucine repeat-rich kinase 2 gene (LRRK2). The mechanism of the disease due to LRRK2 mutations is unknown. The signaling cascades regulated by LRRK2 are difficult to study because the physiological substrates of the enzyme are unidentified. The G2019S substitution has been found to be the most common LRRK2 mutation, facilitating a search for patients with LRRK2-associated PD in various populations. The review considers the effects of LRRK2 mutations on protein and, in particular, α-synuclein aggregation, cytoskeletal dynamics, the inflammatory response, and the induction of apoptosis as revealed in both in vitro experiments and studies in PD patients. Investigation of rare hereditary PD forms with known etiology provides for a better understanding of the mechanism of neurodegeneration in more common sporadic PD forms.     

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages

Mutations in the leucine‐rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol‐3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2‐dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.     

Abberant protein synthesis in G2019S LRRK2 Drosophila Parkinson disease-related phenotypes

LRRK2 mutations are a frequent cause of familial Parkinson disease (PD) and are also found in a number of sporadic PD cases. PD-linked G2019S and I2020T mutations in the kinase domain of LRRK2 result in elevated kinase activity, which is required for the toxicity of these pathogenic variants in cell and animal models of PD. We recently reported that LRRK2 interacts with and phosphorylates a number of mammalian ribosomal proteins, several of which exhibit increased phosphorylation via both G2019S and I2020T LRRK2. Blocking the phosphorylation of ribosomal protein s15 through expression of phospho-deficient T136A s15 prevents age-associated locomotor deficits and dopamine neuron loss caused by G2019S LRRK2 expression in Drosophila indicating that s15 is a pathogenic LRRK2 substrate. We previously described that G2019S LRRK2 causes an induction of bulk mRNA translation that is blocked by T136A s15 or the protein synthesis inhibitor anisomycin. Here, we report the protective effects of the eIF4E/eIF4G interaction inhibitor 4EGI-1, in preventing neurodegenerative phenotypes in G2019S LRRK2 flies, and discuss how our findings and those of other groups provide a framework to begin investigating the mechanistic impact of LRRK2 on translation.     

The R1441C mutation of LRRK2 disrupts GTP hydrolysis

Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the leading genetic cause of Parkinson's disease (PD). LRRK2 is predicted to contain kinase and GTPase enzymatic domains, with recent evidence suggesting that the kinase activity of LRRK2 is central to the pathogenic process associated with this protein. The GTPase domain of LRRK2 plays an important role in the regulation of kinase activity. To investigate how the GTPase domain might be related to disease, we examined the GTP binding and hydrolysis properties of wild type and a mutant form of LRRK2. We show that LRRK2 immunoprecipitated from cells has a detectable GTPase activity that is disrupted by a familial mutation associated with PD located within the GTPase domain, R1441C.     

Dispensable role of Drosophila ortholog of LRRK2 kinase activity in survival of dopaminergic neurons

Background

Parkinson's disease (PD) is the most prevalent incurable neurodegenerative movement disorder. Mutations in LRRK2 are associated with both autosomal dominant familial and sporadic forms of PD. LRRK2 encodes a large putative serine/threonine kinase with GTPase activity. Increased LRRK2 kinase activity plays a critical role in pathogenic LRRK2 mutant-induced neurodegeneration in vitro. Little is known about the physiological function of LRRK2.

Results

We have recently identified a Drosophila line with a P-element insertion in an ortholog gene of human LRRK2 (dLRRK). The insertion results in a truncated Drosophila LRRK variant with N-terminal 1290 amino acids but lacking C-terminal kinase domain. The homozygous mutant fly develops normally with normal life span as well as unchanged number and pattern of dopaminergic neurons. However, dLRRK mutant flies were selectively sensitive to hydrogen peroxide induced stress but not to paraquat, rotenone and β-mercaptoethanol induced stresses.

Conclusion

Our results indicate that inactivation of dLRRK kinase activity is not essential for fly development and suggest that inhibition of LRRK activity may serve as a potential treatment of PD. However, dLRRK kinase activity likely plays a role in protecting against oxidative stress.     

Discovery of novel indolinone-based,potent, selective and brain penetrant inhibitors of LRRK2

Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common genetic cause of Parkinson’s disease (PD). The most frequent kinase-enhancing mutation is the G2019S residing in the kinase activation domain. This opens up a promising therapeutic avenue for drug discovery targeting the kinase activity of LRRK2 in PD. Several LRRK2 inhibitors have been reported to date. Here, we report a selective, brain penetrant LRRK2 inhibitor and demonstrate by a competition pulldown assay in vivo target engagement in mice.