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LRRK2, a puzzling protein: Insights into Parkinson's disease pathogenesis
... This mutation was reported to have a modulatory role in the kinase activity of the protein. According to several studies, G2019S mutations enhance the kinase activity by two-to three-fold by increasing the catalysis rate and not enhancing the substrate affinity [95]. Similarly, other pathogenic PD mutations, such as R1441C/G/H, Y1699C, and I2020T, were also reported to moderately enhance the kinase activity of the protein [95]. ...
... According to several studies, G2019S mutations enhance the kinase activity by two-to three-fold by increasing the catalysis rate and not enhancing the substrate affinity [95]. Similarly, other pathogenic PD mutations, such as R1441C/G/H, Y1699C, and I2020T, were also reported to moderately enhance the kinase activity of the protein [95]. The pathogenic LRRK2 PD mutations discussed above interfere with the modulation of LRRK2 cellular phosphorylation sites. ...
... The pathogenic LRRK2 PD mutations discussed above interfere with the modulation of LRRK2 cellular phosphorylation sites. In the G2019S mutation, Ser910, Ser935, Ser955, and Ser973 are completely phosphorylated, whereas these sites are hypo-phosphorylated in the presence of R1441G/C, Y1699C, and I2020T mutations [95,96]. ...
Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease, characterized by the specific loss of dopaminergic neurons in the midbrain. The pathophysiology of PD is likely caused by a variety of environmental and hereditary factors. Many single-gene mutations have been linked to this disease, but a significant number of studies indicate that mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a potential therapeutic target for both sporadic and familial forms of PD. Consequently, the identification of potential LRRK2 inhibitors has been the focus of drug discovery. Various investigations have been conducted in academic and industrial organizations to investigate the mechanism of LRRK2 in PD and further develop its inhibitors. This review summarizes the role of LRRK2 in PD and its structural details, especially the kinase domain. Furthermore, we reviewed in vitro and in vivo findings of selected inhibitors reported to date against wild-type and mutant versions of the LRRK2 kinase domain as well as the current trends researchers are employing in the development of LRRK2 inhibitors.
... Possible LRRK2-induced abnormalities in corticostriatal synaptic transmission are suggested by the key role this protein plays in synaptogenesis and synaptic function (Esteves et al., 2014;Benson et al., 2018;Chen et al., 2018; Figure 1). Several studies, some of which investigating synaptic fraction preparations (Biskup et al., 2006;Piccoli et al., 2011), showed that LRRK2 is highly expressed in cerebral cortex and dorsal striatum compared to other brain areas (Taymans et al., 2006;Westerlund et al., 2008;Mandemakers et al., 2012;Davies et al., 2013;Giesert et al., 2013;West et al., 2014). ...
... Many of the proteins encoded by genes causing recessive, atypical forms of PD, such as parkin, PINK1, and DJ-1, are involved in mitochondrial homeostatic processes (Lin and Beal, 2006;McCoy and Cookson, 2012;Kalia and Lang, 2015;Bose and Beal, 2016). The investigation of the pathophysiological consequences of LRRK2 mutations also unveiled a mitochondrial regulatory role for this protein (Esteves et al., 2014;Singh et al., 2019). Indeed, the presence of LRRK2 mutations has been linked to abnormalities in mitochondrial ATP and reactive oxygen species (ROS) production, mitochondrial fusion and fission dynamics, mitophagy, mitochondrial DNA (mtDNA) damage, and calcium homeostasis (Figure 2). ...
... The investigation of the molecular pathways linking LRRK2 and α-syn has attracted a lot of attention (Esteves et al., 2014;Schapansky et al., 2015;Cresto et al., 2019;Outeiro et al., 2019). A possible role for LRRK2 in the formation of abnormally folded α-syn aggregates was suggested by histopathological studies showing that LRRK2 could be found in the context of LBs. ...
The pathogenesis of Parkinson’s disease (PD) is thought to rely on a complex interaction between the patient’s genetic background and a variety of largely unknown environmental factors. In this scenario, the investigation of the genetic bases underlying familial PD could unveil key molecular pathways to be targeted by new disease-modifying therapies, still currently unavailable. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are responsible for the majority of inherited familial PD cases and can also be found in sporadic PD, but the pathophysiological functions of LRRK2 have not yet been fully elucidated. Here, we will review the evidence obtained in transgenic LRRK2 experimental models, characterized by altered striatal synaptic transmission, mitochondrial dysfunction, and α-synuclein aggregation. Interestingly, the processes triggered by mutant LRRK2 might represent early pathological phenomena in the pathogenesis of PD, anticipating the typical neurodegenerative features characterizing the late phases of the disease. A comprehensive view of LRRK2 neuronal pathophysiology will support the possible clinical application of pharmacological compounds targeting this protein, with potential therapeutic implications for patients suffering from both familial and sporadic PD.
... Indeed all 7 of our validated hits represent highly interesting molecular targets to follow up on. We chose, however, to focus on LRRK2, a seven domain, 285kDa cytosolic protein implicated in lysosomal degradation 33,34 . Previous work by us and others suggests that patients harbouring Z-AAT mutations develop liver disease when their polymer disposal mechanisms (autophagy -lysosomal -degradation pathway; ALP) are overrun. ...
... Interestingly, genetic variation at the LRRK2 locus has been identified as a key contributor to the risk of another protein misfolding disorder, Parkinson's disease 34 . This association led to the development of LRRK2 kinase inhibitors and antisense oligonucleotides as potential new medicines for PD, with leading candidates expected to enter late-stage clinical trials this year 17 . ...
Alpha-1 antitrypsin deficiency (A1ATD) is a life-threatening condition caused by inheritance of the SERPINA1-Z genetic variant (PiZ) leading to protein misfolding and liver toxicity. There remain no approved medicines for this disease. Here we report the results of a small molecule screen performed in patient-derived iPSC hepatocytes that identified Leucine-rich repeat kinase-2 (LRRK2), as a potentially new therapeutic target. Genetic deletion of LRRK2 in PiZ mice reduced both intrahepatic misfolded polymers and fibrosis leading us to test a library of commercially available LRRK2 inhibitors across several cell models. One of these molecules, CZC-25146, a candidate with favourable pharmacokinetic properties, reduced polymer load, increased normal AAT secretion and reduced inflammatory cytokines in both cells and the PiZ mouse. Mechanistically, this effect was achieved via inhibition of LRRK2 kinase activity and autophagy induction. These findings support the use of CZC-25146 (and potentially LRRK2 inhibitors more broadly) in hepatic proteinopathy disease research and as potential new therapies.
... These results suggest a role for the LRRK2 pathway in compulsive alcohol intake. LRRK2 is a multifunctional protein with kinase and GTPase activities (24), involved in neuronal vesicle trafficking (25) and synaptic plasticity (26). In this regard, LRRK2 regulates the subcellular distribution of protein kinase A (PKA) and the phosphorylation of its targets, thus influencing glutamatergic neurotransmission (27). ...
... More recently, SNPs in LRRK2 were associated with cancer (55,56) and inflammatory conditions (including infectious and autoimmune diseases) (57). LRRK2 is a complex protein with different functional regions, including protein-protein interaction, GTPase, and kinase activity domains (24). Alterations in LRRK2 kinase function have been shown to affect dopaminergic function (26,28,58,59) via disruption of synaptic vesicle formation and trafficking (60), signal transmission (26), and receptor function (28). ...
Background: Genetics influence the vulnerability to alcohol use disorders, and among the implicated genes, three previous studies have provided evidences for the involvement of LRRK2 in alcohol dependence (AD). LRRK2 expression is broadly dysregulated in postmortem brain from AD humans, as well as in the brain of mice with alcohol dependent-like behaviors and in a zebrafish model of alcohol preference. The aim of the present study was to evaluate the association of variants in the LRRK2 gene with AD in multiethnic populations from South and North America.
Methods: Alcohol-screening questionnaires [such as CAGE and Alcohol Use Disorders Identification Test (AUDIT)] were used to determine individual risk of AD. Multivariate logistic regression analyses were done in three independent populations (898 individuals from Bambuí, Brazil; 3,015 individuals from Pelotas, Brazil; and 1,316 from the United States). Linkage disequilibrium and conditional analyses, as well as in silico functional analyses, were also conducted.
Results: Four LRRK2 variants were significantly associated with AD in our discovery cohort (Bambuí): rs4768231, rs4767971, rs7307310, and rs1465527. Two of these variants (rs4768231 and rs4767971) were replicated in both Pelotas and US cohorts. The consistent association signal (at the LRRK2 locus) found in populations with different genetic backgrounds reinforces the relevance of our findings.
Conclusion: Taken together, these results support the notion that genetic variants in the LRRK2 locus are risk factors for AD in humans.
... Current evidence suggests that the increased kinase activity caused by the LRRK2 G2019S -mutation (LRRK2 G2019S ), associated with the regulation of mitochondrial dynamics, vesicle trafficking and chaperone mediated autophagy [7][8][9], plays a pivotal role in the pathogenesis of LRRK2 G2019S -associated PD, but the molecular mechanisms leading to neurodegeneration remain largely unknown [10]. ...
... Neuronal function is strongly dependent on oxidative metabolism and efficient organelle clearance, and mitochondrial homeostasis greatly depends on adequate mitochondrial dynamics, turnover and renewal through autophagy, which makes the study of mitochondrial function and autophagy highly relevant in the elucidation of pathological pathways leading to neurodegeneration [7,8]. Recent studies have characterized mitochondrial dysfunction and autophagy impairment in LRRK2 G2019Sassociated PD [9][10][11]. Furthermore, therapeutic strategies in LRRK2 G2019S -associated PD consisting of drug testing in experimental models directed to reverse mitochondrial alterations, and LRRK2 inhibition as a therapeutic target for impaired autophagy in PD, have been tested with positive outcomes [12][13][14]. ...
Background:
Mutations in leucine rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD). Mitochondrial and autophagic dysfunction has been described as etiologic factors in different experimental models of PD. We aimed to study the role of mitochondria and autophagy in LRRK2 G2019S -mutation, and its relationship with the presence of PD-symptoms.
Methods:
Fibroblasts from six non-manifesting LRRK2 G2019S -carriers (NM-LRRK2 G2019S ) and seven patients with LRRK2 G2019S -associated PD (PD-LRRK2 G2019S ) were compared to eight healthy controls (C). An exhaustive assessment of mitochondrial performance and autophagy was performed after 24-h exposure to standard (glucose) or mitochondrial-challenging environment (galactose), where mitochondrial and autophagy impairment may be heightened.
Results:
A similar mitochondrial phenotype of NM-LRRK2 G2019S and controls, except for an early mitochondrial depolarization (54.14% increased, p = 0.04), was shown in glucose. In response to galactose, mitochondrial dynamics of NM-LRRK2 G2019S improved (- 17.54% circularity, p = 0.002 and + 42.53% form factor, p = 0.051), probably to maintain ATP levels over controls. A compromised bioenergetic function was suggested in PD-LRRK2 G2019S when compared to controls in glucose media. An inefficient response to galactose and worsened mitochondrial dynamics (- 37.7% mitochondrial elongation, p = 0.053) was shown, leading to increased oxidative stress. Autophagy initiation (SQTSM/P62) was upregulated in NM-LRRK2 G2019S when compared to controls (glucose + 118.4%, p = 0.014; galactose + 114.44%, p = 0.009,) and autophagosome formation increased in glucose media. Despite of elevated SQSTM1/P62 levels of PD-NM G2019S when compared to controls (glucose + 226.14%, p = 0.04; galactose + 78.5%, p = 0.02), autophagosome formation was deficient in PD-LRRK2 G2019S when compared to NM-LRRK2 G2019S (- 71.26%, p = 0.022).
Conclusions:
Enhanced mitochondrial performance of NM-LRRK2 G2019S in mitochondrial-challenging conditions and upregulation of autophagy suggests that an exhaustion of mitochondrial bioenergetic and autophagic reserve, may contribute to the development of PD in LRRK2 G2019S mutation carriers.
... A third protein associated with PD is LRRK2, which under normal circumstances forms a complex with RHOT1/Miro1 essential for its proteasomal degradation, but not when carrying a PDassociated mutation [134]. LRRK2 downregulation increases autophagy, and causes accumulation of autophagic structures [135]. Therefore, LRRK2 could also genetically link PD development with impairment in mitophagy. ...
... Therefore, LRRK2 could also genetically link PD development with impairment in mitophagy. Yet, while LRRK2 is found on mitochondria, and the G2019S variant alters mitochondrial morphology, ROS levels and mitochondrial membrane potential, there is no direct link between this protein and mitophagy [135]. While the roles of PINK1, PRKN and LRRK2 in preventing PD can be linked to mitophagy, it remains unclear why other proteins involved in this pathway are not found mutated in PD patients and why related mutations lead to ALS rather than PD. ...
The catabolic process of macroautophagy, through the rapid degradation of unwanted cellular components, is involved in a multitude of cellular and organismal functions that are essential to maintain homeostasis. Those functions include adaptation to starvation, cell development and differentiation, innate and adaptive immunity, tumor suppression, autophagic cell death, and maintenance of stem cell stemness. Not surprisingly, an impairment or block of macroautophagy can lead to severe pathologies. A still increasing number of reports, in particular, have revealed that mutations in the autophagy-related (ATG) genes, encoding the key players of macroautophagy, are either the cause or represent a risk factor for the development of several illnesses. The aim of this review is to provide a comprehensive overview of the diseases and disorders currently known that are or could be caused by mutations in core ATG proteins but also in the so-called autophagy receptors, which provide specificity to the process of macroautophagy. Our compendium underlines the medical relevance of this pathway and underscores the importance of the eventual development of therapeutic approaches aimed at modulating macroautophagy.
... P62 is a receptor for ubiquitinated substrates sequestered into autophagosomes, and it regulates protein aggregate formation 26 . Indeed, P62 is the major component of the ubiquitin-containing inclusions in various neurodegenerative diseases such as PD 27,28 . The adaptor protein P62 regulates Nrf2 levels. ...
Chronic neuroinflammation is implicated in the pathogenesis of Parkinson’s disease (PD), one of the most common neurodegenerative diseases. Itaconate, an endogenous metabolite derived from the tricarboxylic acid cycle via immune‐responsive gene 1 activity, may mediate anti-inflammatory responses by activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway. This study investigates the neuroprotective potential of 4-octyl itaconate (OI), a cell-permeable derivative of itaconate, in cellular models of PD. OI not only suppressed lipopolysaccharide-induced proinflammatory cascades of inducible nitric oxide synthase, cyclooxygenase-2, and cytokines release in mouse BV2 microglial cells but also activated the Nrf2 signaling pathway and its downstream targets in these cells. Conditioned medium derived from OI-treated BV2 cells protected against rotenone- and MPP⁺-induced neurotoxicity in Neuro 2A cells. Overall, our findings support the anti-inflammatory neuroprotective potential of OI in PD.
... Mutations in LRRK2 are an important genetic cause of familial PD (fPD) overall with 1 to 40% of fPD cases associated with LRRK2 depending on the population under study (Bras et al., 2005;Greggio et al., 2008;Lesage et al., 2006;Ozelius et al., 2006). Since 2004, when a missense change on the LRRK2 gene was firstly associated with fPD, numerous coding and non-coding variants of LRRK2 have been identified in PD families, among which the G2019S and R1441C/G mutations are the 2 most common pathogenic variants occurring on the kinase and GTPase domains of the LRRK2 protein respectively (Dauer & Ho, 2010;Esteves et al., 2014;Giesert et al., 2017;Henry et al., 2015;Paisán-Ruiz et al., 2013). However, how these pathogenic changes contribute to fPD is still unclear. ...
Mutations in the LRRK2 gene are an important genetic cause for familial Parkinsons Disease (LRRK2-PD) and clinical trials are ongoing to evaluate the benefits associated with the therapeutical reduction of LRRK2 kinase activity. In this study, we aimed at modelling the molecular milieu surrounding LRRK2 and describe the changes that occur during disease with the aim of contrasting and comparing sporadic PD and LRRK2-PD. We analysed the molecular expression profiles (whole blood mRNA) of LRRK2s protein interactors in the sporadic vs familial PD conditions and found interesting differences between the 2 scenarios. Our results showed that LRRK2 interactors not only presented different alterations in expression levels in sporadic and familial PD while compared to controls; they also exhibited distinct co-expression behaviours in the 2 PD conditions. These results suggest that, albeit being classified as the same disease based on clinical features, LRRK2-PD and sporadic PD show significant differences from a molecular perspective.
... For instance, the p.Gly2019Ser mutation, the most common LRRK2 variant, has an 85% penetrance by age 80 [67]. However, penetrance can vary among different ethnic groups and is even lower for mutations in codon 1441 [68]. In general, it is believed that monogenic forms of PD account for 5-10% of cases, with LRRK2 and PRKN being the major contributors to autosomal dominant and autosomal recessive forms, respectively [21,69]. ...
Brain disorders pose a significant global healthcare challenge due to their complexity and profound impact on individuals and society. This review explores recent advancements in brain disease research, deepening our understanding and progress towards treatments. Technological innovations, like high-resolution imaging and optogenetics, play a pivotal role in illuminating cellular structures and dissecting neural circuits. Genetic analysis identifies dis-ease-associated genes and mutations, yet translating findings to clinical applications remains challenging. Epigenomics, transcriptomics, and proteomics provide molecular insights, un-covering disrupted pathways in conditions like Alzheimer's disease. Stem cell models elucidate developmental mechanisms, and immunotherapies, including CAR T cells, emerge as prom-ising strategies for brain cancers like glioblastoma. Genome editing tools, exemplified by CRISPR, enable precise modification of disease genes in models, deepening our understanding of pathogenesis. Multifaceted approaches integrating human studies, animal models, and bi-oinformatics propel the field forward. However, complexities persist, as disease specificity for cell types or brain regions remains an enigma, and genetic factors interact with environmental influences, highlighting disease intricacies. Ultimately, interdisciplinary efforts combining bi-ophysical and psychospiritual perspectives are essential to fully understand brain health and unlock transformative treatments for these debilitating disorders. A comprehensive under-standing of brain disorders is vital to improve the quality of life for millions and alleviate the global burden of neurological afflictions. Continued collaborative research will lead to an era of improved brain health worldwide.
... There is some evidence supporting interactions between PD and AD associated proteins such as APP and LRRK2 . The LRRK2 gene encodes a protein with armadillo repeats (ARM) region, an ankyrin repeat (ANK) region, a leucine-rich repeat (LRR) domain, a kinase domain, a RAS domain, a GTPase domain, and a WD40 domain and has diverse cellular mechanisms such as alpha-synuclein phosphorylation, involvement in the autophagy-lysosomal pathway, microtubule dynamics, synthesis and trafficking of vesicles, mitochondrial function, and the ubiquitinproteasome system (UPS) (Paisán-Ruíz et al., 2004;Zimprich et al., 2004;Esteves et al., 2014;Steger et al., 2016). The interaction between LRRK2 and APP occurs at the intracellular domain of APP, and causes phosphorylation of APP at Thr 668 which promotes the transcriptional activity of the APP intracellular domain and its translocation to the nucleus . ...
Alzheimer's disease (AD) is the most common disorder associated with cognitive impairment. Recent observations emphasize the pathogenic role of multiple factors inside and outside the central nervous system, supporting the notion that AD is a syndrome of many etiologies rather than a "heterogeneous" but ultimately unifying disease entity. Moreover, the defining pathology of amyloid and tau coexists with many others, such as α-synuclein, TDP-43, and others, as a rule, not an exception. Thus, an effort to shift our AD paradigm as an amyloidopathy must be reconsidered. Along with amyloid accumulation in its insoluble state, β-amyloid is becoming depleted in its soluble, normal states, as a result of biological, toxic, and infectious triggers, requiring a shift from convergence to divergence in our approach to neurodegeneration. These aspects are reflected-in vivo-by biomarkers, which have become increasingly strategic in dementia. Similarly, synucleinopathies are primarily characterized by abnormal deposition of misfolded α-synuclein in neurons and glial cells and, in the process, depleting the levels of the normal, soluble α-synuclein that the brain needs for many physiological functions. The soluble to insoluble conversion also affects other normal brain proteins, such as TDP-43 and tau, accumulating in their insoluble states in both AD and dementia with Lewy bodies (DLB). The two diseases have been distinguished by the differential burden and distribution of insoluble proteins, with neocortical phosphorylated tau deposition more typical of AD and neocortical α-synuclein deposition peculiar to DLB. We propose a reappraisal of the diagnostic approach to cognitive impairment from convergence (based on clinicopathologic criteria) to divergence (based on what differs across individuals affected) as a necessary step for the launch of precision medicine.
... The LRRK2 mutations G2019S and R1441C (hereafter referred to as GS or RC) are commonly found in familial PD [9][10][11] . These two mutations are located in the kinase and ROC (Ras of complex GTPase) domains of the LRRK2 protein, respectively (Fig. 1a) 12 . ...
LRRK2 mutations are closely associated with Parkinson’s disease (PD). Convergent evidence suggests that LRRK2 regulates striatal function. Here, by using knock-in mouse lines expressing the two most common LRRK2 pathogenic mutations—G2019S and R1441C—we investigated how LRRK2 mutations altered striatal physiology. While we found that both R1441C and G2019S mice displayed reduced nigrostriatal dopamine release, hypoexcitability in indirect-pathway striatal projection neurons, and alterations associated with an impaired striatal-dependent motor learning were observed only in the R1441C mice. We also showed that increased synaptic PKA activities in the R1441C and not G2019S mice underlie the specific alterations in motor learning deficits in the R1441C mice. In summary, our data argue that LRRK2 mutations’ impact on the striatum cannot be simply generalized. Instead, alterations in electrochemical, electrophysiological, molecular, and behavioral levels were distinct between LRRK2 mutations. Our findings offer mechanistic insights for devising and optimizing treatment strategies for PD patients.
... This suggests that a series of age-related disfunctions may contribute to the development of PD. Proteins accumulation, genetic factors, autophagy, mitophagy, and lower protection against oxidative stress have been proposed as possible age-related PD factors [6][7][8][9]. ...
Background
Parkinson’s Disease (PD) is the second most frequent degenerative disorder, the risk of which increases with age. A preclinical PD diagnostic test does not exist. We identify PD blood metabolites and metabolic pathways significantly correlated with age to develop personalized age-dependent PD blood biomarkers.
Results
We found 33 metabolites producing a receiver operating characteristic (ROC) area under the curve (AUC) value of 97%. PCA revealed that they belong to three pathways with distinct age-dependent behavior: glycine, threonine and serine metabolism correlates with age only in PD patients; unsaturated fatty acids biosynthesis correlates with age only in a healthy control group; and, finally, tryptophan metabolism characterizes PD but does not correlate with age.
Conclusions
The targeted analysis of the blood metabolome proposed in this paper allowed to find specific age-related metabolites and metabolic pathways. The model offers a promising set of blood biomarkers for a personalized age-dependent approach to the early PD diagnosis.
... LRRK2 est une protéine qui possède de nombreux sites d'autophosphorylation (Kamikawaji et al., 2009;Lobbestael et al., 2012;Webber et al., 2011). Six sites putatifs d'autophosphorylation sont présents dans le domaine LRR, tous orientés vers la face extérieure de la protéine (T1024, S1025, S1124, S1253, S1283, S1292 (Esteves et al., 2014). ...
Les protéines alpha-synucléine (α-syn) et leucine-rich repeat kinase 2 (LRRK2) sont deux protéines ayant un rôle majeur dans la physiopathologie de la maladie de Parkinson (MP) et interviennent aussi bien dans les formes dites sporadiques que dans les formes familiales. La mutation G2019S du gène codant pour LRRK2 est la mutation la plus fréquente. Cette mutation induit une augmentation de l’activité kinase de LRRK2 qui conduit à sa toxicité. Plusieurs hypothèses convergent vers l’idée que LRRK2 et l’α-syn interagiraient pour conduire à la dysfonction et/ou la mort des neurones dopaminergiques (DA) de la substance noire (SNc) dans la MP. Dans la première partie de cette étude, différentes formes sauvage (WT) ou mutée (G2019S) de LRRK2 ont été surexprimées spécifiquement dans les neurones de la SNc via l’utilisation de vecteurs lentiviraux (LV) et adéno-viraux associés (AAV). La question principale de cette étude était d’évaluer si l’expression spécifiquement neuronale de LRRK2 induisait la dégénérescence des neurones DA de la SNc. Nous avons généré des constructions comportant uniquement la partie C-terminale de LRRK2 (ΔLRRK2) en aval du domaine LRR. In vitro, le fragment ΔLRRK2G2019S présente une activité kinase supérieure au fragment ΔLRRK2WT avec une augmentation d’activité comparable à la forme entière de LRRK2. In vivo, six mois après l’injection (PI) de ΔLRRK2 WT ou G2019S dans la SNc, les mesures du nombre de neurones montrent que seul le fragment ΔLRRK2G2019S induit une mort neuronale significative (30%) comparé à la forme ΔLRRK2WT, uniquement lorsque l’expression est générée via des vecteurs AAV. Ces résultats suggèrent que l’expression purement neuronale d’un fragment contenant le domaine kinase de LRRK2 est suffisante pour induire une dégénérescence de la SN. Dans la seconde partie du projet, nous avons étudié l’hypothèse que ΔLRRK2G2019S via son activité kinase amplifiée, pourrait augmenter la toxicité le l’α-syn mutée A53T. Pour répondre à cette question, les vecteurs AAV codant pour ΔLRRK2 G2019S ou une forme inactive de la kinase (ΔLRRK2G2019S/D1994A), et celui codant pour l’α-syn A53T ont été co-injectés dans la SNc. Les analyses réalisées à 6 et 15 semaines PI montrent que ΔLRRK2G2019S augmente la mort neuronale induite par l’α-syn A53T d’une manière kinase dépendante. Tous ces résultats supportent l’hypothèse que l’existence d’une interaction fonctionnelle entre LRRK2 et l’α-syn pourrait jouer un rôle fondamental dans la physiopathologie de la MP offrant des possibilités de stratégie de neuroprotection ciblant l’interaction LRRK2/α-syn.
... Proteins encoded by the human genes of the monogenic forms of PD, SNCA, VPS35, LRRK2, and ATP13A2 serve to maintain normal neuronal transport functions [36,[51][52][53][54][55][56]. Mutations in these genes can contribute to the disruption of endo-and exocytosis and intracellular transport, which results in the incorrect redistribution of neurotransmitters, primarily dopamine (DA), in the synaptic gap and presynaptic terminals. ...
Processes of intracellular and extracellular transport play one of the most important roles in the functioning of cells. Changes to transport mechanisms in a neuron can lead to the disruption of many cellular processes and even to cell death. It was shown that disruption of the processes of vesicular, axonal, and synaptic transport can lead to a number of diseases of the central nervous system, including Parkinson’s disease (PD). Here, we studied changes in the expression of genes whose protein products are involved in the transport processes (Snca, Drd2, Rab5a, Anxa2, and Nsf) in the brain tissues and peripheral blood of mice with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced models of PD. We detected changes in the expressions of Drd2, Anxa2, and Nsf at the earliest modeling stages. Additionally, we have identified conspicuous changes in the expression level of Anxa2 in the striatum and substantia nigra of mice with MPTP-induced models of PD in its early stages. These data clearly suggest the involvement of protein products in these genes in the earliest stages of the pathogenesis of PD.
... LRRK2 encodes a serine/threonine kinase called dardarin, after the Basque word for tremor. The native protein appears to transit between a monomeric and dimeric form [83,84]. LRRK2 is involved in many cellular functions such as regulation of neurite growth and cytoskeletal dynamics, maintenance of lysosomal function, and synaptic vesicle endocytosis (SVE) [85]. After neurotransmission, the replenishment of synaptic vesicles with neurotransmitters is ensured by the SVE [86]. ...
Parkinson’s disease (PD) is a disorder characterized by a triad of motor symptoms (akinesia, rigidity, resting tremor) related to loss of dopaminergic neurons mainly in the Substantia nigra pars compacta. Diagnosis is often made after a substantial loss of neurons has already occurred, and while dopamine replacement therapies improve symptoms, they do not modify the course of the disease. Although some biological mechanisms involved in the disease have been identified, such as oxidative stress and accumulation of misfolded proteins, they do not explain entirely PD pathophysiology, and a need for a better understanding remains. Neurodegenerative diseases, including PD, appear to be the result of complex interactions between genetic and environmental factors. The latter can alter gene expression by causing epigenetic changes, such as DNA methylation, post-translational modification of histones and non-coding RNAs. Regulation of genes responsible for monogenic forms of PD may be involved in sporadic PD. This review will focus on the epigenetic mechanisms regulating their expression, since these are the genes for which we currently have the most information available. Despite technical challenges, epigenetic epidemiology offers new insights on revealing altered biological pathways and identifying predictive biomarkers for the onset and progression of PD.
... This increase was associated with poorer CMA efficiency since the clearance of lysosomal specific substrates with a KFERQ motif was lower on LRRK2 knock-in midbrain cells. This is consistent with evidence that demonstrates that LRRK2 regulates the activity of a subset of Rab GTPases, which are responsible for membrane mobilization, vesicle assembly, and transportation [198,199]. G2019S is the most common mutation in LRRK2-associated PD, and mutated LRRK2 is thought to halt the autophagic process. For example, differentiated SH-SY5Y neuronal cells expressing this mutant form of LRRK2 presented much smaller neurites and an aberrant accumulation of LC3 vesicles [200]. ...
Macroautophagy, a quality control mechanism, is an evolutionarily conserved pathway of lysosomal degradation of protein aggregates, pathogens, and damaged organelles. As part of its vital homeostatic role, macroautophagy deregulation is associated with various human disorders, including neurodegenerative diseases. There are several lines of evidence that associate protein misfolding and mitochondrial dysfunction in the etiology of Alzheimer's, Parkinson's, and Huntington's diseases. Macroautophagy has been implicated in the degradation of different protein aggregates such as Aβ, tau, alpha-synuclein (α-syn), and mutant huntingtin (mHtt) and in the clearance of dysfunctional mitochondria. Taking these into consideration, targeting autophagy might represent an effective therapeutic strategy to eliminate protein aggregates and to improve mitochondrial function in these disorders. The present review describes our current understanding on the role of macroautophagy in neurodegenerative disorders and focuses on possible strategies for its therapeutic modulation.
... LRRK2 has been linked to a wide range of biological processes and several LRRK2 kinase substrates have been proposed (Boon et al., 2014;Wallings et al., 2015;Roosen and Cookson, 2016;Rosenbusch and Kortholt, 2016;Tang, 2016). LRRK2 signaling is involved in mitochondrial regulation, autophagy and cell death, actin and microtubule dynamics, synthesis and transport of vesicles as well as neurotransmitter, immune responses, and the activity of the intestinal network (Esteves et al., 2014). Impaired mitochondrial activity is a common characteristic of both, iPD and familial PD, and several genes linked to PD play an important role in mitochondrial quality control (Narendra et al., 2008;Youle and van der Bliek, 2012;Liu et al., 2017;Toyofuku et al., 2019;Delcambre et al., 2020;Weindel et al., 2020). ...
Parkinson’s Disease (PD) is the second most common neurodegenerative disease world-wide. Mutations in the multidomain protein Leucine Rich Repeat Kinase 2 (LRRK2) are the most frequent cause of hereditary PD. Furthermore, recent data suggest that independent of mutations, increased kinase activity of LRRK2 plays an essential role in PD pathogenesis. Isolated mitochondria of tissue samples from PD patients carrying LRRK2 mutations display a significant impairment of mitochondrial function. However, due to the complexity of the mitochondrial signaling network, the role of LRRK2 in mitochondrial metabolism is still not well understood. Previously we have shown that D. discoideum Roco4 is a suitable model to study the activation mechanism of LRRK2 in vivo . To get more insight in the LRRK2 pathways regulating mitochondrial activity we used this Roco4 model system in combination with murine RAW macrophages. Here we show that both Dictyostelium roco4 knockout and cells expressing PD-mutants show behavioral and developmental phenotypes that are characteristic for mitochondrial impairment. Mitochondrial activity measured by Seahorse technology revealed that the basal respiration of D. discoideum roco4- cells is significantly increased compared to the WT strain, while the basal and maximal respiration values of cells overexpressing Roco4 are reduced compared to the WT strain. Consistently, LRRK2 KO RAW 264.7 cells exhibit higher maximal mitochondrial respiration activity compared to the LRRK2 parental RAW264.7 cells. Measurement on isolated mitochondria from LRRK2 KO and parental RAW 264.7 cells revealed no difference in activity compared to the parental cells. Furthermore, neither D. discoideum roco4- nor LRRK2 KO RAW 264.7 showed a difference in either the number or the morphology of mitochondria compared to their respective parental strains. This suggests that the observed effects on the mitochondrial respiratory in cells are indirect and that LRRK2/Roco proteins most likely require other cytosolic cofactors to elicit mitochondrial effects.
... It was observed that locomotor defects have occurred due to low levels of striatal mitochondrial complex I (NDUFS4), striatal synaptosomes, and synaptic vesicular proton pump protein (V-ATPase H) that lowered dopamine uptake in PD mouse than wildtype mouse (31). Moreover, LRRK2 mutants reported significant mitochondrial abnormalities, including damaged mitochondrial DNA, abnormal morphology, movement, and homeostasis, low ATP production, decreased membrane potential, reduced respiratory chain complex IV activity, increased mitochondrial proton leakage, disturbed calcium handling, and impaired mitochondrial degradation (32)(33)(34)(35). ...
Mitochondrial dysfunction is one of the crucial factors involved in PD’s pathogenicity, which emerges from a combination of genetic and environmental factors. These factors cause differential molecular expression in neurons, such as varied transcriptional regulation of genes, elevated oxidative stress, α‐synuclein aggregation and endogenous neurotoxins release, which induces epigenetic modifications and triggers energy crisis by damaging mitochondria of the dopaminergic neurons (DN). So far, these events establish a complicated relationship with underlying mechanisms of mitochondrial anomalies in PD, which has remained unclear for years and made PD diagnosis and treatment extremely difficult. Therefore, in this review, we endeavored to discuss the complex association of epigenetic modifications and other associated vital factors in mitochondrial dysfunction. We propose a hypothesis that describes a vicious cycle in which mitochondrial dysfunction and oxidative stress act as a hub for regulating DA neuron's fate in PD. Oxidative stress triggers the release of endogenous neurotoxins (CTIQs) that lead to mitochondrial dysfunction along with abnormal α‐synuclein aggregation and epigenetic modifications. These disturbances further intensify oxidative stress and mitochondrial damage, amplifying the synthesis of CTIQs and works vice versa. This vicious cycle may result in the degeneration of DN to hallmark Parkinsonism. Furthermore, we have also highlighted various endogenous compounds and epigenetic marks (neurotoxic and neuroprotective), which may help for devising future diagnostic biomarkers and target specific drugs using novel PD management strategies. Impact of the epigenetic regulator on mitochondrial dysfunction in Parkinson's disease.
... Consistent with other investigations, Chen et al. had confirmed the therapeutic role of lncRNA MALAT1 in PD models [69]. Moreover, the leucine-rich repeat kinase (LRRK2) gene could encode a serine-threonine protein kinase, and its mutations were linked to familial and sporadic PD [70]. Some studies had further indicated that the LRRK2 overexpression could cause oxidative stress damage and induce the apoptosis of dopaminergic neurons [71]. ...
Parkinson’s disease (PD) is known as one of the most common degenerative disorders related to the damage of the central nervous system (CNS). This brain disorder is also characterized by the formation of Lewy bodies in the cytoplasm of the dopaminergic neurons in the substantia nigra pars compacta (SNc), which consequently leads to motor and non-motor symptoms. With regard to the growing trend in the number of cases with PD and its effects on individuals, families, and communities, immediate treatments together with diagnostic methods are required. In this respect, long non-coding ribonucleic acids (lncRNAs) represent a large class of ncRNAs with more than 200 nucleotides in length, playing key roles in some important processes including gene expression, cell differentiation, genomic imprinting, apoptosis, and cell cycle. They are highly expressed in the CNS and previous studies have further reported that the expression profile of lncRNAs is disrupted in human diseases such as neurodegenerative disorders. Since the levels of some lncRNAs change over time in the brains of patients with PD, a number of previous studies have examined their potentials as biomarkers for this brain disorder. Therefore, the main purpose of this study was to review the advances in the related literature on lncRNAs as diagnostic, therapeutic, and prognostic biomarkers for PD.
... The LRRK2 gene contains approximately 144 kilobases with 51 exons, which encodes a 2527-amino acid protein leucine-rich repeat serine/threonine-protein kinase 2 (LRKK2) (also known as dardarin, which means trembling or unsteady) with both kinase and GTPase activity [90]. LRRK2 gene is identified to be the most common cause of both familial and sporadic PD. ...
Parkinson’s disease (PD) is one of the most common types of neurological disorder prevailing worldwide and is rapidly increasing in the elderly population across the globe. The cause of PD is still unknown, but a number of genetic as well as environmental factors contributing to the pathogenesis of Parkinson’s disease have been identified. The hallmark of PD includes dopamine deficiency (neurotransmitter imbalance) due to the gradual loss of dopaminergic nerves in the substantia nigra in the midbrain. Studying the mutation of associated genes is particularly informative in understanding the fundamental molecular and pathogenic changes in PD. Intracellular accumulation of misfolded or degraded protein due to mutated gene leads to the manifestation of mitochondrial dysfunction, oxidative stress followed by multifaceted patho-physiologic symptoms. Other studies include the appearance of motor and non-motor responses like resting tremor, muscle stiffness, slow movement and anxiety, anaemia, constipation, rapid eye movement and sleep behaviour disorder. Many bioactive natural compounds have shown positive pharmacological results in treating a number of extensive disease models of PD. Despite the availability of an end number of potent medicinal plants around the world, limited research has been done associated with various neurological disorders, including PD. The currently available dopamine-based drug treatments have several side effects; further, they are not effective enough to combat PD completely. Therefore, various plant-based compounds with medicinal benefits have grabbed lots of attention from researchers to deal with various life-threatening neurodegenerative disorders like PD. On the basis of literature available to date, here, we have discussed and addressed the molecular basis, current scenario, and the best possible treatment of PD for the future with minimal or no side-effects using various key bioactive compounds from natural origin/medicinal plants.
... It is highly expressed in the lung, spleen, kidney and peripheral immune cells (neutrophils, monocytes and dendritic cells), while in the brain, there is a comparatively low level of LRRK2 expression [10][11][12]. In the cell, the protein is mainly cytoplasmic and prevalently associated with microtubules, endoplasmic reticulum and Golgi apparatus, trans-Golgi network (TGN), endosomes and lysosomes, where it has been linked to different processes, including cytoskeleton remodeling, neurite morphology, cell processes outgrowth, mitochondrial functions, protein synthesis, proteostasis, autophagy, vesicular trafficking and inflammation [13,14]. In the years several LRRK2 kinase substrates [9,[15][16][17][18][19] and binding partners have been identified [20][21][22][23][24]. Interestingly, many of these binding partners interact with the Roc domain, indicating ...
Missense mutations in the leucine-rich repeat kinase-2 (LRRK2) gene represent the most common cause of autosomal dominant Parkinson's disease (PD). In the years LRRK2 has been associated with several organelles and related pathways in cell. However, despite the significant amount of research done in the past decade, the contribution of LRRK2 mutations to PD pathogenesis remains unknown. Growing evidence highlights that LRRK2 controls multiple processes in brain immune cells, microglia and astrocytes, and suggests that deregulated LRRK2 activity in these cells, due to gene mutation, might be directly associated with pathological mechanisms underlying PD. In this brief review, we recapitulate and update the last LRRK2 functions dissected in microglia and astrocytes. Moreover, we discuss how dysfunctions of LRRK2-related pathways may impact glia physiology and their cross-talk with neurons, thus leading to neurodegeneration and progression of PD.
... Mutations in the gene encoding LRRK2 have been linked to familial and sporadic Parkinson disease (PD) cases. [1] Trafficking of vesicles: LRRK2 localizes to vesicles, where it interacts with vesicular proteins [2]In presynaptic vesicles, LRRK2 silencing leads to a redistribution of vesicles, alters recycling dynamics, and increases vesicle kinetics. ...
... Of note, mitochondrial impairment occurs early in PD pathogenesis, especially at the level of complex I, and animal models of PD are generated after administration of complex I inhibitors such as 1-methyl-4-phenylpyridinium (MPP+) [104]. Relevantly, familial PD is mainly characterized by mutations in genes involved in mitochondrial dysfunction, such as Parkin, α-synuclein, and leucine-rich repeat kinase 2 (LRRK2) [103,[105][106][107]. On the other hand, exposure to pesticides that disrupt the mitochondrial function increases the likelihood of developing PD [108]. ...
Mitochondria are the main source of reactive oxygen species (ROS), most of them deriving from the mitochondrial respiratory chain. Among the numerous enzymatic and non-enzymatic antioxidant systems present in mitochondria, mitochondrial glutathione (mGSH) emerges as the main line of defense for maintaining the appropriate mitochondrial redox environment. mGSH's ability to act directly or as a co-factor in reactions catalyzed by other mitochondrial enzymes makes its presence essential to avoid or to repair oxidative modifications that can lead to mitochondrial dysfunction and subsequently to cell death. Since mitochondrial redox disorders play a central part in many diseases, harboring optimal levels of mGSH is vitally important. In this review, we will highlight the participation of mGSH as a contributor to disease progression in pathologies as diverse as Alzheimer's disease, alcoholic and non-alcoholic steatohepatitis, or diabetic nephropathy. Furthermore, the involvement of mitochondrial ROS in the signaling of new prescribed drugs and in other pathologies (or in other unmet medical needs, such as gender differences or coronavirus disease of 2019 (COVID-19) treatment) is still being revealed; guaranteeing that research on mGSH will be an interesting topic for years to come.
... Figure 10. Implication du gène LRRK2 dans les mécanismes cellulaires [165]. LRRK2 peut affecter la fonction mitochondriale, le système ubiquitine-protéasome, la voie autophagie-lysosomale, la dynamique des microtubules ainsi que le trafic de vésicules et de protéines, la phosphorylation de l'alpha-synucléine et les cellules du système immunitaire. ...
La Tunisie a vu une grande variété d'envahisseurs et de migrants allant de la population berbère autochtone aux Arabes et Européens. Cette région est caractérisée par les grands pedigrees, les faibles taux de migration et les taux élevés de consanguinité qui augmentent le risque des maladies autosomiques récessives y compris les maladies neuro-dégénératives. Dans notre pays, la prévalence de la maladie de Parkinson (MP) augmente jusqu’à 43/100000 personnes et devient un problème de santé majeur. La MP est une maladie neuro-dégénérative dont la forme idiopathique débute en moyenne vers 60 ans. Au cours des 20 dernières années, plusieurs gènes ont été identifiés chez des patients qui ont développé leurs premiers symptômes avant l’âge de 40 ans. Notre étude a montré que la structure génétique de la MP en Tunisie est distincte des autres populations, puisque plus de 50% des cas avaient une origine génétique. La fréquence des formes monogéniques chez les patients avec un âge de début tardif de la MP était relativement élevée et similaire à celle des patients avec un âge de début précoce. Ces formes étaient essentiellement dues à la mutation LRRK2-p.G2019S (identifiée chez 44.4% des cas) qui s'avère être une mutation fondatrice apparue chez un seul ancêtre commun d’origine berbère. Sur le plan clinique, nous avons montré que les patients porteurs de la mutation LRRK2-p.G2019S avaient un âge de début de la MP plus précoce mais avec un phénotype plus bénin que celui des formes idiopathiques. Une deuxième mutation fondatrice d’origine berbère (p.Q456*) a été identifiée sur le gène PINK1. La fréquence élevée inhabituelle des mutations sur ce gène peut être limitée à la population tunisienne berbère. Nos résultats ont aussi confirmé que les mutations sur le gène PARK2 et sur le gène GBA ne constituent pas un facteur de risque fréquent de la MP en Tunisie. En plus de ces mutations sur les gènes connus, nous avons identifié 4 nouveaux gènes candidats dans la MP. Cette structure génétique particulière de la MP en Tunisie pourrait être principalement le résultat de l'origine historique berbère de notre population Tunisienne.
... The precise biological role of LRRK2 is not fully understood. However, to date, the protein has been shown to be involved in different cellular processes such as the regulation of cytoskeleton, neurite morphology, inflammatory processes, regulation of mitochondrial fission, protein synthesis, proteostasis, and vesicular trafficking (Esteves et al., 2014). The multitude of proposed functions can be summarized by findings from protein interaction network analysis that point to a role for LRRK2 in intracellular organization, intracellular transport, and protein localization (Manzoni et al., 2015;Porras et al., 2015;Tomkins et al., 2018). ...
Mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) gene are linked to autosomal dominant Parkinson's disease (PD), and genetic variations at the LRRK2 locus are associated with an increased risk for sporadic PD. This gene encodes a kinase that is physiologically multiphosphorylated, including clusters of both heterologous phosphorylation and autophosphorylation sites. Several pieces of evidence indicate that LRRK2's phosphorylation is important for its pathological and physiological functioning. These include a reduced LRRK2 heterologous phosphorylation in PD brains or after pharmacological inhibition of LRRK2 kinase activity as well as the appearance of subcellular LRRK2 accumulations when this protein is dephosphorylated at heterologous phosphosites. Nevertheless, the regulatory mechanisms governing LRRK2 phosphorylation levels and the cellular consequences of changes in LRRK2 phosphorylation remain incompletely understood. In this review, we present current knowledge on LRRK2 phosphorylation, LRRK2 phosphoregulation, and how LRRK2 phosphorylation changes affect cellular processes that may ultimately be linked to PD mechanisms.
... The LRRK2 protein has been found to localize, among other sites, to mitochondrial structures where it interacts with a number of key regulators of mitochondrial fission/fusion, as well as to mitochondrial autophagy and motility (Biskup et al., 2006, Wang et al., 2012, Rosenbusch and Kortholt, 2016. Several other studies have found prominent signs of mitochondrial dysfunction relating both to size and distribution as a result of the G2019S mutation (MacLeod et al., 2006, Mortiboys et al., 2010, Li et al., 2014, Esteves et al., 2014, Singh et al., 2019, which strongly implies a role for LRRK2 in mitochondrial homeostasis. The initiating process leading to these mitochondrial alterations, however, are still elusive. ...
Mutations in the LRRK2 gene have been widely linked to Parkinson's disease. The G2019S variant has been shown to contribute uniquely to both familial and sporadic forms of the disease. LRRK2-related mutations have been extensively studied, yet the wide variety of cellular and network events directly or indirectly related to these mutations remain poorly understood. In this study, we structured multi-nodal human neural networks carrying the G2019S mutation using custom-designed microfluidic chips coupled to microelectrode-arrays. By applying live imaging approaches, immunocytochemistry and computational modelling, we have revealed alterations in both the structure and function of the resulting neural networks when compared to controls. We provide first evidence of increased neuritic density associated with the G2019S LRRK2 mutation, while previous studies have found either a strong decrease, or no change, compared to controls. Additionally, we corroborate previous findings regarding increased baseline network activity compared to control neural networks. Furthermore, we can reveal additional network alterations attributable to the specific mutation by selectively inducing transient overexcitation to confined parts of the structured multi-nodal networks. These alterations, which we were able to capture both at the micro- and mesoscale manifested as differences in relative network activity and correlation, as well as in mitochondria activation, neuritic remodelling, and synaptic alterations. Our study thus provides important new insights into early signs of neural network pathology significantly expanding upon the current knowledge relating to the G2019S Parkinson's disease mutation.
... The LRRK2 mutations G2019S and R1441C (hereafter referred to as GS or RC) are commonly found in familial PD [9][10][11] . These two mutations are located in the kinase and ROC (Ras of complex GTPase) domains of the LRRK2 protein, respectively (Fig. 1a) 12 . ...
LRRK2 mutations are associated with both familial and sporadic forms of Parkinsons disease (PD). Convergent evidence suggests that LRRK2 plays critical roles in regulating striatal function. Here, by using knock-in mouse lines that express the two most common LRRK2 pathogenic mutations (G2019S and R1441C) we investigated how pathogenic LRRK2 mutations altered striatal physiology. We found that R1441C mice displayed a reduced nigrostriatal dopamine release and hypoexcitability in indirect-pathway striatal projection neurons. These alterations were associated with impaired striatal-dependent motor learning in the R1441C knock-in mice. In contrast, no detectable alterations were observed in the G2019S knock-in mice. In summary, our data argue that the impact of individual LRRK2 mutations cannot be simply generalized. Our findings have far-reaching implications for devising treatment strategies for PD patients.
... LRRK2 gene product is a large multi-domain protein with two catalytic domains: a GTPase (ROC-COR) domain and a serine/threonine-directed protein kinase domain. Pathogenic mutations are found predominantly in these two domains, suggesting that LRRK2 enzymatic activities are involved in PD pathogenesis (Cookson, 2010), (Esteves, Swerdlow and Cardoso, 2014). Yet, how LRRK2 mutations in the two distinct functional domains contribute to PD pathogenesis and whether they act through a common mechanism is unknown. ...
LRRK2 is a kinase expressed in striatal spiny projection neurons (SPNs), cells which lose dopaminergic input in Parkinson disease (PD). R1441C and G2019S are the most common pathogenic mutations of LRRK2. How these mutations alter the structure and function of individual synapses on direct and indirect pathway SPNs is unknown and may reveal pre-clinical changes in dopamine-recipient neurons that predispose towards disease. Here, R1441C and G2019S knock-in mice enabled thorough evaluation of dendritic spines and synapses on pathway-identified SPNs. Biochemical synaptic preparations and super-resolution imaging revealed increased levels and altered organization of glutamatergic AMPA receptors in LRRK2 mutants. Relatedly, decreased frequency of excitatory post-synaptic currents accompanied changes in dendritic spine nano-architecture, and single-synapse currents, evaluated using 2-photon glutamate uncaging. Overall, LRRK2 mutations reshaped synaptic structure and function, an effect exaggerated in R1441C dSPNs. These data open the possibility of new neuroprotective therapies aimed at SPN synapse function, prior to disease onset.
... As mentioned above, several gene mutations causing familial PD are associated with mitochondrial dysfunction. In particular, the autosomal dominant G2019S mutation of the common late-onset familial PD gene LRRK2 is involved in the reduction of the mitochondrial membrane potential and ATP levels, thus leading to mitochondrial dysfunction [71] as well as other functions such as neuronal differentiation [72]. Although the physiological role of SNCA is still unclear, a series of experimental evidence has shown that it is implicated in synaptic transmission, gene expression in the nucleus, as well as mitochondrial function [73]. ...
... It has already been reported that LRRK2 is expressed in neurons, astroglia, and microglia in the central nervous system (CNS), and several types of immune cells (Higashi et al., 2007;Kubo et al., 2010;Liu et al., 2011;Moehle et al., 2012). Accumulated evidence suggests that LRRK2 plays a key role in endocytosis, axonal extension, autophagy, proliferation, and survival of neurons (Esteves et al., 2014;Maekawa et al., 2016a,b). While many studies have been focusing on the role of LRRK2 in the pathogenesis of motor dysfunction in PD, some groups have reported an association between LRRK2 and the subtle non-motor phenotypes of PD. ...
Leucine-rich repeat kinase 2 (LRRK2) is a molecule associated with familial and sporadic Parkinson’s disease. It regulates many central neuronal functions, such as cell proliferation, apoptosis, autophagy, and axonal extension. Recently, it has been revealed that LRRK2 is related to anxiety/depression-like behavior, implying an association between LRRK2 and stress. In the present study, we investigated for the first time the stress pathway and its relationship to gastrointestinal motility in LRRK2-knockout (KO) mice. The mice were subjected to acute restraint stress, and analyzed for activation of the paraventricular nucleus of the hypothalamus (PVN) using an immunohistochemical approach. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was assessed by Western blotting. The KO mice showed a lower number of c-Fos-positive cells and disruption of the ERK signaling pathway in the PVN in the presence of restraint stress. Stress responses in terms of both upper and lower gastrointestinal motility were alleviated in the mice, accompanied by lower c-Fos immunoreactivity in enteric excitatory neurons. Our present findings suggest that LRRK2 is a newly recognized molecule regulating the stress pathway in the PVN, playing a role in stress-related gastrointestinal dysmotility.
... Recent evidence has shown that there is interaction between APP and PD-related protein, called Leucine-rich repeat kinase 2 (LRRK2). LRRK2 is a protein which has been associated with diverse cellular mechanisms such as alpha-synuclein phosphorylation, autophagy-lysosomal pathway, microtubule dynamics, synthesis and trafficking of vesicles, mitochondrial function and ubiquitin-proteasome system (UPS) [27,28]. The interaction between LRRK2 and APP occurs at intracellular domain of APP(AICD), causing phosphorylation of APP at Thr 668 which promotes the transcriptional activity of AICD and its translocation to the nucleus. ...
Parkinson’s disease (PD) is the second commonest neurodegenerative disorder in the world with a rising prevalence. The pathophysiology is multifactorial but aggregation of misfolded α-synuclein is considered to be a key underpinning mechanism. Amyloid-β (Aβ) and tau deposition are also comorbid associations and especially Aβ deposition is associated with cognitive decline in PD. Some existing evidence suggests that low cerebrospinal fluid (CSF) Aβ42 is predictive of future cognitive impairment in PD. Recent studies also show that CSF Aβ is associated with the postural instability and gait difficulties (PIGD) or the newly proposed cholinergic subtype of PD, a possible risk factor for cognitive decline in PD. The glial-lymphatic system, responsible for convective solute clearance driven by active fluid transport through aquaporin-4 water channels, may be implicated in brain amyloid deposition. A better understanding of the role of this system and more specifically the role of Aβ in PD symptomatology, could introduce new treatment and repurposing drug-based strategies. For instance, apomorphine infusion has been shown to promote the degradation of Aβ in rodent models. This is further supported in a post-mortem study in PD patients although clinical implications are unclear. In this review, we address the clinical implication of cerebral Aβ deposition in PD and elaborate on its metabolism, its role in cognition and motor function/gait, and finally assess the potential effect of apomorphine on Aβ deposition in PD.
... These findings suggest that presenilin and PINK1 play important roles in the RO/PQ-induced neurotoxicity through the mechanisms involved in mitochondria-associated membranes. Esteves et al., 2014;Surmeier et al., 2017). Dysfunction of mitochondrial respiratory chain components was found in the brain, skeletal https://doi. ...
... Neuropathologisch zeigt sich ein heterogenes Bild: Interessanterweise wurden sowohl dem iPS ähnliche Befunde erhoben, mit Verlust von dopaminergen Neuronen und Lewy-Körper-Aggregation im Hirnstamm, als auch von einem Fehlen von Lewy-Körper-Ablagerungen berichtet [13]. Das LRRK2-Gen kodiert für eine Serin-Threonin-Kinase, pathologisch werden Auswirkungen u. a. auf endosomale und synaptische Transportund Recyclingprozesse diskutiert [8]. Aktuell wird eine Phase 1-Studie zur Inhibition der LRRK2-Kinase-Funktion durchgeführt. ...
Zusammenfassung
Monogene, also auf einem einzelnen Gendefekt beruhende Parkinson-Syndrome (PS), machen ca. 5 % aller Parkinson-Erkrankungen aus. Hierbei konnten in den letzten 20 Jahren drei autosomal-dominant ( SNCA, LRRK2, VPS35 ) und drei autosomal-rezessiv ( Parkin, PINK1, DJ-1 ) vererbte kausale Parkinson-Gene identifiziert und validiert werden. Während pathogene Veränderungen in SNCA sehr selten sind, früh beginnen und mit einer dementiellen Entwicklung einhergehen können, sind pathogene Varianten in LRRK2 unter den monogenen PS am häufigsten und Patienten klinisch nicht vom idiopathischen PS zu unterscheiden. Bei Patienten mit Erkrankungsbeginn vor dem 40. Lebensjahr sollte zunächst an Veränderungen im Parkin- und PINK1- Gen gedacht werden und, ebenso wie bei Patienten mit positiver Familienanamnese, eine genetische Beratung erfolgen. In jüngerer Zeit haben die dynamischen Entwicklungen auf dem Gebiet der Parkinson-Genetik zu neuen therapeutischen Ansätzen und ersten aktuell durchgeführten genspezifischen klinischen Studien geführt. Neben den etablierten monogenen PS existieren zum jetzigen Zeitpunkt noch nicht validierte Parkinson-Kandidatengene und gut charakterisierte genetische Risikofaktoren. Da monogene PS auch für das idiopathische PS Modellerkrankungen darstellen, sind in der Zukunft sowohl für monogene PS als auch für das idiopathische PS weitere Fortschritte auf dem Weg zur personalisierten Medizin zu erwarten.
... Consisting of 2527 amino acids, the LRRK2 protein is a serine-threonine kinase, which can catalyze the phosphorylation of cellular proteins involved in the MAPK signaling pathway [21]. Numerous studies have shown that the LRRK2 gene is associated with autosomal dominant PD [22]. In vivo and in vitro experiments demonstrated that LRRK2 modulates the eIF4E/4E-BP signaling pathway to stimulate eIF4E-mediated protein translation. ...
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), as a long chain non-coding RNA (lncRNA), has been reported to be upregulated in Parkinson's disease (PD). However, the mechanisms underlying this process remain unknown. Hence, to investigate the role of MALAT1 in PD, N-methyl-4-phenylpyridinium (MPP+) was used to induce PDin vitroin the MN9D dopaminergic neuronal cell line and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to induce PDin vivoin C57BL/6 mice. Quantitative Real-Time PCR (qRT-PCR) and western blot assay showed that the expression levels of MALAT1 and leucine-rich repeat kinase (LRRK2) were increased, and that of miR-205-5p was decreased in the midbrains of mice in which PD was induced by MPTP. MALAT1 suppressed the expression of miR-205-5p in MN9D cells. The results of luciferase reporter assay indicated that LRRK2 was a direct target of miR-205-5p. Transfection with the miR-205-5p mimics decreased, whereas transfection with miR-205-5p inhibitor increased the expression levels of LRRK2 mRNA and protein. The cell counting kit-8 (CCK-8) and flow cytometry assays showed that overexpression of LRRK2 reduced the viability and promoted apoptosis in MN9D cells treated with MPP+. MALAT1 knockdown exerted a protective effect on the viability and apoptosis of MN9D cells treated with MPP+, which was abrogated by LRRK2 overexpression and miR-205-5p inhibition. Our study demonstrates that the MALAT1/miR-205-5p axis regulates MPP+-induced apoptosis in MN9D cells by targeting LRRK2, thereby improving our understanding of the molecular pathogenesis of PD.
... In the case of the ROCO proteins, the literature-derived PPI network incorporates a potential bias toward the LRRK2 interactome, due to extensive investigation into LRRK2 in relation to PD. [44,45] This is evidenced by the nearly twofold increase in the number of LRRK2 interactors (from 62 to 113) compared to a previous analysis performed in 2014 using an earlier version of the same data processing pipeline. [4] Conversely, the distribution of nodes amongst the other ROCO proteins highlights the comparative neglect of research into characterizing the DAPK1, LRRK1, and MASL1 interactomes. ...
Signal transduction cascades governed by kinases and GTPases are a critical component of the command and control of cellular processes, with the precise outcome partly determined by direct protein‐protein interactions (PPIs). Here, we use the human ROCO proteins as a model for investigating PPI signalling events – taking advantage of the unique dual kinase/GTPase activities and scaffolding properties of these multidomain proteins. We report PPI networks that encompasses the human ROCO proteins, developed using two complementary approaches. First, using our recently developed weighted PPI network analysis (WPPINA) pipeline, a confidence‐weighted overview of validated ROCO protein interactors was obtained from peer‐reviewed literature. Second, novel ROCO PPIs were assessed experimentally via protein microarray screens. We compared the networks derived from these orthologous approaches to identify common elements within the ROCO protein interactome; functional enrichment analysis of this common core of the network identified stress response and cell projection organisation as shared functions within this protein family. Despite the presence of these commonalities, our results suggest that many unique interactors and therefore some specialised cellular roles have evolved for different members of the ROCO proteins. Overall, this multi‐approach strategy to increase the resolution of protein interaction networks represents a prototype for the utility of PPI data integration in understanding signalling biology.
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Parkinson's disease is a progressive neurodegenerative disorder that is associated with motor and nonmotor symptoms. Accumulation of misfolded α-synuclein is considered a key pathological feature during disease initiation and progression. While clearly deemed a synucleinopathy, the development of amyloid-β plaques, tau-containing neurofibrillary tangles, and even TDP-43 protein inclusions occur within the nigrostriatal system and in other brain regions. In addition, inflammatory responses, manifested by glial reactivity, T-cell infiltration, and increased expression of inflammatory cytokines, plus other toxic mediators derived from activated glial cells, are currently recognized as prominent drivers of Parkinson's disease pathology. However, copathologies have increasingly been recognized as the rule (>90%) and not the exception, with Parkinson's disease cases on average exhibiting three different copathologies. While microinfarcts, atherosclerosis, arteriolosclerosis, and cerebral amyloid angiopathy may have an impact on disease progression, α-synuclein, amyloid-β, and TDP-43 pathology do not seem to contribute to progression.
Neurodegenerative diseases manifest as progressive loss of neuronal structures and their myelin sheaths and lead to substantial morbidity and mortality, especially in the elderly. Despite extensive research, there are few effective treatment options for the diseases. MicroRNAs have been shown to be involved in the developmental processes of the central nervous system. Mounting evidence suggest they play an important role in the development of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. However, there are few reviews regarding the roles of miRNAs in neurodegenerative diseases. This review summarizes the recent developments in the roles of microRNAs in neurodegenerative diseases and presents the application of microRNA-based methods in the early diagnosis of these diseases.
Alterations to the LRRK2 gene have been associated with Parkinson’s disease and alcohol consumption in animals and humans. Furthermore, these disorders are strongly related to anxiety disorders (ADs). Thus, we investigated how the LRRK2 gene might influence anxiety in humans and mice. We elaborated a systematic review based on the PRISMA Statement of studies that investigated levels of anxiety in animal or human models with alterations in the LRRK2 gene. The search was conducted in the PubMed, Scopus, and Web of Science databases, and in reference lists with descriptors related to ADs and the LRRK2. From the 62 articles assessed for eligibility, 16 were included: 11 conducted in humans and seven, in mice. Lrrk2 KO mice and the LRRK2 G2019S, LRRK2 R1441G, and LRRK2 R1441C variants were addressed. Five articles reported an increase in anxiety levels concerning the LRRK2 variants. Decreased anxiety levels were observed in two articles, one focusing on the LRRK2 G2019S and the other, on the Lrrk2 KO mice. Eight other articles reported no differences in anxiety levels in individuals with Lrrk2 alterations compared to their healthy controls. This study discusses a possible influence between the LRRK2 gene and anxiety, adding information to the existing knowledge respecting the influence of genetics on anxiety.
Parkinson’s disease is a relatively common neurological disorder with incidence increasing with age. Since current medications only relieve the symptoms and do not change the course of the disease, therefore, finding disease-modifying therapies is a critical unmet medical need. However, significant progress in understanding how genetics underpins Parkinson's disease (PD) has opened up new opportunities for understanding disease pathogenesis and identifying possible therapeutic targets. One such target is leucine-rich repeat kinase 2 (LRRK2), an elusive enzyme implicated in both familial and idiopathic PD risk. As a result, both academia and industry have promoted the development of potent and selective inhibitors of LRRK2. In this review, we have summarized recent progress on the discovery and development of LRKK2 inhibitors as well as the bioactivity of several small-molecule LRRK2 inhibitors that have been used to inhibit LRRK2 kinase activity in vitro or in vivo.
Mutations in leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease with a similar clinical presentation and progression to idiopathic Parkinson’s disease, and common variation is linked to disease risk. Recapitulation of the genotype in rodent models causes abnormal dopamine release and increases the susceptibility of dopaminergic neurons to insults, making LRRK2 a valuable model for understanding the pathobiology of Parkinson’s disease. It is also a promising druggable target with targeted therapies currently in development. LRRK2 mRNA and protein expression in the brain is highly variable across regions and cellular identities. A growing body of work has demonstrated that pathogenic LRRK2 mutations disrupt striatal synapses before the onset of overt neurodegeneration. Several substrates and interactors of LRRK2 have been identified to potentially mediate these pre-neurodegenerative changes in a cell-type-specific manner. This review discusses the effects of pathogenic LRRK2 mutations in striatal neurons, including cell-type-specific and pathway-specific alterations. It also highlights several LRRK2 effectors that could mediate the alterations to striatal function, including Rabs and protein kinase A. The lessons learned from improving our understanding of the pathogenic effects of LRRK2 mutations in striatal neurons will be applicable to both dissecting the cell-type specificity of LRRK2 function in the transcriptionally diverse subtypes of dopaminergic neurons and also increasing our understanding of basal ganglia development and biology. Finally, it will inform the development of therapeutics for Parkinson’s disease.
Synapses are critical for neuronal communication and brain function. To maintain neuronal homeostasis, synapses rely on autophagy. Autophagic alterations cause neurodegeneration and synaptic dysfunction is a feature in neurodegenerative diseases. In Parkinson's disease (PD), where the loss of synapses precedes dopaminergic neuron loss, various PD-causative proteins are involved in the regulation of autophagy. So far only a few factors regulating autophagy at the synapse have been identified and the molecular mechanisms underlying autophagy at the synapse is only partially understood. Here, we describe Endophilin-B (EndoB) as a novel player in the regulation of synaptic autophagy in health and disease. We demonstrate that EndoB is required for autophagosome biogenesis at the synapse, whereas the loss of EndoB blocks the autophagy induction promoted by the PD mutation LRRK2G2019S. We show that EndoB is required to prevent neuronal loss. Moreover, loss of EndoB in the Drosophila visual system leads to an increase in synaptic contacts between photoreceptor terminals and their post-synaptic synapses. These data confirm the role of autophagy in synaptic contact formation and neuronal survival.
LRRK2 is a kinase expressed in striatal spiny projection neurons (SPNs), cells which lose dopaminergic input in Parkinson’s disease (PD). R1441C and G2019S are the most common pathogenic mutations of LRRK2. How these mutations alter the structure and function of individual synapses on direct and indirect pathway SPNs is unknown and may reveal pre-clinical changes in dopamine-recipient neurons that predispose towards disease. Here, R1441C and G2019S knock-in mice enabled thorough evaluation of dendritic spines and synapses on pathway-identified SPNs. Biochemical synaptic preparations and super-resolution imaging revealed increased levels and altered organization of glutamatergic AMPA receptors in LRRK2 mutants. Relatedly, decreased frequency of miniature excitatory post-synaptic currents accompanied changes in dendritic spine nano-architecture, and single-synapse currents, evaluated using 2-photon glutamate uncaging. Overall, LRRK2 mutations reshaped synaptic structure and function, an effect exaggerated in R1441C dSPNs. These data open the possibility of new neuroprotective therapies aimed at SPN synapse function, prior to disease onset.
The ubiquitin-proteasome system is the major pathway for the maintenance of protein homeostasis. Its inhibition causes accumulation of ubiquitinated proteins; this accumulation has been associated with several of the most common neurodegenerative diseases. Several genetic factors have been identified for most neurodegenerative diseases, however, most cases are considered idiopathic, thus making the study of the mechanisms of protein accumulation a relevant field of research. It is often mentioned that the biggest risk factor for neurodegenerative diseases is aging, and several groups have reported an age-related alteration of the expression of some of the 26S proteasome subunits and a reduction of its activity. Proteasome subunits interact with proteins that are known to accumulate in neurodegenerative diseases such as α-synuclein in Parkinson's, tau in Alzheimer's, and huntingtin in Huntington's diseases. These interactions have been explored for several years, but only until recently, we are beginning to understand them. In this review, we discuss the known interactions, the underlying patterns, and the phenotypes associated with the 26S proteasome subunits in the etiology and progression of neurodegenerative diseases where there is evidence of proteasome involvement. Special emphasis is made in reviewing proteasome subunits that interact with proteins known to have an age-related altered expression or to be involved in neurodegenerative diseases to explore key effectors that may trigger or augment their progression. Interestingly, while the causes of age-related reduction of some of the proteasome subunits are not known, there are specific relationships between the observed neurodegenerative disease and the affected proteasome subunits.
Background:
The gender effect in the prevalence of leucine-rich repeat kinase 2 (LRRK2) associated Parkinson disease (PD) remains controversial. Herein, we conducted a meta-analysis to investigate the gender effect among these patients.
Methods:
PubMed and EMBASE databases were searched to identify the potential related studies published before December 2017. Case-control studies with separated data of sex and mutation status were included in further analyses. We pooled relative risk (RR) using fixed-effect model. The publication bias and sensitivity analyses were also performed.
Results:
Sixty-four studies with 32452 patients diagnosed with PD were included. Higher prevalence of female patients with LRRK2-associated PD was observed with a pooled RR of 1.22 (95% CI 1.14-1.30, P＜0.001). Further subgroup analyses showed that higher prevalence of female patients was only obtained in G2019S mutation patients (RR = 1.32, 95% CI 1.23-1.43, P＜0.001), but not in G2385R variant patients (RR = 1.03, 95% CI 0.91-1.17, P = 0.651). No significant heterogeneity and publication bias were observed in additional analyses.
Conclusions:
Higher female prevalence of LRRK2 mutation suggests roles of gender-related risk factors in PD patients, especially who carried G2019S mutation. Contrary to idiopathic PD, no sex difference was observed in prevalence of patients carried G2385R variant.
Leucine-rich repeat kinase 2 (LRRK2) is a serine-threonine kinase involved in multiple cellular processes and signaling pathways. LRRK2 mutations are associated with autosomal-inherited Parkinson's disease (PD), and evidence suggests that LRRK2 pathogenic variants generally increase kinase activity. Therefore, inhibition of LRRK2 kinase function is a promising therapeutic strategy for PD treatment. The search for drug-like molecules capable of reducing LRRK2 kinase activity in PD led to the design of selective LRRK2 inhibitors predicted to be within the CNS drug-like space. This review highlights the journey that translates chemical tools for interrogating the role of LRRK2 in PD into promising drug candidates, addressing the challenges in discovering selective and brain-penetrant LRRK2 modulators and exploring the structure–activity relationship of distinct LRRK2 inhibitors.
Neuronal microtubules are key determinants of cell morphology, differentiation, migration and polarity, and contribute to intracellular trafficking along axons and dendrites. Microtubules are strictly regulated and alterations in their dynamics can lead to catastrophic effects in the neuron. Indeed, the importance of the microtubule cytoskeleton in many human diseases is emerging. Remarkably, a growing body of evidence indicates that microtubule defects could be linked to Parkinson’s disease pathogenesis. Only a few of the causes of the progressive neuronal loss underlying this disorder have been identified. They include gene mutations and toxin exposure, but the trigger leading to neurodegeneration is still unknown. In this scenario, the evidence showing that mutated proteins in Parkinson’s disease are involved in the regulation of the microtubule cytoskeleton is intriguing. Here, we focus on α-Synuclein, Parkin and Leucine-Rich Repeat Kinase 2 (LRRK2), the three main proteins linked to the familial forms of the disease. The aim is to dissect their interaction with tubulin and microtubules in both physiological and pathological conditions, in which these proteins are overexpressed, mutated or absent. We highlight the relevance of such an interaction and suggest that these proteins could trigger neurodegeneration via defective regulation of the microtubule cytoskeleton.
Parkinson disease (PD) treatment options have conventionally focused on dopamine replacement and provision of symptomatic relief. Current treatments cause undesirable adverse effects, and a large unmet clinical need remains for treatments that offer disease modification and that address symptoms resistant to levodopa. Advances in high-throughput drug screening methods for small molecules, developments in disease modelling and improvements in analytical technologies have collectively contributed to the emergence of novel compounds, repurposed drugs and new technologies. In this Review, we focus on disease-modifying and symptomatic therapies under development for PD. We review cellular therapies and repurposed drugs, such as nilotinib, inosine, isradipine, iron chelators and anti-inflammatories, and discuss how their success in preclinical models has paved the way for clinical trials. We provide an update on immunotherapies and vaccines. In addition, we review non-pharmacological interventions targeting motor symptoms, including gene therapy, adaptive deep brain stimulation (DBS) and optogenetically inspired DBS. Given the many clinical phenotypes of PD, individualization of therapy and precision of treatment are likely to become important in the future.
Parkinson disease-associated mutations within the GTPase domain Ras of complex proteins (ROC) of leucine rich repeat kinase 2 (LRRK2) result in an abnormal over-activation of its kinase domain. However, the mechanisms involved remain unclear. Recent study has shown that LRRK2 G-domain cycles between monomeric and dimeric conformations upon binding to GTP or guanosine diphosphate, and that the Parkinson's disease (PD)-associated R1441C/G/H mutations impair the G-domain monomer-dimer dynamics and trap the G-domain in a constitutive monomeric conformation. That led us to question whether other disease-associated mutations in G-domain would also affect its conformation. Here, we report that another PD-associated N1437H mutation also impairs its monomer-dimer conformational dynamics and GTPase activity. In contrast with mutations at R1441, ROCN1437H was found to be locked in a stable dimeric conformation in solution and its GTPase activity was ∼4-fold lower than that of the wild-type. Furthermore, the N1437H mutation reduced the GTP binding affinity by ∼2.5-fold when compared with other pathogenic G-domain mutations. Moreover, ROCN1437H was found to have a slower GTP dissociation rate, indicating that N1437H might interrupt the nucleotide exchange cycle. Taken together, our data support that conformational dynamics is important for LRRK2 GTPase activity and that the N1437H mutation impairs GTPase activity by locking the ROC domain in a persistently dimeric state.-Huang, X., Wu, C., Park, Y., Long, X., Hoang, Q. Q., Liao, J. The Parkinson's disease-associated mutation N1437H impairs conformational dynamics in the G domain of LRRK2.
Many neurodegenerative diseases feature mitochondrial dysfunction. This includes both common sporadic and rare Mendelian disorders. In some cases, mitochondrial defects mediate disease pathology, and in others, the defects actually initiate and drive the disease. Understanding the causes and consequences of mitochondrial dysfunction provides insight into neurodegenerative diseases and reveals potential therapeutic targets that could lead to new treatments. This chapter provides an overview of mitochondrial function and its role in cell physiology, presents current perceptions about the relevance of mitochondrial and bioenergetic dysfunction to neurodegenerative diseases, and discusses targeting mitochondria for therapeutic purposes.
Leucine-rich repeat kinase 2 (LRRK2) G2019S (glycine to serine) is the most common mutation associated with sporadic and familial Parkinson's disease (PD) with 80% penetrance by age 70. This mutation is found worldwide, with up to 40% of individuals in the North African Arab population carrying the mutation. Induced pluripotent stem cells (iPSCs) derived from fibroblasts of patients carrying the LRRK2 G2019S mutation have been a critical source of cells for generating dopaminergic neurons and studying G2019S-related pathology. These studies have elucidated LRRK2-related mechanisms of mitochondrial dysregulation, increased reactive oxygen species, truncated and simplified neurites, and cell death. These phenotypes are thought to result from the G2019S mutation increasing substrate access and therefore increasing the catalytic rate of the serine/threonine kinase. In this article, we critically review the contributions of in vitro modeling to the current knowledge on LRRK2 G2019S. We also analyze the role of patient-derived cell lines for the identification and validation of therapeutic targets, emphasizing their importance as part of a 3R approach to translational research and personalized medicine.
Degenerating neurons of Parkinson’s disease (PD) patient brains exhibit granules of phosphorylated extracellular signal-regulated protein kinase 1/2 (ERK1/2) that localize to autophagocytosed mitochondria. Here we show that 6-hydroxydopamine (6-OHDA) elicits activity-related localization of ERK1/2 in mitochondria of SH-SY5Y cells, and these events coincide with induction of autophagy and precede mitochondrial degradation. Transient transfection of wild-type (WT) ERK2 or constitutively active MAPK/ERK Kinase 2 (MEK2-CA) was sufficient to induce mitophagy to a degree comparable with that elicited by 6-OHDA, while constitutively active ERK2 (ERK2-CA) had a greater effect. We developed green fluorescent protein (GFP) fusion constructs of WT, CA, and kinase-deficient (KD) ERK2 to study the role of ERK2 localization in regulating mitophagy and cell death. Under basal conditions, cells transfected with GFP-ERK2-WT or GFP-ERK2-CA, but not GFP-ERK2-KD, displayed discrete cytoplasmic ERK2 granules of which a significant fraction colocalized with mitochondria and markers of autophagolysosomal maturation. The colocalizing GFP-ERK2/mitochondria granules are further increased by 6-OHDA and undergo autophagic degradation, as bafilomycin-A, an inhibitor of autolysosomal degradation, robustly increased their detection. Interestingly, increasing ERK2-WT or ERK2-CA expression was sufficient to promote comparable levels of macroautophagy as assessed by analysis of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3). In contrast, the level of mitophagy was more tightly correlated with ERK activity levels, potentially explained by the greater localization of ERK2-CA to mitochondria compared to ERK2-WT. These data indicate that mitochondrial localization of ERK2 activity is sufficient to recapitulate the effects of 6-OHDA on mitophagy and autophagic cell death.
By combining experimental neuron models and mathematical tools, we developed a "systems" approach to deconvolve cellular mechanisms of neurodegeneration underlying the most common known cause of Parkinson's disease (PD), mutations in leucine-rich repeat kinase 2 (LRRK2). Neurons ectopically expressing mutant LRRK2 formed inclusion bodies (IBs), retracted neurites, accumulated synuclein, and died prematurely, recapitulating key features of PD. Degeneration was predicted from the levels of diffuse mutant LRRK2 that each neuron contained, but IB formation was neither necessary nor sufficient for death. Genetic or pharmacological blockade of its kinase activity destabilized LRRK2 and lowered its levels enough to account for the moderate reduction in LRRK2 toxicity that ensued. By contrast, targeting synuclein, including neurons made from PD patient-derived induced pluripotent cells, dramatically reduced LRRK2-dependent neurodegeneration and LRRK2 levels. These findings suggest that LRRK2 levels are more important than kinase activity per se in predicting toxicity and implicate synuclein as a major mediator of LRRK2-induced neurodegeneration.
Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2, are a common cause of Parkinson's disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson's disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4 and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine-362 and alanine-364 of β-tubulin. Molecular modelling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine-40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knockout mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson's disease.
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.
Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson’s disease (PD). The neuropathology of LRRK2-related PD is heterogeneous and can include aberrant tau phosphorylation or neurofibrillary tau pathology. Recently, LRRK2 has been shown to phosphorylate tau in vitro; however, the major epitopes phosphorylated by LRRK2 and the physiological or pathogenic consequences of these modifications in vivo are unknown. Using mass spectrometry, we identified multiple sites on recombinant tau that are phosphorylated by LRRK2 in vitro, including pT149 and pT153, which are phospho-epitopes that to date have been largely unexplored. Importantly, we demonstrate that expression of transgenic LRRK2 in a mouse model of tauopathy increased the aggregation of insoluble tau and its phosphorylation at T149, T153, T205, and S199/S202/T205 epitopes. These findings indicate that tau can be a LRRK2 substrate and that this interaction can enhance salient features of human disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-013-1188-4) contains supplementary material, which is available to authorized users.
Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most important genetic contributors to Parkinson's disease. LRRK2 has been implicated in a number of cellular processes, including macroautophagy. To test whether LRRK2 has a role in regulating autophagy, a specific inhibitor of the kinase activity of LRRK2 was applied to human neuroglioma cells and downstream readouts of autophagy examined. The resulting data demonstrate that inhibition of LRRK2 kinase activity stimulates macroautophagy in the absence of any alteration in the translational targets of mTORC1, suggesting that LRRK2 regulates autophagic vesicle formation independent of canonical mTORC1 signaling. This study represents the first pharmacological dissection of the role LRRK2 plays in the autophagy/lysosomal pathway, emphasizing the importance of this pathway as a marker for LRRK2 physiological function. Moreover it highlights the need to dissect autophagy and lysosomal activities in the context of LRRK2 related pathologies with the final aim of understand their aetiology and identify specific target for disease modifying therapies in patients.
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known cause of Parkinson's disease (PD). Whether loss of LRRK2 function accounts for neurodegeneration of dopamine neurons in PD is not known, nor is it known whether LRRK2 kinase activity modulates the susceptibility of dopamine (DA) neurons to the selective dopaminergic toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To better understand the role of LRRK2 in DA neuronal survival and its role in the susceptibility of DA neurons to MPTP, we generated LRRK2 knock-out (KO) mice lacking the kinase domain of LRRK2. Here, we show that LRRK2 KO mice are viable and have no major abnormalities and live to adulthood. The dopaminergic system is normal in LRRK2 KO mice as assessed via HPLC for DA and its metabolites and via stereologic assessment of DA neuron number in young and aged mice. Importantly, there is no significant difference in the susceptibility of LRRK2 KO and wild-type mice to MPTP. These results suggest that LRRK2 plays little if any role in the development and survival of DA neurons under physiologic conditions. Thus, PD due to LRRK2 mutations are likely not due to a loss of function. Moreover, LRRK2 is not required for the susceptibility of DA neurons to MPTP.
Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). The most prevalent LRRK2 mutation is the G2019S coding change, located in the kinase domain of this complex multi-domain protein. The majority of G2019S autopsy cases feature typical Lewy Body pathology with a clinical phenotype almost indistinguishable from idiopathic PD (iPD). Here we have investigated the biochemical characteristics of α-synuclein in G2019S LRRK2 PD post-mortem material, in comparison to pathology-matched iPD. Immunohistochemistry with pS129 α-synuclein antibody showed that the medulla is heavily affected with pathology in G2019S PD whilst the basal ganglia (BG), limbic and frontal cortical regions demonstrated comparable pathology scores between G2019S PD and iPD. Significantly lower levels of the highly aggregated α-synuclein species in urea-SDS fractions were observed in G2019S cases compared to iPD in BG and limbic cortex. Our data, albeit from a small number of cases, highlight a difference in the biochemical properties of aggregated α-synuclein in G2019S linked PD compared to iPD, despite a similar histopathological presentation. This divergence in solubility is most notable in the basal ganglia, a region that is affected preclinically and is damaged before overt dopaminergic cell death.
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. We found LRRK2 to be degraded in lysosomes by chaperone-mediated autophagy (CMA), whereas the most common pathogenic mutant form of LRRK2, G2019S, was poorly degraded by this pathway. In contrast to the behavior of typical CMA substrates, lysosomal binding of both wild-type and several pathogenic mutant LRRK2 proteins was enhanced in the presence of other CMA substrates, which interfered with the organization of the CMA translocation complex, resulting in defective CMA. Cells responded to such LRRK2-mediated CMA compromise by increasing levels of the CMA lysosomal receptor, as seen in neuronal cultures and brains of LRRK2 transgenic mice, induced pluripotent stem cell-derived dopaminergic neurons and brains of Parkinson's disease patients with LRRK2 mutations. This newly described LRRK2 self-perpetuating inhibitory effect on CMA could underlie toxicity in Parkinson's disease by compromising the degradation of α-synuclein, another Parkinson's disease-related protein degraded by this pathway.
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial Parkinson's disease (PD). Although biochemical studies have shown that certain PD mutations confer elevated kinase activity in vitro on LRRK2, there are no methods available to directly monitor LRRK2 kinase activity in vivo. We demonstrate that LRRK2 autophosphorylation on Ser(1292) occurs in vivo and is enhanced by several familial PD mutations including N1437H, R1441G/C, G2019S, and I2020T. Combining two PD mutations together further increases Ser(1292) autophosphorylation. Mutation of Ser(1292) to alanine (S1292A) ameliorates the effects of LRRK2 PD mutations on neurite outgrowth in cultured rat embryonic primary neurons. Using cell-based and pharmacodynamic assays with phosphorylated Ser(1292) as the readout, we developed a brain-penetrating LRRK2 kinase inhibitor that blocks Ser(1292) autophosphorylation in vivo and attenuates the cellular consequences of LRRK2 PD mutations in vitro. These data suggest that Ser(1292) autophosphorylation may be a useful indicator of LRRK2 kinase activity in vivo and may contribute to the cellular effects of certain PD mutations.
Mutations in the LRRK2 gene cause autosomal dominant Parkinson's disease (PD). LRRK2 encodes a multi-domain protein containing a Ras-of-complex (Roc) GTPase domain, a C-terminal of Roc domain and a protein kinase domain. LRRK2 can function as a GTPase and protein kinase although the interplay between these two enzymatic domains is poorly understood. While guanine nucleotide binding is critically required for the kinase activity of LRRK2, the contribution of GTP hydrolysis is not known. In general, the molecular determinants regulating GTPase activity and how the GTPase domain contributes to the properties of LRRK2 remains to be clarified. Here, we identify a number of synthetic missense mutations in the GTPase domain that functionally modulate GTP binding and GTP hydrolysis and we employ these mutants to comprehensively explore the contribution of GTPase activity to the kinase activity and cellular phenotypes of LRRK2. Our data demonstrate that guanine nucleotide binding and, to a lesser extent, GTP hydrolysis are required for maintaining normal kinase activity and both activities contribute to the GTP-dependent activation of LRRK2 kinase activity. Guanine nucleotide binding but not GTP hydrolysis impairs the dimerization, structure and stability of LRRK2. Furthermore, GTP hydrolysis regulates the LRRK2-dependent inhibition of neurite outgrowth in primary cortical neurons but is unable to robustly modulate the effects of the familial G2019S mutation. Our study elucidates the role of GTPase activity in regulating kinase activity and cellular phenotypes of LRRK2 and has important implications for validation of the GTPase domain as a molecular target for attenuating LRRK2-mediated neurodegeneration.
Mutations in the leucine-rich repeat kinase 2 (LRRK2) have been associated with familial and sporadic cases of Parkinson disease. Mutant LRRK2 causes in vitro and in vivo neurite shortening, mediated in part by autophagy, and a parkinsonian phenotype in transgenic mice; however, the underlying mechanisms remain unclear. Because mitochondrial content/function is essential for dendritic morphogenesis and maintenance, we investigated whether mutant LRRK2 affects mitochondrial homeostasis in neurons. Mouse cortical neurons expressing either LRRK2 G2019S or R1441C mutations exhibited autophagic degradation of mitochondria and dendrite shortening. In addition, mutant LRRK2 altered the ability of the neurons to buffer intracellular calcium levels. Either calcium chelators or inhibitors of voltage-gated L-type calcium channels prevented mitochondrial degradation and dendrite shortening. These data suggest that mutant LRRK2 causes a deficit in calcium homeostasis, leading to enhanced mitophagy and dendrite shortening.
Mutations in the genes encoding leucine-rich repeat kinase 2 (LRRK2) and α-synuclein are associated with both autosomal dominant and idiopathic forms of Parkinson’s disease (PD). α-Synuclein is the main protein in Lewy bodies, hallmark inclusions present in both sporadic and familial PD. We show that in PD brain tissue, the levels of LRRK2 are positively related to the increase in α-synuclein phosphorylation and aggregation in affected brain regions (amygdala and anterior cingulate cortex), but not in the unaffected visual cortex. In disease-affected regions, we show co-localization of these two proteins in neurons and Lewy body inclusions. Further, in vitro experiments show a molecular interaction between α-synuclein and LRRK2 under endogenous and over-expression conditions. In a cell culture model of α-synuclein inclusion formation, LRRK2 co-localizes with the α-synuclein inclusions, and knocking down LRRK2 increases the number of smaller inclusions. In addition to providing strong evidence for an interaction between LRRK2 and α-synuclein, our results shed light on the complex relationship between these two proteins in the brains of patients with PD and the underlying molecular mechanisms of the disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-012-0984-y) contains supplementary material, which is available to authorized users.
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent known cause of late-onset Parkinson's disease (PD). To explore the therapeutic potential of small molecules targeting the LRRK2 kinase domain, we characterized two LRRK2 kinase inhibitors, TTT-3002 and LRRK2-IN1, for their effects against LRRK2 activity in vitro and in C. elegans models of LRRK2-linked neurodegeneration. TTT-3002 and LRRK2-IN1 potently inhibited in vitro kinase activity of LRRK2 wild-type and mutant proteins, attenuated phosphorylation of cellular LRRK2, and rescued neurotoxicity of mutant LRRK2 in transfected cells. To establish whether LRRK2 kinase inhibitors can mitigate pathogenesis caused by different mutations including G2019S and R1441C located within and outside of the LRRK2 kinase domain, respectively, we evaluated effects of TTT-3002 and LRRK2-IN1 against R1441C- and G2019S-induced neurodegeneration in C. elegans models. TTT-3002 and LRRK2-IN1 rescued the behavioral deficit characteristic of dopaminergic impairment in transgenic C. elegans expressing human R1441C- and G2019S-LRRK2. The inhibitors displayed nanomolar to low micromolar rescue potency when administered either pre-symptomatically or post-symptomatically, indicating both prevention and reversal of the dopaminergic deficit. The same treatments also led to long-lasting prevention and rescue of neurodegeneration. In contrast, TTT-3002 and LRRK2-IN1 were ineffective against the neurodegenerative phenotype in transgenic worms carrying the inhibitor-resistant A2016T mutation of LRRK2, suggesting that they elicit their neuroprotective effects in vivo by targeting LRRK2 specifically. Our findings indicate that the LRRK2 kinase activity is critical for neurodegeneration caused by R1441C and G2019S mutations, suggesting that kinase inhibition of LRRK2 may represent a promising therapeutic strategy for PD.
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent known cause of late-onset Parkinson's disease (PD). To explore the therapeutic potential of small molecules targeting the LRRK2 kinase domain, we characterized two LRRK2 kinase inhibitors, TTT-3002 and LRRK2-IN1, for their effects against LRRK2 activity in vitro and in C. elegans models of LRRK2-linked neurodegeneration. TTT-3002 and LRRK2-IN1 potently inhibited in vitro kinase activity of LRRK2 wild-type and mutant proteins, attenuated phosphorylation of cellular LRRK2, and rescued neurotoxicity of mutant LRRK2 in transfected cells. To establish whether LRRK2 kinase inhibitors can mitigate pathogenesis caused by different mutations including G2019S and R1441C located within and outside of the LRRK2 kinase domain, respectively, we evaluated effects of TTT-3002 and LRRK2-IN1 against R1441C- and G2019S-induced neurodegeneration in C. elegans models. TTT-3002 and LRRK2-IN1 rescued the behavioral deficit characteristic of dopaminergic impairment in transgenic C. elegans expressing human R1441C- and G2019S-LRRK2. The inhibitors displayed nanomolar to low micromolar rescue potency when administered either pre-symptomatically or post-symptomatically, indicating both prevention and reversal of the dopaminergic deficit. The same treatments also led to long-lasting prevention and rescue of neurodegeneration. In contrast, TTT-3002 and LRRK2-IN1 were ineffective against the neurodegenerative phenotype in transgenic worms carrying the inhibitor-resistant A2016T mutation of LRRK2, suggesting that they elicit their neuroprotective effects in vivo by targeting LRRK2 specifically. Our findings indicate that the LRRK2 kinase activity is critical for neurodegeneration caused by R1441C and G2019S mutations, suggesting that kinase inhibition of LRRK2 may represent a promising therapeutic strategy for PD.
Mutations in LRRK2 (leucine-rich repeat kinase 2) have been linked to inherited forms of PD (Parkinson's disease). Substantial pre-clinical research and drug discovery efforts have focused on LRRK2 with the hope that small-molecule inhibitors of the enzyme may be valuable for the treatment or prevention of the onset of PD. The pathway to develop therapeutic or neuroprotective agents based on LRRK2 function (i.e. kinase activity) has been facilitated by the development of both biochemical and cell-based assays for LRRK2. LRRK2 is phosphorylated on Ser910, Ser935, Ser955 and Ser973 in the N-terminal domain of the enzyme, and these sites of phosphorylation are likely to be regulated by upstream enzymes in an LRRK2 kinase-activity-dependent manner. Knowledge of these phosphorylation sites and their regulation can be adapted to high-throughput-screening-amenable platforms. The present review describes the utilization of LRRK2 phosphorylation as indicators of enzyme inhibition, as well as how such assays can be used to deconvolute the pathways in which LRRK2 plays a role.
Mutations in leucine-rich repeat kinase 2 (LRRK2) are a major cause of familial Parkinsonism, and the G2019S mutation of LRRK2 is one of the most prevalent mutations. The deregulation of autophagic processes in nerve cells is thought to be a possible cause of Parkinson's disease (PD). In this study, we observed that G2019S mutant fibroblasts exhibited higher autophagic activity levels than control fibroblasts. Elevated levels of autophagic activity can trigger cell death, and in our study, G2019S mutant cells exhibited increased apoptosis hallmarks compared to control cells. LRRK2 is able to induce the phosphorylation of MAPK/ERK kinases (MEK). The use of 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a highly selective inhibitor of MEK1/2, reduced the enhanced autophagy and sensibility observed in G2019S LRRK2 mutation cells. These data suggest that the G2019S mutation induces autophagy via MEK/ERK pathway and that the inhibition of this exacerbated autophagy reduces the sensitivity observed in G2019S mutant cells.
Parkinson's disease (PD) is a common neurodegenerative disorder caused by genetic and environmental factors that results in degeneration of the nigrostriatal dopaminergic pathway in the brain. We analyzed neural cells generated from induced pluripotent stem cells (iPSCs) derived from PD patients and presymptomatic individuals carrying mutations in the PINK1 (PTEN-induced putative kinase 1) and LRRK2 (leucine-rich repeat kinase 2) genes, and compared them to those of healthy control subjects. We measured several aspects of mitochondrial responses in the iPSC-derived neural cells including production of reactive oxygen species, mitochondrial respiration, proton leakage, and intraneuronal movement of mitochondria. Cellular vulnerability associated with mitochondrial dysfunction in iPSC-derived neural cells from familial PD patients and at-risk individuals could be rescued with coenzyme Q(10), rapamycin, or the LRRK2 kinase inhibitor GW5074. Analysis of mitochondrial responses in iPSC-derived neural cells from PD patients carrying different mutations provides insight into convergence of cellular disease mechanisms between different familial forms of PD and highlights the importance of oxidative stress and mitochondrial dysfunction in this neurodegenerative disease.
The G2019S leucine rich repeat kinase 2 (LRRK2) mutation is the most common genetic cause of Parkinson's disease (PD), clinically and pathologically indistinguishable from idiopathic PD. Mitochondrial abnormalities are a common feature in PD pathogenesis and we have investigated the impact of G2019S mutant LRRK2 expression on mitochondrial bioenergetics. LRRK2 protein expression was detected in fibroblasts and lymphoblasts at levels higher than those observed in the mouse brain. The presence of G2019S LRRK2 mutation did not influence LRRK2 expression in fibroblasts. However, the expression of the G2019S LRRK2 mutation in both fibroblast and neuroblastoma cells was associated with mitochondrial uncoupling. This was characterized by decreased mitochondrial membrane potential and increased oxygen utilization under basal and oligomycin-inhibited conditions. This resulted in a decrease in cellular ATP levels consistent with compromised cellular function. This uncoupling of mitochondrial oxidative phosphorylation was associated with a cell-specific increase in uncoupling protein (UCP) 2 and 4 expression. Restoration of mitochondrial membrane potential by the UCP inhibitor genipin confirmed the role of UCPs in this mechanism. The G2019S LRRK2-induced mitochondrial uncoupling and UCP4 mRNA up-regulation were LRRK2 kinase-dependent, whereas endogenous LRRK2 levels were required for constitutive UCP expression. We propose that normal mitochondrial function was deregulated by the expression of G2019S LRRK2 in a kinase-dependent mechanism that is a modification of the normal LRRK2 function, and this leads to the vulnerability of selected neuronal populations in PD.
Mutations in the LRRK2 gene are the most common cause of genetic Parkinson’s disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation.
We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis.
Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes.
Parkinson's disease (PD) is a neurodegenerative disease in which the etiology of 90 percent of the patients is unknown. Pesticide exposure is a major risk factor for PD, and paraquat (PQ), pyridaben (PY) and maneb (MN) are amongst the most widely used pesticides. We studied mRNA expression using transcriptome sequencing (RNA-Seq) in the ventral midbrain (VMB) and striatum (STR) of PQ, PY and paraquat+maneb (MNPQ) treated mice, followed by pathway analysis. We found concordance of signaling pathways between the three pesticide models in both the VMB and STR as well as concordance in these two brain areas. The concordant signaling pathways with relevance to PD pathogenesis were e.g. axonal guidance signaling, Wnt/β-catenin signaling, as well as pathways not previously linked to PD, e.g. basal cell carcinoma, human embryonic stem cell pluripotency and role of macrophages, fibroblasts and endothelial cells in rheumatoid arthritis. Human PD pathways previously identified by expression analysis, concordant with VMB pathways identified in our study were axonal guidance signaling, Wnt/β-catenin signaling, IL-6 signaling, ephrin receptor signaling, TGF-β signaling, PPAR signaling and G-protein coupled receptor signaling. Human PD pathways concordant with the STR pathways in our study were Wnt/β-catenin signaling, axonal guidance signaling and G-protein coupled receptor signaling. Peroxisome proliferator activated receptor delta (Ppard) and G-Protein Coupled Receptors (GPCRs) were common genes in VMB and STR identified by network analysis. In conclusion, the pesticides PQ, PY and MNPQ elicit common signaling pathways in the VMB and STR in mice, which are concordant with known signaling pathways identified in human PD, suggesting that these pathways contribute to the pathogenesis of idiopathic PD. The analysis of these networks and pathways may therefore lead to improved understanding of disease pathogenesis, and potential novel therapeutic targets.
(G2019S) mutation of leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of both familial and sporadic Parkinson's disease (PD) cases. Twelve- to sixteen-month-old (G2019S) LRRK2 transgenic mice prepared by us displayed progressive degeneration of substantia nigra pars compacta (SNpc) dopaminergic neurons and parkinsonism phenotypes of motor dysfunction. LRRK2 is a member of mixed lineage kinase subfamily of mitogen-activated protein kinase kinase kinases (MAPKKKs). We hypothesized that (G2019S) mutation augmented LRRK2 kinase activity, leading to overphosphorylation of downstream MAPK kinase (MKK) and resulting in activation of neuronal death signal pathway. Consistent with our hypothesis, (G2019S) LRRK2 expressed in HEK 293 cells exhibited an augmented kinase activity of phosphorylating MAPK kinase 4 (MKK4) at Ser(257), and protein expression of active phospho-MKK4(Ser257) was upregulated in the SN of (G2019S) LRRK2 transgenic mice. Protein level of active phospho-JNK(Thr183/Tyr185) and phospho-c-Jun(Ser63), downstream targets of phospho-MKK4(Ser257), was increased in the SN of (G2019S) LRRK2 mice. Upregulated mRNA expression of pro-apoptotic Bim and FasL, target genes of phospho-c-Jun(Ser63), and formation of active caspase-9, caspase-8 and caspase-3 were also observed in the SN of (G2019S) LRRK2 transgenic mice. Our results suggest that mutant (G2019S) LRRK2 activates MKK4-JNK-c-Jun pathway in the SN and causes the resulting degeneration of SNpc dopaminergic neurons in PD transgenic mice.
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.
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 hydroxylase-positive 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.
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.
Mutations in LRRK2 are associated with familial and sporadic Parkinson's disease (PD). Subjects with PD caused by LRRK2 mutations show pleiotropic pathology that can involve inclusions containing α-synuclein, tau or neither protein. The mechanisms by which mutations in LRRK2 lead to this pleiotropic pathology remain unknown.
To investigate mechanisms by which LRRK2 might cause PD.
We used systems biology to investigate the transcriptomes from human brains, human blood cells and Caenorhabditis elegans expressing wild-type LRRK2. The role of autophagy was tested in lines of C. elegans expressing LRRK2, V337M tau or both proteins. Neuronal function was measured by quantifying thrashing.
Genes regulating autophagy were coordinately regulated with LRRK2. C. elegans expressing V337M tau showed reduced thrashing, as has been noted previously. Coexpressing mutant LRRK2 (R1441C or G2019S) with V337M tau increased the motor deficits. Treating the lines of C. elegans with an mTOR inhibitor that enhances autophagic flux, ridaforolimus, increased the thrashing behavior to the same level as nontransgenic nematodes.
These data support a role for LRRK2 in autophagy, raise the possibility that deficits in autophagy contribute to the pathophysiology of LRRK2, and point to a potential therapeutic approach addressing the pathophysiology of LRRK2 in PD.
LRRK2, a Parkinson's disease associated gene, is highly expressed in microglia in addition to neurons; however, its function in microglia has not been evaluated. Using Lrrk2 knockdown (Lrrk2-KD) murine microglia prepared by lentiviral-mediated transfer of Lrrk2-specific small inhibitory hairpin RNA (shRNA), we found that Lrrk2 deficiency attenuated lipopolysaccharide (LPS)-induced mRNA and/or protein expression of inducible nitric oxide synthase, TNF-α, IL-1β and IL-6. LPS-induced phosphorylation of p38 mitogen-activated protein kinase and stimulation of NF-κB-responsive luciferase reporter activity was also decreased in Lrrk2-KD cells. Interestingly, the decrease in NF-κB transcriptional activity measured by luciferase assays appeared to reflect increased binding of the inhibitory NF-κB homodimer, p50/p50, to DNA. In LPS-responsive HEK293T cells, overexpression of the human LRRK2 pathologic, kinase-active mutant G2019S increased basal and LPS-induced levels of phosphorylated p38 and JNK, whereas wild-type and other pathologic (R1441C and G2385R) or artificial kinase-dead (D1994A) LRRK2 mutants either enhanced or did not change basal and LPS-induced p38 and JNK phosphorylation levels. However, wild-type LRRK2 and all LRRK2 mutant variants equally enhanced NF-κB transcriptional activity. Taken together, these results suggest that LRRK2 is a positive regulator of inflammation in murine microglia, and LRRK2 mutations may alter the microenvironment of the brain to favor neuroinflammation.