RESUMO
Although mutations in mitochondrial-associated genes are linked to inflammation and susceptibility to infection, their mechanistic contributions to immune outcomes remain ill-defined. We discovered that the disease-associated gain-of-function allele Lrrk2G2019S (leucine-rich repeat kinase 2) perturbs mitochondrial homeostasis and reprograms cell death pathways in macrophages. When the inflammasome is activated in Lrrk2G2019S macrophages, elevated mitochondrial ROS (mtROS) directs association of the pore-forming protein gasdermin D (GSDMD) to mitochondrial membranes. Mitochondrial GSDMD pore formation then releases mtROS, promoting a switch to RIPK1/RIPK3/MLKL-dependent necroptosis. Consistent with enhanced necroptosis, infection of Lrrk2G2019S mice with Mycobacterium tuberculosis elicits hyperinflammation and severe immunopathology. Our findings suggest a pivotal role for GSDMD as an executer of multiple cell death pathways and demonstrate that mitochondrial dysfunction can direct immune outcomes via cell death modality switching. This work provides insights into how LRRK2 mutations manifest or exacerbate human diseases and identifies GSDMD-dependent necroptosis as a potential target to limit Lrrk2G2019S-mediated immunopathology.
Assuntos
Mitocôndrias , Necroptose , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Animais , Humanos , Inflamassomos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Macrófagos , Camundongos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) are commonly implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 regulates critical cellular processes at membranous organelles and forms microtubule-based pathogenic filaments, yet the molecular basis underlying these biological roles of LRRK2 remains largely enigmatic. Here, we determined high-resolution structures of full-length human LRRK2, revealing its architecture and key interdomain scaffolding elements for rationalizing disease-causing mutations. The kinase domain of LRRK2 is captured in an inactive state, a conformation also adopted by the most common PD-associated mutation, LRRK2G2019S. This conformation serves as a framework for structure-guided design of conformational specific inhibitors. We further determined the structure of COR-mediated LRRK2 dimers and found that single-point mutations at the dimer interface abolished pathogenic filamentation in cells. Overall, our study provides mechanistic insights into physiological and pathological roles of LRRK2 and establishes a structural template for future therapeutic intervention in PD.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Sequência de Aminoácidos , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/ultraestrutura , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of inflammatory pathways. Here, we studied how microglia handle and cope with α-synuclein (α-syn) fibrils and their clearance. We found that microglia exposed to α-syn establish a cellular network through the formation of F-actin-dependent intercellular connections, which transfer α-syn from overloaded microglia to neighboring naive microglia where the α-syn cargo got rapidly and effectively degraded. Lowering the α-syn burden attenuated the inflammatory profile of microglia and improved their survival. This degradation strategy was compromised in cells carrying the LRRK2 G2019S mutation. We confirmed the intercellular transfer of α-syn assemblies in microglia using organotypic slice cultures, 2-photon microscopy, and neuropathology of patients. Together, these data identify a mechanism by which microglia create an "on-demand" functional network in order to improve pathogenic α-syn clearance.
Assuntos
Estruturas da Membrana Celular/metabolismo , Microglia/metabolismo , Proteólise , alfa-Sinucleína/metabolismo , Actinas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Citoesqueleto/metabolismo , Regulação para Baixo , Feminino , Humanos , Inflamação/genética , Inflamação/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Microglia/patologia , Microglia/ultraestrutura , Mitocôndrias/metabolismo , Nanotubos , Agregados Proteicos , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma/genéticaRESUMO
Parkinson's disease (PD) is a leading cause of neurodegeneration that is defined by the selective loss of dopaminergic neurons and the accumulation of protein aggregates called Lewy bodies (LBs). The unequivocal identification of Mendelian inherited mutations in 13 genes in PD has provided transforming insights into the pathogenesis of this disease. The mechanistic analysis of several PD genes, including α-synuclein (α-syn), leucine-rich repeat kinase 2 (LRRK2), PTEN-induced kinase 1 (PINK1), and Parkin, has revealed central roles for protein aggregation, mitochondrial damage, and defects in endolysosomal trafficking in PD neurodegeneration. In this review, we outline recent advances in our understanding of these gene pathways with a focus on the emergent role of Rab (Ras analog in brain) GTPases and vesicular trafficking as a common mechanism that underpins how mutations in PD genes lead to neuronal loss. These advances have led to previously distinct genes such as vacuolar protein-sorting-associated protein 35 (VPS35) and LRRK2 being implicated in a common signaling pathway. A greater understanding of these common nodes of vesicular trafficking will be crucial for linking other PD genes and improving patient stratification in clinical trials underway against α-syn and LRRK2 targets.
Assuntos
Doença de Parkinson/metabolismo , Animais , Autofagia , Vesículas Citoplasmáticas/metabolismo , Humanos , Mitocôndrias/metabolismo , Doença de Parkinson/genética , Agregados Proteicos , Transporte ProteicoRESUMO
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by degeneration of the substantia nigra pars compacta and by accumulation of α-synuclein in Lewy bodies. PD is caused by a combination of environmental factors and genetic variants. These variants range from highly penetrant Mendelian alleles to alleles that only modestly increase disease risk. Here, we review what is known about the genetics of PD. We also describe how PD genetics have solidified the role of endosomal, lysosomal, and mitochondrial dysfunction in PD pathophysiology. Finally, we highlight how all three pathways are affected by α-synuclein and how this knowledge may be harnessed for the development of disease-modifying therapeutics.
Assuntos
Doença de Parkinson , Humanos , Lisossomos , Doença de Parkinson/genética , alfa-Sinucleína/genéticaRESUMO
Mutations in LRRK2 are the most common genetic causes of Parkinson's disease (PD). While the enzymatic activity of LRRK2 has been linked to PD, previous work has also provided support for an important role of elevated LRRK2 protein levels, independent of enzymatic activity, in PD pathogenesis. However, the mechanisms underlying the regulation of LRRK2 protein levels remain unclear. Here, we identify a role for the purine biosynthesis pathway enzyme ATIC in the regulation of LRRK2 levels and toxicity. AICAr, the precursor of ATIC substrate, regulates LRRK2 levels in a cell-type-specific manner in vitro and in mouse tissue. AICAr regulates LRRK2 levels through AUF1-mediated mRNA decay. Upon AICAr treatment, the RNA binding protein AUF1 is recruited to the AU-rich elements (ARE) of LRRK2 mRNA leading to the recruitment of the decapping enzyme complex DCP1/2 and decay of LRRK2 mRNA. AICAr suppresses LRRK2 expression and rescues LRRK2-induced dopaminergic neurodegeneration and neuroinflammation in PD Drosophila and mouse models. Together, this study provides insight into a novel regulatory mechanism of LRRK2 protein levels and function via LRRK2 mRNA decay that is distinct from LRRK2 enzymatic functions.
Assuntos
Doença de Parkinson , Animais , Camundongos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , MutaçãoRESUMO
Activating leucine-rich repeat kinase 2 (LRRK2) mutations cause Parkinson's and phosphorylation of Rab10 by pathogenic LRRK2 blocks primary ciliogenesis in cultured cells. In the mouse brain, LRRK2 blockade of primary cilia is highly cell type specific: For example, cholinergic interneurons and astrocytes but not medium spiny neurons of the dorsal striatum lose primary cilia in LRRK2-pathway mutant mice. We show here that the cell type specificity of LRRK2-mediated cilia loss is also seen in human postmortem striatum from patients with LRRK2 pathway mutations and idiopathic Parkinson's. Single nucleus RNA sequencing shows that cilia loss in mouse cholinergic interneurons is accompanied by decreased glial-derived neurotrophic factor transcription, decreasing neuroprotection for dopamine neurons. Nevertheless, LRRK2 expression differences cannot explain the unique vulnerability of cholinergic neurons to LRRK2 kinase as much higher LRRK2 expression is seen in medium spiny neurons that have normal cilia. In parallel with decreased striatal dopaminergic neurite density, LRRK2 G2019S neurons show increased autism-linked CNTN5 adhesion protein expression; glial cells show significant loss of ferritin heavy chain. These data strongly suggest that loss of cilia in specific striatal cell types decreases neuroprotection for dopamine neurons in mice and human Parkinson's.
Assuntos
Cílios , Neurônios Dopaminérgicos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Neuroproteção , Doença de Parkinson , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Cílios/metabolismo , Animais , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Humanos , Camundongos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Neuroproteção/genética , Mutação , Corpo Estriado/metabolismo , Corpo Estriado/patologia , MasculinoRESUMO
Variations in the LRRK2 gene represent one of the strongest genetic factors for Parkinson's disease (PD). It has become clear that structural knowledge of the encoded large multidomain LRRK2 protein will cast light on its biological function. The new study from Myasnikov, Zhu, et al. provides a high-resolution structure of the full-length LRRK2.
Assuntos
Doença de Parkinson , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Mutação , Doença de Parkinson/genética , Doença de Parkinson/metabolismoRESUMO
Mutations in Leucine-rich repeat kinase 2 (LRRK2) are responsible for late-onset autosomal dominant Parkinson's disease. LRRK2 has been implicated in a wide range of physiological processes including membrane repair in the endolysosomal system. Here, using cell-free systems, we report that purified LRRK2 directly binds acidic lipid bilayers with a preference for highly curved bilayers. While this binding is nucleotide independent, LRRK2 can also deform low-curvature liposomes into narrow tubules in a guanylnucleotide-dependent but Adenosine 5'-triphosphate-independent way. Moreover, assembly of LRRK2 into scaffolds at the surface of lipid tubules can constrict them. We suggest that an interplay between the membrane remodeling and signaling properties of LRRK2 may be key to its physiological function. LRRK2, via its kinase activity, may achieve its signaling role at sites where membrane remodeling occurs.
Assuntos
Doença de Parkinson , Proteínas Serina-Treonina Quinases , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Fosforilação , MutaçãoRESUMO
PPM1H phosphatase reverses Parkinson's disease-associated, Leucine Rich Repeat Kinase 2-mediated Rab GTPase phosphorylation. We show here that PPM1H relies on an N-terminal amphipathic helix for Golgi localization. The amphipathic helix enables PPM1H to bind to liposomes in vitro, and small, highly curved liposomes stimulate PPM1H activity. We artificially anchored PPM1H to the Golgi, mitochondria, or mother centriole. Our data show that regulation of Rab10 GTPase phosphorylation requires PPM1H access to Rab10 at or near the mother centriole. Moreover, poor colocalization of Rab12 explains in part why it is a poor substrate for PPM1H in cells but not in vitro. These data support a model in which localization drives PPM1H substrate selection and centriolar PPM1H is critical for regulation of Rab GTPase-regulated ciliogenesis. Moreover, Golgi localized PPM1H may maintain active Rab GTPases on the Golgi to carry out their nonciliogenesis-related functions in membrane trafficking.
Assuntos
Doença de Parkinson , Monoéster Fosfórico Hidrolases , Humanos , Fosforilação , Monoéster Fosfórico Hidrolases/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Lipossomos , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Fosfoproteínas Fosfatases/metabolismoRESUMO
Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.
Assuntos
Repetição de Anquirina , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , Proteínas rab de Ligação ao GTP , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Humanos , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Células HEK293 , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Fosforilação , Microscopia Crioeletrônica , Ligação ProteicaRESUMO
Human brain organoid models that recapitulate the physiology and complexity of the human brain have a great potential for in vitro disease modeling, in particular for neurodegenerative diseases, such as Parkinson disease. In the present study, we compare single-cell RNA-sequencing data of human midbrain organoids to the developing human embryonic midbrain. We demonstrate that the in vitro model is comparable to its in vivo equivalents in terms of developmental path and cellular composition. Moreover, we investigate the potential of midbrain organoids for modeling early developmental changes in Parkinson disease. Therefore, we compare the single-cell RNA-sequencing data of healthy-individual-derived midbrain organoids to their isogenic LRRK2-p.Gly2019Ser-mutant counterparts. We show that the LRRK2 p.Gly2019Ser variant alters neurodevelopment, resulting in an untimely and incomplete differentiation with reduced cellular variability. Finally, we present four candidate genes, APP, DNAJC6, GATA3, and PTN, that might contribute to the LRRK2-p.Gly2019Ser-associated transcriptome changes that occur during early neurodevelopment.
Assuntos
Substituição de Aminoácidos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Neurogênese/genética , Organoides/metabolismo , Doença de Parkinson/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular , Citocinas/genética , Citocinas/metabolismo , Embrião de Mamíferos , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glicina/química , Glicina/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Mesencéfalo , Modelos Biológicos , Mutação , Organoides/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Análise de Sequência de RNA , Serina/química , Serina/metabolismo , Análise de Célula Única/métodos , TranscriptomaRESUMO
Rab proteins are small GTPases that regulate a myriad of intracellular membrane trafficking events. Rab29 is one of the Rab proteins phosphorylated by leucine-rich repeat kinase 2 (LRRK2), a Parkinson's disease-associated kinase. Recent studies suggest that Rab29 regulates LRRK2, whereas the mechanism by which Rab29 is regulated remained unclear. Here, we report a novel phosphorylation in Rab29 that is not mediated by LRRK2 and occurs under lysosomal overload stress. Mass spectrometry analysis identified the phosphorylation site of Rab29 as Ser185, and cellular expression studies of phosphomimetic mutants of Rab29 at Ser185 unveiled the involvement of this phosphorylation in counteracting lysosomal enlargement. PKCα and PKCδ were deemed to be involved in this phosphorylation and control the lysosomal localization of Rab29 in concert with LRRK2. These results implicate PKCs in the lysosomal stress response pathway comprised of Rab29 and LRRK2, and further underscore the importance of this pathway in the mechanisms underlying lysosomal homeostasis.
Assuntos
Lisossomos , Proteínas rab de Ligação ao GTP , Fosforilação , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Lisossomos/metabolismo , MutaçãoRESUMO
Estimates of the spectrum and frequency of pathogenic variants in Parkinson's disease (PD) in different populations are currently limited and biased. Furthermore, although therapeutic modification of several genetic targets has reached the clinical trial stage, a major obstacle in conducting these trials is that PD patients are largely unaware of their genetic status and, therefore, cannot be recruited. Expanding the number of investigated PD-related genes and including genes related to disorders with overlapping clinical features in large, well-phenotyped PD patient groups is a prerequisite for capturing the full variant spectrum underlying PD and for stratifying and prioritizing patients for gene-targeted clinical trials. The Rostock Parkinson's disease (ROPAD) study is an observational clinical study aiming to determine the frequency and spectrum of genetic variants contributing to PD in a large international cohort. We investigated variants in 50 genes with either an established relevance for PD or possible phenotypic overlap in a group of 12 580 PD patients from 16 countries [62.3% male; 92.0% White; 27.0% positive family history (FH+), median age at onset (AAO) 59 years] using a next-generation sequencing panel. Altogether, in 1864 (14.8%) ROPAD participants (58.1% male; 91.0% White, 35.5% FH+, median AAO 55 years), a PD-relevant genetic test (PDGT) was positive based on GBA1 risk variants (10.4%) or pathogenic/likely pathogenic variants in LRRK2 (2.9%), PRKN (0.9%), SNCA (0.2%) or PINK1 (0.1%) or a combination of two genetic findings in two genes (â¼0.2%). Of note, the adjusted positive PDGT fraction, i.e. the fraction of positive PDGTs per country weighted by the fraction of the population of the world that they represent, was 14.5%. Positive PDGTs were identified in 19.9% of patients with an AAO ≤ 50 years, in 19.5% of patients with FH+ and in 26.9% with an AAO ≤ 50 years and FH+. In comparison to the idiopathic PD group (6846 patients with benign variants), the positive PDGT group had a significantly lower AAO (4 years, P = 9 × 10-34). The probability of a positive PDGT decreased by 3% with every additional AAO year (P = 1 × 10-35). Female patients were 22% more likely to have a positive PDGT (P = 3 × 10-4), and for individuals with FH+ this likelihood was 55% higher (P = 1 × 10-14). About 0.8% of the ROPAD participants had positive genetic testing findings in parkinsonism-, dystonia/dyskinesia- or dementia-related genes. In the emerging era of gene-targeted PD clinical trials, our finding that â¼15% of patients harbour potentially actionable genetic variants offers an important prospect to affected individuals and their families and underlines the need for genetic testing in PD patients. Thus, the insights from the ROPAD study allow for data-driven, differential genetic counselling across the spectrum of different AAOs and family histories and promote a possible policy change in the application of genetic testing as a routine part of patient evaluation and care in PD.
Assuntos
Testes Genéticos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , Humanos , Doença de Parkinson/genética , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Testes Genéticos/métodos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Glucosilceramidase/genética , alfa-Sinucleína/genética , Predisposição Genética para Doença , Ubiquitina-Proteína Ligases/genética , Estudos de Coortes , Proteínas Quinases/genética , Mutação , AdultoRESUMO
Variants in seven genes (LRRK2, GBA1, PRKN, SNCA, PINK1, PARK7 and VPS35) have been formally adjudicated as causal contributors to Parkinson's disease; however, individuals with Parkinson's disease are often unaware of their genetic status since clinical testing is infrequently offered. As a result, genetic information is not incorporated into clinical care, and variant-targeted precision medicine trials struggle to enrol people with Parkinson's disease. Understanding the yield of genetic testing using an established gene panel in a large, geographically diverse North American population would help patients, clinicians, clinical researchers, laboratories and insurers better understand the importance of genetics in approaching Parkinson's disease. PD GENEration is an ongoing multi-centre, observational study (NCT04057794, NCT04994015) offering genetic testing with results disclosure and genetic counselling to those in the US (including Puerto Rico), Canada and the Dominican Republic, through local clinical sites or remotely through self-enrolment. DNA samples are analysed by next-generation sequencing including deletion/duplication analysis (Fulgent Genetics) with targeted testing of seven major Parkinson's disease-related genes. Variants classified as pathogenic/likely pathogenic/risk variants are disclosed to all tested participants by either neurologists or genetic counsellors. Demographic and clinical features are collected at baseline visits. Between September 2019 and June 2023, the study enrolled 10 510 participants across >85 centres, with 8301 having received results. Participants were: 59% male; 86% White, 2% Asian, 4% Black/African American, 9% Hispanic/Latino; mean age 67.4 ± 10.8 years. Reportable genetic variants were observed in 13% of all participants, including 18% of participants with one or more 'high risk factors' for a genetic aetiology: early onset (<50 years), high-risk ancestry (Ashkenazi Jewish/Basque/North African Berber), an affected first-degree relative; and, importantly, in 9.1% of people with none of these risk factors. Reportable variants in GBA1 were identified in 7.7% of all participants; 2.4% in LRRK2; 2.1% in PRKN; 0.1% in SNCA; and 0.2% in PINK1, PARK7 or VPS35 combined. Variants in more than one of the seven genes were identified in 0.4% of participants. Approximately 13% of study participants had a reportable genetic variant, with a 9% yield in people with no high-risk factors. This supports the promotion of universal access to genetic testing for Parkinson's disease, as well as therapeutic trials for GBA1 and LRRK2-related Parkinson's disease.
Assuntos
Testes Genéticos , Glucosilceramidase , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , alfa-Sinucleína , Humanos , Doença de Parkinson/genética , Doença de Parkinson/diagnóstico , Testes Genéticos/métodos , Masculino , Feminino , Glucosilceramidase/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , alfa-Sinucleína/genética , Idoso , Pessoa de Meia-Idade , Ubiquitina-Proteína Ligases/genética , Proteínas Quinases/genética , Proteína Desglicase DJ-1/genética , Proteínas de Transporte Vesicular/genética , América do Norte , Variação Genética/genética , Predisposição Genética para Doença/genética , Adulto , Revelação , Aconselhamento Genético , Canadá , Estados UnidosRESUMO
The LRRK2 G2019S variant is the most common cause of monogenic Parkinson's disease (PD); however, questions remain regarding the penetrance, clinical phenotype and natural history of carriers. We performed a 3.5-year prospective longitudinal online study in a large number of 1286 genotyped LRRK2 G2019S carriers and 109 154 controls, with and without PD, recruited from the 23andMe Research Cohort. We collected self-reported motor and non-motor symptoms every 6 months, as well as demographics, family histories and environmental risk factors. Incident cases of PD (phenoconverters) were identified at follow-up. We determined lifetime risk of PD using accelerated failure time modelling and explored the impact of polygenic risk on penetrance. We also computed the genetic ancestry of all LRRK2 G2019S carriers in the 23andMe database and identified regions of the world where carrier frequencies are highest. We observed that despite a 1 year longer disease duration (P = 0.016), LRRK2 G2019S carriers with PD had similar burden of motor symptoms, yet significantly fewer non-motor symptoms including cognitive difficulties, REM sleep behaviour disorder (RBD) and hyposmia (all P-values ≤ 0.0002). The cumulative incidence of PD in G2019S carriers by age 80 was 49%. G2019S carriers had a 10-fold risk of developing PD versus non-carriers. This rose to a 27-fold risk in G2019S carriers with a PD polygenic risk score in the top 25% versus non-carriers in the bottom 25%. In addition to identifying ancient founding events in people of North African and Ashkenazi descent, our genetic ancestry analyses infer that the G2019S variant was later introduced to Spanish colonial territories in the Americas. Our results suggest LRRK2 G2019S PD appears to be a slowly progressive predominantly motor subtype of PD with a lower prevalence of hyposmia, RBD and cognitive impairment. This suggests that the current prodromal criteria, which are based on idiopathic PD, may lack sensitivity to detect the early phases of LRRK2 PD in G2019S carriers. We show that polygenic burden may contribute to the development of PD in the LRRK2 G2019S carrier population. Collectively, the results should help support screening programmes and candidate enrichment strategies for upcoming trials of LRRK2 inhibitors in early-stage disease.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/genética , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Estudos Longitudinais , Predisposição Genética para Doença/genética , Adulto , Estudos Prospectivos , Heterozigoto , Penetrância , Idoso de 80 Anos ou mais , Transtorno do Comportamento do Sono REM/genética , MutaçãoRESUMO
The identification of multiple genes linked to Parkinson's disease (PD) invites the question as to how they may co-operate. We have generated isogenic cell lines that inducibly express either wild-type or a mutant form of the retromer component VPS35 (D620N), which has been linked to PD. This has enabled us to test proposed effects of this mutation in a setting where the relative expression reflects the physiological occurrence. We confirm that this mutation compromises VPS35 association with the WASH complex, but find no defect in WASH recruitment to endosomes, nor in the distribution of lysosomal receptors, cation-independent mannose-6-phosphate receptor and Sortilin. We show VPS35 (D620N) enhances the activity of the Parkinson's associated kinase LRRK2 towards RAB12 under basal conditions. Furthermore, VPS35 (D620N) amplifies the LRRK2 response to endolysosomal stress resulting in enhanced phosphorylation of RABs 10 and 12. By comparing different types of endolysosomal stresses such as the ionophore nigericin and the membranolytic agent l-leucyl-l-leucine methyl ester, we are able to dissociate phospho-RAB accumulation from membrane rupture.
Assuntos
Doença de Parkinson , Humanos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Mutação , Lisossomos/genética , Lisossomos/metabolismo , Endossomos/genética , Endossomos/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismoRESUMO
Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and mutations in LRRK2 are key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to explore intra-domain cross-talk in LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is common in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and thus serves as a multi-functional protein : protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and used peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB domain (CORB loop) using a combination of Gaussian accelerated molecular dynamics simulations and peptide arrays. This Ct-Motif contains two auto-phosphorylation sites (T2483 and T2524), and T2524 is a 14-3-3 binding site. The Ct helix, CORB loop, and the CORB-kinase linker together form a part of a dynamic 'CAP' that regulates the N-lobe of the kinase domain. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will also mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.
Assuntos
Proteínas de Repetições Ricas em Leucina , Proteínas Serina-Treonina Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Domínios Proteicos , Mutação , Peptídeos/metabolismo , FosforilaçãoRESUMO
Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson's disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/metabolismo , Anticorpos de Domínio Único , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Mapeamento de Epitopos , Células HEK293 , Humanos , Camundongos , Microtúbulos/metabolismo , Fosforilação , Ligação Proteica , Células RAW 264.7 , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease. A plethora of evidence has indicated a role for LRRK2 in endolysosomal trafficking in neurons, while LRRK2 function in glia, although highly expressed, remains largely unknown. Here, we present evidence that LRRK2/dLRRK mediates a lysosomal pathway that contributes to glial cell death and the survival of dopaminergic (DA) neurons. LRRK2/dLRRK knockdown in the immortalized microglia or flies results in enlarged and swelling lysosomes fewer in number. These lysosomes are less mobile, wrongly acidified, exhibit defective membrane permeability and reduced activity of the lysosome hydrolase cathepsin B. In addition, LRRK2/dLRRK depletion causes glial apoptosis, DA neurodegeneration, and locomotor deficits in an age-dependent manner. Taken together, these findings demonstrate a functional role of LRRK2/dLRRK in regulating the glial lysosomal pathway; deficits in lysosomal biogenesis and function linking to glial apoptosis potentially underlie the mechanism of DA neurodegeneration, providing insights on LRRK2/dLRRK function in normal and pathological brains.