RESUMO
Genetic variation in LRRK2 associates with the susceptibility to Parkinson's disease, Crohn's disease, and mycobacteria infection. High expression of LRRK2 and its substrate Rab10 occurs in phagocytic cells in the immune system. In mouse and human primary macrophages, dendritic cells, and microglia-like cells, we find that Rab10 specifically regulates a specialized form of endocytosis known as macropinocytosis, without affecting phagocytosis or clathrin-mediated endocytosis. LRRK2 phosphorylates cytoplasmic PI(3,4,5)P3-positive GTP-Rab10, before EEA1 and Rab5 recruitment to early macropinosomes occurs. Macropinosome cargo in macrophages includes CCR5, CD11b, and MHCII, and LRRK2-phosphorylation of Rab10 potently blocks EHBP1L1-mediated recycling tubules and cargo turnover. EHBP1L1 overexpression competitively inhibits LRRK2-phosphorylation of Rab10, mimicking the effects of LRRK2 kinase inhibition in promoting cargo recycling. Both Rab10 knockdown and LRRK2 kinase inhibition potently suppress the maturation of macropinosome-derived CCR5-loaded signaling endosomes that are critical for CCL5-induced immunological responses that include Akt activation and chemotaxis. These data support a novel signaling axis in the endolysosomal system whereby LRRK2-mediated Rab10 phosphorylation stalls vesicle fast recycling to promote PI3K-Akt immunological responses.
Assuntos
Proteínas de Transporte/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Macrófagos/metabolismo , Fagócitos/imunologia , Pinocitose/genética , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Membrana Celular/metabolismo , Quimiocina CCL5/farmacologia , Quimiotaxia/genética , Células Dendríticas/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Macrófagos/efeitos dos fármacos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Mutação , Fagócitos/efeitos dos fármacos , Fagócitos/metabolismo , Fosforilação , Pinocitose/efeitos dos fármacos , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Pathogenic missense variants in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified through linkage analysis in familial Parkinson disease (PD). Subsequently, other missense variants with lower effect sizes on PD risk have emerged, as well as non-coding polymorphisms (e.g. rs76904798) enriched in PD cases in genome-wide association studies. Here we leverage recent whole-genome sequences from the Accelerating Medicines Partnership-Parkinson's Disease (AMP-PD) and the Genome Aggregation (gnomAD) databases to characterize novel missense variants in LRRK2 and explore their relationships with known pathogenic and PD-linked missense variants. Using a computational prediction tool that successfully classifies known pathogenic LRRK2 missense variants, we describe an online web-based resource that catalogs characteristics of over 1200 LRRK2 missense variants of unknown significance. Novel high-pathogenicity scoring variants, some identified exclusively in PD cases, tightly cluster within the ROC-COR-Kinase domains. Structure-function predictions support that some of these variants exert gain-of-function effects with respect to LRRK2 kinase activity. In AMP-PD participants, all p.R1441G carriers (N = 89) are also carriers of the more common PD-linked variant p.M1646T. In addition, nearly all carriers of the PD-linked p.N2081D missense variant are also carriers of the LRRK2 PD-risk variant rs76904798. These results provide a compendium of LRRK2 missense variants and how they associate with one another. While the pathogenic p.G2019S variant is by far the most frequent high-pathogenicity scoring variant, our results suggest that ultra-rare missense variants may have an important cumulative impact in increasing the number of individuals with LRRK2-linked PD.
Assuntos
Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação de Sentido Incorreto , Doença de Parkinson/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/genética , Adulto JovemRESUMO
The accumulation of aggregated α-synuclein in susceptible neurons in the brain, together with robust activation of nearby myeloid cells, are pathological hallmarks of Parkinson's disease (PD). While microglia represent the dominant type of myeloid cell in the brain, recent genetic and whole-transcriptomic studies have implicated another type of myeloid cell, bone-marrow derived monocytes, in disease risk and progression. Monocytes in circulation harbor high concentrations of the PD-linked enzyme leucine-rich repeat kinase 2 (LRRK2) and respond to both intracellular and extracellular aggregated α-synuclein with a variety of strong pro-inflammatory responses. This review highlights recent findings from studies that functionally characterize monocytes in PD patients, monocytes that infiltrate into cerebrospinal fluid, and emerging analyses of whole myeloid cell populations in the PD-affected brain that include monocyte populations. Central controversies discussed include the relative contribution of monocytes acting in the periphery from those that might engraft in the brain to modify disease risk and progression. We conclude that further investigation into monocyte pathways and responses in PD, especially the discovery of additional markers, transcriptomic signatures, and functional classifications, that better distinguish monocyte lineages and responses in the brain from other types of myeloid cells may reveal points for therapeutic intervention, as well as a better understanding of ongoing inflammation associated with PD.
Assuntos
Doença de Parkinson , Humanos , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Monócitos/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Encéfalo/metabolismo , MutaçãoRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of late-onset, autosomal-dominant familial Parkinson's disease (PD). LRRK2 functions as both a kinase and GTPase, and PD-linked mutations are known to influence both enzymatic activities. While PD-linked LRRK2 mutations can commonly induce neuronal damage in culture models, the mechanisms underlying these pathogenic effects remain uncertain. Rodent models containing familial LRRK2 mutations often lack robust PD-like neurodegenerative phenotypes. Here, we develop a robust preclinical model of PD in adult rats induced by the brain delivery of recombinant adenoviral vectors with neuronal-specific expression of human LRRK2 harboring the most common G2019S mutation. In this model, G2019S LRRK2 induces the robust degeneration of substantia nigra dopaminergic neurons, a pathological hallmark of PD. Introduction of a stable kinase-inactive mutation or administration of the selective kinase inhibitor, PF-360, attenuates neurodegeneration induced by G2019S LRRK2. Neuroprotection provided by pharmacological kinase inhibition is mediated by an unusual mechanism involving the robust destabilization of human LRRK2 protein in the brain relative to endogenous LRRK2. Our study further demonstrates that G2019S LRRK2-induced dopaminergic neurodegeneration critically requires normal GTPase activity, as hypothesis-testing mutations that increase GTP hydrolysis or impair GTP-binding activity provide neuroprotection although via distinct mechanisms. Taken together, our data demonstrate that G2019S LRRK2 induces neurodegeneration in vivo via a mechanism that is dependent on kinase and GTPase activity. Our study provides a robust rodent preclinical model of LRRK2-linked PD and nominates kinase inhibition and modulation of GTPase activity as promising disease-modifying therapeutic targets.
Assuntos
Neurônios Dopaminérgicos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Modelos Animais de Doenças , Dopamina/metabolismo , Feminino , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Camundongos , Camundongos Knockout , Mutação , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doença de Parkinson/patologia , Fenótipo , Projetos Piloto , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Substância NegraRESUMO
BACKGROUND: Pathogenic leucine-rich repeat kinase 2 LRRK2 mutations may increase LRRK2 kinase activity and Rab substrate phosphorylation. Genetic association studies link variation in LRRK2 to idiopathic Parkinson disease (iPD) risk. OBJECTIVES: Through measurements of the LRRK2 kinase substrate pT73-Rab10 in urinary extracellular vesicles, this study seeks to understand how LRRK2 kinase activity might change with iPD progression. METHODS: Using an immunoblotting approach validated in LRRK2 transgenic mice, the ratio of pT73-Rab10 to total Rab10 protein was measured in extracellular vesicles from a cross-section of G2019S LRRK2 mutation carriers (N = 45 participants) as well as 485 urine samples from a novel longitudinal cohort of iPD and controls (N = 85 participants). Generalized estimating equations were used to conduct analyses with commonly used clinical scales. RESULTS: Although the G2019S LRRK2 mutation did not increase pT73-Rab10 levels, the ratio of pT73-Rab10 to total Rab10 nominally increased over baseline in iPD urine vesicle samples with time, but did not increase in age-matched controls (1.34-fold vs. 1.05-fold, 95% confidence interval [CI], 0.004-0.56; P = 0.046; Welch's t test). Effect estimates adjusting for sex, age, disease duration, diagnosis, and baseline clinical scores identified increasing total Movement Disorder Society-Sponsored Revision of the Unified (MDS-UPDRS) scores (ß = 0.77; CI, 0.52-1.01; P = 0.0001) with each fold increase of pT73-Rab10 to total Rab10. Lower Montreal Cognitive Assessment (MoCA) score in iPD is also associated with increased pT73-Rab10. CONCLUSIONS: These results provide initial insights into peripheral LRRK2-dependent Rab phosphorylation, measured in biobanked urine, where higher levels of pT73-Rab10 are associated with worse disease progression. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , Proteínas rab de Ligação ao GTP , Animais , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Fosforilação , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/urinaRESUMO
α-Synuclein aggregation underlies pathological changes in Lewy body dementia. Recent studies highlight structural variabilities associated with α-synuclein aggregates in patient populations. Here, we develop a quantitative real-time quaking-induced conversion (qRT-QuIC) assay to measure permissive α-synuclein fibril-templating activity in tissues and cerebrospinal fluid (CSF). The assay is anchored through reference panels of stabilized ultra-short fibril particles. In humanized α-synuclein transgenic mice, qRT-QuIC identifies differential levels of fibril activity across the brain months before the deposition of phosphorylated α-synuclein in susceptible neurons. α-Synuclein fibril activity in cortical brain extracts from dementia with Lewy bodies (DLB) correlates with activity in matched ventricular CSF. Elevated α-synuclein fibril activity in CSF corresponds to reduced survival in DLB. α-Synuclein fibril particles amplified from cases with high fibril activity show superior templating in the formation of new inclusions in neurons relative to the same number of fibril particles amplified from DLB cases with low fibril activity. Our results highlight a previously unknown broad heterogeneity of fibril-templating activities in DLB that may contribute to disease phenotypes. We predict that quantitative assessments of fibril activities in CSF that correlate to fibril activities in brain tissue will help stratify patient populations as well as measure therapeutic responses to facilitate the development of α-synuclein-targeted therapeutics.
Assuntos
Técnicas de Química Analítica/métodos , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/patologia , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Fenótipo , alfa-Sinucleína/análiseRESUMO
The mechanisms by which parkin protects the adult human brain from Parkinson disease remain incompletely understood. We hypothesized that parkin cysteines participate in redox reactions and that these are reflected in its posttranslational modifications. We found that in post mortem human brain, including in the Substantia nigra, parkin is largely insoluble after age 40 years; this transition is linked to its oxidation, such as at residues Cys95 and Cys253. In mice, oxidative stress induces posttranslational modifications of parkin cysteines that lower its solubility in vivo. Similarly, oxidation of recombinant parkin by hydrogen peroxide (H2O2) promotes its insolubility and aggregate formation, and in exchange leads to the reduction of H2O2. This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. In prkn-null mice, H2O2 levels are increased under oxidative stress conditions, such as acutely by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxin exposure or chronically due to a second, genetic hit; H2O2 levels are also significantly increased in parkin-deficient human brain. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at the primate-specific residue Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation in vitro. By probing sections of adult, human midbrain from control individuals with epitope-mapped, monoclonal antibodies, we found specific and robust parkin reactivity that co-localizes with neuromelanin pigment, frequently within LAMP-3/CD63+ lysosomes. We conclude that oxidative modifications of parkin cysteines are associated with protective outcomes, which include the reduction of H2O2, conjugation of reactive dopamine metabolites, sequestration of radicals within insoluble aggregates, and increased melanin formation. The loss of these complementary redox effects may augment oxidative stress during ageing in dopamine-producing cells of mutant PRKN allele carriers, thereby enhancing the risk of Parkinson's-linked neurodegeneration.
Assuntos
Envelhecimento/metabolismo , Dopamina/metabolismo , Mesencéfalo/metabolismo , Degeneração Neural/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Animais , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Mesencéfalo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Degeneração Neural/patologia , Oxirredução , Adulto JovemRESUMO
Human genetic studies implicate LRRK2 and RAB7L1 in susceptibility to Parkinson disease (PD). These two genes function in the same pathway, as knockout of Rab7L1 results in phenotypes similar to LRRK2 knockout, and studies in cells and model organisms demonstrate LRRK2 and Rab7L1 interact in the endolysosomal system. Recently, a subset of Rab proteins have been identified as LRRK2 kinase substrates. Herein, we find that Rab8, Rab10, and Rab7L1 must be membrane and GTP-bound for LRRK2 phosphorylation. LRRK2 mutations that cause PD including R1441C, Y1699C, and G2019S all increase LRRK2 phosphorylation of Rab7L1 four-fold over wild-type LRRK2 in cells, resulting in the phosphorylation of nearly one-third the available Rab7L1 protein in cells. In contrast, the most common pathogenic LRRK2 mutation, G2019S, does not upregulate LRRK2-mediated phosphorylation of Rab8 or Rab10. LRRK2 interaction with membrane and GTP-bound Rab7L1, but not Rab8 or Rab10, results in the activation of LRRK2 autophosphorylation at the serine 1292 position, required for LRRK2 toxicity. Further, Rab7L1 controls the proportion of LRRK2 that is membrane-associated, and LRRK2 mutations enhance Rab7L1-mediated recruitment of LRRK2 to the trans-Golgi network. Interaction studies with the Rab8 and Rab10 GTPase-activating protein TBC1D4/AS160 demonstrate that LRRK2 phosphorylation may block membrane and GTP-bound Rab protein interaction with effectors. These results suggest reciprocal regulation between LRRK2 and Rab protein substrates, where Rab7L1-mediated upregulation of LRRK2 kinase activity results in the stabilization of membrane and GTP-bound Rab proteins that may be unable to interact with Rab effector proteins.
Assuntos
Guanosina Trifosfato/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Mutação , Fosforilação , Transporte ProteicoRESUMO
Competencies can guide outcomes assessment in veterinary medical education by providing a core set of specific abilities expected of new veterinary graduates. A competency-guided evaluation of Colorado State University's (CSU) equine veterinary curriculum was undertaken via an alumni survey. Published competencies for equine veterinary graduates were used to develop the survey, which was distributed to large animal alumni from CSU's Doctor of Veterinary Medicine program. The results of the survey indicated areas for improvement, specifically in equine business, surgery, dentistry, and radiology. The desire for more hands-on experiences in their training was repeatedly mentioned by alumni, with the largest discrepancies between didactic knowledge and hands-on skills in the areas of business and equine surgery. Alumni surveys allow graduates to voice their perceived levels of preparation by the veterinary program and should be used to inform curriculum revisions. It is proposed that the definition and utilization of competencies in each phase of a curricular review process (outcomes assessment, curriculum mapping, and curricular modifications), in addition to faculty experience and internal review, is warranted.
Assuntos
Currículo , Educação em Veterinária , Animais , Colorado , Currículo/normas , Currículo/tendências , Educação em Veterinária/normas , Educação em Veterinária/tendências , Docentes/normas , Cavalos , Inquéritos e QuestionáriosRESUMO
Virtual microscopy (VM) using scanned slides and imaging software is increasingly used in medical curricula alongside instruction in conventional microscopy (CM). Limited reports suggest that VM is useful in the veterinary education setting, and generally well-received by students. Whether students can apply knowledge gained through VM to practical use is unknown. Our objective was to determine whether instruction using VM, compared to CM, is a successful method of training veterinary students for the application of cytology in practice (i.e., using light microscopes). Seventy-one veterinary students from Colorado State University who attended a voluntary 3-hour cytology workshop were randomized to receive the same instruction with either VM (n = 35) or CM (n = 36). We compared these students to a control group (n = 22) of students who did not attend a workshop. All students took a post-workshop assessment involving the interpretation of four cases on glass slides with CM, designed to simulate the use of cytology in general practice. Students also took an 18-question survey related to the effectiveness of the workshop, providing their opinions on cytology instruction in the curriculum and their learning preference (VM or CM). The mean assessment score of the VM group (14.18 points) was significantly higher than the control group (11.33 points, p = .003), whereas the mean of the CM group (12.77 points) was not statistically significantly different from controls (p = .170). Not only is VM an effective method of teaching cytology to veterinary students that can be translated to a real-world case scenario, but it outperformed CM instruction in this study.
Assuntos
Instrução por Computador , Educação em Veterinária , Animais , Humanos , Colorado , Microscopia/veterinária , Estudantes , EnsinoRESUMO
Content expertise in basic science and clinical disciplines does not assure proficiency in teaching. Faculty development to improve teaching and learning is essential for the advancement of veterinary education. The Consortium of West Region Colleges of Veterinary Medicine established the Regional Teaching Academy (RTA) with the focus of "Making Teaching Matter." The objective of the RTA's first effort, the Faculty Development Initiative (FDI), was to develop a multi-institutional faculty development program for veterinary educators to learn about and integrate effective teaching methods. In 2016, the Veterinary Educator Teaching and Scholarship (VETS) program was piloted at Oregon State University's College of Veterinary Medicine. This article uses a case study approach to program evaluation of the VETS program. We describe the VETS program, participants' perceptions, participants' teaching method integration, and lessons learned. A modified Kirkpatrick Model (MKM) was used to categorize program outcomes and impact. Quantitative data are presented as descriptive statistics, and qualitative data are presented as the themes that emerged from participant survey comments and post-program focus groups. Results indicated outcomes and impacts that included participants' perceptions of the program, changes in participant attitude toward teaching and learning, an increase in the knowledge level of participants, self-reported changes in participant behaviors, and changes in practices and structure at the college level. Lessons learned indicate that the following are essential for program success: (1) providing institutional and financial support; (2) creating a community of practice (COP) of faculty development facilitators, and (3) developing a program that addresses the needs of faculty and member institutions.
Assuntos
Educação em Veterinária , Animais , Docentes , Docentes de Medicina , Bolsas de Estudo , Humanos , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Desenvolvimento de Pessoal , Inquéritos e Questionários , EnsinoRESUMO
Our understanding of the mechanisms underlying Parkinson's disease, the once archetypical nongenetic neurogenerative disorder, has dramatically increased with the identification of α-synuclein and LRRK2 pathogenic mutations. While α-synuclein protein composes the aggregates that can spread through much of the brain in disease, LRRK2 encodes a multidomain dual-enzyme distinct from any other protein linked to neurodegeneration. In this review, we discuss emergent datasets from multiple model systems that suggest these unlikely partners do interact in important ways in disease, both within cells that express both LRRK2 and α-synuclein as well as through more indirect pathways that might involve neuroinflammation. Although the link between LRRK2 and disease can be understood in part through LRRK2 kinase activity (phosphotransferase activity), α-synuclein toxicity is multilayered and plausibly interacts with LRRK2 kinase activity in several ways. We discuss common protein interactors like 14-3-3s that may regulate α-synuclein and LRRK2 in disease. Finally, we examine cellular pathways and outcomes common to both mutant α-synuclein expression and LRRK2 activity and points of intersection. Understanding the interplay between these two unlikely partners in disease may provide new therapeutic avenues for PD.
Assuntos
Encéfalo/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/efeitos adversos , Degeneração Neural/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/efeitos adversos , Animais , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Mutação , Neurônios/metabolismo , Doença de Parkinson/genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a frequent genetic cause of late-onset Parkinson's disease (PD) and a target for therapeutic approaches. LRRK2 protein can influence vesicle trafficking events in the cytosol, with action both in endosomal and lysosomal pathways in different types of cells. A subset of late endosomes harbor intraluminal vesicles that can be secreted into the extracellular milieu. These extracellular vesicles, called exosomes, package LRRK2 protein for transport outside the cell into easily accessed biofluids. Both the cytoplasmic complement of LRRK2 as well as the exosome-associated fraction of protein appears regulated in part by interactions with 14-3-3 proteins. LRRK2 inside exosomes have disease-linked post-translational modifications and are relatively stable compared with unprotected proteins in the extracellular space or disrupted cytosolic compartments. Herein, we review the biology of exosome-associated LRRK2 and the potential for utility in diagnosis, prognosis, and theragnosis in PD and other LRRK2-linked diseases.
Assuntos
Biomarcadores/metabolismo , Exossomos/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Animais , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismoRESUMO
Primary cilia play central roles in signaling during metazoan development. Several key regulators of ciliogenesis and ciliary signaling are mutated in humans, resulting in a number of ciliopathies, including Joubert syndrome (JS). ARL13B is a ciliary GTPase with at least three missense mutations identified in JS patients. ARL13B is a member of the ADP ribosylation factor family of regulatory GTPases, but is atypical in having a non-homologous, C-terminal domain of â¼20 kDa and at least one key residue difference in the consensus GTP-binding motifs. For these reasons, and to establish a solid biochemical basis on which to begin to model its actions in cells and animals, we developed preparations of purified, recombinant, murine Arl13b protein. We report results from assays for solution-based nucleotide binding, intrinsic and GTPase-activating protein-stimulated GTPase, and ARL3 guanine nucleotide exchange factor activities. Biochemical analyses of three human missense mutations found in JS and of two consensus GTPase motifs reinforce the atypical properties of this regulatory GTPase. We also discovered that murine Arl13b is a substrate for casein kinase 2, a contaminant in our preparation from human embryonic kidney cells. This activity, and the ability of casein kinase 2 to use GTP as a phosphate donor, may be a source of differences between our data and previously published results. These results provide a solid framework for further research into ARL13B on which to develop models for the actions of this clinically important cell regulator.
Assuntos
Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/isolamento & purificação , Fatores de Ribosilação do ADP/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Caseína Quinase II/metabolismo , Cerebelo/anormalidades , Cerebelo/metabolismo , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Humanos , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Camundongos , Mutação de Sentido Incorreto , Retina/anormalidades , Retina/metabolismoRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common known cause of inherited Parkinson's disease (PD), and LRRK2 is a risk factor for idiopathic PD. How LRRK2 function is regulated is not well understood. Recently, the highly conserved 14-3-3 proteins, which play a key role in many cellular functions including cell death, have been shown to interact with LRRK2. In this study, we investigated whether 14-3-3s can regulate mutant LRRK2-induced neurite shortening and kinase activity. In the presence of 14-3-3θ overexpression, neurite length of primary neurons from BAC transgenic G2019S-LRRK2 mice returned back to wild-type levels. Similarly, 14-3-3θ overexpression reversed neurite shortening in neuronal cultures from BAC transgenic R1441G-LRRK2 mice. Conversely, inhibition of 14-3-3s by the pan-14-3-3 inhibitor difopein or dominant-negative 14-3-3θ further reduced neurite length in G2019S-LRRK2 cultures. Since G2019S-LRRK2 toxicity is likely mediated through increased kinase activity, we examined 14-3-3θ's effects on LRRK2 kinase activity. 14-3-3θ overexpression reduced the kinase activity of G2019S-LRRK2, while difopein promoted the kinase activity of G2019S-LRRK2. The ability of 14-3-3θ to reduce LRRK2 kinase activity required direct binding of 14-3-3θ with LRRK2. The potentiation of neurite shortening by difopein in G2019S-LRRK2 neurons was reversed by LRRK2 kinase inhibitors. Taken together, we conclude that 14-3-3θ can regulate LRRK2 and reduce the toxicity of mutant LRRK2 through a reduction of kinase activity.
Assuntos
Proteínas 14-3-3/fisiologia , Neuritos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Crescimento Celular , Células Cultivadas , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Neuritos/enzimologia , Neurônios/citologia , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Fosforilação , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas/genética , Serina/metabolismoRESUMO
UNLABELLED: Pathologic inclusions define α-synucleinopathies that include Parkinson's disease (PD). The most common genetic cause of PD is the G2019S LRRK2 mutation that upregulates LRRK2 kinase activity. However, the interaction between α-synuclein, LRRK2, and the formation of α-synuclein inclusions remains unclear. Here, we show that G2019S-LRRK2 expression, in both cultured neurons and dopaminergic neurons in the rat substantia nigra pars compact, increases the recruitment of endogenous α-synuclein into inclusions in response to α-synuclein fibril exposure. This results from the expression of mutant G2019S-LRRK2, as overexpression of WT-LRRK2 not only does not increase formation of inclusions but reduces their abundance. In addition, treatment of primary mouse neurons with LRRK2 kinase inhibitors, PF-06447475 and MLi-2, blocks G2019S-LRRK2 effects, suggesting that the G2019S-LRRK2 potentiation of inclusion formation depends on its kinase activity. Overexpression of G2019S-LRRK2 slightly increases, whereas WT-LRRK2 decreases, total levels of α-synuclein. Knockdown of total α-synuclein with potent antisense oligonucleotides substantially reduces inclusion formation in G2019S-LRRK2-expressing neurons, suggesting that LRRK2 influences α-synuclein inclusion formation by altering α-synuclein levels. These findings support the hypothesis that G2019S-LRRK2 may increase the progression of pathological α-synuclein inclusions after the initial formation of α-synuclein pathology by increasing a pool of α-synuclein that is more susceptible to forming inclusions. SIGNIFICANCE STATEMENT: α-Synuclein inclusions are found in the brains of patients with many different neurodegenerative diseases. Point mutation, duplication, or triplication of the α-synuclein gene can all cause Parkinson's disease (PD). The G2019S mutation in LRRK2 is the most common known genetic cause of PD. The interaction between G2019S-LRRK2 and α-synuclein may uncover new mechanisms and targets for neuroprotection. Here, we show that expression of G2019S-LRRK2 increases α-synuclein mobility and enhances aggregation of α-synuclein in primary cultured neurons and in dopaminergic neurons of the substantia nigra pars compacta, a susceptible brain region in PD. Potent LRRK2 kinase inhibitors, which are being developed for clinical use, block the increased α-synuclein aggregation in G2019S-LRRK2-expressing neurons. These results demonstrate that α-synuclein inclusion formation in neurons can be blocked and that novel therapeutic compounds targeting this process by inhibiting LRRK2 kinase activity may slow progression of PD-associated pathology.
Assuntos
Corpos de Inclusão/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação/genética , Neurônios/metabolismo , Transcitose/fisiologia , alfa-Sinucleína/metabolismo , Animais , Regulação da Expressão Gênica/genética , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligorribonucleotídeos Antissenso/farmacologia , Fotodegradação , Ratos , Sinucleínas/metabolismo , Transcitose/genética , Tubulina (Proteína)/metabolismo , Canais de Ânion Dependentes de Voltagem/genética , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
The Leucine rich repeat kinase 2 (LRRK2) gene is genetically and biochemically linked to several diseases that involve innate immunity. LRRK2 protein is highly expressed in phagocytic cells of the innate immune system, most notably in myeloid cells capable of mounting potent pro-inflammatory responses. Knockdown of LRRK2 protein in these cells reduces pro-inflammatory responses. However, the effect of LRRK2 pathogenic mutations that cause Parkinson's disease on myeloid cell function is not clear but could provide insight into LRRK2-linked disease. Here, we find that rats expressing G2019S LRRK2 have exaggerated pro-inflammatory responses and subsequent neurodegeneration after lipopolysaccharide injections in the substantia nigra, with a marked increase in the recruitment of CD68 myeloid cells to the site of injection. While G2019S LRRK2 expression did not affect immunological homeostasis, myeloid cells expressing G2019S LRRK2 show enhanced chemotaxis both in vitro in two-chamber assays and in vivo in response to thioglycollate injections in the peritoneum. The G2019S mutation enhanced the association between LRRK2 and actin-regulatory proteins that control chemotaxis. The interaction between G2019S LRRK2 and actin-regulatory proteins can be blocked by LRRK2 kinase inhibitors, although we did not find evidence that LRRK2 phosphorylated these interacting proteins. These results suggest that the primary mechanism of G2019S LRRK2 with respect to myeloid cell function in disease may be related to exaggerated chemotactic responses.
Assuntos
Actinas/metabolismo , Imunidade Inata/genética , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/genética , Actinas/genética , Animais , Quimiotaxia/genética , Modelos Animais de Doenças , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mutação , Células Mieloides/metabolismo , Células Mieloides/patologia , Doença de Parkinson/patologia , Ligação Proteica/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Substância Negra/metabolismo , Substância Negra/patologiaRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson's disease, and LRRK2 is also linked to Crohn's and Hansen's disease. LRRK2 is expressed in many organs in mammals but is particularly abundant in the kidney. We find that LRRK2 protein is predominantly localized to collecting duct cells in the rat kidney, with much lower expression in other kidney cells. While genetic knockout (KO) of LRRK2 expression is well-tolerated in mice and rats, a unique age-dependent pathology develops in the kidney. The cortex and medulla of LRRK2 KO rat kidneys become darkly pigmented in early adulthood, yet aged animals display no overt signs of kidney failure. Accompanying the dark pigment we find substantial macrophage infiltration in LRRK2 KO kidneys, suggesting the presence of chronic inflammation that may predispose to kidney disease. Unexpectedly, the dark kidneys of the LRRK2 KO rats are highly resistant to rhabdomyolysis-induced acute kidney injury compared with wild-type rats. Biochemical profiling of the LRRK2 KO kidneys using immunohistochemistry, proteomic and lipidomic analyses show a massive accumulation of hemoglobin and lipofuscin in renal tubules that account for the pigmentation. The proximal tubules demonstrate a corresponding up-regulation of the cytoprotective protein heme oxygenase-1 (HO-1) which is capable of mitigating acute kidney injury. The unusual kidney pathology of LRRK2 KO rats highlights several novel physiological roles for LRRK2 and provides indirect evidence for HO-1 expression as a protective mechanism in acute kidney injury in LRRK2 deficiency.