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1.
Mov Disord ; 35(11): 2095-2100, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32652692

RESUMO

BACKGROUND: Leucine-rich repeat kinase 2 kinase inhibitors are being vigorously pursued as potential therapeutic options; however, there is a critical need for sensitive and quantitative assays of leucine-rich repeat kinase 2 function and target engagement. OBJECTIVES: Our objective was to compare collection and storage protocols for peripheral blood mononuclear cells, and to determine the optimal conditions for downstream analyses of leucine-rich repeat kinase 2 in PD cohorts. METHODS: Here, we describe enzyme-linked immunosorbent assay-based assays capable of detecting multiple aspects of leucine-rich repeat kinase 2 function at endogenous levels in human tissues. RESULTS: In peripheral blood mononuclear cells from both healthy and affected carriers of the G2019S mutation in leucine-rich repeat kinase 2, we report, for the first time, significantly elevated in vitro kinase activity, while detecting a significant increase in pS935/leucine-rich repeat kinase 2 in idiopathic PD patients. CONCLUSIONS: Quantitative assays such as these described here could potentially uncover specific markers of leucine-rich repeat kinase 2 function that are predictive of disease progression, aid in patient stratification, and be a critical component of upcoming clinical trials. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.


Assuntos
Leucócitos Mononucleares , Doença de Parkinson , Ensaio de Imunoadsorção Enzimática , Humanos , Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação/genética
2.
Biochem J ; 476(3): 559-579, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30670570

RESUMO

The Parkinson's disease (PD) protein leucine-rich repeat kinase 2 (LRRK2) exists as a mixture of monomeric and dimeric species, with its kinase activity highly concentrated in the dimeric conformation of the enzyme. We have adapted the proximity biotinylation approach to study the formation and activity of LRRK2 dimers isolated from cultured cells. We find that the R1441C and I2020T mutations both enhance the rate of dimer formation, whereas, the G2019S kinase domain mutant is similar to WT, and the G2385R risk factor variant de-stabilizes dimers. Interestingly, we find a marked departure in the kinase activity between G2019S-LRRK2 homo-dimers and wild-type-G2019S hetero-dimers. While the homo-dimeric G2019S-LRRK2 exhibits the typical robust enhancement of kinase activity, hetero-dimers comprised of wild-type (WT) and G2019S-LRRK2 exhibit kinase activity similar to WT. Dimeric complexes of specific mutant forms of LRRK2 show reduced stability following an in vitro kinase reaction, in LRRK2 mutants for which the kinase activity is similar to WT. Phosphorylation of the small GTPase Rab10 follows a similar pattern in which hetero-dimers of WT and mutant LRRK2 show similar levels of phosphorylation of Rab10 to WT homo-dimers; while the levels of pRab10 are significantly increased in cells expressing mutant homo-dimers. Interestingly, while the risk variant G2385R leads to a de-stabilization of LRRK2 dimers, those dimers possess significantly elevated kinase activity. The vast majority of familial LRRK2-dependent PD cases are heterozygous; thus, these findings raise the possibility that a crucial factor in disease pathogenesis may be the accumulation of homo-dimeric mutant LRRK2.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Mutação de Sentido Incorreto , Doença de Parkinson/enzimologia , Multimerização Proteica , Substituição de Aminoácidos , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fosforilação/genética , Estrutura Quaternária de Proteína , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
3.
Proc Natl Acad Sci U S A ; 114(15): 3999-4004, 2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28348207

RESUMO

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic (DAergic) neurons in the substantia nigra and the gradual depletion of dopamine (DA). Current treatments replenish the DA deficit and improve symptoms but induce dyskinesias over time, and neuroprotective therapies are nonexistent. Here we report that Nuclear receptor-related 1 (Nurr1):Retinoid X receptor α (RXRα) activation has a double therapeutic potential for PD, offering both neuroprotective and symptomatic improvement. We designed BRF110, a unique in vivo active Nurr1:RXRα-selective lead molecule, which prevents DAergic neuron demise and striatal DAergic denervation in vivo against PD-causing toxins in a Nurr1-dependent manner. BRF110 also protects against PD-related genetic mutations in patient induced pluripotent stem cell (iPSC)-derived DAergic neurons and a genetic mouse PD model. Remarkably, besides neuroprotection, BRF110 up-regulates tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), and GTP cyclohydrolase I (GCH1) transcription; increases striatal DA in vivo; and has symptomatic efficacy in two postneurodegeneration PD models, without inducing dyskinesias on chronic daily treatment. The combined neuroprotective and symptomatic effects of BRF110 identify Nurr1:RXRα activation as a potential monotherapeutic approach for PD.


Assuntos
Antiparkinsonianos/farmacologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Doença de Parkinson/tratamento farmacológico , Receptor X Retinoide alfa/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Antiparkinsonianos/química , Antiparkinsonianos/farmacocinética , Encéfalo/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Dopamina/genética , Estabilidade de Medicamentos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/fisiologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/agonistas , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Multimerização Proteica , Ratos , Receptor X Retinoide alfa/agonistas , Receptor X Retinoide alfa/química , Receptor X Retinoide alfa/genética
4.
Biochem J ; 475(7): 1271-1293, 2018 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-29519959

RESUMO

Autosomal-dominant, missense mutations in the leucine-rich repeat protein kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson's disease (PD). LRRK2 kinase activity is increased in several pathogenic mutations (N1437H, R1441C/G/H, Y1699C, G2019S), implicating hyperphosphorylation of a substrate in the pathogenesis of the disease. Identification of the downstream targets of LRRK2 is a crucial endeavor in the field to understand LRRK2 pathway dysfunction in the disease. We have identified the signaling adapter protein p62/SQSTM1 as a novel endogenous interacting partner and a substrate of LRRK2. Using mass spectrometry and phospho-specific antibodies, we found that LRRK2 phosphorylates p62 on Thr138 in vitro and in cells. We found that the pathogenic LRRK2 PD-associated mutations (N1437H, R1441C/G/H, Y1699C, G2019S) increase phosphorylation of p62 similar to previously reported substrate Rab proteins. Notably, we found that the pathogenic I2020T mutation and the risk factor mutation G2385R displayed decreased phosphorylation of p62. p62 phosphorylation by LRRK2 is blocked by treatment with selective LRRK2 inhibitors in cells. We also found that the amino-terminus of LRRK2 is crucial for optimal phosphorylation of Rab7L1 and p62 in cells. LRRK2 phosphorylation of Thr138 is dependent on a p62 functional ubiquitin-binding domain at its carboxy-terminus. Co-expression of p62 with LRRK2 G2019S increases the neurotoxicity of this mutation in a manner dependent on Thr138. p62 is an additional novel substrate of LRRK2 that regulates its toxic biology, reveals novel signaling nodes and can be used as a pharmacodynamic marker for LRRK2 kinase activity.


Assuntos
Embrião de Mamíferos/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Neurônios/patologia , Proteína Sequestossoma-1/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/metabolismo , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação , Neurônios/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Proteína Sequestossoma-1/genética
5.
Biochem Soc Trans ; 45(1): 123-129, 2017 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-28202665

RESUMO

Autosomal dominantly inherited mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease. While considerable progress has been made in understanding its function and the many different cellular activities in which it participates, a clear understanding of the mechanism(s) of the induction of neuronal death by mutant forms of LRRK2 remains elusive. Although several in vivo models have documented the progressive loss of dopaminergic neurons of the substantia nigra, more complete interrogations of the modality of neuronal death have been gained from cellular models. Overexpression of mutant LRRK2 in neuronal-like cell lines or in primary neurons induces an apoptotic type of cell death involving components of the extrinsic as well as intrinsic death pathways. While informative, these studies are limited by their reliance upon isolated neuronal cells; and the pathways triggered by mutant LRRK2 in neurons may be further refined or modulated by extracellular signals. Nevertheless, the identification of specific cell death-associated signaling events set in motion by the dominant action of mutant LRRK2, the loss of an inhibitory function of wild-type LRRK2, or a combination of the two, expands the landscape of potential therapeutic targets for future intervention in the clinic.


Assuntos
Apoptose/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação , Neurônios/metabolismo , Transdução de Sinais , Animais , Caspases/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Modelos Biológicos , Neurônios/citologia , Doença de Parkinson/enzimologia , Doença de Parkinson/genética
6.
Hum Mol Genet ; 21(4): 890-9, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22080837

RESUMO

Dominant missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic causes of Parkinson disease (PD) and genome-wide association studies identify LRRK2 sequence variants as risk factors for sporadic PD. Intact kinase function appears critical for the toxicity of LRRK2 PD mutants, yet our understanding of how LRRK2 causes neurodegeneration remains limited. We find that most LRRK2 PD mutants abnormally enhance LRRK2 oligomerization, causing it to form filamentous structures in transfections of cell lines or primary neuronal cultures. Strikingly, ultrastructural analyses, including immuno-electron microscopy and electron microscopic tomography, demonstrate that these filaments consist of LRRK2 recruited onto part of the cellular microtubule network in a well-ordered, periodic fashion. Like LRRK2-related neurodegeneration, microtubule association requires intact kinase function and the WD40 domain, potentially linking microtubule binding and neurodegeneration. Our observations identify a novel effect of LRRK2 PD mutations and highlight a potential role for microtubules in the pathogenesis of LRRK2-related neurodegeneration.


Assuntos
Microtúbulos/metabolismo , Mutação/genética , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica/genética , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína
7.
Neurochem Res ; 39(3): 576-92, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-23729298

RESUMO

Leucine-rich repeat kinase 2 (LRRK2) is a large, widely expressed protein of largely unknown function. Mutations in the gene encoding LRRK2 have been linked to multiple diseases, including a prominent association with familial and sporadic Parkinson's disease (PD), as well as inflammatory bowel disorders such as Crohn's disease. The LRRK2 protein possesses both kinase and GTPase signaling domains, as well as multiple protein interaction domains. Experimental studies in both cellular and in vivo models of mutant LRRK2-induced neurodegeneration have given clues to potential function(s) of LRRK2, yet much remains unknown. For example, while it is known that intact kinase and GTPase activity are required for mutant forms of the protein to trigger cell death, the specific targets of these enzymatic activities that mediate the death of neurons are not known. In this review, we discuss the evidence linking LRRK2 to various cellular/neuronal activities such as extrinsic death and inflammatory signaling, lysosomal protein degradation, the cytoskeletal system and neurite outgrowth, vesicle trafficking, mitochondrial dysfunction, as well as multiple points of interaction with several other genes linked to the pathogenesis of PD. In order for more effective therapeutic strategies to be envisioned and implemented, the mechanisms underlying LRRK2-mediated neurodegeneration need to be better characterized. Furthermore, insights into LRRK2-associated PD pathogenesis can potentially advance our understanding of the more common sporadic forms of PD.


Assuntos
Neurônios/metabolismo , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , GTP Fosfo-Hidrolases/metabolismo , Humanos , Inflamação/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mutação/genética , Doença de Parkinson/etiologia , Doença de Parkinson/genética
8.
J Cachexia Sarcopenia Muscle ; 14(3): 1322-1336, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36905126

RESUMO

BACKGROUND: Sarcopenia, the age-associated decline in skeletal muscle mass and strength, has long been considered a disease of muscle only, but accumulating evidence suggests that sarcopenia could originate from the neural components controlling muscles. To identify early molecular changes in nerves that may drive sarcopenia initiation, we performed a longitudinal transcriptomic analysis of the sciatic nerve, which governs lower limb muscles, in aging mice. METHODS: Sciatic nerve and gastrocnemius muscle were obtained from female C57BL/6JN mice aged 5, 18, 21 and 24 months old (n = 6 per age group). Sciatic nerve RNA was extracted and underwent RNA sequencing (RNA-seq). Differentially expressed genes (DEGs) were validated using quantitative reverse transcription PCR (qRT-PCR). Functional enrichment analysis of clusters of genes associated with patterns of gene expression across age groups (adjusted P-value < 0.05, likelihood ratio test [LRT]) was performed. Pathological skeletal muscle aging was confirmed between 21 and 24 months by a combination of molecular and pathological biomarkers. Myofiber denervation was confirmed with qRT-PCR of Chrnd, Chrng, Myog, Runx1 and Gadd45ɑ in gastrocnemius muscle. Changes in muscle mass, cross-sectional myofiber size and percentage of fibres with centralized nuclei were analysed in a separate cohort of mice from the same colony (n = 4-6 per age group). RESULTS: We detected 51 significant DEGs in sciatic nerve of 18-month-old mice compared with 5-month-old mice (absolute value of fold change > 2; false discovery rate [FDR] < 0.05). Up-regulated DEGs included Dbp (log2 fold change [LFC] = 2.63, FDR < 0.001) and Lmod2 (LFC = 7.52, FDR = 0.001). Down-regulated DEGs included Cdh6 (LFC = -21.38, FDR < 0.001) and Gbp1 (LFC = -21.78, FDR < 0.001). We validated RNA-seq findings with qRT-PCR of various up- and down-regulated genes including Dbp and Cdh6. Up-regulated genes (FDR < 0.1) were associated with the AMP-activated protein kinase signalling pathway (FDR = 0.02) and circadian rhythm (FDR = 0.02), whereas down-regulated DEGs were associated with biosynthesis and metabolic pathways (FDR < 0.05). We identified seven significant clusters of genes (FDR < 0.05, LRT) with similar expression patterns across groups. Functional enrichment analysis of these clusters revealed biological processes that may be implicated in age-related changes in skeletal muscles and/or sarcopenia initiation including extracellular matrix organization and an immune response (FDR < 0.05). CONCLUSIONS: Gene expression changes in mouse peripheral nerve were detected prior to disturbances in myofiber innervation and sarcopenia onset. These early molecular changes we report shed a new light on biological processes that may be implicated in sarcopenia initiation and pathogenesis. Future studies are warranted to confirm the disease modifying and/or biomarker potential of the key changes we report here.


Assuntos
Fenômenos Biológicos , Sarcopenia , Feminino , Camundongos , Animais , Sarcopenia/etiologia , Transcriptoma , Estudos Transversais , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo
9.
NPJ Parkinsons Dis ; 8(1): 73, 2022 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-35676398

RESUMO

Despite several advances in the field, pharmacodynamic outcome measures reflective of LRRK2 kinase activity in clinical biofluids remain urgently needed. A variety of targets and approaches have been utilized including assessments of LRRK2 itself (levels, phosphorylation), or its substrates (e.g. Rab10 or other Rab GTPases). We have previously shown that intrinsic kinase activity of LRRK2 isolated from PBMCs of G2019S carriers is elevated, irrespective of disease status. In the present study we find that phosphorylation of Rab10 is also elevated in G2019S carriers, but only those with PD. Additionally, phosphorylation of this substrate is also elevated in two separate idiopathic PD cohorts, but not in carriers of the A53T mutation in α-synuclein. In contrast, Rab29 phosphorylation was specifically reduced in urinary exosomes from A53T and idiopathic PD patients. Taken together, our findings highlight the need for the assessment of multiple complimentary targets for a more comprehensive picture of the disease.

10.
J Neurochem ; 119(3): 630-43, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21883213

RESUMO

Proteasome-mediated proteolysis is a major protein degradation mechanism in cells and its dysfunction has been implicated in the pathogenesis of several neurodegenerative diseases, each with the common features of neuronal death and formation of ubiquitinated inclusions found within neurites, the cell body, or nucleus. Previous models of proteasome dysfunction have employed pharmacological inhibition of the catalytic subunits of the 20S proteasome core, or the genetic manipulation of specific subunits resulting in altered proteasome assembly. In this study, we report the use of dominant negative subunits of the 19S regulatory proteasome complex that mediate the recognition of ubiquitinated substrates as well as the removal of the poly-ubiquitin chain. Interestingly, while each mutant subunit-induced inclusion formation, like that seen with pharmacological inhibition of the 20S proteasome, none was able to induce apoptotic death, or trigger activation of macroautophagy, in either dopaminergic cell lines or primary cortical neurons. This finding highlights the dissociation between the mechanisms of neuronal inclusion formation and the induction of cell death, and represents a novel cellular model for Lewy body-like inclusion formation in neurons.


Assuntos
Marcação de Genes/métodos , Corpos de Inclusão/enzimologia , Neurônios/enzimologia , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Ubiquitinação/genética , Animais , Morte Celular/genética , Células Cultivadas , Corpos de Inclusão/genética , Corpos de Inclusão/patologia , Camundongos , Neurônios/patologia , Células PC12 , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos
11.
Brain Res ; 1771: 147639, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34492263

RESUMO

Parkinson's disease (PD) is the second most common neurodegenerative disease, comprised of both familial and idiopathic forms, behind only Alzheimer's disease (AD). The disease is characterized, regardless of the pathogenesis, primarily by a loss of DA neurons in the ventral midbrain as well as noradrenergic neurons of the locus coeruleus; however, by the time symptoms manifest, considerable neuronal loss in both areas has occurred. Neuroprotective strategies thus have to be paired with more sensitive and specific biomarker assays that can identify early at-risk patients in order to initiate disease-modifying therapies at an earlier stage in the disease. Complicating this is the fact that multiple forms of cell death mediate the neuronal loss; however, with a common underlying element that the cell death is considered a "regulated" form of cell death, in contrast to an un-controlled necrotic cell death process. In this review we focus our discussion on several categories of regulated cell death in the context of PD: apoptosis, necroptosis, pyroptosis, and autophagic cell death. In clinical studies as well as experimental in vivo models of PD, there is evidence for a role of each of these forms of cell death in the loss of midbrain DA neurons, and specific therapeutic strategies have been proposed and tested. What remains unclear however is the relative contributions of these distinct forms of cell death to the overall loss of DA neurons, whether they occur at different stages of the disease, or whether specific sub-regions within the midbrain are more susceptible to specific death triggers and pathways.


Assuntos
Morte Celular , Neurônios/patologia , Doença de Parkinson/patologia , Animais , Neurônios Dopaminérgicos/patologia , Humanos
12.
J Neurosci ; 29(4): 1011-6, 2009 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-19176810

RESUMO

Neurodegenerative illnesses such as Parkinson and Alzheimer disease are an increasingly prevalent problem in aging societies, yet no therapies exist that retard or prevent neurodegeneration. Dominant missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson disease (PD), but the mechanisms by which mutant forms of LRRK2 disrupt neuronal function and cause cell death remain poorly understood. We report that LRRK2 interacts with the death adaptor Fas-associated protein with death domain (FADD), and that in primary neuronal culture LRRK2-mediated neurodegeneration is prevented by the functional inhibition of FADD or depletion of caspase-8, two key elements of the extrinsic cell death pathway. This pathway is activated by disease-triggering mutations, which enhance the LRRK2-FADD association and the consequent recruitment and activation of caspase-8. These results establish a direct molecular link between a mutant PD gene and the activation of programmed cell death signaling, and suggest that FADD/caspase-8 signaling contributes to LRRK2-induced neuronal death.


Assuntos
Apoptose/fisiologia , Caspase 8/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Neurônios/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Transporte/genética , Linhagem Celular Transformada , Peptídeos Penetradores de Células , Proteína de Domínio de Morte Associada a Fas/genética , Proteínas de Fluorescência Verde/genética , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mutagênese Sítio-Dirigida/métodos , Neurônios/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/farmacologia , Transfecção/métodos
13.
Biomolecules ; 10(6)2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32560161

RESUMO

Biomarkers and disease-modifying therapies are both urgent unmet medical needs in the treatment of Parkinson's disease (PD) and must be developed concurrently because of their interdependent relationship: biomarkers for the early detection of disease (i.e., prior to overt neurodegeneration) are necessary in order for patients to receive maximal therapeutic benefit and vice versa; disease-modifying therapies must become available for patients whose potential for disease diagnosis and prognosis can be predicted with biomarkers. This review provides an overview of the milestones achieved to date in the therapeutic strategy development of disease-modifying therapies and biomarkers for PD, with a focus on the most common and advanced genetically linked targets alpha-synuclein (SNCA), leucine-rich repeat kinase-2 (LRRK2) and glucocerebrosidase (GBA1). Furthermore, we discuss the convergence of the different pathways and the importance of patient stratification and how these advances may apply more broadly to idiopathic PD. The heterogeneity of PD poses a challenge for therapeutic and biomarker development, however, the one gene- one target approach has brought us closer than ever before to an unprecedented number of clinical trials and biomarker advancements.


Assuntos
Biomarcadores , Terapia Genética/tendências , Terapia de Alvo Molecular/tendências , Doença de Parkinson/terapia , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Heterogeneidade Genética , Terapia Genética/métodos , História do Século XXI , Humanos , Terapia de Alvo Molecular/métodos , Mutação , Doença de Parkinson/diagnóstico , Doença de Parkinson/genética , Doença de Parkinson/metabolismo
14.
Front Neurosci ; 14: 865, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33013290

RESUMO

Evidence is mounting that LRRK2 function, particularly its kinase activity, is elevated in multiple forms of Parkinson's disease, both idiopathic as well as familial forms linked to mutations in the LRRK2 gene. However, sensitive quantitative markers of LRRK2 activation in clinical samples remain at the early stages of development. There are several measures of LRRK2 activity that could potentially be used in longitudinal studies of disease progression, as inclusion/exclusion criteria for clinical trials, to predict response to therapy, or as markers of target engagement. Among these are levels of LRRK2, phosphorylation of LRRK2 itself, either by other kinases or via auto-phosphorylation, its in vitro kinase activity, or phosphorylation of downstream substrates. This is advantageous on many levels, in that multiple indices of elevated kinase activity clearly strengthen the rationale for targeting this kinase with novel therapeutic candidates, and provide alternate markers of activation in certain tissues or biofluids for which specific measures are not detectable. However, this can also complicate interpretation of findings from different studies using disparate measures. In this review we discuss the current state of LRRK2-focused biomarkers, the advantages and disadvantages of the current pallet of outcome measures, the gaps that need to be addressed, and the priorities that the field has defined.

15.
J Neurochem ; 105(3): 653-65, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18194212

RESUMO

Proteasomal dysfunction has been linked to neurodegeneration. Pharmacological proteasomal inhibitors may have pro-survival or pro-death effects in neuronal cells. We have previously found that application of such agents to mouse sympathetic neurons leads to activation of the intrinsic apoptotic pathway. We show here that in rat sympathetic neurons proteasomal inhibition leads to a form of death that is morphologically non-apoptotic, with features of autophagy. The intrinsic apoptotic pathway is activated in a delayed fashion compared with mouse neurons, and is in part responsible for death, as evidenced by the partial protective effects of bcl-xL and the general caspase inhibitor Boc-aspartyl-fluoromethylketone. Death is accompanied by induction of Bim and caspase activation, but caspase 3 activation is lacking; 3-methyl-adenine inhibits macroautophagy, but has a relatively small pro-survival effect. We conclude that a complex array of pro- and anti-apoptotic effects elicited by proteasomal inhibition in rat sympathetic neurons leads to partial engagement of the intrinsic apoptotic pathway and a morphologically non-apoptotic, autophagic form of death. The species difference with mouse neurons is underscored by the fact that proteasomal inhibitors are protective against apoptosis elicited by nerve growth factor deprivation in rat, but not mouse, sympathetic neurons. The type of death described herein may be relevant to neurodegenerative diseases, where morphological evidence for apoptosis has been scant.


Assuntos
Caspases/metabolismo , Gânglios Simpáticos/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Transdução de Sinais/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Proteína 11 Semelhante a Bcl-2 , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Forma Celular/efeitos dos fármacos , Forma Celular/fisiologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Gânglios Simpáticos/citologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Especificidade da Espécie , Proteína bcl-X/metabolismo
17.
Sci Rep ; 8(1): 3455, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29472595

RESUMO

In experimental models, both in vivo and cellular, over-expression of Parkinson's linked mutant leucine-rich repeat kinase 2 (LRRK2) is sufficient to induce neuronal death. While several cell death associated proteins have been linked to LRRK2, either as protein interactors or as putative substrates, characterization of the neuronal death cascade remains elusive. In this study, we have mapped for the first time the domain within LRRK2 that mediates the interaction with FADD, thereby activating the molecular machinery of the extrinsic death pathway. Using homology modeling and molecular docking approaches, we have identified a critical motif within the N-terminal armadillo repeat region of LRRK2. Moreover, we show that co-expression of fragments of LRRK2 that contain the FADD binding motif, or deletion of this motif itself, blocks the interaction with FADD, and is neuroprotective. We further demonstrate that downstream of FADD, the mitochondrial proteins Bid and Bax are recruited to the death cascade and are necessary for neuronal death. Our work identifies multiple novel points within neuronal death signaling pathways that could potentially be targeted by candidate therapeutic strategies and highlight how the extrinsic pathway can be activated intracellularly in a pathogenic context.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Animais , Proteínas do Domínio Armadillo/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Morte Celular , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Camundongos , Simulação de Acoplamento Molecular , Neurônios/citologia , Cultura Primária de Células , Domínios e Motivos de Interação entre Proteínas/genética , Mapeamento de Interação de Proteínas , Ratos , Sequências Repetitivas de Aminoácidos , Transdução de Sinais , Proteína X Associada a bcl-2/metabolismo
18.
Adv Neurobiol ; 14: 193-208, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28353285

RESUMO

Since its cloning and identification in 2004, considerable gains have been made in the understanding of the basic functionality of leucine-rich repeat kinase 2 (LRRK2), including its kinase and GTPase activities, its protein interactors and subcellular localization, and its expression in the CNS and peripheral tissues. However, the mechanism(s) by which expression of mutant forms of LRRK2 lead to the death of dopaminergic neurons of the ventral midbrain remains largely uncharacterized. Because of its complex domain structure, LRRK2 exhibits similarities with multiple protein families including ROCO proteins, as well as the RIP kinases. Cellular models in which mutant LRRK2 is overexpressed in neuronal-like cell lines or in primary neurons have found evidence of apoptotic cell death involving components of the extrinsic as well as intrinsic death pathways. However, since the expression of LRRK2 is comparatively quite low in ventral midbrain dopaminergic neurons, the possibility exists that non-cell autonomous signaling also contributes to the loss of these neurons. In this chapter, we will discuss the different neuronal death pathways that may be activated by mutant forms of LRRK2, guided in part by the behavior of other members of the RIP kinase protein family.


Assuntos
Apoptose/fisiologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Humanos , Mutação
19.
PLoS One ; 11(11): e0166053, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27832104

RESUMO

BACKGROUND: Despite the plethora of sequence variants in LRRK2, only a few clearly segregate with PD. Even within this group of pathogenic mutations, the phenotypic profile can differ widely. OBJECTIVE: We examined multiple properties of LRRK2 behavior in cellular models over-expressing three sequence variants described in Greek PD patients in comparison to several known pathogenic and non-pathogenic LRRK2 mutations, to determine if specific phenotypes associated with pathogenic LRRK2 can be observed in other less-common sequence variants for which pathogenicity is unclear based on clinical and/or genetic data alone. METHODS: The oligomerization, activity, phosphorylation, and interaction with FADD was assessed in HEK293T cells over-expressing LRRK2; while the induction of neuronal death was determined by quantifying apoptotic nuclei in primary neurons transiently expressing LRRK2. RESULTS: One LRRK2 variant, A211V, exhibited a modest increase in kinase activity, whereas only the pathogenic mutants G2019S and I2020T displayed significantly altered auto-phosphorylation. We observed an induction of detergent-insoluble high molecular weight structures upon expression of pathogenic LRRK2 mutants, but not the other LRRK2 variants. In contrast, each of the variants tested induced apoptotic death of cultured neurons similar to pathogenic LRRK2 in a FADD-dependent manner. CONCLUSIONS: Overall, despite differences in some properties of LRRK2 function such as kinase activity and its oligomerization, each of the LRRK2 variants examined induced neuronal death to a similar extent. Furthermore, our findings further strengthen the notion of a convergence on the extrinsic cell death pathway common to mutations in LRRK2 that are capable of inducing neuronal death.


Assuntos
Proteína de Domínio de Morte Associada a Fas/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação , Neurônios/patologia , Doença de Parkinson/genética , Transdução de Sinais , Morte Celular , Linhagem Celular , Células Cultivadas , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosforilação , Mapas de Interação de Proteínas
20.
J Neurosci ; 23(4): 1237-45, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12598612

RESUMO

Growing evidence suggests that the proteasome may be dysfunctional in a number of neurodegenerative disorders, including Lewy body diseases. We have reported previously that application of pharmacological inhibitors of the proteasome to cultured cortical neurons leads to apoptotic death and formation of ubiquitinated cytoplasmic inclusions. A number of cell cycle regulatory proteins are known to be degraded by the proteasome. In light of the emerging role of aberrant cell-cycle activation in neuronal cell death, we have assessed the involvement of cell-cycle components in the effects induced by proteasomal inhibitors in cortical neurons. Death and mitochondrial dysfunction induced by lactacystin and other pharmacological inhibitors of the proteasome were prevented by flavopiridol, a specific inhibitor of cyclin-dependent kinases (Cdks). Molecular expression of the Cdk inhibitors p16 or p27, or of dominant-negative Cdk2, Cdk4, or Cdk6 was also protective against lactacystin-induced death. Flavopiridol blocked the induction of retinoblastoma protein (pRb) phosphorylation that occurred after lactacystin application, and expression of a mutant pRb that lacked phosphorylation sites was neuroprotective. These results suggest that in cortical neurons, proteasomal inhibition leads to a cell death pathway that is dependent on Cdk activation and pRb inactivation. Although cyclins D1 and E were sequestered within the ubiquitinated inclusions formed at late time points after lactacystin application, the formation of ubiquitinated inclusions was unaffected by Cdk inhibition. This suggests that there are parallel pathways regulating neuronal death and inclusion formation elicited by proteasomal inhibition in cortical neurons.


Assuntos
Acetilcisteína/análogos & derivados , Apoptose , Quinases relacionadas a CDC2 e CDC28 , Córtex Cerebral/enzimologia , Quinases Ciclina-Dependentes/metabolismo , Complexos Multienzimáticos/antagonistas & inibidores , Neurônios/enzimologia , Proteínas Proto-Oncogênicas , Acetilcisteína/antagonistas & inibidores , Acetilcisteína/farmacologia , Transporte Ativo do Núcleo Celular , Animais , Ciclo Celular , Núcleo Celular/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/fisiologia , Ciclinas/análise , Ciclinas/metabolismo , Cisteína Endopeptidases , Inibidores de Cisteína Proteinase/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Corpos de Inclusão/química , Neurônios/química , Neurônios/efeitos dos fármacos , Piperidinas/farmacologia , Complexo de Endopeptidases do Proteassoma , Proteínas Serina-Treonina Quinases/fisiologia , Ratos , Proteína do Retinoblastoma/metabolismo , Ubiquitina/metabolismo
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