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1.
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
2.
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
3.
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
4.
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
5.
Cell Res ; 29(4): 313-329, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30858560

RESUMO

Missense mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) cause the majority of familial and some sporadic forms of Parkinson's disease (PD). The hyperactivity of LRRK2 kinase induced by the pathogenic mutations underlies neurotoxicity, promoting the development of LRRK2 kinase inhibitors as therapeutics. Many potent and specific small-molecule LRRK2 inhibitors have been reported with promise. However, nearly all inhibitors are ATP competitive-some with unwanted side effects and unclear clinical outcome-alternative types of LRRK2 inhibitors are lacking. Herein we identify 5'-deoxyadenosylcobalamin (AdoCbl), a physiological form of the essential micronutrient vitamin B12 as a mixed-type allosteric inhibitor of LRRK2 kinase activity. Multiple assays show that AdoCbl directly binds LRRK2, leading to the alterations of protein conformation and ATP binding in LRRK2. STD-NMR analysis of a LRRK2 homologous kinase reveals the contact sites in AdoCbl that interface with the kinase domain. Furthermore, we provide evidence that AdoCbl modulates LRRK2 activity through disrupting LRRK2 dimerization. Treatment with AdoCbl inhibits LRRK2 kinase activity in cultured cells and brain tissue, and prevents neurotoxicity in cultured primary rodent neurons as well as in transgenic C. elegans and D. melanogaster expressing LRRK2 disease variants. Finally, AdoCbl alleviates deficits in dopamine release sustainability caused by LRRK2 disease variants in mouse models. Our study uncovers vitamin B12 as a novel class of LRRK2 kinase modulator with a distinct mechanism, which can be harnessed to develop new LRRK2-based PD therapeutics in the future.


Assuntos
Cobamidas/farmacologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/tratamento farmacológico , Vitamina B 12/análogos & derivados , Complexo Vitamínico B/farmacologia , Regulação Alostérica , Animais , Caenorhabditis elegans , Modelos Animais de Doenças , Drosophila melanogaster , Reposicionamento de Medicamentos , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ratos
6.
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
7.
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
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