RESUMO
BACKGROUND: Mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene cause late-onset, autosomal dominant Parkinson's disease (PD), with a single missense mutation (Asp620Asn, D620N) known to segregate with disease in families with PD. The VPS35 gene encodes a core component of the retromer complex, involved in the endosomal sorting and recycling of transmembrane cargo proteins. VPS35-linked PD is clinically indistinguishable from sporadic PD, although it is not yet known whether VPS35-PD brains exhibit α-synuclein-positive brainstem Lewy pathology that is characteristic of sporadic cases. Prior studies have suggested a functional interaction between VPS35 and the PD-linked gene product α-synuclein in lower organisms, where VPS35 deletion enhances α-synuclein-induced toxicity. In mice, VPS35 overexpression is reported to rescue hippocampal neuronal loss in human α-synuclein transgenic mice, potentially suggesting a retromer deficiency in these mice. METHODS: Here, we employ multiple well-established genetic rodent models to explore a functional or pathological interaction between VPS35 and α-synuclein in vivo. RESULTS: We find that endogenous α-synuclein is dispensable for nigrostriatal pathway dopaminergic neurodegeneration induced by the viral-mediated delivery of human D620N VPS35 in mice, suggesting that α-synuclein does not operate downstream of VPS35. We next evaluated retromer levels in affected brain regions from human A53T-α-synuclein transgenic mice, but find normal levels of the core subunits VPS35, VPS26 or VPS29. We further find that heterozygous VPS35 deletion fails to alter the lethal neurodegenerative phenotype of these A53T-α-synuclein transgenic mice, suggesting the absence of retromer deficiency in this PD model. Finally, we explored the neuroprotective capacity of increasing VPS35 expression in a viral-based human wild-type α-synuclein rat model of PD. However, we find that the overexpression of wild-type VPS35 is not sufficient for protection against α-synuclein-induced nigral dopaminergic neurodegeneration, α-synuclein pathology and reactive gliosis. CONCLUSION: Collectively, our data suggest a limited interaction of VPS35 and α-synuclein in neurodegenerative models of PD, and do not provide support for their interaction within a common pathophysiological pathway.
Assuntos
Doença de Parkinson , Animais , Humanos , Camundongos , Ratos , alfa-Sinucleína/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Transgênicos , Doença de Parkinson/metabolismo , Transporte Proteico , Roedores/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
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
Mutations in a number of genes cause familial forms of Parkinson's disease (PD), including mutations in the vacuolar protein sorting 35 ortholog (VPS35) and parkin genes. In this study, we identify a novel functional interaction between parkin and VPS35. We demonstrate that parkin interacts with and robustly ubiquitinates VPS35 in human neural cells. Familial parkin mutations are impaired in their ability to ubiquitinate VPS35. Parkin mediates the attachment of an atypical poly-ubiquitin chain to VPS35 with three lysine residues identified within the C-terminal region of VPS35 that are covalently modified by ubiquitin. Notably, parkin-mediated VPS35 ubiquitination does not promote the proteasomal degradation of VPS35. Furthermore, parkin does not influence the steady-state levels or turnover of VPS35 in neural cells and VPS35 levels are normal in the brains of parkin knockout mice. These data suggest that ubiquitination of VPS35 by parkin may instead serve a non-degradative cellular function potentially by regulating retromer-dependent sorting. Accordingly, we find that components of the retromer-associated WASH complex are markedly decreased in the brain of parkin knockout mice, suggesting that parkin may modulate WASH complex-dependent retromer sorting. Parkin gene silencing in primary cortical neurons selectively disrupts the vesicular sorting of the autophagy receptor ATG9A, a WASH-dependent retromer cargo. Parkin is not required for dopaminergic neurodegeneration induced by the expression of PD-linked D620N VPS35 in mice, consistent with VPS35 being located downstream of parkin function. Our data reveal a novel functional interaction of parkin with VPS35 that may be important for retromer-mediated endosomal sorting and PD.
Assuntos
Proteínas Relacionadas à Autofagia/genética , Proteínas de Membrana/genética , Degeneração Neural/genética , Doença de Parkinson/genética , Ubiquitina-Proteína Ligases/genética , Proteínas de Transporte Vesicular/genética , Animais , Autofagia/genética , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Endossomos/genética , Humanos , Camundongos , Camundongos Knockout , Degeneração Neural/patologia , Doença de Parkinson/patologia , Poliubiquitina/genética , Transporte Proteico/genéticaRESUMO
Parkinson's disease (PD) is defined by the loss of dopaminergic neurons in the substantia nigra and the formation of Lewy body inclusions containing aggregated α-synuclein. Efforts to explain dopamine neuron vulnerability are hindered by the lack of dopaminergic cell death in α-synuclein transgenic mice. To address this, we manipulated both dopamine levels and α-synuclein expression. Nigrally targeted expression of mutant tyrosine hydroxylase with enhanced catalytic activity increased dopamine levels without damaging neurons in non-transgenic mice. In contrast, raising dopamine levels in mice expressing human A53T mutant α-synuclein induced progressive nigrostriatal degeneration and reduced locomotion. Dopamine elevation in A53T mice increased levels of potentially toxic α-synuclein oligomers, resulting in conformationally and functionally modified species. Moreover, in genetically tractable Caenorhabditis elegans models, expression of α-synuclein mutated at the site of interaction with dopamine prevented dopamine-induced toxicity. These data suggest that a unique mechanism links two cardinal features of PD: dopaminergic cell death and α-synuclein aggregation.
Assuntos
Corpo Estriado/metabolismo , Dopamina/biossíntese , Neurônios Dopaminérgicos/metabolismo , Doenças Neurodegenerativas/metabolismo , Substância Negra/metabolismo , alfa-Sinucleína/biossíntese , Animais , Caenorhabditis elegans , Células Cultivadas , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Feminino , Humanos , Levodopa/farmacologia , Levodopa/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Substância Negra/efeitos dos fármacos , Substância Negra/patologiaRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). LRRK2 contains functional GTPase and kinase domains. The most common G2019S mutation enhances the kinase activity of LRRK2 in vitro whereas G2019S LRRK2 expression in cultured neurons induces toxicity in a kinase-dependent manner. These observations suggest a potential role for kinase activity in LRRK2-associated PD. We have recently developed a novel rodent model of PD with progressive neurodegeneration induced by the adenoviral-mediated expression of G2019S LRRK2. In the present study, we further characterize this LRRK2 model and determine the contribution of kinase activity to LRRK2-mediated neurodegeneration. Recombinant human adenoviral vectors were employed to deliver human wild-type, G2019S or kinase-inactive G2019S/D1994N LRRK2 to the rat striatum. LRRK2-dependent pathology was assessed in the striatum, a region where LRRK2 protein is normally enriched in the mammalian brain. Human LRRK2 variants are robustly expressed throughout the rat striatum. Expression of G2019S LRRK2 selectively induces the accumulation of neuronal ubiquitin-positive inclusions accompanied by neurite degeneration and the altered distribution of axonal phosphorylated neurofilaments. Importantly, the introduction of a kinase-inactive mutation (G2019S/D1994N) completely ameliorates the pathological effects of G2019S LRRK2 in the striatum supporting a kinase activity-dependent mechanism for this PD-associated mutation. Collectively, our study further elucidates the pathological effects of the G2019S mutation in the mammalian brain and supports the development of kinase inhibitors as a potential therapeutic approach for treating LRRK2-associated PD. This adenoviral rodent model provides an important tool for elucidating the molecular basis of LRRK2-mediated neurodegeneration.
Assuntos
Adenoviridae/fisiologia , Corpo Estriado/patologia , Mutação/genética , Doença de Parkinson/genética , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/genética , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Feminino , Membro Anterior/fisiopatologia , Regulação da Expressão Gênica/genética , Glicina/genética , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson/fisiopatologia , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos Wistar , Serina/genética , Fatores de Tempo , Transdução Genética , Tubulina (Proteína)/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Mutations in the ATP13A2 (PARK9) gene cause early-onset, autosomal recessive Parkinson's disease (PD) and Kufor-Rakeb syndrome. ATP13A2 mRNA is spliced into three distinct isoforms encoding a P5-type ATPase involved in regulating heavy metal transport across vesicular membranes. Here, we demonstrate that three ATP13A2 mRNA isoforms are expressed in the normal human brain and are modestly increased in the cingulate cortex of PD cases. ATP13A2 can mediate protection toward a number of stressors in mammalian cells and can protect against α-synuclein-induced toxicity in cellular and invertebrate models of PD. Using a primary cortical neuronal model combined with lentiviral-mediated gene transfer, we demonstrate that human ATP13A2 isoforms 1 and 2 display selective neuroprotective effects toward toxicity induced by manganese and hydrogen peroxide exposure through an ATPase-independent mechanism. The familial PD mutations, F182L and G504R, abolish the neuroprotective effects of ATP13A2 consistent with a loss-of-function mechanism. We further demonstrate that the AAV-mediated overexpression of human ATP13A2 is not sufficient to attenuate dopaminergic neurodegeneration, neuropathology, and striatal dopamine and motoric deficits induced by human α-synuclein expression in a rat model of PD. Intriguingly, the delivery of an ATPase-deficient form of ATP13A2 (D513N) to the substantia nigra is sufficient to induce dopaminergic neuronal degeneration and motor deficits in rats, potentially suggesting a dominant-negative mechanism of action. Collectively, our data demonstrate a distinct lack of ATP13A2-mediated protection against α-synuclein-induced neurotoxicity in the rat nigrostriatal dopaminergic pathway, and limited neuroprotective capacity overall, and raise doubts about the potential of ATP13A2 as a therapeutic target for PD.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Peróxido de Hidrogênio/toxicidade , Manganês/toxicidade , Fármacos Neuroprotetores/farmacologia , Transtornos Parkinsonianos/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Bancos de Tecidos , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/genética , Gravidez , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). The clinical and neurochemical features of LRRK2-linked PD are similar to idiopathic disease although neuropathology is somewhat heterogeneous. Dominant mutations in LRRK2 precipitate neurodegeneration through a toxic gain-of-function mechanism which can be modeled in transgenic mice overexpressing human LRRK2 variants. A number of LRRK2 transgenic mouse models have been developed that display abnormalities in dopaminergic neurotransmission and alterations in tau metabolism yet without consistently inducing dopaminergic neurodegeneration. To directly explore the impact of mutant LRRK2 on the nigrostriatal dopaminergic pathway, we developed conditional transgenic mice that selectively express human R1441C LRRK2 in dopaminergic neurons from the endogenous murine ROSA26 promoter. The expression of R1441C LRRK2 does not induce the degeneration of substantia nigra dopaminergic neurons or striatal dopamine deficits in mice up to 2years of age, and fails to precipitate abnormal protein inclusions containing alpha-synuclein, tau, ubiquitin or autophagy markers (LC3 and p62). Furthermore, mice expressing R1441C LRRK2 exhibit normal motor activity and olfactory function with increasing age. Intriguingly, the expression of R1441C LRRK2 induces age-dependent abnormalities of the nuclear envelope in nigral dopaminergic neurons including reduced nuclear circularity and increased invaginations of the nuclear envelope. In addition, R1441C LRRK2 mice display increased neurite complexity of cultured midbrain dopaminergic neurons. Collectively, these novel R1441C LRRK2 conditional transgenic mice reveal altered dopaminergic neuronal morphology with advancing age, and provide a useful tool for exploring the pathogenic mechanisms underlying the R1441C LRRK2 mutation in PD.
Assuntos
Nucléolo Celular/patologia , Neurônios Dopaminérgicos/ultraestrutura , Mesencéfalo/citologia , Mutação/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Arginina/genética , Células Cultivadas , Cisteína/genética , Neurônios Dopaminérgicos/patologia , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Camundongos TransgênicosRESUMO
Mutations in the vacuolar protein sorting 35 homolog (VPS35) gene at the PARK17 locus, encoding a key component of the retromer complex, were recently identified as a new cause of late-onset, autosomal dominant Parkinson's disease (PD). Here we explore the pathogenic consequences of PD-associated mutations in VPS35 using a number of model systems. VPS35 exhibits a broad neuronal distribution throughout the rodent brain, including within the nigrostriatal dopaminergic pathway. In the human brain, VPS35 protein levels and distribution are similar in tissues from control and PD subjects, and VPS35 is not associated with Lewy body pathology. The common D620N missense mutation in VPS35 does not compromise its protein stability or localization to endosomal and lysosomal vesicles, or the vesicular sorting of the retromer cargo, sortilin, SorLA and cation-independent mannose 6-phosphate receptor, in rodent primary neurons or patient-derived human fibroblasts. In yeast we show that PD-linked VPS35 mutations are functional and can normally complement VPS35 null phenotypes suggesting that they do not result in a loss-of-function. In rat primary cortical cultures the overexpression of human VPS35 induces neuronal cell death and increases neuronal vulnerability to PD-relevant cellular stress. In a novel viral-mediated gene transfer rat model, the expression of D620N VPS35 induces the marked degeneration of substantia nigra dopaminergic neurons and axonal pathology, a cardinal pathological hallmark of PD. Collectively, these studies establish that dominant VPS35 mutations lead to neurodegeneration in PD consistent with a gain-of-function mechanism, and support a key role for VPS35 in the development of PD.
Assuntos
Neurônios Dopaminérgicos/patologia , Mutação/genética , Degeneração Neural/genética , Degeneração Neural/patologia , Doença de Parkinson/genética , Proteínas de Transporte Vesicular/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular , Células Cultivadas , Dependovirus/metabolismo , Neurônios Dopaminérgicos/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Doença de Parkinson/patologia , Estabilidade Proteica , Transporte Proteico , Ratos Sprague-Dawley , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Vesículas Transportadoras/metabolismoRESUMO
Mutations in LRRK2 cause autosomal dominant Parkinson's disease (PD). LRRK2 encodes a multi-domain protein containing GTPase and kinase domains, and putative protein-protein interaction domains. Familial PD mutations alter the GTPase and kinase activity of LRRK2 in vitro. LRRK2 is suggested to regulate a number of cellular pathways although the underlying mechanisms are poorly understood. To explore such mechanisms, it has proved informative to identify LRRK2-interacting proteins, some of which serve as LRRK2 kinase substrates. Here, we identify common interactions of LRRK2 with members of the dynamin GTPase superfamily. LRRK2 interacts with dynamin 1-3 that mediate membrane scission in clathrin-mediated endocytosis and with dynamin-related proteins that mediate mitochondrial fission (Drp1) and fusion (mitofusins and OPA1). LRRK2 partially co-localizes with endosomal dynamin-1 or with mitofusins and OPA1 at mitochondrial membranes. The subcellular distribution and oligomeric complexes of dynamin GTPases are not altered by modulating LRRK2 in mouse brain, whereas mature OPA1 levels are reduced in G2019S PD brains. LRRK2 enhances mitofusin-1 GTP binding, whereas dynamin-1 and OPA1 serve as modest substrates of LRRK2-mediated phosphorylation in vitro. While dynamin GTPase orthologs are not required for LRRK2-induced toxicity in yeast, LRRK2 functionally interacts with dynamin-1 and mitofusin-1 in cultured neurons. LRRK2 attenuates neurite shortening induced by dynamin-1 by reducing its levels, whereas LRRK2 rescues impaired neurite outgrowth induced by mitofusin-1 potentially by reversing excessive mitochondrial fusion. Our study elucidates novel functional interactions of LRRK2 with dynamin-superfamily GTPases that implicate LRRK2 in the regulation of membrane dynamics important for endocytosis and mitochondrial morphology.
Assuntos
Dinamina III/metabolismo , Dinamina II/metabolismo , Dinamina I/metabolismo , Neuroblastoma/metabolismo , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Western Blotting , Estudos de Casos e Controles , Células Cultivadas , Endocitose , Feminino , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Camundongos Knockout , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Mutação , Neuritos/metabolismo , Neuroblastoma/patologia , Neurônios/citologia , Neurônios/metabolismo , Doença de Parkinson/patologia , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Ratos , Ratos Sprague-Dawley , Saccharomyces cerevisiae , Técnicas do Sistema de Duplo-HíbridoRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2, PARK8, OMIM 607060) gene represent the most common known cause of hereditary Parkinson's disease (PD) with late-onset and dominant inheritance. LRRK2 protein is composed of multiple domains including two distinct enzymatic domains, a kinase and a Ras-of-complex (Roc) GTPase, connected by a C-terminal-of-Roc (COR) domain, and belongs to the ROCO protein family. Disease-causing mutations located in the kinase domain enhance kinase activity (i.e., G2019S) whereas mutations clustering within the Roc-COR tandem domain impair GTPase activity (i.e., R1441C/G and Y1699C). Familial LRRK2 mutations commonly induce neuronal toxicity that, at least for the frequent G2019S variant, is dependent on kinase activity. The contribution of GTPase activity to LRRK2-dependent neuronal toxicity is not yet clear. Therefore, both GTPase and kinase activity may be important for mediating neurodegeneration in PD due to familial LRRK2 mutations. At present, the physiological function of LRRK2 in the mammalian brain and the regulation of its enzymatic activity are incompletely understood. In this review, we focus on the GTPase domain of LRRK2 and discuss the recent advances in elucidating its function and its interplay with the kinase domain for the regulation of LRRK2 activity and toxicity. GTPase activity is an important feature of LRRK2 biology and pathophysiology and represents an underexplored yet potentially tractable therapeutic target for treating LRRK2-associated PD.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/genética , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Polimorfismo de Nucleotídeo Único , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de ProteínaRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset Parkinson's disease (PD). Emerging evidence suggests a role for LRRK2 in the endocytic pathway. Here, we show that LRRK2 is released in extracellular microvesicles (i.e. exosomes) from cells that natively express LRRK2. LRRK2 localizes to collecting duct epithelial cells in the kidney that actively secrete exosomes into urine. Purified urinary exosomes contain LRRK2 protein that is both dimerized and phosphorylated. We provide a quantitative proteomic profile of 1673 proteins in urinary exosomes and find that known LRRK2 interactors including 14-3-3 are some of the most abundant exosome proteins. Disruption of the 14-3-3 LRRK2 interaction with a 14-3-3 inhibitor or through acute LRRK2 kinase inhibition potently blocks LRRK2 release in exosomes, but familial mutations in LRRK2 had no effect on secretion. LRRK2 levels were overall comparable but highly variable in urinary exosomes derived from PD cases and age-matched controls, although very high LRRK2 levels were detected in some PD affected cases. We further characterized LRRK2 exosome release in neurons and macrophages in culture, and found that LRRK2-positive exosomes circulate in cerebral spinal fluid (CSF). Together, these results define a pathway for LRRK2 extracellular release, clarify one function of the LRRK2 14-3-3 interaction and provide a foundation for utilization of LRRK2 as a biomarker in clinical trials.
Assuntos
Proteínas 14-3-3/metabolismo , Exossomos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células Epiteliais/metabolismo , Humanos , Túbulos Renais Coletores/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Mutação , Neurônios/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/líquido cefalorraquidiano , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Ratos , Ratos TransgênicosRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of autosomal dominant familial Parkinson's disease (PD). LRRK2 encodes a multi-domain protein containing GTPase and kinase enzymatic domains. Disease-associated mutations in LRRK2 variably influence enzymatic activity with the common G2019S variant leading to enhanced kinase activity. Mutant LRRK2 induces neuronal toxicity through a kinase-dependent mechanism suggesting that kinase activity is important for mediating the pathogenic effects of LRRK2 mutations. A number of LRRK2 kinase substrates have been identified in vitro but whether they represent authentic physiological substrates in mammalian cells or tissues is not yet clear. The eukaryotic initiation factor 4E (eIF4E)-binding protein, 4E-BP1, was recently identified as a potential substrate of LRRK2 kinase activity in vitro and in Drosophila with phosphorylation occurring at Thr37 and Thr46. Here, we explore a potential interaction of LRRK2 and 4E-BP1 in mammalian cells and brain. We find that LRRK2 can weakly phosphorylate 4E-BP1 in vitro but LRRK2 overexpression is not able to alter endogenous 4E-BP1 phosphorylation in mammalian cells. In mammalian neurons LRRK2 and 4E-BP1 display minimal co-localization, whereas the subcellular distribution, protein complex formation and covalent post-translational modification of endogenous 4E-BP1 are not altered in the brains of LRRK2 knockout or mutant LRRK2 transgenic mice. In the brain, the phosphorylation of 4E-BP1 at Thr37 and Thr46 does not change in LRRK2 knockout or mutant LRRK2 transgenic mice, nor is 4E-BP1 phosphorylation altered in idiopathic or G2019S mutant PD brains. Collectively, our results suggest that 4E-BP1 is neither a major nor robust physiological substrate of LRRK2 in mammalian cells or brain.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Mamíferos/metabolismo , Mutação/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/patologia , Proteínas de Ciclo Celular , Fatores de Iniciação em Eucariotos , Feminino , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Camundongos , Complexos Multiproteicos/metabolismo , Doença de Parkinson/enzimologia , Doença de Parkinson/patologia , Fosforilação , Fosfotreonina/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Frações Subcelulares/metabolismoRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene represent the most common cause of familial Parkinson's disease (PD), whereas common variation at the LRRK2 locus is associated with an increased risk of idiopathic PD. Considerable progress has been made toward understanding the biological functions of LRRK2 and the molecular mechanisms underlying the pathogenic effects of disease-associated mutations. The development of neuronal culture models and transgenic or viral-based rodent models have proved useful for identifying a number of emerging pathways implicated in LRRK2-dependent neuronal damage, including the microtubule network, actin cytoskeleton, autophagy, mitochondria, vesicular trafficking, and protein quality control. However, many important questions remain to be posed and answered. Elucidating the molecular mechanisms and pathways underlying LRRK2-mediated neurodegeneration is critical for the identification of new molecular targets for therapeutic intervention in PD. In this review we discuss recent advances and unanswered questions in understanding the pathophysiology of LRRK2.
Assuntos
Predisposição Genética para Doença , Mutação/genética , Doenças Neurodegenerativas/genética , Proteínas Serina-Treonina Quinases/genética , Autofagia/genética , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mitocôndrias/genética , Mitocôndrias/patologia , Modelos Moleculares , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797-17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes.
Assuntos
Encéfalo/metabolismo , Eritrócitos/metabolismo , Proteínas Recombinantes/metabolismo , alfa-Sinucleína/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Sistema Nervoso Central/metabolismo , Cromatografia em Gel , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Conformação Proteica , Estrutura Secundária de Proteína , Desdobramento de Proteína , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/química , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMO
UNLABELLED: Mitochondria are highly dynamic, multifunctional organelles. Aside from their major role in energy metabolism, they are also crucial for many cellular processes including neurotransmission, synaptic maintenance, calcium homeostasis, cell death, and neuronal survival. SIGNIFICANCE: Increasing evidence supports a role for abnormal mitochondrial function in the molecular pathophysiology of Parkinson's disease (PD). For three decades we have known that mitochondrial toxins are capable of producing clinical parkinsonism in humans. PD is the most common neurodegenerative movement disorder that is characterized by the progressive loss of substantia nigra dopaminergic neurons leading to a deficiency of striatal dopamine. Although the neuropathology underlying the disease is well defined, it remains unclear why nigral dopaminergic neurons degenerate and die. RECENT ADVANCES: Most PD cases are idiopathic, but there are rare familial cases. Mutations in five genes are known to unambiguously cause monogenic familial PD: α-synuclein, parkin, DJ-1, PTEN-induced kinase 1 (PINK1), and leucine-rich repeat kinase 2 (LRRK2). These key molecular players are proteins of seemingly diverse function, but with potentially important roles in mitochondrial maintenance and function. Cell and animal-based genetic models have provided indispensable tools for understanding the molecular basis of PD, and have provided additional evidence implicating mitochondrial dysfunction as a primary pathogenic pathway leading to the demise of dopaminergic neurons in PD. CRITICAL ISSUES: Here, we critically discuss the evidence for mitochondrial dysfunction in genetic animal models of PD, and evaluate whether abnormal mitochondrial function represents a cause or consequence of disease pathogenesis. FUTURE DIRECTIONS: Mitochondria may represent a potential target for the development of disease-modifying therapies.
Assuntos
Mitocôndrias/genética , Mitocôndrias/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Animais , Modelos Animais de Doenças , Genes Dominantes , Genes Recessivos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/patologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
The Pdss2 gene product is needed for the isoprenylation of benzoquinone to generate coenzyme Q (CoQ). A fatal kidney disease occurs in mice that are homozygous for a missense mutation in Pdss2, which can be recapitulated in conditional Pdss2 knockouts targeted to glomerular podocytes. We now report that homozygous missense mutants also demonstrate significant neuromuscular deficits, as validated by behavioral and coordination assays, and these deficits are recapitulated in conditional Pdss2 knockouts targeted to dopaminergic neurons. Both conditional knockout and missense mutant mice demonstrate deficiencies in tyrosine hydroxylase-positive neurons in the substantia nigra, implicating a pathology similar to sporadic Parkinson's disease (PD).
Assuntos
Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Doenças Neuromusculares/patologia , Animais , Técnicas de Inativação de Genes , Homozigoto , Masculino , Camundongos , Doenças Mitocondriais/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Doenças Neuromusculares/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Mutations in the ATP13A2 gene (PARK9, OMIM 610513) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome and early-onset parkinsonism. ATP13A2 is an uncharacterized protein belonging to the P(5)-type ATPase subfamily that is predicted to regulate the membrane transport of cations. The physiological function of ATP13A2 in the mammalian brain is poorly understood. Here, we demonstrate that ATP13A2 is localized to intracellular acidic vesicular compartments in cultured neurons. In the human brain, ATP13A2 is localized to pyramidal neurons within the cerebral cortex and dopaminergic neurons of the substantia nigra. ATP13A2 protein levels are increased in nigral dopaminergic and cortical pyramidal neurons of Parkinson's disease brains compared with normal control brains. ATP13A2 levels are increased in cortical neurons bearing Lewy bodies (LBs) compared with neurons without LBs. Using short hairpin RNA-mediated silencing or overexpression to explore the function of ATP13A2, we find that modulating the expression of ATP13A2 reduces the neurite outgrowth of cultured midbrain dopaminergic neurons. We also find that silencing of ATP13A2 expression in cortical neurons alters the kinetics of intracellular pH in response to cadmium exposure. Furthermore, modulation of ATP13A2 expression leads to reduced intracellular calcium levels in cortical neurons. Finally, we demonstrate that silencing of ATP13A2 expression induces mitochondrial fragmentation in neurons. Oppositely, overexpression of ATP13A2 delays cadmium-induced mitochondrial fragmentation in neurons consistent with a neuroprotective effect. Collectively, this study reveals a number of intriguing neuronal phenotypes due to the loss- or gain-of-function of ATP13A2 that support a role for this protein in regulating intracellular cation homeostasis and neuronal integrity.
Assuntos
Adenosina Trifosfatases/metabolismo , Vesículas Citoplasmáticas/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/fisiologia , Doença de Parkinson/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Adenosina Trifosfatases/imunologia , Animais , Autofagia , Encéfalo/metabolismo , Encéfalo/patologia , Cálcio/metabolismo , Células Cultivadas , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Corpos de Lewy/ultraestrutura , Proteínas de Membrana/imunologia , Camundongos , Mitocôndrias/ultraestrutura , Neuritos/fisiologia , Neuritos/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Doença de Parkinson/patologia , ATPases Translocadoras de Prótons/imunologia , Células Piramidais/metabolismo , Interferência de RNA , Ratos , Substância Negra/metabolismo , Substância Negra/patologiaRESUMO
Aggregation of alpha-synuclein (alpha-syn), a process that generates oligomeric intermediates, is a common pathological feature of several neurodegenerative disorders. Despite the potential importance of the oligomeric alpha-syn intermediates in neuron function, their biochemical properties and pathobiological functions in vivo remain vastly unknown. Here we used two-dimensional analytical separation and an array of biochemical and cell-based assays to characterize alpha-syn oligomers that are present in the nervous system of A53T alpha-syn transgenic mice. The most prominent species identified were 53 A detergent-soluble oligomers, which preceded neurological symptom onset, and were found at equivalent amounts in regions containing alpha-syn inclusions as well as histologically unaffected regions. These oligomers were resistant to SDS, heat, and urea but were sensitive to proteinase-K digestion. Although the oligomers shared similar basic biochemical properties, those obtained from inclusion-bearing regions were prominently reactive to antibodies that recognize oxidized alpha-syn oligomers, significantly accelerated aggregation of alpha-syn in vitro, and caused primary cortical neuron degeneration. In contrast, oligomers obtained from non-inclusion-bearing regions were not toxic and delayed the in vitro formation of alpha-syn fibrils. These data indicate that specific conformations of alpha-syn oligomers are present in distinct brain regions of A53T alpha-syn transgenic mice. The contribution of these oligomers to the development of neuron dysfunction appears to be independent of their absolute quantities and basic biochemical properties but is dictated by the composition and conformation of the intermediates as well as unrecognized brain-region-specific intrinsic factors.
Assuntos
Encéfalo/metabolismo , Corpos de Lewy/metabolismo , Doenças Neurodegenerativas/metabolismo , Fragmentos de Peptídeos/metabolismo , alfa-Sinucleína/metabolismo , Amiloide/genética , Amiloide/metabolismo , Animais , Anticorpos/farmacologia , Especificidade de Anticorpos/fisiologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Células Cultivadas , Feminino , Humanos , Corpos de Lewy/patologia , Doença por Corpos de Lewy/genética , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peso Molecular , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/fisiopatologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/toxicidade , Polímeros/metabolismo , Proteínas PrPC/genética , Regiões Promotoras Genéticas/genética , Conformação Proteica , Solubilidade , alfa-Sinucleína/genética , alfa-Sinucleína/toxicidadeRESUMO
Parkinson's disease (PD) is the most common neurodegenerative movement disorder. A pathological hallmark of PD is the presence of intraneuronal inclusions composed of fibrillized alpha-synuclein (alpha-syn) in affected brain regions. Mutations in the gene, PARK7, which encodes DJ-1, can cause autosomal recessive early-onset PD. Although DJ-1 has been shown to be involved in diverse biological processes, several in vitro studies suggest that it can inhibit the formation and protect against the effects of alpha-syn aggregation. We previously established and characterized transgenic mice expressing pathogenic Ala53Thr human alpha-syn (M83 mice) that develop extensive alpha-syn pathologies in the neuroaxis resulting in severe motor impairments and eventual fatality. In the current study, we have crossbred M83 mice on a DJ-1 null background (M83-DJnull mice) in efforts to determine the effects of the lack of DJ-1 in these mice. Animals were assessed and compared for survival rate, distribution of alpha-syn inclusions, biochemical properties of alpha-syn protein, demise and function of nigral dopaminergic neurons, and extent of gliosis in the neuroaxis. M83 and M83-DJnull mice displayed a similar onset of disease and pathological changes, and none of the analyses to assess for changes in pathogenesis revealed any significant differences between M83 and M83-DJnull mice. These findings suggest that DJ-1 may not function to directly modulate alpha-syn nor does DJ-1 appear to play a role in protecting against the deleterious effects of expressing pathogenic Ala53Thr alpha-syn in vivo. It is possible that alpha-syn and DJ-1 mutations may lead to PD via independent mechanisms.
Assuntos
Mutação de Sentido Incorreto , Proteínas Oncogênicas/deficiência , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/toxicidade , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Atividade Motora , Proteínas Oncogênicas/genética , Doença de Parkinson/genética , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Peroxirredoxinas , Proteína Desglicase DJ-1 , alfa-Sinucleína/metabolismoRESUMO
While numerous hypotheses have been proposed to explain the molecular mechanisms underlying the pathogenesis of neurodegenerative diseases, the theory of oxidative stress has received considerable support. Although many correlations have been established and encouraging evidence has been obtained, conclusive proof of causation for the oxidative stress hypothesis is lacking and potential cures have not emerged. Therefore it is likely that other factors, possibly in coordination with oxidative stress, contribute to neuron death. Using Parkinson's disease (PD) as the paradigm, this review explores the hypothesis that oxidative modifications, mitochondrial functional disruption, and impairment of protein degradation constitute three interrelated molecular pathways that execute neuron death. These intertwined events are the consequence of environmental exposure, genetic factors, and endogenous risks and constitute a "Bermuda triangle" that may be considered the underlying cause of neurodegenerative pathogenesis.