RESUMO
BACKGROUND: Recently, a novel mutation in exon 24 of DNAJC13 gene (p.Asn855Ser, rs387907571) has been reported to cause autosomal dominant Parkinson's disease (PD) in a multi-incident Mennonite family. METHODS: In the present study the mutation containing exon of the DNAJC13 gene has been sequenced in a Caucasian series consisting of 1938 patients with clinical PD and 838 with pathologically diagnosed Lewy body disease (LBD). RESULTS: Our sequence analysis did not identify any coding variants in exon 24 of DNAJC13. Two previously described variants in intron 23 (rs200204728 and rs2369796) were observed. CONCLUSION: Our results indicate that the region surrounding the DNAJC13 p.Asn855Ser substitution is highly conserved and mutations in this exon are not a common cause of PD or LBD among Caucasian populations.
Assuntos
Doença por Corpos de Lewy/genética , Chaperonas Moleculares/genética , Doença de Parkinson/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Europa (Continente) , Éxons , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
Mutations in the LRRK2 gene represent the most common genetic cause of late onset Parkinson's disease. The physiological and pathological roles of LRRK2 are yet to be fully determined but evidence points towards LRRK2 mutations causing a gain in kinase function, impacting on neuronal maintenance, vesicular dynamics and neurotransmitter release. To explore the role of physiological levels of mutant LRRK2, we created knock-in (KI) mice harboring the most common LRRK2 mutation G2019S in their own genome. We have performed comprehensive dopaminergic, behavioral and neuropathological analyses in this model up to 24months of age. We find elevated kinase activity in the brain of both heterozygous and homozygous mice. Although normal at 6months, by 12months of age, basal and pharmacologically induced extracellular release of dopamine is impaired in both heterozygous and homozygous mice, corroborating previous findings in transgenic models over-expressing mutant LRRK2. Via in vivo microdialysis measurement of basal and drug-evoked extracellular release of dopamine and its metabolites, our findings indicate that exocytotic release from the vesicular pool is impaired. Furthermore, profound mitochondrial abnormalities are evident in the striatum of older homozygous G2019S KI mice, which are consistent with mitochondrial fission arrest. We anticipate that this G2019S mouse line will be a useful pre-clinical model for further evaluation of early mechanistic events in LRRK2 pathogenesis and for second-hit approaches to model disease progression.
Assuntos
Encéfalo/enzimologia , Dopamina/metabolismo , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Autofagia/genética , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Neurônios Dopaminérgicos/metabolismo , Feminino , Técnicas de Introdução de Genes , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/ultraestrutura , Atividade Motora/genética , Teste de Desempenho do Rota-Rod , Proteínas tau/metabolismoRESUMO
Parkinson's disease is a neurodegenerative movement disorder that is caused, in part, by the loss of dopaminergic neurons within the substantia nigra pars compacta of the basal ganglia. The presence of intracellular protein aggregates, known as Lewy bodies and Lewy neurites, within the surviving nigral neurons is the defining neuropathological feature of the disease. Accordingly, the identification of specific genes mutated in families with Parkinson's disease and of genetic susceptibility variants for idiopathic Parkinson's disease has implicated abnormalities in proteostasis, or the handling and elimination of misfolded proteins, in the pathogenesis of this neurodegenerative disorder. Protein folding and the refolding of misfolded proteins are regulated by a network of interactive molecules, known as the chaperone system, which is composed of molecular chaperones and co-chaperones. The chaperone system is intimately associated with the ubiquitin-proteasome system and the autophagy-lysosomal pathway which are responsible for elimination of misfolded proteins and protein quality control. In addition to their role in proteostasis, some chaperone molecules are involved in the regulation of cell death pathways. Here we review the role of the molecular chaperones Hsp70 and Hsp90, and the cochaperones Hsp40, BAG family members such as BAG5, CHIP and Hip in modulating neuronal death with a focus on dopaminergic neurodegeneration in Parkinson's disease. We also review current progress in preclinical studies aimed at targetting the chaperone system to prevent neurodegeneration. Finally, we discuss potential future chaperone-based therapeutics for the symptomatic treatment and possible disease modification of Parkinson's disease.
Assuntos
Antiparkinsonianos/farmacologia , Antiparkinsonianos/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Chaperonas Moleculares/efeitos dos fármacos , Degeneração Neural/tratamento farmacológico , Doença de Parkinson/tratamento farmacológico , Animais , Humanos , Degeneração Neural/metabolismoRESUMO
Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) are neurodegenerative disorders that share progressive dementia as the common major clinical symptom. Damages to memory-related brain structures are the likely pathological correlate, and in both illnesses deposition of amyloidogenic proteins are present mainly within these limbic structures. Amyloid-beta-positive plaques and phospho-tau-positive neurofibrillary tangles are the main feature of AD and alpha-synuclein-positive Lewy bodies and Lewy neurites are found in DLB. Interestingly the associated proteins also interfere with synaptic function and synaptic plasticity. Here, we propose that the same neuronal circuits are disturbed within the hippocampal formation in AD and DLB and that in both diseases the associated proteins might lead to changes in synaptic plasticity and function. Thus both classic neuropathological changes and cellular dysfunctions might contribute to the cognitive impairments in AD and DLB.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/patologia , Neuritos/patologia , Plasticidade Neuronal , Transmissão Sináptica , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Doença por Corpos de Lewy/fisiopatologia , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Transtornos da Memória/fisiopatologia , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais/metabolismo , Vias Neurais/patologia , Vias Neurais/fisiopatologia , Sinucleínas , alfa-SinucleínaRESUMO
Alpha-synuclein, a protein in which two mutations have been identified that cause autosomal dominant Parkinson's disease, is thought to serve as a nidus for the development of a Lewy body. We hypothesized that alpha-synuclein would display different intra- and intermolecular associations in Lewy bodies than it does in its normal intracellular compartments. Using sensitive fluorescence resonance energy transfer (FRET) techniques, we found evidence that alpha-synuclein is more compact and in closer association with other alpha-synuclein molecules in Lewy bodies than it is in the neuropil. In addition, we found evidence of a close, direct intermolecular interaction between the N terminus of alpha-synuclein and ubiquitin. These observations provide support for the hypothesis that in Lewy bodies alpha-synuclein adopts an altered three-dimensional structure and undergoes N-terminal ubiquitination.
Assuntos
Corpos de Lewy/patologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/patologia , Ubiquitina/metabolismo , Hipocampo/patologia , Humanos , Corpos de Lewy/química , Neurópilo/patologia , Conformação Proteica , Espectrometria de Fluorescência , Substância Negra/patologia , Sinucleínas , alfa-SinucleínaRESUMO
TorsinA, a novel protein in which a mutation causes dominant, early onset torsion dystonia, may serve as a chaperone for misfolded proteins that require refolding or degradation. It has been hypothesized that misfolded alpha-synuclein, a protein in which two mutations cause autosomal dominantly inherited Parkinson's disease, serves as a nidus for the development of a Lewy body. We hypothesized that torsinA plays a role in the cellular processing of alpha-synuclein. We demonstrate that anti-torsin antibodies stain Lewy bodies and Lewy neurites in the substantia nigra and cortex. Using sensitive fluorescent resonance energy transfer (FRET) techniques, we find evidence of a close association between torsinA and alpha-synuclein in Lewy bodies.
Assuntos
Proteínas de Transporte , Hipocampo/metabolismo , Corpos de Lewy/metabolismo , Chaperonas Moleculares , Proteínas do Tecido Nervoso/metabolismo , Substância Negra/metabolismo , Transferência de Energia , Humanos , Imuno-Histoquímica , Doença por Corpos de Lewy/metabolismo , Microscopia Confocal , Valores de Referência , Espectrometria de Fluorescência , Sinucleínas , alfa-SinucleínaRESUMO
Alpha-synuclein accumulates in the brains of sporadic Parkinson's disease patients as a major component of Lewy bodies, and mutations in alpha-synuclein are associated with familial forms of Parkinson's disease. The pathogenic mechanisms that precede and promote the aggregation of alpha-synuclein into Lewy bodies in neurons remain to be determined. Here, we constructed a series of alpha-synuclein-enhanced green fluorescent protein (alpha-synucleinEGFP, SynEGFP) fusion proteins to address whether the Parkinson's disease-associated mutations alter the subcellular distribution of alpha-synuclein, and to use as a tool for experimental manipulations to induce aggregate formation. When transfected into mouse cultured primary neurons, the 49-kDa alpha-synucleinEGFP fusion proteins are partially truncated to a approximately 27-kDa form. This non-fluorescent carboxy-terminally modified fusion protein spontaneously forms inclusions in the neuronal cytoplasm. A marked increase in the accumulation of inclusions is detected following treatment with each of three proteasome inhibitors, n-acetyl-leu-leu-norleucinal, lactacystin and MG132. Interestingly, Ala30Pro alpha-synucleinEGFP does not form the cytoplasmic inclusions that are characteristic of wild-type and Ala53Thr alpha-synucleinEGFP, supporting the idea that the Ala30Pro alpha-synuclein protein conformation differs from wild-type alpha-synuclein. Similar inclusions are formed if alpha-synuclein carboxy-terminus is modified by the addition of a V5/6xHistidine epitope tag. By contrast, overexpression of unmodified alpha-synuclein does not lead to aggregate formation. Furthermore, synphilin-1, an alpha-synuclein interacting protein also found in Lewy bodies, colocalizes with the carboxy-terminally truncated alpha-synuclein fusion protein in discrete cytoplasmic inclusions.Our finding that manipulations of the carboxy-terminus of alpha-synuclein lead to inclusion formation may provide a model for studies of the pathogenic mechanisms of alpha-synuclein aggregation in Lewy bodies.
Assuntos
Corpos de Inclusão/metabolismo , Proteínas Luminescentes/metabolismo , Complexos Multienzimáticos/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Proteínas de Transporte/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Crioprotetores/farmacologia , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Dimetil Sulfóxido/farmacologia , Feto , Genes Reporter/fisiologia , Proteínas de Fluorescência Verde , Humanos , Imuno-Histoquímica , Corpos de Inclusão/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Leupeptinas/farmacologia , Proteínas Luminescentes/genética , Camundongos , Complexos Multienzimáticos/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Complexo de Endopeptidases do Proteassoma , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes de Fusão/genética , Sinucleínas , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , alfa-SinucleínaRESUMO
alpha-Synuclein is a major component of Lewy bodies, a neuropathological feature of Parkinson's disease. Two alpha-synuclein mutations, Ala53Thr and Ala30Pro, are associated with early onset, familial forms of the disease. Recently, synphilin-1, a protein found to interact with alpha-synuclein by yeast two hybrid techniques, was detected in Lewy bodies. In this study we report the interaction of alpha-synuclein and synphilin-1 in human neuroglioma cells using a sensitive fluorescence resonance energy transfer technique. We demonstrate that the C-terminus of alpha-synuclein is closely associated with the C-terminus of synphilin-1. A weak interaction occurs between the N-terminus of alpha-synuclein and synphilin-1. The familial Parkinson's disease associated mutations of alpha-synuclein (Ala53Thr and Ala30Pro) also demonstrate a strong interaction between their C-terminal regions and synphilin-1. However, compared with wild-type alpha-synuclein, significantly less energy transfer occurs between the C-terminus of Ala53Thr alpha-synuclein and synphilin-1, suggesting that the Ala53Thr mutation alters the conformation of alpha-synuclein in relation to synphilin-1.
Assuntos
Proteínas de Transporte/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/genética , Western Blotting , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Transferência de Energia , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Glioma , Humanos , Imuno-Histoquímica , Microscopia Confocal , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/química , Conformação Proteica , Relação Estrutura-Atividade , Frações Subcelulares/química , Sinucleínas , Transfecção , Células Tumorais Cultivadas , alfa-SinucleínaRESUMO
Numerous recent observations have implicated alpha-synuclein in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies and multiple-system atrophy. Two missense mutations in the gene for alpha-synuclein have been identified in some cases of familial Parkinson's disease and it is thought that these may disrupt the normal structure of the protein and thus promote aggregation into Lewy body filaments. Here, we examine the subcellular localization of alpha-synuclein in primary cortical neurons maintained in a monolayer culture. The protein has widespread expression throughout neurons, including the nucleus, and has a discete localization in the neurites of more mature neurons, reminiscent of synaptic specializations. Interestingly, in a subpopulation of cortical neurons transfected at 13 days in vitro, we find that alpha-synuclein appears to aggregate into distinct punctate inclusions in the cytoplasm and proximal neurites. Unlike Lewy bodies, these structures are not ubiquitin positive. These regions of alpha-synuclein accumulation are observed following transfections with wild-type, Ala30Pro or Ala53Thr alpha-synuclein; neither mutation alters their frequency.
Assuntos
Mutação de Sentido Incorreto/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Frações Subcelulares/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Citoplasma/metabolismo , Embrião de Mamíferos , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Neuritos/metabolismo , Sinucleínas , Distribuição Tecidual , Transfecção , alfa-SinucleínaRESUMO
The ability of nerve cells to regulate the expression of specific neurotransmitter receptors is of central importance to nervous system function, but little is known about the DNA elements that mediate neuron specific gene expression. The type A gamma-aminobutyric acid (GABA(A)) receptor alpha6-subunit gene, which is expressed exclusively in cerebellar granule cells, presents a unique opportunity to study the cis elements involved in restricting gene expression to a distinct neuronal population. In an effort to identify the regulatory elements that govern cerebellar granule cell-specific gene expression, the proximal 5' flanking regions for the human, rat, and mouse alpha6 genes were cloned and sequenced, and a major transcriptional initiation site was identified in the rodent genes. Functional analysis of rat alpha6 gene-reporter constructs in primary neuronal cultures reveals that a 155-bp TATA-less promoter region (-130 to +25 bp) constitutes a minimal promoter that can drive cerebellar granule cell-specific expression. Internal deletion and decoy competition studies demonstrate that the minimal promoter contains a 60-bp region (-130 to -70 bp) that is critical for enhanced promoter activity in cerebellar granule cells. Activity of the compromised promoter containing the deletion cannot be rescued by placing the 60-bp region downstream of the reporter gene, demonstrating that it is not a classical enhancer but rather a positionally dependent regulator. An additional cerebellar-specific activating sequence is located between -324 and -130 bp, and a downstream negative regulatory region (+158 to +294) has been shown to be active in fibroblasts but inactive in cerebellar granule cells. Taken together, the results suggest a possible mechanism for the control of cerebellar granule cell-specific expression of the GABA(A) receptor alpha6 subunit gene.
Assuntos
Regiões Promotoras Genéticas/fisiologia , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Animais , Sequência de Bases/genética , Células Cultivadas , Cerebelo/citologia , Cerebelo/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Neurônios/fisiologia , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Especificidade por Substrato , Transcrição Gênica/fisiologiaRESUMO
Two missense mutations (Ala-30 --> Pro and Ala-53 --> Thr) in the gene encoding alpha-synuclein are associated with rare autosomal dominant forms of familial Parkinson's disease. In addition, alpha-synuclein is an abundant component of Lewy bodies in sporadic Parkinson's disease and diffuse Lewy body disease. However, the normal conformation of alpha-synuclein, its cellular localization in neurons, and the effects of the mutations remain to be determined. In the present study, we examine these questions using sensitive fluorescence resonance energy transfer techniques. Transient transfection of alpha-synuclein expression constructs into primary cortical neurons and counterstaining with the lipophilic fluorescent marker, DiI, demonstrates a close association between alpha-synuclein and cellular membranes. Both the N- and C-terminal regions of alpha-synuclein are tightly associated with membranes. A weak interaction also occurs between the N and C termini themselves. The Parkinson's disease-associated mutations have no effect on membrane interaction; however, the Ala-30 --> Pro mutation alters the three-dimensional conformation of alpha-synuclein, as measured by significantly increased fluorescence resonance energy transfer between the N and C termini.
Assuntos
Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Doença de Parkinson/genética , Animais , Células Cultivadas , Córtex Cerebral/citologia , Ligação Genética , Hipocampo/citologia , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Conformação Proteica , Sinucleínas , alfa-SinucleínaRESUMO
We demonstrated previously that an alpha 1-beta 2-gamma 2 gene cluster of the gamma-aminobutyric acid (GABAA) receptor is located on human chromosome 5q34-q35 and that an ancestral alpha-beta-gamma gene cluster probably spawned clusters on chromosomes 4, 5, and 15. Here, we report that the alpha 4 gene (GABRA4) maps to human chromosome 4p14-q12, defining a cluster comprising the alpha 2, alpha 4, beta 1, and gamma 1 genes. The existence of an alpha 2-alpha 4-beta 1-gamma 1 cluster on chromosome 4 and an alpha 1-alpha 6-beta 2-gamma 2 cluster on chromosome 5 provides further evidence that the number of ancestral GABAA receptor subunit genes has been expanded by duplication within an ancestral gene cluster. Moreover, if duplication of the alpha gene occurred before duplication of the ancestral gene cluster, then a heretofore undiscovered subtype of alpha subunit should be located on human chromosome 15q11-q13 within an alpha 5-alpha x-beta 3-gamma 3 gene cluster at the locus for Angelman and Prader-Willi syndromes.