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2.
Cell Rep ; 17(5): 1369-1382, 2016 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-27783950

RESUMO

The polycomb repressive complex 2 (PRC2) methylates lysine 27 of histone H3 (H3K27) through its catalytic subunit Ezh2. PRC2-mediated di- and tri-methylation (H3K27me2/H3K27me3) have been interchangeably associated with gene repression. However, it remains unclear whether these two degrees of H3K27 methylation have different functions. In this study, we have generated isogenic mouse embryonic stem cells (ESCs) with a modified H3K27me2/H3K27me3 ratio. Our findings document dynamic developmental control in the genomic distribution of H3K27me2 and H3K27me3 at regulatory regions in ESCs. They also reveal that modifying the ratio of H3K27me2 and H3K27me3 is sufficient for the acquisition and repression of defined cell lineage transcriptional programs and phenotypes and influences induction of the ESC ground state.


Assuntos
Linhagem da Célula , Histonas/metabolismo , Lisina/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Diferenciação Celular/genética , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação da Expressão Gênica , Genoma , Metilação , Camundongos , Neurônios/citologia , Edição de RNA , Sequências Reguladoras de Ácido Nucleico/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Transcrição Gênica
3.
Methods Enzymol ; 547: 75-96, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25416353

RESUMO

Mitochondria are cellular power plants that supply ATP to power various biological activities essential for neuronal growth, survival, and function. Due to extremely varied morphological features, neurons face exceptional challenges to maintain energy homeostasis. Neurons require specialized mechanisms distributing mitochondria to distal synapses where energy is in high demand. Axons and synapses undergo activity-dependent remodeling, thereby altering mitochondrial distribution. The uniform microtubule polarity has made axons particularly useful for exploring mechanisms regulating mitochondrial transport. Mitochondria alter their motility under stress conditions or when their integrity is impaired. Therefore, research into the mechanisms regulating mitochondrial motility in healthy and diseased neurons is an important emerging frontier in neurobiology. In this chapter, we discuss the current protocols in the characterization of axonal mitochondrial transport in primary neuron cultures isolated from embryonic rats and adult mice. We also briefly discuss new procedures developed in our lab in analyzing mitochondrial motility patterns at presynaptic terminals and evaluate their impact on synaptic vesicle release.


Assuntos
Transporte Axonal , Mitocôndrias/metabolismo , Biologia Molecular/métodos , Neurônios/citologia , Neurônios/metabolismo , Animais , Transporte Biológico/fisiologia , Técnicas de Cultura de Células , Células Cultivadas , Embrião de Mamíferos/citologia , Corantes Fluorescentes , Hipocampo/citologia , Hipocampo/embriologia , Processamento de Imagem Assistida por Computador , Camundongos , Mitocôndrias/química , Ratos , Transfecção
4.
Cell Metab ; 20(1): 145-57, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24882066

RESUMO

Neurodegenerative diseases represent an increasing burden in our aging society, yet the underlying metabolic factors influencing onset and progression remain poorly defined. The relationship between impaired IGF-1/insulin-like signaling (IIS) and lifespan extension represents an opportunity to investigate the interface of metabolism with age-associated neurodegeneration. Using data sets of established DAF-2/IIS-signaling components in Caenorhabditis elegans, we conducted systematic RNAi screens in worms to select for daf-2-associated genetic modifiers of α-synuclein misfolding and dopaminergic neurodegeneration, two clinical hallmarks of Parkinson's disease. An outcome of this strategy was the identification of GPI-1/GPI, an enzyme in glucose metabolism, as a daf-2-regulated modifier that acts independent of the downstream cytoprotective transcription factor DAF-16/FOXO to modulate neuroprotection. Subsequent mechanistic analyses using Drosophila and mouse primary neuron cultures further validated the conserved nature of GPI neuroprotection from α-synuclein proteotoxicity. Collectively, these results support glucose metabolism as a conserved functional node at the intersection of proteostasis and neurodegeneration.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Envelhecimento , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Cultivadas , Citocinas/antagonistas & inibidores , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/citologia , Drosophila/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Glucose/metabolismo , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Glucose-6-Fosfato Isomerase/genética , Glicólise , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor de Insulina/antagonistas & inibidores , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo
5.
Metallomics ; 6(3): 476-90, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24452053

RESUMO

Parkinson's disease (PD) is a neurodegenerative brain disorder characterized by selective dopaminergic (DAergic) cell loss that results in overt motor and cognitive deficits. Current treatment options exist to combat PD symptomatology, but are unable to directly target its pathogenesis due to a lack of knowledge concerning its etiology. Several genes have been linked to PD, including three genes associated with an early-onset familial form: parkin, pink1 and dj1. All three genes are implicated in regulating oxidative stress pathways. Another hallmark of PD pathophysiology is Lewy body deposition, associated with the gain-of-function genetic risk factor α-synuclein. The function of α-synuclein is poorly understood, as it shows both neurotoxic and neuroprotective activities in PD. Using the genetically tractable invertebrate Caenorhabditis elegans (C. elegans) model system, the neurotoxic or neuroprotective role of α-synuclein upon acute Mn exposure in the background of mutated pdr1, pink1 or djr1.1 was examined. The pdr1 and djr1.1 mutants showed enhanced Mn accumulation and oxidative stress that was reduced by α-synuclein. Moreover, DAergic neurodegeneration, while unchanged with Mn exposure, returned to wild-type (WT) levels for pdr1, but not djr1.1 mutants expressing α-synuclein. Taken together, this study uncovers a novel, neuroprotective role for WT human α-synuclein in attenuating Mn-induced toxicity in the background of PD-associated genes, and further supports the role of extracellular dopamine in exacerbating Mn neurotoxicity.


Assuntos
Aldeído Oxirredutases/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Manganês/metabolismo , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genética , alfa-Sinucleína/metabolismo , Aldeído Oxirredutases/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Mutação , Estresse Oxidativo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , alfa-Sinucleína/genética
6.
Hum Mol Genet ; 21(17): 3785-94, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22645275

RESUMO

Lysosomes are responsible for degradation and recycling of bulky cell material, including accumulated misfolded proteins and dysfunctional organelles. Increasing evidence implicates lysosomal dysfunction in several neurodegenerative disorders, including Parkinson's disease and related synucleinopathies, which are characterized by the accumulation of α-synuclein (α-syn) in Lewy bodies. Studies of lysosomal proteins linked to neurodegenerative disorders present an opportunity to uncover specific molecular mechanisms and pathways that contribute to neurodegeneration. Loss-of-function mutations in a lysosomal protein, ATP13A2 (PARK9), cause Kufor-Rakeb syndrome that is characterized by early-onset parkinsonism, pyramidal degeneration and dementia. While loss of ATP13A2 function plays a role in α-syn misfolding and toxicity, the normal function of ATP13A2 in the brain remains largely unknown. Here, we performed a screen to identify ATP13A2 interacting partners, as a first step toward elucidating its function. Utilizing a split-ubiquitin membrane yeast two-hybrid system that was developed to identify interacting partners of full-length integral membrane proteins, we identified 43 novel interactors that primarily implicate ATP13A2 in cellular processes such as endoplasmic reticulum (ER) translocation, ER-to-Golgi trafficking and vesicular transport and fusion. We showed that a subset of these interactors modified α-syn aggregation and α-syn-mediated degeneration of dopaminergic neurons in Caenorhabditis elegans, further suggesting that ATP13A2 and α-syn are functionally linked in neurodegeneration. These results implicate ATP13A2 in vesicular trafficking and provide a platform for further studies of ATP13A2 in neurodegeneration.


Assuntos
Dobramento de Proteína/efeitos dos fármacos , ATPases Translocadoras de Prótons/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidade , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Ligação Proteica/efeitos dos fármacos , Reprodutibilidade dos Testes , Técnicas do Sistema de Duplo-Híbrido , alfa-Sinucleína/química
7.
Cell ; 146(1): 37-52, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21700325

RESUMO

Parkinson's disease (PD), an adult neurodegenerative disorder, has been clinically linked to the lysosomal storage disorder Gaucher disease (GD), but the mechanistic connection is not known. Here, we show that functional loss of GD-linked glucocerebrosidase (GCase) in primary cultures or human iPS neurons compromises lysosomal protein degradation, causes accumulation of α-synuclein (α-syn), and results in neurotoxicity through aggregation-dependent mechanisms. Glucosylceramide (GlcCer), the GCase substrate, directly influenced amyloid formation of purified α-syn by stabilizing soluble oligomeric intermediates. We further demonstrate that α-syn inhibits the lysosomal activity of normal GCase in neurons and idiopathic PD brain, suggesting that GCase depletion contributes to the pathogenesis of sporadic synucleinopathies. These findings suggest that the bidirectional effect of α-syn and GCase forms a positive feedback loop that may lead to a self-propagating disease. Therefore, improved targeting of GCase to lysosomes may represent a specific therapeutic approach for PD and other synucleinopathies.


Assuntos
Doença de Gaucher/metabolismo , Glucosilceramidase/metabolismo , alfa-Sinucleína/metabolismo , Animais , Encéfalo/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Retroalimentação Fisiológica , Doença de Gaucher/patologia , Glucosilceramidas/metabolismo , Humanos , Lisossomos/metabolismo , Camundongos , Neurônios/metabolismo
8.
Methods ; 53(3): 220-5, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21195766

RESUMO

Protein misfolding and aggregation are key pathological features observed in numerous neurodegenerative diseases, including the misfolding of α-synuclein (α-syn) in Parkinson's disease (PD) and ß-amyloid in Alzheimer's disease. While this phenomenon is widely observed, the etiology and progression of these diseases is not fully understood. Furthermore, there is a lack of therapeutic treatments directed at halting the progression and neurodegeneration associated with these diseases. This demands a need for an inexpensive, easy to manipulate multicellular organism to conduct both genetic and chemical screens within to identify factors that may play a pivotal role in the pathology of these diseases. Herein, we describe methodology involved in identifying genetic modifiers of α-syn misfolding and toxicity in the nematode roundworm, Caenorhabditis elegans. Transgenic nematodes engineered to express human α-syn in the body wall muscles or dopaminergic (DA) neurons result in formation of cytoplasmic puncta or DA neurodegeneration, respectively. Using these models, we describe the use of RNA interference (RNAi) and transgenic gene expression to functionally elucidate potential therapeutic gene targets that alter α-syn misfolding and DA neurotoxicity.


Assuntos
Caenorhabditis elegans/fisiologia , Dopamina/metabolismo , Neurônios/metabolismo , Doença de Parkinson/patologia , Proteínas Recombinantes/metabolismo , alfa-Sinucleína/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Morte Celular , Clonagem Molecular , Modelos Animais de Doenças , Humanos , Músculos/metabolismo , Neurônios/citologia , Especificidade de Órgãos , Dobramento de Proteína , Interferência de RNA
10.
J Vis Exp ; (18)2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-19066505

RESUMO

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosome. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.


Assuntos
Caenorhabditis elegans/genética , Microinjeções/métodos , Transgenes , Animais , Animais Geneticamente Modificados , DNA/genética , Plasmídeos/genética
11.
J Vis Exp ; (17)2008 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-19066512

RESUMO

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration. Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets. Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.


Assuntos
Dopamina/fisiologia , Degeneração Neural/patologia , Neurônios/fisiologia , Doença de Parkinson/genética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Modelos Animais de Doenças , Dopamina/metabolismo , Vetores Genéticos/genética , Humanos , Degeneração Neural/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , alfa-Sinucleína/biossíntese , alfa-Sinucleína/genética
12.
Proc Natl Acad Sci U S A ; 105(2): 728-33, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-18182484

RESUMO

Genomic multiplication of the locus-encoding human alpha-synuclein (alpha-syn), a polypeptide with a propensity toward intracellular misfolding, results in Parkinson's disease (PD). Here we report the results from systematic screening of nearly 900 candidate genetic targets, prioritized by bioinformatic associations to existing PD genes and pathways, via RNAi knockdown. Depletion of 20 gene products reproducibly enhanced misfolding of alpha-syn over the course of aging in the nematode Caenorhabditis elegans. Subsequent functional analysis of seven positive targets revealed five previously unreported gene products that significantly protect against age- and dose-dependent alpha-syn-induced degeneration in the dopamine neurons of transgenic worms. These include two trafficking proteins, a conserved cellular scaffold-type protein that modulates G protein signaling, a protein of unknown function, and one gene reported to cause neurodegeneration in knockout mice. These data represent putative genetic susceptibility loci and potential therapeutic targets for PD, a movement disorder affecting approximately 2% of the population over 65 years of age.


Assuntos
Doença de Parkinson/genética , Interferência de RNA , Animais , Caenorhabditis elegans , Modelos Animais de Doenças , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/química , Humanos , Modelos Biológicos , Degeneração Neural , Doenças Neurodegenerativas/genética , Fármacos Neuroprotetores/farmacologia , Peptídeos/metabolismo , Dobramento de Proteína , Sinucleínas/metabolismo
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