Agradecemos a membros dos laboratórios Kopito, Luo e Pearce para muitas discussões útil durante o desenvolvimento desses métodos. Agradecemos também o Brian Temsamrit pela leitura crítica deste manuscrito. Este trabalho foi financiado por fundos da Universidade das Ciências e as confianças Charitable W.W. Smith.

Os autores não têm nada para divulgar.

Como o número de pacientes que sofrem de doenças neurodegenerativas continua a aumentar, há uma necessidade urgente de aumentar a compreensão da patogênese molecular dessas doenças para que melhor terapias podem ser desenvolvidas. Aqui, descrevemos os métodos que permitem a monitorização do prião, como transmissão de agregados de proteínas patogênicas entre diferentes tipos de células no organismo modelo, Drosophila melanogaster. Recentemente usamos esta metodologia para demonstrar prião, como tra...

A hipótese do príon afirma que o agente infeccioso responsável para as encefalopatias espongiformes transmissíveis (por exemplo, doença de Creutzfeldt - Jakob nos seres humanos, o tremor epizoótico dos ovinos, doença desperdiçando crônica em cervos e alces e "doença da vaca louca" em bovinos ) é unicamente composto de proteína e desprovidos de ácidos nucleicos1. Em doenças de príon, a proteína príon celular (PrPC) pressupõe uma dobra não-nativas, estável (PrPSc) que é altamente beta folha-rico e pode propagar self convertendo e recrutamento monomérico PrPC moléculas em amiloide estável agregados. PrPSc agregados usam esse mecanismo de auto-replicação para espalhar entre diferentes células em um organismo e mesmo entre organismos individuais2.
Enrolamento de proteínas e agregação é também uma característica central da maioria das doenças neurodegenerativas (a doença de Alzheimer (AD), a doença de Parkinson (PD), a doença de Huntington (HD) e esclerose lateral amiotrófica (ela))3. Formação das assembleias intra ou extra cellular agregados da proteína nestas doenças está intimamente associada com a citotoxicidade4 e progride ao longo de caminhos altamente reprodutíveis e doenças específicas, através do cérebro ao longo do tempo5, 6. estes padrões de propagação sugerem que agregados patogénicos associados a esses transtornos tem príon-como propriedades. Forte apoio agora existe para transmissão de príons, como dos agregados associados AD, PD, HD e ALS - espalham de célula para célula e modelo a mudança conformacional monomérico formas da mesma proteína em células anteriormente afetado7, 8.
A maioria dos estudos investigando o prião, como propagação de agregados de proteínas até à data têm sido realizada usando modelos de cultura de células de mamíferos, onde agregados transferir para o citoplasma das células ingênuo do espaço extracelular ou de outra célula citoplasma9,10,11,12,13,14,15, ou pela injeção de material agregado contendo em cérebro de rato e monitoramento agregar aparência fora a injeção local16,17,18,19,20,21,22, 23. mais recentemente, os animais transgénicos têm sido utilizados para demonstrar que agregados intracelulares se espalhar para outras células no cérebro intacto24,25,26,27, 28,29,30. Aqui, descrevemos um método para a visualização directa de transferência agregada entre células individuais do cérebro intacto de Drosophila melanogaster. Drosófila modelos de HD/polyglutamine (polyQ) doenças foram desenvolvidos primeiramente há quase duas décadas de31,32 e forneceram muitos insights inestimáveis sobre os mecanismos patogénicos subjacentes a estes distúrbios 33. HD é uma desordem neurodegenerativa hereditária causada por uma mutação autossômica dominante no gene que códigos para a proteína huntingtina (Htt)34. Esta mutação resulta na expansão de um trecho de polyQ perto N-terminal do Htt, além de um limite de patogenicidade de ~ 37 glutaminas, fazendo com que a proteína misfold e agregado35,36. Proteínas de Htt do selvagem-tipo contendo < 37 glutaminas neste estiramento alcançar seu aprisco nativo, mas pode ser induzidas a agregada ao contacto físico directo com um Htt agregada "semente"12,,27,37. Exploramos este homotypic, nucleada agregação do selvagem-tipo Htt como uma leitura para transferência de príon-como e citoplasmática entrada dos mutantes Htt agregados originários de outras células.
Determinar os mecanismos pelos quais agregados de príon-como viagens entre células podem levar à identificação de novos alvos terapêuticos para doenças neurodegenerativas incuráveis. Levamos vantagem do ciclo de vida rápido, facilidade de uso e rastreabilidade genética da Drosophila melanogaster para definir mecanismos moleculares para propagação de célula para célula do mutant Htt agregados. Nossa estratégia experimental emprega dois sistemas de expressão binário disponíveis em Drosophila, o bem-estabelecida Gal4 específicas upstream ativando sequência (Gal4-UAS) sistema38 e o recentemente desenvolvido QF-QUAS sistema39. Estes dois sistemas independentes de acoplamento permite restringir a expressão dos mutantes e selvagem-tipo Htt transgenes para populações de células distintas dentro da mesma voar40. Usando essa abordagem, nós examinamos príon como difusão do mutant Htt, monitorando a redistribuição de citoplasmática Htt selvagem-tipo de estado solúvel, normalmente difuso a um estado de agregados, uma consequência direta do contato físico com um mutante pré-formado Htt agregado "semente". Conversão de tipo selvagem Htt por mutant Htt pode ser confirmado usando bioquímicas ou transferência de técnicas biofísicas que relatam as interações da proteína-proteína, tais como energia de ressonância de fluorescência (FRET)9,,27,41 .
Importante, também podemos acessar um grande número de ferramentas genéticas em Drosophila para identificar genes e/ou caminhos que medeiam príon como propagação de agregados de proteínas. Temos recentemente usado essa abordagem para desvendar um papel-chave para o receptor de superfície ao tesouro de célula,42,Draper43, na transferência de mutante Htt agregados de axônios neuronais para glia fagocítica nas proximidades em Drosophila central sistema nervoso (SNC)27. Assim, a abordagem genética e imagem latente-baseada que descrevemos aqui pode revelar importantes informações biológicas básicas sobre um fenômeno doença relevantes no simples de usar mas organismo modelo poderoso, Drosophila.

<table><tbody><tr><td>Phosphate buffered saline (PBS), 10X, pH 7.4</td><td>ThermoFisher Scientific</td><td>AM9625</td><td>Dilute to 1X</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284-1L</td><td></td></tr><tr><td>Kimwipes</td><td>Thomas Scientific</td><td>2904F24</td><td></td></tr><tr><td>20% paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15713-S</td><td></td></tr><tr><td>Normal Goat Serum (NGS), filtered</td><td>Lampire Biological Laboratories</td><td>7332500</td><td>Aliquot and freeze upon receipt</td></tr><tr><td>Chicken anti-GFP</td><td>Aves Labs</td><td>GFP-1020</td><td>Use at 1:500 dilution</td></tr><tr><td>Rabbit anti-DsRed</td><td>Clontech</td><td>632496</td><td>Use at 1:2000 dilution; can recognize DsRed-based fluorescent proteins (e.g. mCherry, mStrawberry, tdTomato, etc.)</td></tr><tr><td>Mouse anti-Bruchpilot</td><td>Developmental Studies Hybridoma Bank</td><td>nc82</td><td>Use at 1:100 dilution; will label active pre-synaptic structures thoughout the fly brain</td></tr><tr><td>FITC anti-chicken</td><td>ThermoFisher Scientific</td><td>SA1-7200</td><td>Use at 1:250 dilution</td></tr><tr><td>Alexa Fluor 568 anti-rabbit</td><td>Life Technologies</td><td>A11011</td><td>Use at 1:250 dilution</td></tr><tr><td>Alexa Fluor 647 anti-mouse antibody</td><td>Life Technologies</td><td>A21235</td><td>Use at 1:250 dilution</td></tr><tr><td>Slowfade Gold Antifade Reagent</td><td>Life Technologies</td><td>S36936</td><td></td></tr><tr><td>Microscope Slides (25 x 75 x 1.0 mm)</td><td>Fisher Scientific</td><td>12-550-143</td><td></td></tr><tr><td>Cover Glass (22 x 22 mm)</td><td>Globe Scientific</td><td>1404-15</td><td></td></tr><tr><td>Dumont Biology Grade Forceps, Style 3</td><td>Ted Pella</td><td>503</td><td>use in non-dominant hand</td></tr><tr><td>Dumont Biology Grade Forceps, Style 5</td><td>Ted Pella</td><td>505</td><td>use in dominant hand</td></tr><tr><td>LAS X image analysis software</td><td>Leica</td><td></td><td></td></tr><tr><td>Imaris image analysis software</td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

monitoring cell-to-cell transmission of prion-like protein aggregates in drosophila melanogaster

A agregação da proteína é uma característica central da maioria das doenças neurodegenerativas, incluindo a doença de Alzheimer (AD), a doença de Parkinson (PD), a doença de Huntington (HD) e esclerose lateral amiotrófica (ela). Agregados de proteína estão intimamente associados com neuropatologia destas doenças, embora o mecanismo exato pelo qual a agregação da proteína aberrante interrompe a homeostase celular normal não é conhecido. Emergentes dados fornecer forte apoio para a hipótese que agregados patogénicos em AD, PD, HD, e ALS tem muitas semelhanças com os prions, que são somente proteína agentes infecciosos responsáveis para as encefalopatias espongiformes transmissíveis. Priões auto replicam por modelagem a conversão das versões nativamente-dobrado da mesma proteína, causando a propagação do fenótipo agregação. Como príons e proteínas de príon, como no anúncio, PD, HD e ALS mover de uma célula para outra é atualmente uma área de intensa investigação. Aqui, um modelo de Drosophila melanogaster que permite o monitoramento da transmissão príon-like, célula a célula do mutant huntingtin (Htt) agregados associados com HD é descrito. Este modelo aproveita-se de poderosas ferramentas para manipular a expressão do transgene em muitos tecidos diferentes da drosófila e utiliza uma proteína citoplasmática marcados fluorescentemente diretamente transferência de prião, como relatório de mutante Htt agregados. Importante, a abordagem que descrevemos aqui pode ser usada para identificar novos genes e vias que medeiam o espalhamento de agregados de proteínas entre célula diversos tipos em vivo. Informações obtidas a partir desses estudos irão expandir a compreensão limitada dos mecanismos patogénicos que fundamentam a doenças neurodegenerativas e revelar novas possibilidades de intervenção terapêutica.

Os métodos descritos aqui produzem dados robustos, demonstrando o prião, como transferência de agregados de proteína de Htt da população de uma célula para outra na mosca da intacta CNS. Conversão de tipo selvagem Htt de difusa para punctate é observado por fluorescência direta desta proteína de fusão YFP na glia destinatário como resultado da expressão de HttQ91-mCherry em ORNs doador (Figura 2A C e a Figur...

Watch this Scientific Journal Video about Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster at JoVE.com

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Acumular evidências suporta a ideia que agregados de proteínas patogênicas associam com doenças neurodegenerativas espalhados entre as células com o prião-como propriedades. Aqui, descrevemos um método que permite a visualização de propagação de célula para célula de príon como agregados no organismo modelo, Drosophila melanogaster.

Monitoramento de transmissão de celular para celular da proteína príon como agregados em Drosophila Melanogaster.

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

monitoring-cell-to-cell-transmission-prion-like-protein-aggregates

Research

JoVE Journal

Neuroscience

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

7.9K visualizações.  University of the Sciences. Acumular evidências suporta a ideia que agregados de proteínas patogênicas associam com doenças neurodegenerativas espalhados entre as células com o prião-como propriedades. Aqui, descrevemos um método que permite a visualização de propagação de célula para célula de príon como agregados no organismo modelo, Drosophila melanogaster.

Vídeo: Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

Monitoramento células imunes Tráfico Rods Horas Prion fluorescente após a infecção intraperitoneal

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

Imunologia e Infecção

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Avaliando fenótipos Neurodegenerativas em<em&gt; Drosophila</em&gt; Neurônios dopaminérgicos subindo Ensaios e toda Imunocoloração Cérebro

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Neurociência

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Uma técnica simples para avaliar a atividade locomotora em Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

Comportamento

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

Mass Histologia para quantificar neurodegeneração em<em&gt; Drosophila</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

Biologia celular quantitativa de neurodegeneração em Drosophila , através de uma análise imparcial das proteínas fluorescente etiquetadas usando o ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700&#181;&#948;6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700&#181;&#948;6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700&#181;&#948;6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

Medição da captação de glicose em modelos de Drosophila de Proteinopatia TDP-43

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Investigando as proteínas príon-like espalhando e de toxicidade de usar o Metazoan Organismo Modelo<em&gt; C. elegans</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Biologia

Monitoramento de eventos epileptiformes em Drosophila por meio de imagens de cálcio

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

Autor em destaque: insights sobre as técnicas e descobertas de avanços recentes na pesquisa da epilepsia 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Ex Vivo Imagem de cálcio para Drosophila Modelo de Epilepsia

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...

Monitoramento de transmissão de celular para celular da proteína príon como agregados em Drosophila Melanogaster.

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