Name: monitoring cell-to-cell transmission of prion-like protein aggregates in drosophila melanogaster
Uploaded: 2018-03-12T14:45:00.000+00:00
Duration: 10 min 26 s
Description: Watch this Scientific Journal Video about Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster at JoVE.com

Agradecemos a los miembros de los laboratorios Kopito, Luo y Pearce para muchas discusiones útiles durante el desarrollo de estos métodos. También agradecemos a Brian Temsamrit para la lectura crítica de este manuscrito. Este trabajo fue financiado por los fondos de la Universidad de las Ciencias y las confianzas caritativas de W.W. Smith.

Los autores no tienen nada que revelar.

Sigue aumentando el número de pacientes que sufren de enfermedades neurodegenerativas, hay una necesidad urgente de aumentar la comprensión de la patogenia molecular de estas enfermedades que se pueden desarrollar mejores terapias. Aquí, describimos los métodos que permiten monitoreo de transmisión del prión-como de agregados de proteínas patogénicas entre diferentes tipos de células en el organismo modelo Drosophila melanogaster. Recientemente hemos utilizado esta metodología para demostrar la transmi...

La hipótesis del prión afirma que el agente infeccioso responsable de las encefalopatías espongiformes transmisibles (p. ej., enfermedad de Creutzfeldt - Jakob en los seres humanos, scrapie en ovejas, la caquexia crónica en ciervos y alces y "enfermedad de las vacas locas" en bovinos ) está exclusivamente compuesto de proteína y desprovista de ácidos nucleicos1. En enfermedades por prión, la proteína priónica celular (PrPC) asume un pliegue (PrPSc) no nativos, estable que es altamente beta ricos en hoja y puede uno mismo-propagar por conversión y reclutar moléculas monoméricas de PrPC en amiloide estable agregados. Agregados de PrPSc utilizan este mecanismo uno mismo-repliegue para difundir entre las diferentes células en un organismo e incluso entre los organismos individuales2.
Agregación y mal plegamiento de la proteína también es una característica central de la mayoría de las enfermedades neurodegenerativas (enfermedad de Alzheimer (EA), enfermedad de Parkinson (EP), enfermedad de Huntington (EH) y esclerosis lateral amiotrófica (ELA))3. Formación de asambleas de cellular intra o extra proteína agregada en estas enfermedades está íntimamente relacionada con la citotoxicidad4 y progresa a lo largo de caminos altamente reproducibles y específica de la enfermedad a través del cerebro en tiempo5, 6. estos patrones de propagación sugieren que agregados patógenos asociados con estos trastornos tienen propiedades similares a los priones. Ahora existe fuerte apoyo para transmisión del prión-como de agregados asociados a AD, PD, HD y la ELA - transmite de célula a célula y la plantilla el cambio conformacional de la forma monomérica de la misma proteína en células previamente inafectados7, 8.
La mayoría de los estudios que investigan propagación de prion-como de agregados de proteínas hasta la fecha se han realizado utilizando modelos de cultivo de células de mamífero, donde transferencia de agregados en el citoplasma de las células ingenuas desde el espacio extracelular o desde otra celda citoplasma9,10,11,12,13,14,la15, o por inyección de material que contiene el agregado en cerebros de ratón y control agregado apariencia fuera la inyección sitio16,17,18,19,20,21,22, 23. más recientemente, se han utilizado animales transgénicos para demostrar que agregados intracelulares se extensión a otras células en el cerebro intacto24,25,26,27, 28,29,30. Aquí, describimos un método para la visualización directa de la transferencia total entre las células individuales del cerebro intacto de Drosophila melanogaster. En primer lugar se desarrollaron modelos de Drosophila de HD/polyglutamine (poliQ) enfermedades hace casi dos décadas31,32 y han proporcionado muchos conocimientos invaluables en los mecanismos patogénicos que subyacen a estos trastornos 33. HD es un trastorno neurodegenerativo hereditario causado por una mutación dominante autosómica en el gen que codifica para la proteína huntingtina (Htt)34. Esta mutación da lugar a la ampliación de un tramo de la poliQ cerca N-terminal de Htt, más allá de un umbral patógeno de ~ 37 glutaminas, haciendo que la proteína misfold y agregado35,36. Proteínas de Htt de tipo salvaje que contienen < 37 glutaminas en este tramo lograr su pliegue natural, pero puede ser inducidas a agregado al contacto físico directo con un Htt agregada "la semilla"12,27,37. Aprovechamos este homotípicos, nucleada agregación de Htt de tipo salvaje como una lectura para transferencia-como prión y entrada citoplasmática de agregados de Htt mutantes procedentes de otras células.
Determinar los mecanismos por los que agregados-como prión viajes entre células pueden conducir a la identificación de nuevas dianas terapéuticas para enfermedades neurodegenerativas incurable. Tomamos ventaja del ciclo de vida rápido, facilidad de uso y maleabilidad genética de Drosophila melanogaster para definir mecanismos moleculares para la propagación de célula a célula de agregados Htt mutantes. Nuestra estrategia experimental emplea dos sistemas de expresión binaria disponibles en Drosophila, la bien establecida Gal4 específicos aguas arriba activar secuencia (Gal4-UAS) sistema38 y el recientemente desarrollado QF-QUAS sistema39. Estos dos sistemas independientes de acoplamiento permite restringir la expresión de los transgenes de Htt mutantes y el salvaje-tipo poblaciones celulares distintas dentro de la misma mosca40. Usando este acercamiento, examinamos prion-como separarse del mutante Htt mediante el control de la redistribución de los citoplásmico Htt de tipo salvaje de su estado soluble, normalmente difuso a un estado agregado, consecuencia directa del contacto físico con una forma mutante Htt agregado "semilla." Conversión de tipo salvaje Htt por mutante Htt puede confirmarse mediante bioquímica o transferencia de técnicas biofísicas que informe las interacciones proteína-proteína, como la energía de resonancia de fluorescencia (FRET)9,27,41 .
Lo importante, también podemos acceder a un gran número de herramientas genéticas en Drosophila para identificar genes o vías que median la propagación del prión-como de agregados de proteína. Recientemente hemos utilizado este enfoque para desvelar un papel clave para el receptor scavenger superficial, Draper42,43, en la transferencia agregados Htt mutantes de axones neuronales a glia fagocitaria cercana en Drosophila central sistema nervioso (CNS)27. Así, el enfoque basado en la proyección de imagen y genética que se describe aquí puede revelar importante información biológica básica sobre un fenómeno de enfermedad relevante en simple-to-use pero organismo modelo de gran alcance, 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

Agregación de la proteína es una característica central de la mayoría las enfermedades neurodegenerativas, como esclerosis lateral amiotrófica (ELA), enfermedad de Alzheimer (EA), enfermedad de Parkinson (EP) y la enfermedad de Huntington (HD). Agregados de proteína están estrechamente asociados con neuropatología en estas enfermedades, aunque no se conoce el mecanismo exacto por el cual la agregación de la proteína aberrante interrumpe homeostasis celular normal. Surgiendo datos proporcionar fuerte apoyo para la hipótesis de patógenos agregados en TDA, PD, y ALS tienen muchas similitudes a los priones, que son sólo proteínas agentes infecciosos responsables de las encefalopatías espongiformes transmisibles. Priones autoreplicarse por plantillas la conversión de forma nativa doblado versiones de la misma proteína, causando la propagación del fenotipo de agregación. Cómo los priones y las proteínas del prión-como en AD, PD, HD y ALS se mueven de una celda a otra es actualmente un área de intensa investigación. Aquí, se describe un modelo de Drosophila melanogaster que permite monitoreo de transmisión del prión-como, célula a célula de agregados de la huntingtina (Htt) asociados con el HD. Este modelo se aprovecha de potentes herramientas para manipular la expresión de transgenes en muchos diversos tejidos de Drosophila y utiliza una proteína citoplásmica fluorescencia etiquetada transferencia directamente de prion-como del informe de agregados Htt mutantes. Lo importante, el enfoque se describe aquí puede utilizarse para identificar nuevos genes y vías que median la separarse de agregados de proteína entre célula diversos tipos en vivo. Información obtenida de estos estudios será ampliar la limitada comprensión de los mecanismos patogénicos que subyacen a las enfermedades neurodegenerativas y revelar nuevas oportunidades para la intervención terapéutica.

Los métodos descritos aquí producen datos robustos demostrando a-como prión transferencia de agregados de la proteína Htt de población de una célula a otra en la mosca intacta del CNS. Conversión de tipo salvaje Htt de difuso a punteado se observa por fluorescencia directa de esta proteína de fusión YFP en glia receptor como resultado de la expresión HttQ91-mCherry en donantes ORNs (A-C de lafigura 2y figura 4</...

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

Acumulación de evidencia apoya la idea de que agregados de proteína patógena asociada a enfermedades neurodegenerativas entre células con propiedades similares a los priones. Aquí, describimos un método que permite la visualización de la propagación de célula a célula del prion-como los agregados en el organismo modelo Drosophila melanogaster.

Monitoreo de la transmisión de célula a célula de los agregados de la proteína Prion-como en 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

8.0K vistas.  University of the Sciences. Acumulación de evidencia apoya la idea de que agregados de proteína patógena asociada a enfermedades neurodegenerativas entre células con propiedades similares a los priones. Aquí, describimos un método que permite la visualización de la propagación de célula a célula del prion-como los agregados en el organismo modelo Drosophila melanogaster.

Video: 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.

Monitoreo células inmunes Tráfico Varillas fluorescente priones horas de la infección intraperitoneal

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

Investigación

Inmunología e Infección

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.

La evaluación de fenotipos neurodegenerativas en<em&gt; Drosophila</em&gt; Las neuronas dopaminérgicas mediante ensayos de Escalada y inmunotinción de todo el cerebro

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

Neurociencias

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.

Una técnica simple para evaluar la actividad locomotora en Drosophila

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

Comportamiento

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.

La histología de masas para cuantificar la neurodegeneración en<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.

Biología celular cuantitativa de neurodegeneración en Drosophila mediante análisis imparcial de fluorescencia etiquetas proteínas con 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.

Medición de la absorción de glucosa en modelos de Drosophila de proteinopatía por 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.

La investigación de las proteínas priónicas, como la extensión y la toxicidad de Uso del 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 ...

Biología

Monitoreo de eventos epileptiformes en Drosophila a través de imágenes de calcio

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 destacado: Perspectivas sobre las técnicas y los hallazgos de los avances recientes en la investigación de la 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 Imágenes de calcio para el modelo de epilepsia de Drosophila

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

Monitoreo de la transmisión de célula a célula de los agregados de la proteína Prion-como en Drosophila Melanogaster

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