Wir danken Mitgliedern von Kopito, Luo und Pearce Labs für viele hilfreiche Diskussionen während der Entwicklung dieser Methoden. Wir danken auch Brian Temsamrit für kritische Lektüre des Manuskripts. Diese Arbeit wurde durch Mittel von Universität der Wissenschaften und der w. Smith Stiftungen unterstützt.

Die Autoren haben nichts preisgeben.

Da die Zahl der Patienten mit neurodegenerativen Erkrankungen weiter zu erhöhen, gibt es eine dringende Notwendigkeit, das Verständnis der molekularen Pathogenese dieser Erkrankungen erhöhen, so dass bessere Therapien entwickelt werden können. Hier beschreiben wir Methoden, die für die Überwachung der Prion-artigen Übertragung von pathogenen Proteinaggregaten zwischen verschiedenen Zelltypen in der Modellorganismus Drosophila Melanogaster. Wir haben vor kurzem diese Methode verwendet, um Prion-wie Getrieb...

Die Prion-Hypothese besagt, dass der Erreger verantwortlich für die transmissiblen spongiformen Enzephalopathien (z.B.Creutzfeldt - Jakob-Krankheit beim Menschen, Scrapie bei Schafen, verschwenden chronisch im Hirschen und Elchen und "Rinderwahnsinn" bei Rindern ) besteht ausschließlich aus Protein und frei von Nukleinsäuren1. Prion-Krankheiten übernimmt die zellulären Prion-Protein (PrPC) eine nicht-Native, stabile Falte (PrP-Sc), die ist sehr Beta Blatt-reich und kann selbst durch die Umwandlung und Rekrutierung von Monomeren PrPC Moleküle in stabilen Amyloid ausbreiten Aggregate. PrP-Sc -Aggregate nutzen diesen selbstreplizierende Mechanismus zwischen verschiedenen Zellen in einem Organismus und sogar zwischen einzelnen Organismen2zu verbreiten.
Protein misfolding und Aggregation ist auch ein zentrales Merkmal der meisten neurodegenerativen Erkrankungen (Alzheimer-Krankheit (AD), Parkinson-Krankheit (PD), Chorea Huntington (HD) und Amyotrophe Lateralsklerose (ALS))3. Bildung von Intra oder extra zellulare aggregierten Protein Baugruppen bei diesen Erkrankungen ist eng verknüpft mit Zytotoxizität4 und schreitet hoch reproduzierbare und krankheitsspezifische Pfaden durch das Gehirn über Zeit5, 6. diese Muster der Ausbreitung legen nahe, dass Pathogene Aggregate mit diesen Erkrankungen assoziiert sind Prion-ähnliche Eigenschaften haben. Starke Unterstützung existiert nun für Prion-wie Übertragung der Aggregate zugeordnete AD, PD, HD und ALS - sie breitete sich von Zelle zu Zelle und Vorlage der Konformationsänderung der Monomere Formen des gleichen Proteins in zuvor nicht betroffen Zellen7, 8.
Die Mehrheit der Studien, die untersuchen Prion-artigen Ausbreitung von Proteinaggregaten bisher wurden durchgeführt mit Säugerzelle Kultur Modelle, bei denen ein Übergang Aggregate in das Zytoplasma der naive Zellen aus dem extrazellulären Raum oder von einer anderen Zelle Zytoplasma9,10,11,12,13,14,15, oder durch Einspritzen von Aggregat-haltigen Material in Mäusegehirnen und Überwachung aggregieren Auftritt außerhalb der Injektion Seite16,17,18,19,20,21,22, 23. in jüngerer Zeit, transgene Tiere verwendet wurden, um nachzuweisen, dass die intrazelluläre Aggregate auf andere Zellen im intakten Gehirn24,25,26,27, verbreiten 28,29,30. Hier beschreiben wir eine Methode für die direkte Visualisierung der aggregierten Transfer zwischen den einzelnen Zellen im intakten Gehirn von Drosophila Melanogaster. Drosophila -Modelle HD/Polyglutamin-(PolyQ) Krankheiten wurden vor fast zwei Jahrzehnten entwickelt31,32 und haben viele wertvolle Einblicke in die pathogenen Mechanismen, die diese Störungen zugrunde liegen 33. HD ist eine vererbte Neurodegenerative Erkrankung, verursacht durch eine autosomal dominante Mutation in dem gen, das für das Protein Huntingtin (Htt)34kodiert. Diese Mutation führt zu Ausdehnung von einer PolyQ Strecke in der Nähe von Htts N-Terminus jenseits einer pathogenen Schwelle des ~ 37 Glutaminen, verursacht das Protein Misfold und insgesamt35,36. Wildtyp Htt Proteine mit < 37 Glutaminen in diesem Küstenabschnitt erreichen ihre heimischen Herde, sondern induziert werden können, um aggregierte bei direktem Körperkontakt mit einer Htt Aggregat "seed"12,27,37. Wir nutzen diese homotypische kernhaltigen Aggregation von Wildtyp Htt als einer Anzeige für Prion-wie Transfer und zytoplasmatischen Eintrag von mutierten Htt-Aggregate mit Ursprung in anderen Zellen.
Bestimmung der Mechanismen durch die Prion-ähnliche Aggregate kann Reisen zwischen Zellen zur Identifizierung der neue therapeutische Targets für unheilbare Neurodegenerative Erkrankungen führen. Wir nutzen Sie den schnellen Lebenszyklus, Benutzerfreundlichkeit und genetische Lenkbarkeit von Drosophila Melanogaster , molekulare Mechanismen für die Zell-Zell-Ausbreitung von mutierten Htt Aggregate zu definieren. Unsere experimentelle Strategie beschäftigt zwei binäre Expressionssysteme in Drosophila, etablierten Gal4-spezifische vorgelagerten aktivierende Sequenz (Gal4-UAS) System38 und vor kurzem entwickelt QF-QUAS System39zur Verfügung. Diese zwei unabhängige Kupplungssysteme ermöglicht Ausdruck von Mutanten und Wildtyp Htt transgenen auf unterschiedliche Zellpopulationen innerhalb der gleichen fliegen40zu beschränken. Mit diesem Ansatz untersuchen wir Prion-ähnliche Verbreitung der mutierte Htt durch Überwachung der Umverteilung der zytoplasmatischen Wildtyp Htt von seinem normalerweise diffuse, löslichen Zustand zu einem aggregierten Zustand, eine direkte Folge der Körperkontakt mit einer vorgeformten mutierte Htt aggregierte "Samen". Umwandlung von Wildtyp Htt durch mutierte Htt kann bestätigt werden, mit biochemischen und biophysikalische Techniken, die berichten von Protein-Protein-Wechselwirkungen, wie Fluoreszenz Resonanz Energie transfer (FRET)9,27,41 .
Wichtig ist, greifen wir auch eine große Anzahl von genetischen Werkzeuge in Drosophila , Gene und/oder Wege, die Prion-artigen Ausbreitung von Proteinaggregaten vermitteln zu identifizieren. Wir haben vor kurzem dieser Ansatz verwendet, um eine Schlüsselrolle für die Zelle Oberfläche Scavenger Rezeptor, Draper42,43, bei der Übertragung von mutierten Htt Aggregate von neuronalen Axone zum nahe gelegenen phagocytic Glia in Drosophila zentrale enthüllen Nervensystem (ZNS)27. So kann die genetischen und Imaging-basierte Ansatz, den wir hier beschreiben wichtige biologische Basisinformationen über ein krankheitsrelevante Phänomen in der einfach zu bedienenden aber mächtige Modellorganismus Drosophilaoffenbaren.

<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

Proteinaggregation ist ein zentrales Merkmal der meisten neurodegenerativen Krankheiten, einschließlich der Alzheimer-Krankheit (AD), Parkinson-Krankheit (PD), Chorea Huntington (HD) und Amyotrophe Lateralsklerose (ALS). Protein-Aggregate sind eng verbunden mit Neuropathologie bei diesen Erkrankungen, obwohl der genaue Mechanismus, mit dem aberranten Proteinaggregation normale zelluläre Homöostase stört, nicht bekannt ist. Daten liefern starke Unterstützung für die Hypothese, dass Pathogene Aggregate in AD, PD, HD und ALS haben viele Ähnlichkeiten mit Prionen, die nur-Protein Krankheitserreger für den transmissiblen spongiformen Enzephalopathien verantwortlich sind. Prionen replizieren selbst von Template die Konvertierung nativ gefaltet Versionen des gleichen Proteins, Ausbreitung des Phänotyps Aggregation verursacht. Wie Prionen und Prion-ähnliche Proteine in AD, PD, HD und ALS Übergang von einer Zelle zur anderen ist derzeit eine Fläche von intensiver Untersuchung. Hier wird ein Drosophila Melanogaster -Modell, das erlaubt, Überwachung der Prion-Like, Zell-Zell-Übertragung von mutiertem Huntingtin (Htt) Aggregaten verbunden mit HD beschrieben. Dieses Modell nutzt die leistungsstarke Werkzeuge zur Manipulation der Transgene Ausdruck in vielen verschiedenen Geweben in Drosophila und nutzt ein Eindringmittel getaggt zytoplasmatischen Protein direkt Bericht Prion-artigen Übertragung von mutierten Htt-Aggregate. Wichtig ist, kann der Ansatz, den wir hier beschreiben, verwendet werden, identifizieren neue Gene und Wege, die Verbreitung von Proteinaggregaten zwischen unterschiedlichen Zelle Arten in Vivozu vermitteln. Erkenntnisse aus diesen Studien werden die begrenzte Verständnis der pathogenen Mechanismen erweitern, die neurodegenerativen Erkrankungen zu Grunde liegen und zeigen neue Möglichkeiten für eine therapeutische Intervention.

Die hier beschriebenen Methoden produzieren robuste Daten, die zeigen Prion-artigen Übertragung von Proteinaggregaten Htt aus einer Zellpopulation zum anderen in der intakten Fliege CNS. Konvertierung von Wildtyp Htt von diffusen auf punktförmige wird durch direkte Fluoreszenz dieser YFP Schmelzverfahren Protein im Empfänger Glia infolge HttQ91 mCherry Ausdruck im Spender ORNs (Abbildung 2A-C und Abbildung 4<s...

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

Beweise sammeln, unterstützt die Idee, dass Pathogene Protein-Aggregate im Zusammenhang mit neurodegenerativen Erkrankungen verteilt zwischen den Zellen mit Prion-ähnliche Eigenschaften. Hier beschreiben wir eine Methode, die ermöglicht die Visualisierung von Zelle zu Zelle ausbreiten von Prion-ähnlichen Aggregaten im Modellorganismus Drosophila Melanogaster.

Überwachung von Zelle zu Zelle Übertragung von Prion-wie Protein-Aggregate in 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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

7.9K Aufrufe.  University of the Sciences. Beweise sammeln, unterstützt die Idee, dass Pathogene Protein-Aggregate im Zusammenhang mit neurodegenerativen Erkrankungen verteilt zwischen den Zellen mit Prion-ähnliche Eigenschaften. Hier beschreiben wir eine Methode, die ermöglicht die Visualisierung von Zelle zu Zelle ausbreiten von Prion-ähnlichen Aggregaten im Modellorganismus Drosophila Melanogaster.

Serie: 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.

Überwachung Immunzellen Trafficking Fluorescent Prion Rods Stunden nach intraperitonealer Infektion

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

Forschung

Immunologie und Infektion

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.

Beurteilung Neurodegenerative Phänotypen in<em&gt; Drosophila</em&gt; Dopaminergen Neuronen durch Klettern Assays und 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 ...

Neurowissenschaften

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.

Eine einfache Technik zur Bestimmung der lokomotorischen Aktivität in Drosophila

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

Verhalten

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.

Massen Histologie zu Quantifizieren Neurodegenerative Erkrankungen in<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.

Quantitative Zellbiologie der Neurodegeneration in Drosophila durch unvoreingenommene Analyse der Gewebekulturen Tagged Proteine mit 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.

Messung der Glukoseaufnahme in Drosophila-Modellen der TDP-43-Proteinopathie

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.

Untersuchung der Verbreitung und Toxizität von Prion-ähnlichen Proteine ​​Mit dem Metazoan Modellorganismus<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 ...

Biologie

Überwachung epileptiformer Ereignisse in Drosophila mittels Calcium-Bildgebung

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 im Rampenlicht: Einblicke in die Techniken und Erkenntnisse der jüngsten Fortschritte in der Epilepsieforschung 

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 Kalzium-Bildgebung für Drosophila-Modell der Epilepsie

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

Überwachung von Zelle zu Zelle Übertragung von Prion-wie Protein-Aggregate in Drosophila Melanogaster

Zusammenfassung

Weitere Videos entdecken

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Beurteilung Neurodegenerative Phänotypen in

Eine einfache Technik zur Bestimmung der lokomotorischen Aktivität in Drosophila

Massen Histologie zu Quantifizieren Neurodegenerative Erkrankungen in

Quantitative Zellbiologie der Neurodegeneration in Drosophila durch unvoreingenommene Analyse der Gewebekulturen Tagged Proteine mit ImageJ

Messung der Glukoseaufnahme in Drosophila-Modellen der TDP-43-Proteinopathie

Untersuchung der Verbreitung und Toxizität von Prion-ähnlichen Proteine Mit dem Metazoan Modellorganismus

Ex Vivo Kalzium-Bildgebung für Drosophila-Modell der Epilepsie

Ex Vivo Kalzium-Bildgebung für Drosophila-Modell der Epilepsie

Ex Vivo Kalzium-Bildgebung für Drosophila-Modell der Epilepsie

Überwachung von Zelle zu Zelle Übertragung von Prion-wie Protein-Aggregate in Drosophila Melanogaster

Zusammenfassung

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Untersuchung der Verbreitung und Toxizität von Prion-ähnlichen Proteine ​​Mit dem Metazoan Modellorganismus

Ex Vivo Kalzium-Bildgebung für Drosophila-Modell der Epilepsie

Ex Vivo Kalzium-Bildgebung für Drosophila-Modell der Epilepsie

Ex Vivo Kalzium-Bildgebung für Drosophila-Modell der Epilepsie

Untersuchung der Verbreitung und Toxizität von Prion-ähnlichen Proteine Mit dem Metazoan Modellorganismus