Bu yöntemlerin geliştirilmesi sırasında birçok yararlı tartışmalar Kopito, Luo ve Pearce Labs'in üyeleri teşekkür ediyoruz. Biz de bu el yazması eleştirel okuma için Brian Temsamrit teşekkür ederim. Bu eser Üniversitesi Bilimler ve chilling Smith hayırsever güven fonlarından desteklenen bir durumdu.

Yazarlar ifşa gerek yok.

Nörodejeneratif hastalıklardan muzdarip hastaların sayıları artmaya devam ettikçe, böylece daha iyi tedaviler geliştirilen bu hastalıkların moleküler patogenezi anlayışı artırmak için acil bir ihtiyaç vardır. Burada, patojenik protein toplamları model organizma, Drosophila melanogasterfarklı hücre türleri arasında deli dana gibi iletimi izlemek için izin yöntemleri açıklanmaktadır. Biz son zamanlarda mutant Htt toplamları vivo içinde iletimini deli dana gibi göstermek ve n...

Deli dana hipotez devletlerin bulaşıcı transmissible spongiform Encephalopathy (örneğin, Creutzfeldt - Jakob hastalığı insanlarda, koyun, geyik ve elk kronik israf hastalığı ve "deli dana hastalığı" sığır büyük sorumlu ) sadece protein ve nükleik asitler1yoksun oluşmaktadır. Prion hastalıkları, hücresel prion protein (PrPC) son derece beta sayfa zengini ve dönüştürme ve monomeric PrPC molekülleri istikrarlı amiloid işe alma tarafından kendi kendine yayabilirsiniz bir yerel olmayan, istikrarlı kat (PrPSc) varsayar. toplar. PrPSc toplamları bu kendini çoğaltan mekanizması bir canlı farklı hücreler arasında ve hatta bireysel organizmaların2arasında yaymak için kullanın.
Protein misfolding ve toplama Ayrıca merkezi bir özelliğidir çoğu nörodejeneratif hastalıklar (Alzheimer hastalığı (Ah), Parkinson hastalığı (PD), Huntington hastalığı (HD) ve amyotrofik lateral skleroz (ALS))3. Bu hastalıklar derlemelerde içi veya ekstra cellular toplanan protein oluşumunu saat5, yakından ile sitotoksisite4 ve beyin ile son derece tekrarlanabilir ve hastalığa özgü yolları boyunca ilerledikçe ilişkilidir 6. patojen toplamları bu bozuklukları ile ilişkili deli dana gibi özelliklere sahip bu kalıpları yayılan öneririz. Şimdi güçlü desteği vardır deli dana gibi iletim reklam ile ilgili toplamları için PD, HD ve ALS - onlar yaymak hücre-hücre ve şablon monomeric formları daha önce etkilenmemiş hücreleri7, aynı proteinin konformasyon Değiştir 8.
Deli dana gibi protein toplamları bugüne yayılmasını araştıran çalışmalar çoğunluğu memeli hücre kültür modelleri, ekstrasellüler boşluk veya başka bir hücrenin toplamları saf hücre sitoplazma nerede Aktarım'ı kullanarak gerçekleştirilen sitoplazma9,10,11,12,13,14,15, veya toplama içeren malzeme fare beyin enjekte ve izleme Toplu Görünüm dışında Enjeksiyon Makinası16,17,18,19,20,21,22, 23. son zamanlarda Transjenik hayvanlar sağlam beynini24,25,26,27, içinde diğer hücrelere yayılan hücre içi toplamları göstermek için kullanılmıştır 28,29,30. Burada, doğrudan görselleştirme Drosophila melanogastersağlam beynini tek tek hücreleri arasında toplu aktarım için bir yöntem açıklanmaktadır. Drosophila modelleri HD/polyglutamine (polyQ) hastalıkların ilk geliştirilmiş olup, tüm yaklaşık iki yıl önce31,32 ve bu bozuklukların altında yatan patojenik mekanizmaların birçok çok değerli anlayışlar hazırladık 33. HD için protein huntingtin (Htt)34kodları gen bir otozomal dominant mutasyon neden bir devralınan nörodejeneratif bozukluğu olduğunu. Genişleme polyQ abartılı Htt'ın N-terminus bir patojen eşiğinde ötesinde yakınındaki bu mutasyon sonucunda ~ misfold ve toplam35,36protein neden 37 glutamines. Vahşi-türü Htt protein içeren < bu streç içinde 37 glutamines kendi yerel kat elde, ama için indüklenen bir Htt toplama "tohum"12,27,37ile doğrudan fiziksel temas üzerine toplamak. Biz bu homotipik, vahşi tipi Htt çekirdekli toplama deli dana benzeri transfer için bir okuma olarak ve diğer hücrelerde kaynaklanan mutant Htt toplamları sitoplazmik giriş yararlanmak.
Mekanizmaları göre hangi deli dana gibi toplamları belirleme seyahat hücreler arasında tedavi edilemez nörodejeneratif hastalıklar için yeni tedavi hedefleri tanımlaması yol açabilir. Biz almak avantajı hızlı yaşam döngüsünün, kullanım kolaylığı ve hücre-hücre mutant Htt toplamları yayılması için moleküler mekanizmaları tanımlamak için Drosophila melanogaster genetik tractability. Deneysel stratejimiz iki ikili ifade sistemleri Drosophila, köklü Gal4 özgü ters yönde harekete geçirmek sıra (Gal4-UAS) sistem38 ve son zamanlarda geliştirilen QF-QUAS sistemi39uygun kullanmaktadır. Bu iki bağımsız sistem kaplin mutant ve vahşi-type Htt transgenes ifadesi aynı sinek40içinde farklı hücre popülasyonlarının sınırlama sağlar. Bu yaklaşımı kullanarak, biz normalde diffüz, çözünür durumuna gelen sitoplazmik vahşi tipi Htt toplanan bir devlet, önceden biçimlendirilmiş bir mutant Htt ile fiziksel temas doğrudan bir sonucu yeniden dağıtılması izleyerek mutant Htt deli dana gibi yayılan incelemek toplama "tohum." Biyokimyasal kullanarak dönüşüm vahşi tipi Htt mutant Htt tarafından onaylanabilir veya floresan rezonans enerji gibi protein-protein etkileşimleri rapor biyofiziksel teknikleri (FRET)9,27,41 transfer .
Da önemlisi, aynı zamanda genetik genler ve/veya protein toplamları yayılmasını deli dana benzeri aracılık yolları tanımlamak için Drosophila araçlarında çok sayıda erişebilirsiniz. Biz son zamanlarda önemli bir rol için hücre yüzey çöpçü reseptör, mutant Htt toplamları Drosophila Merkezi yakındaki fagositik glia nöronal aksonlar aktarılması içinde Draper42,43, açıklayacak bu yaklaşım kullanılan sinir sistemi (MSS)27. Böylece, biz burada tarif genetik ve görüntüleme tabanlı yaklaşım için kullanımı kolay ama güçlü model organizma, Drosophilahastalık ilgili bir fenomen hakkında önemli temel biyolojik bilgi ortaya çıkarabilir.

<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

Protein toplama Alzheimer hastalığı (Ah), Parkinson hastalığı (PD), Huntington hastalığı (HD) ve amyotrofik lateral skleroz (ALS) de dahil olmak üzere çoğu nörodejeneratif hastalıkların merkezi bir özellik olur. Protein toplamları nevropatoloji bu hastalıklarda ile yakından ilişkili olmakla birlikte hangi normal hücresel homeostazı anormal protein toplama bozan kesin mekanizması bilinmiyor. Veri ortaya çıkan güçlü bir destek hipotez için reklam, PD, HD o patojenik toplamları sağlayan, ve ALS Prionlar, sadece protein bulaşıcı ajanlar için bulaşıcı spongiform Encephalopathy sorumlu birçok benzerlikler. Prionlar şablonu tarafından yerel olarak katlanmış sürümleri toplama fenotip yayılmasını neden aynı Protein çevrimi kendini çoğaltır. Nasıl Prionlar ve deli dana gibi proteinler yılında, PD, HD ve ALS bir hücreden diğerine harekettir şu anda yoğun araştırma alanıdır. Burada, deli dana gibi hücre hücre iletim HD ile ilişkili mutant huntingtin (Htt) toplamları izleme izni Drosophila melanogaster modeli kullanılmıştır. Bu model birçok farklı Drosophila dokularda transgene ifade değiştirmek için güçlü araçlar yararlanır ve doğrudan rapor deli dana benzeri transfer mutant Htt toplamları fluorescently öğesini sitoplazmik protein kullanır. Önemlisi, biz burada tarif yaklaşım roman genler ve protein toplamları farklı hücre türleri içinde vivoarasında yayılan aracılık yollar tanımlamak için kullanılabilir. Bu çalışmalardan elde edilen bilgilerden nörodejeneratif hastalıkların altında yatan ve terapötik müdahale için yeni fırsatlar ortaya patojenik mekanizmaların sınırlı anlayış genişletir.

Yöntem tanımlamak burada sağlam veri bozulmamış anında CNS alanında bir hücre popülasyondan deli dana benzeri transfer Htt protein toplamları gösteren üretmek. Dönüştürülmesine ilişkin yaban tipi Htt diffüz punctate için bu YFP füzyon proteinin doğrudan Floresans tarafından alıcı glia donör ORNs (Resim 2A-C ve şekil 4A, B) HttQ91-mCherry ifade sonucu olarak görülmekte...

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

Kanıt biriken patojenik protein toplamları deli dana gibi özelliklere sahip hücreleri arasında yaymak nörodejeneratif hastalıklar ile ilgili fikir destekler. Burada, hücre hücre yayılma model organizma, Drosophila melanogasterdeli dana gibi toplamları görselleştirme sağlar bir yöntemi açıklanmaktadır.

Drosophila Melanogaster deli dana gibi Protein toplamları izleme hücre hücre iletim

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 Görüntüleme.  University of the Sciences. Kanıt biriken patojenik protein toplamları deli dana gibi özelliklere sahip hücreleri arasında yaymak nörodejeneratif hastalıklar ile ilgili fikir destekler. Burada, hücre hücre yayılma model organizma, Drosophila melanogasterdeli dana gibi toplamları görselleştirme sağlar bir yöntemi açıklanmaktadır.

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.

İntraperitoneal Enfeksiyon sonra Bağışıklık Hücreleri Ticareti Floresan Prion Çubuklar Saat İzlenmesi

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

İmmünoloji ve Enfeksiyon

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.

Içinde Nörodejeneratif fenotipleri değerlendirilmesi<em&gt; Drosophila</em&gt; Tırmanma Tahliller tarafından Dopaminerjik nöronlar ve Tüm Beyin immün

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

Nörobilim

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.

Drosophila'da Lokomotor Aktiviteyi Test Etmek İçin Basit Bir Teknik

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

Davranış

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.

Kitle Histoloji nörodejenerasyon belirlememize<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.

Ateş Drosophila ImageJ kullanarak Fluorescently Tagged proteinlerin tarafsız analizi ile yılında niceliksel hücre biyolojisi

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.

TDP-43 Proteinopatisinin Drosophila Modellerinde Glikoz Alımının Ölçülmesi

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.

Metazoan Model Organizma kullanma Serim ve toksisitesi prion-benzeri Proteinler incelenmesi<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 ...

Biyoloji

Drosophila'da Epileptiform Olayların Kalsiyum Görüntüleme ile İzlenmesi

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.

Yazar Spotlight: Epilepsi Araştırmalarındaki Son Gelişmelerin Tekniklerine ve Bulgularına İlişkin Görüşler 

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 (Canlı Canlı) Drosophila Epilepsi Modeli için Kalsiyum Görüntüleme

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

Drosophila Melanogaster deli dana gibi Protein toplamları izleme hücre hücre iletim

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Ex Vivo (Canlı Canlı) Drosophila Epilepsi Modeli için Kalsiyum Görüntüleme

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Ex Vivo (Canlı Canlı) Drosophila Epilepsi Modeli için Kalsiyum Görüntüleme