これらのメソッドの開発の間に多くの役に立つ議論の Kopito、羅、ピアースのラボのメンバーに感謝いたします。我々 はまた、この原稿の批判的読解のブライアン Temsamrit を感謝します。この作品は、大学科学大戦スミスの公益信託からの資金によって支えられました。

著者が明らかに何もありません。

神経変性疾患の患者数は増加し続けているより良い治療法を開発することができますので、これらの疾患の病態の理解を高めるための緊急の必要性があります。ここでは、監視モデル生物、ショウジョウバエの異なる細胞型の間の病原性タンパク質凝集体のプリオンのような通信を可能にする方法をについて説明します。最近、突然変異体 Htt 集計生体内でのプリオンのような?...

プリオン仮説では、感染性病原体 (例えばヒトのクロイツ フェルト ・ ヤコブ病、スクレイピー羊、鹿、ヘラジカ、慢性消耗性疾患や牛の「狂牛病」の伝達性海綿状脳症を担当) 蛋白質と核酸1を欠いているだけで構成されています。プリオン病の細胞プリオンタンパク質 (PrPC) ではネイティブではない、安定した倍 (PrPSc) 高ベータ版シートが豊富な自己に変換し、安定したアミロイドに単量体の PrPC分子を募集を反映することを前提としています集計します。PrPSc集合体は、生物に異なる細胞の間とも個々 の生物2を広げるためこの自己複製機構を使用します。
タンパクはまたほとんどの神経変性疾患 (アルツハイマー病 (AD)、パーキンソン病 (PD)、(HD)、ハンチントン病、筋萎縮性側索硬化症 (ALS)) の中央特徴3。これらの疾患の内、または極細胞集計タンパク質複合体の形成は時間5,細胞毒性4と脳を高い再現性で疾患固有のパスに沿って進行と密接に関連6。 普及のこれらのパターンはプリオンのような性質があるこれらの疾患に関連付けられている病原性の骨材をお勧めします。広告に関連付けられている集計のプリオンのような伝達のための強力なサポートが今存在する PD、HD、および ALS - 彼らは細胞間およびテンプレート以前影響を受けない細胞7、同じ蛋白質の単量体の形態の構造変化から広がる 8。
集計は、ナイーブ細胞の細胞質に転送場所細胞外スペースから、またはから別のセルの哺乳類の細胞培養モデルを使用して実行された日付にタンパク質凝集体のプリオンのような広がりを調査し、研究の大半細胞質9,10、11,12,13,14,15、または骨材含有材料をマウスの脳に注入し、監視インジェクション サイト16,17,18,19,20,21,22,の外の出現を集計23. 最近では、トランスジェニック動物の細胞内凝集体は、そのまま脳24,25,26,27、内の他のセルに広がったことを示すに使用されています。 28,29,30。ここでは、ショウジョウバエのまま脳の個々 のセルの間の転送の直接可視化の手法について述べる。HD/ポリグルタミン (polyQ) 症ショウジョウバエモデル最初ほぼ二十年前に開発された31,32これらの疾患の根底にある病原性のメカニズムに多くの非常に貴重な洞察力を提供しています。33. HD はタンパク質 huntingtin (Htt)34をコードする遺伝子の常染色体の優性突然変異によって引き起こされる継承した神経変性疾患。この突然変異の結果、polyQ ストレッチの病原性のしきい値を超えて Htt の N 末端付近の展開 〜 37 glutamines、ミスフォールドタンパクし集計35,36タンパク質を引き起こしています。野生型 Htt 蛋白質を含む < このストレッチで 37 glutamines そのネイティブの倍を実現するに誘導することができます、Htt 集計「シード」12,27,37直接物理的な接触時に集計。このエネルギーは、プリオンのような転送用読み出しとして野生型 Htt の核の集約と変異 Htt 集計元の他のセルの細胞内のエントリを利用します。
プリオンのような集計でメカニズムを決定する細胞間の旅行は難治性神経変性疾患の新たな治療標的の同定に します。我々 は、迅速なライフ サイクルの利点は、使いやすさと変異 Htt 集計の細胞間伝播の分子メカニズムを定義するショウジョウバエの遺伝学的トレーサビリティを取る。私たちの実験的戦略では、ショウジョウバエ、老舗 Gal4 固有上流活性化シーケンス (Gal4 UAS) システム38および最近開発された QF 擬システム39で利用可能な 2 つの二項式システムを採用しています。これらの 2 つの独立したシステムを結合には、同じフライ40内の異なる細胞集団に突然変異体と野生型の Htt 遺伝子の表現を制限することができます。このアプローチを使用して、集約の状態に前もって形成された変異体 Htt と物理的に接触の直接の結果、通常拡散反射光の可溶性の状態から細胞質の野生型 Htt の再分配を監視することによって変異体 Htt の広がっているプリオンのようなを調べた「seed」を集計生化学的変異 Htt 野生型 Htt の転換を確認ことができますまたは蛍光共鳴エネルギーなどの蛋白質蛋白質の相互作用の報告生物物理技術移転 (FRET)9,27,41.
重要なは、我々 も多数の遺伝子やタンパク質凝集体のプリオンのような広がりを仲介する経路を識別するためにショウジョウバエの遺伝的ツールにアクセスできます。我々 は最近細胞表面のスカベン ジャー受容体、ドレーパー42,43,ショウジョウバエ中心部の近くに貪食グリアに神経細胞の軸索から突然変異体 Htt 集計を転送するのに重要な役割を明らかにするのにこのアプローチを使用しています。神経系 (CNS)27。したがって、ここで述べる遺伝とイメージング ベースのアプローチは、使用する簡単な強力なモデル生物、ショウジョウバエが病気に関連する現象についての重要な基本的な生物学的情報を表示できます。

<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

ほとんどの神経変性疾患、アルツハイマー病 (AD)、パーキンソン病 (PD)、ハンチントン病 (HD)、筋萎縮性側索硬化症 (ALS) などの中枢機能が蛋白質の集計です。異常な蛋白質の集合、通常生体のホメオスタシスを混乱させる正確なメカニズムは知られていないが、タンパク質凝集体が密接に関連付けられてこれらの疾患の神経病理学。データを新たな広告、PD、HD でその病原性骨材仮説のための強力なサポートを提供し、ALS プリオンは、タンパク質だけ感染性病原体伝染性海綿状脳症の責任は、多くの類似点があります。プリオンは自己テンプレートによって集約表現の広がりを引き起こす同じ蛋白質のネイティブに折り返されているバージョンの変換を複製します。どのようにプリオンとプリオン様タンパク質の広告、PD、HD、および ALS 1 つのセルから別への移行が強烈な調査の領域では現在。ここでは、HD に関連する変異ハンチンチン (Htt) 集計のプリオンのような細胞間伝達の監視を許可するショウジョウバエモデルを説明します。このモデルは多くの異なるショウジョウバエのティッシュの遺伝子発現を操作するための強力なツールを活用して、直接変異 Htt 骨材のプリオンのような転送を報告する蛍光タグ細胞質蛋白質を利用しています。重要なは、我々 はここで説明するアプローチは、新規遺伝子および蛋白質集積体はさまざまな細胞のタイプの生体間の広がりを仲介する経路を識別するために使用できます。これらの研究から得られる情報は、神経変性疾患の根底にある、治療的介入のための新しい機会を明らかにする病原性のメカニズムの限られた理解を拡大します。

ここで説明する方法は、そのままフライ中枢神経系内の別の 1 つのセルの人口から Htt タンパク質凝集体のプリオンのような転送を示す堅牢なデータを生成します。点状に拡散反射光から野生型 Htt の変換は、この YFP 融合タンパク質の直接蛍光 HttQ91 mCherry ドナー ORNs (A ~ Cの図 2と図 4A, B) に式の結?...

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

病原性タンパク質凝集体がプリオンのようなプロパティを持つセルの間に広がって神経変性疾患に関連する考えを支持する証拠を蓄積します。ここでは、モデル生物、ショウジョウバエのプリオンのような骨材の細胞-細胞拡散の可視化を可能にする手法について述べる。

キイロショウジョウバエにおけるプリオン様蛋白質集合体の細胞間伝達を監視

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 ビュー.  University of the Sciences. 病原性タンパク質凝集体がプリオンのようなプロパティを持つセルの間に広がって神経変性疾患に関連する考えを支持する証拠を蓄積します。ここでは、モデル生物、ショウジョウバエのプリオンのような骨材の細胞-細胞拡散の可視化を可能にする手法について述べる。

ビデオ: 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.

腹腔内感染後に免疫細胞の人身売買蛍光プリオンロッドの時間を監視

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

免疫・感染学

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.

神経変性表現型を評価する<em&gt;ショウジョウバエ</em&gt;クライミングアッセイによりドーパミン作動性ニューロンと脳全体の免疫染色

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

神経科学

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.

ショウジョウバエの自発運動活性をアッセイする簡単な技術

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

行動学

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.

に神経変性を定量化するために、マス組織学<em&gt;ショウジョウバエ</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.

ImageJ を用いた蛍光タグ付きタンパク質の公平な分析によるショウジョウバエの神経変性の定量細胞生物学

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蛋白症の ショウジョウバエ モデルにおけるグルコース取り込みの測定

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.

拡散とプリオン様タンパク質の毒性は後生動物モデル生物を用いて調査<em&gt; C。エレガンス</em

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

生物学

カルシウムイメージングによる ショウジョウバエ のてんかん様イベントのモニタリング

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.

著者スポットライト:てんかん研究の最近の進歩の技術と調査結果への洞察 

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(エクス・ビボ )てんかんのショウ ジョウバエ モデルに対するカルシウムイメージング

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

キイロショウジョウバエにおけるプリオン様蛋白質集合体の細胞間伝達を監視

要約

さらに動画を探す

この動画の章

腹腔内感染後に免疫細胞の人身売買蛍光プリオンロッドの時間を監視

神経変性表現型を評価する

ショウジョウバエの自発運動活性をアッセイする簡単な技術

に神経変性を定量化するために、マス組織学

ImageJ を用いた蛍光タグ付きタンパク質の公平な分析によるショウジョウバエの神経変性の定量細胞生物学

TDP-43蛋白症のショウジョウバエモデルにおけるグルコース取り込みの測定

拡散とプリオン様タンパク質の毒性は後生動物モデル生物を用いて調査

Ex Vivo(エクス・ビボ )てんかんのショウジョウバエモデルに対するカルシウムイメージング

Ex Vivo(エクス・ビボ )てんかんのショウジョウバエモデルに対するカルシウムイメージング

Ex Vivo(エクス・ビボ )てんかんのショウジョウバエモデルに対するカルシウムイメージング

キイロショウジョウバエにおけるプリオン様蛋白質集合体の細胞間伝達を監視

要約

さらに動画を探す

この動画の章

腹腔内感染後に免疫細胞の人身売買蛍光プリオンロッドの時間を監視

神経変性表現型を評価する

ショウジョウバエの自発運動活性をアッセイする簡単な技術

に神経変性を定量化するために、マス組織学

ImageJ を用いた蛍光タグ付きタンパク質の公平な分析によるショウジョウバエの神経変性の定量細胞生物学

TDP-43蛋白症の ショウジョウバエ モデルにおけるグルコース取り込みの測定

拡散とプリオン様タンパク質の毒性は後生動物モデル生物を用いて調査

Ex Vivo(エクス・ビボ )てんかんのショウ ジョウバエ モデルに対するカルシウムイメージング

Ex Vivo(エクス・ビボ )てんかんのショウ ジョウバエ モデルに対するカルシウムイメージング

Ex Vivo(エクス・ビボ )てんかんのショウ ジョウバエ モデルに対するカルシウムイメージング

TDP-43蛋白症のショウジョウバエモデルにおけるグルコース取り込みの測定

Ex Vivo(エクス・ビボ )てんかんのショウジョウバエモデルに対するカルシウムイメージング

Ex Vivo(エクス・ビボ )てんかんのショウジョウバエモデルに対するカルシウムイメージング

Ex Vivo(エクス・ビボ )てんかんのショウジョウバエモデルに対するカルシウムイメージング