Abstract
The aggregation of α-synuclein (Syn or S) to form insoluble fibrils is important in the pathogenesis of Parkinson’s disease, but key risk factors remain ill-defined. We have developed Fluorescence Resonance Energy Transfer (FRET)-based assays for α-synuclein aggregation, using Green Fluorescent Protein variants Cerulean (C) or Venus (V), fused to each other (CV, VC) or to human synuclein (SC, SV etc). Bacterially expressed proteins were purified to homogeneity, and C-terminal fusions SC and SV largely retained their ability to aggregate in vitro. FRET signals from mixtures of SC and SV were used to monitor aggregation. These fusion genes were linked to the C. elegans unc-54 myosin promoter to generate integrated transgenic strains. Increased FRET signals, indicative of S aggregation, were observed following treatment of unc-54::SC + unc-54::SV double transgenic worms with low concentrations of mercury or chlorpyrifos, or with RNAi against hsp-70 and hip-1. Opposite changes in Yellow Fluorescent Protein (YFP) fluorescence in an unc-54::SV strain (NL5901) are likely to reflect FRET from Yellow Fluorescent Protein to aggregates of Syn fusion protein. This could provide the basis for a high throughput screening assay, which could be used for studying the effects of toxic chemicals and environmental pollutants on the aggregation of proteins such as Syn in vivo.
Keywords: FRET, C. elegans, α-synuclein, aggregation, pollutants, fluorescent proteins.
CNS & Neurological Disorders - Drug Targets
Title:A Fluorescence Resonance Energy Transfer Assay For Monitoring α- Synclein Aggregation in a Caenorhabditis Elegans Model For Parkinson’s Disease
Volume: 14 Issue: 8
Author(s): Archana Nagarajan, Rakesh Bodhicharla, Jody Winter, Charumathi Anbalagan, Kevin Morgan, Mark Searle, Aamir Nazir, Ademola Adenle, April Fineberg, Declan Brady, Kelly Vere, Jo Richens, Paul O'Shea, David Bell and David de-Pomerai
Affiliation:
Keywords: FRET, C. elegans, α-synuclein, aggregation, pollutants, fluorescent proteins.
Abstract: The aggregation of α-synuclein (Syn or S) to form insoluble fibrils is important in the pathogenesis of Parkinson’s disease, but key risk factors remain ill-defined. We have developed Fluorescence Resonance Energy Transfer (FRET)-based assays for α-synuclein aggregation, using Green Fluorescent Protein variants Cerulean (C) or Venus (V), fused to each other (CV, VC) or to human synuclein (SC, SV etc). Bacterially expressed proteins were purified to homogeneity, and C-terminal fusions SC and SV largely retained their ability to aggregate in vitro. FRET signals from mixtures of SC and SV were used to monitor aggregation. These fusion genes were linked to the C. elegans unc-54 myosin promoter to generate integrated transgenic strains. Increased FRET signals, indicative of S aggregation, were observed following treatment of unc-54::SC + unc-54::SV double transgenic worms with low concentrations of mercury or chlorpyrifos, or with RNAi against hsp-70 and hip-1. Opposite changes in Yellow Fluorescent Protein (YFP) fluorescence in an unc-54::SV strain (NL5901) are likely to reflect FRET from Yellow Fluorescent Protein to aggregates of Syn fusion protein. This could provide the basis for a high throughput screening assay, which could be used for studying the effects of toxic chemicals and environmental pollutants on the aggregation of proteins such as Syn in vivo.
Export Options
About this article
Cite this article as:
Nagarajan Archana, Bodhicharla Rakesh, Winter Jody, Anbalagan Charumathi, Morgan Kevin, Searle Mark, Nazir Aamir, Adenle Ademola, Fineberg April, Brady Declan, Vere Kelly, Richens Jo, O'Shea Paul, Bell David and de-Pomerai David, A Fluorescence Resonance Energy Transfer Assay For Monitoring α- Synclein Aggregation in a Caenorhabditis Elegans Model For Parkinson’s Disease, CNS & Neurological Disorders - Drug Targets 2015; 14 (8) . https://dx.doi.org/10.2174/1871527314666150821110538
DOI https://dx.doi.org/10.2174/1871527314666150821110538 |
Print ISSN 1871-5273 |
Publisher Name Bentham Science Publisher |
Online ISSN 1996-3181 |
- Author Guidelines
- Graphical Abstracts
- Fabricating and Stating False Information
- Research Misconduct
- Post Publication Discussions and Corrections
- Publishing Ethics and Rectitude
- Increase Visibility of Your Article
- Archiving Policies
- Peer Review Workflow
- Order Your Article Before Print
- Promote Your Article
- Manuscript Transfer Facility
- Editorial Policies
- Allegations from Whistleblowers
Related Articles
-
Treatment for Obsessive Compulsive Disorder
Current Psychiatry Reviews A Critical Evaluation of Adenosine A2A Receptors as Potentially “Druggable” Targets in Huntingtons Disease
Current Pharmaceutical Design The 5-HT1B Receptor: A Novel Target for the Pathophysiology of Depression
Current Drug Targets Probabilistic Cost-Effectiveness Analysis of Vaccination for Mild or Moderate Alzheimer’s Disease
Current Alzheimer Research Structure and Function of Small Non-Peptide CRF Antagonists and their Potential Clinical Use
Current Molecular Pharmacology Substrate Specificity of Rat DESC4, a Type II Transmembrane Serine Protease
Protein & Peptide Letters Induction of Tumour Cell Senescence: A New Strategy in Anticancer Treatment
Medicinal Chemistry Reviews - Online (Discontinued) Clinical Use of Aspirin in Ischemic Heart Disease: Past, Present and Future
Current Pharmaceutical Design Dysregulation of RNA Mediated Gene Expression in Motor Neuron Diseases
CNS & Neurological Disorders - Drug Targets L-Glutamate and its Ionotropic Receptors in the Nervous System of Cephalopods
Current Neuropharmacology How and Why to Screen for CYP2D6 Interindividual Variability in Patients Under Pharmacological Treatments
Current Drug Metabolism Discovery of Novel Lead in the Group of N-substituted Piperazine Ether Derivatives with Potential Histamine H<sub>3</sub> Receptor Activity
Medicinal Chemistry Ethosomes as Novel Vesicular Carrier: An Overview of the Principle, Preparation and its Applications
Current Drug Delivery Impulse Control Disorders in Parkinson’s Disease: A Review
Current Psychiatry Reviews Targeting Neurotrophic Signal Transduction Pathways in the Treatment of Mood Disorders
Current Signal Transduction Therapy Recent Pharmacoproteomics Studies of Warfarin in the Asia-Pacific: A New Strategy for Personalized Medicine?
Current Pharmacogenomics and Personalized Medicine Statins and Carotid Intima-Media Thickness Reduction: An Up-to-Date Review
Current Medicinal Chemistry In Silico Modeling of P450 Substrates, Inhibitors, Activators, and Inducers
Combinatorial Chemistry & High Throughput Screening Recent Developments on Multi-Target-Directed Tacrines for Alzheimer's Disease. I. The Pyranotacrines
Current Topics in Medicinal Chemistry Potential of Photobiomodulation to Induce Differentiation of Adipose- Derived Mesenchymal Stem Cells into Neural Cells
Current Stem Cell Research & Therapy