Western Blot Cell-Based Dose Response to Identify Inhibitors of Binding of Alpha-Synuclein Translation in H4 Cells
Jack T. Rogers, Massachusetts General Hospital, firstname.lastname@example.org, 617-726-8838, Charlestown, MA ..more
BioActive Compound: 1
Broad Institute MLPCN Alpha-Synuclein 5'UTR Project
Project ID: 2023
Keywords: alpha-synuclein, Parkinson's disease, translation, RNA stem-loop, 5'-untranslated region
Primary Collaborators (and laboratory where this assay was performed):
Jack T. Rogers, Massachusetts General Hospital, email@example.com, 617-726-8838, Charlestown, MA
The goal of this project is to identify novel small molecule probes that inhibit alpha-synuclein translational expression in dopaminergic neurons by targeting the 5'-untranslated region (5'UTR) stem-loop of alpha-synuclein as a major new therapeutic target to retard the progression of Parkinson's disease (PD). The 5'UTR of alpha-synuclein mRNA can interact with Iron Regulatory Protein-1 (IRP1), which upon interaction causes an increase in alpha-synuclein mRNA translation. Probes that can successfully reduce alpha-synuclein expression levels as measured in this luciferase reporter assay will be further tested for specificity in cells lacking the alpha-synuclein 5'UTR stem-loop to confirm that probes are acting through the intended target. Probe selectivity will be tested in cells containing the prion protein 5'UTR mRNA stem-loop, which is fused to a luciferase reporter.
In the Western blot orthogonal screen confirmation of alpha-synuclein translation inhibitors was sought. The Western used H4 neuroblastoma cells that were not transfected. Compound was tested in 4-point dose in an effort to observe a dose-response effect.
Inhibitors of alpha-synuclein translation should decrease alpha-synuclein protein levels without affecting actin levels.
Cell Culture and Preparation of Lysates:
1. Human H4 neuroglioblastoma cells were cultured in Dulbeccos modified essential medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and penicillin/streptomycin (Bio-Whittaker, Walkerville, MD). Cells were exposed to increasing concentrations of compound (0, 0.1, 1, 10, 100 micromolar) for 48 hours.
2. Cytoplasmic protein lysates were prepared by homogenizing the cells in midRIPA buffer (25 mM Tris pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 15 mM NaCl, protease inhibitors, RNase inhibitor and 10 mM DTT).
1. Western blotting for alpha-synuclein was performed using mouse monoclonal anti-alpha-synuclein (BD Transduction Laboratories), and anti-beta-actin (Chemicon).
2. The blots were developed using chemiluminescence (PIERCE) and visualized with a PhosphoImager (BioRad, Hercules, CA), and bands were quantified using QuantityOne software (BioRad).
Dose Response Data Analysis:
Negative control condition (DMSO) was included.
SNCA Correction Factor was calculated as:
Density SNCA at neg/ Density Actin at neg
Percent inhibition of SNCA expression was calculated based on the assumption that complete (100% ) inhibition is attainable, as follows:
100 - ((100/Correction Factor) * Density SNCA at dose/ Density Actin at dose)
EC50 was determined to be the lowest dose that supported >= 50% of Maximum Observed Activity.
Inactive compounds = 0
Active compounds = -10 * Log(EC50)
Activity_Outcome = 1 (inactive)
Dose Response is not observed.
Activity_Outcome = 2 (active)
Dose Response is observed with an EC50 < 10uM.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Cell Type: H4
Assay Type: Binding
* Activity Concentration. ** Test Concentration.
Data Table (Concise)