Project: Research project

Project Details


Background: The major protein component of the inclusions found in amyotrophic lateral sclerosis (ALS) was recently identified as TAR DNA-binding protein-43 (TDP-43). Several mutations in the gene encoding TDP-43 have since been identified in sporadic and familial ALS patients. Studies in yeast reveal that C-terminal TDP-43 fragments are prone to aggregate, and that only TDP-43 species that form inclusions are toxic. Work from our laboratory has shown that the aggregation of C-terminal TDP-43 fragments corresponding to caspase truncation products is detrimental to mammalian cells through a toxic gain of function. These results indicate that the aggregation of TDP-43 may play an important role in the pathogenesis of ALS.Objective/Hypothesis: We hypothesize that compounds that prevent TDP-43 aggregation will provide neuroprotection in ALS. The objective of this project is to employ our novel cell model system that mimics the aggregation of TDP-43 observed in ALS to screen a small-molecule library for the identification of agents that block TDP-43 aggregation and toxicity.Specific Aims:1) Refine our inducible cell model and validate its use for high-throughput screening of compounds with therapeutic potential for the treatment of ALS, and establish secondary and tertiary screening models. We have established a human neuroblastoma cell line (M17D3) that inducibly expresses an enhanced GFP-tagged C-terminal TDP-43 truncation product, GFP-TDP220-414. GFP-TDP220-414 expression leads to the formation of cytoplasmic, caspase-3-positive, inclusions. The goal of this aim is to establish sensitive, nonbiased, and rapid methods to measure inclusion burden and toxicity in M17D3 cells. Also, secondary or tertiary screening models to be using in Specific Aims 2 and 3, will be produced and characterized.2) Conduct high-throughput screens with our inducible cell model to identify lead agents for ALS treatment. M17D3 cells will be used to screen our proprietary small-molecule library for the identification of anti-aggregative and cytoprotective compounds. This library was assembled for us by Chembridge Corporation and is composed of ~58,000 compounds that were specifically chosen because they meet Lipinski's rules and have structural characteristics consistent with increased probability of oral bioavailability and blood brain barrier penetration.3) Validate compounds of interest using secondary and tertiary cell models. A subset of anti-aggregative and cytoprotective compounds will subsequently be validated in human neuroblastoma cells expressing GFP-TDP208-414 and in primary cortical and motor neuron cultures expressing GFP-TDP220-414.Study Design: To establish sensitive and rapid methods to measure inclusion burden in Specific Aim 1, we will compare two different approaches. GFP levels in M17D3 cells in 96-well plates will be measured by fluorescence microplate-reader or by in-cell Western, a method we previously developed for drug screening. We will determine which assay is the most robust for drug screening by calculating the Z-factor for each. A LDH assay will be used to quantify cytotoxicity. In Specific Aim 2, ~58,000 drugs will be screened for their ability to both decrease GFP fluorescence and LDH levels in M17D3 cells. Of those compounds showing anti-aggregation and cytoprotective properties, a carefully designed decision tree will be used to select a subset of compounds based on potency, safety/toxicity, specificity and structural diversity. In Specific Aim 3, this subset of compounds will be validated in neuroblastoma cells expressing GFP-TDP208-414 or in primary neuronal cultures expressing GFP-TDP220-414.Innovation: This project will take advantage of our novel cell model that recapitulates a pathological hallmark of ALS, namely the aggregation of TDP-43, to screen a small-molecule library for the identification of agents that attenuate TDP-43 aggregation and toxicity.Impact: Since the majority of ALS cases show TDP-43-positive inclusions, we believe that the identification of compounds that target TDP-43 aggregation and toxicity will lead to the development of therapeutic agents that will benefit most ALS patients, including those that may have developed ALS due to their service in the military.

Effective start/end date1/1/097/31/13


  • Congressionally Directed Medical Research Programs: $1,377,000.00
  • U.S. Department of Defense: $1,377,000.00


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