Functional RNAi screen to identify pathways essential for protein aggregate clearance and degradation

2008 Pilot Project Read More

Investigators

Principal Investigator: Conrad Weihl, MD, PhD (WUSTL Neurology)
Co-investigator: David Piwnica-Worms, MD, PhD (formerly WUSTL Radiology)

Description

Protein aggregation is the pathologic basis of many neurodegenerative diseases. Protein aggregates form when a protein, either mutant or endogenous, assumes an abnormal conformation and escapes
normal degradative pathways. This results in the accumulation of protein inclusions that are deleterious to the cell and underlies disease pathogenesis. Approaches aimed at decreasing protein
aggregate formation or disaggregating preformed protein aggregates have proven to be reasonable therapeutic targets in some neurodegenerative disease models. However, an alternative approach
may be to enhance the degradation or “clearance” of preformed protein aggregates.  This proposal aims to identify novel pathways involved in the sequestration and clearance of intracellular protein aggregates via autophagy. These novel pathways will serve as potential therapeutic targets aimed at decreasing aggregate burden in a cell.

To do this we will utilize a prototypical aggregate prone protein, the expanded polyglutamine (polyQ) repeat containing protein. Expanded polyQ repeats are present in many aggregate prone proteins associated with neurodegenerative diseases such as mutant huntingtin protein in Huntington’s disease, ataxin-3 in spinocerebellar ataxia type-3 and the androgen receptor in spinobulbar muscular atrophy. While an unexpanded polyQ repeat containing protein is soluble, once a polyQ repeat expands to a critical number, it develops the propensity to misfold, form fibrils and aggregate into inclusion bodies. We constructed two luciferase based reporter constructs 1) a non-aggregating polyQ19-luciferase and 2) an aggregate prone expanded polyglutamine containing luciferase, polyQ80-luciferase. Enhancement of autophagy via the mTOR inhibitor rapamycin or nutrient deprivation selectively decreases the polyQ80-luciferase activity as compared with polyQ19-luciferase in cells. Our preliminary studies show that these reporter constructs are sensitive to autophagic activation and inhibition in a time and concentration dependent manner. Moreover, these reporters can be used in vivo to measure the autophagic clearance of protein aggregates in live animal tissue.

Publications

Fuentealba RA, Marasa J, Diamond MI, Piwnica-Worms D, Weihl CC. An aggregation sensing reporter identifies leflunomide and teriflunomide as polyglutamine aggregate inhibitors. Human Molecular Genetics. 21(3):664-80 (2012).

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Hope Center Investigator

Conrad (Chris) Weihl