Parameters Affecting the Pathogenicity of Huntingtin Protein

Abstract

This work explores mechanisms of pathology in Huntington's Disease (HD) in an attempt to identify attractive therapeutic targets. By inserting the gene for human huntingtin (HTT) into the genome of the fruit fly Drosophila melanogaster, we have reproduced the main features of HD. Using this model, we sought to explore the inherent qualities of specific fragments of mutant huntingtin protein (mHtt), and the role of modifier genes on the progression of pathology in the context of an intact, living organism. These studies contribute to a basic understanding of the molecular basis of HD pathogenic progression as well as providing a guide to selection of targets of therapeutic value.

Flies expressing mutant huntingtin display a range of pathology, including developmental abnormalities, neurodegeneration, movement deficits, reduction in longevity, biochemically detectable accumulation of mutant huntingtin protein, and microscopically visible inclusion body formation. Previous research has established that the 3144 aa Htt protein is processed by various endogenous proteolytic enzymes, releasing a series of N-terminal fragments of the huntingtin protein. Many of these fragments have been studied, utilizing a variety of model systems, and it has been suggested that some fragments can play a critical role in HD pathology while others do not. In this work I have compared the most commonly studied fragments side-by-side in flies with a common genetic background to determine their intrinsic pathogenic potential and biophysical behavior. Our findings indicate that the exon 1 fragment is clearly the most toxic of the naturally occurring fragments.

Previous research has also suggested that the small ubiquitin-like modifying protein SUMO plays an important role in HD pathology. We pursued this by genetically modifying the activity of SUMO in flies. Our results suggest that while SUMO does play a role, it is not primarily in the direct modification of huntingtin, and that modulation of SUMO-specific isopeptidases is unlikely to be an attractive therapeutic strategy.

Our studies also led to the investigation of the SUMO-related protein NEDD8. We found that while the NEDD8 pathway may play a role in HD pathology, it also affects the mechanics of the GAL4/UAS transgenic expression system. This is an important caveat for future investigations of this nature.

Finally, to address the long-standing problem of quantifying the levels of Htt protein in the face of aggregation and anomalous effects on secondary structure, we compared a number of methods of quantifying the amount of huntingtin in our samples. We find that time-resolved fluorescence resonance energy transfer detects greater relative levels of monomeric small fragments compared to western blotting and immunostaining, raising important questions about what parameters influence measurements of Htt in the different methods.