Evaluating the concordance of N-terminal and full length Huntington's disease modifiers and identifying potential therapeutic targets in Drosophila

Huntington’s disease (HD) is one of nine polyglutamine diseases and it is caused by a CAG expansion in the HTT gene. HD is an autosomal, dominantly inherited neurodegenerative disease affecting between 2 and 5 individuals per 100,000 worldwide and it is currently untreatable. HD spreads from the striatum to the rest of the brain and causes widespread motor, cognitive, and psychiatric symptoms, including Huntington’s chorea. A fruitful approach to identifying potential therapeutic targets for HD is to modify genes in a model organism in an unbiased manner and screen the effect by testing the model in a functional assay. Drosophila models of HD have emerged as key tools for these large scale genetic screens thanks to their combination of ease of maintenance, and breeding in large numbers and their ability to be tested neurobehaviorally. During the course of HD pathogenesis in mammals, the FL-HTT protein is cleaved by many proteases including caspase-6. This cleavage leads to the co-existence of N-terminal (NT) as well as full-length (FL) forms of mutant HTT in the HD neurons. Drosophila lacks caspase-6 therefore FL-HTT is not naturally cleaved at its target site, this allows us to express either the FL mutant HTT or its cleaved NT fragment independently to characterize their differential pathogenic contribution. This study aims to test the concordance of a sample of 75 NT-HTT modifiers identified through a directed screen by testing them in a FL-HTT model. In doing so, we hope to identify shared modifiers and shared functional genetic networks, which may be particularly central to HD progression and useful areas in which to discover therapeutic targets. Further, this study may help to determine what types of models are necessary for future screens to adequately understand the genetic networks that underpin HD progression. In order to assess the impact of the modifier, flies expressing both the modifier and FL-HTT were tested in a climbing assay that measures motor function taking advantage of the model’s innate negative geotactic behavior. Motor performance is measured as the percentage of flies of each genotype that climb up to a 9 cm threshold in a given time interval. Flies were tested at 6 time points on days 18, 19, 20, 21, 22, and 25 of age in order to observe their level of neurobehavioral function in comparison to a positive control of flies with FL-HTT and no modifier and a negative control of flies without mutant HTT. When NT modifiers were tested in the FL model, there was an enrichment in modifiers relative to what is seen by chance. The NT suppressor sample was significantly enriched in modifier genes that effected motor performance in the FL model. Meanwhile, NT HD enhancers were not enriched with modifiers in the FL model. Some modifiers demonstrated contradictory effects on motor performance depending on the HD model tested. This could be caused by different mechanisms of toxicity inherent to NT versus FL HD or from secondary toxicity as the FL experiment occurred over a longer time period and flies were aged at a higher temperature. There was particular enrichment of modifiers in the calcium signaling and inflammation and cytoskeleton stress response pathways, which are robust functional gene networks identified by previous gene screening. These findings suggest that these shared networks are particularly central to HD progression and both are involved in inhibiting a cell’s ability to cope with stress and promoting excitotoxicity. The aforementioned pathogenic features are associated with impaired autophagy, which many see as the key to HTT clearance and ultimately rescuing neurons from degradation and curing HD. As genetic screening continues in Drosophila, shared networks between models have the potential to reveal new therapeutic targets and broaden our understanding of the mechanisms that lead to HD progression. These lessons will be essential as whole genome unbiased screenings in Drosophila continue and our networks become more robust and interconnected. Despite the enrichment in shared modifiers, our results show not infrequent contradictory effects on motor performance when NT modifiers are tested in the FL model. As such, we suggest that future screens test both FL and NT models independently to best study the causes of HD and to help identify the shared modifiers and networks, which are promising areas to mine for therapeutic targets