The Fruit Fly as a Possible Model of DRPLA: Directed Gene Expression to Drive the Rescue of Atrophin Deficient D. melanogaster

Because of its short generation time and the powerful genetic tools available, the fruit fly has proven itself an invaluable tool for the study of human neurological disorders. Drosophila melanogaster models have been created for several neurodegenerative disease including Alzheimer’s, Parkinson’s and Huntington’s. Dentatorubral-pallidoluysian atrophy (DRPLA) is a rare disorder, inducing neurodegeneration by expansion of the Atrophin1 (Atr) protein in human brains. Flies produce their own variant of Atr, designated DAtr, sharing 38% homology with the human Atr. While this is a high conservation for an invertebrate, it is currently unknown if Drosophila would be an effective model for the disease. This study aims to assess the correlation of function between human Atrophin and fly DAtr. At the core of the project are a series of rescue crosses utilizing directed gene expression i.e. the Upstream Activating Sequence (UAS)/Gal4 scheme. This system relies on one line of the transgenic animal containing the Gal4 activator being crossed to a gene of interest inserted with the UAS in another line; the progeny of aforementioned lines will express the gene of interest. The Gal4 line was also matched with a mutant allele of DAtr, atroe46-2, while the UAS lines were matched with a different mutant, gug35. In a heterozygous state, these mutations are viable with life, but in trans heterozygotes, they are lethal at an embryonic stage. The progeny of the UAS lines and the Gal4 lines contain both mutations, and thus should die unless rescued by expression of the UAS transgene. We created seven stocks, each with a different gene of interest behind the UAS: (1) DAtr (positive control), (2) EFGP (negative control), (3) Wild type Atr2, (4) Wild type Atr2 short, (5) short DAtr (lacking 270 a. acids) and (6) short DAtr (lacking 169 a. acids). Lines 3 and 4 are wildtype mammalian alleles while lines 5 and 6 each lack part of the N terminal of DAtr. Each of these transgenic lines were crossed to the Gal4 enhancer line, and the progeny analyzed for rescues. The positive control has been rescued to a very late pupa stage, showing virtually perfect morphology. In the confined context of the UAS scheme, this is the best rescue achieved. In contrast, the EFGP cross produced no rescues. It is expected that the remaining variants show rescues in between. Rescue 4, the short mammalian atrophin showed no rescue while the full version, Rescue 3, surprisingly rescued to a pupal stage, almost as fully developed as our positive control. Additionally, Rescue 6 may have shown a slight larval rescue, while Rescue 5 was identical to the negative. The conclusion of these results suggest that (1) Mammalian and Drosophila Atrophin may share similar functions in the developing organism, and (2) the 101 a. acid difference from Rescues 5 and 6 play a crucial role in the development of the fly. It is our hope that these findings pioneer research into weather the fly can serve as a viable model for DRPLA. Supported by NIH-MHIRT Grant MD-01485