Characterization of the Sirt6 Knockout in Drosophila Melanogaster

Abstract

Parkinson’s disease (PD) is a movement disorder that is characterized by the loss of dopaminergic neurons in the midbrain. It is known that midbrain dopaminergic neurons demonstrate differential vulnerability to Parkinson’s disease pathology and aging. Here, I took a novel approach to elucidate the molecular mechanisms of this differential vulnerability to PD-induced pathology. Specifically, I carried out a meta-analysis of published human-derived microarray datasets to identify gene candidates that show a similar expression signature in populations of resistant neurons, such as the medial substantia nigra and the ventral tegmental area (VTA) neurons, and an altered signature in vulnerable neurons, such as those of the lateral substantia nigra. I identified Sirt6, which codes for a component of the DNA repair complex, as a candidate neuroprotective gene as it has high levels of expression in the VTA and the medial substantia nigra and low expression levels in the lateral substantia nigra. I used immunohistochemical analysis to confirm this differential expression pattern of Sirt6 in the midbrain of wild type mice. To further investigate the effects of altered Sirt6 expression on the nervous system, I used CRISPR genome editing tools to create a Sirt6 knockout Drosophila melanogaster model. I use histological analyses to show that the recessive mutation leads to early-onset neurodegeneration, as evidenced by a doubling of neuropil vacuolization in 30-day-old flies. I use behavioral studies to demonstrate that mutant flies have significant climbing activity loss compared to age-matched control flies. In addition, I demonstrate that overexpression of Sirt6 significantly attenuates loss of locomotor function and neurodegeneration in an alpha-synuclein transgenic fly model. Lastly, I discuss the promise of our findings with regards to gaining a deeper understanding of the aging brain and Parkinson’s disease pathology.