Parkinson disease (PD) is the second most common progressive neurodegenerative disorder and is only sur- passed in frequency by Alzheimer disease [1] . The pathophysiological hallmarks of PD include the loss of do- paminergic (DA) neurons in the substantia nigra pars compacta (SNc) and the presence of intra-neuronal inclu- sions known as Lewy bodies (LB) in surviving cells. Affected individuals have both motor and non-motor symptoms ranging from bradykinesia, resting tremor, and muscular rigidity to dementia, depression and olfac- tory dysfunction. Initially believed to be an entirely sporadic disease, linkage studies identified α-synuclein (PARK 1 and 4) as the first gene related to PD [2] . The human α-synuclein gene (SNCA) encodes a 140 amino acid peripheral membrane protein that localizes to the pre-synaptic region of neurons [3] . Both point mutations and duplications of its gene locus result in autosomal-dominant PD, the latter causing a more severe early-onset form of the disease. Additionally, both LBs and Lewy neurites, located in the perikarya and neuronal processes, respectively, stain positively for α-synuclein. α-synuclein is associated with both sporadic and familial PD and seems to play a critical role in its origin.

Oxidative stress is consistently associated with the pathogenesis of PD; however, its role in disease progres- sion remains unclear. A cell undergoes oxidative stress when the net balance between the generated reactive oxygen species (ROS) and the available antioxidant defense mechanisms favour the former. Post-mortem anal- ysis of PD patient brains reveals higher levels of oxidative stress biomarkers like dysfunctional mitochondria, decreased levels of reduced glutathione, and deficiencies in antioxidant enzymes in the SN of affected individu- als [4] . Recent evidence suggests pluripotent stem cell-derived DA neurons from a PD patient with a SNCA trip- lication accumulate α-synuclein and are susceptible to oxidative stress [5] . This data suggest that the combina- tion of oxidative stress and excess α-synuclein may play a pivotal role in the progression of PD.

The toxicity of excess α-synuclein appears to be enhanced under conditions of oxidative stress. Studies in Drosophila melanogaster have been especially helpful in elucidating this relationship. Neural expression of α-synuclein in Drosophila brains, especially DA neurons, recapitulates the locomotor dysfunctions, age-depen- dent degeneration of DA neurons, and formation of LBs characteristic of human PD [6] . Decreased lifespan and retinal degeneration have been observed in Drosophila with increased neuronal levels of α-synuclein [7] . Co-expression of methionine sulfoxide reductase A (MSRA) or PTEN-induced putative kinase 1 (PINK1), pro- teins involved in ROS neutralization and damaged mitochondrion turnover, respectively, with α-synuclein im- proves PD-related phenotypes [8] . Given their versatility, Drosophila can help unravel the role of oxidative stress in PD and unveil potential antioxidant therapies.

Blueberries are an excellent source of dietary antioxidants. The therapeutic potential of blueberries in cancer and vascular disease has been described and recent studies in Drosophila suggest that plant extracts may be beneficial to individuals suffering from neurodegenerative diseases [9] [10] . In this study we describe the restor- ative effects of blueberry extract (BBE) concentrate on a Drosophila model of PD. Enhanced mortality and eye degeneration caused by directed expression of α-synuclein in the DA neurons and developing eye, respectively, is improved by supplementing growth media with BBE.

Recent evidence has suggested that a diet rich in blueberries may help slow the age-related degeneration of neu- rons. In a human study, blueberry juice supplementation improved memory function in older adults with early

Groups with different superscripted letters are significantly different from each other (p < 0.05).

memory decline [16] . Furthermore, it has been demonstrated that short-term blueberry supplementation in- creased heat shock protein 70 (HSP70)-mediated protection against inflammation in aged rat hippocampal cells [17] . These studies suggest that the neuroprotective effects of blueberries or BBE are not confined to D. mela- nogaster and may extend to mammals.

The Free Radical/Oxidative Stress theory of ageing originated in the 1950’s and suggests that an organism ages due, in part, to the accumulation of free radical-induced damage to cellular macromolecules. Previous stu- dies have shown that BBE is capable of ex tending lifespan in D. melanogaster [18] . Our results are novel as we have shown that BBE supplementation can extend lifespan in a Drosophila model of a neurodegenerative dis- ease. The additional antioxidants provided by BBE supplementation may alleviate some of the excess ROS gen- erated during the progression of PD-like cell death resulting in less cellular damage and a longer median surviv- al time in affected flies.

The Drosophila compound eye consists of multiple subunits, or ommatidia, composed of several neurons and peripheral mechanosensory bristle cells. Directed expression of α-synuclein during early eye development re- sults in premature degeneration of the retina and abnormal development of external eye morphology [8] [19] Our results suggest that a diet containing BBE protects neurons in the eye against α-synuclein-dependent deve- lopmental defects. Both the amount of atypical ommatidia and total number of bristles were improved in flies fed a BBE-supplemented diet. A link exists between BBE supplementation and protein turnover via the ubiqui- tin proteasome system (UPS) as flies fed BBE have increased levels of regulatory particle non-ATPase 11 (Rpn11) mRNA, an essential lid component of the 26S proteasome structure [18] . In humans, the parkin gene (PARK2) encodes an E3 ubiquitin ligase and expression of parkin in Drosophila eyes suppresses α-synuclein- induced retinal degeneration in older flies [19] . Protein aggregation/turnover is a point of major interest in PD aetiology and the neuroprotective effects of BBE on α-synuclein-induced damage in the Drosophila eye may be due in part to increased activity of the UPS system.

Our findings may be attributed to a strengthened overall antioxidant defense mechanism in α-synuclein-ex- pressing flies. BBE extract can increase the expression of intrinsic antioxidant defense enzymes in D. melano- gaster that include catalase (Cat), Cu-Zn superoxide dismutase (Sod1), and Mn superoxide dismutase (Sod2) [18] . Additionally, BBE extends lifespan and partially protects wild-type flies under conditions of increased oxidative stress, whereas no effect was reported for Cat or Sod loss-of-function mutants. Similar results have been documented for several other foods high in dietary antioxidants, including extracts of apple polyphenols [20] , green tea catechins [21] and black tea [22] . Although dietary antioxidants likely provide an invaluable secondary support to cells undergoing oxidative stress, it appears their protective effects are dependent on in- trinsic enzyme antioxidant defense systems.

Here we present the first demonstration of the neuroprotective effects of BBE in a Drosophila model of α-syn- ucleinpathic disease. Previous findings have demonstrated that grape extract improved both the early mortality and premature decline in locomotion in a similar Drosophila model of PD [9] . Taken together, these results epi- tomize the value of using Drosophila to study PD aetiology. Though the literature is relatively new, studies in this versatile organism have helped develop interest in the potential neuroprotective effects of dietary antioxi- dants in medical research. Future studies could aim towards unraveling the interaction of dietary antioxidants and the activity of cellular mechanisms, such as the UPS and enzymatic antioxidant pathways.

This research was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) Canada Graduate Scholarship (NSERC CGS-M) to DBL and an NSERC Discovery Grant to BES. Funding was pro- vided by the School of Graduate Studies at Memorial University of Newfoundland to DBL. We thank Dr. Liqui Men (Bio-molecular Imaging Cluster, Memorial University of Newfoundland) for technical assistance with scanning electron microscopy.