Grape-Seed Polyphenolic Extract Improves the Eye Phenotype in a Drosophila Model of Tauopathy

Drosophila models of tauopathies have been developed by transgenically overexpressing the disease-associated forms of tau. In this paper we report for the first time that a recently developed Grape-Seed Polyphenolic Extract (GSPE) improves the eye phenotype of a Drosophila eye model of R406W tau. GSPE-mediated improvements in this distinct in vivo neurodegeneration model for protein misfolding/aggregation suggest that GSPE may have therapeutic value in disorders involving aberrant protein aggregation.


Introduction
Abnormal protein conformations resulting in misfolded and/or abnormally aggregated protein species are a characteristic feature of several neurodegenerative disorders. For example, the microtubule-associated protein tau adopts abnormal conformations resulting in protein helical filament/immunopositive neurofibrillary tangles (NFTs) and is a characteristic neuropathological feature in Alzheimer's Disease (AD), Pick's disease, progressive supranuclear palsy, and corticobasal ganglionic degeneration (CBD). Tau mutations cause frontotemporal dementia with Parkinsonism-(FTDP-) 17, proving that tau dysfunction can directly promote neurodegeneration [1].
We previously reported that moderate consumption of the red wine, Cabernet Sauvignon (Vitis vinifera), prevented abnormal β-amyloid (Aβ) oligomerization coincidental with a significant attenuation of spatial memory impairment in a mouse model of AD-type amyloid neuropathology [9]. Most importantly, we identified a polyphenolic compound highly concentrated in grape-seed polyphenolic extract (GSPE) as potentially responsible for the beneficial role of moderate consumption of red wine (Vitis vinifera) [10,11].
More recently, Ho et al. reported that, using an in vitro aggregation assay, GSPE can significantly inhibit tau peptide Ac(306)VQIVYK(311) aggregation. Moreover, GSPE can also disaggregate preexisting aggregated tau peptides [12]. These results strongly suggest that GSPE might provide beneficial disease-modifying activity in tau-associated neurodegenerative disorders by modulating tau-mediated neuropathologic mechanisms. In this paper, we use the eye phenotype of a Drosophila model of mutant R406W tau to further evaluate the beneficial role of GSPE in tau-mediated neuropathology in vivo. We report for the first time that treatment with GSPE significantly benefits Drosophila phenotypes carrying mutant tau (R406W), further supporting the potential use in vivo of GSPE in abnormal tau aggregation, as previously demonstrated in vitro [12].

R406W Tau Experiments and Visual Scoring of Eye
Abnormality. eygal4/SM6-TM6B flies were crossed to UAS R406W flies to generate ey > R406W flies. ey > R406W eggs were laid in and reared on instant fly medium formula 4-24 supplemented with 2.8 μg/mL GSPE ("GSPE food"), the concentration used in our previous study [13], or control food supplemented with an equivalent volume of water (GSPE solvent, vehicle control). Flies of the indicated genotypes were examined side by side under a dissecting scope. Eye regions which formed no ommatidia (therefore lacked eye tissue completely) were considered "0 = no eye" and required no comparison. For eye areas which did form obvious ommatidia/eye tissue, wild-type control flies were examined at the same time to establish the "4 = almost wild-type" eye size, shape, and pattern upper limit. Eyes that formed but did not reach the "4 = almost wild-type" category were lined up under the scope by size and grouped into categories based on the amount of eye tissue present. Because eyes were grouped side by side under the microscope, relative sizes were easy to establish. When additional conditions or repeated experiments were examined, representative eyes of each category from the previous experiments were examined again in parallel to ensure that the same designations/scoring were maintained.

Statistical Analyses.
The distribution of eye sizes was reviewed and found to have negative kurtosis, indicating a distribution that was more uniform over the range from 0 to 4 than a normal distribution. Negative kurtosis does not substantially impair analysis of variance.
In addition to overall analysis of variance (with trials nested within gender and crossed by treatment), separate ttests were performed for each trial tested.

GSPE Treatment
Improves the Eye Phenotype of ey > R406W Flies. ey > R406W flies show a range of phenotypes from no eye to small, abnormal eyes. The reduced size and abnormal morphology of ey > R406W eyes were improved by GSPE (F(1,531) = 57.29; P < .0005). The incidence of the worst outcomes (visual score of 0 or 1) decreased upon GSPE treatment in each trial while the incidence of the best outcomes (visual score of 3 or 4) increased upon GSPE treatment in each trial. There were also differences in the size of male and female eyes (F(1,531) = 58.62; P < .0005). Representative eyes are shown in Figures 1(a)-1(f) ((a)-(c) male; (d)-(f) female). Nonetheless, there was no interaction between gender and treatment (F(1,531) = 0.30; P = .59); the difference between treated and untreated flies was similar in male and female flies. Separate t-tests showed that each of the six trials had a statistical difference with GSPE treatment characterized by increased visual scoring relative to untreated controls (Figure 1(g)). However, within the combinations of gender and treatment, the trials did not differ significantly (F(4,531) = 1.90; P < .11).

GSPE Effects on Gal4/UAS-Mediated Protein Expression.
To address the concern that GSPE could disrupt the production of the toxic proteins by the Gal4/UAS system, we performed control experiments using the Gal4/UAS system to determine whether GSPE treatment prevents expression of GFP using a UAS-GFP transgene and TubGal4 (constitutively expressed as Gal4). We found no detectable alteration in the production of GFP both by fluorescent imaging of GFP (not shown) and by Western analysis (Figure 2(a)).

GSPE Does Not Affect the Small-Eye Phenotype Caused by
Overexpression of the Proapoptotic Gene Grim. To investigate if GSPE acts on cell death pathways directly, we examined the effects of GSPE treatment on flies overexpressing the proapoptotic gene grim in differentiating cells in the eye (which causes a very small eye). GMR-grim flies (which overexpress the pro-apoptotic gene grim in differentiating cells in the eye) laid eggs on control food or GSPE food. GMR-grim flies reared entirely on GSPE food showed no effect of reduced eye size (Figures 2(b)-2(c)).

Discussion
We have demonstrated that GSPE treatment benefits a distinct Drosophila model of neurodegeneration involving aggregation in vivo (Figures 1 and 2). GSPE showed no effect on Gal4-mediated expression of GFP (Figure 2(a)), indicating that GSPE does not block Gal4/UAS-dependent gene expression. GSPE had no effect on eye size in GMRgrim flies (which overexpress in the eye the pro-apoptotic gene grim which activates caspases and severely reduces eye size [14]) (Figures 2(b)-2(c)). Thus, although the mechanism remains unknown, GSPE-mediated improvements in the Drosophila eye model likely occur subsequent to production of the toxic protein but upstream of caspase activation.
Presently there is no treatment for the devastating progressive neurodegenerative disorders involving tau abnormalities. Continuing investigations suggest that oligomeric forms of these misfolded proteins may play a seminal role in disease etiology. Results from our studies show that GSPE may rescue abnormal tau phenotype in a Drosophila model of tauopathy. This study is consistent with our previous study, which provided evidence that GSPE could inhibit tau peptide aggregation [12] and prevent oligomerization of Aβ peptides into high molecular weight species in models of AD-amyloid neuropathology [10,11]. The study supports further investigations of GSPE in models of tauopathies, for example, frontotemporal dementia and CBD, among others.