Sir-2.1 mediated attenuation of α-synuclein expression by Alaskan bog blueberry polyphenols in a transgenic model of Caenorhabditis elegans

Abstract

Misfolding and accumulation of cellular protein aggregates are pathological hallmarks of aging and neurodegeneration. One such protein is α-synuclein, which when misfolded, forms aggregates and disrupts normal cellular functions of the neurons causing Parkinson's disease. Nutritional interventions abundant in pharmacologically potent polyphenols have demonstrated a therapeutic role for combating protein aggregation associated with neurodegeneration. The current study hypothesized that Alaskan bog blueberry (Vaccinum uliginosum), which is high in polyphenolic content, will reduce α-synuclein expression in a model of Caenorhabditis elegans (C. elegans). We observed that blueberry extracts attenuated α-synuclein protein expression, improved healthspan in the form of motility and restored lipid content in the transgenic strain of C. elegans expressing human α-synuclein. We also found reduced gene expression levels of sir-2.1 (ortholog of mammalian Sirtuin 1) in blueberry treated transgenic animals indicating that the beneficial effects of blueberries could be mediated through partial reduction of sirtuin activity. This therapeutic effect of the blueberries was attributed to its xenohormetic properties. The current results highlight the role of Alaskan blueberries in mediating inhibition of sir-2.1 as a novel therapeutic approach to improving pathologies of protein misfolding diseases. Finally, our study warrants further investigation of the structure, and specificity of such small molecules from indigenous natural compounds and its role as sirtuin regulators.

Figure 1

Crude extract of Alaskan blueberry reduced expression of α-synuclein in the OW13 strain of C. elegans. (A) Graphical representation of fluorescence intensity of the OW13 day 7 animals fed on different concentrations of Alaskan blueberry crude extract (0, 100, 200 and 400 μg/ml) from Larval L4 stage. The data represent the mean ± SEM (n = 20–25 animals per group) with significant differences between the control and extract treatments (0 and 100 μg/ml, 0 and 400 μg/ml) *p < 0.01, ***p < 0.001. Dose 200 μg/ml was not significantly different from control (p > 0.4). Each experiment was repeated three times. (B) Representative confocal images of the α-synuclein/YFP expression in the head region of day 7 OW13 animals, magnification 40X and scale bar 50 μm. (C) Original western blot images of α-synuclein protein of the OW13 day 7 animals fed on concentrations of Alaskan blueberry crude extract (0 and 400 μg/ml). Blots of actin were used as a protein loading control. (D) Graphical representation of quantification of α-synuclein protein bands from Western blots. The intensity of α-synuclein protein was normalized to actin and presented as a percentage. Each experiment was repeated twice. For each treatment of each experiment, protein samples were pooled from three biological replicates. The data represent the mean ± SEM with significant differences between control and extract treatments (0 and 400 μg/ml, *p < 0.02).

Figure 2

Alaskan blueberry polyphenols reduced expression of α-synuclein in the OW13 strain of C. elegans. (A) Graphical representation of fluorescence intensity of the OW13 day 7 animals fed on different polyphenols found in Alaskan bog blueberry: CA = Chlorogenic acid, PAC = Proanthocyanidins, ANT = Anthocyanins from Larval L4 stage. A concentration of 400 μg/ml was used for all extracts and 0.1% DMSO in deionized water was used as a control. The data represent the mean ± SEM (n = 20–25 animals per group) with significant differences between the control and treatments, **p < 0.001, ***p < 0.0001. Each experiment was repeated three times. (B) Representative confocal images of the α-synuclein/YFP expression in the head region of day 7 OW13 animals, magnification 40X and scale bar 50 μm.

Figure 3

Alaskan blueberry polyphenols reduced expression of α-synuclein in the OW13 strain of C. elegans through a Sirtuin mediated pathway. (A) Graphical representation of fluorescence intensity of the day 7 OW13 animals fed on different genetic RNA interference treatments (L4440, sir-2.1 and daf-16) and Alaskan bog blueberry (0 and 400 μg/ml) from larval L4 stage. Empty vector L4440 was considered as a control. The data represent the mean ± SEM (n = 20–25 animals per group) with significant differences between the control and treatments at **p < 0.001 and ***p < 0.0001. Each experiment was repeated three times. (B) Representative confocal images of the α-synuclein/YFP expression in the head region of day 7 OW13 animals, magnification 40X and scale bar 50 μm.

Figure 4

Alaskan bog blueberry polyphenols improved motility in aged (Day 12) OW13 strain of C. elegans. Percent per motility class of middle age (day 5 adults; (A)) and old age (day 12 adults; (B)) animals treated with different concentrations of Alaskan bog blueberry extract (0, 100, 200 and 400 μg/ml) from larval L4 stage. Class A animals (dark gray) moved normally and spontaneously, class B animals (light gray) moved abnormally and may have required prodding, and class C animals (black) were unable to move or moved just head or tail in response to touch (n = 20 animals per group). Each experiment was repeated at least three times.

Figure 5

Reduction of sir-2.1 expression reverses the motility improving effects of Alaskan bog blueberry polyphenols in aged (Day 12) OW13 strain of C. elegans. Percent per motility class of middle age ((day 5 adults; (A) and old age (day 12 adults; (B)) animals treated with different genetic RNA interference treatments (L4440, sir-2.1 and daf-16) and Alaskan bog blueberry extract (0 and 400 μg/ml) from larval L4 stage. Empty vector L4440 was used as control. Class A animals (dark gray) moved normally and spontaneously, class B animals (light gray) moved abnormally and may have required prodding, and class C animals (black) were unable to move or moved just head or tail in response to touch (n = 20 animals per group). Each experiment was repeated at least three times.

Figure 6

Alaskan bog blueberry did not alter lifespan in OW13 strain of C. elegans. (A) Representative survival curves for animals treated with crude extract of blueberries (0, 100, 200 and 400 μg/ml) from larval L4 stage. There was no significant difference in lifespan extension (p > 0.05; Kaplan–Meier log-rank test) among the groups. Each experiment was repeated at least three times (n = 44 animals per treatment). (B) Representative survival curves for animals treated with RNA interference treatments (L4440 and sir-2.1) and crude extract of blueberries/BB (0 and 400 μg/ml) from larval L4 stage. There was no significant difference in lifespan extension (p > 0.05; Kaplan–Meier log-rank test) among the groups. Each experiment was repeated at least three times (n = 44 animals per treatment).

Figure 7

Alaskan bog blueberry and/or RNA interference treatments did not influence total fecundity in OW13 strain of C. elegans. (A) Graphical representation of total fecundity of the OW13 animals fed on different concentrations of Alaskan bog blueberry crude extract (0, 100, 200 and 400 μg/ml) from larval L4 stage. The data represent the mean ± SEM (n = 10 animals per group) with no significant differences among the control and treatments, p > 0.05. Each experiment was repeated three times. (B) Graphical representation of total fecundity of the OW13 animals fed on different genetic RNA interference treatments (L4440 and sir-2.1) and concentrations of Alaskan blueberry crude extract (0 and 400 μg/ml). The data represent the mean ± SEM (n = 10 animals per group) with no significant differences between the control and treatments, p > 0.05. Each experiment was repeated three times.

Figure 8

Alaskan bog blueberry increased lipid content by a marginal increase in reactive oxygen species in OW13 strain of C. elegans. (A) Graphical representation for fluorescence intensity for Nile red staining of day 7 wild-type N2 (control) and OW13 animals fed Alaskan bog blueberry extract (0 and 400 μg/ml) from larval L3 stage. The data represent the mean ± SEM (n = 20–25 animals per group) with significant differences between the control and treatments at ***p < 0.001. Each experiment was repeated at least three times. (B) Representative confocal images of the intestine of day 7 wild-type N2 (control) and OW13 animals fed Alaskan bog blueberry extract (0 and 400 μg/ml), magnification 20X and scale bar 50 μm. (C) The change in endogenous ROS was measured by DCF-DA assay at day 7 of adulthood after treatment with crude extract of blueberries (0 and 400 μg/ml). Bars represent mean ± SEM of each replicate, with three technical replicates for the DCF-DA assay. #p = 0.07.

Figure 9

Alaskan bog blueberry extract reduced the gene expression of sir-2.1 in OW13 strain of C. elegans. qPCR analysis of mRNA levels of sir-2.1, daf-16 and cep-1 (400 μg/ml of crude blueberry or BB extract) in day 7 OW13 animals. qPCR reactions were run in triplicates for each gene. Each experiment was repeated at least three times. The data represent the mean ± SEM at **p < 0.01 and ***P < 0.001.