Molecular mechanisms underlying protective effects of quercetin against mitochondrial dysfunction and progressive dopaminergic neurodegeneration in cell culture and MitoPark transgenic mouse models of Parkinson's Disease

Abstract

Quercetin, one of the major flavonoids in plants, has been recently reported to have neuroprotective effects against neurodegenerative processes. However, since the molecular signaling mechanisms governing these effects are not well clarified, we evaluated quercetin's effect on the neuroprotective signaling events in dopaminergic neuronal models and further tested its efficacy in the MitoPark transgenic mouse model of Parkinson's disease (PD). Western blot analysis revealed that quercetin significantly induced the activation of two major cell survival kinases, protein kinase D1 (PKD1) and Akt in MN9D dopaminergic neuronal cells. Furthermore, pharmacological inhibition or siRNA knockdown of PKD1 blocked the activation of Akt, suggesting that PKD1 acts as an upstream regulator of Akt in quercetin-mediated neuroprotective signaling. Quercetin also enhanced cAMP response-element binding protein phosphorylation and expression of the cAMP response-element binding protein target gene brain-derived neurotrophic factor. Results from qRT-PCR, Western blot analysis, mtDNA content analysis, and MitoTracker assay experiments revealed that quercetin augmented mitochondrial biogenesis. Quercetin also increased mitochondrial bioenergetics capacity and protected MN9D cells against 6-hydroxydopamine-induced neurotoxicity. To further evaluate the neuroprotective efficacy of quercetin against the mitochondrial dysfunction underlying PD, we used the progressive dopaminergic neurodegenerative MitoPark transgenic mouse model of PD. Oral administration of quercetin significantly reversed behavioral deficits, striatal dopamine depletion, and TH neuronal cell loss in MitoPark mice. Together, our findings demonstrate that quercetin activates the PKD1-Akt cell survival signaling axis and suggest that further exploration of quercetin as a promising neuroprotective agent for treating PD may offer clinical benefits.

Keywords: MitoPark; PGC-1α; PKD1; Parkinson's disease; mitochondrial biogenesis; quercetin.

Figure 1. Activation of pro-survival PKD1 and Akt kinases by quercetin

A–D, MN9D dopaminergic neuronal cells were treated with varying concentrations of quercetin (0.3–30 µM) for 24 h and cell lysates were prepared and subjected to Western blot analysis. Representative immunoblots of PKD1 S744/748 and S916 phosphorylation (A). B, Densitometric analysis of phospho-PKD1 S744/748 levels (n=3). Total Akt and Akt S473 phosphorylation (C). D, Densitometric analysis of phospho-Akt S473 levels (n=3). Values expressed as mean ± SEM of three independent experiments comparing control and quercetin-treated groups (*p<0.05; **p<0.01). E, MN9D cells were pre-treated with 50 µM PKD1 inhibitor CID 755673 for 1 h and then co-treated with 10 µM quercetin for 24 h and phospho-Akt (S473) levels were determined by Western blot analysis. F, MN9D cells were transfected either with control-siRNA (scrambled) or PKD1-siRNA. Cells were treated 48 h after transfection with 10 µM quercetin for 24 h and total PKD1 and phospho-Akt (S473) levels were analyzed by Western blot (n=2–3).

Figure 2. Quercetin induces expression of the CREB target gene BDNF

A, MN9D cells were treated with varying concentrations of quercetin (0.3–30 µM) for 24 h and cell lysates were prepared and subjected to Western blot analysis. Representative immunoblots of phospho-CREB (S133), total CREB, and BDNF (n=2–3). B, MN9D cells were treated with 10 and 30 µM quercetin for 24 h and real-time RT-PCR analysis of BDNF mRNA level was performed (n=9). 18S rRNA served as an internal control. Values represented as a percentage of the activity of control and are expressed as means ± SEM of three independent experiments performed in triplicate (***p<0.001 comparing control and quercetin-treated samples).

Figure 3. Quercetin induces PGC-1α promoter activity, mRNA and protein expression in MN9D cells

A, MN9D cells were transfected with PGC-1α promoter-reporter construct, and then 24 h post-transfection they were treated with 1 and 10 µM quercetin for 24 h (n=3). B, MN9D cells were transfected with PGC-1α-FL and PGC-1α-ΔCRE promoter-reporter constructs, and then 24 h post-transfection they were treated with 10 µM quercetin for 24 h (n=3). Luciferase activities were measured and normalized to β-galactosidase activity. Values represented as a percentage of the activity of control and are expressed as means ± SEM of three independent experiments comparing control and quercetin-treated groups (**p<0.01; ***p<0.001). C, MN9D cells were treated with 10 and 30 µM quercetin for 24 h and real-time RT-PCR analysis of PGC-1α mRNA levels was performed (n=6). 18S rRNA served as an internal control. Values represented as a percentage of the activity of control and are expressed as the means ± SEM of three independent experiments performed in duplicate comparing control and quercetin-treated groups (**p<0.01; ***p<0.001). D, Representative immunoblot of PGC-1α after Western blot analysis of cell lysates prepared from MN9D cells treated with varying concentrations of quercetin for 24 h (n=2–3).

Figure 4. Quercetin stimulates mitochondrial biogenesis in MN9D cells

A and B, MN9D cells were treated with 10 and 30 µM quercetin for 24 h and real-time RT-PCR analyses of cytochrome B (CYTB) (A) and TFAM (B) mRNA levels were performed (n=6). 18S rRNA served as an internal control. Values represented as a percentage of the activity of control and are expressed as the means ± SEM of three independent experiments performed in duplicate comparing control and quercetin-treated groups (p<0.01; ***p<0.001). C, Representative immunoblot of TFAM after Western blot analysis of cell lysates prepared from MN9D cells treated with varying concentrations of quercetin for 24 h. D, Densitometric analysis of TFAM levels (n=3). Values expressed as the mean ± SEM of three independent experiments comparing control and quercetin-treated (*p<0.05). E, MN9D cells were treated with 10 and 30 µM quercetin for 24 h and stained with 200 nM MitoTracker Green FM, which was detected on a fluorescence microplate reader (n=6). Values represented as percentage of the activity of control and are expressed as the means ± SEM of two independent experiments performed in triplicate comparing control and quercetin-treated groups (***p<0.001). F, MN9D cells were treated with 10 and 30 µM quercetin for 24 h. Genomic DNA was isolated and mtDNA content was determined by quantitative PCR with SYBR green (n=6). Primers for mitochondrial cytochrome b and nuclear β-actin genes were used to amplify 10 ng nuclear DNA and 1 ng mitochondrial DNA. Values represented as percentage of the activity of control and are expressed as the means ± SEM of three independent experiments performed in duplicate comparing control and quercetin-treated groups (**p<0.01; ***p<0.001).

Figure 5. Effect of quercetin on mitochondrial respiration rate in N27 dopaminergic cells

A–E, N27 dopaminergic neuronal cells were treated with 1 and 10 µM quercetin for 24 h and the N27 cells-containing culture plates were loaded into the Seahorse XF96 analyzer for OCR measurement. Mitochondrial dynamics were measured using the sequential injection of oligomycin A (1 µg/ml), FCCP (1 µM), and antimycin A (10 µM) (A). Basal OCR (B), ATP-linked respiration (C), maximal OCR (D), and spare respiratory capacity (E) were calculated from the output OCR values (n=6). Values expressed as the means ± SEM of three independent experiments performed in duplicate comparing control and quercetin-treated groups (**p<0.01; ***p<0.001).

Figure 6. Quercetin protects against 6-OHDA-induced neurotoxicity

A–C, MN9D dopaminergic cells were pre-treated with 10 µM quercetin for 1 h and then co-treated with 6-OHDA (50 µM) for 24 h and assayed for cytotoxicity using the Muse cell analyzer (A). The percentage of live cells (B) and total apoptotic cells (C) were quantified by flow cytometry (n=4). Values represented as percentage of the activity of control and are expressed as means ± SEM of four independent experiments (*p<0.05; ***p<0.001).

Figure 7. Quercetin protects against dopaminergic neurodegeneration in MitoPark mice

MitoPark and C57BL/6 mice were gavaged with either 25 mg/kg quercetin or vehicle from ages 12 to 18 weeks (n=8). Locomotor activities were assessed using a VersaMax system. A, moving track of mice. B, horizontal activity. C, vertical activity. D, levels of striatal dopamine (n=4). E, Treatment schedule of MitoPark and C57BL6 littermate mice with QB3C, wherein 12-week-old animals were treated either with vehicle (50% propylene glycol) or QB3C via oral gavage for 8 weeks (n=9). After the treatment, animals were perfused and 30-µm sections were cut and processed for TH-DAB immunostaining in the striatum and substantia nigra (SN) (F). G, stereological counting of TH⁺ neurons in the SN. Values expressed as means ± SEM of four mice per group (*p<0.05; **p<0.01). C57BL/6-LC are littermate controls from the MitoPark breeding colony.

Figure 8. Quercetin reduces striatal neurotransmitter depletion in MitoPark mice

A–C, twelve-week-old MitoPark and C57BL/6-LC littermate mice were treated either with vehicle (50% propylene glycol) or QB3C via oral gavage for 8 weeks. Mice were sacrificed one day after the last dose of QB3C and the levels of striatal dopamine (A) and its metabolites DOPAC (B) and HVA (C) were measured by HPLC. Values expressed as means ± SEM of five to six mice per group (*p<0.05; ***p<0.001).

Figure 9. Quercetin reverses behavioral deficits in MitoPark mice

Twelve-week-old MitoPark and C57BL6-LC littermate mice were treated either with vehicle (50% propylene glycol) or QB3C via oral gavage for 8 weeks. Locomotor activities were measured using a VersaMax system and a rotarod instrument one day prior to sacrifice. Representative maps tracking the movements of mice (A). Group analysis of movement time (B), total distance travelled (C), number of movements (D), and time spent on rotarod (E). Values expressed as means ± SEM of nine mice per group (*p<0.05; **p<0.01).