Αnti-prion effects of anthocyanins

Abstract

Prion diseases, also known as Transmissible Spongiform Encephalopathies (TSEs), are protein-based neurodegenerative disorders (NDs) affecting humans and animals. They are characterized by the conformational conversion of the normal cellular prion protein, PrP^C, into the pathogenic isoform, PrP^Sc. Prion diseases are invariably fatal and despite ongoing research, no effective prophylactic or therapeutic avenues are currently available. Anthocyanins (ACNs) are unique flavonoid compounds and interest in their use as potential neuroprotective and/or therapeutic agents against NDs, has increased significantly in recent years. Therefore, we investigated the potential anti-oxidant and anti-prion effects of Oenin and Myrtillin, two of the most common anthocyanins, using the most accepted in the field overexpressing PrP^Scin vitro model and a cell free protein aggregation model. Our results, indicate both anthocyanins as strong anti-oxidant compounds, upregulating the expression of genes involved in the anti-oxidant response, and reducing the levels of Reactive Oxygen Species (ROS), produced due to pathogenic prion infection, through the activation of the Keap1-Nrf2 pathway. Importantly, they showcased remarkable anti-prion potential, as they not only caused the clearance of pathogenic PrP^Sc aggregates, but also completely inhibited the formation of PrP^Sc fibrils in the Cerebrospinal Fluid (CSF) of patients with Creutzfeldt-Jakob disease (CJD). Therefore, Oenin and Myrtillin possess pleiotropic effects, suggesting their potential use as promising preventive and/or therapeutic agents in prion diseases and possibly in the spectrum of neurodegenerative proteinopathies.

Keywords: Anthocyanins; Anti-oxidant; Neuroprotection; PrP(Sc); Prion; Proteinopathies.

Graphical abstract

Fig. 1

Anti-oxidant effects of Oenin and Myrtillin in N2a22L cells. ROS levels were measured in N2a22L cells after a 48-h treatment with Oenin or Myrtillin (250 μΜ each), without or following pre-treatment with (A) 6.25 μM H₂O₂ and (B) 200 μM H₂O₂, to induce oxidative stress. Controls received DMSO at concentrations matching those delivered with the compounds. For ROS measurement, H₂DCFDA, dissolved in DMSO, was added in the cell medium at a final concentration of 20 μΜ and cells were further incubated for 30 min at room temperature. Then, fluorescence was measured using a Tecan fluorometer. The % ROS was calculated based on the maximum ROS production value. (C) Oenin and Myrtillin induce the expression of Keap1-Nrf2 pathway gene targets in N2a22L cells. The Keap1-Nrf2 pathway is a key cellular defense mechanism against oxidative stress, that protects cells by reducing the risk of ROS-mediated damage through the activation of cytoprotective enzymes. More specifically, Nrf2 binds to Antioxidant Response Elements (AREs) in the promoters of anti-oxidant genes, aiming to restore redox homeostasis [68]. The expression of NFE2L2 (encoding Nrf2), GCLM and HMOX1 in Oenin and Myrtillin treated cells (250 μΜ, 48 h) is assessed in N2a22L cells, relative to controls (administered DMSO at the same concentration as those delivered with the compounds). Data represent Standard Error of Mean (SEM) of three independent experiments. Stars denote statistical significance (unpaired, one-tailed, T-test); *: p-value <0.05, **: p-value <0.01.

Fig. 2

Oenin and Myrtillin reduce PrP^Sc aggregation in N2a22L cells. (A) Representation of the structural rearrangement taking place during the PrP^C–PrP^Sc conversion. During this process, the α-helix rich PrP^C (in which C stands for the cellular form of the normal prion protein and is expressed in neurons and the spinal cord) is transformed into the β-sheet enriched PrP^Sc (Sc stands for scrapie, the prion disease of sheep and goats). This results in physio- and bio-chemical properties distinct from PrP^C, including reduced solubility in mild detergents, enhanced resistance to partial proteolysis by PK. (B) Representative Western blot results for each compound, along with the densitometric analysis from three independent experiments are depicted. Cell lysates from Oenin and Myrtillin treated N2a22L cells as well as controls (administered DMSO at the same concentrations as those delivered to the ACN treated cells) were processed for PrP immunodetection. A fraction of each lysate (150 μg total protein) was treated with proteinase K (PK+, 1.25 μg PK/mg total protein) for 1 h at 37 °C, to allow the identification of the partially resistant to PK, PrP^Sc. Due to its conformation, PrP^Sc is not accessible for enzymatic treatment, except a segment at its amino-terminal site which is digested resulting in the characteristic band shift of PrP immunopositive bands towards lower molecular weights. Analysis of non-PK treated (PK-) material (50 μg) from the same sample allowed total PrP detection (PrP^C and PrP^Sc). For PrP immunodetection the monoclonal 6H4 antibody (7500997, Invitrogen, Waltham, MA, USA) was used (0.2 μg/mL). PK activity degrades β-Actin, thus it is not visible in PK(+) samples. Blots were developed on autoradiography films using chemiluminesence. Densitometric analysis was performed with ImageJ. Bar graphs show the conversion rate of each ACN treated sample (PrP^Sc/Total PrP) relative to the control conversion rate (PrP^Sc %). Data represent Standard Error of Mean (SEM). Stars denote statistical significance (unpaired, one-tailed, T-test); *: p value < 0.05, **: p value < 0.01.

Fig. 3

Oenin and Myrtillin inhibit recPrP^C fibrillation in RT-QuIC assays seeded with human PrP^Sc. (A) Summary of RT-QuIC steps: (1) A sample containing PrP^Sc (such as CSF from CJD patients) is mixed with a recombinant PrP^C (recPrP^C) monomers and Th-T, which specifically binds to β-sheets, leading to fluorescence. (2) The recPrP^C monomers are recruited by the PrP^Sc oligomers. (3) Recruited recPrP^C monomers are transformed into recPrP^Sc and the PrP^Sc oligomers are elongated. (4) Creation of PrP^Sc -recPrP^Sc fibrils. (5) Quaking induces fragmentation of the PrP^Sc fibrils. (6) The process is repeated [83,91,92]. (B) Aggregation of recPrP^C in RT-QuIC was assessed in the CSF from twelve different CJD patients. Oenin and Myrtillin were added in the reaction mixture of RT-QuIC in three different concentrations (2.5, 5 and 10 μM) and the results were compared with that from CSF only and CSF with DMSO. Reactions were set using 15 μL of diluted seed material and performed at 42 °C for 80 h with intermittent rest and shaking cycles. Th-T fluorescence, as a measure of protein aggregation, was recorded every 30 min. The graph depicts combined (mean) data from the results acquired from the twelve independent patients CSFs used as seed. sCJD: positive control; RT-QuIC assays performed with no anthocyanin supplementation. Oenin, Myrtillin: RT-QuIC assays performed in the presence of Oenin or Myrtillin. Both compounds block PrP aggregates formation. (C) Quantification of Oenin and Myrtillin effects on PrP conversion and aggregation inhibition. Box plots represent the Standard Error of Mean (SEM) of the Area Under Curve (AUC) calculated for the individual fluorescence curves of each replicate reaction. AUC values were used as a measure of protein conversion and aggregation. Stars indicate statistical significance (unpaired, one-tailed, T-test). **: p value < 0.01, ***: p value < 0.001.

Fig. 4

Oenin and Myrtillin protect cells from PrP^Sc mediated oxidative stress through Keap1-Nrf2 activation. Treatment with either, Oenin or Myrtillin, disrupts the Keap1/Nrf2 dimer, resulting in Nrf2 nuclear translocation. This leads to the activation of a series of anti-oxidant genes, that are related to the glutathione and thioredoxin anti-oxidant systems, NADPH regeneration, iron metabolism, quinone reduction and superoxide neutralization [75,[135], [136], [137]]. Consequently, excessive ROS production is inhibited, and cellular homeostasis is restored, inducing neuroprotection.