Αnti-prion effects of anthocyanins

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, PrPC, into the pathogenic isoform, PrPSc. 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 PrPScin 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 PrPSc aggregates, but also completely inhibited the formation of PrPSc 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.


Introduction
Prion diseases are progressive and fatal Neurodegenerative Diseases (NDs), such as Creutzfeldt-Jakob disease (CJD), that affect humans and animals [1][2][3].The fundamental event underlying scrapie infection seems to be a conformational change in the prion protein.Transmissible Spongiform Encephalopathies (TSEs) share a common pathogenic mechanism, which involves the autocatalytic conversion of the normal prion protein, PrP C , to its disease associated variant, PrP Sc .PrP C molecules are repeatedly recruited and misfolded by PrP Sc , resulting in the formation of protease-resistant aggregates, known as amyloid fibrils [1][2][3].Accumulation of PrP Sc fibrils, results in Endoplasmic Reticulum (ER) stress, dysregulated calcium signaling, mitochondrial disfunction, and eventually neuronal cell death [4][5][6].
Oenin (Malvidin-3-glucoside) and Myrtillin (Delphinidin 3-glucoside) are two of the most prevalent ACNs present in grapes and red wine [47].While, they have previously showcased anti-oxidant and anti-inflammatory activity [48][49][50][51][52][53], their potential effect in prion diseases has not yet been investigated.In this study, the anti-prion potential of Oenin and Myrtillin (Supplementary Fig. 2) is described for the first time.In scrapie-infected murine neuroblastoma N2a (ScN2a) cells, we addressed the effect of ACNs for the reduction of ROS levels through the activation of the Keap1-Nrf2 pathway, and the reduction of PrP Sc aggregates in ScN2a22L cells, and also the inhibition of the formation of PrP Sc fibrils in the Cerebrospinal fluid (CSF) of CJD patients.Therefore, our results highlight the strong potential of Oenin and Myrtillin against prion diseases and possibly other neurodegenerative proteinopathies.

In vitro estimation of ROS amounts
N2a22L cells were incubated with Oenin or Myrtillin (250 μМ) for 48 h.Cells were incubated for 30 min at room temperature with H 2 DCFDA (2′,7′-Dichloro-dihydro-fluorescein, D399, Invitrogen, Waltham, MA, USA) dissolved in DMSO, and fluorescence was measured using a Tecan fluorometer.Controls received DMSO at concentrations matching those delivered with the compounds.The same set of experiments were performed after pre-treatment with two different concentrations of H 2 O 2 , 6.25 μM and 200 μM for 30 min before addition of Oenin and Myrtillin.Analysis was done in triplicates and relative fluorescence was expressed as "% of maximum emission", determined with Tecan Magellan software (https://lifesciences.tecan.com/software-magellan, accessed December 19, 2023).

In vitro assessment of PrP Sc aggregation
Oenin-, Myrtillin-treated and control N2a22L cells were lysed in icecold lysis buffer (10 mM Tris pH 7.5, 100 mM NaCl, 10 mM EDTA, 0.5% v/v Triton-X-100, 0.5% w/v sodium deoxycholate) and centrifuged (1 min, 14,000×g).Total protein in the supernatant was estimated with Bradford reagent (A6932, 0250, AppliChem, Darmstadt, Germany).One fraction of each lysate was digested with PK (1.24569.0100,Merck, Darmstadt, Germany) in 1% w/v N lauryl-sarcosine.Phenylmethylsulfonyl Fluoride (PMSF) (5 mM final concentration) was used to stop the reaction.PK treated samples (PK+) and non-PK treated samples (PK-) were resolved on 12% w/v poly-acrylamide gels, electrotransferred onto Polyvinylidene Fluoride (PVDF) membranes and subjected to Western Blot analysis using the monoclonal antibody 6H4.Chemiluminescence was used for development on X-ray films.Films were digitized and relative protein levels were estimated with ImageJ (available at https://imagej.net/ij/index.html,accessed on May 08, 2023), utilizing exposures within the linear dynamic range of the film.
For each sample, the ratio of the intensity of PrP-immunopositive bands in the PK-resistant fraction (PK+) to the intensity of total PrP in the non-PK (PK-) treated fraction was estimated and expressed relative to controls using the formula: Where PrP( RES )ACN and PrP( TOT )ACN are the intensity of PrP bands in the PK-treated and non-PK-treated fractions respectively in Oenin or Myrtillin treated samples.PrP( RES )Cntr and PrP( TOT )Cntr show the intensity of PrP bands in the PK-treated and non-PK treated controls, respectively.In order to verify that PK treatment conditions resulted in complete digestion of PrP C , cell lysates from N2a58 cells that were not

NDs
Neurodegenerative  4).The immunoreactive bands detected in N2a22L cells that were treated with PK do not correspond to partially digested PrP C , but rather to PrP Sc .

Cell free detection of de novo PrP Sc fibrillation through RT-QuIC
Real-time quaking-induced conversion reactions, RT-QuIC [67] were performed using CSF containing PrP Sc seed material from patients with confirmed sCJD diagnosis, originating from the National Reference Center for TSEs, Göttingen, Germany.15 μL CSF (diluted 1000 times) was mixed with 85 μL reaction buffer (5 × PBS pH 6.9, 170 mM NaCl, 1 mM EDTA, 10 μM Thioflavin-T and 0.1 mg/mL recPrP C ). Oenin or Myrtillin were added to a final concentration of 2.5 μM.Reactions were set in 96-well black bottom optical plates and carried out in a BMG Labtech FluoO Star OPTIMA plate reader at 42 • C for 80 h with intermittent rest and shaking cycles.Thioflavin-T (Th-T) fluorescence was measured every 30 min.Analysis was performed in triplicates.

Statistical analysis
GraphPad Prism version 8.0.2 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com,Accessed on May 08, 2023) was used.Non-linear regression analysis was applied to the dose-response equations for LD 50 determination.Differences in gene expression and PrP Sc accumulation between untreated and treated cells were estimated with unpaired, one-tailed T-tests.Data represent Standard Error of Mean (SEM) of three independent experiments and P-values of 0.05 or lower were considered statistically significant.

Assessment of LD 50 of Oenin and Myrtillin in N2a22L cell line
The viability assay showed Oenin as less toxic.Oenin LD 50 values 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.μM (Supplementary Fig. 3).A concentration of 250 μM for each ACN was used for the rest of the study, in which both compounds presented no cytotoxicity to N2a22L cells, and cell treatment entailed 48-h incubation.

Oenin and Myrtillin reduced ROS levels in ScN2a22L cells
Prion diseases are associated with elevated oxidation and ROS production [8][9][10]22].Owing to their known anti-oxidant activity [41,48], it was tested whether Oenin and Myrtillin could affect ROS levels in ScN2a22L cells.Both compounds significantly reduced the endogenous ROS levels and the ROS produced after H 2 O 2 administration (Fig. 1).It is worth noting that, Myrtillin in most cases (without H 2 O 2 administration and with 6.25 μM) neutralized better the amount of generated ROS when compared with Oenin.These results showcased that Oenin and especially Myrtillin have strong anti-oxidant action in prion affected cells.

Oenin and Myrtillin decrease the levels of PrP Sc aggregates
The PrP Sc leads to enhanced resistance against PK and higher propensity to polymerize into amyloid fibrils, the primary cause of prion diseases [78][79][80][81].Consequently, reducing the amount of PrP Sc is of paramount importance for any potential anti-prion and neuroprotective compound.For that reason, the ability of Oenin and Myrtillin to increase the PK sensitivity of PrP Sc aggregates, was tested.Both compounds significantly reduced the amount of PrP Sc aggregation in N2a22L cells, providing further support for their potential anti-prion action (Fig. 2).

Oenin and Myrtillin inhibit the de novo PrP Sc aggregation
Oenin and Myrtillin promoted the clearance of PrP Sc aggregates.As a result, it was tested whether they could also block the de novo PrP Sc fibrillation.For that purpose, RT-QuIC [82], a highly sensitive technique that is routinely used for the diagnosis of prion diseases and similar neurodegenerative disorders, that it is able to detect the presence of misfolded proteins with almost 100% accuracy, was utilized [83][84][85].Consequently, it has also been deployed for the screening of anti-prion compounds [67,[86][87][88][89][90].Both compounds showcased remarkable anti-aggregation capacity, as they completely inhibited the formation of PrP Sc fibrils at concentrations of 5 and 10 μM for both compounds, and Myrtillin maintained moderate anti-aggregation action even at 2.5μМ (Fig. 3).

Discussion
Prion diseases belong to a group of NDs known as proteinopathies, or prion-like diseases, in which pathologic protein misfolding and accumulation, plays a crucial role in disease development and progression [93][94][95][96].In the case of prion diseases, this is due to the transformation of normal prion protein, PrP C , into the pathologic PrP Sc [78,79].In this study, the strong anti-prion ability of Oenin and Myrtillin are described for the first time.Treatment with Oenin and Myrtillin for just 48 h was able to significantly decrease the number of PrP Sc aggregates in N2a22L cells.Additionally, both compounds completely inhibited the de novo formation of PrP Sc fibrils in the CSF of CJD patients, at a concentration of 10 μM and 5 μM, in the case of Myrtillin, maintained robust anti-prion action even at 2.5 μM.
Additionally, Oenin and Myrtillin could be directly interacting with the prion protein.Indeed, other flavonoids are capable of directly binding to PrP C .For example, Quercetin, interaction with PrP Sc fibrils renders them vulnerable to protein degradation, leading to deaggregation [123,124].Moreover, Apigenin and Nepetin managed to inhibit the fibrillation of the PrP 106-126 peptide and also depolymerize the already formed fibrils [125].It is also worth noting that, Oenin and Myrtillin might exert their anti-prion action with a combination of different mechanisms.Quercetin can simultaneously bind to the C-terminal region of murine prion protein and also act as an anti-oxidant [124,126].A similar observation was made with Curcumin, which can bind to PrP Sc fibrils, as well as intermediate aggregates of the PrP C -PrP Sc conversion, while also exerting anti-oxidant action [127,128].
Oxidative stress has been identified as a hallmark of neurodegeneration [32,[129][130][131].In accordance with other anti-oxidant compounds exhibiting anti-prion activity [72,124,126,128,132,133], Oenin and Myrtillin successfully decreased the levels of ROS in prion-infected cells.While Oenin didn't affect Nrf2 expression levels, it managed to activate the Keap1-Nrf2 pathway (albeit less effectively compared to Myrtillin).Indeed, the important step for the activation of Keap1-Nrf2 target genes is nuclear translocation of Nrf2 [70,73,75,114,134,135].As a result, one potential explanation for the activity of Oenin is that it successfully triggered the nuclear translocation of Nrf2, but due to the fact that it didn't lead to upregulation of Nrf2 itself, the activation of the Keap1-Nrf2 pathway was less potent.

Conclusions
To summarize, our results of the current study provide promising evidence regarding the anti-prion neuroprotective potential of Oenin and Myrtillin.Both compounds are able to de-aggregate pre-existing PrP Sc fibrils, and also severely inhibit the process of de novo PrP Sc fibrillation.Moreover, they acted as potent anti-oxidants, decreasing ROS levels through the activation of the Keap1-Nrf2 pathway, leading to neuroprotection (Fig. 4).

Funding
This work was supported by the European Regional Development Fund, 2021-2023, Investment Research Plans for Business Research and Development of Central Macedonia (Grant number: KMP6-0079465).

Financial interests
The authors declare they have no financial interest.

Non-financial interests
None.

Fig. 1 .
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 μМ each), without or following pre-treatment with (A) 6.25 μM H 2 O 2 and (B) 200 μM H 2 O 2 , to induce oxidative stress.Controls received DMSO at concentrations matching those delivered with the compounds.For ROS measurement, H 2 DCFDA, dissolved in DMSO, was added in the cell medium at a final concentration of 20 μМ 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 μМ, 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 .
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

N
. Christoudia et al. estimated at 506.8 μM as opposed to Myrtillin LD 50 estimated at 293.1

Fig. 3 . 4 )
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 .
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.