*3.1. Safety of Long-Term Supplementation with Grape Polyphenols and Their E*ff*ect on Life Span in Mice*

Pallauf et al. [25] reviewed the data on life span extension by flavonoids in worms, flies and mice, describing possible mechanisms that may underpin longer life spans of model organisms treated with flavonoids: energy-restriction-like effects, inhibition of insulin-like-growth-factor signaling, induction of antioxidant activity, hormesis and antimicrobial properties.

In the experiments, with mice, their life span increased when the diets were supplemented with combined extracts of blueberry, green tea and pomegranate powder [26] or green tea polyphenols containing approximately 70% epigallocatechins, epicatechins and gallocatechins [27]. In other studies, however, experimenting with blackcurrant juice (containing anthocyanins, quercetin and quercetin glycosides), epimedium flavonoids (containing 20% icariin) [28], green tea extracts [29,30], blueberry extract [30], triple combination of green tea extract, black tea extract and morin [30] or pomegranate powder [30] failed to induce a longer life span in mice. Overall, several issues should be taken into account when the potential effect of flavonoids on animal or human life span in discussed. When different flavonoids are consumed simultaneously, they may have an additive or even synergistic impact on life expectancy. Moreover, other compounds in extracts may either attenuate or improve the life-extending effect of flavonoids. Besides, synergy was observed between polyphenols, drugs, and hormones [16]. The duration of treatment and the age when the supplementation starts also matter and may lead to different outcomes.

In our study, grape polyphenol concentrate (containing a wide range of anthocyanins, flavan-3-ols, hydroxycinnamic acids, high molecular weight, oligomeric and condensed procyanidins plus 121.2 mg/L, of quercetin, 46.0 mg/L of quercetin-3-O-glycoside,928.4 mg/L of gallic acid and 5.6 mg/L of trans-resveratrol; Supplementary Materials: Table S1) increased life span (Figure 1). A conclusion can be made that *GPC* was safe for mice during almost life-long supplementation starting from an early age. The long-term treatment did not produce any visible harmful effects on the general condition of the animals, nor disturbed their behavioral performance or cognitive function. This observation is quite important, since a preventive long-term intervention at the asymptomatic preclinical and early stages of the disease progression is considered the most promising in the management of neurodegenerative disorders [31].

#### *3.2. Polyphenols Attenuate Neuropathological Changes Associated with Aging and PD-Like Disturbances*

Our experiments with a transgenic PD model, demonstrated the grape polyphenols enhanced memory reconsolidation and abated memory extinction in the passive avoidance test (Figure 3). It should be mentioned that the open field test revealed no significant alterations in the *GPC*-treated groups. Hence, the observed effect of *GPC* on cognitive functions was specific and did not depend on general changes in locomotor or exploratory behavior. The beneficial effect of grape polyphenols on cognitive functions agrees well with the previous findings on the restoration of impaired cognition in the mouse models of aging or Alzheimer disease [32–35].

Low bioavailability of flavonoids questioned their direct effects on the central nervous system. However, when their bioactive metabolites are taken into account, including those associated with the activity of the gut microbiota and interaction products, bioavailability appears to be much higher [36]. For example, anthocyanins and their metabolites were found in almost all organs and tissues including the brain of animals fed with anthocyanin-rich feeds. The latter observation attests to active absorption and the ability to overcome the blood–brain barrier [37–39]. Hence, the results obtained on biological activity and impact mechanisms of the intact compounds produced in vitro should be interpreted with a certain caution and need to be confirmed by in vivo findings. Moreover, the dosage is also an essential issue. For example, high doses of resveratrol applied to overcome its low bioavailability caused various side effects [40]. Moreover, some components of the *GPC* mixture, such as resveratrol and epigallocatechin-3-gallate, are regarded as pan-assay-interference (PAINS) compounds [41]. Hence, their effect on an organism might be nonspecific, including cell membrane perturbations, rather than specific protein binding [42], especially when high supraphysiological doses are applied.

Nevertheless, preclinical animal studies revealed that grape polyphenols might affect certain pathogenetic mechanisms involved in the aging-related cognitive decline and neurodegenerative disorders, such as neuroinflammatory response, oxidative stress, protein homeostasis, and apoptotic signaling [17,32,33]. A systematic review of 43 publications performed by de Andrade Teles et al. [43] summarized that the main targets of action for the flavonoid-based PD therapy were the reduction of the cellular oxidative potential and activation of neuronal death mechanisms. Strathearn et al. [44] suggested that anthocyanin- and proanthocyanidin-rich plant extracts could alleviate PD-induced neurodegeneration by enhancing the mitochondrial function.

Although pronounced motor disturbances occur in transgenic mice with overexpression of mutant human α-synuclein at the age of 9–13 months [23], certain behavioral and cognitive alterations appear at early stages of the pathology course including memory deficit [22]. Those non-motor symptoms are associated with the accumulation of α-synuclein and neuroinflammation in the forebrain regions. Indeed, we revealed the deposits of α-synuclein and enhanced expression of inflammatory markers (IBA1 and CD54) in the frontal cortex of transgenic mice. *GPC* supplementation significantly decreased the α-synuclein accumulation and reduced the expression of the neuroinflammatory markers in the frontal cortex (Figure 4). We did not find any d α-synuclein deposition in the hippocampus while the neuroinflammatory response was less pronounced. However, *GPC* treatment produced e similar effects attenuating the inflammatory markers in the hippocampal regions (Figure 5). Many studies

reported on the anti-inflammatory effect of polyphenols, including the recent study of a mouse PD model induced by 6-OHDA neurotoxin [20]. At the same time, the effect of polyphenols on α-synuclein aggregation and neurotoxicity was observed in vitro in cellular models [45,46]. Specifically, the major metabolite among anthocyanins cyanidin 3-glucoside inhibited aggregation and fibril formation of α-synuclein [47,48]. The present study confirmed the neuroprotective activity of grape polyphenols against α-synuclein in vivo. Thus, *GPC* supplementation has a potential therapeutic effect in preventing and treating Parkinson disease.

#### **4. Materials and Methods**

#### *4.1. Experimental Animal and Design*

Experiments were performed using male mice: (1) non-SPF mice of the C57Bl/6 strain born and reared in a conventional animal facility at the Federal State Budgetary Scientific Institution "Scientific Research Institute of Neurosciences and Medicine" (SRINM; former Scientific Research Institute of Physiology and Basic Medicine) (Novosibirsk, Russia); (2) mice of the B6.Cg-Tg(Prnp-SNCA\*A53T)23Mkle/J) strain (hereinafter: mut(PD)) and control WT mice acquired from the SPF-vivarium of the Institute of Cytology and Genetics SB RAS (Novosibirsk, Russia). Hemizygous mut (PD) mice were produced by inserting the human A53T missense mutant form of alpha-synuclein cDNA in the mouse genome downstream of a mouse prion Prnp promoter [49].

Animals were housed in groups of 4–5 animals per cage (40 × 25 × 15 cm) under standard conditions (light–dark cycle: 14 h light and 10 h dark (lights off at 15:00); temperature: 18–22 ◦C; relative humidity: 50–60%). All experimental procedures were carried out in accordance with the guidelines of the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the SRINM. Every effort was made to minimize the number of animals used and their suffering.

In each experiment, mice of each strain were subdivided into two groups and prescribed one of the following diets. The mice of the control groups received a standard granulated chow for laboratory mice (Ssniff R/M-H V1534-300, Soest, Germany) and pure water (Rosinka, Novosibirsk, Russia) ad libitum. The mice of the *GPC* groups were given the standard chow and the grape polyphenol aqueous solution of the concentrate *Enoant* (produced by RESSFOOD, Company, Yalta, Russia) [50] ad libitum. *Enoant* was diluted with the pure water to a concentration of 0.5–0.8% taking into account a mouse body weight and liquid consumption to provide the daily averaged dosage of 1.5 mL/kg. Fresh solution was prepared every other day. To adjust the dosage, liquid consumption per cage was measured daily and mice were weighed weekly. Concentrations of polyphenolic compounds in *Enoant* are described in Supplementary Materials: Table S1.

The experiment with non-SPF mice of the C57Bl/6 strain born and reared at the conventional animal facility started when mice reached an age of 6–8 weeks and lasted until the death of the last mouse (control, *n* = 15; *GPC*, *n* = 8). Mut(PD) mice, a genetic model of PD, and control WT mice born and reared under SPF conditions were fed with the *GPC* supplement since the age of six weeks (WT control, *n* = 9; WT + GPC, *n* = 9; mut(PD) control, *n* = 8; mut(PD) + *GPC*, *n* = 9). After four months of *GPC* feeding mut(PD) and WT mice were tested for behavior and then sacrificed for further immunohistochemical analysis of their brains.

#### *4.2. Behavioral Tests*

Each animal was handled for 5 min/day on three consecutive days, prior to being taken into the experiment. Open field and passive avoidance tests were performed. Observations were made during the dark phase between 15:00 and 22:00 h. For behavioral testing, the animals were placed individually in a clean cage (25 × 40 × 20 cm), and transported to a dim observation room (28 lux of the red light) with sound isolation reinforced by a masking white noise of 70 dB. Performance in the behavioral tests was monitored using a video camera Panasonic WV-CL930 (Panasonic System Networks Suzhou

Co.,Ltd., Suzhou, China) positioned above an apparatus and processed with original EthoVision XT software (Noldus, Wageningen, The Netherlands). The test equipment was cleaned using 20% ethanol and thoroughly dried before each test trial.

#### 4.2.1. The Open Field Test

This test was carried out in an apparatus with a square arena (40 × 40 cm) and plastic walls 37.5 cm high brightly lit from above (1000 lux). A mouse was placed in the center of the arena, and its movements were recorded for 10 min. The following parameters were assessed: general locomotion (distance travelled in cm); vertical locomotor and exploratory activity (number of rearing); anxiety (time spent in the central part of the arena); and emotionality (number of defecations).

#### 4.2.2. The Passive Avoidance Test

Training on the passive avoidance reaction was performed by a standard single-session method in an experimental chamber with dark and light compartments and an automated Gemini Avoidance System apparatus (San Diego Instruments, San Diego, CA, USA)) as described in detail earlier [51]. The Gemini software automatically recorded the latency of the transfer to the dark compartment and the data of testing served as a measure of acquisition of the conditioned passive avoidance reaction. Memory extinction was measured during the next ten days.

#### *4.3. Immunohistochemical Analysis*

On the day of euthanasia, mice were culled with CO2. The animals were perfused transcardially with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS, then the brains were rapidly excised and postfixed in PBS containing 30% sucrose at 4 ◦C until further neuromorphological analysis. The analysis was performed on 30-μm-thick cryosections according to a protocol described in detail previously [18]. Coronal slices along the frontal cortex (AP: 2.93 to −2.45 mm) or hippocampus (AP: −2.03 to −2.15 mm) of each mouse brain were made. We applied a rabbit polyclonal antibody (NB110-61645, 1:1000 dilution, Novus Biologicals, Littleton, CO, USA) as the primary antibody to detect humanα-synuclein, a goat polyclonal antibody (NB100-1028, 1:200 dilution, Novus Biologicals, Littleton, CO, USA) as the primary antibody to detect the AIF-1/IBA1 microglial marker, or a rat monoclonal antibody (catalog # 16-0542-81, 1:300 dilution, Invitrogen, Carlsbad, CA, USA) as the primary antibody to detect the CD54(ICAM-1) inflammatory marker. A fluorescently labeled (Alexa Fluor 488-conjugated) goat anti-rabbit IgG antibody (ab150077, 1:600 dilution, Abcam, Cambridge, UK), Alexa Fluor 488-conjugated donkey anti-goat IgG antibody (ab150129, 1:200 dilution, Abcam, Cambridge, UK), or Alexa Fluor 594-conjugated goat anti-rat IgG antibody (ab150160, 1:500 dilution, Abcam, Cambridge, UK) served as the secondary antibodies, respectively. Fluorescent images were finally obtained by means of an Axioplan 2 (Carl Zeiss) imaging microscope and then analyzed in Image Pro Plus Software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Fluorescence intensity was measured as background-corrected optical density (OD) with subtraction of staining signals of the non-immunoreactive regions in the images converted to grayscale. The area of interest was 7423 μm2 (IBA1 or CD54) or 30,014 μm2 (α-synuclein) in the frontal cortex; 19,353 μm2, 26,100 μm2, and 50,868 μm2 in the hippocampal CA1, CA3 areas, and dentate gyrus, respectively.

#### *4.4. Data Analysis*

Survival analysis was performed using Gehan's Wilcoxon test and presented as a Kaplan–Meier diagram; average life span for each diet was compared with the Mann–Whitney U-test. The results on the PD model were presented as mean ± SEM and compared using a two-way ANOVA followed by Fisher LSD post-hoc test. The independent variables for the two-way ANOVA were Genotype (WT or mut(PD)) and Diet (control or *GPC*). Repeated Measures ANOVA followed by Fisher LSD post-hoc comparison was applied to analyze the data of the passive avoidance test with Genotype and Diet as between-subject variables and Time (Training, Test, or Extinction days) as a repeated measure. The level of significance was defined as *p* < 0.05. STATISTICA 10.0 software was used to perform all statistical analyses.
