**Maternal Diet Influences the Reinstatement of Cocaine-Seeking Behavior and the Expression of Melanocortin-4 Receptors in Female O**ff**spring of Rats**

### **Dawid Gawli ´nski , Kinga Gawli ´nska , Małgorzata Frankowska and Małgorzata Filip \***

Maj Institute of Pharmacology Polish Academy of Sciences, Department of Drug Addiction Pharmacology, Sm ˛etna Street 12, 31-343 Kraków, Poland; gawlin@if-pan.krakow.pl (D.G.); kingaw@if-pan.krakow.pl (K.G.); frankow@if-pan.krakow.pl (M.F.)

**\*** Correspondence: mal.fil@if-pan.krakow.pl

Received: 12 April 2020; Accepted: 14 May 2020; Published: 19 May 2020

**Abstract:** Recent studies have emphasized the role of the maternal diet in the development of mental disorders in offspring. Substance use disorder is a major global health and economic burden. Therefore, the search for predisposing factors for the development of this disease can contribute to reducing the health and social damage associated with addiction. In this study, we focused on the impact of the maternal diet on changes in melanocortin-4 (MC-4) receptors as well as on behavioral changes related to cocaine addiction. Rat dams consumed a high-fat diet (HFD), high-sugar diet (HSD, rich in sucrose), or mixed diet (MD) during pregnancy and lactation. Using an intravenous cocaine self-administration model, the susceptibility of female offspring to cocaine reward and cocaine-seeking propensities was evaluated. In addition, the level of MC-4 receptors in the rat brain structures related to cocaine reward and relapse was assessed. Modified maternal diets did not affect cocaine self-administration in offspring. However, the maternal HSD enhanced cocaine-seeking behavior in female offspring. In addition, we observed that the maternal HSD and MD led to increased expression of MC-4 receptors in the nucleus accumbens, while increased MC-4 receptor levels in the dorsal striatum were observed after exposure to the maternal HSD and HFD. Taken together, it can be concluded that a maternal HSD is an important factor that triggers cocaine-seeking behavior in female offspring and the expression of MC-4 receptors.

**Keywords:** cocaine self-administration; high-fat diet; high-sugar diet; maternal diet; pregnancy and lactation; melanocortin-4 receptor; offspring brain; rat offspring

#### **1. Introduction**

In the global population, every fourth person suffers from mental illnesses throughout their life, the most common of which are depression, anxiety, substance (alcohol, drugs) use disorder, and schizophrenia [1–3]. Substance use disorder is a chronic brain disorder associated with the uncontrolled exploration and use of drugs, leading to devastating health consequences and a destructive impact on the familial, social, and professional aspects of a patient's life. In addition, even after long periods of abstinence, this disease is characterized by high susceptibility to relapse in response to stress and to cues or contexts associated with drugs [4].

Recent studies have focused on the contribution of a properly balanced maternal diet during intrauterine growth and early childhood in the development of the central nervous system of offspring. In fact, exposure to excessive or insufficient amounts of macronutrients (fats, sugars, proteins) can lead to morphological, molecular, and functional changes in the brains of offspring, predisposing them to the occurrence of behavioral disorders and mental diseases, such as increased impulsiveness, depression, or anxiety, which further predispose them to the risk of developing substance use disorders later in life [5–9]. A maternal diet rich in fat provokes increased consumption and preference for palatable but unhealthy food in offspring [10–12], increased nicotine and ethanol self-administration [13,14], and disturbed behavioral reactions in animals regarding the administration of psychostimulant substances (reduced locomotor activity and amphetamine-induced behavioral sensitization) [15]. In turn, a maternal diet rich in fructose or sucrose induces an increase in the amount of alcohol consumed by the offspring [16]. Together, these studies confirm the positive relationship between the maternal diet and the offspring's susceptibility to drugs; however, there is still a lack of data on the relationship between the composition of the maternal diet and the development of cocaine use disorder (one of the most used drugs in the world among psychostimulants) [17]. Increasing knowledge about the predisposing factors for the development of mental disorders, including psychostimulant addiction, may contribute to reducing the health, social, and economic damage associated with these diseases in the future. Most literature provide data on the role of maternal obesity induced by high-calorie food on the development of the central nervous system in offspring. By limiting the modified types of diets only to pregnancy and lactation, our research will allow to indicate the period of intrauterine development and early childhood more specifically as a key factor in the development of the brain and behavioral disorders in adult offspring. Because the melanocortin system in the brain acts through melanocortin-4 (MC-4) receptors, which are located in brain regions that represent part of the reward system [18,19] and control (among other things) nutritional behavior, memory, positive enhancement, and emotions [20–22], these receptors were selected as a potential molecular biomarker of mechanisms underlying the cocaine susceptible phenotype in rat offspring induced by maternal nutrition. The relationship between maternal diet, MC-4 receptors and cocaine self-administration was demonstrated in recent studies by our group [23]. We showed, among other results, that a maternal high sugar-diet (HSD) significantly affects the expression of MC-4 receptors in the brains of male offspring following chronic cocaine self-administration or an abstinence period. Moreover, administration of an MC-4 receptor antagonist reduced cocaine- and cue-induced reinstatement of cocaine-seeking behavior; concurrently, male offspring that had received maternal nutrition from an HSD were more sensitive to the anti-relapsing effects of an MC-4 receptor antagonist than control male offspring [23].

In light of the above data and the small number of studies on the pathogenesis of developing addiction in females compared to studies in males, the purpose of this study was to determine the role of a maternal high-fat diet (HFD), HSD, and mixed diet (MD; rich in carbohydrate and fat) during pregnancy and lactation (critical periods in early life) on the behavioral and neurochemical changes in female offspring in the aspect of cocaine addiction. For this purpose, using the animal model of intravenous self-administration, we have comprehensively characterized the impact of a modified maternal diet on changes in the phenotype of offspring assessed at various stages of addiction: the acquisition and maintenance of cocaine addiction; abstinence; and the strength of cocaine-seeking behavior to cue- and cocaine-induced priming. In addition, at the molecular level, we evaluated the role of diet modifications during pregnancy and lactation on changes in MC-4 receptor protein expression in the synaptosomal fraction of brain structures related to cocaine addiction (the prefrontal cortex, dorsal striatum, nucleus accumbens, ventral tegmental area, and hypothalamus) in young adult female offspring.

#### **2. Materials and Methods**

#### *2.1. Animals and Diets*

All experiments were performed in accordance with the guidelines of the European Directive 2010/63/EU and were approved by the 2nd Local Institutional Animal Care and Use Committee (Maj Institute of Pharmacology Polish Academy of Sciences, Kraków, Poland; approval number 1270/2015; 42/2017). Every effort was made to minimize suffering and the number of animals used.

Wistar rats from Charles River (Germany) were housed in standard plastic rodent cages in a room maintained at 22 ± 2 ◦C and 55 ± 10% humidity under a 12 h light–dark cycle (lights on at 6:00 a.m.). Unless otherwise specified, animals had free access to water and food. Nulliparous female rats (200–240 g), after the acclimatization period and during the proestrus phase (smears from females were collected daily to determine the estrous cycle phase), were mated with males overnight, and pregnancy was confirmed by examining vaginal smears for the presence of sperm. Pregnant females (*n* = 10/group) were then individually housed and randomly assigned to four groups: control diet (CD; cat# VRF1; Special Diets Services, UK) or special diets purchased from Altromin (Germany): HFD (cat# C1057 mod.), HSD (cat# C1010), or MD (cat# C1011). The composition of the diets used in the study is presented in Table 1.


**Table 1.** Macronutrient profiles (expressed as a percentage of energy) and energy values of the control and modified diets used in this study.

Dams consumed these diets ad libitum during the gestation (21 days) and lactation periods (21 days). Dam body weight and food intake were monitored every third day. Litter sizes were normalized to 9–12 pups. After weaning, offspring were separated according to sex, housed six per cage, and fed a CD. Female offspring were used in the present study. The experimental design and timeline of the study are illustrated in Figure 1.

**Figure 1.** Experimental design and timeline. Dams were fed a control diet (CD) or one of the three modified diets: high-fat (HFD), high-sugar (HSD), or mixed (MD; rich in carbohydrate and fat) during pregnancy and lactation. Female offspring were divided into three cohorts and at postnatal day (PND) 63, they were assessed by tissue isolation and biochemical analyses, via the cocaine self-administration study and for locomotor activity.

### *2.2. Behavioral Procedures*

#### 2.2.1. Drugs

Cocaine hydrochloride (Toronto Research Chemicals, North York, ON, Canada) was dissolved in sterile 0.9% NaCl. The cocaine solution was administered intravenously or intraperitoneally. In a volume of 0.1 mL/infusion or 1 mL/kg, respectively.

#### 2.2.2. Cocaine Self-Administration Procedure

#### Catheter Implantation and Initial Lever-Press Training

At PND 53, female rats (*n* = 12 for each group) were anaesthetized with ketamine HCl (75 mg/kg; Bioketan; Biowet, Puławy, Poland) and xylazine (5 mg/kg; Sedazin; Biowet, Puławy, Poland) and chronically implanted with a silastic catheter in the external jugular vein, as described previously [24]. After surgery, meloxicam subcutaneously (0.5 mg/kg; Metacam; Boehringer Ingelheim Vetmedica GmbH, Ingelheim am Rhein, Germany) was administered, and animals were kept individually in standard home cages water and food ad libitum. Each day, catheters were flushed with 0.1 mL of saline solution containing heparin (70 U/mL; Polfa, Warszawa, Poland) or 0.1 mL of cephazolin solution (10 mg/mL; Biochemie GmbH, Kundl, Austria). After seven days of recovery, animals were food-deprived for 18–20 h (with free access to water) and were then trained to press a lever during 2-h daily sessions under the fixed ratio (FR) 1 schedule of sweetened milk reinforcement for two days. Throughout the two training days, food was limited to 70% of the rats' free-feeding amount.

#### Cocaine Self-Administration

Female offspring at PND 63 began lever pressing for cocaine reinforcement during the 2-h daily sessions performed six days/week (maintenance) for a total of three weeks, and from that time, they were given free access to food throughout the remainder of the experiment. Rats were trained to self-administer cocaine in described previously [25] standard operant chambers (Med-Associates, Fairfax, VT, USA) in contact with an infusion pump (Model 3.33 RPM, Med-Associates, Fairfax, VT, USA) according to two experimental protocols: (a) a stable dose of cocaine (0.5 mg/kg/infusion) and an increased schedule of reinforcement (FR1–5) or (b) increased cocaine doses (0.25–1 mg/kg/infusion) and a stable FR1 schedule of reinforcement. Each schedule was completed by pressing the "active" lever resulting in a 5 s infusion of cocaine and a 5 s presentation of the stimulus complex, which consisted of an activation of the white stimulus light directly above the "active" lever and a tone from the generator (2000 Hz; 15 dB above the ambient noise level). Following each infusion, there was a 20 s time-out period during which the response was recorded but had no programmed consequences. An "inactive" lever response never activated the infusion pump.

#### Progressive Ratio Test

After the final experimental cocaine session described above, rats were tested for the self-administration of cocaine under the progressive ratio (PR) schedule of reinforcement, and this session lasted 4 h. During the PR session, the delivery of intravenous cocaine was contingent on an increasing number of responses incremented through the following progression: 1–603 [26]. Breakpoints were defined as the number of completed ratios in the series over 4 h.

#### Extinction

After cocaine self-administration (see above), the rats previously used for the two different cocaine self-administration protocols were used in the extinction training/reinstatement tests. During the extinction sessions, the animals had 1-h daily sessions with neither a cocaine delivery (exchanged to saline) nor tone and light stimuli (conditioned stimuli).

#### Reinstatement of Cocaine-Seeking

After 10 days of extinction training, animals were evaluated for the response reinstatement induced by either a conditioned cue (the tone and light associated with cocaine self-administration) or a noncontingent presentation of a self-administered reinforcement (2.5 or 10 mg/kg cocaine, intraperitoneal). The order of the cocaine priming injections was counterbalanced according to a Latin square design, and the test sessions were separated by at least 2–3 baseline sessions of extinction training. During the 2-h reinstatement tests, presses of the active lever resulted in the intravenous infusion of saline.

#### 2.2.3. Locomotor Activity

Spontaneous motor activity was recorded individually for each drug-naïve rat from the subset at PND 63 in Opto-Varimex cages (43 cm × 44 cm, Columbus Instruments, Columbus, OH, USA) and analyzed using Auto-Track software (Columbus Instruments, Columbus, OH, USA) as described previously [24]. The locomotor activity of rats was defined as horizontal activity and was presented as the distance traveled in cm during 5-, 30-, and 120-min trials.

#### *2.3. Biochemical Analysis*

#### 2.3.1. Brain Tissue Collection

For biochemical analysis, at PND 63, a subset of drug-naïve female offspring rats was sacrificed by rapid decapitation, and the brains were promptly removed. Relevant brain structures were dissected according to the rat brain atlas [27] and isolated on ice-cold glass plates, immediately frozen on dry ice and stored at −80 ◦C for enzyme-linked immunosorbent assay (ELISA) analyses. To avoid the potential effect of stress on molecular changes in the brain, animals were not fasting before decapitation. All samples were collected between 9:00–12:00 (a.m.).

#### 2.3.2. Melancortin-4 Receptor Expression

MC-4 receptor expression was determined in the synaptosomal fraction The brain tissue samples were homogenized using a sonicator (EpiShear™ Probe Sonicator; Active Motif, Carlsbad, CA, USA) in 10% (*w*/*v*) of 0.32 M sucrose HEPES buffer (containing 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 5 mM HEPES) with a protease inhibitor cocktail (Complete, Roche, Mannheim, Germany). Later, suspended tissue was homogenized with a Dounce tissue grinder. The homogenate was centrifuged at 4 ◦C for 10 min at 600× *g*. The supernatant was then diluted 1:1 with 1.3 M sucrose HEPES buffer to obtain a suspension at a final concentration of 0.8 M sucrose. This suspension was further centrifuged twice in a series of washes with HEPES buffer at 4 ◦C for 15 min at 12,000× *g*. The supernatant was discarded each time. The pellet was suspended in RIPA buffer (containing a protease inhibitor, PMSF and 0.2% Triton X-100) and centrifuged at 4 ◦C for 30 min at 20,000× *g* [28]. The supernatant containing the synaptosomal fraction was frozen overnight at −20 ◦C and was used for further analyses the following day.

The levels of MC-4 receptors in the synaptosomal fraction of the prefrontal cortex (including the infralimbic, prelimbic, and cingulate cortices; Bregma: 5.2–2.7 mm), dorsal striatum, nucleus accumbens, ventral tegmental area, and hypothalamus were measured using ELISA kits (cat# E11964R; Wuhan EIAab Science Co., Wuhan, China) according to the manufacturer's protocols. Duplicate aliquots of 100 µL of each sample along with MC-4 receptor standards (0, 0.312, 0.625, 1.25, 2.5, 5, 10, and 20 ng/mL) were transferred to precoated 96-well ELISA plates. The absorbance was measured at a wavelength of λ = 450 nm using a Multiskan Spectrum spectrophotometer (Thermo LabSystems, Philadelphia, PA, USA). The concentration of MC-4 receptors was calculated from a standard curve and expressed as ng/mg of protein. Bicinchoninic acid assay (BCA) protein assay kits (Thermo Scientific, Rockford, IL, USA) were used (the Pierce™ BCA Protein Assay Kit for the prefrontal cortex, dorsal striatum and nucleus accumbens or the Micro BCA™ Protein Assay kit for the ventral tegmental area and hypothalamus) to determine the protein concentrations.

#### *2.4. Statistical Analysis*

Animals that had problems with the catheters during the recovery or experimental periods were excluded from the data analysis. All data are expressed as the mean ± standard error of mean (SEM). Statistical analyses were performed with either one-, two-, or multi-way analysis of variance (ANOVA), with the terms of the repeated measure analysis dependent on the experiment, using Statistica version 12 software (StatSoft, Tulsa, OK, USA). Post hoc Dunnett's or Newman–Keuls tests were used to analyze differences between group means. *p* < 0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. Maternal Body Weight, Caloric Intake and Litter Size*

The effects of the modified diets on changes in dam body weight and caloric intake during pregnancy and lactation are shown in Supplemental Figure S1. A two-way ANOVA for repeated measures showed significant effects of maternal diet × day interactions (F(51, 1324) = 2.515, *p* < 0.01). We observed that dams consumed MD had lower body weight gain in the last days of lactation compared to that of the control group. In addition, it was shown that modified diet consumption resulted in a change in caloric intake during pregnancy (F(3, 36) = 4.754, *p* < 0.01) and lactation (F(3, 36) = 20.660, *p* < 0.001). Females fed an HSD consumed more calories during pregnancy (*p* < 0.05), while during lactation, significantly more calories were consumed by dams from the HFD group (*p* < 0.01), in contrast to females fed MD, in which a decrease in the average daily caloric intake was observed (*p* < 0.01; Figure S1b). At the same time, modified maternal diets did not affect the litter size (F(3, 36) = 0.873, *p* = 0.464; Figure S1c) or birth weight of female offspring (F(3, 124) = 1.585, *p* = 0.196; Figure S1d).

#### *3.2. Expression of MC-4 Receptors*

The influence of maternal diet on the level of MC-4 receptors in the synaptosomal fraction of brain structures related to cocaine addiction (the prefrontal cortex, dorsal striatum, nucleus accumbens, ventral tegmental area, and hypothalamus) at PND 63 in the naïve female offspring was assessed (Figure 2).

**Figure 2.** Effects of maternal diet during pregnancy and lactation on melanocortin-4 (MC-4) receptor expression in the synaptosomal fraction of the prefrontal cortex, nucleus accumbens, dorsal striatum, ventral tegmental area, and hypothalamus in female offspring rats at PND 63. The results are expressed as the mean (±SEM). *n* = 8 rats/group. Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. \* *p* < 0.05, \*\*\* *p* < 0.001 versus the control diet (CD) group. HSD, high-sugar diet; HFD, high-fat diet; MD, mixed diet.

We observed that modified maternal diets during pregnancy and lactation evoked changes in MC-4 receptors in the synaptosomal fraction in the nucleus accumbens (F(3, 28) = 6.604, *p* < 0.01) and dorsal striatum (F(3, 28) = 3.359, *p* < 0.05), but not in the prefrontal cortex (F(3, 28) = 2.493, *p* = 0.081), ventral tegmental area (F(3, 28) = 0.148, *p* = 0.930) or hypothalamus (F(3, 28) = 2.128, *p* = 0.119) in female offspring. Post hoc analysis showed that a maternal HSD or MD during pregnancy and lactation increased the expression of MC-4 receptors in the female nucleus accumbens (*p* < 0.05 and *p* < 0.001, respectively), and a maternal HFD or HSD increased its expression within the dorsal striatum (*p* < 0.05).

#### *3.3. Locomotor Activity*

Spontaneous locomotor activity, recorded for 5, 30, and 120 min, did not differ between female offspring whose mothers consumed different diets, CD, HFD, HSD, or MD, during pregnancy and lactation (Table 2).


**Table 2.** Locomotor activity of female offspring at postnatal day 63.

Distance traveled (cm) was measured after 5, 30, and 120 min in female offspring whose mothers were fed a high-fat (HFD), high-sugar (HSD), or mixed diet (MD) during pregnancy and lactation. The results are expressed as the mean (±SEM). *n* = 12 rats/group. Data were analyzed by one-way analysis of variance (ANOVA) versus the control diet (CD) group.

#### *3.4. Cocaine Self-Administration*

The impact of maternal diets during pregnancy and lactation on cocaine self-administration in female offspring rats in two experimental protocols (stable dose of drug reinforcement and an increased reinforcement schedule, or an increased dose of the drug with a stable reinforcement schedule) was studied.

#### 3.4.1. Stable Cocaine Dose and Increased Reinforcement Schedule

Figure 3a shows the number of active and inactive lever presses (upper panels) and the number of infusions (lower panels) during three sequential weeks with increasing FR schedules of reinforcement (FR1–5) and a stable dose of cocaine (0.5 mg/kg/infusion) for the female offspring from the CD, HFD, HSD, and MD groups. Multi-way ANOVA for repeated measures did not show significant effects of maternal diet × session × lever interactions during the eighteen days of cocaine self-administration (F(51, 1324) = 0.515, *p* = 0.998). In addition, we did not find differences between the effect of maternal diet on the number of cocaine infusions during the three weeks of self-administration, as demonstrated by a two-way ANOVA for repeated measures (F(51, 663) = 1.112, *p* = 0.280). Despite the lack of statistically significant differences, a reduced amount of infusions can be seen in the first and second weeks of self-administration in female offspring exposed to a maternal HSD.

#### 3.4.2. Increased Cocaine Dose and Stable Reinforcement Schedule

The number of active and inactive lever presses (upper panels) and the number of cocaine infusions (lower panels) for the increasing doses of cocaine (0.25, 0.5 and 1 mg/kg/infusion) with a stable FR1 reinforcement schedule during the entire experiment are shown in Figure 4a. We observed that the active and inactive lever presses of female offspring whose mothers were fed modified diets during pregnancy and lactation did not differ significantly from the lever presses conducted by the control diet group in this protocol. Multi-way ANOVA for repeated measures did not reveal significant maternal diet × session × lever interactions during the eighteen sessions of cocaine self-administration (F(51, 1428) = 0.896, *p* = 0.682). A two-way ANOVA for repeated measures also showed no significant changes in the number of cocaine infusion (F(51, 714) = 0.989, *p* = 0.497) in offspring.

**Figure 3.** (**a**) Effects of a maternal high-fat (HFD), high-sugar (HSD), or mixed (MD) diet during pregnancy and lactation on the acquisition/maintenance of cocaine (0.5 mg/kg/infusion) self-administration with increasing fixed ratio (FR) schedules of reinforcement (FR1–5) in female offspring rats. (**b**) Effects of the modified maternal diets on cocaine self-administration with a progressive ratio schedule in female offspring day after the last session of cocaine self-administration under an increasing schedule of reinforcement (FR1–5) and a stable dose of cocaine (0.5 mg/kg/infusion). (**c**) Effects of the modified maternal diets upon self-administration extinction with a fixed dose of cocaine (0.5 mg/kg/infusion) and an increased schedule of reinforcement (FR1–5) in female offspring rats. Numbers of active and inactive lever presses and cocaine infusions are expressed as the mean (±SEM). The number of animals in each group was as follows: CD (*n* = 11), HFD (*n* = 10), HSD (*n* = 10), MD (*n* = 12). CD, control diet.

90

a)

**Figure 4.** (**a**) Effects of a maternal high-fat (HFD), high-sugar (HSD), or mixed (MD) diet during pregnancy and lactation on the acquisition/maintenance of cocaine (0.25–1 mg/kg/infusion) self-administration with a stable FR1 schedule of reinforcement in female offspring rats. (**b**) Effects of the modified maternal diets on cocaine self-administration with a progressive ratio schedule in female offspring day after the last session of cocaine self-administration with a stable FR1 schedule of reinforcement and an increasing dose of cocaine (0.25–1 mg/kg/infusion). (**c**) Effects of the modified maternal diets on self-administration extinction with increased doses of cocaine (0.25–1 mg/kg/infusion) and a stable FR1 schedule of reinforcement in female offspring rats. Numbers of active and inactive lever presses and cocaine infusions are expressed as the mean (±SEM). The number of animals in each group was as follows: cocaine self-administration CD (*n* = 12), HFD (*n* = 11), HSD (*n* = 11), MD (*n* = 12); extinction training CD (*n* = 11), HFD (*n* = 10), HSD (*n* = 10), MD (*n* = 12). CD, control diet.

#### *3.5. PR Schedule of Cocaine Reinforcement*

The female offspring from the HFD, HSD, and MD groups did not differ from the control group in the break point during cocaine self-administration under the PR reinforcement schedule following an increasing schedule of reinforcement and a stable dose of cocaine (F(3, 39) = 1.222, *p* = 0.315). Animals did not differ in active and inactive lever presses during cocaine (0.5 mg/kg/infusion) self-administration in the PR protocol (F(3, 78) = 0.637, *p* = 0.594; Figure 3b).

Maternal exposure to HFD, HSD, or MD did not change behavioral readouts in female offspring in cocaine (1 mg/kg/infusion) self-administration in the PR scheme following a stable FR1 reinforcement schedule and an increasing dose of cocaine (number of cocaine infusions (F(3, 42) = 0.406, *p* = 0.749); number of active and inactive lever presses (F(3, 84) = 0.804, *p* = 0.495; Figure 4b).

#### *3.6. Extinction Training*

Extinction training was introduced to all rats after cocaine self-administration. After substituting saline for cocaine, a progressive drop in lever responses was observed over the extinction sessions for the CD, HFD, HSD, and MD groups. In the female offspring trained to self-administer cocaine (0.5 mg/kg/infusion) with the increased FR1–5 schedule of reinforcement, a multi-way ANOVA for repeated measures did not show significant effects of maternal diets on extinguished active lever pressing (F(27, 702) = 0.545, *p* = 0.972; Figure 3c). Rats from mothers fed different diets did not differ in their extinction of active lever responses (F(27, 702) = 0.442, *p* = 0.994) following cocaine (from 0.25 to 1 mg/kg/infusion) with the FR1 reinforcement schedule (Figure 4c).

#### *3.7. Reinstatement of Cocaine-Seeking Behavior*

Following 10 days of extinction training, all groups of female rats were tested for response reinstatement induced by the cocaine-associated cue or cocaine (2.5 or 10 mg/kg, intraperitoneal).

#### 3.7.1. Relapse after Stable Cocaine Dose and Increased Reinforcement Schedule

Figure 5a shows the number of active and inactive lever presses in offspring that were previously subjected to self-administered cocaine (0.5 mg/kg/infusion) with the increased FR schedule of reinforcement. Statistical analysis demonstrated that in female offspring, modified maternal diets changed cue-induced reinstatement (F(3, 78) = 3.453, *p* < 0.05) as well as cocaine-induced reinstatement for a cocaine dose of 2.5 mg/kg (F(3, 78) = 4.670, *p* < 0.01). Furthermore, there was no significant difference in the strength of the recurrence of cocaine-seeking behavior between the CD group and the tested diets for cocaine doses of 10 mg/kg (F(3, 78) = 1.661, *p* = 0.182). Post hoc tests indicated an increase in active lever responses only in female offspring from the HSD group upon re-exposure on cue or intraperitoneal cocaine administration at a dose of 2.5 mg/kg (*p* < 0.01 and *p* < 0.001, respectively).

#### 3.7.2. Relapse after Increased Cocaine Dose and Stable Reinforcement Schedule

The number of active and inactive lever responses in animals that were previously trained to self-administer increasing cocaine doses (0.25–1 mg/kg/infusion) with a stable FR1 schedule of reinforcement is shown in Figure 5b. We found that a maternal HFD, HSD, and MD did not significantly change the female offspring relapse strength after re-exposure to cocaine-associated cues (F(3, 78) = 1.895, *p* = 0.448) and cocaine (2.5 mg/kg) compared to that of the control animals (F(3, 78) = 1.990, *p* = 0.122). However, differences in active lever presses were observed after cocaine was administered at a dose of 10 mg/kg (F(3, 78) = 3.043, *p* < 0.05). The HSD female offspring pressed the active lever more times during the test (*p* < 0.05) compared to the number of presses performed by the CD group.

**Figure 5.** Effects of a maternal high-fat (HFD), high-sugar (HSD), or mixed (MD) diet during pregnancy and lactation on reinstatement of the cocaine-seeking behavior induced by the cue (CUE; tone + light) and the drug in female rat offspring. Drug-induced reinstatement was triggered by the administration of cocaine (2.5 or 10 mg/kg, i.p.). (**a**) Tests were performed after cocaine (0.5 mg/kg/infusion) self-administration with an increasing schedule of reinforcement (FR1–5) and 10 days of extinction training. (**b**) Tests were performed after cocaine (0.25–1 mg/kg/infusion) self-administration with a stable FR1 schedule of reinforcement and 10 days of extinction training. The numbers of active and inactive lever presses are expressed as the mean (±SEM). The number of animals each group was as follows: CD (*n* = 11), HFD (*n* = 10), HSD (*n* = 10), MD (*n* = 12). Data were analyzed by two-way ANOVA and the post hoc Newman–Keuls test. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 versus the control diet (CD) group.

#### **4. Discussion**

In this study, we showed that a modified maternal diet during pregnancy and the lactation period is an important factor inducing impairments at the behavioral and neurochemical levels and a cocaine-seeking prone phenotype in female offspring. We focused on females due to the observed differences in addiction to psychoactive substances in relation to sex (e.g., women are more likely to relapse than men) [29] as well as the smaller number of studies assessing the effect of maternal nutrition on behavioral changes in female offspring compared to the number of studies regarding male

offspring. The results of this work are the first to indicate that a modification in the composition of maternal diet, particularly an increased amount of carbohydrates (mainly sucrose) consumed during fetal development and early childhood, results in an increased relapse of cocaine-seeking behavior and in increased levels of MC-4 receptors in selected brain structures in female offspring.

Human research indicates that both obesity and the consumption of an HFD increases total food consumption and the risk of alcohol [30,31] and nicotine addictions [32]. Recently, an increasing number of preclinical studies focused mainly on maternal HFDs have proven that nutrition may predispose offspring to an increased intake of palatable food [10,11,33], drinking alcohol [16,34,35], nicotine [14], or simultaneous ethanol and nicotine [13] self-administration. In this paper, a series of behavioral tests using an animal model of intravenous cocaine self-administration to determine the female offspring phenotype after exposure to modified maternal diets were performed. We showed that the predisposition to cocaine self-administration in female rats from a CD, HFD, HSD, or MD groups was similar and did not depend on the schedule of reinforcement or the dose range of cocaine. In fact, the animals did not differ significantly in the number of presses on the active and inactive levers or the number of drug infusions during the acquisition/maintenance phase in either the cocaine motivational scoring protocol (FR1–5) while testing the rewarding effects of the drug (cocaine 0.25–1 mg/kg/infusion) or during the following test using the PR schedule to assess animals' motivation. Moreover, following three weeks of exposure to cocaine self-administration, no changes in female behavior were noted during the drug abstinence period. In other words, modified maternal diets during pregnancy and lactation did not influence either the rewarding or motivational aspects of cocaine intake in female offspring.

Substance use disorder is a debilitating chronic brain disease characterized by high susceptibility to relapse in response to stress or stimuli associated with previous drug use, even after a long period of abstinence [4]. Approximately 40–60% of human addicts entering therapy return to using addictive substances within the first year of treatment [36]. Due to the serious problem of relapse concerning effective addiction therapy, our main finding was that in comparison to the control groups, the females consuming an HSD, depending on the protocol of cocaine self-administration, evoked a higher reinstatement following re-exposure to the cue associated with the previous cocaine infusions or the presentation of the cocaine-priming dose (10 mg/kg). Interestingly, cocaine at a dose of 2.5 mg/kg, which normally does not cause relapse (subthreshold dose), also significantly enhanced the active lever presses in the female offspring of mothers that had consumed an HSD. It seems, therefore, that the increased potency of cocaine relapse in offspring exposed to a maternal HSD may depend not only on the conditions of individual development, but also on the pattern of drug use before abstinence. Moreover, in both schedule protocols, there was no change in the rats' responses to the inactive lever, which suggests the specificity of the observed behavioral responses. Additionally, naïve female offspring from diet-exposed groups showed similar spontaneous locomotor activity, which means that the rats' motor activity was not affected by the maternal diets and that the observed changes in cocaine-seeking behavior are specific. Our findings showed that the effect of a maternal HSD during pregnancy and lactation on the severity of a cocaine relapse is not dependent on sex, as a similar increase in the reinstatement of drug-seeking behavior was found in male offspring [23].

Behavioral studies emphasize that prenatal exposure to cocaine causes increased self-administration of cocaine [37–39] or alcohol [40] in offspring. Data from human observations indicate that exposure to cocaine in utero leads to the earlier and more frequent use of drugs (cocaine, marijuana, alcohol, and nicotine) by offspring in adolescence and early adulthood [41–44]. On the other hand, the literature provides evidence that the consumption of a natural reward, such as sugar, manifests similar neurochemical effects in the brain as the use of most addictive psychoactive substances. Within the central reward system, an increased dopamine level was observed [45–48], as was the altered expression of dopamine receptors [49–51] or the adaptive reduction of dopamine levels as a result of chronic exposure to a sweet, natural reward [52]. In addition, behavioral changes characteristic of drug addiction were observed similar to the neurochemical responses to natural rewards and psychoactive

substances. These included, among others, increased sugar consumption [53] or increased animal responses to a conditional stimulus associated with prior sucrose self-harvesting [54] after a period of abstinence. Hence, the abuse of tasty foods or drugs is not the only way to induce similar neurochemical and behavioral changes. Preclinical studies emphasize that exposure to a modified maternal diet during intrauterine development, as well as addictive substances, also significantly affects the offspring reward system being formed and its behavior later in life. Exposure to a diet rich in energy (e.g., an HFD or western-type diet) during pregnancy and lactation induced increased cocaine-induced place preference in the conditioned place preference (CPP) test, alcohol consumption, sensitivity to amphetamine administration, and preference for fat, and at the molecular level changes in dopaminergic brain signaling in juvenile and adult offspring [10,12,35,55]. There is also evidence of the interaction of natural rewards (e.g., sucrose) and addictive substances such as cocaine [56–58], methamphetamine [59], or alcohol [60]. For example, sensitization of behavioral responses occurs following cocaine administration and prolonged activity involving addictive substances in rats having intermittent access to granulated sucrose compared to the behavioral responses in animals consuming only standard laboratory feed [56]. This may suggest that maternal exposure to an increased amount of sucrose relative to a CD during pregnancy and early childhood, which is crucial for normal brain development, may lead to hypersensitivity between the natural reward (sugars) and the psychostimulants in offspring. Hence, female offspring of mothers that consumed an HSD may be more sensitive to conditional and unconditional stimuli associated with previous positive enhancements experienced during cocaine self-administration.

One of the neurochemical mechanisms that can potentially explain the offspring's behavior to cocaine may be associated with changes in the melanocortin system induced by a modified maternal diet. In fact, female offspring of mothers that consumed an HSD demonstrated increased cocaine-seeking behavior irrespective of the protocol, and the drug-naïve young adults female originating from mothers that consumed an HSD showed an upregulation of MC-4 receptors in the synaptosomal fraction of the nucleus accumbens and dorsal striatum, the brain regions linked to drug-seeking behavior [61,62]. Similarly, increased levels of accumbal MC-4 receptors were observed in female offspring exposed to an MD or in the dorsal striatum in the HFD group, but such changes did not provoke behavioral changes. This indicates the complexity of the factors involved in cocaine-seeking behavior or that behavioral changes manifest only when disturbance within MC-4 receptors occurs in several structures of the reward system. The simultaneous change in the expression of MC-4 receptors within the nucleus accumbens and dorsal striatum may lead to different activities of these structures than that observed in members of the CD group after exposure to conditional and unconditional stimuli, which may contribute to the observed intensification of the reinstatement of cocaine-seeking behavior in these animals. Previous studies assessing the effect of HFD intake by mothers before conception and during pregnancy and lactation showed reduced MC-4 mRNA expression in the female hypothalamus of offspring immediately after lactation (PND 20) [63]. Our findings demonstrate that switching to a CD after weaning, on one hand, can restore the basal level of MC-4 receptors in the hypothalamus, but the maternal diet leading to distant neurochemical consequences occurring in the nucleus accumbens and dorsal striatum does not disappear during the lifetime of the offspring.

The changes within the MC-4 receptors observed in the rat brain regions associated with the dopamine mesocorticolimbic system seem to be particularly important, as the brain structure is strongly associated with psychostimulant reward effects [64] and is involved in cue-, cocaine-, or stress-inducing reinstatement [65,66], supplementing existing knowledge about the interaction of psychoactive substances and MC-4 receptors. Recent studies have shown a decrease in the activity of agouti-related protein (AgRP) and proopiomelanocortin (POMC) neurons in the hypothalamus after the administration of cocaine, amphetamine, and nicotine, which suggests that not only reward pathways but also neuronal pathways associated with maintaining homeostasis affect the enhancement of these substances [67]. Typically, passively administered cocaine resulted in an increase in MC-4 receptor mRNA levels in the striatum, hippocampus, and hypothalamus [68,69]. Moreover, administration

of the MC-4 receptor antagonist SHU-9119 to the nucleus accumbens reduced low-dose cocaine self-administration (0.125–0.25 mg/kg/infusion), cocaine-induced CPP, and the reinstatement of cocaine-seeking behavior [69]. On the other hand, the blockade of central MC-3/4 receptors with AgRP resulted in the blockade of acute and sensitized locomotor responses to cocaine [70]. Our recent data showed differences in the levels of MC-4 receptors within the prefrontal cortex, nucleus accumbens, dorsal striatum, and amygdala between CD and HSD male offspring after three weeks of cocaine self-administration and 10 days of extinction [23]. The data suggest that a maternal HSD during pregnancy and lactation interferes with the adaptive mechanisms in the brains of male offspring, during which abstinence can restore melanocortin signaling homeostasis after cocaine exposure. Current evidence suggests that MC-4 receptor activity may affect behavioral aspects of the cocaine response through interaction and modulation of the dopaminergic system [71–75].

In summary, the described results emphasize the important role of a maternal diet rich in sugars during fetal development and early childhood in the predisposition of female offspring to cocaine-seeking behavior in adult life. Moreover, an altered amount of macronutrients in the maternal diet disrupts the proper expression of MC-4 receptors in brain structures involved in cocaine relapse in female offspring, thus leading to a stronger response to exposure to conditioned and unconditioned stimuli combined with earlier cocaine self-administration.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/12/5/1462/s1, Figure S1: Effects of a high-fat (HFD), high-sugar (HSD), or mixed (MD) diet on (a) maternal body weight changes (as a percentage of weight gain compared to start weight) and (b) average daily energy intake during pregnancy and lactation. Effects of the modified maternal diets on (c) litter size and (d) birth body weight of female offspring.

**Author Contributions:** D.G. designed and performed the study, analyzed the data and writing the manuscript. K.G. and M.F. (Małgorzata Frankowska) performed the study, analyzed the data and contributed to writing the manuscript. M.F. (Małgorzata Filip) conceived, designed and coordinated the study and contributed to writing the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by National Science Centre (Kraków, Poland), research grant UMO-2016/21/B/NZ4/00203.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Beneficial Effects of Exogenous Ketogenic Supplements on Aging Processes and Age-Related Neurodegenerative Diseases**

**Zsolt Kovács <sup>1</sup> , Brigitta Brunner 1,2 and Csilla Ari 3,4, \***


<sup>3</sup> Behavioral Neuroscience Research Laboratory, Department of Psychology, University of South Florida, 4202 E. Fowler Ave, PCD 3127, Tampa, FL 33620, USA

<sup>4</sup> Ketone Technologies LLC, 2780 E. Fowler Ave. #226, Tampa, FL 33612, USA

**\*** Correspondence: csari2000@yahoo.com; Tel.: +1-(813)-2409925

**Abstract:** Life expectancy of humans has increased continuously up to the present days, but their health status (healthspan) was not enhanced by similar extent. To decrease enormous medical, economical and psychological burden that arise from this discrepancy, improvement of healthspan is needed that leads to delaying both aging processes and development of age-related diseases, thereby extending lifespan. Thus, development of new therapeutic tools to alleviate aging processes and related diseases and to increase life expectancy is a topic of increasing interest. It is widely accepted that ketosis (increased blood ketone body levels, e.g., β-hydroxybutyrate) can generate neuroprotective effects. Ketosis-evoked neuroprotective effects may lead to improvement in health status and delay both aging and the development of related diseases through improving mitochondrial function, antioxidant and anti-inflammatory effects, histone and non-histone acetylation, β-hydroxybutyrylation of histones, modulation of neurotransmitter systems and RNA functions. Administration of exogenous ketogenic supplements was proven to be an effective method to induce and maintain a healthy state of nutritional ketosis. Consequently, exogenous ketogenic supplements, such as ketone salts and ketone esters, may mitigate aging processes, delay the onset of age-associated diseases and extend lifespan through ketosis. The aim of this review is to summarize the main hallmarks of aging processes and certain signaling pathways in association with (putative) beneficial influences of exogenous ketogenic supplements-evoked ketosis on lifespan, aging processes, the most common age-related neurodegenerative diseases (Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis), as well as impaired learning and memory functions.

**Keywords:** ketogenic supplement; ketosis; aging; lifespan; neurodegenerative disease; learning; memory

#### **1. Introduction**

Aging processes result in irreversible decline of normal physiological functions (timedependent functional decline) and age-related diseases. It has been demonstrated that several genes and environmental factors can modulate cellular functions leading to the appearance of ageing hallmarks, such as cellular senescence, mitochondrial dysfunction, loss of proteostasis, telomere attrition, deregulated nutrient sensing, stem cell exhaustion and epigenetic alterations [1,2]. These changes may generate, for example, chronic inflammation and aging that leads to increased risk for age-related chronic diseases, such as neurodegenerative diseases (e.g., Alzheimer's disease), osteoporosis, cardiovascular diseases, cancer, diabetes, sarcopenia and osteoarthritis [1,2].

A worldwide increase in elderly population has been predicted, as about 9% of people were over the age of 65 in 2019, which number was predicted to increase to approximately 17% by 2050 [3,4]. Human lifespan is increasing, as a result of more and more effective

**Citation:** Kovács, Z.; Brunner, B.; Ari, C. Beneficial Effects of Exogenous Ketogenic Supplements on Aging Processes and Age-Related Neurodegenerative Diseases. *Nutrients* **2021**, *13*, 2197. https:// doi.org/10.3390/nu13072197

Academic Editor: M. Hasan Mohajeri

Received: 26 May 2021 Accepted: 24 June 2021 Published: 26 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

therapeutic tools and improvement in living conditions, but the health status of patients is not improving by the same intensity. Thus, the prevalence of age-related diseases, such as neurodegenerative diseases are continuously increasing each year [5,6] and the consequences of aging processes and related diseases generate enormous medical, psychological and economical burden for humanity [7]. To decrease the negative consequences of aging processes and related diseases, thereby to mitigate their negative effects on health and the economy, several drugs were developed that are undergoing clinical trials. For example, rapamycin and its analogues [8–10], metformin [11,12], sirtuin (SIRT) activators [13,14] and senolytics (for elimination of senescent cells) [15] can modulate aging mechanisms, and, as a consequence, increase lifespan and decrease risk for age-related diseases. However, to prevent, alleviate and delay age-related processes and diseases, to extend health span and to improve the quality of life of elderly population, development of safer and more effective drugs and therapeutic tools are needed.

Exogenous ketogenic supplements (EKSs), such as ketone esters (KEs, e.g., *R*,*S*-1,3 butanediol—acetoacetate diester), ketone salts (KSs, e.g., Na+/K+—β-hydroxybutyrate/βHB mineral salt), and medium chain triglycerides (MCTs/MCT oils containing, e.g., about 60% caprylic triglyceride and 40% capric triglyceride) have been proven effective when used together with normal diet to induce and maintain an increased blood ketone body level (ketosis) [16–20]. It has been demonstrated that the level of EKSs-induced ketosis may change by age and gender [21]. Ketone bodies (e.g., βHB and acetoacetate) can enter to the central nervous system (CNS) via monocarboxylate transporters and can be used for ATP (adenosine triphosphate) synthesis via the Krebs-cycle in brain cells [22–25]. It has been demonstrated that EKSs can generate rapid (0.5–6 h after administration) and mild to moderate [19,26–29] therapeutic ketosis (about 1–7 mM) [30,31]. In order to sustain therapeutic ketosis leading to positive outcome, administration of different amounts of EKSs must be repeated for several days or up to several months depending on the disease, the dose and type of EKSs. For example, administration of 30 g MCT drink/day for 6 months and 75 g KE/day for 4 weeks were able to evoke beneficial effects in patients with mild cognitive impairment and type 2 diabetes, respectively [32,33]. However, it has been suggested that not only these, but other EKSs may be effective and safe ketone body precursors for the treatment of diseases in humans through increased βHB level (ketosis) [29,32,34,35]. It has been demonstrated that EKSs are well-tolerated and safe (with mild adverse effects, if any) [19,26,28,29,33,36]. Moreover, administration of EKSs can circumvent both dietary restrictions and adverse effects of ketogenic diets (e.g., nephrolithiasis, constipation and hyperlipidemia) [37]. Thus, administration of EKSs may be a safe and effective alternative metabolic therapy to the ketogenic diet.

It has also been demonstrated that administration of EKSs-generated therapeutic ketosis may evoke beneficial effects on CNS diseases [34,38,39]. For example, KEs, KSs and MCT oils can evoke anti-seizure and anti-epileptic effects [36,40–42], anxiolytic influence [26,43,44], regeneration of nervous system injuries [45] and alleviating effects on neurodegenerative diseases (such as Alzheimer's disease) [41,46–48]. These beneficial effects were induced likely through ketosis-evoked neuroprotective effects, for example, by improved mitochondrial functions, enhanced ATP levels, decreased inflammatory processes and decreased oxidative stress [23,24,34,49,50]. Moreover, ketone bodies may modulate aging processes thereby extend lifespan and delay the development of agerelated diseases, such as neurodegenerative diseases. In fact, it has been demonstrated that not only ketogenic diets, but also administration of EKSs can increase and maintain blood ketone body level [19,26–29], which ketone bodies, such as βHB, may promote anti-aging effects [35,51,52]. Moreover, it was demonstrated that βHB, as an endogenous ligand molecule, can activate the hydroxycarboxylic acid receptor 2 (HCAR2 or GPR109A receptor) [53,54]. HCAR2 receptors are expressed not only in macrophages, but also in the brain cells, mainly in microglia, as well as astrocytes and neurons [54–56]. Thus, βHB molecule via, for example, HCAR2 receptors can modulate not only physiological, but also pathophysiological processes in the brain that are connected to aging and neurode-

generative diseases [55,57,58]. Based on the literature, increase of βHB level may be the main factor contributing to the beneficial effects on aging, lifespan and age-related diseases after administration of EKSs. Indeed, it has been demonstrated that βHB decreased the senescence associated secretory phenotype (SASP) of mammals [59] and extended the lifespan of *C. elegans* [60]. Consequently, in this review paper we focused on βHB-generated alleviating effects. Although limited evidence supports the alleviating influence of EKSs on lifespan, aging processes and related CNS diseases, we can hypothesize that EKSs-evoked increase in blood βHB level can modulate (alleviate) aging processes and improve symptoms of age-related diseases through their neuroprotective effects, therefore may delay both aging and the development of related diseases and extend lifespan.

This review discusses the hallmarks of aging and putative anti-aging molecular mechanisms (pathways) by which EKSs may be able to exert their beneficial effects on lifespan, healthspan, aging, the most common age-related neurodegenerative diseases (Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis), as well as learning and memory.

#### **2. Main Features of Aging Processes**

It has been demonstrated that aging is the most common risk factor for emergence of neurodegenerative diseases [2]. Indeed, as life expectancy of humans increase, more and more people suffer from different types of neurodegenerative diseases, such as Alzheimer's disease [61]. Moreover, it has been demonstrated that development and incidence of the most common neurodegenerative diseases, Alzheimer's disease (e.g., characterized by extracellular senile, amyloid-β/Aβ plaque and neurofibrillary tangle/hyperphosphorylated and misfolded Tau accumulation in the brain; impairment of learning and memory), Parkinson's diseases (e.g., characterized by the accumulation of α-synuclein and the loss of dopaminergic neurons; tremors and muscle rigidity) and amyotrophic lateral sclerosis (e.g., accumulation of TAR DNA-binding protein 43; progressive degeneration of motor neurons a motor defects; muscle weakness) are promoted by aging [6,62–64]. It has also been also demonstrated that aging hallmarks, such as reduced telomere length and/or genomic instability, epigenetic alterations, mitochondrial dysfunction, cellular senescence, loss of proteostasis, changes in activity of nutrient sensing pathways and intercellular communication, as well as stem cell exhaustion can be detected in Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. However, in amyotrophic lateral sclerosis, the reduced telomere length, genomic instability, cellular senescence and changes in intercellular communication may be the main contributing factors [63,64]. Thus, in this chapter, we shortly characterize the main aging hallmarks and their connection with the development of the above-mentioned age-related neurodegenerative diseases. Moreover, based on the literature (e.g., administration and effects of senomorphic drugs and caloric restriction) we present the main signaling pathways contributing to the modulation of aging processes, suggesting that inhibition or activation of these pathways may be used for delaying not only aging, but also related neurodegenerative diseases, improve impaired learning and memory functions, as well as to promote lifespan.

#### *2.1. Nutrient Sensing Pathways*

Changes in activity of nutrient sensing pathways may have a role in aging and development of age-related diseases. It has been demonstrated that caloric restriction and fasting can attenuate aging, expand lifespan, generate neuroprotective effects and prevent age-related diseases through energy (nutrient) sensing insulin/insulin-like growth factor (IGF) 1 (IIS) pathway, AMP (adenosine monophosphate) activated serine-threonine protein kinase (AMPK), Sirtuin 1 (SIRT1) and transcriptional factor FOXOs (Forkhead box Os) [65–68]. Previous studies show that caloric restriction can decrease IGF, insulin, glucose and amino acid levels, whereas increase NAD<sup>+</sup> (nicotinamide adenine dinucleotide) and AMP levels (Figure 1). These alterations are sensed by the (i) IIS pathway, activated by increased IGF and glucose levels; (ii) AMPK, which senses low energy states via increased –

AMP levels; (iii) SIRT1, which also senses low energy states via increased NAD<sup>+</sup> levels (NAD<sup>+</sup> -dependent protein deacetylase); and (iv) mechanistic target of rapamycin (mTOR), which senses high amino acid levels leading to stress resistance, oxidative metabolism, enhanced DNA repair, epigenetic stability and increase in longevity [69–71]. –

evoked ketosis (βHB) can imβHB, beta κB, nuclear factor kappa 1α activated receptor gamma (PPARγ) coactivator 1α; **Figure 1.** Main downstream signaling pathways and some effects, by which different senomorphic drugs (e.g., metformin), interventions (e.g., caloric restriction) and, theoretically, exogenous ketogenic supplements-evoked ketosis (βHB) can improve age-dependent impaired processes (aging hallmarks). Abbreviations: Akt, Akt kinase/protein kinase B; AMPK, AMP activated serine-threonine protein kinase; ATP, adenosine triphosphate; βHB, beta-hydroxybutyrate; FOXOs, Forkhead box Os; HCAR2, hydroxycarboxylic acid receptor 2; IGF 1, insulin-like growth factor 1; mTORC1, mechanistic target of rapamycin C1; NAD<sup>+</sup> , nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide (NAD) + hydrogen (H); NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; Nrf2, nuclear factor erythroid 2-related factor 2; PGC-1α, peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α; PI3K, phosphatidyl inositol-3-kinase; ROS, reactive oxygen species; SASP, senescence associated secretory phenotype; SIRT1, Sirtuin 1.

mediated toxicity of the Aβ1– β – abnormal aggregation of proteins in neurodegenerative diseases, such as Alzheimer's dis-Reduced activity of the IIS pathways can extend lifespan [72], similarly to the mTOR inhibitor rapamycin-evoked increase in lifespan [9]. It was also demonstrated that decreased IIS signaling reduced the aggregation-mediated toxicity of the Aβ1–42 (amyloid β-peptide 1–42), suggesting that decreased insulin signaling may be protective against abnormal aggregation of proteins in neurodegenerative diseases, such as Alzheimer's disease [73]. Moreover, mTOR (a serine/threonine protein kinase) is the main regulator of cellular growth and mass accumulation, which contains mTORC1 and mTORC2 complexes [6]. mTORC1 is able to integrate signals from nutrients, growth factors, energy, and oxygen level in order to promote cell proliferation and growth (e.g., enhancement of energy metabolism/glycolysis and nucleotide, protein, as well as lipid synthesis and inhibition of catabolism/autophagy) [74,75] (Figure 1). Indeed, for example, mTORC1 supports protein synthesis by phosphorylation of S6K1 (ribosomal protein S6 kinase 1) and 4EBP1 (eukaryotic translation initiation factor 4E binding protein 1) molecules, which processes may be activated by Akt kinase (protein kinase B) [6,75,76] (Figure 1). Moreover, mTORC1 can suppress autophagy via inhibition of ULK1 (Uncoordinated/Unc-51-like kinase 1) by which impedes the cellular homeostasis maintaining processes (e.g., providing nutrients under starvation and removing damaged organelles and misfolded proteins) [75,77]. Thus, inhibition of mTORC1 effects on autophagy may be an important tool to decrease age-dependent

processes (aging hallmarks, such as loss of proteostasis) and promoting longevity [6] (Figure 1). It was also demonstrated that mTORC2 has a role in the cytoskeleton reorganization (connected to cell growth) and cell survival modulation [75,78].

SIRTs and AMPK also have a role in the modulation of lifespan. Activation of AMPK mediated pathways by low energy levels has a role in inhibition of glucose production, increase in activity of beta-oxidation (fat burning) and promotion of mitochondrial functions and mitochondrial biogenesis [79,80] (Figure 1). AMPK exerts its effect on energy metabolism by phosphorylation of, for example, (i) ACCs (acetyl-CoA carboxylases), such as ACC1, which ACC1 inhibition lead to enhancement of fatty acid oxidation/mitochondrial-oxidation and suppression of lipogenesis; and (ii) the transcription factor SREBP1 (sterol regulatory element-binding protein 1). The inhibitory effect of AMPK results in reduced fatty acid synthesis [80]. It was suggested that AMPK activation may be a promising anti-aging therapeutic target, for example, by improvement of mitochondrial dysfunction. AMPK activation not only decreased the activity of anabolic pathways and increased activity of catabolic pathways leading to increase of activity of energy (ATP)-generating pathways and decrease in energy (ATP)-consuming processes, but also increased lifespan in diabetic patients [79,80]. Moreover, increase in AMPK activity decreases the expression of proinflammatory cytokines, therefore modulate intercellular communication (Figure 1) by inhibition of advanced-glycation end products (AGEs)-evoked increase in the level of transcription factor NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) mRNA and protein [81]. However, AMPK activation may suppress inflammation through the inflammatory response inducer NF-κB by other pathways, for example, through triggering of inhibitory activity of SIRT1, PGC-1α (peroxisome proliferator-activated receptor γ/PPARγ coactivator 1α), FOXOs and p53 (transcription factor tumor suppressor protein 53) on NF-κB-signaling or via inhibition of NF-κB activator ER (endoplasmic reticulum) stress and oxidative stress [82]. Moreover, AMPK is able to increase PGC-1α activity not only directly (by phosphorylation, before subsequent deacetylation of PGC1-α by SIRT1) [83], but also via arrest of PGC-1α inhibitory effect of mTORC1 [66] (Figure 1).

It has also been demonstrated that caloric restriction may exert its effect on lifespan through SIRTs [84], thus SIRTs are considered as putative anti-aging factors. SIRTs, such as SIRT1 and SIRT3 are able to sense low energy levels via detection of high NAD<sup>+</sup> levels. SIRTs are Class III HDACs histone deacetylases, which enzymes use coenzyme NAD<sup>+</sup> to remove acyl groups of proteins, such as acetyl-lysine residues of histones and non-histones, such as PGC-1α, FOXOs, p53 and NF-κB [69,85]. Under nutrient deprivation (caloric restriction), the level of a nutrient-sensing deacetylase SIRT1 is elevated (which, e.g., increases hepatic glucose production through PGC-1α), but its level reduced by overfeeding [86,87]. It has been demonstrated that activation (overexpression) of SIRT1 may increase lifespan and have an alleviating role in all age-related processes (hallmarks) (Figure 1) and several diseases, such as neurodegenerative diseases [88–90]. Indeed, SIRT1 expression was found to decrease with age, for example in the brain [91]. Moreover, it was also demonstrated that decreased level of SIRT1 in microglia can lead to cognitive decline (Tau-mediated memory deficits) in aging and neurodegeneration by upregulation of IL-1β (interleukin-1β) [91]. It was also demonstrated that caloric restriction can attenuate Alzheimer's disease progression, for example, by decreasing the accumulation of Aβ plaque [92] and promote longevity and healthy aging [93] likely via SIRT1 activation [93–95], whereas higher caloric intake may increase the risk of the development of Alzheimer's disease [96]. Reduction of SIRT1 levels was also demonstrated in parietal cortex in patients with Alzheimer's disease, which was associated with the accumulation of Aβ and Tau [97], whereas activation of SIRT1 can suppress α-synuclein aggregation [98]. It has been demonstrated that SIRT1-evoked neuroprotection may evoke not only decrease in excitotoxicity and neurodegeneration [99,100], but also improved healthspan and extended lifespan likely through activation of PGC-1α (regulation of mitochondrial biogenesis) (Figure 1) and FOXOs (enhancing stress response via autophagy, resistance to oxidative stress and DNA damage and FOXO3′ s ability to

induce cell cycle arrest), as well as inhibition of p53 (regulation of apoptosis and cell cycle) and SREBP1 (regulation of lipid metabolism) activation [6,88,101,102]. These pathways can lead to alleviating effects in neurodegenerative diseases, such as Alzheimer's disease and amyotrophic lateral sclerosis via, for example, SIRT1-generated deacetylation (and activation) of PGC-1α [94]. It has been demonstrated that SIRT1 is able to inhibit cell aging via p53 (deacetylation thereby inhibition of both p53 and its proapoptotic activity) [103] and can modulate development (fate) of neural progenitor cells [104]. It was also demonstrated that cellular NAD<sup>+</sup> level decreased with age (evoked by, e.g., accumulated DNA damages during aging) leading to decreased SIRT activity, mitochondrial dysfunction [88,105] and development of age-related diseases, such as neurodegenerative diseases [106]. Consequently, therapeutic tools, such as administration of different drugs and metabolic therapies, which increase NAD<sup>+</sup> level can evoke alleviating effects on aging-related processes and diseases, as well as promote longevity [6,106] (Figure 1).

It was also demonstrated that mutation, lacking, genetic variants or inactivation of insulin/IGF-1 receptor, as well as caloric restriction (inhibiting insulin/IGF-1 signaling) (Figure 1) extends the lifespan, not only in different animals, such as mice, but also in humans [6,107,108] via PI3K (phosphatidyl inositol-3-kinase)/Akt/FOXOs pathway promoting stress defense. Under these conditions (e.g., caloric restriction-evoked decrease in insulin level) unphosphorylated FOXOs can be transported to the nucleus to promote the transcription of several genes (namely, their phosphorylation impedes their translocation to the nucleus) leading to increased stress resistance, cell cycle arrest, damage repair and increased longevity (lifespan) [72,109].

#### *2.2. Telomere Shortening and Genome Instability*

Reduced length of repetitive ribonucleoprotein sequences at the distal ends of eukaryotic chromosomes (telomere) during cell division was demonstrated during physiological ("natural") aging of mammals [110]. However, if the length of telomeres is too short it can cause damage of the DNA molecules, cellular senescence, mitochondrial dysfunctions (decreased mitochondrial biogenesis and functions, as well as increased ROS/reactive oxygen species level via p53-evoked repression of PGC-1α/β), and inflammation thereby aging [110–112]. It was also suggested that activation of telomerase activity not only enhances the survival time and increase lifespan of mammals [3,113], but also may be favorable for cancer cell development (by decreased senescence and immortalization) [2,114]. Thus, shorter telomeres- and low (if any) telomerase activity-evoked senescence can prevent tumorigenesis at least in animals with long lifespan [2]. It was also suggested that telomere attrition may have a role in development of age-related neurodegenerative diseases, such as Alzheimer's disease [111]. AMPK and SIRT1 can attenuate age-related telomere shortening through PGC-1α (Figure 1) suggesting beneficial role of AMPK/SIRT1 activation on neurodegenerative diseases [115].

Not only telomere shortening, but also chromosomal aneuploidy, somatic mutations and copy mutations may have a role in DNA damage [116]. Moreover, defects of DNA repair mechanisms (such as base excision repair), mitochondrial DNA mutation and perturbations of the nuclear lamina may also generate genome instability (accumulation of genetic damage), cell dysfunction and aging via senescence [63,117–119], which processes may evoke (or have a role in) age-related diseases [78]. Indeed, DNA damage can trigger the onset of neurodegenerative diseases, such as Parkinson's disease and amyotrophic lateral sclerosis [120]. Changes in integrity and stability of DNA can be evoked through both exogenous effects (e.g., by chemical, physical and biological agents) and endogenous influences (e.g., by increase in ROS level and DNA replication errors) [118]. SIRT1 have a positive influence on DNA repair thereby genomic instability (Figure 1), suggesting alleviating effect of SIRT1 activation on neurodegenerative diseases [115].

#### *2.3. Epigenetic Alterations*

The epigenome contains molecular switches by which genes may be activated or inhibited during the entire lifetime [121]. It was demonstrated that epigenetic alterations, such as changes in DNA methylation patterns (which methylation is inversely proportional to gene activation), chromatin remodeling, expression of non-coding RNAs and posttranslational histone modifications may also promote aging processes [78,122]. For example, it has been demonstrated that (hyper)methylation of promoter sequences of the genes (and in general on the DNA) can lead to silencing of genes related to, for example, apoptosis [123], whereas DNA hypomethylation promotes gene activation [124,125]. It was also demonstrated that changes in the pattern of DNA methylation (hypermethylation or hypomethylation) by age may be important in the mechanism of aging [126] and used as an aging clock (e.g., a link between methylcytosine/DNA methylation and age was demonstrated) [125,127]. Both global decrease of DNA methylation (which hypomethylation may induce age-associated genomic instability and loss of telomere integrity) and site-specific hypermethylation of promoter sequences were observed by age [122–124,128]. A previous study showed that age-induced hypomethylation was corrected by caloric restriction [129].

It has been suggested that caloric restriction can upregulate SIRT1 transcription leading to increase in histone deacetylation and methylation of DNA, which effects may compensate the decrease in both SIRT1 activity and DNA methylation, as well as increase in histone acetylation by age and increase lifespan (e.g., by maintenance of adequate DNA methylation pattern and genomic stability) [90,130] (Figure 1). Histone acetyl transferases (HATs) can attach acetyl groups to histones leading to increased positive charge, and attenuation of interaction with DNA, and thereby enhancing DNA transcription. Conversely, HDACs can remove acetyl groups from histones, which effect enhances interaction between histones and DNA resulting decreased transcription. Consequently, antagonists of HDACs may facilitate DNA transcription [131,132]. Based on these results above, expression of genes can be blocked (silenced) through not only methylation of DNA (e.g., methylation of promoter sequences of genes), but also deacetylation of histones, which continuous silencing of genes may be an important factor in progressive aging [123]. Moreover, histone methylation and demethylation (by histone methyl transferases and demethylases) and histone acetylation and deacetylation (by HATs and HDACs) can modulate lifespan, aging and age-related diseases [124,133,134]. For example, SIRT1-evoked deacetylation of Nk2 homeobox 1 can extend lifespan and delay aging processes in mice [133]. It has been demonstrated that inhibitors of HDACs (Classes I, II and IV HDACs), such as Trichostatin A, may be effective in the treatment of neurodegenerative diseases and the extension of lifespan [135,136]. Moreover, HDAC inhibitors decreased death of motor neurons, enhanced motor performance, increased the survival time and resulted in life extension in a mice model of amyotrophic lateral sclerosis [137], restored fear learning, decreased Aβ accumulation and improved cognitive performance in mouse models of Alzheimer's disease [138,139] and generated neuroprotection in a model of Parkinson's disease [140]. It was also suggested that miRNAs (microRNAs; a class of small non-coding silencing RNAs, which have a role in regulation of mRNA translation) may promote longevity and have a role in both neurodegeneration and age-related neurodegenerative diseases [141,142]. For example, hippocampal upregulation of miR-181 and related decrease of SIRT1 expression and, as a result, reduction of synaptic plasticity was demonstrated in a mouse model of Alzheimer's disease [143]. As a response to severe, persistent DNA damage (e.g., by oxidative stress), activated poly(ADP-ribose)-polymerase-1 (PARP-1) adds ADP-ribose units to histones leading to the promotion of chromatin relaxation [144], enhances PARylation (generating PAR polymers as epigenetic effect) at sites of DNA damage (alteration) [63] and induce neuronal cell death via modulation of gene expression and mitochondrial dysfunction [145]. Moreover, excess PARP1 activation was demonstrated in aging and neurodegenerative diseases resulting mitochondrial dysfunction, neuroinflammation and dysregulation of autophagy (and mitophagy; e.g., via mTOR activation) [144,146]. For example, PARP1 enhances inflammation via NF-κB, decreased NAD<sup>+</sup> level and SIRT1 activity

and has a role in telomere shortening and, as a consequence, enhances senescence, leading to neurodegeneration and reduced lifespan [144,146,147]. As SIRT1 activity decreased by age [91], under this condition, both acetylation (activation) of PARP1, and PAPR1-evoked neuroinflammation may be increased. However, to retain its own functions via preservation of NAD<sup>+</sup> levels, SIRT1 is able to deactivate (deacetylate) Parp1 [148]. Moreover, increased expression and excessive activation of PARP1 was demonstrated in Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis [145,149,150]. As it was demonstrated, Aβ and α-synuclein accumulation may generate activation of PAPR1 via, for example, increased level of ROS; thus, enhanced PARP1 activity aggravates Alzheimer's disease and Parkinson's disease symptoms by promotion of Aβ and α-synuclein aggregation, respectively [145,149]. Consequently, PARP1 inhibition can alleviate neuroinflammation, dysregulation of autophagy and mitochondrial dysfunction thereby inhibit development of inflammation(age)-related neurodegenerative diseases (or alleviate their symptoms), for example via SIRT1 activation [146,151]. It was also demonstrated that increase in βHB level can evoke epigenetic (posttranslational) gene regulation by β-hydroxybutyrylation of histones resulting regulation of gene expression thereby adaptation of cells to altered cellular energy source [152].

#### *2.4. Mitochondrial Dysfunction*

Mitochondrial dysfunction is associated with the decline of mitochondrial activity, such as defect of respiratory chain, decrease in ATP synthesis and level, as well as increase in ROS production. This hallmark of aging may be evoked by, for example, decreased mitochondrial biogenesis, defective mitophagy and mtDNA mutations leading to processes (e.g., enhancement of inflammatory processes), which can reduce lifespan, enhance aging and the risk of age-related diseases [69,153]. Indeed, it has been demonstrated that decrease in mitochondrial functions or damage of mitochondria may also be in the background of the development of neurodegenerative diseases [154] through excessive ROS formation leading to inflammation and genomic instability. These processes can enhance cellular senescence, aging processes and development of age-related diseases [154]. It was also demonstrated, that increased level of ROS may generate protective, homeostatic (alleviating) processes (e.g., on lifespan limiting cellular processes via ROS-dependent, protective, stress-response pathways), but, by aging progress, above a certain level ROS can evoke (aggravate) age-related damages [155]. It was demonstrated that autophagy (and mitophagy) declined with age [156], which can generate accumulation of damaged mitochondria thereby increased inflammation (e.g., via increased ROS level-evoked activation of NLRP3/NOD-like receptor pyrin domain 3 and NF-κB), cell death (e.g., through activation of caspases and mitochondrial permeability transition/mPT pore by excess ROS) and DNA damage (by ROS leading to increase in apoptotic signaling, such as p53) [153]. Moreover, it has been demonstrated that defects in mitochondria and autophagy (thereby aggregation of not only α-synuclein and Aβ peptide, but also impaired mitochondria) may have a role in development of neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease [153,156–158]. Thus, drugs or interventions, such as caloric restriction, which are able to promote autophagy and mitophagy, therefore inhibit mitochondrial dysfunction, ROS production, aggregation of toxic proteins, inflammation, cell death and cell senescence, can delay age-related degeneration, extend healthy lifespan and alleviate neurodegenerative diseases [159–161]. Indeed, for example, it was demonstrated that SIRT1 has a role in elimination of damaged mitochondria via autophagy (by enhanced activity of autophagy proteins) [162–164] and in mitochondrial biogenesis (increase in mitochondrial biogenesis) via increased transcriptional cofactor PGC-1α activity [87] (Figure 1), whereas a mitochondrial deacetylase SIRT3 controls (decreases) ROS level by enhancement of antioxidant activity of superoxide dismutase 2 (SOD2) during caloric restriction, leading to increased oxidative stress resistance [165]. Moreover, it was also demonstrated that increased SIRT3 activity can suppress mPT pore formation by which it can prevent mitochondrial dysfunctions [166]. It was also demonstrated that PGC-1α activation can enhance

mitochondrial biogenesis and improve mitochondrial energy metabolism, therefore increasing lifespan and protecting against neurodegenerative diseases [167]. PGC-1α can bind and co-activate the transcription factor PPARγ (belongs to the superfamily of nuclear receptors) and promotes not only mitochondrial biogenesis, but also SOD and catalase activity, glucose metabolism and oxidative phosphorylation [162,168–170], whereas reduces the level of NF-κB and pro-inflammatory cytokines [171,172], as well as Aβ generation [173,174]. Indeed, reduced level of PGC1-α can result in decreased mitochondrial respiration and enhanced inflammatory processes [175]. Moreover, mitochondrial uncoupling via the overexpression of uncoupling protein 1 (UCP1) may also increase the lifespan [176].

#### *2.5. Altered Intercellular Communication: Increased Inflammatory Processes*

Aging processes are also connected to dysregulation of cell-cell connectivity and intercellular communication leading to, among others, sterile (activation of immune response without appearance of pathogens), chronic, low-grade inflammation (named "inflammaging") with activation of NF-κB, as well as increased synthesis and release of proinflammatory cytokines (e.g., IL-1β and TNF-α/tumor necrosis factor-α) [69,125,177,178]. Increase in inflammatory processes and proinflammatory cytokine levels can also enhance (trigger) aging processes, for example, through increased activation of intracellular multiprotein sensor NLRP3 inflammasome, senescent cells-evoked release of proinflammatory cytokines and NF-κB level and signaling [177,179,180]. Autophagy failure in old organisms (e.g., decrease in activity of autophagy), and in patients with Alzheimer's disease and Parkinson's disease [181,182] were also demonstrated. It was suggested that aging (e.g., decreased autophagy by age) can stimulate NF-κB signaling, which transcription factor NF-κB (similar to increase in ROS by mitochondria and aggregation of Aβ) stimulate inflammatory processes, for example, via increased NLRP3 expression and IL-1β release [161,179,183–185], whereas autophagic uptake of damaged mitochondria (resulting decrease in ROS level) suppresses NLRP3 stimulation [161]. Thus, it was suggested that autophagy may generate an anti-inflammatory effect by inhibition of NLRP3 inflammasome thereby mitigating the NLRP3-evoked cleavage of pro-IL-1β to its active form/IL-1β by caspase-1 [186,187] leading to delay in aging processes [180].

Moreover, responsiveness of AMPK signaling decreased by age [180,188], which mitigates its inhibitory activity on NF-κB signaling [82] (Figure 1) and impairs autophagic activity leading to increased oxidative stress and activation of inflammasomes [180] and can attenuate lifespan [82]. As mTORC1 is able to inhibit autophagy (e.g., mitophagy or macroautophagy of altered proteins) all of drugs or interventions, which are able to inhibit mTORC1 (e.g., caloric restriction leading to mTOR inhibition) may be potent delayer of aging processes and enhancer of lifespan via inhibition of inflammation [180] (Figure 1), by which can alleviate not only neuroinflammation, but also neurodegeneration and related diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis [183,189]. Indeed, inhibition of NF-κB signaling was able to prevent age-associated features in mouse models extending their longevity [190].

#### *2.6. Cellular Senescence*

Cellular senescence can be evoked by intracellular and extracellular, genomic and epigenomic harmful stimuli and damages resulting hallmarks of aging (e.g., age-related stress: oxidative stress and telomere shortening; metabolic, as well as ER stress; mitochondrial dysfunction, loss of proteostasis) [191–193]. One of the main features of aging is the enhancement of cellular senescence (irreversible cell-cycle arrest regulated by, e.g., telomere attrition/DNA damage-evoked p53-dependent DNA-damage response, in which p53 is activated). Excessive accumulation of senescent cells, which cells decrease tissue regeneration and resistant to apoptosis (e.g., by upregulation of antiapoptotic Bcl-2/B cell lymphoma-2 family proteins resulting resistance to apoptosis-inducing signals), can evoke harmful processes on surrounding cells by secretion of proinflammatory agents (SASP factors, e.g., IL-1β,) and other components (e.g., IGF-1) [2,191,194,195]. For example, previous studies show that acute administration of IGF-1 can promote cell proliferation and survival whereas prolonged administration of IGF-1 promotes cell growth arrest and senescence (and the latter, enhances aging processes and inhibits tumorigenesis) via through SIRT1 inhibition and increased p53 activity (by increased acetylation) [196] and suppression of autophagy (e.g., via mTOR) [197] (Figure 1). Indeed, SIRT1 can inhibit not only DNA-damage, but also cellular senescence via deacetylation (inhibition) of p53 resulting anti-aging effects [198]. In contrast with cellular senescence, cellular quiescence occurs when nutrition or growth factor levels are very low (or lack) leading to a reversible cell-cycle arrest. In this state the cells may impede initiation of cell senescence [199] and has a role in maintenance of stemness [200]. However, in relation to maintaining cellular balance, senescence of cells is a double-edged sword [2]. For example, cellular senescence can reduce liver fibrosis [201], promote tissue repair and has a role in not only physiological, but also pathophysiological processes (e.g., embryogenesis and wound healing) [195] and prevent cancer development [202], but exaggerated attenuation of processes of cell senescence and accumulation of senescent cells can generate (or enhance) aging, and, as a consequence, development of age related diseases, such as Alzheimer's disease and cancer [192,195,203–205]. Thus, medication of cellular senescence needs careful attention. Under glucose deprived condition, AMPK-induced p53 activation potentiates cellular survival (p53-dependent metabolic arrest), but excessive (lasting) AMPK activation leads to enhanced p53-dependent cellular senescence [206,207]. However, not only SIRT1, but also AMPK activation can improve cellular senescence via, for example, inhibition of proinflammatory mediators [5,81,82] (Figure 1).

#### *2.7. Loss of Proteostasis and Stem Cell Exhaustion*

Impaired protein homeostasis (loss of proteostasis) by age may also be in the background of aging and related diseases (e.g., neurodegenerative diseases) leading to dysregulation of protein synthesis, degradation and protein aggregation, disaggregation, assembly, folding and trafficking [208]. For example, activity of ubiquitin-proteasome system and autophagy decreased with age [209], whereas increased activity of proteostasis network (e.g., enhanced autophagy) extended the healthspan and lifespan [210]. Inhibition of mTOR pathways (e.g., by caloric restriction through decreased protein synthesis and activation of autophagy) may improve protein homeostasis and extend lifespan [211,212] (Figure 1). It has been demonstrated that maintained mitochondrial proteostasis prolonged lifespan and reduced Aβ protein aggregation in Alzheimer's disease models [213]. Moreover, decreased activity of autophagy-lysosomal pathway may have a role in the development of both Alzheimer's disease and Parkinson's disease and other neurodegenerative diseases [77]. Indeed, activation of mitophagy (by which autophagy-lysosomal pathway remove damaged/dysfunctional mitochondria) was able to increase lifespan in worms and reverse cognitive deficits in models of Alzheimer's disease [214,215]. AMPK activation may participate in maintenance of proteostasis via inhibition of mTOR and phosphorylation of eIF2α (eukaryotic initiation factor 2α; resulting attenuation of protein synthesis) and via activation of autophagy [79,80] (Figure 1). Moreover, it was also demonstrated that autophagy may be enhanced via inhibition of mTOR by SIRT1 [216] (Figure 1). Thus, activation of AMPK/SIRT1 and inhibition of mTOR (mTORC1, but not mTORC2 because the latter is required for autophagy) activity may be a promising target in anti-aging therapy [77]. Indeed, aging and age-associated diseases can upregulate mTORC1 [69].

Stem cell exhaustion may have a role in aging and appearance of age-related diseases through loss of regenerative ability of cells, tissues and organs. For example, activity and number of hematopoietic cells and intestinal stem cells are decreased by age leading to decrease in lymphoid cell number and adaptive immune response, increase in risk of anemia development and myeloid cell number, as well as malfunctions in intestinal functions [217,218]. Moreover, age-dependent decrease in function of other stem cells, such as neuronal stem cells was also demonstrated [71]. It was suggested that stem cell aging may be evoked by several factors, such as DNA damage and mutation, cellular senescence, defects in proteostasis, mitochondrial dysfunction and telomere attrition [63,71].

Thus, we can conclude that activation of AMPK/SIRTs-modulated signaling pathways, inhibition of mTOR effects (e.g., by inhibition of IIS pathway) and modulation of gene expression (e.g., by HDAC inhibitors) can alleviate aging processes (hallmarks) through direct and indirect manner (e.g., improvement of one of aging hallmarks, such as telomere attrition can improve other aging hallmarks, such as senescence and mitochondrial dysfunction), leading to extended lifespan and delay the appearance of neurodegenerative diseases.

#### *2.8. Effects of Senotherapeutic Drugs on Aging Hallmarks and Neurodegenerative Diseases: Main Signaling Pathways*

It has been demonstrated that elimination of senescent cells by senolytics (such as senolytic cocktails containing quercetin and dasatinib) can evoke alleviating effects on age-related diseases, such as Alzheimer's disease and Parkinson's disease and improve healthspan in aged humans [192,203,219]. Another senotherapeutic strategy is the administration of senomorphics (e.g., metformin and rapamycin) to alleviate (abolish) features of senescence (e.g., decrease in production and release of SASP factors) (Figure 1) without elimination of senescent cells, which may delay both aging and development of age-related diseases [194]. It was suggested that mTOR has a role in, among others, lifespan control [220]. Indeed, rapamycin (sirolimus; as an mTOR inhibitor) (Figure 1) is able to decrease the risk of development of age-related diseases, such as neurodegenerative diseases, to improve age-related decrease in memory and learning functions and to extend longevity [74,220]. Rapamycin decreased accumulation of Aβ and Tau leading to decreased loss of neurons, attenuated neuroinflammation and alleviated cognitive dysfunction in mouse models of Alzheimer's disease [221]. Resveratrol also promotes clearance of Aβ peptides [95], likely via inhibition of mTOR and activation of AMPK [5] and prevents cognitive impairment [222] in different cell lines and models of Alzheimer's disease. Thus, these results suggest that resveratrol and rapamycin exert its neuroprotective, alleviating effect on health span, lifespan and age-associated diseases likely by modulation of autophagy and proteostasis (via mTOR inhibition), as well as inflammation, among others [211,212,220] (Figure 1). Rapamycin and metformin (an antidiabetic drug, which reduces IGF levels, insulin resistance and insulin level) reduced accumulation of α-synuclein and improved behavioral impairments in models of Parkinson's disease [74,220,223]. Moreover, metformin inhibits the mitochondrial electron transport chain (ETC complex I: NADH/ubiquinone oxidoreductase; thereby oxidative phosphorylation), consequently, cytoplasmic AMP/ATP and ADP/ATP ratios were increased resulting direct activation (phosphorylation) of AMPK [224,225] and decrease in ROS level [226]. Activation of AMPK (e.g., by metformin) (Figure 1) enhances mitochondrial biogenesis (via SIRT1/PGC-1α) and lipid beta-oxidation (via ACCs), inhibits hepatic glucose production and alleviates proteostasis (via mTOR inhibition), enhances autophagy (via mTOR inhibition and activation of ULK1), evokes hypoglycemia (decreasing plasma glucose levels, e.g., via improved hepatic insulin sensitivity leading to decrease in hepatic glucose production), improves nutrient sensing (via IIS/mTOR/SIRT1 pathways), inhibits NF-κB, improves DNA repair and decreases the level of proinflammatory cytokines (e.g., via activation of SIRT1) [6,12,225–228] leading to alleviating effects on aging-processes and related neurodegenerative diseases. As AMPK-independent influences, metformin is able to inhibit ROS production (via, e.g., inhibition of mitochondrial ETC and activation of antioxidant transcription factor nuclear factor erythroid 2-related factor 2/Nrf2) (Figure 1), enhance autophagy (through direct inhibition of mTOR), enhance SIRT1 activity (especially when NAD<sup>+</sup> level highly reduced), activate DNA-damage-like response (and facilitates DNA repair likely via p53), attenuate NF-κB signaling and synthesis (release) of proinflammatory cytokines, inhibit SASP factors via Nrf2 and decrease level of insulin and IGF-1 levels thereby insulin/IGF-1 signaling (by which decreases mTOR activity) [66,86,225,229–232]. All of these processes can increase lifespan and evoke alleviating effects on both ageing and

age-related diseases, such as Alzheimer's disease and Parkinson's disease [224,225,233]. Moreover, metformin is able to inhibit premature stem cell aging (via Nrf2), enhance stem cell rejuvenation (through AMPK) [234], affect histone modifications (e.g., via activation of SIRT1, inhibition of Class II HDACs and HAT phosphorylation) through AMPK-dependent and independent pathways [235], increase the levels of several miRNAs, which are implicated in the regulation of aging and cellular senescence, likely via AMPK [236] and reduce telomere shortening (e.g., via AMKP/PGC-1α/telomeric repeat-containing RNA/TERRA pathway; TERRA is transcribed from telomeres and has an important role in protection of telomere integrity) [225,237,238] (Figure 1). Indeed, it was demonstrated that, activation of AMPK can both enhance gene expression (e.g., by phosphorylation/inactivation of HDACs and activation of HAT1-evoked acetylation of histones) and inhibit gene transcription (e.g., via enhanced cellular NAD<sup>+</sup> levels, and, as a consequence, increased SIRT1 deacetylation activity) [235]. Moreover, resveratrol can also extend lifespan and prevent neurodegenerative diseases [239]. For example, resveratrol can generate anti-inflammatory and anti-oxidative effects (e.g., decreases level of ROS, p53, NF-κB and proinflammatory cytokines, such as TNF-α and IL-1β) [5] and increase mean life expectancy and maximal life span in models of Alzheimer's disease [240]. Moreover, resveratrol improved motor neuron function and extended the lifespan in a mouse model of amyotrophic lateral sclerosis [241]. It was suggested that resveratrol may exert its effects via activation of AMPK/SIRT1-modulated pathways [242] (Figure 1) by which this drug can deacetylate several substrates, such as p53, PGC-1α, FOXOs (e.g., FOXO3) and SREBP1 leading to induction of cell cycle arrest, mitochondrial biogenesis, DNA repair, oxidative stress response, autophagy and regulation of lipid metabolism [6,101,243]. For example, SIRT1 can decrease ROS and NF-κB-evoked effects (e.g., neuroinflammation) via Nrf2 [5] (Figure 1). However, it was also suggested that not only SIRT1, but also PGC1-α can increase the expression of Nrf2 [244,245] and AMPK enhances the nuclear translocation of the Nrf2 [246]. Based on these results, more effective sirtuin-activating compounds were developed, such as SRT2104, which drug may be a promising anti-aging drug (e.g., it increased lifespan and decreased inflammatory processes) [247]. Other natural products, such as curcumin, berberine and quercetin [6] can also generate positive effects on lifespan (by slowing aging), age and age-related diseases, for example, through AMPK activation and mTOR inhibition (e.g., to induce autophagy), activation of SIRT1 (to promote mitochondrial biogenesis) and anti-inflammatory effects [74,248–250].

Thus, administration of senotherapeutic drugs suggests that therapeutic tools and drugs, which are able to modify aging processes through activation or inhibition of certain signaling pathways can also delay development (or improve symptoms) of neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis), improve memory and learning functions, as well as extend longevity.

#### **3. Alleviating Effects of Ketosis on Lifespan, Aging and Age-Related Neurodegenerative Diseases**

#### *3.1. Ketosis-Evoked Neuroprotective Effects and Downstream Signaling Pathways*

It has been demonstrated that ketosis and administration of βHB (as an alternative energy fuel to glucose) can increase mitochondrial ATP production and ATP release leading to increased extracellular level of purine nucleoside adenosine (via metabolism of ATP) [251–253]. Adenosine can activate its receptors leading to reduced oxidative stress (ROS level) [254] and reduce inflammatory processes [255]. Indeed, as enhanced level of ROS may activate (open) mPT pore thereby uncouple electron transport system from ATP production, βHB-evoked decrease in ROS production [94] can improve mitochondrial respiration and ATP production [49]. It was also suggested that therapeutic ketosis can increase the inhibitory GABAergic effects [22,256], decrease glutamate release and glutamate-induced neuronal excitability [256,257] and modulate (increase) the level of dopamine, adrenaline, noradrenaline and serotonin [258,259].

As an epigenetic gene regulator, βHB can inhibit the activity of the classical HDAC family (Class I and Class IIa HDACs) leading to enhanced acetylation of histone residues,

thereby DNA can be accessed for transcription factors, such as FOXO3A [53,132,260]. FOXO3A generates enhanced expression of various antioxidants genes, enhances mitochondrial homeostasis (e.g., by regulation of mitochondrial biogenesis and ATP synthesis) and decreases oxidative stress [260,261]. Moreover, decrease in oxidative stress can also be generated by βHB-evoked inhibition of HDACs via attenuation of ER stress [262]. It has also been demonstrated, that the expression of brain-derived neurotrophic factor (BDNF) may be increased through βHB-evoked inhibition of HDACs [263] by which βHB evokes anti-inflammatory effects (via inhibition of both NLRP3, NF-κB and proinflammatory cytokine levels) [264,265], increases mitochondrial respiration and ATP levels [266], enhances the activity of anti-oxidant enzymes (such as SOD), and protects tissues against glutamate-induced excitotoxicity [267,268]. It has been also demonstrated that βHB can modulate gene expression through promotion of histone and non-histone acetylation by HATs [266,269]. Moreover, βHB is able to directly bind to an RNA-binding protein hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1), which protein regulates, for example, RNA processing and function, as well as stabilization of mRNA [59,270,271].

Previous studies showed that βHB, through HCAR2, activates AMPK leading to NAD<sup>+</sup> generation, which increases activity of SIRTs (e.g., SIRT1 and SIRT3; βHB/HCAR2/NAD+/ SIRTs pathways) [272] (Figure 1) and thereby evoke neuroprotective effects [53,83,273,274]. Through both βHB/HCAR2/AMPK/SIRT1/NF-κB pathway and βHB/HCAR2/AMPK/mTOR pathway, βHB may generate anti-inflammatory effects by, for example, inhibition of proinflammatory transcription factor NF-κB and enhancement of autophagy, respectively [55,272,275], leading to decreased level of proinflammatory agents (e.g., TNF-α, IL-1β) [50,55,57,276]. βHB/HCAR2/AMPK/SIRT1/FOXO3A pathway can evoke antioxidant influences, thereby decrease in oxidative stress by increased expression of genes of the antioxidants (e.g., manganese superoxide dismutase/MnSOD: βHB/HCAR2/AMPK/SIRT1/FOXO3A/MnSOD pathway) [164,277]. Ketone bodies increase the expression of not only HCAR2 [278,279], but also SIRTs (e.g., SIRT1 and SIRT3) and PGC1-α [164,278,280]. These results suggest that both βHB/HCAR2/AMPK/SIRT1/PGC1-α and βHB/HCAR2/AMPK/SIRT3/PGC1-α pathways can function in the CNS. Indeed, neuroprotective influences of PGC1-α (e.g., anti-inflammatory effects and promotion of mitochondrial functions) can be modulated through not only SIRT1, but also SIRT3 [278,281–283]. It was also suggested that βHBevoked effects on mitochondrial functions (e.g., mitochondrial biogenesis) may be generated through βHB/HDAC/BDNF/PGC1-α pathway [284]. Moreover, ketosis can enhance the expression of PPARs and the activity of the Nrf2 in the brain likely through βHB/ HCAR2/AMPK/Nrf2 or βHB/HCAR2/AMPK/SIRTs/PGC1-α/Nrf2 pathway [285–287]. It has been suggested that ketosis may enhance expression of UCPs, therefore decrease the production of ROS [23,288,289] and defend mitochondria and mitochondrial functions (e.g., by reduction of oxidative stress) through activation of βHB/HCAR2/AMPK/ SIRT3/PGC1-α/UCP1 pathway [283] and/or βHB/HCAR2/AMPK/SIRT3/PGC1-α/UCP2 pathway [278]. Moreover, not only ketosis (βHB), but also decrease in glucose level can mitigate inflammatory processes through decreased NLRP3 inflammasome activity. Namely, βHB is an endogenous inhibitor of NLRP3 inflammasome, likely via βHB/NLRP3/IL-1R (IL-1 receptor)/NF-κB pathway, whereas increased glucose level may enhance activity of NLRP3 and inflammatory processes. In addition, enhanced glucose level generally increases insulin level leading to decrease in ketone body synthesis [290–292]. EKSs were proven to decrease glucose levels [21,26,28,36,293], thereby they may increase activity of AMPK/SIRTs signaling pathways and inhibit mTOR-evoked effects (Figure 1).

Thus, based on previous studies, βHB/HCAR2/AMPK/SIRT1/NF-κB, βHB/HCAR2/ AMPK/mTOR and βHB/NLRP3/IL-1R/NF-κB pathways (anti-inflammatory effects), βHB/HCAR2/AMPK/SIRT1/FOXO3A pathway (improving mitochondrial functions, anti-oxidant influences), βHB/HCAR2/AMPK/SIRT1/PGC1-α/Nrf2, HCAR2/AMPK/ SIRT3/PGC1-α/Nrf2 and HCAR2/AMPK/Nrf2 pathways (improving mitochondrial functions, anti-oxidant and anti-inflammatory effects), βHB/HDAC/BDNF/PGC1-α pathway (improving mitochondrial functions; anti-oxidant and anti-inflammatory influences),

βHB/HCAR2/AMPK/SIRT3/PGC1-α/UCP1 and/or βHB/HCAR2/AMPK/SIRT3/PGC1 α/UCP2 pathways (anti-oxidant and anti-inflammatory effects, improving mitochon-

osis (βHB) may extend threonine protein kinase; βHB, beta κB, nuclear facα activated receptor gamma (PPARγ) coactivator 1α; *βHB* **Figure 2.** Signaling pathways and effects by which exogenous ketogenic supplements-generated ketosis (βHB) may extend lifespan, delay both aging and development of neurodegenerative diseases, and improve learning and memory dysfunctions. Abbreviations: AMPK, AMP activated serine-threonine protein kinase; βHB, beta-hydroxybutyrate; BDNF, brain-derived neurotrophic factor; FOXO, Forkhead box O; HATs, histone acetyltransferases; HCAR2, hydroxycarboxylic acid receptor 2; HDAC, histone deacetylase; IL-1R, IL-1 receptor; mTOR, mechanistic target of rapamycin; NF-κB, nuclear factor kappalight-chain-enhancer of activated B cells; NLRP3, NOD-like receptor pyrin domain 3; Nrf2, nuclear factor erythroid 2-related factor 2; PGC1-α, peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α; ROS, reactive oxygen species; SIRT, sirtuin; UCP, uncoupling protein.

#### dministration of βHB generated anti *3.2. Beneficial Effects of EKSs-Evoked Ketosis (βHB) on Lifespan, Aging, Age-Related Diseases, as Well as Learning and Memory Dysfunctions*

. This result suggests that lifespan extension by βHB may also be mediated in mam-Administration of βHB generated anti-aging and life-extending effects in *C. elegans* [22,60]. This result suggests that lifespan extension by βHB may also be mediated in

and βHB

generated ketosis/elevated blood βHB, at least partly) can reduce midli

and KSs. For example, in mammals, this effect may be mediated through βHB

mammals through signaling pathways similar to *C. elegans* [60,294], likely by activation of AMPK/SIRT1/mTOR/FOXOs/Nrf2 pathways, HDAC inhibition (and related increase in FOXOs activity) or reduction of insulin signaling pathway activity (Figures 1 and 2). Indeed, for example, it was demonstrated that inhibition of IIS pathways, thereby activation of FOXOs are important processes for lifespan extension [295] and FOXO3A gene is strongly associated with human longevity [296]. Increase in autophagy by caloric (or dietary) restriction can enhance lifespan not only in *C. elegans,* but also in mammals through similar pathways, which may also be activated by administration of EKSs, such as KEs and KSs. For example, in mammals, this effect may be mediated through βHB-evoked inhibition of mTOR activity, activation of FOXOs (via both activation of SIRT1 and direct inhibition of Akt), and ketone body metabolism-evoked decrease in blood glucose and insulin levels, which also decrease the activity of IIS pathways [52,77,180,297,298]. Moreover, long-lived animals showed decrease in mitochondrial ROS production [299] suggesting both inverse correlation between longevity and mitochondrial ROS production (and mitochondrial DNA damage) [52,299] and βHB-evoked enhancement of longevity (lifespan) (Figures 1 and 2). It was also suggested that ketogenic diet (likely through ketogenic dietgenerated ketosis/elevated blood βHB, at least partly) can reduce midlife mortality [300], extends longevity and healthspan in adult mice [51], increased lifespan in *Kcna1*-null mice [301] and decreased senescence may be partly through β-hydroxybutyrylation-evoked decrease in p53 activity (in addition, β-hydroxybutyrylation also can attenuate acetylation of p53, because β-hydroxybutyrylation interferes with acetylation) [302]. These results suggest that βHB-generated activation of different signaling pathways may have a role in modulation of aging processes, thereby both lifespan and healthspan. Indeed, it was demonstrated that βHB can alleviate cellular senescence through increased autophagy and decreased plasma insulin level and inflammatory processes in male rats [303], likely through AMPK/SIRT1 pathways (Figure 1). It has also been demonstrated that increased level of blood βHB can delay the age-related processes, for example, by inhibition of SASP, thereby senescence, likely through βHB/hnRNP A1-binding-evoked increase in binding of hnRNP A1 and Oct4 (embryonic stem cell regulator octamer-binding transcriptional factor 4) leading to stabilization of Oct4 mRNA (complex formation with Oct4 mRNA and upregulation of Oct4 expression) and SIRT1 mRNAs [59,304]. βHB-evoked activation of Oct4 not only triggers (maintains) quiescent state of cells (e.g., AMPK activation and mTOR inhibition), but also decreases induction of senescent state of cells (e.g., reduction of the blood level of a pro-senescence marker IL-1α and SASP expression) leading to protection of cells against senescence, and likely, induction of autophagy [59]. These results above suggest that, indeed, EKSs(βHB)-evoked ketosis can alleviate aging-processes (aging hallmarks), at least theoretically, through βHB-evoked activation of AMPK/SIRT1 or SIRT3 downstream signaling pathways (e.g., βHB/HCAR2/AMPK/SIRT1/NF-κB pathway), inhibition of mTOR- (e.g., βHB/HCAR2/AMPK/mTOR pathway) and NLRP3/IL-1R-generated effects, HDAC inhibition, β-hydroxybutyrylation and hnRNP A1-binding (Figures 1 and 2) leading to improved healthspan, delayed aging, thereby extended lifespan.

A great deal of evidence suggests that progression of aging processes by age can lead to not only emergence of aging hallmarks, but also enhanced risk for development of neurodegenerative diseases and impaired learning and memory functions through, for example, mitochondrial dysfunction, epigenetic alterations and enhanced inflammation, which processes may be alleviated by EKSs-generated ketosis (βHB) (Figures 1 and 2). For example, impaired mitochondrial functions, increased oxidative stress and neuronal injury were demonstrated in different CNS diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis [305–308]. Moreover, mitochondrial dysfunctionevoked increase in ROS level may enhance inflammatory processes [309,310], leading to impaired cognitive functions, for example in patients with neurodegenerative diseases (e.g., Alzheimer's disease) [311–313]. It has been suggested that ketogenic diet- and EKSs-evoked ketosis can improve or prevent impaired cognitive functions, learning and memory, for example, via enhanced mitochondrial respiration and antioxidant mecha-

nisms [49,314–317]. Indeed, not only ketogenic diet (and related ketosis) and βHB, but also KE, KS and MCT supplementation improved cognitive functions, learning and memory, as well as their age-related decline in animal models of Alzheimer's disease and patients with Alzheimer's disease or mild cognitive impairment [32,43,47,50,317–320] (Table 1), in a mouse model of Angelman syndrome [41] and in old animals and elderly humans [321,322]. EKSs may exert these beneficial effects via increased ketone body level, which can improve mitochondrial functions. For example, increased level of βHB can compensate glucose hypometabolism-generated decrease in energy source in human and restore ATP synthesis [16,289,318,319,323]. In fact, glucose hypometabolism may contribute to the development of, for example, Alzheimer's disease [324,325]. It has also been demonstrated that MCT supplementation-evoked improvement in cognitive functions was observed in patients with mild to moderate Alzheimer's disease or mild cognitive impairment without apolipoprotein E (*APOE*) ε4 allele [326,327], but the mechanism of action of *APOE*-*ε*4 status on MCT/ketosis-generated alleviating effects was not identified. Moreover, improved learning and memory was also demonstrated in relation to ketone bodies-evoked decrease in both oxidative stress and intracellular Aβ<sup>42</sup> accumulation, and increased mitochondrial complex I activity in models of Alzheimer's disease [50,328,329] (Table 1). It was demonstrated that βHB can protect neurons and alleviate symptoms in models of not only Alzheimer's disease, but also Parkinson's disease [328,330], likely via improvement of mitochondrial function (e.g., by increased ATP synthesis) and activation of other neuroprotective mechanisms, leading to improvement (or protection) in neurodegeneration, motor functions (e.g., tremor) and impaired cognition [258,259,328,331]. Moreover, indeed, βHB administration can decrease aggregation of α-synuclein and delay the toxicity of Aβ [60]. Ketogenic diet- and EKSs-generated ketosis, βHB or the Deanna protocol, containing (among others) MCTs, can also generate alleviating effects on (i) motor neurons and motor performance in preclinical rodent models, such as animal models of amyotrophic lateral sclerosis [48,332–336] and (ii) dopaminergic neurons and motor performance in animal models of Parkinson's disease [55,258] likely through improved mitochondrial function and ATP synthesis (Table 1). Dysregulation of different neurotransmitter systems may have a role in the pathophysiology of neurodegenerative diseases, for example, in animal models and patients with impaired motor function (e.g., dopaminergic dysfunction; GABA and glutamate imbalance) [337–340], Parkinson's disease (e.g., decrease in serotonin level and increase in glutamatergic transmission), Alzheimer's disease (decreased cholinergic neurotransmission) and both Alzheimer's disease and Parkinson's disease (deficits in dopaminergic signaling) [337,339,341–343]. Moreover, dysfunctions in neurotransmitter systems (e.g., GABAergic, glutamatergic and cholinergic) can lead to impaired learning and memory [340,342,344]. It has also been demonstrated that dysregulation of acetylation and deacetylation can lead to neurodegenerative diseases (such as Alzheimer's disease, Parkinson's diseases, amyotrophic lateral sclerosis) and learning and memory deficits [345–348]. Moreover, HDAC inhibitors can improve symptoms or impede development of Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and restore learning and memory functions [347,349–352]. Low BDNF levels were demonstrated in patients with Alzheimer's disease, which decrease in BDNF level correlates with loss of cognitive functions [353,354], suggesting that ketosis (elevated blood βHB levels) can exert its beneficial effects on Alzheimer's disease and cognitive functions, among others, through HDAC/BDNF system leading to enhancement of alleviating BDNF effects (e.g., by stimulation of hippocampal neurogenesis) [355]. Thus, EKSs (via ketosis/βHB) can exert alleviating effects on neurodegenerative diseases, learning and memory functions through modulation of not only mitochondrial functions and inflammatory processes, but also neurotransmitter systems and via epigenetic modification (Figure 2). Indeed, for example, it was suggested that EKSs may be able to prevent or improve neurodegenerative diseases and learning and memory, among others, through HDAC inhibition [30].

**Table 1.** Beneficial effects of beta-hydroxybutyrate (βHB), ketone esters (KEs) and medium chain triglycerides (MCTs) on neurodegenerative diseases as well as impaired motor, memory and learning functions in in vivo studies.



**Table1.***Cont.*

Abbreviations: 5XFAD, β-amyloid precursor protein and presenilin-1 double-transgenic mouse; Aβ, amyloid β; *APOE*, apolipoprotein E; APPSwInd, a transgenic mouse, express a mutant form of the human amyloid protein precursor (APP) with the APP KM670/671NL (Swedish) and APP V717F (Indiana) mutations; LPS, lipopolysaccharide; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; *UBE3Atm1Alb*, a mouse with *Ube3A* (ubiquitin protein ligase E3A) knock-out mutation; 3xTgAD, a mouse with APP KM670/671NL (Swedish), MAPT P301L and PSEN1 M146V mutations; SOD1-G93A, a transgenic mouse with a G93A mutant form of human superoxide dismutase (SOD1).

HCAR2 ligands can generate alleviating effects on Parkinson's disease, Alzheimer's disease, impaired learning, memory and motor functions, as well as amyotrophic lateral sclerosis via anti-inflammatory effects [43,50,57,258], suggesting that EKSs-evoked ketosis (βHB) exerts its alleviating effects on learning, memory, as well as age and agerelated diseases through βHB/HCAR2-evoked downstream signaling (Figure 2). Indeed, previous studies show that ketosis (βHB) may evoke therapeutic effects in the treatment of Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis and enhance learning and memory through anti-inflammatory effects induced by HCAR2 [50,55,57,58,275,279]. It was also demonstrated that enhanced expression of proinflammatory cytokines and oxidative stress have a role in the development of Alzheimer's disease [276,356,357], Parkinson's disease [55,276,356,357], amyotrophic lateral sclerosis [356–358], impaired motor functions [337,359] and impairment of learning and memory [309,310,360]. Thus, ketosis may also improve symptoms of neurodegenerative diseases, motor, learning and memory dysfunctions through anti-inflammatory and anti-oxidative effects via HCAR2 [50,275,361] (Figure 2). It has been demonstrated that SIRT1 levels were decreased in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease [97,362] suggesting alleviating effects of SIRT1 activation-modulated pathway(s) in the treatment of neurodegenerative diseases [363]. It was also suggested that activation of SIRT1-dependent pathways can modulate learning and memory by which ketone bodies may be able to improve both learning and memory functions [327]. Indeed, overexpression of SIRT1 was protective against learning and memory impairment in animal models of Alzheimer's disease [364,365] and increased SIRT1 activity could promote memory processes, whereas SIRT1 knockout animals showed impaired cognitive abilities [366,367]. Moreover, activation of SIRT1 generated protective influences in mouse models of amyotrophic lateral sclerosis (e.g., enhanced biogenesis of mitochondria and suppressed deterioration of motor neurons) [94,368,369], preserved dopaminergic neurons in a mouse model of Parkinson's disease [370] and evoked protection against Aβ plaque formation in mouse models of Alzheimer's disease [94,371] likely via, for example, SIRT1/PGC1-α/MnSOD pathway [173,372]. In fact, it has been demonstrated that PGC1 α-deficiency may be in connection with neurodegenerative lesions [373], and decreased PGC1-α expression may be one of the most important factors in the development of both Parkinson's disease [374,375] and Alzheimer's disease [174,376]. Moreover, PPARγ agonist pioglitazone (an antidiabetic agent) and overexpression of PGC1-α were able to improve symptoms of amyotrophic lateral sclerosis in mouse models [377,378] and other PPARγ agonists can improve not only symptoms of neurodegenerative diseases (e.g., Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis), but also impaired cognitive functions, learning and memory [379,380]. As oxidative stress has a role in the pathophysiology of neurodegenerative diseases, such as Parkinson's disease, Nrf2 thereby, for example, AMPK/SIRT1/Nrf2 pathway may be an important therapeutic target in the treatment of these diseases [381,382]. Moreover, it was also suggested that activation of SIRT3/PGC1-α/MnSOD pathways could also generate alleviating effect on Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis [383–385]. Consequently, indeed, EKSs-generated ketosis (βHB) can alleviate or delay development of neurodegenerative diseases, and improve learning and memory dysfunctions likely through different βHB/HCAR2/AMPK-modulated downstream signaling pathways (Figure 2).

#### **4. Conclusions**

A great deal of evidence suggests that EKSs-generated ketosis may improve healthspan, therefore can delay ageing and the onset of age-related neurodegenerative diseases, as well as learning and memory dysfunctions through neuroprotective effects. In spite of the overwhelming amount of promising mechanistic findings, only a limited number of studies focused on and demonstrated the beneficial effects of EKSs-evoked ketosis on lifespan, aging-processes, age-related diseases and impaired learning and memory functions. However, their beneficial effects on healthspan and lifespan—likely through improving

mitochondrial functions, anti-oxidant effects, anti-inflammatory influences, and modulation of histone and non-histone acetylation, as well as neurotransmitter systems-, can be hypothesized. Indeed, it has been suggested that EKSs-evoked ketosis may alter the activity of different downstream signaling pathways (e.g., AMPK-, SIRTs- and mTOR-modulated pathways) and modulatory effects, through which not only senotherapeutic drugs, but also ketosis (βHB) can improve symptoms and delay development of age-related hallmarks, age-associated neurodegenerative diseases and learning and memory dysfunctions, and extend lifespan. Consequently, administration of EKSs may be a potential therapeutic tool as an adjuvant therapeutics in combination with different therapeutic drugs (such as metformin and rapamycin) for regenerative medicine to enhance effectivity of drugs to rejuvenate aging hallmarks, decrease the risk for age-related neurodegenerative diseases and increase the healthspan of the aging human population. However, modulating ageing processes and related diseases by administration of EKSs needs careful attention, because insufficient clinical data is available currently on its positive effects, efficacy and safety, regarding this specific application. Thus, long-term studies are needed to investigate the exact mechanisms of action by which EKSs-evoked ketosis modulate aging processes, agerelated diseases, learning and memory functions, healthspan and lifespan. Moreover, in order to develop effective treatments for patients with different age-related diseases more studies are needed to identify the most effective doses, administration routes, treatment duration and different formulations of EKSs.

**Author Contributions:** Writing—original draft, Z.K., and B.B.; Writing—review and editing, C.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by ELTE BDPK Excellence Program 12/2020 (to Zsolt Kovács) and Ketone Technologies LLC. The funding body had no influence on writing the manuscript.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** Patent: #10980764, University of South Florida, C.A., D.P.D. "Exogenous ketone supplements for reducing anxiety-related behavior"; Non-provisional patents: Ari, C., Arnold P., D'Agostino, D.P. Technology Title: "Elevated Blood Ketone Levels by Ketogenic Diet or Exogenous Ketone Supplements Induced Increased Latency of Anesthetic Induction" USF Ref. No. 16A018PR ; Ari, C., Arnold P., D'Agostino, D.P. Technology Title: "Exogenous Ketone Supplementation Improved Motor Function in Sprague-Dawley Rats." USF Ref. No: 16A019; Ari, C., Arnold P., D'Agostino, D.P. Technology Title: "Lowering of Blood Glucose in Exercising and Non-Exercising Rats Following Administration of Exogenous Ketones and Ketone Formulas." USF Ref. No: 16A049; Ari, C., Arnold P., D'Agostino, D.P. Technology Title: "Neuroregeneration improved by ketone." USF Ref. No: 16B128 (provisional patent); Ari, C., D'Agostino, D.P. Dean, J.B. Technology Title: "Delaying latency to seizure by combinations of ketone supplements." USF Ref. No: 16B138PR. C. Ari is co-owner of Ketone Technologies LLC, and owner of Fortis World LLC. These interests have been reviewed and managed by the University in accordance with its Institutional and Individual Conflict of Interest policies. All authors declare that there are no additional conflict of interest.

#### **Abbreviations**

Aβ: amyloid-β; ACCs, acetyl-CoA carboxylases; Akt, Akt kinase/protein kinase B; AMPK, AMP activated serine-threonine protein kinase; BDNF, brain-derived neurotrophic factor; βHB, beta-hydroxybutyrate; CNS, central nervous system; EKSs, exogenous ketogenic supplements; ER, endoplasmic reticulum; ETC, electron transport chain; FOXOs, Forkhead box Os; HATs, histone acetyltransferases; HCAR2, hydroxycarboxylic acid receptor 2; HDACs, histone deacetylases; hnRNP A1, heterogeneous nuclear ribonucleoprotein A1; IGF 1, insulin-like growth factor 1; IIS pathway, insulin/insulin-like growth factor (IGF) 1 pathway; IL-1β, interleukin-1β; IL-1R, IL-1 receptor; KE, ketone ester; KS, ketone salt; MCT, medium chain triglyceride; miRNAs, microRNAs; MnSOD, manganese superoxide dismutase; mPT pore, mitochondrial permeability transition pore; mTOR,

mechanistic target of rapamycin; NAD<sup>+</sup> , nicotinamide adenine dinucleotide; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3, NOD-like receptor pyrin domain 3; Nrf2, nuclear factor erythroid 2-related factor 2; Oct4, embryonic stem cell regulator octamer-binding transcriptional factor 4; p53, transcription factor tumor suppressor protein 53; PARP-1, poly(ADP-ribose) polymerase-1; PGC-1α, peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α; ROS, reactive oxygen species; SASP, senescence associated secretory phenotype; SIRT, Sirtuin; SOD, superoxide dismutase; SREBP1, sterol regulatory element-binding protein 1; TNF-α, tumor necrosis factor-α; UCP, uncoupling protein; ULK1, Uncoordinated/Unc-51-like kinase 1.

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