Multicomponent Training Prevents Memory Deficit Related to Amyloid-β Protein-Induced Neurotoxicity

Abstract

Background:

Alzheimer’s disease (AD) is characterized by the accumulation of the amyloid-β peptide in the brain, leading to early oxidative stress and neurotoxicity. It has been suggested that physical exercise could be beneficial in preventing AD, but studies with multicomponent training are scanty.

Objective:

Verify the effects of multicomponent exercise training to prevent deficits in recognition memory related to Aβ neurotoxicity.

Methods:

We subjected Wistar rats to multicomponent training (including aerobic and anaerobic physical exercise and cognitive exercise) and then infused amyloid-β peptide into their hippocampus.

Results:

We show that long-term multicomponent training prevents the amyloid-β-associated neurotoxicity in the hippocampus. It reduces hippocampal lipid peroxidation, restores antioxidant capacity, and increases glutathione levels, finally preventing recognition memory deficits.

Conclusion:

Multicomponent training avoids memory deficits related to amyloid-β neurotoxicity on an animal model.

Keywords

Alzheimer’s disease cognitive dysfunction exercise therapy oxidative stress

INTRODUCTION

Alzheimer’s disease (AD) involves progressive degeneration in areas from the hippocampus and cerebral cortex, being characterized as the most common form of dementia [1]. Nowadays, there is no pharmacological curative treatment, and there is a continuous search for preventive strategies and treatments [2]. AD leads to memory-related deficits resultant from synaptic loss, selective neuronal death, decrease in specific neurotransmitters’ levels, and abnormal protein deposits in extracellular spaces, mainly the amyloid-β protein (Aβ) [3].

Aβ induces neurotoxicity by multiple mechanisms, including the generation of reactive oxygen species (ROS), leading to a cascade of cellular imbalances [4]. Pro-inflammatory responses, mitochondrial dysfunction, and oxidative stress are also directly related to Aβ neurotoxicity [5]. The brain is highly susceptible to oxidative imbalance due to its high-energy demand, high oxygen consumption, rich abundance of easily peroxidable polyunsaturated fatty acids, and the relative scarcity of antioxidants and related enzymes [6]. Besides, there is a significant decrease in antioxidant enzyme activity such as glutathione reductase, glutathione peroxidase, catalase, among others, in response to Aβ neurotoxicity [7]. Thus, the ROS overproduction and the impaired antioxidant system lead the cellular redox state to oxidative imbalance [8]. Evidence of the oxidative damage caused by Aβ includes the high levels of lipid peroxidation measured as malondialdehyde (MDA) in the hippocampus from AD patients [8, 9].

The hippocampus is a brain region with high levels of neurotrophic factors related to neural plasticity and closely linked to learning and memory [10]. Also, it is one of the first brain areas affected by AD [3]. The Aβ neurotoxicity induces changes that interfere with hippocampal function, promoting oxidative stress and apoptosis and decreasing brain-derived neurotropic factor (BDNF) levels. The interference in neural plasticity and neurogenesis regulated by the BDNF pathway results in cognitive decline [11, 12]. Physical exercise or cognitive stimulation drives neurotrophic factors (such as BDNF), resulting in neuroprotection associated with age-related disorders and diseases [13]. Also, the elevation of BDNF can modulate oxidative stress [14].

Physical exercise is considered a potent non-pharmacological preventive agent of AD. Aerobic exercise promotes hippocampal neurogenesis and cell proliferation [15, 16], contributing to memory persistence through neurotransmitter modulation, increase of pre and postsynaptic protein levels in the hippocampus [17, 18], and increase of BDNF levels and insulin-like growth factors (IGF-1) [16, 19]. Furthermore, the neuroprotective effect of aerobic exercise is also related to the preservation of the energetic metabolic function in the hippocampus [20, 21]. Physical exercise promotes an increase in blood flow in the brain, representing a greater supply of oxygen and, therefore, energy, favoring the improvement of cognitive performance [22]. Exercises involving anaerobic stimuli can also decrease neuroinflammation and oxidative stress, preventing the toxic effects of Aβ in the hippocampus [23, 24].

Cognitive exercise is another strategy that helps to maintain neural functions, promoting cognitive flexibility, and improving patients’ quality of life [25]. Using an Aβ neurotoxicity rats’ model, we recently showed that, despite the related effects on neuroplasticity, cognitive exercise prevents memory deficits by decreasing hippocampal oxidative stress and maintaining the tissue organization [26]. While the beneficial independent effect of aerobic and anaerobic physical exercise is well-known, the effects of cognitive exercises are less noticed by the literature. Only a few studies consider the effects of combining exercises on cognition [27, 28].

The combination of exercises effectively improves muscle strength, gait speed, and physical performance, including endurance, aerobics, balance, and flexibility tasks [28 –30]. Considering functional performance, multicomponent exercise training is more effective than isolated aerobic exercise training [27]. It may result in the increased stimulus from aerobic and anaerobic tasks on strength, for example, resulting in a higher training load associated with better results. However, it is still unclear if these possible cumulative effects may positively impact cognition.

Here we demonstrate the beneficial effects of multicomponent exercise training to prevent deficits in recognition memory related to Aβ neurotoxicity. We also show that the multicomponent exercise training prevents hippocampal oxidative stress and the decline in the antioxidant capacity.

MATERIALS AND METHODS

Animals and experimental design

Adult male Wistar rats (3 months old; n = 48), purchased from the vivarium of the Federal University of Santa Maria (RS/Brazil), were used. They were housed four per cage and maintained under controlled light and environmental conditions (12 h light/12 h dark cycle at a temperature of 23±2°C and humidity of 50±10%) with food and water ad libitum. All experiments were conducted following the “Principles of laboratory animal care” (NIH publication n. 80–23, revised 1996) and approved by the Animal Use Ethics Committee of the Federal University of Pampa (RS/Brazil) (Protocol 031/2018).

Initially, the rats were divided into two large groups, according to the intervention: Sedentary (control) and Multicomponent training (MT). The MT group animals were submitted to 8 weeks of exercise, including three different modalities (aerobic exercise, anaerobic exercise, and cognitive exercise). The training was carried out 6 times a week, with one different modality each day, resulting in 2 exercise sessions of each modality per week (see details in Table 1).

Table 1

Details of the multicomponent training. The training protocol involved three components (endurance/aerobic, strength/anaerobic, and cognitive flexibility), performed in 6 sessions per week, 1 different modality each day, resulting in 2 exercise sessions of each modality per week, for 8 weeks

Days of week Training	Day 1 Aerobic	Day 2 Aerobic	Day 3 Cognitive	Day 4 Aerobic	Day 5 Anaerobic	Day 6 Cognitive	Day 7 Rest
Multicomponent training
Aerobic exercise	Endurance/running training on a treadmill at 60–70%of maximal indirect VO₂ at 0%inclination and lasting 40 min.
Anaerobic exercise	Strength training performed on a ladder with workload progression from 50%, 75%, 90%to 100%of body mass. For each session, 2 series for each load with 60s rest interval between series.
Cognitive exercise	Cognitive flexibility training using an adaptation of the Barnes Maze. Every ten days, the escape cage was changed to a new location.

After the intervention period, the rats were submitted to stereotaxic surgery for Aβ or saline infusion and subdivided into 4 groups (n = 12/group): Sedentary (Control), Amyloid-β (Aβ), Multicomponent training (MT), and Multicomponent training +Amyloid-β (MT + Aβ). Ten days after the surgery, the time required for beta-amyloid protein aggregation [31], the rats were subjected to behavioral tests to assess memory and control behavioral parameters. In the end, the rats were euthanized, and brain tissue samples were collected for biochemical (n = 6/group) analyses (see Fig. 1).

Fig. 1

Experimental Design. In the first week, the rats were divided into groups according to the intervention of multicomponent exercise training (MT) or no (Sedentary/control). The MT was conducted for 8 weeks. Afterward, all rats were submitted to stereotaxic surgery to hippocampal beta-amyloid protein or saline (vehicle) infusion; 10 days after surgery, animals were submitted to memory and behavioral control tests. After the tests, the rats were euthanized, and the hippocampal tissue was removed for further biochemical analysis.

Amyloid-β preparation and infusion

Aβ peptide 25–35 (A4559; Sigma Aldrich) was dissolved in saline solution (i.e., vehicle) at a concentration of 100μM and incubated at 37°C for 4 days to induce Aβ 25–35 aggregation initiation [30]. The animals were anesthetized intraperitoneally with ketamine and xylazine (75 mg/kg and 10 mg/kg, respectively) and submitted to stereotaxic surgery. Based on the Paxinos and Watson atlas coordinates for the hippocampus (anterior-posterior = – 4.2 mm; lateral-lateral, ±3.0 mm; ventromedial, – 3.0 mm) [31], using a Hamilton syringe and an infusion pump, 2.0μl of Aβ protein or vehicle were infused bilaterally in the hippocampal CA1 region. Afterward, the rats were returned to their cages and monitored for 10 days to recovery and Aβ aggregation [31].

Multicomponent training (MT)

The MT protocol involved three components: aerobic/endurance, anaerobic/strength, and cognitive. Training sessions were performed 6 times/week, over 8 weeks, and each day one of the components was trained (Table 1). Each component followed a specific protocol, as described below.

Aerobic exercise

For the aerobic training, the rats were familiarized with a treadmill specific for rodents (Insight Ltda, São Paulo, Brazil). Familiarization aimed to avoid stress effects and was conducted for 5 days (treadmill velocity starts from 0 to 5 m/min for 10 min/day) as described in previous researches [23, 32]. On the last day of familiarization, an indirect oxygen consumption test (VO₂) was performed to determine the individual training intensity of each animal. To do this, each rat started at a slow speed and gradually increased by 5 m/min every 3 min until the rat was not long able to run. Fatigue time (min) and workload (m/min) were considered as an indirect measure of maximal VO₂ [33].

The aerobic exercise started in the following week and was performed at an intensity of 60–70%of maximal indirect VO₂, 2 times a week, once a day, for 40 min, with the treadmill at 0%inclination, for 8 weeks. After 4 weeks of aerobic training and again in the last week of training, the same indirect VO₂ test was repeated to adjust exercise intensity and/or verify aerobic gains [34].

Anaerobic exercise

The apparatus used for the exercise of strength was a vertical ladder with an inclination of 80 degrees (110 cm high, 18 cm wide, 2 cm between the steps). There was a box at the top of the stairs (25×25×20 cm) that gave shelter to the animal. Before starting training, the animals were familiar with the device, making 4 attempts a day for 3 days. In the first trial, the rat was kept in the chamber for 60 s; in the second trial, the rat was placed 35 cm below the top; at the third attempt, at the bottom half of the ladder, and at the last attempt, at the bottom of the ladder.

After familiarization, anaerobic training started, and 8 series were performed, with 12 repetitions/climb with a progressive load on the proximal part of the animal’s tail. In the first week, the training load was 50%of the animal’s body mass. In the second week, the load was determined through the maximum load test (MCL), which consists of climbing the stairs initially with 75%of body weight and then an additional 30 grams for each new climbing repetition. The maximum load was determined and recorded when the animal could not perform the repetitions. After the maximum load test, the training series were performed with a progressively increased percentage of load (50%, 75%, 90%, 100%of the maximum load test), with two series being performed for each percentage. A rest interval of 60 s was defined between the series. The MCL test was performed every two weeks of training to adapt the training protocol to the animals’ gains [35].

Cognitive exercise (CE)

The CE was performed according to an adaptation of the Barnes Maze Spatial Memory Task, as proposed by Daré et al. (2019). The Barnes Maze device is a circular platform with 20 potential exhaust holes, equally distributed on the periphery of the platform; only one of these holes leads to an escape cage. The rat was placed in the center of the apparatus, and negative reinforcement (bright lights at the top of the platform) was used to motivate the animal to escape to the cage hidden under one of the holes. Geometric clues around the maze were used as visual clues to make spatial learning possible. After finding the escape, the animal was removed from the escape cage and returned to its housing box. The maximum time to meet the platform was limited to 180 s; if the rat did not find the escape at this time, the animal was gently guided to it.

The training was performed twice a week, for 8 weeks, with only 1 session per day. With each new training session, the rats can perform the CE more efficiently, which means that they can find the escape cage more quickly using the space cues. Thus, every ten days, the escape cage was changed to another location. Consequently, the rats had to reorganize spatial memory, which required cognitive flexibility [36].

Control behavioral tasks

Considering that memory assessment depends on behavioral observation, behavioral control tasks were used to ensure that surgery, MT, or other procedures did not impair exploratory and motor performance, which could alter the performance of memory tests. The elevated plus maze (PM) and the open field (OF) tests were used to evaluate anxiety state and to analyze locomotor activities, respectively.

In the PM test, the rats were placed in the center of the maze. The maze consisted of two open arms, 50×10 cm, and two enclosed arms, 50×10×40 cm, with an open roof, arranged in a way that the two open arms were opposite to each other. The maze was elevated to a height of 50 cm. The total number of entries and the time spent in the four arms were recorded over a 5 min session [37]. For the OF test, the rats were placed in the left quadrant of a 50×50×39 cm box made of white painted wood with a front glass wall. Black lines were drawn on the floor to divide it into 12 equal quadrants. The number of crossings, as measures of locomotor activity, was measured over 5 min [38].

Memory tests

Object recognition memory task (OR)

Initially, the rats were individually habituated to the OR apparatus and were free to explore it for 20 min during four consecutive days before the training session. On the fifth day, OR training was performed. In training, two different objects (named A and B) were placed in the apparatus, and rats were allowed to freely explore them for 5 min. The short (STM) and long-term (LTM) memories were evaluated 3 h and 24 h after, respectively [39]. In each testing session, one of the objects was randomly replaced by a novel object (C and D, respectively), and the rats were reintroduced into the apparatus for an additional 5 min period of free exploration. The time spent exploring the new and the familiar object was recorded.

Social recognition memory task (SR)

The SR memory task is an adaptation of the social interaction test [32]. The task was completed in 3 days. First, the rats were placed in an arena for habituation with two small cages during 20 min of free exploration. On the following day, a training session was performed with the inclusion of one unfamiliar rat in one of the cages for 60 min of free exploration. After 24 h, a testing session was performed, when the same rat from the training (now a familiar rat) and a new rat was placed for exploration for 60 min. The time spent exploring the new and the familiar rat was recorded. Exploration of the conspecific animal was defined as sniffing or touching the small cages with the nose and/or forepaws.

Oxidative stress status

After the euthanasia, the brain tissues of some animals (n = 6/group) were quickly removed on an iced surface. The hippocampal tissues were immediately isolated from the brain and cleaned using ice-cold saline. Tissue samples were frozen in liquid nitrogen and stored at –80°C until biochemical analysis. For biochemical experiments, the tissue samples were homogenized in 50 mM Tris-HCl, pH 7.4. The homogenates were centrifuged at 2,400 g for 20 min at 4°C to obtain supernatants used to analyze all biochemical variables.

Reactive oxygen species (ROS)

The hippocampal ROS levels were measured by a spectrofluorometric method using 20,70-dichlorofluorescein diacetate (DCFH-DA) [40]. The sample was incubated in darkness with 5μL of DCFH-DA (1 mM). The oxidation of DCHF-DA to fluorescent dichlorofluorescein (DCF) was measured to detect intracellular ROS. The formation of the oxidized fluorescent derivative (i.e., DCF), measured by DCF fluorescence intensity, was recorded at 520 nm (480 nm excitation) 30 min after adding DCFH-DA to the medium. Results were expressed as arbitrary units.

Detection of lipid peroxidation (TBARS)

Hippocampal lipid peroxidation levels were evaluated by the TBARS test [41]. Samples were incubated with a 0.8%thiobarbituric acid solution, acetic acid buffer (pH 3.2), and SDS solution (8%) at 95°C for 2 h, and the color reaction was measured at 532 nm. Results were expressed as nanomoles of malondialdehyde per milligrams protein.

Ferric reducing/antioxidant power (FRAP) assay

The total antioxidant capacity was measured by FRAP assay (ferric reducing/antioxidant power). The working FRAP reagent was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution, and 2.5 mL FeCl3·6H2O solution. The homogenate (10μL) was added to the 300μL working FRAP reagent in the microplate [42]. Additionally, a standard curve with 10μL Trolox concentrations (15, 30, 60, 120, and 240 mM) and 300μL working FRAP reagent was used. The microplate was incubated at 37°C for 15 min before reading in a SpectraMax M5 Microplate Reader at 593 nm.

Glutathione (GSH) levels

GSH levels were determined by fluorometry [43]. An aliquot of the homogenized sample was mixed (1:1) with perchloric acid (HClO₄) and centrifuged at 3000 for 10 min. This mixture was centrifuged, the protein pellet was discarded, and free thiols (SH) groups were determined in the clear supernatant. An aliquot of the supernatant was incubated with orto-phthaladehyde, and fluorescence was measured at an excitation of 350 nm and emission of 420 nm. Results were expressed as nmol g^–1 of tissue.

Statistical analysis

The normality of data distribution was checked by the Shapiro-Wilk test. Object exploration time in OR task and exploration time in SR task were converted to a percentage of total exploration time, and a one-sample t-test was used to compare the percentage of the total time of exploration spent on each object/rat with a theoretical mean of 50%. Besides, the %of the total exploration time of the new object was used for comparison between the groups using the one-way ANOVA followed by Tukey’s post-hoc.

The OF and PM data were analyzed by a one-way ANOVA. The maximum running speed and MCL data were analyzed by repeated-measures ANOVA followed by Tukey post hoc (to compare the initial parameters with the final ones). The Mann-Whitney test was used to compare groups in each day. Biochemical results were compared using a two-way ANOVA test followed by Sidak’s post hoc. Results are expressed as mean and standard deviation. The significance level was set at 0.05 for all analyses.

RESULTS

Strength and oxygen uptake (VO₂) gains

The aerobic gains were estimated by indirect VO₂ measures repeated three times during the MT protocol (Fig. 2A). Strength gains were estimated by the maximum carrying load (MCL) measured every two weeks (Fig. 2B, data showed represents only 3 measures). Trained animals from both groups MT and MT + Aβ presented an increase in the maximum running speed (MT: p = 0.0262; MT + Aβ: p = 0.0099; Fig. 2A) and MCL (MT: p = 0.0010; MT + Aβ: p < 0.0001; Fig. 2B) towards the end of the training, indicating that the training improved aerobic resistance and strength. There were no differences between groups on any test day (p > 0.05).

Fig. 2

The maximum running speed and the MLC of trained animals increased along the MT, indicating that the training improved aerobic resistance and strength. A) Progression of the maximum running speed (m/min), an indirect measurement of VO₂, considering the first, the fifth, and the last week of training. B) Progression of the load (grams), measured by MLC test in the first, the fifth, and the last week of training. The data were analyzed by repeated-measures ANOVA and Tukey post hoc. The Mann-Whitney test was used to compare groups. ^*p < 0.05.

Memory tasks

Object recognition memory (OR)

In the OR training session the rats explored each object (A and B) for about 50%of the total exploration time (in the figures, training is the mean of all groups; p > 0.05 for all groups versus a theoretical mean of 50%; Control: t₍₄₎ = 1.086, p = 0.3384; Aβ: t₍₅₎ = 0.3077, p = 0.7707; MT: t₍₇₎ = 1.452, p = 0.1897; MT + Aβ: t₍₁₀₎ = 2.206, p = 0.0632; Fig. 3A, B).

Fig. 3

The hippocampal Aβ infusion promotes OR memory deficit. Multicomponent training prevents the OR memory deficit caused by hippocampal Aβ infusion. A) OR short-term memory (STM) test; B) OR long-term memory (LTM) test. ^*p < 0.05; one-sample Student t-test (theoretical mean 50%). Data are presented as mean±SD (n = 5–12/group).

In the STM test, the control rats spent significantly more than 50%of the total exploration time exploring the new object (Control: t₍₄₎ = 2.788, p = 0.0494; Fig. 3A); it suggests that they remembered the familiar object, i.e., formed an STM. The same was observed in the MT group, in which rats spent a long time exploring the new object (MT: t₍₇₎ = 5.597, p = 0.0008; Fig. 3A). The Aβ infusion impaired STM, since the rats spent about 50%of the total exploration time exploring each object (Aβ: t₍₅₎ = 2.073, p = 0.0929, Fig. 3A). The MT was able to prevent the deficits caused by Aβ neurotoxicity since the animals submitted to Aβ infusion but previously to the MT were able to form OR STM, i.e., they explored more than 50%of the total exploration time of the new object (MT + Aβ: t₍₇₎ = 4.460, p = 0.0342; Fig. 3A). No differences were found in the comparison between the groups’ percentage of time exploring the new object (F_(3,23) = 0.5589; p = 0.6475).

Twenty-four hours after the training, the LTM was tested; the results are quite similar to the STM. Rats from control and MT groups explored the novel object for more than 50%of the total exploration time (Control: t₍₄₎ = 3.444, p = 0.0262; MT: t₍₇₎ = 3.190, p = 0.0153; LTM, Fig. 3B). On the other hand, animals from Aβ group presented impaired LTM, since they spent about 50%of total exploration time in each object (LTM; Aβ: t₍₅₎ = 0.1525, p = 0.1878, Fig. 3B). Multicomponent training prevented Aβ protein-induced damage, as the Aβ+ MT group spent more than 50%of the total exploration time exploring the new object (MT + Aβ: t₍₇₎ = 2.623, p = 0.0342; Fig. 3B). No differences were found in the comparison between the groups’ percentage of time exploring the new object (F_(3,23) = 0.8110; p = 0.5008).

Social recognition memory (SR)

In the SR test session, control and MT rats explored the new rat for more than 50%of the total exploration time (Control: t₍₅₎ = 4.458, p = 0.0066; MT: t₍₁₁₎ = 4.893, p = 0.0005; Fig. 4). Animals from Aβ group, however, explored for ∼50%of the total exploration time each rat (Aβ: t₍₁₁₎ = 0.6406, p = 0.5394; Fig. 4), demonstrating memory deficit. Multicomponent training prevented the deleterious effect of Aβ protein on SR memory, since the Aβ trained animals spent more than 50%of the total exploration time exploring the new rat (MT + Aβ: t₍₁₁₎ = 4.239, p = 0.0014; Fig. 4). No differences were found in the comparison between the groups’ percentage of time exploring the new rat (F_(3,38) = 1.739; p = 0.1754).

Fig. 4

The hippocampal infusion of Aβ promotes SR memory deficit. Multicomponent training prevents SR memory deficits caused by Aβ infusion into the hippocampus. ^*p < 0.05; one-sample Student t-test (theoretical mean 50%). Data are presented as mean±SD (n = 5–11/group).

Control behavioral tasks

The intrahippocampal infusion of Aβ or saline and the other procedures involved in the experimental design did not affect the exploratory behavior on OR (p > 0.05; total exploration time on OR training and tests; Table 2) and the locomotor activity during the 5 min free exploration session in the OF (p = 0.58; number of crossings; Table 2). Additionally, no effects were detected for anxiety in the PM test (p = 0.39; time spent at open arms; see Table 2).

Table 2

The infusion of Aβ or vehicle and other procedures involved in the study design did not affect locomotor activity evaluated in an open field, exploratory behavior evaluated in OR training and test, and anxiety evaluated in the PM test (p > 0.05; one-way ANOVA). Data are expressed as mean±SD (n = 8–12 per group)

	Control	Aβ	MT	MT + Aβ	p
OR total exploration time (s) Training	82.75±26.85	81.78±31.86	69.42±23.94	74.92±26.86	0.66
Test (3 h)	70.38±26.35	77.78±33.70	75.50±30.89	67.75±29.27	0.87
Test (24 h)	81.63±30.84	76.22±25.99	69.83±27.29	72.75±23.32	0.79
OF Crossings (n)	86.13±29.43	76.33±21.90	78.33±18.30	87.08±16.84	0.58
PM Time in open arms (s)	71.25±63.97	109.8±22.86	99.83±57.26	104.5±43.78	0.39

Oxidative stress status

The ROS levels measured by the DCFH test showed effects of MT (F_(1,20) = 8.552; p = 0.0084; Fig. 5A). The MT + Aβ group increased ROS levels compared to the control group (p = 0.0197; Fig. 5A). There were no significant differences in ROS levels in the hippocampus between other groups.

Fig. 5

The hippocampal infusion Aβ does not promote the increase of ROS (A) but increases lipid peroxidation (B) and decreases the total antioxidant capacity (C); Aβ does not alter GSH levels (D). Multicomponent training performed for 8 weeks generates changes in ROS (A), prevents the increase of lipid peroxidation (B), and the reduction of antioxidant capacity (C); also, MT increases the levels of GSH (D). Data are presented as mean±SD and were analyzed by two-way ANOVA, followed by Sidak post hoc. Groups whose bars are with different letters are statistically different (p < 0.05).

Lipid peroxidation determined by TBARS showed an effect for the Aβ infusion (F_(1,19) = 8.007; p =0.0107; Fig. 5B) and MT (F_(1,19) = 6.643; p = 0.0185; Fig. 5B). Additionally, there was an interaction between Aβ and MT (F_(1,19) = 14.63; p = 0.0011; Fig. 5B). Aβ group presented increased lipid peroxidation in comparison to control (p = 0.0012; Fig. 5B), MT (p = 0.0056; Fig. 5B) and MT + Aβ group (p = 0.0015; Fig. 5B). Aβ rats that underwent to MT did not present increase of the lipid peroxidation (p = 0.8569 Control versus MT + Aβ group; Fig. 5B).

In the total antioxidant capacity (i.e., ferric reducing/antioxidant power –FRAP) an effect of intervention (MT) was observed (F_(1,18) = 13.00; p =0.0020; Fig. 5C). Additionally, the interaction between the Aβ and MT was significant (F_(1,18) =39.74; p = < 0.0001; Fig. 5C). Aβ infusion decreased antioxidant capacity (p = 0.0075 Aβ versus control group; Fig. 5C). Aβ rats that performed the MT increased antioxidant capacity compared to the control (p = 0.0075 control versus MT + Aβ group; Fig. 5C).

Considering the GSH levels, an effect of the MT was observed (F_(1,16) = 5.290; p = 0.0352; Fig. 5D). Additionally, the interaction between the factors was significant (F_(1,16) = 7.607; p = 0.0140; Fig. 5D). Aβ-infused animals that previously were submitted to MT increased GHS levels in comparison to Aβ (p = 0.0099; Fig. 5D) and MT rats (p = 0.0258; Fig. 5D).

DISCUSSION

Our results show that MT avoids recognition memory deficits related to Aβ neurotoxicity. We suggest that these outcomes are associated with the capacity of MT to prevent the increase of hippocampal lipid peroxidation and the decrease of hippocampal antioxidant capacity induced by Aβ neurotoxicity.

AD is the most common manifestation of dementia. Despite there is no single animal model of AD that reproduces all the disease’s characteristics, the animal models available are important to the study of AD pathophysiology and preventive and therapeutic strategies. Considering that the extracellular Aβ deposition is one of the main features of AD [3], brain Aβ protein infusion is an important model that can contribute to the understanding of the main biological aspects of AD. The use of Aβ 25–35 peptide in animal models contribute to the understanding of its effects on mechanisms related to Aβ toxicity [44], since its administration in the temporal cortex, dorsal hippocampus, or intracerebroventricularly increases the oxidative stress [45], the membrane lipid peroxidation [46], and the neuroinflammation [47], processes that may contribute to synaptic dysfunction, neuronal death and consequently, the cognitive decline [48]—outcomes observed in the AD patients’ brain.

A high density of Aβ deposits, lipid peroxidation, and protein oxidation were found in AD patients’ hippocampus and cortex with cognitive deficits [49, 50]. Similarly, in our study, the infusion of Aβ in the hippocampus altered memory processes by promoting oxidative damage. If, on the one hand, we observed that the MT + Aβ group showed a slight increase in DCFH levels compared to the control, which could be justified by the Aβ peptide infusion generating an increase of the hippocampal oxidative stress; on the other hand, the MT seems to be able to control the damage induced by oxidative stress, as promoted the increase of antioxidant capacity and the reduction of lipid peroxidation, what could be related to positive effects on memory.

The molecular mechanisms and signaling pathways involved in exercise benefits are complex and not completely clear. Here we demonstrate that the combination of exercises (aerobic, anaerobic, and cognitive) effectively acts in redox regulation. Anabolic and catabolic muscle pathways are strongly influenced by physical exercise and by stimulating muscle anabolism; exercise inhibits protein degradation, an effect probably mediated by lower levels of oxidative stress after training [51]. The intensity of the modulation by exercise depends on the training’s duration and intensity. Initially, physical exercise raises oxidative stress to a significant level, but when performed regularly, it may be able to induce positive adaptations in cellular antioxidant systems and stimulate oxidative damage repair systems [52 –55]. Thus, unlike what happens after acute exercise sessions, regular exercise is associated with low levels of oxidative damage markers and increased enzymatic and non-enzymatic antioxidant capacity in young, middle-aged, and elderly individuals [56 –59].

Different physical training models affect oxidative parameters and memory [24 , 61]. In this study, we observed that after eight weeks of training, the combination of exercises probably became part of a beneficial adaptive process, since it was observed a decrease in lipid peroxidation, an increase in total antioxidant capacity, and an increase in GSH, demonstrating the strong modulation of the combined training on oxidative stress. Also, these effects are supported by the improvement of cognitive deficits in animals infused with Aβ peptide and submitted to multicomponent exercise (group MT + Aβ), a result observed in short and long-term object and social recognition memory. Furthermore, the animals that performed the MT gradually increased their aerobic resistance and their workload gain.

It is known that running exercise at moderate intensity, as performed here (60–70%of VO₂ max), results in cognitive benefits in different conditions of brain degeneration, significantly increasing BDNF and promoting brain health [32 , 62]. Similarly, the maximum load protocol sought to adjust the exercise intensity to the animal’s capacity. The researchers recommend that strength training protocols contain sufficient volume for each muscle group with intensities between 50 and 80%of a maximum repetition [63]. Our strength protocol was designed to be an intermittent exercise, a training set that has shown benefits in cognition considering different conditions [62, 64]. These muscular and functional adaptations are closely linked to mitochondrial regulation [56] that directly impact the redox imbalance.

Glutathione (GSH) is an important endogenous antioxidant found in millimolar concentrations in the brain. GSH levels decrease in various degenerative diseases, including AD [65]. Our Aβ rats did not show significant hippocampal GSH alterations, but the MT increased GSH levels suggesting an adaptive brain response due to the exercise in Aβ rats [66]. The MT was also able to improve the total antioxidant capacity, which Aβ decreased. Supporting these findings, some previous studies highlight that regular physical training can lead to favorable adaptations to the antioxidant system [67], and some antioxidant enzymes such as glutathione-peroxidase and glutathione reductase are increased after six weeks of training [68], protecting tissues from oxidative damage. Previous studies using aerobic exercise protocols have also reported increased antioxidant gene expression [69].

Here we observed that combining aerobic, anaerobic, and cognitive exercises in MT prevents deficits in short and long-term memory caused by Aβ. The hippocampus is one of the main structures involved in the learning and memory processes, responsible for signaling the cascade involved in the acquisition and formation of new memories [70]. Oxidative stress in the hippocampus region leads to cellular dysfunction and is related to memory impairment, reducing the production of new neurons and altering dendritic structures [26, 71]. The biochemical results support the benefits of MT seen in memory tests. The hippocampus of MT rats did not show the same oxidative imbalance found in untrained rats. Besides, physical exercise or cognitive stimulation generates positive regulation of neurotrophic factors, like BDNF, improving synaptic plasticity and stimulating neurogenesis [13]. It is known that higher levels of BDNF can decrease oxidative stress and increase antioxidant proteins acting in the control of neuronal apoptosis through the modulation of mechanisms involved in the cellular ROS and CREB pathway [14, 72]. So, MT may increase mitochondrial energy production efficiency by limiting oxidative stress and supporting the BDNF-mediated synaptic plasticity process [13], which would result in memory improvements.

Taken together, our results demonstrate that the MT acted in the dysfunctions caused by oxidative stress avoiding deficits in the acquisition and consolidation of new memories. Yet, we believe that the MT could be advantageous, as it results in multiple benefits beyond cognition, such as improved muscle, metabolic and functional parameters, considering that it involves the training of other components, in addition to aerobic [17 , 73]. These results support the idea that the combination of physical and cognitive exercise therapies promotes memory improvement by biochemical mechanisms related to oxidative balance. It is important to highlight that many factors can influence exercise (combined or not) effects, as intensity, duration, frequency, and training organization [74, 75].

CONCLUSIONS

Our results highlighted that multicomponent training, combining aerobic, anaerobic, and cognitive exercises, avoids memory deficits related to amyloid-β neurotoxicity on an animal model. Additionally, multicomponent training reduces hippocampal lipid peroxidation, restores hippocampal antioxidant capacity, and increases glutathione levels in the hippocampus.

Footnotes

ACKNOWLEDGMENTS

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior –CAPES/Brazil [finance code 001 and grant number PROCAD 88881.068493/2014-01] and by the Federal University of Pampa. CBS is supported by Fundação de Amparo à Pesquisa do Rio Grande do Sul –FAPERGS/Brazil. KRL is supported by CAPES/Brazil. LFL, HLS, FPC and PBM-C are supported by Conselho Nacional de Pesquisa –CNPq/Brazil.

Authors’ disclosures available online ().

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Multicomponent Training Prevents Memory Deficit Related to Amyloid-β Protein-Induced Neurotoxicity

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Animals and experimental design

Amyloid-β preparation and infusion

Multicomponent training (MT)

Aerobic exercise

Anaerobic exercise

Cognitive exercise (CE)

Control behavioral tasks

Memory tests

Object recognition memory task (OR)

Social recognition memory task (SR)

Oxidative stress status

Reactive oxygen species (ROS)

Detection of lipid peroxidation (TBARS)

Ferric reducing/antioxidant power (FRAP) assay

Glutathione (GSH) levels

Statistical analysis

RESULTS

Strength and oxygen uptake (VO2) gains

Memory tasks

Object recognition memory (OR)

Social recognition memory (SR)

Control behavioral tasks

Oxidative stress status

DISCUSSION

CONCLUSIONS

Footnotes

ACKNOWLEDGMENTS

References

Strength and oxygen uptake (VO₂) gains