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O P E N A C C E S S S O U R C E : Redox Biology @ Science Direct
Highlights
• Hippocampus-dependent learning and memory are impaired with age, which correlated with synaptic mitochondrial dysfunction.
• Synaptic mitochondria fail before non-synaptic mitochondria, indicating premature synaptic mitochondrial damage in aging.
• Reducing synaptic mitochondrial dysfunction, with MitoQ or Curcumin, decrease age-associated hippocampal memory impairment.
• Age-related changes in ATP production of synaptic mitochondria correlated with decreased hippocampal memory.
• Maintenance of functional synaptic mitochondria is critical to prevent memory loss during aging.
Abstract
Aging is a process characterized by cognitive impairment and mitochondrial dysfunction. In neurons, these organelles are classified as synaptic and non-synaptic mitochondria depending on their localization. Interestingly, synaptic mitochondria from the cerebral cortex accumulate more damage and are more sensitive to swelling than non-synaptic mitochondria. The hippocampus is fundamental for learning and memory, synaptic processes with high energy demand. However, it is unknown if functional differences are found in synaptic and non-synaptic hippocampal mitochondria; and whether this could contribute to memory loss during aging. In this study, we used 3, 6, 12 and 18 month-old (mo) mice to evaluate hippocampal memory and the function of both synaptic and non-synaptic mitochondria. Our results indicate that recognition memory is impaired from 12mo, whereas spatial memory is impaired at 18mo. This was accompanied by a differential function of synaptic and non-synaptic mitochondria. Interestingly, we observed premature dysfunction of synaptic mitochondria at 12mo, indicated by increased ROS generation, reduced ATP production and higher sensitivity to calcium overload, an effect that is not observed in non-synaptic mitochondria. In addition, at 18mo both mitochondrial populations showed bioenergetic defects, but synaptic mitochondria were prone to swelling than non-synaptic mitochondria. Finally, we treated 2, 11, and 17mo mice with MitoQ or Curcumin (Cc) for 5 weeks, to determine if the prevention of synaptic mitochondrial dysfunction could attenuate memory loss. Our results indicate that reducing synaptic mitochondrial dysfunction is sufficient to decrease age-associated cognitive impairment. In conclusion, our results indicate that age-related alterations in ATP produced by synaptic mitochondria are correlated with decreases in spatial and object recognition memory and propose that the maintenance of functional synaptic mitochondria is critical to prevent memory loss during aging.
1. Introduction
Aging is a multifactorial process, characterized by deterioration of physiological and cellular functions [1], including brain function [2]. One of the most affected functions is memory, requiring more time to carry out the learning and memory process [3]. The hippocampus plays an important role in memory [4]; storing information associated with the recognition of an event (recognition memory), as well as spatiotemporal context (spatial memory) [5]. However, hippocampal atrophy is observed in aging, which could explain the age-associated memory deficit [6,7].
Studies have shown the importance of mitochondria in synaptic communication as well as to hippocampus-dependent learning and memory [8]. Mitochondria supply energy, maintain calcium homeostasis and regulate the redox balance [9]. The internal mitochondrial membrane contains the electron transport chain (ETC) that generates ATP [10] and as a secondary product form reactive oxygen species (ROS) [11]. Oxidative molecules act as cellular regulators [12]; however, its overproduction generates oxidative stress, which is strongly associated with aging [[13], [14], [15]]. In addition, mitochondrial calcium regulation is mediated by transient mitochondrial permeability transition pore (mPTP) opening [16]. Nevertheless, in conditions of high mitochondrial calcium, the mPTP opening is induced, generating mitochondrial swelling and apoptosis [17]. During the last few decades, it has been suggested that mitochondrial dysfunction plays an important role in aging [18]. Aged mitochondria are incapable of regulating calcium; they present decreased ATP production, and increased ROS generation; which result in bioenergetic defects and oxidative damage [14,19,20]. In addition, mitochondrial dysfunction is considered a hallmark of aging [14,21] and could contribute to the loss of cognitive abilities observed with age [14,22].
In the brain, mitochondria have been classified into non-synaptic and synaptic mitochondria [23]. Non-synaptic mitochondria come from neuronal and glial cells, whereas synaptic mitochondria are exclusively found in neurons, specifically in the synapses [24]. Pre-synaptic mitochondria are necessary to produce ATP required for the release of neurotransmitters [25]; whereas post-synaptic mitochondria are fundamental to the synaptic transmission [26]. Increasing evidence suggests that synaptic mitochondrial impairment is strongly associated with neuronal failure in Alzheimer's Disease (AD) [27]. In AD, synaptic mitochondria show increased ROS production, decreased respiration rate, and impaired calcium regulation; which occur before the alterations in non-synaptic mitochondria and the appearance of the AD pathology [27]. Interestingly, synaptic mitochondria from the cerebral cortex of 3month-old (mo) rats are more susceptible to high calcium concentrations than non-synaptic mitochondria [28] and fail earlier than non-synaptic mitochondria at advanced age [29,30]. Considering this evidence and the importance of the hippocampus to learning and memory, we proposed that hippocampal synaptic mitochondria failure could occur before non-synaptic mitochondria during aging, contributing to age-associated cognitive impairment.
Here, we studied the function of hippocampal synaptic and non-synaptic mitochondria from 3, 6, 12 and 18mo mice, and its contribution to hippocampus-dependent memory loss. We observed that 12mo mice present recognition memory impairment, while the loss of spatial memory was observed at 18mo. Interestingly, regarding mitochondrial function, we observed reduced ATP production only in the synaptic mitochondria of 12mo mice; whereas 18mo mice showed bioenergetic defects in both populations. Similarly, calcium sensibility was higher in synaptic mitochondria from 12 and 18mo mice than non-synaptic mitochondria, indicating that synaptic mitochondria fail in a premature manner compared with non-synaptic mitochondria. In addition, to validate that synaptic mitochondrial dysfunction contributes to memory impairment, 2, 11, and 17mo mice were treated with the mitochondria-targeted antioxidant MitoQ, or Curcumin (Cc) for 5 weeks. MitoQ consists of a ubiquinone moiety linked to a triphenyl-phosphonium moiety by a 10-carbon alkyl chain [31,32]; which improves behavior in mice after brain damage [33,34] and in a mouse model of AD [35]. Additionally, we studied the beneficial effects of Cc, because has been described as an anti-inflammatory and antioxidant molecule, improving inflammatory and neurodegenerative diseases [36,37]. Interestingly, we observed that treatment was sufficient to ameliorate the cognitive impairment, exclusively improving synaptic mitochondrial function. In fact, we observed a correlation between the concentrations of ATP produced by synaptic mitochondria and the cognitive performance in the Novel Object Recognition (NOR) and Morris Water Maze (MWM) tests. In conclusion, synaptic mitochondrial dysfunction occurs before that non-synaptic fail and contributes to memory loss during aging; therefore, molecules that preserve synaptic mitochondrial function could be used to prevent the development of age-associated diseases.
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3. Results
3.1. Impairment of object recognition memory occurs before object localization memory
For several years, researchers have studied memory loss during aging [51]. Recognition memory is a type of hippocampus-dependent memory, specifically of the CA3 region [52], which is affected during aging [53]. Here, we evaluated changes in recognition memory with age. We performed the Novel Object Localization (NOL) and Novel Object Recognition (NOR) test (Supplementary Fig. 1A and 1B) [49] in 3, 6, 12, and 18mo C57BL/6 mice. To carry out these tests, we first exposed the animals to a habituation phase, in which each animal explored the empty chamber (without objects present) for 5 min. The next day, the mice were subject to the familiarization phase. In this stage, each animal had 10 min to explore the chamber, which contained two identical objects. After 2 h, the NOL stage was performed. In this phase, the animals explored the same objects for 5 min, but one object was localized in other position in the chamber (Supplementary Figure 1A). We observed that the 3, 6, and 12mo mice exhibited more time exploring the novel localization of the object, as indicated by the time that the animal's head spent in this area (Fig. 1A). In contrast to 18mo mice, which showed no preference by the novel localization, observing that this group spent similar time exploring both object locations (Fig. 1A). This was more evident when we analyzed the Recognition Index, which represents the time spent exploring the localization of the novel object relative to the total time exploring both localizations (Fig. 1B). We observed that 3, 6, and 12mo mice showed a higher preference for the localization of the novel object compared to 18mo mice (Fig. 1B). The differences in explorative behavior are shown in the representative traces of each group (Fig. 1C) and in the heat maps (Fig. 1D), where only 18mo mice showed no preference for the new location of the object, remaining similar time exploring both object locations. These results indicate that 18mo aged mice are incapable of recognizing the novel localization of the object, suggesting that at 18mo object localization memory is impaired.
Fig. 1. Object localization memory and object recognition memory are differentially impaired during aging.
(A) Time that the animals explore old and novel localization of the object. (B) Recognition Index of each group. © Representative track of one animal per group in NOL test. (D) Heat maps of each group in the NOL test. (E) Time that the animals explore old and novel objects. (F) Recognition Index of each group. (G) Representative track of one animal from the group in the NOR test. (H) Heat maps of each group in the NOR test. Graph bars represent means ± SEM. *p < 0.05. **p < 0.01; ***p < 0.001.
Diverse studies performed in rats, monkeys, and humans indicate that recognition memory is impaired at an advanced age [54]. Considering this, object recognition memory also was evaluated (NOR test). For this, 2 h after the NOL test, a familiar object was replaced by a novel object (Supplementary Figure 1B) [49]. In this phase, animals explored both old and novel objects for 5 min. We observed that 3 and 6mo mice spent more time exploring the novel object compared with 12 and 18mo mice, which spent a similar time exploring both objects (Fig. 1E). Similarly, this is observed in the Recognition Index (Fig. 1F), the representative track (Fig. 1G) and the heat map (Fig. 2H), where 12 and 18mo mice presented significantly reduced novel object recognition. Thus, these results indicate a loss of object recognition memory since 12mo in this mouse line. Altogether, our findings indicate that both object localization and recognition memory are impaired with age; however, defects in object recognition memory appear before localization memory.
Fig. 2. Spatial memory loss is observed in animals of 18 month-old.
(A) Time that the animals spent to find the escape chamber during BM training. (B) Representative track of one animal per group during BM training. © Time that each group stayed in the escape area. (D) Representative track of one animal per group during the BM test. (E) Escape latency during the MWM test. Significant differences during the (F) 5th day, (G) 8th day and (H) tenth day. (I) Representative track of one animal per group during the 10th day of MWM. (J) Time that each group spent in the area of the platform during the Probe test. (K) Heat maps of each group in the Probe test. Graph bars represent means ± SEM. *p < 0.05. **p < 0.01; ***p < 0.001.
3.2. Loss of spatial memory is observed in 18 month-old mice
The hippocampus is a crucial structure for spatial memory, associated with mental images that help to recognize characteristics of the environment [55]. For several years, researchers have shown that the loss of spatial memory is associated with aging [56]. Here, we evaluated spatial memory using the Barnes Maze (BM) (Supplementary Figure 1C) [46] and Morris Water Maze (MWM) task (Supplementary Figure 1D) [47] (Fig. 2). In the Barnes Maze test, the animals were exposed to a training phase, where animals had to find the location of a hole containing an escape chamber within 2 min (Fig. 2A and B). After 48 h of the last training session, animals had to find the location of the escape hole in the absence of the escape chamber (Fig. 2C and D). Our results showed that in the training stage, the 18mo animals took a longer time to find the escape chamber compared to 3, 6, and 12mo animals (Fig. 2A). The training track is observed in Fig. 2B. Finally, after 48 h the 18mo animals remembered the location of the escape chamber significantly less compared to other groups (Fig. 2C). This was also evident in the representative track (Fig. 2D). Therefore, in this test we observed that 3, 6, and 12mo mice learned and remember the spatial location of the escape chamber, in contrast to 18mo animals; suggesting that spatial memory is reduced with age, specifically at 18mo.
To validate this last observation, we used the MWM test, where each animal was placed 3 times per day in a pool to find the hidden escape platform guided by spatial cues, for 10 days. We observed that during the first 5 days of training 3, 6, and 12mo mice quickly learn the location of the hidden platform, in contrast to 18mo mice; nevertheless, it was also observed that the 6 and 12mo mice reduced their learning from day 3 of the MWM test (Fig. 2E). After a 48 h break, 3, 6, and 12mo mice remembered the location of the platform, meanwhile, 18mo mice had higher escape latency (Fig. 2E). Statistical analyses revealed that during the 5th day of training 3 and 12mo mice found the platform in less time than 18mo mice (Fig. 2F). Similarly, on the 8th day of training, 3, 6 and 12mo groups presented significant differences compared to 18mo mice (Fig. 2G). Also, it was possible to observe that 12mo mice spend more time to find the platform compared to the 3mo group, suggesting that the 48 h delay negatively affected the memory of 12mo animals (Fig. 2G). Interestingly, on the last day of training, all experimental groups showed significant differences compared to the 18mo group, which spent more time finding the hidden platform (Fig. 2H). Analyzing the track of the 10th day, we observed that the 3, 6 and 12mo animals showed a shorter path towards the platform than the 18mo group (Fig. 2I). Finally, on the 11th day, we performed the Probe test, which consisted of removing the platform to evaluate the time that the animals explored the platform zone. 3mo mice spent significantly more time in the platform area compared to 18mo animals (Fig. 2J). There was also a gradual reduction in the time spent exploring the platform area as age increased (Fig. 2J), which was shown by the heat maps of Fig. 2K. Thus, the MWM test also revealed an impairment in spatial memory at 18mo. Therefore, these results indicate an impairment of learning and spatial memory during aging, specifically at 18mo.
In summary, our behavior studies showed that hippocampal-dependent memory is affected with age; the object recognition memory was first impaired at 12mo; whereas the localization and spatial memory were affected at 18mo in this mouse background.
3.3. Synaptic mitochondrial dysfunction occurs before non-synaptic mitochondria in the hippocampus during aging
Due to the high energetic demand, mitochondria are fundamental for the functioning of hippocampal neurons [57,58]. Mitochondrial dysfunction contributes to aging-related alterations [14,59]. In the brain, there are at least two mitochondrial populations; which differ according to their origin [24,60]. Non-synaptic mitochondria originate from glial and neuronal cells; meanwhile, synaptic mitochondria are obtained exclusively from synaptic regions of the neuron (synaptosomes) [61]. Here, we evaluated the bioenergetics function and the calcium buffering capacity of synaptic and non-synaptic mitochondria from the hippocampus. We dissected the hippocampus of 3, 6, 12, and 18mo mice, and we isolated the synaptic (contained in synaptosomes) and non-synaptic mitochondria using a Percoll Gradient (Fig. 3A) [28]. We measured the bioenergetic function of the ETC of both mitochondrial populations, by measuring: i) ROS and ii) ATP production, 30 min after the addition of oxidative substrates [48,49]. Interestingly, when we evaluated ROS production after exposure to pyruvate-malate substrates in the synaptic mitochondria, we observed that 6mo mice showed a tendency to increase the amount of ROS compared to 3mo mice, an effect that is significant at 12mo; whereas 18mo mice did not present differences with 3mo (Fig. 3B). In contrast, non-synaptic mitochondria did not present significant differences in ROS production between all groups (Fig. 3C). To demonstrate whether these changes in ROS result in defects in ATP production, we evaluated ATP concentration in the medium of synaptic and non-synaptic mitochondria after exposure to pyruvate-malate substrates, using a bioluminescent assay. Surprisingly, we observed that the synaptic mitochondria obtained from 12 and 18mo mice had a significantly lower ATP production rate compared with 3mo animals (Fig. 3D); meanwhile, non-synaptic mitochondria only presented a significant reduction in 18mo animals (Fig. 3E). Altogether, these results indicate that both synaptic and non-synaptic mitochondria from the hippocampus reduce their bioenergetics function with age, but synaptic mitochondria fail prematurely at 12mo, generating an increase in ROS production and a deficit in ATP formation.
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