AD, the primary cause of dementia, begins in the temporal lobe and encroaches on all areas of the brain. Memory impairment is a major symptom because the hippocampus, the central organ involved in memory, is located in the temporal lobe. Similar to many diseases, treatment at the initial stage of AD is crucial. Various studies have shown that 3xTg-AD mice begin to develop deficits in short-term and long-term meomory, spatial learning and memory at 4 months , 6 months, and 6.5 months of age [23, 24]. Although these findings provide insight into the starting point of various cognitive deficits, they develop continuously over time . Consistent with previous studies, we detected the impairment of spatial learning, memory, and long-term memory in the AD group. These AD-related cognitive dysfunctions support the amyloid cascade hypothesis, in which disease onset is characterized by changes in Aβ, followed by a series of events, including the accumulation of toxic forms of tau, which induces apoptosis . Amyloid plaque deposition begins in the neocortex several years before the onset of symptoms and gradually spreads to the hippocampus, diencephalon, striatum, brainstem, and finally the cerebellum . In both the human brain and animal models, the expression of APP, presenilin mutations, and plaques, which are related to AD, are not only associated with synaptic loss but also with a deficiency of memory and synaptic plasticity [26–29]. Pathological forms of Aβ and tau as well as glia-mediated neuroinflammation play roles in synatptotoxicity .
Microglial cells and astrocytes exhibit changes in gene expression, morphology, and secretion in response to toxic stimuli in the brain, and these alterations affect other cells, including neurons . The activation of microglial cells and astrocytes in the early stage of AD results in microgliosis, astrogliosis, impairments in amyloid and tau removal, neurotoxin release, and pro-inflammatory cytokine release [31–35]. Previous studies have reported that the frequencies of GFAP-positive astrocytes and Iba-1 microglial cells are increased in the 3xTg-AD hippocampus at 6 and 10 months [36–38]. Microglial cells and astrocytes associated with amyloids secrete pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α . In various animal models of AD, the entire brain, including the hippocampus and hypothalamus, shows increases in IL-6 and TNF-α at the mRNA and protein levels [40–42]. In particular, IL-6 results in the release of a cascade of pro-inflammatory cytokines by microglia and astrocytes . Consistent with previous results, in this study, tau hyperphosphorylation and APP expression were higher, p-Akt and p-GSK3β in the hippocampus were lower, and Aβ-positive cells in CA1 were higher in the AD group than in the control group. These results suggested that the dysregulation of Tau and Aβ increased the expression of TNF-α and IL-6 by activating astrocyte and microglia in the hippocampus. As such, Aβ and tau and phosphorylation are associated with increases in pro-inflammatory cytokine levels via the activation of microglia and astrocytes and contribute to Ca2+ dysregulation , ROS , and cell death . Mitochondria provide an important buffer to regulate the calcium concentration during signaling, which is particularly important for excitatory cells, such as neurons .
Mitochondrial dysfunction related to Aβ, such as ROS release [47, 48] and the disruption of calcium homeostasis , is frequently observed in patients with AD and in animal models. The mPTP is associated with Aβ-induced mitochondrial dysfunction, such as a disturbance of intracellular calcium regulation, ROS generation, and the release of pro-apoptotic factors . In previous studies, mitochondria from mice with AD had much lower calcium capacities than those of non-transgenic mouse mitochondria, and the impaired Ca2+ uptake capacity, starting from 6 months, decreased gradually. This decreased Ca2+ retention capacity increased the expression of cyclophilin D (cyp-D), a mitochondrial matrix component, and mPTP components, such as voltage-dependent anion channel 1 (VDAC1) of the outer membrane [51, 52]. Adenine nucleotide translocator (ANT), a component of the inner membrane, binds to cyp-D, and cyp-D and ANT interact with Aβ. ANT strongly interacts with Aβ to change mPTP regulation and is related to mitochondrial dysfunction . Additionally, H2O2 is an important ROS that induces oxidative damage, and an increase in mitochondrial H2O2 is significantly associated with the onset of AD [51, 54]. Mitochondrial dysfunction, including increases in mPTP and ROS, can induce increased apoptosis. Increased levels of Cyp-D, VDAC1, and ANT increase apoptosis [51–53], and H2O2 penetrates all tissue compartments, triggers oxidative toxicity, and induces apoptosis . Mitochondria play an important role in the regulation of fundamental processes of neuroplasticity , and alterations in mitochondrial function are associated with changes in synaptic plasticity .
Mitochondrial dysfunction caused by Aβ may be related to neuroplasticity changes. Synapse-related proteins, such as BDNF, PSD95, and synaptophysin, in the hippocampus in the early stage, DCX, and neurogenesis are decreased in various AD animal models [58–62]. In this study, the Ca2+ retention capacity was lower and H2O2 emission was higher in hippocampal mitochondria in the AD group than in the control group, and mPTP-related proteins were overexpressed, indicating a decline in mitochondrial function. Furthermore, cell death increased, as determined by increases in Bax, a pro-apoptotic factor, cytochrome c, and cleaved caspase-3 and decreases in Bcl-2, an anti-apoptotic factor. Additionally, synapse-related proteins, such as BDNF, PSD95, and synaptophysin, as well as DCX and neurogenesis decreased. In patients with AD, decreased levels of BDNF in the blood and brain and impaired neurogenesis have been observed in the early stages of the disease with decreased cognitive function [63–65]. There is a positive correlation between the BDNF concentration and cognitive function . As such, it is believed that Aβ production or aggregation and tau phosphorylation, via direct or indirect pathways, affect neuroinflammation, mitochondrial function, apoptosis, and synapses in various brain regions, including the hippocampus, leading to cognitive decline.
Altered gamma oscillations have been observed in several brain regions in various neurological and mental disorders, including a decrease of spontaneous gamma synchronization in patients with AD and a decrease in gamma power in several AD mouse models [67–70]. Although gamma oscillations could not be measured in this study, they are associated with functional decline in AD, as established in an APP/PS1 model of AD . Gamma oscillations in the hippocampus are degraded according to the concentration and time of Aβ , and damaged mitochondrial function abolishes gamma oscillations in the hippocampal network . In a transgenic mouse model of AD, theta-gamma coupling was found to be defective in the subiculum, the major output area of the hippocampus . Recent work has shown that 40-Hz light flicker stimulation, a non-invasive treatment method, reduces Aβ and phosphorylated tau in various AD animal models by entraining gamma oscillations through the visual cortex. This improves spatial learning and memory by reducing the progression of the degenerative state of neurons, improving synaptic function, enhancing neuronal protective factors, reducing DNA damage, and reducing the inflammatory response of microglia [19, 20, 75].
Furthermore, 40-Hz auditory stimulation boosts hippocampal function by gamma entrainment, and combined visual and auditory stimulation improves cognitive function by reducing amyloid pathology . In this study, Aβ and phosphorylated tau in the hippocampus were attenuated in the 40-Hz light flicker group, as were Iba-1-positive microglial cells and GFAP-positive astrocytes. Long-term 40-Hz light flicker stimulation through the visual cortex stabilized gamma oscillations and alleviated Aβ, tau, and inflammatory responses in the hippocampus.
In the hippocampus, the mitochondrial Ca2+ retention capacity and alleviated H2O2 emissions may have improved cognitive function via the inhibition of apoptosis, increased neurogenesis, and improved neuroplasticity, including increased synaptic protein expression. Exercise, another non-invasive intervention, has a positive effect on brain function and is a candidate lifestyle intervention for reducing the incidence of dementia and AD . Physical exercise decreases Aβ and tau levels and increases memory, and high-quality exercise is associated with decreased Aβ in the plasma and brain of patients with AD [77–79]. Additionally, exercise is considered a powerful tool to prevent neuroinflammation and prevent the decline of cognitive function .
Liu et al.  showed that exercise increases the expression of Akt/GSK3β pathway members in the 3xTg-AD model, decreases Aβ deposition, and decreases Iba-1-positive microglial cells and GFAP-positive astrocytes in the hippocampal DG. Exercise at the early stage of AD has a protective effect on cognitive function by reducing Aβ plaques and GFAP-positive astrocytes in the hippocampus and increasing neurogenesis . Exercise with anti-inflammatory effects inhibits pro-inflammatory cytokines, such as TNF-α, IL-6, and IL1β, in the tau-transgenic hippocampus .
In this study, the AD exercise group showed increases in Akt/GSK3β pathway expression, decreases in tau and Aβ, and decreases in excessive Iba-1-positive microglial cells and GFAP-positive astrocytes, as well as decreases in TNF-α and IL-6 expression in the hippocampus. Exercise induces an antioxidant defense system, which reduces ROS levels, and adaptation to long-term regular exercise directly decreases ROS production, reduces oxidative damage , and may improve mitochondrial function. In brain regions, including the hippocampus, cortex, and cerebellum, increased mitochondrial Ca2+ accumulation and calcium-induced mPTP opening by exercise increased resistance to exercise and reduced apoptosis, suggesting that exercise has a protective effect against mitochondrial degeneration and cell death [84, 85]. Furthermore, exercise inhibits the overexpression of various membrane-related proteins, such as ANT1/2, cyp-D, and VDAC1, and H2O2, an ROS marker . Therefore, in an AD transgenic and systemic model, Bax, cytochrome c, caspase 3, and caspase 9 levels are decreased and Bcl-2 levels are increased after exercise [86, 87]. Brain mitochondrial function increases in a BDNF concentration-dependent manner , and BDNF expression increases in response to exercise [89, 90]. Neurotrophin is associated with learning, memory, neuronal activity, and plastic responses [91, 92]. Decreases in synapse-related proteins, such as PSD95 and synaptophysin, in the hippocampus in the early stage of AD are also increased by exercise [93, 94]. Exercise, especially through increased neurogenesis with decreased Aβ in the AD hippocampus and increased BDNF, improves cognitive function .
In the exercise group in this study, mitochondrial function improved with an increased Ca2+ retention capacity, decreased mPTP proteins, including ANT1/2, VDAC1, and cyp-D, decreased H2O2 emissions in the hippocampal mitochondria, and reduced cell death via decreased Bax, cytochrome c, and caspase-3 and increased Bcl-2. Additionally, BDNF, synaptophysin, PSD95, and neurogenesis were increased. In early AD, exercise seems to have a protective effect on cognitive function by improving the neuroplasticity of the mitochondria and hippocampus.
Under specific environmental conditions, such as 40-Hz light flicker, a non-invasive method, exercise seemed to have a greater effect. A limitation of this study was the inability to analyze gamma oscillations. However, under an environment where gamma oscillations are entrained through 40-Hz light flicker by the visual cortex and improve Aβ and tau pathology by inducing a variety of positive cellular changes, exercise may result in the stimulation of various brain regions via various myokines secreted from the muscles.