Cognitive–exercise dual-task intervention ameliorates cognitive decline in natural aging rats via inhibiting the promotion of LncRNA NEAT1/miR-124–3p on caveolin-1-PI3K/Akt/GSK3β Pathway

Aging-related cognitive impairment (ARCI) is rapidly becoming a healthcare priority. However, there is currently no excellent cure for it. Cognitive-exercise dual-task intervention (CEDI) is a promising method to improve ARCI, while the underlying mechanisms remain unclear. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are involved in the onset, development, and rehabilitation of ARCI. This study aimed to investigate the effects of CEDI and the role of regulation of the lncRNA NEAT1/miR-124-3p on the caveolin-1-PI3K/Akt/GSK3β pathway in CEDI improving cognitive function. Forty 18-month-old natural aging rats were randomly assigned to four groups: exercise training group, cognitive training group, CEDI group, and aging control group, and underwent 12 weeks of intervention. A novel object recognition test was performed to determine the cognitive function, and the hippocampus was separated three days after the behavioral tests for further molecular detection. In an in vitro study, the mouse hippocampal neuronal cell line HT22 was cultured. MiR-124-3p and lncRNA NEAT1 were over-expressed or down-expressed, respectively. The expressions of related proteins, lncRNA, and miRNA were examined by WB and/or qRT-PCR. The results showed that compared with the aging control group, the CEDI group had a higher discrimination index, and significantly decreased the expressions of lncRNA NEAT1, and the protein expressions of caveolin-1 and p-GSK3β, while significantly increased the expressions of miR-124-3p, and the protein expressions of p-PI3K and p-Akt. Inhibition of the lncRNA NEAT1 could significantly increase the protein expressions of p-PI3K and p-Akt in HT22 cells. Upregulation of miR-124-3p decreased the protein expressions of caveolin-1 and p-GSK3β, and increased the protein expressions of p-PI3K and p-Akt significantly. Inhibition of miR-124-3p had the opposite effects. Our study demonstrated that CEDI improved cognitive function in aging rats better than a single intervention. The mechanisms of cognitive improvement could be related to the regulation of the lncRNA NEAT1/miR-124-3p on the caveolin-1-PI3K/Akt/GSK3β pathway.

* Corresponding author at: Beijing Rehabilitation Hospital, Beijing Rehabilitation Medicine Academy, Capital Medical University, Beijing, China.E-mail address: gwj197104@ccmu.edu.cn(W.Gong). 1 These authors have contributed equally to this work and share the first authorship.
which impair the quality of life for the elderly and impose a heavy burden on families and society.Current pharmacological interventions for aging-related cognitive impairment (ARCI) are limited and less effective.A systematic review showed that drugs like cholinesterase inhibitors and memantine had no effect on reducing the risk of dementia in people with mild cognitive impairment (MCI), but might have adverse effects (Russ and Morling, 2012;Patnode et al., 2020).As a result, there is an urgent need for safer, easier, and more effective treatment methods.Evidence suggests that both exercise and cognitive training can help prevent and delay ARCI, as well as mitigate the negative effects of aging on cognitive function, and such simultaneous dual tasks are superior to certain single training for improving ARCI (Lauenroth et al., 2016;Joubert and Chainay, 2018;Brehmer et al., 2014).However, the underlying mechanisms of cognitive-exercise dual-task intervention (CEDI) to improve ARCI remain unclear.Thus, further research on CEDI is required to clarify the relevant mechanisms.
ARCI is associated with disruption of membrane lipid raft (MLR) structure and function, obstruction of cell membrane extension and protrusion, damage to long-term potentiation (LTP), reduction of synaptic plasticity, and an increase in neuroinflammation and oxidative stress in key brain regions such as the hippocampus (Maren et al., 2013;Fjell et al., 2014;Bradburn et al., 2019).Our group has found that CEDI could alleviate cognitive impairment in natural aging rats by activating the BDNF-TrkB signaling pathway, increasing GSH-Px and SOD activity, decreasing MDA content to reduce oxidative stress, and increasing neuroplasticity (Li et al., 2022a).Noncoding RNAs (ncRNAs) are emerging as important regulators of cognitive function.MicroRNAs (miRNAs) are a class of short, single-stranded ncRNAs, that are approximately 21-22 nucleotides in length, account for approximately 1% of all human genes, and are the most abundant class of small RNAs in animals.MiRNAs are post-transcriptional regulators of gene expression that modulate the expression of protein-coding genes (Bartel, 2004).Previous studies have shown that miRNAs could indirectly affect neurogenesis by regulating the proliferation and self-renewal of neural stem cells, which are key regulators of a variety of biological functions, including synaptic plasticity and neurogenesis (Grasso et al., 2014).For example, upregulation of miR-132 expression in the hippocampus and cortex could induce synaptic plasticity, which could thus be involved in learning novel tasks (Hansen et al., 2013).Brain-specific deletion of SIRT1 could increase the expression of miR-134 and decrease synaptic plasticity through the inhibition of CREB and BDNF (Gao et al., 2010).Although ncRNAs of different types are abundant in the central nervous system (CNS) and have complicated regulatory roles on brain neurons, synapses, and other chemicals (Earls et al., 2014), little is known about how ncRNAs change and impact learning and memory during aging.Therefore, one of the main goals of our research is to investigate the role ncRNAs in CEDI for improving ARCI and the underlying mechanisms.
NEAT1, a long non-coding RNA (lncRNA) identified on human chromosome 11, participates in the onset of disease by binding to various downstream sequences via the 3′ non-coding region of its nucleic acid sequence (Xie et al., 2019).A study reported that the lncRNA NEAT1 could impair memory development and maintenance by preventing histone methylation, which results in memory loss and exacerbates ARCI (Butler et al., 2019).However, the mechanism of interaction between the lncRNA NEAT1 and miR-124-3p in ARCI is unknown.As a result, whether this interaction could play a significant role in CEDI to ameliorate ARCI would be an important aspect of this research.
Caveolin-1, which makes up a small portion of the fossil membrane, is essential for the structure of mammalian cell microvesicles, and functions as a scaffolding protein in a variety of signal transduction pathways (Nwosu et al., 2016).It not only promotes the progression of cancer but is also linked to the pathogenesis of neurological illnesses (Cha et al., 2015).Increased expression of caveolin-1 may promote Alzheimer's disease (AD) progression by enhancing Tau protein-induced hyperphosphorylation of apoptotic cells (van Helmond et al., 2007;Ikezu et al., 1998), and caveolin-1 has been found to play a crucial role in PI3K/Akt pathway inactivation (Kang et al., 2017).Glycogen synthase kinase-3β (GSK3β) is a constitutive serine/threonine kinase that regulates cell division, cell differentiation, development, and apoptosis through many various signaling pathways (Maqbool et al., 2016).GSK3β, a downstream target of the PI3K/Akt signaling pathway and a major Tau kinase, regulates Tau phosphorylation and Aβ production in AD (Qi et al., 2017).Therefore, it seems reasonable to speculate that caveolin1-PI3K/Akt/GSK3β could be a downstream pathway of LncRNA NEAT1 acting on miR-124-3p.
In this study, we hypothesized that dual-task training improves cognitive function in natural aging rats by regulating the lncRNA NEAT1/miR-124-3p on the caveolin1-PI3K/Akt/GSK3β pathway and conducted the experiment to test this hypothesis.Our data suggested that dual-task training might be a safe and promising novel therapeutic strategy for the treatment or prevention of ARCI.

Animals
Forty 18-month-old Sprague-Dawley (SD) rats were purchased from Beijing Sibeifu Biotechnology Co., Ltd.(Beijing, China).Rats were housed in a room with a 12-hour light/dark cycle and free access to water and food.Each cage contained 5 rats of the same sex.The room temperature was controlled at 23-25 ℃.All of the animal experiments were approved by the Institutional Animal Research Ethics Committee of Beijing Rehabilitation Hospital (Approval No. 2021bkky018).

Experimental design
All rats were randomly divided into four groups (computerized random number generation): cognitive intervention (CI) group, exercise intervention (EI) group, dual-task intervention (DI) group, and aged control (AC) group, with 10 rats in each group, half male and half female.The four rat groups received treatment using the previously described procedure by our team (Li et al., 2022b).The males and females were trained one after another during the intervention period.To reduce hormonal interference, the males were trained first.The CI group performed attention box training, and the EI group performed running wheel training.The DI group performed both tasks simultaneously, while the AC group did not perform any tasks.Prior to the formal intervention, the rats in the CI, EI, and DI groups were placed in a Skinner box or running wheel for 10 min a day for one week, respectively, according to their subsequent intervention, to familiarize themselves with the field and training process.To stimulate the rats to feel hungry in order to find food once placed into the Skinner box, the rats in the CI group were subjected to a 20% caloric restriction at the start of CI.The rats in the CI group were placed in a Skinner box that measured mm in length, 265 mm in width, and 325 mm in height and trained for 10 min every time.On one wall, there was a pellet dispenser, and on the opposite wall, there were five nasal contacts with infrared probes and built-in indicator lights.The indicator light was turned on at random and automatically turned off after 10 s.The rats were trained to receive one food pellet if they touched the nasal contact within 10 s of the light being turned on.If they did not make contact with the nasal contact within s of the light being turned on, the light would go out and the rats would receive nothing.Rats in the EI group underwent a preparatory experiment before the formal EI.The preparatory experiment determined the appropriate exercise intensity for older rats.Briefly, three natural aging rats in each preparatory group were trained at a speed of 1, 2, 3, or circles per minute, respectively.The results showed that when the speed was too high, the rats were easy to be carried high by the wheel and easy to fall down, and they were easy to fatigue if trained for a long time.While the speed was too low, the expected training effect could not be achieved.Therefore, the speed of 2 circles per minute was determined.In the formal EI, individually seated on a running wheel with a diameter of 355 mm, the rats in the EI group underwent passive activity at a speed of 2 circles per minute for 10 min every time.For the rats in the DI group, 10-minute exercise training was administered first.Then the door between the Skinner box and the runner opened.The rats entered the Skinner box through a channel between the boxes and received another 10 min of cognitive training.The corresponding training methods for each were described above.The duration of each intervention was 12 weeks, once a day from Monday to Friday, with weekends off.The four rat groups were tested on their learning and memory skills using the novel object recognition (NOR) test after 12 weeks.The interventions continued until the end of the behavioral tests.The rats were euthanized by intraperitoneal injection of sodium pentobarbital (200 mg/kg) three days after the behavioral tests.The expression of relevant pathway proteins, lncRNA, and miRNA was then examined in the hippocampus using Western blotting (WB) or reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), respectively.

Novel object recognition test
A large open test box (830 mm long, 580 mm wide, and 510 mm high) was placed in a quiet room with a low-wattage light bulb.All rats were put into the empty test box in turn for 10 min to become familiar with the environment for 3 consecutive days.In the first part of the experiment (TI), two identical objects were placed in two corners on the same side of the test box, and the rat was allowed to explore for 5 min.In the second part of the experiment (T2), 1 h after the T1 phase, one object was replaced with another object of a different shape, and the rat was allowed to explore for 5 min.Effective detection was defined as pointing the nose at an object at a distance of less than 2 cm and/or touching the object with the nose.Turning around or sitting on the object was not considered an exploration.Animals with less than 6 s of total exploration time during T1 or T2 are excluded.In view of the actual exploration of animals in the T1 stage, DI = 1 was considered a preference and was excluded.The time to explore familiar and novel objects in T2 is denoted by F and N, respectively.Discrimination index = (N -F) / (N + F) × 100%.

Cell Culture and plasmid transfection
The HT22 mouse hippocampal neuronal cells were cultured in DMEM containing 1% double antibodies, and 10% FBS, at 37 ℃. 5% CO 2 was maintained in saturated humidity, and the cells were routinely passaged at approximately 80% cell density.The expression changes of proteins in the caveolin1-PI3K/Akt/GSK3β pathway were observed by transfecting shNEAT1, miR-124-3p mimics, and miR-124-3p inhibitors into neuronal cells to interfere with the expression of lncRNA NEAT1 and miR-124-3p, respectively.HT22 cells were spread at a density of 4 × 10 5 to 8 × 10 5 cells/cm 2 on a 10 cm cell culture dish and incubated in a 37 ℃ incubator with 5% CO 2 for 8-24 h.Transfection was started after complete cell wall attachment.Briefly, empty plasmid-pLKO.1-puro,interference plasmid, and lentivirus packing plasmid (12 µg lentivirus plasmid and 12 µg Lenti-Mix, where Lenti-Mix was composed of pMDLg/pRRE: pVSV-G: pRSV-Rev =5:3:2) were mixed into a 5 mL-EP tube, then 1 mL 0.25 M CaCl 2 was added, then another 1 mL 2 ×HBS was added, gently blown well with a gun after every addition operation, and maintained at room temperature for 10 min.Finally, the Lenti-DNA-Mix transfection system liquid was introduced drop by drop into the cell culture dish that needed to be transfected.After transfection, the medium was incubated in a 37 ℃ incubator with 5% CO 2 for 8-12 h.Then the medium was replaced with serum-containing medium pre-warmed at 37 ℃ and continued to incubate for 48 h.The lncRNA shNEAT1 and miR-124-3p mimics, and miR-124-3p inhibitor primer sequences are shown in Table 1.

Western Blotting
After collecting the cells and rat hippocampal tissue for lysis and centrifugation, the supernatants were collected, and the protein concentrations were detected using a BCA protein assay kit.Protein extracts were resolved by SDS-PAGE and transferred to PVDF membranes.The PVDF membranes were blocked at room temperature for 1 h, followed by a wash with TBST.The primary antibody diluted to the appropriate concentration was placed into the PVDF membrane overnight at 4 ℃, then washed 3 times with TBST and once with double-distilled water.Then, the PVDF membranes were incubated with a secondary antibody labeled with horseradish peroxidase (HRP) at room temperature for 1 h.Finally, the membranes were washed 5 times with TBST and once with double-distilled water before detection using a Western chemiluminescent HRP substrate.The protein levels were analyzed using ImageJ software.The primary antibodies are Anti-caveolin-1 (1:1000; Abcam; England; ab32577), Anti-P-Akt (1:000; Cell Signaling Technology (CST); USA; 40160), Anti-P-PI3k (1:000; Affinity; USA; AF4372), and Anti-P-GSK3β (1:1000; CST; USA; 5558 S).

Reverse Transcription Quantitative Real-time Polymerase Chain Reaction (RT-qPCR)
Total RNA was extracted from cells and rat brain tissue and reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit.The cDNA obtained by reverse transcription, specific primers (Table 2), and 2 × UltraSYBR Mixture were used in a 20 µL reaction system according to 10 µL 2 × UltraSYBR Mixture, Primers (2 µL Tabe (F/R mix) (0.2 µM), and 8 µL cDNA.Reaction conditions were: 10 min at 95 • C, then 40 cycles of 15 s at 95 • C, 20 s at 60 • C, 25 s at 72 • C, and finally 5 min at 72 • C.

Statistical analysis
The data were analyzed by IBM SPSS Statistics 20 and expressed as the mean ± SEM.The normality of the data was determined using Shapiro-Wilk normality tests.If the data fit a normal distribution, a oneway ANOVA was used.The LSD post-hoc test was then performed.Otherwise, a non-parametric Kruskal-Wallis test was applied.When p < 0.05, differences were deemed significant.

Effects of CI, EI, and DI on cognitive function in natural aging rats
The NOR test was performed to investigate the effects of CI, EI, and DI on cognitive function in natural aging rats.The discrimination index of rats in each group without differentiating the sex [F (3, 36) = 4.358] was shown in Fig. 1A.The discrimination index of the rats in the DI group was significantly increased compared with that of the rats in the AC (P < 0.01) and CI groups (P < 0.05), indicating that rats in the DI group had a better ability to recognize old and new objects compared with rats in the AC and CI groups.The discrimination index of the rats in the EI group was also significantly higher compared with that of the rats in the AC group (P < 0.05).As shown in Fig. 1B [male: F (3, 16) = 3.272, female: F (3, 16) = 1.547], the discrimination index of the rats in the DI group was still significantly higher than that of the rats in the AC (P < 0.05) and the CI groups (P < 0.01) for male rats but not significantly different from that of the EI group, while the discrimination index of the rats in the EI group was not significantly different from that of the remaining three groups, and the difference between the EI and CI groups approached significance (P = 0.064).There was no significant difference in the discrimination index among the groups for female rats.

Effects of CI, EI, and DI on LncRNA NEAT1/miR-124-3p in natural aging rats
We assessed the expression of the lncRNA NEAT1 and mi-124-3p in the hippocampus of the rats in each group.As shown in Fig. 2, the expression levels of the lncRNA NEAT1 in the hippocampus of rats in the DI group were significantly reduced compared with those of the rats in the AC group (P < 0.05).The expression levels of the lncRNA NEAT1 in the EI and CI groups were not significantly different from the other intervention groups [Fig.2A, total: F (3, 30) = 2.050].The expression of lncRNA NEAT1 in male rats was not significantly different among groups, while that in female rats was significantly lower in the DI, EI, and CI groups compared with the AC group (P < 0.05) [Fig.2B, male: F (3, 13) = 1.457, female: F (3, 13) = 13.045].The expression of miR-124-3p was significantly increased in the DI group compared with that in the AC group (P < 0.01).The expression of miR-124-3p was significantly higher in the EI group compared with that in the AC group (P < 0.05) [Fig.2C, total: F (3, 32) = 3.872].The expression of miR-124-3p was significantly higher in all intervention groups for male rats compared with that in the AC group (P < 0.05), with the most significant increase in the DI group (P < 0.01).In contrast, there was no significant difference between the female groups [Fig.2D

Effects of CI, EI, and DI on caveolin-1-PI3K/Akt/GSK3β in Natural Aging Rats
A previous study reported the relationship between miR-124-3p and the caveolin-1-PI3K/Akt/GSK3β pathway (Kang et al., 2017), thus the expression of the related proteins in the caveolin-1-PI3K/Akt/GSK3β pathway was further detected using WB assays (Fig. 3).We found that the protein expression level of caveolin-1 in the rats was significantly decreased in the DI group compared with that of the rats in the AC and CI groups (P < 0.05), but not significantly different from that in the EI group [Fig.3A, total: F (3, 36) = 2.510].The protein expression of caveolin-1 was significantly lower in the DI group compared with that in the CI group for male rats (P < 0.01) and in the DI group compared with the AC group for female rats (P < 0.05) [Fig.3B, male: F (3,16) = 3.145, female: F (3,16) = 3.163].The protein expression of p-PI3K was significantly increased in the DI group compared with that in the AC, EI, and CI groups (P < 0.001) [Fig.3C, total: F (3, 36) = 14.355].Similarly, the protein expression of p-PI3K in the DI group was significantly higher (P < 0.01) compared to the other three groups for both females and males [Fig.3D, male: F (3,16) = 6.270, female: F (3,16) = 8.326].Compared with the AC group, the protein expression of p-Akt was significantly higher in the DI, EI, and CI groups, with the most significant increase in the DI group (P < 0.001), and there was also a significant difference between the DI group and the EI or CI groups (P < 0.05) [Fig.3E, total: F (3, 36) = 7.016].The protein expression of p-Akt was significantly higher in the DI group compared with other groups for male rats (EI, CI: P < 0.05; AC: P < 0.01).For female rats, the protein expression of p-Akt in the DI group was significantly different from that in the AC group only (P < 0.05) [Fig.3F, male: F (3,16) = 5.663, female: F (3,16) = 2.000].The protein expression of p-GSK3β was significantly lower in the DI group compared with other groups (AC: P < 0.05; EI, CI: P < 0.01) [Fig.3G, total: F (3, 36) = 4.026].The protein expression of p-GSK3β was significantly lower in the DI group compared with the CI group in male rats (P < 0.01), while for female rats, the protein expression of p-GSK3β was significantly lower in the DI group compared with the other groups (P < 0.01) [Fig.3H, male: F (3,16) = 2.556, female: F (3,16) = 2.704].

Inhibition of LncRNA NEAT1 Prevents the Expression of caveolin-1-PI3K/Akt/GSK3β Pathway
To further clarify the role lncRNA NEAT1 played in the caveolin-1-PI3K/Akt/GSK3β pathway, we inhibited the expression of lncRNA NEAT1 in HT22 mouse hippocampal neuronal cells.WB was performed to detect related pathway proteins (Fig. 4E), and the results showed that the expression of p-PI3K and p-Akt proteins in the shNEAT1 group was significantly increased (P < 0.05) compared with the control group (Fig. 4B, C), while the protein expression of caveolin-1 and GSK3β was significantly decreased (P < 0.05) (Fig. 4A, D).

Regulation of miR-124-3p disturbs the expression of caveolin-1-PI3K/Akt/GSK3β pathway
In our study, the expression of related proteins in the caveolin-1-PI3K/Akt/GSK3β pathway in HT22 mouse hippocampal neuronal cells was detected using WB to investigate the role of miR-124-3p in this pathway.The results showed that the protein expression of caveolin-1 and p-GSK3β was significantly lower in the miR-124-3p mimics group (P < 0.05) compared with the empty vector (Fig. 5 A, D), while it was significantly higher in the miR-124-3p inhibitor group (P < 0.05) compared with the empty vector (Fig. 5 A, D).The protein expression of p-PI3K and p-Akt was significantly higher in the miR-124-3p mimics group compared with the empty vector group (P < 0.05) (Fig. 5 B, C), and significantly lower in the miR-124-3p inhibitor group (P < 0.05) (Fig. 5 B, C).

Discussion
ARCI has a significant negative impact on the quality of life for the elderly and places a great burden on families and society.In this study, the effects of CEDI on ARCI and the underlying mechanisms were investigated.The results showed that CEDI could significantly improve the cognitive performance of natural aging rats in the NOR test.CEDI improved cognitive performance and caveolin-1-PI3K/Akt/GSK3β pathway significantly better than single CI and/or EI interventions.According to the current work, the influence of lncRNA NEAT1/miR-124-3p on the caveolin-1-PI3K/Akt/GSK3β pathway may be responsible for the enhancement of cognitive performance in rats following CEDI.
Several studies have shown that the lncRNA NEAT1 regulates the expression of miR-124-3p in a sequence-specific and binding-dependent manner (Pang et al., 2019;Zhao et al., 2022;Cui et al., 2019;Zhou et al., 2022;Yuan et al., 2022).MiR-124-3p expression was significantly increased after knockdown of lncRNA NEAT1, and miR-124-3p expression was significantly decreased after overexpression of lncRNA NEAT1 (Liu et al., 2018).In the present study, the expression of the lncRNA NEAT1 was significantly decreased in the DI group of rats, whereas the expression of miR-124-3p was significantly increased.Our previous study confirmed the protection of neuroplasticity and inhibition of oxidative stress in rats by dual-task training (Li et al., 2022a), and miR-124-3p plays an important role in the protective properties of the CNS, such as inhibition of neuroinflammation and improvement of synaptic plasticity (Liu et al., 2021;Garcia et al., 2022).
Caveolin-1, a multifunctional membrane lipid raft (MLR) scaffolding protein, is involved in numerous signaling pathways and plays a complex role in cognitive decline in aging.Numerous studies have explored the molecular mechanisms of caveolin-1 in different cognitive decline diseases.Numerous studies have demonstrated that caveolin-1 can safeguard cognition by fostering neurogenesis and restoring the structural and functional flexibility of neurons.By modifying the caveolin-1/ VEGF pathway in ischemic areas, treadmill exercise may aid in the restoration of neurological function after a stroke (Pang et al., 2017).In both the adult and aged hippocampus, neuronal overexpression of caveolin-1 would enhance MLR function and improve hippocampal function and neuronal structural plasticity (Mandyam et al., 2017), and loss of caveolin-1 in young animals would accelerate aging and neurodegeneration (Park et al., 2003).Contrary to the aforementioned evidence, caveolin-1 was upregulated with aging in the hippocampus, cerebral cortex, and elderly cerebral cortex of aged rats (Kang et al., 2006), and caveolin-1 deficiency ameliorates cognitive deficits in some aging-related diseases associated with cognitive decompensation.The possible explanation for this discrepancy was that caveolin-1 functions in various pathogenetic mechanisms in various diseases (Tang et al., 2021).It has been shown that caveolin-1 is linked to amyloid precursor protein (APP) in aging neurons, that its overexpression causes APP processing through secretase and results in upregulated APP secretion involved in AD progression, and that it is even possible to gauge the rate of AD progression in elderly individuals by measuring fossa protein-1 expression (Kang et al., 2006).Therefore, a plausible explanation is that as the degree of aging increases and cognitive function decreases, the portion of cognitive impairment due to caveolin-1 in the progression to AD becomes increasingly important.In our in vivo study, the protein expression of caveolin-1 was indeed somewhat elevated, but there was no significant difference compared to the AC group.We hypothesized that inactivity in naturally aged rats might affect the effect of CI.
MiR-124-3p has neuroprotective functions, such as suppressing neuroinflammation and promoting axonal recovery (Xl et al., 2022), and may be involved in the long-term regulation of hippocampal gene expression networks (Vuokila et al., 2018).Caveolin-1 is negatively regulated by miR-124-3p (Butz et al., 2015) and is involved in the inactivation of the PI3K/Akt pathway.The PI3K/Akt signaling pathway enhances cell survival and can protect brain cells.Swimming exercise has been shown to prevent aging-induced apoptosis and inflammatory Fig. 3. Effects of CI, EI, and DI on caveolin-1-PI3K/Akt/GSK3β in Natural Aging Rats.(A, C, E, G) Differences in caveolin-1-PI3K/Akt/GSK3β pathway protein expression among groups without differentiating between sex.(B, D, F, H) Differences in protein expression of caveolin-1-PI3K/Akt/GSK3β pathway between male and female rats after differentiation of sex.(I-J) Representative immunoblotting of caveolin-1-PI3K/ Akt/GSK3β pathway proteins in hippocampal tissue of rats in each group.Data for different sexes were analyzed separately.Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. the AC group; # P < 0.05; ## P < 0.01; ### P < 0.001 vs. the DI group.AC , aged control; CI, cognitive intervention; EI, exercise intervention; DI, dual-task intervention.
signaling activity by boosting the brain IGF1/PI3K/Akt pathway to inhibit the overexpression of inflammatory pathway markers (Bliss and Collingridge, 1993).Exercise also raises SIRT1 activity in the hippocampus and increases its expression in several brain regions, such as the cortex, hippocampus, and hypothalamus (Gerlai, 2001;Murai and Pasquale, 2004).In this investigation, we discovered that CEDI dramatically decreased the protein expression of caveolin-1 and p-GSK3 in natural aging rats, whereas single-task training had no discernible impact on the expression of these genes when compared to untrained rats.Dual-task training significantly increased the PI3K/Akt pathway's activity compared to exercise training alone and cognitive training alone.Dual-task training was more neuroprotective than single-task training, according to behavioral outcomes and pathway protein expression.
To confirm that the caveolin1-PI3K/Akt/GSK3β pathway was indeed regulated by lncRNA NEAT1 and miR-124-3p, we performed in vitro experiments on cultured hippocampal neurons and found that silencing lncRNA NEAT1 significantly decreased the protein expression of caveolin-1 and p-GSK3β while significantly increasing the protein expression of p-PI3K and p-Akt.We discovered that upregulating miR-124-3p expression promoted p-PI3K and p-Akt protein expression while inhibiting caveolin-1 and p-GSK3β protein expressions.Downregulating miR-124-3p had the opposite effect.The findings suggest that the action of lncRNA NEAT1 on the caveolin1-PI3K/Akt/GSK3β pathway may be one of the mechanisms underlying decreased neuroplasticity and inflammatory response activation in the rat hippocampus.Unlike many previous studies, lncRNA NEAT1, a cancer-associated lncRNA, has been shown to activate the PI3K/Akt pathway in many tumor-related studies (Zhang et al., 2019).For example, lncRNA NEAT1 is involved in the development of multiple myeloma by promoting cell proliferation through activation of the PI3K/Akt pathway (Xu et al., 2018).LncRNA NEAT1 also activates the PI3K/Akt signaling pathway and regulates epithelial-mesenchymal transition (EMT) through the miR-186-5p/PTP4A1 axis, which is involved in cell proliferation, migration, and invasion in cholangiocarcinoma (Li et al., 2021).Overexpression of lncRNA NEAT1 enhanced the viability and migration of gastric cancer cells through upregulation of miR-17 while activating the PI3K/Akt and GSK3β pathways (Wang et al., 2018).As far as the results of this study were concerned, inhibition of the lncRNA NEAT1 in the hippocampal tissue of aging rats promoted the PI3K/Akt pathway and enhanced its neuroprotective effects.One possible explanation for this opposite scenario was that miR-124-3p might play a key role in the regulation of the PI3K/Akt pathway by lncRNA NEAT1 in the CNS.Inhibiting the lncRNA NEAT1 reduced inhibition of miR-124-3p while inhibiting its direct target, caveolin-1, resulting in reduced inactivation of the PI3K/Akt pathway and inhibition of GSK3β expression.Therefore, the results of this experiment demonstrated that dual-task training could improve cognition in aging rats by inhibiting the lncRNA NEAT1 and thus reducing the inhibition of miR-124-3p and activating the PI3K/Akt/GSK3β pathway.
It should be noted that in the in vivo experiment, we also analyzed the differences in indicators between each intervention group by sex separately.The results showed that the discrimination index, miR- 124-3p and p-Akt of male rats, lncRNA NEAT and p-GSK3β of female rats, and p-PI3K of both sexes were significantly consistent with those of no differentiation of sex.These sex differences may be due to differences in baseline cognitive function.Studies have reported that in new object recognition tasks, while both sexes prefer familiar objects, females learn spatial tasks more frequently, showing longer decision delays and faster error correction (Wallace and Hofmann, 2021).This suggests that there may be differences in search or memory strategies between the sexes, which requires us to further refine the analysis of behavioral indicators.Furthermore, sex differences in physiology and morphology may become apparent in specific responses or degrees of response to various training regimens, such as oxygen carrying ability (Lewis et al., 1986), which may lead to different outcomes and require more personalized interventions in further studies.Sex may also play a key role in the molecular heterogeneity of diseases associated with neurological disorders (Wang et al., 2017).Recent studies reported that changes in p-GSK3β showed a sex-dependent effect on cognitive decline induced by cerebral microhemorrhages (Barus et al., 2021).In addition, this discrepancy in results is also likely due to the small sample size of each group in the case of the inclusion of both sexes and the possible influence of the sexual cycle of the female animals (Yuen et al., 2016), which is also a major shortcoming of our study.We hope that more sample sizes can be included in future studies for further analysis and verification.
This study showed that CEDI was more effective than a single training modality in improving cognitive performance in aging rats.This effectiveness may be attributed to the inhibition of the promotion of lncRNA NEAT1/miR-124-3p on the caveolin-1-PI3K/Akt/GSK3β Pathway.By examining the role of lncRNA-miRNA interactions in cognitive function, our findings provide a deeper perspective on dual training to ameliorate aging cognitive decline.It would aid in the search for a more widely used general intervention that is safe, efficient, and easy for the treatment or prevention of ARCI in older adults.

Conclusion
We used in vivo and in vitro experiments to show that CEDI improved cognitive function in aging rats.Especially, CEDI improved cognitive performance and the caveolin-1-PI3K/Akt/GSK3β pathway significantly better than single CI and/or EI interventions.CEDI could upregulate miR-124-3p by inhibiting the expression of the lncRNA NEAT1, reducing the expression of caveolin-1, p-GSK3β, and increasing the expression of p-PI3K and p-Akt to exert neuroprotective effects, which may be one of the important mechanisms of cognitive function improvement in aging rats.

Fig. 1 .
Fig. 1.Effects of CI, EI, and DI on Cognitive Function in Natural Aging Rats.(A) The discrimination index of different groups.(B) The discrimination index of rats of different sexes.Data for different sexes were analyzed separately.Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01 vs. the AC group; # P < 0.05 vs. the DI group.AC, aged control; CI, cognitive intervention; EI, exercise intervention; DI, dual-task intervention.

Table 1
Primers used for reporter gene vector.

Table 2
Primer sets used for qPCR.