Trifluoperazine upregulates ACSS2-related autophagy and promotes cerebral cognitive function in rats after sleep deprivation

Summary

Cognitive function impairment following sleep deprivation (SD) can induce significant aftereffects. Elevated calmodulin (CaM) expression following stroke causes calcium overload—a key contributor to cognitive function impairment. Trifluoperazine (TFP), a CaM inhibitor, reduces CaM overexpression following ischemic stroke. However, it remains unclear whether TFP has influences on cognitive function impairment following SD. We administered TFP to rats subjected to SD. TFP treatment in SD rats reduced cerebral CaM expression and alleviated cognitive function impairment. Improved cognitive function was coincident with increased CaM protein levels and reduced acetyl-CoA synthetase 2 (ACSS2) protein levels after SD. TFP treatment reversed these changes. Our results showed that TFP administration in rats inhibited CaM protein following SD by upregulating ACSS2 protein expression, thereby improving ACSS2-related autophagy and alleviating cognitive function impairment. Consequently, this treatment may promote cerebral cognitive function recovery after SD.

Graphical abstract

Highlights

• •CaM is implicated in the pathogenesis of cognitive impairment in rats following SD
• •TFP attenuates cognitive impairment in sleep-deprived rats
• •ACSS2-associated autophagy is implicated in SD-induced cognitive impairment

Behavioral neuroscience; Cognitive neuroscience; Molecular biology; Neuroscience; Sensory neuroscience

Introduction

Sleep and sleep deprivation

Sleep is a natural and cyclical fundamental activity, characterized by a complex and dynamically changing state.¹ This process encompasses multiple stages, each exhibiting distinct patterns in brain waves, eye movements, and bodily functions, along with unique electroencephalogram (EEG) patterns and physiological characteristics.^1,2 Sleep deprivation (SD) is a prominent and widespread issue in modern society.³ Chronic SD is defined as obtaining 6 h of sleep or less per night over the past 4 weeks. SD leads to excessive daytime sleepiness in 9%–24% of individuals, with over 20% of adults estimated to experience chronic SD.⁴ Epidemiological surveys in China indicate a prevalence of chronic insomnia of 9.3% among young adults, with rates increasing progressively with age, reaching 38.2% among the elderly. In the United States, 35% of adults sleep less than 7 h within a typical 24-h period, 73% of high school students report insufficient sleep (<8 h per night), and 58% of middle school students report sleeping less than 9 h.⁵ SD may impair learning capacity and the ability to form new memories, particularly affecting the consolidation of hippocampus-dependent memory tasks.⁶ Accumulating evidence suggests that SD may induce neuroinflammation, oxidative stress, alterations in synaptic plasticity, neurotransmitter systems, gene expression, and protein synthesis, thereby contributing to cognitive and memory declines.⁷ SD not only adversely affects multiple systems, including the cardiovascular, nervous, immune, and endocrine systems,^8,9,10 but also increases the risk of the development of sleep disorders, mild cognitive impairment, and Alzheimer disease (AD). Concurrently, SD exerts significant negative impacts on cognitive function, with numerous studies demonstrating its effects on memory, attention, alertness, and other cognitive domains.^{11,12,13,14,15} Recent studies by Holth, Benedict, and colleagues, utilizing clinical insomnia cases and animal models of SD to simulate insomnia, have also revealed that SD leads to elevated levels of β-amyloid and tau proteins in cerebrospinal fluid and plasma.^16,17 Given the severity of cognitive impairment induced by SD and its associated risk of increasing dementia prevalence, exploring the mechanisms underlying SD-induced cognitive dysfunction is of paramount importance. Such exploration will provide a theoretical foundation for the prevention and treatment of SD-induced impairments in learning and memory, as well as the development of relevant therapeutic agents.¹⁸

Hippocampus in SD

In recent years, a growing body of research has highlighted the pivotal role of the hippocampus in sleep disorders and cognitive dysfunction, with close ties to the initiation and maintenance of sleep.^19,20 As a critical neural structure within the brain, the hippocampus plays an indispensable role in memory formation, spatial perception, and emotional regulation. During the induction and maintenance of sleep, the hippocampus modulates neuronal firing patterns and synaptic transmission, facilitating memory consolidation and clearance processes that ultimately influence sleep quality and duration.²¹ Furthermore, studies have demonstrated that SD disrupts neural signal transmission and synaptic plasticity within the hippocampus, thereby impairing memory function. Notably, the hippocampal CA1 region holds particular significance in memory consolidation and retrieval. It receives informational output from areas such as the CA3 region and maintains extensive connections with other brain regions, including the prefrontal cortex. This connectivity enables the integration of hippocampal memory traces with functional outputs from other brain areas, thereby supporting memory expression and application. In the context of episodic memory, the CA1 region facilitates the matching and association of contextual information (e.g., scenes and events) stored in memory with present circumstances. Additionally, the CA1 region is equally critical for spatial memory, participating in the construction of spatial location and navigation-related memories. By integrating and processing information from diverse sources, it enables the brain to form coherent memory representations and cognitive maps, thus comprehensively supporting memory formation and information processing functions.²²

CaM in SD

Calmodulin (CaM), encoded by the CALM1 gene, is a ubiquitous Ca²⁺ signaling sensor present in various eukaryotic cells and serves as the most rapidly Ca²⁺-binding receptor highly expressed in the brain. CaM facilitates Ca²⁺ influx rates and modulates Ca²⁺-related proteins, imposing a greater calcium overload burden on neuronal cells and thereby accelerating neuronal damage.²³ Nuclear Ca²⁺ transients that initiate DNA transcription may hold significant implications for learning and memory. However, more intense or sustained neuronal stimulation can overactivate nuclear Ca²⁺ regulatory processes, triggering inappropriate gene expression and leading to neurodegeneration.²⁴ CaM can fuse with multiple Ca²⁺-binding proteins to form diverse Ca²⁺ complexes/CaM complexes, including the Ca²⁺-CaM complex. In the brain, Ca²⁺/CaM-activated adenylate cyclase activity and the subsequent production of cyclic adenosine monophosphate (cAMP) are involved in memory acquisition and retention. cAMP enhances the phosphorylation of Ca²⁺ channels by activating protein kinase A (PKA), disrupting feedback control over Ca²⁺ influx. This results in sustained Ca²⁺ entry into neuronal cells, causing Ca²⁺ overload, neurodegeneration, and memory loss. Concurrently, studies have shown that domoic acid induces brain injury and central nervous system dysfunction by causing Ca²⁺ overload and reducing cAMP levels.^25,26 Furthermore, CaMKII, a key molecule in the CaM/CaMKII signaling pathway, is phosphorylated and activated by activated CaM. It serves as a critical regulator for maintaining long-term potentiation (LTP) and synaptic plasticity in the hippocampus, acting as a well-established molecular substrate for memory storage. CaMKII participates in learning and memory formation through multiple pathways and mechanisms.^27,28 The aforementioned evidence suggests that the Ca²⁺/CaM/CaMKII signaling pathway may be involved in SD-induced cognitive impairments, particularly in learning and memory. Further investigation into the Ca²⁺ and CaM signaling pathways, along with their associated enzymatic processes, holds substantial significance. Such research could facilitate the development of therapeutic strategies for neurodegenerative diseases and central nervous system dysfunction, particularly addressing deficits in memory and LTP.

ACSS2 in SD

Acetyl-CoA synthetase 2 (ACSS2) is a crucial member of the acetyl-CoA synthetase family²⁹ and is widely distributed across various human tissues. ACSS2 exhibits a dual role as both a fatty acid synthesis enzyme and a stress response regulator.³⁰ Under conditions of sufficient energy, ACSS2 promotes lipid synthesis and storage. Conversely, during ketogenesis triggered by food deprivation or exposure to stressors such as injury, hypoxia, and infection, ACSS2 induces fatty acid oxidation and autophagy to help maintain energy homeostasis. In the adult rat brain, ACSS2 is primarily localized in the nucleus. Reduced levels of ACSS2 in the hippocampus lead to the downregulation of memory-related neuronal genes, thereby affecting LTP. The LTP of excitatory synaptic transmission constitutes a fundamental component of the cellular substrate for memory,³¹ suggesting that ACSS2 may play a potential role in memory consolidation by influencing LTP. ACSS2 contributes to hippocampal memory through multiple mechanisms, most notably by promoting histone acetylation, which facilitates the expression of a series of downstream genes involved in memory consolidation. However, acetyl-CoA generated by ACSS2 for acetate synthesis not only provides acetyl groups for histone acetylation but also supplies acetyl groups to other nuclear proteins, such as hypoxia-inducible factor 2α (HIF-2α),^32,33 transcription factor EB (TFEB),³⁴ and interferon regulatory factor 4 (IRF4),³⁵ all of which play significant roles in memory. This suggests the possible involvement of additional mechanisms or ACSS2-related regulatory factors in memory function. TFEB^36,37 is a protein that plays a pivotal role in various physiological processes, including cellular metabolism and autophagy. It promotes autophagosome formation and lysosomal biogenesis. Genes involved in the autophagy-lysosome pathways are critical for regulating lysosomal biogenesis and autophagy.³⁸ TFEB translocates to the nucleus with the assistance of importin 8 (IPO8), where it specifically binds to nuclear ACSS2 to form a complex. This complex then binds to the promoter regions of lysosomal and autophagic genes (i.e., TFEB target genes), promoting the expression of key factors (such as CLEAR network genes, RRAGC, UVRAG, CSTB, M6PR, and IGF2R) that enhance lysosome-related gene expression.³⁹ Upregulation of these genes strengthens lysosomal functions and promotes autophagy. Autophagy plays a significant role in synaptic plasticity, dendritic neurons, and cultured neurons⁴⁰ and negatively regulates axonal extension.⁴¹ Given the extensive link between synaptic plasticity and memory,^42,43 the upregulation of these genes may inhibit synaptic plasticity and memory function by promoting autophagy, leading to impaired memory consolidation.⁴⁴ Additionally, UV radiation resistance-associated gene (UVRAG) is a key regulator in autophagosome formation, mediating the maturation of autophagosomes.⁴⁵ It binds to Beclin 1, enhancing the activity of the Beclin 1-Vps34 complex, which is essential for the nucleation and elongation of autophagosomal membranes, thereby facilitating autophagosome formation. UVRAG also regulates autophagic flux—the entire process of autophagosome formation, maturation, and fusion with lysosomes.⁴⁶ Autophagy is a self-degradative process induced under various forms of stress. The autophagy-lysosome degradation pathway plays a crucial role in adapting to metabolic stress, clearing dangerous cargo (such as protein aggregates, damaged organelles, and intracellular pathogens), facilitating renewal during differentiation and development, and preventing genomic damage.⁴⁷ However, the specific relationship between ACSS2-related autophagy and memory consolidation in human neurons remains unclear. ACSS2-related autophagy factors may play a significant role in SD-induced cognitive impairment, warranting further investigation.

Based on support from domestic and international literature, we hypothesize that CaM plays a critical role in SD-induced cognitive impairment and is also involved in neuronal autophagy. This study aims to determine the efficacy of trifluoperazine (TFP), a CaM inhibitor, in cognitive impairment induced by SD and to clarify its underlying biological mechanism.

Results

Cognitive impairment is induced in rats after SD

To investigate the effects of SD on cognitive functions, including learning and memory, in rats, we established an SD model in rats, using the modified multiple platform method. The rats were allowed to sleep for 4 h from 8:00 to 12:00 each day and were subjected to 20 h of SD from 12:00 noon to 8:00 the next morning, continuously for 21 days. After the model was established, we employed the Morris water maze, Y-maze, and novel object recognition tests to evaluate the changes in cognitive functions of rats following SD. We observed that compared with the control group (Con group), rats in the SD group showed statistically significant differences in escape latency in the Morris water maze test (p < 0.05). Specifically, differences in escape latency emerged from the third day of the place navigation trial, with the escape latency of rats in the SD group being significantly prolonged (Figure 1A). During the spatial probe trial of the Morris water maze, compared with the Con group, rats in the SD group exhibited a significant decrease in the number of effective entries (p < 0.05) (Figure 1C) and the number of crossings over the original escape platform (p < 0.05) (Figure 1B). Meanwhile, compared with the Con group, rats in the SD group demonstrated significant differences in the novel object recognition index in the novel object recognition test (p < 0.05) (Figure 1D) and in the spontaneous alternation rate in the Y-maze test (p < 0.05) (Figure 1E). The above results indicated that SD leads to cognitive impairment in rats.

Figure 1.

Cognitive impairment is induced in rats after SD

(A) Escape latency from the first to the fifth day of the Con group and the SD group in Morris water maze test.

(B) Number of entries into the escape platform of the Con group and the SD group in Morris water maze test.

(C) Number of entries into the effective area of the Con group and the SD group in Morris water maze test.

(D) Novel object discrimination index of the Con group and the SD group in novel object recognition test.

(E) Spontaneous alternation rate of the Con group and the SD group in Y-maze test.

Data are represented as the mean ± SEM (n = 6 per group). ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01.

Upregulated expression of CaM and decreased expression of ACSS2 protein in hippocampal CA1 region of sleep-deprived rats

To investigate whether CaM and ACSS2 proteins are involved in the cognitive dysfunction of sleep-deprived rats, we selected hippocampal tissue samples from rats that exhibited the most pronounced cognitive-behavioral alterations on the basis of the number of escape platform crossings and effective area entries in the Morris water maze test after 21 consecutive days of SD. Specifically, we isolated hippocampal CA1 region tissues from these samples, extracted proteins, and conducted western blot (WB) experiments to detect and analyze differences in the expression levels of CaM and ACSS2 proteins in rats with SD-induced cognitive impairment. Compared with the Con group, the western blot results revealed that CaM expression was upregulated (p < 0.05) (Figures 2A and 2B) and ACSS2 protein expression was downregulated (p < 0.05) (Figures 2C and 2D) in the hippocampal CA1 region of rats with SD-induced cognitive impairment. These findings suggest that CaM and ACSS2 proteins are implicated in the cognitive dysfunction caused by SD in rats.

Figure 2.

Upregulated expression of CaM and decreased expression of ACSS2 protein in hippocampal CA1 region of sleep-deprived rats

(A) Western blotting analysis of ACSS2 in the hippocampal CA1 region in Con and SD groups.

(B) Quantitative analysis of the expression levels of ACSS2 in (A).

(C) Western blotting analysis of CaM in the hippocampal CA1 region in Con and SD groups.

(D) Quantitative analysis of the expression levels of CaM in (C).

Data are represented as the mean ± SEM (n = 6 per group). ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01.

SD and the CaM inhibitor TFP do not affect the motor ability and swimming speed of rats

Given that the motor ability and swimming speed of rats can influence the outcomes of cognitive-behavioral experiments, leading to diverse results, we aimed to investigate the impact of SD on these parameters in rats. To this end, we employed the open-field test and the Morris water maze clear-platform test to evaluate relevant indicators. Additionally, we verified whether the administration of the CaM inhibitor TFP in an SD model altered the motor ability and swimming speed of rats. Comparative analysis of the results from the open-field test across four groups of rats revealed that, after 21 days of SD, no significant differences were observed in the activity time, total distance traveled, or movement speed among the Con group, SD group, Con + CaMi group (control group with TFP intervention), and SD + CaMi group (SD group with TFP intervention) (p > 0.05) (Figure 3A). Similarly, in the Morris water maze clear-platform test, no significant differences in swimming speed were noted among the four groups (p > 0.05) (Figure 3B). The findings from these experiments indicate that 21 days of SD induce cognitive impairment in rats without significantly affecting their motor ability or swimming speed, thereby supporting the continuation of subsequent behavioral experiments. Our results demonstrated that neither SD nor intervention with the CaM inhibitor TFP had a discernible impact on the motor ability or swimming speed of rats across the experimental groups (Figure S2).

Figure 3.

SD and the CaM inhibitor TFP do not affect the motor ability and swimming speed of rats

(A) Comparison of the ratio of the total distance traveled to the total activity duration in open-field test, representing the movement speed in rats in the Con, SD, Con + CaMi, and SD + CaMi groups.

(B) Comparison of the swimming time taken to locate the platform in Morris water maze clear-platform test, representing the swimming speed in rats in the Con, SD, Con + CaMi, and SD + CaMi groups.

Data are represented as the mean ± SEM (n ≥ 6 per group). ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01.

The intervention with the CaM inhibitor TFP attenuates cognitive impairment in sleep-deprived rats

To further investigate whether the intervention with a CaM inhibitor (CaMi) ameliorates cognitive impairment in sleep-deprived rats, we selected TFP as the CaM inhibitor for the intervention. The drug was administered according to the time points and dosage requirements reported in the literature, followed by cognition-related behavioral tests, including the Morris water maze test, Y-maze test, and novel object recognition test. The results of the Morris water maze test showed that, compared with the Con group, in the place navigation trial, the escape latency of rats in the SD group was significantly different (p < 0.05) (Figure 4D), with a marked prolongation (Figure 4G). Compared with the SD group, the escape latency in the SD + CaMi group increased more slowly, indicating that CaMi could slow down the decline in learning and memory abilities in the SD group. Meanwhile, during the spatial probe trial of the Morris water maze test, compared with the Con group, the number of entries into the third quadrant, the number of effective entries, and the number of crossings over the original escape platform in the SD group were all significantly reduced (p < 0.05). In contrast, compared with the SD group, these parameters in the SD + CaMi group were significantly increased (p < 0.05) (Figures 4A–4C). The results of the Y-maze test revealed that, compared with the Con group, the spontaneous alternation rate of rats in the SD group was significantly decreased (p < 0.05); and compared with the SD group, the spontaneous alternation rate in the SD + CaMi group was significantly increased (p < 0.05) (Figure 4E). In the novel object discrimination test, we observed that, compared with the Con group, the novel object discrimination index of rats in the SD group was significantly decreased (p < 0.05); and compared with the SD group, the novel object discrimination index in the SD + CaMi group was significantly increased (p < 0.05) (Figure 4F). The above results indicate that cognitive function is impaired in rats after SD, and the intervention with the CaM inhibitor TFP alleviates cognitive impairment in sleep-deprived rats.

Figure 4.

The intervention with the CaM inhibitor TFP attenuates cognitive impairment in sleep-deprived rats

(A) Across escape platform times of the Con, SD, Con + CaMi, and SD + CaMi groups in Morris water maze test.

(B) Across effective area times of the Con, SD, Con + CaMi, and SD + CaMi groups in Morris water maze test.

(C) Across the third quadrant times of the Con, SD, Con + CaMi, and SD + CaMi groups in Morris water maze test.

(D) Escape latency from the first to the fifth day of the Con, SD, Con + CaMi, and SD + CaMi groups in Morris water maze test.

(E) Spontaneous alternation rate of the Con, SD, Con + CaMi, and SD + CaMi groups in Y-maze test.

(F) Novel object discrimination index of the Con, SD, Con + CaMi, and SD + CaMi groups in novel object recognition test.

(G) Representative images of moving routes of the Con, SD, Con + CaMi, and SD + CaMi groups on day 5 and day 6 in Morris water maze test; scale bars, 30 cm.

Data are represented as the mean ± SEM (n ≥ 6 per group). ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01.

The CaM inhibitor TFP reverses the elevated expression of CaM and the reduced expression of ACSS2 protein in the hippocampal CA1 region of rats with cognitive impairment induced by SD

To further investigate the impact of CaM and ACSS2 proteins on cognitive functions such as learning and memory in rats following SD, we selected the CaM inhibitor TFP for intervention and conducted western blot experiments on the hippocampal CA1 region of rats with SD-induced cognitive impairment. The results revealed that, compared with the Con group, the expression of ACSS2 protein in the hippocampal CA1 region of rats in the SD cognitive impairment group was significantly downregulated (p < 0.05). Conversely, compared with the SD group, the expression of ACSS2 protein in the hippocampal CA1 region of rats in the drug-administered experimental group was significantly upregulated (p < 0.05) (Figures 5A and 5B). Additionally, compared with the Con group, the expression of CaM protein in the hippocampal CA1 region of rats in the SD group was significantly upregulated (p < 0.05), and compared with the SD group, the expression of CaM protein in the hippocampal CA1 region of rats in the drug-administered experimental group was significantly downregulated (p < 0.05) (Figures 5C and 5D). These findings indicate that SD leads to the upregulation of CaM expression and the downregulation of ACSS2 protein expression in the hippocampal CA1 region of rats, while the CaM inhibitor TFP can reverse these changes and improve cognitive impairment caused by SD.

Figure 5.

The CaM inhibitor TFP reverses the elevated expression of CaM and the reduced expression of ACSS2 protein in the hippocampal CA1 region of rats with cognitive impairment induced by SD

(A) Western blotting analysis of ACSS2 in the hippocampal CA1 region in the Con, SD, Con + CaMi, and SD + CaMi groups.

(B) Quantitative analysis of the expression levels of ACSS2 in (A).

(C) Western blotting analysis of CaM in the hippocampal CA1 region in the Con, SD, Con + CaMi, and SD + CaMi groups.

(D) Quantitative analysis of the expression levels of CaM in (C). Data are represented as the mean ± SEM (n ≥ 6 per group). ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01.

The CaM inhibitor TFP elevates the expression of ACSS2-associated autophagy factors in the hippocampal CA1 region of rats following SD-induced cognitive impairment

Meanwhile, to further elucidate the roles of CaM and ACSS2 proteins in cognitive dysfunction induced by SD and to verify the involvement of ACSS2-related autophagy in memory regulation, we selected the CaM inhibitor TFP for intervention. We prepared homogenates from the hippocampal CA1 region of rats with SD-induced cognitive impairment, collected the supernatants, and conducted enzyme-linked immunosorbent assay (ELISA) to measure expression changes of the ACSS2 downstream autophagy factors TFEB and UVRAG. The ELISA results demonstrated that, compared with the Con group, the concentrations of UVRAG (p < 0.05) (Figure 6A) and TFEB (p < 0.05) (Figure 6B) in the hippocampal CA1 region of rats with SD-induced cognitive impairment were significantly decreased. Conversely, compared with the experimental group without drug treatment, the expression levels of UVRAG (p < 0.05) (Figure 6A) and TFEB (p < 0.05) (Figure 6B) in the hippocampal CA1 region of rats in the drug-administered experimental group were significantly increased. These findings suggest that ACSS2-related autophagy is involved in cognitive dysfunction induced by SD in rats, and the CaM inhibitor TFP can reverse the expression changes of ACSS2-related autophagy factors in the hippocampal CA1 region of rats with SD-induced cognitive impairment.

Figure 6.

The CaM inhibitor TFP elevates the expression of ACSS2-associated autophagy factors in the hippocampal CA1 region of rats with SD-induced cognitive impairment

(A) ELISA results for UVRAG expression in the hippocampal CA1 region in the Con, SD, Con + CaMi, and SD + CaMi groups.

(B) ELISA results for TFEB expression in the hippocampal CA1 region in the Con, SD, Con + CaMi, and SD + CaMi groups.

Data are represented as the mean ± SEM (n ≥ 6 per group). ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01.

Discussion

SD disrupts the daily lives of millions of individuals globally, significantly impairing learning efficiency and the ability to form new memories. Notably, it exerts a particularly profound inhibitory effect on the consolidation of hippocampus-dependent memory tasks. A wealth of research indicates that SD may lead to cognitive impairments, including memory deficits, by triggering neuroinflammatory responses, exacerbating oxidative stress, disrupting synaptic plasticity, and altering neurotransmitter levels, gene expression patterns, and protein synthesis mechanisms.⁴⁸ In this study, we employed the multi-platform water environment method to establish a chronic SD model in rats, aiming to investigate the expression patterns of CaM, ACSS2, and ACSS2-related autophagy factors in the hippocampal CA1 region of rats experiencing cognitive impairment due to SD. Subsequently, we administered the CaM inhibitor TFP to downregulate CaM expression and further examined the expression of ACSS2 and its associated autophagy factors, thereby delving deeper into the specific mechanisms underlying cognitive impairment in rats following SD. Our findings shed light on the intricate relationships among SD, memory and cognitive impairment, and autophagic responses in rats. SD markedly reduced the learning and memory capacities of rats, a result consistent with those of previous studies on the cognitive effects of SD. Additionally, we observed elevated CaM expression, alongside reduced expressions of ACSS2 and ACSS2-related autophagy factors in the hippocampal CA1 region of rats with cognitive impairment induced by SD. These alterations likely triggered neuroautophagic responses in the rats. Notably, intervention with the CaM inhibitor TFP ameliorated cognitive impairment in sleep-deprived rats and reversed the expression changes of ACSS2 and its related autophagy factors in the hippocampal CA1 region. By integrating behavioral research with molecular biological analysis, this study aimed to elucidate the specific mechanisms by which SD leads to cognitive impairment and to gain deeper insights into the roles of expression changes in CaM, ACSS2, and ACSS2-related autophagy factors in the pathogenesis of cognitive impairment caused by SD. The results of this study further enhance our understanding of the extensive neurobiological alterations that may arise from SD.

The hippocampus, a pivotal brain region governing learning and memory, is situated medially within the temporal lobe and plays a crucial role in cognitive function, spatial navigation, and emotional regulation.⁴⁹ Its anatomical structure can be further divided into subregions such as CA1, CA3, and the dentate gyrus (DG), each possessing distinct neural circuits and functional characteristics.^49,50 In this study, we selected rats as our research subjects and analyzed tissue from the hippocampal CA1 region, which plays a central role in spatial memory encoding and retrieval.⁵¹ Within the brain’s memory system, the CA1 region holds a pivotal position, particularly in memory consolidation, retrieval, and spatial memory, where it exerts an irreplaceable and critical function.²² Following SD, the rats exhibited significant cognitive impairment. Using western blot analysis, we discovered that in rats with cognitive impairment induced by SD, CaM expression was increased and ACSS2 protein levels were decreased in the hippocampal CA1 region. These findings align with our research group’s previous studies on cerebrovascular diseases. Our team has long been exploring the associations between CaM, cerebrovascular diseases, and sleep-related disorders. In our earlier research, by constructing a Calm1 gene knockout mouse model and employing proteomic techniques, we observed a notable upregulation of ACSS2 protein expression in the hippocampus when CaM was absent, indicating a significant correlation between the two (Figure S1). We further validated this mechanism in the context of ischemia-reperfusion (I/R) injury through studies using rat MCAO/R (middle cerebral artery occlusion/reperfusion) and HT22 cell OGD/R (oxygen-glucose deprivation/reperfusion) models. Our results revealed that in the ischemic penumbra tissue of MCAO/R rats, CaM expression increased, while ACSS2 protein levels decreased. Conversely, specific intervention targeting CaM led to a dose-dependent increase in ACSS2 expression, suggesting a potential negative regulatory relationship between the two that jointly participate in the pathological process of I/R injury. CaM, as the primary effector molecule of Ca²⁺, mediates the regulation of downstream kinases through Ca²⁺-CaM complexes. During I/R injury, intracellular Ca²⁺ overload triggers conformational changes in CaM, activating pathways for the release of vasoconstrictive substances and exacerbating vasospasm and tissue damage. Although Ca²⁺ serve as a central hub for signal transduction across multiple systems, including the nervous, cardiovascular, and vascular systems, and the Ca²⁺ signaling pathway has become a research hotspot in basic medicine, existing studies still face multiple challenges: the complex regulatory network of Ca²⁺ homeostasis imbalance remains incompletely elucidated; the clinical application of Ca²⁺ channel blockers is limited by their interactions with other drugs and individual differences; and the translation of basic research findings into clinical practice still requires validation through large-scale, multi-center trials.

This study primarily focuses on the pathological mechanisms of CaM and ACSS2 proteins in rats with cognitive impairment induced by SD. Research has indicated that ACSS2 can function as an epigenetic regulator, inducing histone acetylation in the nucleus to enhance the transcription of early genes and consolidate hippocampal memory. It can also participate in memory consolidation by promoting the expression of autophagy-related genes.⁵² ACSS2 influences memory through multiple mechanisms, primarily by regulating epigenetics via histone acetylation. Histone acetylation primarily relies on acetyl-CoA, whose metabolism is complex and involves numerous mechanisms, potentially participating in various underlying mechanisms of memory consolidation. Activators of ACSS2-related pathways can serve as memory enhancers, facilitating memory consolidation. Conversely, ACSS2 inhibitors can be employed to alleviate the consolidation of fear memories and slow down their formation.⁵³ ACSS2 may emerge as a solid therapeutic target for specific memory dysfunctions, holding promising potential applications. Modulating ACSS2 activity in memory dysfunctions could offer relevant insights for drug research aimed at improving memory and cognition. Through ELISA experiments, we observed a decrease in the expression levels of TFEB and UVRAG, which are downstream autophagy factors related to ACSS2, in the hippocampal CA1 region of rats with cognitive impairment induced by SD. TFEB is a protein that plays a crucial role in various physiological processes, including cellular metabolism and autophagy, promoting the formation of autophagosomes and lysosomal biogenesis. Mechanistic studies have shown that TFEB translocates to the nucleus with the assistance of IPO8, where it specifically binds to nuclear ACSS2 to form a complex. This complex then binds to the promoter regions of lysosomal and autophagosomal genes (i.e., TFEB target genes), promoting the expression of numerous key factors (such as CLEAR, RRAGC, UVRAG, CSTB, M6PR, and IGF2R) to enhance the expression of lysosome-related genes.³⁹ This suggests that the upregulation of these genes can enhance lysosomal function and promote autophagy. Meanwhile, autophagy plays a significant role in synaptic plasticity, dendritic neurons, and cultured neurons⁴⁰ and can negatively regulate axonal extension.⁴¹ There is a broad connection between synaptic plasticity and memory.^42,43 Consequently, the upregulation of these genes can inhibit synaptic plasticity and memory function by promoting autophagy, leading to memory consolidation dysfunction.⁴⁴ Immunohistochemical and double-fluorescence labeling experiments have confirmed that TFEB nuclear translocation activates autophagy in the early stages of ischemia, while the subsequent stagnation of nuclear-cytoplasmic translocation may be associated with autophagy dysfunction. Further research has revealed that protein phosphatase 3/calcineurin is significantly activated in the early stages of ischemia and may participate in the regulation of autolysosomal function by modulating TFEB nuclear translocation. Additionally, exercise intervention experiments have shown that long-term exercise can enhance lysosomal function and accelerate Aβ clearance in AD mouse models by promoting TFEB nuclear translocation and its interaction with ACSS2.^54,55 In glioblastoma cell models, ACSS2 that translocated to the nucleus bound to TFEB, regulating histone acetylation levels through local acetyl-CoA generation and influencing the transcription of autophagy-related genes.⁵⁵ Furthermore, UVRAG is a key regulator in the formation of autophagosomes, mediating the maturation of autophagic vesicles.⁴⁵ UVRAG can bind to Beclin 1, thereby enhancing the activity of the Beclin 1-Vps34 complex. This complex plays an indispensable role in the nucleation and elongation of autophagosomal membranes, driving the gradual formation of autophagosomes. Moreover, UVRAG plays a significant role in regulating autophagic flux, including the entire process from autophagosome generation and maturation to fusion with lysosomes.⁴⁶ In some neurodegenerative diseases, such as AD and Parkinson disease, UVRAG dysfunction may lead to autophagy defects, resulting in the ineffective clearance of abnormal protein aggregates within cells and exacerbating neuronal damage.

In this study, TFP was found to mitigate the adverse effects of SD on cognitive impairment in rats and reverse the expression changes of CaM, ACSS2 protein, and ACSS2-related autophagy factors in their hippocampal CA1 region. TFP is a well-recognized CaM inhibitor, and previous experiments by our research group have confirmed that multiple intraperitoneal injections of TFP can reduce CaM levels in tissues and serum. Additionally, TFP regulates blood-brain barrier permeability through the MLCK-p-MLC pathway, promoting recovery from ischemic stroke.⁵⁶ Studies have shown that TFP induces conformational changes in CaM by directly binding to it, thereby blocking its biological functions. This interaction significantly inhibits CaM activity, particularly its regulatory effect on the CaM-Inositol 1,4,5-triphosphate receptor (IP3R) complex. Mechanistic research indicates that TFP binding may interfere with CaM conformation, affecting IP3R channel function and Ca²⁺ release kinetics, while Ca²⁺-bound CaM negatively regulates IP3R channel opening.⁵⁷ Therefore, TFP exhibits promising clinical application prospects in treating ischemic brain injury and sleep-related cognitive disorders, although its specific mechanisms require further investigation. Furthermore, a large number of studies have shown that insomnia can lead to cognitive impairment and increase the risk of AD.^15,58 CaM activation can improve neural plasticity and cognitive function by phosphorylating CaMKII.^27,28 Furthermore, based on the findings of this study, TFP is worthy of in-depth investigation for the treatment of AD-related cognitive impairment. One of the primary findings of this study is that TFP inhibits CaM and improves cognitive impairment in rats following SD by upregulating the expression of ACSS2 and ACSS2-related autophagy factors. This not only underscores the detrimental impact of SD on memory and cognitive function in rats but also reveals the potential mechanism by which CaM inhibitors may influence brain function through neuroautophagy pathways. This is crucial for deepening our understanding of the role of autophagy in maintaining normal sleep and cognitive function. Furthermore, our study suggests that the neuroautophagic response triggered by SD may represent a pathway through which SD affects brain function and leads to cognitive impairment. This discovery provides a biological basis for developing interventions aimed at improving sleep quality. In-depth exploration of the roles of expression changes in CaM, ACSS2, and ACSS2-related autophagy factors in the pathogenesis of cognitive impairment induced by SD offers solid theoretical evidence for the prevention and treatment of cognitive dysfunction following SD. Such insights could pave the way for relevant therapeutic strategies targeting these molecular pathways to mitigate the cognitive consequences of SD.

Our results suggest that CaM is involved in cognitive impairment in rats following SD. Furthermore, our findings indicate that the CaM inhibitor TFP ameliorates SD-induced cognitive impairment, possibly by upregulating ACSS2 and the autophagy-related factors UVRAG and TFEB.

Limitations of the study

There are remarkable differences in physiological characteristics, behavioral patterns, and cognitive environments between rats and humans, imposing non-negligible limitations on the translational application of animal models to humans. Additionally, this study evaluated rat cognitive function by using behavioral assays, which inherently involve a degree of subjectivity. In experimental studies, male rats are commonly selected as research subjects. Sex also exert certain effects on sleep, and its potential influence on both drug efficacy and sleep outcomes cannot be ruled out but was not investigated in this study, which constitutes a limitation of the current work.

To better understand the impact of SD on cognitive function and its underlying mechanisms, future studies could focus on the following directions: (1) investigate the intervention effects of TFP at different dosages and administration timings on SD-induced cognitive impairment; (2) explore the expression of CaM and ACSS2 in brain regions other the CA1 region of the hippocampus, which was focused in this study; (3) further clarify how CaM and ACSS2 interact during SD, including their potential regulatory mechanisms and molecular signaling pathways; (4) elucidate whether ACSS2 improves learning and memory impairments caused by SD by regulating hippocampal synaptic plasticity through autophagy, and explore its underlying mechanisms.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Mei Yuan (2012020002@usc.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

All data reported in this paper will be shared by the lead contact upon request.

This paper does not report original code.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This work was supported by the Natural Science Foundation of Hunan Province (2025JJ50615), the University of South China Clinical Research 4310 Program (20224310NHYCG08), the Clinical Medical Research Center of Hunan Province (2023SK4050), the Project of Guangxi Health Commission (Z-A20221278), and the Major Scientific Research Projects for Advanced Talents of Hunan Provincial Health Commission (R2023150). We thank the Institute of Neuroscience, Hengyang Medical School, University of South China and the Medical Innovation Experimental Center of the Second Affiliated Hospital of the University of South China for providing the experimental conditions.

Author contributions

Conceptualization, M.Y. and C.H.; methodology, C.H., B.W., X.C., J.X., and Y.Y.; investigation, C.H., B.W., and X.C.; visualization, C.H.; supervision, M.Y.; writing – original draft, C.H.; writing – review & editing, C.H. and M.Y. All authors have reviewed and approved the final version of the manuscript.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Calmodulin	sigma	Cat#05-173
ACSS2	CST	Cat#3658S
Beta Tubulin	Proteintech	Cat#10068-1-AP
Goat anti-mouse IgG (H + L)	Proteintech	Cat#SA00001-1
Goat anti-rabbit IgG (H + L)	Proteintech	Cat#SA00001-2
GAPDH	Proteintech	Cat#60004-1-Ig
Chemicals, peptides, and recombinant proteins
Chloral hydrate	Macklin	Lot C804539
PMSF	Solarbio	Lot P0100
RIPA buffer	Solarbio	Lot R0010
Primary Antibody Dilution Buffer	Beyotime	Lot P0256
Rat TFEB ELISA KIT	Zcibio	Cat#ZC-57643
Rat UVRAG ELISA KIT	Zcibio	Cat#ZC-57671
Trifluoperazine	Sigma	Cat#T8516
DMSO	Thermo Fisher Scientific	Lot 11965092
Triton x-100	Sangon	Cat#A100777
ECL	Abbkine	Cat#BMU102-CN
Software and algorithms
GraphaPad Prism V8.02	GraphaPad	N/A
Image Lab	Bio-rad	N/A
Image J	National Institutes of Health	N/A

Experimental model and study participant details

Animals

The methods for the feeding, care, and testing of experimental animals were approved by the Research Ethics Committee of University of South China (Ethics No:2023-022-1). To ensure the reliability and consistency of experimental results, we selected SPF-grade Sprague Dawley rats aged 8–10 weeks and weighing (260–280) g from Changsha Tianqin Biotechnology Co., Ltd. (Hunan, China) as the research subjects. This study used single male rats as experimental subjects, aiming to exclude the potential interference of fluctuating estrogen levels in female individuals on experimental data, thereby improving the statistical efficiency of the data. The experimental animals were housed in a barrier environment animal laboratory, maintained at a constant temperature of 22 ± 2°C and a relative humidity of 40%–60%, and subjected to a strict 12-h light/dark cycle. The experimental animals were assigned to the blank control group, model experimental group, drug intervention group, and drug control group according to the random number table method.

Rat model for SD

The chronic SD model was established using a modified multi-platform water environment method. A stainless-steel water tank (170 cm length × 70 cm width × 50 cm height) with transparent walls was used to ensure light penetration. The tank was filled with water and equipped with 10 small platforms (6.5 cm diameter), with the water level set 1 cm below the platform surface. Standard food and water were placed on the cage cover above the tank, allowing rats to freely eat and drink during SD. The laboratory temperature was maintained at (23 ± 2) °C, water temperature at (22 ± 1) °C, and a 12-h light/dark cycle was regulated by 40 W fluorescent lamps to simulate daytime lighting. All rats were group-housed in the same cage before the experiment. One week prior to modeling, rats were acclimated to the platform setup for 6 h daily. When rats entered rapid eye movement (REM) sleep, decreased skeletal muscle tone caused their bodies to touch the water surface, triggering awakening and SD. SD rats were allowed to sleep and rest at fixed times (08:00–12:00) daily, followed by 20 h of platform-based SD for 21 consecutive days. To control for environmental factors, non-SD rats were placed on wide platforms (12 cm diameter) within the tank, ensuring no water contact during REM sleep. Control group rats were housed in standard cages in the same room for 21 days with ad libitum access to food and water.

Experimental grouping and drug administration

In this study, according to the experimental design, rats were randomly divided into four subgroups, with 10 rats in each subgroup. The grouping was as follows: (a): Normal control group (Control group). This group served as the model control group, receiving normal feeding without SD intervention or drug intervention, but given a vehicle control. (b): SD experimental group (SD group, SD group). This group was the pure SD experimental group, receiving normal feeding and SD intervention, without drug intervention but given a vehicle control. (c): SD + CaM inhibitor group (SD + CaMi group). This group received normal feeding and SD intervention, and was given the CaM inhibitor TFP to explore the changes in cognitive function of rats after SD intervention and conduct subsequent experiments. (d): Con + CaM inhibitor group (Here, it should be more accurately named as Drug control group, Con + CaMi group as stated in the original text might cause confusion, but we’ll keep it as per the original for now). This group was the drug control group, receiving normal feeding without SD intervention but with drug intervention to explore the effects of the drug on normal rats.

To prepare an appropriate amount of the drug at the corresponding concentration, we used 2% dimethyl sulfoxide (DMSO) to prepare a TFP solution at a dose of 10 mg/kg for the CaM inhibitor groups. One hour before model construction, rats in the drug-administered groups were gently grasped and received an intraperitoneal injection of the corresponding drug. In contrast, rats in the SD group received an injection of an equal volume of 2% DMSO solution as a control. TFP can specifically inhibit the activity of CaM. After 7 days of SD, TFP was used as a therapeutic agent, and rats in the SD + CaMi group and the drug control group were given a daily intraperitoneal injection of TFP (10 mg/kg)^56,57 for a total of 14 days.⁵⁹

Method details

Morris water maze test

The Morris water maze was evenly divided into four quadrants, which were then filled with water. A circular platform with a diameter of approximately 10–12 cm was placed at the center of the third quadrant, with its surface 1–2 cm below the water level, making it invisible to the animals directly but accessible for escape from buoyancy by climbing onto it. Non-food-grade titanium dioxide was added to the water, and the temperature was maintained at (25 ± 1)°C. Video tracking equipment was employed to continuously monitor and record the animals’ movement trajectories, swimming speeds, and other parameters within the pool. During the place navigation trial, rats were placed into the water facing the pool wall from each of the four entry points multiple times per day. The time taken to locate the hidden platform beneath the water surface (escape latency) was recorded. Over the five-day trial period, animals underwent approximately four trials per day. Rats that failed to find the platform within 120 s were guided to it and allowed to remain there for 10–15 s. On the sixth day after the place navigation trial, the platform was removed. Rats were then placed into the water from the center of the first quadrant, adjacent to the wall, and the number of times they crossed the former platform location and the duration spent in the third quadrant within 120 s were recorded. The primary assessment metrics included: escape latency, swimming speed, number of platform crossings, number of entries into the effective zone, and time spent in the third quadrant.

Y-maze spontaneous alternation test

The Y-maze consists of three identical arms arranged in a Y-shape, with each arm forming a 120° angle relative to the others. Each arm measures approximately 40–50 cm in length, 10–15 cm in width, and 20–30 cm in height, ensuring unrestricted movement for the animals within the arms while preventing them from seeing external cues outside the maze. Prior to the experiment, animals are placed inside the Y-maze and allowed to freely explore for 5-10 min to familiarize themselves with the environment, thereby minimizing stress responses to the novel setting and ensuring that subsequent experimental results are not influenced by unfamiliarity with the surroundings. After the acclimation period, each rat is individually placed in the triangular central area of the maze and permitted to explore freely for 5 min. During this time, the animal’s movement trajectory, total number of arm entries, and sequence of arm entries within the 5-min period are recorded using a video tracking system or through direct observation. A correct alternation is defined as three consecutive entries into different arms, whereas any other sequence is considered incorrect. The spontaneous alternation accuracy rate for each rat is then calculated using the formula: Accuracy Rate (%) = (Number of Correct Alternations/Total Number of Trials) × 100%.

Novel object recognition test

The experimental site is a relatively open, quiet, and evenly lit square open-field device. In the experiment, two objects with different appearances are used as stimuli. These objects have obvious differences in shape, color, texture, etc., and have no harmful effects or special odors and other interfering factors on the experimental animals. During the adaptation period, the experimental animals are placed in the experimental device and allowed to freely explore for 6 min to familiarize themselves with the experimental environment and reduce the stress response to the new environment. After the adaptation period, two identical Objects A are placed in the device. The rats are placed from the center point of the wall opposite the blocks with their backs to the blocks, allowing the animals to freely explore for 6 min to form a memory of Object A, which is the training period. After the training period, after an interval (generally ranging from 30 min to 24 h), one of the Objects A is replaced with a new Object B. The rats are then placed again from the center point of the wall opposite the blocks with their backs to the blocks, allowed to freely explore for 6 min, and the exploration behaviors of the animals toward Object A and Object B are observed and recorded. The novel object recognition index is defined as the ratio of the exploration time for the novel object to the total exploration time for both novel and familiar objects. This index can intuitively reflect the animal’s discrimination ability toward novel objects. A higher value indicates a more obvious preference for the novel object, demonstrating stronger recognition ability.

Open field test

The open-field test apparatus for rats is a white acrylic square chamber (100 cm × 100 cm × 50 cm), with the bottom evenly divided into nine zones. Before the experiment, ensure the proper installation and operation of the SuperMaze behavioral analysis software. The camera should be stably mounted on a bracket and correctly connected to a dedicated computer, while top lighting must be kept soft. Rats from each group should be preplaced in the open-field test room for 60 min in advance to acclimate to the indoor environment. Once ready, gently place the rat at the center of the chamber to start the test. Simultaneously activate the software recording function, which automatically captures and records activity data for 5 min, including the number of rears, total distance traveled, and immobility time. Before testing the next rat, thoroughly clean the entire open field with 75% alcohol and paper towels to eliminate odor traces from the previous rat, preventing interference with behavioral judgment of subsequent subjects.

Swimming test

The Morris water maze was used as the experimental field, filled with clear water. A circular platform with a diameter of approximately 10–12 cm was placed at the center of the third quadrant, with its surface 1–2 cm below the water level, allowing the animals to directly see the platform and escape the buoyancy of the water by climbing onto it. The water temperature was maintained at (25 ± 1)°C. Video tracking equipment was employed to monitor and record the movement trajectory, swimming speed, and other parameters of the animals in the pool in real time. At the beginning of the experiment, the rats were placed in the water with their backs facing the platform and on the opposite quadrant. The time it took for them to find the platform, the distance they swam, and their swimming speed were recorded.

Western blotting

The protein samples were collected from brain tissues of Sprague-Dawley rats for western blot analysis. Total protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer. Equal quantities of protein from each sample were mixed with the loading buffer and boiled for 5 min 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the sample proteins, which were then transferred to a polyvinylidene fluoride (PVDF) membranes. The membrane was blocked for 2 h at room temperature with 5% defatted milk, then incubated overnight at 4°C with primary antibodies against ACSS2 (1:1000, CST, USA), CaM (1:1000, sigma, USA), GAPDH (1:1000, Proteintech, USA). After washing the membrane in TBST, it was incubated for 1.5 h at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, Proteintech, USA). The enhanced chemiluminescence (ECL) kit was used to see the blotting protein bands. Chemiluminescence detection of proteins was imaged with the Tanon-5600 gel imager (Tanon, China).

ELISA experiment

The concentration of the factor to be measured in the tissue was quantitatively detected in vitro by the “one-step” sandwich enzyme-linked immunosorbent assay (ELISA). The target antibody was coated in a 96-well microplate to form a solid-phase carrier. Standard substances or specimens were added to the Wells respectively. The target was bound to the antibody on the solid-phase carrier. Then, horseradish peroxidase-labeled antibody was added. The unbound antibody was washed off and thoroughly washed again before adding TMB substrate for color development. TMB is converted into blue under the catalysis of peroxidase and then into the final yellow under the action of acid. The depth of the color is positively correlated with the target in the sample. The absorbance (O.D. value) was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the actual concentration of the sample was calculated based on the obtained formula.

Quantification and statistical analysis

All experimental data were repeated at least three times. All statistical analyses were performed using IBM SPSS Statistics 26.0 software. All statistical graphs were plotted using GraphPad Prism 9.0 software. All data were expressed as mean ± standard deviation ($\overline{\mathrm{x}}$±s). To compare the differences between the two groups, the Student’s t test was adopted. To compare the differences among multiple groups, one-way analysis of variance and Tukey’s post hoc test were adopted. Statistical significance was defined as p < 0.05. Data are represented as mean ± SEM. ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01.

Published: March 30, 2026