Cognitive impairment following maternal separation in the rat is regulated by effects of the NAD + /SIRT3 axis on hippocampal synaptic plasticity

Cognitive impairment following maternal separation in the rat is regulated by effects of the NAD + /SIRT3 axis on hippocampal synaptic plasticity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cognitive impairment following maternal separation in the rat is regulated by effects of the NAD + /SIRT3 axis on hippocampal synaptic plasticity Keke Hao, Fashuai Chen, Shilin Xu, Ying Xiong, Rui Xu, Huan Huang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4207040/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Maternal separation during early life can induce behaviors in adult animals that resemble those seen in schizophrenia, manifesting cognitive deficits. This may be indicative of oxidative stress linked to mitochondrial dysfunction. However, there is limited understanding of the molecular mechanisms regulating mitochondria in neural circuits that govern cognitive impairment relevant to schizophrenia, and their impact on neuronal structure and function. A 24-hour maternal separation (MS) rat model was utilized to simulate features associated with schizophrenia. Schizophrenia-associated behaviors and cognitive impairment were assessed using the open field test, pre-pulse inhibition, novel object recognition test, and Barnes maze test. The levels of mitochondrial proteins were measured using Western blot analysis. Additionally, alterations in mitochondrial morphology, reduced hippocampal neuronal spine density, and impaired LTP in the CA1 region were observed. Nicotinamide (NAM) supplementation, administration of honokiol (HNK) (a SIRT3 activator), or overexpression of SIRT3 could inhibit this process. Conversely, administration of 3-TYP (a SIRT3 inhibitor) in control and NAM-treated MS rats led to deficits in behavior, mitochondrial morphology, and the hippocampal neuronal phenotype. Our findings suggested a causal role for the NAD+/SIRT3 axis in modulating cognitive behaviors via effects on hippocampal neuronal synaptic plasticity. The NAD+/SIRT3 axis could be considered a promising therapeutic target for addressing cognitive-related behavioral disturbances, such as those seen in schizophrenia. schizophrenia maternal separation cognitive impairment LTP mitochondria NAD+/SIRT3 axis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Cognitive impairment is a common feature of severe mental illness. Maternal separation (MS) is an early life intervention that can produce in behaviors of adult animals reminiscent of schizophrenia, including cognitive deficits [ 1 ]. Cognitive impairment has been considered as a core symptom domain of schizophrenia [ 2 ] in which the hippocampus has been strongly implicated [ 3 – 6 ]. Hippocampal neuronal dysfunction is also associated with cognitive deficits in animal models of schizophrenia. In particular, the impairment of hippocampal neuronal dendritic complexity has been shown to contribute causally to stress-induced cognitive deficits [ 7 – 9 ]. Given that neuronal morphology is a strong determinant of synaptic connectivity and strength, understanding the factors that control hippocampal neuronal dendritic regulation may help in developing treatments to ameliorate cognitive deficits. Mitochondria appear to be ideally suited to contribute. Increasing evidence points to a central role of mitochondria in the etiology of psychiatric disorders [ 10 ]. In neurons, mitochondria supports metabolic demands through energy supply [ 11 , 12 ]. Cell cultures and neurodevelopmental studies have implicated mitochondria in the regulation of both dendritic arborization [ 13 – 17 ] and spine and synapse formation [ 18 , 19 ]. However, whether mitochondrial dysfunction impairs hippocampal neuronal dendritic complexity and consequently, contributes to cognitive deficits associated with schizophrenia remains unknown. Our previous studies have suggested that degradation of nicotinamide adenine dinucleotide (NAD+) and associated bioenergetics failure of cellular metabolism may be one of the major factors leading to neuronal damage [ 20 , 21 ]. NAD + is an essential cofactor in most enzymatic reactions supporting fundamental mitochondrial functions including oxidative phosphorylation and enzymatic reactions of the tricarboxylic acid cycle [ 22 – 24 ]. When NAD + is degraded, mitochondria become incapable of ATP synthesis. Previous study has demonstrated that when naïve mice were treated with an NAD + precursor, neuronal mitochondrial function recovers [ 25 ], and an increase in mitochondrial NAD + will reduce acetylation of mitochondrial proteins and ROS generation in hippocampal tissue [ 26 ]. Furthermore, a high NAD + level facilitates the activation of proteins involved in mitochondrial quality control, such as Sirtuin3 (SIRT3). SIRT3 reinforces mitochondrial antioxidant defense by deacetylating and increasing the activity of superoxide dismutase 2 (SOD2) [ 27 , 28 ]. However, it is not clear whether the mitochondria-related NAD+/SIRT3 axis is involved in hippocampal neuronal synaptic plasticity and the schizophrenia-associated cognitive impairment. Mother-infant interaction may be a key factor in brain maturation, and stress associated with MS may induce the development of psychosis or susceptibility to psychotic diseases [ 20 , 21 ]. The abnormal behaviors and molecular changes taking place after a single 24 h period of MS on a postnatal day (PND) 9 in rats successfully recapitulate several features of schizophrenia [ 29 – 31 ]. Therefore, the MS rat model has become a powerful tool for exploring the neurobiological bases of schizophrenia. 2. Materials and Methods 2.1 Animals and m aternal s eparation Forty nulliparous female and forty male eight-week-old Wistar rats were obtained from Beijing Vital Rival Laboratory Animal Technology Co., Ltd. (Beijing, China). Rats of the same sex were caged together, with 3 or 4 per cage. The animals were mated at age of 3 months and the males were removed one week later. The mated female rats were housed individually in ventilated plastic cages in a temperature- and humidity-controlled (22 ± 20℃, 50 ± 10%) holding facility with a constant 12h day-night cycle (lights: 08:00 – 20:00). All animals had free access to food and tap water. The MS protocols were performed according to previous research [30, 32]. Females were checked twice daily for delivery (08:00 and 17:00). The day of delivery was considered as PND 0. Each pregnant rat provided on average 10 ± 2 offspring. On PND9, litters were randomly assigned to either the MS or control groups. In brief, the mothers were removed at 10:00. The pups remained in their home cages with the heated mat for 24h, after which the mothers were returned to their cages. The control groups grew naturally to adulthood. All the litters were otherwise left undisturbed except for the routine cleaning of the cages. On PND 21, the MS and the control groups of pups were weaned, and then group-housed by sex (3-4 per cage). All the following determinations were carried out only on male offspring avoiding the effects of estrogen in regulating neuronal activity and animal behavior [33, 34]. All procedures involving animals were approved and carried out according to the guidelines of the Institutional Animals Care Committee of Renmin Hospital of Wuhan University. 2.2 Experimental design Experiment 1: On postnatal day 9 (PND9), the dams and their pups were randomly assigned to either the control group or the maternal separation (MS) groups. On PND10 after MS, each infant group was further divided into several subgroups with approximately 20 pups in each subgroup. The resulting four groups were as follows: control group receiving vehicle (Control+Vehicle), MS group receiving vehicle (MS+Vehicle), control group receiving nicotinamide (NAM) (Control+NAM), and MS group receiving NAM (MS+NAM). The NAM groups received oral gavage of NAM (100mg/kg/d, diluted in vehicle) for 30 days from PND56 to PND85. The dosage and treatment protocol of NAM were based on previous studies [35, 36]. The vehicle groups received daily saline (1ml/kg) from PND56 to PND85 (see supplementary Figure 1). Experiment 2: Pups in both the MS and control groups were randomly divided into two subgroups of 15. The MS animals received intraperitoneal (i.p.) injections of either honokiol (HNK) (10mg/kg/d) or vehicle, while the control animals received 3-TYP (10mg/kg/d) or vehicle for 15 days. The vehicle solution consisted of 90% saline and 10% DMSO (1ml/kg/d) (see supplementary Figure 1). Experiment 3: The 36 MS pups were divided into three groups: MS+Vehicle, MS+NAM, and MS+NAM+3-TYP. The MS+NAM+3-TYP group received NAM by gavage for 30 days and intraperitoneal (i.p.) injections of 3-TYP (10mg/kg) during the last 15 days. The MS+NAM group received NAM by gavage for 30 days. The MS+Vehicle group received saline (1ml/kg/d) by gavage for 30 days and the vehicle solution (90% saline and 10% DMSO at 1ml/kg/d i.p.) for the last 15 days (see supplementary Figure 1). Experiment 4: AAV9-SIRT3 and AAV9-Nc were stereotactically injected into mice under isoflurane anesthesia. The bilateral hippocampal CA1 region injections were performed at the following coordinates: −2.4 mm anteroposterior, −3.75 mm mediolateral from the bregma, and −2.6 mm dorsoventral from the dural surface. A viral suspension (1μl) containing 2 × 109 vector genomes per μL was infused into each site at a rate of 0.25μl/min using a 10μl glass syringe with a fixed needle. After the injection, the needle was left in place for 10 min and then slowly removed over 2 min. Rats were kept on a heating pad until they fully recovered from anesthesia. Three weeks following the stereotactic injection, a multiple behavior test was conducted on the rats (see supplementary Figure 1). For the rest of the detailed methods, please refer to the Supplemental Information of this paper, which covers the following aspects: 2.3 Behavioral testing of animals 2.3.1 Open-field Test (OFT) 2.3.2 Novel object recognition test 2.3.3 Barnes maze test 2.3.4 Elevated-Plus Maze (EPM) 2.3.5 Sucrose preference Test (SPT) 2.3.6 PPI test 2.4 NAD + quantification 2.5 Protein extraction and western blot analysis 2.6 Electron microscopy 2.7 Immunofluorescent staining assay 2.8 Golgi-cox staining 2.9 Electrophysiology 3. Data analysis and statistics The data are reported as means ± standard error of the mean (SEM) and were analyzed using SPSS Statistics version 20.0 (SPSS Inc.). Significance in the datasets was assessed through Student’s unpaired two-tailed t-tests for comparisons between two groups, while one-way or two-way analysis of variance (ANOVA) was utilized for comparisons involving three or more groups. Statistical significance was considered at a p-value below 0.05. 4. Results 4.1 Reduced SIRT3 expression observed in the individuals with schizophrenia. To investigate changes in SIRT3 gene expression in the postmortem hippocampus of schizophrenia patients, an analysis was conducted using the GEO database ( https://www.ncbi.nlm.nih.gov/geo/ ). The database GSE53987 included gene expression data from 15 schizophrenia patients and 18 matched healthy controls. In comparison to age- and sex-matched controls, a significant decrease in SIRT3 mRNA levels was observed in the hippocampus (Supplementary Fig. 2A), but not in the prefrontal cortex (Supplementary Fig. 2B), suggesting potential involvement of hippocampal SIRT3 in schizophrenia pathology. Furthermore, a clinical study revealed reduced SIRT3 protein expression in peripheral blood mononuclear cells (PBMCs) among schizophrenia patients as compared to the healthy controls (Supplementary Fig. 2C, D). 4.2 NAM normalized cognitive impairment and schizophrenia-associated behaviors induced by MS in rats. To investigate the role of SIRT3 in the pathogenesis of schizophrenia, we employed the MS rat model, known for its ability to replicate key features of schizophrenia [ 29 – 31 ]. No differences in body weight (Supplementary Fig. 3A) or anxiety/depression-like behaviors (Supplementary Fig. 3B-H) were observed between the MS and control groups. However, MS induced schizophrenia-associated behavioral phenotypes in rats, including impairments in memory, cognition, hyperlocomotion, and sensory gating defects. Remarkably, all these phenotypes were reversed by administering NAM at 100 mg/kg/d for 30 days. In the Barnes maze test, MS rats displayed deficits in spatial learning and memory, as evidenced by an elevated latency to escape from day 1 to day 3 (Fig. 1 A), which was normalized by NAM treatment. Importantly, NAM did not reduce latency in the absence of MS treatment (Fig. 1 A). Cognitive function in MS rats was further evaluated using the novel object recognition test. All groups exhibited similar exploration times for two identical objects during the adaptation stage (Fig. 1 B, left panel). However, the MS group spent relatively less time exploring a novel object when one of the objects was replaced after 24 hours (Fig. 1 B, right panel). NAM reversed the reduced exploration time of the novel object in the MS group after 24 hours, but had no effect on the exploration time in the group without MS treatment (Fig. 1 B, right panel). Similarly, in the OFT, NAM reversed the MS-induced increase in total movement distance, without affecting the untreated MS group (Fig. 1 C). Finally, MS rats exhibited impaired PPIs at various pre-pulse intensities, which were fully reversed following NAM administration (Fig. 1 D). 4.3 NAM administration restored LTP and hippocampal neuronal dendritic complexity in MS rats. In terms of the synaptic transmission and plasticity characteristics of CA1 synapses, MS rats displayed impaired LTP (Fig. 1 E-G) and elevated paired-pulse ratio (PPR) (Fig. 1 H). However, NAM treatment effectively reversed the LTP impairment (Fig. 1 E-G) and normalized the PPR (Fig. 1 H). Consistent with these findings, neuronal structure analysis revealed a reduced spine density in MS rats compared to control rats (Fig. 1 I, J), with spine density recovering after NAM treatment in MS + NAM rats (Fig. 1 I, J). 4.4 NAM administration normalized microglial engulfment of hippocampal neuronal spines of MS rats. Microglia play a critical role in the synaptic pruning process [ 37 ]. To investigate the underlying cause of reduced hippocampal neuronal spines, we assessed the phagocytic function of microglial cells through immune co-labeling of CD68 and SYN in the hippocampus. Our findings demonstrated an elevated co-labeled area of SYN + and CD68 + in the hippocampus of the MS + Vehicle group compared to the Control + Vehicle rats (Supplementary Fig. 4A-D). However, daily administration of NAM via gavage (from PND56 to PND85) in MS + NAM rats significantly reduced the co-labeled area of SYN + and CD68 + in the hippocampus compared to the MS + Vehicle group (Supplementary Fig. 4A-D). 4.5 MS rats displayed lower SIRT3 expression level in the hippocampus and normalized by NAM administration. To assess the role of the SIRT3 in hippocampal neuronal phenotypes, we conducted co-labeling of SIRT3 and NeuN. Our findings revealed a notable decrease in the co-stained area in the hippocampus of MS + Vehicle rats during SIRT3 and NeuN co-labeling, which was mitigated by NAM treatment in MS + NAM rats (Fig. 2 A-D). 4.6 NAM administration normalized mitochondrial morphology in the hippocampal neurons of MS rats. Subsequently, we investigated the impact of early life stress on mitochondrial morphology in hippocampal neurons. No significant differences in mitochondrial density were observed among the four groups of hippocampal neurons (Fig. 2 E, F). However, the mitochondrial area was notably overexpressing in hippocampal neurons of MS rats (Fig. 2 E, J). Remarkably, NAM administration attenuated mitochondrial swelling in MS + NAM rats, thereby restoring the mitochondrial characteristics of MS + NAM rats to levels comparable to those of control rats (Fig. 2 E-G). 4.7 SIRT3 regulated microglial engulfment of hippocampal neuronal spines. In Experiment 2, our findings revealed that HNK administration augmented the proportion of co-stained SIRT3 and NeuN positive cells compared to the MS + Vehicle group (Fig. 3 A-D). No significant differences in mitochondrial density among hippocampal neurons were observed across the four groups (Fig. 3 E, F). However, the mitochondrial area notably reduced following HNK administration compared to the MS + Vehicle group (Fig. 3 E, G). Conversely, 3-TYP administration elevated the mitochondrial area in Control + 3-TYP rats compared to Control + Vehicle rats (Fig. 3 E, G). Moreover, 3-TYP administration elevated the co-labeled area of SYN + and CD68 + in the Control + 3-TYP group compared to Control + Vehicle rats (Supplementary Fig. 5A-D). HNK administration in MS + HNK rats suppressed the elevated co-labeled area of SYN + and CD68 + compared to the MS + Vehicle groups in the hippocampus (Supplementary Fig. 5A-D). 4.8 SIRT3 mediated NAD + stabilization influencing hippocampal neuronal dendritic complexity, LTP, and behavioral phenotypes. Neuronal analysis revealed spine restoration following 15 days of HNK administration in MS + HNK rats (Fig. 4 A, B), while Control + 3-TYP rats exhibited reduced spine density and less intricate dendritic arborization after 15 days of 3-TYP administration compared to Control + Vehicle rats (Fig. 4 A, B). Impaired LTP and heightened PPR were observed in Control + 3-TYP rats (Fig. 4 C-E, F), which were fully rescued by HNK administration (Fig. 4 C-E) with subsequent PPR restoration in MS + HNK rats (Fig. 4 F). Additionally, HNK administration reduced latency entering the target hole in the Barnes maze test for MS + HNK rats compared to MS + Vehicle rats (Fig. 4 G), while 3-TYP administration led to spatial learning and memory deficits with elevated latency in the Control + 3-TYP group versus Control + Vehicle rats (Fig. 4 G). During the novel object recognition test, all groups displayed similar exploration times for identical objects initially (Fig. 4 H, left panel). Post 24 hours, MS + HNK rats exhibited elevated interest in the novel object compared to MS + Vehicle rats, whereas Control + 3-TYP rats showed reduced exploration time when one object was replaced (Fig. 4 H, right panel). In the OFT, HNK administration reversed MS-induced total movement distance increments seen in MS + Vehicle rats (Fig. 4 I), while 3-TYP administration significantly elevated total distance moved in Control + 3-TYP rats compared to Control + Vehicle rats (Fig. 4 I). In the PPI test, HNK administration partially ameliorated impaired PPI in MS + HNK rats, whereas Control + 3-TYP rats displayed varying degrees of impaired PPI at different pre-pulse levels, indicating reduced startle stimulus inhibition compared to Control + Vehicle rats (Fig. 4 J). 4.9 SIRT3 inhibitor blocked NAM-induced restoration of microglial engulfment of hippocampal neuronal spines. In experiment 3, we sought to investigate the vital role of SIRT3 in enhancing hippocampal neuronal dendritic complexity and cognitive function associated with schizophrenia in NAM-induced MS rats. Our findings revealed that no significant differences in mitochondrial density were observed among the three groups of hippocampal neurons (Supplementary Fig. 6E, F). However, mitochondria in MS + NAM + 3-TYP rats exhibited swelling compared to those in MS + NAM rats (Supplementary Fig. 6E, G). Furthermore, 3-TYP administration led to an elevated co-labeled area of SYN + and CD68 + in MS + NAM + 3-TYP rats compared to the MS + NAM groups (Supplementary Fig. 7A-D). 4.10 SIRT3 inhibitor effectively blocked the NAM-induced restoration of hippocampal neuronal dendritic complexity, LTP, and behavioral phenotypes. Our findings demonstrated that 3-TYP administration in the MS + NAM + 3-TYP group hindered the restoration of spine numbers (Supplementary Fig. 8A, B), LTP (Supplementary Fig. 8C-E), and PPR (Supplementary Fig. 8F). Additionally, the results indicated that 3-TYP administration significantly impeded the restoration of cognitive impairment and schizophrenia-associated behavior in the MS + NAM rats. In the Barnes maze test, the MS + NAM + 3-TYP rats exhibited a longer escape latency to reach the target hole from day 1 to day 2 compared to the MS + NAM rats (Supplementary Fig. 8G). Results from the novel object recognition test revealed that the MS + NAM + 3-TYP rats spent less time exploring the novel object when one of the objects was replaced after 24 hours, in comparison to the MS + NAM rats (Supplementary Fig. 8H, right panel). In the OFT, the MS + NAM + 3-TYP rats showed an elevated total distance of spontaneous movement compared to the MS + NAM rats (Supplementary Fig. 8I). In the PPI test, the MS + NAM + 3-TYP rats exhibited impaired PPIs at varying pre-pulse intensities (Supplementary Fig. 8J). 4.11 SIRT3 signaling impact on neuronal plasticity and cognitive behavior. Knocking down SIRT3 in the CA1 region of the hippocampus resulted in reduced spine density (Fig. 5 C, D). This intervention also led to a reduction in latency in reaching the target hole during the Barnes maze test (Fig. 5 E). Moreover, the novel object recognition test showed that SIRT3 knockdown elevated the time rats spent investigating the novel object after 24 hours (Fig. 5 F). Additionally, the open field test indicated an increase in total movement distance following SIRT3 knockdown (Fig. 5 G). PPI testing revealed impaired prepulse inhibition after SIRT3 knockdown (Fig. 5 H). Conversely, neuronal analysis demonstrated a restoration in spine density upon SIRT3 overexpression in MS rats (Fig. 6 C, D). Furthermore, overexpressing of SIRT3 in MS rats reduced latency in reaching the target hole during the Barnes maze test (Fig. 6 E) and elevated exploration time of the novel object in the novel object recognition test after 24 hours (Fig. 6 F). Overexpression of SIRT3 also reversed the MS-induced increase in total movement distance in the open field test (Fig. 6 G) and partially ameliorated impaired prepulse inhibition in the PPI test in MS rats (Fig. 6 H). 5. Discussion The main finding of this study was the identification of the involvement of the mitochondrial NAD+/SIRT3 axis in the pathophysiological mechanisms associated with schizophrenia. The study also provided a mechanistic account of the neurobiological intermediaries from the organelle to the cellular and synaptic levels. We observed alterations in the mitochondrial NAD+/SIRT3 axis in the hippocampus, reduced hippocampal neuronal spine density, impaired LTP in the CA1 region, and cognitive behavior deficits in the MS rat model. Furthermore, our results demonstrated that supplementing NAD + or activating/overexpressing of SIRT3 can restore hippocampal neuronal synaptic plasticity and mitigate cognitive impairment associated with schizophrenia in MS rats. Conversely, inhibiting or knocking down SIRT3 activity can lead to deficits in hippocampal neuronal and behavioral phenotypes. Our data strongly suggested that the NAD+/SIRT3 axis could be a crucial therapeutic target for schizophrenia and its associated cognitive deficits. Mitochondrial dysfunction is a commonly reported phenomenon in schizophrenia [ 38 , 39 ]. Early life stress can induce various behavioral changes [ 29 – 32 , 40 ] and lead to alterations in gene expression and metabolism of mitochondria in different brain regions [ 41 , 42 ]. However, the specific causal role of mitochondrial mechanisms in stress-induced behavioral changes remains unclear. MS, a widely-used and well-documented rodent model, has been extensively reported to induce schizophrenia-related behaviors and cognitive deficits in adulthood [ 29 – 32 , 40 ]. Our findings supported these studies by demonstrating cognitive deficits in MS animals using the novel object recognition test and Barnes maze tests. The reductions in PPI further confirmed the validity of the schizophrenia model, as PPI impairments are observed in various neuropsychiatric disorders, including schizophrenia [ 43 , 44 ], and are commonly used as behavioral assays in animal models of schizophrenia [ 45 , 46 ]. Administration of NAM (a precursor of NAD+) normalized cognitive deficits, spontaneous activity, and impaired sensorimotor gating induced by MS. In addition, the SIRT3 activator or overexpression of SIRT3 attenuated most abnormal behaviors caused by MS. Meanwhile, the SIRT3 inhibitor or knocking down of SIRT3 led to spontaneous activity increase, abnormal spontaneous activity, PPI and impaired cognitive behaviors in adult control and NAM-treated MS rats. Therefore, a distinct contribution of our study was to have identified a novel role for the mitochondrial NAD+/SIRT3 axis in the regulation of early life stress-induced variation in behavior. Spine remodeling, an important biological process shaping brain connectivity, has also been suggested to underlie complex behaviors and cognitive functions, such as learning and memory [ 47 ]. Reduced spine densities and elevated immature spines on the subiculum and CA1 regions have been previously found in patients with schizophrenia [ 48 , 49 ]. The involvement of mitochondrial function in dendritic and spine complexity has been previously acknowledged for early development [ 13 , 14 , 17 – 19 ] and neurodegeneration [ 50 , 51 ]. Our study showed that early life stress induced a reduced dendritic spine density in hippocampal neurons at the adult stage. Reduced dendritic spine numbers may induce reduced postsynaptic transmission, which may cause a significant reduction in LTP induction and LTP maintenance [ 52 ]. Consistent with previous study, our results showed that early life stress induced impaired LTP in these neurons in the hippocampal CA1 region. However, the mitochondrial contribution to molecular mechanisms regulating spine stabilization and maturation have not been fully understood. SIRT3, the primary mitochondrial NAD+-dependent protein deacetylase, plays a crucial role in maintaining mitochondrial redox homeostasis by regulating the function of electron transport chain complexes I and III, thus preventing ROS generation within the mitochondria [ 53 , 54 ]. Our previous study has showed that SIRT3 inhibitor could induce ace-SOD2, ROS increase and mitochondria damage in the HT22 cells [ 21 ]. In this animal study, our results demonstrated that MS led to a decrease in SIRT3 levels in hippocampal neurons compared to control animals. Additionally, we observed mitochondrial swelling in the hippocampal neurons of MS rats. Studies suggest that mitochondria with a more rounded shape are often associated with less efficient bioenergetics [ 55 , 56 ] and ATP levels [ 57 ]. Additionally, we observed an increase in microglial contacts with dendrites and microglial phagocytosis of spines under early life stress. Microglia in the developing brain are highly mobile phagocytic cells with remarkably adaptable and versatile characteristics [ 58 ]. Apart from their notable functional adaptability, these cells are distinguished by a very low activation threshold, enabling them to carry out surveillance and scavenging functions effectively [ 59 ]. Studies suggest that the functional behavior of microglial cells may be influenced by neuronal activity [ 60 ]. Therefore, we hypothesized that alterations in the NAD+/SIRT3 axis within hippocampal neurons may contribute to the excessive engulfment of spines by microglia, leading to impaired LTP in the CA1 region and cognitive deficits in MS rats. To validate this hypothesis, we administered NAD + to adult MS rats. Our findings demonstrated the restoration of cognitive deficits, hippocampal neuronal synaptic plasticity, mitochondrial morphology, and NAD+/SIRT3 axis function in MS rats. Additionally, in order to further confirm the pivotal role of the NAD+/SIRT3 axis in regulating synaptic plasticity, we modulated the activity of SIRT3 in the animal models. Remarkably, in line with our hypothesis, the administration of a SIRT3 inhibitor to control and NAM-treated MS rats resulted in deficits in mitochondrial morphology, as well as in hippocampal neuronal and behavioral phenotypes. Activation of SIRT3 was able to reverse the mitochondrial, hippocampal neuronal, and behavioral phenotypes in MS rats. Furthermore, we conducted loss-of-function experiments through virus injection to investigate whether specific reduction of SIRT3 expression in hippocampal neurons replicated the behavioral and neuronal phenotypes observed in MS rats, which aligned with our initial predictions. Our findings indicated that dysfunction of the NAD+/SIRT3 axis in hippocampal neurons leads to excessive microglial engulfment of neuronal spines, ultimately resulting in impaired synaptic plasticity. However, this intricate process involves various molecules, such as the classic complement cascade-dependent phagocytic signaling, transforming growth factor β, chemokine signaling, and brain-derived neurotrophic factor, which either promote or inhibit the elimination of specific synaptic connections [ 37 , 61 – 65 ]. Further research is warranted to elucidate the precise mechanisms underlying the interactions between microglial cells and hippocampal neurons. 6. Conclusions Our study provided evidence that the NAD+/SIRT3 axis plays a crucial role in mediating mitochondrial and neuronal synaptic plasticity in response to early-life stress, and it significantly influenced cognitive behaviors. Moreover, we identified the NAD+/SIRT3 axis as a promising therapeutic target for addressing cognitive deficits associated with schizophrenia. Declarations Acknowledgements We thank Professor Gavin Reynolds for providing the assistance and advice in interpreting the findings and preparing the manuscript. Authors’ contributions G-H W, H-L W, Y-S L, F-S C and K-K H designed the study and wrote the protocol. K-K H and F-S C performed the experiments and analyzed the data. K-K H, H-H, R-X, Y-X, C-S, and S-L X performed literature searches and drew the figures. F-S C and K-K H wrote the manuscript. G-H W, H-L W, F-S C revised the manuscript. Funding The study was supported by the Medical Science Advancement Program of Wuhan University (NO. TFLC2018001). The Interdisciplinary Innovative Talents Foundation from Renmin Hospital of Wuhan University (JCRCFZ-2022-003) and the Key Research and Development Program of Hubei Province (2020BCA064). Availability of data and materials Data generated during the current study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate This work was carried out based on the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China, with the approval and consent to participate of the Ethics Committee in Renmin Hospital of Wuhan University. Consent for publication Not applicable. 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Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor. Cell. 2013;155:1596–609. Stephan AH, Barres BA, Stevens B. The complement system: an unexpected role in synaptic pruning during development and disease. Annu Rev Neurosci. 2012;35:369–89. Additional Declarations No competing interests reported. Supplementary Files SupplementalInformation.docx Supplementfigure1.png Supplementfigure2.png Supplementfigure3.png Supplementfigure4.png Supplementfigure5.png Supplementfigure6.png Supplementfigure7.png Supplementfigure8.png fulluncroppedGelsandBlotsimages.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4207040","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":289207634,"identity":"4b17385f-b833-449d-8877-ab761f63e029","order_by":0,"name":"Keke Hao","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Keke","middleName":"","lastName":"Hao","suffix":""},{"id":289207635,"identity":"bd5d45ef-e63f-4054-b38d-513d6c40fdcc","order_by":1,"name":"Fashuai Chen","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Fashuai","middleName":"","lastName":"Chen","suffix":""},{"id":289207637,"identity":"8200cf91-501e-46ed-8a91-87238c5938a3","order_by":2,"name":"Shilin Xu","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Shilin","middleName":"","lastName":"Xu","suffix":""},{"id":289207638,"identity":"cd000fe7-80a5-467f-baff-ac65b41963b4","order_by":3,"name":"Ying Xiong","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Xiong","suffix":""},{"id":289207639,"identity":"135f219e-c8f5-488c-952c-ef75023225b3","order_by":4,"name":"Rui Xu","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Xu","suffix":""},{"id":289207641,"identity":"d868bfd1-a8c8-4a24-8335-3c7b9cdf41db","order_by":5,"name":"Huan Huang","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Huang","suffix":""},{"id":289207645,"identity":"44136064-a5ce-431b-8b43-c260aa2b8195","order_by":6,"name":"Chang Shu","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Chang","middleName":"","lastName":"Shu","suffix":""},{"id":289207647,"identity":"d7810ab4-941d-4a7e-a43a-9fe4bd84abd1","order_by":7,"name":"Yisheng Lv","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yisheng","middleName":"","lastName":"Lv","suffix":""},{"id":289207648,"identity":"1f955845-5c85-4e50-be17-47a3e06ab7ad","order_by":8,"name":"Gaohua Wang","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Gaohua","middleName":"","lastName":"Wang","suffix":""},{"id":289207650,"identity":"a2ad54c5-c48e-49b4-9ea3-946781b10d32","order_by":9,"name":"Huiling Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACA3YwZcNgAKJ4iNLCDKbSSNdymAQt5sw8hp8Lfp1P3C6RwPjgbRuDvDkhLZbNPMbSM/tuJ+6ckcBsOLeNwXBnAyGHHebdIM3bczt3w40ENmneNoYEgwOEtWz+zdtzDqSF/TexWrZJ8/w4ALaFmUgt/N+seRuS6zecedgsOeechOEGglqOtyXf5vljZ2xwPPnghzdlNvIEbQEDxjYw2QAkJIhRDwJ/iFU4CkbBKBgFIxIAAHQuQQJDyUYPAAAAAElFTkSuQmCC","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":true,"prefix":"","firstName":"Huiling","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-04-02 13:30:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4207040/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4207040/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54527786,"identity":"8fe13054-f13b-484d-ab1a-ac09235ae741","added_by":"auto","created_at":"2024-04-11 21:37:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1121913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdministration of NAM restored behavioral and hippocampal cellular phenotypes affected by MS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) NAM administration in the CA1 region MS induced memory deficit, evaluated by Barnes maze test (n = 15, per group).\u003csup\u003e \u0026amp; \u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e\u0026amp;\u0026amp; \u003c/sup\u003ep \u0026lt; 0.01 as CON+Vehicle rats compared to the MS+Vehicle rats, \u003csup\u003e# \u003c/sup\u003ep \u0026lt; 0.05 as MS+Vehicle rats compared to the MS+NAM rats.\u003c/p\u003e\n\u003cp\u003e(B) NAM administration reversed MS induced memory deficit, evaluated by the novel object recognition test. The left panel presented the time spent exploring the two identical sample objects within the 10-min period on day 2. The right panel presented the time spent exploring novel and old objects during the 10-min period on day 3 (n = 15, per group).\u003c/p\u003e\n\u003cp\u003e(C) NAM reversed MS induced hyperlocomotion, revealed by the total distances traveled in the OFT test (n = 15, per group).\u003c/p\u003e\n\u003cp\u003e(D) NAM reversed MS induced PPI deficits at different pre-pulse intensities (n = 12, per group).\u003c/p\u003e\n\u003cp\u003e(E) Representative trajectories of baseline (top) and post-HFS (down) fEPSP, recorded from hippocampal CA1 region in brain slices at the adult rats. Scales: 0.02 mV, 2 ms.\u003c/p\u003e\n\u003cp\u003e(F) NAM administration reversed MS induced LTP impairment. And NAM administration not change LTP in group CON+NAM compared with control. Arrows indicate LTP induction. (n = 4 per group).\u003csup\u003e \u0026amp;\u0026amp;\u0026amp; \u003c/sup\u003ep \u0026lt; 0.001 as CON+Vehicle rats compared to the MS+Vehicle rats, \u003csup\u003e### \u003c/sup\u003ep \u0026lt; 0.001 as MS+Vehicle rats compared to the MS+NAM rats.\u003c/p\u003e\n\u003cp\u003e(G) Quantitative analysis of data in (F).\u003c/p\u003e\n\u003cp\u003e(H) NAM administration reversed MS induced PPR impairment (n = 4 per group).\u003c/p\u003e\n\u003cp\u003e(I) The representative micrographs of hippocampal dendrites in the CA1 region. Scale bar, 2 μm.\u003c/p\u003e\n\u003cp\u003e(J) NAM administration reversed MS induced spine density decrease (n = 5, per group).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4207040/v1/b8cb367c0dd7669cfb984c04.png"},{"id":54527788,"identity":"5606ba63-8e04-4613-87db-bf61b459d05a","added_by":"auto","created_at":"2024-04-11 21:37:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7711554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNAM administration restored SIRT3 expressions in neuronal cells and mitochondrial morphology in the hippocampus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative immunofluorescence images show the expression of NeuN+ (green pixels), SIRT3+ (red pixels), and DAPI (blue) in the hippocampus of the four groups.\u003c/p\u003e\n\u003cp\u003e(B) NAM administration did not affect the percentage of neuronal cells area in the hippocampus (n = 4, per group).\u003c/p\u003e\n\u003cp\u003e(C) NAM administration normalized the percentage of SIRT3+ area in the hippocampus induced by MS (n = 4, per group).\u003c/p\u003e\n\u003cp\u003e(D) NAM administration normalized the percentage of NeuN+ and SIRT3+ co-labeling area in the hippocampus induced by MS (n = 4, per group).\u003c/p\u003e\n\u003cp\u003e(E) Representative electron micrographs from the hippocampal neurons in CON+Vehicle, MS+Vehicle, MS+NAM, and CON+NAM rats.\u003c/p\u003e\n\u003cp\u003e(F) Mitochondria density was comparable in the four groups (n = 4, per group).\u003c/p\u003e\n\u003cp\u003e(G) Mitochondria area was comparable in the four groups (n = 4, per group).\u003c/p\u003e\n\u003cp\u003eThe data are presented as mean ± SEM for each group. n.s. was not significant; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4207040/v1/99c6088ad861833402d42045.png"},{"id":54527789,"identity":"809fc52c-18b9-4b87-aedd-dc64182224a9","added_by":"auto","created_at":"2024-04-11 21:37:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8626157,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of SIRT3 activation and inhibition on neuronal cells and mitochondrial morphology in the hippocampus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative immunofluorescence images show the expression of NeuN+ (green pixels) and SIRT3+ (red pixels) in the hippocampus.\u003c/p\u003e\n\u003cp\u003e(B) HNK and 3-TYP administration had no effect on the percentage of neuronal cells area in the hippocampus (n = 4, per group).\u003c/p\u003e\n\u003cp\u003e(C) Elevated percentage of SIRT3+ area in the hippocampus after HNK administration on MS rats (n = 4, per group).\u003c/p\u003e\n\u003cp\u003e(D) Elevated percentage of NeuN+ and SIRT3+ co-labeling area in the hippocampus after HNK administration on MS rats (n = 4, per group).\u003c/p\u003e\n\u003cp\u003e(E) Representative electron micrographs from the hippocampal neurons in CON+Vehicle, MS+Vehicle, MS+HNK, and CON+3-TYP rats.\u003c/p\u003e\n\u003cp\u003e(F) Mitochondria density was comparable in the four groups (n = 4, per group).\u003c/p\u003e\n\u003cp\u003e(G) Mitochondria area was comparable in the four groups (n = 4, per group).\u003c/p\u003e\n\u003cp\u003eThe data are presented as mean ± SEM for each group. n.s. was not significant; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4207040/v1/02bc1143fece173338bc56a5.png"},{"id":54527790,"identity":"8f3f7394-2d64-4103-8de6-b13e2568eb7f","added_by":"auto","created_at":"2024-04-11 21:37:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1188594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of SIRT3 activation and inhibition on cellular and behavioral phenotypes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The representative micrographs of hippocampal dendrites in the CA1 region. Scale bar, 2 μm.\u003c/p\u003e\n\u003cp\u003e(B) Restoration of spine density after HNK administration on MS rats. And reduced spine density after 3-TYP administration on control rats (n = 5, per group).\u003c/p\u003e\n\u003cp\u003e(C) Representative trajectories of baseline (top) and post-HFS (down) fEPSP, recorded from hippocampal CA1 region in brain slices at the adult rats. Scales: 0.02 mV, 2 ms.\u003c/p\u003e\n\u003cp\u003e(D) Restoration of LTP after HNK administration on MS rats. And impairment of LTP after 3-TYP administration on control rats. Arrows indicate LTP induction. (n = 4 per group). \u003csup\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/sup\u003ep \u0026lt; 0.001 as CON+Vehicle rats compared to the MS+Vehicle rats,\u003csup\u003e ##\u003c/sup\u003ep \u0026lt; 0.01 as MS+Vehicle rats compared to the MS+HNK rats, \u003csup\u003e$$\u003c/sup\u003ep \u0026lt; 0.01 as CON+Vehicle rats compared to the CON+3-TYP rats.\u003c/p\u003e\n\u003cp\u003e(E) Quantitative analysis of data in (D).\u003c/p\u003e\n\u003cp\u003e(F) Effects of HNK and 3-TYP administration on the PPR (n = 4 per group).\u003c/p\u003e\n\u003cp\u003e(G) Reduced latency to the target hole in the Barnes maze test after HNK administration on MS rats. And elevated latency to the target hole in the Barnes maze test after 3-TYP administration on control rats (n = 15, per group).\u003csup\u003e \u0026amp;\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003ep \u0026lt; 0.01 as CON+Vehicle rats compared to the MS+Vehicle rats,\u003csup\u003e ##\u003c/sup\u003ep \u0026lt; 0.01, and \u003csup\u003e####\u003c/sup\u003ep \u0026lt; 0.001 as MS+Vehicle rats compared to the MS+HNK rats. \u003csup\u003e$\u003c/sup\u003ep \u0026lt; 0.05, and \u003csup\u003e$$\u003c/sup\u003ep \u0026lt; 0.01 as CON+Vehicle rats compared to the CON+3-TYP rats.\u003c/p\u003e\n\u003cp\u003e(H) Elevated discrimination ratio in the novel object recognition test after HNK administration on MS rats. And reduced discrimination ratio in the novel object recognition test after 3-TYP administration on control rats (n = 15, per group).\u003c/p\u003e\n\u003cp\u003e(I) Reduced total distances traveled during the OFT after HNK administration on MS rats. And elevated total distances traveled during the OFT after 3-TYP administration on control rats (n = 15, per group).\u003c/p\u003e\n\u003cp\u003e(J) Restoration of PPI deficits after HNK administration on MS rats. And impairment of PPI at different pre-pulse intensities after 3-TYP administration on control rats (n = 12, per group).\u003c/p\u003e\n\u003cp\u003eThe data are represented as the mean ± SEM. n.s. was not significant; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001.\u003csup\u003e \u003c/sup\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4207040/v1/3d3e3081f8749b4f6ed32a4c.png"},{"id":54527993,"identity":"f09d6175-7fc4-404a-a268-8c9a2dcdab68","added_by":"auto","created_at":"2024-04-11 21:45:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1057303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnocking down SIRT3 in the CA1 region of the hippocampus induced deficits in neuronal plasticity and cognitive behavior.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) AAV carrying EGFP was injected into the CA1 region of 8-wk-old rats and was observed after 3 weeks. The EGFP was expressed in different coronal sections of the hippocampus limited to the CA1 region.\u003c/p\u003e\n\u003cp\u003e(B) Representative immunoblots of SIRT3 protein expression in the hippocampus from the above rats after AAV-Nc or AAV-SIRT3 injection.\u003c/p\u003e\n\u003cp\u003e(C) The representative micrographs of hippocampal dendrites in the CA1 region. Scale bar, 2 μm.\u003c/p\u003e\n\u003cp\u003e(D) Quantitative analysis of the spine density about hippocampal dendrites in the CA1 region (n = 5, per group).\u003c/p\u003e\n\u003cp\u003e(E) Knocking down of SIRT3 in the CA1 region of the hippocampus of CON rats elevated latency to the target hole in the Barnes maze test (CON+saline n = 6, CON+si-Nc n = 6, CON+si-SIRT3 n = 13).\u003c/p\u003e\n\u003cp\u003e(F) Knocking down of SIRT3 in the CA1 region of the hippocampus of CON rats reduced discrimination ratio in the novel object recognition test (CON+saline n = 6, CON+si-Nc n = 6, CON+si-SIRT3 n = 13).\u003c/p\u003e\n\u003cp\u003e(G) Knocking down of SIRT3 in the CA1 region of the hippocampus of CON rats elevated total distances traveled during the OFT (CON+saline n = 6, CON+si-Nc n = 6, CON+si-SIRT3 n = 13).\u003c/p\u003e\n\u003cp\u003e(H) Knocking down of SIRT3 in the CA1 region of the hippocampus of CON rats induced PPI deficits (CON+saline n = 6, CON+si-Nc n = 6, CON+si-SIRT3 n = 13).\u003c/p\u003e\n\u003cp\u003eThe data are represented as the mean ± SEM. n.s. was not significant; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4207040/v1/e24dc6513cc952b9f0957b49.png"},{"id":54527791,"identity":"7c482e14-1736-41ce-bc78-af491af3aae3","added_by":"auto","created_at":"2024-04-11 21:37:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1250437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpressing of SIRT3 in the CA1 region of the hippocampus alleviated ELS-induced deficits in neuronal plasticity and cognitive behavior in MS rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) AAV carrying EGFP was injected into the CA1 region of 8-wk-old rats and was observed after 3 weeks. The EGFP was expressed in different coronal sections of the hippocampus limited to the CA1 region.\u003c/p\u003e\n\u003cp\u003e(B) Representative immunoblots of SIRT3 protein expression in the hippocampus from the above rats after AAV-Nc or AAV-SIRT3 injection.\u003c/p\u003e\n\u003cp\u003e(C) The representative micrographs of hippocampal dendrites in the CA1 region. Scale bar, 2 μm.\u003c/p\u003e\n\u003cp\u003e(D) Quantitative analysis of the spine density about hippocampal dendrites in the CA1 region (n = 5, per group).\u003c/p\u003e\n\u003cp\u003e(E) Overexpressing of SIRT3 in the CA1 region of the hippocampus of MS rats reduced latency to the target hole in the Barnes maze test (CON+aav9-Nc n = 9, MS+aav9-Nc n = 8, MS+aav9-SIRT3 n = 13, CON+aav9-SIRT3 n = 7).\u0026nbsp; \u003csup\u003e\u0026amp;\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003ep \u0026lt; 0.01 as MS+aav9-Nc rats compared to the CON+aav9-Nc rats,\u003csup\u003e #\u003c/sup\u003ep \u0026lt; 0.005,\u003csup\u003e ##\u003c/sup\u003ep \u0026lt; 0.01, and \u003csup\u003e####\u003c/sup\u003ep \u0026lt; 0.001 as MS+aav9-SIRT3 rats compared to the MS+aav9-Nc rats.\u003c/p\u003e\n\u003cp\u003e(F) Overexpressing of SIRT3 in the CA1 region of the hippocampus of MS rats elevated discrimination ratio in the novel object recognition test (CON+aav9-Nc n = 9, MS+aav9-Nc n = 8, MS+aav9-SIRT3 n = 13, CON+aav9-SIRT3 n = 7).\u003c/p\u003e\n\u003cp\u003e(G) Overexpressing of SIRT3 in the CA1 region of the hippocampus of MS rats reduced total distances traveled during the OFT (CON+aav9-Nc n = 9, MS+aav9-Nc n = 8, MS+aav9-SIRT3 n = 13, CON+aav9-SIRT3 n = 7).\u003c/p\u003e\n\u003cp\u003e(H) PPI deficits recovered after overexpressing of SIRT3 in the CA1 region of the hippocampus of MS rats (CON+aav9-Nc n = 9, MS+aav9-Nc n = 8, MS+aav9-SIRT3 n = 13, CON+aav9-SIRT3 n = 7).\u003c/p\u003e\n\u003cp\u003eThe data are represented as the mean ± SEM.\u0026nbsp; n.s. was not significant; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4207040/v1/55afe58fc4ee8b1c4b475fe9.png"},{"id":55264475,"identity":"3986dffc-e604-401d-9c73-80caca783ef9","added_by":"auto","created_at":"2024-04-25 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21:37:48","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":24325509,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementfigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4207040/v1/f526098900ecd57fa5489fb8.png"},{"id":54527792,"identity":"8b869d33-92f5-4874-80a3-d83e5b8e4338","added_by":"auto","created_at":"2024-04-11 21:37:46","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":992272,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementfigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4207040/v1/67325d8741508259a7f60ce2.png"},{"id":54527793,"identity":"abc16f39-6d73-4a7c-83a0-b9fb9817900b","added_by":"auto","created_at":"2024-04-11 21:37:47","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":92316,"visible":true,"origin":"","legend":"","description":"","filename":"fulluncroppedGelsandBlotsimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4207040/v1/fb8acac4690fb6f9bddb8edd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cognitive impairment following maternal separation in the rat is regulated by effects of the NAD + /SIRT3 axis on hippocampal synaptic plasticity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCognitive impairment is a common feature of severe mental illness. Maternal separation (MS) is an early life intervention that can produce in behaviors of adult animals reminiscent of schizophrenia, including cognitive deficits [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Cognitive impairment has been considered as a core symptom domain of schizophrenia [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] in which the hippocampus has been strongly implicated [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Hippocampal neuronal dysfunction is also associated with cognitive deficits in animal models of schizophrenia. In particular, the impairment of hippocampal neuronal dendritic complexity has been shown to contribute causally to stress-induced cognitive deficits [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Given that neuronal morphology is a strong determinant of synaptic connectivity and strength, understanding the factors that control hippocampal neuronal dendritic regulation may help in developing treatments to ameliorate cognitive deficits.\u003c/p\u003e \u003cp\u003eMitochondria appear to be ideally suited to contribute. Increasing evidence points to a central role of mitochondria in the etiology of psychiatric disorders [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In neurons, mitochondria supports metabolic demands through energy supply [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Cell cultures and neurodevelopmental studies have implicated mitochondria in the regulation of both dendritic arborization [\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and spine and synapse formation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, whether mitochondrial dysfunction impairs hippocampal neuronal dendritic complexity and consequently, contributes to cognitive deficits associated with schizophrenia remains unknown.\u003c/p\u003e \u003cp\u003eOur previous studies have suggested that degradation of nicotinamide adenine dinucleotide (NAD+) and associated bioenergetics failure of cellular metabolism may be one of the major factors leading to neuronal damage [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. NAD\u0026thinsp;+\u0026thinsp;is an essential cofactor in most enzymatic reactions supporting fundamental mitochondrial functions including oxidative phosphorylation and enzymatic reactions of the tricarboxylic acid cycle [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. When NAD\u0026thinsp;+\u0026thinsp;is degraded, mitochondria become incapable of ATP synthesis. Previous study has demonstrated that when na\u0026iuml;ve mice were treated with an NAD\u0026thinsp;+\u0026thinsp;precursor, neuronal mitochondrial function recovers [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and an increase in mitochondrial NAD\u0026thinsp;+\u0026thinsp;will reduce acetylation of mitochondrial proteins and ROS generation in hippocampal tissue [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Furthermore, a high NAD\u0026thinsp;+\u0026thinsp;level facilitates the activation of proteins involved in mitochondrial quality control, such as Sirtuin3 (SIRT3). SIRT3 reinforces mitochondrial antioxidant defense by deacetylating and increasing the activity of superoxide dismutase 2 (SOD2) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, it is not clear whether the mitochondria-related NAD+/SIRT3 axis is involved in hippocampal neuronal synaptic plasticity and the schizophrenia-associated cognitive impairment.\u003c/p\u003e \u003cp\u003eMother-infant interaction may be a key factor in brain maturation, and stress associated with MS may induce the development of psychosis or susceptibility to psychotic diseases [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The abnormal behaviors and molecular changes taking place after a single 24 h period of MS on a postnatal day (PND) 9 in rats successfully recapitulate several features of schizophrenia [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, the MS rat model has become a powerful tool for exploring the neurobiological bases of schizophrenia.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.1 Animals and m\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eaternal \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003es\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eeparation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForty nulliparous female and forty male eight-week-old Wistar rats were obtained from Beijing Vital Rival Laboratory Animal Technology Co., Ltd. (Beijing, China). Rats of the same sex were caged together, with 3 or 4 per cage. The animals were mated at age of 3 months and the males were removed one week later. The mated female rats were housed individually in ventilated plastic cages in a temperature- and humidity-controlled (22 \u0026plusmn; 20℃, 50 \u0026plusmn; 10%) holding facility with a constant 12h day-night cycle (lights: 08:00 \u0026ndash; 20:00). All animals had free access to food and tap water. The MS protocols were performed according to previous research [30, 32]. Females were checked twice daily for delivery (08:00 and 17:00). The day of delivery was considered as PND 0. Each pregnant rat provided on average 10 \u0026plusmn; 2 offspring. On PND9, litters were randomly assigned to either the MS or control groups. In brief, the mothers were removed at 10:00. The pups remained in their home cages with the heated mat for 24h, after which the mothers were returned to their cages. The control groups grew naturally to adulthood. All the litters were otherwise left undisturbed except for the routine cleaning of the cages. On PND 21, the MS and the control groups of pups were weaned, and then group-housed by sex (3-4 per cage). All the following determinations were carried out only on male offspring avoiding the effects of estrogen in regulating neuronal activity and animal behavior [33, 34]. All procedures involving animals were approved and carried out according to the guidelines of the Institutional Animals Care Committee of Renmin Hospital of Wuhan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.2 Experimental design\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiment 1: On postnatal day 9 (PND9), the dams and their pups were randomly assigned to either the control group or the maternal separation (MS) groups. On PND10 after MS, each infant group was further divided into several subgroups with approximately 20 pups in each subgroup. The resulting four groups were as follows: control group receiving vehicle (Control+Vehicle), MS group receiving vehicle (MS+Vehicle), control group receiving nicotinamide (NAM) (Control+NAM), and MS group receiving NAM (MS+NAM). The NAM groups received oral gavage of NAM (100mg/kg/d, diluted in vehicle) for 30 days from PND56 to PND85. The dosage and treatment protocol of NAM were based on previous studies [35, 36]. The vehicle groups received daily saline (1ml/kg) from PND56 to PND85 (see supplementary Figure 1). \u003c/p\u003e\n\u003cp\u003eExperiment 2: Pups in both the MS and control groups were randomly divided into two subgroups of 15. The MS animals received intraperitoneal (i.p.) injections of either honokiol (HNK) (10mg/kg/d) or vehicle, while the control animals received 3-TYP (10mg/kg/d) or vehicle for 15 days. The vehicle solution consisted of 90% saline and 10% DMSO (1ml/kg/d) (see supplementary Figure 1).\u003c/p\u003e\n\u003cp\u003eExperiment 3: The 36 MS pups were divided into three groups: MS+Vehicle, MS+NAM, and MS+NAM+3-TYP. The MS+NAM+3-TYP group received NAM by gavage for 30 days and intraperitoneal (i.p.) injections of 3-TYP (10mg/kg) during the last 15 days. The MS+NAM group received NAM by gavage for 30 days. The MS+Vehicle group received saline (1ml/kg/d) by gavage for 30 days and the vehicle solution (90% saline and 10% DMSO at 1ml/kg/d i.p.) for the last 15 days (see supplementary Figure 1). \u003c/p\u003e\n\u003cp\u003eExperiment 4: AAV9-SIRT3 and AAV9-Nc were stereotactically injected into mice under isoflurane anesthesia. The bilateral hippocampal CA1 region injections were performed at the following coordinates: \u0026minus;2.4 mm anteroposterior, \u0026minus;3.75 mm mediolateral from the bregma, and \u0026minus;2.6 mm dorsoventral from the dural surface. A viral suspension (1\u0026mu;l) containing 2 \u0026times; 109 vector genomes per \u0026mu;L was infused into each site at a rate of 0.25\u0026mu;l/min using a 10\u0026mu;l glass syringe with a fixed needle. After the injection, the needle was left in place for 10 min and then slowly removed over 2 min. Rats were kept on a heating pad until they fully recovered from anesthesia. Three weeks following the stereotactic injection, a multiple behavior test was conducted on the rats (see supplementary Figure 1).\u003c/p\u003e\n\u003cp\u003eFor the rest of the detailed methods, please refer to the Supplemental Information of this paper, which covers the following aspects:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.3 Behavioral testing of animals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2.3.1 Open-field Test (OFT) \u003c/p\u003e\n\u003cp\u003e2.3.2 Novel object recognition test\u003c/p\u003e\n\u003cp\u003e2.3.3 Barnes maze test\u003c/p\u003e\n\u003cp\u003e2.3.4 Elevated-Plus Maze (EPM)\u003c/p\u003e\n\u003cp\u003e2.3.5 Sucrose preference Test (SPT)\u003c/p\u003e\n\u003cp\u003e2.3.6 PPI test\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.4 NAD\u003csup\u003e+\u003c/sup\u003e quantification\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.5 Protein extraction and western blot analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.6 Electron microscopy\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.7 Immunofluorescent staining assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.8 Golgi-cox staining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.9 Electrophysiology\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"3. Data analysis and statistics","content":"\u003cp\u003eThe data are reported as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) and were analyzed using SPSS Statistics version 20.0 (SPSS Inc.). Significance in the datasets was assessed through Student\u0026rsquo;s unpaired two-tailed t-tests for comparisons between two groups, while one-way or two-way analysis of variance (ANOVA) was utilized for comparisons involving three or more groups. Statistical significance was considered at a p-value below 0.05.\u003c/p\u003e"},{"header":"4. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Reduced SIRT3 expression observed in the individuals with schizophrenia.\u003c/h2\u003e \u003cp\u003eTo investigate changes in SIRT3 gene expression in the postmortem hippocampus of schizophrenia patients, an analysis was conducted using the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The database GSE53987 included gene expression data from 15 schizophrenia patients and 18 matched healthy controls. In comparison to age- and sex-matched controls, a significant decrease in SIRT3 mRNA levels was observed in the hippocampus (Supplementary Fig.\u0026nbsp;2A), but not in the prefrontal cortex (Supplementary Fig.\u0026nbsp;2B), suggesting potential involvement of hippocampal SIRT3 in schizophrenia pathology. Furthermore, a clinical study revealed reduced SIRT3 protein expression in peripheral blood mononuclear cells (PBMCs) among schizophrenia patients as compared to the healthy controls (Supplementary Fig.\u0026nbsp;2C, D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.2 NAM normalized cognitive impairment and schizophrenia-associated behaviors induced by MS in rats.\u003c/h2\u003e \u003cp\u003eTo investigate the role of SIRT3 in the pathogenesis of schizophrenia, we employed the MS rat model, known for its ability to replicate key features of schizophrenia [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. No differences in body weight (Supplementary Fig.\u0026nbsp;3A) or anxiety/depression-like behaviors (Supplementary Fig.\u0026nbsp;3B-H) were observed between the MS and control groups. However, MS induced schizophrenia-associated behavioral phenotypes in rats, including impairments in memory, cognition, hyperlocomotion, and sensory gating defects. Remarkably, all these phenotypes were reversed by administering NAM at 100 mg/kg/d for 30 days. In the Barnes maze test, MS rats displayed deficits in spatial learning and memory, as evidenced by an elevated latency to escape from day 1 to day 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), which was normalized by NAM treatment. Importantly, NAM did not reduce latency in the absence of MS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Cognitive function in MS rats was further evaluated using the novel object recognition test. All groups exhibited similar exploration times for two identical objects during the adaptation stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, left panel). However, the MS group spent relatively less time exploring a novel object when one of the objects was replaced after 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, right panel). NAM reversed the reduced exploration time of the novel object in the MS group after 24 hours, but had no effect on the exploration time in the group without MS treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, right panel). Similarly, in the OFT, NAM reversed the MS-induced increase in total movement distance, without affecting the untreated MS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Finally, MS rats exhibited impaired PPIs at various pre-pulse intensities, which were fully reversed following NAM administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.3 NAM administration restored LTP and hippocampal neuronal dendritic complexity in MS rats.\u003c/h2\u003e \u003cp\u003eIn terms of the synaptic transmission and plasticity characteristics of CA1 synapses, MS rats displayed impaired LTP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G) and elevated paired-pulse ratio (PPR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). However, NAM treatment effectively reversed the LTP impairment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G) and normalized the PPR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Consistent with these findings, neuronal structure analysis revealed a reduced spine density in MS rats compared to control rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J), with spine density recovering after NAM treatment in MS\u0026thinsp;+\u0026thinsp;NAM rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.4 NAM administration normalized microglial engulfment of hippocampal neuronal spines of MS rats.\u003c/h2\u003e \u003cp\u003eMicroglia play a critical role in the synaptic pruning process [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. To investigate the underlying cause of reduced hippocampal neuronal spines, we assessed the phagocytic function of microglial cells through immune co-labeling of CD68 and SYN in the hippocampus. Our findings demonstrated an elevated co-labeled area of SYN\u0026thinsp;+\u0026thinsp;and CD68\u0026thinsp;+\u0026thinsp;in the hippocampus of the MS\u0026thinsp;+\u0026thinsp;Vehicle group compared to the Control\u0026thinsp;+\u0026thinsp;Vehicle rats (Supplementary Fig.\u0026nbsp;4A-D). However, daily administration of NAM via gavage (from PND56 to PND85) in MS\u0026thinsp;+\u0026thinsp;NAM rats significantly reduced the co-labeled area of SYN\u0026thinsp;+\u0026thinsp;and CD68\u0026thinsp;+\u0026thinsp;in the hippocampus compared to the MS\u0026thinsp;+\u0026thinsp;Vehicle group (Supplementary Fig.\u0026nbsp;4A-D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.5 MS rats displayed lower SIRT3 expression level in the hippocampus and normalized by NAM administration.\u003c/h2\u003e \u003cp\u003eTo assess the role of the SIRT3 in hippocampal neuronal phenotypes, we conducted co-labeling of SIRT3 and NeuN. Our findings revealed a notable decrease in the co-stained area in the hippocampus of MS\u0026thinsp;+\u0026thinsp;Vehicle rats during SIRT3 and NeuN co-labeling, which was mitigated by NAM treatment in MS\u0026thinsp;+\u0026thinsp;NAM rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.6 NAM administration normalized mitochondrial morphology in the hippocampal neurons of MS rats.\u003c/h2\u003e \u003cp\u003eSubsequently, we investigated the impact of early life stress on mitochondrial morphology in hippocampal neurons. No significant differences in mitochondrial density were observed among the four groups of hippocampal neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). However, the mitochondrial area was notably overexpressing in hippocampal neurons of MS rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, J). Remarkably, NAM administration attenuated mitochondrial swelling in MS\u0026thinsp;+\u0026thinsp;NAM rats, thereby restoring the mitochondrial characteristics of MS\u0026thinsp;+\u0026thinsp;NAM rats to levels comparable to those of control rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.7 SIRT3 regulated microglial engulfment of hippocampal neuronal spines.\u003c/h2\u003e \u003cp\u003eIn Experiment 2, our findings revealed that HNK administration augmented the proportion of co-stained SIRT3 and NeuN positive cells compared to the MS\u0026thinsp;+\u0026thinsp;Vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). No significant differences in mitochondrial density among hippocampal neurons were observed across the four groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). However, the mitochondrial area notably reduced following HNK administration compared to the MS\u0026thinsp;+\u0026thinsp;Vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, G). Conversely, 3-TYP administration elevated the mitochondrial area in Control\u0026thinsp;+\u0026thinsp;3-TYP rats compared to Control\u0026thinsp;+\u0026thinsp;Vehicle rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, G). Moreover, 3-TYP administration elevated the co-labeled area of SYN\u0026thinsp;+\u0026thinsp;and CD68\u0026thinsp;+\u0026thinsp;in the Control\u0026thinsp;+\u0026thinsp;3-TYP group compared to Control\u0026thinsp;+\u0026thinsp;Vehicle rats (Supplementary Fig.\u0026nbsp;5A-D). HNK administration in MS\u0026thinsp;+\u0026thinsp;HNK rats suppressed the elevated co-labeled area of SYN\u0026thinsp;+\u0026thinsp;and CD68\u0026thinsp;+\u0026thinsp;compared to the MS\u0026thinsp;+\u0026thinsp;Vehicle groups in the hippocampus (Supplementary Fig.\u0026nbsp;5A-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.8 SIRT3 mediated NAD\u0026thinsp;+\u0026thinsp;stabilization influencing hippocampal neuronal dendritic complexity, LTP, and behavioral phenotypes.\u003c/h2\u003e \u003cp\u003eNeuronal analysis revealed spine restoration following 15 days of HNK administration in MS\u0026thinsp;+\u0026thinsp;HNK rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B), while Control\u0026thinsp;+\u0026thinsp;3-TYP rats exhibited reduced spine density and less intricate dendritic arborization after 15 days of 3-TYP administration compared to Control\u0026thinsp;+\u0026thinsp;Vehicle rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Impaired LTP and heightened PPR were observed in Control\u0026thinsp;+\u0026thinsp;3-TYP rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E, F), which were fully rescued by HNK administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E) with subsequent PPR restoration in MS\u0026thinsp;+\u0026thinsp;HNK rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Additionally, HNK administration reduced latency entering the target hole in the Barnes maze test for MS\u0026thinsp;+\u0026thinsp;HNK rats compared to MS\u0026thinsp;+\u0026thinsp;Vehicle rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), while 3-TYP administration led to spatial learning and memory deficits with elevated latency in the Control\u0026thinsp;+\u0026thinsp;3-TYP group versus Control\u0026thinsp;+\u0026thinsp;Vehicle rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). During the novel object recognition test, all groups displayed similar exploration times for identical objects initially (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, left panel). Post 24 hours, MS\u0026thinsp;+\u0026thinsp;HNK rats exhibited elevated interest in the novel object compared to MS\u0026thinsp;+\u0026thinsp;Vehicle rats, whereas Control\u0026thinsp;+\u0026thinsp;3-TYP rats showed reduced exploration time when one object was replaced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, right panel). In the OFT, HNK administration reversed MS-induced total movement distance increments seen in MS\u0026thinsp;+\u0026thinsp;Vehicle rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI), while 3-TYP administration significantly elevated total distance moved in Control\u0026thinsp;+\u0026thinsp;3-TYP rats compared to Control\u0026thinsp;+\u0026thinsp;Vehicle rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). In the PPI test, HNK administration partially ameliorated impaired PPI in MS\u0026thinsp;+\u0026thinsp;HNK rats, whereas Control\u0026thinsp;+\u0026thinsp;3-TYP rats displayed varying degrees of impaired PPI at different pre-pulse levels, indicating reduced startle stimulus inhibition compared to Control\u0026thinsp;+\u0026thinsp;Vehicle rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.9 SIRT3 inhibitor blocked NAM-induced restoration of microglial engulfment of hippocampal neuronal spines.\u003c/h2\u003e \u003cp\u003eIn experiment 3, we sought to investigate the vital role of SIRT3 in enhancing hippocampal neuronal dendritic complexity and cognitive function associated with schizophrenia in NAM-induced MS rats. Our findings revealed that no significant differences in mitochondrial density were observed among the three groups of hippocampal neurons (Supplementary Fig.\u0026nbsp;6E, F). However, mitochondria in MS\u0026thinsp;+\u0026thinsp;NAM\u0026thinsp;+\u0026thinsp;3-TYP rats exhibited swelling compared to those in MS\u0026thinsp;+\u0026thinsp;NAM rats (Supplementary Fig.\u0026nbsp;6E, G). Furthermore, 3-TYP administration led to an elevated co-labeled area of SYN\u0026thinsp;+\u0026thinsp;and CD68\u0026thinsp;+\u0026thinsp;in MS\u0026thinsp;+\u0026thinsp;NAM\u0026thinsp;+\u0026thinsp;3-TYP rats compared to the MS\u0026thinsp;+\u0026thinsp;NAM groups (Supplementary Fig.\u0026nbsp;7A-D).\u003c/p\u003e \u003cp\u003e4.10 SIRT3 inhibitor effectively blocked the NAM-induced restoration of hippocampal neuronal dendritic complexity, LTP, and behavioral phenotypes.\u003c/p\u003e \u003cp\u003eOur findings demonstrated that 3-TYP administration in the MS\u0026thinsp;+\u0026thinsp;NAM\u0026thinsp;+\u0026thinsp;3-TYP group hindered the restoration of spine numbers (Supplementary Fig.\u0026nbsp;8A, B), LTP (Supplementary Fig.\u0026nbsp;8C-E), and PPR (Supplementary Fig.\u0026nbsp;8F). Additionally, the results indicated that 3-TYP administration significantly impeded the restoration of cognitive impairment and schizophrenia-associated behavior in the MS\u0026thinsp;+\u0026thinsp;NAM rats. In the Barnes maze test, the MS\u0026thinsp;+\u0026thinsp;NAM\u0026thinsp;+\u0026thinsp;3-TYP rats exhibited a longer escape latency to reach the target hole from day 1 to day 2 compared to the MS\u0026thinsp;+\u0026thinsp;NAM rats (Supplementary Fig.\u0026nbsp;8G). Results from the novel object recognition test revealed that the MS\u0026thinsp;+\u0026thinsp;NAM\u0026thinsp;+\u0026thinsp;3-TYP rats spent less time exploring the novel object when one of the objects was replaced after 24 hours, in comparison to the MS\u0026thinsp;+\u0026thinsp;NAM rats (Supplementary Fig.\u0026nbsp;8H, right panel). In the OFT, the MS\u0026thinsp;+\u0026thinsp;NAM\u0026thinsp;+\u0026thinsp;3-TYP rats showed an elevated total distance of spontaneous movement compared to the MS\u0026thinsp;+\u0026thinsp;NAM rats (Supplementary Fig.\u0026nbsp;8I). In the PPI test, the MS\u0026thinsp;+\u0026thinsp;NAM\u0026thinsp;+\u0026thinsp;3-TYP rats exhibited impaired PPIs at varying pre-pulse intensities (Supplementary Fig.\u0026nbsp;8J).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.11 SIRT3 signaling impact on neuronal plasticity and cognitive behavior.\u003c/h2\u003e \u003cp\u003eKnocking down SIRT3 in the CA1 region of the hippocampus resulted in reduced spine density (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). This intervention also led to a reduction in latency in reaching the target hole during the Barnes maze test (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Moreover, the novel object recognition test showed that SIRT3 knockdown elevated the time rats spent investigating the novel object after 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Additionally, the open field test indicated an increase in total movement distance following SIRT3 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). PPI testing revealed impaired prepulse inhibition after SIRT3 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Conversely, neuronal analysis demonstrated a restoration in spine density upon SIRT3 overexpression in MS rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). Furthermore, overexpressing of SIRT3 in MS rats reduced latency in reaching the target hole during the Barnes maze test (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) and elevated exploration time of the novel object in the novel object recognition test after 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Overexpression of SIRT3 also reversed the MS-induced increase in total movement distance in the open field test (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) and partially ameliorated impaired prepulse inhibition in the PPI test in MS rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"5. Discussion","content":"\u003cp\u003eThe main finding of this study was the identification of the involvement of the mitochondrial NAD+/SIRT3 axis in the pathophysiological mechanisms associated with schizophrenia. The study also provided a mechanistic account of the neurobiological intermediaries from the organelle to the cellular and synaptic levels. We observed alterations in the mitochondrial NAD+/SIRT3 axis in the hippocampus, reduced hippocampal neuronal spine density, impaired LTP in the CA1 region, and cognitive behavior deficits in the MS rat model. Furthermore, our results demonstrated that supplementing NAD\u0026thinsp;+\u0026thinsp;or activating/overexpressing of SIRT3 can restore hippocampal neuronal synaptic plasticity and mitigate cognitive impairment associated with schizophrenia in MS rats. Conversely, inhibiting or knocking down SIRT3 activity can lead to deficits in hippocampal neuronal and behavioral phenotypes. Our data strongly suggested that the NAD+/SIRT3 axis could be a crucial therapeutic target for schizophrenia and its associated cognitive deficits.\u003c/p\u003e \u003cp\u003eMitochondrial dysfunction is a commonly reported phenomenon in schizophrenia [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Early life stress can induce various behavioral changes [\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and lead to alterations in gene expression and metabolism of mitochondria in different brain regions [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, the specific causal role of mitochondrial mechanisms in stress-induced behavioral changes remains unclear. MS, a widely-used and well-documented rodent model, has been extensively reported to induce schizophrenia-related behaviors and cognitive deficits in adulthood [\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our findings supported these studies by demonstrating cognitive deficits in MS animals using the novel object recognition test and Barnes maze tests. The reductions in PPI further confirmed the validity of the schizophrenia model, as PPI impairments are observed in various neuropsychiatric disorders, including schizophrenia [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and are commonly used as behavioral assays in animal models of schizophrenia [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Administration of NAM (a precursor of NAD+) normalized cognitive deficits, spontaneous activity, and impaired sensorimotor gating induced by MS. In addition, the SIRT3 activator or overexpression of SIRT3 attenuated most abnormal behaviors caused by MS. Meanwhile, the SIRT3 inhibitor or knocking down of SIRT3 led to spontaneous activity increase, abnormal spontaneous activity, PPI and impaired cognitive behaviors in adult control and NAM-treated MS rats. Therefore, a distinct contribution of our study was to have identified a novel role for the mitochondrial NAD+/SIRT3 axis in the regulation of early life stress-induced variation in behavior.\u003c/p\u003e \u003cp\u003eSpine remodeling, an important biological process shaping brain connectivity, has also been suggested to underlie complex behaviors and cognitive functions, such as learning and memory [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Reduced spine densities and elevated immature spines on the subiculum and CA1 regions have been previously found in patients with schizophrenia [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The involvement of mitochondrial function in dendritic and spine complexity has been previously acknowledged for early development [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and neurodegeneration [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Our study showed that early life stress induced a reduced dendritic spine density in hippocampal neurons at the adult stage. Reduced dendritic spine numbers may induce reduced postsynaptic transmission, which may cause a significant reduction in LTP induction and LTP maintenance [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Consistent with previous study, our results showed that early life stress induced impaired LTP in these neurons in the hippocampal CA1 region. However, the mitochondrial contribution to molecular mechanisms regulating spine stabilization and maturation have not been fully understood.\u003c/p\u003e \u003cp\u003eSIRT3, the primary mitochondrial NAD+-dependent protein deacetylase, plays a crucial role in maintaining mitochondrial redox homeostasis by regulating the function of electron transport chain complexes I and III, thus preventing ROS generation within the mitochondria [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Our previous study has showed that SIRT3 inhibitor could induce ace-SOD2, ROS increase and mitochondria damage in the HT22 cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this animal study, our results demonstrated that MS led to a decrease in SIRT3 levels in hippocampal neurons compared to control animals. Additionally, we observed mitochondrial swelling in the hippocampal neurons of MS rats. Studies suggest that mitochondria with a more rounded shape are often associated with less efficient bioenergetics [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] and ATP levels [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Additionally, we observed an increase in microglial contacts with dendrites and microglial phagocytosis of spines under early life stress.\u003c/p\u003e \u003cp\u003eMicroglia in the developing brain are highly mobile phagocytic cells with remarkably adaptable and versatile characteristics [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Apart from their notable functional adaptability, these cells are distinguished by a very low activation threshold, enabling them to carry out surveillance and scavenging functions effectively [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Studies suggest that the functional behavior of microglial cells may be influenced by neuronal activity [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Therefore, we hypothesized that alterations in the NAD+/SIRT3 axis within hippocampal neurons may contribute to the excessive engulfment of spines by microglia, leading to impaired LTP in the CA1 region and cognitive deficits in MS rats. To validate this hypothesis, we administered NAD\u0026thinsp;+\u0026thinsp;to adult MS rats. Our findings demonstrated the restoration of cognitive deficits, hippocampal neuronal synaptic plasticity, mitochondrial morphology, and NAD+/SIRT3 axis function in MS rats. Additionally, in order to further confirm the pivotal role of the NAD+/SIRT3 axis in regulating synaptic plasticity, we modulated the activity of SIRT3 in the animal models. Remarkably, in line with our hypothesis, the administration of a SIRT3 inhibitor to control and NAM-treated MS rats resulted in deficits in mitochondrial morphology, as well as in hippocampal neuronal and behavioral phenotypes. Activation of SIRT3 was able to reverse the mitochondrial, hippocampal neuronal, and behavioral phenotypes in MS rats. Furthermore, we conducted loss-of-function experiments through virus injection to investigate whether specific reduction of SIRT3 expression in hippocampal neurons replicated the behavioral and neuronal phenotypes observed in MS rats, which aligned with our initial predictions.\u003c/p\u003e \u003cp\u003eOur findings indicated that dysfunction of the NAD+/SIRT3 axis in hippocampal neurons leads to excessive microglial engulfment of neuronal spines, ultimately resulting in impaired synaptic plasticity. However, this intricate process involves various molecules, such as the classic complement cascade-dependent phagocytic signaling, transforming growth factor β, chemokine signaling, and brain-derived neurotrophic factor, which either promote or inhibit the elimination of specific synaptic connections [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan additionalcitationids=\"CR62 CR63 CR64\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Further research is warranted to elucidate the precise mechanisms underlying the interactions between microglial cells and hippocampal neurons.\u003c/p\u003e"},{"header":"6. Conclusions","content":"\u003cp\u003eOur study provided evidence that the NAD+/SIRT3 axis plays a crucial role in mediating mitochondrial and neuronal synaptic plasticity in response to early-life stress, and it significantly influenced cognitive behaviors. Moreover, we identified the NAD+/SIRT3 axis as a promising therapeutic target for addressing cognitive deficits associated with schizophrenia.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Professor Gavin Reynolds for providing the assistance and advice in interpreting the findings and preparing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Authors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG-H W, H-L W, Y-S L, F-S C and K-K H designed the study and wrote the protocol. K-K H and F-S C performed the experiments and analyzed the data. K-K H, H-H, R-X, Y-X, C-S, and S-L X performed literature searches and drew the figures. F-S C and K-K H wrote the manuscript. G-H W, H-L W, F-S C revised the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by the Medical Science Advancement Program of Wuhan University (NO. TFLC2018001). The Interdisciplinary Innovative Talents Foundation from Renmin Hospital of Wuhan University (JCRCFZ-2022-003) and the Key Research and Development Program of Hubei Province (2020BCA064).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData generated during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was carried out based on the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People\u0026rsquo;s Republic of China, with the approval and consent to participate of the Ethics Committee in Renmin Hospital of Wuhan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePerrin M, Kleinhaus K, Messinger J, Malaspina D. 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Annu Rev Neurosci. 2012;35:369\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"schizophrenia, maternal separation, cognitive impairment, LTP, mitochondria, NAD+/SIRT3 axis","lastPublishedDoi":"10.21203/rs.3.rs-4207040/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4207040/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMaternal separation during early life can induce behaviors in adult animals that resemble those seen in schizophrenia, manifesting cognitive deficits. This may be indicative of oxidative stress linked to mitochondrial dysfunction. However, there is limited understanding of the molecular mechanisms regulating mitochondria in neural circuits that govern cognitive impairment relevant to schizophrenia, and their impact on neuronal structure and function. A 24-hour maternal separation (MS) rat model was utilized to simulate features associated with schizophrenia. Schizophrenia-associated behaviors and cognitive impairment were assessed using the open field test, pre-pulse inhibition, novel object recognition test, and Barnes maze test. The levels of mitochondrial proteins were measured using Western blot analysis. Additionally, alterations in mitochondrial morphology, reduced hippocampal neuronal spine density, and impaired LTP in the CA1 region were observed. Nicotinamide (NAM) supplementation, administration of honokiol (HNK) (a SIRT3 activator), or overexpression of SIRT3 could inhibit this process. Conversely, administration of 3-TYP (a SIRT3 inhibitor) in control and NAM-treated MS rats led to deficits in behavior, mitochondrial morphology, and the hippocampal neuronal phenotype. Our findings suggested a causal role for the NAD+/SIRT3 axis in modulating cognitive behaviors via effects on hippocampal neuronal synaptic plasticity. The NAD+/SIRT3 axis could be considered a promising therapeutic target for addressing cognitive-related behavioral disturbances, such as those seen in schizophrenia.\u003c/p\u003e","manuscriptTitle":"Cognitive impairment following maternal separation in the rat is regulated by effects of the NAD + /SIRT3 axis on hippocampal synaptic plasticity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-11 21:37:41","doi":"10.21203/rs.3.rs-4207040/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"89a140fb-1dd8-4822-bcc6-e70d7c9e0ed5","owner":[],"postedDate":"April 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[{"value":"featured","date":"2024-04-12 17:03:31"}],"updatedAt":"2024-07-29T09:21:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-11 21:37:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4207040","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4207040","identity":"rs-4207040","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}