Introduction: Inflammation in early life is a risk factor for the development of neuropsychiatric diseases later in adolescence and adulthood, yet the underlying mechanism remains elusive. In the present study, we performed an integrated proteomic and phosphoproteomic analysis of the hippocampus to identify potential molecular mechanisms of early life inflammation-induced cognitive impairment. Methods: Both female and male mice received a single intraperitoneal injection of 100 μg/kg lipopolysaccharide (LPS) on postnatal day 10 (P10). Behavioral tests, including open field, elevated plus-maze, and Y-maze tests, were performed on P39, P40, and P41, respectively. After behavioral tests, male mice were sacrificed. The whole brain tissues and the hippocampi were harvested on P42 for proteomic, phosphoproteomic, Western blot, and Golgi staining. Results: Early life LPS exposure induced cognitive impairment in male mice but not in female mice, as assessed by the Y-maze test. Therefore, following biochemical tests were conducted on male mice. By proteomic analysis, 13 proteins in LPS group exhibited differential expression. Among these, 9 proteins were upregulated and 4 proteins were downregulated. For phosphoproteomic analysis, a total of 518 phosphopeptides were identified, of which 316 phosphopeptides were upregulated and 202 phosphopeptides were downregulated in the LPS group compared with the control group. Furthermore, KEGG analysis indicated that early life LPS exposure affected the glutamatergic synapse and neuroactive ligand-receptor interaction, which were associated with synaptic function and energy metabolism. Increased level of brain protein i3 (Bri3), decreased levels of PSD-95 and mGLUR5, and dendritic spine loss after early life LPS exposure further confirmed the findings of proteomic and phosphoproteomic analysis. Conclusions: Our findings demonstrated that neuroinflammation and impaired synapse may be involved in early life inflammation-induced cognitive impairment. Future studies are required to confirm our preliminary results.

© 2023 The Author(s). Published by S. Karger AG, Basel

Introduction

The early postnatal period is a key stage for the neural development, maturation of synaptic transmission, and functional receptor expression [1-3]. It has been demonstrated that early adversity experience is a risk factor for neuropsychiatric diseases in adolescence and adulthood [2, 3]. A previous study indicated that bacterial infection in the immature brain increased the occurrence of neurological diseases later in life [4]. Synaptic transmission impairment [1], oxidative stress [2], and neural circuit abnormality [3] are the potential mechanisms of early life inflammation-induced cognitive impairment. However, the precise mechanism remains unclear.

Lipopolysaccharide (LPS), a Gram-negative bacteria endotoxin, can induce systematic adverse effects through multiple mechanisms such as disruption of the blood-brain barrier (BBB), microglia activation, and the release of proinflammatory cytokines [5]. Studies in rodents have shown that postnatal 2 weeks is a crucial period for brain network maturation [6, 7]. During this sensitive period, microglia actively engulf redundant synapses, playing a key role in synaptic pruning during postnatal development [6, 7]. An inflammatory event during this period may contribute to synaptic plasticity abnormalities, which have been noted in many neurodevelopmental disorders [6-8].

Proteomics aims to systematically study the characteristics of proteins on a large scale and explain the molecular networks under the protein level [9]. Proteomic approach is a powerful tool in identifying disease phenotypes, drug targets, and disease biomarkers [9]. So far, proteomics has been applied to clarify the protein expression pattern and examine possible molecular pathways in many neurological and neuropsychiatric diseases, including Parkinson’s disease (PD) [10], Alzheimer’s disease (AD) [11], and major depressive disorder (MDD) [12]. Furthermore, about one-third of all proteins are likely to be phosphorylated; thus, phosphoproteomic analysis offers an excellent approach for the identification of candidate regulatory proteins in many cellular processes. As most studies focus on the specific genes or proteins to elucidate the underlying mechanisms that contribute to the pathogenesis of early life inflammation-induced cognitive impairment, systematic analysis of the hippocampal proteomic profile is still lacking.

In this study, we performed an integrated analysis of proteomics and phosphoproteomics to identify potential molecular mechanisms of early life inflammation-induced cognitive impairment. The results will help to improve understanding of the pathogenesis of cognitive impairment induced by early life inflammation.

Materials and MethodsAnimals and the Experimental Procedure

All the experiments were approved by the Ethics Committee of Nanjing Medical University (SYXK [Su] 2020-0020) and all procedures were performed in accordance with the approved guidelines. Pregnant C57BL/6 mice were purchased from Sibeifu (Beijing) Biotechnology Company and arrived on gestational day 15. Pregnant mice were individually housed in standard cages and maintained at 22–25°C and 40–60% humidity on a 12 h light/dark cycle. Food and water were obtained ad libitum. After weaning at postnatal day 21 (P21), pups from different dams were allocated to control group or LPS group according to their neonatal treatment. A total of 60 male and 24 female pups from 16 pregnant C57BL/6 mice were used.

Previous studies have shown that P7-P14 is a crucial period for brain neuronal network maturation [6, 7]. P10 becomes a vulnerable time point when suffering inflammation or other stressors [6, 7]. Therefore, the pups received a single intraperitoneal injection of 40 μL of LPS (100 μg/kg, L2630, Merck-sigma) or the same volume of normal saline (NS) on P10 as previously described [13]. Behavioral tests, including open field, elevated plus-maze, and Y-maze tests, were performed on P39, P40, and P41, respectively. Female mice did not display neurobehavioral impairment in adolescence; thus, we only chose male mice for further biochemical tests. On P42, mice were sacrificed. Then the whole brain tissues and the hippocampi were harvested for biochemical tests.

Behavioral TestsOpen Field Test

Mice were placed in the center of a white experimental box (50 cm × 50 cm × 50 cm) and allowed to explore the area freely for 5 min. The arena was divided into sixteen squares of equal areas. The center area was defined as the middle four squares of the arena. The total distance traveled and the time spent in center were automatically recorded by a video tracking system (SMART V3.0; RWD Life Science Co.). At the end of each test, the arena was cleaned with 75% alcohol to eliminate olfactory cues.

Elevated Plus-Maze Test

The elevated plus-maze consisted of two open arms and closed arms extending from a common central platform. Before the test, mice were put in the open field for 5 min. Subsequently, mice were individually placed in the central platform of the maze and allowed to explore spontaneously for 5 min. The total distance traveled and the time spent in the open arms were automatically recorded by a video tracking system (SMART V3.0; RWD Life Science Co.). At the end of each test, the arena was cleaned with 75% alcohol to eliminate olfactory cues.

Y-Maze Test

The Y-maze test measures the willingness of rodents to explore new environments to evaluate the spatial working memory. The Y-maze consisted of three arms and each arm was labeled as A, B, and C. The test was used to evaluate the spontaneous alternation performance during an 8-min session. Mice were placed in the center of the maze and explored freely throughout three arms. The hind paw of the mouse completely entry the arm was recorded. The alternation was defined as successive consecutive entries to three different arms. The number of maximum spontaneous alternations was considered the total number of arms entered minus 2. The percentage of spontaneous alternation is the number of triads containing entries into all three arms divided by the maximum spontaneous alternations ×100. At the end of each test, the arena was cleaned with 75% alcohol to eliminate olfactory cues.

Western Blot Analysis

Western blot was used to verify the expressions of brain protein i3 (Bri3), TNF-α, Gastrin releasing peptide (Grp), PSD-95, and mGLUR5 in the hippocampi. After behavioral tests, six mice per group were anesthetized by 5% chloral hydrate (0.1 mL/10 g), and the brain samples were removed on ice subsequently. The hippocampi were separated from the brain tissues on ice. The hippocampi were dissociated completely by RIPA lysis buffer (P0013B, Beyotime) and a phosphatase inhibitor cocktail (ST505, Beyotime). Protein concentration was determined by BCA protein assay kit (P0010, Beyotime). Approximately 30-µg proteins were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes. Then the membranes were blocked by 3% skimmed milk for 1 h at room temperature, and incubated with primary antibodies overnight at 4°C, including anti-PSD-95 (1:1000, ab238135, Abcam), anti-TNF-ɑ (1:1000, 60291, Proteintech), Grp (1:2000, PA5-115272, Thermo), Bri3 (1:1000, PA5-68115, Thermo), and mGLUR5 (1:500, sc-293442, Santa Cruz). These antibodies were validated by the company using blocking peptide or knock out validation, please refer to the manufacturer description: anti-PSD-95: http://www.abcam.com/psd-95-antibody-epr23124-118-ab238135.html; anti-TNF-α: http://www.ptgcn.com/products/TNF-a-Antibody-60291-1-Ig.htm; anti Grp: http://www.thermofisher.cn/cn/zh/antibody/product/Bombesin-Antibody-Polyclonal/PA5-115272; anti-Bri3: http://www.thermofisher.cn/cn/zh/antibody/product/BRI3-Antibody-Polyclonal/PA5-68115; anti-mGLUR5: http://www.scbt.com/zh/p/mglur-5-antibody-1b3. The PVDF membranes were incubated with second antibodies for 2 h at room temperature. The protein bands were detected by enhanced chemiluminescence and with Image J software.

Golgi Staining

Three fresh brain tissues from male mice per group were treated with the FD Rapid Golgi Stain Kit (#PK401, FD NeuroTechnologies, Columbia, MD, USA) in accordance with manufacturer’s instructions. Briefly, non-perfused mouse brains were immersed in impregnation solution for 2–3 weeks in the dark at room temperature (22–25°C) and then transferred to solution C for 5 days in the dark at room temperature. Then, the brains were cut into 120-μm-thick sections from bregma −1.70 to −2.30 with freezing microtome and mounted on gelatin-coated slides for staining. After staining and alcohol dehydration, the tissue sections were cleared in xylene and cover-slipped. Neuronal morphology and dendritic spines were captured with a microscope (Nikon 1901680s, Japan) at 200 and 1,000 magnifications under an oil-immersion objective, respectively. At least three pyramidal neurons in the hippocampal CA1 region per mouse were randomly selected. The tracings of neurons, quantification of total dendritic lengths, and number of dendritic spines were performed using Neuron J software. Sholl analysis was used to examine the number of dendritic intersections per 30-mm concentric radial interval from the cell body.

Proteomic and Phosphoproteomic Analysis

Male mice were deeply anesthetized by 5% chloral hydrate (0.1 mL/10 g) and rapidly sacrificed. To make the results more repeatable, we mixed 3 hippocampi from the control group (n = 9) or the LPS group (n = 9) into one sample, providing 3 biological replicates for each group. The hippocampi were dissolved using a SDT buffer (4% SDS, 100 mM Tris-HCl, 1 mM DTT, pH 7.6). The amount of protein was quantified with the BCA Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Then, protein digestion by trypsin was performed according to filter-aided sample preparation (FASP) procedure described by Matthias Mann and labeled using TMT reagent according to the manufacturer’s instructions (Thermo Scientific). Approximately 200-μg proteins for each sample were mixed with 30 μL of STD buffer. The subsequent procedures included peptide fractionation with strong cation exchange (SCX) chromatography, liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS/MS), and sequence database searching and data analysis. The labeled peptides were fractionated by High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific). LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific) that was coupled to Easy nLC (Thermo Fisher Scientific) for 60/90 min. The protein sequences of the selected differentially expressed proteins were locally searched using the NCBI BLAST + client software (ncbi-blast-2.2.28+ win32.exe) and InterProScan to find homolog sequences, then gene ontology (GO) terms were mapped and sequences were annotated using the software program Blast2GO. The GO annotation results were plotted by R scripts. Following annotation steps, the studied proteins were blasted against the online Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://geneontology.org/) to retrieve their KEGG orthology identifications and were subsequently mapped to pathways in KEGG. The MS raw data for each sample were searched using the MASCOT engine (version 2.2; Matrix Science, London, UK) embedded into Proteome Discoverer 1.4 software for identification and quantitation analysis. The final proteins that were deemed to be differentially expressed were filtered as a p value < 0.05 and 1.2-fold changes (>1.20 or <0.83) relative to the control group.

Statistical Analysis

For behavioral tests and biochemical experiments, statistical analysis was analyzed by the GraphPad Prism version 8.0. Data are presented as mean ± SEM. The results between two groups were assessed with unpaired t test. A significant difference was considered as p < 0.05.

ResultsEarly Life LPS Exposure-Induced Cognitive Impairment in Male Mice

The flow chart of study procedures is shown in Figure 1. First, we examined the effects of early life LPS exposure on cognitive function. In the open field and elevated plus-maze tests, there was no difference in total distance between the two groups (Fig. 2a: male: t = 1.410, p = 0.1725; female: t = 0.7444, p = 0.4639; Fig. 2d: male: t = 0.8101, p = 0.4265; female: t = 1.651, p = 0.1112). There was no difference in time in center and time in open arm (Fig. 2e: male: t = 0.9678, p = 0.3437; female: t = 1.795, p = 0.0858), indicating that early life LPS exposure did not induce anxiety-like behavior. Compared with the control group, male mice in the LPS group showed lesser spontaneous alternation in the Y-maze test, suggesting early life LPS exposure induced impaired working memory in male mice (Fig. 2c: male: t = 2.417, p = 0.0244). However, no difference in spontaneous alternation in the Y-maze test was found in female mice (Fig. 2c: female: t = 2.021, p = 0.0556). We therefore selected male offspring for further biochemical tests.

Fig. 1.

Flow chart of study procedures.

Fig. 2.

a Early life LPS exposure-induced cognitive impairment in male offspring. There was no significant difference in total distance in the open field test between control and LPS groups in both male and female offspring. b There was no significant difference in time spend in the center between control and LPS groups in both male and female offspring. c The LPS group displayed lesser spontaneous alteration than control in male offspring. d–e There was no significant difference in total distance and time spent in the open arm in the elevated plus-maze test between control and LPS groups in both male and female offspring. Data are presented as the mean ± SEM (n = 12 mice/group). *p < 0.05 compared to control group.

Identification of Altered Proteins in the Hippocampus of LPS-Treated Mice

A total of 6,118 proteins were identified with at least one unique peptide and 1% FDR. According to the criteria of p < 0.05 and fold changes >1.20 or <0.83, 13 proteins exhibited significant differential expression between the two groups. Of these, 9 proteins were upregulated (Table 1) and 4 downregulated (Table 2) in LPS group compared with control group. The magnitude and significance of changes in the protein level between control and LPS groups are presented in hierarchical clustering heat maps and a volcano plot (Fig. 3a, b).

Table 1.

Upregulated proteins in the hippocampus of LPS-treated mice compared to control mice

Table 2.

Downregulated proteins in hippocampus of LPS-treated mice compared to control mice

Fig. 3.

Changes of proteins in the hippocampus of LPS-treated mice. a Hierarchical clustering heat maps of changes in the protein level between control and LPS groups. b The volcano plot of changes in the protein level after LPS exposure.

Bioinformatics Analysis of Differentially Expressed Proteins of LPS-Treated Mice

According to GO analysis (Fig. 4a), the proteins were found to be associated with metabolic process, cellular process, and multicellular organismal process. According to the KEEG pathway (Fig. 4b), early life LPS exposure might affect the ribosome biogenesis in eukaryotes, PPAR signaling pathway, neuroactive ligand-receptor interaction, peroxisome, and insulin resistance.

Fig. 4.

Bioinformatics analysis of differentially expressed proteins of LPS-treated mice. a GO functional enrichment analysis of differentially expressed proteins after LPS exposure. b KEEG pathway enrichment of differentially expressed proteins after LPS exposure.

Changes of Phosphoproteins in the Hippocampus of LPS-Treated Mice

A total of 11,393 phosphopeptides corresponding to 3,626 phosphoproteins were identified. After setting the cutoff fold change to >1.2 or <0.83, 316 and 202 phosphopeptides increased and decreased in LPS group, respectively, when compared to the control group. The top 20 phosphoproteins are listed in Tables 3 and 4.

Table 3.

Top 20 upregulated phosphorylated proteins in hippocampus of LPS-treated mice compared to control mice

Table 4.

Top 20 downregulated phosphorylated proteins in hippocampus of LPS-treated mice compared to control mice

Classification of Phosphoproteins into Functional Groups

Changes of the protein phosphorylation status are implicated in the regulation of biological functions. To understand the biological significance of the up- or downregulated phosphoproteins observed in LPS group, we performed gene ontology (GO) analysis (Fig. 5), in which proteins and their coding genes are classified on the basis of their cellular component, molecular function, and biological processes. GO analysis of molecular function indicated that the protein phosphorylation changes may be involved in the protein binding, structural molecule activity, structural constituent of synapse, kinase binding, and structural constituent of postsynaptic density. Furthermore, a large percentage of the proteins were associated with biological processes, including anterograde trans-synaptic signaling, chemical synaptic transmission, trans-synaptic signaling, synaptic signaling, and behavior. Cell component analysis indicated that changes of the protein phosphorylation were enriched in dendrite, dendritic tree, glutamatergic synapse, somatodendritic compartment, and postsynaptic specialization.

Fig. 5.

Classification of phosphoproteins into functional groups. A GO functional enrichment analysis of differentially expressed phosphoproteins after LPS exposure.

Pathway Analysis of Differentially Expressed Phosphoproteins

To further understand the biological roles of identified phosphoproteins and the signaling events they regulate, we performed KEEG pathway analysis of the differentially expressed phosphopeptides. We defined significant enrichment as FDR <0.05 and identified five candidate pathways (Fig. 6). Most of the top KEGG pathways, such as nicotine addiction, Rap1 signaling pathway, carbohydrate digestion and absorption, glutamatergic synapse, and neuroactive ligand-receptor interaction, most of which are closely associated with changes in synaptic functions and cognitive performance (Table 5).

Table 5.

Comparison of functional enrichment characteristics of phosphorylated proteins in the hippocampus between two groups

Fig. 6.

Pathway analysis of differentially expressed phosphoproteins. A KEEG pathway enrichment of differentially expressed phosphoproteins after LPS exposure.

Validation of Results from Proteomic and Phosphoproteomic Analysis by Western Blot

To validate the results from proteomic and phosphoproteomic analysis, we performed Western blot to examine changes in these protein expressions in the hippocampus. Compared to control group, the protein levels of Bri3 and TNF-α were significantly increased in the LPS group (Fig. 7d: p = 0.0365; Fig. 7f: p = 0.0022). The synaptic related protein such as PSD-95 and mGLUR5 were significantly decreased after early life LPS exposure (Fig. 7i: p = 0.0054; Fig. 7j: p = 0.0101).

Fig. 7.

a Validation of results from proteomics and phosphoproteomic analysis by Western blot. Representative Western blots of Bri3 in the hippocampi. b Representative Western blots of Grp in the hippocampi. c Representative Western blots of TNF-α in the hippocampi. d Quantitative analysis of Bri3 level in the hippocampi. e Quantitative analysis of Grp level in the hippocampi. f Quantitative analysis of TNF-α levels in the hippocampi. g Representative Western blots of PSD-95 in the hippocampi. h Representative Western blots of mGLUR5 in the hippocampi. i Quantitative analysis of PSD-95 level in the hippocampi. j Quantitative analysis of the mGLUR5 level in the hippocampi. Data are presented as the mean ± SEM (n = 6 mice/group). *p < 0.05 compared to the control group.

Early Life LPS Exposure-Induced Hippocampal Dendritic Spine Loss

Finally, we detected early life LPS exposure on the neuronal morphological changes and spine density in the CA1 of hippocampus. The dendritic spine density was significantly reduced in the LPS group compared to the control group (Fig. 8f: t = 4.210, p = 0.0005). However, there was no difference in the total dendritic length between the two groups (Fig. 8d: t = 1.165, p = 0.2635).

Fig. 8.

Early life LPS exposure-induced hippocampal dendritic spine loss. a Hippocampal profile image of Golgi staining. b Images of tracings of hippocampal neurons. c Quantification of dendritic intersections of hippocampal neurons. d Quantification of the total dendritic length of hippocampal neurons. e Representative dendritic spine density of hippocampal neurons. f Quantification of the dendritic spine density of hippocampal neurons. Scale bar = 150 μm. Data are presented as the mean ± SEM (n = 3 mice/group). *p < 0.05 compared to control group.

Discussion

In the present study, we used proteomic and phosphoproteomic technology to evaluate changes of proteins and phosphoproteins in the hippocampus of LPS-exposed mice. Our results revealed that early life LPS exposure induced significant changes in proteins related to glutamatergic synapse loss. Thus, synaptic impairment may be involved in early life inflammation-induced cognitive impairment.

Early life inflammation has consistently been demonstrated to cause negative effects on cognitive impairment in adolescent and adult mice [1-3, 14]. Clinical studies have provided substantial findings that early life inflammation can lead to an increased risk of subsequent neurodevelopmental disorders in children and adults [4, 15, 16]. Thus, it is necessary to clarify the changes in protein expressions for future targeted therapy in early life inflammation-induced cognitive impairment.

LPS is an endotoxin released from the cell wall of gram-negative bacteria, which is widely used for induction of inflammation [5]. Previous studies have shown that early life exposure to LPS can lead to anxiety-like behavior and cognitive impairment [17, 18]. In our study, we showed that early life LPS exposure induced working memory impairment in adolescent male mice as assessed by Y maze, which is critically dependent on the hippocampus. This brain region is critical for learning and memory and is particularly vulnerable to inflammation. Therefore, the observed cognitive impairment in our study is likely to be caused by damage to this structure, although other brain regions cannot be excluded. This is also the reason why we selected the hippocampus for determination of changes in protein expressions by proteomic and phosphoproteomic technology. However, additional cognitive tests including novel object recognition and Morris water maze tests that involved in the hippocampus should be performed in our future study. Interestingly, female mice did not show obvious behavioral phenotypes in adolescence in our study, indicating male mice are more susceptible to early life adversity. Supporting our observation, Danielle S. Macedo et al. [19] found that early life LPS exposure caused depressive-like, anxiety-like, and repetitive behaviors with working memory deficits in male mice, while female mice did not present these behavioral phenotypes. Moreover, one previous study provided evidence that androgens play a key role in the susceptibility to stressors, which can explain why male offspring may be more susceptible to early life LPS exposure [20].

In proteomics analysis, 13 proteins were differentially expressed between control and LPS groups, which mainly involved in the PPAR signaling pathway and TNF-α mediated cell death. We have selected several significantly altered proteins related to neurodegeneration for further verification. Growing evidence suggests that neuroinflammation contribute to abnormal neurogenesis and synaptic plasticity [21-23]. Bri3, which is highly expressed in the cerebral cortex, amygdala, and hippocampus [24, 25], has been suggested to play a role in TNF-α mediated inflammation, the interaction with Alzheimer amyloid plaques, and neuronal differentiation [26-28]. It has been showed that overexpression of Bri3 induced apoptosis in L929 cells [29]. Additionally, according to KEEG enrichment analysis, significantly altered proteins were involved in the PPAR signaling pathway, which was closely associated with cell death [30]. In the present study, we confirmed that early life inflammation induced the increased levels of Bri3 and TNF-α, which was consistent with previous studies [26, 29]. Moreover, synaptic plasticity is the cellular process involved in learning and memory, its impairment is observed in neurodegenerative disorders such as AD [31] and sepsis associated encephalopathy [32]. It has been shown that TNF impaired LTP induction or maintenance in the hippocampus, by preventing the initial reduction of potentiation and by inhibiting the late increased potentiation [33]. Grp has been reported to bind to the bombesin receptor which is expressed on inhibitory GABAergic interneurons [34]. In a mouse model of vascular dementia, Grp has been found to have effects on the interactions between theta and gamma oscillations in the hippocampal CA3-CA1 pathway [35]. These results suggested that neuroinflammation and synaptic impairment might play important roles in early life inflammation-induced cognitive impairment.

Protein phosphorylation is an important mechanism for regulating protein activity. In phosphoproteomic analysis, the differentially expressed proteins were enriched in nicotine addiction, the Rap1 signaling pathway, carbohydrate digestion and absorption, glutamatergic synapse, and neuroactive ligand-receptor interaction. Furthermore, early postnatal 2 weeks is a key period for the process of glutamatergic synapse development and is vulnerable to inflammation [36]. For these reasons, we focused on glutamatergic synapse associated proteins in our study. In the present study, we showed the decreased levels of PSD-95 and mGLUR5 in early life inflammation-induced cognitive impairment, which was consistent with previous studies [37]. PSD-95, a scaffolding protein located on excitatory synapses, is involved in the stabilization, recruitment, and trafficking of glutamate receptor to the postsynaptic membrane [38]. During neurodevelopment, PSD-95 has been demonstrated to be involved in dendritic spine morphogenesis [38-40], which was consistent with our results that early life LPS exposure induced hippocampal dendritic spine loss. Early studies have demonstrated that protein phosphorylation is a key mechanism for regulating the function of glutamate receptors [41]. By regulating phosphorylation levels, protein kinases control the biochemistry and physiology of glutamate receptors, thus modulating the synaptic plasticity. mGluR5, enriched in the postsynaptic density, has been demonstrated to be important for synapse formation, synaptic plasticity, and long-term potentiation [42]. In addition, mGluR5 agonists have been studied as potential therapeutic agents in schizophrenia [43], which further explain the importance of mGluR5 signaling in neuropsychiatric diseases.

Conclusion

In summary, our study suggested that neuroinflammation and synaptic impairment should be the potential molecular mechanisms underlying early life inflammation-induced cognitive impairment. Since our result is descriptive, more studies are required to confirm these results.

Acknowledgments

We thank the Institute of Neuroscience of Zhengzhou University for technical support.

Statement of Ethics

All the animal experiments were approved by the Ethics Committee of Nanjing Medical University (SYXK [Su] 2020-0020).

Conflict of Interest Statement

The authors declare no conflict of interest.

Funding Sources

This work was supported by the grants from the National Natural Science Foundation of China (81772126, 81971892, 81971020).

Author Contributions

Xin-miao Wu and Yu-zhu Gao contributed to performing the experiment and writing the manuscript together with Ting-ting Zhu and Han-wen Gu. Jian-hua Tong established the animal model and analyzed the data. Jie Sun edited original draft. Jian-jun Yang and Mu-huo Ji contributed to the design of experiment.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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