Abstract

   Autism spectrum disorder (ASD) is a multifactorial neurodevelopmental
   disorder, with lead (Pb) exposure increasingly linked to its risk.
   However, the molecular mechanisms linking Pb to ASD remain poorly
   understood. This study established a postnatal Pb-exposed mouse model
   and employed the three-chamber social test and the marble-burying test
   to assess ASD-like behavioral phenotypes. The Pb levels in both blood
   and the hippocampus were quantified, and hippocampal neurons were
   assessed for morphological alterations. Moreover, a Tandem Mass Tag
   (TMT)-based quantitative proteomics approach was applied to elucidate
   the underlying mechanisms. Neurobehavioral experiments revealed
   Pb-exposed C57BL/6 offspring exhibited reduced social interaction and
   novelty preference along with increased repetitive marble-burying
   behavior. The Pb levels in both the blood and hippocampus of Pb-treated
   mice were significantly elevated compared with those of control
   animals. Postnatal Pb exposure resulted in a reduction in the neuronal
   numbers and disorganized neuronal arrangement in the hippocampus. A
   total of 66 proteins were identified as being differentially expressed
   after postnatal Pb exposure. Among them, 34 differentially expressed
   proteins were common in both Pb exposure groups, with 33 downregulated
   and 1 upregulated. Bioinformatic analysis revealed multi-pathway
   regulation involved in Pb-induced neurodevelopmental disorders,
   including dysregulation of synaptic signaling, abnormal activation of
   neuron apoptosis, and neuroinflammation. Notably, the SYT10/IGF-1
   signaling pathway may play a potential key role. These findings enhance
   understanding of Pb-induced autism-like behaviors, providing novel
   proteomic insights into the etiology of ASD.

   Keywords: autism spectrum disorder (ASD), lead (Pb), postnatal,
   hippocampus, proteomics

1. Introduction

   Autism spectrum disorder (ASD) encompasses a group of
   neurodevelopmental conditions characterized by impaired social
   interaction coupled with repetitive behaviors. The Centers for Disease
   Control and Prevention (CDC) reported that 1 in 36 children in the
   United States was diagnosed with ASD in 2020 [[40]1]. The prevalence of
   ASD in Chinese children is estimated at 7‰ (almost 1/142) [[41]2]. Boys
   are, on average, four times more likely to be diagnosed with ASD than
   girls. ASD accounts for 11.5 million all-age disability-adjusted
   life-years (DALYs), with an age-standardized DALY rate of 147.6 per
   100,000 people. Notably, ASD was ranked among the top 10 causes of
   non-fatal health burden for people under 20 years old [[42]3]. Due to
   its high prevalence and substantial socioeconomic costs, more attention
   should be paid to the etiology of ASD for the purposes of early
   screening and intervention.

   While both genetic and environmental factors likely contribute to the
   development of ASD, research has suggested that lead (Pb) is recognized
   as an important environmental risk factor for ASD
   [[43]4,[44]5,[45]6,[46]7]. A large number of studies have demonstrated
   that Pb levels are higher in children with than in those without ASD
   [[47]8,[48]9,[49]10,[50]11,[51]12,[52]13]. Our previous study showed
   that there is a positive correlation between Pb levels and symptom
   severity in children with ASD (p < 0.001) [[53]14], which is consistent
   with the findings of earlier research [[54]15,[55]16]. Childhood
   neurodevelopment is marked by critical periods that determine brain
   plasticity. It has been shown that exposure to heavy metals during 10
   to 30 weeks after birth increases the risk of ASD occurrence [[56]17].
   Compared with non-ASD control twin pairs, Pb levels in ASD discordant
   twins were 1.5 times higher at 15 weeks postnatally [[57]18].
   Additionally, Zhou et al. [[58]19] reported that the postnatal period
   represents a sensitive window for perturbations in the methylation of
   the promoter of methyl-CpG-binding protein 2 (MECP2), a gene strongly
   implicated in ASD. Hence, identifying the potential threats of Pb,
   particularly postnatal exposure, to childhood neurodevelopment is
   crucial for exploring the etiology of ASD and safeguarding children’s
   health. Exposure to Pb has recently been associated with an increase in
   the levels of inflammatory cytokines, proinflammatory markers, and
   immunoglobulins, which may contribute to the development of autism-like
   symptoms in a mouse model of autism [[59]20,[60]21,[61]22]. Pb can
   induce ASD-like behavior by triggering oxidative stress [[62]23]. These
   findings highlight the need for in-depth studies on the mechanisms
   underlying the effects of Pb exposure on ASD development. However, the
   relevant research information remains limited. This study aimed to
   systematically explore the association between postnatal Pb exposure
   and the development of ASD as well as its underlying molecular
   mechanisms.

   Quantitative proteomic analysis helps us to develop a more thorough
   understanding at the protein level for neurotoxicity, and some of the
   key proteins identified can also potentially be used as biomarkers in
   epidemiologic studies [[63]24,[64]25,[65]26]. Using TMT-based
   quantitative proteomics, the synaptic proteomes across five mouse
   models for ASD were analyzed, and significant dysregulations were
   identified in multiple cellular and molecular pathways at the synaptic
   level, including oxidative phosphorylation and Rho family small GTPase
   signaling [[66]27]. In this study, we employed TMT-based proteomics to
   screen for protein profiling in male offspring subjected to postnatal
   Pb exposure and investigate their potential connections to ASD. The aim
   was to further elucidate the underlying molecular mechanisms linking Pb
   exposure and ASD and to screen the potential intervention targets.

2. Materials and Methods

2.1. Establishment of Animal Models and Grouping

   C57BL/6 mice were purchased from the Experimental Animal Centre of
   Xiamen University (Xiamen, China). The mice were housed with laboratory
   chow and distilled water and individually housed in an ambient
   temperature (20 ± 2 °C) and relative humidity (50 ± 10%) controlled
   environment on a 12 h:12 h light/dark cycle. Mice had free access to
   food and water. All animal experiments complied with the Animal
   Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The
   experiments were conducted in accordance with the National Institutes
   of Health Guide for the Care and Use of Laboratory Animals and were
   approved by the Medical Ethics Committee of Xiamen Medical College
   (Approval No. 2021041311).

   Male and female mice (20 ± 2 g, 1: 2) were mated overnight, and the
   vaginal secretion was collected the next morning. Pregnant females were
   raised independently. The day of birth was considered postnatal day 0
   (PND0) ([67]Figure 1). Male offspring were randomly divided into a
   control group, a low-Pb group (15 mg/kg bodyweight PbAc), and a high-Pb
   group (30 mg/kg bodyweight PbAc). Each group included 15 male
   offspring. The experimental design and the Pb exposure doses were based
   on a previous study [[68]28,[69]29]. Male offspring were
   intraperitoneally injected with PbAc dissolved in saline solution every
   two days from PND7 until PND21 to evaluate the effect of Pb during
   critically developmental windows. The control group received saline
   solution (sterile 0.9% NaCl). After Pb exposure, the male offspring
   were housed as normal until used for follow-up experiments.

Figure 1.

   [70]Figure 1
   [71]Open in a new tab

   Visual representation of the comprehensive research protocol timeline.

2.2. Three-Chamber Social Test

   The three-chamber social test was conducted to evaluate the sociability
   of male offspring. The test was performed as previously described
   [[72]30,[73]31] with minor modifications. The apparatus consisted of a
   box with three chambers (length 65 cm, width 45 cm, height 20 cm) with
   small rectangular openings allowing access to each chamber. A wire cage
   was placed in the middle chamber. As shown in [74]Figure 2A, in stage 1
   (adaption period), an empty wire cage was placed in both the left and
   right chambers. In stage 2 (social preference test period), a
   never-before-encountered mouse (Stranger 1) was placed under the wire
   cage in the left chamber, while the cage in the right chamber remained
   empty. Finally, in stage 3 (social novelty preference test period), a
   second never-before-encountered mouse (Stranger 2) was placed under the
   wire cage in the right chamber. In each stage, the test mouse was
   placed in the middle chamber and allowed to freely explore for 10 min.
   The time spent exploring the empty wire cage or the one with another
   mouse was measured, and the social index (SI) and social novelty
   preference index (SNI) were subsequently calculated. Data acquisition
   and analysis were performed using SMART software (Version 3.0.06).

Figure 2.

   [75]Figure 2
   [76]Open in a new tab

   Influence of postnatal Pb exposure on social deficits in offspring mice
   (n = 8/each group). (A) Experimental protocol of three-chamber social
   test. (B) Track of male offspring in the three-chamber test. The traces
   on the left side of each group represent the behavior activities in
   stage 2, while those on the right side represent the behavior
   activities in stage 3. In stage 2, the green circle means a mouse, and
   the purple circle means an empty cage; while in stage 3, the green
   circle means a familiar mouse, and the purple circle means a strange
   mouse. (C) Quantification of the social index and (D) the social
   novelty preference index in the three-chamber test showed decreased
   social behaviors of Pb-exposed offspring. * p ≤ 0.05, ** p ≤ 0.01, ***
   p ≤ 0.001.

2.3. Marble-Burying Test

   As detailed in previous studies [[77]32,[78]33], the marble-burying
   test was used for the assessment of restricted repetitive behaviors in
   Pb exposed male offspring mice. The mouse was placed in a plastic
   container (length 40 cm, width 40 cm, height 40 cm) filled with clean
   woodchip bedding to a height of 5 cm. Twenty glass marbles (previously
   washed with 90% alcohol) were placed equidistant in the testing area.
   After the acclimation period, mice were allowed to freely bury marbles
   for 30 min. At the end of the test, the number of buried marbles was
   counted. Marbles covered at least two-thirds by bedding were considered
   buried. After each experiment, the marbles were thoroughly cleaned, and
   new bedding was placed in the testing chamber.

2.4. Pb Level Determination in Blood and Hippocampus

   Pb level determination in blood and the hippocampus was performed as
   previously described [[79]34,[80]35] with minor modifications. At the
   end of the social tests, fresh blood and hippocampal tissue were
   immediately collected. Blood (0.25 mL) or hippocampal tissue (0.1 g)
   was placed in 5 mL of 65% nitric acid at room temperature for 6 h,
   followed by digestion with a microwave digestion system (MARSXpress,
   CEM Corporation, Matthewss, NC, USA). Finally, the Pb level was
   detected using inductively coupled plasma mass spectrometry (ICP-MS;
   Agilent 7500cx, Palo Alto, CA, USA).

2.5. Histopathological Examination

   To observe neuronal changes in the hippocampus of mice in the three
   groups, the hippocampus was collected on the day after the behavioral
   tests concluded. The hippocampus was washed with normal saline and
   immediately fixed in 4% paraformaldehyde. The tissue was dehydrated,
   embedded in paraffin (Leica AG, Frankfurt, Germany), sliced into 5
   mm-thick sections, and stained with hematoxylin and eosin (H&E). The
   hippocampal subregions, namely, the cornu ammonia 1 (CA1), CA2, CA3,
   and dentate gyrus (DG), play distinct roles in socialization, learning,
   and memory. The sections were examined in detail under a light
   microscope (Olympus Co., Tokyo, Japan) to identify morphological
   changes in any of the above-mentioned regions.

   The hippocampal neurons were quantitatively analyzed using Image J
   software (Version 1.53e). Hippocampal neurons were identified by
   recognizing blue-purple stained cell nuclei and manually counted in
   each visual field. Two visual fields were observed for each section,
   with a total of six visual fields observed in each group. Finally, the
   total numbers of hippocampal neurons in each group were statistically
   analyzed.

2.6. TMT-Based Quantitative Proteomics

2.6.1. Protein Sample Preparation

   Hippocampal protein was prepared as previously reported
   [[81]36,[82]37,[83]38]. For protein extraction, the samples were placed
   in lysis buffer and homogenized using an automatic grinder. The
   supernatant was collected by centrifugation at 25,000× g for 15 min at
   4 °C. Then, protein samples (100 μg) were reduced, alkylated, and
   precipitated by adding 5× the volume of chilled acetone and placing at
   −20 °C for 2 h. The precipitated proteins were resolved with SDS-free
   lysis buffer and centrifugated at 25,000× g for 15 min at 4 °C. The
   collected protein was quantitated using the Bradford assay, and protein
   integrality was assessed by SDS-PAGE. Precipitated proteins were
   digested for 4 h at 37 °C in 100 μL of TEAB buffer containing
   sequence-grade trypsin at a trypsin-to-protein mass ratio of 1:20.
   Enzymatic cleavage was terminated by adding 5 μL of 10% formic acid,
   and the collected peptide fractions were freeze-dried.

2.6.2. TMT Labeling

   TMT labeling was conducted to reconstitute and process peptides based
   on the protocol of TMT Multiplex Kit (Thermo Fisher, Waltham, MA, USA).
   The TMT reagent was warmed to room temperature; then, 41 μL (0.8
   mg/tube) acetonitrile was added to the TMT reagent and shaken
   thoroughly. Digested peptide (100 μg) was mixed with 41 μL of TMT
   reagent. The reaction was terminated by shaking followed by
   centrifugation and incubation at room temperature for 2 h. TMT-labeled
   mixed peptides were fractionated using liquid chromatography (LC-20AB,
   Shimadzu, Kyoto, Japan) coupled with a Gemini C18 column (250 mm × 4.6
   mm × 5 μm). In the binary solvent system, the mobile phase A contained
   5% acetonitrile (pH 9.8), whereas mobile phase B contained 95%
   acetonitrile (pH 9.8). The dried peptide samples were reconstituted
   with mobile phase A and injected. LC separation was performed at a flow
   rate of 500 µL/min by the following gradients: 5% B for 10 min, 5–35% B
   for 40 min, 35–95% B for 1 min, 100% B for 3 min, and 5% B for 10 min.
   The elution peak was monitored at a wavelength of 214 nm, one component
   was collected per minute, and the samples were combined according to
   the chromatographic elution peak map to obtain 20 fractions, which were
   then vacuum-dried.

2.6.3. LC-MS/MS Analysis

   Dried polypeptides were reconstituted in mobile phase A (2%
   acetonitrile, 0.1% formic acid) and centrifuged at 20,000× g for 10
   min. One microliter of supernatant was injected into a Thermo UltiMate
   3000 UPLC. Each sample was separated at a flow rate of 300 nL/min using
   the following effective gradient: 5% mobile phase B (98% acetonitrile,
   0.1% formic acid) in 5 min, 5–25% B over 40 min, 25–35% B over 5 min,
   35–80% B over 2 min, 80% B over 2 min, and 5% B for 6 min. The peptides
   separated by liquid chromatography were ionized using a nanoESI source
   and then analyzed in a Q-Exactive HF X tandem mass spectrometer (Thermo
   Fisher Scientific, San Jose, CA, USA) in data-dependent acquisition
   (DDA) mode. The main parameters were set as follows: ion source
   voltage, 2 kV; MS1 scanning range, 350–1500 m/z; resolution, 60,000;
   MS2 starting m/z, 100, with a resolution of 15,000. MS2 fragmentation
   was performed on ions with charges ranging from 2+ to 6+, selecting the
   top 20 parent ions with peak intensities exceeding 20,000. High-energy
   collisional dissociation (HCD) was used for ion fragmentation with
   Orbitrap detection. The dynamic exclusion time was set to 30 s, and the
   automatic gain control was set to 1E5 for MS1 and 2E4 for MS2.

2.6.4. Processing and Analysis of the Spectral Data

   The raw files obtained from LC-MS/MS were searched against the UniProt
   Protein Database (version 2.4, Thermo Scientific, San Jose, CA, USA)
   using the following parameters: Precursor ion mass tolerance: 20 ppm;
   Fragment ion mass tolerance: 20 ppm; Maximum missed cleavages: 2. The
   false discovery rate (FDR) was set at 1%. DEPs were identified based on
   a fold change (FC) greater than 1.5 between Pb-treated group and
   control group with a p-value less than 0.05. The DEPs were annotated
   through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes
   (KEGG) databases. In GO annotation, the DEPs were classified into
   subcategories, namely, biological process (BP), cellular component
   (CC), and molecular function (MF). The functions of DEPs were explored
   through GO and KEGG enrichment analysis. A heatmap was plotted on an
   online platform ([84]https://www.bioinformatics.com.cn, last accessed
   on 10 December 2024) for data analysis and visualization.
   Protein–protein interaction (PPI) was completed by Markov Clustering
   (MCL) with the inflation parameter of more than 3 by using the STRING
   database ([85]https://cn.string-db.org/, last accessed on 5 April
   2025).

2.7. Western Blotting Analysis

   To further investigate the potential targets for ASD, we analyzed the
   protein expression levels of key synapse-related proteins identified in
   proteomics, including synaptotagmin-10 (SYT10) and syntaxin-16 (STX16).
   Additionally, the expression level of insulin-like growth factor 1
   (IGF-1), a downstream signaling factor of SYT10, was assessed via
   Western blotting.

   Total protein was extracted from hippocampal tissue. Equal amounts of
   protein were separated by sodium dodecyl sulfate–polyacrylamide gel
   electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride
   (PVDF) membrane via wet transfer, blocked, incubated with primary
   antibodies against SYT10 (1:2000, BioSource, Muskego, WI, USA), STX16
   (1:1000, Invitrogen, Waltham, MA, USA), IGF-1 (1:1000, Cell Signaling
   Technology, Danvers, MA, USA), glyceraldehyde-3-phosphate dehydrogenase
   (GAPDH, 1:10,000, Proteintech, Rosemont, IL, USA), or β-actin
   (1:10,000, Abbkine, Wuhan, China) at 4 °C overnight, and then with
   peroxidase-conjugated goat anti-rabbit or anti-mouse immunoglobulin G
   secondary antibody (1:10,000, Abcam, Cambridge, UK) at room temperature
   for 1 h. After exposure using an ECL reagent, the bands were visualized
   and imaged using the ECL imaging system (Bio-Rad, Berkeley, CA, USA).

2.8. Statistical Analysis

   All results for each group were expressed as means ± SEM. Differences
   among groups were analyzed using one-way or two-way ANOVA, followed by
   Tukey’s multiple-comparison test. Post hoc tests were performed when F
   in ANOVA achieved the necessary level of statistical significance (p <
   0.05) and there was no significant variance in homogeneity. Student’s
   t-tests were used to compare two sets of data.

3. Results

3.1. Postnatal Pb Exposure Induced Autism-like Behaviors in Mice

   The three-chamber social test was employed to elucidate the social
   behavior deficits in mice exposed postnatally to Pb ([86]Figure 2A). In
   the sociability stage of the test, control mice spent more time in the
   chamber containing the stranger than in the empty chamber. In contrast,
   mice in both the 15 and 30 mg/kg PbAc groups showed no significant
   difference for either chamber ([87]Figure 2B). Accordingly, mice in the
   Pb exposure groups demonstrated a significant decrease in the SI and
   the SNI compared with those in the control group (p < 0.01, [88]Figure
   2C,D).

   The marble-burying test was employed to quantify restricted repetitive
   behaviors ([89]Figure 3A). Compared with control animals, Pb-treated
   mice showed a significant increase in the number of marbles buried
   ([90]Figure 3B, p < 0.0001). These findings imply that mice exposed to
   Pb during the postnatal period display social deficits and repetitive
   behaviors, which are characteristic features of an ASD-like phenotype.

Figure 3.

   [91]Figure 3
   [92]Open in a new tab

   Influence of postnatal Pb exposure on repetitive behaviors in offspring
   mice (n = 8/each group). (A) Experimental demonstration diagram of the
   marble-burying test. (B) The Pb-exposed mice induced a significant
   increase in the number of buried marbles compared with the control
   mice. **** p ≤ 0.0001.

3.2. Pb Levels in Blood and the Hippocampus

   To assess the validity of the model established in this study, the Pb
   levels in blood and the hippocampus of mice were analyzed. The results
   indicated that blood Pb levels in the Pb-exposed groups were
   significantly elevated compared with those in the control group (p <
   0.05, [93]Figure 4). However, there was no significant difference in
   blood Pb levels between the Pb treatment groups (15 and 30 mg/kg PbAc;
   p > 0.05, [94]Figure 4A). Additionally, the Pb levels in the
   hippocampal tissue of Pb-treated groups (15 and 30 mg/kg PbAc) were
   significantly higher than those in the control group (p < 0.01,
   [95]Figure 4B).

Figure 4.

   [96]Figure 4
   [97]Open in a new tab

   Pb levels in both the blood and hippocampus of offspring mice followed
   by Pb exposure (n = 3/each group). (A) Blood Pb levels. (B) Hippocampus
   Pb levels. * p ≤ 0.05, ** p ≤ 0.01. “ns” means no significant
   difference.

3.3. Postnatal Pb Exposure Induced Morphological Alterations in the
Hippocampus

   We employed H&E staining to detect morphological changes in hippocampal
   subregions. In the control group, neurons in the CA1, CA2, CA3, and DG
   regions of the hippocampus were arranged in an orderly manner, with
   well-defined layers, distinct nucleoli, pale red cytoplasm, and no
   apparent pathological alterations ([98]Figure 5A). In contrast, in the
   Pbexposed groups, there was a significant reduction in the number of
   hippocampal neurons ([99]Figure 5B), the neurons showed a disordered
   arrangement, and the boundary with the surrounding tissues was unclear.
   Moreover, Pb exposure led to neuronal shrinkage and hyperchromatism,
   nuclear condensation, and a decrease in the number of nuclei. Nuclear
   condensation was most pronounced in the 30 mg/kg PbAc group, and there
   were fewer nuclei in the 30 mg/kg PbAc group than in the 15 mg/kg PbAc
   group, indicative of more extensive pathological changes.

Figure 5.

   [100]Figure 5
   [101]Open in a new tab

   Postnatal Pb exposure induced morphological changes in the hippocampus
   of offspring mice (n = 3/each group). (A) H&E staining analysis.
   Low-magnification images (×100); high-magnification images (×400).
   Orange arrows represent neurons. (B) The neuron numbers in the
   hippocampus. * p ≤ 0.05, ** p ≤ 0.01.

3.4. Postnatal Pb Exposure Affected the Proteome Profiling

   To further elucidate the underlying molecular mechanisms linking
   postnatal Pb exposure and ASD-like behaviors, TMT-based proteomic
   analysis was employed to detect protein expression (n = 3/each group).
   A total of 7900 proteins were identified and quantified by referencing
   the Mus musculus database. Principal component analysis (PCA) of the
   proteomic profiling results indicated that there were distinct overall
   differences among the control, 15 mg/kg PbAc, and 30 mg/kg PbAc groups,
   with samples clustering within each respective group ([102]Figure 6A).
   Compared with the control group, a total of 66 proteins were identified
   as being differentially expressed after postnatal Pb exposure, and
   then, a cluster analysis was performed as described by Tang et al.
   [[103]39] ([104]Figure 6B, [105]Table S1). A total of 46 DEPs were
   identified in the 15 mg/kg PbAc group (37 downregulated and 9
   upregulated), while 54 DEPs (51 downregulated and 3 upregulated) were
   detected in the 30 mg/kg PbAc group ([106]Figure 6C). Among the DEPs
   identified in both Pb exposure groups, 34 were common to both groups
   ([107]Figure 7A). Of these, 33 were downregulated and 1 was upregulated
   ([108]Table S1).

Figure 6.

   [109]Figure 6
   [110]Open in a new tab

   Differentially expressed proteins (DEPs) identified by TMT-based
   proteomic technology. (A) Principal component analysis (PCA). (B)
   Heatmap of DEPs. (C) Volcano plot of DEPs. The dots in red represent
   significantly upregulated DEPs, dots in green represent significantly
   downregulated DEPs, and dots in gray represent proteins with no
   significant difference in expression levels.

Figure 7.

   [111]Figure 7
   [112]Open in a new tab

   Bioinformatic analysis of DEPs. (A) Venn diagram of DEPs in Pb-exposed
   groups vs. control. (B) The top-ranked GO terms for a minimum of two
   DEPs in 15 mg/kg PbAc group. (C) The top-ranked GO terms for a minimum
   of two DEPs in 30 mg/kg PbAc group. (D) The significant enriched KEGG
   pathways in 15 mg/kg PbAc group. (E) The significant enriched KEGG
   pathways in 30 mg/kg PbAc group. (F) Protein–protein interaction (PPI)
   network diagram of target proteins.

3.5. Postnatal Pb Exposure Triggered Multi-Pathway Alterations

   GO functional enrichment analysis was performed to identified DEPs.
   Among the 46 DEPs in the 15 mg/kg PbAc group, the top-ranked GO terms
   in the biological process (BP) category were mainly concentrated on the
   negative regulation of transcription and brain development.
   Furthermore, a total of 17 DEPs were annotated as integral components
   of the membrane in the cellular component (CC) category, while in the
   molecular function (MF) category, the top-ranked GO terms encompassed
   enzyme binding and transcription coregulator activity ([113]Figure 7B).
   In the 30 mg/kg PbAc group, meanwhile, the top-ranked GO terms in the
   BP category were concentrated on the regulation of transcription by RNA
   polymerase II and peptidyl-serine phosphorylation. Within the CC
   category, the preeminent GO terms were centered around chromatin,
   transcription factor complex, and PML body. For MF, the top-ranked GO
   terms encompassed activities related to enzyme binding, ubiquitin
   protein ligase binding, transcription factor binding, and transcription
   coregulator activity ([114]Figure 7C).

   KEGG pathway enrichment analysis was used to identify major biochemical
   metabolic pathways and signal transduction pathways associated with the
   above DEPs. Significant enrichment of signaling pathways was observed
   in both Pb-exposure groups, with 8 and 10 pathways identified in the 15
   mg/kg PbAc group and in the 30 mg/kg PbAc group ([115]Figure 7D,E),
   respectively. Among them, six enriched KEGG signaling
   pathways—glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan
   sulfate, non-homologous end-joining, cell cycle, apoptosis, longevity
   regulating pathway, and hepatitis C—were common to both Pb exposure
   groups. In addition, two signaling pathways were specifically enriched
   in the 30 mg/kg PbAc group, namely, transcriptional dysregulation in
   cancer and neutrophil extracellular trap formation. These results
   suggested that there are multiple pathways involved in Pb-induced
   ASD-like behaviors.

   To further explore the connections among these pathways, a total of 66
   DEPs were submitted to the STRING tool and constructed into an
   interaction network. As shown in [116]Figure 7F, there was a close
   cluster between biogenesis of lysosome-related organelles complex 1
   subunit 4 (BLOC1S4), syntaxin-16 (STX16), and Yip1 domain family member
   6 (YIPF6), which are involved in Golgi-associated vesicle biogenesis
   and/or the BLOC-1 complex.

3.6. Postnatal Pb Exposure Decreased the Protein Levels of SYT10, STX16, and
IGF-1

   Western blotting analysis was performed to quantify the protein levels
   of SYT10, STX16, and IGF-1 in the hippocampus after postnatal Pb
   exposure (n = 3/each group). Pb exposure resulted in a significant
   downregulation in protein levels of SYT10 and STX16, which were
   consistent with those obtained in the proteomic analysis ([117]Figure
   8). Meanwhile, the protein expression of IGF-1 was significant
   downregulated in both the 15 mg/kg PbAc group and the 30 mg/kg PbAc
   group ([118]Figure 9).

Figure 8.

   [119]Figure 8
   [120]Open in a new tab

   Postnatal Pb exposure inhibited the expression of SYT10 and STX16. (A)
   The expressed abundance of SYT10 and STX16 was detected with TMT-based
   proteomic analysis. (B) The protein level of SYT10 and STX16 was
   detected with Western blotting analysis.

Figure 9.

   [121]Figure 9
   [122]Open in a new tab

   Postnatal Pb exposure inhibited the expression of IGF-1. * p ≤ 0.05, **
   p ≤ 0.01.

4. Discussion

   The pathogenesis of ASD remains elusive, which impedes the development
   of therapeutic and preventive strategies. Several lines of evidence
   have revealed a critical role of the hippocampus in the social memory
   dysfunction associated with ASD [[123]40,[124]41]. In the present
   study, postnatal Pb exposure in male mice not only led to a notable
   alteration in the hippocampus, but also significantly induced ASD-like
   phenotypes such as sociability deficits and repetitive behaviors. These
   findings further suggest a positive correlation between Pb exposure and
   the incidence of ASD. In the classic trisynaptic circuit, information
   proceeds from the entorhinal cortex (EC) to DG, then to CA3, and
   finally to CA1, which serves as the primary output of the hippocampus.
   The alterations in the CA2 subregion have received less attention in
   previous studies. Recent studies have shown the CA2 subregion is
   crucial for socially cognitive memory [[125]42,[126]43]. Notably, we
   also observed that, following Pb exposure, the CA2 subregion of the
   hippocampus exhibited a substantial decrease in neuron numbers and
   increased morphological abnormalities. These changes may represent
   potential mechanisms underlying the social deficits by Pb exposure.

   ASD is typically diagnosed in early life, mainly before age three—a
   critical developmental phase of intense synaptogenesis. Disruption of
   synaptic plasticity is a key mechanism in Pb-induced neuropsychiatric
   disorders [[127]5,[128]44] and is linked to ASD pathogenesis
   [[129]45,[130]46]. Our proteomic finding revealed that Pb exposure
   significantly downregulated the protein expression of DEPs associated
   with synaptic regulation. This observation further implies a reduction
   in synaptic signaling, which may be linked to ASD-like behaviors. SYT10
   belongs to the SYT family, which mainly participates in the regulation
   of synaptic vesicle fusion with target plasma membrane. However, the
   biomolecular functions of SYT10 remain poorly understood. Cao et al.
   reported that SYT10 functions as the Ca^2+-sensor, which triggers
   insulin-like growth factor 1 (IGF-1) exocytosis in olfactory bulb
   neurons [[131]47]. Deletion of SYT10 impaired activity-dependent IGF-1
   secretion, resulting in smaller neurons and an overall decrease in
   synapse numbers. In a different study, it was demonstrated that the
   conditional deletion of SYT10 inhibited IGF-1 secretion, thereby
   preventing socially relevant GABAergic long-term potentiation (LTP) and
   inducing neuropsychiatric disorders [[132]48,[133]49]. In the current
   study, employing proteomics and Western blotting analysis, we found
   that Pb exposure significantly downregulated protein levels of SYT10.
   Importantly, the protein level of IGF-1 was markedly downregulated in
   Pb-exposed groups (p < 0.05) based on Western blotting analysis. These
   results suggest that the SYT10/IGF-1 signaling pathway plays important
   roles in Pb-induced neurodevelopmental toxicity and may also be
   involved in impairing GABAergic neurotransmission and synaptic
   plasticity. Nevertheless, further research is needed to elucidate the
   underlying mechanisms of neurodevelopmental disorders.

   In the present study, through STRING analysis and MCL cluster, we
   identified a cluster closely related to synaptic vesicle function. The
   cluster pertains to Golgi-associated vesicle biogenesis and the BLOC-1
   complex, which consists of biogenesis of lysosome-related organelles
   complex 1 subunit 4 (BLOC1S4), syntaxin-16 (STX16), and Yip1 domain
   family member 6 (YIPF6). The BLOC-1 complex, in conjunction with
   synaptic soluble N-ethylmaleimide-sensitive factor attachment protein
   receptor (SNARE) proteins, is also proposed to play a role in neurite
   extension and be involved in intracellular vesicle trafficking
   [[134]50]. BLOC1S4, a constituent of the biogenesis of lysosome-related
   organelles complex 1 (BLOC-1), is thought to participate in
   vesicle-mediated transport and vesicle budding from the trans-Golgi
   network [[135]51]. STX16, a SNARE protein, was observed to play a key
   role in regulating polarized neurite growth, particularly dendritic
   extension [[136]52]. The loss of STX16 function can limit dendritic
   branching or affect localized transport processes within dendritic
   trees or spines, thus affecting neural transmission and potentially
   contributing to detectable behavioral abnormalities [[137]53].
   Furthermore, YIPF6 is a five-transmembrane-spanning protein associated
   with Golgi compartments [[138]54]. It is hypothesized that YIPF
   proteins play a role in vesicle budding and/or fusion within the Golgi
   apparatus [[139]55]. These proteins establish a core physical
   interaction network that is linked to other gene products involved in
   membrane trafficking, such as SNAREs [[140]55]. The inhibition of the
   BLOC-1 complex suggested Pb exposure might disrupt the Golgi-associated
   synaptic vesicle trafficking. The disruption, in turn, could affect
   neurite extension and ultimately the dysregulation of neural
   transmission.

   In addition to the above-mentioned DEPs, despite the fact that KEGG
   analysis did not result in its enrichment, we identified a protein,
   DENN domain-containing protein 2B (DENND2B), which is closely related
   to synaptic vesicle function. In the Pb exposure groups, DENND2B was
   significantly downregulated. Notably, Göhring et al. reported that a
   loss-of-function mutation in DENND2B disrupts vesicle formation and
   neurotransmitter trafficking [[141]56,[142]57]. This provides further
   evidence for elucidating that Pb induces developmental disorders by
   interfering with the function of synaptic vesicles. The dysregulation
   of synaptic signaling may, in part, account for the molecular pathology
   of ASD associated with Pb exposure.

   Studies have indicated that cell apoptosis may be pivotal in the
   pathogenesis of ASD [[143]58,[144]59]. In the present study, using KEGG
   enrichment analysis, the apoptosis pathway was identified, suggesting
   its key role in ASD-like behaviors by Pb exposure. Pb induced apoptosis
   not only by reducing the protein level of B-cell lymphoma 2 (Bcl-2), a
   typical anti-apoptotic marker protein [[145]60], but also by triggering
   the accumulation of reactive oxygen species (ROS) [[146]61]. Our
   proteomic analysis demonstrated a critical anti-apoptotic protein,
   namely Bcl-2-associated athanogene 3 (BAG3), was significantly
   downregulated by Pb exposure. Its downregulation may imply that
   neuronal apoptosis was abnormally activated. Moreover, we observed that
   sodium/myo-inositol cotransporter (SLC5A3) was the only protein that
   was significantly upregulated in both the 15 mg/kg PbAc and 30 mg/kg
   PbAc treatment groups. SLC5A3, a member of the solute carrier family 5,
   is recognized as a sodium-coupled myo-inositol transporter.
   Functionally, SLC5A3 can positively modulate ROS biosynthesis and
   oxidative stress responses [[147]62]. Its upregulation suggested that
   postnatal Pb exposure may also induce ROS accumulation. This, in turn,
   leads to the abnormal activation of hippocampal neuron apoptosis and
   triggers the emergence of ASD-like behaviors.

   Based on the integrative omics analysis, we have reported that
   neuroimmune response during development is critically involved in ASD
   [[148]63]. In the current work, a crucial regulator of immune response,
   namely Myb-like, SWIRM, and MPN domains containing protein 1 (MYSM1),
   was identified by proteomic analysis. The deficiency of MYSM1 induced
   the activation of the NOD2/RIP2 complex [[149]64], and then resulted in
   excessive inflammation [[150]65]. As for a deubiquitinating enzyme, the
   deletion of MYSM1 also stimulated PI3K/AKT signaling [[151]66] and
   exhibited developmental abnormalities [[152]67]. The downregulation of
   MYSM1 in Pb exposure groups revealed that neuroinflammation may play a
   key role involved in ASD-like behaviors. Interestingly, through KEGG
   pathway enrichment, one pathway related with neuroimmune signaling was
   identified, that is, glycosaminoglycan biosynthesis–chondroitin
   sulfate/dermatan sulfate. Chondroitin sulfate (CS) serves as a potent
   inhibitor of the immune response [[153]68,[154]69]. Additionally, it
   inhibits the activation of human mast cells, and such activation has
   been associated with ASD [[155]70]. Genetically, reduction in the
   activity of chondroitin sulfate synthase 1 (CHSS1) leads to
   neuroinflammation in the mouse hippocampus [[156]71]. Notably, our
   proteomic analysis found that chondroitin sulfate synthase 2 (CHSS2)
   was significantly downregulated by Pb exposure, suggesting the
   possibility of the occurrence of neuroinflammation.

5. Conclusions

   In summary, we established a mouse model of postnatal Pb exposure,
   which gave rise to prominent social deficits, repetitive behaviors, and
   hippocampal neuronal damage. These results clearly demonstrated that
   postnatal Pb exposure induced autism-like behaviors through alteration
   of key protein profiling and multi-pathway regulation, including
   dysregulation of synaptic signaling, abnormal activation of neuron
   apoptosis, and neuroinflammation ([157]Figure 10). The SYT10/IGF-1
   signaling pathway may play a potential key role in Pb-induced
   neurodevelopmental disorders. However, further research, both in vivo
   and in vitro, is needed to comprehensively elucidate the mechanism
   through which Pb disrupts neurodevelopmental integrity. Nevertheless,
   these findings heighten our understanding of the potential association
   between environmental heavy metals and neurodevelopmental disorders.

Figure 10.

   [158]Figure 10
   [159]Open in a new tab

   Potential molecular pathology of ASD associated with Pb exposure. White
   arrow means down-regulation, red arrow means up-regulation, and green
   arrow means down-regulation.

Acknowledgments