Hippocampal Proteomics Reveals the Novel Molecular Profiling of Postnatal Lead (Pb) Exposure on Autism-like Behaviors

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); hippocampus; lead (Pb); postnatal; proteomics.

Figure 1

Visual representation of the comprehensive research protocol timeline.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

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

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.

Figure 8

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

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

Figure 10

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.