Low-Dose Copper Exposure Exacerbates Depression-Like Behavior in ApoE4 Transgenic Mice

Abstract

Depression is one of the most common neuropsychiatric disorders. Although the pathogenesis of depression is still unknown, environmental risk factors and genetics are implicated. Copper (Cu), a cofactor of multiple enzymes, is involved in regulating depression-related processes. Depressed patients carrying the apolipoprotein ε4 allele display more severe depressive symptoms, indicating that ApoE4 is closely associated with an increased risk of depression. The study explored the effect of low-dose Cu exposure and ApoE4 on depression-like behavior of mice and further investigates the possible mechanisms. The ApoE4 mice and wild-type (WT) mice were treated with 0.13 ppm CuCl₂ for 4 months. After the treatment, ApoE4 mice displayed obvious depression-like behavior compared with the WT mice, and Cu exposure further exacerbated the depression-like behavior of ApoE4 mice. There was no significant difference in anxiety behavior and memory behavior. Proteomic analysis revealed that the differentially expressed proteins between Cu-exposed and nonexposed ApoE4 mice were mainly involved in the Ras signaling pathway, protein export, axon guidance, serotonergic synapse, GABAergic synapse, and dopaminergic synapse. Among these differentially expressed proteins, immune response and synaptic function are highly correlated. Representative protein expression changes are quantified by western blot, showing consistent results as determined by proteomic analysis. Hippocampal astrocytes and microglia were increased in Cu-exposed ApoE4 mice, suggesting that neuroglial cells played an important role in the pathogenesis of depression. Taken together, our study demonstrated that Cu exposure exacerbates depression-like behavior of ApoE4 mice and the mechanisms may involve the dysregulation of synaptic function and immune response and overactivation of neuroinflammation.

Figure 1

The 4 months of low-dose copper exposure on depression-like behavior in mice. (a, b) The percentage of immobility time. The data was shown as mean ± SEM. ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001. n = 10-12 for each group.

Figure 2

Low-dose Cu exposure did not induce anxiety-like behavior, spatial learning, and memory impairment in ApoE4 mice. (a) Total distance (mm). (b) The time spent in the open arms (%). (c) The latency 5-day training period (s). (d) The representative track diagram. (e) The distance traveled in the platform quadrant (%). (f) The time spent in the platform quadrant (%). The data was shown as mean ± SEM. n = 10-12 for each group.

Figure 3

Comprehensive analysis of DEPs in the three groups of mice: (a) approaches used to evaluate hippocampal proteins; (b) clusters representing the typical expression profiles were colored accordingly to the DEPs; (c) numbers of the upregulation and downregulation of DEPs.

Figure 4

Clue GO and heat map analysis of the differentially expressed proteins. Differentially expressed proteins identified in (a) upregulations and (c) downregulations. The enriched GO terms were organized in different colors; gene names are linked to related items. Heat map analysis of DEPs localized to (b) cholesterol metabolism, endocytosis, immune response, and synaptic function and (d) synaptic function, immune response, and apoptosis. Blue color represents low abundance, and red represents high abundance.

Figure 5

The KEGG pathway analysis for DEPs. (a, b) The enriched pathways in upregulation proteins of WT, ApoE4, and Cu-treated ApoE4 mice. (c, d) Downregulation proteins of WT, ApoE4, and Cu-treated ApoE4 mice.

Figure 6

Axon guidance pathway analysis for the DEPs. Heat map displayed as nodes labeled with gene names. The red and blue boxes represent upregulation and downregulation, respectively.

Figure 7

Western blot analysis validation for the changed proteins. (a, d) Quantified synaptic proteins, such as synaptophysin, PSD95, EphB2, p38 MAPK, and phospho-p38 MAPK expression, were validated. (b–d) The expression level and quantified proteins of the GFAP and Iba1. The data was shown as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01. n = 3-4 for each group.

Figure 8

Low-dose copper exposure increased the number of astrocytes and microglia. Immunoreactivity of astroglia (GFAP) and microglia (Iba1) in the CA1 of the hippocampus. (a–c) The representative image of positive staining of GFAP (astrocytes) and Iba1 (microglia) in the CA1 hippocampus and quantitative analysis. The scale: 100 μm and 50 μm. The data was shown as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01. n = 3-4 for each group.