Differential changes in Neuregulin-1 signaling in major brain regions in a lipopolysaccharide-induced neuroinflammation mouse model

Abstract

Neuregulin 1 (Nrg1) is involved in multiple biological processes in the nervous system. The present study investigated changes in Nrg1 signaling in the major brain regions of mice subjected to lipopolysaccharide (LPS)-induced neuroinflammation. At 24 h post‑intraperitoneal injection of LPS, mouse brain tissues, including tissues from the cortex, striatum, hippocampus and hypothalamus, were collected. Reverse transcription‑polymerase chain reaction was used to determine the expression of Nrg1 and its receptors, Neu and ErbB4, at the mRNA level. Western blotting was performed to determine the levels of these proteins and the protein levels of phosphorylated extracellular signal-regulated kinases (Erk)1/2 and Akt1. Immunohistochemical staining was utilized to detect the levels of pNeu and pErbB4 in these regions. LPS successfully induced sites of neuroinflammation in these regions, in which changes in Nrg1, Neu and ErbB4 at the mRNA and protein levels were identified compared with controls. LPS induced a reduction in pNeu and pErbB4 in the striatum and hypothalamus, although marginally increased pErbB4 levels were found in the hippocampus. LPS increased the overall phosphorylation of Src but this effect was reduced in the hypothalamus. Moreover, increased phosphorylation of Akt1 was found in the striatum and hippocampus. These data suggest diverse roles for Nrg1 signaling in these regions during the process of neuroinflammation.

Figure 1

Systemic LPS injection induced neuroinflammation in major brain regions. Immunohistochemical staining demonstrated a wide distribution of Iba-1-positive activated microglia in the cortex, striatum and hippocampus. By contrast, the number of activated microglia was limited in the hypothalamus. LPS, lipopolysaccharide.

Figure 2

Evaluation of the effects of LPS on Nrg1 expression and Neu and ErbB4 expression and activation in mouse cortex, striatum, hippocampus and hypothalamus. (A) Reverse transcription-polymerase chain reaction analysis of the influence of LPS on the mRNA levels of Nrg1, Neu and ErbB4. (B) Western blot analysis of the effect of LPS on the protein levels of multiple isoforms of Nrg1. (C) Western blot analysis of the effect of LPS on the phosphorylation-induced activation of Neu and ErbB4. Quantification of the (D) Neu and (E) ErbB4 westernblot results. Independent Student's t-test was applied. ^*P<0.05 vs. the control group (control group, n=3; LPS group, n=4). LPS, lipopolysaccharide; Nrg1, neuregulin-1; C, control group; L, LPS group; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p-, phosphorylated.

Figure 3

Immunohistochemical staining of pNeu and pErbB4 in the cortex, striatum, hippocampus and hypothalamus in mice treated with either saline (con) or LPS. Original magnification, ×400 for cortex, striatum and hypothalamus; ×100 for hippocampus. LPS, lipopolysaccharide; p-, phosphorylated.

Figure 4

Evaluation of the effects of LPS on the signaling pathway downstream of Nrg1. (A) Western blot analysis of the effect of LPS on the phosphorylation of Src, Akt1 and Erk in the cortex, striatum, hippocampus and hypothalamus in mice. Graphs presenting quantification of (B) Src, (C) Akt1 and (D) Erk western blot results. Independent Student's t-test was applied. ^*P<0.05 and ^**P<0.01 vs. the control group (control group, n=3; LPS group, n=4). LPS, lipopolysaccharide; Nrg1, neuregulin 1; Erk, extracellular signal-regulated kinase; p-, phosphorylated; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.