Neuregulin-1 (Nrg1) signaling has a preventive role and is altered in the frontal cortex under the pathological conditions of Alzheimer's disease

Abstract

Alzheimer's disease (AD), one of the neurodegenerative disorders that may develop in the elderly, is characterized by the deposition of β‑amyloid protein (Aβ) and extensive neuronal cell death in the brain. Neuregulin‑1 (Nrg1)‑mediated intercellular and intracellular communication via binding to ErbB receptors regulates a diverse set of biological processes involved in the development of the nervous system. In the present study, a linear correlation was identified between Nrg1 and phosphorylated ErbB (pNeu and pErbB4) receptors in a human cortical tissue microarray. In addition, increased expression levels of Nrg1, but reduced pErbB receptor levels, were detected in the frontal lobe of a patient with AD. Western blotting and immunofluorescence staining were subsequently performed to uncover the potential preventive role of Nrg1 in cortical neurons affected by the neurodegenerative processes of AD. It was observed that the expression of Nrg1 increased as the culture time of the cortical neurons progressed. In addition, H2O2 and Aβ1‑42, two inducers of oxidative stress and neuronal damage, led to a dose‑dependent decrease in Nrg1 expression. Recombinant Nrg1β, however, was revealed to exert a pivotal role in preventing oxidative stress and neuronal damage from occurring in the mouse cortical neurons. Taken together, these results suggest that changes in Nrg1 signaling may influence the pathological development of AD, and exogenous Nrg1 may serve as a potential candidate for the prevention and treatment of AD.

Figure 1

Linear correlation analyses for Nrg1 with either pErbB4 (A and B) or pNeu (C and D) based on integrated fluorescence intensity of the human frontal cortex tissue array. Double immunofluorescence staining of Nrg1 with either pErbB4 (A) or pNeu (C) at each tissue point is shown. Linear correlation graph depicting the association between Nrg1 and pErbB4 (B) or pNeu (D). Each corresponding Pearson correlation coefficient (r) is shown. Nrg1, neuregulin 1; pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu.

Figure 2

Changes in the Nrg1 signaling pathway molecules in the frontal lobe of a human AD brain. (A) Congo red staining and abnormal aggregation of Aβ_1–42, indicating the formation of the amyloid plaques (indicated by the arrowheads) in the frontal cortical gray matter of a human AD brain. The scale bar represents 20 μm. (B) Western blotting analysis of Nrg1, phosphorylation levels of ErbB4, Neu and Erk1/2 in the frontal lobe of a human AD patient brain. (C) Immunohistochemical detection of Nrg1, pErbB4 and pNeu (indicated by the arrowheads) in the frontal cortical gray matter from either the normal indivdual or the human AD patient. Double immunofluorescence staining images are shown for co-localization of Nrg1 with either (D) pErbB4 or (E) pNeu in the frontal cortical gray matter from either the normal individual or the human AD patient. The scale bar represents 20 μm. AD, Alzheimer's disease; Aβ, β-amyloid; Nrg1, neuregulin 1; pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu; Erk, extracellular-signal regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3

Western blot analysis of Nrg1, pNeu and pErbB4 in primary mouse cortical neurons in response to cell senescence. (A) Protein levels of Nrg1 isoforms and (B) the phosphorylation levels of Neu and ErbB4 in cortical neurons cultured for 0 to 10 days (n=5, one-way analysis of variance with Tukey's post-hoc test; data are expressed as the mean ± standard error of the mean). ^*P<0.05, ^**P<0.01 and ^***P<0.001 compared with the 0-day control. Nrg1, neuregulin 1; pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

Protective role of rNrg1β in primary mouse cortical neurons in response to oxidative stress and neuraxon damage. (A) Protein levels of Nrg1 isoforms and (B) phosphorylation levels of Neu and ErbB4 receptors in primary cortical neurons after a 24 h treatment with 0-20 μM H₂O₂ (n=6, one-way ANOVA with Tukey's post-hoc test; data are expressed as the mean ± SEM). ^*P<0.05, ^**P<0.01 and ^***P<0.001 compared with the control group. (C) The phosphorylation levels of Neu and ErbB4, and (D) the pAkt1 levels and (E) the pErk1/2 levels in primary cortical neurons pretreated with rNrg1β at a concentration of 5 or 10 nM for 2 h prior to a 24-h treatment with 2.5 μM oligomeric H₂O₂ (n=5, one-way ANOVA with Tukey's post-hoc test; data are expressed as the mean ± SEM). ^##P<0.01 and ^###P<0.001 vs. the vehicle control and ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. the H₂O₂-treated group. Double immunofluorescence staining of Nrg1 with either (F) pErbB4 or (G) pNeu in the cortical neurons pretreated with rNrg1β at a concentration of 5 or 10 nM for 2 h prior to a 24 h treatment with 2.5 μM oligomeric H₂O₂. Scale bars=20 μm. ANOVA, analysis of variance; SEM, standard error of the mean; H₂O₂, hydrogen peroxide; rNRG1β, recombinant neuregulin 1β, pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu; pErk1/2, phosphorylated extracellular-regulated signal kinase 1/2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Effects of rNrg1β pretreatment on the Nrg1 signaling in primary mouse cortical neurons exposed to Aβ_1–42. The (A) relative levels of the Nrg1 isoforms, (B) phosphorylation levels of Neu and ErbB4, (C) pAkt1 levels and (D) pErk1/2 levels in primary cortical neurons pretreated with rNrg1β at a concentration of 5 or 10 nM for 2 h prior to a 24-h treatment with 10 μM oligomeric Aβ_1–42 are shown (n=5, one-way analysis of variance with Tukey's post-hoc test; data are expressed as the mean ± standard error of the mean). Double immunofluorescence staining of β-III tubulin with either (E) pErbB4 or (F) pNeu in the cortical neurons pretreated with rNrg1β at a concentration of 5 or 10 nM for 2 h prior to a 24 h treatment with 10 μM oligomeric Aβ_1–42. Scale bars=20 μm. rNRG1β, recombinant neuregulin 1β, pErbB4, phosphorylated ErbB4; pNeu, phosphorylated Neu; pErk1/2, phosphorylated extracellular-regulated signal kinase 1/2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Aβ, amyloid-beta; DAPI, 4′,6-diamidino-2-phenylindole. ^#P<0.05 and ^##P<0.01 vs. vehicle control and ^*P<0.05, ^**P<0.01 and ^***P<0.001 compared with the control group.