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. 2017 Nov;23(11):855-865.
doi: 10.1111/cns.12757. Epub 2017 Sep 20.

Androgen alleviates neurotoxicity of β-amyloid peptide (Aβ) by promoting microglial clearance of Aβ and inhibiting microglial inflammatory response to Aβ

Peng-Le Yao  1 , Shu Zhuo  1 , Hong Mei  1 , Xiao-Fang Chen  1 , Na Li  1 , Teng-Fei Zhu  1 , Shi-Ting Chen  1 , Ji-Ming Wang  2 , Rui-Xing Hou  3 , Ying-Ying Le  1   3
Affiliations

Androgen alleviates neurotoxicity of β-amyloid peptide (Aβ) by promoting microglial clearance of Aβ and inhibiting microglial inflammatory response to Aβ

Peng-Le Yao et al. CNS Neurosci Ther. 2017 Nov.

Abstract

Aims: Lower androgen level in elderly men is a risk factor of Alzheimer's disease (AD). It has been reported that androgen reduces amyloid peptides (Aβ) production and increases Aβ degradation by neurons. Activated microglia are involved in AD by either clearing Aβ deposits through uptake of Aβ or releasing cytotoxic substances and pro-inflammatory cytokines. Here, we investigated the effect of androgen on Aβ uptake and clearance and Aβ-induced inflammatory response in microglia, on neuronal death induced by Aβ-activated microglia, and explored underlying mechanisms.

Methods: Intracellular and extracellular Aβ were examined by immunofluorescence staining and Western blot. Amyloid peptides (Aβ) receptors, Aβ degrading enzymes, and pro-inflammatory cytokines were detected by RT-PCR, real-time PCR, and ELISA. Phosphorylation of MAP kinases and NF-κB was examined by Western blot.

Results: We found that physiological concentrations of androgen enhanced Aβ42 uptake and clearance, suppressed Aβ42 -induced IL-1β and TNFα expression by murine microglia cell line N9 and primary microglia, and alleviated neuronal death induced by Aβ42 -activated microglia. Androgen administration also reduced Aβ42 -induced IL-1β expression and neuronal death in murine hippocampus. Mechanistic studies revealed that androgen promoted microglia to phagocytose and degrade Aβ42 through upregulating formyl peptide receptor 2 and endothelin-converting enzyme 1c expression, and inhibited Aβ42 -induced pro-inflammatory cytokines expression via suppressing MAPK p38 and NF-κB activation by Aβ42 , in an androgen receptor independent manner.

Conclusion: Our study demonstrates that androgen promotes microglia to phagocytose and clear Aβ42 and inhibits Aβ42 -induced inflammatory response, which may play an important role in reducing the neurotoxicity of Aβ.

Keywords: Alzheimer's disease; androgen; microglia; β-amyloid peptide.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Dihydrotestosterone promotes uptake and clearance of Aβ42 by microglia. N9 cells (A,C,D) or murine primary microglia (B) were treated with different concentrations of dihydrotestosterone (DHT) for 24 hours followed by incubation with 1 μmol/L Aβ42 for 1 hour (A‐D) or 24 hours (D). Intracellular Aβ was examined by immunofluorescence staining (A,B, red) and Western blot (C,D), respectively. Cell nuclei were stained with Hoechst (A,B, blue). Amyloid peptides (Aβ) levels in supernatant were detected with Western blot (D). Data are presented as mean ± SD, n = 3. *P < 0.05, ***P < 0.001 vs Aβ42 treatment alone for 1 hour (C) or 24 hours (D). Images are representative results of three independent experiments
Figure 2
Figure 2
Dihydrotestosterone promotes Aβ42 uptake by microglia through upregulating formyl peptide receptor 2 (Fpr2). (A) N9 cells were treated with 1 nmol/L dihydrotestosterone (DHT) for 6 hours and examined for mRNA levels of Fpr2, TLR2, TLR4, and CD36 by RT‐PCR. (B) Mouse primary microglia from wild‐type mice (WT) or Fpr2 knockout mice (Fpr2−/−) were treated with or without 1 nmol/L DHT for 24 hours; then cells were incubated with 1 μmol/L Aβ42 for 1 hour. Amyloid peptides (Aβ) in cell lysate was examined by Western blot. (C) Expression of androgen receptor (AR) in N9 cells and murine primary microglia was examined by RT‐PCR, AR expression in kidney tissues was shown as a positive control. (D) N9 cells pretreated with 10 μmol/L Flutamide (Flu) for 1 hour were treated with 1 nmol/L DHT for 24 hours and with 1 μmol/L Aβ42 for another 1 hour, intracellular Aβ was examined by Western blot. Data are mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs untreated control cells (NC) (A) or cells incubated with Aβ42 alone (C). Images are representative results of three independent experiments
Figure 3
Figure 3
Dihydrotestosterone promotes Aβ42 clearance by microglia through upregulating ECE‐1c expression. (A) N9 cells were treated with 1 nmol/L dihydrotestosterone (DHT) for 6 hours and examined for Aβ42 degrading enzymes expression by RT‐PCR. (B) N9 cells pretreated with 10 μmol/L Flutamide (Flu) for 1 hours were treated with 1 nmol/L DHT for 24 hours and with 1 μmol/L Aβ42 for another 1 or 24 hours, intracellular and supernatant Aβ42 was examined by Western blot. Data are mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs untreated cells (A) or cells incubated with Aβ42 alone for 24 hours (B). Images are representative results of three independent experiments
Figure 4
Figure 4
Androgen inhibits Aβ42‐induced pro‐inflammatory cytokines expression in microglia and attenuates neurotoxicity caused by Aβ42‐activated microglia. (A‐B) Serum starved N9 cells (A) or murine primary microglia (B) were stimulated with Aβ42 (4 μmol/L for N9 cells, 2 μmol/L for primary microglia) for 6 hours and examined for pro‐inflammatory cytokine expression by real‐time PCR. (C‐D) Murine primary microglia pretreated with 10 nmol/L dihydrotestosterone (DHT) or 100 nmol/L testosterone (T) in serum‐free culture medium for 24 hours were stimulated with 2 μmol/L Aβ42 for 6 (C) or 24 hours (D), and examined for IL‐1β and TNFα expression at mRNA (C) and protein (D) levels, respectively. (E) N9 cells pretreated with or without androgen (10 nmol/L DHT or 100 nmol/L T) for 24 hours were stimulated with 4 μmol/L Aβ42 for another 24 hours, the supernatant (conditional medium) was collected to incubate N2a cells for 36 hours. The viability of N2a cells was examined by immunofluorescence staining with anti‐MAP2 antibody. Images are representative of three independent experiments. Data are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 vs untreated control cells (NC); # P < 0.05, ## P < 0.01, ### P < 0.001 vs microglia treated with Aβ42 alone (C,D), or N2a cells treated with conditional medium from Aβ42 stimulated N9 cells (E)
Figure 5
Figure 5
Androgen inhibits Aβ42‐induced pro‐inflammatory cytokines expression in microglia through suppressing MAP kinase p38 and NF‐κB activation. (A) Serum starved murine primary microglia were stimulated with 2 μmol/L Aβ42 for 1 hour, the phosphorylation of MAP kinases and IκBα was examined by Western blot. (B) Murine primary microglia pretreated with 10 μmol/L SB203580 (SB) or 100 μmol/L PDTC for 1 hour were stimulated with 2 μmol/L Aβ42 for 6 hours and examined for expression of IL‐1β and TNFα by real‐time RT‐PCR. (C) Serum starved N9 cells pretreated with 10 nmol/L dihydrotestosterone (DHT) or 100 nmol/L testosterone (T) for 1 hour were stimulated with 4 μmol/L Aβ42 for 0.5 hours, then examined for p38 and NF‐κB p65 phosphorylation by Western blot. (D‐E) N9 cells pretreated with 10 μmol/L Flutamide (Flu) for 1 hour were incubated with 10 nmol/L DHT or 100 nmol/L T for 24 hours followed by stimulation with Aβ42 for 24 hours, IL‐1β and TNFα levels in cell lysate were examined by ELISA (D,E). Data are presented as mean ± SD, n = 3. *P < 0.05, ***P < 0.001, compared with untreated control cells (NC); # P < 0.05, ## P < 0.01, ### P < 0.001, compared with cells treated with Aβ42 alone. Images are representative results of three independent experiments
Figure 6
Figure 6
Androgen inhibits Aβ42‐induced inflammation and neurotoxicity in mouse brain. Mice were subcutaneously injected with different doses of dihydrotestosterone (DHT), testosterone (T), or vehicle every other day for 2 weeks. 2 μL Aβ42 (2 μg) or same volume of PBS was injected into CA1 region of hippocampus. After 24 hours, IL‐1β expression in hippocampus was examined by ELISA (A), neurons in brain section were stained with Hoechst and neurons in CA1 region of hippocampus were quantified with Image‐Pro Plus 6.0 (C). Data are mean ± SD, n = 3~7/per group. Images in (B) are representative results of three independent experiments. Scale bar: 200 μm (upper panel), 20 μm (bottom panel). **P < 0.01 vs PBS injected group; # P < 0.05, ## P < 0.01 vs Aβ42 injected alone
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