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

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 3, Rui‐Xing Hou 2, Ying‐Ying Le 1,2,✉
PMCID: PMC6492702  PMID: 28941188

Summary

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: β‐amyloid peptide, Alzheimer's disease, androgen, microglia

1. INTRODUCTION

Alzheimer's disease (AD) is a chronic progressive neurodegenerative disease characterized by neurofibrillary tangles, amyloid plaques, and neuronal loss. Activated microglia and astrocytes are usually found to infiltrate in or surround amyloid plaques.1 β amyloid peptides (Aβ), the main components of amyloid plaques, are capable of inducing neuronal death directly or indirectly through activating glial cells.2 Microglia can phagocytose and degrade Aβ. However, excessive activation of microglia leads to production of pro‐inflammatory cytokines, nitric oxide, and reactive oxygen species, which contributes to neuronal death.3, 4

During normal aging, men experience a significant decline in testosterone levels. Epidemiological studies show that lower testosterone level in elderly men is a risk factor for the development of AD.5, 6, 7 Male AD patients have lower brain and serum testosterone levels than that of normal persons.5, 8 In male AD patients with mild neuropathological changes, there is a negative correlation between brain levels of testosterone and soluble Aβ in brain.8 These data suggest that androgen may modulate Aβ production and/or clearance in brain tissues. Studies with animal AD model showed that depletion of androgen in male AD mice through gonadectomy significantly increased Aβ deposition in brain. Treatment of gonadectomized male AD mice with testosterone prevented the increased Aβ accumulation in brain and improved working memory.9, 10 In vitro studies showed that treatment of neuronal cells with testosterone increased the secretion of soluble amyloid precursor protein α and decreased the secretion of Aβ.11 Dihydrotestosterone (DHT) promotes Aβ degradation through increasing neprilysin (NEP) expression in neuronal cells.12 These results indicate that androgen inhibits Aβ production and enhances Aβ clearance in neurons. However, it is not clear whether androgen could modulate uptake and clearance of Aβ by microglia.

It has been reported that androgen possesses antiinflammatory property.13 Dihydrotestosterone (DHT) is capable of suppressing oxidized low density lipoprotein or lipopolysaccharide (LPS)‐induced expression of pro‐inflammatory cytokines (IL‐1β and TNFα) in macrophages.14, 15 DHT also inhibits LPS‐induced TNFα expression in ApoE3 expressing mouse microglia.16 However, whether androgen could inhibit Aβ‐induced inflammatory response in microglia is unknown.

In this study, we investigated the effect of androgen on Aβ42 uptake and clearance, Aβ42‐induced pro‐inflammatory cytokines expression by microglia and explored the underlying mechanisms. We further examined the effect of androgen on neuronal death induced by mediators released by Aβ42‐activated microglia in vitro, and the effect of androgen administration on Aβ42‐induced pro‐inflammatory cytokine expression and neuronal death in murine hippocampus.

2. MATERIALS AND METHODS

2.1. Reagents

Dihydrotestosterone (DHT), testosterone, SB203580, and flutamide (Flu) were purchased from Sigma, St. Louis, MI, USA. Aβ42 was provided by California Peptide Research. Pyrrolidine dithiocarbamate (PDTC) was purchased from Beyotime Biotechnology, Shanghai, China. Antibodies against microtubule‐associated protein 2 (MAP2), phosphorylated and total forms of NF‐κB p65, IκBα and MAP kinases (ERK1/2, p38, JNK), and FITC labeled secondary antibodies were obtained from Cell Signaling Technology, Danvers, MA, USA. Anti‐Aβ antibody (6E10) was purchased from Covance Princeton, NJ, USA. Anti‐β actin antibody was from Invitrogen, Waltham, MA, USA. HRP tagged secondary antibodies were purchased from Proteintech, Rosemont, IL, USA. Fetal bovine serum (FBS), DMEM, DMEM/F12 without phenol red, and 0.25% trypsin (with 0.53 mmol/L EDTA) were purchased from Thermo Fisher Scientific, Waltham, MA, USA. IMDM and nonessential amino acids were obtained from Hyclone, Pittsburgh, PA, USA. Insulin was from Novo Nordisk , Bagsvard, Denmark.

2.2. Murine microglia isolation and culture

Primary mouse microglia were isolated from brain of newborn C57BL/6 mice according to literature with minor modification.17 Briefly, brains from <1‐day‐old C57BL/6 mice were excised, removed meninges, minced into small pieces, and incubated with trypsin/DNase. The cell suspension was filtered through a 40‐μm cell strainer to remove debris. Cells were centrifuged, washed with cold phosphate‐buffered saline (PBS), and resuspended in culture medium (IMDM with 10% FBS, 100 μmol/L nonessential amino acids and 5 μg/mL insulin) at 2 × 106 cells/mL and cultured at 37°C/5% CO2. Medium was replaced every 2‐3 days. After 14 days, cells were incubated with a trypsin solution (0.25% trypsin/0.53 mmol/L EDTA) diluted 1:4 in DMEM‐F12 to detach the upper layer of cells. The remained attached cells (microglia) were treated with 0.25% trypsin/0.53 mmol/L EDTA for 5 minutes. The cells were collected and cultured in IMDM containing 10% FBS for future experiments.

2.3. RT‐PCR and real‐time RT‐PCR

Total RNA was extracted from cells with TRIzol reagent (Invitrogen) and depleted of contaminating genomic DNA with RNase‐free DNase I. Reverse transcription of RNA was carried out with M‐MLV reverse transcriptase (Takara, Shimogyo‐ku, Kyoto, Japan) and oligo (dT)12‐18 (Invitrogen) according to the manufacturer's protocol. The primers for RT‐PCR and real‐time RT‐PCR are listed in Table S1 and Table S2. PCR products were visualized by GoldView staining in 1% agarose gel and quantified using Image‐Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Real‐time PCR was performed with ABI 7900 sequence detection system (Applied Biosystems, Waltham, MA, USA) with SYBR Green PCR Master Mix (Applied Biosystems). Transcriptional levels for mRNA were normalized to 36b4. The relative expression of mRNA was calculated using the 2−ΔΔCT method.

2.4. Western blot

Western blotting was carried out according to standard protocols. Target proteins were detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Primary antibodies against phosphorylated and total forms of NF‐κB p65, IκBα and MAP kinases (p38, JNK and ERK1/2) were used. The phosphorylated proteins were quantified with Image‐Pro Plus 6.0 (Media Cybernetics, Inc.) and normalized to corresponding total proteins.

To examine Aβ42 uptake and degradation by N9 cells and murine primary microglia, cells were treated with different concentrations of DHT for 24 hours, then incubated with 1 μmol/L Aβ for 1 or 24 hours. Amyloid peptides (Aβ) levels in the supernatant and cell lysate were detected by Western blot with 15% SDS‐PAGE. Intracellular and supernatant Aβ levels were quantified with Image‐Pro Plus 6.0 (Media Cybernetics, Inc.) and normalized to β–actin levels.

2.5. Immunofluorescence staining

To examine Aβ uptake by N9 or primary microglial cells, cells cultured in 12‐well plates at 2 × 105 cells/mL were incubated with different concentrations of DHT in FBS‐free IMDM medium for 24 hours, followed by treatment with 1 μmol/L Aβ42 for 1 hour. Intracellular Aβ was examined by immunofluorescence staining with antibody against Aβ (6E10, Covance). Cell nuclei were stained with Hoechst.

N9 cells pretreated with 10 nmol/L DHT or 100 nmol/L testosterone in FBS‐free IMDM medium for 24 hours were stimulated with 4 μmol/L Aβ42 for 24 hours. The supernatant was collected to incubate N2a cells for 36 hours. Cell viability was examined by immunofluorescence staining with anti‐MAP2 antibody.

For immunofluorescence staining, cells were fixed with 4% paraformaldehyde, washed with PBS and incubated with blocking buffer (5% normal goat serum/0.3% Triton™ X‐100 BBI Life Sciences; Shanghai, China in PBS) for 1 hour at room temperature. Then cells were incubated with primary antibody against Aβ or MAP2 overnight at 4°C, washed and incubated with FITC tagged secondary antibody for 1 hour at room temperature. After washing with PBS, immunofluorescence labeling was observed under a fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan). Microtubule‐associated protein 2 (MAP2) positive cells were quantified with Image‐Pro Plus 6.0 (Media Cybernetics, Inc.).

2.6. Cytokine determination by ELISA

IL‐1β and TNFα levels in supernatants of cell culture or hippocampus tissue lysate were measured using ELISA kits (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer's instructions.

2.7. Animal treatment

Male C57BL/6 mice (8 weeks old) were used in the experiments. Dihydrotestosterone (DHT) and testosterone were dissolved in ethanol with 3% ethanoic acid at concentrations of 100 mg/mL and 200 mg/mL, respectively. Dihydrotestosterone (DHT) and testosterone were diluted with sesame oil and subcutaneously injected into mice every other day. Control mice were injected with same volume of sesame oil. After seven times of injection, mice were anesthetized intraperitoneally (i.p.) with 6% chloral hydrate (BBI Life Sciences, Shanghai, China) diluted in PBS. 2 μg oliger Aβ42 (1 μg/μL) or same volume of solvent was injected into CA1 region of hippocampus using a microsyringe. The coordinates were determined according to literature18 as follows: −2.0 mm anteroposterior, 1.8 mm mediolateral, 2.0 mm dorsoventral to bregma. Aβ42 was oligomerized as described.19 Briefly, Aβ42 stock solution (2 mmol/L in DMSO) was diluted with DMEM/F12 medium (without phenol red) at concentration of 1 μg/μL, and incubated at 4°C for 24 hours before use. After 24 hours of Aβ42 injection, mice were sacrificed, and the brains were removed to dissect out hippocampi for measuring pro‐inflammatory cytokines or freeze in liquid nitrogen. Coronal 20 μm thick brain sections were cut and stained with Hoechst to examine neuronal death in hippocampus. Neurons in CA1 region of hippocampus were quantified with Image‐Pro Plus 6.0 (Media Cybernetics, Inc.). All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

2.8. Statistical analysis

Data were presented as mean ± SD. Student's t test was used to determine the differences between two groups. P < 0.05 was considered to be statistically significant.

3. RESULTS

3.1. Dihydrotestosterone promotes uptake and clearance of Aβ42 by microglia

We first examined the effect of androgen on Aβ uptake by microglia. Immunofluorescence staining with antibody against Aβ showed that pretreatment of murine microglia cell line N9 with DHT for 24 hours significantly enhanced Aβ42 uptake, in a dose‐dependent manner, at physiological concentrations (Figure 1A). Dihydrotestosterone (DHT) also promoted primary microglia to phagocytose Aβ42 in a dose‐dependent manner (Figure 1B). Western blot analysis confirmed that DHT could enhance Aβ42 uptake by microglia at physiological concentrations (Figure 1C,D). We then asked whether DHT could also enhance Aβ42 clearance by microglia. We exposed DHT‐treated N9 cells to Aβ42 for different periods of time and detected intracellular and supernatant Aβ levels by Western blot. As shown in Figure 1D, DHT significantly increased intracellular Aβ when cells were incubated with Aβ42 for 1 hour and remarkably decreased intracellular and extracellular Aβ levels when cells were incubated with Aβ42 for 24 hours. All together, these results demonstrate that DHT not only promotes Aβ42 uptake but also enhances Aβ42 clearance by microglia.

Figure 1.

Figure 1

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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

3.2. Dihydrotestosterone promotes Aβ42 uptake by microglia through upregulating Fpr2

It has been reported that many receptors mediate Aβ uptake by microglia,19, 20, 21 such as formyl peptide receptor 2 (FPR2), Toll‐like receptor 2 (TLR2), TLR4 and CD36. To explore the mechanisms underlying the enhancement of microglial phagocytic activity by DHT, we examined the effect of DTH on the expression of these receptors by microglia. We found that DHT significantly increased the mRNA level of Fpr2, the mouse homologue of human FPR2, but not other receptors (Figure 2A). Further studies showed that DHT enhanced Aβ42 uptake by microglia from wild‐type mice but not from Fpr2 knockout mice (Figure 2B). These results indicate that DHT increases Aβ42 uptake by microglia through upregulating Fpr2 expression.

Figure 2.

Figure 2

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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

Androgen regulates cell functions through androgen receptor (AR) dependent and independent pathways.22 To investigate whether DHT enhances Aβ42 uptake by microglia through AR, we examined the expression of AR in N9 cells and murine primary microglia, and found that no AR mRNA was detectable (Figure 2C). Furthermore, we found that AR antagonist flutamide (Flu) had no significant effect on DHT‐induced Aβ42 uptake by N9 cells (Figure 2D). These results indicate that DHT promotes Aβ42 uptake by microglia independent of AR.

3.3. Dihydrotestosterone upregulates ECE‐1c expression

Amyloid peptides (Aβ) is degraded by enzymes expressed and released by neurons and glial cells.23 To explore the mechanisms involved in the enhancement of Aβ42 clearance by DHT‐treated microglia, we examined the effect of DHT on the expression of Aβ degrading enzymes in microglia, including insulin degrading enzyme (IDE), matrix metalloproteinase 2 (MMP2), MMP9, neprilysin (NEP), and endothelin‐converting enzyme 1 (ECE‐1). As shown in Figure 3A, treatment of N9 cells with DHT had no significant effect on IDE, MMP2, MMP9, and NEP expressions. ECE‐1 can degrade Aβ in both cultured cells and animal models.24, 25 Among four isoforms of ECE‐1, DTH significantly increased the expression of ECE‐1c but had no effect on the expression of other isoforms. Murine ECE‐1c is a homologue of human ECE‐1c which is localized mainly on plasma membrane with detectable intracellular expression.26, 27 Thus, the upregulation of ECE‐1c expression in microglia by DHT may contribute to the enhancement of Aβ clearance by DHT. Further study showed that pretreatment of N9 cells with Flu had no significant effect on the enhancement of Aβ42 clearance by DHT (Figure 3B), indicating that DHT promotes Aβ42 clearance by microglia through AR‐independent pathway.

Figure 3.

Figure 3

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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

3.4. Androgen inhibits Aβ42‐induced pro‐inflammatory cytokine expression in microglia and protects neurotoxicity caused by Aβ42‐activated microglia

We further investigated whether androgen could inhibit pro‐inflammatory cytokines expression induced by Aβ42 in microglia. Stimulation of N9 cells with Aβ42 significantly enhanced IL‐1β and TNFα expression, which was confirmed in mouse primary microglial cells (Figure 4A,B). Pretreatment of microglia with DHT or testosterone significantly suppressed Aβ42‐induced expression of IL‐1β and TNFα at both mRNA and protein levels (Figure 4C,D). These results demonstrate that androgen inhibits pro‐inflammatory factors expression induced by Aβ42 in microglia.

Figure 4.

Figure 4

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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)

It is well known that Aβ42 induces neurotoxicity directly or indirectly through activating microglia to produce pro‐inflammatory cytokines, such as IL‐1β and TNFα.3 As we found that androgen not only enhanced Aβ42 uptake and clearance, but also suppressed Aβ42‐induce IL‐1β and TNFα expression, we speculate that androgen could inhibit the neurotoxicity resulted from microglia activation by Aβ42. By immunofluorescence staining with MAP2, a neuron marker, we found that incubation of N2a, a mouse neuroblastoma cell line, with culture medium from Aβ42‐treated N9 cells significantly reduced cell number. Pretreatment of N9 cells with DHT or testosterone significantly attenuated neurotoxicity of Aβ42‐simulated N9 cells (Figure 4E). These results support that androgen could suppress neurotoxicity of Aβ42‐activated microglia.

3.5. Androgen inhibits Aβ42‐induced pro‐inflammatory cytokines expression in microglia through suppressing MAP kinase p38 and NF‐κB activation

It has been reported that Aβ induces pro‐inflammatory cytokines expression through activating MAP kinase p38 and NF‐κB.28, 29, 30 Consistently, we found that Aβ42 significantly induced p38 and Iκ‐Bα phosphorylation in murine primary microglia but had no effect on ERK1/2 and JNK phosphorylation (Figure 5A). Pretreatment of microglia with p38 inhibitor SB203580 or NF‐κB inhibitor PDTC significantly inhibited Aβ42‐induced IL‐1β and TNFα expression (Figure 5B). To explore the mechanisms mediating the inhibitory effect of androgen on pro‐inflammatory cytokines expression induced by Aβ42, we first examined the effect of androgen on Aβ42‐induced p38 and NF‐κB activation. As shown in Figure 5C, pretreatment of N9 cells with DHT or testosterone significantly reduced Aβ42‐induced p38 and NF‐κB p65 phosphorylation. These results indicate that androgen inhibits Aβ42‐induced pro‐inflammatory factors expression in microglia through suppressing MAP kinase p38 and NF‐κB activation. We then examined the contribution of AR in the inhibitory effect of androgen on Aβ42‐induced pro‐inflammatory cytokine expression and found that pretreatment of N9 cells with Flu could not reverse the inhibition of pro‐inflammatory cytokines expression by DHT or testosterone in Aβ42‐stimulated N9 cells (Figure 5D,E). Collectively, these results indicate that androgen inhibits pro‐inflammatory cytokines expression in Aβ42‐stimulated microglia independent of AR.

Figure 5.

Figure 5

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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

3.6. Androgen inhibits Aβ42‐induced inflammation and neurotoxicity in mouse brain

Finally, we checked whether androgen could inhibit Aβ42‐induced pro‐inflammatory cytokines expression and neuronal death in vivo. As shown in Figure 6, injection of Aβ42 into CA1 region of mouse hippocampus significantly induced IL‐1β expression and reduced neurons in this area. Subcutaneous administration of DHT or testosterone not only significantly suppressed Aβ42‐induced IL‐1β expression in hippocampus (Figure 6A), but also reversed Aβ42‐induced neuron loss in CA1 region (Figure 6B,C). These results indicate that androgen may attenuate the neurotoxicity of Aβ42 through suppressing Aβ42‐induced inflammation in brain.

Figure 6.

Figure 6

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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

4. DISCUSSION

In this study, we demonstrated that DHT and testosterone enhanced Aβ42 uptake and clearance, suppressed Aβ42‐induced IL‐1β and TNFα expression by microglia, and alleviated microglia mediated neurotoxicity of Aβ42. Furthermore, we found that DHT increased uptake and clearance of Aβ42 by microglia through upregulating Fpr2 and ECE‐1c expression, DHT and testosterone inhibited Aβ42‐induced pro‐inflammatory cytokines expression via suppressing MAP kinase p38 and NF‐κB activation by Aβ42. These results indicate that androgen alleviates neurotoxicity of Aβ by promoting microglial clearance of Aβ and inhibiting microglial inflammatory response to Aβ.

Microglia is resident brain phagocytes with functions similar to peripheral macrophages. It has been reported that activated microglia can reduce Aβ accumulation by increasing its phagocytosis, clearance and degradation.31, 32 Microglia contribute to Aβ immunization‐induced reduction of Aβ deposits in brains of AD mouse models.33, 34 Several receptors have been reported to mediate the uptake and clearance of Aβ by microglia, such as FPR2, TLR2, TLR4, and CD36.19, 20, 21 Our previous studies revealed that human FPR2 and its mouse homologue Fpr2 are functional receptors for Aβ.21, 35, 36 Formyl peptide receptor 2 (FPR2) is expressed at high levels by mononuclear (microglial) cells infiltrating amyloid plaques in brain tissues from AD patients.35 FPR2/Fpr2 mediates the uptake of Aβ by macrophages/microglia.21, 35, 36 Formyl peptide receptor 2 (Fpr2) is up‐regulated by pathogen‐associated molecular patterns and pro‐inflammatory cytokines, such as LPS, peptidoglycan, CpG, and TNFα, which was associated with a markedly increased Aβ42 endocytosis.37, 38, 39, 40 These results suggest that Fpr2 contributes to the clearance of Aβ by microglia. Our previous studies demonstrated that norepinephrine (noradrenaline), a neurotransmitter in brain, promoted microglial uptake of Aβ through upregulating Fpr2 expression.41 Our present study reveals that DHT is able to induce the expression of Fpr2 but not TLR2, TLR4 and CD16. Dihydrotestosterone (DHT) promotes Aβ42 uptake by microglia through upregulating Fpr2 expression, which may contribute to the protective effect of androgen on neurotoxicity induced by Aβ.

Amyloid peptides (Aβ) is produced continuously in brain and degraded by several enzymes, such as NEP, IDE, ECE‐1, ECE‐2, MMP9, and MMP2.23 Human ECE‐1 is expressed in neurons in different areas of the CNS, including areas relevant to AD.42, 43 It is also expressed in astrocytes and macrophages.44, 45 Human ECE‐1 has four different isoforms, ECE‐1a, ECE‐1b, ECE‐1c, and ECE‐1d, which differ only in their N‐terminal regions and are derived from a single gene through the use of alternative promoters.26 Human ECE‐1a is localized predominantly in plasma membrane, ECE‐1b is expressed exclusively intracellularly, ECE‐1c is localized both on plasma membrane and in intracellular compartments, ECE‐1d shows an intracellular location but limited only to endosomes.26, 46, 47 Mouse ECE‐1c is a homologue of human ECE‐1c.27 ECE‐1 can degrade both intracellular and extracellular Aβ.24, 48 In mice deficient for ECE‐1, both Aβ40 and Aβ42 levels in brain were significantly higher than that of wild‐type littermate controls.25 Adeno‐associated viruses mediated expression of ECE‐1 in the hippocampus and the right anterior cortex of APP/PS1 transgenic mice significantly reduced Aβ deposition.49 These results suggest that ECE‐1 is a physiological regulator of Aβ metabolism in the brain, ECE‐1 could be a potential target for reducing Aβ in AD brain. In the current study, we found that microglia express ECE‐1a, ECE‐1b, ECE‐1c, and low level of ECE‐1d, androgen induced ECE‐1c expression in microglia and enhanced Aβ clearance by microglia. In general, enzyme‐mediated cleavage of Aβ results in fragments that are generally less toxic, less likely to aggregate, and more easily to be cleared. Therefore, our results indicate that androgen‐induced ECE‐1c expression in microglia may promote Aβ42 clearance and reduce the neurotoxicity of Aβ42.

Although microglial uptake and clearance of Aβ are helpful for reducing neurotoxicity of Aβ, microglial activation has become increasingly regarded as a contributor to AD pathogenesis by producing neurotoxins, including reactive oxygen species and pro‐inflammatory cytokines.30, 50, 51 Aβ stimulates microglia to produce pro‐inflammatory cytokines, such as TNFα and IL‐1β, through activating MAP kinases and NF‐κB.29, 52 Our study confirmed that Aβ42 induced TNFα and IL‐1β production through activation of p38 and NF‐κB, and further demonstrated that androgen exhibited significant inhibitory activity on Aβ42‐induced microglial activation most probably through inhibiting p38 MAP kinase and NF‐κB signaling pathways. The antiinflammatory activity of androgen may contribute to its protective effect on activated microglia‐induced neurotoxicity.

Multiple epidemiological studies suggest that long‐term usage of nonsteroidal antiinflammatory drugs (NSAIDs) decreases risk of AD development,53 suggesting that antiinflammatory agents might be useful in the treatment of AD. Since then, NSAIDs have been investigated for treatment of AD. However, meta‐analyzes showed that clinical trials with NSAIDs did not significantly attenuate progression of dementia of AD patients.54, 55 NSAIDs inhibits the inflammatory cyclooxygenases 1 (COX‐1) and COX‐2, enzymes that generate prostaglandin from arachidonic acid. The failure of NSAIDs in clinical studies with AD patients may be due to the inhibition of both toxic and beneficial downstream prostaglandin signaling pathways. The disease phase of AD, treatment time, and dosage of NSAIDs may also affect the effect of NSAIDs on AD. In addition to NSAIDS, a range of antiinflammatory agents, such as PPARγ agonists, cannabinoids, TNFα inhibitor/antibody, resveratrol, curcumin, and tea catechins, has been evaluated and shown antiinflammatory effect and therapeutic promise in pre‐clinical AD models, and cognitive enhancement by some of these agents was confirm in early AD patients.56, 57 As androgen has been reported to prevent the increased Aβ accumulation in brain and improve working memory in AD mouse models,9, 10 and our current study demonstrated that androgen inhibits Aβ‐induced inflammation in brain, androgen may be a potential therapeutic agent for the prevention and treatment of AD.

Testosterone and its active metabolite DHT act on cells through AR‐dependent and independent pathways.22 We found that AR mRNA was not detectable in both N9 cells and murine primary microglial cells, and AR antagonist Flu had no effect on the enhancement of androgen on Aβ uptake and clearance by microglia and on the inhibitory effect of DHT or testosterone on Aβ‐stimulated pro‐inflammatory cytokines expression. Therefore, androgen modulates microglial response to Aβ through AR‐independent pathways. The underlying mechanisms remain further investigation. Testosterone can be converted to estradiol by aromatase. Estradiol is capable of promoting Aβ uptake by microglia.58, 59 It has been reported that microglia had no detectable expression of aromatase.60 Therefore, the modulation of testosterone on microglial response to Aβ may not be mediated through estradiol.

Advancing age is the most significant risk factor for the development of AD. In men, one normal consequence of aging is a robust decline in circulating and brain levels of the testosterone. Lower testosterone level is a risk of AD in the elderly men.5, 6, 7 Previous studies showed that androgen deficiency accelerated Aβ deposition in brain tissues of AD mouse models,9 androgen could reduce Aβ production and increase Aβ clearance by neuron.11, 12 In the current study, we found that androgen enhanced Aβ42 uptake through upregulation of Fpr2, increased Aβ42 clearance through induction of ECE‐1c, and inhibited Aβ42‐induced pro‐inflammatory cytokine expression through suppressing p38 MAP kinase and NF‐κB activation. The modulation of androgen on microglia phagocytic activity and inflammatory response to Aβ42 may protect neurotoxicity caused directly by Aβ42 or indirectly by Aβ42‐activated microglia. Our findings suggest that androgen is needed to maintain adequate Aβ uptake and clearance by microglia and androgen deficiency may contribute to defective Aβ clearance, neuroinflammation, and neurodegeneration in AD.

5. CONCLUSIONS

In summary, our study demonstrates that androgen ameliorates microglia mediated neurotoxicity of Aβ42. The protective effect of androgen on neuronal death may be mediated through enhancing uptake and clearance of Aβ42 by microglia and inhibiting Aβ42‐induced pro‐inflammatory cytokines production by microglia. Our findings indicate that deficiency of androgen may contribute to the pathogenesis of AD in men, and support the potential therapy of androgen in the prevention and perhaps treatment of AD.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Supporting information

 

Click here for additional data file. (43KB, doc)

ACKNOWLEDGMENTS

This research was supported by grant from the Science & Technology Commission of Shanghai Municipality (13JC1404003).

Yao P‐L, Zhuo S, Mei H, et al. Androgen alleviates neurotoxicity of β‐amyloid peptide (Aβ) by promoting microglial clearance of Aβ and inhibiting microglial inflammatory response to Aβ. CNS Neurosci Ther. 2017;23:855–865. 10.1111/cns.12757

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