Progesterone attenuates Aβ25–35-induced neuronal toxicity by activating the Ras signalling pathway through progesterone receptor membrane component 1

Zhigang Wu, Hang Wu, Shuang Sun, Honghai Wu, Wenjing Shi, Jing Song, Jianfang Liu, Yunhao Zhang, Fang Bian, Pengpeng Jia, Yanning Hou

PII: S0024-3205(20)30107-7
DOI: https://doi.org/10.1016/j.lfs.2020.117360

Reference: LFS 117360

To appear in: Life Sciences

Received date: 11 November 2019
Revised date: 16 January 2020
Accepted date: 24 January 2020

Please cite this article as: Z. Wu, H. Wu, S. Sun, et al., Progesterone attenuates Aβ25–35-induced neuronal toxicity by activating the Ras signalling pathway through progesterone receptor membrane component 1, Life Sciences(2020), https://doi.org/ 10.1016/j.lfs.2020.117360

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© 2020 Published by Elsevier.

Progesterone attenuates Aβ25-35-induced neuronal toxicity by activating the Ras signalling pathway through progesterone receptor membrane component 1

Zhigang Wua, Hang Wua, Shuang Suna,c, Honghai Wub, Wenjing Shia Jing Songb, Jianfang Liub, Yunhao Zhangb, Fang Biana, Pengpeng Jiaa, and Yanning Houa,b,*

a Hebei Medical University, Shijiazhuang 050017, Hebei Province, China
b Department of Pharmacy, Bethune International Peace Hospital of Chinese PLA,
Shijiazhuang 050082, Hebei Province, China
c School of Chemical Engineering, Shijiazhuang University, Shijiazhuang 050035, Hebei Province, China

*Corresponding author at: Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang 050017, Hebei Province, China.

Tel.: +86 311 87978503; fax: +86 311 87978480.
E-mail address: [email protected] (Y. Hou).

Abstract

Aims: Progesterone receptor membrane component 1 (PGRMC1) has been reported to mediate the neuroprotective effect of progesterone, but the exact mechanism has not been elucidated. Therefore, the purpose of this study was to investigate the signalling pathway downstream of PGRMC1 in progesterone-induced neuroprotection. Recognition of the mechanism of progesterone opens novel perspectives for the treatment of diseases of the nervous system.
Main methods: The PGRMC1 protein level was knocked down in rat primary cortical neurons, and Aβ25-35 was used to establish an Alzheimer’s disease cell model. The neuroprotective effect of progesterone was assessed by Hoechst 33258 staining and a cell counting kit-8 (CCK-8) assay. Then, proteomic and bioinformatic methods were used to analyse the proteins altered in response to PGRMC1 silencing to identify target proteins and signalling pathways involved in PGRMC1-mediated progesterone-induced neuroprotection. These findings were further verified by using signalling pathway inhibitors and western blotting.

Key findings: The neuroprotective effect of progesterone was significantly attenuated with PGRMC1 silencing. The expression of many proteins in the Ras signalling pathway was significantly changed in response to PGRMC1 silencing. FTI-277 inhibited progesterone-induced neuroprotection. Progesterone increased the expression of total Ras and Grb2.

Significance: These findings provide new perspectives for understanding the mechanism of and role of PGRMC1 in progesterone-induced neuroprotection. The Ras signalling pathway is the signalling pathway downstream of PGRMC1 in the mediation of progesterone-induced neuroprotection.

Keywords: Alzheimer’s disease, Progesterone, Progesterone receptor membrane component 1, Tandem Mass Tags, Proteomics, Ras signalling pathway

1. Introduction

Alzheimer’s disease (AD), which is characterized by the progressive deterioration of memory, cognition, and behaviour, is the most common age-related neurodegenerative disorder [1]. Epidemiological studies revealed that two-thirds of AD patients are women, and the drop in sex steroid hormone levels that occurs after menopause has been proposed as one risk factor for AD [2]. Sex steroid hormones are synthesized in the central and peripheral nervous system from cholesterol or steroidal precursors imported from peripheral sources and are termed neurosteroids. They are important endogenous modulators of several brain-related functions [3]. The classical neurosteroid progesterone has been reported to exert neuroprotective effects in various experimental models. Progesterone reduces ischaemic brain injury and ameliorates the acute stage of the stroke-induced injury cascade. It reduces cerebral oedema and cerebellar infarct size, improving cognitive function [4]. In addition, physiological levels of progesterone significantly reduce oxidative damage caused by glutamate and toxicity caused by glucose deprivation [5-7], also abolishing the toxicity of Aβ to primary hippocampal neurons [8].

Progesterone is generally assumed to exert genetic effects by acting on classical nuclear receptors and to exert rapid effects on nongenetic pathways by acting on membrane receptors [9]. Progesterone receptor membrane component 1 (PGRMC1) has a single transmembrane structure in which the intracellular region has potential Src2 and Src3 homologous structural domains that transmit extracellular signals to intracellular areas [10].
Numerous studies have shown that PGRMC1 may be an important
target through which progesterone exerts its neuroprotective effects. Progesterone has been reported to inhibit apoptosis and play a role in maintaining calcium homeostasis via PGRMC1 [11-13]. In addition,

progesterone activates extracellular regulated protein kinases 5 (ERK5) via PGRMC1, promotes brain-derived neurotrophic factor (BDNF) secretion, and exerts further neuroprotective effects [14, 15]. In addition, previous studies in our laboratory found that progesterone reduces Aβ-induced mitochondrial damage through PGRMC1 [16]. Moreover, progesterone regulates the PI3K/Akt-GSK-3β pathway via PGRMC1 and inhibits Aβ-induced hyperphosphorylation of tau and endoplasmic reticulum stress in astrocytes. Although many studies have addressed PGRMC1-mediated progesterone-induced neuroprotection, the exact mechanism underlying this effect remains unclear. In particular, the downstream signalling of PGRMC1 needs to be identified.

Proteomics comprehensively examines all proteins expressed in various types of samples, including tissues, cells or organelles, in an unbiased manner and is receiving increasing attention for studying mechanisms of drug action [17, 18]. In the present study, proteomics was used to analyse protein changes in response to PGRMC1 silencing in order to investigate the downstream signalling of PGRMC1.

2. Material and methods

2.1. Primary Culture of Cortical Neuron and Lentivirus Infection

Cerebral cortices were separated from a newborn Sprague-Dawley rat and dissociated followed by at 37℃ for 15 min in trypsin (1.25 g/L) (Solarbio, China). Cells were plated on polylysine-coated multi well plates at 1.5×106 cells/ml in DMEM medium (Gibco, USA) containing 10% FBS (Gibco). After 4 h, the medium was replaced with neurobasal medium (Gibco) with 10% B27 supplement (Gibco). Then, half of the culture medium was changed every other day. Neurons cultured for 7 days were used for experiments [16]. Cells were incubated with lentivirus (Genechem, China) at different multiplicities of infection (MOI) for 24 h.

The fluorescence intensity of green fluorescent protein (GFP) was measured. Expression of PGRMC1 was examined to determine which lentivirus exerted the strongest silencing effect. All experiments were performed in accordance with the Guidance Suggestion for the Care and Use of Laboratory Animals. The protocol was approved by the Animal Care and Use Committee of Bethune International Peace Hospital of Chinese PLA (approval number: 2019-KY-48, Shijiazhuang, China).

2.2. Assessment of Cell Viability

Cell viability was determined using a cell counting kit-8 (CCK-8) (Dojindo, Japan) according to the manufacturer’s protocol. For each well, we added 10 µL CCK-8 reagents. Ninety-six-well plates were placed in the incubator at 37℃ for 1 h, and absorbance was subsequently measured by a microplate reader (SPECTROstar®Nano, BMG LABTECH, Germany) at a wavelength of 450 nm.

2.3. Apoptosis Analysis

Apoptotic cells were identified with the nuclear dye Hoechst 33258 (Solarbio) in accordance with the manufacturer’s instructions. Cells were stained with Hoechst 33258 for 10 min at room temperature and imaged under a fluorescence microscope (Olympus BX41, Japan) [16].

2.4. Sample Preparation and Tandem Mass Tag (TMT) Labelling

Urea (8M) (Sigma-Aldrich, USA) supplemented with 1% phenylmethanesulfonyl fluoride protease inhibitor was used to lyse cells. Supernatants containing protein were collected after centrifugation (FRESCO 21, Thermo Scientific, USA) at 12,000 × g for 10 min. After protein quantification, 50 µg of protein from each sample was subjected

to disulfide bond reduction. Then, proteins were digested using lysyl endopeptidase (1:150 w:w) (Promega, USA) at 37°C. After 3 h, trypsin (1:100 w:w) (Promega) was added to the samples and incubated overnight at 37°C. Enzymatic hydrolysis was terminated by the addition of 5 µL of 10% formic acid to a final concentration of 1%. All samples were centrifuged at 15,000×g at 4°C for 10 min, and the precipitates were then removed. The supernatant was dried in a centrifugal evaporator (ZLS-2, Hunanherexi Instrument & Eqipment Co., Ltd, China) under a vacuum and stored at -40°C until use.
TMT labelling (Thermo Fisher, USA) was used to perform relative protein quantification according to previously reported procedures. Samples containing negative control siRNA lentivirus were labelled with TMT-129C, TMT-130N and TMT-130C, and samples containing the PGRMC1 interference sequence for infection were labelled with TMT-128N, TMT-128C and TMT-129N. TMT-labelled peptides were resuspended in 100 μL of buffer (3% acetonitrile (ACN); 97% 20 mM ammonium formate, HCOONH4, pH=10.0) and separated in a Dionex UltiMate 3000 rapid separation liquid chromatography (RSLC) system (Thermo Scientific) [19].We collected 15 fractions over the gradient at 1 min intervals. All fractions were desalted on Zip-Tip C18 columns and dried under a vacuum.

2.5. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis

LC-MS/MS analysis was performed using an Easy-nLC 1000 liquid chromatography system (Thermo Scientific) connected to an Orbitrap Fusion mass spectrometer (Thermo Scientific). Fifteen fractions were redissolved in 0.1% formic acid in water and loaded onto an Acclaim PepMap100 C18 75 µm×2 mm trap column (injection volume, 2 μL) (Thermo Scientific) with buffer A (0.1% formic acid in water) at a flow

rate of 3 μL/min. Peptides were separated at a flow rate of 300 nL/min using an Acclaim PepMap C18 RSLC (1.7 μm, 75 μm × 50 cm) column. The gradient was as follows: 5% B (98% ACN and 2% water containing 0.1% formic acid) from 0 to 3 min, 5-7% B from 3 to 5 min, 7-25% B
from 5 to 105 min, 25-60% B from 105 to 125 min, 60-95% B from 125
to 126 min, and 95-95% B from 126 to 140 min [19].
The Orbitrap Fusion mass spectrometer was operated in positive ion mode. The electrospray voltage was set to 2.1 kV, and the ion transfer tube temperature was set to 300°C. The mass spectrometer was operated in the data-dependent mode for the MS2 method. A full scan was obtained over the 350-1550 m/z range at a nominal resolution of 120,000, and the most abundant but not sequential precursor ions above the intensity threshold of 5.0e4 were selected for high-energy collisional dissociation (HCD) fragmentation. For MS2 events, ions were filtered by using the quadrupole isolation mode with a transmission window of 1.6 m/z. The collision energy used for HCD fragmentation was 37%, and fragment ions were subsequently analysed in the Orbitrap with a nominal resolution of 30,000. The automatic gain control (AGC) was set at 5.0e4. The maximum ion injection times were set at 50 ms for full scans and 70 ms for MS2 scans. The dynamic exclusion time was set at 40 s [19].

2.6. MS/MS Data Analysis

For protein identification by MS, raw files were processed using Proteome Discoverer Software 2.1 (Thermo Scientific). The raw files of the 15 fractions were merged and searched via the SEQUEST HT search engine with the rat UniProt database (uniprot_proteome_rat20180708. fasta). The parameters for the search were set as follows: The enzyme name was set to “Trypsin (Full)”, and a maximum of two missed cleavages was allowed. The precursor mass tolerance was set to 10 ppm for all MS1 spectra acquired, and the fragment ion mass tolerance was set

at 0.02 Da for all MS2 spectra acquired. The dynamic modifications included TMT 6plex/+229.163 Da (N-terminal) and acetylation (N-terminal). Carbamidomethylation / +57.021 Da (C-terminal) was used as the static modification. The coisolation threshold value was set at 50. The abundance values for each channel were scaled by the total intensity of all identified peptides. A false discovery rate (FDR) of 1% was applied at the peptide and protein levels.

2.7. Bioinformatic Analysis of Differentially Expressed Proteins

Significantly differentially expressed proteins (fold change>1.2 or<0.83) were demonstrated with volcano plots and hierarchical clustering analysis. Significantly differentially expressed proteins were subjected to Gene Ontology (GO) enrichment analysis via the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/). The enrichment analysis includes biological processes (BP), cellular components (CC), and molecular functions (MF). In addition, these significantly differentially expressed proteins were further subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/tool/map_pathway2) signalling pathway enrichment analysis. A network of protein pathway interactions was constructed based on the STRING database (https://string-db.org/), and Cytoscape software (http://www.cytoscape.org/) was used for visual presentation and network analysis.

2.8. Ras Activation Assay

The activation state of Ras was determined using a Ras Activation Assay Biochem Kit (Cytoskeleton, USA). Cell lysates were prepared according to the kit instructions. Ras-GTP in the lysates was pulled down using the Ras binding domain (RBD) region of the Ras effector protein,

Raf kinase. The RBD protein motif has been shown to bind specifically to the GTP-bound FTI 277 form of Ras proteins. The presence of Ras-GTP was detected by western blotting using an anti-Ras antibody.

2.9. Western Blotting Analysis

Western blotting was performed for PGRMC1, Ras, PI3K, Akt, p-Akt, Bax, Bcl-2, SOS1, and Grb2. Total cellular proteins were electrophoresed on a 10% or 12% sodium dodecyl sulfate polyacrylamide gel and transfered to polyvinylidene fluoride (PVDF) membranes (Milipore, USA). The membrane was sealed with protein sealant for 2 h, rinsed with TTBS, incubated with primary antibody overnight at 4°C (anti-PGRMC1, anti-Ras, anti-PI3K, anti-Akt, anti-p-Akt anti-Bax, anti-Bcl-2 (CST, USA), anti-SOS1, anti-Grb2, anti-actin, and anti-GAPDH, 1:1000) (Abways, China). After washing with TBST, membranes were incubated with secondary antibody for 1 h at room temperature. Blots were developed using FTI 277 an ECL western blotting detection kit (Santa Cruz, USA), and integrated band densities were measured by ImageJ (1.46r, NIH, USA).

2.10. Statistical Analysis

The data were expressed as the means ± SD. One-way ANOVA or two-way ANOVA was used for statistical analysis of the data of each group. When differences were considered statistically significant, the Dunnett’s modified t-test was performed to compare between the two groups. P< 0.05 was considered statistically significant.

3. Results

3.1. Lentiviral Infection Conditions and Silencing Effects

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ImageBright green fluorescence was observed under the fluorescence microscope, indicating that cells were successfully infected with lentivirus. As the MOI increased, the fluorescence strengthened. The infection efficiency was approximately 81.3%, and cells infected at MOI=20 appeared healthy 72 h following infection (Fig. 1A, B). The silencing efficiency of three lentiviral vectors harbouring RNA interference (RNAi) sequences targeting the PGRMC1 gene was assessed by western blotting. LV-PGRMC1-RNAi (61634) had the highest interference efficiency, 67.7% (Fig. 1C). The LV-PGRMC1-RNAi (61634) infection conditions were used in subsequent experiments.

Fig. 1. Infection conditions and silencing effects of lentivirus. A. Expression of green

fluorescent protein in neurons after infection with lentiviruses at different multiplicities of infection (MOI) for 72 h. B. Infection efficiency of lentiviruses with different MOIs for 72 h. C. Expression of PGRMC1 in neurons after infection with three lentiviruses harboring different RNAi sequences targeting the PGRMC1 gene (MOI=20) for 72 h.

3.2. Effect of PGRMC1 on the Protective Effect of Progesterone Against Aβ25-35-induced Neuronal Toxicity

After exposure to 25 μM Aβ25-35 for 48 h, a large number of neurons were injured in both the siScramble and siPGRMC1 groups. After progesterone treatment, the survival rate of neurons in the siScramble group was significantly increased, and the neuronal apoptosis rate was significantly decreased. The neuroprotective effect of progesterone was inhibited by PGRMC1 siRNA. Progesterone at a concentration of only 10 μM increased neuronal survival in the siPGRMC1 group. The rate and apoptosis rates in the 1 μM progesterone treatment group before and after PGRMC1 interference were significantly different. The quantitative assessment is shown in Fig. 2.

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Fig. 2. Neuroprotective effects of progesterone are inhibited by PGRMC1 siRNA. Neurons after being infected by siPGRMC1 or siScramble lentiviruses were exposed to 25 μM Aβ25-35 with different concentration of progesterone (0.1, 1, 10 μM) for 48 h and then assayed for percentage of apoptotic cells and cell viability. A. Apoptotic cells were determined with nuclear dye Hoechst 33258 that characterized fragmented or intensely stained nuclei. B. The percentage of apoptotic cells were quantified from 5 fields per well. C. Neuronal viability was measured by cell counting kit-8. Data are expressed as the mean±SD (n = 6). **P<0.01 vs control, #P<0.05, ##P<0.01 vs Aβ,