Mediation of insulin growth factor-1 in Alzheimer's disease and the mechanism of PRNP genetic expression and the PI3K/Akt signaling pathway.

The aim of the study was to examine the mediation of insulin growth factor-1 (IGF-1) in Alzheimer's disease (AD), as well as the underlying mechanism of the PRNP genetic expression and PI3K/Akt signaling pathway. The Aβ25-35-incubated rat adrenal pheochromocytoma cell (PC12) in vitro was established, constituting the AD model. Different doses (0, 20, 40 and 80 ng/ml) of IGF-1 were used in PC12 cells and the level of PRNP mRNA was tested after 24 h using the quantitative PCR method and the level of APP protein was assessed using western blot analysis. PC12 cells were divided into the control group (PC12 cells without Aβ25-35 treatment), model group (PC12 cells with Aβ25-35 treatment), IGF-1 80 ng/ml group, IGF-1 80 ng/ml+PI3K inhibitor LY294002 25 µmol/l group, and IGF-1 80 ng/ml+LY294002 50 µmol/l group, whose PRNP mRNA level and Akt, pAkt and APP protein level were tested 24 h later. As the dose of IGF-1 increases, the expression levels of PRNP mRNA and APP protein were more highly expressed. The difference between them was significant (P<0.05). In addition, regarding Akt protein, the expression levels of PRNP mRNA, APP protein and pAkt protein in the IGF-1 groups were significantly higher than those in the control and model groups. With the LY concentration increasing, the levels of expression of the three substances gradually decreased significantly (P<0.05). In conclusion, IGF-I can mediate the expression of the PRNP gene and APP protein through the PI3K/Akt signaling pathway, in a rat model.

Introduction

Alzheimer's disease (AD), a neurodegenerative disease, is the most common type of dementia affecting the quality of life of elderly individuals (1). AD's most prominent neurohistopathological characteristic is neuro-entanglement inside neurons and amyloid plaques in vitro (2). The incidence of AD is related to the deposition of β-amyloid (Aβ) peptides, a high level of phosphorylation of microtubule protein (τ protein), ApoE4, neurofibrillary tangles and many other features (3). It has been found that prion protein was significantly involved in regulating the process of Aβ-induced AD (4). Previous findings showed that the polymorphic coding by the PRNP gene of prion protein (PrPc) was important in the incidence of AD (5). However, there is no unified conclusion and the exact mechanism remains unclear.

Recent findings showed that the pathologic change in AD features was associated with the transferring disorder of signals mediated by insulin in brain and insulin growth factor-1 (IGF-I), an important neuro-nutrition factor (6). Indeed, IGF-1 and its receptors were prevalently expressed in the neuro system (7). IGF-1 affects the eradication of IGF-1 and the phosphorylation of τ protein, which was related to the PI3K/Akt and mitogen-activated protein kinase (MAPK)/ERK1/2 pathway (8). However, the adjustment by IGF-1 to PRNP genetic expression remains unclear. Accordingly, the present study analyzes the mediation of IGF-1 in AD PRNP genetic expression and PI3K/Akt signaling pathway mechanism, through constructs of AD cell models in vitro.

Materials and methods

Constructing of AD cell model in vitro

The PC12 cells (AD cell model) induced by Aβ25–35 were provided by Shanghai Jimian Bioengineering Co., Ltd. PC 12 cells were cultivated in DMEM cell-culture media (Solarbio Science & Technology Co., Ltd., Beijing, China) containing 10% heat-inactivated FBS (fetal bovine serum) and 1% mixture of penicillin and streptomycin in a 37°C incubator (Beijing Liyi Apparatus Factory) with 5% CO2 and 100% humidity. When 80% of the cells merged, the cell-culture media were discarded and the digestion was proceeded with trypsin for cell passages. When the cells entered the logarithmic growth phase, the culture media were discarded and the cells were washed with PBS twice. The cell was cultured with DMEM without serum. After 24 h, DMSO was added at a final concentration of 0.1%. After a further 24 h, the supernatant liquid was collected and the concentrated protein was stored at −20°C.

Group division

Different doses of IGF-1 (0, 20, 40 and 80 ng/ml) were incubated in PC12 cells and the levels of PRNP mRNA and APP protein were tested 24 h later using quantitative PCR and western blotting, respectively. PC12 cells were divided into the control group (PC12 cells without Aβ25–35 treatment), model group (PC12 cells with Aβ25–35 treatment), IGF-1 80 ng/ml group, IGF-1 80 ng/ml+PI3K inhibitor LY294002 25 µmol/l group, and IGF-1 80 ng/ml+LY294002 50 µmol/l group. PRNP mRNA level and Akt, pAkt and APP protein levels were tested 24 h later. Cells disintegrated with RIPA lysis solution were centrifuged for 30 min in 4°C (10,000 × g), the supernatant liquid was removed and then stored separately at 4°C.

Real-time quantitative PCR

TRIzol, the RNA extraction reagent (Invitrogen, Waltham, MA, USA), dNTP, reverse transcriptase, Taq enzyme (Promega, Madison, WI, USA), PCR markers (Shanghai Huamei Bioengineering Co., Ltd). The main steps were as follows: Total RNA was extracted using the TRIzol one-step method. Total RNA (1 µg) was used for reverse transcription with Oligo18 as the primer. The reaction system was 20 µl. Oligo18 was produced by Shanghai Boya Bioengineering Co., Ltd. The primers included: PRNP (F): 5′-AGGTGGTTCTCATTCTTGC-3′, (R): 5′-GTGGCTTCTTTGGTTGTA-3′, 307 bp; and internal reference GAPDH (F): 5′-ACCACAGTCCATGCCATCAC-3′, (R): 5′-CACCACCCTGTTGCTGTA-3′, 432 bp. The reaction conditions included pre-denaturation at 95°C for 5 min, denaturation in 94°C for 30 sec, annealing in 58°C for 30 sec (GAPDH: annealing at 60°C for 30 sec), extension at 70°C for 40 sec with 25 cycles in total (GAPDH: 28 cycles in total). The final extension lasted for 7 min at 72°C. After 20g/l agarose gel electrophoresis, UAP gel density scanner should be used to scan the electrophoretic band of the amplification products of the target and internal reference genes. The results should be demonstrated using the 2−∆∆Cq method.

Western blot analysis

Cells were reacted with RIPA lysis solution on ice for 30 min and vibrated intensely for 1 min every 5 min. The sample was centrifuged at 4°C for 20 min at 1,500 × g. The supernatant liquid was the total proteins. The quantity of protein was calculated using the Bradford method. The kit was provided by Jiangsu Biyuntian Bioengineering Science and Technology Research Institute (Jiangsu, China). Then SDS-PAGE gel electrophoresis was performed, followed by 50 V of stacking gel and 100 V to separate 100 V gel for 3 h in total. Proteins were then transferred to nitrocellulose membranes. Sealing liquid (5% skimmed milk) was added to the membranes at 0.1 ml/cm2. The membranes were incubated at room temperature for 4 h after being sealed by a welding machine. After discarding the blocking liquid, diluted primary antibodies (1:500 APP, Akt, pAkt and 1:2,000 β-actin) were added and the membranes were incubated at 37°C for 1 h. Rabbit polyclonal APP antibody (dilution, 1:500; cat. no. ab95195), rabbit polyclonal Akt antibody (dilution, 1:500; cat. no. ab8805) and rabbit monoclonal pAkt antibody (dilution, 1:500; cat. no. ab81283) were all purchased from Abcam (Cambridge, MA, USA). The membranes were washed with TBST three times for 10 min. The goat anti-rat secondary antibody (dilution, 1:2,000; cat. no. ab6721; Abcam) was added. Then the membrane was incubated in 37°C for 1 h. The membranes were further washed three times with TBST for 10 min. The result was revealed with ECL staining and scanned. Image analysis software IPP6.0 was used for gray analysis.

Statistical analysis

Data of the study were analyzed using SPSS19.0 software (Chicago, IL, USA). Quantitative data were presented as means ± standard deviation. Comparison among groups was analyzed with single-factor ANOVA analysis. P<0.05 indicated that the difference was statistically significant.

Results

Intervention of different-dose IGF

As the dose of IGF-1 increased, the expression levels of PRNP mRNA and APP protein were highly expressed in a significant manner (P<0.05) (Table I, and Figs. 1 and 2).

Table I.

Intervention result of different-dose IGF.

Groups	PRNP mRNA	APP protein
0	0.0243±0.0055	0.08±0.02
20 ng/ml	0.1156±0.0322	0.16±0.09
40 ng/ml	0.2478±0.0483	0.33±0.10
80 ng/ml	0.4316±0.0695	0.75±0.18
F-value	15.632	13.428
P-value	<0.001	<0.001

IGF, insulin growth factor.

Figure 1.

PRNP mRNA expression detected by RT-PCR in groups using different concentrations of IGF-1. IGF-1, insulin growth factor-1.

Figure 2.

APP protein expression detected by western blot analysis in groups using different concentrations of IGF-1. IGF-1, insulin growth factor-1.

Results of the intervention by PI3K inhibitor

Regarding Akt protein, the levels of expression of PRNP mRNA, APP protein and pAkt protein in the IGF-1 groups were significantly higher than those in the control and model groups. With the increase in LY concentrations, the levels of expression of the three substances gradually decreased, which was statistically significantly (P<0.05) (Table II, and Figs. 3 and 4).

Table II.

Results of the intervention by PI3K inhibitor.

Groups	PRNP mRNA	APP protein	Akt protein	pAkt protein
Control	0.0056±0.0012	0.02±0.01	0.24±0.06	0.06±0.01
Model	0.0312±0.0078	0.07±0.02	0.22±0.05	0.15±0.04
IGF-1	0.4867±0.0152	0.72±0.13	0.23±0.07	0.88±0.16
IGF-1+LY 25 µmol/l	0.2516±0.0123	0.53±0.10	0.29±0.10	0.63±0.13
IGF-1+LY 50 µmol/l	0.1028±0.0257	0.39±0.08	0.27±0.12	0.42±0.11
F-value	19.654	16.527	2.306	20.325
P-value	<0.001	<0.001	0.548	<0.001

IGF, insulin growth factor.

Figure 3.

PRNP mRNA expression of PC12 cells after different treatments detected by RT-PCR. Lane 1, control group; lane 2, model group; lane 3, IGF-1 group; lane 4, IGF-1+LY 25 µmol/l group; and lane 5, IGF-1+LY 50 µmol/l group. IGF-1, insulin growth factor-1.

Figure 4.

AKT and APP protein expression of PC12 cells after different treatments detected by western blot analysis. Lane 1, control group; lane 2, model group; lane 3, IGF-1 group; lane 4, IGF-1+LY 25 µmol/l group; and lane 5, IGF-1+LY 50 µmol/l group. IGF-1, insulin growth factor-1.

Discussion

PRNP gene is a single copy gene located at the short arm of chromosome 13 (5). In the 5′ end activation domain, there are two SP1 transcription factors binding sites but no TATA boxes determining the starting site for transcription, which is a typical housekeeping gene structure. Such a gene is very conservative and there is little difference between spices. The open reading frame of men's gene is included in a complete exon. The gene of rat and sheep consists of 3 exons. The study on the 3′ end gene shows different poly(A) areas in this domain for different categories, which can impact the speed of protein formation and further the incidence of disease (9). Prion protein genes are expressed in the central neuro-system of animals and peripheral tissues, but its expression features such as when and where it is expressed remain unclear. A study on gene rat demonstrated that the levels of expression of PRNP gene is positively correlated with the body's susceptibility to prion (10). Prion protein can function as membrane-anchored glycoprotein in the incidence of AD. It can function as the receptor of signal modular and induce the pathway inside of the cell, by combining with Aβ oligomer in vitro, and further becomes toxic and contains neuroprotective effects by impacting Aβ formation. In fact, due to the structural features of prion protein and the complexity of glycosylation modifications, it has multiple functions, two of which can coexist and exert a certain impact on the body (11).

IGF-1R is a member of the receptor tyrosine kinase family. Phosphorylated by IGF-1, it activates and stimulates the transcription of insulin receptor substrate series and other series of signals, including the MAPK and PI3K/PKB pathways (12). Adlerz et al found that IGF-1 stimulates α secretase to lessen Aβ formation (13). A previous result confirmed in PC12 cells by Zhang et al also found that IGF-1 treatment significantly decreased the β secretase (BACE-1) mRNA and protein levels, in order to reduce Aβ formation, which was related to the PI3K/Akt and MAPK/ERK1/2 signaling pathways (14). The disorder of the signaling pathway was involved in the incidence of AD. Wang et al used Aβ25–35 protein to induce the damaged PC12 cells to construct τ protein over-phosphorylation cell model and carried out IGF-1 intervention (15). Those authors found that IGF-1 can hinder the apoptosis of PC12 cells and τ protein phosphorylation, which was achieved through the PI3K/Akt signaling pathway. Our data showed that as the dose of IGF-1 increased, the expression of PRNP mRNA and APP protein were significantly increased. Regarding the Akt protein, the levels of expression of PRNP mRNA, APP protein and pAkt protein in the IGF-1 groups were significantly higher than those in the control and model groups. The increase in LY concentrations led to the expression of the three substances gradually decreasing, a difference that was statistically significant (P<0.05). Altogether, the data suggest that IGF-1 may mediate the expression of PRNP gene and APP protein through the PI3K/Akt signaling pathway, in the rat model.