Mol Neurobiol DOI 10.1007/s12035-015-9241-z
Aspirin Promotes Oligodendroglial Differentiation Through Inhibition of Wnt Signaling Pathway Nanxin Huang 1 & Dong Chen 1,2 & Xiyan Wu 1 & Xianjun Chen 1 & Xuesi Zhang 1 & Jianqin Niu 1 & Hai-Ying Shen 3 & Lan Xiao 1
Received: 3 February 2015 / Accepted: 26 May 2015 # Springer Science+Business Media New York 2015
Abstract Aspirin, one of the most commonly used antiinflammatory drugs, has been recently reported to display multiple effects in the central nervous system (CNS), including neuroprotection and upregulation of ciliary neurotrophic factor (CNTF) expression in astrocytes. Although it was most recently reported that aspirin could promote the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) after white matter lesion, the underlying mechanisms remain unclear. To dissect the effects of aspirin on oligodendroglial development and explore possible mechanisms, we here demonstrated the following: (i) in vitro treatment of aspirin on OPC cultures significantly increased the number of differentiated oligodendrocytes (OLs) but had no effect on the number of proliferative OPCs, indicating that aspirin can promote OPC differentiation but not proliferation; (ii) in vivo treatment of aspirin on neonatal (P3) rats for 4 days led to a nearly twofold increase in the expression of myelin basic protein
Electronic supplementary material The online version of this article (doi:10.1007/s12035-015-9241-z) contains supplementary material, which is available to authorized users. * Jianqin Niu
[email protected] * Lan Xiao
[email protected] 1
Department of Histology and Embryology, Chongqing Key Laboratory of Neurobiology, Third Military Medical University, Chongqing 400038, China
2
Department of Biological Technique, Third Military Medical University, Chongqing 400038, China
3
Robert Stone Dow Neurobiology Laboratories, Legacy Research Institute, Portland, OR 97232, USA
(MBP), devoid of change in OPC proliferaion, in the corpus callosum (CC); (iii) finally, aspirin treatment increased the phosphorylation level of β-catenin and counteracted Wnt signaling pathway synergist QS11-induced suppression on OPC differentiation. Together, our data show that aspirin can directly target oligodendroglial lineage cells and promote their differentiation through inhibition of Wnt/β-catenin signaling pathway. These findings suggest that aspirin may be a novel candidate for the treatment of demyelinating diseases. Keywords Aspirin . Oligodendrocyte . Differentiation . Oligodendrocyte precursor cell . Wnt/β-catenin
Introduction Oligodendrocytes (OLs), the myelinating cells in the central nervous system (CNS), play an essential role in maintaining neural signal conduction. OLs are generated from the oligodendrocyte progenitor cells (OPCs) through distinguished stages controlled by intrinsic and extrinsic regulatory mechanisms [1–3]. Maldevelopment of myelin and/or demyelination have been suggested to be involved in the pathogenesis of various devastating diseases, including multiple sclerosis (MS) and/or schizophrenia [4–6]. Because only mature OLs are able to form normal myelin sheaths, previous studies were aimed to seek approaches that can promote oligodendroglial differentiation under pathological conditions [7]. It has been shown that various factors, including thyroxine 3 (T3), neurotrophin 3 (NT3), as well as ciliary neurotrophic factor (CNTF), can promote oligodendroglial differentiation through activation or inhibition of signaling pathways such as sonic hedgehog (Shh), bone morphogenetic protein (BMP), and others [8–11]. However, to date, there is no effective therapy
Mol Neurobiol
clinically available due to the low efficiency of OPC differentiation. Aspirin (ASA), a widely used anti-inflammatory and antipyretic-analgesic drug, is considered to have neuroprotective properties. Accordingly, ASA has also been utilized for the treatment of neurodegenerative diseases or neurological disorders such as Alzheimer’s disease and schizophrenia [12, 13]. Recently, ASA was reported to upregulate the secretion of astrocytic CNTF, which has the capacity to enhance the survival of oligodendroglial lineage cells and to promote expression of myelin-associated proteins, e.g., proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) [14, 15]. A most recent study using white matter lesion (WML) model demonstrated that low concentrations of aspirin can promote OPC proliferation while high concentrations of aspirin usage induce OPC differentiation [16], suggesting potential effects of ASA on oligodendroglial lineage. However, the underlying mechanisms of ASA on oligodendroglial development remain unclear. To address this issue, we firstly investigated the effects of aspirin on proliferation and differentiation of oligodendroglial cells using OPC cultures. Secondly, we evaluated the in vivo effects of ASA on proliferation and differentiation in postnatal rats. Finally, we explored potential underlying mechanisms of ASA-mediated changes in OPC number or differentiation via Erk and Wnt/β-catenin signaling—two pathways which have been implicated in the regulation of oligodendroglial differentiation and subsequent myelination [17–19].
Materials and Methods
OPC Purification and Drug Treatment Rat OPCs were purified as previously described [20]. To examine gene expression during differentiation, 500 μL of dissociated cells were seeded into 24-well plates with poly-Dlysine-coated glass cover slips (4.5×104 cells/well). Meanwhile, another 3 mL (3×105 cells/mL) of dissociated cells were plated into 60-mm dishes. After a 12-h incubation, OPCs were (i) treated with ASA at final concentrations ranging from 0.5 to 10 μM in OPC proliferation medium to test proliferation and (ii) treated with ASA (0.5 μM), QS11, a selective Wnt/βcatenin signaling pathway synergist (2.5 μM) or ASA+QS11 (0.5 μM ASA and 2.5 μM QS11) in OPC differentiation medium to test differentiation. At designated time points (i.e., day 1, day 2, or day 3 for the proliferation test; day 1, day 3, and day 5 for the differentiation test), the cells on coverslips were fixed with 4 % PFA for immunofluorescence (IF) staining. Cells in culture dishes were then harvested for Western blot assay. MTS Assay The number of viable cells was measured using an MTS assay. Briefly, OPCs were cultured at a density of 1.5×104 cells/well in poly-D-lysine-coated 96-well plates with 100 μL OPC proliferation medium as previously described [20]. After 12 h, OPCs were treated with ASA at final concentrations ranging from 0.5 to 10 μM. At designated time points (i.e., day 1, day 2, and day 3), a 10 μL of MTS solution reagent (Promega, Madison, WI) was added to each well of OPCs and allowed to incubate for 4 h at 37 °C. The viable cell number was determined by measuring the absorbance at 490 nm using a microplate reader (Bio-RAD, Model 680).
Animals and Drug Administration IF Staining, Morphometric Analysis, and Quantification Sprague–Dawley (SD) rats were obtained from the Animal Facility Centre of the Third Military Medical University (TMMU), People’s Republic of China. All animal studies were performed in accordance with the guide of Institutional Animal Care and Use Committee of the TMMU. For ASA treatment, ASA (Sigma, A6810) was dissolved into 0.9 % saline to make a workingsolution (ASA, 1 mg/ml). One cohort of postnatal day 3 (P3) rats was subjected to gastric gavage of increased dosages of ASA (i.e., 2, 4, and 8 mg/kg/day) for 4 days, respectively. Another cohort of littermates was used as vehicle controls and received gastric gavage of 0.9 % saline based on their body weight ratio. At P7, all neonatal rats were anesthetized with an intraperitoneal (i.p.) injection of 1 % pentobarbital and transcardially perfused with 4 % paraformaldehyde (PFA). The rat brains were collected and dehydrated in 30 % sucrose at 4 °C for 24–48 h. Serial coronal sections (30 μm) were obtained using a cryostat microtome (MS 1900, Leica) for histological study.
To detect myelin-related molecular changes, IF staining and evaluation were performed as previously described [21]. Brain sections or cells cultured on coverslips were incubated in primary antibodies overnight at 4 °C, followed by a 1-h incubation with fluorescein-conjugated secondary antibodies at room temperature. All primary antibodies and the respective dilutions used are listed in Table 1. Cell nuclei were stained with DAPI (0.1 μg/mL in PBS). Immunoreactivity was determined using a confocal laser-scanning microscope (PV100, Olympus) with excitation wavelengths appropriate for FITC (488 nm), Cy3 (552 nm), or Cy5 (625 nm). Images obtained were processed and analyzed using Image-Pro Plus 5.0. Myelin basic protein (MBP) positive reactions were determined by measuring mean optical density. Cell counting was conducted on nine randomly chosen fields for each coverslip. Morphometric analysis was performed using the densitometer and Image-Pro Plus image analysis system as previously
Mol Neurobiol Table 1
Primary antibodies
Western Blot
Antigen
Source
Dilution
Supplier
PDGFaR
Rabbit
Santa Cruz
PDGFaR MBP
Goat Goat
Ki67 P-β-catenin β-Catenin P-GSK3β(Ser9) P-GSK3β(Tyr216) GSK3β P-Erk1/2 Erk1/2 Menin β-Actin
Rabbit Rabbit Mouse Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Mouse
1:250 (ICC) 1:500 (WB) 1:150 (ICC) 1:150 (ICC) 1:700 (WB) 1:1000 (ICC) 1:500 (WB) 1:1000 (WB) 1:500 (WB) 1:800(WB) 1:500 (WB) 1:500 (WB) 1:1000 (WB) 1:1000 (WB) 1:2000 (WB)
Santa Cruz Santa Cruz Dako Millipore Abmart Beyotime Bioss Beyotime Cell Signalling Millipore Bethyl Santa Cruz
ICC immunocytochemistry, WB Western blot
described [21]. For each experiment, three independent repeats were performed. Real-Time Polymerase Chain Reaction Total ribonucleic acid (RNA) was isolated from OPC cultures or astrocyte cultures, treated with or without 0.5 μM ASA, using TRIzol (Invitrogen) and the RNeasyPlus Mini Kit (Qiagen). To detect CNTF expression, real-time polymerase chain reaction (PCR) was performed with the C1000 Touch™ Real-time PCR Detection System (Bio-Rad) and GoTaq® qPCR Master Mix (Promega). The oligonucleotide primers, amplification procedure, and melt curve analysis were performed as described by Jahromy et al. [22]. For each sample, three independent repeats were performed. Enzyme-Linked Immunosorbent Assay Enzyme-linked immunosorbent assay (ELISA) was used to detect CNTF releases in OPC cultures treated with or without ASA. At appropriate time points, differentiation culture medium was collected for detection according to the manufacturer’s instructions in the CNTF ELISA kit (Kmaels Biotechnology). The 48-well plate was read using a microplate reader (Bio-Rad). To eliminate the influence of different cell numbers on the result, cells were lysed using RIPA lysis buffer with freshly added 1 % PMSF solution (Beyotime) and protein concentrations were estimated by Coomassie brilliant Blue G-250. The final value was calculated through division of ELISA result by the protein concentration. For each sample, three independent repeats were performed.
The nuclear/cytoplasmic fractionation, SDS-PAGE, and Western blot were performed as previously described [21]. Proteins were transferred to polyvinylidenedifluoride membranes and visualized by chemiluminescence (ECL Plus, GE Healthcare) after incubation with the antibodies (see Table 1). Band intensity was quantified with the Image-Pro Plus image analysis system. β-Actin was used as a loading control for total protein and menin was used as an internal control for the nuclear protein. The ratio of phosphorylated protein to total protein was calculated to evaluate the activation of Wnt signaling pathway. For each experiment, three independent repeats were performed. Statistical Analysis Statistical analyses were performed using one-way or twoway analysis of variance (ANOVA) followed by a Tukey post hoc test. Comparisons between two experimental groups were performed using Student’s t test. p0.05 at each time point). Noteworthy,10-μM ASA treatment resulted in a significant decrease of viable cell numbers at day 3, indicating a sign of cytotoxicity (Fig. 1a). Thus, the treatment of 10 μM ASA was excluded in successive experiments. Furthermore, the percentage of Ki67 + /PDGFαR + doublelabeled OPCs was not significantly different (p>0.05) between ASA-treated cultures and controls (Fig. 1b, c). These results suggest that ASA does not affect the proliferation of OPCs. To evaluate the effect of ASA on oligodendroglial differentiation, the cultured OPCs were treated with or without ASA during their differentiation period. OPCs and mature OLs were labeled with anti-PDGFαR or anti-MBP antibodies, respectively. Data from confocal photomicrographs indicated that at day 1, the cell number of early-stage OPCs in ASAtreated cultures was not significantly different from controls (Fig. 2a). However, the cell number of MBP+ OLs was significantly increased in ASA-treated cultures at day 3 and day 5.
Mol Neurobiol
To exclude the possibility that the ASA-induced enhancement of oligodendroglial differentiation is caused by increased release of CNTF from astrocytes, we measured messenger RNA (mRNA) levels of CNTF in OPC cultures. Real-time PCR results showed that the mRNA levels of CNTF were extremely low in both vehicle-treated and ASA-treated (0.5 μM) OPC cultures, and a slight increase in CNTF mRNA level in astrocyte cultures post-ASA (0.5 μM) treatment (Fig. 3a), though a much higher concentration of ASA (i.e., 5 μM) could significantly increase CNTF mRNA levels in astrocyte cultures (Fig. 3b), that agreed with the previous report [14]. The ELISA analysis of the culture media further confirmed that ASA did not significantly upregulate CNTF secretion in OPCs (Fig. 3c). These results extinguished the possibility of a CNTF-mediated differential effect in our study conditions and suggest that ASA may exert direct effects to promote oligodendroglial differentiation. Aspirin Accelerates Oligodendroglial Differentiation in the Corpus Callosum
Fig. 1 ASA shows no effect on OPC proliferation. a The viable cell numbers of OPCs were measured using an MTS assay and normalized to vehicle treatment at day 1, day 2 and day 3. The normalized ratio of viable cell numbers indicate proliferation of OPCs on designated posttreatment time points. b Representative IF staining showing proliferative status of OPCs at day 3. Cells with double-positive staining of Ki67 (shown as green) and PDGFαR (shown as red) indicating proliferative OPCs. c Quantification assay of cell counting for Ki67+/ PDGFαR+ OPCs on designated posttreatment time points. Data are displayed as mean±SEM (three independent experiments were performed in triplicate). **p
