Contributed equally
In Parkinson's disease (PD), microglial activation-mediated neuroinflammation is associated with dopaminergic neurons degeneration in the substantia nigra pars compacta. Previous studies that have investigated this neurodegenerative disease have reported that the Sonic hedgehog (SHH) signaling pathway, through inhibiting the inflammatory processes, exerts a beneficial neuroprotective effect. However, the mechanisms underlying the anti-inflammatory and neuroprotective effects of this signaling pathway remain poorly understood. The present study aimed to further investigate these mechanisms
Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by a selective loss of DA neurons in the substantia nigra pars compacta (SNpc) (
The Sonic hedgehog (SHH) diffusible protein, a member of the hedgehog family, become active in binding Patched receptors (Ptch). This binding relieves Ptch-mediated inhibition exerted on Smoothened (Smo) receptor (a key transducer of the Hedgehog, HH, signaling pathway). Subsequently, the Smo receptor promotes the transcription factor ‘Glioma-associated oncogene’ (Gli) which translocate to the nucleus and regulates the transcription and expression of target genes (
In the present study, by way of
The following reagents were used in the present study: Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS), from Gibco (Grand Island, NY, USA); TH antibody, p-AKt antibody and AKt antibody, from Abcam (Cambridge, UK); Ionized calcium binding adaptor molecule 1 (Iba1) antibody, from Wako (Osaka, Japan); β-actin antibody, HRP-conjugated goat polyclonal anti-rabbit IgG antibody, Purmorphamine and Cyclopamine, from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); LPS and MPTP, from Sigma-Aldrich (St. Louis, MO, USA); LY294002, from Cell Signaling Technology, Inc. (Danvers, MA, USA).
The murine BV2 microglial cell line was grown in DMEM supplemented with 10% FBS (Gibco), 100 U/ml penicillin, and 100 mg/ml (Sigma, St. Louis, MO, USA). In a humidified 5% CO2 incubator maintained at 37°C, streptomycin and the culture medium were renewed every day. Cells were plated at 5×105 concentration and grown for 24 h prior to the experiments.
Dishes of cultured BV2 cells were randomly divided into six groups: Including: i) Control group, ii) LPS group, iii) PM+LPS group, iv) Cyclopamine+PM+LPS group, v) LY294002+PM+LPS group and vi) LY294002+LPS group. For LPS group: LPS (1 µg/ml) was employed during 24 h to obtain an inflammatory response with BV2 cells. For PM+LPS group: Purmorphamine (PM, 1.5 µmol/l) was used to activate the SHH pathway in BV2 cells 24 h before LPS treatment; For Cyclopamine+PM+LPS group: BV2 cells were pretreated with a specific SHH signal inhibitor (Cyclopamine) to further explore the role of PI3K/Akt pathway on the effects of SHH pathway; in this respect, Cyclopamine (20 µmol/l) was administered to block the SHH pathway (1 h before PM treatment); then, PM was used to treat BV2 cells for 24 h; then after, a LPS treatment was applied. For LY294002+PM+LPS group: A selective inhibitor of PI3K/AKt, LY294002 (20 µmol/l) was used during 30 min in order to block PI3K/Akt pathway before PM treatment; then PM was used to treat BV2 cells for 24 h; this pharmacological situation was completed with a LPS treatment. For LY294002+LPS group: LY294002 was used to treat BV2 cells without PM treatment; then after, a LPS treatment was applied.
All animal studies were approved by the Institutional Animal Care and Use Committee at Guangzhou Medical University. Male C57BL/6 mice (8–10 weeks, 22-25 g) were housed under a 12-h light/dark cycle with free access to food and water. All animals were randomly divided into five group, including: i) Control (n=10); ii) MPTP (n=10); iii) PM+MPTP (n=11, PM+MPTP); iv) LY294002+PM+MPTP (n=11, LY+PM+MPTP); and v) LY294002+MPTP (n=11, LY+MP). For the MPTP group, mice received four intraperitoneally (i.p.) injections of MPTP (20 mg/kg) in a 2 h interval (
Traction behavior (TR) test was described previously (
For qPCR analysis, total RNA was isolated from BV2 cells and from the brain substantia nigra by TRIzol reagent (Takara Bio, Inc., Otsu, Japan). Total RNA (1 µg) was reverse transcribed into cDNA using PrimerScript™ RT reagent kit (Takara Bio, Inc.). The mRNA expression levels were measured to use SYBR® Premix Ex Tag™ and primers. The sequences for qPCR promers were as follows: IL-1β forward, 5′-TGCCACCTTTTGACAGTGATG-3′; reverse, 5′-GGAAG for each GTCCACGGGAAAGAC-3′; TNF-α forward, 5′-ATGGCCTCCCTCTCATCAGT-3′; reverse, 5′-ATAGCAAATCGGCTGACGGT-3′; Nurr1 forward, 5′-AAGACCTTCTCCCAAGCACG-3′; reverse, 5′-GAACTGGACACTTCAACCAGC-3′; TGF-β1 forward, 5′-GGTCCTTGCCCTCTACAACC- 3′; reverse, 5′-CCACGTAGTAGACGATGGGC-3′; β-actin forward, 5′-GTTACAGGAAGTCCCTCACCC-3′; reverse, 5′-CAGAAGCAATGCTGTCACCTT-3′. Cycling condition included one cycle at 95°C for 5 sec, 30 cycles at 95°C for 30 sec and 60°C for 1 min, followed by one cycle 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. β-actin was used as an internal control and the transcript levels are expressed as 2−∆∆Cq values.
BV2 cells and substantia nigra were lysed in ice cold lysis buffer (1X PBS, 1% Nonidet P-40, 05% sodium deoxycholate and 0.1% SDS; RIPA) containing phosphatase inhibitor protease inhibitor. Lysates (50 µg protein) from each sample were resolved on SDS 10% polyacrylamide gel (10% PAGE) and electrotransferred to PVDF membrane. The membrane was incubated in 5% non-fat dry milk to block non-specific antibody binding site and then incubated overnight at 4°C with rabbit anti-p-AKt (1:1,000), rabbit anti-AKt (1:1,000), rabbit anti-TH (1:200), rabbit anti-Iba1 (1 µg/ml), rabbit anti-β-actin (1:1,000). After washing in TBST (0.01 M TBS and 0.1% Tween-20), membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit immunoglobulin G [IgG], 1:1,000) for 2 h. This step was followed by a washing in TBST and protein visualized. In this respect, chemiluminescence (ECL) reagents were employed.
Mice were anesthetized and perfused transcardially with phosphate-buffered saline (PBS) followed by a 4% fresh paraformaldehyde (PFA) solution (pH 7.4). Brains were removed and kept in 4% PFA overnigth at 4°C. Then after, they were inserted in a 30% sucrose solution for 48 h at 4°C. Afterwards, brains were cutted (slices of 30 m depth) with a freezing microtome (Leica, Germany) and incubated during 30 min in 3% H2O2 solution. Then, slices were rinsed three times and during 5 min in PBST (0.01 M PBS and 0.3% Triton-X 100) and incubated with a 5% normal goat serum at room temperature for 1 h. Brain section were then incubated with polyclonal rabbit anti-TH (1:750; cat. no. ab112; Abcam), Ibal (0.5 µg/ml; Wako) overnight at 4°C. The next day, the sections were washed with PBST three times for 5 min and incubated with corresponding secondary antibodies at room temperature for 2 h. They were washed again in PBS and subsequently incubated with streptavidin-peroxidase for 30 min. After a new wash in PBS (3×5 min.), they were incubated with a DAB chromogenic substrate (Fuzhou Maixin Biotech Co., Ltd. (Fuzhou, China) for 5 min and then after washed again in distilled water. Afterwards, brain sections were mounted on gelatin-coated slides, air-dried, dehydrated, and covered with a glass plate. Brain sections were examined using a light-field microscope. Finally, TH+ cells were identified with the Image-Pro Plus software and visually counted.
The Statistical Package for the Social Sciences (SPSS version 13.0) was used for the statistical analyses. All data were expressed as mean ± standard deviation (SD) for three independent experiments, at least. The analysis of variance (ANOVA) was performed for all tests, followed by post hoc Fisher's LSD multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.
Data obtained indicate that LPS-induced inflammatory response in BV2 microglial cells produces a significant reduction in p-AKt protein content when compared with the saline group of animals. This effect, however, does not reach significance vs. total AKt proteins. Activation of SHH pathway by PM enhances the expression of p-AKt protein, which can be reversed by the SHH pathway inhibitor cyclopamine. No change are observed in total AKt proteins after PM and cyclopamine treatments (
We also checked whether the SHH pathway regulated the expression of p-AKt protein in the area of SNpc in mice model of PD. As shown in
As shown in
Western blot reveals that LY294002 blocked the effect of PM treatment on microglial cells: The Iba1 protein is significantly increased in the MPTP group. This effect, however, is also observed when the PM group is inhibited by LY294002. LY294002 increases the expression of Iba1 protein compared with MPTP group alone (
To check this aspect, SNpc tissue homogenate from our five group of animals were performed. As shown in
We also investigated the role of PI3K/Akt signaling
TH immunohistochemistry reveals a significant cell reduction in MPTP group compared with the saline one. Treatment with PM protects dopaminergic neurons of the SNpc and enhances their rate of survival. However, pretreatment with LY294002 abolishes the neuroprotection exerted by PM in the model of PD and also decreases the number of TH-positive neurons compared with MPTP group (
With TR test, mice treated with MPTP spend significantly less time on the steel compared with those treated with saline (
In the present study, we further investigated the part played by the SHH signaling
Data of literature, in keeping with our results, now point out that neuroinflammation, triggered by microglial activation, may play a central role in the progression of the dopaminergic neurons degeneration in PD pathogenesis (
Beside above considerations, our results also point out that SHH signaling negatively regulated the activation of microglia through PI3K/Akt pathway. A study of literature indicating that SHH signaling can alleviate inflammatory response (
Finally, our data indicating that the activation of the SHH signaling is essential to prevent, via the PI3K/AKt signaling pathway, the MPTP-induced loss of dopaminergic neurons and the motor deficit are in keeping with those of literature (
Apart from the data discussed above, in the full statement of our results it remains to be further understood: How the LY294002 inhibitor of the PI3K/Akt signaling pathway may downregulate the expression of TGF-β1 induced by PM; how this inhibitor can upregulate the expression of TGF-β1 induced by LPS
In conclusion, our study demonstrates that SHH-PI3K/Akt coupled signalings are capable to protect DA neurons, inhibit microglia activation and improve motor performances. While additive experiments remains, obviously, necessary to clarify the results reported, our research appears promising for a better understanding of the neurodegenerative pathologies.
This study was supported by grants from the Guangdong Provincial Development of Science and Technology (no. 201301) and the Medical Scientific Research Foundation of Guangdong Province (no. A2013239). PR Raymond CESPUGLIO contributed to the manuscript improvement.
Purmorphamine (PM) and LY294002 respectively activates and inhibits Sonic hedgehog (SHH) signaling by way of PI3K/AKt pathway either
SHH signaling inhibits microglia activation through PI3K/Akt pathway. (A) We performed immunohistochemical staining with Iba1 to visualize the activation of microglia in SNpc of five mice groups: i) Control group; ii) MPTP group; iii) PM+MPTP group; iv) LY+PM+MPTP group; and v) LY+MPTP group. A1 scale bar=500 µm; A2 scale bar=200 µm; A3 scale bar=50 µm. (B) The Iba1 protein in SNpc were determined using western blotting and analysis of Iba1. (C) Relative level of Iba1 protein: *P<0.01 compared with control group; **P<0.01 compared with MPTP group; ***P<0.01 compared with PM+MPTP group. SHH, Sonic hedgehog; Iba1, Ionized calcium binding adaptor molecule 1; LY, LY294002.
SHH signaling mediates anti-inflammatory effects through PI3K/Akt pathway. (A and B) Expression of IL-1β and TNF-α mRNA in SNpc by quantitative real time PCR analysis. (C-F) Expression of IL-1β, TNF-α, TGF-β, 1 and Nurr1 mRNA in BV2 cells by quantitative real time PCR analysis. *P<0.01 compared with control group; **P<0.01 compared with LPS or MPTP group; ***P<0.01 compared with PM+LPS or PM+MPTP group.
SHH signaling mediates neuroprotection through the PI3K/Akt pathway. (A) Immunohistochemical staining was performed to visualize TH-positive dopaminergic neurons in the SNpc. A1 scale bar=500 µm; A2 scale bar=200 µm; A3 scale bar=400 µm. (B) Number of TH-positive dopaminergic neurons in SNpc. (C) Western blotting and analysis of TH protein. (D) Relative level of TH protein. *P<0.01 compared with control group; **P<0.01 compared with MPTP group; ***P<0.01 compared with PM+MPTP group. TH, Tyrosine hydroxylase.
SHH signaling induces behavioral change through PI3K/Akt pathway in MPTP mice model of PD. Traction behavior (TR) test was applied to test the motor function. Scale given in Material and methods was employed to evaluate performances. *P<0.01 compared with control group; **P<0.01 compared with MPTP group; ***P<0.01 compared with PM+MPTP group. TR, Traction behavior.