Gracilin A Derivatives Target Early Events in Alzheimer's Disease: in Vitro Effects on Neuroinflammation and Oxidative Stress

Abstract

The search for compounds capable of targeting early pathological changes of Alzheimer̀s disease (AD), such as oxidative stress and neuroinflammation, is an important challenge. Gracilin A derivatives were recently synthesized, using a pharmacophore-directed retrosynthesis (PDR) strategy, and found to possess potent neuroprotective effects. In this work, the previously described derivatives 1-7 which demonstrated mitochondrial-mediated, antioxidant effects were chosen for further study. The ability of compounds to modulate the expression of antioxidant genes (CAT, GPx, SODs, and Nrf2) was determined in SH-SY5Y cells, and the simplified derivatives 2 and 3 were found to be the most effective. The anti-neuroinflammatory properties of all derivatives were assessed in BV2 microglial cells activated with lipopolysaccharide (LPS). Several derivatives decreased the release of cytokines (Il-1β, IL-6, GM-CSF, and TNF-α) and other damaging molecules (ROS, NO) and also regulated the translocation of Nrf2 and NFκB, and reduced p38 activation. These protective effects were confirmed in a trans-well coculture with BV2 and SH-SY5Y cells and several derivatives increased SH-SY5Y survival. This present work demonstrates the neuroprotective properties of gracilin A derivatives, making them promising candidate drugs for AD. Particularly, derivatives 2 and 3 showed the greatest potential as lead compounds for further development.

Keywords: Alzheimer’s disease; Gracilin; Nrf2; antioxidant; neuroinflammation; neuroprotection.

Figure 1.

Chemical structures of gracilin A and derivatives

Figure 2.

Relative expression of anti-oxidant genes in neuroblastoma cells treated with gracilin A derivatives. 150 μM H₂O₂ and compounds at μM concentrations were added to SH-SY5Y cells for 6 h and their effect on the expression of (a) catalase, (b) glutathione peroxidase 1, (c) superoxide dismutase 1, (d) superoxide dismutase 2 and (e) Nrf2 was evaluated. RPL0 was used as internal normalization control. Relative gene expression was calculated with ΔΔCt method. Cells treated only with H₂O₂ were used as calibrator and are represented by x-axes. Data are expressed as mean ±SEM of three independent replicates performed by duplicate and compared to H₂O₂ control cells by one way ANOVA and Dunnett’s tests. *p<0.05. **p<0.01, *** p<0.001

Figure 3.

Effect of Spongionella synthetic derivatives on cytokine release by microglia. BV2 cells were pre-treated with compounds at μM concentrations for 1 h and activated with 500 ng/mL LPS for 24 h. (a) IL-1β, (b) IL-6, (c) TNF-α and (d) GM-CSF levels in the medium of microglial cells were analysed with a Mouse Inflammatory 4-Plex Panel. Data are presented as percentage of LPS control cells. Results are mean± SEM of three independent experiments and compared to cells treated with LPS alone by one way ANOVA and Dunnett’s tests. *p<0.05, **p<0.01, ***p<0.001. LPS control cells are compared to inactivated control cells. ###p<0.001

Figure 4.

Compounds decreased ROS and NO produced by microglia. BV2 cells were stimulated with 500 ng/mL LPS after pre-treatment with derivatives at μM concentrations for 1 h. After this time, the levels of (a) ROS and (b) NO were measured. Values are mean± SEM of three replicates performed by triplicate and expressed as percentage of LPS control cells. Statistical significance was determined with one way ANOVA test followed by Dunnett’s post-hoc test. LPS control cells are compared to inactivated microglia (##p<0.01, ###p<0.001). Treatments with compounds are compared to cells treated only with LPS (*p<0.05, **p<0.01, ***p<0.001).

Figure 5.

Effects of gracilin A derivatives on iNOS and p38 MAPK. Microglial cells were treated with compounds at μM concentrations and 500 ng/mL LPS. Then, BV2 cells were lysed and the protein expression levels of (a) iNOS and (b) p38 were analysed by western blot. The activation of the kinase was determined as the ratio between phosphorylated and total protein levels. Protein expression levels were corrected by β-actin. Treatments are reordered to match with blots. Values are mean± SEM of three independent experiments carried out by duplicate. Treatments with derivatives are compared to LPS control cells by one way ANOVA and Dunnett’s test (*p<0.05, **p<0.01, ***p<0.001). Cells treated with LPS alone are compared to untreated control cells (#p<0.05, ###p<0.001)

Figure 6.

Gracilin A derivatives modulated the expression of Nrf2 and NFκB-p65 in microglia. BV2 murine cells were pre-treated with compounds at μM concentrations for 1 h. Then, LPS was added for 24 h in order to activate the cells. The expression levels of Nrf2 were measured in (a) the nucleus and (b) the cytosol. In the same way, NFκB-p65 expression was determined in (c) nuclear and (d) cytosolic fractions. Protein levels were normalized with lamin B1 and β-actin in the nucleus and the cytosol, respectively. Results are mean± SEM of three replicates performed by duplicate. Statistical differences were calculated with one way ANOVA and Dunnett’s test. Treatments with compounds are compared to LPS control cells (*p<0.05, **p<0.01, ***p<0.001) and LPS control cells are compared to inactivated cells (#p<0.05, ##p<0.01, ###p<0.001)

Figure 7.

Gracilin A derivatives protected SH-SY5Y cells from activated microglia in a trans-well co-culture. BV2 cells were seeded in inserts placed above SH-SY5Y cells. Microglia was pre-treated with compounds at μM concentrations for 1 h and activated with 500 ng/mL LPS during 24 h. Then, the viability of neuroblastoma cells was determined with MTT assay. Values are mean± SEM of three independent experiments carried out by duplicate. Results are expressed as percentage of SH-SY5Y cells co-cultured with inactivated microglia (control). One way ANOVA and Dunnett’s tests were used to analyse the statistical differences between treatments and neuroblastoma cells co-cultured with BV2 cells treated with LPS alone (LPS) (*p<0.05). LPS cells are compared to control cells (##p<0.01).