Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Feb;55(2):1676-1691.
doi: 10.1007/s12035-017-0432-7. Epub 2017 Feb 13.

Cofilin Mediates LPS-Induced Microglial Cell Activation and Associated Neurotoxicity Through Activation of NF-κB and JAK-STAT Pathway

Affiliations
Free PMC article

Cofilin Mediates LPS-Induced Microglial Cell Activation and Associated Neurotoxicity Through Activation of NF-κB and JAK-STAT Pathway

Qasim Alhadidi et al. Mol Neurobiol. .
Free PMC article

Abstract

Microglial cells are activated in response to different types of injuries or stress in the CNS. Such activation is necessary to get rid of the injurious agents and restore tissue homeostasis. However, excessive activation of microglial cells is harmful and contributes to secondary injury. Pertinently, microglial cell activity was targeted in many preclinical and clinical studies but such strategy failed in clinical trials. The main reason behind the failed attempts is the complexity of the injury mechanisms which needs either a combination therapy or targeting a process that is involved in multiple pathways. Cofilin is a cytoskeleton-associated protein involved in actin dynamics. In our previous study, we demonstrated the role of cofilin in mediating neuronal apoptosis during OGD conditions. Previous studies on microglia have shown the involvement of cofilin in ROS formation and phagocytosis. However, additional studies are needed to delineate the role of cofilin in microglial cell activation. Therefore, in the current study, we investigated the role of cofilin in LPS-induced microglial cell activation using cofilin siRNA knockdown paradigms. The viability of differentiated PC12 cells was used as a measure of the neurotoxic potential of conditioned medium derived from cofilin siRNA-transfected and LPS-activated microglial cells. Cofilin knockdown significantly inhibited LPS-induced microglial cell activation through NF-κB and JAK-STAT pathways. The release of proinflammatory mediators (NO, TNF-α, iNOS, and COX2) as well as microglial proliferation and migration rates were significantly reduced by cofilin knockdown. Furthermore, differentiated PC12 cells were protected from the neurotoxicity induced by conditioned medium derived from cofilin-transfected and LPS-activated microglial cells. In conclusion, we demonstrated that cofilin is involved in the cascade of microglial cell activation and further validates our previous study on cofilin's role in mediating neuronal apoptosis. Together, our results suggest that cofilin could present a common target in neurons and microglial cells and might prove to be a promising therapy for different brain injury mechanisms including stroke.

Keywords: Cofilin; Inflammation; LPS; Microglia; Neurotoxicity; ROS.

Figures

Figure 1
Figure 1. Phosphocofilin and cofilin expression levels in response to OGD, OGD/R and LPS
SIM-A9 microglial cells were treated with OGD for 1 h, OGD/R for 24 h and LPS 100 ng/ml for 24 h and then protein expression level was evaluated by WB. Although cytosolic (A) and nuclear (D) total cofilin level did not show any significant change in all treatment groups, p-cofilin level was significantly downregulated relative to total cofilin (B) and α-tubulin (C). Furthermore, p-cofilin level was significantly downregulated in LPS treated group compared with OGD/R (B and C). α- tubulin and histone H3 were used as a loading control for cytosolic and nuclear proteins, respectively. E. Representative immunoflorescence pictures show cofilin (stained red) is localized predominantly in the cell periphery. After OGD and LPS treatment, cofilin is redistributed in the whole cell area. Scale bar is 25 μm. Data are expressed as mean±SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 relative to control group. #P < 0.05, ##P < 0.01, ###P < 0.001 relative to OGD/R treated groups.
Figure 2
Figure 2. Cofilin knockdown by siRNA and its associated effect on microglial cell morphology
A. Microglial cells were transfected with scrambled/cofilin siRNA at 25 and 50 nM concentration for 72 h and then harvested for WB analysis. Cofilin siRNA at 25 and 50 nM concentrations reduced cofilin expression level significantly 72 h posttransfection. Although no significant difference was detected between the two concentrations used, the trend with cofilin siRNA 50 nM was higher. α- tubulin was used as a loading control for cytosolic proteins. B and C. Microglial cells were transfected with scrambled/cofilin siRNA at 50 nM for 72 h and then the changes in cell morphology associated with cofilin knockdown were imaged (C) and quantified (B). Cofilin knockdown reduced the radius ratio (maximum radius over minimum radius) significantly relative to scrambled littermate (B). Furthermore, cofilin transfected cells imaged at 10× and 20× magnification were almost round in shape while their control showed different morphology (C). The 20× magnification images were used to measure the radius ratio in B. Data are expressed as mean±SEM of three independent experiments. *P < 0.05, **P < 0.01, relative to scrambled siRNA control group.
Figure 3
Figure 3. The effect of cofilin knockdown on microglial cells proliferation and viability
A. Summary of the experimental design. B. Microglial cells were transfected with scrambled/cofilin siRNA at 50 nM concentration and then MTT proliferation assay was performed after 12 h, 60 h and 84 h. While the cell number was almost same for cofilin and scrambled groups after 12 h, cofilin knockdown reduced the cell number significantly after 60 and 84 h. C and D. Microglial cells were transfected with scrambled/cofilin siRNA at 50 nM concentration for 72 h and then treated with LPS (100 ng/ml) for 24 h (C) or OGD for 1 h (D) followed by MTT viability assay. LPS induced significant microglial cells death in scrambled siRNA transfected group but not in cofilin group (C). Similarly, OGD induced significant cell death in scrambled but not in cofilin group (D). As shown in B, cofilin knockdown reduced cell numbers in all treatment groups (C and D). The data was expressed as Mean±SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 relative to scrambled siRNA group without treatment (LPS and OGD). ns: non-significant.
Figure 4
Figure 4. The effect of cofilin knockdown on LPS induced microglial cells activation
A and B. Microglial cells were transfected with scrambled/cofilin siRNA at 50 nM concentration for 72 h and then treated with LPS (100 ng/ml) or OGD for 1 h. Nitrite release assay was done after that by mixing 100 μl of cell culture medium with 100 μl of griess reagent. Relative to LPS treated control, NO release was significantly reduced by cofilin knockdown. Furthermore, no significant difference in NO release was detected between LPS untreated groups (scrambled and cofilin) and LPS treated cofilin transfected group (A). In OGD treated groups, no significant increase in NO release was detected (B). C–F. Microglial cells were transfected with scrambled/cofilin siRNA at 50 nM concentration for 72 h and then treated with LPS (100 ng/ml) for further 24 h. Cell culture medium was used for ELISA assay of TNF-α and IL-1β (E and F) and cell lysate was used for WB analysis of iNOS and COX2 expression (C and D). Relative to LPS stimulated control, cofilin knockdown reduced LPS induced iNOS (C) and COX2 (D) expression significantly. Furthermore, no significant differences in iNOS and COX2 levels was detected between LPS untreated groups (scrambled and cofilin) and LPS treated cofilin transfected group. α-tubulin was used as a loading control. (E and F). In ELISA assay, LPS treatment increased TNF-α level significantly in both groups (scrambled and cofilin transfected), compared to unstimulated scrambled control group. In LPS treated groups, TNF-α level was significantly less in cofilin transfected group (E). Similarly, IL-1β release was also reduced by cofilin knockdown but the reduction did not reach the level of significance (P = 0.2) (F). Data are represented as Mean±SEM of three independent experiments. **P < 0.01, ***P < 0.001 relative to scrambled siRNA group without LPS treatment. #P < 0.05, ###P < 0.001 relative to scrambled transfected and LPS treated group.
Figure 5
Figure 5. The effect of cofilin knockdown on microglial cells migration and phagocytic activity
Microglial cells were transfected with scrambled/cofilin siRNA at 50 nM concentration for 72 h and then a scratch was made in the middle of each well of 24-well plate. Microglial cells migrated to the scratched area were imaged at 20× magnification (B) and then the percentage of area occupied by migrated cells was calculated at 6 h, 12 h and 24 h (A) using image J software. Cofilin knockdown reduced microglial cell migration significantly at all time intervals tested (A). C–E. Microglial cells were transfected with scrambled/cofilin siRNA at 50 nM concentration for 72 h and then treated with LPS (100 ng/ml) for 24 h more. After that FITC-labeled Ecoli bioparticles was added at 0.125 mg/ml and incubated for 6 h. Trypan blue was used to quench unphagocytosed particles and the fluorescence intensity of phagocytosed particles was imaged using fluorescent microscope (E) and quantified using Synergy H1 Hybrid Reader at fluorescence excitation/emission of 494/518 (C and D). Cofilin knockdown reduced microglial cell phagocytic activity significantly (C). However, by dividing FITC- fluorescence over MTT absorbance (to consider cell density), phagocytic activity was higher in cofilin transfected and LPS treated group (D). In general, LPS treatment increased phagocytic activity in all groups as shown in D and E. Furthermore, in cofilin transfected groups, phagocytic activity was significantly higher in LPS treated one (C and D). Data are expressed as mean±SEM of three independent experiments. *P <0.05, **P < 0.01, ***P < 0.001 relative to scrambled siRNA group without LPS treatment. #P < 0.05, ##P < 0.01 relative to cofilin transfected and LPS untreated group. ns: non-significant.
Figure 6
Figure 6. The effect of conditioned medium derived from cofilin transfected and LPS treated microglia on differentiated PC12 cells viability
Microglial cells were transfected with scrambled/cofilin siRNA at 50 nM concentration for 72 h and then treated with LPS (100 ng/ml) for 24 h more. Afterwards microglial cells conditioned medium was added to differentiated PC12 cells and incubated for 24 h. MTT viability assay was done to assay the neurotoxic potential of the conditioned medium. Differentiated PC12 cells viability was significantly reduced by treatment with the conditioned medium derived from scrambled transfected and LPS activated microglia. However, cofilin knockdown in microglial cells protect differentiated PC12 cells from the neurotoxic effect of the conditioned medium. Data are expressed as mean±SEM of three independent experiments. **P < 0.01 relative to scrambled siRNA group without LPS treatment. ##P < 0.01 relative to scrambled transfected and LPS treated group.
Figure 7
Figure 7. The effect of cofilin knockdown in microglia on LPS induced NF-κB, JNK and STAT1 activation
Microglial cells were transfected with scrambled/cofilin siRNA at 50 nM concentration for 72 h and then treated with LPS (100 ng/ml) for another hour. Protein expression levels for cytosolic NF-κB (A), nuclear NF-κB (B), p-JNK (C) and p-STAT1 (D) was evaluated using WB. Relative to LPS treated and scrambled transfected group, cofilin knockdown reduced nuclear translocation of NF-κB significantly (B). STAT1 activation was also significantly reduced by cofilin knockdown (D). However, no significant effect for cofilin knockdown on JNK activation (C) and cytosolic NF-κB expression (A) was detected. Data are expressed as mean±SEM of three independent experiments. *P < 0.05, ***P < 0.001 relative to scrambled siRNA group without LPS treatment. ##P < 0.01, ###P < 0.001 relative to scrambled transfected and LPS treated group. ns: non-significant.
Figure 8
Figure 8. The proposed role of cofilin in mediating LPS stimulatory effect on microglia
LPS binding to TLR-4 initiates Src activation which subsequently leads to induction of p-65, NF-κB, STAT1 and cofilin. Basically, active cofilin regulate actin dynamics and control cell morphology, proliferation, migration and phagocytosis. Moreover, active cofilin has a signaling role through controlling the activity of Src activated transcription factors, p-65 NF-κB and STAT1. These factors initiate neurotoxicity by regulating the expression of different cytotoxic materials like TNF-α, IL-1β, ROS and RNS. Dashed arrows indicate that these pathways are established in cell types other than microglia and macrophage. Solid arrows indicate that these pathways are established in microglia or macrophage.
Active Pathway
Inactive Pathway

Similar articles

See all similar articles

Cited by 13 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback