Abstract
Purpose : Microglia modulate inflammation and neurodegeneration in common neurodegenerative diseases. However, the signalling systems that induce dysregulation of microglial homeostasis are incompletely understood. We have recently identified that tropomyosin 1 (TPM1), an actin binding protein, regulates inflammation and neuronal remodelling in the aging retina. Here, we further investigated the role of TPM1 in regulating microglia activity in BV2 cells, a murine microglia cell line.
Methods : To study the role of TPM1 in inflammation, we first transfected BV2 cells with TPM1 overexpressed plasmids for two days, and then the molecular and morphological alterations of cells were evaluated by qRT-PCR, Western blotting and immunohistochemistry. To study if inhibition of TPM1 could reverse LPS-induced inflammation, we treated cells with TPM1 specific siRNA for one days followed by LPS treatment, and then the inflammatory cytokines were detected. To further study the PKA/CREB signaling pathway, we treated cells with dbCAMP (an activator of PKA) in TPM1 plasmids treated cells, or H89 (an inhibitor of PKA) in siTPM1 treated cells following LPS treatment.
Results : TPM1 overexpression in BV2 cells significantly activated microglia and promoted the release of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 and chemokines COX-2 and iNOS, and downregulated phosphorylated PKA and CREB. TPM1 overexpression failed to regulated p-CREB after treatment with dbCAMP in BV2 cells. When transfecting with TPM1 siRNA in BV2 cells following LPS treatment, we found that TPM1 knockdown remarkably inhibited microglia activation and inflammatory cytokines release, and reversed the decrease of PKA/CREB signaling pathway which was induced by LPS treatment. By applying H89 in BV2 cells, we found that TPM1 knockdown failed to upregulate p-CREB in LPS-treated cells.
Conclusions : Our data demonstrated that TPM1 mediated microglia activation and inflammation by regulating PKA/CREB signaling pathway. The therapeutic approaches targeting the inhibition of TPM1 could be a potential strategy to study microglia homeostasis and inflammation-related diseases.
Methods : To study the role of TPM1 in inflammation, we first transfected BV2 cells with TPM1 overexpressed plasmids for two days, and then the molecular and morphological alterations of cells were evaluated by qRT-PCR, Western blotting and immunohistochemistry. To study if inhibition of TPM1 could reverse LPS-induced inflammation, we treated cells with TPM1 specific siRNA for one days followed by LPS treatment, and then the inflammatory cytokines were detected. To further study the PKA/CREB signaling pathway, we treated cells with dbCAMP (an activator of PKA) in TPM1 plasmids treated cells, or H89 (an inhibitor of PKA) in siTPM1 treated cells following LPS treatment.
Results : TPM1 overexpression in BV2 cells significantly activated microglia and promoted the release of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 and chemokines COX-2 and iNOS, and downregulated phosphorylated PKA and CREB. TPM1 overexpression failed to regulated p-CREB after treatment with dbCAMP in BV2 cells. When transfecting with TPM1 siRNA in BV2 cells following LPS treatment, we found that TPM1 knockdown remarkably inhibited microglia activation and inflammatory cytokines release, and reversed the decrease of PKA/CREB signaling pathway which was induced by LPS treatment. By applying H89 in BV2 cells, we found that TPM1 knockdown failed to upregulate p-CREB in LPS-treated cells.
Conclusions : Our data demonstrated that TPM1 mediated microglia activation and inflammation by regulating PKA/CREB signaling pathway. The therapeutic approaches targeting the inhibition of TPM1 could be a potential strategy to study microglia homeostasis and inflammation-related diseases.
| Original language | English |
|---|---|
| Title of host publication | Investigative Ophthalmology & Visual Science |
| Pages | 4125 |
| Volume | 63 |
| ISBN (Electronic) | 1552-5783 |
| Publication status | Published - Jun 2022 |