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Methods for the Modulation and Analysis of NF-κB-dependent Adult Neurogenesis
Published on: February 13, 2014
[Effects of different nuclear factor kappaB dimers on the survival of immortalized neural progenitor cells]
Ling-Li Gui1, Chuan-Han Zhang, Zhi-Heng Liu
1Department of Anesthesiology, Tongji Hospital, Tongi Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Objective:
To investigate the effects of different nuclear factor (NF)-KB dimers on the survival of immortalized neural progenitor cells (INPCs).
Methods:
The control vector RC/CMV, containing the promoter of cytomegalovirus (CMV), and the expression vectors, RcCMV-p50 and RcCMV-p65, containing the coding regions of NF-KB subunits p50 and p65 genes, were transfected into the INPCs by liposome respectively. Stably transfected clones were screened out following G418 selection. Subsequently, the plasmid RcCMV-p50 was transiently transfected into the INPCs which had been stably transfected with the plasmid RcCMV-p65. The expression of p50 or p65 gene was detected in each cell strain by Western blotting. And the NF-KB DNA binding activity in the cell nuclear extracts was measured by electrophoresis mobility shift assay (EMSA). The expression of IkappaBalpha in the cytoplasm was detected by Western blotting. After oxygen and glucose deprivation for 13 h, the cell survival rate was measured by MTT assay.
Results:
After gene transfection, five different cell strains were obtained: INPC, INPC/CMV, INPC/p50, INPC/p65, and INPC/p50p65. p50 or p65 gene was translated correctly and efficiently in the cell strains which had been transfected with the corresponding plasmids. EMSA showed that the INPC/p50, INPC/p65, and INPC/p50p65 cells all gave rise to NF-kappaB specific bands, which were composed of p50 homodimer, p65 homodimer, and p50 p65 heterodimer and p50 homodimer respectively. The expression of IkappaBbeta was increased significantly in the cytoplasm of the INPC/p65 and INPC/p50p65 cells. Games-Howell test showed that after oxygen and glucose deprivation for 13 h, the survival rates of the NPC/p65 and INPC/p50p65 cells were (6.0 +/- 1.0)% and (4.6 +/- 0.6)% respectively, both significantly lower than those of the INPC, INPC/CMV, and INPC/p50 cells [(72.5 +/- 6.2)%, (70.1 +/- 4.3)%, and (70.4 +/- 7.3)% respectively, all P < 0.05].
Conclusions:
Overexpression of p50 gene and p65 gene directly enhance the DNA binding activities of different NF-kappaB dimers in the nuclei. In neural progenitor cells, NF-kappaB dimers with transcriptive activity decreases the cellular survival after oxygen and glucose deprivation, but NF-kappaB dimers without transcriptive activity don't prevent the cells from death.
Insights
Nuclear factor-kappa B (NF-κB) dimers impact neural progenitor cell survival. Transcriptionally active NF-κB dimers decrease cell survival after oxygen-glucose deprivation, while inactive dimers do not prevent cell death.
Area of Science:
- Molecular Biology
- Neuroscience
- Cell Biology
Context:
- Neural progenitor cells (INPCs) are crucial for brain development and repair.
- Nuclear factor-kappa B (NF-κB) signaling plays a role in cellular responses to stress.
- Understanding NF-κB dimer function is vital for neuroprotection strategies.
Purpose:
- To investigate the differential effects of nuclear factor-kappa B (NF-κB) dimers on immortalized neural progenitor cell (INPC) survival.
- To elucidate the role of specific NF-κB subunits (p50 and p65) in cellular response to ischemic conditions.
Summary:
- Different NF-κB dimers were expressed in INPCs, and their DNA binding activities were assessed.
- Cells overexpressing transcriptionally active NF-κB dimers (p65 homodimers and p50/p65 heterodimers) showed significantly reduced survival rates following oxygen and glucose deprivation.
- Cells with transcriptionally inactive NF-κB dimers (p50 homodimers) did not exhibit decreased survival.
Impact:
- This study reveals that transcriptionally active NF-κB dimers exacerbate cell death in neural progenitor cells under ischemic stress.
- Findings suggest that targeting specific NF-κB dimers could be a therapeutic strategy for stroke or other neurological disorders involving oxygen-glucose deprivation.
- The differential roles of NF-κB dimers provide insights into the complex regulation of cell survival in the central nervous system.
