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Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection
Published on: September 21, 2014
NFkappaB activation is required for the neuroprotective effects of pigment epithelium-derived factor (PEDF) on
T Yabe1, D Wilson, J P Schwartz
1Neurotrophic Factors Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892-4126, USA.
Insights
Pigment epithelium-derived factor (PEDF) activates the NF-kappaB pathway, protecting neurons from cell death. This neuroprotection involves upregulating anti-apoptotic and neurotrophic genes, crucial for neuronal survival.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Pigment epithelium-derived factor (PEDF) is known to protect neurons, but its mechanism remains unclear.
- Nuclear factor kappa B (NF-kappaB) is a transcription factor that inhibits cell death pathways, including neuronal apoptosis.
Purpose of the Study:
- To investigate whether PEDF exerts its neuroprotective effects through the activation of NF-kappaB.
- To elucidate the downstream molecular targets of PEDF-mediated NF-kappaB activation in neuronal cells.
Main Methods:
- Western blotting to assess IkappaBalpha phosphorylation and protein levels.
- Immunofluorescence to detect p65 (RelA) nuclear translocation.
- Electrophoretic mobility shift assay (EMSA) for NF-kappaB DNA binding activity.
- Inhibition studies using IKK inhibitor BAY 11-7082 and proteasome inhibitor.
- Reverse transcription-polymerase chain reaction (RT-PCR) to analyze gene expression.
Main Results:
- PEDF treatment increased IkappaBalpha phosphorylation, decreased IkappaB levels, and promoted p65 nuclear translocation in both immature and mature neurons.
- NF-kappaB activation was essential for PEDF's protective effects against apoptosis and glutamate toxicity, as confirmed by inhibitor studies.
- PEDF upregulated anti-apoptotic genes (Bcl-2, Bcl-x, MnSOD) in immature neurons and neurotrophic factor genes (NGF, BDNF, GDNF) in mature neurons.
Conclusions:
- PEDF promotes neuronal survival by activating the NF-kappaB signaling pathway.
- NF-kappaB activation by PEDF leads to the differential expression of anti-apoptotic and neurotrophic factor genes, contributing to neuroprotection.
Abstract:
Pigment epithelium-derived factor (PEDF) protects immature cerebellar granule cells (1-3 days in vitro) against induced apoptosis and mature cells (5+ days in vitro) against glutamate toxicity, but its precise mechanism is still unknown. Because the transcription factor NFkappaB blocks cell death, including neuronal apoptosis, we have investigated the ability of PEDF to exert its effects via NFkappaB activation. PEDF induced an increased phosphorylation of IkappaBalpha, decreased levels of IkappaB proteins, and translocation of p65 (RelA) to the nucleus followed by a time-dependent increase of NFkappaB-DNA binding activity in both immature and mature neurons. The protective effects of PEDF against both induced apoptosis and glutamate toxicity were blocked by the addition of either the IkappaB kinase inhibitor BAY 11-7082, which inhibits the phosphorylation of IkappaB, or N-acetyl-Leu-Leu-norleucinal, which blocks proteosome degradation of IkappaB, demonstrating that NFkappaB is required for the neuroprotective effects of PEDF. Reverse transcription-polymerase chain reaction analysis revealed that up-regulation of the anti-apoptotic genes for Bcl-2, Bcl-x, and manganese superoxide dismutase was observed in PEDF-treated immature but not mature neurons. Up-regulation of nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor mRNA was long-lasting in mature neurons. These results suggest that PEDF promotes neuronal survival through activation of NFkappaB, which in turn induces expression of anti-apoptotic and/or neurotrophic factor genes.
