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Unique aspects of transcriptional regulation in neurons--nuances in NFkappaB and Sp1-related factors.

Mao XR, Moerman-Herzog AM, Chen Y, Barger SW - J Neuroinflammation (2009)

Bottom Line: These studies led us to conclude that this population of cells is nearly incapable of activating the NFkappaB that is nonetheless expressed at reasonable levels.A subset of the kappaB cis elements are instead bound by members of the Sp1 family in neurons.Also surprising was our discovery that Sp1 itself, typically described as ubiquitous, is severely restricted in expression within forebrain neurons; Sp4 seems to be substituted during neuronal differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, Washington University School of Medicine, St Louis, MO 63110, USA. maox@morpheus.wustl.edu

ABSTRACT
The unique physiology and function of neurons create differences in their cellular physiology, including their regulation of gene expression. We began several years ago exploring the relationships between the NFkappaB transcription factor, neuronal survival, and glutamate receptor activation in telencephalic neurons. These studies led us to conclude that this population of cells is nearly incapable of activating the NFkappaB that is nonetheless expressed at reasonable levels. A subset of the kappaB cis elements are instead bound by members of the Sp1 family in neurons. Also surprising was our discovery that Sp1 itself, typically described as ubiquitous, is severely restricted in expression within forebrain neurons; Sp4 seems to be substituted during neuronal differentiation. These findings and their implications for neuronal differentiation--as well as potential dedifferentiation during degenerative processes--are discussed here.

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RelA nuclear translocation in neurons after glutamate treatment. Nearly pure neocortical neurons were treated with glutamate (50 μM) for the indicated times and cells were harvested for fractionation (Cyto.: cytosolic fraction; Nuc.: nuclear fraction); these were probed by western blot for RelA. Whole-cell lysates were also prepared from either astrocyte or neuron cultures; these were probed by western blot for IκBα (Whole: neuron whole cell lysate). TNFα was applied to the astrocytes at 100 ng/ml for the indicated times (min).
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Figure 1: RelA nuclear translocation in neurons after glutamate treatment. Nearly pure neocortical neurons were treated with glutamate (50 μM) for the indicated times and cells were harvested for fractionation (Cyto.: cytosolic fraction; Nuc.: nuclear fraction); these were probed by western blot for RelA. Whole-cell lysates were also prepared from either astrocyte or neuron cultures; these were probed by western blot for IκBα (Whole: neuron whole cell lysate). TNFα was applied to the astrocytes at 100 ng/ml for the indicated times (min).

Mentions: Many of the original reports of NFκB activation in neurons were based on nuclear translocation of the transcription factor as an endpoint. We tested this parameter in our highly enriched cortical neuron cultures. Cells treated with glutamate and then lysed and separated into a cytosolic fraction (supernatant) and a nuclear fraction (whole nuclear pellet sonicated) for analysis by western blotting (note: this is different from the nuclear extract used in EMSA, which only includes the salt-extractable components). Glutamate was indeed found to evoke nuclear translocation of RelA under these conditions (Figure 1). This is consistent with previous findings we obtained with immunocytochemistry [20]. Translocation of RelA-containing NFκB complexes is typically dependent upon degradation of an IκB, so we assessed the levels of IκBα in whole-cell lysates obtained after an identical glutamate treatment. No significant levels of other IκB proteins can be detected in cultured cortical neurons, although multiple antibodies have been tested for each. Interestingly, only a small reduction of IκBα levels could be observed in neurons (Figure 1). This is particularly apparent when compared to the nearly complete loss of IκBα we observed in astrocytes treated with TNFα (Figure 1).


Unique aspects of transcriptional regulation in neurons--nuances in NFkappaB and Sp1-related factors.

Mao XR, Moerman-Herzog AM, Chen Y, Barger SW - J Neuroinflammation (2009)

RelA nuclear translocation in neurons after glutamate treatment. Nearly pure neocortical neurons were treated with glutamate (50 μM) for the indicated times and cells were harvested for fractionation (Cyto.: cytosolic fraction; Nuc.: nuclear fraction); these were probed by western blot for RelA. Whole-cell lysates were also prepared from either astrocyte or neuron cultures; these were probed by western blot for IκBα (Whole: neuron whole cell lysate). TNFα was applied to the astrocytes at 100 ng/ml for the indicated times (min).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2693111&req=5

Figure 1: RelA nuclear translocation in neurons after glutamate treatment. Nearly pure neocortical neurons were treated with glutamate (50 μM) for the indicated times and cells were harvested for fractionation (Cyto.: cytosolic fraction; Nuc.: nuclear fraction); these were probed by western blot for RelA. Whole-cell lysates were also prepared from either astrocyte or neuron cultures; these were probed by western blot for IκBα (Whole: neuron whole cell lysate). TNFα was applied to the astrocytes at 100 ng/ml for the indicated times (min).
Mentions: Many of the original reports of NFκB activation in neurons were based on nuclear translocation of the transcription factor as an endpoint. We tested this parameter in our highly enriched cortical neuron cultures. Cells treated with glutamate and then lysed and separated into a cytosolic fraction (supernatant) and a nuclear fraction (whole nuclear pellet sonicated) for analysis by western blotting (note: this is different from the nuclear extract used in EMSA, which only includes the salt-extractable components). Glutamate was indeed found to evoke nuclear translocation of RelA under these conditions (Figure 1). This is consistent with previous findings we obtained with immunocytochemistry [20]. Translocation of RelA-containing NFκB complexes is typically dependent upon degradation of an IκB, so we assessed the levels of IκBα in whole-cell lysates obtained after an identical glutamate treatment. No significant levels of other IκB proteins can be detected in cultured cortical neurons, although multiple antibodies have been tested for each. Interestingly, only a small reduction of IκBα levels could be observed in neurons (Figure 1). This is particularly apparent when compared to the nearly complete loss of IκBα we observed in astrocytes treated with TNFα (Figure 1).

Bottom Line: These studies led us to conclude that this population of cells is nearly incapable of activating the NFkappaB that is nonetheless expressed at reasonable levels.A subset of the kappaB cis elements are instead bound by members of the Sp1 family in neurons.Also surprising was our discovery that Sp1 itself, typically described as ubiquitous, is severely restricted in expression within forebrain neurons; Sp4 seems to be substituted during neuronal differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, Washington University School of Medicine, St Louis, MO 63110, USA. maox@morpheus.wustl.edu

ABSTRACT
The unique physiology and function of neurons create differences in their cellular physiology, including their regulation of gene expression. We began several years ago exploring the relationships between the NFkappaB transcription factor, neuronal survival, and glutamate receptor activation in telencephalic neurons. These studies led us to conclude that this population of cells is nearly incapable of activating the NFkappaB that is nonetheless expressed at reasonable levels. A subset of the kappaB cis elements are instead bound by members of the Sp1 family in neurons. Also surprising was our discovery that Sp1 itself, typically described as ubiquitous, is severely restricted in expression within forebrain neurons; Sp4 seems to be substituted during neuronal differentiation. These findings and their implications for neuronal differentiation--as well as potential dedifferentiation during degenerative processes--are discussed here.

Show MeSH
Related in: MedlinePlus