Unique aspects of transcriptional regulation in neurons--nuances in NFkappaB and Sp1-related factors - PubMed (original) (raw)

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

Xianrong R Mao et al. J Neuroinflammation. 2009.

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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Figures

Figure 1

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).

Figure 2

Figure 2

Glutamate is unable to activate neuronal NFκB in mixed cell cultures. Neocortical neurons from κB/β-gal transgenic mice were either plated on the top of wild-type astrocytes ("+glia") or retained as nearly pure neuronal cultures ("-glia"). A. Glutamate was applied at 50 μM for 10 min, then chased for the times indicated (h). B. Glutamate was applied at 20 μM continuously for the times indicated (h). β-gal activity was determined by a luminescent assay. Values represent activity relative to untreated cultures ± SEM in quadruplicate cultures. ANOVA followed by Scheffe post-hoc test showed significance between control and each treatment, and between the "+glia" and "-glia" in all comparisons except the 6 h timepoint in A. (p < 0.02).

Figure 3

Figure 3

Differential levels of mRNA for key components of the NFκB pathway in neurons versus glia. RNA was harvested from rat primary neocortical neurons and astrocytes. Semiquantitive RT-PCR was performed on equal amounts of total mRNA to survey expression of genes in the NFκB pathway, housekeeping genes, and Sp-family factors [see Additional file 1]. Each lane represents one of four individual cultures.

Figure 4

Figure 4

NFκB activity in neurons cannot be evoked by supplementing key components of its pathway. IKK1, IKK2, IKK3, NIK, β-TrCP1, and TRAF2 were transfected either alone or in combination as indicated to test NFκB activity in neocortical neurons by reporter assays with a κB-driven firefly luciferase construct. Some transfection conditions were also further treated with rat TNFα (1–100 ng/ml). Rat RelA transfection served as a positive control. After 24 h, firefly luciferase activities were determined relative to Renilla luciferase reference. Values reflect the mean ± SEM of triplicate cultures. Except RelA transfection, no condition created a significant change compared to control.

Figure 5

Figure 5

Upstream components of NFκB pathway cannot boost exogenous RelA activity in neurons. Primary neocortical neurons were transfected with a κB-driven firefly luciferase reporter, along with RelA and other key factors in the NFκB pathway. (The dose of RelA plasmid was selected to be intermediate with respect to the maximal level of the assay's dynamic range.) In some conditions, cells were treated with rat TNFα (10 ng/ml) after transfection. Relative luciferase activity from RelA (100 ng) transfection was 111.05 ± 8.9 (mean ± SEM) (not shown). All plasmids were 100 ng/well, except those labeled otherwise. After 24 h, firefly luciferase activities were determined relative to Renilla luciferase reference. Values reflect the mean ± SEM of triplicate cultures. *p < 0.05 (t-test; other significant differences are not labeled).

Figure 6

Figure 6

ApoE declines with Sp1 during neuronal differentiation but is induced by glutamate. A. NTera2 cells were harvested undifferentiated (U) or after incubation as neurospheres in retinoic acid for 14 days (RA). Subsequent to RA treatment, additional cultures underwent selection by incubation with a cocktail of mitotic inhibitors (MI) for 7 days. Equal amounts of protein were resolved by SDS-PAGE, and the levels of ApoE, Sp1, and Sp4 were assessed by western blot analysis. B. Differentiated NTera2 cells were untreated or treated with 20 μM glutamate for 20 h, then levels of ApoE and actin were assessed by western blot.

Figure 7

Figure 7

Induction of neuronal expression of cyclin D1 in βAPP-deficient mice. Brain sections from wild-type ("WT") and βAPP-knockout ("APP-KO") mice were subjected to immunofluorescence detection of cyclin D1 (green). The sections were counterstained with DAPI to visualize nuclei (blue). Representative images are shown. The pixel intensity of specific staining was randomly sampled in the CA1 and dentate cell body layer. The mean values and standard errors are shown in graphically. Statistical comparison between the two genotypes by t-test indicated p < 0.05 (n = 4) in each hippocampal region.

Figure 8

Figure 8

Schematic for the roles of Sp1 and Sp4 in neuronal differentiation and degeneration. Immature neuroblasts appear to similar levels of Sp1 and Sp4, perhaps competing for regulatory control of genes not strongly related to differentiated neuronal function. After differentiation, mature neurons have much lower levels of Sp1; Sp4 competitively inhibits DNA binding and exerts transcriptional repression on genes related to mitosis and other genes inappropriate for neurons, such as ApoE. Upon excessive stimulation of glutamate receptors, a large calcium influx activates calpain, which digests Sp4. This may relieve the transcriptional repression of mitotic genes, permitting their inappropriate expression in terminally differentiated neurons; the latter appears sufficient to trigger apoptosis.

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