The moonlighting protein c-Fos activates lipid synthesis in neurons, an activity that is critical for cellular differentiation and cortical development

Abstract

Differentiation of neuronal cells is crucial for the development and function of the nervous system. This process involves high rates of membrane expansion, during which the synthesis of membrane lipids must be tightly regulated. In this work, using a variety of molecular and biochemical assays and approaches, including immunofluorescence microscopy and FRET analyses, we demonstrate that the proto-oncogene c-Fos (c-Fos) activates cytoplasmic lipid synthesis in the central nervous system and thereby supports neuronal differentiation. Specifically, in hippocampal primary cultures, blocking c-Fos expression or its activity impairs neuronal differentiation. When examining its subcellular localization, we found that c-Fos co-localizes with endoplasmic reticulum markers and strongly interacts with lipid-synthesizing enzymes, whose activities were markedly increased in vitro in the presence of recombinant c-Fos. Of note, the expression of c-Fos dominant-negative variants capable of blocking its lipid synthesis-activating activity impaired neuronal differentiation. Moreover, using an in utero electroporation model, we observed that neurons with blocked c-Fos expression or lacking its AP-1-independent activity fail to initiate cortical development. These results highlight the importance of c-Fos-mediated activation of lipid synthesis for proper nervous system development.

Keywords: AP-1; AP1 transcription factor (AP-1); c-Fos; central nervous system; central nervous system (CNS); hippocampus; lipid synthesis; neurodevelopment; neurodifferentiation; oncogene; oncogene.; phospholipid metabolism.

Figure 1.

c-Fos is expressed in primary rat hippocampal neuronal cultures. A, rat primary hippocampal neurons were fixed at different times of culture and immunostained with an anti-c-Fos antibody (red). Scale bar, 20 μm. B, Western blotting of hippocampal cell lysates harvested at different times of culture. Membranes were immunostained with an anti c-Fos antibody (top) and an anti-tubulin (Tub) antibody (bottom) as a loading control and secondary antibodies suitable for near-IR fluorescence. The obtained images were converted to gray scale and inverted. The results of one of three independent experiments are shown.

Figure 2.

Blocking the activity or the expression of c-Fos impairs neuronal differentiation. A, cells were profected with anti c-Fos (c-Fos Ab) or with a nonrelated anti-mouse IgG antibody as a control (Control Ab) 2 h after seeding using BioPORTER and then were fixed after 48 h of culture. The different morphological aspects were quantified from microscopy images, using ImageJ software, and are shown as the mean ± S.D. (error bars) in each case. Student's t test statistical analysis was performed using GraphPad Software. ***, p < 0.001; n.s., nonsignificant; in each experiment, n = 40 cells from each condition were examined. Results of one of three independent experiments performed are shown. B, cells were infected at the initiation of the culture with lentiviral particles designed to express an anti c-Fos shRNA or an shRNA with a scrambled sequence of c-Fos as a control. After 48 h of culture, cells were fixed and immunostained with an anti-c-Fos antibody (red, first column) and an anti βIII-tubulin antibody (green, second column). The third column shows the merge between both labels. Scale bar, 20 μm. C, morphological quantification of neuronal differentiation stages in both c-Fos and scrambled infected cells was performed using ImageJ software. The graph shows the mean number of cells ± S.D. in each case. Student's t test statistical analysis was performed using GraphPad software. *, p < 0.05; ***, p < 0.001; n.s., nonsignificant; n = 198 cells for scrambled shRNA–infected cells, n = 254 cells for c-Fos shRNA-infected cells. Results of one of three independent experiments performed are shown.

Figure 3.

c-Fos co-localizes with ER markers. Immunocytochemistry of c-Fos (red), the ER marker calnexin (green), and βIII-tubulin (cyan) of rat hippocampal neurons at 48 h of culture. The fourth panel shows the merged images between the three labels. A Pearson's r value of 0.81 was calculated for co-localization of both labels (Coloc2, ImageJ). An inset is shown with a co-localization analysis between c-Fos and the ER, where the co-localizing pixels are colored in white (Colocalization Finder, ImageJ). Scale bar, 20 μm. The results of one of three independent experiments are shown.

Figure 4.

c-Fos activates and physically interacts with CDS. A, top row, neurons were co-transfected at 24 h of culture to express c-Fos-CFP (first panel) and CDS-YFP (second panel) and examined by confocal microscopy at 48 h. FRET images were obtained by the sensitized emission method and pseudocolored using ImageJ software (third panel). The bottom row shows a negative control with cells co-transfected with the CFP empty vector and CDS-YFP. The scale goes from no FRET (black) to maximum FRET (yellow). Scale bar, 30 μm. B, the graphic shows the quantification of the mean efficiencies ± S.D. (error bars) for the donor/acceptor pairs shown in the images, with one-way ANOVA and Tukey's post-test. ***, p < 0.001; n = 25 cells for each condition. The results of one of three independent experiments are shown. C, evaluation of CDS activity through measurement of the incorporation of [³H]CTP into CDP-DAG in neuron homogenates in the presence of c-Fos (+ c-Fos). Elution buffer was used as a control (– c-Fos). Results are the mean of three independent experiments performed in triplicate. The results are expressed as the mean ± S.D., with Student's t test analysis. *, p < 0.05.

Figure 5.

NA deletion mutant of c-Fos impairs differentiation in neuronal cultures and abrogates c-Fos-dependent lipid synthesis activation. A, neuronal cultures were transfected at seeding with NA-YFP (pseudocolored red, bottom row), NB-YFP (pseudocolored red, middle row), or the empty vector as a control (pseudocolored red, top row) and were fixed after 48 h of culture. Cells were subjected to immunofluorescence against βIII-tubulin (green, second column). Scale bar, 20 μm. B, morphological quantification of neuronal differentiation stages in both NA-YFP– and NB-YFP–transfected cells at different fixation times. The results of one of three independent experiments are shown. A normality Kolmogorov–Smirnov test was performed, where the deviation from the distribution with respect to the NB-transfected cells was evaluated. *, p < 0.05; ***, p < 0.001; n.s., nonsignificant; n = 15 from each condition were examined. C, evaluation of ³²P-phospholipid labeling capacity of neuron homogenates in the presence of c-Fos (+c-Fos), NA (+NA), or both (+c-Fos +NA 1:1 and +c-Fos +NA 1:3). Elution buffer was used as a control (Control). Results are the mean of three independent experiments performed in triplicate. The results are expressed as the mean ± S.D. (error bars), with one-way ANOVA. ***, p < 0.001 with respect to control conditions.

Figure 6.

Expression of c-Fos is involved in cortical development. A, embryo brains were electroporated with a specifically designed shRNA to block c-Fos expression (sh c-Fos) (right) or a control scrambled shRNA (left) at E15 and analyzed at E19. IUE, in utero electroporation; MZ, marginal zone. Scale bar, 100 μm. B, quantification of the distribution of dsRed-positive cells in the CP, IZ, and VZ/SVZ. The mean ± S.D. of the quantification of at least 20 coronal cryosections from three independent experiments performed independently is shown, with two-way ANOVA with Bonferroni post-test. ***, p < 0.001; n.s., nonsignificant. C, embryo brains were electroporated with a vector designed to express the NA domain of c-Fos fused to CFP or the empty vector as a control at E15 and analyzed at E19. D, quantification of the distribution of dsRed-positive cells in the CP, IZ, and VZ/SVZ. The mean ± S.D. of the quantification of at least 20 coronal cryosections from three independent experiments performed independently is shown, with two-way ANOVA with Bonferroni post-test. ***, p < 0.001; **, p< 0.001.