Lactoferrin Modulates Induction of Transcription Factor c-Fos in Neuronal Cultures

Abstract

Lactoferrin (Lf) is a multifunctional protein from the transferrin family. Of particular interest is the ability of Lf to affect a wide range of neuronal processes by modulating the expression of genes involved in long-term neuroplasticity. The expression of the immediate early gene c-fos that is rapidly activated in response to external influences, and its product, transcription factor c-Fos, is widely used as a marker of long-term neuronal plasticity. The present study aims to examine the effect of human Lf on the induction of transcription factor c-Fos in the primary mouse neuronal cultures after stimulation and to determine the cellular localization of human Lf and its colocalization with induced c-Fos protein. Primary dissociated cultures of hippocampal cells were obtained from the brains of newborn C57BL/6 mice (P0-P1). On day 7 of culturing, human Lf was added to the medium. After 24 h (day 8 in culture), c-Fos protein was induced in cells by triple application of 50 mM KCl. c-Fos content was analyzed using the immunofluorescent method 2 h after stimulation. Stimulation promoted exogenous Lf translocation into the nuclei of cultured neuronal cells, which correlated with increased induction of transcription factor c-Fos and was accompanied by nuclear colocalization of these proteins. These results attest to the potential of Lf as a modulator of neuronal processes and open up new prospects in studying the mechanisms of the regulatory effects of lactoferrin on cell function.

Keywords: cytoplasmic and nuclear localization; human lactoferrin; neuronal cultures; stimulation; transcription factor c-Fos.

Figure 1

Immunofluorescent detection of NeuN, a marker of mature neurons (dark blue) in hippocampal cell cultures on day 7 of culturing. Primary dissociated cultures of hippocampal cells were obtained from the brains of newborn C57BL/6 mice (P0-P1). Nuclei were poststained with Hoechst (blue). Representative micrographs of cultures. Scale bar = 10 μm.

Figure 2

The dynamics of Lf penetration into the cytoplasm and nucleus of cells in the primary culture of the hippocampus. Primary dissociated cultures of hippocampal cells were obtained from the brains of newborn C57BL/6 mice (P0-P1). On day 8 of culturing, human Lf was added to the medium to make a final concentration of 1 mg/mL for 2, 24, or 72 h. Immunofluorescent staining was performed using monoclonal antibodies to human Lf. No hLf-positive cells were detected before Lf addition. (a). On day 8, culturing stimulation was carried out by adding 50 mM KCl (3 × 2 min with 5 min intervals) to the culture medium with complete medium replacement between the stimulations. Human Lf (to a final concentration of 1 mg/mL) was preliminary added for 24 h. Immunocytochemical analysis was performed 2 h after stimulation (b). n = 4–7 for each group. The data are presented as the mean ± SEM. * p < 0.05; ** p < 0.01; and **** p < 0.0001.

Figure 3

TUNEL staining (red) of hippocampal cell cultures 2 and 24 h after stimulation with 50 mM KCl. Representative micrographs of cultures in groups PC (passive control) and KCl. Nuclei were poststained with Hoechst (blue). Primary dissociated cultures of hippocampal cells were obtained from the brains of newborn C57BL/6 mice (P0-P1). On day 8 of culturing, stimulation was carried out by adding 50 mM KCl (3 × 2 min with 5 min intervals) to the culture medium with complete medium replacement between the stimulations. The PC groups were fixed simultaneously with other cultures without additional manipulations. Scale bars = 30 μm (a). Quantitative analysis of TUNEL-positive cells. n = 4 for each group The data are presented as the mean ± SEM (b).

Figure 4

Immunofluorescent detection of c-Fos protein (green) and Lf (red) in hippocampal cell cultures on day 8 of culturing 2 h after stimulation. Nuclei were poststained with Hoechst (blue). Representative micrographs of cultures in group KCl+Lf. Primary dissociated cultures of hippocampal cells were obtained from the brains of newborn C57BL/6 mice (P0-P1). On day 8 of culturing, stimulation was carried out by adding 50 mM KCl (3 × 2 min with 5 min intervals) to the culture medium with complete medium replacement between the stimulations. Human Lf (to a final concentration of 1 mg/mL) was preliminary added for 24 h. Immunofluorescent staining was performed using monoclonal antibodies to hLf. Presence of hLf in the cytoplasm of all c-Fos-positive cells in the KCl+Lf group 2 h after stimulation (a). Colocalization of hLf inclusion and c-Fos protein was observed in some nuclei (b). Scale bars = 10 μm.

Figure 5

Immunofluorescent detection of c-Fos protein (green) and NeuN (dark blue) in hippocampal cell cultures on day 8 of culturing 2 h after stimulation. Nuclei were poststained with Hoechst (blue). Representative micrographs of cultures in group KCl+Lf. The arrows indicate NeuN-positive/c-Fos-negative cells. Primary dissociated cultures of hippocampal cells were obtained from the brains of newborn C57BL/6 mice (P0-P1). On day 8 of culturing, stimulation was carried out by adding 50 mM KCl (3 × 2 min with 5 min intervals) to the culture medium with complete medium replacement between the stimulations. Human Lf (to a final concentration of 1 mg/mL) was preliminary added for 24 h. Scale bar = 20 μm.

Figure 6

Immunofluorescent detection of c-Fos protein (green) in hippocampal cell cultures on day 8 of culturing 2 h after stimulation. Nuclei were poststained with Hoechst (blue). Representative micrographs of cultures in groups PC+Lf, AC, KCl, and KCl+Lf. Primary dissociated cultures of hippocampal cells were obtained from the brains of newborn C57BL/6 mice (P0-P1). On day 8, culturing stimulation was carried out by adding 50 mM KCl (3 × 2 min with 5 min intervals) to the culture medium with complete medium replacement between the stimulations. Human Lf was added to the culture medium of KCl+Lf, KCl+Lfconst, AC+Lf, and PC+Lf groups to a final concentration of 1 mg/mL 24 h before stimulation, medium changing, or fixation. In the KCl+Lfconst group, the concentration of hLf during medium changes between the stimulations was maintained constant until the fixation of the cultures. Cultures with 3-fold medium replacement were used as active controls. The passive control group was fixed simultaneously with other cultures without additional manipulations. Scale bars = 20 μm (a). Quantitative analysis of c-Fos protein induction in hippocampal cell cultures on day 8 of culturing 2 h after stimulation with 50 mM KCl. ** p < 0.0001—differences from the corresponding passive control groups; && p < 0.0001—differences from the corresponding active control groups; ## p < 0.0001—differences from the KCl group. n = 5–6 for each group. Preliminary administration of hLf increases the number of cells with induced c-Fos protein (b). The presence of hLf in the culture medium during and after stimulation does not significantly affect the number of c-Fos-positive cells. ## p < 0.0001—differences from the KCl group. n = 5–6 for each group (c). The data are presented as the mean ± SEM. PC—passive control, AC—active control.

Figure 6

Immunofluorescent detection of c-Fos protein (green) in hippocampal cell cultures on day 8 of culturing 2 h after stimulation. Nuclei were poststained with Hoechst (blue). Representative micrographs of cultures in groups PC+Lf, AC, KCl, and KCl+Lf. Primary dissociated cultures of hippocampal cells were obtained from the brains of newborn C57BL/6 mice (P0-P1). On day 8, culturing stimulation was carried out by adding 50 mM KCl (3 × 2 min with 5 min intervals) to the culture medium with complete medium replacement between the stimulations. Human Lf was added to the culture medium of KCl+Lf, KCl+Lfconst, AC+Lf, and PC+Lf groups to a final concentration of 1 mg/mL 24 h before stimulation, medium changing, or fixation. In the KCl+Lfconst group, the concentration of hLf during medium changes between the stimulations was maintained constant until the fixation of the cultures. Cultures with 3-fold medium replacement were used as active controls. The passive control group was fixed simultaneously with other cultures without additional manipulations. Scale bars = 20 μm (a). Quantitative analysis of c-Fos protein induction in hippocampal cell cultures on day 8 of culturing 2 h after stimulation with 50 mM KCl. ** p < 0.0001—differences from the corresponding passive control groups; && p < 0.0001—differences from the corresponding active control groups; ## p < 0.0001—differences from the KCl group. n = 5–6 for each group. Preliminary administration of hLf increases the number of cells with induced c-Fos protein (b). The presence of hLf in the culture medium during and after stimulation does not significantly affect the number of c-Fos-positive cells. ## p < 0.0001—differences from the KCl group. n = 5–6 for each group (c). The data are presented as the mean ± SEM. PC—passive control, AC—active control.

Figure 7

Experimental timeline. Primary dissociated cultures of hippocampal cells were obtained from the brains of newborn C57BL/6 mice (P0-P1). On day 7 of culturing, human Lf was added to the medium. After 24 h (day 8 in culture), c-Fos protein was induced in cells by triple application of 50 mM KCl. Cultures with 3-fold medium replacement were used as an active control (AC). The passive control group (PC) was fixed simultaneously with other cultures without additional manipulations. Immunocytochemical analysis (ICC) was performed 2 h after stimulation. DIV—day in vitro. PC—passive control, AC—active control.

Figure 8

Experimental timeline for the analysis of the dynamics of human Lf penetration into the cytoplasm and nucleus of cells in the primary culture of the hippocampus. Primary dissociated cultures of hippocampal cells were obtained from the brains of newborn C57BL/6 mice (P0-P1). On day 8 of culturing, hLf was added to the medium. After 2, 24, and 72 h incubation, the cultures were washed and fixed, and immunocytochemical analysis (ICC) was performed using monoclonal antibodies to hLf.