Expression profiling upon Nex1/MATH-2-mediated neuritogenesis in PC12 cells and its implication in regeneration

Abstract

The expression of Nex1 peaks during brain development when neurite outgrowth and synaptogenesis are highly active. We previously showed that Nex1 is a critical effector of the nerve growth factor (NGF) pathway and its overexpression results in spontaneous neuritogenesis. Furthermore, the PC12-Nex1 cells exhibit accelerated neurite extension upon NGF exposure, and have the capacity to regenerate neurites in the absence of NGF. In this study, we identify the repertoire of genes targeted by Nex1 to unravel the molecular mechanisms by which Nex1 promotes differentiation and regeneration. Our transcriptional analysis reveals that Nex1 modulates a wide spectrum of genes with diverse functions, many of them being key downstream regulators of the NGF pathway, and critical to neuritogenesis, such as microtubules, microtubule-associated proteins (MAPs) and intermediate filaments. We also provide the first evidence that a basic helix-loop-helix (bHLH) protein stimulates the expression of the cyclin-dependent kinase (CDK) inhibitors belonging to the INK4 family, which plays a role in promoting cell-cycle arrest. Finally, we show a dramatic synergistic effect between Nex1 and cAMP, resulting in an impressive regeneration of an elaborate and dense neurite network. Thus, Nex1 has endowed the PC12-Nex1 cells with a distinct combination of gene products that takes part in the complex regulation of neuritogenesis and regeneration.

Fig. 1

Overexpression of Nex1 results in increased expression of specific intermediate filaments in the absence of NGF. The control PC12 and PC12-Nex1 cells were grown on collagen I-coated plates, and cell extracts (40 μg) were subjected to immunoblot analysis with appropriate antibodies as indicated at the bottom of each panel. The antigen-antibody complexes were detected by chemiluminescence. Equal loading was verified by visualization of total proteins by Ponceau S staining and by blotting with the anti-peripherin antibody. The immune-specific polypeptides are indicated by an arrow with the corresponding molecular weight (in kDa) next to it, whereas the non-specific immune reactive polypeptides are indicated by an asterisk. Relevant molecular weight markers are indicated on the left side of each figure. Data shown are representative of at least three independent experiments.

Fig. 2

Overexpression of Nex1 induces the expression of microtubules and microtubule-associated proteins independently of NGF. The control PC12 and PC12-Nex1 cells were grown on collagen I-coated plates, and cell extracts (40 μg) were subjected to immunoblot analysis with appropriate antibodies as indicated at the bottom of each panel. The antigen-antibody complexes were detected by chemiluminescence. The immune-specific polypeptides are indicated by an arrow with the corresponding molecular weight (in kDa) next to it, or a bracket for the five bands of Tau spanning 52-60 kDa, whereas the non-specific immune reactive polypeptides are indicated by an asterisk. Relevant molecular weight markers are indicated on the left side of each figure. Data shown are representative of at least three independent experiments.

Fig. 3

Overexpression of Nex1 induces the expression of several inhibitors of G1 cyclin-dependent kinases. (a) Nex1 only induces the expression of the member of the CIP/KIP family, p^21CIP1. The control PC12 and PC12-Nex1 cells were grown on collagen I-coated plates, and cell extracts (40 μg) were subjected to immunoblot analysis with appropriate antibodies as indicated at the bottom of each panel. The antigen-antibody complexes were detected by chemiluminescence. Equal loading was verified by visualization of total proteins by Ponceau S staining and by blotting with the anti-p27^Kip1 antibody. The immune-specific polypeptides are indicated by an arrow with the corresponding molecular weight (in kDa) next to it, whereas the non-specific immune reactive polypeptides are indicated by an asterisk. Relevant molecular weight markers are indicated on the left side of each figure. Data shown are representative of at least three independent experiments. (b) Nex1 stimulates the expression of the members of the INK4 family. The control PC12 and PC12-Nex1 cells were treated and analyzed as described in (a). Equal loading was verified by visualization of total proteins by Ponceau S staining and by blotting with the anti-p27^Kip1 antibody. The immune-specific polypeptides are indicated by an arrow with the corresponding molecular weight (in kDa) next to it, whereas the non-specific immune reactive polypeptides are indicated by an asterisk. Relevant molecular weight markers are indicated on the left side of each figure. Data shown are representative of at least three independent experiments.

Fig. 4

Constitutive expression of Nex1 accelerates cAMP-mediated neuritogenesis. (a) Phase-contrast micrographs of control PC12 and PC12-Nex1 cells treated with dbcAMP (1 mM). The cells were grown on collagen I-coated plates and exposed to dbcAMP for different periods of time, as indicated at the side of each panel. The phase-contrast micrograph of PC12-Nex1 cells at To is shown as an inset figure in the panel. The phase-contrast micrographs are representative of at least three independent experiments. (b) Quantification of neurite-bearing cells in the presence of dbcAMP (1 mM). The percentage of neurite-bearing cells was scored on at least 300 cells for each experiment, and three independent experiments were carried out. The open bars represent the control PC12 cells treated with dbcAMP (1 mM), whereas the hatched bars represent the PC12-Nex1 cells treated with dbcAMP (1 mM). This is a representative graph from a triplicate experiment that was repeated three times. The error bar is the standard deviation of the mean.

Fig. 5

Constitutive expression of Nex1 in conjunction with dbcAMP treatment results in dramatic neurite regeneration. (a) Phase-contrast micrographs of NGF-treated control PC12 and PC12-Nex1 cells in the presence or absence of dbcAMP. Control PC12 and PC12-Nex1 cells were grown on collagen I-coated plates and treated with NGF (50 ng/mL) for 7 days, after which the cells were thoroughly washed with NGF-free medium. Neurites were then sheared as described in Materials and methods. The cells were re-plated on collagen I-coated plates in the absence or presence of dbcAMP (1 mM). The regeneration process was analyzed for 48 h. The phase-contrast micrographs are representative of at least three independent experiments. (b) Quantification of neurite regeneration in the absence or presence of dbcAMP. Cells were treated and processed as described above. The percentage of neurite-bearing cells was scored on at least 300 cells from three distinct experiments. This is a representative graph from a triplicate experiment that was repeated three times. The error bar is the standard deviation of the mean. The open bars and the hatched bars represent neurite regeneration from NGF-differentiated control PC12 cells in the absence or presence of dbcAMP (1 mM), respectively. The dotted bars and the shaded bars represent neurite regeneration from NGF-treated PC12-Nex1 cells in the absence or presence of dbcAMP (1 mM), respectively.