Reduced expression of MECP2 affects cell commitment and maintenance in neurons by triggering senescence: new perspective for Rett syndrome

The neural differentiation process is studied in mesenchymal stem cells obtained from Rett patients and in neuroblastoma cells carrying a partially silenced MECP2 gene. The data suggest that neural cell fate and neuronal maintenance might be perturbed by senescence triggered by impaired MECP2 protein activity either before or after neural differentiation.


MAP2 NSE
Arbitrary Units ** C A -NeuN staining. The table shows the mean expression percentages of NeuN positive cells (± SD, n = 3). The symbol (*) indicates statistically differences (p<0.05) among mutated-MECP2 and wild type cells in RTT patient with respect to control MSCs. B -Expression analysis of neural differentiation markers by RT-PCR. The mRNA levels were normalized to HPRT as an internal control. The histogram shows the mean expression values (± SD, n = 3) of NSE and MAP2 in both proliferating and in differentiated MSCs. Statistical analysis was carried out comparing the same cell type among RTT patient with respect to control MSCs. The symbol (**) indicates highly statistically differences (p<0.01). The comparative cycle threshold (Ct) method was employed for quantitative analysis. C -Senescence assay in differentiated MSCs. The table shows the percentage of senescent cells for each cell type (± SD, n = 3). Statistical analysis was carried out comparing the same cell type among mutated-MECP2 and wild type cells in RTT patient with respect to control MSCs. The symbol (*) indicates statistically differences (p<0.05). The table shows the percentage of senescent and apoptotic cells for each cell type (± SD, n = 3). Statistical analysis was carried out comparing the same cell type among samples with silenced MECP2 and control. The symbol (*) indicates statistically differences (p<0.05).

SUPPLEMENTAL FILE 11 Detailed immunostaining procedures
BrdU and MECP2 immunodetection (see figure 1B) For BrdU immunostaining, the cells were grown on glass coverslips and incubated for 10 hours with 10 μM BrdU (Sigma Aldrich, MO, USA). Briefly, the cells were rinsed with PBS, fixed with 100% methanol at 4°C for 10 min, air-dried, and then incubated with 2 N HCl for 30 min at 37°C. HCl was neutralized with 0.1 M borate buffer (pH 8.5). Following additional PBS washes, the slides were incubated with an anti-BrdU rabbit polyclonal antibody (1:200) (Bioss Inc., MA, USA).
After 60 min at RT, the cells were washed with PBS and incubated with goat anti-rabbit secondary antibodies conjugated to peroxidase (Santacruz Biotech, CA, USA) for 60 min at RT. Finally, after additional washes in PBS, the slides were treated with DAB substrate (Roche, Germany). Following BrdU staining, cells were washed three-five times with PBS and were incubated over-night at 4°C Senescence-associated beta-galactosidase and MECP2 immunodetection (see figure 1D) The cells were fixed for 5 min with a solution of 2% formaldehyde and 0.2% glutaraldehyde. The cells were then washed with PBS and incubated at 37°C for at least 2 hours with a staining solution, according to the manufacturer's protocol (Roche, Italy). Following senescence staining, cells were washed three-five times with PBS and were incubated over-night at 4°C with anti-MECP2 (clone Mec-168) primary antibody (1:200) (Abcam, UK). Subsequently, cells were washed with PBS and then incubated with goat anti-mouse secondary antibodies, conjugated to peroxidase (Santacruz Biotech, CA, USA) and then treated with the DAB substrate (Vector Laboratories, CA, USA). The percentage of MECP2-positive and/or beta-galactosidase-positive cells was calculated by counting at least 500 cells in different microscopic fields. Beta-galactosidase-positive cells appeared blue and MECP2-positive cells were brown.
Neuronal Nuclei (NeuN) and MECP2 detection (see figure 2B) Cells were fixed for 15 min with 4% paraformaldehyde. NeuN was detected by immunocytochemistry using the anti-NeuN primary rabbit polyclonal antibody (Millipore Italy, Italy) according to the manufacturer's protocol. Cells were incubated with goat anti-rabbit secondary antibodies conjugated to FITC (Jackson Laboratories, MA, USA). Following NeuN staining, cells were washed with PBS and fixed again for 5 min with 4% paraformaldehyde. Cells then were incubated over-night at 4°C with anti-MECP2 (clone Mec-168) primary antibody (1:200) (Abcam, UK). Subsequently, cells were washed with PBS and then were incubated with goat anti-mouse secondary antibodies, conjugated to Texas Red (Jackson Laboratories, MA, USA), and the nuclei were counterstained with Hoechst 33342. In each experiment, at least 1,000 cells were counted in different fields to calculate the percentage of MECP2-positive and NeuN-positive cells in each culture. NeuN-positive cells appeared green and MECP2-positive cells were red. Nuclei were blue stained.
Annexin, MECP2 and NeuN immunodetection (see figure 2C) Apoptotic cells were detected using fluorescein-conjugated Annexin V (Roche, Italy) on unfixed cells according to the manufacturer's instructions. Following annexin staining, cells were washed three-five times with PBS and were fixed with 4% paraformaldehyde for 15 min. Cells then were incubated over-night at 4°C with anti-MECP2 (clone Mec-168) primary antibody (1:200) (Abcam, UK). Subsequently, cells were washed with PBS and then were incubated with goat anti-mouse secondary antibodies, conjugated to Texas Red (Jackson Laboratories, MA, USA). Following MECP2 staining, cells were washed with PBS and fixed again for 5 min with 4% paraformaldehyde. NeuN was detected by immunocytochemistry using the anti-NeuN primary rabbit polyclonal antibody (Millipore Italy, Italy) according to the manufacturer's protocol. Cells were incubated with goat anti-rabbit secondary antibodies conjugated to AMCA (Jackson Laboratories, MA, USA). At the end of immunodetection procedures, nuclei were counterstained with hematoxylin. In each experiment, at least 1,000 cells were counted in different fields to calculate the percentage of MECP2-positive, NeuN positive and annexin positive-cells. Annexin V positive cells appeared green, MECP2-positive cells were red and NeuN cells were blue stained. Nuclei appeared purple in light microscopy fields.
Senescence-associated beta-galactosidase, MECP2 and NeuN immunodetection (see figure 2D) The cells were fixed for 5 min with a solution of 2% formaldehyde and 0.2% glutaraldehyde. The cells were then washed with PBS and incubated at 37°C for at least 2 hours with a staining solution, according to the manufacturer's protocol (Roche, Italy). Following senescence staining, cells were washed three-five times with PBS and were incubated over-night at 4°C with anti-MECP2 (clone Mec-168) primary antibody (1:200) (Abcam, UK). Subsequently, cells were washed with PBS and then incubated with goat anti-mouse secondary antibodies, conjugated to peroxidase (Santacruz Biotech, CA, USA) and then treated with the DAB substrate (Vector Laboratories, CA, USA). Following MECP2 and beta-galactosidase staining, cells were washed with PBS and incubated with the anti-NeuN primary rabbit polyclonal antibody (Millipore Italy, Italy) according to the manufacturer's protocol. Then cells were incubated with goat anti-rabbit secondary antibodies, conjugated to peroxidase (Santacruz Biotech, CA, USA) and treated with the ImmPACT VIP substrate (Vector Laboratories, CA, USA). USA).
In each experiment, at least 1,000 cells were counted in different fields to calculate the percentage of MECP2-positive, NeuN positive and beta-galactosidase positive-cells. Beta-galactosidase-positive cells appeared blue, MECP2-positive cells were brown and NeuN cells were purple. MECP2 and NeuN positive cells were blackish.
Annexin and NeuN immunodetection (see figure 3E and 4D) Apoptotic cells were detected using fluorescein-conjugated Annexin V (Roche, Italy) on unfixed cells according to the manufacturer's instructions. Following annexin staining, cells were washed three-five times with PBS and were fixed with 4% paraformaldehyde for 15 min.
NeuN was detected by immunocytochemistry using the anti-NeuN primary rabbit polyclonal antibody (Millipore Italy, Italy) according to the manufacturer's protocol. Cells were incubated with goat anti-rabbit secondary antibodies conjugated to Texas red (Jackson Laboratories, MA, USA). At the end of immunodetection procedures, nuclei were counterstained with Hoechst 33342. In each experiment, at least 1,000 cells were counted in different fields to calculate the percentage of NeuN positive and annexin positive-cells. Annexin V positive cells appeared green, NeuN-positive cells were red. Nuclei were blue stained. Senescence-associated beta-galactosidase and NeuN immunodetection (see figure 3D and 4C) The cells were fixed for 5 min with a solution of 2% formaldehyde and 0.2% glutaraldehyde. The cells were then washed with PBS and incubated at 37°C for at least 2 hours with a staining solution, according to the manufacturer's protocol (Roche, Italy). Following senescence staining, cells were washed three-five times with PBS and were incubated over-night at 4°C the anti-NeuN primary antibody (Millipore Italy, Italy) according to the manufacturer's protocol. Subsequently, cells were washed with PBS and then incubated with goat anti-rabbit secondary antibodies, conjugated to peroxidase (Santacruz Biotech, CA, USA) and then treated with the DAB substrate (Vector Laboratories, CA, USA). The percentage of NeuN-positive and/or beta-galactosidase-positive cells was calculated by counting at least 500 cells in different microscopic fields. Beta-galactosidase-positive cells appeared blue and NeuN-positive cells were brown.