Impact of protein kinase CK2 inhibitors on proliferation and differentiation of neural stem cells

Abstract

Background: Protein kinases play central roles in cell and tissue development. Protein kinase CK2, an ubiquitously expressed serine/threonine kinase has severe impacts on embryo- and spermatogenesis. Since its role in neurogenesis has so far only been investigated in very few studies, we analysed the role of CK2 in neural stem cells by using two specific inhibitors.

Methods: Neural stem cells were isolated from the subventricular zone of neonatal mice, using a neurosphere approach. Proliferation of the neurospheres, as well as their differentiation was investigated with and without inhibition of CK2. Changes in proliferation were assessed by counting the number and measuring the diameter of the neurospheres. Furthermore, the absolute cell numbers within the neurospheres were estimated. Differentiation was induced by retinoic acid in single cells after dissociation of the neurospheres. CK2 was inhibited at consecutive time points after induction of the differentiation process.

Results: CK2 inhibition reduced the amount and size of proliferating neurospheres dose dependently. Adding the CK2 inhibitor CX-4945 at the start of differentiation we observed a dose-dependent effect of CX-4945 on cell viability and glia cell differentiation. Adding quinalizarin, a second CK2 inhibitor, at the start of differentiation led to an elevated level of apoptosis, which was accompanied by a reduced neural differentiation. Adding the CK2 inhibitors at 72 h after the start of differentiation had no effect on stem cell differentiation. Conclusion: Inhibition of CK2 influences early gliogenesis in a time point and concentration dependent manner.

General significance: The use of a CK2 inhibitor significantly affects the neural stem cell niche.

Keywords: Biochemistry; Biological sciences; Cell biology; Developmental biology; Health sciences; Neuroscience.

Fig. 1

Neurosphere growth in dependence of CK2-inhibition. Size of SVZ neurospheres after 72 h treatment with DMSO (dimethylsulfoxide) as a control (A) and increasing concentrations of the CK2-inhibitor CX-4945: 10 μM (B), 20 μM (C) and 25 μM (D). Tables E and F display two-dimensional diameter of the individual spheres of one representative experiment in μm (E) and the calculated number of cells within a sphere (F), thus underlining the large differences between the untreated, DMSO treated and CK2-inhibited neurospheres. The graph in G shows normalised neurosphere diameters after 24, 48 and 72 h averaged over all experiments. The diameters were normalised against DMSO (control). For the analysis of a statistical significance a non-parametric Kruskal-Wallis test was applied. Lower limit of measured neurosphere sizes was ≤20 μm. Scale bar 500 μm.

Fig. 2

Assessment of CK2 kinase activity. CK2 inhibitors efficiently inhibit CK2 kinase activity in SVZ cells without altering expression of CK2 subunits. (A) Endogenous CK2 kinase activity in undissociated SVZ cells was determined 24 h after treatment with DMSO, 10 μM or 20 μM CX-4945 or 40 μM quinalizarin. Untreated cells were used as a control. Relative CK2 kinase activities measured in DMSO-treated cells were set to 100%. The mean of at least two independent experiments is shown. (B) SVZ cells were kept in normal medium (untreated) or treated with DMSO (solvent control), 10 μM or 20 μM CX-4945 or 40 μM quinalizarin for 24 h. Cell lysates were prepared and analysed on a 12.5% SDS-polyacrylamide gel followed by Western Blotting using anti-CK2α, anti-CK2α’ and anti-CK2β specific antibodies. GAPDH or actin was used as a loading control. One representative of at least 2 Western Blots is shown here. Full, uncropped versions of the Western Blots are available as supplementary material.

Fig. 3

FACS analysis of DMSO-control and inhibitor treated cells. Proliferating SVZ cells were seeded at an equal density and treated with DMSO (control), 10 μM or 20 μM CX-4945 in proliferation medium for 24 h. Then cells were harvested, carefully dissociated, fixed with ethanol and stained for 15–30 min using the FxCycle PI/RNase staining kit (Life Technologies). 10.000 cells per treatment were counted in a flow cytometer. Percentages of individual cell cycle phases were calculated according to the total of all phases.

Fig. 4

Comparison of stem cell and differentiation markers, as well as expression and activity of CK2 in undifferentiated and differentiated neurospheres. (A-D) Analysis of GFAP-, βIII-tubulin- and nestin expression in undifferentiated and 5 days differentiated neurospheres. Neurospheres were seeded on matrix-coated coverslips and either fixated directly after sedimentation (undifferentiated) or differentiated by adding retinoic acid to the medium (differentiated, day 5). Neurospheres were stained for GFAP (red) and βIII-tubulin (green) (A, B) or for total nestin (green) and phosphoThr316-nestin (red) (C, D). Exposure time was adjusted to the strongest signal and was identical for undifferentiated and differentiated samples. Scale bar 50 μm. (E) Neurospheres were harvested during proliferation (undifferentiated) or after 1, 3, 5, 14 or 21 days of differentiation with retinoic acid. Cell lysates were analysed for GFAP, βIII-tubulin, PGP9.5 and nestin expression (E) and for the expession of the CK2 subunits (F) by Western Blot. Actin was used as loading control. Cell lysates were also analysed for CK2 activity (G).Full, uncropped versions of the Western Blots are available as supplementary material.

Fig. 5

Influence of CK2 inhibition on neural and glial differentiation. GFAP- and βIII-tubulin staining of differentiated neurospheres at day 5 in vitro. SVZ cells were seeded on matrix-coated coverslips and differentiation was induced by adding retinoic acid (RA) to the medium. 10 μM or 20 μM CX-4945 or 40 μM quinalizarin was added to the RA-containing medium at the indicated time points after start of differentiation. DMSO served as solvent control. (A) GFAP+ and βIII-tubulin+ cells compared to the total cell number (DAPI). (B) to (I) demonstrate the impact of CK2 inhibition upon glial (red GFAP staining) and neural (green βIII-tubulin staining) differentiation: (B) RA alone, (C) RA plus DMSO, (D) RA plus 10 μM CX-4945: 0 h, (E) RA plus 10 μM CX-4945: 72 h, (F) RA plus 20 μM CX-4945: 0 h, (G) RA plus 20 μM CX-4945: 72 h. (H) RA plus 40 μM quinalizarin: 0 h, (I) RA plus 40 μM quinalizarin: 72 h. Scale bar 50 μm.

Fig. 6

Dose and time dependent inhibition of CK2 and its influence on glial and neurogenic differentiation. (A) Endogenous CK2 kinase activity in SVZ cells was determined at day 5 of differentiation after treatment with retinoic acid (RA control), RA plus DMSO or RA plus 10 μM or 20 μM of CK2 inhibitor CX-4945. CX-4945 was added either directly with the differentiation mix (0 h) or 72 h after start of differentiation (72 h). Relative CK2 kinase activities measured in DMSO-treated cells were set to 100%. The mean of at least two independent experiments is shown. (B) SVZ cells were differentiated for 5 days with retinoic acid (RA) alone or with RA plus DMSO or 10 μM or 20 μM CX- 4945 as indicated. Cell lysates were prepared and analysed on a 12.5% SDS-polyacrylamide gel followed by Western Blotting using anti-GFAP and anti-βIII-tubulin specific antibodies. GAPDH or actin was used as a loading control. One representative of at least 2 Western Blots is shown. Full, uncropped versions of the Western Blots are available as supplementary material.

Fig. 7

Influence of CK2 inhibition on apoptosis induction. TUNEL-staining of differentiated neurospheres at day 5 in vitro. SVZ cells were seeded on matrix-coated coverslips and differentiation was induced by adding retinoic acid (RA) to the medium. 10 or 20 μM CX-4945 or 40 μM quinalizarin was added to the RA-containing medium at the indicated time points after start of differentiation. DMSO served as solvent control. (A) TUNEL positive cells compared to the total cell number (DAPI), normalized to DMSO. (B) to (H) demonstrate the impact of CK2 inhibition upon apoptosis induction: (B) RA plus DMSO, (C) RA plus 10 μM CX-4945: 0 h, (D) RA plus 10 μM CX-4945: 72 h, (E) RA plus 20 μM CX-4945: 0 h, (F) RA plus 20 μM CX-4945: 72 h, (G) RA plus 40 μM quinalizarin: 0 h, (H) RA plus 40 μM quinalizarin: 72 h. Scale bar 50 μm.

Fig. 8

Influence of CK2 inhibition on neural stem cells. Nestin staining of differentiated neurospheres after 5 days in vitro. SVZ cells were plated on matrix-coated coverslips and differentiation was induced by adding retinoic acid (RA) to the medium. 10 μM or 20 μM CX-4945 or 40 μM quinalizarin was added to the RA-containing medium at the indicated time points after start of differentiation. DMSO served as solvent control. (A) Nestin-positive cells compared to the total cell number (DAPI). (B) retinoic acid (RA), (C) RA plus DMSO, (D) RA plus 10 μM CX-4945: 0 h, (E) RA plus 10 μM CX-4945: 72 h, (F) RA plus 20 μM CX-4945: 0 h, (G) RA plus 20 μM CX-4945: 72 h, (H) RA plus 40 μM quinalizarin: 0 h, (I) RA plus 40 μM quinalizarin: 72 h. Scale bar 50 μm.

Fig. 9

CK2 inhibition at late time points (14 days) of differentiation. SVZ cells were differentiated with retinoic acid (RA) for 14 days. Then DMSO or 20 μM CX-4945 were added to the RA-containing medium for 3 additional days. Neither GFAP- nor βIII-tubulin or nestin-expression were altered when CK2 was inhibited at late time points of differentiation (14 days) (A, E). GFAP- and βIII-tubulin staining of neurospheres after 14 days of undisturbed differentiation and three more days of either RA alone (B), RA plus DMSO (C) or RA plus 20 μM CX-4945 (D) treatment. Nestin-staining of parallel experiments did also not show any difference between RA (F), RA plus DMSO (G) or RA plus 20 μM CX- 4945 (H) treatment. Scale bar 50 μm.