Targeting p38 mitogen-activated protein kinase signaling restores subventricular zone neural stem cells and corrects neuromotor deficits in Atm knockout mouse

Abstract

Ataxia-telangiectasia (A-T) is a progressive degenerative disorder that results in major neurological disability. In A-T patients, necropsy has revealed atrophy of cerebellar cortical layers along with Purkinje and granular cell loss. We have previously identified an oxidative stress-mediated increase in phospho-p38 mitogen-activated protein kinase (MAPK) and the resultant downregulation of Bmi-1 and upregulation of p21 as key components of the mechanism causing defective proliferation of neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of Atm(-/-) mice. However, the in vivo aspect of alteration in SVZ tissue and the functional significance of p38MAPK activation in NSCs for neuropathogenesis of ATM deficiency remain unknown. Here we show that the NSC population was abnormally decreased in the SVZ of 3-month-old Atm(-/-) mice; this decrease was accompanied by p38MAPK activation. However, after a 2-month treatment with the p38MAPK inhibitor SB203580, starting at 1 month old, Atm(-/-) mice showed restoration of normal levels of Bmi-1 and p21 with the rescue of NSC population in the SVZ. In addition, treated Atm(-/-) mice exhibited more Purkinje cells in the cerebellum. Most importantly, motor coordination of Atm(-/-) mice was significantly improved in the treatment group. Our results show for the first time in vivo evidence of depleted NSCs in the SVZ of Atm(-/-) mice and also demonstrate that pharmacologic inhibition of p38MAPK signaling has the potential to treat neurological defects of A-T. This study provides a promising approach targeting the oxidative stress-dependent p38 signaling pathway not only for A-T but also for other neurodegenerative disorders.

Figure 1.

NSC depletion in the subventricular zone (SVZ) of Atm^−/− mice. (A): Paraformaldehyde-fixed frozen sections of SVZ tissue of adult (postnatal day 90 [P90]) Atm^+/+ and Atm^−/− mice were stained with antibodies against nestin (red) to identify NSCs and against Ki67 (green) to detect proliferating cells in the SVZ. Cells were counterstained by DAPI (blue), which identifies the nuclei of the NSCs. Scale bars = 50 μm. (B): Total RNA was purified from SVZ tissue of adult (P90) Atm^+/+ and Atm^−/− mice and subjected to quantitative reverse transcription-polymerase chain reaction analysis for nestin and Ki-67 mRNA expression. Probing for gapdh was used as an internal control. (C): After injection of adult (P90) Atm^+/+ and Atm^−/− mice with BrdU for a period of 5 days, SVZ sections were stained with an antibody against BrdU to label proliferating cells. Scale bars = 100 μm. BrdU-positive cells in the SVZ of adult (P90) Atm^+/+ and Atm^−/− mice were counted. Values represent BrdU-positive cell numbers in an SVZ section of Atm^+/+ mice ± SD (three independent countings of three sections). Abbreviations: BrdU, bromodeoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 2.

p38 activation is responsible for abnormalities in the subventricular zone (SVZ) of Atm^−/− mice. (A): SVZ sections of adult (postnatal day 90 [P90]) Atm^+/+ and Atm^−/− mice were stained with antibody against phospho-p38 (green). Cells were counterstained by DAPI. The right panel is an enlargement of the small gray framed area. Scale bars = 20 μm. (B): Proteins were extracted from SVZ tissue of adult (P90) Atm^+/+ and Atm^−/− mice, and p38 activation was determined by Western blot analysis with an antibody against phospho-p38. (C): 1-month-old Atm^+/+ and Atm^−/− mice were treated with either phosphate-buffered saline (PBS) or SB203580 for 2 months. Atm^+/+, n = 10; Atm^−/−, n = 9; Atm^−/−+SB, n = 10. After treatment, SVZ sections were stained with an antibody against another NSC-specific protein, vimentin, to identify NSCs. Sections were also stained with antibodies against Bmi-1 and p21. Arrow indicates p21-expressing cells. Scale bar = 50 μm. (D): Protein levels of Bmi-1 and p21 were determined by Western blot analysis using protein extracts from SVZ tissue of adult (P90) Atm^+/+, Atm^−/−, and Atm^−/−+SB mice. (E): Expression of bmi-1 and p21 mRNA was determined by quantitative reverse transcription-polymerase chain reaction using total RNA from SVZ tissue of adult (P90) Atm^+/+, Atm^−/−, and Atm^−/−+SB mice. Probing for gapdh was used as an internal control. Abbreviations: Atm^+/+, PBS-treated Atm^+/+; Atm^−/−, PBS-treated Atm^−/−; Atm^−/−+SB, SB203580-treated Atm^−/−; DAPI, 4′,6-diamidino-2-phenylindole; p-p38, phospho-p38; SB, SB203580.

Figure 3.

SB203580 protected Atm^−/− cerebella Purkinje cells. (A): After treatment, cerebellum sections of adult (postnatal day 90 [P90]) Atm^+/+, Atm^−/−, and Atm^−/−+SB mice were stained with H&E to display cerebellum cytoarchitecture. (B): After treatment, cerebellum sections of adult (P90) Atm^+/+, Atm^−/−, and Atm^−/−+SB mice were stained with an antibody against calbindin to detect Purkinje cells. Scale bar = 20 μm. (C): Purkinje cell numbers along the PCL of cerebellar hemispheres of adult (P90) Atm^+/+, Atm^−/−, and Atm^−/−+SB mice were counted. Values represent percentage of Atm^+/+ Purkinje cell number ± SD (three independent counting of 10 fields; ***, p < .001; *, p < .05). (D): Protein levels of the Purkinje cell-specific protein calbindin were determined by Western blot analysis using protein extracts from cerebellar tissue of adult (P90) Atm^+/+, Atm^−/−, and Atm^−/−+SB mice. (E): Expression of calbindin mRNA was determined by quantitative reverse transcription-polymerase chain reaction using total RNA from cerebellar tissue of adult (P90) Atm^+/+, Atm^−/−, and Atm^−/−+SB mice. Probing for gapdh was used as an internal control. Abbreviations: Atm^+/+, PBS-treated Atm^+/+; Atm^−/−, PBS-treated Atm^−/−; Atm^−/−+SB, SB203580-treated Atm^−/−; Calb, calbindin; GL, granular layer; H&E, hematoxylin/eosin; ML, molecular layer; PCL, Purkinje cell layer; SB, SB203580; WT, wild-type.

Figure 4.

SB203580 retained the motor function of Atm^−/− mice. (A): Motor coordination of 2-month-old Atm^+/+ and Atm^−/− mice was analyzed by measuring the time before falling off the rotating beam at 25 rpm with three trials. Values represent means ± SD of time on the rotating rod (Atm^+/+, n = 4; Atm^−/−, n = 4; three independent tests; ***, p < .001). The body weights of 2-month-old Atm^+/+ and Atm^−/− mice were also measured. Values represent means ± SD of body weights (Atm^+/+, n = 4; Atm^−/−, n = 4; three independent tests; *, p < .05). (B): After treatment with antioxidant NAC and MSL, motor coordination of adult (postnatal day 90 [P90]) Atm^+/+, Atm^−/−, Atm^−/−+NAC, and Atm^−/−+MSL mice were analyzed. Values represent means ± SD of time on the rotating beam (n = 6 per group; three independent tests; **, p < .01, *, p < .05). (C): After treatment, the motor coordination of adult (P90) Atm^+/+, Atm^−/−, and Atm^−/−+SB203580 (SB) mice was assessed by measuring the time before falling off the rotating beam at 25 rpm with three trials. Values represent means ± SD of time on the rotating beam (Atm^+/+, n = 10; Atm^−/−, n = 9; Atm^−/−+SB, n = 10; three independent tests; ***, p < .001, **, p < .01). (D): After treatment, the body weights of adult (P90) Atm^+/+, Atm^−/−, and Atm^−/−+SB mice were measured. Values represent means ± SD of body weights (Atm^+/+, n = 10; Atm^−/−, n = 9; Atm^−/−+SB, n = 10; three independent tests; ***, p < .001, *, p < .05). Abbreviations: MSL, monosodium luminol; NAC, N-acetyl-l-cysteine; UT, untreated.