mTOR pathway inhibition alters proliferation as well as differentiation of neural stem cells

Abstract

Introduction: Neural stem cells (NSCs) are essential for both embryonic development and adult neurogenesis, and their dysregulation causes a number of neurodevelopmental disorders, such as epilepsy and autism spectrum disorders. NSC proliferation and differentiation in the developing brain is a complex process controlled by various intrinsic and extrinsic stimuli. The mammalian target of rapamycin (mTOR) regulates proliferation and differentiation, among other cellular functions, and disruption in the mTOR pathway can lead to severe nervous system development deficits. In this study, we investigated the effect of inhibition of the mTOR pathway by rapamycin (Rapa) on NSC proliferation and differentiation.

Methods: The NSC cultures were treated with Rapa for 1, 2, 6, 24, and 48 h. The effect on cellular functions was assessed by immunofluorescence staining, western blotting, and proliferation/metabolic assays.

Results: mTOR inhibition suppressed NSC proliferation/metabolic activity as well as S-Phase entry by as early as 1 h of Rapa treatment and this effect persisted up to 48 h of Rapa treatment. In a separate experiment, NSCs were differentiated for 2 weeks after treatment with Rapa for 24 or 48 h. Regarding the effect on neuronal and glial differentiation (2 weeks post-treatment), this was suppressed in NSCs deficient in mTOR signaling, as evidenced by downregulated expression of NeuN, MAP2, and GFAP. We assume that the prolonged effect of mTOR inhibition is realized due to the effect on cytoskeletal proteins.

Discussion: Here, we demonstrate for the first time that the mTOR pathway not only regulates NSC proliferation but also plays an important role in NSC differentiation into both neuronal and glial lineages.

Keywords: cytoskeletal proteins; differentiation; mTOR; neural stem cells; proliferation.

Figure 1

Treatment algorithm of NSC culture with the mTOR inhibitor rapamycin. NSCs were treated with Rapa in NSC medium without growth factors for 1, 2, 6, 24 and 48 h. At each time point, the expression of mTOR pathway components (total and phosphorylated form of mTOR along with S6K1 and Akt) were analyzed by WB and IFC analysis. Proliferative/metabolic activity of NSC as well as expression of proliferation markers (nestin, CD24 and β1 integrin) were analyzed at a 24-h delay following exposure to Rapa. In a separate set of experiments, differentiation was induced by adding medium containing Shh and retinoic acid after 24 or 48 h of Rapa treatment. Cells were allowed to differentiate for 2 weeks. The expression analysis of differentiation markers (nestin, NF-H, NeuN, MAP2, βIII-tubulin, GFAP, ALDH1L1) was analyzed by WB and IFC analysis. Rapa, rapamycin; Shh, Sonic hedgehog; mTOR, mammalian target of rapamycin; S6K1, ribosomal protein S6 kinase 1; Akt, protein kinase B; NF-M, neurofilament medium chain; NF-H, neurofilament heavy chain; MAP2, microtubule associated protein 2; GFAP, glial fibrillar acid protein; ALDH1L1, aldehyde dehydrogenase 1 family member L1; WB, Western blot; IFC, immunofluorescence.

Figure 2

mTORC1 inhibition by rapamycin affected several upstream and downstream molecules of the mTOR pathway. Densitometry analysis revealed that Rapa treatment resulted in a decrease in pmTOR^Ser2448/mTOR (A) and pS6K1^Ser235/236/S6K1 (C) expression ratio and an increase in pAkt ^Ser473/Akt (G) and in pE4BP1/E4BP1 (E) expression ratio, however only in one time point – after 24 h of Rapa treatment. WB data were supported by the immunofluorescence staining of NSCs for pmTOR^Ser2448 (B), pS6K1^Ser235/236 (D), p4E-BP1 (F) and pAkt^Ser473 (H). All data represent mean ± SEM. The level of statistical significance was marked as follows: ^*p < 0.05; ^**p < 0.01. Scale bar = 20 μm. Rapa, rapamycin; mTOR, mammalian target of rapamycin; S6K1, ribosomal protein S6 kinase 1; Akt, protein kinase B; NSC, neural stem cell; WB, Western blot; IFC, immunofluorescence.

Figure 3

mTORC1 inhibition suppressed NSC proliferation. Proliferative activity of NSCs, as assessed by EdU-incorporation assay at a 24 h delay after Rapa treatment was significantly reduced compared to controls (A). The Alamar blue assay also demonstrated a significant reduction in NSC metabolic activity following mTORC1 inhibition (B). WB and IFC analysis depicting a significant decrease in nestin expression at 6, 24 and 48 h (C,D); an increase in β1integrin expression was observed at 24 and 48 h after Rapa treatment (E,F). Rapa treatment suppressed CD24 expression at 1, 2 and 48 h (G,H). All data represent mean ± SEM. The level of statistical significance was marked as follows: ^*p < 0.05; ^**p < 0.01. Scale bar = 20 μm. Rapa, rapamycin; NSCs, neural stem cells; WB, Western blot; IFC, immunofluorescence.

Figure 4

Neuronal differentiation of rapamycin treated NSCs. Rapa treatment caused alterations in the expression of neuronal markers within 2 weeks of NSC differentiation. The expression of NF-M 180 kDa (A), NF-H 220 kDa (C) and βIII-tubulin (H) was significantly decreased as compared to the corresponding time-matched controls immediately following 24–48 h of Rapa treatment, while the expression of NeuN (D) and MAP2 (F) at these time points remained unaltered. Within 2 weeks of neuronal differentiation (presented as dark and light pink bars), Rapa treated cells displayed a significant reduction in expression of NeuN (D), MAP2 (F) as well as βIII-tubulin (H), while expression of NF-H and NF-M remained unchanged. WB analysis was supported by IFC staining for markers of NSC differentiation: NF-H (B), NeuN (E), MAP2 (G) and βIII-tubulin (I). All data represent mean ± SEM. The level of statistical significance was marked as follows: ^*p < 0.05; ^**p < 0.01. Scale bar = 20 μm. Rapa, rapamycin; NF-M, neurofilament medium chain; NF-H, neurofilament heavy chain; MAP2, microtubule associated protein 2; WB, Western blot; IFC, immunofluorescence.

Figure 5

Glial differentiation of rapamycin-treated NSCs. The expression GFAP (A) as well as ALDH1L1 (C) immediately following 24–48 h of Rapa treatment was significantly reduced compared to the time-matched controls. Within 2 weeks of glial differentiation (presented as dark and light pink bars), Rapa-treated cells displayed a significant reduction in GFAP (A). WB analysis was supported by IFC staining of 2 weeks differentiated cells (B,D). All data represent mean ± SEM. The level of statistical significance was marked as follows: ^*p < 0.05; ^**p < 0.01. Scale bar = 20 μm. Rapa, rapamycin; GFAP, glial fibrillar acid protein; ALDH1L1, aldehyde dehydrogenase 1 family member L1; WB, Western blot; IFC, immunofluorescence.