Limits...
Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signalling pathway.

Li H, Zhong X, Chau KF, Santistevan NJ, Guo W, Kong G, Li X, Kadakia M, Masliah J, Chi J, Jin P, Zhang J, Zhao X, Chang Q - Nat Commun (2014)

Bottom Line: Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons.However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts.Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types.

View Article: PubMed Central - PubMed

Affiliation: 1] Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA [2] Genetics Training Program, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.

ABSTRACT
Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons. The precise control of the phosphorylation status of MeCP2 in neurons is critical for the normal development and function of the mammalian brain. However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts. Here we show that in neural progenitor cells isolated from the adult mouse hippocampus, cell cycle-linked phosphorylation at serine 421 on MeCP2 is directly regulated by aurora kinase B and modulates the balance between proliferation and neural differentiation through the Notch signalling pathway. Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types.

Show MeSH
Phosphorylation of MeCP2 S421 is regulated by cell cycle in aNPCs(a,b) Western blot analysis of MeCP2 S421 phosphorylation and the quantification of total MeCP2 protein level in WT and phosphor-mutant aNPCs. (p=0.718, unpaired t-test with Welch’s correction, n=3 in each group) (c,d) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in WT aNPCs under normal proliferating condition and FGF2/EGF withdrawal for 24 hours. (n=3 in each group) (e) Western blot analysis reveals that MeCP2 S421 phosphorylation is induced by synchronizing aNPCs with nocodazole (150ng/ml). (f,g) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in aNPCs under conditions: 1) DMSO, 2) 36 hours of nocodazole treatment, 3) 24 hours of roscovitine (25 μM) treatment after pre-synchronization of the cells by nocodazole for 12 hours. (n=3 in each group) Numbers next to Western blots are molecular weight markers. The bar graph shows the mean ± s.e.m * p<0.05 ** p<0.01
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4288926&req=5

Figure 1: Phosphorylation of MeCP2 S421 is regulated by cell cycle in aNPCs(a,b) Western blot analysis of MeCP2 S421 phosphorylation and the quantification of total MeCP2 protein level in WT and phosphor-mutant aNPCs. (p=0.718, unpaired t-test with Welch’s correction, n=3 in each group) (c,d) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in WT aNPCs under normal proliferating condition and FGF2/EGF withdrawal for 24 hours. (n=3 in each group) (e) Western blot analysis reveals that MeCP2 S421 phosphorylation is induced by synchronizing aNPCs with nocodazole (150ng/ml). (f,g) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in aNPCs under conditions: 1) DMSO, 2) 36 hours of nocodazole treatment, 3) 24 hours of roscovitine (25 μM) treatment after pre-synchronization of the cells by nocodazole for 12 hours. (n=3 in each group) Numbers next to Western blots are molecular weight markers. The bar graph shows the mean ± s.e.m * p<0.05 ** p<0.01

Mentions: We discovered that S421 was phosphorylated (Fig. 1a) in proliferating aNPCs isolated from the dentate gyrus (DG) of wild type (WT) mouse hippocampus (Supplementary Fig. 1a). This phosphorylation was abolished in phosphor-mutant aNPCs isolated from the Mecp2S421A;S424A/y hippocampus (Fig. 1a). Consistent with our previous report6, the level of total MeCP2 protein in aNPCs was indistinguishable between the wild type and the phosphor-mutant (Fig. 1a). When the WT aNPCs were differentiated into neurons, S421 phosphorylation first decreased, but then increased again as the neurons matured (Supplementary Fig. 1b). Since previous studies on postmitotic neurons have identified calcium influx through VGCCs as the trigger for S421 phosphorylation, we first tested whether they are involved in inducing S421 phosphorylation in aNPCs. Neither membrane depolarization by KCl nor treatment by Bay K8644, a calcium channel agonist, induced S421 phosphorylation in aNPCs (Supplementary Fig. 1c). Consistent with these results, Nimodipine, a VGCC blocker, failed to inhibit S421 phosphorylation in aNPCs (Supplementary Fig. 1d–e). In contrast, roscovitine, a broad-spectrum inhibitor of cyclin-dependent kinases (CDKs), significantly decreased S421 phosphorylation in aNPCs (Supplementary Fig. 1d–e). In light of this surprising result and the fact that aNPCs are dividing cells, we explored the possibility that growth factor and cell cycle regulation are involved in regulating S421 phosphorylation. Withdrawing growth factors led to concurrent decrease of phosphorylation of S421 (Fig. 1b–c) and absence of G2/M phase cells (Supplementary Fig. 2a) in WT aNPCs. These alterations were unlikely to be secondary to potential cell fate changes, because expression of key cell type markers remained similar before and after the withdrawal of growth factors (Supplementary Fig. 2b–c). To investigate whether S421 phosphorylation is linked to cell cycle, we arrested WT aNPCs at the G2/M phase with nocodazole (Supplementary Fig. 2d–e) or colchicine and observed a dramatic increase in the level of phospho-S421 (Fig. 1d,f and Supplementary Fig. 2f). Using a series of pharmacological reagents, we excluded the involvement of VGCCs, CaMKK and CaMKII in regulating cell cycle-linked S421 phosphorylation in aNPCs, because selective inhibitors of VGCCs, CaMKK or CaMKII failed to block cell cycle-linked S421 phosphorylation in these cells (Supplementary Fig. 2g). In contrast, roscovitine, a synthetic molecule with sub-μM IC50 values against CDK1, CDK2, CDK5, CDK7 and CDK916, was highly effective in blocking nocodazole-induced S421 phosphorylation, suggesting that cell cycle dependent kinases (CDKs) were upstream of S421 phosphorylation in aNPCs (Fig. 1e–f). Since nocodazole and roscovitine together arrested slightly more cells at G1 than nocodazole alone (Supplementary Fig. 2d–e), roscovitine may have additional indirect effect on S421 phosphorylation through cell cycle alteration. Finally, such cell cycle-linked MeCP2 S421 phosphorylation was also observed in aNPCs isolated from the subventricular zone (SVZ) and NPCs isolated from neonatal brains (Supplementary Fig. 2h), raising the possibility that this may be a general phenomenon in proliferating cells.


Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signalling pathway.

Li H, Zhong X, Chau KF, Santistevan NJ, Guo W, Kong G, Li X, Kadakia M, Masliah J, Chi J, Jin P, Zhang J, Zhao X, Chang Q - Nat Commun (2014)

Phosphorylation of MeCP2 S421 is regulated by cell cycle in aNPCs(a,b) Western blot analysis of MeCP2 S421 phosphorylation and the quantification of total MeCP2 protein level in WT and phosphor-mutant aNPCs. (p=0.718, unpaired t-test with Welch’s correction, n=3 in each group) (c,d) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in WT aNPCs under normal proliferating condition and FGF2/EGF withdrawal for 24 hours. (n=3 in each group) (e) Western blot analysis reveals that MeCP2 S421 phosphorylation is induced by synchronizing aNPCs with nocodazole (150ng/ml). (f,g) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in aNPCs under conditions: 1) DMSO, 2) 36 hours of nocodazole treatment, 3) 24 hours of roscovitine (25 μM) treatment after pre-synchronization of the cells by nocodazole for 12 hours. (n=3 in each group) Numbers next to Western blots are molecular weight markers. The bar graph shows the mean ± s.e.m * p<0.05 ** p<0.01
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4288926&req=5

Figure 1: Phosphorylation of MeCP2 S421 is regulated by cell cycle in aNPCs(a,b) Western blot analysis of MeCP2 S421 phosphorylation and the quantification of total MeCP2 protein level in WT and phosphor-mutant aNPCs. (p=0.718, unpaired t-test with Welch’s correction, n=3 in each group) (c,d) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in WT aNPCs under normal proliferating condition and FGF2/EGF withdrawal for 24 hours. (n=3 in each group) (e) Western blot analysis reveals that MeCP2 S421 phosphorylation is induced by synchronizing aNPCs with nocodazole (150ng/ml). (f,g) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in aNPCs under conditions: 1) DMSO, 2) 36 hours of nocodazole treatment, 3) 24 hours of roscovitine (25 μM) treatment after pre-synchronization of the cells by nocodazole for 12 hours. (n=3 in each group) Numbers next to Western blots are molecular weight markers. The bar graph shows the mean ± s.e.m * p<0.05 ** p<0.01
Mentions: We discovered that S421 was phosphorylated (Fig. 1a) in proliferating aNPCs isolated from the dentate gyrus (DG) of wild type (WT) mouse hippocampus (Supplementary Fig. 1a). This phosphorylation was abolished in phosphor-mutant aNPCs isolated from the Mecp2S421A;S424A/y hippocampus (Fig. 1a). Consistent with our previous report6, the level of total MeCP2 protein in aNPCs was indistinguishable between the wild type and the phosphor-mutant (Fig. 1a). When the WT aNPCs were differentiated into neurons, S421 phosphorylation first decreased, but then increased again as the neurons matured (Supplementary Fig. 1b). Since previous studies on postmitotic neurons have identified calcium influx through VGCCs as the trigger for S421 phosphorylation, we first tested whether they are involved in inducing S421 phosphorylation in aNPCs. Neither membrane depolarization by KCl nor treatment by Bay K8644, a calcium channel agonist, induced S421 phosphorylation in aNPCs (Supplementary Fig. 1c). Consistent with these results, Nimodipine, a VGCC blocker, failed to inhibit S421 phosphorylation in aNPCs (Supplementary Fig. 1d–e). In contrast, roscovitine, a broad-spectrum inhibitor of cyclin-dependent kinases (CDKs), significantly decreased S421 phosphorylation in aNPCs (Supplementary Fig. 1d–e). In light of this surprising result and the fact that aNPCs are dividing cells, we explored the possibility that growth factor and cell cycle regulation are involved in regulating S421 phosphorylation. Withdrawing growth factors led to concurrent decrease of phosphorylation of S421 (Fig. 1b–c) and absence of G2/M phase cells (Supplementary Fig. 2a) in WT aNPCs. These alterations were unlikely to be secondary to potential cell fate changes, because expression of key cell type markers remained similar before and after the withdrawal of growth factors (Supplementary Fig. 2b–c). To investigate whether S421 phosphorylation is linked to cell cycle, we arrested WT aNPCs at the G2/M phase with nocodazole (Supplementary Fig. 2d–e) or colchicine and observed a dramatic increase in the level of phospho-S421 (Fig. 1d,f and Supplementary Fig. 2f). Using a series of pharmacological reagents, we excluded the involvement of VGCCs, CaMKK and CaMKII in regulating cell cycle-linked S421 phosphorylation in aNPCs, because selective inhibitors of VGCCs, CaMKK or CaMKII failed to block cell cycle-linked S421 phosphorylation in these cells (Supplementary Fig. 2g). In contrast, roscovitine, a synthetic molecule with sub-μM IC50 values against CDK1, CDK2, CDK5, CDK7 and CDK916, was highly effective in blocking nocodazole-induced S421 phosphorylation, suggesting that cell cycle dependent kinases (CDKs) were upstream of S421 phosphorylation in aNPCs (Fig. 1e–f). Since nocodazole and roscovitine together arrested slightly more cells at G1 than nocodazole alone (Supplementary Fig. 2d–e), roscovitine may have additional indirect effect on S421 phosphorylation through cell cycle alteration. Finally, such cell cycle-linked MeCP2 S421 phosphorylation was also observed in aNPCs isolated from the subventricular zone (SVZ) and NPCs isolated from neonatal brains (Supplementary Fig. 2h), raising the possibility that this may be a general phenomenon in proliferating cells.

Bottom Line: Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons.However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts.Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types.

View Article: PubMed Central - PubMed

Affiliation: 1] Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA [2] Genetics Training Program, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.

ABSTRACT
Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons. The precise control of the phosphorylation status of MeCP2 in neurons is critical for the normal development and function of the mammalian brain. However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts. Here we show that in neural progenitor cells isolated from the adult mouse hippocampus, cell cycle-linked phosphorylation at serine 421 on MeCP2 is directly regulated by aurora kinase B and modulates the balance between proliferation and neural differentiation through the Notch signalling pathway. Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types.

Show MeSH