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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.

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Altered proliferation and differentiation of MeCP2 phosphor-mutant aNPC in vitro and in vivo(a) Representative images of aNPCs isolated from WT and Mecp2S421A;S424A/y hippocampus with BrdU pulse labeling, followed by immunocytochemistry analysis (b) Quantification of the percentage of BrdU/Sox2/Nestin triple-labeled cells in WT and Mecp2S421A;S424A/y aNPCs. (n=3 in each group) (c) Representative images of Tuj1+ neurons differentiated from WT and Mecp2S421A;S424A/y aNPCs (d) Quantification of the percentage of Tuj1+ cells in WT and Mecp2S421A;S424A/y aNPCs upon differentiation. (n=3 in each group) (e) Representative images of GFAP+ astrocyte differentiated from WT and Mecp2S421A;S424A/y aNPCs (f) Quantification of the percentage of GFAP+ cells in WT and Mecp2S421A;S424A/y aNPCs upon differentiation. (n=3 in each group) (g) Relative mRNA level of neuronal marker (Tuj1 and NeuroD1) and astrocyte marker (GFAP) in WT and Mecp2S421A;S424A/y aNPCs upon differentiation, assayed by RT-qPCR. (n=3 in each group) (h) Schematics of the design of the in vivo BrdU labeling experiment. (i) Representative images of WT and the Mecp2S421A;S424A/y brain sections stained for BrdU immunoreactivity. (j) Quantification of relative number of BrdU+ cells obtained through stereological counting from WT and Mecp2S421A;S424A/y mice (n=9 in each group). (k) Quantification of the relative number of Ki67+ cells obtained through stereological counting from WT and Mecp2S421A;S424A/y mice (n=6 in each group). (l) Schematics of the design of in vivo BrdU pulse/chase experiment to examine the differentiation profile of the adult-born hippocampal cells. (m) Representative confocal microscopy images to demonstrate how each cell type is identified. Three adult-born neurons (co-stained by BrdU and NeuN) are marked by arrow. One adult-born glial cell (co-stained by BrdU and S100b) is marked by arrowhead. Two adult-born undetermined cells (stained by BrdU only) are marked by asterisk. The rectangle panel to the right of the merged channel image is the y-z view of the same optical stack. The optical size of the z-scan is 0.4μm/step. (n) Quantification of proportions of the cell fate choices made by the dividing aNPCs in the hippocampus of WT and Mecp2S421A;S424A/y mice. All scale bars are 50 μm. The bar graph shows the mean ± s.e.m * p<0.05 ** p<0.01
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Figure 3: Altered proliferation and differentiation of MeCP2 phosphor-mutant aNPC in vitro and in vivo(a) Representative images of aNPCs isolated from WT and Mecp2S421A;S424A/y hippocampus with BrdU pulse labeling, followed by immunocytochemistry analysis (b) Quantification of the percentage of BrdU/Sox2/Nestin triple-labeled cells in WT and Mecp2S421A;S424A/y aNPCs. (n=3 in each group) (c) Representative images of Tuj1+ neurons differentiated from WT and Mecp2S421A;S424A/y aNPCs (d) Quantification of the percentage of Tuj1+ cells in WT and Mecp2S421A;S424A/y aNPCs upon differentiation. (n=3 in each group) (e) Representative images of GFAP+ astrocyte differentiated from WT and Mecp2S421A;S424A/y aNPCs (f) Quantification of the percentage of GFAP+ cells in WT and Mecp2S421A;S424A/y aNPCs upon differentiation. (n=3 in each group) (g) Relative mRNA level of neuronal marker (Tuj1 and NeuroD1) and astrocyte marker (GFAP) in WT and Mecp2S421A;S424A/y aNPCs upon differentiation, assayed by RT-qPCR. (n=3 in each group) (h) Schematics of the design of the in vivo BrdU labeling experiment. (i) Representative images of WT and the Mecp2S421A;S424A/y brain sections stained for BrdU immunoreactivity. (j) Quantification of relative number of BrdU+ cells obtained through stereological counting from WT and Mecp2S421A;S424A/y mice (n=9 in each group). (k) Quantification of the relative number of Ki67+ cells obtained through stereological counting from WT and Mecp2S421A;S424A/y mice (n=6 in each group). (l) Schematics of the design of in vivo BrdU pulse/chase experiment to examine the differentiation profile of the adult-born hippocampal cells. (m) Representative confocal microscopy images to demonstrate how each cell type is identified. Three adult-born neurons (co-stained by BrdU and NeuN) are marked by arrow. One adult-born glial cell (co-stained by BrdU and S100b) is marked by arrowhead. Two adult-born undetermined cells (stained by BrdU only) are marked by asterisk. The rectangle panel to the right of the merged channel image is the y-z view of the same optical stack. The optical size of the z-scan is 0.4μm/step. (n) Quantification of proportions of the cell fate choices made by the dividing aNPCs in the hippocampus of WT and Mecp2S421A;S424A/y mice. All scale bars are 50 μm. The bar graph shows the mean ± s.e.m * p<0.05 ** p<0.01

Mentions: To reveal the functional significance of S421 phosphorylation in aNPCs, we examined proliferation and differentiation in both WT and phosphor-mutant aNPCs. Significantly fewer BrdU -labeled Nestin+Sox2+ (triple positive) aNPCs were observed in phosphor-mutant compared with WT cells (Fig. 3a–b and Supplementary Fig. 3a), indicating a reduced proliferation potential. While the phosphor-mutant aNPCs consistently took more time to grow than the wild type aNPCs at each passage, no significant difference was found in the percentage of cells at each stages of the cell cycle (Supplementary Fig. 3b). Upon differentiation, significantly more Tuj1 or MAP2 positive cells were detected in phosphor-mutant compared with WT cells (Fig. 3c–d and Supplementary Fig. 3c–d), suggesting increased potential in neural differentiation. In contrast, no difference in glial differentiation was observed between the WT and phosphor-mutant aNPCs (Fig. 3e–f). Consistent with the stereological counting data (Fig. 3c–f), the mRNA levels of Tuj1 and NeuroD1, two neuronal genes, were significantly higher in differentiated phosphor-mutant aNPCs than in WT aNPCs; while the mRNA levels of GFAP, a glial gene, remained unchanged in these cells (Fig. 3g). To validate our in vitro observations, we first performed BrdU labeling (Fig. 3h–i) to mark all the proliferating aNPCs in the subgranular zone of the hippocampus in adult mice, and conducted unbiased stereological quantification throughout the dentate gyrus of the hippocampus to compare the numbers of BrdU labeled cells in the dentate gyrus between Mecp2S421A;S424A/y and WT littermates (n=9 in each group). Fewer BrdU labeled cells (89% of WT level) were found in the Mecp2S421A;S424A/y hippocampus than in the WT littermate (Fig. 3j, p=0.002, unpaired t-test with Welch’s correction). Consistent with the BrdU labeling results, fewer Ki67 (a proliferation marker) stained cells (65% of WT level) were found in the Mecp2S421A;S424A/y hippocampus than in the WT littermates in stereological analysis an additional cohort of mice (Fig. 3k, n=6 in each group, p=0.003, unpaired t-test with Welch’s correction). In addition to aNPC proliferation, we also examined the cell fates of these adult-born cells 4 weeks after they were born (as illustrated in Fig. 3l). In the study, dividing aNPCs in the hippocampus of 8–9 weeks old Mecp2S421A;S424A/y mice (n=9) and their WT littermates (n=8) were first labeled by BrdU and given 4 weeks to differentiate. Sections throughout the hippocampus were triple labeled with an anti-BrdU antibody (to mark adult-born cells), an anti-NeuN antibody (to mark neurons), and an anti-S100β antibody (to mark glial cells). Under a confocal microscope, cells were identified as new neurons if they were positive for both BrdU and NeuN (Fig. 3m, indicated by arrows), as new glial cells if they were positive for both BrdU and S100β (Fig. 3m, indicated by an arrowhead), or as undetermined if they were only positive for BrdU. All BrdU positive cells (407±132 in WT vs. 379±81 in phosphor-mutant, unpaired t-test with Welch’s correction, p=0.85) in the subgranular zone from all stained sections were included in the analysis. Our results showed that a higher proportion of the dividing aNPCs in the hippocampus of the Mecp2S421A;S424A/y mice differentiated into neurons (Fig. 3n, p=0.01, unpaired t-test with Welch’s correction) at the expense of undetermined cells (Fig. 3n, p=0.02, unpaired t-test with Welch’s correction). As for the absolute number of BrdU positive newborn neurons, no significant difference was detected between the Mecp2S421A;S424A/y mice and their WT littermate (283±86 in WT vs. 299±61 in phosphor-mutant, unpaired t-test with Welch’s correction, p=0.88). The percentage of glial cells differentiated from the newborn cells in the adult hippocampus did not change significantly in the Mecp2S421A;S424A/y mice when compared with the WT littermates (Fig. 3n, p=0.48, unpaired t-test with Welch’s correction). No difference in the volume of granular cell layer was observed between the WT and phosphor-mutant mice (Supplementary Fig. 3e). Taken together, these in vitro and in vivo observations consistently identify a critical role of S421 phosphorylation in regulating aNPC proliferation and differentiation.


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)

Altered proliferation and differentiation of MeCP2 phosphor-mutant aNPC in vitro and in vivo(a) Representative images of aNPCs isolated from WT and Mecp2S421A;S424A/y hippocampus with BrdU pulse labeling, followed by immunocytochemistry analysis (b) Quantification of the percentage of BrdU/Sox2/Nestin triple-labeled cells in WT and Mecp2S421A;S424A/y aNPCs. (n=3 in each group) (c) Representative images of Tuj1+ neurons differentiated from WT and Mecp2S421A;S424A/y aNPCs (d) Quantification of the percentage of Tuj1+ cells in WT and Mecp2S421A;S424A/y aNPCs upon differentiation. (n=3 in each group) (e) Representative images of GFAP+ astrocyte differentiated from WT and Mecp2S421A;S424A/y aNPCs (f) Quantification of the percentage of GFAP+ cells in WT and Mecp2S421A;S424A/y aNPCs upon differentiation. (n=3 in each group) (g) Relative mRNA level of neuronal marker (Tuj1 and NeuroD1) and astrocyte marker (GFAP) in WT and Mecp2S421A;S424A/y aNPCs upon differentiation, assayed by RT-qPCR. (n=3 in each group) (h) Schematics of the design of the in vivo BrdU labeling experiment. (i) Representative images of WT and the Mecp2S421A;S424A/y brain sections stained for BrdU immunoreactivity. (j) Quantification of relative number of BrdU+ cells obtained through stereological counting from WT and Mecp2S421A;S424A/y mice (n=9 in each group). (k) Quantification of the relative number of Ki67+ cells obtained through stereological counting from WT and Mecp2S421A;S424A/y mice (n=6 in each group). (l) Schematics of the design of in vivo BrdU pulse/chase experiment to examine the differentiation profile of the adult-born hippocampal cells. (m) Representative confocal microscopy images to demonstrate how each cell type is identified. Three adult-born neurons (co-stained by BrdU and NeuN) are marked by arrow. One adult-born glial cell (co-stained by BrdU and S100b) is marked by arrowhead. Two adult-born undetermined cells (stained by BrdU only) are marked by asterisk. The rectangle panel to the right of the merged channel image is the y-z view of the same optical stack. The optical size of the z-scan is 0.4μm/step. (n) Quantification of proportions of the cell fate choices made by the dividing aNPCs in the hippocampus of WT and Mecp2S421A;S424A/y mice. All scale bars are 50 μm. The bar graph shows the mean ± s.e.m * p<0.05 ** p<0.01
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Figure 3: Altered proliferation and differentiation of MeCP2 phosphor-mutant aNPC in vitro and in vivo(a) Representative images of aNPCs isolated from WT and Mecp2S421A;S424A/y hippocampus with BrdU pulse labeling, followed by immunocytochemistry analysis (b) Quantification of the percentage of BrdU/Sox2/Nestin triple-labeled cells in WT and Mecp2S421A;S424A/y aNPCs. (n=3 in each group) (c) Representative images of Tuj1+ neurons differentiated from WT and Mecp2S421A;S424A/y aNPCs (d) Quantification of the percentage of Tuj1+ cells in WT and Mecp2S421A;S424A/y aNPCs upon differentiation. (n=3 in each group) (e) Representative images of GFAP+ astrocyte differentiated from WT and Mecp2S421A;S424A/y aNPCs (f) Quantification of the percentage of GFAP+ cells in WT and Mecp2S421A;S424A/y aNPCs upon differentiation. (n=3 in each group) (g) Relative mRNA level of neuronal marker (Tuj1 and NeuroD1) and astrocyte marker (GFAP) in WT and Mecp2S421A;S424A/y aNPCs upon differentiation, assayed by RT-qPCR. (n=3 in each group) (h) Schematics of the design of the in vivo BrdU labeling experiment. (i) Representative images of WT and the Mecp2S421A;S424A/y brain sections stained for BrdU immunoreactivity. (j) Quantification of relative number of BrdU+ cells obtained through stereological counting from WT and Mecp2S421A;S424A/y mice (n=9 in each group). (k) Quantification of the relative number of Ki67+ cells obtained through stereological counting from WT and Mecp2S421A;S424A/y mice (n=6 in each group). (l) Schematics of the design of in vivo BrdU pulse/chase experiment to examine the differentiation profile of the adult-born hippocampal cells. (m) Representative confocal microscopy images to demonstrate how each cell type is identified. Three adult-born neurons (co-stained by BrdU and NeuN) are marked by arrow. One adult-born glial cell (co-stained by BrdU and S100b) is marked by arrowhead. Two adult-born undetermined cells (stained by BrdU only) are marked by asterisk. The rectangle panel to the right of the merged channel image is the y-z view of the same optical stack. The optical size of the z-scan is 0.4μm/step. (n) Quantification of proportions of the cell fate choices made by the dividing aNPCs in the hippocampus of WT and Mecp2S421A;S424A/y mice. All scale bars are 50 μm. The bar graph shows the mean ± s.e.m * p<0.05 ** p<0.01
Mentions: To reveal the functional significance of S421 phosphorylation in aNPCs, we examined proliferation and differentiation in both WT and phosphor-mutant aNPCs. Significantly fewer BrdU -labeled Nestin+Sox2+ (triple positive) aNPCs were observed in phosphor-mutant compared with WT cells (Fig. 3a–b and Supplementary Fig. 3a), indicating a reduced proliferation potential. While the phosphor-mutant aNPCs consistently took more time to grow than the wild type aNPCs at each passage, no significant difference was found in the percentage of cells at each stages of the cell cycle (Supplementary Fig. 3b). Upon differentiation, significantly more Tuj1 or MAP2 positive cells were detected in phosphor-mutant compared with WT cells (Fig. 3c–d and Supplementary Fig. 3c–d), suggesting increased potential in neural differentiation. In contrast, no difference in glial differentiation was observed between the WT and phosphor-mutant aNPCs (Fig. 3e–f). Consistent with the stereological counting data (Fig. 3c–f), the mRNA levels of Tuj1 and NeuroD1, two neuronal genes, were significantly higher in differentiated phosphor-mutant aNPCs than in WT aNPCs; while the mRNA levels of GFAP, a glial gene, remained unchanged in these cells (Fig. 3g). To validate our in vitro observations, we first performed BrdU labeling (Fig. 3h–i) to mark all the proliferating aNPCs in the subgranular zone of the hippocampus in adult mice, and conducted unbiased stereological quantification throughout the dentate gyrus of the hippocampus to compare the numbers of BrdU labeled cells in the dentate gyrus between Mecp2S421A;S424A/y and WT littermates (n=9 in each group). Fewer BrdU labeled cells (89% of WT level) were found in the Mecp2S421A;S424A/y hippocampus than in the WT littermate (Fig. 3j, p=0.002, unpaired t-test with Welch’s correction). Consistent with the BrdU labeling results, fewer Ki67 (a proliferation marker) stained cells (65% of WT level) were found in the Mecp2S421A;S424A/y hippocampus than in the WT littermates in stereological analysis an additional cohort of mice (Fig. 3k, n=6 in each group, p=0.003, unpaired t-test with Welch’s correction). In addition to aNPC proliferation, we also examined the cell fates of these adult-born cells 4 weeks after they were born (as illustrated in Fig. 3l). In the study, dividing aNPCs in the hippocampus of 8–9 weeks old Mecp2S421A;S424A/y mice (n=9) and their WT littermates (n=8) were first labeled by BrdU and given 4 weeks to differentiate. Sections throughout the hippocampus were triple labeled with an anti-BrdU antibody (to mark adult-born cells), an anti-NeuN antibody (to mark neurons), and an anti-S100β antibody (to mark glial cells). Under a confocal microscope, cells were identified as new neurons if they were positive for both BrdU and NeuN (Fig. 3m, indicated by arrows), as new glial cells if they were positive for both BrdU and S100β (Fig. 3m, indicated by an arrowhead), or as undetermined if they were only positive for BrdU. All BrdU positive cells (407±132 in WT vs. 379±81 in phosphor-mutant, unpaired t-test with Welch’s correction, p=0.85) in the subgranular zone from all stained sections were included in the analysis. Our results showed that a higher proportion of the dividing aNPCs in the hippocampus of the Mecp2S421A;S424A/y mice differentiated into neurons (Fig. 3n, p=0.01, unpaired t-test with Welch’s correction) at the expense of undetermined cells (Fig. 3n, p=0.02, unpaired t-test with Welch’s correction). As for the absolute number of BrdU positive newborn neurons, no significant difference was detected between the Mecp2S421A;S424A/y mice and their WT littermate (283±86 in WT vs. 299±61 in phosphor-mutant, unpaired t-test with Welch’s correction, p=0.88). The percentage of glial cells differentiated from the newborn cells in the adult hippocampus did not change significantly in the Mecp2S421A;S424A/y mice when compared with the WT littermates (Fig. 3n, p=0.48, unpaired t-test with Welch’s correction). No difference in the volume of granular cell layer was observed between the WT and phosphor-mutant mice (Supplementary Fig. 3e). Taken together, these in vitro and in vivo observations consistently identify a critical role of S421 phosphorylation in regulating aNPC proliferation and differentiation.

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