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Ankyrin-G regulates neurogenesis and Wnt signaling by altering the subcellular localization of β-catenin.

Durak O, de Anda FC, Singh KK, Leussis MP, Petryshen TL, Sklar P, Tsai LH - Mol. Psychiatry (2014)

Bottom Line: Recent genome-wide association studies and whole-exome sequencing have identified ANK3, the gene coding for ankyrin-G, to be a risk gene for multiple neuropsychiatric disorders, such as bipolar disorder, schizophrenia and autism spectrum disorder.We found that ankyrin-G regulates canonical Wnt signaling by altering the subcellular localization and availability of β-catenin in proliferating cells.Taken together, these results suggest that ankyrin-G is required for proper brain development.

View Article: PubMed Central - PubMed

Affiliation: Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.

ABSTRACT
Ankyrin-G is a scaffolding protein required for the formation of the axon initial segment in neurons. Recent genome-wide association studies and whole-exome sequencing have identified ANK3, the gene coding for ankyrin-G, to be a risk gene for multiple neuropsychiatric disorders, such as bipolar disorder, schizophrenia and autism spectrum disorder. Here, we describe a novel role for ankyrin-G in neural progenitor proliferation in the developing cortex. We found that ankyrin-G regulates canonical Wnt signaling by altering the subcellular localization and availability of β-catenin in proliferating cells. Ankyrin-G loss-of-function increases β-catenin levels in the nucleus, thereby promoting neural progenitor proliferation. Importantly, abnormalities in proliferation can be rescued by reducing Wnt pathway signaling. Taken together, these results suggest that ankyrin-G is required for proper brain development.

No MeSH data available.


Related in: MedlinePlus

Dampening canonical Wnt-signaling rescues phenotypes associated with ankyrin-G knockdowna) GSK3β overexpression rescues increased Wnt-mediated luciferase activity (in all cases, n=4; Tukey’s Multiple Comparison test). b–d) Images of E16 mouse cortices electroporated at E13 with non-targeting (b, Control) or ankyrin-G-directed small hairpin along with either empty vector (c, AnkG shRNA) or GSK3β overexpression construct (d, AnkG shRNA + GSK3β), and GFP expression plasmid. Images were stained for GFP (green), BrdU (red) and Ki67 (blue). e) Increased BrdU incorporation associated with ankyrin-G knockdown is reduced to control levels when ankyrin-G shRNA is co-expressed with GSK3β (Control + Empty Vector, n=4; AnkG shRNA + Empty Vector, n=2; AnkG shRNA+ GSK3β, n=4; Tukey’s Multiple Comparison Test). f) Co-expression of GSK3β with ankyrin-G shRNA rescues the cell cycle exit phenotype assayed using Ki67 proliferative marker (Control + Empty Vector, n=4; AnkG shRNA + Empty Vector, n=2; AnkG shRNA+ GSK3β, n=4; Tukey’s Multiple Comparison Test). g) Distribution of GFP+ cells in different cortical zones 72 hours after transfection at E16. Co-expression of GSK3β with ankyrin-G shRNA reduced the percentage of GFP+ cells in the VZ compared to ankyrin-G knockdown condition (Control + Empty Vector, n=4; AnkG shRNA + Empty Vector, n=2; AnkG shRNA+ GSK3β, n=4; Tukey’s Multiple Comparison Test). h) Working model for ankyrin-G function during cortical development. Left, in control condition ankyrin-G localizes E-cadherin to plasma membrane where it participates in cadherin-catenin complex. Right, in ankyrin-G knockdown condition E-cadherin localized to plasma membrane is reduced, which in turn results in increased free β-catenin available for Wnt signaling. Upregulation of Wnt signaling results in increased neural progenitor proliferation in developing cortex. *, P<0.05; **, P<0.01; ***, P<0.001. Scale bar: 100 µm
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Figure 4: Dampening canonical Wnt-signaling rescues phenotypes associated with ankyrin-G knockdowna) GSK3β overexpression rescues increased Wnt-mediated luciferase activity (in all cases, n=4; Tukey’s Multiple Comparison test). b–d) Images of E16 mouse cortices electroporated at E13 with non-targeting (b, Control) or ankyrin-G-directed small hairpin along with either empty vector (c, AnkG shRNA) or GSK3β overexpression construct (d, AnkG shRNA + GSK3β), and GFP expression plasmid. Images were stained for GFP (green), BrdU (red) and Ki67 (blue). e) Increased BrdU incorporation associated with ankyrin-G knockdown is reduced to control levels when ankyrin-G shRNA is co-expressed with GSK3β (Control + Empty Vector, n=4; AnkG shRNA + Empty Vector, n=2; AnkG shRNA+ GSK3β, n=4; Tukey’s Multiple Comparison Test). f) Co-expression of GSK3β with ankyrin-G shRNA rescues the cell cycle exit phenotype assayed using Ki67 proliferative marker (Control + Empty Vector, n=4; AnkG shRNA + Empty Vector, n=2; AnkG shRNA+ GSK3β, n=4; Tukey’s Multiple Comparison Test). g) Distribution of GFP+ cells in different cortical zones 72 hours after transfection at E16. Co-expression of GSK3β with ankyrin-G shRNA reduced the percentage of GFP+ cells in the VZ compared to ankyrin-G knockdown condition (Control + Empty Vector, n=4; AnkG shRNA + Empty Vector, n=2; AnkG shRNA+ GSK3β, n=4; Tukey’s Multiple Comparison Test). h) Working model for ankyrin-G function during cortical development. Left, in control condition ankyrin-G localizes E-cadherin to plasma membrane where it participates in cadherin-catenin complex. Right, in ankyrin-G knockdown condition E-cadherin localized to plasma membrane is reduced, which in turn results in increased free β-catenin available for Wnt signaling. Upregulation of Wnt signaling results in increased neural progenitor proliferation in developing cortex. *, P<0.05; **, P<0.01; ***, P<0.001. Scale bar: 100 µm

Mentions: Our findings suggest that ankyrin-G is a negative regulator of the Wnt signaling pathway, since reduced levels of ankyrin-G lead to upregulated TCF/LEF reporter activity and nuclear β-catenin accumulation. To directly test this hypothesis, we examined the effect of the overexpression of GSK3β, which phosphorylates β-catenin and targets it for ubiquitin-dependent proteasomal degradation41,42, upon on the ankyrin-G loss of function phenotype. In P19 cells, increased TCF/LEF luciferase reporter activity, observed as the result of ankyrin-G shRNA knockdown, was completely reversed by the overexpression of GSK3β (Figure 4a). These results indicate that ankyrin-G functionally interacts with other Wnt signaling proteins such as GSK3β and that the effects of ankyrin-G knockdown on Wnt signaling activity can be normalized by reducing β-catenin abundance. Together these results further suggest that ankyrin-G is a negative regulator of Wnt signaling


Ankyrin-G regulates neurogenesis and Wnt signaling by altering the subcellular localization of β-catenin.

Durak O, de Anda FC, Singh KK, Leussis MP, Petryshen TL, Sklar P, Tsai LH - Mol. Psychiatry (2014)

Dampening canonical Wnt-signaling rescues phenotypes associated with ankyrin-G knockdowna) GSK3β overexpression rescues increased Wnt-mediated luciferase activity (in all cases, n=4; Tukey’s Multiple Comparison test). b–d) Images of E16 mouse cortices electroporated at E13 with non-targeting (b, Control) or ankyrin-G-directed small hairpin along with either empty vector (c, AnkG shRNA) or GSK3β overexpression construct (d, AnkG shRNA + GSK3β), and GFP expression plasmid. Images were stained for GFP (green), BrdU (red) and Ki67 (blue). e) Increased BrdU incorporation associated with ankyrin-G knockdown is reduced to control levels when ankyrin-G shRNA is co-expressed with GSK3β (Control + Empty Vector, n=4; AnkG shRNA + Empty Vector, n=2; AnkG shRNA+ GSK3β, n=4; Tukey’s Multiple Comparison Test). f) Co-expression of GSK3β with ankyrin-G shRNA rescues the cell cycle exit phenotype assayed using Ki67 proliferative marker (Control + Empty Vector, n=4; AnkG shRNA + Empty Vector, n=2; AnkG shRNA+ GSK3β, n=4; Tukey’s Multiple Comparison Test). g) Distribution of GFP+ cells in different cortical zones 72 hours after transfection at E16. Co-expression of GSK3β with ankyrin-G shRNA reduced the percentage of GFP+ cells in the VZ compared to ankyrin-G knockdown condition (Control + Empty Vector, n=4; AnkG shRNA + Empty Vector, n=2; AnkG shRNA+ GSK3β, n=4; Tukey’s Multiple Comparison Test). h) Working model for ankyrin-G function during cortical development. Left, in control condition ankyrin-G localizes E-cadherin to plasma membrane where it participates in cadherin-catenin complex. Right, in ankyrin-G knockdown condition E-cadherin localized to plasma membrane is reduced, which in turn results in increased free β-catenin available for Wnt signaling. Upregulation of Wnt signaling results in increased neural progenitor proliferation in developing cortex. *, P<0.05; **, P<0.01; ***, P<0.001. Scale bar: 100 µm
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Figure 4: Dampening canonical Wnt-signaling rescues phenotypes associated with ankyrin-G knockdowna) GSK3β overexpression rescues increased Wnt-mediated luciferase activity (in all cases, n=4; Tukey’s Multiple Comparison test). b–d) Images of E16 mouse cortices electroporated at E13 with non-targeting (b, Control) or ankyrin-G-directed small hairpin along with either empty vector (c, AnkG shRNA) or GSK3β overexpression construct (d, AnkG shRNA + GSK3β), and GFP expression plasmid. Images were stained for GFP (green), BrdU (red) and Ki67 (blue). e) Increased BrdU incorporation associated with ankyrin-G knockdown is reduced to control levels when ankyrin-G shRNA is co-expressed with GSK3β (Control + Empty Vector, n=4; AnkG shRNA + Empty Vector, n=2; AnkG shRNA+ GSK3β, n=4; Tukey’s Multiple Comparison Test). f) Co-expression of GSK3β with ankyrin-G shRNA rescues the cell cycle exit phenotype assayed using Ki67 proliferative marker (Control + Empty Vector, n=4; AnkG shRNA + Empty Vector, n=2; AnkG shRNA+ GSK3β, n=4; Tukey’s Multiple Comparison Test). g) Distribution of GFP+ cells in different cortical zones 72 hours after transfection at E16. Co-expression of GSK3β with ankyrin-G shRNA reduced the percentage of GFP+ cells in the VZ compared to ankyrin-G knockdown condition (Control + Empty Vector, n=4; AnkG shRNA + Empty Vector, n=2; AnkG shRNA+ GSK3β, n=4; Tukey’s Multiple Comparison Test). h) Working model for ankyrin-G function during cortical development. Left, in control condition ankyrin-G localizes E-cadherin to plasma membrane where it participates in cadherin-catenin complex. Right, in ankyrin-G knockdown condition E-cadherin localized to plasma membrane is reduced, which in turn results in increased free β-catenin available for Wnt signaling. Upregulation of Wnt signaling results in increased neural progenitor proliferation in developing cortex. *, P<0.05; **, P<0.01; ***, P<0.001. Scale bar: 100 µm
Mentions: Our findings suggest that ankyrin-G is a negative regulator of the Wnt signaling pathway, since reduced levels of ankyrin-G lead to upregulated TCF/LEF reporter activity and nuclear β-catenin accumulation. To directly test this hypothesis, we examined the effect of the overexpression of GSK3β, which phosphorylates β-catenin and targets it for ubiquitin-dependent proteasomal degradation41,42, upon on the ankyrin-G loss of function phenotype. In P19 cells, increased TCF/LEF luciferase reporter activity, observed as the result of ankyrin-G shRNA knockdown, was completely reversed by the overexpression of GSK3β (Figure 4a). These results indicate that ankyrin-G functionally interacts with other Wnt signaling proteins such as GSK3β and that the effects of ankyrin-G knockdown on Wnt signaling activity can be normalized by reducing β-catenin abundance. Together these results further suggest that ankyrin-G is a negative regulator of Wnt signaling

Bottom Line: Recent genome-wide association studies and whole-exome sequencing have identified ANK3, the gene coding for ankyrin-G, to be a risk gene for multiple neuropsychiatric disorders, such as bipolar disorder, schizophrenia and autism spectrum disorder.We found that ankyrin-G regulates canonical Wnt signaling by altering the subcellular localization and availability of β-catenin in proliferating cells.Taken together, these results suggest that ankyrin-G is required for proper brain development.

View Article: PubMed Central - PubMed

Affiliation: Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.

ABSTRACT
Ankyrin-G is a scaffolding protein required for the formation of the axon initial segment in neurons. Recent genome-wide association studies and whole-exome sequencing have identified ANK3, the gene coding for ankyrin-G, to be a risk gene for multiple neuropsychiatric disorders, such as bipolar disorder, schizophrenia and autism spectrum disorder. Here, we describe a novel role for ankyrin-G in neural progenitor proliferation in the developing cortex. We found that ankyrin-G regulates canonical Wnt signaling by altering the subcellular localization and availability of β-catenin in proliferating cells. Ankyrin-G loss-of-function increases β-catenin levels in the nucleus, thereby promoting neural progenitor proliferation. Importantly, abnormalities in proliferation can be rescued by reducing Wnt pathway signaling. Taken together, these results suggest that ankyrin-G is required for proper brain development.

No MeSH data available.


Related in: MedlinePlus