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Selective regulation of NR2B by protein phosphatase-1 for the control of the NMDA receptor in neuroprotection.

Farinelli M, Heitz FD, Grewe BF, Tyagarajan SK, Helmchen F, Mansuy IM - PLoS ONE (2012)

Bottom Line: The protective effect of PP1 is associated with a selective dephosphorylation on a residue phosphorylated by CaMKIIα on the NMDA receptor subunit NR2B, which promotes pro-survival pathways and associated transcriptional programs.These results reveal a novel contributor to the mechanisms of neuroprotection and underscore the importance of PP1-dependent dephosphorylation in these mechanisms.They provide a new target for the development of potential therapeutic treatment of neurodegeneration.

View Article: PubMed Central - PubMed

Affiliation: Brain Research Institute, University of Zürich, Zürich, Switzerland.

ABSTRACT
An imbalance between pro-survival and pro-death pathways in brain cells can lead to neuronal cell death and neurodegeneration. While such imbalance is known to be associated with alterations in glutamatergic and Ca(2+) signaling, the underlying mechanisms remain undefined. We identified the protein Ser/Thr phosphatase protein phosphatase-1 (PP1), an enzyme associated with glutamate receptors, as a key trigger of survival pathways that can prevent neuronal death and neurodegeneration in the adult hippocampus. We show that PP1α overexpression in hippocampal neurons limits NMDA receptor overactivation and Ca(2+) overload during an excitotoxic event, while PP1 inhibition favors Ca(2+) overload and cell death. The protective effect of PP1 is associated with a selective dephosphorylation on a residue phosphorylated by CaMKIIα on the NMDA receptor subunit NR2B, which promotes pro-survival pathways and associated transcriptional programs. These results reveal a novel contributor to the mechanisms of neuroprotection and underscore the importance of PP1-dependent dephosphorylation in these mechanisms. They provide a new target for the development of potential therapeutic treatment of neurodegeneration.

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NR2B Ser1303 phosphorylation is necessary and sufficient to drive Ca2+ overload.(a) Expression of wild-type NR2B (NR2B S1303 WT, n = 6) increased OGD-induced ΔF/F (% relative to basal level), while expression of NR2B mutated on Ser1303 (NR2B S1303A, n = 5) was not changed compared to control slices (control, n = 6). C-terminus amino-acid sequence of NR2B S1303 WT and NR2B S1303A mutated version with substitution of serine with alanine on residue 1303 in shaded grey (left inset). Relative fluorescence (ΔF/F) traces of individually recorded CA1 pyramidal neurons in control, NR2B S1303A, and NR2B S1303 WT slices subjected to OGD (middle inset). Image of a CA1 pyramidal neuron expressing the calcium sensitive dye TN-XXL with outlined region of interest (cell soma) used to assess fluorescence and [Ca2+]i (right inset). (b) Quantitative histogram illustrating the area under the curve used as a measure of total intracellular Ca2+ load, given by the calculation of ΔF/F integral normalized to control. *p<0.05. (c) Representative Western blot showing co-immunoprecipitation of NR2B and NR1 with PP1α using a PP1α antibody in mouse hippocampus lysates. No protein interaction was observed with non-specific IgG. PP1α and IgG immunoprecipitation was performed using a comparable amount of antibodies, and samples were loaded on the same blot (same exposure). (d) PP1 directly dephosphorylates Ser1303 in vitro. Immunoblot analyses showing the time course of PP1-mediated dephosphorylation on Ser1303 from a GST-fused NR2B C-terminal peptide. GST-NR2B (aa 1221–1482) was incubated with active PP1 following Ser1303 phosphorylation by CaMKII. NR2B was used as loading control.
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pone-0034047-g004: NR2B Ser1303 phosphorylation is necessary and sufficient to drive Ca2+ overload.(a) Expression of wild-type NR2B (NR2B S1303 WT, n = 6) increased OGD-induced ΔF/F (% relative to basal level), while expression of NR2B mutated on Ser1303 (NR2B S1303A, n = 5) was not changed compared to control slices (control, n = 6). C-terminus amino-acid sequence of NR2B S1303 WT and NR2B S1303A mutated version with substitution of serine with alanine on residue 1303 in shaded grey (left inset). Relative fluorescence (ΔF/F) traces of individually recorded CA1 pyramidal neurons in control, NR2B S1303A, and NR2B S1303 WT slices subjected to OGD (middle inset). Image of a CA1 pyramidal neuron expressing the calcium sensitive dye TN-XXL with outlined region of interest (cell soma) used to assess fluorescence and [Ca2+]i (right inset). (b) Quantitative histogram illustrating the area under the curve used as a measure of total intracellular Ca2+ load, given by the calculation of ΔF/F integral normalized to control. *p<0.05. (c) Representative Western blot showing co-immunoprecipitation of NR2B and NR1 with PP1α using a PP1α antibody in mouse hippocampus lysates. No protein interaction was observed with non-specific IgG. PP1α and IgG immunoprecipitation was performed using a comparable amount of antibodies, and samples were loaded on the same blot (same exposure). (d) PP1 directly dephosphorylates Ser1303 in vitro. Immunoblot analyses showing the time course of PP1-mediated dephosphorylation on Ser1303 from a GST-fused NR2B C-terminal peptide. GST-NR2B (aa 1221–1482) was incubated with active PP1 following Ser1303 phosphorylation by CaMKII. NR2B was used as loading control.

Mentions: To confirm the specific effect of NR2B Ser1303 phosphorylation on Ca2+ overload, we next tested whether a loss of this residue abolishes such overload. We co-expressed a native (NR2B S1303 WT) or mutated NR2B subunit carrying a Ser1303 to Ala1303 substitution (NR2B S1303A) that prevents phosphorylation, with a highly sensitive Ca2+ indicator [28] (TN-XXL) using biolistic transfection in organotypic hippocampal slices. The analysis of isolated post-synaptic NMDAR-dependent Ca2+ influx in CA1 pyramidal neurons expressing native NR2B revealed a rapid and transient two-fold increase in somatic [Ca2+]i during OGD (Figure 4a, b). This increase was prevented by blockade of NR2B Ser1303 phosphorylation in slices expressing NR2B S1303A. The effect of NR2B S1303A expression was not resulting from incorrect trafficking to the surface as observed in transfected Neuro-2a cells (Figure S3 and Supporting Methods S1). Moreover, there was no significant difference in TN-XXL expression when comparing cells transfected with the different NR2B variants (Figure S4 and Supporting Methods S1). These data overall strongly suggest that Ser1303 phosphorylation is required for OGD-induced Ca2+ overload.


Selective regulation of NR2B by protein phosphatase-1 for the control of the NMDA receptor in neuroprotection.

Farinelli M, Heitz FD, Grewe BF, Tyagarajan SK, Helmchen F, Mansuy IM - PLoS ONE (2012)

NR2B Ser1303 phosphorylation is necessary and sufficient to drive Ca2+ overload.(a) Expression of wild-type NR2B (NR2B S1303 WT, n = 6) increased OGD-induced ΔF/F (% relative to basal level), while expression of NR2B mutated on Ser1303 (NR2B S1303A, n = 5) was not changed compared to control slices (control, n = 6). C-terminus amino-acid sequence of NR2B S1303 WT and NR2B S1303A mutated version with substitution of serine with alanine on residue 1303 in shaded grey (left inset). Relative fluorescence (ΔF/F) traces of individually recorded CA1 pyramidal neurons in control, NR2B S1303A, and NR2B S1303 WT slices subjected to OGD (middle inset). Image of a CA1 pyramidal neuron expressing the calcium sensitive dye TN-XXL with outlined region of interest (cell soma) used to assess fluorescence and [Ca2+]i (right inset). (b) Quantitative histogram illustrating the area under the curve used as a measure of total intracellular Ca2+ load, given by the calculation of ΔF/F integral normalized to control. *p<0.05. (c) Representative Western blot showing co-immunoprecipitation of NR2B and NR1 with PP1α using a PP1α antibody in mouse hippocampus lysates. No protein interaction was observed with non-specific IgG. PP1α and IgG immunoprecipitation was performed using a comparable amount of antibodies, and samples were loaded on the same blot (same exposure). (d) PP1 directly dephosphorylates Ser1303 in vitro. Immunoblot analyses showing the time course of PP1-mediated dephosphorylation on Ser1303 from a GST-fused NR2B C-terminal peptide. GST-NR2B (aa 1221–1482) was incubated with active PP1 following Ser1303 phosphorylation by CaMKII. NR2B was used as loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316588&req=5

pone-0034047-g004: NR2B Ser1303 phosphorylation is necessary and sufficient to drive Ca2+ overload.(a) Expression of wild-type NR2B (NR2B S1303 WT, n = 6) increased OGD-induced ΔF/F (% relative to basal level), while expression of NR2B mutated on Ser1303 (NR2B S1303A, n = 5) was not changed compared to control slices (control, n = 6). C-terminus amino-acid sequence of NR2B S1303 WT and NR2B S1303A mutated version with substitution of serine with alanine on residue 1303 in shaded grey (left inset). Relative fluorescence (ΔF/F) traces of individually recorded CA1 pyramidal neurons in control, NR2B S1303A, and NR2B S1303 WT slices subjected to OGD (middle inset). Image of a CA1 pyramidal neuron expressing the calcium sensitive dye TN-XXL with outlined region of interest (cell soma) used to assess fluorescence and [Ca2+]i (right inset). (b) Quantitative histogram illustrating the area under the curve used as a measure of total intracellular Ca2+ load, given by the calculation of ΔF/F integral normalized to control. *p<0.05. (c) Representative Western blot showing co-immunoprecipitation of NR2B and NR1 with PP1α using a PP1α antibody in mouse hippocampus lysates. No protein interaction was observed with non-specific IgG. PP1α and IgG immunoprecipitation was performed using a comparable amount of antibodies, and samples were loaded on the same blot (same exposure). (d) PP1 directly dephosphorylates Ser1303 in vitro. Immunoblot analyses showing the time course of PP1-mediated dephosphorylation on Ser1303 from a GST-fused NR2B C-terminal peptide. GST-NR2B (aa 1221–1482) was incubated with active PP1 following Ser1303 phosphorylation by CaMKII. NR2B was used as loading control.
Mentions: To confirm the specific effect of NR2B Ser1303 phosphorylation on Ca2+ overload, we next tested whether a loss of this residue abolishes such overload. We co-expressed a native (NR2B S1303 WT) or mutated NR2B subunit carrying a Ser1303 to Ala1303 substitution (NR2B S1303A) that prevents phosphorylation, with a highly sensitive Ca2+ indicator [28] (TN-XXL) using biolistic transfection in organotypic hippocampal slices. The analysis of isolated post-synaptic NMDAR-dependent Ca2+ influx in CA1 pyramidal neurons expressing native NR2B revealed a rapid and transient two-fold increase in somatic [Ca2+]i during OGD (Figure 4a, b). This increase was prevented by blockade of NR2B Ser1303 phosphorylation in slices expressing NR2B S1303A. The effect of NR2B S1303A expression was not resulting from incorrect trafficking to the surface as observed in transfected Neuro-2a cells (Figure S3 and Supporting Methods S1). Moreover, there was no significant difference in TN-XXL expression when comparing cells transfected with the different NR2B variants (Figure S4 and Supporting Methods S1). These data overall strongly suggest that Ser1303 phosphorylation is required for OGD-induced Ca2+ overload.

Bottom Line: The protective effect of PP1 is associated with a selective dephosphorylation on a residue phosphorylated by CaMKIIα on the NMDA receptor subunit NR2B, which promotes pro-survival pathways and associated transcriptional programs.These results reveal a novel contributor to the mechanisms of neuroprotection and underscore the importance of PP1-dependent dephosphorylation in these mechanisms.They provide a new target for the development of potential therapeutic treatment of neurodegeneration.

View Article: PubMed Central - PubMed

Affiliation: Brain Research Institute, University of Zürich, Zürich, Switzerland.

ABSTRACT
An imbalance between pro-survival and pro-death pathways in brain cells can lead to neuronal cell death and neurodegeneration. While such imbalance is known to be associated with alterations in glutamatergic and Ca(2+) signaling, the underlying mechanisms remain undefined. We identified the protein Ser/Thr phosphatase protein phosphatase-1 (PP1), an enzyme associated with glutamate receptors, as a key trigger of survival pathways that can prevent neuronal death and neurodegeneration in the adult hippocampus. We show that PP1α overexpression in hippocampal neurons limits NMDA receptor overactivation and Ca(2+) overload during an excitotoxic event, while PP1 inhibition favors Ca(2+) overload and cell death. The protective effect of PP1 is associated with a selective dephosphorylation on a residue phosphorylated by CaMKIIα on the NMDA receptor subunit NR2B, which promotes pro-survival pathways and associated transcriptional programs. These results reveal a novel contributor to the mechanisms of neuroprotection and underscore the importance of PP1-dependent dephosphorylation in these mechanisms. They provide a new target for the development of potential therapeutic treatment of neurodegeneration.

Show MeSH
Related in: MedlinePlus