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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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PP1α reverses CaMKIIα Thr286 phosphorylation and mediates the dephosphorylation of NR2B Ser1303 upon OGD.(a) Representative Western blots and corresponding quantitative analysis of CaMKIIα Thr286 phosphorylation. CaMKIIα phosphorylation significantly increases 1 min after OGD (control 1 min post-OGD, n = 8) with no significant change at 16 min (control 16 min post-OGD, n = 6) in control slices. PP1α slices do not display any change in CaMKIIα phosphorylation either 1 min (PP1α 1 min post-OGD, n = 6) or 16 min post-OGD (PP1α 16 min post-OGD, n = 7) compared to control. Phospho-protein levels were normalized to non-phosphorylated protein levels and β-actin was used as a loading control. Quantitative data for each condition were normalized to levels of non-OGD condition (control non-OGD, n = 14) from the same blot and exposure. *p<0.05. (b) Representative Western blots and corresponding quantitative analysis of NR2B Ser1303 phosphorylation. Increased level of phospho-NR2B in control slices 1 min after OGD (control 1 min post-OGD, n = 9), but not 16 min after OGD (control 16 min post-OGD, n = 8). PP1α expression or KN-93 treatment blocks this increase (PP1α 1 min post-OGD, n = 6; KN-93 1 min post-OGD, n = 6), but has no effect 16 min post-OGD (PP1α 16 min post-OGD, n = 8; KN-93 16 min post-OGD, n = 5). Phospho-protein levels were normalized to non-phosphorylated protein levels, and β-actin was used as a loading control. Quantitative data for each condition were normalized to levels of non-OGD condition (control non-OGD, n = 14) from the same blot and exposure. *p<0.05.
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pone-0034047-g003: PP1α reverses CaMKIIα Thr286 phosphorylation and mediates the dephosphorylation of NR2B Ser1303 upon OGD.(a) Representative Western blots and corresponding quantitative analysis of CaMKIIα Thr286 phosphorylation. CaMKIIα phosphorylation significantly increases 1 min after OGD (control 1 min post-OGD, n = 8) with no significant change at 16 min (control 16 min post-OGD, n = 6) in control slices. PP1α slices do not display any change in CaMKIIα phosphorylation either 1 min (PP1α 1 min post-OGD, n = 6) or 16 min post-OGD (PP1α 16 min post-OGD, n = 7) compared to control. Phospho-protein levels were normalized to non-phosphorylated protein levels and β-actin was used as a loading control. Quantitative data for each condition were normalized to levels of non-OGD condition (control non-OGD, n = 14) from the same blot and exposure. *p<0.05. (b) Representative Western blots and corresponding quantitative analysis of NR2B Ser1303 phosphorylation. Increased level of phospho-NR2B in control slices 1 min after OGD (control 1 min post-OGD, n = 9), but not 16 min after OGD (control 16 min post-OGD, n = 8). PP1α expression or KN-93 treatment blocks this increase (PP1α 1 min post-OGD, n = 6; KN-93 1 min post-OGD, n = 6), but has no effect 16 min post-OGD (PP1α 16 min post-OGD, n = 8; KN-93 16 min post-OGD, n = 5). Phospho-protein levels were normalized to non-phosphorylated protein levels, and β-actin was used as a loading control. Quantitative data for each condition were normalized to levels of non-OGD condition (control non-OGD, n = 14) from the same blot and exposure. *p<0.05.

Mentions: Changes in intracellular [Ca2+]i are known to modulate Ca2+-dependent signaling pathways and alter the activity of Ca2+-activated enzymes, in particular Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII is a serine/threonine protein kinase that can autophosphorylate on several residues in the presence of Ca2+[38]. Autophosphorylation on Thr286 confers autonomous Ca2+-independent activity to the enzyme [38], [39], [40], and promotes its association with the PSD [41]. We examined whether CaMKII Thr286 phosphorylation is modulated by OGD in our experimental conditions, and whether PP1α influences this autophosphorylation. Western blot analyses revealed that Thr286 phosphorylation was significantly increased 1 min after OGD (1.4 fold+/−0.1), a time point that corresponds to maximum [Ca2+]i peak, then returned to baseline within 16 min in control slices (Figure 3a). This effect was specific to CaMKIIα phosphorylation and was not accompanied with any change in the total level of the protein. Since in the PSD, CaMKIIα can be dephosphorylated by PP1 [41], we also investigated the effect of PP1α overexpression on Thr286 phosphorylation. We observed that CaMKII Thr286 phosphorylation was significantly reduced by PP1α overexpression 1 min after OGD (Figure 3a). PP1α overexpression did not induce any change in CaMKII Thr286 phosphorylation.


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)

PP1α reverses CaMKIIα Thr286 phosphorylation and mediates the dephosphorylation of NR2B Ser1303 upon OGD.(a) Representative Western blots and corresponding quantitative analysis of CaMKIIα Thr286 phosphorylation. CaMKIIα phosphorylation significantly increases 1 min after OGD (control 1 min post-OGD, n = 8) with no significant change at 16 min (control 16 min post-OGD, n = 6) in control slices. PP1α slices do not display any change in CaMKIIα phosphorylation either 1 min (PP1α 1 min post-OGD, n = 6) or 16 min post-OGD (PP1α 16 min post-OGD, n = 7) compared to control. Phospho-protein levels were normalized to non-phosphorylated protein levels and β-actin was used as a loading control. Quantitative data for each condition were normalized to levels of non-OGD condition (control non-OGD, n = 14) from the same blot and exposure. *p<0.05. (b) Representative Western blots and corresponding quantitative analysis of NR2B Ser1303 phosphorylation. Increased level of phospho-NR2B in control slices 1 min after OGD (control 1 min post-OGD, n = 9), but not 16 min after OGD (control 16 min post-OGD, n = 8). PP1α expression or KN-93 treatment blocks this increase (PP1α 1 min post-OGD, n = 6; KN-93 1 min post-OGD, n = 6), but has no effect 16 min post-OGD (PP1α 16 min post-OGD, n = 8; KN-93 16 min post-OGD, n = 5). Phospho-protein levels were normalized to non-phosphorylated protein levels, and β-actin was used as a loading control. Quantitative data for each condition were normalized to levels of non-OGD condition (control non-OGD, n = 14) from the same blot and exposure. *p<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316588&req=5

pone-0034047-g003: PP1α reverses CaMKIIα Thr286 phosphorylation and mediates the dephosphorylation of NR2B Ser1303 upon OGD.(a) Representative Western blots and corresponding quantitative analysis of CaMKIIα Thr286 phosphorylation. CaMKIIα phosphorylation significantly increases 1 min after OGD (control 1 min post-OGD, n = 8) with no significant change at 16 min (control 16 min post-OGD, n = 6) in control slices. PP1α slices do not display any change in CaMKIIα phosphorylation either 1 min (PP1α 1 min post-OGD, n = 6) or 16 min post-OGD (PP1α 16 min post-OGD, n = 7) compared to control. Phospho-protein levels were normalized to non-phosphorylated protein levels and β-actin was used as a loading control. Quantitative data for each condition were normalized to levels of non-OGD condition (control non-OGD, n = 14) from the same blot and exposure. *p<0.05. (b) Representative Western blots and corresponding quantitative analysis of NR2B Ser1303 phosphorylation. Increased level of phospho-NR2B in control slices 1 min after OGD (control 1 min post-OGD, n = 9), but not 16 min after OGD (control 16 min post-OGD, n = 8). PP1α expression or KN-93 treatment blocks this increase (PP1α 1 min post-OGD, n = 6; KN-93 1 min post-OGD, n = 6), but has no effect 16 min post-OGD (PP1α 16 min post-OGD, n = 8; KN-93 16 min post-OGD, n = 5). Phospho-protein levels were normalized to non-phosphorylated protein levels, and β-actin was used as a loading control. Quantitative data for each condition were normalized to levels of non-OGD condition (control non-OGD, n = 14) from the same blot and exposure. *p<0.05.
Mentions: Changes in intracellular [Ca2+]i are known to modulate Ca2+-dependent signaling pathways and alter the activity of Ca2+-activated enzymes, in particular Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII is a serine/threonine protein kinase that can autophosphorylate on several residues in the presence of Ca2+[38]. Autophosphorylation on Thr286 confers autonomous Ca2+-independent activity to the enzyme [38], [39], [40], and promotes its association with the PSD [41]. We examined whether CaMKII Thr286 phosphorylation is modulated by OGD in our experimental conditions, and whether PP1α influences this autophosphorylation. Western blot analyses revealed that Thr286 phosphorylation was significantly increased 1 min after OGD (1.4 fold+/−0.1), a time point that corresponds to maximum [Ca2+]i peak, then returned to baseline within 16 min in control slices (Figure 3a). This effect was specific to CaMKIIα phosphorylation and was not accompanied with any change in the total level of the protein. Since in the PSD, CaMKIIα can be dephosphorylated by PP1 [41], we also investigated the effect of PP1α overexpression on Thr286 phosphorylation. We observed that CaMKII Thr286 phosphorylation was significantly reduced by PP1α overexpression 1 min after OGD (Figure 3a). PP1α overexpression did not induce any change in CaMKII Thr286 phosphorylation.

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