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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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Inducible and neuron-specific PP1α expression in hippocampus.(a) Schematic representation of the genetic system used to achieve doxycycline-dependent and neuron-specific PP1α or PP1α-EGFP expression in mouse organotypic hippocampal slices. Upon doxycycline treatment, binding of the tetracycline repressor and Krüppel-associated box fusion protein (tTR-KRAB) to the tetracycline response element (tetO) is blocked, which prevents the epigenetic silencing of the human prion (hPrion) promoter and allows PP1α or PP1α-EGFP expression. (b) EGFP (green) and NeuN (red) co-immunostaining in CA1 area showing neuron-specific PP1α-EGFP expression. Scale bar, 400 µm in A–C, 200 µm in D–F, and 25 µm in G–I. (c) Real-time quantitative RT-PCR showing increased PP1α mRNA expression in slices injected with PP1α (n = 14) compared to control slices (n = 8). Data is expressed as relative quantification. *p<0.05. Error bars represent mean SEM.
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pone-0034047-g001: Inducible and neuron-specific PP1α expression in hippocampus.(a) Schematic representation of the genetic system used to achieve doxycycline-dependent and neuron-specific PP1α or PP1α-EGFP expression in mouse organotypic hippocampal slices. Upon doxycycline treatment, binding of the tetracycline repressor and Krüppel-associated box fusion protein (tTR-KRAB) to the tetracycline response element (tetO) is blocked, which prevents the epigenetic silencing of the human prion (hPrion) promoter and allows PP1α or PP1α-EGFP expression. (b) EGFP (green) and NeuN (red) co-immunostaining in CA1 area showing neuron-specific PP1α-EGFP expression. Scale bar, 400 µm in A–C, 200 µm in D–F, and 25 µm in G–I. (c) Real-time quantitative RT-PCR showing increased PP1α mRNA expression in slices injected with PP1α (n = 14) compared to control slices (n = 8). Data is expressed as relative quantification. *p<0.05. Error bars represent mean SEM.

Mentions: To address the importance of PP1 in NMDAR-dependent excitotoxicity in the adult hippocampus, we conditionally expressed PP1α in CA1 hippocampal neurons using a lentivirus approach. We chose CA1 neurons because they are highly vulnerable to excitotoxicity [30]. We generated vectors expressing PP1α alone (PP1α) or PP1α fused to enhanced green fluorescent protein (PP1α-EGFP) under the control of the neuron-specific human prion (hPrion) promoter (Figure 1a). These vectors were used to overexpress PP1 in organotypic hippocampal slices. In the slices injected with the PP1α-EGFP vector, EGFP/NeuN co-staining confirmed the neuronal specificity of PP1α expression, and showed that expression is homogenous and mainly distributed across CA1 neurons but is also present in CA3 and dentate gyrus neurons (Figure 1b). Quantitative real-time RT-PCR showed that the PP1α vector increased PP1α mRNA expression in the hippocampus by 36±11% (Figure 1c). PP1α protein shows a somato-dendritic distribution with enrichment in dendritic spines (Figure S1a and Supporting Methods S1), suggesting its presence at glutamatergic synapses. In contrast, the other major catalytic isoform PP1γ is enriched in the nucleus (Figure S1b and Supporting Methods S1).


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)

Inducible and neuron-specific PP1α expression in hippocampus.(a) Schematic representation of the genetic system used to achieve doxycycline-dependent and neuron-specific PP1α or PP1α-EGFP expression in mouse organotypic hippocampal slices. Upon doxycycline treatment, binding of the tetracycline repressor and Krüppel-associated box fusion protein (tTR-KRAB) to the tetracycline response element (tetO) is blocked, which prevents the epigenetic silencing of the human prion (hPrion) promoter and allows PP1α or PP1α-EGFP expression. (b) EGFP (green) and NeuN (red) co-immunostaining in CA1 area showing neuron-specific PP1α-EGFP expression. Scale bar, 400 µm in A–C, 200 µm in D–F, and 25 µm in G–I. (c) Real-time quantitative RT-PCR showing increased PP1α mRNA expression in slices injected with PP1α (n = 14) compared to control slices (n = 8). Data is expressed as relative quantification. *p<0.05. Error bars represent mean SEM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316588&req=5

pone-0034047-g001: Inducible and neuron-specific PP1α expression in hippocampus.(a) Schematic representation of the genetic system used to achieve doxycycline-dependent and neuron-specific PP1α or PP1α-EGFP expression in mouse organotypic hippocampal slices. Upon doxycycline treatment, binding of the tetracycline repressor and Krüppel-associated box fusion protein (tTR-KRAB) to the tetracycline response element (tetO) is blocked, which prevents the epigenetic silencing of the human prion (hPrion) promoter and allows PP1α or PP1α-EGFP expression. (b) EGFP (green) and NeuN (red) co-immunostaining in CA1 area showing neuron-specific PP1α-EGFP expression. Scale bar, 400 µm in A–C, 200 µm in D–F, and 25 µm in G–I. (c) Real-time quantitative RT-PCR showing increased PP1α mRNA expression in slices injected with PP1α (n = 14) compared to control slices (n = 8). Data is expressed as relative quantification. *p<0.05. Error bars represent mean SEM.
Mentions: To address the importance of PP1 in NMDAR-dependent excitotoxicity in the adult hippocampus, we conditionally expressed PP1α in CA1 hippocampal neurons using a lentivirus approach. We chose CA1 neurons because they are highly vulnerable to excitotoxicity [30]. We generated vectors expressing PP1α alone (PP1α) or PP1α fused to enhanced green fluorescent protein (PP1α-EGFP) under the control of the neuron-specific human prion (hPrion) promoter (Figure 1a). These vectors were used to overexpress PP1 in organotypic hippocampal slices. In the slices injected with the PP1α-EGFP vector, EGFP/NeuN co-staining confirmed the neuronal specificity of PP1α expression, and showed that expression is homogenous and mainly distributed across CA1 neurons but is also present in CA3 and dentate gyrus neurons (Figure 1b). Quantitative real-time RT-PCR showed that the PP1α vector increased PP1α mRNA expression in the hippocampus by 36±11% (Figure 1c). PP1α protein shows a somato-dendritic distribution with enrichment in dendritic spines (Figure S1a and Supporting Methods S1), suggesting its presence at glutamatergic synapses. In contrast, the other major catalytic isoform PP1γ is enriched in the nucleus (Figure S1b and Supporting Methods S1).

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