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Kainate receptor activation induces glycine receptor endocytosis through PKC deSUMOylation.

Sun H, Lu L, Zuo Y, Wang Y, Jiao Y, Zeng WZ, Huang C, Zhu MX, Zamponi GW, Zhou T, Xu TL, Cheng J, Li Y - Nat Commun (2014)

Bottom Line: Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices.This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC.SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

ABSTRACT
Surface expression and regulated endocytosis of glycine receptors (GlyRs) play a critical function in balancing neuronal excitability. SUMOylation (SUMO modification) is of critical importance for maintaining neuronal function in the central nervous system. Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices. This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC. SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels. Altogether, we have identified a SUMOylation-dependent regulatory pathway for GlyR endocytosis, which may have important physiological implications for proper neuronal excitability.

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Kainate induces GlyR internalization in spinal cord neurons.(a) Antibody-feeding assay under non-permeabilized conditions labelled cell surface GlyRs (green) in cultured rat spinal cord neurons untreated (Control) or briefly treated with kainate (KA, 200 μM, 1 min). Neurons were then permeabilized and labelled with MAP2 (blue) to show cell body and processes. Puncta numbers and integrated fluorescence intensities of surface GlyRs from all images analysed by ImageJ and values are normalized to untreated controls. Data are means±s.e.m. from at least three experiments; the total numbers of neurons analysed (n) ranged from 66 to 71 cells per condition. ***P<0.001 compared with control by Student’s t-test. Scale bar, 10 μm. (b) Endocytosis of GlyRs was visualized by the antibody-feeding immunofluorescence assay between untreated (Control) or briefly treated with kainate (KA) for 1 min. Pre-labelled GlyRs remaining on the surface are in green; internalized receptors are in red. Magnified view of the region enclosed by the white square in the lower panel. Scale bar, 10 μm (original, upper) and 1 μm (magnified, lower). (c) Surface biotinylation of GlyRs in cultured rat spinal cord neurons untreated (control) or treated with kainate (KA, 1 min). Endogenous transferrin receptor (TfR) is shown as a surface protein control and endogenous glyceraldehyde 3-phosphate dehydrogenase as a cytoplasmic protein control. S, surface; T, total; Ctrl, control. KA treatment decreased surface GlyRs to 73.6±4.3% (means±s.e.m.) of the untreated control. *P<0.05 compared with untreated control by Student’s paired t-test P=0.0258, n=3. (d) NMDA treatment (30 μM, 1 min) did not evoke significant change in the number and intensity of surface GlyRs as quantified by the antibody-feeding assay under non-permeabilized conditions. Data are shown as in d with the bar graphs representing the means±s.e.m. from at least three experiments; the total numbers of neurons analysed (n) ranged from 28 to 38 cells per condition. Scale bar, 10 μm.
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f1: Kainate induces GlyR internalization in spinal cord neurons.(a) Antibody-feeding assay under non-permeabilized conditions labelled cell surface GlyRs (green) in cultured rat spinal cord neurons untreated (Control) or briefly treated with kainate (KA, 200 μM, 1 min). Neurons were then permeabilized and labelled with MAP2 (blue) to show cell body and processes. Puncta numbers and integrated fluorescence intensities of surface GlyRs from all images analysed by ImageJ and values are normalized to untreated controls. Data are means±s.e.m. from at least three experiments; the total numbers of neurons analysed (n) ranged from 66 to 71 cells per condition. ***P<0.001 compared with control by Student’s t-test. Scale bar, 10 μm. (b) Endocytosis of GlyRs was visualized by the antibody-feeding immunofluorescence assay between untreated (Control) or briefly treated with kainate (KA) for 1 min. Pre-labelled GlyRs remaining on the surface are in green; internalized receptors are in red. Magnified view of the region enclosed by the white square in the lower panel. Scale bar, 10 μm (original, upper) and 1 μm (magnified, lower). (c) Surface biotinylation of GlyRs in cultured rat spinal cord neurons untreated (control) or treated with kainate (KA, 1 min). Endogenous transferrin receptor (TfR) is shown as a surface protein control and endogenous glyceraldehyde 3-phosphate dehydrogenase as a cytoplasmic protein control. S, surface; T, total; Ctrl, control. KA treatment decreased surface GlyRs to 73.6±4.3% (means±s.e.m.) of the untreated control. *P<0.05 compared with untreated control by Student’s paired t-test P=0.0258, n=3. (d) NMDA treatment (30 μM, 1 min) did not evoke significant change in the number and intensity of surface GlyRs as quantified by the antibody-feeding assay under non-permeabilized conditions. Data are shown as in d with the bar graphs representing the means±s.e.m. from at least three experiments; the total numbers of neurons analysed (n) ranged from 28 to 38 cells per condition. Scale bar, 10 μm.

Mentions: KARs modulate neurotransmitter release at the presynaptic terminal and contribute to fast excitatory synaptic transmission at the postsynaptic membrane2728293031323334. Cell surface density/stability of GlyRs is dynamically regulated in a PKC-dependent manner by receptor endocytosis and exocytosis1617. Given that KAR activation can trigger PKC signalling5212535, we hypothesized that KARs may alter the dynamics of GlyR trafficking. To test this possibility, cell surface GlyRs were labelled using antibodies raised against either amino acids 1–10 of the GlyR α1 subunit or the extracellular amino termini of GlyRs on cultured live spinal cord neurons under non-permeabilized conditions. Under control conditions, surface fluorescent immunoreactivity of GlyRs exhibited a punctate distribution over the neuronal dendrites and soma (Fig. 1a). A brief treatment with kainate induced a marked decrease in the number of puncta and fluorescence intensity of surface GlyRs (Fig. 1a). The effect of kainate was dose dependent, being detectable at as low as 0.5 μM and approaching the maximum at around 10 μM (Supplementary Fig. 1a). Visualizing the internalized GlyRs under permeabilized conditions in an antibody-feeding immunofluorescence assay also revealed increases in the number and intensity of GlyR-positive labels in the spinal cord neuron dendrites in response to kainate treatment (Fig. 1b), and the kainate-induced loss of GlyRs from the cell surface was further confirmed by the spinal cord neuron surface biotinylation experiment (Fig. 1c). On the other hand, co-staining with a presynaptic marker glycine transporter 2 showed no obvious loss of synapses (Supplementary Fig. 1c), indicating that the loss of surface GlyRs was solely due to receptor internalization but not a decrease in the number of synapses. In contrast, treatment with NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) did not significantly change the surface number or intensity of GlyRs (Fig. 1d; Supplementary Fig. 1b), suggesting a specific regulation of surface GlyRs by kainate.


Kainate receptor activation induces glycine receptor endocytosis through PKC deSUMOylation.

Sun H, Lu L, Zuo Y, Wang Y, Jiao Y, Zeng WZ, Huang C, Zhu MX, Zamponi GW, Zhou T, Xu TL, Cheng J, Li Y - Nat Commun (2014)

Kainate induces GlyR internalization in spinal cord neurons.(a) Antibody-feeding assay under non-permeabilized conditions labelled cell surface GlyRs (green) in cultured rat spinal cord neurons untreated (Control) or briefly treated with kainate (KA, 200 μM, 1 min). Neurons were then permeabilized and labelled with MAP2 (blue) to show cell body and processes. Puncta numbers and integrated fluorescence intensities of surface GlyRs from all images analysed by ImageJ and values are normalized to untreated controls. Data are means±s.e.m. from at least three experiments; the total numbers of neurons analysed (n) ranged from 66 to 71 cells per condition. ***P<0.001 compared with control by Student’s t-test. Scale bar, 10 μm. (b) Endocytosis of GlyRs was visualized by the antibody-feeding immunofluorescence assay between untreated (Control) or briefly treated with kainate (KA) for 1 min. Pre-labelled GlyRs remaining on the surface are in green; internalized receptors are in red. Magnified view of the region enclosed by the white square in the lower panel. Scale bar, 10 μm (original, upper) and 1 μm (magnified, lower). (c) Surface biotinylation of GlyRs in cultured rat spinal cord neurons untreated (control) or treated with kainate (KA, 1 min). Endogenous transferrin receptor (TfR) is shown as a surface protein control and endogenous glyceraldehyde 3-phosphate dehydrogenase as a cytoplasmic protein control. S, surface; T, total; Ctrl, control. KA treatment decreased surface GlyRs to 73.6±4.3% (means±s.e.m.) of the untreated control. *P<0.05 compared with untreated control by Student’s paired t-test P=0.0258, n=3. (d) NMDA treatment (30 μM, 1 min) did not evoke significant change in the number and intensity of surface GlyRs as quantified by the antibody-feeding assay under non-permeabilized conditions. Data are shown as in d with the bar graphs representing the means±s.e.m. from at least three experiments; the total numbers of neurons analysed (n) ranged from 28 to 38 cells per condition. Scale bar, 10 μm.
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f1: Kainate induces GlyR internalization in spinal cord neurons.(a) Antibody-feeding assay under non-permeabilized conditions labelled cell surface GlyRs (green) in cultured rat spinal cord neurons untreated (Control) or briefly treated with kainate (KA, 200 μM, 1 min). Neurons were then permeabilized and labelled with MAP2 (blue) to show cell body and processes. Puncta numbers and integrated fluorescence intensities of surface GlyRs from all images analysed by ImageJ and values are normalized to untreated controls. Data are means±s.e.m. from at least three experiments; the total numbers of neurons analysed (n) ranged from 66 to 71 cells per condition. ***P<0.001 compared with control by Student’s t-test. Scale bar, 10 μm. (b) Endocytosis of GlyRs was visualized by the antibody-feeding immunofluorescence assay between untreated (Control) or briefly treated with kainate (KA) for 1 min. Pre-labelled GlyRs remaining on the surface are in green; internalized receptors are in red. Magnified view of the region enclosed by the white square in the lower panel. Scale bar, 10 μm (original, upper) and 1 μm (magnified, lower). (c) Surface biotinylation of GlyRs in cultured rat spinal cord neurons untreated (control) or treated with kainate (KA, 1 min). Endogenous transferrin receptor (TfR) is shown as a surface protein control and endogenous glyceraldehyde 3-phosphate dehydrogenase as a cytoplasmic protein control. S, surface; T, total; Ctrl, control. KA treatment decreased surface GlyRs to 73.6±4.3% (means±s.e.m.) of the untreated control. *P<0.05 compared with untreated control by Student’s paired t-test P=0.0258, n=3. (d) NMDA treatment (30 μM, 1 min) did not evoke significant change in the number and intensity of surface GlyRs as quantified by the antibody-feeding assay under non-permeabilized conditions. Data are shown as in d with the bar graphs representing the means±s.e.m. from at least three experiments; the total numbers of neurons analysed (n) ranged from 28 to 38 cells per condition. Scale bar, 10 μm.
Mentions: KARs modulate neurotransmitter release at the presynaptic terminal and contribute to fast excitatory synaptic transmission at the postsynaptic membrane2728293031323334. Cell surface density/stability of GlyRs is dynamically regulated in a PKC-dependent manner by receptor endocytosis and exocytosis1617. Given that KAR activation can trigger PKC signalling5212535, we hypothesized that KARs may alter the dynamics of GlyR trafficking. To test this possibility, cell surface GlyRs were labelled using antibodies raised against either amino acids 1–10 of the GlyR α1 subunit or the extracellular amino termini of GlyRs on cultured live spinal cord neurons under non-permeabilized conditions. Under control conditions, surface fluorescent immunoreactivity of GlyRs exhibited a punctate distribution over the neuronal dendrites and soma (Fig. 1a). A brief treatment with kainate induced a marked decrease in the number of puncta and fluorescence intensity of surface GlyRs (Fig. 1a). The effect of kainate was dose dependent, being detectable at as low as 0.5 μM and approaching the maximum at around 10 μM (Supplementary Fig. 1a). Visualizing the internalized GlyRs under permeabilized conditions in an antibody-feeding immunofluorescence assay also revealed increases in the number and intensity of GlyR-positive labels in the spinal cord neuron dendrites in response to kainate treatment (Fig. 1b), and the kainate-induced loss of GlyRs from the cell surface was further confirmed by the spinal cord neuron surface biotinylation experiment (Fig. 1c). On the other hand, co-staining with a presynaptic marker glycine transporter 2 showed no obvious loss of synapses (Supplementary Fig. 1c), indicating that the loss of surface GlyRs was solely due to receptor internalization but not a decrease in the number of synapses. In contrast, treatment with NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) did not significantly change the surface number or intensity of GlyRs (Fig. 1d; Supplementary Fig. 1b), suggesting a specific regulation of surface GlyRs by kainate.

Bottom Line: Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices.This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC.SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

ABSTRACT
Surface expression and regulated endocytosis of glycine receptors (GlyRs) play a critical function in balancing neuronal excitability. SUMOylation (SUMO modification) is of critical importance for maintaining neuronal function in the central nervous system. Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices. This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC. SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels. Altogether, we have identified a SUMOylation-dependent regulatory pathway for GlyR endocytosis, which may have important physiological implications for proper neuronal excitability.

Show MeSH
Related in: MedlinePlus