SUMOylation Is Required for Glycine-Induced Increases in AMPA Receptor Surface Expression (ChemLTP) in Hippocampal Neurons

Multiple pathways participate in the AMPA receptor trafficking that underlies long-term potentiation (LTP) of synaptic transmission. Here we demonstrate that protein SUMOylation is required for insertion of the GluA1 AMPAR subunit following transient glycine-evoked increase in AMPA receptor surface expression (ChemLTP) in dispersed neuronal cultures. ChemLTP increases co-localisation of SUMO-1 and the SUMO conjugating enzyme Ubc9 and with PSD95 consistent with the recruitment of SUMOylated proteins to dendritic spines. In addition, we show that ChemLTP increases dendritic levels of SUMO-1 and Ubc9 mRNA. Consistent with activity dependent translocation of these mRNAs to sites near synapses, levels of the mRNA binding and dendritic transport protein CPEB are also increased by ChemLTP. Importantly, reducing the extent of substrate protein SUMOylation by overexpressing the deSUMOylating enzyme SENP-1 or inhibiting SUMOylation by expressing dominant negative Ubc9 prevent the ChemLTP-induced increase in both AMPAR surface expression and dendritic SUMO-1 mRNA. Taken together these data demonstrate that SUMOylation of synaptic protein(s) involved in AMPA receptor trafficking is necessary for activity-dependent increases in AMPAR surface expression

ChemLTP increases GluA1 surface expression.

<p>A) Schematic of the ChemLTP protocol. Dissociated hippocampal neuronal cultures (DIV 17–20) were treated with glycine (200 µM) for 3 min, then incubated in media without glycine for 5, 20 or 40 min. B) ChemLTP increases surface GluA1. Graph shows quantification of the ratio of surface to total GluA1. Statistical significance was determined using ANOVA. n = 5 ***p<0.005, ***p<0.001. C) ChemLTP Increases surface expression of endogenous GluA1-containing AMPARs in dissociated hippocampal neurons expressing EGFP. Surface and internal GluA1 were sequentially labeled under non-permeabilised and permeabilised conditions, respectively, with different coloured secondary antibodies. Representative images are shown. Graph shows surface:internal ratio relative to unstimulated control. n = 12–15 cells per condition in 3 independent experiments. ANOVA **p<0.005 and ***p<0.001. Scale bars – 20 µm.</p

ChemLTP increases SUMO-1 and Ubc9 mRNA.

<p>A) Fluorescent in situ hybridization using cRNA probes for SUMO-1, SUMO-2 and Ubc9 (red) in hippocampal neurons co-stained with the dendritic marker MAP-2 (green). ChemLTP increased levels of SUMO-1 and Ubc9 but not SUMO-2 mRNA in soma and dendrites. Mean fluoresence intensity was quantified in at least 3 independent experiments with 10 neurons per condition and 3 ROIs per neuron using ImageJ. B) ChemLTP increases levels of the mRNA binding protein CPEB1 (red) in hippocampal cultured neurons co-immunostained with MAP2 (green). Three independent experiments with 8 neurons per condition and 4 ROIs per neuron were quantified using JACoP plugin in ImageJ. Statistical significance was determined using ANOVA *p<0.02; **p<0.005. Scale bars – 20 µm.</p

ChemLTP upregulates of SUMO-1 and Ubc9 protein levels.

<p>A) ChemLTP increases in SUMO-1 and Ubc9 immunoreactivity with no change in the intensity of SUMO-2 staining. Mean fluorescence intensity was analysed using ImageJ. Graphs show quantification of 3 independent experiments, 10 neurons per condition (ANOVA. *p<0.02; **p<0.005). Scale bars – 20 µm. B) ChemLTP enhances colocalization between SUMO-1 and Ubc9, and SUMO-2/3 and Ubc9 in cultured hippocampal neurons. Example of dendritic segments stained for SUMO-1 (upper panel) or SUMO-2 (lower panel) protein (blue) and Ubc9 (red). Colocalized puncta were quantified using JACoP plugin in ImageJ. Analysis was made in at least 3 different ROIs for each neuron (n = 10), for each condition in 3 independent experiments. (ANOVA. *p<0.02; **p<0.005). C) ChemLTP increases SUMO-1 clusters at synapses. Examples of fluorescence images of dendrites stained for SUMO-1 and PSD95. The colocalized clusters are indicated as yellow puncta. Graph shows quantification of SUMO-1/PSD95 colocalizing pixels. Analysis was performed in at least 3 independent experiments with 10 neurons per condition and 3 ROIs per neuron, using JACoP plugin in ImageJ. Statistical significance was determined using ANOVA. *p<0.02; **p<0.005.</p

Overexpression of SENP-1 diminishes the ChemLTP-mediated increase in SUMO-1 mRNA.

<p>A) Representative images showing effects of ChemLTP in the soma and processes of hippocampal neurons overexpressing SENP-1 C603S (inactive control) or SENP-1 WT (green). Fluorescence <i>in situ</i> hybridization was performed using cRNA probes for SUMO-1 (red). Neurons were then co-stained with MAP-2 (blue). Graph shows quantification of mean fluorescence intensity (arbitrary units) in soma quantified from 4 independent experiments with 8–12 neurons per condition using ImageJ. B) Representative examples of dendritic regions of neurons overexpressing SENP-1 C603S (inactive control) or WT. Graph shows quantification of mean fluorescence intensity (arbitrary units) in dendrites. Quantification from 4 independent experiments; each experiment: 8–12 neurons per condition and 4 dendrites per neuron, done using ImageJ Scale bars – 5 µm. C) <i>In situ</i> hybridization with SUMO-1 negative control (sense probe) yielded virtually no signal compared to the anti-sense (experimental) probe. Scale bars – 20 µm. In all cases statistical significance was determined using ANOVA followed by Bonferroni post-hoc test (*p<0.01).</p

Overexpression of SENP-1 or dominant-negative Ubc9 inhibits the ChemLTP-mediated increase in surface GluA1.

<p>A, B) Overexpression of active SENP-1 (A) catalytic domain, but not inactive SENP-1(C603S) (B), abolishes the ChemLTP-mediated increase in surface expression of GluA1 as determined by surface biotinylation and immunoblotting. The corresponding bar graphs show quantification of surface/total GluA1 ratio. Statistical significance was determined using Student's t-test. C, D) Dominant-negative Ubc9 blocks the ChemLTP-mediated increase in surface GluA1. Neurons were infected with virus expressing dominant-negative Ubc9 (DN-Ubc9) (C) or control GFP (D) and subjected to ChemLTP. Surface biotinylation and western blotting were used to monitor changes in surface expression. Corresponding graphs show quantification of surface/total GluA1 ratio. Statistical significance was determined using Student's t-test. n = 4 (A and B) or n = 3 (C and D) *p<0.02.</p