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FRET-FLIM investigation of PSD95-NMDA receptor interaction in dendritic spines; control by calpain, CaMKII and Src family kinase.

Doré K, Labrecque S, Tardif C, De Koninck P - PLoS ONE (2014)

Bottom Line: We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction.The activity of both CaMKII and calpain were essential for this effect in both developmental stages.Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.

View Article: PubMed Central - PubMed

Affiliation: Institut Universitaire en Santé Mentale de Québec, Université Laval, Québec, QC, Canada.

ABSTRACT
Little is known about the changes in protein interactions inside synapses during synaptic remodeling, as their live monitoring in spines has been limited. We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction. NMDAR stimulation caused a transient decrease in FRET between the NMDAR and PSD95 in spines of young and mature neurons. The activity of both CaMKII and calpain were essential for this effect in both developmental stages. Meanwhile, inhibition of Src family kinase (SFK) had opposing impacts on this decrease in FRET in young versus mature neurons. Our data suggest concerted roles for CaMKII, SFK and calpain activity in regulating activity-dependent separation of PSD95 from GluN2A or GluN2B. Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.

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Calpain mediates the activity-dependent dissociation of PSD95 from the NMDAR and Src family kinases differentially control this process during development.(A) Calpain inhibition with PD150606 (10 µM) prevents the activity-dependent dissociation of PSD95 from the NMDAR both in DIV7 and DIV21 neurons. Light green, control unstimulated; dark green, stimulated for 1–2 min with Glu/Gly. A Kruskal-Wallis test revealed significant differences in the data set (p<0.0001). Dunn's post hoc test showed differences induced by Glu/Gly stimulation and calpain inhibition. * indicates p<0.05, ** p<0.01 and ***p<0.001 through the figure. (N = 10–14 neurons per condition). B) Calpain inhibition also prevents the activity-dependent dissociation of PSD95 from the NMDAR in DIV7 neurons expressing PSD95-S73D-mCherry. One-way ANOVA test was performed, revealing differences in the data sets (p<0.0001). Bonferroni was used as a post hoc test. *** indicates p<0.001. (N = 9–11 neurons per condition). (C) Src family kinase inhibition with PP2 (10 µM) prevents the activity-dependent dissociation of PSD95 from the NMDAR in DIV7 neurons, whereas it increases it in DIV21 neurons. The inactive analog PP3 (10 µM) does not influence the dissociation. One-way ANOVA was performed (p<0.0001) followed by Bonferroni post hoc test. (N = 10–14 neurons per condition). (D) PP2 no longer has an effect in DIV7 neurons overexpressing GluN2A, while it now blocks dissociation in DIV21 neurons overexpressing GluN2B. One-way ANOVA revealed significant differences between the groups (p<0.001), Bonferroni test was used for post hoc comparisons. (N = 10–11 neurons per condition).
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pone-0112170-g004: Calpain mediates the activity-dependent dissociation of PSD95 from the NMDAR and Src family kinases differentially control this process during development.(A) Calpain inhibition with PD150606 (10 µM) prevents the activity-dependent dissociation of PSD95 from the NMDAR both in DIV7 and DIV21 neurons. Light green, control unstimulated; dark green, stimulated for 1–2 min with Glu/Gly. A Kruskal-Wallis test revealed significant differences in the data set (p<0.0001). Dunn's post hoc test showed differences induced by Glu/Gly stimulation and calpain inhibition. * indicates p<0.05, ** p<0.01 and ***p<0.001 through the figure. (N = 10–14 neurons per condition). B) Calpain inhibition also prevents the activity-dependent dissociation of PSD95 from the NMDAR in DIV7 neurons expressing PSD95-S73D-mCherry. One-way ANOVA test was performed, revealing differences in the data sets (p<0.0001). Bonferroni was used as a post hoc test. *** indicates p<0.001. (N = 9–11 neurons per condition). (C) Src family kinase inhibition with PP2 (10 µM) prevents the activity-dependent dissociation of PSD95 from the NMDAR in DIV7 neurons, whereas it increases it in DIV21 neurons. The inactive analog PP3 (10 µM) does not influence the dissociation. One-way ANOVA was performed (p<0.0001) followed by Bonferroni post hoc test. (N = 10–14 neurons per condition). (D) PP2 no longer has an effect in DIV7 neurons overexpressing GluN2A, while it now blocks dissociation in DIV21 neurons overexpressing GluN2B. One-way ANOVA revealed significant differences between the groups (p<0.001), Bonferroni test was used for post hoc comparisons. (N = 10–11 neurons per condition).

Mentions: What NMDAR activity-dependent signaling process, other than CaMKII activation, could also disrupt the NMDAR-PSD95 interaction? It was previously demonstrated that NMDAR stimulation can cause cleavage of the GluN2 c-tails by calpain in cultured hippocampal neurons [8], [9]. To investigate whether calpain regulates the NMDAR-PSD95 interaction in spines, we incubated the neurons with calpain inhibitor PD150606. Figure 4A shows that this treatment completely blocked the activity-dependent dissociation of the NMDAR-PSD95 complex, both in DIV7 and DIV21 cultures. Another organic calpain inhibitor (MDL-28170) also blocked this dissociation (in DIV7 neurons incubated with 50 µM MDL, FRET efficiency was 6.3±0.7 without stimulation (N = 10 neurons) and 7.6±0.7 with 1–2 min Glu/Gly (N = 9 neurons); p = 0.21, unpaired t-test), validating further the specificity of this calpain inhibition. In addition, over-expression of the natural inhibitor calpastatin largely reduced this dissociation (in DIV7 neurons expressing only NR1-GFP and PSD95-mCherry, FRET efficiency dropped by ∼3 fold with Glu/Gly, Fig 4A, whereas in calpastatin-transfected neurons, FRET efficiency dropped only by ∼1.4 fold: 8.1±0.9 without stimulation (N = 10 neurons) vs 5.8±0.7 with 1–2 min Glu/Gly, (N = 10 neurons)). Thus, calpain activity can be another mechanism by which the NMDAR/PSD95 interaction is disrupted, even in mature neurons. It is noteworthy that KN93 was shown not to inhibit calpain activity in cultured neurons [33], suggesting that CaMKII is not acting directly on calpain activity. Furthermore, while we found that phosphorylation of PSD95, as mimicked by the mutation S73D, does not impact on NMDAR-PSD95 interaction in young neurons, the mutant can still dissociate upon NMDAR activation (Fig 3A) and this dissociation is also calpain-dependent (Fig 4B). Since calpain appears to preferentially cleave GluN2 subunits over PSD95 [34], we might expect the receptor to be the first target for cleavage.


FRET-FLIM investigation of PSD95-NMDA receptor interaction in dendritic spines; control by calpain, CaMKII and Src family kinase.

Doré K, Labrecque S, Tardif C, De Koninck P - PLoS ONE (2014)

Calpain mediates the activity-dependent dissociation of PSD95 from the NMDAR and Src family kinases differentially control this process during development.(A) Calpain inhibition with PD150606 (10 µM) prevents the activity-dependent dissociation of PSD95 from the NMDAR both in DIV7 and DIV21 neurons. Light green, control unstimulated; dark green, stimulated for 1–2 min with Glu/Gly. A Kruskal-Wallis test revealed significant differences in the data set (p<0.0001). Dunn's post hoc test showed differences induced by Glu/Gly stimulation and calpain inhibition. * indicates p<0.05, ** p<0.01 and ***p<0.001 through the figure. (N = 10–14 neurons per condition). B) Calpain inhibition also prevents the activity-dependent dissociation of PSD95 from the NMDAR in DIV7 neurons expressing PSD95-S73D-mCherry. One-way ANOVA test was performed, revealing differences in the data sets (p<0.0001). Bonferroni was used as a post hoc test. *** indicates p<0.001. (N = 9–11 neurons per condition). (C) Src family kinase inhibition with PP2 (10 µM) prevents the activity-dependent dissociation of PSD95 from the NMDAR in DIV7 neurons, whereas it increases it in DIV21 neurons. The inactive analog PP3 (10 µM) does not influence the dissociation. One-way ANOVA was performed (p<0.0001) followed by Bonferroni post hoc test. (N = 10–14 neurons per condition). (D) PP2 no longer has an effect in DIV7 neurons overexpressing GluN2A, while it now blocks dissociation in DIV21 neurons overexpressing GluN2B. One-way ANOVA revealed significant differences between the groups (p<0.001), Bonferroni test was used for post hoc comparisons. (N = 10–11 neurons per condition).
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pone-0112170-g004: Calpain mediates the activity-dependent dissociation of PSD95 from the NMDAR and Src family kinases differentially control this process during development.(A) Calpain inhibition with PD150606 (10 µM) prevents the activity-dependent dissociation of PSD95 from the NMDAR both in DIV7 and DIV21 neurons. Light green, control unstimulated; dark green, stimulated for 1–2 min with Glu/Gly. A Kruskal-Wallis test revealed significant differences in the data set (p<0.0001). Dunn's post hoc test showed differences induced by Glu/Gly stimulation and calpain inhibition. * indicates p<0.05, ** p<0.01 and ***p<0.001 through the figure. (N = 10–14 neurons per condition). B) Calpain inhibition also prevents the activity-dependent dissociation of PSD95 from the NMDAR in DIV7 neurons expressing PSD95-S73D-mCherry. One-way ANOVA test was performed, revealing differences in the data sets (p<0.0001). Bonferroni was used as a post hoc test. *** indicates p<0.001. (N = 9–11 neurons per condition). (C) Src family kinase inhibition with PP2 (10 µM) prevents the activity-dependent dissociation of PSD95 from the NMDAR in DIV7 neurons, whereas it increases it in DIV21 neurons. The inactive analog PP3 (10 µM) does not influence the dissociation. One-way ANOVA was performed (p<0.0001) followed by Bonferroni post hoc test. (N = 10–14 neurons per condition). (D) PP2 no longer has an effect in DIV7 neurons overexpressing GluN2A, while it now blocks dissociation in DIV21 neurons overexpressing GluN2B. One-way ANOVA revealed significant differences between the groups (p<0.001), Bonferroni test was used for post hoc comparisons. (N = 10–11 neurons per condition).
Mentions: What NMDAR activity-dependent signaling process, other than CaMKII activation, could also disrupt the NMDAR-PSD95 interaction? It was previously demonstrated that NMDAR stimulation can cause cleavage of the GluN2 c-tails by calpain in cultured hippocampal neurons [8], [9]. To investigate whether calpain regulates the NMDAR-PSD95 interaction in spines, we incubated the neurons with calpain inhibitor PD150606. Figure 4A shows that this treatment completely blocked the activity-dependent dissociation of the NMDAR-PSD95 complex, both in DIV7 and DIV21 cultures. Another organic calpain inhibitor (MDL-28170) also blocked this dissociation (in DIV7 neurons incubated with 50 µM MDL, FRET efficiency was 6.3±0.7 without stimulation (N = 10 neurons) and 7.6±0.7 with 1–2 min Glu/Gly (N = 9 neurons); p = 0.21, unpaired t-test), validating further the specificity of this calpain inhibition. In addition, over-expression of the natural inhibitor calpastatin largely reduced this dissociation (in DIV7 neurons expressing only NR1-GFP and PSD95-mCherry, FRET efficiency dropped by ∼3 fold with Glu/Gly, Fig 4A, whereas in calpastatin-transfected neurons, FRET efficiency dropped only by ∼1.4 fold: 8.1±0.9 without stimulation (N = 10 neurons) vs 5.8±0.7 with 1–2 min Glu/Gly, (N = 10 neurons)). Thus, calpain activity can be another mechanism by which the NMDAR/PSD95 interaction is disrupted, even in mature neurons. It is noteworthy that KN93 was shown not to inhibit calpain activity in cultured neurons [33], suggesting that CaMKII is not acting directly on calpain activity. Furthermore, while we found that phosphorylation of PSD95, as mimicked by the mutation S73D, does not impact on NMDAR-PSD95 interaction in young neurons, the mutant can still dissociate upon NMDAR activation (Fig 3A) and this dissociation is also calpain-dependent (Fig 4B). Since calpain appears to preferentially cleave GluN2 subunits over PSD95 [34], we might expect the receptor to be the first target for cleavage.

Bottom Line: We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction.The activity of both CaMKII and calpain were essential for this effect in both developmental stages.Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.

View Article: PubMed Central - PubMed

Affiliation: Institut Universitaire en Santé Mentale de Québec, Université Laval, Québec, QC, Canada.

ABSTRACT
Little is known about the changes in protein interactions inside synapses during synaptic remodeling, as their live monitoring in spines has been limited. We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction. NMDAR stimulation caused a transient decrease in FRET between the NMDAR and PSD95 in spines of young and mature neurons. The activity of both CaMKII and calpain were essential for this effect in both developmental stages. Meanwhile, inhibition of Src family kinase (SFK) had opposing impacts on this decrease in FRET in young versus mature neurons. Our data suggest concerted roles for CaMKII, SFK and calpain activity in regulating activity-dependent separation of PSD95 from GluN2A or GluN2B. Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.

Show MeSH
Related in: MedlinePlus