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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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CaMKII regulates the NMDAR/PSD95 interaction by distinct mechanisms during synaptic development.(A) CaMKII inhibition with KN93 (10 µM) and PSD95 phosphorylation reduce the activity-dependent dissociation of PSD95 from the NMDAR in DIV21 neurons. The inactive drug KN92 (10 µM) gives results similar to control. NMDAR interaction with PSD95-S73D is much less than with PSD95-WT. In contrast, PSD95-S73A interacts with the NMDAR and FRET does not change upon stimulation. Light green, control unstimulated; dark green, 1–2 min Glu/Gly stimulation. Statistical analysis by Kruskal-Wallis test (p<0.0001), followed by Dunn's post hoc test * indicates p<0.05, ** p<0.01 and *** p<0.001. (N = 10–14 neurons per condition). (B) In DIV7 neurons, CaMKII inhibition also reduces the activity-dependent dissociation of PSD95 from the NMDAR, whereas PSD95-S73D interacts with the NMDAR as well as PSD95-WT does (compare unstimulated CTRL vs PSD95-S73D, p>0.05), the 1–2 min Glu/Gly stimuli disrupting the interaction. PSD95-S73A mutant does not dissociate from the NMDAR upon stimulation. Statistical analysis by one-way ANOVA test (p<0.0001), followed by Bonferroni post hoc test * indicates p<0.05, ** p<0.01 and *** p<0.001. (N = 10–22 neurons per condition).
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pone-0112170-g003: CaMKII regulates the NMDAR/PSD95 interaction by distinct mechanisms during synaptic development.(A) CaMKII inhibition with KN93 (10 µM) and PSD95 phosphorylation reduce the activity-dependent dissociation of PSD95 from the NMDAR in DIV21 neurons. The inactive drug KN92 (10 µM) gives results similar to control. NMDAR interaction with PSD95-S73D is much less than with PSD95-WT. In contrast, PSD95-S73A interacts with the NMDAR and FRET does not change upon stimulation. Light green, control unstimulated; dark green, 1–2 min Glu/Gly stimulation. Statistical analysis by Kruskal-Wallis test (p<0.0001), followed by Dunn's post hoc test * indicates p<0.05, ** p<0.01 and *** p<0.001. (N = 10–14 neurons per condition). (B) In DIV7 neurons, CaMKII inhibition also reduces the activity-dependent dissociation of PSD95 from the NMDAR, whereas PSD95-S73D interacts with the NMDAR as well as PSD95-WT does (compare unstimulated CTRL vs PSD95-S73D, p>0.05), the 1–2 min Glu/Gly stimuli disrupting the interaction. PSD95-S73A mutant does not dissociate from the NMDAR upon stimulation. Statistical analysis by one-way ANOVA test (p<0.0001), followed by Bonferroni post hoc test * indicates p<0.05, ** p<0.01 and *** p<0.001. (N = 10–22 neurons per condition).

Mentions: We next sought to understand the mechanisms by which the interaction between the two proteins is regulated upon NMDAR stimulation. Since CaMKII is known to play a role in regulating PSD95 trafficking [5], we used KN93 to test whether CaMKII influences NMDAR/PSD95 interaction. KN93, but not the inactive analogue KN92, impaired the FRET loss normally seen upon stimulation, both in DIV21 and DIV7 neurons, suggesting that CaMKII activation is needed to disrupt the interaction between NMDAR and PSD95 (Figure 3A and B). Interestingly however, the effect of the competitive inhibitor KN93 seemed less pronounced in DIV21 neurons, which could be due to the important increase in CaMKII expression between DIV7 and DIV21 [32].


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)

CaMKII regulates the NMDAR/PSD95 interaction by distinct mechanisms during synaptic development.(A) CaMKII inhibition with KN93 (10 µM) and PSD95 phosphorylation reduce the activity-dependent dissociation of PSD95 from the NMDAR in DIV21 neurons. The inactive drug KN92 (10 µM) gives results similar to control. NMDAR interaction with PSD95-S73D is much less than with PSD95-WT. In contrast, PSD95-S73A interacts with the NMDAR and FRET does not change upon stimulation. Light green, control unstimulated; dark green, 1–2 min Glu/Gly stimulation. Statistical analysis by Kruskal-Wallis test (p<0.0001), followed by Dunn's post hoc test * indicates p<0.05, ** p<0.01 and *** p<0.001. (N = 10–14 neurons per condition). (B) In DIV7 neurons, CaMKII inhibition also reduces the activity-dependent dissociation of PSD95 from the NMDAR, whereas PSD95-S73D interacts with the NMDAR as well as PSD95-WT does (compare unstimulated CTRL vs PSD95-S73D, p>0.05), the 1–2 min Glu/Gly stimuli disrupting the interaction. PSD95-S73A mutant does not dissociate from the NMDAR upon stimulation. Statistical analysis by one-way ANOVA test (p<0.0001), followed by Bonferroni post hoc test * indicates p<0.05, ** p<0.01 and *** p<0.001. (N = 10–22 neurons per condition).
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Related In: Results  -  Collection

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pone-0112170-g003: CaMKII regulates the NMDAR/PSD95 interaction by distinct mechanisms during synaptic development.(A) CaMKII inhibition with KN93 (10 µM) and PSD95 phosphorylation reduce the activity-dependent dissociation of PSD95 from the NMDAR in DIV21 neurons. The inactive drug KN92 (10 µM) gives results similar to control. NMDAR interaction with PSD95-S73D is much less than with PSD95-WT. In contrast, PSD95-S73A interacts with the NMDAR and FRET does not change upon stimulation. Light green, control unstimulated; dark green, 1–2 min Glu/Gly stimulation. Statistical analysis by Kruskal-Wallis test (p<0.0001), followed by Dunn's post hoc test * indicates p<0.05, ** p<0.01 and *** p<0.001. (N = 10–14 neurons per condition). (B) In DIV7 neurons, CaMKII inhibition also reduces the activity-dependent dissociation of PSD95 from the NMDAR, whereas PSD95-S73D interacts with the NMDAR as well as PSD95-WT does (compare unstimulated CTRL vs PSD95-S73D, p>0.05), the 1–2 min Glu/Gly stimuli disrupting the interaction. PSD95-S73A mutant does not dissociate from the NMDAR upon stimulation. Statistical analysis by one-way ANOVA test (p<0.0001), followed by Bonferroni post hoc test * indicates p<0.05, ** p<0.01 and *** p<0.001. (N = 10–22 neurons per condition).
Mentions: We next sought to understand the mechanisms by which the interaction between the two proteins is regulated upon NMDAR stimulation. Since CaMKII is known to play a role in regulating PSD95 trafficking [5], we used KN93 to test whether CaMKII influences NMDAR/PSD95 interaction. KN93, but not the inactive analogue KN92, impaired the FRET loss normally seen upon stimulation, both in DIV21 and DIV7 neurons, suggesting that CaMKII activation is needed to disrupt the interaction between NMDAR and PSD95 (Figure 3A and B). Interestingly however, the effect of the competitive inhibitor KN93 seemed less pronounced in DIV21 neurons, which could be due to the important increase in CaMKII expression between DIV7 and DIV21 [32].

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