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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 is essential for activity-dependent spine remodeling.(A) FLIM images of control spines (top), spines from neurons stimulated with 0Mg2+/Gly for 5 min (second row), spines from neurons treated with PD150606 (third row), and spines from neurons treated with PD150606 and stimulated with 0Mg2+/Gly for 5 min (last row). Scale bar is 1 µm. Color coding represents GluN1-GFP lifetime from 2 ns to 2.8 ns. (B) Spine area change (area 20 min after stimulation – area before) in control spines kept in blocking solution for the same time (112 spines/14 neurons), 0Mg2+/Gly stimulated spines (99s/15N), PD150606 treated spines (132 s/14 N) and PD150606 treated and stimulated with 0Mg2+/Gly spines (126 s/14 N). Statistical analysis performed by Kruskal-Wallis (p<0.0001) followed by Dunn's test. *indicates p<0.05 and *** =  p<0.001. (C) FRET efficiency change after the 0Mg2+/Gly LTP stimulation (%FRET efficiency after – %FRET efficiency before) in the same neurons as in B. One-way ANOVA (p<0.02) followed by Bonferroni post hoc test, p<0.05 between CTRL and 0Mg2+/Gly.
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pone-0112170-g005: Calpain is essential for activity-dependent spine remodeling.(A) FLIM images of control spines (top), spines from neurons stimulated with 0Mg2+/Gly for 5 min (second row), spines from neurons treated with PD150606 (third row), and spines from neurons treated with PD150606 and stimulated with 0Mg2+/Gly for 5 min (last row). Scale bar is 1 µm. Color coding represents GluN1-GFP lifetime from 2 ns to 2.8 ns. (B) Spine area change (area 20 min after stimulation – area before) in control spines kept in blocking solution for the same time (112 spines/14 neurons), 0Mg2+/Gly stimulated spines (99s/15N), PD150606 treated spines (132 s/14 N) and PD150606 treated and stimulated with 0Mg2+/Gly spines (126 s/14 N). Statistical analysis performed by Kruskal-Wallis (p<0.0001) followed by Dunn's test. *indicates p<0.05 and *** =  p<0.001. (C) FRET efficiency change after the 0Mg2+/Gly LTP stimulation (%FRET efficiency after – %FRET efficiency before) in the same neurons as in B. One-way ANOVA (p<0.02) followed by Bonferroni post hoc test, p<0.05 between CTRL and 0Mg2+/Gly.

Mentions: Since the phosphorylation of PSD95 has been shown to control long-lasting change in spine size and PSD95 trafficking in organotypic slices [5], we wondered whether calpain activity during synaptic activity could play a role in spine plasticity. We showed previously that synaptic NMDAR stimulation and CaMKII activation can induce long-lasting change in spine volume in cultured hippocampal neurons [32]. We thus measured both the area projected from GluN1-GFP expressing spines (see Methods) and FRET change between GluN1-GFP and PSD95-mCherry in the same neurons. Figure 5B shows that while basal activity produced little change in spine size, 5 min of enhanced synaptic NMDAR activity (0Mg2+/Gly) led to a 0.40±0.08 µm2 increase in size 20 min later, an effect that was blocked by inhibiting calpain with PD150606. In the same spines, the FRET efficiency between GluN1-GFP and PSD95-mCherry decreased on average by 1.4% upon stimulation by 0Mg2+/Gly, but not in presence of PD150606 (Figure 5C). These results demonstrate a correlation between the synaptic NMDAR activity- and calpain-dependent disruption of NMDAR/PSD95 and long-lasting change in spine size.


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 is essential for activity-dependent spine remodeling.(A) FLIM images of control spines (top), spines from neurons stimulated with 0Mg2+/Gly for 5 min (second row), spines from neurons treated with PD150606 (third row), and spines from neurons treated with PD150606 and stimulated with 0Mg2+/Gly for 5 min (last row). Scale bar is 1 µm. Color coding represents GluN1-GFP lifetime from 2 ns to 2.8 ns. (B) Spine area change (area 20 min after stimulation – area before) in control spines kept in blocking solution for the same time (112 spines/14 neurons), 0Mg2+/Gly stimulated spines (99s/15N), PD150606 treated spines (132 s/14 N) and PD150606 treated and stimulated with 0Mg2+/Gly spines (126 s/14 N). Statistical analysis performed by Kruskal-Wallis (p<0.0001) followed by Dunn's test. *indicates p<0.05 and *** =  p<0.001. (C) FRET efficiency change after the 0Mg2+/Gly LTP stimulation (%FRET efficiency after – %FRET efficiency before) in the same neurons as in B. One-way ANOVA (p<0.02) followed by Bonferroni post hoc test, p<0.05 between CTRL and 0Mg2+/Gly.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230936&req=5

pone-0112170-g005: Calpain is essential for activity-dependent spine remodeling.(A) FLIM images of control spines (top), spines from neurons stimulated with 0Mg2+/Gly for 5 min (second row), spines from neurons treated with PD150606 (third row), and spines from neurons treated with PD150606 and stimulated with 0Mg2+/Gly for 5 min (last row). Scale bar is 1 µm. Color coding represents GluN1-GFP lifetime from 2 ns to 2.8 ns. (B) Spine area change (area 20 min after stimulation – area before) in control spines kept in blocking solution for the same time (112 spines/14 neurons), 0Mg2+/Gly stimulated spines (99s/15N), PD150606 treated spines (132 s/14 N) and PD150606 treated and stimulated with 0Mg2+/Gly spines (126 s/14 N). Statistical analysis performed by Kruskal-Wallis (p<0.0001) followed by Dunn's test. *indicates p<0.05 and *** =  p<0.001. (C) FRET efficiency change after the 0Mg2+/Gly LTP stimulation (%FRET efficiency after – %FRET efficiency before) in the same neurons as in B. One-way ANOVA (p<0.02) followed by Bonferroni post hoc test, p<0.05 between CTRL and 0Mg2+/Gly.
Mentions: Since the phosphorylation of PSD95 has been shown to control long-lasting change in spine size and PSD95 trafficking in organotypic slices [5], we wondered whether calpain activity during synaptic activity could play a role in spine plasticity. We showed previously that synaptic NMDAR stimulation and CaMKII activation can induce long-lasting change in spine volume in cultured hippocampal neurons [32]. We thus measured both the area projected from GluN1-GFP expressing spines (see Methods) and FRET change between GluN1-GFP and PSD95-mCherry in the same neurons. Figure 5B shows that while basal activity produced little change in spine size, 5 min of enhanced synaptic NMDAR activity (0Mg2+/Gly) led to a 0.40±0.08 µm2 increase in size 20 min later, an effect that was blocked by inhibiting calpain with PD150606. In the same spines, the FRET efficiency between GluN1-GFP and PSD95-mCherry decreased on average by 1.4% upon stimulation by 0Mg2+/Gly, but not in presence of PD150606 (Figure 5C). These results demonstrate a correlation between the synaptic NMDAR activity- and calpain-dependent disruption of NMDAR/PSD95 and long-lasting change in spine size.

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