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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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Related in: MedlinePlus

The interaction between PSD95 and the NMDAR is transiently disrupted upon NMDAR stimulation.(A) Two examples of confocal images of 14 DIV dendrites expressing GluN1-GFP (first column) and PSD95-mCherry (second column). Next are shown corresponding FLIM images of the same dendrites before (third column), just after stimulation (fourth column) and 15 min after washing out the stimulation solution (last column). The arrows point to spines in which the lifetime increased upon stimulation, the arrowheads point to dendritic shaft synapses in which the lifetime is also increased by stimulation. Color-coding represents GluN1-GFP lifetime from 2 ns to 2.8 ns (columns 3 and 4). Scale bar is 1 µm. (B) GluN1-GFP lifetime in PSD95-mCherry clusters increases upon 1–2 min Glu/Gly stimulation and decreases after 15 min wash (gray, 16 live neurons). Repeated measures ANOVA was performed (p<0.0001), followed by Bonferroni post hoc test. ** indicates p <0.01 and ***  =  p<0.001 through the figure. GluN1-GFP lifetime is significantly higher in Homer-mCherry clusters than in PSD95-mCherry clusters (dark blue, 15 live neurons). One-way ANOVA test with 6 groups was performed (p<0.0001), followed by Bonferroni post hoc test. Before stimulation and 15 min after, PSD95-mCherry neurons are statistically different from Homer-mCherry neurons (p<0.001 and p<0.01 respectively). Glu/Gly stimulation and washing had no effect on GluN1-GFP lifetime in Homer-mCherry clusters (Repeated measures ANOVA test was not significant). (C) FRET efficiency in GluN1-GFP/PSD95-mCherry expressing cells (same neurons as in B, calculated with GluN1-GFP/Homer-mCherry as the donor lifetime (experiments done the same day)) decreases upon 1–2 min Glu/Gly stimulation and increases again after 15 min wash. Repeated measures ANOVA was performed (p<0.0001), followed by Bonferroni post hoc test. (D) In DIV7 (light gray) and DIV21 (dark gray) neurons, FRET efficiency decreases after 1–2 min Glu/Gly stimulation and 5 min 0Mg2+/Gly stimulation. Neurons were fixed and mounted prior to imaging (see Methods). After 30 min of wash in High Mg2+ solution, FRET is back to basal levels. MK-801 blocks the FRET loss. Each bar in the histogram is the mean FRET efficiency of at least 10 neurons (one mean value/neuron obtained from ≈40–200 synapses), taken from at least 3 separate animal preparations. Kruskal-Wallis test was performed on both DIV7 and DIV21 groups p<0.0001 for both ages. Dunn's test was used for post hoc comparison. (E) The interaction between the NMDAR and PSD95 is mediated via an interaction with GluN2A or GluN2B and is dissociated by Ca2+ influx in HEK293 cells. The cells were co-transfected with the indicated constructs and treated as described in the methods. To confirm the differences between groups, Kruskal-Wallis test was performed (p<0.0001), followed by Dunn's post hoc test.
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pone-0112170-g002: The interaction between PSD95 and the NMDAR is transiently disrupted upon NMDAR stimulation.(A) Two examples of confocal images of 14 DIV dendrites expressing GluN1-GFP (first column) and PSD95-mCherry (second column). Next are shown corresponding FLIM images of the same dendrites before (third column), just after stimulation (fourth column) and 15 min after washing out the stimulation solution (last column). The arrows point to spines in which the lifetime increased upon stimulation, the arrowheads point to dendritic shaft synapses in which the lifetime is also increased by stimulation. Color-coding represents GluN1-GFP lifetime from 2 ns to 2.8 ns (columns 3 and 4). Scale bar is 1 µm. (B) GluN1-GFP lifetime in PSD95-mCherry clusters increases upon 1–2 min Glu/Gly stimulation and decreases after 15 min wash (gray, 16 live neurons). Repeated measures ANOVA was performed (p<0.0001), followed by Bonferroni post hoc test. ** indicates p <0.01 and ***  =  p<0.001 through the figure. GluN1-GFP lifetime is significantly higher in Homer-mCherry clusters than in PSD95-mCherry clusters (dark blue, 15 live neurons). One-way ANOVA test with 6 groups was performed (p<0.0001), followed by Bonferroni post hoc test. Before stimulation and 15 min after, PSD95-mCherry neurons are statistically different from Homer-mCherry neurons (p<0.001 and p<0.01 respectively). Glu/Gly stimulation and washing had no effect on GluN1-GFP lifetime in Homer-mCherry clusters (Repeated measures ANOVA test was not significant). (C) FRET efficiency in GluN1-GFP/PSD95-mCherry expressing cells (same neurons as in B, calculated with GluN1-GFP/Homer-mCherry as the donor lifetime (experiments done the same day)) decreases upon 1–2 min Glu/Gly stimulation and increases again after 15 min wash. Repeated measures ANOVA was performed (p<0.0001), followed by Bonferroni post hoc test. (D) In DIV7 (light gray) and DIV21 (dark gray) neurons, FRET efficiency decreases after 1–2 min Glu/Gly stimulation and 5 min 0Mg2+/Gly stimulation. Neurons were fixed and mounted prior to imaging (see Methods). After 30 min of wash in High Mg2+ solution, FRET is back to basal levels. MK-801 blocks the FRET loss. Each bar in the histogram is the mean FRET efficiency of at least 10 neurons (one mean value/neuron obtained from ≈40–200 synapses), taken from at least 3 separate animal preparations. Kruskal-Wallis test was performed on both DIV7 and DIV21 groups p<0.0001 for both ages. Dunn's test was used for post hoc comparison. (E) The interaction between the NMDAR and PSD95 is mediated via an interaction with GluN2A or GluN2B and is dissociated by Ca2+ influx in HEK293 cells. The cells were co-transfected with the indicated constructs and treated as described in the methods. To confirm the differences between groups, Kruskal-Wallis test was performed (p<0.0001), followed by Dunn's post hoc test.

Mentions: Human embryonic kidney 293 (HEK293) cells were grown to 30–40% on poly-D-lysine coated Aclar coverslips (13 mm in diameter) in DMEM growth media supplemented with 10% fetal bovine serum and Glutamax-1 (Invitrogen). Cells were transfected using Lipofectamine 2000 as described by the manufacturer and treated 16–24 h later. For Figure 2E, cells were incubated in a solution containing HBSS supplemented with 5 mM CaCl2 and 10 µM ionomycin (1∶1000 dilution of a methanol stock) or a solution containing only HBSS for 5 min. Cells were then immediately fixed for 10 min in methanol (−20C), washed in PBS and mounted with Prolong Gold. For FLIM analysis, all detected photons for each cell were summed into a single histogram from which one mean lifetime was obtained.


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)

The interaction between PSD95 and the NMDAR is transiently disrupted upon NMDAR stimulation.(A) Two examples of confocal images of 14 DIV dendrites expressing GluN1-GFP (first column) and PSD95-mCherry (second column). Next are shown corresponding FLIM images of the same dendrites before (third column), just after stimulation (fourth column) and 15 min after washing out the stimulation solution (last column). The arrows point to spines in which the lifetime increased upon stimulation, the arrowheads point to dendritic shaft synapses in which the lifetime is also increased by stimulation. Color-coding represents GluN1-GFP lifetime from 2 ns to 2.8 ns (columns 3 and 4). Scale bar is 1 µm. (B) GluN1-GFP lifetime in PSD95-mCherry clusters increases upon 1–2 min Glu/Gly stimulation and decreases after 15 min wash (gray, 16 live neurons). Repeated measures ANOVA was performed (p<0.0001), followed by Bonferroni post hoc test. ** indicates p <0.01 and ***  =  p<0.001 through the figure. GluN1-GFP lifetime is significantly higher in Homer-mCherry clusters than in PSD95-mCherry clusters (dark blue, 15 live neurons). One-way ANOVA test with 6 groups was performed (p<0.0001), followed by Bonferroni post hoc test. Before stimulation and 15 min after, PSD95-mCherry neurons are statistically different from Homer-mCherry neurons (p<0.001 and p<0.01 respectively). Glu/Gly stimulation and washing had no effect on GluN1-GFP lifetime in Homer-mCherry clusters (Repeated measures ANOVA test was not significant). (C) FRET efficiency in GluN1-GFP/PSD95-mCherry expressing cells (same neurons as in B, calculated with GluN1-GFP/Homer-mCherry as the donor lifetime (experiments done the same day)) decreases upon 1–2 min Glu/Gly stimulation and increases again after 15 min wash. Repeated measures ANOVA was performed (p<0.0001), followed by Bonferroni post hoc test. (D) In DIV7 (light gray) and DIV21 (dark gray) neurons, FRET efficiency decreases after 1–2 min Glu/Gly stimulation and 5 min 0Mg2+/Gly stimulation. Neurons were fixed and mounted prior to imaging (see Methods). After 30 min of wash in High Mg2+ solution, FRET is back to basal levels. MK-801 blocks the FRET loss. Each bar in the histogram is the mean FRET efficiency of at least 10 neurons (one mean value/neuron obtained from ≈40–200 synapses), taken from at least 3 separate animal preparations. Kruskal-Wallis test was performed on both DIV7 and DIV21 groups p<0.0001 for both ages. Dunn's test was used for post hoc comparison. (E) The interaction between the NMDAR and PSD95 is mediated via an interaction with GluN2A or GluN2B and is dissociated by Ca2+ influx in HEK293 cells. The cells were co-transfected with the indicated constructs and treated as described in the methods. To confirm the differences between groups, Kruskal-Wallis test was performed (p<0.0001), followed by Dunn's post hoc test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230936&req=5

pone-0112170-g002: The interaction between PSD95 and the NMDAR is transiently disrupted upon NMDAR stimulation.(A) Two examples of confocal images of 14 DIV dendrites expressing GluN1-GFP (first column) and PSD95-mCherry (second column). Next are shown corresponding FLIM images of the same dendrites before (third column), just after stimulation (fourth column) and 15 min after washing out the stimulation solution (last column). The arrows point to spines in which the lifetime increased upon stimulation, the arrowheads point to dendritic shaft synapses in which the lifetime is also increased by stimulation. Color-coding represents GluN1-GFP lifetime from 2 ns to 2.8 ns (columns 3 and 4). Scale bar is 1 µm. (B) GluN1-GFP lifetime in PSD95-mCherry clusters increases upon 1–2 min Glu/Gly stimulation and decreases after 15 min wash (gray, 16 live neurons). Repeated measures ANOVA was performed (p<0.0001), followed by Bonferroni post hoc test. ** indicates p <0.01 and ***  =  p<0.001 through the figure. GluN1-GFP lifetime is significantly higher in Homer-mCherry clusters than in PSD95-mCherry clusters (dark blue, 15 live neurons). One-way ANOVA test with 6 groups was performed (p<0.0001), followed by Bonferroni post hoc test. Before stimulation and 15 min after, PSD95-mCherry neurons are statistically different from Homer-mCherry neurons (p<0.001 and p<0.01 respectively). Glu/Gly stimulation and washing had no effect on GluN1-GFP lifetime in Homer-mCherry clusters (Repeated measures ANOVA test was not significant). (C) FRET efficiency in GluN1-GFP/PSD95-mCherry expressing cells (same neurons as in B, calculated with GluN1-GFP/Homer-mCherry as the donor lifetime (experiments done the same day)) decreases upon 1–2 min Glu/Gly stimulation and increases again after 15 min wash. Repeated measures ANOVA was performed (p<0.0001), followed by Bonferroni post hoc test. (D) In DIV7 (light gray) and DIV21 (dark gray) neurons, FRET efficiency decreases after 1–2 min Glu/Gly stimulation and 5 min 0Mg2+/Gly stimulation. Neurons were fixed and mounted prior to imaging (see Methods). After 30 min of wash in High Mg2+ solution, FRET is back to basal levels. MK-801 blocks the FRET loss. Each bar in the histogram is the mean FRET efficiency of at least 10 neurons (one mean value/neuron obtained from ≈40–200 synapses), taken from at least 3 separate animal preparations. Kruskal-Wallis test was performed on both DIV7 and DIV21 groups p<0.0001 for both ages. Dunn's test was used for post hoc comparison. (E) The interaction between the NMDAR and PSD95 is mediated via an interaction with GluN2A or GluN2B and is dissociated by Ca2+ influx in HEK293 cells. The cells were co-transfected with the indicated constructs and treated as described in the methods. To confirm the differences between groups, Kruskal-Wallis test was performed (p<0.0001), followed by Dunn's post hoc test.
Mentions: Human embryonic kidney 293 (HEK293) cells were grown to 30–40% on poly-D-lysine coated Aclar coverslips (13 mm in diameter) in DMEM growth media supplemented with 10% fetal bovine serum and Glutamax-1 (Invitrogen). Cells were transfected using Lipofectamine 2000 as described by the manufacturer and treated 16–24 h later. For Figure 2E, cells were incubated in a solution containing HBSS supplemented with 5 mM CaCl2 and 10 µM ionomycin (1∶1000 dilution of a methanol stock) or a solution containing only HBSS for 5 min. Cells were then immediately fixed for 10 min in methanol (−20C), washed in PBS and mounted with Prolong Gold. For FLIM analysis, all detected photons for each cell were summed into a single histogram from which one mean lifetime was obtained.

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