FRET-FLIM Investigation of PSD95-NMDA Receptor Interaction in Dendritic Spines; Control by Calpain, CaMKII and Src Family Kinase

<div><p>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.</p></div

GluN1-GFP and PSD95-mCherry are specific probes for measuring the interaction between PSD95 and the NMDAR with FRET-FLIM.

<p>(A) FLIM images of GluN1-GFP expressing spines, expressed alone (top row), with mCherry (second row), with PSD95-mCherry (third row) or Homer-mCherry (last row). Scale bar is 1 µm (placed in first spine). Color coding represents GluN1-GFP lifetime from 2 ns to 2.8 ns. Black crosses indicate the pixel selected for traces in B. Circles indicate ROI for panel C. (B) Fit curves of fluorescence intensity decays obtained from one pixel (black crosses in the spines shown in A) for a GluN1-GFP expressing spine (green curve) and GluN1-GFP/PSD95-mCherry expressing spine (black curve), see Fig. S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112170#pone.0112170.s001" target="_blank">file S1</a> for raw data. (C) Distribution histograms of GluN1-GFP lifetimes in encircled spines in A (GluN1-GFP, green; GluN1-GFP/PSD95-mCherry, gray), the median (dotted line) of each distribution was averaged across samples to produce a mean lifetime. (D) The mean lifetime in GluN1-GFP/PSD95-mCherry expressing spines (14 neurons (N)/258 spines (s), gray) is significantly shorter than GluN1-GFP alone, indicating FRET between the NMDAR and PSD95. Spines expressing either GluN1-GFP (12 N/160 s, green), GluN1-GFP/mCherry (12 N/123 s, yellow) or GluN1-GFP/Homer-mCherry (13 N/182 s, blue) all have similar lifetimes. Statistical analysis was performed by Kruskal-Wallis test (p<0.0001) followed by Dunn's test. GluN1-GFP/PSD95-mCherry group is different from all the other conditions p<0.001. (E) FRET efficiency in the same spines as in D. Kruskal-Wallis test was performed (p<0.0001), followed by Dunn's test. GluN1-GFP/PSD95-mCherry group is different from all the other conditions p<0.001.</p

Calpain mediates the activity-dependent dissociation of PSD95 from the NMDAR and Src family kinases differentially control this process during development.

<p>(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).</p

Proposed mechanism of NMDAR-PSD95 dissociation during NMDAR activation in young neurons (A) versus mature neurons (B).

<p>(A) In young neurons expressing more GluN2B than GluN2A, the PDZ2 domain of PSD95 supports its binding to GluN2B c-terminus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112170#pone.0112170-Kornau1" target="_blank">[15]</a>. The phosphorylation by CaMKII of S73 in the PDZ1 domain of PSD95, following NMDAR activation and Ca²⁺ influx, would repel this portion of PSD95 from the receptor, possibly where another binding site for PSD95 on GluN2B was recently identified (aa 1149–1157; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112170#pone.0112170-Cousins2" target="_blank">[31]</a>. This may then reduce the protective effect of PSD95 against calpain-mediated cleavage of the ctail of GluN2B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112170#pone.0112170-Dong1" target="_blank">[6]</a>, leading to the separation of PSD95 from the receptor. This scenario reconciles our data showing that inhibiting CaMKII (KN93) prevents the separation of PSD95 from the receptor, but that S73D-PSD95 can still bind GluN2B-containing NMDARs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112170#pone.0112170-Gardoni1" target="_blank">[16]</a>. (B) In mature neurons, PSD95 is mainly bound to GluN2A-containing receptors, via two binding sites on the receptor: i) the c-terminal site binding to PDZ2 and ii) another site (aa 1382–1389) binding to the SH3 domain of PSD95 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112170#pone.0112170-Cousins2" target="_blank">[31]</a>. The phosphorylation of S73-PSD95 alone (S73D mutation) was sufficient to prevent the PSD95-NMDAR interaction in mature synapses, but CaMKII was unable to trigger the complex separation in the presence of a calpain inhibitor, suggesting that a calpain-sensitive PSD component (spectrin is a well know calpain substrate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112170#pone.0112170-Vinade1" target="_blank">[34]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112170#pone.0112170-Jourdi1" target="_blank">[38]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112170#pone.0112170-Lynch1" target="_blank">[39]</a> and is thus shown merely as an example) must be removed to allow CaMKII to access S73 on PSD95.</p

CaMKII regulates the NMDAR/PSD95 interaction by distinct mechanisms during synaptic development.

<p>(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).</p

The interaction between PSD95 and the NMDAR is transiently disrupted upon NMDAR stimulation.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112170#s2" target="_blank">Methods</a>). 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112170#s2" target="_blank">methods</a>. To confirm the differences between groups, Kruskal-Wallis test was performed (p<0.0001), followed by Dunn's post hoc test.</p

Calpain is essential for activity-dependent spine remodeling.

<p>(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.</p

Rem2 and CaMKII redistribution is co-dependent.

<p>(<b>A</b>) CaMKIIN inhibits redistribution of Rem2 and CaMKII. Rat hippocampal neurons coexpressing <b>(</b>left) GFP-Rem2 and (right) mRuby-CaMKII without (left panels) and with (right panels) HA-CaMKIIN. Cells were imaged before stimulation (top panels), then stimulated with 100 µM glutamate/10 µM glycine and imaged again (middle panels). Neurons were fixed and probed for HA to detect CaMKIIN (bottom panel). The scale bar represents 20 µm. (<b>B</b>) CaMKIIN reduces redistribution of coexpressed CaMKII and Rem2. Clustering factor determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041185#pone.0041185-Hudmon1" target="_blank">[2]</a>. Mean ± SEM clustering factor for CaMKII without CaMKIIN: before stimulation, 0.004±0.002; after, 0.021±0.004; with CaMKIIN: before, 0.002±0.0002; after, 0.009±0.002. Rem2 clustering without CaMKIIN: before stimulation, 0.001±0.0002; after, 0.009±0.002; with CaMKIIN: before, 0.001±0.0002; after, 0.003±0.0006. Data is from 4 separate experiments with a total of N = 27 neurons/condition. Error bars are ± SEM; asterisks represent p<0.05 (Kruskal-Wallis test followed by Tukey’s post hoc test). (<b>C</b>) Rem2 and CaMKII redistributions overlap temporally. A timecourse of aggregation of Rem2 and CaMKII shows little clustering before stimulation with glutamate/glycine, and Rem2 and CaMKII clustering occurs at similar rates. N = 27 neurons/condition. (<b>D</b>) CaMKIIN does not interact with Rem2, and expression of CaMKIIN does not interfere with Rem2-CaMKII interaction. HEK cells were transfected with the indicated plasmids. Cell lysates were subjected to co-precipitation assays using a mix of proteinA/G beads and anti-HA,-myc or-GFP. Eluates were separated on SDS-PAGE and probed with anti-HA (top) and anti-myc (bottom). (<b>E</b>) CaMKII aggregates in HEK cells following a pH drop/high Ca protocol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041185#pone.0041185-Hudmon1" target="_blank">[2]</a> (mean clustering factor before simulation 0.030±0.002; after 0.166±0.013). Aggregation is inhibited by CaMKIIN (before stimulation, 0.024±0.002; after, 0.068±0.010) but unaffected by coexpression of Rem2 (before, 0.031±0.003; after, 0.152±0.009). CaMKIIN inhibition is also insensitive to Rem2 coexpression (before, 0.031±0.003; after, 0.071±0.006). N = 20 cells and p<0.05 for all before-after pairs. (<b>F</b>) Rem2 does not aggregate using the same protocol (without CaMKIIN: before stim, 0.037±0.004; after, 0.032±0.004; with CaMKIIN: before, 0.018±0.002; after, 0.022±0.003), unless CaMKII is also present (without CaMKIIN: before stim, 0.012±0.002; after, 0.073±0.008). CaMKII-induced Rem2 aggregation is also inhibited by CaMKIIN (before, 0.008±0.001; after, 0.021±0.004). Error bars are ± SEM; asterisks represent p<0.05 (Kruskal-Wallis test followed by Tukey’s post hoc test).</p

Activity-Dependent Subcellular Cotrafficking of the Small GTPase Rem2 and Ca2+/CaM-Dependent Protein Kinase IIα

<div><h3>Background</h3><p>Rem2 is a small monomeric GTP-binding protein of the RGK family, whose known functions are modulation of calcium channel currents and alterations of cytoskeletal architecture. Rem2 is the only RGK protein found predominantly in the brain, where it has been linked to synaptic development. We wished to determine the effect of neuronal activity on the subcellular distribution of Rem2 and its interacting partners.</p> <h3>Results</h3><p>We show that Rem2 undergoes activity-and N-Methyl-D-Aspartate Receptor (NMDAR)-dependent translocation in rat hippocampal neurons. This redistribution of Rem2, from a diffuse pattern to one that is highly punctate, is dependent on Ca²⁺ influx, on binding to calmodulin (CaM), and also involves an auto-inhibitory domain within the Rem2 distal C-terminus region. We found that Rem2 can bind to Ca²⁺/CaM-dependent protein kinase IIα (CaMKII) a in Ca²⁺/CaM-dependent manner. Furthermore, our data reveal a spatial and temporal correlation between NMDAR-dependent clustering of Rem2 and CaMKII in neurons, indicating co-assembly and co-trafficking in neurons. Finally, we show that inhibiting CaMKII aggregation in neurons and HEK cells reduces Rem2 clustering, and that Rem2 affects the baseline distribution of CaMKII in HEK cells.</p> <h3>Conclusions</h3><p>Our data suggest a novel function for Rem2 in co-trafficking with CaMKII, and thus potentially expose a role in neuronal plasticity.</p> </div

The Rem2 C-terminus directs redistribution.

<p>(<b>A</b>) Schematic of the Rem2 protein. The last 30 residues of the C-terminal extension contain a previously identified PIP lipid binding domain as well as a calmodulin binding site. Small triangles in the N-and C-termini represent 14-3-3 binding sites. We created truncated Rem2 proteins ending at residues 310 and 320 to determine if these interaction sites are relevant for Rem2 redistribution. (<b>B</b>) Effect of Rem2 truncations on redistribution. (<b>Left panel</b>) Full-length Rem2 (top) redistributed into puncta on glutamate/glycine stimulation. 1–310 Rem2 (center) showed no redistribution upon stimulation, while 1–320 Rem2 (bottom) formed puncta constitutively. Scale bars indicate 5 µm. (<b>Right panel</b>) WT Rem2 shows a significant difference in pixel intensity variance after stimulation (normalized mean (± SEM) pixel value variance before stimulation, 1.00±0,08; after, 1.74±0.17; paired t-test p<0.001). Additionally, 1–320 Rem2 distribution is different from WT before but not after stimulation (one-way ANOVA followed by Bonferroni’s test; before, p<0.001; after, p = 0.19). Pixel intensity variances were normalized to the variance of full-length Rem2 before stimulation.</p