Altered surface mGluR5 dynamics provoke synaptic NMDAR dysfunction and cognitive defects in Fmr1 knockout mice

Abstract

Metabotropic glutamate receptor subtype 5 (mGluR5) is crucially implicated in the pathophysiology of Fragile X Syndrome (FXS); however, its dysfunction at the sub-cellular level, and related synaptic and cognitive phenotypes are unexplored. Here, we probed the consequences of mGluR5/Homer scaffold disruption for mGluR5 cell-surface mobility, synaptic N-methyl-D-aspartate receptor (NMDAR) function, and behavioral phenotypes in the second-generation Fmr1 knockout (KO) mouse. Using single-molecule tracking, we found that mGluR5 was significantly more mobile at synapses in hippocampal Fmr1 KO neurons, causing an increased synaptic surface co-clustering of mGluR5 and NMDAR. This correlated with a reduced amplitude of synaptic NMDAR currents, a lack of their mGluR5-activated long-term depression, and NMDAR/hippocampus dependent cognitive deficits. These synaptic and behavioral phenomena were reversed by knocking down Homer1a in Fmr1 KO mice. Our study provides a mechanistic link between changes of mGluR5 dynamics and pathological phenotypes of FXS, unveiling novel targets for mGluR5-based therapeutics.

Fig. 1

Cell-surface mGluR5 displays an increased lateral diffusion rate within the synaptic compartment of hippocampal Fmr1 KO neurons. (a) Experimental setup. Upper panel: schematic representation of endogenous mGluR5 in the dendritic membrane labeled with a QD-antibody complex targeting the extracellular domain of the receptor. Lower panel: representative images of the dendrites of hippocampal neurons shown in phase contrast (left), and their MitoTracker-labeled synaptic sites (right; green) overlaid with reconstructed trajectories of surface mGluR5-QD complexes (depicted in red) in the dendritic membrane of the same neurons. Scale bar = 5 μm. (b) Representative trajectories of single surface mGluR5-QD in WT and Fmr1 KO neurons. The synaptic sites are represented by the green areas. Scale bar = 1 μm. (c) Cumulative distribution (left panel) and cumulative frequency distribution (right panel) of the instantaneous diffusion coefficient of mGluR5-QDs in the synaptic compartment. The lateral diffusion is significantly higher in Fmr1 KO neurons (WT, 0.017 ± 0.001 µm²/s, n = 1632 trajectories from 16 dendritic fields of 3 different cultures); Fmr1 KO, 0.024 ± 0.001 µm²/s, n = 1451 trajectories (14 dendritic fields from 3 cultures); ***P < 0.001 by Mann–Whitney test on cumulative distribution; ***P < 0.001 by Kolmogorov–Smirnov test on cumulative frequency distribution). (d) Cumulative distribution (left panel) and cumulative frequency distribution (right panel) of the instantaneous diffusion coefficient of mGluR5-QDs in the extrasynaptic area of WT and Fmr1 KO neurons (WT, 0.045 ± 0.002 µm²/s, n = 1907 trajectories from 16 dendritic fields of 3 different cultures; Fmr1 KO, 0.048 ± 0.002 µm²/s, n = 1347 trajectories from 14 dendritic fields of 3 different cultures; P = 0.106 by Mann–Whitney test on cumulative distribution; P = 0.649 by Kolmogorov–Smirnov test on cumulative frequency distribution)

Fig. 2

Disruption of the link between mGluR5 and Homer in WT neurons mimics the Fmr1 KO phenotype. (a) Schematic illustration of the effect of the cell-permeable TAT-mGluR5ct peptide. This peptide disrupts the mGluR5/Homer link, mimicking the situation in Fmr1 KO neurons. (b) Representative trajectories of single surface mGluR5-QD in WT and Fmr1 KO neurons treated with TAT-mGluR5ct and its mutated TAT-mGluR5mu control peptide (both peptides 5 µM, 1 h). The synaptic sites are represented by the green areas. Scale bar = 1 μm. (c) Normalized cumulative distribution of the instantaneous diffusion coefficient of mGluR5-QDs in the synaptic area of WT and Fmr1 KO neurons treated with TAT-mGluR5mu and TAT-mGluR5ct. The lateral diffusion rate of mGluR5-QDs in WT neurons treated with TAT-mGluR5ct peptide is comparable to that in Fmr1 KO neurons under basal conditions (WT, 0.016 ± 0.002 µm²/s, n = 636 trajectories from 12 dendritic fields of 3 cultures; WT TAT-mGluR5mu, 0.016 ± 0.002 µm²/s, n = 1798 trajectories from 12 dendritic fields of 3 cultures; WT TAT-mGluR5ct, 0.026 ± 0.002 µm²/s, n = 1444 trajectories from 19 dendritic fields of 3 cultures; Fmr1 KO, 0.025 ± 0.002 µm²/s, n = 797 trajectories from 16 dendritic fields of 3 cultures; Fmr1 KO TAT-mGluR5mu, 0.023 ± 0.001 µm²/s, n = 1419 trajectories from 13 dendritic fields of 3 cultures; Fmr1 KO TAT-mGluR5ct, 0.024 ± 0.002 µm²/s, n = 489 trajectories from 9 dendritic fields of 3 cultures; WT TAT-mGluR5mu vs. WT TAT-mGluR5ct ***P < 0.001 by Kruskal–Wallis test with Dunn’s multiple comparison test; WT TAT-mGluR5ct vs. Fmr1 KO P > 0.999 by Kruskal–Wallis test with Dunn’s multiple comparison test). (d) Cumulative frequency distribution of the instantaneous diffusion coefficient of mGluR5-QDs within the synaptic area of WT and Fmr1 KO neurons treated with TAT-mGluR5mu and TAT-mGluR5ct (***P < 0.001 by Kolmogorov–Smirnov test. For statistical analysis each condition was separately compared to WT TAT-mGluR5mu)

Fig. 3

mGluR5 and GluN1 are more confined within the synaptic compartment in Fmr1 KO neurons. (a) Representative surface distribution of mGluR5-QD (upper panel) and GluN1-QD (lower panel) in a 500-frame stack (each dot represents the detection of a single receptor during 50-ms acquisition time), revealing the synaptic site as a trapping zone (green). (b) Relative fractions of synaptic mGluR5-QD (left panel) and GluN1-QD (right panel) particles. These values are increased in Fmr1 KO neurons (mGluR5-QD: WT, 8.053 ± 0.504 %, n = 16 dendritic fields from 3 cultures; Fmr1 KO, 15.95 ± 0.685 %, n = 14 dendritic fields from 3 cultures; ***P < 0.001, t = 9.44, df = 28, unpaired Student’s t-test; GluN1-QD: WT, 8.318 ± 0.382 %, n = 63 dendritic fields from 6 cultures; Fmr1 KO, 10.02 ± 0.446 %, n = 51 dendritic fields from 6 cultures; **P < 0.01, t = 2.91, df = 112, unpaired Student’s t-test)

Fig. 4

Disruption of the mGluR5/Homer scaffold increases surface mGluR5/NMDAR co-clustering. (a) Cultured WT and Fmr1 KO hippocampal neurons were triple-labeled for mGluR5, GluN1 and Homer1. (b, c) Left: Representative image of Homer/mGluR5 and Homer/GluN1 co-localization; middle: Distribution of co-localized mGluR5/Homer1 or GluN-/Homer1 clusters; right: mGluR5/Homer1 and GluN1/Homer1 clusters as percentage of total mGluR5 or GluN1 signal (mGluR5: WT, 9.53 ± 0.959 %, n = 27; Fmr1 KO, 34.14 ± 4.598 %, n = 20; ***P < 0.001, t = 6, df = 45, unpaired Student’s t-test; GluN1: WT 15.75 ± 1.841 %, n = 29; Fmr1 KO 37.15 ± 5.324 %, n = 18; ***P < 0.001, t = 4.47, df = 45, unpaired Student’s t-test). (d) Left: Representative image showing mGluR5/GluN1/Homer1 colocalization; middle: Distribution of co-localized mGluR5/GluN1/Homer1 labeling; right: Co-localized mGluR5/GluN1/Homer1 clusters as percentage of synaptic GluN1 signal (WT, 63.97 ± 2.414 %, n = 26; Fmr1 KO, 72.14 ± 2.081 %, n = 23; *P < 0.05, t = 2.53, df = 47, unpaired Student’s t-test). (e) TAT-mGluR5ct peptide increased mGluR5/GluN1 co-clustering at synapses in WT neurons, whereas TAT-mGluR5mu or TAT-mGluR5ct (both 5 µM, 1 h) had no effect in Fmr1 KO neurons; Left: Representative images and distribution of mGluR5/GluN1/Homer1-co-labeling signal in control and TAT-mGluR5mu or TAT-mGluR5ct treated WT and Fmr1 KO neurons. Right: Co-localized mGluR5/GluN1/Homer1-positive signals as percentage of synaptic GluN1 signal (WT: 63.97 ± 2.414 %, n = 26; WT TAT-mGluR5mu: 61.19 ± 3.489 %, n = 14; WT TAT-mGluR5ct: 71.79 ± 1.528 %, n = 22; WT vs. WT TAT-mGluR5ct, *P = 0.043; WT TAT-mGluR5mu vs. WT TAT-mGluR5ct *P  = 0.017, F (2, 59) = 4.87; Fmr1 KO: 72.14 ± 2.081 %, n = 23; Fmr1 KO TAT-mGluR5mu: 67.32 ± 2.832 %, n = 18; Fmr1 KO TAT-mGluR5ct: 67.58 ± 2.69 %, n = 26; Fmr1 KO vs. Fmr1 KO TAT-mGluR5ct P = 0.391; Fmr1 KO TAT-mGluR5mu vs. Fmr1 KO TAT-mGluR5ct P = 0.997, F (2, 64) = 1.13). P values by one-way ANOVA test with Tukey’s Multiple Comparison test. n = dendritic fields from 3 cultures. Scale bar = 10 μm (a) 2 μm (b, c, d, e)

Fig. 5

Disruption of mGluR5/Homer coupling alters synaptic NMDAR function and plasticity. (a) NMDAR-mediated excitatory post-synaptic currents (EPSCs_NMDA) were recorded from CA1 pyramidal neurons. Scale bar = 100 μm (b) Left: representative EPSCs_NMDA traces from WT, Fmr1 KO, WT treated with either TAT-mGluR5ct or TAT-mGluR5mu (both 5 µM, 4 h). Histograms: EPSCs_NMDA amplitude (F_{(3, 35)} = 6.26; **P = 0.0016; one-way ANOVA with Tukey’s Multiple Comparison). Compared to WT (175.7 ± 21.9 pA, n = 11 neurons from 7 animals), EPSCs_NMDA were lower in Fmr1 KO (46.4 ± 8.4 pA, n = 7 neurons from 4 animals, **P < 0.01) and in WT treated with TAT-mGluR5ct (88.1 ± 12.7 pA, n = 13 neurons from 7 animals, *P < 0.05) but not with TAT-mGluR5mu (125.9 ± 16.8 pA n = 8 neurons from 3 animals, P = 0.14). (c) NMDA/AMPA ratio in WT, Fmr1 KO, WT treated with TAT-mGluR5ct or TAT-mGluR5mu (F_{(3, 32)} = 4.1; *P = 0.013; one-way ANOVA with Tukey’s Multiple Comparison). NMDA/AMPA ratio (in WT 1.37 ± 0.17, n = 9 neurons from 4 animals) was reduced in Fmr1 KO (0.86 ± 0.05, n = 8 neurons from 3 animals, *P = 0.02) and in WT treated with TAT-mGluR5ct (0.88 ± 0.10, n = 9 neurons from 3 animals, *P = 0.02). (d, f) The mGluR1/5 agonist DHPG (100 µM, 5 min) induced long-term depression (mGluR-LTD) of EPSCs_NMDA in WT (EPSCs_NMDA amplitude: 24.9 ± 2 % of baseline, n = 8 neurons from 5 animals) but not in Fmr1 KO (EPSCs_NMDA 107.8 ± 25 %, n = 6 neurons from 4 animals, **P < 0.01, WT vs. Fmr1 KO). (e, f) mGluR-LTD of EPSCs_NMDA was abolished in WT treated with TAT-mGluR5ct (EPSCs_NMDA 100.6 ± 15 %, n = 8 neurons from 7 animals, **P < 0.01, WT vs. TAT-mGluR5ct) but not with TAT-mGluR5mu (EPSCs_NMDA 56.6 ± 11 %, n = 7 neurons from 3 animals). (f) mGluR-LTD magnitude in all conditions (F_{(3, 19)} = 7.27, **P = 0.0019, one-way ANOVA with Tukey’s Multiple Comparison)

Fig. 6

Homer1a knockdown rescues synaptic NMDAR dysfunction and cognitive defects in Fmr1 KO mice. (a) Experimental procedure: electrophysiology (upper panel), novel object-recognition task (NOR) on L-maze (middle panel), contextual fear-conditioning task (CFC) (lower panel). WT and Fmr1 KO mice (21 days) were bilaterally injected into the dorsal hippocampus with AAV-shH1a or AAV-Scr expressing GFP, and tested at 7–8 weeks (electrophysiology; b, c) or 12–13 weeks of age (behavior; d, e). (b) The EPSC_NMDA amplitude of Fmr1 KO AAV-sh H1a mice (311.3 ± 58 pA) was larger than that of Fmr1 KO AAV-scr mice (160.4 ± 27.8 pA, n = 12 neurons from 4 animals for both conditions, *P < 0.05, t = 2.35, df = 22, unpaired Student’s t-test). (c) AAV-sh H1a infection in Fmr1 KO mice rescues LTD of EPSC_NMDA induced by DHPG (100 µM, 10 min) (EPSC_NMDA amplitude 40 min after LTD induction: 70.4 ± 6 % of control, n = 6 neurons from 5 animals). mGluR-LTD of EPSC_NMDA was absent in Fmr1 KO AAV-scr mice (EPSC_NMDA amplitude: 106.8 ± 7 % of control, n = 7 neurons from 6 animals, ***P < 0.001, t = 7.73, df = 117 by unpaired Student’s t-test). (d) Discrimination index of NOR on test day: WT AAV-scr, 0.285 ± 0.040, n = 8; Fmr1 KO AAV-scr, −0.030 ± 0.032, n = 9; Fmr1 KO AAV-sh H1a, 0.235 ± 0.022, n = 8; F (2, 22) = 29.02 (***P < 0.001, one-way ANOVA); Fmr1 KO AAV-sh H1a mice performed better than Fmr1 KO AAV-scr, while Fmr1 KO AAV-scr were impaired cf. WT AAV-scr (***P < 0.001, one-way ANOVA test with Tukey’s test). (e) Percentage of time freezing on test day in the CFC (WT AAV-scr, 32.623 ± 3.637 %, n = 9; Fmr1 KO AAV-scr, 13.038 ± 2.203 %, n = 8; Fmr1 KO AAV-sh H1a, 29.743 ± 4.371 %, n = 8, F (2, 22) = 8.836, **P < 0.01, one-way ANOVA). Fmr1 KO AAV-scr exhibited an impaired CFC memory cf. WT AAV-scr, whereas Fmr1 KO AAV-sh H1a showed rescued CFC memory (**P < 0.01, one-way ANOVA test with Tukey’s test)

Fig. 7

Model for dysfunction of the NMDAR/mGluR5 crosstalk in Fmr1 KO neurons. In WT neurons, long Homer proteins anchor mGluR5 to a chain of PSD proteins in the synapse and prevent a direct interaction with NMDAR. Under those conditions, a co-clustering of mGluR5 and NMDAR is prevented. In Fmr1 KO neurons, mGluR5 is less associated with the long Homer proteins and more associated with the short isoform, Homer1a. This disengagement from the long Homer protein–containing complex increases the lateral diffusion of mGluR5 and promotes its interaction with synaptic NMDAR. This configuration at the synapse prevents boosting of NMDAR currents under control conditions and their LTD following mGluR5 stimulation