Modulation of AMPA receptor surface diffusion restores hippocampal plasticity and memory in Huntington’s disease models

Impaired hippocampal synaptic plasticity contributes to cognitive impairment in Huntington’s disease (HD). However, the molecular basis of such synaptic plasticity defects is not fully understood. Combining live-cell nanoparticle tracking and super-resolution imaging, we show that AMPAR surface diffusion, a key player in synaptic plasticity, is disturbed in various rodent models of HD. We demonstrate that defects in the brain-derived neurotrophic factor (BDNF)–tyrosine receptor kinase B (TrkB) signaling pathway contribute to the deregulated AMPAR trafficking by reducing the interaction between transmembrane AMPA receptor regulatory proteins (TARPs) and the PDZ-domain scaffold protein PSD95. The disturbed AMPAR surface diffusion is rescued by the antidepressant drug tianeptine via the BDNF signaling pathway. Tianeptine also restores the impaired LTP and hippocampus-dependent memory in different HD mouse models. These findings unravel a mechanism underlying hippocampal synaptic and memory dysfunction in HD, and highlight AMPAR surface diffusion as a promising therapeutic target.

in which its kinase domain is exposed, thereby decreasing FRET and increasing the fluorescence lifetime of mEGFP. (b) Representative lifetime image of rat hippocampal neurons expressing PDS95-GFP, REACh-CaMKIIα plus FL-wHTT, and REACh-CaMKIIα plus FL-polyQ-HTT. Blue color indicates strong FRET and short lifetime, while red color represents weak FRET and long lifetime. (c, d) Quantification of lifetime in dendritic puncta (c) or in dendritic shaft (d) in randomly-selected regions of rat hippocampal neurons expressing PDS95-GFP, REACh-CaMKIIα plus FL-wHTT, or REACh-CaMKIIα plus FL-polyQ-HTT. PDS95-GFP-expressing neurons showed long lifetime (≥ 2.4 ns) in both dendritic puncta and shaft indicating no FRET. Lower lifetime indicates stronger FRET and reduced CamKIIα activity; data are mean ± s.e.m; n = 178, 231, and 238 regions for dendritic puncta, and n = 93, 115 and 188 regions for dendritic shaft in neurons expressing PDS95-GFP, REACh-CaMKIIα plus FL-wHTT, and REACh-CaMKIIα plus FL-polyQ-HTT, respectively. Significance was assessed by One-way ANOVA followed by Tukey's multiple comparison test; *** P < 0.001.

Supplementary Figure 4
Tianeptine facilitated BDNF intracellular transport in wHTT-expressing rat hippocampal neurons and neurons from WT mice. (a, b) Anterograde and retrograde BDNF transport velocity in all neurites of vehicle-or tianeptine-treated wHTT-expressing rat hippocampal neurons (a), and in the axon of hippocampal neurons from WT and Hdh Q111/Q111 mice (b); values are mean ± s.e.m; n = 5569, 2522, 5227 and 2542 trajectories for anterograde and retrograde BDNF velocity in vehicle and tianeptine-treated wHTT-expressing neurons, respectively; n = 236, 157, 194 and 110 trajectories for anterograde and retrograde BDNF velocity in vehicle and tianeptinetreated neurons from WT and Hdh Q111/Q111 mice. Significance was determined by unpaired two-tailed Student's t-test; *P < 0.05, ***P < 0.001.

Supplementary Figure 5
Tianeptine did not affect moving velocity of R6/1 mice in Open Field test, either change ambulatory distance or food assumption of CAG140 mice in elevated plus maze (EPM) and novelty-suppressed feeding (NSF) tests, respectively. (a) Moving velocity in open field was significantly different between genotypes but not between treatments; values are mean ± s.e.m; n = 25, 28, 33 and 32 mice for vehicle-and tianeptine-treated WT and R6/1 mice, respectively. (b) In EPM, there is no significant difference in the locomotor activity between genotypes or between treatments, which is revealed by ambulatory distance. (c) In NSF, food consumption was not significantly different between genotypes or between treatments; values are mean ± s.e.m; n = 12, 9, 14, and 13 mice for vehicle-and tianeptine-treated WT and HTT CAG140 mice, respectively. Significance was assessed by twoway ANOVA followed by Tukey's multiple comparison test (a, b, c). ***P < 0.001; ns, not significant.

Immunostaining
For surface GluA2-AMPAR staining, live neurons were incubated with mouse anti-Nterminal GluA2 antibody (a kind gift from E. Gouaux, Oregon Health and Science Unieristy, USA)(1:1000) for 7 minutes followed by fixation and Alexa Fluor 568 goat-anti-mouse secondary antibodies (Thermo Scientific). For total GluA2-AMPAR or HA staining, cells were fixed and permeabilized before the incubation with anti-GluA2 antibody and anti-HA antibody (Cell Signaling Technology) and the following Alexa Fluor 568 secondary antibodies.

Fluorescence Resonance Energy Transfer (FRET)-Fluorescence-lifetime imaging microscopy (FLIM)
A FRET-based CamKIIα, named REACh-CamKII is a kind gift from R. Yasuda (Max Planck Insitute, Florida, USA). The amino and carboxy termini of CamKIIa are labeled with the FRET pair of monomeric enhanced green fluorescent protein (mEGFP) and resonance energy-accepting chromoprotein (REACh), a non-radiative yellow fluorescent protein variant. 1,2 . FLIM experiments were performed at 37 °C using an incubator box with an air heater system (Life Imaging Services) installed on an inverted Leica DMI6000B (Leica Microsystem) spinning disk microscope and using the LIFA frequency domain lifetime attachment (Lambert Instruments, Roden, The Netherlands) and the LI-FLIM software. Cells were imaged with an HCX PL Apo X 100 oil NA 1.4 objective using an appropriate GFP filter set. Cells were excited using a sinusoidally modulated 1-W 477nm LED (lightemitting diode) at 40 MHz under wild-field illumination. Emission was collected using an intensified CCD LI2CAM camera (FAICM; Lambert Instruments). The phase and modulation were determined from a set of 12 phase settings using the manufacturer's LI-FLIM software.
Lifetimes were referenced to a 1µM solution of fluorescein in in Tris-HCl (pH 10) that was set at 4.00 ns lifetime. Signals were recorded with a back-illuminated Evolve EMCCD camera (Photometrics). Acquisitions were carried out on the software MetaMorph (Molecular Devices).