Impaired alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking and function by mutant huntingtin

Abstract

Emerging evidence from studies of Huntington disease (HD) pathophysiology suggests that huntingtin (htt) and its associated protein HAP1 participate in intracellular trafficking and synaptic function. However, it is largely unknown whether AMPA receptor trafficking, which is crucial for controlling the efficacy of synaptic excitation, is affected by the mutant huntingtin with polyglutamine expansion (polyQ-htt). In this study, we found that expressing polyQ-htt in neuronal cultures significantly decreased the amplitude and frequency of AMPAR-mediated miniature excitatory postsynaptic current (mEPSC), while expressing wild-type huntingtin (WT-htt) increased mEPSC. AMPAR-mediated synaptic transmission was also impaired in a transgenic mouse model of HD expressing polyQ-htt. The effect of polyQ-htt on mEPSC was mimicked by knockdown of HAP1 and occluded by the dominant negative HAP1. Moreover, we found that huntingtin affected mESPC via a mechanism depending on the kinesin motor protein, KIF5, which controls the transport of GluR2-containing AMPARs along microtubules in dendrites. The GluR2/KIF5/HAP1 complex was disrupted and dissociated from microtubules in the HD mouse model. Together, these data suggest that AMPAR trafficking and function is impaired by mutant huntingtin, presumably due to the interference of KIF5-mediated microtubule-based transport of AMPA receptors. The diminished strength of glutamatergic transmission could contribute to the deficits in movement control and cognitive processes in HD conditions.

FIGURE 1.

Synaptic AMPAR responses are enhanced by WT-htt and impaired by polyQ-htt. A and B, cumulative distribution plot of the mEPSC amplitude and inter-event interval in cortical neurons transfected with GFP, WT-htt (17Q) or polyQ-htt (68Q). C, representative mEPSC traces. Scale bar: 25 pA, 2 s. D and E, summary data (mean ± S.E.) of mEPSC amplitude and frequency in cortical or striatal neurons with different transfections. *: p < 0.05, ANOVA. F, summarized input-output curves of AMPAR-EPSC evoked by a series of stimulus intensities in striatal MSNs taken from WT versus N171–82Q mice (4.5-month-old). Inset, representative AMPAR-EPSC traces. Scale bars: 50 pA, 20 ms. **, p < 0.01, ANOVA.

FIGURE 2.

Knockdown of HAP1 causes a loss of synaptic AMPAR responses. A and B, cumulative distribution plot of the mEPSC amplitude and inter-event interval in cortical neurons transfected with GFP, HAP1 siRNA, or a scrambled control siRNA. C, representative mEPSC traces. Scale bar: 25 pA, 2 s. D, summary data (mean ± S.E.) of mEPSC amplitude and frequency in cortical neurons with different transfections. *, p < 0.05, ANOVA.

FIGURE 3.

Dominant negative HAP1 (DN-HAP1) occludes the reducing effect of polyQ-htt on synaptic AMPAR responses. A and B, cumulative distribution plot of the mEPSC amplitude and inter-event interval in cortical neurons transfected with GFP, polyQ-htt, DN-HAP1, or DN-HAP1 plus polyQ-htt. C, representative mEPSC traces. Scale bar: 25 pA, 2 s. D, summary data (mean ± S.E.) of mEPSC amplitude and frequency in cortical neurons with different transfections. *, p < 0.05, ANOVA.

FIGURE 4.

The polyQ-htt-induced impairment of synaptic AMPAR responses is occluded by knockdown of KIF5, but not KIF-17. A, representative Western blots in HEK293 cells transfected with FLAG-tagged rat KLC1 in the absence or presence of a control siRNA or a KLC1 siRNA. B–D, representative mEPSC traces (B) and cumulative distribution plot of the mEPSC amplitude (C) and inter-event interval (D) from cortical neurons transfected with control siRNA, polyQ-htt, KLC1 siRNA, or KLC1 siRNA plus polyQ-htt. Scale bar: 25 pA, 1 s. E and F, summary data (mean ± S.E.) of mEPSC amplitude and frequency in cortical neurons with different transfections. *, p < 0.05, ANOVA. NS, no significance.

FIGURE 5.

The WT-htt-induced enhancement of synaptic AMPAR responses is blocked by knockdown of KIF5. A–C, representative mEPSC traces (A) and cumulative distribution plot of the mEPSC amplitude (B) and inter-event interval (C) from cortical neurons transfected with control siRNA, WT-htt, KLC1 siRNA, or KLC1 siRNA plus WT-htt. Scale bar: 25 pA, 1 s. D and E, summary data (mean ± S.E.) of mEPSC amplitude and frequency in cortical neurons with different transfections. *, p < 0.05, ANOVA. NS, no significance.

FIGURE 6.

PolyQ-htt impairs synaptic AMPAR responses independent of GABA_ARs, and mainly targets GluR2-containing AMPARs. A and B, representative mEPSC traces and summary data (mean ± S.E.) of mEPSC amplitude and frequency from bicuculline-treated cortical neurons transfected with GFP versus polyQ-htt. Bicuculline (10 μm) was added 2 h before transfection and remained throughout the course of expressing polyQ-htt. Scale bar: 10 pA, 2 s. *, p < 0.05, t test. C and D, representative mEPSC traces and summary data (mean ± S.E.) of mEPSC amplitude from cortical neurons transfected with GFP versus polyQ-htt in the absence or presence of Naspm. *, p < 0.05; **, p < 0.005, t test. Scale bar: 10 pA, 10 s. E, summary data (mean ± S.E.) of the percentage block of mEPSC amplitude by Naspm in GFP- versus polyQ-htt-transfected cortical neurons. *, p < 0.05; t test.

FIGURE 7.

HAP1/GluR2/KIF5/microtubule complex is disrupted in the mouse model of HD. A–C, co-immunoprecipitation assays showing the association between tubulin and KIF5 (A), GluR2 and KIF5 (B), tubulin and GluR2 (B), GluR2 and HAP1 (C) from striatal slices of WT versus HD mice (4-month-old). Each experiment was repeated in 3–5 pairs of mice. D, immunoblots and quantification analysis showing the surface and total GluR2 and GluR1 subunits in lysates of striatal slices taken from WT versus HD mice (4-month-old). *, p < 0.01, ANOVA. E, co-immunoprecipitation assays showing the association between KIF5 and GluR1 or GluR2 in brain lysates.

FIGURE 8.

A schematic model demonstrating the potential mechanism for huntingtin regulation of AMPAR trafficking. A, in normal conditions, WT-htt binds to HAP1 and enables the binding of HAP1 to KIF5 motor protein. KIF5 heavy chain interacts with GRIP1/GluR2-containing vesicles, and the whole complex is anterogradely transported along the microtubule (MT) rails. B, in HD conditions, polyQ-htt interacts abnormally with HAP1 (Step 1), thereby interrupting the binding of GluR2/KIF5 (Step 2), and KIF5/MT (Step 3). Consequently, KIF5 is unable to transport the GluR2 cargo along MTs (Step 4), leading to insufficient synaptic delivery of AMPAR channels.