Disrupting stroke-induced GAT-1-syntaxin1A interaction promotes functional recovery after stroke

Abstract

Although stroke is a frequent cause of permanent disability, our ability to promote stroke recovery is limited. Here, we design a small-molecule stroke recovery promoting agent that works by dissociating γ-aminobutyric acid (GABA) transporter 1 (GAT-1) from syntaxin1A (Synt1A), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. Stroke induces an increase in GAT-1-Synt1A interaction in the subacute phase, a critical period for functional recovery. Uncoupling GAT-1-Synt1A reverses stroke-induced GAT-1 dysfunction and cortical excitability decline and enhances synaptic GABAergic inhibition and consequently cortical oscillations and network plasticity by facilitating the assembly of the SNARE complex at the synapse. Based on the molecular mechanism of GAT-1 binding to Synt1A, we design GAT-1-Synt1A blockers. Among them, ZLQ-3 exhibits the greatest potency. Intranasal use of ZLQ-3-1, a glycosylation product of ZLQ-3, substantially lessens impairments of sensorimotor and cognitive functions in rodent models. This compound, or its analogs, may serve as a promoting agent for stroke recovery.

Keywords: GAT-1; functional recovery; network plasticity; stroke; syntaxin1A.

Graphical abstract

Figure 1

Stroke-induced GAT-1-Synt1A interaction hinders stroke recovery (A–C) Coimmunoprecipitation (coIP) showing the amounts of Synt1A-GAT-1 complex and GAT-1 in the peri-infarct cortex on indicated days after stroke. (A) IgG control of coIP experiment (left) and representative immunoblots of coIP experiment (right). Input GAT-1 and input β-actin refers to total GAT-1 and β-actin level in the sample, respectively. (B) The amount of Synt1A-GAT-1 complex. One-way ANOVA followed by post hoc Scheffe test, F_{(4, 20)} = 18.18, n = 5. (C) The level of GAT-1 coupling with Synt1A as a fraction of total GAT-1 protein. One-way ANOVA followed by post hoc Scheffe test, F_{(4, 20)} = 16.21, n = 5. (D) Representative images showing Synt1A-GAT-1 interaction detected by proximity ligation assay (PLA) in samples from the indicated groups. (E) PLA quantification showing the amount of Synt1A-GAT-1 complex per 50 nuclei. Two-tailed t test, t₍₂₂₎ = −8.23, n = 12. (F) CoIP showing Synt1A-GAT-1 complex level in the neurons after treating with Synt1A_251-265 or peptides clipped from Synt1A_251-265. One-way ANOVA followed by post hoc Scheffe test, F_{(4, 20)} = 17.03, n = 5. (G) CoIP showing the effect of Tat-Synt1A_251-265mut on Synt1A-GAT-1 interaction in cultured neurons. Two-tailed t test, t₍₁₀₎ = 0.21, n = 6. (H) CoIP showing the effect of AAV-Synt1A_251-265 on stroke-induced Synt1A-GAT-1 interaction. One-way ANOVA followed by post hoc Scheffe test, F_{(2, 12)} = 5.85, n = 5. (I) Left, foot faults of the left forelimb in the grid-walking task. Middle, foot faults of the left hindlimb in the grid-walking task. Right, forelimb symmetry in the cylinder task. Two-way repeated-measures ANOVA followed by post hoc Bonferroni test. Left, F_{(2, 31)} = 312.827; middle, F_{(2, 31)} = 77.627; right, F_{(2, 31)} = 233.376. (J) Left, foot faults of the left forelimb in the grid-walking task. Middle, foot faults of the left hindlimb in the grid-walking task. Right, forelimb symmetry in the cylinder task. Two-way repeated-measures ANOVA followed by post hoc Bonferroni test. Left, F_{(3, 37)} = 231.769; middle, F_{(3, 37)} = 22.897; right, F_{(3, 37)} = 122.262. See also Figure S1.

Figure 2

Dissociating Synt1A from GAT-1 reverses stroke-induced decrease in cortical excitability (A) Bar graph showing the effect of Tat-Synt1A_251-265 on the concentrations of extracellular GABA in the peri-infarct cortex. One-way ANOVA followed by post hoc Scheffe test, F_{(2, 15)} = 23.29, n = 6. (B) Bar graph showing the effect of Tat-Synt1A_251-265 on GABA uptake in the cultured neurons. Two-tailed t test, t₍₈₎ = −6.72, n = 5. (C) Hypothesis: stroke-induced GAT-1-Synt1A association causes GAT-1 dysfunction and consequent tonic inhibition increase, dissociating Synt1A from GAT-1 by Synt1A_251-265 reverses stroke-induced GAT-1 dysfunction and tonic inhibition. (D) Representative traces and bar graphs showing tonic inhibitory currents from indicated groups. Tonic current was the change in baseline holding current after bath-application of BMI, represented as the distance between the red and blue dashed lines. One-way ANOVA followed by post hoc Scheffe test, F_{(4, 51)} = 8.37. (E) Normalized GCaMP6s fluorescence of pyramidal neurons of mouse before and during spontaneously rearing. (F) Quantification of fluorescence changes after spontaneously rearing. One-way ANOVA followed by post hoc Scheffe test, F_{(3, 23)} = 34.98. (G) Representative mEPSC traces. (H) mEPSC amplitude recorded in the peri-infarct pyramidal neurons from mice of indicated groups. One-way ANOVA followed by post hoc Scheffe test, F_(3, 45) = 2.04. (I) mEPSC frequency recorded in the peri-infarct pyramidal neurons from mice of indicated groups. One-way ANOVA followed by post hoc Scheffe test, F_(3, 45) = 26.07. (J) Representative sEPSC traces. (K) sEPSC amplitude recorded in the peri-infarct pyramidal neurons from mice of indicated groups. One-way ANOVA followed by post hoc Scheffe test, F_(3, 36) = 0.11. (L) sEPSC frequency recorded in the peri-infarct pyramidal neurons from mice of indicated groups. One-way ANOVA followed by post hoc Scheffe test, F_(3, 36) = 19.9. See also Figure S2.

Figure 3

Dissociating Synt1A from GAT-1 enhances synaptic GABAergic inhibition through facilitating the assembly of SNARE complex (A) Representative immunoblots (upper) and bar graphs showing the effect of Tat-Synt1A_251-265 on the amount of Synt1A-SNAP-25 (middle) and Synt1A-VAMP-2 (lower) complexes. For Synt1A-SNAP-25 coupling, one-way ANOVA followed by post hoc Scheffe test, F_{(2, 12)} = 30.58; for Synt1A-VAMP-2 coupling, one-way ANOVA followed by post hoc Scheffe test, F_{(2, 12)} = 27.86. n = 5. (B) Representative immunoblots (left) and bar graphs showing SNARE complexes (middle) and SNAP-25 monomer (right). For SNARE complexes, one-way ANOVA followed by post hoc Scheffe test, F_{(2, 12)} = 20.37; for SNAP-25 monomer, one-way ANOVA followed by post hoc Scheffe test, F_{(2, 12)} = 1.84. n = 5. (C) Representative immunoblots (left) and bar graphs showing SNARE complexes (middle) and Synt1A monomer (right). For SNARE complexes, one-way ANOVA followed by post hoc Scheffe test, F_{(2, 15)} = 13.25; for Synt1A monomer, one-way ANOVA followed by post hoc Scheffe test, F_{(2, 15)} = 0.8. n = 6. (D) Representative immunoblots (left) and bar graphs showing SNARE complexes (middle) and VAMP-2 monomer (right). For SNARE complexes, one-way ANOVA followed by post hoc Scheffe test, F_{(2, 15)} = 6.31; for VAMP-2 monomer, one-way ANOVA followed by post hoc Scheffe test, F_{(2, 15)} = 1.21. n = 6. (E) Hypothesis: dissociating Synt1A from GAT-1 reverses stroke-induced impairment of SNARE complex formation. (F) Representative mIPSC traces. (G) mIPSC amplitude recorded in the peri-infarct pyramidal neurons from mice of indicated groups. One-way ANOVA followed by post hoc Scheffe test, F_(3, 38) = 1.67. (H) mIPSC frequency recorded in the peri-infarct pyramidal neurons from mice of indicated groups. One-way ANOVA followed by post hoc Scheffe test, F_(3, 38) = 13.69. (I) Representative sIPSC traces. (J) sIPSC amplitude recorded in the peri-infarct pyramidal neurons from mice of indicated groups. One-way ANOVA followed by post hoc Scheffe test, F_(3, 39) = 0.9. (K) sIPSC frequency recorded in the peri-infarct pyramidal neurons from mice of indicated groups. One-way ANOVA followed by post hoc Scheffe test, F_(3, 39) = 7.7. See also Figure S2.

Figure 4

Dissociating Synt1A from GAT-1 enhances cortical oscillations, neural population activity, and plasticity of projections (A) Left, percentage of change power spectrum of the peri-infarct cortex LFPs during movement. Solid lines represent the average and shaded areas indicate SEM. Right, percentage of change in LFP power of each waveband during movement. For delta, theta, beta, low-gamma, and high-gamma oscillations, one-way ANOVA followed by post hoc Scheffe test, F_{(3, 43)} = 28.69, 38.78, 33.77, 12.38, and 2.08, respectively. (B) Raster plots showing neural population firing within the layer 5 of the peri-infarct cortex during movement. Bottom: bar graphs of firing events. (C) Percentage of change in firing events during movement. One-way ANOVA followed by post hoc Scheffe test, F_{(3, 18)} = 12.61. (D) Right: representative traces and input-output curve peak amplitude of LFPs in the contralateral motor cortex. Two-way repeated-measures ANOVA followed by post hoc Bonferroni test, F_{(3, 35)} = 14.435, n = 9 slices from 6 animals for stroke + Tat-Synt1A_251-265mut (GAT-1 WT) group, n = 10 slices from 6 to 7 animals for other groups. Middle: representative traces and input-output curve peak amplitude of LFPs in the ipsilateral striatum. Two-way repeated-measures ANOVA followed by post hoc Bonferroni test, F_{(3, 36)} = 20.519, n = 10 slices from 6 to 7 animals for each group. Right: representative traces and input-output curve peak amplitude of LFPs in the spinal cord. Two-way repeated-measures ANOVA followed by post hoc Bonferroni test, F_{(3, 34)} = 76.282, n = 9 slices from 6 to 7 animals for sham + Tat-Synt1A_251-265mut (GAT-1 WT) and stroke + Tat-Synt1A_251-265 (GAT-1 CKO) groups, n = 10 slices from 6 to 7 animals for other groups. See also Figure S3.

Figure 5

Design of small-molecule GAT-1-Synt1A blockers by targeting the N-terminal of GAT-1 (A) Synt1A-GAT-1 complex level in the cultured neurons after overexpressing Synt1A with site-directed point mutations at residues 251–256. One-way ANOVA followed by post hoc Scheffe test, F_{(5, 24)} = 13.72, n = 5. (B) Synt1A-GAT-1 complex level after overexpressing the mutation of GAT-1_{K33A/K36A/K37A}. Two-tailed t test, t₍₁₀₎ = 2.67, n = 6. (C) GABA uptake after overexpressing the mutation of GAT-1_{K33A/K36A/K37A}. Two-tailed t test, t₍₈₎ = −2.62, n = 5. (D) Hypothesis: stroke induces Synt1A-GAT-1 association, which plays negative roles in the extracellular reuptake of GABA into neurons. (E) Upper, general structure of ZLQ series of compounds designed to uncouple Synt1A-GAT-1; lower, structure of ZLQ-3. (F) Molecular docking of ZLQ-3 to the N-terminal of GAT-1. See also Figure S4.

Figure 6

Small-molecule Synt1A-GAT-1 blockers promote stroke recovery (A) Structure of ZLQ-3-1. (B) GABA uptake after ZLQ-3-1 treatment in the cultured neurons. Two-tailed t test, t₍₈₎ = −4.02, n = 5. (C) Experimental design for (D–K). (D) The time course of concentrations of ZLQ-3-1 in the brain tissue. (E) Left: coIP showing Synt1A-GAT-1 complexes in the peri-infarct cortex. One-way ANOVA followed by post hoc Scheffe test, F_{(2, 12)} = 26.21. Right: coIP showing Synt1A-GAT-1 complexes in the hippocampus. One-way ANOVA followed by post hoc Scheffe test, F_{(2, 12)} = 54.09. (F) The concentrations of extracellular GABA in cultured neurons. One-way ANOVA followed by post hoc Scheffe test, F_{(2, 12)} = 23.49, n = 5. (G) Left: foot faults of the left forelimb in the grid-walking task. Middle: forelimb symmetry in the cylinder task. Right: forelimb sticky-tape ratio in modified sticky-tape test. Two-way repeated-measures ANOVA followed by post hoc Bonferroni test. Left: F_{(2, 36)} = 514.555. Middle: F_{(2, 36)} = 242.291. Right: F_{(2, 36)} = 131.507. (H) Escape latency to the platform during the training trails in a Morris water maze. Two-way repeated-measures ANOVA followed by post hoc Bonferroni test, F_(2, 36) = 19.383. (I) Target (platform) entries in the probe test. One-way ANOVA followed by post hoc Scheffe test, F_{(2, 36)} = 9.01. (J) Time spent in target quadrant in the probe test. One-way ANOVA followed by post hoc Scheffe test, F_{(2, 36)} = 15.58. (K) Mean swimming speed of rats. One-way ANOVA followed by post hoc Scheffe test, F_{(2, 36)} = 0.04. See also Figures S5 and S6.