Secreted amyloid-β precursor protein functions as a GABABR1a ligand to modulate synaptic transmission

Abstract

Amyloid-β precursor protein (APP) is central to the pathogenesis of Alzheimer's disease, yet its physiological function remains unresolved. Accumulating evidence suggests that APP has a synaptic function mediated by an unidentified receptor for secreted APP (sAPP). Here we show that the sAPP extension domain directly bound the sushi 1 domain specific to the γ-aminobutyric acid type B receptor subunit 1a (GABA_BR1a). sAPP-GABA_BR1a binding suppressed synaptic transmission and enhanced short-term facilitation in mouse hippocampal synapses via inhibition of synaptic vesicle release. A 17-amino acid peptide corresponding to the GABA_BR1a binding region within APP suppressed in vivo spontaneous neuronal activity in the hippocampus of anesthetized Thy1-GCaMP6s mice. Our findings identify GABA_BR1a as a synaptic receptor for sAPP and reveal a physiological role for sAPP in regulating GABA_BR1a function to modulate synaptic transmission.

Fig. 1.. sAPP selectively binds the sushi 1 domain of GABA_BR1a

(A) Cartoon illustrating AP-MS workflow. (B) Spectral counts of proteins identified by mass spectrometry from 2 independent sAPPα-Fc pull-downs on rat synaptosome extracts. Only proteins which were absent in the Fc controls and present with > 2 spectral counts in a single trial are included. Cell-surface proteins are highlighted in blue. (C) Cartoon of GABA_BR subunits and isoforms. (D) Confocal images (upper) and quantifications (lower) of immunostaining for sAPPα-Fc or Fc binding to GABA_BR1a- , 1b- , or 2-expressing HEK293T cells (n=24). (E) Binding of sAPPα purified protein to immobilized Fc-tagged sushi 1, sushi 2, or sushi 1+2 peptides by biolayer interferometry. (F) Confocal images (upper) and quantifications (lower) of immunostaining for Fc control or sAPPα-Fc binding to GABA_BR1a-expressing HEK293T cells in the presence of increasing concentrations of untagged sushi 1 peptide (n=24–31). (G) Binding of purified sAPPα and sushi 1 proteins (Fc-tag enzymatically removed from both constructs) by isothermal titration calorimetry (ITC). The number of total cells from 3–4 independent experiments is defined by n. Graphs show means ± SEM. Two-way (D) or one-way (F) ANOVA with Bonferroni’s post hoc analysis. ***P < 0.001. Scale bar 10 μm.

Fig. 2.. The extension domain of sAPP binds GABA_BR1a

(A) Cartoon of sAPPα domains. (B) Confocal images (upper) and quantifications (lower) of immunostaining for sAPPα-Fc, GFLD-Fc, CuBD-Fc, ExD-AcD-Fc, ExD-Fc, AcD-Fc, E2-Fc or sAPPαΔExD-Fc binding to GFP- or GABA_BR1a-expressing HEK293T cells (n=24–32). (C) Binding of purified ExD-AcD-Fc and sushi 1 proteins by ITC. (D) Confocal images (upper) and quantifications (lower) of immunostaining for Fc control, sAPPα-Fc, sAPPβ-Fc binding to GABA_BR1a-expressing HEK293T cells (n=24–30). (E) Confocal images (upper) and quantifications (lower) of immunostaining for sAPPα-Fc, sAPLP1-Fc, of sAPLP2-Fc (red) binding to GFP or GABA_BR1a-expressing HEK293T cells (green) (n=24). The number of total cells from 3–5 independent experiments is defined by n. Graphs show means ± SEM. Two-way (B,E) or one-way (D) ANOVA with Bonferroni’s post hoc analysis. ***P < 0.001. Scale bar 10 μm.

Fig. 3.. sAPPα reduces the release probability of synaptic vesicles via presynaptic GABA_BR1a

(A) Cartoon of mPSC measurements in cultured hippocampal mouse neurons reported in B-E. (B,C) Example traces of mEPSCs (green arrowheads) and mIPSCs (red arrowheads) (B) and average mEPSC frequency (C) normalized to baseline recorded from primary neurons before (baseline) and after treatment with sAPPα (250 nM, Fc-tag enzymatically removed, n=13, N = 3, paired t-test). (D) Same as C but with either ExD-AcD, or sAPPαΔExD (Fc-tag enzymatically removed, n=17–20, N=3, one way ANOVA with Dunnett’s post hoc analysis). (E) Same as C but with sAPP and either without (blue) or with (green) preincubation with CGP55845 (CGP, 5 μM), a GABA_BR antagonist. Dotted line denotes baseline (n=14–17, N=3 unpaired t-test). (F) Cartoon of FM1–43 measurements in cultured hippocampal mouse neurons reported in G-I. (G) High-magnification ΔF images before and after application of sAPPα (Fc-tag enzymatically removed, 1 μM) to primary neurons. (H) Summary of the dose-dependent inhibitory effect of sAPPα on presynaptic strength (S) (N= 5–8, one way ANOVA analysis with post hoc Tukey’s analysis). (I) Summary of sAPPα effect on presynaptic vesicle recycling in hippocampal neurons with or without CGP (normalized to control (ctrl)) (N =8). The number of neurons is defined as n, and the number of independent experiments or mice is defined as N. Graphs show means ± SEM. * P < 0.05, ** P < 0.1 *** P < 0.001.

Fig. 4.. sAPP enhances short-term plasticity at Schaffer collateral synapses in a GABA_BR1a-dependent manner

(A) Cartoon of fEPSC measurements in acute mouse hippocampal slices reported in B-G. (B) Representative traces (upper) and average fEPSP amplitude (lower) recorded at Schaffer collaterals (SC) in response to high-frequency burst stimulation at 20 Hz in mouse hippocampal slices incubated without (n = 12, N = 7) or with sAPPα (1 μM, Fc-tag enzymatically removed) (n = 10, N = 7). fEPSPs were normalized to the peak amplitude of the first response. (C) Paired-pulse ratios (PPR) for the first two pulses at each frequency (20 Hz, 50 Hz, and 100 Hz). (D) Same as B but in slices incubated without (n = 10, N = 4) or with sAPPαΔExD (1 μM, Fc-tag enzymatically removed, n = 9, N = 4). (E) Same as C. (F) Same as B but in slices incubated with CGP 54626 (CGP, 10μM) alone (n = 9, N =4) and slices incubated with CGP + sAPPα (n = 8, N = 4). (G) Same as C. The number of slices is defined as n, and the number of independent experiments or mice is defined as N. Graphs show means ± SEM. * P < 0.05, ** P < 0.1 *** P < 0.001. Two-way ANOVA analysis.

Fig. 5.. A short peptide within the APP ExD suppresses synaptic vesicle release via GABA_BR1a

(A) Sequence alignment for the extension domain (ExD) of human APP with APLPs and with 7 vertebrate APP sequences. (B,C) ITC binding experiments of purified sushi 1 and synthetic peptides within the ExD corresponding to (B) 204–220AA or (C) 204–212AA of APP695. (D) An ensemble of 20 lowest-energy NMR structures of the sushi 1 domain of GABA_BR1a when bound to the APP 9mer peptide. (E) A structural model of the complex between the sushi 1 domain of GABA_BR1a (green) and the APP 9mer peptide (cyan) shown as the molecular surface. Protein termini are indicated by the labels. (F) Average mEPSC frequency normalized to baseline recorded from mouse primary neurons before (baseline) and after treatment with 17mer APP peptide (250 nM, APP695 204–220AA) (n= 20, N=3) or scrambled 17mer control peptide (250 nM, n= 18, N= 4) (one way ANOVA with Dunnett’s post hoc analysis). (G) Quantification of the effect of 250 nM 17mer APP peptide (APP695 204–220AA) on mEPSC frequency normalized to baseline (K) either without (n=14; N=3) or with preincubation with CGP55845 (CGP, 5 μM; n=16, N=3) (unpaired t-test). Dotted line denotes baseline. The number of neurons is defined by n. The number of independent experiments is defined by N. Graphs show means ± SEM. * P < 0.05, ** P < 0.1 *** P < 0.001.

Fig. 6.. A 17AA peptide corresponding to the GABA_BR1a binding region within APP suppresses neuronal activity in vivo

(A) Cartoon of in vivo 2-photon calcium imaging of CA1 hippocampus of anesthetized Thy1-GCaMP6s mice with superfusion of APP 17mer, or scrambled control 17mer. (B) in vivo image of CA1 hippocampal neurons of Thy1-GCaMP6s mice. Representative neurons indicated with dotted outline. (C) Calcium traces of five representative neurons, labeled in panel A, before (baseline) and during bath application of 5 μM APP 17mer peptide corresponding to the GABA_BR1a binding region within APP (APP 17mer). (D) Cumulative distribution of the frequency of calcium transients at baseline (black line) and during APP 17mer bath application (blue line) (n=277; N=3). (E) in vivo image of CA1 hippocampal neurons of Thy1-GCaMP6s mice. (F) Calcium traces of five representative neurons, labeled in panel D, before (baseline) and during bath application of 5μM scrambled 17mer control peptide (scrambled 17mer). (G) Cumulative distribution of the frequency of calcium transients at baseline (black line) and during scrambled 17mer bath application (red line) (n=183; N=3). Wilcoxon rank sum test. The number of neurons is defined by n. The number of mice is defined by N. *** P < 0.001; NS P>0.05