Tuning of Glutamate, But Not GABA, Release by an Intrasynaptic Vesicle APP Domain Whose Function Can Be Modulated by β- or α-Secretase Cleavage

Abstract

APP, whose mutations cause familial Alzheimer's disease (FAD), modulates neurotransmission via interaction of its cytoplasmic tail with the synaptic release machinery. Here we identified an intravesicular domain of APP, called intraluminal SV-APP interacting domain (ISVAID), which interacts with glutamatergic, but not GABAergic, synaptic vesicle proteins. ISVAID contains the β- and α-secretase cleavage sites of APP: proteomic analysis of the interactome of ISVAID suggests that β- and α-secretase cleavage of APP cuts inside the interaction domain of ISVAID and destabilizes protein-protein interactions. We have tested the functional significance of the ISVAID and of β-/α-secretase-processing of APP using various ISVAID-derived peptides in competition experiments on both female and male mouse and rats hippocampal slices. A peptide encompassing the entire ISVAID facilitated glutamate, but not GABA, release acting as dominant negative inhibitor of the functions of this APP domain in acute hippocampal slices. In contrast, peptides representing the product of β-/α-secretase-processing of ISVAID did not alter excitatory neurotransmitter release. These findings suggest that cleavage of APP by either β- or α-secretase may inactivate the ISVAID, thereby enhancing glutamate release. Our present data support the notion that APP tunes glutamate release, likely through intravesicular and extravesicular interactions with synaptic vesicle proteins and the neurotransmitter release machinery, and that β-/α cleavage of APP facilitates the release of excitatory neurotransmitter.SIGNIFICANCE STATEMENT Alzheimer's disease has been linked to mutations in APP. However, the biological function of APP is poorly understood. Here we show that an intravesicular APP domain interacts with the proteins that control the release of glutamate, but not GABA. Interfering with the function of this domain promotes glutamate release. This APP domain contains the sites cleaved by β- and α-secretases: our data suggest that β-/α cleavage of APP inactivates this functional APP domain promoting excitatory neurotransmitter release.

Keywords: APP; Alzheimer disease; GABA; glutamate; secretases; synaptic vesicles.

Figure 1.

The intraluminal APP-SV interactome. A, β- and/or α-secretases may: (1) destroy a binding domain of APP, thereby downregulating interaction with putative binding partners, schematically represented as A; and (2) generate APP metabolites, sAPPβ, sAPPα, αCTF, and βCTF, with new docking sites for proteins, schematically represented as a–d, which do not interact with full-length APP. B, Baits used for the proteomic experiments shown in this manuscript. Casp. indicates the caspase cleavage site present in the cytosolic tail of APP. β and arrow, β' and arrow indicate the two cleavage sites of APP by β-secretase; α and arrow indicates the cleavage site of APP by α-secretase. C, Brain lysates were affinity purified on StrepTactin columns bound to the indicated St peptides. Bound proteins were analyzed by Western blot. Syp, Vamp2, Syt1, and Vglut1 bound APP-derived baits with this quantitative order: Ex/TM-St > β/TM-St [tmt] α/TM-St. None of these four proteins bounds the negative control peptide St.

Figure 2.

APP localizes at presynaptic vesicles. A, TIF and TSF were prepared from mouse cortex and analyzed by SDS-PAGE and Western analysis. Postnuclear supernatant is labeled S1. S2 fractions have been depleted of synaptic markers, present only in P2 and/or subsequent fractions. SPs contain both presynaptic and postsynaptic markers. The TIF is enriched in postsynaptic markers PSD95 and Nmdar2b, and the presynaptic markers Syp, Vamp2, Nsf, SNAP25, Stx1b, Stxbp1, SV2A, SV2B, SV2C, VGlut1, and VIAAT are enriched in the TSF. Fractions were analyzed for the presence of APP by blotting against the C terminus, which recognizes full-length APP (∼100 kDa) and all C-terminal fragments (∼10–15 kDa), for the C terminus of PS1, Nct, and β-secretase. Immuno-EM using secondary antibody alone (B) or an anti-APP-C-terminal antibody Y188 in mouse cerebral cortex (C) or hippocampal CA1 (D) shows that APP-positive signals are closely associated with presynaptic vesicles. We selected Y188 for I-EM because it has demonstrated specificity for APP in immunofluorescence experiments. Synapses were identified by morphology: SV (arrowheads), clefts (SC), the AZ, and the postsynaptic density (PSD). Larger arrows pointing to gold particles (10 nm) indicate distribution of APP predominately on SVs (SV-A in D). Mitochondria (Mito) are also indicated. Scale bars: B, C, 500 nm; D, 200 nm.

Figure 3.

Ex/TM is uploaded into cells. Pen1-JCasp is present extracellularly, in SVs, and in the cytosol (A), whereas JCasp can only reside in extracellularly and in SVs (B). The evidence that Pen1-JCasp, but not JCasp, reduces glutamate release (Fanutza et al., 2015) demonstrates that JCasp must be delivered to the cytosol to exert this biological activity. C, We postulate that Ex/TM is biologically active inside the SV and that Ex/TM can be uploaded in SV during SV recycling. D, Confocal microscopy analysis of FITC-Ex/TM in brain slices. Brain slices from 12-week-old mice were treated with 10 μm FITC-Ex/TM in ACSF, fixed, and stained with DAPI. Hippocampi (CA1 shown here) were visualized by confocal microscopy. The images were merged in the right two panels. FITC-ExTM is taken up into cells.

Figure 4.

Ex/TM increases glutamate release: APP is required for this effect. A, Sequence of the Ex/TM peptide. Ex indicates the arbitrarily chosen NH₂ terminus for our analysis; β and α indicate the sites cleaved by β- and α-secretase, respectively; and TM indicates the beginning of the transmembrane region of APP. B, Average PPF (second EPSP/first EPSP) at 50 and 200 ms ISI. Representative traces of EPSPs are shown. Ex/TM significantly decreases PPF in WT, but not App-KO, rats. C, Cumulative probability of AMPAR-mediated mEPSC interevent intervals. Inset in cumulative probability graphs represents average mEPSC frequency. mEPSC frequency was significantly increased by Ex/TM in WT, but not App-KO, mice. Amplitudes and decay time of mEPSCs were not changed by Ex/TM. Representative recording traces of mEPSCs are shown. Data were analyzed by ordinary one-way ANOVA followed by post hoc Tukey's multiple-comparisons test when ANOVA showed statistically significant differences. The number of recordings analyzed for each group are indicated inside the bars. Data are mean ± SEM. ****p < 0.0001.

Figure 5.

The regions of Ex/TM NH₂- and COOH-terminal to the β cleavage site are required to increase glutamate release. A, Sequence of Ex/TM, β/TM, Ex/β, and Ex/α. B, Average PPF at 50 and 200 ms ISI and representative traces of EPSPs evoked at 50 ms ISI are shown. Ex/TM and Ex/α decrease PPF. C, Ex/TM and Ex/α increase mEPSC frequency. Amplitudes and decay time were not changed. Representative recording traces of mEPSCs are shown. D, AMPA/NMDA ratio was not changed by any peptide. Data were analyzed by ordinary one-way ANOVA followed by post hoc Tukey's multiple-comparisons test when ANOVA showed statistically significant differences. The number of recordings analyzed for each group are indicated inside the bars. Data are mean ± SEM.

Figure 6.

Ex/TM increases synaptic strength. Input–output relationships for AMPA receptor-mediated EPSCs in vehicle (n = 6) and Ex/TM (n = 6) group. Traces showing averages of 15 sweeps of AMPA receptor-mediated EPSCs at each stimulation intensity. Data were analyzed by two-way repeated-measures ANOVA followed by post hoc Tukey's multiple-comparisons test. The number of recordings analyzed for each group are indicated inside the bars. Data are mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 7.

Ex/TM does not alter GABA transmission at inhibitory CA1 synapses. A, Ex/TM increases mEPSC frequency, but not amplitude and decay time. Representative recording traces of mEPSCs are shown. B, Ex/TM does not alter frequency, amplitude, or decay time of mIPSCs. Representative recording traces of mIPSCs are shown. Data were analyzed by unpaired t test. The number of recordings analyzed for each group are indicated inside the bars. Data are mean ± SEM. ***p < 0.001.