Amyloid-β Oligomers Interact with Neurexin and Diminish Neurexin-mediated Excitatory Presynaptic Organization

Alzheimer’s disease (AD) is characterized by excessive production and deposition of amyloid-beta (Aβ) proteins as well as synapse dysfunction and loss. While soluble Aβ oligomers (AβOs) have deleterious effects on synapse function and reduce synapse number, the underlying molecular mechanisms are not well understood. Here we screened synaptic organizer proteins for cell-surface interaction with AβOs and identified a novel interaction between neurexins (NRXs) and AβOs. AβOs bind to NRXs via the N-terminal histidine-rich domain (HRD) of β-NRX1/2/3 and alternatively-spliced inserts at splicing site 4 of NRX1/2. In artificial synapse-formation assays, AβOs diminish excitatory presynaptic differentiation induced by NRX-interacting proteins including neuroligin1/2 (NLG1/2) and the leucine-rich repeat transmembrane protein LRRTM2. Although AβOs do not interfere with the binding of NRX1β to NLG1 or LRRTM2, time-lapse imaging revealed that AβO treatment reduces surface expression of NRX1β on axons and that this reduction depends on the NRX1β HRD. In transgenic mice expressing mutated human amyloid precursor protein, synaptic expression of β-NRXs, but not α-NRXs, decreases. Thus our data indicate that AβOs interact with NRXs and that this interaction inhibits NRX-mediated presynaptic differentiation by reducing surface expression of axonal β-NRXs, providing molecular and mechanistic insights into how AβOs lead to synaptic pathology in AD.

Note that cells expressing SEP-NRX1βS4(-) ∆HRD have no signal of bound Aβ.
Scale bar represents 30 μm .Data are presented as mean ± SEM.

Animals
All animal experiments were carried out in accordance with the Canadian Council on Animal Care guidelines and approved by the IRCM Animal Care Committee and the McGill University Animal Care Committee. We used heterozygous transgenic adult C57BL/6 mice (6 months old, mixed sex) expressing the human amyloid precursor protein (hAPP) carrying the Swedish (K670N, M671L) and Indiana (V717F) familial AD mutations driven by the platelet-derived growth factor (PDGF) β-chain promoter (APP mice, J20 line) 3 and age-matched wild-type (WT) littermates.

Preparation of Aβ 42 oligomers
Aβ(1-42) (r-peptide, A-1002-2, 1 mg) and biotin-tagged Aβ(1-42) (Anaspec, AS-23523-05, 0.5 mg) were used to generate oligomeric forms essentially as described previously 4 . Briefly, lyophilized peptides were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; Sigma-Aldrich, Cat # 52517) to ensure that the starting material was in a homogenous non-aggregated monomeric state, then aliquots containing peptide were placed in low-binding polypropylene microcentrifuge tubes for 2 hours at room temperature for peptide monomerization. The HFIP was evaporated in a vacuum centrifuge concentrator (SPD131, Thermo Scientific) and the resulting Aβ peptide films were stored at −80°C with desiccant. Prior to use, each peptide film was reconstituted in dimethylsulfoxide (DMSO, Sigma-Aldrich, Hybri-Max D-2650) to obtain a 1 mM Aβ stock solution, which was then incubated in a bath sonicator for 10 minutes. The peptide stock was then diluted to a concentration of 100 μM with 10 mM Tris-HCl, pH 7.4, and incubated for 48 hours at 22°C to facilitate the formation of higher molecular weight oligomers. These preparations were stored at −80°C or used in experiments immediately. Individual Aβ oligomer stocks were never thawed and re-frozen. To confirm oligomer formation, the preparation was run on a 4-20% TGX precast gel (Biorad) and immunoblotted with anti-β-Amyloid 1-16 (1:5000; mouse IgG1; clone 6E10; Covance).

Neuron culture, coculture-based artificial synapse formation assay and immunocytochemistry
Cultures of rat hippocampal neurons, COS-7 cells, HEK293 cells, coculture-based artificial synapse formation assays, and immunocytochemistry were performed essentially as reported previously 1,2 .

Time-lapse imaging
For time-lapse imaging, hippocampal neurons cultured on 18-mm coverslips were cotransfected with a SEP-NRX construct and mCherry at 10 days in vitro (DIV) and used for imaging at 20-22 DIV. During imaging, the live transfected neurons were mounted in a Chamlide CMB magnetic chamber (Live Cell Instrument) and maintained in ECS at 37°C controlled by a Tempcontrol 37-2 device (Pecon Germany) without perfusion. Aβ 42 oligomers (500 nM, monomer equivalent) were manually added into ECS in the chamber 5 minutes after taking the first image. Fluorescent imaging was performed using a Leica DMIRE2 inverted microscope (Leica Germany) equipped with an Orca ER CCD camera (Hamamatsu Japan) and a 63X 1.4 NA oil objective lens. All images were acquired by Volocity software (Perkin Elmer) at 1344 × 1024 resolution with 12 bits/pixel.

Fluorescence quantification
All imaging and image analysis were done while blind to the experimental condition. Analysis was performed by using Metamorph 7.8 software (Molecular Devices), Microsoft Excel, and GraphPad Prism 6. For binding of biotin-Aβ 42 oligomers and Fc-fusion proteins, the average intensity of bound protein per COS-7 cell area minus off-cell background was normalized to the average intensity of the surface HA signal on COS-7 cells expressing the indicated HA-tagged proteins. For cocultures, fields for imaging were chosen using only the HA and phase contrast channels to locate HA-positive HEK293 cells in neurite-rich regions. The VGLUT1 or VGAT channel was thresholded and the total intensity of the puncta within HA-positive HEK293 cell regions was measured. For time-lapse imaging, the average background intensity of the image before Aβ treatment was measured, and this value was subtracted from the intensity of each frame of the time-lapse image sequences. The axons of transfected neurons were defined based on the morphology of mCherry-expressing neurons. In the image before Aβ treatment, the areas corresponding to puncta of SEP-NRX1β in mCherry-positive axons were manually traced as regions of interest (ROIs) using Metamorph 7.8. The average intensity of SEP and mCherry signals in these ROIs in each frame was measured. To quantify the effects of Aβ treatment on NRX surface expression, the SEP signal was normalized to the mCherry signal. Correction of the image shift in the x-y plane was done by comparing mCherry and SEP images. Pseudo-color images were created based on the fluorescence intensity range of the image prior to the Aβ treatment by Metamorph 7.8.

Synaptosome preparation
Preparation of synaptosome fractions from mice was performed essentially as described previously 5 . All steps were performed at 4°C. The cerebral cortex of each mouse was homogenized in 2 mL of Buffer A (5 mM HEPES, pH 7.4, 1 mM MgCl 2 , 0.5 mM CaCl 2 , 1 mM DTT, 0.32 M sucrose, supplemented with protease inhibitors) by passing the lysate 9 times through a 1-mL syringe without needle and then 5 times through a 1-mL syringe with a 18-gauge needle. The hippocampi from each two mice were homogenized together in 1 mL of Buffer A by passing the lysate as described above to obtain enough material as a single sample. The suspension was centrifuged for 10 minutes at 1,400 g, and the supernatant was set aside. The pellet was resuspended in Buffer A (2 mL in cortex and 1 mL in hippocampus) using a 1-mL syringe with a 23-gauge needle 3-5 times, and the suspension was centrifuged for 10 minutes at 750-1,000 g. The supernatant was pooled with the supernatant collected after the first centrifugation (fraction: S1). The pooled fractions were centrifuged for 10 minutes at 12,000 g, and the supernatant was removed (fraction, Cytoplasm). The pellet was resuspended in 1 mL (cortex) or 0.5 mL (hippocampus) of Buffer B (6 mM Tris, pH 8.1, 0.32 M sucrose, 1 mM EDTA, 1 mM EGTA, 1