Local and Use-Dependent Effects of β-Amyloid Oligomers on NMDA Receptor Function Revealed by Optical Quantal Analysis

Abstract

Beta amyloid (A␤) triggers the elimination of excitatory synaptic connections in the CNS, an early manifestation of Alzheimer's disease. Oligomeric assemblies of A␤ peptide associate with excitatory synapses resulting in synapse elimination through a process that requires NMDA-type glutamate receptor activation. Whether A␤ affects synaptic NMDA receptor (NMDAR) function directly and acts locally at synapses to which it has bound and whether synaptic activity influences A␤ synaptic binding and synaptotoxicity have remained fundamental questions. Here, we used subcellular Ca 2ϩ imaging in rat hippocampal neurons to visualize NMDAR function at individual synapses before and after A␤ application. A␤ triggered a robust impairment of NMDAR Ca 2ϩ entry at most, but not all, synapses. NMDAR function was more severely impaired at highly active synapses and synapses with bound A␤, but activity was not required for A␤ synapse binding. Blocking NMDARs during A␤ exposure prevented A␤-mediated impairment. Finally, A␤ impaired NMDAR Ca 2ϩ entry at doses much lower than those required for NMDAR internalization, revealing a novel, potent mode of NMDAR regulation by A␤.

L-Type Voltage-Gated Ca2+ Channels Regulate Synaptic Activity-Triggered Recycling Endosome Fusion in Neuronal Dendrites

“…This protocol relies on synaptic NMDA receptor activation by spontaneous quantal glutamate release (Lu et al, 2001). We confirmed that NMDA receptors are activated under these conditions using GCaMP6s to monitor intracellular Ca 2+ dynamics (Chen et al, 2013; Sinnen et al, 2016). Upon removal of Mg 2+ from the extracellular solution, we observed robust Ca 2+ transients originating in dendritic spines (Figure 3A).…”

Simultaneous Live Imaging of Multiple Endogenous Proteins Reveals a Mechanism for Alzheimer’s-Related Plasticity Impairment

“…Indeed, block of CaMKII translocation and induction of NMDA-receptor internalization have the same dependence on the Aβ dose, with both effects seen only in response to concentrations higher than 5 nM (see Figure 3E and Sinnen et al, 2016). Additionally, in experiments using surface receptor biotinylation, both effects are seen only after incubation times longer than 5 min (Figures 4F and S3A; compare to Figure 3F).…”

“…CaMKII binding to GluN2B is induced by Ca 2+ / CaM-stimulation (Bayer et al, 2001; O’Leary et al, 2011), which is provided during LTP-stimuli by NMDA-receptor activation. Recent results indicate that Aβ can reduce the Ca 2 currents through the NMDA receptor (Sinnen et al, 2016), which in turn could reduce the Ca 2+ -induced CaMKII movement. However, our results show that Aβ also inhibits CaMKII movement that is triggered when the Ca 2+ -stimulus is instead directly induced by ionomycin.…”

“…HiLyte Fluor 647-labeled and unlabeled Aβ 1–42 (AnaSpec) were prepared in aliquots as a dried 1,1,1,3,3,3-hexafluoro-2-propanol film and stored at 80 C, as described previously (Klein, 2002), however, the peptides were diluted in PBS instead of F12 medium (Sinnen et al, 2016). The peptide film was dissolved in 4.4 μL of anhydrous DMSO and diluted to 50 μM with 1X PBS and allowed to oligomerize at 4°C overnight.…”

Optogenetic Control of Synaptic Composition and Function

“…Because our mEPSC measurements were made under spontaneous release conditions (1 μM TTX), we also measured the rate of spontaneous neurotransmitter release with an independent method that reports spontaneous quantal release. Using the genetically encoded Ca 2+ indicator jRGECO1a expressed in postsynaptic neurons (along with SEP-GluA1-CIB and untagged CRY2-homer1c), we can observe Ca 2+ entry through activated NMDA receptors in response to spontaneous quantal neurotransmission (Dana et al, 2016; Sinnen et al 2016). Thus we can quantify the frequency of spontaneous neurotransmitter release events at the same synaptic sites before and after GluA1 recruitment.…”