Inhibition of Calcineurin-mediated Endocytosis and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Prevents Amyloid β Oligomer-induced Synaptic Disruption

Wei-Qin Zhao,Francesca Santini,Robert Breese,Dave Ross,Xiaohua Douglas Zhang,David J Stone,Marc Ferrer,Matthew Townsend,Abigail L Wolfe,Matthew A Seager,Gene G Kinney,Paul J Shughrue,William J Ray

doi:10.1074/jbc.m109.057182

Abstract

Synaptic degeneration, including impairment of synaptic plasticity and loss of synapses, is an important feature of Alzheimer disease pathogenesis. Increasing evidence suggests that these degenerative synaptic changes are associated with an accumulation of soluble oligomeric assemblies of amyloid beta (Abeta) known as ADDLs. In primary hippocampal cultures ADDLs bind to a subpopulation of neurons. However the molecular basis of this cell type-selective interaction is not understood. Here, using siRNA screening technology, we identified alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits and calcineurin as candidate genes potentially involved in ADDL-neuron interactions. Immunocolocalization experiments confirmed that ADDL binding occurs in dendritic spines that express surface AMPA receptors, particularly the calcium-impermeable type II AMPA receptor subunit (GluR2). Pharmacological removal of the surface AMPA receptors or inhibition of AMPA receptors with antagonists reduces ADDL binding. Furthermore, using co-immunoprecipitation and photoreactive amino acid cross-linking, we found that ADDLs interact preferentially with GluR2-containing complexes. We demonstrate that calcineurin mediates an endocytotic process that is responsible for the rapid internalization of bound ADDLs along with surface AMPA receptor subunits, which then both colocalize with cpg2, a molecule localized specifically at the postsynaptic endocytic zone of excitatory synapses that plays an important role in activity-dependent glutamate receptor endocytosis. Both AMPA receptor and calcineurin inhibitors prevent oligomer-induced surface AMPAR and spine loss. These results support a model of disease pathogenesis in which Abeta oligomers interact selectively with neurotransmission pathways at excitatory synapses, resulting in synaptic loss via facilitated endocytosis. Validation of this model in human disease would identify therapeutic targets for Alzheimer disease.

Highlights

Alzheimer disease (AD)2 likely begins with deficits in synaptic transmission in brain regions that are critical for higher cognitive function [1], as stereological analyses of post-mortem samples show that synaptic loss correlates with cognitive dysfunction better than amyloid plaque or neurofibrillary tangle load [2,3,4,5]
Employing an unbiased functional genomics screen in murine neuroblastoma N2A cells followed by verification in primary hippocampal neuronal cultures, we showed that the surface expression and function of AMPAR are required for ADDLsynaptic interaction, which in turn results in the surface amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor loss
AMPA Receptor Subunit and Calcineurin Implicated in biotin-labeled ADDLs (bADDLs) Binding via High Throughput siRNA Screen—To generate neuroactive oligomeric A␤ assemblies, monomeric A␤ was aggregated into ADDLs and bADDLs

Summary

Introduction

Alzheimer disease (AD)2 likely begins with deficits in synaptic transmission in brain regions that are critical for higher cognitive function [1], as stereological analyses of post-mortem samples show that synaptic loss correlates with cognitive dysfunction better than amyloid plaque or neurofibrillary tangle load [2,3,4,5]. SiRNAs targeting Gria4, a gene encoding the type 4 AMPAR subunit (GluR4), showed a reproducible statistically significant reduction in bADDL binding to N2A cells (Fig. 1B, panel 3).

Results

Conclusion