Amyloid-β oligomers cause deregulation of NMDAR function, initially disrupting calcium homeostasis and triggering neuronal oxidative stress (Kelly and Ferreira 2006; De Felice et al. 2007), followed by removal of NMDARs from dendrites (Snyder et al. 2005; Lacor et al. 2007; Shankar et al. 2007; De Felice et al. 2009). These responses are thought to contribute to plasticity failure and memory dysfunction in AD. We recently found that antibodies against the extracellular domain of the NR1 subunit of NMDARs markedly reduce Aβ oligomer binding to neurons (De Felice et al. 2007). This suggests that Aβ oligomers bind to synapses in close proximity to NMDARs, raising the possibility that NMDARs are necessary components of or are required for the assembly of the receptor complex that binds oligomers.
In the present study, we have used a herpes virus-derived amplicon vector expressing an antisense sequence for the NR1 subunit (Adrover et al. 2003; Cheli et al. 2006) to knock-down the expression of NMDARs in cultured hippocampal neurons. Results showed that although control neurons attacked by Aβ oligomers exhibit abnormally elevated reactive oxygen species (ROS) levels, this pathological response was abolished in NMDAR knock-down neurons. Significantly, knock-down of NMDARs abolished the binding of Aβ oligomers to dendrites, suggesting that NMDARs might be the targets of oligomer binding. Additional results, however, indicated that, although required for dendritic targeting of Aβ oligomers, NMDARs are likely not the molecules to which oligomers directly bind. Identifying the molecular targets of Aβ oligomers may pave the way for development of new approaches to prevent oligomer binding to synapses, effectively halting the progression of AD.
- Top of page
- Material and methods
- Supporting Information
Progress in AD research has been hampered by two fundamental issues with broad implications for our understanding of the molecular mechanisms of pathogenesis and for the rational development of novel and effective therapeutics. The first problem comprises the nature of the toxic species responsible for the initial memory impairment and for neuronal dysfunction in AD. In this regard, considerable advance has been made in the past decade with increased recognition that soluble Aβ oligomers are the proximal neurotoxins that attack synapses and cause synapse failure in the early phases of pathology. The other major unknown is the molecular identity of the receptor(s) that bind Aβ oligomers with high specificity at excitatory synapses. Identification of Aβ oligomer-binding receptor(s) may open the way for development of novel approaches to prevent neuronal targeting of Aβ oligomers.
Aβ oligomers are ligands for post-synaptic spines and co-localize with PSD-95 (Lacor et al. 2004). The specific binding of Aβ oligomers to synapses is lost upon controlled trypsin treatment of neurons, suggesting the involvement of cell-surface proteins (Lambert et al. 1998). A number of candidate oligomer-binding proteins have been proposed in the past few years. For example, an interesting recent study showed that Aβ oligomers bind with nanomolar affinity to cellular prion protein (PrPC) and that anti-PrP antibodies reduce oligomer binding to neurons and rescue synaptic plasticity (Lauren et al. 2009). These findings suggest that PrPC is involved in synaptic binding and mediates synaptotoxicity of Aβ oligomers (Gimbel et al. 2010). However, other studies have shown that PrP-expressing and PrP-knock-out mice are equally susceptible to long-term memory impairment induced by Aβ oligomers (Balducci et al. 2010; Calella et al. 2010), supporting the notion that, at least in part, the deleterious effects of Aβ oligomers on plasticity are independent of PrPC (Kessels et al. 2010).
Co-immunoprecipitation and photoactivated amino acid cross-linking studies indicated that Aβ oligomers interact with complexes containing the GluR2 subunit of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors, a conclusion that was corroborated by the observation that pharmacological inhibition or removal of surface AMPA receptors reduced Aβ oligomer binding to neurons (Zhao et al. 2010). Moreover, using single-particle tracking of quantum dot-labeled Aβ oligomers, a very recent study has demonstrated the participation of metabotropic glutamate receptors (mGluR5) in Aβ oligomer binding and clustering at synapses (Renner et al. 2010).
In addition, interaction of Aβ oligomers with the extracellular domain of p75 neurotrophin receptor (p75NTR) has been reported to mediate Aβ oligomer-induced neuritic dystrophy in vitro and in vivo (Knowles et al. 2009). Other putative receptors involved in neuronal binding of Aβ oligomers include the receptor for advanced glycation end products (Sturchler et al. 2008), nicotinic acetylcholine receptors (Magdesian et al. 2005) and Frizzled (Magdesian et al. 2008), which leads to inhibition of Wnt/β-catenin signaling.
Until now, however, the identity of the specific receptor(s) that bind oligomers at synapses has remained elusive. Current results establish that NMDARs are required for oligomer binding to dendrites. We have previously demonstrated that NMDARs co-immunoprecipitate with Aβ oligomers from detergent-extracted oligomer-treated rat synaptosomal membranes and that an N-terminal anti-NR1 antibody markedly reduced oligomer binding to dendrites (De Felice et al. 2007). Consistent with those findings, we now show that oligomer binding is virtually abolished in dendrites of NMDAR knock-down neurons. One possible explanation for this result could be that Aβ oligomers bind directly to NMDARs at synapses. Interestingly, however, we found that similar NMDAR levels are found in both oligomer-attacked and non-attacked neurons, suggesting that presence of NMDARs alone is not sufficient for oligomer binding.
To further address the question whether Aβ oligomers directly bind NMDARs, we took advantage of our previous finding that insulin signaling down-regulates Aβ oligomer-binding sites (De Felice et al. 2009). Insulin plays a key role in plasticity mechanisms in the CNS (Zhao et al. 1999; Zhao and Alkon 2001) and has been shown to improve cognitive performance in patients with early stage AD (Reger et al. 2008). Insulin acts through the insulin receptor (IR), a protein tyrosine kinase with pivotal roles in regulation of peripheral glucose metabolism and energy homeostasis. IRs also occur in the brain, where they are abundantly distributed in synaptic membranes of the cerebral cortex and hippocampus (Heidenreich et al. 1983, 1988; Matsumoto and Rhoads 1990; Zhao et al. 1999). Of relevance to the current study, we have recently shown that insulin blocks Aβ oligomer-binding to synapses (De Felice et al. 2009). Decreased binding is the result of down-regulation of Aβ oligomer-binding sites by insulin, through a mechanism requiring IR tyrosine kinase activity.
Based on these previous findings, we have employed insulin as a tool to down-regulate Aβ oligomer-binding sites in hippocampal neurons and to examine the possibility that NMDARs might be down-regulated in parallel, as one would expect if NMDARs were the oligomer-binding protein at synapses. Interestingly, pre-treatment of hippocampal neurons with insulin caused a significant reduction in oligomer binding in the absence of a parallel reduction in NMDAR levels. This indicates that NMDARs are not the oligomer binding receptor that is down-regulated by insulin. We thus conclude that, although required for binding of Aβ oligomers, NMDARs are not sufficient for binding. Results suggest that NMDARs are either constituents of a multi-protein receptor complex that binds Aβ oligomers (which may include other protein components, such as those mentioned above) or they are required for the assembly/surface expression of the Aβ oligomer-binding receptor.
Recent studies have shown that Aβ oligomers disrupt neuronal calcium homeostasis via aberrant activation of NMDARs (Kelly and Ferreira 2006; De Felice et al. 2007). NMDARs have also been implicated in other neuronal pathologies induced by oligomers, for example, tau hyperphosphorylation (De Felice et al. 2008), synapse loss (Lacor et al. 2007) and impairment of fast axonal transport of organelles (Decker et al. 2010). In a previous study, we investigated the connection between Aβ oligomer binding, NMDAR dysregulation and NMDAR-dependent neuronal oxidative stress (De Felice et al. 2007). In that study, we showed that memantine, a moderate affinity NMDAR blocker, potently inhibited Aβ oligomer-induced formation of reactive oxygen species (ROS) in hippocampal neurons. Similarly, MK-801, a higher affinity NMDAR blocker, and D-AP5, a competitive antagonist, completely blocked Aβ oligomer-induced ROS formation. Importantly, however, memantine and MK-801 had no effect on Aβ oligomer binding to hippocampal neurons, and D-AP5 caused partial reduction in binding. We also investigated the effect of the AMPA receptor antagonist, 6,7-Dinitroquinoxaline-2,3-dione, on binding and toxicity of Aβ oligomers. Treatment of hippocampal cultures with 6,7-Dinitroquinoxaline-2,3-dione caused only a slight inhibition of ROS formation and had no effect on Aβ oligomer binding (De Felice et al. 2007). Together with the current results, these previous findings indicate that surface expression of NMDARs (but not necessarily their activity) is required for Aβ oligomer binding, and that dysregulated NMDAR activity triggers pathways leading to oligomer-induced neuronal dysfunction and toxicity.
We now show that oligomers failed to induce oxidative stress in NMDAR knock-down neurons, whereas non-infected or mock-infected neurons exhibited robust ROS response to oligomers. These observations support the idea that, in addition to their role in oligomer binding, NMDARs also mediate aberrant signaling that culminates in neuronal damage in AD.
NMDAR function is essential for learning, memory and for the induction of LTP (Morris 2001; Nakazawa et al. 2004; Lau and Zukin 2007). Moreover, specific subunits of NMDARs, such as NR2B, mediate synaptic plasticity in vivo (Hu et al. 2009; Ronicke et al. 2010). Thus, new drugs optimized as blockers of Aβ oligomer binding that do not impair physiological NMDAR function potentially could provide improved AD therapeutics. In this regard, we recently showed that antibodies blocking the N-terminal extracellular domain of NMDARs significantly reduced Aβ oligomer binding to neurons without interference with receptor response to glutamate (De Felice et al. 2007).
In conclusion, current results demonstrate that NMDARs play a pivotal role in the assembly and/or expression of the neuronal receptor that binds Aβ oligomers. This discovery supports the new concept of a multi-component assembly required for stabilizing the toxic accumulation of Aβ oligomers at the synaptic membrane. Whether oligomers target a pre-assembled receptor complex that already exists at the membrane or whether they act as scaffolding molecules to promote the assembly of such a complex (Renner et al. 2010) remains to the determined. Whatever the case, a key concept that derives from this notion is that clustering of Aβ oligomer-bound receptors may be required for toxicity, for example, by triggering excessive calcium influx and ROS formation. From a therapeutic point of view, this suggests the possibility that clustering and toxicity could be blocked by interfering with different molecular components of the receptor complex. Uncovering the identity of the synaptic targets of Aβ oligomers may provide insight into the complexity of the mechanisms that underlie synapse failure in AD and may pave the way for development of novel and more effective AD therapeutics.
- Top of page
- Material and methods
- Supporting Information
Figure S1. Characterization of AβO preparations.
Figure S2. Knockdown efficiency and cell viability.
As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.