The role of AMPA receptor trafficking in synaptic plasticity under physiological conditions is well established; however, its role in ischemia‐induced synaptic remodeling and/or neuronal death is unclear. In the present study, we show direct NP1‐regulation of surface GluR1 expression and synaptic clustering of NP1 with GluR1 in hippocampal neurons. OGD exposure promotes redistribution of GluR1 at the postsynaptic membrane, significantly increasing NP1–GluR1 interaction and ischemic neuronal death. We also found that this clustering activity involves physical association between NP1 and GluR1 and that NP1 exhibited profound synaptic coclustering with GluR1 following OGD. The most important findings in this report are the knocking down of NP1 gene (Ntpx1) blocked OGD‐induced enhanced surface GluR1 expression, its synaptic clustering, and both OGD‐ and AMPA‐mediated neuronal death. These results indicate that NP1 plays a critical role in surface clustering of AMPAR GluR1 at excitatory synapses and excitotoxicity‐mediated ischemic neuronal death.
Hypoxic‐ischemic neuronal injury is triggered by the activation of the glutamatergic excitotoxic cascade6
and several downstream cytotoxic pathways.4,17
At excitatory synapses of central neurons, PSD‐95 couples AMPA receptors to intracellular proteins necessary for synaptic targeting of AMPARs.11
Also, synaptic activation requires surface delivery of AMPARs.29
We found enhanced surface clustering of GluR1 with a simultaneous decrease of internalized GluR1 on the dendrites of hippocampal neurons after OGD and significantly increased neuronal death. It is known that GluR1 surface insertion is regulated by phosphorylation of GluR1 at Ser‐845.30
Thus, the increased levels of phospho‐GluR1 (Ser‐845) observed in this study further corroborate enhanced surface GluR1 trafficking in response to OGD. This surface localization of GluR1 was also documented by surface cross‐linking with BS3
, showing higher levels of surface GluR1. NP1, which is closely related to Narp, has been reported to play a role in excitatory synaptic plasticity in developing and adult brain, and both NP1 and NP2 selectively accumulate at excitatory synapses in primary hippocampal cultures.31
We found that NP1 colocalizes and physically associates with GluR1 and that OGD significantly enhances NP1–GluR1 interactions at synaptic sites, as evident by increased NP1‐GluR1‐PSD‐95 colocalization and coimmunoprecipitation of NP1 with GluR1 and PSD‐95. It appears that OGD recruits NP1 protein to GluR1 subunits to form clusters at excitatory synapses and that increased NP1–GluR1 interaction sensitizes neurons to OGD‐ and AMPA‐induced neuronal death. Our results clearly indicate specific involvement of NP1 in controlling synaptic clustering of GluR1 and excitotoxic function.
The family of long pentraxin proteins has several characteristics: ability to form side‐to‐side and head‐to‐head multimeric aggregates and to bind other proteins via a lectin‐like domain.32
Unlike PSD‐95, NP1 is an extracellular protein with no PDZ domains and no access to the intracellular domains on AMPAR subunits.7,32
This suggests that NP1 interacts with extracellular domains on the receptor proteins. Sia et al showed that knockdown of both NP1 and NPR decreases axonal ability to recruit AMPA GluR4,33
and knockout of NP1 significantly reduced NPR levels, suggesting dependence on each other for synaptic stability.15
We propose that presynaptic NPR binds to NP1, allowing NP1 to trans‐synaptically attach to the extracellular domain of GluR1 at the postsynaptic specialization and thereby facilitating glutamate binding and enhancing excitotoxicity. In contrast, NP1−/− neurons showed reduced excitotoxicity by limiting synaptic GluR1 cluster formation. Xu et al suggested that NP1 and NP2 cofunction to induce AMPAR aggregation and integration into NP1/NP2 heteromultimers, which have a greater ability to induce receptor coclustering.31
Furthermore, NP1 has also been reported to participate in AMPAR GluR4 recruitment in developing postsynaptic specialization.33
Thus, it appears that formation of NP2 clusters alone and NP1–NP2 heterocomplexes with GluR1 may be required for physiological functions in developmental and activity‐dependent synaptic plasticity, but not for pathological brain injury mechanisms. Therefore, differential regulation of NP1 and NP2 provides a mechanism by which neuronal cells can tune their expression and the magnitude of AMPAR clustering during physiological functions and in pathological conditions.
Although excessive activation of glutamate receptors mediates ischemic brain injury, glutamate receptors also mediate essential neuronal excitation under physiological conditions to maintain normal neuronal functions.4,17–18
Most glutamate receptor antagonists indiscriminately block essential glutamate functions and have failed in clinical trials despite their therapeutic potential.34
Most recently, Aarts et al suggested a potential treatment option for ischemic brain damage by perturbing NMDA receptor–PSD‐95 interactions rather than directly blocking NMDARs.20
However, PSD‐95 also plays an equally important role in synaptic plasticity and development in the brain by regulating AMPAR trafficking under normal physiological conditions.17–18,20
Thus, complete blockade of interactions between NMDAR and PSD‐95 is therapeutically impractical. In this study, we show that NP1 interacts with both GluR1 and PSD‐95 and enhances synaptic clustering of GluR1, leading to excitotoxicity and ischemic neuronal death, whereas NP1‐KO neurons were protected against AMPA‐induced neuronal death, demonstrating a direct link between NP1 function and AMPAR‐mediated cell death. Thus, interfering with NP1 and GluR1 interactions would neither compromise AMPAR‐mediated essential neuronal functions nor completely abolish AMPAR–PSD‐95 interactions; rather, it would interrupt signaling downstream of AMPAR, which leads to neuronal death. It is probable that suppression of NP1 induction by suitable pharmacological compounds might hold potential for treating stroke and other diseases, which is a subject of our ongoing investigations.
In summary, our study points to a novel mechanism by which NP1 regulates synaptic clustering of GluR1, an event that has profound effects on signaling pathways downstream of AMPAR activation and excitotoxicity and on ischemic neuronal death. Genetic deletion of NP1 restricts recruitment of GluR1 at synapses, thereby preventing AMPA‐mediated excitotoxicity and ischemic neuronal death. Our results reveal a critical role of NP1 in modulating synaptic AMPAR clustering and its efficacy after OGD, suggesting NP1 as a practical target for preventing ischemic neuronal injury.