INTERLEUKIN-1BETA AND NMDA RECEPTOR: A BRIDGE BETWEEN INFLAMMATION AND THE GLUTAMATERGIC SYSTEM

Interleukin (IL)-1β, originally described as an immune cell mediator in the periphery, has been involved in the modulation of several neurological functions and dysfunctions (Rothwell and Hopkins, 1995; Viviani et al., 2007). IL-1β is involved in processes like regulation of sleep-wake cycle, control of synaptic activity, LTP maintenance/inhibition, and is implicated in several pathological conditions like ischemia, excitotoxic injury, Alzheimer’s disease, HIV-dementia complex, epilepsy, neuropathic pain. Recently IL-1β has been indicated as important mediator in neuroendocrine and neurobehavioral stress response (Goshen and Yirmya, 2009) and to play a role in psychiatric disorders like schizophrenia (Meyers et al. 2011). While the initial trigger for acute injury or chronic disease may differ between neurological disorders, the resulting pathology may involve overlapping, if not identical, mechanisms. As such, a better understanding of the molecular mechanisms that underlie the action of this cytokine within the CNS might facilitate the development of promising therapeutics in the field of CNS disorders. The biochemical pathways by which this cytokine contributes to brain dysfunction and injury remains largely unidentified. Substantial evidence suggests the existence of a reciprocal functional interaction between IL-1β and NMDA receptors (NMDARs) (Fogal et al. 2008; Hagan et al., 1996; Loddick and Rothwell, 1996 Vezzani et al., 1999). NMDARs are glutamate-gated ion channel widely expressed in the central nervous system (CNS) and play key roles in excitatory synaptic transmission. They are essential mediators of many forms of synaptic plasticity and molecular mechanisms of cognition (Aamodt, 1999; Bliss et al., 1993). NMDARs are also key mediators of glutamate exicitotoxicity associated in acute neurological traumas as stroke, or in chronic neurodegeneration disease, including Huntington’s disease, Alzheimer’s diseases (Triller and Coquet, 2005). Based on these observations, in 2003 we hypothesized the existence of a functional relationship between IL-1β and the NMDAR that could in a way provide a molecular mechanism to several features common to both neurodegenerative and psychiatric disorders. We actually demonstrated, that recombinant IL-1β induces the activation of Src family kinases and the subsequent phosphorylation at Tyr-1472 of GluN2B subunit of NMDAR (Viviani et al., 2003) in primary hippocampal neurons. The activation of this pathway potentiates NMDA-induced intracellular Ca2+ increase and also exacerbates NMDA-induced neuronal death in vitro (Viviani et al., 2003). Thus, these results confirmed our hypothesis suggesting that hippocampal neurons exposed to IL-1β are more susceptible to glutamatergic excitation through the NMDA receptor component. Furthermore, this findings suggest that the recruitment of IL-1β/NMDAR cross-talk could provide the missing link to understand the events implicated in the convergence of these to systems. Due to: (i) the relevance of these two systems in the regulation of neuronal functions and in inducing susceptibility of neuronal impairment and decline, and (ii) the potential therapeutic implications, we thought to better define the molecular mechanisms that regulate the IL-1β/NMDAR cross talk by using both in vitro and in vivo approaches. We thus investigated (i) the effect of native IL-1β generated in an in vitro model of neurotoxicity on NMDAR and the impact on neuronal organization and survival; (ii) the distribution of IL-1RI and the associated signalling proteins, IL-1RAcP and MyD88, in rat hippocampal subcellular compartments, both in physiological and pathological conditions and (iii) the dynamical interaction existing between IL-1RI and NMDAR (iv) the possible recruitment of IL-1β/NMDAR cross talk in an animal model of early life stress. (i) Using a sandwich co-cultures of primary hippocampal neurons and glia exposed to HIV-1 envelope glycoprotein gp120, we demonstrate that native IL-1β released by activated glia increases tyrosine phosphorylation of NR2B Tyr1472, enriches NR2B at the post-synaptic site, and also promotes the synaptic localization of IL-1RI. This biochemical cascade triggered by gp120 resulted in a significant loss of spine density in the presence of undamaged dendrites which preceded neuronal death, evident 72 hours after gp120 treatment. This damage was completely prevented in its different stages by either the inhibitor of the src family tyrosine kinases and the IL-1 receptor antagonist (IL-1ra). Thus, tyrosine phosphorylation may contribute to the sensitization of the NMDAR increasing its function and synaptic localization. Both these effects are relevant for neurodegeneration. Furthermore, the ability of IL-1ra to counteract the effects of gp120 on neurons suggests the recruitment of IL-1 receptor type I (IL-1RI) by native IL-1β. (ii) The binding of IL-1β to IL-1RI in the immune system leads to its association with the IL-1R accessory protein (IL-1RAcP) (Korherr et al., 1997) and the myeloid differentiation primary response protein 88 (MyD88) (Burns et al., 1998) to form the core of the IL-1β/IL-1RI signalling complex. However, little information is currently available concerning the molecular composition of the members of the IL-1R complex, or their subcellular distribution and relation with NMDARs in neuronal cells. We thus investigated the distribution of IL-1RI, IL-1RAcP and MyD88, and NR2B subunits, in different subcellular compartments purified both from primary hippocampal neurons and adult rats hippocampus by means of western blotting and/or confocal microscopy. IL-1RI is enriched at the synaptic sites where it co-localizes with the NR2B subunits of the NMDAR. MyD88 is also enriched at the synaptic sites, while only traces of IL-1RAcP are present only indicating a differential subcellular distribution of the IL-1R complex protein members in neuronal cells. Furthermore, immunoprecipitation studies reveal that IL-1RI forms a complex together with NR2B subunit of the NMDAR. (iii) It is well known that the synaptic localization of receptors and ion channels, together with their protein-protein interactions, are modulated in response to various stimuli, and that they undergo dynamic changes under physiological and pathological conditions (Newpher et al., 2008; Groc et al., 2009). In our experimental model gp120 enriches the post-synaptic site with both the NR2B subunits of the NMDAR and IL-1RI. Since gp120 acts through the release of IL-1β and the overactivation of NMDA receptor, we investigated whether IL-1β and NMDA stimulation contributes to the re-distribution of IL-1R1 and interaction with the NMDAR. Treatments of primary hippocampal neurons with NMDA increase IL-1RI interaction with NMDA receptors, as well as the surface expression and localization of IL-1RI at synaptic membranes. IL-1β also increases IL-1RI levels at synaptic site, without affecting the total amount of receptor in the plasma membrane. Our results show for the first time the existence of a functional and dynamical interaction between IL-1RI system and NMDAR. In fact, IL-1β modulates NMDA functions and receptor trafficking inducing an enrichment of the post synaptic membrane with both NMDAR and IL-1RI; also NMDA contributes partly to these events. This suggests a new molecular mechanism by means of which IL-1β system and NMDA system may contribute to an alteration of the synaptic organization, leading to neuronal death. (iv) Stressful challenge experienced in early-life could shape the development and functions of the immune system. These events could change the immunoreactivity in the brain for the remainder of the lifespan and play a fundamental role in promoting susceptibility to central nervous system dysfunctions from poor cognitive disabilities to neuropsychiatric disorders (Hornig et al., 1999; Nelson and Willoughby, 2000; Rantakallio et al., 1997; Shi et al., 2003). The alteration of cognitive and behavior functions are due to an alteration of glutamatergic system. This implicates that in this model the relation between IL-β signalling and NMDAR system could be involved in the alteration of behavioural and cognitive functions. It has been performed a long-time (24 hours) event of maternal deprivation (MD) during at post-natal day 9 (PND9) of rats and then the long-lasting modifications induced by maternal deprivation were evaluated at PND45. MD at PND9 significantly modulates three key events of IL-1β/IL-1RI/NMDAR connection; MD increases the phosphorylation at Tyr-1472 of the GluN2B, increase the levels of IL-1RI at the synapse and the amount of IL-1RI associated to GluN2B. This modulation still evident at PND45, specifically in the hippocampus, while no variation occurs at the prefrontal cortex. Thus, an early life stress induces a long-lasting modifications in synaptic setting by altering IL-1RI/NMDAR interaction. These final results reveal that the dynamic and functional interaction between IL-1RI and NMDAR is enduringly modulated early in life. The enrichment of IL-1RI at the synapse of hippocampal neurons may contribute to prime the neuronal synapse to the action of IL-1β and could provide a molecular basis on the critical role for the immune system in early life programming of later in life brain functions and behaviour REFERENCES Aadmodt, S.M., Constantine-Paton, M. 1999. 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