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

Interleukin (IL)-1\u3b2, 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-1\u3b2 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\u2019s disease, HIV-dementia complex, epilepsy, neuropathic pain. Recently IL-1\u3b2 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-1\u3b2 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\u2019s disease, Alzheimer\u2019s diseases (Triller and Coquet, 2005). Based on these observations, in 2003 we hypothesized the existence of a functional relationship between IL-1\u3b2 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-1\u3b2 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-1\u3b2 are more susceptible to glutamatergic excitation through the NMDA receptor component. Furthermore, this findings suggest that the recruitment of IL-1\u3b2/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-1\u3b2/NMDAR cross talk by using both in vitro and in vivo approaches. We thus investigated (i) the effect of native IL-1\u3b2 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-1\u3b2/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-1\u3b2 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-1\u3b2. (ii) The binding of IL-1\u3b2 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-1\u3b2/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-1\u3b2 and the overactivation of NMDA receptor, we investigated whether IL-1\u3b2 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-1\u3b2 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-1\u3b2 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-1\u3b2 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-\u3b2 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-1\u3b2/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-1\u3b2 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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Interleukin-1beta immunoreactivity and microglia are enhanced in the rat hippocampus by focal kainate application: functional evidence for enhancement of electrographic seizures. J Neurosci 19: 5054-5065. Viviani, B., Bartesaghi, S., Gardoni, F., Vezzani, A., Behrens, M.M., Bartfai, T., Binaglia, M., Corsini, E., M. Di Luca, M., Galli, C.L., Marinovich, M., 2003. Interleukin-1beta enhances NMDA receptor-mediate intracellular calcium increase through activation of the src family kinases. J Neurosci 23: 8692-8700. Viviani, B., Gardoni, F., Marinovich, M. 2007. Cytokines and neuronal ions channels in health and disease. Int Rev Neurobiol 82: 8213-8217

Early maternal deprivation immunologically primes hippocampal synapses by redistributing interleukin-1 receptor type I in a sex dependent manner

Challenges experienced in early life cause an enduring phenotypical shift of immune cells towards a sensitised state that may lead to an exacerbated reaction later in life and contribute to increased vulnerability to neurological diseases. Peripheral and central inflammation may affect neuronal function through cytokines such as IL-1. The extent to which an early life challenge induces long-term alteration of immune receptors organization in neurons has not been shown. We investigated whether a single episode of maternal deprivation (MD) on post-natal day (PND) 9 affects: (i) the synapse distribution of IL-1RI together with subunits of NMDA and AMPA receptors; and (ii) the interactions between IL-1RI and the GluN2B subunit of the NMDAR in the long-term, at PND 45. MD increased IL-1RI levels and IL-1RI interactions with GluN2B at the synapse of male hippocampal neurons, without affecting the total number of IL-1RI or NMDAR subunits. Although GluN2B and GluN2A were slightly but not significantly changed at the synapse, their ratio was significantly decreased in the hippocampus of the male rats who had experienced MD; the levels of the GluA1 and GluA2 subunits of the AMPAR were also decreased. These changes were not observed immediately after the MD episode. None of the observed alterations occurred in the hippocampus of the females or in the prefrontal cortex of either sex. These data reveal a long-term, sex-dependent modification in receptor organisation at the hippocampal post-synapses following MD. We suggest that this effect might contribute to priming hippocampal synapses to the action of IL-1\u3b2

U and Th content in the Central Apennines continental crust: a contribution to the determination of the geo-neutrinos flux at LNGS

The regional contribution to the geo-neutrino signal at Gran Sasso National Laboratory (LNGS) was determined based on a detailed geological, geochemical and geophysical study of the region. U and Th abundances of more than 50 samples representative of the main lithotypes belonging to the Mesozoic and Cenozoic sedimentary cover were analyzed. Sedimentary rocks were grouped into four main "Reservoirs" based on similar paleogeographic conditions and mineralogy. Basement rocks do not outcrop in the area. Thus U and Th in the Upper and Lower Crust of Valsugana and Ivrea-Verbano areas were analyzed. Based on geological and geophysical properties, relative abundances of the various reservoirs were calculated and used to obtain the weighted U and Th abundances for each of the three geological layers (Sedimentary Cover, Upper and Lower Crust). Using the available seismic profile as well as the stratigraphic records from a number of exploration wells, a 3D modelling was developed over an area of 2^{\circ}x2^{\circ} down to the Moho depth, for a total volume of about 1.2x10^6 km^3. This model allowed us to determine the volume of the various geological layers and eventually integrate the Th and U contents of the whole crust beneath LNGS. On this base the local contribution to the geo-neutrino flux (S) was calculated and added to the contribution given by the rest of the world, yielding a Refined Reference Model prediction for the geo-neutrino signal in the Borexino detector at LNGS: S(U) = (28.7 \pm 3.9) TNU and S(Th) = (7.5 \pm 1.0) TNU. An excess over the total flux of about 4 TNU was previously obtained by Mantovani et al. (2004) who calculated, based on general worldwide assumptions, a signal of 40.5 TNU. The considerable thickness of the sedimentary rocks, almost predominantly represented by U- and Th- poor carbonatic rocks in the area near LNGS, is responsible for this difference.Comment: 45 pages, 5 figures, 12 tables; accepted for publication in GC

Epidemiology of intra-abdominal infection and sepsis in critically ill patients: “AbSeS”, a multinational observational cohort study and ESICM Trials Group Project

Purpose: To describe the epidemiology of intra-abdominal infection in an international cohort of ICU patients according to a new system that classifies cases according to setting of infection acquisition (community-acquired, early onset hospital-acquired, and late-onset hospital-acquired), anatomical disruption (absent or present with localized or diffuse peritonitis), and severity of disease expression (infection, sepsis, and septic shock). Methods: We performed a multicenter (n = 309), observational, epidemiological study including adult ICU patients diagnosed with intra-abdominal infection. Risk factors for mortality were assessed by logistic regression analysis. Results: The cohort included 2621 patients. Setting of infection acquisition was community-acquired in 31.6%, early onset hospital-acquired in 25%, and late-onset hospital-acquired in 43.4% of patients. Overall prevalence of antimicrobial resistance was 26.3% and difficult-to-treat resistant Gram-negative bacteria 4.3%, with great variation according to geographic region. No difference in prevalence of antimicrobial resistance was observed according to setting of infection acquisition. Overall mortality was 29.1%. Independent risk factors for mortality included late-onset hospital-acquired infection, diffuse peritonitis, sepsis, septic shock, older age, malnutrition, liver failure, congestive heart failure, antimicrobial resistance (either methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, extended-spectrum beta-lactamase-producing Gram-negative bacteria, or carbapenem-resistant Gram-negative bacteria) and source control failure evidenced by either the need for surgical revision or persistent inflammation. Conclusion: This multinational, heterogeneous cohort of ICU patients with intra-abdominal infection revealed that setting of infection acquisition, anatomical disruption, and severity of disease expression are disease-specific phenotypic characteristics associated with outcome, irrespective of the type of infection. Antimicrobial resistance is equally common in community-acquired as in hospital-acquired infection

Glia-neuron sandwich cocultures: an in vitro approach to evaluate cell-to-cell communication in neuroinflammation and neurotoxicity

Glia-neuron sandwich cocultures are "in vitro" cell systems suitable to evaluate cell-to-cell interactions relaying on the release of soluble factors, such as proinflammatory cytokines. This chapter presents a protocol to obtain a sandwich coculture from primary rat glial cells and hippocampal neurons. Furthermore, it provides details to evaluate the release of tumor necrosis factor-\u3b1 by a biological assay and neuronal cell survival, as well as examples of practical application in the investigation of neuroinflammation and its impact on neuronal function

Cytokines and neuronal channels : a molecular basis for age-related decline of neuronal function?

The achievement that cytokines and all the related "transducing machinery" are expressed within the central nervous system and play a consistent role in the modulation of neurological functions and dysfunctions, has allowed a novel interpretation of brain behaviour. In this view, neuroinflammation and cytokines production has been acknowledged as potential triggers of the functional changes occurring in the brain during "normal" and "pathological" aging. In particular the aged brain seems to be characterized by increased levels of pro-inflammatory cytokines. A growing number of reports show that cytokines may specifically interact with neuronal channels regulating neuronal excitability, synaptic plasticity and responses to injury. By reviewing literature and data obtained in our laboratory, we discuss the hypothesis that cytokines modulation of neuronal channels might occur during the aging process and play a role in aged related decline of neuronal function

Software cost estimation: an experimental study of model performances

The accurate prediction of software development costs may have a large economic impact. As a consequence, considerable research attention is now directed to understand better the software development process. The objective of this paper is to provide an experimental evaluation of the applicability, universality and accuracy of some algorithmic software cost estimating models (COCOMO, TUCOMO, PUTNAM, COPMO, ESSE and Function Points). Data on nine Italian MIS (Management Information Systems) projects were collected and used to evaluate the performance of the models. The evaluation of the estimates was based on the Mean Magnitude Relative Error ( MRE) and Prediction at level 25 % (PRED(25%)) criteria. Results indicated that the models provided interesting performances, better ˝if recalibrated with local data

Perspectives on neuroinflammation and excitotoxicity: A neurotoxic conspiracy?

Emerging evidences underline the ability of several environmental contaminants to induce an inflammatory response within the central nervous system, named neuroinflammation. This can occur as a consequence of a direct action of the neurotoxicant to the CNS and/or as a response secondary to the activation of the peripheral inflammatory response. In both cases, neuroinflammation is driven by the release of several soluble factors among which pro-inflammatory cytokines. IL-1b and TNF-a have been extensively studied for their effects within the CNS and emerged for their role in the modulation of the neuronal response, which allow the immune response to integrate with specific neuronal functions, as neurotransmission and synaptic plasticity. In particular, it has been evidenced a potential detrimental link between these cytokines and the glutamatergic system that seems to be part of increased brain excitability and excitotoxicity occurring in different pathological conditions. Aim of this mini-review will be to present experimental evidence on the way IL-1b and TNF-a impact neurons, focusing on the glutamatergic signalling, to provide a perspective on novel pathways possibly involved in environmental contaminants neurotoxicity

Neuroactive steroids, their metabolites, and neuroinflammation

Neuroinflammation represents a common feature of many neurodegenerative diseases implicated both in their onset and progression. Neuroactive steroids act as physiological regulators and protective agents in the nervous system. Therefore, the attention of biomedical research has been recently addressed in evaluating whether neuroactive steroids, such as progestagens, androgens, and estrogens may also affect neuroinflammatory pathways. Observations so far obtained suggest a general anti-inflammatory effect with a beneficial relapse on several neurodegenerative experimental models, thus confirming the potentiality of a neuroprotective strategy based on neuroactive steroids. In this scenario, neuroactive steroid metabolism and the sophisticated machinery involved in their signaling are becoming especially attractive. In particular, because metabolism of neuroactive steroids as well as expression of their receptors is affected during the course of neurodegenerative events, a crucial role of progesterone and testosterone metabolites in modulating neuroinflammation and neurodegeneration may be proposed. In the present review, we will address this issue, providing evidence supporting the hypothesis that the efficacy of neuroactive steroids could be improved through the use of their metabolites

Somatostatin Modulates Insulin-Degrading-Enzyme Metabolism: Implications for the Regulation of Microglia Activity in AD

The deposition of b-amyloid (Ab) into senile plaques and the impairment of somatostatin-mediated neurotransmission are key pathological events in the onset of Alzheimer’s disease (AD). Insulin-degrading-enzyme (IDE) is one of the main extracellular protease targeting Ab, and thus it represents an interesting pharmacological target for AD therapy. We show that the active form of somatostatin-14 regulates IDE activity by affecting its expression and secretion in microglia cells. A similar effect can also be observed when adding octreotide. Following a previous observation where somatostatin directly interacts with IDE, here we demonstrate that somatostatin regulates Ab catabolism by modulating IDE proteolytic activity in IDE gene-silencing experiments. As a whole, these data indicate the relevant role played by somatostatin and, potentially, by analogue octreotide, in preventing Ab accumulation by partially restoring IDE activity