Marco Pedrazzi

Chronic inflammation is a major driver of lung damage in cystic fibrosis (CF), yet the molecular mechanisms that sustain this self-reinforcing inflammatory state remain incompletely understood. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD biosynthesis, can also act extracellularly (eNAMPT) as a pro-inflammatory mediator. Here, we investigated the relationship between NAMPT and matrix metalloproteinase-9 (MMP-9) in CF. Peripheral blood mononuclear cells (PBMCs) from people with CF (pwCF) displayed increased intracellular NAMPT and MMP-9 expression, together with enhanced AKT and ERK1/2 phosphorylation, compared with non-CF controls. Circulating eNAMPT levels were elevated in pwCF and positively correlated with MMP-9 levels. Although baseline eNAMPT levels were not significantly associated with FEV1% predicted, in a small longitudinal cohort, eNAMPT decreased following one year of elexacaftor/tezacaftor/ivacaftor (ETI) treatment, while FEV1% predicted increased in six of seven patients with paired measurements. In vitro, recombinant NAMPT increased MMP-9 expression and enzymatic activity in PBMCs from healthy donors. Pharmacological treatment with FK866 reduced endogenous eNAMPT release and attenuated NAMPT-induced MMP-9 production. Mechanistically, eNAMPT triggered rapid AKT activation followed by sustained ERK1/2 phosphorylation. Pharmacological inhibition of the MEK/ERK pathway attenuated NAMPT-induced MMP-9 activity, supporting a functional contribution of ERK1/2 signaling to NAMPT-mediated MMP-9 activation. Together, these findings identify a functional NAMPT–MMP-9 axis associated with inflammatory signaling in CF. Further studies are needed to establish its contribution to pulmonary inflammation and to determine the clinical significance of circulating eNAMPT in larger patient cohorts.

Oxytocin’s capacity to affect the glial cell functions is increasingly recognized. We previously reported that oxytocin could cause both excitation and inhibition of Ca²⁺ signals and glutamate release in the processes of adult rodent astrocytes. Our purpose here was to investigate oxytocin receptor expression and oxytocin effects in astrocytes. In primary cortical astrocytes, we assessed the presence of oxytocin receptors by confocal imaging, and the effects of oxytocin receptor activation on intracellular Ca²⁺ signals and glutamate release. We found that oxytocin receptors are expressed in both the soma and processes of astrocytes; oxytocin at nanomolar concentrations could induce dual responses in astrocytes, namely facilitation and inhibition of Ca²⁺ signals and glutamate release; the oxytocin facilitatory and inhibitory effects were duplicated by the biased agonists carbetocin and atosiban, respectively; and the facilitatory and the inhibitory effect were dependent on activation of a G_q and a G_i pathway, respectively. It is concluded that oxytocin effects in astrocytes could duplicate the effects in processes prepared from astrocytes matured in neuron-astrocyte networks, substantiating the use of astrocytes to study astrocytic oxytocin molecular signaling.

The ability of the neuropeptide oxytocin to affect glial cell function is receiving increasing attention. We previously reported that oxytocin at a low nanomolar concentration could inhibit both astrocytic Ca²⁺ signals and glutamate release. Here, we investigate the ability of oxytocin receptors to couple both inhibitory and stimulatory pathways in astrocytes, as already reported in neurons. We assessed the effects of oxytocin at concentrations ranging from low to high in the nanomolar range on intracellular Ca²⁺ signals and on the glutamate release in astrocyte processes freshly prepared from the striatum of adult rats. Our main findings are as follows: oxytocin could induce dual responses in astrocyte processes, namely the inhibition and facilitation of both Ca²⁺ signals and glutamate release; the inhibitory and the facilitatory response appeared dependent on activation of the G_i and the G_q pathway, respectively; both inhibitory and facilitatory responses were evoked at the same nanomolar oxytocin concentrations; and the biased agonists atosiban and carbetocin could duplicate oxytocin’s inhibitory and facilitatory response, respectively. In conclusion, due to the coupling of striatal astrocytic oxytocin receptors to different transduction pathways and the dual effects on Ca²⁺ signals and glutamate release, oxytocin could also play a crucial role in neuron–astrocyte bi-directional communication through a subtle regulation of striatal glutamatergic synapses. Therefore, astrocytic oxytocin receptors may offer pharmacological targets to regulate glutamatergic striatal transmission, which is potentially useful in neuropsychiatric disorders and in neurodegenerative diseases.

The receptor–receptor interaction (RRI) of G protein-coupled receptors (GPCRs) leads to new functional entities that are conceptually distinct from the simple addition of signals mediated by the activation of the receptors that form the heteromers. Focusing on astrocytes, there is evidence for the existence of inhibitory and facilitatory RRIs, including the heteromers formed by the adenosine A2A and the dopamine D2 receptors, by A2A and the oxytocin receptor (OTR), and the D2-OTR heteromers. The possible involvement of these receptors in mosaicism has never been investigated in striatal astrocytes. By biophysical and functional approaches, we focused our attention on the existence of an A2A-D2-OTR high-order receptor complex and its role in modulating cytosolic calcium levels and endogenous glutamate release, when striatal astrocyte processes were stimulated with 4-aminopyridine. Functional data indicate a permissive role of OTR on dopamine signaling in the regulation of the glutamatergic transmission, and an inhibitory control mediated by A2A on both the D2-mediated signaling and on the OTR-facilitating effect on D2. Imaging biochemical and bioinformatic evidence confirmed the existence of the A2A-D2-OTR complex and its ternary structure in the membrane. In conclusion, the D2 receptor appears to be a hotspot in the control of the glutamate release from the astrocytic processes and may contribute to the regulation and integration of different neurotransmitter-mediated signaling in the striatum by the A2A-D2-OTR heterotrimers. Considering the possible selectivity of allosteric interventions on GPCRs organized as receptor mosaics, A2A-D2-OTR heterotrimers may offer selective pharmacological targets in neuropsychiatric disorders and neurodegenerative diseases.

High-pressure and temperature extraction (HPTE) can effectively recover bioactive compounds from olive pomace (OP). HPTE extract obtained by extracting OP with ethanol and water (50:50 v/v) at 180 °C for 90 min demonstrated a pronounced ability to preserve intracellular calcium homeostasis, shielding neurons from the harmful effects induced by N-methyl-d-aspartate (NMDA) receptor (NMDAR) overactivation, such as aberrant calpain activation. In this study, the extraction temperature was changed from 37 to 180 °C, and the extracts were evaluated for their antioxidant potency and ability to preserve crucial intracellular Ca²⁺-homeostasis necessary for neuronal survival. Additionally, to verify the temperature-induced activity of the extract, further extractions on the exhausted olive pomace were conducted, aiming to identify variations in the quality and quantity of extracted phenolic molecules through HPLC analysis. The results revealed a significant increase in bioactive compounds as a function of temperature variation, reaching 6.31 ± 0.09 mgCAE/mL extract for the extraction performed at 180 °C. Subsequent extraction of the exhausted residues yielded extracts that remained active in preventing calcium-induced cell death. Moreover, despite increased antiradical power, extracts re-treated at 180 °C did not display cell protection activity. Our results indicate that the molecules able to maintain physiological Ca²⁺-homeostasis in murine cortical neurons in conditions of cytotoxic stimulation of NMDAR are wholly recovered from olive pomace only following extraction performed at 180 °C.

Background: The most recent modulator combination, elexacaftor/tezacaftor/ivacaftor (Trikafta^®), has been shown to improve clinical outcomes in most patients with cystic fibrosis (PwCF). Unfortunately, the clinical benefits are sometimes variable; thus, improving our knowledge of the possible causes of this variability can help reduce it. Methods: Circulating mononuclear cells (CMCs) and plasma were collected from 16 PwCF (including those on Trikafta^® therapy) and 4 non-CF subjects. Cystic fibrosis transmembrane conductance regulator (CFTR) activity and matrix metalloprotease 9 (MMP9) expression were monitored before and after therapy, together with some clinical parameters. The relationship between MMP9 expression and the modulation of the extracellular-regulated 1/2 (ERK1/2) and nuclear factor-kB (NF-kB) pathways was also analyzed. Results: MMP9, markedly expressed in the CMCs and plasma of all the patients included in the study, was downregulated in the clinically responsive PwCF. In the non-responder, the MMP9 levels remained high. The modulation of MMP9 following treatment with Trikafta^® may be controlled by the NF-kB pathway. Conclusions: These data strongly suggest that MMP9 downregulation is a potential biomarker of therapy efficacy and that it could be useful in understanding the molecular events underlying the variable clinical responses of patients to Trikafta^®. This knowledge could be helpful for future studies of personalized medicine and thereby ensure improvements in individual responses to therapies.

The ability of oxytocin (OT) to interact with the dopaminergic system through facilitatory D2-OT receptor (OTR) receptor-receptor interaction in the limbic system is increasingly considered to play roles in social or emotional behavior, and suggested to serve as a potential therapeutic target. Although roles of astrocytes in the modulatory effects of OT and dopamine in the central nervous system are well recognized, the possibility of D2-OTR receptor-receptor interaction in astrocytes has been neglected. In purified astrocyte processes from adult rat striatum, we assessed OTR and dopamine D2 receptor expression by confocal analysis. The effects of activation of these receptors were evaluated in the processes through a neurochemical study of glutamate release evoked by 4-aminopyridine; D2-OTR heteromerization was assessed by co-immunoprecipitation and proximity ligation assay (PLA). The structure of the possible D2-OTR heterodimer was estimated by a bioinformatic approach. We found that both D2 and OTR were expressed on the same astrocyte processes and controlled the release of glutamate, showing a facilitatory receptor-receptor interaction in the D2-OTR heteromers. Biochemical and biophysical evidence confirmed D2-OTR heterodimers on striatal astrocytes. The residues in the transmembrane domains four and five of both receptors are predicted to be mainly involved in the heteromerization. In conclusion, roles for astrocytic D2-OTR in the control of glutamatergic synapse functioning through modulation of astrocytic glutamate release should be taken into consideration when considering interactions between oxytocinergic and dopaminergic systems in striatum.

In mammalian cells, the content of polyamines is tightly regulated. Polyamines, including spermine, spermidine and putrescine, are involved in many cellular processes. Spermine oxidase specifically oxidizes spermine, and its deregulated activity has been reported to be linked to brain pathologies involving neuron damage. Spermine is a neuromodulator of a number of ionotropic glutamate receptors and types of ion channels. In this respect, the Dach-SMOX mouse model overexpressing spermine oxidase in the neocortex neurons was revealed to be a model of chronic oxidative stress, excitotoxicity and neuronal damage. Reactive astrocytosis, chronic oxidative and excitotoxic stress, neuron loss and the susceptibility to seizure in the Dach-SMOX are discussed here. This genetic model would help researchers understand the linkage between polyamine dysregulation and neurodegeneration and unveil the roles of polyamines in the crosstalk between astrocytes and neurons in neuroprotection or neurodegeneration.

Background: Roles of astrocytes in the modulatory effects of oxytocin (OT) in central nervous system are increasingly considered. Nevertheless, OT effects on gliotransmitter release have been neglected. Methods: In purified astrocyte processes from adult rat striatum, we assessed OT receptor (OTR) and adenosine A2A receptor expression by confocal analysis. The effects of receptors activation on glutamate release from the processes were evaluated; A2A-OTR heteromerization was assessed by co-immunoprecipitation and PLA. Structure of the possible heterodimer of A2A and OT receptors was estimated by a bioinformatic approach. Results: Both A2A and OT receptors were expressed on the same astrocyte processes. Evidence for A2A-OTR receptor-receptor interaction was obtained by measuring the release of glutamate: OT inhibited the evoked glutamate release, while activation of A2A receptors, per se ineffective, abolished the OT effect. Biochemical and biophysical evidence for A2A-OTR heterodimers on striatal astrocytes was also obtained. The residues in the transmembrane domains 4 and 5 of both receptors are predicted to be mainly involved in the heteromerization. Conclusions: When considering effects of OT in striatum, modulation of glutamate release from the astrocyte processes and of glutamatergic synapse functioning, and the interaction with A2A receptors on the astrocyte processes should be taken into consideration.

Background: In the brain, polyamines are mainly synthesized in neurons, but preferentially accumulated in astrocytes, and are proposed to be involved in neurodegenerative/neuroinflammatory disorders and neuron injury. A transgenic mouse overexpressing spermine oxidase (SMOX, which specifically oxidizes spermine) in the neocortex neurons (Dach-SMOX mouse) was proved to be a model of increased susceptibility to excitotoxic injury. Methods: To investigate possible alterations in synapse functioning in Dach-SMOX mouse, both cerebrocortical nerve terminals (synaptosomes) and astrocytic processes (gliosomes) were analysed by assessing polyamine levels, ezrin and vimentin content, glutamate AMPA receptor activation, calcium influx, and catalase activity. Results: The main findings are as follows: (i) the presence of functional calcium-permeable AMPA receptors in synaptosomes from both control and Dach-SMOX mice, and in gliosomes from Dach-SMOX mice only; (ii) reduced content of spermine in gliosomes from Dach-SMOX mice; and (iii) down-regulation and up-regulation of catalase activity in synaptosomes and gliosomes, respectively, from Dach-SMOX mice. Conclusions: Chronic activation of SMOX in neurons leads to major changes in the astrocyte processes including reduced spermine levels, increased calcium influx through calcium-permeable AMPA receptors, and stimulation of catalase activity. Astrocytosis and the astrocyte process alterations, depending on chronic activation of polyamine catabolism, result in synapse dysregulation and neuronal suffering.