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Monica FILAFERRO

Personale tecnico amministrativo
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

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Pubblicazioni

2023 - Human microglia synthesize neurosteroids to cope with rotenone-induced oxidative stress [Articolo su rivista]
Lucchi, Chiara; Codeluppi, Alessandro; Filaferro, Monica; Vitale, Giovanni; Rustichelli, Cecilia; Avallone, Rossella; Mandrioli, Jessica; Biagini, Giuseppe
abstract

We obtained evidence that mouse BV2 microglia synthesize neurosteroids dynamically to modify neurosteroid levels in response to oxidative damage caused by rotenone. Here, we evaluated whether neurosteroids could be produced and altered in response to rotenone by the human microglial clone 3 (HMC3) cell line. To this aim, HMC3 cultures were exposed to rotenone (100 nM) and neurosteroids were measured in the culture medium by liquid chromatography with tandem mass spectrometry. Microglia reactivity was evaluated by measuring interleukin 6 (IL-6) levels, whereas cell viability was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. After 24 hours (h), rotenone increased IL-6 and reactive oxygen species levels by approximately +37% over the baseline, without affecting cell viability; however, microglia viability was significantly reduced at 48 h (p < 0.01). These changes were accompanied by the downregulation of several neurosteroids, including pregnenolone, pregnenolone sulfate, 5α-dihydroprogesterone, and pregnanolone, except for allopregnanolone, which instead was remarkably increased (p < 0.05). Interestingly, treatment with exogenous allopregnanolone (1 nM) efficiently prevented the reduction in HMC3 cell viability. In conclusion, this is the first evidence that human microglia can produce allopregnanolone and that this neurosteroid is increasingly released in response to oxidative stress, to tentatively support the microglia's survival.

2022 - Acute cytotoxicity of mineral fibres observed by time-lapse video microscopy [Articolo su rivista]
Di Giuseppe, D.; Scarfi, S.; Alessandrini, A.; Bassi, A. M.; Mirata, S.; Almonti, V.; Ragazzini, G.; Mescola, A.; Filaferro, M.; Avallone, R.; Vitale, G.; Scognamiglio, V.; Gualtieri, A. F.
abstract

Inhalation of mineral fibres is associated with the onset of an inflammatory activity in the lungs and the pleura responsible for the development of fatal malignancies. It is known that cell damage is a necessary step for triggering the inflammatory response. However, the mechanisms by which mineral fibres exert cytotoxic activity are not fully understood. In this work, the kinetics of the early cytotoxicity mechanisms of three mineral fibres (i.e., chrysotile, crocidolite and fibrous erionite) classified as carcinogenic by the International Agency for Research on Cancer, was determined for the first time in a comparative manner using time-lapse video microscopy coupled with in vitro assays. All tests were performed using the THP-1 cell line, differentiated into M0 macrophages (M0-THP-1) and exposed for short times (8 h) to 25 μg/mL aliquots of chrysotile, crocidolite and fibrous erionite. The toxic action of fibrous erionite on M0-THP-1 cells is manifested since the early steps (2 h) of the experiment while the cytotoxicity of crocidolite and chrysotile gradually increases during the time span of the experiment. Chrysotile and crocidolite prompt cell death mainly via apoptosis, while erionite exposure is also probably associated to a necrotic-like effect. The potential mechanisms underlying these different toxicity behaviours are discussed in the light of the different morphological, and chemical-physical properties of the three fibres.

2022 - Disclosing the Antioxidant and Neuroprotective Activity of an Anthocyanin-Rich Extract from Sweet Cherry (Prunus avium L.) Using In Vitro and In Vivo Models [Articolo su rivista]
Filaferro, M.; Codeluppi, A.; Brighenti, V.; Cimurri, F.; Gonzalez-Paramas, A. M.; Santos-Buelga, C.; Bertelli, D.; Pellati, F.; Vitale, G.
abstract

In this study, an autochthonous variety of sweet cherry (Prunus avium L.), namely “Moretta di Vignola”, was processed to prepare extracts rich in polyphenols, which were characterized by high-performance liquid chromatography (HPLC) separation coupled to UV/DAD and ESI-MSn analysis. Then, a sweet cherry anthocyanin-rich extract (ACE) was prepared, fully characterized and tested for its activity against Parkinson’s disease (PD) in cellular (BV2 microglia and SH-SY5Y neuroblastoma) and in Drosophila melanogaster rotenone (ROT)-induced model. The extract was also evaluated for its antioxidant activity on Caenorhabditis elegans by assessing nematode resistance to thermal stress. In both cell lines, ACE reduced ROT-induced cell death and it decreased, alone, cellular reactive oxygen species (ROS) content while reinstating control-like ROS values after ROT-induced ROS rise, albeit at different concentrations of both compounds. Moreover, ACE mitigated SH-SY5Y cell cytotoxicity in a non-contact co-culture assay with cell-free supernatants from ROT-treated BV-2 cells. ACE, at 50 µg/mL, ameliorated ROT (250 µM)-provoked spontaneous (24 h duration) and induced (after 3 and 7 days) locomotor activity impairment in D. melanogaster and it also increased survival and counteracted the decrease in fly lifespan registered after exposure to the ROT. Moreover, heads from flies treated with ACE showed a non-significant decrease in ROS levels, while those exposed to ROT markedly increased ROS levels if compared to controls. ACE + ROT significantly placed the ROS content to intermediate values between those of controls and ROT alone. Finally, ACE at 25 µg/mL produced a significant increase in the survival rate of nematodes submitted to thermal stress (35 °C, 6–8 h), at the 2nd and 9th day of adulthood. All in all, ACE from Moretta cherries can be an attractive candidate to formulate a nutraceutical product to be used for the prevention of oxidative stress-induced disorders and related neurodegenerative diseases.

2021 - In vitro toxicity of fibrous glaucophane [Articolo su rivista]
Gualtieri, A. F.; Zoboli, A.; Filaferro, M.; Benassi, M.; Scarfi, S.; Mirata, S.; Avallone, R.; Vitale, G.; Bailey, M.; Harper, M.; Di Giuseppe, D.
abstract

The health hazard represented by the exposure to asbestos may also concern other minerals with asbestos-like crystal habit. One of these potentially hazardous minerals is fibrous glaucophane. Fibrous glaucophane is a major component of blueschist rocks of California (USA) currently mined for construction purposes. Dust generated by the excavation activities might potentially expose workers and the general public. The aim of this study was to determine whether fibrous glaucophane induces in vitro toxicity effects on lung cells by assessing the biological responses of cultured human pleural mesothelial cells (Met-5A) and THP-1 derived macrophages exposed for 24 h and 48 h to glaucophane fibres. Crocidolite asbestos was tested for comparison. The experimental configuration of the in vitro tests included a cell culture without fibres (i.e., control), cell cultures treated with 50 μg/mL (i.e., 15.6 μg/cm2) of crocidolite fibres and 25-50−100 μg/mL (i.e., 7.8−15.6–31.2 μg/cm2) of glaucophane fibres. Results showed that fibrous glaucophane may induce a decrease in cell viability and an increase in extra-cellular lactate dehydrogenase release in the tested cell cultures in a concentration dependent mode. Moreover, it was found that fibrous glaucophane has a potency to cause oxidative stress. The biological reactivity of fibrous glaucophane confirms that it is a toxic agent and, although it apparently induces lower toxic effects compared to crocidolite, exposure to this fibre may be responsible for the development of lung diseases in exposed unprotected workers and population.

2020 - BV-2 Microglial Cells Respond to Rotenone Toxic Insult by Modifying Pregnenolone, 5alpha-Dihydroprogesterone and Pregnanolone Level [Articolo su rivista]
Avallone, Rossella; Lucchi, Chiara; Puja, Giulia; Codeluppi, Alessandro; Filaferro, Monica; Vitale, Giovanni; Rustichelli, Cecilia; Biagini, Giuseppe
abstract

Neuroinflammation, whose distinctive sign is the activation of microglia, is supposed to play a key role in the development and progression of neurodegenerative diseases. The aim of this investigation was to determine levels of neurosteroids produced by resting and injured BV-2 microglial cells. BV-2 cells were exposed to increasing concentrations of rotenone to progressively reduce their viability by increasing reactive oxygen species (ROS) production. BV-2 cell viability was significantly reduced 24, 48 and 72 h after rotenone (50–1000 nM) exposure. Concomitantly, rotenone (50–100 nM) determined a dose-independent augmentation of ROS production. Then, BV-2 cells were exposed to a single, threshold dose of rotenone (75 nM) to evaluate the overtime release of neurosteroids. In particular, pregnenolone, pregnenolone sulfate, progesterone, 5alpha-dihydroprogesterone (5-DHP), allopregnanolone, and pregnanolone, were quantified in the culture medium by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. BV-2 cells synthesized all the investigated neurosteroids and, after exposure to rotenone, 5DHP and pregnanolone production was remarkably increased. In conclusion, we found that BV-2 cells not only synthesize several neurosteroids, but further increase this production following oxidative damage. Pregnanolone and 5alpha-DHP may play a role in modifying the progression of neuroinflammation in neurodegenerative diseases.

2020 - CCAP regulates feeding behavior via the NPF pathway in Drosophila adults [Articolo su rivista]
Williams, M. J.; Akram, M.; Barkauskaite, D.; Patil, S.; Kotsidou, E.; Kheder, S.; Vitale, G.; Filaferro, M.; Blemings, S. W.; Maestri, G.; Hazim, N.; Vergoni, A. V.; Schioth, H. B.
abstract

The intake of macronutrients is crucial for the fitness of any animal and is mainly regulated by peripheral signals to the brain. How the brain receives and translates these peripheral signals or how these interactions lead to changes in feeding behavior is not well-understood. We discovered that 2 crustacean cardioactive peptide (CCAP)-expressing neurons in Drosophila adults regulate feeding behavior and metabolism. Notably, loss of CCAP, or knocking down the CCAP receptor (CCAP-R) in 2 dorsal median neurons, inhibits the release of neuropeptide F (NPF), which regulates feeding behavior. Furthermore, under starvation conditions, flies normally have an increased sensitivity to sugar; however, loss of CCAP, or CCAP-R in 2 dorsal median NPF neurons, inhibited sugar sensitivity in satiated and starved flies. Separate from its regulation of NPF signaling, the CCAP peptide also regulates triglyceride levels. Additionally, genetic and optogenetic studies demonstrate that CCAP signaling is necessary and sufficient to stimulate a reflexive feeding behavior, the proboscis extension reflex (PER), elicited when external food cues are interpreted as palatable. Dopaminergic signaling was also sufficient to induce a PER. On the other hand, although necessary, NPF neurons were not able to induce a PER. These data illustrate that the CCAP peptide is a central regulator of feeding behavior and metabolism in adult flies, and that NPF neurons have an important regulatory role within this system.

2020 - Partial versus radical nephrectomy in very elderly patients: a propensity score analysis of surgical, functional and oncologic outcomes (RESURGE project) [Articolo su rivista]
Mir, M. C.; Pavan, N.; Capitanio, U.; Antonelli, A.; Derweesh, I.; Rodriguez-Faba, O.; Linares, E.; Takagi, T.; Rha, K. H.; Fiori, C.; Maurer, T.; Zang, C.; Mottrie, A.; Umari, P.; Long, J. -A.; Fiard, G.; De Nunzio, C.; Tubaro, A.; Tracey, A. T.; Ferro, M.; De Cobelli, O.; Micali, S.; Bevilacqua, L.; Torres, J.; Schips, L.; Castellucci, R.; Dobbs, R.; Quarto, G.; Bove, P.; Celia, A.; De Concilio, B.; Trombetta, C.; Silvestri, T.; Larcher, A.; Montorsi, F.; Palumbo, C.; Furlan, M.; Bindayi, A.; Hamilton, Z.; Breda, A.; Palou, J.; Aguilera, A.; Tanabe, K.; Raheem, A.; Amiel, T.; Yang, B.; Lima, E.; Crivellaro, S.; Perdona, S.; Gregorio, C.; Barbati, G.; Porpiglia, F.; Autorino, R.
abstract

Purpose: To compare the outcomes of PN to those of RN in very elderly patients treated for clinically localized renal tumor. Patients and methods: A purpose-built multi-institutional international database (RESURGE project) was used for this retrospective analysis. Patients over 75 years old and surgically treated for a suspicious of localized renal with either PN or RN were included in this database. Surgical, renal function and oncological outcomes were analyzed. Propensity scores for the predicted probability to receive PN in each patient were estimated by logistic regression models. Cox proportional hazard models were estimated to determine the relative change in hazard associated with PN vs RN on overall mortality (OM), cancer-specific mortality (CSM) and other-cause mortality (OCM). Results: A total of 613 patients who underwent RN were successfully matched with 613 controls who underwent PN. Higher overall complication rate was recorded in the PN group (33% vs 25%; p = 0.01). Median follow-up for the entire cohort was 35 months (interquartile range [IQR] 13–63 months). There was a significant difference between RN and PN in median decline of eGFR (39% vs 17%; p < 0.01). PN was not correlated with OM (HR = 0.71; p = 0.56), OCM (HR = 0.74; p = 0.5), and showed a protective trend for CSM (HR = 0.19; p = 0.05). PN was found to be a protective factor for surgical CKD (HR = 0.28; p < 0.01) and worsening of eGFR in patients with baseline CKD. Retrospective design represents a limitation of this analysis. Conclusions: Adoption of PN in very elderly patients with localized renal tumor does not compromise oncological outcomes, and it allows better functional preservation at mid-term (3-year) follow-up, relative to RN. Whether this functional benefit translates into a survival benefit remains to be determined.

2019 - Outcomes of Partial and Radical Nephrectomy in Octogenarians – A Multicenter International Study (Resurge) [Articolo su rivista]
Antonelli, A.; Veccia, A.; Pavan, N.; Mir, C.; Breda, A.; Takagi, T.; Rha, K. H.; Maurer, T.; Zhang, C.; Long, J. -A.; De Nunzio, C.; Lima, E.; Ferro, M.; Micali, S.; Quarto, G.; Linares, E.; Celia, A.; Schips, L.; Bove, P.; Larcher, A.; Fiori, C.; Mottrie, A.; Bindayi, A.; Trombetta, C.; Silvestri, T.; Palou, J.; Faba, O. R.; Tanabe, K.; Yang, B.; Fiard, G.; Tubaro, A.; Torres, J. N.; De Cobelli, O.; Bevilacqua, L.; Castellucci, R.; Tracey, A.; Hampton, L. J.; Montorsi, F.; Perdona, S.; Simeone, C.; Palumbo, C.; Capitanio, U.; Derweesh, I.; Porpiglia, F.; Autorino, R.
abstract

OBJECTIVE: To analyze the outcomes of partial nephrectomy (PN) and radical nephrectomy (RN) in octogenarian patients. METHODS: The RESURGE (REnal SUrgery in the Eldely) multi-institutional database was queried to identify patients ≥80 years old who had undergone a PN or RN for a renal tumor. Multivariable binary logistic regression estimated the association between type of surgery and occurrence of complications. Multivariable Cox regression model assessed the association between type of surgery and All-Causes Mortality. RESULTS: The study analyzed 585 patients (median age 83 years, IQR 81-84), 364 of whom (62.2%) underwent RN and 221 (37.8%) PN. Patients undergoing RN were older (P = .0084), had larger tumor size (P < .0001) and higher clinical stage (P < .001). At multivariable analysis for complications, the only significant difference was found for lower risk of major postoperative complications for laparoscopic RN compared to open RN (OR: 0.42; P = .04). The rate of significant (>25%) decrease of eGFR in PN and RN was 18% versus 59% at 1 month, and 23% versus 65% at 6 months (P < .0001). After a median follow-up time of 39 months, 161 patients (31%) died, of whom 105 (20%) due to renal cancer. CONCLUSION: In this patient population both RN and PN carry a non-negligible risk of complications. When surgical removal is indicated, PN should be preferred, whenever technically feasible, as it can offer better preservation of renal function, without increasing the risk of complications. Moreover, a minimally invasive approach should be pursued, as it can translate into lower surgical morbidity.

2019 - Structure Model and Toxicity of the Product of Biodissolution of Chrysotile Asbestos in the Lungs [Articolo su rivista]
Gualtieri, A. F.; Lusvardi, G.; Pedone, A.; Di Giuseppe, D.; Zoboli, A.; Mucci, A.; Zambon, A.; Filaferro, M.; Vitale, G.; Benassi, M.; Avallone, R.; Pasquali, L.; Lassinantti Gualtieri, M.
abstract

Asbestos is a commercial term indicating six natural silicates with asbestiform crystal habit. Of these, five are double-chain silicates (amphibole) and one is a layer silicate (serpentine asbestos or chrysotile). Although all species are classified as human carcinogens, their degree of toxicity is still a matter of debate. Amphibole asbestos species are biopersistent in the human lungs and exert their chronic toxic action for decades, whereas chrysotile is not biopersistent and transforms into an amorphous silica structure prone to chemical/physical clearance when exposed to the acidic environment created by the alveolar macrophages. There is evidence in the literature of the toxicity of chrysotile, but its limited biopersistence is thought to explain the difference in toxicity with respect to amphibole asbestos. To date, no comprehensive model describing the toxic action of chrysotile in the lungs is available, as the structure and toxic action of the product formed by the biodissolution of chrysotile are unknown. This work is aimed at fulfilling this gap and explaining the toxic action in terms of structural, chemical, and physical properties. We show that chrysotile's fibrous structure induces cellular damage, mainly through physical interactions. Based on our previous work and novel findings, we propose the following toxicity model: inhaled chrysotile fibers exert their toxicity in the alveolar space by physical and biochemical action. The fibers are soon leached by the intracellular acid environment into a product with residual toxicity, and the dissolution process liberates toxic metals in the intracellular and extracellular environment.

2018 - In vitro and in vivo characterization of the bifunctional μ and δ opioid receptor ligand UFP-505 [Articolo su rivista]
Dietis, N.; Niwa, H.; Tose, R.; Mcdonald, J.; Ruggieri, V.; Filaferro, M.; Vitale, G.; Micheli, L.; Ghelardini, C.; Salvadori, S.; Calo', Giovanni Fabrizio; Guerrini, Remo; Rowbotham, D. J.; Lambert, D. G.
abstract

Background and Purpose: Targeting more than one opioid receptor type simultaneously may have analgesic advantages in reducing side-effects. We have evaluated the mixed μ opioid receptor agonist/ δ opioid receptor antagonist UFP-505 in vitro and in vivo. Experimental Approach: We measured receptor density and function in single μ, δ and μ /δ receptor double expression systems. GTPγ35S binding, cAMP formation and arrestin recruitment were measured. Antinociceptive activity was measured in vivo using tail withdrawal and paw pressure tests following acute and chronic treatment. In some experiments, we collected tissues to measure receptor densities. Key Results: UFP-505 bound to μ receptors with full agonist activity and to δ receptors as a low efficacy partial agonist At μ, but not δ receptors, UFP-505 binding recruited arrestin. Unlike morphine, UFP-505 treatment internalized μ receptors and there was some evidence for internalization of δ receptors. Similar data were obtained in a μ /δ receptor double expression system. In rats, acute UFP-505 or morphine, injected intrathecally, was antinociceptive. In tissues harvested from these experiments, μ and δ receptor density was decreased after UFP-505 but not morphine treatment, in agreement with in vitro data. Both morphine and UFP-505 induced significant tolerance. Conclusions and Implications: In this study, UFP-505 behaved as a full agonist at μ receptors with variable activity at δ receptors. This bifunctional compound was antinociceptive in rats after intrathecal administration. In this model, dual targeting provided no advantages in terms of tolerance liability. Linked Articles: This article is part of a themed section on Emerging Areas of Opioid Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.14/issuetoc.

2017 - Effects of [Nphe1, Arg14, Lys15] N/OFQ-NH2 (UFP-101), a potent NOP receptor antagonist, on molecular, cellular and behavioural alterations associated with chronic mild stress. [Articolo su rivista]
Vitale, Giovanni; Filaferro, Monica; Micioni Di Bonaventura, Maria Vittoria; Ruggieri, Valentina; Cifani, Carlo; Guerrini, Remo; Simonato, Michele; Zucchini, Silvia
abstract

The present study investigated the effect of [Nphe1] Arg14, Lys15-N/OFQ-NH2 (UFP-101), a selective NOP receptor antagonist, in chronic mild stress (CMS) in male Wistar rats. NOP receptor antagonists were reported to elicit antidepressant-like effects in rodents. Our aim was to investigate UFP-101 effects on CMS-induced anhedonia and impairment of hippocampal neurogenesis. UFP-101 (10 nmol/rat intracerebroventricularly) did not influence sucrose intake in non-stressed animals, but reinstated basal sucrose consumption in stressed animals from the second week of treatment. UFP-101 also reversed stress effects in forced swimming test and in open field. Fluoxetine (10 mg/kg intraperitoneally) produced similar effects. Moreover, we investigated whether UFP-101 could affect CMS-induced impairment in hippocampal cell proliferation and neurogenesis, and in fibroblast growth factor (FGF-2) expression. Our data confirm that CMS reduced neural stem cell proliferation and neurogenesis in adult rat hippocampus. Chronic UFP-101 treatment did not affect the reduced proliferation (bromodeoxyuridine-positive cells) observed in stressed animals. However, UFP-101 increased the number of doublecortin-positive cells, restoring neurogenesis. Finally, UFP-101 significantly increased FGF-2 expression, reduced by CMS. These findings support the view that blockade of NOP receptors produces antidepressant-like effects in CMS associated with positive effects on neurogenesis and FGF-2 expression. Therefore, NOP receptors may represent a target for innovative antidepressant drugs.

2015 - Acute isoproterenol induces anxiety-like behavior in rats and increases plasma content of extracellular vesicles [Articolo su rivista]
Leo, Giuseppina; Guescini, Michele; Genedani, Susanna; Stocchi, Vilberto; Carone, Chiara; Filaferro, Monica; Sisti, Davide; Marcoli, Manuela; Maura, Guido; Cortelli, Pietro; Guidolin, Diego; Fuxe, Kjell; Agnati, Luigi Francesco
abstract

Several clinical observations have demonstrated a link between heart rate and anxiety or panic disorders. In these patients, β-adrenergic receptor function was altered. This prompted us to investigate whether the β-adrenergic receptor agonist isoproterenol, at a dose that stimulates peripheral β-adrenergic system but has no effects at the central nervous system, can induce anxiety-like behavior in rats. Moreover, some possible messengers involved in the peripheral to brain communication were investigated. Our results showed that isoproterenol (5 mg kg(-1) i.p.) increased heart rate, evoked anxiety-like behavior, did not result in motor impairments and increased extracellular vesicle content in the blood. Plasma corticosterone level was unmodified as well as vesicular Hsp70 content. Vesicular miR-208 was also unmodified indicating a source of increased extracellular vesicles different from cardiomyocytes. We can hypothesize that peripheral extracellular vesicles might contribute to the β-adrenergic receptor-evoked anxiety-like behavior, acting as peripheral signals in modulating the mental state.

2015 - Collagen VI regulates peripheral nerve regeneration by modulating macrophage recruitment and polarization [Articolo su rivista]
Chen, Peiwen; Cescon, Matilde; Zuccolotto, Gaia; Nobbio, Lucilla; Colombelli, Cristina; Filaferro, Monica; Vitale, Giovanni; Feltri, M. Laura; Bonaldo, Paolo
abstract

Macrophages contribute to peripheral nerve regeneration and produce collagen VI, an extracellular matrix protein involved in nerve function. Here, we show that collagen VI is critical for macrophage migration and polarization during peripheral nerve regeneration. Nerve injury induces a robust upregulation of collagen VI, whereas lack of collagen VI in Col6a1(-/-) mice delays peripheral nerve regeneration. In vitro studies demonstrated that collagen VI promotes macrophage migration and polarization via AKT and PKA pathways. Col6a1(-/-) macrophages exhibit impaired migration abilities and reduced antiinflammatory (M2) phenotype polarization, but are prone to skewing toward the proinflammatory (M1) phenotype. In vivo, macrophage recruitment and M2 polarization are impaired in Col6a1(-/-) mice after nerve injury. The delayed nerve regeneration of Col6a1(-/-) mice is induced by macrophage deficits and rejuvenated by transplantation of wild-type bone marrow cells. These results identify collagen VI as a novel regulator for peripheral nerve regeneration by modulating macrophage function.

2015 - In vitro effects of cocaine on tunneling nanotube formation and extracellular vesicle release in glioblastoma cell cultures [Articolo su rivista]
Carone, Chiara; Genedani, Susanna; Leo, Giuseppina; Filaferro, Monica; Fuxe, Kjell; Agnati, Luigi Francesco
abstract

The effects of cocaine (150 nM, 300 nM, and 150 μM) on human glioblastoma cell cultures were studied on tunneling nanotube formation (1-h cocaine treatment) and extracellular vesicle release (1-, 3-, and 8-h cocaine treatment). Cocaine significantly increased the number of tunneling nanotubes only at the lowest concentration used. The release of extracellular vesicles (mainly exosomes) into the medium was stimulated by cocaine at each concentration used with a maximum effect at the highest concentration tested (150 μM). Moreover, cocaine (150 nM) significantly increased the number of vesicles with 61-80 nm diameter while at concentrations of 300 nM and 150 μM, and the smaller vesicles (30-40 nm diameter) were significantly increased with a reduction of the larger vesicles (41-60 nm diameter). A time dependence in the release of extracellular vesicles was observed. In view of the proposed role of these novel intercellular communication modes in the glial-neuronal plasticity, it seems possible that they can participate in the processes leading to cocaine addiction. The molecular target/s involved in these cocaine effects could be specific molecular components of plasma membrane lipid rafts and/or cocaine-induced modifications in cytoplasmic lipid composition.

2014 - Functional antagonism between nociceptin/orphanin FQ and corticotropin-releasing factor in rat anxiety-related behaviors: involvement of the serotonergic system [Articolo su rivista]
Filaferro, Monica; Ruggieri, Valentina; Novi, C; Calò, G; Cifani, C; Micioni Di Bonaventura, M. V; Sandrini, Maurizio; Vitale, Giovanni
abstract

Nociceptin/orphanin FQ (N/OFQ) acts as an anxiolytic-like agent in the rat and behaves as a functional antagonist of corticotropin-releasing factor (CRF) due to its ability to oppose CRF biological actions. In response to stress, CRF triggers changes in neurotransmitter systems including serotonin (5-HT). The role of 5-HT1A receptor in anxiety has been supported by preclinical and clinical studies. The present study investigated the possible functional antagonism between N/OFQ (1nmol/rat) and CRF (0.2nmol/rat) in anxiety-related conditions in rats, using elevated plus maze and defensive burying tests, in order to confirm previous literature results. Moreover, possible changes in the serotonergic system were studied in areas rich of serotonergic neurons: frontal cortex and pons. In both tests N/OFQ showed anxiolytic-like effects while CRF displayed anxiogenic-like effects. N/OFQ before CRF treatment counteracted the anxiogenic-like effects evoked by CRF. In frontal cortex, N/OFQ significantly decreased 5-HT levels but did not modify the hydroxyindoleacetic acid (5-HIAA) ones; CRF modified neither 5-HT nor 5-HIAA content but counteracted changes induced by N/OFQ alone. In pons, N/OFQ induced no change in serotonergic activity while CRF significantly decreased 5-HT levels and increased 5-HIAA content. The two peptides' combination reinstated serotonergic parameters to controls. In frontal cortex, N/OFQ increased the 5HT1A receptor density but reduced its affinity, while CRF alone did not induce any change. In pons, CRF decreased 5HT1ABmax and KD whereas N/OFQ was ineffective. All biochemical modifications were reverted by N/OFQ plus CRF treatment. The present study confirms that N/OFQ counteracts CRF anxiogenic-like effects in the behavioral tests evaluated. These effects may involve central serotonergic mechanisms since N/OFQ plus CRF induces a reversion of serotonergic changes provoked by single peptide. Our data support the hypothesis that N/OFQ may behave as functional CRF antagonist, this action being of interest for the treatment of anxiety disorders.

2013 - Neuropeptide S stimulates human monocyte chemotaxis via NPS receptor activation. [Articolo su rivista]
Filaferro, Monica; Novi, Chiara; Ruggieri, Valentina; Genedani, Susanna; Alboni, Silvia; Malagoli, Davide; Caló, G; Guerrini, R; Vitale, Giovanni
abstract

Neuropeptide S (NPS) produces several biological actions by activating a formerly orphan GPCR, now named NPS receptor (NPSR). It has been previously demonstrated that NPS stimulates murine leukocyte chemotaxis in vitro. In the present study we investigated the ability of NPS, in comparison with the proinflammatory peptide formyl-Met-Leu-Phe (fMLP), to stimulate human monocyte chemotaxis. At a concentration of 10(-8)M fMLP significantly stimulated chemotaxis. NPS produced a concentration dependent chemotactic action over the concentration range 10(-12) to 10(-5)M. The NPSR antagonists [D-Cys((t)Bu)(5)]NPS, [(t)Bu-D-Gly(5)]NPS and SHA 68 were used to pharmacologically characterize NPS action. Monocyte chemoattractant effect of NPS, but not fMLP, was completely blocked by either peptide antagonists or SHA with the nonpeptide molecule being more potent. None of the NPSR antagonists modified per se random cell migration. Thus, the present study demonstrated that NPS is able to stimulate human monocyte chemotaxis and that this effect is entirely due to selective NPSR activation.

2012 - [tBu-D-Gly5]NPS, a pure and potent antagonist of the neuropeptide S receptor: in vitro and in vivo studies. [Articolo su rivista]
Ruzza, C; Rizzi, A; Camarda, V; Pulga, A; Marzola, G; Filaferro, Monica; Novi, Chiara; Ruggieri, Valentina; Marzola, E; Vitale, Giovanni; Salvadori, S; Guerrini, R; Calo', G.
abstract

Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Recently, the NPSR ligand [(t)Bu-D-Gly(5)]NPS was generated and in vitro characterized as a pure antagonist at the mouse NPSR. In the present study the pharmacological profile of [(t)Bu-D-Gly(5)]NPS has been investigated. [(t)Bu-D-Gly(5)]NPS activity was evaluated in vitro in the calcium mobilization assay at the rat NPSR and in vivo in the locomotor activity and righting reflex tests in mice and in the elevated plus maze and defensive burying assays in rats. In vitro, [(t)Bu-D-Gly(5)]NPS was inactive per se while it inhibited the calcium mobilization induced by 30 nM NPS (pK(B) 7.42). In Schild analysis experiments [(t)Bu-D-Gly(5)]NPS (0.1-10 μM) produced a concentration-dependent rightward shift of the concentration-response curve to NPS, showing a pA(2) value of 7.17. In mouse locomotor activity experiments, supraspinal injection of [(t)Bu-D-Gly(5)]NPS (1-10 nmol) dose dependently counteracted NPS (0.1 nmol) stimulant effects. In the mouse righting reflex assay [(t)Bu-D-Gly(5)]NPS (0.1-10 nmol) fully prevented the arousal-promoting action of the natural peptide (0.1 nmol). Finally, [(t)Bu-D-Gly(5)]NPS (3-30 nmol) was able to completely block NPS (1 nmol) anxiolytic-like actions in rat elevated plus maze and defensive burying assays. Collectively, the present results demonstrated that [(t)Bu-D-Gly(5)]NPS behaves both in vitro and in vivo as a pure and potent NPSR antagonist. This compound represents a novel and useful tool for investigating the pharmacology and neurobiology of the NPS/NPSR system.

2011 - In vitro characterization of the cytokine Drosophila Helical factor [Abstract in Rivista]
Malagoli, Davide; A., Accorsi; D., Conklin; Filaferro, Monica; Mandrioli, Mauro; Pinti, Marcello; S., Sacchi; Ottaviani, Enzo
abstract

Drosophila Helical factor (Hf) is a protein discovered through the QT method, an algorithm specifically designed for finding helical cytokines. Since in vivo experiments suggested the involvement of Hf in Drosophila melanogaster immunity, we have proceeded with the characterization of Hf functions in the macrophage-like Drosophila embryonic hemocytes, SL2 cell line. qPCR results demonstrated that Hf gene is induced in the SL2 cell line, after either 6 or 24 h incubation with Escherichia coli-purified peptidoglycan. The silencing of Hf expression through RNAi resulted in the reduced capability of synthesizing antimicrobial peptides (AMP) after exposure to heat-inactivated E. coli. The effects of the recombinant peptide rHf have also been tested in the SL2 cell line. rHf promotes the expression and triggers the release of Hf from the hemocytes, and stimulates the synthesis of the antimicrobial peptides (AMP) Defensin and Drosomycin, without any further immune stimulation. Consistent with the output of the QT method, which predicts Hf as a secreted protein, chromatin immune-precipitation experiments confirmed that Hf does not bind DNA, excluding that it acts as an immune-regulated transcription factor. Finally, rHf neither exerts chemotactic action nor triggers bacterial phagocytosis in SL2 cells.Altogether, our data supports the prediction that Hf is an helical cytokine produced and secreted by the hemocytes and it is mainly involved in the regulation of the humoral component of the immune response of D. melanogaster.

2010 - Beta-amyloid fibrillation and/or hyperhomocysteinemia modify striatal patterns of hyaluronic acid and dermatan sulfate: possible role in the pathogenesis of Alzheimer's disease. [Articolo su rivista]
Genedani, Susanna; Agnati, Luigi Francesco; Leo, Giuseppina; Buzzega, D.; Maccari, Francesca; Carone, Chiara; Andreoli, Nicola; Filaferro, Monica; Volpi, Nicola
abstract

A key event in Alzheimer's disease (AD) pathogenesis is the formation of insoluble peptides -amyloid aggregates and this process is favoured by a condition of hyperhomocysteinemia. To date, there is growing evidence that implicates glycosaminoglycans (GAGs) in the pathophysiology of amyloidosis but no data are available on the characterization of brain GAGs involved in the enhancing -amyloid fibrillogenesis in relationship to their structure and physico-chemical properties. Furthermore, few studies have been performed on the relationship between hyperhomocysteinemia and extracellular matrix (ECM) modifications. The aim of this study was to evaluate the amount and chemical structure of GAGs in rat striatal areas where -amyioid fibrillogenesis was induced, and in conditions of hyperhomocysteinemia. The intrastriatal injection of -amyloid produced a significant decrease (-40.8%) in the hyaluronic acid (HA) percentage and an increase (+14.5%) in the dermatan sulfate (DS) with a total charge density increasing of 14.9%. A significant decrease (-19.5%) in the HA percentage and an increase (+6.9%) in the DS % was also observed in striata obtained from the hyperhomocysteinemic animals. The total charge density increased by 6.8%. Quite the same trend was observed in rats after intrastriatal injection of -amyloid and in a condition of hyperhomocysteinemia. The observed increase of DS concentration and the correspondent decrease of the nonsulfated polymer HA after in vivo treatment with -amyloid and in a condition of hyperhocysteinemia support the hypothesis that an increase in local production of sulfated GAGs may reduce -amyloid neurotoxicity. However, the consequent modification of the ECM network might impair the extracellular diffusion pathways of different signal molecules and participate in the progression of AD.

2010 - Differential Sensitivity of A(2A) and Especially D (2) Receptor Trafficking to Cocaine Compared with Lipid Rafts in Cotransfected CHO Cell Lines. Novel Actions of Cocaine Independent of the DA Transporter. [Articolo su rivista]
Genedani, Susanna; Carone, Chiara; D., Guidolin; Filaferro, Monica; D., Marcellino; K., Fuxe; Agnati, Luigi Francesco
abstract

The effects of low and high concentrations of cocaine have been studied in vitro on the trafficking of plasma membrane A2A and D2 immunoreactivities in previously characterized A2A-D2 CHO cell lines. Receptor double immunofluorescence staining was performed with D2 and A2A antibodies, planar lipid rafts immunolabeling with biotinylated cholera toxin subunit B and membrane invaginations with an anti-caveolin-1 antibody. A computer-assisted image analysis demonstrated a substantial and highly significant rise of membrane-associated D2 immunoreactivity (IR) after 8 h of exposure to a low concentration of cocaine (150 nM). At this low concentration of cocaine, there was also an increase of membrane associated A2A immunoreactivity but smaller and less significant. However, this increase became considerably larger and highly significant at 150 µM at which concentration the rise of D2 immunoreactivity had begun to disappear. It may be suggested that an allosteric action of cocaine at 150 nM on the D2 receptors may primarily increase the insertion of D2 monomers, homomers and also of a subpopulation of A2A-D2 heteromers from the cytoplasm into the plasma membrane due to the conformational change induced by cocaine in the D2 receptor. The planar lipid rafts and the caveolae are only affected by the higher concentrations of cocaine. It is proposed that changes in D2 and A2A-D2 trafficking induced by allosteric actions of cocaine at D2 receptors may contribute to the alterations of D2 signaling found in cocaine abusers.

2010 - Further studies on the pharmacological profile of the neuropeptide S receptor antagonist SHA 68. [Articolo su rivista]
Ruzza, C; Rizzi, A; Trapella, C; Pela', M; Camarda, V; Ruggieri, Valentina; Filaferro, Monica; Cifani, C; Reinscheid, Rk; Vitale, Giovanni; Ciccocioppo, R; Salvadori, S; Guerrini, R; Calo', G.
abstract

Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Previous studies demonstrated that the non-peptide molecule SHA 68 acts as a selective NPSR antagonist. In the present study the pharmacological profile of SHA 68 has been further investigated in vitro and in vivo. In cells expressing the mouse NPSR SHA 68 was inactive per se up to 10 μM while it antagonized NPS-stimulated calcium mobilization in a competitive manner showing a pA2 value of 8.06. In the 10–50 mg/kg range of doses, SHA 68 counteracted the stimulant effects elicited by NPS, but not those of caffeine, in mouse locomotor activity experiments. In the mouse righting reflex assay SHA 68 fully prevented the arousal-promoting action of the peptide. The anxiolytic-like effects of NPS were slightly reduced by SHA 68 in the mouse open field, fully prevented in the rat elevated plus maze and partially antagonized in the rat defensive burying paradigm. Finally, SHA 68 was found poorly active in antagonizing the NPS inhibitory effect on palatable food intake in rats. In all assays SHA 68 did not produce any effect per se. In conclusion, the present study demonstrated that SHA 68 behaves as a selective NPSR antagonist that can be used to characterize the in vivo actions of NPS. However the usefulness of this research tool is limited by its poor pharmacokinetic properties.

2010 - The antinociceptive effect of acetylsalicylic acid is differently affected by a CB1 agonist or antagonist and involves the serotonergic system in rats [Articolo su rivista]
Ruggieri, Valentina; Vitale, Giovanni; Filaferro, Monica; Frigeri, Claudio; Pini, Luigi Alberto; Sandrini, Maurizio
abstract

Combinations of non steroidal anti-inflammatory drugs (NSAIDs) and cannabinoids are promising because of their potential synergistic effects in analgesia, resulting in a reduction in dosage and minimizing adverse reactions. The analgesic effect of Acetylsalicylic acid (ASA), probably due to a central mechanism, also implicates changes in the central monoaminergic system. Therefore, we decided to evaluate the antinociceptive interaction between the CB1 receptor agonist, HU210, and ASA in tests involving central pain in rats as well as the implication of the central serotonergic system thereon.

2009 - Chronic treatment with the selective NOP receptor antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101) reverses the behavioural and biochemical effects of unpredictable chronic mild stress in rats [Articolo su rivista]
Vitale, Giovanni; Ruggieri, Valentina; Filaferro, Monica; Frigeri, Claudio; Alboni, Silvia; Tascedda, Fabio; Brunello, Nicoletta; Guerrini, R; Cifani, C; Massi, M.
abstract

Introduction The present study was designed to assess the antidepressant effects of UFP-101, a selective nociceptin/orphanin FQ peptide (NOP) receptor antagonist, in a validated animal model of depression: the chronic mild stress (CMS). Materials and methods and Results UFP-101 (5, 10 and 20 nmol/rat; i.c.v., once a day for 21 days) dose- and time-dependently reinstated sucrose consumption in stressed animals without affecting the same parameter in non-stressed ones. In the forced swimming test, UFP-101 reduced immobility of stressed rats from day 8 of treatment. After a 3-week treatment, rats were killed for biochemical evaluations. UFP-101 abolished increase in serum corticosterone induced by CMS and reverted changes in central 5-HT/5-HIAA ratio. The behavioural and biochemical effects of UFP-101 mimicked those of imipramine, the reference antidepressant drug, administered at the dose of 15 mg/kg (i.p.). Co-administration of nociceptin/orphanin FQ (5 nmol/rat, from day 12 to 21) prevented the effects of UFP-101. Brain-derived neurotrophic factor mRNA and protein in hippocampus were not reduced by CMS nor did UFP-101 modify these parameters. Discussion and Conclusion This study demonstrated that chronic treatment with UFP-101 produces antidepressant-like effects in rats subjected to CMS supporting the proposal that NOP receptors represent a candidate target for the development of innovative antidepressant drugs.

2008 - ANXIOLYTIC LIKE EFFECT OF NEUROPEPTIDE S IN THE RAT DEFENSIVE BURYING. [Articolo su rivista]
Vitale, Giovanni; Filaferro, Monica; Ruggieri, Valentina; S., Pennella; Frigeri, Claudio; A., Rizzi; R., Guerrini; G., Calò
abstract

Neuropeptide S (NPS) has been recently identified as the endogenous ligand of a previously orphan G-protein-coupled receptor now named NPSR. Both NPS and its receptor are expressed in the brain, where they modulate different functions. In particular, it has been demonstrated that intracerebroventricular (i.c.v.) injection of NPS in rodents increases wakefulness and promotes anxiolytic-like effects. In the present study we used the defensive burying (DB) test in rats to further investigate the action of human NPS (0.1–10 nmol, i.c.v.) on anxiety-related behaviors. Diazepam (1.5 mg/kg, i.p.) and caffeine (20 mg/kg, i.p.) were used in parallel experiments as standard anxiolytic and anxiogenic drugs, respectively. None of the tested drugs produced statistical differences in the latency to contact the probe, burying behavior latency, number of shocks received or immobility/freezing duration. Caffeine increased cumulative burying behavior and the buried bedding height in a statistically significant manner thus promoting anxiogenic like effects. Opposite results were obtained with diazepam that significantly reduced these behavioral parameters. The anxiolytic-like action of diazepam was mimicked by NPS that reduced cumulative burying behavior in a dose dependent manner. Collectively, robust anxiolytic-like effects were recorded in response to NPS in the DB test. These results are of particular interest since the outcome of this assay is marginally influenced by drug effects on locomotor activity. In conclusion, we provide further evidence that NPS evokes genuine anxiolytic-like effects in the rat; therefore NPSR selective agonists are worthy of development as innovative drugs for the treatment of anxiety disorders.

2008 - Influence of f-MLP, ACTH(1-24) and CRH on in vitro chemotaxis of monocytes from centenarians [Articolo su rivista]
Genedani, Susanna; Filaferro, Monica; Carone, Chiara; Ostan, R; Bucci, L; Cevenini, E; Franceschi, C; Monti, D.
abstract

OBJECTIVE: The lifelong exposure to a variety of stressors activates a plethora of defense mechanisms, including the hypothalamic-pituitary-adrenal axis which releases neuropeptides affecting the immune responses. Here, we report data on the capability of monocytes from young subjects and centenarians to migrate towards chemotactic stimuli (formyl-methionyl-leucyl-phenylalanine, f-MLP; adrenocorticotropic hormone, ACTH, and corticotrophin-releasing hormone, CRH). Plasma levels of ACTH, CRH and cortisol were measured as an index of ongoing stress response. METHODS: Monocyte chemotaxis towards f-MLP (10(-8)M), ACTH(1-24) (10(-14) and 10(-8)M) and CRH (10(-14) and 10(-8)M) was evaluated in vitro in young subjects (n = 8, age range 25-35 years) and centenarians (n = 9, age >100 years) and expressed as chemotactic index. In 9 young subjects and 6 centenarians, plasma levels of cortisol, ACTH and CRH were measured. RESULTS: Monocyte chemotaxis towards f-MLP, ACTH(1-24) and CRH (10(-8)M) was well preserved in centenarians, except when the lowest concentration of CRH was used. CRH, ACTH and cortisol plasma levels were significantly higher in centenarians than in young subjects. CONCLUSIONS: The capability of monocytes from centenarians to respond to chemotactic neuropeptides is well preserved. The decreased responsiveness to the lowest concentration of CRH might be due to downregulation of CRH receptors or to defects in the intracellular signal transduction pathway. The high plasma levels of cortisol, CRH and ACTH in centenarians indicate an activation of the entire stress axis, likely counteracting the systemic inflammatory process occurring with age. This activation fits with the hypothesis that lifelong low-intensity stressors activate ancient, hormetic defense mechanisms, favoring healthy aging and longevity.

2007 - A beta peptides as one of the crucial volume transmission signals in the trophic units and their interactions with homocysteine. Physiological implications and relevance for Alzheimer's disease [Articolo su rivista]
Agnati, Luigi Francesco; Genedani, Susanna; Leo, Giuseppina; Forni, Arrigo; A. S., Woods; Filaferro, Monica; R., Franco; K., Fuxe
abstract

Amyloid peptides (Aβ) can operate as volume transmission (VT) signals since they are continuously released from cells of the central nervous system and diffuse in the extra-cellular space of the brain. They have both regulatory and trophic functions on cellular networks. In agreement with Aβ regulatory actions on glial-neuronal networks, the present paper reports new findings demonstrating that intrastriatal injections of Aβ peptides reduce striatal tyrosine hydroxylase, increase striatal GFAP immunoreactivities and lower pain threshold in experimental rats. Furthermore, it has been demonstrated that exogenous homocysteine (Hcy) binds Aβ(1-40) favouring its β-sheet conformation both in vitro and in vivo and hence the formation of β-fibrils and development of neurotoxicity.Thus, the hypothesis is discussed that Aβ peptides represent crucial VT-signals in the brain and their action is altered by dysmetabolic signals such as high Hcy extra-cellular levels, known to be an important risk factor for Alzheimer’s disease.

2007 - Hyper-homocysteinemia alters amyloid peptide-clusterin interactions and neuroglial network morphology and function in the caudate after intrastriatal injection of amyloid peptides [Articolo su rivista]
Leo, Giuseppina; Genedani, Susanna; Filaferro, Monica; Carone, Chiara; Andreoli, Nicola; Astancolle, Serenella; Davalli, Pierpaola; Fuxe, K; Agnati, Luigi Francesco
abstract

Amyloid peptides (Aβ) are fragments of the Amyloid Precursor Protein (APP), an integral membrane protein. Aβ peptides are continuously generated by neurons and non-neuronal cells via sequential cleavage of APP by secretases. In particular, Aβ1-42 is the main component of the senile plaques associated with Alzheimer's disease (AD). Glial cells participate in the uptake of soluble extra-cellular Aβ and in the clearance of this material at localized sites where the Aβ are concentrated. It has been shown that clusterin (Apo J) and apolipoprotein E (ApoE) exert important additive effects in reducing Aβ deposition. In agreement with the fact that homocysteine (Hcy) potentiates Aβ peptide neurotoxicity, and Hcy brain levels increase with age, it has been demonstrated that high plasma levels of Hcy are a risk factor for AD.In the present paper, we used animals subjected to chronic intake of methionine (1 g/kg/day) in the drinking water, since this treatment can increase plasma Hcy levels by 30%. By means of this animal model, interactions between the Aβ β- sheet rich fibrils and clusterin, have been evaluated in striata of animals after Aβ injection. Furthermore, it has been demonstrated that Aβ peptides are not only signals capable of activating astrocytes but also capable of reducing tyrosinehydroxylase immunoreactivity in the basal ganglia probably leading to a reduction of volume transmission. These alterations in the neuroglial network morphology and function can, at least in part, explain the enhanced pain threshold observed in the Aβ intra-striatally injected animals.

2006 - Allosteric modulation of dopamine D-2 receptors by homocysteine [Articolo su rivista]
Agnati, Luigi Francesco; S., Ferre; Genedani, Susanna; Leo, Giuseppina; D., Guidolin; Filaferro, Monica; P., Carriba; V., Casado; C., Lluis; R., Franco; A. S., Woods; K., Fuxe
abstract

It has been suggested that L-DOPA-induced hyperhomocysteinemia can increase the risk of stroke, heart disease, and dementia and is an additional pathogenetic factor involved in the progression of Parkinson's disease. In Chinese hamster ovary (CHO) cells stably cotransfected with adenosine A(2A) and dopamine D-2 receptors, homocysteine selectively decreased the ability of D-2 receptor stimulation to internalize adenosine A(2A)-dopamine D-2 receptor complexes. Radioligand-binding experiments in the same cell line demonstrated that homocysteine acts as an allosteric D-2 receptor antagonist, by selectively reducing the affinity of D-2 receptors for agonists but not for antagonists. Mass spectrometric analysis showed that, by means of an arginine (Arg)-thiol electrostatic interaction, homocysteine forms noncovalent complexes with the two Arg-rich epitopes of the third intracellular loop of the D-2 receptor, one of them involved in A(2A)-D-2 receptor heteromerization. However, homocysteine was unable to prevent or disrupt A(2A)-D-2 receptor heteromerization, as demonstrated with Fluorescence Resonance Energy Transfer (FRET) experiments in stably cotransfected HEK cells. The present results could have implications for Parkinson's disease.

2005 - Computer-assisted image analysis of Caveolin-1 involvement in the iternalization process of adenosine A2A-dopamine D2 Receptor heterodimers [Articolo su rivista]
Genedani, Susanna; D., Guidolin; Leo, Giuseppina; Filaferro, Monica; M., Torvinen; A. S., Woods; K., Fuxe; S., Ferre; Agnati, Luigi Francesco
abstract

A functional aspect of horizontal molecular networks has been investigated experimentally, namely the heteromerization between adenosine A(2A) and doparnine D-2 receptors and the possible role of caveolin-1 in the cotrafficking of these molecular complexes. This study has been carried out by means of computer-assisted image analysis procedure of laser images of membrane immunoreactivity of caveolin-1, A(2A), D-1, and D-2 receptors obtained in two clones of Chinese hamster ovary cells-one transfected with A(2A) and dopamine D, receptors and the other one with A(2A) and D-2 receptors. Cells were treated for 3 h with 10 mu M D-1 receptor agonist SKF 38393,50 mu M D-2-D-3 receptor agonist quinpirole, and 200 nM A(2A) receptor agonist CGS 21680. In A(2A)-D-1-cotransfected cells, caveolin-1 was found to colocalize with both A2A and D, receptors and treatment with SKF 38393 induced internalization of caveolin-1 and D-1 receptors, with a preferential internalization of D, receptors colocalized. with caveolin-1. In A(2A)-D-2-cotransfected cells, caveolin-1 was found to colocalize with both A2A and D2 receptors and either CGS 21680 or quinpirole treatment induced internalization of caveolin-1 and A2A and D2 receptors, with a preferential internalization of A2A and D2 receptors colocalized with caveolin-1. The results suggest that A2A and D2 receptors and caveolin-1 likely interact forming a macrocomplex that internalizes upon agonist treatment. These observations are discussed in the frame of receptor oligomerization and of the possible functional role of caveolin-1 in the process of co-internalization and, hence, in controlling the permanence of receptors at the plasma membrane level (prerequisite for receptor mosaic organization and plastic adjustments) and in the control of receptor desensitization.

2005 - Studies on homocysteine plasma levels in Alzheimer's patients. Relevance for neurodegeneration [Articolo su rivista]
Agnati, Luigi Francesco; Genedani, Susanna; G., Rasio; Galantucci, Maria; Saltini, Sabrina; Filaferro, Monica; R., Franco; F., Mora; S., Ferre; K., Fuxe
abstract

Homocysteine (HC) may work inter alia as a Volume Transmission signal since HC is present in the brain and cerebrospinal fluid and binds to NMDA receptors. Furthermore, in cell cultures increased HC formation increases its export. In the present study we have shown that after intravenous injection in intact animals HC penetrates the blood-brain barrier. Hence, it works as a blood-born humoral signal. Furthermore, we have studied HC plasma levels in a group of Alzheimer's (AD) patients and compared with a group of age-matched patients. It has been confirmed that a positive correlation exists between age and HC plasma levels in the control group, but not in the AD patients. These results may depend on the fact that in AD patients high HC plasma levels (possibly associated with high glycine levels and/or excessive glutamate release) have favored neurodegeneration and, once this pathological process has been triggered off, the plasma HC levels become independent of the physiological aging-induced increase of HC plasma levels.

2002 - Pharmacological manipulation of brain galaninergic system and sexual behavior in male mice [Articolo su rivista]
Benelli, Augusta; Bertolini, Alfio; Zoli, Michele; Leo, Giuseppina; Filaferro, Monica; S., Saltini; Genedani, Susanna
abstract

Available data suggest a complex role for the brain galaninergic system in male sexual behavior; however, the results so far obtained in animals with either galanin or galanin antagonists are conflicting. Objective: To define the better influence of galanin on male sexual behavior by studying, in mice, (i) the effect of galanin and of the chimeric galanin peptide M40 on the copulatory performance, and (ii) galanin mRNA levels in hypothalamic arcuate and dorso-medial nuclei. Methods: For the behavioral testing, only sexually sluggish male mice were used. Galanin mRNA levels were studied in both sexually potent and impotent mice by means of in situ hybridization. Standard behavioral parameters for sexual behavior were recorded or calculated. Synthetic galanin (0.05, 0.1 or 1 mug/mouse) and M40 (5 or 20 mug/mouse) were intracerebroventricularly (ICV) injected, 15 min before the copulatory test. Galanin mRNA levels were evaluated. Results: In sexually sluggish male mice, both galanin (0.1 and 1 mug/mouse ICV) and M40 (20 mug/mouse ICV), significantly increased intromission frequency and ejaculation latency; M40 also improved copulatory efficacy. On the other hand, in the hypothalamic arcuate and dorso-medial nuclei, the levels of galanin mRNA were not significantly different in sexually potent and impotent male mice. Conclusions.-These results show that in sexually sluggish male mice the ICV injection of either galanin or the chimeric analogue M40 greatly prolongs the duration of the copulation; without a reduction of the sexual drive or of the copulatory performance. On the other hand, the hybridization experiments seem to rule out an important physiological role of the brain galaninergic system in the regulation of male sexual behavior, at least in mice.

2000 - [In vitro study of periodontal ligament cells]. [Articolo su rivista]
R., Galetti; Benelli, Augusta; G., Bruzzesi; Consolo, Ugo; Filaferro, Monica; Genedani, Susanna; S., Palazzini; G., Rasio; DE POL, Anto
abstract

The aim of this research is to outline a procedure able to promote specific cellular differentiation and proliferation with consequent periodontal regeneration. To achieve this goal, use was made of various compounds supposed to have the capacity of aiding periodontal regeneration.The cells utilised for this study were obtained from explants of human periodontal ligaments. Their proliferation and differentiation capacity was examined in the presence of: coral granules (350, 500 mu), collagene type 1, growth factors (Platelet derived growth factor, PDGF and Transforming growth factor beta 1, TGF beta 1), both on their own and in different combination with one another. The differentiation activity was evaluated by ultrastructural morphological method (Transmission electron microscope-TEM) and by spectrophotometric investigation of the alkaline phosphatasis (ALP).The data show that the coral granules and among the growth factors used only TGF beta 1 stimulate the differentiation activity of the periodontal ligament cells valued on the basis of their capacity of producing ALP. These data are supported by the observation with TEM.From these results it is suggested that there may be therapeutic efficiency in the periodontal field of substances promoting cellular proliferation and differentiation.

1999 - Influence of S-adenosyl-L-methionine on chronic mild stress-induced anhedonia in castrated rats [Articolo su rivista]
Benelli, Augusta; Filaferro, Monica; Bertolini, Alfio; Genedani, Susanna
abstract

1 S-adenosyl-L-methionine (SAMe) is the most important methyl donor in the brain and is essential for polyamine synthesis. Methyl group deficiency in the brain has been implicated in depression; on the other hand, polyamines enhance phosphorylation processes, and phosphorylation of functional proteins in neurons is involved in the therapeutic mechanisms of antidepressants. 2 The effect of SAMe in an animal model of 'depression', the chronic mild stress-induced anhedonia, was studied using long-term castrated male and female Lister hooded rats. 3 Chronic daily exposure to an unpredictable sequence of mild stressors produced, within 3 weeks, a significant reduction of the consumption of a sucrose solution. SAMe (100, 200 or 300 mg kg(-1) daily i.m.) while having no influence on sucrose intake in non-stressed animals, dose-dependently reinstated sucrose consumption within the first week of treatment, both in male and in female stressed rats. Imipramine (10 mg kg(-1) daily i.p.) produced a similar effect after a 3 week treatment. 4 Similarly, a palatable food reward-induced place preference conditioning was developed in SAMe (200 or 300 mg kg(-1) daily i.m.)- and in imipramine (10 mg kg(-1) daily i.p.)-treated chronically stressed animals (males and females), whilst it could not be obtained in vehicle-treated rats. 5 Moreover, the same doses of SAMe (but not of imipramine) restored the exploratory activity and curiosity for the environment (rearing), in the open-field test. 6 While imipramine caused a blockade of the growth throughout the treatment, SAMe produced only a transient growth arrest during the first week of treatment. 7 These results show that SAMe reverses an experimental condition of 'depression-like' behaviour in rats, the effect being more rapid and complete than that of imipramine, and without apparent side effects.