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GIOVANNA RIGILLO

Ricercatore Legge 240/10 - t.det.
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

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Pubblicazioni

2023 - A Novel Perilla frutescens (L.) Britton Cell-Derived Phytocomplex Regulates Keratinocytes Inflammatory Cascade and Barrier Function and Preserves Vaginal Mucosal Integrity In Vivo [Articolo su rivista]
Pressi, G.; Rigillo, G.; Governa, P.; Borgonetti, V.; Baini, G.; Rizzi, R.; Guarnerio, C.; Bertaiola, O.; Frigo, M.; Merlin, M.; Paltrinieri, S.; Zambonin, R.; Pandolfo, S.; Biagi, M.
abstract

: In the last years, the medicinal plant Perilla frutescens (L.) Britton has gained scientific interest because leaf extracts, due to the presence of rosmarinic acid and other polyphenols, have shown anti-allergic and skin protective potential in pre-clinical studies. Nevertheless, the lack of standardized extracts has limited clinical applications to date. In this work, for the first time, a standardized phytocomplex of P. frutescens, enriched in rosmarinic acid and total polyphenols, was produced through innovative in vitro cell culture biotechnology and tested. The activity of perilla was evaluated in an in vitro inflammatory model of human keratinocytes (HaCaT) by monitoring tight junctions, filaggrin, and loricrin protein levels, the release of pro-inflammatory cytokines and JNK MAPK signaling. In a practical health care application, the perilla biotechnological phytocomplex was tested in a multilayer model of vaginal mucosa, and then, in a preliminary clinical observation to explore its capacity to preserve vaginal mucosal integrity in women in peri-menopause. In keratinocytes cells, perilla phytocomplex demonstrated to exert a marked activity in epidermis barrier maintenance and anti-inflammatory effects, preserving tight junction expression and downregulating cytokines release through targeting JNK activation. Furthermore, perilla showed positive effects in retaining vaginal mucosal integrity in the reconstructed vaginal mucosa model and in vivo tests. Overall, our data suggest that the biotechnological P. frutescens phytocomplex could represent an innovative ingredient for dermatological applications.

2023 - ADOLESCENCE AS A CRITICAL TIME-WINDOW FOR NEUROINFLAMMATION IN THE MOUSE: WHY SEX MATTERS [Abstract in Rivista]
Toscano, Ylenia; Benatti, Cristina; Alboni, Silvia; Ciani, Miriam; Rigillo, Giovanna; Tascedda, Fabio; Blom, Johanna Maria Catharina; Brunello, Nicoletta
abstract

2023 - ATTIVITÀ DEL FITOCOMPLESSO BIOTECNOLOGICO PERILLA FRUTESCENS (L.) BRITTON NEL PRESERVARE L’INTEGRITÀ DELLA MUCOSA VAGINALE MEDIANTE UN EFFETTO ANTI-INFIAMMATORIO E PROTETTIVO DELLA BARRIERA CUTANEA [Poster]
Rigillo, Giovanna; Pressi, Giovanna; Governa, Paolo; Borgonetti, Vittoria; Baini, Giulia; Guarnerio, Chiara; Bertaiola, Oriana; Frigo, Marco; Merlin, Matilde; Zambonin, Roberto; Pandolfo, Stefano; Biagi, Marco
abstract

Negli ultimi anni, la pianta medicinale Perilla frutescens (L.) Britton ha ricevuto notevole interesse per il ricco contenuto di acido rosmarino e altri polifenoli che hanno dimostrato un’attività anti-allergica e potenzialmente protettiva della cute in diversi studi preclinici e clinici. Tuttavia, la mancanza di estratti standardizzati ha limitato il suo utilizzo in clinica. Questo studio ha avuto l’obiettivo di produrre e testare l’attività di un fitocomplesso P. frutescens, standardizzato nel contenuto di acido rosmarinico e polifenoli totali, ottenuto mediante l’innovativo approccio biotecnologico della cultura vegetale in vitro. L’attività di P. frutescens è stata valutata inizialmente in un modello infiammatorio in vitro di cheratinociti umani (HaCaT) in cui è stato monitorato il rilascio di citochine pro-infiammatorie, i livelli di espressione delle Tight-Junction (TJs), e delle proteine strutturali filaggrina e loricrina. Inoltre, è stata analizzata la via di segnalazione JNK-MAPKs come possibile meccanismo intracellulare coinvolto. Allo scopo di valutare una possibile applicazione cosmetica nella cura e benessere intimo, il fitocomplesso biotecnologico è stato testato prima su un modello di mucosa vaginale ricostruita e, successivamente, in uno studio clinico osservazionale, che ha coinvolto donne volontarie in età pre-menopausa, mediante l’applicazione di perilla nelle formulazioni di crema e maschera. Nel modello in vitro di cheratinociti, il fitocomplesso ha dimostrato: i) un effetto anti-infiammatorio, reprimendo il rilascio di citochine pro-infiammatorie attraverso l’attivazione della via di JNK; ii) una buona efficacia nel mantenimento della barriera cutanea preservando l’espressione delle TJs e delle proteine strutturali dell’epidermide. In questo contesto, perilla ha mostrato effetti positivi nel mantenimento della integrità della mucosa vaginale nel modello in vitro e nei test in vivo. I dati di efficacia e sicurezza ottenuti dallo studio suggeriscono che l’innovativo fitocomplesso di P. frutescens potrebbe rappresentare un ottimo ingrediente nelle formulazioni cosmetiche per uso topico, nel campo della dermatologia e nella cura dell’igiene intima.

2023 - Dysregulation of NF–Y splicing drives metabolic rewiring and aggressiveness in colon cancer [Poster]
Belluti, S.; Mularoni, V.; Campani, Virginia; Rigillo, G.; Cuoghi, L.; Ronzio, M.; Miserocchi, G.; Dolfini, D.; Righi, Valeria; Alessandrini, A.; Zappavigna, V.; Imbriano, C.
abstract

NF-Y is an evolutionarily conserved transcription factor that binds specifically to the CCAAT elements of eukaryotic genes, most of which frequently deregulated in cancer. NF-YA, the regulatory subunit of the NF-Y complex, has two isoforms generated by alternative splicing, NF-YAl and NF-YAs, which differ in the transactivation domain. Transcriptomic data from The Cancer Genome Atlas (TCGA) database highlighted a significant increase in the expression of NF-YAs at the expense of NF-YAl in colorectal cancer (CRC), compared to healthy tissues. Despite this, high NF-YAl levels predict lower patients’ survival and distinguish the mesenchymal molecular subtype CMS4, which is characterized by the worst prognosis. Through the analysis of 3D cellular models, we demonstrated that altered expression of genes related to extracellular matrix and epithelial-mesenchymal transition sustains enhanced migratory and invasive behavior of NF-YAl-transduced cells. Moreover, the integration of metabolomics, bioenergetics and transcriptional analyses demonstrated a direct role for NFYAl in metabolic flexibility of cancer cells that adjust their metabolism in response to environmental changes to potentiate migration. The zebrafish xenograft model confirmed the metastatic potential triggered by NF-YAl in CRC cells. Altogether, our data highlight the transcriptional role of NF-YAl in CRC aggressiveness and suggest splice-switching strategies to hinder NF-YAl-induced metastatic dissemination.

2023 - Environmental enrichment influences mouse hippocampal neuroinflammatory response [Abstract in Rivista]
Rigillo, Giovanna; Benatti, Cristina; Toscano, Ylenia; Tascedda, Fabio; Pani, Luca; Brunello, Nicoletta; Alboni, Silvia
abstract

Background: Many studies have shown the positive effects of environmental enrichment on brain plasticity with significant implications for development, behavior, learning, memory, and recovery from brain damage (1). Experimental and clinical studies report that one’s living environment can modulate cellular and molecular responses in the brain, counteracting cognitive decline, alleviating anxiety, and depressive behaviours, as well as moderating the outcome of pharmacological treatments (2). Neuroinflammation has been well established as an important factor in the aetiopathogenesis and progression of brain disorders. It can affect neural development and alters hippocampal plasticity thus resulting in cognitive impairments. Neuroinflammation is characterized by a dysregulation of the NLRP3 inflammasome activation, an increase in the expression of inflammatory cytokines, and a decrease of neurotrophic factors (3). Behavioral and neurochemical changes, caused by neuroinflammation, have been most frequently investigated through peripheral administration of lipopolysaccharide (LPS), which can, directly and indirectly, affect the central nervous system (4). Based on these premises, the aim of this study was to explore the molecular effects of the quality of the living environment in modulating the LPS-induced neuroinflammatory response in the hippocampus of wild-type mice. Methods: Male C57BL6J mice (13 weeks-old) were randomly housed in Impoverished (IE) or Enriched Environment (EE) condition for 28 days, then exposed to LPS (0.830 mg/Kg, i.p.) or saline (SAL). Twenty-four hours after injection hippocampi were removed for gene expression analysis performed by means of qRT-PCR. Data from groups were analyzed by Two-way ANOVA followed by Tukey’s post hoc test. Results: The analysis of the environmental effects on the LPS signaling system highlighted the downregulation of the membrane-bound protein LBP, the receptor TLR4 and the co-receptor cluster CD14 expression levels in EE-housed animals compared to their counterparts. The exposure to an EE condition was able to attenuate the LPS-induced increase of TLR4 and NLRP3 inflammasome mRNA levels. Gene expression analysis revealed a significant downregulation of the pro-inflammatory cytokines IL-1β and TNFα levels in EE-housed mice while LPS exposure strongly increased IL-1β and TNFα mRNA levels irrespective of the housing conditions. Moreover, EE-exposed mice showed a significant upregulation of BDNF hippocampal mRNA levels, although no effects were observed after LPS treatment in both conditions. Conclusions: Our results displayed the beneficial effect of EE in regulating the expression of inflammatory mediators involved in the LPS-induced response in the hippocampus, a key area for learning, memory, and emotion. These data suggest that living environment may exert a positive and protective role on the brain by reducing susceptibility toward neurodegenerative or neuropsychiatric disorders.

2023 - FROM PRECLINICAL TO CLINICAL EVIDENCE: EXPLORING THE MULTIPLE PERSPECTIVES AND HEALING POWER OF BOSWELLIA SERRATA ROXB. EX COLEBR [Articolo su rivista]
Biagi, M.; Rigillo, G.; Sarill, M.; Collotta, D.; Di Giacomo, S.; Di Sotto, A.; Grilli, M.; Luceri, C.; Milella, L.; Sangiovanni, E.; Vitalone, A.; Montopoli, M.; Testai, L.
abstract

Boswellia serrata Roxb. ex Colebr. is a species belonging to the Burseraceae family, typical of dry environments of the Indian region. The oil-gum-resin, obtained from the trunk and thick branches, is known in phytotherapy for the volatile fraction which contains up to 70% terpenes. The most important and characteristic constituents are represented by pentacyclic triterpenes, named boswellic acids. B. serrata is known for multiple beneficial effects, mainly correlated to anti-inflammatory activity. This review aims to provide a comprehensive overview on the activities and potential applications of B. serrata based on clinical and preclinical evidence. An up-to-date literature review of preclinical and clinical studies related to the applications of B. serrata preparations in different pathological conditions was conducted using the main databases of scientific literature. A body of evidence point out the role of B. serrata extracts and its active constituents in the treatment of several inflammatory diseases. In particular, clinical trials revealed its use as a topical remedy of skin diseases, such as eczema and psoriasis, and internally in the treatment of asthma, intestinal and osteoarticular inflammatory diseases. Preclinical findings highlighted the positive effects of B. serrata extracts in cardiovascular and neurodegenerative diseases, and in cancer. Finally, B. serrata finds application as a feed additive in veterinary use. Although some limitations must be overcome, such as poor bioavailability, evidence supports that B. serrata is a promising medicinal plant. Furthermore, the use of B. serrata appears to have a favorable toxicological profile, but caution may be necessary regarding potential botanical-drug interactions.

2023 - Invertebrates as models of learning and memory: investigating neural and molecular mechanisms [Articolo su rivista]
Rivi, Veronica; Benatti, Cristina; Rigillo, Giovanna; Blom, Johanna M C
abstract

: In this Commentary, we shed light on the use of invertebrates as model organisms for understanding the causal and conserved mechanisms of learning and memory. We provide a condensed chronicle of the contribution offered by mollusks to the studies on how and where the nervous system encodes and stores memory and describe the rich cognitive capabilities of some insect species, including attention and concept learning. We also discuss the use of planarians for investigating the dynamics of memory during brain regeneration and highlight the role of stressful stimuli in forming memories. Furthermore, we focus on the increasing evidence that invertebrates display some forms of emotions, which provides new opportunities for unveiling the neural and molecular mechanisms underlying the complex interaction between stress, emotions and cognition. In doing so, we highlight experimental challenges and suggest future directions that we expect the field to take in the coming years, particularly regarding what we, as humans, need to know for preventing and/or delaying memory loss. This article has an associated ECR Spotlight interview with Veronica Rivi.

2023 - LPS-Induced Garcia Effect and Its Pharmacological Regulation Mediated by Acetylsalicylic Acid: Behavioral and Transcriptional Evidence [Articolo su rivista]
Rivi, V.; Batabyal, A.; Lukowiak, K.; Benatti, C.; Rigillo, G.; Tascedda, F.; Blom, J. M. C.
abstract

Lymnaea stagnalis learns and remembers to avoid certain foods when their ingestion is followed by sickness. This rapid, taste-specific, and long-lasting aversion—known as the Garcia effect—can be formed by exposing snails to a novel taste and 1 h later injecting them with lipopolysaccharide (LPS). However, the exposure of snails to acetylsalicylic acid (ASA) for 1 h before the LPS injection, prevents both the LPS-induced sickness state and the Garcia effect. Here, we investigated novel aspects of this unique form of conditioned taste aversion and its pharmacological regulation. We first explored the transcriptional effects in the snails’ central nervous system induced by the injection with LPS (25 mg), the exposure to ASA (900 nM), as well as their combined presentation in untrained snails. Then, we investigated the behavioral and molecular mechanisms underlying the LPS-induced Garcia effect and its pharmacological regulation by ASA. LPS injection, both alone and during the Garcia effect procedure, upregulated the expression levels of immune- and stress-related targets. This upregulation was prevented by pre-exposure to ASA. While LPS alone did not affect the expression levels of neuroplasticity genes, its combination with the conditioning procedure resulted in their significant upregulation and memory formation for the Garcia effect.

2023 - Narrative Review of the Complex Interaction between Pain and Trauma in Children: A Focus on Biological Memory, Preclinical Data, and Epigenetic Processes [Articolo su rivista]
Rivi, V.; Rigillo, G.; Toscano, Y.; Benatti, C.; Blom, J. M. C.
abstract

The incidence and collective impact of early adverse experiences, trauma, and pain continue to increase. This underscores the urgent need for translational efforts between clinical and preclinical research to better understand the underlying mechanisms and develop effective therapeutic approaches. As our understanding of these issues improves from studies in children and adolescents, we can create more precise preclinical models and ultimately translate our findings back to clinical practice. A multidisciplinary approach is essential for addressing the complex and wide-ranging effects of these experiences on individuals and society. This narrative review aims to (1) define pain and trauma experiences in childhood and adolescents, (2) discuss the relationship between pain and trauma, (3) consider the role of biological memory, (4) decipher the relationship between pain and trauma using preclinical data, and (5) examine the role of the environment by introducing the importance of epigenetic processes. The ultimate scope is to better understand the wide-ranging effects of trauma, abuse, and chronic pain on children and adolescents, how they occur, and how to prevent or mitigate their effects and develop effective treatment strategies that address both the underlying causes and the associated physiological and psychological effects.

2023 - Safety and Efficacy of Red Yeast Rice Phytocomplex and Lovastatin: a Comparative Analysis [Relazione in Atti di Convegno]
Rigillo, G; Baini, G; Miraldi, E; Pani, L; Tascedda, F; Biagi, M
abstract

In the last 20 years, the demand for red yeast rice-based food supplements has significantly increased as consumers have been looked for natural alternatives to manage blood cholesterol levels without the adverse effects associated with synthetic statins. Actually, monacolin K, derived from red yeast rice (RYR) and chemically identical to lovastatin, inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, offering a natural alternative to synthetic drugs. In 2018, despite the health claim approved in 2011, the European Food Safety Authority (EFSA) raised concerns about the safety of monacolin K consumption, especially for vulnerable populations, and highlighted the lack of knowledge on RYR phytocomplex. Consequently, in 2021, the European Commission established a new maximum intake limit < 2.99 mg/day for monacolins. This study aimed to investigate the safety and efficacy of RYR phytocomplex compared to the sole monacolin K (=lovastatin) by analyzing eight different RYR samples with different monacolin K and secondary monacolins content. Efficacy was evaluated through a validated cell-free enzymatic assay, while an integrated in vitro simulated digestion and in silico ADME prediction were employed to compare the pharmacokinetics of different samples and lovastatin. The safety of RYR was assessed by monitoring cytotoxicity in intestinal, hepatic, kidney, and skeletal muscle cells using cell viability assays. Furthermore, muscle damage-related targets and myokines were measured by qRT-PCR in myoblasts exposed to prolonged non-toxic stimuli. Results demonstrated that RYR samples have a large chemical variability not only related to monacolin K content, in lactone and hydroxy-acid form, but also as regards pigments, secondary monacolins, polyphenols, and triterpenes. The enzymatic test revealed that all samples were more effective in inhibiting HMG-CoA activity than lovastatin at equivalent monacolin K content. Although secondary monacolins had a weaker effect than monacolin K, a synergistic effect was observed within the phytocomplex. Additionally, the phytocomplex facilitated higher bioaccessibility of monacolin K in RYR compared to lovastatin. Regarding cell viability, lovastatin and RYR samples exhibited no toxic effects up to 150 g/mL on intestinal, hepatic, and kidney cells; however, variable effects were observed on skeletal muscle cells at medium-high concentrations. Nevertheless, at physiological non-toxic concentrations, RYR samples did not impact the expression levels of myokine IL-6, autophagy factors, and catabolic markers of protein balance, unlike synthetic statin. These results suggest that RYR and lovastatin share similar biological activities, but the phytocomplex's contribution from secondary monacolins, pigments, polyphenols, and triterpenes ensure a superior safety profile for this botanical food supplement. However, it is important to note that the composition of RYR phytocomplex can vary significantly between different products on the market. This variability highlights the need for standardization and quality control measures to ensure the safety and efficacy of this botanical food supplement. In conclusion, our study provides evidence supporting the enhanced efficacy, bioaccessibility, and safety profile of RYR phytocomplex compared to lovastatin demonstrating its potential as a natural and effective option for hypercholesterolemia management.

2023 - The NF-Y splicing signature controls hybrid EMT and ECM-related pathways to promote aggressiveness of colon cancer [Articolo su rivista]
Rigillo, Giovanna; Belluti, Silvia; Campani, Virginia; Ragazzini, Gregorio; Ronzio, Mirko; Miserocchi, Giacomo; Bighi, Beatrice; Cuoghi, Laura; Mularoni, Valentina; Zappavigna, Vincenzo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

: Aberrant splicing events are associated with colorectal cancer (CRC) and provide new opportunities for tumor diagnosis and treatment. The expression of the splice variants of NF-YA, the DNA binding subunit of the transcription factor NF-Y, is deregulated in multiple cancer types compared to healthy tissues. NF-YAs and NF-YAl isoforms differ in the transactivation domain, which may result in distinct transcriptional programs. In this study, we demonstrated that the NF-YAl transcript is higher in aggressive mesenchymal CRCs and predicts shorter patients' survival. In 2D and 3D conditions, CRC cells overexpressing NF-YAl (NF-YAlhigh) exhibit reduced cell proliferation, rapid single cell amoeboid-like migration, and form irregular spheroids with poor cell-to-cell adhesion. Compared to NF-YAshigh, NF-YAlhigh cells show changes in the transcription of genes involved in epithelial-mesenchymal transition, extracellular matrix and cell adhesion. NF-YAl and NF-YAs bind similarly to the promoter of the E-cadherin gene, but oppositely regulate its transcription. The increased metastatic potential of NF-YAlhigh cells in vivo was confirmed in zebrafish xenografts. These results suggest that the NF-YAl splice variant could be a new CRC prognostic factor and that splice-switching strategies may reduce metastatic CRC progression.

2023 - The NF-YA Splicing Signature Controls Aggressiveness of Colon Cancer by Regulating Cell Metabolism and Different Types of Cell Migration [Relazione in Atti di Convegno]
Belluti, Silvia; Mularoni, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Miserocchi, Giacomo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

NF-Y is a transcription factor composed of NF-YA and NF-YB/NF-YC subunits. The two NF-Y isoforms, NFYAs and NF-YAl, differentially control cell proliferation and differentiation. The analysis of patient’s transcriptome profiles from The Cancer Genome Atlas database highlight increased NF-YA expression, specifically NF-YAs, in colorectal cancer (CRC), the second most deadly cancer worldwide. Despite this, patients with high NF-YAl mRNA have a lower overall survival probability. We investigated the role of NF-YA in the metabolism of CRC cells, and the measurement of mitochondrial fuel usage in live cells shows that NF-YAl overexpression enhances the capacity for glutamine pathway, one of the key metabolic pathways involved in EMT and cell dissemination. Specifically, we identified NF-YAl as direct transcriptional regulator of GLS1 glutaminase and GLUL glutamine synthetase. Moreover, we demonstrate that NF-YAl overexpression can generate a hybrid epithelial-mesenchymal transition (EMT) state in CRC cells by direct transcriptional regulation of key EMT, extracellular matrix and adhesion genes. Consistently, NF-YAl enhances cell migration, both in 2D and 3D culture conditions, as highlighted by live imaging investigations. While collective migration characterizes NFYAs-cells, fast single-cell and amoeboid-like migration marks NF-YAl cells. In agreement with these results, NF-YAl overexpression promotes cell dissemination in zebrafish xenografts. Our observations imply that the two NF-YA variants can be potentially novel markers for CRC patients’ stratification. Higher NF-YAl expression can be a hallmark of cancer cell dissemination by affecting cell metabolism and cell migratory abilities.

2023 - VORTIOXETINE ATTENUATES NEUROINFLAMMATION BY MODULATING THE NOD-LIKE RECEPTOR FAMILY PYRIN DOMAIN CONTAINING 3 INFLAMMASOME ACTIVATION IN MICROGLIA: IMPLICATIONS FOR COGNITIVE FUNCTION [Abstract in Rivista]
Rigillo, G.; Ciani, M.; Benatti, C.; Blom, J. M. C.; Tascedda, F.; Pani, L.; Alboni, S.; Brunello, N.
abstract

Vortioxetine (VTX) is a multimodal antidepressant with an extensive pharmacological profile that includes modulation of various neurotransmitter systems, neuroprotective activity, and beneficial effects on cognitive functions. Recent research has revealed a novel aspect of VTX's activity - its antiinflammatory effects - that suggests an intriguing molecular mechanism may underpin its therapeutic benefits. Neuroinflammation, dysfunctional neurogenesis and neurotransmission, and dysregulation of the hypothalamus– pituitary–adrenal (HPA) axis are all pivotal in the onset and progression of depression. One particular immune-inflammatory pathway overactivated in brain disorders is the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, a multiprotein complex. This complex's activation is mediated by NF-kB and reactive oxygen species (ROS) signalling pathways, leading to caspase-1-dependent release of the proinflammatory cytokines, IL-1β and IL-18. Mounting evidence implicates NLRP3 inflammasome in neuroinflammation- related disorders, with its activation associated with cognitive function impairment. Of note, microglia, the resident immune cells crucial for brain plasticity, express high levels of the NLRP3-inflammasome components. Our initial findings indicate that VTX exerts a region-dependent modulatoryeffect on the NLRP3-inflammasome system in a LPS-induced memory impairment in vivo model. Furthermore, VTX's ability to modulate immune response suggests that microglia could be a direct target of the drug. AIM: In light of the compelling evidence surrounding the role of the NLRP3 inflammasome in cognitive dysfunctions and the recent discovery of VTX's anti-inflammatory activity, we aimed to investigate the molecular effects induced by VTX pre-treatment in the presence or absence of the inflammasome-inducer LPS in a well-established in vitro model of mouse microglia: BV2 cells. METHODS: To dissect the influence of VTX pre-treatment (24h) on the NLRP3 inflammasome signaling pathway and microglial polarization, we analyzed gene and protein expression in BV2 cells stimulated with LPS or vehicle for 6h. We also scrutinized the activation/translocation of NF-kB and ROS release under these conditions. We applied one-way or two-way ANOVA followed by Tukey’s post hoc test for statistical analysis based on the experimental design. RESULTS: Our data demonstrate that short-term exposure to LPS significantly induces the activation/translocation of NF-kB signaling and ROS release in BV2 cells. We observed a time-dependent transcriptional upregulation of the inflammasome complex, IL-1β and IL-18, and microglial pro-inflammatory targets post-LPS stimulation, alongside a downregulation of the anti-inflammatory factors. Interestingly, a pre-treatment with VTX (10 nM) for 24h effectively modulated the LPS-induced NF-kB translocation and ROS production compared to control cells. Cells pre-treated with VTX exhibited lower levels of LPS-induced NLRP3 inflammasome- and microglia pro-inflammatory-related targets. However, VTX did not influence the expression of anti-inflammatory factors in both unstimulated and LPS-stimulated BV2 cells. CONCLUSIONS: Our findings reinforce the emerging evidence that supports VTX's anti-inflammatory activity. This activity is mediated via modulation of the inflammasome signaling pathway, which plays a pivotal role in the inflammatory response of microglia cells.

2022 - Alternative splicing of the transcription factor NF-Y promotes cell migration and invasion in colon cancer [Abstract in Atti di Convegno]
Rigillo, Giovanna; Belluti, Silvia; Campani, Virginia; Ronzio, Mirko; Miserocchi, Giacomo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

The heterotrimeric transcription factor NF-Y directly controls the expression of genes involved in cellular pathways commonly altered in cancer cells, such as cell cycle, apoptosis and metabolism. Consistently, the binding site for NF-Y is highly enriched in the regulatory regions of genes overexpressed in tumors, and mRNA levels of NF-Y subunits are altered in cancer tissues and cells. In particular, the DNA binding subunit NF-YA is up-regulated in various tumors, among which gastric, lung, breast, ovarian, osteosarcoma and prostate cancers. Moreover, a switch between the two alternatively NF-YA spliced transcripts, NF-YAs and NF-YAl, occurs in tumor tissues compared to normal ones. Colorectal cancer (CRC) is the third most common malignancy worldwide. Four internationally approved consensus molecular subtypes (CMS) represent the best current description of CRC heterogeneity at the gene-expression level: the CMS1 group is characterized by the immune infiltration signature, CMS2 is the canonical epithelial subtype, CMS3 represents the metabolic group, and CMS4 is the mesenchymal one, associated with a worse prognosis and poor response to therapies compared to other subtypes. Here we show that increased levels of NF-YA characterize CRC versus healthy tissues. We identified a significant association between NF-YA isoforms and CRC subtypes: NF-YAs is up-regulated in all CMSs in opposition to NF-YAl, which is down-regulated in all subtypes with the exception of aggressive and metastatic CMS4 group. By using in vitro cell models, we confirmed that NF-YAs is the predominant isoform in CRC cell lines, while NF-YAl levels proportionally increase from epithelial to hybrid and mesenchymal cells. The modulation of NF-YA isoforms in CRC cells significantly affects cancer cell behavior by modulating differently, even oppositely, the transcription of genes associated to extracellular-matrix (ECM) and epithelial-to-mesenchymal transition (EMT). We described different modes of migration and invasion properties for NF-YAs and NF-YAl overexpressing cells by using 2D and 3D culture conditions, time-lapse imaging of CRC cells and intravascular distribution of NF-YAs/l transduced CRC cells in the embryonic zebrafish xenograft model. Altogether, our data highlight the direct role of the longer NF-YA isoform in CRC cell dissemination and suggest its possible use as biomarker for molecular stratification predictive of progressive disease in CRC patients.

2022 - In Vitro Cell Culture of Rhus coriaria L.: A Standardized Phytocomplex Rich of Gallic Acid Derivatives with Antioxidant and Skin Repair Activity [Articolo su rivista]
Pressi, G.; Bertaiola, O.; Guarnerio, C.; Barbieri, E.; Rigillo, G.; Governa, P.; Biagi, M.; Guzzo, F.; Semenzato, A.
abstract

This study focused on the biological evaluation and chemical characterization of a new ingredient obtained by in vitro cell culture of Rhus coriaria L. An in vitro plant cell culture method permits to cultivate plant in a short period of time and to obtain extract with a high safety profile for the consumer, free from heavy metals, pesticides, aflatoxins, bacterial or fungal contamination. Through the selection of specific cell culture media, it was possible to obtain a Rhus coriaria cell line with a high content of gallic acid derivatives. The Rhus coriaria L. phytocomplex (RC-P), containing 7.6% w/w of acid gallic derivatives, was obtained by drying of plant cell biomass after 14 days of growth in the final selected culture medium. UPLC-ESI-MS and UPLC-DAD analysis allowed to identify numerous gallic acid derivatives, such as galloyl hexose, trigalloyl hexose and high molecular weight galloyl derivatives, and to quantify their overall content. The antioxidant activity of the RC-P was tested by DPPH assay and the wound healing activity was evaluated using a scratch wound healing test on human keratinocytes and fibroblasts. This work showed that RC-P could be a new effective cosmetic ingredient with antioxidant and skin repair activity.

2022 - Non-psychotropic Cannabis sativa L. phytocomplex modulates microglial inflammatory response through CB2 receptors-, endocannabinoids-, and NF-κB-mediated signaling [Articolo su rivista]
Borgonetti, V.; Benatti, C.; Governa, P.; Isoldi, G.; Pellati, F.; Alboni, S.; Tascedda, F.; Montopoli, M.; Galeotti, N.; Manetti, F.; Miraldi, E.; Biagi, M.; Rigillo, G.
abstract

Cannabis sativa L. is increasingly emerging for its protective role in modulating neuroinflammation, a complex process orchestrated among others by microglia, the resident immune cells of the central nervous system. Phytocannabinoids, especially cannabidiol (CBD), terpenes, and other constituents trigger several upstream and downstream microglial intracellular pathways. Here, we investigated the molecular mechanisms of a CBD- and terpenes-enriched C. sativa extract (CSE) in an in vitro model of neuroinflammation. We evaluated the effect of CSE on the inflammatory response induced by exposure to lipopolysaccharide (LPS) in BV-2 microglial cells, compared with CBD and β-caryophyllene (CAR), CB2 receptors (CB2r) inverse and full agonist, respectively. The LPS-induced upregulation of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α was significantly attenuated by CSE and only partially by CBD, whereas CAR was ineffective. In BV-2 cells, these anti-inflammatory effects exerted by CSE phytocomplex were only partially dependent on CB2r modulation and they were mediated by the regulation of enzymes responsible for the endocannabinoids metabolism, by the inhibition of reactive oxygen species release and the modulation of JNK/p38 cascade with consequent NF-κB p65 nuclear translocation suppression. Our data suggest that C. sativa phytocomplex and its multitarget mechanism could represent a novel therapeutic strategy for neuroinflammatory-related diseases.

2022 - The NF-YA splicing signature controls aggressiveness of colon cancer by regulating different modes of cell migration and cell metabolism. [Poster]
Belluti, Silvia; Rigillo, Giovanna; Mularoni, Valentina; Ronzio, Mirko; Miserocchi, Giacomo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

NF-Y is a transcription factor composed of NF-YA and NF-YB/NF-YC subunits. The two NF-YA isoforms, NF-YAs and NF-YAl, differentially control cell proliferation and differentiation. TCGA data highlight increased NF-YA expression, specifically NF-YAs, in colorectal cancer (CRC), the second most deadly cancer worldwide. Despite this, patients with high NF-YAl mRNA levels have a lower overall survival probability. We demonstrate that NF-YAl overexpression can generate a hybrid epithelial-mesenchymal transition (EMT) state in CRC cells by direct transcriptional regulation of key EMT genes. Consistently, NF-YAl enhances cell migration, both in 2D and 3D culture conditions, as highlighted by live imaging investigations. While collective migration characterizes NF-YAs-cells, fast single-cell and amoeboid-like migration marks NF-YAl cells. In agreement with these results, NF-YAl overexpression promotes cell dissemination in zebrafish xenografts. Since metabolic reprogramming is a hallmark of cancer and metastasis, we also investigated the role of NF-YAl in the metabolism of CRC cells. The measure of mitochondrial fuel usage in live cells showed that NF-YAl overexpression enhances the dependency and capacity for glutamine pathway, one of the key metabolic pathways involved in EMT and cell dissemination. Specifically, we identified NF-YAl as direct transcriptional regulator of GLS1 glutaminase and GLUL glutamine synthetase. Our observations imply that the two NF-YA variants can be potentially novel markers for CRC patient stratification. Higher NF-YAl expression can be a hallmark of cancer cell dissemination by affecting cell metabolism and cell migratory abilities.

2021 - Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients [Articolo su rivista]
Belluti, Silvia; Semeghini, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Benati, Daniela; Torricelli, Federica; Reggiani Bonetti, Luca; Carnevale, Gianluca; Grisendi, Giulia; Ciarrocchi, Alessia; Dominici, Massimo; Recchia, Alessandra; Dolfini, Diletta; Imbriano, Carol
abstract

Approaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patient outcomes and response to treatments. The transcription factor NF-Y participates to the progression from benign epithelium to both localized and metastatic PCa and is associated with aggressive transcriptional profile. The gene encoding for NF-YA, the DNA-binding subunit of NF-Y, produces two alternatively spliced transcripts, NF-YAs and NF-YAl. Bioinformatic analyses pointed at NF-YA splicing as a key transcriptional signature to discriminate between different tumor molecular subtypes. In this study, we aimed to determine the pathophysiological role of NF-YA splice variants in PCa and their association with aggressive subtypes.

2021 - Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients [Poster]
Belluti, Silvia; Semeghini, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Benati, Daniela; Torricelli, Federica; REGGIANI BONETTI, Luca; Carnevale, Gianluca; Grisendi, Giulia; Ciarrocchi, Alessia; Dominici, Massimo; Recchia, Alessandra; Dolfini, Diletta; Imbriano, Carol
abstract

2021 - Nutraceuticals and herbal food supplements for weight loss: Is there a prebiotic role in the mechanism of action? [Articolo su rivista]
Bertuccioli, A.; Cardinali, M.; Biagi, M.; Moricoli, S.; Morganti, I.; Zonzini, G. B.; Rigillo, G.
abstract

Numerous nutraceuticals and botanical food supplements are used with the intention of modulating body weight. A recent review examined the main food supplements used in weight loss, dividing them according to the main effects for which they were investigated. The direct or indirect effects exerted on the intestinal microbiota can also contribute to the effectiveness of these substances. The aim of this review is to evaluate whether any prebiotic effects, which could help to explain their efficacy or ineffectiveness, are documented in the recent literature for the main nutraceuticals and herbal food supplements used for weight loss management. Several prebiotic effects have been reported for various nutraceutical substances, which have shown activity on Bifidobacterium spp., Lactobacillus spp., Akkermansia muciniphila, Faecalibacterium prausnitzi, Roseburia spp., and the Firmicutes/Bacteroidetes ratio. Different prebiotics have beneficial effects on weight and the related metabolic profile, in some cases even acting on the microbiota with mechanisms that are completely independent from those nutraceuticals for which certain products are normally used. Further studies are necessary to clarify the different levels at which a nutraceutical substance can exert its action.

2021 - The transcription factor NF-Y is required for satellite stem cell myogenic progression and skeletal muscle tissue repair [Poster]
Rigillo, Giovanna; Basile, Valentina; Belluti, Silvia; Imbriano, Carol
abstract

2021 - The transcription factor NF-Y participates to stem cell fate decision and regeneration in adult skeletal muscle [Articolo su rivista]
Rigillo, Giovanna; Basile, Valentina; Belluti, Silvia; Ronzio, Mirko; Sauta, Elisabetta; Ciarrocchi, Alessia; Latella, Lucia; Saclier, Marielle; Molinari, Susanna; Vallarola, Antonio; Messina, Graziella; Mantovani, Roberto; Dolfini, Diletta; Imbriano, Carol
abstract

2020 - Transcription Factors in Cancer: When Alternative Splicing Determines Opposite Cell Fates [Articolo su rivista]
Belluti, Silvia; Rigillo, Giovanna; Imbriano, Carol
abstract

Alternative splicing (AS) is a finely regulated mechanism for transcriptome and proteome diversification in eukaryotic cells. Correct balance between AS isoforms takes part in molecular mechanisms that properly define spatiotemporal and tissue specific transcriptional programs in physiological conditions. However, several diseases are associated to or even caused by AS alterations. In particular, multiple AS changes occur in cancer cells and sustain the oncogenic transcriptional program. Transcription factors (TFs) represent a key class of proteins that control gene expression by direct binding to DNA regulatory elements. AS events can generate cancer-associated TF isoforms with altered activity, leading to sustained proliferative signaling, differentiation block and apoptosis resistance, all well-known hallmarks of cancer. In this review, we focus on how AS can produce TFs isoforms with opposite transcriptional activities or antagonistic functions that severely impact on cancer biology. This summary points the attention to the relevance of the analysis of TFs splice variants in cancer, which can allow patients stratification despite the presence of interindividual genetic heterogeneity. Recurrent TFs variants that give advantage to specific cancer types not only open the opportunity to use AS transcripts as clinical biomarkers but also guide the development of new anti-cancer strategies in personalized medicine.

2019 - Cannabidiol-enriched Cannabis sativa L. extract modulates inflammatory-induced human peripheral mononuclear cells response [Abstract in Atti di Convegno]
Rigillo, G; Borgonetti, V; Benatti, C; Governa, P; Tascedda, F; Biagi, M
abstract

2019 - Potent Anti-Cancer Properties of Phthalimide-Based Curcumin Derivatives on Prostate Tumor Cells [Articolo su rivista]
Belluti, Silvia; Orteca, Giulia; Semeghini, Valentina; Rigillo, Giovanna; Parenti, Francesca; Ferrari, Erika; Imbriano, Carol
abstract

Metastatic castration-resistant prostate cancer is commonly treated with chemotherapy, whose effect is less than satisfactory. This raised the need for novel agents for the treatment of prostate cancer. In the present study, five phthalimide-based curcumin derivatives were synthesized and completely characterized to assess improved stability, pharmacodynamics, and radical scavenging ability. To investigate the potential application in anti-cancer therapy, the anti-proliferative activity of the synthesized molecules was determined on aggressive prostate tumor cells. We demonstrated that the K3F21 derivative has increased potency compared to curcumin, in terms of GI50, anti-proliferative and anti-migrating activities. K3F21 inhibits anchorage-dependent and -independent growth of prostate cancer cells by altering the expression of key genes controlling cell proliferation, such as Cylins D1, B1 and B2, and apoptosis, among which Puma, Noxa, and Bcl-2 family members. Finally, the anti-cancer activity of K3F21 was demonstrated by the analysis of cancer-associated PI3K/AKT, ERK, and p38 signaling pathways.

2019 - Scouting Sigma Receptor Ligands As New Tools For The Treatment Of Neurodegenerative Diseases And Cancer [Relazione in Atti di Convegno]
Sorbi, C.; Linciano, P.; Tait, A.; Atene, C. G.; Guglielmo, L.; Di Rocco, G.; Ronsisvalle, S.; Denora, N.; Imbriano, C.; Rigillo, G.; Fossa, P.; Cichero, E.; Benassi, L.; Vaschieri, C.; Marocchi, F.; Lanfrancone, M. L.; Brasili, L.; Franchini, S.
abstract

Sigma receptors (Rs) are nowadays recognized as an unique class of membrane receptors divided into two subtypes, R and R. Rs regulate a number of physiological functions and their role has been evaluated in many disorders. Deficits in R are associated with neurodegeneration while their activation may represent a valuable strategy for the treatment of a number of neurodegenerative disorders. Moreover, R is overexpressed in a variety of cancer cells and selective R antagonists are reported to modulate cancer cell viability.1 R are also highly expressed proliferating tumors. R agonists are giving promising results in preclinical studies for the treatment of resistant or hardly treatable tumors and R ligands have been proposed as biomarkers for tumors proliferation.2 However, the identification of potent and selective ligands and the comprehension of the chemical features behind agonism/antagonism still remain a primary challenge in this field. With this aim, following a ligand-based approach, a library of over 120 ligands have been designed and synthesized over the years, by combining different substituted five-membered heterocyclic rings with appropriate R pharmacophoric amines. Compounds were tested for R and R affinity showing Ki values in the micromolar / sub-nanomolar range, with a selectivity mainly shifted toward the R. A detailed SAR, supported by molecular modelling, was drawn up. The intrinsic activity was determined in vivo for the most promising molecules. According to their profile, R agonists were tested for neuroprotection, whereas R antagonists / R agonists for anticancer activity. Preliminary results in SH-SY5Y neuroblastoma cells showed the ability of some compounds to protect neuronal cells from death induced by four toxicity models. Cell viability assays were performed on different cancer cell lines to assess the anti-proliferative potential of selected molecules. In particular, dose and time dependent treatments were done on prostate cancer cells, which express higher levels of both R and R compared to normal samples. Similarly, we assessed the effect of the compounds on melanoma cells: BS148, a potent and selective R agonist, showed anti-proliferative activity on immortalized and PDX (metastatic melanoma patient-derived xenografts) cell lines.3 Confocal microscopy studies with BS148 fluorescent probe revealed the internalization of BS148 within melanoma cells, with a cytoplasmatic localization, mostly in the perinuclear region, according to R distribution. Finally, to verify whether TMEM97 / R mediates BS148-antiproliferative activity, we stably overexpressed the TMEM97 gene in HeLa cells: TMEM97-Hela were more sensitive to BS148 anti-proliferative activity compared to control cells, which express endogenous R levels. Taken together, these results support the idea that R is an innovative target in cancer, paving the way for improved tools for cancer diagnosis, monitoring and therapy.

2019 - Switch of NF-YA splice variants in prostate cancer development and progression [Poster]
Belluti, Silvia; Semeghini, Valentina; Dolfini, Diletta; Rigillo, Giovanna; Imbriano, Carol
abstract

The pioneer transcription factor NF-Y is a heterotrimer composed by NF-YA, -YB and -YC subunits: NF-YA is the limiting subunit and harbors the DNA CCAAT-box binding domain. NF-Y is a master transcriptional regulator that ensures proper cell proliferation, controlling cell cycle, DNA replication, apoptosis and metabolism. Recent analyses pointed out that NF-Y binding site is over-represented in promoters of genes overexpressed during the progression from benign epithelium to Prostate Cancer (PC). Moreover, cell cycle regulation genes have emerged as a signature that can discriminate between metastatic and benign prostate tissues. The analysis of TCGA-RNAseq data highlighted a slight but significant increase of NF-YA levels in PC tumor vs normal tissues, particularly in high Gleason score specimens. Since the NF-YA gene encodes for two splice isoforms, NF-YAl (long) and NF-YAs (short), we generated untransformed and cancer prostate cell lines stably over-expressing NF-YA isoforms, to dissect the functional role of NF-YA in PC development and progression. Our data indicate that NF-YAl and NF-YAs could play different activities in cancer-associated processes, such as clonogenic ability, anchorage-independent growth, 3D cell growth, migration and invasion. In particular, in cancer cells NF-YAl improves invasion ability, while NF-YAs seems to enhance the proliferation of tumor spheroids. These results are consistent with increased NF-YAs/NF-YAl splice ratio observed in the progression from normal to malignant prostatectomy samples. Besides, normal prostate cell lines preferentially express the long NF-YA isoform, while an increase in NF-YAs/NF-YAl ratio can be observed in PC cell lines. These results prompt us to speculate that NF-YAl could participate to metastatization, while NF-YAs could have a major role in tumor growth and colonization. Disclosing this dualism could be crucial to better stratify PC patients and identify new specific anti-cancer treatments.

2019 - The transcription factor NF-Y is required for satellite stem cell proliferation and skeletal muscle tissue repair [Poster]
Rigillo, Giovanna; Basile, Valentina; Belluti, Silvia; Imbriano, Carol
abstract

The transcription factor NF-Y, composed by NF-YA, NF-YB and NF-YC subunits, has an important role in the regulation of cellular proliferation and differentiation in different cell types, among which muscle cells. While NF-YA, the DNA binding subunit of NF-Y, is down-regulated in the adult muscle of WT mice, its expression is observed in the mdx mouse, model for Duchenne Muscular Dystrophy, in which a massive regeneration mediated by resident muscle stem cells, namely Satellite Cells (SCs), occurs. With the aim to investigate the role of NF-YA in the SCs proliferation and differentiation, we generated and characterized a conditional knock out mouse model in which NF-YA is deleted in Pax7+ SCs by Tamoxifen induction in adult NF-YAflox/flox:Pax7CreER mice (NF-YA cKO). Cellular and molecular analysis carried out on isolated myofibers and SCs from WT and NF-YA cKO mice highlighted that NF-Y activity is important for the maintenance of SCs homeostasis. NF-YA loss depletes Pax7+ SCs pool and impairs SCs proliferation. Moreover, SCs-mediated regeneration following muscle damage induced by cardiotoxin is delayed in NF-YA cKO. The effect of NF-YA abrogation was also explored in post-natal muscle growth. Immunohistological analysis showed defects in muscle morphology and a decrease in SCs number in 3 weeks aged NF-YA cKO mice, period of major increment of muscle mass  by SCs-mediated myonuclear accretion.  The molecular mechanism underlying the impairment of SCs activity following NF-YA loss was investigated by AdenoCre-induced NF-YA deletion in ex vivo cultured SCs.   Overall, our results highlight a role of NF-Y in muscle regeneration and in SCs fate, whose modulation could be useful to improve stem cell based therapies to treat muscular dystrophies.

2018 - An autoregulatory loop controls the expression of the transcription factor NF-Y [Articolo su rivista]
Belluti, Silvia; Semeghini, Valentina; Basile, Valentina; Rigillo, Giovanna; Salsi, Valentina; Genovese, Filippo; Dolfini, Diletta; Imbriano, Carol
abstract

The heterotrimeric NF-Y complex is a pioneer factor that binds to CCAAT-genes and regulates their transcription. NF-Y cooperates with multiple transcription factors and co-regulators in order to positively or negatively influence gene transcription. The recruitment of NF-Y to CCAAT box is significantly enriched in cancer-associated gene promoters loci and positively correlates with malignancy. NF-Y subunits, in particular the DNA-binding subunit NF-YA and the histone-fold subunit NF-YC, appear overexpressed in specific types of cancer. Here we demonstrate that NF-Y subunits expression is finely regulated through transcriptional and post-translational mechanisms thus allowing control over basal expression levels. NF-Y negatively regulates the transcription of the genes encoding for its subunits. DNA pull-down/affinity purification assay coupled with Mass Spectrometry identified putative co-regulators, such as Lamin A, involved in NF-YA gene transcription level. We also evidentiate how the stability of the complex is severely affected by the absence of one subunit. Our results identified for the first time one of the mechanisms responsible for NF-Y expression, which may be involved in the aberrant expression and activity observed in tumor cells and other pathological conditions.

2018 - Curcumin derivatives and Aβ-fibrillar aggregates: An interactions’ study for diagnostic/therapeutic purposes in neurodegenerative diseases [Articolo su rivista]
Orteca, Giulia; Tavanti, Francesco; Bednarikova, Zuzana; Gazova, Zuzana; Rigillo, Giovanna; Imbriano, Carol; Basile, Valentina; Asti, Mattia; Rigamonti, Luca; Saladini, Monica; Ferrari, Erika; Menziani, Maria Cristina
abstract

Several neurodegenerative diseases, like Alzheimer's (AD), are characterized by amyloid fibrillar deposition of misfolded proteins, and this feature can be exploited for both diagnosis and therapy design. In this paper, structural modifications of curcumin scaffold were examined in order to improve its bioavailability and stability in physiological conditions, as well as its ability to interfere with β-amyloid fibrils and aggregates. The acid-base behaviour of curcumin derivatives, their pharmacokinetic stability in physiological conditions, and in vitro ability to interfere with Aβ fibrils at different incubation time were investigated. The mechanisms governing these phenomena have been studied at atomic level by means of molecular docking and dynamic simulations. Finally, biological activity of selected curcuminoids has been investigated in vitro to evaluate their safety and efficiency in oxidative stress protection on hippocampal HT-22 mouse cells. Two aromatic rings, π-conjugated structure and H-donor/acceptor substituents on the aromatic rings showed to be the sine qua non structural features to provide interaction and disaggregation activity even at very low incubation time (2h). Computational simulations proved that upon binding the ligands modify the conformational dynamics and/or interact with the amyloidogenic region of the protofibril facilitating disaggregation. Significantly, in vitro results on hippocampal cells pointed out protection against glutamate toxicity and safety when administered at low concentrations (1 μM). On the overall, in view of its higher stability in physiological conditions with respect to curcumin, of his rapid binding to fibrillar aggregates and strong depolymerizing activity, phtalimmide derivative K2F21 appeared a good candidate for both AD diagnostic and therapeutic purposes.

2018 - LPS-induced histone H3 phospho(Ser10)-acetylation(Lys14) regulates neuronal and microglial neuroinflammatory response [Articolo su rivista]
Rigillo, Giovanna; Vilella, Antonietta; Benatti, Cristina; Schaeffer, Laurent; Brunello, Nicoletta; Blom, Johanna M. C.; Zoli, Michele; Tascedda, Fabio
abstract

Epigenetic modifications of DNA and histone proteins are emerging as fundamental mechanisms by which neural cells adapt their transcriptional response to environmental cues, such as, immune stimuli or stress. In particular, histone H3 phospho(Ser10)-acetylation(Lys14) (H3S10phK14ac) has been linked to activation of specific gene expression. The purpose of this study was to investigate the role of H3S10phK14ac in a neuroinflammatory condition. Adult male rats received a intraperitoneal injection of lipopolysaccharide (LPS) (830 μg/Kg/i.p., n = 6) or vehicle (saline 1 mL/kg/i.p., n = 6) and were sacrificed 2 or 6 h later. We showed marked region- and time-specific increases in H3S10phK14ac in the hypothalamus and hippocampus, two principal target regions of LPS. These changes were accompanied by a marked transcriptional activation of interleukin (IL) 1β, IL-6, Tumour Necrosis Factor (TNF) α, the inducible nitric oxide synthase (iNOS) and the immediate early gene c-Fos. By means of chromatin immunoprecipitation, we demonstrated an increased region- and time-specific association of H3S10phK14ac with the promoters of IL-6, c-Fos and iNOS genes, suggesting that part of the LPS-induced transcriptional activation of these genes is regulated by H3S10phK14ac. Finally, by means of multiple immunofluorescence approach, we showed that increased H3S10phK14ac is cell type-specific, being neurons and reactive microglia, the principal histological types involved in this response. Present data point to H3S10phK14ac as a principal epigenetic regulator of neural cell response to systemic LPS and underline the importance of distinct time-, region- and cell-specific epigenetic mechanisms that regulate gene transcription to understand the mechanistic complexity of neuroinflammatory response to immune challenges.

2016 - Disease-induced neuroinflammation and depression [Articolo su rivista]
Benatti, Cristina; Blom, Johanna Maria Catharina; Rigillo, Giovanna; Alboni, Silvia; Zizzi, Francesca; Torta, Riccardo; Brunello, Nicoletta; Tascedda, Fabio
abstract

Progression of major depression, a multifactorial disorder with a neuroinflammatory signature, seems to be associated with the disruption of body allostasis. High rates of comorbidity between depression and specific medical disorders, such as, stroke, chronic pain conditions, diabetes mellitus, and human immunodeficiency virus (HIV) infection, have been extensively reported. In this review, we discuss how these medical disorders may predispose an individual to develop depression by examining the impact of these disorders on some hallmarks of neuroinflammation known to be impaired in depressed patients: altered permeability of the blood brain barrier, immune cells infiltration, activated microglia, increased cytokines production, and the role of inflammasomes. In all four pathologies, blood brain barrier integrity was altered, allowing the infiltration of peripheral factors, known to activate resident microglia. Evidence indicated morphological changes in the glial population, increased levels of circulating pro-inflammatory cytokines or increased production of these mediators within the brain, all fundamental in neuroinflammation, for the four medical disorders considered. Moreover, activity of the kynurenine pathway appeared to be enhanced. With respect to the inflammasome NLRP3, a new target whose role in neuroinflammation is emerging as being important, accumulating data suggest its involvement in the pathogenesis of brain injury following stroke, chronic pain conditions, diabetes mellitus or in HIV associated immune impairment. Finally, data gathered over the last 10 years, indicate and confirm that depression, stroke, chronic pain, diabetes, and HIV infection share a combination of underlying molecular, cellular and network mechanisms leading to a general increase in the neuroinflammatory burden for the individual.

2015 - Molluscs as models for translational medicine [Articolo su rivista]
Tascedda, Fabio; Malagoli, Davide; Accorsi, Alice; Rigillo, Giovanna; Blom, Johanna Maria Catharina; Ottaviani, Enzo
abstract

This paper describes the advantages of adopting a molluscan model for studying the biological basis of some central nervous system pathologies affecting humans. In particular, we will focus on the freshwater snail Lymnaea stagnalis, which is already the subject of electrophysiological studies related to learning and memory, as well as ecotoxicological studies. The genome of L. stagnalis has been sequenced and annotated but the gene characterization has not yet been performed. We consider the characterization of the gene networks that play crucial roles in development and functioning of the central nervous system in L. stagnalis, an important scientific development that comparative biologists should pursue. This important effort would add a new experimental model to the limited number of invertebrates already used in studies of translational medicine, the discipline that seeks to improve human health by taking advantage of knowledge collected at the molecular and cellular levels in non-human organisms.

2015 - RESILIENCY OF CHILDREN AND ADOLESCENTS DURING TREATMENT OF ACUTE LYMPHOBLASTIC LEUKEMIA: THE USE OF 5-HTT AND BDNF POLYMORPHISMS AS BIOMARKERS [Abstract in Rivista]
Blom, Johanna Maria Catharina; Pomicino, L; Migliozzi, C; Montanari, L; Rigillo, Giovanna; Zanazzo, G; Cellini, M; Benatti, Cristina; Tascedda, Fabio
abstract

Background/Objectives: Why are some children more likely than others to develop resilience in the face of similar levels of trauma exposure as compared to others who do not. It is increasingly clear that there are critical roles for predisposing genetic and environmental influences in differentially mediating psychological risk. Resilience differs from traditional concepts of risk and protection in its focus on individual variations in response to comparable experiences. Here, we tested the hypothesis that anxiety and depression as well as neural repair and plasticity related polymorphisms may partly account for the difference in resilience observed during treatment for acute lymphoblastic leukemia (ALL). Design/Methods: Forty-five patients (1-18 yrs old) diagnosed with ALL were enrolled in two centers (protocol AIEOP-BFM-2009) and genotyped for 5HTT and BDNF (val66met). Patients and their family were subjected to a short screening battery, psychosocial testing (PAT2.1) and a specific assessment of their resiliency during treatment. The resiliency scale was composed of three subscales: Sense of Mastery (MAS), Sense of Relatedness (REL), and Emotional Reactivity (REA). Results: Patients with the SL allele of 5HTTLPR had a more compromised score in some areas of resiliency than patients with the LL allele; the presence of the S allele most affected emotional reactivity REA and sense of mastery MAS. Furthermore, age was an important factor, as younger children displayed a reduced trust and tolerance versus their surroundings. This then contributed importantly to an overall reduction in their overall resiliency. Also, resiliency was reduced one year into therapy while vulnerability was significantly enhanced. Conclusion: Genes regulating susceptibility to stress, such as 5HTTLPR and BDNF, may help to predict susceptibility towards the development of resiliency in children and adolescents treated for cancer, and may play a critical role as a predisposing factor in differentially dealing efficiently with the emotional risks related to cancer and its treatment.

2014 - Interleukin 18 activates MAPKs and STAT3 but not NF-κB in hippocampal HT-22 cells [Articolo su rivista]
Alboni, Silvia; Montanari, C; Benatti, Cristina; Sanchez Alavez, M; Rigillo, Giovanna; Blom, Johanna Maria Catharina; Brunello, Nicoletta; Conti, B; Pariante, Mc; Tascedda, Fabio
abstract

Interleukin (IL)-18 is a cytokine previously demonstrated to participate in neuroinflammatory processes. Since the components of the IL-18 receptor complex are expressed in neurons throughout the brain, IL-18 is also believed to directly influence neuronal function. Here we tested this hypothesis on mouse hippocampal neurons by measuring the effects of IL-18 on three pathways previously shown to be regulated by this cytokine in non-neuronal cells: the MAPK pathways, p38 and ERK1/2 MAPKs, STAT3 and NF-κB. Experiments were carried out in vitro using the immortalized hippocampal neuronal line HT-22 or in vivo following i.c.v. injection with recombinant mouse IL-18. We showed that IL-18 did not activate NF-κB in HT-22 cells whereas it induced a rapid (within 15min) activation of the MAPK pathways. Moreover, we demonstrated that IL-18 treatment enhanced P-STAT3 (Tyr705)/STAT3 ratio in the nucleus of HT-22 cells after 30-60min of exposure. A similar increase in P-STAT3 (Tyr705)/STAT3 ratio was observed in the whole hippocampus one hour after i.c.v. injection. These data demonstrate that IL-18 can act directly on neuronal cells affecting the STAT3 pathway; therefore, possibly regulating the expression of specific genes within the hippocampus. This effect may help to explain some of the IL-18-induced effects on synaptic plasticity and functionality within the hippocampal system.

2013 - Epigenetic modification in neurons of the mollusc Pomacea canaliculata after immune challenge [Articolo su rivista]
Ottaviani, Enzo; Accorsi, Alice; Rigillo, Giovanna; Malagoli, Davide; Joan M. C., Blom; Tascedda, Fabio
abstract

In human and rodents, the transcriptional response of neurons to stress is related to epigenetic modifications of both DNA and histone proteins. To assess the suitability of simple invertebrate models in studying the basic mechanisms of stress-related epigenetic modifications, we analyzed epigenetic modifications in neurons of the freshwater snail Pomacea canaliculata after the injection of Escherichia coli-derived lipopolysaccharide (LPS). The phospho-acetylation of histone H3, together with the induction of stress-related factors, c-Fos and HSP70, were evaluated in large and small neurons of the pedal ganglia of sham- and LPS-injected snails. Immunocytochemical investigations showed that after LPS injection, the immunopositivity towards phospho (Ser10)-acetyl (Lys14)-histone H3 and c-Fos increases in the nuclei of small gangliar neurons. Western blot analysis confirmed a significant increase of phospho (Ser10)-acetyl (Lys14)-histone H3 in nuclear extracts from 2h LPS-injected animals. c-Fos protein levels were significantly augmented 6h after LPS injection. Immunocytochemistry and western blot indicated that no changes occurred in HSP70 distribution and protein levels. To our knowledge this is the first demonstration of epigenetic changes in molluscan neurons after an immune challenge and indicate the gastropod P. canaliculata as a suitable model for evolutionary and translational studies on stress-related epigenetic modifications.

2013 - The injection of LPS induces epigenetic changes in Pomacea canaliculata neurons. XIII Meeting of the Italian Association of Developmental and Comparative Immunology, Palermo, Inv. Surv. J., 10: 16, 2013 [Abstract in Rivista]
Accorsi, Alice; Rigillo, Giovanna; Malagoli, Davide; Blom, Johanna Maria Catharina; Tascedda, Fabio; Ottaviani, Enzo
abstract

The injection of LPS induces epigenetic changes in Pomacea canaliculata neurons