Prin PNRR

Dissecting CircadiAn Reprogramming in a model of hEart failure with preserved ejection fraction (HFpEF) (CARE)

Finanziatore: MUR
Responsabile Scientifico: BELLET Marina Maria
Finanziamento:€ 58.555
Durata: 2023-2025

Summary:Cardiovascular diseases (CVDs) are the leading cause of death globally, taking an estimated 17.9 million lives each year. Among CVDs, heart failure with preserved ejection fraction (HFpEF) is a public health problem with only limited cures. The development of new treatments is challenged by the complexity of the disease. Indeed, it is becoming clear that HFpEF is a multiorgan clinical syndrome with several tissue dysfunction contributing to its pathogenesis. Intriguingly, patients suffering from HFpEF display impairment in cognitive performance and an increased risk of developing dementia and neurodegenerative diseases (e.g., Alzheimer disease). Furthermore, alterations of intestinal morphology, permeability and absorption are present in the same patients, reinforcing the concept of HFpEF as a multiorgan syndrome. Despite this evidence, why and how HFpEF influences brain and intestinal functions is not understood. Our project aims at exploring novel pathophysiological mechanisms of HFpEF considering the inter-organ crosstalk between heart-brain and heart-intestine, and the concept of systemic circadian rhythms misalignment caused by the heart pathological condition. The circadian clock is an endogenous time-keeping system encompassing virtually every aspect of human physiology. At organismal level, an integrated network of tissue-specific endogenous clocks ensures a tightly coordinated diurnal physiology across the whole body, individual adaptation to the external environment and survival. Circadian misalignment has been linked to a variety of diseases, ranging from metabolic syndrome to neuropsychiatric disorders and cancer. We hypothesize that in the pathological setting of HFpEF, signaling from the heart rewires circadian physiology of brain and intestine, negatively affecting their function, further worsening the patients’ quality of life. To verify our hypothesis, we will exploit a mouse model of cardiometabolic HFpEF that consists of feeding adult mice with a high fat diet and the NOS inhibitor L-NAME. First, to explore clock function we will perform RNA-seq analysis at 6 time-points along the daily cycle in the left ventricle, cerebral cortex, and small intestine. Then, to investigate the physiological relevance of diurnal transcriptional reprogramming in HFpEF, we will perform global untargeted metabolomics of the same tissues and serum. Moreover, functional assessment of brain and intestinal function will be performed at two circadian times.

Novel cell and gene therapy approach on patient-specific endothelial cells and iPSCs-derived megakaryocytes to correct the dysfunctional release of lysosome-related organelles in Hermansky-Pudlak syndrome

Acronyme: BARRIO
Finanziatore: MUR
Responsabile Scientifico: BURY Loredana
Finanziamento: €112.900
Durata: 2023-2025

Summary:Hermansky-Pudlak syndrome (HPS) is an inherited multisystem disorder caused by a defect of lysosome-related organelles (LRO) invarious tissues, like delta granules in platelets, melanosomes in melanocytes and Weibel Palade Bodies (WPB) in endothelial cells (ECs).HPS is characterized by oculocutaneous albinism with nystagmus and visual acuity loss, bleeding diathesis and, in some cases, extra-hematological complications such as pulmonary fibrosis, granulomatous colitis and immunodeficiency. HPS is caused by genetic variants in 11 different genes, all coding for proteins playing a role in the biogenesis of lysosome-related organelles. The most severe subtype of HPS is HPS-1, in which variants affect the HPS1 protein that interacts with HPS4 to compose the BLOC‐3 complex. Treatment options for inherited platelet disorders (IPD), including HPS, are rather limited, unspecific, and frequently ineffective, therefore further studies are required to define the mechanism of the disease and identify an effective treatment. The mechanisms involved in the pathogenesis of HPS have already been investigated in melanosomes, alveolar epithelial cells and platelet delta granules but little is known about Weibel Palade Bodies (WPB) secretion by ECs. Recently, endothelial colony forming cells (ECFC) have been isolated from an HPS-2 patient, and in parallel with CRISPR-Cas9-engineered ECs, have been used as a cellular model to study the mechanism of maturation of WPBs and their exocytosis in HSP-2 suggesting a link between impaired endothelial cell function and bleedings. However, little is known about the maturation and storage of WPBs in HPS-1. Moreover, induced pluripotent stem cells (iPSCs) have been generated from the reprogramming of somatic cells of HSP-1 patients, but they were not differentiated in any relevant cell to be used in HSP. Therefore, this project aims to shed new light on the pathogenesis of HPS-1 and to develop for the first time a cell and gene approach to correct genetic variants causing HPS-1. Hence, we first aim to investigate the endothelial function and WPB secretion of ECFCs isolated from an HPS-1 patient. Secondly, we will generate iPSCs from an HPS-1 patient, and differentiate them into megakaryocytes and subsequently obtain platelets, to study dense granule content and release. Finally, we will develop a cell and gene therapy strategy, based on the use of lentiviral vectors carrying the HSP1 gene under the control of an endothelial or a megakaryocyte promoter, to correct the genetic defect in HPS-1 ECFCs and iPSCs-derived megakaryocytes. The findings deriving from this project will expand knowledge on a very rare inherited disorder, moreover, the concept that the bleeding phenotype of patients with HPS may be due not only to the well-known platelet defect but also to a defect in endothelial cells is very pioneering and may change the therapeutic approach to this disorder.

Feasibility of InnovaTive approaches for personalized cardiovascular PREVention: randomized controlled pilot trial and multidisciplinary evaluation for National Health Service implementation (FItPreV)

Finanziatore: MUR
Responsabile Scientifico: DE WAURE Chiara
Finanziamento: €85.000
Durata: 2023-2025

Summary:Cardiovascular diseases (CVDs) contribute to the largest burden of mortality and morbidity in Europe and worldwide, and they mainly result from an interaction between lifestyle and genetic determinants. It has been suggested that genetic influence derives from a combined effect of many genetic variants, which can be assessed by computing polygenic risk scores (PRS) to estimate the risk of developing CVDs and eventually to inform personalised prevention strategies. Personalisation drives innovation in health research and development in health systems with limited resources and under pressure due to an ageing population and increasing health needs. To date, no studies have yet evaluated the value of PRS risk prediction alone or in combination with digital technologies to promote lifestyle changes aiming at CVD risk reduction. FItPreV (Feasibility of InnovaTive approaches for personalized cardiovascular PREVention: randomized controlled pilot trial and multidisciplinary evaluation for National Health Service implementation) aims to provide preliminary evidence and guidance to the Italian national health service for the take-up of innovative approaches for personalised primary preventive interventions for CVD. The primary objective of the project is to develop and carry out a feasibility study to address a number of relevant topics around the use of genetic tests and innovative digital tools to encourage people to modify their lifestyles for primary prevention of CVDs. For this purpose, we will set up a pilot trial including 200 participants between 40 and 69 years at a high 10-year risk profile of CVD (2.5-10% risk of the SCORE 2 chart). Enrolled individuals will fill out a questionnaire on socioeconomic status, smoking status, alcohol consumption, dietary and sleep patterns, and physical activity that will define the individual lifestyle pattern. Participants will be randomized to four interventions: 1)traditional risk assessment at baseline; 2) intervention 1 plus dedicated app and wearable device to monitor lifestyle; 3) intervention 1 plus receiving baseline CVD PRS; 4) intervention 1 plus dedicated app and wearable device and baseline CVD PRS. The primary outcome is the evaluation of the feasibility of a larger, more definitive study on innovative approaches for personalised cardiovascular prevention. In this light, the results of the pilot study will be complemented by a multidisciplinary assessment of the potential implementation of innovative approaches for personalised cardiovascular prevention that will take into account the individuals’ preferences, values and facilitators and barriers for the scalability. Two partners (UCSC and UNIPG), with documented expertise in personalized medicine and assessment of health technologies and health systems, will be involved in the project.

Bromodomain-containing protein 4 as a novel positive regulator of gut inflammation and immune target for innovative therapeutics in inflammatory bowel diseases

Finanziatore: MUR
Responsabile Scientifico: Francesca FALLARINO
Finanziamento: € 78.000
Durata: 2023-2025

Summary: Crohn's disease (CD) and ulcerative colitis (UC), the major inflammatory bowel diseases (IBD) in human beings, are lifelong progressive disorders characterized by chronic, recurring inflammation in the gastrointestinal system, which can promote the development of local and extra-intestinal complications thus negatively influencing the quality of life of the patients. The etiology of both CD and UC remains unknown and, therefore, there exists no curative therapy. Recent advances in our understanding the basic mechanisms that induce and sustain IBD-associated tissue damage have facilitated the development of innovative drugs (e.g. antibodies targeting inflammatory cytokines/integrins), which have improved the way we manage such patients. Unfortunately, nearly half of IBD patients are primarily or secondarily resistant to such drugs and some of them develop drug-related adverse events, which lead to the therapy discontinuation. The identification of novel targets for therapeutic purposes is thus worth pursuing. To identify detrimental pathways acting in the gut of IBD patients, we evaluated the expression of bromodomain-containing protein 4 (BRD4) in intestinal biopsies taken from patients with CD, patients with UC, and normal controls. BRD4 is a transcriptional and epigenetic regulator of cell proliferation and cytokine production and studies in other systems have shown that BRD4 can control intracellular pathways that promote detrimental inflammation. BRD4 over-expression was seen in both epithelial cells and lamina propria mononuclear cells (LPMC) of IBD samples as compared to controls, and knockdown of BRD4 in IBD LPMC reduced expression of various inflammatory cytokines, which are supposed to make a valid contribution to IBD-associated mucosal injury. Based on these data, we hypothesize that BRD4 may control multiple inflammatory pathways thereby contributing to the IBD-associated pathological process. By using multiple experimental models of IBD and multi-disciplinary approaches, we aim at examining the role of BRD4 in the control of gut inflammation and ascertain whether BRD4 is a valid target for therapeutic purposes in IBD. We plan to assess BRD4 levels in other cohorts of IBD patients and evaluate whether the enhanced expression of BRD4 is a specific hallmark of IBD and which basic mechanisms account for such an induction. We plan to assess whether BRD4 is a useful marker of disease phenotype/course in IBD. Next, by using RNA-sequence and metabolomic data, we will ascertain how and which cellular pathways are under the control of BRD4 in IBD. Finally, by using several mouse models of IBD-like colitis, we will evaluate whether targeting BRD4 in colitic mice helps prevent and/or cure gut inflammation. Results from the proposed studies will contribute to advancing our understanding the mechanisms underlying IBD-associated tissue damage, thereby bringing important rewards in the therapy of patients with such disorders.

Targeting Leukaemia Inhibitory Factor and Bile Acid Receptors by synthetic small molecules as a new armamentarium in the treatment of pancreatic ductal adenocarcinoma.e

Finanziatore: MUR
Responsabile Scientifico: FIORUCCI Stefano
Finanziamento: € 53.768
Durata: 2023-2025

Summary:Pancreatic ductal adenocarcinoma (PDAC) is projected to become one of the principal causes of cancer death worldwide by 2030. PDAC is a multifactorial disease, driven by multiple environmental factors, including excessive alcohol consuming, obesity, diabetes mellitus and chronic pancreatitis. Unfortunately, PDAC patients have a poor prognosis, making the identification of novel molecular biomarkers and therapeutic targets an urgent need. Recently, the Leukaemia Inhibitory Factor (LIF) has been identified as one of these potential biomarkers and it is also a potential target for a pharmacological-driven intervention. LIF is a pleiotropic cytokine that belongs to the Interleukin (IL)-6 family, and it is overexpressed in a variety of solid tumors including pancreas and stomach and promotes cancer cell proliferation and invasiveness. In target tissues, LIF signaling is mediated by its binding to a heterodimeric receptor complex formed by the LIF-receptor (LIFR) and the glycoprotein (gp) 130 subunit of the IL-6 receptor. To test our multidisciplinary platform encompassing molecular docking, cell-free biophysical and cellular investigations focusing on LIF/LIFR axis, we have performed a similarity in-silico screening of the FDA approved drug database identifying mifepristone (also known as RU-486) as a potential LIFR antagonist. The in-silico strategy deepened the binding mode between the counterparts and it was followed by alpha screen assays to measure the binding affinity and the results were validated by studies on pancreatic cancer cell lines. Our data demonstrate that LIF promotes cancer cells proliferation and migration in a JAK-STAT3 dependent manner and, mainly, that these features were reversed by mifepristone (1). From a chemical point of view, mifepristone is a steroidal molecule and, thus, we speculated if our wide library of in-house steroidal molecules, generated harnessing the bile acid scaffold and thus activating Bile Acid Receptors (BARs) such as GPBAR1 and FXR, could also target LIF/LIFR axis. Among these, BAR502, a bile alcohol identified by our research groups as a potent dual GPBAR1 and FXR agonist and recently advanced in phase 1 clinical trials, has been proved as a LIF/LIFR antagonist (data not published). Since in recent years, our research groups have generated a library of BARs modulators with the identification of diverse scaffolds endowed with selective or dual activities in term of agonism or antagonism toward FXR and GPBAR1, the recent promising results for mifepristone, BAR502 and isoxazole-based derivatives let us speculate about the opportunity to enlarge our in-house library opening the way to the investigation of the therapeutic potential of synthetic small molecules interacting with LIF/LIFR axis and with thedebated pathways activated by endogenous bile acids in cancers.

Clonal evolution in newly defined high-risk acute myeloid leukemia and role of leukemic stem cells as disease reservoir

Finanziatore: MUR
Responsabile Scientifico: MARTELLI Maria Paola
Finanziamento:€ 118.500
Durata: 2023-2025

Summary:In the era of precision medicine, the massive use of molecular biology techniques unveiled a plethora of genomic alterations underpinning the clinical-biological heterogeneity of acute myeloid leukemia (AML), leading to the discovery of novel targeted therapies. Generally, AML is driven by the sequential accumulation of acquired genetic alterations, either cytogenetic abnormalities or gene mutations, occurring in hematopoietic precursors. A currently accepted paradigm posits that a small population of leukemic stem/progenitor cells (LSPCs), also known as leukemia-initiating cells, represents the disease reservoir. LSPCs are heterogeneous in terms of phenotype, gene expression and metabolism, are frequently resistant to treatment, and are ultimately responsible for leukemia relapse. According to the most recent 2022 EuropeanLeukemiaNet (ELN) recommendations and the International Consensus Classification (ICC) of myeloid neoplasms, mutations involving either TP53 or the following genes, i.e. ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2, define two new disease entities characterized by high-risk (HR) features and adverse prognosis. These newly defined HR-AML entities represent an urgent unmet medical need because of the high rate of failure to achieve complete remission and dismal outcomes, with only less than 20% of patients becoming long-term survivors. The present proposal aims at deciphering the genetic landscape and immunophenotypic profile of these newly defined HR-AML subtypes at a single-cell level, by studying clonal evolution and dynamics during the disease course to identify potential therapeutic targets. Specifically, HR-AML, as defined above, will be identified and characterized by deep targeted next generation DNA sequencing (NGS) according to the most modern diagnostic procedures. The tracking of clonal evolution dynamics by repeated NGS over the disease course upon strategic time points (diagnosis, complete remission, disease relapse) will enable a precise characterization of disease dynamics in multiple real-life scenarios of patients undergoing different therapeutics (intensive chemotherapy, CPX-351, or combination of azacitidine/venetoclax). Viable cells from bone marrow aspirates will be cryopreserved at each time point for deeper single-cell DNA sequencing and immunophenotype analyses, in order to clarify the clonal hierarchy of HR-AML and unveil the proteogenomic features of their LSPCs in a prospective proof-of-concept non-interventional study. Our approach will shed light onto the stepwise process of leukemogenesis and clonal evolution, including LSPCs behavior, under a variety of currently used treatment for these HR-AML disease entities. This study will generate data to support larger cooperative efforts aimed at re-defining management and improve treatment and prognosis of such dismal AML categories.

AMICI-SjS Analysis of Microbes, Immune and Clinical Insights in Sjögren’s Syndrome

Finanziatore: MUR
Responsabile Scientifico: PERRICONE Carlo
Finanziamento: 134.945
Durata: 2023-2025

Summary:Primary Sjögren’s Syndrome (SjS) is a systemic autoimmune disease that affects 1–23 persons per 10 000 inhabitants in European countries. The disease is characterized by a wide spectrum of clinical manifestations, dominated by sicca syndrome caused by immune-mediated glandular involvement. Fatigue, musculoskeletal pain, and systemic involvement can occur, lymphoma may affect up to 5% of patients. Antinuclear antibodies are the most frequently detected autoantibodies, anti-Ro/SSA the most specific, while cryoglobulins and hypocomplementaemia are the main prognostic markers. The histological hallmark is a focal infiltration of the exocrine glands by lymphocytes, that can be observed at minor labial salivary gland biopsy. SjS represents a significant social burden having a mean annual total direct cost per patient ranging between £2200 in UK and US$20 000 in the USA. Several microorganisms, including pathogens, pathobionts, symbionts and commensals are able to damage tissue(s) and to trigger different types of immune responses in a balance between elimination and control, in certain cases resulting in the breakdown of tolerance. Infection persistence, molecular mimicry, bystander activation, self-antigens release, exceeding antigen presentation and superantigen presentation, each contribute to infection-triggered immune imbalance. Many associations between infections and autoimmune and non-autoimmune disorders have been described, although proven evidence is often lacking. The literature about the microbial species in the oral cavity of SjS patients shows variable results in quantity and proportions, depending on changes in the pH, buffering capacity, protein, immunological profiles of the saliva, retention of food debris and scarce periodontal health. SjS patients seem to be less affected in the subgingival area, possibly since gingival crevicular fluid rather than saliva provides the bulk fluid to the area. The present project aims at defining not only the associated oral/gingival microbiome to SjS pathogenesis, but also to deepen the possible mechanism through which specific microbes specifically contribute to disease initiation/progression. Moreover, we intend to investigate the balance between immune and microbial, through the immune-histological characterization of the lesions and their potential correlation with colonizing species, aiming at the definition of the impact of the balance between immune response and microbes

Targeting NPM1-mutated Acute Myeloid Leukemia by a dual targeting strategy of Chimeric Antigen Receptor (CAR) engineered cells

Finanziatore: MUR
Responsabile Scientifico: PERRIELLO Vincenzo
Finanziamento: € 130.000
Durata: 2023-2025

Summary:Nucleophosmin (NPM1)-mutated Acute Myeloid Leukemia (AML) is one of the most common acute leukemia in adults and shows unique clinical, molecular and pathological features. Although the prognosis of NPM1-mutated AML is relatively favorable in the absence of FLT3-ITD, still >50% of patients die of the disease. Adoptive cell therapy by chimeric antigen receptor (CAR)-engineered T cells demonstrated a high therapeutic potential in B-ALL and B-NHL, but translation in AML is limited by the absence of an ideal tumor-specific antigen. NPM1-mutated AML may serve as a model for developing CAR-T cells with restricted selectivity since the expression of NPM1-mutant protein is AML-specific and its ectopic cytoplasmic delocalization in AML cells generates a leukaemia-specific neo-epitope presented on cell surface by the HLA-A°0201 allele. Although the NPM1mutant-HLA complex is potentially druggable by anti-NPM1mut/HLA-A2 CAR-T cells, derived from a novel monoclonal antibody (mAb) developed at Massachusetts Institute of Technology (MIT), this approach may be inadequate to trigger full T cell stimulation because of the low density of NPM1 neoepitopes. The aim of this proposal is to overcome this issue through the development of a dual-targeting strategy to enhance CAR-T cell activity, while preserving selectivity against HLA-A2(+) NPM1mut AML. The research unit of Perugia will validate this approach by constructing dual CAR-T cells co-expressing the anti-NPM1mut/HLA-A2 CAR with an anti-CD123 co-stimulatory receptor (CCR) without signalling domains, in order to boost T cell activation. Notably, the absence of activation signals in the anti-CD123 CCR should avoid any potential on-target off-tumor effects against CD123+ normal endothelial and hematopoietic stem cells. CD123 was chosen to enhance CAR-affinity towards leukemic cells since CD123 is characteristically overexpressed in NPM1-mutated AML, including LSCs. Once validated in lentivirally transduced CAR-T cells, the research unit of Bergamo will clone CAR-sequences in transposon plasmids to generate genetically modified CAR-Citokine Induced Killer (CIK) cells. The Bergamo unit long-standing experience on optimized clinical-grade platform for CIK cell differentiation, expansion and non-viral transduction will offer the opportunity of an efficient preclinical in vitro and in vivo comparison between dual anti-NPM1mut-HLA-A2 complex/CD123 CAR -CIK and -T cells. In the meanwhile, the research unit of Naples will dissect by flow-cytometry the co-expression of NPM1mut/HLA-A2 complex and CD123 on bulk and stem cell leukaemic population from NPM1-mutated AML patients, exploiting the mAb available from MIT. Based on these preclinical results, we envision to proceed into a clinical trial in HLA-A2+ patients with r/r NPM1-mutated AML./p>

Pharmacogenitics of Lenvatinib in advanced thyroid cancer patients: correlation with adverse events and clinical outcome)

Finanziatore: MUR
Responsabile Scientifico: PUXEDDU Efisio
Finanziamento: € 53.500
Durata: 2023-2025

Thyroid cancer is the most common malignant tumor of the endocrine system. These tumors frequently have genetic alterations leading to the activation of the mitogen-activated protein kinase (MAPK) signalling pathway, responsible for uncontrolled cell proliferation. Nowadays, tyrosine kinase inhibitors (TKIs) represent the best therapeutic option in advanced thyroid cancer patients for their ability to inhibit tyrosine kinase receptors or other kinases involved in cell growth and tumoral transformation. The therapeutic efficacy and toxicity of TKIs are very heterogeneous and are difficult to predict before starting treatment. In vitro studies evaluated the pharmacokinetics of these TKIs that are mainly metabolized in the liver, by cytochrome P450 (CYP). These enzymes are associated with drug bioavailability by regulating the mechanism of absorption and elimination of the drug. Recent studies demonstrated that there was a large inter-individual pharmacokinetic variability among patients treated with TKIs and that TKIs steady-state was associated with the genetic polymorphisms in genes encoding for metabolism enzymes or transporters. In addition, the rate of development of adverse events during the treatment as well as the drug efficacy, are significantly associated with these genetic polymorphisms in different human tumours. Given the lack of data, additional studies evaluating the possible correlation between polymorphisms in genes encoding metabolism enzymes or transporters, TKIs related adverse events and efficacy, are needed on thyroid cancer patients. Identifying the effects of polymorphisms in genes encoding metabolism enzymes or transporters could help to optimize the therapeutic management strategy and maximize the clinical benefits of TKI treatment in advanced thyroid cancer patients. This project aims at dissecting the complex interplay between lenvatinib side effects, SNPs in genes associated with drug bioavailability, plasma dose of the drug and microbioma alteration by: 1) Retrospectively and prospectively correlating clinical features with the genomic landscape (WP1, WP2, WP3); 2) Prospectively correlating plasma drug concentrations with lenvatinib's adverse events and SNPs (WP4); 3) Investigating the presence of an altered microbiota composition in enrolled patients that experienced more severe diarrhea and eventually correlate the results with those obtained in WP3 and WP4 (WP5); 4) Optimize the therapeutic management strategy and maximize the clinical benefits of TKI treatment in advanced thyroid cancer patients (data management, WP6). Altogether, this project will propose several innovations, by linking drug treatment, disease phenotypes, and potential molecular targets, which could be widely exploited in diagnostic, therapeutic, preventive and health-economic applications.

Fighting WESTERN diet-derived AGEs (advanced glycation end products) with natural compounds to mitigate muscle wasting in sarcobesity (WESTERNAGE).

Finanziatore: MUR
Responsabile Scientifico: RIUZZI Francesca
Finanziamento: € 141.104
Durata: 2023-2025

Summary:Skeletal muscle has a pivotal role in the maintenance of whole-body metabolism, and the loss of muscle mass and functionality (muscle wasting; MW) predisposes to several diseases. MW is a common feature of noncommunicable chronic diseases (NCDs), such as obesity, diabetes, and sarcopenia, contributing to their deleterious outcomes. Sarcobesity, i.e., MW coincident with increased fat tissue, is a growing, urgent and poorly understood complex syndrome with detrimental consequences and elevated healthcare costs. The nutritional transition to a "Western diet" (WD) increases the development of insulin resistance and metabolic inflexibility predisposing to MW and sarcobesity by sustaining systemic/local inflammation and oxidative stress. WD foods contain high advanced glycation end-products (AGEs), a group of glycosylated adducts also endogenously formed. AGE accumulation over time alters the function of tissue cross-linked proteins and sustains oxidative stress and inflammation via the receptor RAGE. Indeed, AGEs/RAGE interactions have been implicated in multiple metabolic disorders, including obesity, insulin resistance, and diabetes. In diabetic and geriatric patients, accumulation of AGEs in skeletal muscle, blood, and skin, has been associated with sarcopenia, and RAGE signaling induces MW in several conditions. The mechanisms underlying WD-dependent MW, including the potential role of dietary AGEs (dAGEs)/RAGE axis have not been investigated so far. We hypothesize that high dAGEs might be mediators of WD-dependent MW and contribute to the onset and progression of sarcobesity, predisposing to earlier and more severe metabolic consequences, including type 2 diabetes (T2D). By using in vitro and in vivo preclinical models, and an observational study in patients with obesity and T2D, our project aims to: i) discover and dissect the role of dAGEs/RAGE axis in WD-induced MW; ii) unravel genes, pathways and metabolomic signature affected by WD, thus identifying therapeutic targets for sarcobesity; iii) test anti-dAGEs natural compounds as an approach to maintain muscle mass and functionality, and reduce the risk of comorbidities associated to WD intake; iv) correlate the level of dietary and endogenous AGEs with the severity of sarcopenia; v) identify dAGEs as potential reliable biomarkers of MW risk in patients with obesity and T2D, with high clinical relevance in prevention and early screening of sarcobesity. The synergy between basic science and clinical research will allow the validation in patients of the main findings obtained in preclinical models conferring to the project high translational potential and future socio-economic impact. The already ongoing collaboration between the research units and the consistent collection of preliminary results ensure the project feasibility that will accelerate the identification of strategies to mitigate the detrimental effects of WD and related NCDs in line with the PNRR objectives and WHO agenda goals.

Neuronal and synaptic dysfunctions caused by TCF20 intellectual disability gene.

Finanziatore: MUR
Responsabile Scientifico: TOZZI Alessandro
Finanziamento: € 102.000
Durata: 2023-2025

Summary:Among several gene mutations identified as high-risk factors for the pathogenesis of neurodevelopmental disorders, de novo mutations of TCF20 (transcription factor 20) were identified in large-scale exome sequencing data of intellectual disability and autism patients. TCF20 encodes a transcriptional coregulator, identified by its ability to bind the stromelysin-1 PDGF-responsive element, an element of the stromelysin-1 (matrix metalloproteinase-3/MMP3) promoter. TCF20 is localized to the nucleus and was found to play a role as transcriptional coactivator in modulating the transcriptional activity of Sp1, c-Jun, Est1, Pax6 and RNF4. TCF20 is highly expressed in several brain areas, involved in high cognitive functions including, cortex hippocampus and cerebellum. An increasing number of patients with TCF20 mutations, including de novo and inherited variants have been recently reported. For these patients, the described clinical features include mild-to moderate intellectual disability with or without ASD and accompanying features such as proportionate overgrowth and muscular hypotonia. The function of TCF20 in the brain development and physiology remains to be clarified. The proposed study will define the role of TCF20 in neuronal and synaptic differentiation. The project will use dual strategy: i) the production and employment of the new animal model in which TCF20 will be deleted by injecting an AAV vector expressing shTCF20 in mice mPFC or hippocampus II) the genereation and the characterization of a model obtained by rodent and human neurons knocked down for TCF20 that will be employed for a detailed characterization in vitro. Mice knocked down for TCF20 will by characterized using complimentary molecular, cell biology and electrophysiological approaches of investigation. Like that of most genetic neurodevelopmental disorders, the treatment of patients is based on symptom management, whereas the aim of this project is to boost or modulate BDNF expression in order to design new strategic therapies aimed at forestalling the neuropathology and behavioral deficits associated with TCF20 deletion or mutation. A first in vitro screening will be used to test the effect of BDNF re-expression and the effect of treatment with AMPAR positive allosteric modulator Tulrampator. If successful, these protocols will be tested in vitro for their potential to rescue neuron dysmorphology, altered synaptic activity, and behavioural symptoms. Improvement of cellular and behavioural phenotype would have a profound impact on our understanding of the role of TCF20 in neurons and would represent a preclinical step toward the development of potentially specific and effective therapies to ameliorate symptoms caused by TCF20 disruption. Finally, trought the transcriptomic analysis of human neurons knocked down for TCF20 we expect to identify new possible therapeutic target to develop treatments for the increasing identified number of patients with TCF20 mutations.