Roberta Misasi

The endocannabinoid system is a neuromodulatory network regulating synaptic plasticity, neuronal activity, and neuroinflammatory responses in both the central and peripheral nervous systems. The endocannabinoid system comprises endogenous ligands, termed endocannabinoids, and two principal receptors, cannabinoid receptor type 1 and type 2 (CB1R and CB2R). While CB1R is predominantly associated with the central nervous system and mediates the psychotropic effects of cannabis-derived compounds, CB2R was initially considered mainly peripheral. However, growing evidence over the last decades has highlighted a pivotal role for CB2R in central nervous system homeostasis and pathology. Importantly, the lack of psychotropic effects associated with CB2R signaling has positioned this receptor as a promising therapeutic target for several brain-related disorders, including neuroinflammatory, neurodegenerative, neuropsychiatric, and neurovascular conditions. Here, we provide a structured review of experimental studies published over the last 3 years investigating CB2R modulation in the central nervous system, with a particular focus on disease mechanisms and emerging therapeutic strategies.

Background/Objectives: In this study, we investigated the effect on antioxidant defenses of a tomato extract obtained by supercritical CO₂ extraction (sCO₂TE), evaluating whether this green extraction method preserves biological activity compared to a conventional tomato extract (CTE) and focusing on superoxide dismutase (SOD) and glutathione peroxidase (GPx) regulation, Nuclear factor erythroid 2-related factor 2 (NRF2) activation, reactive oxygen species (ROS) and lipid peroxidation modulation. Methods: Human glioblastoma astrocytoma U-373 cells were pre-treated with sCO₂TE or conventional tomato extract (CTE) and subsequently exposed to sodium arsenite (AsNaO₂) to induce oxidative stress, or lipopolysaccharide (LPS) to trigger inflammatory signaling. Cell viability was assessed by Trypan Blue and MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide]; cell toxicity by propidium iodide staining. Intracellular ROS and lipid peroxidation were measured by flow cytometry. Gene expression of NRF2, SOD1 and GPX1 was analyzed by qRT-PCR, NRF2 activation and modulation of ERK1/2 (Extracellular Signal-Regulated Kinase 1/2) and NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) were evaluated by Western blot. Results: Pre-treatment with sCO₂TE significantly reduced AsNaO₂-induced ROS production and lipid peroxidation, showing a stronger effect compared to CTE. sCO₂TE enhanced the expression of NRF2 phosphorylation and its downstream targets SOD1 and GPX1, particularly under oxidative stress conditions. In addition, sCO₂TE attenuated LPS-induced phosphorylation of ERK1/2 and NF-κB p65, suggesting anti-inflammatory activity. Conclusions: These findings demonstrate that sCO₂TE preserves the antioxidant and anti-inflammatory properties of tomato-derived bioactives. The comparable efficacy of sCO₂TE and CTE supports the use of sCO₂ as a sustainable and solvent-free extraction method for the development of nutraceutical formulations targeting oxidative stress and neuroinflammation.

Thyroid cancer (TC) incidence is rising, necessitating a refined understanding of its complex biology, particularly for advanced forms. This review synthesizes the state-of-the-art knowledge, guided by the WHO 5th Classification (2022), which incorporates molecular findings and introduces categories like Differentiated High-Grade Thyroid Carcinoma (DHGTC) to better stratify prognosis. The review summarizes the molecular changes in thyroid cancer (TC) by establishing a clear link between specific oncogenic alterations and the resulting tumor phenotype, prognosis, risk stratification and therapeutic vulnerabilities. The central importance of the review lies in its comprehensive integration of these molecular changes with the resulting immunological microenvironment and the rationale for novel, personalized therapies. Moreover, high-level genomic instability within aggressive thyroid malignancies promotes an immunosuppressive tumor microenvironment via the selection and recruitment of suppressive immune components, contributing to immune evasion and poor prognosis. This characteristic immunosuppression identifies the aggressive tumors as prime candidates for targeted immunotherapies. The review implicitly argues that understanding the molecular drivers of this immunosuppression is essential for designing effective clinical trials using these novel agents. Diagnostic advancements, including molecular testing for high-risk mutations (BRAF, TERT) and the integration of Artificial Intelligence (AI) for refined risk stratification, are enabling personalized treatment. The evolving molecular and clinical understanding allows for a paradigm shift toward individualized therapies that balance optimal disease control with minimizing morbidity, especially in the context of high-risk disease.

Proteins tend to misfold upon stressful events that alter their homeostasis, potentially leading to protein aggregation. A tight regulation of synthesis, folding and degradation, defined as proteostasis network (PN), is required to ensure the functionality of the cell. PN is of utmost importance in post-mitotic cells such as neurons, where protein quality must be preserved for their entire lifetime. Most neurodegenerative disorders are associated with dysregulation of this network. Here, we describe the alteration in key components of the PN during chronic stress and link them with the increase in the amyloid burden and with the aggregation of the protein TDP-43, a major player in Amyotrophic Lateral Sclerosis and other neurodegenerative diseases. Neuroblastoma SH-SY5Y cells were treated with a panel of environmental stressors and analyzed after 24 h and 72 h. Treatments resulted in altered PN functionality, including proteasome impairment, halted protein synthesis, engulfed bulk and selective autophagy, in the absence of overt cell death. Thioflavin staining showed increased amyloid burden throughout treatments, associated with phosphorylated TDP-43 (pTDP-43). Biochemical analyses further revealed the cleavage and increased insolubility of pTDP-43. Our results suggest that TDP-43 is a central player during the integrated stress response to chr onic insults and that increased amyloid burden may reflect the global wellfare of a cellular system, pointing toward the alteration of the PN as the main drive for the onset of sporadic neurodegenerative disorders.

Autophagy and apoptosis are two essential mechanisms regulating cell fate. Although distinct, their signaling pathways are closely interconnected through various crosstalk mechanisms. Lipid rafts are described to act as both physical and functional platforms during the early stages of autophagic and apoptotic processes. Only recently has a role for lipid raft-associated molecules in regulating EV biogenesis and release begun to emerge. In particular, lipids of EV membranes are essential components in conferring stability to these vesicles in different extracellular environments and/or to facilitate binding or uptake into recipient cells. In this review we highlight these aspects, focusing on the role of lipid molecules during apoptosis and secretory autophagy pathways. We describe the molecular machinery that connects autophagy and apoptosis with vesicular trafficking and lipid metabolism during the release of EVs, and how their alterations contribute to the development of various diseases, including autoimmune disorders and cancer. Overall, these findings emphasize the complexity of autophagy/apoptosis crosstalk and its key role in cellular dynamics, supporting the role of lipid rafts as new therapeutic targets.

Neuroglobin (NGB) is a hexacoordinated hemeprotein mainly expressed in neurons. Following its upregulation and mitochondrial localization, NGB plays a pro-survival role against neuronal stress. Previously, we built a stable NGB-FLAG-overexpressing neuroblastoma cell line and showed that NGB promotes autophagy and localizes in autophagolysosomes. Here we studied the interactome of NGB-FLAG cells to identify novel autophagy-related NGB-binding partners and investigate how its upregulation could induce autophagy. LC3-II and p62 levels as well as mTORC1 activity were analyzed to evaluate autophagy in NGB-FLAG cells. NGB interactors were identified by affinity purification-mass spectrometry and protein-protein interaction network analysis and validated by immunoprecipitation. The increase of LC3-II and decrease of p62 in NGB-FLAG compared to control confirmed that NGB overexpression promotes autophagy. Interactome analysis identified the Regulatory associated protein of mTOR (RPTOR) as one of 134 putative NGB interactors, further validated by immunoprecipitation. NGB overexpression also determined a consistent increment of RPTOR phosphorylation at Ser792 which is required for mTORC1 inhibition, then confirmed by lower levels of phospho-mTOR and phospho-ULK1 in NGB-FLAG compared to control. Collectively, our data suggests that NGB is a positive regulator of autophagy. Through association with RPTOR, NGB may promote its activation and inhibit mTORC1 repressive activity on autophagy initiation.

Rheumatoid arthritis (RA) and Psoriatic arthritis (PsA) are chronic inflammatory diseases mainly affecting joints. RA primarily targets the synovial joints and is characterized by cartilage and bone erosion, whereas PsA is associated with skin and nail psoriasis and is characterized by erosive bone damage with an exuberant bone formation and soft tissue involvement. Recent evidence described the involvement of the Wnt pathway in the pathogenesis of these diseases. Thus, we aimed to analyze some components of Wnt signaling, i.e. DKK1, Wnt 5a and β-catenin, and their association with disease activity indices, investigating possible differences between the two diseases. Sera from 18 RA patients naïve for biological therapy, 18 PsA patients and 20 matched healthy donors (HD) were tested for DKK1 by ELISA, Wnt 5a and β-catenin by Immunoblotting. Values were correlated with CTX-1, detected by ELISA, and with disease activity indices: Disease Activity Score on 28 joints (DAS28-CRP) for RA and the Disease Activity in Psoriatic Arthritis (DAPSA) score for PsA. This study highlights significant increase in DKK1, Wnt 5a, and β-catenin levels in RA and PsA patients compared to HD, with distinct patterns of correlation with disease activity indices. Indeed, in RA patients, DKK1 levels positively correlated with DAS28-CRP score, whereas in PsA patients, DKK1 levels negatively correlated with DAPSA score. Our findings showed a strong correlation between DKK1 and CTX-1 levels in RA patients, supporting the relationship between DKK1 levels and the presence of joint erosions. Furthermore, a significant positive correlation was found between β-catenin and IL-6 levels in RA, indicating that β-catenin may be involved in the inflammatory cascade. This study compares the involvement of Wnt signaling in RA and PsA, suggesting that Wnt signaling may represent a possible mechanism of disease activity. In particular, it indicates that DKK1 levels are correlated with CTX-1, a marker of bone resorption, and with disease activity in RA patients. These findings underscore the importance of these biomarkers in the potential monitoring of patients, offering insights into disease mechanisms and potential therapeutic targets.

Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.

Since the discovery of graphene, there has been a wide range of the literature dealing with its versatile structure and easy binding of biomolecules as well as its large loading capacity. In the emerging field of immunotherapy, graphene and its derivatives have potential uses as drug delivery platforms directly into tumour sites or as adjuvants in cancer vaccines, as they are internalized by monocytes which in turn may activate adaptive anti-tumoral immune responses. In this study, we expose cells of the innate immune system and a human acute monocytic leukemia cell line (THP-1) to low doses of small-sized GO nanosheets functionalized with bovine serum albumin (BSA) and fluorescein isothiocyanate (FITC), to study their acute response after internalization. We show by flow cytometry, uptake in cells of GO-BSA-FITC reaches 80% and cell viability and ROS production are both unaffected by exposure to nanoparticles. On the contrary, GO-BSA nanosheets seem to have an inhibitory effect on ROS production, probably due to their antioxidant properties. We also provided results on chemotaxis of macrophages derived from peripheral blood monocytes treated with GO-BSA. In conclusion, we showed the size of nanosheets, the concentration used and the degree of functionalization were important factors for biocompatibility of GO in immune cells. Its low cytotoxicity and high adaptability to the cells of the innate immune system make it a good candidate for deployment in immunotherapy, in particular for delivering protein antigens to monocytes which activate adaptive immunity.