Maternal nutrition during pregnancy and lactation represents a crucial determinant of offspring health, with long-term effects on metabolic and neurobehavioral development. This study examined the effects of altered diets during pregnancy and lactation on adult rat pups that received a standard postnatal diet. Since in often altered low-protein diets proteins are supplemented with foods rich in phytoestrogens, such as soy, four groups were compared: low-protein maternal diet (LOW), genistein-enriched diet (GEN), a combination of both (LOWGIN), and a control group (CON). Maternal milk composition and various offspring parameters were analyzed, including metabolic measures, (body weight and intake), behavioral assessments (EPM and OF tests), and neurochemical markers of the hypothalamic POMC system and serotonergic systems (immunohistochemistry for 5-HT in the Raphe, and POMC, 5-HT, and 5-HT₂C in the arcuate nucleus of the hypothalamus). Maternal protein restriction significantly reduced milk protein and fat content, hindered neonatal growth, and decreased POMC expression, while also increasing 5-HT₂C receptors levels, making it more difficult for the neonate to adapt to changes and new stimuli. The phytoestrogen Genistein, increased milk lipid content and neonatal weight, suggesting a potential obesogenic effect in female pups. The long-term effects of genistein were sexspecific: males exhibited enhanced serotonergic sensitivity, characterized by increased expression of the 5-HT system, and an anxiolytic behavioral profile. In contrast, females showed serotonergic hyperactivity. The combination of the two diets displayed milk composition and metabolic parameters similar to those of the LOW group, suggesting a predominant effect of protein restriction that overrides the potential benefits of genistein. Behaviorally, profiles were sexually dimorphic: males showed anxiety, whereas females displayed an anxiolytic profile similar to GEN females, highlighting greater female sensitivity to genistein’s estrogenic effects. Overall, the results demonstrate that early nutritional programming has a stable influence on the development of metabolic and emotional circuits in a sex-specific manner, underscoring the importance of adequate protein intake during pregnancy and lactation, as well as the cautious evaluation of phytoestrogen consumption during critical periods, when maternal dietary choices can have lasting consequences for offspring health.

Endocrine disruptors (EDs) are compounds that affect the endocrine system by interfering with hormone synthesis and action, altering organ development, reproductive and metabolic processes. Bisphenols and perfluoroalkyl compounds are key EDs studied, used in industrial processes to produce everyday products, i.e. plastics for food packaging. This creates a direct pathway for human exposure, with potential risks for pregnant women and their offspring, about which little is known. This study examined how maternal exposure to bisphenol A (BPA), bisphenol S (BPS), and perfluorooctanesulfonic acid (PFOS) affects mothers’ spontaneous behavior and anxiety levels. CD1 mouse mothers were orally treated during pregnancy and lactation and divided into four groups: 1) control, 2) BPA (0.2 ng/kg), 3) BPS (0.2 ng/kg), and 4) PFOS (0.7 ng/kg). The mothers were subsequently tested for maternal and anxiety-related behaviors. Feeding behavior indicated an increase in food intake among PFOS-treated mothers, while their body weight remained unchanged. The pups from these mothers were significantly heavier than the controls, whereas the pups exposed to BPA were smaller. These characteristics persisted throughout development. Behavioral tests (elevated plus maze and open field) revealed increased anxiety in PFOS mothers, while BPA mothers showed hyperactivity. Maternal care analysis from PND2 to PND8 revealed that PFOS mothers exhibited fewer maternal behaviors, spending less time nursing and more time outside the nest. Additionally, immunohistochemical analysis of vasopressin immunoreactivity revealed altered vasopressin content in the paraventricular nucleus of PFOS-treated mothers, suggesting a neuroendocrine basis for behavioral changes. In conclusion, BPA, BPS, and PFOS exposure during pregnancy and lactation resulted in diverse behavioral and metabolic effects. Future analyses will focus on the sexual and anxiety behavior of adult puppies and neural markers linked to these topics.

FLVCR1 (feline leukemia virus subgroup C receptor 1) is a solute carrier protein implicated in choline and ethanolamine transport, highly expressed in the developing brain. Biallelic pathogenic variants in FLVCR1 are associated with a broad spectrum of neurological disorders, including congenital hydrocephalus, microcephaly, developmental delay, epileptic encephalopathy, and adultonset neurodegeneration. While Flvcr1a deletion in neural progenitor cells (NPCs) disrupts mitochondrial calcium homeostasis and energy metabolism leading to defective neurogenesis and congenital hydrocephalus, this does not fully account for the cognitive symptoms observed in patients. Here, we explore the contribution of FLVCR1a to cortical interneuron development, as these GABAergic neurons are essential for the formation of neuronal networks. Using a conditional knockout mouse model lacking Flvcr1a in NPCs, we observed altered interneuron migration, characterized by reduced interneuron migration rates, shortened leading processes, and aberrant direction changes, along with altered morphology in vitro. Additionally, explants of medial ganglionic eminence (MGE) from mutant embryos displayed migration deficits compared to wild-type littermates. To dissect the molecular mechanism, we investigated the FLVCR1a protein interactome and identified DOCK7, a Rac1 guanine nucleotide exchange factor, and CRAD, an inhibitor of F-actin capping proteins, as FLVCR1a interactors. Additionally, by using a FRET biosensor, we reported that FLVCR1a modulates Rac1 activity a key regulator of cytoskeletal organization during migration. These findings link FLVCR1a to the regulation of cytoskeletal dynamics and cell polarity in migrating interneurons. These findings suggest that FLVCR1a deficiency disrupts cellular migration and morphology during development, likely through the modulation of cell bioenergetics and cytoskeletal dynamics. This study contributes to a deeper understanding of neurodevelopmental disorders linked to FLVCR1a mutations.

Intranasal delivery represents a non-invasive and promising route to directly target the central nervous system (CNS), as it enables therapeutic agents to bypass the blood–brain barrier and allows repeated administration with low invasiveness. In this context, extracellular vesicles derived from adipose mesenchymal stem cells (ASC-EVs) are gaining increasing attention for their intrinsic neuroprotective and immunomodulatory properties, making them attractive candidates for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). In this study, we established an in vitro epithelial barrier model using RPMI 2650 cells (derived from nasal epithelium carcinoma) cultured on transwell system and coupled with motor neuron-like NSC-34 cells treated with hydrogen peroxide to mimic oxidative stress-induced injury. This setup was designed to investigate the ability of ASC-EVs to cross the epithelial layer, representing the first step of access to the CNS following intranasal administration, and to reach damaged motor neurons. The study also explores the mechanisms underlying epithelial transport by evaluating both transcellular and paracellular routes through the use of selective pathway inhibitors and by monitoring changes in transepithelial electrical resistance (TEER) and junctional integrity. Our findings demonstrate that ASC-EVs are able to traverse the epithelial barrier and reach damaged motor neurons, where they are internalized and exert significant neuroprotective effects. These results provide a mechanistic basis supporting the potential of intranasally delivered ASC-EVs as a non-invasive therapeutic approach for ALS and other neurodegenerative disorders.

Multiple Sclerosis (MS) is a chronic disease of the Central Nervous System (CNS), where neuroinflammation and autoimmune response against myelin lead to functional impairments and psychiatric symptoms. Exposure to peaks of air pollution – and in particular of particulate matter (PM) - has been associated with increased hospitalizations for MS onset and relapses, pointing to persons with MS (or predisposed to develop MS) as a PM-vulnerable cohort. With the aim to disclose the biological substrate of such vulnerability, and to understand whether individuals primed to develop autoimmunity (as it occurs in MS and in the experimental autoimmune encephalomyelitis - EAE - animal model of MS) respond differently to PM, we firstly characterized plasmatic extracellular vesicles (EVs) and their microRNA (miRNA) cargo in healthy and presymptomatic EAE mice after exposure to PM10, compared to unexposed healthy and EAE mice. The response of EAE mice to PM10 did not differ in terms of EV number or source compared to that of healthy mice. Remarkable differences existed instead in the identity of deregulated EV-associated miRNAs, which in EAE mice were predicted to target several MS-relevant biological processes and nervous system-, immune- and inflammation- related pathways, possibly contributing to disease worsening. To validate our findings, we monitored EAE pathological course. PM10 exposure in the presymptomatic phase did not modify EAE course, as assessed by clinical and neuropathological analyses. Yet, in PM-exposed EAE mice we observed selective behavioral alterations - i.e. increased disinhibited, risk-taking and novelty-seeking behaviors - compatible with neurotransmitter imbalances linked with the detected EV-packaged miRNA deregulation. Overall, these data show a selective vulnerability of immunologically primed mice toward the effects of PM, detected at molecular and behavioral levels as early as in the pre-symptomatic stages of the pathology.

Recreating physiologically relevant spinal cord models in vitro requires not only appropriate cell types but also structural and topographical cues that mimic native tissue. While 2D cultures fail to reproduce the spatial complexity of the central nervous system, 3D bioprinting strategies enable more realistic neural microenvironments. We aim to develop a 3D spinal cord model by combining murine cell lines or human stem cell–derived neural progenitors with a multi-material scaffold system. Murine neuronal/astrocytic (NE-4C) and motor neuron-like (NSC-34) cells were embedded in gelatin methacrylate (GelMA) hydrogels and layered on aligned polycaprolactone (PCL) microfibers produced by Melt Electrowriting via extrusion-based bioprinting. The matrix supported long-term viability, proliferation, and differentiation: neuronal (MAP2, βIII-tubulin) and glial (GFAP) markers increased progressively over 14-21-28 days in vitro (DIV), confirmed by immunofluorescence and western blot, and spontaneous network activity was detected through calcium imaging. In parallel, human neural progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs) were successfully differentiated and later cultured within a composite hydrogel composed of GelMA and Geltrex for 7, 10, and 14 DIV. The NPCs expressed SOX2 and nestin, confirming their neural progenitor identity, and exhibited a characteristic rosette- like morphological organization, indicative of early neuroepithelial architecture. Over time, they showed high viability and progressive neuronal maturation, with MAP2, βIIItubulin, and synapsin expression suggesting neuronal interconnection and emerging network formation. This preliminary 3D system might represent a versatile and biomimetic platform for modeling spinal cord structure and function, with translational potential for studies on spinal cord injury, neurodegeneration, and physiologically relevant drug screening. The work was supported by Fondazione CRT and the European Union – Next Generation EU within the PRIN 2022 program (D.D. 104 – 02/02/2022, Ministero dell’Università e della Ricerca).

Diabetic peripheral neuropathy (DPN) is the most frequent complication of diabetes, involving progressive damage to neurons, Schwann cells, and microvasculature. It results in altered pain perception, either heightened sensitivity or complete sensory loss, raising the risk of injury. Due to the structural complexity of peripheral nerves, the pathogenic mechanisms of DPN remain poorly understood, limiting therapeutic development. Human induced pluripotent stem cell (hiPSC)-based models are emerging as promising tools to recreate the cellular diversity of peripheral nerves in vitro and study DPN pathogenesis. To model DPN in vitro, we first established a rat-based system using primary Schwann cells and primary dorsal root ganglion (DRG) neurons cultures. Schwann cells were then cultured under high-glucose and hypoxic conditions to mimic diabetic stress, showing altered morphology, reduced proliferation, increased oxidative stress, and decreased expression of mature Schwann cell key markers. In parallel, dissociated rat DRG neurons exposed to the same conditions showed reduced neurite complexity, with fewer branches and junctions per cell. These findings confirm the induction of key DPNlike features in our primary cell cultures model. Building on this foundation, we differentiated human induced pluripotent stem cells (hiPSCs) into Sensory Neurons and Schwann cells following previously established protocols. We successfully obtained both iNOCs (induced-nociceptors) and iSCs (induced-Schwann cells) and cultured them under hyperglycemic conditions on the basis of preliminary data obtained on primary cell cultures. These foundational steps are critical for our ultimate goal of integrating both cell types into a multicellular system, fully human-based in vitro model of DPN. Moreover, by validating cellular responses to diabetic stress in both rat and human-derived components, we are laying the groundwork for a translational platform that will enable deeper mechanistic insights into the pathophysiology of DPN and support future personalized and therapeutic development. This work has been conducted under the National Plan for Complementary Investments to the NRRP, project “D34H– Digital Driven Diagnostics, prognostics and therapeutics for sustainable Health care” (project code: PNC0000001), Spoke 4 funded by the Italian Ministry of University.

Olive pomace (OP), a byproduct of olive oil extraction, retains numerous bioactive compounds (in particular, hydroxytyrosol and tyrosol, but also other beneficial flavonoids, phenols and secoiridoids like luteolin, oleocanthal, and oleuropein) found in extra virgin olive oil, which are recognized for their health-promoting properties, particularly in reducing oxidative stress and inflammation. Recent studies have demonstrated that oleuropein and other polyphenols may exert significant neuroprotective effects contributing to improved cognitive function and reducing neuroinflammation, thereby supporting neuronal health. Furthermore, research suggests that polyphenols can promote neurogenesis and synaptic plasticity, essential processes for maintaining brain function during aging. Dietary supplementation (DS) with OP compounds may positively influence gut microbiota, which interacts with the central nervous system, highlighting the intricate relationship between diet and brain health. Following our observation in a previous study that dietary supplementation with OP significantly increased the half-life in both female and male flies, in the present pilot study, using one of the two OP batches previously employed, we evaluated the effects of dietary supplementation with OP on motor abilities and on the maintenance of brain tissue morphological integrity during aging in female flies. Climbing ability, brain size, and quantitative analyses of vacuole-like degeneration associated with specific brain regions were utilized as indicators of brain functional integrity and neurodegeneration during the aging process. Dietary supplementation commenced at 30 of flies age (w1118 strain), and climbing ability was assessed at the beginning (T0) and after 10 days of OP DS (T1). Brain tissue collections were conducted immediately after the T1 climbing assay. Female flies supplemented with OP exhibited significantly improved climbing activity at T1 (p<0.0188), a trend toward larger brain size (p=0.053) along with a significantly reduced total volume of brain vacuoles compared to flies without DS (p<0.05). The gut microbiota modulation by bioactive compounds has been linked to various health benefits through multiple mechanisms. Understanding how OP-derived polyphenols specifically influence these microbial populations and their functional outputs would provide valuable insights into the neuroprotective and longevity-promoting effects observed in our observations.

Psoriasis is a chronic immune-mediated inflammatory disorder characterized by accelerated keratinocyte proliferation and immune dysfunction1. Trichosanthin (TCS) demonstrates potent induction of apoptosis and regulates T-cell activities2. This was the first study to investigate the therapeutic effects of TCS topical application in the treatment of psoriasis. After a series of in vitro and in vivo experiments, we found that TCS significantly alleviated psoriatic lesions in mice, reducing PASI scores comparable to those achieved with tacrolimus. TCS also decreased the protein expression levels of Ki-67, KRT14, and IL-17, as well as reduced T cell infiltration in psoriatic skin tissue, and inhibited the expression of Ki-67, β-catenin, Wnt5a, STAT3, KRT14, and KRT17 in HaCat cells. Furthermore, TCS suppressed the protein levels of TRAF6, IKKα/β, and NF-κB, along with its phosphorylated form, and downregulated the mRNA expression of inflammatory cytokines including IL-17, IL-12, TNF-α, and IFN-γ. In summary, TCS effectively suppresses epithelial hyperproliferation, keratinization, and skin inflammation by modulating Wnt and NF- κB signaling pathways, underscoring its potential as a novel therapeutic agent for psoriasis.

A major goal in regenerative medicine is restoring damaged tissues through cell replacement or by activating endogenous repair pathways. Immune modulation is increasingly recognized as essential for creating a permissive environment for regeneration. Bioactive factors secreted by stem and stromal cells -either as soluble molecules or within extracellular vesicles (EVs)- can reshape immune responses, not just by reducing inflammation but by actively promoting pro-resolving, regenerative profiles. For nearly two decades, our research has focused on the therapeutic potential of human term placenta-derived cells, particularly mesenchymal stromal cells from the amniotic membrane (hAMSC). We have shown that hAMSC and their secretome (CM-hAMSC) exert potent immunomodulatory effects in vitro: suppressing T- and B-cell proliferation, downregulating proinflammatory Th1/Th17 phenotypes, enhancing regulatory T cells, and inhibiting naïve CD8 T cell polarization into memory subsets. They also reduce B-cell differentiation into antibody-secreting cells and steer monocytes away from dendritic cells and M1 macrophages toward anti-inflammatory M2 phenotypes. These effects have translated into therapeutic benefits in numerous preclinical models of inflammation-driven disease (including lung/liver fibrosis, sepsis, IBD, cardiac ischemia, autoimmune encephalomyelitis, rheumatoid arthritis, and traumatic brain injury) by tempering inflammation and promoting resolution. Ongoing studies aim to dissect the mechanisms driving these effects, especially whether they are mediated by soluble molecules, EVs, or both. A key area of interest is their ability to modulate the inflammasome pathway, a central driver of innate immunity linked to aging and degeneration. By regulating this pathway, placental factors may redirect chronic inflammation toward regeneration. Given inflammation’s dual role as trigger and barrier in many degenerative conditions, placental cells -especially those from the amniotic membrane- emerge as a powerful paradigm. This lecture will explore how their unique immunological properties can inspire innovative regenerative strategies.