Intranasal drug delivery is convenient and provides a high bioavailability but requires the use of mucoadhesive nanocarriers. Chitosan is a well-established polymer for mucoadhesive applications but can suffer from poor cytocompatibility and stability upon administration. In this work, we present a method to obtain stable and cytocompatible crosslinked chitosan nanoparticles. We used 2,6-pyridinedicarboxylic acid as a biocompatible crosslinker and compared the obtained particles with those prepared by ionotropic gelation using sodium tripolyphosphate. Nanoparticles were tested to evaluate the size and the surface charge, as well as their stability in storage conditions (4 °C), at the nasal cavity temperature (32 °C), and at the body temperature (37 °C). The crosslinked chitosan nanoparticles showed a size around 150 nm and a surface charge of 10.3 mV ± 0.9 mV, both compatible with the intranasal drug administration. Size and surface charge parameters did not significantly vary over time, indicating the good stability of these nanoparticles. We finally tested their cytocompatibility in vitro using SHSY5Y human neuroblastoma and RPMI 2650 human nasal epithelial cells, with positive results. In conclusion, the proposed synthetic system shows an interesting potential as a drug carrier for intranasal delivery.

Angelman Syndrome (AS) is a neurodevelopmental disorder caused by the deficiency of the UBE3A gene that for a E3 ligase protein part of the ubiquitin–proteasome system (UPS). Autophagy and UPS systems remove abnormal proteins, but any dysfunction in these processes can affect neuronal development and wellbeing. Herein, the involvement of the UPS/autophagy system in the regulation of intracellular signaling pathways related to toxic protein accumulation was investigated in cellular/mice AS models, silenced for UB3A (UB3A⁻). The main findings are as follows: (i) autophagy was upregulated in UBE3A⁻ cells with respect to control cells; (ii) a dysregulation of the AKT/mTOR pathway, linked to autophagy/synaptic development, was evidenced in cellular/animal models of AS with respect to controls; (iii) the ubiquitin ligase MDM2 was downregulated, and the tumor suppressor p53, normally inhibited by MDM2, enhanced its expression and transcriptional activity in UB3A⁻ cells with respect to controls. Finally, UB3A⁻ cells presented a significant alteration in the levels of β-amyloids with respect to control cells, and a reduction of α-synuclein levels, typical of neurodevelopmental disorder. Nevertheless, UB3A⁻ cells do not show evident morphological abnormalities. Overall, these data suggest that AS models presented an altered signaling pathway related to autophagy/UPS systems, potentially leading to the accumulation of toxic proteins affecting synaptic development.

Angelman Syndrome (AS) is a neurodevelopmental disorder caused by the deficiency of the UBE3A gene that for a E3 ligase protein part of the ubiquitin-proteasome system (UPS). Autophagy and UPS systems remove abnormal proteins, but any dysfunction in these processes can affect neuronal development and wellbeing. Herein, the involvement of the UPS/autophagy system in the regulation of intracellular signaling pathways related to toxic protein accumulation was investigated in cellular and mice AS models, silenced for UB3A (UB3A-). The main findings are as follows: i)autophagy markers were upregulated in UBE3A- cells respect to control cells; ii)a dysregulation of AKT/mTOR pathway, which is linked to autophagy/synaptic development, was evidenced in cellular and animal models of AS with respect to controls; iii) the ubiquitin ligase MDM2 was downregulated, and the tumor suppressor p53, normally inhibited by MDM2, enhanced its expression and transcriptional activity in UB3A-cells with respect to controls. Finally, UB3A-cells presented a significant alteration in the levels of β-amyloids with respect to control cells, and a reduction of α-synuclein levels, typical of neurodevelopmental disorder. Overall, these data suggest that AS models presented altered signaling pathway related to autophagy/UPS systems, potentially leading to the accumulation of toxic proteins that impact synaptic development.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of maternal UBE3A expression, leading to disrupted proteostasis and synaptic dysfunction. Adenosine is a ubiquitous neuromodulator whose G protein-coupled receptors (ARs) regulate neuronal differentiation and neurite outgrowth during development. Here, we investigated AR signaling and their influence on survival–autophagy balance and neuronal morphology in an AS cellular model. Using SH-SY5Y cells with silenced UBE3A, we found that UBE3A loss markedly decreased A1AR, A2BAR, and A3AR protein levels while significantly increasing A2AR expression. Ligand affinity was preserved across genotypes, but A1AR and A2AAR desensitization kinetics were significantly slower in UBE3A-deficient cells. These effects were associated with reduced recruitment of G protein-coupled receptor kinase 2 (GRK2) to the plasma membrane and decreased GRK2–AR association in UBE3A-deficient cells, suggesting a possible contribution of altered GRK2 dynamics to prolonged AR signaling. Functionally, A1AR and A2AR agonists preferentially promoted survival of UBE3A-deficient cells and modulated the MDM2–p53 axis and autophagy markers; A1R stimulation also increased neurite density in UBE3A-deficient cells. Together, these results identify AR-level alterations and defective desensitization machinery in AS neuronal cells and link receptor changes to downstream proteostasis and morphological phenotypes relevant to AS pathophysiology.

Angelman syndrome (AS) is a neurodevelopmental disorder for which non-invasive biomarkers suitable for longitudinal monitoring and clinical trials remain limited. Although EEG abnormalities have shown promise as treatment-responsive biomarkers, their applicability may be constrained by tolerability and developmental variability. Here, we identify visually evoked hemodynamic responses as a robust cross-species functional biomarker of AS. Using intrinsic optical signal (IOS) imaging, we show that AS mouse model exhibits enhanced visual cortical responses that persist across development and partially reflect disease severity in adulthood. These alterations are highly sensitive to therapeutic interventions: restoration of UBE3A expression normalizes visual responses and scales with both behavioral recovery and molecular rescue. To assess translational relevance, we performed functional near-infrared spectroscopy (fNIRS) in individuals with AS during visual stimulation, demonstrating the feasibility of fNIRS in behaviorally challenging settings. Individuals with AS display amplified and temporally altered hemodynamic responses that are consistently detected across preprocessing strategies and remaine stable across age. Our findings demonstrate that visually evoked hemodynamic responses capture a disease-specific cortical signature of AS conserved across species. The scalability, non-invasiveness, and feasibility of fNIRS, combined with preclinical sensitivity to therapeutic interventions, position neurovascular imaging as a promising biomarker for future clinical trials in AS.

Angelman syndrome (AS) is a neurogenetic disorder caused by the loss of function in the maternally inherited UBE3A gene, leading to a spectrum of symptoms that include developmental delays, intellectual disability, and motor impairments. While genetic and molecular mechanisms of AS are increasingly well characterized, a critical gap remains in understanding how disruptions in neuronal connectivity at the circuit level contribute to its pathophysiology. Here, we aimed to address this gap by investigating cortical connectivity alterations in a mouse model of AS. Using wide-field calcium imaging, we monitored real-time neuronal activity across the entire dorsal cortex in awake animals. Our results revealed widespread cortical hyperconnectivity in AS mice compared to wild-type controls, particularly in regions involved in motor and sensory processing. Notably, this pattern of aberrant connectivity persisted across two distinct time points in adulthood, suggesting a stable and enduring network dysfunction. These findings indicate that the absence of UBE3A function drives persistent disruptions of functional connectivity, which may underlie key clinical manifestations of AS.