To investigate the role of PML in neuroinflammation, the project was structured around four main goals:
- Characterize the PML–NLRP3–P2X7 axis in neuroinflammatory processes;
- Define the novel function of cytoplasmic PML in modulating immune responses in SE, IS, and MS;
- Establish IL-1β and PML as potential biomarkers of neuroinflammatory diseases;
- Design, synthesize, and evaluate novel compounds capable of attenuating the PML-dependent immune response.
To elucidate the molecular mechanisms by which PML regulates IL-1β release and NLRP3 activity, we examined the intracellular localization of NLRP3 and P2X7 in wild-type (WT) and PML-knockout (KO) models, both at baseline and under inflammasome activation. NLRP3 predominantly localized at the endoplasmic reticulum/mitochondria-associated membranes (ER/MAMs), with increased accumulation in PML-deficient cells. Unexpectedly, P2X7 was also found at MAMs, and its presence was enhanced in the absence of PML. A stronger NLRP3–P2X7 interaction was detected in PML-KO cells, suggesting PML normally limits their association.
Biochemical analyses confirmed the co-localization and interaction of PML, NLRP3, and P2X7 at the ER/MAM interface, supporting the existence of a functional tripartite complex regulated by PML to control immune signaling at the subcellular level.
To further explore PML’s role in CNS immune regulation, we developed in vitro and in vivo models of multiple sclerosis (MS), status epilepticus (SE), and ischemic stroke (IS). In all models, PML deficiency led to markedly increased neuroinflammation, confirming its role as a negative regulator of pathological immune activation in the brain.
We synthesized a new series of aryl sulfonamide derivatives (ASDs) targeting NLRP3. In vitro assays on macrophages identified three lead compounds capable of significantly reducing IL-1β release. Molecular docking simulations, based on cryo-EM structures of NLRP3, confirmed a specific and stable binding mode.
Additionally, we developed novel proteolysis-targeting chimeras (PROTACs) that not only inhibited IL-1β release but also induced selective degradation of NLRP3. These compounds showed high potency at nanomolar concentrations, significantly outperforming traditional inflammasome inhibitors. Preliminary in vivo studies confirmed their efficacy, making them strong candidates for next-generation treatments of inflammasome-driven neuroinflammation.
As part of our biomarker investigations, IL-1β was assessed as a prognostic and predictive marker across neuroinflammatory conditions:
- In MS, IL-1β serum levels were monitored longitudinally from pre-diagnosis to one year post-treatment, showing a gradual decline and supporting its use in disease monitoring;
- In SE, in collaboration with the IRCCS Besta Institute (Milan), IL-1β levels were elevated in epileptogenic brain regions, where PML was also found to be delocalized;
- In IS, in collaboration with the Neurology Department at Ferrara Hospital, a prospective study was launched to assess IL-1β serum levels during acute and post-acute phases.
The project outcomes were disseminated through:
- Three peer-reviewed publications in PNAS, Cell Death & Differentiation, and Biomedicine & Pharmacotherapy, along with several reviews;
- Multiple presentations at international scientific conferences;
- The filing of three national patents on novel NLRP3 inhibitors, now entering the European phase;
- Submission of Proof of Concept proposals awarded the Seal of Excellence by the European Commission.
