Periodic Reporting for period 2 - InflaPML (Promyelocytic leukemia protein (PML) outside the tumor: a new player in the control of inflammation)
Período documentado: 2021-12-01 hasta 2023-05-31
The innate immune response can represent a double-edged sword. It is indispensable for combatting external pathogens, but it can also be triggered inappropriately after post-ischemic reperfusion and in neurodegenerative diseases (NDDs), inducing resistance to therapies and worsening patients’ status. There are still no satisfactory therapeutic approaches for these diseases, all of which are sustained by high levels of inflammation. NDDs affect millions of EU citizens and are forecasted to increase together with the longer life expectancy and growth of the ageing population. Both scientific knowledge and pharmacological approaches have, to date, only very partially resolved this problem. In this project we propose that the expression of the protein PML at the host level can drive an immune response that worsens the pathology rather than ameliorating it, by promoting the release of IL-1β, a well-known proinflammatory cytokine. The release of IL-1β is modulated by two other players, NLRP3 and P2X7, which are responsible for its production and release in the extracellular space. Local sterile inflammation arises in many pathologic states, including several diseases of the nervous system such as brain stroke (IS), multiple sclerosis (MS) and epilepsy (SE). Here we propose that different NDDs can be linked together in a common disease pathway, of which damaged function should be targeted for therapy. We focus our attention on the study of the PML-NLRP3-P2X7 axis, acting at the endoplasmic reticulum mitochondria interfaces (ER/MAMs), in NDDs. The aim of this analysis is to reconstruct the mechanistic action of PML in controlling other inflammatory factors, identifying the potential use of IL-1β and/or of PML amount or variants as a biomarker to better profile the patients. Our final goal is to determine how to properly modulate the PML-NLRP3-P2X7, approaching the preclinical level with an innovative biochemical scenario that will allow novel profiling of patients’ immune status. We believe that a detailed understanding of the role of PML in controlling inflammation will represent a breakthrough and a pillar to sustain future studies in this field, improving the quality of care and tailoring treatments to the individual patient.
To unravel the role of PML in neuroinflammation we divided our project into four different sections:
1) To study the PML-NLRP3-P2X7 axis in neuroinflammation.
2) To define the new role of cytoplasmic PML in controlling the immune response in SE, IS and MS.
3) To define a role for IL-1β and PML as a hallmark of these neuroinflammatory diseases.
4) To design, produce and test new, more efficient and focused drugs that can reduce the PML-dependent immune response.
To clarify the molecular mechanism through which PML modulates NLRP3 and IL-1β release, we investigated the intracellular localization of NLRP3 and P2X7 in wild-type (WT) and PML-depleted (KO) models in both resting condition and after inflammasome activation. We found that NLRP3 localize ate the ER/MAMs compartments, redistributing more to MAMs in KO cells than in WT cells following inflammasome activation. We revealed a previously unknown localization of P2X7R at the ER/MAMs, where it accumulates more in the absence of PML. Furthermore, we observed a stronger interaction between NLRP3 and P2X7R at the ER/MAMs in the absence of PML. We demonstrated that PML, NLRP3, and P2X7R are all present and interact at the ER, indicating that they may form a trimer. Altogether, these data suggest that PML, NLRP3, and P2X7R work as triumvirates at MAMs, depicting a multiprotein complex orchestrated by PML.
To evaluate the involvement of PML in controlling the immune response in the central nervous system’s pathologies, we recapitulate in vitro models of MS, SE and IS. We observed that in all the studied pathologies, the PML KO genotype showed increased levels of neuroinflammation.
In order to identify IL-1β as a possible prognostic and predictive factor for neurodegenerative diseases progression, we adopted different experimental approaches, depending on the studied pathology. Since SM is a progressive neurodegenerative disorder, we decided to evaluate IL-1β levels in patients’ serum at different time points, beginning with the days before the diagnosis until 1-year therapy follow-up. IL-1β levels in patients’ serum revealed that there is a progressive decrease in the expression of this cytokine over time. Regarding SE, we established a collaboration with Besta Institute in Milan, Italy, a hospital specialized in the treatment of epilepsy. Due to the acute manifestation of SE, we decided to analyze the presence of IL-1β in brain specimens collected upon surgical treatments of epileptic patients. Lastly, with the collaboration of the Neurology section at the Hospital of Ferrara, we are enrolling stroke patients in order to collect and analyze IL-1β levels in the blood during the crisis and in the days after.
As for the therapeutical approach, we synthesized and biologically evaluated a novel series of aryl sulfonamide derivatives (ASDs) that were designed as NLRP3. All the molecules were evaluated in vitro, identifying three compounds that exhibited potent activity in inhibiting IL-1β release by macrophages. Molecular docking studies based on the NLRP3 cryogenic electron microscopy structure suggested a consensus binding mode for the new analogues supporting a specific interaction of ASDs with NLRP3. Moreover, we identified velutone F, a compound extracted from the leguminous plant Millettia velutina, as a new NLRP3-inflammasome inhibitor, and we created a photoaffinity probe on it, that worked perfectly, maintaining its inhibition property on NLRP3 activation.
1) To study the PML-NLRP3-P2X7 axis in neuroinflammation.
2) To define the new role of cytoplasmic PML in controlling the immune response in SE, IS and MS.
3) To define a role for IL-1β and PML as a hallmark of these neuroinflammatory diseases.
4) To design, produce and test new, more efficient and focused drugs that can reduce the PML-dependent immune response.
To clarify the molecular mechanism through which PML modulates NLRP3 and IL-1β release, we investigated the intracellular localization of NLRP3 and P2X7 in wild-type (WT) and PML-depleted (KO) models in both resting condition and after inflammasome activation. We found that NLRP3 localize ate the ER/MAMs compartments, redistributing more to MAMs in KO cells than in WT cells following inflammasome activation. We revealed a previously unknown localization of P2X7R at the ER/MAMs, where it accumulates more in the absence of PML. Furthermore, we observed a stronger interaction between NLRP3 and P2X7R at the ER/MAMs in the absence of PML. We demonstrated that PML, NLRP3, and P2X7R are all present and interact at the ER, indicating that they may form a trimer. Altogether, these data suggest that PML, NLRP3, and P2X7R work as triumvirates at MAMs, depicting a multiprotein complex orchestrated by PML.
To evaluate the involvement of PML in controlling the immune response in the central nervous system’s pathologies, we recapitulate in vitro models of MS, SE and IS. We observed that in all the studied pathologies, the PML KO genotype showed increased levels of neuroinflammation.
In order to identify IL-1β as a possible prognostic and predictive factor for neurodegenerative diseases progression, we adopted different experimental approaches, depending on the studied pathology. Since SM is a progressive neurodegenerative disorder, we decided to evaluate IL-1β levels in patients’ serum at different time points, beginning with the days before the diagnosis until 1-year therapy follow-up. IL-1β levels in patients’ serum revealed that there is a progressive decrease in the expression of this cytokine over time. Regarding SE, we established a collaboration with Besta Institute in Milan, Italy, a hospital specialized in the treatment of epilepsy. Due to the acute manifestation of SE, we decided to analyze the presence of IL-1β in brain specimens collected upon surgical treatments of epileptic patients. Lastly, with the collaboration of the Neurology section at the Hospital of Ferrara, we are enrolling stroke patients in order to collect and analyze IL-1β levels in the blood during the crisis and in the days after.
As for the therapeutical approach, we synthesized and biologically evaluated a novel series of aryl sulfonamide derivatives (ASDs) that were designed as NLRP3. All the molecules were evaluated in vitro, identifying three compounds that exhibited potent activity in inhibiting IL-1β release by macrophages. Molecular docking studies based on the NLRP3 cryogenic electron microscopy structure suggested a consensus binding mode for the new analogues supporting a specific interaction of ASDs with NLRP3. Moreover, we identified velutone F, a compound extracted from the leguminous plant Millettia velutina, as a new NLRP3-inflammasome inhibitor, and we created a photoaffinity probe on it, that worked perfectly, maintaining its inhibition property on NLRP3 activation.
Thanks to this project we unveil a new mechanism of action for NLRP3 inflammasome at MAMs. We found that PML acts as a brake regulating the NLRP3-P2X7 axis at the ER/MAMs and the loss of PML promotes an NLRP3-related cytokine storm in response to stress conditions due to the increased redistribution and interaction of NLRP3 and P2X7R at MAMs. These breakthroughs provide new mechanistic insights for the PML-P2X7R-NLRP3 axis, fostering new targeted therapeutic approaches.
In 2019 the molecular target for MCC950 has been published, therefore we adopt new strategies to target the NLRP3 complex. On one side we developed new ASDs, based on the MCC950 structure, making some structural modifications that should make them better and should reduce liver toxicity. On the other side, we created and tested a photoaffinity probe on velutone F for its binding site identification. Though the capacity of these compounds to block NLRP3 signaling is well established, we sought to identify their molecular target and more precisely delineate their mechanisms of action to provide a promising strategy for the development of new treatments for neuroinflammatory diseases.
By the end of the project, we aim to achieve the complete characterization of the extra-nuclear role of PML in neurodegenerative disorders, by describing the role PML-NLRP3-P2X7 axis in controlling neuroinflammation. Moreover, we aim to identify IL-1β and/or PML variants as useful biomarkers for SE and SM diagnosis and prognosis and to define the role of IL-1β as a neurotoxic mediator during the stroke and PML mutation as predisposing factors for patient outcome after stroke injury.
In 2019 the molecular target for MCC950 has been published, therefore we adopt new strategies to target the NLRP3 complex. On one side we developed new ASDs, based on the MCC950 structure, making some structural modifications that should make them better and should reduce liver toxicity. On the other side, we created and tested a photoaffinity probe on velutone F for its binding site identification. Though the capacity of these compounds to block NLRP3 signaling is well established, we sought to identify their molecular target and more precisely delineate their mechanisms of action to provide a promising strategy for the development of new treatments for neuroinflammatory diseases.
By the end of the project, we aim to achieve the complete characterization of the extra-nuclear role of PML in neurodegenerative disorders, by describing the role PML-NLRP3-P2X7 axis in controlling neuroinflammation. Moreover, we aim to identify IL-1β and/or PML variants as useful biomarkers for SE and SM diagnosis and prognosis and to define the role of IL-1β as a neurotoxic mediator during the stroke and PML mutation as predisposing factors for patient outcome after stroke injury.