Francesc Ventura

Biallelic hypomorphic variants in the E3 ubiquitin ligase HERC2 cause a neurodevelopmental disorder clinically resembling Angelman syndrome, characterized by global developmental delay, intellectual disability, autism spectrum features, and movement abnormalities. Defining the target substrates of HERC2 is essential for understanding its biological role and the mechanisms underlying its pathological variants. To this end, we performed a quantitative proteomic analysis using biotinylated ubiquitin to identify HERC2 targets and assess their regulation in cells expressing HERC2 with or without ubiquitin‑ligase activity. This approach revealed extensive sets of subunits from major multimeric complexes, including the proteasome, the tRNA–biosynthesis machinery, microtubule‑associated assemblies, vesicle‑coat complexes, centrosomes, and the Ski and GATOR2 complexes, as substrates of HERC2-dependent ubiquitylation. Among these, the proteasome emerged as the most prominently affected complex. We identified up to eleven proteins required for assembly of the 19S regulatory particle whose ubiquitylation depends on HERC2. Mechanistically, we show that HERC2 recognizes unassembled subunits via chaperone-mediated interactions and targets them for proteasomal degradation. Loss of this mechanism in HERC2‑deficient cells alters proteasomal activity. It is noteworthy that fibroblasts derived from patients carrying the common pathogenic variant c.1781 C > T (p.Pro594Leu) exhibit impaired processing of 19S subunits and an aberrant increase in proteasome activity. Our findings establish a link between HERC2-related neurodevelopmental disorder and impaired proteasome activity, elucidate the molecular mechanisms through which HERC2 regulates proteostasis and how its disruption contributes to human pathology, and underscore potential therapeutic strategies for affected individuals.

Heterotopic ossification (HO) occurs following mechanical trauma and burns, or congenitally in patients suffering from fibrodysplasia ossificans progressiva (FOP). Recently, we demonstrated that inhibitors of phosphatidylinositol 3-kinase alpha (PI3Kα) may be a useful therapy for patients undergoing HO. In this study, using the already marketed BYL719/Alpelisib/Piqray drug, we have further confirmed these results, detailed the underlying mechanisms of action, and optimized the timing of the administration of BYL719. We found that BYL719 effectively prevents HO even when administered up to 3–7 days after injury. We demonstrate in cell cultures and in a mouse model of HO that the major actions of BYL719 are on-target effects through the inhibition of PI3Kα, without directly affecting ACVR1 or FOP-inducing ACVR1^R206H kinase activities. In vivo, we found that a lack of PI3Kα in progenitors at injury sites is sufficient to prevent HO. Moreover, time course assays in HO lesions demonstrate that BYL719 not only blocks osteochondroprogenitor specification but also reduces the inflammatory response. BYL719 inhibits the migration, proliferation, and expression of pro-inflammatory cytokines in monocytes and mast cells, suggesting that BYL719 hampers the hyper-inflammatory status of HO lesions. Altogether, these results highlight the potential of PI3Kα inhibition as a safe and effective therapeutic strategy for HO.

Heterotopic ossification (HO) occurs following mechanical trauma and burns, or congenitally in patients suffering from fibrodysplasia ossificans progressiva (FOP). Recently, we demonstrated that inhibitors of phosphatidyl-inositol 3-kinase alpha (PI3Kα) may be a useful therapy for patients undergoing HO. In this study, using the already marketed BYL719/Alpelisib/Piqray drug, we have further confirmed these results, detailed the underlying mechanisms of action, and optimized the timing of the administration of BYL719. We found that BYL719 effectively prevents HO even when administered up to three to seven days after injury. We demonstrate in cell cultures and in a mouse model of HO that the major actions of BYL719 are on-target effects through the inhibition of PI3Kα, without directly affecting ACVR1 or FOP-inducing ACVR1R206H kinase activities. In vivo, we found that a lack of PI3Kα in progenitors at injury sites is sufficient to prevent HO. Moreover, time course assays in HO lesions demonstrate that BYL719 not only blocks osteochondroprogenitor specification, but also reduces the inflammatory response. BYL719 inhibits the migration, proliferation and expression of pro-inflammatory cytokines in monocytes and mast cells, suggesting that BYL719 hampers the hyper-inflammatory status of HO lesions. Altogether, these results highlight the potential of PI3Kα inhibition as a safe and effective therapeutic strategy for HO.

Background: Fibrodysplasia ossificans progressiva (FOP) is caused by an activating mutation (p.R206H) in the type I BMP receptor ALK2, leading to heterotopic ossification (HO) in soft connective tissues. While aberrant Activin A-induced SMAD signaling is central in FOP pathogenesis, global signaling alterations remain poorly understood. Methods: We performed phosphoproteomics, transcriptomics and biochemical analyses in mesenchymal cells (MSCs) overexpressing wild-type ALK2 ^WT or mutant ALK2 ^R206H receptors and in induced-MSCs derived from FOP patient iPSCs. Findings were validated in vivo using FOP-like mouse models and in vitro via pharmacological interventions. Results: Multi-omics analyses revealed previously unrecognized signaling networks in ALK2 ^R206H cells, including enhanced MAPK, mTOR, RUNX2 and RHO-mediated mechanotransduction pathways. Notably, we identified dysregulated Activator Protein-1 (AP-1) expression and function as a novel contributor to FOP. AP-1 factors were highly enriched in HO lesions in FOP-like animals. Pharmacological inhibition of AP-1 significantly reduced osteochondrogenic differentiation in vitro. Conclusion: This study highlights global signaling dysregulation in FOP and identifies AP-1 as a critical driver and potential therapeutic target for FOP.

Background: The dental pulp's environment is essential for the regulation of mesenchymal stem cells' homeostasis and thus, it is of great importance to evaluate the materials used in regenerative procedures.Aim: To assess in vitro (i) the effect of chitosan nanoparticles, 0.2% chitosan irrigation solution, Dual Rinse®, 17% EDTA, 10% citric acid and 2.5% NaOCl on DSCS viability; (ii) the effect of different concentrations of TGF‐β1 on DCSC proliferation; and (iii) whether treatment with TGF‐β1 following exposure to the different irrigation solutions could compensate for their negative effects.Methodology: (i) DSCS were treated with three dilutions (1:10, 1:100 and 1:1000) of the six irrigation solutions prepared in DMEM for 10 and 60 min to assess the effect on viability. (ii) The effect of different concentrations (0, 1, 5 and 10 ng/mL) of TGF‐β1 on DCSC proliferation was assessed at 1, 3 and 7 days. (iii) The proliferative effect of TGF‐β1 following 10‐min exposure to 1:10 dilution of each irrigation solution was also tested. We used MTT assay to assess viability and proliferation. We performed statistical analysis using Prism software.Results: (i) The different endodontic irrigation solutions tested showed a significant effect on cell viability (p ≤ .0001). Significant interactions between the endodontic irrigation solutions and their dilutions were also found for all parameters (p ≤ .0001). Chitosan nanoparticles and 0.2% chitosan irrigation solution were the least cytotoxic to DSCS whilst 2.5% NaOCl was the most cytotoxic followed by 17% EDTA. (ii) TGF‐β1 at concentrations of 1 and 5 ng/mL resulted in significantly higher proliferation compared to the control group. (iii) Exposure to 17% EDTA or 2.5% NaOCl for 10 min was sufficient to make DSCS cells refractory to the proliferative effects of TGF‐β1. DSCS groups treated with TGF‐β1 following exposure to chitosan nanoparticles, 0.2% chitosan irrigation solution, Dual Rinse® and 10% CA demonstrated significantly higher proliferation compared to non‐TGF‐β1‐treated groups (p ≤ .0001, p ≤ .0001, p ≤ .0001 and p = .01 respectively).Conclusions: The current study offers data that can be implemented to improve the outcome of regenerative endodontic procedures by using less toxic irrigation solutions and adding TGF‐β1 to the treatment protocol.

Sequence variants in the HERC2 gene are associated with a significant reduction in HERC2 protein levels and cause a neurodevelopmental disorder known as the HERC2-related disorder, which shares clinical features with Angelman syndrome, including global developmental delay, intellectual disability, autism, and movement disorders. Remarkably, the HERC2 gene is commonly deleted in individuals with Angelman syndrome, suggesting a potential contribution of HERC2 to the pathophysiology of this disease. Given the known critical role of autophagy in brain development and its implication in neurodevelopmental diseases, we undertook different experimental approaches to monitor autophagy in fibroblasts derived from individuals affected by the HERC2-related disorder. Our findings reveal alterations in the levels of the autophagy-related protein LC3. Furthermore, experiments with lysosomal inhibitors provide confirmation of an upregulation of the autophagy pathway in these patient-derived cells. Mechanistically, we corroborate an interaction between HERC2 and the deubiquitylating enzyme USP20; and demonstrate that HERC2 deficiency leads to increased USP20 protein levels. Notably, USP20 upregulation correlates with enhanced stability of the autophagy initiating kinase ULK1, highlighting the role of HERC2 as an autophagy regulator factor through the USP20-ULK1 axis. Moreover, we show that p38 acts as a modulator of this pathway, since p38 activation disrupts HERC2-USP20 interaction, leading to increased USP20 and LC3-II protein levels. Together, these findings uncover a previously unknown role for HERC2 in autophagy regulation and provide insights into the pathomolecular mechanisms underlying the HERC2-related disorder and Angelman syndrome.

Dental pulp infections are common buccal diseases. When this happens, endodontic treatments are needed to disinfect and prepare the root canal for subsequent procedures. However, the lack of suitable in vitro models representing the anatomy of an immature root canal hinders research on regenerative events crucial in endodontics, such as regenerative procedures. This study aimed to develop a 3D microphysiological system (MPS) to mimic an immature root canal and assess the cytotoxicity of various irrigating solutions on stem cells. Utilizing the Dental Stem Cells SV40 (DSCS) cell line derived from human apical papilla stem cells, we analyzed the effects of different irrigants, including etidronic acid. The results indicated that irrigating solutions diminished cell viability in 2D cultures and influenced cell adhesion within the microphysiological device. Notably, in our 3D studies in the MPS, 17% EDTA and 9% 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) irrigating solutions demonstrated superior outcomes in terms of DSCS viability and adherence compared to the control. This study highlights the utility of the developed MPS for translational studies in root canal treatments and suggests comparable efficacy between 9% HEBP and 17% EDTA irrigating solutions, offering potential alternatives for clinical applications.

Heterotopic ossification (HO) occurs following mechanical trauma and burns, or congenitally in patients suffering from fibrodysplasia ossificans progressiva (FOP). Recently, we demonstrated that inhibitors of phosphatidyl-inositol 3-kinase alpha (PI3Kα) may be a useful therapy for patients undergoing HO. In this study, using the already marketed BYL719/Alpelisib/Piqray drug, we have further confirmed these results, detailed the underlying mechanisms of action, and optimized the timing of the administration of BYL719. We found that BYL719 effectively prevents HO even when administered up to three to seven days after injury. We demonstrate in cell cultures and in a mouse model of HO that the major actions of BYL719 are on-target effects through the inhibition of PI3Kα, without directly affecting ACVR1 or FOP-inducing ACVR1 ^R206H kinase activities. In vivo , we found that a lack of PI3Kα in progenitors at injury sites is sufficient to prevent HO. Moreover, time course assays in HO lesions demonstrate that BYL719 not only blocks osteochondroprogenitor specification, but also reduces the inflammatory response. BYL719 inhibits the migration, proliferation and expression of pro-inflammatory cytokines in monocytes and mast cells, suggesting that BYL719 hampers the hyper-inflammatory status of HO lesions. Altogether, these results highlight the potential of PI3Kα inhibition as a safe and effective therapeutic strategy for HO.

The purpose of this paper was to determine the effect of anodization on the in vitro proliferation and adhesion of immortalized human keratinocytes (HaCats) and mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) in Titanium Grade 23 (Ti6Al4V ELI) discs and to describe the surface topography, roughness, and composition of dental implants (body and collar) and abutments submitted to an area-specific anodization process. HaCat cells and BM-MSCs were seeded onto discs with three different surface treatments: machined, area-specific anodization for abutments, and area-specific anodization for implant collars. Cell proliferation was assessed using a resazurin-based fluorescent dye on days 1, 3, and 7, while cell adhesion was examined using scanning electron microscopy (SEM). Surface topography, roughness, and composition were evaluated for six implant bodies with an anodized rough surface, six anodized implant smooth collars, and six anodized prosthetic abutments. Both HaCats and BM-MSCs showed increased viability over time (p < 0.001) with no statistically significant differences among the different surfaces (p = 0.447 HaCats and p = 0.631 BM-MSCs). SEM analysis revealed an enhanced presence and adhesion of HaCat cells on the anodized surface for the implant collars and an increased adhesion of BM-MSCs on both the anodized and machined surface abutments. The topography characteristics of the treated implants and abutments varied depending on the specific implant region. Chemical analysis confirmed the presence of oxygen, calcium, phosphorus, and sodium on the anodized surfaces. The area-specific anodization process can be utilized to create variable topography, increase the specific surface area, and introduce oxygen, calcium, phosphorus, and sodium to dental implants and abutments. While BM-MSCs and HaCat cells showed similar adhesion and proliferation on anodized and machined surfaces, a positive interaction between anodized Ti6Al4V ELI surfaces and these two cell lines present in the peri-implant mucosa was observed. Due to the limitations of the present study, further research is necessary to confirm these findings.