Jose Luis Rosa

Pathogenic variants in HERC1 are associated with a syndromic neurodevelopmental disorder characterized by macrocephaly, developmental delay, intellectual disability, and psychomotor dysfunction. Although genetic and molecular studies suggest a loss-of-function mechanism, definitive in vivo evidence that HERC1 deficiency is sufficient to cause neurodevelopmental abnormalities is lacking. Using a HERC1 knockout mouse model, we investigated the behavioural, neuronal, and synaptic consequences of complete HERC1 loss. HERC1 deficiency caused motor coordination and cognitive deficits in both sexes, while male mutants additionally displayed hyperactivity and increased anxiety-like behaviour. Histological analyses revealed selective hippocampal abnormalities, including disruption of dentate gyrus organization, whereas Purkinje cells survival and cerebellar compartmentalization remained preserved. HERC1-deficient mice exhibited a marked increase in hippocampal GABAergic interneurons, impaired maturation of dendritic spines in CA1 pyramidal neurons, and altered adult neurogenesis, indicating widespread defects in hippocampal circuit organization. Consistent with these structural abnormalities, glutamatergic synaptic components were significantly altered throughout the hippocampus. These molecular changes showed a sex-dependent pattern, with predominantly presynaptic defects in males and postsynaptic alterations in females. Despite these differences, whole-cell recordings revealed a common functional phenotype in both sexes. At both mossy fibre-CA3 and Schaffer collateral-CA1 synapses, HERC1-deficient neurons exhibited a progressive inability to sustain excitatory neurotransmission during repetitive stimulation, resulting in impaired steady-state glutamatergic transmission. Together, these findings demonstrate that loss of HERC1 function is sufficient to produce a neurodevelopmental phenotype and identify HERC1 as a critical regulator of hippocampal circuit assembly and function. Our results provide mechanistic insight into the pathogenesis of HERC1-associated disorders and establish the Herc1 knockout mouse as a preclinically relevant model for investigating disease mechanisms and therapeutic approaches.

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.

INTRODUCCIÓ. L’estratègia d’aprenentatge basat en equips (TBL) és una metodologia estructurada que fomenta el desenvolupament de competències mitjançant l’aula invertida. Aquest estudi avalua la implementació del TBL en pràctiques de Bioquímica per a estudiants de Ciències de la Salut, analitzant la satisfacció d’estudiants i professors, i les crítiques per a la millora contínua. MÈTODE. Les activitats de TBL van consistir en sessions teòriques i pràctiques de laboratori, seguides d’avaluacions iRAT+tRAT, amb equips formats aleatòriament. Els estudiants van treballar de manera autònoma i col·laborativa i van ser avaluats individualment i en equip. RESULTATS. Els resultats que les puntuacions dels alumnes al tRAT (prova en equip; n = 20; mitjana = 9,27; DE = 0,63; SEM = 0,14) eren consistentment superiors a les de l’iRAT (prova individual; n = 86; mitjana = 6,45; DE = 1,04; SEM = 0,11), amb una diferència de 2,82 punts sobre 10 (IC 95 %: de 2,34 a 3,30). Els resultats de l’enquesta de satisfacció mostren una acollida positiva, amb altes valoracions per a les activitats TBL (9,4/10) i l’avaluació iRAT+tRAT (9,3/10). DISCUSSIÓ. La metodologia va destacar per la participació activa, el treball col·laboratiu i l’avaluació continuada per millorar l’aprenentatge i fomentar l’ús de metodologies actives.

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.

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.

Ubiquitin is a small regulatory protein found in all eukaryotic cells

Protein ubiquitylation acts as a complex cell signaling mechanism since the formation of different mono- and polyubiquitin chains determines the substrate’s fate in the cell. E3 ligases define the specificity of this reaction by catalyzing the attachment of ubiquitin to the substrate protein. Thus, they represent an important regulatory component of this process. Large HERC ubiquitin ligases belong to the HECT E3 protein family and comprise HERC1 and HERC2 proteins. The physiological relevance of the Large HERCs is illustrated by their involvement in different pathologies, with a notable implication in cancer and neurological diseases. Understanding how cell signaling is altered in these different pathologies is important for uncovering novel therapeutic targets. To this end, this review summarizes the recent advances in how the Large HERCs regulate the MAPK signaling pathways. In addition, we emphasize the potential therapeutic strategies that could be followed to ameliorate the alterations in MAPK signaling caused by Large HERC deficiencies, focusing on the use of specific inhibitors and proteolysis-targeting chimeras.

Bone remodeling is a continuous process between bone-forming osteoblasts and bone-resorbing osteoclasts, with any imbalance resulting in metabolic bone disease, including osteopenia. The HERC1 gene encodes an E3 ubiquitin ligase that affects cellular processes by regulating the ubiquitination of target proteins, such as C-RAF. Of interest, an association exists between biallelic pathogenic sequence variants in the HERC1 gene and the neurodevelopmental disorder MDFPMR syndrome (macrocephaly, dysmorphic facies, and psychomotor retardation). Most pathogenic variants cause loss of HERC1 function, and the affected individuals present with features related to altered bone homeostasis. Herc1-knockout mice offer an excellent model in which to study the role of HERC1 in bone remodeling and to understand its role in disease. In this study, we show that HERC1 regulates osteoblastogenesis and osteoclastogenesis, proving that its depletion increases gene expression of osteoblastic makers during the osteogenic differentiation of mesenchymal stem cells. During this process, HERC1 deficiency increases the levels of C-RAF and of phosphorylated ERK and p38. The Herc1-knockout adult mice developed imbalanced bone homeostasis that presented as osteopenia in both sexes of the adult mice. By contrast, only young female knockout mice had osteopenia and increased number of osteoclasts, with the changes associated with reductions in testosterone and dihydrotestosterone levels. Finally, osteocytes isolated from knockout mice showed a higher expression of osteocytic genes and an increase in the Rankl/Opg ratio, indicating a relevant cell-autonomous role of HERC1 when regulating the transcriptional program of bone formation. Overall, these findings present HERC1 as a modulator of bone homeostasis and highlight potential therapeutic targets for individuals affected by pathological HERC1 variants.

HERC2 gene encodes an E3 ubiquitin ligase involved in several cellular processes by regulating the ubiquitylation of different protein substrates. Biallelic pathogenic sequence variants in the HERC2 gene are associated with HERC2 Angelman-like syndrome. In pathogenic HERC2 variants, complete absence or marked reduction in HERC2 protein levels are observed. The most common pathological variant, c.1781C > T (p.Pro594Leu), encodes an unstable HERC2 protein. A better understanding of how pathologic HERC2 variants affect intracellular signalling may aid definition of potential new therapies for these disorders. For this purpose, we studied patient-derived cells with the HERC2 Pro594Leu variant. We observed alteration of mitogen-activated protein kinase signalling pathways, reflected by increased levels of C-RAF protein and p38 phosphorylation. HERC2 knockdown experiments reproduced the same effects in other human and mouse cells. Moreover, we demonstrated that HERC2 and RAF proteins form molecular complexes, pull-down and proteomic experiments showed that HERC2 regulates C-RAF ubiquitylation and we found out that the p38 activation due to HERC2 depletion occurs in a RAF/MKK3-dependent manner. The displayed cellular response was that patient-derived and other human cells with HERC2 deficiency showed higher resistance to oxidative stress with an increase in the master regulator of the antioxidant response NRF2 and its target genes. This resistance was independent of p53 and abolished by RAF or p38 inhibitors. Altogether, these findings identify the activation of C-RAF/MKK3/p38 signalling pathway in HERC2 Angelman-like syndrome and highlight the inhibition of RAF activity as a potential therapeutic option for individuals affected with these rare diseases.