Naringenin Ameliorates Drosophila ReepA Hereditary Spastic Paraplegia-Linked Phenotypes

Defects in the endoplasmic reticulum (ER) membrane shaping and interaction with other organelles seem to be a crucial mechanism underlying Hereditary Spastic Paraplegia (HSP) neurodegeneration. REEP1, a transmembrane protein belonging to TB2/HVA22 family, is implicated in SPG31, an autosomal dominant form of HSP, and its interaction with Atlastin/SPG3A and Spastin/SPG4, the other two major HSP linked proteins, has been demonstrated to play a crucial role in modifying ER architecture. In addition, the Drosophila ortholog of REEP1, named ReepA, has been found to regulate the response to ER neuronal stress. Herein we investigated the role of ReepA in ER morphology and stress response. ReepA is upregulated under stress conditions and aging. Our data show that ReepA triggers a selective activation of Ire1 and Atf6 branches of Unfolded Protein Response (UPR) and modifies ER morphology. Drosophila lacking ReepA showed Atf6 and Ire1 activation, expansion of ER sheet-like structures, locomotor dysfunction and shortened lifespan. Furthermore, we found that naringenin, a flavonoid that possesses strong antioxidant and neuroprotective activity, can rescue the cellular phenotypes, the lifespan and locomotor disability associated with ReepA loss of function. Our data highlight the importance of ER homeostasis in nervous system functionality and HSP neurodegenerative mechanisms, opening new opportunities for HSP treatment

Caratterizzazione dei meccanismi molecolari associati alla patogenesi della Mucopolisaccaridosi di tipo I in un nuovo modello di Drosophila melanogaster

La mucopolisaccaridosi di tipo I (MPS I) è una malattia autosomica recessiva, appartenente al gruppo delle malattie metaboliche pediatriche. È causata da mutazioni nel gene codificante per l’enzima lisosomiale a-L-iduronidasi (IDUA) che degrada i glicosaminoglicani (GAG) eparan- e dermatan-solfato. Di conseguenza,questi si accumulano non degradati in diversi tessuti e organi, causandone un progressivo malfunzionamento. MPS I presenta uno spettro di fenotipi, dalle forme attenuate, sindrome di Scheie e di Hurler-Scheie, a quella severa, la sindrome di Hurler. Le manifestazioni cliniche della malattia sono principalmente rappresentate da facies caratteristica, organomegalia, valvulopatie, alterazioni osteo-articolari, infezioni respiratorie, e, nelle forme severe, da un progressivo coinvolgimento neurologico. Sia la terapia enzimatica sostitutiva (ERT) che il trapianto di cellule staminali ematopoietiche (HSCT) sono oggi disponibili. ERT consiste nell’infusione settimanale dell’enzima ricombinante ed è parzialmente efficace nel trattare molti organi periferici, mentre è inefficace per il trattamento del sistema nervoso centrale (SNC) e per quello di alcuni distretti periferici, come ossa e valvole cardiache. Invece, l’HSCT è efficace nel trattamento del SNC, rallentando la progressione della malattia. Tuttavia, deve essere effettuato molto precocemente e non è in grado di revertere eventuali segni clinici instauratisi nelle fasi precedenti il trapianto. Sebbene clinicamente ben caratterizzata, la malattia rimane ancora poco conosciuta dal punto di vista patogenetico e i meccanismi molecolari coinvolti non sono ancora sufficientemente chiari. L’identificazione di pathway alterati coinvolti nella patogenesi, potrebbe potenzialmente fornire anche nuovi target terapeutici. A tale scopo, in questo progetto è stato sviluppato un modello basato su Drosophila melanogaster, il moscerino della frutta. Drososphila offre diversi vantaggi come animale modello, dato il suo breve ciclo vitale, che permette di effettuare studi rapidi sullo sviluppo, condotti su un numero elevato di animali, così come rapidi screening farmacologici. Drosophila è semplice da maneggiare, con costi di mantenimento contenuti. La disponibilità di linee transgeniche fluorescenti permette di condurre semplici e accurati studi in vivo. Il modello è stato sviluppato utilizzando l’approccio dell’RNA interference (RNAi). La riduzione ubiquitaria dell’omologo del gene umano Idua in Drosophila (D-idua) porta ad una diminuita attività enzimatica nel terzo stadio larvale, e a una completa letalità allo stadio di pupa. La selettiva diminuzione di D-idua in neuroni e glia causa una lieve, ma progressiva, disfunzione motoria e, allo stesso tempo, un aumento dell’aspettativa di vita. Il modello di Drosophila mostra alcune caratteristiche simili a quelle osservate in vitro nei fibroblasti umani e nel topo, quali l’aumentato numero e dimensione dei lisosomi in cervello e tessuto muscolare, e una riduzione della percentuale di lisosomi acidificati. Sono state osservate disfunzioni autofagiche e alterazioni di alcuni pathway metabolici, entrambe migliorate in condizioni di deprivazione di nutrienti. Sono state anche osservate alterazioni della rete mitocondriale e un alterato stato ossidativo dei mitocondri. Infine, l’aumento del volume cerebrale e la morfologia alterata delle terminazioni degli assoni motori riflettono alcune alterazioni strutturali del sistema nervoso. Nel complesso il modello sembra mimare alcuni aspetti associati alla patologia umana, mostra forti alterazioni in alcuni pathways ancora poco caratterizzati e un grande potenziale per approfondire i meccanismi coinvolti nella patogenesi della malattia. A partire dalle alterazioni osservate, il modello di Drosophila offre la possibilità di svolgere futuri screening farmacologici indirizzati a questi pathways alterati.Mucopolysaccharidosis type I (MPSI) is an autosomal recessive disease, belonging to the group of the inborn errors of metabolism. It is due to mutations in the gene encoding for the lysosomal enzyme α-L-iduronidase (IDUA), leading to a deficit of the enzymatic activity. IDUA degrades the glycosaminoglycansheparan- and dermatan-sulfate, which accumulate undegraded in different tissues and organs, leading to a progressive multi-organ impairment. The syndrome presents a spectrum of phenotypes, from the attenuated forms, Scheie and Hurler-Scheie syndromes, to the severe form, Hurler syndrome. Clinical manifestations of the disease are facial dimorphisms, organomegaly, valvuolpathies, osteo-articular abnormalities, respiratory infections and, in the severe form, a progressive neurological impairment. Enzyme replacement therapy (ERT) and hematopoietic stem cells transplantation (HSCT) are available for the treatment. ERT, consisting in weekly infusions of the recombinant enzyme, although quite efficient in treating most peripheral tissues, is inefficient in treating both the CNS disease and some difficult districts, as bones and heart valves. Instead, HSCT is efficient in treating the CNS compartment slowing down the progression of the disease. However, it is effective if carried out very early in life and unable to revert clinical signs established before transplantation. Although clinically well characterized, the disease remains poorly understood from the pathogenic side, and the underlined molecular mechanisms are not enough clarified. Understanding the pathological mechanisms underlying MPSI could help to identify altered pathways involved in the pathogenesis, possibly addressable as new therapeutic targets. To this aim, in this project a Drosophila melanogaster model for MPSI was developed and characterized. Drosophila offers several advantages as an animal model, due to a short life cycle, which permits to perform rapid developmental studies conducted in a high number of flies, as well as rapid pharmacological screenings. In addition, it is easy to handle, and has relatively contained costs of maintenance. Furthermore, the availability of transgenic fluorescent lines allows to conduct simplified and accurate in vivo studies. The model was developed using the RNA interference approach. The ubiquitous downregulation of the Drosophila Idua homologue led to a decreased enzyme activity in the third instar larvae and to a complete lethality at pupal stage. The selective downregulation of D-idua in neurons and glial cells led to a mild, progressive locomotor impairment and, on the other hand, to an increased lifespan. The D-idua model showed some cellular and molecular features similar to those observed in other MPSI models, as an increased number and size of lysosomes, both in the brain and in the muscle tissue, together with a decreased percentage of acidified lysosomes. In addition, autophagy and metabolic pathways impairment were observed, both ameliorated in starvation conditions. Also, alterations of the mitochondrial network, together with impaired mitochondria oxidation status were recorded. Lastly, increased brain volumes and abnormal morphology of motor axon terminations represent signs of nervous system structural alterations. Overall, the MPSI Drosophila model appears to mimic some aspects of the human MPSI pathology, and shows strong alterations in some pathways, still poorly characterized in the disease. The model presents great potential for deepening the mechanisms involved in the pathogenesis of the disease. Starting from the involvement of different metabolic pathways, which in this project were shown to be drastically affected, the MPSI fly model offers the possibility to conduct in the future pharmacological screenings specifically targeted to these altered pathways

Exploiting the potential of drosophila models in lysosomal storage disorders: Pathological mechanisms and drug discovery

none5noLysosomal storage disorders (LSDs) represent a complex and heterogeneous group of rare genetic diseases due to mutations in genes coding for lysosomal enzymes, membrane proteins or transporters. This leads to the accumulation of undegraded materials within lysosomes and a broad range of severe clinical features, often including the impairment of central nervous system (CNS). When available, enzyme replacement therapy slows the disease progression although it is not curative; also, most recombinant enzymes cannot cross the blood-brain barrier, leaving the CNS untreated. The inefficient degradative capability of the lysosomes has a negative impact on the flux through the endolysosomal and autophagic pathways; therefore, dysregulation of these pathways is increasingly emerging as a relevant disease mechanism in LSDs. In the last twenty years, different LSD Drosophila models have been generated, mainly for diseases presenting with neurological involvement. The fruit fly provides a large selection of tools to investigate lysosomes, autophagy and endocytic pathways in vivo, as well as to analyse neuronal and glial cells. The possibility to use Drosophila in drug repurposing and discovery makes it an attractive model for LSDs lacking effective therapies. Here, ee describe the major cellular pathways implicated in LSDs pathogenesis, the approaches available for their study and the Drosophila models developed for these diseases. Finally, we highlight a possible use of LSDs Drosophila models for drug screening studies.openRigon L.; De Filippis C.; Napoli B.; Tomanin R.; Orso G.Rigon, L.; De Filippis, C.; Napoli, B.; Tomanin, R.; Orso, G

Methane dry reforming on Ru perovskites, AZrRuO 3 : Influence of preparation method and substitution of A cation with alkaline earth metals

Dry reforming could become an effective route to mitigate CO 2 emission. The process required high temperatures due to its endothermicity and its accompanied by coke formation; therefore, the development of suitable catalysts is fundamental. In this work, ternary perovskites type oxides, AZrRuO 3 , were synthetized and used in the methane dry reforming. The influences of the preparation method, modified citrate and autocombustion method, and of substitution of the A cation within alkaline earth metals (Ba, Sr and Ca) on the texture, physiochemical characteristics, activity and stability of the catalysts were evaluated. Tests were conducted in a fixed bed bench scale reactor in a temperature range of 600-750 °C. Catalysts synthetized by autocombustion method showed higher reducibility and major surface area and thus better performance in the methane dry reforming. Between them, SrZrRUO 3 gave the best results in terms of conversion and stability during long duration tests (66 h). Furthermore, these catalysts showed very good resistance to thermal stresses as demonstrated by XRD analysis performed on used catalysts

Rh, Ru and Pt ternary perovskites type oxides BaZr(1-x)MexO3 for methane dry reforming

In recent years dry reforming of methane has received considerable attention as a promising alternative to steam reforming for synthesis gas (H2 and CO) production, yielding a syngas with a H2/CO ratio close to 1 and thus suitable for many chemical processes. The major drawback of the process is the endothermicity of the reaction that implies the use of a suitable catalyst to work at relatively low temperatures (923–1023 K). In this work methane dry reforming over three ternary perovskite type oxides BaZr(1-x)MexO3 using Rh, Ru and Pt as metal was studied at atmospheric pressure and in a temperature range 850–1150 K. Experimental tests at different temperatures were performed in order to analyze and compare the performances of the catalysts and to carry out a detailed kinetic study. Furthermore, long duration tests were conducted to evaluate the possible deactivation of the perovskites. Rhodium-perovskite catalyst shows the highest activity for dry methane reforming while the Pt one the lowest. No deactivation of the catalysts was observed meaning that the perovskite structure is very stable and allows to minimize the carbon deposition that is the main responsible of catalyst deactivation in this process

Drosophila D-idua Reduction Mimics Mucopolysaccharidosis Type I Disease-Related Phenotypes

Deficit of the IDUA (α-L-iduronidase) enzyme causes the lysosomal storage disorder mucopolysaccharidosis type I (MPS I), a rare pediatric neurometabolic disease, due to pathological variants in the IDUA gene and is characterized by the accumulation of the undegraded mucopolysaccharides heparan sulfate and dermatan sulfate into lysosomes, with secondary cellular consequences that are still mostly unclarified. Here, we report a new fruit fly RNAi-mediated knockdown model of a IDUA homolog (D-idua) displaying a phenotype mimicking some typical molecular features of Lysosomal Storage Disorders (LSD). In this study, we showed that D-idua is a vital gene in Drosophila and that ubiquitous reduction of its expression leads to lethality during the pupal stage, when the precise degradation/synthesis of macromolecules, together with a functional autophagic pathway, are indispensable for the correct development to the adult stage. Tissue-specific analysis of the D-idua model showed an increase in the number and size of lysosomes in the brain and muscle. Moreover, the incorrect acidification of lysosomes led to dysfunctional lysosome-autophagosome fusion and the consequent block of autophagy flux. A concomitant metabolic drift of glycolysis and lipogenesis pathways was observed. After starvation, D-idua larvae showed a quite complete rescue of both autophagy/lysosome phenotypes and metabolic alterations. Metabolism and autophagy are strictly interconnected vital processes that contribute to maintain homeostatic control of energy balance, and little is known about this regulation in LSDs. Our results provide new starting points for future investigations on the disease’s pathogenic mechanisms and possible pharmacological manipulations

Cannabinoids reduce granuloma-associated angiogenesis in rats by controlling transcription and expression of mast cell protease-5

Background and purpose: Chronic inflammatory conditions, such as granulomas, are associated with angiogenesis. Mast cells represent the main cell type orchestrating angiogenesis, through the release of their granule content. Therefore, compounds able to modulate mast cell behaviour may be considered as a new pharmacological approach to treat angiogenesis-dependent events. Here, we tested the effect of selective cannabinoid ( CB) receptor agonists in a model of angiogenesis-dependent granuloma formation induced by lambda-carrageenin in rats. Experimental approach: Granulomas were induced by lambda-carrageenin-soaked sponges implanted subcutaneously on the back of male Wistar rats. After 96 h, implants were removed and granuloma formation was measured (wet weight); angiogenesis was evaluated by histological analysis and by the measurement of haemoglobin content. Mast cells in the granulomas were evaluated histologically and by RT-PCR and immunoblotting analysis for mast cell-derived proteins (rat mast cell protease-5 (rMCP-5) and nerve growth factor). Selective CB1 and CB2 receptor agonists, ACEA and JWH-015 (0.001- 0.1 mg mL(-1)), were given locally only once, at the time of implantation. Key results: The CB1 and CB2 receptor agonists decreased the weight and vascularization of granulomas after 96 h. This treatment also reduced mast cell number and activation in granulomatous tissue. Specifically, these compounds prevented the transcription and expression of rMCP-5, a protein involved in sprouting and advance of new blood vessels. Conclusion and implications: Modulation of mast cell function by cannabinoids reduced granuloma formation and associated angiogenesis. Therefore cannabinoid-related drugs may be useful in the management of granulomatous diseases accompanied by angiogenesis

BIOLOGICAL INTERACTIONS BETWEEN A NEW SYNTHETIC SURFACTANT (CHF5633) AND SECRETORY PHOSPHOLIPASE A2 SYSTEM

9nonenoneVedovelli, L; Touqui, L; Shankar-Aguilera, S; Salomone, F; Pelizzi, N; de Filippis, C; Carnielli, VP; Cogo, P; De Luca, DVedovelli, L; Touqui, L; Shankar-Aguilera, S; Salomone, F; Pelizzi, N; de Filippis, C; Carnielli, Vp; Cogo, P; De Luca,