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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of the upper and lower motor neurons (MNs). About 10% of patients have a family history (familial, fALS); however, most patients seem to develop the sporadic form of the disease (sALS). SOD1 (Cu/Zn superoxide dismutase-1) is the first studied gene among the ones related to ALS. Mutant SOD1 can adopt multiple misfolded conformation, lose the correct coordination of metal binding, decrease structural stability, and form aggregates. For all these reasons, it is complicated to characterize the conformational alterations of the ALS-associated mutant SOD1, and how they relate to toxicity. In this work, we performed a multilayered study on fibroblasts derived from two ALS patients, namely SOD1L145F and SOD1S135N, carrying the p.L145F and the p.S135N missense variants, respectively. The patients showed diverse symptoms and disease progression in accordance with our bioinformatic analysis, which predicted the different effects of the two mutations in terms of protein structure. Interestingly, both mutations had an effect on the fibroblast energy metabolisms. However, while the SOD1L145F fibroblasts still relied more on oxidative phosphorylation, the SOD1S135N fibroblasts showed a metabolic shift toward glycolysis. Our study suggests that SOD1 mutations might lead to alterations in the energy metabolism.

In three different biological models, the recombinant protein expressed in E. coli, the neuronal cells SH-SY5Y and the nematode Caenorhabditis elegans, cadmium inhibits SOD1 activity without affecting its expression level.

Epidemiological data have related cadmium exposure to neurotoxicity. The combination of multi-level and complementary approaches provides a comprehensive view of mechanistic processes in neurotoxicity.

Il cadmio, elemento chimico ampiamente usato in ambito industriale, è considerato un contaminante ambientale con effetti tossici sugli organismi viventi. Il suo ingresso nel corpo umano può avvenire per inalazione o ingestione di cibi ed acqua contaminati, fumo di sigaretta o impiego professionale, con tratto respiratorio e gastrointestinale principalmente coinvolti nel suo assorbimento cellulare. Anche il cervello è un bersaglio della tossicità del cadmio, che può entrare nel sistema nervoso centrale tramite una maggiore permeabilità della barriera ematoencefalica o attraverso i nervi olfattivi. Infatti, l'esposizione al cadmio è stata correlata sia ad alterazioni funzionali del sistema nervoso sia a malattie neurodegenerative, come la sclerosi laterale amiotrofica (SLA). Il 90-95% dei casi di SLA sono sporadici (sALS), mentre il restante 5-10% ha origine familiare (fALS), di cui il 15-20% è attribuito a mutazioni nel gene dell’enzima antiossidante superossido dismutasi 1 (SOD1). SOD1 è un omodimero di 32 kDa, in cui ciascun monomero presenta un ponte disulfuro e due ioni metallici, il rame con ruolo catalitico e lo zinco con funzione strutturale. Poiché uno dei principali meccanismi con cui il cadmio esercita la propria tossicità è lo stress ossidativo, responsabile di un insieme di eventi avversi che culminano nella morte cellulare, scopo di questa tesi è lo studio dell'effetto neurotossico del cadmio sul metabolismo energetico nella linea cellulare umana SH-SY5Y, sulle difese antiossidanti in cellule LUHMES differenziate e sulla funzione di SOD1 in tre modelli sperimentali (proteina ricombinante in E. coli, linea cellulare SH-SY5Y e nematode Caenorhabditis elegans). La valutazione del metabolismo energetico in cellule SH-SY5Y trattate per 24 ore con dosi sub-letali di CdCl2 ha evidenziato il passaggio ad un metabolismo anaerobico; infatti cellule trattate mostrano un aumento della glicolisi, una maggiore produzione di ATP per via glicolitica e una r, The heavy metal cadmium is a widespread toxic pollutant, released into the environment mainly by anthropogenic activities. Human exposure can occur through different sources: occupationally or environmentally, with its uptake through inhalation of polluted air, cigarette smoking or ingestion of contaminated food and water. It mainly enters the human body through the respiratory and the gastrointestinal tract and it accumulates in liver and kidneys. Brain is also a target of cadmium toxicity, since this toxicant may enter the central nervous system by increasing blood brain barrier permeability or through the olfactory nerves. In fact, cadmium exposure has been related to impaired functions of the nervous system and to neurodegenerative diseases, like amyotrophic lateral sclerosis (ALS). ALS is a fatal motor neuron pathology with the 90-95% of ALS cases being sporadic (sALS), while the remaining 5-10% of familial onset (fALS); among fALS, the 15-20% is attributed to mutations in superoxide dismutase 1 (SOD1). SOD1 is an antioxidant protein responsible for superoxide anions disruption and it is a homodimeric metalloenzyme of 32 kDa mainly located in the cytoplasm, with each monomer binding one catalytic copper ion and one structural zinc ion within a disulfide bonded conformer. Since oxidative stress is one of the major mechanisms of cadmium induced toxicity and an alteration of oxidative homeostasis, through depletion of antioxidant defences, is responsible for a plethora of adverse outcoming mainly leading to cell death; we focused on cadmium effect (1) on the energetic metabolism in human neuroblastoma SH-SY5Y cell line, (2) on the oxidative defences responses in differentiated human LUHMES neural cell line and (3) on the function of human SOD1 in a three models approach (recombinant protein in E. coli, in SH-SY5Y cell line and in the nematode Caenorhabditis elegans). The evaluation of energetic metabolism of SH-SY5Y neural cells treated with sub-lethal CdCl2 doses

Cadmium is a widespread pollutant, which easily accumulates inside the human body with an estimated half-life of 25–30 years. Many data strongly suggest that it may play a role in the pathogenesis of neurodegenerative diseases. In this paper we investigated cadmium effect on human SH-SY5Y neuroblastoma cells metabolism. Results showed that, although SH-SY5Y cells already showed hyperactivated glycolysis, cadmium further increased basal glycolytic rate. Both glycolytic capacity and reserve were also increased following cadmium administration, endowing the cells with a higher compensatory glycolysis when oxidative phosphorylation was inhibited. Cadmium administration also led to an increase in glycolytic ATP production rate, paralleled by a decrease in ATP production by oxidative phosphorylation, due to an impairment of mitochondrial respiration. Moreover, following cadmium administration, mitochondria increased their dependency on glutamine, while decreasing lipids oxidation. On the whole, our data show that cadmium exacerbates the Warburg effect and promotes the use of glutamine as a substrate for lipid biosynthesis. Although increased glutamine consumption leads to an increase in glutathione level, this cannot efficiently counteract cadmium-induced oxidative stress, leading to membrane lipid peroxidation. Oxidative stress represents a serious threat for neuronal cells and our data confirm glutathione as a key defense mechanism.

In this paper, we demonstrate that the antimicrobial activity of L. plantarum PBS067 strain against antagonist microorganisms was mediated by the production of a bacteriocin-like compound secreted at the stationary phase of the growth. The novel bacteriocin-like compound, designed plantaricin P1053, was identified by using sorption–desorption method, butanol extraction and SEC-HPLC. The molecular mass of plantaricin P1053 was shown to be 1053 Da by ESI-MS analysis. Plantaricin P1053 exhibited a broad-spectrum antimicrobial activity against Gram-positive bacteria as S. aureus and Gram-negative bacteria as E. coli. In addition to the antimicrobial activity, the isolated bacteriocin-like compound showed effects on normal and cancerogenic epithelial intestinal cell lines through an enhancing of viability of healthy cells and a proliferation reduction of cancer cells. Moreover, in this paper we demonstrate that the isolated bacteriocin-like compound acts on healthy cells through the epidermal growth factor receptor (EGFR) pathways. In conclusion, plantaricin P1053 isolated from L. plantarum PBS067 strain could represent one of the first multifunctional bacteriocin-like compound acting on human epithelial intestinal cells.