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Microglial cells play a key role in protecting the central nervous system from pathogens and toxic compounds and are involved in the pathogenesis of different neurodegenerative diseases. Cadmium is a widespread toxic heavy metal, released into the environment at a rate of 30,000 tons/year by anthropogenic activities; it is easily uptaken by the human body through diet and cigarette smoke, as well as by occupational exposure. Once inside the body, cadmium enters the cells and substitutes to zinc and other divalent cations altering many biological functions. Its extremely long half-life makes it a serious health threat. Recent data suggest a role for heavy metals in many neurodegenerative diseases; however, the role of cadmium is still to be elucidated. In this work we report the investigation of cadmium toxicity towards murine BV2 microglial cells, a widely used model for the study of neurodegeneration. Results show that increasing cadmium concentrations increase oxidative stress, a proposed mechanism of neurodegeneration, but also that BV2 cells can keep oxidative stress under control by increasing glutathione reduction. Moreover, cadmium induces alterations of cell morphology and metabolism leading to mitochondrial impairment, without switching the cells to Warburg effect. Finally cadmium induces the release of proinflammatory cytokines, but does not markedly switch BV2 cells to M1 phenotype.

La Sclerosi Laterale Amiotrofica è una malattia neurodegenerativa, caratterizzata dalla perdita dei motoneuroni, che causa perdita muscolare con progressiva paralisi. Nonostante sia principalmente idiopatica (casi sporadici, sALS), diversi geni sono stati associati alla patologia e per il 10% è familiare (fALS). I meccanismi molecolari sono per lo più sconosciuti, ma convergono in pathways comuni: difetti mitocondriali, stress ossidativo, metabolismo dell’RNA deregolato, formazione di aggregati proteici tossici e neuro infiammazione con coinvolgimento della glia. 150 geni sono stati associati al rischio di SLA, tra i quali SOD1 (Cu/Zn superossido dismutasi- 1) è il primo e più studiato. Può indurre tossicità attraverso “gain-of-function”, mediata da conformazioni mal ripiegate e formazione di aggregati proteici. Durante il primo anno di dottorato, ho effettuato un’analisi biochimica di fibroblasti SOD1 derivati da due pazienti affetti da SLA, con le mutazioni p.L145F e p.S135N. L’analisi bioinformatica ha predetto diversi effetti delle mutazioni sulla struttura proteica, in accordo con le diverse presentazioni cliniche e progressioni della malattia nei pazienti. Concordemente, i profili metabolici dei fibroblasti, ottenuti tramite analisi Seahorse, sono diversi: mentre i fibroblasti SOD1L145F utilizzano maggiormente la fosforilazione ossidativa, i fibroblasti SOD1S135N mostrano uno shift verso la glicolisi. Questo riarrangiamento metabolico potrebbe essere causato da un aumento dello stress ossidativo dovuto dalla perdita dell’attività di SOD1, suggerendo che il fenotipo più grave a livello proteico e cellulare, corrisponde anche alla forma più aggressiva di SLA. Anche nei casi familiari, le mutazioni possono avere penetranza variabile. Ho caratterizzato il fingerprint metabolico di fibroblasti derivati da un paziente e un membro asintomatico di una famiglia italiana riportante casi di fALS legati alla mutazione p.G376D nel gene TARDBP, che codifica per la, Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that causes degeneration of upper and lower motor neurons (MNs), consequent loss of muscles and progressive paralysis. Although it is predominantly idiopathic (sporadic ALS cases, sALS), several mutated genes have been associated with the pathology and, 10 % of the cases are categorized familial (fALS). The molecular mechanisms are largely unknown, but they converge on common pathways: mitochondria defects, oxidative stress, dysregulated RNA metabolism, formation of toxic protein aggregates, and neuroinflammation with glial contribution. To date, more than 150 distinct genes have been associated with a risk of ALS, among which SOD1 (Cu/Zn superoxide dismutase-1) is the first and most extensively studied one. It can induce toxicity through a gain-of-function mediated by the adoption of misfolded conformations and formation of protein aggregates. During my first year of PhD, I have performed a biochemical profiling of SOD1 fibroblasts derived from two ALS patients, carrying the p.L145F and the p.S135N missense variants. Our bioinformatic analysis, predicted a different effect of the two mutations on the protein structure in accordance with the diverse clinical presentation and disease progression of the two patients. Correspondingly, the metabolic profiles of patients’ fibroblasts, obtained through Seahorse assays, were diverse: while the SOD1L145F fibroblasts still relied more on oxidative phosphorylation, the SOD1S135N fibroblasts showed a metabolic shift toward glycolysis. SOD1S135N cells experiencing metabolic reframing might be caused by increased oxidative stress caused by the loss of SOD1 activity. Of note, the mutation with the most severe phenotypes at the protein and cellular level also corresponded to the most aggressive form of ALS. Even in familial case of ALS, mutations might have a highly variable penetrance. In the third year of my PhD, I have characterized the metabolic fingerprint of

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative condition with phenotypic and genetic heterogeneity. It is characterized by the selective vulnerability and the progressive loss of the neural population. Here, an induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of an individual carrying the p.G376D mutation in the TDP-43 protein. Fibroblasts were reprogrammed using non-integrating episomal plasmids. There were no karyotype abnormalities, and iPSCs successfully differentiated into all three germ layers. This cell line may prove useful in the study of the pathogenic mechanisms that underpin ALS syndrome.

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease affecting both upper and lower motoneurons. The transactive response DNA binding protein (TARDBP) gene, encoding for TDP-43, is one of the most commonly mutated gene associated with familial cases of ALS (10%). We generated a human induced pluripotent stem cell (hiPSC) line from the fibroblasts of an asymptomatic subject carrying the TARDBP p.G376D mutation. This mutation is very rare and was described in a large Apulian family, in which all ALS affected members are carriers of the mutation. The subject here described is the first identified asymptomatic carrier of the mutation.

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.

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.

Among the known causative genes of familial ALS, SOD1 mutation is one of the most common. It encodes for the ubiquitous detoxifying copper/zinc binding SOD1 enzyme, whose mutations selectively cause motor neuron death, although the mechanisms are not as yet clear. What is known is that mutant-mediated toxicity is not caused by loss of its detoxifying activity but by a gain-of-function. In order to better understand the pathogenic mechanisms of SOD1 mutation, a human induced pluripotent stem cell (hiPSC) line was generated from the somatic cells of a female patient carrying a missense variation in SOD1 (L145F).

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative condition with phenotypic and genetic heterogeneity. It is characterized by the selective vulnerability and the progressive loss of the neural population. Here, an induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of an individual carrying the p.G376D mutation in the TDP-43 protein. Fibroblasts were reprogrammed using non-integrating episomal plasmids. There were no karyotype abnormalities, and iPSCs successfully differentiated into all three germ layers. This cell line may prove useful in the study of the pathogenic mechanisms that underpin ALS syndrome.