Alkaptonuria (AKU) is an ultra-rare genetic disease resulting from a deficient activity of the enzyme homogentisate 1,2- dioxygenase (HGD), responsible for the catabolism of the aromatic amino acids Phenylalanine and Tyrosine. This condition leads to the accumulation of a toxic metabolite, HGA and of its product of oxidation BQA, whose polymerization generates melanin-like aggregates, with amyloidogenic properties, leading to the phenomenon of “Ochronosis”. The most affected structures by the deposition of these ochronotic aggregates are joints, which undergo severe arthropathy; therefore, articular cartilage is the main investigated tissue for the comprehension of AKU physiopathology. Articular cartilage homeostasis is maintained by articular chondrocytes, which have an important role in the synthesis and release of extracellular matrix (ECM) components, essential to properly respond to mechanical compression processes the tissue is constantly subjected to. This mechanism is mediated by chondrocyte cytoskeleton, including the microtubule-based organelle primary cilium, which exerts its function through the activation of the Hedgehog (Hh) signaling pathway. On the basis of the previous considerations, for the aims of this thesis, the set-up of in vitro AKU cellular and tissue models, was fundamental to counteract problems related to the collection of AKU samples, due to the rarity of the disease. Both experimental models were properly characterized, by highlighting the role of HGA in mediating pigment deposition and cartilage degradation processes, through histological and cytological analyses and immunofluorescence assays. Results showed that the ochronotic pigment deposits following HGA treatment lead to an oxidative stress status and to the accumulation of serum amyloid A (SAA) protein in the cartilaginous tissue. Another in vitro model was used to analyse the ability of AKU chondrocytes to respond to mechanical loading in terms of production of ECM components, by applying a 10% strain on adherent cell cultures. AKU chondrocytes resulted unable to respond properly to mechanical strain and HGA seemed to be involved in this altered responsiveness. Afterwards, a deep investigation of the cytoskeleton characteristics of AKU chondrocytes was performed, mainly by focusing on the interaction of each cytoskeletal marker with the amyloidogenic protein serum amyloid A (SAA). An in situ proximity ligation assay confirmed the co-localization of cytoskeletal marker with SAA, thus explaining the severe alterations observed in AKU cytoskeleton. Furthermore, another cytoskeletal component was characterized, the primary cilium, both investigating on its structural features and on its function in terms of Hedgehog (Hh) signaling activation. Primary cilia of AKU chondrocytes resulted shorter than normal chondrocytes and HGA treatment led to the same ciliary phenotype. In order to investigate the activation of the Hh pathway, the expression of the constitutive activator of the pathway (Gli-1) was evaluated both at the gene and at the protein level. This protein was found overexpressed in AKU/HGA-treated chondrocytes. Finally, given the role of Hh activation in mediating processes of cartilage degradation in osteoarthritis-like diseases, a therapeutic approach based on the inhibition of the Hh signaling was adopted, by using treatment with lithium chloride and with small molecule antagonists of the Hh receptor Smoothened (Smo). All the testing compounds resulted efficient in inhibiting Hh signal and in restoring cilia lengths, suggesting the use of these compounds as a potential therapeutic approach for the treatment of AKU.
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|Titolo:||Novel insights into alkaptonuria physiopathology|
|Citazione:||Gambassi, S. (2016). Novel insights into alkaptonuria physiopathology.|
|Appare nelle tipologie:||8.1 Tesi Dottorato|
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