The investigation of molecular mechanisms of Alkaptonuria through novel cellular and tissue models

Alkaptonuria (AKU) is an ultra-rare genetic disease resulting from a lack in activity of the enzyme Homogentisate 1,2- dioxygenase (HGD) and responsible for one of the steps in the catabolism of the aromatic amino acids Phenylalanine and Tyrosine. This condition leads to the accumulation of Homogentisic Acid (HGA) and of its products caused by oxidation and polymerization which can generate melanin-like aggregates, with amyloidogenic properties related to the phenomenon “Ochronosis”. The most affected parts of the body are the joints with a consequent arthropathy. This work is focused on the investigation of molecular mechanisms of Alkaptonuria through different novel models. The first part is a set up of an osteoblast model and the consequent investigation on the mechanisms which link HGA with the high bone reabsorption and osteoporosis that often occurs in alkaptonuric patients. The results suggest an impairment in the RANK-L/Osteoprotegerin system related to Wnt-Beta catenin signaling inhibition. In the same model an altered redox status rate was found and further investigated in the second part of this work. The further advance of the study evidenced a strong involvement of NADPH and mitochondria in oxidative stress generation and propagation. Ulterior analysis revealed an impairment in the antioxidant system related to Nrf2 which seems to be affected by 4-HNE derived from HGA induced lipid peroxidation. The last part of the work is the set -up of a skin tissue model of alkaptonuria which revealed the activation of NLRP1 related inflammasome. The data was further confirmed using a novel cellular model which includes endothelial cells, fibroblast and keratinocytes.