Recent discoveries on the ultra-rare disease Alkaptonuria

Rare diseases by definition are pathologies that affect small numbers of people in the worldwide. The rarity of some diseases creates important challenges, more for patients and their family but also for researchers and clinicians that attempt to achieve the best care for affected people. Indeed researchers have to face with heterogeneity of rare diseases and their dispersed nature all over the word complicating even more the way that leads to the best knowledge of these pathologies. The aim of this work is to give a contribution to a better knowledge on Alkaptonuria rare disease, the first inborn error of metabolism recognized but still little studied. Alkaptonuria (AKU) is an ultra-rare genetic disease caused by mutations on homogentisate 1,2-dioxygenase (HGD) enzyme responsible for the catabolism of phenylalanine and tyrosine amino acids. The deficient activity of HGD enzyme leads to the failed metabolization of the homogentisic acid (HGA), which accumulates in connective tissues in form of a black pigment called “ochronotic” melanin-like pigment. The system most affected by pigmentations is the osteoarticular one, especially cartilage, which is the main investigated tissue for the study of the AKU disease. In the present project, AKU pathology was studied in different field of the disease (see graphical abstract). The first step of the analysis was an in silico study the HGD structure in order to identify the oxygen pathways that allow O2 molecule to reach the active site and help the enzyme to perform the enzymatic function. A specific route was identified as the principal passage for the oxygen, thanks also to the correspondence of this pathway with a transient pocket, which assumes an important role in the oxygen transition. An AKU cellular model was set up, in order to evaluate the role of HGA in the alteration of the autophagic processes. The application of an AKU in vitro model was important to overcome the rarity of AKU samples to collect. For the first time it was demonstrated that the accumulation of HGA can alter the autophagic processes in human chondrocytic cell line, inducing oxidative stress, mitochondrial damage and activation of chondroptotic processes. Afterwards, an investigation on the role HGA in the alteration of cytoskeleton structure of AKU chondrocytic model was performed, analysing the principal components of the cytoskeletal network, actin, vimentin and tubulin. These alterations have an impact on the extracellular matrix (ECM) organization of alkaptonuric cartilage leading to a pathological tissue. Another aspect analysed in this project is the alteration of the number and disposition of 2 lysosomes in chondrocytic AKU model. After HGA treatment in chondrocytes, lysosomes appeared located in the periphery of cells and, in the perinuclear region of cell, they resulted more acidic in comparison with control. Moreover black spots were found in lysosomes of AKU chondrocytes model, hypothesizing that lysosomes try to eliminate the black pigment contrasting its accumulation, toxic for cells. The toxicity of the ochronotic pigment accumulation was already in depth studied, but less is known about its structure and how HGA polymerizes, creating the ochronotic dark pigment, the main cause of tissue destruction. Through an EPR analysis conducted on AKU dark pigmented cartilage, the formation of two stable radicals on carbon and oxygen were identified. The peak that results only from the EPR analysis of the pigmented black cartilage is due to a signal of melanin-like radicals, probably of a semiquinon structure. It is possible to confirm that the polymerization of HGA takes place by radical reaction. The formation of radical species was also demonstrated after alkalization of AKU urine samples. The addiction of an alkaline solution to AKU urine, in which the concentration of HGA is high, leads to the immediate darkening of the urine colour, activating the instantaneous formation of HGA polymer. Through NMR spectroscopy, this reaction was monitored during the time. Peaks reconnected to the HGA molecule started to decrease in intensity when alkaline solution was added, but no new species were registered with NMR, hypothesising the formation of radical species non visible with NMR. A metabolic analysis was then performed on AKU urine in comparison with control samples. The alteration of the amount of some metabolites, glycine, tyrosine and creatinine, was observed in AKU urine samples, suggesting an increase in the degeneration of articular cartilage and a renal damage of AKU patients, of which glycine and creatinine are markers. Through this metabolic analysis, the measurement of metabolites in AKU urine can be an important method for monitoring kidney and joint damage status by analysing creatinine and glycine concentrations