New biochemical and cellular approaches in the study of alkaptonuria

Alkaptonuria (AKU) is an ultra-rare autosomal recessive genetic disorder involving a deficient homogentisate 1,2-dioxygenase (HGD) activity, essential for phenylalanine and tyrosine catabolism. This results in the accumulation of homogentisic acid (HGA), which polymerizes to form a dark pigment that is progressively deposited in connective tissues, a phenomenon known as “ochronosis” and associated with significant tissue degeneration. While the symptomatology of AKU is well described in the medical literature, the molecular mechanisms of the disease are far less characterized. Therefore, the aim of this PhD thesis was to enhance our understanding of AKU by using in vitro models to explore its pathophysiological mechanisms. We first focused on developing a novel cellular model of AKU by isolating primary human tenocytes from non-AKU individuals and treating them in vitro with exogenous HGA, enabling the detailed study of ochronotic pigment formation and its relationship with amyloid formation. Tenocyte viability, pigment deposition, and structural integrity were analysed, revealing how increasing HGA concentrations correlated with reduced cell viability, altered cell morphology, and intracellular accumulation of ochronotic pigment. Histological staining procedures, including Fontana Masson and Schmorl’s staining, confirmed progressive pigment accumulation within treated tenocytes over time, replicating ochronotic effects observed in AKU patient tissues. Additionally, biochemical assays demonstrated significant amyloid protein deposition in AKU-affected tenocytes, consistent with secondary amyloidosis. Based on mounting evidence linking ochronosis and alkaptonuric arthropathy with HGA-induced oxidative stress, inflammation, and BMI (eventually as a consequence of treatment with NTBC), we then moved to investigate the potential role of resistin in AKU. Resistin levels were found to be elevated in AKU patients’ blood. Furthermore, resistin could promote pro-inflammatory pathways involving the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in an in vitro model based on human chondrocytes. Immunofluorescence and Western blot analyses showed increased expression of NF-κB subunit p50 following HGA treatment, underscoring the contribution of inflammatory response to cellular stress and tissue damage in AKU. The metabolomic investigation of AKU provided additional insights into its systemic effects. Using nuclear magnetic resonance (NMR) spectroscopy, urinary tract stones and urine samples from AKU patients were analysed, revealing distinct metabolic profiles. This analysis identified specific metabolites linked to HGA metabolism and its downstream pathways. Finally, therapeutic approaches were assessed by investigating nitisinone (NTBC) and its analogs, focusing on their capacity to reduce HGA levels while minimizing side effects in a cell model based on primary human AKU chondrocytes. Although NTBC was shown to decrease HGA and ochronotic pigment formation, associated adverse effects necessitated the exploration of analogs with improved safety profiles. In summary, this research establishes a robust in vitro model that simulates the key pathological features of AKU (ochronotic pigment deposition, inflammation, and amyloid accumulation) thus providing a foundation for future studies on the molecular mechanisms of tissue degeneration in AKU and aiding the development of targeted therapeutic interventions to mitigate the progression of the disease.