A Novel Reproducible Cell-Based Model to Investigate Alkaptonuria: Overcoming Biopsy-Related Challenges and Unravelling Hallmark Cellular Events Implicated in Disease Onset and Progression

Alkaptonuria (AKU) is an ultra-rare autosomal recessive metabolic disorder caused by loss-of-function mutations in the HGD gene, which encodes homogentisate 1,2-dioxygenase (HGD), a key enzyme of the tyrosine and phenylalanine catabolic pathway. Enzymatic deficiency leads to lifelong accumulation of homogentisic acid (HGA), which undergoes oxidation and polymerization, forming the melanin-like ochronotic pigment that progressively deposits in connective tissues. Ochronosis drives chronic oxidative stress, inflammation, extracellular matrix degradation, mitochondrial dysfunction, and ultimately severe early onset osteoarthropathy. Increasing evidence also implicates impaired autophagy, lysosomal failure, and Endoplasmatic Reticulum (ER) stress in disease progression, but the scarcity of patient-derived chondrocytes severely limits mechanistic studies. The aim of this thesis was to establish and validate a reliable, reproducible, and human-relevant in vitro AKU model using C20/A4 immortalized human chondrocytes exposed to physiologically relevant HGA concentrations (0.066–0.1 mM). The model was used to investigate ochronotic pigmentation, oxidative and inflammatory responses, amyloidogenic processes, autophagy-lysosome dynamics, and mitochondrial and ER response, under HGA treatment. HGA induced time- and dose-dependent accumulation of ochronotic pigment, confirmed by Fontana Masson staining and ochronotic pigment autofluorescence. Oxidative stress was evident from early ROS production and 4-HNE accumulation after prolonged treatment, paralleled by upregulation of inflammatory mediators, including nitric oxide (NO), Nitric oxide synthase (iNOS), and Cycloxygenase 2 (COX-2). Amyloidogenic events were also reproduced and investigated: Serum Amyloid A (SAA) and Serum Amyloid P (SAP) expression were increased and redistributed into intracellular aggregates, with a marked colocalization under HGA treatment, and Congo red staining revealed amyloid-positive birefringent fibrils exclusively in HGA-treated cells, indicating the presence of amyloid deposition. A key finding of this thesis is the detailed characterization of autophagy impairment. After one week of HGA treatment, LC3, p62, and LAMP1 upregulation, along with increased LC3-LAMP1 and p62-LAMP1 colocalization, indicated active autophagy and efficient autophagosome-lysosome fusion. After two weeks, however, LC3 levels decreased, p62 accumulated, and LC3-LAMP1 colocalization declined sharply, despite persistent LAMP1 upregulation. Live-cell imaging confirmed this biphasic response: LysoTracker and LC3-GFP signals increased at early time points but markedly dropped at two weeks, revealing lysosomal acidification deficiency and defective autophagic flux. These changes were accompanied by progressive mitochondrial dysfunction from early hyperfusion and reduced superoxide production to later fragmentation and elevated mitochondrial ROS, as well as pronounced ER remodeling, with early ER reduction followed by severe ER swelling and structure disorganization. Altogether, these results demonstrate that chronic HGA exposure induces a sequence of pathological events recapitulating AKU progression: pigment formation, oxidative stress, inflammation, amyloidogenesis, and ultimately collapse of cellular quality-control systems due to lysosomal failure, impaired autophagy, mitochondrial dysfunction, and maladaptive ER stress response. This work provides a comprehensive in vitro AKU model and offers an efficient tool for investigating disease mechanisms and testing potential therapeutic strategies.