Study of molecular mechanisms and pharmacological approaches of Alkaptonuria's disease

Alkaptonuria (AKU) is an autosomal recessive disorder, called also Black Bone Disease, for the characteristic dark coloration that some tissues and parts of the body assumed. The pathology is caused by the failure of the enzyme homogentisate 1,2- dioxygenase (HGD), that leads the accumulation of the metabolic intermediate homogentisic acid (HGA), derived by tyrosine. HGA highly reactivity triggers the formation of HGA-derived oxidized products, that react with cellular macromolecules, causing a significant generation of ROS and occurrence of oxidative stress. The ongoing oxidative stress status induces the expression of pro-inflammatory cytokines and the activation of immune cell system, with the consequently occurrence in patients of chronic inflammation and secondary AA amyloidosis. Moreover, HGA molecules bonds generate a dark polymer, called ochronotic pigment, that sticks on several organs, particularly on articular joints. Ochronosis triggers detrimental effects on tissues, as cellular death, extracellular matrix destruction, collagen fibrils rupture, since organs lose their function. Tyrosine and phenylalanine are daily taken by the body with diet, and catabolized by the HGD pathway. Thus, in AKU patients, HGA production is constant, since HGA levels in blood and urine are always elevated. Differently, the ochronotic pigment formation and deposit require more time, indeed symptoms in patients generally appear after the fourth decade of life. The rarity of AKU implies important challenges for its study. Actually, some aspects of the disease are still unexplored, despite it represents the first genetic disorder discovered that follows the principles of Mendelian recessive inheritance. In particular, one of the principal obstacles is the retrieval of AKU samples, that are scarce and generally in bad condition, for the nature of the disease. For this reason, the first step of this thesis project, described in Chapter 3, focused on the set up of in vitro model that allowed to reproduce, in a simple and cheap manner, all the characteristics of pathology. Specifically, it was set up an AKU model based on human primary chondrocytes, that allowed to study the most affected compartment in the disorder. Moreover, it was showed a preliminary study on the development of an in vivo Zebrafish model, with the objective of overcome the numerous limitations related to AKU mouse model. The aim of the present thesis work was dual: explore the molecular characteristics still unknown in AKU, and propose an innovative therapeutic approach, that could be extended to all the chronic inflammatory pathologies (Fig.1). In our lab, it was already showed that HGA administration to cells led the activation of autophagic process. Following this observation, it was explored in Chapter 4 the role of lysosomes in AKU. Indeed, is known that a dysregulation of these organelles frequently occurs in different kind of pathologies, as autoimmune or neurodegenerative disorders. Actually, an increase in lysosomes’ number had been detected in both AKU samples and model. Moreover, AKU lysosomes were localized in the periphery of cells, that represent a not physiologic conformation suggesting a decrease in their activity. Despite the ochronotic pigment deposition on cartilage and collagen fibrils was deeply studied, its formation and intracellular localization was never explicated. Thus, considering lysosomes’ role in the storage and degradation of toxic compounds, it was demonstrated in this work, for the first time, that, when cells were exposed to HGA, the ochronotic pigment was developed intracellularly and concentrated in lysosomes. Obviously, this observation could have enormous impact for the treatment of the disease and the counteraction of ochronotic pigment accumulation. Oxidative detrimental effects of HGA had been already described. Cellular macromolecules, as proteins and lipids, but also organelles, as mitochondria, undergo oxidative reactions, with the occurrence of damages often irreversible. Is known that oxidative stress and ROS target also DNA, with possible deleterious effects for cells and for all the body, considering the potential development of mutations that lead tumors onset. Thus, this aspect needs to be monitored in the progression of disorders. Therefore, the effect of HGA on the genome integrity was studied for the first time, as shown in Chapter 5. It was highlighted that HGA indirectly affected DNA, causing strand breaks and nucleolar stress. This induced the activation of repair mechanisms, on which depended cells destiny. In addition, Chapter 8 was dedicated to the study of inflammatory signal activated in AKU, a crucial characteristic of the disease. For the first time, the disorder was modeled on immune cells, in order to analyze the pattern of cytokines stimulated by HGA. It was demonstrated that the molecule was able to directly induce pro-inflammatory cytokines expression in different immune cell types. This preliminary study provides the basis for deeply understand the key issue of inflammation and immune cells activation in AKU patients. In scientific research, the molecular understanding of biological mechanisms and pathways involved in disorders results fundamental to improve their knowledge. This, beside its crucial significance, provides also the theoretical starting point for the research of possible therapeutic cure. Therefore, frequently these approaches are developed together and strictly connected. Until few years ago, AKU patients were treated only with palliative cure. Recently, EMA (European Medicines Agency) extended the application of the drug nitisinone for the treatment of AKU in adult patients. Despite this represents an important progress for the patients’ care, the drug carries some collateral effects, due to the induction of tyrosinemia, and its inability to counteract inflammation. Hence, in the present project it was studied a new therapeutic approach, described in Chapters 6 and 7, based on the combination of low-doses methotrexate (MTX), a widely used anti-inflammatory drug, with antioxidant molecules. In this way, it was obtained a formulation that combined anti-inflammatory and antioxidants properties, with a stronger effect in the counteraction of these conditions, compared to the effect of single treatments. Moreover, it was proved that the co-administration of drugs allowed to use a low concentration of MTX, with the consequent decrease of its adverse effects, and beneficial impact on patients’ health. The effectiveness of the proposed treatment was tested against typical markers of inflammation, oxidative stress and amyloidosis, proving that its application could be extended to different kind of inflammatory disorders (Chapter 6). It was also studied specifically its effect on AKU model, and demonstrated that the combination of MTX and antioxidants successfully reduced ochronotic pigment and amyloid fibrils (Chapter 7). In summary, the present thesis work gives new insight into molecular mechanisms of AKU and presented a new potential formulate for its treatment.