| Summary: | Alkaptonuria (AKU) is an ultra rare autosomal recessive disorder resulting from a deficiency of the homogentisate 1,2-dioxygenase (Hgd) enzyme and is characterized by accumulation of homogentisic acid (HGA) in plasma. The disorder has three distinct stages of disease beginning with the excretion of large quantities of HGA in the urine, followed by deposition of HGA as a polymerized pigment in collagenous tissues principally in the cartilages of loaded joints (termed ochronosis), and finally the early onset of severe and devastating osteoarthropathy. There is currently no effective treatment available to AKU patients. Studying the extreme osteoarthritis (OA) phenotype seen in AKU is helping to increase understanding of more common OA, and may help elucidate the mechanisms behind the initiation and progression of OA. Although a murine model of AKU has previously been reported, published studies reported that Hgd-/- mice did not develop ochronosis. The aim of this thesis was to make a comprehensive survey of Hgd-/- mice to identify if ochronosis was present, determine the pathogenesis of the disorder, and to establish whether a potential treatment could prevent pigment deposition in tissues. Through studying a large number of Hgd-/- mice, of a wide variety of ages, this thesis has provided novel findings in relation to the presence of ochronosis in these mice. Using a modified version of Schmorl’s stain, which can specifically identify ochronotic pigment, ochronosis was observed in Hgd-/- mice for the first time. The identification of the earliest stages of ochronosis in Hgd-/- mice provided an opportunity to follow the pathogenesis of the disease throughout their lifespan. Pigmentation was initially identified in the pericellular matrix (PCM) of chondrons in the articular calcified cartilage (ACC), before progressing intracellularly. Examination of aged mice revealed widespread pigmentation throughout all areas of the tibio-femoral joint. Quantification of the pigmented chondrons demonstrated a progressive, linear increase in pigmentation with increasing age. Similar to ochronosis observed in AKU patients, Hgd-/- mice exhibited signs of ochronotic osteoarthropathy which became progressively worse with age. The early identification of ochronosis and its associated osteoarthropathy in Hgd-/- mice is helping to investigate the biochemical and pathological changes associated with AKU in humans. Following the identification of ochronosis Hgd-/- mice were treated with nitisinone, which had been identified as a possible therapeutic for AKU. Administration of nitisinone throughout the lifespan completely prevented deposition of ochronotic pigment. When given mid-life, nitisinone stopped any further pigment deposition but was unable to reverse the effects of ochronosis which had already taken place. The results showed nitisinone to be an effective treatment against the initiation and progression of AKU. During the course of investigation to identify ochronotic pigment at the ultrastructural level, high resolution transmission electron microscopy revealed the presence of previously undescribed microanatomical concentric lamellae in the ACC of Hgd-/- and wild type mice. Although the pathogenesis of these structures is still undetermined they may play a role in OA development as they appear to be associated with tidemark advancement and increased cartilage mineralization. In summary the studies reported in this thesis present novel findings on the identification of pigmentation, and on the initiation, progression and mechanism of ochronosis which leads to ochronotic osteoarthropathy in Hgd-/- mice. The prevention of ochronotic pigmentation, using the drug nitisinone, was also reported for the first time.
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