Search

Alkaptonuria (AKU) is an ultra-rare disease caused by the deficient activity of homogentisate 1,2-dioxygenase enzyme, leading the accumulation of homogentisic acid (HGA) in connective tissues implicating the formation of a black pigmentation called "ochronosis." Although AKU is a multisystemic disease, the most affected tissue is the articular cartilage, which during the pathology appears to be highly damaged. In this study, a model of alkaptonuric chondrocytes and cartilage was realized to investigate the role of HGA in the alteration of the extracellular matrix (ECM). The AKU tissues lost its architecture composed of collagen, proteoglycans, and all the proteins that characterize the ECM. The cause of this alteration in AKU cartilage is attributed to a degeneration of the cytoskeletal network in chondrocytes caused by the accumulation of HGA. The three cytoskeletal proteins, actin, vimentin, and tubulin, were analyzed and a modification in their amount and disposition in AKU chondrocytes model was identified. Cytoskeleton is involved in many fundamental cellular processes; therefore, the aberration in this complex network is involved in the manifestation of AKU disease.

Alkaptonuria (AKU) is a rare disease correlated with deficiency of the enzyme homogentisate 1,2 dioxygenase, which causes homogentisic acid (HGA) accumulation. HGA is subjected to oxidation/polymerization reactions, leading to the production of a peculiar melanin-like pigmentation (ochronosis) after chronic inflammation, which is considered as a triggering event for the generation of oxidative stress. Clinical manifestations of AKU are urine darkening, sclera pigmentation, early severe osteoarthropathy, and cardiovascular and renal complication. Despite major clinical manifestations of AKU being observed in the bones and skeleton, the molecular and functional parameters are so far unknown in AKU. In the present study, we used human osteoblasts supplemented with HGA as a AKU cellular model. We observed marked oxidative stress, and for the first time, we were able to correlate HGA deposition with an impairment in the Wnt/β-catenin signaling pathway, opening a range of possible therapeutic strategies for a disease still lacking a known cure.

Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2-dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog-related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU.

Alkaptonuria (AKU) is an ultra-rare autosomal genetic disorder caused by a defect in the activity of the enzyme homogentisate 1,2-dioxygenase (HGD) that leads to the accumulation of homogentisic acid (HGA) and its oxidized product, benzoquinone acetic acid (BQA), in the connective tissues causing a pigmentation called "ochronosis." The consequent progressive formation of ochronotic aggregates generate a severe condition of oxidative stress and inflammation in all the affected areas. Experimental evidences have also proved the presence of serum amyloid A (SAA) in several AKU tissues and it allowed classifying AKU as a secondary amyloidosis. Although AKU is a multisystemic disease, the most affected system is the osteoarticular one and articular cartilage is the most damaged tissue. In this work, we have analyzed for the first time the cytoskeleton of AKU chondrocytes by means of immunofluorescence staining. We have shown the presence of SAA within AKU chondrocytes and finally we have demonstrated the co-localization of SAA with three cytoskeletal proteins: actin, vimentin, and β-tubulin. Furthermore, in order to observe the ultrastructural features of AKU chondrocytes we have performed TEM analysis, focusing on the Golgi apparatus structure and, to demonstrate that pigmented areas in AKU cartilage are correspondent to areas of oxidation, 4-HNE presence has been evaluated by means of immunofluorescence. J. Cell. Physiol. 232: 1728-1738, 2017. © 2016 Wiley Periodicals, Inc.

The mechanisms of B-type Raf kinase (BRAF) V600E mutation in papillary thyroid cancer (PTC) remain to be elucidated. With the aim to investigate the key candidate genes distinctive to BRAF

Alkaptonuria (AKU) results from defective homogentisate1,2-dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT-PCR, mono- and 2D-Western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by Western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy. J. Cell. Physiol. 227: 3254–3257, 2012. © 2011 Wiley Periodicals, Inc.

Alkaptonuria (AKU) is a rare autosomal recessive disease, associated with deficiency of homogentisate 1,2-dioxygenase activity in the liver. This leads to an accumulation of homogentisic acid (HGA) and its oxidized derivatives in polymerized form in connective tissues especially in joints. Currently, AKU lacks an appropriate therapy. Hence, we propose a new treatment for AKU using the antioxidant N-acetylcysteine (NAC) administered in combinations with ascorbic acid (ASC) since it has been proven that NAC counteracts the side-effects of ASC. We established an in vitro cell model using human articular primary chondrocytes challenged with an excess of HGA (0.33 mM). We used this experimental model to undertake pre-clinical testing of potential antioxidative therapies for AKU, evaluating apoptosis, viability, proliferation, and metabolism of chondrocytes exposed to HGA and treated with NAC and ASC administered alone or in combination addition of both. NAC decreased apoptosis induced in chondrocytes by HGA, increased chondrocyte growth reduced by HGA, and partially restored proteoglycan release inhibited by HGA. A significantly improvement in efficacy was found with combined addition of the two antioxidants in comparison with NAC and ASC alone. Our novel in vitro AKU model allowed us to demonstrate the efficacy of the co-administration of NAC and ASC to counteract the negative effects of HGA for the treatment of ochronotic arthropathy.