Cell and tissue models of alkaptonuria

DDMOD-529; No of Pages 8 Drug Discovery Today: Disease Models DRUG DISCOVERY TODAY Vol. xxx, No. xx 2019 Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA DISEASE MODELS Cell and tissue models of alkaptonuria Daniela Braconi, Lia Millucci, Ottavia Spiga, Annalisa Santucci* Department of Biotechnology, Chemistry and Pharmacy (Department of Excellence 2018-2022), University of Siena, Italy Alkaptonuria (AKU) is a rare metabolic disease of historical and medical interest. Despite the identification of gene and protein defects leading to the accumulation of homogentisic acid (HGA), little is known on how HGA is transformed into an ochronotic pig- Section editors: Daniela Braconi – Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Italy. Annalisa Santucci – Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Italy. ment (the hallmark of the disease) leading to a range of clinical manifestations. Major obstacles in tackling the pathological features of AKU are the rarity of biological samples, the invasiveness of sampling techniques and the intrinsic difficulties of studying the pigmented tissues. This review provides an overview of the in vitro and ex vivo cell and tissue models that were recently developed and characterized to fill the above-mentioned gaps in the knowledge of AKU. Introduction Alkaptonuria (AKU, OMIM: 203500) is a rare genetic disease of significant historical and medical interest as it was among the first identified inborn errors of metabolism [1,2,3]. Several gene mutations have been described so far in AKU subjects [4]; they are linked to a deficient activity of the enzyme homogentisate 1,2-dioxygenase (HGD, E.C.1.13.11..5), which is involved in the catabolism of phenylalanine and tyrosine and expressed in a variety of tissues [5,6,7]. HGD deficiency leads to homogentisic aciduria (responsible of urine darkening) and deposition of an ochronotic pigment in connective tissues (ochronosis) [8]. The most disabling manifestations of ochronosis are at the articular level with osteoarthritis-like joint damage causing high morbidity [9]. *Corresponding author: A. Santucci ([email protected]) Cardiac tissues can be affected too, leading to the development of aortic valve diseases [10,11]. Progress in understanding rare diseases has often been limited despite advances in molecular biology. In AKU and ochronotic arthropathy, this lack of knowledge was one of the major obstacles in developing appropriate and timely pharmacological interventions. Ascorbic acid and low protein diet were the first attempts to treat AKU, though with conflicting evidence [12–14]. More recently, clinical trials were run to evaluate the effectiveness and safety of long-term nitisinone in AKU [15] [https://clinicaltrials.gov/ct2/show/ NCT01916382], the results of which are under evaluation. So far, however, there is still no approved pharmacological treatment for AKU and only palliative care and/or surgery are available to address patients’ symptoms. Why developing models in AKU? Today, the exact molecular composition of the ochronotic pigment observed in AKU and the mechanisms for its production are still obscure. A major obstacle in tackling the pathological features of AKU is obviously the rarity of biological samples (incidence of AKU is 1:250000-1:1000000 [5]). Studies on ochronotic pigment are also hindered because invasive sampling techniques are required for the collection of pigmented samples. Furthermore, ochronosis often severely damages tissues, making them intrinsically difficult to study (e.g. tissues have increased stiffness and brittleness, 1740-6757/$ © 2020 Elsevier Ltd. All rights reserved. https://doi.org/10.1016/j.ddmod.2019.12.001 Please cite this article in press as: Braconi D, et al. Cell and tissue models of alkaptonuria, Drug Discov Today: Dis Model (2020), https://doi.org/10.1016/j.ddmod.2019.12.001 1 DDMOD-529; No of Pages 8 Drug Discovery Today: Disease Models | and the pigment is resistant to chemical and enzyme degradation) [16]. However, clarifying the molecular mechanisms of AKU and ochronosis would provide significant advantages. Finding a cure able to address the complications observed in AKU would significantly improve patients’ quality of life. At the same time, since AKU can be considered a model for more common rheumatic conditions such as osteoarthritis (OA) [17], the social and economic impact of studying AKU would be much wider. Therefore, the development of suitable models able to reproduce AKU becomes fundamental. This review will provide a brief discussion of the in vitro and ex vivo cell and tissue models that were recently developed and characterized to fill the above-mentioned gaps in the knowledge of AKU. In particular, in vitro models consisting of human cell lines, primary cells from the osteoarticular compartment, plasma and tissues were used to investigate the molecular effects of excess HGA. Furthermore, ex vivo human cartilage explants treated with HGA offered a disease model mimicking AKU under laboratory conditions, and ex vivo cultured cells from alkaptonuric individuals were instrumental for the molecular characterization of AKU and the validation of in vitro findings. Both in vitro and ex vivo models were also useful for drug testing. The first HGA-treated in vitro models of AKU Human serum treated with HGA In 1994, Hegedus and Nayak showed for the first time the in vitro formation of melanin like pigments in blood or plasma upon addition of HGA (range 1.5–6 mM) [18]. The tested HGA concentrations were higher compared to circulating HGA in AKU (50 400 mM) [19], hence more recently a novel in vitro serum model treated with 330 mM HGA was developed [20]. This latter model provided a non-destructive SDS-PAGEbased method to analyze qualitatively and quantitatively the auto-fluorescence of protein bands containing HGA-induced pigments, and showed the efficacy of some antioxidants in preventing or slowing down HGA-induced ochronotic pigmentation and protein oxidation [20]. The HGAinduced oxidative damage towards serum proteins was analyzed in terms of carbonylation (i.e. irreversible damage) and thiol oxidation (including both reversible and irreversible post-translational modifications) suggesting mechanisms of HGA toxicity and novel therapeutic strategies for AKU. A further proteomic and redox-proteomic analysis of the HGAtreated serum model allowed the identification of serum proteins oxidized by HGA and/or binding the HGA-derived quinone, indicating possible alterations of metal homeostasis and aberrant protein aggregation due to HGA [21]. Human cartilage treated with HGA Tinti et al. developed a tissue-based model of AKU by using thin sections of human cartilage that were cultured in laboratory in HGA-conditioned medium or in control conditions 2 Vol. xxx, No. xx 2019 over a 2-month period [22] offering the possibility of threedimensional tissue studies in an avascular tissue. Microscopy analysis revealed morphological integrity for both control and HGA-treated cartilage, indicating preservation of tissue architectures. After one month, ochronotic deposits varying in location and distribution appeared in HGA-treated cartilage, with areas showing only minute granules and others more severe advanced forms of pigmentation; there were also regions where no pigmentation occurred. This pattern was suggestive of nucleation points (smaller granules) leading to the formation of larger ochronotic deposits, a phenomenon that was already hypothesized in AKU [23], adding validity to the model. The finding of pigment around the lacunae space suggested that chondrocytes found there could undergo significant stress. Later on, the same model was used to show that the exogenous addition of HGA induced the deposition of amyloid fibers co-localizing with the ochronotic pigment and amyloid proteins [24], as discussed in the following sections. Human cells treated with HGA Rabbit and human articular chondrocytes showed different susceptibilities to HGA-toxicity compared to human fibroblasts, as fibroblasts required higher HGA concentrations to obtain a comparable growth inhibition [25]. In these cell models, supplementation with ASC ameliorated growth and prevented the morphological changes observed with HGA treatment [25]. Starting from these very preliminary observations, a novel in in vitro cell model consisting of primary chondrocytes isolated from human articular cartilage and treated with exogenously added HGA was proposed, offering the opportunity to undertake pre-clinical testing of potential antioxidant therapies for AKU [26]. This cell model was first used to set-up culture conditions, in particular to find out the optimal HGA concentration leading to the formation of ochronotic pigment in vitro in a reasonable time with minimal effects on cell viability (0.33 mM). HGA-treated chondrocytes showed decreased proliferation, dose-dependent apoptosis (without necrosis) and a substantial decrease in the release of proteoglycans (used as a marker of chondrocyte anabolism). These effects were counteracted by treatment with N-acetylcysteine (NAC) either alone or in combination with ASC. Protein carbonylation, a widely accepted biomarker of oxidative stress [27], was significantly higher in HGA-treated chondrocytes [26]. Later on, additional in vitro cell-based models of AKU were proposed by using three osteosarcoma cell lines, namely MG63, SaOS-2 and TE85, cultured in medium containing HGA (range 0.1 mM–1 mM) [28]. These cell lines were used because they are known to secrete extra-cellular matrix components including type I collagen, but without matrix mineralization. Light microscopy, transmission electron www.drugdiscoverytoday.com Please cite this article in press as: Braconi D, et al. Cell and tissue models of alkaptonuria, Drug Discov Today: Dis Model (2020), https://doi.org/10.1016/j.ddmod.2019.12.001 DDMOD-529; No of Pages 8 Vol. xxx, No. xx 2019 microscopy, and Schmorl’s stain were used to detect the HGA-induced pigment deposits, which showed similarities with parallel observations in fibroblasts from the knee joint capsule of an alkaptonuric subject. Additional analyses confirmed that HGA-induced pigmentation was much more rapid in vitro than in vivo (where protective mechanisms may exist) and in the presence of cells (discoloration of cell-free medium took longer). Collagen synthesis, assessed through measure of secreted P1NP, increased on treatment with HGA up to 330 mM but there was almost a complete inhibition at 1 mM HGA, which caused severe toxicity. Pigment deposition could be observed even for non-toxic HGA concentrations (from 33 mM to 330 mM). The level of pigmentation varied among the cell lines, being higher in SaOS-2 cells that showed the lowest collagen synthesis. Thus, deposition of ochronotic pigment seemed not to be dependent on HGA toxicity or collagen synthesis [28]. A human articular chondrocyte cell line (C20) was also used as an additional model to study alkaptonuric ochronosis. Cells were challenged with HGA, alone or in combination with ASC. The supplementation with ASC was used to enhance the production of collagen [29] and to assess possible anti-oxidant effects against HGA-mediated toxicity. A temporal analysis of cell proliferation, protein expression and protein oxidation profiles was undertaken [30]. The comparative proteomic approach showed that ASC caused a general under-expression of chondrocyte proteins, especially those involved in the stress response and cell morphology/motility functional classes. The same trend was observed when ASC was administered together with HGA, overall pointing to a pro-oxidant effect of ASC. The under-expression of several proteins with structural functions and the over-expression of proteins assisting in protein folding was found in HGA-treated cells, in good agreement with proteomic studies on osteoarthritic cartilage. In particular, the altered expression of the protein PDIA1 was highlighted: this is a fundamental protein in load bearing joints. The effects generated by the concomitant treatment of cells with HGA and ASC reinforced the hypothesis of an oxidative imbalance, which is a crucial regulator for cartilage integrity, and no beneficial effect of ASC could be identified. The redox-proteomic analysis of carbonylation was undertaken to assess protein oxidation, revealing the presence of oxidized proteins (also as high molecular weight aggregates) in HGA-treated cells. Since oxidized proteins are more prone to aggregation, and in the light of the proteome changes observed, authors were able to speculate on novel mechanisms for ochronosis, and recommended caution in considering ASC a beneficial drug in AKU [30]. In additional works, chondrocytes treated with HGA showed for the first time that HGA could be linked to the presence of amyloid in AKU [24,31] and were instrumental to assess the effects of methotrexate [24] and antioxidants [31] Drug Discovery Today: Disease Models | in reducing the release of pro-inflammatory molecules and deposition of HGA-induced amyloid. An in vitro interaction between HGA and SAA (and other amyloidogenic proteins) was also documented in a cell-free model of AKU. Such an interaction led to a quick aggregation of SAA into polymers of amyloid nature, allowing speculations on possible binding sites for HGA or its metabolites onto SAA molecule [32]. Observations in ex vivo models of AKU The characterization of the HGA-supplemented models described above not only confirmed the pro-oxidant effect of HGA, but also offered novel cues to study AKU pathophysiology. Inflammation, protein aggregation, and impaired cell function were found in these models, suggesting that they can be collectively responsible for the symptomatology of alkaptonuric patients. These findings, together with the availability of patients’ samples prompted the characterization of ex vivo alkaptonuric models, i.e. cells and tissues from diseased individuals, where such findings were validated and novel insights into molecular aspects of AKU were discovered. Inflammation, oxidative stress and amyloidosis The biochemical characterization of alkaptonuric chondrocytes (carried out on two sub-populations termed ‘‘white’’ and ‘‘black’’ according to the macroscopic pigmentation of the hip cartilage they were obtained from), showed that the deposition of ochronotic pigments occurred even in areas of cartilage with no visible pigmentation. Both ‘‘white’’ and ‘‘black’’ chondrocytes had increased apoptosis, released nitric oxide and pro-inflammatory cytokines, and shared similar proteomes [33]. When compared to healthy counterparts, alkaptonuric chondrocytes showed altered expression of proteins involved in protein fate, cell structure and organization, and stress response, as well as increased protein oxidation/ aggregation, in good agreement with preliminary observations in in vitro HGA-treated cell models [30]. Therefore, inflammation, impaired cell functioning, oxidative stress and protein aggregation could be hypothesized in alkaptonuric cells to explain the damage observed in vivo in affected patients. In cartilage, inflammation could be linked to the activation of a catabolic program, production of proteases, apoptosis and release of cytokines, leading to tissue disruption. The proteome alterations observed in alkaptonuric chondrocytes suggested significant alterations of cell structure and organization and were reminiscent of an impaired ability to withstand loading forces and cope with external stresses. The proteomic investigation of alkaptonuric articular cells indicated an aberrant expression of several proteins involved in the control of folding/unfolding and amyloidosis [33], a condition where the abnormal deposition of fibers generated upon aggregation is observed. One of the key molecules that was under-expressed in alkaptonuric chondrocytes was www.drugdiscoverytoday.com Please cite this article in press as: Braconi D, et al. Cell and tissue models of alkaptonuria, Drug Discov Today: Dis Model (2020), https://doi.org/10.1016/j.ddmod.2019.12.001 3 DDMOD-529; No of Pages 8 Drug Discovery Today: Disease Models | cathepsin D, a protein with a protective role against the development of secondary (reactive or type AA) amyloid fibrils due to persistently increased serum amyloid A (SAA) levels. This finding prompted the investigators to assess the levels of circulating SAA and the presence of such amyloid fibrils in tissues from alkaptonuric subjects. Several papers are now available showing that nearly all the alkaptonuric subjects tested so far show pathological circulating levels of SAA [24,34,35,36,37,38]. Notably, patients presenting with higher circulating SAA levels reported more often a decreased quality of life or have worse clinical scores [37], suggesting that SAA may be a useful prognostic biomarker in AKU, currently lacking. The presence of AA amyloidosis was also documented in a variety of tissues from alkaptonuric subjects such as cartilage, synovia and heart valves [11,24,34,35] where there was an interesting co-localization of amyloid and ochronotic pigment. Notably, amyloidosis was found in cartilage and synovial tissues from young, even asymptomatic alkaptonuric subjects, suggesting that the detection of amyloid deposits at an early phase may be important for treatment [39]. Tissues from heart, labial salivary gland, tendon and infrapatellar fat showed amyloid deposition as well [40], which could be important for diagnostic purposes [40]. Oxidative stress and inflammation markers were also investigated by Braconi et al. in the first proteomic profiling of serum and plasma from alkaptonuric individuals [36]. The tested subjects were one female and five males (range 39–66 years) presenting with ochronotic complications treated by surgery and increased circulating levels of SAA and Advanced Oxidation Protein Products (AOPP). Analogies were found with proteomic investigations in the HGA-treated serum model [21] and osteoarthritis (OA), supporting the hypothesis that AKU can be considered a disease model for OA [17]. Since several blood proteins with a well-defined role in oxidative stress were found at aberrant levels, the pro-oxidant role of HGA was confirmed. Possible protein biomarkers to assess disease severity, monitor progression and response to treatment, which are still lacking in AKU, were identified as well [36]. Neoangiogenesis Several findings from either in vitro or ex vivo AKU models suggested that HGA and ochronotic pigment may be linked to inflammation and responsible for macrophage infiltration [31,35,41], release of pro-inflammatory cytokines from articular cells [24,31], impaired chondrocyte functions and chondroptosis (i.e. a typical ultra-structural pattern for dying chondrocytes) [33,35,41] and impaired cartilage homeostasis [26,35,41]. Since angiogenesis is involved in a plethora of inflammatory rheumatic diseases, researchers speculated that this could be the case of AKU too. The analysis of several knee synovium samples from alkaptonuric subjects showed, 4 Vol. xxx, No. xx 2019 besides the macroscopically visible ochronotic pigment, the presence of hyperemic pigmented villi. Microscopy examination revealed lymphocytic inflammation and blood vessels mainly in proximity to ochronotic shards; immunofluorescence analyses confirmed that these vessels were newly formed and showed similarities to observations in OA [42]. Accordingly, inflammation and neoangiogenesis appeared to be part of a vicious crosstalk for AKU progression, supplying oxygen and nutrients for inflammatory cells as well as angiogenic growth factors for novel vessels to be formed [42]. Similarly, it could be speculated that increased HGA levels can be delivered to newly vascularized areas, promoting pigment formation and deposition possibly through interactions with other factors (e.g. SAA). Ciliopathy The comparative proteomics analysis of HGA-treated and alkaptonuric cells showed that several proteins involved in cell organization, and especially those of cytoskeleton and microtubules, have aberrant expression and/or are oxidized due to HGA [30,33,43]. The impairment of cytoskeleton network was confirmed in HGA-treated and alkaptonuric chondrocytes, which showed altered immunofluorescence staining of actin, vimentin and beta tubulin proteins [44]. Interestingly, in these cells the co-localization of cytoskeleton markers, SAA, lipid peroxidation products and ochronotic pigment was found [44]. Primary cilia play an essential role in the homeostasis of articular cartilage through their involvement in mechanosignalling and Hedgehog signalling pathways. In OA, the activation of the Hedgehog signaling promotes cartilage degeneration [45]. Due to the similarities between OA and AKU, the integrity of the cilium was thus investigated also in AKU. Both HGA-treated and alkaptonuric human chondrocytes showed shorter cilia and enhanced expression of Gli-1 protein, which is involved in Hedgehog signaling, suggesting an HGA-induced ciliopathy [46]. Several Smo antagonists could counteract cilium shortening and restore Gli-1 expression, sometimes even at very low concentrations [46]. Furthermore, alkaptonuric and HGA-treated chondrocytes showed disruption of actin contractility and ciliary trafficking. These changes were paralleled by a complete inability to activate Hedgehog signaling in presence of exogenous ligands [47] and confirmed that the cilium may be a novel therapeutic target in AKU. Tissue characterization and mechanical properties of alkaptonuric cartilage Synchrotron Radiation Infrared and X-Ray Fluorescence microscopy techniques were used to characterize the chemical composition and morphology of alkaptonuric cartilage, showing depletion of proteoglycans associated with increased sodium content, accumulation of lipids in the perilacunar www.drugdiscoverytoday.com Please cite this article in press as: Braconi D, et al. Cell and tissue models of alkaptonuria, Drug Discov Today: Dis Model (2020), https://doi.org/10.1016/j.ddmod.2019.12.001 DDMOD-529; No of Pages 8 Vol. xxx, No. xx 2019 Drug Discovery Today: Disease Models | Molecular mechanisms Novel biomarkers Novel drug targets Ochronotic pigment Oxidative stress Amyloid Inflammation Impaired protein/cell/tissue functions Neoangiogenesis Altered cytoskeleton/cilium serum+HGA [18,20,21] cartilage+HGA [22,24] Serum SAA (amyloidosis) Serum chitotriosidase activity (inflammation) cells+HGA [24-26,28,30,31] alkaptonuric cells [29,33,43,44,46] Amyloidosis Ciliopathy Neoangiogenesis alkaptonuric tissues [11,24,34-38,40-42,48] Drug Discovery Today: Disease Models Fig. 1. Early findings in cell- and tissue-based models where the effects of exogenous HGA supplementation were investigated laid the basis for the characterization of novel in vitro alkaptonuric models and patients’ samples, which overall provided further molecular insights into the disease, novel possible biomarkers and drug targets. Input parameters Treatment optimisation In Vivo In Silico New data evaluation Input parameters Treatment optimisation Experimental validation In Vitro Drug Discovery Today: Disease Models Fig. 2. The in silico-in vitro-in vivo chain. The clockwise arrows illustrate data input whereas the anti-clockwise arrows represent the feedback data used for model validation in support of further optimization. www.drugdiscoverytoday.com Please cite this article in press as: Braconi D, et al. Cell and tissue models of alkaptonuria, Drug Discov Today: Dis Model (2020), https://doi.org/10.1016/j.ddmod.2019.12.001 5 DDMOD-529; No of Pages 8 Drug Discovery Today: Disease Models | regions, and confirming a close correlation between amyloid and ochronotic pigment. Lower magnesium was found in perilacunar regions, which could be associated to arthropathy and support the hypothesis of a possible interaction with HGA or its derivatives. A typical feature of alkaptonuric cartilage was also the presence of carbonate moieties in proximity of the most pigmented areas, which could suggest calcification as seen in OA, though the different chemical composition of such calcifications indicated once more the peculiar features of AKU [38]. Thermal and rheological analyses on alkaptonuric cartilage [48] showed dramatic alterations of hydrostatic pressure and a decreased load-bearing capacity, which could be related to an altered trafficking within the tissue matrix. In parallel, increased stiffness and decreased dissipative and lubricant functions were found. A decreased heat capacity suggested an impaired chondrocyte metabolism too. These findings were validated in HGA-treated healthy cartilage, indicating that HGA is the toxic responsible of morphological and mechanical alterations of cartilage in AKU [48]. Conclusions and future perspectives In the last years, our knowledge of the rare orphan disease AKU increased significantly. Early findings on the effects of exogenous HGA obtained in cell- and tissue-based models laid the basis for the characterization of novel in vitro and ex vivo models of AKU, which provided further molecular insights into the disease. In these novel models, inflammation, oxidative stress, secondary amyloidosis [11,16,34,35,36,40,41,20,21,24,26,30–33], alterations of cytoskeleton [44], ciliopathy [46,47], and neoangiogenesis [42] were found to play a role in AKU (Fig. 1). The activity of antioxidants [20,26,31] and methotrexate [24] was assayed in vitro to evaluate their role in counteracting HGArelated oxidative stress and amyloidosis, respectively. Furthermore, the activity of nitisinone analogues was tested in vitro [49], and an silico approach highlighted potential binding sites for drugs acting as pharmacological chaperones for the enzyme HGD [50] (Fig. 1). Similarities between AKU and OA at the molecular level were found [38,46,47], which reinforced the significance of findings in AKU and the potential application of similar workflows for more common diseases. Since the development of the first HGA-treated cell and tissue models of AKU, a significant amount of data have been produced and several biomarkers have been investigated in alkaptonuric samples. These data are now stored in a dedicated database (named ApreciseKUre) [51,52], which is continuously updated and refreshed to highlight significant correlations between data. The overall goal of ApreciseKUre is the development of a Precision Medicine approach in AKU, representing a best practice model for other rare diseases. Future work will be necessary to implement ApreciseKUre, for instance by including data on additional protein post-translational modifications (e.g. lipid peroxidation markers, thiol 6 Vol. xxx, No. xx 2019 modifications) or relevant tissue-specific markers to fully understand their relevance in vivo. This may help the development of therapeutic strategies for AKU or the identification of additional drugs to treat HGA-related inflammation and amyloidosis. Further possibilities are also offered by the recently developed mouse models of AKU [53,54] where hypotheses on molecular mechanisms obtained by in vitro assays may be validated, novel hypotheses generated and drugs (other than nitisinone) tested. In parallel, significant advances should be expected from in silico approaches (Fig. 2) that could be used to search for additional data sets and to identify molecules for in vitro and in vivo screenings. Conflict of interest The authors declared that they have no conflict of interest. Acknowledgements The authors thank aim AKU, Associazione Italiana Malati di Alcaptonuria (ORPHA263402). This work was supported in part by European Commission Seventh Framework Programme funding granted in 2012 (DevelopAKUre, project number: 304985). The funding source was not involved in the study design, collection, analysis and interpretation of data, the writing of the manuscript, or in the decision to submit the manuscript for publication. References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest [1] Garrod AE. About alkaptonuria. Med Chir Trans 1902;85:69–78. [2] Garrod AE. The incidence of alkaptonuria: a study in chemical individuality. 1902. Mol Med 1996;2:274–82. [3] Gaines JJ. The pathology of alkaptonuric ochronosis. Hum Pathol 1989;20:40–6. [4] Nemethova M, Radvanszky J, Kadasi L, Ascher DB, Pires DE V, Blundell TL, et al. Twelve novel HGD gene variants identified in 99 alkaptonuria patients: focus on ‘black bone disease’ in Italy. Eur J Hum Genet 2016;24:66–72. http://dx.doi.org/10.1038/ejhg.2015.60. [5] Phornphutkul C, Introne WJ, Perry MB, Bernardini I, Murphey MD, Fitzpatrick DL, et al. Natural History of Alkaptonuria. N Engl J Med 2002;347:2111–21. http://dx.doi.org/10.1056/NEJMoa021736 This papers provides an accurate description of the disease. [6] Bernardini G, Laschi M, Geminiani M, Braconi D, Vannuccini E, Lupetti P, et al. Homogentisate 1,2 dioxygenase is expressed in brain: implications in alkaptonuria. J Inherit Metab Dis 2015;38. http://dx.doi.org/10.1007/ s10545-015-9829-5. [7] Laschi M, Tinti L, Braconi D, Millucci L, Ghezzi L, Amato L, et al. Homogentisate 1,2 dioxygenase is expressed in human osteoarticular cells: implications in alkaptonuria. J Cell Physiol 2012;227. http://dx.doi. org/10.1002/jcp.24018. [8] La Du BN, Zannoni VG, Laster L, Seegmiller JE. The nature of the defect in tyrosine metabolism in alcaptonuria. J Biol Chem 1958;230:251–60. [9] Laxon S, Ranganath L, Timmis O. Living with alkaptonuria. BMJ 2011;343. http://dx.doi.org/10.1136/bmj.d5155. d5155–d5155. www.drugdiscoverytoday.com Please cite this article in press as: Braconi D, et al. Cell and tissue models of alkaptonuria, Drug Discov Today: Dis Model (2020), https://doi.org/10.1016/j.ddmod.2019.12.001 DDMOD-529; No of Pages 8 Vol. xxx, No. xx 2019 [10] Pettit SJ, Fisher M, Gallagher JA, Ranganath LR. Cardiovascular manifestations of alkaptonuria. J Inherit Metab Dis 2011;34:1177–81. http://dx.doi.org/10.1007/s10545-011-9339-z. [11] Millucci L, Ghezzi L, Braconi D, Laschi M, Geminiani M, Amato L, et al. Secondary amyloidosis in an alkaptonuric aortic valve. Int J Cardiol 2014;172. http://dx.doi.org/10.1016/j.ijcard.2013.12.117. [12] Wolff JA, Barshop B, Nyhan WL, Leslie J, Seegmiller JE, Gruber H, et al. Effects of ascorbic acid in alkaptonuria: alterations in benzoquinone acetic acid and an ontogenic effect in infancy. Pediatr Res 1989;26:140–4. http://dx.doi.org/10.1203/00006450198908000-00015. [13] Forslind K, Wollheim FA, Akesson B, Rydholm U. Alkaptonuria and ochronosis in three siblings. Ascorbic acid treatment monitored by urinary HGA excretion. Clin Exp Rheumatol n.d.; 6:289–92. [14] Morava E, Kosztolányi G, Engelke UFH, Wevers RA. Reversal of clinical symptoms and radiographic abnormalities with protein restriction and ascorbic acid in alkaptonuria. Ann Clin Biochem 2003;40:108–11. http:// dx.doi.org/10.1258/000456303321016268. [15] Ranganath LR, Milan AM, Hughes AT, Dutton JJ, Fitzgerald R, Briggs MC, et al. Suitability of nitisinone in alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid. Ann Rheum Dis 2016;75. http://dx.doi.org/10.1136/annrheumdis2014-206033. [16] Braconi D, Millucci L, Ghezzi L, Santucci A. Redox proteomics gives insights into the role of oxidative stress in alkaptonuria. Expert Rev Proteomics 2013;10. http://dx.doi.org/10.1586/14789450.2013.858020 This paper shows how proteomics and redox proteomics might successfully overcome the difficulties of studying a rare disease such as alkaptonuria and the limitations of the hitherto adopted approaches. [17] Gallagher JA, Ranganath LR, Boyde A. Lessons from rare diseases of cartilage and bone. Curr Opin Pharmacol 2015;22:107–14. http://dx.doi. org/10.1016/j.coph.2015.04.002. [18] Hegedus ZL, Nayak U. Homogentisic acid and structurally related compounds as intermediates in plasma soluble melanin formation and in tissue toxicities. Arch Int Physiol Biochim Biophys 1994;102:175–81. [19] Martin JPJ, Batkoff B. Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis. Free Radic Biol Med 1987;3:241–50. [20] Braconi D, Laschi M, Amato L, Bernardini G, Millucci L, Marcolongo R, et al. Evaluation of anti-oxidant treatments in an in vitro model of alkaptonuric ochronosis. Rheumatology 2010;49:1975–83. http://dx.doi. org/10.1093/rheumatology/keq175. [21] Braconi D, Bianchini C, Bernardini G, Laschi M, Millucci L, Spreafico A, et al. Redox-proteomics of the effects of homogentisic acid in an in vitro human serum model of alkaptonuric ochronosis. J Inherit Metab Dis 2011;34. http://dx.doi.org/10.1007/s10545-011-9377-6. [22] Tinti L, Spreafico A, Chellini F, Galeazzi M, Santucci A. A novel ex vivo organotypic culture model of alkaptonuria-ochronosis. Clin Exp Rheumatol 2011;29:693–6. [23] Taylor AM, Wlodarski B, Prior IA, Wilson PJM, Jarvis JC, Ranganath LR, et al. Ultrastructural examination of tissue in a patient with alkaptonuric arthropathy reveals a distinct pattern of binding of ochronotic pigment. Rheumatology 2010;49:1412–4. http://dx.doi.org/10.1093/ rheumatology/keq027. [24] Millucci L, Spreafico A, Tinti L, Braconi D, Ghezzi L, Paccagnini E, et al. Alkaptonuria is a novel human secondary amyloidogenic disease. Biochim Biophys Acta - Mol Basis Dis 2012;1822. http://dx.doi.org/ 10.1016/j.bbadis.2012.07.011. [25] Angeles AP, Badger R, Gruber HE, Seegmiller JE. Chondrocyte growth inhibition induced by homogentisic acid and its partial prevention with ascorbic acid. J Rheumatol 1989;16:512–7. [26] Tinti L, Spreafico A, Braconi D, Millucci L, Bernardini G, Chellini F, et al. Evaluation of antioxiodant drugs for the treatment of ochronotic alkaptonuria in an in vitro human cell model. J Cell Physiol 2010;225. http://dx.doi.org/10.1002/jcp.22199. Drug Discovery Today: Disease Models | [27] Dalle-Donne I, Rossi R, Giustarini D, Milzani A, Colombo R. Protein carbonyl groups as biomarkers of oxidative stress. Clin Chim Acta 2003;329:23–38. [28] Tinti L, Taylor AM, Santucci A, Wlodarski B, Wilson PJ, Jarvis JC, et al. Development of an in vitro model to investigate joint ochronosis in alkaptonuria. Rheumatology 2011;50:271–7. http://dx.doi.org/10.1093/ rheumatology/keq246. [29] Ibold Y, Lübke C, Pelz S, Augst H, Kaps C, Ringe J, et al. Effect of different ascorbate supplementations on in vitro cartilage formation in porcine high-density pellet cultures. Tissue Cell 2009;41:249–56. http://dx.doi. org/10.1016/j.tice.2008.12.001. [30] Braconi D, Laschi M, Taylor AM, Bernardini G, Spreafico A, Tinti L, et al. Proteomic and redox-proteomic evaluation of homogentisic acid and ascorbic acid effects on human articular chondrocytes. J Cell Biochem 2010;111. http://dx.doi.org/10.1002/jcb.22780. [31] Spreafico A, Millucci L, Ghezzi L, Geminiani M, Braconi D, Amato L, et al. Antioxidants inhibit SAA formation and pro-inflammatory cytokine release in a human cell model of alkaptonuria. Rheumatol (United Kingdom) 2013;52. http://dx.doi.org/10.1093/rheumatology/ ket185. [32] Braconi D, Millucci L, Bernini A, Spiga O, Lupetti P, Marzocchi B, et al. Homogentisic acid induces aggregation and fibrillation of amyloidogenic proteins. Biochim Biophys Acta - Gen Subj 2017;1861. http://dx.doi.org/ 10.1016/j.bbagen.2016.11.026. [33] Braconi D, Bernardini G, Bianchini C, Laschi M, Millucci L, Amato L, et al. Biochemical and proteomic characterization of alkaptonuric chondrocytes. J Cell Physiol 2012;227. http://dx.doi.org/10.1002/ jcp.24033. [34] Millucci L, Ghezzi L, Paccagnini E, Giorgetti G, Viti C, Braconi D, et al. Amyloidosis, inflammation, and oxidative stress in the heart of an alkaptonuric patient. Mediators Inflamm 2014;2014. http://dx.doi.org/ 10.1155/2014/258471. [35] Millucci L, Braconi D, Bernardini G, Lupetti P, Rovensky J, Ranganath L, et al. Amyloidosis in alkaptonuria. J Inherit Metab Dis 2015;38. http:// dx.doi.org/10.1007/s10545-015-9842-8 This paper provides an overview on direct and indirect lines of evidence related to the presence of amyloidosis in alkaptonuria. [36] Braconi D, Bernardini G, Paffetti A, Millucci L, Geminiani M, Laschi M, et al. Comparative proteomics in alkaptonuria provides insights into inflammation and oxidative stress. Int J Biochem Cell Biol 2016;81. http:// dx.doi.org/10.1016/j.biocel.2016.08.016. [37] Braconi D, Giustarini D, Marzocchi B, Peruzzi L, Margollicci M, Rossi R, et al. Inflammatory and oxidative stress biomarkers in alkaptonuria: data from the DevelopAKUre project. Osteoarthr Cartil 2018;26. http:// dx.doi.org/10.1016/j.joca.2018.05.017 This paper shows how established biomarkers might be used as disease activity markers in alkaptonuria and provides data on a very large cohort of alkaptonuric subjects thanks to the set-up of an international consortium (DevelopAKUre). [38] Mitri E, Millucci L, Merolle L, Bernardini G, Vaccari L, Gianoncelli A, et al. A new light on Alkaptonuria: a Fourier-transform infrared microscopy (FTIRM) and low energy X-ray fluorescence (LEXRF) microscopy correlative study on a rare disease. Biochim Biophys Acta Gen Subj 2017;1861:1000–8. http://dx.doi.org/10.1016/j. bbagen.2017.02.008. [39] Millucci L, Bernardini G, Spreafico A, Orlandini M, Braconi D, Laschi M, et al. Histological and ultrastructural characterization of alkaptonuric tissues. Calcif Tissue Int 2017;101. http://dx.doi.org/10.1007/s00223-0170260-9. [40] Millucci L, Ghezzi L, Bernardini G, Braconi D, Lupetti P, Perfetto F, et al. Diagnosis of secondary amyloidosis in alkaptonuria. Diagn Pathol 2014;9. http://dx.doi.org/10.1186/s13000-014-0185-9. [41] Millucci L, Giorgetti G, Viti C, Ghezzi L, Gambassi S, Braconi D, et al. Chondroptosis in alkaptonuric cartilage. J Cell Physiol 2015;230. http:// dx.doi.org/10.1002/jcp.24850. [42] Millucci L, Bernardini G, Marzocchi B, Braconi D, Geminiani M, Gambassi S, et al. Angiogenesis in alkaptonuria. J Inherit Metab Dis 2016;39. http:// dx.doi.org/10.1007/s10545-016-9976-3. www.drugdiscoverytoday.com Please cite this article in press as: Braconi D, et al. Cell and tissue models of alkaptonuria, Drug Discov Today: Dis Model (2020), https://doi.org/10.1016/j.ddmod.2019.12.001 7 DDMOD-529; No of Pages 8 Drug Discovery Today: Disease Models | [43] Bernardini G, Braconi D, Spreafico A, Santucci A. Post-genomics of bone metabolic dysfunctions and neoplasias. Proteomics 2012;12. http://dx. doi.org/10.1002/pmic.201100358. [44] Geminiani M, Gambassi S, Millucci L, Lupetti P, Collodel G, Mazzi L, et al. Cytoskeleton aberrations in Alkaptonuric Chondrocytes. J Cell Physiol 2017;232. http://dx.doi.org/10.1002/jcp.25500. [45] Wang M, Shen J, Jin H, Im H-J, Sandy J, Chen D. Recent progress in understanding molecular mechanisms of cartilage degeneration during osteoarthritis. Ann N Y Acad Sci 2011;1240:61–9. http://dx.doi.org/ 10.1111/j.1749-6632.2011.06258.x. [46] Gambassi S, Geminiani M, Thorpe SD, Bernardini G, Millucci L, Braconi D, et al. Smoothened-antagonists reverse homogentisic acid-induced alterations of Hedgehog signaling and primary cilium length in alkaptonuria. J Cell Physiol 2017;232. http://dx.doi.org/10.1002/jcp.25761. [47] Thorpe SD, Gambassi S, Thompson CL, Chandrakumar C, Santucci A, Knight MM. Reduced primary cilia length and altered Arl13b expression are associated with deregulated chondrocyte Hedgehog signaling in alkaptonuria. J Cell Physiol 2017;232:2407–17. http://dx.doi.org/ 10.1002/jcp.25839. [48] Bernardini G, Leone G, Millucci L, Consumi M, Braconi D, Spiga O, et al. Homogentisic acid induces morphological and mechanical aberration of ochronotic cartilage in alkaptonuria. J Cell Physiol 2019;234. http://dx. doi.org/10.1002/jcp.27416. [49] Laschi M, Bernardini G, Dreassi E, Millucci L, Geminiani M, Braconi D, et al. Inhibition of para-hydroxyphenylpyruvate dioxygenase by Analogues of the herbicide nitisinone As a strategy to decrease 8 Vol. xxx, No. xx 2019 homogentisic acid levels, the causative agent of alkaptonuria. ChemMedChem 2016;11. http://dx.doi.org/10.1002/cmdc.201500578. [50] Bernini A, Galderisi S, Spiga O, Bernardini G, Niccolai N, Manetti F, et al. Toward a generalized computational workflow for exploiting transient pockets as new targets for small molecule stabilizers: application to the homogentisate 1,2-dioxygenase mutants at the base of rare disease Alkaptonuria. Comput Biol Chem 2017;70:133–41. http://dx.doi.org/ 10.1016/j.compbiolchem.2017.08.008. [51] Spiga O, Cicaloni V, Zatkova A, Millucci L, Bernardini G, Bernini A, et al. A new integrated and interactive tool applicable to inborn errors of metabolism: Application to alkaptonuria. Comput Biol Med 2018;103. http://dx.doi.org/10.1016/j.compbiomed.2018.10.002 This paper shows how data on a rare disease such as alakptonuria might be handled in a precision medicine perspective. [52] Spiga O, Cicaloni V, Bernini A, Zatkova A, Santucci A. ApreciseKUre: An approach of Precision Medicine in a Rare Disease. BMC Med Inform Decis Mak 2017;17:42. http://dx.doi.org/10.1186/s12911-017-0438-0. [53] Preston AJ, Keenan CM, Sutherland H, Wilson PJ, Wlodarski B, Taylor AM, et al. Ochronotic osteoarthropathy in a mouse model of alkaptonuria, and its inhibition by nitisinone. Ann Rheum Dis 2014;73:284–9. http://dx. doi.org/10.1136/annrheumdis-2012-202878. [54] Hughes JH, Liu K, Plagge A, Wilson PJM, Sutherland H, Norman BP, et al. Conditional targeting in mice reveals that hepatic homogentisate 1,2dioxygenase activity is essential in reducing circulating homogentisic acid and for effective therapy in the genetic disease alkaptonuria. Hum Mol Genet 2019;28:3928–39. http://dx.doi.org/10.1093/hmg/ddz234. www.drugdiscoverytoday.com Please cite this article in press as: Braconi D, et al. Cell and tissue models of alkaptonuria, Drug Discov Today: Dis Model (2020), https://doi.org/10.1016/j.ddmod.2019.12.001