Alkaptonuria is a novel human secondary amyloidogenic disease

Abstract

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated human chondrocytes and cartilage. SAA and SAP deposition was examined using immunofluorescence and their levels were evaluated in the patients' plasma by ELISA. 2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular tissues. SAA and SAP composition of the deposits assessed secondary type of amyloidosis. High levels of SAA and SAP were found in AKU patients' plasma. Systemic amyloidosis was assessed by Congo Red staining of patients' abdominal fat and salivary gland. AKU is the second pathology after Parkinson's disease where amyloid is associated with a form of melanin. Aberrant expression of proteins involved in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a novel type II AA amyloidosis open new important perspectives for its therapy, since methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid aggregates.

Chondroptosis in Alkaptonuric Cartilage

“…Finally, since we reported that AKU is an amyloidogenic disease (Millucci et al, 2012), at the best of our knowledge, the present work is the first associating chondroptosis and amyloidosis.…”

“…Moreover, ultra‐structural observations were complemented with biochemical and proteomic characterization of chondrocytes isolated from the ochronotic cartilage of AKU patients, indicating that AKU chondrocytes are characterized by HGA‐induced apoptosis, protein aggregation, nitric oxide release, and oxidative stress (Tinti et al, 2011a; Laschi et al, 2012; Millucci et al, 2012; Braconi et al, 2013; Spreafico et al, 2013). AKU patients have high levels of plasma serum amyloid A and pro‐inflammatory cytokines (Tinti et al, 2011b; Laschi et al, 2012; Millucci et al, 2012; Braconi et al, 2013; Millucci et al, 2014). The local expression of HGD in human osteoarticular system (Laschi et al, 2012) dramatically increases the effects in AKU cartilage degeneration.…”

The Pigment in Alkaptonuria Relationship to Melanin and Other Coloured Substances: A Review of Metabolism, Composition and Chemical Analysis

“…SAA amyloidosis is a serious complication of chronic inflammatory conditions such as rheumatoid arthritis, and its amyloid deposition process involves a cleaved product of the acute-phase protein serum amyloid A (SAA) (Momohara et al 2008). Indeed it was suggested that alkaptonuria is a novel-type II AA amyloidosis, if so will open new important perspectives for its therapy, particularly as methotrexate treatment significantly reduced in vitro HGA-induced amyloid A aggregates ( Millucci et al 2012).…”

Redox proteomics gives insights into the role of oxidative stress in alkaptonuria

“…On the one hand, this would help in finding a dedicated cure for AKU, ochronotic arthropathy and other AKU-related severe organ complications; on the other hand, since AKU can be considered as a model for more common rheumatic diseases such as osteoarthritis and rheumatoid arthritis [53], the social and economic relevance of AKU study would definitely be much wider. In this light, developing in vitro and ex vivo models reproducing the disease condition becomes fundamental.…”

“…A range of human serum-, cell-and tissue-based human models have been established in the last years (schematically depicted in FIGURE 2) [30,[33][34][35]53,[60][61][62][63]. These human AKU models are based on exogenous addition of HGA range concentrations analogous to those found in AKU patients' plasma.…”