The microRNA-29 family in cartilage homeostasis and osteoarthritis

MicroRNAs have been shown to function in cartilage development and homeostasis, as well as in progression of osteoarthritis. The objective of the current study was to identify microRNAs involved in the onset or early progression of osteoarthritis and characterise their function in chondrocytes. MicroRNA expression in mouse knee joints post-DMM surgery was measured over 7 days. Expression of miR-29b-3p was increased at day 1 and regulated in the opposite direction to its potential targets. In a mouse model of cartilage injury and in end-stage human OA cartilage, the miR-29 family were also regulated. SOX9 repressed expression of miR-29a-3p and miR-29b-3p via the 29a/b1 promoter. TGFβ1 decreased expression of miR-29a, b and c (3p) in primary chondrocytes, whilst IL-1β increased (but LPS decreased) their expression. The miR-29 family negatively regulated Smad, NFκB and canonical WNT signalling pathways. Expression profiles revealed regulation of new WNT-related genes. Amongst these, FZD3, FZD5, DVL3, FRAT2, CK2A2 were validated as direct targets of the miR-29 family. These data identify the miR-29 family as microRNAs acting across development and progression of OA. They are regulated by factors which are important in OA and impact on relevant signalling pathways

Accelerating functional gene discovery in osteoarthritis

Osteoarthritis causes debilitating pain and disability, resulting in a considerable socioeconomic burden, yet no drugs are available that prevent disease onset or progression. Here, we develop, validate and use rapid-throughput imaging techniques to identify abnormal joint phenotypes in randomly selected mutant mice generated by the International Knockout Mouse Consortium. We identify 14 genes with functional involvement in osteoarthritis pathogenesis, including the homeobox gene Pitx1, and functionally characterize 6 candidate human osteoarthritis genes in mouse models. We demonstrate sensitivity of the methods by identifying age-related degenerative joint damage in wild-type mice. Finally, we phenotype previously generated mutant mice with an osteoarthritis-associated polymorphism in the Dio2 gene by CRISPR/Cas9 genome editing and demonstrate a protective role in disease onset with public health implications. We hope this expanding resource of mutant mice will accelerate functional gene discovery in osteoarthritis and offer drug discovery opportunities for this common, incapacitating chronic disease

Iron-induced oligomerization of human FXN81-210 and bacterial CyaY frataxin and the effect of iron chelators

Patients suffering from the progressive neurodegenerative disease Friedreich’s ataxia have reduced expression levels of the protein frataxin. Three major isoforms of human frataxin have been identified, FXN42-210, FXN56-210 and FXN81-210, of which FXN81-210 is considered to be the mature form. Both long forms, FXN42-210 and FXN56-210, have been shown to spontaneously form oligomeric particles stabilized by the extended N-terminal sequence. The short variant FXN81-210, on other hand, has only been observed in the monomeric state. However, a highly homologous E. coli frataxin CyaY, which also lacks an N-terminal extension, has been shown to oligomerize in the presence of iron. To explore the mechanisms of stabilization of short variant frataxin oligomers we compare here the effect of iron on the oligomerization of CyaY and FXN81-210. Using dynamic light scattering, small-angle X-ray scattering, electron microscopy (EM) and cross linking mass spectrometry (MS), we show that at aerobic conditions in the presence of iron both FXN81-210 and CyaY form oligomers. However, while CyaY oligomers are stable over time, FXN81-210 oligomers are unstable and dissociate into monomers after about 24 h. EM and MS studies suggest that within the oligomers FXN81-210 and CyaY monomers are packed in a head-to-tail fashion in ring-shaped structures with potential iron-binding sites located at the interface between monomers. The higher stability of CyaY oligomers can be explained by a higher number of acidic residues at the interface between monomers, which may result in a more stable iron binding. We also show that CyaY oligomers may be dissociated by ferric iron chelators deferiprone and DFO, as well as by the ferrous iron chelator BIPY. Surprisingly, deferiprone and DFO stimulate FXN81-210 oligomerization, while BIPY does not show any effect on oligomerization in this case. The results suggest that FXN81-210 oligomerization is primarily driven by ferric iron, while both ferric and ferrous iron participate in CyaY oligomer stabilization. Analysis of the amino acid sequences of bacterial and eukaryotic frataxins suggests that variations in the position of the acidic residues in helix 1, β-strand 1 and the loop between them may control the mode of frataxin oligomerization

DLS studies of iron-dependent oligomerization of human frataxin FXN^81-210.

<p><b>A</b>) Measurements after 30 min of incubation with iron at 2:1 equivalents of iron-to-protein (magenta), 4:1 (green), and 10:1 (blue). <b>B</b>) Measurements after 60 min of incubation showing buildup of oligomers. In black is monomeric human FXN^81-210 without the addition of iron. On the x-axis is the hydrodynamic radius of the particles and on the y-axis is the volume percentage of particles.</p

DLS studies of iron-dependent oligomerization of <i>E</i>. <i>coli</i> CyaY.

<p><b>A</b>) CyaY was incubated for 1 h at 2:1 iron-to-protein ratio (green); 6:1, magenta; 10:1, blue. Dashed lines show the same sample after 26 h of incubation. In black, monomeric CyaY without addition of iron. On the x-axis the hydrodynamic radius and on the y-axis the volume-percentage of particles are shown. <b>B</b>) CyaY oligomerization with and without DFO. The protein was incubated at 6:1 (red) and 8:1 (green) iron equivalents; in black the protein without iron addition; dashed line show the sample after the addition of DFO.</p

The interface between subunits in the head-to-tail arrangement.

<p><b>A</b>) Human frataxin interface with residues D112, D115, D122, and D124 marked in magenta. The N-terminus is colored in pink and the different monomers within the dimer are shown in yellow (head) and green (tail). A crystal structure (PDB entry 1EKG) was used for preparing the figure. <b>B</b>) CyaY interface and the residues making up the potential metal binding sites are shown. Residues H7, E19, D22, and D23, which may build up the first metal binding site, are shown as red sticks; D3, H58, and D25 may participate in the second site (yellow sticks); and H70, D29, and E44 (blue sticks) in the third. Gray spheres show Europium ions bound in the 2P1X crystal structure. Residues involved in metal binding in the crystal structures of CyaY in complex with Co and/or Eu are labeled. The different monomers in the dimer are shown in green (head) and blue (tail). Crystal structures (PDB entry 2P1X, 2EFF, and 1EW4) were used in the preparation of the figue.</p

Iron-induced oligomerization of human FXN^81-210 and bacterial CyaY frataxin and the effect of iron chelators

<div><p>Patients suffering from the progressive neurodegenerative disease Friedreich’s ataxia have reduced expression levels of the protein frataxin. Three major isoforms of human frataxin have been identified, FXN^42-210, FXN^56-210 and FXN^81-210, of which FXN^81-210 is considered to be the mature form. Both long forms, FXN^42-210 and FXN^56-210, have been shown to spontaneously form oligomeric particles stabilized by the extended N-terminal sequence. The short variant FXN^81-210, on other hand, has only been observed in the monomeric state. However, a highly homologous <i>E</i>. <i>coli</i> frataxin CyaY, which also lacks an N-terminal extension, has been shown to oligomerize in the presence of iron. To explore the mechanisms of stabilization of short variant frataxin oligomers we compare here the effect of iron on the oligomerization of CyaY and FXN^81-210. Using dynamic light scattering, small-angle X-ray scattering, electron microscopy (EM) and cross linking mass spectrometry (MS), we show that at aerobic conditions in the presence of iron both FXN^81-210 and CyaY form oligomers. However, while CyaY oligomers are stable over time, FXN^81-210 oligomers are unstable and dissociate into monomers after about 24 h. EM and MS studies suggest that within the oligomers FXN^81-210 and CyaY monomers are packed in a head-to-tail fashion in ring-shaped structures with potential iron-binding sites located at the interface between monomers. The higher stability of CyaY oligomers can be explained by a higher number of acidic residues at the interface between monomers, which may result in a more stable iron binding. We also show that CyaY oligomers may be dissociated by ferric iron chelators deferiprone and DFO, as well as by the ferrous iron chelator BIPY. Surprisingly, deferiprone and DFO stimulate FXN^81-210 oligomerization, while BIPY does not show any effect on oligomerization in this case. The results suggest that FXN^81-210 oligomerization is primarily driven by ferric iron, while both ferric and ferrous iron participate in CyaY oligomer stabilization. Analysis of the amino acid sequences of bacterial and eukaryotic frataxins suggests that variations in the position of the acidic residues in helix 1, β-strand 1 and the loop between them may control the mode of frataxin oligomerization.</p></div

Negative staining TEM images of iron-induced FXN^81-210 oligomers.

<p><b>A</b>) Monomeric FXN^81-210. <b>B)</b> FXN^81-210 after 30 min of incubation with iron at 6:1 of iron-to-protein molar ratio. Ring-shaped particles are marked by black circles. <b>C</b>) FXN^81-210 incubated with iron at 6:1 molar ratio of iron-to-protein and with deferiprone added. <b>D</b>) FXN^81-210 incubated with iron at 6:1 molar ratio of iron-to-protein and with DFO added. Ring-shaped particles and larger spherical particles are marked by black circles. <b>E</b>) Class averages of the ring-shaped structures obtained from 1265 particles. The tetrameric character of the ring-structures can be clearly seen on some of the class averages. The EM images have a magnification of 55,000x.</p

Average size and distribution of particles of CyaY obtained from DLS measurements and the effect of iron chelators.

<p>Average size and distribution of particles of CyaY obtained from DLS measurements and the effect of iron chelators.</p

DLS studies of iron-dependent oligomerization of human frataxin FXN^81-210.

<p><b>A</b>) Measurements after 30 min of incubation with iron at 2:1 equivalents of iron-to-protein (magenta), 4:1 (green), and 10:1 (blue). <b>B</b>) Measurements after 60 min of incubation showing buildup of oligomers. In black is monomeric human FXN^81-210 without the addition of iron. On the x-axis is the hydrodynamic radius of the particles and on the y-axis is the volume percentage of particles.</p