Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) resident protein that can be secreted due to an imperfect KDEL motif. MANF plays a cytoprotective role in several soft tissues and is upregulated in conditions resulting from intracellular retention of mutant protein, including two skeletal diseases, metaphyseal chondrodysplasia, Schmid type (MCDS) and multiple epiphyseal dysplasia (MED). The role of MANF in skeletal tissue homeostasis is currently unknown. Interestingly, cartilage-specific deletion of Manf in a mouse model of MED resulted in increased disease severity, suggesting its upregulation may be chondroprotective. Treatment of MED chondrocytes with exogenous MANF led to a decrease in the cellular levels of BiP (GRP78), confirming MANF’s potential to modulate ER stress responses. However, it did not alleviate the intracellular retention of mutant matrilin-3, suggesting that it is the intracellular MANF that is of importance in the pathobiology of skeletal dysplasias. The Col2Cre-driven deletion of Manf from mouse cartilage resulted in a chondrodysplasia-like phenotype. Interestingly, ablation of MANF in cartilage did not have extracellular consequences but led to an upregulation of several ER-resident chaperones including BiP. This apparent induction of ER stress in turn led to dysregulated chondrocyte apoptosis and decreased proliferation, resulting in reduced long bone growth. We have previously shown that ER stress is an underlying disease mechanism for several skeletal dysplasias. The cartilage-specific deletion of Manf described in this study phenocopies our previously published chondrodysplasia models, further confirming that ER stress itself is sufficient to disrupt skeletal growth and thus represents a potential therapeutic target.

Purpose: Multiple epiphyseal dysplasia (MED) is an autosomal chondrodysplasia characterized by early-onset osteoarthritis, epiphyseal hypoplasia with pain and stiffness in the weight-bearing joints and in some cases short stature. Its genetic background is complex and heterogeneous since mutations in several genes coding for extracellular matrix (ECM) components have been identified in patients. Among these genes, are those encoding for the pro-α chains of the Type IX collagen, COL9A1, COL9A2 and COL9A3 where mutations in each lead to the skip of exon 3 and a protein lacking 12 aa. The purpose of this study is to understand, by mean of a mouse model, the pathogenic role of the skipping of exon 3 in COL9A3 gene detected in MED patients in relation to all the abnormal changes of MED cartilage stability and homeostasis. Methods: By CRISPR/Cas9 technology we generated a mouse carrying a deletion of Col9a3 exon 3 (Col9A3Δex3), reproducing the splicing events reported in MED patients. Initial phenotyping of 3 and 9 week old Col9A3Δex3-/- mice is underway including skeletal X-ray and growth plate analysis (through immunohistochemistry and BrdU labelling to monitor chondrocyte proliferation). DMM surgery has been applied on 10-week old mice wild-type and Col9A3Δex3 mice. Mouse joints were collected 8 weeks post-surgery, fixed, decalcified and wax embedded for subsequent histological procedures. Results: CRISPR/Cas9 injection generated mice with the differing genomic deletion of exon 3, the breakpoints of which were confirmed by Sanger sequencing. The phenotyping of offspring through DNA and Xiphoid cartilage RNA analysis had led to the establishment of two transgenic mouse lines, one splicing as predicted (Col9a3Δex3-/-) and a second almost completely lacking the Col9a3 transcript (Col9a3-/-). Immunoblotting confirmed the lack of Col9 protein from predicted null mouse cartilage. From the initial mice and subsequent breeding, animals from both lines are viable. However, only the Col9a3-/- displayed mild detectable phenotypic abnormalities by X-rays analysis; mild short stature and hip dysplasia. Histological/immunological analysis of the growth plate from both lines showed disorganized structure but with overall normal rate of chondrocytes proliferation. DMM surgery on 10-week mice was used to accelerate an osteoarthritic cartilage damage detected in MED patients and histological evaluation of the joints is underway. Conclusions: Having confirmed the production of a shorter RNA from cartilage of Col9A3Δex3-/- mice, lacking only exon 3 and therefore splicing as expected and producing Collagen type IX protein, further work is ongoing to determine whether type IX collagen protein is incorporated into the ECM. Col9a3-/- line instead, shows no transcript and protein, results confirmed by WB and immunohistochemistry of the growth plate. Preliminary results to date confirm that Col9A3Δex3-/- and Col9a3-/- mice are viable and have a mild phenotype. Both mutant mice will represent an important tool to gain insights on collagen IX structure and its role into the matrix. In particular, the exon skipping line, by recapitulating human Col9-MED, can add to our understanding of the disease mechanism responsible for the onset of MED and to more general mechanisms of accelerated cartilage degradation that are the causes of the more common forms of osteoarthritis.

Metaphyseal chondrodysplasia, Schmid type (MCDS) is an orphan disease with highly abnormal endochondral ossification causing shortening and deformities of the limbs, impairment of mobility and chronic pain. As for most skeletal dysplasias, current treatment options remain symptomatic due to the lack in causal therapeutic measures. In the actual study the authors took advantage of the well-described pathogenesis in MCDS: In contrast to other collagenopathies, collagen X mutations do not directly lead to structural deficits but induce ER-stress by accumulation of misfolded protein. The authors hypothesize that by pharmacologically upregulating autophagy, enhanced protein clearance would ameliorate ER-stress and improve associated pro-apoptotic and differentiation-inhibiting effects.

Purpose: Several papers have described the occurrence of ER stress in osteoarthritic chondrocytes although the role of ER stress in either disease onset or progression has not been delineated. However, we have previously demonstrated that increased chondrocyte ER stress plays a pivotal role in chondrodysplasias caused by mutations in cartilage extracellular matrix proteins. The aims of the studies I will present were therefore: i) to characterise the role of chondrocyte ER stress in osteoarthritis; and ii) to demonstrate the clinical potential of alleviating ER stress in an ER stress -related cartilage pathology. Methods: DMM was performed on 10–12 weeks old mice that experienced elevated chondrocyte ER stress due to the chondrocyte-targeted expression of an ER-stress inducing transgene (ColIITgcog). The onset and development of OA was assessed in control and ColIITgcog mice at 2–8 weeks after operation. All histology was assessed in a blinded manner by 3 separate scorers using the Osteoarthritis Research Society International (OARSI) semi-quantitative scoring system for murine OA. Immunohistochemistry was performed on 95% ethanol/5% acetic acid fixed sections and in situ hydridisation using DIG labelled RNA probes. TUNEL assays were performed using a fluorometric TUNEL kit. Cell culture assays of ER stress were performed by qRT-PCR using transiently transfected HeLa cells expressing mutant forms of collagen X and treated with a variety of ER stress reducing drugs. In vivo tests were performed on the mutant pN617K.Col10a1 mouse line. Results and conclusions: I will present data to illustrate that i) increased ER stress is an early event in the development of OA in DMM; ii) chondrocyte apoptosis is an early event in disease onset; iii) increased capacity to cope with ER stress can delay disease onset; however, iv) ER stress does not appear to play a pivotal role in disease progression; v) increased ER stress can be pharmacologically reduced in a number of ways; vi) alleviating ER stress in chondrocytes in vivo can significantly reduce disease severity in an ER-stress related condition. Supported by Grants from ARUK (18253 & 19501) and the EU (7th Framework grant 602300 - SYBIL)

OBJECTIVE:To investigate the in vivo role of the IRE1/XBP1 unfolded protein response (UPR) signaling pathway in cartilage.DESIGN:Xbp1(flox/flox).Col2a1-Cre mice (Xbp1(CartΔEx2)), in which XBP1 activity is ablated specifically from cartilage, were analyzed histomorphometrically by Alizarin red/Alcian blue skeletal preparations and X-rays to examine overall bone growth, histological stains to measure growth plate zone length, chondrocyte organization, and mineralization, and immunofluorescence for collagen II, collagen X, and IHH. Bromodeoxyuridine (BrdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses were used to measure chondrocyte proliferation and cell death, respectively. Chondrocyte cultures and microdissected growth plate zones were analyzed for expression profiling of chondrocyte proliferation or endoplasmic reticulum (ER) stress markers by Quantitative PCR (qPCR), and of Xbp1 mRNA splicing by RT-PCR to monitor IRE1 activation.RESULTS:Xbp1(CartΔEx2) displayed a chondrodysplasia involving dysregulated chondrocyte proliferation, growth plate hypertrophic zone shortening, and IRE1 hyperactivation in chondrocytes. Deposition of collagens II and X in the Xbp1(CartΔEx2) growth plate cartilage indicated that XBP1 is not required for matrix protein deposition or chondrocyte hypertrophy. Analyses of mid-gestation long bones revealed delayed ossification in Xbp1(CartΔEx2) embryos. The rate of chondrocyte cell death was not significantly altered, and only minimal alterations in the expression of key markers of chondrocyte proliferation were observed in the Xbp1(CartΔEx2) growth plate. IRE1 hyperactivation occurred in Xbp1(CartΔEx2) chondrocytes but was not sufficient to induce regulated IRE1-dependent decay (RIDD) or a classical UPR.CONCLUSION:Our work suggests roles for XBP1 in regulating chondrocyte proliferation and the timing of mineralization during endochondral ossification, findings which have implications for both skeletal development and disease.

Multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH) are autosomal dominant chondrodysplasias that have similar phenotypes at both clinical and cytological levels. With the recent mapping of PSACH and one form of MED (EDM1) to the pericentromeric region of chromosome 19, it is likely that the disease mutations are allelic. D19S212 and D19S215, genetic markers flanking the EDM1/PSACH locus, have been localized in a chromosome 19 physical map consisting of cosmid contigs ordered by high-resolution FISH. These two markers define an interval of approximately 3.1 Mb at the 19p13.1-p12 boundary. With as many as five informative crossovers within the D19S212-D19S215 interval in one family with EDM1 and one family with a mild form of PSACH, recombination mapping at greater resolution was undertaken. From cosmid contigs physically mapped within the D19S212-D19S215 interval, four new dinucleotide repeat polymorphisms have been identified. Analysis of recombinant haplotypes in the two families has narrowed the possible location of the ECM1/PSACH gene to an interval of approximately 600 kb.

Mutations causing metaphyseal chondrodysplasia type Schmid (MCDS) (e.g., Col10a1p.N617K) induce the pathology by a mechanism involving increased endoplasmic reticulum (ER) stress triggering an unfolded protein response (UPR) in hypertrophic chondrocytes (Rajpar et al. 2009). Here we correlate the expression of mutant protein with the onset of the UPR and disease pathology (hypertrophic zone [HZ] expansion) in MCDS and ColXTg cog mouse lines from E14.5 to E17.5. Embryos homozygous for the Col10a1p.N617K mutation displayed a delayed secretion of mutant collagen X accompanied by a UPR at E14.5, delayed ossification of the primary center at E15.5, and an expanded HZ at E17.5. Heterozygote embryos expressed mutant collagen X from E14.5 but exhibited no evidence of a UPR or an HZ expansion until after E17.5. Embryos positive for the ER stress-inducing ColXTg cog allele expressed Tgcog at E14.5, but the onset of the UPR was not apparent until E15.5 in homozygous and E17.5 in hemizygous embryos. Only homozygous embryos exhibited an HZ expansion at E17.5. The differential onset of the UPR and pathology, dependent on mutation type and gene dosage, indicates that hypertrophic chondrocytes have a latent capacity to deal with ER stress, which must be exceeded to trigger the UPR and HZ expansion.