The aim of this study is to clarify the implication of ciliary pathway on the onset of the spinal curvature that occurs in Adolescent Idiopathic Scoliosis (AIS) patients through functional studies of two genes: POC5 and TTLL11. Since the genetic implication for AIS is accepted, many association and candidate gene analysis revealed the implication of ciliary genes.

The characterisation of these two proteins was assessed by qPCR, WB and immunofluorescence in vitro using control cells and cells derived from AIS patients. The impact of genetic modification of these genes on the functionality of the proteins in vitro and in vivo was analysed in zebrafish model created by CRISPR/Cas9 using microCT and histologic analysis.

Our study revealed that mutant cells, for both gene, were less ciliated and the primary cilia was significantly shorter compared to control cells. We also observed a default in cilia glutamylation by immunofluorescence and Western Blot. Moreover, we observed in both zebrafish model, a 3D spine curvature similar to the spinal deformation in AIS. Interestingly, our preliminary results of immunohistology showed a retinal defect, especially at the cone cell layer level.

This study strongly supports the implication of the ciliary pathway in the onset of AIS and this is the first time that a mechanism is described for AIS. Indeed, we show that shorter cilia could be less sensitive to environmental factors due to lower glutamylation and result in altered signalling pathway. Identifying the biological mechanism involved is crucial for elucidating AIS pathogenesis.

The etiology of adolescent idiopathic scoliosis (AIS) is largely unknown, but clinical observations revealed the role of hereditary and rapid growth in the development of this condition. More recently, several genes were suspected to cause or contribute to AIS. Our group identified gene variants of POC5 centriolar protein in a French and French-Canadian families with multiple members affected with AIS. We sought to expand on this study and to investigate for the role of POC5 gene and mutated protein. In this work, the potential pathogenic effect of mutated POC5 was investigated in vitro (human osteoblats cell culture) and in vivo in a zebrafish animal model.

To investigate the role of POC5 in AIS, we investigated subcellular localization of POC5 with respect to cilia in cells overexpressing wt or POC5 variants (C1286T, A429V) and in human osteoblasts from scoliotic patients carrying these POC5 variants and normal control cells (in vitro study). We also created a loss-of-function model in zebrafish (in vivo study). The role of POC5 was investigated by: 1) mass spectroscopy analysis and co-immunoprecipitation to identify differences in binding partners between the wild-type (wt POC5 and mut POC5 protein; 2) immunolocalization of POC5 wt and mut proteins at the cellular level; 3) histology and immunohistochemistry performed on tissues from wt (control) and scoliotic (poc5 mut) zebrafish.

Our work identified several interacting partners with POC5, and documented functional connections with respect to cilia and centrosome dysfunction. A number of ciliary proteins were identified to be interacting with wt POC5 but not mut POC5. At the cellular level, localization and co-localisation of wt POC5 and mut POC5 protein with alpha acetylated tubulin (cilia marker), confirmed the consequence of the mutation on subcellular location with respect to cilium structure, length and staining intensity of cilia. In vivo, several defects in the retina were identified in mut poc5 zebrafish compared wt zebrafish. Finally, using different markers for retinal layers and acetylated tubulin, the defects were localized in ganglion cell layer and cones of the retina.

Our findings confirm the involvement of POC5 in scoliosis. A role of POC5 with respect to the primary cilia was attributed. These findings open new avenues for the understanding the primary causes of AIS at the molecular and physiological levels.

Introduction

Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity. It occurs mainly in girls and progresses during pre-pubertal and pubertal growth, which is a crucial period for bone mass acquisition. The cause and molecular mechanisms of AIS are not clear; at present the consensus is that AIS has a multifactor cause, with many genetic factors. During the past 5 years, considerable effort has been devoted to identify a gene or genes that cause a predisposition to AIS. Many loci for this disorder have been mapped to different chromosome regions, but no genes have been clearly identified as being responsible for AIS, and, most importantly, the resulting protein defects remain to be shown. We aimed to identify the gene(s) that could be involved in AIS and to validate their involvement by both genetic and functional analyses.

Introduction

Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity in children, and its cause is unknown. Recently, researchers have traced a defect in the gene CHD7 to AIS. CHD7 encodes for a chromodomain helicase of the DNA-binding domain protein family and is thought to have a crucial role in many basic cellular functions. However, the functional role of CHD7 in AIS is still elusive. In this study, we investigated the potential pathogenic effect of gene defects in CHD7 in vivo by evaluating their effect on spine formation and development in zebrafish.

Introduction

Idiopathic scoliosis is a spine disorder of unknown origin with a prevalence of 1·5-3% in the general population. Apart from the large multifactorial form sample of idiopathic scoliosis, there is a good evidence for the existence of a monogenic subgroup in which the disease is inherited in a dominant manner. However, results from published work suggest a strong heterogeneity in locations of the mutated genes.

Introduction: Adolescent idiopathic scoliosis (AIS) is the most common form of scoliosis, which appears to be caused by a melatonin signalling dysfunction proved recently in osteoblasts. This pathology occurs and progresses during the time of pre-puberty and puberty growth. This period is known to be under the hormonal control and coincides with many biological changes related to the secretion of estrogens, of which estradiol (E2) is the most active. The female prevalence of AIS disease is clearly evident. Indeed, in Quebec the spine deformities considered clinically significant (at least 11° of deformity) are found in a girl:boy ratio of approximately 2:1 for reduced scoliosis, and this ratio increases to 10:1 for scoliosis of more than 30o of deformation. However, the reason for this female prevalence as well as the role of estrogens and estrogen receptors in AIS is not clear despite the fact that these hormones are known for their impact on bone and bone growth, including the spine.

The purpose of the present study was to investigate the role of E2 on the responsiveness of the AIS cells to the melatonin, to determine the expression of estrogens receptors (ERα and ERβ) in AIS tissues and to clarify the impact of estrogen receptor gene polymorphisms in the pathogenesis of AIS.

Methodology: The effects of oestrogen on the AIS osteoblasts (n=10) response to the melatonin was determined by measuring the reduction of forskolin-induced cAMP accumulation. The forskolin treated osteoblasts were incubated in the presence of increasing amounts of melatonin (10–11 to 10-5 M) with or without physiological concentrations (10-10 M) of 17-β-estradiol for 16 hours, and the intracellular cAMP measured by radio-immunoassay using Biotrak Kit. Using RT-PCR, we determined ERα and ERβ mRNA expression in osteoblasts from AIS patients (n=14). Polymorphisms of the first intron of the ERα gene, which contains the XbaI and PvuII polymorphisms, were investigated by PCR following digestion with restriction enzyme and using the genomic DNA from lymphocytes isolated from scoliotic patients (n=33). Using the restriction enzymes XbaI and PvuII, the allelic variants XX, Xx, xx, PP, Pp, and pp were identified in 33 AIS patients (uppercase letters represent absence, and lowercase letters represent presence of restriction sites).

Results: The intracellular level of cAMP was significantly increased (p< 0.01) in the presence of a physiological concentration of 17-β-estradiol (10-10 M) when compared to the level observed in the presence of melatonin alone (10-9 M) (melatonin + estradiol: 109.46 ± 20.07; melatonin 76.09 ± 12.32 (mean ± SD)). As previously described by Dr Moreau’s team, the same pattern (three type of response to melatonin) takes place in the presence of 17-β-estradiol. We observed the loss of ERβ gene expression in 8/ 14 AIS patients contrasting with ERα gene expression that was found in all AIS patients. The XbaI and PvuII polymorphisms were found in 70% (23/33) and 80% (26/33) of the cases respectively. Of the 33 cases, 21 presented both digestion sites, 24 presented PvuII digestion site (6 homozygote, 18 heterozygote) and 23 (8 homozygote, 15 heterozygote) presented XbaI digestion site. The allelic variants were found as follows: XX: n=8, Xx: n=15, xx: n=8, PP: n=6, Pp: n=18 and pp: n=6. Classified by their location in the spine, seven right thoracic, one left thoracic, one right thoracolumbar, three left thoracolumbar and nine right thoracic-left lumbar were found among the patients presenting PvuII positive polymorphism. Among the patients with XbaI positive polymorphism, six right thoracic, one left thoracic, one right thoracolumbar, three left thoracolumbar and eight right thoracic left lumbar were found.

Conclusion: These results show the antagonistic effects of the 17-β-estradiol on AIS osteoblasts response to the melatonin. Thus estrogens interference with melatonin signalling activity would act as a triggering or aggravating factor in the pathogenesis of AIS. At the molecular level, it is possible that estrogens attenuate the response of AIS cells to melatonin through the desensitization of melatonin receptors. The loss of ERβ expression in a significant number of AIS patients appears to be important for the change of the ERα/ERβ receptors ratio that consequently may perhaps alter estrogens signalling pathways. The XbaI and PvuII polymorphisms are present in a significant number of AIS patients but this was not dependant of the curve pattern. These results clearly support the interplays and crosstalk between estrogens and melatonin signalling pathways in AIS aetiopathogenesis.

Supported by the Fondation Yves Cotrel, Institut de France

Purpose: Osteosarcoma (OS) is the most common type of cancer in children. OS demonstrates aggressive growth with a high risk of early, pulmonary metastasis. Here we investigated expression of malignancy-related factors in six osteosarcoma cell lines (SaOS, MNNG/ HOS, MG63, SW1353, SKES, SJSA), ten biopsies of primary OS and OS derived cells.

Methods: For this project, we are using RT-PCR and immunohistochemistry to detect PARP, ET-1, ETA and ETB receptor. We also, examined the expression of osteocrin by in situ hybridization. Zymography and Northern Blot were use to observe the presence of gela-tinases (MMP-2 and MMP-9) and finally, we showed the presence of ET-1(1–31) by Elisa-immunoassay.

Results: In OS tissues and cells, we observed ET-1, MMP-2, and ETA receptor overexpression, in contrast to under-expression of MMP-9 and ETB receptor. Additionally, in high malignant OS cells, MMP-2, MMP-3, MMP-13 and PARP were overexpressed and TIMP-1, TIMP-2 and TIMP-4 expressed at low levels. Using a new specific immunoassay for ET-1 (1–31) we showed presence of this alternative form of ET-1 in OS-derived cells. We also showed that, in cells exposed to ET-1, the inhibition of NF-kB pathway (NF-kB is an ubiquitous transcription factor playing a central role in differentiation, proliferation and malign transformation) drastically blocked MMP-2 production and activity, and that ET-1 induces NF-kB p65 unit translocation to the nucleus.

Conclusions: Thus, we concluded that expression of malignancy-related factors in parallel to the histological evaluation of these tumors, could be beneficial for malignancy understanding. Tumor cells invasion and aggressiveness of OS cells are the results, in part, of the tumorigenic potential of alternative forms of ET-1 generated by MMPs and enhanced in malignant microenvironment. These factors could be predictive for tumoral progression. Funding: Educational Grant from the Canadian Orthopaedic Foundation. Funding Parties: Canadian Orthopaedic Foundation (Carrol A. Laurin Award) and MENTOR program of CIHR

Purpose: Recently, we highlighted a dysfunction in the melatonin signalling pathway in the osteoblasts from adolescent idiopathic scoliosis patients (AIS). The objective of this project is to verify if in the cells coming from the SIA patients, estrogens interfere with melatonin signalling pathways and to identify mechanisms through which these effects are carried out.

Methods: The effects of estrogens on the melatonin signalling pathway, in osteoblasts from AIS patients (n=7), were determined by measuring the capacity of the Gi proteins to inhibit the accumulation of cAMP. The osteoblasts were incubated in the presence of increasing amounts of melatonin (10–11 to 10–5 M) with or without 17-& #946;-estradiol in physiological concentrations (10–10 M) (n=7). Moreover, coimmunoprecipitations using anti-phosphoserine antibodies were carried out and then followed with a Western blot in order to detect melatonin receptors (MT1 and MT2).

Results: The intracellular level of cAMP is higher in the presence of a physiological concentration of 17-& #946;-estradiol among scoliotic patients compared to the level observed in the presence of melatonin alone. Moreover, the preliminary results of the coimmunoprecipitations seem to show an increase in the phosphorylation of proteins interacting with MT1 and MT2 receptors. The precise nature of these proteins remains to be identified.

Conclusions: These results seem to show the antagonistic effects of the 17-& #946;-estradiol on the melatonin signalling pathway in the osteoblasts from AIS patients. However, more cAMP dosages in the presence and absence of 17-& #946;-estradiol are underway so as to increase the number of patients. The results of this study could contribute to the development of the first molecular screening tests as well as the development of new therapeutic approaches.