Dr. Sandra Pohl, PhD
University Medical Center Hamburg-Eppendorf, Germany

Report: Osteoporosis in mucolipidosis II: a potential corrective approach
Funding period (March 2017-February 2018)

Significance and aim
Major symptoms of MLII are severe skeletal defects summarized as Dysostosis multiplex resembled
in an MLII knock-in mouse model generated in our laboratory. The progressive bone loss andosteoporotic phenotype in MLII is a consequence of an increased number of osteoclasts most likely
induced by osteoblast-derived interleukin-6 (IL-6). Soluble gp130 (sgp130) functions as natural
inhibitor of the IL-6 signaling pathway. Therefore sgp130-mediated IL-6 blocking represents apotential treatment in MLII and most likely in MLIII that aims to prevent the bone loss and skeletal
deformities in these diseases.

Progress in the funding period
1. Ex-vivo analysis of MLII osteoblasts
Bone marrow cells were isolated from tibias and femora of 15 weeks old wild-type (WT) andMLII mice and cultured for 5 days. For osteoblast differentiation, ascorbic acid and βglycerophosphate was added to bone marrow cells and cultured for 25 days. Total RNA wereisolated from WT and MLII osteoblasts cultures and analysed by quantitative real-time PCR. MLIIosteoblasts displayed reduced expression of osteoblast differentiation markers, such as alkaline
phosphatase, osteocalcin and collagen. In contrast, the expression of IL-6 mRNA was 10-foldincreased in MLII osteoblasts compared to WT cells. These data confirm our previous results andshow that the impaired bone formation in MLII is caused by dysfunctional osteoblasts, whichhighly produce the osteoclastogenic cytokine IL-6.

2. Generation of MLII mice overexpressing the IL-6 inhibitor sgp130
For sgp130-mediated IL-6 blocking, we have crossed heterozygous MLII mice with transgenic miceoverexpressing human sgp130 that were kindly provided by Dr. Stefan Rose-John (University ofKiel, Germany). For genotyping of the offspring from eight breeding pairs, genomic DNAs fromtail biopsies of a total of 400 mouse pubs were isolated. The subsequent PCR analysis wasperformed using gene-specific primers to amplify genomic fragments of wild-type (WT) or mutant
murine Gnptab and human sgp130.

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The high number of breeding pairs was required to obtain at least 10 animals of each genotype(WT, WT+sgp130, MLII, MLII+sgp130), which are necessary for the further experiments. Theexpression of human sgp130 was determined in the serum of all 40 mice at the age of 6 weeks by aspecific enzyme linked immunosorbent assay (ELISA). As expected, in WT and MLII mice nohuman sgp130 was detectable. In WT+sgp130 and MLII+sgp130 concentrations between 19 and 22µg/ml sgp130 was found confirming the results obtained by sgp130-specific PCR of genomic DNA.
Analysis by cytokine multiplex ELISA revealed an IL-6 level in the range of 5-10 pg/ml in mouseserum. Since overexpressed sgp130 is in molar excess over IL-6, the increased secretion of IL-6 by
MLII osteoblasts will be neutralized by circulating sgp130. These molecular and biochemical
analyses demonstrate that the generation of sufficient MLII+sgp130 mice and respective control
mice was successful.

3. Skeletal analysis of MLII+sgp130 mice
The bone analysis of 15 weeks old mice, i.e. WT, WT+sgp130, MLII and MLII+sgp130, will bestarted in May 2018 and performed in close collaboration with Dr. Thorsten Schinke (Institute forOsteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Germany). In
particular, we will systematically analyse the skeleton of the animals, which includes contact
radiography, cross-sectional microcomputed tomography, and dynamic histomorphometricquantification

oftheboneformation

rate.

Additionally,non-calcified

histology

andhistomorphometry will be applied to quantify trabecular and cortical bone parameters as well as
numbers of osteoclasts and osteoblasts. In addition, we will perform in-situ immunohistological IL6 staining and electron microscopy to analyse bone marrow cells and growth plate chondrocytes of
the tibia of transgenic and WT mice.
We expect an improvement of various bone-morphometric parameters in transgenic MLII+sgp130
mice in comparison with MLII mice.

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