Elucidation of a new pathological mechanism for brittle bone disease

Description

The incidence of the brittle bone disease osteogenesis imperfecta (OI) is estimated to be around 1 in 10,000 individuals. The phenotypic spectrum of OI ranges from mild to perinatal lethal and includes extra-skeletal connective tissue manifestations.

Molecular mechanisms held responsible for OI include insufficiencies or defects in the bone collagenous extracellular matrix (ECM), as well as osteoblast dysfunction caused by overload of protein degradation pathways. Intracellular alterations can include over-modification of the procollagen molecule, activation of branches of the unfolded protein response (UPR) pathway or the integrated stress response, canonical or non-canonical autophagy and cytoskeletal alterations.

Type I collagen is the major structural component of bone and its molecules are predominantly (α1)2(α2)1 heterotrimers derived from the polypeptide gene products of the COL1A1 and COL1A2 genes. The mouse ‘osteogenesis imperfecta murine (oim)’ OI model is caused by deletion of a single guanidine residue in COL1A2, causing a frameshift that alters the last 47 amino acids, and adds an additional residue to the α2 chain of type I procollagen. As the α2 chain is defective, solely homotrimeric (α1)3 type I collagen is produced in oim homozygotes that have brittle bones. However, it is not the homotrimeric type I collagen itself that causes bone fragility (Lee et al., 2022). Intriguingly we did not detect synthesis of the abnormal (pro)α2(I) chain in oim homozygotes, even when autophagy or proteosomal degradation was inhibited, despite the presence of mRNA from the oim allele in both heterozygotes and homozygotes

These findings lead to the question of whether the oim proα2(I) chain is translated and at what rate, and how it affects the rate of synthesis of the normal proα1(I) chain.

Objectives

1. To determine if the oim alpha-2(I) collagen chain is translated in a cell free system.
2. To create N-terminally tagged Col1a2 constructs containing the oim mutation and follow translation and secretion in cell culture.
3. To follow collagen fibril assembly in transfected cells and in primary cells of wild-type, heterozygous and homozygous oim genotypes using high resolution microscopy.
4. To perform protease inhibition experiments in explant culture and determine the effect of the oim mutation on collagen fibril diameter in older samples.

The studentship will include training for the relevant techniques required for the project which include cDNA cloning and mutagenesis, established and primary cell culture, transfection, confocal microscopy and live cell imaging. For the final objective, training will also be provided in tissue dissection for explant culture and electron microscopy.

Understanding the fundamental reasons for brittle bone disease has the potential to lead to the identification of molecular therapies to treat the condition.