Non-canonical cell stress in osteogenesis imperfecta

Abstract

We used this model to demonstrate noncanonical quality control of PC1 folding. Like other secretory proteins, PC1 misfolds and accumulates in the lumen of endoplasmic reticulum (ER), disrupting the function of this crucial cellular compartment. Unlike other proteins, misfolding of which is detected in the ER lumen where it occurs, misfolded PC1 is recognized at ER exit sites (ERESs) away from the ER lumen. We used a G610C mouse model of moderately severe OI to validate this unexpected finding and confirmed it in vivo. Because physical separation between the sites of pathogenic accumulation and recognition of misfolded molecules has not been described before, we had to search for how cells might respond without any a priori knowledge. We therefore analyzed detectable RNA products of all genes in individual cells by single-cell RNA sequencing of mouse osteoblasts. We identified Hspa9 and Atf5 genes encoding chaperone HSPA9 and transcription factor ATF5 as likely regulators of the stress response to PC1 misfolding. Focusing on Atf5, we confirmed its increased expression in osteoblasts directly in bone sections from G610C mice. We discovered that ATF5 might be regulating not only osteoblast response to PC1 misfolding but also stress response of other cells to misfolding of all procollagens. We believe that the tools and the findings from this study lay a foundation for future research and eventual development of new therapies for procollagen misfolding disorders.

Procollagen misfolding underlies or accompanies pathologies from rare congenital diseases to common ailments, yet it is known only that the resulting cellular response and malfunction are different from the response to misfolding of other proteins. Osteogenesis imperfecta (OI) is a heritable disorder of bone development, in which over 80% of severe cases are caused by misfolding of type I procollagen (PC1). Not only PC1 misfolding leads to bone cell (osteoblast) malfunction in severe OI but it is also a likely factor in common osteoporosis and other bone pathologies. Nonetheless, it is not targeted by any of available therapies because it is poorly understood. To address this knowledge gap, we utilized gene editing technology to develop a new cell culture model of osteoblasts that enables visualization of the cellular response by super-resolution imaging of fluorescently tagged endogenous PC1 molecules in live cells.