Collagen-related diseases

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications, trials & patents

Abstract

Type I collagen (Col-I) is the most abundant human protein forming the structural scaffold of bone, skin and other tissues. The most prominent human development pathologies associated with disruptions in Col-I biosynthesis are bone fragility in OI and skin, tendon, and ligament laxity and fragility in EDS. Over 80% of severe OI cases are caused by substitutions of glycine (Gly) required in every third position of a 300-nm-long triple helix that is the main functional unit of Col-I. By altering the helix folding, Gly substitutions cause malfunction of bone producing cells (osteoblasts) and alter formation and function of the collagen scaffold of tissues. Over the years, our studies of mouse models and cells from OI patients revealed both effects to be pathogenic and osteoblast malfunction to be a major factor in bone failure. The latter malfunction results from accumulation of misfolded collagen precursor (procollagen) in osteoblast Endoplasmic Reticulum (ER) that leads to cell stress. We previously described noncanonical features of this cell stress, which were inconsistent with any known molecular mechanism. In last year, we have identified several unexpected pathways underlying the cellular stress response, suggesting both a novel mechanism and novel therapeutic targets. To understand and target osteoblast cell stress in OI, we earlier created and characterized a G610C mouse model of the disorder, mimicking a Gly610 to Cys substitution in the alpha-2 chain of Col-I in a large group of OI patients. We found that misfolding of mutant procollagen in G610C mice does not activate unfolded protein response pathways of canonical ER stress. We then utilized single-cell and spatially resolved in-situ RNA sequencing to identify increased expression of cell stress response genes Atf5 and Hspa9 instead of their homologues Atf4 and Hspa5 activated in canonical ER stress. We have confirmed this response by in-situ RNA hybridization and are currently examining the underlying mechanism and potential treatment targets. While bone fragility and skeletal deformities due to osteoblast malfunction are the most discussed pathologies in OI, deficient lung function is also a common complication and lung failure is the most common cause of mortality in OI, particularly in newborns. Since we observed perinatal lethality of all homozygous and few heterozygous G610C animals due to lung failure, we recently initiated studies of molecular mechanisms underlying this pathology. Surprisingly, we found severe developmental abnormalities in tissue structure, integrity, and performance despite seemingly normal function of all cells producing Col-I in lungs. These findings plus inflammatory and fibrotic lesions we observed to be associated with non-traumatic tissue injuries suggested lung pathology being caused by malfunction of secreted mutant Col-I rather than cells that produce it, unlike the bone pathology. We are investigating whether the resulting tissue weakness is a counterindication for mechanical ventilation, which is the most common approach to treating lung deficiency in OI. We suspect that the tissue injury due to forced ventilation might be causing inflammation and fibrosis like those we observe in G610C mice, leading to life-long lung tissue damage, lung malfunction, and lung failure later in life. Hence, we are exploring whether other ways of normalizing blood oxygenation or drugs suppressing lung inflammation and fibrosis caused by forced ventilation might be beneficial for OI patients. Overall, our translational studies include collaborations with many intramural and extramural scientists and clinicians on mechanisms of pathology in OI and other collagen-related disorders. Over the years, we assisted Dr. Marini in discovering novel forms of OI and characterizing underlying pathology. In collaboration with Dr. Byers, we investigated OI caused by arginine substitutions in type I collagen, demonstrating features similar to Gly substitutions. We assisted Dr. Bonnemann in characterization of a complex connective tissue disorder involving pathology of multiple tissues due to deficient function of prolyl 4 hydroxylase 1, an enzyme hydroxylating proline in Col-I. In collaboration with Dr. Stratakis, we described abnormal maturation and function of osteoblasts caused by deficiencies in catalytic and regulatory subunits of protein kinase A that disrupt cAMP signaling, which was reminiscent of McCune-Albright syndrome (constitutively overactive cAMP signaling). In collaboration with Dr. Leppert, we described abnormal composition of collagen deposited in uterine fibromas, which could be involved in the dysregulation of uterine fibroblasts underlying this pathology. We are currently collaborating with Dr. Otsuru on studies of growth plate pathology and growth deficiency in G610C mice. We are also collaborating with Dr. Forlino on characterization of type I collagen processing in zebra fish models of OI. We are also pursuing more fundamental cell biology of procollagen biosynthesis by osteoblasts and fibroblasts, the goal of which is understanding presently unknown molecular mechanisms for subsequent translation into clinical research. For instance, observations made by us and others suggest that disruptions in secretory trafficking and degradation of procollagen might be involved in a many pathologies spanning the entire lifespan, from skeletal dysplasia in early development to osteoporosis in aging. Better understanding procollagen trafficking and degradation might therefore reveal new therapeutic targets and approaches. To study these mechanisms, we developed novel fluorescent constructs of procollagen for live cell imaging. Contrary to popular models, we found procollagen to be delivered to Golgi from ERES by rapidly moving transport vesicles that have no COPII coat and that are dependent on COPI coat formation. We also discovered that misfolded procollagen is recognized at ERES and rerouted from the secretory pathway to a novel autophagy (lysosomal degradation) pathway we termed ERES micro-autophagy, in which ERESs containing misfolded molecules are directly engulfed by lysosomes. We are investigating the mechanism of the lysosomal recruitment to ERES as a potential target for therapeutic applications. We are also investigating whether ERES micro-autophagy is a general quality control mechanism utilized by cells for many proteins and not just procollagen. To facilitate these studies, we have recently utilized CRISPR/CAS gene editing technology for creating several osteoblast cell lines, in which endogenous procollagen is fluorescently tagged and can be manipulated by Flp-recombinase to introduce mutations and change the fluorescent tags. We have used these cells to confirm and expand our knowledge of procollagen trafficking and autophagy. Together, our translational and fundamental studies suggest that autophagy of misfolded procollagen is a crucial adaptation mechanism in OI. To test this hypothesis, we created an OI mouse model for autophagy manipulation by altering Atg5 expression. Using this model, we discovered that osteoblasts recycle misfolded procollagen primarily by ERES micro-autophagy not just in cell culture but also in vivo. We also observed that 3-4-fold reduction in Atg5 increased perinatal lethality of heterozygous G610C mice due to lung failure from just a few to 50%, yet the lung development was not affected. Apparently, it is the overall animal adaptation to the abnormal lung tissue development that is responsible for the increased lethality. Understanding the underlying adaptation mechanisms may therefore help us to find new treatment targets for improving the survival of OI babies with severely affect lungs as well as avoiding the potential long-terms side effects of forced lung ventilation.