Collagen-related diseases

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

Type I collagen (Col-I) is the most abundant human protein forming the structural scaffold (matrix) of bone, skin and other tissues. Among prominent pathologies associated with disruptions in Col-I biosynthesis are bone fragility and lung failure in OI as well as skin, tendon, and ligament laxity in EDS. Over 80% of severe OI cases are caused by substitutions of glycine (Gly) required in every third position of collagen triple helix, 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 matrix in tissues. Our studies of mouse models and cells from OI patients revealed both effects to be pathogenic. We demonstrated that a key factor in bone failure is cell stress and resulting osteoblast malfunction due to Endoplasmic Reticulum (ER) disruption by mutant procollagen (collagen precursor) misfolding. We described noncanonical features of this stress, but the mechanism of its activation remained unknown. Recently, we identified ER-mitochondria contacts as the likely sensor of the ER disruption in an OI mouse model, suggesting a novel stress mechanism and novel therapeutic targets. We also demonstrated that lung pathology in the same mice is caused by deficient formation and function of collagen matrix incorporating secreted mutant molecules. To understand and target these pathologies, we created and characterized a G610C mouse model of OI, which mimics a Gly610 to Cys substitution in the alpha-2 chain of Col-I in a large group of patients. This model was instrumental in demonstrating that Gly substitutions may disrupt the osteoblast ER and trigger integrated stress response of the cell without activating canonical ER stress pathways. We then utilized single-cell and spatially resolved in-situ RNA sequencing to reveal activation of mitochondrial stress response via disruption of ER-mitochondria contacts. We showed this response to be regulated by mitochondrial HSP70 and ATF5, which are paralogues of BIP (ER HSP70) and ATF4 regulating canonical ER stress. After confirming this novel response to ER disruption, we are developing a new mouse model with the G610C mutation and ATF5 knockout to investigate the underlying molecular events and find potential therapeutic targets. While bone fragility and skeletal deformities due to osteoblast malfunction are the most discussed pathologies in OI, 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 initiated studies of molecular mechanisms underlying this pathology. In contrast to bones, we found lung pathology to be caused by mutant collagen affecting extracellular matrix and response of lung cells to this matrix rather to be caused by mutant collagen disrupting ER of lung cells. We are now combining structural characterization of the matrix with single-cell and spatially resolved RNA sequencing of lung cells to identify potential therapeutic targets and approaches. For instance, we discovered unusual, non-traumatic inflammatory and fibrotic lesions that may contribute to life-long lung tissue damage, lung malfunction, and eventual lung failure. Understanding the exact origin of these lesions may enable us to devise therapeutic approaches to simultaneously suppressing their formation and improving blood oxygenation in the lungs of 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 ER exit sites (ERESs) 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 are using these cells to confirm and expand our knowledge of procollagen trafficking and autophagy. We are also providing them to researchers outside NIH as a useful resource for their studies of collagen-related pathologies. 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 < 10% 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 help finding new treatment targets for improving the survival of OI babies and minimizing long-term lung pathology.