Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Karolinska Institutet

Karolinska University Hospital

Surgery and critical illness often associate with cognitive decline. Surgical trauma or infection can lead independently to learning and memory impairments via similar, but not identical, cellular signaling of the innate immune system that promotes neuroinflammation. In this study we explored the putative synergism between aseptic orthopedic surgery and infection, the latter reproduced by postoperative lipopolysaccharide (LPS) administration. We observed that surgery and LPS augmented systemic inflammation up to postoperative d 3 and this was associated with further neuroinflammation (CD11b and CD68 immunoreactivity) in the hippocampus in mice compared with those receiving surgery or LPS alone. Administration of a selective α7 subtype nicotinic acetylcholine receptor (α7 nAChR) agonist 2 h after LPS significantly improved neuroinflammation and hippocampal-dependent memory dysfunction. Modulation of nuclear factor-kappa B (NF-κB) activation in monocytes and regulation of the oxidative stress response through nicotinamide adenine dinucleotide phosphate (NADPH) signaling appear to be key targets in modulating this response. Overall, these results suggest that it may be conceivable to limit and possibly prevent postoperative complications, including cognitive decline and/or infections, through stimulation of the cholinergic antiinflammatory pathway. 

Stimulation of the α7 nicotinic acetylcholine receptor protects against neuroinflammation after tibia fracture and endotoxemia in mice.

Longitudinal bone growth takes place in epiphyseal growth plates located in the ends of long bones. The growth plate consists of chondrocytes traversing from the undifferentiated (resting zone) to the terminally differentiated (hypertrophic zone) stage. Autophagy is an intracellular catabolic process of lysosome-dependent recycling of intracellular organelles and protein complexes. Autophagy is activated during nutritionally depleted or hypoxic conditions in order to facilitate cell survival. Chondrocytes in the middle of the growth plate are hypoxic and nutritionally depleted owing to the avascular nature of the growth plate. Accordingly, autophagy may facilitate their survival. To explore the role of autophagy in chondrocyte survival and constitutional bone growth, we generated mice with cartilage-specific ablation of either Atg5 (Atg5cKO) or Atg7 (Atg7cKO) by crossing Atg5 or Atg7 floxed mice with cartilage-specific collagen type 2 promoter-driven Cre. Both Atg5cKO and Atg7cKO mice showed growth retardation associated with enhanced chondrocyte cell death and decreased cell proliferation. Similarly, inhibition of autophagy by Bafilomycin A1 (Baf) or 3-methyladenine (3MA) promoted cell death in cultured slices of human growth plate tissue. To delineate the underlying mechanisms we employed ex vivo cultures of mouse metatarsal bones and RCJ3.IC5.18 rat chondrogenic cell line. Baf or 3MA impaired metatarsal bone growth associated with processing of caspase-3 and massive cell death. Similarly, treatment of RCJ3.IC5.18 chondrogenic cells by Baf also showed massive cell death and caspase-3 cleavage. This was associated with activation of caspase-9 and cytochrome C release. Altogether, our data suggest that autophagy is important for chondrocyte survival, and inhibition of this process leads to stunted growth and caspase-dependent death of chondrocytes.

Targeted Deletion of Autophagy Genes Atg5 or Atg7 in the Chondrocytes Promotes Caspase-Dependent Cell Death and Leads to Mild Growth Retardation.

Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H(+)-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A1 or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity. 

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner.

It has been suggested that the lysosomal recycling process called macro-autophagy plays a role in osteoarthritis development. We thus decided to genetically ablate the autophagy-indispensable Atg5 gene specifically in chondrocytes and analyse the development of osteoarthritis upon aging and in a post-traumatic model.

Targeted deletion of Atg5 in chondrocytes promotes age-related osteoarthritis.

Articular cartilage has little regenerative capacity. Recently, genetic lineage tracing experiments have revealed chondrocyte progenitors at the articular surface. We further characterized these progenitors by using  genetic approaches. Histone H2B-green fluorescent protein retention revealed that superficial cells divide more slowly than underlying articular chondrocytes. Clonal genetic tracing combined with immunohistochemistry revealed that superficial cells renew their number by symmetric division, express mesenchymal stem cell markers, and generate chondrocytes  both asymmetric and symmetric differentiation. Quantitative analysis of cellular kinetics, in combination with phosphotungstic acid-enhanced micro-computed tomography, showed that superficial cells generate chondrocytes and contribute to the growth and reshaping of articular cartilage. Furthermore, we found that cartilage renewal occurs as the progeny of superficial cells fully replace fetal chondrocytes during early postnatal life. Thus, superficial cells are self-renewing progenitors that are capable of maintaining their own population and fulfilling criteria of unipotent adult stem cells. Furthermore, the progeny of these cells reconstitute adult articular cartilage  entirely substituting fetal chondrocytes.-Li, L., Newton, P. T., Bouderlique, T., Sejnohova, M., Zikmund, T., Kozhemyakina, E., Xie, M., Krivanek, J., Kaiser, J., Qian, H., Dyachuk, V., Lassar, A. B., Warman, M. L., Barenius, B., Adameyko, I., Chagin, A. S. Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice.

Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice.

Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale.

Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage.

There are several pitfalls associated with research based on transgenic mice. Here, we describe our interpretation and analysis of mTORC1 activation in growth plate chondrocytes and compare these to a recent publication (Yan et al., Nature Communications 2016, 7:11151). Both laboratories employed TSC1-floxed mice crossed with collagen type 2-driven Cre (Col2-Cre), but drew substantially different conclusions. It was reported that activation of mechanistic target of rapamycin complex 1 (mTORC1)  Tsc1 ablation promotes the hypertrophy of growth plate chondrocytes, whereas we observe only disorganization in the resting zone, with no effect on chondrocyte hypertrophy or proliferation. Here, we present our data and discuss the differences in comparison to the earlier phenotypic characterization of  ablation in cartilage. Importantly, we detect Col2-Cre activity in non-cartilaginous tissues (including the brain) and discuss it in relation to other studies reporting non-cartilaginous expression of collagen alpha(1) II. Altogether, we conclude that mouse phenotypes following genetic ablation using Col2-Cre should be interpreted with care. We also conclude that activation of mTORC1 by  ablation in postnatal chondrocytes with inducible Col2-Cre (Col2-CreERt) leads to disorganization of the resting zone but causes no changes in chondrocyte proliferation or differentiation.

Activation of mTORC1 in chondrocytes does not affect proliferation or differentiation, but causes the resting zone of the growth plate to become disordered.

Longitudinal bone growth in children is sustained by growth plates, narrow discs of cartilage that provide a continuous supply of chondrocytes for endochondral ossification. However, it remains unknown how this supply is maintained throughout childhood growth. Chondroprogenitors in the resting zone are thought to be gradually consumed as they supply cells for longitudinal growth, but this model has never been proved. Here, using clonal genetic tracing with multicolour reporters and functional perturbations, we demonstrate that longitudinal growth during the fetal and neonatal periods involves depletion of chondroprogenitors, whereas later in life, coinciding with the formation of the secondary ossification centre, chondroprogenitors acquire the capacity for self-renewal, resulting in the formation of large, stable monoclonal columns of chondrocytes. Simultaneously, chondroprogenitors begin to express stem cell markers and undergo symmetric cell division. Regulation of the pool of self-renewing progenitors involves the hedgehog and mammalian target of rapamycin complex 1 (mTORC1) signalling pathways. Our findings indicate that a stem cell niche develops postnatally in the epiphyseal growth plate, which provides a continuous supply of chondrocytes over a prolonged period.

A radical switch in clonality reveals a stem cell niche in the epiphyseal growth plate.

Colorectal cancer is one of the most common forms of cancer in the world, with more than half a million new cases annually. Amongst the most promising new therapies, niclosamide-an FDA-approved drug for treating tapeworm infections-is being assessed in a stage II clinical trial for the treatment of metastatic colorectal cancer. Despite this advanced stage of research, the underlying mechanisms behind its actions remain uncertain. Niclosamide reduces the growth of colorectal cancer cells by targeting several intracellular signalling pathways, including the β-catenin-dependent WNT signalling pathway. In a recent paper published in the  [ (2019) , 535-546], Wang and colleagues revealed that niclosamide down-regulates β-catenin-dependent WNT signalling in colorectal cancer cells by degrading components of the pathway via autophagy. Autophagy is a catabolic process in which cellular macromolecules and organelles are recycled to their monomer units. This finding provides a further understanding of the actions of niclosamide upon colorectal cancer cells and may yield improved future treatment models for colorectal cancer patients.

Phillip T. Newton

Department of Physiology and Pharmacology

Karolinska Institutet

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Phillip T. Newton

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology and Pharmacology

Karolinska Institutet

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Department of Physiology and Pharmacology