Neuroectoderm phenotypes in a human stem cell model of O-GlcNAc transferase associated with intellectual disability.

Pathogenic variants in the O-GlcNAc transferase gene (OGT) have been associated with a congenital disorder of glycosylation (OGT-CDG), presenting with intellectual disability which may be of neuroectodermal origin. To test the hypothesis that pathology is linked to defects in differentiation during early embryogenesis, we developed an OGT-CDG induced pluripotent stem cell line together with isogenic control generated by CRISPR/Cas9 gene-editing. Although the OGT-CDG variant leads to a significant decrease in OGT and O-GlcNAcase protein levels, there were no changes in differentiation potential or stemness. However, differentiation into ectoderm resulted in significant differences in O-GlcNAc homeostasis. Further differentiation to neuronal stem cells revealed differences in morphology between patient and control lines, accompanied by disruption of the O-GlcNAc pathway. This suggests a critical role for O-GlcNAcylation in early neuroectoderm architecture, with robust compensatory mechanisms in the earliest stages of stem cell differentiation.

An O-GlcNAc transferase pathogenic variant linked to intellectual disability affects pluripotent stem cell self-renewal.

O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential enzyme that modifies proteins with O-GlcNAc. Inborn OGT genetic variants were recently shown to mediate a novel type of congenital disorder of glycosylation (OGT-CDG), which is characterised by X-linked intellectual disability (XLID) and developmental delay. Here, we report an OGTC921Y variant that co-segregates with XLID and epileptic seizures, and results in loss of catalytic activity. Colonies formed by mouse embryonic stem cells carrying OGTC921Y showed decreased levels of protein O-GlcNAcylation accompanied by decreased levels of Oct4 (encoded by Pou5f1), Sox2 and extracellular alkaline phosphatase (ALP), implying reduced self-renewal capacity. These data establish a link between OGT-CDG and embryonic stem cell self-renewal, providing a foundation for examining the developmental aetiology of this syndrome.

Epo/EpoR signaling in osteoprogenitor cells is essential for bone homeostasis and Epo-induced bone loss.

High erythropoietin (Epo) levels are detrimental to bone health in adult organisms. Adult mice receiving high doses of Epo lose bone mass due to suppressed bone formation and increased bone resorption. In humans, high serum Epo levels are linked to fractures in elderly men. Our earlier studies indicated that Epo modulates osteoblast activity; however, direct evidence that Epo acts via its receptor (EpoR) on osteoblasts in vivo is still missing. Here, we created mice lacking EpoR in osteoprogenitor cells to specifically address this gap. Deletion of EpoR in osteoprogenitors (EpoR:Osx-cre, cKO) starting at 5 weeks of age did not alter red blood cell parameters but increased vertebral bone volume by 25% in 12-week-old female mice. This was associated with low bone turnover. Histological (osteoblast number, bone formation rate) and serum (P1NP, osteocalcin) bone formation parameters were all reduced, as were the number of osteoclasts and TRAP serum level. Differentiation of osteoblast precursors isolated from cKO versus control mice resulted in lower expression of osteoblast marker genes including Runx2, Alp, and Col1a1 on day 21, whereas the mineralization capacity was similar. Moreover, the RANKL/OPG ratio, which determines the osteoclast-supporting potential of osteoblasts, was substantially decreased by 50%. Similarly, coculturing cKO osteoblasts with control or cKO osteoclast precursors produced significantly fewer osteoclasts than coculture with control osteoblasts. Finally, exposing female mice to Epo pumps (10 U·d-1) for 4 weeks resulted in trabecular bone loss (-25%) and increased osteoclast numbers (1.7-fold) in control mice only, not in cKO mice. Our data show that EpoR in osteoprogenitors is essential in regulating osteoblast function and osteoblast-mediated osteoclastogenesis via the RANKL/OPG axis. Thus, osteogenic Epo/EpoR signaling controls bone mass maintenance and contributes to Epo-induced bone loss.

Increased EPO Levels Are Associated With Bone Loss in Mice Lacking PHD2 in EPO-Producing Cells.

The main oxygen sensor hypoxia inducible factor (HIF) prolyl hydroxylase 2 (PHD2) is a critical regulator of tissue homeostasis during erythropoiesis, hematopoietic stem cell maintenance, and wound healing. Recent studies point toward a role for the PHD2-erythropoietin (EPO) axis in the modulation of bone remodeling, even though the studies produced conflicting results. Here, we used a number of mouse strains deficient of PHD2 in different cell types to address the role of PHD2 and its downstream targets HIF-1α and HIF-2α in bone remodeling. Mice deficient for PHD2 in several cell lineages, including EPO-producing cells, osteoblasts, and hematopoietic cells (CD68:cre-PHD2f/f ) displayed a severe reduction of bone density at the distal femur as well as the vertebral body due to impaired bone formation but not bone resorption. Importantly, using osteoblast-specific (Osx:cre-PHD2f/f ) and osteoclast-specific PHD2 knock-out mice (Vav:cre- PHD2f/f ), we show that this effect is independent of the loss of PHD2 in osteoblast and osteoclasts. Using different in vivo and in vitro approaches, we show here that this bone phenotype, including the suppression of bone formation, is directly linked to the stabilization of the α-subunit of HIF-2, and possibly to the subsequent moderate induction of serum EPO, which directly influenced the differentiation and mineralization of osteoblast progenitors resulting in lower bone density. Taken together, our data identify the PHD2:HIF-2α:EPO axis as a so far unknown regulator of osteohematology by controlling bone homeostasis. Further, these data suggest that patients treated with PHD inhibitors or EPO should be monitored with respect to their bone status. © 2016 American Society for Bone and Mineral Research.

Soluble Envelope Glycoprotein Trimers from a CD4-Independent HIV-1 Elicit Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies in Guinea Pigs.

CD4-independent HIV-1 variants can infect coreceptor-expressing cells lacking CD4. The envelope (Env) glycoproteins on these HIV-1 variants expose a coreceptor binding site that overlaps some CD4-induced (CD4i) epitopes. Reports have demonstrated that CD4i antibodies mediate antibody-dependent cellular cytotoxicity (ADCC). Here we investigated the immunogenicity of soluble Env trimers (sgp140) from a CD4-independent HIV-1 in guinea pigs and found that the sgp140 elicited ADCC-mediating antibodies. Therefore, these sgp140 might be useful in vaccine regimens aimed at eliciting ADCC responses.