Bone Marrow Adipose Tissue (BMAT) emerges as a distinct fat depot whose importance has been proved in the bone-fat interaction. Indeed it is well-recognized that adipokines and free fatty acids released by adipocytes can directly or indirectly interfere with cells of bone remodelling or hematopoiesis. In pathological states such as osteoporosis, each of adipose tissues -subcutaneous White Adipose tissue (WAT), visceral WAT, Brown Adipose Tissue (BAT), BMAT - is differently associated with Bone Mineral Density (BMD) variations. However, compared to the other fat depots BMAT displays striking features that makes it a substantial actor in bone alterations. BMAT quantity is well-associated with BMD loss in aging, menopause and other metabolic conditions such as anorexia nervosa. Consequently, BMAT is sensed as a relevant marker of a compromised bone integrity. However, analyses of BMAT development in metabolic diseases (obesity and diabetes) are scarce and should be thus more systematically addressed to better apprehend the bone modifications in that pathophysiological contexts. Moreover, bone marrow adipogenesis occurs throughout the whole life at different rates. Following an ordered spatiotemporal expansion, BMAT has turned to be a heterogeneous fat depot whose adipocytes diverge in their phenotype and their response to stimuli according to their location in bone and bone marrow. In vitro, in vivo and clinical studies point to a detrimental role of bone marrow adipocytes throughout the release of paracrine factors which modulate osteoblast and/or osteoclast formation and function. However, the anatomical dissemination and the difficulties to access bone marrow adipocytes still hamper our understanding of the relative contribution of BMAT secretions compared with those of peripheral adipose tissues. A further characterization of the phenotype and the functional regulation of bone marrow adipocytes is ever more required. Based on currently available data and comparison with other fat tissues, this review addresses the originality of the BMAT with regard to its development, anatomy, metabolic properties and response to physiological cues.

Emerging evidence suggests that reactive oxygen species (ROS) can stimulate the Wnt/β-catenin pathway in a number of cellular processes. However, potential sources of endogenous ROS have not been thoroughly explored. Here, we show that growth factor depletion in human neural progenitor cells induces ROS production in mitochondria. Elevated ROS levels augment activation of Wnt/β-catenin signaling that regulates neural differentiation. We find that growth factor depletion stimulates the release of Ca²⁺ from the endoplasmic reticulum stores. Ca²⁺ subsequently accumulates in the mitochondria and triggers ROS production. The inhibition of mitochondrial Ca²⁺ uptake with simultaneous growth factor depletion prevents the rise in ROS metabolism. Moreover, low ROS levels block the dissociation of the Wnt effector Dishevelled from nucleoredoxin. Attenuation of the response amplitudes of pathway effectors delays the onset of the Wnt/β-catenin pathway activation and results in markedly impaired neuronal differentiation. Our findings reveal Ca²⁺-mediated ROS metabolic cues that fine-tune the efficiency of cell differentiation by modulating the extent of the Wnt/β-catenin signaling output.

Canonical WNT/β-catenin signaling is a central pathway in embryonic development, but it is also connected to a number of cancers and developmental disorders. Here we apply a combined in-vitro and in-silico approach to investigate the spatio-temporal regulation of WNT/β-catenin signaling during the early neural differentiation process of human neural progenitors cells (hNPCs), which form a new prospect for replacement therapies in the context of neurodegenerative diseases. Experimental measurements indicate a second signal mechanism, in addition to canonical WNT signaling, being involved in the regulation of nuclear β-catenin levels during the cell fate commitment phase of neural differentiation. We find that the biphasic activation of β-catenin signaling observed experimentally can only be explained through a model that combines Reactive Oxygen Species (ROS) and raft dependent WNT/β-catenin signaling. Accordingly after initiation of differentiation endogenous ROS activates DVL in a redox-dependent manner leading to a transient activation of down-stream β-catenin signaling, followed by continuous auto/paracrine WNT signaling, which crucially depends on lipid rafts. Our simulation studies further illustrate the elaborate spatio-temporal regulation of DVL, which, depending on its concentration and localization, may either act as direct inducer of the transient ROS/β-catenin signal or as amplifier during continuous auto-/parcrine WNT/β-catenin signaling. In addition we provide the first stochastic computational model of WNT/β-catenin signaling that combines membrane-related and intracellular processes, including lipid rafts/receptor dynamics as well as WNT- and ROS-dependent β-catenin activation. The model’s predictive ability is demonstrated under a wide range of varying conditions for in-vitro and in-silico reference data sets. Our in-silico approach is realized in a multi-level rule-based language, that facilitates the extension and modification of the model. Thus, our results provide both new insights and means to further our understanding of canonical WNT/β-catenin signaling and the role of ROS as intracellular signaling mediator. Human neural progenitor cells offer the promising perspective of using in-vitro grown neural cell populations for replacement therapies in the context of neurodegenerative diseases, such as Parkinson’s or Huntington’s disease. However, to control hNPC differentiation within the scope of stem cell engineering, a thorough understanding of cell fate determination and its endogenous regulation is required. Here we investigate the spatio-temporal regulation of WNT/β-catenin signaling in the process of cell fate commitment in hNPCs, which has been reported to play a crucial role for the differentiation process of hNPCs. Based on a combined in-vitro and in-silico approach we demonstrate an elaborate interplay between endogenous ROS and lipid raft dependent WNT/beta-catenin signaling controlling the nuclear beta-catenin levels throughout the initial phase of neural differentiation. The stochastic multi-level computational model we derive from our experimental measurements adds to the family of existing WNT models, addressing major biochemical and spatial aspects of WNT/beta-catenin signaling that have not been considered in existing models so far. Cross validation studies manifest its predictive capability for other cells and cell lines rendering the model a suitable basis for further studies also in the context of embryonic development, developmental disorders and cancers.

Bone marrow adipocytes (BMA-) constitute an original and heterogeneous fat depot whose development appears interlinked with bone status throughout life. The gradual replacement of the haematopoietic tissue by BMA arises in a well-ordered way during childhood and adolescence concomitantly to bone growth and continues at a slower rate throughout the adult life. Importantly, BM adiposity quantity is found well associated with bone mineral density (BMD) loss at different skeletal sites in primary osteoporosis such as in ageing or menopause but also in secondary osteoporosis consecutive to anorexia nervosa. Since BMA and osteoblasts originate from a common mesenchymal stem cell, adipogenesis is considered as a competitive process that disrupts osteoblastogenesis. Besides, most factors secreted by bone and bone marrow cells (ligands and antagonists of the WNT/β-catenin pathway, BMP and others) reciprocally regulate the two processes. Hormones such as oestrogens, glucocorticoids, parathyroid and growth hormones that control bone remodelling also modulate the differentiation and the activity of BMA. Actually, BMA could also contribute to bone loss through the release of paracrine factors altering osteoblast and/or osteoclast formation and function. Based on clinical and fundamental studies, this review aims at presenting and discussing these current arguments that support but also challenge the involvement of BMA in the bone mass integrity.

Bone marrow adipocytes (BMAds) accumulate in aging, menopause, and metabolic diseases such as Type 2 diabetes. These osteoporotic conditions are associated with oxidative stress and hyperglycemia which are both considered as critical factors underlying bone fragility. Glucose excess and reactive oxygen species (ROS) are known to favor adipogenesis over osteoblastogenesis. In this study, we investigated whether high glucose exposure could determine dysfunction of mature BMAds, specifically through ROS production. The effects of low (LG, 5 mM) or high glucose (HG, 25 mM) concentrations were examined using human bone mesenchymal stromal cells (hBMSCs) in the time course of differentiation, and, up to 21 days once adipocytes were mature. HG did not alter the adipocyte differentiation process of hBMSCs. Yet, after 21 days under HG exposure, PPARG, CEBPA, and adiponectin mRNA expressions were decreased. These alterations were also observed following adipogenic inducer withdrawal as well as in adipocytes fully differentiated in LG then cultured in HG for the last 11 days. Without inducers, HG condition also led to decreased leptin mRNA level. Importantly, intracellular and extracellular ROS concentrations measured using Amplex Red were significantly raised by 50% under HG exposure. This rise was observed once adipocytes ended differentiation and was reproduced within the different cell culture settings without any cytotoxicity. Among genes involved in ROS metabolism, the mRNA level of the H₂O₂ generating enzyme NOX4 was found upregulated in the presence of HG. Following cell separation, mature BMAds were shown to overproduce ROS and to display the gene alterations in contrast to non-lipid-laden cells. Finally, a non-lethal treatment with a pro-oxidant agent under LG condition reduces the mRNA levels of PPARG, adiponectin, and leptin as the HG condition does in the absence of inducers, and amplifies the effect of glucose excess on gene expression. HG concentration drives mature BMAds toward altered expression of the main adipokines and transcriptional factors. These perturbations are associated with a rise in ROS generation likely mediated through enhanced expression of NOX4. Mature BMAds are thus responsive to changes in glucose and ROS concentrations, which is relevant regarding with their phenotype and function in age- or metabolic disease-related osteoporosis.

Improving the neuronal yield from in vitro cultivated neural progenitor cells (NPCs) is an essential challenge in transplantation therapy in neurological disorders. In this regard, Ascorbic acid (AA) is widely used to expand neurogenesis from NPCs in cultures although the mechanisms of its action remain unclear. Neurogenesis from NPCs is regulated by the redox-sensitive WNT/β-catenin signaling pathway. We therefore aimed to investigate how AA interacts with this pathway and potentiates neurogenesis. Effects of 200 μM AA were compared with the pro-neurogenic reagent and WNT/β-catenin signaling agonist lithium chloride (LiCl), and molecules with antioxidant activities i.e. N-acetyl-L-cysteine (NAC) and ruthenium red (RuR), in differentiating neural progenitor ReNcell VM cells. Cells were supplemented with reagents for two periods of treatment: a full period encompassing the whole differentiation process versus an early short period that is restricted to the cell fate commitment stage. Intracellular redox balance and reactive oxygen species (ROS) metabolism were examined by flow cytometry using redox and ROS sensors. Confocal microscopy was performed to assess cell viability, neuronal yield, and levels of two proteins: Nucleoredoxin (NXN) and the WNT/β-catenin signaling component Dishevelled 2 (DVL2). TUBB3 and MYC gene responses were evaluated by quantitative real-time PCR. DVL2-NXN complex dissociation was measured by fluorescence resonance energy transfer (FRET). In contrast to NAC which predictably exhibited an antioxidant effect, AA treatment enhanced ROS metabolism with no cytotoxic induction. Both drugs altered ROS levels only at the early stage of the differentiation as no changes were held beyond the neuronal fate commitment stage. FRET studies showed that AA treatment accelerated the redox-dependent release of the initial pool of DVL2 from its sequestration by NXN, while RuR treatment hampered the dissociation of the two proteins. Accordingly, AA increased WNT/β-catenin signaling output i.e. MYC mRNA level, whereas RuR attenuated it. Moreover, AA improved neurogenesis as much as LiCl as both TUBB3-positive cell yield and TUBB3 mRNA level increased, while NAC or RuR attenuated neurogenesis. Markedly, the neurogenesis outputs between the short and the full treatment with either NAC or AA were found unchanged, supporting our model that neuronal yield is altered by events taking place at the early phase of differentiation. Our findings demonstrate that AA treatment elevates ROS metabolism in a non-lethal manner prior to the NPCs commitment to their neuronal fate. Such effect stimulates the redox-sensitive DVL2 activation and WNT/β-catenin signaling response that would enhance the ensuing neuronal cell differentiation.