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Inherited bone marrow failure syndromes (BMFS) are rare, distressing, inherited blood disorders of children. Although the genetic origin of these pathologies involves genes with different functions, all are associated with progressive haematopoietic impairment and an excessive risk of malignancies. Defects in energy metabolism induce oxidative stress, impaired energy production and an unbalanced ratio between ATP and AMP. This assumes an important role in self-renewal and differentiation in haematopoietic stem cells (HSC) and can play an important role in bone marrow failure. Defects in energetic/respiratory metabolism, in particular in FA and SDS cells, have been described recently and seem to be a pertinent argument in the discussion of the haematopoietic defect in BMFS, as an alternative to the hypotheses already established on this subject, which may shed new light on the evolution of these diseases.
The bone marrow failure syndromes (BMFS) include a group of disorders than can be either inherited or acquired, and share phenotypic hallmarks of age-related diseases. The aging process in particular affects haematopoietic stem cells, inducing reduced regenerative potential with a possible increase in genetically unstable cells and malignancy. Although the genetic origin of these pathologies involves genes with different functions1,2 (e.g., DNA repair, ribosome biogenesis, and telomere maintenance) there is a common final pathway, yet still largely elusive, by which HSC are unable to support the production of blood cells.3
Telomere maintenance, DNA repair and oxidative stress are biochemically interrelated processes responsible for cellular aging. Even though the aging mechanism has not yet been clarified and several theories are proposed,4 mitochondrial metabolism, reactive oxygen species (ROS) and cellular antioxidant systems maintain a central role in the aging process.5,6 In fact, oxidative stress and ROS increment are a common factor in BMFS.7
Fanconi's Anaemia (FA), Shwachman-Diamond Syndrome (SDS), Dyskeratosis Congenital (DC) and the Diamond-Blackfan Syndrome (DBA) are the most frequent inherited BMFS disorders. FA cells show chromosomal instability, hypersensitivity to crosslinking agents and defects of DNA repair mechanisms. These characteristics seem strikingly linked to defects in the oxidative stress response. In fact, chromosomal aberrations in FA lymphocytes are positively related to oxygen tension and spontaneous chromosomal instability is reduced by superoxide dismutase and catalase activity. Recently, it has been shown that antioxidant (AO) molecules improve haematopoiesis and reduce spontaneous and induced chromosomal instability in FA. Moreover, oxidative stress is a critical factor in the pathogenesis of bone marrow failure and leukemia progression in FA cells.8,9
SDS cells have a defect in ribosome biogenesis, chemotaxis, mitotic spindle formation and cellular stress responses. Regarding the latter aspect, in SDS cells, endoplasmic reticulum and mitochondrial stress may enhance reactive oxygen species (ROS) production, leading to increased apoptosis and decreased cell growth.10-12 DC is caused by mutations in genes involved in telomere maintenance, which induces sensitivity to DNA damage and increments of oxidative stress, sensitive to NAC treatment.13,14
DBA depends on mutations in one of at least 10 ribosomal proteins necessary for the processing of pre-rRNA and the assembly of ribosomal subunits and show an increment of oxidative stress that regulates the autophagy process.15 Also in this case, antioxidant treatment causes a reduction in autophagy in DBA cells.
Recently, we have demonstrated that FA and SDS are characterized by a decrease in energy production by the oxidative phosphorylation (OXPHOS) machinery.16,17 These alterations cause a decrement in ATP production, determining an unbalanced ratio between ATP and AMP.16 Whatever is the role of FA and SDS, it is important to note that the dysfunction of the OXPHOS machinery determines an increment in oxidative stress production, which induces lipid peroxidation and therefore, an alteration in the mitochondrial membrane, where the OXPHOS machinery resides.16,17 This triggers a vicious cycle that increases the inefficiency of ATP and oxidative stress production.18 To re-establish the correct energetic status, SDS and FA cells augment the glycolysis rate.16,17 Hence, the return to glycolytic metabolism does not represent a Warburg effect, but the only choice of the cell to produce energy without increasing the accumulation of oxidative damage. However, it is also possible to speculate that this metabolic switch in a differentiated cell represents a precancerous condition that may facilitate tumor transformation. In DBA, the metabolic defects seem completely different. In fact, DBA cells show altered expression of several genes involved in energetic metabolism with reduced ATP levels and a switch from glycolytic to OXPHOS metabolism, with an augmentation of oxidative stress.19 However, studies are needed to better understand energy metabolism in DBA cells.
Different topics can be discussed in relation to the energy problem in these cells. Mitochondrial activity is influenced by the integrity of the mitochondrial reticulum, due to the fusion-fission mechanism, and by their correlation with the endoplasmic reticulum, via mitochondria-associated membranes proteins (MAM).20,21 In fact, frequent cycles of fusion and fission adapt the morphology of the mitochondrial compartment to the metabolic needs of the cell. Fusion is particularly important in metabolically active cells because it enables the distribution of metabolites, whereas mitochondrial fission plays an important role in the removal of damaged organelles by autophagy.22 Thus, mitochondrial fusion and fission contribute to the maintenance of mitochondrial function and optimize bioenergetic capacity. In FA lymphocytes, mitochondria are swollen with matrix rarefaction and altered cristae, while in primary fibroblasts, fragmentation of the mitochondrial reticulum has been described.16,23 Moreover, altered mitochondrial structures are also present in cell models of DBA, while human SDS lymphocytes show normal mitochondrial structure when observed under electron microscopy.17,19 Moreover, the energetic function of mitochondria depends on different factors, including several signaling pathways. Among these, AMP-dependent protein kinase (AMPK) and the IP3K/AKT/mTOR pathway play a pivotal role. AMPK is an important sensor of cellular energy status activated by high concentrations of intracellular AMP.24 AMPK activation stimulates alternative catabolic processes (such as glycolytic flux, glucose uptake, and fatty acid oxidation), generating ATP. At the same time, activated AMPK limits energy-consuming processes by antagonizing mTOR kinase, which is critical for translation initiation. In a simplified view, when energy is low, AMPK is active and mTOR is inhibited. mTOR signaling is necessary for the maintenance of mitochondrial oxidative function25 through a complex crosstalk, comprising Bcl-xl and VDAC1 at the mitochondrial outer membrane. It was observed that mTOR activates Bcl-xl, maintaining a sufficient ATP/ADP exchange between the mitochondria and cytosol to sustain coupled respiration, allowing the mitochondria to adapt to changes in metabolic demand.26 In FA and SDS energetic defects of cells induce AMPK hyper-phosphorylation.16,17,19,27 Surprisingly, the IP3K/AKT/mTOR pathway is also hyper-activated.17,28,29 In DBA, hyper-activation of mTOR is congruent with the increment in OXPHOS over glycolytic metabolism, while in FA and SDS, hyper-activation of mTOR might suggest the cells' attempt to overcome the energy defect through glutaminolysis, a pathway used in particular by cancer cells to support their energy requirements. Moreover, hyper-activation of mTOR inhibits autophagy and these data fit with its reduction in FA,30 even though in SDS and DBA, autophagy increases.15
Finally, Ca2+ is a crucial signaling molecule in energy metabolism that regulates many cellular ATP-consuming reactions. Ca2+ signaling appears to be maintained through a finely tuned dynamic interplay between the endoplasmic reticulum, mitochondria and plasma membrane. Ca2+ uptake into mitochondria activates the tricarboxylic acid (TCA) cycle, which supplies NADH for oxidative phosphorylation. Electron transfer, coupled with proton pumping in the inter-mitochondrial membrane space, establishes the electrochemical potential used to convert ADP to ATP. Mitochondria, however, are also the most important source of free-radical production and have a crucial role in intracellular calcium concentration ([Ca2+]i) homeostasis and the consequent negative effects associated with the induction of intrinsic cell death.31 Moreover, in response to energetic stress, increasing intracellular Ca2+ acts as a second messenger, leading to activation of AMPK.32 Therefore, the alterations of the energetic status of FA and SDS cells could be related to an altered calcium level. In FA cells, the [Ca2+]i is dramatically low.33 By contrast, SDS cells display a 2-fold elevation of [Ca2+]i relative to controls.17 These data are very interesting, considering that Ca2+ has a dual effect on energetic metabolism: on the one hand, it enhances OXPHOS function, activating the Krebs' cycle and the mitochondrial substrate transporters34; on the other hand, it inhibits complex IV, stimulating ROS production through the respiratory chain.35 This implies that any alteration in mitochondrial [Ca2+]i impacts on aerobic energy metabolism,31 but also limits oxidative stress production. Therefore, this mechanism, while leading to a decrement in ATP production, might allow, at the same time, an extension of the cell's life, delaying apoptosis events.36
Biochemical defects in energetic/respiratory metabolism described in BMFS (represented in Fig. 1) seem to be a pertinent argument to discuss the haematopoietic defect in these patients. Considering that no mutations related to the function or the organization of mitochondria have been described in the BMFS diseases treated in this work, it is possible to presume that functional mitochondrial impairment is actually a secondary defect with reference to the pathologic trait in the specific disease.
Stem cells are sensitive to increases in oxidative stress production. To counteract the effect of oxidative damage, stem cells are in a state of quiescence within a hypoxic microenvironment (niche) composed of different cells and molecular factors.37 Stem cells possess 2 idiosyncratic characteristics: pluripotency, which allows mature cells to compose specific organs or tissues, and self-renewal, which supplies an organ with an adequate number of cells to maintain the organ's function. During self-renewing stem cell division, asymmetric segregation of mitochondria has been described. The daughter cells that retain pluripotent capacity inherit fewer mitochondria to protect the stem cells from increased oxidative risk, while the other ones increase in number and the activity of mitochondria and start the OXPHOS metabolism needed for the differentiation process.38
In bone marrow, haematopoietic stem cells (HSC) are located in 2 different niches. The osteoblastic niche is closest to the endosteum with low oxygen concentration (hypoxia), which forces HSC to utilize glycolytic metabolism and protects the cells from oxidative stress induced by mitochondrial oxidative phosphorylation. Moving radially toward the longitudinal axis of the marrow into a more vascularised area, the vascular niche, HSC favor proliferation and differentiation, acquiring an oxido-reductive metabolic profile. The calcium ion gradient regulates the quiescent state of HSC by Ca2+ calmodulin (CaM)-dependent protein kinase IV.39 Thus, the duplication of quiescent stem cells represents a delicate event, which must be strictly regulated. The passage from glycolytic to OXPHOS metabolism produces oxidative stress and DNA damage. Therefore, the damage caused by mitochondrial alteration becomes severe during differentiation or when metabolism switches from anaerobic to aerobic. The energetic metabolism assumes, therefore, an important role in self-renewal and differentiation. While under normal conditions, this switch represents a more efficient system to produce energy, its impairment represents a cause of the increment of oxidative stress products and of energetic defect, contributing to cellular damage and aging. Therefore, since HSC are subject to a continuous process of cell division and differentiation, BM represents a target organ in disease in which genetic alteration causes impairment in cellular respiratory and energy metabolism. Walter et al. recently described metabolic stress in HSC, observing that the induction of proliferation causes an increase in DNA damage induced by oxidative stress.40
In particular, in HSC of FA mice, an augmentation of DNA damage was observed, along with cell death and reduced ability of stem cells to renew, underlining the DNA repair function of FA proteins in haematopoiesis. These results, however, underline the importance of DNA repair in FA, apart from the role of oxidative stress in this disease.
Different considerations could be made about the defect of energetic metabolism in DBA, where the glycolytic pathway appears altered, compromising the energetic source of HSC cells. However, the information concerning energy metabolism in these cells is yet incomplete and other studies are needed to better understand the biochemical metabolism of DBA cells, which, despite being a disease whose target is the erythroid lineage, upon differentiation will mandatorily imply glycolytic metabolism.
In conclusion, we can speculate that defects in energetic and respiratory metabolism play an important role in FA and SDS patients. If our assumption is true, the difference in the cellular biochemical phenotype seems to be well represented from the clinical hematologic phenotype. In fact, while in FA patients, progressive bone marrow failure appears during the first decade of life and has an incidence of 50%−90% by 40 y of age,3 in SDS, neutropenia is the most common hematologic abnormality, although anaemia and thrombocytopenia are also common. Moreover, an increased risk of developing myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML) occurs with less frequency and longer latency in SDS than in patients with FA. These differences could be due to the higher oxidative stress in FA, which also involves defects in mitochondrial structure.
The above reported observations are very interesting as they suggest how MDS represents an evolutionary outcome of very different pathological conditions. In the case of FA, the metabolic switch between glycolysis and oxidative phosphorylation might probably be associated with the process of differentiation from HSC progenitors through the modulation of calcium signals and an active communication between mitochondria and endoplasmic reticulum controlled by a modulation of the activity of the SERCA pumps.41 It is interesting to note that we reported that in FA, SERCA cells are inactive,33 while in SDS cells, there is a defect in calcium storage.17 Calcium influx is associated with the processes of maturation and differentiation of HSC. These processes are also associated with the structural and functional maturation of mitochondria and their activation. In the case of SDS, the metabolic switches in favor of glycolysis and the progressive functional inhibition of mitochondrial activity would appear to be the result of a set of events aimed as countermeasures for the survival of cells which, for different reasons -errors in ‘RNA processing, high oxidative stress and autophagy, in the case of SDS- are in extreme danger.
No potential conflicts of interest were disclosed.
Fondo Tumori e Lucemie del Bambino, AIRFA, ERG spa, Cambiaso and Risso, Rimorchiatori Riuniti, Saar Depositi Oleari Portuali, UC Sampdoria are acknowledged for supporting the activity of the Clinical and Experimental Haematology Unit of the G.Gaslini Institute. 5 per mille 2013 to IRCCS AOU San Martino – IST is acknowledged for supporting the activity of PD.