Session: 112. Thalassemia and Globin Gene Regulation: Poster I
Hematology Disease Topics & Pathways:
Translational Research, Thalassemia, hematopoiesis, Hemoglobinopathies, Diseases, metabolism, Biological Processes
Transcriptome analysis of sorted HSC from th3 mice showed a positive enrichment of genes involved in iron homeostasis, such as TfR1, Steap3, Fth1 and Ftl1, suggesting iron uptake and storage. Consistently, BThal HSC displayed higher levels of intracellular free reactive iron, as compared to wild-type (wt) controls (1.7±0.2 calcein MFI fold to wt, p<0.0001). We found that iron specifically accumulates in the mitochondria causing mitochondrial defects, as reduced size and lower membrane potential (0.4±0.05 TMRE/MTG fold to wt, p<0.001). These data, along with the downregulation of mitochondrial biogenesis and mitophagy genes, indicate an accumulation of damaged mitochondria in BThal HSC. In vivo administration of iron dextran to wt mice generated intracellular IO in HSC and was sufficient to decrease the mitochondrial membrane potential at levels comparable to those of th3 HSC, thus suggesting a direct effect of iron on mitochondrial activity.
Since mitochondria are the powerhouse of the cell, we wondered if the mitochondrial dysfunction of BThal HSC could affect their production of metabolic energy. Metabolism indeed control HSC function: active HSC are more cycling and, because of their higher energy request, change the metabolism from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). Interestingly, we found that ATP levels in th3 HSC are lower compared to controls (wt 9.8±2.6 vs. th3 3.7±0.3 ATP nM, p<0.01). In line with mitochondrial dysfunction, in vitro inhibition of OXPHOS did not affect ATP content of BThal HSC since they switch to glycolysis for their energy demand. Consistently, th3 HSC showed positive enrichment of glycolytic genes and higher glucose uptake (wt 1639±163 vs. th3 2401±85 MFI 2NBDG, p<0.05).
To unravel the basis of mitochondrial damage caused by IO, we investigated the role of reactive oxygen species (ROS). Iron is one of the sources of ROS and consistently, th3 HSC showed increased activation of antioxidant genes and higher levels of mitochondrial ROS (wt 2507±756 vs. th3 5205±459 MFI MitoSOX, p<0.01). In vivo reduction of mitochondrial ROS by MitoQ rescued mitochondrial activity (th3 0.3±0.07 vs. th3+MitoQ 0.9±0.2 TMRE/MTG fold to wt, p<0.05), thus demonstrating that mitochondrial dysfunction is reversible in BThal. Ongoing experiments will assess the metabolite profile, the restoration of HSC metabolism and in vivo function upon MitoQ and will also test HSC from BThal patients for mitochondrial defects.
Overall, our study revealed that the multisystemic complications of IO in BThal also affect HSC. Iron has a direct impact on HSC metabolism by inducing oxidative stress and mitochondrial dysfunction. Despite the active cycling state, BThal HSC switch their metabolism to glycolysis because of the mitochondrial defect. Targeting mitochondrial iron or ROS might provide new tools to preserve HSC function in the setting of autologous transplantation upon gene therapy/editing for BThal. Moreover, this research will add novel insight about the regulation of HSC biology linking iron to mitochondrial metabolism.
Disclosures: No relevant conflicts of interest to declare.
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