Reticular Dysgenesis (RD) is one of the most serious forms of severe combined immune deficiency (SCID). It is characterized by complete absence of circulating lymphocytes and neutrophils. In addition, patients suffer from sensorineural hearing loss. Before newborn screening for SCID was implemented, the majority of patients succumbed to infection long before hematopoietic cell transplantation (HCT) could be attempted. To this date, the prognosis for RD remains grim. RD is caused by mutations in the mitochondrial ADP-generator Adenylate Kinase 2 (AK2). AK1 is a cytosolic protein that may compensate in various tissues for the lack of AK2. However, AK1 is not expressed in leukocytes and the stria vascularis of the inner ear . While this observation may explain where AK2 defects manifest, the molecular mechanisms how AK2 defects take effect, remain largely obscure. Significant obstacles to elucidating disease pathology have been the lack of a suitable animal models and the unavailability of patient specimens.
Using skin fibroblasts from an RD-patient we have recently identified at Boston Children’s Hospital , we have generated induced pluripotent stem cells (iPSC) with homozygous loss of function mutation in AK2. In-vitro myeloid differentiation of AK2-mutated iPSCs recapitulates the characteristic maturation arrest at the promyelocyte stage observed in-vivo in patients with this condition. AK2 is expressed in the intermitochondrial space and serves as primary mitochondrial ADP generator by promoting the reversible reaction AMP + ATP = 2 ADP. Maintenance of adequate levels of ADP is critical to support ATP synthase activity. Using Mass Spectrometry, we have shown that decreased AK2 activity leads to an increase in the AMP/ADP ratio in iPS-derived myeloid cells, indicating that AK2 is indispensable in maintaining ADP supply in the myeloid lineage. We have also performed transcriptome analysis of AK2- mutated myeloid cells compared to control and found a significant down regulation of ATP-dependant transporters. Based on this data, we hypothesized that in patients with RD, ADP-depletion in myeloid progenitors leads to stage 4 respiration, a well defined state in mitochondrial biology, in which the ATP-synthase lacks substrate and decreases its activity. This causes a reduction in proton flux from the intermitochondrial space back into the matrix, transient rise in membrane potential, and an escalation in the formation of reactive oxygen species (ROS). The cell responds by activating “inducible uncoupling”, the opening of alternative proton pores, which allows proton flux back into the matrix, bypassing the ATP-synthase and foregoing the use of energy stored in the proton gradient. While this represents a cellular rescue mechanism in response to acute oxidative stress, extended oxidative-stress-induced uncoupling eventually leads to a decline in proton gradient and membrane potential and ultimately in demise of the cell. To test this hypothesis, we have added Glutathione, the primary endogenous cellular antioxidant, to the culture conditions. We also tested G-CSF and all-trans-retinoic acid (ATRA), agents known to promote promyelocyte differentiation to mature neutrophils in other conditions. While G-CSF had no, and ATRA clearly deleterious effects on myeloid maturation in AK2-mutated cells, Glutathione led to a significant improvement in differentiation, allowing development of mature neutrophils in-vitro. Our results suggest that cell fate in RD is linked to oxidative stress and identify antioxidants as a possible therapeutic approach that may help reduce early mortality due to severe infections in patients with RD.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.