The Fountain of Youth for Aging Stem Cells

The functional capacity of hematopoietic stem cells (HSCs) declines with age, due in large part to the consequences of cumulative DNA damage, with age-related deficiency in DNA repair contributing to acquisition of mutations that lead to bone marrow failure and myeloid malignancies. With the aging of the baby boomer generation, the incidence of myeloid disorders is expected to rise. Therefore, understanding the basis of both age-related bone marrow dysfunction and the origins of myeloid neoplasms is essential for developing effective strategies for addressing these disorders. The recent studies of Dr. Johanna Flach and colleagues in the laboratory of Dr. Emmanuelle Passegué at the University of California, San Francisco, provide novel insights into the mechanisms that underlie age-related decline of HSC function and identify molecular targets for rejuvenation strategies. In experiments that compared outcomes in purified HSCs from young and old mice, those investigators presented data suggesting that replication stress contributes to the chromosomal damage and functional decline characteristic of old HSCs (Figure).

The term “replication stress” encompasses a variety of mechanisms that impair DNA copying at replication forks, including delay in cell cycle kinetics, defective prereplication cycle activity, altered replication dynamics, and chromosomal breaks/gaps (Figure). The capacity to circumvent replication stress is central to the maintenance of genomic stability.¹  Replication stress is accompanied by phosphorylation of histone H2AX (γH2AX) — an epigenetic mark that serves as the nidus for recruitment of DNA repair proteins (Figure). Dr. Flach and colleagues found evidence of increased replication stress in old HSCs compared with young HSCs during cell cycling that was accompanied by accumulation of γH2AX foci (Figure, small green stars). Subsequent studies identified decreased expression of minichromosome maintenance (MCM) helicase components Mcm 4 and Mcm 6 as the mechanism underlying the greater replication stress observed in old cycling HSCs. The deficiency of MCM components altered replication fork dynamics and impaired progression through the cell cycle, resulting in chromosome gaps and breaks. Nonetheless, old HSCs remain competent in activating canonical DNA damage repair and thereby survive replication unless confronted by a strong replication challenge such as treatment with a replication stressor drug or transplantation (Figure).

To test the relative sensitivity of young and old HSCs, the investigators treated samples with aphidicolin, a DNA polymerase inhibitor that induces replication stress by stalling the copying process at the replication fork. After treatment with aphidicolin, old HSCs had significantly greater γH2AX accumulation and rates of apoptosis compared with treated young HSCs, demonstrating the relative insensitivity of young HSCs to replication stress compared with their old counterparts. However, compared with untreated young HSCs, transplantation of aphidicolin-treated young HSCs resulted in impaired reconstitution capacity, reduced engraftment, and early-onset bone marrow failure and death in transplant recipients — outcomes that were similar to those observed with treated or untreated old HSCs. Thus, forced induction of replication stress in young HSCs recapitulated the aging defect. 

Once old HSCs re-established quiescence after cycling, aggregated, residual γH2AX is observed in reformed nucleoli of the postmitotic cells (Figure, large green stars). Because replication stress is intrinsically linked to cell proliferation, the authors sought an alternative mechanism that could account for the large γH2AX foci in quiescent old HSCs. The authors discovered that the persistence of γH2AX in old, quiescent HSCs was due to mislocalization of PP4c, a γH2AX phosphatase (Figure). In this case, PP4c remained in the cytoplasm rather than translocating to the nucleolus where it would be available to dephosphorylate γH2AX. In this setting, persistent nucleolar γH2AX functions noncanonically as a histone modifier, epigenetically silencing transcription of ribosomal DNA genes, resulting in decreased ribosome biogenesis in quiescent old HSCs (Figure). Whether this decreased ribosomal DNA synthesis induced by γH2AX accumulation creates a ribosomopathy that contributes to age-related bone marrow failure remains to be determined (Figure).