The Aging Bone Marrow Niche Disrupts Glutamine Metabolism By Upregulating HSC Derived Glutamine Synthetase

As our aging population grows it is becoming increasingly important to better understand the physiological changes that drive that drive the aging process. Aging has long been known to drive haematopoietic stem cell (HSCs) dysfunction and therefore has been implicated in promoting the development of age-related haematopoietic diseases, such as leukaemia and myeloma. It is likely that a combination of intrinsic changes within the HSCs and extrinsic changes within the bone marrow (BM) microenvironment contribute to HSC aging. We have previously shown that the bone marrow niche accumulates senescent cells which contribute to impaired HSC response to stress ¹. Here we investigate the intrinsic changes that can be observed in aged HSCs and how these are influenced by the bone marrow niche.

In order to investigate the intrinsic changes that drive metabolic HSC aging we BM was isolated from young (8-12 weeks) and aged (18-24 months). Single HSCs were then FACS purified and sequenced using the SmartSeq2 protocol. Three transcriptionally distinct populations were identified, HSCs from young mice (Y), HSCs from aged mice with an aging transcriptional signature (O) and HSCs from aged mice that are transcriptionally similar to HSCs from young mice (OY). Assessment of differentially expressed genes between these three populations revealed that glutamine synthetase (glul) is significantly upregulated in the O population compared to both the Y and OY population. Glul is responsible for the conversion of glutamate to glutamine and therefore levels of both were next measured in FACS purified HSCs from aged and young mice. Results show that whilst there was no difference in glutamate levels, the glutamine levels were significantly reduced in HSCs from aged mice. These data suggest that glutamine metabolism is impaired in aged HSC.

Next, to determine the impact of the aged BM niche on glul expression in aged HSCs, HSCs from young and aged PepCboy (CD45.1+) mice were FACS purified and adoptively transferred into young C57Bl/6 mice. After 12 weeks the mice were sacrificed and engraftment of CD45.1+ cells was confirmed by flow cytometry. In addition CD45.1+ HSCs were FACS purified to assess glul expression and measure glutamine and glutamate levels. Results confirm that the mechanism for changes in glul expression and glutamine levels is driven by the aging BM niche and transplantation into a young niche reverses this change.

To understand the importance of dysregulated glutamine metabolism in aged humans we collected BM from older patients undergoing elective hip replacement surgeries. All samples were collected following informed consent and under approval of the UK NHS Health Research Authority (ref07/H0310/146). CD34+ cells were isolated and RNA extracted to measure glul expression. Similarly, to data from aged mice glul expression was elevated in aged CD34+ cells compared to young CD34+ controls. Furthermore, glutamine levels were also low in aged CD34+ cells. This suggests that the changes in HSCs from aged mice correlate with finding in human stem cells and mice are therefore a suitable species to study dysregulated glutamine metabolism in aged HSCs.

Together this work demonstrates that aging causes a disruption of glutamine and glutamate metabolism in HSCs. The cause for this disruption and its direct consequences on overall HSC metabolism remains to be explored. However, it is likely that these metabolic changes will have a knock-on effect on HSC function and may elucidate some of the drivers of metabolic HSC aging.