Transcriptome analysis in aged murine hematopoietic stem cells highlights an axon guidance receptor as a novel regulator of hematopoietic stem cell function and aging

Abstract

Hematopoietic stem cell (HSC) aging is associated with functional decline, yet the molecular changes contributing to this remain unclear. Previous studies have compared gene expression in pooled young and old murine HSCs but there is little consensus among them (3 genes). With the addition of our transcriptome analysis from individual C57BL/6 mice, only one gene, Neogenin, is consistently reported as upregulated with age. Surprisingly, this gene has not been investigated in HSC function or aging. Although frequently described as an axon guidance receptor due to its role in axon migration, it is also implicated in neuronal and embryonic stem cell differentiation. In this study we investigate the function of Neogenin in HSCs. We confirm increased Neogenin expression in long-term hematopoietic stem cells (LT-HSCs) compared to progenitors and validate its protein expression on their cell surface. Neogenin ligands Repulsive Guidance Molecules RGMA and RGMB as well as Netrins Netrin-1 and Netrin-4 are expressed by bone marrow mesenchymal stem cells and osteoblasts, suggesting the presence of Neogenin signalling in the HSC niche. Using intracellular FACS to measure protein at the single cell level we show that the degree of Neogenin expression is positively correlated to another differentially expressed gene, Stat3. Neogenin stimulation by RGMB increases Stat3 gene expression within 1 hour (p < 0.05) and phosphorylation (Tyr705) within 5 minutes (p< 0.0001). Interestingly, aged HSCS show increased basal phosphorylation of Stat3 (p< 0.0001). Neogenin stimulation also activates Focal Adhesion Kinase and Cell division control protein 42 (Cdc42), both of which are associated with HSC aging. With the use of live-cell imaging we monitored chemotaxis of bone marrow cells in response to RGMA. At 500ng/ml bone marrow cells migrate away from RGMA, similar to what has been described in developing axons. It has been reported that old HSCs have defects in homing, increased mobilization and are differentially localized in the bone marrow. Based on our migration data, it is possible that upregulation of Neogenin affects HSC homing and/or localization. Future studies repeat migration experiments with purified LT-HSCS, investigate differential expression of Neogenin ligands in young and old stromal cells and the functional consequence of knock-down of Neogenin in young and old HSCs. In summary, our data reveal multifunctional receptor Neogenin as a novel regulator of HSCs able to affect gene expression, pathway activation and migration.