The Netrin-1 - Neogenin Axis Regulates Hematopoietic Stem Cell Dormancy and Function with Implications for Stem Cell Ageing

Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors that differentiate into lineage-committed progenitors and subsequently mature cells. Recently, we explored the molecular signatures employed by hematopoietic-stem-cells (HSCs) during differentiation by performing quantitative proteome, transcriptome (RNA-seq) and whole genome DNA methylation analyses on dormant and active HSCs as well as multipotent progenitors populations (MPP) 1-4 (Cabezas-Wallscheid et al., Cell 2017; Cabezas-Wallscheid et al., Cell Stem Cell 2014; Klimmeck et al., Stem Cell Reports 2014; Lipka et al., Cell Cycle 2014).

By exploiting these datasets, we found the DCC-like cell surface receptor Neogenin (Neo) almost exclusively expressed in CD34^negCD150⁺CD48^negLSK HSCs with an even higher expression in dormant HSCs. In addition, Neo expression has been reported to be robustly upregulated in aged HSCs (Sun et al., Cell Stem Cell 2015). The Neogenin receptor can bind different neuronal guidance molecules and can function as a BMP co-receptor. Based on this, we hypothesized that Neo and its ligands may preserve HSC dormancy and function. To investigate its role, we analyzed HSCs isolated from Neogenin-mutant mice during homeostasis and in reconstitution assays after transplantation. Neogenin-mutant HSCs show initially a competitive repopulation advantage in chimeras compared to control cells, which was associated with reduced stem cell quiescence. In agreement, RNA-seq analysis of Neogenin-mutant HSCs revealed reduced expression of dormancy related factors including the Egr1 transcription factor encoded by the early growth response gene. In contrast to the initial phases, Neogenin-mutant mice presented reduced HSC numbers associated with massively reduced reconstitution potential in 15 months old aged chimeras, as well as a myeloid differentiation bias. Collectively, these data suggest a role for Neo in preserving HSC dormancy and preventing their premature ageing.

With regard to ligands of the Neo receptor, only stimulation of cultured HSCs with the axon guidance molecule Netrin-1, but not with other known Neogenin ligands affected gene expression and cellular function of HSCs. This effect of Netrin-1 was absent in Neogenin-mutant HSC demonstrating specificity. In support of the hypothesis that Netrin-1- Neo signaling may preserve HSC function, treatment of cultured HSCs with Netrin-1 alone increased the reconstitution capabilities of HSCs after transplantation compared to untreated control cells. Taken together, our results identify the Neogenin receptor as a novel player important for promoting HSC maintenance through dormancy, while its inhibition leads to exhaustion and loss of HSC self-renewal capacity upon ageing. The here identified Netrin-1- Neo axis also raise the possibility that nerves growing into the bone marrow may control HSC dormancy and function by the production of the axon guidance molecule Netrin-1.