Netrin-1 regulates hematopoietic stem cell function via UNC5A Free

Aging is associated with an increased susceptibility to hematopoietic deficiencies and a reduced tolerance to therapeutic myeloablative regimens, which are underpinned by aging-related loss of vascular and hematopoietic stem cell (HSC) function. Rigorous functional assays have established the importance of interactions between the vascular niche and HSCs in the bone marrow (BM) microenvironment for sustaining a properly functioning, youthful hematopoietic system. Recent studies confirm that aging-associated defects within the BM vascular niche result in the phenotypic defects seen in the aged hematopoietic system, however, the ability to rejuvenate aged HSCs or their supportive niche has been a significant challenge.

In this regard, we have recently found that Netrin-1 (NTN1) is a niche-derived pro-HSC factor, and its expression is downregulated during aging in bone marrow endothelial cells (BMECs) and mesenchymal stem cells (MSCs). Generation of murine models which conditionally knocked out NTN1 within MSCs or ECs resulted in BM niche and HSC defects which mimic an aged phenotype, including reduced HSC engraftment, decreased self-renewal, and an increase in DNA damage. Conversely, treatment of aged mice with recombinant NTN1 resulted in the rejuvenation of HSC function indistinguishable from young controls; as well as enhanced self-renewal demonstrated by complete survival following serial regimens of myelosuppression. We also found that NTN1 infusions reactivate HSC DNA damage response (DDR) and resolve the age-related accumulation of DNA damage. These findings demonstrate that NTN1 restores DDR and rejuvenates an aged BM niche and HSC functionality to a youthful state.

Transcriptional analysis on young and aged HSCs revealed that the primary NTN1 receptors expressed on HSCs are Neogenin-1 (NEO1) and UNC5A. Recent data has demonstrated that a gene trap mouse model with incomplete deletion of NEO1 resulted in minor defects in HSC function only following severe stress, and that complete deletion of NEO1 utilizing Mx1-cre did not result in significant HSC dysfunction, thus we focused on the role of UNC5A maintaining NTN1-mediated HSC function. To this end, we performed in-depth analyses on an UNC5A global knockout mouse model (UNC5A^gKO) to determine the role of UNC5A in NTN1-induced rejuvenation.

Peripheral blood and bone marrow analyses demonstrated that UNC5A^gKO mice have decreased blood counts and femur counts, and an increase in HSC frequency. To determine HSC functionality, a competitive HSC transplant was performed where recipients received 100 HSCs from either control or UNC5A^gKO mice. The recipients which received UNC5A^gKO HSCs had significantly decreased engraftment, indicating HSC dysfunction. Furthermore, UNC5A^gKO mice were unable to recover following sublethal chemotherapy (150 mg/kg of 5-fluorouracil). Taken together, these data demonstrate that loss of UNC5A results in HSC dysfunction.

To determine if the HSC dysfunction seen in the UNC5A^gKO mouse model is due to intrinsic HSC defects or extrinsic defects, a reciprocal transplant was performed. Lethally irradiated control or UNC5A^gKO mice received competent CD45.1 whole bone marrow (WBM). The mice were assessed as previously described, 16-weeks post-transplant. The deficiencies previously seen in the steady-state analyses of UNC5A^gKO mice were resolved; there were no significant differences between control and UNC5A^gKO femur counts, HSC frequencies, and recovery following myelosuppression. Furthermore, HSCs were isolated from these mice and competitively transplanted into lethally irradiated C57BL/6 mice. There were no significant engraftment differences between recipients which received HSCs from UNC5A^gKO mice compared to controls. These data show that the HSC defects seen in the UNC5A^gKO mice are intrinsic and not a result of underlying niche defects.Finally, to determine if NTN1 is acting through UNC5A to promote hematopoietic rejuvenation, NTN1 or PBS (vehicle control) was administered to control and UNC5A^gKO mice. Following competitive transplantation, control mice which received NTN1 displayed a 3-fold increase in engraftment compared to their vehicle control counterparts, whereas UNC5A^gKO mice were unresponsive to NTN1. These data demonstrate that NTN1 improves HSC functionality but is unable to do so when UNC5A is knocked out, indicating that NTN1 administration results in hematopoietic rejuvenation by intrinsic HSC signaling via UNC5A.