Sympathetic Neuron Expressed Neuropeptide Y Is Crucial for Bone Marrow Niche Function and Hematopoietic Stem Cell Repopulation

The bone marrow (BM) microenvironment comprises a complex network of blood vessels and mesenchymal stromal cells (MSC) and is innervated by sympathetic nerve fibers. These BM niche components' soluble factors are pivotal for hematopoietic stem/progenitor cell (HSPC) function. Neuropeptide Y (NPY) is a 36-amino-acid polypeptide produced by several cell types in the BM, including nerve fibers, hematopoietic cells, and stromal cells. Using germline NPY knockout (KO) mouse models and pharmacological NPY supplementation in mice, we and others showed that NPY can prevent BM niche damage and support hematopoietic regeneration. However, the germline NPY deficiency in all cell types may lead to compensatory changes in the hematopoietic system, and the function of sympathetic neuron-specific NPY in the BM niche and HSPC is unknown. In this study, we discovered that sympathetic neuron-specific NPY gene deletion in mice impairs HSC repopulation and attenuates BM niche restoration. To test the effect of sympathetic neuron-specific NPY deficiency on BM niche and HSPC function, we first bred NPY floxed (NPY^fl/fl) mice with tamoxifen-inducible tyrosine hydroxylase Cre (TH-2A-CreER) mice and generated sympathetic neuron-specific NPY conditional KO mice (TH-Cre NPY^fl/fl mice). To induce NPY gene deletion, three-month-old NPY^fl/fl control mice and sympathetic neuron-specific NPY KO, TH-Cre NPY^fl/fl mice were treated with tamoxifen (2 mg/mouse; 5 consecutive days). Two weeks post-treatment, the HSPC and BM niche of these mice were evaluated by flow cytometry and functional assays. Compared to NPY^fl/fl mice, TH-Cre NPY^fl/fl mice showed increased HSC-enriched SLAM LSK counts (1.6 fold, p≤ 0.001) and decreased the HPC clonal expansion (1.2 fold, p≤0.01) in the BM. TH-Cre NPY^fl/fl mice SLAM LSK showed increased reactive oxygen species (ROS) production. These alterations in HSPCs of TH-Cre NPY^fl/fl mice were accompanied by a substantial reduction in HSC-supporting factors in the BM, including angiopoietin, VEGF, SDF-1, and HGF (p≤0.02). To examine if sympathetic neuron-specific NPY deficiency influences HSC repopulation ability, we performed competitive transplantation using BM donor cells from NPY^fl/fl or TH-Cre NPY^fl/fl mice (C57BL/6) mixed with competitor cells (Boy J; 1:1 ratio) and transplanted into lethally irradiated wild-type (WT) recipient mice (F1 hybrid mice). TH-Cre NPY^fl/fl mice donor cells showed reduced hematopoietic engraftment than NPY^fl/fl mice donor cells (33%, p≤0.01). Moreover, TH-Cre NPY^fl/fl mice donor cells showed increased myeloid differentiation (1.3 fold higher than NPY^fl/fl , p≤0.05). High-dose irradiation/ chemotherapy routinely used in patients undergoing HSC transplantation causes nerve damage in the BM and impairs hematopoietic regeneration. To test the impact of sympathetic neuron-specific NPY deficiency on BM niche ability to support hematopoietic reconstitution after transplantation, we injected WT donor cells (2 million) into lethally irradiated NPY^fl/fl and TH-Cre NPY^fl/fl recipient mice (NPY deletion prior to transplantation). TH-Cre NPY^fl/fl recipient mice demonstrated significantly fewer SLAM LSK, LSK, and hematopoietic cell recovery than NPY^fl/fl mice at seven days post-transplantation (p≤0.05). We next investigated sympathetic neuron-NPY deficiency impact on WT HSPC homing. Compared to NPY^fl/fl recipient mice, TH-Cre NPY^fl/fl recipient mice exhibited reduced WT donor HSPC homing (2fold, p≤0.01). The reduction in HSPC homing and hematopoietic recovery observed in TH-Cre NPY^fl/fl was accompanied by an increased loss of ECs (CD45^-Ter119^-CD31⁺VE-cadherin⁺) in the BM. To identify the potential molecular mechanism associated with NPY gene deletion contributing to impaired BM niche restoration, we performed RNA sequencing analysis of EC of WT and NPY KO mice. EC from NPY KO mice showed impaired expression of genes involved in pathways regulating antioxidant defense (Gpx3, Prdx4, Gpx3, Sod3, Srxn1, p<0.05) and DNA damage repair (Trp53i11, PARP, Gadd45a, Ddit4l, Rad51, p<0.05) compared to WT mice ECs. These findings revealed the previously unrecognized role of sympathetic neurons produced NPY in regulating BM niche function and HSC repopulation. Thus, preventing the NPY-producing sympathetic neuron damage during myeloablative irradiation/chemotherapy can improve BM restoration and hematopoietic reconstitution.