ALBERT

Description: Hematopoietic stem and progenitor cells (HSPCs) have multi-lineage potential and can be used in transplants as a curative treatment for various hematopoietic diseases. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. One level of regulation comes from the sympathetic nervous system that innervates the niche and releases neurotransmitters to stromal cells. However, the direct regulation of HSPCs via cell surface expression of neural receptors has not been functionally explored. Using imaging mass spectrometry, we detected strong and regionally specific gamma-aminobutyric acid (GABA) neurotransmitter signal in the endosteal region of mouse bone marrow. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation has not been investigated. Based on published mouse HSPC single cell RNA-seq data (Nestorowa et al., Blood 2016), we found that a subset of HSPCs expressed the GABA type B receptor subunit 1 (Gabbr1). We confirmed by surface receptor expression that a subset of mouse bone marrow HSPCs express Gabbr1 protein. Using the same single cell RNA-seq data as above, our own gene set enrichment analysis (GSEA) revealed positive correlation of Gabbr1 expression with genes involved in immune system processes, such as response to type I interferons. We generated a CRISPR-Cas9 Gabbr1 mutant mouse model on a C57/BL6 background suitable for hematopoietic studies. Analysis of Gabbr1 mutant bone marrow cells revealed a reduction in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but no change in the number of long-term hematopoietic stem cells (LT-HSCs). With further hematopoietic profiling, we discovered reduced numbers of white blood cells in peripheral blood that was primarily due to fewer B220+ cells. We show that Gabbr1 null HSPCs display reduced proliferative capacity, as well as diminished reconstitution ability when transplanted in a competitive setting. An in vitro differentiation assay revealed the impaired ability of Gabbr1 null HSPCs to produce B cell lineages. We tested our predicted association with type I interferon response by administration of poly(I:C) and found reduced HSPC proliferation in Gabbr1 null mice. Our results may translate well to humans, as a rare human SNP within the GABBR1 locus was found that correlates with altered leukocyte counts (Astle et al., Cell 2016). Our results indicate an important role for Gabbr1 in the regulation of HSPC proliferation and differentiation, highlighting Gabbr1 as an emerging factor in the modulation of HSPC function and behavior. Disclosures No relevant conflicts of interest to declare.

Description: Hematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the g-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry (IMS) and found that endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1 knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared to wild-type littermates. Moreover, Gabbr1 null hematopoietic stem cells (HSCs) had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1 null HSPCs were less proliferative under steady-state conditions and upon stress. Colony assays demonstrated that almost all Gabbr1 null HSPCs were in a slow or non-cycling state. In vitro differentiation of Gabbr1 null HSPCs in co-cultures, produced fewer overall cell numbers with significant defects in differentiation and expansion of the B cell lineage. To determine if GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared to GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation.

Description: Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells which differentiate to maintain and replenish blood lineages throughout life. Due to these characteristics, HSPC transplants represent a cure for patients with a variety of hematological disorders. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. The nervous system has been shown to indirectly influence hematopoiesis by innervating the niche; however, we present a direct route of HSPC regulation via expression of neurotransmitter receptors on HSPC surface. We have identified Gamma Aminobutyric acid (GABA) receptor B subunit 1 (Gabbr1), a hitherto unknown hematopoietic player, as a regulator of HSPC function. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation remains unknown. Based on published RNA-seq data (Nestorowa et al., Blood 2016), we discovered that Gabbr1 is expressed on a subset of HSPCs. We confirmed this expression using RT-qPCR to assay hematopoietic populations in the bone marrow (BM). Surface receptor expression analysis showed that Gabbr1 protein is expressed on a subset of BM HSPCs. To detect GABA, the ligand for Gabbr1 in the BM microenvironment, we utilized imaging mass spectrometry (IMS). We detected regionally specific GABA signal in the endosteal region of the BM. We further identified B cells as a cellular source of GABA in the BM. To understand the role of Gabbr1 in hematopoiesis, we generated CRISPR-Cas9 Gabbr1 null mutants on a C57/BL6 background suitable for hematopoietic studies and studied their hematopoietic phenotype. We discovered a decrease in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but the long-term hematopoietic stem cells (LT-HSCs) remain unaffected. Further analysis of peripheral blood of Gabbr1 null mutants showed decreased white blood cells due to reduced B220+ cells. This differentiation defect was confirmed in an in vitro differentiation assay where Gabbr1 null HSPCs displayed an impaired ability to produce B cells. We show that Gabbr1 null HSCs show diminished reconstitution ability when transplanted in a competitive setting. Reduced Gabbr1 null HSC reconstitution persisted in secondary transplant recipients indicating a cell autonomous role for Gabbr1 in regulating reconstitution of HSCs in transplant recipients. Our results show a crucial role for Gabbr1 in HSPC regulation and may translate to human health as a rare human SNP within the GABBR1 locus that correlates with altered leukocyte counts has been reported (Astle et al., Cell 2016). Our studies indicate an important role for Gabbr1 in HSPC reconstitution and differentiation into B cell lineages. Disclosures No relevant conflicts of interest to declare.