GDNF family receptor alpha-like (GFRAL) expression is restricted to the caudal brainstem

Abstract

Objective: Growth differentiation factor 15 (GDF15) acts on the receptor dimer of GDNF family receptor alpha-like (GFRAL) and Rearranged during transfection (RET). While Gfral-expressing cells are known to be present in the area postrema and nucleus of the solitary tract (AP/NTS) located in the brainstem, the presence of Gfral-expressing cells in other sites within the central nervous system and peripheral tissues is not been fully addressed. Our objective was to thoroughly investigate whether GFRAL is expressed in peripheral tissues and in brain sites different from the brainstem.

Methods: From Gfral:eGFP mice we collected tissue from 12 different tissues, including brain, and used single molecule in-situ hybridizations to identify cells within those tissues expressing Gfral. We then contrasted the results with human Gfral-expression by analyzing publicly available single-cell RNA sequencing data.

Results: In mice we found readably detectable Gfral mRNA within the AP/NTS but not within other brain sites. Within peripheral tissues, we failed to detect any Gfral-labelled cells in the vast majority of examined tissues and when present, were extremely rare. Single cell sequencing of human tissues confirmed GFRAL-expressing cells are detectable in some sites outside the AP/NTS in an extremely sparse manner. Importantly, across the utilized methodologies, smFISH, genetic Gfral reporter mice and scRNA-Seq, we failed to detect Gfral-labelled cells with all three.

Conclusions: Through highly sensitive and selective technologies we show Gfral expression is overwhelmingly restricted to the brainstem and expect that GDF15 and GFRAL-based therapies in development for cancer cachexia will specifically target AP/NTS cells.

Keywords: Area postrema; GDF15; GFRAL; Nucleus of the solitary tract.

Figure 1

smISH for Gfral mRNA in the brain and peripheral tissues. (A) Schematic of the tissue analysis pipeline carried out in mice. We interrogated immune cells, testes, white adipose tissue, pancreas, stomach, lung, ovary, small intestine, kidney, liver, heart and brain and performed either msISH, FACS-based analysis or RNA scope in-situ hybridization. (B) Representative images of smISH for Gfral (Red, left panel) and Ppib mRNA (Green, center panel) and a merged image (right panel) of mouse area postrema and nucleus of the solitary tract (AP/NTS), hippocampus and medial basal hypothalamus. (C) Representative images of smISH for Gfral (Red) and Ppib mRNA (Green) with a merged image and higher magnification image of region in boxed (far right panels) of mouse kidney, heart, liver and intestine. DAPI (white) was used to stain nuclei in all images. Dashed lines in A represent the boundaries of the area postrema. Abbreviations: eGFP = enhanced green fluorescent protein; WT = wild-type; msISH = single molecule in-situ hybridization; FACS=Fluorescence activated cell sorting; AP = area postrema; NTS = nucleus of the solitary tract; ARC = arcuate nucleus; 3v = third ventricle; CA1 = cornu Ammonis region 1.

Figure 2

Gfral reporter expression in brainstem and peripheral tissues. (A) Representative images of GFP immunoreactivity within the AP/NTS of eGFP-L10a reporter mice lacking Cre recombinase (Gfral^WT::eGFP, left panel), constitutively active GfralCre::eGFP (middle panel) and tamoxifen inducible Gfral^CreERT::eGFP mice (right panel). (B) Representative images of GFP immunoreactivity assessed through immunohistochemistry for GFP-labelled cells in mouse tissues from Gfral^WT::eGFP (left panels), GfralCre::eGFP (center panels), Gfral^CreERT::eGFP (right panels) animals. (C) Combined boxplot and dotplot of the quantification of GFP positive cells in AP/NTS and peripheral tissues. Three samples per tissue per genotype were quantified. The percentage of cells are given regarding total nuclei on each sample. Abbreviations: eGFP = enhanced green fluorescent protein; GFP = enhanced green fluorescent protein; AP = area postrema; NTS = nucleus of the solitary tract; iWAT = inguinal white adipose tissue; WT = wild-type.

Figure 3

Gfral Cre reporter expression in immune cells. (A) Representative flow cytometry analysis of GFP reporter expression in CD45+ immune cells, further sub-gated by differential expression of CD4, CD8, CD11b, and CD19. (B) Mean frequency of GFP+ cells in the pre-gated parent population of cells taken from the inguinal lymph nodes (left) and spleen (right) (n = 5) compared to control (n = 5). (C) Mean count of GFP+ cells adjusted per 1000 cells in the pre-gated parent population of cells taken from the inguinal lymph nodes (left) and spleen (right) (n = 5) compared to controls (n = 5). Numbers in FACS plots indicate the frequencies of gated cells. Error bars, SEM. ∗p-value≤0.05 determined by Two-Way ANOVA. Abbreviations: eGFP = enhanced green fluorescent protein; GFP = enhanced green fluorescent protein; WT = wild-type; ns = non-significant; L.N. = lymph node; FACS=Fluorescence activated cell sorting; SEM = standard error of the mean.

Figure 4

GFRAL expression in human tissues using scRNA-seq data. (A) UMAP plot of the scRNA-seq publicly available data on adult, pediatric and developmental tissues showing Gfral expression (right panels) on each cell type (left panels). (B) Models of the GDF15/GFRAL interaction proposed. The DVC includes the area postrema and the nucleus of the solitary tract. Abbreviations: UMAP = uniform manifold approximation and projection; scRNA-seq = single-cell RNA-sequencing; OPC = oligodendrocyte precursor cell; GABA = gamma-aminobutyric acid; DVC = dorsal vagal complex; EECs = enteroendocrine cells; EC = enterochromaffin cells; ILC2 = type 2 innate lymphoid cells; ILC3 = type 3 innate lymphoid cells; LEC = lymphatic endothelial cells; DC = dentritic cells; MAIT = Mucosal Associated Invariant T; TA = transit amplifying; cDC1 = type-1 conventional dendritic cells; cDC2 = type-2 conventional dendritic cells; gdT = Gamma Delta T cells; mLN = mesenteric lymph node; Tmem = memory T cell; DZ = dark zone; LZ = light zone; GC = germinal centre; FDC = follicular dendritic cells; NK = natural killer; Tfh = T follicular helper; Th = T helper; Treg = regulatory T cells; mLTo = mesenchymal lymphoid tissue organizers; pDC = plasmacytoid dentritic cells; eS = endometrial stroma; dS = decidualized stroma; uSMC = uterine smooth muscle cells; PV = perivascular; GABA = Gamma-aminobutyric acid.