Selective changes in vasopressin neurons and astrocytes in the suprachiasmatic nucleus of Prader-Willi syndrome subjects

Abstract

The hypothalamic suprachiasmatic nucleus (SCN) hosts the central circadian pacemaker and regulates daily rhythms in physiology and behavior. The SCN is composed of peptidergic neuron populations expressing arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP), as well as glial cells. Patients with Prader-Willi Syndrome (PWS) commonly experience circadian disturbances, which are particularly evident in their sleep/wake patterns. Using publicly available single-cell RNA sequencing data, we assessed the cell-type specificity of PWS-causative genes in murine SCN, which revealed the differential presence of PWS-related genes in glial and neural subpopulations. We then investigated neurons and glial cells in the SCN using immunohistochemistry in the postmortem hypothalami of PWS subjects and matched controls. We profiled neural populations characterized by AVP and VIP, astroglia characterized by glial fibrillary acid protein (GFAP), and microglia marked by ionized calcium-binding adapter molecule 1 (Iba1) and NADPH oxidase 2 (NOX2). Our analysis revealed an increased total number, neuronal density, and relative staining intensity of AVP-containing neurons in the PWS compared to controls while VIP-containing cells were unaltered. In contrast, GFAP-expressing astroglial cells were significantly lower in PWS subjects. Moreover, we did not detect any differences in microglia between PWS subjects and controls. Collectively, our findings show that PWS selectively affects AVP-containing neurons and GFAP-expressing astrocytes in the SCN. As each of these cell populations can affect the daily rhythmicity of the SCN biological clock machinery, the disruption of these cells may contribute to the circadian disturbances in patients with PWS.

Keywords: Astroglial cells; Prader‐Willi syndrome; arginine‐vasopressin; biological rhythms.

FIGURE 1

A schematic and simplified representation of the HypoMap interrogation pipeline, as detailed in the methodology section. In the illustration of the murine brain, the hypothalamus is highlighted in pink, while the SCN is shown in yellow.

FIGURE 2

PWS causative genes are expressed in the SCN (A) Schematic representation of the chromosome map of the 15q11–13 region, illustrating the PWS causative genes. (B) UMAP of an SCN subset from the integrated single‐cell atlas HypoMap identifying 20 unique cell clusters. (C) Dot plot representing both the centered log‐normalized average expression (z‐score) and the percentage of positive cells for the PWS causative genes per cluster. (D) Dot plot representing both the centered log‐normalized average expression (z‐score) and the percentage of positive cells for the neuropeptides per cluster. PWS, Prader–Willi Syndrome; Chr, chromosome; Oligo, oligodendrocytes; OPC, oligodendrocyte precursor cell; NFO, Newly formed oligodendrocyte; MO, Myelinating oligodendrocyte; Gfap, glial fibrillary acid protein; Bdnf, Brain‐derived neurotrophic factor; Avp, Arginine‐vasopressin; Vip, vasoactive intestinal polypeptide; Vipr, receptor for vasoactive intestinal peptide; Penk, Proenkephalin; Prokr2, Prokineticin receptor 2; Chodl, Chondrolectin; Nms, Neuromedin S; Grp, Gastrin Releasing Peptide; Cck, Cholecystokinin; C1ql3, Complement C1q Like 3; Trbc1, T Cell Receptor Beta Constant 1; IPW, imprinted in Prader–Willi syndrome simplified representation of snoRNA‐associated region (including Snord116).

FIGURE 3

Increased numbers of AVP‐ir neurons in the SCN of PWS subjects. (A) The distribution of AVP‐ir neurons along the rostral to caudal axis of the SCN of controls and PWS individuals. (B) Total number of AVP‐ir neurons in the SCN of controls and PWS subjects. Quantitative analysis of (C) AVP‐ir soma number/mm² (neuronal density) in the “peak” of the SCN and (D) AVP‐ir relative optical density measurements in the “peak” of the SCN. (E) Representative images of AVP‐ir neurons in the SCN of control and PWS subjects. (F) The distribution of VIP‐ir neurons along the rostral to caudal axis of the SCN of controls and PWS individuals. (G) Total number of VIP‐ir neurons in the SCN of controls and PWS subjects. Quantitative analysis of (H) VIP‐ir soma number/mm² (neuronal density) in the “peak” of the SCN and (I) VIP‐ir relative optical density measurements in the peak of the SCN. (J) Representative images of VIP‐ir neurons in the SCN of control and PWS subjects. Please note the artwork detailing the SCN domains throughout the rostral–caudal axis. Additionally, illustration of a coronal hypothalamic section including the SCN “peak” and other anatomical landmarks. Controls (n = 15) and PWS (n = 9). Data are represented as mean ± SEM. *p < .05; **p < .001. Significance was calculated using Student's t test in B, C, G, H, I and Mann–Whitney test in D. Scale bar: 40 μm in E and J. 3v, third ventricle; AC, anterior commissure; PVN, paraventricular nucleus of the hypothalamus; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus; OC, optic chiasm; AVP, arginine vasopressin; VIP, vasoactive intestinal polypeptide; PWS, Prader–Willi Syndrome.

FIGURE 4

Reduced GFAP‐ir astrocytes and unaltered microglial cells in the SCN of PWS subjects. (A) Representative images of GFAP‐ir astrocytes in the SCN of control and PWS subjects. (B) Quantitative analysis of GFAP‐ir relative masked area. (C) Representative images of Iba1‐ir microglia in the SCN of control and PWS subjects. (D) Quantitative analysis of Iba1‐ir relative masked area (microglial cells). (E) Representative images of NOX2‐ir microglia in the SCN of control and PWS subjects. (F) Quantitative analysis of NOX2‐ir relative masked area. Data are represented as mean ± SEM. **p < .001. Significance was calculated using Student's t test. Scale bars, 50 μm in A, C, and E. Please note the artwork detailing the SCN “peak” domain, in which glial‐related quantifications were performed. This is accompanied by an illustration of a coronal hypothalamic section including the SCN “peak” and other anatomical landmarks. Controls (n = 15) and PWS (n = 9). 3v, third ventricle; AC, anterior commissure; PVN, paraventricular nucleus of the hypothalamus; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus; OC, optic chiasm; AVP, arginine vasopressin; GFAP, glial fibrillary acid protein; Iba1, ionized binding protein 1; NOX2, NADPH oxidase 2.