snRNA-seq profiling of the hypothalamus from O, O.T and Y samples.

(A) UMAP visualization of nuclei colored by 10 cell types: neuron (Neu), astrocyte (Astro), oligodendrocyte (Oligo), oligodendrocyte precursor cell (OPC), tanycyte (Tany), ependymocyte (Epen), microglia (Micro), fibroblast (Fibro), pars tuberalis cell (PTC), and endothelial cell (Endo), from hypothalamus of aged rats (O), 17α-estradiol-treated aged rats (O.T) and young rats (Y). (B) Heatmap showing the classic markers of 10 major cell types in hypothalamus. (C) Cell-type compositions by groups (left panel) or by major cell types with the total cell numbers shown above each column. (D) Circos plot depicting the number of ligand–receptor pairs between Neu and other cell types (color strips) for each group. (E) Dot plot showing significant ligand–receptor interactions between Neurons for each group. Boxes showing the unique ligand–receptor interactions between Neuron.O (black boxes) or between Neuron.O.T (blue boxes). (F) Dot plot of the top 6 enriched GO biological process terms across three groups of neurons via GSEA analysis. (G) The top 15 changed pathways/gene sets according to the ranks of AUC values in selected pathways related to neuronal synapses and axons from Gene Ontology (GO) biological process, GO molecular function and GO cellular component.

Two opposing regulatory signaling networks in neuron metabolism.

(A) Dot plot of the selected pathways representing the prominent changes of overall expression levels across Neuron.O, Neuron.O.T and Neuron.Y in metabolism, signaling and synaptic activity. (B) Correlation heatmap showing transcription factors (TFs) that correlated with the two opposing regulatory signaling networks in the mixed neurons of O, O.T and Y. (C) The shared unique markers of each quarter (c1-c4) in 6 pathways in hypothalamic neurons (O, O.T, and Y). The markers were then collected as c1.up.signature (19 genes) and c4.up.signature (12 genes). (D) The aging-related cell proportions of each quarter shown by 4 pathways. (E) The correlation of c1.up.signature and c2.up.signature with the two opposing regulatory signaling networks.

Screening of neuron subtypes via supervised clustering, which responded distinctly to aging and 17α-estradiol treatment.

(A) Diagram outlining the features of supervised clustering of neurons in the hypothalamus in comparison with traditional unsupervised clustering. (B) The ranks of cell counts in neuropeptide-secreting neuron subclusters (left panel) and subclusters expressing neuropeptide receptors or hormone receptors (right panel) in sample Y. The cell number (n) in each subset is ≥ 10. (C, D) The prioritization of the top 20 neuron subclusters across the 3 types of perturbation (O vs Y, O.T vs Y, and O.T vs O) calculated by the Augur algorithm, in neuropeptide-secreting neurons (C) and neuron subclusters expressing neuropeptide receptors or hormone receptors (D).

Ranking of neuron subtypes with distinct responses to aging and 17α-estradiol treatment.

(A, B) The top 20 and bottom 20 neuron subtypes based on the mean expression values of five signatures or gene sets, ranked by their values in sample O, in neuropeptide-secreting subtypes (A) and in neuron subtypes expressing neuropeptide receptors or hormone receptors (B).

Responses of Crh neurons to long-term 17α-estradiol treatment.

(A) The top 20 and bottom 20 neuropeptide-secreting neuron subtypes, ranked by their mean expression values of five signatures or gene sets in sample O. (B) Expression profiles of selected pathways from two opposing signaling networks in Crh, Kiss1, and Prlh neurons. (C) Downregulated and upregulated differentially expressed genes (DEGs) associated with mitochondria or the adherens junction pathway in Crh neurons, comparing O.T vs O. (D) Top 25 transcription factor (TF) activities in Crh and Gnrh1 neurons. (E) Serum levels of Crh, cortisol, and aldosterone in Y, O, and O.T groups as measured by enzyme immunoassay; two-tailed unpaired t-tests were performed, with p-values indicated.

The response of Oxt neurons to 17α-estradiol and the causal effects of Oxt on other endocrine factors.

(A) The relative cell proportions of peptide-expressing subclusters (upper panel) and receptor-expressing subclusters (lower panel) across Y, O, and O.T (sorted in descending order of proportions in Y). Only subclusters with a cell count of n ≥ 10 in sample Y were included for calculation. (B) Dot plots showing the expression profiles of the selected pathways from the two opposing signaling pathways in four types of food uptake-related neurons, which decreased or increased among the top 10 ranks in (A) or (B). Blue arrows: c1.up.signature and c4.up.signature. (C) Volcanic plots showing the DEGs between Neuron.O.T and Neuron.O in the pathway synaptic membrane. (D) Enzyme immunoassay of the plasma levels of Oxt in three groups. (E) Top 25 TF activities in neuron Oxt. (F) Significant causal effects (p < 0.05, IVW) between exposure OXT (id: prot-a-2159) and 203 endocrine-related outcomes, which were not significant in reverse MR analysis. Significant heterogeneity (Q_pval < 0.05). Significant horizontal pleiotropy (pval < 0.05).

The response of HPG axis in the males to 17α-estradiol and the causal effects of Gnrh on other endocrine factors.

(A) The expression profiles of pathways from the two opposing signaling networks in Gnrh1-, Esr2-, Esr1- or Ar-positive neurons. (B) Enzyme immunoassay of the serum levels of Gnrh, total testosterone (T), and estrogen (E) in Y, O and O.T samples. Two-tail unpaired T-test was performed. (C) Inflammation of seminiferous tubules in testes of O and O.T. Left two panels: representative HE staining of testis inflammation in O and the normal seminiferous tubules of O.T. Right panel: the mean testis inflammation index of O and O.T. (D) The top 25 TF activities in Gnrh1 neurons in three groups. (E) The activities of 14 pathways in Gnrh1-, Esr2-, Esr1- or Ar-positive neurons. (F) Significant causal effects (IVW, p < 0.05) between exposure GNRH1 (id: prot-a-1233) and 203 endocrine-related outcomes, which were not significant in reverse MR analysis. (G) Items with significant causal effects (IVW, p < 0.05) in both directions of MR analysis between GNRH1 (id: prot-a-1233) and 203 endocrine-related outcomes.