Reviewer #1 (Public Review): The effects of 17α-estradiol treatment on endocrine system revealed by single-nucleus transcriptomic sequencing of hypothalamus

In this study, we investigated the role of 17α-estradiol in lifespan extension and its potential side effects for long-term administration. Pooled hypothalami from aged male Norway brown rats treated with 17α-estradiol (O.T), aged male controls (O), and young male controls (Y) were subjected to single-nucleus transcriptomic sequencing (snRNA-seq). To evaluate the effects of 17α-estradiol in anti-aging in neurons, supervised clustering on neurons by neuropeptides and their receptors were used to evaluate the responses of each neuron subtype during aging and after 17α-estradiol treatment. The elevated cellular metabolism, stresses and decreased synaptic activity in neurons initiated by aging were remarkably attenuated by 17α-estradiol. Assessment of changes in neuron populations showed that neurons related to food intake, reproduction, blood pressure, stress response and electrolyte balance were sensitive to 17α-estradiol treatment. 17α-estradiol treatment not only increased Oxytocin (Oxt), but also increased the activity of hypothalamic-pituitary-gonadal (HPG) axis, evidenced by significantly elevated levels of plasma Gnrh, bioavailable testosterone, and decreased estradiol. Elevated Gnrh1 was verified to be one of the causal effects mediating the role of 17α-estradiol in energy homeostasis, neural synapse, and stress response. Notably, Crh neurons displayed most prominently senescent phenotype among all checked neuron subtypes in O.T, which may be a potential side effect of 17α-estradiol treatment. Therefore, HPG axis and energy metabolism may be key targets of 17α-estradiol in male lifespan extension. Additionally, supervised clustering of neurons was shown to be a useful method to assess the responses to treatment among different neuron subtypes in the hypothalamus.