The effects of 17α-estradiol treatment on endocrine system revealed by single-nucleus transcriptomic sequencing of hypothalamus
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eLife assessment
This study demonstrates the potential role of 17α-estradiol in modulating neuronal gene expression in the aged hypothalamus of male rats, identifying key pathways and neuron subtypes affected by the drug. While the findings are useful and provide a foundation for future research, the strength of supporting evidence is incomplete due to the lack of female comparison, a young male control group, unclear link to 17α-estradiol lifespan extension in rats, demonstration of physiological effects of the treatment, and insufficient analysis of glial cells and cellular senescence in CRH neurons.
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Abstract
In this study, we investigated the role of 17α-estradiol in lifespan extension and its potential side effects from 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 on aging neurons, supervised clustering of neurons based on 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, stress and decreased expression levels of pathways involved in synapse formation in neurons initiated by aging were significantly 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 serum Oxytocin (Oxt), but also heightened the activity of hypothalamic-pituitary-gonadal (HPG) axis, as evidenced by significantly elevated levels of plasma Gnrh, total testosterone, and decreased estradiol. Elevated Gnrh1 was confirmed to be one of the causal effects mediating the role of 17α-estradiol in energy homeostasis, neural synapse, and stress response. Notably, Crh neurons exhibited prominent stressed phenotype among all the checked neuron subtypes in O.T, which may indicate a potential side effect of 17α-estradiol treatment. Therefore, the HPG axis and energy metabolism may be key targets of 17α-estradiol in male hypothalamus. Additionally, supervised clustering of neurons was shown to be a useful method for assessing treatment responses among different neuron subtypes in the hypothalamus.
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eLife assessment
This study demonstrates the potential role of 17α-estradiol in modulating neuronal gene expression in the aged hypothalamus of male rats, identifying key pathways and neuron subtypes affected by the drug. While the findings are useful and provide a foundation for future research, the strength of supporting evidence is incomplete due to the lack of female comparison, a young male control group, unclear link to 17α-estradiol lifespan extension in rats, demonstration of physiological effects of the treatment, and insufficient analysis of glial cells and cellular senescence in CRH neurons.
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Reviewer #1 (Public Review):
Summary:
Previous studies have shown that treatment with 17α-estradiol (a stereoisomer of the 17β-estradiol) extends lifespan in male mice but not in females. The current study by Li et al, aimed to identify cell-specific clusters and populations in the hypothalamus of aged male rats treated with 17α-estradiol (treated for 6 months). This study identifies genes and pathways affected by 17α-estradiol in the aged hypothalamus.
Strengths:
Using single-nucleus transcriptomic sequencing (snRNA-seq) on the hypothalamus from aged male rats treated with 17α-estradiol they show that 17α-estradiol significantly attenuated age-related increases in cellular metabolism, stress, and decreased synaptic activity in neurons.
Moreover, sc-analysis identified GnRH as one of the key mediators of 17α-estradiol's effects on …
Reviewer #1 (Public Review):
Summary:
Previous studies have shown that treatment with 17α-estradiol (a stereoisomer of the 17β-estradiol) extends lifespan in male mice but not in females. The current study by Li et al, aimed to identify cell-specific clusters and populations in the hypothalamus of aged male rats treated with 17α-estradiol (treated for 6 months). This study identifies genes and pathways affected by 17α-estradiol in the aged hypothalamus.
Strengths:
Using single-nucleus transcriptomic sequencing (snRNA-seq) on the hypothalamus from aged male rats treated with 17α-estradiol they show that 17α-estradiol significantly attenuated age-related increases in cellular metabolism, stress, and decreased synaptic activity in neurons.
Moreover, sc-analysis identified GnRH as one of the key mediators of 17α-estradiol's effects on energy homeostasis. Furthermore, they show that CRH neurons exhibited a senescent phenotype, suggesting a potential side effect of the 17α-estradiol. These conclusions are supported by supervised clustering by neuropeptides, hormones, and their receptors.
Weaknesses:
However, the study has several limitations that reduce the strength of the key claims in the manuscript. In particular:
(1) The study focused only on males and did not include comparisons with females. However, previous studies have shown that 17α-estradiol extends lifespan in a sex-specific manner in mice, affecting males but not females. Without the comparison with the female data, it's difficult to assess its relevance to the lifespan.
(2) It is not known whether 17α-estradiol leads to lifespan extension in male rats similar to male mice. Therefore, it is not possible to conclude that the observed effects in the hypothalamus, are linked to the lifespan extension.
(3) The effect of 17α-estradiol on non-neuronal cells such as microglia and astrocytes is not well-described (Figure 1). Previous studies demonstrated that 17α-estradiol reduces microgliosis and astrogliosis in the hypothalamus of aged male mice. Current data suggest that the proportion of oligo, and microglia were increased by the drug treatment, while the proportions of astrocytes were decreased. These data might suggest possible species differences, differences in the treatment regimen, or differences in drug efficiency. This has to be discussed.
(4) A more detailed analysis of glial cell types within the hypothalamus in response to drugs should be provided.
(5) The conclusion that CRH neurons are going into senescence is not clearly supported by the data. A more detailed analysis of the hypothalamus such as histological examination to assess cellular senescence markers in CRH neurons, is needed to support this claim.
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Reviewer #2 (Public Review):
Summary:
Li et al. investigated the potential anti-ageing role of 17α-Estradiol on the hypothalamus of aged rats. To achieve this, they employed a very sophisticated method for single-cell genomic analysis that allowed them to analyze effects on various groups of neurons and non-neuronal cells. They were able to sub-categorize neurons according to their capacity to produce specific neurotransmitters, receptors, or hormones. They found that 17α-Estradiol treatment led to an improvement in several factors related to metabolism and synaptic transmission by bringing the expression levels of many of the genes of these pathways closer or to the same levels as those of young rats, reversing the ageing effect. Interestingly, among all neuronal groups, the proportion of Oxytocin-expressing neurons seems to be the one …
Reviewer #2 (Public Review):
Summary:
Li et al. investigated the potential anti-ageing role of 17α-Estradiol on the hypothalamus of aged rats. To achieve this, they employed a very sophisticated method for single-cell genomic analysis that allowed them to analyze effects on various groups of neurons and non-neuronal cells. They were able to sub-categorize neurons according to their capacity to produce specific neurotransmitters, receptors, or hormones. They found that 17α-Estradiol treatment led to an improvement in several factors related to metabolism and synaptic transmission by bringing the expression levels of many of the genes of these pathways closer or to the same levels as those of young rats, reversing the ageing effect. Interestingly, among all neuronal groups, the proportion of Oxytocin-expressing neurons seems to be the one most significantly changing after treatment with 17α-Estradiol, suggesting an important role of these neurons in mediating its anti-ageing effects. This was also supported by an increase in circulating levels of oxytocin. It was also found that gene expression of corticotropin-releasing hormone neurons was significantly impacted by 17α-Estradiol even though it was not different between aged and young rats, suggesting that these neurons could be responsible for side effects related to this treatment. This article revealed some potential targets that should be further investigated in future studies regarding the role of 17α-Estradiol treatment in aged males.
Strengths:
(1) Single-nucleus mRNA sequencing is a very powerful method for gene expression analysis and clustering. The supervised clustering of neurons was very helpful in revealing otherwise invisible differences between neuronal groups and helped identify specific neuronal populations as targets.
(2) There is a variety of functions used that allow the differential analysis of a very complex type of data. This led to a better comparison between the different groups on many levels.
(3) There were some physiological parameters measured such as circulating hormone levels that helped the interpretation of the effects of the changes in hypothalamic gene expression.
Weaknesses:
(1) One main control group is missing from the study, the young males treated with 17α-Estradiol.
(2) Even though the technical approach is a sophisticated one, analyzing the whole rat hypothalamus instead of specific nuclei or subregions makes the study weaker.
(3) Although the authors claim to have several findings, the data fail to support these claims.
(4) The study is about improving ageing but no physiological data from the study demonstrated such a claim with the exception of the testes histology which was not properly analyzed and was not even significantly different between the groups.
(5) Overall, the study remains descriptive with no physiological data to demonstrate that any of the effects on hypothalamic gene expression are related to metabolic, synaptic, or other functions.
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