Glucocorticoids desensitize hypothalamic CRH neurons to norepinephrine and somatic stress activation via rapid nitrosylation-dependent regulation of α1 adrenoreceptor trafficking

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigate the cellular mechanism underlying suppression of adrenergic effects on excitatory transmission onto hypothalamic CRH neurons by stress. Experiments in ex-vivo slices show that this is a long-lasting effect that occurs through endocytosis of receptors. The authors then move into an immortalized hypothalamic cell line to enable investigation of the mechanism of changes in receptor trafficking. They use a series of immunohistochemistry, FRET, and biochemical experiments to show that application of corticosterone increases targeting of alpha1 adrenergic receptors to the late endosome and lysosome rather than the recycling endosome. Perhaps most interesting, they find that alpha1 receptors and glucocortioid receptors form a complex that is ultimately transferred to the nucleus.

Strengths:

Overall, the studies in this manuscript are rigorous and well-conducted. The data supports their conclusions, and they've shown convincingly that glucocorticoid signaling affects trafficking of alpha1 receptors in the culture system they are using. These findings are important for the field of stress research, both in understanding how two components of the stress system (norepinephrine and HPA axis) interact with each other and in neuromodulatory modulation of hypothalamic CRH neurons. Their finding that alpha1 receptors and glucocorticoid receptors form a complex is particularly interesting and maybe impactful outside of the immediate application in the hypothalamus.

Weaknesses:

The study has two primary weaknesses. First, the majority of the experiments were conducted in an immortalized hypothalamic cell line. This was necessary to conduct the type of experiments needed to test the author's hypothesis, but it remains unclear how closely these cells resemble CRH neurons, or how the same mechanism may be preserved or altered in an intact circuit. Further discussion of these points would strengthen the manuscript.
Second, while experiments are generally well-designed, the authors do not show that the effects of corticosterone can be blocked with a glucocorticoid receptor antagonist. This is fairly standard pharmacology and would strengthen confidence in the findings presented in the study.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors report novel and exciting findings delineating a non-transcriptional mechanism whereby glucocorticoids desensitize CRH neurons to NE in response to somatic stress. The authors find that this desensitization induced by CORT 1. persists more than 18h, 2. reduces surface expression of AR1bR (NE receptors) by redirecting trafficking from rapid recycling to late endosomal pools and lysosomes, 3. is dependent on NE binding to the AR1bR, 4. involves cellular nitrosylation, 5. involves ubiquitination of beta-arrestin, and 5. involves interactions between glucocorticoid receptors and AR1bs, glucocorticoid receptors and ubiquitinated beta-arrestin, and AR1b and ubiquitinated beta-arrestin. While the authors do not directly provide evidence for a trimeric complex composed of these three proteins, their data that CORT causes translocation of these dimeric complexes to the cell nucleus suggests it is likely. Overall, these results are highly informative for understanding novel mechanisms mediating glucocorticoid regulation of GPCRs.

Strengths:
- Good rationale for each experiment, which describes many parts of the CORT-NE desensitization mechanism
- Great discussion of limitations of the approaches and the parts of the mechanism we do not fully understand yet
- Appropriate approaches for questions being answered
- Describes a highly novel CORT mechanism that non-transcriptionally switches GPCR trafficking dynamics, something that could have far reaching implications for other GPCRs involved in stress responses

Weaknesses:
- Unclear how this mechanism would generalize to other stressor modalities. Restraint stress is a somatic stressor, but can also be considered a psychological stressor (model of depression-like behavior). A purely somatic stressor might increase the robustness of this phenomenon.
- Remains unknown how nitrosylation plays into the mechanism in terms of specific proteins affected by CORT (GRK2, endophilin, clathrin possibilities)

Reviewer #3 (Public review):

Summary:

In this manuscript, Weiss et al describe a mechanism through which glucocorticoids desensitize CRH neurons in the PVN to norepinephrine. This follows on from previous work from this lab showing rapid glucocorticoid suppression of adrenergic signaling in CRH neurons specific to somatic stress activation, and modality-selective glucocorticoid negative feedback.

Specifically, their previous work shows that:
(1) NE increases glutamate drive to CRH neurons
(2) CORT blunts the effects of NE through a dynamin-dependent mechanism
(3) This contributes to loss of NE signalling after stress (specifically when the second stressor is a physiological one)

Here they extend this line of interrogation by showing that CORT redistributes Ara1b receptors from rapid recycling endosomes to late endosomes and lysosomes. They show a time window of CORT actions and provide additional mechanistic details implicating nitric oxide-dependent nitrosylation in receptor trafficking.

Strengths:

Builds on existing work to provide additional mechanistic details.
The experiments are well done and data are compelling.
The link to nitrosylation is novel (but see below)

Weaknesses:

(1) The link to nitrosylation is interesting, but a bit confusing. If I understand correctly, inhibiting the production of NO or using NEM increases receptor internalization, suggesting that NO-dependent nitrosylation prevents ligand-dependent internalization. What is unclear to me is how CORT is linked to this step. I note the authors show a decrease in nitrosylation with CORT. So, does CORT decrease the activity of NOS and, thus, the production of NO? If so, then exogenously activating this system in the presence of CORT should result in a recovery of NE-dependent increase in glutamate release. Or is the GCR directly decreasing nitrosylation? Linking these elements is critical in terms of furthering our mechanistic understanding of this process.

(2) It's not clear why/how blockade of Ara1 after CORT-induced cytosolic accumulation results in a reversal of effect. Unless I misunderstood something, this requires further explanation.

Author response:

We appreciate the expression of enthusiasm for our paper by the editors and the three reviewers and the suggestions on how to improve the study. Here we outline how we will address the reviewers’ concerns and suggestions in a planned revision of our manuscript.

Reviewer #1 listed two primary weaknesses:

(1) the need for discussion of the extent to which the cell line we used resembles CRH neurons and

(2) that we did not test for the effect of blockade of the glucocorticoid receptor.

(1) As the reviewer acknowledges, our experiments called for the use of a cell line to dissect intracellular trafficking of the α1 adrenoreceptor. We selected the N42 cell line for this purpose because it is an immortalized hypothalamic cell line (developed by Belsham and colleagues, Belsham et al., 2004) that expresses CRH. We have used this cell line successfully in the past to study transcriptional and rapid non-genomic actions of glucocorticoids, which indicated that, in addition to expressing CRH, these cells also express both the nuclear glucocorticoid receptor and a membrane-associated receptor that binds glucocorticoids (Rainville et al., 2019; Weiss et al., 2019). We believe that this hypothalamic cell line is the most closely related to native PVN CRH neurons of any cell line available. As requested, we will add to the Discussion of the manuscript to further justify our choice of cells.

(2) We agree that this experiment should be performed. We will test the classical GR (and progesterone) antagonist RU486 (mifepristone) for its effect on the cort regulation of α1 adrenoreceptor trafficking. Our ex vivo electrophysiology studies have indicated that the rapid glucocorticoid effect in native hypothalamic CRH neurons is not blocked by RU486 and is not, therefore, dependent on activation of the classical nuclear GR (Di et al., 2003; Di et al., 2016).

Reviewer #2 also listed two main weaknesses of the study:

(1) that we did not test whether the adrenoreceptor desensitization by restraint stress generalizes to other stress modalities and might be more robust with a pure somatic stressor, and

(2) the lack of identification of a target protein as a mechanism for the role of nitrosylation.

(1) We used restraint stress as a means to elicit corticosterone release, which desensitized the HPA response to a NE-dependent somatic stressor (lipopolysaccharide injection) but not to a NEindependent psychological stressor (predator odor) (Jiang et al., 2021). We got a near-complete loss of the sensitivity of CRH neurons to NE with restraint (i.e., near ceiling effect), such that a different stressor, including a more purely somatic stressor, should not increase the Cort-induced desensitization further. For that reason, we would argue that testing other stressors would not add value to the current study. That said, we plan and have received new funding to test in the future whether the Cort desensitization of the HPA response to LPS stress generalizes to other somatic stressors. We also have future plans to test for the Cort desensitization of other Gq-coupled receptors.

(2) We agree that finding the molecular target of nitrosylation as the mechanism for Cort desensitization of α1 adrenoreceptors would significant improve the study, but this is a potentially enormous undertaking as it will require the screening and validation of multiple proteins involved in protein trafficking to find the one(s) targeted for nitrosylation by Cort. We tested β-arrestin as a possible target in the paper, but did not find Cort to regulate β-arrestin nitrosylation. We plan to undertake a general nitrosylation screen of proteins to identify multiple possible targets, but prefer to defer this and the validation of possible targets to a future, more thorough analysis.

Reviewer #3 also pointed out two main weaknesses of our study:

(1) that the glucocorticoidnitrosylation link was confusing, and

(2) that it was unclear how blocking α1 adrenoreceptors reversed the Cort-induced cytosolic accumulation of the receptor.

We appreciate the reviewer pointing out these deficiencies in our interpretation and explanation of our findings. We plan to address them directly in the revised version of the paper.

References

Belsham DD, Cai F, Cui H, Smukler SR, Salapatek AMF, Shkreta L (2004) Generation of a phenotypic array of hypothalamic neuronal cell models to study complex neuroendocrine disorders. Endocrinology 145:393–400.

Weiss GL, Rainville JR, Zhao Q, Tasker JG (2019) Purity and stability of the membrane-limited glucocorticoid receptor agonist dexamethasone-BSA. Steroids 142:2-5.

Rainville JR, Weiss GL, Evanson N, Herman JP, Vasudevan N, Tasker JG (2019) Membrane-initiated nuclear trafficking of the glucocorticoid receptor in hypothalamic neurons. Steroids 142:55-64.

Di S, Malcher-Lopes R, Halmos KCs, Tasker JG (2003) Non-genomic glucocorticoid inhibition via endocannabinoid release in the hypothalamus: a fast feedback mechanism. Journal of Neuroscience 23:4850-4857.

Di S, Itoga CA, Fisher MO, Solomonow J, Roltsch EA, Gilpin NW, Tasker JG (2016) Acute stress suppresses inhibition and increases anxiety via endocannabinoid release in the basolateral amygdala. Journal of Neuroscience 36:8461-8470.

Jiang Z, Chen C, Weiss GL, Fu X, Stelly CE, Sweeten BLW, Tirrell PS, Pursell I, Stevens CR, Fisher MO, Begley JC, Harrison LM, Tasker JG (2022) Stress-induced glucocorticoid desensitizes adrenoreceptors to gate the neuroendocrine response to somatic stress in male mice. Cell Reports 41(3):111509.