Abstract
Summary
Noradrenergic afferents to hypothalamic corticotropin releasing hormone (CRH) neurons provide a major excitatory drive for somatic stress activation of the hypothalamic-pituitary-adrenal (HPA) axis. We showed that glucocorticoids rapidly desensitize CRH neurons to norepinephrine and suppress inflammation-induced HPA activation via a glucocorticoid receptor- and endocytosis-dependent mechanism. Here, we show that α1 adrenoreceptor (ARα1) trafficking is regulated by convergent glucocorticoid and nitric oxide synthase signaling mechanisms. Live-cell imaging of ARα1b-eGFP-expressing hypothalamic cells revealed rapid corticosterone-stimulated redistribution of internalized ARα1 from rapid recycling endosomes to late endosomes and lysosomes via a nitrosylation-regulated mechanism. Proximity assay demonstrated interaction of glucocorticoid receptors with ARα1b and β-arrestin, and showed corticosterone blockade of norepinephrine-stimulated ARα1b/β-arrestin interaction, which may prevent ARα1b from entering the rapid recycling endosomal pathway. These findings demonstrate a rapid glucocorticoid regulation of G protein-coupled receptor trafficking and provide a molecular mechanism for rapid glucocorticoid desensitization of noradrenergic signaling in CRH neurons.
Introduction
Increased circulating glucocorticoids induced by activation of the hypothalamic-pituitary-adrenal (HPA) axis play an important role in the metabolic, immunoregulatory, and cognitive effects of stress 1,2. Rapid glucocorticoid-mediated negative feedback regulation of the corticotropin releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) contributes to the termination of the HPA response 3. While the mechanism responsible for negative feedback regulation of the HPA axis has been classically attributed to transcriptional regulation by glucocorticoid receptors 4,5, several studies have shown that glucocorticoids rapidly inhibit PVN CRH neurons (Di et al., 2003; Tasker et al., 2006) and pituitary corticotrophs 9 to mediate fast negative feedback (Evanson et al., 2010). Noradrenergic afferents to the CRH neurons provide a primary excitatory drive for the somatic stress activation of the HPA axis 11 via activation of α1 adrenergic receptors (ARα1) 12–15. We recently reported that glucocorticoid rapidly desensitizes PVN CRH neurons to ARα1 activation by norepinephrine (NE), which suppresses the HPA response to somatic stress, but not psychological stress, due to the NE dependence of somatic stress activation of the HPA axis 11,16. A main source of noradrenergic input to PVN CRH neurons is the brainstem nucleus of the solitary tract (NTS) 17. The NTS is activated by inputs from the vagal nerve and relays to the forebrain somatic interoceptive stress signals such as organ damage, metabolic challenge, and immune activation 18. Stimulation of an immune response by systemic lipopolysaccharide (LPS), for example, activates the HPA axis by way of the NTS noradrenergic system 19,20. Our recent findings suggest that prior priming by acute stress exposure attenuates subsequent LPS activation of the HPA axis via a rapid glucocorticoid-induced desensitization of the CRH neurons to NE activation of ARα1 21, but the cellular and molecular mechanisms of this desensitization are not known.
The stress desensitization of PVN CRH neurons to NE is dependent on the ligand-induced internalization of ARα1 21. ARα1 is internalized by clathrin-mediated endocytosis and is trafficked back to the membrane via recycling endosomes, a process that involves multiple trafficking proteins and that establishes a steady state of surface receptors and NE sensitivity 22. Initial steps of endocytosis following ligand binding and G protein activation include receptor phosphorylation, recruitment of β-arrestin to the phosphorylated receptor, and engagement of clathrin and dynamin to execute endocytosis. β-arrestin and the internalized ARα1 undergo post-translational modifications, including nitrosylation and ubiquitination, which regulate trafficking through the cell’s endosomal pathways 23,24. Here, we tested the cellular mechanism by which glucocorticoids desensitize NE signaling. We found that corticosterone (CORT) desensitizes CRH neurons to NE in a process involving nitric oxide synthesis (NOS)-dependent protein S-nitrosylation that rapidly depletes the cells of surface adrenoreceptors by re-routing the ARα1 out of the rapid membrane recycling endosomal pathway and into late endosomes and lysosomes.
Results
Corticosterone causes a rapid, long-lasting desensitization of ARα1
Our previous study (Chen et al., 2019) showed that in ex vivo brain slices, PVN CRH neurons in the male mouse respond to NE with an increase in excitatory postsynaptic currents (EPSCs) that is mediated by postsynaptic ARα1 activation and stimulation of retrograde glial-neuronal circuits by dendritic volume transmission (see diagram in Fig. 1A). A follow-up study showed that the PVN CRH neuron excitatory response to NE is suppressed by 5-10 min of preincubation of brain slices from unstressed mice in CORT (2 μM) (Fig. 1B) and by prior restraint stress-induced endogenous glucocorticoid exposure in vivo (Fig. 1C) 21. The rapid glucocorticoid-induced desensitization of CRH neurons to NE was blocked by a dynamin inhibitor, indicating that it is dependent on endocytosis (Fig. 1C).
Here, we tested the duration of the stress-induced desensitization of CRH neurons to NE by allowing the mice to recover from a 30-min restraint stress in their home cage for 4 h and 18 h prior to sacrifice and brain slice preparation. In animals allowed to recover from acute restraint for 4 h, the PVN CRH neurons remained insensitive to NE (100 µM), whereas the EPSC response to NE was partially restored 18 h after the restraint stress (Fig. 1D). This indicated that the stress-induced desensitization of PVN CRH neurons to activation by NE takes longer than 18 h to fully reverse and suggests a surprisingly long-lasting diminished CRH neuron sensitivity to NE and to adrenoreceptor activation of the HPA axis following exposure to acute stress 21. Thus, acute glucocorticoid exposure causes a long-lasting desensitization of CRH neurons to NE via a mechanism with rapid onset, which is dependent on dynamin-dependent endocytosis of the ARα1 (Fig. 1E).
ARα1b has been reported to be the dominant α1 adrenoreceptor subtype in CRH neurons 25, and we previously reported evidence of ARα1b internalization in fixed N42 cell cultures 21. Here, we used live-cell imaging to follow the internalization and trafficking of the ARα1b through the endosomal pathway. We used the N42 immortalized hypothalamic cell line 26, which we transiently transfected with an pEGFP-ARα1b construct and imaged live by confocal microscopy to visualize ARα1b receptor trafficking. A low NE concentration (1 μM) caused ARα1b internalization, which was seen by a decrease in membrane pEGFP-ARα1b and an increase in cytosolic pEGFP-ARα1b fluorescence, which reached a peak at ∼30 min (Fig. 2A and B), a time course that coincided approximately with that of the NE-induced increase in sEPSC frequency (see Fig. 1). Following 30 min of NE application, CORT (2 μM) was co-applied with NE, which induced a rapid increase in the ARα1b internalization over the NE-induced steady-state internalization. The onset of the CORT effect occurred within minutes and peaked within 10 min of its introduction into the chamber (Fig. 2A and B), which was consistent with a rapid CORT-induced desensitization of CRH neurons to NE. The combination of NE and CORT caused significantly more internalization than NE alone (Fig. 2B, C). The bulk of the NE- and CORT-induced increases in cytosolic ARα1b appeared to be directed mainly to a single “hot-spot” in the cells (arrows in Fig. 2A), which we targeted for quantitation and used to map the trafficking of the ARα1b receptor in the endocytic pathway with endosomal markers. The dynamic change in ARa1b distribution within the cell during NE and NE+CORT application can be followed in real time in the supplemental video 1.
Cort redirects ARα1b trafficking from rapid recycling endosomes to late endosomes
Receptor-mediated endocytosis forms early endosomes, which either cycle to the plasma membrane as recycling endosomes to replenish surface receptors or develop into late endosomes and are routed out of the recycling pathways. Specific Rab GTPases regulate distinct stages of endosomal trafficking 27. By co-transfecting the N42 cells with pEGFP-ARα1b and constructs for different dsRed-tagged Rab proteins, we were able to use Förster resonance energy transfer (FRET) analysis to detect with 10-nm resolution the colocalization of the ARα1b with the specific markers of different endosomal compartments. We tested for ARα1b association with the early endosomal marker Rab5, the late endosomal marker Rab 7, the rapid recycling endosomal marker Rab4a, and the slow recycling endosomal marker Rab 11 28.
Cells treated with NE (1 µM) were first analyzed for an increase in ARα1b FRET with dsRed-tagged Rab5, a marker of newly internalized cargo being transported to the early endosome. Norepinephrine caused the expected increase in ARα1b localization in the early endosome, serving as a control for ligand-mediated endocytosis of the adrenoreceptor. However, subsequent CORT application failed to increase colocalization of ARα1b with Rab5 compared to that caused by NE alone (Fig. 3A). This indicated that the CORT-induced increase in ARα1b intracellular concentration was mediated by an accumulation of ARα1b in the cytosol following standard ligand-dependent internalization and not by a CORT-induced increase in ARα1b endocytosis. We next tested whether CORT caused an increase in the receptor trafficking to the late endosome using ARα1b FRET with the late endosomal marker Rab7. Norepinephrine alone caused an increase in the ARα1b-Rab7 FRET signal, and subsequent CORT treatment increased the FRET signal compared to that seen with NE alone (Fig. 3B). This indicated that CORT caused an increase in the NE-induced ARα1b trafficking to the late endosome and suggests that the CORT-induced desensitization is not associated with an increase in the amount of ARα1b undergoing internalization, but rather that it causes an increase in the receptor trafficking into the late endosomal pathway. A possible explanation for the accumulation of ARα1b in the late endosome and loss of sensitivity to NE could be a decrease in the trafficking of the receptor back to the plasma membrane.
We tested this using FRET analysis with the rapid recycling endosomal marker Rab4a and with the slow recycling endosomal marker Rab11. Norepinephrine did not cause a change in the ARα1b-Rab4a FRET signal, and the addition of CORT decreased the ARα1b-Rab4a signal compared to NE alone (Fig. 3C), indicating reduced ARa1b association with Rab4a and suggesting a reduction in trafficking of the ARα1b through the rapid recycling endosome. Finally, we tested for changes in the ARα1b interaction with the slow recycling endosomal marker Rab11. Neither NE nor NE+CORT had any effect on the ARα1b-Rab 11 FRET signal (Fig. 3D), suggesting that neither treatment increases the slow recycling of ARα1b to the membrane within the timeframe of the desensitization to NE. Together, these data indicate that CORT influences ARα1b trafficking by redirecting the receptor away from rapidly recycling endosomes and into late endosomes. This would be expected to deplete the membrane of adrenoreceptors and provides, therefore, a molecular substrate for the glucocorticoid-induced desensitization to NE observed in ex vivo recordings.
Cort causes ARα1b trafficking to the lysosome
To determine whether CORT causes ARα1b to travel further down the endocytic pathway from late endosomes to lysosomes, we tested for ARα1b trafficking to the lysosome by imaging the co-localization of pEGFP-ARα1b with the lysosomal marker LAMP1 conjugated to red fluorescent protein (LAMP1-RFP). Application of NE (1 μM) caused an increase in ARα1b-LAMP1 co-staining (Fig. 4A-C), indicating an increase in the co-localization of the two proteins. After 30 min of NE application, CORT co-application (2 µM) further increased the ARα1b-LAMP1 co-staining (Fig. 4A-C), which revealed a CORT facilitation of ARα1b trafficking to the lysosome for degradation. However, a large proportion of the ARα1b was not co-stained with the LAMP1 marker, including the CORT-induced ARα1b “hot spot”, resulting in a low Pearson’s correlation of ARα1b and LAMP1 localization (maximum Pearson’s coefficient ∼0.5) (Fig. 4B), which suggests that, on this time scale (∼50 min), most of the ARα1b receptor was not trafficked to the lysosome for degradation, but rather was sequestered within the cell.
Blocking ARα1 reverses Cort-induced ARα1b trafficking
We found previously that CORT alone without prior NE application has no effect on ARα1b internalization in N42 cells and that inhibiting ARα1 with prazosin during CORT application in hypothalamic slices blocked the glucocorticoid-induced desensitization of CRH neurons to NE 21, suggesting that glucocorticoid-induced desensitization to NE requires ligand (NE)-induced ARα1 internalization. Here, we tested whether N42 cells in which CORT had already caused increased ARα1b cytosolic accumulation would recover after blocking ARα1 with prazosin. As described above (see Fig. 1 and Fig. 2A-C), 1 μM NE caused ARα1b accumulation in the cytosol that was further increased by CORT (2 μM). The addition of the ARα1 antagonist prazosin (1 μM) caused the internalized ARα1b to return to near baseline levels within 10 min (Fig. 5A, B). These data suggest that blocking ARα1 reverses glucocorticoid-induced desensitization to NE.
Nitrosylation dependence of the CRH neuron response to norepinephrine
Since GPCR internalization and trafficking are dependent on S-nitrosylation of proteins associated with receptor trafficking, including β-arrestin 24, we next tested whether the CORT control of ARα1 trafficking is regulated by nitric oxide synthase (NOS) activity. We first tested this with whole-cell recordings of the NE response in CRH neurons in ex vivo hypothalamic slices. Blocking NOS activity with the broad-spectrum NOS inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME, 50 μM) inhibited the NE-induced increase in sEPSC frequency (Fig. 6A, B), which was similar to the glucocorticoid inhibition of the NE-induced increase in sEPSC frequency in CRH neurons (see Fig. 1). Since NOS induces both the production of the gaseous messenger NO and post-translational S-nitrosylation of target proteins, we tested for these two NOS-dependent mechanisms pharmacologically. We first blocked the NO receptor, soluble guanylyl cyclase, with 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) (100 μM), but this failed to inhibit the NE-induced increase in sEPSC frequency in the CRH neurons (Fig. 6A, B). This confirmed our previous finding that NE does not induce an NO retrograde signal at glutamate synapses on the CRH neurons 15. We next tested the effect of the S-nitrosylation inhibitor N-ethylmaleimide (NEM) (50 µM) on the NE response. NEM completely blocked the NE-induced increase in sEPSC frequency in the CRH neurons (Fig. 6A, B). These findings suggested that the NOS dependence of the NE activation of excitatory inputs to the CRH neurons is due to S-nitrosylation of target proteins in the CRH neurons, and not to the release of NO as a transmitter.
Several proteins involved in GPCR trafficking are regulated by nitrosylation, including G protein-coupled receptor kinase 2 (GRK2), β-arrestin, and clathrin 24,29. We first tested for the nitrosylation dependence of intracellular trafficking of the ARα1b with live-cell imaging in N42 cells. We found that N42 cells express endothelial NOS and inducible NOS, but not neuronal NOS (Supplemental Fig. 1). Co-application of the NOS inhibitor L-NAME (50 μM) with NE (1 μM) caused an increase in the ARα1b internalization over that induced by NE application alone (Fig. 6C, D), suggesting that inhibiting S-nitrosylation enhanced ligand-dependent internalization/trafficking of the receptor. Co-application of CORT (2 µM) with L-NAME failed to increase the ARα1b cytosolic concentration compared to L-NAME alone (Fig. 6C, D), indicating that CORT did not cause any additional intracellular ARα1b accumulation. This suggested, therefore, that blocking S-nitrosylation caused an occlusion of the CORT effect, revealing a convergence of the two signaling mechanisms and opposite effects of CORT (i.e., increased internal ARα1b accumulation) and S-nitrosylation (i.e., decreased internal ARα1b accumulation).
We next tested for changes in S-nitrosylation in the N42 cells with a biochemical approach using Western blot analysis and a proximity ligation assay following the application of a modified biotin-switch procedure. The biotin-switch procedure consists of cleaving the NO groups on cysteine residues of nitrosylated proteins and labeling these thiols with biotin or another tag such as a tandem mass tag (TMT) 30. Western blot of TMT-tagged proteins in the cytosolic fraction of N42 cells showed a significant decrease in total S-nitrosylation with a 20-min application of CORT (2 µM) (Fig. 6E). This was likely due to activation of the glucocorticoid receptor because we found a similar decrease in total S-nitrosylation in N42 cells with the synthetic glucocorticoid dexamethasone (1 µM) (data not shown).
Western blot analysis revealed a strong nitrosylation signal at a molecular weight of about 50 kD, the approximate molecular weight of β-arrestin. Since β-arrestin has been shown to be regulated by S-nitrosylation 24,31, we next performed a histological analysis of β-arrestin1/2 S-nitrosylation to test whether the glucocorticoid-induced reduction in total S-nitrosylation is due to changing the nitrosylation state of β-arrestin1/2. We combined the biotin-switch nitrosylation assay with a proximity ligation assay using a biotin antibody and a β-arrestin1/2 antibody to detect nitrosylated β-arrestin. Proximity ligation assay reveals protein-protein interactions with a 40-nm resolution 32. Cells were analyzed for the total number of biotin-β-arrestin interactions (i.e., nitrosylation association with β-arrestin) and for nuclear versus cytosolic localization of the interactions. Treatments with NE (1 µM), CORT (2 µM), and NE + CORT all failed to produce a statistically significant change in the PLA signal with respect to vehicle, suggesting they did not affect the nitrosylation of β-arrestin (Fig. 6F, G). The NE + CORT treatment caused a small decrease in the PLA signal, but this did not reach statistical significance (p = 0.1470, Dunnett’s multiple comparisons test). Similarly, the treatments failed to change the nuclear fraction of nitrosylated β-arrestin (Fig. 6H). These findings suggest that the nitrosylation dependence of the NE and Cort effects on ARa1b trafficking is not mediated by the regulation of β-arrestin1/2 S-nitrosylation and that nitrosylated β-arrestin1/2 is not trafficked to the nucleus in response to NE and/or Cort.
Ubiquitination is critical for endosomal processing and cellular trafficking. If glucocorticoid changes the internal trafficking of ARα1b, then it may do so by changing the ubiquitination state of β-arrestin 33. We tested for changes in the ubiquitination of β-arrestin in response to NE and CORT treatment (20 min). Both NE (1 µM) and CORT (2 µM) alone increased the ubiquitination of immunoprecipitated β-arrestin1/2 approximately two-fold (Supplemental Fig. 2). The increases in ubiquitinated β-arrestin1/2 caused by CORT and NE were additive when the cells were co-treated with the two drugs (Supplemental Fig. 2). Since the ubiquitination pattern of β-arrestin has been shown to affect trafficking of internalized cargo 34, these data suggest that NE and CORT may alter ARα1b receptor trafficking by increasing β-arrestin ubiquitination.
Glucocorticoid receptor interaction with the α1 adrenoreceptor
To interrogate the mechanism by which NE and CORT exert cooperative effects on ARα1b trafficking and β-arrestin ubiquitination, we tested for a direct interaction between ARα1b and the glucocorticoid receptor (GR) with the proximity ligation assay. A human ARα1b construct with a myc-DDK tag (hARα1b-myc-DDK) or an ARα1b-eGFP construct was transiently transfected into N42 cells. Proximity ligation was achieved by using either a mouse anti-myc antibody or a mouse anti-GFP antibody to target ARα1b and a rabbit anti-glucocorticoid receptor antibody (M-20, Santa Cruz) to target GR. Individual cells were analyzed for the number of protein-protein interactions and for nuclear versus cytosolic localization of the interacting proteins. A basal level of interaction between ARα1b and GR was observed in vehicle-treated cells (Fig. 7A). Norepinephrine alone had no effect on ARα1b-GR interaction, whereas CORT treatment caused a small but significant decrease in total ARα1b-GR interaction compared to vehicle and NE treatment, which was unchanged by the combination of CORT + NE (Fig. 7A, B). We next analyzed the nuclear translocation of the ARα1b-GR complex after treatment with NE and/or CORT. Norepinephrine alone had no effect on the nuclear fraction of the ARα1b-GR complex, but CORT caused a significant increase in the nuclear ARα1b-GR PLA signal compared to vehicle and to NE. The increase in nuclear localization of the ARa1b-GR complex caused by CORT was reversed by co-application of NE (Fig. 7A, C). This suggested that NE activation of ARα1b prevented the CORT-induced nuclear translocation of the GR-ARα1b complex. Thus, ARα1b and GR can be found in a protein complex that is trafficked to the nucleus in response to CORT but not in the presence of NE, when the ARα1b is presumably mostly bound to its ligand.
β-arrestin interactions with the α1 adrenoreceptor and glucocorticoid receptor
We next examined interactions of β-arrestin1/2 to determine whether the post-translational S-nitrosylation of β-arrestin is accompanied by changes in its interactions with ARα1b and GR. As expected, NE treatment (1 μM) of the N42 cells for 20 min caused an increase in ARα1b-β-arrestin interaction (Fig. 8A, B). Treatment with CORT alone (2 μM) had no effect on the number of ARα1b-β-arrestin PLA profiles, but it blocked the NE-induced increase in ARα1b-β-arrestin complex formation when co-applied with NE (Fig. 8A, B). CORT treatment alone also caused a significant increase in nuclear localization of the ARα1b/β-arrestin complex, which was blocked by NE when NE and CORT were co-applied (Fig. 8A, C).
Similar to what has been reported previously 35, we found a baseline interaction between GR and β-arrestin1/2. The drug treatments did not affect the total number of GR-β-arrestin1/2 interactions (Fig. 9A, B). CORT application caused the GR-β-arrestin1/2 complex to translocate to the nucleus, and co-application of NE with CORT did not prevent the CORT-induced nuclear translocation of the GR-β-arrestin1/2 complex (Fig. 9A, C).
Discussion
Acute stress desensitizes PVN CRH neurons in male mice to stimulation by noradrenergic afferents via a rapid glucocorticoid signaling mechanism. The glucocorticoid-induced NE desensitization suppresses the HPA response to somatic, but not psychological, stress and is dependent on the NE-mediated endocytosis of α1 adrenoreceptors 21. Here, we tested for the cellular mechanisms of the α1 adrenoreceptor desensitization in slices from male mice (consistent with our previous studies, Chen et al., 2019; Jiang et al., 2022), and in the N42 hypothalamic cell line, which expresses both CRH 26 and a putative membrane receptor that mediates rapid glucocorticoid signaling21,36,37.
Our findings reveal a rapid glucocorticoid regulation of the trafficking of α1 adrenoreceptors that diverts the receptors away from the rapid recycling endosomal pathway and into late endosomes. The glucocorticoid regulation of α1 receptor trafficking requires internalization of the α1 receptors by NE, is dependent on the overall nitrosylation state of the cells and involves ubiquitination of β-arrestin. The rapid glucocorticoid-induced re-routing of the α1 adrenoreceptors out of the rapid recycling endosomal pathway would be expected to deplete the cell of surface receptors and to cause the desensitization of CRH neurons to NE following acute stress exposure 21.
Glucocorticoid secretion after immune activation of the HPA axis is essential for downregulation of the immune response 38. Left unchecked, inflammation and hypotension associated with immune system activation can cause problems beyond the immune challenge itself. One example is toxic shock syndrome, which occurs when bacteria-produced toxins cause over-activation of cytokines and inflammatory cells resulting in fever, rash, hypotension, and, in extreme cases, organ failure and death39. Glucocorticoids constrain the immune response, and glucocorticoid therapy is often employed to inhibit the overactive immune system 40. For example, the cytokine storm occurring in some COVID-19 patients was treated with the synthetic glucocorticoid dexamethasone 41,42.
Previously we reported that both acute restraint stress and CORT pre-exposure induced a desensitization of CRH neurons to the excitatory effect of NE that lasted for the entire period of brain slice recording (2-6 h) 21. However, whether ex vivo brain slice preparation prevented the CRH neurons from recovering from their desensitization to NE by placing them in a suspended desensitized state was not known, nor was it clear how long the desensitization to NE lasts following stress. Here we sacrificed the mice at two time points following a 30-min restraint, at 4 h and 18 h. A 4-h interval falls within the normal recording period following slice preparation and, we reasoned, would determine whether the observed desensitization of the CRH neurons sustained ex vivo is an accurate reflection of what occurs in vivo. The 18-h interval fell on the day after the stress exposure and tested whether the in vivo duration of the NE desensitization was sustained overnight. We found that the 4-h post-stress recovery period was insufficient for the CRH neurons to recover from glucocorticoid-induced NE desensitization, and that the NE response was partially, though not entirely, restored at 18 h after the acute stress. Thus, the desensitization of CRH neurons to noradrenergic activation is long-lasting, persisting for over 18 h.
We showed previously that the CRH neuron response and HPA activation to a somatic stress, LPS exposure, but not to a psychological stress, predator odor, are mediated by NE afferents and susceptible to desensitization by prior stress exposure 21. The long-lasting desensitization to NE appears, therefore, to be a negative feedback filter of the HPA axis that passes acute psychological stress activation while filtering out more long-lasting somatic stress activation (e.g., by immune challenge). This filtering of stress modality-specific stress responsiveness is adaptive in that it reduces the duration of stress exposure, since somatic stressors tend to be prolonged in nature, which would otherwise subject the organism to the damaging effects of sustained glucocorticoid exposure. However, this feature could be maladaptive in modern day, since deprioritizing somatic stressors in favor of psychological stressors may be beneficial in life-or-death situations, such as a looming predator, but those situations are less likely to occur in modern human life. Additionally, many modern stressors could be perceived as severe even though they may not be life threatening. Thus, acute stress-induced desensitization could be a maladaptive mechanism that needlessly deprioritizes somatic stressors and leaves the body susceptible to physiological distress. Further studies are needed to determine whether desensitization of the HPA axis generalizes to other somatic stressors, and to ascertain the role of noradrenergic desensitization of the HPA axis in diseases known to be caused by HPA dysregulation.
Glucocorticoid altered ARα1b trafficking
Live-cell imaging of NE and CORT effects on trafficking of ARα1b revealed that much of the trafficked receptor was directed to a single area of each cell, or a “hot spot”. This receptor-concentrated “hot-spot” co-localized well with the Rab7 marker of late endosomes. FRET analysis confirmed that Rab7 and ARα1b are physically associated within 10 nm of each other, indicating that ARα1b is likely located in the late endosome. The lack of increase in early endosomal ARα1b in the presence of CORT indicates that the desensitization to NE by CORT is not caused by an increase in ARα1b internalization, as we originally hypothesized based on its prevention by blocking dynamin-dependent endocytosis 21. The decrease in ARα1b in rapidly recycling endosomes suggests that the increase in cytosolic receptor is due to a decrease in receptor trafficking back to the membrane. This rapid steroid-driven change in GPCR trafficking is not only novel per se, but also reveals a previously unobserved switch in the speed of receptor trafficking. Receptors of the Class A GPCRs, including the adrenoreceptors, are rapidly recycled back to the plasma membrane in minutes, whereas Class B receptors, which bind more stably to β-arrestin, take several hours 43. The ubiquitination patterns of the β-arrestin that associates with these receptors during endocytosis appear to be specific for the receptor family type and ultimately control the speed of trafficking 34. Our findings reveal a previously unobserved switch in GPCR trafficking kinetics and suggest that CORT could be altering the ubiquitination patterns of β-arrestin to switch GPCRs from rapidly trafficking Class A receptors to slowly trafficking Class B-like receptors. The overall increase in β-arrestin ubiquitination in the presence of CORT suggests that other rapidly trafficking GPCRs may be similarly affected. Thus, this may represent a more universal mechanism of CORT desensitization of multiple neurotransmitters systems, a prospect that needs to be further explored.
While it remains unclear how glucocorticoids cause an increase in β-arrestin ubiquitination, changes in trafficking may also be achieved by S-nitrosylation of trafficking proteins. We found no change in the nitrosylation of β-arrestin 1/2 with NE or CORT treatment, indicating that the convergent regulation of ARα1 by nitrosylation and glucocorticoid is not mediated by a change in the nitrosylation state of β-arrestin. S-nitrosylation regulates multiple other proteins involved in GPCR trafficking in addition to β-arrestin, including G protein-coupled receptor kinase 2, endophilin, and clathrin (Hayashi et al., 2018; Ozawa et al., 2008; Whalen et al., 2007). It is possible, therefore, that rapid CORT regulation of nitrosylation of these proteins may mediate the glucocorticoid modulation of ARα1b intracellular trafficking, a possibility that remains to be determined.
The nuclear glucocorticoid receptor is one possible receptor candidate for the rapid CORT actions in this pathway. However, one or more other membrane receptors may also be candidates for the rapid glucocorticoid effects. Many studies have provided evidence for the existence of a glucocorticoid receptor on the membrane that signals via G protein or tyrosine kinase activity (Di et al., 2003; Rhen and Cidlowski, 2005), and a recent study shows glucocorticoid binding to a membrane adhesion Gαi-coupled orphan receptor 45. Our PLA analysis indicated a close physical association (<40 nm) of GR with ARα1b, which suggests a possible direct interaction of the two receptors. We have found the M-20 GR antibody we used here to recognize proteins in the membrane, cytosolic, and nuclear fractions in Western blot analyses (Figure 7). The GR/ARα1b complex detected by PLA responded to CORT by translocating to the nucleus, an effect that was blocked by concomitant activation of ARα1b by NE. Thus, the receptor complex is differentially regulated depending on its component receptor activation. The interaction between the two receptors suggests this as a possible rapid mechanism whereby glucocorticoids could influence ARα1b intracellular trafficking.
Based on the well-established role of β-arrestin in receptor trafficking, our finding of an additive effect of Cort and NE on β-arrestin ubiquitination suggests that β-arrestin is a locus of the Cort actions that regulate ARα1b trafficking. The proximity ligation assay showed that both GR and ARα1b can individually interact with β-arrestin, and further showed that GR and ARα1b can interact with each other. However, the PLA did not allow for determination of whether GR, ARα1b, and β-arrestin act together in a single complex. The ability of CORT to cause nuclear translocation of the ARα1b-β-arrestin complex, the ARa1b-GR complex, and the GR-β-arrestin complex suggests a trimeric complex that includes the GR, but we have not demonstrated the multimeric association of the three proteins by molecular assays. While adrenoreceptor localization in the nucleus has been previously reported in cardiomyocytes 46 and astrocytes 47, this phenomenon is still relatively novel. ARα1b signaling could be further biased upon entering the nucleus, giving rise to more potential targets for synergistic NE-CORT signaling. Regardless, co-activation of ARα1b and GR results in fundamental changes in the state of β-arrestin, increasing its ubiquitination, which are associated with profound alterations in the trafficking of the ARα1b. The CORT regulation of adrenoreceptor trafficking is likely to be responsible for the stress desensitization of the CRH neurons to the noradrenergic excitatory synaptic input activated by somatic stress.
Finally, the concept of CORT-induced changes in endosomal trafficking is novel. Changes in receptor trafficking could have a broader impact on neuronal function including plasticity via regulation of ionotropic receptors 48,49. Glucocorticoids have been shown to facilitate Rab4 cycling, in turn increasing AMPAR recycling 50. These studies are crucial for understanding the full extent of rapid stress-induced glucocorticoid regulation of synaptic signaling beyond the classically studied transcriptional regulation.
Acknowledgements
We would like to thank Dr. Chris Hague of the University of Washington for his donation of the ARa1b-eGFP plasmid. This work was supported by NIH grant MH119283 and a Carol Lavin Bernick Faculty Grant from Tulane University.
Additional information
Author contributions
Conceptualization: G.L.W, L.M.H., and J.G.T.; methodology: G.L.W., L.M.H, Z.J., and J.G.T.; investigation: G.L.W., L.M.H, Z.J., A.M.N., M.S.F., S.N., P.S.T.; writing – original draft: G.L.W. and L.M.H.; writing –review & editing: J.G.T., G.L.W., L.M.H., Z.J., and G.L.W.; visualization: G.L.W., L.M.H., Z.J., and J.G.T.; supervision, J.G.T.; project administration, J.G.T.; funding acquisition, J.G.T.
Declaration of interest
The authors declare no competing interests.
Methods
Mice
CRH-eGFP transgenic mice were raised in-house from breeders provided by the Mutant Mouse Resource and Research Center at the University of California, Davis (MMRRC, stock: Tg(Crh-eGFP)HS57Gsat/Mm, RRID:MMRRC_017058-UCD). The mice were genotyped at 2-3 weeks using the primer set: CRH-F1 (CTG TCT TGT CGT GGG TGT CCG AT); GFP R2 (TAG CGG CTG AAG CAC TGC A). All animals and procedures were approved by the Tulane Institutional Animal Care and Use Committee (IACUC) and followed National Institutes of Health guidelines. Mice were housed in an AAALAC-accredited animal facility on a 12:12 light/dark cycle (lights on at 7:00 AM) under controlled temperature (20°C) and received food and water ad libitum.
Cell culture
We used an immortalized hypothalamic cell line, mHYPOE-N42 (N42) cells (Cellutions Biosystems), that expresses CRH 26 as well as both the nuclear glucocorticoid receptor and a membrane-associated glucocorticoid receptor 36,37. Cells were plated at a density of 4.5 x 104 cells/well on 12 mm glass coverslips (size 0, Carolina Biological, Burlington, NC) in 24-well plates (Corning, Corning, NY) in 1X Dulbecco’s Modified Eagle’s Medium (DMEM, Millipore Sigma, Burlington, MA) supplemented with 10% fetal bovine serum (FBS, Atlas Biologicals), 1% penicillin-streptomycin solution, and 0.2% Plasmocin Prophylactic® (Invivogen, San Diego, CA). Plates were incubated at 37°C with 5% CO2 until cells were approximately 80% confluent. To remove endogenous hormones, the culture medium was then removed, cells were washed once with 1X Dulbecco’s-phosphate buffered saline (PBS), and fresh DMEM without phenol red supplemented with 5% charcoal-stripped (CS) FBS was added for 16 h at 37°C with 5% CO2. For proximity ligation assays, N42 cells were seeded at a density of 12,000/well in 16-well chamber slides and cultured as described above.
Alpha-1 adrenoreceptor overexpression
For live imaging assays, N42 cells were transiently transfected with a pEGFP-ARα1b construct generously donated by Dr. Chris Hague of the University of Washington. Transfection was achieved by electroporation with two 10 ms, 1400 mV pulses using the NEON® Transfection System (Life Technologies, Carlsbad, CA). Transfected cells were plated onto No. 1 coverslip bottom plates for live-cell imaging with a Nikon A1 confocal microscope with incubator stage (Nikon USA, Melville, NY). For proximity ligation assays, cells were transfected with either pEGFP-ARα1b or Myc-DDK-tagged human ARα1b (Origene, Rockville, MD) using Lipofectamine 3000.
Cell imaging
Intracellular ARα1b trafficking was tracked in live N42 cells using cytosolic intensity analysis, Förster resonance energy transfer (FRET), or pixel-based colocalization. For cytosolic intensity analysis, cells were transiently transfected as described above with pEGFP-ARα1b. Images were captured every 15 seconds and intensity was measured within a manually defined cytosolic region of interest. For FRET, cells were transiently transfected with a combination of pEGFP-ARα1b (the FRET donor) and Rab4a-dsRed, Rab5-dsRed, Rab7-dsRed, or Rab11-dsRed (FRET acceptors) and imaged repeatedly over time. Three images were taken at each time point: 1) donor excitation, donor emission, 2) acceptor excitation, acceptor emission, and 3) donor excitation, acceptor emission. The same set of images was also taken in cells only expressing either donor or acceptor fluorophores and used as bleed-through controls. Images were analyzed using the FRET analysis tool in Image J 51, which yielded images with a FRET intensity for each pixel. A region of interest around each cell expressing both constructs was defined, and the average FRET signal was calculated in the region to quantify the colocalization of ARα1b with each Rab protein. For pixel-based colocalization, cells were transfected with pEGFP-ARα1b and RFP-LAMP1 followed by colocalization analysis using Cell Profiler 52 to calculate each cell’s mean Pearson’s coefficient.
Ubiquitination assay
N42 cells were treated with drugs for 20 min at 37°C before lysis with NP40 lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40 and 5 mM EDTA for 1 h at 4°C. Immunoprecipitation (IP) of the protein of interest was performed using the Catch-and-Release kit (Millipore Sigma, Burlington, MA), eluting with the non-denaturing buffer. Eluates were run on a gradient SDS-PAGE gel (Bio-Rad, Hercules, CA) and transferred to PVDF membrane. Blots were blocked for 1 h in 5% dry milk in Tris-buffered saline with 0.05% tween (TBST). Primary antibodies were applied in 5% BSA in TBST overnight, followed by 4 washes in TSBT. HRP-conjugated secondary antibody (Clean-Blot IP Detection Reagent, Thermo-Fisher, Waltham MA) was applied in 5% dry milk in TBST for 1 h at room temperature followed by 4 washes in TBST. Chemiluminescent substrate (Super Signal West Pico, Bio-Rad, Hercules, CA) was applied for 5 min followed by detection on a ChemiDoc densitometer (Bio-Rad). Densitometry scores for ubiquitin were quantified and normalized against the protein purified by immunoprecipitation. The antibodies used to target GFP, β-arrestin, and ubiquitin are described in Table 1.
Proximity ligation Assay
Proximity ligation assay (PLA) was carried out with the Duolink system (Millipore Sigma). N42 cells were seeded at a density of 12,000 cells/well on chamber slides (Thermo Scientific) and cultured as described above. For some assays, cells were transfected with human ARα1b-myc-DDK (Origene) or pEGFP-ARα1b using Lipofectamine 3000 (Thermo Scientific). Before assay, cells were cultured for 16h in phenol red-free DMEM supplemented with 10% CS FBS, pen-strep, and Plasmocin. Cells were treated for 20 min with the indicated drugs at 37°C, followed by three washes with PBS, fixation in 4% paraformaldehyde, and blocking with Duolink blocking solution. Incubation in primary antibody was carried out overnight at 4°C. For each assay, two primary antibodies were used, one for each protein or post-translational modification. One antibody of each set was prepared in rabbit and the other in mouse in order to use the complementary secondary antibodies of the Duolink system. After washing with PBS, PLA was performed with the Duolink In Situ system according to the manufacturer’s instructions (Millipore Sigma, Burlington, MA). Negative control wells were incubated with only one of the two primary antibodies and generally yielded less than 5 signals per cell (Supplemental Figure 3). For some assays, PLA was followed by incubation in phalloidin-FITC (1:750, Abcam, Boston, MA) to demarcate cells. Cells were imaged at 40x or 60x with a Nikon Eclipse Ti confocal microscope. For each treatment group, Z-stacks were taken from 2-4 fields. Image quantification was performed with NIH ImageJ/FIJI software. Protein-protein interaction signal was quantified as the number of “dots”. For cells expressing the ARα1b-myc-DDK transgene, a threshold of 15 fluorescent dots per cell was used to distinguish the stain from background, and the phalloidin-FITC stain was used to demarcate the cell boundary. For assays employing pEGFP-ARα1b, transfected cells were determined by GFP signal, and these cells were individually analyzed for total number and nuclear fraction of interactions. See Table 1 for antibodies and dilutions.
Nitrosylation assay
For histological nitrosylation assays, cells were cultured on chamber slides as described above. A biotin switch assay was performed according to the manufacturer’s instructions (Cayman Chemical Company, Ann Arbor, MI) to determine nitrosylation of endogenous β-arrestin1/2. After substitution of biotin for the NO group, PLA was performed with mouse anti-biotin and rabbit anti-β-arrestin1/2 antibodies (see Table 1 for antibody information). Phalloidin-FITC staining was not compatible with the biotin switch assay, so entire fields, rather than individual cells, were quantified for the post-translational modification signal. For each field, the total number of nuclei and the total number of PLA signals were determined in their individual channels. An average number of PLA (nitrosylation) signals per cell was determined by dividing the total number of PLA signals by the total number of nuclei. The nuclear outlines were superimposed onto the image of PLA signal, the PLA signal outside the nuclei was cleared, and the remaining PLA signal was determined to calculate the average nuclear fraction for each field.
For assays of nitrosylation in cell lysates, N42 cells were seeded at 0.5 x 106 in 6-well culture plates and grown to confluence. After treatment for 20 min with the indicated drugs at 37°C, cells were lysed, and NO post-translational modifications were substituted with a tandem mass tag (TMT) according to the manufacturer’s instructions (Thermo Pierce). Lysates were subjected to Western blotting as described above and probed with an anti-TMT antibody. Densitometry was performed with Image Lab software (Bio-Rad), and results are presented as the sum density of all bands in each lane.
Electrophysiology
Ex vivo whole-cell patch clamp electrophysiological recordings were conducted in acutely prepared hypothalamic slices from 6-9 week-old male CRH-eGFP mice. On the mornings of experiments, a mouse was gently removed from its home cage to a transfer cage and transported to an adjacent room in the vivarium, where it was immobilized in a flexible plastic decapitation cone (DecapiCone, Braintree Scientific) and, within less than 2 min of removal from its home cage, decapitated without anesthesia using a rodent guillotine. Anesthetics activate the HPA axis and increase circulating levels of ACTH and CORT 53, which otherwise remain low in our hands for ∼3 min from the start of handling. Following decapitation, the brain was quickly removed and cooled in oxygenated, ice-cold artificial cerebrospinal fluid (aCSF) containing (in mM): 140 NaCl, 3 KCl, 1.3 MgSO4, 11 Glucose, 5 HEPES, 1.4 NaH2PO4, 3.25 NaOH, 2.4 CaCl2, pH 7.2-7.4, with an osmolarity of 290-300 mOsm. The forebrain was isolated, the ventral half was blocked, and the caudal surface of the block was glued to a vibratome chuck. Two or three 300 μm-thick coronal slices containing the hypothalamic PVN were then sectioned on a vibrating slicer (Leica) in cooled aCSF, bisected down the midline, and the hemi-slices were transferred to a holding chamber, where they were maintained in oxygenated aCSF at 20°C for at least 1 h to allow for recovery prior to electrophysiological recordings. Single slices were then transferred and weighted with a silver wire to the bottom of a submerged recording chamber on a fixed-stage, upright microscope (Olympus BXW51) equipped with a long working distance, water immersion 40x objective, where they were perfused with fresh aCSF at a rate of ∼2 ml/min at 20°C. eGFP-expressing CRH neurons in the PVN were first identified under epifluorescence illumination, which was then switched to infrared-differential interference contrast optics and the cells were visualized on a monitor using a near-infrared camera to target them for whole-cell patch clamp recordings. Patch pipettes were pulled from borosilicate glass (ID 1.2 mm, OD 1.65 mm) to a resistance of 3-6 MΩ on a horizontal puller (P-97, Sutter Instr.) and filled with an internal patch solution containing (in mM): 120 potassium gluconate, 10 KCl, 1 NaCl, 1 MgCl2, 0.1 CaCl2, 5.5 EGTA, 10 HEPES, 2 Mg-ATP, and 0.3 Na-GTP, with a pH adjusted to 7.3 with KOH and osmolarity adjusted to 300 mOsm with D-sorbitol. The GABAA receptor antagonist picrotoxin (PTX, 50 µM) was applied via the bath perfusion to isolate excitatory postsynaptic currents (EPSCs). While multiple cells from the same mouse were sometimes recorded, only one cell was recorded per hemi-slice. The numbers of recorded CRH neurons are designated as the ‘n’ and numbers of mice used for each experiment are designated as the ‘N’.
Statistical analyses
Data are presented as the mean ± standard error of the mean. Postsynaptic currents were selected and analyzed for changes in frequency, amplitude, and decay time with Minianalysis 6.0 (Synaptosoft Inc.). Baseline values were calculated from 3 min of recording just prior to drug application and compared with the 3 min of recording at the peak of drug responses using a within-cell analysis. For live cell imaging experiments, mean fluorescence values were calculated from the final 3 min of the baseline and of the drug application and were compared using a within-cell analysis. FRET values were taken from single time points at the end of both the baseline and drug applications. Statistical significance was determined with the two-tailed, paired Student’s t-test for within-cell drug effects, or ANOVA with post-hoc Dunnett’s or Tukey’s analysis where indicated (GraphPad Prism 7-9 and SigmaPlot 11.0, Systat Software, Inc.).
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