Chronic stress impairs autoinhibition in neurons of the locus coeruleus to increase asparagine endopeptidase activity

  1. Hiroki Toyoda
  2. Doyun Kim
  3. Byeong Geon Koh
  4. Tomomi Sano
  5. Takashi Kanematsu
  6. Seog Bae Oh
  7. Youngnam Kang

  • Curated by eLife

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    eLife Assessment

    This fundamental study explores a novel cellular mechanism underlying the degeneration of locus coeruleus neurons during chronic restraint stress. The evidence supporting the overexpression of LC neurons after chronic stress is compelling. However, to fully support the broad implications for LC degeneration and Alzheimer's disease, the study would benefit from stronger causal integration and validation in age-relevant models.

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Abstract

Abstract

Impairments of locus coeruleus (LC) are implicated in anxiety/depression and Alzheimer’s disease (AD). Increases in cytosolic noradrenaline (NA) concentration and MAO-A activity initiate the LC impairment through production of NA metabolite, 3,4-dihydroxyphenyl-glycolaldehyde (DOPEGAL), by MAO-A. However, how NA accumulates in soma/dendritic cytosol of LC neurons has never been addressed despite the fact that NA is virtually absent in cytosol while NA is produced exclusively in cytoplasmic vesicles from dopamine by dopamine-β-hydroxylase. Since re-uptake of autocrine-released NA following spike activity is the major source of NA accumulation, we investigated whether and how chronic stress can increase the spike activity accompanied by NA-autocrine. Overexcitation of LC neurons is normally prevented by the autoinhibition mediated by activation of α2A-adrenergic receptor (AR)-coupled inwardly rectifying potassium-current (GIRK-I) with autocrine-released NA. Patch-clamp study revealed that NA-induced GIRK-I in LC neurons was decreased in chronic restraint stress (RS) mice while a similar decrease was gradually caused by repeated excitation. Chronic RS caused internalization of α2A-ARs expressed in cell membrane in LC neurons and decreased protein/mRNA levels of α2A-ARs/GIRKs in membrane fraction. Subsequently, chronic RS increased the protein levels of MAO-A, DOPEGAL-induced asparagine endopeptidase (AEP) and tau N368. These results suggest that chronic RS-induced overexcitation due to the internalization of α2A-ARs/GIRK is accompanied by [Ca2+]i increases, subsequently increasing Ca2+-dependent MAO-A activity and NA-autocrine. Thus, it is likely that internalization of α2A-AR increased cytosolic NA, as reflected in AEP increases, by facilitating re-uptake of autocrine-released NA. The suppression of α2A-AR internalization may have a translational potential for AD treatment.

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  1. Version published to 10.7554/elife.106362.2 on eLife
  2. Version published to 10.7554/elife.106362 on eLife
  3. eLife

    eLife Assessment

    This fundamental study explores a novel cellular mechanism underlying the degeneration of locus coeruleus neurons during chronic restraint stress. The evidence supporting the overexpression of LC neurons after chronic stress is compelling. However, to fully support the broad implications for LC degeneration and Alzheimer's disease, the study would benefit from stronger causal integration and validation in age-relevant models.

  4. eLife

    Reviewer #1 (Public review):

    This study aims to elucidate the mechanisms by which stress-induced α2A-adrenergic receptor (α2A-AR) internalization leads to cytosolic noradrenaline (NA) accumulation and subsequent neuronal dysfunction in the locus coeruleus (LC). While the manuscript presents an interesting but ambitious model involving calcium dynamics, GIRK channel rundown, and autocrine NA signaling, several key limitations undermine the strength of the conclusions.

    First, the revision does not include new experiments requested by reviewers to validate core aspects of the mechanism. Specifically, there is no direct measurement of cytosolic NA levels or MAO-A enzymatic activity to support the link between receptor internalization and neurochemical changes. The authors argue that such measurements are either not feasible or beyond the scope of the study, leaving a significant gap in the mechanistic chain of evidence.

    Second, the behavioral analysis remains insufficient to support claims of cognitive impairment. The use of a single working memory test following an anxiety test is inadequate to verify memory dysfunction behaviors. Additional cognitive assays, such as the Morris Water Maze or Novel Object Recognition, are recommended but not performed.

    Third, concerns regarding the lack of rigor in differential MAO-A expression in fluorescence imaging were not addressed experimentally. Instead of clarifying the issue, the authors moved the figure to supplementary data without providing further evidence (e.g., an enzymatic assay or quantitative reanalysis of Western blot, or re-staining of IF for MAO-A) to support their interpretation.

    Fourth, concerns regarding TH staining remain unresolved. In Figure S7, the α2A-AR signal appears to resemble TH staining, and vice versa, raising the possibility of labeling errors. It is recommended that the authors re-examine this issue by either double-checking the raw data or repeating the immunostaining to validate the staining.

    Overall, the manuscript offers a potentially interesting framework but falls short in providing the experimental rigor necessary to establish causality. The reliance on indirect reasoning and reorganizing of existing data, rather than generating new evidence, limits the overall impact and interpretability of the study.

  5. eLife

    Reviewer #2 (Public review):

    Summary:

    This manuscript investigates the mechanism by which chronic stress induces degeneration of locus coeruleus (LC) neurons. The authors demonstrate that chronic stress leads to the internalization of α2A-adrenergic receptors (α2A-ARs) on LC neurons, causing increased cytosolic noradrenaline (NA) accumulation and subsequent production of the neurotoxic metabolite DOPEGAL via monoamine oxidase A (MAO-A). The study suggests a mechanistic link between stress-induced α2A-AR internalization, disrupted autoinhibition, elevated NA metabolism, activation of asparagine endopeptidase (AEP), and Tau pathology relevant to Alzheimer's disease (AD). The conclusions of this paper are largely well-supported by the data, but some aspects of image acquisition require further examination.

    Strengths:

    This study clearly demonstrates the effects of chronic stimulation on the excitability of LC neurons using electrophysiological techniques. It also elucidates the role of α2-adrenergic receptor (α2-AR) internalization and the associated upstream and downstream signaling pathways of GIRK-1, using a range of pharmacological agents, highlighting the innovative nature of the work. Additionally, the study identifies the involvement of the MAO-A-DOPEGAL-AEP pathway in this process. The topic is timely, the proposed mechanistic pathway is compelling, and the findings have translational relevance, particularly about therapeutic strategies targeting α2A-AR internalization in neurodegenerative diseases.

    Weaknesses:

    (1) The manuscript reports that chronic stress for 5 days increases MAO-A levels in LC neurons, leading to the production of DOPEGAL, activation of AEP, and subsequent tau cleavage into the tau N368 fragment, ultimately contributing to neuronal damage. However, the authors used wild-type C57BL/6 mice, and previous literature has indicated that AEP-mediated tau cleavage in wild-type mice is minimal and generally insufficient to cause significant behavioral alterations. Please clarify and discuss this apparent discrepancy.

    (2) It is recommended that the authors include additional experiments to examine the effects of different durations and intensities of stress on MAO-A expression and AEP activity. This would strengthen the understanding of stress-induced biochemical changes and their thresholds.

    (3) Please clarify the rationale for the inconsistent stress durations used across Figures 3, 4, and 5. In some cases, a 3-day stress protocol is used, while in others, a 5-day protocol is applied. This discrepancy should be addressed to ensure clarity and experimental consistency.

    (4) The abbreviation "vMAT2" is incorrectly formatted. It should be "VMAT2," and the full name (vesicular monoamine transporter 2) should be provided at first mention.

    Comments on revisions:

    The authors have addressed all of the reviewers' comments.

  6. eLife

    Reviewer #3 (Public review):

    Summary:

    The authors present a technically impressive data set showing that repeated excitation or restraint stress internalises somatodendritic α2A adrenergic autoreceptors (α2A ARs) in locus coeruleus (LC) neurons. Loss of these receptors weakens GIRK-dependent autoinhibition, raises neuronal excitability, and is accompanied by higher MAO A, DOPEGAL, AEP, and tau N368 levels. The work combines rigorous whole-cell electrophysiology with barbadin-based trafficking assays, qPCR, Western blotting and immunohistochemistry. The final schematic is appealing and in principle, could explain early LC hyperactivity followed by degeneration in ageing and Alzheimer's disease.

    Strengths:

    Multi-level approach - The study integrates electrophysiology, pharmacology, mRNA quantification, and protein-level analysis.

    Use of barbadin to block β-arrestin/AP-2-dependent internalisation is both technically precise and mechanistically informative

    Well-executed electrophysiology

    translation relevance

    converges to a model that peers discussed (scientists can only discuss models - not data!)

    Weaknesses:

    Nevertheless, the manuscript currently reads as a sequence of discrete experiments rather than a single causal chain

  7. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Reviewer #1 (Public review):

    Weaknesses:

    (1) The manuscript's logical flow is challenging and hard to follow, and key arguments could be more clearly structured, particularly in transitions between mechanistic components.

    We have revised our manuscript so as to make it easy for readers to follow the logical flow in transitions between mechanistic components by adding the descriptions of Figure S1E-J, Figure S2F-K, Figure S3A-H, Figure S4A-F, Figure S5, and Figure S6 in the revised manuscript.

    (2) The causality between stress-induced α2A-AR internalization and the enhanced MAO-A remains unclear. Direct experimental evidence is needed to determine whether α2A-AR internalization itself or Ca2+ drives MAO-A activation, and how they activate MAO-A should be considered.

    We believe that the causality between stress-induced α2A-AR internalization and the enhancement of MAO-A is clearly demonstrated by our current experiments, while our explanations may be improved by making them easier to understand especially for those who are not expert on electrophysiology.

    Firstly, it is well established that autoinhibition in LC neurons is mediated by α2A-AR coupled-GIRK (Arima et al., 1998, J Physiol; Williams et al., 1985, Neuroscience). We found that spike frequency adaptation in LC neurons was also mediated by α2A-AR coupled GIRK-I (Figure 1A-I), and that α2A-AR coupled GIRK-I underwent [Ca2+]i dependent rundown (Figures 2, S1, S2), leading to an abolishment of spike-frequency adaptation (Figures S4). [Ca2+]i dependent rundown of α2A-AR coupled GIRK-I was prevented by barbadin (Figure 2G-J), which prevents the internalization of G-protein coupled receptor (GPCR) channels.

    Abolishment of spike frequency adaptation itself, i.e., “increased spike activity” can increase [Ca2+]i because [Ca2+]i is entirely dependent on the spike activity as shown by [Ca2+]i imaging method in Figure S3.

    Thus, α2A-AR internalization can increase [Ca2+]i through the abolishment of autoinhibition or spike frequency adaptation, and a [Ca2+]i increase drives MAO-A activation as reported previously (Cao et al., 2007, BMC Neurosci). The mechanism how Ca2+ activates MAO-A is beyond the scope of the current study.

    Our study just focused on the mechanism how chronic or sever stress can cause persistent overexcitation and how it results in LC degeneration.

    (3) The connection between α2A-AR internalization and increased cytosolic NA levels lacks direct quantification, which is necessary to validate the proposed mechanism.

    Direct quantification of the relationship between α2A-AR internalization and increased cytosolic NA levels may not be possible, and may not be necessarily needed to be demonstrated as explained below.

    The internalization of α2A-AR can increase [Ca2+]i through the abolishment of autoinhibition or spike frequency adaptation, and [Ca2+]i increases can facilitate NA autocrine (Huang et al., 2007), similar to the transmitter release from nerve terminals (Kaeser & Regehr, 2014, Annu Rev Physiol).

    Autocrine released NA must be re-uptaken by NAT (NA transporter), which is firmly established (Torres et al., 2003, Nat Rev Neurosci). Re-uptake of NA by NAT is the only source of intracellular NA, and NA re-uptake by NAT should be increased as the internalization of NA biding site (α2A-AR) progresses in association with [Ca2+]i increases (see page 11, lines 334-336).

    Thus, the connection between α2A-AR internalization and increased cytosolic NA levels is logically compelling, and the quantification of such connection may not be possible at present (see the response to the comment made by the Reviewer #1 as Recommendations for the authors (2) and beyond the scope of our current study.

    (4) The chronic stress model needs further validation, including measurements of stress-induced physiological changes (e.g., corticosterone levels) to rule out systemic effects that may influence LC activity. Additional behavioral assays for spatial memory impairment should also be included, as a single behavioral test is insufficient to confirm memory dysfunction.

    It is well established that restraint stress (RS) increases corticosterone levels depending on the period of RS (García-Iglesias et al., 2014, Neuropharmacology), although we are not reluctant to measure the corticosterone levels. In addition, there are numerous reports that showed the increased activity of LC neurons in response to various stresses (Valentino et al., 1983; Valentino and Foote, 1988; Valentino et al., 2001; McCall et al., 2015), as described in the text (page 4, lines 96-98). Measurement of cortisol levels may not be able to rule out systemic effects of CRS on the whole brain.

    We had already done another behavioral test using elevated plus maze (EPM) test.By combining the two tests, it may be possible to more accurately evaluate the results of Y-maze test by differentiating the memory impairment from anxiety. However, the results obtained by these behavioral tests are just supplementary to our current aim to elucidate the cellular mechanisms for the accumulation of cytosolic free NA. Therefore, we have softened the implication of anxiety and memory impairment (page 13, lines 397-400 in the revised manuscript).

    (5) Beyond b-arrestin binding, the role of alternative internalization pathways (e.g., phosphorylation, ubiquitination) in α2A-AR desensitization should be considered, as current evidence is insufficient to establish a purely Ca2+ -dependent mechanism.

    We can hardly agree with this comment.

    It was clearly demonstrated that repeated application of NA itself did not cause desensitization of α2A-AR (Figure S1A-D), and that the blockade of b-arrestin binding by barbadin completely suppressed the Ca2a-dependent downregulation of GIRK (Figure 2G-K). These observations can clearly rule out the possible involvement of phosphorylation or ubiquitination for the desensitization.

    Not only the barbadin experiment, but also the immunohistochemistry and western blot method clearly demonstrated the decrease of α2A-AR expression on the cell membrane (Figure 3).

    Ca2+-dependent mechanism of the rundown of GIRK was convincingly demonstrated by a set of different protocols of voltage-clamp study, in which Ca2+ influx was differentially increased. The rundown of GIRK-I was orderly potentiated or accelerated by increasing the number of positive command pulses each of which induces Ca2+ influx (compare Figure S1E-J, Figure S2A-E and Figure S2F-K along with Figure 2A-F). The presence or absence of Ca2+ currents and the amount of Ca2+ currents determined the trend of the rundown of GIRK-I (Figures 2, S1 and S2). Because the same voltage protocol hardly caused the rundown when it did not induce Ca2+ currents in the absence of TEA (Figure S1F; compare with Figure 2B), blockade of Ca2+ currents by nifedipine would not be so beneficial.

    We believe the series of voltage-clamp protocols convincingly demonstrated the orderly involvement of [Ca2+]i in accelerating the rundown of GIRK-I.

    (6) NA leakage for free NA accumulation is also influenced by NAT or VMAT2. Please discuss the potential role of VMAT2 in NA accumulation within the LC in AD.

    It has been demonstrated that reduced VMAT2 levels increased susceptibility to neuronal damage: VMAT2 heterozygote mice displayed increased vulnerability to MPTP as evidenced by reductions in nigral dopamine cell counts (Takahashi et al, 1997, PNAS). Thus, when the activity of VMAT2 in LC neurons were impaired by chronic restraint stress, cytosolic NA levels in LC neurons would increase. We have added such discussion in the revised manuscript (page 12, lines 381-384).

    (7) Since the LC is a small brain region, proper staining is required to differentiate it from surrounding areas. Please provide a detailed explanation of the methodology used to define LC regions and how LC neurons were selected among different cell types in brain slices for whole-cell recordings.

    LC neurons were identified immunohistochemically and electrophysiologically as we previously reported (see Fig. 2 in Front. Cell. Neurosci. 16:841239. doi: 10.3389/fncel.2022.841239). We have added this explanation in the method section of the revised manuscript (page 15, lines 474-475). A delayed spiking pattern in response to depolarizing pulses (Figure S10 in the revised manuscript) applied at a hyperpolarized membrane potential was commonly observed in LC neurons in many studies (Masuko et al., 1986; van den Pol et al., 2002; Wagner-Altendorf et al., 2019).

    Reviewer #2 (Public review):

    Weaknesses:

    (1) The manuscript reports that chronic stress for 5 days increases MAO-A levels in LC neurons, leading to the production of DOPEGAL, activation of AEP, and subsequent tau cleavage into the tau N368 fragment, ultimately contributing to neuronal damage. However, the authors used wild-type C57BL/6 mice, and previous literature has indicated that AEP-mediated tau cleavage in wild-type mice is minimal and generally insufficient to cause significant behavioral alterations. Please clarify and discuss this apparent discrepancy.

    In our study, normalized relative value of AEP-mediated tau cleavage (Tau N368) was much higher in CRS mice than non-stress wild-type mice. It is not possible to compare AEP-mediated tau cleavage between our non-stress wild type mice and those observed in previous study (Zhang et al., 2014, Nat Med), because band intensity is largely dependent on the exposure time and its numerical value is the normalized relative value. In view of such differences, our apparent band expression might have been intensified to detect small changes.

    (2) It is recommended that the authors include additional experiments to examine the effects of different durations and intensities of stress on MAO-A expression and AEP activity. This would strengthen the understanding of stress-induced biochemical changes and their thresholds.

    GIRK rundown was almost saturated after 3-day RS and remained the same in 5-day RS mice (Fig. 4A-G), which is consistent with the downregulation of α2A-AR and GIRK1 expression by 3-day RS (Fig. 3C, F and G; Fig. 4J and K). However, we examined the protein levels of MAO-A, pro/active-AEP and Tau N368 only in 5-day RS mice without examining in 3-day RS mice. This is because we considered the possibility that a high [Ca2+]i condition may have to be sustained for some period of time to induce changes in MAO-A, AEP and Tau N368, and therefore 3-day RS may be insufficient to induce such changes. We have added this in the revised manuscript (page 17, lines 521-525).

    (3) Please clarify the rationale for the inconsistent stress durations used across Figures 3, 4, and 5. In some cases, a 3-day stress protocol is used, while in others, a 5-day protocol is applied. This discrepancy should be addressed to ensure clarity and experimental consistency.

    Please see our response to the comment (2).

    (4) The abbreviation "vMAT2" is incorrectly formatted. It should be "VMAT2," and the full name (vesicular monoamine transporter 2) should be provided at first mention.

    Thank you for your suggestion. We have revised accordingly.

    Reviewer #3 (Public review):

    Weaknesses:

    Nevertheless, the manuscript currently reads as a sequence of discrete experiments rather than a single causal chain. Below, I outline the key points that should be addressed to make the model convincing.

    Please see the responses to the recommendation for the authors made by reviewer #3.

    Reviewer #1 (Recommendations for the authors):

    (1) Improve the clarity and organization of the manuscript, ensuring smoother transitions between concepts and mechanisms.

    Please see the response to the comment raised by Reviewer #1 as Weakness

    (2) Adjust any quantifying method for cytosolic NA levels under different conditions to support the link between receptor internalization and NA accumulation.

    If fluorescent indicator of cytosolic free NA is available, it would be possible to measure changes in cytosolic NA levels. However, at present, there appeared to be no fluorescence probe to label cytosolic NA. For example, NS521 labels both dopamine and norepinephrine inside neurosecretory vesicles (Hettie & Glass et al., 2014, Chemistry), and BPS3 fluorescence sensor labels NA around cell membrane by anchoring on the cell membrane (Mao et al., 2023, Nat Comm). Furthermore, the method reported in “A Genetically Encoded Fluorescent Sensor for Rapid and Specific In Vivo Detection of Norepinephrine” is limited to detect NA only when α2AR is expressed. In the present study, increases in cytosolic NA levels are caused by internalization of α2AR. Cytosolic NA measurements with GRAB NE photometry may not be applicable in the present study. However, we have discussed the availability of such fluorescent methods to directly prove the increase in cytosolic NA as a limitation of our study (page 14, lines 429-436 in the revised manuscript).

    (3) Include validation of the chronic stress model with physiological and behavioral measures (e.g., corticosterone levels and another behavioral test).

    Please see the response to the comment raised by Reviewer #1 as Weakness (4).

    (4) All supplemental figures should be explicitly explained in the Results section. Specifically, clarify and describe the details of Figure S1G-K, Figure S2F-K, Figure S3A-H, Figure S4A-F, Figure S5, and Figure S6 to ensure all supplementary data are fully integrated into the main text.

    We have more explicitly and clearly described the details of Figure S1E-J, Figure S2F-K, Figure S3A-H, Figure S4A-F, Figure S5, and Figure S6 and fully integrated those explanations into the main text in the revised manuscript.

    (5) In Figure 3, the morphology of TH-positive cells differs between panels D and E. Additionally, TH is typically expressed in the cytosol, but in the provided images, it appears to be localized only to the membrane. Please clarify this discrepancy and provide a lower-magnification image to display a larger area, not one cell.

    In a confocal image, TH is not necessarily expressed homogenously in the cytosol, but is expressed in a ring-shaped pattern inside the plasma membrane, avoiding the cell nucleus and its surrounding Golgi apparatus and endoplasmic reticulum (ER) (Henrich et al., 2018, Acta Neuropathol Commun; see Fig. 4a and 6e), especially when the number of z-stack of confocal images is small. This is presumably because LC neurons are especially enriched with numerous Golgi apparatus and ER (Groves & Wilson, 1980, J Comp Neurol).

    In Figure S7, we showed a lower-magnification image of LC and its adjacent area (mesencephalic trigeminal nucleus). In the LC area, there are a variety of LC neurons, which include oval shaped neurons (open arrowhead; similar to Figure 3D) and also rhombus-like shaped neurons (open double arrowheads, similar to Figure 3E). A much lower-magnification image of LC neurons constituting LC nucleus was shown in Figure 5A.

    (6) In Figure 5, the difference in MAO-A expression is not clearly visible in the fluorescence images. Enzymatic assays for AEP and MAO-A should be included to demonstrate the increased activity better.

    In the current study, we did not elaborate to detect the changes in TH, MAO-A and AEP in terms of immunohistochemical method. Instead, we elaborated to detect such changes in terms of western blot method. The main conclusions in the current study were drawn primarily by electrophysiological techniques as we have expended much effort on electrophysiological experiments. Because the relative quantification of active AEP and Tau N368 proteins by western blotting analysis may accurately reflect changes in those enzyme activities, enzymatic assay may not be necessarily required but is helpful to better demonstrate AEP and MAO-A activity. We have described the necessity of enzymatic assay to better demonstrate the AEP and MAO-A activities (page 10, lines 314-315).

    Reviewer #3 (Recommendations for the authors):

    (1) Causality across the pathway

    Each step (α2A internalisation, GIRK rundown, Ca2+ rise, MAO-A/AEP upregulation) is demonstrated separately, but no experiment links them in a single preparation. Consider in vivo Ca2+ or GRAB NE photometry during restraint stress while probing α2A levels with i.p. clonidine injection or optogenetic over excitation coupled to biochemical readouts. Such integrated evidence would help to overcome the correlational nature of the manuscript to a more mechanistic study.

    It is not possible to measure free cytosolic NA levels with GRAB NE photometry when α2A AR is internalized as described above (see the response to the comment made by reviewer #1 as the recommendation for the authors).

    (2) Pharmacology and NE concentration

    The use of 100 µM noradrenaline saturates α and β adrenergic receptors alike. Please provide ramp measurements of GIRK current in dose-response at 1-10 µM NE (blocked by atipamezole) to confirm that the rundown really reflects α2A activity rather than mixed receptor effects.

    It is true that 100 µM noradrenaline activates both α and β adrenergic receptors alike. However, it was clearly showed that enhancement of GIRK-I by 100 µM noradrenaline was completely antagonized by 10 µM atipamezole and the Ca2+ dependent rundown of NA-induced GIRK-I was prevented by 10 µM atipamezole. Considering the Ki values of atipamezole for α2A AR (=1~3 nM) (Vacher et al., 2010, J Med Chem) and β AR (>10 µM) (Virtanen et al., 1989, Arch Int Pharmacodyn Ther), these results really reflect α2A AR activity but not β AR activity (Figure S5). Furthermore, because it is already well established that NA-induced GIRK-I was mediated by α2A AR activity in LC neurons (Arima et al., 1998, J Physiol; Williams et al., 1985, Neuroscience), it is not necessarily need to re-examine 1-10 µM NA on GIRK-I.

    (3) Calcium dependence is not yet definitive

    The rundown is induced with a TEA-enhanced pulse protocol. Blocking L-type channels with nifedipine (or using Cd²⁺) during this protocol should show whether Ca2+ entry is necessary. Without such a control, the Ca2+ link remains inferential.

    The Ca2+ link was precisely demonstrated by a series of voltage clamp experiment, in which Ca2+ influx was orderly potentiated by increasing the number of positive voltage pulses (Figures S1 and S2). As the number of positive voltage pulses was increased, the rundown of GIRK-I was accelerated or enhanced more. The relationship between the number of spikes and the Ca2+ influx detected as Ca2+ transients was well documented in Ca2+ imaging experiments using fura-2 (Figure S3).

    The presence or absence of Ca2+ currents and the amount of Ca2+ currents determined the trend of the rundown of GIRK-I (Figs. 2, S1 and S2). The same voltage protocol hardly caused the rundown when it did not induce Ca2+ currents in the absence of TEA (Fig. S1F; compare with Fig. 2B), and the series of voltage-clamp protocols convincingly demonstrated the orderly involvement of [Ca2+]i in accelerating the rundown of GIRK-I. Therefore, blockade of Ca2+ currents by nifedipine may not be so beneficial.

    (4) Age mismatch and disease claims

    All electrophysiology and biochemical data come from juvenile (< P30) mice, yet the conclusions stress Alzheimer-related degeneration. Key endpoints need to be replicated in adult or aged mice, or the manuscript should soften its neurodegenerative scope.

    As described in the section of Conclusion, we never stress Alzheimer-related degeneration, but might give such an impression. To avoid such a misunderstanding, we have added a description “However, the present mechanism must be proven to be valid in adult or old mice, to validate its involvement in the pathogenesis of AD.” (page 14, lines 448-450).

    (5) Direct evidence for extracellular/cytosolic NE

    The proposed rise in reuptake NA is inferred from electrophysiology. Modern fluorescent sensors (GRAB NE, nLight) or fast scan voltammetry could quantify NE overflow and clearance during stress, directly testing the model.

    Please see the response to the comment made by Reviewer #1 as the Recommendations for the authors (2) as described above.

    (6) Quantitative histology

    Figure 5 presents attractive images but no numerical analysis. Please provide ROI-based fluorescence quantification (with n values) or move the images to the supplement and rely on the Western blots.

    We have moved the immunohistochemical results in Fig. 5 to the supplement as we believe the quantification of immunohistochemical staining is not necessarily correct.

  8. eLife

    Author response:

    Reviewer #1 (Public review):

    Weaknesses:

    (1) The manuscript's logical flow is challenging and hard to follow, and key arguments could be more clearly structured, particularly in transitions between mechanistic components.

    We will revise our manuscript so as to make it easy to follow the logical flow in transitions between mechanistic components.

    (2) The causality between stress-induced α2A-AR internalization and the enhanced MAO-A remains unclear. Direct experimental evidence is needed to determine whether α2A-AR internalization itself or Ca2+ drives MAO-A activation, and how they activate MAO-A should be considered.

    We believe that the causality between stress-induced α2A-AR internalization and the enhancement of MAO-A is clearly demonstrated by our current experiments, while our explanations may be improved by making them easier to understand especially for those who are not expert on electrophysiology.

    Firstly, it is well established that autoinhibition in LC neurons is mediated by α2A-AR coupled-GIRK (Arima et al., 1998, J Physiol; Williams et al., 1985, Neuroscience). We found that spike frequency adaptation in LC neurons was also mediated by α2A-AR coupled GIRK-I (Fig. 1A-I), and that α2A-AR coupled GIRK-I underwent [Ca2+]i-dependent rundown (Figs. 2, S1, S2), leading to an abolishment of spike-frequency adaptation (Figs. S4). [Ca2+]i-dependent rundown of α2A-AR coupled GIRK-I was prevented by barbadin (Fig 2G-J), which prevents the internalization of G-protein coupled receptor (GPCR) channels.

    Abolishment of spike frequency adaptation itself, i.e., “increased spike activity” can increase [Ca2+]i because [Ca2+]i is entirely dependent on the spike activity as shown by Ca2+ imaging method in Figure S3.

    Thus, α2A-AR internalization can increase [Ca2+]i through the abolishment of autoinhibition or spike frequency adaptation, and a [Ca2+]i increase drives MAO-A activation as reported previously (Cao et al., 2007, BMC Neurosci). The mechanism how Ca2+ activates MAO-A is beyond the scope of the current study.

    Our study just focused on the mechanism how chronic or sever stress can cause persistent overexcitation and how it results in LC degeneration.

    (3) The connection between α2A-AR internalization and increased cytosolic NA levels lacks direct quantification, which is necessary to validate the proposed mechanism.

    Direct quantification of the relationship between α2A-AR internalization and increased cytosolic NA levels may not be possible, and may not be necessarily needed to be demonstrated as explained below.

    The internalization of α2A-AR can increase [Ca2+]i through the abolishment of autoinhibition or spike frequency adaptation, and [Ca2+]i increases can facilitate NA autocrine (Huang et al., 2007), similar to the transmitter release from nerve terminals (Kaeser & Regehr, 2014, Annu Rev Physiol).

    Autocrine released NA must be re-uptaken by NAT (NA transporter), which is firmly established (Torres et al., 2003, Nat Rev Neurosci). Re-uptake of NA by NAT is the only source of intracellular NA, and NA re-uptake by NAT should be increased as the internalization of NA biding site (α2A-AR) progresses in association with [Ca2+]i increases (see page 11, lines 334-336).

    Thus, the connection between α2A-AR internalization and increased cytosolic NA levels is logically compelling, and the quantification of such connection may not be possible at present (see the response to the comment made by the Reviewer #1 as Recommendations for the authors (2) and beyond the scope of our current study.

    (4) The chronic stress model needs further validation, including measurements of stress-induced physiological changes (e.g., corticosterone levels) to rule out systemic effects that may influence LC activity. Additional behavioral assays for spatial memory impairment should also be included, as a single behavioral test is insufficient to confirm memory dysfunction.

    It is well established that restraint stress (RS) increases corticosterone levels depending on the period of RS (García-Iglesias et al., 2014, Neuropharmacology), although we are not reluctant to measure the corticosterone levels. In addition, there are numerous reports that showed the increased activity of LC neurons in response to various stresses (Valentino et al., 1983; Valentino and Foote, 1988; Valentino et al., 2001; McCall et al., 2015), as described in the text (page 4, lines 96-98). Measurement of cortisol levels may not be able to rule out systemic effects of CRS on the whole brain.

    We had already done another behavioral test using elevated plus maze (EPM) test.

    By combining the two tests, it may be possible to more accurately evaluate the results of Y-maze test by differentiating the memory impairment from anxiety. However, the results obtained by these behavioral tests are just supplementary to our current aim to elucidate the cellular mechanisms for the accumulation of cytosolic free NA. Its subsequent anxiety and memory impairment are just supplementary to our current study. We will soften the implication of anxiety and memory impairment.

    (5) Beyond b-arrestin binding, the role of alternative internalization pathways (e.g., phosphorylation, ubiquitination) in α2A-AR desensitization should be considered, as current evidence is insufficient to establish a purely Ca2+-dependent mechanism.

    We can hardly agree with this comment.

    It was clearly demonstrated that repeated application of NA itself did not cause desensitization of α2A-AR (Figure S1A-D), and that the blockade of b-arrestin binding by barbadin completely suppressed the Ca2+-dependent downregulation of GIRK (Fig. 2G-K). These observations can clearly rule out the possible involvement of phosphorylation or ubiquitination for the desensitization.

    Not only the barbadin experiment, but also the immunohistochemistry and western blot method clearly demonstrated the decrease of α2A-AR expression on the cell membrane (Fig. 3).

    Ca2+-dependent mechanism of the rundown of GIRK was convincingly demonstrated by a set of different protocols of voltage-clamp study, in which Ca2+ influx was differentially increased. The rundown of GIRK-I was orderly potentiated or accelerated by increasing the number of positive command pulses each of which induces Ca2+ influx (compare Figure S1E-J, Figure S2A-E and Figure S2F-K along with Fig. 2A-F). The presence or absence of Ca2+ currents and the amount of Ca2+ currents determined the trend of the rundown of GIRK-I (Figs. 2, S1 and S2). Because the same voltage protocol hardly caused the rundown when it did not induce Ca2+ currents in the absence of TEA (Fig. S1F; compare with Fig. 2B), blockade of Ca2+ currents by nifedipine would not be so beneficial.

    We believe the series of voltage-clamp protocols convincingly demonstrated the orderly involvement of [Ca2+]i in accelerating the rundown of GIRK-I.

    (6) NA leakage for free NA accumulation is also influenced by NAT or VMAT2. Please discuss the potential role of VMAT2 in NA accumulation within the LC in AD.

    We will discuss the role of VMAT2 in NA accumulation, especially when VMAT2 was impaired. Indeed, it has been demonstrated that reduced VMAT2 levels increased susceptibility to neuronal damage: VMAT2 heterozygote mice displayed increased vulnerability to MPTP as evidenced by reductions in nigral dopamine cell counts (Takahashi et al, 1997, PNAS). Thus, when the activity of VMAT2 in LC neurons were impaired by chronic restraint stress, cytosolic NA levels in LC neurons would increase. We will add such discussion in the revised manuscript.

    (7) Since the LC is a small brain region, proper staining is required to differentiate it from surrounding areas. Please provide a detailed explanation of the methodology used to define LC regions and how LC neurons were selected among different cell types in brain slices for whole-cell recordings.

    LC neurons were identified immunohistochemically and electrophysiologically as we previously reported (see Fig. 2 in Front. Cell. Neurosci. 16:841239. doi: 10.3389/fncel.2022.841239). A delayed spiking pattern in response to depolarizing pulses (Figure S9) applied at a hyperpolarized membrane potential was commonly observed in LC neurons in many studies (Masuko et al., 1986; van den Pol et al., 2002; Wagner-Altendorf et al., 2019).

    Reviewer #2 (Public review):

    Weaknesses:

    (1) The manuscript reports that chronic stress for 5 days increases MAO-A levels in LC neurons, leading to the production of DOPEGAL, activation of AEP, and subsequent tau cleavage into the tau N368 fragment, ultimately contributing to neuronal damage. However, the authors used wild-type C57BL/6 mice, and previous literature has indicated that AEP-mediated tau cleavage in wild-type mice is minimal and generally insufficient to cause significant behavioral alterations. Please clarify and discuss this apparent discrepancy.

    In our study, normalized relative value of AEP-mediated tau cleavage (Tau N368) was much higher in CRS mice than non-stress wild-type mice. It is not possible to compare AEP-mediated tau cleavage between our non-stress wild type mice and those observed in previous study (Zhang et al., 2014, Nat Med), because band intensity is largely dependent on the exposure time and its numerical value is the normalized relative value. In view of such differences, our apparent band expression might have been intensified to detect small changes.

    (2) It is recommended that the authors include additional experiments to examine the effects of different durations and intensities of stress on MAO-A expression and AEP activity. This would strengthen the understanding of stress-induced biochemical changes and their thresholds.

    GIRK rundown was almost saturated after 3-day RS and remained the same in 5-day RS mice (Fig. 4A-G), which is consistent with the downregulation of α2A-AR and GIRK1 expression by 3-day RS (Fig. 3C, F and G; Fig. 4J and K). However, we examine the protein levels of MAO-A, pro/active-AEP and Tau N368 only in 5-day RS mice without examining in 3-day RS mice. This is because we considered the possibility that 3-day RS may be insufficient to induce changes in MAO-A, AEP and Tau N368 and some period of high [Ca2+]i condition may be necessary to induce such changes. We will discuss this in the revised manuscript.

    (3) Please clarify the rationale for the inconsistent stress durations used across Figures 3, 4, and 5. In some cases, a 3-day stress protocol is used, while in others, a 5-day protocol is applied. This discrepancy should be addressed to ensure clarity and experimental consistency.

    Please see our response to the comment (2).

    (4) The abbreviation "vMAT2" is incorrectly formatted. It should be "VMAT2," and the full name (vesicular monoamine transporter 2) should be provided at first mention.

    Thank you for your suggestion. We will revise accordingly.

  9. eLife

    eLife Assessment

    This fundamental study explores a novel cellular mechanism underlying the degeneration of locus coeruleus neurons during chronic restraint stress. The evidence supporting the overexpression of LC neurons after chronic stress is compelling. However, to fully support the broad implications for LC degeneration and Alzheimer's disease, the study would benefit from stronger causal integration and validation in age-relevant models.

  10. eLife

    Reviewer #1 (Public review):

    Summary:

    This study investigates how chronic stress may contribute to LC dysfunction in AD by examining the mechanisms underlying NA accumulation and α2A-AR internalization. Using electrophysiological recordings and molecular analyses, the authors propose that stress-induced receptor internalization impairs autoinhibition, leading to excessive NA accumulation and increased MAO-A activity. The findings have potential implications for understanding the progression of AD-related neurodegeneration and targeting noradrenergic dysfunction as a therapeutic strategy.

    Strengths:

    (1) The study integrates electrophysiology and molecular approaches to explore the mechanistic effects of chronic stress on LC neurons.

    (2) The evidence supporting NA accumulation and α2A-AR internalization as contributing factors to LC dysfunction is novel and relevant to AD pathology.

    (3) The electrophysiological findings, particularly the loss of spike-frequency adaptation and reduction in GIRK currents, provide functional insights into stress-induced changes in LC activity.

    Weaknesses:

    (1) The manuscript's logical flow is challenging and hard to follow, and key arguments could be more clearly structured, particularly in transitions between mechanistic components.

    (2) The causality between stress-induced α2A-AR internalization and the enhanced MAO-A remains unclear. Direct experimental evidence is needed to determine whether α2A-AR internalization itself or Ca2+ drives MAO-A activation, and how they activate MAO-A should be considered.

    (3) The connection between α2A-AR internalization and increased cytosolic NA levels lacks direct quantification, which is necessary to validate the proposed mechanism.

    (4) The chronic stress model needs further validation, including measurements of stress-induced physiological changes (e.g., corticosterone levels) to rule out systemic effects that may influence LC activity. Additional behavioral assays for spatial memory impairment should also be included, as a single behavioral test is insufficient to confirm memory dysfunction.

    (5) Beyond b-arrestin binding, the role of alternative internalization pathways (e.g., phosphorylation, ubiquitination) in α2A-AR desensitization should be considered, as current evidence is insufficient to establish a purely Ca²⁺-dependent mechanism.

    (6) NA leakage for free NA accumulation is also influenced by NAT or VMAT2. Please discuss the potential role of VMAT2 in NA accumulation within the LC in AD.

    (7) Since the LC is a small brain region, proper staining is required to differentiate it from surrounding areas. Please provide a detailed explanation of the methodology used to define LC regions and how LC neurons were selected among different cell types in brain slices for whole-cell recordings.

    Impact:

    This study provides valuable insights into the impact of chronic stress on LC function and its relevance to AD pathogenesis. The proposed mechanism linking NA dysregulation and receptor internalization may have implications for developing therapeutic strategies targeting the noradrenergic system in neurodegenerative diseases. However, additional validation is needed to strengthen the mechanistic claims before the findings can be fully integrated into the field.

  11. eLife

    Reviewer #2 (Public review):

    Summary:

    This manuscript investigates the mechanism by which chronic stress induces locus coeruleus (LC) neuron degeneration. The authors demonstrate that chronic stress leads to internalization of α2A-adrenergic receptors (α2A-ARs) on LC-neurons, causing increased cytosolic noradrenaline (NA) accumulation and subsequent production of the neurotoxic metabolite DOPEGAL via monoamine oxidase A (MAO-A). The study suggests a mechanistic link between stress-induced α2A-AR internalization, disrupted autoinhibition, elevated NA metabolism, asparagine endopeptidase (AEP) activation, and Tau pathology relevant to Alzheimer's disease (AD). The conclusions of this paper are mostly well supported by data, but some aspects of image acquisition need to be extended.

    Strengths:

    This study clearly demonstrates the effects of chronic stimulation on the excitability of LC neurons using electrophysiological techniques. It also elucidates the role of α2-adrenergic receptor (α2-AR) internalization and the associated upstream and downstream signaling pathways of GIRK1 using a range of pharmacological agents, highlighting the innovative nature of the work.

    Additionally, the study identifies the involvement of the MAO-A-DOPEGAL-AEP pathway in this process. The topic is timely, the proposed mechanistic pathway is compelling, and the findings have translational relevance, particularly regarding therapeutic strategies targeting α2A-AR internalization in neurodegenerative diseases.

    Weaknesses:

    (1) The manuscript reports that chronic stress for 5 days increases MAO-A levels in LC neurons, leading to the production of DOPEGAL, activation of AEP, and subsequent tau cleavage into the tau N368 fragment, ultimately contributing to neuronal damage. However, the authors used wild-type C57BL/6 mice, and previous literature has indicated that AEP-mediated tau cleavage in wild-type mice is minimal and generally insufficient to cause significant behavioral alterations. Please clarify and discuss this apparent discrepancy.

    (2) It is recommended that the authors include additional experiments to examine the effects of different durations and intensities of stress on MAO-A expression and AEP activity. This would strengthen the understanding of stress-induced biochemical changes and their thresholds.

    (3) Please clarify the rationale for the inconsistent stress durations used across Figures 3, 4, and 5. In some cases, a 3-day stress protocol is used, while in others, a 5-day protocol is applied. This discrepancy should be addressed to ensure clarity and experimental consistency.

    (4) The abbreviation "vMAT2" is incorrectly formatted. It should be "VMAT2," and the full name (vesicular monoamine transporter 2) should be provided at first mention.

  12. eLife

    Reviewer #3 (Public review):

    Summary:

    The authors present a technically impressive data set showing that repeated excitation or restraint stress internalises somato dendritic α2A adrenergic autoreceptors (α2A ARs) in locus coeruleus (LC) neurons. Loss of these receptors weakens GIRK-dependent autoinhibition, raises neuronal excitability, and is accompanied by higher MAO-A, DOPEGAL, AEP, and tau N368 levels. The work combines rigorous whole-cell electrophysiology with barbadin-based trafficking assays, qPCR, Western blotting, and immunohistochemistry. The final schematic is appealing and could, in principle, explain early LC hyperactivity followed by degeneration in ageing and Alzheimer's disease.

    Strengths:

    (1) Multi-level approach - The study integrates electrophysiology, pharmacology, mRNA quantification, and protein-level analysis.

    (2) The use of barbadin to block β-arrestin/AP-2-dependent internalisation is both technically precise and mechanistically informative.

    (3) Well-executed electrophysiology.

    (4) Translation relevance - converges to a model that can be discussed by peers (scientists can only discuss models - not data!).

    Weaknesses:

    Nevertheless, the manuscript currently reads as a sequence of discrete experiments rather than a single causal chain. Below, I outline the key points that should be addressed to make the model convincing.

  13. Version published to 10.7554/elife.106362.1 on eLife
  14. Version published to 10.1101/2025.03.10.642335 on bioRxiv