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

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.

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.

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.

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.