Sleep is bi-directionally modified by amyloid beta oligomers

  1. Güliz Gürel Özcan
  2. Sumi Lim
  3. Patricia LA Leighton
  4. W Ted Allison
  5. Jason Rihel

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Abstract

Disrupted sleep is a major feature of Alzheimer’s disease (AD), often arising years before symptoms of cognitive decline. Prolonged wakefulness exacerbates the production of amyloid-beta (Aβ) species, a major driver of AD progression, suggesting that sleep loss further accelerates AD through a vicious cycle. However, the mechanisms by which Aβ affects sleep are unknown. We demonstrate in zebrafish that Aβ acutely and reversibly enhances or suppresses sleep as a function of oligomer length. Genetic disruptions revealed that short Aβ oligomers induce acute wakefulness through Adrenergic receptor b2 (Adrb2) and Progesterone membrane receptor component 1 (Pgrmc1), while longer Aβ forms induce sleep through a pharmacologically tractable Prion Protein (PrP) signaling cascade. Our data indicate that Aβ can trigger a bi-directional sleep/wake switch. Alterations to the brain’s Aβ oligomeric milieu, such as during the progression of AD, may therefore disrupt sleep via changes in acute signaling events.

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  1. Version published to 10.7554/elife.53995 on eLife
  2. eLife

    ###Reviewer #3

    The authors report the use of a novel model of intracardiac infusion of Aβ peptides in zebrafish larvae to study the effects of Aβ on sleep and neuronal activity. They provide convincing data that preparations of shorter Aβ oligomers induce neuronal activity and decrease sleep, while longer oligomers suppress neuronal activity and decrease sleep. They then delete known Aβ receptor proteins, and show that the effects of Aβ-short can be blocked by deletion of Adrb2 and Pgrmc1, while the effects of Aβ-long are blocked by prion protein deletion, or specific drugs.

    This is a unique system and the method for administering Aβ that is quite powerful, and the experiments are rigorous and generally use multiple converging approaches (for instance genetic+pharmacologic) to support their findings. The reversibility of the effect, as well as blockade with specific pharmacological agents suggests that these are not non-specific toxic events. The findings provide a framework with which to potentially test other neurodegenerative proteins (such as a-syn), and to inform similar studies in mammalian systems.

    1. While the experiments are well performed and the data intrinsically consistent, the applicability to mammals (and humans) is a consideration. Infusion of Aβ into the heart of larvae is a highly artificial system, and events that occur during sudden changes in Aβ levels may be different that those observed when Aβ is chronically present (as in AD). For example, infusion of Aβ peptide into the brains of mice or rats can induce acute, local neurodegeneration that is not observed in APP transgenic mice with chronically elevated Aβ levels. This is a fundamental shortcoming of the model, and there is little that can be done to address it, but it should be perhaps mentioned in the Discussion.

    2. The implications of this bidirectional effect of short and long oligomers for sleep phenotypes in AD are also a bit unclear, as oligomers of all sizes are likely present in AD brain (though perhaps in different ratios as the disease progresses). It would be helpful to determine which pathway is dominant when both short and long oligomers are infused together, perhaps in different ratios. This is the only experiment I would suggest.

  3. eLife

    ###Reviewer #2

    The use of zebrafish to investigate the role of beta amyloid polymers on sleep/wake regulation is potentially interesting as AD patients suffer from insomnia. Here Ozcan and colleagues inject oligomers synthesized in vitro into the fish neonate hearts and fish motion was then recorded and used as a proxy for sleep and wake states. The authors found a correlation between the polymer length and the impact on fish motor and brain activity.

    While the findings are potentially interesting, several points are unclear or concerning to the reviewer:

    1. First, all the experiments and interpretations rely on overexpression of Abeta polymers; there is no description or investigation in this study of the normal baseline of Abeta accumulation in this species. One would expect to see such data in Fig. 1 and S1 for example. Is there in fish a night vs. day, sleep vs. night rhythm of Abeta accumulation/expression?

    2. The fish undergo anesthesia and heart perforation and are recorded a few hours later. How can handling, surgical stress, and confounds of prior anesthesia be eliminated from "sleep-wake" data interpretation?

    3. It is hard for the reader to distinguish a specific effect on sleep/wake. Increased or decreased motion could be due to toxicity or specific stimulation of neuronal circuits due to non physiological presence of exogenous oligomers. The authors try to tackle this issue with cfos and ERK staining, but Fig. 2 shows at least 6 different staining patterns, none of them compared to a sleep/wake baseline of staining. It is quite worrisome to see such a broad over expression of cfos throughout the brain when A beta is accumulated. Are the fish having a seizure? Toxicity could lead to reduced motion and even if it's reversible it can still be transient toxicity until oligomers are washed out. Hyperactivity could be due to a specific overstimulation of neurons as illustrated by cfos and ERK staining.

    4. Injections in mutant backgrounds indeed show some specificity in binding/interaction but still it does not demonstrate that the impact is on wake or sleep regulation per se. Again only motion or broad brain staining (at one time point) are shown. An alternative interpretation is that adrb2a, pgrmc, prp1 can indeed bind Abeta but relay the toxic or aspecific impact of oligomers over expression in a brain that normally does not accumulate such molecules.

    This study has the potential to be extremely interesting but many controls and demonstration of endogenous Abeta role on sleep-wake cycle are needed.

  4. eLife

    ###Reviewer #1

    There is a growing appreciation about the fundamental bidirectional link between sleep and Alzheimer's disease. Here Rihel and colleagues use a zebrafish model coupled to the injection of amyloid beta oligomers (the initiating pathogenic species for AD) to examine the link between Abeta and sleep. They demonstrate that the length of the oligomers determines whether Abeta induces wake (short Abeta) or sleep (long Abeta), providing novel insights into the role of different forms on sleep/wake. Importantly, they extend their findings to reveal novel molecular insights into the mechanisms into how Abeta exerts these sleep/wake effects. Overall, the findings make an important advance that will be of interest to a broad readership.

    I have one significant concern relating to claims that these studies reveal novel functions for the endogenous Abeta. A key missing experiment in this regard is manipulation of the endogenous Abeta gene/protein (or even assessment of endogenous Ab) and thus it is unclear if exogenous (intracardiac) injection of Abeta faithfully reproduces how an endogenous neuronal pathway would deliver Abeta in terms of location, local concentrations and kinetics. I think the findings are significant and important on their own without having to make this claim, which in this case is highly speculative. I would suggest either addressing experimentally or rewording and de-emphasizing this point in the text to make clear the speculative possibilities. In any case, these shortcomings should be more forthrightly noted.

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to Version 2 of the preprint: https://www.biorxiv.org/content/10.1101/610014v2

    ###Summary

    This study describes the use of intracardiac infusion of various sized amyloid-beta (Aβ) peptides in zebrafish larvae to study the effects of Aβ on sleep and neuronal activity and dissect the molecular mechanism of their action. They show that short Aβs induce neuronal activity and decrease sleep, while long Aβs suppress neuronal activity and decrease sleep. They use genetic perturbations to show that short Aβs act through Adrb2 and Pgrmc1, while long Aβs act via PrP.

    As described below, the reviewers consider this manuscript to be a potentially important methodological and conceptual advance, but recommend that the authors address the following concerns:

    The model is based on intracardiac injection of Abeta, so the phenotypes result from exogenous expression/overexpression. Given this, the authors should refrain from drawing conclusions about endogenous Abeta. At the same time, the manuscript would benefit from minimal characterization of the endogenous molecules. For instance, is there a rhythm of Abeta expression over the sleep:wake cycle?

    The fish undergo anesthesia and heart perforation and are recorded a few hours later. What are the controls for handling, surgical stress, and confounds of prior anesthesia? On a related note, can the authors exclude toxicity, which could affect motion? They address this point by showing cfos and ERK staining, but many different patterns are observed and none are compared to staining under baseline sleep:wake conditions. It is also concerning that the c-fos expression is so widespread. The reversibility of the effect is important and the role of specific molecules is interesting, but these still do not demonstrate impact on wake or sleep regulation per se.

    Given that AD brains likely have oligomers of all sizes, it would be good to know what happens when short and long oligomers are infused together.

  6. Version published to 10.1101/610014 on bioRxiv