Could poor sleep be an early indicator of Alzheimer’s disease? – A replication study

1. Overview

Replication studies are important because many scientific findings cannot be reproduced, there are estimates in some fields that 70% of experiments could not be reproduced^1,2, which raises concerns about their reliability. Therefore, we believe that replication studies are important to independently validate previous findings. In this pre-registration, we propose to repeat recent work suggesting that poor sleep may appear before memory decline in models of Alzheimer’s disease, to test whether sleep disruption could serve as an early biomarker. We estimate to complete this project within 3-4 months.

2. Introduction

Sleep disturbance has been increasingly recognized as an early biomarker and possible driver of Alzheimer’s disease (AD).³ Experimental models suggest a potential bidirectional relationship between β-amyloid (Aβ) accumulation and sleep disruption/fragmentation, but causal mechanisms remain poorly understood. In a recent study, Drosophila melanogaster flies were used to model AD-related pathology by neuronal expression of human Aβ42, which produced fragmented sleep prior to cognitive decline.⁴ These findings have also been covered by major news outlets as evidence that sleep loss may play a role in AD progression.⁵

In this pre-registration, we propose to replicate the main experiment from the original study to test whether sleep fragmentation can be an early biomarker of AD pathology. The experiments will be performed by our independent Contract Research Organization (CRO), Tracked Biotechnologies, which will apply automated experimental set-up to enhance the reproducibility of these findings.

Objective:

To replicate the finding that neuronal Aβ42 expression in Drosophila causes fragmented sleep, as assessed by the amount of sleep, and by how continuous or interrupted the sleep is (measured by the number and length of sleep episodes).

Project summary of original paper:

Flies expressing human Aβ42 under the pan-neuronal driver elav-GAL4 were compared with control flies carrying only the driver (elav-GAL4/+). Adult flies were assayed for sleep behavior using an infrared beam monitoring system under standard light–dark conditions. Sleep was defined as periods of immobility of 5 minutes or longer, as described in the paper.

Replication Figure:

Figure 4 (panels C–J): Daytime and nighttime sleep quantity and bout structure in Aβ42-expressing flies.

3. Methods

1. Experimental set-up

We will use Drosophila expressing human Aβ42 under the elav-GAL4 driver (elav-GAL4 > UAS-Aβ42), dFabp flies, dFabp combined with UAS-Aβ42 flies, and driver-only controls (elav-GAL4/+). Each group will include 35–40 flies at age 2–3 days, similar sample size and age with the original paper.

2. Sleep Assay

We will use an adapted TrackedFlyBox™ system to monitor adult flies under standard 12:12 light–dark cycles. The TrackedFlyBox™ will record continuous video, and custom analysis scripts will quantify immobility and sleep patterns. Sleep will be defined as ≥5 minutes of inactivity, following the criteria established by Gerstner et al. (2024).

3. Data Readouts

From the TrackedFlyBox™ output, the following parameters will be extracted:

- Total sleep (Figure 4B)

- Daytime sleep (Figure 4C)

- Nighttime sleep (Figure 4G)

- Number of sleep episodes at night (Figure 4J)

- Sleep episode length at night (Figure 4H)

All readouts will be formatted to ensure consistency with the metrics presented in Figure 4C–J of Gerstner et al. (2024).

4. Analysis

Statistical comparisons (t-tests or ANOVA) will be performed between Aβ42-expressing flies and appropriate controls, following the approach of the original study. Estimated completion within 3-4 months from initiation to final report and data delivery. Success of the replication will be defined if at least 2 of the extracted 5 criteria will be replicated showing a p < 0.05 in the performed statistical tests.

5. Budget

Total Project Cost: $10,000

These costs will cover 1) purchase and maintenance of Drosophila lines under standard laboratory conditions. 2) the use of the TrackedFlyBox™ system, video recording, and automated behavioral analysis, 3) CRO staff time for experimental setup, monitoring, data analysis 4) preparation of figures, final report generation, and data formatting for open sharing. This budget has been approved by the Tracked Biotechnologies CRO.

6. References

1. Baker, M. 1,500 scientists lift the lid on reproducibility. Nature 533, 452–454 (2016).

2. Collaboration, O. S. Estimating the reproducibility of psychological science. Science (2015) doi:10.1126/science.aac4716.

3. Gottesman, R. F. et al. Impact of Sleep Disorders and Disturbed Sleep on Brain Health: A Scientific Statement From the American Heart Association. Stroke (2024) doi:10.1161/STR.0000000000000453.

4. Gerstner, J. R. et al. Amyloid-β induces sleep fragmentation that is rescued by fatty acid binding proteins in Drosophila. J. Neurosci. Res. 95, 1548–1564 (2017).

5. Tiny fruit flies offer a few clues to sleep-related Alzheimer’s mysteries. https://www.spokesman.com/stories/2017/mar/13/tiny-fruit-flies-offer-a-few-clues-to-sleep-relate/.

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Could poor sleep be an early indicator of Alzheimer’s disease? – A replication study

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