Sleep and memory consolidation are linked by RNA processing genes in the Drosophila mushroom body

Reviewer #1 (Public Review):

The authors investigated the molecular underpinnings of sleep-related memory consolidation and explored transcriptional changes in memory-related neurons, specifically the α′β′-Kenyon cells in the mushroom bodies (MB) of fruit flies in different states of memory consolidation. Their experiments identified changes in several genes and subsequently conducted functional experiments on sleep and memory. These findings are useful for further characterization of the molecular pathways that may link sleep to long-term memory formation.

The functional characterization of the identified genes revealed that the perturbation of two genes alters sleep: 1) Polr1f knockdown reduces sleep and increases pre-ribosome and translation. 2) In contrast, knockdown of Regnase-1 decreases sleep. Furthermore, Regnase-1 knockdown impairs all forms of appetitive memory. Although the findings are generally interesting, the functional relationship between these genes, sleep, and memory does not entirely become clear from the presented work. Some conclusions are not completely supported by the data, and alternative explanations are not considered.

Reviewer #2 (Public Review):

Sleep and memory are intertwined processes, with sleep-deprivation having a negative impact on long-term memory in many species. Recently, the authors showed that fruit flies form sleep-dependent long-term appetitive memory only when fed. They showed that this context-dependent memory trace maps to the anterior-posterior (ap) α'β' mushroom body neurons (MBNs) (Chouhan et al., (2021) Nature). However, the molecular cascades induced during training that promote sleep and memory have remained enigmatic.

Here the authors investigate this issue by combining cell-specific transcriptomics, genetic perturbations, and measurements of sleep and memory. They identify an array of genes altered in expression following appetitive training. These genes are mainly downregulated, and predominantly encode regulators of transcription and RNA biosynthesis. This is a conceptually attractive finding given that long-term memory requires de novo protein translation.

The authors then screen these genes for novel regulators of sleep and memory. They show that one of these genes (Polr1F) acts in ap α'β' MBNs to promote wakefulness, while another (Regnase-1) promotes sleep. They also identify a specific role for Regnase-1 in ap α'β' MBNs in regulating short- and long-term memory formation, and demonstrate that Pol1rF inhibits translation throughout the fly brain.

The analyses of molecular alterations in ap α'β' MBNs are interesting and impressive. However, caveats remain regarding the effect of Polr1F and Regnase-1 on sleep. There are significant differences in the impact of Polr1F knockdown on sleep between datasets, and from the data currently presented, it is unclear whether Polr1F and Regnase-1 might also play important developmental roles in ap α'β' MBNs that influence sleep. These caveats can be readily addressed by additional experiments that would enhance the robustness of the manuscript.

Reviewer #3 (Public Review):

A landmark work (Chouhan et al., 2022) from the Sehgal group previously investigated the relationship between sleep and long-term memory formation by dissecting the role of mushroom body intrinsic neurons, extrinsic neurons, and output neurons during sleep-dependent and sleep-independent memory consolidation. In this manuscript, Li et al., profiled transcriptome in the anterior-posterior (ap) α'/β' neurons and identified genes that are differentially expressed after training in fed condition, which supports sleep-dependent memory formation. By knocking down candidate genes systematically, the authors identified Polr1F and Regnase-1 as two important hits that play potential roles in sleep and memory formation. What is the function of sleep and how to create a memory are two long-standing questions in science. The present study used a creative approach to identify novel components that may link sleep and memory consolidation in a specific type of neuron. Importantly, these components implicated that RNA processing may play a role in these processes.

While I am enthusiastic about the innovative approach employed to identify RNA processing genes involved in sleep regulation and memory consolidation, I feel that the data presented in the manuscript is insufficient to support the claim that these two genes establish a definitive link between sleep and memory consolidation. First, the developmental role of Regnase-1 in reducing sleep remains unclear because knocking down Regnase-1 using the GeneSwitch system produced neither acute nor chronic sleep loss phenotype. To address potential confounding issues caused by the GeneSwtich system, I would suggest considering alternative methods, such as Gal80ts, to restrict the RNAi knockdown to adulthood. In addition, QPCR or other expression-measuring methods should be used to validate the specificity and efficiency of the knockdown. Further testing of additional RNAi fly lines and conducting overexpression experiments would also lend credibility to the phenotypes. Second, while constitutive Regnase-1 knockdown produced robust phenotypes for both sleep-dependent and sleep-independent memory, it also led to a severe short-term memory phenotype. This raises the possibility that flies with constitutive Regnase-1 knockdown are poor learners, thereby having little memory to consolidate. The defect in learning could be simply caused by chronic sleep loss before training. Thus, this set of results does not substantiate a strong link between sleep and long-term memory consolidation. Lastly, the discussion on the sequential function of training, sleep, and RNA processing on memory consolidation appears to be speculative based on the present data. While the novel approach did provide novel candidate genes with functions in sleep, memory, and potentially their link, the manuscript would greatly benefit from carefully adjusting the conclusions and incorporating rigorous validations for the RNAi knockdown experiments.

Author Response:

We would like to express our gratitude for the valuable feedback provided by the editors and reviewers. In response to the reviewer's comments, we have outlined a plan to carry out additional experiments to bolster our paper's strength. Our primary objectives are to explore the developmental roles of both Porl1F and Regnase-1 and to provide further clarification regarding the involvement of Regnase-1 in memory consolidation. We will utilize the newly available Polr1F RNAi transgenes and confirm the efficacy of both the previous and new RNAi lines through quantitative polymerase chain reaction (qPCR) analysis. Additionally, we aim to investigate the impact of Regnase-1 overexpression on sleep and memory consolidation. We will also clarify some points that may not have been clear to reviewers.