Magnesium and the magnesium transporter UEX regulate sleep via Ca 2+ -dependent CREB signaling and a CNK-ERK pathway
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eLife assessment
This manuscript provides valuable evidence for the role of magnesium homeostasis and relevant signaling pathway in Drosophila sleep regulation. It will be of interest to cellular biologists and neuroscientists interested in sleep:wake behavior and the potential role of magnesium in promoting sleep. Nevertheless, the evidence for the key claims of the manuscript is incomplete and is not fully supported by the data as reasonable alternative explanations exist.
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Abstract
Magnesium and its related preparations are already in medical use and have recognized therapeutic effects on sleep disorders. However, its underlying molecular mechanisms remain unclear. Here, using Drosophila as a model, we found that RNAi-mediated knockdown of Uex , the homologous gene of magnesium transporters of the Cyclin M family (CNNM) causes increased daily total sleep. Ectopic-expression of CNNM1 can rescue the sleep phenotype in Uex knockdown flies. UEX exhibits rhythmic oscillations in the brain and affects the efflux of cellular Mg 2+ . Knockdown of Uex in the nervous system influences Ca 2+ -mediated CREB signaling and neuroplasticity. Additionally, Uex physically interacts with CNK, the upstream regulator of ERK pathway. Similar effects on sleep are observed with knockdown of Cnk in flies. We propose that the UEX regulates sleep through its downstream Ca 2+ -dependent CREB signaling and a CNK-ERK pathway. Our findings may provide new insight into mechanisms of magnesium and magnesium transporter related sleep disorder.
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eLife assessment
This manuscript provides valuable evidence for the role of magnesium homeostasis and relevant signaling pathway in Drosophila sleep regulation. It will be of interest to cellular biologists and neuroscientists interested in sleep:wake behavior and the potential role of magnesium in promoting sleep. Nevertheless, the evidence for the key claims of the manuscript is incomplete and is not fully supported by the data as reasonable alternative explanations exist.
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Reviewer #1 (Public Review):
Yuan et al. propose that the magnesium transporter unextended (uex) controls Drosophila sleep via Mg2+ efflux, Ca2+-dependent CREB signaling, and a CNK-ERK pathway. UEX protein levels display daily oscillations in fly heads, whereas UEX-depleted flies show long sleep with low levels of Ca2+ and synaptic plasticity. Transgenic expression of wild-type UEX or the mammalian uex homolog CNNM1 possibly rescues the uex mutant sleep, supporting their evolutionary conservation. UEX forms a protein complex with the ERK signaling suppressor CNK, and UEX depletion appears to de-repress ERK activation. As expected, pan-neuronal CNK depletion phenocopies uex mutant sleep. Taken together, the authors suggest a novel mechanism whereby magnesium shapes animal sleep through the specific MAPK pathway. Overall, the authors …
Reviewer #1 (Public Review):
Yuan et al. propose that the magnesium transporter unextended (uex) controls Drosophila sleep via Mg2+ efflux, Ca2+-dependent CREB signaling, and a CNK-ERK pathway. UEX protein levels display daily oscillations in fly heads, whereas UEX-depleted flies show long sleep with low levels of Ca2+ and synaptic plasticity. Transgenic expression of wild-type UEX or the mammalian uex homolog CNNM1 possibly rescues the uex mutant sleep, supporting their evolutionary conservation. UEX forms a protein complex with the ERK signaling suppressor CNK, and UEX depletion appears to de-repress ERK activation. As expected, pan-neuronal CNK depletion phenocopies uex mutant sleep. Taken together, the authors suggest a novel mechanism whereby magnesium shapes animal sleep through the specific MAPK pathway. Overall, the authors reported quite a few exciting phenotypes in UEX-depleted flies using a range of analyses (e.g., behaviors, gene expression, Mg2+/neural imaging, and biochemistry). However, evidence for their causal link to sleep regulation is missing, and some key conclusions remain justified by more rigorous analyses. It is noteworthy that Wu et al. have previously demonstrated the Mg2+ efflux transporter activity of UEX and mapped its memory-enhancing function to a specific group of adult neurons in the fly brain (https://pubmed.ncbi.nlm.nih.gov/33242000/). Given the intimate interactions between sleep and memory, these findings may have broad implications in sleep-relevant physiology and disorders. However, a number of important control studies and statistical assessments are not included, but are necessary to conclusively interpret the data.
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Reviewer #2 (Public Review):
The authors described the analysis of a magnesium transporter UEX as a sleep-regulating gene in Drosophila melanogaster. They also proposed the UEX regulates sleep through its downstream Ca2+-dependent CREB signaling and a CNK-dependent ERK pathway. The involvement of UEX in sleep regulation is novel and potentially interesting, but the data presented in the manuscript does not fully support the conclusions the authors proposed. Most of the data are derived from elav-GAL4, which is a non-specific pan-neuronal GAL4 driver. Since as the authors described, UEX functions to alter sleep in various brain regions, the relationship between UEX and other molecules in Ca2+-dependent CREB signaling and a CNK-dependent ERK pathway may be indirect in the sleep-regulating pathway, which means it may involve multiple …
Reviewer #2 (Public Review):
The authors described the analysis of a magnesium transporter UEX as a sleep-regulating gene in Drosophila melanogaster. They also proposed the UEX regulates sleep through its downstream Ca2+-dependent CREB signaling and a CNK-dependent ERK pathway. The involvement of UEX in sleep regulation is novel and potentially interesting, but the data presented in the manuscript does not fully support the conclusions the authors proposed. Most of the data are derived from elav-GAL4, which is a non-specific pan-neuronal GAL4 driver. Since as the authors described, UEX functions to alter sleep in various brain regions, the relationship between UEX and other molecules in Ca2+-dependent CREB signaling and a CNK-dependent ERK pathway may be indirect in the sleep-regulating pathway, which means it may involve multiple regions of the brain using different pathways, and the sleep phenotype is the summation of different functions of UEX.
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Reviewer #3 (Public Review):
In this manuscript, Yuan et al. examined the relationship between a magnesium transporter and sleep behavior. They find that the knockdown of a magnesium efflux transporter (uex) in neurons increases bout length of inactivity and recovery activity of the flies with neuronal knockdown of uex with a human homolog CNNM1. The authors suggest a model in which Mg2+ promotes sleep through the inhibition of Ca2+ levels that are wake-promoting in the mushroom body and PDF+ neurons. Overall, the idea explored here that ion homeostasis in the neurons contributes to behavior is an area that is timely and interesting to the neuroscience community. The transgenic lines of human CNNMs could be a useful tool for scientists studying metal transport and ion homeostasis in flies. Unfortunately, the results of the experiments …
Reviewer #3 (Public Review):
In this manuscript, Yuan et al. examined the relationship between a magnesium transporter and sleep behavior. They find that the knockdown of a magnesium efflux transporter (uex) in neurons increases bout length of inactivity and recovery activity of the flies with neuronal knockdown of uex with a human homolog CNNM1. The authors suggest a model in which Mg2+ promotes sleep through the inhibition of Ca2+ levels that are wake-promoting in the mushroom body and PDF+ neurons. Overall, the idea explored here that ion homeostasis in the neurons contributes to behavior is an area that is timely and interesting to the neuroscience community. The transgenic lines of human CNNMs could be a useful tool for scientists studying metal transport and ion homeostasis in flies. Unfortunately, the results of the experiments do not entirely support the authors' conclusions.
The authors fall short of showing that the increased inactivity is sleep behavior as Mg2+ changes in neurons could be affecting the mobility of the fly. To validate that the increased inactivity is sleep, the authors should have used a combination of negative geotaxis, arousal threshold, or multibeam/video monitoring. Another characteristic of sleep is the presence of compensatory rebound following sleep deprivation. Here, when the authors sleep deprive the flies with uex knockdown, the flies do not have increased rebound sleep over control flies. Together the current data suggest that the increased inactivity may not be sleep and more evidence to the contrary should be shown.
In Fig 1, the authors show that there is a huge developmental effect on rest:activity rhythms when using the elav-gal4>uex RNAi compared to the inducible elav-geneswitch > uex RNAi, but in Fig 2, the authors use gal4 drivers rather than an inducible system. Use of an inducible system such as geneswitch, AGES, or TARGET is important to rule out developmental effects. Again, in Fig 4, the authors use the gal4 rather than the geneswitch for knocking down the other magnesium channels/transporters so it is unclear whether any sleep increase may be due to the role magnesium plays in development. In Fig 6 elav-gal4 was also used instead of GS. According to previously published work on UEX in fly neurons (Wu et al. eLife 2020 PMID: 33242000), UEX is primarily in the mushroom body and much lower expression in the PI or PDF+ neurons of the adult brains, further suggesting that sleep increases in the PDF+ and PI gal4s driving uex RNAi may be developmental.
From this work, the authors suggest that Mg2+ is sleep-promoting, and in the absence of uex efflux transporter to remove the Mg2+, Mg2+ increases to the point of inhibiting Ca2+, a wake-promoting signal; however, not all the Mg2+ transporters assayed efflux out Mg2+, but rather regulate the influx of Mg2+ into the cells. If a channel regulating Mg2+ influx is inhibited, the prediction would be that Mg2+ would be decreased and thus the flies should sleep less. But in Fig 4H that was not the case. All the Mg2+ transports/channel RNAi lines increased sleep. The authors do not reconcile this data with their proposed model. It is possible the Mg2+ transporter RNAi lines result in increased Mg2+ in the relevant neuronal subgroup in which case Mg2+ levels should be measured in the RNAi lines.
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