Author(s): Matthew Perez, Mounia Hammadi
Mentor(s): Ren L. Guerriero, Interdisciplinary Program in Neuroscience
AbstractSleep is a vital part of human life known for rejuvenation and memory consolidation, but the molecular processes of this state are poorly understood. Drosophila melanogaster has been shown to exhibit this similar behavior under circadian and homeostatic control. Thus, to perform genetic manipulations that will enhance or degrade sleep, this model organism will be utilized to present a better understanding of the molecular aspect of sleep in humans. The three novel sleep-related genes analyzed within this study include Ptx1, stj (straightjacket), and Spn (Spinophilin). A UAS-Gal4 system will be utilized to carry either an RNAi or knockdown mutation of the gene. The Gal4 targets known sleep-regulating areas in the Drosophila brain, including the mushroom body, fan-shaped body, and ellipsoid-shaped body. Drosophila Activity Monitors (DAM2) will be used to record their locomotor activity over time. Sleep is determined if the infrared beam has not been broken in 5 minutes. Preliminary data has shown that stj in mice (Cacna2d3) codes for a calcium channel protein that interacts with various circadian genes that control the internal clock (Joshi et al., 2019). The Cacna2d3 knockout mice were shown to have reduced sleep during their rest phase. Thus, since fruit flies are diurnal, this dip in sleep is expected during the dark phase. Pilot data has shown in Ptx1 RNAi flies there was an increase in sleep time in males and females. Spn’s pilot data has shown increased total sleep times, more specifically around ZT12 and ZT21 in both sexes. The importance of validating these novel sleep-related genes is to provide scientists with an understanding of the molecular processes of the circadian rhythm and even the homeostatic responses to sleep. This molecular understanding will allow scientists to better develop medicines or therapies that can counteract sleep-related diseases, including insomnia and restless leg syndrome (RLS).
Audio TranscriptHello, my name is Mounia Hammadi and this summer, with the help of Dr. Ren Guerriero and Matthew Perez, I set out to understand the molecular processes of novel sleep-related genes in fruit flies, also known as Drosophila melanogaster.
So, what are novel sleep-related genes? Well, they are genes that acquire evidence that shows that they may be somehow associated with the physiological and molecular processes of this vigilant state.
Some genes that have been found to play a role in sleep include PERIOD and CLOCK, both of which are associated with the circadian rhythm of this vigilant state as well as the sleep-wake cycle in general.
There are three novel sleep-related genes that are of interest for this experiment: the straightjacket, the Ptx1, and Spinophilin. These three genes can be found in the Drosophila melanogaster, also known as the fruit fly. The main reason that we’ve decided to use the fruit flies for this experiment is due to the plethora of data behind this model organism in regards to genetic research.
And due to the fact that their genes are orthologs to that of mammalian organisms, such as us and house mice.
In house mice, they do not acquire the straightjacket gene, but they do acquire a gene that is an ortholog to that, also known as cacna2d3.
An ortholog, or an orthologous gene is one that descends from a common ancestor.
Thus, not only are the flies inexpensive to maintain and acquire a very simple genetic structure, they also follow the six conditions of sleep.
The first one is behavioral quiescence, where it shows inactivity or dormancy. The second is homeostatic regulation with their ability to make up for sleep that has been lost if they have been sleep deprived. Third is circadian control where, even if put in light-light conditions or dark-dark conditions, they still follow a cycle for sleep and wake. Fourth is arousal threshold which is reduced responsiveness to stimuli. Fifth is reversibility where, with a large enough stimuli, they are able to be woken up, unlike in a coma or a vegetative state. And then sixth is posture where they acquire a distinct posture with their heads down and their wings down.
Thus, with all this background information, this summer I went out to see if these novel sleep-related genes were indeed novel sleep-related genes and if so what were their molecular processes.
I hypothesize that the genes are in fact associated with sleep somehow.
When we look at the straightjacket’s ortholog in mice, we see that it is a calcium channel protein that interacts with circadian genes that controls the mice’s’ internal clock. Thus, when the gene is disturbed, they acquired a reduction in sleep overall.
Preliminary data shows something similar with the Spinophilin gene and the Ptx1 gene in the Drosophila melanogaster, but instead of seeing a decrease in sleep, we see an increase in sleep in both sexes, males and females.
But in order to begin experimentation, we needed to acquire a large stock of flies. Thus, we made a plethora of food that was molasses mixed with propionic acid. The propionic acid was added to mitigate the risk of contamination from bacteria or fungi, as well as constantly flipping vials to separate the generations and keep overcrowding to a minimum.
The flies were housed in an incubator kept at 21 degrees Celsius and put at 12 hours light and 12 hours dark conditions.
To understand the sleeping habits of our flies, we ran [and are still running], three trials in the Drosophila Activity Monitors. If the flies did not cross the beam within five minutes, it was dictated as sleep. Each monitor held 32 flies, each within 5 mm tubes.
The data that we will acquire will be able to be drawn as a line graph that will show the trend of sleep over time. Thus, I have developed a code that takes the raw data from the DAM2 monitors and puts them into the RStudio system, thus outputting the results as a line graph that is easy to read and easy to follow.
The pandemic has made it rather difficult for us to pursue with our experimentation due to the constant shipping delays and back orders. We’ve only now acquired our CO2 that separates the virgin females from the males that can then be utilized to cross the proper genes that will be silenced or reduced.
Nevertheless, we have begun to collect a plethora of data on our parental lines and hope to continue to move forward.
With the trials currently being run, with the code functioning, and the plethora of stocks that we have acquired, I would like to thank my coworkers for assisting me with all that we have done so far. The interdisciplinary program of [in] neuroscience at George Mason University, Dr. Ren Guerriero’s lab for taking me in to pursue this project, and the OSCAR URSP for providing me with the funding and support to do this throughout the summer.
Thank you for taking the time to watch my presentation. Hopefully in the next few semesters we will acquire some more concrete data on these novel sleep-related genes as well as their molecular processes in the Drosophila melanogaster. I appreciate your time.
So, what are novel sleep-related genes? Well, they are genes that acquire evidence that shows that they may be somehow associated with the physiological and molecular processes of this vigilant state.
Some genes that have been found to play a role in sleep include PERIOD and CLOCK, both of which are associated with the circadian rhythm of this vigilant state as well as the sleep-wake cycle in general.
There are three novel sleep-related genes that are of interest for this experiment: the straightjacket, the Ptx1, and Spinophilin. These three genes can be found in the Drosophila melanogaster, also known as the fruit fly. The main reason that we’ve decided to use the fruit flies for this experiment is due to the plethora of data behind this model organism in regards to genetic research.
And due to the fact that their genes are orthologs to that of mammalian organisms, such as us and house mice.
In house mice, they do not acquire the straightjacket gene, but they do acquire a gene that is an ortholog to that, also known as cacna2d3.
An ortholog, or an orthologous gene is one that descends from a common ancestor.
Thus, not only are the flies inexpensive to maintain and acquire a very simple genetic structure, they also follow the six conditions of sleep.
The first one is behavioral quiescence, where it shows inactivity or dormancy. The second is homeostatic regulation with their ability to make up for sleep that has been lost if they have been sleep deprived. Third is circadian control where, even if put in light-light conditions or dark-dark conditions, they still follow a cycle for sleep and wake. Fourth is arousal threshold which is reduced responsiveness to stimuli. Fifth is reversibility where, with a large enough stimuli, they are able to be woken up, unlike in a coma or a vegetative state. And then sixth is posture where they acquire a distinct posture with their heads down and their wings down.
Thus, with all this background information, this summer I went out to see if these novel sleep-related genes were indeed novel sleep-related genes and if so what were their molecular processes.
I hypothesize that the genes are in fact associated with sleep somehow.
When we look at the straightjacket’s ortholog in mice, we see that it is a calcium channel protein that interacts with circadian genes that controls the mice’s’ internal clock. Thus, when the gene is disturbed, they acquired a reduction in sleep overall.
Preliminary data shows something similar with the Spinophilin gene and the Ptx1 gene in the Drosophila melanogaster, but instead of seeing a decrease in sleep, we see an increase in sleep in both sexes, males and females.
But in order to begin experimentation, we needed to acquire a large stock of flies. Thus, we made a plethora of food that was molasses mixed with propionic acid. The propionic acid was added to mitigate the risk of contamination from bacteria or fungi, as well as constantly flipping vials to separate the generations and keep overcrowding to a minimum.
The flies were housed in an incubator kept at 21 degrees Celsius and put at 12 hours light and 12 hours dark conditions.
To understand the sleeping habits of our flies, we ran [and are still running], three trials in the Drosophila Activity Monitors. If the flies did not cross the beam within five minutes, it was dictated as sleep. Each monitor held 32 flies, each within 5 mm tubes.
The data that we will acquire will be able to be drawn as a line graph that will show the trend of sleep over time. Thus, I have developed a code that takes the raw data from the DAM2 monitors and puts them into the RStudio system, thus outputting the results as a line graph that is easy to read and easy to follow.
The pandemic has made it rather difficult for us to pursue with our experimentation due to the constant shipping delays and back orders. We’ve only now acquired our CO2 that separates the virgin females from the males that can then be utilized to cross the proper genes that will be silenced or reduced.
Nevertheless, we have begun to collect a plethora of data on our parental lines and hope to continue to move forward.
With the trials currently being run, with the code functioning, and the plethora of stocks that we have acquired, I would like to thank my coworkers for assisting me with all that we have done so far. The interdisciplinary program of [in] neuroscience at George Mason University, Dr. Ren Guerriero’s lab for taking me in to pursue this project, and the OSCAR URSP for providing me with the funding and support to do this throughout the summer.
Thank you for taking the time to watch my presentation. Hopefully in the next few semesters we will acquire some more concrete data on these novel sleep-related genes as well as their molecular processes in the Drosophila melanogaster. I appreciate your time.