Fly on the Wall

Making fly science approachable for everyone

Author: Bethany Christmann (page 2 of 4)

Fly Life: Watching fruit flies sleep

December 19, 2014 / / 0 Comments

Did you know that fruit flies sleep? There are actually a lot of similarities between sleep in fruit flies and sleep in humans and other mammals. For example…

Caffeinated flyImage modified from Colwell, 2007
  • Like us, fruit flies get most of their sleep at night, and they also have an afternoon slump (although unlike us, they actually give in to their sleep desires instead of running for coffee).
  • Their sleep is affected by the same drugs: caffeine, cocaine, and modafinil (an alertness drug) keep them awake, while antihistamines (allergy medication) make them drowsy just like us.
  • They sleep more at higher temperatures (think of lazy summer days)
  • If they get a bad night’s sleep, they’ll try to sleep more the next day to make up for it. In fact, sleep deprivation even affects their memory performance (have you ever noticed that your memory isn’t so great after a bad night’s sleep?)

And the comparisons don’t stop there. Over the years, fruit flies have proven to be a great animal model for studying sleep, and researchers have used them to improve our understanding of why sleep is important. In fact, earlier this year a group of researchers found that young flies need sleep for normal brain development.

But, how do fly researchers actually study sleep in these tiny flies?

DAM system

The most common method is called the Drosophila Activity Monitoring (DAM) system. Flies are loaded into individual tubes (Figures A-C) and placed in a DAM machine (Figure D). An infrared beam (marked as a red line in Figure D) crosses each fly tube, and the machine notes when the beam is blocked by the fly. If the beam isn’t broken for five or more minutes (meaning the fly hasn’t moved), it’s counted as sleep. Using this method, researchers can analyze how long a fly sleeps, how many times it goes to sleep and wakes back up (called sleep bouts) and the duration of each sleep bout.

divider line

DAM system in incubatorClick here for a larger version of the picture

To conduct the sleep experiment, the machines are hooked up to a computer and placed in an incubator, which controls the humidity, temperature, and lights. The light cycle is usually set to lights-on for 12 hours (day) and then lights-off for 12 hours (night), and experiments often run for several days or even weeks. In this picture, a newer version of DAM machines is shown (one is circled in red).

divider line

Sleepless dataSleepless mutants sleep much less than normal (control) flies. Image modified from Koh et al, 2008

This system allows researchers to study the sleep habits of dozens of flies at once, and it’s relatively quick and easy (compared to studying sleep in mammals). So what’s an example of a real sleep experiment? In 2008, the Seghal lab used this system to find sleep-related genes. They ran a genetic screen in which they exposed a population of flies to toxic chemicals that caused random mutations in their DNA. Then, they tested the flies to see if any had sleep abnormalities. They found that flies with a mutation in one gene (which they aptly named sleepless) slept about 80% less! Isn’t it crazy that a single gene can affect such a complex behavior so dramatically?

Sleepless dataA common way to display fly sleep habits over the day and night period for one day. The amount of sleep within 30 minute intervals (y axis) is plotted for the time of day (x axis). Note that Sleepless mutants (open circles) sleep much less than control flies (filled circles). The bar at the bottom indicates time: white = day, black = night. Image modified from Koh et al, 2008

divider line
In another experiment that same year, the Guo lab was interested in finding out how sleep affects memory. They tested the sleeping habits of a fly mutant called amnesiac, which can’t form long-term memories. They found that amnesiac mutants had very fragmented sleep (meaning that they woke up a lot during the night). The fact that the same gene is involved in both sleep and memory provides a link that researchers can use for future studies about how these behaviors are connected.

Tracker setupA schematic of the Tracker setup. Image modified from Gilestro, 2012

Although studies have shown that the DAM system is relatively accurate for measuring fly sleep, more sophisticated methods have recently been developed for tracking fly movement. These systems (sometimes called the “Tracker” program) rely on cameras and sensitive tracking software to measure movement at a much higher resolution. As use of the Tracker program becomes more widespread, I have no doubt that we will gain yet more insights into how and why we sleep.

 
 

References:

  • Potdar S. (2013). Lessons From Sleeping Flies: Insights from Drosophila melanogaster on the Neuronal Circuitry and Importance of Sleep , Journal of Neurogenetics, 27 (1-2) 23-42. DOI: http://dx.doi.org/10.3109/01677063.2013.791692
  • Koh K., M. N. Wu, Z. Yue, C. J. Smith & A. Sehgal (2008). Identification of SLEEPLESS, a Sleep-Promoting Factor, Science, 321 (5887) 372-376. DOI: http://dx.doi.org/10.1126/science.1155942
  • Liu W., Beika Lu & Aike Guo (2008). amnesiac regulates sleep onset and maintenance in Drosophila melanogaster, Biochemical and Biophysical Research Communications, 372 (4) 798-803. DOI: http://dx.doi.org/10.1016/j.bbrc.2008.05.119
  • Donelson N., Kim E.Z., Slawson J.B., Vecsey C.G., Huber R. & Griffith L.C. (2012). High-resolution positional tracking for long-term analysis of Drosophila sleep and locomotion using the “tracker” program., PloS one, PMID: http://www.ncbi.nlm.nih.gov/pubmed/22615954
  • Gilestro G.F. (2012). Video tracking and analysis of sleep in Drosophila melanogaster, Nature Protocols, 7 (5) 995-1007. DOI: http://dx.doi.org/10.1038/nprot.2012.041

Breaking Research: A new technique for studying axon death using fruit fly wings

December 12, 2014 / / 0 Comments
labeled neuronThe axon is the part of a neuron that carries outgoing information. (cb = cell body)

In neurodegenerative diseases such as Parkinson’s disease or amyotrophic lateral sclerosis (ALS), a genetic mutation leads to widespread neuron damage. When a neuron is damaged, its axon—the part of the neuron that carries outgoing signals—is actively broken down and cleared away by maintenance cells (called glia). This destructive process is known as Wallerian degeneration (WD) and disrupts signaling between neurons due to the loss of their axons. By studying WD in animal models, scientists hope to figure out how to interfere with it and possibly slow the progression of the neurodegenerative diseases.

Because Wallerian degeneration also occurs after physical trauma such as in a spinal cord injury, scientists usually study it by cutting or crushing bundles of axons. Traditionally, researchers have induced random genetic mutations in a population of animals (a forward genetic screen), dissected and damaged a bundle of axons in individual animals, and watched the degradation process to see if the mutation had an effect. This process allows them to determine which genes are involved in breaking down and/or clearing away the damaged axons. So far, studies in mammals and fruit flies have found three genes that are required for WD (Wld, Sarm, and highwire/Phr1). Mutating these genes slows WD significantly, and researchers may be able to target these genes in the future to slow axon death.

But progress so far has been slow due to limitations in the current methods used for studying this phenomenon. In a recent paper published in PNAS by the Freeman lab, researchers describe a new technique for studying Wallerian degeneration in fruit flies that overcomes many of these limitations. Previous work has already shown that WD occurs in fruit fly axons and uses the same known genes, so they are an ideal model for quickly identifying the genes involved in axon death.

The authors developed a system in the fly wing that fulfilled three key requirements they believed were necessary for efficiency:

  1. Rapid observation of individual axons (rather than an entire axon bundle) with minimal dissection
  2. The opportunity to sever only a subset of axons so that one could observe axon death alongside healthy, uninjured axons
  3. The ability to initiate and visualize axon death without killing the fly
fly wingTop: A fly wing with sensory neurons labeled in green. (cb = cell body). Inset: a bundle of labeled axons makes up the L1 vein. Bottom: A schematic of the fly wing and cutting a subset of axons (dashed line). Axons with cell bodies closer to the fly’s body are spared.

The Drosophila wing is transparent and contains a large bundle of sensory neuron axons known as the L1 vein. The cell bodies of the neurons are located around the edge of the wing, and their axons extend through the L1 vein back into the body of the fly. Using genetic tools (check out this article on MARCM for details), the authors added a protein called GFP (which glows green) in a small number of these sensory neurons. This setup allows researchers to easily observe individual axons in the wing under a fluorescent microscope without dissection. These characteristics fulfill their first requirement.

This setup also easily allows researchers to sever some of the axons without injuring others by simply snipping a piece from the edge of the wing. Axons whose cell bodies were cut off succumb to WD, while axons with cell bodies were closer to the fly’s body are unharmed. This method of inducing WD in only some of the GFP-labeled neurons fulfills the second requirement.

GFP axon being degradedThree GFP-labeled axons: two healthy, one damaged and undergoing Wallerian degeneration (white arrow).

Finally, because the entire procedure can be performed in the fly’s wing, the fly itself is generally unharmed. This fulfills the final requirement, which is important for speeding up the screening process. Usually, after random mutations are induced in a population of flies, a fly line for each mutation has to be maintained until it can be tested. But if the original fly can live through the testing process, researchers can first determine if fly’s mutation is useful and then allow the fly to mate and create a stock if it is. This characteristic not only reduces the time needed for the screen by weeks, but it also significantly reduces the amount of labor required.

So how can this help us humans? This technique can speed up the process for discovering new genes involved in Wallerian degeneration. After a gene is identified in flies, researchers will know where to look in mammals and can begin studying the gene’s function (this has already happened once, with the Sarm gene). Figuring out how these genes function will provide a target for slowing axon death, which may in turn slow the progression of neurodegenerative diseases. And it all starts with a fruit fly’s wing.

 
Reference:

  • Neukomm L.J., Burdett, T.C., M. A. Gonzalez, S. Zuchner & M. R. Freeman (2014). Rapid in vivo forward genetic approach for identifying axon death genes in Drosophila, Proceedings of the National Academy of Sciences, 111 (27) 9965-9970. DOI: http://dx.doi.org/10.1073/pnas.1406230111

Breaking Research: Glycogen build-up in the brain contributes to aging

December 5, 2014 / / 1 Comment
young vs old brainTotal brain volume decreases as we age. Image modified from brainpowerrelease.

Why is the aging process accompanied by progressive cognitive decline such as impaired memory, decreased focus, and slowed reaction time? Although we don’t fully know what causes it, researchers have found that aging visibly affects the brain, most strikingly as a decrease in total brain volume due to progressive loss of neurons. As we age, it is thought that neurons are irreversibly damaged by cumulative mistakes in building proteins, copying DNA, and other processes.

Sometimes, these mistakes can cause abnormal proteins and other molecules to clump together and form dense deposits inside neurons. In many neurodegenerative diseases such as Alzheimer’s or Lafora’s disease, an excess of deposits is associated with rapid neuron death. Yet although the (albeit much less severe) accumulation of deposits in the brain is a normal part of aging, its role in cognitive decline has been historically ignored. In a new study published in Aging Cell by the Milán and Guinovart labs, researchers investigate why these deposits appear during the normal aging process and what effect they have on neuronal function.

glycogen moleculeGlycogen is a storage molecule made up of thousands of branching glucose units. Image by Mikael Häggström.

Previous research has shown that the deposits present in aged human brains (which are referred to as either corpora amylacea or polyglucosan bodies; I’m just going to call them PGBs) are primarily made up of glycogen molecules, along with some other proteins. Glycogen is an important energy storage molecule in animal cells (second only to fats) and each glycogen molecule is composed of thousands of glucose units. But what causes the glycogen to aggregate and form deposits in a normal aging brain?

PGBs in mouse brainTop: brain (hippocampus) slice of aged normal mouse, stained with PAS, scale bar 200um. Bottom: Zoomed in on black box, arrow points to PGBs. Modified from Sinadinos et al, 2014.

The authors analyzed PGBs in the brains of aged mice to answer this question. They first studied the composition of the mouse PGBs and confirmed that they were similar to the PGBs found in aged human brains, down to the types of proteins bound up among the glycogen molecules. Next, they created a knock-out mouse line that was missing the glycogen synthase gene, which synthesizes glycogen by attaching glucose molecules together. The authors couldn’t find PGBs in the aged brains of these mutant mice, thus demonstrating that the process of synthesizing glycogen contributed to PGB formation. This finding is admittedly unsurprising: if the cells can’t make glycogen, it’s not there to clump together.

Well here’s the interesting part! Not only couldn’t the authors find glycogen deposits, but they also didn’t see accumulation of any of other protein associated with PGBs. One of the proteins they tested was alpha-synuclein, the aggregate-prone protein involved in Parkinson’s disease. In the normal mice, alpha-synclein accumulated along with the PGBs, but there was no accumulation in the mutant mice. Thus, glycogen synthesis seemed to be a prerequisite for the formation of other protein deposits. This finding could have implications for possible treatments to slow aging—if we can interfere with glycogen synthesis, could we stop the accumulation of other damage-causing proteins and reduce the detrimental effects on the brain?

To answer that question, we’d need to first confirm that the PGBs are actually involved in the decline of neuronal function during aging. This leads me to the authors’ next set of experiments, for which they turned to Drosophila melanogaster. Because fruit flies have shorter lifespans, it’s easier to study how PGBs affect them over their entire lives. In addition, fly researchers have developed a vast array of genetic tools for their animal model, which the authors used to knock-out the glycogen synthase gene again, but this time only in the brain and only during adulthood. This allowed the researchers to study how PGB formation affects brain function without altering the development and general health of the fly (a very difficult feat in mice).

As expected, the authors found that the mutant flies had reduced levels of glycogen in the brain. But remarkably, they also found that the mutants lived significantly longer than the normal flies. And this was quality life—the aged mutant flies could climb better and faster than normal flies of the same age. Based on these results, the authors concluded that glycogen synthesis impairs neuronal function and survival with age.

The findings from this paper is a step in the right direction for figuring out why cognitive decline is associated with aging. Before this can be useful for humans, however, there are a lot of questions to answer. How and why does glycogen synthesis cause PGB formation as we age? Will interfering with this process in adulthood extend quality life and cognitive function (and can it be done safely)?  Finally, the finding that glycogen synthesis may be required for other protein deposits was completely unexpected. Future work in this field may therefore provide insights into neurodegenerative diseases associated with protein aggregation, such as Alzheimer’s disease (beta-amyloid), Parkinson’s disease (alpha-synuclein), and Lafora’s disease (glycogen!).
 
 
For more research on aging, click the “aging” tag in the right column.

For another post about how to reduce aging-related protein aggregation, check out this post about the possible use of lithium.

 

 
Reference:

  • Sinadinos C., Laura Boulan, Estel Solsona, Maria F. Tevy, Mercedes Marquez, Jordi Duran, Carmen Lopez-Iglesias, Joaquim Calbó, Ester Blasco, Marti Pumarola, Marco Milán & Joan J. Guinovart (2014). Neuronal glycogen synthesis contributes to physiological aging, Aging Cell, n/a-n/a. DOI: http://dx.doi.org/10.1111/acel.12254

General References:

Thanksgiving Special: Uncovering the link between sleep and food

November 28, 2014 / / 0 Comments
rest and digest turkey

If you ate a big Thanksgiving dinner yesterday, you probably felt drowsy and sluggish afterward, a phenomenon often referred to as a “food coma”. The belief that it’s caused by the tryptophan in turkey is a long busted myth, and in fact it can happen after any carb-heavy meal. The reasons for this post-food slump are relatively well understood from experiments in humans and other mammals. For one, big meals trigger a “rest and digest” response when the food reaches the stomach and small intestine (via activation of the parasympathetic nervous system). This diverts energy to digestion, making you feel sluggish and sleepy.  But wait, there’s more! Eating a meal full of carbs and other sugars also stimulates the release of insulin, which triggers a process to take up nutrients from the bloodstream. But a side effect of this process is an increase in the amount of serotonin and melatonin in the brain, two chemicals that are associated with drowsiness (and often happiness).

So having a full stomach makes you sleepy, but did you know that the relationship between sleep and food goes deeper than that? For example, studies in animals from fruit flies all the way to humans have shown that having an empty stomach can keep you awake. Even worse, chronic sleep deprivation stimulates appetite and can lead to weight gain. Studies in humans have uncovered strong correlations between sleep disorders like insomnia and obesity-related disorders such as diabetes and cardiovascular disease. One of the reasons for this is that sleep loss wreaks havoc on the levels of certain hormones, such as the “hunger hormone” ghrelin.

Thus, even though sleeping and eating are mutually exclusive behaviors (you can’t sleep while you’re eating), they’re also obviously connected. Both are essential for survival, so the brain may often be promoting at least one of them. But how does the brain decide which behavior is more important at any given time? When you’re hungry, it seems more important to stay awake and find food to prevent starvation, but sleep deprivation also has health consequences, so the brain needs to ensure you get enough sleep.

So how are sleep and hunger connected in the brain? Are they independent, so that hunger suppresses sleep, and sleepiness stimulates hunger? Or maybe they arise from the same mechanism, which signals in turn for either sleep or eating depending on your body’s needs. Answering this question will improve our understanding of sleep- and obesity-related disorders and how they’re linked, a necessary step before treatments can be developed.

Fruit flies make a great animal model for answering detailed questions like these, which take place on a molecular and cellular scale. Although much research has yet to be done, there have already been some informative findings. For starters, scientists have found that insulin-producing cells in the fly brain regulate both feeding and sleep, providing a cellular link between these two behaviors (remember that insulin’s activities are partially responsible for the food coma after Thanksgiving dinner). Their findings suggest that these cells integrate information from other brain regions about sleep need and hunger, and may even potentially act as a “behavioral switch” to signal which behavior is more important at any given time.

Other fruit fly researchers have found a link between eating and sleeping in a single molecule called “NPY-like short neuropeptide F” (sNPF). sNPF had already been shown to regulate food intake, but recently it was found that sNPF also promotes sleep. sNPF is similar to a mammalian chemical called “neuropeptide Y” (NPY), which also plays a little-understood role in sleep and eating in mammals. NPY has recently been studied as a possible drug target for obesity treatment in humans even though its role in other behaviors is unclear, so a better understanding of this chemical is essential to account for possible side effects such as sleep disruption.

Research in a variety of animal models (and humans) has shown us that sleep and hunger are related. Overall, the combined results of fruit fly research suggests that sleep and feeding behaviors may arise from shared mechanisms in the brain. Future studies will pin down the exact mechanisms and apply those findings to more complex mammalian systems. Hopefully, this research will lead to the development of drugs that can help with sleep- and obesity-related disorders. In the meantime, I think I’ll try drinking coffee with Thanksgiving dinner.

Happy Thanksgiving!

References:

Fly Life: Fruit flies at SFN 2014

November 21, 2014 / / 0 Comments

Every year, over 30,000 neuroscientists gather at the Society for Neuroscience annual meeting to present their latest research and catch up with long-lost colleagues. This year’s meeting took place November 15-19th at the Walter E. Washington Convention Center in Washington DC. The meeting is often dominated by researchers working with mammalian models (we Drosophila researchers often attend conferences specific to our model instead), but invertebrate scientists still make a good showing every year. And this year’s meeting was a great one for fruit flies!

This year, two of the Special Lectures were dedicated to fruit fly research. First, Dr. Amita Seghal presented her lab’s latest sleep research showing that young flies need more sleep for normal brain development (I reviewed their latest paper). This finding highlights how important it can be for children of all species (including humans) to get enough sleep. Later in the week, Dr. Vivian Budnik presented her lab’s discoveries on the mechanisms neurons use to form stable connections (called synapses) and talked about how problems in these processes in humans can lead to disease.

Fruit fly researchers also presented over 100 posters and gave several short talks on a range of topics, from basic molecular and cellular research to complex behavioral studies, such as how sleep and memory can arise from a network of cells. Although it can sometimes be hard to imagine how fruit fly research could be relevant to humans, research in this animal model often has important implications for human diseases. In particular, fly researchers presented in several sessions related to Alzheimer’s disease this year, and they also showed up in sessions for other human diseases such as schizophrenia, Parkinson’s disease, and autism. In fact, a fly poster about a possible new treatment for a genetic form of autism was highlighted in a SFARI post by blogger Virginia Hughes (also an official blogger for SFN 2014).

Did you see (or present) any fascinating or surprising fruit fly research at SFN this year?  Leave a comment below!

Breaking Research: Lithium may protect against Alzheimer’s and other aging-related diseases

November 14, 2014 / / 0 Comments

As human life expectancy continues to increase at a steady rate in most countries worldwide, the prevalence of aging-related diseases is also increasing. One such example is Alzheimer’s disease (AD), the most common cause of dementia in the aging population. There is currently no cure for AD, and the only treatments that exist temporarily cover up the symptoms without actually slowing the disease itself.

Alzheimer’s disease can be identified by the abnormal accumulation of a protein called amyloid beta (Aβ) in the brain. Aβ is a byproduct of an important cellular process, and is usually cleared away by the cell’s garbage recycling processes. It’s normal for some leftovers to be missed, however, and over time Aβ builds up as we age. But in large enough concentrations—such as in older patients with AD—accumulation leads to the formation of aggregated deposits of Aβ. These deposits damage neurons and cause neurodegeneration (progressive neuron death).

healthy vs Alzheimer's neuronsThe left side shows healthy neurons. The right side shows damaged neurons and Aβ deposits as seen in Alzheimer’s disease patients. Image modified from alz.org

Eventually, this widespread brain tissue damage leads to imbalances in the levels of neurotransmitters, which are chemicals that neurons use to communicate. This imbalance is thought to cause the symptoms of AD, including problems with memory, thinking, or changes in behavior. Current treatments simply alter the amount of these neurotransmitters without doing anything to slow or prevent Aβ aggregation and neuron death. Therefore, research aimed at developing a drug that can interfere with Aβ accumulation is important for treating this disease.

Recent findings in lithium research holds hope for such a treatment. Although lithium is most widely used as a treatment for bipolar disorder, some preliminary research suggests that it might also be able to slow or prevent the symptoms of Alzheimer’s disease in humans if prescribed early enough. However, the results have been mixed and even sometimes contradictory. This is largely due to the fact that lithium’s actions in the brain are not understood, so figuring out how lithium might be helping AD patients is essential before it could be considered as a treatment. A recent paper published in Frontiers of Aging Neuroscience by the Partridge lab has begun to do just that by studying how lithium can reduce Aβ accumulation in a fruit fly model of Alzheimer’s disease.

The authors created a model for AD by introducing a mutated form of human Aβ protein known to cause AD in some families (called Arctic Aβ42) into adult fruit flies. Flies with the Arctic Aβ42 mutation displayed progressive neuron dysfunction and shortened lifespan, which mimicked the symptoms of AD in human patients. They had previously shown that lithium treatment was able to reduce the amount of Aβ (and thus also its toxic effects on neurons) in these flies. But how did it work?  In this study, they treated these mutant flies with lithium again to answer this question.

What they found was surprising. Lithium didn’t just reduce the amount of Aβ protein in the brain, it reduced the amount of all proteins.  It worked by suppressing the activity of the “translation machinery”, which refers to the system that actually assembles and produces proteins from the instructions in the genetic code. So lithium actually reduced the production of all proteins through a mechanism that wasn’t specific to Aβ.

What does this mean for the possibility of using lithium to treat Alzheimer’s disease? The fact that lithium’s effects are more general is actually pretty good news not just for AD, but for all research into the aging process. It is currently thought that normal aging is caused by the accumulation of damaged or mutated proteins that haven’t been cleaned up, and research aimed at increasing lifespan has focused on either improving the cell’s recycling processes or reducing protein production. In fact, lithium treatment has been shown to increase life expectancy in animal models, including fruit flies (and possibly even in humans!).

So if lithium can increase lifespan in animals without Alzheimer’s disease, can it reverse the lifespan reduction in the AD model flies? Yes! The authors found that lithium treatment also improved the life expectancy of their mutant flies compared to ones that did not get a lithium treatment. This result provides further hope that lithium could one day be used to actually slow the progression of AD and give patients more years of quality life.

The findings in this paper are not just promising for Alzheimer’s disease, but also for other aging-related diseases caused by abnormal accumulation of protein in the brain, such as Parkinson’s disease and Huntington’s disease. Lithium also has the advantage of already being an approved drug for treating patients with bipolar disease, so some information on side-effects and dosages already exists. Of course, this doesn’t mean doctors should begin prescribing lithium for AD patients right away; the dosage requirements will likely be different and older adults may experience other side-effects. But research in this field has definitely leapt forward, and we may see a cure for aging-related diseases in our (extended?) lifetime.

Reference:

  • Sofola-Adesakin O., Jorge I. Castillo-Quan, Charalampos Rallis, Luke S. Tain, Ivana Bjedov, Iain Rogers, Li Li, Pedro Martinez, Mobina Khericha, Melissa Cabecinha, Jürg Bähler & Linda Partridge(2014). Lithium suppresses Aβ pathology by inhibiting translation in an adult Drosophila model of Alzheimer’s disease, Frontiers in Aging Neuroscience, 6 DOI: http://dx.doi.org/10.3389/fnagi.2014.00190

General References:

Breaking Research: A method by which invading bacteria avoid detection could also be our key to defeating them

November 7, 2014 / / 0 Comments

Have you ever wondered how our body recognizes when it’s being invaded by harmful bacteria? Nearly all forms of life—from single-celled organisms all the way to humans—have an “innate” immune system, which has evolved to recognize cellular components shared by broad groups of pathogens. One such example is peptidoglycan, a molecule found on the cell walls of virtually all bacteria. Peptidoglycan forms a sort of “load-bearing mesh” required for the bacteria to maintain their shape and is therefore an essential part of their structure. As a result, our immune systems have evolved to recognize peptidoglycan as a danger signal and will trigger an immune response when it is detected.

But just as our innate immune system has evolved to recognize invaders bearing peptidoglycan, bacteria have also evolved to escape detection. In a recent paper published in eLife by the Filipe lab, researchers used fruit flies to study how some bacteria can remain undetected by its host. Understanding how bacteria avoid detection will help us develop new ways to defeat them by preventing them from evading our immune system. This could reduce the need for antibiotics, which is particularly important now that antibiotic-resistant bacteria are becoming painfully common (in 2014, the World Health Organization declared antibiotic resistance a major threat to public health).

Gram-negative and -positive bacteriaThe difference between Gram-negative and Gram-positive bacteria. Image source

So how do bacteria with peptidoglycan conceal themselves from our immune system? Previous work has categorized bacteria into two groups based on the way they cover up their peptidoglycan mesh: Gram-negative bacteria, which have a full membrane surrounding the peptidoglycan molecules, and Gram-positive bacteria, which have the mesh directly outside the cell wall. Instead of a full membrane, Gram-positive bacteria have layers of molecules that stick out of the cell wall and block access to the peptidoglycan. Because of this, it has long been thought that the immune system can only detect peptidoglycan when fragments have been snipped off of the bacterial walls (when bacteria need to grow and divide, they must break down their mesh and then rebuild it).

But now, researchers have uncovered a new twist to this story. Recent findings have suggested that under certain conditions, the immune system can actually recognize peptidoglycan while it’s still a part of the bacterial cell wall. So the authors of this paper asked: Have bacteria evolved other methods to prevent this from happening?

To answer this question, they infected fruit flies with Staphylococcus aureus (S. aureus), a Gram-positive strain of bacteria related to MRSA. They observed how well the fruit fly hosts were able to defend against the invaders, and then mutated parts of the bacteria to determine how each manipulation affected the hosts’ ability to survive. The authors learned that S. aureus releases a molecule called Atl, which they found was responsible for trimming off pieces of peptidoglycan that stick up above the bacteria’s protective layer. When the bacteria couldn’t release Atl, the flies were much more likely to survive the infection because their immune system could more easily recognize the intruders and fight them off.

How can this help us humans?  The mammalian innate immune system is similar to that of flies, and also recognizes peptidoglycan as a trigger for activating an immune response. Thus, if bacteria that infect humans use the same evasive maneuvers, it could be possible to develop a drug that targets and disables Atl and other peptidoglycan-snipping molecules. This would allow our immune systems to better recognize and fight back against bacterial infections and reduce the need for antibiotics.

The authors have already done some of the work toward this goal. To find out if strains of bacteria known to endanger humans use the same avoidance mechanism, they also infected flies with MRSA, a dangerous antibiotic-resistant strain of bacteria often found in hospitals, and Streptococcus pneumoniae, which is a frequent cause of pneumonia in developed countries and a major cause of infant mortality in developing countries. That found that both of these bacteria use Atl to shave their surface and avoid recognition by the immune system. Future research may therefore lead to treatments that prevent these bacteria from going into hiding, allowing our immune system to hunt them down and do its job with ease.

Reference:

  • Atilano M.L., Filipa Vaz, Maria João Catalão, Patricia Reed, Inês Ramos Grilo, Rita Gonçalves Sobral, Petros Ligoxygakis, Mariana Gomes Pinho & Sérgio Raposo Filipe (2014). Bacterial autolysins trim cell surface peptidoglycan to prevent detection by the Drosophila innate immune system, eLife, 3 DOI: http://dx.doi.org/10.7554/elife.02277

Halloween Special: The Drosophila Halloween Genes

October 31, 2014 / / 0 Comments

In the movies, spooks and phantoms are often undead humans with unfinished business. But would you be afraid of a ghostly fruit fly?

In 1995, fruit fly researchers Christiane Nüsslein-Volhard and Eric Wieschaus were awarded a Nobel Prize for their research on development. They were interested in understanding how a fertilized egg develops into a complex organism, and were the first to show that development was controlled by genes. In their famous paper published in 1980, they found a small number of genes that were important in determining the body plan and formation of body segments in fruit fly larva.

Four years later, the same researchers published a set of papers on a group of genes that caused developmental defects in fruit fly embryos. When a gene in this group was mutated, the embryos died before the exoskeleton was created. The mutations somehow disrupted the formation of the embryonic cuticle, the protective outer layer that should form around the embryo. The researchers (not without a sense of humor after long grueling hours in the lab) dubbed them the Halloween genes. The genes earned their name not just because they mutated and killed, but because the mutant embryos took on a ghostly appearance. This resulted in gene names such as disembodied, spook, spookier, shadow, shade, shroud, and phantom.

Phenotypes of two Halloween gene mutationsImages of the cuticles of a normal fruit fly embryo and two with mutations in a Halloween gene. Modified from Gilbert, 2004

So what do the Halloween genes do? Since the 1980s, researchers have discovered that all of the Halloween genes are cytochrome P450 (CYP) enzymes involved in synthesizing a steroid hormone called 20-hydroxyecdysone (20E) from cholesterol. 20E is required for metamorphosis and moulting in arthropods such as insects and crabs. As a result, disruption of 20E synthesis in fruit flies blocked formation of the exoskeleton in embryos. Hmm… I had to look all that up.

20E pathway involving Halloween genesThe Halloween genes encode enzymes that convert cholesterol into the arthropod steroid hormone 20E. Modified from Gilbert, 2008

CYP enzymes are found in most species and are involved in a very large variety of processes. In humans, they are involved in regulating hormones (among other things), including steroid hormones (just like in flies!). Steroid hormones are basically a group of steroids that act as hormones in the body, and they are synthesized from cholesterol (just like in flies!). Interestingly, testosterone and anabolic steroids, such as the ones that athletes may take, are actually steroid hormones. Thus, although mammals do not have 20E, they have other steroid hormones are important for development as well as reproduction, metabolism, and homeostasis, which allows cells to adapt to their changing environment. Because of these similarities, research in the Halloween genes may help us better understand how steroid hormones are synthesized in mammals.

Happy Halloween!

General References:

 

Breaking Research: WIDE AWAKE is a newly identified gene that explains how we become sleepy at night

October 24, 2014 / / 0 Comments

The body’s biological clock is responsible for keeping track of time and synchronizing behavior with the environment, so that you feel alert during daylight hours and sleepy at night. This biological clock (also called the circadian clock or circadian rhythms) consists of three major parts:

  1. The central pacemaker, which oscillates with a period of about 24 hours to keep time
  2. Inputs to the clock, such as light or temperature, that synchronize it with the environment
  3. Outputs from the clock, which signal to your body to indicate when it’s time to sleep, eat, etc

Researchers have made great progress in understanding how the central pacemaker works and how environmental cues can control the clock, but how does the clock signal to the rest of the body to control rhythmic behaviors? A recent fruit fly paper published in Neuron by the Koh and Wu labs investigated this question and discovered a new gene involved in signaling that it’s time to sleep.

How does our body know when it’s time to feel sleepy or alert? The biological clock must have an output pathway for controlling sleep, but the mechanism of exactly how that happens is poorly understood. After analyzing the sleep patterns of thousands of fruit fly colonies with different genetic mutations, the authors of this paper found a group of flies that had trouble falling asleep at night. They called the mutated gene Wide Awake (wake for short) and discovered that while the gene wasn’t necessary for accurate functioning of the circadian clock itself, it was important for signaling a transition from wake to sleep at the end of the day.

The role of WAKE in LNvsDuring the day, GABA signaling from sleep circuits in the fly brain has little effect on LNvs (the wake-promoting clock cells). At dusk, increased amounts of WAKE proteins in clock cells leads to an increase in the number of GABA receptors on the membrane, thus increasing the cells’ sensitivity to GABA. This dampens LNvs activity, allowing flies to fall asleep at night.

So how does this gene translate clock information into a feeling of sleepiness? The authors looked to “clock cells” to answer this question. Clock cells are a group of cells that make up the central pacemaker. In mammals, this pacemaker is located in the suprachiasmatic nucleus (SCN). In flies, the central pacemaker includes a small group of neurons called LNvs, which promote wakefulness. During the day, activity in the LNvs causes flies to be awake and active. But at dusk, the amount of WAKE protein from the wake gene in these cells increases. WAKE makes the cells more susceptible to inhibition from a chemical messenger called GABA, which dampens activity. As the wake-inducing LNvs become more sensitive to GABA, they become less active, which allows the sleep circuits in the brain to exert more control over behavior so the flies can fall asleep. On the other hands, flies with a mutation in the wake gene were not getting enough GABA signal to quiet the LNvs at night, keeping them agitated and awake.

The circadian clock in humans

The authors identified a gene that seems to be a messenger from the circadian clock to the brain, telling it that it’s time to shut down and go to sleep at night. But how can these fruit fly findings help us humans? They also found the same gene in the SCN in mammals, suggesting that it plays the same role in other species as well. The finding that wake mutants had difficulty falling asleep after lights-off is reminiscent of sleep-onset insomnia in humans. Currently, the most common treatment for insomnia are drugs the mimic the effects of GABA in the brain. However, these findings suggest that the effectiveness of this treatment is limited if wake-promoting neurons are not sensitive enough to GABA signaling. Thus, future research in this area may focus on manipulating the wake gene and its proteins to develop a better treatment for sleep disorders such as insomnia in humans.

For more information on circadian rhythm research in fruit flies, check out the related Translational Findings post.

Reference:

  • Liu S., Qili Liu, Masashi Tabuchi, Yong Yang, Melissa Fowler, Rajnish Bharadwaj, Julia Zhang, Joseph Bedont, Seth Blackshaw & Thomas E. Lloyd & (2014). WIDE AWAKE Mediates the Circadian Timing of Sleep Onset, Neuron, 82 (1) 151-166. DOI: http://dx.doi.org/10.1016/j.neuron.2014.01.040

Translational Findings: Fruit fly contributions to research in circadian rhythms

October 17, 2014 / / 0 Comments

What are circadian rhythms, and why are they important to humans?

Over the past century, technological innovations have changed human society dramatically, undeniably for the better. But the advent of jet travel, round-the-clock manufacturing, and internet communication has also had a disruptive effect on our bodies’ circadian rhythms.

The word “circadian” comes from the latin words circa and diem, meaning “about a day”. Circadian rhythms—which are also referred to as our biological or internal clock—keep time at about 24 hours, and are found in most organisms including cyanobacteria, fruit flies, mice, and humans. In the wild, these biological pacemakers allow organisms to predict rhythmic changes in their environment, such as the day-night cycle of light and temperature variations. The clock helps animals to know when to sleep, eat, and when predators might be prowling.

Although the internal clock can remain rhythmic on its own, it regularly synchronizes with environmental changes such as light and temperature. This is important as light and temperature rhythms gradually fluctuate by the season, but sudden unexpected environmental cues can wreak havoc on our bodies until the clock can adapt and reset itself. The role of circadian rhythms in our lives can be best understood by seeing what happens when our clocks become desynchronized from the environment.

Effects of jet lag on baseball

“Jet lag” is the first thing people usually think of for disruptions in circadian rhythms. When we travel through time zones, our body “remembers” the previous time zone, including the sleep-wake pattern, mental alertness and eating habits associated with it. For example, if you take a red-eye flight from California to New York, when you arrive your internal clock might tell you that it’s 3am, but outside, it’s time for breakfast. It can take days to adjust to the sudden shift in environmental cues in the new time zone, and during that time you feel tired and may find it “hard to think”.

In our ever increasing 24-hour society, disruption of normal circadian rhythms is commonplace. It can be caused by working long hours or with international partners, irregular shift-work, or even “social jet lag”, which is caused by having a different sleep schedule on the weekends than during the week. In addition, recent research suggests that teenagers experience a natural forward shift in their biological clock, causing them to become more alert later at night. During this period of life, getting up early every morning for school can be very disruptive.

When the circadian clock desynchronizes from the environment, it can have negative consequences to cognitive function and health. In the short term, a person can experience difficulties with learning and memory, mood disorders, immune system dysfunction, and generally reduced quality of life and well-being. Chronic disruption can lead to health problems such as obesity and diabetes, increased risk of cancer, heart disease, depression, and other conditions. To make matters worse, there are numerous sleep disorders that lead to loss of restful sleep. Could some of these be the result of dysfunction in the biological clock?

Circadian rhythm research is important for understanding the consequences of clock disruption and how it can be treated. There are several questions that need to be answered: How does the biological clock keep time? How does it synchronize with the environment and adjust to new time zones? Why do some individuals seem to have abnormal sleep-wake cycles, and how can these disorders be fixed?

How have fruit flies helped us understand circadian rhythms to answer these questions?

Although the existence of a circadian clock had been agreed upon for decades, until the 1970s no one knew anything about how it worked. Circadian rhythms are a complex behavior, and how do you even begin to piece together such a difficult puzzle? In 1971, a group of fruit fly researchers led by Dr. Seymour Benzer published a paper describing a gene named Period. They found three different mutations in the Period gene that caused flies to have faster, slower, or a complete absence of circadian rhythms. While it is now an accepted fact that all behaviors have a genetic component, at the time many were skeptical that genes could be involved in complex behaviors. Certainly they couldn’t believe that mutating a single gene would have an effect on something as complex as circadian rhythms.

Now more than 40 years later, research in circadian rhythms has made it the best understood complex behavior at the genetic and molecular level. Many of the pioneering experiments on circadian rhythms were performed in fruit flies because they are such an easy-to-use genetic animal model, and the genes and processes involved in mammalian circadian rhythms were often first identified in fruit flies. Once researchers understood the basic components of the clock in flies, they were able to apply that knowledge to the more complex mammalian systems in later studies. The core mechanism for the biological clock is shared between flies and mammals, which spans over 600 million years of evolutionary time!

How does the biological clock keep time?

Double loop circadian clockClick on the picture for a bigger version. Circadian rhythms in both flies and mice are controlled by two negative transcriptional feedback loops. Although there are various differences between the species, the structure is similar. Modified from Hardin, 2000

Since the 1970s, dozens more clock genes have been identified including Timeless, Clock, and Cycle. Researchers have learned that the clock keeps time via daily fluctuations of clock-related proteins which interact in what is called a “negative transcriptional feedback loop”. There are two such loops: the Period/Timeless (Per/Tim) loop and the Clock/Cycle (Clk/Cyc) loop. In the Clk/Cyc loop, levels of both Clock protein and Cycle protein are high during the day, and then decrease after dark. When there is enough of each protein in the cell, Clock and Cycle proteins bind together and attach to strands of DNA to activate the Period and Timeless genes. This allows the cell to create Period and Timeless proteins from the DNA’s instructions. Thus, over the course of the day, the cell starts to make plenty of Period and Timeless proteins, but light-activated mechanisms (involving a gene named Doubletime) in the cell break them down. When night falls, however, levels of these proteins can begin to accumulate. The Per/Tim loop is therefore the reverse of the Clk/Cyc loop in that there are low levels of proteins during the day which increase after dark. When the levels of each protein are high enough, Period and Timeless bind together and inhibit Clock and Cycle, effectively deactivating the Period and Timeless genes so that no new protein is being made. As the next day begins and light begins breaking down the Period and Timeless proteins again, the cycle restarts. This is a simplified explanation of the system, and there are many more genes involved (especially in the mammalian version), but this two-loop clock is found in flies, mammals, and other species, suggesting that it is the optimal system for timekeeping.

For a great visualization of how the molecular clock works, check out the graphic on this website under “How do clock genes work”. This page also gives more information about the mammalian circadian clock if you’re interested.

More recent research has also begun to focus on how networks of cells work together to keep each other synchronized. Although each cell has its own running internal clock, it can become desynchronized by error or damage. Researchers have found that clock cells communicate with each other to maintain an overall synchronized circadian rhythm.

How does the clock synchronize with the environment and adjust to new time zones?

Although circadian rhythms continue even in the absence of environmental stimuli (such as in complete darkness and stable temperature), the clock can also synchronize with the environment based on cues such as light, temperature, or other rhythmic stimuli. The actual circadian period is slightly longer than 24 hours, but the environmental length of day can vary based on location and/or season. Researchers have found that the clock can be reset by unexpected light pulses or fluctuations in temperature, but how do external cues reset the clock? While not much is known yet about the effects of most cues, scientists have made extensive progress in understanding how the clock synchronizes to light.

Research implications for individuals with total blindness

In fruit flies, light entering the brain through the fly head and eyes can break down the Timeless protein, so levels of this protein can’t start to increase until after dark. This contributes to the normal cycling behavior. But when an unexpected light pulse occurs, such as too early in the morning or too late at night, degradation of Timeless causes the brain to think that the next “day” cycle has begun and resets the circadian clock. There are other genes involved in this process as well, such as Crytochrome and the aptly-named Jetlag. In 2006, it was found that a mutation in the Jetlag gene causes defects in flies’ ability to adapt to a new light-dark cycle.  A shared gene in humans might be responsible for differences in individuals’ ability to adapt after flying to a new time zone.

The future of circadian research: Why do some individuals have abnormal sleep-wake cycles, and how can circadian disorders be fixed?

By increasing our understanding of how the circadian clock ticks away the time and responds to environmental cues, we may be able to design drugs to treat jet lag, reset circadian rhythms, or even reverse the consequences of a desynchronized clock.

Research may even help scientists better understand human sleep disorders such as narcolepsy or insomnia and identify underlying circadian components. For example, humans with a sleep disorder called Familial advanced sleep phase syndrome (FASPS) have been described as having a shifted circadian rhythm. Though they have a normal sleep structure, everything is pushed forward by about 4-6 hours. As a result, individuals tend to wake up well before sunrise (around 1-3am) and feel compelled to sleep around 6-8pm. A similar shifted sleep schedule has been reported in Doubletime fruit fly mutants and tau mutants in hamsters. Eventually, researchers found that FASPS is caused by mutations in similar clock genes in humans called PER2 and CSNK1D. A better understanding of the role these genes play in circadian rhythms could help scientists develop medicines to treat this disorder.

 

Interested in recent circadian rhythm research in fruit flies? Check out the Breaking Research post on WIDE AWAKE, a gene that explains why we feel sleepy at night.

 

General References:

 

« Older posts Newer posts »

Pages

Archives

Fly on the Wall

Written by Bethany Christmann

© 2023 Fly on the Wall

Theme by Anders Noren — Up ↑

Protected by Akismet
Blog with WordPress

Welcome Guest | Login (Brandeis Members Only)