Green fluorescent protein (GFP) is expressed in motor neurons in the Drosophila melanogaster larval ventral ganglion. The larva is superimposed on an image of a starry night sky with the North star aligned at the top of the “tree”. The North star stays fixed in the night sky at this time of year, which inspired the tradition of stars on top of evergreen Christmas trees.
Image created by Dr. James Hodge
Image source Griffith lab
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…
Image modified from Colwell, 2007And 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?
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.
Click here for a larger version of the pictureTo 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).
Sleepless mutants sleep much less than normal (control) flies. Image modified from Koh et al, 2008This 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?
A 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
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.
A schematic of the Tracker setup. Image modified from Gilestro, 2012Although 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.
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The 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:
Top: 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.
Three 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.
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Total 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 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?
Top: 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.
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