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Category: Translational Findings

Translational Findings: How fruit flies are helping us find cures for cancer

General Cancer Hope Ribbon

At universities and companies around the world, scientists are studying the mechanisms of cancer and tumors using fruit flies. They hope to identify failures in the genes that lead to cancer, and develop treatments to prevent or reverse these problems. Because approximately 60% of the genes associated with human cancers are shared with fruit flies, these interventions will lead to a better understanding of cancer in humans, a critical step for the development of cures. Fortunately, fruit fly research is already making huge contributions to the field of cancer research and has produced several success stories (with more on the horizon).

What is cancer, and how can flies help?

Cancer is an encompassing term used to refer to a collection of more than 100 related diseases, and is the second leading cause of death in the United States. Although there are many kinds of cancers, all are caused by the uncontrolled growth of abnormal cells. But what causes these abnormal cells to grow out of control?

Usually, when a cell’s DNA is damaged, the cell either repairs itself or dies. But a cell becomes cancerous if it doesn’t (or can’t) repair the damage and then doesn’t die like it should. Instead, it replicates itself unchecked, forming a mass of cancerous cells called a tumor. These tumors continue to grow and invade nearby normal tissue, and can sometimes even “break off” into the bloodstream and circulate into other tissues as well.

Ideally, if researchers can identify the mutation that leads to cancer, they can use it to develop a cure. For example, they can create a drug that recognizes the specific mutation to target and kill cancerous cells, or they can develop a treatment to at least compensate for the mutation. Unfortunately, there are two main reasons why one approach doesn’t work for all cancers. First, there are different types of cancers (such as colon, liver, or lung cancer) which respond to drugs differently, so a different cure would need to be developed for each type. Second, the same type of cancer in one person can often be caused by a different mutation in another. In fact, one cancer may often be caused by mutations in multiple genes, so a drug for a single type of cancer may need to have multiple targets. This variation between and within cancer types means that there can’t be one universal cure.

So how can flies help? There are two very promising avenues for researching cures for cancer.

  1. Identifying “cancer-risk” genes – Although different cancers arise from different and often multiple mutations, scientists have found that certain mutations commonly occur in many types of cancer. For example, some of these cancer-risk genes may be categorized as “repair” genes, which are involved in fixing damaged DNA. When a repair gene is damaged, mutations in other genes are allowed to occur, increasing the chances of cancer. Other categories of cancer-risk genes include “growth” and “cell death” genes which, when mutated, allow or cause the cell to replicate itself without dying. Many of these and other cancer-risk genes were first identified in fruit flies, and further research will help us gain a better understanding of how mutations can lead to cancer and how to prevent that from happening.
  1. Personalized medicine – Many of the drugs we currently have are only effective against specific kinds of cancer, and even then with only moderate success. Doctors must often try multiple courses of treatment before finding one that can effectively target a patient’s cancer. This is due to the fact that the combination of mutations varies between patients. Personalized medicine, in which treatment is tailored for each patient, is therefore becoming more common. After identifying the specific genetic mutations causing cancer in a patient, doctors can prescribe the right combination of drugs to target their cancerous cells. Unfortunately, a lot more work needs to be done to identify every mutation and the appropriate drug to target it (see #1). One group of researchers led by Ross Cagan is attempting to bypass this step with relative success, by growing a copy of each patient’s tumor in flies and testing thousands of drug combinations until one works without killing the fly.
fly cancerA comparison of eyes from a normal fly (A) versus flies with visible signs of cancer (B-D). Image modified from Uhlirova et al., 2005.

Do flies get cancer? Flies generally don’t live long enough to naturally develop sufficient DNA damage to cause cancer, but flies with genetic mutations like those in humans will quickly develop cancer, and we can easily see abnormalities in the eye (see figure). Researchers can use these flies to investigate how mutations in specific genes led to cancer and test thousands of drug candidates to see if the tumor goes away. Even better, they can introduce the specific combination of human mutations from patients in the flies and then test drugs against it, as described in #2.

Here, I’ll go into detail about the accomplishments of fruit fly research so far, and where they might lead in the future.

Identifying cancer-risk genes and understanding how mutations lead to cancer

Dozens of genes that contribute to cancer have been identified in fruit flies. When a mutation occurs in any one of these genes, it can lead to the hallmarks of cancer, such unchecked cell growth and the formation of tumors. For example, a mutation in a gene known as scribbled (scrib) can lead to masses of disorganized cells similar to tumors.  As mentioned previously, identifying these genes and understanding the role they play in cancer is critical for developing drugs to work against them.

But one of the most important insights we’ve gained from cancer research in flies is how multiple mutations in different genes often cooperate with each other, leading to the invasive properties of cancer that are so deadly in humans. For example, a combination of a scrib mutation with a Notch mutation allows the disorganized cells to invade other tissues and spread to other parts of the fly’s body. Research in flies therefore helps us understand which of the sometimes dozens of combined mutations in patients is “driving” the cancer (so to speak), so doctors can target the most aggressive genes.

The Notch gene is part of a group of genes that make up the Notch signaling pathway, which refers to a group of genes required to do a specific job. The Notch signaling pathway is involved in detecting communications from other cells and acting upon them by controlling each cell’s replication or death. Mutations in the genes in this pathway, therefore, can lead to overactive signaling, allowing for uncontrolled growth and subsequently cancer. In humans, Notch mutations contribute to several types of human cancer, including breast, prostate, and pancreas cancers, as well as cancers of the blood (leukemias). Fortunately, identifying the genes involved in the Notch pathway has led to the development of several promising drug candidates that suppress mutated genes in this pathway, the most studied of which is known as gamma-secretase inhibitors (GSIs). Although more work is needed to confirm the effectiveness and safety of this and other drugs against Notch-based cancers in humans, it is a promising step in the right direction.

Perhaps one of the greatest contributions the fly has made to cancer research was the discovery of the Ras signaling pathway, which is also involved in regulating cell growth. Each of the genes involved in this pathway have also been found in mammals, and the Ras gene itself is estimated to play a role in 30-50% of human cancers. Continued research in this pathway is critical for developing drugs to target mutations in these genes, as these drugs may have broader success against human cancers due to the pervasiveness of this pathway in cancer.

Once cancer-risk genes and pathways have been identified, drug candidates can be tested against them. Thousands of drugs can be screened against cancerous cells in petri dishes, but once drugs that successfully target mutations in cancer-risk genes have been found, they need to be tested in live animals for effectiveness and lethality. Fruit flies, with their short lifespans and massive numbers of offspring, are a great resource as a first step for rapidly testing promising drug candidates.

Personalized medicine: Flies with human tumors 

Thanks to improvements in the speed and cost of genome sequencing, doctors can now take a “genetic snapshot” of a person’s cancerous tumors to find out which genes have mutated. Dr. Ross Cagan and his team at the Center for Personalized Cancer Therapeutics at Mt. Sinai Hospital in New York City are using this approach to identify the mutations causing cancer in individual patients, and then introduce that combination of mutations in fruit flies. Essentially, these scientists are growing personalized tumors in fruit flies, which can be seen in the flies’ eyes (see figure above).

Using those flies, the researchers can test thousands of drug combinations until they find a cocktail that works. Usually, drugs approved for certain types of cancer are hit-or-miss—what works for one patient may not work for others. To make matters worse, cancer drugs tend to damage healthy cells at the same time, albeit less so than the cancerous ones. But using this new personalized technique, Dr. Cagan and crew can figure out what combination of drug works best to kill the tumor without killing the flies.

Besides helping cure cancer in some individual patients, this technique has also inspired the development of a new drug called vandetanib. Dr. Cagan and his colleagues found that this drug (originally known by the catchy name of ZD6474) seemed to work against certain types of cancerous growths, and eventually the drug was picked up by AstraZeneca and approved for the treatment of advanced thyroid cancer.

The future of fruit fly cancer research 

The technique pioneered by Dr. Cagan and his colleagues will also improve researchers’ ability to identify new cancer-risk genes and understand how they lead to cancer. As these scientists uncover more mutations in their patients, they may start to see patterns emerging, hopefully leading to treatments that have more success across patients, such as those that target the pervasive Notch and Ras signaling pathways.

Using the techniques described here (and others), fruit fly research will likely revolutionize the way we approach cancer research and think about cures. In humans with cancer, we see only the end point without being able to determine how the cancer began. With flies, researchers can go back and study the genes involved in the triggering event. And with luck, we’ll figure out how to keep that trigger from being pulled.

 

References:

Translational Findings: Fruit fly contributions to research in circadian rhythms

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:

 

Translational Findings: What drunk fruit flies can tell us about alcohol addiction

A study in 2012 found that approximately 7.2% of adults in the United States have an alcohol use disorder (a term that covers any person for whom their drinking causes distress or harm). That adds up to approximately 17 million Americans! Treatments for alcoholism, such as behavioral therapies or medications, can often be ineffective in the long-term due to changes that happen in the brain as a result of addiction. Improved treatment for alcoholism therefore requires an understanding of how and why addiction forms.

Cartoon of the alcohol geneFigure 1. Genes can account for about 50% of the risk for alcoholism. Image by Addiction Blog

Research has shown that genes are responsible for about 50% of the risk for alcoholism, which can explain why alcoholism seems to run in families. Although genes alone don’t determine whether someone will become an alcoholic, they can influence a person’s sensitivity and tolerance for alcohol. Environmental or social interactions account for the remainder of the risk.  A person with increased sensitivity to the stimulant effects of alcohol (increased energy, lowered inhibition) and/or decreased sensitivity to its depressant effects (motor impairment, sedation) might have a more pleasant experience with it, making them more likely to use it regularly. On the other hand, someone who finds the taste or side effects more aversive is unlikely to become addicted. For example, some people of Asian descent carry a gene variant that causes them to metabolize alcohol too fast, and they experience symptoms like flushing, nausea, and rapid heartbeat when they drink.

Studying the genes that increase the risk of alcoholism in animal models could lead to better preventive measures in at-risk families. Alcohol addiction also causes neuronal changes in the brain that can be studied in animal models to gain a better understanding of how the brain is changing and how to reverse it. Such an understanding will inevitably lead to better treatments for alcohol addiction.

Cartoon of fly addictFigure 2. Fruit flies can serve as a model for alcohol addiction. Drawing by the Heberlein Lab

Wait, isn’t this a fruit fly blog?  How can they possibly have anything to do with alcohol addiction? You may have heard the old adage, “You catch more flies with honey than vinegar”, but in truth, fruit flies are more attracted to the smell of vinegar and fermenting fruit. Fruit flies like to feed off of the microbes (such as yeast) that are found in fermenting fruit, and they have evolved to develop a tolerance for the alcohol produced by fermentation.  In fact, researchers have found that fruit flies even prefer to feed and lay their eggs on substances with about 4-5% alcohol—the concentration of an average beer.

One of the reasons fruit flies have learned to love alcohol is because it protects them from parasitic wasps, one of their most dangerous predators. Parasitic wasps inject their eggs into fruit fly larva. Although the larva’s immune system can sometimes resist the wasp growing inside it, most often the wasp larva eats the fly from the inside out, later bursting from it as a mature wasp. But while fruit flies have evolved a resistance to alcohol, the parasitic wasps have not. When a larva has been injected by one of these predators, it will consume almost toxic levels of alcohol in an attempt to kill off the parasite within it. In fact, female fruit flies that have detected the presence of parasitic wasps nearby will seek out the most alcohol-laden foods she can find before laying her eggs, giving her offspring their greatest chance to survive the predators.

But what happens when fruit flies are exposed to levels of alcohol not normally found in their natural environments? Stronger alcohol concentrations than what they have evolved to tolerate can actually be harmful to fruit flies. Does that mean fruit flies are smart enough to avoid such concentrations?

Fruit fly drinking alcohol from a capillary tubeFigure 3. A fruit fly drinking alcohol from a capillary tube during a test for whether flies prefer food laced with alcohol. Photo by G. Shohat-Ophir, UCSD

Researchers have found that fruit flies seem to treat intoxicating levels of alcohol in the same way that some humans do—as a reward they just can’t get enough of. Fruit flies that have never been exposed to alcohol before will initially show a slight preference for it over a non-alcoholic food source, likely because it instinctively knows that alcohol signals food and safety. But with each exposure their preference for the alcohol-laden food gets stronger and stronger, despite its bitter taste and aversive side effects such as motor incoordination and sedation (sound familiar?). Eventually, these fruit flies show signs of alcoholism: they regularly drink until they’re “drunk”, build up a tolerance over time and drink more and more to compensate, and continue drinking despite increasingly dangerous side effects. They’ll keep drinking even if researchers add an aversive stimulus to the alcohol-laden food, such as a repulsive chemical or an electric shock. Fruit flies will also experience symptoms of withdrawal when the alcohol is taken away, and relapse to previous levels of drinking when it’s returned. Perhaps even more surprising is that drunk fruit flies lower their standards when looking for suitable sexual mates, and flies that have been sexually rejected will turn to alcohol to cope. These parallels between human and fruit fly drinking behavior are amazing! Scroll down to see a breakdown of the many ways fruit flies show signs of alcohol addiction.

These findings have led scientists to develop fruit flies as a model organism for alcohol addiction, because although fruit flies and humans may seem very different, many genes and cellular processes are shared between them (and in fact among most species!). Fruit flies are fantastic for genetic research, and could tell us a lot about genetic risks for alcoholism and why alcohol addiction forms. They can even be used to study the changes that occur in the brain as a result of addiction, since fruit flies exhibit many “alcoholic” behaviors.

What have fruit flies taught us so far? Fly scientists have already identified several genes that contribute to the risk of alcoholism (listed below). Despite their funny names, they could provide some very serious information about why some individuals have higher sensitivity or tolerance for alcohol and how these genes can be targeted by drugs to prevent or treat alcoholism.

  • Krasavietz – Researchers have found that fruit flies with a mutation in this gene are completely uninterested in alcohol. They showed that mutations in krasavietz reduced flies’ sensitivity to the “sedation” effect of alcohol, causing flies to quickly become sedated after intoxication, skipping all the fun stimulating side-effects.
  • Cheapdate – Flies with a mutation in this gene experienced increased sensitivity to alcohol, such that much lower doses were able to cause “intoxicated” behaviors.
  • Happyhour – A mutation in this gene causes flies to be less sensitive to the sedative effects of alcohol while maintaining normal sensitivity for the stimulating effects. A mutation in this gene in humans might increase the risk of addiction, because they will have a more pleasant experience when drinking and are more likely to drink regularly.
  • Hangover – Flies with a mutation in the hangover gene don’t develop a tolerance for alcohol. Instead of needing increased doses to achieve the same behavioral effects over time, the same dose always affects them the same way. Because increased consumption over time leads to dependence and addiction, human with a mutation in this gene may be at lower risk of alcoholism.

Researchers have also learned that a desire for alcohol in flies depends upon a certain chemical in the brain called neuropeptide F (NPF), which is very similar to neuropeptide Y (NPY) found in mammals. NPY signaling in mammals has been linked to stress, alcohol consumption, sexual motivation, and sugar satiety, among other things. In flies, reduced NPF levels led to increased alcohol intake. Sexual fulfillment was found to increase NPF levels, while sexual rejection decreased them. NPF levels could therefore indicate general “reward satisfaction”, and reduced levels causes flies to seek out something rewarding, whether it’s sex, drugs, or rock and roll. It is very likely that NPY in mammals plays the same role, which means manipulating NPY levels in humans could be a possible treatment for addiction.

Finally, fruit fly researchers have found that dopamine, a chemical that some neurons in the brain use for communication, was also involved in alcohol addiction. Dopamine has also been implicated in addiction in mammalian research because of the role it plays in the mammalian “reward system”, a brain region that gets hijacked in addiction. Further research in this area in flies may provide clues as to how dopamine signaling can lead to changes in “reward” structures.

No animal model will ever be a perfect model for alcoholism, because it is a largely a human phenomenon influenced by social, cultural, and cognitive factors. But animal models can be used to model important physical and behavioral facets of addiction, and to determine the genetic basis for withdrawal and tolerance. These findings will lead to the development of better medications for the prevention and treatment of alcoholism.

 

Fruit flies exposed to high levels of alcohol show behaviors that indicate they could be “tipsy” and/or “drunk”

Image of the booze-o-matFigure 4. The “booze-o-mat” is where fruit flies are placed in tubes with alcohol vapors to ensure consumption. Photo by the Heberlein Lab

While researchers sometimes provide fruit flies with alcohol by mixing it into their food, they can ensure consistency in their flies’ level of “drunkenness” by putting them in tubes filled with alcohol vapors so they breathe it in (Figure 4). This allows them to assess the behavioral effects of alcohol with less variation.  At lower doses, the alcohol acts as a stimulant and increases their level of activity (comparable to increased energy and lowered inhibitions in humans). This is thought to be the rewarding effects of the alcohol. But increase the dose, and flies start showing signs of motor incoordination. They actually seem to be tipsy—they fall over, bump into each other and the walls, and have difficulties climbing. Even higher doses have a sedating effect.

 

Fruit flies prefer to consume alcohol, even if they don’t need it for nutritional purposes

Cartoon of preference assayFigure 5. Fruit flies are given a choice of capillary tubes containing either regular food or alcohol-laden food. Modified from Devineni & Heberlein, 2009

When fruit flies that have never experienced alcohol before are given a choice between non-alcoholic food and alcohol-laden food, they initially show only a slight preference for it. But the next time they are given a choice, they will overwhelming choose the food laced with alcohol.  The flies will even consume enough alcohol to cause intoxication and alter its behavior as previously described. They will consume an intoxicating amount of alcohol time and time again with increased frequency, much like an alcoholic might.

 

Fruit flies will continue to consume alcohol even if they don’t like the way it tastes

Researchers have shown that while fruit flies like the way alcohol smells (it signals food and safety, after all), they don’t like the way it tastes. Nevertheless, just as humans consume alcohol despite its initially aversive bitter taste (and eventually develop a “taste for it”) fruit flies drink the alcohol anyway.  Even when researchers associate alcohol with an aversive stimulus, such as giving the flies an electric shock whenever they drink or lacing the alcohol with a repulsive chemical called quinine, they will continue to drink up. This shows that flies are willing to overcome an aversive stimulus in order to consume alcohol.

 

Fruit flies develop a tolerance to alcohol and consume more and more each time to compensate

Like humans, fruit flies build up a resistance to the effects of alcohol after repeated exposure, which is known as tolerance. Flies that have been previously exposed to alcohol show an increasing tolerance to its effects over time, and will drink more and more each time to produce the same behavioral effects.

 

Fruit flies develop a physiological dependence on alcohol and experience symptoms of withdrawal

In humans, withdrawal symptoms include dysphoria, anxiety, cognitive impairment, and seizures. Clinically, these symptoms are a sign of alcohol dependence. After flies were chronically exposed to alcohol, they showed some of these same symptoms when it was taken away.

In one study, researchers showed that flies experiencing withdrawal had a lowered threshold for seizures and had more seizures than flies that had never been exposed to alcohol.

Researchers have also shown that fruit fly larva experience cognitive impairment as a symptom of withdrawal. They found that larva who drank too much had difficulties learning at first, but after chronic exposure they adapted and were able to learn almost as well as larva that were not exposed to alcohol.  When the alcohol was taken away, their cognitive abilities were lost, and when the alcohol was returned, the larva also regained their learning abilities. These results suggest that the animals were dependent on alcohol not just physiologically, but also cognitively.

 

Fruit flies “relapse” after abstinence from alcohol

One characteristic of alcohol addiction is relapse, in which an individual will return to similar or greater consumption levels after a period of abstinence. In one study, fruit flies were chronically exposed to alcohol, followed by a period of abstinence. When the researchers provided them with alcohol again, the flies immediately began drinking at the same levels as before, without the gradual increase in preference that is seen when fruit flies are exposed for the first time.

 

Male fruit flies who have been sexually rejected turn to alcohol to cope

Researchers found that male fruit flies who had been unsuccessful in attempting to mate with a female drank more alcohol afterwards than males who were successful. They found that the desire to drink was dependent on levels of neuropeptide F (NPF), which were decreased after rejection but increased after successful mating. Alcohol consumption increased NPF levels again, suggesting that NPF acts as a “reward signal”. NPF is similar to mammalian neuropeptide Y (NPY), which has been linked to stress, anxiety, sexual motivation, alcohol consumption, and sugar satiety in mammals.

 

“Drunk” male fruit flies become hypersexual and lower their standards for suitable sexual mates

Image of courtship chainFigure 6. Drunk male fruit flies sometimes form a “courtship chain”, where male flies follow each other around trying to mate. Photo by Kyung-An Han laboratory, Penn State

Normally, male flies will only try to mate with females, but when they have been exposed to alcohol, they will not only step up their courting of females, but also even try to mate with other males. This effect got worse and worse with each exposure to alcohol. Unfortunately, just as humans have learned over the years, rates of successful mating actually decreased after getting tipsy. Even for fruit flies, getting drunk doesn’t necessarily lead to good sex.

 

 

References:

  1. Milan N. & Todd A. Schlenke (2012). Alcohol Consumption as Self-Medication against Blood-Borne Parasites in the Fruit Fly, Current Biology, 22 (6) 488-493. DOI: http://dx.doi.org/10.1016/j.cub.2012.01.045
  2. Kacsoh B.Z., N. T. Mortimer & T. A. Schlenke (2013). Fruit Flies Medicate Offspring After Seeing Parasites, Science, 339 (6122) 947-950. DOI: http://dx.doi.org/10.1126/science.1229625
  3. Shohat-Ophir G., R. Azanchi, H. Mohammed & U. Heberlein (2012). Sexual Deprivation Increases Ethanol Intake in Drosophila, Science, 335 (6074) 1351-1355. DOI: http://dx.doi.org/10.1126/science.1215932
  4. Lee H.G., Jennifer S. Dunning & Kyung-An Han (2008). Recurring Ethanol Exposure Induces Disinhibited Courtship in Drosophila, PLoS ONE, 3 (1) e1391. DOI: http://dx.doi.org/10.1371/journal.pone.0001391
  5. Robinson B., Anna Kuperman & Nigel S. Atkinson (2012). Neural Adaptation Leads to Cognitive Ethanol Dependence, Current Biology, 22 (24) 2338-2341. DOI: http://dx.doi.org/10.1016/j.cub.2012.10.038
  6. Devineni A.V. (2009). Preferential Ethanol Consumption in Drosophila Models Features of Addiction, Current Biology, 19 (24) 2126-2132. DOI: http://dx.doi.org/10.1016/j.cub.2009.10.070

General references:

Translational Findings: How fruit flies are helping us understand Parkinson’s disease

Parkinson’s disease is the second most common neurodegenerative disorder, and patients experience primarily movement-related symptoms including shaking and rigidity in their limbs, slow movements, and difficulty walking, all of which progressively worsen over time. It was formally recognized as a disease in 18171, but didn’t receive much attention until it was given its name in 1861. Parkinson’s disease was not very well understood, and there were no real treatments for about 100 years. Then, in the 1960s researchers discovered that Parkinson’s patients had low levels of dopamine, a chemical in the brain that some neurons use for communication. This finding led to the use of L-Dopa (also known as levodopa) as a treatment. L-Dopa is taken orally and can reach the brain, where it is converted to dopamine2. Now approximately 40 years later, L-Dopa is still the primary treatment for Parkinson’s disease.

Cartoon of dopamine loss in Parkinson's diseaseFigure 1. Parkinson’s patients show a decrease in dopamine levels in the brain. source

The problem is that L-Dopa doesn’t work forever. Since the 1960s, we have learned that Parkinson’s disease is caused by progressive damage and eventual loss of dopaminergic (DA) neurons (these are the ones that release the dopamine), most severely in an area of the brain important for movement known as the basal ganglia3. As more and more DA neurons become damaged and die off, L-Dopa loses its ability to compensate and the symptoms start to come back.  To make matters worse, L-Dopa has a number of side effects that need to be treated with yet more drugs. Of course, L-Dopa is currently very necessary for treating Parkinson’s disease and can give patients an extra 5-15 years of quality symptom-free life. Even as L-Dopa’s effectiveness begins to decrease, it’s still better than no treatment at all. But newer and better treatments need to be developed.

Unfortunately, we can’t fix the disease until we understand what’s causing it. Why are the DA neurons dying? How can we stop it and then reverse the damage? We’ve learned just about everything we can from (ethical) research in humans. It’s time to bring in the model animals! The humble fruit fly Drosophila melanogaster has emerged as a particularly important model because while most cases of Parkinson’s disease occur randomly, about 10-15% of cases are due to inherited genetic mutations. And for genetic research, flies are our best bet.

Why study the genetic mutations if only 10-15% of cases are caused by them? The easiest answer is because we can introduce mutations into the same genes in fruit flies to find out what those genes do. If researchers can figure out what’s causing damage to the DA neurons in the genetic cases, those findings can be directly applied to the random cases. So even if we don’t yet know what caused them,  treatments that protect DA neurons and compensate for their damage should still work to improve symptoms in both genetic and random cases of Parkinson’s disease.

Since the late 1990s, mutations in five genes have been found to lead to inherited forms of Parkinson’s disease in some families. Each was given a cryptic name: SNCA4, Parkin5, PINK16, DJ-17, and LRRK28-9. Once those genes were identified in human patients, researchers were able to mutate the same genes in animal models to figure out what they do and how their failure leads to Parkinson’s disease. Luckily for us, fruit flies already have four of those genes, so it was relatively easy to make a fly line with a mutation in them.  The remaining gene, SNCA, can still be studied by introducing the human mutation into flies using a binary expression system (UAS/GAL4).

Magnified image of Lewy body in brain of Parkinson's patientFigure 2. alpha-synuclein proteins clump to form masses called Lewy bodies. Figure depicts magnified image of a Lewy body surrounded by neurons in the substantia nigra (a part of the basal ganglia) in a patient with Parkinson’s disease. Photo by Suraj Rajan / CC BY-SA 3.0

So what have we learned so far from our winged friends?  An overview of fruit fly research for each gene is listed below, but if you just want the punchline, here it is: Parkin, PINK1, DJ-1, and LRRK2 are all involved in maintaining and/or protecting mitochondria, which are structures inside cells that create the fuel the cell uses as energy (think of them as little power plants). When any of these four genes is mutated, mitochondria begin to function abnormally and are more sensitive to damage from environmental stressors such as toxins. As more and more damage accumulates, DA neurons begin to die off, which may explain why Parkinson’s disease is progressive and usually begins later in life. On the other hand, although the role of normal SNCA is still unknown, a mutated SNCA gene creates masses in the brain called Lewy bodies that may cause damage to DA neurons. Although it’s not fully understood why mutations in these genes affect DA neurons more severely than other neurons in the brain, it is thought that DA neurons may simply be more sensitive to environmental toxins.

Okay, but what about the random cases?  While the cause of most of the random cases of Parkinson’s disease remains a mystery, results from research in the genetic cases can give us a clue. It is currently thought that this form of Parkinson’s disease is caused by a combination of factors such as accumulated damage and genetic mutations with age, exposure to environmental toxins such as pesticides, and genetic predisposition (meaning that there may be genes that don’t directly cause Parkinson’s disease, but may increase your risk if exposed to certain environmental triggers).

Research in fruit flies has definitely improved our understanding of the underlying causes of Parkinson’s disease, which will ultimately lead to the development of better treatments. Instead of simply compensating for reduced dopamine levels, future treatments may target the Lewy bodies or bolster the cell’s protective mechanisms for mitochondria. Treatments such as these will have longer lasting effects because they could potentially prevent further loss of DA neurons. And fruit flies aren’t our only hope. Many of the findings described above have already been used for designing studies in mammalian models, and research in mammals has also led to important discoveries not mentioned here.

 

Findings in fruit flies specific to each Parkinson-related gene:

  • Parkin/PINK1: I grouped these two mutations together because fruit fly researchers have recently discovered that these genes play similar roles in neurons10-12. PINK1’s protein actually interacts with Parkin’s protein to maintain and protect mitochondria. As a result, a mutation in either PINK1 or Parkin causes mitochondrial defects and increases sensitivity to environmental stress from toxins. The accumulation of damage leads to death in DA neurons and, of course, the resulting impairments in movement as seen in Parkinson’s patients. Further understanding of how PINK1 and Parkin work will allow researchers to develop drugs to compensate for lost function in these genes.
  • SNCA: Many Parkinson’s patients develop dense masses in their brains called Lewy bodies, which are formed when large molecules called proteins accumulate abnormally and bind to each other. Lewy bodies are primarily made up of the protein alpha-synuclein bound to various other proteins (alpha-synuclein is made from the instructions in the SNCA gene). But why is alpha-synuclein clumping together? Researchers introduced the mutated version of the human SNCA gene in fruit flies and found that mutant flies showed progressive DA neuron death and loss of motor skills, just like the symptoms in human Parkinson’s patients13. And of course, they found clumps of protein. Studies in this fly model are now focused on understanding why the mutated version of alpha-synuclein clump together and cause DA neuron death. Once these questions have been answered, researchers can develop treatments that either prevent formation of Lewy bodies, break them down, or prevent them from damaging neurons. Interestingly, researchers have found that when they add extra normal PINK1 or Parkin protein in this SNCA mutant, the extra PINK1 or Parkin actually helps to protect against DA neuron death14-15. So, another treatment option for Parkinson’s patients with Lewy bodies may be to stimulate extra PINK1 or Parkin protein production as a protective measure.
  • DJ-1:  Researchers have found that fruit flies with a mutation in DJ-1β (flies actually have two versions of this gene, and DJ-1β is similar to the human version) are more sensitive to environmental toxins, demonstrating that this gene plays a protective role16. Flies with this mutant gene also showed reduced lifespan and locomotor defects, as seen in Parkinson’s patients17. Finally, mutant DJ-1 protein resulted in abnormal mitochondrial function, suggesting that this gene, like PINK1 and Parkin, is necessary for normal functioning in mitochondria18.
  • LRRK2: Mutations in LRRK2 are likely the most common genetic cause of Parkinson’s disease in humans, but it has so far been the most inconsistent in fruit flies, making it a difficult gene to study. Researchers have performed several kinds of genetic manipulations, including completely knocking-out LRRK2 function, introducing the mutated version of the human gene, or mutating it in a way that changed its function instead of making it completely non-functional. Some researchers found that the mutations led to DA neuron death and severely impaired movement, some researchers found no effect, and others found a result somewhere in the middle19. A few important reliable discoveries have come out of research in this gene, however. First, all of the mutants were more sensitive to various environmental toxins. Second, researchers found that LRRK2 proteins interact with mitochondria, suggesting that it, like PINK1 and Parkin, plays a role in maintaining and/or protecting these structures. These findings support an increasingly convincing conclusion that Parkinson’s disease may be caused by mitochondrial defects or damage that leads to death in DA neurons.

 

Interested in recent Parkinson’s disease research in fruit flies? Check out the Breaking Research post on MUL1.

 

References:

  1. Parkinson J. (1817). An Essay on the Shaking Palsy, Journal of Neuropsychiatry, 2002, 14 (2) 223-236. DOI: http://dx.doi.org/10.1176/appi.neuropsych.14.2.223
  2. <Cotzias G.C. (1967). Dopa and Parkinsonism, BMJ, 3 (5563) 497-497. DOI: http://dx.doi.org/10.1136/bmj.3.5563.497
  3. Blandini F., Cristina Tassorelli & Emilia Martignoni (2000). Functional changes of the basal ganglia circuitry in Parkinson’s disease, Progress in Neurobiology, 62 (1) 63-88. DOI: http://dx.doi.org/10.1016/s0301-0082(99)00067-2
  4. <Polymeropoulos M.H. (1997). Mutation in the -Synuclein Gene Identified in Families with Parkinson’s Disease, Science, 276 (5321) 2045-2047. DOI: http://dx.doi.org/10.1126/science.276.5321.2045
  5. Kitada T., Asakawa S., Hattori N., Matsumine H., Yamamura Y., Minoshima S., Yokochi M., Mizuno Y. & Shimizu N. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism., Nature, PMID: http://www.ncbi.nlm.nih.gov/pubmed/9560156
  6. Valente E.M. (2004). Hereditary Early-Onset Parkinson’s Disease Caused by Mutations in PINK1, Science, 304 (5674) 1158-1160. DOI: http://dx.doi.org/10.1126/science.1096284
  7. Bonifati V. (2003). Mutations in the DJ-1 Gene Associated with Autosomal Recessive Early-Onset Parkinsonism, Science, 299 (5604) 256-259. DOI: http://dx.doi.org/10.1126/science.1077209
  8. Paisán-Ruı́z C., E.Whitney Evans, William P. Gilks, Javier Simón, Marcel van der Brug, Adolfo López de Munain, Silvia Aparicio, Angel Martı́nez Gil, Naheed Khan & Janel Johnson & (2004). Cloning of the Gene Containing Mutations that Cause PARK8-Linked Parkinson’s Disease, Neuron, 44 (4) 595-600. DOI: http://dx.doi.org/10.1016/j.neuron.2004.10.023
  9. Zimprich A., Petra Leitner, Peter Lichtner, Matthew Farrer, Sarah Lincoln, Jennifer Kachergus, Mary Hulihan, Ryan J. Uitti, Donald B. Calne & A.Jon Stoessl & (2004). Mutations in LRRK2 Cause Autosomal-Dominant Parkinsonism with Pleomorphic Pathology, Neuron, 44 (4) 601-607. DOI: http://dx.doi.org/10.1016/j.neuron.2004.11.005
  10. Clark I.E., Changan Jiang, Joseph H. Cao, Jun R. Huh, Jae Hong Seol, Soon Ji Yoo, Bruce A. Hay & Ming Guo (2006). Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin, Nature, 441 (7097) 1162-1166. DOI: http://dx.doi.org/10.1038/nature04779
  11. Park J., Sungkyu Lee, Yongsung Kim, Saera Song, Sunhong Kim, Eunkyung Bae, Jaeseob Kim, Minho Shong, Jin-Man Kim & Jongkyeong Chung & (2006). Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin, Nature, 441 (7097) 1157-1161. DOI: http://dx.doi.org/10.1038/nature04788
  12. Yang Y., Y. Imai, Z. Huang, Y. Ouyang, J.-W. Wang, L. Yang, M. F. Beal, H. Vogel & B. Lu (2006). Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin, Proceedings of the National Academy of Sciences, 103 (28) 10793-10798. DOI: http://dx.doi.org/10.1073/pnas.0602493103
  13. Feany M.B. & Bender W.W. A Drosophila model of Parkinson’s disease., Nature, PMID: http://www.ncbi.nlm.nih.gov/pubmed/10746727
  14. Bell J. & Brian E. Staveley (2008). Pink1 suppresses α-synuclein -induced phenotypes in a Drosophila model of Parkinson’s disease , Genome, 51 (12) 1040-1046. DOI: http://dx.doi.org/10.1139/g08-085
  15. Haywood A.F.M. & Staveley B.E. (2004). Parkin counteracts symptoms in a Drosophila model of Parkinson’s disease., BMC neuroscience, PMID: http://www.ncbi.nlm.nih.gov/pubmed/15090075
  16. Yang Y., Md. E. Haque, Y. Imai, J. Kosek, L. Yang, M. F. Beal, I. Nishimura, K. Wakamatsu, S. Ito & R. Takahashi & (2005). Inactivation of Drosophila DJ-1 leads to impairments of oxidative stress response and phosphatidylinositol 3-kinase/Akt signaling, Proceedings of the National Academy of Sciences, 102 (38) 13670-13675. DOI: http://dx.doi.org/10.1073/pnas.0504610102
  17. Meulener M., Cecilia E. Armstrong-Gold, Patrizia Rizzu, Peter Heutink, Paul D. Wes, Leo J. Pallanck & Nancy M. Bonini (2005). Drosophila DJ-1 Mutants Are Selectively Sensitive to Environmental Toxins Associated with Parkinson’s Disease, Current Biology, 15 (17) 1572-1577. DOI: http://dx.doi.org/10.1016/j.cub.2005.07.064
  18. Hao L.Y. & N. M. Bonini (2010). DJ-1 is critical for mitochondrial function and rescues PINK1 loss of function, Proceedings of the National Academy of Sciences, 107 (21) 9747-9752. DOI: http://dx.doi.org/10.1073/pnas.0911175107
  19. Muñoz-Soriano V. (2011). Drosophila Models of Parkinson’s Disease: Discovering Relevant Pathways and Novel Therapeutic Strategies, Parkinson’s Disease, 2011 1-14. DOI: http://dx.doi.org/10.4061/2011/520640

 

General references and reviews:

Translational Findings: How fruit fly research has already contributed to human health

In my previous post, I described how Drosophila melanogaster serves as an important and relevant model organism for biological research.  But how is fruit fly research actually helping us to better understand ourselves?  In my future “Translational Findings” posts, I will talk about how fruit flies are furthering our understanding of a specific human-related issue. These will include diseases such as Parkinson’s or Alzheimer’s disease, developmental and genetic disorders such as autism or Down syndrome, and other human concerns like addiction, sleep, or aging.

In this first Translational Findings post, however, I would like to give an overview of the history of fruit fly research and how it has already contributed to human health. Fruit flies have been used as a model organism for over a century, so the list is long. To narrow it down, I will focus on describing the important findings that led to Nobel Prizes: four of them since Thomas Hunt Morgan published the first scientific paper using Drosophila melanogaster in 19101!

Comparison of eye color in fruit fliesFigure 1. The fly on the top has a mutation that causes white eyes. The fly on the bottom has normal red eyes. source

The first Nobel Prize was awarded to Thomas Hunt Morgan himself in 1933. He studied heredity and was interested in understanding how physical traits were passed down through generations. Morgan began by searching for visible variations among fruit flies so he could determine how those traits were inherited. Finally, he found white-eyed flies among a stock of normal red-eyed flies (Figure 1). He and his students began studying the pattern of inheritance for the white-eyed trait, and they later found other mutations to study as well. Their findings led to a radical new theory of heredity which suggested that genes (the pieces of DNA that contain the information for the traits) are carried in a linear arrangement on chromosomes, and these chromosomes are passed down through generations. Their findings showed the physical mechanism for genetic inheritance and are now considered the foundation of modern genetics.

The second Nobel Prize was awarded to Hermann Müller, one of Morgan’s students. After leaving Morgan’s lab, Müller began researching methods for inducing mutations in fruit flies instead of waiting for them to occur spontaneously. In the 1920s, he made a breakthrough when he noticed a connection between radiation and lethal mutations and, in 1927, published a paper demonstrating that X-rays damaged chromosomes and caused genetic mutations2. Although the public was beginning to realize that radiation was dangerous (Marie Curie died in 1934 as a result of her own research), this was the first evidence of specific harmful effects. Müller began publicizing the dangers of radiation soon after, and earned a Nobel Prize in 1946 for his work.

In 1995, the third Nobel Prize for fruit fly research was shared by Christiane Nüsslein-Volhard, Eric Wieschaus, and Ed Lewis. Using recently developed techniques that allowed DNA to be more easily manipulated (such as X-ray induced mutations), these scientists screened thousands of mutant flies and identified several genes responsible for development in Drosophila melanogaster3. Their research paved the way for understanding how multicellular organisms develop from single cells, and showed that development is genetically controlled. Shortly after their discoveries, studies in other species found closely related developmental genes in vertebrates, confirming an evolutionary link between fruit fly and human biology.

The final Nobel Prize was award to Jules Hoffmann, Bruce Beutler, and Ralph Steinman in 2011 for their research in immunity. Humans have two methods for defending against infections: innate immunity, which is inherited, and adaptive immunity, which responds to invaders and “learns”. Hoffman’s research in fruit flies showed that a gene called Toll was important for the fly’s innate immune system. He found that the Toll gene contained instructions for a receptor responsible for recognizing certain bacterial and fungal infections and triggering an immune response4. Beutler later found related “Toll-like” receptors with the same function in mammals, demonstrating that this innate immunity control mechanism is shared across species through evolution. A few years later, Steinman showed that Toll-like receptors activate the adaptive immune system in mammals as well.

Fruit fly research has already made huge contributions to understanding human biology, and it shows no signs of stopping. In today’s research environment, research in flies has gone beyond the genetic research it founded and has moved into more complex issues such as disease and behavior. Which new major contribution will earn this little insect its fifth Nobel Prize?

  1. Morgan TH (1910). SEX LIMITED INHERITANCE IN DROSOPHILA. Science (New York, N.Y.), 32 (812), 120-2 PMID: 17759620
  2. Muller HJ (1927). ARTIFICIAL TRANSMUTATION OF THE GENE. Science (New York, N.Y.), 66 (1699), 84-7 PMID: 17802387
  3. Nüsslein-Volhard C, & Wieschaus E (1980). Mutations affecting segment number and polarity in Drosophila. Nature, 287 (5785), 795-801 PMID: 6776413
  4. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, & Hoffmann JA (1996). The dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell, 86 (6), 973-83 PMID: 8808632

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