Too much phosphorylation, time to go to sleep!

It’s Friday night and you are at a concert, wishing you hadn’t woken up at 4:45am to go to spin class. As the night wears on you get more tired and fall asleep on the train ride home. Why do you get tired the longer you stay awake? It’s not your muscles-- they could keep contracting. There are chemical changes to molecules that accumulate the longer we stay awake and they drive this need for sleep. This was shown in a recent paper in Nature by Wang et al. using an interesting mouse mutant.

Sleepy mice

When mice are sleep deprived, they have an increased need for sleep (just like humans who get more tired the longer they stay awake). Sleep need is measured by putting electrodes on the mouse’s scalp that measure brain waves, which are large synchronized and rhythmic patterns of electrical activity in the brain. When mammals sleep, there are characteristic changes in the brain waves, so we can tell what stage of sleep the animal is in. After sleep deprivation in mice, slow wave activity and the duration of non-REM sleep increase, so this is used to measure sleep need in mice. The researchers who did this study used sleep deprived mice, as well as the Sleepy mutant mouse model (I’m not being cute, this is the actual name of the mutant strain).

The Sleepy mice have a mutation in a gene called Sik3 that encodes for an enzyme. The mutation causes the enzyme to work more efficiently and the mice sleep more, but have an elevated need for sleep (as measured by the brain waves). So these mice are always tired due to one amino acid change in one enzyme – that’s powerful.

Phosphorylated proteins drive sleep need

The researchers compared normal mice with the sleep deprived and Sleepy mice, looking at the chemical changes to the proteins in their brains. The sleep deprived and Sleepy mice had more phosphorylated proteins than the mice who had a normal amount of sleep.

Phosphorylated? That’s a mouth full (here’s how to say it). There is a small molecule called a phosphate, made up of a phosphorous atom surrounded by oxygens. This chemical group is big and charged and will change the shape of the rest of the protein when it is added on. Since phosphorylation changes the shape of proteins, that may also change the way the proteins function.

Phosphorylation changes the shape of the protein (from Campbell's "Biology")

The longer the sleep deprived mice stay awake, the more phosphorylated proteins there are. If the mice are allowed to sleep after being deprived, their proteins go back to the unphosphorylated state.  

Sleepy mice are always in need of sleep, regardless of how much sleep they get, so their proteins are always phosphorylated. Why do Sleepy mice have so many phosphorylated proteins? Remember that the Sleepy mice have a mutation that makes the Sik3 enzyme more active. Guess what the function of Sik3 is! It is a kinase enzyme, which adds phosphates to proteins. So the poor Sleepy mice accumulate phosphorylation at a higher rate than normal mice, so they will always have an increased need for sleep.

Many of the proteins that are being phosphorylated during the awake state function at the synapse, where neurons communicate with each other. Some neuroscientists believe that memories are encoded while we are awake by changes to synaptic function. These synaptic changes are refined during sleep to consolidate the memories in long-term storage. The authors suggest that the accumulating phosphorylation regulates synapse function and memory formation, though they don’t show evidence for the connection with memory.

In conclusion, next time you are getting tired at that concert, just tell your friends, “My synaptic proteins are too phosphorylated, I need to go home.” They’ll understand.

Videos!

Cellways, it's been awhile! Here are a couple of videos I made in the last years:


A Ted-Ed video about X-chromosome inactivation and some interesting consequences of that.

A Science Sketches video about the basics of stem cell biology.

Microbiome accelerates neurodegeneration

Parkinson disease (PD) is a neurodegenerative disease characterized by motor deficits and aggregates of a protein called α-synuclein (α-syn) in the brain (pronounced sin-NU-clee-in). Genetics plays a role in PD, because there are some early-onset forms of PD that are caused by mutations in α-syn that cause it to more readily clump together and form the protein aggregates. The purely genetic forms of the disease, though, are relatively rare, so the environment must also play a role in most cases. A recent paper published in Cell by Sampson et al. explores how the microbiome in the gut affects development of PD symptoms.

The microbiome is the community of bacteria and fungi living in and on us (watch this awesome video about the microbiome). It has previously been shown that the normal gut microbiome is disrupted in various diseases such as autism and in Parkinson’s patients. It’s always hard to know, though, what is the cause and what is the effect. Does the disease cause the microbiome to change, or does the change in the microbiome cause the disease? Maybe a little of both.

Mouse model

To address the role of the microbiome in Parksinson Disease, the authors relied on an established mouse model of PD. These mice overexpress the normal human form of α-syn in all their neurons. Even though this isn’t the mutant form of the gene, the fact that it is overexpressed all over the brain causes the characteristic α-syn aggregates. These mice are slow in motor tasks, including removing a piece of tape from their noses (sounds like a frustrating, but also adorable behavioral task). They also have impaired gastrointestinal function, which is to say they don’t produce as much poo as other mice. [An aside: normal mice apparently drop about 7 fecal pellets every 15 minutes!]

The researchers took these mice with mouse-Parkinson’s and raised half of them in a super sterile environment where they have no microbiome (called “germ free” mice), and the other half got all dirty so they had a microbiome (I will call these “dirty mice”). The PD mice with a microbiome had way more motor impairments than the mice without a microbiome! Yes, I wrote that correctly. I thought the microbiome was supposed to help its host? Well, not in these mice overexpressing α-syn.

Get this: if you give the dirty mice antibiotics from age 5-13 weeks old and then test them, they were more like the germ free mice – no motor impairments and better fecal output. Not that you would want to give humans antibiotics for their entire lives (that could cause some autoimmune diseases and serious digestive issues), but this does demonstrate that it is the gut microbiome that is affecting the symptoms of Parkinson Disease.

Short chain fatty acids

The bacteria living in our gut produce all sorts of chemicals that can get into our blood and nervous system. Bacteria produce short-chain fatty acids (SCFA), which are basically just little fats that can cross over the intestinal lining and get into our bodies. Parkinson’s patients produce more SCFAs, so the authors tested the role of SCFAs in their mouse model.

Germ free mice overexpressing α-syn are relatively normal, right? The authors fed these mice a bunch of SCFAs to mimic what the gut bacteria would be making and the mice became impaired like the dirty mice (can’t get that tape off their nose). This is amazing to me. So short-chain fatty acids that are normally made by the gut bacteria are sufficient to cause the Parkinson’s symptoms. Note that feeding SCFAs to normal mice without all that α-syn did not cause Parkinson's symptoms.

           α-syn mice no microbiome + SCFAs = impairments of α-syn mice with microbiome

Microbiome and the immune system

What are the short-chain fatty acids doing to the nervous system? One important role of the microbiome is to train the host’s immune system so it knows what to attack and what to ignore. This is why the microbiome plays a role in the development of autoimmune diseases, where the body attacks the wrong things (like a harmless pollen molecule or the body’s own cells like in type I diabetes). SCFAs can get up into the brain and regulate the immune cells of the nervous system, called the microglia (pronounced micro-GLEE-a). Indeed, the dirty mice with a full microbiome had more activated microglia in the brain than the germ free mice. Likewise, the germ free mice fed SCFAs also had activated microglia.

An overactive immune system promotes protein aggregation, so here’s the model: something causes the microbiome to become unhealthy, which causes the release of a lot of SCFAs, which activate the immune system in the brain, leading to neuron death and protein aggregation. The diagram below has some extra information in it, but the pathway in black is what they showed in this paper.

What about human patients?

Okay, so the microbiome plays a role in this one particular mouse model of PD, but what about in humans? Remember that the microbiome and the amount of short-chain fatty acids in Parkinson’s patients are different than in healthy humans. The authors took the microbes from human feces and transplanted it into the guts of the germ free α-syn mice. Amazingly, the germ free mice that got the bacteria from Parkinson’s patients had more severe motor impairments than the mice that got bacteria from the healthy humans. So there’s something going on in the microbiome of humans with PD that enhances the symptoms.

The authors raise the point that two things were needed for these mice to have the symptoms of Parkinson disease:

1) Overexpression of α-syn (genetics)

2) Disordered microbiome, also known as dysbiosis (environment)

This is a great example of a complex disease that is caused by the interplay of genetics and environment. Perhaps this information can be used to come up with new treatments to correct the dysbiosis and slow down the progression of Parkinson disease. 

Human language in dog brains

Spoken language conveys meaning in two ways: the meaning of the words (semantics or lexical knowledge) and the intonation that the speaker uses. We can sense questions by the rising pitch at the end of the sentence. Likewise, we can tell if someone is upset or being sarcastic based on how they say the words. The patterns of intonation in language is known as prosody. There are areas of the brain that are specialized for decoding the semantic meaning of language and different areas for interpreting prosody. In fact, you can have damage to one area during a stroke, while the other area remains intact. There are great examples of this in “The President’s Speech” in Oliver Sacks' book The Man who Mistook his Wife for a Hat.

In most people, word meanings are processed by the left side of the brain and prosody is localized to the right side of the brain. Some animals also use the left side of their brains to understand meaningful and familiar sounds of their species (like alert calls or bird songs). What about for animals, like dogs, which can understand the sounds of another species (i.e. commands from humans). Is the dog brain really processing the intonations of praise “good dog!” or are they responding to the words? Do they process meaning and intonation separately like humans do?

Dogs in MRI machines

In the latest issue of Science, Andies et al. published their studies of language processing in dog brains. My first thought when I read the abstract was “how do you get a dog into an MRI machine?” We commonly study which areas of human brains are active during different tasks using a technique called functional MRI (or fMRI). fMRI was done on these dogs while they listened to their trainers speak. If you have ever had an MRI scan, you know they strap you in and you cannot move your head at all. Same thing with these dogs. Needless to say, they were very well trained dogs. If you still can’t believe it, check out this video the researchers made and the cute photo of dogs in an MRI machine below.

Really well trained dogs lying still before their MRIs. (Image from phys.org)

Dogs process language like humans

Okay, so they got the dogs in the MRI machine and scanned their brains while they heard their trainer say different things. The trainer would either say words of praise, like “good boy” (in Hungarian), or neutral words. And they used either a neutral, flat intonation or they raised the pitch of their voice to create a praising intonation. This created four possibilities:

• Praise words with praising intonation
• Praise words with neutral intonation
• Neutral words with praising intonation
• Neutral words with neutral intonation

They compared the brain responses to each combination and found that the left side of the brain responded to words of praise regardless of the intonation. This is amazing, right? The dogs have heard “good boy” enough times that their brains responded specifically to that phrase regardless of how it was said. It’s like they sort of know what it means. It would be interesting to see if they respond to the same phrase spoken by a stranger.

The researchers also found that the right side of the brain had active areas when praising intonation was used, regardless of the word meaning. So dogs also understand how our voices change when we praise them.

Finally, the researchers looked at areas of the brain associated with reward. These areas are active in a variety of animals when they receive natural rewards like food or during sex, but the reward pathways are also active if the animal is given an addictive drug like cocaine. Alternatively, you can put an electrode into a mouse brain that stimulates the reward pathway and the mouse will push a lever to receive an electrical shock in this area of the brain over and over until it starves.

Andies et al. found that praising words spoken in a praising intonation activated the reward pathway in the dogs. Praise words alone and praise intonation alone had no effect. So dogs really do feel good when you say “good dog” in a high pitched voice.

Notice the organization of language processing in the dog brain. Just like in humans, language semantics (praise vs neutral words) was processed on the left side and prosody (praise vs neutral intonation) was processed on the right side. What does this tell us about the evolution of language? Language lateralization has likely been around a long time and is not uniquely human. The authors end the article with this gem: “What makes lexical items uniquely human is thus not the neural capacity to process them, but the invention of using them.”