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.

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.”

Bigger brains with Frizzled HARE

We have all heard that the sequence of human DNA differs from chimpanzee DNA by only about 1%.  Yet humans are capable of building complex civilizations while the chimps are still eating bugs in the forest.  If you compare the human brain to the brain of any other primate, it’s easy to see where our sophisticated cognitive abilities come from. 

From thebrain.mcgill.ca

DNA is the blueprint for making proteins, cells and organs, so is there something special hidden in that 1% sequence difference that gives humans bigger brains?  In particular, scientists have focused on regions in the human genome that have undergone rapid sequence changes in the human lineage, but not in other primates.  Besides looking for differences in genes that make proteins, we can also look for changes in regulatory regions, like enhancers, that control when and where the genes are expressed.

A recent paper in Current Biology by Boyd et al. explores these questions by studying a human-accelerated regulatory enhancer (HARE5), which differs significantly between humans and chimps.

Enhancer activity of HARE5

How do you study enhancers?  One way is to use a reporter gene.  Enhancers drive expression of nearby genes, so what if you swapped out a nearby gene and replaced it with a gene for a fluorescent protein?  Then you can look at your organism and wherever you see the fluorescent protein, the enhancer is active, meaning that the normal “nearby gene” is normally expressed in those cells.  Instead of doing these experiments with chimps and humans, which would take forever and be unethical in some cases, the authors put these reporter constructs into mice.  The enhancers from the chimps and humans drove expression of the reporter gene in the embryonic mouse brains.  The gene adjacent to the human enhancer was expressed earlier in development and more strongly than when placed next to the chimp enhancer (in other words, a lot more protein is being made).

Reporter gene experiment.  The mouse brain images are actual results from Figure 2 in Boyd et al. (2015).

This tells us that whatever normal gene is near HARE5, it is probably expressed earlier and way more in humans than in chimps.  There are just 10 sequence differences in the human HARE5 (i.e. mutations), which is enough to affect the way the enhancer functions and activates expression of genes. 

Frizzled expression is regulated by HARE5

So which genes are near the HARE5 sequence?  The closest gene is called Frizzled 8 and it is a receptor that responds to signals sent by other cells.  Frizzled 8 (FZD8) is a well known component of the Wnt signaling pathway that regulates many aspects of embryonic development, including neurogenesis (formation of new neurons).  The authors demonstrate that the mouse HARE5 physically interacts with Fzd8, which is a necessary  first step of gene expression, so Fzd8 is likely affected by the HARE5 sequence differences in humans and chimps.

The authors wanted to see what would happen to development of the mouse brain when Fzd8 is expressed in the same pattern as in humans or chimps.  They repeated the earlier experiments, but this time instead of using a reporter gene, they put the mouse Fzd8 gene next to the chimp or human HARE5 sequence.  They injected these DNA constructs into mice and waited to see what would happen to embryonic brain development.  When the chimp-HARE5 was driving expression of Fzd8, not much changed in terms of mouse brain development.  However, when the human-HARE5 sequence was activating the mouse Fzd8 gene, the mouse brain grew 12% bigger!! 

Let me be clear here-- they are not expressing the human Fzd gene in mice.  No, they are using the human enhancer to drive expression of the mouse Fzd8 gene, so presumably it is expressed more and earlier in development (like they saw in the reporter gene experiment).  The neural progenitor cells (pre-neurons) divided faster than in a normal mouse, leading to formation of more neurons, and a bigger brain! 

10 sequence changes in an enhancer may be one reason why I am able to write and you are able to read and understand this blog.  Whoa.  No news yet about whether these mice with bigger brains are also able to read and write… I’m sure they’re saving that for another paper.

I should say too, that there are probably a number of other similar changes to other enhancers and genes that all led to the rapid development of the big ol’ human brain.

Here's another blogger's take on this paper 

Smart phone use changes the brain

One of the most remarkable things about our brains is how organized they are. Sensory information from our eyes, mouth, skin, nose and ears goes to different locations in the brain. For example, visual signals are processed first in the very back of the brain, whereas sensations of touch and pain activate the middle region of the brain called the somatosensory cortex.

Functional organization of brain cortex. (Source: imgarcade.com/1/sensorycortex)

Remarkably, the brain gets even more organized from there. Within the visual cortex, there are columns of neurons that only respond to light that is horizontal and others that only respond to lines that are tilted 45 degrees. The somatosensory cortex is also highly organized, with different parts of the body represented by specific sets of neurons. If you were to send electrical shocks into one specific area of the somatosensory cortex to activate those neurons, you may elicit feelings of touch from the right thumb, even though the subject is not being touched at all. Move those electrical signals over slightly to another area, and the subject may feel touch instead coming from the palm of their hand.

Wow, right? But here’s the real mind blower: this organization can change over time as the person experiences different sensory inputs. If you are a violin player, you feel the strings with your fingertips a lot, so the fingertip part of the somatosensory cortex is super active. This extra activity allows the fingertip representation in the brain to grow and recruit nearby neurons to also respond to touch in the fingertips. The cortical representations are “plastic” and always changing with use.

A violin player may practice this one particular skill a lot, but what about other activities we do everyday with less intensity, like using smart phones? Think about how often you are swiping the screen with your thumb. That’s a lot of sensory information being sent to the thumb part of your somatosensory cortex. Would this increase the thumb representation in your brain? A recent paper by Gindrat et al. addressed this exact question using EEG to record brain activity in smart phone users versus people with the old-style cell phones.

Electroencephalography

How can you actually measure the area of body representations in the somatosensory cortex? You could stick electrodes into people’s brains and record the activity in their neurons, but that’s a little invasive. You could put them into a MRI machine and measure brain activity when you touch their thumbs, but that is time consuming for so many subjects (37 total). Instead, the authors used a method known as electroencephalography, or EEG, which consists of 62 surface electrodes placed on the scalps of the subjects. Each electrode records the summed electrical activity from all the neurons positioned right under the electrode. Before an experiment, all the electrodes would be picking up a baseline of activity from lots of different neurons firing asynchronously. However, during an experiment, there is a single stimulus (like touching the subject’s thumb), which elicits activity in a lot of neurons all at the same time. This activity summates to give one large response called the event related potential (ERP), which is recorded by the nearest electrodes.

EEG electrodes record brain activity (source: Wikimedia commons)

Finger representations in smart phone users

The ERPs for the thumb, index finger and middle finger were larger for the smart phone users than for the non-touchscreen users. There was a correlation between the amount of phone use per hour and the ERP, so the more use, the greater the ERP, which is to say the more activity in the somatosensory cortex. The number of electrodes recording the ERP was greater in the touchscreen users, so when you touch the thumb of a touchscreen user, a larger part of the somatosensory cortex responds. In other words, the thumb representation was larger in smartphone users who use their thumbs more often.

The more recently the subjects had used their phones intensely, the larger the ERP for the thumb, which indicates that brain remodeling occurs on a very short time scale (within 10 days in this experiment). Interestingly, there was no correlation between ERPs and the age at which the subject started using a touchscreen. This is in contrast to the previous experiments done with trained violin players, which did show a correlation between the size of the finger representations and the age at which they first started playing. The authors suspect that a trained violinist develops a more stable sensory representation than touchscreen users who are casually using their phones (as opposed to years of disciplined practice).

So the take-home message is that normal day-to-day activities can influence brain plasticity and the way our sensory representations are organized in our brains. This could be a good thing, because subjects develop better touchscreen skills. On the other hand, the enlarged thumb representation could cause focal dystonia, which is characterized by involuntary muscle contractions and sometimes pain, as the various body part representations lose their distinct boundaries and start to overlap. This probably won’t be a problem for most phone users, but be forewarned all you smart phone addicts out there.

Ctenophores come before

Three months ago, if I had seen this article about the ctenophore genome, I would have moved right passed it without a second look.  What is a ctenophore and why would I care about the sequence of its DNA?  But then I taught Bio 2 this spring and learned about animal diversity and the evolutionary tree (a day before I taught it).  This is a great example that the more you learn, the more interested you become in the subject.  Today’s article by Moroz et al. was published recently online in Nature (this one is open access, so take a look).  The results totally change the roots of the animal tree and invalidate what we taught to our students this semester.  Before we get into the paper, let me answer my own questions:

What is a ctenophore?

Ctenophores are also known as comb jellies, because they look like jellyfish and have a comb-like structure that they wiggle around to move through the water.  They have sensory organs to sense light and gravity.  They have tentacles that they move with their nervous system in order to catch prey.

Comb jelly (from Wikimedia Commons)

Although they look like jellyfish, they are in a totally different phylum, or branch of the evolutionary tree (also known as a phylogenetic tree).  Jellyfish are in the phylum Cnidaria, along with sea anemones, coral and hydras.  Comb jellies are in their own phylum known as Ctenophora. 

Why should we care about ctenophores? 

Ctenophores, along with cnidarians and sponges, represent some of the most ancient lineages of animals.  Studying them can give us a clue about how animals evolved.  All the rest of the animals are in the large taxonomic group called Bilateria, because they have bilateral symmetry (which is symmetry across a single axis).  This includes humans, insects, crustaceans, worms, fish, molluscs, etc.  Think about a jellyfish or a sea anemone; they have radial symmetry, which means they can be bisected in lots of different axes and you would still have symmetrical halves.  The bilateral animals are more complicated in lots of other ways, such as having a greater variety of tissues and more complex physiology.

The phylogenetic tree according to the biology textbook

Sponges don’t have organized tissues and they don’t have a nervous system, so based on that, researchers have considered them to be the most ancient lineage (i.e. the “basal” animals).  So if we are building a phylogenetic tree based on morphological characteristics, we are going to put them as the first branch.

Cnidarians and ctenophores look very similar, so it would make sense to put them right next to each other, followed by the bilateral animals.  Thus, based on morphological observations, the tree should look something like this:

No one ever explained to me why cnidarians get to be closer to the bilaterals than ctenophores.  Perhaps this is because the cnidarians come in so many different body plans, so maybe they are considered to be more complex and thus, an evolutionarily “newer” animal.

We told our students over and over: “Sponges are the most basal animals”.  But like many phylogenetic theories that have come before, new DNA sequencing data is challenging this view.

What does the ctenophore genome tell us?

First off, for the non-biologists out there, you need to understand one fundamental thing about gene evolution.  Two species that are highly related will have very similar DNA sequences.  The further apart two species are in evolutionary time, the more time there is for mutations to change the DNA sequences and the gene functions.

Moroz et al. sequenced one of the ctenophore genomes and then compared it with the genomes of sponges and cnidarians.  One of the main findings was that there are many missing animal-specific genes that are involved in animal development (Hox genes), regulating gene expression (no miRNAs!) and innate immunity.  Although some animal-specific genes are absent, the ctenophores have many unique genes that are not found in other animals, indicating that these genes evolved independently in the ctenophore lineage. 

The researchers devoted a lot of the paper to looking at genes involved in nervous system function.  Ctenophores, like cnidarians, have neural nets, as opposed to organized bundles of nerves.  Bilateral animals have many different neurotransmitters, which are the signals that get sent between nerve cells.  The ctenophores only have genes for making the neurotransmitter glutamate (and GABA), but they have a ton of glutamate receptors, more than other animals.

All of these findings led the authors to conclude that ctenophores are the most basal animals, not sponges.  Alternatively, it is still possible to keep the same phylogenetic tree, but there would need to have been massive gene loss in the ctenophore lineage.  The most parsimonious explanation is shown here:

The main difference between these two trees is that the sponges and ctenophores have swapped positions.  Note that this would require a nervous system to have developed twice independently.  That’s totally insane.  The ctenophores and the cnidarians “needed” a method of controlling their body to capture prey and both lineages “came up” with the same solution (of course evolution is random and doesn’t have a particular goal in mind).  When similar structures evolve independently, this is known as convergent evolution.

Next year, instead of devoting half a lecture to sponges and tossing in a single slide on ctenophores, I think I’ll have to give ctenophores their due, as potentially the most ancient lineage of animals still in existence. 

Carl Zimmer always beats me to the punch, so here’s his take on the same article.  Better writing, but fewer trees!

The mosaic female brain

Female mammals have two copies of the X chromosome while males have only one copy (because they have a Y chromosome instead).  Chromosomes contain genes and genes are the instructions for making proteins, so if females have twice as many copies of each gene on the X chromosome, will they make twice as much protein?  The answer to that is mostly “no”.  In young female embryos, one X chromosome is randomly inactivated and will remain that way through her life.  The chromosome gets compacted into a structure known as a Barr body.  However, when X inactivation occurs there are many embryonic cells and each one can inactivate one copy or the other.  Why does this matter?  Well, remember that one X chromosome came from dad and one from mom, so there may be different variants for each gene; different versions of proteins can be made depending on which X chromosome is still active in that cell.  In other words, females are genetic mosaics, where each cell may express one X chromosome or the other.  That’s cool!

What if the female embryo inherits one good copy of a gene and one bad copy that is non-functional and disease-causing?  Some of her cells would express the good copy of the gene and be fine and other cells would express the bad copy and be messed up.  The severity of the disease for this female will depend on how many cells inactivated the good copy and where these cells are located in the body.  Imagine that X inactivation occurred at the 4 cell stage, where two cells inactivate the good chromosome and the other two cells inactivate the bad chromosome.  Once an X chromosome is inactivated, it will stay that way in all the cells that are formed from that original cell in the 4 cell stage (see the figure below).  If each one of those 4 cells divides the same amount to form the final adult form, then you would expect half of her cells to be messed up and half of them to be fine.  But what if the two cells with the active bad chromosome happen to be cells that will divide way more and make way more future tissues of the body?  Then in the adult form, she would have tons of messed up cells and probably have a much more severe version of the disease.

In females one X chromosome is inactivated early in development (image from www.scoop.it)

As I mentioned earlier, X inactivation actually happens later on in embryonic development when there are more cells and each one can choose to inactivate one chromosome or the other.  If we consider the disease scenario, this random nature of X inactivation can lead to huge variability in X-linked disease expression in females.  It’s also important to think about how certain types of cells and tissues develop.  If an entire tissue develops from a single cell after X inactivation, then all of the cells in that tissue will have the same inactivated chromosome. 

Researchers at John Hopkins University visualized X inactivation by marking expression from each X chromosome with a different fluorescent protein.  Wu et al published their beautiful images in a recent article in the journal Neuron.

Marking X chromosomes

The authors created two types of mice, which each had an extra inserted gene on the X chromosome.  One type had a gene that encodes a red fluorescent protein called tdTomato.  The other mice had a gene for the green fluorescent protein, or GFP, which was originally discovered in jellyfish.  They then mated these two mice together and used the female offspring that had one X with tdTomato (X_t) and one X with GFP (X_G).  If the “red” chromosome is inactivated, then only GFP will be expressed and this cell will look green, as will all of its daughter cells.  This way they can look at the heterogeneity of X chromosome expression in different parts of the body. 

The results

Overall, the mice came in all different amounts of red and green.  For instance one mouse might be nearly all green while its sibling is all red, again indicating that X inactivation is a random process.  In the mice that had both red and green, it was interesting to see the different patterns in the body.  For instance, in the intestine, cells of the same color were found in columns.  That’s because the cells in the column originate from one single stem cell, so they should all contain the same active X chromosome.

X inactivation appears in columns in intestinal tissue, because cells from a single stem cell migrate together

 Another interesting finding was that skeletal muscle cells expressed both red and green fluorescent proteins.  This would seem to indicate that there is no X inactivation in muscle, but this is not the case.  Skeletal muscles are actually formed by muscle progenitor cells (myoblasts) that fuse together, creating cells with multiple nuclei and copies of the genome.  If a cell with an active “green” X chromosome and a cell with an active “red” chromosome fuse together, then the muscle will express both proteins.  This only works for skeletal muscle; cardiac muscle in the heart does not develop by cell fusion, so these muscle cells are either red or green.  This is a great demonstration of the differences in muscle development.

Skeletal muscle cells express both X chromosomes, because they are formed via cell fusion

 They also noticed clear differences between the left and right side of the body, like in the tongue, retinas and brain.  This indicates that progenitor cells stay segregated to either the left or right side during development.  In other words, there is not a lot of migration between the two sides of the body, where a cell on the right side would make cells for the left side of the body, and vice versa.

The mosaic brain

The main focus of this paper is on the heterogeneity in the nervous system.  They looked at two different cell types in the brain: excitatory pyramidal cells and inhibitory interneurons.  These two types of neurons develop from different areas of the embryonic brain.  They found that inhibitory interneurons were highly mixed.  When they quantified the fraction of red inhibitory cells in two different parts of the brain, the values were very similar.  On the other hand, when looking at excitatory neurons, there was a lot of variability of which X chromosome was inactivated, across different parts of the brain and in different animals.  If there was an X-linked gene that affected excitatory neuron function, then the effects on neuronal circuits would be different for different regions of the brain in an individual.  The authors suggest that this could actually be a good thing, because it would allow females with different genetic variants to respond to a range of stimuli, increasing the dynamic range. 

So there are bad aspects of X chromosome inactivation, like the expression of X-linked diseases, but there are also some good points, like increased functional diversity of neurons.  The authors suggest that X inactivation “may represent one of the more significant mechanisms by which individual differences in central nervous system function are generated.”  It is crazy to think that random inactivation of a chromosome in the early embryo might give us our future individual personalities. 

Stop seizures with a brain graft

There are two types of neurons in the brain: excitatory and inhibitory neurons.  They do exactly what you think they would.  Excitatory neurons release chemical messengers, which activate other neurons, which may eventually lead to some sort of perception or action.  Inhibitory neurons release chemicals that silence other neurons.  Why would you want inhibitory neurons in your brain?  Well, if all your neurons were excitatory and interconnected, all your neurons would be active all the time and the signals would be meaningless.  In fact, this sort of overactivation in the brain can lead to seizures.  It’s been shown in numerous cases of epilepsy that there is some sort of dysfunction of the inhibitory neurons.  The excitatory neurons have free reign and go crazy, leading to a seizure.

How is epilepsy treated?  Medications that potentiate the inhibitory neurons can help, but they activate all inhibitory neurons throughout the brain, when maybe the problem is more localized to one spot.  Just as all excitatory neurons is a bad thing, too much inhibition is also bad and can lead to cognitive side effects.  Another treatment is to open up the patient’s head, try to find the overactive area and cut it out or zap those neurons with a laser.  Destroying brain cells is always a last resort, though.

In a recent paper published in Nature Neuroscience by Hunt et al., the authors propose another potential treatment: adding new inhibitory neurons into the epileptic brain.  Like all new medical ideas, the story starts with mice.  They can create a model of human epilepsy in these mice by treating them with a potent drug.  These epileptic mice have seizures just like humans do.

Where do you get new inhibitory neurons?

The researchers obtained progenitor cells from mice embryos.  In other words, these weren’t inhibitory neurons yet, but they were destined to turn into them as the mice developed.  They grafted these progenitors into adult epileptic mice in the hippocampal region of the brain (a common area for seizures).   Amazingly, these pre-neurons migrated throughout the brain region, as far as 1.5 mm (that’s a lot… think about how small a mouse brain is).  Then the progenitors differentiated into inhibitory neurons, as if they were in a normal developing brain.  One week later, the epileptic mice with extra inhibitory neurons had hardly any seizures, whereas the untreated mice were having about 2 a day.  Not only that, but the treated mice showed cognitive improvements compared to the untreated epileptic mice. 

So they seemed to “cure” the epileptic mice by giving them some new inhibitory neurons that were able to make functional connections with the existing neurons.  This isn’t as invasive as brain surgery and it’s much more localized than medication.  If the epilepsy were focused in a different part of the brain, then they could transplant the cells there instead.

Is this possible to try in humans?  Maybe so, but the first problem is that we can’t take inhibitory progenitor cells from human embryos.  There are some ethical issues with growing clones to harvest parts from them.  However, you could use embryonic stem cells, or induced pluripotent stem cells.  Pluri-what?  Recent technology allows researchers to take a skin biopsy, do some genetic engineering to these cells and push them back in developmental time to a stem cell.  Pluripotent means that these stem cells have the potential to become any type of cell, like an inhibitory neuron.  All it takes is turning on the right genes in these cells to push them to a particular fate, and if that isn’t already known for inhibitory neurons, I bet it’s not too far off.  Plus there’s the benefit that the transplanted cells will have the same genome as all the patient’s other cells, because they originated from their skin cells.  Just wait, regenerative medicine is moving ahead at lightning speed.

A biological reason for aging weight gain

There is a growing epidemic of obesity in the aging population.  Of course a lot of this has to do with our cultural lifestyle, but could there also be a biological explanation related to the way our bodies age?  One clue comes from the fact that older lab mice have a tendency to become obese, without the added influence of fast food restaurants.  Although there certainly are differences in metabolism as you age, the older mice also intake more food; it’s as if their body isn’t telling them the “I’m full” signal.  Yang et al examine the biological mechanism underlying this age-dependent obesity in the newest edition of Neuron. 

The Hypothalamus

There is a region towards the interior of the brain called the hypothalamus which controls all sorts of basic physiological parameters.  For instance, it sets the body temperature, monitors blood pressure and the water content of the blood, and initiates the feeling of thirst and hunger.  There are a group of neurons in the hypothalamus called POMC neurons, which release a hormone, called a-MSH, which decreases appetite (the feeling of “I’m done eating”).  Could it be that these neurons don’t function properly in older mice, so they aren’t getting enough a-MSH to signal them to stop eating?

Problems with the POMC neurons

The authors find that as the mice get older, their POMC neurons get more negative inside.  Remember that active neurons fire action potentials, which are basically short bursts of positive ions rushing into the cell.  If the POMC neurons are more negative than usual, they will have further to go to fire an action potential and will be less active.  The older POMC neurons are in fact much less active and therefore release less a-MSH.

What makes the older POMC neurons more negative?  The authors find that the neurons are overexpressing a potassium channel (K channel), which will mean there are more open pores in the membrane for K to escape the cell.  As the positive K ions leave the neuron, it will make the inside more negative.  Okay, but why are K channels overexpressed in older neurons?  Turns out this whole cascade is initiated by a key signaling protein called TOR.  Increased TOR levels have been associated with various aspects of aging before, and an inhibitor of TOR (called rapamycin) can increase the life span of mice and other animals.  Check out the diagram below, which puts all these steps together.

Summary: For whatever reason, POMC neurons overexpress TOR, which makes these neurons less active.  They release less of the a-MSH hormone, so the mice don’t get the “stop eating” signal and continue to intake food, leading to obesity.

Two complimentary experiments

To test that this pathway is actually correct, the authors did two complimentary experiments.

1) If TOR is artificially increased in young mice (to mimic older mice), will they intake more food and gain weight?

2) If TOR levels are decreased in older mice (to mimic younger mice), will they lose weight?

For experiment #1, they raised TOR levels in young mice by knocking out an upstream inhibitor of TOR.  TOR levels are normally controlled within a certain range by inhibitors, so if you get rid of that inhibition, there will be more TOR present.  Over many weeks, these mutant mice did in fact get fatter than the controls and their POMC neurons were too negative and didn’t function properly, just like older mice.

For experiment #2, they wanted to decrease TOR in older mice.  This is actually pretty easy to do by injecting the older mice with the drug rapamycin, which inhibits TOR (TOR actually stands for Target Of Rapamycin).  Rapamycin is made by bacteria, which were first discovered in soil samples from Easter Island.  It is currently approved for human use, as an immunosuppressant for organ transplant patients.  When the older mice were injected with rapamycin for a few weeks, the POMC neurons came back to life and fired many action potentials.  The cells weren’t so negative because there were less K channels being expressed.  And yes, rapamycin caused the older mice to eat less and lose a considerable amount of weight.

So there you have it: rapamycin is the wonder drug—it will make you live longer and healthier, but there will be a price to pay with a lowered immune system.  There may be other ways to tap into this dysfunction in the older POMC neurons to help prevent midlife obesity.