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.

Go go gadget extendo filopodia

I’m back from an intense semester of learning and teaching Developmental Biology.  One theme that emerged from my studies was that the development of organisms is centered around gene expression and cell to cell signaling.  Often times, one cell will differentiate into its mature form, and then release a signaling protein that tells neighboring cells what to develop into.  For instance, the nervous system is induced by signals released from the embryonic backbone.  There are a number of common signals that are used over and over throughout development, like BMP, Wnt and Shh.

A recent paper by Sanders et al., published in Nature, looked at how distant cells can signal to each other via the Shh pathway.  Unfortunately for Developmental Biology teachers everywhere, Shh stands for Sonic Hedgehog.  Oftentimes, strange or humorous gene names like this can be blamed on the fruit fly researchers who first discovered the gene, but in this case everyone is to blame.  This gene was originally discovered by researchers studying fruit fly embryonic development; they named the gene hedgehog because the mutant embryos had lots of tiny bristles all over, kind of like a hedgehog.  The mammalian researchers took it to the next ridiculous level, by naming the mammalian version of this gene Sonic Hedgehog.  The Shh protein is a secreted signal that binds receptors on other cells, which activate gene expression in the receiving cell.  Shh signaling is important for specifying many different cell fates, such as the different neurons in the spinal cord, the cells that become the vertebrae, as well as the formation of the digits of the hand.

Although Shh is secreted from the cell, it has chemical modifications that make it stick to the plasma membrane that surrounds the cell that released Shh.  How then can Shh induce the development of cells that are located at a distance?  Well, the answer is by stretching out long cellular extensions with Shh localized at the tip.

Shh Filopodia

Sanders et al. did live imaging of cells in the developing limb of the chicken using fluorescent proteins.  They did some genetic trickery so only a few cells were labeled in red and others in green.  This way they could detect individual cells in a sea of unlabeled cells and examine their structure in real time.  They observed individual cells extending long protrusions, called filopodia, from the cell bodies.  These filopodia could stretch long distances (150 micrometers, like 3-5 cell widths) and were dynamic-- retracting and growing over time. 

How to think about filopodia?  Imagine a stretchy balloon with a stick inside of it.  If you could push that stick into the wall of the balloon, the balloon would protrude from that one spot as the stick pushes it out.  That is like a filopodia, where the balloon wall is the plasma membrane and the stick is a protein called Actin.  Actin forms long chains that can grow, pushing out the membrane in front of it. 

The thin, string-like extensions from this cell are filopodia and are filled with Actin.  Image from proteopedia.org

The authors then labeled the Shh protein with another fluorescent marker and saw that it localized to the tips of filopodia.  Not only that, but the filopodia expressing Shh were more stable and did not retract as often.  In order for Shh to act as a signaling molecule, it has to bind a receptor on another cell.  Using a different color, the authors observed two co-receptors for Shh localized to filopodia from other cells.  They even saw filopodia from two different cells make contact with each other, where one cell expressed Shh and the other expressed the receptors.

This is amazing!  Instead of releasing a signal out of the cell with the hope that it goes to the right place and isn’t degraded, the cells literally grow to the right place with the signal on their membranes.  This is like hand delivering a note to your coworker, rather than making the note into a paper airplane and throwing it in the direction of their desk.

This is how I imagine this working.  Two cells that are located at a distance, reach out extensions and meet somewhere in the middle. The Shh signal would bind the receptor, causing changes to the pink cell.

A study like this could not have been done before recent innovations in live imaging and molecular biology to introduce the fluorescent proteins into the cells.  The filopodia are not preserved during the more traditional, static method of fixing cells with formaldehyde and then staining them.  Who knows what other tricks live cells use during embryonic development.  I suspect this is only the beginning.

Swapping eggs

This week’s paper describes a new technique that could be used to manipulate human oocytes (i.e. eggs) to prevent a group of diseases called mitochondrial diseases.  The paper was presented by Tachibana et al. in Nature along with a similar paper by Paull et al.  For the sake of brevity, I will only discuss the findings from the first paper.

Mitochondria

So what are mitochondria?  Mitochondria are little compartments in the cell that make cellular energy.  They convert the energy stored in food into an energy source that the cell can use to drive chemical reactions.  In other words, they are absolutely essential for our survival.  The oxygen that we breathe in goes to the mitochondria to aid in this energy conversion, and we all know how vital oxygen is. 

There are two other interesting facts about mitochondria that relate to our story:

1) All the mitochondria in our body are duplicates of the mitochondria that were in our mother’s egg.  In other words, embryonic mitochondria are not made from our genomic DNA (gDNA) or from sperm contributions.

2) Mitochondria have their own DNA , which directs the synthesis of proteins that are necessary for their function.  This DNA is known as mitochondrial DNA (mtDNA) and it is only inherited from the mother, since all mitochondria originate from the egg.

If there are mutations in the mtDNA, then this can lead to problems with the synthesis of cellular energy, which can lead to human diseases known as mitochondrial diseases.  There are different types of mutations, which can affect people in different ways and with differing severities.  In this paper, the authors propose a way to prevent mitochondrial diseases from being inherited from generation to generation.  Let’s see how that works.

Nuclear transplantation

Let’s say you have a female patient with a mitochondrial disease, who wants to have a healthy child.  She is guaranteed to pass this disease on to her child via the mitochondria in her oocytes.  However, most of what makes the child “hers” is what lies in the mother’s genomic DNA, not in the mitochondrial DNA.  What if you could take the mother’s genomic DNA (plus the DNA from the father) and stick it into a healthy “enucleated” oocyte from a donor who has good, functioning mitochondria?  All the genomic DNA will have to be cleared out of the donated oocyte first, creating an enucleated egg.  The embryo that results from this nuclear transplantation will have genomic DNA from its mother and father, but its mitochondria will originate from the donor oocyte.  This would circumvent the mutated mtDNA that is in the real mother’s oocyte.

Tachibana et al. obtained human oocytes from volunteers and transfered the genomic DNA from one into another.  They then injected these oocytes with sperm DNA (like during real fertilization) and observed what happened.  Some oocytes failed to be fertilized and others died soon after, but a handful of oocytes survived into the blastula stage of development.  You can’t really grow a human embryo in a dish beyond the blastula stage and they are not allowed (yet) to implant these into women, so we don’t know what would happen to a child born from this procedure. 

They did carry out the above scenario with monkeys.  They transplanted the genomic DNA from one oocyte into another and implanted the blastula into another female monkey who carried the embryo to term.  The monkey youths are 3 years old now and doing just fine.  Their maternal genomic DNA is from one mother and their mitochondria are from a different oocyte donor. 

Isn’t this amazing?  I seriously doubt this procedure will be approved for human use anytime soon, because it’s too much like cloning, which basically follows the same procedure of putting genomic DNA into an enucleated egg.  It's a cool idea, though. 

Isolation and drug addiction

We all know that adverse, early life experiences can affect normal development and the ability to lead a happy and healthy adult life.  A number of recent studies have shown that rodents which are mistreated as pups have long lasting changes to their gene expression (i.e. epigenetics).  They are more anxious and have a harder time forming new memories.  A paper this week in Neuron builds upon these results, by studying the effects of social isolation on the “reward pathway” in the brain.

Reward pathway

What is a reward pathway?  Deep in the brain is a region known as the ventral tegmental area (VTA), which makes connections to the nucleus accumbens and prefrontal cortex.  When we do something that is naturally good, like eating or sex, the neurons in the VTA release dopamine onto the nucleus accumbens and we interpret that as “feeling good”.  This is our reward for doing something that will help us survive and procreate. 

The Reward Pathway in a brain cross section (from brainfacts.org)

Many drugs of abuse like cocaine, amphetamines and alcohol increase the amount of dopamine signaling in this pathway; this is one reason why drugs produce a “high”.  When this pathway gets overstimulated by increased drug use, the brain will try to compensate by making the pathway less efficient.  This is why drug users feel depressed when not on drugs and why higher and higher concentrations of drugs are necessary to produce the same high feeling.  This is a neurological explanation for drug addiction.  Drug abusers also start to make connections in their lives, and in their brains, between environments (a certain room, certain people, etc) and the feeling of reward.  Getting sober is so difficult because the brain has to unlearn these connections and the reward system has to recover back to its normal level of activity.

Plasticity

Before we talk about the paper, I need to introduce one more concept.  Neurons become activated when channels in their membranes open and positive ions rush in.  They can then pass on this signal to another neuron by releasing neurotransmitters (like dopamine) onto the next neuron.  The activity in the neuron and the amount of transmitter it releases into the synapse can change over time, based on that neuron’s previous experiences.  This is known as synaptic plasticity.  There are short-term changes, like facilitation, and longer-term changes (we’re talking hours and days here).  One of the more famous types of long-term plasticity is called long term potentiation (LTP) and is thought to underlie learning and memory.  When drug users start to become addicted, these types of long term changes to neuronal activity are occurring throughout the reward pathway.

Social isolation and VTA neurons

The experiment begins when young male rats were either housed together in groups of 3 or alone.  The researchers then recorded neuronal activity of VTA neurons under various conditions.  They found that rats that were isolated for more than 3 weeks, specifically during the equivalent of the rats’ early adolescence, can more easily induce LTP in the VTA neurons.  In other words, rats that had no social interactions during a critical period had more sensitive VTA neurons.  That is to say, their reward pathway is primed to be overstimulated, just like during repeated drug use.

What are the behavioral manifestations of having a sensitive reward pathway?

The next set of experiments they did is called conditioned place preference.  The rats were placed in a cage that had two different compartments, with different wall colors and floor textures.  The rats were then injected with amphetamine in one of those particular compartments, so they learned to associate the drug high with that environment.  The rats were then given a choice between the two compartments and inevitably they went to the room that was associated with the drug.  The researchers found that isolated rats had a greater preference for the drug room and developed the preference sooner than the control rats.  Social isolation as an adolescent causes an increased rate of learning an association between drugs and environment.  This could make these rats more vulnerable to drug addiction. 

What about unlearning the drug association?

After the drug testing, the rats were exposed repeatedly to the drug room, but this time they didn’t receive any drugs.  This is called extinction of a memory and it is measured by the rats losing their preference for the former drug compartment.   Socially isolated animals had a significantly slower rate of unlearning the preference.  Their memory was more resistant to extinction.  If their VTA neurons are overly sensitive, then it may be harder to rewrite that connection in the brain between environment and reward.

In the context of drug addiction, these findings are big.  An adverse early adolescence can prime the brain to develop addiction more easily and make it harder to sober up.  If the VTA neurons start firing every time you go through an environment associated with drugs, you’re going to want to take a hit again.  The authors bring up an interesting point that social isolation generally causes a depression of neuronal activity in places like the hippocampus (the site of learning), so maybe the increased activity in the VTA is the way for the brain to maintain some sort of homeostasis – some areas increase, some decrease, but overall the brain may have normal amounts of activity.  This is an interesting way of looking at this problem.  I suspect that social isolation offers little in the way of rewards, so the reward pathway is trying to compensate by getting more sensitive.  It will be interesting to see if there is also a connection with changes in gene expression.  The authors explain how the VTA neurons get overactive, from a cellular point of view, but what actually initiates those changes?  And how can social interactions feed into the biology of the cell?