Someday with a Scientist

Parkinson’s Disease and Mutations in the Protein DJ-1

I always struggle with talking about science, especially to the lay audience. It’s something I actually want to perfect. So, what better than to bring up the research closest to my heart? When I say heart there, I really mean my thesis project. Practically the same thing at this point in time!

Before entering Mark Wilson’s lab at University of Nebraska-Lincoln, the lab published a paper called “Structural impact of three parkinsonism-associated missense mutations on human DJ-1” in the journal Biochemistry back in 2008. If you are really curious and want to read the paper in it’s entirety, which I recommend because, quite frankly, it’s a great paper, you can go here.

This paper pretty much sets up my thesis work, in a nutshell. (I really do mean nutshell here. All of my blood, sweat and tears over the past 5-6 years will boil down to a single slide in my boss’s presentations, and eventually maybe even a single bullet point. This is just too sad to focus on, so let’s talk DJ-1.)

When DJ-1, a small conserved protein of 189 amino acids, is absent or genetically mutated thereby making the protein inactive, humans get Parkinsonism. Parkinsonism differs from Parkinson’s disease in semantics only, really. You see, you can’t be diagnosed with Parkinson’s disease (henceforth called PD) until autopsy. At this stage, they will look in specific regions of your brain for some hallmarks of PD (such as loss of dopaminergic neurons in the region of your midbrain called the Substantia Nigra pars compacta). It always helps to illustrate what this all means in science. So, here are a few very neat pictures.

DJ1
[Source: This is DJ-1! The image in (a) looks like a crazy person staring you down. If you can imagine yourself hitting this person with your palm, and the head rotating away from you, revealing underneath the chin area, that is what you see in (b). If this is too complicated a visual, then think of a 90 degree rotation in to the screen of (a) and you will get (b).]

I work in a lab that visualizes proteins by means of X-ray Crystallography. So, the model representation of the structure I showed above was solved by my lab! Pretty nifty, right?

PD_brain
[Source: I really like this image, and I often show it in my data presentations. Not only does it show you exactly what (small) area of your brain is affected, but it shows you how it’s affected! In a normal brain, you have a dark substantia nigra (the top right picture), and this is because you are producing plenty of dopamine. This pigmentation becomes lesser when you have Parkinson’s disease (the image just below the normal one), and this is what results in loss of motor functions, etc. If you are interested in more information on Parkinson’s disease, feel free to visit these websites: MJF Foundation, National Parkinson Foundation, Parkinson’s Disease Foundation, or the Mayo Clinic, among many other sources.]

Back to this paper!

Several mutations in DJ-1 have been implicated in Parkinsonism in humans. One of these mutations has been extensively studied and is called L166P, meaning in the normal, or wild-type, DJ-1 protein, we see a leucine at position 166 and in the Parkinsonism-associated version of the same protein, this leucine is mutated to a proline. This amino acid can be found in the above DJ-1 structure as a red stick figure in the “smile”. In technical terms, this mutation lies on the dimer interface, and when it’s mutated, it disrupts the ability of DJ-1 to form the dimer, which causes the protein to just unravel. When it’s unraveled, it can’t protect the cells anymore, and you present with Parkinsonism. What this boils down to is this: you don’t want this mutation. It’s bad news.

But what about other lesser studied mutations? How do they cause Parkinsonism? Is it by the same mechanism as L166P? These are great questions, my readers, and I am soo00 glad you asked them! In short, we don’t know yet, and this is my thesis project (so I can’t really spill the beans yet, because it’s not published. But, it’s all very exciting, I assure you!)

This paper really starts looking at some other mutations that are known to cause Parkinsonism, and they are A104T, E163K, and M26I. Again, the first letter in each of these instances is what should be there to have a normal protein, but alas, they are mutated to the second letter in an abnormal protein.

What did this paper find out about these three mutants? First of all, that these mutations all reduce thermal stability of DJ-1 (meaning, it falls apart at lower temperatures), but the structure of each mutant (because that is what my lab does) barely changes at all! This is BIG news because the structure of L166P can’t be figured out by X-ray Crystallography because it’s a jumbled heap of a mess and has no structure to determine. Luckily, other methods (primarily NMR) has illustrated this nicely! So, now we have the normal protein and 3 mutations in that protein, and all 4 of these versions looks nearly identical.

This basically sets up my thesis project, which aims to understand the mechanisms of action of some of these mutations. Basically, I’m trying to figure out why people get Parkinson’s disease. No big deal.

Science basics that EVERYONE should know

Yesterday, I found myself in the same old rut; perusing Facebook when Lo! and behold, my cousin Crystal posted a video saying “1 in 4 Americans unaware that earth circles sun”. I immediately thought “NO! This can’t be … I must watch this video (and hope it’s not a virus.)” To find out, IT’S TRUE!! Don’t believe me? Watch it here.

Needless to say, today, I find myself inspired. Maybe I’m going to far in to science with my science posts? Maybe, I need to take a step back so everyone is on the same page – and, just in case you get stopped on the street for a science pop quiz, you will know the answer! Plus, trivia night, anyone?

1. All of the planets orbit (revolve around) the sun. (Feel free to watch the video again). Lighter objects always orbit heavier objects. This is why satellites and the moon orbit earth. Do you remember anything about Sir Isaac Newton? It was Newton who realized that the planets must orbit the sun in much the same way an object falls to the earth after we drop it. Basically, the sun’s gravity pulls on all of the planets, causing the planets to orbit it. It is also important to note that the bigger an object is, the larger it’s gravitational pull. It goes without saying that all of you know that the sun is the largest object in our solar system. NASA has some amazing resources for this subject. I should also mention that it takes 1 year for the earth to orbit the sun.
Sun
[Source]

2. There is a difference between a scientific hypothesis, theory and law. And those differences aren’t what the general public seems to think they are. Let’s start at the beginning. A hypothesis is an educated guess that is based on observations. You may remember making “If … then …” statements back in middle school. If not, they kind of sound like this: If I punch my little brother, then he will cry. This statement has to be testable. In our example, I could walk up to my little brother and give him a punch. Then, I will observe the aftermath. Does he cry? What’s wonderful about a hypothesis is, you could be wrong, BUT you will get a scientific answer. In our case, my brother could cry, he could do nothing, or he might yell for mom. Our observations can change our hypothesis, to make it better, so we can get better answers. To hammer home this idea, I might be able to punch my brother 10 times with him crying each time. On the 11th try, he might punch me back!  A theory summarizes a group of hypotheses made by lots of people. It’s important to re-state that these hypotheses have undergone a lot of testing and re-testing by a lot of scientists. A theory is accepted as fact as long as there is no evidence that disputes it. Once this evidence arises, the theory is not thrown out, but changed to accommodate the new observation. Basically, a theory explains why and how something happens. For example, when we talk about gravity, we use Einstein’s theory of general relativity. A law explains things, but does not describe them and it does this by generalizing a body of observations from many hypotheses that have been tested and re-tested by many scientists. A great way to distinguish a law from a theory is to see which one answers why something happens. For example, Sir Isaac Newton had a Law of Gravity. This law could predict that an object would fall, but it couldn’t tell us why it fell. For a list of 10 laws and theories everyone should at least be acquainted with, check out this article here.

3. Evolution. I was debating whether or not I should include this … as I really don’t enjoy talking about controversial things. However, everyone should at least know about it, whether you decide to believe in it or not. If you want to read more about all the intricacies of Evolution, check out Darwin’s Origin of Species (it’s even free for the Kindle App!) Many of you have heard of, and perhaps even said, the phrase “survival of the fittest!” – that’s evolution you are talking about! There are many genetic changes that can be inherited, and these small changes, over the course of time, add up to larger changes. This is why every new generation of people differs slightly from the one before it. Many of these changes have negative or neutral effects, but sometimes, there are positive ones – hence, survival of the fittest. The rate of evolution varies a lot and it depends on the organism and environment. Recently, Bill Nye (the science guy!) and Ken Ham debated evolution and creationism (see the entire debate here). When it came down to evolution and creationism, both of them said the same thing, just using different words. Ken Ham said Noah saved 7000 couples on his little arc, and because of that, we have all the ones we have today. I found the below picture to really hammer home the math. The picture’s link will take you to someone else’s perspective on the debate.
evolution
[Source]

4. The sky is blue because of scattering. It’s not blue because it’s a reflection of the ocean waters (like what I believed when I was in elementary school). Let’s think about this. We have a sun, that we orbit around (see #1 above), and in order for that sun’s rays to reach earth, the rays have to penetrate the earth’s atmosphere. This atmosphere includes things like gas and particles that cause the sunlight to bounce (or scatter) off of them in many directions, and this sunlight is actually a lot of different colours. Have you ever seen a light flashing on a prism or maybe Pink Floyd’s Dark side of the moon album cover? All of the colours in sunlight have different wavelengths, with blue being very short. So, blue light can make it’s way through the “filter” (i.e. atmosphere) easier than colours with larger wavelengths, and, as a result, the blue light is scattered very widely. This is why the sky is blue when the sun is at the highest point in the sky, in relation to where you are standing. So, how do we explain the beauty of sunsets and sunrises? At these points, the sun is not above where you are standing, it’s much farther away. The sunlight has to cross a great distance to reach you, and this distance allows the blue colour to, essentially, fade away, allowing us to see the reds and oranges and yellows of the light!
prism
[Source]

I think we will stick with these four things for now. If you are curious about anything, feel free to post your questions in the comments section below. Remember, we aren’t debating anything here (there are other blogs and forums for that).

The first ever plague

Time to talk about one of my favourite things: Infectious diseases. Seriously, I got in to science because I fell in love with the movie Outbreak! I know you would probably have never guessed, considering I’ve been studying neurodegenerative diseases for the past 8 years (Sheesh, am I really that old?), but I do really love them. Well … infectious diseases, serial killers, and mother nature. What can I say? I’m obsessed with destruction.

A new article was just published in The Lancet Infectious Diseases journal (you can sign up for a free account and gain access to all of their articles here) called “Yersinia pestis and the Plague of Justinian 541–543 AD: a genomic analysis.” This article is so recent, that it’s not actually in print yet, but you can find it in the “Online first” section. There is also a Comment made on this same article (kind of like a short ~1 page synopsis) that you can read too.

Lancet
[Source]

Anyway – on to the amazing things found in this article!

To begin, it is important to mention that there have been at least three human plagues: The Justinian Plague (6-8th century), Black Death (14-17th century) and the Third plague (19-20th century). I guess we ran out of cool names for plagues – but because they found that it came from the Yunnan Province of China, they could have called it the Yunnan Plague. Anyway, if you want to know more about plagues in general, visit the CDC and read away! This current journal article is going to focus on the Justinian Plague because of a few reasons: it’s not clear where it originated, the link between the first plague and the other two is not clear, it’s not clear whether or not the second two plagues evolved from the first one (did the plague get stronger or weaker over time?), and it’s not clear whether or not all of the plagues were just the same disease but continuously mutated (like our flu viruses) or brand new versions.

These researchers came upon a medieval burial site that contained many bodies (indicative of people dying from an outbreak) and when they radiocarbon dated the goods in the burial site, they found they dated back 1500 years – during the Justinian Plague! The coolest part is this: the researchers pulled some teeth and they still had blood in them and they were subjected to DNA sequencing! How amazing is that? Teeth, bones AND DNA surviving 1500 years?!

They found that the first pandemic was spread by fleas that bit some rats and, like the other plagues, they originated in China! They actually followed the Silk Road Trade Route!

Silk_Road
[Source]

Another finding? The first pandemic is a unique strain passed from rodents in to humans. It is not like the other Plague strains!

Discovery of a new cell death mechanism: Ferroptosis (fair-oh-toe-sis)

I have decided that I want a section of this blog to be about science, not just my frustrations with it. I will summarize some awesome papers (awesome to me) one at a time in as simple of a way as I can. I won’t be able to dismiss all the science jargon, but I will do my best. This topic is very dear to my heart because it is what granted me candidacy (where I went from a graduate student to a Ph.D. Candidate …. super cool title). Here, I present to you, a new cell death – but before we go in to that, I explain cell death first. I wrote this research summary for the undergraduates here at University of Nebraska-Lincoln, and I wanted to share it with all of you (assuming you don’t fall asleep). If you ever want a science-y topic to be discussed, hit me up in the comments below or send me an e-mail!

***

There are many types of cell death mechanisms. The one you are probably most familiar with is apoptosis (a-poe-toe-sis), or programmed cell death. In trying to understand apoptosis, more cell death mechanisms were discovered because certain things were just not consistent with apoptosis. Take necrosis, another type of cell death, as an example. In apoptosis, the cells shrink because components of the nucleus (DNA and proteins called chromatin) were condensing. Once everything gets so small, it starts packaging itself into smaller bodies, and breaks away from the original cell. Eventually, all of these dead bodies are taken in by other types of cells and completely destroyed. In necrosis, the cells get larger because the membrane is breaking which allows some of the extracellular fluid to get inside of the cell. This starts happening to all of the compartments within the cell, and eventually all of the contents, including enzymes and proteases, spill in to the extracellular environment. The released enzymes and proteases then chew away at the cell until it is destroyed. The differences between these 2 types of cell death can be seen in the figure.

Cellular differences between 2 major cell death pathways

Cellular differences between 2 major cell death pathways

In May 2012, a group recently started characterizing a new form of cell death, which they decided to call Ferroptosis [1] , mainly because it involves iron (Ferro) and they see death (so they just stuck with the ‘ptosis’). They came across this unique cell death pathway when they were trying to use a drug called Erastin to selectively kill certain types of mutants (RAS family of small GTPases) that cause cancer in 20-30% of all cancer patients. First, they took their cancer cells and incubated them with erastin, and in just 6 hours they noticed a lot of their cells were dead. They then decided to incubate the cancer cells with an iron inhibitor (called deferroxamine, or DFO), and they didn’t see any death at all. Next, they wondered what would happen if they put BOTH the erastin and the DFO on the cells. There was no death! This means that the death caused by erastin depended on iron being present! This is the very first time any cell death mechanism has required iron! Using these same scenarios, they measured the toxic products generated via the mitochondrial electron transport chain, called reactive oxygen species (ROS). This group did some measurements with a flow cytometer and found out that erastin also caused a lot of ROS to build up and the ROS were also dependent on the iron.

In science, just because you have a neat discovery doesn’t mean you neglect some controls. So, this group had to prove ferroptosis was in fact different from apoptosis and other types of cell death. So, they used a different type of cells and chemically induced the different types of cell death. After enough time had gone by, they took those cells and looked at them under a power microscope. They noticed the mitochondria were smalled in ferroptosis but any of the other cell deaths. So far, so good! They did a lot of complicated tests with many cell death inhibitors, and cell death inducers, to really hammer home the idea that ferroptosis was indeed unique.

Now, we have ferroptosis, which isn’t like any other type of cell death, and it requires iron and ROS. This group decided to figure out what makes ferroptosis genetically distinct. To do this, they used 8 different cell lines, and lowered the mRNA levels (with something called short hairpin RNA, or shRNA) for 6 genes they recently found in a screen. They found some iron proteins, and proteins important for translation, to be important in ferroptosis!

Because ferroptosis is new, this group had to find a way to restore growth so they could look at this death mechanism inside of a mouse, and they did this by finding an inhibitor of ferroptosis. They are a witty group and decided to call it Ferrostatin-1. When they were trying to figure out how ferrostatin-1 inhibited cell death, they realized that lipid (fat) generated ROS were crucial for this death mechanism.

Overall, this group found a new form of cell death, which they decided to name Ferroptosis. Ferroptosis requires iron-dependent production of ROS, NADPH-dependent oxidases (proteins that can transfer electrons across a membrane to an oxygen thereby generating superoxide, a type of ROS), and lipid peroxidation (oxygen takes away an electron from the lipids, thereby creating more ROS). With the use of a power microscope, it could be seen that the mitochondria were much smaller in cells that died from ferroptosis. Therefore, ferroptosis is a truly unique way for cells to die!

REFERENCES

1.                   Dixon, S.J., et al., Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell, 2012. 149(5): p. 1060-72.
IMAGE:      http://www2.uah.es/daviddiaz/Apoptosis/caracteristicas.htm