Journal Club – A mutation in mice to study ALS

Amyotrophic Lateral Sclerosis (ALS) is a debilitating neurodegenerative disease that affects neurons in the brain, brain stem, and spinal cord. When these neurons die, it affects the connections that they have to muscles throughout the body resulting in muscle weakness that affects speaking, swallowing and breathing leading to paralysis and eventual dead.  The cause of ALS isn’t known in 90-95% of cases.  However, recently scientists have identified a mutation in a gene nondescriptly called “chromosome 9 open reading frame 72,” which is abbreviated to C9ORF72.  This mutation results in six nucleotides, GGGGCC, being repeating up to 1000 times within this gene in ALS patients.  Even though this is the most common mutation found in ALS patients, it’s still unclear exactly how these repeats affect neurons or the progression of the disease. The two hypotheses that are most studied are that the mutated C9ORF72 makes mutated RNA transcripts (RNA is the molecule that usually helps DNA be translated into proteins) or makes unusual proteins called dipeptide repeat proteins (DRPs), each of which can aggregate (clump) together and disrupt the normal activity of the neurons leading to neurodegeneration.

neuron_als_c9ORF72

The pink dots show clumped up RNA from the many many repeats in the C9ORF72 gene. Each panel shows these clumps in different areas of the mouse brain. Taken from the article Peters et al. 2015 Neuron

In two recent publications in the journal Neuron, researchers have used mice as a model system to look at how these large GGGGCC repeats in C9ORF72 affect the mouse nervous system.  Why use a mouse? First, scientists know how to experimentally change the mouse genome in order to add hundreds or thousands of repeats to a single gene, like C9ORF72.  Second, mouse and human genes are about 85% identical, so if scientists can understand how a gene like the mutated C9ORF72 affects neurons in mice, it may also help scientists understand how it works in humans. Third, by creating a mouse “model” of ALS, scientists can use this model to better understand ALS and to test potential future therapies (it’s worth noting that only one drug currently exists for ALS and it typically extends life only by a few months).

In each article, the mouse model was slightly different – one had a mutated C9ORF72 with 500 GGGGCC repeats and the other varied between 100-1000 repeats. However, both sets of researchers found the same results.  The mice had aggregated RNA transcripts and DRPs in their neurons just like what are found in human patients with ALS, but none of the mice had behavioral changes or neurodegeneration that are seen in human ALS patients.  So why didn’t the clumped up RNA and proteins cause neurodegeneration in mice like they do in humans?  There are lots of potential reasons – including the fact that even though mice and humans are similar, there are still lots of differences and mice may respond to these aggregated RNA and proteins differently than humans.  However, the authors of these papers suggest that other environmental and/or genetic factors along with the aggregates caused by the C9ORF72 mutation must be involved in developing the neurodegeneration.  It may also mean that getting rid of these aggregates before neurodegeneration occurs may prevent development of ALS.  Now that these mice are available to study, they should help in identifying the other factors involved in ALS development along with developing possible treatments for this debilitating disease.

Want to read the articles?  Unfortunately, they are behind a paywall, but you can see the abstracts here:

O’Rourke et al. (2015) C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD. Neuron. Volume 88, Issue 5, p892–901, 2 December 2015 Article

Peters et al. (2015) Human C9OFR72 Hexanucleotide Expansion Reproduces RNA Foci and Dipeptide Repeat Proteins but Not Neurodegeneration in BAC Transgenic Mice. Neuron. Volume 88, Issue 5, p902–909, 2 December 2015 Article

Read the Article from PN News that I contributed to here

Did your steak sit on the counter overnight? What experiments I did at work last week

I was at a party last night telling my non-scientist friends about my week at work.  For the first time since I left my post-doc 8 years ago, I spent multiple days in the lab doing experiments!! We had a few drinks at this point, and in their lapse of judgement (thinking I may not give them a descriptive answer at a party) they asked me what my experiments were about.  I did not disappoint, and told them all about my experiments.

This party was a BBQ and even though I’m a vegetarian, most of my friends are huge fans of meat.  So I started by asking them about steak.  If you bought a steak that you weren’t going to throw on the grill right away, you might freeze it and cook it later.  When you take it out of the freezer, how do you know if the steak is still of high quality?  Maybe it sat on the counter overnight before being put in the freezer.  Maybe the freezer lost power during a storm, the steak defrosted and when the power came back on, the steak froze again.  Maybe the steak wasn’t even good when it got to your house – maybe it sat on the butcher’s counter for a few days, forgotten about, before being put into the display case for you to buy. It may be nearly impossible to tell by just looking at your frozen steak if any of these events happened and whether or not these events would affect the steak’s quality.

frozen tissue

Steak image courtesy of Steven Depolo under a Creative Commons License

This is something that we deal with daily as biobankers. To remind you, we collect human tumor samples for researchers to use to better understand diseases and to develop improved treatments. Ideally these tissue samples are very quickly frozen (as described in this blog post) and kept frozen.  Like the steak, there are a lot of “variables” that may affect the quality of the tumor tissue sample – including the same issues that a steak may have – like sitting at room temperature for too long or freezing and then thawing and then freezing again. And also similar to the steak, it’s nearly impossible to tell by just looking at the tissue sample if any of these things have happened. Except, instead of affecting whether or not your dinner tastes good, with tumor tissue samples, research results that are essential for drug or biomarker development for brain tumors are affected.

How do we handle this little problem?  We have to use a proxy for quality – something that we can analyze directly that will tell us whether or not the tissue sample is of good enough quality to be used for certain research purposes.  In this case, the proxy is RNA.  RNA is a great molecule to look at because it’s found in every cell, but it’s also unstable because of its natural enemy RNases. When these RNases are active (when the tissue sample is warm or at room temperature) they will function like little PacMans to chomp on the RNA, turning it into smaller and smaller pieces. When frozen, the RNases are inactive and can’t chomp on RNA.  So if you look at how chomped up/small the RNA is in tissue samples, you can figure out whether the quality of the sample is good for your experiments.

rneasy column

A tiny filter (seen as the white line inside the pink tube) binds to the RNA.

This is what I did last week in the lab. How did I do this? First, I had to get the RNA out of the tissue sample. To do this, you have to separate the cells from one another, which essentially like grinding up a steak in a meat grinder. You then have to bust open the cells to get to the RNA.  You do that by adding something that works a lot like soap that opens up the outer coating of the cells.  From there, you can isolate the RNA by adding ethanol (an alcohol) that makes the RNA no longer dissolved in the liquid (what we call “precipitating” the RNA).  From there we isolate the RNA on a column – exactly like the pink one you see on the right. The white line inside the pink tube is like an RNA filter that traps RNA while letting all the other cell bits flow through. Then you use water to get the RNA out of the filter.  How much RNA do you get at the end?  Imagine an eye drop worth of liquid that contains 10 micrograms of RNA.  That’s 0.0001 grams and for comparison, a grain of rice weighs 0.015 grams.  It’s not a lot, but it’s enough to know if your sample is good or not.

screentapes

These ScreenTapes can analyze 16 RNA samples at one time. They are about the size of a long box of a glass slide or a ling box of matches

You still can’t “see” the RNA to know if it is all in one piece or if it’s been chomped up by RNases just by looking at this tiny bit of liquid in a tube.  You still have to analyze it, and to do this, I used a cute little machine called a TapeStation (I honestly have no idea why it’s called this, since the “ScreenTapes” that you use for the analysis – in the photo at left – look nothing like Tapes to me). This machine separates the RNA based on size (you can see that by the black lines in the image below).  There are two main sizes it looks at – these separate into to peaks (called 16S and 28S). If these RNAs have been chomped up, there won’t just be two peaks (or two black lines), but lots of little peaks of smaller sizes. This will indicate that the RNases were activated for some reason and the quality isn’t as good. In my experiments, the results were awesome and the RNA was of good enough quality for most experiments.  It also meant that the tumor tissue was handled really well when it was collected and didn’t sit on the counter or get thawed and frozen again, for example.

TapeStation

The two peaks are a graphical image of the separation of the RNA shown by the dark lines on the right.

Now you may be wondering, “what about my steak?” I honestly cannot encourage you to do this level of analysis to see if your steak’s RNA is high quality.  Then again, you just want to eat the steak – not do thousands of dollars of important experiments with it.  So let’s just consider the steak a useful scientific analogy and go start your grill – I’ve heard there are some steaks in your freezer.

Thanks to Kelli and Alia for asking me what experiments I’ve been doing and inspiring this blog post. 

How does a gene make a protein? Introducing RNA!

Genes are pieces of DNA that code for a protein.  That sounds great…but how does it do this? DNA is made out of nucleotides (A, T, C, and G) and proteins are made out of amino acids.  To “crack the code” and transform the DNA code into the protein code, you need an intermediary.  That’s a molecule called RNA!  messenger RNA  (or mRNA, for short) to be precise, because it is the message that communicates the information from the DNA to the protein.

Let’s revisit our car blueprint analogy to discuss the role of mRNA.  If DNA is the blueprint for the steering wheel, and the protein is the steering wheel, mRNA is the person in the middle who reads the blueprint and builds the steering wheel.

gene_mRNA_protein

So let’s see how this actually works.  The first step is to understand what RNA is actually made out of. DNA stands for DeoxyriboNucleic Acid and RNA stands for RiboNucleic Acid, and if they sound like they are related, you are correct. Here are the three main differences

  1. DNA is double-stranded and RNA is single stranded (see the photo here)
  2. DNA is made from A, T, C, and G and RNA uses A, (standing for uracil), C, and G
  3. In DNA, A always matches with T, whereas with RNA, A always matched with U

Because they are made up of similar components, the cell can copy the code from the DNA into the single-stranded mRNA molecule.  Also because these two molecules are related, this process is call transcription – like what you would do when copying the words in a book from one place (DNA) to another (mRNA).

The mRNA is then what codes for the protein.  It does this just like you would expect any code to – using a key.  In this case, each set of 3 nucleotides codes for an amino acid.  The cells have machinery (called a ribosome) that “reads” the mRNA strand.  The combination of “ATG” always tells the ribosome to START making a protein.  It then reads the three digit code, adding the appropriate amino acids along the way, until it reads one of the three codons that tells the ribosome to STOP making protein.  At this point, the protein can fold up to make the 3D structure so it will function.  Because this process takes nucleotides and codes them into amino acids (a totally different molecule), this process is called translation.  Just like translating a book from English into French, this process translates mRNA into a protein.

translation

 

Wow what a lot to take in! This process from DNA (the gene) to mRNA to protein is called the “central dogma” of molecular biology.  This process was originally described in 1956 by Francis Crick (the same guy who along with Watson discovered the structure of DNA).  Why is it called this? Probably because when it was described, they realized that this was a fundamental process for all life.  It’s also important for us to know for so many reasons.  If you know that DNA codes for RNA which codes for protein, any changes in this process can result in a protein that isn’t quite right.  What if you have change the code of the DNA?  This will change the protein!  What if you change the amount of the mRNA? You will change the amount of protein!  And what can this do?  Cause problems in your cell that cause disease!

transcription-translation