The 11th Annual ENCALS meeting highlights how TDP-43 spreads in MND

The European Network for a cure of ALS (ENCALS) held its 11th Annual meeting in Sheffield from 31 May to the 2 June. The weekend was full of glorious British sunshine and more than 200 international scientists and clinicians were also able to enjoy a range of incredibly interesting talks about the latest developments in MND research.

A particular talk caught my attention on the first day by Dr Johannes Brettschneider from the University of Ulm in Germany. Dr Brettschneider explained how his research had shown the stages and spread of the protein TDP-43 in ALS (the commonest form of MND).

Dr Brian Dickie, Director of Research Development, said: “The key to defeating MND lies in fostering strong collaborations between neurologists, healthcare professionals, research scientists, early career investigators and students in the field of MND and the 11th Annual ENCALS meeting in Sheffield provided that opportunity. The MND Association was proud to support this event.”

‘Special’ staining

At the end of an afternoon of talks on the MND- causing genes C9orf72, FUS and SOD1, Dr Brettschneider engrossed over 200 delegates with his talk on the TDP-43 protein and how it spreads in ALS.

Although TDP-43 genetic mistakes are a rare cause of MND, scientists are especially interested in the TDP-43 protein because in the vast majority of cases of MND (irrespective of whether it was caused by an inherited genetic mistake), TDP-43 protein forms pathological clumps inside motor neurons.

The study (which is a collaboration between Dr. John Trojanowski and Dr. Virginia Lee from the Penn University Center of Neurodegenerative Disease Research in Philadelphia, America and the group of Dr. Heiko Braak in Ulm) used a technique known as ‘immunohistochemistry’.  This technique involves taking tissue samples of the brain and spinal cord from people who have died from ALS. The researchers would then make extremely thin slices of the tissue, which could then be stained using a ‘special stain’ and viewed under a microscope.

The stain used by Dr Brettschneider only ‘stained’ the TDP-43 protein in the samples, meaning that he could see the amount of TDP-43 in different areas of the brain and spinal cord.

Using the clinical information and TDP-43 staining this would allow Dr Brettschneider to stage the disease.

Image kindly provided by Dr Robin Highley, SITraN: (top left) a motor neurone with a skein-like neuronal cytoplasmic inclusion, next to a normal motor neurone (bottom left) on TDP-43 immunohistochemistry.

Image kindly provided by Dr Robin Highley, SITraN: (top left) a motor neurone with a skein-like neuronal cytoplasmic inclusion, next to a normal motor neurone (bottom left) on TDP-43 immunohistochemistry.

Axonal ‘telephone wires’ do more than just talking

Dr Brettschneider showed that TDP-43 increased in different areas of the brain and spinal cord during different stages of the disease. Amazingly, he also showed how ALS (characterized by clumps of TDP-43) spreads from one are of the body to another.

A motor neurone consists of three parts; the cell body, axon and nerve ending. The cell body contains the nucleus, or the control centre of the cell. When a message travels from the brain the cell body sends the message down the axon. Like telephone wires, the axon carries the message to the muscle, where the nerve endings cause the muscle to move.

However, in ALS it seems that these ‘telephone wires’ do more than just carry a message. The protein TDP-43 forms ‘clumps’ in the motor neurones and it seems that these clumps use the axon to travel from one motor neurone to the next (possibly explaining why someone get’s weakness in their arm and then their hand).

Another key finding was that TDP-43 clumps develop in the front part of the brain (prefrontal cortex), which is responsible for personality and may explain the development of cognitive symptoms.

Dr Brettschneider explained the importance of this research While spreading of disease-related proteins has been described for other neurodegenerative diseases like Alzheimer’s disease or Parkinson’s disease, this had not been previously shown in ALS. Now, we can show evidence that supports a spreading of the major disease protein TDP-43 in ALS across specific regions of the brain and spinal cord with ongoing disease.

 If these findings can be confirmed (for example in cell culture or mouse model studies) then this could lead to the design of new treatments specifically aiming to impair the spread of TDP-43 protein clumps.

Dr Johannes Brettschneider from the University of Ulm in Germany at ENCALS

Dr Johannes Brettschneider from the University of Ulm in Germany at ENCALS

Furthermore, we believe that our findings offer a better understanding of disease progression in ALS.  Our data implies that TDP-43 spreads throughout the prefrontal cortex with ongoing disease, thereby lending support to the idea that all ALS patients could eventually develop “frontal type” cognitive deficits.”

The future

Dr Brettschneider commented why this research is important to people living with MND explaining that “If these stages can be reproduced in patients with ALS they could offer a new way to assess disease progression and response to new treatments. We hope that our study provides the essential groundwork for strategies designed to prevent pTDP-43 spread.”

This research is only the beginning and more work is needed, Dr Brettschneider also explained what he hoped to do next with these exciting results. “There were restrictions in time and availability of the tissue samples during this study, so we were unable to determine how and where exactly ALS begins in the very early stage of the disease. Therefore, an important next step in our work would be to analyze very early cases with ALS to look at TDP -43 spread as this offers the most promising window for therapeutic intervention.”

Reference

Brettschneider J, Del Tredici K, Toledo JB, Robinson JL, Irwin DJ, Grossman M, Suh E, Van Deerlin VM, Wood EM, Baek Y, Kwong L, Lee EB, Elman L, McCluskey L, Fang L, Feldengut S, Ludolph AC, Lee VM, Braak H, Trojanowski JQ. Stages of pTDP-43 pathology in amyotrophic lateral sclerosis. Ann Neurol. 2013 May 20. doi: 10.1002/ana.23937. [Epub ahead of print]

The C9orf72 mystery begins to unravel even more of its secrets

In 2011 an international team of scientists, including three MND Association-funded researchers, identified the elusive C9orf72 gene located on Chromosome 9. Since this ground-breaking discovery, researchers from around the world have been trying to find a way to open-up and reveal more about this MND-causing gene.

Determined to get inside and unravel the secrets behind C9orf72, the Association is funding a number of new and exciting research projects to help solve the mystery. These projects look at, not one, but a number of different aspects to try and understand more about C9orf72.

In order to solve this mystery our C9orf72 researchers are following the clues using zebrafish, mice, flies and DNA samples.

How the C9orf72 MND mystery began

We each contain copies of 23 pairs of chromosomes, including the X and Y sex chromosomes. These chromosomes contain thousands of genes that portray our characteristics such as hair and eye colour. These genes are made up of DNA which can either be ‘coding’ to make a protein, or ‘non-coding’. For details of how genes make a protein see our earlier blog post.

Before C9orf72 was identified researchers had focused on an area on Chromosome 9 that appeared to be connected with both the rare inherited form of MND and the related neurodegenerative disease frontotemporal dementia (FTD).

Using a number of cutting-edge techniques the international team isolated the C9orf72 gene expanded GGGGCC hexanucleotide repeat as being a crucial player in both inherited MND and FTD. Not only did the researchers find a link between MND and FTD, they also found that C9orf72 was found in approximately 40% of cases of inherited MND (where there is a strong family history). This means that we now know 70% of the genes that cause the rare inherited form of MND. For more details on C9orf72 see our earlier blog post.

For more information on inherited MND please see our website.

So, researchers found C9orf72. The next question was ‘What does it do? Is the gene defect repeat itself, or the protein it makes responsible for causing MND? And what goes wrong in MND?’

Detective

Following the clues to solve C9orf72

Two recent research clues

Since 2011 researchers have been trying to answer these questions and find out more about C9orf72. This has led to a dramatic increase in research, including two papers published in February and March this year!

Prof Christian Haass (Munich Centre for Neurosciences, Germany), who recently presented at our 23rd International Symposium on ALS/MND in December 2012, published a paper on the 7 February in the journal Science. The second paper lead by Prof Leonard Petrucelli (Mayo Clinic, USA) was published open access in the journal Neuron on the 20 February.

In a big surprise, both researchers found that the presumed ‘non-coding’ C9orf72 GGGGCC repeat expansion actually made a protein. Normally these ‘non-coding’ regions do not make proteins so this was a very big surprise indeed!

The researchers found that these proteins formed large clumps in the brains, and throughout the central nervous system (CNS), of people with C9orf72 MND and/or FTD. Importantly, they did not find these clumps in healthy individuals or those with other neurological disorders.

It is currently unknown as to whether these protein clumps are involved in MND and/or FTD, but they may be a potential biomarker or a therapeutic target in this most common type of MND. The next step is for the researchers to find out whether these proteins actually cause MND and/or FTD.

Finding more evidence to piece together the clues

In addition to these two papers looking into the mystery behind C9orf72, the Association is funding some exciting new research projects, each looking at different things, to further understand more about this gene.

Dr Johnathan Cooper-Knock

Dr Johnathan Cooper-Knock, MRC/MND Association Lady Edith Wolfson Clinical Research Fellow

Dr Johnathan Cooper-Knock (Sheffield Institute for Translational Neuroscience, UK) is already trying to identify how C9orf72 causes MND by utilising a genetic technique known as gene expression profiling. He is using samples from the Association’s DNA bank which are positive for the C9orf72 genetic mistake. Gene expression profiling is a technique which allows researchers to understand how the activity of genes contributes towards causing MND. (Traditional genetic studies are designed to look at which genes are affected, rather than their activity – ie when and how). Read more about Johnathan’s project here.

Developing new disease models enables us to understand the causes of MND and to test new therapies. One way to understand the function of C9orf72 and how this goes wrong in MND is to create a model. Our current research projects are developing new C9orf72 models in flies, mice and zebrafish.

Dr Frank Hirth (Kings College London, UK) will be producing a fly model, Dr Javier Alegre Abarrategui (University of Oxford) will be making a mouse model and Dr Andrew Grierson (University of Sheffield, UK) will be creating a zebrafish model.

All of these models aim to understand the function of C9orf72 and what goes wrong. The researchers hope to study what happens in MND and how this occurs by looking at behaviour and what happens when C9orf72 is ‘switched’ on and off. For more information about these exciting research projects please see our website.

Solving the mystery

All of our C9orf72 Association-funded research projects are using different approaches to look at C9orf72 in different ways as we are still unsure whether the protein or the repeat is the problem. From mice to flies all of these research projects together are helping to solve the mystery of C9orf72 and MND.

With the proteins formed by C9orf72 likely to be a potential biomarker or therapeutic target the two recent papers are adding to the growing number of clues, pointing researchers in the right direction to unravelling and solving the secrets of C9orf72.

References:

Mori, K. et al. The C9orf72 GGGGCC Repeat Is Translated into Aggregating Dipeptide-Repeat Proteins in FTLD/ALS. Science. 339(6125): 1335-1338. 2013 DOI: 10.1126/science.1232927

Ash, P. E. A. et al. Unconventional Translation of C9ORF72 GGGGCC Expansion Generates Insoluble Polypeptides Specific to c9FTD/ALS. Neuron. 77(4): 639-646. 2013 DOI: 10.1016/j.neuron.2013.02.004

Baking with proteins, mRNA and DNA

Each and every one of us is made up of thousands of different ingredients, which all combine together to create something amazing; life. Perhaps the most important of these are proteins.

Each protein in the body has its own special job to do. From making our muscles contract to controlling blood sugar, proteins are an essential ingredient in life.

In MND research we have identified a number of MND causing genes. These are genes that are found to be mutated in some people living with MND, which somehow causes the motor neurones to die. But, how does this happen? How does a gene form a protein? This blog post explains how an MND causing gene becomes a protein.

As simple as baking a cake

Here at the MND Association we love our cake. So, I thought what better way is there to describe how we make proteins?

cheesecake

cheesecake

Every cell in our body contains 23 pairs of chromosomes (46 in total), except for the egg and sperm cells that contain 23 chromosomes each.

Like a recipe book, these chromosomes hold all of our genetic material in the form of genes, in which everyone inherits two sets of (one from each parent).

Humans have approximately 24,000 genes, which each consist of their own DNA recipe to make a protein. Like cakes, proteins come in a range of different shapes and sizes, that come together to create you and me.

These DNA recipes are read by different cells to create the right protein for the job. For example, you would only make a wedding cake for a wedding, and the type of cake (chocolate or fruit) would depend on the wedding couple.

This is what happens in nerve cells (or motor neurones). A motor neurone will make a specific type of protein to help it grow, or to help it survive in low oxygen levels.

Following the recipe

A nerve cell creates a protein by finding the exact DNA recipe amongst the genes within the cell’s control centre, known as the nucleus. Once the recipe has been found the cell has a problem… The nucleus does not have the right tools to make a protein! The cell instead needs a specialised machine, or food mixer, which is only found outside of the nucleus called a ribosome.

In order to make the protein the DNA recipe needs to travel from the nucleus to the ribosome and this is done by means of a messenger. The DNA recipe can’t leave the nucleus so the cell ‘copies’ it into a messenger version, called mRNA.

The cell does this by removing certain parts of the DNA that do not affect the finished protein which are known as introns or ‘non-coding DNA’. This is known as ‘RNA splicing’ and is the same as removing raisons from a fruit cake. The cake is still made and still contains fruit, but the raisons are not essential in the finished cake.

mRNA can then travel the DNA recipe safely from the nucleus to the ribosome, where it can be finally made into a protein. Once made, this protein can then go on to do its specific job role (or in cake terms, be a wedding cake!).

ruined cake

ruined cake

Changing and ruining the recipe

Sometimes the DNA recipe in our genes can change through means of a mutation. Most of these are harmless spelling mistakes (sugarr instead of sugar) that do not affect the finished protein. However, sometimes these mutations can be so big and harmful (salt instead of sugar) that they do.

These kind of mutations are so big that the size, shape and structure of the protein can be changed – meaning that the protein can no longer do the job it was designed to do (our wedding cake is now no longer sweet and tasty, but ruined and salty!)

This is what happens in some of the MND causing genes. A big mutation occurs in the DNA recipe in a specific gene that causes the structure and shape of that protein to change. This change can then cause the proteins to ‘clump’ together in the motor neurones as they can no longer do the job they were designed to do.

Bake MND history

Bake MND history

Expert bakers

An understanding of genes and how proteins are connected is essential for understanding how they can go wrong in MND. The Association funds a number of exciting research projects investigating the MND causing genes, along with the proteins they form.

To help raise awareness of MND you can bake your own cake as part of our ‘Bake it!’ fundraising campaign. For more information and to request a fundraising pack please see our website.

Sharing and networking in Liverpool

From Sunday morning to Tuesday evening last week, there was a lot of talk of MND research going on in Liverpool. The reason for this ‘hotspot’ of discussions was due to the annual meeting of an international consortium of MND researchers taking place at the University of Liverpool. The 10th International Consortium on SOD1 and ALS (ICOSA) meeting took place last weekend (4 – 5 March).

In 2001, five laboratories came together to form ICOSA, where the aim was to share knowledge to design better-informed experiments to understand the rare, inherited SOD1 form of MND. MND Association grantee, Prof Samar Hasnain was one of its founding members. Success of this philosophy of sharing knowledge prior to publication has resulted in several leading groups joining the effort, looking at other causes of inherited MND too.

A tradition of ICOSA meetings is to hold an open meeting for sharing latest results with a wider audience, following their closed meeting. Thus, on Tuesday 6 March, an open meeting was held to allow the exchange of the latest results and ideas between ICOSA members and the UK MND research community.

I attended this one day meeting in Liverpool and I’ve written a mini report on the meeting below, including a couple of highlights.

The first few presenters demonstrated the truly international nature of this collaboration – they had travelled from the snowy landscape of northern Sweden, the sweltering heat (at least in August!) of mid-state Florida and from RIKEN, the large natural sciences research centre, in Japan .

The researchers represented were a mixture of physicists, biochemists and neurologists – an unusually broad spectrum of knowledge and speciality for an MND research meeting. Essentially, their core, joint interest was in understanding how the structure of a protein has such a marked change leading to MND developing or the disease progressing.

The structure of a protein is essentially about folding. The correct folding will mean that the protein can do its job. Folded incorrectly the protein won’t be able to work. An example of incorrectly folded protein is the protein clumps or ‘aggregates’ seen within motor neurones in MND. There is a whole chain of events that lead the appearance of these clumps of protein – and researchers at the meeting discussed every step along the way.

How do proteins fold and why is it important?

When the instructions for making a protein (ie genes) are read and edited by DNA and RNA respectively, they are reading or editing instructions to arrange a set of building blocks in a particular order – there are 20 different types of building block – our amino acids. ALL of our proteins within our bodies are made from specific arrangements of this core set of 20 building blocks. The arrangement of the building blocks determines where the protein folds, in which direction and the shape it makes. There are many possible folding arrangements a protein could make, but it will always try and fold itself into the lowest energy shape (a good way to think about this is the shape where the protein is ‘most comfortable’).

Geneticists know a lot about the beginning of the process (what the sequence of building blocks will be) and biochemists and pathologists know a lot about the end of this process (what the protein does and a what it looks like in the cell when it clumps together) – but the physicists of the MND research world are working on the bit in the middle (precisely where which building block is, in the folded protein).

A change to the sequence of the building blocks, as seen in the proteins made from mutated genes that cause MND, will lead to unusual folding, and damage to the cell – due to the loss of normal function or a trigger for toxicity. So having a complete picture of a protein ‘lifespan’ is really important in understanding what goes wrong in MND and how to fix it.

Unravelling questions about SOD1

People with the SOD1 form of the rare, inherited type of MND have a mistake in the assembly of one building block in the instruction to make the SOD1 protein. Over 160 different, single building block mistakes have been found in this form of MND so far. All of them lead to the development of MND. So that means 160 damaging variations in the folding of the SOD1 protein.

Over 70 other delegates and I heard the latest on how mimicking the effects of these mutations (by changing building blocks of the protein) in SOD1 mouse models tells us more about this cause of MND. It’s even possible to study the different effects of the toxic protein on different cell types essential for motor neurone function. (Although motor neurones carry the messages, they are supported by groups of ‘glia’ cells around them).

Where (the) ‘FUS’ is

Prof Larry Hayward presented his research on a protein called ‘FUS’; mutations in this gene causes another form of the rare inherited MND. The damaged ‘FUS’ protein is found in a completely different place in motor neurones than usual. Images of motor neurones where the FUS is in the centre of motor neurones, as usual, looked a bit like fried eggs; but the location of the damaged FUS in the outside of the cell reminded me of ring donuts! By stressing motor neurones, he showed a video of the proteins moving from the centre to the outside of the cell; and back to the centre when the stress was removed. This all happens very quickly, in a matter of minutes!

C9orf72 – a hot topic

Another highlight of the meeting was the presentation by MND Association grantee Prof Huw Morris on both how the C9orf72 gene mistake was found last year, and also on what’s happened since the results of this finding were announced. In the five and a half months since the 21 September announcement, another 26 reports have been published in this area of MND research. That’s slightly more than one report a week! (To put this in context there are roughly 36 MND reports published a week, total, across a broad range of topics). He commented that one factor that kept him focussed in the long search for this gene defect was the people with MND in his care.

Drug scaffolding to correct damaged folding

Above I mentioned that the physicists work out the precise folding of proteins, knowing where each of the building blocks is within its final shape. They do this by isolating the protein they want to study and placing it in increasingly high concentrations of salt solution to remove literally every molecule of water, until the protein itself comes out of solution and forms crystals. These crystals are then analysed by x-ray crystallography and other analytical chemistry techniques.

For a protein made from a mutated SOD1 gene, x-ray crystallography studies found a hole in the protein folding that may explain why it forms clumps within motor neurones. MND Association funded researcher Dr Neil Kershaw from the University of Liverpool presented the latest results from his research in designing a drug that will ‘prop up’ incorrectly folded SOD1, in the hope that this will remove its damaging effects.

I hope that this report demonstrates that in between the ‘big news’ stories about MND research, steady progress continues to be made in understanding MND and searching for treatments for it.