Researchers from the Flinders University, Australia and University of Miami have discovered a new protein that can act as a biomarker to track disease progression in people with MND. A paper written under the leadership of Dr Shepheard and Dr Rogers was published today in the research journal ‘Neurology’.
What is p75 and what do we know so far
The biomarker is a protein called p75, which initially
supports the growth of neurones during embryonic development and its levels markedly decrease after birth. Throughout our lives, p75 only reappears in higher levels when the body detects injury of the nervous system, and shows its presence in urine.
The researchers have previously shown that, after birth, mice with a mutation in the SOD1 gene, known to cause MND, had high levels of p75 after about 40 days from the onset of MND. This also coincided with increased levels of p75 in motor neurones found in tissue of people with MND after death.
With all the talk of new gene discoveries in recent years, the Sunday morning scientific session returned to the original discovery in 1993 that mutations in the SOD1 gene were responsible for around a fifth of familial (inherited) MND cases and 2-3% of all cases of the disease.
Although much of our understanding of MND in the past two decades comes from SOD1 laboratory models of the disease, we still don’t know exactly how SOD1 kills motor neurons. But that hasn’t stopped several groups from working on a number of innovative ways of protecting motor neurons from SOD1 toxicity. Although focused on a relatively rare form of MND, some of the strategies being followed could potentially also be applicable to other forms of the disease.
A number of articles were published in various news sources on 11 July 2014, highlighting how scientists in Sheffield are working towards testing a promising treatment for a rare inherited form of MND caused by the SOD1 gene. Here we write about the research and what it means for people living with MND.
The Sheffield Institute for Translational Neuroscience (SITraN) specialises in research into MND and other neurodegenerative diseases. Recently the institute received an anonymous donation of £2.2 million to help translate their research from the lab to the clinic. This is a huge amount of money into MND research and this donation will enable the researchers to further our understanding of the disease.
We know that approximately 10% of cases of MND are inherited. This means that they are characterised by a strong family history and the disease is caused directly by a mistake in a specific gene. Of these 10% of cases, 2% are caused by the SOD1 gene (meaning that for every 100 cases of MND, 10 cases are inherited and of these, only 2 are directly caused by the faulty SOD1 gene).
Prof Mimoun Azzouz’s research at SITraN was reported in a number of news outlets, highlighting how his research is paving the way to a treatment for a rare form of MND. His research is at a relatively early stage, where he has only just begun investigating the use of a technique known as ‘gene therapy’ in mice affected by the SOD1 inherited form of MND. If the research goes to plan, he will be able to submit a proposal for regulatory approval by August 2015.Read More »
The 24th International Symposium on ALS/MND began in Milan today with a record number of over 950 delegates attending to hear the latest news in MND research.
Inherited MND is a rare form of MND characterised by a family history of the disease. Over recent years more and more genes have been discovered, which has lead to an increase in individuals wishing to pursue genetic testing.
A genetic test consists of a sample, which is then sent off to a genetic laboratory. Here the blood sample is then screened for the MND-causing genes.
The gene that is faulty in inherited MND can differ between one affected family and another. Mistakes in genes called SOD1, TARD-BP, FUS and C9ORF72 between them account for about 65 – 70% of cases of inherited MND. Scientists have yet to identify the gene defects that cause the remaining 30%.
The exact course, duration and rate of progression of MND often varies greatly from person to person; even when there is a known family history of the disease caused by a specific MND-causing gene (eg SOD1).
This same variability also occurs in mice. Researchers, funded by the MND Association, took two mice with the same SOD1 gene mutation from two different families (strains). By using these two mice the researchers identified a number of key changes in motor neurones that differ between fast and slow progressing forms of the disease.
Two mice… One gene
Developing new disease models enables us to both understand the causes of MND and test potential new therapies.
Mice are commonly used in MND research and for the past 10 years or more, the SOD1 mouse model has been one of the most important research tools for scientists working in the field, particularly with testing potential new therapies.
Research published in September 2013 was carried out in a joint collaboration between Dr Caterina Bendotti (Mario Negri Institute for Pharmacological Research, Milan Italy) and Prof Pam Shaw (University of Sheffield, UK).
Our bodies need to be able to make new proteins, to maintain long term memory. So if the ability to make new proteins is switched off, does this cause Alzheimer’s Disease? New research findings published yesterday by scientists based in Leicester take us closer to answering this question. Journalist are describing this as a step forward for all neurodegenerative disease, so I wanted to explain what the researchers found, and what it might mean for MND.
What’s the story?
The activated form of a chemical called ‘eIF2’, is found in higher levels than normal in the brains of Alzheimer’s Disease patients. (In it’s turn, eIF2 is activated by an enzyme called PERK – hence the name of the blog post.. !).
Last month (September 2013) researchers found that genetically blocking the activation eIF2 prevented memory problems in a mouse model of Alzheimer’s Disease. The research published yesterday showed that in a mouse model of prion disease, chemically blocking eIF2 (as opposed to genetically blocking it) helped prevent the development of prion disease (Variant CJD or ‘mad cow disease’ is an example of a prion disease).
The chemical block was given to mice orally (one of way of doing this is to give it to them in their food). It got to the brain OK and effectively blocked eIF2, but the chemical did have serious side effects. So it’s a possible turning point for drug treatment for Alzheimer’s Disease and prion disease, but not the answer.
Omega-3 fatty acids have been in the media a great deal over recent years. They can lower our risk of heart disease and they may even have neuroprotective properties (for example limit damage to the brain and spinal cord after acute injuries).
But, what about in MND? Could this dietary supplement have an effect?
MND Association-funded researchers have found out, rather unexpectedly, that the omega-3 fatty acid eicosapentaenoic acid (EPA) actually accelerates disease progression in an animal model of MND which is based on a SOD1 mutation, when EPA is given before symptoms first appear (this is sometimes known as the pre-symptomatic stage).
Why omega-3 might have had an effect
Omega-3 polyunsaturated fatty acids are natural compounds primarily found in oily fish such as sardines, mackerel or salmon. They have been widely associated with significant health benefits and researchers have reported that some long-chain polyunsaturated fatty acids may be beneficial in several neurological conditions.
Previous research in rats has shown that dietary food supplements, containing omega-3 long-chain polyunsaturated fatty acids (including EPA), reversed age-related problems in neurones (nerve cells) and also enabled the growth of new neurons.
The neuroprotective properties of EPA could occur through a variety of mechanisms such as reducing oxidative stress (damage due to low oxygen levels), reducing neuroinflammation and the activation of anti-‘cell death’ pathways. These are all factors that are relevant in MND.
A number of studies have found that high blood lipids (the breakdown product of dietary fats) are a common feature in ALS (the commonest form of MND), and are correlated with increased survival. High-fat diets have been studied in the lab to further investigate this and have been shown in mice to delay motor neurone death and extend lifespan.
What the researchers found
Due to these previous studies the researchers decided to study one omega-3 long-chain polyunsaturated fatty acid in particular, EPA, to assess whether it had neuroprotective effects in a mouse model of ALS based on a mutation in the enzyme SOD1.
Mouse models are commonly used to study the causes of the disease and investigate potential treatments in MND. A SOD1 mouse model is a mouse that has been given a faulty MND-causing gene producing an enzyme known as ‘SOD1’, which is known to cause 20% of cases of the rare inherited form of MND.
The researchers intended to study the effects of dietary EPA when given at disease onset (the symptomatic stage when symptoms first appear) or at the pre-symptomatic stage.
The mice were fed either a standard rodent powdered diet (control) or a diet supplemented with EPA-enriched oil. The researchers then looked at a number of factors such as: disease progression, survival and body weight to find out if there were any differences.
When a diet supplemented with EPA was given at the symptomatic stage there was no significant difference in the development of MND compared to the control group (mice who were fed a standard rodent powdered diet). However, rather unexpectedly, when the EPA diet was given at the pre-symptomatic stage, the researchers found that the diet accelerated the progression of MND, but did not affect disease onset.
Glial cells (such as astrocytes and microglia) were also affected, and found in reduced levels when the mice were given the EPA diet.
Overall, the researchers found that long-chain omega-3 fatty acid EPA-enriched diets have no impact on disease onset or survival. Unexpectedly, if dietary EPA is given before symptoms appear it can actually accelerate the progression of MND.
What this means for people living with MND
The omega-3 fatty acid EPA, although it may have other health benefits, appears to have the potential to be more damaging rather than protective in this specific MND mouse model. The results from this study have highlighted the need for caution by those who are at risk of developing MND, who may use these long-chain omega-3 fatty acids dietary supplements, which are freely available, over prolonged periods of time.
This study has shown that individuals who carry the SOD1 inherited form of MND in particular should take extreme caution with diets enriched in long-chain omega-3 fatty acids such as EPA. For the future, it remains to be seen if EPA has also negative effects in other models of MND (eg zebrafish or flies with the C9orf72 mutation).
Dr Adina Michael-Titus (Blizzard Institute, Queen Mary University of London), one of the researchers involved in the study, commented “The most important point, in my view, is to be aware that we do not have yet the scientific evidence to prove that EPA or any other omega-3 fatty acids are dangerous for all forms of MND. The only experimental evidence we have so far is for a particular SOD1 mutation which leads to this disease (where the faulty SOD1 mutation is greatly overexpressed). More work is required and future research will help us fully assess and understand the potential or the risk associated with omega-3 fatty acids in people living with MND.”
Dr Andrea Malaspina (a member of our Biomedical Research Advisory Panel) also commented on the results. “EPA was found to accelerate the progression of MND when given at the pre-symptomatic stage in a SOD1 mouse model. To fully assess the potential risk of EPA further research is needed in other animal models, with different MND mutations (as different mutations cause different metabolic changes) to see if a similar effect is observed. At present we can only say that EPA accelerates the progression of MND in a SOD1 mouse model and it is not known whether it accelerates the progression of all forms of MND.”
For more information about the rare inherited form of MND please see our website
References: Yip PK, Pizzasegola C, Gladman S, Biggio ML, Marino M, et al. (2013) The Omega-3 Fatty Acid Eicosapentaenoic Acid Accelerates Disease Progression in a Model of Amyotrophic Lateral Sclerosis. PLoS ONE 8(4): e61626. doi:10.1371/journal.pone.0061626
Results from a phase I clinical trial of a drug known as ISIS 333611 have been published open-access online in the scientific journal Lancet Neurology on 29 March 2013.
This is the first time researchers have tested the effects of delivering an antisense oligonucleotide directly into the human cerebral spinal fluid (the fluid between the spinal cord) showing that it is both safe and well tolerated in people with the SOD1 form of inherited MND. For information on inherited MND please see our website.
This work suggests that this ‘antisense’ approach may be a good strategy for other neurological disorders.
What is antisense?
Antisense is a type of therapy that causes the ISIS 333611 to directly interfere with the faulty instructions for making a SOD1 protein, thus stopping the production of the disease-causing substance. This is called ‘gene silencing’ as that part of the gene is not ‘heard’ when the final protein is made.
ISIS 333611 works by targeting mRNA, the ‘messenger’ that carries the genetic instructions from the SOD1 gene to the protein-making machinery (for more about mRNA and how proteins are made see our earlier blog post). Instructions in the mRNA for making the SOD1 protein (sometimes called a ‘sense’ sequence) are faulty in people with SOD1 inherited MND, which leads to harmful SOD1 proteins being made.
So if the levels of harmful SOD1 can be reduced, might this be protective? That’s the thinking behind the treatment. By binding to the SOD1 mRNA, ISIS 333611 prevents the production of a harmful SOD1 protein. Indeed, studies in SOD1 positive animal models indicated that reducing the level of SOD1 by antisense therapy increased lifespan. However, targeting the SOD1 gene in this way is a very ‘personalised’ treatment strategy – if it does work it will only work for people who have the SOD1 from of MND.
Results from the trial
Based on the encouraging animal studies, the researchers and ISIS Pharmaceuticals conducted a phase I trial of the antisense oligonucleotide ISIS 333611.
Twenty-one people with SOD1 MND were involved in the study and results from the trial have shown that there were no toxic effects due to increased dosing of the drug and that the drug was safe and well tolerated.
In animal models antisense therapy is found to spread well throughout the central nervous system (brain and spine). However, unlike animal models, the researchers showed that concentrations of ISIS 333611 were lower in the upper end of the spinal cord and brain compared to the injection site. Due to this the delivery site of the drug will probably need to be revisited in future trials.
As this was only a short-term ‘Phase I’ trial it was not designed to test whether this antisense therapy had an effect on MND. This would only be seen with long term treatment and future trials. However, the results are encouraging as they show that this type of therapy is both safe and well tolerated in people with SOD1 MND.
Results make sense
Dr Pietro Fratta (University College London), who is a recipient of a Medical Research Council/MND Association’s Lady Edith Wolfson Clinical Research Fellowship, has written an accompanying commentary on the paper. He said that this study “paves the way for applying antisense oligonucleotides to other forms of genetically determined MND” such as the C9orf72 form of the disease.
However, he stressed that “many hurdles still need to be overcome to bring this treatment to the clinic”.Dr Fratta also cautioned that the longer-term implications of lowering SOD1 protein levels had to be examined. The antisense approach not only targets the harmful mutated SOD1 protein, but will also lower levels of ‘healthy’ normally functioning SOD1, which plays an important role in protecting neurons from damage. So, the antisense treatment approach may be a ‘double-edged sword’ that will require very careful handling.
Miller, T. M. et al. An antisense oligonucleotide against SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral sclerosis: a phase 1, randomised, first-in-man study. Lancet Neurology 2013 DOI: 10.1016/s1474-4422(13)70061-9 Read the full article here.
Fratta P. Antisense makes sense for amyotrophic lateral sclerosis C9orf72 Lancet Neurology 2013 DOI: 10.1016/s1474-4422(13)70059-0
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.
The Opening Session theme on how the disease progresses within the Central Nervous System (CNS) continued with the presentation by Prof Stan Appel from Baylor College of Medicine, Huston on neuroinflammation.
Examination of post-mortem brain and spinal cords from people with MND shows clear evidence of inflammation (although Prof Appel was quick to point out that this is not the same as occurs in ‘primary’ inflammatory conditions such as multiple sclerosis). Similar patterns are seen in human MND spinal cord and in SOD1 mice, suggesting that at least for this aspect of the disease, SOD1 mice may be a good model of human MND.
He went on to explain how migroglia, the ‘innate’ immune cells of the CNS, help mediate a delicate balance between protection and damage. The speed of progression in MND appears to be dictated by this balance.
Prof Appel showed that SOD1 mice exhibit two phases of disease: an early slow phase, where the microglia release a series of protective factors, and a rapid secondary progressive phase where levels of these protective markers fall and are replaced by a rise in ‘pro-inflammatory’ toxic factors. Of course, strains of lab mice are so inbred that they are genetically very similar and develop the disease in a uniform manner. Humans on the other hand are very different, as is the way the disease progresses between one individual and the next, so the two stages of disease are not easy to demonstrate in MND patients. However, by examining the inflammatory factors present in patients with very rapid progression against those with slower progression, he was able to show that the factors associated with the second ‘rapid progression’ phase in mice were also present in the rapidly progressing patients. He suggested that this may assist clinicians in predicting how the disease is likely to progress in patients at an early stage in the disease.
It is relatively easy in cell culture studies to tilt this balance from protective to toxic, but could the balance be tilted the other way in patients, as a therapeutic strategy? Certainly, in response to a question from the floor, he suggested that greater attempts should be made in this direction, commenting, “The whole issue of immunosuppressant drugs in MND needs to be re-opened. But – you can’t just take down all immune responses in an uncontrolled way. You need drugs that are much more selective”.