Energy and metabolism in MND cells answer the burning question

Despite the winter chill, there is a warm fuzzy feeling today with the news of a paper published in the journal ‘Brain’ by an MND Association funded Research Fellow, Dr Scott Allen. Based at the Sheffield Institute for Translational Neuroscience (SITraN), Dr Allen was awarded a Senior Non-Clinical Research Fellowship by the Association in 2016, and we are immensely proud to have been able to play a supporting role in his work.

Dr. Allen giving a platform presentation at the International Symposium on MND/ ALS in December.
Dr Allen giving a talk at the International Symposium on MND/ ALS in December 2018.

In his paper, Dr Allen and his colleagues took a novel approach to understanding how MND affects the pathways that are important for making energy in cells of the central nervous system (CNS), that are crucial to keep motor neurons functioning and alive. Specifically, his work has pinpointed a specific mechanism that is changed in MND. The team also demonstrated that there is the potential to tackle this issue by circumventing the problem in order to maintain a critical energy balance in the CNS, and therefore potentially identifying a significant new target in the development of future treatment.

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It’s not just about the neurones

Long before the latest wave of cellular and molecular biology advances started to give us new information on what was going on at the cellular level in MND, some doctors had observed that if the disease started in one particular part of the body, it would be neighbouring parts that became affected next.  This suggested that the disease usually starts in a single part of the brain or spinal cord before spreading further, like ripples in a pond.

How this happens is not well understood. It is likely that there are a number of processes going on, but they can broadly be divided into two theories. One of these is that damaged proteins can leak out of sick neurons and ‘infect’ their neighbours – a subject we have discussed at previous international Symposia.Read More »

Stem cells and growth factors: from bench to bedside

After a brilliant first day at ENCALS, which included a talk by Dr Johannes Brettschneider, the second day began with a talk by Thierry Latran Speaker Prof Clive Svendsen (Director of the Cedars-Sinai Regenerative Medicine Institute) arriving directly from attending at the Anne Rowling Regenerative Neurology Symposium.

Prof Svendsen gave a riveting talk to over 200 delegates, explaining his research on treating ALS (the commonest form of MND) with stem cells and growth factors, and the journey taken from bench to bedside.

The talk began with Prof Svendsen explaining his earlier research into Spinal Muscular Atrophy (SMA) – a genetic disease which causes severe paralysis in children. He explained how he and his collaborators took skin cells that had been banked for over 10 years from a patient with SMA and ‘reprogrammed’ them back into stem cells which were then pushed forward again into motor neurones. Stem cells are ‘immature’ cells, which have not yet ‘matured’ into a specific cell type (eg nerve cell or heart cell). Prof Svendsen’s research was similar to that by Prof Chandran (who did a post-doc with Prof Svendsen) who took skin cells from an MND patient.

Mo Le Cule on our stand at ENCALS
Mo Le Cule on our stand at ENCALS

A little bit of everything is good for you

Like red wine and chocolate (which are both allegedly good for us in moderation) Prof Svendsen highlighted that “a little bit of everything is good for you ” particulary with regards to radiation.

Radiation is a word that people associate with cancer and being dangerous but Prof Svendsen explained that low doses of radiation actually increases DNA repair.  Work by Dr. Seigo Hatada at the Cedars-Sinai Regenerative Medicine Institute has shown that when induced pluripotent stem (IPS) cells are given a low dose of radiation in the lab this enhances the ability to put new genes into the stem cells (homologous recombination) an important technique needed to either label the cells or correct bad mutations.   This is a very important new finding that may help the stem cell field in the future.

Ageing astrocytes

Astrocytes are support cells that are known to play an important role in keeping motor neurones healthy. SOD1 astrocytes (positive for the SOD1 MND-causing gene) were previously found to be toxic to motor neurones but TDP-43 astrocytes were found not to be toxic. Prof Svendsen showed that aged wild type (normal ‘healthy’) astrocytes were also toxic to motor neurones, suggesting that ageing of these cells may have an important role in MND.

Not only were the aged adult wild type cells toxic, they were almost as toxic  as a SOD1 astrocyte (upto 40% more than foetal wild type astrocytes)!

Astrocytes are the key

Replacing damaged motor neurones with stem cells, or healthy motor neurones, is just not possible today”. This is because motor neurones have incredibly long connections and replacing them in the body is a hard thing for researchers to do.

Prof Svendsen explained that replacing astrocytes offered a much better alternative. This is because astrocytes are easy to transplant and are sick and aged in MND. His approach, as described previously at the Anne Rowling Regenerative Neurology symposium, involves a combination of gene therapy and stem cells. Prof Svendsen converted human stem cells into astrocytes and then genetically modified them to produce large quantities of a nerve protecting factor called glial-derived neurotrophic factor (GDNF).

Genetic modification of these astrocytes was carried out by infecting them with a harmless virus. This virus then inserts a gene into the astrocyte, which enables it to produce and secrete GDNF. These modified astrocytes are then inserted into one side of the spinal cord of a SOD1 rat (expressing signs of MND). Prof Svendsen successfully showed that these astrocytes secreted GDNF and protected the motor neurones in the rat at the side of the transplant.

Prof Svendsen explained that the modified astrocytes do not seem to cross to the other side of the spinal cord and are only a ‘partial protection mode’ which means they don’t affect paralysis. They do, however, protect the healthy motor neurones. It is important to note that these experiments used the SOD1 rat model. Only 20% of inherited MND cases have the SOD1 MND-causing gene so this model is not a complete representation of other inherited and sporadic MND cases.   It is now important to try these exciting new stem cell and growth factor treatments directly in patients – they are the only real representation of the disease.

A phase I clinical trial after twelve years of research

Prof Svendsen concluded his talk by mentioning that with funding from the California Institute for Regenerative Medicine (CIRM) he is seeking U.S Food and Drug Administration (FDA) approval for a phase I/IIa clinical trial, which aims to transplant these genetically modified astrocytes into the lumbar (lower) spinal cord of ALS patients.

This trial plans to begin in 2015 by transplanting the GDNF secreting astrocytes into one side of the spinal cord to see the effects on the patient’s legs. Because, the astrocytes can’t cross the spinal cord, this will mean that the researchers will be able to compare both legs to look for differences in disease progression. The trial is double-blinded (with only the surgeon knowing which side the astrocytes are transplanted) and is across three centers in America. Prof Svendsen mentioned that he is on track for the first patient in 2015 providing the safety studies in animals work out as planned.

Thierry Latran Speaker Prof Clive Svendsen (Director of the Cedars-Sinai Regenerative Medicine Institute)
Thierry Latran Speaker Prof Clive Svendsen (Director of the Cedars-Sinai Regenerative Medicine Institute)

Prof Svendsen stressed that this has been a long road and shows just how long it takes to go from making observations in the lab to a clinical trial (he started this work back in 2003).

What next?

Prof Svendsen’s research has shown a great deal of work; including how he converted stem cells into astrocytes, showed that aged wild type and SOD1 astrocytes are toxic to motor neurones, found that GDNF prevented motor neurone death and the start of his clinical trial in 2015.

Prof Sevndsen commented on what the future might be. “If this therapy is found to be effective in ALS patients during this phase I/IIa trial we plan a much bigger trial!! We would aim to move from protecting the legs to protecting respiration – as we have shown the cells can work there too.”

Finally, Prof Svendsen stated what this research means to people living with MND with two simple words. “New hope”

Anne Rowling Regenerative Neurology Symposium

The sun was (uncharacteristically!) shining on Edinburgh last week for a symposium to celebrate the launch of the new Anne Rowling Regenerative Neurology Clinic. The clinic, which opened to patients earlier this year, was founded following a donation by the author JK Rowling, in memory of her mother, who died from complications related to multiple sclerosis (MS).  Run by Professors Siddarthan Chandran and Charles ffench-Constant, the clinic aims to translate laboratory research into clinical trials for neurodegenerative diseases such as MS and MND.

Anne rowling logo

The programme for the two-day meeting was packed with ‘big hitters’ from the world of neurology. In keeping with the regenerative neurology theme, the opening session was chaired by Sir John Gurdon, recent co-winner of the Nobel Prize for physiology and Medicine, whose pioneering work on cell cloning set the foundations for the more recent development of induced pluripotential stem cells, which are currently revolutionising medical research.

Different diseases, common challenges

The first day was given over to research areas such as multiple sclerosis, Parkinson’s disease and Alzheimer’s disease, as well as spinal injury and pain. What was also apparent is that different fields of neurology are wrestling with similar challenges: to diagnose disease earlier, ideally even before symptoms occur; to find biomarkers that tell us about the changes occurring in the Central Nervous System(CNS) at different stages of disease; to really understand the order in which these different aspects of pathology (the study and diagnosis of disease) occur and, given the theme of the conference, to sift the cellular changes caused by disease from the body’s attempts at cellular repair. All of these feed into the greatest challenge – how to take this accumulated knowledge from bench to bedside.

We can learn a lot from diseases that are further ahead in this process, such as the excellent overview by Prof Alastair Compston (Cambridge) on MS. It’s becoming clear that MS has distinct disease stages, starting off as an inflammatory disease, but progressing to a more ‘traditional’ neurodegenerative disease in more advanced stages. Whist there has been some considerable success in treating the former, the approaches to the latter have, as with MND, met with very limited success.

The use of imaging techniques to work out what is happening within the brain has been a vital factor in drug development for MS. As Prof David Miller (University College London) pointed out, magnetic resonance imaging (MRI) can pick up positive changes in small MS drug trials that are not large enough to show changes in disability. This sort of biomarker-based evidence gives drug companies the confidence to invest in the larger, much more expensive trials needed to show a clinical effect.

A presentation on the imaging of pain by Prof Irene Tracey (Oxford) provided a fascinating insight into the power of the placebo effect. She explained how neuroimaging has helped researchers to identify the brain regions associated with placebo effects and also gave examples of studies where the placebo effect has performed as well as (and even outperformed) commonly used painkillers! The power of placebo can be very strong indeed and it is important to always ensure that trials are rigorously performed to account for this.

Parkinson’s disease has always been viewed as a promising candidate for cell transplantation therapy, but clinical studies over the past 30 years have produced mixed results. Profs Roger Barker (Cambridge) and Anders Bjorklund (Lund University) discussed the various reasons for this ‘heterogeneity of response’ and how these are being addressed in the plans for a pan-European study.

In terms of cell transplantation, the approaches that will need to be taken for MND are very different from those for Parkinson’s disease. In Parkinson’s disease the strategy is to try and replace some of the key neurons that have died, but due to the immense length of human motor neurons, such a strategy of rewiring the nervous system is highly unlikely to work for MND. However, there are other approaches that can be taken, as Prof Clive Svendsen (Cedars-Sinai Medical Center) explained.

His approach involves a combination of gene therapy and stem cell therapy. By converting human stem cells into astrocytes, which are cells known to play an important role in keeping neurons healthy. By genetically modifying these cells to produce large quantities of a nerve protecting factor called glial-derived neurotrophic factor, and injecting them into the spinal cord of SOD1 rats, he has shown that the surviving motor neurons can be protected. He is in the process of gearing up for a phase 1 therapeutic trial in up to 18 carefully selected MND patients.neuron

Disease in the dish

Prof Svendsen also briefly spoke about the promising research arising from the use of induced pluripotential stem cells (iPSCs) to study MND – a topic taken up in much more detail by Prof Jeff Rothstein (Johns Hopkins University) who highlighted recent advances in understanding the C9orf72 form of the disease.

It may be possible to create specially tailored gene therapy approaches for some forms of familial (inherited) MND, as is currently being attempted in SOD1 MND. Prof Rothstein’s initial work using iPSC-derived motor neurons suggests that this approach is also worth considering for the more common C9orf72 from as well.

Prof Steve Finkbeiner (University of California) who is collaborating in the Association-funded international stem cell initiative elaborated on the use of iPSCs as a tool for drug discovery, demonstrating how fully-automated robot-based systems can be used to follow the fate of thousands of individual human motor neurons in the dish over a prolonged time period. The great thing about robots is that they don’t need sleep, so can analyse the cells at all times of day and night. They do, however, have Twitter accounts, so they can report in to the centre staff when they have completed their experiments!

One of the exiting prospects of using these automated systems is the potential to screen thousands of compounds. If human motor neurons can be protected in the dish, there are no guarantees, but it at least shortens the odds that the human motor neurons can be protected in the human as well. There are still many improvements that can be made to the process, but screening work is underway, with a particular focus on drugs that stimulate cellular process called autophagy (a process in which a cell breaks down damaged components), which is believed to be protective across a number of neurodegenerative diseases.

There were many take home messages from this meeting, but what was abundantly clear from all the work presented was the enthusiasm of each speaker for their field of research and an optimism that we are on the cusp of major advances in understanding neurological conditions. Sharing of new knowledge across the various diseases and disciplines can only bring those advances closer.

Reading the stars – why are ‘astrocytes’ toxic?

On the last day of the 23rd International Symposium on ALS/MND in Chicago last week there was an excellent session on ‘the role of non-neuronal cells’ – it was an exciting session – below is a flavour of some of the topics discussed.

As its name suggests, motor neurone disease causes the degeneration of motor neurones – the long nerve cells that carry messages from the brain to the muscles via the spinal cord. But motor neurones don’t exist in isolation. Particularly in the last five years or so we have learnt a lot about the contribution of glia to the development of MND. In health, the different cell types that are collectively known as glia (eg astrocytes, microglia and oligodendrocytes) protect and support motor neurones. We know that this changes in MND. It’s an exciting and fast moving area of MND research, where there is lots still to find out. So it was a treat to have a session of the Symposium dedicated to the discussion of the latest results.

It opened with a great overview of what we know so far about the role of astrocytes in MND from Serge Przedborski. (Astrocytes are called astrocytes due to their star shape when seen down the microscope). Leading on from the studies showing that the medium (fluid) that astrocytes grow in can damage healthy motor neurones, he set out to find out whether astrocytes (and the chemicals that they emit) are toxic or whether there is a lack of benefit. (Bearing in mind glia are sometimes called ‘support’ cells – this comment about the lack of benefit is pertinent).

 Using a clever assay, where it is pulled through a filter by spinning it, he showed that the astrocyte medium is toxic. So the next question was, what is it in the medium that makes it toxic? He and members of his lab looked at many possible components to check for their toxicity to motor neurones. The studies took over two years and were all negative “it’s too painful to list them all” he commented. “I had to change approach as I was risking the health of members of my lab!”

As he was describing the new approach I was reminded of the guessing game ‘animal, vegetable or mineral”. Is it a protein? was his first question, then the next was ‘is there an overall positive or negative charge to the protein?’ (some of the protein building blocks – amino acids – have a positive or negative charge, so the use of charge is a common way to separate them) and finally ‘how much does this protein weigh?’. The answers to these questions provided the first sort through before a second approach narrowed the search for the ‘toxic protein’ in astrocytes down to a choice of just nine possibilities. After looking at all nine in more detail, he found that a receptor on the surface of the astrocyte known as DR6 was responsible for its toxicity.

Dr Dan Blackburn from the Sheffield Institute of Translational Research, UK, described another approach to uncovering clues about why astrocytes are toxic – using an approach that looks at which proteins are made at a particular time called ‘gene expression’ profiling.

 Although the genes in each cell are there all the time, they are not read all at once. (In the same way that you won’t try and make every single dish in your recipe book simultaneously). So looking at which genes are read (known as gene expression) over the course of the disease leaves a detective trail to find out what caused the motor neurones to die.

Following on from earlier work in their lab, Dr Blackburn presented the trail of evidence from when a mouse model of MND first begins to show symptoms, and from a later time point, when the disease is far more advanced. He pointed the finger at abnormalities in cholesterol transport.

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Thinking outside the neurone and toward the stars

Neural progenitor cells, courtesy of Chandran lab, University of Edinburgh
Neural Progenitor cells, courtesy of Prof Chandran lab, University of Edinburgh

By using spinal cord donations from people with MND, an American group of researchers have created a new, human ‘in a dish’ model of MND. Their results were published in the journal Nature Biotechnology.

Human neuronal progenitor cells, which have the potential to turn into brain cells but not other types of cell, were extracted from post-mortem spinal cords and programmed to turn into living neurone support cells called astrocytes. The research group, led by Dr Brian Kaspar from Ohio USA, is hopeful that these human astrocytes grown in the laboratory can be used to learn more about the causes of MND.

Astrocytes and MND

Astrocytes, so called because of their star-like appearance, have been known to be involved with MND for many years now. Normally, astrocytes support and nourish nerve cells but in MND, they can become toxic to motor neurones, causing them to degenerate. However, before now, there hasn’t been a way to prove that this really does happen in people as it’s impossible to extract these cells while the person is still alive.

Using post mortem samples to find the answers

This research group used post-mortem spinal cord donations from people with MND to create a new, human astrocyte model, grown in laboratory dishes.

They were then able to demonstrate in the lab that these human astrocytes are toxic to healthy human motor neurones, causing them to degenerate. This verifies that astrocytes can cause MND in people, and not just in animal models.

They then went on to show that the healthy motor neurones could be protected if they stopped a protein called SOD1, which can be faulty in MND, from being created. This was found in astrocytes created from a person with a SOD1 form of inherited MND and remarkably, also from astrocytes created from people with the randomly occurring, sporadic form.

Reaching for the stars

By using post-mortem spinal cord donations from people with MND, and thinking ‘outside the neurone’ this research group were able to successfully create a new human ‘in a dish’ model of disease. There are still many unanswered questions left to explore with this new model, such as how the astrocytes cause the motor neurones to degenerate. By understanding more about this process, new treatments could be investigated to stop astrocytes from being toxic and slow down, or stop MND.

Find out more about tissue donation for general research purposes.


Published in Nature Biotechnology: doi:10.1038/nbt.1957

Stem cell conference part four: Laying the foundations for future stem cell clinical trials

The remainder of the morning covered some of the preclinical research that has laid the foundations for current and forthcoming clinicalstudies. Prof Clive Svendsen, from University Wisconsin-Madison, gave an overview of the strategy that helped lay the foundations for the current Neuralstem trial. This strategy involves the implantation of support cells (astrocytes) into the spinal cord. These astrocytes produce important nourishing (neurotrophic) factors that are essential to maintain the health of neurones. The strategy therefore is not about rewiring the nervous system, but instead providing the surviving motor neurones with a ‘boost’ to aid their survival.

He stressed the need for long and detailed study of the astrocyte cell lines if they are to be seriously considered as candidates for transplantation studies, using the comment “rubbish in, rubbish out”. He also provided very useful cautionary information in that some of the human cell lines show a tendency towards developing genetic changes over time, reminiscent of some types of tumours. By careful characterisation of the cell lines, his team was able to select only cells that demonstrated they were extremely stable. He has grafted these cells into the spinal cords of SOD1 rats, which does indeed help to protect the motor neurones.

However, it does not markedly alter the survival of the animals, probably due to the fact that the implanted cells can take months to mature into functional astrocytes, plus the fact that the motor neurones were still drawing back from the muscles and losing their connection. He is therefore looking at a ‘two-pronged attack’, treating both ends of the motor neurone through astrocyte implantation into the spinal cord, combined with nerve growth factor injections into the muscle.

Why stem cells derived astrocytes?
Astrocytes vastly outnumber neurones in the brain and spine: they are the cells that make up most of the ‘cellular neighbourhood’ and it is believed that in diseases such as MND, that neighbourhood is toxic to motor neurones. Prof Don Cleveland from University of California San Diego believes that if healthier, correctly functioning, astrocytes can be implanted into the spinal cord, it could turn the cellular neighbourhood into one that will protect the motor neurones and alter disease progression. The question is whether we can engraft enough cells to radically change the neighbourhood for good?

In colaboration with Prof Larry Goldstein, also from University California San Diego, his studies in SOD1 rats showed that injection of embryonic stem cell-derived human astrocytes can ‘clear the hurdles’ that need to be overcome in order to get astrocyte implantation studies into the clinic. Studies will move to a larger animal model and further work on producing, purifying and screening the cells needs to be done, in order to satisfy strict regulatory conditions.

He stressed the importance of setting milestones and getting the administration in place to deal with these hurdles. If all goes well in achieving these milestones, the plan is to be able to perform the first clinical studies in the next four years.