Isabella Lambert-Smith (PhD Candidate, Illawarra Health and Medical Research Institute, International BSc (Honours), University of Wollongong NSW) has provided us with a special summary of the key MND research hot topics from the 27th International Symposium on ALS/MND held in December 2016.
The 27th International Symposium on ALS/MND was off to an exciting start with Roel Vermeulen (Utrecht, The Netherlands) taking us on a tour of the ALS/MND ‘exposome’ in the joint opening session.
The exposome is the measure of all the exposures of an individual in their lifetime and how those exposures relate to their health. This holistic view of an individual’s health was developed because our understanding of the environmental stresses that contribute to MND is still limited despite years of research. And the contribution of environmental factors to MND is not trivial; it’s estimated that 40-50% of sporadic MND cases have an environmental origin.
Richard Bedlack (Duke University, USA) sparked the crowd’s intrigue when he walked onto the podium sporting a tiger-stripe jacket, sunglasses and a beaming smile. He launched into his talk with a brief overview of the X-files TV series and how the story of Agent Fox Mulder began – the mysterious disappearance of his sister and his ensuing search for her and extra-terrestrial life. This was his segue into the X-files of MND; the pursuit of alternative and off-label treatments (AOTs) by more than 50% of people with MND, the harms that can occur with AOTs and, amazingly, the rare examples of dramatic improvements that have coincided with starting AOTs (‘reversals’). When diagnosed with MND it’s natural for people to resort to searching for MND treatments on the internet. Referencing Mulder’s poster hanging on his office wall, Richard emphasised how strongly patients ‘want to believe’. And so with more than 50% of people looking into AOTs, Richard has been involved in a collaborative international program called ‘ALSUntangled’ (ALSU). ALSU uses social networking (Twitter) to bring together patients, clinicians and scientists to help untangle AOTs and assist patients in evaluating their options. Richard has also designed two programs to try and understand MND reversals, the ‘Replication of ALS Reversals’ program (ROAR) in which small pilot trials of the exact AOTs associated with rare MND reversals are conducted, and the ‘Study of ALS Reversals’ program (StAR).
RNA and RBPs
The scientific talks continued with the focus zooming way down to the molecular level and the role of RNA abnormalities in MND. RNA is one of the three major biological molecules that all modern life depends on (with DNA and proteins being the other two major molecules). RNA abnormalities have become a major focus of MND research in recent years with the discovery of genetic defects in several RNA-binding proteins (RBPs) in people with MND. Information stored in the DNA genetic code is translated into the proteins that build and function in our cells through RNA molecules and the interactions between RNA and RBPs.
Jernej Ule (University College London, UK) described to the audience how his group is using a technique called iCLIP to study the interactions between RNA and RBPs that are implicated in MND, focusing particularly on the RBP TDP-43, one of the major MND-linked RBPs. Using iCLIP they’ve discovered the region of TDP-43’s structure that acts as a docking platform for the RNA molecules it regulates. This particular region, called the low complexity (LC) domain, is common to most RBPs and is where most MND-causing genetic mutations occur. This indicates that these mutations interfere with TDP-43 and other RBPs’ function in regulating RNA molecules in our cells.
Nadine Bakkar (Phoenix, AZ, USA) showed how her group is using an artificial intelligence system called IBM Watson to identify other RBPs associated with MND. IBM Watson scoured prior published information and came up with a set of candidate RBP genes. Nadine’s group then examined the distribution of these RBPs in patient tissue and detected alterations in five RBPs; hnRNPU, SYNCRIP, SRSF2, Caprin1 and RBMS3. SYNCRIP in particular was abnormal in tissue from both patients with family history of MND and those with no known genetic mutations (sporadic MND).
The RBP Matrin-3 has been the focus of Edgardo Rodriguez and his colleagues (Florida, USA). They wanted to map out Matrin-3’s role in the brain and spinal cord and how it functions in MND as five different mutations have recently been discovered in it in people with MND. Their analysis showed that Matrin-3 regulates the levels of TDP-43, and binds to two other MND-associated RBPs. TDP-43, and Matrin-3’s interactions with it, thus appear to be important players in people with Matrin-3 mutations. Mutations in the RBP FUS are another cause of MND in about 5% of people with MND, and Udai Pandey (Pittsburgh, PA, USA) discovered a novel RBP called muscleblind that has a role in FUS-mediated motor neuron degeneration. They found that depleting the levels of muscleblind in a fruit fly model of FUS-MND rescued the flies from degeneration. It is clear then that RBPs and the regulation of RNA is crucial for normal motor neuron function and that RBPs play strong roles in MND.
The focus of the presented research then switched to how different types of RNA are involved in MND. MicroRNAs (miRNAs) play critical roles in maintaining brain tissue integrity, and it’s known that TDP-43 and FUS affect the production of miRNAs in cells. Eran Hornstein (Rehovot, Israel) presented evidence for miRNA failure in MND with the finding that the loss of a motor neuron-specific microRNA, miR-218, causes disconnection of motor neurons from muscle tissue. Alex Parker (Montréal, Québec, Canada) described how his group have used a worm-based model of TDP-43-linked MND to find that a miRNA molecule is responsible for the spread of degeneration between motor neurons. What now needs to be done is for researchers to develop a way to therapeutically target this mechanism.
A newly discovered class of RNA, circular RNAs (CircRNAs), was introduced to the audience by Stefano Dini Modigliani (Rome, Italy) in his presentation. CircRNAs are very important in neurons, and Stefano has found that many CircRNAs depend on the MND-causing protein FUS in order to function properly. MND-linked genetic mutations in FUS caused major disturbances in the functioning of these CircRNAs. This discovery opens up a new research focus into how FUS-mediated CircRNA abnormalities cause damage in motor neurons.
Messenger RNA (mRNA) molecules are the little molecular interpreters that translate the genetic language encoded in DNA into the building blocks of proteins. Edward Lee (Philadelphia, PA, USA) and his group have found that non-coding RNA molecules (RNA that doesn’t code for proteins) are processed and chopped up abnormally when TDP-43 is absent from its normal home in the cell nucleus. These chopped up non-coding RNAs appear to be toxic to motor neurons by capturing and sequestering important RBPs, stopping them from carrying out their crucial functions.
Darren Saunders (UNSW, AUS) started off the final session of the day, covering for Justin Yerbury (UOW, AUS) to tell us about “supersaturated” proteins in MND. In all people with MND, their diseased motor neurons are found to contain clumps of misshapen, aggregated proteins, which are termed ‘inclusions’. In people carrying genetic defects in SOD1, TDP-43 and FUS, there have so far been 73 different proteins found in these inclusions. Justin and his collaborators in Cambridge, UK, and Chicago, USA, wanted to know why these proteins end up being associated with inclusions in diseased motor neurons. What they discovered is that these proteins are “supersaturated” in motor neurons; that is, they are present in cells at very high concentrations that put them in danger of becoming solid and sticky, and this is specific to motor neurons (compared to other cell types in the body). So when motor neurons are exposed to extra stress, such as a genetic defect, or if a person is exposed to a damaging environmental stress, these supersaturated proteins are liable to clumping and forming aggregates while the rest of the affected motor neuron becomes unable to maintain control of all its cellular machinery, leading to its death.
Edward Pokrishevsky (Vancouver, BC, Canada) told us about a toxic pas de deux (a dance of two) that occurs between SOD1 and TDP-43 in MND. His group have demonstrated that TDP-43 can trigger SOD1 to change shape, becoming sticky and clumping into inclusions, and that TDP-43 is able to do this because one of the building blocks of TDP-43, an amino acid called tryptophan, is the same as one in SOD1, and these identical tryptophans interact. Furthermore, a drug that targets the tryptophan was able to block this interaction and stop SOD1 from aggregating, and thus may be a viable target for MND therapy.
The audience was then introduced to a new gene associated with MND, called CCNF. Ian Blair (Macquarie University, AUS) revealed to us his group’s genetic discovery and how this genetic defect appears to cause damage in motor neurons by interfering with the cell’s ability to destroy and remove unwanted, garbage proteins. These garbage proteins are then able to wreak havoc in motor neurons, and clump into inclusions. Marco Morsch (Macquarie University, AUS) followed on to demonstrate to us how these inclusions are able to persist in the space surrounding motor neurons after the motor neuron dies. Marco’s experiments were done inside zebrafish, and he was able to image and record the release of inclusions from individual motor neurons after killing them using a laser. If this wasn’t scary enough, the persistent inclusions were then taken up by neighbouring cells. Further work to understand this mechanism better is important as it may lead to the development of a treatment to stop the disease from spreading through the nervous system.
Brian Drawert (Santa Barbara, CA, USA) has also been working on better understanding the spread of motor neuron degeneration in MND. He’s developed a mathematical model that realistically recapitulates the spread of MND through the nervous system, and he’s working on extending his model to examine the spread of misshapen proteins in the brain and spinal cord, variability in cell vulnerability to MND and to help in developing a treatment to stop the spread of inclusions and cell death.
The day was rounded off nicely with a stimulating evening of poster presentations and discussions amongst researchers who’d travelled from many different countries around the world. My fellow lab member (who’s very recently moved to work with a MND research group in Vancouver) Luke McAlary and I received a lot of interest in the work we were presenting. Luke showed how he has pinpointed the exact tryptophan building block of SOD1 that is responsible for the clumping of SOD1 into inclusions. I revealed proteins I’d identified to be changed in response to MND-causing SOD1, TDP-43 and FUS and spoke to other researchers about how I’m trying to figure out the mechanisms by which these proteins are involved in MND.
Day two kicked off with a session devoted to potential therapeutic strategies that are in the works. Emily Lowry (New York, USA) has been working on developing and testing potential therapeutic compounds that target a mechanism called ER stress that’s thought to be involved in MND. ER (short for the endoplasmic reticulum, a special bit of cell machinery that manufactures and packages fats and proteins) stress leads to a special kind of cell death (called apoptosis) that the cell programs in to carry out when there’s an unhealthy build-up of sticky garbage proteins. In simple cell-in-a-dish experiments this set of compounds were effective at preventing programmed cell death, so Emily and her colleagues are moving onto test them in animal MND models. Hopefully these more thorough tests confirm their results, and clinical trials in patients can start.
Similarly, York Rudhard (Hamburg, Germany) told us how he is carrying out tests on compounds that target ER stress and protect motor neurons from its effects.
Focusing on a different set of cell machinery that’s known to be involved in MND, the cytoskeleton, Ashkan Javaherian (San Francisco, CA, USA) has been testing out compounds that target disease-associated changes in this machinery. The cytoskeleton is essentially the tiny skeleton that supports the cell and also aids in the transport of molecules around the cell. Ashkan and his colleagues had previously identified genetic defects in two cytoskeletal genes, PFN1 and TUBA4A, in people with MND. So Ashkan tested out the candidate compounds in models of defective PFN1, TUBA4A as well as TDP-43. He ended up discovering that one of the compounds rescued motor neurons in all three genetic models, suggesting that overlapping mechanisms occur in these three genetic forms of MND.
A very real challenge in the development of effective treatments for MND is the inaccessibility of the brain and spinal cord (the central nervous system, CNS); small molecule drugs are more often than not unable to cross through a natural barrier that exists between the blood vessels and the CNS. Paul Brennan (Oxford, UK), a drug discovery expert, enlightened the room of MND researchers to the effectiveness of therapeutic antibodies for CNS diseases. Antibodies are large Y-shaped proteins that the immune system produces to target foreign objects that cause disease, such as bacteria and viruses, and are very specific in the molecules they target. Some Alzheimer’s disease researchers have harnessed these natural molecules and their specificity and used them to target the amyloid plaques (a type of inclusion) that are responsible for Alzheimer’s. Their effectiveness at accessing the CNS sparked the interest of the audience, so we may see the development of therapeutic antibodies that are able to treat the misshapen proteins involved in MND.
An aspect of MND that many researchers aren’t aware of is the dysfunction that occurs in the blood vessels as disease progresses. Jochen Prehn (Dublin, Ireland) has been studying a protein called angiogenin, genetic defects in which are associated with MND. Angiogenin is involved in the growth of new blood vessels, and interestingly is present at high levels in motor neurons. Jochen’s group found that delivery of extra angiogenin to MND model mice reversed the breakdown of blood vessels and slowed down disease progression. His group’s work highlights the importance of maintaining the health of the blood vessels as MND progresses, an eye-opener for many of us in the audience.
Upper motor neurons (UMNs)
Challenging our current theories about MND further was the work presented by Pembe Hande Ozdinler (Chicago, IL, USA). Many researchers focus on the damage and death that occurs in the lower motor neurons (LMNs) in MND, which are the nerve cells that connect the spinal cord to the muscles. Pembe, however, brought our attention to the importance of the upper motor neurons (UMNs), which connect the brain to the spinal cord and are thus essential for messages from the brain to be relayed onto the lower motor neurons and muscles. She has found that the UMNs are affected very early in the development of MND, and that research effort should be made to improve the health of UMNs as well as the LMNs.
Peter Crouch (Melbourne, AUS) showed us how important the essential element copper is for the normal functioning of our motor neurons, and that therapies that restore the levels of copper in the nervous system may be a viable treatment option.
The most commonly identified genetic cause of MND (as well as a form of dementia) is a genetic defect in the C9ORF72 gene. This defect causes the production of unusual tiny proteins (dipeptide repeat proteins, DPRs) that are toxic to motor neurons. Guillaume Hautbergue (Sheffield, UK) discovered that it’s possible to rescue motor neurons from this toxicity by depleting a gene called SRSF1, which controls the export of RNA molecules that code for DPRs from the cell nucleus. RNA that’s exited the nucleus is then able to produce DPRs, so by depleting SRSF1 and stopping the RNA, Guillaume was thus able to stop the production of the toxic DPRs. Genetically manipulating SRSF1 and similar genes may thus provide a promising strategy to treat MND caused by C9ORF72 genetic defects. Manal Farg and her colleagues (La Trobe & Macquarie University, AUS and Tokyo, Japan) uncovered further insight into the effects of the C9ORF72 defect, with their findings that it induces extensive DNA damage.
Rounding off this session of diverse and insightful talks was Diane Moujalled’s (Melbourne, AUS) finding that a RBP, hnRNP K, forms a link between the damage caused by TDP-43 genetic defects and disrupted antioxidant activity in diseased motor neurons.
A critical part of being able to treat MND patients effectively is understanding the unique biology of each individual patient; the combination of factors, genetic and otherwise, that caused their specific case of MND and that affect their disease progression. To help in diagnosing a person, determining how their disease will progress and monitoring the effects of any therapeutic drugs used, scientists are on the hunt for biomarkers in patient tissues. Biomarkers essentially are biological entities such as genes and different molecules that can be measured in blood or cerebrospinal fluid samples taken from patients and that show changes that are specific to MND patients.
Marka Van Blitterswijk (Mayo Clinic, FL, USA) told the audience how her team of researchers have discovered two genes that can be used as biomarkers in people with C9ORF72-linked MND. These genes, HOXA5 and TTR, are involved in neuronal development and neuroprotection and shed light on how these mechanisms may influence disease progression in people carrying C9ORF72 genetic defects. Jonathan Cooper-Knock (Sheffield, UK) told us how he has established a molecular signature of TDP-43-linked MND, in which genes involved in inflammation of the nervous system (neuroinflammation) are more active than in people without MND.
NEK1, C21ORF2 and ARPP21
A person’s susceptibility to MND is believed to be determined by their genetics, and despite the identification of several different genetic defects that cause MND, the genetic defects causing disease in 30% of people with inherited MND and 90% of people with sporadic MND cannot currently be accounted for.
Kevin Kenna (MA, USA) is one of the researchers working to discover the genetic defects that cause disease in these people, and he told the audience in Dublin about a new gene he has identified; NEK1.
Wouter Van Rheenan (Utrecht, the Netherlands) then introduced us to C21ORF2 and the mutations in this gene that they found in a huge cohort of MND patients from Project MinE. Topping off the session of talks was Simon Topp (London, UK), who revealed yet another novel genetic defect, in ARPP21, that his group discovered in MND patients from the UK, Spain, Italy and the USA.
In the last few years it has come to light that motor neurons aren’t the only cells involved in MND; it’s become clear that the cells that surround, support and protect motor neurons, called glia, have multiple central roles in the changes that occur with disease. There are four types of glial cells in the CNS, but the two involved in MND are astrocytes and microglia. Astrocytes maintain the levels of neurotransmitters around motor neurons, while microglia are the immune cells of the CNS and defend motor neurons against harmful material.
Najwa Ouali Alami (Ulm, Germany) showed the audience that as disease progresses in MND patients, the roles of astrocytes and microglia, and the neuroinflammatory reactions they cause, change over time. At early stages of disease, the neuroinflammation they cause is beneficial, but at later stages it becomes detrimental. Najwa raised the idea that if we can identify the exact molecules involved in the beneficial phase, these may offer new ways to therapeutically intervene in disease.
Stanley Appel (Houston, TX, USA) shared additional insight into the changes that occur with neuroinflammation, talking about the T regulatory cells (Tregs; a type of white blood cell) that infiltrate the CNS at sites of damage to promote neuroprotection. In MND, Tregs are found to be decreased and thus their ability to protect motor neurons is severely compromised. With this understanding, Stanley’s group tested whether or not it would be therapeutic to take out samples of Tregs from MND patients, correct the Tregs by working on them in the lab, and then transplanting them back into the same patient. Amazingly this method slowed disease progression in the patients involved in the study and Stanley’s team are now investigating if they can sustain the beneficial effects.
Offering yet more treatment hope, Luis Barbeito (Montevideo, Uruguay) told the audience how his research team have found that an anti-cancer drug that acts on the immune system, Masitinib, was able to slow disease progression and prolong post-paralysis survival in MND model rats. This is a very encouraging finding as it shows that Masitinib was effective even once the model rats had become paralysed. In 2017 we should see significant progress in the testing of Masitinib as a treatment option for MND.
The role of astrocytes in MND isn’t only limited to neuroinflammatory reactions, as Amit Chouhan (St Andrews, UK) revealed to us. Together with his fellow researchers, they found that astrocytes that carried genetically defective C9ORF72 induced motor neurons to lose their ability to transmit information coming from their neighbouring neurons in the communication lines that make up the nervous system. Amit suggested that the next thing to be investigated is how to therapeutically target astrocyte-neuron signalling as a novel treatment for MND.
This mammoth of a conference ended with a joint closing session focused on the era of precision medicine, which medical research is transitioning into. As put by Winston Hide (Sheffield, UK), “precision medicine offers delivery of optimally targeted, precisely timed interventions tailored to an individual’s molecular drivers of disease”. This is critical for MND, as it is a complex disease and is highly variable from patient to patient. For precision medicine to become a reality for MND, individual research groups need to combine into a collaborative community that fosters the sharing of new discoveries, technologies and resources. Currently this is progressing well, with ongoing efforts in the genome sequencing of DNA from large cohorts of MND patients in Project MinE.
2017 holds much promise for MND research to progress at an unprecedented rate, given the hope that MND researchers from around the world feel about the truly exciting discoveries that were presented at this stimulating 2016 meeting in Dublin.
Isabella Lambert-Smith, PhD Candidate
Illawarra Health and Medical Research Institute, International BSc (Honours)
University of Wollongong NSW