Categories: Featured , Roundtable

25th Annual Symposium on ALS/MND Research: Day 2

Posted by author Jessica Sullivan

clock January 14, 2015

The second day of the International ALS/MND Research Symposium started off with a talk from Kevin Eggan of Harvard University (pictured). For many, Dr. Eggan is recognized as the first person to create an induced pluripotent stem cell line from a person diagnosed with ALS (the technology and process to create iPS cells was invented previously by Shinya Yamanaka, M.D., Ph.D. of Kyoto University in 2006).  Dr. Eggan’s talk dove deep into asking whether or not these type of stem cells were living up to their potential as drug development tools, and he provided some interesting results from his analysis of the field to date.

According to Dr. Eggan, his lab can coax mature adult fibroblasts to revert to a state of pluripotency using the Yamanaka factors, inserting into the nucleus of those cells several genes which induce their revision into stem cells.  One of the challenges with this method is that these genes randomly insert themselves into the genome of the cell and remain there in perpetuity. However, the cells do become stem cell like.  Newer methods to induce to revert to a stem cell state are being used by other labs, including ALS TDI, which don’t rely on random gene insertion approach. Regardless, the Eggan lab differentiated its iPS cells into motor neurons over a period of 24 days on average, creating a disease-relevant cell line to study. Before subjecting this cell line to experiments, the team spends about 30 days confirming its status and stability.

Of interest to the Eggan lab was to look at whether or not there were differences in the way electrical currents traveled across the ALS motor neurons.  They used sophisticated technology to shoot a current across several different lines created from PALS with different ALS-associated gene mutations, including SOD1.  Interestingly, they found that the SOD1 A4V lines had the greatest number of action potential spikes (a way to measure the amount/speed at which neurons communicate with surrounding cells, such as other neurons or astrocytes) of all the lines they looked at, suggesting that perhaps different forms of familial ALS may have specific cellular phenotypes, which could help explain the different trajectories of disease and be used in drug development experiments.

Speaking of drug development, the Eggan Lab earlier in 2014 announced that it had taken this information on action potential and begun to screen potential drugs which may modify its expression or amplification back to what is seen in non-ALS cell lines. One such drug screened, retigabine, showed the greatest benefit in the lab’s cell based screen.

Retigabine (pictured) is thought to open potassium acid channels and correct the sodium channel regulation in motor neurons.  It is an FDA approved compound for the treatment of certain forms of epilepsy and, together with the Northeast ALS  Consortium, a 120 person clinical trial is being planned at 12 NEALS sites.  Enrolled participants will have the opportunity to be placed into either the treatment group (which will include two different dosing groups) or a placebo control group. No date was given for when patient screenings will begin, site locations, or when exclusion criteria will be available for this trial.  When the trial does begin, it will be the first trial to be launched on the basis of information gathered from iPS lines only in ALS.  It is not known whether iPS cell lines are “better” tools for drug screening than more common models including fruit flies, mice, and other animals. However, this trial will provide an opportunity to begin to answer that question for the first time, at least in ALS research.

There were several other speakers during this first session of the day which focused on in vitro modeling. Two such talks were given regarding the characterization of iPS cells created from C9orf72 patient samples. Roxanne Mutihac, Ph.D. of the Nuffield Department of Clinical Neurosciences at the University of Oxford (pictured), provided data from her research on two C9orf72 iPSc lines she created. In one of her lines, she reported that it maintained a total of 1380 hexanucleotide repeats and in the other, 510 were maintained through the reprogramming process. After confirming that the expansion was stable, she began studying the cells and found a key hallmark of C9orf72 pathology- the presence of toxic RNA foci within the cells. Other interesting findings reported included the phenomenon of high levels of calcium in C9orf72 motor neurons versus cortical neurons, which may suggest a unique trait for further study. Finally, it is worth noting that in the age of oligogenetics in ALS, TDP43 proteins mislocalized in the C9orf72 iPSc lines created in Mutihac’s lab.    

ALS in China, the US Military and Head Injury

An entire session at this year’s International Symposium was devoted to the epidemiology of ALS. It included important reports on the landscape of ALS in China, new data on military incidence of ALS from the US, and a conversation on whether head injuries (ie: concussions) alter the progression of ALS.

China is the world’s most populous country with more than 1.3 billion people. It is an immensely diverse demographic, and results reported at this year’s meeting from Liying Cui, Ph.D. of the Peking Union Medical College in Beijing, suggest that the population of PALS in China may be very different from that found in other nations across the globe. According to Cui, 95% of ALS cases in China are sporadic, and the ALS-associated genes found in Chinese PALS are very different from those found in western countries. However, there are some similar characteristics. The mean age of onset of ALS in China is about 52 years of age in PALS, and the time from symptom onset to diagnosis is generally about 13 months- statistics not wholly unlike what we see in many other countries.

Recently, Cui and her colleagues in 10 different research hospitals created an ALS registry, recruiting and documenting 461 PALS from across China.  According to China ALS Registry data, only 29% of PALS take rilutek versus 39% which opt for traditional herbs to treat the disease. In a separate study of 680 PALS, Cui reported on the site of onset and spread of disease in Chinese PALS.  More than half of all PALS in the study were found to have their first symptoms in upper limbs generally (cervical), with another 1 in 5 cases experiencing the first symptoms in either bulbar or lumbar regions.  According to this new study, 85% of PALS developed symptoms in the upper and lower motor neurons, with 10% lower motor neuron only. The mean survival of PALS in this study was 34 months with bulbar onset ALS (PALS survive only 12 months on average in China). As seen and reported in other epidemiological studies of ALS, Cui suggested that the data confirmed that the number of regions of the spinal cord involved in the first three months of disease onset seem to correlate with the overall pace of the disease.

For many years, ALS was not thought to be related to dementia. However, more recent studies have now shown different cognitive and executive dysfunctions in PALS.  In the US, it is now thought that at least 10% of PALS develop frontotemporal dementia in addition to the progressive neurodegenerative disease. However, according to Cui’s research, the percentage of ALS/FTD in China is only 2% overall. Similarly, it seems ALS-associated genes in China vary greatly from those found in western nations. Less than 1% of FALS cases in China are carriers of the C9orf72 expansion, according to Cui.  More commonly found are SOD1 (26%), TDP43 (5.6%), and FUS (12%).

It is a commonly cited fact in the US that deployed military service members have a greater chance of developing ALS than the civilian population. However, little additional data has been reported since the original work was presented more than a decade ago. Marc Weisskopf, PhD, ScD (pictured) of the School of Public Health at Harvard University provided his analysis of the National Longitudinal Mortality Study, a sampling of households within the US.  In this data, he identified 800 ALS deaths, including 221 military service members through 2002. Nearly two thirds of these PALS served in the Second World War (WWII), with the others serving in the Korean, Vietnam, or other wars except World War I.  Weisskopf’s review of the Mortality Study found there were different rates of ALS within the military over time, with WWII veterans developing ALS at a great rate when compared to veterans from Korea or Vietnam.  He suggests there may be other deployment-related linkages which need to be explored.

Before moving on to head injury and ALS, it is worth noting an important talk from Daniela Mariosa, a Ph.D. student at the Karolinska Institute in Stockholm, Sweden. Mariosa’s research concerned diabetes as a risk factor in the development of ALS, which was originally suggested in a paper from Martin Turner, M.D., Ph.D. last year. Mariosa decided to look at the 1990 Swedish census, in which she identified 5108 newly diagnosed ALS cases. Controlling nearly 5:1 with non-ALS cases in the census, Mariosa found that an insulin-dependent diabetes diagnosis before age 30 increased the likelihood of developing ALS.

There has been great attention paid to the topic of head injury in the US as of late, with the National Football League (NFL) recently settling a class-action lawsuit of former players claiming the injuries they suffered while playing caused them to develop several different neurodegenerative diseases, including ALS. An original research study done by Everett Lehman at the National Institute of Occupational Safety and Health found a greater incidence rate of neurodegeneration in former NFL players as compared to the general population, a summary of which was provided via webinar by the ALS Therapy Development Institute in 2012. Notable former NFL players diagnosed with ALS include Philadelphia Eagles fullback Kevin Turner (Kevin Turner Foundation), New Orleans Saint’s hero Stephen Gleason (Team Gleason) and journey-man linebacker Tim Shaw (pictured, @TShawsTruth). Despite the evidence of some kind of correlation between ALS and the NFL, Lehman and most others have been careful to clearly state their studies are not intended to measure if concussions or other type of injuries play a direct role in ALS onset.

Instead, the goal of their studies was to explore if head injuries affect the progression rate of the disease. Christina Fournier, M.D., an instructor at Emory University, reported on her research into this question. She presented data suggesting no correlation between a head injury (which she defined as causing the loss of consciousness or hospitalization) and the progression rate of ALS.  Using data from Emory University, she found 24 PALS which met her criteria for head injury and compared them to 76 cases which didn’t. Fournier also looked at autopsy samples from PALS with and without head injury, and found no major differences in the frequency of proteinopathies in TDP-43, Tau, and others. In summary, more research is needed before a definitive connection between head injury and ALS can be identified.

Neurofilaments as ALS Biomarker

One of the greatest challenges in ALS drug development is the lack of biomarkers to predict disease progression. Biomarkers are typically defined as a measureable indicator of the presence or severity of a disease in patients.  Most of the time, ALS progression is measured using a patient-reported and rather subjective scale known as the revised ALS Function Rating Scale, a questionnaire used by clinicians to note changes in a PALS’ ability to move, eat, or breathe independently. Earlier in 2014, the ALS Therapy Development Institute called together more than 30 scientists for a biomarker research meeting, co-hosted with The ALS Association and NINDS, to discuss the latest data- some of which was later presented at the International Symposium in December.

According to Robert Bowser, Ph.D. (pictured), one of the foremost leaders in biomarker research in ALS, there is a huge gap between the discovery and validation of biomarkers. A review of the published work in this field conducted by Bowser showed there have been more than 20,000 publications on potential biomarkers in neurodegenerative disorders such as ALS. However, only 318 of those publications specifically aimed to validate a proposed biomarker.  This important gap highlights the difference between basic, observational research and the application of those observations in a drug development or clinical setting.

At this year’s International Symposium, a special session on biomarkers focused on the changes in the presence of neurofilaments in PALS overtime as a valid biomarker of disease progression. Filaments play a major role in the structural health of cells, and a specific class of them known as neurofilaments can be found in motor neurons. As any cell degenerates, its shape, size, and overall structure (cytoskeleton) changes.  Neurofilaments have been looked at in ALS as a potential way to measure the overall health of motor neurons for many years; however since they are primarily found in cells within the spinal cord, assessing neurofilament levels is both costly and difficult. 

Two of the presenters during the biomarker session speaking on neurofilaments in ALS, Martin Turner, Ph.D. of Oxford and Andrea Malaspina, M.D., Ph.D., decided to skip protocol and to present back-to-back and then to take questions together from the audience. Similar in topic, the data they would be presenting resulted from a collaborative effort on their parts.

Malaspina began with an overview of neurofilaments and review of previous research. Neurofilament auto-antibodies increase as ALS progresses and the neurons degenerate.  However, there are many different forms of neurofilament chains. Typically, they are separated based on weight into three different categories or subunits: light, medium, and heavy. The loss of neurofilaments is not unique to ALS, and decreases have been associated with all sorts of disorders of the spinal cord which are associated with degeneration or damage to motor neurons, including spinal cord injuries. According to Malaspina’s research, ALS stands out- at least when it comes to the light subunit of neurofilament (NfL). In his research on plasma and cerebrospinal fluid (CSF) samples, he discovered a correlation between the increase and decrease in the level of NfL present.  This may be an important finding as blood samples are easier to collect and analyze than CSF; both for the patient as well as the researcher.

The collaborative group has been collecting samples and tracking a group of patients for more than three years now. The data suggests that changes in NfL levels measured in the blood do match up to those found in CSF as the disease progresses. Further, Malaspina and team suggest that high blood NfL levels may correlate with survival time with ALS; meaning that the higher the levels, the longer the survival.

Turner followed Malaspina with additional research from the collaborative, which used magnetic resonance imaging (MRI) technology to attempt to corroborate this data. Looking at the white matter in PALS’ brains, Turner and his team determined the fractional anisotrophy in the corticospinal tract, and found that it correlates with disease progression- further suggesting that NfL levels may be a useful biomarker in ALS.

Following the tag-team event, Bowser took to the podium and offered a different view of neurofilament measurement in ALS.  His teams at both the Barrows Institute and his spin-off Iron Horse Diagnostics have been seeking to validate earlier published work suggesting that neurofilament heavy chain (NfH) may be a more appropriate biomarker for ALS, specifically as a diagnostic tool. Bowser has been developing an assay, or test, to measure the presence of NfH in biological samples with the idea that it could be used to speed up the diagnostic process.  On average it takes more than a year to diagnose ALS, generally because it is a process of excluding all other possible disorders.  This presents a major problem for patients, their attending medical teams, as well as researchers seeking to develop treatments; by the time a person is diagnosed, their disease has progressed significantly.

To attempt to address this issue, Bowser conducted, in partnership with NEALS, a prospective validation study of 126 CSF and 204 plasma samples from 23 different states or provinces in US and Canada.  The samples were sent blind to Bowser’s lab, and his team applied their assay to measure the level of NfH in the sample to determine if it was from a PALS or a healthy volunteer.  His team failed to identify only four PALS in the CSF samples (92% specificity) with slightly lower efficacy in the plasma samples. Bowser and his team are focused on improving the assay and have already created a modified version which has greater specificity, especially in identifying NfH levels in plasma.  However, in closing, and during questioning, he suggested that the test isn’t ready for primetime use in the clinic, and that further work needs to be done to take it from an “academic adventure to industry standards.”

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Antisense and ALS sensibility

clock December 19, 2013

C9ORF72 Rademakers RNA foci repeat expansion motor neuron ALS MND FTD FTLD


Repeat offenders Expanded RNAs appear to tightly fold up (RNA foci, above) in the nuclei of motor neurons in people with C9orf72-linked ALS - keeping key regulatory proteins out of action. Image: Courtesy of Nature Publishing Group.

With the discovery of disruptions in C9orf72 in 2011, ALS began to emerge as a whole brain disease. Key network glitches, detected by MRI, could be traced back to structural changes in cognitive centers of the brain – potentially explaining key cognitive challenges including problem solving and complex decision making (executive function). And, key behavioral signs including apathy and assessing risk, characteristic of frontotemporal dementia (FTD), became more widely appreciated in people with ALS – further blurring the line between these diseases.

“We used to think of ALS and FTD as different diseases. But now it is becoming clear that it is a disease continuum,” says University of Leuven’s Wim Robberecht MD PhD.

C9orf72 is now one of ten genes implicated in both ALS and ALS/FTD according to University of Massachusetts’ Bob Brown MD PhD. And, repeat expansions in the gene appear to be linked to at least 1 out of 20 cases of ALS – including more than 40% of people with familial ALS (fALS) – across the globe.

“This is clearly the most common cause of neurodegeneration,” says Brown.

Key questions remain. Expanded RNAs appear to accumulate in people with C9orf72-linked ALS – potentially keeping regulatory proteins out on the cytoplasmic sidelines. But according to recent studies led by Deutsches Zentrum für Neurodegenerative Erkrankungen’s Christian Haass PhD and Mayo Clinic’s Len Petrucelli PhD, the disease may instead be a perfect storm of proteins and RNAs brewing in people with the disease. Repeat-associated proteins called C9RANTs also appear to build up in motor neurons in people with C9orf72-linked ALS – potentially fueling their destruction. And, the production of C9orf72 protein appears to be reduced in people with C9orf72-linked ALS and may also contribute to the disease.

But research teams led by University of California San Diego’s Don Cleveland PhD and Johns Hopkins University School of Medicine's Jeff Rothstein MD PhD remain undaunted – developing treatment strategies that aim to destroy expanded RNAs in people with the disease.


The Whole C9 Yards Explore our interactive timeline to learn more about C9orf72, the most common form of ALS identified to date.

Ahead of the 2013 meeting of the Society of Neuroscience, researchers gathered at the RNA Metabolism and Neurological Disease conference in San Diego to talk about the latest advances in C9orf72-linked ALS and emerging treatment strategies for the disease going forward.

Modeling workshop

The race is on to create a model of C9orf72-linked ALS – a key first step in generating a treatment strategy for the disease.

University of Massachusetts’ Bob Brown MD PhD is leading the pack - developing a mouse model of the disease. 

The transgenic mice, which harbors 580 repeats in the first intron of the C9orf72 gene, do not appear to develop paralysis.  But according to Brown, may have other emerging hallmarks of the disease.

“It is early days,” says Brown.

But whether these mice actually exhibit key symptoms of ALS makes no difference according to University of California San Diego’s Don Cleveland PhD, who is also developing them.

“We don’t care whether these mice develop full or partial disease,” says Cleveland.  “It is not a key feature to move forward.”

Researchers, however, need to keep trying to develop mouse models of C9orf72-linked ALS, says ALS Therapy Development Institute’s Fernando Vieira MD.

Although these mice will be helpful in validating emerging antisense treatment strategies for C9orf72-linked ALS, says Vieira, we don’t know what drives the disease. And, therefore we cannot be sure which therapy to develop.

“It is important to keep striving for models that actually recapitulate elements of ALS symptomatology,” says Vieira. “Then, we start to learn which pieces of the C9orf72 puzzle actually lead to the disease.”

Meanwhile, Johns Hopkins University School of Medicine’s Jeff Rothstein MD PhD is turning to stem cells to develop a treatment strategy for C9Orf72-linked ALS.

fibroblast reprogramming stem cell ALS


Skin to skin A growing number of scientists are turning to stem cells, recreated from skin cells (above) from people with C9orf72-linked ALS, in hopes to discover more effective medicines for the disease. Image: Ankur Singh PhD, Georgia Institute of Technology.

Motor neurons are recreated from people with ALS by reprogramming their skin cells. And, the cells scoured for key signs of disease.

The approach aims to identify key phenotypes that can be used to develop medicines that help weather these motor neurons from the C9orf72 storm by detoxifying them. 

The strategy is the same strategybeing developed by Cedar Sinai’s Bob Baloh MD PhD and University of California San Diego’s Clotilde Lagier-Tourenne PhD.

“We do not need a mouse to move a treatment forward,” says Rothstein.

Motor neurons do appear to exhibit tell tale signs of disease. Expanded C9orf72 RNAs and C9RANTs can be detected according to Mayo Clinic's Len Petrucelli PhD and University of Florida College of Medicine's Laura Ranum PhD.  And, according to Johns Hopkins University School of Medicine’s Chris Donnelly PhD, the motor neurons may also be more vulnerable – at least to increased levels of glutamate which occurs in many people with the disease.

But whether this model alone can be used to develop treatments for the disease remains an open question according to Mayo Clinic’s Rosa Rademakers PhD. Thousands of repeats appear to be embedded in the C9orf72 gene in key cells in the brain and spinal cord.  But only at most 700 – 800 repeats can be detected in skin cells – the cells researchers use to model C9orf72-linked disease.

“I think it is extremely important to keep this in mind,” says Rademakers.

Storm Troopers

In 2011, research teams led by Mayo Clinic’s Rosa Rademakers PhD and National Institutes of Health’s Bryan Traynor MD PhD fingered toxic RNA as the prime suspect behind C9orf72-linked ALS.

Repeat-rich RNAs appear to be detected in motor neurons of people with C9orf72-linked ALS.  And, suspected researchers, kept key regulatory proteins out of commission – leading to the disease.

The same underlying mechanism that leads to progressive muscle weakness in people with myotonic dystrophy.

c9rants ran translation C9ORF72 ALS MND


RANT and rave? RNAs appear to tightly fold up in people with C9orf72-linked ALS - enabling the synthesis of potentially toxic short proteins called C9orf72 non-ATG translated peptides (C9RANTs). RANTs are emerging as a potentially key feature in a growing number of diseases including myotonic dystrophy (above). Image: Courtesy of the Public Library of Science.

With the discovery of short dipeptide repeat proteins in people with C9orf72-linked ALS in 2012, however, researchers began to realize that the disease is much more complicated.

As many as six dipeptides appear to accumulate in key neurons in the brain in people with C9orf72-linked ALS and ALS/FTD according to studies led by University of Florida College of Medicine’s Laura Ranum PhD. And, at least one of these short proteins according to cell culture studies presented by Mayo Clinic’s Len Petrucelli PhD may damage them.

The short dipeptides are emerging as a characteristic feature of a growing number of RNA repeat-associated diseases including myotonic dystrophy I (DM1) and fragile X-associated tremor/ataxia syndrome (FXTAS).

“We need to start thinking about RAN translation,” says University of Florida College of Medicine’s Laura Ranum PhD. “And, their role in these diseases.”

But although there are a lot of lessons that can be learned from these RNA repeat-associated diseases, says Hospital of Sick Kids’ Christopher Pearson PhD, researchers still need to prove that the same underlying mechanisms lead to ALS.

At least 7 regulatory proteins appear to be kept out of action by C9orf72 expanded RNAs in the brain and spinal cord according to Johns Hopkins University School of Medicine’s Chris Donnelly PhD.  But whether the loss of any of them contributes to ALS remains an open question. 

What’s more, researchers remain unsure what role if any C9RANTs play in the disease.

“Toxicity is a question that is looming over all of us,” says Johns Hopkins School of Medicine’s Jeff Rothstein MD PhD.


Eye of the C9 Storm Researchers hope to identify key proteins that rescue the rough eye phenotype (top, right) of C9orf72 fruit flies in hopes to unravel underlying mechanisms of C9orf72-linked ALS. Image: Courtesy of the National Academy of Sciences.

To begin to tackle this question, the Johns Hopkins University School of Medicine team is turning to an emerging model of C9orf72-linked ALS in hopes to identify key sequestered proteins that may contribute to the disease. 

The transgenic fruit flies, which produce expanded repeats in key regions of the eye, sustain a progressive loss of key ocular neurons which leads to a “rough” appearance.  

The team hopes to identify key proteins benched by repeat-rich C9orf72 RNAs by increasing their levels in the eye.  And, looking to see whether their eyes are restored.

“Some RNA-binding proteins appear to reduce the rough eye phenotype,” says Johns Hopkins’ Christopher Donnelly PhD.

The GAL4 UAS fruit fly model, developed by Zihui Xu PhD, now at Huazhong University of Science and Technology, is one of the first to recreate neuron loss, a key feature of the disease.

Meanwhile, scientists including Mayo Clinic’s Len Petrucelli PhD are trying to figure out whether dipeptide repeat proteins also contribute to the disease.

“We are trying to understand what C9RANTs are doing,” says Mayo Clinic’s Len Petrucelli PhD, “whether they are toxic.”

Target Practice

A potential treatment strategy for C9orf72-linked ALS appears to be safe and well tolerated – at least in mice according to University of California San Diego’s Clotilde Lagier-Tourenne PhD.


Search and destroy Antisense oligonucleotides directed against the repeat expanded form of C9orf72 RNA bind these potentially toxic molecules- tagging them for destruction. Image: Courtesy of Rockefeller University Press.

The approach, developed by ISIS Pharmaceuticals, deploys antisense oligonucleotides (ASOs) known as gapmers to search for expanded C9orf72 RNAs and target them for destruction.

The emerging treatment strategy does appear to reduce levels of repeat-rich C9orf72 RNAs in motor neurons recreated from people with the disease.

Treatment with antisense oligonucleotides appear to reduce levels of expanded C9orf72 RNAs (RNA foci) to nearly 50% according to University of California San Diego’s Clotilde Lagier-Tourenne PhD. And, according to Johns Hopkins University School of Medicine’s Chris Donnelly PhD, may help protect them from potentially toxic buildup of glutamate (excitotoxicity) – a potential contributor to the disease.

What's more, many of these antisense oligonucleotides do not appear to further reduce expression of the C9orf72 gene.

The results add to growing evidence that this emerging treatment strategy may be helpful to people with C9orf72-linked disease.

“It is really encouraging – going forward with this strategy in C9orf72 ALS,” says Lagier-Tourenne.

But whether antisense is the best approach to treat C9orf72-linked ALS remains an open question. Existing dipeptide repeat proteins remain untouched. Expanded RNAs tightly fold up into stable quadruplexes according to studies led by Hospital of Sick Kids’ Christopher Pearson PhD – making them potentially less vulnerable to oligo attack. And, the battle may need to be waged in the nervous system.  ASOs are deployed intrathecally – directly into the spinal cord.

Small molecules may be a better choice to keep key regulatory proteins in action according to Scripps Institute’s Matthew Disney PhD.  Small molecules can often be taken by mouth.  And, may bind more tightly – burying themselves in the expanded RNA’s unique folds.


Foursquare Researchers hope to design small molecules that hide out in the unique folds of the expanded C9orf72 RNA 'quadruplexes' in people with ALS -to keep regulatory proteins in action. Image: Reprinted with permission from Gunaratnam, M. et al. (2009). Copyright 2009 American Chemical Society.

“We are targeting secondary structure,” explains Disney.  “This is more selective than oligonucleotides.”

But designing small molecules that target these RNAs may be tricky to do.  Small molecule libraries contain few compounds that bind RNAs according to Disney.  And, no antimicrobial or antiviral substance (natural product) has been discovered to date that target RNAs - providing no clues to create them.

To overcome these challenges, the Scripps Institute team introduced a new method to design molecules that target expanded RNAs to treat disease.

The library vs library approach involves the identification of key small molecules that target the RNA. And, tweaking them using computational-based methods to optimize their abilities to bind and detoxify them.

The resulting small molecules can then be displayed on peptide-like scaffolds to enable them to bind cooperatively to the repeat-rich RNAs – maximizing their efficacy.

The approach is currently being used to develop potential treatments for rare muscles diseases known as myotonic dystrophies.

Efforts to create expanded C9orf72 repeat-rich RNA-targeted small molecules are now underway.


To learn more about antisense and its potential for people with C9orf72-linked ALS, tune into our podcast with Jeff Rothstein MD PhD. To find out more about C9orf72-linked ALS and potential stem cell treatments for the disease, check out Astrocytes for the people?


Donnelly, C.J. et al. (2013)  RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention.  Neuron 80(2), 415-420.  Abstract  |  Full Text  (Subscription Required)

Lagier-Tourenne, C. et al. (2013) Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration.  Proceedings of the National Academy of Sciences 110(47), E4530-4539.  Abstract  |  Full text  (Subscription Required)

Sareen, D. et al. (2013) Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion.  Science Translational Medicine 5(208), 208ra149.  Abstract  |  Full Text (Subscription Required)

Zu, T. et al. (2013)  RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proceedings of the National Academy of Sciences 110(51), E4968-E4977.  Abstract  |  Full Text

Mori, K. et al. (2013) The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS.  Science 339(6125), 1335 – 1338.  Abstract  |   Full Text  (Subscription Required)

Ash, P.E. et al. (2013)  Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS.  Neuron 77(4), 639-646.  Abstract  |  Full Text  (Subscription Required)

Xu, Z.  et al. (2013)  Expanded GGGGCC repeat RNA associated with amyotrophic lateral sclerosis and frontotemporal dementia causes neurodegeneration.  Proceedings of the National Academy of Sciences  110(19), 7778–7783.  Abstract  |  Full Text

Reddy, K., Zamiri, B., Stanley, S.Y., Macgregor, R.B. Jr and Pearson, C.E. (2013) The disease-associated r(GGGGCC)n repeat from the C9orf72 gene forms tract length-dependent uni- and multimolecular RNA G-quadruplex structures.  Journal of Biological Chemistry 288(14), 9860-9866.Abstract|Full Text (Subscription Required)

Majounie, E. et al. (2012) Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study. Lancet Neurology 11(4):323-30. Abstract | Full Text  

DeJesus-Hernandez, M. et al. (2011) Expanded GGGGCC hexanucleotide repeat in a noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS Neuron 72(2), 245-256. Abstract | Full Text (Subscription Required)

Renton, A.E. et al. (2011) A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72(2), 257-268. Abstract | Full Text (Subscription Required)

Further Reading

van Blitterswijk, M., DeJesus-Hernandez, M. and Rademakers R. (2012)  How do C9ORF72 repeat expansions cause amyotrophic lateral sclerosis and frontotemporal dementia: can we learn from other noncoding repeat expansion disorders?  Current Opinion in Neurology 25(6), 689-700.  Abstract  |  Full Text  (Subscription Required)

Disney, M.D. (2013)  Rational design of chemical genetic probes of RNA function and lead therapeutics targeting repeating transcripts.  Drug Discovery Today 18(23-24), 1228-1236.  Abstract  |  Full Text (Subscription Required)



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SfN13: Astrocytes for the people?

clock November 19, 2013

astrocyte als


A starry knight? The introduction of healthy astrocytes in people with ALS may help keep existing motor neurons 'alive' by reducing levels of potentially toxic substances including glutamate. Image: Jonathan Cohen PhD, National Institute of Child Health.

More than 10 stem cell strategies are being developed for ALS across the globe.  One of these potential treatments, known as astrocyte replacement, is approaching the clinic. The strategy aims to protect motor neurons by introducing healthy astrocytes in people with ALS in hopes to detoxify the nervous system.

But according to recent preclinical studies, this approach may be limited because astrocytes may be a key player in ALS in only certain forms of the disease.

Now, a US team led by Nationwide Children’s Hospital’s Brian Kaspar PhD reports that astrocytes may contribute to motor neuron loss in people with C9ORF72-linked ALS.

The results suggest that strategies targeting astrocytes might benefit many people with ALS – including those with the most common form of the disease identified to date.

Nationwide Children’s Hospital neuroscientist Katherine Meyer PhD presented the results last week at the 2013 meeting of the Society of Neuroscience in San Diego.

Clinicians first turned to stem cells in the late 1990s in hopes to treat ALS. The strategy aimed to help people with ALS keep moving by replacing motor neurons destroyed by the disease.

But this approach turned out to be too tricky to do. Motor neurons must plug into the central nervous system. And, their axons need to be extended nearly 3 feet to reach the muscles.

astrocyte als


Appetite for destruction? Astrocytes (green) may contribute to ALS in part by failing to regulate levels of glutamate in motor neurons. Image: Hajime Hirase PhD, RIKEN Brain Science Institute.

This stem-cell based procedure could take 2-3 years by some estimates to potentially take effect– too late for many people with the disease.

In the mid 2000s, researchers turned to astrocytes as a more feasible approach to treat the disease. The potential stem cell treatment, known as astrocyte replacement, involves injection of astrocyte precursors directly into the spinal cord.

The strategy aims to protect existing motor neurons in people with ALS by restoring the balance of key neuronal substances including glutamate in the brain and spinal cord.

One approach, known as Q cells, according to preclinical studies led by Johns Hopkins University School of Medicine's Nicholas Maragakis MD appears to delay onset and extend survival of the G93A SOD1 mouse model of ALS. 

Astrocyte replacement is now at the IND-enabling stage - the final step before entering the clinic.  At least three strategies are being developed.   An IND could be filed as early as February 2014 according to Cedar Sinai's Clive Svendsen PhD.

But which people might benefit from astrocyte replacement remains an open question. 

Astrocytes regenerated from skin cells from people with SOD1-linked ALS appear to contribute to motor neuron loss.  But according to studies led by Johns Hopkins University School of Medicine’s Nicholas Maragakis MD and University of Edinburgh’s Siddharthan Chandran PhD, motor neurons appear to emerge unscathed when co-cultured with astrocytes harboring mutations in TDP-43, a protein mislocalized in most people with the disease.

astrocyte als ips stem cell


Replacement, Ltd? Astrocytes harboring mutations in TDP-43 appear to leave motor neurons untouched - at least in mice. But according to preliminary co-culture results from Nationwide Children’s Hospital’s Katherine Meyer PhD, these astrocytes may contribute to motor neuron loss – at least under certain conditions. Image: Robert Krencik, University of Wisconsin.

Now, researchers from Nationwide Children’s Hospital report that astrocytes in people with C9Orf72-linked ALS may be a key player in the disease.

The “induced” astrocytes, directly converted from skin cells from three people with C9Orf72-linked ALS, reduced survival of mouse motor neurons by at least 50%.

The preliminary co-culture findings come at the heels of previous studies from Amanda-Haidet Phillips PhD, now at Johns Hopkins University School of Medicine, and Columbia University’s Virginia Le Verche PhD which suggest that astrocytes may contribute to motor neuron loss in certain forms of sporadic ALS.

Together, the results suggest that astrocytes may be a key player in ALS in many people with the disease. And, potential treatments directed at astrocytes may be of benefit to them.

Clinicians hope to launch phase I studies of astrocyte replacement sometime next year. 


To learn more about astrocyte replacement, tune into our podcast with Johns Hopkins University School of Medicine’s Nicholas Maragakis MD, A new player in the stem cell Q? To find out about other stem cell strategies being developed the disease, check out our report from the 2013 meeting of the International Society of Stem Cell Research, ALS, Stem to Stern.  



Haidet-Phillips, A.M., Gross, S.K., Williams, T., Tuteja, A., Sherman, A., Ko, M., Jeong, Y.H., Wong, P.C. and Maragakis NJ (2013) Altered astrocytic expression of TDP-43 does not influence motor neuron survival.  Experimental Neurology doi: 10.1016/j.expneurol.2013.10.004.  Abstract |  Full Text  (Subscription Required)

Serio, A. et al. (2013) Astrocyte pathology and the absence of non-cell autonomy in an induced pluripotent stem cell model of TDP-43 proteinopathy.  Proceedings of the National Academy of Sciences 110(12): 4697-4702.   Abstract  |  Full Text

Haidet-Phillips, A.M. et al. (2011) Astrocytes from familial and sporadic ALS patients are toxic to motor neurons. Nature Biotechnology,29(9), 824-8. Abstract Full Text (Subscription Required)

Lepore, A.C., Rauck, B., Dejea, C., Pardo, A.C., Rao, M.S., Rothstein, J.D., and Maragakis, N.J. (2008). Focal transplantation-based astrocyte replacement is neuroprotective in a model of motor neuron disease. Nature Neuroscience, 11(11), 1294-1301.  Abstract Full Text  

Yamanaka, K., Chun, S.J., Boillee, S., Fujimori-Tonou, N., Yamashita, H., Gutmann, D.H., Takahashi, R., Misawa, H. and Cleveland DW. (2008)  Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis.  Nature Neuroscience 11(3), 251-253.  Abstract  |  Full Text

Further Reading

Robberecht, W. and Philips, T. (2013)  The changing scene of amyotrophic lateral sclerosis.  Nature Reviews Neuroscience 14(4), 248-264.  Abstract  |  Full Text  (Subscription Required)

Papadeas, S.T. and Maragakis, N.J. (2009) Advances in stem cell research for Amyotrophic Lateral Sclerosis.  Current Opinion in Biotechnology 20(5), 545-551.  Abstract  |  Full Text  (Subscription Required)

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Categories: Featured , Roundtable

ALS clinical trials, powering forward

clock January 16, 2013

mitochondria dysfunction ALS


Power up Biogen Idec's dexpramipexole hoped to slow ALS by keeping the power on in the deteriorating motor nerves. Image: Judith Stoffer, NIGMS.

Biogen Idec pulled the plug on its potential energy booster, dexpramipexole, this month due to its inability to show efficacy at the phase III stage in the clinic. 

The drug is one of two emerging medicines that appeared to slow ALS by more than 30% at phase II. And, one of at least 9 drugs at phase III that were unable to show substantial benefit in the clinic.

Nearly 1000 people with ALS participated in the phase III clinical trial.

The results suggest that experts might need to rethink the design of phase II clinical trials for ALS to better inform go/no go decisions.

ALS Today’s Michelle Pflumm PhD talked with Biogen Idec’s Director of Neurodegenerative Research Doug Kerr MD PhD about dexpramipexole, the lessons learned and the development of future medicines for ALS going forward.


To find out about other emerging treatment strategies for ALS, check out our 2012 International Symposium on ALS/MND meeting review ALS Trials and Tribulations.


Cudkowicz, M. et al. (2011) The effects of dexpramipexole (KNS-760704) in individuals with amyotrophic lateral sclerosis.  Nature Medicine. doi: 10.1038/nm 2579  Abstract Full Text (Subscription Required)

Gribkoff, V.K. and Bozik, M.E. (2008) KNS-760704 [(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine dihydrochloride monohydrate] for the Treatment of Amyotrophic Lateral Sclerosis CNS Neuroscience and Therapeutics 14, 215-226.  Abstract Full Text (Subscription Required)

Alavian K.N. et al. (2012) Effects of dexpramipexole on brain mitochondrial conductances and cellular bioenergetic efficiency. Brain Research 1446, 1-11. Abstract Full Text (Subscription Required)

Further Reading

Gladman, M., Cudkowicz, M. and Zinman, L. (2012) Enhancing clinical trials in neurodegenerative disorders: lessons from amyotrophic lateral sclerosis. Current Opinion in Neurology 25, 735-742. Abstract Full Text (Subscription Required)

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Categories: Roundtable

The Eligible Patient

clock October 19, 2011

Since the discovery of the first ALS-associated gene, superoxide dismutase 1 (SOD1) in 1993, scientists tested more than 30 experimental ALS drugs in clinical trials.  Only Sanofi’s Rilutek is FDA approved and at best moderately treats the disease.

A big part of the problem say ALS experts is that the disease is extremely variable and the underlying mechanism of the disease is not understood.  But despite these obstacles, University of Torino scientists argue based on findings of a new study that more can be done now to increase the chances for a new drug to be successfully developed simply by selecting patients that participate in clinical trials differently.

”I think what we have shown,” says lead author Adriano Chiò MD, “indicates that we are making mistakes.  It is time to correct the mistakes.”

The study is published this month in the journal Neurology.

The University of Torino team compared the local ALS patient population that participated in clinical trials to those diagnosed with the disease between 2003 and 2008.  The researchers found that those enrolled in clinical trials tended to be younger, male and half as likely to have the bulbar form of the disease.


ALS clinical trial participants measure for measure. A comparison between Italian ALS patients in the metropolitan Torino area that participated in clinical trials and those diagnosed with the disease between 2003 and 2008.  Adapted from Chio, A. et al. Sept. 28; Neurology doi: 10.1212/WNL.0b013e318232ab9b

These results suggest that experimental ALS medicines may be more difficult to demonstrate to be effective because the people participating in clinical trials tend to be healthier and have a more slowly progressing form of the disease.

The University of Torino team suggests clinical trial investigators should accommodate a broad range of people with ALS by dropping forced vital capacity minimums to 60%.  And these researchers say that clinical trials should instead include people with possible ALS, people with probable ALS or people who are recently diagnosed with the disease.  The researchers argue that by sticking to these patients, the interventions also have a much better chance to be effective since these people are in the early stages of the disease.

“The inclusion of patients in the earliest phase of the disease, when more motor neurons are still present, increase the probability that a drug may be demonstrated to be effective,” explains Chiò.

Rethink the possible

Many of these recommendations have already been put in place.  Within the last two years, clinical trials started to include broader populations of people with ALS by reducing their forced vital capacities to as low as 50%.  And researchers are opening their doors to people suspected to have the disease.  Ongoing trials such as Biogen Idec’s Empower (dexpramipexole), Cytokinetics’ CK-2017357 and GlaxoSmithKline’s “NOGO” trials include people with only a possible ALS diagnosis.

“There has been significant changes in how the trials are designed,” says Massachusetts General Hospital ALS Clinic Director Merit Cudkowicz MD, MSc.  “We already recognize that inclusion criteria were too strict.”

The reason drug developers feel comfortable with including people with possible ALS says Director of Duke University’s ALS Clinic, Rick Bedlack MD, PhD, MS, is that there is growing evidence that these people are extremely like to get the disease.  “Once you get to the category of possible ALS the chances that you are going to move into one of those other areas is incredibly high,” says Bedlack.

And says Knopp Biosciences’ Valentin Gribkoff, who heads the dexpramipexole team, opening the door to people in earlier stages gives them the best chance of detecting efficacy.

“What we think that did for us is allow for a wider-dynamic range for seeing a drug effect,” explains Gribkoff.


Diagnosis 101. ALS is often diagnosed in stages which are defined by the number and location of areas affected and the degree of disease spread. Called El Escorial criteria, a combination of clinical, electrical, and imaging measures are typically used. LMN, lower motor neurons.  UMN, upper motor neurons.  EMG, electomyography.

Reality Check

While more and more clinical trials open their doors to people with the first signs of ALS, some researchers however continue to keep them open to prevalent cases – people up to two years into ALS - even when an intervention is expected to be most effective early in the disease. 

One reason says Cudkowicz is that the diagnosis of definite ALS with today’s tools typically takes 12 to 14 months.  It is just not possible to test interventions in people with definite ALS, at least in the U.S., within the first year of experiencing symptoms of the disease.

But this is not the only reason researchers are hesitant to focus clinical trials on so-called incident cases of the disease.  With only a small percentage of people participating in clinical trials, some ALS experts worry that these restrictions will make it that much harder to develop promising medicines for the disease.

“We are really going to magnify the enrollment problem,” says Bedlack. “It’s probably going to be even slower and more difficult to recruit patients.”

Chiò disagrees.  He says that testing early interventions under these restrictions is doable – particularly in large cities in which 100s of cases of ALS are diagnosed annually– and is necessary to give the drugs their best chance of being demonstrated to be effective

“I hope that trials will be modified and will take into account the problems I have found,” says Chiò.

Trials and Tribulations

With the prospect of a growing number of trials however, being restricted to people who experience their first foot drop within 24 months, people with ALS are understandably frustrated.  But says Cudkowicz this is not going to be the case for each and every drug being developed for the disease.  Such decisions are made case by case based on when the medicines are most likely to work during the course of the disease. As researchers learn more about ALS, new treatment strategies may emerge that target more advanced stages of ALS - treatment strategies that must be evaluated in those populations.   

“I don’t think there is one model fits all clinical trials,” says Cudkowicz.


Chiò, A., Canosa, A., Gallo, S., Cammarosano, S., Moglia, C., Fuda, G., Calvo. A, and Gabriele, M.. (2011) ALS clinical trials: Do enrolled patients accurately represent the ALS population? Neurology, 77(15), 1432-1437. Abstract Full Text (Subscription Required)

Further Reading

Beghli, E. et al. (2011). The epidemiology and treatment of ALS: focus on the heterogeneity of the disease and critical appraisal of therapeutic trials. Amyotrophic Lateral Sclerosis, 12(1), 1-10Abstract Full Text (Subscription Required)

Bedlack, R.S., Wicks, P., Heywood, J. and Kasarskis E. (2010). Modifiable barriers to enrollment in American ALS research studies. Amyotrophic Lateral Sclerosis, 11(6), 502-507Abstract Full Text (Subscription Required)


UPDATED 11/21/11:  Knopp Biosciences' Val Gribkoff joins the conversation.

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