Therapies for amyotrophic lateral sclerosis (ALS) have proven hard to develop, in part because of inadequate disease models. A new stem cell-based screening method may help. Researchers led by Lee Rubin, Harvard University, Cambridge, Massachusetts, screened for compounds that sustain motor neurons derived from mouse embryonic stem cells (ESCs) and ALS patients. As reported in the April 18 Cell Stem Cell online, the kinase inhibitor kenpaullone topped the list of potential drug candidates. It kept motor neurons alive for weeks longer than expected after trophic support was withdrawn. Kenpaullone could point to a new therapeutic target for ALS, and the screening model provides a better way to seek out future compounds, suggested the authors. “When disease-specific cells are part of the process to validate drugs, you are more likely to obtain better results,” Rubin told ARF. Others pointed out that a big leap still exists between cell models and human disease.

Before heading to clinical trials, most drugs are tested on mouse models, but that does not always guarantee success in people. ALS has seen a string of clinical trial failures. To better approximate disease processes, several groups have recently developed human induced pluripotent stem cell (iPSC) models to generate different cell types for drug screens. Although motor neurons have been tricky to obtain from iPS cells in the past (see ARF related news story), researchers now have more reliable methods (see Makhortova et al., 2011) and have used human-derived motor neurons to test a handful of compounds that might treat ALS (see ARF related news story on Egawa et al., 2012).

First author Yin Yang and colleagues grew motor neurons from the embryonic stem cells of wild-type mice or mice carrying the human superoxide dismutase 1 (SOD1/G93A) mutation. SOD1 variants account for a fifth of familial ALS mutations and its misfolded protein aggregates in the disease. To mimic conditions that might cause neurodegeneration in vivo, the group starved the cells of trophic factors or inhibited the PI3K/Akt pathway, which promotes cell survival. The researchers then tested to see if any of the 5,000 compounds in their chemical library kept the cells alive.

A variety of anti-apoptotic compounds did. One in particular—kenpaullone, an inhibitor of glycogen synthase kinase-3 (GSK-3) and cyclin-dependent kinases—performed better than the rest. After three days of treatment, three- to fourfold more wild-type and mutant SOD1 motor neurons survived with kenpaullone treatment compared to untreated controls. Kenpaullone also maintained neuronal synapse number, structural morphology, and electrophysiological characteristics. The compounds also suppressed SOD1/G93A protein levels in neuronal cultures and reduced its aggregation and ubiquitination. Kenpaullone was first synthesized in the early 1990s, but has never been clinically tested for any disease, said Rubin. GSK-3 activation has been implicated in neurodegeneration, but no GSK-3 inhibitor has succeeded in clinical trials.

Kenpaullone may do more than just inhibit GSK-3, however, as it boosted motor neuron survival more than did other inhibitors of this enzyme or small RNAs that silence the kinase gene. Yang and colleagues found it reduced phosphorylation of both c-Jun N-terminal kinase (JNK) and c-Jun, which help drive stress-induced neuronal apoptosis. Kenpaullone also blocked activation of their upstream regulators, including MKK4, Tak1, and HPK1/GCK-like kinase (HGK), which is expressed in motor neurons. Together, the data suggest that kenpaullone tones down a cell-death signaling cascade, the authors wrote. They suggested that HGK may be a new drug target for ALS.

How does kenpaullone cut the level of SOD1 and prevent it from accumulating? “My guess is that it does not affect protein aggregation directly,” Rubin told Alzforum. Given that the compound curtails both protein levels and its ubiquitination, it probably affects general accumulation processes, he explained. “By keeping cells alive and healthier, it diminishes whatever processes cause the protein to aggregate.”

Yang and colleagues wondered if kenpaullone would affect human motor neurons the same way. To find out, the researchers made motor neurons from the iPSCs of one healthy person and two ALS patients carrying either a SOD1 or TDP-43 mutation. Kenpaullone enhanced cell survival in all three cell types. The researchers compared the compound to two others that recently failed in Phase 3 clinical trials for ALS—olesoxime (see ARF related news story) and dexpramipexole (see ARF related news story). Dexpramipexole did not enhance motor neuron survival. Olesoxime did, but less than kenpaullone and less consistently across cell lines. “We might have been able to predict the clinical trial failure if we had tried the compounds in this human cell system,” said Rubin.

Rubin’s group plans to test iPSC-derived motor neurons from other ALS patients, including those with sporadic disease and non-SOD1 mutations. He also plans to evaluate the compound in an ALS mouse model, though the kinase inhibitor has to be directly delivered into the spinal cord, as it does not easily get into the brain. Rubin has no plans to optimize kenpaullone by medicinal chemistry, but is open to doing so with interested collaborators. His group will also look into HGK as a drug target, he said.

More broadly, these experiments aim to validate this system as a way to screen for new drugs. Techniques for growing human cells are improving, and it may soon be practical to use them for high-throughput screens, said Rubin. Drugs could then be routinely tested on human cells before going into clinical trials, he said.

This comes at a time when some are questioning the value of mouse models of neurodegenerative disorders. “The use of human-derived neural cells is an important addition to drug discovery to help bridge the preclinical-clinical translational gap,” wrote Rebecca Pruss of the pharmaceutical company Trophos in Marseille, France, in an e-mail (see full comment below). Current animal models come with a high translational risk, she wrote. However, Pruss cautioned that it may be difficult to define how activity in such cell models will translate into treatments, pointing out that olexisome showed no benefit in an 18-month trial despite activity in this model. Pruss would have liked to know what riluzole—the only approved drug for ALS—did in this cell model. Rubin said they did test riluzole and it had no effect.

Richard Smith, Center for Neurologic Study, La Jolla, California, also offered cautions. “It is too early to know whether stem cell technology offers a better way to predict clinical results in trials,” he told Alzforum. “Leaping from any model, be it an animal or embryonic stem cell model, is still quite a leap. Nevertheless, anything that advances the identification of molecules that have therapeutic benefit and prove to be useful in clinical trials is extremely welcome.”—Gwyneth Dickey Zakaib


  1. This is an exciting paper showing the ability to use a specific class of stem cell-derived neurons—motor neurons—to screen for potential neuroprotective small molecules. While the mouse ESC-derived neural cultures appear suitable for systematic screening, the authors have been able to then evaluate selected hits on human motor neurons derived from ESC- as well as control- and patient-derived iPSC cultures. I was delighted to see that olesoxime was used as a comparator and clearly promoted survival of trophic factor-deprived human motor neurons with similar potency as we found using trophic factor-deprived primary rodent motor neurons. Even though it appears less active than the selected hit, kenpaullone, the large variability in the response to trophic factors, the gold standard, makes it hard to define a maximum response or calibrate the relative response of compounds in this assay. Having human-derived neurons to use as a way of selecting compounds is an important advance for CNS drug discovery, which suffers exceptionally from the high translational risk associated with current animal models. It may still be difficult to define how activity in this model will translate in terms of clinical significance, particularly for such a rapidly progressing neurodegenerative disease as ALS. Despite its activity in these cell assays, olesoxime did not have a statistically significant benefit over riluzole in an 18-month trial in ALS patients. One wonders what effect riluzole, the only approved drug for ALS, might have in this model?

    In terms of technical achievement, this paper is a real tour de force. A screening assay was possible using mouse-derived GFP-expressing motor neurons, which allowed the researchers to overcome a number of challenges. Motor neurons represent typically only 30-50 percent of the cells in the mixed cultures (GFP expression is driven by a motor neuron-specific promoter), and new progenitor-derived motor neurons can appear during the assay, giving rise to false positives. Fluorescent-activated cell sorting showed that the compounds acted directly on motor neurons.

    The authors selected trophic factor deprivation to trigger robust and rapid motor neuron death and stringently selected hits from a 5,000-compound library based on activity in two separate assays derived from “control” or mutant SOD1 mouse motor neurons. Testing in a number of secondary assays (PI3K/Akt inhibition, "toxic astrocytes," morphology, functionality) further classified those hits, and a number of compounds affecting known “survival promoting pathways” were identified. The authors then focused on one of these, kenpaullone, and carefully dissected its mechanism of action as an inhibitor of a kinase cascade culminating in c-Jun-activated apoptosis and fingered a particular kinase as a potential drug target for ALS. Interestingly, kenpaullone also suppressed expression of mutant SOD1 with longer treatment. Further analysis of the role of this kinase cascade and apoptosis pathway in normal and pathological processes, as well as other off-target effects of this class of compounds, will be needed to evaluate the risk/benefit of targeting this pathway and to base a chemical optimization strategy on this compound family. Nevertheless, despite questions about the target itself, the use of human- and especially patient-derived neural cells is an important addition to CNS drug discovery to help bridge the preclinical-clinical translational gap.

    Regarding the stem cell motor neuron screening models, one could envisage testing combinations of compounds with different mechanisms of action to see if their effects are additive, or even synergistic, and potentially define a mixture that would provide maximal activity. Most diseases are best treated with combination therapies. Finding a regulatory pathway to test two or more investigational drugs could be the way forward to find effective treatments for neurodegenerative diseases.

    It is intriguing to think that olesoxime might provide additional benefit to compounds acting on the kenpaullone-activated pathway, as its mechanism of action is probably different. We have recently compared olesoxime's mechanism of activity with BDNF, a trophic factor (see Gouarné et al., 2013). Using a cortical neuron neurotoxicity model, we showed that olesoxime did not activate the PI3K/Akt pathways but appears to have a direct effect to stabilize mitochondria and prevent release of apoptotic factors—olesoxime binds to two outer mitochondrial membrane proteins, VDAC and TSPO. Taking all the data on olesoxime accumulated by ourselves and others, it appears to have clear neuroprotective effects. Trophos believes it has a place for the treatment of neurodegenerative diseases, especially where treatment could be started earlier, before neurodegeneration is advanced and accelerating, as in genetic or neuroinflammatory conditions . For this reason, olesoxime is currently being tested in a Phase 2 study in type 2 and type 3 SMA patients and a Phase 1b study in relapsing remitting multiple sclerosis patients as an add-on to β interferon (both trials are registered on


    . Olesoxime protects embryonic cortical neurons from camptothecin intoxication by a mechanism distinct from BDNF. Br J Pharmacol. 2013 Apr;168(8):1975-88. PubMed.

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News Citations

  1. iPSC Disease Models Up and Coming for AD, Down’s, ALS
  2. News Brief: ALS Drug Olesoxime Fails in Phase 3 Trial
  3. Chicago—ALS Clinical Trials: New Hope After Phase 3 Setbacks

Paper Citations

  1. . A screen for regulators of survival of motor neuron protein levels. Nat Chem Biol. 2011 Aug;7(8):544-52. PubMed.
  2. . Drug screening for ALS using patient-specific induced pluripotent stem cells. Sci Transl Med. 2012 Aug 1;4(145):145ra104. PubMed.

Other Citations

  1. ARF related news story

Further Reading


  1. . The effects of dexpramipexole (KNS-760704) in individuals with amyotrophic lateral sclerosis. Nat Med. 2011 Dec;17(12):1652-6. PubMed.
  2. . Stem cells and drug discovery: the beginning of a new era?. Cell. 2008 Feb 22;132(4):549-52. PubMed.
  3. . Drug screening for ALS using patient-specific induced pluripotent stem cells. Sci Transl Med. 2012 Aug 1;4(145):145ra104. PubMed.

Primary Papers

  1. . A Small Molecule Screen in Stem-Cell-Derived Motor Neurons Identifies a Kinase Inhibitor as a Candidate Therapeutic for ALS. Cell Stem Cell. 2013 Jun 6;12(6):713-26. PubMed.