Researchers need to meet each year to advance the cause of research into ALS,” was the message of Monica Lalancette, neurologist and researcher at CHU Université Laval, and of Jean-Pierre Julien, a professor at Université Laval. The gathering of experts from around the world became a reality at the 5th Symposium on Amyotrophic Lateral Sclerosis. Some 100 invited guests attended a series of talks on the clinical promise of stem cells, organogenesis and genetics. “The symposium also included students,” Nicolas Dupré, neurologist at Hôpital de l’Enfant-Jésus and associate professor at Université Laval, points out. “It’s a way of encouraging them to consider a career in ALS research, rather than in another field.
Québec: Stem Cells in ALS Update
Stem cell research made a strong showing at the André-Delambre Foundation Symposium on amyotrophic lateral sclerosis (ALS) in Québec City September 25-26, 2009, with an entire session devoted to progress in the field. Researchers are turning embryonic stem cells and induced pluripotent stem (iPS) cells into motor neurons and astrocytes, and using those cells to develop new in vitro model systems for ALS as well as to hunt for treatments. In related news, the first Phase 1 trial for stem cell therapy for ALS, recently approved by the U.S. Food and Drug Administration, will soon begin in clinics.
Much of the potential for iPS cells lies in the possibility of new models for human disease (see ARF related news story). Researchers studying ALS rely heavily on mice carrying mutant superoxide dismutase 1 (SOD1), which exhibit many symptoms of the disease. However, only a few percent of ALS cases are caused by SOD1 mutations. The majority of people with the disease have a sporadic form with no known genetic cause, and even among familial cases, SOD1 explains only one-fifth.
Enter iPS cells: patient-specific cell lines that can be nudged in the direction of a number of cell fates. With a little Sonic hedgehog, and a little retinoic acid, pluripotent cells can become motor neurons. Kevin Eggan of Harvard University reported on progress in turning fibroblasts from people with ALS—both familial and sporadic—into iPS cells. “We really see this as one more arrow in the quiver” for ALS research, Eggan said. Last year, Eggan’s group reported on an iPS line from an 82-year-old woman with ALS (see ARF related news story on Dimos et al., 2008). Those cells expressed the SOD1-L144F mutant; Eggan has added to his repertoire SOD1-G85R and SOD1-D90A cells. In addition, the group has generated five iPS lines from people with sporadic ALS. They are working on another SOD1 mutant line, SOD1-A4V, and a TDP-43 mutant line.
However, much work remains, Eggan noted, in the “painstaking and important, but rather mind-numbing” process of validation. The researchers must show that the cells are truly pluripotent, by checking biochemical markers and assaying their ability to form teratomas. They must confirm that the retroviral genes they used to induce pluripotency are truly silenced. And, they must verify that the cells can indeed differentiate into motor neurons. Of the 12 lines tried so far, all formed motor neurons, Eggan said, but he plans to test them further using electrophysiology. Overall, he said, the process is going well: “Version 1.0 of the technology is ending up being more robust than I had hoped for.”
Once valid iPS lines are available, they could be a testing ground for drug screening, and Haruhisa Inoue, of Kyoto University, reported on his progress with that approach. Inoue focused his search on iPS-derived, mSOD1-overexpressing astrocytes. These support cells are important players in ALS (Yamanaka et al., 2008). Inoue and colleagues engineered cells to express luciferase under the human SOD1 promoter, and screened 12,000 drugs for an effect on expression. They also examined SOD1 protein expression in cells treated with promising candidates.
The work led to a potential therapeutic Inoue is calling “Drug X,” as its identity is not yet public. The compound, which he said is currently used in hospitals for a non-neurological condition, decreased levels of both mutant and wild-type SOD1. The researchers are now testing the drug in mSOD1 mice, as well as researching its mechanism of action in iPS cells, Inoue wrote in an e-mail to ARF.
With a new preponderance of stem and iPS cell-derived lines poised to enter the ALS research laboratory, a key question is how well these lines model the “real thing,” motor neurons in a person or animal. Certainly, Eggan said, “These cells have not experienced exactly the same experiences” as natural motor neurons. However, he said they are more like the body’s motor neurons than any other model system.
Victor Rafuse of Dalhousie University in Halifax, Canada, also argued that differentiated stem cells are close facsimiles of motor neurons. In a paper last year, Rafuse and colleagues showed that embryonic stem cell-derived motor neurons, when injected into adult mice, form functional synapses (Yohn et al., 2008). “These cells live very happily in the peripheral nerve, and become myelinated,” Rafuse said. He also reported that when differentiated motor neurons are injected into chick embryos, they migrate to the correct location to form neuromuscular junctions.
François Berthod of Laval University in Québec City, who chaired the stem cell session, found the presentations encouraging. “The iPS cells will certainly transform our way to study these diseases in vitro in a couple of years,” he said. “That really will help to understand the disease.”
Rafuse suggested that stem cell-derived saviors might someday home to the right place in humans needing motor neuron replacements. With FDA approval now in place, other clinicians are poised to begin a safety study of spinal cord stem cells in people with ALS. The trial, led by Eva Feldman of the University of Michigan in Ann Arbor and Jonathan Glass of Emory University in Atlanta, Georgia, will test a patented neural stem cell line from Neuralstem, Inc. of Rockville, Maryland. These cells have already shown promise in mSOD1 rats, where they differentiated into motor neurons and formed synapses with host cells, and delayed disease onset and progression (Xu et al., 2006).
A similar strategy using purified motor neurons derived from stem cells was recently shown to improve pathology in mouse models of spinal muscular atrophy with respiratory distress (see ARF related news story). For the first stage of the human ALS trial, 12 participants will receive five to 10 spinal injections of neural stem cells. Safety is the primary outcome, but the researchers will also be looking for a slowing of disease. Final results are expected in two years.—Amber Dance.
Québec: Transporters, Antibodies Offer Potential ALS Therapies
The doctor’s toolkit for amyotrophic lateral sclerosis is pathetically small—only one drug, riluzole, is approved for the disease, and its effects are hardly blockbuster. However, researchers are constantly coming up with new strategies to rescue motor neurons from degeneration. At the André-Delambre Symposium in Québec City, held September 25-26, 2009, two scientists presented ideas, still in the early stages, which might lead to new therapeutics down the line.
Brett Morrison of Johns Hopkins University in Baltimore, Maryland, discussed a strategy aimed at a monocarboxylate transporter, MCT1, which shuttles lactate out of the cell. Previous work in the laboratory of collaborator Jeff Rothstein, also at Johns Hopkins University, suggested MCT1 was reduced in animals with mSOD1. The transporter is particularly important in astrocytes, the motor neurons’ support cells which have been implicated in the disease (Yamanaka et al., 2008). Astrocytes have long, thin processes where mitochondria cannot fit; those tentacles must rely solely on glycolysis for energy. Lactate is a byproduct of this process, and MCT1 pumps it out of the cell.
Neurons express another transporter, MCT2, which is able to take up lactate into the cell. A longstanding theory says that motor neurons may then extract energy from the lactate via oxidative respiration (Aubert et al., 2005). Using lactate might be important, Morrison suggested, if the cells were low on glucose. “There are not many cells in the body that would be more metabolically active than a motor neuron,” he said.
Morrison showed that MCT1 levels were reduced in spinal cord astrocytes of people with ALS and in the same cells in a rat model of the disease. In cultured spinal cord slices, RNA inhibition of MCT1 expression caused motor neuron degeneration. To further study the importance of these transporters, Morrison used a specific small molecule inhibitor of MCT1 (Murray et al., 2005). The inhibitor’s effects on cultured spinal cord cells mirrored that of the RNAi: up to one-third of motor neurons died after three weeks. Under glucose starvation, the effect was even more dramatic, with inhibitor-treated motor neuron numbers dropping by more than half after two hours of glucose deprivation. When the researchers added extracellular lactate—to compensate for loss of MCT1-driven export of lactate from astrocytes—the motor neurons survived in the presence of the inhibitor. The results suggest that motor neurons depend on lactate, normally supplied by astrocytes via MCT1, for survival.
In vivo, the story was similar. RNA inhibition of MCT1 in the spinal cords of wild-type mice caused motor neuron numbers to drop by approximately one-half by four weeks after the RNAi treatment. The next step will be to augment MCT1 activity and look for a positive effect on motor neuron survival and disease progression. Collaborators are performing those experiments now, Morrison said. Simply injecting lactate would not work, Morrison noted, because the molecule is broken down too quickly.
François Gros-Louis of Laval University in Québec City presented another therapeutic possibility aimed at people with ALS mutations in superoxide dismutase 1. If too much aggregated SOD1 is the problem, Gros-Louis reasoned, then antibodies to the protein might induce the immune system to get rid of them. Similar strategies are being pursued for other neurodegenerative diseases, including Alzheimer disease, where both active and passive immunotherapies are in clinical trials (see ARF related news story). In previous work, immunizing mice with SOD1 protein delayed disease by a month in one model of the disease, although mice expressing the highest levels of mutant SOD1 were not helped. However, passive immunization by infusing mSOD1 antibodies was effective in the mice with the most severe disease (Urushitani et al., 2007). “We believe that passive immunization would be safer” for eventual use in people, Gros-Louis said.
Gros-Louis tested two antibodies, specific for only the mutant form of SOD1, as well as control IgG in 65 mSOD1 mice. The animals received antibody infusions from external pumps for 25 or 40 days. One antibody failed to give any benefit, but the other extended lifespan by up to nine days. Those treated animals had 25 percent less misfolded SOD1 than did control mice.
Gros-Louis also found that Fab fragments of the antibody extended survival. This suggests that smaller, single-chain fragments, which offer advantages such as ease of optimization and very little immunogenicity in the host, might work in the case of mSOD1. Gros-Louis is currently testing single-chain fragments in animals.
“For patients with the SOD1 mutation, there are very interesting approaches,” Morrison said of Gros-Louis’s work. But there is a caveat: “You are only going to help the 1 to 2 percent of patients with ALS with that mutation in SOD1.”—Amber Dance.
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Québec: STOMPing on Inclusions May Reveal their True Identity
The nervous systems of people with neurodegenerative disease are often pocked with tiny inclusions, which are packed with a variety of proteins, many still unidentified. Many of those proteins may be involved in disease pathology, and figuring out what they are is the first step to discovering how they might relate to disease. Researchers at the University of Toronto are working on a new method to collect proteins from small inclusions for identification by mass spectrometry. Kevin Hadley presented their progress in a poster at the André-Delambre Foundation Symposium on amyotrophic lateral sclerosis, held 25-26 September in Québec City. They call their technique STOMP: Spatially Targeted Optical Microproteomics.
Mass spectrometry requires less than a nanogram of protein, but with inclusions measuring less than 10 microns across, acquiring even a nanogram of material is challenging, Hadley said. Current techniques are not good enough to collect and identify the contents of these tiny aggregates. One option, laser microdissection, lacks the fine resolution needed to capture them. Alternatively, researchers can homogenize tissues and collect insoluble material. However, this brute-force approach may miss some inclusion components and collect other, non-inclusion but insoluble material. By simply grinding up tissue and collecting the insoluble fraction in a centrifuge, “we are probably losing a lot of interesting stuff,” Hadley said.
Along with principal investigator Avijit Chakrabartty, also at the University of Toronto, and Rishi Rakhit, now at Stanford University in Palo Alto, California, Hadley is working on a method to isolate and identify small inclusions with resolution and specificity that supersedes microdissecting out the protein globs or simply collecting insoluble bits. The technique relies on the strong and specific interaction between the vitamin biotin and the protein streptavidin to pull out proteins from inclusions.
First, Hadley soaks tissue samples in a photoactivatable biotin analog. This compound crosslinks biotin to proteins only when exposed to ultraviolet light. If Hadley were to wash the sample at this stage, all proteins would remain unlabeled. The key to the technique is to only biotin-tag proteins in inclusions.
To do that, the researchers must first find the inclusions. They rely on immunofluorescence to pick out aggregates under the microscope—so an antibody must be available for at least one inclusion component. Hadley uses a confocal microscope to obtain multiple images of the tissue along the vertical axis, resulting in a three-dimensional record of where all the inclusions are.
Next, he targets an ultraviolet laser only at the inclusions, leaving the surrounding tissue unlit. This ensures that only proteins in the aggregates will be biotin-tagged. The tedious way to do this would be to manually define each area, then zap it with the laser. Since this is too time-consuming, the researchers developed a computer program to do it for them. With the picture of the inclusions as input, the program figures out where to aim the laser to light up only the labeled areas.
Then, it is time to turn on the UV and link inclusion proteins to biotin. A standard laser lacks the resolution to finely define tiny aggregates, so Hadley relies on two-photon excitation, which allows two red photons to stand in for a single UV photon. UV radiation, the kind needed to activate the biotin tagging, contains twice as much energy as red light. But very occasionally, with a red laser, two red photons will hit the same spot at the same time, and the double hit packs a punch similar to a single UV photon. “It may be one of those things that is forbidden in physics, but works in real life,” Hadley quipped. Because the doubling of the red photons is a rare event, it is much more likely to occur in the very center of the laser beam than out on the edges. Therefore, it hits the sample with a much finer resolution, and allows Hadley to label the tiniest aggregates without blasting the adjacent tissue.
Thus far, the researchers have achieved the fine resolution they need to tag inclusions. To test the system, Hadley showed that he could hit a six-micron-diameter fluorescent bead dead center with the laser. The next step is to try it out in real tissue samples. First, the researchers will validate the system in tissue samples from a mouse model of Alzheimer disease. They know exactly what the inclusions should contain: Aβ.
If the techniques works, proteins in the inclusions will be biotin-tagged, but peptides in the surrounding tissue will not. Hadley will homogenize the tissue and apply it to a column of streptavidin-coated beads. The streptavidin will grab the biotin-tagged proteins and let the rest wash through. Then, Hadley can collect the proteins that remained in the column, run them out on a gel, and send the bands in for mass spectrometry to identify them. With the Alzheimer model tissue, they are looking to see Aβ in their results, but Aβ inclusions contain a wide variety of other proteins as well, some potentially unknown. “If we find something else, that would be interesting, too,” Hadley said.
Once they have verified their approach, the researchers will move on to tissue from people with ALS. They can expect to see well-known aggregate components such as SOD1 and TDP-43—but they may see something else that gives new clues to the disease. And since aggregates are common in many neurodegenerative diseases, the technique has far-reaching potential.—Amber Dance.
Mutant superoxide dismutase 1 (SOD1) has been linked to one-fifth of inherited cases of amyotrophic lateral sclerosis (ALS), where its misfolded form appears to damage motor neurons. But SOD1 does not act alone. Work presented by Jean-Pierre Julien at the André-Delambre Foundation Symposium on ALS, held 25-26 September in Québec City, builds on previous evidence for an accomplice, the chromogranins. Julien, Samer Abou Ezzi, and colleagues at Laval University in Québec City found that mice carrying mutant SOD1 and an excess of chromogranin A got sick earlier than mSOD1 animals with normal levels of chromogranins. They also found evidence for chromogranin B involvement in risk for ALS in people. The work suggests chromogranins act to exacerbate the toxicity of misfolded SOD1.
Chromogranins A and B are secretory vesicle components of unknown function in the nervous system. Julien’s group became interested in the proteins when a yeast two-hybrid screen using mSOD1 as bait pulled out chromogranin A. Both chromogranins immunoprecipitated with mutant SOD1, but not wild-type SOD1 (see ARF related news story on Urushitani et al., 2006). The chromogranins appear to require a domain similar to heat-shock protein sequences to bind mSOD1, suggesting they may recognize the mutant protein’s improper conformation. In human studies by another group, chromogranins co-localized with SOD1 in intracellular aggregates in tissue from people who had ALS (Schrott-Fischer et al., 2009). Chromogranins have also been found in Aβ plaques in Alzheimer disease (Marksteiner et al., 2000), and in prion deposits in Creutzfeld-Jakob disease (Rangon et al., 2003).
To further investigate the chromogranin-mSOD1 interaction in disease, Julien and colleagues engineered mice with the chromogranin A gene driven by the human Thy1 promoter, which is active in the nervous system. The mice express twice the normal amount of chromogranin A in neurons. They crossed these mice with animals expressing human SOD1-G37R. The double mutant mice exhibited symptoms of motor neuron disease one month earlier than their mSOD1 single-mutant parents. The double mutants had increased degeneration of motor neurons, compared to SOD1-G37R mice, as well.
However, the excess chromogranin A did not speed up the final stages of the disease; the double mutant mice survived for the same length of time as single mutants. Julien hypothesized that early on, the animal’s full complement of motor neurons produced plenty of chromogranin A and mSOD1, accelerating disease onset. But as motor neurons died, there were fewer of them to produce the damaging proteins. In later stages, then, the rate of disease slowed to normal.
How does chromogranin A accelerate motor neuron degeneration? Using an antibody specific for misfolded SOD1, the researchers found that the double mutants had more of the malformed species than did single mutant mice. Julien hypothesized that chromogranin A binds and stabilizes the misfolded protein, allowing it to hang around longer and do more damage. Alternatively, he suggested, the excess chromogranin A may promote secretion of misfolded SOD1 into the extracellular space where it is cytotoxic.
Previous results indicated that chromogranins promote secretion of mSOD1 (Urushitani et al., 2006), and aggregated mSOD1 is found in the endoplasmic reticulum and Golgi, along the secretory roadway (Urushitani et al., 2008). Abou Ezzi suggested that the increased chromogranin A might capture mSOD1, causing it to aggregate in the ER and Golgi, leading to ER stress. Other researchers have shown that motor neurons are especially vulnerable to endoplasmic reticulum stress (see ARF related news story on Saxena et al., 2009), so stressing the ER or secretory pathways could be a common theme in the disease, Julien suggested.
The scientists also crossed the SOD1-G37R mouse with a chromogranin A knockout strain. These double mutants showed little difference in disease phenotype from single SOD1 mutants, perhaps because chromogranin B compensates for the loss of chromogranin A, Julien suggested. However, the double mSOD1/chromogranin A knockout mutants did have more motor axons in the ventral root than their mSOD1 counterparts at a late stage of disease, suggesting reduced motor neuron degeneration.
Do these findings in mice have any bearing on human disease? Julien and colleagues also looked for evidence of chromogranin involvement in people with ALS. They discovered a chromogranin B variant, not yet published, that was present at higher levels in a French ALS population than in controls. This variant appears to be a risk factor for the disease, and to cause onset seven years earlier. “It resembles very much ApoE in Alzheimer’s,” Julien said. The researchers are currently working with cell cultures and mice to decipher the mechanism behind the increased risk.
The research supports current hypotheses about mSOD1’s damaging effects, said Christine Vande Velde of the University of Montréal, who was not involved in the study. “It lends more strength to the idea that misfolded SOD1, if you let it stick around or if you stabilize it, then it is more toxic,” she said.—Amber Dance.
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