Distinct growth factors promote the survival of specific types of motor neurons in the spinal cord, according to a study led by Georg Haase, of Aix-Marseille University in Marseille, France. The results suggest that these factors may work together to provide trophic support to motor neurons in the CNS and therefore, a combination of them may be needed to protect motor neurons damaged by disease.
“Growth factors have always been tantalizingly attractive in ALS,” said Nicholas Boulis of Emory University Medical School, who was not involved in the study. “But the problem is, there has been a failure of growth factors to perform [in the clinic].” This study “provides tangible evidence that you may be able to get a bigger effect by combining growth factors.”
The study appeared on March 16 in the Proceedings of the National Academy of Sciences.
Neurotrophic Factors in ALS: The power of two+
Researchers first turned to neurotrophic factors (NTFs) in the early 1990s as a potential therapy for ALS in hopes to promote the survival of motor neurons damaged by the disease. But initial therapies proved ineffective in part due to delivery challenges (see Rogers, 2014).
In more recent years, neuroscientists discovered that many of these growth factors may work together to provide trophic support for motor neurons and promote their survival – at least in the developing spinal cord (see Gould and Enomoto, 2009). But how these substances orchestrate this process remains an open question.
A growing number of researchers suspect that there may be distinct classes of motor neurons that are protected by distinct NTFs during development. To test this hypothesis, Haase’s team at Aix-Marseille University in France isolated motor neurons from the developing lumbar spinal cord in the mouse and determined which growth factors supported them.
To carry out this analysis, first author Sébastien Schaller and colleagues dissected out lumbar spinal cords at day E12 and suspended the tissue. Then, they used fluorescence-activated cell sorting (FACS) to isolate the motor neurons, cultured them and exposed them to combinations of neurotrophic substances.
The technique enabled motor neurons to be specifically captured from embryos by using Hb9:GFP mice, originally developed by Columbia University’s Thomas Jessell in New York, which express GFP in motor neurons in the developing central nervous system.
100% of the cells expressed the motor neuronal markers ChAT and SMI 32, and none expressed interneuronal markers, indicating the “exquisite purity” of the isolated cells, said Haase. That, combined with the method’s speed and degree of automation, make FACS-derived motor neurons a promising platform for future studies, he said, including screening for potential ALS therapies.
Next, the team exposed motor neurons to 12 different neurotrophic factors (BDNF, NT3, GDNF, neurturin, artemin, persephin, CNTF, CT1, LIF, HGF, IGF1, and VEGF), alone or in combination. Individually, all NTFs promoted neuronal survival after 3 days in culture, with GDNF being the most effective (43%). HGF, however, protected only about 20% of motor neurons in culture. But when HGF, CNTF and artemin were combined, motor neuron survival reached nearly 50%.
The effects were additive, explained Haase. “That suggested to us that each [of these growth factors] were supporting a subset of motor neurons.”
To test that hypothesis, the researchers used subtype cell surface-specific antibodies to label three major subsets of motor neurons from the lumbar spinal cord—the medial motor column, which innervate axial muscles, the lateral motor column, which innervate limb muscles, and preganglionic, which synapse with downstream neurons of the autonomic motor system. They then used FACS to separate each subtype, and exposed them to HGF, CNTF or artemin.
They found that each of these NTFs promoted the survival of distinct classes of motor neurons in the lumbar spinal cord. For example, HGF preferentially supported survival of motor neurons in the lateral motor column neurons, key motor neurons affected by ALS.
The effects were mediated by distinct neurotrophic factor receptors decorating the surface of each type of motor neuron, explained Haase. “When we blocked the HGF receptor, we completely blocked the survival effect of HGF. That means these motor neurons depend on this particular factor for their survival.”
Additional analysis indicated that CNTF and artemin protected other types of motor neurons located elsewhere in the spinal cord.
Together, the findings suggest that these substances provide trophic support and promote the survival of specific types of motor neurons in the developing spinal cord.
“This is a very high-quality paper that helps clarify the field,” said Clive Svendsen of Cedars-Sinai in Los Angeles, California. Until now, it was not clear that distinct subsets of motor neurons may respond to their own subsets of growth factors.
Motor neurons that could potentially include those that descend from the brainstem, and those involved in breathing, also affected by the disease.
The results suggest that combining growth factors may offer more therapeutic benefit than single factors in ALS according to Nicholas Boulis.
Svendsen agreed. “This is suggesting that for therapies, if you want to protect motor neurons, you may have to expand to include multiple growth factors,” Svendsen said. However, he noted, and as confirmed in this study, GDNF by itself is still perhaps the most powerful all-around survival factor for motor neurons.
Svendsen is now developing a potential therapy for ALS that uses genetically engineered neural stem cells to deliver GDNF to the spinal cord. The Phase 1 clinical trial is soon to be launched (see October 2016 news).
Neuroprotective therapies: the next generation?
The next big question, which this paper leaves open, according to Svendsen is whether the growth factors identified in this study protect motor neurons in the adult nervous system.
Haase agreed. “This is a critical question, and we are adapting our method to look at this now.”
“Some neural circuits change drastically during adulthood, while others stay pretty much the same, so we’ve got to do the experiments to find out, “explained Svendsen. “But I will probably be trying HGF soon in my own experiments.”
In the meantime, said Haase, it is important to keep in mind that the growth factors found to be less effective in this study should not be ruled out as potential therapies. “They may act sequentially during development, or may require co-factors to exert their effect which were not present in our growth medium,” he said.
It is also important to keep in mind that this study did not evaluate the ability of any of these substances to regenerate axons, a key goal in terms of developing therapies for ALS and other motor neuron diseases including SMA.
The challenges of delivery, which have stymied the field to date, remain paramount, Haase also noted. Gene delivery approaches with adeno-associated vectors have been studied for single growth factors, but if several are needed, a larger-capacity vector, such as lentivirus, may be required, according to Boulis. Multiple rounds of ex vivo gene therapy to equip stem cells with multiple growth factor genes, would be another option, followed by surgical implantation of the modified cells.
Further exploration in in vivo models and patient-derived iPS cells are an important next step to determine which combination of these substances could be of the most benefit, added Boulis.
But despite these challenges, Boulis agrees this approach is worth considering. “As a surgeon who does translational work on the application of growth factors to ALS, this may be an ‘Aha!’ moment.”
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