It is the million-dollar question in research on amyotrophic lateral sclerosis: Why do only motor neurons fail when the cause of pathology, such as mutant protein, exists in many different cells? In a Nature Neuroscience paper published March 29, scientists from the Friedrich Miescher Institute in Basel, Switzerland, provide a clue—some motor neurons are particularly vulnerable because they are quicker to succumb to endoplasmic reticulum stress. By dissecting out and examining gene expression in neurons that degenerate first in a mouse model of ALS, the authors found that ER stress markers were upregulated in these most sensitive cells. They also discovered that a drug that subdues the ER stress response extended survival in the animals.
Principal investigator Pico Caroni and colleagues at the Friedrich Miescher Institute had previously shown that in a common ALS model—mice overexpressing mutant superoxide dismutase 1—the degeneration of motor neurons follows a distinct order. First to suffer are the largest, fast-fatigable motor neurons, responsible for quick movements such as leaping and sprinting, which lose neuromuscular connections before the animal exhibits visible symptoms. At symptom onset, the fast-fatigue-resistant motor neurons—which also generate quick signals, but are slow to tire—are affected. Slow motor neurons, responsible for prolonged muscle activity like standing and walking, are resistant to the effects of mutant SOD1 and some are still connected to muscle at the time of death (Pun et al., 2006).
In the current study, Caroni, along with first author Smita Saxena and Erik Cabuy, used gene expression data to analyze how pathology progresses in vulnerable, fast-fatigable motor neurons versus resistant motor neurons, encompassing fast-fatigue-resistant and slow neurons. To acquire pure populations of both, they took advantage of the fact that parts of the lateral gastrocnemius are exclusively innervated by vulnerable neurons, while the soleus is only served by resistant ones. They injected rhodamine-labeled dextran as a tracer into those muscles, and the tracer entered the neurons by retrograde transfer. When the scientists sacrificed the mice a few days later, the motor neurons of interest lit up under fluorescent light and the researchers were able to dissect them away from other tissue. “The beauty of the experiment is that we are always comparing the exact same 10-11 vulnerable and 10-11 resistant motoneurons in all our experiments,” Caroni wrote in an e-mail to ARF.
The vulnerable motor neurons of SOD1-G93A mice showed upregulation of a variety of stress genes, in comparison with resistant neurons, as early as 12 days of age. The upregulated genes included players in protein ubiquitination, hypoxia, and NRF2-mediated response to oxidative stress. Later on, around their fifth week, the animals’ vulnerable motor neurons upregulated genes for the ER-mediated unfolded protein response such as ATF4. Vulnerable motor neurons, but not resistant ones, also overexpressed the ER stress protein BiP at day 28. “What [they have] done is unprecedented in terms of detailed analysis of the gene changes,” said Christopher Henderson, co-director of the Motor Neuron Center at Columbia University in New York City. “[They have] analyzed changes in a very small subset of the motor neurons.”
Resistant neurons appeared to undergo similar changes but at a much later time; these cells did not upregulate ER stress markers until 25 to 30 days after the vulnerable cells did. “The results suggest that all motor neurons are particularly sensitive to mutant SOD1, but that subtypes of motor neurons differ in their sensitivities to ER stress, and it is that sensitivity that determines the time course of pathology and denervation,” Caroni wrote.
The scientists then used the drug salubrinal, which maintains the activity of a translation initiation factor that promotes production of stress-relieving proteins, to damp down ER stress in the mutant animals, and found it prolonged life by 25-30 days. These animals typically survive approximately 135 days. “It’s not a blockbuster, but it certainly improved the survival of these animals,” said Serge Przedborski, whose laboratory at Columbia University has found evidence linking ER stress and mSOD1 in mice. However, he noted that salubrinal is a “dirty” drug of uncertain specificity, and it may have effects beyond the ER.
These experiments confirmed the suspected relationship between ER stress and ALS. Previous work had shown ER stress in mSOD1 mice (Kikuchi et al., 2006), but only made the correlation, not a causal link, Przedborski said. Although mSOD1 is responsible for approximately 2 percent of human ALS cases, spinal cord ER stress markers have also been reported in people who died from sporadic ALS (Ilieva et al., 2007). ER stress pathways may be promising therapeutic targets, Caroni wrote, as well as potentially provide biomarkers to monitor disease.
“This builds on earlier reports…that not all motor neurons are equal when faced with the disease,” Henderson said. The problem is a general one, he noted; for example, different parts of the substantia nigra exhibit different pathology in Parkinson disease. That makes it important for scientists to select small neural populations to study, Henderson said, as Saxena and colleagues have done.
Caroni suggested that ER stress response might peak when motor neuron toxins such as mSOD1, compounded by the animal’s age, exceed a particular threshold. “Above the threshold cells would trigger more robust stress responses (unfolded protein response), which can get rid of the protein, but are not compatible with a healthy neuron if they are maintained for too long,” he wrote. However, the research simply leads to another question: Why are certain motor neurons more vulnerable to ER stress? Those million dollars are still up for grabs.—Amber Dance