HONOLULU—One or more proteins released from astrocytes contribute to motor neuron death in amyotrophic lateral sclerosis (ALS), according to new work described here at the Frontiers in Clinical Neuroscience plenary session at the AAN annual meeting last month. The finding is aligned with a major shift in focus on ALS pathogenesis within the past several years, away from neurons alone and to the entire neighborhood within the CNS.
“Motor neurons do not die or live in clinical isolation. They are surrounded by a huge number of non-neuronal cells,” said Serge Przedborski, MD, PhD, who offered an overview of this shift in perspective. Dr. Przedborski discussed the latest research results from his lab at Columbia University in New York City, where he is a professor of neurology and pathology at the College of Physicians and Surgeons.
“The major problem we face in both the basic and clinical understanding of neurodegenerative diseases,” he said, “is that even when we know the etiology, we know very little about the pathogenesis. That lack of knowledge is a major challenge to developing therapies.”
“One way to gain a better insight is to broaden the scope of our investigations” to those non-neuronal cells, including astrocytes, the most abundant cell type in the CNS. Mutant superoxide dismutase 1 (SOD1), a common cause of familial ALS and the basis of most ALS animal models, is expressed in every cell, including astrocytes.
This raises two important questions, Dr. Przedborski said. “What is the site of action of mutant SOD1: neurons, astrocytes, or both? And if mutant SOD1 does play a deleterious role in both, is the mechanism the same in the two? I believe that understanding that question may have far-reaching implications for both understanding the disease and developing treatments.”
Experiments in cell culture and animal models have suggested that astrocytes are central to the ALS disease process. When normal astrocytes are plated with SOD1-mutant neurons, the neurons display some alterations in shape, but remain as viable as normal motor neurons, a result that was “quite surprising and unexpected, to say the least,” he said.
In contrast, when normal motor neurons are grown with mutant astrocytes, half the neurons die within a week, which Dr. Przedborski called “a stunning finding.” And the toxic effect is specifically from astrocytes to motor neurons. Other cell types neither exert nor suffer toxicity in the model.
These and related findings have shifted the attention of the ALS research community to a “systems approach,” to understand the factors and signals that motor neurons exchange with their neighbors to maintain their health or succumb to disease. “It takes a neighborhood” has become the dominant view of ALS pathogenesis within the past five years.
Those discoveries led quickly to a search for the toxic factor. In a key experiment, Dr. Przedborski's lab grew mutant astrocytes in isolation, and then transferred the growth medium to a dish of normal neurons. The neurons died just as if they had been in contact with the astrocytes, suggesting the medium contained a soluble toxic factor.
Astrocytes from sporadic ALS patients exerted the same effect, Dr. Przedborski said, “exactly as we saw in the rodent model. We may have discovered a generic property of diseased astrocytes. It doesn't seem to be specific to rodents, or to mutant SOD1 models of ALS.”
The lab is now working hard to identify the toxic factor. Treatment with a protease knocks out the toxic effect, indicating a protein or peptide is involved. Using mass spectrometry and other techniques, Dr. Przedborski indicated they were looking for one or more negatively charged proteins in the range of 10 to 14 kilodaltons in weight, and had narrowed the search down to a group of ten proteins.
They are also currently working to develop a high-throughput screen to test compounds that influence the production of this factor, and to determine downstream effects within the motor neuron. “We are most interested to use this approach to identify the signaling network recruited by the astrocytic toxic factor,” Dr. Przedborski said.
Currently the focus is on the signaling pathway involving the kinase JNK3, he continued. [JNK3 kinases, which are part of the mitogen-activated protein kinase family, are thought to be involved in a wide variety of cellular processes, including regulatory roles in the signaling pathways during neuronal apoptosis. They are responsive to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, as well as play a role in T cell differentiation.]
Inhibiting this molecule abrogates the astrocytic toxicity, he said, and the investigators are now testing chronic administration of JNK3 inhibitors to determine their clinical potential.
Motor neurons likely die from both intrinsic (autonomous) and extrinsic (non-autonomous) causes, Dr. Przedborski concluded. “Astrocytes are instrumental in the demise of neighboring neurons. From a therapeutic standpoint, the most likely chance we have to obtain neuroprotection is to use a polypharmacy approach to target these key elements of the non-autonomous process.”
“It is eminently clear that neurons don't die alone in ALS,” and the evidence that astrocytes are involved is “definitive,” said Stanley Appel, MD, chair of neurology at the Weill Cornell Medical College of Cornell University in New York, in an interview with Neurology Today.
The demonstration that something is released from the astrocytes that kills motor neurons “is an extremely important experiment,” he said.
“The reason that this is potentially exciting is that if there are toxic factors, we know how to shut off genes for all sorts of toxic factors, or use antibodies, or block receptors. This is a potential pathway for therapy if astrocytes are toxic.”
“The great thing about Dr. Przedborski is that when he says it, you know it is terrific science,” Dr. Appel said. Nonetheless, Dr. Appel is “still a little skeptical” that the story on ALS pathogenesis is close to an end. Diminishment of activity with a protease, he said, means it is likely that a protein is involved, “but until you demonstrate those factors are toxic, you haven't completed the argument.”
Dr. Appel also noted that the association of the factor with astrocytes, rather than motor neurons themselves, is potentially important therapeutically.
In ALS, he said, “the first changes are likely to be in the motor neuron,” with the consequence that neuron-specific changes are most important for determining disease onset. These changes then signal to astrocytes to respond, either to protect or destroy the motor neuron. It is those responses, he suggested, that may determine disease progression and duration.
“By the time we see patients, it's beyond the onset of the disease,” Dr. Appel said. “That's why we want to know what is coming out of astrocytes, in order to prolong the duration of disease and to provide a better quality of life.”