ARTICLE IN BRIEF
Working with a mouse model of amyotrophic lateral sclerosis (ALS), researchers used DNA microarray profiling to compare tens of thousands of genes found in neurons resistant to ALS with genes found in affected neurons. Of the “susceptibility” genes found only in vulnerable motor neurons, the gene for MMP-9 was strongly active into adulthood.
Amyotrophic lateral sclerosis (ALS) lays waste to the vast majority of the body's motor neurons, but preserves a few islands of function, such as eye movement, urination, defecation, and sexual function. Also, slow motor neurons that control posture are far more resistant to the ravages of ALS than the fast-fatigable motor neurons, which initiate quick and forceful muscle contractions.
This discrepancy has enabled researchers at the Columbia University Medical Center to show that a protein known as matrix metalloproteinase-9, or MMP-9, is present in motor neurons susceptible to ALS, but not in those resistant to the disease. This correlation, termed “perfect” by study leader Christopher E. Henderson, PhD, suggests treatments that could slow the inexorable progression of ALS, and perhaps halt it.
“It's an amazing correlation,” said Dr. Henderson, the Gurewitsch and Vidda Foundation professor of rehabilitation and regenerative medicine, professor of pathology and cell biology and neuroscience (in neurology), and co-director of Columbia's Motor Neuron Center, who reported the findings in the Jan. 22 issue of Neuron. “The moment we saw it, I was certain that MMP-9 was at the very least the first marker we had for the vulnerable motor neurons in this model of ALS.”
THE EXPERIMENTAL APPROACH
Working with a mouse model of ALS, the researchers used DNA microarray profiling to compare tens of thousands of genes found in neurons resistant to ALS with genes found in affected neurons. Of the “susceptibility” genes found only in vulnerable motor neurons, the gene for MMP-9 was strongly active into adulthood, when ALS begins. The protein was found in greatest abundance in fast-fatigable neurons, and not found at all in neurons resistant to ALS. This makes the protein “a reliable predictor” of which neurons will die, at least in the mouse model of the disease, according to Dr. Henderson.
The researchers curbed the activity of MMP-9 in the mice by injecting an enzyme inhibitor directly into the central nervous system. “That showed some benefit, but it certainly was not a complete rescue, perhaps because the drug we used is far from perfect,” Dr. Henderson said. “It could also be because MMP-9 may act in two ways to cause neuron degeneration — both by enzymatically digesting specific substrates, and by triggering degeneration pathways by interacting with other proteins. The enzyme inhibitor would only block the first of these. It may be that we need both to inhibit activity and to find ways of reducing levels of MMP-9 protein.”
In another experiment, the researchers crossed MMP-9 knockout mice with the standard mouse model of ALS, which carries a mutant form of superoxide dismutase 1, or SOD1, linked to familial ALS. Progeny with no MMP-9 exhibited an 80-day delay in the loss of fast-fatigable motor neuron function, and lived 25 percent longer than mice carrying two copies of the gene for MMP-9. This benefit is believed to be the largest ever produced in a mouse model of ALS.
“What this says unambiguously, is that reducing the amounts of MMP-9 is a good thing — in the mouse model (of ALS),” Dr. Henderson said. “We need to examine expression in human material much more closely now, and we need to be certain that removing MMP-9 has benefits not only in SOD1 cases, but also in the much more frequent cases of ALS caused by other genes or by sporadic causes.”
The effect of MMP-9 removal on survival of ALS mice was reported in a 2007 paper in Experimental Neurology, which contradicted earlier findings that MMP-9 played no role in ALS. The authors of that paper found that knocking out MMP-9 increased the survival of a mouse model of ALS by 31 percent, and reduced neuronal loss. The ALS knockout mice also showed reductions in the immunoreactivity within lumbar spinal cord neurons for tumor necrosis factor-alpha and Fas ligand (FasL), suggesting that MMP-9 exerts toxic effects by upregulating these immune regulators, according to lead author M. Flint Beal, MD, professor of neurology and neuroscience at the Weill Medical College of Cornell University.
The 2007 findings dovetail with results reported in the Neuron paper, in Dr. Beal's view.
MMP-9: BACK ON THE RADAR
“I think this will put MMP-9 back on the radar as an important player in disease pathogenesis,” he said of the Neuron paper. “I thought their carefully controlled experiment was superb; very well done. Basically, it covered all the bases. And it's always nice when someone replicates your own data.”
MMPs, which help to degrade proteins, are upregulated by brain injury caused by head trauma, stroke, and various diseases. MMP inhibitors exist and have undergone trials in cancer treatment, but aggressive, chronic inhibition could produce serious side-effects, according to Dr. Beal.
“I don't think you could knock (MMPs) out completely, but I think you might be able to dial them down,” he said. “I think you could use an agent that reduced their activity without deleterious effects. Or perhaps you could do it genetically with siRNAs [small interfering RNAs]. In our case we saw increases in TNF-alpha and FasL that were ameliorated by knockout of MMP-9. So you could potentially go after those and determine if they are critical in mediating cell death.”
A group of Chinese researchers recently reported in PLoS One a “significant association” between a polymorphism of MMP-9 and both ALS and Parkinson's disease (PD). Like Dr. Beal and his colleagues, they speculate that this polymorphism promotes disease by upregulating inflammatory cytokines like IL-1 and TNF-alpha, which are among the most potent transcriptional activators of MMP-9.
The Chinese researchers found a significant association between the C(-1562)T polymorphism in the MMP-9 gene and the risk of sporadic ALS (odds ratio = 2.163, 95% CI 1.233–3.796, p=0.006) and PD (odds ratio = 2.268, 95% CI 1.506–3.416, p<0.001).
They compared 351 PD patients and 226 ALS patients to equal numbers of controls, all living in southwest China. “To the best of our knowledge, this is the first report investigating the association between the C(21562)T polymorphism in the MMP-9 gene and risk of PD, and the first study to confirm a positive association between the C(21562)T polymorphism and risk of (sporadic) ALS,” the researchers wrote. They also pointed out that MMP-9 has been implicated in Alzheimer's and Huntington's diseases, which “may suggest that MMP-9 is involved in neurodegenerative disorders per se, or that these seemingly different diseases are part of the same neurodegenerative spectrum.”
Dr. Henderson and his colleagues also noted that vulnerability genes like MMP-9 may play a role in both ALS and PD. Like ALS, PD devastates certain neurons while leaving others unaffected. Also, differences in gene expression appear to confer protection on some neurons. “Blocking selective vulnerability therefore might be a viable strategy in multiple therapeutic contexts,” they concluded.
Teepu Siddique, MD, a professor of neurology at Northwestern University's Feinberg School of Medicine, praised the Neuron paper for finding such a tight correlation between specific gene expression and ALS pathology.
“Others have attempted to do this, but I think Dr. Henderson and colleagues have taken it to its conclusion,” he said. “I would like to congratulate them for doing a very nice piece of elegant work.”