Research Brief: Blocking Apoptosis Delays ALS in Mice

In amyotrophic lateral sclerosis and related diseases, damaged motor neurons commit cellular suicide, calling on the apoptosis program to finish their lives. But what has been unclear is whether apoptosis is a core process early in the disease, or merely the swan song of cells that have lost the ability to do anything other than make a graceful exit. In the October 1 Journal of Clinical Investigation, scientists from the University of California in San Francisco report that blocking apoptosis in ALS model mice keeps motor neurons alive, suggesting apoptosis is a central part of the pathological process.

First author Nichole Reyes and senior author Scott Oakes led the study. They reasoned that if apoptosis happens early in ALS, and helps to push motor neurons to their final hour, then blocking apoptosis should slow the disease. Conversely, if apoptosis simply finishes off cells that are already beyond repair, then blocking the process should not affect symptoms.

To get to the answer they sought, Reyes and colleagues chose to knock out apoptosis at a key gateway: mitochondrial permeability mediated by BCL2-associated X protein (Bax) and BCL2-homologous antagonist/killer (Bak). Bax and Bak share a similar function, sitting in the mitochondrial membrane and waiting for cell death signals. Upon receiving those signals, they form pores, allowing cytochrome c to leak out and initiating cellular suicide.

Other studies have also targeted the apoptotic pathway. Blocking caspases or other cell death genes, or overexpressing BCL2 (which puts a damper on apoptosis) increases lifespan in ALS model mice (see ARF related news story on Li et al., 2000; Friedlander et al., 1997; Kostic et al., 1997). And the drug minocycline, which prevents cytochrome c release, also prolongs survival (Zhu et al., 2002).

The scientists generated mice that lack the Bak gene, but carry a floxed version of Bax. They expressed Cre recombinase under the nervous system-specific rat nestin promoter. Around day 12 of embryonic development, when nestin turns on, the recombinase cuts Bax out of brain and spinal cord tissue. We could really, truly, for the first time, completely block the apoptotic pathway in vivo, Oakes said. They crossed these mice with animals overexpressing human superoxide dismutase 1 (SOD1)-G93A, a mutant associated with familial ALS. As controls, they used SOD1-G93A mice carrying only the nestin-Cre construct, with no changes to their Bak or Bax levels. These control animals evince apoptosis in spinal cord motor neurons and paralysis starting at 100 days of age. They die within a month of symptom onset.

In the transgenic Bax/Bak double knockouts, disease onset was delayed by more than three weeks, and the animals survived for nearly a month longer than controls. When the control mice were dying, the double knockouts were still walking around, pretty much fine, Oakes said. The double knockout transgenic mice not only had more motor neurons than age-matched controls, they also preserved neuromuscular junctions for longer. The motor neurons they had were also functional, the scientists concluded, because the double knockouts maintained their ability to balance on a rotarod. Oakes and colleagues concluded that apoptosis must contribute directly to the disease process.

It is terrific that they have been able to look into this very specific trigger of mitochondrial apoptosis, said Robert Friedlander of the University of Pittsburgh, Pennsylvania, who noted the work confirms previous results on the importance of mitochondria and mitochondrial cell death pathways in ALS. For example, SOD1-G93A mice deficient in only Bax also exhibit extended survival (Gould et al., 2006).

The study also poses further questions, said Serge Przedborski of Columbia University in New York. Now, he wonders what pathway leads from ALS, to Bak and Bax, and beyond to motor neuron death. They have a very elegant model that will allow us, perhaps, to dissect a little further, and in more detail, what is the molecular cascade, he said.

Przedborski added, It re-stimulates the idea that targeting things like Bak and Bax for therapy may be a valuable strategy. However, it could also be a dangerous strategy. A lot of cells, in the brain and outside the brain, rely on the machinery of apoptosis to be eliminated, he said. You run the risk to have major side effects, including tumorigenesis.

For that reason, Oakes is interested in targeting apoptosis at a different point than the Bax/Bak gateway. This experiment, he said, was a proof of principle that blocking apoptosis can extend motor neuron survival. Now, he and his colleagues are searching for upstream signals—such as those from the unfolded protein response—that activate Bak and Bax in ALS. Those upstream signals should be amenable to drugs, he said, and might allow doctors to prevent apoptosis specifically in struggling neurons.

Reyes NA, Fisher JK, Austgen K, VandenBerg S, Huang EJ, Oakes SA. Blocking the mitochondrial apoptotic pathway preserves motor neuron viability and function in a mouse model of amyotrophic lateral sclerosis. J Clin Invest. 2010 Oct 1;120(10):3673-9. Abstract

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