Researchers home in on biological cause of ALS

A defect in how proteins are transported inside cells may be at least partially responsible for some symptoms of amyotrophic lateral sclerosis, according to a study by researchers at the Stanford University School of Medicine.

The researchers found that cells with a mutation associated with the disease were hampered in their ability to move proteins into and out of the nucleus, a cell’s command center. The findings are among the first to open a window into the molecular causes of the deadly condition.

Also known as Lou Gehrig’s disease, ALS is a progressive neuromuscular disorder. Although it’s unknown what causes many cases of the disease, a genetic stutter in a region of DNA called C9orf72 has been associated with the development of both ALS and another related neurodegenerative disorder called frontotemporal dementia. In this region, the DNA sequence is made up of a six-nucleotide pattern, which repeats abnormally in some people with ALS or FTD.

“Healthy people have two to five repeats of this six-nucleotide pattern,” said Aaron Gitler, PhD, associate professor of genetics. “But in some people, this region is expanded into hundreds or thousands of copies. This mutation is found in about 40 to 60 percent of ALS inherited within families and in about 10 percent of all ALS cases. This is by far the most common cause of ALS, so everyone has been trying to figure out how this expansion of the repeat contributes to the disease.”

Although the repeat occurs in a region of the gene called an intron, which does not normally carry protein-making instructions, researchers have speculated that a molecular miscue may cause a cell to mistakenly make RNAs and proteins from the expanded repeat.

Gitler is the senior author of the study, which was published online Aug. 26 in Nature Neuroscience. Postdoctoral scholar Ana Jovičić, PhD, is the lead author. The Stanford research is complemented by similar findings from two other groups that was published in the Aug. 27 issue of Nature.

Toxic proteins

Previous research has shown that proteins made from the expanded section of nucleotides are toxic to fruit fly and mammalian cells and trigger neurodegeneration in animal models. However, it’s not been clear why. Gitler and Jovičić used a yeast-based system to understand what happens in these cells. Although yeast are a single-celled organism without nerves, Gitler has shown that, because they share many molecular pathways with more-complex organisms, they can be used to model some aspects of neuronal disease.

Cells make proteins by copying a nucleotide sequence from DNA. This copy, called RNA, is then shuttled to the cell’s protein-making factories. Proteins are made, or translated, from the RNA by assembling building blocks called amino acids in a sequence dictated by the order of nucleotides in the RNA string. Every three-nucleotide combination either encodes a particular amino acid or carries a cellular command such as “start” and “stop” to facilitate protein production.

This is by far the most common cause of ALS, so everyone has been trying to figure out how this expansion of the repeat contributes to the disease.

Often, genes include strings of intervening nucleotides called introns that don’t contain instructions to make proteins. These introns are usually removed from the RNA before it is translated. However, researchers have speculated that cells with introns harboring expanded repeats (as in the C9orf72 gene) may mistakenly make protein from the repeated section even in the absence of a traditional “start” command. Although it’s not known exactly why this abnormal protein translation may occur, previous research has shown it does sometimes happen in regions of the genome with repeated nucleotide sequences.

Jovičić first constructed a series of yeast strains, each of which expressed one of the proteins thought to be made from the expanded repeat. She found that one of the proteins, made up of the amino acids proline and arginine, was particularly toxic to the yeast cells. Another, with glycine and arginine, was also toxic. The particular toxicity of the arginine-containing proteins in the yeast mirrors what had previously been found in mammalian cells and in fruit flies.

Using a technique known as a genomewide screen, Jovičić then systematically tested whether expressing higher-than-normal amounts of any one of the yeast’s proteins could suppress the toxicity of the arginine-containing proteins and allow the mutated yeast to grow like its unmutated peers. Conversely, she used another technique to investigate whether the loss of any one protein could influence the protein’s toxicity.

Potential targets for drugs

Jovičić found 27 genes that, when expressed at high levels, suppressed the toxicity of the proline-arginine protein. Mutated yeast strains with higher-than-normal levels of any one of these proteins grew better in the presence of the arginine-containing proteins.  She also identified 35 genes that, when highly expressed, made the arginine-containing proteins even more toxic. Finally, she discovered 16 genes that, when missing from the yeast, rendered the arginine-containing protein less toxic. These deleted genes are of particular interest because they could be potential targets for drug interventions or therapies.

When Jovičić and Gitler examined all the genes identified in the yeast screens, they found several that encoded proteins responsible for ferrying molecules in and out of the nucleus. In particular, a class of nuclear import proteins called karyopherins appeared important in modulating the effect of the toxic protein. One, called KAP122, was one of the strongest suppressors of toxicity when overexpressed.

When Jovičić and Gitler sought to confirm their results in mammalian cells, they found that overexpression in rat neurons of a mammalian gene similar to KAP122 more than doubled the survival of neurons expressing the toxic protein.

The researchers then turned their attention to human cells. They were able to directly convert skin cells from three people without the extended nucleotide repeat and two with the extended repeat into neurons that could be studied in the laboratory. They compared the location of a protein called RCC1, which is normally found in the cell’s nucleus, between the two groups of cells. RCC1 is the human version of one of the yeast proteins that Jovičić found worsened the toxicity of the proline-arginine proteins when overexpressed.

We hope that our research may one day lead to new potential therapies for these devastating, progressive conditions.

They discovered that although RCC1 was in the nucleus in nearly all the unmutated neurons, it was found primarily in the cytoplasm in about 70 to 80 percent of neurons with the expanded repeat.

Although much remains to be learned about how these genes are involved in the development of ALS and FTD, the fact that the researchers were able to identify specific types of protein families common among yeast, rodent and human cells represents an important step forward in the field, they said. Future work will also focus on disentangling the relative contributions of the RNAs and repeat proteins produced by the mutated C9orf72 gene.

“Neurodegenerative diseases are very complicated,” said Jovičić. “They likely occur as a result of a defect or defects in basic biology, which is conserved among many distantly related species. We hope that our research may one day lead to new potential therapies for these devastating, progressive conditions.”

Other Stanford-affiliated authors of the paper are graduate students Noori Chai, Shizuka Yamada, Gregor Bieri and Nicholas Kramer, and undergraduate student Joseph Paul III.

The research was supported by the National Institutes of Health (grants 1R01NS065317, 1R01NS073660 and NS091538), the Packard Center for ALS Research at Johns Hopkins, a Target ALS Springboard Fellowship, the J.P.B. Foundation, the Helmsley Foundation, the Mathers Foundation, the University of Leuven, the European Research Council, the Fund for Scientific Research Flanders, the Belgian Science Policy Office, the Association Belge contre les Maladies Neuro-Musculaires and the ALS League Belgium.

Stanford’s Department of Genetics also supported the work.