29 June 2010. Sifting through the genome has netted several mutations responsible for motor neuron diseases, but scientists still cannot explain the cause for most incidences. For amyotrophic lateral sclerosis (ALS), an adult-onset condition, genetics explains about one-tenth of cases, and researchers have identified a handful of mutations that cause those inherited forms. Spinal muscular atrophy (SMA), a neuromuscular degeneration that commonly affects infants, is frequently the result of mutations in the gene SMN1, but not all affected children carry such a mutation.
Papers in PNAS online provide one new connection—linking microRNAs to motor neuron degeneration—and one disappointment—showing that a gene previously associated with survival times in ALS may not be meaningful after all. In an upcoming issue, researchers from the Weizmann Institute of Science in Rehovot, Israel, report that preventing motor neurons from making microRNA (miRNA) causes motor neuron disease (MND) reminiscent of ALS and SMA. In the June 21 early edition online, researchers based at the National Institutes of Health, Bethesda, Maryland, and in Italy suggest that the motor accessory protein KIFAP3, previously linked to ALS survival rates in a genomewide association study, has no effect on survival in their sample of Italians with ALS.
MicroRNA Malfunction in MND
Several lines of research point to an miRNA connection in MNDs. SMN, a protein linked to SMA, shows up in miRNA-protein complexes, where it could conceivably alter the production of miRNA-9 (miR-9) and other RNA regulators (Dostie et al., 2003). The ALS-linked proteins FUS and TDP-43 interact with the miRNA processor Drosha (Gregory et al., 2004; Fukuda et al., 2007), and TDP-43 knockdown alters miRNA levels in cultured cells (Buratti et al., 2010).
“I think that there are good chances that some of the motor neuron diseases…are actually RNA regulatory diseases,” said Eran Hornstein, senior author on the paper from the Weizmann Institute. Along with first author Sharon Haramati and co-senior author Alon Chen, he sought to understand the role of miRNAs in MNDs.
MicroRNA precursors become active miRNAs when digested by Drosha and Dicer. To investigate the role of miRNAs in spinal motor neurons, the authors generated a mouse that, via a Cre/Lox system, selectively knocks out Dicer, and thus miRNA production, in lower motor neurons. From seven weeks on, the Dicer knockouts struggled with standard motor tests, and moved around less than their wild-type littermates. While lab mice normally survive for two years or more, the mutant mice lived for a median of 29 weeks, with the oldest reaching only 50 weeks. In pathological analyses, the researchers observed denervation and neuron degeneration characteristic of MNDs. “It provides the first evidence for microRNAs being potentially related to ALS using an in vivo model,” Hornstein said.
Next, the researchers searched for specific reasons that Dicer-deficient motor neurons might struggle. Neurons tightly coordinate expression of neurofilament, a cytoskeletal protein in axons that comes in light, medium, and heavy varieties. The three must be present in the proper proportions, Hornstein said. The researchers discovered that the heavy neurofilament gene encodes nine binding sites for miR-9, which is known to regulate neural gene expression. MiR-9, the scientists reasoned, may dampen heavy neurofilament expression to match levels of the other neurofilament components. Without miR-9, too much heavy neurofilament could throw the system out of whack.
To test their theory, Haramati and her coauthors studied motor neurons carrying the SMA-associated SMN1 mutation. They discovered a few miRNAs were downregulated in the SMA cells, with miR-9 among those most affected. Mutant cells had as much as 15 times less miR-9 as control cells. This loss of miR-9 could potentially lead to degeneration.
Although miR-9 was the most strikingly downregulated miRNA in the authors’ analysis, there are plenty more miRNAs to examine. “I trust that there would be quite a few relevant microRNAs,” Hornstein said. In addition, the researchers are working on an miR-9 loss-of-function mouse, to see if it has a motor neuron disease like SMN1 or Dicer mutants.
The study “adds to a growing list of evidence that dysregulated miRNA expression contributes to neurodegenerative disease,” wrote Andrew Williams of the University of Texas Southwestern Medical Center in Dallas, who was not involved in the study, in an e-mail to ARF. “Upregulating the expression of miR-9 (or other microRNAs) could be a viable therapeutic strategy for treating neurodegenerative diseases like SMA and ALS,” he speculated.
Motor’s Role Minimized
Neurofilament is essential for forming and maintaining axons, leading to another theme in research on neurodegenerative disease: axonal transport (see ARF related news story). Last year, researchers at the University of Massachusetts Medical School in Worcester reported that kinesin-associated protein 3 (KIFAP3), a subunit of a microtubule motor protein, is associated with survival time in ALS. Using a GWAS, they found a KIFAP3 variant that, when present in both copies of the gene, increased survival by 14 months in people (see ARF related news story on Landers et al., 2009).
GWASs often provide tantalizing results that fail to hold up to further scrutiny. In the second PNAS paper, researchers used a new ALS cohort, hoping to confirm or deny the 2009 results. The team included first author Bryan Traynor of the National Institute on Aging in Bethesda, Maryland; joint senior authors Bagriella Restagno of the Azienda Ospedaliera Ospedale Infantile Regina Margherita-S.Anna in Turin, Italy; Gabriele Mora of the Fondazione Salvator Maugeri in Milan, Italy; and Adriano Chiò of the University of Turin.
The researchers analyzed 140 single-nucleotide polymorphisms in the KIFAP3 locus in 504 people from the Piemonte and Valle d’Aosta Registry for ALS. “Unluckily, we could not confirm” the KIFAP3 association, Chiò said. It is possible, the authors suggested, that differing populations could account for the discrepancy. The Italian ALS registry includes anyone in the region with ALS, while the Massachusetts team focused on people who came to specialized ALS clinics. These people tend to be more motivated, receive the best treatment, and may live longer, Chiò said. In addition, he noted, most of the participants in the original paper were of Northern European descent, as opposed to the southerners in the Italian sample.
Another possibility is that the original GWAS simply yielded a false-positive hit, as happens routinely in these kinds of screens. “Our results reduce the possibility that the gene has a real effect on ALS,” Chiò said. However, he said, researchers have only analyzed a few thousand ALS samples for SNPs, and more data are required to make a final judgment. John Landers and Robert Brown, authors of the 2009 paper on KIFAP3, were unavailable for comment.
The GWAS was not the only evidence of a role for KIFAP3 in motor neuron disease, noted Toshiyuki Araki of the National Center of Neurology and Psychiatry in Tokyo, Japan, in an e-mail to ARF. His research group has found KIFAP3 associated with spinal aggregates in some people with familial ALS (Tateno et al., 2009). “It is possible that dysfunction of KIFAP3 does not affect survival at all, or has some impact on survival only on some specific types of ALS,” Araki wrote.—Amber Dance.
Haramati S, Chapnik E, Sztainberg H, Eilam-Altstadter R, Zwang R, Gershoni N, McGlinn E, Heiser PW, Wills AM, Wirguin I, Rubin L, Misawa H, Tabin CJ, Brown R Jr., Chen A, Hornstein E. miRNA malfunction causes spinal motor neuron disease. Proc Natl Acad Sci U. S. A. 2010 June. Abstract
Traynor BJ, Nalls M, Lai SL, Gibbs RJ, Schymick JC, Arepalli S, Hernandez D, van der Brug MP, Johnson JO, Dillman A, Cookson M, Moglia C, Calvo A, Restagno G, More G, Chiò A. Kinesin-associated protein 3 (KIFAP3) has no effect on survival in a population-based cohort of ALS patients. Proc Natl Acad Sci U S A. 2010 June 21. Abstract