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Dec 1

A new genetic explanation for ALS heterogeneity

Research Bits
Packard investigator Udai Pandey of the University of Pittsburgh led an international team that found a protein called muscleblind modifies the toxicity of ALS-linked mutations in a second protein called FUS (Fused in Sarcoma).

Although all cases of ALS are characterized by motor neuron degeneration, there can be huge variability in the age of onset and the rapidity of disease progression. New research by Packard scientists in Nature Communications may help explain some of this heterogeneity.

Neuroscientist Udai Pandey of the University of Pittsburgh led an international team that found a protein called muscleblind modifies the toxicity of ALS-linked mutations in a second protein called FUS (Fused in Sarcoma). Understanding the other genes and proteins that impact motor neuron degeneration tells scientists not just about the molecular causes of disease, but it can also point towards potential treatments, Pandey says.

FUS mutations were first linked to ALS in 2009. The role of FUS as an RNA-binding protein helped to spur the scientific community’s appreciation of the role of RNA metabolism in the onset and progression of ALS. Although FUS mutations are thought to be responsible for only 5% of ALS cases, FUS-linked ALS shows a high level of heterogeneity.

One FUS mutation, known as P525L, causes a rapidly progressing, juvenile-onset form of ALS. Another mutation, R521C, doesn’t typically cause disease until middle age or beyond. Other mutations, such as those found in SOD1 or C9ORF72, have their own unique patterns of illness. Sorting through this genetic and phenotypic diversity can provide clues about what factors may hasten ALS onset and what might slow disease progression.

Pandey has spent much of his career investigating FUS using fruit flies, which show distinctive eye changes as part of their ALS-like disease. When he searched for genes that could reduce signs of eye degeneration in fruit flies engineered to carry a mutant FUS protein, Pandey identified one gene. Known as muscleblind, researchers had previously linked this RNA splicing protein to myotonic dystrophy, Fragile X, and Huntington’s, although it had never been associated with ALS. Muscleblind also contains long introns (portions of the gene that are later snipped out of the RNA), something to which FUS frequently binds.

When Ian Casci, a graduate student in the Pandey lab and the leading author, looked more closely, he found that knocking down muscleblind levels by 49% significantly reduced the toxic effects of FUS on fruit fly eyes. Increasing muscleblind expression, on the other hand, enhanced FUS toxicity. Additional experiments by Sukhleen Kour, a postdoc in the Pandey lab, revealed that FUS mutations did not affect normal muscleblind levels, and studies in human embryonic kidney cells revealed that FUS and muscleblind physically interact. Their work also showed that muscleblind affected FUS toxicity in fly motor neurons, as reducing muscleblind levels in FUS-ALS flies significantly increased the number of flies able to climb.

Lowering muscleblind levels in human embryonic kidney cells also decreased FUS inclusion in stress granules (dense aggregates of RNA and protein that form when a cell is stressed). In collaboration with Dr. Piera Pasinelli, scientific director of the Packard Center, experiments in rat cortical neurons revealed that muscleblind prevents the misloalization of FUS to the cytoplasm, which consequently keeps it from being incorporated into stress granules. Karthik Krishnamurthy, a postdoc in Pasinelli’s lab, utilized human cortical neurons to demonstrate that knockdown of muscleblind reduced morphological and molecular signs of FUS toxicity. Follow-up studies by Jared Sterneckert of the Technical University of Dresden revealed that muscleblind reduction also decreased the number of stress granules in motor neurons derived from induced pluripotent stem cells expressing FUS P525L mutation.

The research also revealed connections to SMN protein, which has been associated with both spinal muscular atrophy and ALS and is known to physically interact with FUS. Muscleblind knockdown rescued SMN trapped in the cytoplasm by mutant FUS and increasing SMN expression decreased FUS toxicity in primary motor neurons.

Pandey says that these results are important because they highlight the role of muscleblind in a variety of neurodegenerative conditions as well as providing explanations for some of the heterogeneity seen in ALS. The researchers want to test whether muscleblind and SMN are important in the pathogenesis of disease and a candidate for interventions.

All of this work, Pandey says, was made possible by support from the Packard Center. An initial grant let him apply for NIH funding to start his lab and to begin experiments in fruit flies. Ongoing Packard funds have enabled his team to keep working.

“I’m very, very grateful to the Packard Center,” Pandey said.

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