Profilin Gene Is Actin’ in ALS

Another handful of familial amyotrophic lateral sclerosis cases are explained, with a July 15 report in Nature, as being caused by mutations in profilin. Profilin catalyzes the formation of actin filaments. The new mutations, which inhibit axonal outgrowth, point toward cytoskeletal defects. Four different profilin mutations seem to account for 1-2 percent of inherited ALS, said senior author John Landers of the University of Massachusetts Medical School in Worcester.

The multi-institute, international collaboration, led by first author Chi-Hong Wu, started with two families in which ALS was inherited in a dominant negative fashion. Carriers tended to fall ill during their early forties, and exhibited "fairly typical ALS," Landers said, with no signs of dementia. Notably, the disease always started in the spinal cord of 22 cases examined, never in the bulbar region, as is typical in one-quarter of ALS cases.

The researchers sequenced the exomes of two affected members of each kindred. After eliminating known variants, there was only one likely candidate gene common to the four people: profilin 1 (PFN1). One family had a cysteine-71-glycine (C71G) mutation, the other a methionine-114-threonine (M114T) substitution. Sequencing an additional 272 people with familial ALS uncovered two more mutations: glycine-118-valine (G118V) and glutamic acid-117-glycine (E117G). None of the first three appeared in sporadic ALS cases or large genome databases; E117G showed up in two of 816 sporadic cases and three out of 7,560 control samples. Landers suspects E117G is less penetrant than the other three mutations.

The scientists have not yet been able to access autopsy tissue from anyone who had a profilin mutation, but they used cell culture to study the effects of the variants. They transfected N2A mouse neuroblastoma cells and mouse primary motor neurons with the four mutants or wild-type profilin. The team found, by Western blots, that like many neurodegeneration-linked proteins, the three most penetrant mutants formed large, insoluble profilin structures. E117G stayed mostly soluble, as did the wild-type, although light microscopy revealed that E117G assembled into aggregates in the N2A cells.

These aggregates were ubiquitinated and frequently contained TDP-43, another ALS-linked protein. However, when Wu and colleagues examined spinal cord sections from people who died of sporadic ALS with TDP-43 pathology, they did not observe profilin colocalizing with TDP-43. "Perhaps [profilin] is not part of the common mechanism," said Jackie de Belleroche of Imperial College London, U.K., who was not involved in the study (see full comment below). "Perhaps this is in a category of its own with other cytoskeletal abnormalities."

All four of the ALS-associated mutations occur in the actin-binding domain of profilin, which assembles actin monomers into filaments. The researchers investigated the ability of each mutant to bind actin by immunoprecipitating profilin from transfected HEK293 human embryonic kidney cells. The three most penetrant mutants picked up less actin than either wild-type or E117G profilin. Expressed in primary motor neurons, the strongest three mutations inhibited axon outgrowth, while E117G shortened axons slightly, but the effect did not reach statistical significance.

The team examined the dynamics of monomeric and filamentous actin in the growth cone of primary motor neurons for C71G and G118V, which most strongly affected axon outgrowth. With the mutants, growth cones were short, lacked filopodia, and had less filamentous actin compared to cells transfected with wild-type profilin. Other ALS proteins, superoxide dismutase 1 (SOD1) and TDP-43, also stunt axon outgrowth (Takeuchi et al., 2002; Duan et al., 2011). While it is certainly tempting to conclude that faulty profilin directly interferes with outgrowth and sickens neurons, the researchers still have more work to do to confirm that or any other mechanism, Landers said. Profilin binds dozens of other proteins, any of which also might contribute to its misbehavior in ALS. "I keep an open mind," de Belleroche said.

Wu CH, Fallini C, Ticozzi N, Keagle PJ, Sapp PC, Piotrowska K, Lowe P, Koppers M, McKenna-Yasek D, Baron DM, Kost JE, Gonzalez-Perez P, Fox AD, Adams J, Taroni F, Tiloca C, Leclerc AL, Chafe SC, Mangroo D, Moore MJ, Zitzewitz JA, Xu ZS, van den Berg LH, Glass JD, Siciliano G, Cirulli ET, Goldstein DB, Salachas F, Meininger V, Rossoll W, Ratti A, Gellera C, Bosco DA, Bassell GJ, Silani V, Drory VE, Brown RH Jr., Landers JE. Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis. Nature. 2012. Jul 15.

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