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.—Amber Dance.

Reference:
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. Abstract

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  1. John Landers and colleagues have successfully used exome capture followed by deep sequencing to identify novel mutations in the profilin gene (PFN1) that cause familial amyotrophic lateral sclerosis (FALS). The application of this methodology has greatly speeded up the identification of pathogenic mutations. Data obtained from affected members of two kindreds revealed different coding mutations, C71G and M114T, both being present in PFN1, that segregated with disease and had not been previously reported in available SNP databases. Subsequent screening of this gene for mutations in 273 further FALS and 816 sporadic ALS cases revealed two more FALS index cases with the C71G mutation and one other case with the M114T mutation. Two new mutations, G118V and E117G, were found in familial cases, and the E117G mutation was also found in two sporadic cases. The E117G mutation also occurred in three out of 7,560 controls and must be viewed with caution. No coding changes in PFN2 and PFN3 were seen in FALS cases.

    Quite a lot of research has been performed on profilin, and it is known to function in the regulation of actin structure, transforming a globular monomer, known as G-actin, to long helical polymer, F-actin. This transformation is crucial in cytoskeletal dynamics, important in neurite outgrowth, growing axons, and synapse formation. Furthermore, the FALS-associated mutations lie in close proximity to the actin binding residues. The mutations were convincingly shown in this study to reduce axon outgrowth. Growth cone size and morphology were also affected by the mutations, with greatly reduced F-actin-rich filopodia being present.

    Functional studies carried out by the authors showed that the three novel mutations had a propensity to aggregate and, when transfected into a neuronal cell line (Neuro2A) or primary motor neurons, formed ubiquitinated aggregates. Whilst these aggregates did not show co-aggregation with FUS or SMN, co-staining of aggregates with PFN1 and TDP-43 occurred in about one-third of cells. However, no abnormal PFN pathology was seen in spinal cord sections from sporadic cases of ALS.

    Abnormalities in a number of cytoskeletal proteins are found in motor neuron diseases, but tend to be quite rare. At present, no autopsy cases harboring these PFN1 mutations are available, but information about the neuropathological features of this condition will be extremely valuable. The pathogenic mechanism remains to be elucidated and could involve effects on actin polymerization, or may yet result from one of the other numerous protein interactions reported for PFN.

  2. There remain many families with ALS where the causative genes are yet to be discovered. In our Department of Neurology at the University of Tokyo, causative genes are unidentified in approximately half of ALS families.

    In PFN1-associated familial ALS (FALS), the mutations were found in seven out of 274 families, meaning the frequency of families with mutations in PFN1 is rare. Nonetheless, I think this finding is important. Surely, most of the FALS families may have mutations in orphan genes. We need to identify all the causative genes for FALS, which should bring insight into sporadic ALS, which is more common compared to FALS.

    I believe this discovery provides better understanding of the pathophysiology of ALS. Abnormality in conversion of monomeric (G)-actin to filamentous (F)-actin is a new mechanism in the disease.

    We are pursuing similar approaches, and hope to contribute to better understanding ALS in the very near future.

References

Paper Citations

  1. . Hsp70 and Hsp40 improve neurite outgrowth and suppress intracytoplasmic aggregate formation in cultured neuronal cells expressing mutant SOD1. Brain Res. 2002 Sep 13;949(1-2):11-22. PubMed.
  2. . MG132 enhances neurite outgrowth in neurons overexpressing mutant TAR DNA-binding protein-43 via increase of HO-1. Brain Res. 2011 Jun 23;1397:1-9. PubMed.
  3. . Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis. Nature. 2012 Aug 23;488(7412):499-503. PubMed.

Further Reading

Papers

  1. . Novel FUS Deletion in a Patient With Juvenile Amyotrophic Lateral Sclerosis. Arch Neurol. 2012 Jan 16; PubMed.
  2. . Secreted VAPB/ALS8 major sperm protein domains modulate mitochondrial localization and morphology via growth cone guidance receptors. Dev Cell. 2012 Feb 14;22(2):348-62. PubMed.
  3. . Accumulation of TDP-43 and alpha-actin in an amyotrophic lateral sclerosis patient with the K17I ANG mutation. Acta Neuropathol. 2009 Oct;118(4):561-73. PubMed.
  4. . Axonopathy and cytoskeletal disruption in degenerative diseases of the central nervous system. Brain Res Bull. 2009 Oct 28;80(4-5):217-23. PubMed.
  5. . Phosphorylation of profilin by ROCK1 regulates polyglutamine aggregation. Mol Cell Biol. 2008 Sep;28(17):5196-208. PubMed.
  6. . Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis. Nature. 2012 Aug 23;488(7412):499-503. PubMed.

Primary Papers

  1. . Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis. Nature. 2012 Aug 23;488(7412):499-503. PubMed.