Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder resulting from motor neuron death. Approximately 10% of cases are familial (FALS), typically with a dominant inheritance mode. Despite numerous advances in recent years1-9, nearly 50% of FALS cases have unknown genetic etiology. Here we show that mutations within the profilin 1 (PFN1) gene can cause FALS. PFN1 is critical for monomeric (G)-actin conversion to filamentous (F)-actin. Exome sequencing of two large ALS families revealed different mutations within the PFN1 gene. Additional sequence analysis identified 4 mutations in 7 out of 274 FALS cases. Cells expressing PFN1 mutants contain ubiquitinated, insoluble aggregates that in many cases contain the ALS-associated protein TDP-43. PFN1 mutants also display decreased bound actin levels and can inhibit axon outgrowth. Furthermore, primary motor neurons expressing mutant PFN1 display smaller growth cones with a reduced F-/G-actin ratio. These observations further document that cytoskeletal pathway alterations contribute to ALS pathogenesis.

Leucine zipper peptides provide simple model systems for studying both the intramolecular and intermolecular interactions that govern protein folding. The synthetic 33-residue peptide GCN4-p1, derived from the yeast transcriptional activator GCN4, forms a stable biomolecular coiled-coil structure [O'Shea, E. K., Klemm, J. D., Kim, P. S., & Alber, T. (1991) Science 254, 539-544]. The guanidine-HCl induced equilibrium unfolding of this peptide at 5 degrees C and pH 7.0 yields a standard state free energy of 10.49 +/- 0.23 kcal (mol dimer)-1 when fit to a two-state model involving the native dimer and the unfolded monomer. The unfolding and refolding kinetics of GCN4-p1 were monitored by stopped-flow circular dichroism spectroscopy as a function of both peptide concentration and final denaturant concentration. The unfolding kinetics displayed single-exponential behavior, consistent with a unimolecular reaction. The refolding kinetics, which are dependent on both peptide and guanidine concentration, are well described by a simple bimolecular association reaction. A simultaneous fit of all of the unfolding and refolding kinetic data to the model, N2[symbol: see text]2U, yields refolding and unfolding rate constants in the absence of denaturant of 4.2 x 10(5) M-1 S-1 and 3.3 x 10(-3) S-1, respectively. The equilibrium unfolding curve is accurately predicted from these rate constants, providing further support for the validity of the two-state kinetic model.

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Mutations in profilin 1 (PFN1) are associated with amyotrophic lateral sclerosis (ALS); however, the pathological mechanism of PFN1 in this fatal disease is unknown. We demonstrate that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutationinduced destabilization can account for the high propensity of ALSlinked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization is illuminated by the X-ray crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis.amyotrophic lateral sclerosis | profilin 1 | protein stability | X-ray crystallography | protein misfolding M utations in the profilin 1 gene (PFN1) were recently associated with both familial and sporadic forms of amyotrophic lateral sclerosis (ALS) (1, 2), an incurable and fatal neurodegenerative disease that primarily targets motor neurons (3). The etiology of sporadic ALS is poorly understood, whereas familial ALS is caused by inheritable genetic defects in defined genes such as PFN1 (3). PFN1 is a 15-kDa protein that is best known for its role in actin dynamics in the context of endocytosis, membrane trafficking, cell motility, and neuronal growth and differentiation (4). In addition to binding monomeric or G-actin, PFN1 also binds to a host of different proteins through their poly-L-proline motifs and to lipids such as phosphatidylinositol 4,5-bisphosphate (4, 5). However, little is known about the mechanism(s) associated with PFN1-mediated ALS pathogenesis. The observation that most ALS-linked PFN1 variants are highly prone to aggregation in mammalian cultured cells suggests that diseasecausing mutations induce an altered, or misfolded, conformation within PFN1 (2). Protein misfolding is a hallmark feature of most neurodegenerative diseases, including ALS (3), and can contribute to disease through both gain-of-toxic-function and loss-of-normalfunction mechanisms (6). Although mutations in PFN1 cause ALS through a dominant inheritance mode (2), there is some evidence supporting a loss-of-function mechanism for mutant PFN1. For example, ALS-linked mutations were shown to abrogate the binding of PFN1 to actin (2) and to impair the incorporation of PFN1 into cytoplasmic stress granules during arsenite-induced stress (7) in cultured cells. Moreover, ectopic expression of these variants in murine motor neurons led to a reduction in both axon outgrowth and growth cone size, consistent with a loss of function through a dominant-negative mechanism (2).Although ALS-linked mutations were shown to induce PFN1 aggregation, the ef...

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