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.

The midbody (MB) is a singular organelle formed between daughter cells during cytokinesis and required for their final separation. MBs persist in cells long after division as midbody derivatives (MBds), but their fate is unclear. Here we show that MBds are inherited asymmetrically by the daughter cell with the older centrosome. They selectively accumulate in stem cells, induced pluripotent stem cells (iPSCs) and potential cancer ‘stem cells’ (CSCs) in vivo and in vitro. MBd loss accompanies stem cell differentiation, and involves autophagic degradation mediated by binding of the autophagic receptor, NBR1, to the MB protein Cep55. Differentiating cells and normal dividing cells do not accumulate MBds and possess high autophagic activity. Stem cells and cancer cells accumulate MBds by evading autophagosome encapsulation and exhibit low autophagic activity. MBd enrichment enhances reprogramming to iPSCs and increases in vitro tumorigenicity of cancer cells. These results suggest unexpected roles for MBds in stem cells and CSCs.

Cilia and flagella are highly conserved, complex organelles involved in a variety of important functions. Flagella are required for motility of several human pathogens and ciliary defects lead to a variety of fatal and debilitating human diseases. Many of the major structural components of cilia and flagella are known, but little is known about regulation of flagellar beat. Trypanosoma brucei, the causative agent of African sleeping sickness, provides an excellent model for studying flagellar motility. We have used comparative genomics to identify a core group of 50 genes unique to organisms with motile flagella. These genes, referred to as T. brucei components of motile flagella (TbCMF) include 30 novel genes, and human homologues of many of the TbCMF genes map to loci associated with human ciliary diseases. To characterize TbCMF protein function we used RNA interference to target 41 TbCMF genes. Sedimentation assays and direct observation demonstrated clear motility defects in a majority of these knockdown mutants. Epitope tagging, fluorescence localization and biochemical fractionation demonstrated flagellar localization for several TbCMF proteins. Finally, ultrastructural analysis identified a family of novel TbCMF proteins that function to maintain connections between outer doublet microtubules, suggesting that they are the first identified components of nexin links. Overall, our results provide insights into the workings of the eukaryotic flagellum, identify several novel human disease gene candidates, reveal unique aspects of the trypanosome flagellum and underscore the value of T. brucei as an experimental system for studying flagellar biology.

BackgroundAmyotrophic lateral sclerosis (ALS)-linked fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is concentrated within cytoplasmic stress granules under conditions of induced stress. Since only the mutants, but not the endogenous wild-type FUS, are associated with stress granules under most of the stress conditions reported to date, the relationship between FUS and stress granules represents a mutant-specific phenotype and thus may be of significance in mutant-induced pathogenesis. While the association of mutant-FUS with stress granules is well established, the effect of the mutant protein on stress granules has not been examined. Here we investigated the effect of mutant-FUS on stress granule formation and dynamics under conditions of oxidative stress.ResultsWe found that expression of mutant-FUS delays the assembly of stress granules. However, once stress granules containing mutant-FUS are formed, they are more dynamic, larger and more abundant compared to stress granules lacking FUS. Once stress is removed, stress granules disassemble more rapidly in cells expressing mutant-FUS. These effects directly correlate with the degree of mutant-FUS cytoplasmic localization, which is induced by mutations in the nuclear localization signal of the protein. We also determine that the RGG domains within FUS play a key role in its association to stress granules. While there has been speculation that arginine methylation within these RGG domains modulates the incorporation of FUS into stress granules, our results demonstrate that this post-translational modification is not involved.ConclusionsOur results indicate that mutant-FUS alters the dynamic properties of stress granules, which is consistent with a gain-of-toxic mechanism for mutant-FUS in stress granule assembly and cellular stress response.

SummaryStuck in reverse: loss of LC1 in Trypanosoma brucei disrupts outer dynein arms and leads to reverse flagellar beat and backward movement

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Edited by Paul E. Fraser Excitotoxic levels of glutamate represent a physiological stress that is strongly linked to amyotrophic lateral sclerosis (ALS) and other neurological disorders. Emerging evidence indicates a role for neurodegenerative disease-linked RNAbinding proteins (RBPs) in the cellular stress response. However, the relationships between excitotoxicity, RBP function, and disease have not been explored. Here, using primary cortical and motor neurons, we found that excitotoxicity induced the translocation of select ALS-linked RBPs from the nucleus to the cytoplasm within neurons. RBPs affected by excitotoxicity included TAR DNA-binding protein 43 (TDP-43) and, most robustly, fused in sarcoma/translocated in liposarcoma (FUS/ TLS or FUS). We noted that FUS is translocated through a calcium-dependent mechanism and that its translocation coincides with striking alterations in nucleocytoplasmic transport. Furthermore, glutamate-induced up-regulation of glutamate ionotropic receptor ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type subunit 2 (GRIA2) in neurons depended on FUS expression, consistent with a functional role for FUS in excitotoxic stress. These findings reveal molecular links among prominent factors in neurodegenerative diseases, namely excitotoxicity, disease-associated RBPs, and nucleocytoplasmic transport. This work was supported by National Institutes of Health Grants R21 NS091860 (to D. A. B.) and R01 NS078145 (to D. A. B.) from NINDS; ALS Association Grant 18-IIA-418 (to C. F.), and a Zelda Haidek Memorial Scholarship from University of Massachusetts Medical School (to M. T.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Figs. S1-S6.

Aberrant translational repression is a feature of multiple neurodegenerative diseases. The association between disease-linked proteins and stress granules further implicates impaired stress responses in neurodegeneration. However, our knowledge of the proteins that evade translational repression is incomplete. It is also unclear whether disease-linked proteins inf luence the proteome under conditions of translational repression. To address these questions, a quantitative proteomics approach was used to identify proteins that evade stress-induced translational repression in arsenite-treated cells expressing either wild-type or amyotrophic lateral sclerosis (ALS)-linked mutant FUS. This study revealed hundreds of proteins that are actively synthesized during stress-induced translational repression, irrespective of FUS genotype. In addition to proteins involved in RNA-and protein-processing, proteins associated with neurodegenerative diseases such as ALS were also actively synthesized during stress. Protein synthesis under stress was largely unperturbed by mutant FUS, although several proteins were found to be differentially expressed between mutant and control cells. One protein in particular, COPBI, was downregulated in mutant FUS-expressing cells under stress. COPBI is the beta subunit of the coat protein I (COPI), which is involved in Golgi to endoplasmic reticulum (ER) retrograde transport. Further investigation revealed reduced levels of other COPI subunit proteins and defects in COPBI-related 2144