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...

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.

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Cyanobacteria are capable of producing a wide range of bioactive compounds with many considered to be toxins. Although there are a number of toxicological outcomes with respect to cyanobacterial exposure, this review aims to examine those which affect the central nervous system (CNS) or have neurotoxicological properties. Such exposures can be acute or chronic, and we detail issues concerning CNS entry, detection and remediation. Exposure can occur through a variety of media but, increasingly, exposure through air via inhalation may have greater significance and requires further investigation. Even though cyanobacterial toxins have traditionally been classified based on their primary mode of toxicity, increasing evidence suggests that some also possess neurotoxic properties and include known cyanotoxins and unknown compounds. Furthermore, chronic long-term exposure to these compounds is increasingly being identified as adversely affecting human health.

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

The relationship between gene dosage imbalance and phenotypes associated with Trisomy 21, including the etiology of abnormal bone phenotypes linked to Down syndrome (DS), is not well understood. The Ts65Dn mouse model for DS exhibits appendicular skeletal defects during adolescence and adulthood but the developmental and genetic origin of these phenotypes remains unclear. It is hypothesized that the postnatal Ts65Dn skeletal phenotype originates during embryonic development and results from an increased Dyrk1a gene copy number, a gene hypothesized to play a critical role in many DS phenotypes. Ts65Dn embryos exhibit a lower percent bone volume in the E17.5 femur when compared to euploid embryos. Concomitant with gene copy number, qPCR analysis revealed a  ~1.5 fold increase in Dyrk1a transcript levels in the Ts65Dn E17.5 embryonic femur as compared to euploid. Returning Dyrk1a copy number to euploid levels in Ts65Dn, Dyrk1a(+/-) embryos did not correct the trisomic skeletal phenotype but did return Dyrk1a gene transcript levels to normal. The size and protein expression patterns of the cartilage template during embryonic bone development appear to be unaffected at E14.5 and E17.5 in trisomic embryos. Taken together, these data suggest that the dosage imbalance of genes other than Dyrk1a is involved in the development of the prenatal bone phenotype in Ts65Dn embryos.

Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of neuromuscular disease such as Duchenne muscular dystrophy has been an advance for the splice-switching field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. To overcome limitations of existing technologies, we engineered chimeric stereopure oligonucleotides with phosphorothioate (PS) and phosphoryl guanidine-containing (PN) backbones. We demonstrate that these chimeric stereopure oligonucleotides have markedly improved pharmacology and efficacy compared with PS-modified oligonucleotides, preventing premature death and improving median survival from 49 days to at least 280 days in a dystrophic mouse model with an aggressive phenotype. These data demonstrate that chemical optimization alone can profoundly impact oligonucleotide pharmacology and highlight the potential for continued innovation around the oligonucleotide backbone. More specifically, we conclude that chimeric stereopure oligonucleotides are a promising splice-switching modality with potential for the treatment of neuromuscular and other genetic diseases impacting difficult to reach tissues such as the skeletal muscle and heart.

<b><i>Introduction:</i></b> Amyotrophic lateral sclerosis (ALS) is a fatal, neuromuscular disease with no cure. ALS incidence rates have not been assessed specifically in Ohio, yet the state contains both metropolitan and rural areas with a variety of environmental factors that could contribute to disease etiology. We report the incidence of ALS in Ohio residents diagnosed from October 2016 through September 2018. <b><i>Methods:</i></b> We engaged practitioners from 9 Ohio sites to identify newly diagnosed ALS patients and to complete case report forms with demographic and clinical information. ALS was diagnosed according to the Awaji criteria and classified as either definite, probable, or possible. We developed a method to estimate missing cases using a Poisson regression model to impute cases in counties with evidence of undercounting. <b><i>Results:</i></b> We identified 333 newly diagnosed ALS patients residing in Ohio during the 2-year index period and found incidence rates varied in the 88 state counties. After incorporating the estimated 27% of missing cases, the corrected crude annual incidence was 1.96/100,000 person-years, and the age- and gender-standardized incidence was 1.71/100,000 person-years (standardized to the 2010 US census). <b><i>Discussion/Conclusion:</i></b> The estimated Ohio incidence of ALS is overall similar to that reported in other states in the USA. This study reveals a geospatial variation in incidence within the state, and areas with higher rates warrant future investigation.