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Summary RNA-binding protein aggregation is a pathological hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To gain better insight into the molecular interactions underlying this process, we investigated FUS, which is mutated and aggregated in both ALS and FTLD. We generated a Drosophila model of FUS toxicity and identified a previously unrecognized synergistic effect between the N-terminal prion-like domain and the C-terminal arginine-rich domain to mediate toxicity. Although the prion-like domain is generally considered to mediate aggregation of FUS, we find that arginine residues in the C-terminal low-complexity domain are also required for maturation of FUS in cellular stress granules. These data highlight an important role for arginine-rich domains in the pathology of RNA-binding proteins., Graphical Abstract, Highlights • Both QGSY and RGG2 domains are necessary for FUS-induced neurodegeneration in flies • Arginine-rich domains interact with QGSY hydrogels and liquid droplets • RGG2 arginines promote phase separation of FUS in vitro and in cells • FUS phase separation behavior in vitro correlates with neurodegeneration in vivo, Protein aggregation is a hallmark of ALS. Bogaert et al. describe the molecular interactions between disordered regions of the FUS protein driving its liquid phase behavior, maturation, and neurotoxicity. These findings highlight the physicochemical interactions driving FUS phase separation and give us insights into its misregulation in disease.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases characterized by the progressive loss of specific groups of neurons. Due to clinical, genetic and pathological overlap, both diseases are considered as the extremes of one disease spectrum and in a number of ALS and FTD patients, fused in sarcoma (FUS) aggregates are present. Even in families with a monogenetic disease cause, a striking variability is observed in disease presentation. This suggests the presence of important modifying genes. The identification of disease-modifying genes will contribute to defining clear therapeutic targets and to understanding the pathways involved in motor neuron death. In this study, we established a novel in vivo screening platform in which new modifying genes of FUS toxicity can be identified. Expression of human FUS induced the selective apoptosis of crustacean cardioactive peptide (CCAP) neurons from the ventral nerve cord of fruit flies. No defects in the development of these neurons were observed nor were the regulatory CCAP neurons from the brain affected. We used the number of CCAP neurons from the ventral nerve cord as an in vivo read-out for FUS toxicity in neurons. Via a targeted screen, we discovered a potent modifying role of proteins involved in nucleocytoplasmic transport. Downregulation of Nucleoporin 154 and Exportin1 (XPO1) prevented FUS-induced neurotoxicity. Moreover, we show that XPO1 interacted with FUS. Silencing XPO1 significantly reduced the propensity of FUS to form inclusions upon stress. Taken together, our findings point to an important role of nucleocytoplasmic transport proteins in FUS-induced ALS/FTD. ispartof: HUMAN MOLECULAR GENETICS vol:27 issue:23 pages:1-14 ispartof: location:England status: Published online

The exact mechanism underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) associated with the GGGGCC repeat expansion in C9orf72 is still unclear. Two gain-of-function mechanisms are possible: repeat RNA toxicity and dipeptide repeat protein (DPR) toxicity. We here dissected both possibilities using a zebrafish model for ALS. Expression of two DPRs, glycine-arginine and proline-arginine, induced a motor axonopathy. Similarly, expanded sense and antisense repeat RNA also induced a motor axonopathy and formed mainly cytoplasmic RNA foci. However, DPRs were not detected in these conditions. Moreover, stop codon-interrupted repeat RNA still induced a motor axonopathy and a synergistic role of low levels of DPRs was excluded. Altogether, these results show that repeat RNA toxicity is independent of DPR formation. This RNA toxicity, but not the DPR toxicity, was attenuated by the RNA-binding protein Pur-alpha and the autophagy-related protein p62. Our findings demonstrate that RNA toxicity, independent of DPR toxicity, can contribute to the pathogenesis of C9orf72-associated ALS/FTD. ispartof: Acta Neuropathologica vol:135 issue:3 pages:427-443 ispartof: location:Germany status: published

Patients with Charcot-Marie-Tooth disease with predominant axonal loss (CMT2) show extensive genetic heterogeneity. Benoy et al. demonstrate a link between CMT2 and histone deacetylase 6 (HDAC6), which controls the acetylation of α-tubulin, and propose that pharmacological inhibition of HDAC6 has therapeutic potential in CMT2 genetic variants., Peripheral nerve axons require a well-organized axonal microtubule network for efficient transport to ensure the constant crosstalk between soma and synapse. Mutations in more than 80 different genes cause Charcot-Marie-Tooth disease, which is the most common inherited disorder affecting peripheral nerves. This genetic heterogeneity has hampered the development of therapeutics for Charcot-Marie-Tooth disease. The aim of this study was to explore whether histone deacetylase 6 (HDAC6) can serve as a therapeutic target focusing on the mutant glycyl-tRNA synthetase (GlyRS/GARS)-induced peripheral neuropathy. Peripheral nerves and dorsal root ganglia from the C201R mutant Gars mouse model showed reduced acetylated α-tubulin levels. In primary dorsal root ganglion neurons, mutant GlyRS affected neurite length and disrupted normal mitochondrial transport. We demonstrated that GlyRS co-immunoprecipitated with HDAC6 and that this interaction was blocked by tubastatin A, a selective inhibitor of the deacetylating function of HDAC6. Moreover, HDAC6 inhibition restored mitochondrial axonal transport in mutant GlyRS-expressing neurons. Systemic delivery of a specific HDAC6 inhibitor increased α-tubulin acetylation in peripheral nerves and partially restored nerve conduction and motor behaviour in mutant Gars mice. Our study demonstrates that α-tubulin deacetylation and disrupted axonal transport may represent a common pathogenic mechanism underlying Charcot-Marie-Tooth disease and it broadens the therapeutic potential of selective HDAC6 inhibition to other genetic forms of axonal Charcot-Marie-Tooth disease.

Hexanucleotide repeat expansions in C9orf72 are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) (c9ALS/FTD). Unconventional translation of these repeats produces dipeptide repeat proteins (DPRs) that may cause neurodegeneration. We performed a modifier screen in Drosophila and discovered a critical role for importins and exportins, Ran-GTP cycle regulators, nuclear pore components and arginine methylases in mediating DPR toxicity. These findings provide evidence for an important role for nucleocytoplasmic transport in the pathogenic mechanism of c9ALS/FTD.

Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease. One of the proposed disease mechanisms is excitotoxicity, in which excessive cytosolic calcium causes neuronal death. Although most calcium may originate from the extracellular space through activation of calcium-permeable AMPA receptors, we investigated in this study the contribution of endoplasmic reticulum calcium release by blocking the ryanodine receptor (RyR) using dantrolene. In vitro , dantrolene provides a significant protection to motor neurons exposed to a brief excitotoxic insult. However, daily administration of dantrolene to mice overexpressing superoxide dismutase 1 glycine to alanine at position 93 (SOD1 G93A ) does affect neither survival nor the number of motor neurons and ubiquitin aggregates indicating that calcium release through RyRs does not contribute to the selective motor neuron death in this animal model for ALS.

Influx of Ca(2+) ions through the α-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptors is toxic to neurons and contributes to motor neuron degeneration observed in amyotrophic lateral sclerosis (ALS). The Ca(2+) permeability of the AMPA receptor depends on its subunit composition. If the GluR2 subunit is present in the receptor complex, the AMPA receptor is impermeable to Ca(2+). In this study, we identified vascular endothelial growth factor-A (VEGF) as a GluR2 inducing molecule. Cultured motor neurons pretreated with VEGF displayed higher GluR2 levels. This resulted in AMPA receptor currents with a low relative Ca(2+) permeability and in motor neurons that were less vulnerable to AMPA receptor-mediated excitotoxicity. This effect of VEGF was mediated through the VEGFR2 present on the motor neurons and was due to stimulation of GluR2 transcription. Intracerebroventricular treatment with VEGF similarly induced GluR2 expression in the ventral spinal cord of rats and this mechanism contributes to the protective effect of VEGF on motor neurons.

Influx of Ca 2+ ions through α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors contributes to neuronal damage in stroke, epilepsy, and neurodegenerative disorders such as ALS. The Ca 2+ permeability of AMPA receptors is largely determined by the glutamate receptor 2 (GluR2) subunit, receptors lacking GluR2 being permeable to Ca 2+ ions. We identified a difference in GluR2 expression in motor neurons from two rat strains, resulting in a difference in vulnerability to AMPA receptor-mediated excitotoxicity both in vitro and in vivo . Astrocytes from the ventral spinal cord were found to mediate this difference in GluR2 expression in motor neurons. The presence of ALS-causing mutant superoxide dismutase 1 in astrocytes abolished their GluR2-regulating capacity and thus affected motor neuron vulnerability to AMPA receptor-mediated excitotoxicity. These results reveal a mechanism through which astrocytes influence neuronal functioning in health and disease.

Both in mice and humans, low expression levels of vascular endothelial growth factor (VEGF) are linked to adult-onset motor neuron disease or amyotrophic lateral sclerosis (ALS). The mechanism through which reduced VEGF levels result in this phenotype is unknown. We therefore examined the direct effects of VEGF on motor neurons and found VEGF to have a direct neurotrophic effect on motor neurons in vitro. Survival and vulnerability to excitotoxicity of motor neurons from VEGF(delta/delta) mice was however similar to that of motor neurons from non-transgenic littermates. The VEGF concentration in the spinal cord of mutant (G93A) SOD1 mice was not different from that found in wild-type SOD1 overexpressing mice. Upregulation of VEGF in the spinal cord, by housing mutant (G93A) SOD1 mice in hypoxic conditions, did not affect their life span. Our results show that VEGF is a neurotrophic factor for motor neurons in vitro, and shortage of this neurotrophic factor may contribute to the motor neuron death observed in humans and animals with low VEGF expression levels.

Progranulin (PGRN) is a growth factor involved in wound healing, inflammation, tumor growth, and neurodegeneration. Mutations in the gene encoding PGRN give rise to shortage of PGRN and cause familial frontotemporal lobar degeneration. PGRN exerts neurotrophic functions and binding of PGRN to the membrane receptor sortilin (SORT1) mediates the endocytosis of PGRN. SORT1-mediated uptake plays an important role in the regulation of extracellular PGRN levels. We studied the role of SORT1 in PGRN-mediated neuroprotection in vitro and in vivo. The survival-enhancing effect of PGRN seemed to be dependent on the granulin E (GRN E) domain. Pharmacologic inhibition of the GRN E-SORT1 interaction or deletion of the SORT1 binding site of GRN E did not abolish its neurotrophic function. In addition, the in vivo phenotype of PGRN knockdown in zebrafish embryos was not phenocopied by SORT1 knockdown. These results suggest that GRN E mediates the neurotrophic properties of PGRN and that binding to SORT1 is not required for this effect.