Dipeptide repeat peptides on the attack: Certain neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), are associated with expanded dipeptides translated from RNA transcripts of disease-associated genes (see the Perspective by West and Gitler). Kwon et al. show that the peptides encoded by the expanded repeats in the C9orf72 gene interfere with the way cells make RNA and kill cells. These effects may account for how this genetic form of ALS causes disease. Working in Drosophila , Mizielinska et al. aimed to distinguish between the effects of repeat-containing RNAs and the dipeptide repeat peptides that they encode. The findings provide evidence that dipeptide repeat proteins can cause toxicity directly. Science , this issue p. 1139 and p. 1192 ; see also p. 1118

Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinson's disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation ofPsmc1(Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome andPsmc1conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and α-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinson's disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.

Mutations in FUS are causative for amyotrophic lateral sclerosis with a dominant mode of inheritance. In trying to model FUS-amyotrophic lateral sclerosis (ALS) in mouse it is clear that FUS is dosage-sensitive and effects arise from overexpression per se in transgenic strains. Novel models are required that maintain physiological levels of FUS expression and that recapitulate the human disease—with progressive loss of motor neurons in heterozygous animals. Here, we describe a new humanized FUS-ALS mouse with a frameshift mutation, which fulfils both criteria: the FUS Delta14 mouse. Heterozygous animals express mutant humanized FUS protein at physiological levels and have adult onset progressive motor neuron loss and denervation of neuromuscular junctions. Additionally, we generated a novel antibody to the unique human frameshift peptide epitope, allowing specific identification of mutant FUS only. Using our new FUSDelta14 ALS mouse-antibody system we show that neurodegeneration occurs in the absence of FUS protein aggregation. FUS mislocalization increases as disease progresses, and mutant FUS accumulates at the rough endoplasmic reticulum. Further, transcriptomic analyses show progressive changes in ribosomal protein levels and mitochondrial function as early disease stages are initiated. Thus, our new physiological mouse model has provided novel insight into the early pathogenesis of FUS-ALS.

Liquid–liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease.

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease resulting from a complex interplay between genetics and environment. Impairments in axonal transport have been identified in several ALS models, but in vivo evidence remains limited, thus their pathogenetic importance remains to be fully resolved. We therefore analyzed the in vivo dynamics of retrogradely transported, neurotrophin-containing signaling endosomes in nerve axons of two ALS mouse models with mutations in the RNA processing genes TARDBP and FUS. TDP-43M337V mice, which show neuromuscular pathology without motor neuron loss, display axonal transport perturbations manifesting between 1.5 and 3 months and preceding symptom onset. Contrastingly, despite 20% motor neuron loss, transport remained largely unaffected in FusΔ14/+ mice. Deficiencies in retrograde axonal transport of signaling endosomes are therefore not shared by all ALS-linked genes, indicating that there are mechanistic distinctions in the pathogenesis of ALS caused by mutations in different RNA processing genes.