SummaryThe mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.

To investigate lipid rafts as a site where amyloid ␤ protein (A␤) oligomers might accumulate and cause toxicity in Alzheimer's disease (AD), we analyzed A␤ in the Tg2576 transgenic mouse model of AD. A␤ was highly concentrated in lipid rafts, which comprise a small fraction of brain volume but contain 27% of brain A␤42 and 24% of A␤40 in young mice. In the Tg2576 model, memory impairment begins at 6 months before amyloid plaques are visible. Here we show that A␤ dimers appear in lipid rafts at 6 months and that raft A␤, which is primarily dimeric, rapidly accumulates reaching levels Ͼ500ϫ those in young mice by 24 -28 months. A similar large accumulation of dimeric A␤ was observed in lipid rafts from AD brain. In contrast to extracellular amyloid fibrils, which are SDS-insoluble, virtually all A␤ in lipid rafts is SDS soluble. Coupled with recent studies showing that synthetic and naturally occurring A␤ oligomers can inhibit hippocampal long-term potentiation, the in vivo age-dependent accumulation of SDS-soluble A␤ dimers in lipid rafts at the time when memory impairment begins in Tg2576 mice provides strong evidence linking A␤ oligomers to memory impairment. After dimeric A␤ began to accumulate in lipid rafts of the Tg2576 brain, apolipoprotein E (ApoE) and then phosphorylated tau accumulated. A similar increase in ApoE and a large increase in phosphorylated tau was observed in lipid rafts from AD brain. These findings suggest that lipid rafts may be an important site for interaction between dimeric A␤, ApoE, and tau.

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It is unclear whether mutations in fused in sarcoma (FUS) cause familial amyotrophic lateral sclerosis via a loss-of-function effect due to titrating FUS from the nucleus or a gain-of-function effect from cytoplasmic overabundance. To investigate this question, we generated a series of independent Caenorhabditis elegans lines expressing mutant or wild-type (WT) human FUS. We show that mutant FUS, but not WT-FUS, causes cytoplasmic mislocalization associated with progressive motor dysfunction and reduced lifespan. The severity of the mutant phenotype in C. elegans was directly correlated with the severity of the illness caused by the same mutation in humans, arguing that this model closely replicates key features of the human illness. Importantly, the mutant phenotype could not be rescued by overexpression of WT-FUS, even though WT-FUS had physiological intracellular localization, and was not recruited to the cytoplasmic mutant FUS aggregates. Our data suggest that FUS mutants cause neuronal dysfunction by a dominant gain-of-function effect related either to neurotoxic aggregates of mutant FUS in the cytoplasm or to dysfunction in its RNA-binding functions.

We examined the distribution of Pael-R, a newly identified substrate for Parkin, in Parkinson's disease (PD) and multiple system atrophy (MSA). Pael-R, Parkin, alpha-synuclein, and ubiquitin accumulated in Lewy bodies (LBs) and neurites. Pael-R was localized in the core of LBs. Parkin and alpha-synuclein accumulated in the halo, neuronal cell bodies, and processes. These findings potentially suggest the involvement of Pael-R in LB formation, and protection role of Parkin in Pael-R-mediated neurotoxicity in PD. The absence of Pael-R and Parkin in glial cytoplasmic inclusions (GCIs) in MSA implies a distinct pathway involved in the formation of LBs and GCIs.

Missense mutations of the tau gene cause autosomal dominant frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an illness characterized by progressive personality changes, dementia, and parkinsonism. There is prominent frontotemporal lobe atrophy of the brain accompanied by abundant tau accumulation with neurofibrillary tangles and neuronal cell loss. Using a hamster prion protein gene expression vector, we generated several independent lines of transgenic (Tg) mice expressing the longest form of the human four-repeat tau with the R406W mutation associated with FTDP-17. The TgTauR406W 21807 line showed tau accumulation beginning in the hippocampus and amygdala at 6 months of age, which subsequently spread to the cortices and subcortical areas. The accumulated tau was phosphorylated, ubiquitinated, conformationally changed, argyrophilic, and sarcosyl-insoluble. Activation of GSK-3beta and astrocytic induction of mouse tau were observed. Astrogliosis and microgliosis correlated with prominent tau accumulation. Electron microscopic examination revealed the presence of straight filaments. Behavioral tests showed motor disturbances and progressive acquired memory loss between 10 to 12 months of age. These findings suggested that TgTauR406W mice would be a useful model in the study of frontotemporal dementia and other tauopathies such as Alzheimer's disease (AD).

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The purpose of this study was to examine mitochondrial changes in the spinal cord of transgenic mice of a relatively low transgenic copy number (gene copy 10) expressing a G93A mutant human Cu/Zn superoxide dismutase (SOD1) that were generated in our own laboratories by electron and immunoelectron microscopy from presymptomatic to symptomatic stages. Age-matched non-transgenic mice served as controls at each stage. Ultrastructurally, at the early presymptomatic stage, many mitochondria in large myelinated axons exhibited swelling with an increased number of cristae, and bore small vacuoles in the matrix, cristae or both, in the anterior root exit zone, anterior root, and in the neuropils of the ventral portion of the anterior horn. At the late presymptomatic stage, vacuoles of various sizes (including large ones) were observed in the same regions as in the previous stage. The intermembrane space of mitochondria was also vacuolated. In mitochondria with advanced vacuolation, the vacuolar space was filled with a granular or amorphous substance. At the symptomatic stage, mitochondrial vacuolation seen in the late presymptomatic stage persisted, although to a lesser extent. These vacuolated mitochondria were predominantly seen in the axons, but not in the somata of normal-looking neurons or dendrites at any stage, which differs from that described in other reports. Non-transgenic littermates occasionally exhibited vacuolar changes in the axons of anterior horns. However, they were smaller both in size and number than those in transgenic mice. By immunoelectron microscopy using an immunogold labeling method, at the presymptomatic and symptomatic stages both SOD1 and ubiquitin determinants were localized in vacuolated mitochondria, particularly in the granular or amorphous substance of large vacuoles, but were not detected in most normal-appearing mitochondria. The SOD1-immunoreactive mitochondria were exclusively observed in the axons, and not in proximal dendrites or somata. These findings suggest that the toxicity of mutant SOD1 directly affects mitochondria in the axons and increases with the disease progression. Thus, the mutant SOD1 toxicity might disrupt axonal transport of substrates needed for neuronal viability, leading to motor neuron degeneration. The localization of both ubiquitin and SOD1 in vacuolated mitochondria indicates that protein degradation by ubiquitin-proteasomal system may be also disrupted by several pathomechanisms, such as decreased processing of ubiquitinated proteins due to impairment of mitochondrial function or of proteasomal function, both of which are caused by mutant SOD1. Moreover, giant mitochondrial vacuoles occupying almost the entire axonal caliber could be another contributing factor in motor neuron degeneration, in that they could physically block axonal transport.