ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function.

The 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.

ALS mutations in FUS cause neuronal dysfunction and death in Caenorhabditis elegans by a dominant gain-of-function mechanism.

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.

Factors responsible for neurofibrillary tangles and neuronal cell losses in tauopathy.

TgTauP301L mice that overexpress the mutant human tauP301L present in FTDP-17 reproduce neurofibrillary tangles (NFTs), neuronal cell losses, memory disturbance, and substantial phenotypic variation. To demonstrate factors responsible for NFT formation and neuronal cell losses, sets of TgTauP301L for comparison with or without NFTs and neuronal cell losses were studied with oligonucleotide microarrays. Gene expressions were altered in biological pathways, including oxidative stress, apoptosis, mitochondrial fatty acid betaoxidation, inflammatory response pathway, and complement and coagulation cascade pathways. Among 24 altered genes, increased levels of apolipoprotein D (ApoD) and neuronal PAS domain protein 4 (Npas4) and decreased levels of doublecortin (DCX) and potassium channel, voltage-gated, shaker-related subfamily, ß member 1 (Kcnab1) were found in the TgTauP301L with NFTs and neuronal cell losses, Alzheimer's brains, and tauopathy brains. Thus, many biological pathways and novel molecules are associated with NFT formation and neuronal cell losses in tauopathy brains.

Amyloid ß accelerates phosphorylation of tau and neurofibrillary tangle formation in an amyloid precursor protein and tau double-transgenic mouse model.

In Alzheimer's disease, Aß deposits are considered the initial cardinal events that induce tauopathy secondarily. However, the relationship between Aß amyloidosis and tauopathy has not been determined in detail. We produced double transgenic mice, 2×TgTau(+/-) APP(+/-) , by mating Tg2576 mice that exhibit Aß amyloidosis and TgTauP301L mice that show tauopathy, and statistically analyzed the effect of Aß accumulation on tauopathy. There was no significant difference in theprogression of Aß accumulation among 2×TgTau(+/-) APP(+/-) and 1×TgTau(-/-) APP(+/-) , and tau accumulation among 2×TgTau(+/-) APP(+/-) and 1×Tg Tau(+/-) APP(-/-) . The appearance rates of phosphorylated tau developing in neurons and processes were significantly accelerated in 2×TgTau(+/-) APP(+/-) mice compared with those in 1×TgTau(+/-) APP(-/-) mice at 23 months of age. Accumulation of phosphorylated and confomationally altered tau and GSK3ß in neuronal processes was accelerated in the white matter in 2×TgTau(+/-) APP(+/-) . The level of phosphorylated tau in the sarkosyl-insoluble fraction was increased in 2×TgTau(+/-) APP(+/-) brains compared with that in 1×TgTau(+/-) APP(-/-) brains. Thus, Aß amyloid partially enhances tauopathy through accumulation of insoluble, phosphorylated, and conformationally changed tau in neuronal cytoplasm and processes in the late stage.

Changes of Nogo-A and receptor NgR in the lumbar spinal cord of ALS model mice.

Detailed assessment of Nogo-A and its receptor NgR in the spinal cord of amyotrophic lateral sclerosis (ALS) models or patients has not been reported previously, and we examined the expression and distribution patterns of Nogo-A and NgR in an ALS mouse model to determine whether these molecules play a role in this disease. As compared with wild-type (WT) mice, transgenic (Tg) mice showed that the expression levels of Nogo-A transiently increased in motor neurons at an age of 10 weeks old (W), while it progressively decreased from 15 to 18 W. NgR expression in motor neurons of the Tg mice increased at 10 W, then progressively decreased from 15 to 18 W. In contrast, there was no significant change in the dorsal lumbar cord or the cerebellum of Tg mice throughout the progression of ALS. This study suggests that the function of Nogo-A may alter under certain conditions and locations, and thus transient overexpression of Nogo-A and NgR in motor neurons of this ALS mouse model at 10 W may represent a survival reaction of these cells under stressful conditions.

Motor impairment and aberrant production of neurochemicals in human alpha-synuclein A30P+A53T transgenic mice with alpha-synuclein pathology.

Missense point mutations, duplication and triplication in the alpha-synuclein (alphaSYN) gene have been identified in familial Parkinson's disease (PD). Familial and sporadic PD show common pathological features of alphaSYN pathologies, e.g., Lewy bodies (LBs) and Lewy neurites (LNs), and a loss of dopaminergic neurons in the substantia nigra that leads to motor disturbances. To elucidate the mechanism of alphaSYN pathologies, we generated TgalphaSYN transgenic mice overexpressing human alphaSYN with double mutations in A30P and A53T. Human alphaSYN accumulated widely in neurons, processes and aberrant neuronal inclusion bodies. Sarcosyl-insoluble alphaSYN, as well as phosphorylated, ubiquitinated and nitrated alphaSYN, was accumulated in the brains. Significantly decreased levels of dopamine (DA) were recognized in the striatum. Motor impairment was revealed in a rotarod test. Thus, TgalphaSYN is a useful model for analyzing the pathological cascade from aggregated alphaSYN to motor disturbance, and may be useful for drug trials.

Early detachment of neuromuscular junction proteins in ALS mice with SODG93A mutation.

The transgenic animals with mutant copper/zinc superoxide dismutase (SOD1) DNA develop paralytic motor neuron disease resembling human amyotrophic lateral sclerosis (ALS) patients and are commonly used as models for ALS. In the transgenic (Tg) mice with the G93A mutation of the human SOD1 gene SOD1(G93A) mice), the loss of ventral root axons and the synapses between the muscles and the motor neurons suggested that the motor neuron degeneration might proceed in a dying-back degeneration pattern. To reveal the relationship between axonal degeneration and the progression of the muscle atrophy in the SOD1(G93A) mice, we investigated the status of the neuromuscular junction along the disease progression. As a presynaptic or postsynaptic marker of neuromuscular junction (NMJ), anti-synaptic vesicle protein 2 (anti-SV2) antibody and α-bungarotoxin (α-BuTX) were chosen in this study and, as a marker of synaptic cleft, anti-agrin antibody was chosen in this study. In the immunohistochemistry of α-BuTX and anti-SV2 antibody, the percentages of double positive NMJs among α-BuTX single positive were decreased in Tg mice through time from ten weeks. The number of postsynaptic acethylcholine receptor (AChR) clusters did not decrease in Tg mice even at the end stage. Immunohistochemistry of α-BuTX and anti-agrin antibody revealed that the increase of immunopositive area of anti-agrin antibody around the muscle fiber in Tg mice from ten weeks of age. In this study, we revealed that the detachment of nerve terminals started at ten weeks in Tg mice. The levels of AChR did not change throughout 5-20 weeks of age in both groups of mice, and AChR remains clustering at NMJs, suggesting that the muscle abnormality is the result of detachment of nerve terminals.

Differentiation of PA from early PSP with different patterns of symptoms and CBF reduction.

OBJECTIVE: To clarify the features of pure akinesia (PA) and progressive supranuclear palsy (PSP) in the early stage of disease. METHODS: We investigated 15 PA and 41 PSP patients' clinical and radiologic features including head MRI, ethyl cysteinate dimmer-single photon emission-computed tomography (ECD-SPECT) and iodine-123 meta-iodobenzyl guanidine (123I-MIBG) myocardial scintigraphy. In ECD-SPECT study, cerebral blood flow (CBF) reduction was quantitatively expressed as Z-score, and that in the frontal lobe was evaluated. RESULTS: Many PSP patients claimed falls as the initial symptom but no PA patients did. Eye movement, as well as optokinetic nystagmus elicitation, was more frequently disturbed in PSP. Dementia, dysarthria and rigidity were also more frequent in PSP than in PA. Midbrain tegmentum atrophy in head MRI was more frequently observed in PSP. CBF in the frontal lobe, especially in the frontal eye field, was significantly lower in PSP than in PA. MIBG myocardial scintigraphy showed no difference between two groups. DISCUSSION: PA and PSP show distinct symptoms from the early stage, indicating that they are distinct disorders. The occurrence of falls and eye movement disturbance, as well as CBF reduction at the frontal eye field, is very important for distinguishing these disorders.

Plasma antibodies to Abeta40 and Abeta42 in patients with Alzheimer's disease and normal controls.

Antibodies to amyloid beta protein (Abeta) are present naturally or after Abeta vaccine therapy in human plasma. To clarify their clinical role, we examined plasma samples from 113 patients with Alzheimer's disease (AD) and 205 normal controls using the tissue amyloid plaque immunoreactivity (TAPIR) assay. A high positive rate of TAPIR was revealed in AD (45.1%) and age-matched controls (41.2%), however, no significance was observed. No significant difference was observed in the MMS score or disease duration between TAPIR-positive and negative samples. TAPIR-positive plasma reacted with the Abeta40 monomer and dimer, and the Abeta42 monomer weakly, but not with the Abeta42 dimer. TAPIR was even detected in samples from young normal subjects and young Tg2576 transgenic mice. Although the Abeta40 level and Abeta40/42 ratio increased, and Abeta42 was significantly decreased in plasma from AD groups when compared to controls, no significant correlations were revealed between plasma Abeta levels and TAPIR grading. Thus an immune response to Abeta40 and immune tolerance to Abeta42 occurred naturally in humans without a close relationship to the Abeta burden in the brain. Clarification of the mechanism of the immune response to Abeta42 is necessary for realization of an immunotherapy for AD.

Therapeutic benefits of intrathecal protein therapy in a mouse model of amyotrophic lateral sclerosis.

When fused with the protein transduction domain (PTD) derived from the human immunodeficiency virus TAT protein, proteins can cross the blood-brain barrier and cell membrane and transfer into several tissues, including the brain, making protein therapy feasible for various neurological disorders. We have constructed a powerful antiapoptotic modified Bcl-X(L) protein (originally constructed from Bcl-X(L)) fused with PTD derived from TAT (TAT-modified Bcl-X(L)), and, to examine its clinical effectiveness in a mouse model of familial amyotrophic lateral sclerosis (ALS), transgenic mice expressing human Cu/Zn superoxide dismutase (SOD1) bearing a G93A mutation were treated by intrathecal infusion of TAT-modified Bcl-X(L). We demonstrate that intrathecally infused TAT-fused protein was effectively transferred into spinal cord neurons, including motor neurons, and that intrathecal infusion of TAT-modified Bcl-X(L) delayed disease onset, prolonged survival, and improved motor performance. Histological studies show an attenuation of motor neuron loss and a decrease in the number of cleaved caspase 9-, cleaved caspase 3-, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the lumbar cords of TAT-modified Bcl-X(L)-treated G93A mice. Our results indicate that intrathecal protein therapy using a TAT-fused protein is an effective clinical tool for the treatment of ALS.

Microglial activation in brain lesions with tau deposits: comparison of human tauopathies and tau transgenic mice TgTauP301L.

The aim of this study is to clarify the relationship of microglia to phosphorylated tau accumulation and the characteristics of microglial activation in brain lesions of human tauopathies in comparison to mutant tau transgenic (TG) mice. We performed immunocytochemical analyses of brains from six patients with tauopathies, and 24 mice (18 TG mice expressing mutant tau P301L and six non-TG control mice, 11 to 27 months of age) using anti-tau antibodies and various microglial markers. In the tau TG, both semiquantitative severity ratings of microglial activation and an ultrastructural study were performed. In human tauopathies, Iba1- and major histocompatibility complex (MHC) class II-positive activated microglia increased in regions of phosphorylated tau (AT8) accumulation. The immunoreactivity of scavenger receptor class A (SRA) was present in some activated microglia, including phagocytic microglia in Alzheimer's disease (AD). Double-immunofluorescent analysis under a confocal microscope showed activated microglia at the vicinity of AT8-positive cells. Semiquantitative data of the TG and control mice indicated that the immunopositivity of AT8 was closely associated with the number of Iba1-positive microglia in the cortical area. Tau-associated microglia showed rare immunoreactivity for MHC class II antigen and SRA in the TG mice. Ultrastructurally, activated microglia with enlarged cytoplasm were located near neurons containing abnormal cytoskeletons. This comparative study of human tauopathies and tau TG mice indicated that microglial activation was closely related to phosphorylated tau accumulation, and that activated microglia of the TG mice may have the low expression of MHC class II and SRA compared with those of human tauopathies.

Elevation of MCP-1 and MCP-1/VEGF ratio in cerebrospinal fluid of amyotrophic lateral sclerosis patients.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with progressive cell death of upper and lower motor neurons. In this study, we measured monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) levels in cerebrospinal fluid (CSF) and serum by enzyme-linked immunosorbent assay (ELISA) in 42 ALS patients, and compared these levels with those of control subjects with other neurodegenerative disorders or with those of normal controls. MCP-1 levels in CSF were significantly higher in ALS patients than in the control group. VEGF levels in CSF tended to be lower in ALS patients than in the control group, but not significantly. A positive correlation was found between MCP-1 levels in CSF of ALS patients and the total Norris scale. The elevation of MCP-1/VEGF ratio in CSF was more specific to ALS patients compared to other neurological diseases such as Parkinson's disease (PD) and spinocerebellar ataxia (SCA) and to controls. Our data suggested that both MCP-1 levels and MCP-1/VEGF ratio in CSF may be useful markers for the clinical diagnosis of ALS.

Increased ER stress during motor neuron degeneration in a transgenic mouse model of amyotrophic lateral sclerosis.

The endoplasmic reticulum (ER), which plays important roles in apoptosis, is susceptible to oxidative stress. ER stress is also thought to be involved in the pathogenesis of neurodegenerative diseases. In this study, we investigated whether ER stress is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) using the anterior part of the lumbar spinal cord of transgenic mice carrying a mutation (G93A) in the superoxide dismutase 1 (SOD1) gene. Western blot and immunohistochemical analyses demonstrated that the expressions of p-PERK and p-eIF2alpha were increased in the microsome fraction (P3) of the lumbar spinal cord at the pre-symptomatic age of 12 weeks (12W), while the expression of activated caspase-12 was increased in the cytoplasmic fraction (S3) of the lumbar spinal cord at both the pre-symptomatic age of 12W and the late symptomatic age of 20W. In contrast, GRP78 did not show any increases in the microsome fraction (P3) of the lumbar spinal cord at either the pre-symptomatic or symptomatic ages. Thus, the present results strongly suggest that the balance between anti- and pro-apoptotic proteins related to ER stress is impaired from the pre-symptomatic stage in this ALS mouse model, and that this imbalance may be related to the pathogenesis of motor neuron degeneration in ALS.

Increased autophagy in transgenic mice with a G93A mutant SOD1 gene.

Autophagy, like the ubiquitin-proteasome system, is considered to play an important role in preventing the accumulation of abnormal proteins. Rat microtubule-associated protein 1 light chain 3 (LC3) is important for autophagy, and the conversion from LC3-I into LC3-II is accepted as a simple method for monitoring autophagy. We examined a SOD1G93A transgenic mouse model for amyotrophic lateral sclerosis (ALS) to consider a possible relationship between autophagy and ALS. In our study we analyzed LC3 and mammalian target of rapamycin (mTOR), a suppressor of autophagy, by immunoassays. The level of LC3-II, which is known to be correlated with the extent of autophagosome formation, was increased in SOD1G93A transgenic mice at symptomatic stage compared with non-transgenic or human wild-type SOD1 transgenic animals. Moreover, the ratio of phosphorylated mTOR/Ser2448 immunopositive motor neurons to total motor neurons was decreased in SOD1G93A-Tg mice. The present data show the possibility of increased autophagy in an animal model for ALS. And autophagy may be partially regulated by an mTOR signaling pathway in these animals.

Two cases of spinocerebellar ataxia accompanied by involvement of the skeletal motor neuron system and bulbar palsy.

We report two patients with spinocerebellar ataxia (SCA) with cranial and spinal motor neuron involvement. They initially presented with cerebellar ataxia, followed by bulbar palsy and limb motor neuron sign. One of the patients had a brother with allied disorder. SCA type 1 (SCA1), SCA3 and SCA6 have been reported to involve the motor neuron system, but they were excluded by DNA analyses in the present two patients. These two patients may form a distinct disease entity among SCAs.

Enhanced accumulation of phosphorylated alpha-synuclein in double transgenic mice expressing mutant beta-amyloid precursor protein and presenilin-1.

A recent report showed that the accumulation of alpha-synuclein (alpha-syn) was detected in the brains of one-third of Alzheimer's disease and Down syndrome patients. However, the relationship between amyloid-beta protein (Abeta) and alpha-syn remains unclear. We analyzed the relation between the mutation of presenilin-1 (PS-1) and the pathological features of beta-amyloidosis and alpha-synucleinopathy. We generated doubly transgenic mice overexpressing mutant beta-amyloid precursor protein (betaAPP; Tg2576) and mutant PS-1 (PS1L286Vtg; line 198) and analyzed 19 double Tg betaAPP(+)/PS(+) mice at 5-23 months (young to old), 23 age-matched single Tg betaAPP(+)/PS(-) mice, and 11 non-Tg littermates. Immunohistochemical comparison was performed in these three groups by counting the area and the number of alpha-syn- or phosphorylated alpha-syn (palpha-syn)-positive dystrophic neurites per plaque (ASPDN, pASPDN). The acceleration of Abeta pathology was found with earlier onset and exaggerated numbers in double Tg betaAPP(+)/PS(+) compared with single Tg betaAPP(+)/PS(-) mouse brains. The accumulation of ASPDN and pASPDN was also accelerated in double Tg betaAPP(+)/PS(+) compared with single Tg betaAPP(+)/PS(-) mouse brains, especially in pASPDN. The number and area of alpha-syn and palpha-syn, and the ratio of palpha-syn positive neurites were significantly higher in double Tg betaAPP(+)/PS(+) than in single Tg betaAPP(+)/PS(-) mouse brains in middle-aged and old groups. Additional overexpression of mutant PS-1 accelerated Abeta-induced alpha-synucleinopathy and further facilitated the phosphorylation of alpha-syn, suggesting a direct association between mutant PS-1 and phosphorylation of alpha-syn.

Early decrease of mitochondrial DNA repair enzymes in spinal motor neurons of presymptomatic transgenic mice carrying a mutant SOD1 gene.

Growing evidence has recently shown that mutant SOD1 accumulate in the mitochondria and cause vacuolation in transgenic mice carrying mutant SOD1, an animal model of amyotrophic lateral sclerosis (ALS). In this study, the expressions of DNA repair enzymes, oxoguanine glycosylase 1 (ogg1), DNA polymerase beta (polbeta), and DNA polymerase gamma (polgamma) were examined in transgenic mice with an ALS-linked mutant SOD1 gene, a valuable model for human ALS. In presymptomatic Tg mice, the nuclear form of ogg1 was upregulated, whereas mitochondrial ogg1 remained at the same level. DNA polymerase was selectively downregulated in the mitochondria. This study suggests an impaired protective mechanism against oxidative stress in mitochondria. The expressions of these enzymes are predominant in spinal motor neurons, suggesting a mechanism of selective motor neuron death in this animal model of ALS.

Cortical neuronal and glial pathology in TgTauP301L transgenic mice: neuronal degeneration, memory disturbance, and phenotypic variation.

Recapitulation of tau pathologies in an animal model has been a long-standing goal in neurodegenerative disease research. We generated transgenic (TgTauP301L) mice expressing a frontotemporal dementia with parkinsonism linked to chromosome 17 (FTPD-17) mutation within the longest form of tau (2N, 4R). TgTauP301L mice developed florid pathology including neuronal pretangles, numerous Gallyas-Braak-positive neurofibrillary tangles, and glial fibrillary tangles in the frontotemporal areas of the cerebrum, in the brainstem, and to a lesser extent in the spinal cord. These features were accompanied by gliosis, neuronal loss, and cerebral atrophy. Accumulated tau was hyperphosphorylated, conformationally changed, ubiquitinated, and sarkosyl-insoluble, with electron microscopy demonstrating wavy filaments. Aged TgTauP301L mice exhibited impairment in hippocampally dependent and independent behavioral paradigms, with impairments closely related to the presence of tau pathologies and levels of insoluble tau protein. We conclude that TgTauP301L mice recreate the substantial phenotypic variation and spectrum of pathologies seen in FTDP-17 patients. Identification of genetic and/or environmental factors modifying the tau phenotype in these mice may shed light on factors modulating human tauopathies. These transgenic mice may aid therapeutic development for FTDP-17 and other diseases featuring accumulations of four-repeat tau, such as Alzheimer's disease, corticobasal degeneration, and progressive supranuclear palsy.

Intrathecal injection of epidermal growth factor and fibroblast growth factor 2 promotes proliferation of neural precursor cells in the spinal cords of mice with mutant human SOD1 gene.

We investigated three steps of neural precursor cell activation--proliferation, migration, and differentiation--in amyotrophic lateral sclerosis spinal cord treated with intrathecal infusion of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) into the lumbar spinal cord region of normal and symptomatic transgenic (Tg) mice with a mutant human Cu/Zn superoxide dismutase (SOD1) gene. We observed that 5-bromodeoxyuridine (BrdU) + nestin double-labeled neural precursor cells increased in the spinal cords of Tg mice compared with non-Tg mice, with a much greater increase produced by EGF and FGF2 treatment. The number of BrdU + nestin double-labeled cells was larger than that of BrdU + ionized calcium-binding adapter molecule-1 (Iba1), BrdU + glial fibrillary acidic protein (GFAP), or BrdU + highly polysialylated neural cell adhesion molecule (PSA-NCAM) double-labeled cells, but none expressed neuronal nuclear antigen (NeuN). On further analysis of the gray matter of Tg mice, the number of BrdU + nestin and BrdU + PSA-NCAM double-labeled cells increased more in the ventral horns than the dorsal horns, which was again greatly enhanced by EGF and FGF2 treatment. Because neural precursor cells reside close to the ependyma of central canal, the present study suggests that proliferation and migration of neural precursor cells to the ventral horns is greatly activated in symptomatic Tg mice and is further enhanced by EGF and FGF2 treatment and, furthermore, that the neural precursor cells preferentially differentiate into neuronal precursor cells instead of astrocytes in Tg mice with EGF and FGF2 treatment.