Shu Hui Yen

Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein.

Neurofibrillary tangles (NFT) composed of the microtubule-associated protein tau are prominent in Alzheimer disease (AD), Pick disease, progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Mutations in the gene (Mtapt) encoding tau protein cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), thereby proving that tau dysfunction can directly result in neurodegeneration. Expression of human tau containing the most common FTDP-17 mutation (P301L) results in motor and behavioural deficits in transgenic mice, with age- and gene-dose-dependent development of NFT. This phenotype occurred as early as 6.5 months in hemizygous and 4.5 months in homozygous animals. NFT and Pick-body-like neuronal lesions occurred in the amygdala, septal nuclei, pre-optic nuclei, hypothalamus, midbrain, pons, medulla, deep cerebellar nuclei and spinal cord, with tau-immunoreactive pre-tangles in the cortex, hippocampus and basal ganglia. Areas with the most NFT had reactive gliosis. Spinal cord had axonal spheroids, anterior horn cell loss and axonal degeneration in anterior spinal roots. We also saw peripheral neuropathy and skeletal muscle with neurogenic atrophy. Brain and spinal cord contained insoluble tau that co-migrated with insoluble tau from AD and FTDP-17 brains. The phenotype of mice expressing P301L mutant tau mimics features of human tauopathies and provides a model for investigating the pathogenesis of diseases with NFT.

Mutations in tau reduce its microtubule binding properties in intact cells and affect its phosphorylation.

In vitro evidence has suggested a change in the ability of tau bearing mutations associated with fronto‐temporal dementia to promote microtubule assembly. We have used a cellular assay to quantitate the effect of both isoform differences and mutations on the physiological function of tau. Whilst all variants of tau bind to microtubules, microtubule extension is reduced in cells transfected with 3‐relative to 4‐repeat tau. Mutations reduce microtubule extension with the P301L mutation having a greater effect than the V337M mutation. The R406W mutation had a small effect on microtubule extension but, surprisingly, tau with this mutation was less phosphorylated in intact cells than the other variants.

Multiple system atrophy: A sporadic synucleinopathy

Multiple system atrophy (MSA) is a sporadic neurodegenerative disease characterized clinically by varying degrees of Parkinsonism, cerebellar ataxia and autonomic dysfunction and pathologically by degeneration in the substantia nigra, putamen, olivary nucleus, pontine nuclei and cerebellum. In addition to selective neuronal loss, iron pigment accumulation and gliosis, myelin pathology is increasingly recognized. In affected white matter, myelin displays signs of degeneration and oligodendroglia contain argyrophilic inclusion bodies, so‐called glial cytoplasmic inclusions (GCI). GCI are composed of 10–15‐nm diameter coated filaments that are immunoreactive for ubiquitin and α‐synuclein. Similar inclusions are occasionally found in neuronal cell bodies and cell processes in MSA. Given the presence of inclusion bodies composed of synuclein, it is reasonable to assume that biochemical alterations would be detected in synuclein in MSA and indeed this is the case. In MSA synuclein has biophysical properties that suggest increasing insolubility such as sedimentation in dense fractions in sucrose gradients and ready extraction into detergents and formic acid. Surprisingly, these biochemical modifications in synuclein are more widespread in the brain that the obvious pathology and suggest a fundamental molecular characteristic of the disorder. Similar neuronal, and less frequently glial, inclusions are detected in Lewy body disease, where there is also evidence for biophysical alterations in synuclein. Thus, MSA and LBD are both synucleinopathies, and they may comprise different poles of a disease spectrum that includes sporadic disorders as well as genetically determined disorders such as familial Lewy body Parkinsonism.

Autophagic‐lysosomal perturbation enhances tau aggregation in transfectants with induced wild‐type tau expression

The intracellular assembly of tau aggregates is a pathological hallmark shared by Alzheimers disease and other neurodegenerative disorders known collectively as tauopathies. To model how tau fibrillogenesis evolves in tauopathies, we previously established transfectant M1C cultures from human neuroblastoma BE(2)‐M17D cells that inducibly express human tau. In the present study, these cells were used to determine the role of the autophagic‐lysosomal system in the degradation and aggregation of wild‐type tau. Tau induction for 5 days led to the accumulation of tau with nominal assembly of tau aggregates within cells. When the lysosomotropic agent, chloroquine (CQ), was added following the termination of tau induction, tau clearance was delayed. Decreased tau truncation and increased levels of intact tau were observed. When present during tau induction, CQ led to tau accumulation and promoted the formation of sarkosyl‐insoluble aggregates containing both truncated and full‐length tau. CQ treatment significantly decreased the activities of cathepsins D, B and L, and the inhibition of cathepsins B and L mimicked the effect of CQ and increased tau levels in cells. Additionally, exposure of cells to the autophagy inhibitor, 3‐methyladenine, led to tau accumulation and aggregation. These results suggest that the autophagic‐lysosomal system plays a role in the clearance of tau, and that dysfunction of this system results in the formation of tau oligomers and insoluble aggregates.

Distinctive neuropathology revealed by alpha-synuclein antibodies in hereditary parkinsonism and dementia linked to chromosome 4p.

Abstract The identification of the α-synuclein gene on chromosome 4q as a locus for familial Lewy-body parkinsonism and of α-synuclein as a component of Lewy bodies has heralded a new era in the study of Parkinson’s disease. We have identified a large family with Lewy body parkinsonism linked to a novel locus on chromosome 4p15 that does not have a mutation in the α-synuclein gene. Here we report the clinical and neuropathological findings in an individual from this family and describe unusual high molecular weight α-synuclein-immunoreactive proteins in brain homogenates from brain regions with the most marked neuropathology. Distinctive histopathology was revealed with α-synuclein immunostaining, including pleomorphic Lewy bodies, synuclein-positive glial inclusions and widespread, severe neuritic dystrophy. We also discuss the relationship of this familial disorder to a Lewy body disease clinical spectrum, ranging from Parkinson’s disease to dementia with psychosis.

Phosphate analysis and dephosphorylation of modified tau associated with paired helical filaments.

We performed phosphate analysis of tau proteins isolated from normal human brain, tau proteins associated with paired helical filaments (PHF-tau), and Alzheimer tau not associated with PHF. These tau fractions were of high purity. Normal and Alzheimer tau were purified by heat treatment, acid extraction and calmodulin-affinity chromatography with or without HPLC. Fractions containing primarily PHF-tau polypeptides of 60, 64 and 68 kDa and their degraded fragments were purified either on a sucrose density gradient as filaments (PHF) or by heat treatment and acid extraction as amorphous proteins (PHF-tau). PHF and PHF-tau were found to contain 6-8 mol phosphate/mol protein while normal and Alzheimer tau proteins contained 1.9 and 2.6 mol phosphate/mol protein, respectively. Upon 2-h incubation with alkaline phosphatase, PHF lost two of the phosphate groups without apparent changes in the stability and morphology of PHF. The released phosphate originated from the N-terminal half of PHF-tau as determined by immunoblotting with antibodies to epitopes blocked by phosphorylation. Tau-1 and E-2, and by a prominent shift in the electrophoretic mobility of some fragments of PHF-tau. The shift in mobility was not observed with the C-terminal fragments of 25-26 kDa, which retained the epitope to Tau 46. The results suggest that the phosphorylation sites not affected by phosphatase may be located in the 25-26 kDa C-terminal region of PHF-tau and may play a role in structural stability of PHF.

Structural stability of paired helical filaments requires microtubule-binding domains of tau: a model for self-association.

Highly purified and SDS-soluble paired helical filaments (PHFs) were immunogold labeled and immunoblotted with antibodies to tau: Tau 14 (N-terminal half), AH-1 (microtubule-binding domain), and Tau 46 (C-terminal end). The main component of PHFs was modified tau of 68, 64, and 60 kd, also called A68 or PHF-tau. Trypsin digestion reduced the maximum width of PHFs by 10%-20%, increased aggregation of filaments, and abolished the binding of Tau 14, but had no effect on the binding of AH-1. The smallest tau-reactive tryptic fragments were 13 and 7-8 kd, positive with AH-1, and negative with Tau 46. Our results and the model of Crowther and Wischik suggest that by self-association and anti-parallel arrangement of the microtubule-binding domains, PHF-tau forms the backbone of PHFs.

Assembly of two distinct dimers and higher‐order oligomers from full‐length tau

Abnormal accumulation of tau as filamentous structures is a neuropathological hallmark of neurodegenerative diseases referred to as tauopathies. Little is known about the role of native cysteine residues in tau assembly because their substitution with other amino acids has no effect on tau filament morphology. To understand the process involved in tau oligomerization, we analysed both heparin‐induced assembly of different forms of recombinant human tau and assembly of tau from COS‐7 cells transiently expressing different human tau constructs. Here, we demonstrated that tau assembly involves two distinct dimers (cysteine‐dependent and cysteine‐independent) that differ in resistance to reduction. During assembly, an increase of cysteine‐dependent tau oligomer was observed prior to detection of increased thioflavin T fluorescence signals. The latter event was accompanied by an increase of cysteine‐independent dimer. Fewer higher‐order oligomers and aggregates were assembled from four‐repeat tau containing two amino‐terminus inserts that have either the C291A/C322A mutation (cysless‐4R2N) or a hexapeptide deletion at residues 306–311 (ΔPHF6–4R2N) compared with those assembled from wild‐type tau. Assembly of distinct types of dimers was also observed in lysates from COS‐7 cells expressing wild‐type 4R2N and brain extracts from mice expressing P301L mutant tau. In contrast, COS‐7 cells expressing cysless‐ or ΔPHF6–4R2N tau contained very little cysteine‐dependent dimer. Together, the results indicate that intermolecular disulfide crosslinking along with PHF6 hexapeptide facilitates tau oligomerization and that this event is accompanied by cysteine‐independent intermolecular bridging of microtubule‐binding domain, leading to assembly of higher‐order oligomers. The levels of these dimers may be used to gauge the potential for tau assembly.

Assembly of tau in transgenic animals expressing P301L tau: alteration of phosphorylation and solubility.

Transgenic mice (JNPL3), which develop neurofibrillary degeneration and express four‐repeat human tau with P301L missense mutation, were characterized biochemically to determine whether the development of aggregated tau from soluble tau involves an intermediate stage. Homogenates from mice of different ages were separated into buffer‐soluble (S1), sarkosyl‐ and salt‐extractable (S2) and sarkosyl‐insoluble pellet (P3) fractions, and analyzed for human tau distribution, phosphorylation and filament formation. S1 and S2 fractions contained 50–60‐kDa tau whereas the S2 fraction also had 64‐kDa tau. The level of tau in the P3 fraction increased in an age‐dependent manner and correlated positively with the soluble tau concentration. The P3 fraction from 2.5–6.5‐month‐old mice contained 64‐ and 50–60‐kDa tau, whereas that from 8.5‐month and older transgenic animals contained mostly 64‐kDa and higher molecular weight tau. The S2 and P3 fractions contained comparable amounts of 64‐kDa tau. The 64‐kDa tau was predominantly human, and phosphorylated at multiple sites: Thr181, Ser202/Thr205, Thr212, Thr231, Ser262, Ser396/Ser404, Ser409 and Ser422. Most of these sites were phosphorylated to a lesser extent in S2 than in P3 fractions. Tau polymers were detected in P3 fractions from 3‐month and older female JNPL3 mice, but not in non‐transgenic controls. The results suggest that tau in S2 represents an intermediate from which insoluble tau is derived, and that phosphorylation may play a role in filament formation and/or stabilization.

Accumulation of Filamentous Tau in the Cerebral Cortex of Human Tau R406W Transgenic Mice

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 Alzheimers disease (AD).