R. Anthony Crowther

Transmission and spreading of tauopathy in transgenic mouse brain

Hyperphosphorylated tau makes up the filamentous intracellular inclusions of several neurodegenerative diseases, including Alzheimers disease. In the disease process, neuronal tau inclusions first appear in the transentorhinal cortex from where they seem to spread to the hippocampal formation and neocortex. Cognitive impairment becomes manifest when inclusions reach the hippocampus, with abundant neocortical tau inclusions and extracellular β-amyloid deposits being the defining pathological hallmarks of Alzheimers disease. An abundance of tau inclusions, in the absence of β-amyloid deposits, defines Picks disease, progressive supranuclear palsy, corticobasal degeneration and other diseases. Tau mutations cause familial forms of frontotemporal dementia, establishing that tau protein dysfunction is sufficient to cause neurodegeneration and dementia. Thus, transgenic mice expressing mutant (for example, P301S) human tau in nerve cells show the essential features of tauopathies, including neurodegeneration and abundant filaments made of hyperphosphorylated tau protein. By contrast, mouse lines expressing single isoforms of wild-type human tau do not produce tau filaments or show neurodegeneration. Here we have used tau-expressing lines to investigate whether experimental tauopathy can be transmitted. We show that injection of brain extract from mutant P301S tau-expressing mice into the brain of transgenic wild-type tau-expressing animals induces assembly of wild-type human tau into filaments and spreading of pathology from the site of injection to neighbouring brain regions.

Filamentous α-synuclein inclusions link multiple system atrophy with Parkinson's disease and dementia with Lewy bodies

α-Synuclein forms the major component of Lewy bodies and Lewy neurites, the defining neuropathological characteristics of Parkinsons disease and dementia with Lewy bodies. Here we show that α-synuclein is also the major component of the filamentous inclusions of multiple system atrophy which comprises several neurodegenerative diseases with a shared filamentous pathology in nerve cells and glial cells. These findings provide an unexpected link between multiple system atrophy and Lewy body disorders and establish that α-synucleinopathies constitute a major class of human neurodegenerative disorder.

Abundant Tau Filaments and Nonapoptotic Neurodegeneration in Transgenic Mice Expressing Human P301S Tau Protein

The identification of mutations in the Tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has made it possible to express human tau protein with pathogenic mutations in transgenic animals. Here we report on the production and characterization of a line of mice transgenic for the 383 aa isoform of human tau with the P301S mutation. At 5–6 months of age, homozygous animals from this line developed a neurological phenotype dominated by a severe paraparesis. According to light microscopy, many nerve cells in brain and spinal cord were strongly immunoreactive for hyperphosphorylated tau. According to electron microscopy, abundant filaments made of hyperphosphorylated tau protein were present. The majority of filaments resembled the half-twisted ribbons described previously in cases of FTDP-17, with a minority of filaments resembling the paired helical filaments of Alzheimers disease. Sarkosyl-insoluble tau from brains and spinal cords of transgenic mice ran as a hyperphosphorylated 64 kDa band, the same apparent molecular mass as that of the 383 aa tau isoform in the human tauopathies. Perchloric acid-soluble tau was also phosphorylated at many sites, with the notable exception of serine 214. In the spinal cord, neurodegeneration was present, as indicated by a 49% reduction in the number of motor neurons. No evidence for apoptosis was obtained, despite the extensive colocalization of hyperphosphorylated tau protein with activated MAP kinase family members. The latter may be involved in the hyperphosphorylation of tau.

Synthetic filaments assembled from C‐terminally truncated α‐synuclein

Recently two point mutations in the α‐synuclein gene have been found in familial Parkinsons disease. The characteristic fibrous neuropathological lesions of Parkinsons and other neurodegenerative diseases have been shown to stain strongly with antibodies against α‐synuclein and extracted filaments have been labelled with anti‐α‐synuclein antibodies. In view of the close involvement of α‐synuclein filaments with pathology, it was important to establish an in vitro assembly system. We report here that C‐terminally truncated recombinant α‐synuclein readily assembles into filaments resembling those isolated from diseased brain and suggest that truncation by proteolysis may play a role in the pathological process.

Brain homogenates from human tauopathies induce tau inclusions in mouse brain.

Filamentous inclusions made of hyperphosphorylated tau are characteristic of numerous human neurodegenerative diseases, including Alzheimer’s disease, tangle-only dementia, Pick disease, argyrophilic grain disease (AGD), progressive supranuclear palsy, and corticobasal degeneration. In Alzheimer’s disease and AGD, it has been shown that filamentous tau appears to spread in a stereotypic manner as the disease progresses. We previously demonstrated that the injection of brain extracts from human mutant P301S tau-expressing transgenic mice into the brains of mice transgenic for wild-type human tau (line ALZ17) resulted in the assembly of wild-type human tau into filaments and the spreading of tau inclusions from the injection sites to anatomically connected brain regions. Here we injected brain extracts from humans who had died with various tauopathies into the hippocampus and cerebral cortex of ALZ17 mice. Argyrophilic tau inclusions formed in all cases and following the injection of the corresponding brain extracts, we recapitulated the hallmark lesions of AGD, PSP and CBD. Similar inclusions also formed after intracerebral injection of brain homogenates from human tauopathies into nontransgenic mice. Moreover, the induced formation of tau aggregates could be propagated between mouse brains. These findings suggest that once tau aggregates have formed in discrete brain areas, they become self-propagating and spread in a prion-like manner.

Tau Mutations Cause Frontotemporal Dementias

have recently been identified in over a dozen families Arnold Pick provided the first clinical description of fronwith FTDP-17 (Figure 1). The known exonic mutations totemporal dementia in 1892, and since then this class are missense mutations located in the microtubuleof disease has been shown to account for a significant binding repeat region or close to it (Figure 1a), whereas proportion of dementias. In 1911, Alois Alzheimer dethe intronic mutations are located close to the splicescribed the neuropathological lesions characteristic of donor site of the intron following exon 10, where they Pick’s disease. These so-called Pick bodies contain abdestabilize a predicted RNA stem-loop (Figure 1b) (Hutnormal filaments, which consist of hyperphosphorylated ton et al., 1998; Poorkaj et al., 1998; Spillantini et al., 1998b). microtubule-associated protein tau. They resemble the Exonic mutations in exons 9 (G272V), 12 (V337M), and 13 neurofibrillary lesions described by Alzheimer in 1907 (R406W) affect all six tau isoforms, whereas a mutation in the disease subsequently named after him. Unlike located in exon 10 (P301L) only affects tau isoforms Alzheimer’s disease, Pick’s disease lacks significant amywith four repeats. Recombinant tau proteins with these loid Ab pathology. missense mutations show a reduced ability to promote Frontotemporal dementias occur in familial forms and microtubule assembly, which is more marked for threemore commonly as sporadic cases. Neuropathologirepeat than for four-repeat isoforms (Hasegawa et al., cally, they are characterized by a remarkably circum1998). The likely primary effect of the exonic mutations scribed atrophy of the frontal and temporal lobes of is thus a reduced ability of mutated tau to interact with the cerebral cortex, often with additional, subcortical microtubules. The consequences of a reduced ability to changes. In 1994, an autosomal-dominantly inherited interact with microtubules may be equivalent to a partial familial form of frontotemporal dementia with Parkinsonloss of function, with resultant microtubule destabilizaism was linked to chromosome 17q21.2, the same region tion and deleterious effects on cellular processes such that contains the tau gene (Wilhelmsen et al., 1994). This

EFFECTS OF FRONTOTEMPORAL DEMENTIA FTDP-17 MUTATIONS ON HEPARIN-INDUCED ASSEMBLY OF TAU FILAMENTS

Missense mutations and intronic mutations in the gene for microtubule‐associated protein tau cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP‐17). Most missense mutations have as likely primary effect a reduced ability of tau to interact with microtubules. We report here an additional effect of several missense mutations, namely the stimulation of heparin‐induced filament assembly of recombinant tau, despite the absence of any change in structure indicated by circular dichroism. These findings indicate that missense mutations in tau lead to frontotemporal dementia through potentially multiple mechanisms.

Mutation E46K increases phospholipid binding and assembly into filaments of human α-synuclein

Missense mutations (A30P and A53T) in α‐synuclein and the overproduction of the wild‐type protein cause familial forms of Parkinsons disease and dementia with Lewy bodies. α‐Synuclein is the major component of the filamentous Lewy bodies and Lewy neurites that define these diseases at a neuropathological level. Recently, a third missense mutation (E46K) in α‐synuclein was described in an inherited form of dementia with Lewy bodies. Here, we have investigated the functional effects of this novel mutation on phospholipid binding and filament assembly of α‐synuclein. When compared to the wild‐type protein, the E46K mutation caused a significantly increased ability of α‐synuclein to bind to negatively charged liposomes, unlike the previously described mutations. The E46K mutation increased the rate of filament assembly to the same extent as the A53T mutation. Filaments formed from E46K α‐synuclein often had a twisted morphology with a cross‐over spacing of 43 nm. The observed effects on lipid binding and filament assembly may explain the pathogenic nature of the E46K mutation in α‐synuclein.

Tau filaments from human brain and from in vitro assembly of recombinant protein show cross-β structure

Abnormal filaments consisting of hyperphosphorylated microtubule-associated protein tau form in the brains of patients with Alzheimers disease, Downs syndrome, and various dementing tauopathies. In Alzheimers disease and Downs syndrome, the filaments have two characteristic morphologies referred to as paired helical and straight filaments, whereas in tauopathies, there is a wider range of morphologies. There has been controversy in the literature concerning the internal molecular fine structure of these filaments, with arguments for and against the cross-β structure demonstrated in many other amyloid fibers. The difficulty is to produce from brain pure preparations of filaments for analysis. One approach to avoid the need for a pure preparation is to use selected area electron diffraction from small groups of filaments of defined morphology. Alternatively, it is possible to assemble filaments in vitro from expressed tau protein to produce a homogeneous specimen suitable for analysis by electron diffraction, x-ray diffraction, and Fourier transform infrared spectroscopy. Using both these approaches, we show here that native filaments from brain and filaments assembled in vitro from expressed tau protein have a clear cross-β structure.

Tau gene mutation G389R causes a tauopathy with abundant pick body-like inclusions and axonal deposits.

Exonic and intronic mutations in Tau cause familial neurodegenerative syndromes characterized by frontotemporal dementia and dysfunction of multiple cortical and subcortical circuits. Here we describe a G389R mutation in exon 13 of Tau. When 38 years old, the proband presented with progressive aphasia and memory disturbance, followed by apathy, indifference, and hyperphagia. Repeated magnetic resonance imaging showed the dramatic progression of cerebral atrophy. Positron emission tomography revealed marked glucose hypometabolism that was most severe in left frontal, temporal, and parietal cortical regions. Rigidity, pyramidal signs and profound dementia progressed until death at 43 years of age. A paternal uncle, who had died at 43 years of age, had presented with similar symptoms. The probands brain showed numerous tau-immunoreactive Pick body-like inclusions in the neocortex and the fascia dentata of the hippocampus. In addition, large numbers of tau-positive filamentous inclusions were present in axons in the frontal, temporal, and parietal lobes. Immunoblot analysis of sarkosyl-insoluble tau showed 2 major bands of 60 and 64 kDa. Upon dephosphorylation, these bands resolved into 4 bands consisting of three- and four-repeat tau isoforms. Most isolated tau filaments were straight and resembled filaments found in Alzheimer disease and some frontotemporal dementias with tau mutations. A smaller number of twisted filaments was also observed. Biochemically, recombinant tau proteins with the G389R mutation showed a reduced ability to promote microtubule assembly, suggesting that this may be the primary effect of the mutation. Taken together, the present findings indicate that the G389R mutation in Tau can cause a dementing condition that closely resembles Picks disease.