Filamentous nerve cell inclusions in neurodegenerative diseases: tauopathies and alpha-synucleinopathies.

Alzheimer's disease and Parkinson's disease are the most common neurodegenerative diseases. They are characterized by the degeneration of selected populations of nerve cells that develop filamentous inclusions before degeneration. The neuronal inclusions of Alzheimer's disease are made of the microtubule-associated protein tau, in a hyperphosphorylated state. Recent work has shown that the filamentous inclusions of Parkinson's disease are made of the protein alpha-synuclein and that rare, familial forms of Parkinson's disease are caused by missense mutations in the alpha-synuclein gene. Besides Parkinson's disease, the filamentous inclusions of two additional neurodegenerative diseases, namely dementia with Lewy bodies and multiple system atrophy, have also been found to be made of alpha-synuclein. Abundant filamentous tau inclusions are not limited to Alzheimer's disease. They are the defining neuropathological characteristic of frontotemporal dementias such as Pick's disease, and of progressive supranuclear palsy and corticobasal degeneration. The recent discovery of mutations in the tau gene in familial forms of frontotemporal dementia has provided a direct link between tau dysfunction and dementing disease. The new work has established that tauopathies and alpha-synucleinopathies account for most late-onset neurodegenerative diseases in man. The formation of intracellular filamentous inclusions might be the gain of toxic function that leads to the demise of affected brain cells.     

From genetics to pathology: tau and alpha-synuclein assemblies in neurodegenerative diseases

The most common degenerative diseases of the human brain are characterized by the presence of abnormal filamentous inclusions in affected nerve cells and glial cells. These diseases can be grouped into two classes, based on the identity of the major proteinaceous components of the filamentous assemblies. The filaments are made of either the microtubule-associated protein tau or the protein alpha-synuclein. Importantly, the discovery of mutations in the tau gene in familial forms of frontotemporal dementia and of mutations in the alpha-synuclein gene in familial forms of Parkinson's disease has established that dysfunction of tau protein and alpha-synuclein can cause neurodegeneration.     

Tau mutations in frontotemporal dementia FTDP-17 and their relevance for Alzheimer's disease

Alzheimer's disease is characterised by the degeneration of selected populations of nerve cells that develop filamentous inclusions prior to degeneration. The neuronal inclusions of Alzheimer's disease are made of the microtubule-associated protein tau, in a hyperphosphorylated state. Abundant filamentous tau inclusions are not limited to Alzheimer's disease. They are the defining neuropathological characteristic of frontotemporal dementias, such as Pick's disease, and of progressive supranuclear palsy and corticobasal degeneration. The discovery of mutations in the tau gene in familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has provided a direct link between tau dysfunction and dementing disease. Known mutations produce either a reduced ability of tau to interact with microtubules, or an overproduction of tau isoforms with four microtubule-binding repeats. This leads in turn to the assembly of tau into filaments similar or identical to those found in Alzheimer's disease brain. Several missense mutations also have a stimulatory effect on heparin-induced tau filament formation. Assembly of tau into filaments may be the gain of toxic function that is believed to underlie the demise of affected brain cells.     

Mutations causing neurodegenerative tauopathies

Tau is the major component of the intracellular filamentous deposits that define a number of neurodegenerative diseases. They include the largely sporadic Alzheimer's disease (AD), progressive supranuclear palsy, corticobasal degeneration, Pick's disease and argyrophilic grain disease, as well as the inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). For a long time, it was unclear whether the dysfunction of tau protein follows disease or whether disease follows tau dysfunction. This was resolved when mutations in Tau were found to cause FTDP-17. Currently, 32 different mutations have been identified in over 100 families. About half of the known mutations have their primary effect at the protein level. They reduce the ability of tau protein to interact with microtubules and increase its propensity to assemble into abnormal filaments. The other mutations have their primary effect at the RNA level and perturb the normal ratio of three-repeat to four-repeat tau isoforms. Where studied, this resulted in a relative overproduction of tau protein with four microtubule-binding domains in the brain. Individual Tau mutations give rise to diseases that resemble progressive supranuclear palsy, corticobasal degeneration or Pick's disease. Moreover, the H1 haplotype of Tau has been identified as a significant risk factor for progressive supranuclear palsy and corticobasal degeneration. At a practical level, the new work is leading to the production of experimental animal models that reproduce the essential molecular and cellular features of the human tauopathies, including the formation of abundant filaments made of hyperphosphorylated tau protein and nerve cell degeneration.     

Abnormal tau-containing filaments in neurodegenerative diseases

It has been known for some time that the neurofibrillary pathology in Alzheimer's disease consists of so-called paired helical and straight filaments made up of the microtubule-associated protein tau. The degree of dementia observed in the disease correlates better with the extent of neurofibrillary pathology than with the Abeta amyloid deposits, the other characteristic defining pathological fibrous deposit in Alzheimer's disease. However, no familial cases of Alzheimer's disease have been genetically linked to the tau protein locus. Recently a group of frontotemporal dementias with parkinsonism linked to chromosome 17 has been shown to be caused by mutations in the tau gene. Some are missense mutations giving altered tau proteins, whereas others affect the splicing of the pre-mRNA and change the balance between different tau isoforms. Histologically these diseases are all characterised by various kinds of filamentous tau protein deposits, mostly in the complete absence of Abeta deposits. The abnormal tau filaments show different morphologies, depending on the nature of the tau mutation. These diseases show that tau mutations can be a prime cause of inherited dementing illness and may throw some light on the pathological process in the much larger number of sporadic cases of Alzheimer's disease.     

High prevalence of mutations in the microtubule-associated protein tau in a population study of frontotemporal dementia in the Netherlands.

Mutations in microtubule-associated protein tau recently have been identified in familial cases of frontotemporal dementia (FTD). We report the frequency of tau mutations in a large population-based study of FTD carried out in the Netherlands from January 1994 to June 1998. Thirty-seven patients had >/=1 first-degree relative with dementia. A mutation in the tau gene was found in 17.8% of the group of patients with FTD and in 43% of patients with FTD who also had a positive family history of FTD. Three distinct missense mutations (G272V, P301L, R406W) accounted for 15.6% of the mutations. These three missense mutations, and a single amino acid deletion (DeltaK280) that was detected in one patient, strongly reduce the ability of tau to promote microtubule assembly. We also found an intronic mutation at position +33 after exon 9, which is likely to affect the alternative splicing of tau. Tau mutations are responsible for a large proportion of familial FTD cases; however, there are also families with FTD in which no mutations in tau have been found, which indicates locus and/or allelic heterogeneity. The different tau mutations may result in disturbances in the interactions of the protein tau with microtubules, resulting in hyperphosphorylation of tau protein, assembly into filaments, and subsequent cell death.     

Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins

Tau protein is the major component of the intraneuronal filamentous inclusions that constitute defining neuropathological characteristics of Alzheimer's disease and other tauopathies. The discovery of tau gene mutations in familial forms of frontotemporal dementia has established that dysfunction of the tau protein is sufficient to cause neurodegeneration and dementia. Here we have tested 42 compounds belonging to nine different chemical classes for their ability to inhibit heparin-induced assembly of tau into filaments in vitro. Several phenothiazines (methylene blue, azure A, azure B, and quinacrine mustard), polyphenols (myricetin, epicatechin 5-gallate, gossypetin, and 2,3,4,2',4'-pentahydroxybenzophenone), and the porphyrin ferric dehydroporphyrin IX inhibited tau filament formation with IC(50) values in the low micromolar range as assessed by thioflavin S fluorescence, electron microscopy, and Sarkosyl insolubility. Disassembly of tau filaments was observed in the presence of the porphyrin phthalocyanine. Compounds that inhibited tau filament assembly were also found to inhibit the formation of Abeta fibrils. Biochemical analysis revealed the formation of soluble oligomeric tau in the presence of the inhibitory compounds, suggesting that this may be the mechanism by which tau filament formation is inhibited. The compounds investigated did not affect the ability of tau to interact with microtubules. Identification of small molecule inhibitors of heparin-induced assembly of tau will form a starting point for the development of mechanism-based therapies for the tauopathies.     

Regulation of tau isoform expression and dementia

In the central nervous system (CNS), aberrant changes in tau mRNA splicing and consequently in protein isoform ratios cause abnormal aggregation of tau and neurodegeneration. Pathological tau causes neuronal loss in Alzheimer's disease (AD) and a diverse group of disorders called the frontotemporal dementias (FTD), which are two of the most common forms of dementia and afflict more than 10% of the elderly population. Autosomal dominant mutations in the tau gene cause frontotemporal dementia with parkinsonism-chromosome 17 type (FTDP-17). Just over half the mutations affect tau protein function and decrease its affinity for microtubules (MTs) or increase self-aggregation. The remaining mutations occur within exon 10 (E10) and intron 10 sequences and alter complex regulation of E10 splicing by multiple mechanisms. FTDP-17 splicing mutations disturb the normally balanced levels of distinct protein isoforms that result in altered biochemical and structural properties of tau. In addition to FTDP-17, altered tau isoform levels are also pathogenically associated with other FTD disorders such as progressive supranuclear palsy (PSP), corticobasal degeneration and Pick's disease; however, the mechanisms remain undefined and mutations in tau have not been detected. FTDP-17 highlights the association between splicing mutations and the pronounced variability in pathology as well as phenotype that is characteristic of inherited disorders.     

The neuropathological spectrum of neurodegenerative tauopathies

Abundant neurofibrillary lesions made of abnormal and hyperphosphorylated microtubule-associated protein tau constitute one of the defining neuropathological features of Alzheimer's disease. However, tau containing filamentous deposits in neurons and/or glial cells also define a heterogeneous group of neurodegenerative disorders clinically characterized by dementia and/or motor syndromes. Thus, all these disorders are collectively grouped under the generic term of tauopathies. In the present review we outline the morphological and biochemical characteristics of some major tauopathies, including Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration and argyrophilic grain disease. The second part will deal with the recent discovery of tau gene mutations in frontotemporal dementia and parkinsonism linked to chromosome 17 which demonstrates that tau dysfunction can lead to neurodegeneration. Finally, we will discuss the very recent finding of 'tau-deficient' tauopathy in a subset of frontotemporal dementia cases.     

Slow development of ALS-like spinal cord pathology in mutant valosin-containing protein gene knock-in mice

Pathological features of amyotrophic lateral sclerosis (ALS) include, in addition to selective motor neuron (MN) degeneration, the occurrence of protein aggregates, mitochondrial dysfunction and astrogliosis. SOD1 mutations cause rare familial forms of ALS and have provided the most widely studied animal models. Relatively recent studies implicating another protein, TDP-43, in familial and sporadic forms of ALS have led to the development of new animal models. More recently, mutations in the valosin-containing protein (VCP) gene linked to the human genetic disease, Inclusion Body Myopathy associated with Paget''s disease of bone and frontotemporal dementia (IBMPFD), were found also to be associated with ALS in some patients. A heterozygous knock-in VCP mouse model of IBMPFD (VCP^R155H/+) exhibited muscle, bone and brain pathology characteristic of the human disease. We have undertaken studies of spinal cord pathology in VCP^R155H/+ mice and find age-dependent degeneration of ventral horn MNs, TDP-43-positive cytosolic inclusions, mitochondrial aggregation and progressive astrogliosis. Aged animals (∼24–27 months) show electromyography evidence of denervation consistent with the observed MN loss. Although these animals do not develop rapidly progressive fatal ALS-like disease during their lifespans, they recapitulate key pathological features of both human disease and other animal models of ALS, and may provide a valuable new model for studying events preceding onset of catastrophic disease.     

Identification and characterization of the Drosophila tau homolog

A pathological hallmark of neurodegenerative tauopathies, including Alzheimer's disease and a group of clinically heterogeneous frontotemporal dementias, is the presence of intracellular neurofibrillary protein lesions (reviewed in Spillantini and Goedert, TINS 10 (1998) 428). The principal component of these structures is the microtubule-associated protein tau. Although tau is normally a highly soluble protein enriched in axons, in these deposits, it is abnormally hyperphosphorylated, insoluble, and redistributed to the somatodendritic compartments of neurons. Through ultrastructual analyses, it has been determined that the tau protein in these lesions is filamentous and organized into paired-helical filaments, straight filaments, or ribbon-like filaments (Goedert et al., The Molecular and Genetic Basis of Neurological Disease (1997) 613). By the dynamic binding of microtubules, tau is thought to promote the structural stability of axons, but whether tau aggregates contribute to neurodegeneration through a direct toxicity on normal cellular functions such as organelle transport or an indirect effect on microtubule stability, is currently unknown. The identification of mutations in the tau locus in patients with familial frontotemporal dementia and Parkinsonism linked to chromosome 17 has demonstrated that mutations in tau are sufficient to cause neurodegenerative disease (Poorkaj et al., Ann. Neurol. 43 (1998) 815; Hutton et al., Nature 393 (1998) 702). To elucidate the mechanisms by which tau dysfunction contributes to neuronal loss, we have sought to model human tauopathies in a genetically tractable organism. Here we describe the isolation of a Drosophila tau cDNA (GenBank accession number AY032977), the production of antibodies that recognize the encoded protein, and their use in determining the expression and subcellular localization of the fly tau protein.     

Tau protein and neurodegeneration

Tau protein is the major component of the intracellular filamentous deposits that define a number of neurodegenerative diseases. They include the largely sporadic Alzheimer's disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), argyrophilic grain disease, as well as the inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). The identification of mutations in Tau as the cause of FTDP-17 established that dysfunction or misregulation of tau protein is sufficient to cause neurodegeneration and dementia. At an experimental level, the new understanding is leading to the development of good transgenic animal models of the tauopathies.     

Functional effects of tau gene mutations deltaN296 and N296H

Mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome-17 (FTDP-17). Functionally, about half of the known mutations increase the alternative mRNA splicing of exon 10 of the tau gene, resulting in the overproduction of tau isoforms with four microtubule-binding repeats. The other mutations reduce the ability of tau to interact with microtubules, with some mutations also increasing the propensity of tau to assemble into filaments. Here we have examined the functional effects of the recently described tau gene mutations deltaN296 and N296H. Both mutations reduced the ability of tau to promote microtubule assembly, without having a significant effect on tau filament formation. By exon trapping, they increased the splicing of exon 10. DeltaN296 and N296H thus define a class of tau mutations with effects at both the RNA and the protein level.     

A Raman optical activity study of rheomorphism in caseins, synucleins and tau. New insight into the structure and behaviour of natively unfolded proteins.

The casein milk proteins and the brain proteins alpha-synuclein and tau have been described as natively unfolded with random coil structures, which, in the case of alpha-synuclein and tau, have a propensity to form the fibrils found in a number of neurodegenerative diseases. New insight into the structures of these proteins has been provided by a Raman optical activity study, supplemented with differential scanning calorimetry, of bovine beta- and kappa-casein, recombinant human alpha-, beta- and gamma-synuclein, together with the A30P and A53T mutants of alpha-synuclein associated with familial cases of Parkinson's disease, and recombinant human tau 46 together with the tau 46 P301L mutant associated with inherited frontotemporal dementia. The Raman optical activity spectra of all these proteins are very similar, being dominated by a strong positive band centred at approximately 1318 cm(-1) that may be due to the poly(l-proline) II (PPII) helical conformation. There are no Raman optical activity bands characteristic of extended secondary structure, although some unassociated beta strand may be present. Differential scanning calorimetry revealed no thermal transitions for these proteins in the range 15-110 degrees C, suggesting that the structures are loose and noncooperative. As it is extended, flexible, lacks intrachain hydrogen bonds and is hydrated in aqueous solution, PPII helix may impart a rheomorphic (flowing shape) character to the structure of these proteins that could be essential for their native function but which may, in the case of alpha-synuclein and tau, result in a propensity for pathological fibril formation due to particular residue properties.     

Cdk5 is involved in NFT-like tauopathy induced by transient cerebral ischemia in female rats

Although neurofibrillary tangle (NFT) formation is a central event in both familial and sporadic Alzheimer's disease (AD), neither cellular origin nor functional consequence of the NFTs are fully understood. This largely is due to the lack of available in vivo models for neurofibrillary degeneration (NFD). NFTs have only been identified in transgenic mice, bearing a transgene for a rare hereditary neurodegenerative disease, frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP17). Epidemiological evidence suggests a much higher occurrence of dementia in stroke patients. This may represent the underlying cause of the pathogenesis of sporadic AD, which accounts for the majority of AD cases. We examined pathological markers of AD in a rodent stroke model. Here we show that after transient cerebral ischemia, hyperphosphorylated tau accumulates in neurons of the cerebral cortex in the ischemic area, forms filaments similar to those present in human neurodegenerative tauopathies and colocalizes with markers of apoptosis. As a potential underlying mechanism, we were able to determine that transient ischemia induced tau hyperphosphorylation and NFT-like conformations are associated with aberrant activation of cyclin dependent kinase 5 (Cdk5) and can be rescued by delivery of a potent, but non-specific cyclin dependent kinase inhibitor, roscovitine to the brain. Our study further indicates that accumulation of p35 and its calpain-mediated cleavage product, p25 may account for the deregulation of Cdk5 induced by transient ischemia. We conclude that Cdk5 may be the principal protein kinase responsible for tau hyperphosphorylation and may be a hallmark of the tauopathies in this stroke model.     

The natural osmolyte trimethylamine N-oxide (TMAO) restores the ability of mutant tau to promote microtubule assembly

Coding region and intronic mutations in the gene for microtubule-associated protein tau cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Most coding region mutations effect a reduced ability of tau protein to interact with microtubules and lead to the formation of a filamentous pathology made of hyperphosphorylated tau. Here we show that trimethylamine N-oxide (TMAO) restores the ability of tau with FTDP-17 mutations to promote microtubule assembly. To mimic phosphorylation, serine and threonine residues in tau were singly or multiply mutated to glutamic acid, resulting in a reduced ability of tau to promote microtubule assembly. With the exception of the most heavily substituted protein (27 glutamic acid residues), TMAO increased the ability of mutant tau to promote microtubule assembly. However, it had no significant effect on heparin-induced assembly of tau into filaments.     

Accelerated filament formation from tau protein with specific FTDP-17 missense mutations

Tau is the major component of the neurofibrillar tangles that are a pathological hallmark of Alzheimers' disease. The identification of missense and splicing mutations in tau associated with the inherited frontotemporal dementia and Parkinsonism linked to chromosome 17 demonstrated that tau dysfunction can cause neurodegeneration. However, the mechanism by which tau dysfunction leads to neurodegeneration remains uncertain. Here, we present evidence that frontotemporal dementia and Parkinsonism linked to chromosome 17 missense mutations, P301L, V337M and R406W, cause an accelerated aggregation of tau into filaments. These results suggest one mechanism by which these mutations can cause neurodegeneration and frontotemporal dementia and Parkinsonism linked to chromosome 17.     

Age-dependent emergence and progression of a tauopathy in transgenic mice overexpressing the shortest human tau isoform

Filamentous tau aggregates are hallmarks of tauopathies, e.g., frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC). Since FTDP-17 tau gene mutations alter levels/functions of tau, we overexpressed the smallest human tau isoform in the CNS of transgenic (Tg) mice to model tauopathies. These mice acquired age-dependent CNS pathology similarto FTDP-17 and ALS/PDC, including insoluble, hyperphosphorylated tau and argyrophilic intraneuronal inclusions formed by tau-immunoreactive filaments. Inclusions were present in cortical and brainstem neurons but were most abundant in spinal cord neurons, where they were associated with axon degeneration, diminished microtubules (MTs), and reduced axonal transport in ventral roots, as well as spinal cord gliosis and motor weakness. These Tg mice recapitulate key features of tauopathies and provide models for elucidating mechanisms underlying diverse tauopathies, including Alzheimer's disease (AD).     

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.