Tau and _-Synuclein in Neurodegenerative Diseases
7.3. TAU EXPRESSION AND FUNCTION
proteolysis of _-synuclein may form “seeds” that could initiate _-synuclein filament assembly.
tau to bind and modulate MT assembly is negatively regulated by phospho-rylation (90,93,104–106).
Surprisingly, tau is not essential for MT function because disruption of expression by a genetically engineered null mutation does not result in an overt phenotype (107). Moreover, depletion of axonal tau in rat sympathetic neurons by microinjection of anti-tau antibodies had no detectable effects on the dynamics of axonal MTs (108). Thus, it seems likely that the ability of tau to modulate MT assembly can be compensated for by other MAPs.
The distribution of tau in cultured rat sympathetic and hippocampal neurons suggests that it may serve functions other than the stabilization of MTs. In these cultures, tau is more concentrated at the distal end than the proximal end of axons even though axonal MTs in the distal end are less stable and turn over more rapidly (109,110). It is possible that these apparent discrepancies may be the result of differential phosphorylation of tau within these axonal regions, but these observations also suggest that the abundance of tau at the growth cone neck may reflect an alternative role for tau. Tau Fig. 2. Schematic of exon organization and the six brain tau isoforms generated by alternative splicing. Alternative splicing of exons 2, 3, and 10 produce the six alternative products. Putative exons 6 and 8 are not used in brain. Exon 4A, which is also not used in the brain, is included in the PNS leading to the translation of larger tau isoforms, termed “big tau” (see text). Black bars depict the 18–amino–
acid MT–binding repeats.
expression can inhibit kinesin-dependent trafficking of organelles such as mitochondria and vesicles (111). Additionally, the amino-terminal projection domain of tau interacts with the plasma membrane, although the importance of this observation is still unknown (112). Furthermore, tau has been shown to exist in complex with phospholipase C-a (113) and to increase the activity of this enzyme (114).
Tau Pathogenesis in Alzheimer’s Disease
In AD, tau aggregates into cytoplasmic inclusions in the form of neurofibrillary tangles (NFTs) in neuronal cell bodies, and neuropil threads and dystrophic neurites of senile plaques in neuronal processes (115). Ultra-structurally, these aberrant structures are comprised of 8- to 20-nm twisted double-helical ribbons, referred to as paired helical filaments (PHFs) (116,117) and the less abundant 15-nm-wide straight filaments (SFs) (118,119). Compelling biochemical and immuno-electron microscopic studies have demonstrated that PHFs are comprised of tau (120–124). SFs are a structural variant of PHFs and are likely entirely composed of tau (118).
Abnormally aggregated tau isolated from AD brain, referred to as PHF-tau (or A68), contains all six CNS tau isoforms (125) aberrantly hyperphosphorylated at >25 Ser or Thr residues (126–128). It is still unclear which enzymes are responsible for this hyperphosphorylation of tau, as numerous kinases and phosphatases can modulate tau phosphorylation in vivo and/or in vitro (129,130). It is likely that a change in a combination of enzymatic activities is involved in generating hyperphosphorylated PHF-tau.
The mechanism of PHF-tau formation in neurons remains enigmatic.
Because hyperphosphorylation is the most prominent difference between PHF-tau and normal tau, it would be reasonable to hypothesize that phosphorylation may induce tau filament formation. However, there is no direct evidence to support this model, and nonphosphorylated, recombinant tau can assemble into filaments in vitro (131). It is more likely that abnormal phosphorylation increases the pool of MT-unbound tau, which then becomes available for PHF formation. Supporting this notion are the findings that (1) hyperphosphorylation of tau precedes PHF formation (132,133), (2) phosphorylation inhibits MT binding (90,93,106,134), and (3) the ability of PHF-tau to bind to MTs is greatly impaired, but this loss of function can be overcome by dephosphorylation (93,105,135).
Interestingly, tau filament assembly in vitro can be facilitated by long polyanionic molecules such as strongly or moderately sulfated glycosami-noglycans and nucleic acids (136–140). Moreover, sulfated glycosaminogly-cans and nucleic acids has been shown to prevent tau MT-binding, and
heparin sulfate, chondroitin sulfate, and dermatan sulfate proteoglycans have been colocalized with NFTs of AD brains (136,141,142). It is unclear how sulfated glycosaminoglycans appear within the cytoplasm, although a likely explanation would involve leakage from membrane-bound organelles.
PHF-tau also is modified by ubiquitination (143), glycation (144,145), and N-linked glycosylation (146). Ubiquitination occurs after aggregation, probably as an attempt by the cellular machinery to degrade these protein aggregates, and it is unlikely to contribute to PHF formation (133,147).
Glycation is a nonenzymatic addition of reduced carbohydrates, and the pres-ence of this modification is likely to result from the slow turnover of PHFs.
The importance of N-linked gycosylation is undetermined, although it may contribute to the maintenance of PHF structure (146).
FTDP-17: Direct Genetic Evidence for the Importance of Tau in Disease
FTDP-17 refers to a group of autosomal dominant hereditary neurodegenerative disorders characterized by behavioral changes with subsequent cognitive disturbance and, in some cases, parkinsonism (148).
Most, if not all, FTDP-17 families show tau deposits either in neurons or in both neurons and glia without accompanying amyloid deposition (149–157).
Genetic analysis has revealed 12 different mutations in the tau gene in at least 26 FTDP-17 families, establishing that in FTDP-17 kindreds, tau muta-tions are pathogenic for the disease (Table 1). The mutamuta-tions can be divided into two functional groups: missense mutations that impair the ability of tau to bind to MTs and promote MT assembly, and exonic or intronic mutations that alter the inclusion of exon 10 during splicing. Missense mutations G272V, 6280, P301L, V337M, and R406W belong to the former category (81,155,158,159).
These mutations may lead to pathogenesis through an initial loss of function, followed by a gain of toxic effect. The reduced capacity of these mutants to stabilize MTs may lead to a loss of MT function, such as fast axonal transport.
Pathology may subsequently be compounded by a progressive accumulation of tau in the cytoplasm and eventual aggregation into insoluble filaments. More-over, mutations P301L, V337M, and R406W may accelerate tau filament formation (160,161). Consistent with the location of these mutations within tau, aggregated tau from cases with mutation V337M or R406W is predominantly comprised of all six CNS tau isoforms, whereas only 4R-tau is present in the case of P301L (81,162).
Some pathogenic missense mutations and silent mutations at or close to exon 10 can alter the splicing efficiency of this exon, as demonstrated by exon-trapping analysis (155,163,164). The mutations may affect splicing via three different mechanisms. First, in cases of known intronic mutations
Giasson et al.
Table 1
Tau Mutations in FTDP–17
Mutations Exon/intron location Protein domain Functional impact Ref.
G272V Exon 9 Repeat 1 Reduced MT binding 159,163
N279K Exon 10 Interrepeat 1–2 Altered splicing 162
6280 Exon 10 Interrepeat 1–2 Altered splicing/reduced MT binding 159
L284L (T to C) Exon 10 Interrepeat 1–2 Altered splicing 155
P301L Exon 10 Repeat 2 Reduced MT binding 159,162,163,183
S305N Exon 10 Interrepeat 2–3 Altered splicing 149
V337M Exon 12 Interrepeat 3–4 Reduced MT binding 184
R406W Exon 13 C–terminus Reduced MT binding 163
E10+3 (G to A) Intron 10 N/A Altered splicing 171
E10+13 (A to G) Intron 10 N/A Altered splicing 163
E10+14 (C to T) Intron 10 N/A Altered splicing 163
E10+16 (C to T) Intron 10 N/A Altered splicing 154,163,185
Note: The positions of the mutations are assigned according to the longest brain tau isoform (441 amino acid long).
and the missense mutation S305N, it has been proposed that altered splicing efficiency may be due to the disruption of a putative inhibitory RNA stem loop structure at the 5' boundary of the intron following exon 10 (Fig. 3) (155,163). This secondary structure may compete with the U1 snRNP or other splicing factors for the binding of the splice donor site, and its destabi-lization leads to the increased inclusion of exon 10. However, attempts to rescue the putative function of the stem-loop structure with compensatory double mutants were not successful, suggesting that other elements beside the secondary structure are involved (155). The S305N mutation also changes the 5' splice site of intron 10 to a stronger splice site (GUguga to AUguga) (165), which likely also contributes to the effect on splicing by this mutation. Consistent with this pathogenic mechanism, an increased ratio of exon 10+/exon 10– tau RNA in the brains of patients with intronic muta-tions has been reported (163). A second mechanism by which splicing is affected is demonstrated by the N279K mutation, which may enhance the insertion of exon 10 by improving an exon-splicing enhancer. At the RNA level, this mutation changes a nucleotide stretch from TAAGAA to GAAGAA. The latter sequence is a repeat of GARs (where R is a purine), which can act as an exon-splice enhancer (166–169). The notion that this nucleotide stretch is a splicing enhancer is supported by the finding that the deletion of nucleotides AAG by the 6280 mutation obliterates exon 10 inclusion (155). Finally, a third mechanism is demonstrated by the silent mutation L284L (CTTACTC), which likely affects splicing because it disrupts the sequence UUAG that can act as a putative exon-splicing silencer (170), and thereby increases the ratio of exon 10+/exon 10– tau mRNA (155).
Consistent with the notion that an alteration in RNA splicing is the cause of pathogenesis, biochemical postmortem analysis of the brains of affected patients with mutations predicted to increase exon 10 splicing (i.e., E10+14, N279K, E10+3 mutations) showed an increase in the abundance of 4R-tau over 3R-tau and the specific accumulation of aggregated 4R-tau (81,150,154,162,171).
The mechanism by which changes in the 3R/4R-tau ratio lead to neu-ronal and, in some cases, glial dysfunction and death is still nebulous.
Four repeat-tau and 3R-tau may bind to distinct sites on MTs (101), and the overproduction of one group of isoforms may result in a pool of MT-unbound tau that may polymerize into filaments over time. It is also possible that a specific ratio of tau isoforms is required for the normal maintenance and function of MTs. Although speculative, the possibility that specific isoforms might have other, undetermined functions should not be overlooked.
Other Tauopathies Involving Specific Isoforms of Tau
Pick’s disease is a fronto-temporal-type dementia characterized by the presence of Pick bodies, round-shaped neuronal inclusions composed of granular material together with 10- to 20-nm diameter filaments (172). These disease specific filamentous tau inclusions contain 3R-tau isoforms exclusively (173,174). The reasons for this selective aggregation of 3R-tau isoforms is unknown, but a possible explanation is that neurons expressing specifically these forms of tau are more vulnerable in Pick’s disease. The restricted expression of 3R-tau in the granule cell layer of the dentate gyrus demonstrates that expres-sion of tau isoforms can be cell-type specific (79). This concept has not been extensively studied and further evaluation is certainly warranted.
Progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) are late onset neurodegenerative disorders characterized by both neuronal and glial tau inclusions. Aggregated tau in these diseases is predominantly comprised of 4R-tau isoforms (175). In CBD, tau precipitates in the form of astrocytic plaques, oligodendroglial “coil bodies,” and neuronal inclusions sometimes termed corticobasal bodies (176,177). Neuronal tau in PSP brains aggregates in the form of classical flame-shaped NFTs or globose NFTs (176). PSP also features distinctive glial tau inclusions termed oligodendroglial “coiled bodies,”
tufted astrocytes, and thorn-shaped astrocytes (177).
Genetic changes in the tau gene may contribute to the risk of developing PSP. Conrad et al. (178) reported a link between PSP and a polymorphic dinucleotide repeat region found between exons 9 and 10 of the tau gene.
Subsequent studies confirmed this correlation (179–181), and it was recently demonstrated that this association is the result of a specific haplotype that also contains at least eight single nucleotide polymorphisms (182).
Fig. 3. Structure of the putative inhibitory RNA stem loop structure at the 5' boundary of the intron following exon 10. Pathogenic mutations that can affect the stability of this secondary structure are depicted.