Relevance of Mutations in Tau for Understanding the Tauopathies

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Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, 3, 341-348 341 Relevance of Mutations in Tau for Understanding the Tauopathies

Michel Goedert*

Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

Abstract: 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, corticobasal degeneration, Pick’s disease and argyrophilic grain disease, as well as the inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Until recently, it was unclear whether the dysfunction of tau protein follows disease or whether disease follows the dysfunction of tau protein. The identification of mutations in Tau as the cause of FTDP-17 has resolved this issue by showing that the dysfunction of tau protein is sufficient to cause neurodegeneration and dementia. 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. Surprisingly, the other mutations have their primary effect at the RNA level, thus perturbing the normal ratio of three-repeat to four-repeat tau isoforms. Where studied, this resulted in the relative overproduction of tau protein with four microtubule-binding repeats in brain. Several 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 shown to be a significant risk factor for progressive supranuclear palsy and corticobasal degeneration. At an experimental level, the work on FTDP-17 is rapidly leading to the development of good transgenic mouse models for the human tauopathies.

INTRODUCTION evidence implicating dysfunction of tau protein in the neurodegenerative process. This changed with the discovery Recent progress in our understanding of some of the most of tau gene mutations in a familial form of frontotemporal common neurodegenerative diseases was made possible by dementia and parkinsonism [3-5]. Here I review the evidence the coming together of two independent lines of research. On implicating tau protein in this and other neurodegenerative one hand, the biochemical study of the neuropathological diseases. lesions that define these diseases led to the identification of their main molecular components. On the other hand, the TAU ISOFORMS IN HUMAN BRAIN AND THEIR study of familial forms of disease led to the identification of INTERACTIONS WITH MICROTUBULES gene defects that cause the inherited variants of the different diseases. Remarkably, in most cases, the defective genes Tau is a microtubule-binding protein that is believed to have been found to encode or increase the expression of the be important for the assembly and stabilization of main components of the neuropathological lesions. It has microtubules [2]. In nerve cells, tau is normally found in therefore been established that the basis of the familial forms axons, but in the tauopathies it is redistributed to the cell of these diseases is a toxic property conferred by mutations body and dendrites. In normal adult human brain, there are in the proteins that make up the filamentous lesions. A six isoforms of tau, produced from a single gene by corollary of this insight is that a similar toxic property might alternative mRNA splicing [6-9]. They differ from one also underlie the sporadic disease forms. another by the presence or absence of a 29- or 58-amino acid insert in the amino-terminal half of the protein and by the Alzheimer’s disease is the most common neurodegenera- inclusion, or not, of a 31-amino acid repeat, encoded by exon tive disease. Neuropathologically, it is defined by the 10 of , in the carboxy-terminal half of the protein (Fig. presence of abundant extracellular neuritic plaques made of Tau ). The exclusion of exon 10 leads to the production of the beta-amyloid protein and intraneuronal neurofibrillary 1a three isoforms, each containing three repeats, and its lesions made of the microtubule-associated protein tau [1]. inclusion leads to a further three isoforms, each containing Similar tau lesions, in the absence of beta-amyloid deposits, four repeats. The repeats constitute the microtubule-binding are also the defining characteristic of a number of other region of tau protein. In normal adult human cerebral cortex, neurodegenerative diseases, the best known of which are there are similar levels of three-repeat and four-repeat progressive supranuclear palsy, corticobasal degeneration isoforms. In developing human brain, only the shortest tau and Pick’s disease [2]. Until recently, there was no genetic isoform (three repeats and no amino-terminal inserts) is expressed. *Address correspondence to this author at the Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK; The tau molecule can be subdivided into an amino- Tel: 0044 1223 402036; Fax: 0044 1223 402197; terminal domain that projects from the microtubule surface E-mail: [email protected] and the carboxy-terminal microtubule-binding domain.

Recent structural work using gold-labelled tau co-assembled nucleotide change at position +19 was reported in a patient with microtubules has begun to shed light on the way that with frontotemporal dementia [33]. tau protein interacts with microtubules [10,11]. When bound to taxol-stabilized microtubules, tau was found to localize FUNCTIONAL EFFECTS OF TAU MUTATIONS along the outer ridges of the microtubule protofilaments [10]. A different conclusion was reached when tau was Tau mutations fall into two largely nonoverlapping bound to microtubules in the absence of taxol [11]. The tau categories – those whose primary effect is at the protein level repeats were now found to bind to the inner surface of the and those that influence the alternative splicing of tau pre- microtubule, close to the taxol-binding site on b-tubulin. mRNA. Several mutations in exon 10 of Tau, such as Taxol binds to a site on b-tubulin, where a -tubulin has a DK280, DN296 and N296H have effects at both protein and conserved extra loop of eight amino acids. Interestingly, this RNA levels [26,27,34,35]. In accordance with their location extended loop has significant sequence homology with the in the microtubule-binding region of tau, most missense tau repeats. Since tubulin cannot have evolved to bind taxol, mutations reduce the ability of tau protein to interact with this work may answer the question of what type of natural microtubules, as reflected by a reduction in the ability of substrate binds to this pocket in b-tubulin. In this model, part mutant tau to promote microtubule assembly [36,37]. Similar of the proline-rich region of tau must provide the link effects on microtubule function are observed when tau is between the amino-terminal projection domain on the expressed in a number of cell types. Expression of tau with a outside of the microtubule and the repeat motifs on the inside variety of mutations caused varying degrees of reduced surface. It could thread through one of the holes between microtubule binding and stability, as well as disorganized protofilaments. microtubule morphology [38-41]. The same was true when Xenopus oocyte maturation was used as an indicator of microtubule function [42]. Following microinjection of wild- MUTATIONS CAUSING TAUOPATHY type or mutant human tau proteins, oocyte maturation was Frontotemporal dementias occur as familial forms and, inhibited to variable degrees. A good correlation was more commonly, as sporadic diseases. Neuropathologically, observed between the effects of individual tau mutations in they are characterized by a remarkably circumscribed atro- this system [42] and in the in vitro experiments [36]. phy of the frontal and temporal lobes of the cerebral cortex, A number mutations in may cause FTDP-17, at least often with additional, subcortical changes. In 1994, an auto- Tau in part, by promoting the aggregation of tau protein [43-47]. somal-dominantly inherited form of frontotemporal dementia Several studies have demonstrated that some of these with parkinsonism was linked to chromosome 17q21.2 [12]. mutations, including R5L, K257T, G272V, K280, P301L, This observation was followed by the identification of other D P301S, V337M and R406W, promote heparin- or arachido- familial forms of frontotemporal dementia that were linked nic acid-induced filament formation of tau relative to to this region, resulting in the denomination “frontotemporal in vitro wild-type tau. This effect is particularly marked for the dementia and parkinsonism linked to chromosome 17” P301L and P301S mutations. Furthermore, the assembly of (FTDP-17) for this class of disease. All cases of FTDP-17 mutant tau into filaments following its conditional have so far shown a filamentous pathology made of expression in human neuroglioma cells has also been hyperphosphorylated tau protein. In 1998, the first mutations reported [48]. Additional mechanisms may play a role in the in Tau in FTDP-17 patients were identified [3-5,13]. Currently, 31 different mutations have been described in case of some coding region mutations. For instance, protein phosphatase 2A is known to be the major tau phosphatase in over 80 families with FTDP-17 (Fig. 1). brain and to bind to the tandem repeats in tau. Accordingly, Tau mutations in FTDP-17 are either missense, deletion several FTDP-17 mutations have been found to result in the or silent mutations in the coding region, or intronic reduced binding of protein phosphatase 2A to tau [49]. mutations located close to the splice-donor site of the intron The intronic mutations and most coding region mutations following the alternatively spliced exon 10 (Fig. 1). in exon 10 (N279K, L284L, N296, N296N, N296H, S305N Missense mutations have been found in exons 1, 9, 10, 11, D and S305S) increase the splicing of exon 10, thus changing 12 and 13 [3,4,7,14-25]. Using the numbering of the 441 the ratio between three- and four-repeat isoforms, resulting amino acid isoform of tau, they comprise R5H and R5L in the overproduction of four-repeat tau [4,5,7,19,26, (exon 1); K257T, I260V, L266V and G272V (exon 9); N279K, N296H, P301L, P301S and S305N (exon 10); 28,29,31,32,34,35,50,51]. Mutation DK280 in exon 10 is an apparent exception, since it decreases the splicing of exon 10 L315R and S320F (exon 11); V337M, E342V, S352L and in transfection experiments [26]. However, it remains to be K369I (exon 12); and G389R and R406W (exon 13). There are also two mutations with a single amino acid deletion, seen whether this mutation leads to an overproduction of three-repeat tau in human brain. It is one of the few known DK280 and DN296, and three silent mutations, L284L, mutations that has effects at both the RNA [26] and the N296N and S305S, all in exon 10 [26-30]. The seven protein level [27]. Approximately half of the known reported mutations in exon 10 that change the amino acid Tau mutations have their primary effect at the RNA level. Thus, sequence will be present only in four-repeat protein to a significant degree, FTDP-17 is a disease of the isoforms; those lying outside exon 10 will be present in all alternative mRNA splicing of exon 10 of the tau gene. It six tau protein isoforms. Intronic mutations are located at positions +3, +11, +12, +13, +14 and +16 of the intron follows that a correct ratio of three-repeat to four-repeat tau isoforms is essential for preventing neurodegeneration and following exon 10, with the first nucleotide of the splice- dementia in mid-life. donor site taken as +1 [4,5,31,32]. Recently, an additional Relevance of Mutations in Tau for Understanding the Tauopathies Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 343

Accordingly, the regulation of the alternative splicing of G-C base pair that is separated from a double helix of six exon 10 is an area of great interest. It is known to involve base pairs by an unpaired adenine. As is often the case with multiple cis-acting regulatory elements that either enhance or single nucleotide purine bulges, the unpaired adenine at inhibit ultilization of the 5’-splice site of exon 10 [26,52-56]. position –2 does not extrude into solution, but intercalates These elements are located in exon 10 itself and in the intron into the double helix. The apical loop consists of six following exon 10. Splicing regulatory elements within exon nucleotides that adopt multiple conformations in rapid 10 include two exon splicing enhancers (ESEs) separated by exchange. Downstream of this intron splicing silencer (ISS), an exon splicing silencer (ESS). Sequences located at the end an intron splicing modulator has been described [56]. It of exon 10 and at the beginning of the intron following exon mitigates exon 10 expression by the ISS. 10 inhibit the splicing of exon 10, probably because of the Pathogenic mutations in the tau gene may alter exon 10 presence of a stem-loop structure that limits access of the splicing by affecting several of the regulatory elements splicing machinery to the 5’-splice site [4,5]. The described above. Thus, the intronic mutations (+3, +11, +12, determination of the three-dimensional structure of a 25- +14 and +16) and the exonic mutations at codon 305 (S305N nucleotide-long RNA from the exon 10-5’-intron junction and S305S) destabilize the inhibitory stem-loop structure. has shown that this sequence forms a stable, folded stem- The S305N mutation and the +3 intronic mutation may also loop structure (Fig. ) [57,58]. The stem consists of a single 1b enhance exon 10 splicing by increasing the strength of the

Fig. (1). Mutations in the tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). (a) Schematic diagram of the six tau isoforms (352 to 441 amino acids) that are expressed in adult human brain, with mutations in the coding region indicated using the numbering of the 441 amino acid isoform. Nineteen missense mutations, two deletion mutations and three silent mutations are shown. The six tau isoforms are produced by alternative mRNA splicing from a single gene. They differ by the presence or absence of three inserts, shown in red (encoded by exon 2), green (encoded by exon 3) and yellow (encoded by exon 10), respectively. (b) Stem-loop structure in the pre-mRNA at the boundary between exon 10 and the intron following exon 10. Nine mutations are shown, two of which (S305N and S305S) are located in exon 10. Exon sequences are boxed and shown in capital, with intron sequences being shown in lower-case letters. 344 Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 Michel Goedert

5’-splice site. However, the finding that the S305S mutation, [75]. The K257T, L266V, G272V, L315R, S320F, E342V, which weakens the exon 10 5’-splice site, leads to a K369I and G389R mutations lead to a tau pathology similar predominance of four-repeat tau argues against this as the or identical to that of Pick’s disease [16-18,20,21,24,25, primary effect of these mutations. The N279K mutation may 66,69]. These findings indicate that depending on the improve the function of the first ESE, thus enhancing exon positions of Tau mutations in exons 9-13, and perhaps the 10 splicing. Mutation L284L, which enhances exon 10 nature of these mutations, a filamentous tau pathology splicing, may do so by disrupting a potential ESS or by ensues that resembles either that of progressive supranuclear lengthening the first ESE. The effects of the three mutations palsy, corticobasal degeneration, Alzheimer’s disease or at codon 296 (DN296, N296N and N296H) are probably due Pick’s disease. to disruption of the ESS. In general, the high fidelity of splice-site selection is PATHOGENESIS OF FTDP-17 believed to result from the cooperative binding of transacting The pathway leading from a mutation in Tau to factors to cis-acting sequences [59]. The heterogeneous neurodegeneration is unknown. The likely primary effect of nuclear ribonucleoproteins (hnRNPs) and the serine-arginine most missense mutations is a change in conformation that rich (SR) proteins are involved in splice-site selection. The results in a reduced ability of tau protein to interact with SR domain-containing protein Tra2beta has been found to microtubules. It can be overcome by natural osmolytes, such interact with the first ESE in exon 10 and to function as an as trimethylamine N-oxide, probably through the promotion activator of the alternative splicing of exon 10 [60]. In trans- of tubulin-induced folding of tau [76]. The primary effect of fection experiments, several other SR proteins have been these mutations may be equivalent to a partial loss of shown to promote the exclusion of exon 10 [54]. Moreover, function, with resultant microtubule destabilization and phosphorylation of SR proteins by CDC2-like kinases has deleterious effects on cellular processes, such as rapid axonal also been found to result in the skipping of exon 10 [61]. transport. However, in the case of mutations whose primary effect is at the RNA level, this appears unlikely. The net NEUROPATHOLOGY OF FTDP-17 effect of these mutations is a simple overproduction of tau with four repeats, which is known to interact more strongly All cases with Tau mutations that have been examined to with microtubules than tau with three repeats [12]. It is date have shown the presence of a filamentous pathology therefore possible that in cases of FTDP-17 with intronic made of hyperphosphorylated tau protein [62,63]. To a large mutations and those coding region mutations whose primary extent, the morphologies of tau filaments and their isoform effect is at the RNA level, microtubules are more stable than compositions are determined by whether Tau mutations in brain from control individuals. Moreover, some mutations, affect mRNA splicing of exon 10, or whether they are such as P301L and P301S in exon 10, will only affect 20- missense mutations located inside or outside exon 10. 25% of tau molecules, with 75-80% of tau being wild-type, Mutations in Tau that result in the increased splicing of arguing against a simple loss of function of tau as a decisive exon 10 lead to the formation of wide twisted ribbon-like mechanism. Mice without tau protein are largely normal and filaments that contain only four-repeat tau isoforms [65]. exhibit no signs of neurodegeneration [77]. Where examined, the tau pathology was found to be widespread and present in both nerve cells and glial cells, A correct ratio of wild-type three-repeat to four-repeat with an abundant glial component. This has been shown for tau may be essential for the normal function of tau in human brain. However, the fact that the tau isoform composition in the intronic mutations [31,32,51,64-66], as well as for brain is not conserved between humans, rodents and mutations N279K, L284L, S305N, S305S, DN296, N296H and N296N in exon 10 [15,19,26,28,29,67,68]. Mutations in chickens argues against this possibility [78,79]. An alternative hypothesis is that a partial loss of function of tau exon 10 of Tau that do not affect alternative mRNA splicing is necessary for setting in motion the “gain of toxic function” lead to the formation of narrow twisted ribbons that contain four-repeat tau isoforms. This has been shown for the P301L mechanisms that will lead to neurodegeneration. This mutation [37,69,70]. Using an antibody specific for mutant hypothesis requires that an overproduction of tau isoforms with four repeats results in an excess of tau over available tau, biochemical studies have demonstrated that filaments binding sites on microtubules, thus resulting in the extracted from the brains of patients with the P301L cytoplasmic accumulation of unbound four-repeat tau. It mutation contain predominantly mutant tau [71,72]. Tau would probably necessitate the existence of different binding pathology is widespread and present in both nerve cells and sites on microtubules for three-repeat and four-repeat tau. glial cells [69,70]. Compared with mutations that affect the Validation of this hypothesis will probably require structural splicing of exon 10, the glial component is less pronounced. information at the atomic level. Most coding region mutations located outside exon 10 From the above, a reduced ability of tau to interact with lead to a tau pathology that is neuronal, without a significant microtubules emerges as the most likely primary effect of glial component. Exceptions are mutations R5H and R5L in the FTDP-17 mutations. It will lead to the accumulation of exon 1, L266V in exon 9 and L315R in exon 11 [22-25]. tau in the cytoplasm of brain cells and result in its hyper- Two mutations, V337M in exon 12 and R406W in exon 13, phosphorylation. Over time, hyperphosphorylated tau protein lead to the formation of paired helical and straight filaments will assemble into abnormal filaments. It is at present that contain all six tau isoforms, like the tau filaments of unknown whether the filaments themselves cause nerve cell Alzheimer’s disease [73,74]. Using an antibody specific for loss, or whether non-assembled, conformationally altered tau tau protein with the R406W mutation, both wild-type and protein is toxic. mutant proteins were detected in the abnormal filaments Relevance of Mutations in Tau for Understanding the Tauopathies Curr. Med. Chem. – Immun., Endoc. & Metab. Agents, 2003, Vol. 3, No. 4 345

RELEVANCE FOR THE SPORADIC TAUOPATHIES S305S in Tau presented with a clinical picture similar to progressive supranuclear palsy [23,29,30,103]. Moreover, The study of FTDP-17 has established that dysfunction mutations N296N and P301S gave rise to a clinical and of tau protein can cause neurodegeneration and dementia. It neuropathological picture resembling corticobasal degenera- follows that tau dysfunction is most probably also of central tion [14,28]. importance in the pathogenesis of sporadic diseases with a filamentous tau pathology, such as Alzheimer’s disease, All in all, this work strongly suggests that dysfunction of progressive supranuclear palsy, corticobasal degeneration, tau protein is of central importance in progressive supranu- argyrophilic grain disease and Pick’s disease. This is further clear palsy and corticobasal degeneration, as it is in Pick’s underlined by the fact that the aforementioned diseases are disease. partially or completely phenocopied by cases of FTDP-17 [2]. TRANSGENIC ANIMAL MODELS OF THE TAUO- PATHIES Eight missense mutations in Tau have been shown to give rise to a clinical and neuropathological phenotype that Animal models of the tauopathies are essential for is closely related to Pick’s disease [16-18,20,21,24,25,69]. elucidating the mechanisms by which dysfunction of tau The finding that overproduction of four-repeat tau causes protein leads to neurodegeneration. In addition, they may disease and leads to the assembly of tau with four repeats in prove useful for the development and testing of novel nerve cells and glial cells may shed light on the pathogenesis therapies. of progressive supranuclear palsy, corticobasal degeneration Several reports have described transgenic mouse lines and argyrophilic grain disease. Neuropathologically, all three that express wild-type three-repeat or four-repeat human tau diseases are characterized by a neuronal and glial tau protein in nerve cells [78,104-108]. These mice developed pathology, with the tau filaments comprising four-repeat tau numerous abnormal tau-immunoreactive nerve cell bodies isoforms, with at best only a small amount of three-repeat and dendrites, as well as large numbers of pathologically tau [66,80-83]. An association between progressive enlarged axons containing tau- and neurofilament- supranuclear palsy and a dinucleotide repeat polymorphism immunoreactive spheroids. These changes were most in the intron between exons 9 and 10 of Tau has been prominent in spinal cord, but were also seen in the brain. described [84]. The alleles at this locus carry 11-15 repeats. They were accompanied by histological and behavioural The A0 allele, with 11 repeats, has a frequency of over 90% signs of amyotrophy. However, abundant tau filaments were in patients with progressive supranuclear palsy and about not observed. This work has shown that overproduction of 70% in controls. Subsequently, two common tau haplotypes non-filamentous tau is sufficient to lead to axonopathy and that differ at the nucleotide level, but not at the level of the amyotrophy. However, unlike the human diseases with tau protein coding sequence, have been reported [85]. Homo- filaments, there was no sign of nerve cell loss. More zygosity of the more common allele H1 predisposes to recently, mouse lines were produced that expressed the three progressive supranuclear palsy and corticobasal degeneration, human tau isoforms with three repeats in both nerve cells but not to Alzheimer’s disease or Pick’s disease [85-90]. and glial cells [109]. Over time, tau filaments formed in Somewhat unexpectedly, it has also been reported to increase astrocytes and oligodendrocytes, but not in nerve cells. This the risk for sporadic frontotemporal dementia [91,92] and was accompanied by the degeneration of some glial cells. some studies have reported an association between the H1 haplotype and idiopathic Parkinson’s disease [93-96]. The discovery of mutations in Tau in FTDP-17 is leading However, other studies have failed to find an association to the production of transgenic mouse lines that express with Parkinson’s disease [97,98]. Future work will have to mutant human tau protein in nerve cells and glial cells. To show whether tau protein, which is not deposited in either date, studies using tau with mutations P301L, G272V, sporadic frontotemporal dementias or idiopathic Parkinson’s V337M, R406W, P301S and the triple mutation G272V, disease, is nonetheless involved in the aetiology and P301L and V337M have been published [110-118]. Of these, pathogenesis of these diseases. tau proteins with the V337M and R406W mutations were tagged at both ends, complicating the interpretation of the More recently, the existence of , a gene embed- Saitohin reported findings. Abundant tau filaments were found in the ded in the intron between exons 9 and 10 of , was Tau lines expressing four-repeat P301L human tau under the reported [99]. It encodes a 128 amino acid protein of control of the murine prion protein promoter [110] and four- unknown function. This gene contains a single nucleotide repeat P301S human tau under the control of the murine polymorphism responsible for a glutamine to arginine Thy1 promoter [115]. Filamentous tau protein was hyper- substitution at position 7 (Q7R). The initial report found that phosphorylated in a similar way to the human diseases and the RR genotype was associated with a higher risk for late- hyperphosphorylation at most sites appeared to precede the onset Alzheimer’s disease. However, subsequent studies assembly of tau into filaments. However, it remains to be failed to confirm this association [100-102]. The Q allele seen whether hyperphosphorylation of tau is necessary for forms part of the H1 haplotype and the R allele of the Tau filament assembly in brain and spinal cord. Widely differing H2 haplotype. It follows that the QQ allele is a risk factor for conclusions have been drawn from in vitro studies [119- progressive supranuclear palsy and corticobasal degeneration. 121]. In the transgenic mice, filamentous tau deposits This work has led to the suggestion that progressive contained mostly the mutant human tau protein [115,118], in supranuclear palsy and corticobasal degeneration may be line with in vitro findings showing that the P301L and P301S caused by an imbalance between three- and four-repeat tau mutations strongly enhance the heparin-induced assembly of isoforms. 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