A novel tau mutation in exon 9 (1260V) causes a four-repeat tauopathy

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Experimental Neurology 184 (2003) 131–140

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A novel tau mutation in exon 9 (1260V) causes a four-repeat tauopathy Andrew Grover,a,b Elisabet England,c Mathew Baker,a Naruhiko Sahara,d Jennifer Adamson,a Brian Granger,d Henry Houlden,a,e Ulla Passant,f Shu-Hui Yen,d Michael DeTure,d and Michael Huttona,* a

b

Laboratory of Neurogenetics, Neuroscience Department, Mayo Clinic, Jacksonville, FL 32224, USA Department of Psychiatry, Division of Neuroscience, School of Medicine, Queen Elizabeth Psychiatric Hospital, University of Birmingham, Birmingham B15 2QZ, UK c Department of Pathology, Division of Neuropathology, University of Lund, Lund, Sweden d Laboratory of Biochemistry, Neuroscience Department, Mayo Clinic, Jacksonville, FL 32224, USA e Laboratory of Neurogenetic, Institute of Neurology, Queens Square, London WC2, UK f Department of Psychogeriatrics, University of Lund, Lund, Sweden Received 28 March 2003; revised 10 July 2003; accepted 22 July 2003

Abstract A novel mutation in exon 9 of tau, I260V, is associated with a clinical syndrome consistent with frontotemporal dementia with extensive tau pathology; however, neurofibrillary tangles and Pick bodies are absent. Significantly, Sarkosyl-insoluble tau extracted from affected brain tissue consisted almost exclusively of four-repeat isoforms. Consistent with these findings, in vitro biochemical assays demonstrated that the I260V mutation causes a selective increase in tau aggregation and a decrease in tau-induced microtubule assembly with four-repeat isoforms only. The contrasting pathology and biochemical effects of this mutation suggest a different disease mechanism from the other exon 9 mutations and demonstrates the critical role for the first microtubule-binding domain in tau-promoted microtubule assembly and the pathogenic aggregation of tau. © 2003 Elsevier Inc. All rights reserved. Keywords: Tau; Four-repeat; Isoform; Aggregation; Microtubule assembly; Axonal; Glial tau; Neurodegeneration; Dementia; Pick body

Introduction Aggregated tau protein is the major component of the neurofibrillary lesions that characterize a group of neurodegenerative disorders referred to as tauopathies. These include, among others, the autosomal dominant syndrome known as frontotemporal dementia linked to chromosome 17 (FTDP-17), corticobasal degeneration (CBD), Pick disease (PiD), and progressive supranuclear palsy (PSP). Frontotemporal dementia (FTD) is a heterogeneous neuropsychiatric disorder characterized by profound personality change and alterations in social conduct, with deterioration of speech and language, often followed by memory problems and Parkinsonian features later in the course of the * Corresponding author. E-mail address: [email protected] (M. Hutton). 0014-4886/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0014-4886(03)00393-5

disease (Lund and Manchester Groups, 1994). FTDP-17 was subsequently defined (Foster et al., 1997) as a subset of FTD where linkage to chromosome 17 had been identified. The discovery of mutations in the MAPT gene that cause FTDP-17 identified the major cause of this syndrome and demonstrated that perturbation of tau function is sufficient to cause neurodegeneration (Hutton et al., 1998; Poorkaj et al., 1998; Spillantini et al., 1998b). This further suggested that tau dysfunction is likely to play a major role in other tauopathies, including Alzheimer’s disease and the sporadic disorders PSP, CBD, and PiD. Tau is a soluble microtubule-associated protein thought to regulate the assembly (Weingarten et al., 1975) and dynamics (Bre and Karsenti, 1990) of microtubules and has been shown to influence rapid anterograde (Stamer et al., 2002) but not slow (Utton et al., 2002) axonal microtubule transport in vitro. Six major isoforms of tau are expressed in

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the brain, as a result of alternative splicing of exons 2, 3, and 10 (Goedert et al., 1989a). Exons 9 –12 encode four imperfect-repeat microtubule-binding domains; alternative splicing of exon 10 therefore gives rise to isoforms with either three (3R) or four (4R) microtubule-binding domains, with an overall ratio in normal adult human brain of approximately 1:1 (Goedert et al., 1989b). Missense, deletion, and silent mutations have been reported in the coding region of tau, with additional mutations in the intronic region proximal to the 5⬘ splice site of exon 10 (Lee et al., 2001). Most missense mutations appear to disrupt tau protein function, while some missense and silent mutations in exon 10 and the intronic mutations alter the ratio of tau mRNA species with or without exon 10. This disruption of exon 10 alternative splicing results in a shift in the ratio of 3R to 4R tau sufficient to cause neurodegeneration. The clinical and pathological features of FTDP-17 are variable, with some cases bearing a resemblance to PSP (Hayashi et al., 2002; Pastor et al., 2001; Poorkaj et al., 2002), CBD (Spillantini et al., 2000), and PiD (Kobayashi et al., 2003; Murrell et al., 1999; Neumann et al., 2001; Pickering-Brown et al., 2000; Rizzini et al., 2000; Rosso et al., 2002; Spillantini et al., 1998a). Three of the mutations reported in FTDP-17 cases with pathology resembling PiD, K257T (Pickering-Brown et al., 2000; Rizzini et al., 2000), L266V (Kobayashi et al., 2003), and G272V (Hutton et al., 1998; Spillantini et al., 1998a), occur in exon 9 of the tau gene. Clinical symptoms associated with these mutations were reportedly consistent with FTD (Lund and Manchester Groups, 1994), and microscopic and immunohistochemical analysis revealed numerous spherical, neuronal inclusions resembling Pick bodies for each of these three exon 9 mutations. Detergent-insoluble tau protein extracted from brain tissue with the K257T mutation (Rizzini et al., 2000) contained predominantly 3R tau, consistent with the majority of reported sporadic PiD cases (Arai et al., 2001; Delacourte et al., 1998). Furthermore, the K257T mutation was shown to selectively increase the aggregation of three-repeat tau isoforms in vitro, consistent with the isoform content of the insoluble tau extracted from the brains of the K257T patient (Rizzini et al., 2000). In contrast, the L266V mutation in exon 9 is associated with the deposition of both 3R and 4R tau, although there is evidence that the Pick bodies in these patients contain exclusively 3R tau, with the deposited 4R tau occurring largely in glial lesions (Dr. Dennis W. Dickson, personal communication). Biochemical studies of tau deposition in patients with the G272V mutation have not yet been performed due to the lack of suitable frozen brain tissue. In this article we report a case with a clinical diagnosis of FTD carrying a novel mutation, I260V, in exon 9 of the tau gene. In contrast to the previously identified exon 9 tau mutations the pathological phenotype for the I260V mutation bears little resemblance to PiD. We further show that

the sarkosyl-insoluble tau deposited in the brain of an I260V patient contains almost exclusively 4R tau and that in vitro the I260V mutation causes a selective increase in aggregation and a reduction in microtubule assembly for four-repeat tau only. This mutation therefore extends the phenotypic spectrum of mutations occurring in exon 9 of the tau gene. Patient and methods Clinical history The female patient presented at age 68 with depression, anxiety, and increasing speech problems. She was clinically diagnosed as FTD of non-Alzheimer type, based on neuropsychological and psychiatric evaluation and brain imaging, according to routine investigation procedures performed in Lund, Sweden (Lund and Manchester Groups, 1994). She subsequently developed a tendency to fall, resulting in a closed head trauma. Other conditions were type II diabetes, hypertension, and repeated venous thrombi, which were treated with warfarin. She died at age 77 from bronchopneumonia and anoxic brain damage with swelling and uncal herniation. The family history of the patient was unavailable. Neuropathology/immunohistochemistry Following postmortem macroscopic analysis, whole brain coronal sections were cut and hematoxylin– eosin and myelin staining was performed. Modified Gallyas silver staining was performed on selected sections. For immunohistochemistry, phosphorylation-dependent anti-tau AT8 and anti-ubiquitin antibodies were applied to selected sections. Genetic analysis Genomic DNA was extracted from frozen brain tissue, and exons 7–13 of the tau gene were sequenced from the proband’s genomic DNA using previously described protocols and primers designed to the flanking intronic regions of each exon (Hutton et al., 1998). A 60 –50°C touchdown PCR program over 35 cycles was used to amplify each exon, and PCR products were confirmed by agarose gel electrophoresis. Sequencing was performed on an ABI PRISM 377 DNA Sequencer (Perkin–Elmer Applied Biosystems, Foster City, CA 94404) using the rhodamine dye terminator cycle sequencing kit (Perkin–Elmer). RT–PCR analysis of tau exon 10 alternative splicing in vivo Total RNA was isolated from tissue from the affected frontal lobe cortical region (Trizol, Life Technologies) and RT–PCR was performed with the Superscript preamplification kit (Life Technologies). PCR was performed using tau primers in exons 9 and 11, as described previously (Hutton

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et al., 1998). PCR products were run on 4% nondenaturing acrylamide gels and visualized with SYBR green (Molecular Probes). Quantification was performed using the ImageQuant 5.0 program and ratios of exon 10⫹ to exon 10⫺ tau RNA were calculated for both the exon 9 –11 and 9 –13 amplifications. Mean tau ratios were based on results from three independent amplifications. Tau mRNA ratios in the I260V case were compared with those determined in cortical RNA samples from unaffected individuals. Tau biochemistry Sarkosyl-insoluble and -soluble tau protein fractions were extracted from frontal lobe brain tissue according to the method of Greenberg and colleagues (Greenberg and Davies, 1990). Samples were run on a 10% SDS–polyacrylamide gel (SDS–PAGE), transferred to a nitrocellulose membrane, and probed with the anti-tau antibody E1 that recognizes an epitope encoded by exon 1 of tau. A portion of each protein sample was dephosphorylated by hydrofluoric acid treatment as described previously (Togo et al., 2002). To improve signal detection, prior to SDS–PAGE the soluble tau samples were boiled for 5 min and centrifuged at 14;000g to remove non-tau proteins, and the supernatant was retained for Western blotting. Filaments were adsorbed onto carbon/Formvar grids from a sample of the Sarkosyl-insoluble tau fraction and stained with 2% uranyl acetate and images taken using a Phillips EM208S electron microscope and camera. Immunogold labeling was performed using polyclonal anti-tau antibodies WKS44 (rabbit) and Tau46 (mouse), epitopes being residues 162–176 and 420 – 436, respectively, of the longest, 441 (2N4R) amino acids, isoform of tau. Rabbit and mouse secondary antibodies conjugated with 5- and 10-nm colloidal gold particles respectively were used to label the anti-tau antibodies. Recombinant tau protein generation Site-directed mutagenesis (Transfomer mutagenesis kit, Clontech CA) was used to generate the K257T, I260V mutations in 2N3R (htau39) and 2N4R (htau40) tau cDNA isoforms in the pET30a vector. 2N4R tau containing the ⌬K280 mutation (D’Souza et al., 1999) was also generated as a positive control. Recombinant tau protein was expressed in BL21(DE3)pLysS bacteria (Stratagene) and purification was performed using standard techniques of heat treatment followed by separation on an anion exchange column as previously reported (DeTure et al., 2002). Heparin-induced tau aggregation Aggregation reactions using recombinant tau were performed as previously described (Grover et al., 2002). Briefly, 0.2 mg/ml of tau and 0.03 mg/ml of heparin were set up in 30 mM Mops at pH 7.4. Samples were incubated

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at 37°C and analyzed at intervals of from 1 h to 4 days. Aggregation was measured by thioflavin-S fluorescence as previously described (Friedhoff et al., 1998). Experiments were repeated using four different recombinant protein preparations. Fluorescence values in each experiment were normalized to the mean of all values within each experiment, averaged, and analyzed by two-tailed Student’s t test. Filament formation was confirmed by adsorption of 10 ␮l of reaction mixture onto a carbon/Formvar grid and stained with 2% uranyl acetate, and images were taken using a Phillips EM208S electron microscope and camera. Tau-promoted microtubule assembly assay Microtubule assembly with recombinant tau was performed as previously described (Grover et al., 2002). Icecold tubulin (1 mg/ml (20 ␮M)) (Cytoskeleton Inc.) was added to 0.1 mg/ml (2.3 ␮M) of recombinant tau protein in assembly buffer (100 mM Mes, 1 mM GTP, 1 mM DTT) and immediately transferred to a quartz cuvette in a spectrophotometer equilibrated to 35°C, and absorbance was measured at 350 nm (A350) on a Cary 3 Bio UV spectrophotometer (Varian, Palo Alto, CA). Estimates of the initial rate of microtubule assembly and of the extent of assembly for each tau protein sample and tubulin only controls were based on results from four separate assembly reactions. The experiment was further repeated three times using different preparations of recombinant tau protein. Rate and extent values were assessed for significance using a two-tailed Student’s t test.

Results Neuropathological investigation showed mild macroscopic atrophy of the frontal lobes and dilatation of the anterior lateral ventricles. Also, bilateral subdural hematomas had formed. Microscopically, neurodegeneration with gliosis, mild microvacuolation, and neuronal atrophy and loss were seen in superficial layers of the frontal lobes. The degeneration was symmetrical, accentuated in the parasagittal regions of the frontal convexities but not engaging the precentral gyri and mildly in the basal forebrain and temporal lobes. No Pick cells or swollen, achromatic neurons of the type seen in CBD were noted. Modified Gallyas staining revealed dystrophic neuritelike threads and astrocytic processes throughout the affected cortex, staining more extensively with the AT8 tau antibody (Fig. 1A). Tau-positive microglia were also seen, most readily identified in the molecular layer. AT8 immunostained many nonargyrophilic neurons in the affected cortex and also in the pyramidal cell layer and fascia dentata of the hippocampus (Fig. 1B). However, the hippocampus showed no marked atrophy or delineated inclusions and little gliosis. In the white matter argyrophilic axonal threads were AT8positive, with an even distribution of nonargyrophilic AT8-

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Fig. 3. Sarkosyl-insoluble (A,B) and soluble (C, D) tau analysis. Immunoblot of Sarkosyl-insoluble tau before (A) and after (B) dephosphorylation with hydrofluoric acid (HF). Immunoblotting of soluble tau before (C) and after (D) dephosphorylation with HF. Immunoblotting was performed using the phosphorylation-independent anti-tau antibody E1. rTau, recombinant tau; 260, I260V proband; AD, Alzheimer’s Disease case; PiD, sporadic PiD case. All samples were from the frontal cortex.

positive glial cell bodies (Fig. 1C). A small number of tau-positive astrocytes and oligodendroglia were visible, although fewer were visible with silver staining. Demyelination and moderate gliosis were seen in the frontal and parietal white matter, partly of the ischemic type. No amyloid plaques were visible, and there was virtually no ubiquitin staining in gray or white matter. A mild vascular component with a number of minute lesions was noted. Moderate cell loss, depigmentation, and gliosis were visible in the substantia nigra, without inclusions. The basal ganglia, amygdala, and cerebellum showed no marked changes. Neurofibrillary lesions in neuronal cell bodies (tangles) were not observed. Because of the clinical and pathological diagnosis of

FTD, DNA was extracted from brain tissue and exons 7–13 of the tau gene were sequenced, revealing an A to G transition mutation in exon 9, at position 778 of the longest (2N4R) tau isoform (Fig. 2A). This results in a change from isoleucine to valine (ATC3 GTC) at codon 260 that falls within the first microtubule-binding domain of tau. The isoleucine at this position in the microtubule-binding repeat is highly conserved across species (Fig. 2B), although semiconserved in the four microtubule-binding domains of human tau. Given that both isoleucine and valine are hydrophobic residues and the physical size of the valine side chain is smaller than isoleucine, this alteration in the encoded amino acid sequence is relatively conservative. Additional samples from the family of the proband were not

Fig. 1. Neuropathological findings in the I260V proband’s brain. Immunostaining of the frontal cortex with phosphorylation-dependent antibody AT8 shows numerous tau-positive neurons and abundant diffuse tau-positive threads (A), also visible in the hippocampus along with tau-positive neurons in the pyramidal cell layer and fascia dentata (B). At higher magnification the AT8-positive neurons are clearly visible in the cortex (D), pyramidal cell layer (E), and fascia dentata (F) of the hippocampus. In the white matter (C) axonal threads were tau-positive, as were some large astrocytes and some of the oligodendroglial cell bodies (AT8 staining), shown at a higher magnification in (G). Scale bars: A–C, 75 ␮m; D–G, 25 ␮m. Fig. 2. (A) Chromatograph showing genomic sequence from exon 9 of the I260V case. The position of the mutation is marked with an arrow. (B) Schematic diagram of the longest isoform of tau showing the sequence of the first microtubule-binding repeat and the positions of the exon 9 mutations reported to date, showing the relative conservation between microtubule-binding repeats in human tau, and in the first microtubule-binding repeat across species. (C) Alternative splicing of exon 10 of tau in the I260V brain showed normal ratios of exon 10 alternative splicing compared to that in unaffected individuals. 260, I260V case; Unaff, unaffected individuals; I10⫹16, mRNA from ⫹16 mutation carrier.

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Fig. 4. Electron micrographs of tau filaments isolated from the Sarkosyl-insoluble fraction from the proband’s brain show mainly straight (A) filaments ⬃15 nm, in width, with occasional twisted filaments (B) with a periodicity of ⬃500 nm. Filaments labeled with WKS44 (small gold particles) and Tau46 (large gold particles) antibodies (C). Scale bar, 50 nm.

available to check for segregation of the I260V mutation with disease; however, more than 100 Scandinavian controls (⬎200 chromosomes) were screened without detecting this mutation. RT–PCR analysis of tau exon 10 splicing in mRNA extracted from the brain of the I260V case revealed normal ratios of exon 10⫹ exon 10⫺ transcripts (Fig. 2C). Sarkosylinsoluble tau was extracted from tissue from the affected region of the proband’s frontal lobe and also from an Alzheimer’s Disease (AD) case and a sporadic PiD case. Western blot analysis of the insoluble tau fraction from the AD brain (Fig. 3A) revealed a triplet of bands at 60, 64, and 68 kDa, consistent with hyperphosphorylated insoluble tau detected in previous studies in AD brain tissue (Greenberg and Davies, 1990). The I260V sample had two major bands at 64 and 68 kDa, corresponding to the upper two bands of the

AD triplet. This pattern of sarkosyl-insoluble hyperphosphorylated tau is also observed with 4R tauopathies such as PSP and CBD and with mutations in exon 10 of tau that cause FTDP-17 (Lee et al., 2001). The PiD case showed a doublet of bands at 60 and 64 kDa, consistent with the previously reported banding pattern of tau from PiD brain (Delacourte et al., 1996). Dephosphorylation of the Sarkosyl-insoluble tau (Fig. 3B) from the I260V sample revealed a triplet of bands corresponding only to the recombinant tau isoforms with four microtubule-binding repeats. Again the similarity to the isoform pattern of tauopathies such as PSP and CBD that selectively deposit 4R tau is clear. In contrast to the I260V case but consistent with previous reports, all six isoforms were visible in the AD sample (Greenberg et al., 1992), white the PiD sample resolved into mainly threerepeat isoforms (Arai et al., 2001). In the Sarkosyl-soluble

Fig. 5. The I260V mutation selectively increases aggregation and reduces microtubule assembly in four-repeat tau in vitro. (A/B) Heparin-induced aggregation of wild-type (WT), K257T (257), and I260V (260) recombinant tau (A, 2N3R, and B, 2N4R) measured by thioflavin-S fluorescence (arbitrary units) over time. Filament formation was confirmed by EM. (C/D) Polymerization of the tubulin dimer promoted by wild-type (WT), K257T (257), and I260V (260) recombinant 2N3R (C) and 2N4R (D) tau measured by absorbance at 350 nm over time (A350). In both experiments the ⌬K280 mutation was included as a positive control (280).

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fraction from the AD case the triplet of hyperphosphorylated pathological tau was again visible (Fig. 3C). In contrast, similar soluble species were absent in the I260V sample, implying that hyperphosphorylated tau that migrates at 64 and 68 kDa is present almost exclusively in aggregates in this case. Dephosphorylation of the soluble tau fraction (Fig. 3D) showed all six tau isoforms present in approximately normal ratios in the I260V case, the AD case, and the PiD case, consistent with the results from the tau RT– PCR analysis. These results confirm that the I260V mutation is associated with a 4R tauopathy, a very different phenotype from that associated with the previously reported exon 9 mutations (K257T and L266V). The morphology of tau filaments in the Sarkosyl-insoluble extract from the I260V case was examined by electron microscopy. Electron micrographs showed mainly straight filaments (Fig. 4A) ⬃15 nm in width, with occasional twisted filaments (Fig. 4B) with a width of ⬃30 nm and a periodicity of ⬃500 nm. This is a pattern consistent with filaments associated with other tau mutations (Lee et al., 2001). Immunogold labeling of the filaments showed that both polyclonal anti-tau antibodies WKS44 (small gold particles) and Tau46 (large gold particles) bound to filaments (Fig. 4C). It has been demonstrated that recombinant tau can be induced to self-assemble in the presence of the sulfated glycosaminoglycan heparin (Goedert et al., 1996). Given the unexpected 4R isoform content in the insoluble tau, the effect of the I260V mutation on heparin-induced aggregation of the longest tau isoforms, 2N3R and 2N4R, was investigated. Thioflavin-S fluorescence, as an indicator of ␤-sheet formation, was used to quantify the rate of aggregation (Friedhoff et al., 1998). and filament assembly was confirmed by EM (data not shown). For comparison, recombinant protein carrying the K257T mutation in both 2N3R and 2N4R isoforms was included in the experiment. The extent of four-repeat tau aggregation was significantly increased (70% at Day 4, P ⫽ 0.0099) for the I260V mutant protein over the wild-type tau (Fig. 5B). However, there was no significant difference in the extent of aggregation between wild-type and I260V in 2N3R tau (Fig. 5A). This is consistent with the insoluble tau biochemistry data and indicates that the I260V mutation confers a selective enhancement of 4R tau aggregation. In contrast, we observed only a small increase in the aggregation of 2N3R and 2N4R tau with the K257T mutant compared to wild-type tau; however, this increase did not achieve significance for either isoform. The difference between our aggregation results with the K257T mutation and those reported by Rizzini et al. (2000), which showed a robust selective increase in aggregation with the 1N3R isoform, may reflect differences in the conditions and isoforms used to perform the aggregation studies. Most tau missense mutations have previously been shown to reduce the ability of tau to bind to microtubules and to assemble microtubules from tubulin dimers in vitro

(Hong et al., 1998). Consistent with these previous studies, the I260V mutation, in recombinant 2N4R tau, caused a reduction of 23% (P ⫽ 0.05) in the maximum rate of tubulin assembly compared to wild-type 2N4R tau (Fig. 5D), although there was not a significant change in the extent of assembly (P ⫽ 0.078). In contrast, the I260V mutation had no significant effect on the rate (P ⫽ 0.69) or extent (P ⫽ 0.69) of microtubule assembly induced by 2N3R tau (Fig. 5C). The I260V mutation therefore appears to selectively inhibit 4R tau-induced tubulin assembly, as well as enhance 4R tau aggregation, although the relative effect of the I260V mutation on tubulin assembly is small, compared to the previously reported tau mutations K257T and ⌬K280. For the K257T mutation, a 43% (P ⬍ 0.002) decrease in the maximum rate of microtubule formation for the 2N3R isoform was observed, while in the 2N4R isoform the K257T mutant tau showed a 54% reduction in maximum rate (P ⫽ 0.001). The extent of assembly with the K257T mutant tau was reduced by 15% (P ⫽ 0.048) for the 2N3R isoform and by 21% (P ⫽ 0.0005) for the 2N4R isoform. These data for the K257T mutation are consistent with previous reports that this mutation significantly reduces tubulin assembly with both the 3R and 4R tau isoforms (Pickering-Brown et al., 2000; Rizzini et al., 2000). As previously reported, the ⌬K280 mutation caused a reduction in both the maximum rate (80%, P ⫽ 0.0001) and the extent (31% P ⫽ 0.002) of tubulin assembly.

Discussion In this paper we report the identification of a novel mutation, I260V, in exon 9 of the tau gene that is associated with a frontal lobar degeneration featuring abundant neuritic and glial tau pathology. Sarkosyl-insoluble tau extracted from the brain of a single patient contains almost exclusively 4R tau isoforms; however, analysis of RNA and soluble tau demonstrates that this mutation does not disrupt exon 10 alternative splicing. In contrast, in vitro studies demonstrate that the I260V mutation selectively increases 4R tau aggregation and decreases the rate of microtubule assembly. Little or no effect was observed in the assays with 3R tau. This selective effect of the I260V mutation likely explains the almost exclusive deposition of 4R tau observed in the brain of this patient. I260V is the fourth pathogenic mutation to be identified in the first microtubule-binding domain of tau, encoded by exon 9, the previously reported mutations being K257T (Pickering-Brown et al., 2000; Rizzini et al., 2000), L266V (Kobayashi et al., 2003), and G272V (Hutton et al., 1998; Spillantini et al., 1998a). However, in stark contrast to these previously reported exon 9 mutations I260V is the first to be associated with selective 4R tau deposition and with a pathological phenotype that lacks Pick body-like lesions. The I260V proband’s symptoms were consistent with the Lund/Manchester criteria for FTD (Lund and Manchester

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Groups, 1994). However, the age of onset of 68 is higher than that for the majority of FTDP-17 cases (Foster et al., 1997). The other exon 9 FTDP-17 mutation carriers have much earlier ages of onset, ranging from 34 to 51 (Kobayashi et al., 2003; Pickering-Brown et al., 2000; Rizzini et al., 2000; van Swieten et al., 1999). The markedly accentuated degeneration of the medial frontal lobes in the I260V case is in direct contrast to the other exon 9 cases, where temporal lobe degeneration is more pronounced (Kobayashi et al., 2003; Pickering-Brown et al., 2000; Rizzini et al., 2000). Silver and immunohistochemical staining revealed extensive neuritic pathology in the affected region of the frontal cortex and the associated white matter of the I260V brain. The lack of argyrophilic inclusions in neuronal cell bodies (Pick bodies or tangles) in the cortex or hippocampus implies that the majority of aggregated protein is in neuronal processes. In contrast, the other reported exon 9 mutations show extensive argyrophilic tau-positive inclusions resembling Pick bodies in the cytoplasm of neurons in the hippocampus and cortex. The Sarkosyl-insoluble tau isoforms deposited in the I260V case are composed almost exclusively of 4R tau; this is similar to other 4R tauopathies, such as PSP and CBD (Lee et al., 2001). Comparison with other exon 9 mutations reveals a spectrum of tau isoform incorporation into pathologic lesions, with K257T associated predominantly with 3R tau deposition (Rizzini et al., 2000). L266V having similar amounts of 3R and 4R tau (Kobayashi et al., 2003), and I260V depositing almost exclusively 4R tau. A similar spectrum of insoluble tau isoforms has been described by Zhukareva et al. in a series of sporadic PiD cases (Zhukareva et al., 2002). In tau aggregation experiments the I260V mutation caused a highly significant and selective increase in the aggregation of 4R over wild-type tau. In addition, the I260V mutation reduced the rate of 4R tau-induced tubulin assembly, although the relative effect on tubulin assembly was small. These in vitro data are entirely consistent with the in vivo Sarkosyl-insoluble tau data and suggest that I260V causes the selective deposition of 4R tau isoforms through a direct effect on the properties of 4R tau protein. In contrast, the K257T mutation has previously been reported to cause the selective aggregation of 3R tau, consistent with the deposition of 3R tau in patients with this mutation (Pickering-Brown et al., 2000; Rizzini et al., 2000). In this study, we were unable to reproduce this finding; however, this may well reflect the differences in the conditions and isoforms used in the aggregation experiments. We have demonstrated that the I260V mutation is associated with a disease that is pathologically and biochemically distinct from that of the previously reported exon 9 mutations. The contrast in the isoform-specific effects of these mutations, both in vivo and in vitro, demonstrates that the I260V mutation fundamentally differs from the other exon 9 mutations in its impact on the functioning of the first microtubule-binding domain of tau. In addition, these data

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imply that this domain plays a key role in the pathologic process of tau aggregation, which requires further investigation.

Acknowledgments This work was supported by the Mayo Foundation, the NIA (PO1 AG17216 to M.H. and S.H.Y.), and the Wellcome Trust. We thank Dr. Viginia M. Lee (University of Pennsylvania) for the anti-tau antibody Tau46.

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