A Novel Mutation at Position 112 in theIntron following Exon 10 of the Tau Gene

in Familial Frontotemporal Dementia(FTD-Kumamoto)

Minoru Yasuda, MD,* Junichi Takamatsu, MD,† Ian D’Souza, PhD,‡§ R. Anthony Crowther, PhD,i

Toshio Kawamata, MD,* Masato Hasegawa, PhD,¶ Hiroshi Hasegawa, MD,* Maria Grazia Spillantini, PhD,#Satoshi Tanimukai, MD,* Parvoneh Poorkaj, PhD,‡§ Luca Varani,i Gabriele Varani, PhD,i

Takeshi Iwatsubo, MD,¶ Michel Goedert, MD, PhD,i Gerard D. Schellenberg, PhD,‡§**and Chikako Tanaka, MD*

Exonic and intronic mutations in the tau gene cause familial frontotemporal dementia and parkinsonism linked tochromosome 17. Here, we describe a new mutation, consisting of a C-to-T transition at position 112 of the intronfollowing exon 10 of the tau gene in the Kumamoto pedigree, showing frontotemporal dementia. The mutation causeda marked reduction in melting temperature of the tau exon 10–splicing regulatory element RNA and a large increase inexon 10–containing transcripts. Brain tissue from affected individuals showed an abnormal preponderance of exon10–containing transcripts that was reflected at the protein level by an overproduction of tau isoforms with fourmicrotubule-binding repeats. Immunostaining revealed the presence of tau aggregates in degenerating neurons and glialcells. Isolated tau filaments had a twisted ribbon-like morphology and were made of hyperphosphorylated four-repeat tauisoforms. The additional mutation located close to the splice-donor site of the intron following exon 10 of the tau genesupports the view that intronic mutations exercize their pathogenic effect by destabilizing RNA secondary structure.

Yasuda M, Takamatsu J, D’Souza I, Crowther RA, Kawamata T, Hasegawa M, Hasegawa H, Spillantini MG,Tanimukai S, Poorkaj P, Varani L, Varani G, Iwatsubo T, Goedert M, Schellenberg GD, Tanaka C.

A novel mutation at position 112 in the intron following exon 10 of the tau gene in familialfrontotemporal dementia (FTD-Kumamoto). Ann Neurol 2000;47:422–429

Abundant neurofibrillary lesions made of microtubule-associated protein tau constitute a defining neuro-pathological characteristic of Alzheimer’s disease.1 Fila-mentous tau protein deposits are also the definingneuropathological characteristic of other neurodegen-erative diseases, many of which are frontotemporal de-mentias or movement disorders that have been sub-sumed under the heading of “Pick complex.”2 Recentwork has shown that mutations in the tau gene causefamilial frontotemporal dementia and parkinsonismlinked to chromosome 17 (FTDP-17).3–21 Known taumutations are either intronic mutations located close tothe splice-donor site of the intron following exon 10 ormissense, deletion, or silent mutations in the codingregion.

Six tau isoforms are produced in adult human brainby alternative mRNA splicing from a single gene.22

They differ from each other by the presence or absenceof 29- or 58-amino acid inserts located in the amino-terminal half and an additional 31-amino acid repeatlocated in the carboxy-terminal half. Inclusion of thelatter, which is encoded by exon 10, produces the threeisoforms with four repeats each23; the other three iso-forms have three repeats each. The repeats and someadjoining sequences constitute the microtubule-bindingdomains of tau.24,25 Similar levels of three-repeat andfour-repeat tau isoforms are found in normal cerebralcortex,26 and the tau filaments from Alzheimer’s dis-ease brain contain all six tau isoforms in a hyperphos-phorylated state.27

From the *Hyogo Institute for Aging Brain and Cognitive Disor-ders, Himeji, †Division of Clinical Research, Kikuchi National Hos-pital, Kumamoto, and ¶Department of Neuropathology and Neu-roscience, Graduate School of Pharmaceutical Sciences, Universityof Tokyo, Tokyo, Japan; ‡Geriatric Research Education ClinicalCenter, Veterans Affairs Puget Sound Health Care System, and§Division of Gerontology and Geriatric Medicine, Department ofMedicine, and **Departments of Pharmacology and Neurology,University of Washington, Seattle, WA; and iMedical Research

Council Laboratory of Molecular Biology and #Department of Neu-rology, Adrian Building, University of Cambridge, Cambridge, UK.

Received Oct 14, 1999, and in revised form Nov 29. Accepted forpublication Nov 30, 1999.

Address correspondence to Dr Yasuda, Hyogo Institute for AgingBrain and Cognitive Disorders, 520 Saisho-ko, Himeji 670-0981,Japan.

ORIGINAL ARTICLES

422 Copyright © 2000 by the American Neurological Association

Coding region mutations are missense, deletion, orsilent mutations. Most of these reduce the ability of tauto interact with microtubules,28–30 whereas others in-fluence splicing in exon 10.4,14,31 Moreover, severalmissense mutations also stimulate heparin-induced as-sembly of tau into filaments.32,33 Coding region mu-tations are located in the microtubule-binding repeatregion or close to it. They are found in exon 9(G272V), exon 10 (N279K, L284L, DK280, P301L,P301S, and S305N), exon 12 (V337M), and exon 13(R406W). The mutations in exon 10 that reduce theability of tau to interact with microtubules lead to apathology made of narrow, twisted ribbons that consistpredominantly of four-repeat tau isoforms and that arefound in nerve cells and glial cells.34 By contrast, themissense mutations located outside exon 10 that havebeen examined lead to a neuronal tau pathology madeof Alzheimer-type paired helical and straight filaments,which consist of all six tau isoforms.21,35

Four intronic mutations have been described inFTDP-17 at positions 13, 113, 114, and 116 ofthe intron following exon 10 of the tau gene, withthe first nucleotide of the splice-donor site taken as11.4,5 The S305N mutation in exon 10 is also lo-cated close to this splice-donor site.9 All five muta-tions lead to an increase in exon 10 –containing tautranscripts, resulting in the overproduction of four-repeat tau isoforms.4,5,11,14,19,29,36,37 Destabilizationof a stem-loop structure located at the exon 10 –intron boundary is the likely primary effect of thesemutations. Where analyzed, intronic mutations lead toa tau pathology consisting of wide, twisted ribbonsmade of four-repeat tau isoforms in nerve cells andglial cells. This has been shown for familial multiplesystem tauopathy with presenile dementia with the 13mutation,5,38 as well as for familial progressive subcor-tical gliosis11 and Duke family 1684,19 both with the116 mutation.

Here, we describe a novel mutation at position 112of the intron following exon 10 of the tau gene in theKumamoto frontotemporal dementia pedigree and wepresent the ensuing biochemical and morphologicalabnormalities.

Patients and MethodsPedigreeThe kindred, called the frontotemporal dementia-Kumamoto(FTD-K) family, was ascertained through the Kikuchi Na-tional Hospital, Kumamoto, Japan. Some of the clinical andneuropathological features of FTD-K have been described.39

The pedigree is shown in Figure 1. The proband’s mother,who had 8 children by two different husbands, had beenhealthy until death. The proband’s father (Patient II-4), 1sister (Patient III-4), and 3 brothers (Patients III-1, III-5,and III-6) exhibited parkinsonism and dementia in their

early 50s. The mean age of onset of disease was 53 years andthe mean duration of illness was 7 years (n 5 6).

At age 56, the proband (Patient III-3) experienced prob-lems in doing his job and memory impairment was noticed.He had difficulty controlling his behavior and became occa-sionally aggressive. The symptoms worsened gradually, sothat he could no longer care for himself. When the probandwas admitted to the hospital at age 59, neurological exami-nation showed diminished spontaneous speech, bradykinesiawith muscle rigidity, increased deep tendon reflexes, andforced grasping. Magnetic resonance imaging revealed ven-tricular dilatation and marked brain atrophy that was mostpronounced in the frontal and temporal lobes of the cerebralcortex, as presented before.39 The proband’s condition wors-ened significantly over the next 3 years, by which time hewas severely demented and bedridden. He died of recurrentpneumonia at age 65.

NeuropathologyFourteen blocks were taken from the right hemisphere of thebrain, fixed in 10% formalin, and embedded in paraffin;8-mm sections were cut, stained with hematoxylin–eosin,Luxol Fast Blue–-cresyl violet, Holzer, Bodian, periodic ac-id–Schiff, and Alcian blue. Two antibodies directed againstphosphorylated tau protein were used.40 They were specificfor phosphorylated Ser199/202 and phosphorylated Ser396,respectively (in the numbering of the 441-amino acid iso-form of human brain tau22). The left hemisphere was frozenfor biochemical analysis.

Mutation DetectionGenomic DNA was extracted from the temporal cortex ofthe proband’s brain. Tau exons and flanking intronic se-quences were amplified, as described.3 Polymerase chain re-action (PCR) products were purified by using the QiagenPCR purification kit (GmbH, Hilden, Germany). Each exonwas sequenced on both strands by using the BigDye termi-nator sequencing kit (Perkin-Elmer, Norwalk, CT) and rel-evant PCR primers. Sequencing was performed on anABI310 DNA sequencer. In some experiments, the amplifiedDNA was ligated to pCR2.1 (Invitrogen, Carlsbad, CA) andsubjected to T7 autoread sequencing by using an ALFredSequencer (Amersham Pharmacia Biotech, Uppsala, Sweden).To confirm the C-to-T transition at position 112 of theintron following tau exon 10, a corresponding fragment was

Fig 1. Frontotemporal dementia-Kumamoto pedigree. Thearrowhead indicates the proband of the family (Patient III-3).Individuals who developed frontotemporal dementia are indi-cated by black symbols. At-risk children and grandchildren inmore recent generations are not included.

Yasuda et al: Tau 112 Mutation 423

amplified by PCR from 100 ng of genomic DNA from Pa-tient III-3 and from controls by using the forward primer se-quence 10F, 59-GCGTGTCACTCATCCTTTTT-39 and thereverse missense sequence 10T primer, 59-CACAGCACGG-CGCATGGGACGTTT-39, with a single nucleotide mis-match (G to T, underlined) that creates a DraI restriction sitein the mutant sequence. A total of 100 control individualsand 50 patients who fulfilled the clinical criteria of fronto-temporal dementia41 were screened for the FTD-K mutation.For each individual, tau exon 10 was amplified, digestedwith DraI, and examined on 2% agarose gels.

Reverse Transcription–PCR AnalysisTotal RNA was extracted from 200 mg of the proband’sfrontal cortex and the frontal cortex of 10 control individualsby using the RNeasy Mini kit (Qiagen). Reverse transcrip-tion (RT) used 1 mg of total RNA and the cDNA cycle kit(InVitrogen). Tau exons 9 to 11 and 9 to 13 were amplified,as described.4 The 59 ends of the forward primers were la-beled with Cy,5 to allow quantitation of the amplified prod-ucts by using the ALFred DNA sequencer. In preliminaryexperiments, 38 cycles and an annealing temperature of 60°Chad been found to be optimal. After amplification, the PCRproducts were applied to the ALFred DNA sequencer, andtwo peaks were detected for each PCR reaction (327 and418 bp for exons 9–11; 487 and 578 bp for exons 9–13).

Ultraviolet MeltingRNA oligonucleotides extending from nucleotides 25 to119 were prepared by in vitro transcription, using T7 RNApolymerase and synthetic DNA templates. RNA was purifiedby polyacrylamide gel electrophoresis, followed by electroelu-tion, extensive dialysis, and size-exclusion chromatography.Before ultraviolet (UV) melting, the samples were dialyzedagainst 10 mM sodium phosphate buffer, pH 6.7, 0.1 mMEDTA. The final buffer for melting experiments contained ei-ther 150 mM NaCl or no added salt. Melting temperatureswere the same in the presence or absence of added sodiumchloride. UV melting experiments were recorded on a CARYUV/VIS spectrophotometer (Varian, Harbor City, CA)equipped with a temperature-controlled heating unit, as de-scribed.37

Exon TrappingThe pSPL3 vector (Gibco-BRL, Life Technologies, Inc,Rockville, MD) was modified by deleting a 500-bp fragmentbetween the EcoRV (nucleotide 1050) and StuI (nucleotide1550) sites to generate pSPES. This deletion removes a pre-viously described cryptic exon.42 A 177-bp genomic frag-ment that contains tau exon 10 in pSP/WT14 was excisedwith XhoI/BamHI and inserted into pSPES to generate pES/WT1. Nucleotide changes and deletions in tau were per-formed by PCR mutagenesis and sequence alterations con-firmed by dye terminator cycle sequencing with TaqFSDNApolymerase (Perkin-Elmer) and the ABI1377 DNA se-quencer. COS-7 cells were maintained in Dulbecco’s modi-fied Eagle’s medium supplemented with 10% fetal calf se-rum. Transient transfections were performed in triplicate byusing Lipofectamine (Gibco-BRL) in 35-mm wells. Totalcellular RNA was extracted 48 hours later by using the

Trizol reagent (Gibco-BRL). RNA samples were treated withDNAse I before RT; 2 mg of total RNA was reverse tran-scribed with random hexamers by using the GeneAmp RNAPCR kit (Perkin-Elmer). Spliced products were amplified byPCR by using the vector-specific primers SD6 and SA2(Gibco-BRL) and 1 ng of 32P-labeled SA2. PCR reactionswere performed for 18 cycles to obtain linear amplification.Products were resolved on 5% acrylamide gels and quantifiedby using a PhosphorImager.

ImmunoblottingSarkosyl-insoluble tau was extracted from temporal cortex ofthe proband as described.27 Soluble tau was extracted as de-scribed.26 Dephosphorylation with E. coli alkaline phospha-tase (10 U/ml; Sigma Chemical Corp, St Louis, MO) wasperformed as described.43 Tau proteins were run on 10%sodium dodecyl sulfate–polyacrylamide gel electrophoresisand transferred to Immobilon P (Millipore). They wereprobed with the phosphorylation-independent anti-tau se-rum BR133 (1:3,000)22 and the phosphorylation-dependentanti-tau antibodies AT8, M4, C5, and AP422 and detectedby using avidin-biotin-peroxidase (Vector Laboratories, Bur-lingame, CA).

Electron MicroscopyAliquots of resuspended sarkosyl-insoluble tau preparationswere placed on carbon-coated 400-mesh grids and stainedwith 1% lithium phosphotungstate. Micrographs were re-corded at a nominal magnification of 340,000 on a PhilipsEM208S microscope, as described.44 Anti-tau antibodyBR13422 and the phosphorylation-dependent antibody againsttau, AT8, were used at a dilution of 1:100. AT8 recognizestau protein phosphorylated at Ser202 and Thr205.45 Proce-dures for immunoelectron microscopy were as described.44

ResultsNeuropathological FindingsMacroscopically, the brain of the proband (Patient III-3; seeFig 1) exhibited a severe atrophy of frontal and temporallobes with a moderate degree of ventricular dilatation. Mi-croscopic examination revealed extensive neuronal cell losswith concomitant neuropil microvacuolation and fibrillaryastrocytosis, especially in the frontal cortex (Fig 2A). Loss ofnerve cells was most severe in the second, third, fifth, andsixth cortical layers. Nerve cell loss, microvacuolation, andfibrillary astrocytosis were also present in the brainstem, cer-ebellum, and subcortical gray matter, including the caudatenucleus, putamen, globus pallidus, thalamus, hypothalamus,amygdala, and substantia nigra. Degenerating neurons con-tained abnormal inclusions that were immunoreactive withantibodies specific for phosphorylated tau protein (see Fig2B). As reported previously,39 tau-positive inclusions werealso present in glial cells. Classic Pick bodies, neurofibrillarytangles, and neuritic plaques were not observed.

Mutation at Position 112 of the Intron followingExon 10 of the Tau Gene in FTD-KThe proband’s tau gene was analyzed for mutations. Se-quencing of exon 10 and the adjacent intron revealed a het-

424 Annals of Neurology Vol 47 No 4 April 2000

erozygous C-to-T transition located 12 bp from the begin-ning of the intron (Fig 3A). To confirm the presence of thischange, a mismatch primer for PCR–restriction fragmentlength polymorphism was synthesized (see Fig 3B). ThePCR–restriction fragment length polymorphism analysis con-firmed the heterozygote substitution. This change was notobserved in 100 control subjects or in 50 patients with spo-radic frontotemporal dementia.

Thermodynamic Stability of Mutant Tau Exon 10Splicing Regulatory Element RNAWe recorded a set of UV melting curves (optical density 5260 nm as a function of temperature) for the wild-type splic-ing regulatory element RNA and the element with the 112intronic mutation (Fig 4). The melting temperature, definedas the midpoint of the transition from fully structured tofully unstructured RNA, was obtained from the maximum inthe derivative of the UV melting profile. The wild-type reg-

ulatory element had a melting temperature of 72 6 2°C(n 5 4). There was a drop of 15°C in melting temperaturebetween the wild-type sequence and the element with the112 mutation, which had a melting temperature of 57 61°C (n 5 4).

RNA AnalysesBy RT-PCR on control and FTD-K brain, two distinctbands were seen when tau exons 9 to 11 or exons 9 to 13were amplified (Fig 5). By DNA sequencing, the largerbands (487 and 578 bp) were shown to correspond to exon

Fig 2. Neuropathological findings in frontotemporal dementia-Kumamoto brain. (A) Holzer staining of frontal cortex show-ing fibrillary gliosis in layer 2 (arrows), as well as numerousmicrovacuoles (arrowheads) and neuronal cell loss. (B) Nervecells in the caudate nucleus that are immunoreactive with anantibody that recognizes tau protein phosphorylated at Ser199/202. Some of these nerve cells contain tau-immunoreactiveinclusions. Scale bar in B 5 50 mm (for A and B).

Fig 3. Mutation at position 112 of the intron following tauexon 10 in frontotemporal dementia-Kumamoto (FTD-K).(A) DNA sequence of exon 10 and the flanking intron frompatient of the FTD-K pedigree. Asterisk indicates a heterozy-gote of a C and a T nucleotide at position 112. (B) Confir-mation of the mutation by using a forward polymerase chainreaction (PCR) primer of wild-type sequence and a reverseprimer with a single nucleotide mismatch to create a DraI sitein the mutant sequence (see Patients and Methods). The PCRproduct from a normal control (C) was 163 bp long andcould not be digested by DraI. The patient (P) from theFTD-K pedigree was heterozygous for the 112 mutation. Af-ter digestion of the PCR product with DraI, an additional139-bp band appeared. Lane M 5 100-bp ladder.

Yasuda et al: Tau 112 Mutation 425

10–containing tau bands. The shorter bands (327 and 418bp) were tau bands that lacked exon 10. In the FTD-K pa-tient, tau mRNA with exon 10 was predominant, whereas incontrols, tau mRNA without exon 10 was more abundant(see Fig 5).

Exon trapping in transfected COS-7 cells was used to in-vestigate the effect of the 112 intronic mutation on thesplicing in exon 10 (Fig 6). When compared with a wild-type tau exon 10 construct, the construct with the 112 mu-tation led to a significant increase in the proportion of exon10–containing transcripts (see Fig 6).

Sarkosyl-Insoluble Tau and Soluble Tau inFTD-K BrainImmunoblotting of sarkosyl-insoluble tau extracted fromFTD-K brain with anti-tau serum BR133 showed two major

Fig 4. Ultraviolet (UV) melting experiments on wild-type (WT) and 112 mutant human tau exon 10 splicing regulatory elementRNAs. Normalized UV absorbance (Rel. Abs) is shown as a function of temperature. The reduced thermodynamic stability of mu-tant RNA leads to an increase in UV absorbance at a lower temperature than for the WT structure, because of unfolding of theRNA secondary structure. A typical experiment is shown. Similar results were obtained in four separate experiments. The sequencesshown extend from nucleotides 25 to 119, with the first nucleotide of the splice-donor site taken as 11. Exonic sequences areboxed and shown in capital letters. Intronic sequences are shown in small letters, with the 112 mutation circled.

Fig 5. Reverse transcription–polymerase chain reaction(RT-PCR) analysis of tau exon 10 alternative mRNA splicingin brain of patient with frontotemporal dementia-Kumamoto(FTD-K). Agarose gel electrophoresis of PCR products extend-ing from exons 9 to 11 (top) and exons 9 to 13 (bottom).Lane 1 5 100 bp ladder; lanes 2–11 5 controls; lane 12 5FTD-K. Arrows point to the RT-PCR bands.

Š

426 Annals of Neurology Vol 47 No 4 April 2000

bands of 64 and 68 kd apparent molecular mass and a minorband of 72 kd (Fig 7, left). Phosphorylation-dependent anti-tau antibodies AT8, C4, M5, and AP422 also labeled thesebands, indicating that sarkosyl-insoluble tau from FTD-Kbrain is highly phosphorylated (data not shown). After de-phosphorylation with alkaline phosphatase, sarkosyl-insolubletau appeared as two major bands and a minor band thataligned with the recombinant four-repeat tau isoformsof 383 (4R, 0), 412 (4R, 29N), and 441 (4R, 58) aminoacids (see Fig 7, left). Three-repeat tau isoforms were notobserved.

Soluble tau was extracted from temporal cortex of FTD-Kbrain and an age-matched control brain. After alkaline phos-phatase treatment, tau from control brain resolved into fourstrong and two weak bands that aligned with the six recom-binant human brain tau isoforms, in agreement with earlierfindings26 (see Fig 7, right). As shown before,5,11,26,29 similarlevels of three-repeat and four-repeat tau isoforms were de-tected. Soluble tau from FTD-K brain also resolved into sixbands after dephosphorylation. However, when comparedwith tau from control brain, the levels of four-repeat tau iso-forms were increased and those of three-repeat tau isoformsreduced (see Fig 7, right).

Isolated Tau FilamentsIsolated filaments from FTD-K brain appeared as twistedribbons with a more or less regular structure (Fig 8A and B).The ribbon had a maximum width of about 23 nm and aminimum width of about 5 nm, where the ribbon wasviewed edge-on at the position of maximum twist. In themore regular filaments, a central stain-penetrated dark linewas visible, dividing the ribbon into two subfilaments. Thefilaments were strongly labeled by antiserum BR134, whichis directed against the carboxy-terminus of tau (see Fig 8Cand D), and by AT8, a phosphorylation-dependent antibodyagainst tau (see Fig 8E and F).

DiscussionDisease in the FTD-K pedigree begins with progressive per-sonality changes in the sixth decade. Principal clinical fea-tures consist of aggressive behavior, cognitive impairmentprogressing to dementia, pyramidal tract signs, and extrapy-ramidal parkinsonian symptoms. Neuropathologically, thefrontal and temporal lobes of the cerebral cortex, several sub-cortical nuclei, and the substantia nigra are mainly affected.Microscopic features include neuronal cell loss, neuropil vac-uolation, as well as gray and white matter fibrillary gliosis.These clinical and pathological features are typical of FTDP-17. The discovery of a mutation in the tau gene in FTD-K,together with the presence of an abundant filamentous taupathology, indicates that FTD-K does indeed belong to thegroup of FTDP-17.

The FTD-K mutation is located at position 112 of theintron following exon 10 of the tau gene. It is located withina stem-loop structure that is believed to be important for theregulation of alternative mRNA splicing of exon 10.4,5,36,37

Previously, mutations have been described at positions 21,13, 113, 114, and 116 of the stem-loop structure inFTDP-17 families.4,5,9–11,19 The clustering of mutations inthe upper part of the stem37 strongly suggests that this regionis crucial for the quantitative regulation of the splicing in

Fig 6. Exon trapping analysis of the effects of the 112 in-tronic mutation on the splicing in tau exon 10. (A) Autora-diograph of reverse transcription–polymerase chain reaction(RT-PCR) products. Exon 10–containing and exon 10–lack-ing transcripts yield 354-bp and 261-bp bands, respectively.Products were resolved on a 5% acrylamide gel. Lane 1 5empty vector; lane 2 5 wild-type (WT) construct; lane 3 5construct with 112 mutation. (B) Quantitation of exon 10–containing and exon 10–lacking transcripts. Each bar repre-sents the mean 6 SD of three separate transfection experi-ments (p , 0.001 ). WT 5 wild-type.

Fig 7. Sarkosyl-insoluble tau and soluble tau from frontotem-poral dementia-Kumamoto (FTD-K) brain. (Left) Sarkosyl-insoluble tau extracted from temporal cortex of affectedmember from the FTD-K pedigree before (lane 1) and after(lane 2) alkaline phosphatase treatment. The six recombinanthuman brain tau isoforms are shown in lane 3. Tau bandswere visualized by immunoblotting with antiserum BR133.(Right) Soluble tau extracted from temporal cortex of a controlbrain (lane 1) and an affected brain from the FTD-K kin-dred (lane 2). The six recombinant human brain tau isoformsare shown in lane 3. Tau bands were visualized by immuno-blotting with antiserum BR133.

Yasuda et al: Tau 112 Mutation 427

exon 10. As is the case of the other mutations in the stem-loop structure,37 the thermodynamic stability of a synthetictau exon 10 splicing regulatory element RNA was greatly re-duced by the 112 mutation. The reduction in melting tem-perature is likely to be the result of the substitution of themore stable g-c with a wobble g z u pair. By exon trapping,the 112 mutation led to a pronounced increase in the pro-portion of exon 10–containing transcripts, when comparedwith the wild-type construct. Similar results have been ob-tained for other mutations that increase splicing in exon10.4,14,17,31,36,37

By RT-PCR, a large increase in the proportion of exon10–containing transcripts was found in FTD-K brain. At theprotein level, this was reflected by a change in the ratio ofthree-repeat to four-repeat tau isoforms, resulting in a netoverproduction of tau isoforms with four microtubule-binding repeats. Similar findings have been reported for the13, 114, and 116 mutations.5,11,19,29 Overproduction offour-repeat tau is the primary effect of the known mutationsin the intron following exon 10.46 It is believed to lead tothe assembly of four-repeat tau into abnormal filaments in

both nerve cells and glial cells. As expected, sarkosyl-insoluble tau from FTD-K brain contained wide, twisted rib-bons made of hyperphosphorylated tau isoforms with fourrepeats. Wide, twisted ribbons made of four-repeat tau havealso been described in families with the 13 and 116 in-tronic mutations.5,11,19,38 The tau pathology of FTD-K isboth neuronal and glial,39 as is that in other families withmutations in the intron following tau exon 10.1,46

The discovery of mutations in the tau gene in FTDP-17has shown that dysfunction of tau causes neurodegeneration.A major implication that derives from this study is that anormal ratio of functional three-repeat to four-repeat tau iso-forms seems to be essential for preventing neurodegenerationand that overproduction of four-repeat tau is sufficient tolead to neurodegeneration and to cause dementia.46 Thepresent study, which describes a novel mutation in the intronfollowing exon 10, emphasizes the remarkable uniformity ofthe biochemical and morphological abnormalities resultingfrom increased splicing in exon 10 of the tau gene. Themechanisms by which overproduction of four-repeat tauleads to degeneration of nerve cells and glial cells remain tobe discovered.

This study was supported in part by grants from the Hyogo Cre-ation and Technology Association (M.Y.), the U.K. Medical Re-search Council (R.A.C., L.V., G.V., and M.G.), the UK Parkinson’sDisease Society (M.G.S.), and RO1 AG11762 from the NationalInstitute on Aging (G.D.S.).

Presented in part at the International Symposium on Dementia,Kobe, Japan, September 11–13, 1999.

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Fig 8. Electron micrographs of tau filaments isolated fromfrontotemporal dementia-Kumamoto brain. (A and B) Unla-beled filaments showing regular looking ribbon with centralstain-penetrated dark line (A) and less regular twisted ribbon(B). (C–F) The two kinds of filament labeled with anti-tauserum BR134 (C and D) or with anti-tau antibody AT8(E and F). Scale bar 5 100 nm.

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