An R5L Mutation in a Subject with a Progressive Supranuclear Palsy Phenotype Parvoneh Poorkaj, PhD,1,2 Nancy A. Muma, PhD,3 Victoria Zhukareva, PhD,4 Elizabeth J. Cochran, MD,5,6 Kathleen M. Shannon, MD,6 Howard Hurtig, MD,7 William C. Koller, MD,8 Thomas D. Bird, MD,1,9 John Q. Trojanowski, MD, PhD,4 Virginia M.-Y. Lee, PhD,4 and Gerard D. Schellenberg, PhD1,2,9,10 MAPT, the gene encoding tau, was screened for mutations in 96 progressive supranuclear palsy subjects. A point mutation (R5L) was identified in a single progressive supranuclear palsy subject that was not in the other progressive supranuclear palsy subjects or in 96 controls. Functionally, this mutation alters the ability of tau to promote microtubule assembly. Analysis of soluble tau from different brain regions indicates that the mutation does not affect the ratio of tau isoforms synthesized. Aggregated insoluble tau from subcortical regions was predominantly four-repeat tau with no or one amino terminal insert (0N4R and 1N4R). Insoluble tau from cortical regions also contained 1N3R tau. Thus, the R5L mutation causes a progressive supranuclear palsy phenotype, presumably by a gain-of-function mechanism. Ann Neurol 2002;52:511–516 Progressive supranuclear palsy (PSP) is clinically distinguished by vertical supranuclear gaze palsy, pseudobulbar palsy, axial dystonia, parkinsonism, postural insta- From the 1Geriatric Research Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle Division; 2 Division of Gerontology and Geriatric Medicine, University of Washington, Seattle, WA; 3Department of Pharmacology, Loyola University Medical Center, Maywood, IL; 4Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; Departments of 5Pathology and 6Neurological Sciences, Rush-Presbyterian St. Luke’s Medical Center, Chicago, IL; 7Department of Neurology, University of Pennsylvania, Philadelphia, PA; 8 Department of Neurology, University of Miami, Miami, FL; and 9 Departments of Neurology and 10Pharmacology, University of Washington, Seattle, WA. Received Aug 8, 2001, and in revised form Dec 26, 2001, and Mar 1 and Jun 21, 2002. Accepted for publication Jun 21, 2002. Published online Aug 25, 2002, in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.10340 Address correspondence to Dr Schellenberg, GRECC 182-B, Veterans Affairs Puget Sound Health Care System, 1660 S. Columbian Way, Seattle, WA 98195. E-mail: email@example.com This article is a US Government work and, as such, is in the public domain in the United States of America. bility, and progressive subcortical dementia.1,2 Neuropathological findings include subcortical neuronal degeneration, gliosis, and aggregated tau protein in the form of neurofibrillary tangles (NFTs), glial tangles, and neuropil threads.3 This aggregated tau consists primarily of the 4R isoform. Mutations in MAPT, the gene encoding tau, cause frontotemporal dementia with parkinsonism chromosome 17 type (FTDP-17).4 – 6 Some MAPT mutations (eg, R406W, S305S, and ⌬296N-homozygous) result in a phenotype resembling PSP.7–9 We sequenced MAPT in 96 PSP subjects and identified a single mutation, R L 5 , in 1 subject. This mutation site is unique, because all other FTDP-17 mutations are either in, or near, the tau microtubule (MT) binding region. The R5L subject had PSP by clinical, neuropathological, and biochemical criteria. We investigated this mutation to determine whether it is responsible for the PSP phenotype in this subject. Subjects and Methods Subjects PSP subjects, predominantly white (mean age, 71 ⫹ 6.4 years), were diagnosed by established criteria.10 Subjects with clinical features of multiple system atrophy or other causes of parkinsonism were excluded. Controls included white, healthy, nondemented, young (mean age, 36 ⫾ 12.9 years; n ⫽ 96) and elderly (mean age, 75.0 ⫾ 8.84 years; n ⫽ 106) subjects. R L 5 Subject A 62-year-old female white with a history of poorly defined collagen vascular disease, hyperthyroidism, and uterine fibroids developed gait disorder, postural instability with falling, dysarthria, and micrographia. Magnetic resonance imaging scans showed cerebral atrophy and multiple areas of increased signal intensity in the white matter of both cerebral hemispheres. By the third year of illness, she had normal cognitive function (Mini-Mental State Examination score, 28/30), vertical supranuclear gaze palsy, lid retraction, and a “worried” facial expression. She had severe neck rigidity, with less severe symmetrical rigidity in the arms and legs, moderately severe bradykinesia, and a severe gait disorder, walking only with assistance. She developed severe dysphagia in the fourth year, became bed bound, and died 5 years after onset. There was no cogwheel rigidity, no resting tremor, and no response to L-dopa. No family history information was available. Neuropathology and Biochemistry Paraffin-embedded fixed sections were stained with hematoxylin and eosin, modified Bielschowsky, Gallyas, and thioflavin S, and labeled with anti-tau antibodies. Soluble and sarcosyl-insoluble tau were extracted, and immunoblotting using antibodies Tau14 and Tau46 was performed as described.6 For quantitative immunoblotting, duplicate lanes were incubated with anti-tau (Tau46, 1:1,000) and antisynuclein (LB509, 1:1,000) MAbs, and 125I-labeled mouse IgG was as the secondary antibody.11 Published 2002 by Wiley-Liss, Inc. 511 Results We sequenced all MAPT exons in 96 PSP subjects and 4 controls. Twenty biallelic polymorphical sites were found (Fig 1) where both alleles were observed in PSP and in controls.5,12 One PSP case had a mutation (CGC3 CTC) in exon 1 resulting in leucine replacing arginine at amino acid 5 (R5L; see Fig 1A). This mutation was not found in the other PSP subjects or when exon 1 was sequenced in 198 additional controls. The functional effect of the R5L mutation was eval- uated using an MT assembly assay (see Fig 1B). The mutation delayed assembly initiation and lowered the mass of MTs formed. The assembly rate was faster for R L 5 tau (0.03 U/min) compared with normal tau (0.01 U/min). The R5L brain (1,280 gm, 6.5-hour postmortem interval) did not show cortical atrophy, but ventricles were slightly dilated. The basal ganglia and the cerebellum were not significantly atrophied. The brainstem appeared normal, including pigmentation of the sub- Fig 1. Location and function of the R5L PSP mutation. (A) Mutation analysis. The R5L mutation was detected by DNA sequencing analysis of MAPT that included sequencing in 19 progressive supranuclear palsy (PSP) subjects, all coding exons with flanking intronic sequences (60 nucleotides), all of introns 9 and 10, and the 3⬘ untranslated region (UTR). For the remaining 77 PSP subjects, exons 1 to 4 and 9 to 12 with flanking intronic regions were sequenced.5 Four controls were sequenced for all regions. Some polymorphic sites observed in PSP subjects were previously observed in frontotemporal dementia with parkinsonism chromosome 17 type families. These are a 5⬘ UTR site, exon 4A sites P78L, V165A, D161D, D161N, exon 6 sites H47Y and S53P, an exon 7 site P176P, an exon 8 site T2T, exon 9 site N255N, and a 3⬘ UTR site at nucleotide 290 in exon 13.5 Additional polymorphisms, numbered by nucleotide (nt) location in sequence AC091628, are an intron 1 T/C polymorphism (nt 85,172), 3 intron 2 polymorphisms (C/T at nt 85,368; T/G at nt 87,628; A/G at nt 87,651), 1 intron 3 site (A/G at nt 87,885), and an intron 8 polymorphism (G/A at nt 109,779).12 Sequences presented are from GenBank: human, AAH0058; baboon, AF281310; goat, AAB50785; cow, QRBOT1; rat, JS0306; mouse, AAA40165. (B) Functional analysis of the R5L mutation in MT assembly assays. Normal and R5L recombinant tau (15M), or no tau, were added to tubulin under conditions in which MT assembly is tau dependent. The tau isoform used was the longest form (2N4R, see Fig 3) found in adult human brain. Duplicate preparations of normal and R5L recombinant protein were prepared, and each preparation was examined in at least three independent experiments. A representative result is shown. Recombinant tau was purified and MT assembly assays conducted as previously described.11 512 Annals of Neurology Vol 52 No 4 October 2002 stantia nigra; however, no pigmentation of the locus ceruleus was seen. Microscopic examination showed neuronal loss and astrocytosis in the substantia nigra, locus ceruleus, and cerebellar dentate nucleus. Grumose degeneration was identified in the dentate nucleus, and there was patchy loss of Purkinje cells with sparse torpedoes in the cerebellum. No cortical neuronal loss, ballooned cells, Lewy bodies, or Pick bodies were seen. The centrum semiovale exhibited moderate arteriolosclerosis and focal increased perivascuular spaces containing sparse hemosiderin-laden macrophages, but no infarcts were noted. No myelin or axonal loss was seen on Kluver staining and neurofilament immunohistochemistry, respectively. Using modified Bielschowsky and thioflavin S stains, we found that there were one to two NFTs in the cornu ammonis 1 region of the hippocampus sector, sparse neuritic plaques in the midfrontal cortex, and a single focus of a moderate neuritic plaque density in the in- ferior parietal cortex. No neocortical NFTs were present. The subject did not meet National Institute on Aging/Reagan criteria for Alzheimer’s disease, and the white matter findings could not explain the clinical phenotype. Globose NFTs were identified with Gallyas staining and with tau antibody T46 and phosphorylation-dependent tau antibodies AT8 and PHF-1 (Fig 2A–C). These were most numerous in the putamen, pallidum, subthalamic nucleus, thalamus, substantia nigra, locus ceruleus, and basis pontis. Astrocytic tufts, neuropil threads, and oligodendroglial tangles (coiled bodies) were observed (see Fig 2D). Tau-positive tufted astrocytes were most numerous in the caudate, putamen, and thalamus. Coiled bodies and threads were most numerous in the periaqueductal gray matter and basis pontis. These findings fulfill the pathological criteria for PSP.13 Isolated tau filaments from R5L brain appeared mostly straight with different diameters as described in Fig 2. Examples of neurofibrillary tangles and tau filaments from the R5L brain. (A) PHF1 immunostaining in pons (⫻100 magnification). (B) AT8 immunostaining in the substantia nigra (⫻100 magnification). (C) T46 immunostaining in the thalamus (⫻50 magnification). (D) A tufted astrocyte in putamen labeled with PHF1 (⫻142 magnification). (E) Conventional electron microphotograph of tau filaments isolated from caudate nucleus (⫻120,000 magnification). Dispersed tau filaments were isolated and negatively stained, and immunoelectron microscopy was performed as previously described.11,19 (F) Tau filaments decorated with PHF1 (⫻120,000 magnification). Antibodies dilutions were 1 to 500 for AT8, 1 to 250 for PHF1, and 1 to 5,000 for T46. Poorkaj et al: Progressive Supranuclear Palsy 513 sporadic PSP cases14 (see Fig 2E). The isolated filaments were immunodecorated with PHF1 (see Fig 2F). Similar filaments also were obtained from the temporal cortex and caudate nucleus (not shown). Soluble and insoluble tau were isolated from the R5L brain (Fig 3). For soluble tau, the six isoforms normally found in adults were observed in the R5L case, and the 4R to 3R isoform ratios were similar to normal and Alzheimer’s disease subjects. The amount of soluble tau in the frontal and temporal cortices was 1.5- to 2-fold higher for R5L compared with sporadic PSP and controls (see Fig 3C and D). In parietal and occipital R L 5 cortices, soluble tau levels were not different from controls (not shown). Fig 3. Biochemical analysis of tau from the R5L brain. Soluble (A) and sarcosyl-insoluble tau (B) were purified from the following brain regions: frontal (F), temporal (T), parietal (P), occipital (Oc) cortices, caudate nucleus (Caud), basal forebrain (BF), substantia nigra (N), and thalamus (Thal). For some regions, tau was isolated from gray (g) and white (w) matter, separately. Samples were analyzed with (⫹) or without (⫺) treatment with Escherichia coli alkaline phosphatase (18U/ml, 67°C for 1 hours20) to dephosphorylate tau. Recombinant tau (RT) was a mixture of the six isoforms found in adult human brain. The shortest form, 0N3R, is the protein encoded by tau exons 1, 4, 5, 7, 9, and 11 to 13. 4R forms include exon 10, 1N includes exon 2, and 2N includes exons 2 and 3. Soluble tau in gray matter from frontal and temporal cortices for the R5L brain was quantitated by immunoblot analysis (C) and normalized to endogenous ␣-synuclein. In D, the amount of soluble tau in each brain region was normalized to ␣-synuclein, and the ratio of tau to synuclein is expressed as a percentage of the first control in each brain region analyzed. 514 Annals of Neurology Vol 52 No 4 October 2002 Insoluble tau levels in R5L brain was higher in subcortical areas compared with neocortex (see Fig 3B), and this biochemical neuropathology correlated with the morphological neuropathology. In frontal, temporal, and occipital regions, higher amounts of insoluble tau were found in white compared with gray matter. Cortical gray and white matter insoluble tau consisted of three major bands corresponding to 1N4R, 1N3R, and 0N4R tau. For subcortical areas, there were only two major bands corresponding to 0N4R and 1N4R tau isoforms (see Fig 3B), a pattern typically seen in sporadic PSP.3 Discussion The R5L mutation was identified in a subject who met clinical research criteria for definite PSP.10 Five points of evidence indicate that the R5L mutation is pathogenic for the PSP phenotype. First, the mutation is at a highly conserved position (see Fig 1A). Second, the amino acid substitution is nonconservative. Third, the mutation alters the interaction of tau with tubulin and MTs (see Fig 1B). Fourth, the mutation is not observed in 202 controls or 95 other PSP subjects. Fifth, a different missense mutation, R5H, at the same site, results in PSP in a Japanese subject.15 It is surprising that a substitution at the amino terminal end of tau would affect interactions with MTs, because deletion analysis of tau showed that only the tau MT binding domain region is required for MT binding.16 However, epitope mapping studies suggest an intramolecular interaction between the amino terminal and MT binding domain of tau in PHF tau but not in normal tau.17 The R5L mutation may act by a gain-of function mechanism, resulting in an interaction between the amino terminal segment and the MT binding domain of tau, causing reduced MT binding and a tau conformation resembling PHF tau. Analysis of tau isoforms in soluble fractions from multiple R5L brain regions shows no differences in 4R to 3R ratio compared with control and Alzheimer’s disease samples.11 Thus, the R5L mutation does not alter MAPT alternative splicing. In sporadic PSP subjects, there is an increase in 4R relative to 3R mRNA in brainstem but not frontal cortex.18 Thus, the pathogenic mechanism in the R5L case may be somewhat different from that of typical PSP. The tau isoform pattern in the insoluble fraction, corresponding to the aggregated hyperphosphorylated tau, differs depending on the R5L brain region. In the cortical areas, where minimal pathology is present, insoluble tau consists of 0N4R, 1N3R, and 1N4R (see Fig 3B). In more heavily involved subcortical regions, primarily 0N4R and 1N4R are found. This same pattern of regional difference in insoluble tau isoforms is also observed in sporadic PSP (V. Zhukareva and V.M.-Y. Lee, manuscript in preparation). Interestingly, soluble tau in R5L brain was significantly increased in several cortical areas compared with controls even though these brain regions contained only limited neuropathology. Possibly the R5L mutation alters transcriptional regulation, though additional data is needed to make this a plausible hypothesis. The subject reported here has a mutation in the tau gene and the clinical and pathological phenotypes of PSP. With increasing reports of disease-associated tau mutations with widely varying clinical and pathological features, the nosology of these diseases has become very confused. The classification of a particular patient as PSP or FTDP-17 may be of less significance than an awareness of the variety of clinical and pathological manifestations associated with these mutations. This work was supported by grants from the National Institute on Aging (AG17583, V.M.Y.L., AG11762, G.D.S.; and AG10124, J.Q.T.) and an Eloise H. Troxel Memorial Grant from the Society for Progressive Supranuclear Palsy (P.P.). We thank the families of the patients studied here who made this work possible. References 1. Litvan I. Progressive supranuclear palsy revisited. Acta Neurol Scand 1998;98:468 – 484. 2. Steele JC, Richardson JC, Olszewski J. Progressive supranuclear palsy. Arch Neurol 1964;10:333–359. 3. Sergeant N, Wattez A, Delacourte A. Neurofibrillary degeneration in progressive supranuclear palsy and corticobasal degeneration: tau pathologies with exclusively “exon 10” isoforms. J Neurochem 1999;72:1243–1249. 4. Hutton M, Lendon CL, Rizzu P, et al. Association of missense and 5⬘-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 1998;393:702–705. 5. Poorkaj P, Bird TD, Wijsman E, et al. Tau is a candidate gene for chromosome 17 frontotemporal dementia. Ann Neurol 1998;43:815– 825. 6. Spillantini MG, Murrell JR, Goedert M, et al. Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc Natl Acad Sci USA 1998;95:7737–7741. 7. Pastor P, Pastor E, Carnero C, et al. Familial atypical progressive supranuclear palsy associated with homozygosity for the delN296 mutation in the tau gene. Ann Neurol 2001;49: 263–267. 8. Reed LA, Grabowski TJ, Schmidt ML, et al. Autosomal dominant dementia with widespread neurofibrillary tangles. Ann Neurol 1997;42:564 –572. 9. Stanford PM, Halliday GM, Brooks WS, et al. Progressive supranuclear palsy caused by a novel silent mutation in exon 10 of the tau gene. Brain 2000;123:880 – 893. 10. Litvan I, Agid Y, Calne D, et al. Clinical research criteria for the diagnosis of progressive supranuclear palsy (SteeleRichardson-Olszewski syndrome): report of the NINDS-SPSP international workshop. Neurology 1996;47:1–9. 11. Hong M, Zhukareva V, Vogelsberg-Ragaglia V, et al. Mutationspecific functional impairments in distinct Tau isoforms of hereditary FTDP-17. Science 1998;282:1914 –1917. 12. Poorkaj P, Kas A, D’Souza I, et al. A genomic sequence analysis of the mouse and human microtubule-associated protein tau. Mamm Genome 2001;12:700 –712. Poorkaj et al: Progressive Supranuclear Palsy 515 13. Litvan I, Hauw JJ, Bartko JJ, et al. Validity and reliability of the preliminary NINDS neuropathologic criteria for progressive supranuclear palsy and related disorders. J Neuropathol Exp Neurol 1996;55:97–105. 14. Tomonaga M. Ultrastructure of neurofibrillary tangles in progressive supranuclear palsy. Acta Neurpathol 1977;37: 177–181. 15. Hayashi S, Toyoshima Y, Hasegawa M, et al. Late-onset frontotemporal dementia with a novel exon 1 (Arg5His) tau gene mutation. Ann Neurol 2002;51:525–530. 16. Goode BL, Feinstein SC. Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau. J Cell Biol 1994;124:769 –782. 17. Carmel G, Mager EM, Binder LI, Kuret J. The structural basis of monoclonal antibody Alz50’s selectivity for Alzheimer’s disease pathology. J Biol Chem 1996;271:32789 –32795. 18. Chambers CB, Lee JM, Troncoso JC, et al. Overexpression of four-repeat tau mRNA isoforms in progressive supranuclear palsy but not in Alzheimer’s disease. Ann Neurol 1999;46: 325–332. 19. Iijima M, Tabira T, Poorkaj P, et al. A distinct familial presenile dementia with a novel missense mutation in the tau gene. Neuroreport 1999;10:497–501. 20. Goedert M, Spillantini MG, Cairns NJ, Crowter RA. Tau proteins of Alzheimer paired helical filaments: abnormal phosphorylation of all six brain isoforms. Neuron 1992;8:159 –168. A Novel Mutation in the GNE Gene and a Linkage Disequilibrium in Japanese Pedigrees Aki Arai, MD,1 Keiko Tanaka, MD,1 Takeshi Ikeuchi, MD,1 Shuichi Igarashi, MD,1 Hisashi Kobayashi, MD,1 Tomoya Asaka, MD,2 Hidetoshi Date, MS,1 Masaaki Saito, MD,1 Hajime Tanaka, MD,1 Sari Kawasaki, MD,1 Eiichiro Uyama, MD,3 Hidehiro Mizusawa, MD,4 Nobuyoshi Fukuhara, MD,5 and Shoji Tsuji, MD1 Distal myopathy with rimmed vacuoles (DMRV) is an autosomal recessive muscular disorder characterized by weakness of the anterior compartment of the lower limbs with onset in early adulthood and sparing of the quadricep muscles. The UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) gene was recently identified as the causative gene for hereditary inclusion body myopathy (HIBM). To investigate whether DMRV and HIBM are allelic diseases, we conducted mutational analysis of the GNE gene of six Japanese DMRV pedigrees and found that all the pedigrees share a homozygous mutation (V572L) associated with a strong linkage disequilibrium, suggesting a strong founder effect in Japanese DMRV pedigrees. Ann Neurol 2002;52:516 –519 Distal myopathy with rimmed vacuoles (DMRV), also known as Nonaka distal myopathy (OMIM 605820), is an autosomal recessive muscular disorder.3 The disease develops in early adulthood characterized by weakness of the anterior compartment of the lower limbs and hamstring muscles and sparing the quadricep muscles until its late stage. This disease usually progresses slowly, although sometimes rather rapidly. No laboratory findings such as mildly elevated serum creatine kinase levels or mixed characteristics of myopathic and neuropathic patterns in electromyograms are spe- From the 1Department of Neurology, Brain Research Institute, Niigata University, Niigata; 2Department of Neurology, School of Medicine, Kanazawa University, Kanazawa; 3Department of Neurology, School of Medicine, Kumamoto University, Kumamoto; 4 Department of Neurology, Tokyo Medical and Dental University, Tokyo; and 5Department of Neurology, National Saigata Hospital, Niigata, Japan. Received Jan 3, 2002, and in revised form Jun 11. Accepted for publication Jun 21, 2002 Published online Aug 25, 2002, in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.10341 Address correspondence to Dr Tsuji, Department of Neurology, Brain Research Institute, Niigata University, 1 Asahimachi, Niigata 951-8585, Japan. E-mail: firstname.lastname@example.org 516 Published 2002 by Wiley-Liss, Inc.