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An R5L mutation in a subject with a progressive supranuclear palsy phenotype.

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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;
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;
Department of Neurology, University of Miami, Miami, FL; and
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
( 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:
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,
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
Subjects and Methods
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)
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.
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
5 tau (0.03 U/min) compared with normal tau (0.01
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 (15␮M), 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
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
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
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.
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
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.
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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;
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
( 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:
Published 2002 by Wiley-Liss, Inc.
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