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Apolipoprotein E affects the central nervous system response to injury and the development of cerebral edema.

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Apolipoprotein E Affects
the Central Nervous System
Response to Injury and
the Development of
Cerebral Edema
John R. Lynch, MD,1,2 Jose A. Pineda, MD,1,3
Duncan Morgan, BS,2 Lin Zhang, MD,4
David S. Warner, MD,1,4 Helen Benveniste, MD, PhD,4
and Daniel T. Laskowitz, MD1,2
Apolipoprotein E has been implicated in modifying neurological outcome after traumatic brain injury, although
the mechanisms by which this occurs remain poorly defined. To investigate the role of endogenous apolipoprotein E following acute brain injury, noninvasive magnetic
resonance imaging was performed on anesthetized mice
following closed head injury. Effacement of the lateral
ventricle was used as a radiographic surrogate for cerebral
edema. At 24 hours following injury, apolipoprotein
E-deficient animals had a greater degree of cerebral
edema as compared to matched controls. In addition, the
brains of apolipoprotein E-deficient animals had a significantly greater upregulation of tissue necrosis factor ␣
messenger ribonucleic acid as compared to controls as
early as 1-hr post injury. Thus, modulation of the endogenous central nervous system inflammatory response may
be one mechanism by which apolipoprotein E affects outcome following acute brain injury.
Ann Neurol 2002;51:113–117
There are three common human isoforms of apolipoprotein E (apoE), designated apoE2, apoE3, and
apoE4.1 Numerous clinical reports have demonstrated
that apoE modifies neurological recovery following
closed head injury in an isoform-specific manner.2–5
To model the effect of endogenous apoE on acute
brain trauma, C57BL/6 mice and apoE-deficient mice
matched for age, sex, and genetic background were
subjected to closed head injury using a stereotactically
guided pneumatic compression device to exert an acute
acceleration/deceleration injury. This injury causes ce-
From the 1Multidisciplinary Neuroprotection Laboratory, and Departments of 2Medicine (Neurology), 3Pediatrics, and 4Anesthesiology, Duke University Medical Center, Durham, NC.
Received Apr 10, 2001, and in revised form Aug 15, 2001. Accepted for publication Sep 5, 2001.
Published online Dec 3, 2001
Address correspondence to Dr Laskowitz, Department of Medicine
(Neurology), Box 2900, Duke University Medical Center, Durham,
NC 27710. E-mail:
rebral edema, which can increase intracranial hypertension and lead to herniation of brain tissue. The latter
represents a significant source of neurological morbidity in the clinical setting following closed head injury.
In order to obtain additional information regarding the
effect of cerebral edema in producing anatomic distortions in situ, we utilized magnetic resonance imaging
(MRI) as a noninvasive assessment measurement of
edema. Astrocytic and microglial activation with the resultant secretion of inflammatory mediators is believed
to promote breakdown of the blood brain barrier and
subsequent development of cerebral edema. In particular, TNF␣ has been suggested to play an important
role in this regard.7 This is of particular interest given
recent in vitro and in vivo data suggesting that one role
of apoE in the injured central nervous system may be
to downregulate glial activation and the endogenous
inflammatory response.8 –12,15 We measured the differential upregulation of TNF␣ messenger RNA (mRNA)
after closed head injury in apoE-deficient mice compared to wild-type controls.
Materials and Methods
All experiments were performed with the approval of the Duke
University Animal Care and Use Committee. Briefly, 8 –16week-old male C57-BL/6 mice and apoE-deficient mice
matched for gender and age that had been previously back bred
for 10 generations to C57BL/6 background were obtained commercially from Jackson Laboratories (Bar Harbor, ME).
Head Injury Model with Controlled Pneumatic
Impact Device
Mice were anesthetized with 4.3% isoflurane in oxygen at a
FiO2 of 50% in an anesthesia induction box for 90 seconds.
The trachea was intubated and the lungs were mechanically
ventilated and anesthetized with 1.4% isoflurane in 50% O2
and 50% N2. Body temperature was maintained at 37°C using surface heating/cooling. Each mouse was positioned in a
stereotactic device. The top of the skull was exposed to identify anatomical landmarks. Animals were subjected to controlled skull impact with a pneumatic impactor (Air-Power,
High Point, NC) using a 2.0mm steel tip impounder at a
controlled velocity (6.0 ⫾ 0.2m/sec) and vertical displacement (3.0mm). The animals were allowed to recover spontaneous ventilation prior to extubation. Following recovery,
mice were allowed free access to food and water.
Assessment of Functional Effects of apoE Following
Head Injury
Neurological severity score: Motor function was assessed at 1
and 24 hours following injury using a modified neuroseverity
score that included beam walking on 3cm, 2cm, and 1cm
beams, ability to exit from circle (30cm in diameter), presence of seeking behavior, hemiplegia, ability to walk straight,
and presence of a startle reflex. This generated a score from 0
(normal) to 10 (moribund). This scale is weighted toward
© 2001 Wiley-Liss, Inc.
DOI 10.1002/ana.10098
motor function, and minor modifications of this scale have
been described previously.6
Magnetic Resonance Imaging
Anesthetized, spontaneously breathing mice were scanned using a 7.1, 15cm bore superconducting Oxford magnet with
shielded gradient coils controlled by a Signa console (General
Electric Medical Systems, Milwaukee, WI). We used a threedimensional diffusion-weighted spin-echo pulse sequence
with the following acquisition parameters: (a) 2.4-hour scan
time; (b) 20ms echo time; (c) 600ms repetition time; (d)
diffusion gradients of 35 Gauss/cm applied in the slice direction; and (e) b value of 965mm2/sec. All images were acquired at a spatial resolution of 58␮m ⫻ 58␮m ⫻
469␮m ⫽ 1.6 ⫻ 10⫺3mm3. The raw data were reconstructed by Fourier transform and displayed as magnitude
Ventricular volumes were measured on the threedimensional diffusion data sets by manually outlining regions
of interest in each hemisphere using National Institutes of
Health Image 1.68.
Wet-to-Dry Method of Assessing Brain
Water Content
Brains were removed from mice 24 hours after injury, dissected along the midline saggital plane, and immediately
weighed. As previously described,17 to determine water content, the brains were then placed in a desiccating oven at
105°C for 48 hours and then reweighed.
Measurement of Cytokine mRNA
At 1 and 24 hours following injury, cohort animals were perfused with normal saline and decapitated. The left and right
hemispheres were separated and placed in a 3cc volume of
Trizol. Prompt homogenization was performed by rapidly
passing the tissue through an 18g and 22g needle respectively
until virtually no tissue debris was visible (mRNA was quantified by RNAse protection assay [RPA]). RNA isolation was
performed using Trizol reagent manufactured by Gibco-BRL
(Carlesbad, CA) and their recommended protocol was followed. RPA results were resolved using a 5% acrylamide gel,
which was subsequently dried and placed on a phosphorimaging screen for overnight exposure. Numerical data were
collected and analyzed using the Molecular Dynamics
Storm威 phosphorimaging system and the ImageQuant威
(Sunnyvale, CA) software package provided. To normalize
for any potential differences in loading conditions, mRNA
was expressed as ratio of gene of interest to the housekeeping
gene GAPDH.
Statistical Analysis
All results were analyzed using Student’s two-tailed t test.
Values are expressed as mean ⫾ standard error of the mean.
Prior to injury, there were no significant differences between C57BL/6 (n ⫽ 7) and apoE-deficient (n ⫽ 7)
mice with respect to weight (32 ⫾ 1gm vs 31 ⫾ 1gm;
p ⫽ 0.3) or functional neurological status as deter-
Annals of Neurology
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mined by a blinded observer on a 10-point neurological scale.6
There was no asymmetry between left and right ventricular volumes in the two noninjured control groups.
However, at 24 hours following injury, we found that
both apoE-deficient and C57BLJ/6 mice had significant effacement of the lateral ventricle ipsilateral to the
closed head injury side compared to the right, noninjured hemisphere (Fig 1). The apoE-deficient mice had
an average decrease in left lateral ventricular volume of
30%, as compared to 15% in C57BL6/J mice ( p ⬍
0.01). Figure 2 shows diffusion images at the level of
dorsal hippocampus from 3 apoE-deficient and 3
C57BLJ/6 mice. All apoE-deficient mice show effacement of the left lateral ventricle ipsilateral to the injury
site and a subtle midline shift. In contrast, the only
one of the C57BLJ/6 mice shows clear ventricle effacement (see Fig 2, top). At 6-week follow-up, there was
no longer any evidence of ventricular asymmetry in either group. Ventricular volumes were symmetric and
similar to controls, suggesting a complete resolution of
cerebral edema.
To demonstrate that radiographic evidence of ventricular effacement is a valid surrogate for cerebral
edema, we directly measured water content in the injured and control hemispheres. Using the standard
wet-to-dry method, we found significantly increased
water content in the injured hemisphere of the apoEdeficient animals as compared to the wild-type controls
(80.12 ⫾ 1.22mg vs 78.35 ⫾ 1.02mg in apoEdeficient and wild-type controls, respectively; n ⫽ 12,
p ⫽ 0.038. Data expressed as mean ⫾ standard error
of the mean). However, there were no significant differences in the control nonlesioned hemisphere of
apoE-deficient and wild-type animals.
To determine whether animals received a uniform
injury, a blinded observer using a 10-point neurological scale assessed neurological function after controlled
head injury, in which 0 represents a normal exam, and
increasing scores represent progressive motor deficit.6
At 1 hour following injury, there were no significant
differences in neurological function between groups
(median ⫽ 5 ⫾ 1 vs 5 ⫾ 2 in wild-type versus apoEdeficient animals, respectively; p ⫽ 0.5). Animals were
reassessed at 24 hours and again there was no difference in motor function between groups (3 ⫾ 1 vs 3 ⫾
1 in wild-type vs apoE-deficient animals, respectively;
p ⫽ 0.9), although neurological function had improved from baseline in both groups.
To determine whether TNF␣ was downregulated as
a function of endogenous apoE in our model of controlled head injury, cohort animals were sacrificed and
perfusion fixed at different time points following injury. Messenger RNA was extracted from brain homogenates, and TNF␣ mRNA levels were quantified
in the injured hemisphere by RNAse protection assay
Fig 1. Eight representative diffusion magnetic resonance images from a three-dimensional data set obtained 24 hours after closed
head injury in a C57BLJ/6 mouse. The injury site is clearly seen as a high signal intensity area in the most superior part of the
cortex (slices 1 and 2). More ventrally the effacement of the left lateral ventricle is clearly seen. We have demonstrated how measuring left and right lateral ventricle volume indirectly assessed the amount of edema. The apoE-deficient mice had an average decrease
in left lateral ventricular volume of 30%, as compared to 15% in C57BL6/J mice (p ⬍ 0.01). Absolute ventricular volumes were
2.83 ⫾ 0.10mm3 at baseline. After injury the volumes were 2.38 ⫾ 0.17mm3 in the wild-type mice and 2.09 ⫾ 0.27mm3 in
the apoE-deficient mice. Seven animals were used per group. Data expressed as mean ⫾ standard error of the mean.
Fig 2. Diffusion images at the level of the dorsal hippocampus acquired 24 hours after closed head injury from 3 apoE-deficient
and 3 C57BLJ/6 mice are shown. Left lateral effacement and a subtle midline shift (as indirectly indicated by the red line) can be
appreciated in all 3 apoE-deficient mice. In contrast, only 1 (no. 9) of the 3 C57BLJ/6 mice shown demonstrates such changes.
(RPA). We found that as early as 1 hour following injury, apoE-deficient mice had significantly greater
TNF␣ mRNA levels compared to control mice (ratio
TNF␣/GAPDH 0.0018 vs 0.0034; p ⫽ 0.006; Fig 3).
Our observations suggest an increase in hemispheric
edema in apoE-deficient animals as compared to wildtype controls at 24 hours following closed head injury.
This is consistent with a prior report demonstrating that
histological damage and long-term behavioral sequelae
were more severe in apoE-deficient mice after head in-
Annals of Neurology
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January 2002
jury.16 The two groups of mice in this study received
the same initial insult using the pneumatic compression
device, but the apoE-deficient mice suffered greater injuries compared to C57BLJ/6 as evaluated by MRI. One
potential mechanism by which this may occur is that
apoE downregulates glial activation and subsequent secretion of inflammatory cytokines such as TNF␣. This
function of apoE is consistent with prior in vitro and in
vivo data.8 –12,15 We did observe a more robust increase
in the levels of TNF␣ mRNA in the brains of apoEdeficient animals, which more than likely reflects an elevation of TNF␣ in the brain after closed head injury.
following acute brain injury, as well as designing novel
therapeutic strategies in this clinical setting.
This work was supported by the National Institutes of Health
(1K08NS01949, T32GM08600, R01NS37235), a Novartis Pilot
Grant, and two Paul Beeson Physician Faculty Awards to D. Laskowitz and H. Benveniste. Imaging was performed at Center for in
vivo Microscopy supported by funds from the National Institutes of
Health Research Grants NCRR P41 RR05959 (Principle Investigator Dr. G. Allan Johnson).
Fig 3. Baseline brain TNF␣ messenger RNA levels were not
significantly different in apoE-deficient vs wild-type mice. At 1
hour following closed head injury, TNF␣ messenger RNA had
greater expression in the brains of apoE-deficient mice as compared to matched wild-type controls (**p ⬍ 0.01). To control
for loading conditions, quantification of genes of interest were
represented as a ratio to the housekeeping gene GAPDH. Six
animals were used per time point. Data represented as
mean ⫾ standard error of the mean.
Although the role of TNF␣ in the injured central
nervous system has not been fully elucidated, it is upregulated by neurons and astrocytes following human
traumatic brain injury and appears to play an integral
role in the breakdown of the blood brain barrier and
subsequent development of cerebral edema in humans.13 In addition, inhibition of TNF␣ in a rodent
model of closed head injury resulted in decrease of
blood brain barrier breakdown and the subsequent development of cerebral edema.14 In this study we demonstrate that apoE-deficient mice exhibit enhancement
of TNF␣ mRNA synthesis following closed head injury relative to wild-type controls.
These results are consistent with prior observations
that apoE suppresses microglial and astrocytic activation in vitro,8,9,11,12,15 and the inflammatory cytokines, including TNF␣, are more robustly upregulated
after injury in apoE-deficient mice in vivo.10 This excess production of TNF␣ might promote more severe
blood brain barrier breakdown and thus more extensive
Our results suggest that the presence of endogenous
apoE might be protective in closed head injury by
modulating glial activation, TNF␣ production and
thus decrease the amount of cerebral edema in the
injured brain. The results of this study may serve as
a framework for understanding clinically relevant
isoform-specific differences in neurological outcome
1. Weisgraber KH. Apolipoprotein E: structure-function relationships. Adv Protein Chem 1994;45:249 –302.
2. Sorbi S, Nacmias N, Piacentini S, et al. ApoE as a prognostic
factor for post-traumatic coma. Nature Med 1995;1:852.
3. Teasdale, GM, Nicoll, JA, Murray, G, et al. Association of apolipoprotein E polymorphism with outcome after head injury.
Lancet 1997;350:1069 –1071.
4. Friedman, G, Froom, P, Sazbon, L, et al. Apolipoprotein
E-epsilon 4 genotype predicts a poor outcome in survivors of
traumatic brain injury. Neurology 1999;52:244 –248.
5. Jordan, BD, Relkin NR, Raydin LD, et al. Apolipoprotein E
epsilon-4 associated with chronic traumatic brain injury in boxing. JAMA 1997;278:136 –140.
6. Chen Y, Constantini S, Trembovler V, et al. An experimental
model of closed head injury in mice: pathophysiology, histopathology, and cognitive deficits. J Neurotrauma 1996;13:557–568.
7. Ramilo O, Saez-Llorens X, Mertsola J, et al. Tumor necrosis
factor alpha/cachectin and interleukin 1 beta initiate meningeal
inflammation. J Exp Med 1990;172:497–507.
8. Laskowitz D, Goel S, Bennett ER, et al. Apolipoprotein E suppresses glial cell secretion of TNF␣. J Neuroimmunology 1997;
76:70 –74.
9. Laskowitz DT, Thekdi A., Thekdi S, et al. Downregulation of
glial activation by apolipoprotein E and apoE-mimetic peptides.
Exp Neurol 2001;167:74 – 85.
10. Lynch JR, Morgan D, Mance J, et al. Apolipoprotein E modulates glial activation and the endogenous central nervous system response. J Neuroimmunology 2001;114:107–113.
11. Barger SW, Harmon AD. Microglial activation by Alzheimer
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Nature 1997;388:878 – 881.
12. Hu J, Ladu MJ, Van Eldik LJ. Apolipoprotein E attenuates
beta-amyloid-induced astrocyte activation. J Neurochem 1998;
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13. Ott L, McClain CJ, Gillespie M, Toung B. Cytokines and metabolic dysfunction after severe head injury. J Neurotrauma
1994;11:447– 472.
14. Shohami E, Bass R, Wallach D, et al. Inhibition of tumor necrosis factor alpha (TNFalpha) activity in rat brain is associated
with cerebroprotection after closed head injury. J Cereb Blood
Flow Metab 1996;16:378 –384.
15. Laskowitz DT, Matthew WD, Bennett ER, et al. Endogenous
apolipoprotein E suppresses LPS-stimulated microglial nitricoxide production. Neuroreport 1998;9:615– 618.
16. Chen Y, Lomnitski L, Michaelson DM, Shohami E. Motor and
cognitive deficits in apolipoprotein-E deficient mice after closed
head injury. Neuroscience 1997;80:1255–1262.
17. Pineda JA, Aono M, Sheng H, et al. Extracellular superoxide
dismutase overexpression improves behavioral outcome from
closed head injury in the mouse. J Neurotrauma 2001;18:
625– 634.
Lynch et al: Apolipoprotein E Affects the Central Nervous System
Novel Heteroplasmic
mtDNA Mutation in a
Family with Heterogeneous
Clinical Presentations
P. Corona, MSc,1 E. Lamantea, MSc,1 M. Greco, PhD,1
F. Carrara, BSc,1 A. Agostino, MSc,1 D. Guidetti, MD,2
M. T. Dotti, MD,3 C. Mariotti, MD,1
and M. Zeviani, MD, PhD1
The protean manifestations of a novel maternally inherited point mutation of the mitochondrial genome are reported. The proband showed isolated, spastic paraparesis.
A brother, who had suffered from a multisystem progressive disorder, ultimately died of cardiomyopathy. Another brother is healthy. The proband’s mother showed
truncal ataxia, dysarthria, severe hearing loss, mental regression, ptosis, ophthalmoparesis, distal cyclones, and
diabetes mellitus. A muscle biopsy performed in the proband failed to show the morphological abnormalities typical of mitochondrial disorders; the activities of respiratory chain complexes were normal. However, complex I
and IV activities were low in the muscle homogenate of
the affected mother and brother. Sequence analysis of
mtDNA showed a heteroplasmic mutation of the tRNAIle
gene (G4284A). The mutation load was approximately
55%, 80%, and 90% in the muscle mtDNA of the proband, his mother, and his affected brother, respectively.
Mutation was undetected in the healthy brother, as well
as in 100 control samples. Several cybrid clones containing homoplasmic mutant mtDNA from the proband
showed significant reductions of complex IV activity and
maximum oxygen consumption rate, compared with homoplasmic wild-type clones derived from the same subject.
Ann Neurol 2002;51:118 –122
Mutations of mitochondrial DNA (mtDNA) are responsible for a wide spectrum of syndromes, often
characterized by a combination of symptoms that affect
muscle, brain, and peripheral nerves, and occasionally
the heart.1,2 In several cases, syndromes are rather well
From the 1Division of Biochemistry and Genetics, National Neurological Institute C. Besta, Milan; 2Division of Neurology, Public
Health Hospital Santa Maria Nuova, Reggio Emilia; 3Neurometabolic Unit, Institute of Neurological Sciences, University of Siena,
Siena; Italy.
Received Jul 24, 2001, and in revised form Sep 7, 2001. Accepted
for publication Sep 10, 2001.
Published online Dec 28, 2001
Address correspondence to Dr Zeviani, Divisione di Biochimica e
Genetica, Istituto Nazionale Neurologico Carlo Besta, via Celoria
11, 20133 Milano, Italy. E-mail:
© 2001 Wiley-Liss, Inc.
DOI 10.1002/ana.10059
defined. In other cases, overlap presentations or the unusual combination of symptoms, slow progression, and
qualitative and quantitative variations in different
members of the same family can complicate the diagnosis.2,3 Additional difficulty can be attributable to the
absence or scarcity of diagnostic clues that are considered typical of mitochondrial encephalomyopathies,
such as morphological abnormalities in the muscle biopsy.4 The family reported exemplifies these diagnostic
Case Report
The family tree is reported in Figure 1. The proband,
subject III-3, is a 35-year-old man with an 8-year history of progressive spastic paraparesis. He was reported
to have bilateral genu valgum and what was characterized as a “clumsy walk” since childhood. A brain magnetic resonance image (MRI) performed when he was
31 years old disclosed the presence of a poroencephalic
cavitation in the medial part of the left frontal lobe,
associated with moderate atrophy of the anterior segment of the corpus callosum. The lesion was attributed
to an old, possibly perinatal, infarction in the territory
of the left callosomarginal artery. Nevertheless, the patient’s psychomotor development was reported to be
normal. He obtained a master’s degree in life sciences
and, in spite of walking problems, practiced several
sports during adolescence. The clinical examination,
carried out when he was 35 years of age, disclosed the
presence of a “pure” spastic paraparesis with bilateral
ankle clonus and extensor plantar reflexes. No other
neurological or systemic abnormalities were found at
physical examination. An electrocardiogram (EKG) and
echocardiogram were both normal. Electromyography
showed a myopathic pattern; the latencies of visual and
Fig 1. Family tree. The proband is indicated by an arrow.
Black symbols indicate the three affected subjects reported in
this study. Asterisk indicates subjects reported to be affected by
a neurologic disorder which could not be investigated further
(see text). The presence of hearing loss or diabetes mellitus in
other members of the pedigree is also indicated.
Table. Respiratory Chain Activities in Muscle Homogenate
NADH:CoQ1 reductase/CS (complex I)
Succinate dehydrogenase/CS (SDH)
Succinate:CoQ1 reductase/CS (complex II)
DBH2:cyt. c reductase/CS (complex III)
Cytochrome c oxidase/CS (complex IV)
ATPase/CS (complex V)
Citrate synthase (CS)a
Pt II-3
Pt III-2
Pt III-3
Control Range
Expressed as nanomoles/min/mg.
CoQ1 ⫽ coenzyme Q1; DBH2 ⫽ decyl-ubiquinol; cyt. c ⫽ cytochrome c.
brainstem-evoked potentials were moderately delayed.
Blood lactate and pyruvate levels were both normal at
rest and after standard exercise. A muscle biopsy was
morphologically normal, as were the activities of the
mitochondrial respiratory chain (RC) complexes in
both muscle homogenate (Table) and cultured fibroblasts (not shown). An older brother of the proband,
subject III-2, had died at 27 years of age of heart failure as a result of rapidly progressive dilating cardiomyopathy. This patient had suffered from petit mal epilepsy during infancy and had a single episode of
generalized tonic–clonic seizures when he was 17; myoclonic jerks of the distal segments of upper limbs, eyelids, and facial muscles developed from age 16 on. Additional neurological symptoms ensued during the
subsequent years, including severe truncal ataxia, moderate dysarthria and dysmetria, bilateral sensorineural
hearing loss, markedly slowed saccades, and change in
mood and personality, which evolved into overt mental
deterioration. Bilateral macular degeneration was noted
when he was 23 years old, and an episode of transitory
hemiparesis on the right side was reported at age 26
years. Computed tomography (CT) scan and MRI of
the brain displayed marked cortical and subcortical atrophy. The electroencephalogram showed the presence
of diffuse slow waves with no paroxysms. Visual and
brainstem-evoked potentials were reduced with severely
delayed latencies; nerve conduction velocity was also
moderately reduced. Additional symptoms included diabetes mellitus type 2 since age 18 years, the presence
of a single lipoma on the back of the neck, and a hypogonadal appearance. Endocrine tests suggested a hypogonadotropic hypogonadism attributable to hypothalamic dysfunction. At age 25, rapidly progressive
heart failure developed due to hypertrophic-dilating
cardiomyopathy, leading to death at age 27 years. An
EKG performed at age 25 showed the presence of a
complete left bundle branch block and ischemic abnormalities. The echocardiogram showed a reduced ejection fraction with hypomotility of a dilated and hypertrophic left ventricle. Blood lactate at rest was 3.5
mg% (normal values ⬍2 mg%). A muscle biopsy failed
to show ragged-red fibers. Histochemical analysis was
not performed. However, biochemical assays, carried
out years later in our laboratory, disclosed a dramatic
decrease in complex IV (COX) activity and a less profound decrease of complex I and complex V activities
The mother of the proband, subject II-3, is a 64year-old woman with a 10-year history of profound bilateral hearing loss, type 2 diabetes mellitus, and a
complex neurological syndrome, including truncal
ataxia, mild dysarthria, myoclonic jerks of the hands
and forearms, proximal muscle weakness, severe ophthalmoparesis, and mild mental deterioration. Blood
lactate at rest was normal (0.9 mg%). Both the EKG
and the echocardiogram showed no abnormalities.
MRI of the brain disclosed cortical and subcortical diffuse atrophy. A muscle biopsy showed that the homogenate disclosed a combined reduction of the activities
of complex I and complex IV (Table).
A younger brother of the proband (subject III-4) is a
33-year-old, apparently healthy, man. Other members
of the maternal lineage of this family were reported to
be affected. In particular, subject I-2, the proband’s
maternal grandmother, had adult diabetes mellitus and
profound hearing loss. The latter was the major complaint of the older maternal aunt of the proband (II-1),
whose only daughter (III-1) was reported to be affected
by muscular dystrophy. A maternal uncle (III-5) had
died at 8 years as a result of “encephalopathy.”
Morphological and Biochemical Analyses
Morphological examination of skeletal muscle and biochemical assays of the individual RC and citrate synthase (CS) on
muscle homogenate and digitonin-treated fibroblasts were
carried out as described.5 The maximum oxygen consumption rate was measured in intact cells by polarography6 in the
presence of 25 ␮M dinitrophenol (DNP).
Analysis of mtDNA
Southern blot analysis of linearized mtDNA, single-strand
conformation polymorphism analysis of the 22 mitochon-
Corona et al: Novel Mutation in tRNAIle mtDNA Gene
Fig 2. (A) Sequence analysis of muscle
mtDNA from patient II-3. Asterisk indicates the mutant A at position 4284; note
the smaller underneath peak corresponding
to the wild-type G. (B) Proposed tRNA Ile
cloverleaf-like secondary structure. Arrow
indicates the 4284G. (C) Tsp509I–restriction fragment length polymorphism
(RFLP) analysis of muscle mtDNA of a
control muscle (C) and of affected subjects
(III-3, II-3, III-2), as well as lymphocyte
mtDNA of the proband’s healthy brother
(III-4). (D) Biochemical activities in cybrid clones. Scattergrams of COX-to-CS
activity ratios (left panel) and maximum
oxygen consumption rate (right panel) in
143B.206-derived cybrid clones containing
100% mutant (m) and 100% wild-type
(wt) mtDNA, both derived from the proband. Horizontal bars indicate the mean
drial tRNA genes, and automated sequence analysis were carried out as described.7,8 A 150-bp fragment encompassing
nucleotides (nt) 4230 – 4380 of mtDNA9 was polymerase
chain reaction (PCR)-amplified, using suitable primers. The
4284G3 A transition creates a novel Tsp509I restriction site
(AATT). A second Tsp509I site is present at nt4369. Thus,
Tsp509I cuts the wild-type (wt) fragment into two fragments
of 140 and 10 bp, while the mutant fragment is cut into
three fragments of 86, 54, and 10 bp. The fragments were
separated by 8% acrylamide–tris-borate-EDTA gel electrophoresis and visualized under ultraviolet (UV) light by
ethidium bromide staining. The proportion of mutant versus
total mtDNA was calculated by densitometry.
Fibroblast and Cybrid Cell Cultures
Transmitochondrial cybrids were obtained by polyethylene
glycol fusion of 143B.206-derived ␳° cells with probandderived enucleated fibroblasts, followed by selection in a
uridine-free medium.6,10
Respiratory Chain Complexes in Muscle Homogenate
The Table reports the values of the specific activities of
the RC complexes normalized to the specific activity of
CS, measured in muscle homogenates. No abnormality
was detected in the proband’s sample, while combined
reduction of different mtDNA-dependent RC complexes was present in the muscle homogenate of his
brother and mother.
Identification and Quantitative Analysis of a
4284G3 A Transition
The biochemical results suggested the presence of an
mtDNA mutation, likely involving a tRNA gene.
Southern blot analysis of muscle mtDNA excluded the
presence of large-scale rearrangements. SSCP and nu-
Annals of Neurology
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January 2002
cleotide sequence analysis failed to show mutations in
all the 22 mtDNA tRNA genes with the exception of a
G3 A heteroplasmic transition at nucleotide position
4284 (Fig 2A), in the tRNAIle gene (see Fig 2B).
Tsp509I–RFLP analysis (see Fig 2C) showed that the
relative mutant load was approximately 50%, 80% and
90% in the muscle samples of the proband, his mother
and affected brother, respectively. No mutation was detected in the blood lymphocyte DNA and urinary epithelial cells of the healthy brother of the proband, as
well as in 100 blood DNA samples from healthy, unrelated Italian subjects. The 4284G3 A mutation load
was also measured in the proband’s hair follicles
(⬃50%), urinary mucosal cells (⬃40%), skin fibroblasts (⬃30%), buccal smear (⬃20%), and blood lymphocytes (⬃20%), and was ⬃20% in the urinary cells
and ⬃30% in blood lymphocytes of the mother.
Biochemical Assays in Cybrids
A highly significant mean reduction of COX-to-CS activity ratio was demonstrated in a group of 10 homoplasmic mutant clones (20 ⫾ 7 SD) compared with
10 homoplasmic wt clones (33.8 ⫾ 7.6, unpaired twotailed Student’s t test p ⫽ 0.0005) (see Fig 2D, left
panel). Likewise, the maximum oxygen consumption
rate was significantly reduced in a group of eight 100%
mutant clones (3.74 fmol O2/cell ⫾ 1.22 SD) compared with eight 100% wt clones (6.29 fmol O2/cell ⫾
1.89 SD, Student’s t test p ⫽ 0.0062) (see Fig. 2D,
right panel).
The 4284G3 A is the tenth mutation in the mtDNA
tRNAIle gene to be associated with disease (see www. Four mutations have
been reported in patients affected by chronic progressive external ophthalmoplegia,11–14 while five additional mutations were found in individuals affected by
cardiomyopathy of variable severity.15–19 Although the
pathogenetic mechanisms of some of these mutations
have not been characterized completely, the tRNAIle
gene can well be considered a major mutational hotspot in mtDNA disorders. The 4284G3 A transition
affects the boundary between the DHU and the anticodon stems of the putative tRNAIle cloverleaf, a region of uncertain functional significance, and involves
a poorly conserved nucleotide. Thus, its functional
consequences cannot be deduced by an obvious deleterious effect on the structure or function of tRNAIle or
by evolutionistic considerations. However, several lines
of evidence support the pathogenicity of the
4284G3 A transition:
1. It was heteroplasmic and, as often observed in
pathogenic mutations of mtDNA, the mutant
load was higher in a postmitotic tissue (ie, muscle), than in rapid turnover tissues.
2. The degree of heteroplasmy in muscle was concordant with the clinical severity, which was relatively low in the proband, who was affected by
isolated spastic paraparesis, higher in his mother,
affected by a late-onset multisystem neurological
disorder, and even higher in the proband’s
brother, who suffered from juvenile-onset rapidly
progressive encephalocardiomyopathy.
3. The mutation segregated with the disease, which
was undetectable in the blood lymphocytes of a
cohort of 100 control subjects, and in the blood
and urinary epithelium of a normal family member (subject III-4), although the presence of the
mutation cannot be excluded in other tissues of
this subject.
4. The mutation load was correlated with impairment of respiratory chain activities in muscle homogenates. In addition, statistically significant
differences in the activities of COX and maximum oxygen consumption rate were obtained in
two populations of cybrid clones carrying homoplasmic mutant and homoplasmic wt mitochondrial genomes, respectively. As frequently
observed in mitochondrial disorders, the threshold at which the mutant load leads to an overt
biochemical impairment appears to be higher
than the threshold at which clinical symptoms
are produced, as the RC activities were within
the normal range in the muscle sample from a
clinically affected patient, ie, the proband. In addition, some of the 100% mutant cybrid clones
displayed biochemical activities within the normal range, suggesting a relatively mild deleterious
effect of the mutation on the RC biochemistry,
as measured by our standard assays.
5. The complex clinical and laboratory features in
the mother and affected brother of the proband
were indeed suggestive of a mitochondrial cause.
The onset of rapidly progressive heart failure in subject III-2 confirms the frequent association between
mutations of tRNAIle gene and cardiomyopathy. Patient III-3, the proband, was affected by isolated spastic
paraparesis. In this patient, morphological examination
of the muscle biopsy was completely uninformative.
The possibility of a mitochondrial cause was considered only for the presence of a suggestive family history. A causative link between the poroencephalic lesion shown by MRI and the mtDNA mutation found
in this patient remains an interesting, but unproved,
hypothesis. Although pyramidal signs, including spastic
paraparesis, have been reported in mtDNA-related disease, they are usually accompanied by other neurological and extraneurological symptoms. We cannot exclude that additional clinical features will present in the
future in our patient, but it is tempting to speculate
that his monosymptomatic, relatively benign, clinical
course can be correlated with the lower mutation load
found in this subject, compared with the mutation
load detected in his more severely affected relatives.
This work was supported by Fondazione Telethon-Italy (1180) (to
M.Z.), by Ricerca Finalizzata Ministero Sanitá. (ICS 030.3/
RF98.37), and by Fondazione Pierfranco e Luisa Mariani.
We are indebted to Ms B Geehan for revising the manuscript. We
thank Dr Thomas Klopstock for critical discussion and Dr Marina
Mora for technical advice.
1. Hirano M, Davidson M, DiMauro S. Mitochondria and the
heart. Curr Opin Cardiol 2001;16:201–210.
2. Smeitink J, van den Heuvel L, DiMauro S. The genetics and
pathology of oxidative phosphorylation. Nature Rev Genet
3. DiMauro S, Andreu AL. Mutations in mtDNA: are we scraping
the bottom of the barrel? Brain Pathol 2000;10:431– 441.
4. Zeviani M, Tiranti V, Piantadosi C. Mitochondrial disorders.
Medicine 1998;77:59 –72.
5. Tiranti V, Munaro M, Sandonà D, et al. Nuclear DNA origin
of cytochrome c oxidase deficiency in Leigh’s syndrome: genetic
evidence based on patient’s derived rho° transformants. Hum
Mol Genet 1995;4:2017–2023.
6. Mariotti C, Tiranti V, Carrara F, et al. Defective respiratory
capacity and mitochondrial protein synthesis in transformant
cybrids harboring the tRNA leu (UUR) mutation associated
with maternally inherited myopathy and cardiomyopathy.
J Clin Invest 1994;93:1102–1107.
7. Zeviani M, Moraes CT, DiMauro S, et al. Deletions of mitochondrial DNA in Kearns-Sayre syndrome. Neurology 1988;38:
1339 –1346.
Corona et al: Novel Mutation in tRNAIle mtDNA Gene
8. Tiranti V, Carrara F, Confalonieri P, et al. A novel mutation
(8342G3 A) in the mitochondrial tRNA(Lys) gene associated
with progressive external ophthalmoplegia and myoclonus.
Neuromusc Disord 1999;9:66 –71.
9. Anderson S, Bankier AT, Barrell BG, et al. Sequence and organization of the human mitochondrial genome. Nature 1981;
290:457– 465.
10. Tiranti V, Munaro M, Sandonà D, et al. Nuclear DNA origin
of cytochrome c oxidase deficiency in Leigh’s syndrome: genetic
evidence based on patient’s derived rho0 transformants. Hum
Mol Genet 1995;4:2017–2023.
11. Chinnery PF, Johnson MA, Taylor RW, et al. A novel mitochondrial tRNA isoleucine gene mutation causing chronic progressive
external ophthalmoplegia. Neurology 1997;49:1166–1168.
12. Silvestri G, Servidei S, Rana M, et al. A novel mitochondrial
DNA point mutation in the tRNA(Ile) gene is associated with
progressive external ophtalmoplegia. Biochem Biophys Res
Commun 1996;220:623– 627.
13. Taylor RW, Chinnery PF, Bates MJ, et al. A novel mitochondrial DNA point mutation in the tRNA(Ile) gene: studies in a
patient presenting with chronic progressive external ophthalmoplegia and multiple sclerosis. Biochem Biophys Res Commun
14. Franceschina L, Salani S, Bordoni A, et al. A novel mitochondrial tRNA(Ile) point mutation in chronic progressive external
ophthalmoplegia. J Neurol 1998;245:755–758.
15. Taniike M, Fukushima H, Yanagihara I, et al. Mitochondrial
tRNA Ile mutation in fatal cardiomyopathy. Biochem Biophys
Res Commun 1992;186:47–53.
16. Merante F, Myint T, Tein I, et al. An additional mitochondrial
tRNA(Ile) point mutation (A-to-G at nucleotide 4295) causing
hypertrophic cardiomyopathy. Hum Mutat 1996;8:216 –222.
17. Casali C, Santorelli FM, D’Amati G, et al. A novel mtDNA
point mutation in maternally inherited cardiomyopathy. Biochem Biophys Res Commun 1995;213:588 –593.
18. Tanaka M, Ino H, Ohno K, et al. Mitochondrial mutation in
fatal infantile cardiomyopathy. Lancet 1990;336:1452.
19. Santorelli FM, Mak SC, Vazquez-Acevedo M, et al. A novel
mitochondrial DNA point mutation associated with mitochondrial encephalocardiomyopathy. Biochem Biophys Res Commun 1995;216:835– 840.
Increase in Hand Muscle
Strength of Stroke Patients
after Somatosensory
Adriana B. Conforto, MD, Alain Kaelin-Lang, MD,
and Leonardo G. Cohen, MD
It has been proposed that somatosensory input in the
form of peripheral nerve stimulation can influence functional measures of motor performance. We studied the
effects of median nerve stimulation on pinch muscle
strength (a function mediated predominantly by median
nerve innervated muscles) in the affected hand of chronic
stroke patients. A 2-hour period of median nerve stimulation elicited an increase in pinch strength that outlasted
the stimulation period. The improvement in muscle
strength correlated with stimulus intensity and was identified in the absence of motor training. These results suggest that somatosensory stimulation may be a promising
adjuvant to rehabilitation of the motor deficits in stroke
Ann Neurol 2002;51:122–125
Somatosensory input can modulate reorganization in
sensorimotor cortical and subcortical structures.1– 4 It
has been suggested that somatosensory input in the
form of peripheral nerve stimulation (PNS) could contribute to improved motor function after stroke.5 Here,
we studied the effects of median nerve stimulation
(MNS) on pinch strength in the affected hand of
chronic stroke patients in a randomized, crossover design.
Patients and Methods
Eight patients (7 men, 1 woman; mean age 65 years; range
38 – 81 years) with hemiparesis caused by ischemic stroke
participated in this study. The protocol was approved by the
NINDS Investigational Review Board, and all subjects gave
written informed consent. Lesions identified on computerized tomography or magnetic resonance imaging were located in the left internal capsule (3), left pons (1), right in-
From the Human Cortical Physiology Section, NINDS, National
Institutes of Health, Bethesda, MD.
Received Jun 29, 2001, and in revised form Sep 13. Accepted for
publication Sep 14, 2001.
Published online Dec 3, 2001
Address correspondence to Dr Cohen and Dr Kaelin-Lang, Human
Cortical Physiology Section, National Institutes of Health, Building
10, Room 5N23, 10 Center Drive, MSC 1430, Bethesda, MD
20892-1430. E-mail:
© 2001 Wiley-Liss, Inc.
DOI 10.1002/ana.10070
Fig 1. Representation of hand areas where patients reported
paresthesias during the median nerve stimulation session.
ternal capsule and basal ganglia (2), right frontoparietal
cortex, basal ganglia and internal capsule (1), and right pons
(1). The average time after stroke was 5 years and 6 months
(range 14 months to 7 years).
Previously, we performed a pilot study and found that a
2-hour period of median nerve stimulation resulted in an
average increase of 2.55 ⫾ 0.9 Newtons (N) (mean ⫾ standard error [SE]) in pinch strength in 5 chronic stroke patients (unpublished observations). Based on these preliminary
results, we planned the present study in a randomized, prospective, crossover design.
Each patient participated in two different sessions separated by at least 24 hours: 2-hour MNS and 2-hour control
stimulation (CS). Patients were comfortably seated and were
instructed to remain at rest. Background EMG activity recorded from surface electrodes was continuously monitored
during the experiments. In both sessions, silver-silver surface
chloride electrodes (diameter 10mm) were optimally placed
to stimulate the median nerve at the wrist in the affected
arm. Trains of electrical stimulation were delivered at 1Hz
(Grass stimulator S8800 with stimulus isolation unit [SIU],
Grass Instrument Division, Astro-Med Inc., West Warwick,
RI). Each train consisted of five single pulses of 1ms duration delivered at 10Hz.5
These stimulus parameters are thought to preferentially
activate large cutaneous and proprioceptive sensory fibers.6
In the MNS session, stimulus intensity was gradually increased until the patients reported strong paresthesias in the
median nerve territory in the absence of pain (Fig 1). Average stimulus intensity in the MNS session was 47 ⫾ 8.3%
above the minimal stimulus intensity required to elicit paresthesias (mean ⫾ SE; range 22– 83%). This intensity of
stimulation usually elicited compound muscle action potentials smaller than 100␮V from the abductor pollicis brevis
muscle in the absence of visible finger movements. In the CS
session, stimulus intensity was kept immediately below that
required to elicit paresthesias. None of the patients reported
paresthesias in the absence of electrical stimulation. In the
neurological exam, Patient 1 had slightly decreased joint position sense, and Patient 8 had tactile hypoesthesia in the
affected hand. Joint position sense, vibration, tactile, and
pain sensation were normal in the affected hand of the other
6 patients.
Maximal key pinch strength7 of the affected hand was
measured according to a protocol that exhibits good validity
and test-retest reliability7,8 (Jamar dynamometer, Sammons
Preston, Inc., Bolingbrook, IL). Patients held the arm of the
dynamometer between the lateral aspect of the middle phalanx of the index finger and the thumb pad and were instructed to squeeze as hard as they could. Muscle strength
measurements of five consecutive trials were averaged. One
investigator recorded and analyzed muscle strength measurements blind to the intervention type while another investigator administered the interventions.
The order of the sessions was randomized. In the first session, 5 patients received CS and 3 patients received MNS.
Two-tailed, paired t tests were used to compare muscle
strength measurements before and after interventions (data
normally distributed according to Shapiro-Wilk test, significance level set to 0.05). Patient 8 was excluded from this
main comparison because of poor compliance during the CS
session. Linear regression analysis was used to investigate the
relation between changes in muscle strength and stimulus intensity in the MNS sessions (n ⫽ 8).
Muscle strength measurements recorded before the CS
and MNS sessions were comparable: 54.40 ⫾ 10.63N
Fig 2. Absolute changes in muscle strength
(Newtons) in the control stimulation session and the median nerve stimulation
session (n ⫽ 7). Error bars represent standard errors of the mean.
Conforto et al: Somatosensory Stimulation of Stroke Patients
Fig 3. Relation between stimulus intensity
(intensity of median nerve stimulation
[MNS] relative to the threshold to elicit
paresthesias in each individual) and
changes in pinch muscle strength (pinch
muscle strength after median nerve stimulation relative to prestimulation levels) in
the MNS session (n ⫽ 8).
(mean ⫾ SE; range 22.56 –103.59N) and 49.72 ⫾
8.09N (mean ⫾ SE; range 24.53–91.43N), respectively ( p ⫽ 0.22), indicating similar baseline conditions in both sessions. Stimulation resulted in an increase in pinch strength of 2.41 ⫾ 0.74N ( p ⫽ 0.017)
in the MNS session and in a nonsignificant decrease of
1.07 ⫾ 2.4N ( p ⫽ 0.67) in the CS session (Fig 2). In
an “intent to treat analysis,” in the control intervention
there were no significant changes in strength with a
paired t test ( p ⫽ 0.62), whereas in the median nerve
stimulation session there was a significant increase of
2.57N ( p ⫽ 0.006).
Following the MNS session, 2 patients spontaneously
reported that they could “write better” and “hold objects and play cards more accurately”; this perception
lasted for approximately 24 hours. No patients reported any changes after the CS session. The increased
pinch muscle strength identified in the MNS session
correlated well with the intensity of MNS (r ⫽ 0.729,
p ⫽ 0.04, Fig 3).
The main result of this study is that a 2-hour period of
median nerve stimulation increased pinch muscle
strength in the affected hand of stroke patients. This
effect was clearly identifiable in 6 of 8 individuals and
outlasted the stimulation period. The magnitude of the
improvement in pinch strength was similar in this randomized crossover study and in the open-label pilot experiments that we had previously performed. Additionally, increase in muscle strength was proportional to
the intensity of sensory stimulation, suggesting a causal
relationship. These results are consistent with emerging
Annals of Neurology
Vol 51
No 1
January 2002
evidence that somatosensory input can modify motor
Prior experiments demonstrated motor improvement
in the hand of stroke patients after a combination of
motor training and manipulation of sensory input
(Muellbacher and colleagues, unpublished observations) or electrical muscular stimulation over a period
of several weeks or months.11,12
Hand motor deficits play an important role in stroke
disability.13 In some patients, rehabilitative strategies
that are based on motor practice can be difficult or
impossible to implement because of severe muscle
weakness. Our findings in a small sample of stroke patients indicate that a single session of predominantly
sensory nerve stimulation can improve pinch muscle
strength in the absence of practice. This type of intervention may be a promising adjuvant to enhance neurorehabilitative strategies when hand weakness makes
motor training difficult or impossible.
Supported by the National Institutes of Neurological Diseases and
Stroke Intramural Program.
We thank Mark Hallett and Carolyn Wu for helpful discussion and
Devera G. Schoenberg for skillful editing.
1. Asanuma H. Functional role of sensory inputs to the motor
cortex. Prog Neurobiol 1981;16:241–262.
2. Kaas JH. Plasticity of sensory and motor maps in adult mammals. Annu Rev Neurosci 1991;14:137–167.
3. Recanzone GH, Allard TT, Jenkins WM, Merzenich MM. Re
ceptive-field changes induced by peripheral nerve stimulation in
SI of adult cats. J Neurophysiol 1990;63:1213–1225.
4. Fox K, Glazewski S, Schulze S. Plasticity and stability of somatosensory maps in thalamus and cortex. Curr Opin Neurobiol 2000;10:494 – 497.
5. Ridding MC, Brouwer B, Miles TS, et al. Changes in muscle
responses to stimulation of the motor cortex induced by peripheral nerve stimulation in human subjects. Exp Brain Res 2000;
6. Panizza M, Nilsson J, Roth BJ, et al. Relevance of stimulus
duration for activation of motor and sensory fibers: implications
for the study of H-reflexes and magnetic stimulation. Electroencephalogr Clin Neurophysiol 1992;85:22–29.
7. Mathiowetz V, Kashman N, Volland G, et al. Grip and pinch
strength: normative data for adults. Arch Phys Med Rehabil
1985;66:69 –74.
8. Mathiowetz V, Weber K, Volland G, Kashman N. Reliability
and validity of grip and pinch strength evaluations. J Hand
Surg [Am] 1984;9:222–226.
9. Fraser C, Power M, Hobday D, et al. Driving plasticity in adult
human motor cortex improves functional performance after cerebral injury [abstract]. In: Proceedings of the 15th International Congress of Clinical Neurophysiology, May 2001; Buenos Aires, Argentina.
10. Struppler A, Jakob C, Muller-Barna P, et al. Eine neue Methode zur Fruhrehabilitation zentralbedingter Lahmungen von
Arm und Hand mittels Magnetstimulation. Z EEG EMG
11. Cauraugh J, Light K, Kim S, et al. Chronic motor dysfunction
after stroke: recovering wrist and finger extension by
electromyography-triggered neuromuscular stimulation. Stroke
2000;31:1360 –1364.
12. Dimitrijevic MM, Stokic DS, Wawro AW, Wun CC. Modification of motor control of wrist extension by mesh-glove electrical afferent stimulation in stroke patients. Arch Phys Med
Rehabil 1996;77:252–258.
13. Whitall J, McCombe Waller S, Silver KH, Macko RF. Repetitive bilateral arm training with rhythmic auditory cueing improves motor function in chronic hemiparetic stroke. Stroke
2000;31:2390 –2395.
Selective Hippocampal
Neuron Loss in Dementia
with Lewy Bodies
Antony J. Harding, PhD, Bronwyn Lakay, BSc(Hons),
and Glenda M. Halliday, PhD
Hippocampal volume and neuron number were measured
using stereological techniques in pathologically confirmed dementia with Lewy bodies (n ⴝ 8), Parkinson’s
disease only (n ⴝ 4), and controls (n ⴝ 9). We, and
others, have previously shown considerable cell loss in
the CA1 and subiculum subregions in Alzheimer’s disease. In contrast, these regions were spared in dementia
with Lewy bodies where a selective loss of lower presubiculum pyramidal neurons was found. These findings
suggest a selective loss of frontally projecting hippocampal neurons in dementia with Lewy bodies versus those
projecting to temporal lobe regions in Alzheimer’s disease.
Ann Neurol 2002;51:125–128
The hippocampus is a complex structure with multiple
anatomical and functional compartments. Degeneration of the CA1 and subiculum subregions underlies
the dementia found in Alzheimer’s disease (AD),1–3
with gross atrophy of the hippocampus a marker of
this disease.4 – 8 At end stage, similar hippocampal atrophy is found in dementia with Lewy bodies (DLB),9
but the CA1 and subiculum are spared.2,10 The hippocampal atrophy in DLB correlates with atrophy and
Lewy body formation in the frontal lobes, as well as
with the severity of Lewy neurite formation in the
CA2/3 subregions of the hippocampus.9 This suggests
that the subregions of the hippocampus affected in
DLB differ significantly from those affected in AD.
The present study quantifies the density and number
of neurons in the different hippocampal subdivisions
(CA1, CA2, CA3, CA4, dentate gyrus, subiculum, and
presubiculum) comparing cases with DLB to age- and
sex-matched controls, nondemented cases of Parkinson’s disease (PD) only, and previous data published
on cases with AD.1–3
From the Prince of Wales Medical Research Institute and University
of New South Wales, Sydney, Australia.
Received Jun 8, 2001, and in revised form Sep 20. Accepted for
publication Sep 20, 2001.
Published online Dec 3, 2001
Address correspondence to Dr Harding, Prince of Wales Medical
Research Institute, Barker Street, Randwick, Sydney, NSW 2031,
Australia. E-mail:
© 2001 Wiley-Liss, Inc.
DOI 10.1002/ana.10071
Table. Demographics, Quantitative Data and Results of Statistical Analyses
PMD (hr)
Volume (mm3)
Grey matter
White matter
Dentate gyrus
Neuron number (⫻106)
Dentate gyrus
PD only
ANOVA p values
73 ⫾ 3
14 ⫾ 3
76 ⫾ 3
18 ⫾ 8
76 ⫾ 2
23 ⫾ 7
4,967 ⫾ 273
1,772 ⫾ 101
3,195 ⫾ 238
649 ⫾ 56
123 ⫾ 13
168 ⫾ 15
465 ⫾ 55
310 ⫾ 16
56 ⫾ 4
4,050 ⫾ 313
1,768 ⫾ 231
2,282 ⫾ 336
522 ⫾ 30
135 ⫾ 14
198 ⫾ 22
548 ⫾ 51
276 ⫾ 30
58 ⫾ 3
3,796 ⴞ 259
1,603 ⫾ 70
2,193 ⴞ 282
499 ⫾ 30
132 ⫾ 14
171 ⫾ 10
516 ⫾ 41
234 ⴞ 10
51 ⫾ 5
6.1 ⫾ 0.5
2.2 ⫾ 0.2
1.1 ⫾ 0.1
4.7 ⫾ 0.3
10.7 ⫾ 0.7
100 ⫾ 11
100 ⫾ 7
13.2 ⫾ 1.1
37.8 ⫾ 1.0
5.9 ⫾ 0.6
2.4 ⫾ 0.5
1.3 ⫾ 0.2
4.9 ⫾ 0.7
9.8 ⫾ 1.5
100 ⫾ 8
88 ⫾ 19
15.8 ⫾ 1.1
37.1 ⫾ 1.5
6.1 ⫾ 0.3
2.1 ⫾ 0.2
1.1 ⫾ 0.1
4.3 ⫾ 0.6
6.8 ⴞ 0.5
100 ⫾ 11
50 ⴞ 6
13.9 ⫾ 1.4
34.2 ⫾ 1.8
Values in bold are significantly different from control values, p ⬍ 0.05.
Values given as a proportion of control values ⫾ standard error of the mean.
Materials and Methods
Cases were selected using standardized clinicopathological
procedures from our regional brain donor program (approved by Human Ethics Committees) as previously detailed.9 Cases were excluded if they had significant neuropathology (eg, cerebral infarction, head injury, or hepatic
encephalopathy) other than those related to Parkinson’s disease (PD) or AD. Four cases without dementia had PD only,
8 cases had DLB,11,12 and 9 age- and sex-matched controls
were selected. Neocortical neurofibrillary tangles were absent
in all cases and all PD only and 2 DLB cases had no neuritic
plaque. Three DLB cases reached Consortium to Establish a
Registry for Alzheimer’s Disease (CERAD) criteria for probable AD and the remaining three reached CERAD criteria
for definite AD. There were no significant differences in age
or postmortem delay between the groups (Table).
Brains were fixed in 15% buffered formalin for 2 weeks,
the anteroposterior dimensions of each hemisphere recorded,
and the cerebrum embedded in agar, prior to being cut into
3mm thick coronal slices. All tissue blocks containing the
right hippocampus were dissected from the coronal brain
slices, cryoprotected and 50␮m-thick sections cut from the
more posterior end of each hippocampal block using a freezing microtome. Sections were mounted onto glass slides,
stained with cresyl violet, and coverslipped using DPX
All tissue analyses were performed blinded to classification. Quantitation was performed using well-established
published methods.3,9,13 Briefly, the boundaries of the hippocampal subdivisions were delineated (dentate gyrus, CA1,
Annals of Neurology
Vol 51
No 1
January 2002
CA2-3, CA4, subiculum, and presubiculum) according to
the criteria of Duvernoy14 and Amaral and Insausti.15 The
cross-sectional areas of the hippocampal subdivisions in each
section were determined by tracing their boundaries using a
microfiche reader at 19 to 45⫻ magnification, and applying
point counting techniques. The volume of each hippocampal
subdivision was determined by multiplying the sum of the
cross-sectional areas by the distance between the sections using Cavalieri’s principle. The optical disector technique, using the full section thickness (50␮m) as the disector height,
was used to estimate neuronal number for each hippocampal
region, as previously described in detail.3,13 Between cases,
the number of sections quantified varied from 9 to 12, and
the number of disector frames per subregion varied from 14
to 136. The number of nucleolated neurons within the inclusion boundaries of the frame varied between 93 and 276
for the dentate gyrus; 97 and 627 for the CA1; 84 and 293
for the CA2-3; 102 and 243 for the CA4; 87 and 287 for
the subiculum; and 93 and 218 for the presubiculum. Repeated measures of the neuronal number in multiple sections
from multiple cases always gave similar results, even between
different investigators. Neuron density (coefficient of error
range 0.035– 0.054) was determined from the total number
of neurons within the sample volume. Regional neuronal
number was estimated by multiplying the density and volume.
Statistical analysis was performed on Statview 5.0 program
(Abacus Concepts, Berkeley, CA). Group differences were
analyzed using analysis of variance (ANOVA) with post-hoc
Fisher’s least significant difference test. Means ⫾ standard
Fig. (A) Schematic diagram demonstrating
the hippocampus subdivisions: CA1,
CA2–3, CA4, subiculum (SUB), presubiculum (PS), and dentate gyrus (thick black
line around CA4). WM ⫽ white matter.
(B–D) Photomicrographs of 50␮m-thick
frozen cresyl violet-stained sections of the
lower pyramidal layers of presubiculum in
(B) a control case, (C) a nondemented
case with PD only, and (D) a case with
dementia with Lewy bodies (DLB). Note
the obvious decrease in density of the lower
pyramidal neurons in the D compared
with compared to B and C. Scale in D is
equivalent for B and C.
errors are given for all variables and a p value less than 0.05
accepted as statistically significant.
Gross hippocampal atrophy was confined to the white
matter (see Table; ANOVA ⫽ 4.4, p ⫽ 0.027) rather
than the gray matter (ANOVA ⫽ 1.2, p ⫽ 0.32).
However, one hippocampal subregion, the presubiculum, was selectively atrophic in DLB compared with
controls and cases with PD only (ANOVA ⫽ 6.5, p ⫽
0.008). Cellular analysis revealed that the total estimated number of presubicular neurons was significantly decreased in DLB (ANOVA ⫽ 8.1, p ⫽ 0.003).
A separate analysis of the upper clouds of small pyramidal neurons versus the lower larger pyramidal cell
layer of the presubiculum, revealed no significant differences in the number of neurons contained within
the upper clouds. There was, however, a significant
50% decrease in the number of neurons in the lower
pyramidal layer of the presubiculum in DLB (Fig;
ANOVA ⫽ 11, p ⫽ 0.0008). Neuron loss in the presubiculum was similar between cases, regardless of the
presence or absence of neuritic plaque. There was no
other significant neuropathology within the presubiculum, nor was there any significant loss of neurons in
any of the other hippocampal subdivisions in any
group (see Table).
In vivo atrophy of the hippocampus in DLB is less
than that observed for AD,16,17 possibly because of the
selective subregional cell loss observed in the present
study. In DLB, neuronal loss was confined to the pre-
subiculum and Lewy neurites concentrate in the CA2-3
subregion. The presubiculum has not been previously
evaluated in DLB and, together with the CA2-3 subregion, makes up about 25% of hippocampal gray matter volume (see Table). By contrast, in AD the hippocampal cell loss and neuritic pathology concentrates
in the CA1 and subicular subregions,1–3 which together account for over 60% of hippocampal gray matter volume (see Table). Only neuronal densities have
been previously evaluated in the hippocampus of cases
with PD and no dementia.10 However, consistent with
the findings of this and other volumetric studies,9,18
hippocampal neuron number and volume were not reduced in cases with PD only. This data confirms that
hippocampal pyramidal cell loss occurs only in neurodegenerative disorders with clinical dementia syndromes, and that the selective pattern of hippocampal
cell loss may contribute to the different clinical features
observed in these dementia syndromes.
Different hippocampal subregions have significantly
different projections and functions.14,15 The presubiculum differs from other hippocampal regions in that it
has considerable reciprocal connections with the dorsolateral prefrontal cortex,19 whereas the CA1 and subiculum have major reciprocal connections with the entorhinal cortex and innervate the cingulate cortex
through the thalamus.14 The direct prefrontalhippocampal connections are thought to coordinate
working memory tasks,19 whereas the thalamic relays
are important for memory consolidation and retrieval.14 The present study suggests that DLB cases would
have a disruption to working memory because of the
Harding et al: Hippocampal Neuron Loss in Lewy Body Dementia
considerable pyramidal cell loss in the direct hippocampal output to the dorsolateral prefrontal cortex.
Our previous work showing strong correlations between Lewy body formation in the frontal lobe and
frontal and hippocampal atrophy in DLB9 also support
this thesis, and further suggest that frontal afferent
connections with the hippocampus may also be disrupted by Lewy body formation. It would appear that
the direct connections between the frontal lobe and
hippocampus are significantly affected in DLB. Loss of
this pathway would significantly affect memory circuits
in a different manner to the loss of the temporal lobe
connections found in AD.20
We acknowledge support from Parkinson’s New South Wales, Australian Brain Foundation, National Health and Medical Research
Council, Clive and Vera Ramaciotti Foundation, and the Ian Potter
We thank Heidi Cartwright for the figure work and Dr. Jasmine
Henderson for her comments on the manuscript.
1. West MJ, Coleman PD, Flood DG, Troncoso JC. Differences
in the pattern of hippocampal neuronal loss in normal ageing
and Alzheimer’s disease. Lancet 1994;344:769 –772.
2. Lippa CF, Smith TW, Swearer JM. Alzheimer’s disease and
Lewy body disease: a comparative clinicopathological study.
Ann Neurol 1994;35:81– 88.
3. Harding AJ, Wong A, Svoboda M, et al. Chronic alcohol consumption does not cause hippocampal neuron loss in humans.
Hippocampus 1997;7:78 – 87.
4. Laakso M, Partanen K, Riekkinen P, et al. Hippocampal volumes in Alzheimer’s disease, Parkinson’s disease with and without dementia, and in vascular dementia: an MRI study. Neurology 1996;46:679 – 681.
5. Nagy Z, Jobst K, Esini M, et al. Hippocampal pathology reflects memory deficit and brain imaging measurements in Alzheimer’s disease: clinicopathologic correlations using three sets
of pathologic diagnostic criteria. Dementia 1996;7:76 – 81.
6. O’Brien JT, Paling S, Barber R, et al. Progressive brain atrophy
on serial MRI in dementia with Lewy bodies, AD, and vascular
dementia. Neurology 2001;56:1386 –1388.
7. Bobinski M, Wegiel J, Wisniewski HM, et al. Atrophy of hippocampal formation subdivisions with stage and duration of
Alzheimer’s disease. Dementia 1995;6:205–210.
8. Lehtovirta M, Laakso MP, Soininen H, et al. Volumes of hippocampus, amygdala and frontal lobe in Alzheimer patients
with different apolipoprotein E genotypes. Neuroscience 1995;
9. Cordato NJ, Halliday GM, Harding AJ, et al. Regional brain
atrophy in progressive supranuclear palsy and Lewy body disease. Ann Neurol 2000;47:718 –728.
10. Churchyard A, Lees AJ. The relationship between dementia and
direct involvement of the hippocampus and amygdala in Parkinson’s disease. Neurology 1997;49:1570 –1576.
11. Harding A, Halliday G. Simplified neuropathological diagnosis
of dementia with Lewy bodies. Neuropathol Appl Neurobiol
12. McKeith IG, Galasko D, Kosaka K, et al. Consensus guidelines
for the clinical and pathologic diagnosis of dementia with Lewy
bodies (DLB): report of the consortium on DLB international
workshop. Neurology 1996;47:1113–1124.
Annals of Neurology
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13. Harding AJ, Halliday GM, Kril JJ. Variation in hippocampal
neuron number with age and brain volume. Cereb Cortex
1998; 8:710 –718.
14. Duvernoy HM. The human hippocampus. Functional anatomy, vascularization and serial sections with MRI. Berlin:
Springer-Verlag, 1998.
15. Amaral D, Insausti R. Hippocampal formation. In: Paxinos G,
ed. The human nervous system. Melbourne: Academic, 1990:
16. Barber R, Ballard C, McKeith IG, et al. MRI volumetric study
of dementia with Lewy bodies: a comparison with AD and vascular dementia. Neurology 2000;54:1304 –1309.
17. Barber R, McKeith IG, Ballard C, et al. A comparison of medial and lateral temporal lobe atrophy in dementia with Lewy
bodies and Alzheimer’s disease: magnetic resonance imaging
volumetric study. Dement Geriatr Cogn Disord 2001;12:
198 –205.
18. Double KL, Halliday GM, McRitchie DA, et al. Regional brain
atrophy in idiopathic Parkinson’s disease and diffuse Lewy body
disease. Dementia 1996;7:304 –313.
19. Goldman-Rakic P, Selemon L, Schwartz M. Dual pathways
connecting the dorsolateral prefrontal cortex with the hippocampal formation and parahippocampal cortex in the rhesus
monkey. Neuroscience 1984;12:719 –743.
20. Braak H, Braak E, Yilmazer D, et al. Pattern of brain destruction in Parkinson’s and Alzheimer’s disease. J Neural Trans
1996;103:455– 490.
A Novel, Blood-Based
Diagnostic Assay for Limb
Girdle Muscular Dystrophy
2B and Miyoshi Myopathy
Mengfatt Ho, DPhil,1 Eduard Gallardo, PhD,2
Diane McKenna-Yasek, RN, BSN,1
Noemi De Luna, PhD,2 Isabel Illa, MD,2
and Robert H. Brown, Jr., MD, DPhil1
Limb girdle muscular dystrophy 2B and Miyoshi myopathy were recently found to be allelic disorders arising
from defects in the dysferlin gene. We have developed a
new diagnostic assay for limb girdle muscular dystrophy
2B and Miyoshi myopathy, which screens for dysferlin
expression in blood using a commercially available
monoclonal antibody. Unlike current methods that require muscle biopsy for immunodiagnosis, the new
method is simple and entails a significantly less invasive
procedure for tissue sampling. Moreover, it overcomes
some of the problems associated with the handling and
storage of muscle specimens. In our analysis of 12 patients with limb girdle muscular dystrophy 2B or Miyoshi myopathy, the findings obtained using the new assay
are fully consistent with the results from muscle immunodiagnosis.
Ann Neurol 2002;51:129 –133
Limb girdle muscular dystrophy type 2B (LGMD 2B)
and Miyoshi myopathy (MM) have been considered distinct clinical entities because different muscle groups are
involved at onset. LGMD 2B begins with predominantly proximal muscle weakness, whereas initial weakness in MM patients invariably affects the distal musculature. However, both disorders are characterized by
autosomal recessive inheritance, adult onset, and marked
elevations of the muscle enzyme creatine kinase.1
LGMD 2B and MM were recently shown to arise
from defects in a novel gene that encodes dysferlin.2,3
Remarkably, the same mutation in the dysferlin gene
From the 1Day Laboratory for Neuromuscular Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts; 2Department of Neurology, Neuromuscular Diseases
Section, Hospital Santa Creu i St Pau, Universitat Autonoma de
Barcelona, Barcelona, Spain
Received Aug 16, 2001, and in revised form Aug 16, 2001. Accepted for publication Aug 16, 2001.
Published online Dec 28, 2001
Address correspondence to Dr Brown, Day Laboratory for Neuromuscular Research, Massachusetts General Hospital, Harvard Medical School, Bldg. 114, 16th Street, Rm 3125, Charlestown, MA
can cause different clinical presentation, even among
members of the same family.4 – 6 In addition, an anterior distal myopathy was recently linked to dysferlin
mutation.7 Thus, there is considerable clinical heterogeneity in dysferlinopathy. The dysferlin gene is large,
comprising 55 exons that span a genomic region of
⬎150kb.8 It encodes the 237kDa dysferlin protein,
composed of 2,080 amino acids. The function of dysferlin is unknown, but its homology to a Caenorhabitis
elegans protein, fer-1, suggests that it might function in
calcium-mediated membrane fusion or trafficking.9
This hypothesis is supported by recent findings that
dysferlin is membrane associated.10 –12
An accurate diagnosis of dysferlinopathy requires a
combination of clinical evaluation, protein studies (immunoblot or immunohistochemical analysis), or direct
gene analysis. Although the latter approach provides the
most definitive diagnosis, it is costly, time-consuming,
and labor intensive because of the large size of the dysferlin gene. Moreover, defects in the dysferlin gene are
predominantly single nucleotide changes with no evidence of recurrent mutations, gross rearrangements, or
mutational hotspots to aid detection.3,8,10 For these reasons, DNA-based diagnosis is difficult as an initial
screening strategy to distinguish dysferlinopathies from
the other forms of muscular dystrophy.
By contrast, screening for defective protein expression
by immunoblot analysis has proved to be a reliable and
rapid means for differential diagnosis in muscular dystrophies.13 However, current methods require muscle biopsy samples. To avoid this painful and invasive procedure, we sought to develop a new protein-based
diagnosis using nonmuscle tissues and a less invasive
sampling technique. We have previously shown that dysferlin is expressed in multiple tissues,3,11 we therefore investigated its expression in peripheral blood cells. In the
present study, we report that dysferlin is expressed specifically in monocytes and we describe a novel bloodbased diagnostic assay for LGMD 2B and MM.
Patients and Methods
Isolation of Peripheral Blood Mononuclear Cells and
Immunoblot Analysis
Peripheral blood mononuclear cells (PBMC) were isolated
from whole blood of patients and healthy controls by FicollHypaque gradient centrifugation according to the manufacturer’s instructions (Amersham, Buckinghamshire, UK).
PBMC were washed twice in phosphate-buffered saline
(PBS) and lysed in 10 volumes of protein extraction buffer,
M-PER (Pierce, Rockford, IL). Approximately 20␮g of protein was separated on a 4 to 15% gradient sodium dodecyl
sulfate-polyacrylamide gel electrophoresis gel. Immunoblotting was performed according to standard methods, using
primary anti-dysferlin monoclonal antibodies (NCL-Hamlet
from Novacastra, UK) at 1:300 dilution. Immunoreactive
bands were detected with the enhanced chemiluminescence
system (Amersham).
© 2001 Wiley-Liss, Inc.
DOI 10.1002/ana.10080
Fig 1. Dysferlin expression in peripheral blood mononuclear cells (PBMC). (A) Northern blot analysis of dysferlin gene expression in
peripheral blood cells from a healthy individual, with a probe corresponding to nucleotides 5364 –5732 of the dysferlin cDNA. A
7.5kb band corresponding to the dysferlin transcript is detected in peripheral blood cells and skeletal muscle. Additional transcripts
of ⬃4.5kb and 2.0kb are also present in peripheral blood. (B) Western blot analysis of dysferlin expression in multiple tissues, using
the NCL-Hamlet monoclonal antibody. A prominent ⬃230kDa protein is detected in PBMC, but not in white blood cells (WBC),
of two unrelated, healthy individuals. The positive controls include skeletal muscle and brain tissues from mouse and human. (C to
E) Immunocytochemical analysis of dysferlin expression in CD14⫺ cells (C) and CD14⫹ cells (D). (E) Western blot analysis of dysferlin expression in CD14⫹ cells (lane 1) and CD14⫺ cells (lane 2). Immunoblotting of the 42 kDa actin antigen served as a
positive control in both preparations (lanes 1, 2, bottom panel).
Separation of PBMC into CD14⫹ and CD14⫺ cell
PBMC (approximately 107 cells) were mixed with 20␮l of
CD14-coated microbeads (Milteny Biotec, Germany) and incubated at 6 to 12°C for 30 minutes. Unbound microbeads
were removed by washing cells in excess PBS buffer followed
by centrifugation at 300g for 10 minutes. The cell pellet was
resuspended in PBS buffer to a concentration of 2 ⫻ 108
Annals of Neurology
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cells/ml before separation on a MACS apparatus according to
manufacturer’s instructions (Milteny Biotec, Germany).
PBMC were spun onto microscopic slides using a Cytospin
(Shandon, UK) centrifuge. Acetone-fixed preparations were
preincubated in PBS buffer containing 0.5% bovine serum
albumin and 5% normal goat serum. The sections were in-
cubated with NCL-Hamlet (Novacastra, UK), followed by
horseradish peroxidase-labeled secondary antibodies (Jackson
Immunoresearch, PA) and visualized by diaminobenzidine
(Vector Laboratories, Burlingame, CA).
Detection of Dysferlin Expression in Peripheral Blood
Mononuclear Cells
As a first step to develop a nonmuscle diagnostic assay
for dysferlinopathies, we examined dysferlin gene expression in blood cells of healthy individuals by northern blot analysis. A weak but distinct band of ⬃7.5kb,
corresponding to the dysferlin transcript, was detected
in total RNA isolated from blood (Fig 1A). Smaller
transcripts of ⬃4kb and 2.0kb were also detected in
blood cells, suggesting the presence of tissue-specific
spliced variants.
To determine whether the dysferlin protein is expressed, a Western blot of total white blood cells
(WBC), peripheral blood mononuclear cells (PBMC),
brain, and skeletal muscle was screened with an antidysferlin monoclonal antibody (NCL-Hamlet). Despite
the lower levels of dysferlin gene expression in blood
compared with muscle, a prominent ⬃230kDa band
corresponding to the dysferlin protein was detected in
PBMC, skeletal muscle, and brain but was absent in
total WBC (Fig 1B). As PBMC consist primarily of
lymphocytes and monocytes, this result suggests that
dysferlin is expressed in these cell types.
Immunocytochemical Analysis of Dysferlin Expression
To define further the cell type that expresses dysferlin,
we separated PBMC into CD14⫹ and CD14⫺ cells,
using magnetic beads coated with CD14 antibodies.
This approach distinguishes the two main cell types in
PBMC, because CD14 is a specific marker for monocytes. As shown in Figure 1C and D, immunocytochemical analysis showed dysferlin staining only in
CD14⫹ cells, but not in CD14⫺ cells. This result was
confirmed by Western blot analysis (see Fig 1E). Taken
together, these findings indicate that dysferlin is expressed primarily in monocytes. As monocytes constitute only 3 to 7% of total WBC, it is therefore not
surprising that we were unable to detect dysferlin expression in total WBC (see Fig 1A).
Diagnostic Applications of Dysferlin Expression
To evaluate the accuracy of a blood-based diagnostic
assay for dysferlinopathies, we compared dysferlin expression in PBMC and skeletal muscle in 12 patients
with LGMD 2B or MM. In each case, dysferlin deficiency in skeletal muscle was confirmed by Western
Fig 2. Diagnostic screening of dysferlin expression in peripheral
blood mononuclear cells (PBMC) by Western blot analysis.
Lanes 1, 2, 3, 7, 8 contain PBMC from normal healthy controls; lanes 4, 5, 6 (RB 2329, RB 3533, and RB 2079, respectively) are from Miyoshi myopathy (MM) patients (see Table 1 for details of patient genotypes). The effect of delayed
processing of blood samples on the stability of the dysferlin
protein can be seen by comparing samples that were left for 48
hours (lane 1), 36 hours (lane 2), 24 hours (lanes 3, 7), and
6 hours (lane 8) before being processed for immunoblot analysis.
blotting. Conversely, immunoblot analyses of PBMC
showed dysferlin reactivity in controls (all 6 tested) but
was absent in all 12 patients tested (Fig 2, Table), indicating there is an excellent correlation between dysferlin expression in PBMC and skeletal muscle.
We also investigated the effect of delayed processing
of blood samples on the stability of the dysferlin protein. Samples that were processed within 36 hours
showed no evidence of dysferlin degradation while minor degradation can be seen in samples left for 48
hours (see Fig 2). A lower band of ⬃55kDa was detected in control samples that were left for ⱖ24 hours
but not in 6-hour-old samples (see Fig 2), suggesting
that this is a partially degraded dysferlin product, as it
is present only in control, but not in patient, samples.
Thus, only the full-length dysferlin protein should be
used as a diagnostic indicator for dysferlin deficiency.
We report the novel finding that dysferlin is expressed
in monocytes. More importantly, we showed that dysferlin expression in monocytes correlates with its expression in skeletal muscle (see Table). These findings
have led us to develop a new protein-based diagnostic
assay for LGMD 2B and MM patients, without the
need for a muscle biopsy.
The new blood-based diagnostic assay offers several
advantages over current methods of muscle immunodiagnosis. This simple and rapid technique does not require specialized equipment or training. In addition, it
overcomes some of the inherent problems associated
with the handling and storage of muscle specimens.
Ho et al: Limb Girdle Muscular Dystrophy 2B and Miyoshi Myopathy
Table. Evaluation of Dysferlin Deficiency by Immunodiagnosis in Peripheral Blood Mononuclear Cells and Skeletal Muscle
RB 2329
RB 1859
RB 2079
II 1101
II 1102
II 1103
II 1104
II 1303
II 1304
II 1305
II 1306
RB 3533
Dysferlin Expression
Dysferlin Expression
in Skeletal Muscle
Analysis of PBMC
E1883X, 6319 ⫹ 1G
Deletion AG, at 6071
Deletion G at 5966
For positive controls, all 6 PBMC samples obtained from healthy individuals showed positive dysferlin reactivity by Western blot analysis.
MM ⫽ Miyoshi myopathy; LGMD 2B ⫽ limb girdle muscular dystrophy; DACM ⫽ distal anterior compartment myopathy (ref 7); X ⫽ stop
codon; n.d. ⫽ not determined; PBMC ⫽ peripheral blood mononuclear cells; (⫺) ⫽ absence of dysferlin staining.
The most significant improvement offered by the new
assay is the considerably less invasive tissue sampling
method compared to muscle biopsy. Thus, it should
become easier to obtain samples from patients to confirm diagnosis or to guide further testing. Moreover,
collection of patient materials will no longer require
the operating theater, which will be a significant costsavings benefit.
In addition to diagnostic applications, dysferlin expression in monocytes provides a new paradigm to
study the biological function of this protein in a nonmuscle cell type that is readily accessible and amenable
to in-vitro functional assays. Also, for experimental
therapies intended to increase the levels of dysferlin, we
could now track efficacy by monitoring dysferlin levels
in blood, rather than skeletal muscle. LGMD 2B is not
uncommon, accounting for 1 to 25% of limb girdle
dystrophies.14,15 Unlike MM patients, LGMD 2B patients are not readily diagnosed because there is considerable genetic heterogeneity in LGMD, and precise
DNA diagnostic studies are costly and difficult. Thus,
the new assay will be particularly useful as a rapid
screen to identify the dysferlin-deficient subgroup
within LGMDs. More complex DNA analysis can then
be performed selectively in this subgroup, saving both
time and costs.
To date, all reports of dysferlin deficiency are associated with defects in the dysferlin gene.8,10,11 However, it is unknown whether other forms of muscular
dystrophies could cause a secondary reduction in dysferlin levels. Therefore, the new assay will be a useful
tool to address this question. Identification of patients
with reduced dysferlin expression but without defects
in the dysferlin gene could lead to the discovery of proteins that normally associate with dysferlin and help
elucidate the pathophysiological pathway involved in
Annals of Neurology
Vol 51
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January 2002
The authors are very grateful to all the patients who have participated in this study. We thank Drs K. Bejaoui and B. Hosler for the
critical reading of this manuscript.
This work is supported in part by the Muscular Dystrophy Association (USA), the C. B. Day Investment Company, and Fondo de
Investigacı̂on Sanitaria (FIS 99/019-01; FIS 01/0979).
1. Bushby KM. Making sense of the limb-girdle muscular dystrophies. Brain 1999;122:1403–1420.
2. Bashir R, Britton S, Strachan T, et al. A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in
limb-girdle muscular dystrophy type 2B. Nat Genet 1998;20:
37– 42.
3. Liu J, Aoki M, Illa I, et al. Dysferlin, a novel skeletal muscle
gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy. Nat Genet 1998;20:31–36.
4. Illarioshkin SN, Ivanova-Smolenskaya IA, Greenberg CR, et
al. Identical dysferlin mutation in limb-girdle muscular dystrophy type 2B and distal myopathy. Neurology 2000;55:
5. Weiler T, Bashir R, Anderson LV, et al. Identical mutation in
patients with limb girdle muscular dystrophy type 2B or Miyoshi myopathy suggests a role for modifier gene(s). Hum Mol
Genet 1999;8:871– 877.
6. Weiler T, Greenberg CR, Nylen E, et al. Limb-girdle muscular
dystrophy and Miyoshi myopathy in an aboriginal Canadian
kindred map to LGMD2B and segregate with the same haplotype. Am J Hum Genet 1996;59:872– 878.
7. Illa I, Serrano-Munuera C, Gallardo E, et al. Distal anterior
compartment myopathy: a dysferlin mutation causing a new
muscular dystrophy phenotype. Ann Neurol 2001;49:
130 –134.
8. Aoki M, Liu J, Richard I, et al. Genomic organization of the
dysferlin gene and novel mutations in Miyoshi myopathy. Neurology 2001;57:271–278.
9. Achanzar W.E, Ward S. A nematode gene required for sperm
vesicle fusion. J Cell Sci 1997;110:1073–1081.
10. Anderson LV, Davison K, Moss JA, et al. Dysferlin is a plasma
membrane protein and is expressed early in human development. Hum Mol Genet 1999;8:855– 861.
11. Matsuda C, Aoki M, Hayashi YK, et al. Dysferlin is a surface
membrane-associated protein that is absent in Miyoshi myopathy. Neurology 1999;53:1119 –1122.
12. Selcen D, Stilling G, Engel AG. The earliest pathologic alterations in dysferlinopathy. Neurology 2001;56:1472–1481.
13. Anderson LV, Davison K. Multiplex Western blotting system
for the analysis of muscular dystrophy proteins. Am J Pathol
14. Passos-Bueno MR, Vainzof M, Moreira ES, et al. Seven autosomal recessive limb-girdle muscular dystrophies in the Brazilian population: from LGMD2A to LGMD2G. Am J Med
Genet 1999;82:392–398.
15. Fanin M, Pegoraro E, Matsuda-Asada C, et al. Calpain-3 and
dysferlin protein screening in patients with limb-girdle dystrophy and myopathy. Neurology 2001;56:660 – 665.
Association Studies of
Multiple Candidate Genes
for Parkinson’s Disease
using Single
Nucleotide Polymorphisms
Yoshio Momose, MD,1,2 Miho Murata, MD, PhD,2
Kazuhiro Kobayashi, PhD,1 Masaji Tachikawa, MSc,1
Yuko Nakabayashi, BSc,1 Ichiro Kanazawa, MD, PhD,2
and Tatsushi Toda MD, PhD1
We studied 20 single nucleotide polymorphisms in 18
candidate genes for association with Parkinson’s disease.
We found that homozygosity for the V66M polymorphism of the brain-derived neurotrophic factor (BDNF)
gene occurs more frequently in patients with Parkinson’s
disease than in unaffected controls (␹2 ⴝ 5.46) and confirmed an association with the S18Y polymorphism of
the UCH-L1 gene. Our results provide genetic evidence
supporting a role for BDNF in the pathogenesis of Parkinson’s disease.
Ann Neurol 2002;51:133–136
Single nucleotide polymorphisms (SNPs) are singlebase differences in DNA sequences that can be ob-
From the 1Division of Functional Genomics, Department of PostGenomics and Diseases, Osaka University Graduate School of Medicine, Osaka, Japan; 2Department of Neurology, Graduate School of
Medicine, University of Tokyo, Tokyo, Japan.
Received Jun 18, 2001, and in revised form Sep 24, 2001. Accepted
for publication Sep 24, 2001.
Published online Dec 28, 2001
Address correspondence to: Dr Toda, Division of Functional
Genomics, Department of Post-Genomics and Diseases, Osaka University Graduate School of Medicine, 2-2-B9 Yamadaoka, Suita,
Osaka 565-0871, Japan. E-mail:
served between individuals in the population. As such,
they represent high-potential polymorphic markers useful in the study of susceptibility genes for multifactorial
diseases such as Parkinson’s disease (PD) or those related to drug responsiveness.1
PD, one of the most common human neurodegenerative diseases, is characterized by resting tremor, cogwheel rigidity, bradykinesia, and impaired postural reflexes. This disease affects 1 to 2% of persons older
than 65 years of age. Recent studies have indicated the
presence of genetic factors to the pathogenesis of PD.
For example (1) there are large families with a history
of PD, in which the existence of heredity is suggested;
(2) approximately 10% of patients with PD have a
positive family history2; (3) a recent large-scale survey
in Iceland showed that the risk ratio for PD was 6.7
for siblings, 3.2 for offspring, and 2.7 for nephews and
nieces of patients with PD3; and (4) a twin study using
[18F]-dopa PET showed that the concordance rate for
PD, including subclinical cases, is approximately three
times higher in monozygotic twins (55%) than in dizygotic twins (18%).4 Causal genes for Mendelianinherited PD have been reported, including ␣-synuclein
(4q21–23, autosomal dominant [AD]),5 parkin
(6q25.2–27, autosomal recessive [AR]),6 UCH-L1
(4p14, AD),7 PARK3 (2p13, AD),8 PARK4 (4p15,
AD),9 PARK6 (1p35–36, AR),10 PARK7 (1p36, AR).11
Thus far, 84 association studies on 14 genes have examined relationships between PD and polymorphisms12; however, SNPs that influence PD as strongly
as APOE-ε4 influences Alzheimer’s disease have not
been identified.
To identify susceptibility genes for PD and obtain
information helpful for establishing tailor-made medicines, we initiated large-scale association studies using
SNPs in various candidate genes and further surveyed
the correlation between SNPs and susceptibility to various side effects.
Patients and Methods
A total of 232 patients (114 men and 118 women) with PD
and 249 normal controls (127 men and 122 women) were
examined in this study. The mean age of onset was 54.1 ⫾
10.5 (mean ⫾ SD) years [range:24 – 80]; 18 patients showed
early onset of PD (⬍40 years of age), and 13 patients had a
positive family history of PD. Patients who carried parkin
mutations were excluded. All patients and controls were of
Japanese ancestry. Patients were evaluated by certified neurologists specializing in PD.
The diagnosis of idiopathic PD was based on the presence
of two or more of the cardinal features of PD (tremor, rigidity, bradykinesia, and postural instability) and a lack of
other signs or clinical history that might indicate alternative
diagnoses or causes. We were careful to exclude secondary
parkinsonism. Informed consent was obtained from each patient, and approval for the study was obtained from the University Ethical Committees.
© 2001 Wiley-Liss, Inc.
DOI 10.1002/ana.10079
Table 1. Results of Association Studies
Nerve growth factorrelated
Metabolism or transportation of toxins
Familial parkinsonism
SNP Positionb
PD : Control (Allele)
(df ⫽ 1)
G910T (A304S)
A649G (M217V)
G214T (A72S)
A25G (S9G)
G196A (V66M)
G/T ⫽ 447/5 : 472/6
A/G ⫽ 26/438 : 32/464
G/T ⫽ 424/38 : 444/40
A/G ⫽ 319/145 : 339/155
G/A ⫽ 249/213 : 282/190
A545G (H182R)
T116C (V39A)
A1075C (I359L)
A/G ⫽ 450/12 : 486/10
T/C ⫽ 69/391 : 96/396
A/C ⫽ 449/15 : 473/13
T163A (L55M)
A1286C (E429A)
C559T (P187S)
C3435T (I1145I)
C53A (S18Y)
T/A ⫽ 427/29 : 443/45
A/C ⫽ 369/95 : 399/91
C/T ⫽ 270/188 : 300/192
C/T ⫽ 285/179 : 290/204
C/A ⫽ 261/199 : 244/252
SNP names follow nomenclature of the referenced SNP databases.
A of the initial translation site ATG is designated ⫹1. Amino acid changes are indicated in parentheses.
Genomic DNA was extracted from whole blood using
standard methods. Samples were genotyped by allele-specific
oligonucleotide (ASO) hybridization. Briefly, each polymerase chain reaction (PCR) product containing a SNP locus
was transferred to a nylon membrane (Biodyne, Pall, Port
Washington, NY), chemically denatured, and fixed by ultraviolet light. 32P-labeled allele-specific probes were hybridized
to PCR products on the membranes, and washed at 38 to
52°C in 6⫻ SSC. Chi-square analysis was used to evaluate
the associations and Yates correction was carried out to avoid
type I error, when there was a variable of ⬍10. Differences
at p ⬍ 0.05 were considered significant.
Most SNPs were obtained from the SNP database maintained by the Center for Genome Research at the Whitehead
Institute for Biomedical Research, Cambridge, Massachusetts
( and by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee (
We selected 18 genes with potential involvement in
PD on the basis of their actual or potential functions
in the following categories:
1. Dopamine metabolism, receptors, and transportation:
Aromatic L-amino acid decarboxylase (AADC);
catechol-O-methyltransferase (COMT); dopamine
␤-hydroxylase (DBH); dopamine receptor D3
(DRD3); dopamine receptor D5 (DRD5); and
solute carrier family 6, member 3 ⫽ dopamine
transporter (SLC6A3 ⫽ DAT1)
2. Nerve growth factors and their receptors: Brainderived neurotrophic factor (BDNF); ciliary neurotrophic factor (CNTF); nerve growth factor,
␤-polypeptide (NGFB); and neurotrophic tyrosine kinase, receptor, type1 (NTRK1)
Annals of Neurology
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January 2002
3. Metabolism or transportation of toxins: Cytochrome P450 1A2 (CYP1A2); cytochrome
P450 2E1 (CYP2E1); cytochrome P450 2C9
(CYP2C9); PON1 (paraoxonase 1); 5,10methylenetetrahydrofolate reductase (MTHFR);
diaphorase 4 (DIA4); and ATP-binding cassette, subfamily B, member 1 ⫽ multidrugresistance 1 (ABCB1).
4. Familial parkinsonism: Ubiquitin C-terminal hydrolase L1 (UCH-L1)
We genotyped 20 SNPs in the 18 candidate genes.
All SNPs studied are coding SNPs that result in amino
acid changes, except for the ABCB1 SNP, which affects mRNA stability.
Table 1 shows the results of our study. Seven of 20
SNPs (AADCd4, COMTu4, SLC6A3d13, DRD5u15,
NTRK1u7, CYP2E1*2, CYP1A2*2) showed only one
allele in our samples (data not shown). Of the 20 SNPs
tested, two showed association with PD. We found
that homozygosity for the V66M polymorphism of the
BDNF gene is more frequent in PD patients than in
unaffected controls (␹2 ⫽ 5.46, p ⫽ 0.019). In addition, we observed that homozygosity for the S18Y
polymorphism of the UCH-L1 gene is less frequent in
PD patients (␹2 ⫽ 5.41, p ⫽ 0.020) (Table 2).
We examined the association between each SNP and
susceptibility to adverse L-dopa-associated effects such
as dyskinesia, wearing-off-type motor fluctuation, and
neuroleptic malignant syndrome, but we found no significant associations (data not shown).
Our analysis shows that homozygosity for the V66M
polymorphism in the BDNF gene (BDNFu1) is more
Table 2. Frequency of the BDNF Gene Polymorphism (G196A, V66M) and UCH-L1 Gene Polymorphism (C53A, S18Y)
AG or GG
(␹2 ⫽ 3.26, df ⫽ 1, p ⫽ 0.071)
(␹ ⫽ 5.45, df ⫽ 1, p ⫽ 0.020)
(␹2 ⫽ 5.46, df ⫽ 1, p ⫽ 0.019)
(␹2 ⫽ 5.41, df ⫽ 1, p ⫽ 0.020)
PD ⫽ Parkinson’s disease.
frequent in PD patients. The neurotrophin BDNF aids
the survival, growth, and maintenance of neurons in
the central and peripheral nervous systems. It protects
dopaminergic neurons from stresses such as deprivation
of serum or MPTP-induced damage.13 Studies have
shown that BDNF concentrations are reduced in the
nigrostriatal region of the brain in PD as compared
with controls.14
The V66M polymorphism lies within the proBDNF
sequence, which is cleaved posttranslationally. Although how this polymorphism might influence susceptibility to PD remains unclear, some possible scenarios have been suggested. First, the polymorphism
may affect production of mature, biologically active
BDNF by altering proBDNF processing. Lower levels
of mature BDNF may reduce protection of dopaminergic neurons from environmental toxins such as pesticides. Second, Mowla and colleagues15 recently reported that some proBDNF is released extracellularly
and is biologically active, as demonstrated by its ability
to mediate receptor activation. Thus, the V66M polymorphism has the potential to affect the activity of extracellular proBDNF activity as well. Finally, other
SNPs that associate with a more powerful influence on
the onset of PD might exist in linkage disequilibrium
with BDNFu1.
UCH-L1 represents 1 to 2% of total soluble brain
protein and is thought to hydrolyze polymeric ubiquitin and ubiquitin conjugates to produce monomeric
ubiquitin. Immunoreactivity for this protein is found
in Lewy bodies. A missense mutation in the UCH-L1
gene has been reported in relation to PD.7 In that paper, Leroy and colleagues showed that this mutation,
I93M, causes a partial loss of catalytic thiol protease
activity of this enzyme, which may lead to aberrations
in the proteolytic pathway and aggregation of proteins.
Some association studies demonstrated that carriers of
the S18Y polymorphism have a significantly lower risk
of PD.16 –18 Our results are in accordance with these
studies. The amino acid sequence of UCH-L1 is highly
conserved among rat, mouse, and humans, with approximately 95% sequence identity. Isoleucine (I) at
position 93 is conserved in all three species, and mutation of this residue to methionine (M) is causative in
familial PD in a German family. The amino acid at
position 18 is serine (S) or tyrosine (Y) in humans and
alanine (A) in rat and mouse. The amino acid change
at this position may exert a milder functional influence
than the change at position 93. Another study has suggested that the S18Y polymorphism in UCH-L1 does
not influence the risk of the development of PD in the
Australian population.19 Further investigation is
needed to clarify the potential role of this polymorphism in PD. Another coding SNP of UCH-L1,
M124L, was recognized in a French family with PD.20
Although it did not co-segregate and was considered
not to be pathogenic in this family, it would be of
interest to know whether this SNP has an association
with sporadic PD. Considering the significance of the
ubiquitin-protease system in neurodegenerative diseases, the role of UCH-L1 is of great interest.
Recent research has established that SNPs influence
the variability in patients’ responses to drugs. Although
we found no significant results in our current stratified
analyses, we consider it important to establish tailormade treatments by utilizing SNPs for patients with
PD. We are increasing the number of patients and
SNPs in our studies to test for association.
In summary, we report the first genetic association
between BDNFu1 and the onset of PD. This finding
provides the evidence supporting a role for BDNF in
the pathogenesis of PD and information that may
prove useful for future gene therapy approaches using
neurotrophic factors.
This work was supported by research grants from the Ministry of
Health, Labor, and Welfare (H12-Genome-025) and the Ministry
Momose et al: Association Studies for PD by SNPs
of Education, Culture, Sports, Science, and Technology (12204035)
in Japan.
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