Neurofilament inclusion body disease: a new proteinopathy

Neuro®lament inclusion body disease: a newproteinopathy?

Keith A. Josephs,1,5,7 Janice L. Holton,7 Martin N. Rossor,5 Hans Braendgaard,8 Tetsutaro Ozawa,6,7

Nick C. Fox,5 Ronald C. Petersen,1 Gary S. Pearl,4 Milan Ganguly,7 Pedro Rosa,8 Henning Laursen,9

Joseph E. Parisi,1,2 Gunhild Waldemar,9 Niall P. Quinn,6 Dennis W. Dickson1,3 and Tamas Revesz7

Departments of 1Neurology, 2Laboratory Medicine and

Pathology, Mayo Clinic, Rochester, MN and 3Department

of Pathology (Neuropathology), Mayo Clinic, Jacksonville,

FL, 4Department of Pathology, Orlando Regional Medical

Center, Orlando, FL, USA, 5Dementia Research Group,6Department of Motor Neuroscience and Movement

Disorders and 7Queen Square Brain Bank, Department of

Molecular Neuroscience and Division of Neuropathology,

Institute of Neurology, University College London, London,

UK, 8Aarhus Kommunehospital, Aarhus and 9Copenhagen

University Hospital, Copenhagen, Denmark

Correspondence to: Tamas Revesz, MD, Division of

Neuropathology, Institute of Neurology, University College

London, London WC1N 3BG, UK

E-mail: t.revesz@ ion.ucl.ac.uk

SummaryWe describe four cases of a new clinicopathologicalentity presenting with either a frontotemporal dementiaor corticobasal degeneration syndrome with a mean ageof onset of 45 years (range 41±50) characterized patho-logically by deposition of neuro®lament proteins. Allfour patients had a rapidly progressive course and havebecome mute and non-ambulatory, and three have diedafter mean illness duration of only 3 years (range 21

2±4).±4).

Both structural (MRI) and functional (PET andBoth structural (MRI) and functional (PET andSPECT) imaging demonstrated frontal and temporalSPECT) imaging demonstrated frontal and temporallobe and basal ganglia involvement. Gross neuropatho-lobe and basal ganglia involvement. Gross neuropatho-logical examination in the three deceased patients (thelogical examination in the three deceased patients (thefourth patient, still alive, was diagnosed by brainfourth patient, still alive, was diagnosed by brainbiopsy) revealed changes affecting predominantly thebiopsy) revealed changes affecting predominantly thefrontal and temporal cortices, basal ganglia and brain-frontal and temporal cortices, basal ganglia and brain-stem. There was super®cial linear spongiosis affectingstem. There was super®cial linear spongiosis affectingthe frontal lobes in all three autopsied patients, andthe frontal lobes in all three autopsied patients, andsevere caudate atrophy was noted in two of them andsevere caudate atrophy was noted in two of them anddemonstrated on MRI in the living patient. On routinedemonstrated on MRI in the living patient. On routine

staining, there were numerous intracytoplasmic inclu-staining, there were numerous intracytoplasmic inclu-

sions, which ranged from eosinophilic to basophilic.sions, which ranged from eosinophilic to basophilic.

Some had a clearly de®ned basophilic margin, whileSome had a clearly de®ned basophilic margin, while

others were granular with a hyaline core. With modi-others were granular with a hyaline core. With modi-

®ed Bielschowsky silver technique, a small number of®ed Bielschowsky silver technique, a small number of

the inclusions were intensely stained. Inclusions werethe inclusions were intensely stained. Inclusions were

not labelled with other silver stains. Immuno-not labelled with other silver stains. Immuno-

histochemistry revealed that the inclusions were immu-histochemistry revealed that the inclusions were immu-

noreactive with antibodies to neuro®lament heavy andnoreactive with antibodies to neuro®lament heavy and

light chain subunits and to ubiquitin, but not with anti-light chain subunits and to ubiquitin, but not with anti-

bodies to tau andbodies to tau and aa-synuclein. These neuro®lament- and-synuclein. These neuro®lament- and

ubiquitin-positive inclusions were widespread, speci®cubiquitin-positive inclusions were widespread, speci®c

to neurons and occasionally intranuclear. The frequencyto neurons and occasionally intranuclear. The frequency

and distribution of the inclusions and the silver andand distribution of the inclusions and the silver and

immunohistochemical pro®les in these four cases isimmunohistochemical pro®les in these four cases is

novel and has not been described in detail before. Wenovel and has not been described in detail before. We

propose the term neuro®lament inclusion body diseasepropose the term neuro®lament inclusion body disease

for this entity.for this entity.

Key words: neuro®lament inclusion body disease; frontotemporal dementia; corticobasal degeneration; neuro®lament;

ubiquitin

Abbreviations: AD = Alzheimer's disease; BIBD = basophilic inclusion body disease; CBD = corticobasal degeneration;

CJD = Creutzfeldt±Jakob disease; DLB = dementia with Lewy bodies; DUI 6 MND = dementia with ubiquitin-positive

(tau- and a-synuclein-negative) inclusions with or without motor neuron disease; FTD = frontotemporal dementia; iPD =

idiopathic Parkinson's disease; MSA = multiple system atrophy; NIBD = neuro®lament inclusion body disease; NF =

neuro®lament; PiD = Pick's disease; PSP = progressive supranuclear palsy

Brain 126 ã Guarantors of Brain 2003; all rights reserved

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IntroductionA signi®cant group of neurodegenerative diseases is char-

acterized by intraneuronal inclusions, which may be cyto-

plasmic or intranuclear. An evolving molecular classi®cation

of such neurodegenerative disorders is based upon the

biochemical nature of the protein deposits forming the

inclusions. The microtubule-associated protein tau and

the synaptic vesicle protein a-synuclein account for most of

these neurodegenerative disorders (Goedert et al., 2001).

Abnormal deposition of tau de®nes neuro®brillary tangles in

the most common neurodegenerative disease, Alzheimer's

disease (AD). Furthermore, abnormalities of tau characterize

corticobasal degeneration (CBD), progressive supranuclear

palsy (PSP) and frontotemporal dementia with parkinsonism

linked to chromosome 17q (FTDP-17q), in which glial tau

deposits are also frequently found (Lee et al., 2001). The

morphologically distinct Pick bodies characteristic of Pick's

disease (PiD) are also composed of tau (Lowe, 1998; Lee

et al., 2001; McKhann et al., 2001). The diseases associated

with a-synuclein deposition are idiopathic Parkinson's dis-

ease (iPD) and dementia with Lewy bodies (DLB), in which

the pathological hallmark is the Lewy body (Spillantini et al.,

1997). Lewy bodies are also commonly found in AD,

especially in familial forms (Lippa et al., 1998). The glial

cytoplasmic inclusions and intraneuronal inclusions of mul-

tiple system atrophy (MSA) also contain a-synuclein (Lippa

et al., 1998; Spillantini et al., 1998). Many of the de®ning

intracellular inclusions are also labelled by ubiquitin, which is

an essential component of the proteasomal system, respon-

sible for non-lysosomal degradation of intracellular proteins

(Hershko and Ciechanover, 1998). The presence of ubiquitin

in the inclusions is less speci®c, although it is the only

identi®ed component in the inclusions of dementia with

ubiquitin-positive (tau- and a-synuclein-negative) inclusion

bodies with or without motor neuron disease (DUI 6 MND)

(Rossor et al., 2000). The term DUI 6 MND incorporates

motor neuron disease±inclusion dementia (Jackson et al.,

1996; Holton et al., 2002); dementia with inclusions tau- and

synuclein-negative, ubiquitinated (Kertesz et al., 2000); and

motor neuron disease-type dementia (Okamoto et al., 1991;

Wightman et al., 1992).

The neuropathological differential diagnosis of the neuro-

degenerative disorders described above is complex and takes

into account clinical information, the anatomical distribution

of gross and microscopic changes, and the protein compos-

ition of the intracellular inclusions assessed by immunohisto-

chemical and biochemical means.

We have identi®ed four novel cases presenting with either

the frontotemporal dementia (FTD) variant of frontotemporal

lobar degeneration (Neary et al., 1998) or CBD syndrome,

with microscopy demonstrating the presence of widespread

intraneuronal inclusions. Unlike the pathological features

described above, the inclusions were intensely immunoreac-

tive to neuro®lament (NF) antibodies, yet were not recog-

nized by tau or a-synuclein antibodies. We have studied the

clinical features, molecular composition, distribution and

localization of the NF inclusions by performing a review of

the medical records, semiquantitative immunohistochemical

analysis and electron microscopy in these four cases. We

propose the term NF inclusion body disease (NIBD) to

describe these unique cases.

Material and methodsPatientsFour patients with microscopic demonstration of neurode-

generation and unique features characterized by the presence

of diffuse and intensely stained NF-positive inclusion bodies

were recognized by three of the authors (T.R., J.L.H. and

D.W.D.) over a 4 year period (1998±2002). Two of the

patients resided in the United States (cases US1 and US2) and

one each in Denmark (case D1) and the United Kingdom

(case UK1). Three had complete post-mortem analysis, and

one (D1) had a frontal lobe biopsy. As controls, we studied

haematoxylin and eosin (H&E), NF and ubiquitin staining

pro®les in two normal control brains and pathological

controls including three cases of DUI 6 MND, two cases

of AD and one case each of DLB and PiD. For each control

case, we reviewed anterior frontal and temporal cortices and

hippocampal sections.

Neuropathological procedureIn the post-mortem cases, consent to brain examination was

obtained. Half of each brain was ®xed in 10% formalin for a

minimum of 4 weeks, while the other half was stored frozen.

Each ®xed half-brain hemisphere was sliced coronally and

tissue blocks were taken from representative areas, including

the frontal, temporal, parietal and occipital cortices, amygda-

la, hippocampus, basal ganglia, thalamus, midbrain, pons,

medulla oblongata and cerebellum. Cervical cord was also

available in cases US1 and US2. Following processing and

paraf®n wax embedding, 7 mm sections were cut and stained

with routine methods including H&E, Nissl, periodic acid±

Schiff/haematoxylin (PAS/H), Congo Red, thio¯avin-S, luxol

fast blue/cresyl violet, and silver impregnations (Bodian,

modi®ed Bielschowsky, and Gallyas methods).

In case D1, a biopsy of the right frontal lobe was

performed, ®xed in 10% formalin and processed for paraf®n

wax using standard procedures. Sections were cut and stained

as described above.

Immunohistochemistry and antibodiesImmunohistochemistry was carried out with special emphasis

on NF subunits. This involved the use of antibodies to

phosphorylated NF (pNF) light, medium and heavy (L, M and

H) subunits and antibody to non-phosphorylated NF heavy

2292 K. A. Josephs et al.



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subunit (non-pNF-H). An NF-cocktail antibody recognizing

L and H subunits was also utilized.

Sections (7 mm) were cut from selected blocks. The

sections were de-paraf®nized in xylene and subsequently

rehydrated in graded alcohol solutions. Endogenous perox-

idase activity was neutralized in methanol/H2O2 solution for

10 min. Some sections were pretreated by pressure cooking

(10 min) to unmask antigenic activity. Non-speci®c antigen

binding was blocked in normal horse serum (Vectastain elite

ABC kit; Vectastain, Peterborough, UK). The primary

antibodies made up in phosphate-buffered saline (PBS, 1%)

to the dilutions stated below were applied to the sections and

incubated for 1 h at room temperature. After several washes

in PBS solution, the sections were incubated in prediluted

biotinylated secondary antibody (Vectastain elite ABC kit; 30

min, room temperature). Following washes as described

previously, the ABC reagent (Vectastain elite ABC kit) was

applied to sections (30 min, room temperature). Visualization

of immunolabelled structures was achieved by incubating

sections in diaminobenzidine/H2O2 solution until coloration

was visible (usually 3±4 min). Washing and counterstaining

(Mayer's haematoxylin; WWR International, Poole, UK),

clearing and mounting followed.

Primary antibody type (monoclonal unless stated), dilution,

pretreatment and source were as follows: pNF-H (SMI 31, 1:

5000, pressure cooked) and non-pNF-H (SMI 32, 1: 500,

pressure cooked; Sternberger Monoclonals, Lutherville, UK);

pNF-M (BF10, 1: 400, pressure cooked; Af®nity Research

Products, Aurora, USA); pNF-L (Ab1, 1: 100, pressure

cooked; Oncogene Research Products, Boston, USA); NF

cocktail (1: 20, no pretreatment; ICN Pharmaceuticals,

Aurora, USA); ubiquitin (1: 300 polyclonal, pressure cooked;

DAKO, Ely, UK); tau (rabbit anti-tau Cat. No. A0024, reacts

with both phosphorylated and non-phosphorylated tau, 1:

200, no pretreatment; DAKO); tau (AT8, recognizing phos-

phorylated Ser202/Thr205, 1: 600, no microwaved; Autogen

Bioclear, Calne, UK); GFAP (1: 1000, trypsin 0.1%; DAKO);

1C2 (1: 2000, formic acid and pressured cooked; Chemicon,

Chandlers Ford, UK); Ab (1: 100, formic acid and pressure

cooked; DAKO); a-synuclein (N-19, goat polyclonal, 1:

1000, formic acid; Autogen Bioclear); aB-crystallin (1: 1200,

no pretreatment; Novocastra Laboratories, Newcastle Upon

Tyne, UK); prion protein (i) KG9 (1: 50, formic acid and

pressure cooked; obtained from Institute of Animal Health,

Compton, UK) and (ii) 3F4 (1: 2000, formic acid and pressure

cooked; obtained from DAKO).

Assessment of inclusion frequencyThe frequency of the inclusions was assessed using H&E and

both Ab1 and ubiquitin immunohistochemistry in a number of

brain regions (see Table 3). A semiquantitative approach was

used and a grading scale established, in which grade 0 was

used for regions with no inclusions, + for a small number, ++

for a moderate number, +++ for numerous and ++++ for the

most severely affected areas.

Electron microscopyElectron microscopy was performed in all three post-mortem

cases. Formalin-®xed frontal and temporal cortices were post-

®xed in buffered 1% osmium tetroxide, processed by

conventional techniques and ®nally embedded in epoxy

resin (Agar, Stantead, UK). Sections were cut on an LKB

ultramicrotome, stained with uranyl acetate and lead citrate

and viewed with a JEOL 1200 EX electron microscope

(JEOL, Peabody, USA).

ResultsClinical ®ndingsThe demographic data of all four cases are listed in Table 1.

Clinical features of all four cases are outlined in Table 2, and

individual case histories are reported in the Supplementary

data (available at Brain Online). Two cases (UK1 and US2)

presented with asymmetric cortical and basal ganglia symp-

toms, characterized by asymmetric loss of hand dexterity,

apraxia and early L-dopa-unresponsive parkinsonism. The

other two cases (US1 and D1), however, presented with

frontal and temporal lobe features including poor organ-

ization and planning, loss of initiative, reduced hygiene, and

personality change. Parkinsonism was also present in these

cases but developed late and was asymmetric in one (US1).

Prominent features common to all cases included rapid

progression of symptoms with early falling, mutism and loss

of mobility. In some of the cases, headaches (UK1 and US1),

swallowing dif®culties (D1), dystonia (US1 and US2),

supranuclear gaze palsy (UK1), obsessive±compulsive beha-

viours (US1 and US2), pathological laughter (UK1 and D1)

and pathological crying (US2) were also noted to be present

during the course of the illness.

Past medical history was signi®cant for autoimmune

disease (UK1, US1 and US2), a history of headaches (UK1,

Table 1 Demographic features of four cases of neuro®lament inclusion body disease

Feature UK1 US1 US2 D1

Country of residence United Kingdom United States United States DenmarkSex F F M FAge of onset (years) 41 50 44 43Age at death (years) 44 54 47 *Duration of illness (years) 2.5 4 3.5 3

*Patient still alive, now terminal.

Neuro®lament inclusion body disease 2293



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US1 and D1) and a history of depression (UK1 and D1). One

case (US2) had a positive family history of a neurodegen-

erative disorder (iPD followed by dementia in his father).

MRI of the brain in all four cases demonstrated mainly

frontal and temporal lobe and caudate atrophy (Fig. 1). In

case US1, in whom serial MRI scans 1 year apart were

obtained, the most signi®cant changes were noted in the

posterior and lateral orbital gyri and limen insulae (Fig. 1). In

addition, case D1 showed bilateral increased striatal signal

(Fig. 2). [18F]¯uorodeoxyglucose PET scan (UK1 and D1)

and HMPAO single photon emission computer tomography

(SPECT) scan (US1) demonstrated frontotemporal (UK1,

US1 and D1) and basal ganglia (UK1 and D1) hypometabo-

lism (Fig. 3).

Neuropsychometric testing (UK1, US1 and D1) demon-

strated cognitive dysfunction with preserved performances on

testing of visuospatial and visual perceptual function and

impaired performance on tests sensitive to frontal and

temporal lobe functions.

Neuropathological ®ndingsMacroscopic ®ndingsGross neuropathological ®ndings are summarized, with

individual ®ndings reported separately. The calculated total

brain weights ranged from 930 to 1170 g. Macroscopically,

there was predominantly frontal and anterior temporal lobe

atrophy (Fig. 4A). There was a degree of ventricular

enlargement involving the lateral and third ventricles. Two

cases (UK1 and US1) showed severe atrophy of the caudate

nucleus (Fig. 4B), and one case (UK1) had globus pallidus

discoloration. There was variable atrophy in subcortical

regions including the hippocampus, amygdala, substantia

nigra and basal ganglia. The thalamus, subthalamus, pons,

medulla and cerebellum appeared to be within the normal

limits.

Table 2 Clinical features of four cases of neuro®lament inclusion body disease

Feature UK1 US1 US2 D1

Presenting symptoms Reduced left-hand dexterity Poor organization andplanning, reduced hygiene

Reduced right-handdexterity, slowmovements

Loss of memory andinitiative, personality change

Parkinsonism[early]/(late)

[PI, akinetic-rigid gait,bradykinesia]

(Rigidity, hypophonia,hypomimia, bradykinesia)

[Hypomimia, rigidity,PI, reduced right-armswing]

[Rigidity, PI, bradykinesia,slow gait]

Prominent clinicalfeatures duringcourse of illness

Mute, falls, HA, NA,alien limb, supranuclear gazepalsy, L-dopa failure

Mute, falls, NA, torticollis,HA

Mute, falls, NA,spasticity, dystonia,L-dopa failure

Falls, voice change, mutedisinhibited, swallowingdif®culty, NA

Progression Rapid Rapid Rapid RapidAsymmetric signs L > R rigidity, apraxia,

brisk re¯exesL upper limb rigidity R upper limb rigidity,

apraxia, brisk re¯exesNR

Other Pathological laughter,increased sound sensitivity

OCB, 15 lbs weight gain OCB, pathologicalcrying, family history

Pathological laughter

Past medical history Pulmonary sarcoidosis, HA,depression

Hashimoto's thyroiditis,HA, haemochromatosis

Raynaud's syndrome,`lupus'

Hemicrania, depression

MRI ®nding FT and caudate atrophy FT and caudate atrophy FT atrophy FT and caudate atrophy andsignal changes in striatum

PI = postural instability; HA = headaches; NA = non-ambulatory; L = left; R = right; NR = not reported; OCB = obsessive±compulsivebehaviour; FT = frontotemporal.

Table 3 Semiquantitation of inclusion frequency inneuro®lament inclusion body disease in cases UK1 andUS1

Brain area H&E Ab1 Ubiquitin*

Anterior frontal cortex ++++ +++ +++Posterior frontal cortex +++ ++ ++Superior temporal gyrus + + +Middle temporal gyrus ++ ++ ++Inferior temporal gyrus +++ +++ +++Temporal pole +++ + +++Parietal cortex +++ ++ +++Occipital cortex + 0 0Insular cortex +++ +++ +Anterior cingulate gyrus ++++ ++ +++Hippocampus CA1±CA4 +++ ++ ++Dentate fascia ++++ ++ +++Subiculum +++ ++ +++Amygdala +++ ++ +Caudate nucleus + + +Putamen + + 0Globus pallidus + 0 0Thalamus + ++ 0Subthalamus + + 0Substantia nigra 0 0 0Pontine base + + 0Inferior olive 0 ++ 0Cerebellar Purkinje cells + 0 ++Dentate nucleus 0 0 0

++++ = severely affected; +++ = numerous; ++ = moderatenumber; + = small number; 0 = no inclusions. *Staining is weak.




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Histological ®ndingsThe histological ®ndings were similar for all cases and are

therefore summarized with exceptions reported. Inclusions

are depicted in Fig. 5.

Cerebral cortex. On routine microscopic examination, there

was super®cial linear spongiosis with thinning of the cortical

ribbon affecting frontal, temporal and, to a lesser extent, the

parietal lobes in all post-mortem cases. There was severe

subpial and white matter gliosis and mild gliosis affecting

cortical layers I±IV. H&E staining revealed intracytoplasmic

inclusions ranging in size from 3 to 20 mm in all layers of the

neocortex affecting all morphological types of neurons and

often situated towards their apical dendrite (Fig. 5A). The

appearance of the inclusions, irrespective of their localiza-

tion, ranged from eosinophilic to basophilic, sometimes with

a clearly de®ned basophilic margin (Fig. 5B). Inclusions

appeared to be granular or mesh-like and often contained a

hyaline, argyrophilic core resembling a `cherry'; therefore,

we shall describe them as `cherry spots'. Such structures were

found in several locations (Fig. 5C). The SMI 32 and Ab1 NF

antibodies intensely stained most of what appeared to

correspond to the cherry spots (Fig. 5K and L, respectively),

while these were stained less frequently and less intensely by

SMI 31 and rarely by BF10. The NF-cocktail antibody

usually stained a larger part of the inclusions than the Ab1 and

SMI 32 antibodies (Fig. 5M). Most of the inclusions were

weakly immunoreactive for ubiquitin, although a proportion,

often those situated in the super®cial cortical laminae, were

more strongly stained (Fig. 5J). Occasional inclusions were

intranuclear, eosinophilic, up to 6 mm in diameter and

frequently located in cortical layer II. Intranuclear inclusions

(UK1 and US1 only) were strongly stained with both

ubiquitin and Ab1 antibodies (Fig. 5O) and less strongly

stained with SMI 32 and 1C2 antibodies (Fig. 5Q). Glial

inclusions were not found.

Hippocampus. The hippocampal formation showed variable

neuronal loss and spongiosis in the CA1 subregion with

relative preservation of the CA2, CA3 and CA4 subregions.

Many of the residual hippocampal neurons contained

cytoplasmic inclusions that were often large and basophilic

(Fig. 5B) and within which there was a well-de®ned

eosinophilic hyaline cherry spot with a surrounding pale

halo (Fig. 5C). The inclusions in the CA1±CA4 subregions

had similar immunohistochemical staining characteristics to

those in the neocortex. Some axons in CA1 showed fusiform

dilations (Fig. 5N). In the granule cells of the dentate fascia,

frequent neuronal cytoplasmic inclusions were noted

(Fig. 5D), which appeared as single or occasionally multiple

small and faintly eosinophilic. Rod-shaped intranuclear

inclusions were also noted (US1 and UK1). The cytoplasmic

inclusions of the granule cells were more frequently posi-

tively stained with the antibody to ubiquitin (Fig. 5E) than

Fig. 1 Case US1. Coronal T1 images demonstrate signi®cant atrophic changes between 1995 (A) and1996 (B), mainly affecting posterior and lateral orbital gyri and limen insulae, with less atrophyoccurring in hippocampi. Caudate atrophy is also present.




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with the Ab1 or SMI 32 (Fig. 5F) antibodies, while the

intranuclear inclusions were positive for ubiquitin but only

rarely stained with SMI 32. There was variable neuronal loss

and ®brillary gliosis in the subiculum, entorhinal and

transentorhinal cortices as well as the amygdala. Frequent

neuronal cytoplasmic inclusions were found within these four

anatomical areas. In the subiculum, SMI 32 seems to have

stained more inclusions than Ab1 (UK1 only).

Subcortical grey nuclei. The basal nucleus of Meynert had a

normal neuronal population, and ubiquitin, SMI 32 and SMI

31 immunohistochemistry revealed very scanty cytoplasmic

inclusions. The caudate nucleus exhibited severe atrophy

with status spongiosis and gliosis in its medial aspect, most

marked in cases UK1 and US1. The lateral aspect of the

nucleus was less severely affected. Ubiquitin and SMI 32

stained rare inclusions within the ventrolateral caudate in

cases UK1 and US1. There was moderate to severe gliosis in

the globus pallidus and putamen, as well as a small number of

axonal spheroids. The internal globus pallidus was affected

more than the external globus pallidus, and there was an

anteroposterior gradient of severity, with the anterior level

most severely affected. A similar gradient was observed in

the putamen, with the anterior and medial parts of the nucleus

affected more severely than the posterior and lateral aspects.

Few cytoplasmic inclusions were seen in the basal ganglia,

and a proportion of these was positive with Ab1 antibody.

The thalamus showed patchy rarefaction, with neuronal

loss, gliosis and scattered intracytoplasmic inclusions most

frequent in the medial thalamus. The subthalamic nucleus

was well preserved with occasional intracytoplasmic inclu-

sions.

Brainstem. The red nucleus contained large NF-positive

spheroids and showed mild gliosis. The substantia nigra had

mild neuronal loss with extracellular neuromelanin pigment

and gliosis that was most marked in the ventral and lateral

neuronal groups. A number of axonal spheroids were seen in

the pars reticularis. The third nerve had a signi®cant number

of very thick axons. No inclusions were found in the third and

fourth nerve nuclei. The rapheÂ nuclei contained scanty

inclusions in case US1 only. There was mild neuronal loss

Fig. 3 Case D1. An [18F]¯uorodeoxyglucose PET scan revealshypometabolism in the right frontotemporal region and rightthalamus.

Fig. 2 Case D1. An axial FLAIR (¯uid attenuated inversionrecovery) image demonstrates increased signal changes in striatumbilaterally.




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in the locus coeruleus and some of the neurons had NF-

positive hyaline inclusions (US2 only). Rare Ab1-positive

intraneuronal inclusions (UK1 only) and axonal spheroids

were seen in the pontine base (UK1 and US1). In the inferior

olive, there were large inclusions, some appearing ®la-

mentous (Fig. 5H), which were stained with both Ab1

immunohistochemistry and Bielschowsky's silver impregna-

tion but were negative for ubiquitin.

Cerebellum. In the cerebellar cortex, there was variable

Purkinje cell loss and frequent torpedoes in two of the cases

(UK1 and US1). These ®ndings were not noted in the other

post-mortem case (US2). In case UK1, the Purkinje cells

often contained one or more ubiquitin-positive, but NF-

negative, intranuclear inclusions (Fig. 5P), measuring 1±6 mm

in diameter. No cytoplasmic inclusions were seen on routine

stains or with immunohistochemistry.

Spinal cord. Where spinal cord sections were available (US1

and US2), no inclusions were seen in motor neurons. There

was no degeneration of the corticospinal or other tracts.

A moderate number of inclusions were weakly stained with

the modi®ed Bielschowsky silver stain, while a small number,

which mostly appeared to correspond in size to cherry spots,

were intensely stained. Weakly stained inclusions could be

demonstrated in the neocortex, hippocampus, basal ganglia

and thalamus, while intensely stained inclusions were mainly

found in the frontal cortex and hippocampus. The inclusions

were not stained with Gallyas or Bodian silver impregnations.

The inclusions were not labelled by antibodies to either a-

synuclein or tau or stained with PAS/H, Nissl, Congo Red or

thio¯avin-S methods. No cortical or brainstem Lewy bodies

were found using routine stains or a-synuclein immuno-

histochemistry. a-Synuclein immunohistochemistry in the

amygdala (UK1) was negative. None of the cases showed

neuro®brillary pathology on tau immunohistochemistry.

There were no glial inclusions identi®ed on tau, a-synuclein

or ubiquitin immunohistochemistry. No Ab or prion protein

deposition was observed. In the control cases stained with the

same panel of anti-NF and ubiquitin antibodies, no inclusions

with a similar staining pro®le were demonstrated.

Inclusion frequencySemiquantitation of the inclusions found in cases UK1 and

US1 revealed a few important patterns (Table 3). The

inclusions were widely distributed, but they were most

common in the anterior frontal and cingulate cortices and the

hippocampal dentate fascia. Inclusions were also numerous,

but less so, in the posterior frontal, temporal, parietal and

insular cortices, subiculum, hippocampus proper and amyg-

dala. They were scanty to moderate in the occipital cortex,

basal ganglia and brainstem and were absent in the deep

cerebellar nuclei. In the temporal cortex, there was a

gradation whereby the superior temporal gyrus had fewer

inclusions than the inferior temporal gyrus. Overall, the

inclusions were most readily found on the H&E preparations,

with the exception of the granule cells of the hippocampal

dentate fascia and the inferior olive.

Electron microscopyIn all three post-mortem cases, cytoplasmic inclusions of the

cortical neurons were noted to be composed of dense

aggregates of ®laments 11±16 nm in diameter and appeared

consistent with NFs (Fig. 6A). These were surrounded by

granule-coated ®brils and cellular organelles. In cell pro-

cesses an occasional structure of ~3 mm diameter containing

bundles of ®lamentous material, similar to those found in

neurons, was seen (Fig. 6B).

Fig. 4 Case UK1. Gross pathology demonstrates frontal and anterior temporal atrophy (A) and severecaudate atrophy (B).




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Discussion

Clinical ®ndings

The clinical presentation and progression of the cases we

have described, for which we propose the term NIBD, differs

from other neurodegenerative disorders. The presenting

features of cases UK1 and US2 share many similarities to

CBD syndrome, in that they both had asymmetric cortical

features with parkinsonism, L-dopa resistance and apraxia. In

a study of 14 patients with pathologically con®rmed CBD,

asymmetric hand clumsiness was the most common

presenting symptom (Wenning et al., 1998). Similarly, the

presenting symptoms and signs of cases US1 and D1 are in

keeping with the features that help to de®ne FTD, as both

cases had insidious onset at age <65 years, prominent frontal

features and prominent language dysfunction (Neary et al.,

1998; see Supplementary data for histories). Parkinsonism

develops in perhaps the majority of affected cases as FTD

progresses (Knopman et al., 1990).

Most clinicopathological studies that have examined the

progression of patients with CBD and FTD, as well as the

duration of illness, have revealed a difference from our cases.

The mean duration of illness until death for both CBD and

FTD is generally >6 years (Boeve et al., 1999; Snowden et al.,

Fig. 5 Cases UK1, US1 and D1. Photomicrographs demonstrating the main microscopic abnormalitiesin neuro®lament (NF) inclusion body disease. Neuronal intracytoplasmic inclusions (A±C) werenumerous in the frontal and temporal cortices, hippocampus proper and dentate granule cells (D±F).Some of the inclusions had a basophilic margin (B), while others appeared to contain a core resemblinga cherry (`cherry spot') (C). In the hippocampal formation, a small number of inclusions were stronglyargyrophilic (G) and intensely stained with NF antibodies (K and L). Some inclusions were diffuselystained with ubiquitin (E and J) and NF (M) without a visible cherry spot. Argyrophilic inclusions wereseen in neuronal processes in the hippocampus (H), and occasional axons showed fusiform dilatations,often intensely stained with NF antibodies (N). Large inclusions, some with a ®lamentous appearance,were seen in the inferior olive (I). Intranuclear inclusions (O) were rare in cortical neurons andsubiculum. Some of the intranuclear inclusions were highlighted with 1C2 antibody (arrow). Anintracytoplasmic inclusion is unstained (double arrow) (Q). Intranuclear inclusions, some multiple, wereseen in cerebellar Purkinje cells (P). Haematoxylin and eosin (A±D and O), Bielschowsky's silverimpregnation (G±I), SMI 32 (F and K), NF cocktail (M), Ab1 (L and N), ubiquitin (E, J and P) and1C2 (Q). The scale bar represents 20 mm on A±H, J and L±Q; 10 mm on I; and 40 mm on K.




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2002), onset is insidious and progression is usually slow. Of

all the clinical features of our four cases of NIBD, it was the

rapidity of the progression that was most impressive. The

mean duration of illness in our cases was only 3 years, well

below the range reported for CBD and FTD. There are only

two other neurodegenerative disorders that commonly pro-

gress with this ferocity and would need to be considered in the

differential diagnosis: Creutzfeldt±Jakob disease (CJD) and

DUI 6 MND. However, the duration of illness and clinical

presentation in CJD is very different from our cases. In CJD

there is usually prominent myoclonus and the mean duration

of illness is ~6 months, although cases with a longer duration

of illness have been reported (Parchi et al., 1999); MRI, EEG

and CSF studies show basal ganglia and cortical changes,

periodic complexes and increased 14-3-3 protein, respect-

ively (Schroter et al., 2000; Zerr et al., 2000). These ®ndings

were not present in any of our cases except for D1, in whom

MRI demonstrated striatal hyperintensity similar to that

reported in CJD (Fig. 2). However, EEG ®ndings and CSF

results (see Supplementary data) were not consistent with a

diagnosis of CJD. Furthermore, prion protein immunohisto-

chemistry and western blotting (UK1, data not shown) were

negative. DUI 6 MND can have presenting features similar

to our cases (Jackson et al., 1996; Rossor et al., 2000);

however, in those reports, survival from ®rst symptom was 8

and 9 years, respectively. Nevertheless, DUI can be rapidly

progressive with duration of illness of ~3 years, but only in

the setting of MND (i.e. when there is clinical and/or EMG

evidence of motor neuron disease; Mitsuyama, 1993). None

of our cases had any clinical or electophysiological evidence

for MND.

The other features that were prominent in our cases of

NIBD were mutism, falls and terminal non-ambulation. It is

dif®cult to determine the exact mechanism for the mutism, as

the localization of mutism is diverse and has been described

in many patients with bilateral cortical lesions (e.g. bilateral

anterior cerebral infarcts; Bogousslavsky and Regli, 1990)

and subcortical lesions (e.g. bilateral globus pallidus lesions;

Ure et al., 1998). One clue, however, may have been the MRI

®ndings in case US1, in which serial imaging 1 year apart

demonstrates severe and more focal destruction of the inferior

and posterior frontal lobes containing Broca's area, out of

proportion to other cortical areas and subcortical areas

(Fig. 1). During this time, the patient had signi®cantly

reduced verbal output. A comparison with patients with FTD

suggests that these ®ndings may be signi®cant, as mutism is

common in FTD. Falls and terminal non-ambulation most

likely result from progressive destruction of multiple path-

ways and structures that are required for ambulation. These

include the cerebellar and frontal cortical±basal ganglia

pathways. Pathological ®ndings in NIBD revealed Purkinje

cell loss with torpedoes and intranuclear inclusions within

cerebellar Purkinje cells (UK1), while basal ganglia damage

was present, sometimes severe (as in UK1 and US1, in whom

the caudate nuclei were severely atrophic). Frontal cortical

destruction with super®cial linear spongiosis and numerous

NF-positive intraneuronal inclusions were also a common

feature.

Finally, there were other clinical ®ndings including

saccadic eye movement abnormality (UK1), increased

sound sensitivity (UK1), obsessive±compulsive behaviours

(US1 and US2), pathological laughter or crying (UK1, US2

and D1) and past medical history of headache (UK1, US1 and

D1), depression (UK1 and D1) and autoimmune disorders

(UK1, US1 and UK2). These ®ndings were limited to one or a

few cases and at present are of uncertain signi®cance.

Fig. 6 Cases US2 and UK1. Ultrastructural examinationdemonstrates dense aggregates of ®laments of 11±16 nmsurrounded by granule-coated ®brils and cellular organelles bothin neuronal cytoplasm (A) and a cellular process (B). Arrows in Bpoint to surrounding cell membrane. The scale bar represents 0.9mm on A and 0.7 mm on B.




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Only one case (US2) had a positive family history of an

early onset neurodegenerative disease (see Supplementary

data). The patient's father had developed signs and symptoms

of iPD in his early 50s, followed a few years later by

dementia. Unfortunately, post-mortem examination was not

performed. The development of iPD in his early 50s followed

by dementia is very intriguing. The differential diagnosis of

parkinsonism and dementia is wide. A diagnosis of iPD,

however, is very unlikely. More likely would be a diagnosis

of DLB (McKeith et al., 1996). We know his father was

treated with L-dopa therapy, but we do not know whether he

responded to the treatment. With the young age of onset and

short duration of illness (<5 years), NIBD is clearly a

diagnostic possibility.

MRI studies in all four cases revealed prominent frontal

and temporal lobe atrophy and increased signal in the striatum

in one case (D1). The distribution of atrophy is in keeping

with the clinical presentation. The increased signal in the

striatum in case D1 is unusual, as this is uncommon in CBD

and FTLD syndromes (Hauser et al., 1996; Larsson et al.,

2000), although it is more common in CJD (as discussed

above).

The ®ndings on functional neuroimaging (PET and

SPECT) and neuropsychological tests are also in keeping

with the clinical presentation and can be useful ancillary

studies.

Pathological ®ndingsPathological study of all four cases showed similar ®ndings of

NF-containing neuronal cytoplasmic inclusions in several

brain areas, including the neocortex and limbic structures,

with moderate numbers in deep grey and brainstem nuclei. In

at least two of the cases (UK1 and US1), we were able to

demonstrate neocortical ubiquitin-positive intranuclear inclu-

sions, which were also frequent in the cerebellar Purkinje

cells in one case (UK1).

A number of conditions can be considered in the differ-

ential diagnosis of NIBD (Table 4). The absence of a-

synuclein and tau pathology in all our cases excludes the

diagnosis of a number of neurodegenerative conditions with

neuronal inclusions, including PD, DLB, MSA, PiD, CBD,

PSP or AD (Lowe, 1998). The presence of ubiquitin- and NF-

positive inclusions excludes the diagnosis of dementia

lacking distinctive histology (Knopman et al., 1990; Mann,

1998). The immunohistochemical pro®le of NIBD is also

different from that seen in basophilic inclusion body disease

(BIBD) (Munoz-Garcia and Ludwin, 1984; Munoz, 1998;

Holton et al. 2002), as in this condition the basophilic

inclusions are negative or weakly positive for NFs (Munoz-

Garcia and Ludwin, 1984; Tsuchiya et al., 2001). The

distribution of the basophilic inclusions is also different from

that of the NIBD inclusions, as they are found mainly in the

basal ganglia and brainstem (Munoz-Garcia and Ludwin,

1984), sparse in the cortex and absent in the hippocampus. As

the inclusions in NIBD are variably positive for ubiquitin, a

comparison with DUI 6 MND is also necessary. In DUI 6MND, the inclusions are limited largely to the cortex and

hippocampus, in which only dentate granule cells are

involved (Okamoto et al., 1991; Jackson et al., 1996;

Rossor et al., 2000; Holton et al., 2002). In contrast,

inclusions are frequently found not only in the dentate

granule cells, but also throughout the hippocampal formation,

including subiculum in NIBD. Furthermore, the inclusions in

NIBD are more prominent than those in DUI 6 MND and can

be easily seen in H&E-stained sections. Most importantly, the

Table 4 Pathological features of NIBD compared with BIBD, DUI and PiD-UNI

Feature NIBD* BIBD² DUI*³ PiD-UNI§

Number of cases 4 3 3 1Age range of onset (years) 41±50 29±60 43±59 48Mean duration of illness (years) 3 6 11 13Clinical diagnosis FTLD/CBD FTLD FTLD FTLDLobar atrophy Yes Yes Yes YesCaudate atrophy Yes Yes Yes YesSuper®cial cortical spongiosis Yes Yes Yes YesNigral pallor Yes Yes No YesNigral cell loss Yes Yes Yes YesNeuronal intracytoplasmic inclusions Yes Yes Yes YesNeuronal intranuclear inclusions Yes No Yes¶ NoArgyrophilia of inclusions Yes (O) Yes (O) No Yes (F)Immunohistochemical pro®le UB, NF (I) NF (W) UB NF (I)Main distribution of inclusions FT, HP, DG BS, BG FT, DG FT, HP

NIBD = neuro®lament inclusion body disease; BIBD = basophilic inclusion body disease; DUI = dementia with ubiquitin-positiveinclusions; PiD-UNI = Pick's disease with unusual neuronal inclusions; FTLD = frontotemporal lobar degeneration; CBD = corticobasaldegeneration; O = occasional; F = frequent; UB = ubiquitin; NF = neuro®lament; I = intense staining; W = weak or negative staining; FT= frontotemporal; HP = hippocampus CA1±CA4; DG = dentate granule cells; BS = brainstem; BG = basal ganglia.*Analysis of our data; ²adapted from Munoz-Garcia and Ludwin (1984); ³adapted from Holton et al. (2002); §adapted from Yokoo et al.(1994); ¶Woulfe et al. (2001).




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inclusions in DUI 6 MND are negative for NFs, which is also

con®rmed by the current study. A necessary consideration of

our NF inclusions is their possible relationship with NF-

positive inclusions within motor neurons in MND (Sobue

et al., 1990) or the eosinophilic and basophilic inclusions,

labelled with NF and ubiquitin antibodies and described in a

single case reported by Arima et al. (1998). There were no

MND-type inclusions with ubiquitin and NF immunohisto-

chemistry within motor neurons of either the cranial nerve

nuclei, which were available for our studies in all three post-

mortem cases (UK1, US1 and US2), or the cervical cord

studied in two of the cases (US1 and US2). Furthermore,

neuronal inclusions, similar to those seen in NIBD, are not

seen in the cerebral cortices and deep grey nuclei in MND

(Lowe and Leigh, 2002). It could be of interest that, in the

case of Arima et al. (1998) diagnosed clinically and

con®rmed by post-mortem as MND, the intracytoplasmic

ubiquitin-positive inclusions were found in cortical neurons

and hippocampal dentate granule cells. However, these were

reported to be only weakly positive or negative for NF using

the SMI 31 antibody recognizing pNF-H. The entity

described by Yokoo et al. (1994) as `Pick's disease with

unusual inclusions' (PiD-UNI) in 1994 is, with a few

differences, the most similar to our four cases. In their case,

the inclusions were NF-positive with an immunohistochem-

ical pro®le and distribution similar to ours. However, in PiD-

UNI, ubiquitin studies were reported as negative and neither

intranuclear inclusions nor axonal spheroids were reported. A

further difference is that, clinically, unlike our cases, their

case had a long disease duration of 13 years, even though the

early disease course was rapidly progressive. Pietrini et al.

(1993) described a case of the panencephalitic type of CJD

with neuropathological features similar to PiD. In their case,

there were numerous NF-positive swollen neurons and a

small number of Pick body-like argyrophilic intraneuronal

inclusions. However, there is no reference to the presence of

NF-positive intraneuronal inclusions of the kind we observed

in our cases. The only other report of signi®cant interest was a

recent report of ®ve patients with a novel neuro®lamento-

pathy (Cairns et al., 2003). Similar to our cases, their cases

had an early-onset dementia and intraneuronal, cytoplasmic,

NF-positive inclusions. Little clinical information was given,

but their patients seem all to have had an FTD. No mention of

a CBD syndrome was made. Unlike in our cases, NF-L

staining was less impressive than NF-H staining. Also, no

mention was made of intranuclear inclusions, inclusions

within the inferior olive, axonal swellings outside the

cerebellum or the presence of ®lamentous structures in cell

processes, which we were able to demonstrate with electron

microscopy.

One of the striking features noted in our cases was the

presence of neuronal ubiquitin-positive intranuclear inclu-

sions. Although such intranuclear inclusions were only rarely

found in most areas, they were numerous in the hippocampal

dentate granule cells and the cerebellar Purkinje cells (UK1).

Intranuclear inclusions are rather rare in cases with dementia,

although neuronal ubiquitinated intranuclear inclusions have

been described in three cases of DUI 6 MND (Woulfe et al.,

2001) and a single case report of a demented woman

(Weidenheim and Dickson, 1995). Intranuclear neuronal

inclusions are a common feature of polyglutamine disorders

(Davies et al., 1997; Hayashi et al., 1998; Holmberg et al,

1998; Li et al., 1998; Pang et al., 2002) and neuronal

intranuclear hyaline inclusion disease (Munoz-Garcia and

Ludwin, 1986), which may also be a polyglutamine disorder

(Lieberman et al., 1998; Takahashi et al., 2000). Case D1

underwent genetic testing for Huntington's disease and

dentatorubral-pallidolusian atrophy and tested negative (see

Supplementary data). The relationship of these disorders to

our four cases of NIBD is not clear at present and requires

further investigation, especially since the antibody 1C2,

which identi®es polyglutamine sequences, occasionally

labelled cortical intranuclear inclusions in our NIBD cases.

Two of the four cases (UK1 and US2) of NIBD were

treated with dopamine therapy and both failed to improve

symptomatically. This is not surprising, as pathology was not

limited to the substantia nigra, but was also extensive and

severe in the basal ganglia. Replacement anticholinesterase

therapy is also unlikely to be bene®cial in NIBD, as the

nucleus basalis of Meynert is preserved. This is not to say that

trials with either category of medication are not worth

pursuing.

The mechanism for NF deposition in NIBD is currently

unknown, but there are a few theoretical possibilities based

on results from mouse genetic and human NF studies (Nixon,

1993; Julien, 1999). These include `sporadic' NF gene

mutations, post-translational modi®cations including abnor-

mal phosphorylation of the subunit components of NF (NF-L,

NF-M and NF-H) and failure of axonal NF transport.

In summary, NIBD is a unique neurodegenerative disorder

with overlapping clinical features of frontotemporal lobar

degeneration and CBD syndrome. A diagnosis of NIBD

should be considered in patients who present with either a

CBD or FTD syndrome, with rapid progression without

clinical or EMG evidence of MND, with early falls, mutism,

akinesia and terminal immobility. MRI evidence of frontal

and temporal lobe atrophy would support the diagnosis. The

main morphological features that support the pathological

diagnosis of NIBD is a-synuclein- and tau-negative neuronal

intracytoplasmic inclusions, many of which contain NFs and

variable amounts of ubiquitin, and some of which are

argyrophilic with modi®ed Bielschowsky. Such inclusions

are strikingly numerous in neocortices and limbic structures.

Further characterization of NIBD is needed to clarify the

signi®cance and mechanism of the NF deposition in this

disorder.

AcknowledgementsK.A.J. is currently a Mayo Foundation Scholar and Visiting

Researcher at the Institute of Neurology, Queen Square,

London, UK. Dr J.L.H. is partly funded by the Reta Lila




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Weston Foundation. We wish to thank Dr John Stevens for

assistance with MRI and PET interpretation; Dr Eric M.

Rohren for assistance with PET imaging; Dr Brendan

McClean, who referred case UK1; and all the general

practitioners and consultant neurologists who participated in

the care of these patients and documented the clinical

histories and examinations. We appreciate Ms Hardev

Sangha, Ms Kerrie Venner, Ms Catherine Strand and Dr

Wen-Lang Lin for their technical assistance in the patho-

logical studies. We would like to extend our appreciation to

all the family members who extended their support by giving

additional clinical information and allowing us to report

accurately on these four novel cases.

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Received February 26, 2003. Revised April 28, 2003.

Accepted May 9, 2003




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