(American Journal of Pathology. 1999;155:1453-1457.)
© 1999 American Society for Investigative Pathology
Selective Neuronal Vulnerability in Human Prion Diseases
Fatal Familial Insomnia Differs from Other Types of PrionDiseases
Marin Guentchev,
Julia Wanschitz,
Till Voigtländer,
Helga Flicker and
Herbert Budka
From the Institute of Neurology, University of Vienna,
Vienna, Austria
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Abstract
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Human transmissible spongiform encephalopathies (TSEs) or prion
diseases are neurodegenerative disorders of infectious,
inherited or sporadic origin and include Creutzfeldt-Jakob disease
(CJD), Gerstmann-Sträussler-Scheinker disease
(GSS), kuru and fatal familial insomnia (FFI).
Clinicopathologic features of FFI differ markedly from other human
TSEs. Previous studies demonstrated selective neuronal vulnerability of
parvalbumin positive (PV+) GABAergic inhibitory interneurons in
sporadic CJD and experimental TSEs. In this report we show uniform
severe loss of PV+ neurons also in other TSEs such as GSS,
kuru, new variant and familial CJD. In contrast, these
neurons are mostly well preserved, or only moderately
reduced, in FFI. Only PV+ neurons surrounded by isolectin-B4
positive perineuronal nets were severely affected in TSEs,
suggesting a factor residing in this type of extracellular matrix
around PV+ neurons as modulator for the selective neuronal
vulnerability.
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Introduction
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Transmissible spongiform encephalopathies (TSEs) or prion diseases
are neurodegenerative disorders of infectious, inherited, or sporadic
origin (for reviews see Refs. 1-3
). Human TSEs include
Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker
disease (GSS), kuru, and fatal familial insomnia (FFI). CJD is
traditionally categorized into the most common sporadic as well as
iatrogenic, familial (fCJD) and new variant (nvCJD) disease, which
emerged in the past years in the United Kingdom.4
Spongiform change, astrogliosis, and
neuronal loss are the classical neuropathological triad of tissue
lesions in TSEs. Whereas CJD, GSS, and kuru display overlapping
clinical and neuropathological features, the clinical presentation and
neuropathological hallmarks of FFI significantly differ from all other
human TSEs.5
Recent studies have observed selective vulnerability of distinct
neuronal subsets in sporadic CJD, experimental CJD, and experimental
scrapie. These reports showed early, severe and selective loss of
parvalbumin positive (PV+) neurons,6,7
which are a subset
of GABAergic interneurons; correlation between tissue pathology and
distribution of PV+ neurons,8
aberrant neuropeptide Y mRNA
induction, and decrease of neuropeptide Y Y2 receptor binding sites in
the hippocampus of scrapie-infected mice.9
These subset
specific changes were suggested as possible basis for some of the
clinical symptoms in TSEs. However, subset-specific pathology has not
been investigated in human TSEs apart from sporadic CJD, such as GSS,
kuru, FFI, fCJD, and nvCJD. In this study, we investigate PV+ neurons
in these less common types of human TSEs.
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Materials and Methods
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We used formol-fixed, paraffin-embedded human brain tissues
obtained at autopsy. Five FFI (three male and two female patients, age
range 26–56 years, prion protein (PrP) gene (PRNP)
genotype D178N; four homozygous at the polymorphic PRNP
codon 129 for methionine (M), one unknown, including four cases from a
new Austrian family reported previously10,11
), four GSS
(two male and two female patients, age range 37–62 years, with
PRNP genotype P102L (4/4) and M129M (3/4, one unknown);
including two cases from the original Austrian family reported
previously12
), four fCJD with the PRNP mutation
E200K (two male and two female patients, age range 50–66
years, including one case with codon V129V reported
previously13
), 1 kuru (16-year-old male, V129V; NIH case
kindly provided by Dr. P. Liberski, Lodz, Poland, reported
previously14
), and two nvCJD cases (51-year-old male and
27-year-old female, M129M; kindly provided by Dr. J. Ironside,
Edinburgh, UK; reported previously4
). The following
regions were investigated: frontal cortex, cerebellum, hippocampus, and
adjacent temporal cortex. Hippocampal blocks from two nvCJD, two fCJD,
and one FFI cases were not available for investigation. As
controls, eight formol-fixed human autopsy brains (four male and
four female patients, age range 27–66 years) with normal
histology were used.
We performed immunohistochemistry for parvalbumin (PV) and lectin
staining with isolectin-B4 (ILB4) to decorate perineuronal nets (PNNs)
of extracellular matrix around PV+ neurons.15
Two
monoclonal antibodies against PV (PV-235, PARV-19, both diluted 1:5000,
Sigma Chemical Co., St. Louis, MO) were used. Sections for PV labeling
were boiled 10 minutes in target retrieval solution (pH 9.9) (Dako,
Glostrup, Denmark). Lectin staining (ILB4-peroxidase labeled, 10
µg/ml Tris-buffered saline, 1 hour; Sigma) was performed on sections
boiled for 10 minutes in citrate-buffered saline (pH 6.0). Both
anti-PV antibodies showed a similar distribution of PV-like
immunoreactivity, but the PV-235 immunostained sections had a better
signal- to-background ratio and thus were mainly used for evaluation.
Quantification was made blindly by two of us (M. G. and J.
W.). Counting by both showed very similar results; mean values were
entered into final statistical evaluation. PV+ neurons were assessed in
the same regions (frontal and temporal cortex) of control, FFI, GSS,
fCJD, and nvCJD cases by counting immunopositive cell bodies in 3
representative fields with a x20 objective. The mean of all 3 fields
was entered into statistical evaluation. The total numbers of neurons
were evaluated in H&E-stained sections of control, FFI, GSS, fCJD, and
nvCJD cases by counting unequivocal neuronal cell bodies in the 3
fields examined with a x40 objective. The mean of all 3 fields was
entered into statistical evaluation. Neuronal cell bodies were well
distinguishable from the homogeneously red cytoplasm of reactive
astrocytes, which are abundant in TSEs. The mean regional numbers of
all and of PV+ neurons were calculated and tested for significant
differences in all groups (control, FFI, and other TSEs) using a
Kruskal-Wallis one-way followed by a two-tailed Mann-Whitney
U-test.
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Results
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Controls
Controls had a similar profile of region-specific distribution of
PV+ neurons, with highest numbers in pre- and parasubiculum,
frontal (Figure 1A)
and temporal cortex,
moderate numbers in entorhinal cortex and subiculum, and low numbers in
hippocampal sectors CA1–4. In the cerebellum, Purkinje cells were
strongly positive for parvalbumin (Figure 2C)
. Neurons with ILB4+ PNNs were
distributed in the cortex and hippocampal formation; in contrast, ILB4+
PNNs were not detectable around Purkinje cells (Figure 2A)
. The number
of ILB4+ PNNs was higher in the frontal cortex than in the temporal
cortex.
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Figure 1. Parvalbumin immunostaining (A–C,
G–I) and isolectin-B4 staining for
perineuronal nets of extracellular matrix
(D–F, K–M)
in frontal cortex in control (A,
D), FFI
(B, E),
GSS (C,
F), nvCJD
(G, K),
fCJD (H,
L), and kuru
(I, M)
brains. Severe loss of PV+ neurons and PNNs in GSS, nvCJD, fCJD, and
kuru. In FFI, these neurons are better preserved than in all other
TSEs. Original magnification, x128.
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Figure 2. ILB4 staining
(A) of a
control cerebellum and parvalbumin immunostaining of a kuru
(B) and
control (C)
cerebellum. There is lack of ILB4+ PNNs around PV+ Purkinje cells in
normal brain; Purkinje cells are preserved in kuru. Original
magnification: A, x64; B and C,
x128.
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Gerstmann-Sträussler-Scheinker Disease, Kuru, Familial,
and New Variant Creutzfeldt-Jakob Disease
All cases showed TSE-specific pathology: spongiform change,
astrogliosis, neuronal loss (in the frontal cortex ~22% ± 2.3 SEM
as compared to controls) and PrP deposition. These different TSE types
showed a uniform subtotal to total loss of PV+ neurons and severe loss
of PV neuropil staining in the frontal (~71% ± 5.0 SEM,
P 
0.001; Figure 1C, G, H, I
; Figure 3
), temporal (~57.4% ±
16.7 SEM, P 0.02) and entorhinal cortex, pre- and
parasubiculum and all regions of the hippocampus, irrespective of the
local severity of spongiform change or astrogliosis. Only one GSS case
showed a normal number of PV+ neurons in the temporal cortex. The loss
of PV+ neuropil and synaptic buttons appeared more severe than the loss
of PV+ cell bodies (Figure 1C, G, H, I)
. The numbers and morphology of
cerebellar PV+ neurons (Purkinje cells) and neuropil appeared well
preserved (Figure 2B)
. Neurons with ILB4+ PNNs were severely depleted
in all investigated regions (Figure 1F, K, L, M)
, confirming the loss
of PV+ cells. The loss of ILB4+ PNNs seemed to be more severe than the
loss of PV+ neurons (Figure 1F, K, L, M)
.
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Figure 3. Graphic representation of the numbers of PV+ neurons in the frontal
cortex. The columns depict the average numbers of PV+ neurons in the
frontal cortex (P 0.001,
Kruskal-Wallis test; *P < 0.02, compared to
controls; **P < 0.01, compared to FFI or controls,
respectively; Mann-Whitney
U-test). The numbers of PV+
neurons in FFI differ from all other TSEs. Scale bars represent SEM;
the y axis shows the average numbers of PV+ neurons in 3
representative fields (with a x20
objective) of the frontal cortex.
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Fatal Familial Insomnia
All cases showed slight but variable TSE-specific pathology:
spongiform change (restricted to thalamus and one cortical focus each
in three of four brains), astrogliosis, neuronal loss (in the frontal
cortex ~14% ± 1.0 SEM as compared to controls), and in two cases
focal PrP deposition. All cases showed moderate loss (~38% ± 8.1
SEM; P 0.02) of PV+ neurons in the frontal cortex
(Figures 1B and 3)
where the neuropil staining appeared only slightly
reduced (Figure 1B)
. There was a statistically significant difference
between frontal cortical numbers of PV+ neurons in FFI and all other
TSEs (P 0.005) (Figure 3)
. In temporal
(~16.6% ± 7.25 SEM loss as compared to controls, P
= 0.7) and entorhinal cortex, pre- and parasubiculum, all regions of
the hippocampus and the cerebellum, PV+ neurons were well preserved.
Morphology of PV+ neurons in all investigated regions was normal
(Figure 1B)
. ILB4+ PNNs appeared to be slightly reduced in the frontal
cortex (Figure 1E)
but were dense in all other investigated regions,
especially in the temporal cortex.
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Discussion
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Neuronal subsets demarcated by calcium binding proteins (eg,
parvalbumin, calbindin, calretinin) have been recently demonstrated to
be selectively vulnerable or resistant in neurodegenerative disorders
such as Alzheimer’s disease,16,17
Parkinson’s
disease,18
Pick’s disease,19
and others.
Previous reports by our and other groups have shown a severe and
selective loss of PV+ neurons in sporadic CJD,6-8
which
developed already very early in experimental TSEs.7
This
was suggested as a possible substrate of typical symptoms in these
disorders such as myoclonus and characteristic EEG
pattern.6-8
The subset-specific neuronal vulnerability
was recently corroborated by severe loss of PNNs in sporadic
CJD20
and experimental TSEs.7
Here we
describe loss of the PV+ subset of GABAergic neurons in other types of
TSEs. The decrease of a second marker for the PV+ subset of GABAergic
neurons, ILB4, confirms the loss of this neuronal subset. PV is
localized in most cortical GABAergic neurons21
(20 to 25%
of all neurons in the frontal cortex are GABAergic22
), and
the total neuronal loss varies from 14 to 22% in our counts. This
result suggests that the PV+ subset forms the main group of neurons
lost in TSEs. The disproportionate loss of PV+ neurons to the total
neuronal loss reported here and in previous reports7,8
suggests that damage of this particular subset is not consequence
of the local severity of the pathological process, but more likely a
primary event.
Clinically and neuropathologically, FFI clearly differs from all other
human TSEs. Tissue pathology in FFI focuses on the thalamus, and there
is little or no detectable tissue pathology in the
cortex.11
Surprisingly, FFI shows only moderate (as
compared to all other human TSEs in our series) loss of PV+ neurons and
neuropil staining in the frontal cortex, appearing to be the only
unequivocal diffuse pathological change in the FFI cortex. This
moderate loss of cortical PV+ neurons likely accounts for the slight
total neuronal cortical loss in FFI. However, this neuronal subset is
well preserved in the temporal cortex and adjacent hippocampus, in
striking contrast to all other TSE types. This adds another element to
the exceptional position of FFI among human TSEs. It is possible that
some differences in clinical presentation of FFI, as compared with
other TSEs, might be due to the better preservation of PV+ cortical
neurons.
Cortical but not cerebellar PV+ neurons (Purkinje cells)7
are selectively vulnerable in TSEs. Two attributes discriminate
Purkinje cells from all other PV+ neurons: co-expression of another
calcium binding protein, calbindin,7
and lack of ILB4+
PNNs. The fact that PV+ neurons devoid from ILB4+ PNNs such as Purkinje
cells are usually well preserved in TSEs and that loss of ILB4+ PNNs
appeared to be even more severe than loss of PV+
neurons7,20
suggests a factor residing in the
extracellular matrix around cortical or hippocampal PV+ neurons as
modulator for their vulnerability. It remains to be established how
calcium binding proteins and the extracellular matrix interact to
confer such selective neuronal vulnerability or resistance in TSEs,
respectively.
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Acknowledgements
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We thank especially Dr. James Ironside, Edinburgh, UK for allowing
us to use slides from two nvCJD brains. Ms. C. Karner is kindly
acknowledged for excellent technical assistance, Dr. Peter Birner for
helping with statistics, and Dr. Johannes Hainfellner for help and
support.
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Footnotes
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Address reprint requests to Dr. H. Budka, Institute of Neurology, University of Vienna, AKH 04J, Währinger Gürtel 18–20, POB 48, A-1097 Wien, Austria. E-mail: h.budka{at}akh-wien.ac.at
Supported by European Union Biomed-2 Concerted Action "Human Transmissible Spongiform Encephalopathies (Prion Diseases): Neuropathology and Phenotypic Variation" (Project Leader: H. Budka) and European Union Biomed-2 Shared Cost Action "Molecular Biology of Prion Diseases" (Project Leader: J. Collinge, London).
M. Guentchev and J. Wanschitz contributed equally to this work.
Accepted for publication July 21, 1999.
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Abstract
Introduction
