Upregulated Expression of 14-3-3 Proteins in Astrocytes
From Human Cerebrovascular Ischemic Lesions
Yasuhiro Kawamoto, MD; Ichiro Akiguchi, MD; Hidekazu Tomimoto, MD;
Yoshitomo Shirakashi, MD; Yasuyuki Honjo, MD; Herbert Budka, MD
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Background and Purpose—Several types of chaperone proteins, such as heat shock proteins, have been reported to be
associated with brain ischemia. The purpose of this study was to investigate whether an abnormal expression of 14-3-3
proteins, a novel type of molecular chaperones, occurs in human gray and white matter ischemic lesions.
Methods—We prepared formalin-fixed, paraffin-embedded sections from 33 autopsied brains, consisting of 7 normal controls,
4 cases with cerebral thrombosis, 5 cases with cerebral embolism, 8 cases with multiple lacunar infarctions, and 9 cases with
Binswanger disease. Deparaffinized sections from all cases were immunostained with anti–14-3-3 antibodies using the
avidin-biotin-peroxidase complex method, and some sections were also double-immunostained for 14-3-3 and glial markers.
Results—In the normal control brains, 14-3-3 immunoreactivity was mainly localized to the neuronal somata and
processes. Strongly 14-3-3–immunopositive astrocytes were distributed in the infarct lesions and were particularly
abundant in infarcts at the chronic stage. Intensely 14-3-3–immunolabeled astrocytes were also observed in the ischemic
white matter lesions, and in the severely affected white matter lesions from patients with Binswanger disease, dense
14-3-3 immunoreactivity was found in clasmatodendritic astroglia as well as in reactive astrocytes.
Conclusions—Our results suggest that 14-3-3 proteins may be induced mainly in astrocytes from human cerebrovascular
ischemic lesions, and that the upregulated expression of 14-3-3 proteins in astrocytes may be involved in the formation
of astrogliosis. (Stroke. 2006;37:830-835.)
Key Words: astrocytes 䡲 Binswanger disease 䡲 cerebral infarction 䡲 immunohistochemistry
1
4-3-3 proteins constitute a family of highly conserved
molecules expressed in a wide range of eukaryotic cells.1,2
The name of 14-3-3 was derived from their fraction number
on DEAE-cellulose chromatography and their migration pattern on starch gel electrophoresis.2 There are 7 mammalian
14-3-3 isoforms named by the Greek letters ␤, ␥, ⑀, ␨, ␩, ␴,
and ␪, and the isotypes originally designated as ␣ and ␦ have
been confirmed to be the phosphorylated forms of ␤ and ␨,
respectively.2,3 14-3-3 proteins are involved in various kinds
of signal transduction pathways through phosphorylationdependent protein–protein interactions.2
Several types of chaperone proteins, including heat shock
protein 70 (HSP70),4 HSP27,5 and 150-kDa oxygen-regulated
protein,6 have been demonstrated to be associated with ischemia-induced brain damage. Because ischemic rat brains have
been reported to contain strong immunoreactivity for 14-3-3,7
we hypothesized that 14-3-3 proteins, a novel type of molecular chaperones, would be expressed abnormally in brains
from patients with cerebrovascular disease (CVD). In the
present study, we performed immunohistochemical studies on
14-3-3 proteins in autopsied human brain tissues from patients with CVD, including Binswanger disease. We found
the increased expression of 14-3-3 immunoreactivity in astrocytes from human cerebrovascular ischemic lesions.
Materials and Methods
Tissue Preparation
Tissue materials were obtained from autopsied brains collected at the
Neuropathology Laboratories of Kyoto University and the University
of Vienna. We selected a total number of 33 cases consisting of 7
normal controls (5 men, 2 women; age range 68 to 84 years;
mean⫾SD, 75.9⫾5.6 years), 4 patients with cerebral thrombosis (3
men, 1 woman; age range 74 to 87 years; mean⫾SD, 80.3⫾6.2
years), 5 patients with cerebral embolism (3 men, 2 women; age
range 63 to 87 years; mean⫾SD, 76.0⫾9.4 years), 8 patients with
multiple lacunar infarctions (5 men, 3 women; age range 72 to 86
years; mean⫾SD, 78.6⫾4.6 years), and 9 patients with Binswanger
disease (6 men, 3 women; age range 64 to 86 years; mean⫾SD,
76.1⫾8.2 years). Binswanger disease is a form of vascular dementia,
and diffuse white matter (WM) lesions are the main pathological
features of brains with Binswanger disease. All autopsied brains
were fixed in 10% neutral formalin for ⬇2 weeks at room temperature. Several paraffin-embedded tissue blocks from each case were
prepared and were cut into 6-␮m-thick sections on a microtome. For
routine pathological evaluation, deparaffinized sections from all
cases were stained with hematoxylin and eosin, Klüver-Barrera, and
modified Bielschowsky stains. The stages of the infarcted lesions
Received October 16, 2005; final revision received November 11, 2005; accepted November 30, 2005.
From the Department of Neurology (Y.K., I.A., H.T., Y.S., Y.H.), Faculty of Medicine, Kyoto University, Japan; and the Institute of Neurology (H.B.),
Medical University Vienna, Austria.
Correspondence to Yasuhiro Kawamoto, MD, Department of Neurology, Faculty of Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyoku,
Kyoto 606-8507, Japan. E-mail [email protected]
© 2006 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
DOI: 10.1161/01.STR.0000202587.63936.37
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Kawamoto et al
14-3-3 Proteins in Human Cerebrovascular Lesions
831
Figure 1. Schematic drawings of brain
areas examined in the present study. B1,
B2, and B3 indicate basal ganglia; F1,
F2, F3, and F4, frontal areas; O, occipital
area; P, parietal area; T1 and T2, temporal areas; Th, thalamus.
Downloaded from http://stroke.ahajournals.org/ by guest on June 16, 2017
were classified into 3 types as follows: acute (within 1 week after
onset), subacute (1 to 4 weeks), and chronic (over 4 weeks). The
WM lesions were graded as normal (grade 0), low (grade I; reduced
meshwork density with scattered, irregularly widened axons), moderate (grade II; further reduction in meshwork density compared with
grade I, mainly composed of relatively short axons), and high (grade
III; depletion of axon meshwork with a few remaining long axons)
according to the modified criteria proposed by Englund and Brun.8
The areas examined in this study are schematically presented in
Figure 1, and the clinicopathological profiles from all cases are
summarized in Table 1. All procedures followed were in accordance
with institutional guidelines, and informed consent was obtained
from relatives of all subjects.
Primary Antibodies
To examine the immunohistochemical localization of 14-3-3 proteins in autopsied brains, we used several types of anti–14-3-3
antibodies as follows: a mouse monoclonal anti–14-3-3␤ antibody
(H-8; Santa Cruz Biotechnology [SCB]; diluted 1:1000), a rabbit
polyclonal anti–14-3-3␤ antibody (C-20; SCB; diluted 1:400), a
rabbit polyclonal anti–14-3-3␥ antibody (C-16; SCB; diluted 1:400),
a rabbit polyclonal anti–14-3-3⑀ antibody (T-16; SCB; diluted
1:400), a rabbit polyclonal anti–14-3-3␨ antibody (C-16; SCB;
diluted 1:400), a rabbit polyclonal anti–14-3-3␪ antibody (C-17; SCB;
diluted 1:400), a rabbit polyclonal anti–14-3-3␩ antibody (ImmunoBiological Laboratories [IBL]; diluted 5 ␮g/mL), and a rabbit polyclonal anti–14-3-3␴ antibody (IBL; diluted 2 ␮g/mL). The anti–14-3-3␤
antibody (H-8) recognizes all human 14-3-3 isoforms,9,10 whereas the
other antibodies are 14-3-3 isoform specific.10,11
Immunohistochemistry
Formalin-fixed, paraffin-embedded sections were deparaffinized in
xylene, followed by rehydration in a series of ethanol solutions of
decreasing concentration. The deparaffinized sections were pretreated with 0.3% H2O2 (Santoku) in 0.1 mol/L PBS for 30 minutes
at room temperature to inhibit endogenous peroxidase activity. After
washing with 0.1 mol/L PBS, the sections were blocked with 0.1
mol/L PBS plus 3% skimmed milk for 2 hours at room temperature.
After rinsing with 0.1 mol/L PBS, the primary antibody diluted in 0.1
mol/L PBS was applied onto the sections, and these sections were
maintained at room temperature overnight in a humidified chamber.
After washing with 0.1 mol/L PBS, bound antibodies were visualized
by the avidin-biotin-peroxidase complex (ABC) method using bio-
tinylated secondary antibodies (Vector Laboratories), Vectastain ABC
kits (Vector), and diaminobenzidine tetrahydrochloride (Dojin) as a
chromogen. Some sections were incubated with either nonimmune
mouse or rabbit serum, and no specific immunopositive staining was
detected in these negative control sections (data not shown).
Double Immunostaining for 14-3-3
and Glial Markers
To evaluate the relationship between 14-3-3 proteins and glial cells
in cerebrovascular ischemic lesions, we selected a total number of 55
areas from both control and CVD groups, consisting of 5 acute
infarcted areas, 5 subacute infarcted areas, 5 chronic infarcted areas,
10 WM areas of grade 0, 10 WM areas of grade I, 10 WM areas of
grade II, and 10 WM areas of grade III. These area-containing
sections were double-immunostained using antibodies directed
against 14-3-3 (H-8) and glial markers. The following antibodies
were prepared: an anti– glial fibrillary acidic protein (GFAP) antibody (DAKO; rabbit polyclonal; diluted 1:1000) as a major marker
for astrocytes, an anti-vimentin antibody (DAKO; mouse monoclonal; diluted 1:1000) as an another marker for astrocytes, an antitransferrin antibody (DAKO; rabbit polyclonal; diluted 1:100) as a
marker for oligodendrocytes, and an anti-CD11b antibody (SCB;
goat polyclonal; diluted 1:100) as a marker for microglia. After an
incubation with the primary antibodies, the sections were reacted
with secondary antibodies consisting of fluorescein isothiocyanate–
labeled goat anti-mouse IgG (DAKO) and rhodamine-conjugated
swine anti-rabbit IgG (DAKO) or rhodamine-conjugated donkey
anti-goat IgG (Chemicon International). After washing with 0.01
mol/L PBS, the slides were coverslipped with Vectashield (Vector)
and viewed with the aid of a fluorescence microscope. The 14-3-3–
immunolabeled glial density was assessed by calculating the average
percentage of 14-3-3–positive glial cells in various types of ischemic
lesions and was divided into 5 categories: high (ⱖ80%), moderate (from
50% inclusive to 80% exclusive), low (from 10% inclusive to 50%
exclusive), very low (⬍10%), or absent. The density of vimentinpositive astrocytes in the ischemic lesions was evaluated using the same
double-immunofluorescence staining for GFAP and vimentin.
Results
In the normal control brains, 14-3-3 immunoreactivity was mainly
localized to the neuronal somata and processes (Figure 2A). In
contrast to this intense neuronal immunolabeling pattern, the glial
832
Stroke
March 2006
TABLE 1.
Clinicopathological Profiles From All Cases
Case
No.
Age, y/
Sex
Diagnosis
Postmortem
Delay, h
WM
Lesions
Examined Areas
(Figure 1)
1
2
68/M
Rheumatoid arthritis
2.0
0
B2, F2, F3, T2
73/M
Hepatocellular carcinoma
4.5
0
F2, F3, T2, P
3
75/M
Pulmonary emphysema
2.0
0
B1, F2, F3
4
77/M
Renal cell carcinoma
2.3
0
B2, F2, T1
5
72/F
Cervical spondylosis
UD
0
B2, F3, Th
6
84/M
Renal failure
3.0
0
F1, F2, T2, P
7
82/F
Myocardial infarction
UD
I
F2, F3, T1, T2
8
84/M
Cerebral thrombosis
3.1
I
F3, F4, P, O
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9
87/M
Cerebral thrombosis
0.9
II
B3, F2, F3, T2
10
74/F
Cerebral thrombosis
9.4
I
B3, F1, F2, T2
11
76/M
Cerebral thrombosis
UD
I
F1, F2, F3, P
12
73/M
Cerebral embolism
1.3
0
B1, F2, F3, T2
13
83/M
Cerebral embolism
UD
0
F2, F3, F4, P
14
87/F
Cerebral embolism
2.6
I
B1, B2, F2, F3
15
63/M
Cerebral embolism
2.4
I
B1, F2, F3, Th
16
74/F
Cerebral embolism
UD
I
B2, F1, F3, O
17
81/M
Multiple lacunar infarctions
UD
I
B2, B3, F1, F2
18
72/M
Multiple lacunar infarctions
6.0
II
B1, B2, F1, F2
19
79/M
Multiple lacunar infarctions
5.0
II
B2, B3, F1, F2
20
82/F
Multiple lacunar infarctions
UD
II
B2, F3, O, Th,
21
77/F
Multiple lacunar infarctions
UD
II
B2, F2, T2, Th
22
86/M
Multiple lacunar infarctions
4.7
I
B2, B3, O, T2
23
79/F
Multiple lacunar infarctions
1.9
I
B2, F2, F4, Th
24
73/M
Multiple lacunar infarctions
4.5
II
B1, B2, F2, T1
25
77/M
Binswanger disease
6.0
III
F2, F3, F4, T2
26
80/M
Binswanger disease
2.5
III
F2, F3, F4, T2
27
64/M
Binswanger disease
4.5
III
F2, F3, O, P
28
86/F
Binswanger disease
UD
III
F1, F2, F4, P
29
76/F
Binswanger disease
1.2
III
F1, F2, T1, P
30
82/F
Binswanger disease
UD
III
B2, F2, F3, P
31
70/M
Binswanger disease
UD
III
F1, T2
32
65/M
Binswanger disease
8.0
III
F2, T2
85/M
Binswanger disease
3.0
III
F2, T1, T2
33
M indicates male; F, female; UD, undetermined; WM, white matter.
elements generally showed faint or no 14-3-3 immunoreactivity in
the normal control cortical and WM areas (Figure 2B).
In the acute infarcted lesions, these were many neurons in
which an excessive accumulation of 14-3-3 immunoreactivity
Figure 2. 14-3-3 immunoreactivity (H-8) in the normal frontal
cortex (A, case 2) and WM (B, case 2). Note that only weak
immunoreactivity was detected in some glial cells in the normal
cerebral and WM areas. Bar⫽50 ␮m in A and B.
was found in the perinuclear areas (Figure 3A), and moderately immunopositive glial cells, including astrocytes, were
sparsely scattered (Figure 3B). At the subacute stage, 14-3-3
immunoreactivity was well-preserved in most remaining
neurons (Figure 3C). Intensely immunostained reactive astrocytes were distributed around the infarcts (Figure 3D), and
immunolabeled macrophages accumulated in the center of
these infarcts (Figure 3E). In the chronic infarcted lesions,
intensely immunopositive ballooned neurons were scattered
(Figure 3F). Numerous reactive astrocytes showed strong
14-3-3 immunoreactivity (Figure 3G), and densely immunopositive gemistocytic astrocytes with hypertrophic cell
bodies (Figure 3H) and fibrillary astrocytes (Figure 3I) were
observed in some areas of the chronic infarcts.
In the mildly affected WM lesions (grade I), some glial
cells and axons exhibited moderate 14-3-3 immunoreactivity
Kawamoto et al
14-3-3 Proteins in Human Cerebrovascular Lesions
833
Figure 3. 14-3-3 immunoreactivity (H-8)
in the cerebral infarcted lesions at the
acute (A and B, case 14), subacute (C
through E, case 13), and chronic stages
(F and G, case 10; H, case 22; I, case
19). We evaluated 68 types of sections
from 17 brains, consisting of 4 cases
with cerebral thrombosis, 5 cases with
cerebral embolism, and 8 cases with
multiple lacunar infarctions. 14-3-3–
immunopositive astrocytes were
detected in and around the infarcted
lesions from all 17 brains. Bar⫽50 ␮m in
B, D, G, H and I. Bar⫽20 ␮m in A, C, E,
and F.
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(Figure 4A). In the moderately affected WM lesions (grade
II), the number of immunopositive glial cells, some of which
were strongly immunostained, had increased (Figure 4B), and
immunolabeled glial cells were abundant in some areas of
these WM lesions (Figure 4C). In the severely affected WM
lesions (grade III) from Binswanger disease brains (Figure
4G), numerous reactive astrocytes were intensely immunopositive for 14-3-3 (Figure 4D). In addition, in the Binswanger
disease cases with severe rarefactions (Figure 4H and 4I), the
so-called “clasmatodendritic astroglia,” were also densely immunostained (Figure 4E), and their cell bodies were swollen and
vacuolated, with their processes disintegrated (Figure 4F).
Immunohistochemical studies on the 14-3-3 isoforms in
several sorts of human cerebrovascular ischemic lesions
Figure 4. 14-3-3 immunoreactivity (H-8)
in ischemic WM lesions of grade I (mildly
affected; A, case 15), grade II (moderately affected; B, case 21; C, case 20),
and grade III (severely affected; D, case
25; E and F, case 26). We evaluated 124
types of sections from all normal and
disease brains. Enhanced immunoexpression of 14-3-3 proteins in reactive
astrocytes was confirmed in the WM
lesions from all of 26 brains with cerebrovascular disease. Clasmatodendritic
astroglia, which were detected in 4
brains with Binswanger disease, were
intensely immunostained. Decreased
number of oligodendroglial nuclei was
evident in hematoxylin and eosin–stained
sections (G, case 25; H and I, case 26;
arrowheads indicate an identical clasmatodendritic astroglia). Bar⫽50 ␮m in A
through E. Bar⫽20 ␮m in F through I.
showed that the astrocytes were immunoreactive for all types
of 14-3-3 isoforms. In the severely affected WM lesions, not
only reactive astrocytes (Figure 5A through 5E) but also
clasmatodendritic astroglia (Figure 5F) were immunolabeled
by all of the 14-3-3 isoform-specific antibodies. Further
immunohistochemical studies of serial sections immunostained
by the different 14-3-3 isoform-specific antibodies demonstrated
that most reactive astrocytes contained ⱖ2 14-3-3 isoform
immunoreactivities, suggesting that several kinds of 14-3-3
isoforms may be induced into each reactive astrocyte.
The double-immunostaining studies revealed that the majority
of 14-3-3–immunopositive glial cells were astrocytes in the
infarcted areas and ischemic WM lesions (Figure 6A through
6C), and some oligodendrocytes (Figure 6D through 6F) and
834
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March 2006
Discussion
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Figure 5. 14-3-3 isoform immunoreactivity in the severely
affected WM lesions (A through E, case 29; F, case 26). The
reactive astrocytes were intensely immunopositive for 14-3-3␤
(C-20; A), 14-3-3␥ (C-16; B), 14-3-3⑀ (T-16; C), and 14-3-3␪
(C-17; D), and dense 14-3-3␳ immunoreactivity was observed in
the reactive astrocytes (E) and clasmatodendritic astroglia (F).
We evaluated 63 types of sections from 17 brains, consisting of
8 cases with multiple lacunar infarctions and 9 cases with
Binswanger disease. Reactive astrocytes were found in all of 17
brains, and clasmatodendritic astroglia were detected in 4
brains with Binswanger disease, and both types of astrocytes
were immunoreactive for all of the 14-3-3 isoforms. Bar⫽50 ␮m
in A through F.
microglia (Figure 6G through 6I) were also immunoreactive for
14-3-3 in both types of ischemic lesions. The data for the density
of 14-3-3–positive glial cells and vimentin-positive astrocytes
(figure not shown18 –20) is summarized in Table 2.
Figure 6. Double-immunofluorescence
staining for glial markers (A, GFAP; D,
transferrin; G, CD-11b) and 14-3-3 (B, E,
and H; A through I, case 29). Many
GFAP-immunopositive astrocytes were
immunopositive for 14-3-3 (C), and
some transferrin-immunolabeled oligodendrocytes and CD-11b–immunolabeled microglia were immunoreactive
for 14-3-3 (F and I, respectively).
Bar⫽25 ␮m in A through I.
In the present study, we performed immunohistochemical studies on 14-3-3 proteins in autopsied human brains with CVD. The
most remarkable finding of our results was the upregulated
expression of 14-3-3 immunoreactivity in astrocytes from both
cerebral infarctions and ischemic WM lesions, suggesting that
14-3-3 proteins may be induced mainly in astrocytes by ischemic stress. The enhanced astroglial immunoexpression of 14-3-3
has been documented in demyelinated lesions from patients with
multiple sclerosis 11,12 and in cortical and subcortical lesions
from patients with Creutzfeldt-Jakob disease.13 Moreover, similarly to multiple sclerosis and Creutzfeldt-Jakob disease, reactive astrocytes were immunoreactive for all kinds of 14-3-3
isoforms in the cerebrovascular ischemic lesions. These data
suggest that the increased expression of 14-3-3 immunoreactivity in astrocytes is not specific for ischemia, and that 14-3-3
proteins may be induced in astrocytes by several kinds of
pathological conditions.
GFAP and vimentin, both of which are the markers most
often used for identifying astrocytes, increase in reactive
astrocytes.14 Satoh et al found the differential immunoexpression of 14-3-3 isoforms in reactive astrocytes from demyelinating lesions in patients with multiple sclerosis and demonstrated
that the 14-3-3 isoforms, particularly 14-3-3⑀, interacted with
GFAP and vimentin in cultured human astrocytes.11 On the
basis of the fact that GFAP and vimentin are coexpressed and
copolymerized in assembled filaments in astrocytes,15,16 they
proposed the possibility that 14-3-3 proteins may play an
organizing role in the intermediate filament network in
reactive astrocytes from the demyelinating lesions of multiple
sclerosis.11 Not only GFAP, but also vimentin are expressed
in reactive astrocytes of human cerebral infarcts,17 and our
present study showed the immunohistochemical localization
of 14-3-3 isoforms, including 14-3-3⑀, in reactive astrocytes
from infarcted lesions. Our results suggest that similarly to
the demyelinating lesions of multiple sclerosis, 14-3-3 proteins may act as an adaptor that connects GFAP and vimentin
in reactive astrocytes of human cerebral infarcts. Although
Kawamoto et al
14-3-3 Proteins in Human Cerebrovascular Lesions
835
TABLE 2. Density of 14-3-3–Positive Glial Cells and Vimentin-Positive Astrocytes
in Cerebrovascular Ischemic Lesions
14-3-3–Positive Glial Cells
Astrocytes
Oligodendrocytes
Microglia
Vimentin-Positive
Astrocytes
⫾⬃⫹
⫾
⫺
⫾⬃⫹
⫹⫹
⫾
⫺
⫹⫹
Infarction (chronic)
⫹⫹⫹
⫹
⫹
⫹⫹
WM lesions grade 0
⫾
⫾
⫺
⫾
WM lesions grade I
⫹
⫾
⫺
⫹⫹
WM lesions grade II
⫹⫹
⫹
⫾
⫹⫹
WM lesions grade III
⫹⫹⫹
⫹
⫹
⫹⫹
Infarction (acute)
Infarction (subacute)
⫹⫹⫹ indicates high (ⱖ80%); ⫹⫹, moderate (from 50% inclusive to 80% exclusive); ⫹, low
(from 10% inclusive to 50% exclusive); ⫾, very low (⬍10%); ⫺, absent.
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neurons contain intense 14-3-3 immunoreactivity, they are
generally sensitive to ischemic insults, suggesting that the
network formation of GFAP and vimentin by 14-3-3 proteins
may be closely related to the survival of astrocytes under
ischemic conditions.
In ischemic WM lesions, some astrocytes undergo morphological alterations in which their cell bodies become
swollen and vacuolated, and their disintegrated processes are
identified as granules of 1 to 3 ␮m in diameter.18 –20 These
regressive astrocytes are termed “clasmatodendritic astroglia,” and their cytochemical and immunohistochemical features suggest that clasmatodendritic astroglia incorporate
edematous fluid and phagocytose cellular debris and eventually degenerate as a result of cerebral edema.18,20 In the
present study, we found strong 14-3-3 immunoreactivity not
only in reactive astroglia but also in clasmatodendritic astroglia in the severely affected WM lesions from patients with
Binswanger disease. Both reactive and clasmatodendritic
astroglia have been reported to be immunoreactive for GFAP
and vimentin.18,20 Thus, our findings suggest that both GFAP
and vimentin may be target proteins for 14-3-3 proteins in
clasmatodendritic astroglia as well as in reactive astrocytes in
the ischemic WM lesions, and that 14-3-3 proteins may be
associated with the regulation of the intermediate filament
network, in even these regressive astrocytes, but 14-3-3 overexpression mechanisms might be insufficient to protect them
from ischemic damage under severe ischemic conditions.
Acknowledgments
This work was supported by a grant from the Ministry of Education,
Culture, Sports, Science and Technology of Japan, and by a
grant-in-aid for scientific research on priority areas from the Ministry
of Education, Culture, Sports, Science and Technology of Japan.
This work was also supported by a grant from Takeda Medical
Research Foundation (Osaka, Japan). The authors thank Hitomi
Nakabayashi for her excellent technical assistance.
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Upregulated Expression of 14-3-3 Proteins in Astrocytes From Human Cerebrovascular
Ischemic Lesions
Yasuhiro Kawamoto, Ichiro Akiguchi, Hidekazu Tomimoto, Yoshitomo Shirakashi, Yasuyuki
Honjo and Herbert Budka
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Stroke. 2006;37:830-835; originally published online January 19, 2006;
doi: 10.1161/01.STR.0000202587.63936.37
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