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Intranuclear localization and isoform-dependent translocation of 14-3-3 proteins in human brain with infarction
Journal of the Neurological Sciences 260 (2007) 159 – 166 www.elsevier.com/locate/jns
Intranuclear localization and isoform-dependent translocation of 14-3-3 proteins in human brain with infarction Takahiko Umahara a,b,⁎, Toshiki Uchihara b , Kuniaki Tsuchiya c , Ayako Nakamura b , Toshihiko Iwamoto a a
c
Department of Geriatric Medicine, Tokyo Medical University, 6-7-1 Nishi-Shinjuku, Shinjuku-ku, Tokyo, 160-0023, Japan b Department of Neurology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan Department of Laboratory Medicine and Pathology, Tokyo Metropolitan Matsuzawa Hospital, Setagaya-ku, Tokyo, Japan Received 18 November 2006; received in revised form 21 April 2007; accepted 25 April 2007 Available online 11 June 2007
Abstract Immunolocalization of 14-3-3 proteins in human brains with infarction was investigated using isoform-specific antibodies. Neurons around acute or subacute ischemic foci exhibited an enhanced immunoreactivity for 14-3-3 proteins either in the cytoplasm (especially for its sigma isoform) or in the nucleus (especially for its beta isoform), and sometimes in both. 14-3-3-like immunoreactivity was evaluated in each neuron, which enabled us to identify into three patterns: intense cytoplasmic staining with or without nuclear staining; a predominant nuclear staining with weak cytoplasmic staining; and an exclusive nuclear staining without cytoplamic staining. Quantification of 1500 neurons in relation to the severity of ischemia estimated by the relative distance from ischemic foci clarified that nuclear immunoreactivity for 14-3-3 proteins was more frequent in neurons near the ischemic core. Although the cytoplasm of astrocytes was similarly positive for the sigma and the epsilon isoform, their nuclei were only immunopositive for the gamma isoform. In the cerebral white matter with ischemia, axonal swelling and some nuclei of oligodendrocytes were positive for the zeta isoform. Isoform-specific translocation of 14-3-3 proteins into nuclei is a cellular reaction to ischemic stress that may be related to survival of neurons and their protection against cell death. © 2007 Elsevier B.V. All rights reserved. Keywords: 14-3-3 proteins; Apoptosis; Brain infarction; Ischemic penumbra; Neuronal nucleus; Isoform-dependent translocation
1. Introduction 14-3-3 proteins belong to a family of a highly conserved 30 kDa molecule [1,2], and are mainly localized neuronal cytoplasm in physiological conditions. They have now been recognized to have a wide range of potential functions and pathological relevance [reviewed by [2–7]]. The proteins regulate intercellular signal transduction to bind the target molecules including transcription co-factor [8,9]. In addition, they can prevent or mediate apoptosis, by controlling potential signaling molecules [10–12] in the nucleus or mitochondria and cytoplasm. Possible influences of 14-3-3 ⁎ Corresponding author. Department of Geriatric Medicine, Tokyo Medical University, 6-7-1 Nishi-Shinjuku, Shinjuku-ku, Tokyo, 160-0023, Japan. Tel.: +81 3 3342 6111; fax: +81 3 3342 2305. E-mail address: [email protected] (T. Umahara). 0022-510X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2007.04.053
proteins on cell survival or death are attracting increasing attention because they are colocalized to a variety of specific pathological deposits, such as neurofibrillary tangles [13,14], Pick bodies [15], Lewy bodies [16]. Moreover, 7 isoforms (β: beta, γ: gamma, ε: epsilon, ζ: zeta, η: eta, τ: tau, and σ: sigma) of the protein have been so far identified in mammals. There have been several reports concerning possible relevance of 14-3-3 proteins in isoform-specific fashion [17–21]. However, physiological functions or pathological relevance of each isoform are yet to be clarified. To rescue the damaged neuron in ischemic penumbra is one of the major therapeutic concerns in case of ischemic stroke. Potential functions of 14-3-3 proteins, which mediate cell survival, prompted us to examine the possible involvement of 14-3-3 protein isoforms in ischemic penumbra. Although there have been several reports of 143-3 proteins (or molecules) in animal models and human
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brains of ischemia [21–25], detailed immunohistochemical investigations in human brain samples on 14-3-3 proteins are still limited. In the present study, all known isoforms of 14-33 were studied and we demonstrated that nuclear shift of 143-3 immunoreactivity in neurons after ischemia is isoform dependent.
2. Methods Specimens were obtained from 3 control subjects (66– 82 years old), 8 patients (49–93 years old) with cerebral infarction (5 acute or subacute cases, including 3 frontal and 2 parietal lobes; duration 8–13 days, 3 chronic cases; duration
Fig. 1. Immunolocalization of 14-3-3 proteins in the brains with infarction. Epitopes were probed with an anti-14-3-3 antibody that recognized all the isoforms (14-3-3 COM) A: Many neuronal nuclei in the area surrounding acute infarction (the so-called periinfarct area or ischemic penumbra) were immunopositive for 14-3-3 proteins (common: COM). (Bar 100 μm) IC: ischemic core, N: apparently normal (unaffected) area. P: area surrounding acute infarction (so-called periinfarct area or ischemic penumbra), which was divided into three zones; az: adjacent zone, iz: intermediate zone, dz: distant zone. B: Shrunken and/or triangularly shaped neurons (so-called dark neuron or ischemic cell change) were observed at the periinfarct area, and many of those neurons were positive for 14-3-3-COM-like IR that was found in the entire structure of nucleus. (Bar 10 μm) C: Some of markedly shrunken neurons showed 14-3-3-COM-like IR exclusively in nucleus. (Bar 10 μm) D: Some neurons exhibited either mainly cytoplasmic intense immunostaining (arrowhead) or copresence of cytoplasmic and nuclear staining (arrow) for 14-3-3 COM. (Bar 10 μm) E: There were a few strongly immunopositive neurites. (Bar 10 μm); F: In a cerebellar infarction case, some Purkinje cells in unaffected areas on the same section were only weakly immunolabeled with anti-14-3-3 COM antibody. (Bar 50 μm) G: Purkinje cells including nucleus in the ischemic area were constantly and intensely immunolabeled with anti-14-3-3 COM antibody. (Bar 50 μm) H, I: Immunohistochemical labeling (H) was abolished when the primary antibody (14-3-3 COM) was coincubated with the corresponding antigen peptide (I). (Bar 50 μm).
T. Umahara et al. / Journal of the Neurological Sciences 260 (2007) 159–166 Table 1 Number of 14-3-3 proteins positive neurons IR pattern
Regions Adjacent zone Intermediate zone Distant zone Total cells
Type A 69 (13.8%) Type B 220 (44.0%) Type C 211 (42.2%) Total 500 (100%) Cells
91 (18.2%) 345 (69.0%) 64 (12.8%) 500 (100%)
273 (54.6%) 204 (40.8%) 23 (4.6%) 500 (100%)
433 (28.9%) 769 (51.3%) 298 (19.9%) 1500 cells (100%)
Chi-square test: p b 0.0001.
7–12 month) and 2 patients (79, 89 years old) with cerebellar infarction. Five micronmeter-thick sections were obtained from formalin-fixed, paraffin-embedded blocks. After being heated in a citrate buffer in a pressure cooker for 20 min and treated with formic acid for 5 min, sections were treated with
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1% hydrogen peroxide for 30 min. They were then incubated with one of the primary antibodies made in rabbits (ImmunoBiological Laboratories, Gunma, Japan) against human 14-3-3 protein (1:2000, anti-14-3-3 COM, KDSTLIMQLLRDNLT, a target sequence shared by all the isoforms) or each isoform [anti-beta: MTMDKSELVQ, 1:500; anti-gamma: QQDDDGGEGNN, 1:200; anti-epsilon: MGDREQLLQR, 1:500; anti-zeta: MDKNELVQK, 1:200; anti-sigma (C): EEGGEAPQEPQS, 1:300; and anti-eta MGDREQLLQR isoforms], and used anti-14-3-3 tau isoform monoclonal antibody (raised against recombinant human 14-3-3 tau isoform) at 4 °C for 2 days. The specificity of these antibodies had been established previously on Western blot [14,15,18] and absorption studies [14,15]. There was little or no crossreaction among isoform antibodies. They were then incubated with the appropriate biotinylated secondary antibody for 2 h. After incubation with the avidin–
Fig. 2. Immunolocalization of beta, sigma, gamma, zeta and tau isoforms of 14-3-3 proteins at the periinfarct area. A: The nuclear IR of the beta isoform was most prominent among those 7 isoforms and mostly equal to those of 14-3-3COM, however beta isoform-like IR in cytoplasm was less intense than those of 14-33COM. (Bar 10 μm) B: Neuronal cytoplasm intensely immunolabeled with the anti-sigma antibody, however staining of neuronal nuclei of those was relatively moderate. (Bar 10 μm) C: Neuropil of the cerebral cortex was homogeneously stained with the anti-epsilon antibody. Only a few nuclei were labeled by this antiepsilon antibody. Some astrocytic cells were also stained. (Bar 10 μm) D: Gamma isoform like IR were observed in nuclei of glial-like cells around ischemic core. (Bar 25 μm) E: In the ischemic area in the white matter of cerebrum, axonal swelling and some nucleus of oligodendrocyte were immunolabeled with the antizeta isoform antibody. (Bar 25 μm) F: Tau-isoform immunopositive glia located only near the zone of peripheral region of ischemic core. (Bar 100 μm) G–I: Immunohistochemical labeling was abolished when the primary antibodies (gamma; G, H and zeta; I, J isoforms) were coincubated with each corresponding antigen peptides (H, J). (Bar 50 μm).
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biotin–peroxidase complex (1:1,000, ABC Elite, Vector, Burlingame, CA) for 1 h, peroxidase labeling was visualized with 0.03% 3,3-diaminobenzidine, 0.6% nickel ammonium sulfate, 0.05 M imidazole and 0.00015% hydrogen peroxide. A deep purple immunoreaction product appeared after 15– 20 min. For double immunolabeling with anti-gamma isoform and anti-glial fibrillary acidic protein (GFAP, 1:2000, mouse monoclonal antibody clone 6F2; DAKO, Glostrup, Denmark), the sections stained with the anti-gamma isoform were subjected to second-cycle immunostaining with the antiGFAP antibody. They were then treated similarly except that diaminobenzidine was used without nickel ammonium sulfate to yield brown reaction products. In this study, the ischemic core means the area where almost all neurons disappeared and neuropil were necrotic due to acute infarction. The areas around the ischemic core correspond to “the periinfarct area or ischemic penumbra” which are between the outer edge of necrotic area (ischemic core) and the inner margin of the apparently normal (unaffected) area. In order to determine the subcellular localization of 14-3-3 epitopes, areas around the ischemic core were evenly divided into three zones (one example in Fig. 1A): “adjacent”, “intermediate” and “distant” relative to the ischemic core. The localization of the 14-3-3 COM epitope was classified as nuclear or cytoplasmic and its intensity as intense or weak. At least 100 neurons from these three zones of each section were classified. For in-situ labeling of fragmented DNA, terminal deoxynucleotidyl transferase-mediated dUTP nick end labellimg (TUNEL) Kit (Invitrogen Corp. Carlsbad, CA, U.S.A.) was used. Briefly, all sections were incubated with terminal deoxynucleotidyl transferase and biotinylated dUTP according to the manufacturer’s instructions. The labeling was visualized with 0.03% 3,3-diaminobenzidine, 0.6% nickel ammonium sulfate.
“dark neuron” [26]. When extremely shrunken, some neurons exhibited 14-3-3-like IR exclusively in the nucleus (Fig. 1C). Neuronal cell without this ischemic feature exhibited either mainly cytoplasmic staining (Fig. 1D arrowhead) or coexistent cytoplasmic and nuclear staining (Fig. 1D arrow) for 14-3-3 proteins. There were a few strongly immunopositive neurites (Fig. 1E). In apparently normal (unaffected) area on the same section with infarction, 14-3-3-like IR in neurons (Fig. 1A) was similar to those in control cases. In necrotic areas with profound ischemia (ischemic core), where neurons disappeared, there was no 14-3-3-like IR (Fig. 1A). In the cerebellum, some Purkinje cells in unaffected area were at most weakly immunolabeled with the anti-14-3-3 COM antibody (Fig. 1F). In contrast, Purkinje cells in ischemic areas exhibited an intense 14-3-3 COM-IR in the cytoplasm and nucleus (Fig. 1G). Immunohistochemical labeling was abolished when the primary antibody (14-3-3 COM) was coincubated with the corresponding antigen peptide (Fig. 1H, I) In cases with chronic cerebral infarction, 14-3-3 COM-IR was weak and restricted to a small number of neurons (data not shown). In each of the 5 acute cases, 100 neurons immunopositive for 14-3-3 from each of the three zones (adjacent, intermediate and distant) were classified based on the 14-3-3-COM IR as follows; Type A: intense cytoplasmic staining without (Type A1) or with (Type A2) nuclear staining; Type B: predominantly nuclear staining with weak cytoplasmic staining, and Type C: exclusively nuclear staining without cytoplasmic staining. In total 1500 immunopositive neurons were classified into the three patterns (Table 1, chi-square test: p value b 0.0001). In the adjacent zone, Type C was predominant (42.2%) and Type Awas least frequent (13.8%), indicating that the intense nuclear staining was more frequent near the ischemic focus. In the distant zone, however, this staining
3. Results In cerebral sections from control subjects, weak and finegranular 14-3-3-like immunoreactivity (IR) (anti-14-3-3 COM) was observed in the neuronal cytoplasm and processes of neurons in all cases as described previously [14,15]. In cerebellar sections from control subjects, 14-3-3like IR (anti-14-3-3 COM) was more intense than that in the cerebral cortex neurons and was restricted to the cytoplasm of Purkinje cells (data not shown). In acute to subacute cerebral infarction cases, neuronal nuclei exhibited an intense or granular 14-3-3-like IR at the periphery of ischemic focus (so-called periinfarct area or ischemic penumbra) (Fig. 1A). This IR was similarly observed in frontal and temporal cortices. Frequently, the entire nucleus was filled with this IR (Fig. 1B). Less intense IR in the cytoplasm was very often associated with an intense 14-3-3-like IR in the nucleus of neurons. These neuronal cells with an intense 14-3-3-like IR in the nucleus were frequently shrunken and triangular, representing so-called
Fig. 3. Double immunolabeling with anti-gamma isoform antibody (purple) and anti-glial fibrillary acidic proteins (GFAP) antibody (brown). The cytoplasm of gamma isoform-positive glial nuclei was immunolabeled with the anti-GFAP antibody. (Bar 10 μm).
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Fig. 4. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-labeled neuronal nuclei. A: Several neuronal nuclei in the area surrounding acute infarction (so-called periinfarct area or ischemic penumbra) were labeled with TUNEL technique, although a few TUNEL-labeled cells were seen in both the ischemic core and apparently normal (unaffected) area. (Bar 10 μm) P: periinfarct area or ischemic penumbra. IC: ischemic core. N: apparently normal (unaffected) area. B: Apparently normal (unaffected) area. (Bar 10 μm) C: Area surrounding acute infarction (so-called periinfarct area or ischemic penumbra). Several TUNEL positive shrunken and/or the triangular-shaped neurons were observed. (Bar 10 μm) D: A few TUNEL positive neuronal nuclei exhibited fragmentation, which may show apoptotic appearance. (Bar 4 μm) E: In chronic infarction cases, fewer TUNEL-labeled cells were detected. (Bar 10 μm).
profile was reversed, Type C (4.6%) was least frequent and Type A (54.6%) as most prevalent, indicating cytoplasmic staining, reminiscent of normal neurons, is more frequent in area distant from ischemic focus. In the intermediate zone, Type B was predominant (69.0%), suggesting a transition between Type A and Type C. These trends were observed regardless of the location (frontal or parietal lobes) and both in acute and in subacute cases of infarction. In control cerebral sections, beta, gamma, sigma, tau, eta, and zeta isoforms specific antibodies showed findings almost similar to those with anti-14-3-3 COM [14,15]. In acute to subacute cerebral infarction cases, isoform-specific IRs for most of the 7 isoforms were similarly noted in the nuclei, although their intensities were generally less than that of 143-3 COM and varied greatly. Among them, beta isoform-like IR was concentrated in the nucleus (Fig. 2A) similar to that of 14-3-3COM (Fig. 1B) as seen at the periphery of the ischemic core. In contrast, sigma isoform-like IR was more intense in neuronal cytoplasm without nuclear recruitment (Fig. 2B). The cytoplasm of some astrocytes was also immunopositive for the sigma isoform. The neuropil of the cerebral cortex was homogeneously stained with the antiepsilon antibody, while neuronal soma was relatively spared. Number of nucleus labeled by this anti-epsilon antibody was limited. Many astrocytic cells were also immunolabeled with this anti-epsilon antibody (Fig. 2C). On the other hand, gamma isoform-like IR was observed in the nucleus of glial cells (Fig. 2D). In the ischemic area of the white matter, swollen axons and some nuclei of oligodendrocytes were
immunolabeled with the anti-zeta isoform antibody (Fig. 2E). Tau isoform-positive glia located only at the outer border zone of ischemic necrosis (Fig. 2F). Immunohistochemical labeling was abolished when each of the primary antibodies (7 isoforms) was coincubated with the corresponding antigen peptide (Fig. 2G, H). Double immunostaining with the anti-GFAP antibody demonstrated that glial cell with gamma isoform-immunopositive nuclei were astrocytes (Fig. 3). Several neuronal nuclei in areas surrounding acute to subacute infarction (so-called periinfarct area or the ischemic penumbra) were labeled intensely with the TUNEL technique (Fig. 4 A), although a few TUNEL-labeled nuclei were seen in both apparently normal (unaffected) area (Fig. 4A, B) and ischemic core (Fig. 4A). In area surrounding acute infarction, several TUNEL-labeled shrunken and/or triangularly shaped neurons were observed (Fig. 4C). A few TUNEL-labeled neuronal nuclei were fragmented, which may show apoptotic appearance (Fig. 4D). In chronic infarction cases, fewer TUNEL-labeled cells were detected (Fig. 4E). 4. Discussion In the present study, we demonstrated that 14-3-3-like IR is increased in both nuclei and cytoplasm of neuronal and glial cells mainly located in the periinfarct area (or ischemic penumbra) in human brains. Thus, 14-3-3 proteins are upregulated not only in neuronal cytoplasm but also in nuclei in surviving ischemic cortical neurons and Purkinje cells.
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4.1. A possible correlation between the subcellular localization of 14-3-3 IR and the severity of ischemic insult Because the subcellular localization of 14-3-3 IR varied, we tried to identify a possible correlation between its subcellular localization and the severity of ischemic insult. The relative severity of ischemic insult was defined according to the relative distance from the ischemic focus. We then classified at least four different types of 14-3-3 IR in neurons as follows (Fig. 5), A1: nuclei-negative, cytoplasm-positive, less atrophic neurons (Fig. 1D arrowhead); A2: nuclei-positive, cytoplasmpositive, less atrophic neurons (Fig. 1D arrow); B: nucleipositive, weakly positive cytoplasm, atrophic (triangular) neurons corresponding to ‘dark neurons’ (Fig. 1B) and C: nuclei-positive, cytoplasm-negative, severely atrophic neurons (Fig. 1C). Because distinction between Type A1 and A2 was occasionally difficult due to intense cytoplasmic staining, which veiled the nuclear contour, A1 and A2 were combined as Type A for quantification. Classically, atrophic neurons with triangular nuclei in ischemic areas are identified as dark neurons [26]. Many neurons with intense nuclear staining for 14-3-3COM corresponded to such neurons, represented as Type B, which was the most frequent (51.3%) among the 1500 neurons quantified. Neurons with more intense nuclear staining (Type C) were frequent near ischemic focus. In contrast, neurons with cytoplasm-dominant staining (Type A) were prevalent in areas distant from ischemic foci. This suggests that upregulation (Type A and B) and nuclear translocation (Type C) of 14-3-3 proteins are both induced by ischemia but are dependent on the severity of ischemia. As summarized in Fig. 5, upregulation of 14-3-3 proteins is first restricted in the cytoplasm of non-atrophic neurons (Fig. 5, A1), where the ischemic insult is the slightest. Gradual neuronal shrinkage was accompanied by further translocation
Fig. 5. 14-3-3-proteins immunopositive patterns in affected neurons. N: Normal IR pattern of neuron. Cytoplasm is weakly immunopositive for 143-3 proteins. A1: Cytoplasm-immunopositive/nucleus-fine-immunopositive neuron. A2: Cytoplasm-immunopositive/nucleus-immunopositive neuron. B: Cytoplasm-fine-immunopositive/nucleus-immunopositive atrophic neuron. C: Cytoplasm-immunonegative/nucleus-immunopositive atrophic neuron.
into the nucleus (Fig. 5, B). Finally, most atrophic neurons exhibited 14-3-3-like IR exclusively in the nucleus (Fig. 5, C). We also reported nuclear immunoexpression of 14-3-3 proteins in brains with spinocerebellar ataxia type 1 [27]. There have been several investigations about 14-3-3 proteins of animal model of brain infarction[21–24], one [22] is a preliminary report, and Gao et al. [21] reported that phosphorylated sigma isoform was increased after ischemia in a murine model. Recently, Kawamoto et al. [25] demonstrated 14-3-3 immunopositive reactive astrocytes in human brains with cerebral infarction, although astrocytes were also immunolabeled in other pathological conditions [14,28]. However, they paid little attention to neurons and only described briefly that ‘in the acute infarcted lesion, these were many neurons in which an excessive accumulation of 14-3-3 immunoreactivity was found in the perinuclear area, and at the subacute stage the reactivity was wellpreserved’. We were, however, successful in demonstrating specific 14-3-3 IR in neuronal nuclei (not the ‘perinuclear area’) after the pretreatement. More importantly, we demonstrated that this nuclear shift is dependent on the isoform and the relative severity of ischemia, corroborating its functional relevance related to nuclear translocation. 4.2. How are 14-3-3 proteins involved in ischemia or postischemic reactions? Nuclear accumulation of 14-3-3 proteins in surviving neurons suggests their relevance to intranuclear processes, for example, transcription. Indeed, a number of molecules responsible for transcription and its regulation have been found to be binding partners to 14-3-3 proteins. Examples include histone deacetylases [9], TATA-binding protein [8]. Interestingly, another key molecule in transcription, phosphorylated cAMP response element binding protein (pCREB) was found to be accumulated in the nucleus under experimentally induced ischemia [29], and the CREB coactivator called transducer of regulated CREB activity can interact 14-3-3 proteins [30]. These lines of evidence suggest that involvement of 14-3-3 proteins in ischemic processes is mediated through modifications of transcriptions in several ways. Translocation of 14-3-3 proteins into or out of nuclei is regulated by what is known as a nuclear shuttle and mediated by multiple factors, possibly related to apoptosis [2–7]. For example, dephosphorylated Fkhrl 1 (Forkhead rhabdomyosarcoma-like 1) is translocated from the cytoplasm into the nucleus, resulting in activation of apoptotic genes. Efficient export of Fkhrl 1 from the nucleus requires binding of 14-3-3 proteins [11]. It is then possible that nuclear accumulation represents possible association of 14-3-3 proteins to Fkhrl 1 for their export from the nucleus, counteracting apoptosis. Another possibility is some relation with another mechanism of apoptosis. One of the binding partners of 14-3-3 proteins is BAD [10,12], an inducer of apoptosis upon translocation into mitochondria through inactivation of Bcl-family molecules. Abundant 14-3-3 proteins in the cytoplasm, as
T. Umahara et al. / Journal of the Neurological Sciences 260 (2007) 159–166 Table 2 Differences in cellular and subcellular immunolocalization dependent on isoforms of 14-3-3 protein in cases with acute infarction Antibodies
Particularly immunopositive region
14-3-3 COM Beta isoform Sigma isoform Gamma isoform Zeta isoform
Neuronal nuclei and cytoplasm Neuronal nuclei Neuronal cytoplasm Nuclei of astrocytes Nuclei of oligodendrocyte
we demonstrated in mild ischemia as type A1, might counteract this apoptotic process by trapping BAD in the cytoplasm. We showed TUNEL-labeled neuronal nuclei in acute to subacute infarction cases, possibly related to apoptosis [31]. Although the number of TUNEL positive cells was fewer than those of 14-3-3 positive neuronal nuclei, the dominant part of those TUNEL positive neurons was in the area surrounding acute to subacute infarction, which is in agreement with a recent report [32] that showed predominance of apoptosis in the periphery of human cerebral infarction. 4.3. Isoform-dependent cellar and subcellar accumulation of 14-3-3 proteins Among them (Table 2), it was the beta isoform that intensely immunolabeled neuronal nuclei. This suggests that the beta isoform is the major isoform distributed or transported into nuclei. On the other hand, the sigma isoform tended to be located in the cytoplasm. This is compatible with an observation that the sigma isoform is rapidly exported from the nucleus [20]. Indeed, sigma isoform [21] is the main cytoplasmic sequestration protein of Bax, which is translocated to mitochondria after ischemia and contributes to the apoptosis-signaling pathway. Our finding of the cytoplasmicdominant localization of sigma isoform after ischemia might be associated with this condition. IR for the epsilon isoform was found in the neuropil of the gray matter in control and AD brains [14]. Astrocytes in brains with infarct were also immunolabeled with this isoform-specific antibody, as seen in AD brains [14]. On the other hand, IR for the gamma isoform was seen in the nuclei of astrocytes. Recently, it was reported that the gamma isoform mRNA and protein were upregulated in ischemic cortical astrocytes in vitro, and were selectively induced during ischemia, protecting astrocytes from apoptosis through p-BAD-related signaling [12]. Zeta isoform-immunopositive oligodendrocyte nuclei were located in ischemic lesions and easily detected in the white matter. These findings suggest that some isoforms of 14-3-3 proteins are present in glial nuclei and that they mediate glial cell survival and glial–neuronal interaction. In conclusion, we demonstrated upregulation of 14-3-3 proteins in human brain with ischemia. Nuclear and/or cytoplasmic accumulation was dependent on both the severity of ischemia and the isoform.
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Intranuclear localization and isoform-dependent translocation of 14-3-3 proteins in human brain with infarction Takahiko Umahara a,b,⁎, Toshiki Uchihara b , Kuniaki Tsuchiya c , Ayako Nakamura b , Toshihiko Iwamoto a a
c
Department of Geriatric Medicine, Tokyo Medical University, 6-7-1 Nishi-Shinjuku, Shinjuku-ku, Tokyo, 160-0023, Japan b Department of Neurology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan Department of Laboratory Medicine and Pathology, Tokyo Metropolitan Matsuzawa Hospital, Setagaya-ku, Tokyo, Japan Received 18 November 2006; received in revised form 21 April 2007; accepted 25 April 2007 Available online 11 June 2007
Abstract Immunolocalization of 14-3-3 proteins in human brains with infarction was investigated using isoform-specific antibodies. Neurons around acute or subacute ischemic foci exhibited an enhanced immunoreactivity for 14-3-3 proteins either in the cytoplasm (especially for its sigma isoform) or in the nucleus (especially for its beta isoform), and sometimes in both. 14-3-3-like immunoreactivity was evaluated in each neuron, which enabled us to identify into three patterns: intense cytoplasmic staining with or without nuclear staining; a predominant nuclear staining with weak cytoplasmic staining; and an exclusive nuclear staining without cytoplamic staining. Quantification of 1500 neurons in relation to the severity of ischemia estimated by the relative distance from ischemic foci clarified that nuclear immunoreactivity for 14-3-3 proteins was more frequent in neurons near the ischemic core. Although the cytoplasm of astrocytes was similarly positive for the sigma and the epsilon isoform, their nuclei were only immunopositive for the gamma isoform. In the cerebral white matter with ischemia, axonal swelling and some nuclei of oligodendrocytes were positive for the zeta isoform. Isoform-specific translocation of 14-3-3 proteins into nuclei is a cellular reaction to ischemic stress that may be related to survival of neurons and their protection against cell death. © 2007 Elsevier B.V. All rights reserved. Keywords: 14-3-3 proteins; Apoptosis; Brain infarction; Ischemic penumbra; Neuronal nucleus; Isoform-dependent translocation
1. Introduction 14-3-3 proteins belong to a family of a highly conserved 30 kDa molecule [1,2], and are mainly localized neuronal cytoplasm in physiological conditions. They have now been recognized to have a wide range of potential functions and pathological relevance [reviewed by [2–7]]. The proteins regulate intercellular signal transduction to bind the target molecules including transcription co-factor [8,9]. In addition, they can prevent or mediate apoptosis, by controlling potential signaling molecules [10–12] in the nucleus or mitochondria and cytoplasm. Possible influences of 14-3-3 ⁎ Corresponding author. Department of Geriatric Medicine, Tokyo Medical University, 6-7-1 Nishi-Shinjuku, Shinjuku-ku, Tokyo, 160-0023, Japan. Tel.: +81 3 3342 6111; fax: +81 3 3342 2305. E-mail address: [email protected] (T. Umahara). 0022-510X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2007.04.053
proteins on cell survival or death are attracting increasing attention because they are colocalized to a variety of specific pathological deposits, such as neurofibrillary tangles [13,14], Pick bodies [15], Lewy bodies [16]. Moreover, 7 isoforms (β: beta, γ: gamma, ε: epsilon, ζ: zeta, η: eta, τ: tau, and σ: sigma) of the protein have been so far identified in mammals. There have been several reports concerning possible relevance of 14-3-3 proteins in isoform-specific fashion [17–21]. However, physiological functions or pathological relevance of each isoform are yet to be clarified. To rescue the damaged neuron in ischemic penumbra is one of the major therapeutic concerns in case of ischemic stroke. Potential functions of 14-3-3 proteins, which mediate cell survival, prompted us to examine the possible involvement of 14-3-3 protein isoforms in ischemic penumbra. Although there have been several reports of 143-3 proteins (or molecules) in animal models and human
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brains of ischemia [21–25], detailed immunohistochemical investigations in human brain samples on 14-3-3 proteins are still limited. In the present study, all known isoforms of 14-33 were studied and we demonstrated that nuclear shift of 143-3 immunoreactivity in neurons after ischemia is isoform dependent.
2. Methods Specimens were obtained from 3 control subjects (66– 82 years old), 8 patients (49–93 years old) with cerebral infarction (5 acute or subacute cases, including 3 frontal and 2 parietal lobes; duration 8–13 days, 3 chronic cases; duration
Fig. 1. Immunolocalization of 14-3-3 proteins in the brains with infarction. Epitopes were probed with an anti-14-3-3 antibody that recognized all the isoforms (14-3-3 COM) A: Many neuronal nuclei in the area surrounding acute infarction (the so-called periinfarct area or ischemic penumbra) were immunopositive for 14-3-3 proteins (common: COM). (Bar 100 μm) IC: ischemic core, N: apparently normal (unaffected) area. P: area surrounding acute infarction (so-called periinfarct area or ischemic penumbra), which was divided into three zones; az: adjacent zone, iz: intermediate zone, dz: distant zone. B: Shrunken and/or triangularly shaped neurons (so-called dark neuron or ischemic cell change) were observed at the periinfarct area, and many of those neurons were positive for 14-3-3-COM-like IR that was found in the entire structure of nucleus. (Bar 10 μm) C: Some of markedly shrunken neurons showed 14-3-3-COM-like IR exclusively in nucleus. (Bar 10 μm) D: Some neurons exhibited either mainly cytoplasmic intense immunostaining (arrowhead) or copresence of cytoplasmic and nuclear staining (arrow) for 14-3-3 COM. (Bar 10 μm) E: There were a few strongly immunopositive neurites. (Bar 10 μm); F: In a cerebellar infarction case, some Purkinje cells in unaffected areas on the same section were only weakly immunolabeled with anti-14-3-3 COM antibody. (Bar 50 μm) G: Purkinje cells including nucleus in the ischemic area were constantly and intensely immunolabeled with anti-14-3-3 COM antibody. (Bar 50 μm) H, I: Immunohistochemical labeling (H) was abolished when the primary antibody (14-3-3 COM) was coincubated with the corresponding antigen peptide (I). (Bar 50 μm).
T. Umahara et al. / Journal of the Neurological Sciences 260 (2007) 159–166 Table 1 Number of 14-3-3 proteins positive neurons IR pattern
Regions Adjacent zone Intermediate zone Distant zone Total cells
Type A 69 (13.8%) Type B 220 (44.0%) Type C 211 (42.2%) Total 500 (100%) Cells
91 (18.2%) 345 (69.0%) 64 (12.8%) 500 (100%)
273 (54.6%) 204 (40.8%) 23 (4.6%) 500 (100%)
433 (28.9%) 769 (51.3%) 298 (19.9%) 1500 cells (100%)
Chi-square test: p b 0.0001.
7–12 month) and 2 patients (79, 89 years old) with cerebellar infarction. Five micronmeter-thick sections were obtained from formalin-fixed, paraffin-embedded blocks. After being heated in a citrate buffer in a pressure cooker for 20 min and treated with formic acid for 5 min, sections were treated with
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1% hydrogen peroxide for 30 min. They were then incubated with one of the primary antibodies made in rabbits (ImmunoBiological Laboratories, Gunma, Japan) against human 14-3-3 protein (1:2000, anti-14-3-3 COM, KDSTLIMQLLRDNLT, a target sequence shared by all the isoforms) or each isoform [anti-beta: MTMDKSELVQ, 1:500; anti-gamma: QQDDDGGEGNN, 1:200; anti-epsilon: MGDREQLLQR, 1:500; anti-zeta: MDKNELVQK, 1:200; anti-sigma (C): EEGGEAPQEPQS, 1:300; and anti-eta MGDREQLLQR isoforms], and used anti-14-3-3 tau isoform monoclonal antibody (raised against recombinant human 14-3-3 tau isoform) at 4 °C for 2 days. The specificity of these antibodies had been established previously on Western blot [14,15,18] and absorption studies [14,15]. There was little or no crossreaction among isoform antibodies. They were then incubated with the appropriate biotinylated secondary antibody for 2 h. After incubation with the avidin–
Fig. 2. Immunolocalization of beta, sigma, gamma, zeta and tau isoforms of 14-3-3 proteins at the periinfarct area. A: The nuclear IR of the beta isoform was most prominent among those 7 isoforms and mostly equal to those of 14-3-3COM, however beta isoform-like IR in cytoplasm was less intense than those of 14-33COM. (Bar 10 μm) B: Neuronal cytoplasm intensely immunolabeled with the anti-sigma antibody, however staining of neuronal nuclei of those was relatively moderate. (Bar 10 μm) C: Neuropil of the cerebral cortex was homogeneously stained with the anti-epsilon antibody. Only a few nuclei were labeled by this antiepsilon antibody. Some astrocytic cells were also stained. (Bar 10 μm) D: Gamma isoform like IR were observed in nuclei of glial-like cells around ischemic core. (Bar 25 μm) E: In the ischemic area in the white matter of cerebrum, axonal swelling and some nucleus of oligodendrocyte were immunolabeled with the antizeta isoform antibody. (Bar 25 μm) F: Tau-isoform immunopositive glia located only near the zone of peripheral region of ischemic core. (Bar 100 μm) G–I: Immunohistochemical labeling was abolished when the primary antibodies (gamma; G, H and zeta; I, J isoforms) were coincubated with each corresponding antigen peptides (H, J). (Bar 50 μm).
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biotin–peroxidase complex (1:1,000, ABC Elite, Vector, Burlingame, CA) for 1 h, peroxidase labeling was visualized with 0.03% 3,3-diaminobenzidine, 0.6% nickel ammonium sulfate, 0.05 M imidazole and 0.00015% hydrogen peroxide. A deep purple immunoreaction product appeared after 15– 20 min. For double immunolabeling with anti-gamma isoform and anti-glial fibrillary acidic protein (GFAP, 1:2000, mouse monoclonal antibody clone 6F2; DAKO, Glostrup, Denmark), the sections stained with the anti-gamma isoform were subjected to second-cycle immunostaining with the antiGFAP antibody. They were then treated similarly except that diaminobenzidine was used without nickel ammonium sulfate to yield brown reaction products. In this study, the ischemic core means the area where almost all neurons disappeared and neuropil were necrotic due to acute infarction. The areas around the ischemic core correspond to “the periinfarct area or ischemic penumbra” which are between the outer edge of necrotic area (ischemic core) and the inner margin of the apparently normal (unaffected) area. In order to determine the subcellular localization of 14-3-3 epitopes, areas around the ischemic core were evenly divided into three zones (one example in Fig. 1A): “adjacent”, “intermediate” and “distant” relative to the ischemic core. The localization of the 14-3-3 COM epitope was classified as nuclear or cytoplasmic and its intensity as intense or weak. At least 100 neurons from these three zones of each section were classified. For in-situ labeling of fragmented DNA, terminal deoxynucleotidyl transferase-mediated dUTP nick end labellimg (TUNEL) Kit (Invitrogen Corp. Carlsbad, CA, U.S.A.) was used. Briefly, all sections were incubated with terminal deoxynucleotidyl transferase and biotinylated dUTP according to the manufacturer’s instructions. The labeling was visualized with 0.03% 3,3-diaminobenzidine, 0.6% nickel ammonium sulfate.
“dark neuron” [26]. When extremely shrunken, some neurons exhibited 14-3-3-like IR exclusively in the nucleus (Fig. 1C). Neuronal cell without this ischemic feature exhibited either mainly cytoplasmic staining (Fig. 1D arrowhead) or coexistent cytoplasmic and nuclear staining (Fig. 1D arrow) for 14-3-3 proteins. There were a few strongly immunopositive neurites (Fig. 1E). In apparently normal (unaffected) area on the same section with infarction, 14-3-3-like IR in neurons (Fig. 1A) was similar to those in control cases. In necrotic areas with profound ischemia (ischemic core), where neurons disappeared, there was no 14-3-3-like IR (Fig. 1A). In the cerebellum, some Purkinje cells in unaffected area were at most weakly immunolabeled with the anti-14-3-3 COM antibody (Fig. 1F). In contrast, Purkinje cells in ischemic areas exhibited an intense 14-3-3 COM-IR in the cytoplasm and nucleus (Fig. 1G). Immunohistochemical labeling was abolished when the primary antibody (14-3-3 COM) was coincubated with the corresponding antigen peptide (Fig. 1H, I) In cases with chronic cerebral infarction, 14-3-3 COM-IR was weak and restricted to a small number of neurons (data not shown). In each of the 5 acute cases, 100 neurons immunopositive for 14-3-3 from each of the three zones (adjacent, intermediate and distant) were classified based on the 14-3-3-COM IR as follows; Type A: intense cytoplasmic staining without (Type A1) or with (Type A2) nuclear staining; Type B: predominantly nuclear staining with weak cytoplasmic staining, and Type C: exclusively nuclear staining without cytoplasmic staining. In total 1500 immunopositive neurons were classified into the three patterns (Table 1, chi-square test: p value b 0.0001). In the adjacent zone, Type C was predominant (42.2%) and Type Awas least frequent (13.8%), indicating that the intense nuclear staining was more frequent near the ischemic focus. In the distant zone, however, this staining
3. Results In cerebral sections from control subjects, weak and finegranular 14-3-3-like immunoreactivity (IR) (anti-14-3-3 COM) was observed in the neuronal cytoplasm and processes of neurons in all cases as described previously [14,15]. In cerebellar sections from control subjects, 14-3-3like IR (anti-14-3-3 COM) was more intense than that in the cerebral cortex neurons and was restricted to the cytoplasm of Purkinje cells (data not shown). In acute to subacute cerebral infarction cases, neuronal nuclei exhibited an intense or granular 14-3-3-like IR at the periphery of ischemic focus (so-called periinfarct area or ischemic penumbra) (Fig. 1A). This IR was similarly observed in frontal and temporal cortices. Frequently, the entire nucleus was filled with this IR (Fig. 1B). Less intense IR in the cytoplasm was very often associated with an intense 14-3-3-like IR in the nucleus of neurons. These neuronal cells with an intense 14-3-3-like IR in the nucleus were frequently shrunken and triangular, representing so-called
Fig. 3. Double immunolabeling with anti-gamma isoform antibody (purple) and anti-glial fibrillary acidic proteins (GFAP) antibody (brown). The cytoplasm of gamma isoform-positive glial nuclei was immunolabeled with the anti-GFAP antibody. (Bar 10 μm).
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Fig. 4. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-labeled neuronal nuclei. A: Several neuronal nuclei in the area surrounding acute infarction (so-called periinfarct area or ischemic penumbra) were labeled with TUNEL technique, although a few TUNEL-labeled cells were seen in both the ischemic core and apparently normal (unaffected) area. (Bar 10 μm) P: periinfarct area or ischemic penumbra. IC: ischemic core. N: apparently normal (unaffected) area. B: Apparently normal (unaffected) area. (Bar 10 μm) C: Area surrounding acute infarction (so-called periinfarct area or ischemic penumbra). Several TUNEL positive shrunken and/or the triangular-shaped neurons were observed. (Bar 10 μm) D: A few TUNEL positive neuronal nuclei exhibited fragmentation, which may show apoptotic appearance. (Bar 4 μm) E: In chronic infarction cases, fewer TUNEL-labeled cells were detected. (Bar 10 μm).
profile was reversed, Type C (4.6%) was least frequent and Type A (54.6%) as most prevalent, indicating cytoplasmic staining, reminiscent of normal neurons, is more frequent in area distant from ischemic focus. In the intermediate zone, Type B was predominant (69.0%), suggesting a transition between Type A and Type C. These trends were observed regardless of the location (frontal or parietal lobes) and both in acute and in subacute cases of infarction. In control cerebral sections, beta, gamma, sigma, tau, eta, and zeta isoforms specific antibodies showed findings almost similar to those with anti-14-3-3 COM [14,15]. In acute to subacute cerebral infarction cases, isoform-specific IRs for most of the 7 isoforms were similarly noted in the nuclei, although their intensities were generally less than that of 143-3 COM and varied greatly. Among them, beta isoform-like IR was concentrated in the nucleus (Fig. 2A) similar to that of 14-3-3COM (Fig. 1B) as seen at the periphery of the ischemic core. In contrast, sigma isoform-like IR was more intense in neuronal cytoplasm without nuclear recruitment (Fig. 2B). The cytoplasm of some astrocytes was also immunopositive for the sigma isoform. The neuropil of the cerebral cortex was homogeneously stained with the antiepsilon antibody, while neuronal soma was relatively spared. Number of nucleus labeled by this anti-epsilon antibody was limited. Many astrocytic cells were also immunolabeled with this anti-epsilon antibody (Fig. 2C). On the other hand, gamma isoform-like IR was observed in the nucleus of glial cells (Fig. 2D). In the ischemic area of the white matter, swollen axons and some nuclei of oligodendrocytes were
immunolabeled with the anti-zeta isoform antibody (Fig. 2E). Tau isoform-positive glia located only at the outer border zone of ischemic necrosis (Fig. 2F). Immunohistochemical labeling was abolished when each of the primary antibodies (7 isoforms) was coincubated with the corresponding antigen peptide (Fig. 2G, H). Double immunostaining with the anti-GFAP antibody demonstrated that glial cell with gamma isoform-immunopositive nuclei were astrocytes (Fig. 3). Several neuronal nuclei in areas surrounding acute to subacute infarction (so-called periinfarct area or the ischemic penumbra) were labeled intensely with the TUNEL technique (Fig. 4 A), although a few TUNEL-labeled nuclei were seen in both apparently normal (unaffected) area (Fig. 4A, B) and ischemic core (Fig. 4A). In area surrounding acute infarction, several TUNEL-labeled shrunken and/or triangularly shaped neurons were observed (Fig. 4C). A few TUNEL-labeled neuronal nuclei were fragmented, which may show apoptotic appearance (Fig. 4D). In chronic infarction cases, fewer TUNEL-labeled cells were detected (Fig. 4E). 4. Discussion In the present study, we demonstrated that 14-3-3-like IR is increased in both nuclei and cytoplasm of neuronal and glial cells mainly located in the periinfarct area (or ischemic penumbra) in human brains. Thus, 14-3-3 proteins are upregulated not only in neuronal cytoplasm but also in nuclei in surviving ischemic cortical neurons and Purkinje cells.
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4.1. A possible correlation between the subcellular localization of 14-3-3 IR and the severity of ischemic insult Because the subcellular localization of 14-3-3 IR varied, we tried to identify a possible correlation between its subcellular localization and the severity of ischemic insult. The relative severity of ischemic insult was defined according to the relative distance from the ischemic focus. We then classified at least four different types of 14-3-3 IR in neurons as follows (Fig. 5), A1: nuclei-negative, cytoplasm-positive, less atrophic neurons (Fig. 1D arrowhead); A2: nuclei-positive, cytoplasmpositive, less atrophic neurons (Fig. 1D arrow); B: nucleipositive, weakly positive cytoplasm, atrophic (triangular) neurons corresponding to ‘dark neurons’ (Fig. 1B) and C: nuclei-positive, cytoplasm-negative, severely atrophic neurons (Fig. 1C). Because distinction between Type A1 and A2 was occasionally difficult due to intense cytoplasmic staining, which veiled the nuclear contour, A1 and A2 were combined as Type A for quantification. Classically, atrophic neurons with triangular nuclei in ischemic areas are identified as dark neurons [26]. Many neurons with intense nuclear staining for 14-3-3COM corresponded to such neurons, represented as Type B, which was the most frequent (51.3%) among the 1500 neurons quantified. Neurons with more intense nuclear staining (Type C) were frequent near ischemic focus. In contrast, neurons with cytoplasm-dominant staining (Type A) were prevalent in areas distant from ischemic foci. This suggests that upregulation (Type A and B) and nuclear translocation (Type C) of 14-3-3 proteins are both induced by ischemia but are dependent on the severity of ischemia. As summarized in Fig. 5, upregulation of 14-3-3 proteins is first restricted in the cytoplasm of non-atrophic neurons (Fig. 5, A1), where the ischemic insult is the slightest. Gradual neuronal shrinkage was accompanied by further translocation
Fig. 5. 14-3-3-proteins immunopositive patterns in affected neurons. N: Normal IR pattern of neuron. Cytoplasm is weakly immunopositive for 143-3 proteins. A1: Cytoplasm-immunopositive/nucleus-fine-immunopositive neuron. A2: Cytoplasm-immunopositive/nucleus-immunopositive neuron. B: Cytoplasm-fine-immunopositive/nucleus-immunopositive atrophic neuron. C: Cytoplasm-immunonegative/nucleus-immunopositive atrophic neuron.
into the nucleus (Fig. 5, B). Finally, most atrophic neurons exhibited 14-3-3-like IR exclusively in the nucleus (Fig. 5, C). We also reported nuclear immunoexpression of 14-3-3 proteins in brains with spinocerebellar ataxia type 1 [27]. There have been several investigations about 14-3-3 proteins of animal model of brain infarction[21–24], one [22] is a preliminary report, and Gao et al. [21] reported that phosphorylated sigma isoform was increased after ischemia in a murine model. Recently, Kawamoto et al. [25] demonstrated 14-3-3 immunopositive reactive astrocytes in human brains with cerebral infarction, although astrocytes were also immunolabeled in other pathological conditions [14,28]. However, they paid little attention to neurons and only described briefly that ‘in the acute infarcted lesion, these were many neurons in which an excessive accumulation of 14-3-3 immunoreactivity was found in the perinuclear area, and at the subacute stage the reactivity was wellpreserved’. We were, however, successful in demonstrating specific 14-3-3 IR in neuronal nuclei (not the ‘perinuclear area’) after the pretreatement. More importantly, we demonstrated that this nuclear shift is dependent on the isoform and the relative severity of ischemia, corroborating its functional relevance related to nuclear translocation. 4.2. How are 14-3-3 proteins involved in ischemia or postischemic reactions? Nuclear accumulation of 14-3-3 proteins in surviving neurons suggests their relevance to intranuclear processes, for example, transcription. Indeed, a number of molecules responsible for transcription and its regulation have been found to be binding partners to 14-3-3 proteins. Examples include histone deacetylases [9], TATA-binding protein [8]. Interestingly, another key molecule in transcription, phosphorylated cAMP response element binding protein (pCREB) was found to be accumulated in the nucleus under experimentally induced ischemia [29], and the CREB coactivator called transducer of regulated CREB activity can interact 14-3-3 proteins [30]. These lines of evidence suggest that involvement of 14-3-3 proteins in ischemic processes is mediated through modifications of transcriptions in several ways. Translocation of 14-3-3 proteins into or out of nuclei is regulated by what is known as a nuclear shuttle and mediated by multiple factors, possibly related to apoptosis [2–7]. For example, dephosphorylated Fkhrl 1 (Forkhead rhabdomyosarcoma-like 1) is translocated from the cytoplasm into the nucleus, resulting in activation of apoptotic genes. Efficient export of Fkhrl 1 from the nucleus requires binding of 14-3-3 proteins [11]. It is then possible that nuclear accumulation represents possible association of 14-3-3 proteins to Fkhrl 1 for their export from the nucleus, counteracting apoptosis. Another possibility is some relation with another mechanism of apoptosis. One of the binding partners of 14-3-3 proteins is BAD [10,12], an inducer of apoptosis upon translocation into mitochondria through inactivation of Bcl-family molecules. Abundant 14-3-3 proteins in the cytoplasm, as
T. Umahara et al. / Journal of the Neurological Sciences 260 (2007) 159–166 Table 2 Differences in cellular and subcellular immunolocalization dependent on isoforms of 14-3-3 protein in cases with acute infarction Antibodies
Particularly immunopositive region
14-3-3 COM Beta isoform Sigma isoform Gamma isoform Zeta isoform
Neuronal nuclei and cytoplasm Neuronal nuclei Neuronal cytoplasm Nuclei of astrocytes Nuclei of oligodendrocyte
we demonstrated in mild ischemia as type A1, might counteract this apoptotic process by trapping BAD in the cytoplasm. We showed TUNEL-labeled neuronal nuclei in acute to subacute infarction cases, possibly related to apoptosis [31]. Although the number of TUNEL positive cells was fewer than those of 14-3-3 positive neuronal nuclei, the dominant part of those TUNEL positive neurons was in the area surrounding acute to subacute infarction, which is in agreement with a recent report [32] that showed predominance of apoptosis in the periphery of human cerebral infarction. 4.3. Isoform-dependent cellar and subcellar accumulation of 14-3-3 proteins Among them (Table 2), it was the beta isoform that intensely immunolabeled neuronal nuclei. This suggests that the beta isoform is the major isoform distributed or transported into nuclei. On the other hand, the sigma isoform tended to be located in the cytoplasm. This is compatible with an observation that the sigma isoform is rapidly exported from the nucleus [20]. Indeed, sigma isoform [21] is the main cytoplasmic sequestration protein of Bax, which is translocated to mitochondria after ischemia and contributes to the apoptosis-signaling pathway. Our finding of the cytoplasmicdominant localization of sigma isoform after ischemia might be associated with this condition. IR for the epsilon isoform was found in the neuropil of the gray matter in control and AD brains [14]. Astrocytes in brains with infarct were also immunolabeled with this isoform-specific antibody, as seen in AD brains [14]. On the other hand, IR for the gamma isoform was seen in the nuclei of astrocytes. Recently, it was reported that the gamma isoform mRNA and protein were upregulated in ischemic cortical astrocytes in vitro, and were selectively induced during ischemia, protecting astrocytes from apoptosis through p-BAD-related signaling [12]. Zeta isoform-immunopositive oligodendrocyte nuclei were located in ischemic lesions and easily detected in the white matter. These findings suggest that some isoforms of 14-3-3 proteins are present in glial nuclei and that they mediate glial cell survival and glial–neuronal interaction. In conclusion, we demonstrated upregulation of 14-3-3 proteins in human brain with ischemia. Nuclear and/or cytoplasmic accumulation was dependent on both the severity of ischemia and the isoform.
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