- Email: [email protected]
Transient cerebral ischemia induces site-specific hyperphosphorylation of tau protein
Brain Research 1022 (2004) 30 – 38 www.elsevier.com/locate/brainres
Research report
Transient cerebral ischemia induces site-specific hyperphosphorylation of tau protein Yi Wen, Shaohua Yang, Ran Liu, James W. Simpkins * Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699, USA Accepted 24 May 2004 Available online
Abstract Neurofibrillary tangles (NFTs) are a pathological hallmark of many neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP17). However, the cellular origin and the consequence of the NFT formation are poorly understood. Epidemiological evidence suggests a much higher occurrence of dementia in stroke patients. This may represent the pathogenesis of sporadic AD, which accounts for the majority of AD occurrence. Here we show that after a transient cerebral ischemia, hyperphosphorylated tau accumulates in cortical neurons in a site-specific manner. The hyperphosphorylated tau presents a conformation similar to those present in human tauopathies, and colocalizes largely with signs of apoptosis. Our current study suggests that tau hyperphosphorylation may contribute to the brain damage induced by transient cerebral ischemia, and may be involved in the pathogenesis of neurodegenerative disorders in patients after stroke. Further, these results indicate that ischemic neuronal damage and apoptosis associates with tau hyperphosphorylation, and potentially NFTs formation. Finally, our results also suggest that neuronal apoptosis may be a therapeutic target in preventing tauopathy-related neurodegenerative diseases. D 2004 Elsevier B.V. All rights reserved. Keywords: Cerebral ischemia; Hyperphosphorylation; Tau protein
1. Introduction Dysfunctional and filamentous microtubule-binding tau proteins are key markers of both sporadic and familial neurodegenerative pathologies. A large number of neurodegenerative diseases, including AD, Pick’s disease, progressive supranuclear palsy, corticobasal degeneration, and FTDP-17, showed aberrant aggregation of hyperphosphorylated tau protein [38]. Collectively, these diseases are known as neurodegenerative tauopathies. In AD, NFTs are found intracellularly in conjunction with the deposition of extracellular h-amyloid (Ah) fibrils [53]. The dominant component of neurofibrillary lesions in AD is hyperphosphorylated tau that forms paired helical filaments (PHFs) and straight filaments [30,31,33]. Tau proteins are microtubule-associated proteins that are expressed abundantly in soma and axons. Both in vivo and
* Corresponding author. Tel.: +1-817-735-0498. E-mail address: [email protected] (J.W. Simpkins). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.05.106
in vitro studies indicate that tau proteins bind to microtubules, promoting their polymerization [12,58]. Several isoforms of tau are present in the central nervous system (CNS), likely produced by alternative mRNA splicing [20,40]. The isoforms of tau proteins in AD are similar to those observed in normal adult human brain [21,57]. In tau protein, there are 79 potential serine and threonine phosphorylation sites on the longest human tau isoform. Phosphorylation of more than 30 of the phosphorylation sites has been reported [7,10]. These phosphorylation sites are involved in both embryonic development and neurodegeneration [28]. These phosphorylation sites cluster in the flanking region of the tubulin-binding domain and negatively regulate tau’s binding affinity to microtubules [6,8,17]. However, the relative importance of individual phosphorylation sites is still controversial. The prevalence of dementia in ischemic stroke patients is nine times higher than controls at 3 months [55] and 4– 12 times higher than controls at 4 years after a lacunar infarct [36]. Many of these dementias develop progressively, and cerebral damage is not the direct cause of the subsequent
Y. Wen et al. / Brain Research 1022 (2004) 30–38
dementia in at least half of the cases [56]. AD is the most prevalent dementia [42] and shares common neuropathology features with stroke. Alz-50-immunoreactive granules were found around cerebral infarction after a stroke [24], amyloid precursor protein (APP) accumulates following transient cerebral focal ischemia [49], and ApoE4 is a genetic risk factor for both AD and stroke [13]. In the present study, we assessed the induction of tau phosphorylation and epitopes with PHF-like conformation in a rodent model for transient cerebral ischemia. We also examined the potential phosphorylation sites and their degree of phosphorylation induced by transient cerebral ischemia.
31
Briefly, the animal was anesthetized with ketamine (60 mg/ kg) and xylazine (10 mg/kg), then the internal carotid artery (ICA) was exposed, and a 3-0 monofilament nylon suture was introduced into the ICA lumen through a puncture and gently advanced to the distal internal carotid artery (ICA) until proper resistance was felt. After 1 h, the suture was withdrawn from the ICA and the distal ICA was immediately cauterized. At the desired time after the onset of reperfusion, animals were sacrificed and sampled for immunoblotting, immunohistochemistry, or 2% 2,3,5Triphenyl-2H-tetrazolium chloride (TTC) stain, as described below. 2.4. TTC staining
2. Materials and methods 2.1. Animals Four- to six-week-old female Sprague – Dawley (SD) rats were purchased from Charles Rivers (Wilmington, MA) and maintained in our animal facility in a temperature-controlled room (22 –25 jC) with 12-h dark –light cycles. All rats have free access to laboratory chow and tap water. All animal procedures were reviewed and approved by the University of North Texas Health Science Center Institutional Animal Care and Use Committee. 2.2. Chemicals and antibodies The immunoblotting and in vitro phosphorylation screening analysis used antibodies directed against different phospho-epitopes on tau protein, which includes pT181, pS202, pT205, pT212, pS214, pT231, pS262, pS396, pS404, and pS422 (Biosource, Camarillo, CA) at a dilution of 1:500. The following antibodies were used to label the conformationand phosphorylation-dependent tau epitopes in the brain sections: MC1 (PHF-conformation), TG3 (Phospho-tau 231 and AD conformation), CP13 (Phospho-tau 202/205), CP3 (Phospho-tau 214), PHF-1 (Phospho-tau 396/404), and CP9 (Phospho-tau 231) at a dilution of 1:10 in the immunohistochemistry. The above-mentioned phosphorylation/NFT conformation-dependent antibodies were characterized by and were the kind gifts of Dr. Peter Davies (Albert Einstein College of Medicine). All Chemicals were from SigmaAldrich (St. Louis, MO) unless otherwise specified. 2.3. Animal surgeries To avoid fluctuations of estrogens, which are neuroprotective [50,60], all animals received bilateral ovariectomies at least 2 weeks before any surgical procedure. Animals were anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/ kg), then a small cut was made through the skin and muscle. The ovaries were externalized and removed. For middle cerebral artery (MCA) occlusion and reperfusion, an intraluminal filament model was used [3,50].
Brains were removed, immediately after sacrificing the animal, and dissected coronally into 2-mm sections using a metallic brain matrix (ASI Instruments, Warren, MI) and stained by incubation in a 2% TTC in a 0.9% saline solution at 37 jC for 30 min. Photographs were taken with Kodak digital camera. Each treatment group included at least three to four animals. 2.5. In vitro phosphorylation Frontoparietal cortex from either ischemic (n = 4) or the contralateral brains (n = 4) was dissected from other brain tissues and homogenized in modified RIPA buffer [1 PBS, 1% Nonidet P-40 or Igepal CA-630, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 10 Ag/ml Aprotinin, 100 Ag/ml phenylmethyl sulphonyl fluoride (PMSF)]. Samples were incubated on ice for 20 min and then centrifuged at 12,000 g for 20 min. Supernatant was collected for further kinase activity analysis or immunoblotting. For phosphorylation reaction, 1 Ag recombinant human tau protein from Sigma-Aldrich and 10-Ag brain extracts were added to reaction buffer, containing 50 mM HEPES, 0.1 mM EDTA, 0.1 mg/ml BSA, 0.1% h-mercaptoethanol, 0.15 M NaCl, 50 AM ATP, and 1 ACi 32P-labeled g-ATP from ICN Biomedicals (Irvine, CA). The tube was incubated for 30 min in a 30 jC water bath with moderate shaking. SDS-PAGE loading buffer was then added, and the tube was boiled for 10 min to terminate the reaction. Samples from the phosphorylation reaction were separated on 10% or 8– 16% gradient SDS-polyacrylamide gels. The gels were stained with Coomassie Brilliant Blue R250 and then subjected to autoradiography. 2.6. Immunoblotting and in vitro phosphorylation screening Immunoblotting analysis with various antibodies was performed according to the antibody manufacturer’s instructions with slight modification. Briefly, samples (n = 4) from cortex (peri-infarct area) in the ischemic or the corresponding contralateral areas were separated on 10% or 8 – 16% gradient SDS-polyacrylamide gels and
32
Y. Wen et al. / Brain Research 1022 (2004) 30–38
electrophoretically transferred to a nitrocellulose membrane. Membranes were blocked by incubation with 5% dry milk in phosphate-buffered saline (PBS) and 0.02% Tween 20 (PBST) for 1 h at room temperature, followed by overnight incubation at 4 jC with the appropriate primary antibody in blocking buffer. After thorough washing, the membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, followed by another thorough wash in PBST. The membrane was then incubated and developed. The blots were developed with an enhanced chemiluminescent kit (Pierce, Rockford, IL). The in vitro phosphorylation products without 32P-labeled g-ATP were used for phosphorylation site analysis. 2.7. Immunohistochemistry and TUNEL staining For immunohistochemical analysis, animals were perfused transcardially with 4% paraformaldehyde in sodium phosphate buffer (pH 7.4) (n = 3). Brains were paraffinembedded and sectioned at 8 – 10 Am. Immunohistochemical analysis was performed using an HRP-DAB kit, following the manufacturer’s recommended procedure (Zymed, South San Francisco, CA). Some of the sections were pretreated with 100 Ag/ml proteinase K in PBS for 15 min for antigen retrieval and signal enhancement. After staining, sections were dehydrated in a gradient of ethanol and xylene and
then sealed with cover slips. All photographs were taken using a Nikon Diaphot 300 microscope and a 12-bit CCD monochrome camera with Labworks software (UVP, Upland, CA). DNA fragmentation was detected using TdT-mediated dUTP Nick-End Labeling (TUNEL) method with The DeadEndk Fluorometric TUNEL System (Promega, Madison, WI) according to the manufacturer’s instructions. Sections were counterstained with 4V,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Molecular Probes, Eugene, OR). Colocalization of TUNEL with specific antigens was performed with highly specific Alexa-Fluorophore conjugated secondary antibodies (Molecular Probes), whose excitation/emission spectra do not overlap with that of the FITC label used in the TUNEL stain. 2.8. Densitometric and statistical analysis The images of autoradiography and immunoblotting were digitalized and imported using a calibrated scanner at 16-bit gray mode to provide 65,535 gray levels. The area density was quantified using Labworks software (UVP). The unit given is an arbitrary unit that stands for the total units per band in the analyzed samples. The results were assayed with Student’s t-test or one-way ANOVA statistical method using Prism software.
Fig. 1. Induction of apoptosis by transient cerebral ischemia in OVX rats. TUNEL-stained sections from various brain regions after 1-h MCA occlusion followed by 24-h reperfusion: (A) contralateral cortex, (B) ischemic cortex, (C) penumbra area. (D) TTC-stained brain sections indicating area of damage. The letters in (D) indicate the approximate region of A – C. Scale bar = 100 Am.
Y. Wen et al. / Brain Research 1022 (2004) 30–38
33
3. Results 3.1. Evaluation of apoptosis induced by transient cerebral ischemia MCA occlusion is a widely used focal ischemic stroke model [9]. This in vivo model for neuronal death can rapidly induce a synchronized apoptotic process in a large number of neurons [35]. We examined the effects of this transient cerebral focal ischemia on neuronal apoptosis using a TUNEL assay. As early as 2 h after the initiation of reperfusion, positive TUNEL staining appeared in the core ischemic region near the basal ganglion area (data not shown). After 24 h of reperfusion, TUNEL-positive cells were widespread in the ischemic region, which includes the frontoparietal cerebral cortex (Fig. 1B), but were undetectable in the corresponding contralateral regions (Fig. 1A). In the penumbra areas, where the ischemic region is separated from the fully perfused area, TUNEL signal was observed only in the ischemic side (Fig. 1C). In the striatal regions that received the strongest ischemia, many cells showed positive TUNEL signals in a variety of cell types, which include neurons, astrocytes, glia, and some endothelial cells of blood vessels, as determined from morphological evaluation (data not shown). However, in the neocortex area, most TUNEL-positive cells represent the morphology of cortical pyramidal neurons. The distribution of TUNEL
Fig. 3. Screening of hyperphosphorylation sites in tau protein induced by transient cerebral ischemia. (A) Potential hyperphosphorylation sites were analyzed with a panel of antibodies that recognize site-specific phosphorylation in tau protein. The numbers on the x-axis indicates the amino acid residuals in tau protein (n = 6). * indicates significant difference between the indicated group and the contralateral cortex of ovariectomized animal at p < 0.05. (B) Some representative immunoblotting assay in randomly selected duplicates indicating the specific phosphorylation sites of tau protein. Tau 5 recognizes tau protein independent of its phosphorylation status. (C) Representative immunoblotting analysis of in vivo tau hyperphosphorylation. Numbers above indicate the examined phosphorylation site based on human 4-repeat tau protein (I: ischemic extracts, C: contralateral extracts).
signal was consistent with the result of TTC staining for brain slices (Fig. 1D). 3.2. Aberrant tau hyperphosphorylation occurs in the ischemic cortex
Fig. 2. Transient cerebral ischemia-induced tau hyperphosphorylation. (A) Autoradiography (upper lane) and Coomassie brilliant blue-stained SDS-PAGE gel (lower lane) of protein kinase assay, using recombinant tau as a substrate. Each lane represents an individual experimental animal. (B) The densitometric analysis of tau hyperphosphorylation induced by transient cerebral ischemia. * indicates significant difference between the ipsilateral and the contralateral cortex of ovariectomized animals at p < 0.05.
To characterize the phosphorylation status of tau in the ischemic regions, we examined total tau-kinase activity. This assay examines the transfer of 32P-labeled phosphate from ATP to recombinant tau protein. Tau kinase activity increased 2.87-fold in the ischemic region versus the contralateral regions (Fig. 2A and B). The hyperphosphorylation induced apparent multiplicity, represented by multiple bands in the Coomassie brilliant blue-stained
34
Y. Wen et al. / Brain Research 1022 (2004) 30–38
SDS-PAGE (Fig 2A), which is consistent with previous reports [27]. We used a panel of phosphorylation-dependent anti-tau antibodies to screen the potential hyperphosphorylation sites of tau protein induced by this transient ischemia. We observed that the tau hyperphosphorylation was sitespecific, at 24 h of reperfusion. Collectively, pS202, pS214, pT231, and pS402 showed a significant increase in their phosphorylation status (Fig. 3A and B). These hyperphosphorylated sites were confirmed with immunoblotting with endogenous brain cortical extract (Fig. 3C). The rodent tau represents multiple bands ranging from 45 to 64 kd, indicating the presence of multiple isoforms, similar to those in human brain extracts. Our results are consistent with previous research on tau hyperphosphorylation which indicates that rodent tau generally migrates primarily at 40 – 50 kDa, with minor forms at 68 and 100 kDa. However, antibodies that recognize phosphorylated tau appear to emphasize the higher molecular weight isoforms, potentially due to the presence of isoform content alteration, preference in phosphorylation sites, or the hyperphosphorylation [15,25]. To confirm the aberrant hyperphosphorylation of tau, we also examined the ischemic area using immunohistochemistry with a panel of phosphospecific monoclonal antibodies. While the contralateral cortex was almost absent of the specific phosphospecific immunoreactivity (Fig. 4A), we observed extensive phosphospecific immunoreactivity in the ischemic regions, including PHF-1 (Fig. 4B and C), CP3 (Fig. 4D), CP13 (Fig. 4E), and CP9 (Fig. 4F). To further investigate the spatial relationship of hyperphosphorylated tau and apoptosis, we double-stained PHF-1 and TUNEL to evaluate their spatial relationship. We observed that most of the phospho-tau epitopes co-localized with the apoptotic marker, TUNEL (Fig. 5).
3.3. Ischemia-induced tau hyperphosphorylation causes conformational changes similar to those in neurodegenerative tauopathy Tauopathies in neurodegeneration involve not only the hyperphosphorylation, but also conformational change of tau protein. To further characterize the phosphorylation and conformational status of tau in the ischemic regions, we used two conformation-dependent anti-tau antibodies. Antibody TG3 detects both phosphorylation and the unique conformation of PHF-tau [22], while MC1 is a conformation-dependent antibody that detects the Alz-50 epitope of AD tau [26]. In the ischemic region, a large number of pyramidal neurons were immunopositive for TG3 and MC1 in the frontoparietal cortex, but not in the contralateral regions (Fig. 6). The strongest immunoreactivity of MC1 was also observed in the frontoparietal neocortex (Fig. 6B and C). TG3 showed a staining pattern similar to MC1 in the ischemic cortical area, but not in the contralateral areas (Fig. 6D – F).
4. Discussion The results of this study show that transient cerebral ischemia induces a hyperphosphorylation of microtubuleassociated protein tau in a site-specific manner and can further induce NFT-like conformational epitopes in wildtype ovx female rats. These results were confirmed by immunoblotting and immunohistochemistry. Four sites, including pS202, pS214, pT231, pS422, were identified in the in vitro screening of the potential phosphorylation sites, and these sites were confirmed with immunoblotting. The current study may help to clarify the molecular and cellular mechanisms of higher occurrence of dementia after stroke and provides a convenient non-transgenic animal model to
Fig. 4. Immunohistochemical staining with antibodies that detect hyperphosphorylation of tau protein in vivo. (A – C) Representative coronal sections of the frontoparietal cerebral cortex showing immunoreactivity of PHF-1 (detecting P-396/404) in the contralateral (A), ipsilateral (B), and ipsilateral at high magnification (C) brain sections. (D – F) Representative photographs of immunohistochemical staining in the ipsilateral frontoparietal cortex with CP3 (detecting P-214) (D), CP9 (detecting P-231) (E), and CP13 (detecting P-202/205) (F), in the frontoparietal cortex. Scale bar = 100 Am.
Y. Wen et al. / Brain Research 1022 (2004) 30–38
Fig. 5. Colocalization of tau hyperphosphorylation and apoptosis in vivo. Confocal microscopy imaging indicates the colocalization of PHF-1 immunostaining with many TUNEL-positive cells in the ischemic cortical region. (A) TUNEL stain (green), (B) PHF-1 immunofluorescence (red), and (C) the merged image of (A) and (B).
investigate the hyperphosphorylation of tau, which is one of the hallmarks of AD pathology. However, a variety of in vivo and in vitro neuronal insults, including heat shock [41], permanent focal cerebral ischemia in the cat [16], and amyloid treatment [18], induces hyperphosphorylation of tau. Interestingly, in response to the ischemia/reperfusion process, phosphorylation of tau protein appears to be dynamically regulated. Oxygen and glucose deprivation in rat and human neocortical brain slice induces a rapid dephosphorylation within 30 – 60 min during the ischemia [11]; also, during transient forebrain
35
ischemia in the rat, dephosphorylation was detected within 5 min, and restoration occurs in as a little as 15 min after reperfusion [48]. Cardiac arrest-induced ischemia/reperfusion appears to induce a similar rapid dephosphorylation during ischemia and restoration beginning less than 2 h after reperfusion [39]. In general, a rapid dephosphorylation of tau occurs during ischemia or immediately upon reperfusion, potentially due to the activation of phosphatases. During continued reperfusion, tau proteins are slowly phosphorylated and accumulate, potentially due to the downregulation of phosphatases or induction of specific kinases. This may reflect the dynamic regulation of phosphatases and/or the corresponding tau kinases, including Cdk5, Cdc2, and MAPK [46], in response to the ischemia/reperfusion process. Our observations are also consistent and supported by many epidemiological and postmortem clinical studies, as well as other animal studies. Alz-50-immunoreactive granules were found around cerebral infarction after a stroke [24]. Alzheimer-type pathology is often associated with cerebral amyloid angiopathy and infarcts [36]. Further, AD and stroke have common genetic risk factors, including e 4 allele of the apolipoprotein E gene (APOE), which associates with a higher risk of ischemic stroke, coronary heart diseases, and late-onset AD [13]. We investigated 10 out of 30 potential phosphorylation sites on tau protein. Among the 10 potential phosphorylation sites studied, 4 showed statistically significant increases in phosphorylation in response to ischemia, including pS202, pS214, pT231, pS422, and this finding was confirmed by in vivo studies. These four phosphorylation sites are localized in the flanking regions, outside the microtubule binding domain. Hyperphosphorylations of tau have been reported to reduce tau’s ability of binding and promoting tubulin polymerization [10]. However, the critical significance of individual phosphorylation sites and their corresponding kinase are still controversial. Previous research that assessed the correlation of phosphorylation of tau and severity of AD cases suggests that there are events prior to filament formation that are specific to particular phosphorylated tau epitopes, leading to conformational changes and cytopathological alterations [2]. Phosphorylation on S231 can result in the association of pin1, a prolyl isomerase, and may help to restore some of the tau’s function [37]. pS214 is a potential substrate of PKA and appears to detach tau protein from microtubules and protects it against aggregation into Alzheimer-paired helical filaments [47]. The specificity of phosphorylation sites induced by transient ischemia implies that a specific set of kinases is activated during this relatively short period of 24 h. However, another possibility is an alteration of phosphatases. Further, the mechanisms of the tau hyperphosphorylation, in this ischemia/reperfusion injury, may not necessarily be the same as tauopathy-related neurodegenerative diseases, which generally require decades to progress. However, this can provide a convenient animal model to study the pathological tau hyperphosphorylation
36
Y. Wen et al. / Brain Research 1022 (2004) 30–38
Fig. 6. Immunohistochemical staining with antibodies that detect PHF-like conformation of tau protein. Tau phosphorylation induces conformational changes after 24 h of reperfusion in MCA occlusion rats. Antibody MC1, a conformation-dependent antibody, detected tau in the ischemic cerebral cortical region in low (B) and high (C) magnification, but not in the contralateral cortex (A). Most of the positive immunoreactivity was localized to the frontoparietal cortex, with only a few detected in subcortical areas. Antibody TG3, a phosphorylation- and conformation-dependent antibody, stained a subset of pyramidal neurons of the ipsilateral cortex at low (E) and high (F) magnification, but not in the contralateral cortex (D).
and for screening for pharmacological inhibitors of tau phosphorylation. Immunohistochemical studies with monoclonal antibodies directed against the phospho-epitopes or NFT-like conformation recognized a number of cortical neurons in ischemic brain sections, but not on the contralateral side. Most of the immunoreactivity concentrated in the frontoparietal cortex within peri-infarct area. Striatal regions within the core ischemic area, including globus pallidus and caudate putamen, showed only weak signals that appear to originate from glia or potentially infiltrated neutrophils. On the contralateral side, only a diffusive staining was observed and can be regarded as negative. Double-labeling studies of phospho-tau and an apoptotic marker indicate apparent co-localization. In the peri-infarct areas, more than half of the PHF-1 positive cells showed strong TUNEL staining. However, current studies did not determine a definitive relationship between tau-hyperphosphorylation and neuronal apoptosis induced by the transient cerebral ischemia. Our current observation suggests that this tauhyperphosphorylation is the consequence of the activation/ inactivation of a variety of phosphatases and kinases in response to the ischemia/reperfusion damage. The hyperphosphorylation of tau may result in the reduction of its affinity to microtubule protein and promote microtubule depolymerization, which will inhibit axonal transport, destabilize neuronal cytoskeleton, and may contribute to the apoptotic process induced by this ischemia/reperfusion insult. Further, phosphorylated, but not native, tau protein is able to form polymers after the reaction with 4-hydroxy2-nonenal [43]. These observations are consistent with previous reports that phosphorylation and oxidative modification by hydroxynonenal, a product of oxidative stress, are required for tau filament formation [44].
Although it is tempting and exciting to expect this ischemia-induced tau hyperphosphorylation to share significant similarities with those observed in AD brains, there is currently insufficient evidence to demonstrate that the phosphorylated tau detected in the ischemic animal brains is in the aggregated form such as that found in human neurodegenerative tauopathies. Additional studies including Gallyas silver staining, thioflavin-S staining, ultrastructural examination, and analysis of sarcosyl-insoluble brain extracts by electronic microscopy analysis are required to answer these questions. A long-term study is also required for further characterization on the effects of this transient focal ischemia. However, our study supports the hypothesis that NFT formation may be a consequence of a preceding apoptotic event, including amyloid deposit, which results in the aberrant activation of protein kinases and further induction of tau hyperphosphorylation and tangle formation [5,32]. A number of studies support the conclusion that the apoptotic signaling pathways are involved in neuronal loss in AD brains [1,4,14]. Pro-apoptotic factors, including reactive oxygen species [51,52], h-amyloid [61], and growth factors reduction [19], may serve as the initiator of NFT-formation. Tau hyperphosphorylation and tangle formation is associated with apoptosis in AD brains [29,45,54] and can be induced or enhanced by amyloid deposits [23,34]. The MCA occlusion model has been widely used for the evaluation of neuropathology of stroke and neuroprotective compounds, such as estrogen [50,59]. Our results indicate that this transient ischemic model may be used for evaluating tau hyperphosphorylation and tauopathy formation, as well as for pharmaceutical screening for NFT inhibitors in vivo. Finally, our data also suggest that sporadic hypoperfusion of the brain may initiate and contribute to the progression of neuropathology in nonfamilial AD patients and provide a
Y. Wen et al. / Brain Research 1022 (2004) 30–38
mechanism for the long-observed correlation between ischemic events and the increased prevalence of AD [36]. As such, prevention of ischemic episodes may serve as an additional therapeutic target to prevent the initiation and progression of AD.
References [1] A.J. Anderson, J.H. Su, C.W. Cotman, DNA damage and apoptosis in Alzheimer’s disease: colocalization with c-Jun immunoreactivity, relationship to brain area, and effect of postmortem delay, J. Neurosci. 16 (1996) 1710 – 1719. [2] J.C. Augustinack, A. Schneider, E.M. Mandelkow, B.T. Hyman, Specific tau phosphorylation sites correlate with severity of neuronal cytopathology in Alzheimer’s disease, Acta Neuropathol. (Berl.) 103 (2002) 26 – 35. [3] J.B. Bederson, L.H. Pitts, M. Tsuji, M.C. Nishimura, R.L. Davis, H. Bartkowski, Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination, Stroke 17 (1986) 472 – 476. [4] C. Behl, Apoptosis and Alzheimer’s disease, J. Neural Transm. 107 (2000) 1325 – 1344. [5] C. Behl, B. Moosmann, Oxidative nerve cell death in Alzheimer’s disease and stroke: antioxidants as neuroprotective compounds, Biol. Chem. 383 (2002) 521 – 536. [6] J. Biernat, N. Gustke, G. Drewes, E.M. Mandelkow, E. Mandelkow, Phosphorylation of Ser262 strongly reduces binding of tau to microtubules: distinction between PHF-like immunoreactivity and microtubule binding, Neuron 11 (1993) 153 – 163. [7] M.L. Billingsley, R.L. Kincaid, Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration, Biochem. J. 323 (Pt 3) (1997) 577 – 591. [8] G.T. Bramblett, M. Goedert, R. Jakes, S.E. Merrick, J.Q. Trojanowski, V.M. Lee, Abnormal tau phosphorylation at Ser396 in Alzheimer’s disease recapitulates development and contributes to reduced microtubule binding, Neuron 10 (1993) 1089 – 1099. [9] A.M. Bremer, O. Watanabe, R.S. Bourke, Artificial embolization of the middle cerebral artery in primates. Description of an experimental model with extracranial technique, Stroke 6 (1975) 387 – 390. [10] L. Buee, T. Bussiere, V. Buee-Scherrer, A. Delacourte, P.R. Hof, Tau protein isoforms, phosphorylation and role in neurodegenerative disorders, Brain Res. Brain Res. Rev. 33 (2000) 95 – 130. [11] K.K. Burkhart, D.C. Beard, R.A. Lehman, M.L. Billingsley, Alterations in tau phosphorylation in rat and human neocortical brain slices following hypoxia and glucose deprivation, Exp. Neurol. 154 (1998) 464 – 472. [12] D.W. Cleveland, S.Y. Hwo, M.W. Kirschner, Purification of tau, a microtubule-associated protein that induces assembly of microtubules from purified tubulin, J. Mol. Biol. 116 (1977) 207 – 225. [13] J.H. Contois, D.E. Anamani, G.J. Tsongalis, The underlying molecular mechanism of apolipoprotein E polymorphism: relationships to lipid disorders, cardiovascular disease, and Alzheimer’s disease, Clin. Lab. Med. 16 (1996) 105 – 123. [14] C.W. Cotman, J.H. Su, Mechanisms of neuronal death in Alzheimer’s disease, Brain Pathol. 6 (1996) 493 – 506. [15] J.C. Cruz, H.C. Tseng, J.A. Goldman, H. Shih, L.H. Tsai, Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles, Neuron 40 (2003) 471 – 483. [16] D. Dewar, D.I. Graham, G.M. Teasdale, J. McCulloch, Alz-50 and ubiquitin immunoreactivity is induced by permanent focal cerebral ischaemia in the cat, Acta Neuropathol. (Berl.) 86 (1993) 623 – 629. [17] D.N. Drechsel, A.A. Hyman, M.H. Cobb, M.W. Kirschner, Modula-
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32] [33]
[34]
[35] [36]
37
tion of the dynamic instability of tubulin assembly by the microtubule-associated protein tau, Mol. Biol. Cell 3 (1992) 1141 – 1154. A. Ferrari, F. Hoerndli, T. Baechi, R.M. Nitsch, J. Gotz, Beta-amyloid induces paired helical filament-like tau filaments in tissue culture, J. Biol. Chem. 278 (2003) 40162 – 40168. L. Gasparini, W.J. Netzer, P. Greengard, H. Xu, Does insulin dysfunction play a role in Alzheimer’s disease? Trends Pharmacol. Sci. 23 (2002) 288 – 293. M. Goedert, C.M. Wischik, R.A. Crowther, J.E. Walker, A. Klug, Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: identification as the microtubule-associated protein tau, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 4051 – 4055. M. Goedert, R. Jakes, M.G. Spillantini, R.A. Crowther, P. Cohen, E. Vanmechelen, A. Probst, J. Gotz, K. Burki, Tau protein in Alzheimer’s disease, Biochem. Soc. Trans. 23 (1995) 80 – 85. J. Gotz, F. Chen, R. Barmettler, R.M. Nitsch, Tau filament formation in transgenic mice expressing P301L tau, J. Biol. Chem. 276 (2001) 529 – 534. J. Gotz, F. Chen, J. van Dorpe, R.M. Nitsch, Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils, Science 293 (2001) 1491 – 1495. K. Ikeda, H. Akiyama, T. Arai, H. Kondo, C. Haga, K. Tsuchiya, S. Yamada, S. Murayama, A. Hori, Neurons containing Alz-50-immunoreactive granules around the cerebral infarction: evidence for the lysosomal degradation of altered tau in human brain? Neurosci. Lett. 284 (2000) 187 – 189. N.D. James, D.R. Davis, J. Sindon, D.P. Hanger, J.P. Brion, C.C. Miller, M.P. Rosenberg, B.H. Anderton, F. Propst, Neurodegenerative changes including altered tau phosphorylation and neurofilament immunoreactivity in mice transgenic for the serine/threonine kinase Mos, Neurobiol. Aging 17 (1996) 235 – 241. G.A. Jicha, R. Bowser, I.G. Kazam, P. Davies, Alz-50 and MC-1, a new monoclonal antibody raised to paired helical filaments, recognize conformational epitopes on recombinant tau, J. Neurosci. Res. 48 (1997) 128 – 132. Y. Kanai, R. Takemura, T. Oshima, H. Mori, Y. Ihara, M. Yanagisawa, T. Masaki, N. Hirokawa, Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA, J. Cell Biol. 109 (1989) 1173 – 1184. K. Kanemaru, K. Takio, R. Miura, K. Titani, Y. Ihara, Fetal-type phosphorylation of the tau in paired helical filaments, J. Neurochem. 58 (1992) 1667 – 1675. K. Kobayashi, H. Nakano, M. Hayashi, M. Shimazaki, Y. Fukutani, K. Sasaki, K. Sugimori, Y. Koshino, Association of phosphorylation site of tau protein with neuronal apoptosis in Alzheimer’s disease, J. Neurol. Sci. 208 (2003) 17 – 24. J. Kondo, T. Honda, H. Mori, Y. Hamada, R. Miura, M. Ogawara, Y. Ihara, The carboxyl third of tau is tightly bound to paired helical filaments, Neuron 1 (1988) 827 – 834. K.S. Kosik, L.D. Orecchio, L. Binder, J.Q. Trojanowski, V.M. Lee, G. Lee, Epitopes that span the tau molecule are shared with paired helical filaments, Neuron 1 (1988) 817 – 825. M.S. Lee, L.H. Tsai, Cdk5: one of the links between senile plaques and neurofibrillary tangles? J. Alzheimer’s Dis. 5 (2003) 127 – 137. V.M. Lee, B.J. Balin, L. Otvos Jr., J.Q. Trojanowski, A68: a major subunit of paired helical filaments and derivatized forms of normal Tau, Science 251 (1991) 675 – 678. J. Lewis, D.W. Dickson, W.L. Lin, L. Chisholm, A. Corral, G. Jones, S.H. Yen, N. Sahara, L. Skipper, D. Yager, C. Eckman, J. Hardy, M. Hutton, E. McGowan, Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP, Science 293 (2001) 1487 – 1491. Y. Li, M. Chopp, C. Powers, N. Jiang, Apoptosis and protein expression after focal cerebral ischemia in rat, Brain Res. 765 (1997) 301 – 312. C. Loeb, C. Gandolfo, R. Croce, M. Conti, Dementia associated with lacunar infarction, Stroke 23 (1992) 1225 – 1229.
38
Y. Wen et al. / Brain Research 1022 (2004) 30–38
[37] P.J. Lu, G. Wulf, X.Z. Zhou, P. Davies, K.P. Lu, The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein, Nature 399 (1999) 784 – 788. [38] R.B. Maccioni, J.P. Munoz, L. Barbeito, The molecular bases of Alzheimer’s disease and other neurodegenerative disorders, Arch. Med. Res. 32 (2001) 367 – 381. [39] C. Mailliot, V. Podevin-Dimster, R.E. Rosenthal, N. Sergeant, A. Delacourte, G. Fiskum, L. Buee, Rapid tau protein dephosphorylation and differential rephosphorylation during cardiac arrest-induced cerebral ischemia and reperfusion, J. Cereb. Blood Flow. Metab. 20 (2000) 543 – 549. [40] R.L. Neve, P. Harris, K.S. Kosik, D.M. Kurnit, T.A. Donlon, Identification of cDNA clones for the human microtubule-associated protein tau and chromosomal localization of the genes for tau and microtubule-associated protein 2, Brain Res. 387 (1986) 271 – 280. [41] S.C. Papasozomenos, Y. Su, Altered phosphorylation of tau protein in heat-shocked rats and patients with Alzheimer disease, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 4543 – 4547. [42] F. Pasquier, D. Leys, Why are stroke patients prone to develop dementia? J. Neurol. 244 (1997) 135 – 142. [43] M. Perez, R. Cuadros, M.A. Smith, G. Perry, J. Avila, Phosphorylated, but not native, tau protein assembles following reaction with the lipid peroxidation product, 4-hydroxy-2-nonenal, FEBS Lett. 486 (2000) 270 – 274. [44] M. Perez, F. Hernandez, A. Gomez-Ramos, M. Smith, G. Perry, J. Avila, Formation of aberrant phosphotau fibrillar polymers in neural cultured cells, Eur. J. Biochem. 269 (2002) 1484 – 1489. [45] G. Perry, A. Nunomura, P. Lucassen, H. Lassmann, M.A. Smith, Apoptosis and Alzheimer’s disease, Science 282 (1998) 1268 – 1269. [46] M.P. Sanchez, V. Alvarez-Tallada, J. Avila, The microtubule-associated protein tau in neurodegenerative diseases. Tauopathies, Rev. Neurol. 33 (2001) 169 – 177. [47] A. Schneider, J. Biernat, M. von Bergen, E. Mandelkow, E.M. Mandelkow, Phosphorylation that detaches tau protein from microtubules (Ser262, Ser214) also protects it against aggregation into Alzheimer paired helical filaments, Biochemistry 38 (1999) 3549 – 3558. [48] D.A. Shackelford, R.Y. Yeh, Dephosphorylation of tau during transient forebrain ischemia in the rat, Mol. Chem. Neuropathol. 34 (1998) 103 – 120.
[49] J. Shi, S.H. Yang, L. Stubley, A.L. Day, J.W. Simpkins, Hypoperfusion induces overexpression of beta-amyloid precursor protein mRNA in a focal ischemic rodent model, Brain Res. 853 (2000) 1 – 4. [50] J.W. Simpkins, G. Rajakumar, Y.Q. Zhang, C.E. Simpkins, D. Greenwald, C.J. Yu, N. Bodor, A.L. Day, Estrogens may reduce mortality and ischemic damage caused by middle cerebral artery occlusion in the female rat, J. Neurosurg. 87 (1997) 724 – 730. [51] A.F. Slater, C. Stefan, I. Nobel, D.J. van den Dobbelsteen, S. Orrenius, Signalling mechanisms and oxidative stress in apoptosis, Toxicol. Lett. 82 – 83 (1995) 149 – 153. [52] M.A. Smith, P.L. Richey Harris, L.M. Sayre, J.S. Beckman, G. Perry, Widespread peroxynitrite-mediated damage in Alzheimer’s disease, J. Neurosci. 17 (1997) 2653 – 2657. [53] M.G. Spillantini, M. Goedert, R. Jakes, A. Klug, Different configurational states of beta-amyloid and their distributions relative to plaques and tangles in Alzheimer disease, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 3947 – 3951. [54] J.H. Su, A.J. Anderson, B.J. Cummings, C.W. Cotman, Immunohistochemical evidence for apoptosis in Alzheimer’s disease, NeuroReport 5 (1994) 2529 – 2533. [55] T.K. Tatemichi, D.W. Desmond, R. Mayeux, M. Paik, Y. Stern, M. Sano, R.H. Remien, J.B. Williams, J.P. Mohr, W.A. Hauser, et al., Dementia after stroke: baseline frequency, risks, and clinical features in a hospitalized cohort, Neurology 42 (1992) 1185 – 1193. [56] T.K. Tatemichi, M. Paik, E. Bagiella, D.W. Desmond, Y. Stern, M. Sano, W.A. Hauser, R. Mayeux, Risk of dementia after stroke in a hospitalized cohort: results of a longitudinal study, Neurology 44 (1994) 1885 – 1891. [57] J.Q. Trojanowski, V.M. Lee, Paired helical filament tau in Alzheimer’s disease. The kinase connection, Am. J. Pathol. 144 (1994) 449 – 453. [58] M.D. Weingarten, A.H. Lockwood, S.Y. Hwo, M.W. Kirschner, A protein factor essential for microtubule assembly, Proc. Natl. Acad. Sci. U. S. A. 72 (1975) 1858 – 1862. [59] P.M. Wise, Estrogens and neuroprotection, Trends Endocrinol. Metab. 13 (2002) 229 – 230. [60] P.M. Wise, D.B. Dubal, M.E. Wilson, S.W. Rau, M. Bottner, Minireview: neuroprotective effects of estrogen-new insights into mechanisms of action, Endocrinology 142 (2001) 969 – 973. [61] B.A. Yankner, New clues to Alzheimer’s disease: unraveling the roles of amyloid and tau, Nat. Med. 2 (1996) 850 – 852.
Research report
Transient cerebral ischemia induces site-specific hyperphosphorylation of tau protein Yi Wen, Shaohua Yang, Ran Liu, James W. Simpkins * Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699, USA Accepted 24 May 2004 Available online
Abstract Neurofibrillary tangles (NFTs) are a pathological hallmark of many neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP17). However, the cellular origin and the consequence of the NFT formation are poorly understood. Epidemiological evidence suggests a much higher occurrence of dementia in stroke patients. This may represent the pathogenesis of sporadic AD, which accounts for the majority of AD occurrence. Here we show that after a transient cerebral ischemia, hyperphosphorylated tau accumulates in cortical neurons in a site-specific manner. The hyperphosphorylated tau presents a conformation similar to those present in human tauopathies, and colocalizes largely with signs of apoptosis. Our current study suggests that tau hyperphosphorylation may contribute to the brain damage induced by transient cerebral ischemia, and may be involved in the pathogenesis of neurodegenerative disorders in patients after stroke. Further, these results indicate that ischemic neuronal damage and apoptosis associates with tau hyperphosphorylation, and potentially NFTs formation. Finally, our results also suggest that neuronal apoptosis may be a therapeutic target in preventing tauopathy-related neurodegenerative diseases. D 2004 Elsevier B.V. All rights reserved. Keywords: Cerebral ischemia; Hyperphosphorylation; Tau protein
1. Introduction Dysfunctional and filamentous microtubule-binding tau proteins are key markers of both sporadic and familial neurodegenerative pathologies. A large number of neurodegenerative diseases, including AD, Pick’s disease, progressive supranuclear palsy, corticobasal degeneration, and FTDP-17, showed aberrant aggregation of hyperphosphorylated tau protein [38]. Collectively, these diseases are known as neurodegenerative tauopathies. In AD, NFTs are found intracellularly in conjunction with the deposition of extracellular h-amyloid (Ah) fibrils [53]. The dominant component of neurofibrillary lesions in AD is hyperphosphorylated tau that forms paired helical filaments (PHFs) and straight filaments [30,31,33]. Tau proteins are microtubule-associated proteins that are expressed abundantly in soma and axons. Both in vivo and
* Corresponding author. Tel.: +1-817-735-0498. E-mail address: [email protected] (J.W. Simpkins). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.05.106
in vitro studies indicate that tau proteins bind to microtubules, promoting their polymerization [12,58]. Several isoforms of tau are present in the central nervous system (CNS), likely produced by alternative mRNA splicing [20,40]. The isoforms of tau proteins in AD are similar to those observed in normal adult human brain [21,57]. In tau protein, there are 79 potential serine and threonine phosphorylation sites on the longest human tau isoform. Phosphorylation of more than 30 of the phosphorylation sites has been reported [7,10]. These phosphorylation sites are involved in both embryonic development and neurodegeneration [28]. These phosphorylation sites cluster in the flanking region of the tubulin-binding domain and negatively regulate tau’s binding affinity to microtubules [6,8,17]. However, the relative importance of individual phosphorylation sites is still controversial. The prevalence of dementia in ischemic stroke patients is nine times higher than controls at 3 months [55] and 4– 12 times higher than controls at 4 years after a lacunar infarct [36]. Many of these dementias develop progressively, and cerebral damage is not the direct cause of the subsequent
Y. Wen et al. / Brain Research 1022 (2004) 30–38
dementia in at least half of the cases [56]. AD is the most prevalent dementia [42] and shares common neuropathology features with stroke. Alz-50-immunoreactive granules were found around cerebral infarction after a stroke [24], amyloid precursor protein (APP) accumulates following transient cerebral focal ischemia [49], and ApoE4 is a genetic risk factor for both AD and stroke [13]. In the present study, we assessed the induction of tau phosphorylation and epitopes with PHF-like conformation in a rodent model for transient cerebral ischemia. We also examined the potential phosphorylation sites and their degree of phosphorylation induced by transient cerebral ischemia.
31
Briefly, the animal was anesthetized with ketamine (60 mg/ kg) and xylazine (10 mg/kg), then the internal carotid artery (ICA) was exposed, and a 3-0 monofilament nylon suture was introduced into the ICA lumen through a puncture and gently advanced to the distal internal carotid artery (ICA) until proper resistance was felt. After 1 h, the suture was withdrawn from the ICA and the distal ICA was immediately cauterized. At the desired time after the onset of reperfusion, animals were sacrificed and sampled for immunoblotting, immunohistochemistry, or 2% 2,3,5Triphenyl-2H-tetrazolium chloride (TTC) stain, as described below. 2.4. TTC staining
2. Materials and methods 2.1. Animals Four- to six-week-old female Sprague – Dawley (SD) rats were purchased from Charles Rivers (Wilmington, MA) and maintained in our animal facility in a temperature-controlled room (22 –25 jC) with 12-h dark –light cycles. All rats have free access to laboratory chow and tap water. All animal procedures were reviewed and approved by the University of North Texas Health Science Center Institutional Animal Care and Use Committee. 2.2. Chemicals and antibodies The immunoblotting and in vitro phosphorylation screening analysis used antibodies directed against different phospho-epitopes on tau protein, which includes pT181, pS202, pT205, pT212, pS214, pT231, pS262, pS396, pS404, and pS422 (Biosource, Camarillo, CA) at a dilution of 1:500. The following antibodies were used to label the conformationand phosphorylation-dependent tau epitopes in the brain sections: MC1 (PHF-conformation), TG3 (Phospho-tau 231 and AD conformation), CP13 (Phospho-tau 202/205), CP3 (Phospho-tau 214), PHF-1 (Phospho-tau 396/404), and CP9 (Phospho-tau 231) at a dilution of 1:10 in the immunohistochemistry. The above-mentioned phosphorylation/NFT conformation-dependent antibodies were characterized by and were the kind gifts of Dr. Peter Davies (Albert Einstein College of Medicine). All Chemicals were from SigmaAldrich (St. Louis, MO) unless otherwise specified. 2.3. Animal surgeries To avoid fluctuations of estrogens, which are neuroprotective [50,60], all animals received bilateral ovariectomies at least 2 weeks before any surgical procedure. Animals were anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/ kg), then a small cut was made through the skin and muscle. The ovaries were externalized and removed. For middle cerebral artery (MCA) occlusion and reperfusion, an intraluminal filament model was used [3,50].
Brains were removed, immediately after sacrificing the animal, and dissected coronally into 2-mm sections using a metallic brain matrix (ASI Instruments, Warren, MI) and stained by incubation in a 2% TTC in a 0.9% saline solution at 37 jC for 30 min. Photographs were taken with Kodak digital camera. Each treatment group included at least three to four animals. 2.5. In vitro phosphorylation Frontoparietal cortex from either ischemic (n = 4) or the contralateral brains (n = 4) was dissected from other brain tissues and homogenized in modified RIPA buffer [1 PBS, 1% Nonidet P-40 or Igepal CA-630, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 10 Ag/ml Aprotinin, 100 Ag/ml phenylmethyl sulphonyl fluoride (PMSF)]. Samples were incubated on ice for 20 min and then centrifuged at 12,000 g for 20 min. Supernatant was collected for further kinase activity analysis or immunoblotting. For phosphorylation reaction, 1 Ag recombinant human tau protein from Sigma-Aldrich and 10-Ag brain extracts were added to reaction buffer, containing 50 mM HEPES, 0.1 mM EDTA, 0.1 mg/ml BSA, 0.1% h-mercaptoethanol, 0.15 M NaCl, 50 AM ATP, and 1 ACi 32P-labeled g-ATP from ICN Biomedicals (Irvine, CA). The tube was incubated for 30 min in a 30 jC water bath with moderate shaking. SDS-PAGE loading buffer was then added, and the tube was boiled for 10 min to terminate the reaction. Samples from the phosphorylation reaction were separated on 10% or 8– 16% gradient SDS-polyacrylamide gels. The gels were stained with Coomassie Brilliant Blue R250 and then subjected to autoradiography. 2.6. Immunoblotting and in vitro phosphorylation screening Immunoblotting analysis with various antibodies was performed according to the antibody manufacturer’s instructions with slight modification. Briefly, samples (n = 4) from cortex (peri-infarct area) in the ischemic or the corresponding contralateral areas were separated on 10% or 8 – 16% gradient SDS-polyacrylamide gels and
32
Y. Wen et al. / Brain Research 1022 (2004) 30–38
electrophoretically transferred to a nitrocellulose membrane. Membranes were blocked by incubation with 5% dry milk in phosphate-buffered saline (PBS) and 0.02% Tween 20 (PBST) for 1 h at room temperature, followed by overnight incubation at 4 jC with the appropriate primary antibody in blocking buffer. After thorough washing, the membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, followed by another thorough wash in PBST. The membrane was then incubated and developed. The blots were developed with an enhanced chemiluminescent kit (Pierce, Rockford, IL). The in vitro phosphorylation products without 32P-labeled g-ATP were used for phosphorylation site analysis. 2.7. Immunohistochemistry and TUNEL staining For immunohistochemical analysis, animals were perfused transcardially with 4% paraformaldehyde in sodium phosphate buffer (pH 7.4) (n = 3). Brains were paraffinembedded and sectioned at 8 – 10 Am. Immunohistochemical analysis was performed using an HRP-DAB kit, following the manufacturer’s recommended procedure (Zymed, South San Francisco, CA). Some of the sections were pretreated with 100 Ag/ml proteinase K in PBS for 15 min for antigen retrieval and signal enhancement. After staining, sections were dehydrated in a gradient of ethanol and xylene and
then sealed with cover slips. All photographs were taken using a Nikon Diaphot 300 microscope and a 12-bit CCD monochrome camera with Labworks software (UVP, Upland, CA). DNA fragmentation was detected using TdT-mediated dUTP Nick-End Labeling (TUNEL) method with The DeadEndk Fluorometric TUNEL System (Promega, Madison, WI) according to the manufacturer’s instructions. Sections were counterstained with 4V,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Molecular Probes, Eugene, OR). Colocalization of TUNEL with specific antigens was performed with highly specific Alexa-Fluorophore conjugated secondary antibodies (Molecular Probes), whose excitation/emission spectra do not overlap with that of the FITC label used in the TUNEL stain. 2.8. Densitometric and statistical analysis The images of autoradiography and immunoblotting were digitalized and imported using a calibrated scanner at 16-bit gray mode to provide 65,535 gray levels. The area density was quantified using Labworks software (UVP). The unit given is an arbitrary unit that stands for the total units per band in the analyzed samples. The results were assayed with Student’s t-test or one-way ANOVA statistical method using Prism software.
Fig. 1. Induction of apoptosis by transient cerebral ischemia in OVX rats. TUNEL-stained sections from various brain regions after 1-h MCA occlusion followed by 24-h reperfusion: (A) contralateral cortex, (B) ischemic cortex, (C) penumbra area. (D) TTC-stained brain sections indicating area of damage. The letters in (D) indicate the approximate region of A – C. Scale bar = 100 Am.
Y. Wen et al. / Brain Research 1022 (2004) 30–38
33
3. Results 3.1. Evaluation of apoptosis induced by transient cerebral ischemia MCA occlusion is a widely used focal ischemic stroke model [9]. This in vivo model for neuronal death can rapidly induce a synchronized apoptotic process in a large number of neurons [35]. We examined the effects of this transient cerebral focal ischemia on neuronal apoptosis using a TUNEL assay. As early as 2 h after the initiation of reperfusion, positive TUNEL staining appeared in the core ischemic region near the basal ganglion area (data not shown). After 24 h of reperfusion, TUNEL-positive cells were widespread in the ischemic region, which includes the frontoparietal cerebral cortex (Fig. 1B), but were undetectable in the corresponding contralateral regions (Fig. 1A). In the penumbra areas, where the ischemic region is separated from the fully perfused area, TUNEL signal was observed only in the ischemic side (Fig. 1C). In the striatal regions that received the strongest ischemia, many cells showed positive TUNEL signals in a variety of cell types, which include neurons, astrocytes, glia, and some endothelial cells of blood vessels, as determined from morphological evaluation (data not shown). However, in the neocortex area, most TUNEL-positive cells represent the morphology of cortical pyramidal neurons. The distribution of TUNEL
Fig. 3. Screening of hyperphosphorylation sites in tau protein induced by transient cerebral ischemia. (A) Potential hyperphosphorylation sites were analyzed with a panel of antibodies that recognize site-specific phosphorylation in tau protein. The numbers on the x-axis indicates the amino acid residuals in tau protein (n = 6). * indicates significant difference between the indicated group and the contralateral cortex of ovariectomized animal at p < 0.05. (B) Some representative immunoblotting assay in randomly selected duplicates indicating the specific phosphorylation sites of tau protein. Tau 5 recognizes tau protein independent of its phosphorylation status. (C) Representative immunoblotting analysis of in vivo tau hyperphosphorylation. Numbers above indicate the examined phosphorylation site based on human 4-repeat tau protein (I: ischemic extracts, C: contralateral extracts).
signal was consistent with the result of TTC staining for brain slices (Fig. 1D). 3.2. Aberrant tau hyperphosphorylation occurs in the ischemic cortex
Fig. 2. Transient cerebral ischemia-induced tau hyperphosphorylation. (A) Autoradiography (upper lane) and Coomassie brilliant blue-stained SDS-PAGE gel (lower lane) of protein kinase assay, using recombinant tau as a substrate. Each lane represents an individual experimental animal. (B) The densitometric analysis of tau hyperphosphorylation induced by transient cerebral ischemia. * indicates significant difference between the ipsilateral and the contralateral cortex of ovariectomized animals at p < 0.05.
To characterize the phosphorylation status of tau in the ischemic regions, we examined total tau-kinase activity. This assay examines the transfer of 32P-labeled phosphate from ATP to recombinant tau protein. Tau kinase activity increased 2.87-fold in the ischemic region versus the contralateral regions (Fig. 2A and B). The hyperphosphorylation induced apparent multiplicity, represented by multiple bands in the Coomassie brilliant blue-stained
34
Y. Wen et al. / Brain Research 1022 (2004) 30–38
SDS-PAGE (Fig 2A), which is consistent with previous reports [27]. We used a panel of phosphorylation-dependent anti-tau antibodies to screen the potential hyperphosphorylation sites of tau protein induced by this transient ischemia. We observed that the tau hyperphosphorylation was sitespecific, at 24 h of reperfusion. Collectively, pS202, pS214, pT231, and pS402 showed a significant increase in their phosphorylation status (Fig. 3A and B). These hyperphosphorylated sites were confirmed with immunoblotting with endogenous brain cortical extract (Fig. 3C). The rodent tau represents multiple bands ranging from 45 to 64 kd, indicating the presence of multiple isoforms, similar to those in human brain extracts. Our results are consistent with previous research on tau hyperphosphorylation which indicates that rodent tau generally migrates primarily at 40 – 50 kDa, with minor forms at 68 and 100 kDa. However, antibodies that recognize phosphorylated tau appear to emphasize the higher molecular weight isoforms, potentially due to the presence of isoform content alteration, preference in phosphorylation sites, or the hyperphosphorylation [15,25]. To confirm the aberrant hyperphosphorylation of tau, we also examined the ischemic area using immunohistochemistry with a panel of phosphospecific monoclonal antibodies. While the contralateral cortex was almost absent of the specific phosphospecific immunoreactivity (Fig. 4A), we observed extensive phosphospecific immunoreactivity in the ischemic regions, including PHF-1 (Fig. 4B and C), CP3 (Fig. 4D), CP13 (Fig. 4E), and CP9 (Fig. 4F). To further investigate the spatial relationship of hyperphosphorylated tau and apoptosis, we double-stained PHF-1 and TUNEL to evaluate their spatial relationship. We observed that most of the phospho-tau epitopes co-localized with the apoptotic marker, TUNEL (Fig. 5).
3.3. Ischemia-induced tau hyperphosphorylation causes conformational changes similar to those in neurodegenerative tauopathy Tauopathies in neurodegeneration involve not only the hyperphosphorylation, but also conformational change of tau protein. To further characterize the phosphorylation and conformational status of tau in the ischemic regions, we used two conformation-dependent anti-tau antibodies. Antibody TG3 detects both phosphorylation and the unique conformation of PHF-tau [22], while MC1 is a conformation-dependent antibody that detects the Alz-50 epitope of AD tau [26]. In the ischemic region, a large number of pyramidal neurons were immunopositive for TG3 and MC1 in the frontoparietal cortex, but not in the contralateral regions (Fig. 6). The strongest immunoreactivity of MC1 was also observed in the frontoparietal neocortex (Fig. 6B and C). TG3 showed a staining pattern similar to MC1 in the ischemic cortical area, but not in the contralateral areas (Fig. 6D – F).
4. Discussion The results of this study show that transient cerebral ischemia induces a hyperphosphorylation of microtubuleassociated protein tau in a site-specific manner and can further induce NFT-like conformational epitopes in wildtype ovx female rats. These results were confirmed by immunoblotting and immunohistochemistry. Four sites, including pS202, pS214, pT231, pS422, were identified in the in vitro screening of the potential phosphorylation sites, and these sites were confirmed with immunoblotting. The current study may help to clarify the molecular and cellular mechanisms of higher occurrence of dementia after stroke and provides a convenient non-transgenic animal model to
Fig. 4. Immunohistochemical staining with antibodies that detect hyperphosphorylation of tau protein in vivo. (A – C) Representative coronal sections of the frontoparietal cerebral cortex showing immunoreactivity of PHF-1 (detecting P-396/404) in the contralateral (A), ipsilateral (B), and ipsilateral at high magnification (C) brain sections. (D – F) Representative photographs of immunohistochemical staining in the ipsilateral frontoparietal cortex with CP3 (detecting P-214) (D), CP9 (detecting P-231) (E), and CP13 (detecting P-202/205) (F), in the frontoparietal cortex. Scale bar = 100 Am.
Y. Wen et al. / Brain Research 1022 (2004) 30–38
Fig. 5. Colocalization of tau hyperphosphorylation and apoptosis in vivo. Confocal microscopy imaging indicates the colocalization of PHF-1 immunostaining with many TUNEL-positive cells in the ischemic cortical region. (A) TUNEL stain (green), (B) PHF-1 immunofluorescence (red), and (C) the merged image of (A) and (B).
investigate the hyperphosphorylation of tau, which is one of the hallmarks of AD pathology. However, a variety of in vivo and in vitro neuronal insults, including heat shock [41], permanent focal cerebral ischemia in the cat [16], and amyloid treatment [18], induces hyperphosphorylation of tau. Interestingly, in response to the ischemia/reperfusion process, phosphorylation of tau protein appears to be dynamically regulated. Oxygen and glucose deprivation in rat and human neocortical brain slice induces a rapid dephosphorylation within 30 – 60 min during the ischemia [11]; also, during transient forebrain
35
ischemia in the rat, dephosphorylation was detected within 5 min, and restoration occurs in as a little as 15 min after reperfusion [48]. Cardiac arrest-induced ischemia/reperfusion appears to induce a similar rapid dephosphorylation during ischemia and restoration beginning less than 2 h after reperfusion [39]. In general, a rapid dephosphorylation of tau occurs during ischemia or immediately upon reperfusion, potentially due to the activation of phosphatases. During continued reperfusion, tau proteins are slowly phosphorylated and accumulate, potentially due to the downregulation of phosphatases or induction of specific kinases. This may reflect the dynamic regulation of phosphatases and/or the corresponding tau kinases, including Cdk5, Cdc2, and MAPK [46], in response to the ischemia/reperfusion process. Our observations are also consistent and supported by many epidemiological and postmortem clinical studies, as well as other animal studies. Alz-50-immunoreactive granules were found around cerebral infarction after a stroke [24]. Alzheimer-type pathology is often associated with cerebral amyloid angiopathy and infarcts [36]. Further, AD and stroke have common genetic risk factors, including e 4 allele of the apolipoprotein E gene (APOE), which associates with a higher risk of ischemic stroke, coronary heart diseases, and late-onset AD [13]. We investigated 10 out of 30 potential phosphorylation sites on tau protein. Among the 10 potential phosphorylation sites studied, 4 showed statistically significant increases in phosphorylation in response to ischemia, including pS202, pS214, pT231, pS422, and this finding was confirmed by in vivo studies. These four phosphorylation sites are localized in the flanking regions, outside the microtubule binding domain. Hyperphosphorylations of tau have been reported to reduce tau’s ability of binding and promoting tubulin polymerization [10]. However, the critical significance of individual phosphorylation sites and their corresponding kinase are still controversial. Previous research that assessed the correlation of phosphorylation of tau and severity of AD cases suggests that there are events prior to filament formation that are specific to particular phosphorylated tau epitopes, leading to conformational changes and cytopathological alterations [2]. Phosphorylation on S231 can result in the association of pin1, a prolyl isomerase, and may help to restore some of the tau’s function [37]. pS214 is a potential substrate of PKA and appears to detach tau protein from microtubules and protects it against aggregation into Alzheimer-paired helical filaments [47]. The specificity of phosphorylation sites induced by transient ischemia implies that a specific set of kinases is activated during this relatively short period of 24 h. However, another possibility is an alteration of phosphatases. Further, the mechanisms of the tau hyperphosphorylation, in this ischemia/reperfusion injury, may not necessarily be the same as tauopathy-related neurodegenerative diseases, which generally require decades to progress. However, this can provide a convenient animal model to study the pathological tau hyperphosphorylation
36
Y. Wen et al. / Brain Research 1022 (2004) 30–38
Fig. 6. Immunohistochemical staining with antibodies that detect PHF-like conformation of tau protein. Tau phosphorylation induces conformational changes after 24 h of reperfusion in MCA occlusion rats. Antibody MC1, a conformation-dependent antibody, detected tau in the ischemic cerebral cortical region in low (B) and high (C) magnification, but not in the contralateral cortex (A). Most of the positive immunoreactivity was localized to the frontoparietal cortex, with only a few detected in subcortical areas. Antibody TG3, a phosphorylation- and conformation-dependent antibody, stained a subset of pyramidal neurons of the ipsilateral cortex at low (E) and high (F) magnification, but not in the contralateral cortex (D).
and for screening for pharmacological inhibitors of tau phosphorylation. Immunohistochemical studies with monoclonal antibodies directed against the phospho-epitopes or NFT-like conformation recognized a number of cortical neurons in ischemic brain sections, but not on the contralateral side. Most of the immunoreactivity concentrated in the frontoparietal cortex within peri-infarct area. Striatal regions within the core ischemic area, including globus pallidus and caudate putamen, showed only weak signals that appear to originate from glia or potentially infiltrated neutrophils. On the contralateral side, only a diffusive staining was observed and can be regarded as negative. Double-labeling studies of phospho-tau and an apoptotic marker indicate apparent co-localization. In the peri-infarct areas, more than half of the PHF-1 positive cells showed strong TUNEL staining. However, current studies did not determine a definitive relationship between tau-hyperphosphorylation and neuronal apoptosis induced by the transient cerebral ischemia. Our current observation suggests that this tauhyperphosphorylation is the consequence of the activation/ inactivation of a variety of phosphatases and kinases in response to the ischemia/reperfusion damage. The hyperphosphorylation of tau may result in the reduction of its affinity to microtubule protein and promote microtubule depolymerization, which will inhibit axonal transport, destabilize neuronal cytoskeleton, and may contribute to the apoptotic process induced by this ischemia/reperfusion insult. Further, phosphorylated, but not native, tau protein is able to form polymers after the reaction with 4-hydroxy2-nonenal [43]. These observations are consistent with previous reports that phosphorylation and oxidative modification by hydroxynonenal, a product of oxidative stress, are required for tau filament formation [44].
Although it is tempting and exciting to expect this ischemia-induced tau hyperphosphorylation to share significant similarities with those observed in AD brains, there is currently insufficient evidence to demonstrate that the phosphorylated tau detected in the ischemic animal brains is in the aggregated form such as that found in human neurodegenerative tauopathies. Additional studies including Gallyas silver staining, thioflavin-S staining, ultrastructural examination, and analysis of sarcosyl-insoluble brain extracts by electronic microscopy analysis are required to answer these questions. A long-term study is also required for further characterization on the effects of this transient focal ischemia. However, our study supports the hypothesis that NFT formation may be a consequence of a preceding apoptotic event, including amyloid deposit, which results in the aberrant activation of protein kinases and further induction of tau hyperphosphorylation and tangle formation [5,32]. A number of studies support the conclusion that the apoptotic signaling pathways are involved in neuronal loss in AD brains [1,4,14]. Pro-apoptotic factors, including reactive oxygen species [51,52], h-amyloid [61], and growth factors reduction [19], may serve as the initiator of NFT-formation. Tau hyperphosphorylation and tangle formation is associated with apoptosis in AD brains [29,45,54] and can be induced or enhanced by amyloid deposits [23,34]. The MCA occlusion model has been widely used for the evaluation of neuropathology of stroke and neuroprotective compounds, such as estrogen [50,59]. Our results indicate that this transient ischemic model may be used for evaluating tau hyperphosphorylation and tauopathy formation, as well as for pharmaceutical screening for NFT inhibitors in vivo. Finally, our data also suggest that sporadic hypoperfusion of the brain may initiate and contribute to the progression of neuropathology in nonfamilial AD patients and provide a
Y. Wen et al. / Brain Research 1022 (2004) 30–38
mechanism for the long-observed correlation between ischemic events and the increased prevalence of AD [36]. As such, prevention of ischemic episodes may serve as an additional therapeutic target to prevent the initiation and progression of AD.
References [1] A.J. Anderson, J.H. Su, C.W. Cotman, DNA damage and apoptosis in Alzheimer’s disease: colocalization with c-Jun immunoreactivity, relationship to brain area, and effect of postmortem delay, J. Neurosci. 16 (1996) 1710 – 1719. [2] J.C. Augustinack, A. Schneider, E.M. Mandelkow, B.T. Hyman, Specific tau phosphorylation sites correlate with severity of neuronal cytopathology in Alzheimer’s disease, Acta Neuropathol. (Berl.) 103 (2002) 26 – 35. [3] J.B. Bederson, L.H. Pitts, M. Tsuji, M.C. Nishimura, R.L. Davis, H. Bartkowski, Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination, Stroke 17 (1986) 472 – 476. [4] C. Behl, Apoptosis and Alzheimer’s disease, J. Neural Transm. 107 (2000) 1325 – 1344. [5] C. Behl, B. Moosmann, Oxidative nerve cell death in Alzheimer’s disease and stroke: antioxidants as neuroprotective compounds, Biol. Chem. 383 (2002) 521 – 536. [6] J. Biernat, N. Gustke, G. Drewes, E.M. Mandelkow, E. Mandelkow, Phosphorylation of Ser262 strongly reduces binding of tau to microtubules: distinction between PHF-like immunoreactivity and microtubule binding, Neuron 11 (1993) 153 – 163. [7] M.L. Billingsley, R.L. Kincaid, Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration, Biochem. J. 323 (Pt 3) (1997) 577 – 591. [8] G.T. Bramblett, M. Goedert, R. Jakes, S.E. Merrick, J.Q. Trojanowski, V.M. Lee, Abnormal tau phosphorylation at Ser396 in Alzheimer’s disease recapitulates development and contributes to reduced microtubule binding, Neuron 10 (1993) 1089 – 1099. [9] A.M. Bremer, O. Watanabe, R.S. Bourke, Artificial embolization of the middle cerebral artery in primates. Description of an experimental model with extracranial technique, Stroke 6 (1975) 387 – 390. [10] L. Buee, T. Bussiere, V. Buee-Scherrer, A. Delacourte, P.R. Hof, Tau protein isoforms, phosphorylation and role in neurodegenerative disorders, Brain Res. Brain Res. Rev. 33 (2000) 95 – 130. [11] K.K. Burkhart, D.C. Beard, R.A. Lehman, M.L. Billingsley, Alterations in tau phosphorylation in rat and human neocortical brain slices following hypoxia and glucose deprivation, Exp. Neurol. 154 (1998) 464 – 472. [12] D.W. Cleveland, S.Y. Hwo, M.W. Kirschner, Purification of tau, a microtubule-associated protein that induces assembly of microtubules from purified tubulin, J. Mol. Biol. 116 (1977) 207 – 225. [13] J.H. Contois, D.E. Anamani, G.J. Tsongalis, The underlying molecular mechanism of apolipoprotein E polymorphism: relationships to lipid disorders, cardiovascular disease, and Alzheimer’s disease, Clin. Lab. Med. 16 (1996) 105 – 123. [14] C.W. Cotman, J.H. Su, Mechanisms of neuronal death in Alzheimer’s disease, Brain Pathol. 6 (1996) 493 – 506. [15] J.C. Cruz, H.C. Tseng, J.A. Goldman, H. Shih, L.H. Tsai, Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles, Neuron 40 (2003) 471 – 483. [16] D. Dewar, D.I. Graham, G.M. Teasdale, J. McCulloch, Alz-50 and ubiquitin immunoreactivity is induced by permanent focal cerebral ischaemia in the cat, Acta Neuropathol. (Berl.) 86 (1993) 623 – 629. [17] D.N. Drechsel, A.A. Hyman, M.H. Cobb, M.W. Kirschner, Modula-
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32] [33]
[34]
[35] [36]
37
tion of the dynamic instability of tubulin assembly by the microtubule-associated protein tau, Mol. Biol. Cell 3 (1992) 1141 – 1154. A. Ferrari, F. Hoerndli, T. Baechi, R.M. Nitsch, J. Gotz, Beta-amyloid induces paired helical filament-like tau filaments in tissue culture, J. Biol. Chem. 278 (2003) 40162 – 40168. L. Gasparini, W.J. Netzer, P. Greengard, H. Xu, Does insulin dysfunction play a role in Alzheimer’s disease? Trends Pharmacol. Sci. 23 (2002) 288 – 293. M. Goedert, C.M. Wischik, R.A. Crowther, J.E. Walker, A. Klug, Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: identification as the microtubule-associated protein tau, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 4051 – 4055. M. Goedert, R. Jakes, M.G. Spillantini, R.A. Crowther, P. Cohen, E. Vanmechelen, A. Probst, J. Gotz, K. Burki, Tau protein in Alzheimer’s disease, Biochem. Soc. Trans. 23 (1995) 80 – 85. J. Gotz, F. Chen, R. Barmettler, R.M. Nitsch, Tau filament formation in transgenic mice expressing P301L tau, J. Biol. Chem. 276 (2001) 529 – 534. J. Gotz, F. Chen, J. van Dorpe, R.M. Nitsch, Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils, Science 293 (2001) 1491 – 1495. K. Ikeda, H. Akiyama, T. Arai, H. Kondo, C. Haga, K. Tsuchiya, S. Yamada, S. Murayama, A. Hori, Neurons containing Alz-50-immunoreactive granules around the cerebral infarction: evidence for the lysosomal degradation of altered tau in human brain? Neurosci. Lett. 284 (2000) 187 – 189. N.D. James, D.R. Davis, J. Sindon, D.P. Hanger, J.P. Brion, C.C. Miller, M.P. Rosenberg, B.H. Anderton, F. Propst, Neurodegenerative changes including altered tau phosphorylation and neurofilament immunoreactivity in mice transgenic for the serine/threonine kinase Mos, Neurobiol. Aging 17 (1996) 235 – 241. G.A. Jicha, R. Bowser, I.G. Kazam, P. Davies, Alz-50 and MC-1, a new monoclonal antibody raised to paired helical filaments, recognize conformational epitopes on recombinant tau, J. Neurosci. Res. 48 (1997) 128 – 132. Y. Kanai, R. Takemura, T. Oshima, H. Mori, Y. Ihara, M. Yanagisawa, T. Masaki, N. Hirokawa, Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA, J. Cell Biol. 109 (1989) 1173 – 1184. K. Kanemaru, K. Takio, R. Miura, K. Titani, Y. Ihara, Fetal-type phosphorylation of the tau in paired helical filaments, J. Neurochem. 58 (1992) 1667 – 1675. K. Kobayashi, H. Nakano, M. Hayashi, M. Shimazaki, Y. Fukutani, K. Sasaki, K. Sugimori, Y. Koshino, Association of phosphorylation site of tau protein with neuronal apoptosis in Alzheimer’s disease, J. Neurol. Sci. 208 (2003) 17 – 24. J. Kondo, T. Honda, H. Mori, Y. Hamada, R. Miura, M. Ogawara, Y. Ihara, The carboxyl third of tau is tightly bound to paired helical filaments, Neuron 1 (1988) 827 – 834. K.S. Kosik, L.D. Orecchio, L. Binder, J.Q. Trojanowski, V.M. Lee, G. Lee, Epitopes that span the tau molecule are shared with paired helical filaments, Neuron 1 (1988) 817 – 825. M.S. Lee, L.H. Tsai, Cdk5: one of the links between senile plaques and neurofibrillary tangles? J. Alzheimer’s Dis. 5 (2003) 127 – 137. V.M. Lee, B.J. Balin, L. Otvos Jr., J.Q. Trojanowski, A68: a major subunit of paired helical filaments and derivatized forms of normal Tau, Science 251 (1991) 675 – 678. J. Lewis, D.W. Dickson, W.L. Lin, L. Chisholm, A. Corral, G. Jones, S.H. Yen, N. Sahara, L. Skipper, D. Yager, C. Eckman, J. Hardy, M. Hutton, E. McGowan, Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP, Science 293 (2001) 1487 – 1491. Y. Li, M. Chopp, C. Powers, N. Jiang, Apoptosis and protein expression after focal cerebral ischemia in rat, Brain Res. 765 (1997) 301 – 312. C. Loeb, C. Gandolfo, R. Croce, M. Conti, Dementia associated with lacunar infarction, Stroke 23 (1992) 1225 – 1229.
38
Y. Wen et al. / Brain Research 1022 (2004) 30–38
[37] P.J. Lu, G. Wulf, X.Z. Zhou, P. Davies, K.P. Lu, The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein, Nature 399 (1999) 784 – 788. [38] R.B. Maccioni, J.P. Munoz, L. Barbeito, The molecular bases of Alzheimer’s disease and other neurodegenerative disorders, Arch. Med. Res. 32 (2001) 367 – 381. [39] C. Mailliot, V. Podevin-Dimster, R.E. Rosenthal, N. Sergeant, A. Delacourte, G. Fiskum, L. Buee, Rapid tau protein dephosphorylation and differential rephosphorylation during cardiac arrest-induced cerebral ischemia and reperfusion, J. Cereb. Blood Flow. Metab. 20 (2000) 543 – 549. [40] R.L. Neve, P. Harris, K.S. Kosik, D.M. Kurnit, T.A. Donlon, Identification of cDNA clones for the human microtubule-associated protein tau and chromosomal localization of the genes for tau and microtubule-associated protein 2, Brain Res. 387 (1986) 271 – 280. [41] S.C. Papasozomenos, Y. Su, Altered phosphorylation of tau protein in heat-shocked rats and patients with Alzheimer disease, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 4543 – 4547. [42] F. Pasquier, D. Leys, Why are stroke patients prone to develop dementia? J. Neurol. 244 (1997) 135 – 142. [43] M. Perez, R. Cuadros, M.A. Smith, G. Perry, J. Avila, Phosphorylated, but not native, tau protein assembles following reaction with the lipid peroxidation product, 4-hydroxy-2-nonenal, FEBS Lett. 486 (2000) 270 – 274. [44] M. Perez, F. Hernandez, A. Gomez-Ramos, M. Smith, G. Perry, J. Avila, Formation of aberrant phosphotau fibrillar polymers in neural cultured cells, Eur. J. Biochem. 269 (2002) 1484 – 1489. [45] G. Perry, A. Nunomura, P. Lucassen, H. Lassmann, M.A. Smith, Apoptosis and Alzheimer’s disease, Science 282 (1998) 1268 – 1269. [46] M.P. Sanchez, V. Alvarez-Tallada, J. Avila, The microtubule-associated protein tau in neurodegenerative diseases. Tauopathies, Rev. Neurol. 33 (2001) 169 – 177. [47] A. Schneider, J. Biernat, M. von Bergen, E. Mandelkow, E.M. Mandelkow, Phosphorylation that detaches tau protein from microtubules (Ser262, Ser214) also protects it against aggregation into Alzheimer paired helical filaments, Biochemistry 38 (1999) 3549 – 3558. [48] D.A. Shackelford, R.Y. Yeh, Dephosphorylation of tau during transient forebrain ischemia in the rat, Mol. Chem. Neuropathol. 34 (1998) 103 – 120.
[49] J. Shi, S.H. Yang, L. Stubley, A.L. Day, J.W. Simpkins, Hypoperfusion induces overexpression of beta-amyloid precursor protein mRNA in a focal ischemic rodent model, Brain Res. 853 (2000) 1 – 4. [50] J.W. Simpkins, G. Rajakumar, Y.Q. Zhang, C.E. Simpkins, D. Greenwald, C.J. Yu, N. Bodor, A.L. Day, Estrogens may reduce mortality and ischemic damage caused by middle cerebral artery occlusion in the female rat, J. Neurosurg. 87 (1997) 724 – 730. [51] A.F. Slater, C. Stefan, I. Nobel, D.J. van den Dobbelsteen, S. Orrenius, Signalling mechanisms and oxidative stress in apoptosis, Toxicol. Lett. 82 – 83 (1995) 149 – 153. [52] M.A. Smith, P.L. Richey Harris, L.M. Sayre, J.S. Beckman, G. Perry, Widespread peroxynitrite-mediated damage in Alzheimer’s disease, J. Neurosci. 17 (1997) 2653 – 2657. [53] M.G. Spillantini, M. Goedert, R. Jakes, A. Klug, Different configurational states of beta-amyloid and their distributions relative to plaques and tangles in Alzheimer disease, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 3947 – 3951. [54] J.H. Su, A.J. Anderson, B.J. Cummings, C.W. Cotman, Immunohistochemical evidence for apoptosis in Alzheimer’s disease, NeuroReport 5 (1994) 2529 – 2533. [55] T.K. Tatemichi, D.W. Desmond, R. Mayeux, M. Paik, Y. Stern, M. Sano, R.H. Remien, J.B. Williams, J.P. Mohr, W.A. Hauser, et al., Dementia after stroke: baseline frequency, risks, and clinical features in a hospitalized cohort, Neurology 42 (1992) 1185 – 1193. [56] T.K. Tatemichi, M. Paik, E. Bagiella, D.W. Desmond, Y. Stern, M. Sano, W.A. Hauser, R. Mayeux, Risk of dementia after stroke in a hospitalized cohort: results of a longitudinal study, Neurology 44 (1994) 1885 – 1891. [57] J.Q. Trojanowski, V.M. Lee, Paired helical filament tau in Alzheimer’s disease. The kinase connection, Am. J. Pathol. 144 (1994) 449 – 453. [58] M.D. Weingarten, A.H. Lockwood, S.Y. Hwo, M.W. Kirschner, A protein factor essential for microtubule assembly, Proc. Natl. Acad. Sci. U. S. A. 72 (1975) 1858 – 1862. [59] P.M. Wise, Estrogens and neuroprotection, Trends Endocrinol. Metab. 13 (2002) 229 – 230. [60] P.M. Wise, D.B. Dubal, M.E. Wilson, S.W. Rau, M. Bottner, Minireview: neuroprotective effects of estrogen-new insights into mechanisms of action, Endocrinology 142 (2001) 969 – 973. [61] B.A. Yankner, New clues to Alzheimer’s disease: unraveling the roles of amyloid and tau, Nat. Med. 2 (1996) 850 – 852.