J Neural Transm (2009) 116:345–350 DOI 10.1007/s00702-008-0181-x
DEMENTIAS - ORIGINAL ARTICLE
PHF-like tau phosphorylation in mammalian hibernation is not associated with p25-formation Jens Thorsten Stieler Æ Torsten Bullmann Æ Franziska Kohl Æ Brian M. Barnes Æ Thomas Arendt
Received: 28 October 2008 / Accepted: 19 December 2008 / Published online: 28 January 2009 Ó Springer-Verlag 2009
Abstract In Alzheimer’s disease and related disorders, hyperphosphorylation of tau is associated with an increased activity of cyclin dependent kinase 5 (cdk5). Elevated cdk5 activity is thought to be due to the formation of p25 and thereby represents a critical element in the dysregulation of tau phosphorylation under pathological conditions. However, there is still a controversy regarding the correlation of p25 generation and tau pathology. Recently, we demonstrated physiological, paired helical filament-like tau phosphorylation that reversibly occurs in hibernating mammals. Here we used this model to test whether the tau phosphorylation in hibernation is associated with the formation of p25. Analysing brain material of arctic ground squirrels and Syrian hamsters we found no evidence for a hibernation dependent generation of p25. Hence, we suppose that phosphorylation of tau does not require the formation of p25. Instead we suggest that the truncation of p35 to p25 represents a characteristic of pathological alterations and may contribute to aggregation and deposition of hyperphosphorylated tau. Keywords Tau phosphorylation Hibernation Spermophilus parryii Mesocricetus auratus cdk5 p25
J. T. Stieler (&) T. Bullmann T. Arendt Department of Molecular and Cellular Mechanisms of Neurodegeneration, Paul Flechsig Institute of Brain Research, University of Leipzig, Jahnallee 59, 04109 Leipzig, Germany e-mail:
[email protected] F. Kohl B. M. Barnes Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK, USA
Introduction An increase of cyclin dependent kinase 5 (cdk5) activity is suggested as a key element of neurofibrillary degeneration in Alzheimer’s disease (AD) and related disorders (Noble et al. 2003; Cruz and Tsai 2004). Cdk5 is a proline-directed, serine/threonin kinase that is ubiquitously expressed in mammalian tissues. However, kinase activity is predominantly restricted to the brain as its main activators, p35 and p39, are expressed primarily in neurons (Lew et al. 1995). Cdk5 phosphorylates a large number of proteins involved in a variety of cellular processes (Dhariwala and Rajadhyaksha 2008). It plays an important role in regulation of neuronal migration (Ohshima et al. 1996; Chae et al. 1997), neurite growth (Nikolic et al. 1996; Paglini et al. 1998), and synaptic function (Floyd et al. 2001; Fischer et al. 2003). Dysregulation of cdk5 activity has been linked to pathological mechanisms. In this model, the truncation of p35 to p25 represents a pivotal event. Induced by a disturbed calcium homoeostasis, calpain, the calciumdependent cystein protease, cleaves off the N-terminal domain of p35 to form p25 (Lee et al. 2000). In contrast to p35, p25 shows an increased half-life time and an elevated affinity to cdk5. Furthermore, it lacks a myristylation signal responsible for the membrane-association of p35 (Patrick et al. 1999). As a result the enzymatic cdk5–p25 complex exhibits an increased, prolonged, and locally altered kinase activity that results hyperphosphorylation of tau, the formation of paired helical filaments and neurofibrillary tangles (NFT), and neuronal apoptosis (Camins et al. 2006). However, there is still a controversy about whether the cdk5/p25 complex acts directly as the major tau kinase or triggers and regulates the tau hyperphosphorylation by activation of other kinases, or is caused by a post mortem activation of calpain.
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These questions have been hard to address because of the lack of models that demonstrate PHF-like tau phosphorylation under non-pathological, physiological conditions. Recent studies have demonstrated the formation of highly phosphorylated tau protein in the brains of hibernating mammals (Arendt et al. 2003; Ha¨rtig et al. 2007; Stieler et al. 2008; Su et al. 2008). Hibernating animals in torpor with low body temperatures and depressed metabolism express phosphorylation of brain tau protein to an extent typically seen in PHFs. Most interestingly, this tau phosphorylation is fully reversed after animals arouse to normal temperatures and metabolism. Therefore, hibernating mammals may be natural and useful model organisms to analyse the mechanisms and regulation of tau phosphorylation and dephosphorylation. In the present study we investigated whether the physiological tau phosphorylation in hibernating rodents is associated with the formation of p25.
Materials and methods Animals Arctic ground squirrels (Spermophilus parryii) trapped in summer in the Alaska Range or on the North Slope of Alaska near Toolik Lake were transferred to University of Alaska Fairbanks. In total 31 animals were included in this study. In summer, animals were housed individually at 20°C, fed Mazuri Rodent Chow, sunflower seeds, carrots and apple and provided with water. Once per week they were assessed for reproductive and molt status. At least 1 month prior to the start of hibernation, temperaturesensitive radiotransmitters (Minimitter, Inc., Sunriver OR) were implanted into the peritoneal cavity of each animal (body mass range 650–950 g). Before implant, transmitters were sealed in heat shrink tubing and triple coated in Elvax (Minimitter, Inc) creating a package weight of 17–20 g, calibrated to the nearest 0.1°C at 0 and 20°C against a precision mercury glass thermometer, and gas sterilized (Long et al. 2007). In September animals were transferred to a chamber held at ?2°C with an 8:16 h light dark cycle where they entered hibernation. Animals were compared at five stages during their annual cycle. Euthermic animals (A; n = 9) were sampled in May and June after ending hibernation and having gone through their reproductive phase and entered spring molt. Animals in hibernation were sampled after 3–5 days in a torpor bout (early torpor, B n = 5) and after 10–12 days in a torpor bout (late torpor, C; n = 5). Animals that naturally aroused from prolonged torpor were sampled 2–3 h (early arousal D; n = 7) and 10–12 h (late arousal E; n = 5) after body temperature rose above 30°C as indicated by the
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radiotransmitter. For sampling, summer active and naturally aroused animals were anesthetized with isoflurane, euthanized with sodium pentobarbital and decapitated. Torpid animals were euthanized by decapitation after verification of core body temperatures \7°C. Brains were removed from the skull, rapidly dissected, frozen in liquid nitrogen, and stored at -80°C. Animal protocols were approved by the University of Alaska Fairbanks Institutional Animal Care and Use Committee (IACUC 06-06, 06-25). Male and female Syrian hamsters (Mesocritecetus auratus) purchased from Harkan were bred and housed at the Medizinisch Theoretisches Zentrum of the Medical Faculty of the University of Leipzig. The animals had free access to food and water and were maintained on an artificial 12:12 h light dark cycle under conditions of constant temperature (22°C) and humidity. All experimental procedures on animals were carried out in accordance with the European Council Directive of November 24th 1986 (86/609/EEC) and had been approved by the local authorities (T74/05). Twenty-five animals were subjected to hibernation conditions (Ueda and Ibuka, 1995; Oklejewicz et al. 2001). Briefly, they were transferred to the Paul Flechsig Institute where they were maintained in an animal incubator (8:16 h light dark cycle; 23–26°C) for 4–8 weeks and then in the cold room (4:20 h light dark cycle; 5–7°C). General locomotor activity was monitored with custom build infrared detectors mounted on top of each cage allowing the discrimination between euthermic phases and torpor. Hibernating animals show torpor phases (over 24 h of inactivity) whereas non-hibernating euthermic animals did not. The status of the animals was confirmed by body temperature measurements (rectal), ranging between *7°C for hibernating animals and *34°C for euthermic hamsters (A; n = 5). For sampling hamsters that have shown torpor at least three times were designated depending on their time of inactivity after an arousal episode as torpor early (B; 8 h of inactivity; n = 5) or torpor late (C; 36–48 h of inactivity; n = 5), according to the time after induction of arousal as arousal early (D; 2.5 h; n = 5) and arousal late (E; 24–36 h; n = 5). Animals aged approximately 11 month when they were killed by CO2 and decapitated. Subsequently the brain was removed, dissected, immediately frozen in liquid nitrogen and stored at -80°C. Protein extraction and western blot Frozen brain samples were homogenized in nine volumes (w/v) protein extraction buffer D (20 mM Tris–HCl [pH 7.2]; 150 mM NaCl; 2 mM MgCl2; 2 mM EDTA; 2 mM EGTA; 1%NP40, 1 mM activated Na3VO4; 5 mM
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NaF; 1 mM PMSF; 1 lg/ml leupeptin; Complete protease inhibitor [Roche Diagnostics GmbH]) using Ultra Turrax (Polytron). Homogenates were centrifuged 30 min at 50,000g and 4°C and supernatants were removed. To analyse subcellular alterations of p35 and p25, frozen brain samples were sequentially homogenized in nine volumes (w/v) protein extraction buffer W (protein extraction buffer D without the detergent NP40) using Ultra Turrax (Polytron). Homogenates were centrifuged as described above and supernatants representing the water-soluble protein fraction were removed. The pellets were consequently homogenised in six volumes protein extraction buffer D, centrifuged as described above and supernatants representing the detergent-soluble protein fraction were removed. Protein concentration was determined according to (Bradford 1976). Samples were adjusted to a final protein concentration of 1 mg/ml, separated in SDS-polyacrylamide gels (10%) and subsequently transferred to a PVDF-membrane (Polyscreen, Perkin Elmer). Blots were treated with blocking buffer (TBS, 2% BSA, 0.05% Tween 20) at room temperature and probed with primary antibodies (p35 (C-19), Santa Cruz Biotechnologies [sc 820], 1:500; AT180, Pierce [MN1040], 1:1,000; anti-beta-actin, Sigma–Aldrich [A5316], 1:20,000 diluted in blocking buffer each). The antibody p35 (C-19) is able to bind both, p35 and p25 (Kamei et al. 2007; Fischer et al. 2005). Detection of bound primary antibody was performed with HRP-conjugated secondary antibodies (donkey-anti-rabbit, GE Healthcare [NA934 V], 1:10,000; sheep-anti-mouse, GE Healthcare [NA931 V], 1:10,000 diluted in blocking buffer each). Immunoreactivity was detected by enhanced chemiluminescence (0.23 mg/ml luminol, 0.1 mg/ml p-coumaric acid and 0.6 mg/ml sodium perborate in 0.1 M Tris–HCL [pH 8.6]) and acquired with KODAK Image Station 2000R. Prior to detection of the reference antigen blots were stripped (0.2 M glycine, pH 2.1; 1% Tween 20; 0.1% SDS) 2 h at room temperature and subsequent immunoreaction was performed as described above.
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during hibernation. Major p35 immunoreactivity in the detergent-soluble protein fraction occurred as a band at about 40 kDa, indicating a slightly higher molecular weight of p35 in the analysed species compared to human p35.
Discussion Tau is an axonally located protein (Binder et al. 1985) that binds to microtubules and thereby promotes its assembly and stability (Cleveland et al. 1977). The binding capacity of tau is predominantly regulated by its phosphorylation, with elevated levels leading to decreased microtubule affinity (Biernat et al. 1993; Bramblett et al. 1993; Goedert et al. 1992). The interaction between tau and microtubules is a dynamic process in structural remodelling of the cytoskeleton during neuronal plasticity (Samsonov et al. 2004). However, a variety of neurological disorders, called tauopathies, are characterised by the development of
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Results Tau phosphorylation, as indicated by AT180-immunoreactivity, increased substantially during torpor in both arctic ground squirrels and Syrian hamsters and then was reversed shortly following their arousal to normal body temperatures (Fig. 1). However, this PHF-like tau phosphorylation was not associated with a formation of p25 prior to or during hibernation. Water-soluble and detergentsoluble protein fractions of neocortical extracts analysed separately by western blot showed no signal using a C-terminal directed p35 antibody that is able to bind both p35 and p25 (Fig. 2). Levels of p35 did not change
Fig. 1 PHF-like tau phosphorylation in hibernating animals. The phosphorylation degree of tau protein was determined in arctic ground squirrels (Spermophilus parryii) (a) and Syrian hamsters (Mesocricetus auratus) (b) using the phospho-site specific antibody AT180 (upper panel; lanes: A euthermic animals, B early torpor, C late torpor, D early arousal, E late arousal). Data show highly phosphorylated tau in the states of torpor in both species. The lower panel shows the detection of b-actin representing the internal loading control. The approximate molecular weights in kDa are indicated on the left of the according panel
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Fig. 2 Analysis of a potential hibernation-related formation of p25 in arctic ground squirrels (Spermophilus parryii) (a) and Syrian hamsters (Mesocricetus auratus) (b). The C-terminal directed p35 antibody (p35 C-19) was used to determine a potential truncation of p35 to p25. Since p25 lacks a myristylation signal responsible for the membrane-association of p35 we analysed the water-soluble (upper panel) as well as the detergent-soluble protein fraction (lower panel) of neocortical brain extracts (lanes: A euthermic animals, B early torpor, C late torpor, D early arousal, E late arousal). We found no evidence for a state related formation of p25. No change of p35 expression was observed during hibernation. The approximate molecular weights in kDa are indicated on the left of the according panel
intracellular deposits of phosphorylated tau protein. In AD hyperphosphorylated tau is found aggregated to paired helical filaments in NFT, and the phosphorylation of tau protein may be an early event in the pathogenesis of AD and a key factor in neurofibrillary degeneration (Lee et al. 2001). Recent studies have shown paired helical filamentlike tau phosphorylation under physiological conditions in hibernating mammals (Arendt et al. 2003; Ha¨rtig et al. 2007; Stieler et al. 2008; Su et al. 2008). Hibernation is an adaptive process that represents a powerful physiological strategy of endotherms to compensate for periodically limited energy supply (Heldmaier et al. 2004). During hibernation animals enter a hypometabolic state (torpor) that is characterised by a substantial reduction of basal metabolic rate followed by a decline of body temperature that can attain values below the freezing point (Barnes 1989). In the state of torpor these animals show increased tau phosphorylation levels that rapidly reverse after
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arousal. The elevated AT180 immunoreactivity is not the result of an altered tau protein expression as the application of a phospho-independent antibody revealed no differences (data not shown). This finding is consistent with previous studies demonstrating that tau expression is not changed during hibernation (Arendt et al. 2003, Ha¨rtig et al. 2007, Stieler et al. 2008; Su et al. 2008). Consequently, hibernation represents a model suited to analyse the regulation of physiological tau phosphorylation. As the mechanisms that underlie neurofibrillary degeneration remain largely unknown the hibernation model potentially allows the discrimination between disease related physiological and pathological aspects in the pathogenesis of tauopathies. Tau phosphorylation is regulated by a variety of kinases including glycogen synthase kinase 3 beta (GSK3 beta) (Ishiguro et al. 1988, 1993), stress-activated protein kinase/ Jun-amino-terminal kinase (SAPK/JNK) (Goedert et al. 1997; Reynolds et al. 1997), mitogen activated protein kinases (MAPK) (Drewes et al. 1992), and cdk5 (Kobayashi et al. 1993; Baumann et al. 1993). Nevertheless, none of these enzymes give rise to PHF-tau on its own (ZhengFischhofer et al. 1998). Among these kinases, cdk5 has been identified as a key enzyme in regulation of tau phosphorylation in AD (Hisanaga and Saito 2003; Camins et al. 2006). It was shown that cdk5 operates as priming kinase responsible for initialising tau phosphorylation consequently triggering tau hyperphosphorylation by e.g. GSK3 beta (Li et al. 2006). Increased cdk5 activity may result from the calpain-induced cleavage of its regulative subunit p35. Consequently, the proteolytic fragment p25 shows an elevated affinity to cdk5, increased half-life time and lacks a myristylation signal responsible for the membrane-association of p35. As a result the cdk5–p25 complex exhibits an increased, prolonged and locally altered activity. However, there is still a controversy whether the formation of p25 is a characteristic of, or even a prerequisite for tau hyperphosphorylation in tauopathies. While initial studies showed a strong and reproducible association between the generation of p25 and tau pathology (Patrick et al. 1999, 2001; Tseng et al. 2002) others found no evidence for a potential correlation (Yoo and Lubec 2001; Tandon et al. 2003). Furthermore, studies of transgenic mice overexpressing p25 could not demonstrate a clear coherence of increased p25 levels and tau phosphorylation although the majority of p25-transgenic mice show a hyperphosphorylation of tau protein (Giese et al. 2005). In the present study, we analysed whether the hibernation-related phosphorylation of tau protein is associated with the formation of p25. The study was performed with arctic ground squirrels (Spermophilus parryii) and Syrian hamsters (Mesocricetus auratus). These species belong to
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the order rodentia but are members of different suborders. Regardless of the phylogenetic distance, both species show reversible, PHF-like tau phosphorylation during hibernation supporting the theory that phosphorylation of tau is a common characteristic of hibernation. Our results demonstrate that the PHF-like tau phosphorylation itself does not require the truncation of p35 to p25. Even the putative pathological sites, such as AT8 and AT180, were phosphorylated in absence of p25. However, this fact does not imply that cdk5 is not involved in the regulation of physiological tau phosphorylation during hibernation. Since both investigated species revealed similar results we suppose that physiological tau phosphorylation underlies analogous mechanisms that are generally not associated with a p25-evoked increase of cdk5 activity. Therefore, the calpain-mediated cleavage of p35 potentially represents a characteristic of pathophysiological alterations in tauopathies like AD that may contribute to tau phosphorylation but is rather associated with aggregation and the generation of PHF’s. In this context an increased cdk5 activity resulting from the binding to p25 may regulate mechanisms promoting the formation of NFT and neuronal cell death. Acknowledgements We thank Sabine Seifert for technical assistance. This study was supported by grants from the US Army Medical Research and Materiel Command (Proposal No. 05178001) and the NSF (0076039) to B.M. Barnes.
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