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Nandrolone-induced hippocampal phosphorylation of NMDA receptor subunits and ERKs
Biochemical and Biophysical Research Communications 357 (2007) 1028–1033 www.elsevier.com/locate/ybbrc
Nandrolone-induced hippocampal phosphorylation of NMDA receptor subunits and ERKs Uwe L.W. Rossbach a, Pia Steensland a, Fred Nyberg a, Pierre Le Greve`s a
b,*
Department of Pharmaceutical Bioscience, Division of Biological Research on Drug Dependence, Uppsala University, BMC, Box 591, S-751 24 Uppsala, Sweden b Department of Neuroscience, Division of Neurobiology, Uppsala University, BMC, Box 587, S-751 23 Uppsala, Sweden Received 5 April 2007 Available online 17 April 2007
Abstract The age-related decline in gonadal steroids is associated with changes in mood and memory function. It appears that normal physiological concentrations of the steroids are required for adequate synaptic plasticity. However, the effects of high levels of androgens subsequent to misuse of anabolic androgenic steroids (AAS) are largely unknown. In this study, rats were given i.m. nandrolone as a single dose or daily for 14 days and the effects on synaptic components in hippocampal synaptoneurosomes were measured 24 h after the last injection. Western blot analysis revealed that a single injection of AAS increased phosphorylation of the NMDA receptor subunits NR2A and NR2B and ERK1/2, while the levels of phosphorylated CaMKIIa were unaltered. No changes were seen in other synaptic proteins tested, i.e., BDNF, Arc, TUC-4, and b-tubulin III. Daily administration of nandrolone for 2 weeks did not affect the content of any of the proteins tested. From this in vivo study, it is concluded that important synaptic components respond to a single high dose of nandrolone, an effect that may influence synapse function. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Anabolic androgenic steroids; Nandrolone; Phosphorylation; ERK; NMDA receptor subunits; Hippocampus; Rat; Western blot
In recent years, the role of gonadal steroids in cognitive performance has received increasing attention. This heightened interest is mainly the result of the discoveries that reduced memory function is correlated with age-dependent loss of the hormones, and that replacement hormone therapy has been reported to restore some of the dysfunctions [1]. Several studies have demonstrated that androgens and estrogens regulate the function and morphology of the hippocampus [2], an important brain area for cognitive processes. Learning and memory are highly dependent on synaptic plasticity, which involves structural changes in neurons and synapses. The underlying mechanisms, believed to be longterm potentiation (LTP) and long-term depression (LTD), occur in certain brain structures, of which excitatory syn*
Corresponding author. Fax: +46 18 559017. E-mail address: [email protected] (P. Le Greve`s).
0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.04.037
apses in the hippocampus are of particular importance. The glutamate receptor N-methyl-D-aspartate subtype (NMDAR) plays a crucial role in LTP and LTD. Activation of the receptor results in increased concentrations of intracellular calcium which in turn regulates downstream signaling pathways such as the mitogen-activated protein kinase (MAPK) pathway, which involves phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) [3]. There is now convincing evidence that many forms of synaptic plasticity require ERK activation [4]. Among the gonadal steroids, estrogens have a well documented impact on morphological and biochemical components at hippocampal synapses, affecting cognition, mood, and memory (for review see [2]). The hormone increases NMDAR excitatory activity in rat hippocampal slices [5] and its stimulatory effect on MAPK and src tyrosine kinase pathways has been extensively studied and reviewed [6]. The effects of androgens in this respect have
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been less well studied; however, dihydrotestosterone (DHT) is known to increase glutamate binding to the NMDAR in the CA1 region of the male rat hippocampus [7] and androgens can rapidly phosphorylate ERK in cultured embryonic hippocampal neurons [8]. Misuse of anabolic androgenic steroids (AAS) results in supraphysiological levels of androgens that may affect mood and aggression [9]. In view of the regulatory role of gonadal hormones in synaptic plasticity, it is of interest to examine the impact of high doses of AAS on synaptic correlates. We have previously reported that repeated administration of AAS affect the gene regulation of NMDAR subunits in different brain areas [10,11]. In this study, we investigated the in vivo effects of supratherapeutic doses of AAS on selected proteins in rat hippocampal synaptoneurosomes. The content and phosphorylated (p) states of ERK, calcium/calmodulin-dependent protein kinase IIa (CaMKIIa) and the NMDAR subunits NR2A and NR2B were determined. Other components implicated in synaptic plasticity, differentiation or proliferation (brainderived neurotrophic factor (BDNF), b-tubulin III, activity-regulated cytoskeleton-associated protein (Arc) and TOAD/Ulip/CRMP (TUC-4)) were also studied. Materials and methods
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was filtered through two layers of 80 lm nylon net filter disks (NY8002500, Millipore) and the filtrates were then passed through a 5 lm DuraporeÒ membrane (SVLP01300, Millipore). The filtered solutions were centrifuged at 1000g for 15 min at 4 °C, the pellets were resuspended in five volumes of homogenisation buffer and aliquots were kept at 80 °C. The protein concentration was determined by use of a commercial kit (2-D Quant Kit, GE Healthcare). Electrophoresis was used to resolve 10–20 lg of protein on 10–15% polyacrylamide gels (depending on the protein studied) in the presence of 0.1% sodium dodecyl sulphate (SDS) and the protein components were then transferred to a 0.45 lm nitrocellulose membrane (Amersham HybondÒ-ECL, GE Healthcare). Blots were incubated with specific antibodies recognising the following proteins: ERK1/2 (#9102, 1:3000, New England Biolabs) and p-ERK1/2 (1:3000, #9101, New England Biolabs); TUC-4 (1:40000, AB5454, Chemicon); CaMKIIa (1:3000, NB-12, Calbiochem); p-CaMKIIa (1:10000, NB-13, Calbiochem); Arc (1:2000, #612602, BD Biosciences); b-tubulin III (1:30000, T2200, Sigma–Aldrich); p-NR2A (Ser1232) (1:10000, #2056, Tocris Bioscience); p-NR2B (Tyr1472) (1:3000, SIG-9063, Signet Laboratories); BDNF (1:2000, sc-546, Santa Cruz), NR2A (1:20000, sc-1468, Santa Cruz), NR2B (1:10000, sc-1469, Santa Cruz). Blots were probed for actin (anti-actin, 1:3000, sc-1615, Santa Cruz) which was used for the normalisation of protein loaded. The preparations were subsequently incubated with the corresponding secondary horseradish peroxidase conjugated antibodies (Santa Cruz). The immunoblots were visualised using enhanced chemiluminescence reagent (ECL Detection Reagent, GE Healthcare) and exposed to Cronex 5 light-sensitive film (Agfa Gevert). ImageJ 1.34p software (NIH, USA) was used to quantify the band density of the scanned films. When stripped, the filters were washed with a buffer containing 100 mM 2-mercaptoethanol, 2% SDS and 62.5 mM Tris–HCl, pH 6.8, at 50 °C for 20 min.
Animal experiments
Data analysis and statistics
The procedure was approved by the local experimental animal committee. Male Sprague–Dawley rats (Alab, Sollentuna, Sweden), weighing 225–250 g, were housed in air-ventilated rooms (humidity 50–60%, temperature 22–24 °C) under a 12 h-dark/12 h-light cycle with food and water provided ad libitum. The study comprised two separate experiments in which the rats received intramuscular (i.m.) injections of nandrolone either as a single dose or daily over 2 weeks. Experiment 1. The animals were randomly divided into two groups, each consisting of ten rats. The first (control) group was given an oilvehicle injection. The second group was administered one dose of nandrolone decanoate in sterile arachidis oleum (Deca-DurabolÒ Organon, Oss, Netherlands) (15 mg/kg body weight). This dose (40–50 times greater than those used clinically) was chosen in order to mimic the doses used by human steroid abusers. The rats were decapitated 24 h after administration of the drug (at peak plasma concentrations [12]) and the hippocampus was rapidly dissected out on ice, using a rat brain matrix (Activational System Inc., Mortella Drive Warren, MI, USA), and placed on dry ice. The tissues were kept at 80 °C until further processing. Experiment 2. Over a period of 14 days, one group of eight animals was injected daily with oil-vehicle while a second group of eight rats received daily injections of nandrolone decanoate (15 mg/kg body weight). The animals were decapitated on day 15 and dissected as above.
Total and phosphorylated proteins of ERK1/2, NR2A, NR2B, and CaMKIIa were densitometrically determined. The ratio of phosphorylated to total protein was calculated for each individual animal. The mean values from the treated groups were expressed as a percentage of the mean value from the respective control group. Data are expressed as mean ± SEM. Differences between means were calculated using the paired or unpaired Student’s t-test (as appropriate) as indicated in the figures. P < 0.05 was considered to be statistically significant.
Protein preparation and analysis A preparation of rat synaptoneurosomes, enriched for synaptic components, was made according to the method of Hollingsworth et al. [13]. In brief, using a Teflon pestle homogeniser, individual rat hippocampi were homogenised in six volumes of ice-cold buffer pH 7.4 (Hepes 50 mM, NaCl 124 mM, NaHCO3 26 mM, glucose 10 mM, MgCl2 1.3 mM, CaCl2 2.5 mM, KCl 3.2 mM, KH2PO4 1.06 mM, chloramphenicol 0.7 mM, saturated with 95% O2/5% CO2) containing various inhibitors. The homogenate was diluted, mixed with an additional six volumes of the ice-cold homogenisation buffer and incubated on ice for 10 min. The preparation
Results Western blot analysis revealed that both the NR2A (Fig. 1A) and NR2B (Fig. 2A) subunits of the NMDAR showed significant increases in phosphorylation (p-NR2A (Ser1232); 137.3 ± 13.4%, P < 0.05; p-NR2B (Tyr1472); 132.6 ± 7.8%, P < 0.05; unpaired Student’s t-test) 24 h after a single dose of nandrolone decanoate when compared to results in control animals receiving oil-vehicle. Phosphorylation of these receptor subunits in animals receiving 14 days’ administration was not significantly different from that in the control group (p-NR2A (Ser1232); 107.6 ± 16.18%, P = 0.64; p-NR2B (Tyr1472); 100.7 ± 2.95%, P = 0.92; unpaired Student’s t-test) (Figs. 1A and 2A, respectively). The content of total NR2A or total NR2B was not changed in any of the animal groups (Figs. 1B and 2B). Twenty-four hours after the single dose, p-ERK1 and pERK2 were significantly elevated compared with those in the control group (144.8 ± 4.1%, P < 0.001 and 116.3 ± 4.3%, P < 0.01, respectively; unpaired Student’s
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Fig. 1. The effects of nandrolone decanoate on the phosphorylation of the NMDA receptor subunits. (A) p-NR2A (Ser1232) and (B) NR2A. In the single-dose experiment, animals (n = 10/group) received an i.m. injection of 15 mg/kg nandrolone decanoate or oil-vehicle (control) and were sacrificed after 24 h. Multiple-dose consisted of daily injections of 15 mg/ kg nandrolone decanoate or oil-vehicle (control) for 14 days and the rats (n = 8/group) were sacrificed 24 h after the last injection. Representative immunoblots for each group of rats are shown (top). The values are means ± SEM. *P < 0.05; unpaired Student’s t-test.
Fig. 3. The effects of nandrolone decanoate on (A) p-ERK1/ERK1 and (B) p-ERK2/ERK2. In the single-dose experiment, animals (n = 10/ group) received an i.m. injection of 15 mg/kg nandrolone decanoate or oilvehicle (control) and were sacrificed after 24 h. Multiple-dose consisted of daily injections of 15 mg/kg nandrolone decanoate or oil-vehicle (control) for 14 days and the rats (n = 8/group) were sacrificed 24 h after the last injection. Representative immunoblots for each group of rats are shown (top). The values are means ± SEM of two independently conducted Western blot detections. **P < 0.01; ***P < 0.001 versus control; unpaired Student’s t-test.
of the steroid, a tendency towards slight decreases in the phosphorylation of ERK was noted, especially for pERK2 (p-ERK1: 92.3 ± 7.8%, P = 0.51; p-ERK2: 85.6 ± 4.1%, P = 0.06; unpaired Student’s t-test), although the difference was not quite statistically significant (Fig. 3). Neither single- nor multiple-dose administration of nandrolone significantly affected any of the other proteins tested (Fig. 4). Discussion
Fig. 2. The effects of nandrolone decanoate on the phosphorylation of the NMDA receptor subunits. (A) p-NR2B (Tyr1472) and (B) NR2B. In the single-dose experiment, animals (n = 10/group) received an i.m. injection of 15 mg/kg nandrolone decanoate or oil-vehicle (control) and were sacrificed after 24 h. Multiple-dose consisted of daily injections of 15 mg/ kg nandrolone decanoate or oil-vehicle (control) for 14 days and the rats (n = 8/group) were sacrificed 24 h after the last injection. Representative immunoblots for each group of rats are shown (top). The values are means ± SEM. *P < 0.05; unpaired Student’s t-test.
t-test) (Fig. 3). The effect on p-ERK1 levels was significantly greater than that on p-ERK2 levels (P < 0.001, paired Student’s t-test). After 14 days of daily injections
These findings indicate that a single high dose of the AAS nandrolone can affect important synaptic components involved in hippocampal plasticity. This is, to our knowledge, the first study to demonstrate in vivo effects of an androgenic steroid on the phosphorylation of NMDAR and ERK in the rat hippocampus. The increase in phosphorylation appeared 24 h after a single dose of the steroid. None of the proteins studied were significantly altered by 14 days’ administration. A role for androgens in cognitive performance and neurogenesis associated with the hippocampus has recently been suggested. We determined the effect of relatively high doses of nandrolone on proteins located in synaptoneurosomes, consisting of a sub-cellular compartment enriched with synaptic elements. This preparation is suitable for studies of the synaptic events underlying synaptic plasticity, including regulation of proteins and downstream
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Fig. 4. The effects of nandrolone decanoate on (A) p-CaMKIIa/ CaMKIIa, (B) Arc, (C) BDNF, (D) TUC-4, and (E) b-tubulin III (b-T III). In the single-dose experiment, animals (n = 10/group) received an i.m. injection of 15 mg/kg nandrolone decanoate (filled boxes) or oilvehicle (control, open boxes) and were sacrificed after 24 h. Multiple-dose consisted of daily injections of 15 mg/kg nandrolone decanoate (filled boxes) or oil-vehicle (control, open boxes) for 14 days and the rats (n = 8/ group) were sacrificed 24 h after the last injection. Representative immunoblots for each group of rats are shown (top). The values are means ± SEM.
signaling pathways in close proximity to the synapse. Changes in synaptic strength are regulated through phosphorylation of synaptic proteins, of which NMDAR is of particular importance. The activity of this protein is regulated by protein kinases and, in the hippocampus, this is closely associated with the formation of LTP and LTD (for review see [14]). By using specific antibodies against
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the NR2A subunit phosphorylated at Ser1232 or the NR2B at Tyr1472, we detected increased phosphorylation at these sites after a single injection of nandrolone. Phosphorylation of NR2A (Ser1232) by cyclin-dependent kinase 5 (Cdk5) in hippocampal CA1 neurons is reported to regulate NMDA-induced LTP [15] and mediate Cdk5-induced cell death [16]. In fact, it was recently shown that high levels of testosterone induce apoptosis in the neuroblastoma cell line SH-SY5Y [17]. Tyr1472 is the main NR2B phosphorylation site in vitro. It is phosphorylated by the src family member Fyn tyrosine kinase, reportedly with LTP [18], resulting in blockade of NMDAR internalisation [19]. Thus, decreased internalisation would be expected to result in increased NR2B-containing receptors at the membrane. However, the elevated phosphorylation of Tyr1472 observed in this study was not reflected in higher NR2B subunit content. Thus, the androgen-induced increase of phosphorylated NMDAR subunits indicates stimulated glutaminergic signaling in hippocampal synapses. A single injection of nandrolone increased ERK phosphorylation in the synaptoneurosomal preparation. Active ERK is mostly associated with transcriptional regulation in the cell nucleus; however, proteomic analysis has also revealed its presence in synaptoneurosomes [20] where ERK-dependent phosphorylation and protein synthesis modulate synaptic plasticity locally [21]. BDNF can activate ERK in hippocampal synaptic sub-cellular preparations [22]. However, 24 h after a single dose of nandrolone, there were no changes in BDNF in our material, which indicates that the increased ERK phosphorylation may have been associated with potentiated NMDAR function following its phosphorylation. NMDAR-stimulated ERK activation can be mediated through a mechanism involving inhibition by active (phosphorylated) CaMKII of the MAPK pathway inhibitor SynGAP (a Ras-GTPase-activating protein) [23]. CaMKII and SynGAP are present in the postsynaptic density [24], a subfraction of the synaptoneurosomal preparations. However, the increased ERK phosphorylation seen after in this study was not linked to a simultaneous elevation of p-CaMKIIa suggesting an alternative pathway(s) for ERK activation in synaptoneurosomes after androgen stimulation. Androgens have been ascribed a neurotrophic role modulating synaptic plasticity in the hippocampus [2]. The activity-regulated gene Arc is utilised as a presumed correlate of informational processing and plasticity in the brain [25]. Since its expression depends on NMDAR and ERK activation, as shown in primary cultures of hippocampal neurons [26], we investigated whether Arc might be an effector of the nandrolone-induced activation of this signaling cascade. The protein was present and clearly detectable in the hippocampal synaptoneurosomes; however, Arc levels were not altered by either treatment regimen. Supraphysiological levels of nandrolone have been shown to decrease proliferation and differentiation of neuronal stem cells in the rat dentate gyrus [27]. ERK activation stimulates these processes [28], but conflicting results
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have been reported regarding the role of NMDAR activation [29]. TUC-4 and b-tubulin III are expressed in early [30] and late [31] postmitotic neurons, respectively. Both proteins are utilised as markers for proliferation and differentiation, and the expression of b-tubulin III has recently been demonstrated to be ERK dependent in hippocampal neuronal progenitor cells [32]. However, we did not detect any nandrolone-induced changes in the content of these proteins. Neurogenesis is restricted to the dentate gyrus (DG), an area constituting approx. 20% of the hippocampal volume [33]. One could argue against analysing DG-specific events in a preparation obtained from whole hippocampus, due to the dilution effect. Nevertheless, both TUC-4 and b-tubulin III were clearly visualised in our Western blot analysis of synaptoneurosomal proteins. AAS in humans are reported to acutely influence mood, confidence and fear, effects that are sometimes exploited by criminals to prime themselves prior to a violent act such as a bank robbery [34]. In view of our results from this study, it is interesting to note that both NR2B (Tyr1472) and ERK phosphorylation are involved in the response to fear [35,36]. In conclusion, we have demonstrated that a single high dose of the AAS nandrolone elicits significant in vivo phosphorylation of both NMDAR and ERK1/2 in hippocampal synaptoneurosomes. These effects were not seen after a 2-week treatment period, indicating adaptation to high steroid levels. We did not detect any changes in BDNF or p-CaMKIIa levels, which suggest that these signaling components were not involved in the effects of the single injected dose of nandrolone. Moreover, we found no changes in effectors of NMDAR or ERK activation, such as the proteins associated with synaptic plasticity (Arc) or proliferation and differentiation (TUC-4, b-tubulin III). Additional studies should be carried out to further disclose the impact of nandrolone-induced NMDAR and ERK phosphorylation and how these effects are mediated. Acknowledgments This study was supported by the Swedish Research Council (Grant 9459). We thank Madeleine Le Greve`s (supported by Torsten och Ragnar So¨derbergs Stiftelser) for supplying us with antibodies. References [1] O.T. Wolf, Cognitive functions and sex steroids, Ann. Endocrinol. (Paris) 64 (2003) 158–161. [2] A. Parducz, T. Hajszan, N.J. Maclusky, Z. Hoyk, E. Csakvari, A. Kurunczi, J. Prange-Kiel, C. Leranth, Synaptic remodeling induced by gonadal hormones: neuronal plasticity as a mediator of neuroendocrine and behavioral responses to steroids, Neuroscience 138 (2006) 977–985. [3] L.B. Rosen, D.D. Ginty, M.J. Weber, M.E. Greenberg, Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras, Neuron 12 (1994) 1207–1221. [4] G.M. Thomas, R.L. Huganir, MAPK cascade signalling and synaptic plasticity, Nat. Rev. Neurosci. 5 (2004) 173–183.
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Nandrolone-induced hippocampal phosphorylation of NMDA receptor subunits and ERKs Uwe L.W. Rossbach a, Pia Steensland a, Fred Nyberg a, Pierre Le Greve`s a
b,*
Department of Pharmaceutical Bioscience, Division of Biological Research on Drug Dependence, Uppsala University, BMC, Box 591, S-751 24 Uppsala, Sweden b Department of Neuroscience, Division of Neurobiology, Uppsala University, BMC, Box 587, S-751 23 Uppsala, Sweden Received 5 April 2007 Available online 17 April 2007
Abstract The age-related decline in gonadal steroids is associated with changes in mood and memory function. It appears that normal physiological concentrations of the steroids are required for adequate synaptic plasticity. However, the effects of high levels of androgens subsequent to misuse of anabolic androgenic steroids (AAS) are largely unknown. In this study, rats were given i.m. nandrolone as a single dose or daily for 14 days and the effects on synaptic components in hippocampal synaptoneurosomes were measured 24 h after the last injection. Western blot analysis revealed that a single injection of AAS increased phosphorylation of the NMDA receptor subunits NR2A and NR2B and ERK1/2, while the levels of phosphorylated CaMKIIa were unaltered. No changes were seen in other synaptic proteins tested, i.e., BDNF, Arc, TUC-4, and b-tubulin III. Daily administration of nandrolone for 2 weeks did not affect the content of any of the proteins tested. From this in vivo study, it is concluded that important synaptic components respond to a single high dose of nandrolone, an effect that may influence synapse function. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Anabolic androgenic steroids; Nandrolone; Phosphorylation; ERK; NMDA receptor subunits; Hippocampus; Rat; Western blot
In recent years, the role of gonadal steroids in cognitive performance has received increasing attention. This heightened interest is mainly the result of the discoveries that reduced memory function is correlated with age-dependent loss of the hormones, and that replacement hormone therapy has been reported to restore some of the dysfunctions [1]. Several studies have demonstrated that androgens and estrogens regulate the function and morphology of the hippocampus [2], an important brain area for cognitive processes. Learning and memory are highly dependent on synaptic plasticity, which involves structural changes in neurons and synapses. The underlying mechanisms, believed to be longterm potentiation (LTP) and long-term depression (LTD), occur in certain brain structures, of which excitatory syn*
Corresponding author. Fax: +46 18 559017. E-mail address: [email protected] (P. Le Greve`s).
0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.04.037
apses in the hippocampus are of particular importance. The glutamate receptor N-methyl-D-aspartate subtype (NMDAR) plays a crucial role in LTP and LTD. Activation of the receptor results in increased concentrations of intracellular calcium which in turn regulates downstream signaling pathways such as the mitogen-activated protein kinase (MAPK) pathway, which involves phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) [3]. There is now convincing evidence that many forms of synaptic plasticity require ERK activation [4]. Among the gonadal steroids, estrogens have a well documented impact on morphological and biochemical components at hippocampal synapses, affecting cognition, mood, and memory (for review see [2]). The hormone increases NMDAR excitatory activity in rat hippocampal slices [5] and its stimulatory effect on MAPK and src tyrosine kinase pathways has been extensively studied and reviewed [6]. The effects of androgens in this respect have
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been less well studied; however, dihydrotestosterone (DHT) is known to increase glutamate binding to the NMDAR in the CA1 region of the male rat hippocampus [7] and androgens can rapidly phosphorylate ERK in cultured embryonic hippocampal neurons [8]. Misuse of anabolic androgenic steroids (AAS) results in supraphysiological levels of androgens that may affect mood and aggression [9]. In view of the regulatory role of gonadal hormones in synaptic plasticity, it is of interest to examine the impact of high doses of AAS on synaptic correlates. We have previously reported that repeated administration of AAS affect the gene regulation of NMDAR subunits in different brain areas [10,11]. In this study, we investigated the in vivo effects of supratherapeutic doses of AAS on selected proteins in rat hippocampal synaptoneurosomes. The content and phosphorylated (p) states of ERK, calcium/calmodulin-dependent protein kinase IIa (CaMKIIa) and the NMDAR subunits NR2A and NR2B were determined. Other components implicated in synaptic plasticity, differentiation or proliferation (brainderived neurotrophic factor (BDNF), b-tubulin III, activity-regulated cytoskeleton-associated protein (Arc) and TOAD/Ulip/CRMP (TUC-4)) were also studied. Materials and methods
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was filtered through two layers of 80 lm nylon net filter disks (NY8002500, Millipore) and the filtrates were then passed through a 5 lm DuraporeÒ membrane (SVLP01300, Millipore). The filtered solutions were centrifuged at 1000g for 15 min at 4 °C, the pellets were resuspended in five volumes of homogenisation buffer and aliquots were kept at 80 °C. The protein concentration was determined by use of a commercial kit (2-D Quant Kit, GE Healthcare). Electrophoresis was used to resolve 10–20 lg of protein on 10–15% polyacrylamide gels (depending on the protein studied) in the presence of 0.1% sodium dodecyl sulphate (SDS) and the protein components were then transferred to a 0.45 lm nitrocellulose membrane (Amersham HybondÒ-ECL, GE Healthcare). Blots were incubated with specific antibodies recognising the following proteins: ERK1/2 (#9102, 1:3000, New England Biolabs) and p-ERK1/2 (1:3000, #9101, New England Biolabs); TUC-4 (1:40000, AB5454, Chemicon); CaMKIIa (1:3000, NB-12, Calbiochem); p-CaMKIIa (1:10000, NB-13, Calbiochem); Arc (1:2000, #612602, BD Biosciences); b-tubulin III (1:30000, T2200, Sigma–Aldrich); p-NR2A (Ser1232) (1:10000, #2056, Tocris Bioscience); p-NR2B (Tyr1472) (1:3000, SIG-9063, Signet Laboratories); BDNF (1:2000, sc-546, Santa Cruz), NR2A (1:20000, sc-1468, Santa Cruz), NR2B (1:10000, sc-1469, Santa Cruz). Blots were probed for actin (anti-actin, 1:3000, sc-1615, Santa Cruz) which was used for the normalisation of protein loaded. The preparations were subsequently incubated with the corresponding secondary horseradish peroxidase conjugated antibodies (Santa Cruz). The immunoblots were visualised using enhanced chemiluminescence reagent (ECL Detection Reagent, GE Healthcare) and exposed to Cronex 5 light-sensitive film (Agfa Gevert). ImageJ 1.34p software (NIH, USA) was used to quantify the band density of the scanned films. When stripped, the filters were washed with a buffer containing 100 mM 2-mercaptoethanol, 2% SDS and 62.5 mM Tris–HCl, pH 6.8, at 50 °C for 20 min.
Animal experiments
Data analysis and statistics
The procedure was approved by the local experimental animal committee. Male Sprague–Dawley rats (Alab, Sollentuna, Sweden), weighing 225–250 g, were housed in air-ventilated rooms (humidity 50–60%, temperature 22–24 °C) under a 12 h-dark/12 h-light cycle with food and water provided ad libitum. The study comprised two separate experiments in which the rats received intramuscular (i.m.) injections of nandrolone either as a single dose or daily over 2 weeks. Experiment 1. The animals were randomly divided into two groups, each consisting of ten rats. The first (control) group was given an oilvehicle injection. The second group was administered one dose of nandrolone decanoate in sterile arachidis oleum (Deca-DurabolÒ Organon, Oss, Netherlands) (15 mg/kg body weight). This dose (40–50 times greater than those used clinically) was chosen in order to mimic the doses used by human steroid abusers. The rats were decapitated 24 h after administration of the drug (at peak plasma concentrations [12]) and the hippocampus was rapidly dissected out on ice, using a rat brain matrix (Activational System Inc., Mortella Drive Warren, MI, USA), and placed on dry ice. The tissues were kept at 80 °C until further processing. Experiment 2. Over a period of 14 days, one group of eight animals was injected daily with oil-vehicle while a second group of eight rats received daily injections of nandrolone decanoate (15 mg/kg body weight). The animals were decapitated on day 15 and dissected as above.
Total and phosphorylated proteins of ERK1/2, NR2A, NR2B, and CaMKIIa were densitometrically determined. The ratio of phosphorylated to total protein was calculated for each individual animal. The mean values from the treated groups were expressed as a percentage of the mean value from the respective control group. Data are expressed as mean ± SEM. Differences between means were calculated using the paired or unpaired Student’s t-test (as appropriate) as indicated in the figures. P < 0.05 was considered to be statistically significant.
Protein preparation and analysis A preparation of rat synaptoneurosomes, enriched for synaptic components, was made according to the method of Hollingsworth et al. [13]. In brief, using a Teflon pestle homogeniser, individual rat hippocampi were homogenised in six volumes of ice-cold buffer pH 7.4 (Hepes 50 mM, NaCl 124 mM, NaHCO3 26 mM, glucose 10 mM, MgCl2 1.3 mM, CaCl2 2.5 mM, KCl 3.2 mM, KH2PO4 1.06 mM, chloramphenicol 0.7 mM, saturated with 95% O2/5% CO2) containing various inhibitors. The homogenate was diluted, mixed with an additional six volumes of the ice-cold homogenisation buffer and incubated on ice for 10 min. The preparation
Results Western blot analysis revealed that both the NR2A (Fig. 1A) and NR2B (Fig. 2A) subunits of the NMDAR showed significant increases in phosphorylation (p-NR2A (Ser1232); 137.3 ± 13.4%, P < 0.05; p-NR2B (Tyr1472); 132.6 ± 7.8%, P < 0.05; unpaired Student’s t-test) 24 h after a single dose of nandrolone decanoate when compared to results in control animals receiving oil-vehicle. Phosphorylation of these receptor subunits in animals receiving 14 days’ administration was not significantly different from that in the control group (p-NR2A (Ser1232); 107.6 ± 16.18%, P = 0.64; p-NR2B (Tyr1472); 100.7 ± 2.95%, P = 0.92; unpaired Student’s t-test) (Figs. 1A and 2A, respectively). The content of total NR2A or total NR2B was not changed in any of the animal groups (Figs. 1B and 2B). Twenty-four hours after the single dose, p-ERK1 and pERK2 were significantly elevated compared with those in the control group (144.8 ± 4.1%, P < 0.001 and 116.3 ± 4.3%, P < 0.01, respectively; unpaired Student’s
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Fig. 1. The effects of nandrolone decanoate on the phosphorylation of the NMDA receptor subunits. (A) p-NR2A (Ser1232) and (B) NR2A. In the single-dose experiment, animals (n = 10/group) received an i.m. injection of 15 mg/kg nandrolone decanoate or oil-vehicle (control) and were sacrificed after 24 h. Multiple-dose consisted of daily injections of 15 mg/ kg nandrolone decanoate or oil-vehicle (control) for 14 days and the rats (n = 8/group) were sacrificed 24 h after the last injection. Representative immunoblots for each group of rats are shown (top). The values are means ± SEM. *P < 0.05; unpaired Student’s t-test.
Fig. 3. The effects of nandrolone decanoate on (A) p-ERK1/ERK1 and (B) p-ERK2/ERK2. In the single-dose experiment, animals (n = 10/ group) received an i.m. injection of 15 mg/kg nandrolone decanoate or oilvehicle (control) and were sacrificed after 24 h. Multiple-dose consisted of daily injections of 15 mg/kg nandrolone decanoate or oil-vehicle (control) for 14 days and the rats (n = 8/group) were sacrificed 24 h after the last injection. Representative immunoblots for each group of rats are shown (top). The values are means ± SEM of two independently conducted Western blot detections. **P < 0.01; ***P < 0.001 versus control; unpaired Student’s t-test.
of the steroid, a tendency towards slight decreases in the phosphorylation of ERK was noted, especially for pERK2 (p-ERK1: 92.3 ± 7.8%, P = 0.51; p-ERK2: 85.6 ± 4.1%, P = 0.06; unpaired Student’s t-test), although the difference was not quite statistically significant (Fig. 3). Neither single- nor multiple-dose administration of nandrolone significantly affected any of the other proteins tested (Fig. 4). Discussion
Fig. 2. The effects of nandrolone decanoate on the phosphorylation of the NMDA receptor subunits. (A) p-NR2B (Tyr1472) and (B) NR2B. In the single-dose experiment, animals (n = 10/group) received an i.m. injection of 15 mg/kg nandrolone decanoate or oil-vehicle (control) and were sacrificed after 24 h. Multiple-dose consisted of daily injections of 15 mg/ kg nandrolone decanoate or oil-vehicle (control) for 14 days and the rats (n = 8/group) were sacrificed 24 h after the last injection. Representative immunoblots for each group of rats are shown (top). The values are means ± SEM. *P < 0.05; unpaired Student’s t-test.
t-test) (Fig. 3). The effect on p-ERK1 levels was significantly greater than that on p-ERK2 levels (P < 0.001, paired Student’s t-test). After 14 days of daily injections
These findings indicate that a single high dose of the AAS nandrolone can affect important synaptic components involved in hippocampal plasticity. This is, to our knowledge, the first study to demonstrate in vivo effects of an androgenic steroid on the phosphorylation of NMDAR and ERK in the rat hippocampus. The increase in phosphorylation appeared 24 h after a single dose of the steroid. None of the proteins studied were significantly altered by 14 days’ administration. A role for androgens in cognitive performance and neurogenesis associated with the hippocampus has recently been suggested. We determined the effect of relatively high doses of nandrolone on proteins located in synaptoneurosomes, consisting of a sub-cellular compartment enriched with synaptic elements. This preparation is suitable for studies of the synaptic events underlying synaptic plasticity, including regulation of proteins and downstream
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Fig. 4. The effects of nandrolone decanoate on (A) p-CaMKIIa/ CaMKIIa, (B) Arc, (C) BDNF, (D) TUC-4, and (E) b-tubulin III (b-T III). In the single-dose experiment, animals (n = 10/group) received an i.m. injection of 15 mg/kg nandrolone decanoate (filled boxes) or oilvehicle (control, open boxes) and were sacrificed after 24 h. Multiple-dose consisted of daily injections of 15 mg/kg nandrolone decanoate (filled boxes) or oil-vehicle (control, open boxes) for 14 days and the rats (n = 8/ group) were sacrificed 24 h after the last injection. Representative immunoblots for each group of rats are shown (top). The values are means ± SEM.
signaling pathways in close proximity to the synapse. Changes in synaptic strength are regulated through phosphorylation of synaptic proteins, of which NMDAR is of particular importance. The activity of this protein is regulated by protein kinases and, in the hippocampus, this is closely associated with the formation of LTP and LTD (for review see [14]). By using specific antibodies against
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the NR2A subunit phosphorylated at Ser1232 or the NR2B at Tyr1472, we detected increased phosphorylation at these sites after a single injection of nandrolone. Phosphorylation of NR2A (Ser1232) by cyclin-dependent kinase 5 (Cdk5) in hippocampal CA1 neurons is reported to regulate NMDA-induced LTP [15] and mediate Cdk5-induced cell death [16]. In fact, it was recently shown that high levels of testosterone induce apoptosis in the neuroblastoma cell line SH-SY5Y [17]. Tyr1472 is the main NR2B phosphorylation site in vitro. It is phosphorylated by the src family member Fyn tyrosine kinase, reportedly with LTP [18], resulting in blockade of NMDAR internalisation [19]. Thus, decreased internalisation would be expected to result in increased NR2B-containing receptors at the membrane. However, the elevated phosphorylation of Tyr1472 observed in this study was not reflected in higher NR2B subunit content. Thus, the androgen-induced increase of phosphorylated NMDAR subunits indicates stimulated glutaminergic signaling in hippocampal synapses. A single injection of nandrolone increased ERK phosphorylation in the synaptoneurosomal preparation. Active ERK is mostly associated with transcriptional regulation in the cell nucleus; however, proteomic analysis has also revealed its presence in synaptoneurosomes [20] where ERK-dependent phosphorylation and protein synthesis modulate synaptic plasticity locally [21]. BDNF can activate ERK in hippocampal synaptic sub-cellular preparations [22]. However, 24 h after a single dose of nandrolone, there were no changes in BDNF in our material, which indicates that the increased ERK phosphorylation may have been associated with potentiated NMDAR function following its phosphorylation. NMDAR-stimulated ERK activation can be mediated through a mechanism involving inhibition by active (phosphorylated) CaMKII of the MAPK pathway inhibitor SynGAP (a Ras-GTPase-activating protein) [23]. CaMKII and SynGAP are present in the postsynaptic density [24], a subfraction of the synaptoneurosomal preparations. However, the increased ERK phosphorylation seen after in this study was not linked to a simultaneous elevation of p-CaMKIIa suggesting an alternative pathway(s) for ERK activation in synaptoneurosomes after androgen stimulation. Androgens have been ascribed a neurotrophic role modulating synaptic plasticity in the hippocampus [2]. The activity-regulated gene Arc is utilised as a presumed correlate of informational processing and plasticity in the brain [25]. Since its expression depends on NMDAR and ERK activation, as shown in primary cultures of hippocampal neurons [26], we investigated whether Arc might be an effector of the nandrolone-induced activation of this signaling cascade. The protein was present and clearly detectable in the hippocampal synaptoneurosomes; however, Arc levels were not altered by either treatment regimen. Supraphysiological levels of nandrolone have been shown to decrease proliferation and differentiation of neuronal stem cells in the rat dentate gyrus [27]. ERK activation stimulates these processes [28], but conflicting results
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have been reported regarding the role of NMDAR activation [29]. TUC-4 and b-tubulin III are expressed in early [30] and late [31] postmitotic neurons, respectively. Both proteins are utilised as markers for proliferation and differentiation, and the expression of b-tubulin III has recently been demonstrated to be ERK dependent in hippocampal neuronal progenitor cells [32]. However, we did not detect any nandrolone-induced changes in the content of these proteins. Neurogenesis is restricted to the dentate gyrus (DG), an area constituting approx. 20% of the hippocampal volume [33]. One could argue against analysing DG-specific events in a preparation obtained from whole hippocampus, due to the dilution effect. Nevertheless, both TUC-4 and b-tubulin III were clearly visualised in our Western blot analysis of synaptoneurosomal proteins. AAS in humans are reported to acutely influence mood, confidence and fear, effects that are sometimes exploited by criminals to prime themselves prior to a violent act such as a bank robbery [34]. In view of our results from this study, it is interesting to note that both NR2B (Tyr1472) and ERK phosphorylation are involved in the response to fear [35,36]. In conclusion, we have demonstrated that a single high dose of the AAS nandrolone elicits significant in vivo phosphorylation of both NMDAR and ERK1/2 in hippocampal synaptoneurosomes. These effects were not seen after a 2-week treatment period, indicating adaptation to high steroid levels. We did not detect any changes in BDNF or p-CaMKIIa levels, which suggest that these signaling components were not involved in the effects of the single injected dose of nandrolone. Moreover, we found no changes in effectors of NMDAR or ERK activation, such as the proteins associated with synaptic plasticity (Arc) or proliferation and differentiation (TUC-4, b-tubulin III). Additional studies should be carried out to further disclose the impact of nandrolone-induced NMDAR and ERK phosphorylation and how these effects are mediated. Acknowledgments This study was supported by the Swedish Research Council (Grant 9459). We thank Madeleine Le Greve`s (supported by Torsten och Ragnar So¨derbergs Stiftelser) for supplying us with antibodies. References [1] O.T. Wolf, Cognitive functions and sex steroids, Ann. Endocrinol. (Paris) 64 (2003) 158–161. [2] A. Parducz, T. Hajszan, N.J. Maclusky, Z. Hoyk, E. Csakvari, A. Kurunczi, J. Prange-Kiel, C. Leranth, Synaptic remodeling induced by gonadal hormones: neuronal plasticity as a mediator of neuroendocrine and behavioral responses to steroids, Neuroscience 138 (2006) 977–985. [3] L.B. Rosen, D.D. Ginty, M.J. Weber, M.E. Greenberg, Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras, Neuron 12 (1994) 1207–1221. [4] G.M. Thomas, R.L. Huganir, MAPK cascade signalling and synaptic plasticity, Nat. Rev. Neurosci. 5 (2004) 173–183.
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