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Secreted amyloid precursor protein-α upregulates synaptic protein synthesis by a protein kinase G-dependent mechanism
Neuroscience Letters 460 (2009) 92–96
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Secreted amyloid precursor protein-␣ upregulates synaptic protein synthesis by a protein kinase G-dependent mechanism Ana M. Claasen a,c,d , Diane Guévremont a,d , Sara E. Mason-Parker b,d , Katie Bourne c,d , Warren P. Tate c,d , Wickliffe C. Abraham b,d , Joanna M. Williams a,d,∗ a
Department of Anatomy and Structural Biology, Otago School of Medical Sciences, P.O. Box 913, Dunedin, New Zealand Department of Psychology, University of Otago, P.O. Box 56, Dunedin, New Zealand Department of Biochemistry, Otago School of Medical Sciences, P.O. Box 913, Dunedin, New Zealand d Brain Health and Repair Research Centre, University of Otago, Dunedin, New Zealand b c
a r t i c l e
i n f o
Article history: Received 24 February 2009 Received in revised form 13 May 2009 Accepted 14 May 2009 Keywords: Secreted APP␣ Protein synthesis Synaptic plasticity Synaptoneurosome cGMP-dependent protein kinase Aging
a b s t r a c t Secreted amyloid precursor protein-␣ (sAPP␣) is a neuroprotective and neurotrophic protein derived from the parent APP molecule. We have shown that sAPP␣ enhances long-term potentiation in vivo and can restore spatial memory in rats whose endogenous sAPP␣ production is impaired. These observations imply that the reduction of sAPP␣ levels seen in Alzheimer’s disease, which occurs alongside increased levels of toxic amyloid-, may be aetiologically significant. The mechanism by which sAPP␣ brings about changes in plasticity at synapses remains unresolved. We hypothesised that sAPP␣ may stimulate changes in synaptodendritic protein synthesis, an important mechanism for normal plasticity. To test this hypothesis, we investigated the effect of sAPP␣ on protein synthesis in synaptoneurosomes prepared from the hippocampi of adult male Sprague–Dawley rats. sAPP␣ (10 nM) significantly increased de novo protein synthesis as measured by the incorporation of [35 S]-methionine into acid-insoluble proteins. This was dose-dependent and blocked completely by inhibitors of protein synthesis (cycloheximide) and of cGMPdependent protein kinase (KT5823). Inhibitors of calcium/calmodulin-dependent protein kinases (KN62) and mitogen-activated protein kinase (PD98059) partially blocked the response. Further, the sAPP␣induced increase in protein synthesis was significantly attenuated when measured in synapses isolated from aged rats. These observations imply de novo protein synthesis at synapses may contribute to the long-lasting modulatory effects of sAPP␣ on synaptic plasticity. © 2009 Elsevier Ireland Ltd. All rights reserved.
The toxic amyloid-beta (A) peptide, which is strongly implicated in Alzheimer’s disease (AD) aetiology, is derived from amyloid precursor protein (APP). APP is processed by two opposing cleavage pathways: the amyloidogenic pathway involving and ␥-secretases that also produces a large secreted ectodomain, secreted APP- (sAPP), and a non-amyloidogenic pathway involving ␣-secretases that precludes A formation and yields a slightly longer APP ectodomain, secreted APP-␣ (sAPP␣). In models of AD, the balance in these two cleavage pathways is shifted to favour A production, concomitantly reducing the production of sAPP␣ [4,11]. While the toxicity of A, particularly its soluble oligomeric forms ranging from dimers to higher order structures, has been well documented [e.g. 15], much less is known about the potential damaging effects of decreased sAPP␣ levels in AD.
∗ Corresponding author at: Department of Anatomy and Structural Biology, Otago School of Medical Sciences, P.O. Box 913, Dunedin, New Zealand. Tel.: +64 3 479 8821; fax: +64 3 479 7254. E-mail address: [email protected] (J.M. Williams). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.05.040
Although the physiological role of sAPP␣ in the brain is still uncertain, studies have suggested a range of cellular functions, including neuroprotection, neurotrophism and neurogenesis [10,24]. Furthermore, sAPP␣ has been shown to potentiate spatial memory in mice [8], enhance long-term potentiation (LTP) [33], enhance N-methyl-d-aspartate (NMDA) receptor function [33], and restore normal electrophysiological and behavioural profiles in APP-deficient mice [28]. Together, this literature suggests that sAPP␣ plays key physiological roles in plasticity and memory processes. Localised protein synthesis occurring at or near synapses is emerging as a significant mechanism underpinning synaptic plasticity. The long-term maintenance of both LTP and its counterpart long-term depression (LTD) is dependent on protein synthesis, a proportion of which occurs at synapses [1,27,32]. Additionally, several modulators of synaptic plasticity, such as brain-derived neurotrophic factor (BDNF) and dopamine stimulate synaptodendritic protein synthesis [19,30], suggesting that translation of new proteins at synapses is important for their effects on synaptic plasticity. Indeed, a number of proteins are synthesized in the synaptoden-
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dritic compartment in association with LTP induction, including glutamate receptor subunits [18], calcium/calmodulin-dependent protein kinase II (CaMKII)-␣ subunit [25], and the cytoskeletalassociated protein, Arc [31]. Given that sAPP␣ modulates LTP in vitro and in vivo [17,33], we set out to test the hypothesis that sAPP␣ regulates the synthesis of proteins at synapses, in a similar manner to BDNF and dopamine using synaptoneurosomes, an accepted preparation for the modelling of synaptodendritic mechanisms. We report here that recombinant sAPP␣ [35] increased the basal incorporation of radioactive amino acids into acid-precipitated proteins within synaptoneurosomes prepared from adult rat hippocampi. The effect of sAPP␣ on synaptic protein synthesis was age- and concentration-dependent and blocked by an inhibitor of protein kinase G (PKG). We propose that regulation of synaptic protein synthesis by sAPP␣ may be an early step in the pathway that mediates sAPP␣’s effects on synaptic plasticity and memory. In this study, synaptoneurosomes were isolated from individual hippocampi of young adult (8–12 weeks) and aged (22–23 months) male Sprague–Dawley rats as previously described [38]. These animals were deeply anaesthetised with halothane and decapitated, using a protocol approved by the University of Otago Animal Ethics Committee to ensure minimal animal suffering. Synaptoneurosomes were collected by centrifugation (1000 × g/15 min/4 ◦ C) and resuspended in ice-cold HEPES buffer (50 mM HEPES pH 7.4, 124 mM NaCl, 3.2 mM KCl, 1.3 mM MgCl2 , 2.5 mM CaCl2 , 1.06 mM KH2 PO4 , 26 mM NaHCO3 , 1 M glucose, Complete protease inhibitor (Roche, Indianapolis, IN, USA) and 0.2 mg/mL chloramphenicol (Sigma–Aldrich, St. Louis, MO, USA). Previous experiments have shown that synaptoneurosomes prepared in this way are enriched for synapse-associated proteins (e.g., GluA1, PSD-95, CaMKII␣) [39] and exhibit a basal level of protein synthesis [26], against which drug treatments may be normalised. All experiments were conducted in the presence of chloramphenicol to eliminate any contribution from mitochondrial protein synthesis. Prior to assessment of synaptic protein synthesis, synaptoneurosomes isolated from adult rat hippocampi were characterised by transmission electron microscopy (TEM) and Western Blot analysis as previously described [38]. TEMs showed that the preparation contained profiles that could be identified as synaptoneurosomes by the presence of a characteristic resealed presynapse containing synaptic vesicles and an attached postsynaptic region interfaced by the electron dense postsynaptic density (Fig. 1A, right panel). For Western Blot analysis a fixed amount of the whole tissue homogenate or final synaptoneurosome fraction (20 g, as estimated using a Bradford protein assay (BioRad, Hercules, CA, USA) was separated by SDS-PAGE (9%) and transferred to nitrocellulose membrane (Whatman, Maidstone, England, UK). Membranes were probed either with an antibody recognizing PSD-95 (BD Biosciences, San Jose, CA, USA; P-45320), ␣-tubulin (Abcam Inc., Cambridge, MA, USA; ab4074) or calnexin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; sc-11397). Antibodies were detected using appropriate HRP-conjugated secondary antibodies and Supersignal West Pico (Pierce, Rockford, IL, USA). Western Blot analysis documented that the synaptoneurosome fraction was highly enriched for the postsynaptic marker protein PSD-95 relative to the total cellular homogenate, while the homogenate showed contrasting enrichment for the intracellular membrane marker, calnexin and the microtubule-associated protein, tubulin (Fig. 1A, left panel), thus replicating our previous findings [38]. In order to measure synthesis of new synaptic proteins, synaptoneurosomes (100 g) were incubated (30 min/37 ◦ C) in the presence of [35 S]-methionine (10 Ci; Amersham Biosciences, Piscataway, NJ, USA). To assess the effect of pharmacological treatment, synaptoneurosomes were preincubated with or without drugs for 15 min at 37 ◦ C. The experimental period was followed by a chase period (10 min, 200 g ice-cold, non-radioactive methio-
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Fig. 1. sAPP␣ increases the basal rate of synaptic protein synthesis in synaptoneurosomes in a dose-dependent manner. (A) Typical TEM profile of synaptoneurosomes (right panel), showing a vesicle-filled presynaptic terminal and a postsynaptic terminal containing a postsynaptic density. Western Blots (left panel) showing synaptoneurosomes (N, 20 g) are enriched for PSD-95 (95 kDa), but not calnexin (90 kDa) or tubulin (50 kDa) when compared to whole hippocampal homogenate (H, 20 g). (B) Summary graph showing that basal protein measured in synaptoneurosomes can be downregulated by cycloheximide (Cxm) and upregulated by BDNF and SKF38393). Mean ± SEM% change in incorporation of [35 S]-methionine into acid-insoluble proteins from drug-treated synaptoneurosomes, relative to a no-drug control. *p < 0.01, paired t-test (Cxm, n = 7; BDNF, n = 5; SKF38393, n = 9). (C) Summary graph showing that sAPP␣ treatment increased the basal rate of synaptic protein synthesis in synaptoneurosomes in a cycloheximide and dosedependent manner. *p < 0.05, ANOVA of log-transformed data with Dunnett’s post hoc test, compared to no-drug control (0.1 and 1 nM, n = 5; 10 nM, n = 6; 100 nM, n = 3; 10 nM + Cxm, n = 5).
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nine). To precipitate the protein component, synaptoneurosomes were collected by centrifugation (1000 × g/5 min/4 ◦ C) and resuspended in 10% (w/v) TCA/0.1% (w/v) methionine. The acid-insoluble material was recovered by centrifugation (13,400 × g/5 min/4 ◦ C) and the resultant pellet washed three times in 5% (w/v) TCA/0.1% methionine (13,400 × g/5 min/4 ◦ C). The pellets were solubilised in 1 M NaOH (37 ◦ C/overnight) and then neutralised with 1 M HCl. The incorporation of 35 S-methionine into newly synthesized proteins was quantified by liquid scintillation counting using biodegradable counting scintillant (Amersham Biosciences) and a Wallac MicroBeta Trilux (PerkinElmer, Waltham, MA, USA) scintillation counter. Pharmacological treatments used were recombinant human sAPP␣ and sAPP (0.1–100 nM; prepared using the human APP695 sequence. according to previously described protocols [35]), cycloheximide (60 M; Sigma, St. Louis, MO), BDNF (1.2 nM; Millipore Upstate, Temecula, CA, USA), SKF38393 (100 M; Tocris Bioscience, Bristol, UK), KN62 (10 M; Tocris Bioscience), KT5823 (10 M; Tocris Bioscience), and PD98059 (50 M; Tocris Bioscience). All protein synthesis data were expressed relative to the withinexperiment no-drug control samples and data used for statistical analysis were log-transformed using natural logs. For ease of understanding, however, the data in the figures are presented on linear scales. Analyses of variance with Dunnett’s post hoc test and independent t-tests were performed using GraphPad Prism version 5.0a for Mac OS X (Graphpad Software, San Diego, CA, USA). To confirm that we could detect basal protein synthesis, synaptoneurosomes were incubated in the presence of [35 S]-methionine with and without cycloheximide, an inhibitor of eukaryotic protein synthesis. Cycloheximide treatment resulted in a 45 ± 4% (mean ± SEM (n = 7), p < 0.01, paired t-test) reduction in the incorporation of [35 S]-methionine into acid-insoluble material (Fig. 1B). To show that it was possible to upregulate protein synthesis, synaptoneurosomes were incubated in the presence of two neuromodulators known to promote synaptic protein synthesis [30,40]. We found that BDNF (1.2 nM) or D1/D5 dopamine receptor agonist SKF38393 (100 M) both increased the measured protein synthesis but to different degrees (BDNF: 71 ± 15% (n = 5), p < 0.01, paired t-test; SKF38393: 11 ± 6%, n = 9, p < 0.01; Fig. 1B). Having confirmed that upregulation of protein synthesis could be detected in hippocampal synaptoneurosomes, we next tested the effect of sAPP␣. Treatment of hippocampal synaptoneurosomes with 10 nM sAPP␣ induced a significant increase in the incorporation of [35 S]methionine relative to the no-drug control (66 ± 12%; n = 6; p < 0.01; Fig. 1C), which was attenuated by co-application of cycloheximide (−8 ± 3%; n = 5; Fig. 1C). This indicates that incubation of synaptoneurosomes with 10 nM sAPP␣ produces an upregulation of synaptic protein synthesis that is comparable to BDNF. Previous literature has indicated that sAPP␣ demonstrates neuroprotective and neuromodulatory effects over a wide range of concentrations (0.1–100 nM) [24,33]. Accordingly, we determined the effective range of sAPP␣ concentrations with respect to enhancing synaptic protein synthesis. The sAPP␣-induced upregulation of synaptic protein synthesis was dose-dependent, showing an inverted U-shaped dose–response curve with a peak at 10 nM (Fig. 1C). Analysis of variance confirmed the dose–response effect of sAPP␣ treatment, with Dunnett’s post-test indicating that sAPP␣ significantly increased protein synthesis at 1 and 10 nM but not at the 0.1 or 100 nM concentrations, as indicated. Subsequent experiments were carried out using 10 nM sAPP␣. Synaptic protein synthesis is a highly regulated process, which involves numerous signal transduction pathways [9], including those involving CaMKII and p42–44 mitogen-activated protein kinase (MAPK). The neuroprotective effects sAPP␣ can be blocked by inhibitors of PKG [7] and MAPK [12]. Thus, we tested inhibitors of these kinases to determine whether they also block sAPP␣stimulated protein synthesis. Inhibition of PKG activity with
Fig. 2. (A) The increase in synaptic protein synthesis stimulated by 10 nM sAPP␣ was abolished by inhibition of PKG (KT5823, 10 M), and significantly reduced by inhibitors of CaMKII (KN62, 10 M) and MAPK (PD98059, 50 M). (B) Protein kinase inhibitors had no effect on the basal rate of synaptic protein synthesis. Mean ± SEM% change in incorporation of [35 S]-methionine into acid-insoluble proteins from drugtreated synaptoneurosomes, relative to a no-drug control. *p < 0.05, ANOVA of logtransformed data with Dunnett’s post hoc test, compared to 10 nM sAPP␣ control (10 nM sAPP␣, n = 15; KN62, n = 10; KT5823 and PD98059, n = 5).
the specific inhibitor KT5823 (10 M) completely eliminated the upregulation of synaptic protein synthesis induced by 10 nM sAPP␣ (−13 ± 7%, n = 5, p < 0.001, compared to the 10 nM sAPP␣ treatment group within this experiment; Fig. 2A). By contrast, incubation with either KN62, an inhibitor of CaMKII and other related kinases, or PD98059, an inhibitor of p42–44 MAPK attenuated but did not abolish the upregulation of protein synthesis (10 M KN62: 18 ± 8%, n = 10; 50 M PD98059: 13 ± 5%, n = 5; both p < 0.05 compared to 10 nM sAPP␣ treatment; Fig. 2A). Treatment of synaptoneurosomes with each inhibitor drug on its own had no effect on the basal rate of synaptic protein synthesis (Fig. 2B). To investigate whether the sAPP␣-induced upregulation of synaptic protein synthesis was dependent on the age of the animal, we compared sAPP␣-induced protein synthesis in synaptoneurosomes isolated from aged rats (20–22 months) to that measured in young adult rats (8–12 weeks). In these studies, we found the upregulation of synaptic protein synthesis was significantly reduced in synaptoneurosomes from aged compared to young adult rats (young: 49 ± 9%, n = 10; aged: 22 ± 3%, n = 6; p = 0.02, independent t-test; Fig. 3).
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Fig. 3. APP derivatives differentially modulate synaptic protein synthesis depending on age. Summary graph showing that 10 nM sAPP␣ produced less enhancement of synaptic protein synthesis in aged animals (22–23 mo), whereas 10 nM sAPP had no significant effect on protein synthesis in either age group. *p = 0.02, independent t-test of log-transformed data (young: 10 nM sAPP␣, n = 10; 10 nM sAPP, n = 10; aged: 10 nM sAPP␣, sAPP, n = 6).
To determine whether the measured effect on synaptic protein synthesis was specific to sAPP␣, we investigated the effect of the shorter protein, sAPP, that is produced from -secretase cleavage of APP and is missing 16 amino acids at the C-terminus. Treatment with 10 nM sAPP did not significantly increase synaptic protein synthesis in either young or aged animals (young: 9 ± 4%; aged: 4 ± 2%; Fig. 3). Together, these data show that the stimulatory effect on synaptic protein synthesis was specific to sAPP␣, at the concentration used. In the normal brain, sAPP␣ is thought to be neurotrophic, neuroprotective, neurogenic and a modulator of synaptic plasticity. For example, exogenous sAPP␣ enhances LTP [17,33] and reduces the frequency-dependence of LTD [17]. Endogenous sAPP␣ also promotes LTP, possibly through the enhancement of tetanically evoked NMDAR currents [33]. However, the intracellular signalling mechanisms by which sAPP␣ elicits these various effects are poorly understood. Because synaptodendritic protein synthesis has recently become recognised as being important for neural plasticity, we tested here whether this mechanism is regulated by sAPP␣. Indeed, we found that exogenous sAPP␣ upregulates the synthesis of new proteins in synaptoneurosomes in a dose-dependent manner. Notably, sAPP, which is shortened at the C-terminus, failed to affect protein synthesis when administered at the concentration that was maximally effective for sAPP␣. This is in keeping with other reports of its reduced potency as a neuroprotective agent [21,35] and as a regulator of LTP [33]. PKG has not previously been implicated in the pathways leading to synaptodendritic protein synthesis but we found that stimulation of synaptic protein synthesis by sAPP␣ was dependent on the activity of this protein kinase. The previously documented effectiveness of PKG inhibitors against the diverse actions of sAPP␣ suggests that it is activated early in the sAPP␣ signal transduction cascade. Consistent with our observations, multiple studies have shown that sAPP␣ signalling in neurons involves elevated cyclic GMP and PKG activation. For example, sAPP␣ stimulation of NFBdependent transcription, regulation of NMDA and K+ currents, and neuroprotection against glutamate toxicity and glucose deprivation are blocked by PKG inhibitors, and mimicked by cGMP analogues [7,13,14] and the enhancing effect of sAPP␣ on LTP in hippocampal slices is also mimicked by a cGMP analogue [17]. Our data suggest that this link should be examined in more detail, and for its contribution to other signal transduction pathways leading to protein synthesis.
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Inhibitors of MAPK and CaMKII on the other hand caused only partial inhibition of protein synthesis in synaptoneurosomes suggesting that their involvement in sAPP␣ signalling may be more indirect. The neuroprotective action of sAPP␣ against glutamate toxicity and trophic factor deprivation has been shown to be dependent on the MAPK/ERK system [12]. Unlike PKG, the participation of MAPK in the regulation of synaptic protein synthesis is now well established. For example, MAPK activation by LTP-inducing highfrequency stimulation of the hippocampus leads to activation of the mammalian target of rapamycin (mTOR), a key regulator of translation [34]. Similarly, Group I metabotropic glutamate receptor (mGluR) activation leads to a long-term depression in the hippocampus, mediated in part by a MAPK-mediated increase in synaptodendritic protein synthesis [6]. The reduction by a CaMKII inhibitor of the enhancement of protein synthesis by sAPP␣ appears on the one hand to be contrary to previous evidence that sAPP␣ reduces the basal intracellular concentration of Ca2+ [24], and reduces the Ca2+ influx resulting from a glutamate or A insult in cultured neuron preparations [20,23]. However, it is consistent with the demonstrated upregulation of LTP by sAPP␣ in acute slices and in vivo [17,33]. Furthermore, CaMKII can regulate synaptic protein synthesis through phosphorylation of the cytoplasmic polyadenylation element-binding protein, which regulates translation [5], a process which occurs in NMDAstimulated synaptic protein synthesis [37]. CaMKII inhibitors also block Group I mGluR-stimulated protein synthesis in synaptoneurosomes (Guévremont and Williams, unpublished findings). Thus, the ability of a CaMKII inhibitor to significantly reduce sAPP␣-triggered protein synthesis is consistent with its general contribution to synaptodendritic protein synthesis, with the caveat that the inhibitor used is not completely selective for CaMKII, and may also have effects on other CaMKs. Because endogenous sAPP␣ contributes to memory mechanisms, and sAPP␣ levels are decreased in both familial and sporadic AD [3,29], it has been proposed that reduced sAPP␣ signalling contributes to AD-related cognitive decline [22,33,36]. In the present study, the ability of exogenous sAPP␣ to stimulus protein synthesis in aged synaptoneurosomes was significantly reduced. This is consistent with the trend toward a reduced effectiveness of the less potent sAPP, and with our finding that other activators of local protein synthesis, such as Group I mGluRs, are also less effective at stimulating protein synthesis in aged synaptoneurosomes [2]. Thus, the deleterious effects of reduced sAPP␣ production during AD may be compounded by an aging-related reduction in the efficiency of sAPP␣ signalling, at least in the case of stimulating local protein synthesis. In summary, we have found that sAPP␣ reliably promotes protein synthesis in a synaptoneurosome preparation in a concentration-dependent manner that is also dependent on the activity of a number of protein kinases, especially PKG. The contribution of protein synthesis in the synaptodendritic compartment to neural plasticity has in recent times become recognised as another significant mechanism for neural plasticity, including LTP. BDNF enhances LTP through a synaptodendritic protein synthesis mechanism [19], and Group I mGluR-dependent LTD also requires local protein synthesis [16]. It is possible therefore, that sAPP␣ acts in a similar way by increasing the synthesis of proteins required for plastic changes. Identification of the specific changes to the synaptic proteome induced by sAPP␣ may indicate the crucial mechanism by which sAPP␣ mediates effects on the brain. Acknowledgements This research was supported by grants from the New Zealand Health Research Council to JMW, WCA and WPT and the Marsden Fund to JMW and WCA.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Secreted amyloid precursor protein-␣ upregulates synaptic protein synthesis by a protein kinase G-dependent mechanism Ana M. Claasen a,c,d , Diane Guévremont a,d , Sara E. Mason-Parker b,d , Katie Bourne c,d , Warren P. Tate c,d , Wickliffe C. Abraham b,d , Joanna M. Williams a,d,∗ a
Department of Anatomy and Structural Biology, Otago School of Medical Sciences, P.O. Box 913, Dunedin, New Zealand Department of Psychology, University of Otago, P.O. Box 56, Dunedin, New Zealand Department of Biochemistry, Otago School of Medical Sciences, P.O. Box 913, Dunedin, New Zealand d Brain Health and Repair Research Centre, University of Otago, Dunedin, New Zealand b c
a r t i c l e
i n f o
Article history: Received 24 February 2009 Received in revised form 13 May 2009 Accepted 14 May 2009 Keywords: Secreted APP␣ Protein synthesis Synaptic plasticity Synaptoneurosome cGMP-dependent protein kinase Aging
a b s t r a c t Secreted amyloid precursor protein-␣ (sAPP␣) is a neuroprotective and neurotrophic protein derived from the parent APP molecule. We have shown that sAPP␣ enhances long-term potentiation in vivo and can restore spatial memory in rats whose endogenous sAPP␣ production is impaired. These observations imply that the reduction of sAPP␣ levels seen in Alzheimer’s disease, which occurs alongside increased levels of toxic amyloid-, may be aetiologically significant. The mechanism by which sAPP␣ brings about changes in plasticity at synapses remains unresolved. We hypothesised that sAPP␣ may stimulate changes in synaptodendritic protein synthesis, an important mechanism for normal plasticity. To test this hypothesis, we investigated the effect of sAPP␣ on protein synthesis in synaptoneurosomes prepared from the hippocampi of adult male Sprague–Dawley rats. sAPP␣ (10 nM) significantly increased de novo protein synthesis as measured by the incorporation of [35 S]-methionine into acid-insoluble proteins. This was dose-dependent and blocked completely by inhibitors of protein synthesis (cycloheximide) and of cGMPdependent protein kinase (KT5823). Inhibitors of calcium/calmodulin-dependent protein kinases (KN62) and mitogen-activated protein kinase (PD98059) partially blocked the response. Further, the sAPP␣induced increase in protein synthesis was significantly attenuated when measured in synapses isolated from aged rats. These observations imply de novo protein synthesis at synapses may contribute to the long-lasting modulatory effects of sAPP␣ on synaptic plasticity. © 2009 Elsevier Ireland Ltd. All rights reserved.
The toxic amyloid-beta (A) peptide, which is strongly implicated in Alzheimer’s disease (AD) aetiology, is derived from amyloid precursor protein (APP). APP is processed by two opposing cleavage pathways: the amyloidogenic pathway involving and ␥-secretases that also produces a large secreted ectodomain, secreted APP- (sAPP), and a non-amyloidogenic pathway involving ␣-secretases that precludes A formation and yields a slightly longer APP ectodomain, secreted APP-␣ (sAPP␣). In models of AD, the balance in these two cleavage pathways is shifted to favour A production, concomitantly reducing the production of sAPP␣ [4,11]. While the toxicity of A, particularly its soluble oligomeric forms ranging from dimers to higher order structures, has been well documented [e.g. 15], much less is known about the potential damaging effects of decreased sAPP␣ levels in AD.
∗ Corresponding author at: Department of Anatomy and Structural Biology, Otago School of Medical Sciences, P.O. Box 913, Dunedin, New Zealand. Tel.: +64 3 479 8821; fax: +64 3 479 7254. E-mail address: [email protected] (J.M. Williams). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.05.040
Although the physiological role of sAPP␣ in the brain is still uncertain, studies have suggested a range of cellular functions, including neuroprotection, neurotrophism and neurogenesis [10,24]. Furthermore, sAPP␣ has been shown to potentiate spatial memory in mice [8], enhance long-term potentiation (LTP) [33], enhance N-methyl-d-aspartate (NMDA) receptor function [33], and restore normal electrophysiological and behavioural profiles in APP-deficient mice [28]. Together, this literature suggests that sAPP␣ plays key physiological roles in plasticity and memory processes. Localised protein synthesis occurring at or near synapses is emerging as a significant mechanism underpinning synaptic plasticity. The long-term maintenance of both LTP and its counterpart long-term depression (LTD) is dependent on protein synthesis, a proportion of which occurs at synapses [1,27,32]. Additionally, several modulators of synaptic plasticity, such as brain-derived neurotrophic factor (BDNF) and dopamine stimulate synaptodendritic protein synthesis [19,30], suggesting that translation of new proteins at synapses is important for their effects on synaptic plasticity. Indeed, a number of proteins are synthesized in the synaptoden-
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dritic compartment in association with LTP induction, including glutamate receptor subunits [18], calcium/calmodulin-dependent protein kinase II (CaMKII)-␣ subunit [25], and the cytoskeletalassociated protein, Arc [31]. Given that sAPP␣ modulates LTP in vitro and in vivo [17,33], we set out to test the hypothesis that sAPP␣ regulates the synthesis of proteins at synapses, in a similar manner to BDNF and dopamine using synaptoneurosomes, an accepted preparation for the modelling of synaptodendritic mechanisms. We report here that recombinant sAPP␣ [35] increased the basal incorporation of radioactive amino acids into acid-precipitated proteins within synaptoneurosomes prepared from adult rat hippocampi. The effect of sAPP␣ on synaptic protein synthesis was age- and concentration-dependent and blocked by an inhibitor of protein kinase G (PKG). We propose that regulation of synaptic protein synthesis by sAPP␣ may be an early step in the pathway that mediates sAPP␣’s effects on synaptic plasticity and memory. In this study, synaptoneurosomes were isolated from individual hippocampi of young adult (8–12 weeks) and aged (22–23 months) male Sprague–Dawley rats as previously described [38]. These animals were deeply anaesthetised with halothane and decapitated, using a protocol approved by the University of Otago Animal Ethics Committee to ensure minimal animal suffering. Synaptoneurosomes were collected by centrifugation (1000 × g/15 min/4 ◦ C) and resuspended in ice-cold HEPES buffer (50 mM HEPES pH 7.4, 124 mM NaCl, 3.2 mM KCl, 1.3 mM MgCl2 , 2.5 mM CaCl2 , 1.06 mM KH2 PO4 , 26 mM NaHCO3 , 1 M glucose, Complete protease inhibitor (Roche, Indianapolis, IN, USA) and 0.2 mg/mL chloramphenicol (Sigma–Aldrich, St. Louis, MO, USA). Previous experiments have shown that synaptoneurosomes prepared in this way are enriched for synapse-associated proteins (e.g., GluA1, PSD-95, CaMKII␣) [39] and exhibit a basal level of protein synthesis [26], against which drug treatments may be normalised. All experiments were conducted in the presence of chloramphenicol to eliminate any contribution from mitochondrial protein synthesis. Prior to assessment of synaptic protein synthesis, synaptoneurosomes isolated from adult rat hippocampi were characterised by transmission electron microscopy (TEM) and Western Blot analysis as previously described [38]. TEMs showed that the preparation contained profiles that could be identified as synaptoneurosomes by the presence of a characteristic resealed presynapse containing synaptic vesicles and an attached postsynaptic region interfaced by the electron dense postsynaptic density (Fig. 1A, right panel). For Western Blot analysis a fixed amount of the whole tissue homogenate or final synaptoneurosome fraction (20 g, as estimated using a Bradford protein assay (BioRad, Hercules, CA, USA) was separated by SDS-PAGE (9%) and transferred to nitrocellulose membrane (Whatman, Maidstone, England, UK). Membranes were probed either with an antibody recognizing PSD-95 (BD Biosciences, San Jose, CA, USA; P-45320), ␣-tubulin (Abcam Inc., Cambridge, MA, USA; ab4074) or calnexin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; sc-11397). Antibodies were detected using appropriate HRP-conjugated secondary antibodies and Supersignal West Pico (Pierce, Rockford, IL, USA). Western Blot analysis documented that the synaptoneurosome fraction was highly enriched for the postsynaptic marker protein PSD-95 relative to the total cellular homogenate, while the homogenate showed contrasting enrichment for the intracellular membrane marker, calnexin and the microtubule-associated protein, tubulin (Fig. 1A, left panel), thus replicating our previous findings [38]. In order to measure synthesis of new synaptic proteins, synaptoneurosomes (100 g) were incubated (30 min/37 ◦ C) in the presence of [35 S]-methionine (10 Ci; Amersham Biosciences, Piscataway, NJ, USA). To assess the effect of pharmacological treatment, synaptoneurosomes were preincubated with or without drugs for 15 min at 37 ◦ C. The experimental period was followed by a chase period (10 min, 200 g ice-cold, non-radioactive methio-
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Fig. 1. sAPP␣ increases the basal rate of synaptic protein synthesis in synaptoneurosomes in a dose-dependent manner. (A) Typical TEM profile of synaptoneurosomes (right panel), showing a vesicle-filled presynaptic terminal and a postsynaptic terminal containing a postsynaptic density. Western Blots (left panel) showing synaptoneurosomes (N, 20 g) are enriched for PSD-95 (95 kDa), but not calnexin (90 kDa) or tubulin (50 kDa) when compared to whole hippocampal homogenate (H, 20 g). (B) Summary graph showing that basal protein measured in synaptoneurosomes can be downregulated by cycloheximide (Cxm) and upregulated by BDNF and SKF38393). Mean ± SEM% change in incorporation of [35 S]-methionine into acid-insoluble proteins from drug-treated synaptoneurosomes, relative to a no-drug control. *p < 0.01, paired t-test (Cxm, n = 7; BDNF, n = 5; SKF38393, n = 9). (C) Summary graph showing that sAPP␣ treatment increased the basal rate of synaptic protein synthesis in synaptoneurosomes in a cycloheximide and dosedependent manner. *p < 0.05, ANOVA of log-transformed data with Dunnett’s post hoc test, compared to no-drug control (0.1 and 1 nM, n = 5; 10 nM, n = 6; 100 nM, n = 3; 10 nM + Cxm, n = 5).
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nine). To precipitate the protein component, synaptoneurosomes were collected by centrifugation (1000 × g/5 min/4 ◦ C) and resuspended in 10% (w/v) TCA/0.1% (w/v) methionine. The acid-insoluble material was recovered by centrifugation (13,400 × g/5 min/4 ◦ C) and the resultant pellet washed three times in 5% (w/v) TCA/0.1% methionine (13,400 × g/5 min/4 ◦ C). The pellets were solubilised in 1 M NaOH (37 ◦ C/overnight) and then neutralised with 1 M HCl. The incorporation of 35 S-methionine into newly synthesized proteins was quantified by liquid scintillation counting using biodegradable counting scintillant (Amersham Biosciences) and a Wallac MicroBeta Trilux (PerkinElmer, Waltham, MA, USA) scintillation counter. Pharmacological treatments used were recombinant human sAPP␣ and sAPP (0.1–100 nM; prepared using the human APP695 sequence. according to previously described protocols [35]), cycloheximide (60 M; Sigma, St. Louis, MO), BDNF (1.2 nM; Millipore Upstate, Temecula, CA, USA), SKF38393 (100 M; Tocris Bioscience, Bristol, UK), KN62 (10 M; Tocris Bioscience), KT5823 (10 M; Tocris Bioscience), and PD98059 (50 M; Tocris Bioscience). All protein synthesis data were expressed relative to the withinexperiment no-drug control samples and data used for statistical analysis were log-transformed using natural logs. For ease of understanding, however, the data in the figures are presented on linear scales. Analyses of variance with Dunnett’s post hoc test and independent t-tests were performed using GraphPad Prism version 5.0a for Mac OS X (Graphpad Software, San Diego, CA, USA). To confirm that we could detect basal protein synthesis, synaptoneurosomes were incubated in the presence of [35 S]-methionine with and without cycloheximide, an inhibitor of eukaryotic protein synthesis. Cycloheximide treatment resulted in a 45 ± 4% (mean ± SEM (n = 7), p < 0.01, paired t-test) reduction in the incorporation of [35 S]-methionine into acid-insoluble material (Fig. 1B). To show that it was possible to upregulate protein synthesis, synaptoneurosomes were incubated in the presence of two neuromodulators known to promote synaptic protein synthesis [30,40]. We found that BDNF (1.2 nM) or D1/D5 dopamine receptor agonist SKF38393 (100 M) both increased the measured protein synthesis but to different degrees (BDNF: 71 ± 15% (n = 5), p < 0.01, paired t-test; SKF38393: 11 ± 6%, n = 9, p < 0.01; Fig. 1B). Having confirmed that upregulation of protein synthesis could be detected in hippocampal synaptoneurosomes, we next tested the effect of sAPP␣. Treatment of hippocampal synaptoneurosomes with 10 nM sAPP␣ induced a significant increase in the incorporation of [35 S]methionine relative to the no-drug control (66 ± 12%; n = 6; p < 0.01; Fig. 1C), which was attenuated by co-application of cycloheximide (−8 ± 3%; n = 5; Fig. 1C). This indicates that incubation of synaptoneurosomes with 10 nM sAPP␣ produces an upregulation of synaptic protein synthesis that is comparable to BDNF. Previous literature has indicated that sAPP␣ demonstrates neuroprotective and neuromodulatory effects over a wide range of concentrations (0.1–100 nM) [24,33]. Accordingly, we determined the effective range of sAPP␣ concentrations with respect to enhancing synaptic protein synthesis. The sAPP␣-induced upregulation of synaptic protein synthesis was dose-dependent, showing an inverted U-shaped dose–response curve with a peak at 10 nM (Fig. 1C). Analysis of variance confirmed the dose–response effect of sAPP␣ treatment, with Dunnett’s post-test indicating that sAPP␣ significantly increased protein synthesis at 1 and 10 nM but not at the 0.1 or 100 nM concentrations, as indicated. Subsequent experiments were carried out using 10 nM sAPP␣. Synaptic protein synthesis is a highly regulated process, which involves numerous signal transduction pathways [9], including those involving CaMKII and p42–44 mitogen-activated protein kinase (MAPK). The neuroprotective effects sAPP␣ can be blocked by inhibitors of PKG [7] and MAPK [12]. Thus, we tested inhibitors of these kinases to determine whether they also block sAPP␣stimulated protein synthesis. Inhibition of PKG activity with
Fig. 2. (A) The increase in synaptic protein synthesis stimulated by 10 nM sAPP␣ was abolished by inhibition of PKG (KT5823, 10 M), and significantly reduced by inhibitors of CaMKII (KN62, 10 M) and MAPK (PD98059, 50 M). (B) Protein kinase inhibitors had no effect on the basal rate of synaptic protein synthesis. Mean ± SEM% change in incorporation of [35 S]-methionine into acid-insoluble proteins from drugtreated synaptoneurosomes, relative to a no-drug control. *p < 0.05, ANOVA of logtransformed data with Dunnett’s post hoc test, compared to 10 nM sAPP␣ control (10 nM sAPP␣, n = 15; KN62, n = 10; KT5823 and PD98059, n = 5).
the specific inhibitor KT5823 (10 M) completely eliminated the upregulation of synaptic protein synthesis induced by 10 nM sAPP␣ (−13 ± 7%, n = 5, p < 0.001, compared to the 10 nM sAPP␣ treatment group within this experiment; Fig. 2A). By contrast, incubation with either KN62, an inhibitor of CaMKII and other related kinases, or PD98059, an inhibitor of p42–44 MAPK attenuated but did not abolish the upregulation of protein synthesis (10 M KN62: 18 ± 8%, n = 10; 50 M PD98059: 13 ± 5%, n = 5; both p < 0.05 compared to 10 nM sAPP␣ treatment; Fig. 2A). Treatment of synaptoneurosomes with each inhibitor drug on its own had no effect on the basal rate of synaptic protein synthesis (Fig. 2B). To investigate whether the sAPP␣-induced upregulation of synaptic protein synthesis was dependent on the age of the animal, we compared sAPP␣-induced protein synthesis in synaptoneurosomes isolated from aged rats (20–22 months) to that measured in young adult rats (8–12 weeks). In these studies, we found the upregulation of synaptic protein synthesis was significantly reduced in synaptoneurosomes from aged compared to young adult rats (young: 49 ± 9%, n = 10; aged: 22 ± 3%, n = 6; p = 0.02, independent t-test; Fig. 3).
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Fig. 3. APP derivatives differentially modulate synaptic protein synthesis depending on age. Summary graph showing that 10 nM sAPP␣ produced less enhancement of synaptic protein synthesis in aged animals (22–23 mo), whereas 10 nM sAPP had no significant effect on protein synthesis in either age group. *p = 0.02, independent t-test of log-transformed data (young: 10 nM sAPP␣, n = 10; 10 nM sAPP, n = 10; aged: 10 nM sAPP␣, sAPP, n = 6).
To determine whether the measured effect on synaptic protein synthesis was specific to sAPP␣, we investigated the effect of the shorter protein, sAPP, that is produced from -secretase cleavage of APP and is missing 16 amino acids at the C-terminus. Treatment with 10 nM sAPP did not significantly increase synaptic protein synthesis in either young or aged animals (young: 9 ± 4%; aged: 4 ± 2%; Fig. 3). Together, these data show that the stimulatory effect on synaptic protein synthesis was specific to sAPP␣, at the concentration used. In the normal brain, sAPP␣ is thought to be neurotrophic, neuroprotective, neurogenic and a modulator of synaptic plasticity. For example, exogenous sAPP␣ enhances LTP [17,33] and reduces the frequency-dependence of LTD [17]. Endogenous sAPP␣ also promotes LTP, possibly through the enhancement of tetanically evoked NMDAR currents [33]. However, the intracellular signalling mechanisms by which sAPP␣ elicits these various effects are poorly understood. Because synaptodendritic protein synthesis has recently become recognised as being important for neural plasticity, we tested here whether this mechanism is regulated by sAPP␣. Indeed, we found that exogenous sAPP␣ upregulates the synthesis of new proteins in synaptoneurosomes in a dose-dependent manner. Notably, sAPP, which is shortened at the C-terminus, failed to affect protein synthesis when administered at the concentration that was maximally effective for sAPP␣. This is in keeping with other reports of its reduced potency as a neuroprotective agent [21,35] and as a regulator of LTP [33]. PKG has not previously been implicated in the pathways leading to synaptodendritic protein synthesis but we found that stimulation of synaptic protein synthesis by sAPP␣ was dependent on the activity of this protein kinase. The previously documented effectiveness of PKG inhibitors against the diverse actions of sAPP␣ suggests that it is activated early in the sAPP␣ signal transduction cascade. Consistent with our observations, multiple studies have shown that sAPP␣ signalling in neurons involves elevated cyclic GMP and PKG activation. For example, sAPP␣ stimulation of NFBdependent transcription, regulation of NMDA and K+ currents, and neuroprotection against glutamate toxicity and glucose deprivation are blocked by PKG inhibitors, and mimicked by cGMP analogues [7,13,14] and the enhancing effect of sAPP␣ on LTP in hippocampal slices is also mimicked by a cGMP analogue [17]. Our data suggest that this link should be examined in more detail, and for its contribution to other signal transduction pathways leading to protein synthesis.
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Inhibitors of MAPK and CaMKII on the other hand caused only partial inhibition of protein synthesis in synaptoneurosomes suggesting that their involvement in sAPP␣ signalling may be more indirect. The neuroprotective action of sAPP␣ against glutamate toxicity and trophic factor deprivation has been shown to be dependent on the MAPK/ERK system [12]. Unlike PKG, the participation of MAPK in the regulation of synaptic protein synthesis is now well established. For example, MAPK activation by LTP-inducing highfrequency stimulation of the hippocampus leads to activation of the mammalian target of rapamycin (mTOR), a key regulator of translation [34]. Similarly, Group I metabotropic glutamate receptor (mGluR) activation leads to a long-term depression in the hippocampus, mediated in part by a MAPK-mediated increase in synaptodendritic protein synthesis [6]. The reduction by a CaMKII inhibitor of the enhancement of protein synthesis by sAPP␣ appears on the one hand to be contrary to previous evidence that sAPP␣ reduces the basal intracellular concentration of Ca2+ [24], and reduces the Ca2+ influx resulting from a glutamate or A insult in cultured neuron preparations [20,23]. However, it is consistent with the demonstrated upregulation of LTP by sAPP␣ in acute slices and in vivo [17,33]. Furthermore, CaMKII can regulate synaptic protein synthesis through phosphorylation of the cytoplasmic polyadenylation element-binding protein, which regulates translation [5], a process which occurs in NMDAstimulated synaptic protein synthesis [37]. CaMKII inhibitors also block Group I mGluR-stimulated protein synthesis in synaptoneurosomes (Guévremont and Williams, unpublished findings). Thus, the ability of a CaMKII inhibitor to significantly reduce sAPP␣-triggered protein synthesis is consistent with its general contribution to synaptodendritic protein synthesis, with the caveat that the inhibitor used is not completely selective for CaMKII, and may also have effects on other CaMKs. Because endogenous sAPP␣ contributes to memory mechanisms, and sAPP␣ levels are decreased in both familial and sporadic AD [3,29], it has been proposed that reduced sAPP␣ signalling contributes to AD-related cognitive decline [22,33,36]. In the present study, the ability of exogenous sAPP␣ to stimulus protein synthesis in aged synaptoneurosomes was significantly reduced. This is consistent with the trend toward a reduced effectiveness of the less potent sAPP, and with our finding that other activators of local protein synthesis, such as Group I mGluRs, are also less effective at stimulating protein synthesis in aged synaptoneurosomes [2]. Thus, the deleterious effects of reduced sAPP␣ production during AD may be compounded by an aging-related reduction in the efficiency of sAPP␣ signalling, at least in the case of stimulating local protein synthesis. In summary, we have found that sAPP␣ reliably promotes protein synthesis in a synaptoneurosome preparation in a concentration-dependent manner that is also dependent on the activity of a number of protein kinases, especially PKG. The contribution of protein synthesis in the synaptodendritic compartment to neural plasticity has in recent times become recognised as another significant mechanism for neural plasticity, including LTP. BDNF enhances LTP through a synaptodendritic protein synthesis mechanism [19], and Group I mGluR-dependent LTD also requires local protein synthesis [16]. It is possible therefore, that sAPP␣ acts in a similar way by increasing the synthesis of proteins required for plastic changes. Identification of the specific changes to the synaptic proteome induced by sAPP␣ may indicate the crucial mechanism by which sAPP␣ mediates effects on the brain. Acknowledgements This research was supported by grants from the New Zealand Health Research Council to JMW, WCA and WPT and the Marsden Fund to JMW and WCA.
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