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Glucagon-like peptide-1 (GLP-1) is an endogenous intestinal peptide that enhances glucose-stimulated insulin secretion. Its natural cleavage product GLP-1 (9-36)amide possesses distinct properties and does not affect insulin secretion. Here we report that pretreatment of hippocampal slices with GLP-1(9-36)amide prevented impaired long-term potentiation (LTP) and enhanced long-term depression (LTD) induced by exogenous amyloid β peptide (Aβ1-42). Similarly, hippocampal LTP impairments in APP/PS1 mutant mice that model Alzheimer’s disease (AD) were prevented by GLP-1(9-36)amide. In addition, treatment of APP/PS1 mice with GLP-1 (9-36)amide at an age where they display impaired spatial and contextual fear memory resulted in a reversal of their memory defects. At the molecular level, GLP-1 (9-36)amide reduced elevated levels of mitochondrial-derived reactive oxygen species (ROS) and restored dysregulated Akt-GSK3β signaling in the hippocampus of APP/PS1 mice. Our findings suggest that GLP-1(9-36)amide treatment may have therapeutic potential for AD and other diseases associated with cognitive dysfunction.
Despite recent impressive progress in basic research, an effective therapy is still lacking for Alzheimer’s disease (AD), a devastating neurodegenerative disease that is the most common form of dementia and a leading cause of death in the elderly (Alzheimer’s Association, 2011). With the incidence of AD rising due to population aging, it is imperative to design and validate disease-modifying treatments targeting multiple cellular and molecular mechanisms (Dyer et al., 2006; Ma and Klann, 2012; Querfurth and LaFerla, 2010; Selkoe, 2011). The amyloid beta (Aβ) hypothesis, one of the leading theories for AD etiology, has resulted in current research focusing on lowering levels of brain Aβ, a small peptide derived from amyloid precursor protein (APP) (Haass and Selkoe, 2007). Meanwhile, the deleterious effects of Aβ on downstream signaling also are being vigorously pursued as potential disease-modifying targets (Pimplikar et al., 2010; Selkoe, 2011). One such target is mitochondrial-derived reactive oxygen species (ROS). There is substantial evidence linking ROS with neurodegenerative diseases (Lin and Beal, 2006), and it previously was reported that impairments in hippocampal synaptic plasticity and memory in AD model mice can be alleviated by decreasing mitochondrial ROS (Dumont et al., 2009; Massaad et al., 2009; Ma et al., 2011a).
Glucagon-like peptide-1 (GLP-1) is a primary incretin hormone released from the small intestine in response to nutrient ingestion. The majority of circulating “bioactive GLP-1” is in the form of GLP-1 (7-36)amide, which stimulates glucose-dependent insulin secretion (Baggio and Drucker, 2007; Drucker, 2001). GLP-1 has an extremely short half life (<2 min) and is rapidly cleaved into its “inactive” truncated form, GLP-1 (9-36)amide, by the ubiquitous proteolytic enzyme dipeptidyl peptidase-4 (DPP-4) (Baggio and Drucker, 2007). Recent studies have shown that GLP-1 (9-36)amide, initially considered to be an inactive degradation product of GLP-1, carries out important physiological functions distinct from its precursor (Tomas et al., 2011). For example, GLP-1 (9-36)amide does not affect either insulin secretion or glucose homeostasis (Rolin et al., 2004). Furthermore, GLP-1 (9-36)amide directly prevents increased mitochondrial production of superoxide induced by either high glucose or high free fatty acids in human arterial endothelial cells, whereas intact GLP-1, in the presence of inhibitors of the GLP-1-degrading proteases DPP-4 and neutral endopeptidase (NEP) 24.11, does not (Brownlee M., unpublished observation). GLP-1 (9-36)amide also exerts cytoprotective actions on mouse cardiomyocytes exposed to hydrogen peroxide (H2O2) (Ban et al., 2010). We therefore hypothesized that GLP-1 (9-36)amide could reduce hippocampal ROS and their deleterious effects on hippocampal synaptic function and memory in AD model mice.
We investigated the effects of the natural GLP-1 degradation product GLP-1 (9-36)amide on AD-associated molecular, synaptic, and memory deficits. AD-associated impairments of hippocampal synaptic plasticity and memory both were improved by GLP-1 (9-36)amide treatment. At the molecular level, elevated mitochondrial superoxide and dysregulated Akt-GSK3β signaling in the APP/PS1 mice were normalized by GLP-1 (9-36)amide. Our findings suggest that GLP-1 (9-36)amide has potential as a novel therapeutic for treatment of AD and other disorders associated with cognitive dysfunction.
All mice were housed in the Transgenic Mouse Facility of New York University, compliant with the NIH Guide for Care and Use of Laboratory Animals. The facility is kept on a 12-hour light/dark cycle, with a regular feeding and cage cleaning schedule. APP/PS1 transgenic mice were purchased from the Jackson Laboratory, being created via incorporation of a human/murine APP construct bearing the Swedish double mutation and the exon-9-deleted PSEN1 mutation (APPswe + PSEN1/ΔE9) (Jankowsky et al., 2001).
400 μm hippocampal slices were prepared using a vibratome as described previously (Hu et al., 2006). The slices were maintained at room temperature in artificial cerebrospinal fluid (ACSF) for at least two hours before removal for experiments. For electrophysiology experiments, monophasic, constant-current stimuli (100 μsec) were delivered with a bipolar silver electrode placed in the stratum radiatum of area CA3, and the field excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum radiatum of area CA1. LTP was induced with a high-frequency stimulation (HFS) protocol consisting of two one-second long 100 Hz trains, separated by 60 sec, delivered at 70-80 % of the intensity that evoked spiked fEPSPs (Tsokas et al., 2007). LTD was induced with 300 pulses of low-frequency stimulation (LFS) at 1 Hz (Li et al., 2009).
Except for the behavioral tests, drugs were prepared as stock solutions and were diluted to the final concentration before use. The final concentrations and sources of the drugs were as follows: GLP-1 (9-36)amide (100 pM, Bachem), MitoQ 10 Methanesulfonate (MitoQ, 500 nM, Antipodean Pharmaceuticals), Aβ(1-42) (100 μM, Bachem). Aβ(1-42) was prepared in water and stored at −20 °C for at least 24 hours before use at a final concentration of 500 nM. This type of preparation protocol yields ample Aβ oligomers (Ma et al., 2010). For behavioral tests, GLP-1(9-36)amide (500 ng/g/day) was freshly made and incorporated into Alzet micro-osmotic pump model 1002 (Alzet).
Upon completion of either drug or vehicle treatment, hippocampal slices were incubated with 5 μM MitoSOX™ Red, a mitochondrial superoxide indicator (Invitrogen, prepared as 5 mM stock solution immediately prior to the experiments) for 10 min. Slices then were fixed with ice-cold 4% paraformaldehyde in PBS overnight at 4°C. Slices were further cut into 40 μm sections and mounted onto pre-subbed slides with Vectashield mounting medium with DAPI (Vector laboratories). The sections were imaged using a Leica TCS SP5 confocal microscope (Leica, Germany) at 630X. All parameters (pinhole, contrast, gain, offset) were held constant for all sections from the same experiment.
For all behavioral tasks, APP/PS1 and wild-type littermates (male and female, 10–12 months of age) were used, and the experimenter was blind to the genotypes. Experiments were performed as described previously (Banko et al., 2005). The training paradigm for the hidden platform version of the Morris water maze consisted of four trials (60 sec maximum; interval 15 min) each day for five consecutive days. The probe trial was carried two hours after the completion of training on day five. The visible platform task consisted of four trials each day for two consecutive days with the escape platform marked by a visible cue and moved randomly between four locations. The trajectories were recorded with a video tracking system (Ethovision XT).
The training sessions for contextual and cued fear conditioning consisted of a three minute exploration period, followed by two conditioned stimulus (CS) – unconditioned stimulus (US) pairings separated by one minute (foot shock intensity: 0.6 mA, two second duration; tone: 85 dB white noise, 30 second duration). Contextual memory tests were performed in the training chamber 24 hours after training. Cued memory tests were performed in a distinct chamber 24 hours after training.
Lysates were prepared as described previously (Banko et al., 2005). Sample were loaded on 4-12% Tris-Glycine SDS-PAGE (Invitrogen) gels. Membranes were probed overnight at 4°C using primary antibodies for phospho-Akt (Ser473) and Akt (Cell Signaling), phospho-GSK3β (Ser9) and GSK3β (Cell Signaling), Aβ 6E10 (Covance), Actin (Sigma). Densitometric analysis was performed using Scion Image software (Scion Corporation).
Slices were fixed overnight in ice-cold 4% paraformaldehyde in PBS. Free-floating sections were blocked with 10% normal goat serum, 1% BSA, and 0.1% Na azide in PBS for two hours, and incubated with primary antibodies including: phospho-GSK3β (Ser9) (Cell Signaling), Aβ(1-42) (Millipore). Alexa Fluor 568 secondary antibodies (Invitrogen) were used. The sections were imaged using a Leica TCS SP5 confocal microscope (Leica) at 630X. All parameters (pinhole, contrast, gain, offset) were held constant for all sections from the same experiment.
Data are presented as mean ± SEM. Summary data were presented as group means with standard error bars. For comparison between two groups, a two-tailed independent Student’s t-test was used. For comparisons between multiple groups, ANOVA was used followed by individual post hoc tests when applicable. Error probabilities of p < 0.05 were considered statistically significant.
Previous studies have established that exogenous Aβ application results in impairment of hippocampal long-term potentiation (LTP) (Ma et al., 2011a; Shankar et al., 2008). We confirmed that LTP induced by high-frequency stimulation (HFS) was not expressed in hippocampal slices treated with Aβ(1-42) (500 nM) (Fig. 1A & B). Next we pre-treated slices with GLP-1 (9-36)amide (100 pM) for 30 min and found that HFS now was capable of inducing LTP in the presence of Aβ (Fig. 1A & B). We further observed that GLP-1 (9-36)amide by itself did not affect HFS-induced LTP when compared to control slices (Fig. 1A & B). These data indicate that GLP-1 (9-36)amide prevents Aβ-induced impairments in LTP.
Aβ also is known to facilitate long-term depression (LTD), another form of hippocampal synaptic plasticity (Kim et al., 2001; Li et al., 2009). We confirmed that Aβ facilitated induction of LTD with a sub-threshold low-frequency stimulation (LFS) protocol (Fig. 1C). In slices pre-treated with GLP-1 (9-36)amide, the Aβ-induced facilitation of LTD was absent (Fig. 1D). We recently reported that MitoQ, a mitochondria-targeted antioxidant (Smith and Murphy, 2010), improved hippocampal LTP deficits associated with Aβ (Ma et al., 2011a), which is similar to the effects of GLP-1 (9-36)amide on LTP shown here (Fig. 1A & B). Interestingly, Aβ-induced facilitation of LTD also was prevented by MitoQ (500 nM) (Fig. 1D). In addition, MitoQ alone did not alter fEPSPs following the sub-threshold LFS protocol (fEPSPs slope 60 min post-LFS: 99±14% of baseline, n=5). Taken together, these findings indicate that GLP-1 (9-36)amide and MitoQ can prevent Aβ-induced facilitation of LTD.
To further explore the effects of GLP-1 (9-36)amide on AD-related impairments in hippocampal synaptic plasticity, we examined slices from 10-12 month old APP/PS1 mutant mice, a well established AD mouse model (Jankowsky et al., 2001). We found that LTP impairments in APP/PS1 mice were reversed by GLP-1 (9-36)amide treatment (Fig. 1F). These results are in agreement with the protective effects of GLP-1 (9-36)amide on exogenous Aβ-induced alterations in hippocampal synaptic plasticity and suggest that GLP-1 (9-36)amide would be effective in reversing memory phenotypes displayed by APP/PS1 mutant mice.
We next examined whether GLP-1 (9-36)amide could rescue AD-associated learning and memory deficits. APP/PS1 mice (10-12 months old) were treated with GLP-1 (9-36)amide continuously for two weeks (500 ng/g/day) and tested on the hidden platform version of the Morris water maze, a hippocampus-dependent learning and memory task. During acquisition, wild-type mice showed a day-to-day decrease in escape latency whereas APP/PS1 mice displayed significantly higher escape latency (Fig. 2A). Furthermore, APP/PS1 mice spent significantly less time within the target quadrant on probe trial tests (Fig. 2B). Strikingly, the impaired spatial learning and memory deficits exhibited by the APP/PS1 mice were rescued by treatment with GLP-1 (9-36) amide, as indicated by reduced escape latency as well as increased target quadrant occupation that were comparable to those displayed by wild-type mice (Fig. 2A & B). To examine the possibility that the effects of GLP-1 (9-36)amide on improving spatial learning and memory in the APP/PS1 mice were attributable to effects on vision, motivation, and/or swimming ability, we tested mice on the visible platform task, which is independent of spatial learning and memory (Banko et al., 2005). There were no observable differences between the four groups of mice in the latency to find the visible platform (Fig. 2C).
We next investigated the effects of GLP-1 (9-36)amide on associative learning and memory by training mice with a standard conditioned fear memory paradigm (Banko et al., 2005). For both contextual and cue fear conditioning (tested 24 hours after training), APP/PS1 mutant mice exhibited impaired associative memory, as measured by decreased freezing time, compared to wild-type mice (Fig. 2D & E). The conditioned fear memory of GLP-1 (9-36) amide-treated APP/PS1 mutant mice was significantly improved as indicated by augmented freezing time in both contextual and cue fear conditioning tests (Fig. 2D & E). All together, these findings indicate that treatment with GLP-1 (9-36)amide can reverse memory deficits displayed by APP/PS1 mutant mice.
It was reported that peripheral injection of the diabetes drug liraglutide, a novel, non-degradable GLP-1 analog, has neuroprotective effects and decreases the levels of both APP and Aβ in APP/PS1 mice (McClean et al., 2011). Therefore, we examined whether GLP-1(9-36)amide could affect APP and Aβ levels in APP/PS1 mutant mice. Interestingly, we did not detect any significant difference in the brain levels of either APP or Aβ in APP/PS1 mice treated with GLP-1 (9-36)amide (Fig. 3A and B). Using immunofluorescence combined with confocal microscopy, we also examined the effects of GLP-1 (9-36)amide on levels of intra-neuronal Aβ. Consistent with the Western blot data, no differences in intra-neuronal Aβ staining was detected in hippocampal area CA1 (Fig. 3C). These findings indicate that treatment of APP/PS1 mutant mice with GLP-1 (9-36)amide for two weeks reverses memory defects without altering levels of either APP or Aβ in the hippocampus.
To begin to ascertain the underlying molecular mechanisms responsible for the effects of GLP-1(9-36)amide on AD-associated aberrations in synaptic plasticity and memory, we stained live slices with MitoSOX Red, a fluorogenic dye that is selective for the detection of superoxide in mitochondria. MitoSOX Red is chemically targeted to mitochondria and exhibits red fluorescence when oxidized (Ma et al., 2011a). Both wild-type hippocampal slices treated with Aβ and hippocampal slices from APP/PS1 mice demonstrated a significant enhancement in red fluorescence signal (Fig. 4A & B). This AD-associated increase in mitochondrial superoxide was reversed by GLP-1 (9-36) amide, as indicated by the normalizing of the MitoSOX fluorescent signal (Fig. 4A & B). These data provide evidence for a role of GLP-1(9-36)amide as a mitochondrial antioxidant, which may contribute to the correction of aberrant synaptic plasticity induced by Aβ and displayed by APP/PS1 mutant mice.
Mounting evidence suggests a critical role of GSK3β in regulating synaptic plasticity (Ma et al., 2011b; Peineau et al., 2008) and dysregulation of GSK3β signaling has been associated with AD (Hooper et al., 2008). Moreover, regulation of GSK3β activity has been linked to mitochondrial function and recently was proposed as a target of Aβ in AD pathogenesis (Jo et al., 2011). We demonstrated that GLP-1 (9-36)amide normalized mitochondrial ROS levels in the hippocampus of APP/PS1 mutant mice (Fig. 4B), so we asked whether normalization of hippocampal ROS could affect abnormal activation of GSK3β. Hippocampal slices from in vivo experiments in which APP/PS1 mice were treated with GLP-1 (9-36)amide were prepared and stained for the phosphorylation of GSK3β on serine 9, which leads to inhibition of its activity. Utilizing immunofluorescence combined with confocal microscopy, we observed that GSK3β activity was elevated in hippocampal CA1 regions of APP/PS1 mice as measured by decreased immunostaining of phosphorylation on serine 9, and was restored to levels comparable to that of wild-type mice after GLP-1 (9-36)amide treatment (Fig. 5). These findings suggest that increased GSK3β signaling in APP/PS1 mutant mice is induced by increased mitochondrial superoxide production, which is reversed by treatment with GLP-1 (9-36) amide.
To further explore the molecular signaling mechanisms impacted by GLP-1 (9-36) amide, we proceeded to examine hippocampal slices prepared from APP/PS1 mice that had been treated with GLP-1 (9-36)amide. In agreement with the immunofluorescence data (Fig. 5), we also found significant reductions of phosphorylated GSK3β levels in slices from APP/PS1 mice that were reversed by treatment with GLP-1 (9-36)amide (Fig. 6B). In addition, there was no change in GSK3β phosphorylation in GLP-1 (9-36) amide-treated slices from wild-type mice (Fig. 6A).
An established upstream regulator of GSK3β is Akt, a serine/threonine kinase that inhibits the activity of GSK3β via phosphorylation on serine 9 (Manning and Cantley, 2007). Akt regulation in synaptic plasticity also has been connected to mitochondrial activity (Li et al., 2010). We found that in slices from APP/PS1 mice, Akt activity was inhibited as indicated by decreased levels of phosphorylation on serine 473 (Fig. 6D), consistent with the observed increase in GSK3β activity. Importantly, the decreased phosphorylation of Akt was reversed by GLP-1 (9-36) amide, consistent with its action on reducing GSK3β activity (increasing its phosphorylation). Similar to GSK3β, slices from wild-type mice that had been treated with GLP-1 (9-36)amide showed no alteration in Akt phosphorylation (Fig. 6C). A recent study suggested a link between caspase-3 activation and Akt inhibition by Aβ (Jo et al., 2011). Consistent with this report, we found increased levels of active (cleaved) caspase-3 in hippocampal slices from APP/PS1 mice (211±45% of wild-type levels, n=4, p < 0.05 compared to wild-type). However, although there was a trend toward a decrease, the increased levels of active caspase-3 in the APP/PS mice were not altered significantly in slices treated with GLP-1 (9-36), (153+/−22% of wild-type levels, n=4, p=0.35 compared to the APP/PS1 group). Taken together, these findings suggest that GLP-1 (9-36)amide reverses abnormal activation levels of GSK3β in APP/PS1 mutant mice by reversing an ROS-induced decrease in Akt activity.
In the present study, we show for the first time that GLP-1 (9-36) amide, the main endogenously formed cleavage product of the incretin hormone GLP-1, reverses existing impairments in synaptic plasticity and deficits in learning and memory in a mouse model of AD. Hippocampal LTP impairments in APP/PS1 mutant mice were rescued by GLP-1(9-36)amide and treatment of APP/PS1 mice for two weeks with GLP-1 (9-36)amide reversed impairments in spatial and conditioned fear memory. At the molecular level, GLP-1 (9-36)amide reduced elevated levels of mitochondrial ROS and restored dysregulated Akt-GSK3β signaling in the hippocampus of APP/PS1 mice. These findings suggest that GLP-1 (9-36)amide treatment may have therapeutic potential for AD and other diseases associated with cognitive dysfunction. Interestingly, the rescue of synaptic plasticity failure and memory deficits in AD model mice does not correlate with either the reduction of brain Aβ or changes in APP, which is consistent with the recently proposed idea of targeting AD via an Aβ-independent strategy (Pimplikar et al. 2010).
The GLP-1 receptor is expressed widely in the CNS, including the hypothalamus, brainstem, and hippocampus (Alvarez et al., 2005). Thus, it is not surprising that activation of the GLP-1 in brain is associated with a wide variety of physiologic effects, including regulation of feeding, vagus nerve-mediated hormone secretion, hormone action, metabolite flux in peripheral tissues, and decreased arterial blood flow (Cabou et al., 2008; Knauf et al., 2005; Nogueiras et al., 2009; Turton et al., 1996). In normal rodents, signaling through the GLP-1 receptor either maintains or enhances learning and memory. Homozygous GLP-1 receptor knockout mice exhibit learning deficits, which is restored by hippocampal Glp1r gene transfer (During et al., 2003). Central administration of a DPP-4 resistant GLP-1 N-terminal nonapeptide, which activates the GLP-1 receptor, to wild-type rats also enhanced associative and spatial learning (During et al., 2003). The LTP impairments and histological changes that characterize AD were not described in these animal models.
Administration of the GLP-1 receptor agonist liraglutide, an acetylated form of GLP-1 (7-36), to APP/PS1 mice produced functional improvements in memory and other behavioral outcomes, increased synaptic plasticity, and reduced evidence of neuronal damage, plaques, and oligomer formation in the CNS (McClean et al., 2011). Although a reduction in Aβ was not observed in our experiments with GLP-1 (9-36)amide (Fig. 3), the treatment duration was 25% of that used in the liraglutide study. Liraglutide, unlike peptide GLP-1 receptor agonists such as either exendin-4 or its N-terminal homologue [Ser(2)]exendin (1-9) (During et al., 2003), shares a potential C-terminal mitochondrial-targeting sequence with GLP-1(9-36)amide (Tomas et al., 2011). Furthermore, both GLP-1(9-36)amide and liraglutide share the same potential NEP cleavage site. It has been proposed that the nonapeptide remaining after DPP-4 and NEP cleavage targets mitochondria and suppresses oxidative stress (Tomas et al., 2011). Further studies will be required to test this proposition.
The overall mechanism by which GLP-1 (9-36)amide corrects aberrant synaptic plasticity and memory deficits in AD model mice requires further elucidation. However, the observation that GLP-1 (9-36)amide normalizes increased levels of mitochondrial ROS in the APP/PS1 mice (Fig. 4B) is consistent with a body of data linking mitochondrial ROS to synaptic pathology associated with aging and neurodegenerative diseases (Massaad and Klann, 2011). It has been proposed that high levels of ROS produced in pathological situations like AD causes synaptic dysfunction (Balaban et al., 2005; Ma and Klann, 2012). Direct evidence consistent with this idea comes from studies showing that the mitochondria-specific antioxidant MitoQ prevents both LTP (Ma et al., 2011a) and LTD (Fig. 1D) in the APP/PS1 AD model mice. However, MitoQ is poorly absorbed, whereas GLP-1-related peptides administered by nasal inhalation cross the blood brain barrier easily (During et al., 2003). Thus, GLP-1-related peptides may be easier to deliver as a therapeutic to reduce increased levels of mitochondrial-derived ROS in AD.
Mechanistically, the observation that GLP-1 (9-36)amide restores dysregulated Akt-GSK3β signaling in APP/PS1 mice (Fig. 5 & 6) is consistent with previous reports in the AD literature. Originally identified as a regulator of glycogen metabolism (Embi et al., 1980), GSK3 is now widely recognized as a critical signaling molecule involved in many cellular processes, and GSK3β dysfunction has been implicated in the pathogenesis of AD (Hooper et al., 2008; Jope and Johnson, 2004; Ma et al., 2011b). Importantly, recent studies show that abnormally high GSK3β activity undermines normal synaptic function and conversely, GSK3β inhibition facilitates LTP induction (Jo et al., 2011; Ma et al., 2010; Ma et al., 2011b; Peineau et al., 2007). GLP-1 (9-36)amide restored GSK3β inhibitory phosphorylation to normal levels in APP/PS1 mice (Fig. 5, 6A & B), consistent with its efficacy in ameliorating impaired synaptic plasticity associated with AD (Fig. 1). Given the current view that memory deficits in AD are due to synaptic failure (Selkoe, 2002), our studies with GLP-1 (9-36)amide suggest that this peptide may have therapeutic potential for AD and other diseases associated with cognitive dysfunction.
We thank Dr. Michael P. Murphy for generously providing MitoQ and Dr. Hanoch Kaphzan for excellent technical advice. We thank Alyse J. Wexler for technical assistance on mouse behavioral experiments, Dr. Emanuela Santini for advice concerning statistical analysis of the behavioral experiments, and all other members of the Klann laboratory for critical comments on the manuscript. This work was supported by National Institutes of Health grants NS034007 and NS047834, and Alzheimer’s Association Investigator grant IIRG-09-131756 to E.K.
Conflict of interest:
The authors declare no conflict of interest.
The authors declare no competing financial interests.