The Effects of Cardiotrophin-1 on Early Synaptic Mitochondrial Dysfunction and Synaptic Pathology in APPswe/PS1dE9 Mice

Abstract

The coexistence of neuronal mitochondrial pathology and synaptic dysfunction is an early pathological feature of Alzheimer’s disease (AD). Cardiotrophin-1 (CT-1) has been shown to exhibit impressive neuroprotective effects. Previous studies have shown positive effects of CT-1 on brain glucose metabolism and cognition in APPswe/PS1dE9 transgenic mice; however, little is known about the effects of CT-1 on early synaptic mitochondrial dysfunction and resultant synaptic pathology in the brain. In this study, 4-month-old transgenic mice with brain tissue-specific CT-1 expression were used alone or in combination with APPswe/PS1dE9 transgenic mice to evaluate the effect of CT-1 on synaptic mitochondrial dysfunction and resultant synaptic pathology, and cryptic memory deficits in the APPswe/PS1dE9 transgenic mice. The potential mechanism of action of CT-1 was also examined. Young CT-1×APPswe/PS1dE9 transgenic mice exhibited improvements in long-term learning and memory ability and ameliorations of synaptic mitochondrial/synaptic impairments compared to young APPswe/PS1dE9 transgenic mice. Moreover, CT-1 upregulated the expression of AMPAR and increased AMP-activated protein kinase (AMPK) activity in the hippocampus of APPswe/PS1dE9 transgenic mice. However, AMPK inhibition through shRNA knockdown of AMPKα blocked the neuroprotective effects of CT-1 on the expression of AMPAR and mitochondrial/synaptic dysfunction in Aβ-treated mouse neurons. These results suggest that CT-1 may be a potent candidate for the early prevention and treatment of AD.

Keywords

Alzheimer’s disease AMPK CT-1 mitochondrial pathology synaptic dysfunction

INTRODUCTION

Alzheimer’s disease (AD) is a degenerative neurological disease that is clinically characterized by progressive cognitive dysfunction [1, 2]. Synaptic failure is an early neuropathological hallmark in AD patients and AD animal models. Clinical and pathological studies have shown that the extent of synaptic deficits correlates with symptom severity in AD [3, 4]. Occurring along with synaptic failure, brain mitochondrial dysfunction is also an early manifestation of AD [5]. Human AD and AD animal models demonstrate mitochondrial pathologies including impaired oxidative phosphorylation [6 –8], increased generation/accumulation of free radicals [9 –11], and changes in mitochondrial dynamics [12, 13]. The coexistence of mitochondrial alterations with synaptic perturbation warrants investigation of a link between synaptic failure and mitochondrial pathology in AD.

Cardiotrophin-1 (CT-1), a member of the IL-6 family, is expressed not only in peripheral tissues but also in the postnatal and adult central nervous system [14]. CT-1 has been shown to exhibit potential neuroprotective effects in animal models of motor neurodegenerative disorders including amyotrophic lateral sclerosis [15], progressive motor neuropathy [16, 17], and spinal muscular atrophy [18] and spinal cord injuries [19]. Furthermore, CT-1 has been shown to protect against hydroxyl radical and nitric oxide induced neuronal injury in vitro [20] and in vivo [21]. Recently, interest in the role of CT-1 in metabolism has been increasing. Cardiotrophin-1 is known to be a key regulator of energy homeostasis, as well as the glucose and lipid metabolism [22, 23]. Mitochondria, the cellular power factory, act as central organelles in the regulation of aging and age-related neurodegeneration since they control cellular energy status. Synaptic mitochondria, a subpopulation of neuronal mitochondria, play a critical role in maintaining synaptic strength and activity, which are important mechanisms in the processes of learning and memory. Furthermore, several lines of evidences have indicated that synaptic mitochondrial dysfunction is among the earliest manifestations of AD [24 –26]. Previous studies have demonstrated that CT-1 might increase glucose uptake in the brain and ameliorate the disturbances in brain energy metabolism in APPswe/PS1dE9 transgenic mice [27]. In addition, CT-1 has been found to increase the expressions of the post-synaptic related proteins and alleviates obesity-induced cognitive impairment [28]. However, little is known about the effects of CT-1 on the early mitochondrial/synaptic dysfunction. Therefore, the purpose of the present study is to examine the effect of CT-1 on early synaptic mitochondrial dysfunction and synaptic impairment in APPswe/PS1dE9 transgenic mice and to identify potential mechanisms related to the protective effects of CT-1.

MATERIALS AND METHODS

Animals

APPswe/PS1dE9 double transgenic mice, which are on C57BL/6 background, carried the human APPswe (Swedish mutations K594N/M595L) and presenilin-1 with the exon-9 deletion (PS1-dE9) under control of the mouse prion protein promoter [29 –31]. The cDNA encoding human CT-1 was cloned into an expression plasmid under the PDGF beta-chain promoter. This construct was microinjected into the male pronuclei of fertilized mouse oocytes that were implanted into pseudo-pregnant females to generate the transgenic mouse lines. CT-1 transgenic mice were maintained on a C57BL/6J genetic background. CT-1 transgenic mice from the F2 generation were crossed with APPswe/PS1dE9 transgenic mice to generate CT-1×APPswe/PS1dE9 transgenic mice. Non-transgenic littermates and the APPswe/PS1dE9 transgenic mice were used as controls. Four-month-old non-transgenic mice, APPswe/PS1dE9 transgenic mice, CT-1 transgenic mice, and CT-1×APPswe/PS1dE9 transgenic mice, of mixed genders, were studied. All experiments were carried out blinded with respect to the genetic status of mice. All the mice were housed individually in plastic rodent cages and maintained on a 12 h light/dark cycle with ad libitum access to conventional standard rodent chow and water, with the constant temperature (23±1°C) and relative humidity (65%). Protocols were conducted according to the University Policies on the Use and Care of Animals and were approved by the Institutional Animal Experiment Committee of Henan University of Science and Technology, China.

Morris water maze

Spatial learning and memory was tested using the Morris water maze. The protocol for the Morris water maze test was modified from previously reported methods [32, 33]. Briefly, the apparatus included a pool with a diameter of 100 cm that was filled with opaque water at approximately 22±1°C. An escape platform (15 cm in diameter) was placed 0.5 cm below the water surface. Geometric objects with contrasting colors were set at the remote ends of the water tank as references. Room temperature was constant, and the lighting was even throughout the room. Spatial memory is assessed by recording the latency time for the animal to escape from the water onto a submerged escape platform during the learning phase. The mice were subjected to four trials per day for 5 consecutive days. 24 h after the learning phase, the mice swam freely in the water tank without the platform for 60 s, and the time spent in the region, and number of passes through the region and the quadrant of the original platform were recorded. To examine the effects of CT-1 on long-term spatial memory, mice underwent a second test on the 7th day after the initial test. Performance was monitored with a video tracking system (Noldus Ltd., Ethovision XT, Wageningen, The Netherland).

Mitochondria preparation

Synaptic and nonsynaptic mitochondria were isolated from tissue as previously described [34]. Hippocampus tissues were homogenized in ice cold isolation buffer (225 mM mannitol, 75 mM sucrose, 2 mM K₂PO₄, 0.1% BSA, 5 mM Hepes, 1 mM EGTA (pH 7.2)) with a Dounce homogenizer (Wheaton). The resultant homogenate was centrifuged at 1,300 g for 3 min, and the supernatant was layered on a 3 2-ml discontinuous gradient of 15, 23 and 40% (vol/vol) Percoll and centrifuged at 34,000 g for 8 min (flying time) on Beckman Coulter ultracentrifuge (Optima XPN-90 Ultracentrifuge). After centrifugation, the interface between 15 and 23% (Band containing synaptosomes) was collected. Additionally, the interface between 23 and 40% (containing nonsynaptic mitochondria) was removed and collected. The fractions were then resuspended in isolation buffer containing 0.02% digitonin and incubated on ice for 5 min. The suspensions were then centrifuged at 16,500 g for 15 min. The resulting loose pellets were washed for a second time by a centrifugation at 8,000 g for 10 min. Pellets were collected and resuspended in isolation buffer. Percoll density gradient centrifugation was performed as described above for a second time. The interface between 23 and 40% (mitochondria released from synaptosomes) was collected and resuspended in isolation buffer to centrifuge at 16,500 g for 15 min. The resultant pellet was resuspended in isolation buffer followed by a centrifugation at 8,000 g for 10 min. The final synaptic mitochondrial pellet was resuspended in isolation buffer and stored on ice during experiments. Protein concentration was determined using the Bio-Rad DC protein assay (Bio-Rad Laboratories).

Mitochondrial complex estimation

NADH dehydrogenase activity (Complex-I)

Complex-I was measured spectrophotometrically for hippocampus region by the method of King and Howard (1967). The method involves catalytic oxidation of NADH to NAD⁺ with subsequent reduction of cytochrome C. The reaction mixture contained 0.2 M glycyl glycine buffer pH 8.5, 6 mM NADH in 2 mM glycyl glycine buffer, and 10.5 mM cytochrome C. The reaction was initiated by the addition of requisite amount of solubilized mitochondrial sample and followed absorbance change at 550 nm for 2 min.

Succinate dehydrogenase (SDH) activity (Complex-II)

SDH was measured spectrophotometrically for hippocampus according to King (1967). The method involves oxidation of succinate by an artificial electron acceptor, potassium ferricyanide. The reaction mixture contained 0.2 M phosphate buffer pH 7.8, 1% BSA, 0.6 M succinic acid, and 0.03 M potassium ferricyanide. The reaction was initiated by the addition of mitochondrial sample and change in absorbance was recorded at 420 nm.

Cytochrome oxidase assay (Complex-IV)

Cytochrome oxidase activity was assayed in hippocampus region mitochondria as previously described [35]. The assay mixture contained 0.3 mM reduced cytochrome C in 75 mM phosphate buffer. The reaction was started by the addition of solubilized mitochondrial sample and change in absorbance was recorded at 550 nm.

Detection of mitochondrial ATP production

The ATP production in the isolated mitochondria was measured using a bioluminescent ATP detection kit (Promega, USA). After treatment, isolated mitochondria were immediately incubated with ATP synthesizing substrates (2.5 mM ADP, 1 mM L-malic acid and 1 mM pyruvate) for 5 min at 30°C before mixed with luciferin substrate and luciferase enzyme in the kit to produce bioluminesecence. The bioluminescence was assessed on a microplate reader (Envision, Perkin Elmer). The ATP level was calculated as described in the manual of the kit and normalized by control group.

Reactive oxygen species production

Mitochondrial ROS production was measured following incubation of isolated mitochondria with 25 μM 2,7 -dichlorodihydrofluorescein diacetate for 20 min and then the DCF fluorescence (excitation filter 485/20 nm, emission filter 528/20 nm) was read as previously described [36]. In short, 100 μg (0.8 mg/ml final concentration) of isolated mitochondria were added to 120 μl of KCl-based respiration buffer with 5 mM pyruvate and 2.5 mM malate added as respiratory substrates and 25 μM 2,7 -dichlorodihydrofluorescein diacetate. Mitochondrial ROS production in the presence of oligomycin (to increase ROS production) or FCCP (to decrease ROS production) were performed to ensure measurement values were within the range of the indicator.

Membrane potential measurements

A 200 μM stock solution of JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide) was made using DMSO as the solvent. The assay buffer contained mitochondrial isolation buffer with the addition of 5 mM pyruvate and 5 mM malate.150 μl of assay buffer and 20 μl (1.2 mg/ml final concentration) of mitochondria were added to the wells of a 96 well black, clear bottom microplate (Corning) followed by the addition of 1 μM JC-1 and mixed gently. The microplate was covered with aluminum foil and left at room temperature for 20 min before reading. Fluorescence (excitation 530/25 nm, emission 590/35 nm) was then measured.

Synaptic density measurement

Synaptic density of cultured neurons or brain slices was measured by counting PSD95 and vGlut1-labelled clusters attaching to neuronal dendrites and presented as the number of synapses per micron of dendrite as previously described [37]. Briefly, after fixation in 4% paraformaldehyde for 30 min, neurons were blocked in 5% goat serum for 30 min at room temperature. PSD95, vGlut1, and Neuron were labelled by rabbit anti-PSD95 (Cell Signaling, 1:300), guinea pig anti-vGlut1 (Synaptic System, 1:500), and mouse anti-Neuron (Millipore, 1:500) followed by goat anti-rabbit IgG conjugated with Alexa 594 (Invitrogen, 1:500), goat anti-guinea pig IgG conjugated with Alexa 647 (Invitrogen, 1:500), and goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen, 1:500), respectively. Images were collected under a Nikon confocal microscope followed by three-dimensional reconstruction by using Nikon-Elements advanced Research software. The synapses were defined by colocalization of vGlut1 and PSD95.

Oligomeric Aβ preparation

Aβ_1–42 peptide (GenicBio) was diluted in 1,1,1,3,3,3,-hexafluoro-2-propanol to 1 mM using a glass gas-tight Hamilton syringe with a Teflon plunger. The clear solution was then aliquoted in microcentrifuge tubes, and it was dried by vaporation in the fume hood. Peptide film was diluted in DMSO to 5 mM and sonicated for 10 min in bath sonicator. The peptide solution was resuspended in cold HAM’S F-12 to 100 mM and immediately vortexed for 30 s. The solution was then incubated at 4°C for 24 h.

Mouse neuron culture

Hippocampus were dissected from day 0-1 pups in cold Hank’s buffer (without Ca^2 + and Mg^2 +), dissociated with 0.05% trysin at 37°C for 15 min followed by 10 times trituration in ice cold neurobasal A medium. Cells were then passed through 40 mm mesh cell strainer (Corning) and centrifuged for 2 min at 200 g. The pellet was gently resuspended in neuron culture medium (neurobasal A with 2% B27 supplement, 0.5 mM L-glutamine, 50 U ml^-1 penicillin, and 50 mg ml^-1 streptomycin) and plated on poly-D-lysine- (Sigma-Aldrich) coated culture plates (Corning) or Lab-Tek chamber slides (Nunc, 177445) with an appropriate density.

2.10 AMPKα knockdown in mouse primary neuron

The lentiviral shRNAs against mouse AMP-activated protein kinase (AMPK)α were designed, synthesized and verified by Genepharm Co. (Shanghai, China). The scramble lentiviral shRNA (Santa Cruz) was used as a control. Mouse primary neurons were cultured for 3 days before infection with lentivirus at a multiplicity of infection (m.o.i.) of 5. The virus containing medium was removed after 2 h and fresh culture medium was replaced to continue culturing. Neurons were treated with 500 nM Aβ for 24 h and collected for experiments after a further 7 days in culture.

2.11 Western blot analysis

Following behavioral assessment, animals were deeply anesthetized with isoflurane and sacrificed by decapitation. The hippocampus was directly homogenized in RIPA buffer containing 0.1% PMSF and 0.1% protease inhibitor cocktail (Sigma, MO, USA). The lysates were centrifuged at 14,000 g for 30 min at 4°C and the supernatant was used for protein analyses. The protein concentration in supernatants was determined using the BCA method. Equal amounts of soluble protein were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Immobilon NC; Millipore, Molsheim, France). Immunoblotting was performed with antibodies specific for GluA1(1:500), NR1(1:500), α-Tubulin (1:2000) (Abcam), phospho-AMPKα (Thr 172, 1:1000), and AMPK (1:1000) (Cell Signaling Technology). Primary antibodies were visualized using anti-rabbit HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.) and a chemiluminescent detection system (Western blotting Luminal Reagent; Santa Cruz Biotechnology, Inc.). Variations in sample loading were normalized relative to GAPDH.

2.12 Statistical analysis

All data were expressed as the mean±SEM. Data were analyzed by Student’s t test or one-way ANOVA followed by Bonferroni post-hoc test for multiple comparisons. The difference between the first frequency and the second frequency was analyzed with a paired ANOVA test. All analyses were performed with SPSS statistical package (version 13.0 for Windows, SPSS Inc., USA). Differences were considered significant at a p value < 0.05.

RESULTS

CT-1 improves long-term memory of APPswe/PS1dE9 transgenic mice in the Morris water maze

To identify cryptic changes in spatial reference learning and memory function, 4-month-old mice underwent two probe tests with a seven-day interval. Previous studies done by our group have demonstrated that there was a small, but significant difference in the crossing target number in the first probe test of 4-month-old mice. However, when these mice were subjected to a memory retrieval test 7 days after the first probe test, differential forgetting index ((value of first frequency –value of second frequency)/value of first frequency×100%) were unmasked. As shown in Fig. 1A, there was a significant overall group difference in the forgetting index (F (3, 48) = 8.01, p < 0.01) among the four groups in the two probe trials. The forgetting index in the APPswe/PS1dE9 transgenic was 76.3 % higher compared to the WT controls (p < 0.01), while the forgetting index of the CT-1×APPswe/PS1dE9 mice was a significant 33.3% (p < 0.05) lower compared to the APPswe/PS1dE9 transgenic mice. There was no significant difference in the forgetting indexes between WT mice and CT-1 transgenic mice (p > 0.05). In addition, in the second probe test, there was no significant difference in swimming speed (Fig. 1B) or path length (Fig. 1C) between four groups. These results suggest that the positive effects of CT-1 on improved long-term memory in APPswe/PS1dE9 mice were not attributable to non-cognitive factors (decreased activity and/or motivation abnormalities).

Fig.1

The effect of CT-1 on long-term cognitive function as tested using the Morris water maze. Non-transgenic littermates (WT), vehicle control (APP), CT-1 transgenic group (CT-1), and CT-1 crossed with APPswe/PS1dE9 mice (CT-1×APP) were included. The forgetting index (A), the swimming speed (B), and the path (C) in the first probe test were measured. All data are presented as mean±SEM. Analysis was performed using one-way ANOVA with an LSD post hoc test between groups. (WT mice, n = 12; APPswe/PS1dE9 mice, n = 11; CT-1 mice, n = 12; CT-1×APPswe/PS1dE9 mice, n = 14. **p < 0.01, WT mice versus APPswe/PS1dE9 mice; ^#p < 0.05, CT-1×APPswe/PS1dE9 mice versus APPswe/PS1dE9 mice).

CT-1 ameliorates synaptic mitochondrial impairments in APPswe/PS1dE9 mice

To explore the effects of CT-1 on the synaptic mitochondrial dysfunction, we first separated synaptic and nonsynaptic mitochondria from APPswe/PS1dE9 mice at 4 months of age in order to mimic the amyloidopathy and behavioral changes typical of the early-stage of AD. The purity of isolated mitochondria was verified via the detection of a high abundance of mitochondrial protein voltage-dependent anion channel (VDAC) without β-actin or synaptic vesicle contamination (Supplementary Figure 1). Compromised mitochondrial oxidative phosphorylation (OXPHOS) as indicated by a reduction in mitochondrial enzyme complex activity, decreased MMP, ATP deficiency, and enhanced ROS damage are usually direct consequences of mitochondrial dysfunction and have been demonstrated to mitochondrial defects in individuals with AD [7, 38] and in AD animal models [8, 9]. The 4-month-old young APPswe/PS1dE9 mice had an obvious synaptic mitochondrial defects, exhibiting the alterations in mitochondrial enzyme complex activity as indicated by a 39.3% decrease in NADH dehydrogenase activity (p < 0.05), a 40.9% decrease in succinate dehydrogenase activity (p < 0.05), and a 45.5% decrease in COX activity (p < 0.05), a 50.0% decrease in ATP production (p < 0.01), a 1.08-fold increase in ROS production (p < 0.01), and a 52.9% decrease in MMP (p < 0.01) when compared with control mice. In comparison with APPswe/PS1dE9 mice, CT-1 significantly attenuated mitochondrial damage as exhibited by in increasing mitochondrial enzyme complex activity (Fig. 2A), increasing ATP production (Fig. 2B), decreasing ROS production (Fig. 2C), and increasing MMP (Fig. 2D) by 33.3–63.0%, 40.0%, 34.1%, and 43.9%, respectively, in synaptic mitochondria of APPswe/PS1dE9 mice. However, there were no significant nonsynaptic mitochondrial impairments in the APPswe/PS1dE9 mice (Supplementary Figure 2), suggesting that synaptic mitochondria are earlier targets and more susceptible to Aβ.

Fig.2

CT-1 attenuates synaptic mitochondrial dysfunction in APPswe/PS1dE9 mice. Synaptic mitochondria from 4-month-old WT, APPswe/PS1dE9, CT-1, and CT-1×APPswe/PS1dE9 mice were separated and collected. Mitochondrial enzyme complex I, II, and IV activity (n = 5–8 per group) (A), mitochondrial ATP production (n = 6 per group) (B), ROS production (n = 5-6 per group) (C), and MMP (n = 5-6 per group) (D) were examined. All data are presented as mean±S.E.M. (*p < 0.05, **p < 0.01, WT mice versus APPswe/PS1dE9 mice; ^#p < 0.05, ^##p < 0.01, CT-1×APPswe/PS1dE9 mice versus APPswe/PS1dE9 mice).

CT-1 increases synaptic density in APPswe/PS1dE9 mice

Mitochondria play a critical role in maintaining synaptic and neuronal function. To determine the effects of CT-1 on the synaptic dysfunction in the APPswe/PS1dE9 mice, synaptic density was measured by staining for PSD95 and vesicular glutamate transporter 1 (vGlut1). The young APPswe/PS1dE9 mice showed a significant 27.0% reduction in synaptic density when compared with control mice (p < 0.05). However, synaptic density was a significant 24.6% higher in the hippocampal CA1 area of C1xAPPswe/PS1dE9 compared to APPswe/PS1dE9 mice (p < 0.05) (Fig. 3). In addition, there were small, but significant differences in synaptic densities, among the four groups, in the hippocampal CA2 areas, CA3 areas, and dentate gyrus (data not shown).

Fig.3

CT-1 increases synaptic density in APPswe/PS1dE9 mice. Brain tissue from WT, APPswe/PS1dE9 mice, CT-1, and CT-1×APPswe/PS1dE9 mice were utilized in standard pathological procedures. Synapses were visualized by immunostaining with antibody vGLUT1 (blue) and PSD95 (red) to identify the pre- and postsynaptic components of synapses, respectively. Neurons were determined by Neuron (green) (scale bars, 50 μm) (A), and the quantitative analysis of synapses was tabulated (B). All data are presented as mean±S.E.M. (n = 5, *p < 0.05, WT mice versus APPswe/PS1dE9 mice; ^#p < 0.05, CT-1×APPswe/PS1dE9 mice versus APPswe/PS1dE9 mice).

CT-1 upregulates AMPARs through increasing AMPK activity

A-amino-3-hydroxy-5-methyl-4-isoxazolepropio-nic acid receptors (AMPARs) are major groups of glutamate receptors that mediate the majority of fast excitatory transmissions and synaptic plasticity in the brain [39]. To investigate the potential effects of CT-1 overexpression on AMPAR in mice, the CT-1 protein level in the hippocampus was examined. The reduced CT-1 protein usually found in AD-affected brains from APPswe/PS1dE9 transgenic mice was significantly ameliorated by CT-1 overexpression in the CT-1×APPswe/PS1dE9 mice (Supplementary Figure 3). In addition, the total number of GluA1 subunits was significantly higher (39.6%) in the hippocampi of CT1×APPswe/PS1dE9 mice compared with APPswe/PS1De9 mice and was lower in APPswe/PS1De9 mice compared to control mice (p < 0.05). However, CT-1 did not significantly alter the expression of the N-Methyl-d-aspartic acid (NMDA) receptor subunit GluN1 (Fig. 4A) and postsynaptic scaffolding protein, PSD95 (Supplementary Figure 4). In addition to total GluA1 subunits, the amount of synaptic GluA1 in mouse neurons was also examined. Primary hippocampal neurons from day 0-1 control mice (Control) and CT-1 overexpression mice (CT OE) were treated with oligomeric Aβ_1 - 42, and then immunostained for GluA1 together with PSD95 as a synaptic marker. The results showed that CT-1 overexpression significantly ameliorated the Aβ-mediated synaptic GluA1 reduction in control neurons (p < 0.01) (Fig. 4B). Recent studies have demonstrated that AMPK activation is involved in the regulation of the expression of AMPAR protein [40, 41]. Next, phosphorylated AMPK at Thr172, an indicator of AMPK activation, was investigated. The pAMPK172 level was 55.1% higher in CT-1×APPswe/PS1dE9 mice compared with APPswe/PS1dE9 mice (p < 0.05); however, the total AMPK protein levels were not affected (Fig. 4A). To further confirm the involvement of AMPK in the effects of CT-1 the expression of AMPARs, the shRNA strategy was used to knockdown AMPKα to block AMPK activity in mouse primary neurons. AMPKα knockdown abolished AMPK phosphorylation and the resultant AMPAR increase in neurons (Fig. 4C).

Fig.4

CT-1 upregulates AMPARs through increasing AMPK activity. The relative level of GluN1, GluA1, and p-AMPK(172) was detected by western blotting of hippocampal tissues of WT, APPswe/PS1dE9, CT-1, and CT-1×APPswe/PS1dE9 mice and quantitative analysis of GluN1, GluA1, and p-AMPK(172) using Tubulin, Tubulin and AMPK for normalization, respectively. n = 5 per group, *p < 0.05, WT mice versus APPswe/PS1dE9 mice; ^#p < 0.05, CT-1×APPswe/PS1dE9 mice versus APPswe/PS1dE9 mice (A). Primary hippocampal neurons from day 0-1 control mice (Control) and CT-1 overexpression mice (CT OE) were exposed to 500 nM oligomeric Aβ_1 - 42 for 24 h and then collected. Synaptic GluA1 (red) puncta overlapping vGLUT1 (blue) were measured. Neuronal dendrites were identified by staining MAP2 (green). Scale bars, 5 μm. n = 21–36 neurons collected from at least three independent experiments, **p < 0.01 versus the control group; ^#p < 0.05, ^##p < 0.01 versus the control + Aβ group (B). Primary neurons cultured from day 0-1 control mice (Control) and CT-1 overexpression mice (CT OE) were infected with lentivirus carrying AMPKα shRNA or control AMPKα shRNA and then treated with 500 nM oligomeric Aβ for 24 h after 7 days of infection. The relative levels of GluA1 and p-AMPK (172) were detected by western blotting (C). All data are presented as mean±S.E.M. (n = 28–36 neurons from at least three independent experiments, *p < 0.05, **p < 0.01 versus the control group; ^#p < 0.05, versus the control + Aβ group).

AMPK activity is required for the beneficial effects of CT-1 on Aβ-induced mitochondrial/synaptic dysfunction in mouse neurons

The connection between AMPK activation and the positive effects of CT-1 on mitochondrial dysfunction was further addressed via mitochondrial assays. Inhibition of AMPKα activity, via AMPKα shRNA knockdown, almost abolished the protective effects of CT-1 overexpression on impaired mitochondrial function, as indicated by reduced cytochrome c oxidase activity, lowered ATP production, increased ROS, and decreased MMP in Aβ-treated neurons (Fig. 5A-D). Given the critical role of the mitochondria in sustaining synaptic transmission and plasticity and the involvement of AMPK activation in the attenuation of mitochondrial defects by CT-1 overexpression, the role of AMPK activation in the protection provided by CT-1 overexpression on synaptic function against Aβ toxicity was explored. Synaptic density was analyzed via immunofluorescent staining of PSD95 and vGlut1 in control and CT-1 OE neurons exposed to vehicle- or oligomeric Aβ_1 - 42 treatment. There was a significant reduction in synaptic density in oligomeric Aβ_1 - 42-treated control neurons compared to vehicle-treated control neurons. In sharp contrast, the Aβ-induced synaptic loss was significantly ameliorated by CT-1 overexpression. CT-1 overexpression by itself did not markedly affect baseline levels of synaptic density (Fig. 6A-B); however, the protective effects of CT-1 overexpression on synaptic density were blocked by AMPKα knockdown in neurons.

Fig.5

AMPK activity is required for the beneficial effects of CT-1 on Aβ-induced mitochondrial defects in mouse neurons. Primary neurons cultured from day 0-1 control mice (Control) and CT-1 overexpression mice (CT OE) were infected with lentivirus carrying AMPKα shRNA or control AMPKα shRNA, and then treated with 500 nM oligomeric Aβ for 24 h after 7 days of infection. Cytochrome c oxidase activity (A), mitochondrial ATP levels (B), ROS production (C), and MMP (D) were examined. All data are presented as mean±S.E.M. (n = 26–36 neurons from at least three independent experiments, **p < 0.01 versus the control group; ^##p < 0.01 versus the control + Aβ group).

Fig.6

AMPK activity is required for the beneficial effects of CT-1 on Aβ-induced synaptic dysfunctions in mouse neurons. Primary neurons cultured from day 0-1 control mice (Control) and CT-1 overexpression mice (CT OE) were infected with lentivirus carrying AMPKα shRNA or control AMPKα shRNA, and then treated with 500 nM oligomeric Aβ for 24 h after 7 days of infection. Synapses were visualized by staining with antibody PSD95 (red) and vGLUT1 (blue) (A) and quantitative analysis of synapses was performed. Neuronal dendrites were determined by MAP2 (green). Scale bar, 5 μm. (n = 26–36 neurons from at least three independent experiments, **p < 0.01 versus the control group; ^##p < 0.01 versus the control + Aβ group) (B). All data are presented as mean±S.E.M.

DISCUSSION

In this study, the effects of CT-1 on mitochondrial and synaptic dysfunction were investigated in APPswe/PS1dE9 mice. The results showed that CT-1 improved long-term memory deficits, ameliorated the mitochondrial/synaptic pathology, and increased the AMPK activity in this AD animal model. Importantly, the inhibition of AMPK activation blocked the beneficial effects of CT-1 on the mitochondrial/synaptic deteriorations in Aβ-insulted mouse neurons.

Mitochondrial dysfunction has been found in AD patients [42 –45] and AD animal models [46 –48] as well as in Aβ-insulted cell lines [49 –51]. Synaptic mitochondria are a subpopulation of neuronal mitochondria especially residing at synapses. Due to their extremely physical proximity to synapses, synaptic mitochondria play a critical role in maintaining synaptic strength and activity [52]. Increasing evidence has shown that synaptic mitochondrial dysfunction is among the earliest manifestations of AD and occurs prior to severe synaptic injury in early-stage AD [26 , 54] as well as in young AD animal models [24 , 55]. Consistent with this, results of the current study also demonstrate synaptic mitochondrial dysfunction, as indicated by the compromised OXPHOS, the ATP deficiency, the increase in ROS, and the impaired MMP in young APPswe/PS1dE9 mice. AMPK, a key cellular regulator of energy metabolism [56], has been implicated in the regulation of mitochondrial function. Activation of AMPK, or overexpression of constitutively activated AMPK, can enhance mitochondrial biogenesis and improve mitochondrial function in cell lines [57 –59] and animal models [60, 61]. Conversely, the inhibition of AMPK leads to an increase in ROS production, a decrease in mitochondrial biogenesis and thus worsens mitochondrial dysfunction [62, 63]. It is well known that CT-1 interacts with the gp130/leukemia inhibitory factor receptor beta heterodimer and activates at least three different downstream signaling pathways, including the JAK/signal transducer and activators of transcription 3 (STAT3), the PI3K/AKT and the Src-ERK pathways [64]. These pathways have also been shown to be important in CT-1-mediated neuronprotection [65, 66], though no obvious alterations in the levels of p-AKT, p-STAT3, or p-ERK were found in CT-1×APPswe/PS1dE9 transgenic mice. In the present study, CT-1 attenuated synaptic mitochondrial damage (Fig. 2) and decreased AMPK activity (Fig. 4) in young APPswe/PS1dE9 mice compared with control mice. CT-1 has been shown to have protective effects on mitochondrial function [22, 67]. It was recently reported that AMPK activation is involved in the protective effects of CT-1 [22 , 69]. Furthermore, the inhibition of AMPK activity can reverse the beneficial effects of CT-1 on synaptic mitochondrial dysfunction. It could be that CT-1 attenuates impaired mitochondrial dysfunction by increasing the activity of AMPK.

Considering that mitochondrial dysfunction has been identified as a causative factor of synaptic failure in AD, therefore, supporting mitochondrial function seems to be a viable strategy to protect synaptic strength and plasticity to delay the cognitive decline in AD patients [5, 39]. It has been shown that synaptic dysfunction is paralleled by reduced mitochondrial function in Aβ-exposed mouse neurons and in young APPswe/PS1dE9 mice. CT-1 mitigated the Aβ-induced mitochondrial dysfunction and further preserved synaptic function as evidenced by the measures of synaptic density and synaptic transmission. Furthermore, the protective effects of CT-1 on synaptic dysfunction were blocked by the inhibition of AMPK activity, suggesting that the AMPK activation is involved in the positive effect of CT-1 on synaptic activity.

AMPK has been reported to act as a modulator of long-term potentiation [70], and is required for the memory formation [71]. Recent studies have demonstrated a link between the bioenergy sensor AMPK and AMPAR regulation [41, 42]. AMPAR, one of the most important excitatory receptors in the brain, plays a crucial role in synaptic transmission and brain function [40]. Alternations in AMPAR synaptic accumulation serve as the molecular mechanisms underlying the expression of synaptic transmission and synaptic activity [72 –74]. Aberrant AMPAR function and expression have direct impacts on synaptic plasticity and cognitive function [75, 76]. In the present study, CT-1 enhanced AMPAR expression and synaptic accumulation; however, the increase in AMPAR expression was blocked through the inhibition of AMPK activity in Aβ-insulted mouse neurons. Based on these results, it may be that the positive effect of CT-1 on cognition might benefit from attenuating the mitochondrial defects and ameliorating the synaptic dysfunction, by enhancing AMPAR expression via activating AMPK activity. The abundance and turnover of AMPAR are highly regulated by many different molecules and signaling pathways [75, 76]. However, the details regarding the mechanisms related to modulation of AMPK and expression of AMPAR were not examined in this study and should be further investigated.

In conclusion, CT-1 reverses long-term memory cognitive deficits through improving mitochondrial/ synaptic dysfunction in the mouse brain. Based on these findings, CT-1 could be a potential candidate for the prevention and treatment of cognitive deficits in AD.

Footnotes

ACKNOWLEDGMENTS

The present work was supported by National Natural Science Foundation of China (U1304806 and 81601225), the Young Backbone Teachers Assistance Scheme of Henan Province Colleges and Universities.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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