Cardiotrophin-1 (CTF1) ameliorates glucose-uptake defects and improves memory and learning deficits in a transgenic mouse model of Alzheimer's disease

Pharmacology, Biochemistry and Behavior 107 (2013) 48–57

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Cardiotrophin-1 (CTF1) ameliorates glucose-uptake defects and improves memory and learning deficits in a transgenic mouse model of Alzheimer's disease Dongmei Wang a, b, Xiaoying Li a, Kai Gao a, Dan Lu a, Xu Zhang a, Chunmei Ma a, Fei Ye c,⁎, Lianfeng Zhang a,⁎⁎ a Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences & Comparative Medical Center, Peking Union Medical College, China b Department of Pathogen Biology, Medical College, Henan University of Science and Technology, Luoyang, China c Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, China

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Article history: Received 14 July 2012 Received in revised form 28 February 2013 Accepted 7 March 2013 Available online 26 March 2013 Keywords: CTF1 APPswe/PS1dE9 Transgenic mice Cognitive deficits Glucose uptake

a b s t r a c t Cardiotrophin-1 (CTF1) has been reported to act as a trophic factor for a few neurons, such as sensory, cholinergic, dopaminergic, motor and cortical neurons. Studies have indicated that CTF1 delays degenerative disease progression in motor neuron disease. However, little is known about the effects of CTF1 on degenerative disease in the brain. We have shown that expression of CTF1 is strongly down-regulated in the brain of the APPswe/PS1dE9 transgenic mouse model of Alzheimer's disease (AD). Transgenic mice with brain tissue-specific CTF1 expression alone or in combination with APPswe/PS1dE9 transgenic mice were produced to study the effects of CTF1 on AD. CTF1 expressing APPswe/PS1dE9 transgenic mice exhibited improvements in learning and memory, less severe abnormalities in locomotor activity, reduced scattered senile plaques and ameliorated disturbances of brain energy metabolism compared to APPswe/PS1dE9 transgenic mice. Furthermore, CTF1 inhibited the activity of glycogen synthase kinase-3β (GSK-3β) in SH-SY5Y cell line and in the brain tissues of APPswe/PS1dE9 transgenic mice. The transgenic expression of CTF1 compensated for the loss of CTF1 expression and brought about a marked improvement on cognitive functioning in the APPswe/PS1dE9 transgenic mouse model of Alzheimer's disease, suggesting that the inhibition of GSK-3β activity might play an important role. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved.

1. Introduction Alzheimer's disease (AD) is a degenerative neurological disease clinically characterised by progressive cognitive dysfunction (Holmes et al., 2008; Mattson, 2004; Stokin et al., 2005). Previous studies have shown that deficient energy metabolism is involved in the pathogenesis and progression of AD (Kim et al., 2005; Mattson, 2004). Recently, accumulating evidence from epidemiological (de Leon et al., 2001; Mosconi et al., 2008) and clinical studies of family history and genetic susceptibility to AD (Lee et al., 2003; Mosconi et al., 2003; Small et al., 1995) has indicated that altered glucose metabolism presents long before the onset of measurable cognitive decline and may represent an early event in AD (Kim et al., 2005; Mosconi et al., 2008; Mosconi et al., 2006), suggesting altered glucose metabolism plays a pivotal role in triggering neuronal

Abbreviations: AD, Alzheimer's disease; Aβ, amyloid-β; CTF1, Cardiotrophin-1; FDG, Fluoro-2-deoxy-D-glucose; GSK-3β, Glycogen synthase kinase-3β; GLUT-1, glucose transporter-1; GLUT-3, glucose transporter-3; PET, positron emission tomography. ⁎ Correspondence to: F. Ye, Building 1, Xian Nong Tan Street, Beijing 100050, China. Tel.: +86 10 83150495; fax: +86 10 63017751. ⁎⁎ Correspondence to: L. Zhang, Building 5, Panjiayuan Nanli, Chaoyang District, Beijing 100021, P. R. China. Tel.: +86 10 87778442; fax: +86 10 67710812. E-mail addresses: [email protected] (F. Ye), [email protected] (L. Zhang).

death and cognitive decline (Kapogiannis and Mattson, 2011; Santos et al., 2010). Cardiotrophin-1 (CTF1, also known as CT1), a member of the IL-6 family, has been shown to protect cardiac cells against ischemia/ reperfusion injury (Freed et al., 2005; Liao et al., 2002), inhibit apoptotic cell death (Peng et al., 2010) and enhance the liver regeneration after hepatectomy (Ho et al., 2006; Iniguez et al., 2006). CTF1 is expressed at high levels in the embryonic limb bud and is secreted by differentiated myotubes (Pennica et al., 1996a; Sheng et al., 1996). This has been shown to exhibit impressive neuroprotective effects and delay the procession of motor neuron degenerative disorder by prolonging the median neuronal survival time, improving motor function and promoting regeneration in neonatal rat motor neurons (Pennica et al., 1996a) in mouse models of amytrophic lateral sclerosis (ALS) (Bordet et al., 2001), progressive motor neuropathy (PMN) (Bordet et al., 1999; Lesbordes et al., 2002) and spinal muscular atrophy (SMA) (Lesbordes et al., 2003) and in adult rats with spinal cord injuries (Zhang et al., 2003). CTF1 is also expressed in the postnatal and adult CNS (Gard et al., 2004; Sheng et al., 1996). CTF1 treatment protects cortical neuronal cells against free radicalinduced oxidative stress in vitro (Wen et al., 2005), excitatory damage in vitro (Toth et al., 2002) and sodium nitroprusside (SNP)-induced neurotoxic effects in vitro and in vivo (Peng et al., 2010). It has been reported that members of the CTF1 family function as gp130 receptor ligands

0091-3057/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pbb.2013.03.003

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to participate in the regulation of glucose metabolism (Febbraio, 2007; Marcos-Gomez et al., 2008). The APPswe/PS1dE9 transgenic mouse model of Alzheimer's disease exhibits senile plaque formation and amyloid neuropathy (Garcia-Alloza et al., 2006), early glucose uptake defects (Wang et al., 2012; Yuan et al., 2011) and progressive behavioural deficits (Savonenko et al., 2005; Zhang et al., 2011). This mouse model can mimic the progression of the pathology of human AD and therefore is used to comprehensively evaluate the Aβ-induced neurotoxicity, energy metabolism and behavioural deficits. In the present study, we observed down-regulation of CTF1 expression in a transgenic mouse model of AD, suggesting that CTF1 may be involved in the pathogenesis of AD. However, the potential effects of CTF1 and the mechanisms through which it may contribute to degenerative diseases within the CNS have not been evaluated. Thus, the present study sought to investigate the effects of CTF1 on the development of AD by establishing brain tissue-specific CTF1 transgenic mice and crossing them to APPswe/PS1dE9 transgenic mice (Fig. S1).

2. Materials and methods 2.1. Animals All the mice were bred in an AAALAC-accredited facility. This study and the use of animals were approved by the Animal Care and Use Committees of the Institute of Laboratory Animal Science of Peking Union Medical College (SCXK-2005-0013). The mice were individually housed in standard cages (40 × 25 × 12 cm) with food and water available ad libitum. The housing room was maintained at constant room temperature (22 ± 2 °C) and humidity (45%) and kept under a regular light/dark schedule with lights on from 08:00 am to 20:00 h. The APPswe/PS1dE9 transgenic mouse model of Alzheimer's disease has been described in our previous study (Zhang et al., 2011; Zong et al., 2011). Mice express a mouse–human hybrid transgene containing the extracellular and intracellular regions of the mouse sequence and a human sequence within the Aβ domain with Swedish mutations (K594N/M595L), and express the human presenilin-1 deleted exon-9. CTF1 transgenic mice were generated as described below.

2.2. Generation and characterisation of CTF1 transgenic mice The cDNA encoding human CTF1 was cloned into an expression plasmid under the PDGF beta-chain promoter, which was provided by Dr. Tucker Collins of Harvard Medical College. This construct was microinjected into the male pronuclei of fertilised mouse oocytes that were implanted into pseudo-pregnant females to generate the transgenic mouse lines (Gordon and Ruddle, 1981). The genotyping of CTF1 transgenic mice was performed by PCR using the following primers: 5′-AGCCGGAGGGAGGGAAGTCT-3′ and 5′-AGAAGCTGGGCA GCCCGAAG-3′. The PCR to amplify the desired 181 bp fragment of the CTF-1 transgene was conducted using 30 cycles with the following conditions: denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s and extension at 72 °C for 30 s. The expression and distribution of the target gene in the brain was analysed by western blotting and immunohistochemistry using antibodies to CTF1.

2.3. Genetic crosses CTF1 transgenic mice were maintained on a C57BL/6J genetic background. CTF1 transgenic mice from the F2 generation were crossed to APPswe/PS1dE9 transgenic mice to generate CTF1 × APPswe/PS1dE9 transgenic mice. Non-transgenic littermates and the APPswe/PS1dE9 transgenic mice were used as controls. All experiments were carried out blinded with respect to the genetic status of mice.

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2.4. Behavioural tests 2.4.1. Open-field test The spontaneous behavioural changes of mice in a novel environment were measured using the open-field test. Ethovision XT monitoring and analysis software (Noldus Company, Netherlands) was used for scoring. The dimensions of the open field were 50 × 50 cm, and the open-field test was performed according to the specific experimental protocols described by EMPReSS (European Mouse Phenotyping Resource of Standardised Screens). The open field was divided into three areas, including a peripheral zone (Zone 1) and two central zones (Zones 2 and 3). Zone 1 consisted of the area within 8 cm of the outer perimeter of the open field. Zone 3 was located in the centre of the arena and comprised 16% of the total area. The remaining area was Zone 2. Each mouse was allowed to explore the open field freely for 5 min. The time that each mouse stayed in each zone, as well as the frequency that mouse was in the state of immobile, mobile or highly mobile, was recorded. Mobility is the state variable including three different variables: immobile, mobile or highly mobile. The mouse was considered as immobile when the change in area of the mouse between current sample and previous sample (referred to as changed area) is smaller than 20%, as highly mobile when the changed area is larger than 60% and as mobile when the changed area is between 20% and 60%. The amount of time spent in the peripheral zone is a manifestation of thigmotaxis (Meerlo et al., 1996). The room temperature was constant, and white light at a level of 25 lx was evenly distributed across the arena during the testing.. The open field was cleaned with 75% alcohol and dried before each experiment to remove residual odour. 2.4.2. Morris water maze The spatial learning and memory of mice was tested using the Morris water maze, which was conducted one day after the open-field test. The protocol used for the Morris water maze was modified from previously reported methods (Laczo et al., 2009; Liang et al., 1994). Briefly, the apparatus consisted of 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 colours were placed at the remote ends of the water tank as references. The room temperature was constant, and the light levels were even. 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. The mice were allowed to stay on the platform for 15 s before and after each trial. The time that it took for an animal to reach the platform (latency period) and the path length for finding the escape platform were recorded. Twenty-four hours after the learning phase, the mice swam freely in the water tank without the platform for 60 s, the time spent in the region, and number of passes through the region and the quadrant of the original platform were recorded. Monitoring was performed with a video tracking system (Noldus Ltd, Ethovision XT, Holland). 2.5. Histological analysis To demonstrate fibrillar Aβ deposition, Thioflavine-S staining was used, which is commonly used for fluorescent staining of plaques (Kung et al., 2002; Urbanc et al., 2002). Brain tissue from mice (n = 6) following PET/CT scanning was fixed in 10% neutral buffered formalin and mounted in paraffin blocks. After deparaffinisation and hydration, the sections were washed in PBS and incubated in 0.25% potassium permanganate and 1% oxalic acid until they appeared white. The sections were then washed in water and stained for 3 min with a solution of 0.015% Thioflavin-S in 50% ethanol. Finally, the sections were washed in 50% ethanol and in water, then dried, and dipped in Histo-Clear before being cover-slipped with Permount (Bussiere et al., 2004). Sections stained with Thioflavin-S were visualised and images were captured by

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microscope with a digital camera attached to a computer (Nikon, Melville, NY). Images were analysed using Aperio's ImageScope Viewer software (Aperio. Technologies).

24 h prior to treatment and were then left untreated or were treated with recombinant human CTF1 (hCTF1) (5 ng/mL, Sigma, USA). 2.8. Western blotting

2.6. PET/CT image analyses Glucose uptake was examined using PET/CT scans. Briefly, following 6 h fasting time, 6-month-old mice (n = 3 ~ 4) were anaesthetised using 1.5% isoflurane, with 31% O2 inhalation (flow rate: 2.5 L/min), through a nose cone prior to injection of the tracer. Each mouse was imaged on a small-animal scanner (microPET/CT, Inveon, Siemens). Prior to the dynamic small-animal procedure, 18F-FDG tracer (FDG) (at ~14.8–18.5 MBq) was injected as a bolus (~200 μL) through a tail vein catheter, and the animal was kept at room temperature for 45 min. Each mouse was then exposed to a 10 min PET scan, and a 10 min CT scan was obtained for attenuation corrections of small-animal PET images. Images were reconstructed using the filtered back-projection algorithm with CT-based photon-attenuation correction (Chow et al., 2005). The reconstructed images were examined with a 3D display, in axial, coronal and sagittal views. This image was used to define 3 regions of interest (ROI), consisting of the frontal cortex, the temporal cortex, and the hippocampus manually. Injected dose (ID) per gramme of tissue was calculated by Inveon Research Workplace software (IRW) automatically. The field of view was 11.28 × 12.66 cm2.

After PET/CT scanning, the mice (n = 4, each group) were randomly chosen and deeply anaesthetised with sodium pentobarbital (100 mg/kg intraperitoneally). Hippocampus tissues were removed rapidly from freshly dissected brain and directly homogenised in RIPA buffer containing 0.1% PMSF and 0.1% protease inhibitors cocktail (Sigma, MO, USA). The lysates were centrifuged at 14,000 rpm for 30 min at 4 °C. The protein concentrations of the supernatants were determined using the BCA method. Equal amounts of soluble protein were run on SDS-PAGE gels and transferred onto nitrocellulose membranes (Immobilon NC; Millipore, Molsheim, France). Immunoblotting was performed with antibodies specific for phospho-GSK-3β (Ser9, 1:1000), GSK-3β (1:1000), phospho-AKT (Ser473, 1:1000), AKT (1:1000), phospho-STAT3 (Thr705, 1:1000), STAT3 (1:1000), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204, 1:1000), p44/42 MAPK (ERK1/2) (1:1000) (Cell Signaling Technology), GLUT-3 (1:500, Abcam), GLUT-1 (1:250, Abbiotec) and CTF1 (1:500, Bioworld). Primary antibodies were visualised 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 normalised relative to GAPDH.

2.7. Cells and treatments

2.9. Statistical analyses

Human SH-SY5Y neuroblastoma cells (obtained from Cell Culture Center, Peking Union Medical College, China) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco/BRL, Gaithersburg, MD, USA) containing 10% foetal bovine serum (Hyclone, Logan, UT, USA) and antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin; Sigma, St. Louis, MO, USA) at a density not exceeding 5 × 105 cells/mL. The cells were maintained at 37 °C in a humidified incubator with 5% CO2. To examine the effects of CTF1 on the expression of p-GSK-3β in vitro, the cells (approximately 1 × 106 cells/mL) were serum-deprived for

All the measurement data are expressed as the mean ± SEM. For the Morris water maze tests, escape latency in the hidden platform trial were analysed with two-way ANOVA of repeated measures, while one-way ANOVA was conducted on the data obtained from the probe trial. The indexes in the open-field test were analysed by a one-way ANOVA, followed by LSD (equal variances assumed) or Dunnett'sT3 (equal variances not assumed) for a post hoc test between groups. Histological assays and PET/CT data as well as proteins were analysed by one-way ANOVA, followed by LSD. All analyses were performed with

Fig. 1. Expression of CTF1 in APPswe/PS1dE9 hippocampus tissues and the generation of transgenic mice. (A) The protein levels of CTF1 in hippocampus tissues of 3-, 6-, and 9-months-old APPswe/PS1dE9 transgenic mice were detected by western blotting. (B) The quantitative analysis of CTF1 using GADPH as normalisation (n = 6, *p b 0.05, **p b 0.01 versus NTG mice). (C) The CTF1 transgenic construct was generated by inserting the target gene under the control of the brain-specific PDGF beta-chain promoter, and the transgenic mice were created following microinjection. (D) Mouse lines over-expressing CTF1 were selected based on western blotting. (E) The quantitative analysis of CTF1 using GADPH as normalisation (n = 6, *p b 0.05, **p b 0.01 versus WT mice).

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SPSS statistical package (version 13.0 for Windows, SPSS Inc., USA). Differences were considered significant at a p value b 0.05.

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based on its high levels of expression, as determined by western blotting (Fig. 1D, E). 3.2. Behavioural tests

3. Results 3.1. Down-regulation of CTF1 expression in APPswe/PS1dE9 transgenic mice and the generation of CTF1 transgenic mice Hippocampus tissues were obtained from 3-, 6- and 9-month-old WT and APPswe/PS1dE9 transgenic mice, and the relative protein levels of CTF1 were determined. CTF1 protein levels in AD-affected brains from APPswe/PS1dE9 transgenic mice were significantly down-regulated by 28% to 54% compared to those measured in the WT mice (Fig. 1A, B). There is substantial evidence suggesting that the hippocampus plays a central role in memory functions through its interconnections with distributed cortical regions (Amaral et al., 1990; Insausti et al., 1995; Zola-Morgan et al., 1989). This result suggested that CTF1 might be involved and play an important role in the pathophysiology of AD. To study the potential neuroprotective effects of CTF1, C57BL/6J mice overexpressing CTF1 and the CTF1 × APPswe/PS1dE9 mouse line were established. The transgenic plasmid was constructed by inserting human CTF1 cDNA downstream of the PDGF promoter (Fig. 1C). One line of CTF1 transgenic mice (founder #6) was selected for future studies

3.2.1. CTF1 attenuates the abnormal exploratory activity of APPswe/PS1dE9 transgenic mice in the open-field test The exploratory activity of three groups of mice at various age (4-, 6and 9-months-old) in a novel environment was determined using the open-field test. There was a significant overall group difference in rearings (F (3, 48) = 3.13, p b 0.05), total distance moved (F (3, 48) = 3.47, p b 0.05) and thigmotaxis (F (3, 48) = 4.66, p b 0.01) amongst the four groups at 6-month-old mice. Compared to the WT controls, the APPswe/PS1dE9 transgenic mice showed increased numbers of rearings, increased total distance moved and more time spent in the central zone (Zones 2 and 3); they also displayed decreased time spent in the peripheral zone (Zone 1) of the open field (Fig. 2A, B, C). They also exhibited an increased frequency of immobile (F (3, 48) = 3.62, p b 0.05), mobile (F (3, 48) = 3.81, p b 0.05), highly mobile (F (3, 48) = 3.36, p b 0.05) and frequency of total mobility (F (3, 48) = 4.66, p b 0.01) in the open-field test (Fig. 2D, E). The CTF1 × APPswe/PS1dE9 mice exhibited an amelioration of abnormal exploratory activity, as evidenced by their 17.88% decrease in total distance moved, their 8.14% increase in the amount of time spent

Fig. 2. Effects of CTF1 on spontaneous behaviours in the open-field test. (A) Rearings and (B) total distance moved of mice were measured. (C) Thigmotaxis was assessed by the recording the amount of time that each mouse spent in the peripheral zone and in the central zone. (D) The frequency of mice exhibiting immobility, mobility and high mobility and (E) the total frequency of changes in mobility were also measured (WT mice, n = 13; APPswe/PS1dE9 mice, n = 15; CTF1 mice, n = 10; CTF1 × APPswe/PS1dE9 mice, n = 14. *p b 0.05, **p b 0.01, WT mice versus APPswe/PS1dE9 transgenic mice; #p b 0.05, ##p b 0.01, CTF1 × APPswe/PS1dE9 transgenic mice versus APPswe/PS1dE9 transgenic mice).

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in the peripheral zone, and their significantly decreased mobility changes when compared to the APPswe/PS1dE9 transgenic mice. There was no significant difference in rearings, total distance moved, thigmotaxis and mobility (Fig. 2) between WT mice and CTF-1 transgenic mice (p > 0.05). In addition, there was no significant overall group difference in rearings, total distance moved and thigmotaxis amongst the four groups at 4- and 9-months-old mice (Fig. S2). 3.2.2. CTF1 improves the learning and memory of APPswe/PS1dE9 transgenic mice in the Morris water maze To assess spatial reference learning and memory function, three groups of mice at various age (4-, 6- and 9-months-old) underwent testing in the Morris water maze. Spatial learning was assessed in the hidden platform task in all mice. As shown in Fig. 3A, there was a significant overall group difference in escape latency (group effect: F (3, 48) = 12.69, p b 0.01; training day effect: F (4, 192) = 20.27, p b 0.01; group × training day interaction: F (12, 192) = 0.71, p > 0.05) and escape path length (group effect: F (3, 48) = 7.39, p b 0.01; training day effect: F (4, 192) = 65.77, p b 0.01; group × training day interaction: F (12, 192) = 0.58, p > 0.05) amongst the four groups at 6-month-old mice. Both the latency to find the submerged platform (the escape latency) and the escape path length declined every day in both genotypes, but the escape latency (p b 0.01) and the escape path length (p b 0.01) of the APPswe/PS1dE9 transgenic mice were significantly longer than that of the WT animals. The CTF1 × APPswe/PS1dE9 mice showed decreased escape latencies and reduced escape path length (p b 0.05) compared to the APPswe/ PS1dE9 transgenic mice (Fig. 3A, B). In the probe test, the target-quadrant abidance and the crossingtarget number were measured for 60 s on the 6th day after the last

acquisition test. The results indicated that the difference of the crossingtarget number in the probe test began at 4-month-old, increased at 6-month-old and decreased at 9-month-old between the WT and APPswe/PS1dE9 mice (Fig. S3). Based on this, 6-month-old mice were selected for the further investigation. As shown in Fig. 3C, there was a significant overall group difference in the target-quadrant abidance (F (3, 48) = 4.01, p b 0.05) and crossing-target number (F (3, 48) = 3.44, p b 0.05) amongst the four groups at 6-month-old mice. The APPswe/PS1dE9 transgenic mice showed an obvious 40.7% decrease in target-quadrant abidance and a 49.9% decrease in crossingtarget number compared to the WT controls. Compared to the APPswe/PS1dE9 transgenic mice, the target-quadrant abidance and the crossing-target number of the CTF1 × APPswe/PS1dE9 mice were significantly increased by 41.4% and 47.9%, respectively (Fig. 3C). There was no significant difference in the target-quadrant abidance and the crossing-target number between WT mice and CTF1 transgenic mice (p > 0.05). In addition, there was no significant difference in swimming speed (F (3, 48) = 1.49, p > 0.05) and path length (F (3, 48) = 1.50, p > 0.05) in the probe test between the four groups (Fig. 3D). 3.3. CTF1 reduces plaque pathology in APPswe/PS1dE9 mice To investigate whether CTF1 has an effect on amyloid plaque formation in vivo, mice were sacrificed and their brain of mice were stained with Thioavine-S, which specifically binds amyloid plaques. Compared with APPswe/PS1dE9 transgenic mice, the CTF1 × APPswe/PS1dE9 transgenic mice exhibited 35.8%, 41.9% fewer Thioavine-S positive compact plaques (p b 0.01), 18.2% and 26.9% less plaque area (p b 0.05) in hippocampus and cortex (Fig. 4).

Fig. 3. Effects of CTF1 on learning and memory in the Morris water maze test. (A) The Escape latency during the 5 days of hidden platform test. (B) The Escape path length during the 5 days of hidden platform test. (C) The target-quadrant abidance and the crossing-target number in the probe test were tabulated. (D) The swimming speed and path length in the probe test (WT mice, n = 13; APPswe/PS1dE9 mice, n = 15; CTF1 mice, n = 10; CTF1 × APPswe/PS1dE9 mice, n = 14. *p b 0.05, **p b 0.01, WT mice versus APPswe/PS1dE9 transgenic mice; #p b 0.05, ##p b 0.01, CTF1 × APPswe/PS1dE9 transgenic mice versus APPswe/PS1dE9 transgenic mice).

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Fig. 4. CTF1 reduced plaque pathology in APPswe/PS1dE9 mice. (A) Brain tissue from WT mice, APPswe/PS1dE9 mice, and CTF1 × APPswe/PS1dE9 mice were utilised in standard pathological procedures, and sections were stained with Thioflavin-S to visualise the deposition of Aβ. Representative brain sections showing that CTF1 decreased Thioflavin-S immunoreactivity (scale bars = 1 mm). (B) Graphs showing plaque count and the percentage of area occupied by plaques in the hippocampus and cortex (n = 6, *p b 0.05, **p b 0.01, CTF1 × APPswe/PS1dE9 transgenic mice versus APPswe/PS1dE9 transgenic mice).

3.4. CTF1 enhances glucose uptake in APPswe/PS1dE9 mice Early alterations in glucose metabolism (Mosconi et al., 2006) and severe abnormalities in cerebral glucose metabolism (Hoyer, 1991) parallel the worsening of dementia symptoms and play a pivotal role in triggering neuronal death and cognitive decline (Hoyer, 1991; Kapogiannis and Mattson, 2011; Santos et al., 2010). Glucose uptake was examined in vivo in mice using PET scans of 18F-FDG (Fig. 5A). The APPswe/PS1dE9 mice showed an obvious deficiency in energy metabolism; with a 38.3% decrease in 18F-FDG uptake in the frontal lobe (p b 0.05), a 46.4% decrease in the temporal lobe (p b 0.05) and a 44.8% decrease in the hippocampus (p b 0.01) compared to the WT controls. CTF1 expression in the CTF1 × APPswe/PS1dE9 mice significantly reversed these glucose uptake defects and increased 18F-FDG uptake by 37.7% in the frontal lobe, 50.8% in the temporal lobe and 53.2% in the hippocampus (p b 0.05) compared to the APPswe/PS1dE9 controls (Fig. 5B). 3.5. CTF1 up-regulates the phosphorylation of GSK-3β in vivo and in vitro In vivo, the levels of phosphorylated GSK-3β (Ser9) (p-GSK-3β) were significantly decreased in 6-month-old APPswe/PS1dE9 mice compared to the WT controls. The CTF1 × APPswe/PS1dE9 mice exhibited a 57.3% increase in the levels of p-GSK-3β compared to the APPswe/PS1dE9 mice (Fig. 6A, B). In vitro, SH-SY5Y cells responded to CTF1 treatment in a time-dependent manner. Exposure to CTF1 resulted in a timedependent increase in the levels of p-GSK-3β (Fig. 6C, D). 4. Discussion CTF1 was firstly described in cardiac muscle cells, but varying levels of CTF1 expression have been found in a variety of tissues outside of the cardiac compartment, including the liver, lung, kidney, skeletal muscle and brain (Pennica et al., 1996b). CTF1 transcripts and a CTF1-like protein have both been reported to be expressed in the

foetal and adult mouse brains (Ochiai et al., 2001). Meanwhile, Gard et al. (2004) have reported CTF1 protein expression in the human choroid plexus. Consistent with these observations, our results reveal obvious CTF1-positive staining in areas associated with AD (data not shown). Furthermore, our results indicate that CTF1 expression levels are significantly decreased in hippocampus tissues of APPswe/PS1dE9 transgenic mice (Fig. 1A and B), a model of AD. Because circulating levels of CTF1 are associated with glucose levels (Natal et al., 2008) and CTF1 participates in glucose metabolism and energy regulation (Moreno-Aliaga et al., 2011), it has been suggested that the downregulation of CTF1 in AD brains could be related to the energy metabolism disturbances and brain-glucose hypometabolism that exist in cognitive ageing and AD (Cunnane et al., 2011). To understand the effects of CTF1 on the progression of AD in transgenic mice, we produced brain tissue-specific CTF1 transgenic mice and demonstrated that transgenic expression of CTF1 reversed glucose-uptake deficiency in the brain, increasing it by 37.7% in the frontal lobe, 50.8% in the temporal lobe and 53.2% in the hippocampus (Fig. 5). Transgenic CTF1 expression also ameliorated abnormal locomotor activity and cognitive deficits, as indicated by the attenuation of abnormal spontaneous behaviours (i.e., the increased time spent in the peripheral zone and decreased mobility) and by enhancing learning and memory (i.e., inducing a 41.4% increase in target-quadrant abidance and a 47.9% increase in the crossing-target number, see Figs. 2 and 3) in this animal model of AD. Ameliorating energy metabolism disturbances is an effective way of preventing (Colcombe et al., 2006; Geda et al., 2010) or reversing cognitive deficits (Lautenschlager et al., 2008) and attenuating the atrophy observed in AD (Ding et al., 2006; Nichol et al., 2009). Thus, it is possible that the effects of the over-expression of CTF1 in brain tissues on cognitive improvement may be mediated through the reversal of glucose-uptake defects. CTF1 interacts with the gp130/leukaemia inhibitory factor receptor beta heterodimer and activates at least 3 different downstream signalling pathways, including the JAK/signal transducer and activators of transcription 3 (STAT3), the PI3K/AKT and the Src-ERK pathways

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Fig. 5. CTF1 enhances glucose uptake in APPswe/PS1dE9 mice. (A) PET/CT scanning images of WT mice, APPswe/PS1dE9 mice, CTF1 mice and CTF1 × APPswe/PS1dE9 mice. (B) Glucose (as measured by FDG) uptake per gramme of brain tissue (n = 3–4, *p b 0.05, **p b 0.01, WT mice versus APPswe/PS1dE9 transgenic mice; #p b 0.05, ##p b 0.01, CTF1 × APPswe/PS1dE9 transgenic mice versus APPswe/PS1dE9 transgenic mice).

(Fischer and Hilfiker-Kleiner, 2007). These pathways have also been shown to be important in CTF1-mediated protection against cardiac hypertrophy, small-for-size liver transplantation in rats (Song et al., 2008), oxidative damage in PC12 cells (Toth et al., 2002) and focal cerebral ischemia in vitro and in vivo (Peng et al., 2010). In the current study, CTF1 did not alter the levels of p-AKT, p-STAT3 or p-ERK (Fig. S4), all of which have been previously shown to be activated by CTF1 treatment in motor neurons (Dolcet et al., 2001) and cortical neurons (Peng et al., 2010). Emerging evidence indicates that consistent and progressive cerebral glucose hypometabolism occurs during the very early stages of AD (Mosconi, 2005; Mosconi et al., 2006) and that the extent of hypometabolism correlates with symptom severity both in vivo (Desgranges et al., 1998; Haxby et al., 1990). Furthermore, altered brain metabolism predisposes an individual to neuronal damage, dysfunction, and death and leads to various acute or chronic forms of neuropathology (Blass, 2008; Freemantle et al., 2006; Hoyer et al., 1988). GSK-3β, which is known to participate in the pathogenesis

of AD (Avila et al., 2010), was originally identified as a key regulator of glycogen synthesis and has been implicated in the regulation of glucose metabolism. GSK-3β can phosphorylate and inhibit pyruvate dehydrogenase, which has been observed in the AD brain (Sorbi et al., 1983; Yun and Hoyer, 2000), and results in dysfunctions of mitochondria and glycolysis (Hoshi et al., 1996). The inhibition of GSK-3β increases glucose uptake through the up-regulation of glucose transporter (GLUT) expression (Buller et al., 2008) or by enhancing insulin responsiveness (Ciaraldi et al., 2010; Orena et al., 2000). CTF1 can inhibit GSK-3β activity in vivo and in the SH-SY5Y cell line (Fig. 6). We speculate that CTF1 enhances glucose uptake and attenuates impaired energy metabolism by suppressing the activity of GSK-3β. GLUT1 and GLUT3 are the predominant GLUTs responsible for glucose transport (McEwen and Reagan, 2004). GLUT3 is the primary GLUT in neurons and helps transport glucose from the extracellular space into neurons (Dwyer et al., 2002). Our results reveal decreased GLUT3 expression in an animal model of AD, a finding that has also been reported in the AD brain (Harr et al., 1995; Mooradian et al.,

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Fig. 6. CTF1 up-regulates the levels of p-GSK-3β in vivo and in vitro. (A) The relative level of p-GSK-3β was detected by Western blotting from hippocampus tissues of WT mice, APPswe/PS1dE9 mice, CTF1 mice and CTF1 × APPswe/PS1dE9 mice, and a representative experiment was shown. (B) The quantitative analysis of p-GSK-3β using T-GSK-3β as normalisation (n = 4, *p b 0.05, **p b 0.01, WT mice versus APPswe/PS1dE9 transgenic mice; #p b 0.05, ##p b 0.01, CTF1 × APPswe/PS1dE9 transgenic mice versus APPswe/PS1dE9 transgenic mice). (C) SH-SY5Y cells were either untreated or treated for 10 min, 30 min, 45 min, 1 h or 2 h with 5 ng/mL of hCTF1. The relative level of p-GSK-3β was detected by Western blotting from 20 μg cell extracts. (D) The quantitative analysis of p-GSK-3β using T-GSK-3β as normalisation (n = 4, *p b 0.05, **p b 0.01 versus untreated cells).

1997; Simpson et al., 1994). In addition, we demonstrate that CTF1 up-regulates GLUT3 expression in vivo and in the SH-SY5Y cell line (Fig. S5). Furthermore, the GLUT3 inhibitor cytochalasin-B significantly reverses the CTF1-induced increase in glucose uptake in the SH-SY5Y cell line (data not shown). However, we did not observe any changes in GLUT1 expression (Fig. S6). It has been demonstrated that the inhibition of GSK-3β can up-regulate glucose transporter (GLUT) expression in several cell lines (Buller et al., 2008). Consistent with the results, in this present study we observed that CTF1 inhibited the GSK-3β activity and induced GLUT3 expression, suggested that the effects of CTF1 on GLUT-3 expression might be regulated by GSK-3β. However, this hypothesis needs to be investigated further. Our results also indicate that the CTF1 × APPswe/PS1dE9 transgenic mice exhibited 35.8%, 41.9% fewer Thioavine-S positive compact plaques, 18.2% and 26.9% less plaque area in hippocampus and cortex compared with the control APPswe/PS1dE9 mice (Fig. 5). GSK-3β promotes Aβ production (McLoughlin and Miller, 1996) and participates in APP processing (Ryder et al., 2003; Takashima et al., 1998). The inhibition of GSK-3β can decrease Aβ deposition in both animal models (DaRocha-Souto et al., 2012; Ryder et al., 2003; Su et al., 2004) and in cell lines (Sun et al., 2002) and attenuate Aβ-induced neurotoxicity (DaRocha-Souto et al., 2012; Takashima et al., 1993). The positive effect of CTF1 on preventing the formation of senile plaques may be caused by an inhibition of GSK-3β. In conclusion, CTF1 was demonstrated to be down-regulated in the brain of the APPswe/PS1dE9 transgenic mice. The protective effect of induced expression of CTF1 may be mediated through GSK-3β inhibition. Transgenic expression of CTF1 in brain tissue as a means of compensating for the loss in its expression could ameliorate disturbances of brain energy metabolism and improve cognitive deficits. This study supports an important concept that onset of neurodegenerative disease may be delayed or mitigated with use of GSK-3β inhibitors that protect against Aβ plaque formation and energy metabolism deficits. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.pbb.2013.03.003. Acknowledgement This research was supported in part by the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2012BA139B02) and the National Science and

Technology Major Project of the Ministry of Science and Technology of China (2011ZX09307-302-03).

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