International Journal of Neuropsychopharmacology (2014), 17, 1847–1861. doi:10.1017/S1461145714000558

© CINP 2014

ARTICLE

Pro-neurogenesis and anti-dementia properties of tetradecyl 2,3-dihydroxybenzoate through TrkA receptor-mediated signalling pathways Libin Zhou1*, Zihong Lu1*, Lin Li1, Lei Chen1, Jianhua Qi2 and Ling Chen1 1 2

Department of Physiology, Nanjing Medical University, Nanjing, PR China College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, PR China

Abstract Tetradecyl 2,3-dihydroxybenzoate, termed ABG001, has been reported to enhance neurite outgrowth of PC12 cells. Herein, we report that oral administration of ABG001 for five days to adult male mice could dosedependently enhance survival and neurite growth of newborn cells in hippocampal dentate gyrus (DG) without changes in cell proliferation and differentiation of progenitor cells. The ABG001 administration (0.5 mg/kg) enhanced the phosphorylation of tyrosine kinase A (TrkA) receptor, which induced increases in the levels of ERK, Akt and mTOR phosphorylation in hippocampus. The pro-neurogenesis of ABG001 was blocked by the TrkA receptor inhibitor K252a. By contrast, the ERK inhibitor U0126 attenuated only the ABG001-increased number of newborn cells, while the PI3K inhibitor LY294002 prevented mainly the ABG001-enhanced neurite growth. In comparison with control mice, the mice treated with ABG001 showed a more preferential spatial cognitive function as assessed by Morris water maze and Y maze tests, which was sensitive to the blockade of TrkA receptor. In addition, a single injection (i.c.v.) of ‘aggregated’ Aβ25–35 in adult male mice (Aβ25–35mice) impaired spatial memory, survival and neurite growth of newborn cells in the DG with reduced phosphorylation of Akt and mTOR. The treatment of Aβ25–35-mice with ABG001 could protect the survival and neurite growth of newborn cells through increasing TrkA receptor-induced phosphorylation of Akt and mTOR, which was accompanied by the improvement of spatial cognitive performance. Received 24 September 2013; Reviewed 5 January 2014; Revised 17 March 2014; Accepted 24 March 2014; First published online 30 April 2014 Key words: Hippocampus, nerve growth factor, neurogenesis, tetradecyl 2,3-dihydroxybenzoate, TrkA receptor.

Introduction Endogenous neurogenesis continues throughout adulthood within the sub-granular zone (SGZ) of hippocampal dentate gyrus (DG) and the sub-ventricular zone (SVZ) abutting the lateral ventricle (Alvarez-Buylla and Garcia-Verdugo, 2002). The newborn neurons are integrated into hippocampal circuitry after three to four weeks of maturation, where dendritic spines contact perforant path synapses and axons project in the CA3 area (Toni et al., 2008). In addition, the newborn neurons have lower activation thresholds and higher resting potentials, leading to the facilitation of long-term potentiation induction in comparison with mature neurons (van Praag et al., 2002). Several studies indicate that the mature newly generated neuronal cells play an important role in cognitive performance (Rola et al., 2004).

Address for correspondence: Ling Chen, Laboratory of Reproductive Medicine, Department of Physiology, Nanjing Medical University, Hanzhong Road 140, Nanjing, 210029, PR China Tel.: +86 -25-86862878 Fax: +86-25-86260332 Email: [email protected] * These authors contributed equally to this work.

Hippocampal neurogenesis is robust in the young adult and persists into old age even though the aged brain exhibits a reduction in the formation of new hippocampal neurons (Lazarov et al., 2010). This decline in neurogenic capacity has been suggested to underlie cognitive deterioration during aging in rodents (Kempermann and Gage, 1998). Alzheimer’s disease (AD) is characterized by selective learning and memory deficits. Viability of the neural precursor cells harvested from AD patients is significantly lower than that of an age-matched control group (Lovell et al., 2006). The survival of newly generated neuronal cells is selectively impaired in a knock-in mutant mouse model of AD with cognitive and behavioural abnormalities (Zhang et al., 2007). The in vivo over-production and subsequent aggregation of β-amyloid peptides (Aβ) limit the survival of newborn hippocampal neurons (Verret et al., 2007). Our recent study has reported that the treatment with neurosteroid pregnenolone sulfate in APPswe/ PS1dE9 transgenic mice can rescue the Aβ-induced impairment of neurogenesis in hippocampal DG, which is associated with the improvement of spatial cognitive deficit (Xu et al., 2012). Thus, the protection of neurogenesis is expected as a novel therapeutic application in the cognitive deficits of AD.

1848 L. Zhou et al. Nerve growth factor (NGF) promotes the growth, differentiation and survival of cholinergic neurons in the basal forebrain and is thus ideally suited as a cholinergic therapeutic (Winkler et al., 1998). NGF is able to enhance survival of new neurons in hippocampal DG of adult rats (Frielingsdorf et al., 2007). NGF-induced autophosphorylation of tyrosine kinase A (TrkA) receptor through cascading extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)-Akt signalling pathways enhances cognitive function (Terry et al., 2011). In addition, NGF delivery to the brain of AD patients has been reported to exert a potential therapeutic effect on neurodegeneration (Tuszynski et al., 2005). Despite the evidence of beneficial effects of NGF on neurogenesis, therapeutic applications of NGF have several limitations. Because of large molecular size and hydrophilic properties, NGF has minimal penetration across the blood-brain barrier, limiting its use as a therapeutic agent. By screening for neuritogenic substances from Gentiana rigescens Franch, tetradecyl 2,3-dihydroxybenzoate (ABG001), has been found to have a neuritogenic activity comparing to NGF (Luo et al., 2011). ABG001 at 0.03 μM enhances the neurite outgrowth of PC12 cells via cascading ERK signalling. The first purpose in this in vivo study was to examine the effects of ABG001 by oral administration on the process of adult neurogenesis including mitosis of progenitor cells, neurite growth, survival and maturation of newborn neurons in hippocampal DG. Using pharmacological and Western blot methods, the second purpose of this study was to determine the molecular targets and mechanisms of ABG001-regulated neurogenesis. Amyloid beta25–35 (Aβ25–35), as a shortest fragment of Aβ processed in vivo by brain proteases (Kubo et al., 2002), retains the self-aggregation ability and neurotoxicity (Pike et al., 1995). A single injection (i.c.v.) of ‘aggregated’ Aβ25–35 (6 nmol/3 μl) in adult male mice (Aβ25–35-mice) impairs the survival and neurite growth of newborn cells in hippocampal DG with deficits in spatial memory (Li et al., 2010). Thus, the third purpose of this study was to investigate the effects of ABG001 on Aβ25–35-impaired neurogenesis and spatial cognition.

Materials and method Experimental animals All procedures were in accordance with the guidelines of Institute for Laboratory Animal Research of the Nanjing Medical University. The Institutional Animal Care and Use Committee of Nanjing Medical University approved the use of animals. A total of 520 male 12-week-old mice (ICR; Oriental Bio Service Inc., China) were used at the beginning of experiments. All animals were housed under standard conditions (23 ± 2 °C, 40% humidity, inverted 12-h light-dark cycle starting at 07:00) in the Animal Research Centre of Nanjing

University. Four mice were housed in a plastic hanging cage and permitted free access to food and tap water before and after all procedures. All efforts were made to minimize animal suffering and to reduce the number of animals used. Administration of drugs ABG001 was synthesized by esterification of 2,3dihydroxybenzoic acid with tetradecan-1-ol in dry tetrahydrofuran using N,N′-dicyclohexylcarbodiimide as described in a previous paper (Luo et al., 2011). For oral administration (p.o.) ABG001 was first dissolved in 99.5% ethanol and then in 0.9% saline to a final concentration of 0.5% ethanol. ABG001 (0.005∼2.5 mg/kg/day) at 0.2 ml volume was given by syringe (p.o.) as in humans’ administration of drug is most likely via an oral route. Control mice were orally treated with 0.5% ethanol at the same volume. When administered i.p. ABG001 was first dissolved in dimethyl sulfoxide (DMSO) and then in 0.9% saline to a final concentration of 0.5% DMSO. Intraperitoneal injection of DMSO at a concentration of 0.5 mg/kg/day has been observed to have no effect on the adult neurogenesis in the DG of male mice (Zhang et al., 2009). To investigate whether ABG001 gets across the blood-brain barrier, we compared the direct effects by i.c.v. injection of ABG001 on neurogenesis with the effects of it’s oral administration or i.p. injection. Mice were anesthetized with chloral hydrate (400 mg/kg) and were placed in a sterotactic device (Stoelting Co., USA). ABG001 was dissolved in 0.5% DMSO, and then was injected into the right lateral ventricle (0.3 mm posterior 1.0 mm lateral, and 2.5 mm ventral to bregma) using a stepper-motorized micro-syringe (Stoelting Co.) with a rate of 0.2 μl/min. The i.c.v. dosage (0.3 μM/3 μl) of ABG001 used here was based on the report (Luo et al., 2011) that ABG001 at 0.03 μM enhances the neurite outgrowth of PC12 cells via cascading ERK signalling. In addition, ERK inhibitor U0126, PI3K inhibitor LY294002 and Trk inhibitor K252a were purchased from Sigma-Aldrich (USA) and dissolved in 0.5% DMSO. This concentration of DMSO does not affect the neurogenesis (Yang et al., 2011). K252a at 0.2 μM/3 μl/mouse (Camarena et al., 2010), U0126 or LY294002 at 0.3 nmol/ mouse (Yang et al., 2012) were injected (i.c.v.). The mice infused with the same volume of vehicle served as the control group. Administration of the drugs through above any route did not affect body weight relative to vehicle-treated mice. Preparation of Aβ25–35-mice Aβ25–35 (Sigma, USA) was aggregated by incubation in sterile distilled water (1 mg/ml) at 37 °C for four days, and diluted to the final concentration with saline just before each experiment. Mice anesthetized with chloral hydrate (400 mg/kg) were injected with ‘aggregated’

ABG001-enhanced neurogenesis in hippocampal DG Aβ25–35 (6 nmol/3 μl/mouse) into bilateral ventricles (0.3 mm posterior 1.0 mm lateral, and 2.5 mm ventral to bregma) using a stepper-motorized micro-syringe at a rate of 0.2 μl/min. The mice injected with the same volume of vehicle served as the control group (Wang et al., 2007). The injection site was confirmed by injecting Indian ink in preliminary experiments. Neither insertion of the needle nor injection of the vehicle caused changes in cognitive behaviours. Aβ25–35-mice were divided into two different experimental groups of ABG001-administration for eighteen days: in group one the hippocampus was harvested on day 21 post-BrdU for Western blot analysis and group two were perfused with 4% paraformaldehyde on day 28 post-BrdU for immunostaining after cognitive behavioural examination.

Spatial cognitive behaviour tests Morris water maze A pool (diameter = 120 cm) made of black-coloured plastic was prepared, with the water temperature maintained at 20 ± 1 °C. Swimming paths were analysed using a computer system with a video camera (AXIS-90 Target/2; Neuroscience Inc., Japan). Twenty-four hours before water maze training swimming speed was assessed in the absence of a platform. During the initial phase of the test a platform (diameter = 7 cm) was submerged 1 cm below the water surface. Mice were given 90 s in the pool to search for the hidden platform. If the mouse failed to find the platform within 90 s, the trial was terminated and the animal was guided to the platform by the experimenter and allowed to stay there for 10 s. Each mouse started in one of four quadrants in a random manner. Four trials were conducted each day, for five consecutive days. The escape latency was calculated for each training day and analysed over trials using a repeated-measure ANOVA. On day six of water maze training, the platform was removed. The mouse was released from the opposite quadrant of the hidden platform quadrant to allow swimming for 90 s to determine its search patterns. The percentage of total time spent in each quadrant was measured.

Y-maze The Y-maze was made of black painted wood. Each arm was 40 cm long, 13 cm high, 3 cm wide at the bottom, 10 cm wide at the top and converged at an equal angle. Each mouse was placed at the end of one arm and allowed to move freely through the maze during an eight-minute session. The series of arm entries was recorded by camera and arm entry was considered completed when the hind paws of the mouse were completely placed in the arm. Alternation was defined as successive entries into the three arms on overlapping triplet sets. The percentage alternation was calculated as the ratio of actual to possible

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alternations (defined as the total number of arm entries minus two). Examination of neurogenesis BrdU immunostaining Five-bromo-2-deoxyuridine (BrdU) was dissolved in 0.9% saline to make 10 mg/ml solution just prior to injection. All mice received three injections of BrdU (50 mg/kg, i.p.) with an interval of 8 h for mitotic labelling. For BrdU immunostaining, the mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and perfused transcardially with 4% paraformaldehyde. Brains were post-fixed overnight in 4% paraformaldehyde at 4 °C and coronally sectioned (40-μM-thick) using a vibrating microtome (Microslicer DTK 1500; Dousaka EM Co., Japan). Every fifth freefloating section was treated with 2M HCl for 30 min at 37 °C, and then rinsed in standard phosphate-buffer (PBS; 0.1 M, pH 7.4) for 5 min. The sections were treated with 3% normal goat serum for 45 min, and then incubated in mouse monoclonal anti-BrdU antibody (1:1000; Chemicon Int., USA) at 4 °C overnight. After several PBS rinses, sections were incubated in biotin-labelled goat anti-mouse IgG antibody (1:500; Bioworld Technology Inc., USA) under agitation for 2 h. Immunoreactivities were visualized by avidin-biotin horseradish peroxidase complex (ABC Elite; 1:50, Vector Lab., USA) using 3,3′-diaminobenzidine (DAB; Vector Lab.) as chromogen. Two controls were used to check the specific staining: some animals received only saline injections instead of BrdU; in the BrdU-treated mice the primary antibody was omitted from the immuno-histochemistry procedure. BrdU and NeuN or GFAP double-immunostaining Free-floating sections (40-μM-thick) were incubated with rat monoclonal anti-BrdU antibody (1:200, Abcam, UK), revealed using CY3-labeled anti-rat IgG antibody (1:200; Chemicon, USA); mouse monoclonal anti-neuronal nuclei (NeuN) antibody (1:500; Chemicon), revealed using fluorescein-labelled anti-mouse antibody (1:50; Chemicon); mouse polyclonal anti-glial fibrillary acidic protein (GFAP) antibody (1:200, Sigma), revealed using fluorescein-labelled anti-mouse antibody (1:50; Chemicon). After several PBS rinses, the sections were finally mounted onto subbed slides and cover-slipped using Mowiol (Sigma). Doublecortin (DCX) immunostaining Free-floating sections were rinsed extensively in PBS, and then incubated with goat polycolonal anti-DCX antibody (1:500; Santa Cruz, USA) with 2% rabbit serum at 4 °C overnight. Sections were incubated in biotin-labelled rabbit anti-goat IgG antibody (1:500, Santa Cruz) for 2 h at room temperature. The sections were then incubated

1850 L. Zhou et al. with ABC Elite (1:50, Vector Lab.) using DAB (Vector Lab.) as chromogen.

Cell Signalling). Western blot bands were scanned and analysed with the Quantity One image software (Bio-Rad).

Quantitative evaluation of immuno-positive cells Slides were coded before analysis. The experimenter was blind to all samples until they were counted. BrdU immuno-positive (BrdU+) cells in SGZ, defined as a twocell body-wide zone along the border between GCL and hilus and within GCL, DCX immuno-positive (DCX+) cells in SGZ were counted on every fifth section (40-μM-thick) using a conventional light microscope (Olympus DP70, Japan) with a 40 × objective (Stevenson et al., 2009). Profile counting methodology was used as it has previously been shown that profile counting and stereological estimations show identical results in percentage change (Crews et al., 2004). The number of BrdU+ cells obtained from twenty-four stions (bilateral DG) was summed, and then was multiplied by five to give the total number of BrdU+ cells per DG. BrdU+/NeuN+ cells and BrdU+/GFAP+ cells in bilateral hippocampal DG were assessed using a confocal laser-scanning microscope (TCS SP2; Leica, Germany). DCX+ dendrites in the middle third of molecular stratum were observed by conventional light microscope with an oil immersion objective (Plan Apo × 60 oil) (Li et al., 2010). The total number of DCX+ dendrites was divided by the number of DCX+ cells within 1 mm length to obtain the number of DCX+ dendrites per cell. Data were obtained from ten stions (bilateral DG). Western blot analysis Two hours after the last administration of ABG001, the mice were decapitated under deep anaesthesia with ethyl ether. The hippocampus was taken quickly and homogenized in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Roche, Germany). Protein concentration was determined with BCA Protein Assay Kit (Pierce Biotechnology Inc., USA). Total proteins (20 μg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyphorylated difluoride (PVDF) membrane. The membranes were incubated with 5% non-fat dried milk for 60 min and then incubated with rabbit monoclonal antibodies of phosphorylation of TrkA, ERK1/2, Akt or mTOR (1:1000, Cell Signalling Technology, Inc., USA) at 4 °C overnight. The membranes were incubated with an HRP-labelled secondary antibody and developed using the ECL detection Kit (Millipore, USA). Following visualization, the blots were stripped by incubation in stripping buffer (Restore, Pierce Biotechnology Inc.) for 5 min, re-blocked with 5% non-fat dried milk at room temperature for 60 min and then incubated with antibodies of TrkA (1:1000, Abcam), ERK1/2, Akt or mTOR (1:1000,

Reverse transcription-polymerase chain reaction (RT-PCR) Two hours after the last administration of ABG001, the hippocampus was quickly harvested. Real-time PCR was performed as described previously (Wang et al., 2009). Total RNA was isolated from the hippocampus with Trizol reagent (Invitrogen, USA) and was reversetranscribed into cDNA using a Prime Script RT reagent kit (Takara, China) for quantitative PCR (ABI Step One Plus, USA) in the presence of a fluorescent dye (SYBR Green I; Takara). The relative abundance of mRNA was determined using the 2-ΔΔct method with normalization to glyceraldehyde-3-phosphate dehydrogenase ribosomal (GAPDH) mRNA. The primers used for NGF were 5′-TGGGCTTCAGGGACAGAGTC-3′ and 5′-CAGCTTT CTATACTGGCCGCAG-3′; the primers for TrkA were 5′-CTCTTCCTTTCTGCCCTCCT-3′ and 5′-TGATCCCAA ATTTGCTCCTC-3′; the primers for GAPDH were 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-ACCACAG TCCATGCCATCAC-3′. Data analysis/statistics Data were retrieved and processed with the software Microcal Origin 6.1. The group data were expressed as the means±standard error (S.E.M). Differences between the two groups were evaluated using Student’s t-test. When analysing one-variable experiments with more than two groups, significance of difference was evaluated using ANOVA followed by Bonferroni’s post-hoc tests. Statistical analysis was performed using State 7 software (STATA Co., USA). Differences at p < 0.05 were considered statistically significant.

Results Effects of ABG001 on adult neurogenesis in hippocampal DG Oral administration of ABG001 (0.5 mg/kg) in male adult mice (ABG001-mice) was given for five consecutive days before the last BrdU-injection, or on days 2∼6, 9∼13, 16∼20 or 23∼27 after the last BrdU-injection (post-BrdU), respectively (Fig. 1a) in order to evaluate the effects of ABG001 on the mitosis of progenitor cells, survival and maturation of newborn cells. Twenty-four hours after the last administration of ABG001, BrdU+ cells in hippocampal DG were examined and termed 1-, 7-, 14-, 21- and 28-day-old BrdU+ cells (see time chart of experimental procedure in Fig. 1a). In comparison with control mice (p.o. administration of vehicle) per group, ABG001-administration for five days caused an obvious increase in the numbers of

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n = 10). Similarly, the injection (i.c.v.) of ABG001 (0.3 μM/3 μl) or injection (i.p.) of ABG001 (0.5 mg/kg) on days 2∼6 post-BrdU could increase the number of

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0 Fig. 1. Administration of ABG001 enhances survival and neurite growth of newborn cells in hippocampal DG. (a) Time chart of experimental procedure. Black arrows indicate the time of ABG001 administration. Hollow arrows represent the time of BrdU immunostaining. (b) Effects of ABG001 on cell-proliferation and survival of newborn cells. Representative images of 7-day-old BrdU + cells (black arrows). Scale bar = 100 μM. Bar graph shows mean numbers of 1-day-old, 7-day-old, 14-day-old, 21-day-old and 28-day-old BrdU+ cells in control and ABG001-mice. *p < 0.05 and **p < 0.01 vs. controls. (c) Effects of ABG001 injection (i.c.v.) or (i.p.) on survival of newborn cells. *p < 0.05 and **p < 0.01 vs. controls. (d) Dose-dependency of ABG001-increased 7-day-old BrdU+ cells. (e) Representative images of 28-day-old BrdU+ cells (black arrows) in control and ABG001-mice. Scale bar = 100 μM. Bar graph shows mean number of 28-day-old BrdU+ cells. **p < 0.01. (f&g) BrdU+ (red)/NeuN+ (green) cells (yellow) are indicated by white arrows in left panels. Scale bar = 50 μM. Bar graph shows the mean numbers of 28-day-old BrdU+/NeuN+ cells and BrdU+/GFAP+. **p < 0.01. (h) Effects of ABG001 administration on neurite growth of newborn cells. Representative pictures of DCX immunostaining (left panels). Scale bar = 50 μM. Bars indicate mean number of DCX+ dendrites per DCX+ cell. **p < 0.01.

ABG001-enhanced neurogenesis in hippocampal DG 7-day-old BrdU+ cells (i.c.v: p < 0.01; i.p.: p < 0.05, n = 10; Fig. 1c). In addition, the oral administration of ABG001 (0.005∼2.5 mg/kg) on days 2∼6 post-BrdU dosedependently increased the number of 7-day-old BrdU+ cells with EC50 of 0.127 mg/kg (Fig. 1d). Oral administration of ABG001 (0.5 mg/kg) was used in the subsequent experiments. ABG001-administration on days 2∼20 post-BrdU caused a marked increase in the number of 28-day-old BrdU+ cells (p < 0.01, n = 10; Fig. 1e). Following ABG001administration, the number of 28-day-old BrdU+/NeuN+ was significantly increased compared to control mice (p < 0.01, n = 10; Fig. 1f), but the number of 28-day-old BrdU+/GFAP+ cells was not altered (p > 0.05; n = 10; Fig. 1g). The population ratio of BrdU+/NeuN+ cells (76.5 ± 3.81%) and BrdU+/GFAP+ cells (9.2 ± 2.7%) against total 28-day-old BrdU+ cells in ABG001-mice showed no significant difference from those in control mice (80.4 ± 5.32% and 8.6 ± 1.7%, p > 0.05). DCX, a microtubule-associated protein, is expressed specifically in newly generated healthy neurons, reaching a peak during the second week (Rao and Shetty, 2004). Consistent with the results in vitro (Luo et al., 2011), ABG001-administration for five days significantly increased the number of DCX+ dendrites per newborn cell (p < 0.01; n = 10, Fig. 1h). The results indicate that ABG001 can get across the blood-brain barrier to enhance the survival and neurite growth of newly generated neurons, but it does not affect cell-proliferation and differentiation of progenitor cells. Involvement of TrkA receptor in pro-neurogenesis of ABG001 To investigate molecular mechanisms of the ABG001enhanced neurogenesis, the expression and activation of TrkA receptor and NGF in hippocampus were examined after the ABG001 administration for five days. The results showed that the ABG001-administration did not alter the levels of TrkA mRNA (p > 0.05, n = 10; Fig. 2a) and NGF mRNA (p > 0.05, n = 10; Fig. 2b) compared to controls. Interestingly, ABG001 administration significantly elevated the level of TrkA receptor phosphorylation (p < 0.01, n = 10; Fig. 2c), which was blocked by the injection (i.c.v.) of K252a (p < 0.01, n = 10), a selective but not specific inhibitor of TrkA receptor (Camarena et al., 2010). The K252a-injection (i.c.v.) at days 2∼6 post-BrdU decreased the number of 7-day-old BrdU+ cells (p < 0.01; n = 10, Fig. 2d), but it did not alter the number of DCX+ dendrites (p > 0.05; n = 10, Fig. 2e). However, the K252a-injection at 30 min prior to the ABG001 administration could perfectly block the ABG001increased 7-day-old BrdU+ cells (p < 0.01, n = 10) or DCX+ dendrites (p < 0.01, n = 10). The results indicate that ABG001 enhances the survival and neurite growth of newborn neurons through increasing activation of TrkA receptor.

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Influence of ABG001 on TrkA receptor-mediated ERK and PI3K-Akt-mTOR signals The activation of TrkA receptor can trigger ERK and Akt-mTOR signalling pathways (Vaudry et al., 2002). The levels of hippocampal ERK1/2, Akt and mTOR phosphorylation were examined after 2 h of ABG001 administration. A densitometry analysis revealed that the phosphorylation of ERK2, but not ERK1, was increased approximately two-fold by the treatment of ABG001 compared to control (p < 0.01, n = 10; Fig. 3a). In addition, ABG001 administration could significantly elevate the phosphorylation of Akt (p < 0.01, n = 10; Fig. 3b) and mTOR (p < 0.05, n = 10; Fig. 3c). ABG001-increased phosphorylation of ERK2, Akt or mTOR was blocked by the treatment with K252a (p < 0.01, n = 10). The results indicate that ABG001 increases the activation of TrkA receptor to cascade ERK and Akt-mTOR signalling pathways. To further test whether ABG001 through cascading ERK and Akt-mTOR enhances neurogenesis (Jaworski et al., 2005), the ERK inhibitor U0126 or the PI3K inhibitor LY294002 was injected (i.c.v.) 30 min prior to ABG001 administration. In comparison with vehicle mice (i.c.v. injection of vehicle), treatment with U0126 alone at days 2∼6 post-BrdU caused a slight decrease in the number of 7-day-old BrdU+ cells (p < 0.05, n = 10; Fig. 3d), but not in the DCX+ dendrites (p > 0.05, n = 10; Fig. 3e). Pre-treatment with U0126 perfectly blocked the ABG001-increased 7-day-old BrdU+ cells (p < 0.01, n = 10), but ABG001-increased DCX+ dendrites were not affected (p > 0.05, n = 10). In comparison with vehicle mice, treatment with LY294002 at days 2∼6 post-BrdU failed to alter the number of 7-day-old BrdU+ cells (p > 0.05, n = 10), although it reduced the number of DCX+ dendrites (p < 0.01, n = 10). Furthermore, pre-treatment with LY294002 had no effect on the ABG001-increased 7-dayold BrdU+ cells (p > 0.05, n = 10), but it abolished the ABG001-increased DCX+ dendrites (p < 0.01, n = 10). Effects of ABG001-enhanced neurogenesis on spatial memory To test whether ABG001-enhanced neurogenesis can promote spatial memory, the spatial memory was examined (see the time chart of experimental procedure in Fig. 4a) using the space learning from the Morris water maze task and spontaneous alternation behaviour in the Y-maze test. Repeated-measures ANOVA revealed that the escape latency to reach the hidden-platform with training trials progressively decreased in all groups (F(4,180) = 67.20, p < 0.001), which was affected by ABG001-administration (F(1,45) = 4.87, p < 0.05) and K252a (F(1,45) = 18.94, p < 0.001). In comparison with control mice, ABG001-mice needed less time to reach the hidden-platform on days 3–5 after training (p < 0.05, n = 12). Results of the probe trial showed ABG001-mice spent a longer swimming time in the platform quadrant than control mice (p < 0.05, n = 12; Fig. 4c). Furthermore, the alternation

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Fig. 2. Pro-neurogenesis of ABG001 depends on activation of TrkA receptor. (a&b) Effects of ABG001 on TrkA receptor and NGF expression. Bars indicate the levels of TrkA and NGF mRNA in the hippocampus. (c) Representative Western blot of TrkA receptor phosphorylation and protein in the hippocampus. Bars indicate the level of phosphorylated TrkA receptor (p-TrkA) normalized by TrkA receptor protein (TrkA). **p < 0.01 vs. control mice; ##p < 0.01 vs. ABG001-mice (two-way ANOVA). (d) Representative images of 7-day-old BrdU+ cells (black arrows) in control and ABG001-mice (left panels). Scale bar = 100 μM. TrkA receptor-dependent of ABG001-increased 7-day-old BrdU+ cells. (e) Representative pictures of DCX immunostaining (left panels). Scale bar = 50 μM. TrkA receptor-dependent of ABG001-increased DCX+ dendrites. **p < 0.01 vs. control mice; ##p < 0.01 vs. ABG001-mice (two-way ANOVA).

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Fig. 3. ABG001 enhances neurogenesis through promoting TrkA receptor-mediated ERK and PI3K-Akt-mTOR signalling pathways. (a–c) Levels of phosphorylated ERK1/2 (p-ERK1/2), Akt (p-Akt) and mTOR (p-mTOR) were normalized by total ERK1/2, Akt and mTOR, respectively. *p < 0.05 and **p < 0.01 vs. control mice; ##p < 0.01 vs. ABG001-mice (two-way ANOVA). (d&e) Effects of the ERK inhibitor U0126 and the PI3K inhibitor LY294002 on ABG001-increased 7-day-old BrdU+ cells and DCX+ dendrites. **p < 0.01 vs. vehicle-mice; ##p < 0.01 vs. ABG001-mice (two-way ANOVA).

1856 L. Zhou et al. (a)

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Fig. 4. ABG001 administration enhances spatial cognition. (a) Time chart of experimental procedure. (b) Each point represents group mean latency (s) to reach the hidden platform in the Morris water maze task. *p < 0.05 vs. control mice; ##p < 0.01 vs. ABG001-mice. (c) Bars indicate the percentage of time spent in the platform quadrant of the probe trial. *p < 0.05, **p < 0.01 vs. control mice; ##p < 0.01 vs. ABG001-mice (two-way ANOVA). (d) Bars indicate the mean alternative rate in the Y-maze. *p < 0.05, **p < 0.01 vs. vehicle-mice; ##p < 0.01 vs. ABG001-mice (two-way ANOVA).

rate in the Y-maze was significantly increased in ABG001-mice compared to control mice (p < 0.05, n = 12; Fig. 4d). Consistent with ABG001-enhanced neurogenesis, the enhancing effects of ABG001 on spatial cognitive function depended on the activation of TrkA receptor (p < 0.01, n = 12; Fig. 4b–d). The treatment with K252a alone caused the cognitive deficiency compared to control mice (escape latency: p < 0.05; time in platform quadrant: p < 0.01; alternation rate: p < 0.01, n = 12; Fig. 4b–d). Effects of ABG001 on Aβ-impaired spatial memory and neurogenesis Previous studies reported that the neurite growth and survival of newborn neuronal cells were dramatically impaired in AD transgenic mice with Alzheimer’s-type amyloid pathology (Verret et al., 2007; Xu et al., 2012). A single injection (i.c.v.) of Aβ25–35 in mice has been reported to impair the survival and neurite growth of newly generated neurons in hippocampal DG through down-regulating PI3K-Akt-mTOR signalling pathway (Li et al., 2010). A further experiment was designed to

test whether ABG001 has neuroprotective effects against Aβ25–35-impaired neurogenesis. To avoid the influence of Aβ25–35 on cell-proliferation (Li et al., 2010), the injection of Aβ25–35 was given on day three, post-BrdU. At the same time, the ABG001-administration (0.5 mg/kg) was started (see the time chart of experimental procedure in Fig. 5a). In comparison with vehicle mice, Aβ25–35-mice spent a longer time to reach the hidden platform (F(1,44) = 22.08, p < 0.001; Fig. 5b) and a shorter swimming time in the platform quadrant (p < 0.01, n = 12; Fig. 5c). The alternation rate in the Y-maze was less in Aβ25–35-mice than in vehicle mice (p < 0.01, n = 12; Fig. 5d). The ABG001-administration in Aβ25–35-mice could attenuate their latency in reaching the hidden platform (F(1,44) = 18.56, p < 0.001), increase their swimming time in the platform quadrant (p < 0.01, n = 12) and the alternation rate in the Y-maze (p < 0.01, n = 12) compared to Aβ25–35-mice. Moreover, the anti-amnesic effect of ABG001 in Aβ25–35mice required the activation of TrkA receptor (p < 0.01, n = 12; Fig. 5b–d). The treatment of Aβ25–35-mice with ABG001 could rescue the decreased 28-day-old BrdU+ cells (p < 0.01, n = 10; Fig. 5e) and DCX+ dendrites

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(p < 0.01, n = 10; Fig. 5f) in a TrkA receptor-dependent manner (p < 0.01, n = 10). Furthermore, ABG001-administration could prevent Aβ25–35-decreased phosphorylation of Akt (p < 0.01, n = 10; Fig. 5g) and mTOR (p < 0.05, n = 10; Fig. 5h), which was sensitive to the TrkA receptor inhibitor K252a (p < 0.01, n = 10).

Discussion The present study provides, for the first time, in vivo evidence that the oral administration of ABG001 enhances the survival and neurite growth of newborn cells in adult hippocampal DG via TrkA receptor-triggered ERK and

1858 L. Zhou et al. (f )

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Fig. 5. ABG001 administration prevents Aβ-impaired spatial cognition and neurogenesis. (a) Time chart of experimental procedure. (b&c) Effects of ABG001 administration on Aβ25–35-induced spatial cognitive deficit. (b) Each point represents group mean latency (s) to reach the hidden-platform in the Morris water maze task. **p < 0.01 vs. vehicle-mice; ##p < 0.01 vs. Aβ-mice; ++p < 0.01 vs. ABG001-treated Aβ-mice. (c) Bars indicate the percentage of time spent in the platform quadrant of the probe trial. **p < 0.01 vs. vehicle-mice; ##p < 0.01 vs. Aβ-mice; ++p < 0.01 vs. ABG001-treated Aβ-mice (three-way ANOVA). (d) Bars indicate the group mean alternation rate in the Y-maze. **p < 0.01 vs. vehicle-mice; ##p < 0.01 vs. Aβ-mice; ++p < 0.01 vs. ABG001-treated Aβ-mice (three-way ANOVA). (e&f) Representative pictures of BrdU and DCX immunostaining. Bar graph shows the numbers of 28-day-old BrdU+ cells and DCX+ dendrites per DCX+ cell. **p < 0.01 vs. vehicle-mice; ##p < 0.01 vs. Aβ-mice; ++p < 0.01 vs. ABG001-treated Aβ-mice (three-way ANOVA). (g&h) Effects of ABG001 administration on Aβ25–35-reduced Akt-mTOR. Levels of phosphorylated Akt (p-Akt) and mTOR (p-mTOR) were normalized by total Akt and mTOR, respectively. *p < 0.05, **p < 0.01 vs. vehicle-mice; #p < 0.05, ##p < 0.01 vs. Aβ-mice; ++p < 0.01 vs. ABG001-treated Aβ-mice (three-way ANOVA).

PI3K-Akt-mTOR signalling pathways, which lead to a more preferential spatial cognitive function. More importantly, ABG001-administration in Aβ25–35-mice exerts an anti-amnesic effect through protecting neurogenesis deficiency.

In comparison with the in vitro effects of ABG001 in PC12 cells (Luo et al., 2011), this study obtained a lot of interesting in vivo data concerning the pro-neurogenesis of ABG001 in the hippocampal DG of adult male mice. Consistent with the direct application of ABG001

ABG001-enhanced neurogenesis in hippocampal DG by i.c.v. injection or the incubation of PC12 cells (Luo et al., 2011), the oral administration of ABG001 increased the survival and neurite growth of newborn cells, indicating that ABG001 might get across the blood-brain barrier reaching the brain. Oral administration of ABG001 could dose-dependently increase the number of newly generated neuronal cells, whereas it did not affect the cellproliferation and the differentiation of progenitor cells. Inconsistent with ABG001-action in PC12 cells, ABG001 administration could enhance the phosphorylation of TrkA receptor, which was critical for the enhanced survival of newborn cells in hippocampal DG. This discord may arise from the difference in the experimental subjects and environmental factors. In addition to ABG001-increased ERK phosphorylation in PC12 cells, ABG001 could increase the levels of Akt and mTOR phosphorylation in a TrkA receptor-dependent manner, which was responsible for the ABG001-enhanced neurite growth. The results of this study suggest that the oral administration of ABG001, getting across the blood-brain barrier, can mimic the enhancing effects of NGF on the survival and neurite growth of newly generated neuronal cells. This conclusion is deduced mainly from the following observations: consistent with the oral administration of ABG001, the injection (i.c.v.) with NGF can cause a 20% increase of 14-day-old BrdU+ cells, but it does not affect the proliferation rate of progenitor cells in hippocampal DG (Frielingsdorf et al., 2007); NGF promotes the survival of newborn cells by binding to the specific TrkA receptor (Lu et al., 2005). NGF-induced autophosphorylation of TrkA receptor cascades the Ras-dependent protein kinase ERK to induce the phosphorylation of CREB (cAMP-regulated enhancer binding protein) (Riccio et al., 1997), which is essential for neurotrophindependent survival of neurons (Bonni et al., 1999); and similarly, ABG001 administration could significantly elevate the level of TrkA receptor phosphorylation, which was sensitive to the TrkA receptor antagonist. Thus, it is highly likely that ABG001 as an activator of TrkA receptor induces the phosphorylation of TrkA receptor. In addition, the NGF-activated PI3K leads to the phosphorylation of Akt that may be responsible for neurite outgrowth of sensory neurons (Spillane et al., 2012). The activation of the PI3K-Akt-mTOR signalling pathway can phosphorylate p70 ribosomal S6 kinase (p70S6k) to further stimulate F-actin reorganization and phosphorylation of focal adhesion proteins (Liu et al., 2008), which plays a fundamental role in the axonal and dendritic growth. The reduction of NGF in 1,25-dihydroxy vitamin D3 deficiency mice induces the apoptosis of newborn neurons in hippocampal DG (Zhu et al., 2012). At the end stage of AD, the reduction of NGF and the dysfunction of TrkA receptor have been reported to underlie the selective degeneration of the cholinergic neurons (Hock et al., 2000) and death of newly formed

1859

neuronal cells (Xu et al., 2012). The overproduction and aggregation of Aβ through down-regulating mTOR and p70S6k or inhibiting Ras/PI3K signalling can suppress the neurite growth of newborn neurons (Li et al., 2010). NGF administration to the brain may counteract the progressive loss of neuronal cells in AD. The fibrillar Aβ at a non-lethal level is sufficient to interrupt the PI3K-Akt-mTOR signalling pathway (Pei and Hugon, 2008; Chen et al., 2009). Similarly, Lafay-Chebassier et al. (2005) have demonstrated a sustained downregulation in mTOR-p70S6k signalling by Aβ exposure in neuroblastoma cells, in the cortex of APP/PS1 mutant transgenic mice and in lymphocytes of AD patients. Our results showed that the Aβ25–35-infusion decreased the levels of Akt and mTOR phosphorylation, which could be rescued by ABG001 administration in a TrkA receptor-dependent manner. Therefore, oral administration of ABG001, through enhancing TrkA receptortriggered ERK and PI3K-Akt-mTOR signalling, can protect the survival and the neurite growth of newly generated neuronal cells against Aβ-neurotoxicity. An important finding in this study is that the oral administration of ABG001 could protect the survival and neurite growth of newly generated neuronal cells from Aβ25–35-induced damage, which was accompanied by the improvement of spatial cognitive performance. Continual neurogenesis in adult animals is required to maintain the hippocampal-dependent functions (Leuner et al., 2006). The hippocampus-dependent spatial memory depends on the capacity of adult neurogenesis in the hippocampal DG (Thuret et al., 2009). Although newborn cells are a small percentage of the total cells in the granule cell layer, the newborn synapses have lower activation thresholds and higher resting potentials, which leads to the facilitation of LTP induction in comparison with mature neurons (Schmidt-Hieber et al., 2004), since in immature rats, LTP in the DG lasts a longer time than in adults (Bronzino et al., 1994). Several studies have shown that increased neurogenesis exerts a positive effect on cognitive functions (Tanapat et al., 2005). Some aged-rats with preserved spatial memory exhibit a higher number of newborn neurons in comparison with the aged-rats with spatial memory impairments (Drapeau et al., 2003). Thus, it is highly likely that treatment with ABG001 improves the deteriorated cognitive performance in Aβ25–35 mice through recovering the capacity of neuronal replacement in the hippocampus. In summary, several lines of evidence favour a key role of neurogenesis in hippocampus-dependent learning and memory (Aimone et al., 2006). Transplantation of fibroblasts by enhancing NGF release leads to a slowing of cognitive decline in a phase 1 clinical trial performed on patients suffering from mild AD (Tuszynski et al., 2005). The present study provides a possibility for novel therapeutic application of ABG001 for dementia in Alzheimer’s disease through its potent capability to recover the capacity of hippocampal neuronal replacement.

1860 L. Zhou et al. Acknowledgments This work was supported by grants 973 (No. 2014CB943303) and NSFC (No. 81361120247; 31171440) to Lei Chen.

Statement of Interest None

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