Neurobiology of Aging xxx (2014) 1e13
Contents lists available at ScienceDirect
Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Rescue of cognitive-aging by administration of a neurogenic and/or neurotrophic compound Silvia Bolognin a,1, Mario Buffelli a,1, Jukka Puoliväli b, Khalid Iqbal a, * a Department of Neurochemistry, Inge Grundke-Iqbal Research Floor, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA b Department of Behavioral Studies, Charles River Finland, Kuopio, Finland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 December 2013 Received in revised form 18 February 2014 Accepted 26 February 2014
Aging is characterized by a progressive decline of cognitive performance, which has been partially attributed to structural and functional alterations of hippocampus. Importantly, aging is the major risk factor for the development of neurodegenerative diseases, especially Alzheimer’s disease. An important therapeutic approach to counteract the age-associated memory dysfunctions is to maintain an appropriate microenvironment for successful neurogenesis and synaptic plasticity. In this study, we show that chronic oral administration of peptide 021 (P021), a small peptidergic neurotrophic compound derived from the ciliary neurotrophic factor, significantly reduced the age-dependent decline in learning and memory in 22 to 24-month-old Fisher rats. Treatment with P021 inhibited the deficit in neurogenesis in the aged rats and increased the expression of brain derived neurotrophic factor. Furthermore, P021 restored synaptic deficits both in the cortex and the hippocampus. In vivo magnetic resonance spectroscopy revealed age-dependent alterations in hippocampal content of several metabolites. Remarkably, P021 was effective in significantly reducing myoinositol (INS) concentration, which was increased in aged compared with young rats. These findings suggest that stimulating endogenous neuroprotective mechanisms is a potential therapeutic approach to cognitive aging, Alzheimer’s disease, and associated neurodegenerative disorders and P021 is a promising compound for this purpose. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Cognitive aging Alzheimer’s disease Neurogenesis Synaptic plasticity Neurotrophic factor Peptidergic compound
1. Introduction Aging, though physiological in nature, is considered as a critical condition characterized by a progressive deterioration of the overall homeostatic brain mechanisms. In humans, cerebral aging implies a variety of morphologic alterations that include enlargement of ventricles, progressive loss of brain weight (Bertoni-Freddari et al., 2008), and histopathologically significant reduction in the number of synapses (Burke and Barnes, 2006). Hippocampus is the key target of these age-associated changes that affect both its structural and functional integrity, resulting in learning and memory deficits (Driscoll and Sutherland, 2005; Rosenzweig and Barnes, 2003). Aging is also the most important
* Corresponding author at: Department of Neurochemistry, Inge Grundke-Iqbal Research Floor, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA. Tel.: þ1 718 494 5259; fax: þ1 718 494 1080. E-mail address: [email protected] (K. Iqbal). 1 Present address: Department of Neurological and Movement Sciences, Section of Physiology University of Verona, Strada le Grazie 8, 37134 Verona, Italy. 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.02.017
risk factor for the development of neurodegenerative diseases such as Alzheimer’s disease (AD). This has mainly been attributed to alterations of cell microenvironment (neurogenic niche), which compromise the brain milieu. One of the most direct effects of this insufficient microenvironment support is the dramatic decrease of proliferative activity in the aging brain (Drapeau and Nora Abrous, 2008; Miranda et al., 2012). Indeed, the neuronal survival is thought to depend on the surrounding neurotrophic microenvironment. Furthermore, the propensity of newly generated cells to adopt a neuronal phenotype (successful neurogenesis) markedly diminishes with aging (Driscoll et al., 2006; Heine et al., 2004). It is now increasingly believed that adult-generated neurons contribute to the formation of hippocampal-dependent memory (Deng et al., 2009; Shors, 2008) and these cells can be integrated into patterns of memory networks. Notably, evidence suggests that neurons formed during development and adulthood in the dentate gyrus (DG) are most likely integrated into the hippocampal memory circuits at the same rates and equally contribute to hippocampal memory formation (Stone et al., 2011). Accordingly, adult neurogenesis suppression was demonstrated to affect different forms of hippocampal-dependent learning, such as
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Morris water maze and contextual fear conditioning in rodents (Deng et al., 2009; Shors, 2008). Thus, a decline of adult neurogenesis has clearly been correlated to a decline of hippocampal memory functions. The mechanisms underpinning the decrease in successful neurogenesis in the DG are still not well understood (Lazarov and Marr, 2013). However, up-regulation of signals suppressing self-renewal of neural stem cells (Bonaguidi et al., 2008) or decreased neurotrophic factor levels (Bernal and Peterson, 2011; Hattiangady et al., 2005; Shetty et al., 2005) have been both hypothesized as potential causative factors. Consequently, the approaches aiming at recovering the biochemical milieu of the brain might be a promising therapeutic option to enhance healthy aging and eventually to counteract neurodegeneration, especially that seen in AD. Toward this direction, several studies showed the key role of neurotrophins in the promotion on neuronal survival and modulation of synaptic connectivity (Dawbarn and Allen, 2003; Rosenblad, 2004). Among these factors, ciliary neurotrophic factor (CNTF) has shown remarkable neuroprotective properties (Chojnacki et al., 2003; Song et al., 2002). We have previously shown in cell culture that CNTF counteracted the effect of increased fibroblast growth factor-2, which impairs neuronal lineage determination and maturation, resulting in promotion of successful neurogenesis (Chen et al., 2001, 2007). However, attempts to use CNTF (as other neurotrophic factors) as an effective tool to delay deterioration of hippocampal-function have been so far inconclusive. This was mainly because of the lack of effective delivery systems as peripherally administered CNTF poorly reached the central nervous system (Chen et al., 2001) and to the appearance of serious side effects such as anorexia, hyperalgesia, muscle loss, and pain. The use of small CNTF-derived peptides, showing the same neuroprotective properties of trophic factor but without the limitations mentioned previously, represents an important therapeutic challenge. We have previously demonstrated that peripheral administration of a bloodebrain-barrier-permeable 11 mer peptide, peptide 6, corresponding to the active region of CNTF (amino acid residues 146e156) promoted DG neurogenesis and increased synaptic protein expression, which rescued behavioral impairment in rodent models of sporadic (Bolognin et al., 2012) and familial AD (Blanchard et al., 2010b) and Down syndrome (Blanchard et al., 2011). This positive effect of the peptide was mainly achieved by antagonizing the activity of leukemia inhibitory factor and by increasing the transcription of brain derived neurotrophic factor
(BDNF) (Bolognin et al., 2012; Chohan et al., 2011). Moreover, peptide 6 improved cognitive performance in normal adult mice (Chohan et al., 2011). We further reduced the active region of peptide 6 to only 4 amino acids and we added a C-terminally adamantylated group to increase the lipophilicity and stability of the tetrapeptide, which we called peptide 021 (P021) or Compound 021 (DGGLAG). In previous studies both P021 and its parent nonadamantylated peptide increased neurogenesis, synaptic marker expression, and improved cognitive performance in normal adult mice (Blanchard et al., 2010a; Li et al., 2010). The present study shows that oral chronic treatment with Compound P021 for 88 days can rescue age-associated neurogenesis and neuronal plasticity deficits and cognitive impairment in aged female Fisher rats. These positive effects of P021 involved increase in the expression of BDNF and activation of its signaling pathway as well as increase in synaptic activity both in the cortex and hippocampus. Using in vivo magnetic resonance spectroscopy (MRS), we also detected age-dependent alterations in the hippocampal content of several metabolites. Remarkably, P021 was effective in significantly reducing myo-inositol (INS) level, which was increased in the aged rats. 2. Methods 2.1. Structure of P021 and study outline Female aged (19e21 months) Fischer rats were given P021 (Fig. 1A) per os by gavage (10 mL/kg body weight) once a day for 88 days (Fig. 1B). The dose of P021 was 500 nanomoles 289.15 mg/kg body weight daily. As controls, a second group of aged, and a group of young adult (2e3 months) rats were identically treated but with vehicle (normal saline) only. Administration of vehicle and test compound was done at 7e9 AM daily in the pretesting phase. On the days of behavioral testing, the treatment was given minimum 1 hour before the first test trial. At the end of the treatment, the effect of P021 administration was tested by a spatial reference memory task, in vivo 1H-MRS and fluorodeoxyglucose positron emission tomography (FDG-PET). After sacrificing the animals, tissue was processed and assessed for neurogenesis, BDNF-pathway related proteins, synaptic, and dendritic markers. To detect neurogenesis, the cell proliferation specific marker 5-bromodeoxyuridine (BrdU, Sigma) was administered (50 mg/kg BrdU dissolved in saline, intraperitoneally, 5 mL/kg). BrdU injections were started on day 45 and continued once a day for 5 days. Female Fisher rats were
Fig. 1. Schematic representation of the outline of the study. Chemical structure of P021 (A) and design of the study (B).
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behaviorally tested and subjected to in vivo 1H-MRS and FDG-PET in Charles River Facility (Kuopio, Finland). 2.2. Animal housing All animal studies were carried out according to the National Institute of Health guidelines for the care and use of laboratory animals, and approved by the National Animal Experiment Board, Finland. Altogether 49 female Fischer Rats aged 19e21 months (at the time of treatment onset) and 16 young adult female Fischer rats aged 2e3 months (Charles River, France) weighing approximately 300 g were used. Animals were housed at a standard temperature (22 1 C) and in a light-controlled environment (lights on from 7 AM to 8 PM) with ad libitum access to food and water. Rats were assigned to the treatment groups so that the body weight was balanced between the aged rat groups. 2.3. Behavioral tests 2.3.1. Morris water maze task Spatial reference learning and memory were evaluated in the water maze using a procedure adapted from that previously described by Morris (1984). Testing was performed in a large dark-colored tank (200 cm in diameter) filled with clear water at a temperature of 25 1.0 C. A submerged platform (square platform: 10 10 cm; 1.5 cm below water surface) was placed in the middle of the north west (NW) quadrant. The rats were lowered into the pool with their noses pointing toward the wall at one of the starting points. The release point adjacent to platform location (NW) was not used. All rats received 4 visible platform-training trials in one day with inter-trial interval of 15 minutes on day 64. Then, the acquisition was performed days 71e74 and 78e81. 2.3.2. Acquisition training Acquisition trials were executed to determine the ability of the animals to learn the spatial relationship between distant cues and the escape platform (submerged, no cue rod), which remains in the same location for all trials. The starting points were randomized (NW was not used). The rats received 4 trials (15 minutes apart, 60 seconds maximum for each trial) each day for 4 days. Latency, path length, thigmotaxis, and swim speed were recorded. 2.3.3. Probe trialeday 82 A single probe trial was conducted 24 hours after the last place trial to evaluate memory retention. The platform was removed from the pool and rats were placed into the pool in the quadrant opposite to one the platform was placed before. The rats were allowed to swim for 60 seconds during the probe trial. During the probe trial, the time spent in target quadrant was measured. 2.4. Tissue processing For immunohistochemistry and biochemical analyses, animals were anesthetized with an overdose of sodium pentobarbital and then sacrificed by transcardial perfusion with heparinized saline. The left hemisphere was dissected into hippocampus and cortex and kept at 80 C for biochemical analysis while the right half of the brain was immersion-fixed for 24 hours in 4% paraformaldehyde in 0.1% PBS. Following cryoprotection in 30% sucrose in 0.1 M PBS for 2e3 days, 40 mm sagittal sections were cut using a freezing-sliding Microtome.
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2.5. Quantitative real time polymerase chain reaction (RT-qPCR) Total RNA was extracted from the cortex with RNeasy plus mini kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions. Complementary DNA synthesis was achieved using Super script first strand kit (Invitrogen, Carlsbad, CA, USA). Quantitative real time polymerase chain reaction (RT-qPCR) was performed using Brilliant SYBR Green Master Mix (Agilent, Santa Clara, CA, USA) in a Stratagene Mc3000p PCR detection system under the following conditions: 10 minutes at 95 C, 40 cycles of denaturation at 95 C for 30 seconds, and annealing 55 C for 1 minute, extension at 72 C for 1 minute. The primer sequences were the following: forward 50 -gcggcagataaaaagactgc-30 and reverse 50 -gccagccaattctctttttg-30 for BDNF; forward 50 cgccctgtgagctgaactctg-30 and reverse 50 -ctgcttctcagctgcctgacc-30 for tropomyosin receptor kinase B (TrkB); forward 50 -catttctcgaatctccaacctcaca-30 and reverse 50 -ctacccatccagggggatcttatga-30 for TrkB1; forward 50 -gccgcagagcagaagatcgaaagg-3 and reverse 50 gttctcgcaacagaaagcacgaatgag-30 for GADD45b; forward 50 agaatccgaagggaaaggaa-30 and reverse 50 -tgggctgccaaaataaactc-30 for c-fos; forward 50 - gacatgccgcctggagaac-30 and reverse 50 agcccaggatgccctttagt-30 for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative quantification was performed using the DDCt method. 2.6. Western blots Rat hippocampi and cortices were homogenized to generate 10% (wt/vol) homogenate in cold buffer containing 50 mM TrisHCl (pH 7.4), 8.5% sucrose, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM ethylene glycol tetraacetic acid (EGTA), 10 mM b-mercaptoethanol, benzamidine 5 mM, 0.5 mM (2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 8 mg/mL pepstatin A and 20 mg/mL each of aprotinin and leupeptin, 20 mM b-glycerolphosphate, 100 mM sodium fluoride, 1 mM sodium vanadate and 100 nM okadaic acid. After protein assay by modified Lowry method (Bensadoun and Weinstein, 1976), Western blots were carried out by using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The following primary antibodies were used: anti-GluR1 (1:1000; Millipore, Temecula, CA, USA), anti-GluR2/3 (1:5000; Abcam, Cambridge, MA, USA), anti-MAP2a, b (clone SMI52, 1:4000; Sternberger Monoclonals, MD, USA), anti-N-methyl D-aspartate receptor 2B (NR2B, 1:500; Salomone), anti-N-methyl D-aspartate receptor 2A (NR2A, 1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-CREB (1:1000; Cell Signaling Technology), anti-pCREB (ser133, 1:1000; Cell Signaling Technology), anti-BDNF (clone N-20, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-TrkB (1:500; Santa Cruz Biotechnology),anti-synapsin I (1:20,000, Stressgen, Victoria, BC, Canada), anti-synaptophysin (1:1000; Millipore) antiGAPDH (1:1000; Invitrogen). Immunoblots were probed with the corresponding HRP- secondary antibodies (1:5000) and detected using the enhanced chemiluminescence reagents (Thermo Scientific, Rockford, IL, USA). Multi-Gauge V3 software (Fuji Photo Film, Tokyo, Japan) was used to quantify the protein bands. 2.7. Immunohistochemistry Immunohistochemistry was performed on free-floating sections and every 6th brain section was chosen for densitometry and quantification. The primary antibodies against the following proteins were used at the indicated dilution: anti-MAP2a, b (clone SMI52, 1:1000; Sternberger Monoclonals), anti-BrdU (1:400; Accurate, Westbury, NY, USA), anti-NeuN (1:500; Chemicon,
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Temecula, CA, USA). Alexa 488-conjugated goat anti-mouse IgG antibody (1:500; Molecular Probes, Carlsbad, CA, USA), Alexa 594-conjugated goat anti-rat IgG antibody (1:500; Molecular Probes) were used as secondary antibodies. BrdU immunohistochemistry was performed unmasking BrdU antigen by incubating tissue sections for 2 hours in 50% formamide in 0.03 M sodium citrate and 0.3 M NaCl at 65 C, followed by 5 minutes wash in 0.03 M sodium citrate and 0.3 M NaCl and subsequent incubation for 30 minutes at 37 C.
2.8. Densitometry and stereology For densitometry the CA1, the CA3, and the DG of the hippocampus and parietal association cortex were analyzed. Maximum projection images were then generated based on confocal z-stacks, and the antibody staining was quantified by measuring mean pixel intensity with the software Image-ProPlus 5.0 (Media Cybernetics, Silver Spring, MD, USA). Neural progenitor cell proliferation was assessed in the DG by counting the number of BrdU-immunoreactive cells in the granule cell layer (GCL) of the DG. Counting was performed using 40 oil objective of a Nikon 90i fluorescent microscope equipped with Nikon C1 three-laser confocal system and a Nikon DS U1 digital camera. The total number of BrdU and/or NeuN positive cells in subgranular zone of the DG was determined as previously reported (Blanchard et al., 2011). Briefly, the GCL was subdivided into inner and outer halves. The inner GCL consisted of the subgranular zone, defined as a 2- to 3- nuclei-thick layer bordering the inner half of the GCL adjacent to the hilus; the outer GCL (oGCL) was defined as the half of the GCL adjacent to the molecular layer. The molecular layer was defined as the region between the superior limb of GCL and hippocampal fissure and between the inferior limb of the GCL and the inferior borders of the DG. The hilus included the superficial polymorphic layer.
All layers of the DG described previously were analyzed separately for cell counts. For each brain, at least 100 cells were counted. 2.9. 1H-MRS and FDG-PET After the behavioral evaluation, MRS analysis was performed in 10 randomly selected rats from each group at 1 hour postdosing in a horizontal 11.7 T magnet with bore size 160 mm equipped with a gradient set capable of maximum gradient strength 750 mT/m and interfaced to a BrukerAvance III console (BrukerBiospin GmbH, Ettlingen, Germany). A volume coil (BrukerBiospin GmbH) was used for transmission and a surface phased array coil for receiving (Rapid Biomedical GmbH, Rimpar, Germany). Isoflurane-anesthetized rats were fixed to a head holder and positioned in the magnet bore in a standard orientation relative to gradient coils. For anatomic reference magnetic resonance imaging of the hippocampus Turbo-RARE images were collected using a field of view 30 30 mm2 and matrix of 256 256. 1H-MRS data were collected using the same experimental setup. A voxel was placed bilaterally in the hippocampus of the rat based on the anatomic images collected as described previously. Automatic 3D gradient echo shimming was used to adjust B0 homogeneity in the voxel. The water signal was suppressed using variable power RF pulses with optimized relaxation delays to obtain B1 and T1 insensitivity. An STEAM sequence (TE ¼ 3 ms) combined with outer volume suppression was used for the prelocalization. Three outer volume suppression blocks were used interleaved with water suppression pulses. Data were collected by averaging 320 excitations (frequency corrected for each average) with repetition time of 4 seconds, number of points 4096, and spectral width 5 kHz. In addition, a reference spectrum without water suppression was collected from the identical voxel using the same acquisition parameters. Peak area for metabolites was analyzed using LCModel (Stephen Provencher Inc, Oakville,
Fig. 2. Treatment with P021 ameliorated cognitive performance. Weight (g) of the rats during the 88 days of treatment (A). Swim speed during the training (B). Latency to reach the submerged platform across the training (C). Probe trial: % time in the target quadrant (D).
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Fig. 3. P021 increased neurogenesis in the DG. Photomicrographs illustrating the expression of BrdU- (red) and NeuN-positive cells (green) in the DG (AeC). Scale bar 50 mm. Colocalization of BrdU-NeuN-IR cells per section in the oGCL, iGCL, hilus, and molecular layer of the hippocampus (D). Western blot (E) and relative quantification (F) of NeuN normalized against GAPDH in hippocampus homogenate. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iGCL, inner granule cell layer; oGCL, outer granule cell layer.
Canada) and results were expressed relative to water content in tissue. The following metabolites in the hippocampus were analyzed: alanine (Ala), Creatine (Cr), phosphocreatine (Pcr), gamma aminobutyric acid (GABA), glucose (Glu), glutamine (GLN), glutamate (GLU), glycerophosphocholine (GPC), phosphocholine (Pch), glutathione (GSH), myo-inositol (INS), lactate (lac), N-acetyl aspartate (NAA), taurine (TAU), choline (CHO), N-acetyl aspartate þ N-acetyl aspartate glutamate (NAA þ NAAG), creatine þ phosphocreatine (CR þ PCR), glutamate þ glutamine (GLU þ GLN). For the FDG-PET study, 6 rats per treatment group were fasted overnight, anaesthetized with isoflurane, and injected with circa 30 MBq of FDG. After 45 minutes, the rats were scanned with PET (single 15 minutes scan). For the analysis mean standard uptake value (SUV) was analyzed from the following areas: cerebrum, cortex, prefrontal cortex, striatum, and hippocampus. The SUV equation takes into account the dose at the time of injection, animal weight, and scaling factor (measured counts from PET vs. activity in MBq’s). Mean SUV also takes into account the analyzed volume hence standardizing the minor differences between regions of interests between individuals. 2.10. Statistical analysis Data were analyzed with GraphPad software and are presented as mean standard error. Multiple comparisons among groups were
performed using analysis of variance (ANOVA), followed by Bonferroni post hoc test. For all other comparisons (including intergroup comparisons), Student t test was used. * p < 0.05; ** p < 0.01; *** p < 0.001. 3. Results 3.1. Improvement of cognitive performance by P021 administration Aged Fisher rats were chosen in this study as they show loss of neuronal plasticity and cognitive impairment and thus, they represent an appropriate model to study cognitive aging. During the months of the treatment (Fig. 1), the condition of the animals was assessed daily by evaluating physical state and grooming. We did not observe any alteration in general physical state because of P021 treatment and more importantly, we did not observe any weight difference between old-veh and old-P021 animals for all the course of the treatment (Fig. 2A, p > 0.05). The weight of both groups of aged animals was, as expected, significantly higher compared the young rats group (ANOVA, p < 0.001). This clearly suggests that treatment with P021 did not provoke any of the side effects, ie, anorexia, hyperalgesia, muscle loss, and pain, that full length CNTF is reported to cause. Rats were trained in the Morris water maze task, which is commonly used to assess hippocampal dependent learning and memory. We first analyzed the swim speed of the animals and the
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Fig. 4. P021 induced BDNF expression and restored CREB phosphorylation level. Western blots (A) and relative quantification (B) of BDNF, pro-BDNF, TrkB, p-CREB/CREB in the hippocampus and cortex. BDNF and pro-BDNF were normalized against the loading control while p-CREB was normalized against CREB. Messenger RNA expression levels of BDNF, full length (FL) TrkB, truncated TrkB (TrkB-1), c-fos, and GADD45b in the ventral cortex of young-veh and aged rats (C). # p compared with young-veh; * p compared with old-veh. * p and # p < 0.05, ** p and ## p < 0.01. Abbreviation: BDNF, brain derived neurotrophic factor.
statistical analysis did not reveal any difference among groups (Fig. 2B, ANOVA, p > 0.05). Results of the training were analyzed as latency to reach the submerged platform in the water maze (Fig. 2C). During the training of the task, young-veh rats performed better than the aged rats and identified faster the submerge platform location (ANOVA, p < 0.001). This finding indicates that aged rats were impaired in the learning of the task compared with young rats. Remarkably, P021 treated rats showed a significant improvement of behavioral performance compared with the vehicle treated group (ANOVA, p < 0.05). In the probe trial, young rats spent significantly more time in the target quadrant and less time in the other quadrants compared with the aged groups (Fig. 2D ANOVA, Bonferroni post hoc test p < 0.001). Treatment with P021 partially rescued this impairment and old-P021 rats spent more time in the target quadrant than the old-vehicle treated rats (Student t test, p < 0.05). Thus, these results suggest that old Fisher rats performed poorly in a hippocampal-dependent task compared with young animals but P021 administration significantly improved the behavioral outcome. 3.2. Increase in neurogenesis and BDNF activation by P021 Because we observed positive behavioral effects of P021 on a spatial reference memory task, we next investigated whether P021 could increase neurogenesis. Adult neurogenesis occurs only
in the DG and in the subventricular zone. The hippocampus is of particular interest as its neuronal network are involved in key aspects of learning and memory. Neuronal stem cells are located in the subgranular zone, originating neuronal progenitor stem cells that can differentiate into neurons. Newborn neurons have been recently demonstrated to play a crucial role in hippocampaldependent memory. To study neurogenesis acute administration of the thymidine analog BrdU is useful to investigate proliferation of progenitor cells in the DG. The abundance of BrdUþ-NeuNþ-IR cells was assessed by immunofluorescent staining and stereological analysis (Fig. 3AeD). We observed a dramatic reduction in the number of newborn neurons in old rats compared with young rats in the inner GCL (iGCL), outer GCL (oGCL), hilus, and molecular layer. P021 treatment partially restored this decrease in all the investigated hippocampal areas (Student t test, p < 0.05). We also quantified the expression of the neuronal marker NeuN in the hippocampus by quantitative Western blot. We found a trend but not any statistical significant decrease in NeuN level in aged rats, confirming the absence of an appreciable neuronal loss in aged rats. Interestingly, P021 significantly increased the hippocampal NeuN level in the old animals (Fig. 2E and F). As the survival of newborn cells is strictly connected to BDNF signaling (Sairanen et al., 2005), we examined the effect of P021 on BDNF pathway (Fig. 4). We observed a significant increase of
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Fig. 5. Treatment with P021 increased AMPA and NMDA receptor levels. Western blots (A) and relative quantification (B) in the whole hippocampus and cortex of GluR1, GluR2-3, NR2A, and NR2B. # compared with young-veh; * compared with old-veh. * p and # p < 0.05, ** p and ## p < 0.01. Abbreviation: AMPA, a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptor.
BDNF in old-veh rats compared with young-veh in the hippocampus (Bonferroni post hoc
Contents lists available at ScienceDirect
Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Rescue of cognitive-aging by administration of a neurogenic and/or neurotrophic compound Silvia Bolognin a,1, Mario Buffelli a,1, Jukka Puoliväli b, Khalid Iqbal a, * a Department of Neurochemistry, Inge Grundke-Iqbal Research Floor, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA b Department of Behavioral Studies, Charles River Finland, Kuopio, Finland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 December 2013 Received in revised form 18 February 2014 Accepted 26 February 2014
Aging is characterized by a progressive decline of cognitive performance, which has been partially attributed to structural and functional alterations of hippocampus. Importantly, aging is the major risk factor for the development of neurodegenerative diseases, especially Alzheimer’s disease. An important therapeutic approach to counteract the age-associated memory dysfunctions is to maintain an appropriate microenvironment for successful neurogenesis and synaptic plasticity. In this study, we show that chronic oral administration of peptide 021 (P021), a small peptidergic neurotrophic compound derived from the ciliary neurotrophic factor, significantly reduced the age-dependent decline in learning and memory in 22 to 24-month-old Fisher rats. Treatment with P021 inhibited the deficit in neurogenesis in the aged rats and increased the expression of brain derived neurotrophic factor. Furthermore, P021 restored synaptic deficits both in the cortex and the hippocampus. In vivo magnetic resonance spectroscopy revealed age-dependent alterations in hippocampal content of several metabolites. Remarkably, P021 was effective in significantly reducing myoinositol (INS) concentration, which was increased in aged compared with young rats. These findings suggest that stimulating endogenous neuroprotective mechanisms is a potential therapeutic approach to cognitive aging, Alzheimer’s disease, and associated neurodegenerative disorders and P021 is a promising compound for this purpose. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Cognitive aging Alzheimer’s disease Neurogenesis Synaptic plasticity Neurotrophic factor Peptidergic compound
1. Introduction Aging, though physiological in nature, is considered as a critical condition characterized by a progressive deterioration of the overall homeostatic brain mechanisms. In humans, cerebral aging implies a variety of morphologic alterations that include enlargement of ventricles, progressive loss of brain weight (Bertoni-Freddari et al., 2008), and histopathologically significant reduction in the number of synapses (Burke and Barnes, 2006). Hippocampus is the key target of these age-associated changes that affect both its structural and functional integrity, resulting in learning and memory deficits (Driscoll and Sutherland, 2005; Rosenzweig and Barnes, 2003). Aging is also the most important
* Corresponding author at: Department of Neurochemistry, Inge Grundke-Iqbal Research Floor, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA. Tel.: þ1 718 494 5259; fax: þ1 718 494 1080. E-mail address: [email protected] (K. Iqbal). 1 Present address: Department of Neurological and Movement Sciences, Section of Physiology University of Verona, Strada le Grazie 8, 37134 Verona, Italy. 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.02.017
risk factor for the development of neurodegenerative diseases such as Alzheimer’s disease (AD). This has mainly been attributed to alterations of cell microenvironment (neurogenic niche), which compromise the brain milieu. One of the most direct effects of this insufficient microenvironment support is the dramatic decrease of proliferative activity in the aging brain (Drapeau and Nora Abrous, 2008; Miranda et al., 2012). Indeed, the neuronal survival is thought to depend on the surrounding neurotrophic microenvironment. Furthermore, the propensity of newly generated cells to adopt a neuronal phenotype (successful neurogenesis) markedly diminishes with aging (Driscoll et al., 2006; Heine et al., 2004). It is now increasingly believed that adult-generated neurons contribute to the formation of hippocampal-dependent memory (Deng et al., 2009; Shors, 2008) and these cells can be integrated into patterns of memory networks. Notably, evidence suggests that neurons formed during development and adulthood in the dentate gyrus (DG) are most likely integrated into the hippocampal memory circuits at the same rates and equally contribute to hippocampal memory formation (Stone et al., 2011). Accordingly, adult neurogenesis suppression was demonstrated to affect different forms of hippocampal-dependent learning, such as
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Morris water maze and contextual fear conditioning in rodents (Deng et al., 2009; Shors, 2008). Thus, a decline of adult neurogenesis has clearly been correlated to a decline of hippocampal memory functions. The mechanisms underpinning the decrease in successful neurogenesis in the DG are still not well understood (Lazarov and Marr, 2013). However, up-regulation of signals suppressing self-renewal of neural stem cells (Bonaguidi et al., 2008) or decreased neurotrophic factor levels (Bernal and Peterson, 2011; Hattiangady et al., 2005; Shetty et al., 2005) have been both hypothesized as potential causative factors. Consequently, the approaches aiming at recovering the biochemical milieu of the brain might be a promising therapeutic option to enhance healthy aging and eventually to counteract neurodegeneration, especially that seen in AD. Toward this direction, several studies showed the key role of neurotrophins in the promotion on neuronal survival and modulation of synaptic connectivity (Dawbarn and Allen, 2003; Rosenblad, 2004). Among these factors, ciliary neurotrophic factor (CNTF) has shown remarkable neuroprotective properties (Chojnacki et al., 2003; Song et al., 2002). We have previously shown in cell culture that CNTF counteracted the effect of increased fibroblast growth factor-2, which impairs neuronal lineage determination and maturation, resulting in promotion of successful neurogenesis (Chen et al., 2001, 2007). However, attempts to use CNTF (as other neurotrophic factors) as an effective tool to delay deterioration of hippocampal-function have been so far inconclusive. This was mainly because of the lack of effective delivery systems as peripherally administered CNTF poorly reached the central nervous system (Chen et al., 2001) and to the appearance of serious side effects such as anorexia, hyperalgesia, muscle loss, and pain. The use of small CNTF-derived peptides, showing the same neuroprotective properties of trophic factor but without the limitations mentioned previously, represents an important therapeutic challenge. We have previously demonstrated that peripheral administration of a bloodebrain-barrier-permeable 11 mer peptide, peptide 6, corresponding to the active region of CNTF (amino acid residues 146e156) promoted DG neurogenesis and increased synaptic protein expression, which rescued behavioral impairment in rodent models of sporadic (Bolognin et al., 2012) and familial AD (Blanchard et al., 2010b) and Down syndrome (Blanchard et al., 2011). This positive effect of the peptide was mainly achieved by antagonizing the activity of leukemia inhibitory factor and by increasing the transcription of brain derived neurotrophic factor
(BDNF) (Bolognin et al., 2012; Chohan et al., 2011). Moreover, peptide 6 improved cognitive performance in normal adult mice (Chohan et al., 2011). We further reduced the active region of peptide 6 to only 4 amino acids and we added a C-terminally adamantylated group to increase the lipophilicity and stability of the tetrapeptide, which we called peptide 021 (P021) or Compound 021 (DGGLAG). In previous studies both P021 and its parent nonadamantylated peptide increased neurogenesis, synaptic marker expression, and improved cognitive performance in normal adult mice (Blanchard et al., 2010a; Li et al., 2010). The present study shows that oral chronic treatment with Compound P021 for 88 days can rescue age-associated neurogenesis and neuronal plasticity deficits and cognitive impairment in aged female Fisher rats. These positive effects of P021 involved increase in the expression of BDNF and activation of its signaling pathway as well as increase in synaptic activity both in the cortex and hippocampus. Using in vivo magnetic resonance spectroscopy (MRS), we also detected age-dependent alterations in the hippocampal content of several metabolites. Remarkably, P021 was effective in significantly reducing myo-inositol (INS) level, which was increased in the aged rats. 2. Methods 2.1. Structure of P021 and study outline Female aged (19e21 months) Fischer rats were given P021 (Fig. 1A) per os by gavage (10 mL/kg body weight) once a day for 88 days (Fig. 1B). The dose of P021 was 500 nanomoles 289.15 mg/kg body weight daily. As controls, a second group of aged, and a group of young adult (2e3 months) rats were identically treated but with vehicle (normal saline) only. Administration of vehicle and test compound was done at 7e9 AM daily in the pretesting phase. On the days of behavioral testing, the treatment was given minimum 1 hour before the first test trial. At the end of the treatment, the effect of P021 administration was tested by a spatial reference memory task, in vivo 1H-MRS and fluorodeoxyglucose positron emission tomography (FDG-PET). After sacrificing the animals, tissue was processed and assessed for neurogenesis, BDNF-pathway related proteins, synaptic, and dendritic markers. To detect neurogenesis, the cell proliferation specific marker 5-bromodeoxyuridine (BrdU, Sigma) was administered (50 mg/kg BrdU dissolved in saline, intraperitoneally, 5 mL/kg). BrdU injections were started on day 45 and continued once a day for 5 days. Female Fisher rats were
Fig. 1. Schematic representation of the outline of the study. Chemical structure of P021 (A) and design of the study (B).
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behaviorally tested and subjected to in vivo 1H-MRS and FDG-PET in Charles River Facility (Kuopio, Finland). 2.2. Animal housing All animal studies were carried out according to the National Institute of Health guidelines for the care and use of laboratory animals, and approved by the National Animal Experiment Board, Finland. Altogether 49 female Fischer Rats aged 19e21 months (at the time of treatment onset) and 16 young adult female Fischer rats aged 2e3 months (Charles River, France) weighing approximately 300 g were used. Animals were housed at a standard temperature (22 1 C) and in a light-controlled environment (lights on from 7 AM to 8 PM) with ad libitum access to food and water. Rats were assigned to the treatment groups so that the body weight was balanced between the aged rat groups. 2.3. Behavioral tests 2.3.1. Morris water maze task Spatial reference learning and memory were evaluated in the water maze using a procedure adapted from that previously described by Morris (1984). Testing was performed in a large dark-colored tank (200 cm in diameter) filled with clear water at a temperature of 25 1.0 C. A submerged platform (square platform: 10 10 cm; 1.5 cm below water surface) was placed in the middle of the north west (NW) quadrant. The rats were lowered into the pool with their noses pointing toward the wall at one of the starting points. The release point adjacent to platform location (NW) was not used. All rats received 4 visible platform-training trials in one day with inter-trial interval of 15 minutes on day 64. Then, the acquisition was performed days 71e74 and 78e81. 2.3.2. Acquisition training Acquisition trials were executed to determine the ability of the animals to learn the spatial relationship between distant cues and the escape platform (submerged, no cue rod), which remains in the same location for all trials. The starting points were randomized (NW was not used). The rats received 4 trials (15 minutes apart, 60 seconds maximum for each trial) each day for 4 days. Latency, path length, thigmotaxis, and swim speed were recorded. 2.3.3. Probe trialeday 82 A single probe trial was conducted 24 hours after the last place trial to evaluate memory retention. The platform was removed from the pool and rats were placed into the pool in the quadrant opposite to one the platform was placed before. The rats were allowed to swim for 60 seconds during the probe trial. During the probe trial, the time spent in target quadrant was measured. 2.4. Tissue processing For immunohistochemistry and biochemical analyses, animals were anesthetized with an overdose of sodium pentobarbital and then sacrificed by transcardial perfusion with heparinized saline. The left hemisphere was dissected into hippocampus and cortex and kept at 80 C for biochemical analysis while the right half of the brain was immersion-fixed for 24 hours in 4% paraformaldehyde in 0.1% PBS. Following cryoprotection in 30% sucrose in 0.1 M PBS for 2e3 days, 40 mm sagittal sections were cut using a freezing-sliding Microtome.
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2.5. Quantitative real time polymerase chain reaction (RT-qPCR) Total RNA was extracted from the cortex with RNeasy plus mini kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions. Complementary DNA synthesis was achieved using Super script first strand kit (Invitrogen, Carlsbad, CA, USA). Quantitative real time polymerase chain reaction (RT-qPCR) was performed using Brilliant SYBR Green Master Mix (Agilent, Santa Clara, CA, USA) in a Stratagene Mc3000p PCR detection system under the following conditions: 10 minutes at 95 C, 40 cycles of denaturation at 95 C for 30 seconds, and annealing 55 C for 1 minute, extension at 72 C for 1 minute. The primer sequences were the following: forward 50 -gcggcagataaaaagactgc-30 and reverse 50 -gccagccaattctctttttg-30 for BDNF; forward 50 cgccctgtgagctgaactctg-30 and reverse 50 -ctgcttctcagctgcctgacc-30 for tropomyosin receptor kinase B (TrkB); forward 50 -catttctcgaatctccaacctcaca-30 and reverse 50 -ctacccatccagggggatcttatga-30 for TrkB1; forward 50 -gccgcagagcagaagatcgaaagg-3 and reverse 50 gttctcgcaacagaaagcacgaatgag-30 for GADD45b; forward 50 agaatccgaagggaaaggaa-30 and reverse 50 -tgggctgccaaaataaactc-30 for c-fos; forward 50 - gacatgccgcctggagaac-30 and reverse 50 agcccaggatgccctttagt-30 for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative quantification was performed using the DDCt method. 2.6. Western blots Rat hippocampi and cortices were homogenized to generate 10% (wt/vol) homogenate in cold buffer containing 50 mM TrisHCl (pH 7.4), 8.5% sucrose, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM ethylene glycol tetraacetic acid (EGTA), 10 mM b-mercaptoethanol, benzamidine 5 mM, 0.5 mM (2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 8 mg/mL pepstatin A and 20 mg/mL each of aprotinin and leupeptin, 20 mM b-glycerolphosphate, 100 mM sodium fluoride, 1 mM sodium vanadate and 100 nM okadaic acid. After protein assay by modified Lowry method (Bensadoun and Weinstein, 1976), Western blots were carried out by using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The following primary antibodies were used: anti-GluR1 (1:1000; Millipore, Temecula, CA, USA), anti-GluR2/3 (1:5000; Abcam, Cambridge, MA, USA), anti-MAP2a, b (clone SMI52, 1:4000; Sternberger Monoclonals, MD, USA), anti-N-methyl D-aspartate receptor 2B (NR2B, 1:500; Salomone), anti-N-methyl D-aspartate receptor 2A (NR2A, 1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-CREB (1:1000; Cell Signaling Technology), anti-pCREB (ser133, 1:1000; Cell Signaling Technology), anti-BDNF (clone N-20, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-TrkB (1:500; Santa Cruz Biotechnology),anti-synapsin I (1:20,000, Stressgen, Victoria, BC, Canada), anti-synaptophysin (1:1000; Millipore) antiGAPDH (1:1000; Invitrogen). Immunoblots were probed with the corresponding HRP- secondary antibodies (1:5000) and detected using the enhanced chemiluminescence reagents (Thermo Scientific, Rockford, IL, USA). Multi-Gauge V3 software (Fuji Photo Film, Tokyo, Japan) was used to quantify the protein bands. 2.7. Immunohistochemistry Immunohistochemistry was performed on free-floating sections and every 6th brain section was chosen for densitometry and quantification. The primary antibodies against the following proteins were used at the indicated dilution: anti-MAP2a, b (clone SMI52, 1:1000; Sternberger Monoclonals), anti-BrdU (1:400; Accurate, Westbury, NY, USA), anti-NeuN (1:500; Chemicon,
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Temecula, CA, USA). Alexa 488-conjugated goat anti-mouse IgG antibody (1:500; Molecular Probes, Carlsbad, CA, USA), Alexa 594-conjugated goat anti-rat IgG antibody (1:500; Molecular Probes) were used as secondary antibodies. BrdU immunohistochemistry was performed unmasking BrdU antigen by incubating tissue sections for 2 hours in 50% formamide in 0.03 M sodium citrate and 0.3 M NaCl at 65 C, followed by 5 minutes wash in 0.03 M sodium citrate and 0.3 M NaCl and subsequent incubation for 30 minutes at 37 C.
2.8. Densitometry and stereology For densitometry the CA1, the CA3, and the DG of the hippocampus and parietal association cortex were analyzed. Maximum projection images were then generated based on confocal z-stacks, and the antibody staining was quantified by measuring mean pixel intensity with the software Image-ProPlus 5.0 (Media Cybernetics, Silver Spring, MD, USA). Neural progenitor cell proliferation was assessed in the DG by counting the number of BrdU-immunoreactive cells in the granule cell layer (GCL) of the DG. Counting was performed using 40 oil objective of a Nikon 90i fluorescent microscope equipped with Nikon C1 three-laser confocal system and a Nikon DS U1 digital camera. The total number of BrdU and/or NeuN positive cells in subgranular zone of the DG was determined as previously reported (Blanchard et al., 2011). Briefly, the GCL was subdivided into inner and outer halves. The inner GCL consisted of the subgranular zone, defined as a 2- to 3- nuclei-thick layer bordering the inner half of the GCL adjacent to the hilus; the outer GCL (oGCL) was defined as the half of the GCL adjacent to the molecular layer. The molecular layer was defined as the region between the superior limb of GCL and hippocampal fissure and between the inferior limb of the GCL and the inferior borders of the DG. The hilus included the superficial polymorphic layer.
All layers of the DG described previously were analyzed separately for cell counts. For each brain, at least 100 cells were counted. 2.9. 1H-MRS and FDG-PET After the behavioral evaluation, MRS analysis was performed in 10 randomly selected rats from each group at 1 hour postdosing in a horizontal 11.7 T magnet with bore size 160 mm equipped with a gradient set capable of maximum gradient strength 750 mT/m and interfaced to a BrukerAvance III console (BrukerBiospin GmbH, Ettlingen, Germany). A volume coil (BrukerBiospin GmbH) was used for transmission and a surface phased array coil for receiving (Rapid Biomedical GmbH, Rimpar, Germany). Isoflurane-anesthetized rats were fixed to a head holder and positioned in the magnet bore in a standard orientation relative to gradient coils. For anatomic reference magnetic resonance imaging of the hippocampus Turbo-RARE images were collected using a field of view 30 30 mm2 and matrix of 256 256. 1H-MRS data were collected using the same experimental setup. A voxel was placed bilaterally in the hippocampus of the rat based on the anatomic images collected as described previously. Automatic 3D gradient echo shimming was used to adjust B0 homogeneity in the voxel. The water signal was suppressed using variable power RF pulses with optimized relaxation delays to obtain B1 and T1 insensitivity. An STEAM sequence (TE ¼ 3 ms) combined with outer volume suppression was used for the prelocalization. Three outer volume suppression blocks were used interleaved with water suppression pulses. Data were collected by averaging 320 excitations (frequency corrected for each average) with repetition time of 4 seconds, number of points 4096, and spectral width 5 kHz. In addition, a reference spectrum without water suppression was collected from the identical voxel using the same acquisition parameters. Peak area for metabolites was analyzed using LCModel (Stephen Provencher Inc, Oakville,
Fig. 2. Treatment with P021 ameliorated cognitive performance. Weight (g) of the rats during the 88 days of treatment (A). Swim speed during the training (B). Latency to reach the submerged platform across the training (C). Probe trial: % time in the target quadrant (D).
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Fig. 3. P021 increased neurogenesis in the DG. Photomicrographs illustrating the expression of BrdU- (red) and NeuN-positive cells (green) in the DG (AeC). Scale bar 50 mm. Colocalization of BrdU-NeuN-IR cells per section in the oGCL, iGCL, hilus, and molecular layer of the hippocampus (D). Western blot (E) and relative quantification (F) of NeuN normalized against GAPDH in hippocampus homogenate. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iGCL, inner granule cell layer; oGCL, outer granule cell layer.
Canada) and results were expressed relative to water content in tissue. The following metabolites in the hippocampus were analyzed: alanine (Ala), Creatine (Cr), phosphocreatine (Pcr), gamma aminobutyric acid (GABA), glucose (Glu), glutamine (GLN), glutamate (GLU), glycerophosphocholine (GPC), phosphocholine (Pch), glutathione (GSH), myo-inositol (INS), lactate (lac), N-acetyl aspartate (NAA), taurine (TAU), choline (CHO), N-acetyl aspartate þ N-acetyl aspartate glutamate (NAA þ NAAG), creatine þ phosphocreatine (CR þ PCR), glutamate þ glutamine (GLU þ GLN). For the FDG-PET study, 6 rats per treatment group were fasted overnight, anaesthetized with isoflurane, and injected with circa 30 MBq of FDG. After 45 minutes, the rats were scanned with PET (single 15 minutes scan). For the analysis mean standard uptake value (SUV) was analyzed from the following areas: cerebrum, cortex, prefrontal cortex, striatum, and hippocampus. The SUV equation takes into account the dose at the time of injection, animal weight, and scaling factor (measured counts from PET vs. activity in MBq’s). Mean SUV also takes into account the analyzed volume hence standardizing the minor differences between regions of interests between individuals. 2.10. Statistical analysis Data were analyzed with GraphPad software and are presented as mean standard error. Multiple comparisons among groups were
performed using analysis of variance (ANOVA), followed by Bonferroni post hoc test. For all other comparisons (including intergroup comparisons), Student t test was used. * p < 0.05; ** p < 0.01; *** p < 0.001. 3. Results 3.1. Improvement of cognitive performance by P021 administration Aged Fisher rats were chosen in this study as they show loss of neuronal plasticity and cognitive impairment and thus, they represent an appropriate model to study cognitive aging. During the months of the treatment (Fig. 1), the condition of the animals was assessed daily by evaluating physical state and grooming. We did not observe any alteration in general physical state because of P021 treatment and more importantly, we did not observe any weight difference between old-veh and old-P021 animals for all the course of the treatment (Fig. 2A, p > 0.05). The weight of both groups of aged animals was, as expected, significantly higher compared the young rats group (ANOVA, p < 0.001). This clearly suggests that treatment with P021 did not provoke any of the side effects, ie, anorexia, hyperalgesia, muscle loss, and pain, that full length CNTF is reported to cause. Rats were trained in the Morris water maze task, which is commonly used to assess hippocampal dependent learning and memory. We first analyzed the swim speed of the animals and the
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Fig. 4. P021 induced BDNF expression and restored CREB phosphorylation level. Western blots (A) and relative quantification (B) of BDNF, pro-BDNF, TrkB, p-CREB/CREB in the hippocampus and cortex. BDNF and pro-BDNF were normalized against the loading control while p-CREB was normalized against CREB. Messenger RNA expression levels of BDNF, full length (FL) TrkB, truncated TrkB (TrkB-1), c-fos, and GADD45b in the ventral cortex of young-veh and aged rats (C). # p compared with young-veh; * p compared with old-veh. * p and # p < 0.05, ** p and ## p < 0.01. Abbreviation: BDNF, brain derived neurotrophic factor.
statistical analysis did not reveal any difference among groups (Fig. 2B, ANOVA, p > 0.05). Results of the training were analyzed as latency to reach the submerged platform in the water maze (Fig. 2C). During the training of the task, young-veh rats performed better than the aged rats and identified faster the submerge platform location (ANOVA, p < 0.001). This finding indicates that aged rats were impaired in the learning of the task compared with young rats. Remarkably, P021 treated rats showed a significant improvement of behavioral performance compared with the vehicle treated group (ANOVA, p < 0.05). In the probe trial, young rats spent significantly more time in the target quadrant and less time in the other quadrants compared with the aged groups (Fig. 2D ANOVA, Bonferroni post hoc test p < 0.001). Treatment with P021 partially rescued this impairment and old-P021 rats spent more time in the target quadrant than the old-vehicle treated rats (Student t test, p < 0.05). Thus, these results suggest that old Fisher rats performed poorly in a hippocampal-dependent task compared with young animals but P021 administration significantly improved the behavioral outcome. 3.2. Increase in neurogenesis and BDNF activation by P021 Because we observed positive behavioral effects of P021 on a spatial reference memory task, we next investigated whether P021 could increase neurogenesis. Adult neurogenesis occurs only
in the DG and in the subventricular zone. The hippocampus is of particular interest as its neuronal network are involved in key aspects of learning and memory. Neuronal stem cells are located in the subgranular zone, originating neuronal progenitor stem cells that can differentiate into neurons. Newborn neurons have been recently demonstrated to play a crucial role in hippocampaldependent memory. To study neurogenesis acute administration of the thymidine analog BrdU is useful to investigate proliferation of progenitor cells in the DG. The abundance of BrdUþ-NeuNþ-IR cells was assessed by immunofluorescent staining and stereological analysis (Fig. 3AeD). We observed a dramatic reduction in the number of newborn neurons in old rats compared with young rats in the inner GCL (iGCL), outer GCL (oGCL), hilus, and molecular layer. P021 treatment partially restored this decrease in all the investigated hippocampal areas (Student t test, p < 0.05). We also quantified the expression of the neuronal marker NeuN in the hippocampus by quantitative Western blot. We found a trend but not any statistical significant decrease in NeuN level in aged rats, confirming the absence of an appreciable neuronal loss in aged rats. Interestingly, P021 significantly increased the hippocampal NeuN level in the old animals (Fig. 2E and F). As the survival of newborn cells is strictly connected to BDNF signaling (Sairanen et al., 2005), we examined the effect of P021 on BDNF pathway (Fig. 4). We observed a significant increase of
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Fig. 5. Treatment with P021 increased AMPA and NMDA receptor levels. Western blots (A) and relative quantification (B) in the whole hippocampus and cortex of GluR1, GluR2-3, NR2A, and NR2B. # compared with young-veh; * compared with old-veh. * p and # p < 0.05, ** p and ## p < 0.01. Abbreviation: AMPA, a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptor.
BDNF in old-veh rats compared with young-veh in the hippocampus (Bonferroni post hoc
