Enhancement of dentate gyrus neurogenesis, dendritic and synaptic plasticity and memory by a neurotrophic peptide

Neurobiology of Aging 32 (2011) 1420–1434

Enhancement of dentate gyrus neurogenesis, dendritic and synaptic plasticity and memory by a neurotrophic peptide Muhammad Omar Chohan a,1 , Bin Li a , Julie Blanchard a , Yunn-Chyn Tung a , Agnes T. Heaney b , Ausma Rabe b , Khalid Iqbal a , Inge Grundke-Iqbal a,∗ a

b

Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314-6399, USA Department of Developmental Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314-6399, USA Received 24 April 2009; received in revised form 7 August 2009; accepted 17 August 2009 Available online 19 September 2009

Abstract Pharmacological enhancement of hippocampal neurogenesis is a therapeutic approach for improvement of cognition in learning and memory disorders such as Alzheimer’s disease. Here we report the development of an 11-mer peptide that we designed based on a biologically active region of the ciliary neurotrophic factor. This peptide, Peptide 6, induced proliferation and increased survival and maturation of neural progenitor cells into neurons in the dentate gyrus of normal adult C57BL6 mice. Furthermore, Peptide 6 increased the MAP2 and synaptophysin immunoreactivity in the dentate gyrus. Thirty-day treatment of the mice with a slow release bolus of the peptide implanted subcutaneously improved reference memory of the mice in Morris water maze. Peptide 6 has a plasma half life of over 6 h, is blood–brain barrier permeable, and acts by competitively inhibiting the leukemia inhibitory factor signaling. The fact that Peptide 6 is both neurogenic and neurotrophic and that this peptide is effective when given peripherally, demonstrates its potential for prevention and treatment of learning and memory disorders. © 2009 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; Neural progenitor cells; Ciliary neurotrophic factor; CNTFR␣; Leukemia inhibitory factor; LIFR; Learning and memory disorders; Neural proliferation and differentiation; Neurogenesis

1. Introduction Neurogenesis in the brain is a lifelong process taking place primarily in the subgranular zone (SGZ) of the dentate gyrus of the hippocampus and the subventricular zone during adulthood. Although neurogenesis in these brain areas is markedly decreased during aging (Kuhn et al., 1996; Kempermann et al., 1998), dividing neural progenitors have even been found in the brains of octogenarians (Eriksson et al., 1998). Moreover, the contribution of adult hippocampal neuroge-

∗ Corresponding author at: Neuroimmunology Laboratory, NYS Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314-6399, USA. Tel.: +1 718 494 5263; fax: +1 718 494 1080. E-mail address: i g [email protected] (I. Grundke-Iqbal). 1 Present address: Department of Neurosurgery, University of New Mexico, Albuquerque, NM 87131-0001, USA.

0197-4580/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2009.08.008

nesis to memory mechanisms has been suggested both at experimental and theoretical levels. Current literature supports that both stem cells and immature neurons play distinct roles in hippocampal dependent memory tasks (Aimone et al., 2006). Newly born mature cells may have an inherent advantage of being recruited into patterns of new memory networks (Kee et al., 2007). Several neurodegenerative conditions such as Parkinson’s, Huntington’s, and Alzheimer’s diseases have documented abnormal hippocampal neurogenesis (Steiner et al., 2006). Furthermore, hippocampal circuitry is highly involved in memory functions particularly vulnerable to aging and neurodegenerative conditions. Consequently efforts to modulate adult hippocampal neurogenesis are seen as a promising therapeutic approach towards treating neurodegenerative disorders. One such approach is the use of neurotrophic factors (Cameron et al., 1998; Emsley and Hagg, 2003; Scharfman et al., 2005; Frielingsdorf et al., 2007; Henry et al., 2007).

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The ciliary neurotrophic factor (CNTF) is a major determining factor for neurogenesis, both in hippocampus and subventricular zone (Emsley and Hagg, 2003; Yang et al., 2008). In the brain, CNTF is expressed in subsets of astrocytes in the neurogenic regions of the brain (Sendtner et al., 1994), whereas its receptor (CNTFR␣) seems to be expressed mostly in progenitor cells and neurons of the hippocampus and various other areas of the brain including motorcortex and cerebellum (Lee et al., 1997; Emsley and Hagg, 2003). CNTF belongs to the IL-6 family of cytokines which also includes IL-11, leukemia inhibitory factor (LIF), oncostatinM, cardiotrophin-1, and cardiotrophin-like cytokine (Senaldi et al., 1999; Shi et al., 1999). CNTF signaling occurs through the formation of a tripartite complex of CNTFR␣, the LIF␤ receptor (LIFR) and glycoprotein 130 (gp130). CNTF and LIF both signal through tyrosine phosphorylation of the signal transducers and activators of transcription (STAT) proteins by the membrane associated Janus kinase (JAK) (Davis et al., 1993). Upon injury of the brain, the expression of both CNTF and CNTFR␣ increases (MacLennan et al., 1996; Kordower et al., 1997; Lee et al., 1997). Previously we identified CNTF as one of the most prominent neurotrophic activities in a proteolytic brain extract, Cerebrolysin® (Chen et al., 2007), which by itself is neurogenic and neurotrophic and which improves spatial memory in normal rats (Tatebayashi et al., 2003). However, like other neurotrophins (Price et al., 2007), the therapeutic potential of exogenous CNTF is eclipsed by its short half life when administered peripherally requiring invasive mode of administration with unpredictable pharmacokinetics (Chen et al., 2001). Here we report the discovery of an 11-mer peptide, Peptide 6, based on epitope mapping to biologically active regions of the human CNTF molecule. We found that peripheral administration of this peptide in normal adult mice resulted in increased numbers of both proliferating and differentiating adult hippocampal neural progenitor cells into neurons and improvement of reference memory. These findings suggest the therapeutic potential of a small peptide that could be useful in preventing and treating learning and memory disorders.

2. Methods 2.1. Antibodies The following primary antibodies were used for immunohistochemistry: anti-BrdU (1:400; Accurate, Westbury, NY), a rat monoclonal antibody raised against bromo-deoxy uridine (BrdU); anti-DCX (1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA), a goat polyclonal antibody raised against an 18-amino acid peptide representing residues 384–410 of human doublecortin; anti-NeuN (1:500; Chemicon, Temecula, CA), a mouse monoclonal antibody raised against purified cell nuclei from mouse brain; anti-c-Fos (Ab5) (1:500; Calbiochem, San Diego, CA), a rabbit polyclonal

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antibody raised against a synthetic peptide corresponding to amino-acids 4–17 of human c-Fos; SMI52 (1:1000; Sternberger Monoclonals, MD, USA), a mouse monoclonal antibody specific for the mature neuronal marker MAP2a,b; anti-synaptophysin (1:200; Chemicon, Temecula, CA), a mouse monoclonal antibody raised against vesicular fraction of bovine brain; anti-Ferritin (1:2000), a rabbit polyclonal antibody against human brain Ferritin (Grundke-Iqbal et al., 1990); anti-MHCII (1:20, Abcam, Cambridge, MA), a mouse monoclonal antibody against purified full length native protein from rat; anti-STAT3 (mouse monoclonal antibody against STAT3 clone 124H6, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA); anti-p-STAT3, a rabbit polyclonal antibody against STAT3 phosphorylated at Tyr705 (1:1,000; Cell Signaling). The following secondary antibodies were used: Alexa 488-conjugated goat anti-mouse IgG antibody and Alexa 594-conjugated goat anti-rabbit or anti-rat IgG antibodies (Molecular Probes, Carlsbad, CA, USA); biotinylated anti-rat and anti-rabbit IgG antibodies and Cy5-conjugated goat anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA, USA). 2.2. Generation of peptides Based on our previous study (Chen et al., 2007), and epitope mapping of neutralizing antibodies to human CNTF (Pepscan, Lelystad, The Netherlands), we designed a set of ten peptides (Fig. 1A and B). These peptides were synthesized on a commercial basis by the Pan Biotechnology Facility of Stanford University (Palo Alto, CA). 2.3. Animals and housing All in vivo studies for characterization of peptides (stereology and behavioral analysis) were performed on 8–10month-old female retired breeders of C57BL6 background. The animals were acclimatized for at least 3 weeks to exclude occasional pregnant mice from the studies. Mice were grouphoused (3 animals per cage) with a 12:12 light:dark cycle and with free access to food and water. To eliminate any effect of behavioral tests on neurogenesis, animals employed for immunohistochemical/biochemical analyses were separated from those used for behavioral studies. All procedures were conducted in accordance with approved protocols from our institutional Animal Welfare Committee. 2.4. Isolation and culture of progenitor cells from adult rat hippocampus Five-month-old adult male Wistar rats were used for isolation and propagation of adult hippocampal progenitor cells (AHPs). Rats were euthanized with a lethal dose of Nembutal (60 mg/kg) administered intraperitoneally. AHPs were isolated as described previously (Tatebayashi et al., 1999). In short, hippocampi from 5-month-old Wistar rats were quickly dissected and chopped into about 0.5 mm3 pieces

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in Hibernate A containing 2% B27 supplement (Invitrogen, Grand Island, NY) and 0.5 mM glutamine at 4 ◦ C. The tissue pieces were digested with 2 mg/ml papain (Worthington, Freehold, NJ) in Hibernate A/B27 for 30 min at 30 ◦ C. After washing with Hibernate A/B27 once and triturating 15–20 times in Hibernate A/B27, the progenitors were enriched on an Optiprep step density gradient and plated onto a 0.01% poly-D-lysine (70–150 kDa, Sigma–Aldrich, St. Louis, MO) coated plate (Corning, Corning, NY) in Neurobasal A containing 2% B27 supplement and 0.5 mM glutamine. After culture at 37 ◦ C in a CO2 incubator for 1 h, the medium was replaced with fresh medium containing 100 IU/ml of penicillin, 100 ␮g/ml of streptomycin, and 10 ng/ml FGF-2. The medium was changed every other day, and the cells were passaged at confluence by trituration. Cultures from the fourth passage were used for the present study.

15-day treatment group received daily intraperitoneal injections (i.p.) of CNTF peptides (0.05–5 nmol/animal/day) with BrdU (150 mg/kg) treatment beginning 1 day after the first peptide injection and continuing daily for the entire period. In the case of the animals treated for 30 days, Peptide 6 (5 nmol/animal/day) was compounded in slow release depot pellets for continuous dosing over 30 days (Innovative Research of America, Sarasota, FL). For control groups, the pellets consisted of the carrier biopolymer only. For implantation, the mice were anesthetized with 2.5% Avertin (0.38 ml for a 25 g animal). Under sterile conditions, the pellets were then subcutaneously implanted along the anterolateral aspect of the right shoulder with a precision trochar (Innovative Research of America, Sarasota, FL). BrdU was given as two daily i.p. injections (100 mg/kg/dose) for 5 days starting on day 2 of peptide treatment.

2.5. Treatment of AHPs with synthetic peptides in a cell based assay

2.9. Tissue processing and immunohistochemistry for in vivo experiments

This assay measures the survival of dividing progenitors. AHPs were treated with different peptides and 10 ng/ml BrdU (Sigma) for 48 h. Each peptide was tested at final concentrations of 0.01, 0.05, 0.1, 0.5 and 1 ␮M in triplicates. After treatment, the cells were fixed, immunostained for BrdU (Kuhn et al., 1997) and counterstained with DAPI. The relative fluorescence units (RFU) ratio of BrdU and DAPI was computed as an indicator of cell proliferation/survival.

Mice were anesthetized with an overdose of sodium pentobarbital (125 mg/kg), transcardially perfused with 0.1 M PBS, and the right hemisphere was fixed in 4% paraformaldehyde in 0.1 M PBS for at least 24 h at room temperature. Tissues were then stored in 30% sucrose solution at 4 ◦ C until sectioning. The brains were sectioned sagittaly on a freezing sliding microtome at 40 ␮m through the entire hippocampus and the sections were stored in glycol anti-freeze solution (ethylene glycol, glycerol and 0.1 M PBS in 3:3:4 ratio) at −20 ◦ C till further processing. Immunohistochemistry was performed on free floating sections as described elsewhere (Kuhn et al., 1997). Every 5th brain section was chosen for quantification of cell number and every 10th section was chosen for staining intensity scanning.

2.6. Analysis of STAT3 phosphorylation in AHPs AHPs at ∼90% confluence were treated with different concentrations of recombinant human CNTF, Peptide 6, or CNTF combined with Peptide 6 for 10 min. The attached cells were then lysed and subjected to Western blots developed with antibodies to STAT3 and to phospho-Tyr705 STAT3 and quantitated using the ECL detection reagents (Amersham Biosciences Corp., Piscataway, NJ). 2.7. Analysis of haptoglobin secretion from HepG2 cells HepG2 cells (ATCC, Manassas, VA) at 80% confluence were treated with different concentrations of recombinant human LIF (Chemicon International, Inc., Temecula, CA), Peptide 6 or LIF combined with Peptide 6 in serum-free Eagle’s minimal essential medium containing 1 ␮M dexamethasone (Baumann et al., 1993) for 48 h. The amount of haptoglobin secreted into the culture medium was determined by ELISA kit (Immunology Consultants Laboratory, Inc., Newberg, OR). 2.8. Treatment of mice with BrdU and peptides To study neurogenesis, 8–10-month-old female retired breeders of C57BL6 background were treated with peptides or, as a control, with saline for either 15 or 30 days. Mice in the

2.10. Cell death The number of apoptotic cells in the dentate gyrus (DG) was analyzed in every 10th section using the TUNEL Apoptosis Detection Kit (Upstate, CA 92590). The sections were then subjected to confocal microscopy for quantitative analysis in the GCL using a 40× oil objective. 2.11. Assessment of neurogenesis, definitions, stereology and confocal imaging Neurogenesis was assessed in the DG by counting the numbers of BrdU-immunoreactive (BrdU-IR), BrdU-DCXIR and BrdU-NeuN-IR cells in various layers of the DG. The granular cell layer (GCL) was subdivided into an inner and outer half (iGCL and oGCL). The iGCL consisted of the subgranular zone (defined as a 2–3 nuclei thick layer bordering the GCL) and the inner half of the GCL adjacent to the Hilus (Hil); the outer GCL (oGCL) was defined as the half of the GCL adjacent to the molecular layer (Mol).

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A cell in the middle of the GCL was considered part of the iGCL and a cell bordering the GCL in the Mol was included in oGCL counts. Mol 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. Hil included the superficial polymorphic layer. All sections were collected using the random uniform sampling scheme. For BrdU-IR cells, counting was performed on every 5th section using a 40× oil objective of a Nikon 90i fluorescent microscope equipped with Nikon C1 three laser confocal system and a Nikon DS U1 digital camera. Employing the principles of unbiased stereology, the optical fractionator method was used to estimate cell counts for the DG (West et al., 1991). All layers of the DG described above were analyzed separately for cell counting. For each brain, at least 100 cells were counted based on coefficient of error determinations. For BrdU-DCX-, BrdU-NeuN-, and c-Fos-NeuN-IR cells, only GCL (consisting of iGCL and oGCL described above) was counted using 100× oil objective in every 10th section. To ensure objectivity, z-stacks were collected for each double IR cell and analyzed later by generating maximum projection and 3D constructs. A cell was counted only when it showed double IR on 3D reconstructed images. For MAP2 and synaptophysin IR, the entire area of the GCL was outlined on every 10th section. Maximum projection images were then generated based on confocal z-stacks, and the antibody staining was quantitated by measuring mean pixel intensity (MPI) with the help of Image-Pro Plus 5.0 software (Media Cybernetics, Silver Spring, MD). All quantitations based on immunohistochemistry were verified independently on coded slides by a second investigator. 2.12. Behavioral investigation of Peptide 6 effect in the Morris Water Maze The effect of Peptide 6 on learning and memory function was assessed using a spatial reference memory task in the water maze since this task highly entails the hippocampus. Mice were divided in 4 groups of 9 mice each (18 controls and 18 Peptide 6-treated) which received peptide (5 nmol/mouse/day) or placebo for 15 or 30 days. Animals were coded such that the experimentator was blind to the assignment of animals to specific treatment groups. The Morris Water Maze procedure was performed using a 110 cm diameter circular tank. To acquaint the mice to the test situation, each mouse was placed in the tank without the escape platform and allowed to swim for 60 s. Acquisition was started 3 days later with the submerged (invisible) escape platform in the North-East quadrant and each animal was given 60 s to find the submerged escape platform. If the mouse did not find the platform in 60 s, it was guided to it. Five acquisition trials were given on each day, for 4 consecutive days. A test for retention, or probe trial, was given 24 h later. During the probe trial the mouse was allowed to swim in the tank

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without the escape platform for 60 s. A second probe trial was given 4 weeks later. Measures of learning were the time and distance swum to reach the escape platform. For retention during the probe trial, the tank was divided into four imaginary quadrants and a small zone where the escape platform had been (virtual platform). Measures of retention were the percent of time spent and the percent of distance swum in each quadrant, and the number of entries into the platform zone. Mouse behavior in the Morris Water Maze was monitored by a Samsung Digital Camera (SDC 4304) and tracked with the SMART software (Pan Lab/San Diego Instruments). 2.13. Statistics Data are presented as mean ± SEM. Data on haptoglobin secretion were analyzed using a two-way ANOVA followed by Fisher’s least significant difference post hoc analysis to identify significant effects. For stereological and behavioral studies analysis involving multiple groups, ANOVA with post hoc Tukey’s test was used. To further guard against type 1 error, Bonferroni correction was applied to all such comparisons and the accepted level of significance was kept at p < 0.01. For analysis of data with skewed distribution, the nonparametric Mann–Whitney U-test was used. For all other comparisons, Student’s t-test was used. Differences with p < 0.05 were considered significant. 2.14. Stability, pharmacokinetics and blood–brain barrier permeability These studies were carried out through a commercial service (APREDICA, Watertown, MA). For plasma stability, Peptide 6 (5 ␮g) was incubated with mouse plasma at 37 ◦ C and aliquots were collected at 0, 15, 30, 60 and 120 min for analysis by LC/MS/MS. Plasma calibration samples were prepared by diluting Peptide 6 to 5 ␮g/ml in Sprague-Dawley Rat plasma (Lampire Biologics) and serially diluted 3-fold in the same plasma on ice to obtain the following concentrations: 1667, 556, 185, 62, 21, 7, 2.3, 0.8, 0.3 ng/ml. Immediately following dilution of the samples after thawing on ice, the protein was precipitated from the samples by the addition of a 3× volume of ice-cold methanol containing haloperidol internal standard (IS). These samples containing precipitated protein were centrifuged at 3750 rpm, for 5 min, at 4 ◦ C. The supernatants were then filtered through a Varian Captiva vacuum filter plate and the protein-free filtrates were analyzed by LC/MS/MS. For pharmacokinetics and blood–brain barrier permeability studies, adult mice (9–11 mo, C57/Bl6) under lethal anaesthesia (125 mg/kg) were given a single i.p. injection of 6 mg Peptide 6/mouse. Animals were bled 10, 30 and 60 min post injection and plasma was isolated. For blood–brain barrier permeability studies, 10 and 60 min post i.p. injection, each animal was transcardially perfused with PBS followed immediately by the removal of the brain and its homogenization in 1 ml PBS.

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Plasma and homogenates were diluted to 10 ␮g/ml in 10% formic acid and serially diluted 3-fold. Immediately following dilution of the samples after extraction with formic acid, the protein was removed by centrifugation after precipitation from the samples by the addition of a 3× volume of ice-cold methanol containing haloperidol IS. These samples were further processed and analyzed as follows: All samples prepared were analyzed by LC/MS/MS (Agilent 6410 MS coupled with an Agilent 1200 HPLC and a CTC PAL chilled autosampler—all controlled by Agilent MassHunter software). After separation on a C18 reverse phase HPLC column (Zorbax SB-C18 2.1 mm × 30 mm, 3.6 mm) using a 4-min acetonitrile–water with formic acid gradient system, peaks were analyzed by MS using positive ESI ionization in MRM mode. The mass spectrometer gas flows and voltages were individually tuned to provide optimal signal for each compound. 3. Results 3.1. Identification and selection of neurogenic peptides Based on epitope mapping of neutralizing antibodies to human CNTF (Fig. 1A), a total of 10 peptides (7–13 amino acids in length) were synthesized (Fig. 1B). Peptides 1–9 were N-terminally acetylated and C-terminally amidated; Peptide 10 was cyclized by non-oxidative solid phase disulphide bond formation between N- and C-terminal cysteines (Galande et al., 2005). Five of the 10 peptides (Peptides 1, 2, 3, 6 and 10) contained the common sequence GLFE with varying degrees of N and C terminal residues. Peptides 4 and 5 were from identical regions of human and rat CNTF molecules, respectively. These peptides were then individually tested in vitro for their effect on cultured rat AHPs in a cell based assay (Fig. 1C). Peptides 5 and 6 induced a 5- and 3.5-fold increase in the BrdU uptake of AHPs respectively, as indicated by relative fluorescent units (RFU) intensity of BrdU normalized to that of the nuclear stain DAPI. Peptide 4 was less active in cell culture while the rest of the peptides did not have any detectable effect on BrdU uptake. Based on in vitro screening, four CNTF peptides were chosen for testing in vivo. Peptides 5 and 6 were chosen as potentially active peptides while Peptides 9 and 10 were included as negative controls.

Peptides 5, 6, 9 and 10, were tested in vivo for 14 days as a mixture in 8–10-month-old female mice (Fig. 1D). The BrdU-IR cells were seen both as single cells and in clusters of two or more cells. The staining pattern of BrdU-IR cells was variable; some cells had solid nuclear staining whereas others had distinct punctate staining of their nuclei. All types of BrdU-IR cells (single, clusters, solid, punctate) were included in stereological counts. The peptide mixture increased proliferation of BrdU-IR cells in the GCL of the DG by as much as 101% compared to saline-treated animals (Fig. 1E). The absolute cell counts for the 0.05 nmol/peptide group were 1518 ± 147 (SEM); for 0.1 nmol, 1116 ± 104; for 0.5 nmol, 1791 ± 55 and for 5 nmol, 1582 ± 48 compared to control, 788 ± 29 (p < 0.01, ANOVA, post hoc Tukey’s). Since there were no significant differences between different doses of peptides in the mixture, we selected two doses, 0.5 and 5 nmol/animal, for the next set of experiments. Next, each peptide was tested separately at two doses. Thirty-three mice were divided into 10 groups including control (n = 6), peptide mixture (n = 3, 0.5 nmol/peptide/ animal/day) and two groups for each of the four peptides (n = 3 each for 0.5 nmol and 5 mol/peptide/animal/day). Each animal received a daily single i.p. injection of the peptides and BrdU (150 mg/kg, starting on the 2nd day) for 14 days (Fig. 1D). Data from the two doses (0.5 and 5 nmol/animal/day) were combined in statistical analysis because there were no significant differences in BrdU-IR cell counts. Compared to normal saline-treated mice, Peptides 5 and 6 increased BrdU-IR cell counts in the GCL by 66% and 45% respectively (p < 0.001, ANOVA followed by post hoc Tukey’s), whereas Peptides 9 and 10 had no significant effect (Fig. 1F and G, and Table 1). Further examination of the proliferation/survival of progenitors in four sub-regions of the hippocampus (Fig. 2A) revealed that compared to the control group, Peptide 5 increased the number of BrdU-IR cells in the iGCL and oGCL by 67% and 83%, respectively (p < 0.001, ANOVA followed by post hoc Tukey’s), whereas no significant differences were observed in either Mol or Hil (Fig. 2 B–E, Table 1). Similarly, Peptide 6 increased the numbers of proliferating cells in both iGCL and oGCL by 41% and 59%, respectively, but not in Mol or Hil. Peptides 9 and 10 had no effect on BrdU-IR cell numbers in any of the four sub-regions. Together, these data suggested that both Peptides 5 and 6 increased BrdU-IR cells

Table 1 Stereological counts (mean ± SEM) of BrdU-IR cells in various sub-regions of the hippocampus in mice treated with different peptides for 15 days. Control Peptide 5 Peptide 6 Peptide 9 Peptide 10

GCL

iGCL

oGCL

Mol

Hil

952 ± 48 1587 ± 72** (0.0001) 1360 ± 42** (0.0002) 1162 ± 35 (0.088) 948 ± 65 (1)

764 ± 33 1276 ± 31** (0.0001) 1083 ± 47** (0.0003) 953 ± 35 (0.03) 767 ± 55 (0.96)

175 ± 19 320 ± 4** (0.0009) 279 ± 16* (0.01) 209 ± 19 (0.93) 182 ± 21 (1)

1316 ± 119 1092 ± 13 (0.77) 1005 ± 48 (0.93) 1045 ± 72 (0.16) 924 ± 69 (0.015)

183 ± 27 247 ± 31 (0.52) 171 ± 28 (0.99) 208 ± 19 (0.97) 167 ± 15 (0.99)

Asterisks indicate statistically significant differences when compared to the control group. p < 0.01 was the accepted level of significance. Values in parenthesis indicate actual p values. n = 6 animals/group. * p < 0.01, ANOVA followed by post hoc Tukey’s. ** p < 0.001, ANOVA followed by post hoc Tukey’s.

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Fig. 1. Design and characterization of CNTF peptides in vitro and in vivo. (A) CNTF is an ␣-helical molecule with the secondary structure consisting of four anti-parallel ␣-helices. Five regions (represented in different colors) corresponding to the epitopes of neutralizing anti-human CNTF antibodies were employed to design ten synthetic peptides. (B) Peptides 1, 2, 3, 6 and 10 share the sequence GLFE; Peptides 4 and 5 contain the same regions of human and rat CNTF, respectively, while the remaining peptides contain distinct epitopes. (C) In vitro screening of peptides employing a cell based assay to measure proliferation/survival of AHPs isolated from a 5-month-old Wistar rat. Peptides 5 and 6 induced a 5- and 3.5-fold increase in the BrdU uptake of AHPs respectively, as indicated by RFU intensity of BrdU normalized to that of the nuclear stain DAPI. *p < 0.01, **p < 0.001, one-way ANOVA. (D) General schema of the experimental paradigm used for animal studies described in Figs. 1 and 2. (E) Based on data from cell based assay, four peptides, 5, 6, 9 and 10 were administered to 8–10-month-old female C57/BL6 mice as a mixture containing 0.05, 0.1, 0.5 or 5 nmol/peptide/animal/day for 14 days. The peptide mixture induced up to 2-fold increase in the number of BrdU-IR cells in the DG of treated animals (a measure of cell proliferation). (F and G) Peptides 5 and 6 increased the proliferation/survival of DG progenitors. BrdU (red), NeuN (green), merged (yellow). Quantitations are based on unbiased stereological counts of BrdU-IR cells in the DG of treated animals. **p < 0.001, ANOVA, post hoc Tukey’s. Scale bar in (F) is 25 ␮m.

in the GCL. Whether this increase represents proliferation or survival or both, cannot be determined due to the BrdU treatment for several days in this study. Higher number of BrdU-IR cells in the oGCL is also an index of relative migration of progenitors towards the Mol (Emsley and Hagg, 2003). Normally, of the total BrdU-IR

cells in GCL, only 20–25% are present in oGCL. In order to investigate whether the increase in oGCL progenitors due to peptide treatment was a result of migration or “ectopic birth”, a phenomenon that refers to an abnormal and selective increase in the number of oGCL progenitors which may cause abnormal connectivity (Donovan et al., 2006), an ectopic

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Fig. 2. Effects of peptides on cell proliferation/survival and early neuronal commitment in the DG. Proliferation/survival of DG progenitors in response to treatment with peptides in different layers of the DG. The treatment paradigm is the same as described for Fig. 1D. (A) Anatomical boundaries of different layers of the DG as defined in this study. Peptide mixture (Mix) and Peptides 5 and 6 increased cell proliferation in both (B) iGCL and (C) oGCL but not in (D) Mol or (E) Hil in 15 days treated animals. (F) There was no evidence of “ectopic birth” in the oGCL of tested mice. (G) Peptide 6 increased the expression of DCX, an immature neuronal marker, in newly divided progenitors. There were two main types of DCX-IR cells seen based on the orientation of the main process with respect to the GCL. Cells with their main process oriented perpendicularly are shown in the upper right panel, while a cell with its main process oriented horizontally is shown in the lower right panel. DCX (green), BrdU (red). Both types of DCX-IR cells were counted in stereological estimates. (H) Of the total number of newborn cells, early neuronal fate commitment was twice as much in Peptide 6 treated mice as in controls. iGCL: inner granular cell layer; oGCL: outer granular cell layer; Mol: molecular layer of the DG; Hil: hilus. *p < 0.01, **p < 0.001, ANOVA, post hoc Tukey’s. Scale bars in (A) represent 100 ␮m and in (G) 20 ␮m (left panels) and 10 ␮m (right panels).

birth index was generated (oGCL/GCL). None of the peptides induced “ectopic birth” in oGCL (Fig. 2F). To investigate whether any of the four peptides induced early neuronal commitment in dividing progenitors, we quantitated BrdU-IR cells in the GCL (iGCL + oGCL) which co-expressed doublecortin (DCX), a marker for neuroblasts and newly generated neurons (Fig. 2G). Stereological analysis revealed that Peptide 6 increased BrdU-DCX-IR cells by more than 2-fold (338 ± 21 vs. 150 ± 10, p = 0.0002, ANOVA followed by post hoc Tukey’s), whereas none of the other groups showed any significant differences (Fig. 2H). Based

on these data, Peptide 6 was chosen as the “lead peptide” for further studies. 3.2. Neurogenic and neurotrophic properties of Peptide 6 3.2.1. Differentiation and migration of progenitor cells in the dentate gyrus of mice treated with Peptide 6 for 30 days Of the progenitors that reach maturity in the DG, >90% become neurons. However, most of the DG progenitors

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Fig. 3. Effect of Peptide 6 on survival of DG progenitors, induction of mature neuronal phenotype and its neurotrophic activity. (A) Experimental paradigm. To determine neuronal maturation and survival of Peptide 6-induced DG progenitors, the peptide was administered as a subcutaneously implanted slow release depot pellet on day 1 of the experiment to be released over 30 days. BrdU was given as two daily i.p. injections (100 mg/kg) from day 2–6 of peptide treatment. (B) Peptide 6 induced proliferation/survival of BrdU-IR cells across the treatment period in all layers of the DG except Hil and Mol. As in the 15-day treatment group (Figs. 1 and 2), there was no evidence of ectopic neuronal birth in treated animals (inset). (C) Peptide 6 treatment also increased the number of newly generated neurons expressing mature neuronal marker NeuN, by almost 2-fold. NeuN (blue), BrdU (red). *p < 0.01, **p < 0.001, Student’s t-test. (D) Peptide 6 induced expression of the immediate-early gene c-fos, a marker of increased neuronal activity. Arrow shows a triple labeled granule cell (inset) expressing c-fos (green), BrdU (red), and NeuN (blue). (E) Peptide 6 increased the expression of the dendritic marker MAP2 and the synaptic marker synaptophysin (SYN) in the DG of peptide-treated mice in both 15-day and 30-day treatment groups. While MAP2 expression continued to increase from 15 to 30 days, SYN expression stabilized after 15 days. All neurotrophic activity was measured as mean pixel intensity of the scanned area of the DG. *p < 0.05, **p < 0.01, Student’s t-test. (F) There was no indication of microglial activation (MHC-Ferritin-IR cells) or microgliosis (Ferritin, lower panels) in peptide-treated mice, suggesting a non-immune cause of enhanced neurogenesis. There were occasional T-cell activated microglia (MHCII-Ferritin-IR cells) in both groups. (G) Neuroprotective effects of Peptide 6 studied by TUNEL staining of representative DG sections of 15- and 30-day-treated mice. Peptide 6 reduced the number of apoptotic nuclei in the DG in the 30 days treatment group but not in the 15 days treatment group. TUNEL (green); nuclear stain PO-PRO3 (red); merged (yellow); arrow head indicates the same nucleus labeled with TUNEL and PO-PRO3. Arrows indicate apoptotic nuclei in the GCL. *p < 0.05, Student’s t-test. Scale bars: (C) 10 ␮m; (D), 20 ␮m, inset 5 ␮m, (F) 50 ␮m, inset 10 ␮m.

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Table 2 Stereological counts (Mean ± SEM) of BrdU-IR cells in various sub-regions of the hippocampus in mice treated with Peptide 6 for 30 days. Control Peptide 6

GCL

iGCL

oGCL

Mol

Hil

427 ± 37 724 ± 71** (0.0003)

334 ± 27 565 ± 60** (0.0013)

93 ± 23 173 ± 33** (0.009)

526 ± 77 759 ± 121 (0.063)

108 ± 17 138 ± 27 (0.129)

Values in parentheses indicate actual p-values. n = 5 animals/group. ** Statistically significant differences when compared to control group (p < 0.01, Student’s t-test).

undergo apoptosis before they mature (Winner et al., 2002). In order to determine whether Peptide 6 induced survival and differentiation of DG progenitors, mice received subcutaneous implants of 30-day extended release pellets containing either Peptide 6 (5 nmol/animal/day, n = 5) or placebo (n = 5) (Fig. 3A). Dividing cells were labeled with BrdU given i.p. for 5 days, twice a day (100 mg/kg/animal/dose starting on day 2 of treatment. Quantitative analysis of Peptide 6-induced DG neurogenesis in 30-day treatment paradigm revealed an increase in BrdU-IR cells in the GCL by 70% (p < 0.001, Student’s t-test; Fig. 3B and Table 2), with a 69% and 86% increase in iGCL and oGCL, respectively, whereas no differences were observed in Mol or Hil. Again, there was no evidence of “ectopic birth” in the outer margins of the GCL, indicating that the increase of BrdU-IR cells in oGCL was probably due to migration of new born cells towards Mol (Fig. 3B, inset). We next investigated the expression of the mature neuronal marker, NeuN, in the BrdU-IR cells in the GCL. We found a 2-fold increase in new born mature neurons in the GCL in Peptide 6 treated mice (386 ± 26 vs. 166 ± 19, p = 0.004, Student’s t-test; Fig. 3C). 3.2.2. Induction of immediate-early gene expression in resident neurons Changes in the expression of immediate-early genes like c-fos and zif correlate with neuronal firing and are used as indicators of neuronal activity (Guzowski et al., 2005; Kee et al., 2007). It is also known that CNTF can upregulate c-fos mRNA and Fos protein (Alderson et al., 1999). We, therefore, investigated whether Peptide 6 induced an increase in Fos protein expression, providing a biological substrate for spatial learning and memory. We found a ∼68% increase in the number of mature neurons (NeuNIR) co-expressing c-fos in the GCL (282 ± 27 vs. 168 ± 17, p < 0.01, Student’s t-test; Fig. 3D). There was also evidence of increased neuronal activity in newborn mature neurons as some BrdU-NeuN-IR cells in the GCL co-expressed c-fos (Fig. 3D). 3.2.3. Neurotrophy and neuroprotection In order to investigate whether Peptide 6 had neurotrophic effects on dendritic arborization and synapses, we measured the expression of MAP2 (a dendritic marker) and synaptophysin (a synaptic marker) in the GCL of both 15- and 30-day-treated mice. GCL staining was quantitated as mean pixel intensity. We found an increase in

MAP2 and synaptophysin immunoreactivity in the GCL of both 15- and 30-day-treated mice compared to the corresponding placebo groups (Fig. 3E). Whereas synaptophysin immunoreactivity remained unchanged between 15- and 30day treatment groups (an increase of 30% ± 6 and 31% ± 3 respectively, Student’s t-test), MAP2 immunoreactivity continued to increase from 15- to 30-day treatment periods in the GCL (an increase of 22% ± 4 to 49% ± 2, respectively). A number of recent studies have shown a regulatory role of the adaptive immune system in adult hippocampal neurogenesis, particularly through T-cell-directed activation of microglia (Ziv et al., 2006). In order to study whether Peptide 6 increased neurogenesis by activating resident microglia or recruiting activated microglia, we compared microglial profiles in the DG in the 30-day treatment group (Fig. 3F). We found no apparent differences in total microglia as determined by Ferritin immunoreactivity (Kaneko et al., 1989; Grundke-Iqbal et al., 1990). Moreover, activated microglia of neuroprotective nature (as defined by Ferritin-MHCII-IR cells) were infrequent in both groups. These data suggest that the Peptide 6-induced increase in neurogenesis was unlikely due to peripheral immune regulation. In order to evaluate whether Peptide 6 had a neuroprotective effect on the DG, we counted the number of cells undergoing apoptosis in the GCL utilizing TUNEL staining (Fig. 3G). There was a slight decrease in the number of TUNEL-positive nuclei in both treatment groups (98 ± 2 in control vs. 89 ± 3 in 15-day and 77 ± 4 in 30-day groups) with the latter group showing a statistically significant decrease (p < 0.05, Student’s t-test). 3.3. Molecular mechanism of action of Peptide 6 3.3.1. Effect of CNTF and Peptide 6 on phosphorylation of STAT3 in AHPs Neural progenitor cells express on their surface the subunits of the tripartite CNTF receptor complex – CNTF receptor alpha (CNTFR␣), LIF receptor beta (LIFR), and glycoprotein 130 (gp130) (Davis et al., 1993). To study whether Peptide 6 induces CNTF-like JAK/STAT signaling, we treated AHPs with different concentrations of CNTF or Peptide 6 for 10 min to investigate phosphorylation of STAT3 at tyrosine 705. CNTF induced significant phosphorylation at this site in a dose-dependent manner (Fig. 4A) as was also previously reported (Ott et al., 2002; Nagao et al., 2007). But there was no similar effect of Peptide 6 on AHPs (Fig. 4B). These data suggested that Peptide 6 by itself was unable to

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3.3.2. Effect of Peptide 6 on LIF signaling Since Peptide 6 contains the putative LIFR-binding sequence in CNTF, we tested its reactivity with the LIF receptor using the human hepatoma cell line HepG2. This cell line expresses LIFR and gp130 but not CNTFR␣ (Di Marco et al., 1996; Kallen et al., 1999) and signals through the JAK/STAT pathway, resulting in the secretion of haptoglobin. We found that while LIF induced secretion of haptoglobin (Fig. 5A), Peptide 6 by itself had no effect on the HepG2 cells (Fig. 5B). However, when combined with LIF, Peptide 6 effectively inhibited the LIF-mediated signaling and ∼50% inhibition of 10 pM LIF was achieved with 100 pM Peptide 6. Increasing levels of Peptide 6 resulted in further inhibition of LIF up to ∼80% (by 10 nM peptide). But at higher peptide concentrations its inhibitory activity gradually decreased, possibly due to its oligomerization (Fig. 5C). 3.4. Pharmacokinetics and blood–brain barrier permeability of Peptide 6 Peptide 6 was found to be relatively stable; it had an in vitro plasma half life of over 6 h (Fig. 6). In vivo in the mouse, Peptide 6 cleared from the plasma at a rate of ∼1.89% per minute during 10–60 min post intraperitoneal injection with an overall half life of <27 min (Fig. 7, left three bars). In contrast, in the brain, the level of the peptide increased ∼2.5fold during the same period (Fig. 7, middle two bars), yielding a ∼48-fold increase in brain:plasma ratio (Fig. 7, right two bars). These data suggested that Peptide 6 was blood–brain barrier permeable and relatively stable. 3.5. Effect of Peptide 6 on cognition

Fig. 4. Inhibition of CNTF-induced phosphorylation of STAT3 by Peptide 6 in AHPs. Treatment of AHPs with CNTF and/or Peptide 6 for 10 min. (A–C) Representative Western blots of phosphorylated STAT3 at Tyr705 (p-STAT3) and total STAT3 (STAT3). (A) CNTF-induced phosphorylation of STAT3 at Tyr705 in a dose-dependent manner. (B) Peptide 6 alone had no effect on STAT3 phosphorylation. (C) Peptide 6 inhibited CNTF-induced phosphorylation of STAT3 in a dose-dependent manner. (D) Quantitative analysis of phosphorylated STAT3. The p-STAT3 data was normalized to total STAT3 expression. Values are expressed as percentages of the values from cell lysates treated with 2.8 pM CNTF alone (100%) and are the mean ± SEM (n = 6 separate samples of each treatment condition). *p < 0.05; **p < 0.01; ***p < 0.001, two-way ANOVA followed by Fisher’s least significant difference post hoc analysis.

induce CNTF-like JAK/STAT signaling in AHPs. To elucidate the possibility that Peptide 6 might modulate the action of CNTF on AHPs, we treated AHPs with different doses of Peptide 6 in combination with CNTF (2.8 pM) and found that ∼1 ␮M of Peptide 6 inhibited ∼50% of CNTF-induced phosphorylation of STAT3 in AHPs (Fig. 4C and D).

To study the effect of Peptide 6 on cognitive function, we subjected mice to a spatial reference memory task in the Morris Water Maze. Fifteen days treatment with Peptide 6 did not induce any effect on performance, either during training or during probe trial retention tests (data not shown). But when the Peptide 6 treatment lasted 30 days (Fig. 8A), a subtle enhancement of learning was observed (Fig. 8B). While no overall significant effect was observed on the training (effect of treatment: p > 0.05; interaction treatment × day: p > 0.05), a significant difference of the level of performance was observed on the last day of training (treatment effect: p < 0.05). Moreover, treatment with Peptide 6 improved retention performance on all three measures on the first probe trial. Animals treated with Peptide 6 spent a higher percentage of time and covered a higher percentage of distance in the target quadrant than animals treated with placebo (Fig. 6C, p < 0.05). Similarly, the mean number of crossings of the area of the platform was significantly higher for the Peptide 6-treated group (3.7 ± 1.7) than for the control group (2.0 ± 2.2) (p < 0.05, Fig. 6D). However, the enhancement of performance for Peptide 6-treated animals observed in the first probe trial was not observed in the second probe trial after the 30-day washout period (data not

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Fig. 5. Inhibition of LIF-induced haptoglobin secretion by Peptide 6 in HepG2 cells. Treatment of HepG2 cells with LIF and/or Peptide 6 for 48 h. (A) LIFinduced haptoglobin secretion from HepG2 cells in a dose-dependent manner. (B) There was no effect of Peptide 6 on secretion of haptoglobin by HepG2 cells. (C) Peptide 6 inhibited LIF-induced haptoglobin secretion from HepG2 cells. Values are expressed as percentages of the values from control cell lysates (100%) and are the mean ± SEM (n = 4–6 samples of each treatment condition). **p < 0.01, ***p < 0.001, two-way ANOVA followed by Fisher’s least significant difference post hoc analysis.

shown). This latest observation thus confirms the specificity of Peptide 6-treatment since after a 30-day washout period, animals previously treated with Peptide 6 behaved similarly as control animals.

Fig. 7. Pharmacokinetics and blood–brain barrier permeability of Peptide 6. C57/BL6 female mice (9–11 mo) were treated with a single i.p. injection of Peptide 6 (6 mg) and perfused with saline after 10, 30 and 60 min. The amount of Peptide 6 was then determined in the brain and plasma by LC/MS/MS. The peptide cleared from the plasma with a T(1/2) of <27 min. In contrast, in the brain, Peptide 6 almost doubled from 10 to 60 min, while its brain:plasma ratio increased by ∼48-fold during this time.

4. Discussion

Fig. 6. Plasma stability of Peptide 6. Peptide 6 (5 ␮g) was incubated with plasma at 37 ◦ C and aliquots were collected at 0, 15, 30, 60 and 120 min for analysis by LC/MS/MS. (A) Peptide 6 was relatively stable in the plasma for up to 120 min. The projected half life of Peptide 6 in plasma was calculated to be >6 h. (B) Up to 83% of Peptide 6 was detectable in the mouse plasma after 120 min of incubation.

Neurodegenerative diseases like AD show a marked decline in brain cognitive functions, and those associated with the hippocampus fade very early in the disease course. The role of dendritic and synaptic plasticities in cognition is well known (see for e.g. Stranahan et al., 2008; Lee and Silva, 2009). One of the earliest changes in cognitive disorders is the loss of dendritic arborization and synapses (see for e.g. Counts et al., 2006; Dickstein et al., 2007; Arendt, 2009). Due to anatomical location of the neurogenic niche, adult hippocampal neurogenesis has been implicated in hippocampal dependent memory networks (Aimone et al., 2006; Wiskott et al., 2006; Trouche et al., 2009). Consequently, adult hip-

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Fig. 8. Improvement of spatial memory in normal adult mice by Peptide 6. (A) Schema of behavioral evaluation in the Morris Water Maze. Peptide 6 was administered as subcutaneously implanted slow release depot pellets for 30-day delivery. Training of animals was started on day 26 and probe trials were conducted 30 days after implantation of pellets. Dotted lines and circle represent imaginary quadrants and position of the platform, respectively, during training. “×” refers to the positions from where the animals were released in the water tank during training and probe trials. (B) While both control and peptide-treated mice learned, Peptide 6 treated mice performed significantly better on the last day of training as assessed by the distance and escape time to find the platform in the Morris Water Maze. (C and D) In the probe trial, Peptide 6-treated mice showed superior retention as assessed by percent time spent and distance traveled (C) in the target quadrant (*p < 0.05). (D) Similarly, the mean number of crossings of the imaginary platform was significantly higher in the mice treated with Peptide 6 than in the controls.

pocampal neurogenesis and neuronal plasticity appear as key contributors to the brain’s ability to cope with age-associated cognitive decline (Steiner et al., 2006). Therefore, increasing adult hippocampal neurogenesis and stimulating neuronal plasticity pharmacologically could thus be very useful strategies towards inhibiting cognitive decline. The present study demonstrates that such an approach is feasible and that Peptide 6, which stimulates hippocampal neurotrophy and neurogenesis in adult mice with consequential significant enhancement of spatial memory is a promising lead molecule. Of the ten CNTF-based peptides used in this study, Peptides 5 and 6 induced proliferation of AHPs in culture. Moreover, the same peptides also increased BrdU labeling

of endogenous precursors in the adult mouse hippocampus. Fifteen-day treatment of adult mice with Peptide 6 showed a statistically significant increase in the number of BrdUIR cells in both the iGCL and oGCL of the DG. Reports of increased early stages of hippocampal neurogenesis in the AD DG and the PDGF-APP Sw, Lnd transgenic mouse model (Jin et al., 2004a,b) indicate a regenerative attempt by the brain which, in AD, may lack the crucial trophic factors required for terminal differentiation of the neuronal progenitors, as indicated by the striking lack of the mature dendritic marker MAP2a,b in the AD dentate gyrus (Li et al., 2008). It has been also argued that the apparent increase in neurogenesis could, in part, be due to a selective increase of new

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born progenitors in the oGCL, a phenomenon referred to as “ectopic birth” (Donovan et al., 2006). This could theoretically disrupt local connectivity within the DG. None of the peptides tested in the present study produced such a phenomenon. Of the four peptides tested in vivo, only Peptide 6 increased expression of the immature neuronal marker DCX in the DG. Although it has been suggested that such immature neural progenitors are responsive to depolarizing stimuli (Aimone et al., 2006; Zhao et al., 2006) and therefore, could participate in neural coding of memory (Wiskott et al., 2006), we did not find improved performance in 15-day-treated mice on spatial memory tasks. This could be due to two reasons. First, improved behavioral performance may be particularly challenging to demonstrate in normal animals. Moreover, the presence of the immature neuronal marker DCX is not sufficient as an indicator of net neurogenesis (Kempermann et al., 2003). Second, the 15-day-treated animals contained a mixture of newly generated cells of which only a fraction were exposed to Peptide 6 for a maximum of 15 days, the minimum period reported to be required for progenitors to actively participate in encoding of memory as shown by Fos expression (Kee et al., 2007). Indeed, animals treated for 30 days showed a significantly improved performance on spatial memory tasks. These animals also had a 2-fold increase in the number of newly matured BrdU/NeuN-positive neurons, an improvement of neurotrophy reflected by enhanced levels of synaptophysin and MAP2 as well as cell survival (i.e. reduced numbers of TUNEL-positive nuclei) in the DG. Although in this study we did not investigate the effect of Peptide 6 treatment on the dendritic and synaptic plasticity beyond the hippocampus, it is not unreasonable to expect similar effects in the rest of the forebrain neurons. All these hippocampal alterations may account for enhanced neuronal plasticity. Consequently, we observed an increase in c-fos expression in newly generated mature neurons of the DG and enhanced performance in the spatial reference memory task. These results thus provide strong evidence for neurotrophic and neurogenic properties of Peptide 6 when administered as a chronic treatment for 30 days resulting in memory enhancement. This suggests that Peptide 6 could potentially alleviate some impairments resulting from hippocampal damages. Indeed, it is important to highlight that in the present study we may have only observed the strongest effect this treatment could provide since experiments were carried out on normal and healthy animals on which it is particularly challenging to demonstrate drug efficiency and its impact on cognition. Furthermore, we have not studied as yet the effect of Peptide 6 on other brain areas and their contributions to memory. We observed a strong effect of Peptide 6 on retention during the probe trial but only a small effect during the last day of training. However, if mice treated with Peptide 6 benefit from a more plastic hippocampal network to encode a spatial representation, it is obvious that such phenomenon can hardly have consequences on ceiling behavioral performance. Nevertheless, because memory traces become labile

with time and for each new reactivation situation (Nader and Hardt, 2009), it is then possible to observe enhanced performances even in normal animals. In the present study, the probe trial conducted 24 h after the last training session consisted of a reactivation session and showed that the memory trace, which animals treated with Peptide 6 had built, was more resistant to time-dependent decrease. Such effect could be expected for an extended training in the same task, which would have reinforced the strength of the memory trace. Overall, these results confirm the potential effect of Peptide 6 in enhancing formation of neuronal plastic networks to process information. Site directed mutagenesis studies have proposed at least 2 receptor binding regions within the CNTF molecule (Inoue et al., 1995; Di Marco et al., 1996; Kallen et al., 1999; Schuster et al., 2003), one of which, the LIFR-binding site, is the D1 cap region. Two key amino acid residues within this region, Phe-153 and Lys-155, are essential for its biological activity and receptor binding and are present at the LIFR-binding sites of CNTF and LIF of a wide variety of species ranging from human to chicken. The present study shows that Peptide 6, which includes both Phe-153 and Lys-155, most probably binds to the LIF receptors on AHPs and HepG2 cells where it inhibits the binding and signal transduction of CNTF and LIF, respectively. Interestingly, Peptide 6 interferes with the signal transduction of LIF more than with that of CNTF. CNTF and LIF have direct effects on neural progenitor cells and both factors have been found to regulate the maintenance and self-renewal of radial glia-like neural progenitors in the neurogenic areas of the brain (Bauer and Patterson, 2006; Muller et al., 2009). But whereas CNTF induces proliferation of the neural stem cells and different stages of neuronal progenitor cells and neurogenesis in the SGZ of the dentate gyrus (Emsley and Hagg, 2003; Kokoeva et al., 2005), LIF inhibits the proliferation, thus depleting this area of cells in the neuronal lineage and increasing the number of astroglia through its effect on the neuronal stem cells (Shimazaki et al., 2001; Bonaguidi et al., 2005; Bauer and Patterson, 2006). Thus, partial inhibition of LIF activity through Peptide 6 could result in enhanced neurogenesis through CNTF as observed in this study. Together, these data present the generation of a novel small molecule based on CNTF which, due to its antagonistic activity to LIFR, can positively influence forebrain neuronal plasticity and neurogenesis and improve spatial memory in normal adult mice. Peptide 6 and drugs based on this peptide are very attractive candidates for prevention and treatment of age-associated cognitive decline and a wide spectrum of learning and memory disorders ranging from mental retardation to neurodegenerative diseases.

Acknowledgements We are grateful to Dr. George Merz for his help with confocal microscopy and Ms. Janet Murphy for secretarial

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assistance. This work was supported in part by the New York State Office of Mental Retardation and Developmental Disabilities; EBEWE Neuro Pharma, Unterach, Austria; and the T.L.L. Temple Foundation Discovery Award for Alzheimer Disease Research, the Alzheimer Association, Chicago, IL. The authors have no conflicting financial interests. This study was supported in part by a research grant from EBEWE Neuro Pharma, Unterach, Austria, the makers of Cerebrolysin® , and a patent application is pending. There is no other financial, personal or other association between any of the authors of this manuscript and the company and never has been; none of the authors has any financial stake in the company. All sources of funding for the research included in this manuscript have been listed in the acknowledgement section. The use of animals was approved by our Institutional Animal Welfare Committee.

References Aimone, J.B., Wiles, J., Gage, F.H., 2006. Potential role for adult neurogenesis in the encoding of time in new memories. Nat. Neurosci. 9, 723–727. Alderson, R.F., Pearsall, D., Lindsay, R.M., Wong, V., 1999. Characterization of receptors for ciliary neurotrophic factor on rat hippocampal astrocytes. Brain Res. 818, 236–251. Arendt, T., 2009. Synaptic degeneration in alzheimer’s disease. Acta Neuropathol. 118, 167–179. Bauer, S., Patterson, P.H., 2006. Leukemia inhibitory factor promotes neural stem cell self-renewal in the adult brain. J. Neurosci. 26, 12089–12099. Baumann, H., Ziegler, S.F., Mosley, B., Morella, K.K., Pajovic, S., Gearing, D.P., 1993. Reconstitution of the response to leukemia inhibitory factor, oncostatin m, and ciliary neurotrophic factor in hepatoma cells. J. Biol. Chem. 268, 8414–8417. Bonaguidi, M.A., McGuire, T., Hu, M., Kan, L., Samanta, J., Kessler, J.A., 2005. Lif and bmp signaling generate separate and discrete types of gfap-expressing cells. Development 132, 5503–5514. Cameron, H.A., Hazel, T.G., McKay, R.D., 1998. Regulation of neurogenesis by growth factors and neurotransmitters. J. Neurobiol. 36, 287–306. Chen, H., Tung, Y.C., Li, B., Iqbal, K., Grundke-Iqbal, I., 2007. Trophic factors counteract elevated fgf-2-induced inhibition of adult neurogenesis. Neurobiol. Aging 28, 1148–1162. Chen, Z.Y., Cao, L., Wang, L.M., Guo, C., Ye, J.L., Chai, Y.F., Yan, Z.Y., 2001. Development of neurotrophic molecules for treatment of neurodegeneration. Curr. Protein Pept. Sci. 2, 261–276. Counts, S.E., Nadeem, M., Lad, S.P., Wuu, J., Mufson, E.J., 2006. Differential expression of synaptic proteins in the frontal and temporal cortex of elderly subjects with mild cognitive impairment. J. Neuropathol. Exp. Neurol. 65, 592–601. Davis, S., Aldrich, T.H., Stahl, N., Pan, L., Taga, T., Kishimoto, T., Ip, N.Y., Yancopoulos, G.D., 1993. Lifr beta and gp130 as heterodimerizing signal transducers of the tripartite cntf receptor. Science 260, 1805–1808. Di Marco, A., Gloaguen, I., Graziani, R., Paonessa, G., Saggio, I., Hudson, K.R., Laufer, R., 1996. Identification of ciliary neurotrophic factor (cntf) residues essential for leukemia inhibitory factor receptor binding and generation of cntf receptor antagonists. Proc. Natl. Acad. Sci. U.S.A. 93, 9247–9252. Dickstein, D.L., Kabaso, D., Rocher, A.B., Luebke, J.I., Wearne, S.L., Hof, P.R., 2007. Changes in the structural complexity of the aged brain. Aging Cell 6, 275–284. Donovan, M.H., Yazdani, U., Norris, R.D., Games, D., German, D.C., Eisch, A.J., 2006. Decreased adult hippocampal neurogenesis in the

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pdapp mouse model of alzheimer’s disease. J. Comp. Neurol. 495, 70– 83. Emsley, J.G., Hagg, T., 2003. Endogenous and exogenous ciliary neurotrophic factor enhances forebrain neurogenesis in adult mice. Exp. Neurol. 183, 298–310. Eriksson, P.S., Perfilieva, E., Bjork-Eriksson, T., Alborn, A.M., Nordborg, C., Peterson, D.A., Gage, F.H., 1998. Neurogenesis in the adult human hippocampus. Nat. Med. 4, 1313–1317. Frielingsdorf, H., Simpson, D.R., Thal, L.J., Pizzo, D.P., 2007. Nerve growth factor promotes survival of new neurons in the adult hippocampus. Neurobiol. Dis. 26, 47–55. Galande, A.K., Weissleder, R., Tung, C.H., 2005. An effective method of onresin disulfide bond formation in peptides. J. Comb. Chem. 7, 174–177. Grundke-Iqbal, I., Fleming, J., Tung, Y.C., Lassmann, H., Iqbal, K., Joshi, J.G., 1990. Ferritin is a component of the neuritic (senile) plaque in alzheimer dementia. Acta Neuropathol. (Berl.) 81, 105–110. Guzowski, J.F., Timlin, J.A., Roysam, B., McNaughton, B.L., Worley, P.F., Barnes, C.A., 2005. Mapping behaviorally relevant neural circuits with immediate-early gene expression. Curr. Opin. Neurobiol. 15, 599–606. Henry, R.A., Hughes, S.M., Connor, B., 2007. Aav-mediated delivery of bdnf augments neurogenesis in the normal and quinolinic acid-lesioned adult rat brain. Eur. J. Neurosci. 25, 3513–3525. Inoue, M., Nakayama, C., Kikuchi, K., Kimura, T., Ishige, Y., Ito, A., Kanaoka, M., Noguchi, H., 1995. D1 cap region involved in the receptor recognition and neural cell survival activity of human ciliary neurotrophic factor. Proc. Natl. Acad. Sci. U.S.A. 92, 8579–8583. Jin, K., Galvan, V., Xie, L., Mao, X.O., Gorostiza, O.F., Bredesen, D.E., Greenberg, D.A., 2004a. Enhanced neurogenesis in alzheimer’s disease transgenic (pdgf-appsw,ind) mice. Proc. Natl. Acad. Sci. U.S.A. 101, 13363–13367. Jin, K., Peel, A.L., Mao, X.O., Xie, L., Cottrell, B.A., Henshall, D.C., Greenberg, D.A., 2004b. Increased hippocampal neurogenesis in alzheimer’s disease. Proc. Natl. Acad. Sci. U.S.A. 101, 343–347. Kallen, K.J., Grotzinger, J., Lelievre, E., Vollmer, P., Aasland, D., Renne, C., Mullberg, J., Myer zum Buschenfelde, K.H., Gascan, H., Rose-John, S., 1999. Receptor recognition sites of cytokines are organized as exchangeable modules. Transfer of the leukemia inhibitory factor receptor-binding site from ciliary neurotrophic factor to interleukin-6. J. Biol. Chem. 274, 11859–11867. Kaneko, Y., Kitamoto, T., Tateishi, J., Yamaguchi, K., 1989. Ferritin immunohistochemistry as a marker for microglia. Acta Neuropathol. 79, 129–136. Kee, N., Teixeira, C.M., Wang, A.H., Frankland, P.W., 2007. Preferential incorporation of adult-generated granule cells into spatial memory networks in the dentate gyrus. Nat. Neurosci. 10, 355–362. Kempermann, G., Gast, D., Kronenberg, G., Yamaguchi, M., Gage, F.H., 2003. Early determination and long-term persistence of adult-generated new neurons in the hippocampus of mice. Development 130, 391–399. Kempermann, G., Kuhn, H.G., Gage, F.H., 1998. Experience-induced neurogenesis in the senescent dentate gyrus. J. Neurosci. 18, 3206–3212. Kokoeva, M.V., Yin, H., Flier, J.S., 2005. Neurogenesis in the hypothalamus of adult mice: potential role in energy balance. Science 310, 679–683. Kordower, J.H., Yaping, C., Maclennan, A.J., 1997. Ciliary neurotrophic factor receptor alpha-immunoreactivity in the monkey central nervous system. J. Comp. Neurol. 377, 365–380. Kuhn, H.G., Dickinson-Anson, H., Gage, F.H., 1996. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J. Neurosci. 16, 2027–2033. Kuhn, H.G., Winkler, J., Kempermann, G., Thal, L.J., Gage, F.H., 1997. Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain. J. Neurosci. 17, 5820–5829. Lee, M.Y., Deller, T., Kirsch, M., Frotscher, M., Hofmann, H.D., 1997. Differential regulation of ciliary neurotrophic factor (cntf) and cntf receptor alpha expression in astrocytes and neurons of the fascia dentata after entorhinal cortex lesion. J. Neurosci. 17, 1137–1146. Lee, Y.S., Silva, A.J., 2009. The molecular and cellular biology of enhanced cognition. Nat. Rev. Neurosci. 10, 126–140.

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M.O. Chohan et al. / Neurobiology of Aging 32 (2011) 1420–1434

Li, B., Yamamori, H., Tatebayashi, Y., Shafit-Zagardo, B., Tanimukai, H., Chen, S., Iqbal, K., Grundke-Iqbal, I., 2008. Failure of neuronal maturation in alzheimer disease dentate gyrus. J. Neuropathol. Exp. Neurol. 67, 78–84. MacLennan, A.J., Vinson, E.N., Marks, L., McLaurin, D.L., Pfeifer, M., Lee, N., 1996. Immunohistochemical localization of ciliary neurotrophic factor receptor alpha expression in the rat nervous system. J. Neurosci. 16, 621–630. Muller, S., Chakrapani, B.P., Schwegler, H., Hofmann, H.D., Kirsch, M., 2009. Neurogenesis in the dentate gyrus depends on cntf and stat3 signaling. Stem Cells 27, 431–441. Nader, K., Hardt, O., 2009. A single standard for memory: the case for reconsolidation. Nat. Rev. Neurosci. 10, 224–234. Nagao, M., Sugimori, M., Nakafuku, M., 2007. Cross talk between notch and growth factor/cytokine signaling pathways in neural stem cells. Mol. Cell Biol. 27, 3982–3994. Ott, V., Fasshauer, M., Dalski, A., Klein, H.H., Klein, J., 2002. Direct effects of ciliary neurotrophic factor on brown adipocytes: evidence for a role in peripheral regulation of energy homeostasis. J. Endocrinol. 173, R1–8. Price, R.D., Milne, S.A., Sharkey, J., Matsuoka, N., 2007. Advances in small molecules promoting neurotrophic function. Pharmacol. Ther. 115, 292–306. Scharfman, H., Goodman, J., Macleod, A., Phani, S., Antonelli, C., Croll, S., 2005. Increased neurogenesis and the ectopic granule cells after intrahippocampal bdnf infusion in adult rats. Exp. Neurol. 192, 348–356. Schuster, B., Kovaleva, M., Sun, Y., Regenhard, P., Matthews, V., Grotzinger, J., Rose-John, S., Kallen, K.J., 2003. Signaling of human ciliary neurotrophic factor (cntf) revisited. The interleukin-6 receptor can serve as an alpha-receptor for ctnf. J. Biol. Chem. 278, 9528–9535. Senaldi, G., Varnum, B.C., Sarmiento, U., Starnes, C., Lile, J., Scully, S., Guo, J., Elliott, G., McNinch, J., Shaklee, C.L., Freeman, D., Manu, F., Simonet, W.S., Boone, T., Chang, M.S., 1999. Novel neurotrophin-1/b cell-stimulating factor-3: a cytokine of the il-6 family. Proc. Natl. Acad. Sci. U.S.A. 96, 11458–11463. Sendtner, M., Carroll, P., Holtmann, B., Hughes, R.A., Thoenen, H., 1994. Ciliary neurotrophic factor. J. Neurobiol. 25, 1436–1453. Shi, Y., Wang, W., Yourey, P.A., Gohari, S., Zukauskas, D., Zhang, J., Ruben, S., Alderson, R.F., 1999. Computational est database analysis identifies a novel member of the neuropoietic cytokine family. Biochem. Biophys. Res. Commun. 262, 132–138.

Shimazaki, T., Shingo, T., Weiss, S., 2001. The ciliary neurotrophic factor/leukemia inhibitory factor/gp130 receptor complex operates in the maintenance of mammalian forebrain neural stem cells. J. Neurosci. 21, 7642–7653. Steiner, B., Wolf, S., Kempermann, G., 2006. Adult neurogenesis and neurodegenerative disease. Regen. Med. 1, 15–28. Stranahan, A.M., Norman, E.D., Lee, K., Cutler, R.G., Telljohann, R.S., Egan, J.M., Mattson, M.P., 2008. Diet-induced insulin resistance impairs hippocampal synaptic plasticity and cognition in middle-aged rats. Hippocampus 18, 1085–1088. Tatebayashi, Y., Iqbal, K., Grundke-Iqbal, I., 1999. Dynamic regulation of expression and phosphorylation of tau by fibroblast growth factor-2 in neural progenitor cells from adult rat hippocampus. J. Neurosci. 19, 5245–5254. Tatebayashi, Y., Lee, M.H., Li, L., Iqbal, K., Grundke-Iqbal, I., 2003. The dentate gyrus neurogenesis: a therapeutic target for alzheimer’s disease. Acta Neuropathol. (Berl.) 105, 225–232. Trouche, S., Bontempi, B., Roullet, P., Rampon, C., 2009. Recruitment of adult-generated neurons into functional hippocampal networks contributes to updating and strengthening of spatial memory. Proc. Natl. Acad. Sci. U.S.A. 106, 5919–5924. West, M.J., Slomianka, L., Gundersen, H.J., 1991. Unbiased stereological estimation of the total number of neurons in thesubdivisions of the rat hippocampus using the optical fractionator. Anat. Rec. 231, 482–497. Winner, B., Cooper-Kuhn, C.M., Aigner, R., Winkler, J., Kuhn, H.G., 2002. Long-term survival and cell death of newly generated neurons in the adult rat olfactory bulb. Eur. J. Neurosci. 16, 1681–1689. Wiskott, L., Rasch, M.J., Kempermann, G., 2006. A functional hypothesis for adult hippocampal neurogenesis: avoidance of catastrophic interference in the dentate gyrus. Hippocampus 16, 329–343. Yang, P., Arnold, S.A., Habas, A., Hetman, M., Hagg, T., 2008. Ciliary neurotrophic factor mediates dopamine d2 receptor-induced cns neurogenesis in adult mice. J. Neurosci. 28, 2231–2241. Zhao, C., Teng, E.M., Summers Jr., R.G., Ming, G.L., Gage, F.H., 2006. Distinct morphological stages of dentate granule neuron maturation in the adult mouse hippocampus. J. Neurosci. 26, 3–11. Ziv, Y., Ron, N., Butovsky, O., Landa, G., Sudai, E., Greenberg, N., Cohen, H., Kipnis, J., Schwartz, M., 2006. Immune cells contribute to the maintenance of neurogenesis and spatial learning abilities in adulthood. Nat. Neurosci. 9, 268–275.