Lisa Conboy

Andrew G Foley

Noel M O'boyle

Marie Lawlor

Helen C Gallagher

Keith J Murphy

Ciaran M Regan
email: ciaran.regan@ucd.ie

Prof Regan Ciaran

CURCUMIN-INDUCED DEGRADATION OF PKC IS ASSOCIATED WITH ENHANCED DENTATE NCAM PSA EXPRESSION AND SPATIAL LEARNING IN ADULT AND AGED WISTAR RATS

Keywords: PKC degradation enhances NCAM PSA and cognition PKC, PST, NCAM PSA, curcumin, spatial learning

Polysialylation of the neural cell adhesion molecule (NCAM PSA) is necessary for the consolidation processes of hippocampus-based learning. Previously, we have found inhibition of protein kinase C delta (PKC) to be associated with increased polysialyltransferase activity (PST), suggesting inhibitors of this kinase might ameliorate cognitive deficits. Using a rottlerin template, a drug previously considered an inhibitor of PKC, we searched the CAP database with the Accelrys ® Catalyst programme for structurally similar molecules and, using the available crystal structure of the phorbol-binding domain of PKC, found that diferuloylmethane (curcumin) docked effectively into the phorbol site. Curcumin increased NCAM PSA expression in cultured neuro-2A neuroblastoma cells and this was inversely related to PKC protein expression. Curcumin did not directly inhibit PKC activity but formed a tight complex with the enzyme. With increasing doses of curcumin, the Tyr 131 residue of PKC, which is known to direct its degradation, became progressively phosphorylated and this was associated with numerous Tyr 131 -phospho-PKC fragments. Chronic administration of curcumin in vivo also increased the frequency of polysialylated cells in the dentate infragranular zone and significantly improved the acquisition and consolidation of a water maze spatial learning paradigm in both adult and aged cohorts of Wistar rats. These results further confirm the role of PKC in regulating PST and NCAM PSA expression and provide evidence that drug modulation of this system enhances the process of memory consolidation.

involves the attachment of large homopolymers of 2,8-linked polysialic acid (PSA), a modification that is specific to NCAM in the mammalian brain [START_REF] Sadoul | Adult and embryonic mouse neural cell adhesion molecules have different binding properties[END_REF] and [START_REF] Rougon | A monoclonal antibody against meningococcus group B polysaccharides distinguishes embryonic from adult N-CAM[END_REF]. This posttranslational modification of NCAM has been extensively argued to support structural plasticity in the adult nervous system [START_REF] Bonfanti | PSA-NCAM in mammalian structural plasticity and neurogenesis[END_REF], [START_REF] Gascon | Polysialic acid-neural cell adhesion molecule in brain plasticity: From synapses to integration of new neurons[END_REF] and [START_REF] Rutishauser | Polysialic acid in the plasticity of the developing and adult vertebrate nervous system[END_REF] and has been implicated in activity-dependent synaptic remodelling [START_REF] Hoyk | The highly sialylated isoform of the neural cell adhesion molecule is required for estradiol-induced morphological plasticity in the adult arcuate nucleus[END_REF] and [START_REF] Stoenica | In vivo synaptic plasticity in the dentate gyrus of mice deficient in the neural cell adhesion molecule NCAM or its polysialic acid[END_REF]. Moreover, the synaptic remodeling that accompanies task consolidation of spatial learning and avoidance conditioning paradigms [START_REF] O'malley | Ultrastructural analysis reveals avoidance conditioning to induce a transient increase in hippocampal dentate spine density in the 6h posttraining period of consolidation[END_REF], [START_REF] O'malley | Transient spine density increases in the mid-molecular layer of the hippocampal dentate gyrus accompany consolidation of a spatial learning task in the rodent[END_REF] and [START_REF] Eyre | Morphological changes in the hippocampal dentate gyrus synapses following spatial learning in rats are transient[END_REF] is associated with a transient increase in the frequency of dentate granule cells expressing polysialylated NCAM at the 12 h post-training time [START_REF] Fox | Memory consolidation induces a transient and time-dependent increase in the frequency of NCAM-polysialylated cells in the adult rat hippocampus[END_REF], [START_REF] Murphy | Repetitive and transient increases in hippocampal neural cell adhesion molecule polysialylation state following multi-trial spatial training[END_REF] and [START_REF] Sandi | Modulation of hippocampal NCAM polysialylation by stressful experiences[END_REF] and cleavage of PSA with endoneuraminidase-N impairs the development of long-term potentiation (LTP), a cellular analogue of memory and learning, and the consolidation of spatial learning and avoidance conditioning paradigms [START_REF] Becker | The polysialic acid modification of the neural cell adhesion molecule is involved in spatial learning and hippocampal long-term potentiation[END_REF], [START_REF] Muller | PSA-NCAM is required for activity-induced synaptic plasticity[END_REF], [START_REF] Venero | Hippocampal up-regulation of NCAM expression and polysialylation plays a key role on spatial memory[END_REF] and [START_REF] Lópéz-Fernández | Upregulation of polysialylated neural cell adhesion molecule in dorsal hippocampus after contextual fear conditioning is involved in long-term memory formation[END_REF]. Intraventricular infusion of anti-PSA, when administered specifically at the 10 h post-training time, has also been demonstrated to impair the consolidation of spatial and avoidance conditioning tasks, further confirming the time-dependent functional requirement of NCAM PSA in memory formation [START_REF] Seymour | Intraventricular infusions of anti-NCAM PSA impair the process of consolidation of both avoidance conditioning and spatial learning paradigms in Wistar rats[END_REF]. Polysialylation of NCAM occurs in the trans-Golgi compartment and is catalyzed by two enzymes termed sialyltransferase-X (STX or ST8SiaII) [START_REF] Scheidegger | A human STX cDNA confers polysialic acid expression in mammalian cells[END_REF] and [START_REF] Yoshida | Molecular cloning of Sia alpha 2,3Gal beta 1,4GlcNAc alpha 2,8-sialyltransferase from mouse brain[END_REF] and polysialyltransferase (PST or ST8SiaIV) [START_REF] Eckhardt | Molecular characterization of eukaryotic polysialyltransferase-1[END_REF] and [START_REF] Nakayama | Expression cloning of a human polysialyltransferase that forms the polysialylated neural cell adhesion molecule present in embryonic brain[END_REF]. These two polysialyltransferases regulate NCAM polysialylation state differentially during development as STX is the dominant 2,8-polysialyltransferase in the embryonic and early postnatal period whereas PST is mainly associated with 2,8-polysialylation in the postnatal A c c e p t e d M a n u s c r i p t 5 brain [START_REF] Hildebrandt | Heterogenous expression of the polysialyltransferases ST8Sia II and ST8Sia IV during postnatal rat brain development[END_REF] and [START_REF] Ong | Developmental regulation of polysialic acid synthesis in mouse directed by two polysialyltransferases, PST and STX[END_REF]. Pulse-labelling studies in vitro have indicated PSA synthesis to occur in the late-Golgi, or post-Golgi, compartment where NCAM becomes rapidly glycosylated with the newly synthesized 2,8-polysialic acid [START_REF] Alcaraz | Biosynthesis and processing of polysialylated NCAM by AtT20 cells[END_REF].

In general, the molecular events associated with regulation of sialyltransferase-mediated glycosylation events still remain to be fully elucidated. The proposed involvement of PKC in the regulation of PST-mediated NCAM polysialylation state stems from the observation that phorbol-12-myristate-13-acetate, which activates diacylglycerol-dependent PKC isoforms, induces a dose-dependent decrease in NCAM PSA expression in neuro-2A neuroblastoma cells and staurosporine, a pan-specific PKC inhibitor, increases NCAM polysialylation state [START_REF] Gallagher | Regulation of neural cell adhesion molecule polysialylation state by cell-cell contact and protein kinase C delta[END_REF] Moreover, immunoblotting procedures have demonstrated reduced PKC expression to be associated with the enhanced polysialylation of NCAM in vitro and, using a polyclonal antibody directed against a conserved 11-amino acid sequence in E. coli polysialyltransferase, hippocampal PKC has been found to form complexes with PST that increase as animals age [START_REF] Gallagher | Protein kinase C delta regulates neural cell adhesion molecule polysialylation state in the rat brain[END_REF] and NCAM PSA expression decreases in an exponential manner [START_REF] Fox | Polysialylated neural cell adhesion molecule expression by neurons and astroglila processes in the rat dentate gyrus declines dramatically with increasing age[END_REF]. The PST associated with these complexes has also been observed to be phosphorylated on serine residues [START_REF] Gallagher | Protein kinase C delta regulates neural cell adhesion molecule polysialylation state in the rat brain[END_REF], a mechanism known to be associated with the inhibition of other sialyltransferases [START_REF] Gu | Regulation of sialyltransferase activities by phosphorylation and dephosphorylation[END_REF], [START_REF] Gao | Association of a 14-3-3 protein with CMP-NeuNAc: GM1 2,3-sialyltransferase[END_REF], [START_REF] Ma | Sialyltransferase isoforms are phosphorylated in the cis-medial Golgi on serine and threonine residues in their luminal sequences[END_REF] and [START_REF] Breen | The role of protein phosphorylation in 2,6(N)sialyltransferase activity[END_REF]. Phosphorylated tyrosine residues on PST are also found in PST:PKC immunocomplexes [START_REF] Gallagher | Protein kinase C delta regulates neural cell adhesion molecule polysialylation state in the rat brain[END_REF] and this may play a crucial role in regulating PKC expression as phosphorylation of conserved tyrosine residues in the hinged bilobal structure, typical of the PKC family of isozymes, confers a specific post-translational control on the rate of proteolytic cleavage [START_REF] Kishimoto | Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain)[END_REF] and [START_REF] Blake | Courtneidge Src promotes PKC degradation[END_REF].

A c c e p t e d M a n u s c r i p t 6 In previous studies directed to providing evidence for a role of PKC in the regulation of NCAM polysialylation in neuro-2A neuroblastoma cells [START_REF] Gallagher | Regulation of neural cell adhesion molecule polysialylation state by cell-cell contact and protein kinase C delta[END_REF], we employed rottlerin to inhibit PKC, as this had been reported to have an IC 50 value of 3-6uM and to be 5-17 times more potent as a PKC inhibitor over other PKC isozymes [START_REF] Gschwendt | Rottlerin, a novel protein kinase inhibitor[END_REF]. Neuro-2A cells exposed to rottlerin (5M) in the mid-log growth phase, when NCAM PSA expression is dominant, resulted in a rapid and substantial increase in NCAM polysialylation state [START_REF] Gallagher | Regulation of neural cell adhesion molecule polysialylation state by cell-cell contact and protein kinase C delta[END_REF]. Moreover, exposure of the same concentration of rottlerin to cells in contact inhibition, where no NCAM PSA is expressed, resulted in PST activation and expression of polysialylated NCAM. Subsequent studies, however, have failed to confirm rottlerin to be a direct inhibitor of PKC [START_REF] Davies | Specificity and mechanism of action of some commonly used protein kinase inhibitors[END_REF] but that it may modulate the tyrosine phosphorylation state of PKC and indirectly affect enzyme activity [START_REF] Soltoff | Rottlerin: an inappropriate and ineffective inhibitor of PKC[END_REF].

We now demonstrate curcumin to be a chemical similar of rottlerin that indirectly modulates PKC activity by modifying its phosphorylation state to increase the rate of degradation.

A c c e p t e d

M a n u s c r i p t 7

MATERIALS AND METHODS

Materials

All routine laboratory chemicals, secondary antibodies, rottlerin, staurosporine and curcumin were purchased from Sigma-Aldrich, Ireland. All components of the PKC activity assay, including constitutively active recombinant PKC, were purchased from Upstate Biotechnology (Charlottesville, VA). [-32 P]-ATP was purchased from Amersham Biosciences (Buckinghamshire, UK). The BCA assay was purchased from Pierce (Rockford, IL). The monoclonal antibody raised against the meningococcus group B polysaccharides (anti-PSA) was a kind gift of Prof. G. Rougon [START_REF] Rougon | A monoclonal antibody against meningococcus group B polysaccharides distinguishes embryonic from adult N-CAM[END_REF]. Monoclonal primary antibody to PKC was purchased from BD Biosciences (Oxford, UK).

In silico studies

Database searching with Catalyst and protein-ligand docking with Cerius2

Computational studies were carried out using a Silicon Graphics Octane workstation (SGI, Mountain View, CA). Catalyst version 3.4 (Accelrys, Cambridge, UK) was used to search the Compounds Available for Purchase (CAP) database to identify those structurally similar to rottlerin using a template structure based on its side chain (see Figure 1A). Prior to database screening, the search was restricted to drug-like molecules based on the Lipinski rules [START_REF] Lipinski | Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings[END_REF].

Using Cerius2 (version 4.8, Accelrys, UK), the matching compounds were rated using proteinligand docking to the crystal structure of the PKC ligand binding domain which was downloaded from the Brookhaven Protein Data Bank. In preparation for docking, all organic and A c c e p t e d M a n u s c r i p t 8 inorganic cofactors, the phorbol ester ligand, as well as all water molecules, were removed from the PKC crystal structure. Potential PKC inhibitors were sequentially docked to the PKC crystal structure and ligand-enzyme interactions were quantified using the DOCK scoring function of the LigFit module of Cerius2. The top ranking compounds were screened in in vitro assays, described below, to determine their potential to modulate NCAM-PSA.

In vitro analysis of NCAM PSA expression

The mouse neuroblastoma cell line, neuro-2A, was maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, UK) supplemented with 10% (v/v) foetal calf serum (Gibco, UK) and penicillin/streptomycin (100 mg/ml; Sigma, UK) at 37 °C in a humidified atmosphere and 9% CO 2 . To investigate pharmacological modulation of PSA expression, cells were seeded on 96well plates at a density of 10 4 cells/cm 2 and grown until day 3 in vitro (DIV 3). On DIV3, cells were treated with either vehicle (DMSO not exceeding 1% v/v) or test PKC inhibitor ranging in dose from 1M to 500 M for 5 hours. Specifically, curcumin was administered at concentrations of 1, 10, 50, 100 and 500 M. Cells were then fixed with 200 l of 4% paraformaldehyde in DMEM for 60 min at room temperature. This was replaced with undiluted fixative for a further 60 min. Non-specific binding sites were blocked with 2% (w/v) bovine serum albumin (Sigma-Aldrich, Ireland) and 2% (v/v) normal goat serum (Sigma-Aldrich, Ireland) in PBS (blocking solution; in which all antibodies were also diluted). PSA was detected using a monoclonal antibody raised against the meningococcus group B polysaccharides (anti-PSA) prepared as an ascitic fluid and diluted 1:500. Primary antibody was incubated overnight at 4 ºC. Binding of the primary antibody was detected using a secondary goat anti-mouse IgG conjugated to peroxidase diluted 1: 2000 for 1 hour at room temperature. Peroxidase activity was A c c e p t e d M a n u s c r i p t 9 determined using the liquid substrate tetramethylbenzidine (Sigma-Aldrich, Ireland), which was incubated for 30 min at room temperature and the reaction was terminated by acidification with 2 M H 2 SO 4 . Optical density was measured in a microplate reader with a 450 nm measurement filter and 620 nm reference filter.

PKC activity assay

Constitutively active recombinant PKC was derived from Sf21 cells and assay of enzyme activity was performed according to the manufacture's instructions. In brief, 0-20 ng of recombinant PKC was incubated with 50 M PKC substrate peptide (myelin basic protein), in the presence of 200 M ATP, 30 mM magnesium chloride, 0.05 mg/ml phosphatidylserine, 0.005 mg/ml diacylglycerol and [-32 P]-ATP (Amersham Biosciences, UK) for 10 min at 30 ºC.

Following incubation 20 l of each assay was spotted onto phosphocellulose paper and the reaction terminated by washing of the phosphocellulose in 0.75% phosphoric acid for 5 minutes.

Following two subsequent 5 minute washes in phosphoric acid, the samples were washed in acetone for a further 5 min. The phosphocellulose paper was dried, transferred to scintillation fluid and the radioactivity counted. Under these conditions, increasing concentrations of PKC were associated with increasing levels of radioactivity. Using 20 ng of recombinant PKC and the conditions described above, staurosporine (5 nM), rottlerin (10 M and 100 M) and curcumin (10 M and 500 M) were included in the assay and their ability to inhibit PKC kinase activity in a dose-dependent manner was determined. In additional experiments, the assay concentration of ATP was lowered to 40 M ATP to determine potential competition at the ATP binding site.

A c c e p t e d M a n u s c r i p t 10

Immobilisation of curcumin on Epoxy-activated Sepharose 6B

To investigate curcumin-PKC interactions, curcumin was coupled to epoxy-activated Sepharose™ 6B (Sigma-Aldrich, Ireland). Using a method adapted from [START_REF] Godl | An efficient proteomics method to identify the cellular targets of protein kinase inhibitors[END_REF] a 1 gm aliquot of epoxy-activated Sepharose 6B beads was washed in 50 ml of dH 2 O for 5 cycles to give approximately 3.5 ml of swollen beads. For coupling to the epoxy-activated beads, curcumin was prepared as a 20 mM solution in 50% dimethylformamide/0.1 M Na 2 CO 3 /10 mM NaOH and two volumes of this solution was mixed with one volume of swollen beads and incubated overnight at 30 ˚C in the dark. The following day, the curcumin-Sepharose suspension was washed 3 times with coupling buffer and the remaining non-specific binding sites were blocked by incubation with 1M ethanolamine overnight at 30 ˚C. Control beads were prepared by incubating the epoxy-activated Sepharose 6B with 1 M ethanolamine, pH 11. The control and curcumin-coupled beads were finally washed in 3 cycles of alternating low (0.1M acetate buffer, pH 4.0) and high (0.1M Tris-HCl buffer, pH 8, containing 0.5 M NaCl) pH buffers.

Hippocampal PKC was prepared by homogenising rat dentate gyrus in 100 mM Hepes buffer, pH 7.6, containing 300 mM NaCl, 1% Triton-X-100, 2 mM EDTA, 2 mM EGTA and 0.1 mM PMSF, 50mM Na 3 VO 4 and 1g/l aprotinin as protease inhibitors (2X lysis buffer). A 250 l aliquot of cell lysate was incubated with 20 l of curcumin-coupled Sepharose, or control beads, with constant mixing for 3 h at 4 ˚C. Afterwards, the curcumin-Sepharose complex was washed twice with 500 µl of 2X lysis buffer (without protease inhibitor) containing 150 mM NaCl (low salt) followed by 500 µl of 1X lysis buffer containing 1 M NaCl (high salt). Proteins were A c c e p t e d M a n u s c r i p t 11 isolated from the bead-protein complex by resuspending and boiling the washed beads in SDS-PAGE sample buffer. PKC was detected by the immunoblotting procedure described below.

Immunoblot analysis of PKC expression

Neuro-2A cells were seeded in 75 cm 2 plates (10 4 cells/cm 2 ) and exposed to increasing concentrations of curcumin for 5 h during the mid-log growth phase, as described above. The cells were then washed 3 times in phosphate-buffered saline, scraped and pelleted by centrifugation for 10 min at 900 rpm. The pellet was solubilised in RIPA buffer (50 mM Tris, pH 8.0, containing 50 mM NaCl, 1.0 % NP-40, 0.5% sodium deoxycholate, and 0.1% SDS,) containing protease inhibitors for 30 min on ice. The supernatant was then collected by centrifugation at 3000 g and the protein concentrations determined by the BCA assay. Samples, of equal protein concentration, were boiled for 10 min in a reducing sample buffer of 70 mM Tris-HCl, pH 6.8, containing 33 mM NaCl, 1 mM EDTA, 2% (w/v) SDS, 0.01 % (w/v) bromophenol blue, 10% glycerol and 3% (v/v) dithiothreitol reducing agent (New England Biolabs, UK). Samples containing equal amounts of protein were separated on 10 % polyacrylamide minigels and electrophoretically transferred to nitrocellulose membranes and successful transfer was confirmed by ponceau S staining of the membrane (not shown). The nitrocellulose was then blocked using washing buffer (10 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, and 0.05% [v/v] Tween-20) with 5% (w/v) non-fat milk powder for 1 h at room temperature. The monoclonal primary antibody to PKC was diluted 1:1000 in blocking buffer and incubated overnight at 4 C with the nitrocellulose membrane. The primary antibody was then detected using goat anti-mouse IgG peroxidase conjugated secondary antibody. Following membrane washing, the immuno-complexes were detected using a chemiluminescence Equal protein loading was verified by stripping the blots and re-probing them with an anti-actin monoclonal antibody (Sigma-Aldrich, Ireland) diluted 1: 20000. The developed x-ray films were scanned using Adobe Photoshop 7.0.

Quantitative immunohistochemical analysis of NCAM PSA in vivo

Male Wistar rats (postnatal day 80) were obtained from the Biomedical Facility, University College Dublin, and housed singly in a 12 h light/dark cycle at 22 ± 2°C, with ad libitum access to food and water. Curcumin (75 mg/kg), dissolved in a methylcellulose (0.5% w/v) vehicle, was administered daily by gavage for an 8-day period. Control animals received the methylcellulose vehicle for the same 8-day period. At 24 h after the final drug administration, the animals were sacrificed by cervical dislocation and the whole brain was quickly dissected. The brains were coated immediately in an optimal cutting temperature compound (Gurr, Poole, UK), snap-frozen in CO 2 -cooled n-hexane, and stored at -80°C until required for further processing. The frequency of hippocampal polysialylated neurons was determined using PSA immunohistochemistry, as described previously [START_REF] Fox | Memory consolidation induces a transient and time-dependent increase in the frequency of NCAM-polysialylated cells in the adult rat hippocampus[END_REF]. Briefly, cryostat axial sections of 12µm sections were fixed in 70% (v/v) ethanol and incubated overnight with anti-PSA ascitic fluid diluted 1:500 in phosphate buffered saline (PBS). The sections were exposed for 3 h to fluorescein-conjugated goat antimouse IgM diluted 1:100 in PBS and mounted in Citifluor® (Citifluor Ltd., UK), a fluorescenceenhancing medium. The total numbers of PSA-immunopositive neurons at the infragranular cell layer of the dentate gyrus, at -5.6 mm with respect to bregma, were counted in seven alternate 12µm sections. The sections were counter-stained with propidium iodide (60 s; 40 ng/ml) (Sigma-Aldrich, Ireland) to facilitate cell identification and the use of alternate sections precluded A c c e p t e d M a n u s c r i p t 13 double counting of the 5-10 µm perikarya. Cell counts were expressed per unit area of the dentate granule cell layer (0.15mm 2 ) and the mean ± SEM for each treatment group calculated.

Water maze spatial learning paradigm

Separate cohorts of male Wistar rats (postnatal day 80 and postnatal day 540), sourced and maintained as described above, were used in these studies. On each of the 2 days preceding commencement of water maze studies, animals were handled and assessed in an open field arena for locomotor activity, rearing and general behaviour over a 5 min period. Each cohort of animals was treated for 8 days with 75 mg/kg curcumin by gavage. An extended 40-day treatment period did not confer any additional advantage on the parameters under investigation (data not shown). The control groups received vehicle alone. Water maze training was initiated at 24 h following the final drug administration. The water maze apparatus consisted of a large circular pool (1 m diameter, 80 cm high, water temperature 26 ± 1 o C) with a platform (11 cm diameter) submerged 1.5 cm below the water surface [START_REF] Murphy | Repetitive and transient increases in hippocampal neural cell adhesion molecule polysialylation state following multi-trial spatial training[END_REF]. The water was 50 cm deep and the wall of the pool was 30 cm above the level of the water. Both the pool and the platform were constructed of black polyvinyl plastic and offered no intra-maze cues to guide escape behaviour.

The experimental room contained several extra-maze visual cues. During testing the platform was hidden in the same quadrant 30 cm from the side wall. Animals were trained in 4 training sessions separated by 24 h and each session, consisting of 5 trials, was separated by an inter-trial test interval of 300 s. Each trial started with the rat facing the wall of the maze at one of three fixed points A, B, or C arranged triangularly around the pool (separated by 120). The starting positions were used sequentially over the five trials of each training session and continuity in the A c c e p t e d M a n u s c r i p t 14 pattern of starting positions was maintained over different sessions so that all starting points were used the same number of times. Computerised tracking software (Watermaze 3.1) was used to measure relevant parameters that included swim angle, swim speed, escape latency and path length. The time taken by the rat to find the hidden platform within a 60 s criterion period was defined as the escape latency time. On the first trial, rats failing to locate the platform within the 60 s period were placed on it for 10 s. Escape latencies were measured over 5 trials in each training session. Probe trials were performed at 1 and 3 days following the final training session.

In these trials the platform was removed and the time spent in each quadrant of the pool was tracked over a 60 s period.

All experimental procedures were approved by the Animal Research Ethics Committee of University College Dublin and were carried out by individuals holding the appropriate national license issued by the Department of Health.

Statistics

All statistical analysis was carried out using GraphPad Prism (version 4) software. Curcumin effects on NCAM-PSA and PKC expression were analysed by using Student's unpaired twotailed t-test. Kinase activity assay values were analysed by ANOVA followed by Dunnett's posthoc analysis. The effect of drug treatment and trial number on escape latency from the water maze was assessed by repeated measures ANOVA. Specific trials and probe differences were analysed using Student's unpaired two-tailed t-test. In all cases, P values less than 0.05 were considered to be significant.

Identification of curcumin as a chemical similar of rottlerin

Taking rottlerin as our template structure, we used the substructure highlighted in bold in Figure 1A to search the CAP database for structurally similar chemicals using the Accelrys ® Catalyst programme. This allowed the identification of 137 compounds. This list of compounds was further refined using the Accelrys ® Cerius2 modelling and simulation program to test for ligand fit to the crystallised C1B phorbol-binding domain of the PKC molecule (Figure 1Bi). The C1B domain of the regulatory region of PKC, containing the phorbol ester, was obtained from the Protein Data Base (PKC C1B domain RCSB PDB entry 1PTQ; [START_REF] Zhang | Crystal structure of the cys2 activator-binding domain of protein kinase C delta in complex with phorbol ester[END_REF]; Figure 1Bii). The phorbol ester was removed and the binding site defined using the site search function of Cerius2. The energy of the PKC crystal structure was minimised using the Dreiding 2.21 force field and energy minimised rottlerin conformers were docked to the PKC phorbol-binding site. The ligand-enzyme score was quantified using the 'DOCK' score of the Cerius2 LigFit module, which rated interaction energy and internal ligand energy between enzyme and ligand. DOCK scores of the 137 identified compounds with the PKC C1B phorbol-binding domain showed a normal distribution (Figure 1C). Within this distribution, both curcumin and rottlerin exhibited high DOCK scores of 92 and 87, respectively, indicating significant chemical similarity between these compounds. The interaction of curcumin with the PKC C1B phorbol-binding domain is illustrated in Figure 1Biii. Using an ELISA based assay, curcumin was also found to enhance NCAM PSA expression in a dose-dependent manner in neuro-2A neuroblastoma cells (Figure 1D). This confirmed curcumin to have the same biological activity of rottlerin, as reported previously [START_REF] Gallagher | Regulation of neural cell adhesion molecule polysialylation state by cell-cell contact and protein kinase C delta[END_REF], and that both compounds most likely influenced PKC activity in a similar A c c e p t e d M a n u s c r i p t 16 manner. The dose of curcumin required to enhance NCAM PSA expression was, however, an order of magnitude greater than that previously reported for rottlerin (5 M; [START_REF] Gallagher | Regulation of neural cell adhesion molecule polysialylation state by cell-cell contact and protein kinase C delta[END_REF]. This difference in dose-response is most likely related to the unstable nature of curcumin at neutral and basic pH both of which lead to its rapid degradation and formation of ferulic acid, a potent antioxidant [START_REF] Wang | Stability of curcumin in buffer solutions and characterization of its degradation products[END_REF]. Ferulic acid, however, failed to enhance NCAM PSA expression in the neuro-2A neuroblastoma cell line (data not shown) further confirming the primary role of curcumin in this effect.

Influence of curcumin and rottlerin on PKC kinase activity.

Given that previous studies demonstrated rottlerin to be without effect on PKC activity [START_REF] Davies | Specificity and mechanism of action of some commonly used protein kinase inhibitors[END_REF] and [START_REF] Soltoff | Rottlerin: an inappropriate and ineffective inhibitor of PKC[END_REF], we determined if curcumin similarly failed to influence enzyme activity. The in vitro assay employed determined the influence of curcumin on the phosphorylation rate of myelin basic protein using purified human recombinant PKC protein (Figure 2A) in the presence of [-32 P]-ATP and phosphatidylserine and DAG lipid activators. Using this assay, the phosphorylation rate of myelin basic protein substrate was found to be directly related to the concentration of PKC enzyme present (Figure 2B). We further validated the assay using the non-specific PKC inhibitor staurosporine and found it to inhibit PKC kinase activity in a dose-dependent manner (data not shown). Staurosporine exhibited an IC 50 value of approximately 5nM which is in close agreement with the IC 50 value of 3.5nM reported previously [START_REF] Mizuno | Staurosporine-related compounds, K252a and UCN-01, inhibit both cPKC and nPKC[END_REF]. At 5 nM, staurosporine reduced PKC kinase activity to 47.3±9.2% of that observed in the control sample (Figure 2C).

In comparison to rottlerin and curcumin, over concentration ranges of 10-100 M or 100-500M, respectively, only staurosporine inhibited PKC activity (F[5,12]= 11.09; P<0.05). To further preclude the possibility that rottlerin or curcumin may be acting as competitive inhibitors at the A c c e p t e d M a n u s c r i p t 17 PKC ATP binding site, we determined the consequence of lowering available ATP (200 uM to 40M) on kinase activity. At this lower ATP concentration, staurosporine further reduced PKC kinase activity to 29.5±8.4% of the control value but it was without effect on the ability of curcumin or rottlerin to inhibit PKC activity (Figure 2C). These results demonstrated rottlerin to be without any direct effect on PKC activity, confirming previous reports [START_REF] Davies | Specificity and mechanism of action of some commonly used protein kinase inhibitors[END_REF], and that the related compound curcumin similarly failed to influence enzyme activity.

Curcumin binding promotes PKC degradation.

Given curcumin failed to inhibit PKC activity, despite its high DOCK score for the phorbolbinding site of the enzyme, we determined if curcumin formed a complex with the enzyme. To achieve this, we covalently coupled curcumin to epoxy-activated Sepharose 6B, via its available hydroxyl groups, and used these beads to bait PKC in lysates of rat dentate gyrus solubilised with a non-ionic detergent, a procedure employed previously to identify the cellular targets of other protein kinase inhibitors [START_REF] Godl | An efficient proteomics method to identify the cellular targets of protein kinase inhibitors[END_REF]. Incubation of the dentate lysate with curcumin-coupled Sepharose resulted in PKC forming a tight linkage to curcumin. Following removal of weaklybound proteins by a cycle of low-and high-salt wash and rigorous boiling in SDS gel sample buffer, the remaining Coomassie blue-stained electrophoretic protein profile was found to be comparable across all treatment conditions (Figure 3A; equal protein loading). Immunoblot analysis with anti-PKC, however, revealed the enzyme to be associated with the curcumincoupled Sepharose 6B and not with the control Sepharose 6B (Figure 3B; lanes 1 and 2).

Moreover, the curcumin-coupled Sepharose 6B bound all of the PKC in the lysate as immunoblot analysis of the remainder failed to detect any enzyme immunoreactivity (Figure 3B; lane 4) and this was in contrast to the abundant PKC immunoreactivity observed in the lysate A c c e p t e d M a n u s c r i p t 18 following incubation with the control Sepharose 6B (Figure 3B; lane 5). Moreover, the tight association of PKC with curcumin appeared to exert an allosteric effect that rendered the enzyme more amenable to Tyr 311 phosphorylation. Exposure of the neuro-2A neuroblastoma cell line to curcumin for a 5 h period resulted in a dose-dependent decrease in PKC protein expression with no effect being observed on actin protein levels (Figure 4A andB). This increase in PKC protein degradation was paralleled by a dose-dependent increase in the phosphorylation of the Tyr 311 residue of PKC, accompanied by Tyr 311 phosphorylated enzyme protein fragments (Figure 4C), suggesting the association of curcumin with PKC directed its degradation which is in agreement with previous work relating PKC Tyr 311 phosphorylation to its increased proteolysis [START_REF] Blake | Courtneidge Src promotes PKC degradation[END_REF].

Curcumin enhances NCAM polysialylation state and spatial learning in adult Wistar

rats.

Given the transient increase in polysialylated neurons in the hippocampal formation that accompanies consolidation of an avoidance conditioning paradigm [START_REF] Fox | Memory consolidation induces a transient and time-dependent increase in the frequency of NCAM-polysialylated cells in the adult rat hippocampus[END_REF] is associated with a decrease in PKC degradation [START_REF] Gallagher | Protein kinase C delta regulates neural cell adhesion molecule polysialylation state in the rat brain[END_REF], we determined if curcumin, administered in vivo, would similarly increase NCAM polysialylation state in the infragranular zone of the dentate gyrus of adult rats (postnatal day 80). Following daily administration of curcumin (75 mg/kg by gavage) for an 8-day period, we counted a two-fold increase in polysialylated dentate neurons (P<0.05, Student's two-tailed t-test) (Figure 5B). Moreover, the arborisation of these polysialylated neurons in the upper granule cell layers and inner molecular layer was substantially increased, suggesting curcumin induced a substantial remodelling of the dentate neuronal architecture (Figure 5A). two-way repeated measure ANOVA) (Figure 6C). Moreover, curcumin-treatment produced a most significant effect on task recall when measured at both the 24 and 72 h probe trials (Figure 6D), as judged by a two-way repeated measure ANOVA (F [START_REF] Sadoul | Adult and embryonic mouse neural cell adhesion molecules have different binding properties[END_REF][START_REF] Steinberg | Distinctive activation mechanisms and functions for protein kinase Cdelta[END_REF] The ability of curcumin to suppress PKC expression is consistent with our previous in vitro observations demonstrating phorbol activation and/or staurosporine inhibition of PKC to, respectively, suppress and/or enhance PST activity with the direct consequence of regulating the extent to which NCAM is glycosylated with extended chains of 2,8-polysialic acid [START_REF] Eckhardt | Molecular characterization of eukaryotic polysialyltransferase-1[END_REF]. The mechanism by which curcumin induces degradation of PKC appears to be associated with increased phosphorylation of the Tyr 311 residue which lies within the hinge region between the regulatory and catalytic domains of the enzyme. This finding agrees with previous studies which have demonstrated phosphorylation of the Tyr 311 residue to first activate PKC and subsequently render it more susceptible to degradation [START_REF] Blake | Courtneidge Src promotes PKC degradation[END_REF] when the hinge region becomes accessible to proteolytic cleavage during the conformational changes associated with enzyme activation [START_REF] Kishimoto | Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain)[END_REF].

Activation of PKC by phorbol esters is also known to trigger their ubiquitination and degradation [START_REF] Lu | Activation of protein kinase C triggers its ubiquitination and degradation[END_REF]. Moreover, the Tyr 311 residue of PKC is flanked by a sequence that forms an optimal binding substrate for the Src family of kinases that constitutively complex with PKC but not other PKC isoforms, such as PKC or PKC [START_REF] Steinberg | Distinctive activation mechanisms and functions for protein kinase Cdelta[END_REF].

Chronic administration of curcumin in vivo was also demonstrated to enhance the polysialylation of neurons in the infragranular zone of the dentate gyrus and this included their dendritic tree which extends into the inner third of the molecular layer. -Tocopherol, which also reduces PKC expression [START_REF] Ferri | -Tocopherol affects neuronal plasticity in adult rat dentate gyrus: The possible role of PKC[END_REF], has been found to similarly enhance the polysialylation of neurons in the A c c e p t e d M a n u s c r i p t 21 adult dentate gyrus and, moreover, generate greater complexity in their dendritic arbor, as evidenced by increased synaptophysin expression and synapse density in the molecular layer [START_REF] Goel | Curcumin as "Curecumin": From kitchen to clinic[END_REF]. Upregulation of dentate NCAM PSA may also, in part, provide a basis for the neuroprotective actions attributed to curcumin [START_REF] Ebadi | Neuroprotective actions of selegiline[END_REF] as chronic exposure to deprenyl, purported to slow nigral cell degeneration in Parkinson's disease [START_REF] Murphy | Chronic exposure of rats to cognition enhancing drugs produces a neuroplastic response identical to that obtained by complex environment rearing[END_REF], has been demonstrated to increase the frequency of dentate polysialylated cells [START_REF] Duveau | Polysialylation of NCAM is upregulated by hyperthermia and participates in heat shock preconditioning-induced neuroprotection[END_REF]. Moreover, hyperthermia-induced upregulation of NCAM PSA appears to similarly protect against kainate-induced cell death [START_REF] Kim | Curcumin stimulates proliferation of embryonic neural progenitor cells and neurogenesis in the adult hippocampus[END_REF]. Curcumin has been demonstrated to significantly increase the number of newly generated cells in the dentate infragranular zone [START_REF] Foley | The selective 5-HT 6 receptor antagonists SB-271046 and SB-399885 potentiate NCAM-PSA immunolabelling of dentate granule cells, but not neurogenesis, in the hippocampal formation of mature Wistar rats[END_REF], however, not all of these are polysialylated [52]. The ability of curcumin to enhance neuronal polysialylation through suppression of PKC activity may more likely relate to the integration of newborn cells into the dentate neuronal architecture over the 1-2 month period that follows their birth [START_REF] Ge | A critical period for enhanced synaptic plasticity in newly generated neurons of the adult brain[END_REF], [START_REF] Kee | Preferential incorporation of adultgenerated granule cells into spatial memory networks in the dentate gyrus[END_REF] and [START_REF] Toni | Neurons born in the adult dentate gyrus form functional synapses with target cells[END_REF]. This mechanism would be consistent with the transient decrease in PKC expression and associated increase in dentate polysialylated cell frequency that is observed at the 12h post-training time following avoidance conditioning [START_REF] Fox | Memory consolidation induces a transient and time-dependent increase in the frequency of NCAM-polysialylated cells in the adult rat hippocampus[END_REF], [START_REF] Gallagher | Protein kinase C delta regulates neural cell adhesion molecule polysialylation state in the rat brain[END_REF] and with other studies that have failed to implicate increased NCAM PSA-mediated plasticity with neurogenesis [START_REF] Fox | Memory consolidation induces a transient and time-dependent increase in the frequency of NCAM-polysialylated cells in the adult rat hippocampus[END_REF], [START_REF] Pham | Fear learning transiently impairs hippocampal cell proliferation[END_REF], [START_REF] Lópéz-Fernández | Upregulation of polysialylated neural cell adhesion molecule in dorsal hippocampus after contextual fear conditioning is involved in long-term memory formation[END_REF] and [START_REF] Kim | Curcumin stimulates proliferation of embryonic neural progenitor cells and neurogenesis in the adult hippocampus[END_REF].

Chronic administration of curcumin to adult Wistar rats significantly improved their performance in both the acquisition and consolidation of the water maze spatial learning paradigm. This is not surprising given that chronic administration of curcumin produces a two-fold increase in the frequency of polysialylated dentate neurons, the numerical density of which may be directly correlated to improvement in task performance [START_REF] Sandi | Neurobiological correlates of individual differences in spatial learning ability[END_REF] and [START_REF] Duveau | Polysialylation of NCAM is upregulated by hyperthermia and participates in heat shock preconditioning-induced neuroprotection[END_REF]. Environmental enrichment, for example, robustly improves spatial learning and memory and is associated with increased NCAM A c c e p t e d M a n u s c r i p t 22 polysialylation [START_REF] Young | Environmental enrichment inhibits spontaneous apoptosis, prevents seizures and is neuroprotective[END_REF] and [START_REF] Duveau | Polysialylation of NCAM is upregulated by hyperthermia and participates in heat shock preconditioning-induced neuroprotection[END_REF] and also a decreased expression of PKC [START_REF] Gallagher | Protein kinase C delta regulates neural cell adhesion molecule polysialylation state in the rat brain[END_REF]. Moreover, a pyrazole analogue of curcumin [START_REF] Liu | A broadly protective derivative of curcumin[END_REF] has been demonstrated to facilitate the induction of LTP and performance in an object recognition paradigm in Wistar rats through the induction of Ca ++ /calmodulin-dependent protein kinase II [START_REF] Maher | A pyrazole derivative of curcumin enhances memory[END_REF], a mechanism known to be necessary for both spatial learning and NCAM-mediated neuroplasticity [START_REF] Silva | Impaired spatial learning in alphacalcium-calmodulin kinase II mutant mice[END_REF] and [START_REF] Williams | A Ca2+/calmodulin kinase inhibitor, KN-62, inhibits neurite outgrowth stimulated by CAMs and FGF[END_REF]. Although, the dose of curcumin required to enhance NCAM polysialylation and cognition in vivo was relatively high (75 mg/kg), no effect on either parameter was found at a dose of 37.5 mg/kg (data not shown), it was within the range previously reported necessary to induce neuroprotective effects in vivo [START_REF] Jagatha | Curcumin treatment alleviates the effects of glutathione depletion in vitro and in vivo: Therapeutic implications for Parkinson's disease explained via in silico studies[END_REF] and [START_REF] Shukla | Anti-ischemic effect of curcumin in rat brain[END_REF]. Moreover, the dose at which curcumin mediates these neuroplastic actions is likely to be exceptionally low as its bioavailability is exceedingly poor, the majority being excreted in the feces, and only small fraction of the dose penetrates the brain, estimated to be approximately 0.3% of the administered dose in mice [START_REF] Ryu | Curcumin and dihydrozingerone derivatives: synthesis, radiolabelling, and evaluation for beta-amyloid plaque imaging[END_REF] and [START_REF] Ebadi | Neuroprotective actions of selegiline[END_REF]. The lack of any behavioural toxicity in these studies, and those of others in both animals and humans [START_REF] Shankar | Toxicity studies on tumeric (Curcuma longa): acute toxicity studies in rats, guineapigs and monkeys[END_REF] and [START_REF] Lao | Dose escalation of a curcuminoid formulation[END_REF], further implies the effective dose of curcumin necessary to enhance NCAM PSA expression and cognition to be exceptionally small. This unique action of curcumin may have significant implications in facilitating memory formation as the extent of PSA activation is commensurate with the difficulty experienced with task consolidation [START_REF] Sandi | Neurobiological correlates of individual differences in spatial learning ability[END_REF].

Curcumin also significantly improved spatial learning ability in aged animals despite the significant age-dependent decrease in NCAM polysialylation state [START_REF] Fox | Polysialylated neural cell adhesion molecule expression by neurons and astroglila processes in the rat dentate gyrus declines dramatically with increasing age[END_REF] and increase in PKC expression [START_REF] Gallagher | Protein kinase C delta regulates neural cell adhesion molecule polysialylation state in the rat brain[END_REF]. However, extensive NCAM polysialylation is not necessary for the acquisition and/or consolidation of behavioural paradigms in aged animals [START_REF] Abrous | Decrease in highly polysialylated neuronal cell adhesion molecules and in spatial learning during ageing are not correlated[END_REF] and [START_REF] Seymour | Intraventricular infusions of anti-NCAM PSA impair the process of consolidation of both avoidance conditioning and spatial learning paradigms in Wistar rats[END_REF] and appears to relate mainly to the graded, experience-expectant development of explicit memory systems that A c c e p t e d M a n u s c r i p t 23 continue to evolve into maturity [START_REF] Stanton | Multiple memory systems, development and conditioning[END_REF]. Curcumin can also regulate the in vivo expression of several other cell signaling systems required for neuroplastic events necessary for memory formation, notably Notch-1 and NFB [START_REF] Romano | Evolutionarily-conserved role of the NF-kappaB transcription factor in neural plasticity and memory[END_REF], [START_REF] Wang | Involvement of Notch signalling in hippocampal plasticity[END_REF], [START_REF] Chen | Curcumin, both Histone Deacetylase and p300/CBP-Specific Inhibitor, Represses the Activity of Nuclear Factor Kappa B and Notch 1 in Raji Cells[END_REF], [START_REF] Conboy | Notch signalling becomes transiently attenuated during long-term memory consolidation in adult Wistar rats[END_REF] and [START_REF] Shishodia | Modulation of transcription factors by curcumin[END_REF]. Nevertheless, facilitation of PSA activation by curcumin, however minimal, may have implications for the treatment of neurodegenerative conditions. In Alzheimer's disease, the natural autoprotective response to agerelated cognitive deficits is a small, but significant, activation of dentate polysialylated cell frequency [START_REF] Mikkonen | Hippocampal plasticity in Alzheimer's disease: changes in highly polysialylated NCAM immunoreactivity in the hippocampal formation[END_REF] and, over the past few years, an increasing number of studies have highlighted the therapeutic potential of curcumin for the treatment of this condition [START_REF] Wu | Dietary curcumin counteracts the outcome of traumatic brain injury on oxidative stress, synaptic plasticity, and cognition[END_REF] and [START_REF] Xu | Curcumin reverses the effects of chronic stress on behaviour, the HPA axis, BDNF expression and phosphorylation of CREB[END_REF], a suggestion further supported by its ability to reduce -amyloid plaque burden in animal models [START_REF] Begum | Curcumin structure-function, bioavailability, and efficacy in models of neuroinflammation and Alzheimer's disease[END_REF].

Moreover, human populations exposed to curcumin show significantly reduced incidence of Alzheimer's disease, but not ApOE polymorphisms [START_REF] Ganguli | Apolipoprotein E polymorphism and Alzheimer disease[END_REF], and improved cognitive ability as compared to a control group [START_REF] Ng | Curry consumption and cognitive function in the elderly[END_REF]. The chemical structure of rottlerin and curcumin with the pharmacopore side chain in bold is shown in Panel A. Crystal structure of the PKC CIB binding site is shown in Figure 1Bi along with its interaction with the phorbol ester (Figure 1Bii) and curcumin (Figure 1Biii). Figure 1C shows a histogram plot of the 137 rottlerin-like molecules and their strength of their interaction, based on dock score, with the PKC C1B domain (Figure 1C). Figure 1D demonstrates the curcumin-induced, dose-dependent increase in PSA expression in neuro-2A neuroblastoma cells.

A c c e p t e d M a n u s c r i p t 2 ABBREVIATIONS:

 2 ABBREVIATIONS: DMEM: Dulbecco's modified Eagle's medium; NCAM: neural cell

  A significant post-translational modification of the neural cell adhesion molecule (NCAM)

A

  c c e p t e d M a n u s c r i p t 12 peroxidase substrate and exposure of the membranes to x-ray film (Kodak, Dublin, Ireland).

  change in dentate NCAM polysialylation state also correlated with improved acquisition and consolidation of the water maze spatial learning paradigm in cohorts of both adult (postnatal day 80) and aged (postnatal day 540) Wistar rats. Adult animals treated with curcumin exhibited a significant difference in task acquisition over 4 sessions (F[1,72]=4.34, P=0.041; twoway repeated measure ANOVA), as compared to the vehicle-treated controls (Figure6A). These animals also spent significantly more time in the target quadrant during subsequent probe trials (F[1,72]=3.9; P=0.05; two-way repeated measure ANOVA) and this became significant at the 72 h recall time (Figure6B), as judged by Bonferroni post-test and Mann-Whitney U-test (P<0.05).These results demonstrated curcumin treatment to exert a marked improvement on both the acquisition and consolidation of the spatial learning task. Curcumin treatment also resulted in significant improvements in both acquisition and consolidation of the water maze task in the cohort of aged animals. Drug-treated animals exhibited a superior performance to their vehicletreated controls in task acquisition when assessed over 4 training sessions (F[1,40]=6.7; P=0.01;

  These studies have demonstrated increased PKC degradation by curcumin to be associated with an enhanced expression of NCAM polysialylation state in mouse neuro-2A neuroblastoma cells.

Figure 1 :

 1 Figure 1:In silico approach to identification of curcumin as a rottlerin analogue.

Figure 2 :

 2 Figure 2: Influence of curcumin and rottlerin on recombinant PKC activity. Panel A illustrates

Figure 3 :

 3 Figure 3: Association of curcumin-conjugated Sepharose-6B with PKC in lysates from the

Figure 4 :

 4 Figure 4: Influence of curcumin on PKC expression in neuro2A neuroblastoma cells. The

Figure 5 :

 5 Figure 5: Influence of chronic curcumin administration on polysialylated cell frequency in the

Figure 6 :

 6 Figure 6: Influence of chronic administration of curcumin on spatial learning in adult and aged

Figure 1 ;

 1 Figure 1; Conboy et al.

FigureFigure 2 ;

 2 Figure

Figure 3 ;Figure 4 ;Figure 6 ;

 346 Figure 3; Conboy et al.

  polysialylation state through degradation of the PKC inhibitor of polysialyltransferase and improves spatial learning in adult and aged Wistar rats.

  =8.2; P=0.006;) and Bonferroni post-test and Mann-Whitney U-test (P<0.05).

ACKNOWLEDGEMENTS

We would like to thank Suzanne Prenter for technical assistance. This work was supported by Enterprise Ireland.