Elsevier

Biochemical Pharmacology

Volume 77, Issue 7, 1 April 2009, Pages 1254-1265
Biochemical Pharmacology

Curcumin-induced degradation of PKCδ is associated with enhanced dentate NCAM PSA expression and spatial learning in adult and aged Wistar rats

https://doi.org/10.1016/j.bcp.2008.12.011Get rights and content

Abstract

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 (PKCδ) to be associated with increased polysialyltransferase (PST) activity, suggesting inhibitors of this kinase might ameliorate cognitive deficits. Using a rottlerin template, a drug previously considered an inhibitor of PKCδ, we searched the Compounds Available for Purchase (CAP) database with the Accelrys® Catalyst programme for structurally similar molecules and, using the available crystal structure of the phorbol-binding domain of PKCδ, 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 PKCδ protein expression. Curcumin did not directly inhibit PKCδ activity but formed a tight complex with the enzyme. With increasing doses of curcumin, the Tyr131 residue of PKCδ, which is known to direct its degradation, became progressively phosphorylated and this was associated with numerous Tyr131-phospho-PKCδ 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 PKCδ in regulating PST and NCAM PSA expression and provide evidence that drug modulation of this system enhances the process of memory consolidation.

Introduction

A significant post-translational modification of the neural cell adhesion molecule (NCAM) involves the attachment of large homopolymers of α2,8-linked polysialic acid (PSA), a modification that is specific to NCAM in the mammalian brain [1], [2]. This post-translational modification of NCAM has been extensively argued to support structural plasticity in the adult nervous system [3], [4], [5] and has been implicated in activity-dependent synaptic remodelling [6], [7]. Moreover, the synaptic remodelling that accompanies task consolidation of spatial learning and avoidance conditioning paradigms [8], [9], [10] is associated with a transient increase in the frequency of dentate granule cells expressing polysialylated NCAM at the 12 h post-training time [11], [12], [13] 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 [14], [15], [16], [17]. 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 [18]. Polysialylation of NCAM occurs in the trans-Golgi compartment and is catalysed by two enzymes termed sialyltransferase-X (STX or ST8SiaII) [19], [20] and polysialyltransferase (PST or ST8SiaIV) [21], [22]. These two PSTs regulate NCAM polysialylation state differentially during development, as STX is the dominant PST in the embryonic and early postnatal period, whereas PST is mainly associated with α2,8-polysialylation in the postnatal brain [23], [24]. 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 PSA [25].

In general, the molecular events associated with regulation of sialyltransferase-mediated glycosylation 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 [26] Moreover, immunoblotting procedures have demonstrated reduced PKCδ 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 Escherichia coli PST, hippocampal PKCδ has been found to form complexes with PST that increase as animals age [27] and NCAM PSA expression decreases in an exponential manner [28]. The PST associated with these complexes has also been observed to be phosphorylated on serine residues [27], a mechanism known to be associated with the inhibition of other sialyltransferases [29], [30], [31], [32]. Phosphorylated tyrosine residues on PST are also found in PST:PKCδ immunocomplexes [27] and this may play a crucial role in regulating PKCδ 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 [33], [34].

In previous studies directed to providing evidence for a role of PKCδ in the regulation of NCAM polysialylation in neuro-2A neuroblastoma cells [26], we employed rottlerin to inhibit PKCδ, as this had been reported to have an IC50 value of 3–6 μM and to be 5–17 times more potent as a PKCδ inhibitor over other PKC isozymes [35]. Neuro-2A cells exposed to rottlerin (5 μM) in the mid-log growth phase, when NCAM PSA expression is dominant, resulted in a rapid and substantial increase in NCAM polysialylation state [26]. 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 PKCδ[36] but that it may modulate the tyrosine phosphorylation state of PKCδ and indirectly affect enzyme activity [37]. We now demonstrate curcumin to be a chemical similar of rottlerin that indirectly modulates PKCδ activity by modifying its phosphorylation state to increase the rate of degradation.

Section snippets

Materials

All routine laboratory chemicals, secondary antibodies, rottlerin, staurosporine and curcumin were purchased from Sigma–Aldrich, Ireland. All components of the PKCδ activity assay, including constitutively active recombinant PKCδ, were purchased from Upstate Biotechnology (Charlottesville, VA, USA). [γ-32P]-ATP was purchased from Amersham Biosciences (Buckinghamshire, UK). The bicinchoninic acid (BCA) assay was purchased from Pierce (Rockford, IL, USA). The monoclonal antibody raised against

Identification of curcumin as a chemical similar of rottlerin

Taking rottlerin as our template structure, we used the substructure highlighted in bold in Fig. 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 programme to test for ligand fit to the crystallised C1B phorbol-binding domain of the PKCδ molecule (Fig. 1Bi). The C1B domain of the regulatory

Discussion

These studies have demonstrated increased PKCδ degradation by curcumin to be associated with an enhanced expression of NCAM polysialylation state in mouse neuro-2A neuroblastoma cells. The ability of curcumin to suppress PKCδ expression is consistent with our previous in vitro observations demonstrating phorbol activation and/or staurosporine inhibition of PKCδ to, respectively, suppress and/or enhance PST activity with the direct consequence of regulating the extent to which NCAM is

Acknowledgements

We would like to thank Suzanne Prenter for technical assistance. This work was supported by Enterprise Ireland. Marie Lawlor was the recipient of an IRCSET scholarship.

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