Protein kinase C delta regulates neural cell ... - Wiley Online Library

Journal of Neurochemistry, 2001, 77, 425±434

Protein kinase C delta regulates neural cell adhesion molecule polysialylation state in the rat brain Helen C. Gallagher, Keith J. Murphy, Andrew G. Foley and Ciaran M. Regan Department of Pharmacology, The Conway Institute, University College, Dublin, Ireland

Abstract Polysialylation of neural cell adhesion molecule (NCAM PSA) modulates cell±cell homophilic binding and signalling during brain development and the remodelling of discrete brain regions in the adult. Following learning, a transient increase in the frequency of polysialylated neurones occurs in the dentate gyrus of the hippocampal formation, and this has been correlated with the selective retention and/or elimination of synapses that are transiently overproduced during memory consolidation. We now demonstrate that protein kinase C delta (PKCd) negatively regulates polysialyltransferase activity in the rat brain during development and also in the hippocampus during memory consolidation, where its downregulation in the Golgi membrane fraction coincides with the transient increase in NCAM PSA expression. Decreased expression of PKCd was also observed in the hippocampus of rats reared in a complex environment and this directly contrasted the signi®cant increase in frequency of hippocampal polysialylated neurones observed in these animals. These

effects were isoform-speci®c as no change in total PKC enzyme activity was detected during memory consolidation and complex environment rearing had no effect on the hippocampal expression of PKCa, b, g or 1. By sequential immunoprecipitation and immunoblot analysis, phosphorylation of polysialyltransferase protein(s) was (were) demonstrated to occur on both serine and tyrosine residues and this was associated with decreased enzyme activity. Moreover, a similar experimental approach revealed the degree of PKCd co-precipitation with polysialyltransferase protein(s) to be inversely correlated with polysialyltransferase activity. These ®ndings support in vitro evidence indicating PKCd to regulate polysialyltransferase activity and NCAM polysialylation state. Keywords: complex environment, neural cell adhesion molecule, passive avoidance, protein kinase C, polysialyltransferase. J. Neurochem. (2001) 77, 425±434.

Polysialylation of the neural cell adhesion molecule (NCAM) is a unique glycosylation mechanism involving post-translational additions of 2,8-linked polysialic acid (PSA) homopolymers that can exceed 55 sugar units in length (Livingston et al. 1988). The attachment of PSA modulates NCAM-mediated homophilic adhesion and signal transduction events by virtue of its large negative charge (Hoffman and Edelman 1983; Doherty et al. 1990; Williams et al. 1994; Yang et al. 1994). In the developing neural system, NCAM polysialylation is required for cell proliferation and migration (Hildebrandt et al. 1998; Hu et al. 1996) and plays a role in neuritogenesis, axonal path®nding and nerve branching (Doherty et al. 1990; Zhang et al. 1992; Tang et al. 1994). The ubiquitous expression of NCAM PSA in the embryonic brain undergoes a striking down-regulation during development (Abrous et al. 1997; Fox et al. 1995b; Ni Dhuill et al. 1999) without any corresponding decline in

NCAM prevalence (Wagner et al. 1992; Bahr et al. 1993; Linnemann et al. 1993). In the adult, NCAM PSA is restricted to brain regions associated with persisting neurogenesis, such as the hippocampal dentate gyrus and olfactory bulb (Seki and Arai 1993; Miragall et al. 1988). NCAM PSA may also be involved in the selective

Received September 6, 2000; revised manuscript received October 18, 2000; accepted December 14, 2000. Address correspondence and reprint requests to Helen C. Gallagher, Department of Pharmacology, The Conway Institute, University College, Bel®eld, Dublin 4, Ireland. E-mail: [email protected] Abbreviations used: BSA, bovine serum albumin; DIV, days in vitro; LTP, long-term potentiation; NCAM, neural cell adhesion molecule; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PKC, protein kinase C; PSA, polysialic acid; PST1, polysialyltransferase 1; SDS, sodium dodecyl sulfate; STX, sialyltransferase X; TBS-T, 10 mm Tris-HCl, 150 mm NaCl and 0.05% (v/v) Tween-20 (pH 7.4).

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 425±434

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stabilization of synaptic contacts in the adult brain. In the rodent hypothalamus, for example, NCAM polysialylation is progressively down-regulated during lactation and this occurs in parallel with the development of new synaptic contacts (Nothias et al. 1997). Thereafter, NCAM PSA expression in the hypothalamus increases during the subsequent period of synapse elimination. Similarly, during memory consolidation, NCAM polysialylation is activated in granule-like cells of the hippocampal dentate gyrus at the 10±12 h post-training time that follows the acquisition of either avoidance or spatial learning paradigms (Fox et al. 1995a; Murphy et al. 1996). As this follows a period of transient synapse growth in the same brain region and coincides with the initiation of synapse elimination (O'Malley et al. 1998, 2000), it is consistent with the observations in the hypothalamus that directly implicate NCAM polysialylation in synapse selection. NCAM PSA expression is regulated by two polysialyltransferases termed STX and PST that are homologous in their functional domains and possess similar enzyme speci®city (Livingston and Paulson 1993; Eckhardt et al. 1995; Kojima et al. 1995, 1996; Nakayama et al. 1995; Nakayama and Fukuda 1996). Thus, the rapid activation of NCAM PSA that accompanies memory consolidation depends on reactive change in polysialyltransferase activity that must be both transient and reversible. Activation of polysialyltransferase may not necessarily rely on gene transcription, as STX and PST transcripts are detected in neuronal cell populations that do not express PSA in the adult brain (Phillips et al. 1997; Wood et al. 1998), thereby implicating non-transcriptional mechanisms of enzyme regulation. These studies suggest that in the adult brain, STX and PST may be held in a dormant, non-functional state by inhibitory signalling mechanisms, in a manner that facilitates their appropriate reactivation as is required during memory consolidation. Thus, the existence of distinct molecular switching mechanisms that regulate NCAM polysialylation would satisfactorily account for its developmental decline, as well as its transient modulation in the adult brain. Although the mechanisms that control polysialyltransferase activity remain to be fully elucidated, our recent studies on PSA regulation in vitro provide a biochemical basis for the inhibitory regulation of polysialyltransferase in vivo (Phillips et al. 1997; Wood et al. 1998). Using two separate cell culture models, we have demonstrated activation of PKCd to inhibit NCAM polysialylation (Gallagher et al. 2000). In the present study, we have used rodent models of development and learning to investigate if PKCd regulates NCAM polysialylation state in vivo in a manner similar to that observed in vitro.

Experimental procedures Materials Antibodies to PKC isoforms and phosphoproteins were from Transduction Laboratories (Exeter, UK). Citi¯uor was from Agar (Essex, UK). Fluorescein-conjugated anti-mouse IgM and recombinant PKCd were from Calbiochem (Nottingham, UK). Fetal calf serum and medium for cell culture was from Gibco BRL (Paisley, UK). The PKC assay kit, PKC standard and chemiluminescent peroxidase substrate were from Pierce (Chester, UK). Tritiated neuraminic acid was from American Radiolabelled Chemicals (St Louis, MO, USA). Anti-mouse IgG peroxidase and other general laboratory chemicals were from Sigma (Dublin, Ireland). Anti-polysialyltransferase production A polyclonal antibody directed against E. coli K1 polysialyltransferase was synthesized by immunization of a rabbit with a KLHconjugated 11 amino acid peptide. The peptide sequence used (NIFIISNLGQL) corresponded to residues 56±66 of the E. coli polysialyltransferase sequence. This peptide was selected because of the high degree of conservation between it and aligned residues 35±45 in Meninginitis B polysialyltransferase (NLFVISNLGQL; Weisberger et al. 1991). At the time of antibody generation, no mammalian polysialyltransferase sequences had been elucidated and the conserved residues in the bacterial proteins were assumed to be important for polysialyltransferase function. Preimmune serum was collected prior to immunization for use as a control. The antibody was collected as a serum fraction and was not further puri®ed before use. Immunoblot and immunoprecipitation analysis performed with this antibody, on perinatal rat whole brain homogenate, revealed it to recognize two prominent protein bands, one of which precisely corresponded to the predicted molecular weight of the mammalian polysialyltransferases, STX and PST (42 kDa). Furthermore, the immunoprecipitated fraction had polysialyltransferase activity con®rming that this antibody cross-reacts with the mammalian enzyme(s). Passive avoidance training Postnatal day 80 (P80) male Wistar rats (300±350 g) were obtained from the Biomedical Facility, University College Dublin and housed in social groups of 15 animals in standard laboratory conditions with a 12-h light/dark cycle with food and water available ad libitum. Animals were trained in a one-trial, stepthrough, light±dark passive avoidance paradigm as described previously (Fox et al. 1995a). A criterion period of 300 s was used. All experimental procedures were approved by the Review Committee of the Biomedical Facility, University College Dublin and carried out by individuals holding a license issued by the Ministry of Health. Rearing in complex and isolated environments Male Wistar rats were raised from time of weaning at postnatal day 25 (P25) until they were killed at P80, in either a complex environment or isolated condition. Isolated animals were maintained individually under standard facility conditions, however, handling was kept to a minimum. The complex environment consisted of a large (1 1 1 m) stainless steel cage in two sections interconnected by a ramp. The cage was ®lled with numerous stimuli, such as wooden/plastic blocks, wood chips, plastic containers, tunnels and exercise wheels. The arrangement of


PKCd regulates NCAM PSA in the rat brain 427

items and the location of food and water were changed daily and extra walls/levels were randomly inserted into the cage to generate extra variation. These daily alterations were performed during a 1-h period in which the animals were removed to an open arena containing food, water and a few of the above stimuli. PSA immunohistochemistry and quanti®cation of dentate polysialylated neurones The frequency of hippocampal and cortical polysialylated neurones was determined using PSA immunohistochemistry as described previously (Fox et al. 1995a). Brie¯y, cryostat axial sections of 12 mm sections were ®xed in 70% (v/v) ethanol and incubated overnight with anti-PSA ascitic ¯uid diluted 1/500 (Rougon et al. 1986). The sections were exposed for 3 h to ¯uorescein-conjugated goat anti-mouse IgM diluted 1/100 and mounted in Citi¯uorw, a ¯uorescence-enhancing medium. The total number of PSAimmunopositive neurones at the infragranular cell layer of the dentate gyrus was counted in seven alternate 12-mm sections, commencing at 2 5.6 mm below Bregma. Cell identi®cation was facilitated by counter-staining the sections with propidium iodide [60 s; 40 ng/mL phosphate-buffered saline (PBS)] while use of alternate sections precluded double counting of the 5±10 mm perikarya. Cell counts were expressed per unit area (0.15 mm2) of the dentate granule cell layer and the mean ^ SEM for each treatment group calculated. Measurements were performed using a Quantimet 500 Image Analysis System. Dissection and subcellular fractionation of the rat brain The brains of animals killed at postnatal days 1 (P1) and 540 (P540) were homogenized whole, since the P1 brain was too aqueous for accurate dissection of brain substructures. For the P80 animals used in the learning study, the dentate gyrus was dissected from the hippocampus. Light microscopical analysis of the dissected dentate gyrus region indicated it to comprise the dentate proper and, in a few cases, a small portion of the hilus (data not shown). The brains/ brain regions were collected in a suitable volume of ice-cold 0.32 m sucrose containing 1 mm isobutylmethylxanthine and homegenates were prepared with 20 up±down strokes using a hand-held homogenizer with a clearance of 0.25 mm. The homogenate was centrifuged at 170 g for 10 min at 48C (Sorvall RT6000) and the supernatant spun at 6500 g to yield P2 (synaptosome-enriched) and S2 (microsome-enriched) fractions. Golgi membrane fractions were prepared by layering the S2 fraction onto a 1.6-m sucrose cushion and centrifuging at 52 000 g (Beckman TL100, ®xed angle rotor) for 2 h at 48C. Material gathering at the sucrose interface was removed, layered onto a discontinuous gradient (1.6 m, 1.2 m, 0.32 m sucrose) and centrifuged for 3.5 h at 18 000 g, 48C. The Golgi fraction, located at the 1.2±1.6 m sucrose interface, was aspirated and membranes from this fraction were pelleted by centrifugation for 2 h at 52 000 g, 48C. This fractionation procedure has previously been demonstrated to consistently yield material enriched in thiamine pyrophosphate activity ± a Golgi membrane marker (Breen et al. 1987). Cell culture The mouse neuroblastoma cell line, neuro-2 A, was maintained in Dulbecco's modi®ed Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum and penicillin/streptomycin (100 mg/mL) at 378C in a humidi®ed atmosphere and 9% CO2. Cells were seeded at a density of 104 cells/cm2 and grown for 3 or

5 days in vitro (DIV). For preparation of detergent cell extracts, cells were washed three times in PBS, centrifuged for 10 min at 170 g and the resulting pellet was solubilized in 100 mm Tris-HCl pH 7.4, 1% (v/v) nonidet P-40 (NP-40), 1 mm dithiothreitol (DTT), 1 mm phenylmethylsulphonyl ¯uoride (PMSF), 0.01% (v/v) aprotinin, 0.01% (v/v) b-mercaptoethanol for 30 min on ice. The extract was cleared by centrifugation at 6400 g for 10 min. Estimation of protein content Protein content was determined according to the method of Lowry et al. (1951) using bovine serum albumin (BSA) as standard. The absorbance of samples was read at 620 nm in a Beckman DU62 spectrophotometer. Immunoprecipitation For immunoprecipitation with anti-polysialyltransferase, samples containing 500 mg protein in 250 mL volume were diluted 1 : 2 with double strength immunoprecipitation buffer to yield a ®nal buffer composition of 1% (v/v) Triton X-100, 150 mm NaCl, 10 mm Tris pH 7.4, 1 mm EDTA, 1 mm EGTA, 0.2 mm PMSF, 0.5% (v/v) NP-40. Immunoprecipitation with rabbit antipolysialyltransferase IgG (1 : 500) was carried out overnight at 48C. Protein-A sepharose 4-B beads were washed and pre-swollen in immunoprecipitation buffer at 48C. A 100-mL aliquot of a 50% protein-A suspension was added to each tube, and rocked for 2 h at 48C. The immunoprecipitate was pelleted by centrifugation at 6500 g for 10 min at 48C and washed three times in immunoprecipitation buffer. For electrophoresis, the ®nal pellet was resuspended in 50 mL sodium dodecyl sulphate±polyacrylamide gel electrophoreses (SDS±PAGE) sample buffer and boiled for 10 min. The sample was centrifuged at 13 000 g for 2 min and the supernatant was loaded onto SDS±PAGE gels for immunoblot analysis as described below. SDS-PAGE/western blotting Proteins were separated on 10% SDS±PAGE gels and transferred to nitrocellulose membranes using standard techniques. Equal protein loading was veri®ed by Coomassie blue staining. For PKC isoform analysis the membrane was blocked for 1 h in TBS-T [100 mm NaCl, 10 mm Tris-HCl pH 7.4, with 0.1% (v/v) Tween20w] containing 5% non-fat milk powder, and for phosphoprotein analysis in TBS-T containing 5% BSA. In each case primary antibodies were diluted in the respective blocking solution, as recommended by the manufacturer, and incubated overnight at 48C. The membrane was washed and incubated with secondary goat anti-mouse IgG or anti-rabbit IgG peroxidase-conjugated antibody diluted 1 : 2000 in the appropriate blocking solution for 2 h at room temperature. Finally, after rewashing it was incubated for 5±10min with a chemiluminescent peroxidase substrate and exposed to X-ray ®lm for times varying from 30 s to 5 min. Determination of PKC activity PKC activity was assayed using a commercially available kit and a ¯uorescently labelled PKC speci®c substrate, neurogranin, was employed. The ®nal assay mixture contained 0.2 mg/mL phosphatidylserine, 2 mm ATP, 0.36 mm ¯uorescent neurogranin, 10 mm MgCl2, 0.1 mm CaCl2, 0.002% Triton X-100, 20 mm Tris, pH 7.4, and 5±10 mg protein sample, of unknown PKC activity, in a total volume of 25 mL. Incubation proceeded for 30 min at 308C in a circulating waterbath, after which, 20 mL of the assay mixture was



spotted on to the supplied phosphocellulose ®lter, 250 mL of phosphopeptide binding buffer (0.1 m sodium acetate, 0.5 m NaCl, 0.02% sodium azide, pH 5.0) was added and the tube was allowed to stand for 3 min at room temperature. The tube was centrifuged at 600 g for 1 min and this procedure was repeated giving a ®nal binding buffer volume of 500 mL. The membrane unit was transferred to a new receptacle, 250 mL of phosphopeptide elution buffer (15% formic acid) was added and the unit was allowed to stand for 3 min at room temperature. Tubes were centrifuged as above and this procedure was repeated giving a total elution buffer volume of 500 mL. The optical density of the eluted fraction was measured at 570 nm in a spectrophotometer. Standard curves of known PKC activity were constructed using a puri®ed enzyme and PKC activity in unknown samples was determined by extrapolation.

Fig. 1 Modulation of PKCd in the rat hippocampus following avoidance training. (a) shows expression of PKCd in the hippocampal dentate gyrus as revealed by immunoblot analysis of tissue homogenates at increasing times following passive avoidance training. In (b), the speci®c activity of total PKC in the dentate gyrus homogenates is indicated. Activity in the P2 fraction is shown in the solid bars and in S2 fraction in the grey bars. All values are the mean ^ SEM (n 3).

Determination of polysialyltransferase activity The assay was performed in 50 mm potassium phosphate buffer, pH 6.8 with 1 mg of fetuin as acceptor substrate and 4.7 pmol of 3 H-CMP-neuraminic acid as donor substrate per sample. The assay mixture was incubated for 20 h at 378C and the reaction stopped by the addition of an equal volume of 12% trichloroacetic acid with 1% phosphotungstic acid. The sample tubes were rocked on ice for 30 min to precipitate the protein, which was pelleted by centrifugation at 13 000 g for 5 min. The supernatant was removed and discarded and the pellet washed twice by resuspension in distilled water. The ®nal pellet was resuspended in 0.05 m NaOH and incubated overnight at 378C for solubilization. This was transferred to a scintillation vial, 10 mL of scintillation ¯uid was added and the samples were counted in a Beckman LS-6000SC scintillation counter.

Results

Fig. 2 Immunoblot analysis of PKCd expression in subcellular fractions of rat hippocampal dentate gyrus following avoidance training. (a), (b) and (c) illustrate PKCd expression in whole dentate gyrus homogenate and S2 fraction and P2 fraction thereof, respectively. (d) shows PKCd expression in the Golgi membrane. (e) shows NCAM isoform expression in the Golgi membrane fraction (0 h posttraining time) and the polydisperse form that is observed at the 12 h post-training time.

All animals used in the learning study successfully acquired the passive avoidance task, as their recall escape latencies exceeded the 300 s criterion time. Immunoblot analysis of the homogenized dentate gyrus from animals killed at discrete post-training times revealed a marked downregulation of PKCd at 12 h post-training, when PSA is activated in this region (Fig. 1a; Fox et al. 1995a). At the 6 h post-training time a smaller down-regulation of PKCd was apparent in some animals, although this was not signi®cant by scanning densitometry using three separate groups of trained animals (data not shown). Using neurogranin as an enzyme substrate, no change in total PKC activity was observed at 12 h post-training relative to the 0 h control animal (Fig. 1b). This indicates that the differential expression observed with PKCd is likely to be speci®c to this isoform rather than re¯ective of an overall shift in PKC isozyme activities. To further investigate whether down-regulation of PKCd at 12 h post-training was directed to the activation of NCAM polysialylation, its distribution in subcellular fractions of the dentate homogenate was assessed. Initially, the whole dentate homogenate was further fractionated into microsome-enriched (S2) and membrane-enriched (P2)



Fig. 3 The effect of complex environment rearing on the developmental expression of PKCd in rat brain. (a) shows PKCd expression in perinatal (P1; lanes 1±3) and aged (P540; lanes 4±6) rat brain fractions. Whole homogenates were separated in lanes 1 and 4, P2 fractions in lanes 2 and 5, and S2 fractions in lanes 3 and 6. In (b) PKC isoform expression in the hippocampus of P80 animals reared in either a complex or isolated environment is shown. For each condition three different animals were analysed (lanes a, b and c). Immunoblot analysis was performed using monoclonal antibodies to the individual PKC isoforms, their respective molecular weights are indicated.

fractions. In animals killed immediately following training, PKCd was most prominently expressed in the S2 fraction with much lower levels seen in the P2 fraction (Figs 2a±c). However, at the 12 h post training time PKCd was virtually absent in the S2 fraction, and seemed to have translocated to the P2 fraction, although not in its entirety since overall levels of the isozyme in the dentate were much reduced at this time. As polysialyltransferase activity is localized to the Golgi apparatus (Breen et al. 1987), the S2 fraction of the dentate gyrus was layered onto a discontinuous sucrose gradient to isolate the Golgi membrane fraction. NCAM immunoblot analysis, performed prior to boiling these membrane samples, revealed a high degree of NCAM polydispersity in Golgi membranes isolated at the 12 h post-training time, indicating the presence of long PSA homopolymers (Fig. 2e). NCAM polydispersity was not detected in whole brain homogenates or S2 fractions of the dentate gyrus at the

12 h post-training time (data not shown). Similar to observations in the S2, PKCd was found to be expressed in the Golgi fraction of animals killed immediately after training but not in those killed at the 12 h post-training time (Fig. 2d). The differential expression of PKCd observed during memory consolidation supports our in vitro ®nding that this isoform serves as an inhibitory constraint on polysialyltransferase activation. The learning study was performed on animals killed at p80, thus the potential involvement of PKCd at this developmental period had been established. As developmental studies have found high levels of NCAM sialylation during the perinatal period and a signi®cant reduction in old age (Hoffman and Edelman 1983; McCoy et al. 1985; Fox et al. 1995b; Abrous et al. 1997; Ni Dhuill et al. 1999), we extended our study to include these developmental periods. Immunoblot analysis of PKCd in the whole brain and the P2 and S2 fractions thereof failed to detect this protein in the P1 rat brain, although it was expressed in all three fractions at P540, albeit more prominently in the S2 fraction (Fig. 3a). Again, this suggested the involvement of PKCd in polysialyltransferase inhibition during ageing. The ageing study indicated that PKCd undergoes a developmental regulation inversely related to that of PSA. To further investigate the relevance and malleability of this effect, the in¯uence of rearing environment on PKCd expression was determined, as this is known to alter hippocampal PSA expression. Animals were reared in either a complex or isolated environment from weaning (P25) to P80, and their hippocampal polysialylated neurone frequency determined by immunohistochemistry. Similar to the ®ndings of Young et al. (1999), animals reared in a complex environment had a signi®cantly higher number of polysialylated infragranular neurones in the hippocampal dentate gyrus, whereas those reared in isolation had a reduced complement of PSA-positive neurones relative to social controls (complex 85.8 ^ 1.39 vs. social 64.4 ^ 3.9 vs. isolated 39.96 ^ 0.68, mean ^ SEM PSA-positive neurones per unit area). Hippocampal homogenates were prepared from animals reared in either a complex or isolated environment, and their PKCd expression levels analysed by immunoblot analysis. This revealed a markedly reduced level of PKCd in the hippocampus of animals reared in a complex environment, implying that developmental regulation of this enzyme in¯uences, or is in¯uenced by the environmental factors that govern overall neuroplastic state (Fig. 3b). Moreover, investigation of the remaining calciumdependent PKC isoforms, a, b, g and 1, revealed that none were differentially expressed between complex and isolated environment-reared animals, highlighting the speci®city of the effect for PKCd (Fig. 3b). While the above studies provide correlative evidence for PKCd as a polysialyltransferase inhibitor in vivo as well as



Fig. 4 PKCd phosphorylation of mammalian polysialyltransferase. (a) (lane 2) shows the 42 kDa mammalian polysialyltransferase (asterisk) immunoprecipitated from a P1 rat brain homogenate and re-probed with polyclonal anti-E. coli polysialyltransferase. The molecular weight markers are shown in lane 1. Polysialyltransferase phosphorylation on tyrosine and serine residues is shown in (b) and (c). Whole brain homogenate from rats killed at P1 and P540 were immunoprecipitated with anti-polysialyltransferase followed by immunoblot analysis with anti-phosphotyrosine (b) or anti-phosphoserine (c). Where indicated, P1 samples were preincubated with an active, recombinant PKCd enzyme prior to immunoprecipitation.

in vitro, more direct evidence for this mechanism of regulation was required. A polyclonal antibody raised against the E. coli polysialyltransferase was employed to determine the phosphorylation state of the enzyme in P1 and P540 rat brain. Using perinatal (P1) brain as a tissue source,

Fig. 5 Co-precipitation of PKCd with polysialyltransferase. (a) and (b) show anti-polysialyltransferase immunoprecipitates, prepared from P1 and P540 rat brain, respectively, that were re-probed with a monoclonal antibody to PKCd. In (a) where overexposure of the X-ray ®lm resulted in the appearance of the 55 kDa heavy chain IgG band, PKCd could not be detected. However in (b) the 78 kDa PKCd band was detected. (c) shows an immunoblot analysis of PKCd expression in anti-polysialyltransferase immunoprecipitates prepared from neuro 2 A cell in mid log growth (DIV3) and at con¯uency (DIV5).

sequential immunoprecipitation and immunoblot analysis demonstrated this antibody to recognize a 42-kDa protein in P1 rat brain ± the expected molecular weight of the two mammalian polysialyltransferases, PST and STX (Fig. 4a). Furthermore, this antibody immunoprecipitated polysialyltransferase activity, which was not present in immunoprecipitates derived from the corresponding preimmune serum (anti-polysialyltransferase, 1139 ^ 10.6 cpm/500 mg protein vs. preimmune serum, 516 ^ 26 cpm/500 mg protein; n 2), con®rming the antibody to cross-react with the mammalian enzyme(s). To determine polysialyltransferase phosphorylation state, whole brain fractions from perinatal (P1) and aged (P540) animals were immunoprecipitated with the polysialyltransferase antibody and the immunoprecipitated fraction was immunoblotted with antiphosphotyrosine and antiphosphoserine antibodies. This indicated phosphorylation of polysialyltransferase on tyrosine residues to be developmentally regulated in line with decreased activity of the enzyme(s). A phosphotyrosine immunoreactive band appeared at 42 kDa in the P540 immunoprecipitate only (Fig. 4b). The same band was not phosphorylated in the immunoprecipitate from the P1 brain in which NCAM is extensively polysialylated. In contrast, serine phosphorylation of polysialyltransferase protein was not observed in either the perinatal or aged brain. However, phosphorylation of polysialyltransferase in the perinatal brain was inducible. When the brain homogenate was preincubated with an active recombinant PKCd enzyme prior to immunoprecipitation, polysialyltransferase phosphorylation was detected on both serine and tyrosine residues (Figs 4b and c). That recombinant PKCd ± a serine/ threonine kinase ± could induce phosphorylation on both serine and tyrosine indicates that it may directly phosphorylate the polysialyltransferase itself and also activate a tyrosine kinase. This suggests that PKCd may also indirectly mediate the developmentally regulated tyrosine phosphorylation of polysialyltransferase protein that was observed at P540. These ®ndings substantiated the view that PKCd inhibits polysialyltransferase activity by phosphorylation. To investigate whether this is mediated by direct association with polysialyltransferase protein(s), sequential immunoprecipitation and immunoblot analysis was performed, using antipolysialyltransferase and anti-PKCd, respectively. In the perinatal brain, no PKCd was detected in the immunoprecipitated fraction, even when the ®lm was overexposed to reveal the 55 kDa IgG heavy chain band (Fig. 5a). However, in P540 brain homogenate PKCd co-precipitated with the inactive polysialyltransferase, since a speci®c band was observed at 78 kDa (Fig. 5b). Similarly, in one of the in vitro models used to identify polysialyltransferase regulatory elements, the neuroblastoma cell line neuro-2 A, co-precipitation of PKCd was inversely related to NCAM polysialylation state. Signi®cantly more PKCd



co-precipitated with polysialyltransferase in neuro-2 A cells harvested at con¯uency (DIV5) which lack PSA, compared to those in the log growth period (DIV3) which express PSA (Fig. 5c). Discussion This study con®rms a pivotal role for PKCd in the regulation of NCAM polysialylation state in the rat brain. In recent in vitro studies we implicated the PKCd isozyme in the negative regulation of polysialyltransferase activity (Gallagher et al. 2000). Here, to determine if NCAM polysialylation state is regulated similarly in vivo, we employed one trial avoidance learning in which dentate polysialyltransferase is activated at the 10±12 h post-training time (Fox et al. 1995a). We also investigated the regulation of PKCd during the developmental decline of NCAM PSA expression in the rat brain. In both models we found evidence for the same negative regulation of polysialyltransferase activity by PKCd. Furthermore, we demonstrate that phosphorylation of polysialyltransferase(s) is correlated with a state of enzyme inactivation and that this is inducible. Thus an emerging mechanism of NCAM PSA regulation implicates PKCd as a polysialyltransferase negative regulator that mediates transient and reactive changes in NCAM-mediated neuroplasticity in the adult brain via polysialyltransferase phosphorylation and dephosphorylation. This is consistent with mechanisms known to regulate the activity of other sialyltransferase enzymes. PKC has been shown to inhibit ganglioside sialyltransferase activity in a reversible manner (Gu et al. 1995; Gao et al. 1996) and this is consistent with the presence of phosphorylated serine and threonine sites on an a-2,6-sialyltransferase (Ma et al. 1999). The role of PKCd in tyrosine phosphorylation of polysialyltransferase(s) remains unclear. Although induced by recombinant PKCd, this must involve activation of some unidenti®ed tyrosine kinase. PKCd is itself a tyrosine kinase substrate and this phosphorylation event modulates its activity in a substratespeci®c manner (Denning et al. 1996; Li et al. 1994a, 1994b; Haleem-Smith et al. 1995). That tyrosine phosphorylation of polysialyltransferase protein is basally up-regulated with age also suggests a functional involvement of this modi®cation in the age-related decline of NCAM-PSA. Although this does not exclude a similar developmental role for serine/threonine phosphorylation, it is possible that the latter may serve to mediate more acute modulations in polysialyltransferase activity such as those involved in learning. Our observations support this view since no basal developmental regulation of polysialyltransferase serine phosphorylation state was detected, although phosphoserine immunoreactivity was induced by recombinant PKCd.

Although the major carrier of PSA, NCAM is not the only protein that is polysialylated. Notably, the a subunit of the sodium channel (Zuber et al. 1992) and polysialyltransferases have been shown to express PSA (Muhlenhoff et al. 1996; Close and Colley 1998). Although polysialyltransferase autopolysialylation was initially reported to be a requisite for enzyme activity, it now appears that this is not required for, but may enhance, polysialyltransferase activity (Close et al. 2000). Other than PKCd, autopolysialylation is the only additional mechanism proposed to directly regulate polysialyltransferase, but how this serves to enhance PSA synthesis is unclear. Although global expression of NCAM PSA is a general feature of the developing brain, in several brain regions polysialylation persists into adulthood (Ni Dhuill et al. 1999), although by old age it is absent in the rat brain (Fox et al. 1995b). Areas in which the developmental decline in NCAM polysialylation is attenuated, such as the hippocampal dentate gyrus and olfactory bulb, are uniquely associated with persistent neurogenesis (Kuhn et al. 1996; Kempermann et al. 1997). Nevertheless, while the hippocampal polysialylated neurones are relatively young, as they are localized to the ®rst granule cell layer, their increased frequency following learning is not due to the birth of new neurones, as determined by bromodeoxyuridine incorporation (Fox et al. 1995a). Indeed, several independent studies concur that hippocampal neurogenesis is not a consequence of mammalian learning or complex environment rearing, but rather that pre-existing newborn neurones exhibit increased survival and incorporation into this region as a consequence of acquiring novel learning strategies (Gould et al. 1999; Nilsson et al. 1999; Young et al. 1999). Moreover, the polysialylated infragranular neurones of the dentate exhibit mature morphological characteristics, including well developed mossy ®bres and an extensive dendritic outgrowth to the inner molecular layer. That PSA is also expressed on these axons and dendrites, further supports the idea that polysialylation of this neuronal population is critical for the effective maintenance and remodelling of the synaptic circuitry in this region. The synaptic mechanism of memory consolidation is a process reliant on functional hippocampal PSA. Enzymatic removal of PSA interferes with long-term potentiation (LTP) and spatial learning (Becker et al. 1996; Muller et al. 1996), and gene disruption of the PST enzyme selectively perturbs LTP in PSA-expressing neuronal populations (Eckhardt et al. 2000). Furthermore, transient increases in the frequency of polysialylated neurones are task-independent but learning-speci®c, as they are not observed in an avoidance task when the animal is rendered amnesic with scopolamine or when a visible platform is used in a water maze task (Fox et al. 1995a; Murphy et al. 1996). Thus, the learning-associated increase in the frequency of polysialylated neurones in the hippocampus



is both functional and dependent on the reactivation of polysialyltransferase activity in an adult system. The signalling mechanisms regulating PSA synthesis therefore are of prime importance in the overall determination of neuroplastic state and synaptic networking. The down-regulation of PKCd in the hippocampal dentate during the period of increased NCAM polysialylation was most pronounced in the microsomal and Golgi membrane fractions. This observation is consistent with previous studies showing PKCd to be located and functionally active in this subcellular compartment and not necessarily requiring translocation to the plasma membrane as is observed with other isoforms (Goodnight et al. 1995). It also lends support to evidence that PSA is synthesized en bloc in the Golgi apparatus. In rat brain, a developmentally regulated sialyltransferase which catalyses the polysialylation of exogenous NCAM has been localized to the Golgi membrane fraction and recently, autopolysialylated PST and STX have also been localized to the Golgi (McCoy et al. 1985; Breen and Regan 1988; Close and Colley 1998). Pulse-labelling techniques in AtT20 cells indicated that PSA synthesis occurs in a late Golgi or post-Golgi compartment and is a very rapid event, with newly synthesized NCAM being polysialylated within 10 min (Alcaraz and Goridis 1991). Similarly, in the F3 cell line, the appearance of PSA on the cell surface is prevented under conditions in which trans Golgi-plasma membrane transport is blocked (Scheidegger et al. 1994). It is of interest that learning-induced down-regulation of PKCd in the Golgi is associated with an accumulation of this enzyme protein in the membrane-enriched fraction. This may indicate that translocation of this isoform represents an alternative regulatory mechanism for controlling its activity toward certain cellular substrates, localized to speci®c subcellular compartments. For example, PKCd is known to bind and phosphorylate membrane-associated GAP-43 in a calcium-dependent manner, an event crucial to signal transduction in growth cone elaboration and neuritogenesis (Dekker and Parker 1997). While autopolysialylated PST and STX have been localized to the Golgi apparatus, they have also been detected on the cell surface (Close and Colley 1998). It is possible therefore that PKCd translocation to the membrane fraction may inhibit polysialyltransferase autopolysialylation. That the extent of PKCd co-precipitation with polysialyltransferase is developmentally regulated, further supports the idea that differential expression of this PKC isoform mediates the inhibition of NCAM polysialylation during ageing. This is further corroborated by the marked reduction in hippocampal PKCd expression observed in complex environment-reared rats as compared to isolated animals. Co-precipitation of PKCd with polysialyltransferase enzymes in the P540 brain relative to the P1 brain could re¯ect an increase in the relative expression of PKCd, but

this is unlikely as polysialyltransferase prevalence would decrease over time. In vitro, however, a similar inverse relationship exists between NCAM polysialylation state and PKCd co-precipitation. Thus, it is most likely that the amount of PKCd directly associated with polysialyltransferase is developmentally regulated and that this would account for the parallel changes in PSA expression which occur in development. Moreover, evidence from the complex environment study indicates this to be a malleable developmental mechanism that may respond to or regulate environmental in¯uences governing overall neuroplastic state. PKCd belongs to the novel subgroup of PKC isoforms and is ubiquitously expressed in many tissues and cell lines. Various studies, have indicated that this isoform plays a speci®c role in differentiation. For example, in erthyroleukaemia cells, a critical event prior to cell commitment and differentiation is a rapid down-regulation of PKCd (Sparatore et al. 1993), whereas antisense inhibition of this isoform accelerates differentiation (Pessino et al. 1995). Similarly, in the neuronal cell lines PC12 and neuro-2 A we found that PKCd inhibition with rottlerin enhanced differentiation as evidenced by neuritogenesis (Gallagher et al. 2000). With regard to other neural functions, there is relatively little known about PKCd. The calcium-dependent PKCg isoform has been implicated in memory consolidation (Van der Zee et al. 1997a, 1997b) but a role for PKCd in memory consolidation has not been investigated previously. The phosphorylation/dephosphorylation switching mechanism of polysialyltransferase regulation that is mediated by PKCd, however, could satisfactorily account for the rapid and transient changes in PSA that are required for memory consolidation. Acknowledgements We thank Professor G. Rougon for the generous gift of antiMenB and Enterprise Ireland for ®nancial support.

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