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Originally published In Press as doi:10.1074/jbc.M403429200 on May 12, 2004

J. Biol. Chem., Vol. 279, Issue 31, 32603-32613, July 30, 2004
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Sialyltransferase ST8Sia-II Assembles a Subset of Polysialic Acid That Directs Hippocampal Axonal Targeting and Promotes Fear Behavior*

Kiyohiko Angata{ddagger}, Jeffrey M. Long§, Olena Bukalo¶, Wenjau Lee{ddagger}, Alexander Dityatev¶, Anthony Wynshaw-Boris§, Melitta Schachner¶, Minoru Fukuda{ddagger}||, and Jamey D. Marth**{ddagger}{ddagger}

From the **Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, and the §Departments of Pediatrics and Medicine, University of California School of Medicine, San Diego, La Jolla, California 92093, {ddagger}Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037, and Zentrum fuer Molekulare Neurobiologie, University of Hamburg, D-20246 Hamburg, Germany

Received for publication, March 29, 2004 , and in revised form, May 5, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polysialic acid (PSA) is a post-translational protein modification that is widely expressed among neural cell types during development. Found predominantly on the neural cell adhesion molecule (NCAM), PSA becomes restricted to regions of neurogenesis and neuroplasticity in the adult. In the mammalian genome, two polysialyltransferases termed ST8Sia-II and ST8Sia-IV have been hypothesized to be responsible for the production of PSA in vivo. Approaches to discover PSA function have involved the application of endoneuraminidase-N to remove PSA and genetic manipulations in the mouse to deplete either NCAM or ST8Sia-IV. Here we report the production and characterization of mice deficient in the ST8Sia-II polysialyltransferase. We observed alterations in brain PSA expression unlike those observed in mice lacking ST8Sia-IV. This included a PSA deficit in regions of neurogenesis but without changes in the frequency of mitotic neural progenitor cells. In further contrast with ST8Sia-IV deficiency, loss of ST8Sia-II did not impair hippocampal synaptic plasticity but instead resulted in the misguidance of infrapyramidal mossy fibers and the formation of ectopic synapses in the hippocampus. Consistent with studies of animal models bearing these morphological changes, ST8Sia-II-deficient mice exhibited higher exploratory drive and reduced behavioral responses to Pavlovian fear conditioning. PSA produced by the ST8Sia-II polysialyltransferase modifies memory and behavior processes that are distinct from the neural roles reported for ST8Sia-IV. This genetic partitioning of PSA formation engenders discrete neurological processes and reveals that this post-translational modification forms the predominant basis for the multiple functions attributed to the NCAM glycoprotein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polysialic acid (PSA)1 is a post-translational modification consisting of a homopolymer of {alpha}2-8-linked sialic acids that participates in neural development (1-3). Typically attached to N-glycans, PSA is produced in the Golgi apparatus and is found among a limited number of glycoproteins and predominantly on the neural cell adhesion molecule (NCAM). Enzymatic removal of PSA by using endoneuraminidase (endo-N) induces various neurological abnormalities including deficits in hippocampal long term potentiation (LTP) and long term depression (LTD), spatial learning, cell migration, and axonal targeting as observed in NCAM-deficient mice (4-13). PSA may alter the adhesive property of NCAM in mediating these processes and may also influence cell-cell communication involving integrins, cadherins, and members of the immunoglobulin superfamily (14, 15). Expression of PSA is high in embryonic brain and generally reduced in the adult. However, PSA is continuously present in some adult regions such as the olfactory bulb, hippocampus, and hypothalamus, coincident to where neurogenesis and neuronal plasticity persist (16, 17).

Two genes encoding polysialyltransferases ST8Sia-II (STX) and ST8Sia-IV (PST) are independently capable of directing PSA synthesis in vitro (18-22). Although ST8Sia-II and -IV share 59% amino acid identity, they are expressed in different spatial and temporal patterns among neural tissues (23-28). To understand how the mammalian neurological system may be modulated in vivo by altered PSA formation, the genetic bases of PSA formation in vivo must be defined. To date this has been explored by analyzing mice lacking the ST8Sia-IV polysialyltransferase. NCAM expression was unaltered by ST8Sia-IV deficiency, whereas specific brain regions exhibited decreased PSA levels, and adult animals bore a restricted phenotype involving an impairment of LTD and LTP in the hippocampal CA1 region (29). However, unlike NCAM deficiency, no decrease in CA3 LTP was observed, and hippocampal mossy fiber projections were unaltered. Here we report the generation and characterization of ST8Sia-II-deficient mice to investigate the role of this polysialyltransferase in neurological activity and define the biological mechanisms that modulate PSA formation and NCAM function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
ST8Sia-II Mutagenesis—The murine ST8Sia-II gene was isolated from a mouse129/SvJ genomic DNA library and used to construct a targeting vector. An EcoRI-XhoI fragment containing exon 4 was flanked by two loxP sites as described (30). Targeted embryonic stem (ES) clones were identified by PCR and characterized by genomic Southern blotting. ES clones bearing the deleted ({Delta}) ST8Sia-II allele were obtained after transient expression of Cre recombinase. Targeted ES cells were injected into C57BL/6 blastocysts to generate chimeric mice. Mutant ST8Sia-II alleles were bred into the C57BL/6 mouse strain for more than six generations prior to phenotype analysis.

RNA Analyses—Total RNA was purified using Trizol solution (Invitrogen) and analyzed by Northern and RT-PCR as described (26). The oligonucleotide primers (10 µM) used are as follows: mX-Tg1, 5'-CTGGAGGCAGAGGTACAATCAGATC-3' (nucleotides 104-128); mX-Tg2, 5'-CCTCAAAGGCCCGCTGGATGACAGA-3' (nucleotides 646-622); mP-Tg1, 5'-AGGCTGGCTCCACCATCTTCCAACA-3' (nucleotides 173-197); and mP-Tg2, 5'-CTCTGTCACTCTCATTCCGAAAGCC-3' (nucleotides 625-601).

Western Blot Analyses—Brain tissues were isolated and homogenized with RIPA buffer (150 mM NaCl; 50 mM Tris-HCl, pH 7.4; 1% Nonidet P-40; 0.1% SDS; 5 mM EDTA) containing proteinase inhibitor (Roche Applied Science). Protein was analyzed by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore). Some tissue extracts were incubated with endo-N (31) for 1 h at 37 °C prior to analysis. Membranes were blocked with 10% dry milk powder in 20 mM Tris-buffered saline, pH 7.6, containing 0.1% Tween 20 (TBST), and incubated with either mouse anti-PSA 5A5 antibody (Developmental Studies Hybridoma Bank, diluted 1:1000 in TBST) or rat anti-NCAM H28 antibody (Immunotech, diluted 1:200), followed by peroxidase-conjugated anti-mouse IgM (1:4000) or anti-rat IgG (1:3000), and detected by ECL (Amersham Biosciences).

BrdUrd Labeling—BrdUrd (20 mg/ml in 0.007 N NaOH and 0.9% NaCl) was delivered by intraperitoneal injection into ST8Sia-II-deficient and wild-type mice (50 mg/kg body weight) five times at 2-h intervals over an 8-h period. One week after the last administration, the mice were deeply anesthetized with Avertin (0.015 ml/g body weight), and brains were removed, fixed with Carnoy (60% ethanol, 30% chloroform, 10% acetic acid), and embedded in paraffin to cut sagittal or coronal sections at 10 µm. Every third section was collected, and 10 sections, which cover 300 µm in the center of the hippocampus, were stained for BrdUrd. BrdUrd-positive cells were detected by BrdUrd-specific monoclonal antibody (Roche Applied Science) and Alexa Fluor® 488 goat anti-mouse IgG1 (Molecular Probes). To measure distribution of embryonic neural stem cells in brains, BrdUrd was administered into pregnant heterozygous mice (100 mg/kg at embryonic day 16, E16) that were mated with heterozygous male mice. Brains from neonatal mice and 10-day postnatal mice were fixed and stained as described above.

Histology—For Timm's staining, mice were perfused intracardially with 15-50 ml of 0.37% sodium sulfide solution followed by 4% paraformaldehyde in phosphate-buffered saline. Every third section covering 300 µm in the center of the hippocampus was analyzed as described (9). Immunohistochemical or immunofluorescence staining was performed as described (10, 26, 29). To stain sections with antibodies for {beta}-tubulin (Babco), synapsin I (Chemicon), glial fibrillary acidic protein, or MAP2 (Roche Applied Science), brains were fixed with Carnoy; to stain sections with anti-PSA 12F8 (BD Biosciences), anti-NCAM H28, and anti-calbindin D-28K (Chemicon), brains were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4.

Electrophysiology—Hippocampal slices from 5- to 6-month-old ST8Sia-II-deficient mice and their wild-type littermates were used for recordings. After halothane anesthesia, decapitation, and removal of the brain, the hippocampi were cut with a VT 1000 M vibratome (Leica, Nussloch, Germany) in 300-µm-thick slices in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 250 sucrose, 25 NaHCO3, 25 glucose, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, and 1.5 MgCl2, pH 7.3. The slices were then kept at room temperature in a chamber filled with carbogen-bubbled ACSF, containing 125 mM NaCl instead of 250 mM sucrose, for at least 2 h before the start of recordings (modified from Ref. 32). In the recording chamber, slices were continuously superfused with carbogen-bubbled ACSF (2-3 ml/min) at room temperature.

For recordings in the CA3 region, the slices were prepared as for recordings in the CA1 region but with some modifications. Before decapitation, mice were transcardially perfused with ice-cold ACSF, containing (in mM) 250 sucrose, 25 NaHCO3, 25 glucose, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, and 6 MgCl2, pH 7.3. Slices were cut according to Claiborne et al. (33). Exchange of sucrose-containing ACSF with normal ACSF (containing 2.5 mM CaCl2 and 1.5 mM MgCl2) was performed gradually using peristaltic pumps.

Schaffer collateral-CA1 extracellular recordings of focal fEPSPs were obtained from the stratum radiatum of the CA1 region in response to stimulation of Schaffer collaterals by an electrode placed ~400 µm apart from the recording electrode in the stratum radiatum of the CA1 region. Recordings and stimulations were performed with glass pipettes filled with ACSF having a resistance of 2 megohms. Basal synaptic transmission was monitored at 0.033 Hz. The slices were maintained at room temperature.

Homosynaptic LTP in the CA1 region was induced by {theta}-burst stimulation (TBS) applied orthodromically to Schaffer collaterals and recorded extracellularly in the stratum radiatum. A TBS consisted of 10 bursts delivered at 5 Hz. Each burst consisted of four pulses delivered at 100 Hz. Duration of pulses was 0.2 ms, and five TBSs were applied every 20 s to induce LTP (29). The stimulation strength was in the range of 40-70 µA to provide fEPSPs with an amplitude of 50% of the subthreshold maximum. The mean slope of fEPSPs recorded 0-10 min before TBS was taken as 100%. The transient potentiation immediately following TBS (or STP, short term potentiation) was measured as a maximal increase in the fEPSP slope during 1 min after LTP induction. The values of LTP were calculated as increase in the mean slopes of fEPSPs measured 50-60 min after TBS.

Mossy fiber-CA3 extracellular recordings and stimulations were both performed with glass pipettes filled with ACSF and having a resistance of 2 megohms with stimulation strength of ~40 µA. The stimulating electrode was placed close to the internal side of the granule cell layer. The recording pipette was placed in the stratum lucidum of the CA3 region. The mossy fiber responses selected for recording were of 40-60 µV, with a fast rise time and decay of fEPSPs (total duration of fEPSP <10 ms, rise time <3.5 ms), large paired pulse facilitation (>170%), and prominent frequency facilitation (>200%). The selected responses had no hallmarks of polysynaptic activation, such as jagged decay phase with multiple peaks, or variable latencies of fEPSPs.

The LTP-inducing high frequency stimulation (HFS) consisted of one train of stimuli applied at 100 Hz for 1 s one time ("weak" stimulation protocol) or repeated four times with an interval of 20 s ("strong" stimulation protocol). To evoke LTP exclusively in mossy fiber synapses, which are known to undergo LTP in an NMDA receptor-independent manner, the NMDA receptor antagonist AP-5 (50 µM; Tocris, Bristol, UK) was applied 15 min before and during HFS. All recorded mossy fiber responses followed presynaptic stimulation of 100 Hz and showed no changes in the shape of responses after induction of LTP. To additionally confirm that the fEPSPs recorded were evoked by the stimulation of mossy fibers and not by the associational/commissural pathway, an agonist of type II metabotropic glutamate receptors (LCCG1, 10 µM; Tocris), which is known to reduce synaptic transmission in CA3 mossy fiber synapses (34), was applied at the end of each experiment. Slices in which responses were reduced by at least 70% were selected for analysis. Basal synaptic transmission was monitored at 0.033 Hz. The mean amplitude of fEPSPs recorded 0-10 min before HFS was taken as 100%. Post-tetanic potentiation (PTP) was calculated as the maximal increase in the amplitude of fEPSP after HFS. The values of LTP were calculated as increase in the mean amplitude of fEPSPs measured 50-60 min after HFS.

Data acquisition and measurements were performed by using an EPC-9 amplifier and Pulse software (Heka Elektronik, Lambrecht/Pfalz, Germany). Values in electrophysiological experiments are reported as mean ± S.E. Student's t test was used to assess statistical significance using Sigma Plot 5.0 software (Chicago).

Nerve Tract Tracing between the Hippocampus and Amygdala—Neuronal tracing was analyzed in eight mice of each genotype by injecting biotinylated dextran amine (BDA, Molecular Probes) into the amygdala. After the mouse was deeply anesthetized with ketamine (1.25 mg/g) and xylazine (0.07 mg/g), BDA (10% in 0.1 M phosphate buffer, pH 7.4; 0.2 µl) was stereotaxically injected into amygdala by using Picospritzer (Parker Instrumentation) and the following coordinates: Anterior, -0.12; Left, -0.27; Dorsal, -0.4 in cm from Bregma (35). The animals were allowed to recover under close observation and were returned to their cage. Six days after injection, brains were fixed as described above, and cryosections were analyzed immunohisto-chemically using fluorescein isothiocyanate-labeled Avidin (Vector Laboratories).

Metabolic and Behavioral Parameters—Two separate cohorts of 4-month-old male mice were analyzed. The first consisted of 10 wild-type and 9 ST8Sia-II-deficient littermates. These were assessed in a behavioral test battery modified from that used by McIlwain et al. (36) and as described previously (37). This included parameters such as metabolic performance, physical appearance, sensorimotor reflexes, motor activity, nociception, acoustic startle, sensorimotor gating, and assessments of learning and memory. Concern that testing mice in such a large battery could influence behavior in any individual task and that multiple assessments increased the probability of a type I statistical error, a second cohort of mice was also analyzed (wild-type, n = 16; {Delta}/{Delta}, n = 15). In the open field test, activity was measured in a 30-min test period in an area of 45 x 45 cm using a Digiscan apparatus (Accuscan Electronics, Columbus, OH). Vertical activity (rearing) and distance (total and center) were recorded.

Passive avoidance analysis involved a two-compartment light/dark apparatus (35 x 18 x 30 cm, Coulbourn Instruments, Allentown, PA). Each mouse was placed in the lighted compartment. When the animal entered the dark compartment, a guillotine door closed behind and a foot shock of 0.4 mA was delivered through the grid floor of the dark compartment for 3 s. If the mouse did not enter the dark compartment within 10 min, it was excluded from the retrieval test. In the retrieval trial performed 24 h later, the latency for the mice to enter the dark compartment was recorded. The maximum latency was 600 s.

Fear conditioning analyses used chambers (26 x 22 x 18 cm high) made of clear Plexiglas placed in a 2 x 2 array (Med Associates). A video camera was used for recording and analysis (FreezeFrame, Actimetrics, St. Evanston, IL). The conditioned stimulus (CS) was an 85-db, 2,800-Hz, 20-s tone, and the unconditioned stimulus was a scrambled foot shock at 0.75 mA presented during the last 3 s of the CS. Mice were placed in the test chamber for 3 min before recording CS and freezing behavior. Freezing was defined as the absence of movement other than breathing, and thresholds were selected via the software of high correlation with human observers. Three CS/unconditioned stimulus pairings were given with 1-min spacing, and freezing during the CS was also recorded. Each mouse was returned to the shock chamber 24 h later, and freezing responses were recorded for 3 min (context test). The chambers were modified to present a different environmental context (shape, odor, color changes), and 2 h later the mice were placed in this novel environment. Freezing behavior was recorded for 3 min before and during three CS presentations (cued conditioning). The time spent freezing was converted to a percent value.

The water maze task constituted a pretraining phase during which all mice from both cohorts were tested for 2 days in a straight-swim pretraining protocol. Mice received 16 trials (8 trials over 2 days) in a 31 x 60-cm rectangular tank that was located in a different room than the circular tank used in the hidden platform trials. The platform was located 1 cm below the water opposite from the start location. Latency to climb onto the platform was the dependent measure. Criteria for advancing to the hidden platform trials was completing 6 of 8 trials under 10 s on the 2nd day. This pretraining procedure provided experience with swimming and climbing onto a submerged platform without exposing the mice to the spatial cues used in the hidden platform trials. This procedure both screens for mice with severe motor deficits and reduces behavioral variability often seen on the 1st day of hidden platform testing. All mice successfully passed this pretraining phase. Hidden platform testing followed in which extra-maze visual cues were hung from a curtain located around a 1.26-m diameter circular tank. The water was made opaque with the addition of non-toxic paint. The 10-cm diameter escape platform was located 1 cm below the surface of the water, and a Polytrack video-tracking system (San Diego Instruments) was used to collect mouse movement data (location, distance, and latency) during training and probe trials. Each mouse was given eight trials a day, in two blocks of four trials for 4 consecutive days. After 36 trials, each animal was given a 60-s probe trial. During the probe test, the platform was removed, and quadrant search times were measured. Visual cue testing was performed 1 day after the last hidden-platform training trial, wherein mice were trained to locate a visiblecued platform. The visible cue was a gray plastic cube (9 cm) attached to a pole so that it was 10 cm above the platform. On each trial of the visible platform test, the platform was randomly located in one of the four quadrants. Mice were given eight trials, in blocks of four trials, and the latency to find the platform was recorded for each trial.

Metabolic chambers termed CLAMS (Comprehensive Lab Animal Monitoring System; Columbus Instruments, Columbus, OH) automatically recorded metabolic parameters including volume of carbon dioxide produced (VCO2), volume of oxygen consumed (VO2), respiration (respiratory exchange ratio) = VCO2/VO2, and caloric (heat) value ((3.815 + 1.232 x respiratory exchange ratio) x VO2), motion in all three axes in time, and consumption of food and water. Data were collected every 30 min over three 12-h dark cycles and two 12-h light cycles and analyzed as mean values over each 12-h period with the exception of food and water intake which were added to the total during subsequent cycles.

Pulmonary function was scored by measurement of the uptake of CO. A carbon monoxide uptake monitor (Columbus Instruments) measured the CO level in a sealed chamber after exposing the mouse to a 60-s interval of air with 0.17% CO. The mean breath per min was also recorded. Each animal was tested once.

Blood pressure was determined by a noninvasive blood pressure tail-cuff system (Columbus Instruments) that measures systolic blood pressure in addition to heart rate and relative changes in diastolic and mean blood pressure. Individual mice were placed in a small cylinder chamber; occlusion and sensor cuffs were placed on the tail, and the tail was warmed to 37 °C. Mice were first acclimated to the restraining chamber, tail cuffs, and the heat fan for 30 min for 2 days prior to testing. The mean of four measurements on the 3rd day was reported and analyzed by the Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
ST8Sia-II Inactivation in the Mouse Germ Line—The ST8Sia-II gene is highly conserved among mammals and includes multiple exons that compose the catalytic domain (21). Exon 4 encodes a significant portion of the sialyl motif L, a peptide sequence that is essential for sialyltransferase activity (38). DNA encoding exon 4 of the ST8Sia-II gene was targeted for elimination from the mouse genome (Fig. 1A). Targeted ES cells that bore the exon 4 deletion were injected into C57BL/6 blastocysts to obtain chimeric mice. The mutant allele was transmitted into the germ line and bred into the C57BL/6 strain (Fig. 1B). Mice homozygous for the deleted ST8Sia-II allele ({Delta}) were produced by heterozygous parental genotypes at close to Mendelian ratios (wt/wt:wt/{Delta}:{Delta}/{Delta} = 29:50:21%; n = 255). No differences in body weight or brain size were noted despite the fact that highest expression of ST8Sia-II occurs in wild-type embryos at early developmental stages.



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  		FIG. 1.
ST8Sia-II mutagenesis. A, strategy for mutagenesis producing the mutant type I deleted ({Delta}) allele. 129-strain BamHI polymorphism is denoted (*). Exons (black boxes) and loxP sites (triangles) are indicated. E, EcoRI; H, HindIII; X, XhoI. B, genomic DNA from tail tissue digested with BamHI and analyzed by Southern blot using the genomic probe indicated in A. C, total brain RNA isolated from wild-type (wt), heterozygous, and homozygous mutant mice was used in RT-PCR to detect ST8Sia-II and ST8Sia-IV expression. D, RT-PCR analysis was performed from wild-type and {Delta}/{Delta} mice at different ages: post-natal day 0 (P0), 1 month (1 M), 3 months (3 M), and 6 months (6 M).

 
Inactivation of ST8Sia-II because of deletion of exon 4 was investigated by RNA and enzymatic analyses. RT-PCR revealed that the truncated form of ST8Sia-II mRNA lacking exon 4 was produced in both heterozygous and the ST8Sia-II null mice (Fig. 1C). The mutant ST8Sia-II cDNA was isolated and found unable to produce polysialyltransferase activity (data not shown). RNA analyses by RT-PCR at various post-natal ages further demonstrated that the disruption of ST8Sia-II does not affect the level of ST8Sia-IV mRNA (Fig. 1D).

PSA Expression and Neurogenesis—PSA expression was reduced in the olfactory bulb and cerebral cortex of adult ST8Sia-II-deficient mice (Fig. 2). There was no quantitative alteration of PSA among the hypothalamus, hippocampus, and cerebellum, in contrast to the pattern of PSA deficiency found among mice lacking ST8Sia-IV (29). However, closer examination of the hippocampus of ST8Sia-II-deficient mice revealed a PSA deficit in the dentate gyrus. Neurogenesis takes place throughout adulthood in the dentate gyrus, and progenitor cells that migrate into the granule cell layer express high levels of PSA. There is also an association of neuronal precursor cells and PSA expression with other regions supporting neurogenesis in the adult brain, including the subventricular zone. Progenitor cells expressing PSA in the granule layer were greatly reduced and often absent in ST8Sia-II-deficient mice (Fig. 3, A and B). In the subventricular zone, there was no difference in PSA expression in the anterior region; however, PSA expression was reduced among newly generated neural cells along the posterior lateral ventricle (Fig. 3, C-H).



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  		FIG. 2.
Brain PSA and NCAM expression. Regions of 8-week-old mouse brain from wild-type (wt/wt), heterozygous (wt/{Delta}), and homozygous ({Delta}/{Delta}) littermates were dissected, and total protein extracts were subjected to Western blot analysis using anti-polysialic acid (PSA) antibody 5A5 and anti-NCAM antibody H28. A portion of the sample was digested with endo-N before the analysis. OB, olfactory bulb; HY, hypothalamus; HP, hippocampus; CX, cerebral cortex; CB, cerebellum.

 



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  		FIG. 3.
Polysialic acid in the dentate gyrus and subventricular zone of wild-type (WT) and ST8Sia-II-deficient ({Delta}/{Delta}) mice. Cryosections of hippocampus from wild-type mice (WT) (A) and ST8Sia-II-deficient littermates (B) stained with anti-PSA antibody, 12F8 (red). PSA-positive neural precursor cells (arrowheads) are not evident in the dentate gyrus of ST8Sia-II {Delta}/{Delta} mice. C-H, PSA expression (red) in the subventricular zone of WT (C-E) and {Delta}/{Delta} (F-H) brain tissue. Cell nuclei (blue) are shown by Hoechst staining (C and F). D, E, G, and H are magnifications of boxed regions in panels C and F, as indicated. Migrating cells of both genotypes are PSA-positive in the anterior region of the subventricular zone (arrowheads in D and G). However, fewer cells expressing high levels of PSA appear in {Delta}/{Delta} genotypes near the anterior region of the subventricular zone compared with wild type (WT) samples. ST8Sia-II deficiency also results in neural cells with diminished PSA expression in the midposterior part of the subventricular zone (open arrowheads in H). To label mitotic cells in embryonic and adult brains, BrdUrd was injected into a pregnant mouse at embryonic day 16 (I and L) and into adult mice at 2-3 months post-natal age (J, K, M, and N), respectively. BrdUrd-labeled cells were visualized by anti-BrdUrd antibody (green), and nuclei were stained with Hoechst dye (blue). The number of BrdUrd-positive cells in the adult dentate gyrus of ST8Sia-II-deficient mice was not significantly different from that of wild-type mice (wt/wt, 10.9 ± 2.9, n = 4; {Delta}/{Delta}, 8.7 ± 2.0, n = 4; p > 0.05). CC, corpus callosum; DG, dentate gyrus; Gr, granule cell layer; Hl, hilus; LV, lateral ventricle; St, striatum.

 
Deficiency of PSA formation in the context of neurogenesis was further examined by BrdUrd labeling of neural precursors. At embryonic day 16, these cells are distributed in the hippocampus as well as the cortex. BrdUrd-labeled cell numbers and patterns in the dentate gyrus and the pyramidal cell layer were not distinguishable between wild-type and ST8Sia-II-deficient mice. The number of BrdUrd-positive cells in the adult dentate gyrus of ST8Sia-II-deficient mice was not significantly different from that of wild-type mice (wt/wt, 10.9 ± 2.9, n = 4; {Delta}/{Delta}, 8.7 ± 2.0, n = 4; p > 0.05). These results indicate that ST8Sia-II depletion does not affect the frequency of mitotic neuronal precursor cells undergoing DNA replication (Fig. 3, I-N).

Axonal Targeting and Ectopic Synapse Formation—Histological examination of brain structure indicated normal morphology of the olfactory bulb and rostral migratory stream among ST8Sia-II-deficient mice. However, hippocampal infrapyramidal mossy fiber morphology was invariably altered. Hippocampal mossy fibers arise from granule cells in the dentate gyrus and target to the hippocampal CA3 region, fasciculating to become a thick suprapyramidal fiber projection that forms synapses in the stratum lucidum of CA3. In addition, sprouting fibers from the dentate gyrus normally form a much thinner infrapyramidal mossy fiber projection along the border between the pyramidal cell layer and stratum oriens. The infrapyramidal mossy fiber projection is normally shorter than the suprapyramidal mossy fiber projection and terminates or merges with the suprapyramidal projection to synapse at CA3c (9, 10, 39). Among ST8Sia-II-deficient mice, however, the infrapyramidal mossy fibers extended to the far CA3a region at all post-natal ages from 2 weeks through adult life (Fig. 4, A-F). Timm's staining further revealed many fine mossy fibers in ST8Sia-II deficiency invading pyramidal cell layers in the CA3b and CA3c region, forming a web-like structure between these two mossy fiber projections. The calcium-binding protein calbindin is a marker of functional granule cells in the dentate gyrus, as well as interneurons and mossy fibers in the hippocampus (40). Both calbindin and NCAM expression appear among the mistargeted infrapyramidal mossy fibers in ST8Sia-II-deficient mice consistent with the interpretation that these axons retain function (Fig. 4, G-J).



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  		FIG. 4.
Hippocampal mossy fiber topology. Timm's staining (A-F) on brain tissue from denoted genotypes at post-natal ages: 2 weeks (A and B), 3 months (C and D), and 6 months (E and F). The infrapyramidal mossy fiber projection in ST8Sia-II-deficient mice is larger than in wild-type littermates and extends into the CA3a region (arrows). Hippocampal sections from wild-type and {Delta}/{Delta} genotypes (3 months old) were also stained with anti-calbindin (G and H) and anti-NCAM (I and J) antibodies. Arrows in H and J denote NCAM and calbindin expression on mistargeted infrapyramidal mossy fibers of ST8Sia-II-deficient mice. Mistargeted mossy fibers along the ventral side of granule cell layer are indicated by arrowheads in F, H, and J. DG, dentate gyrus.

 
Suprapyramidal mossy fibers continued to express PSA in ST8Sia-II-deficient mice. Moreover, PSA expression was present on infrapyramidal mossy fibers throughout their projection to CA3a (Fig. 5A). The mistargeting of these PSA-bearing infrapyramidal mossy fibers was associated with the presence of ectopic synapses in CA3a where the axons terminated (Fig. 5B, arrows in right middle panel). This is consistent with the finding that endo-N treatment increases transient synapse-like junctions between mossy fibers and pyramidal cells (10). By contrast, such synapses are absent in the CA3a of wild-type mice whose infrapyramidal mossy fibers do not project into this hippocampal region (Fig. 5B, arrowheads in left middle panel). Mossy fibers extending along the ventral side of the granule cell layer were also observed in association with ectopic synapses in ST8Sia-II-deficient mice (Fig. 5B, lower panels). These ectopic synapses are also probably functional because calbindin, NCAM, and synapsin-I are co-expressed among those fibers (arrowheads in Fig. 4, F, H, and J, and Fig. 5B, lower panels). Mistargeting of infrapyramidal mossy fiber axons in ST8Sia-II deficiency was not due to a deficiency per se in PSA expression on mossy fibers (Fig. 5A) or changes in the abundance or spatial pattern of granule and pyramidal cells in the CA3 region (data not shown).



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  		FIG. 5.
Axonal mistargeting of PSA expression mossy fibers with ectopic synapse formation in ST8Sia-II-deficient mice. A, PSA expression (antibody 12F8, brown color) is maintained on mossy fibers of ST8Sia-II-deficient mice. However, the PSA-positive infrapyramidal mossy fiber tract projects to the CA3a region (arrowheads, right panel). Open arrowheads indicate PSA expression among pyramidal cells at stratum oriens of CA3a and CA3b in wild-type mice (left panel). B, anti-synapsin-I antibodies detected the presence of ectopic synapses associated with mistargeted infrapyramidal mossy fibers in ST8Sia-II-deficient mice (arrows, right panels). Middle panels are higher magnification views of CA3a boxed regions. Ectopic synapses also appear in the CA3c region of ST8Sia-II-deficient mice (lower panels), unlike findings among wild-type littermates (arrowheads, left panels).

 
Hippocampal Synaptic Plasticity—The most widely studied form of plasticity, LTP in the CA1 region of the hippocampus, has been reported to be abnormal in NCAM and ST8Sia-IV deficient mice (6, 29). Moreover, removal of PSA from hippocampal cultures of wild-type mice by endo-N prevented both LTP and LTD (6, 7). In our studies, TBS of Schaffer collaterals reliably produced STP and LTP in all slices measured from wild-type animals (Fig. 6A). The mean level of STP measured as maximal potentiation during 1 min after TBS was 216.0 ± 10.7%, and the level of LTP seen 50-60 min after TBS was 129.3 ± 4.3% (number of slices n = 8, number of mice n = 3). The levels of STP and LTP in ST8Sia-II-deficient mice (218.2 ± 7.6 and 127.9 ± 5.3%, respectively; n = 8, n = 3) were not significantly different from wild-type mice. Stimulus-response curves for fEPSPs evoked by stimulation of Schaffer collaterals and the mean amplitudes of responses being 50% of the supra-maximal levels were not different between ST8Sia-II-deficient mice and wild-type littermates, thus demonstrating normal basal levels of excitatory transmission (data not shown).



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  		FIG. 6.
Short and long term potentiation in the hippocampal CA1 and CA3 regions. A, TBS of Schaffer collaterals reliably produced STP and LTP in all slices measured from ST8Sia-II wild-type (WT) and {Delta}/{Delta} littermates. TBS of Schaffer collaterals (marked by arrow) evoked a strong increase in the slopes of fEPSPs recorded in the CA1 region in both genotypes. Traces on the top show averaged fEPSPs recorded before and 50-60 min after induction of LTP. Scale bars, 10 ms and 500 µV. The number of slices (n) was 8, and the number of mice (N) was 3. B, single train of high frequency stimulation of mossy fibers (1x HFS, marked by arrow) evoked a similar increase in amplitude of fEPSPs in the CA3 region. Traces on top show averaged fEPSPs recorded before and 50-60 min after induction of LTP. Scale bars, 10 ms and 50 µV. Data shown are means ± S.E. (wt, n = 6, n = 5; ST8Sia-II-deficient, n = 5, n = 3). Recorded profiles of CA3 LTP resembled those observed in our previous studies (29, 43) and by other groups (44, 45). The values measured 50-60 min after induction of LTP in wild-type and ST8Sia-II-deficient mice were 219.2 ± 24.1% (n = 7, n = 5) and 257.2 ± 32.7% (n = 6, n = 4), respectively. Post-tetanic potentiation induced by a stronger induction protocol (4x HFS) was 1239.0 ± 84.3% (n = 7, n = 5) in wild-type and 1333.5 ± 192.5% (n = 6, n = 4) in ST8Sia-II-deficient mice (not shown).

 
LTP at mossy fiber synapses with CA3 pyramidal cell has been reported to be abnormal in NCAM-deficient mice (11). These synapses are important for hippocampal learning and memory formation (41, 42). CA3 LTP has features clearly distinct from CA1 LTP, being independent of postsynaptic NMDA receptors but mediated by an elevation in cAMP and activation of adenylate cyclase and cAMP-dependent protein kinase (43). fEPSPs evoked in CA3 pyramidal cells by mossy fiber stimulation were identified using a number of criteria (see "Experimental Procedures") and showed properties typical for mossy fiber responses as follows: low frequency stimulation (0.33 Hz) potentiated fEPSPs to ~250% in wild-type and ST8Sia-II-deficient mice. Application of L-CCG1 reduced the amplitude of fEPSPs similarly in both genotypes by 80%. The NMDA receptor antagonist AP-5 did not affect the amplitude of recorded fEPSPs in either wild-type or ST8Sia-II-deficient samples.

A single train of high frequency stimulation (1x HFS, applied in the presence of AP-5) induced robust PTP and LTP in slices from wild-type mice (1133.1 ± 180.4 and 195.8 ± 33.3%, respectively; n = 6, n = 5). The levels of PTP and LTP in ST8Sia-II-deficient mice were 912.3 ± 135.8 and 181.6 ± 13.1%, respectively (n = 5, n = 3) (Fig. 6B). This was not significantly different from values among wild-type littermates. Recorded profiles of CA3 LTP resembled those observed in our previous studies (29, 44) and by other groups (45, 46). PTP induced by a stronger induction protocol (4x HFS) was 1239.0 ± 84.3% (n = 7, n = 5) in wild-type and 1333.5 ± 192.5% (n = 6, n = 4) in ST8Sia-II-deficient mice. The values measured 50-60 min after induction of LTP in wild-type and ST8Sia-II-deficient mice were 219.2 ± 24.1% (n = 7, n = 5) and 257.2 ± 32.7% (n = 6, n = 4), respectively (data not shown). Thus, NMDA receptor-independent LTP in mossy fiber-CA3 synapses was not affected by ST8Sia-II deficiency.

Metabolic and Behavioral Profiles of ST8Sia-II Deficiency—ST8Sia-II-deficient mice were unremarkable in physical, metabolic, and sensory assessments of muscle strength, heart rate, blood pressure, pulmonary function, nociception, and respiration (Table I). In accordance with electrophysiological measurements that indicated normal LTP in hippocampal regions CA1 and CA3, ST8Sia-II-deficient mice were not impaired in spatial learning using the water maze test (Fig. 7A). However, there were consistent and significant differences in exploratory activity, passive avoidance, and fear-conditioning tasks.


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  		TABLE I
Assessments of appearance, locomotor, muscular function, nociception, and metabolic parameters

Male mice were studied, including littermates between 12 and 16 weeks of age. These results were derived from the analyses of 10 wild-type and 9 ST8Sia-II deficient mice as described under "Experimental Procedures."

 



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  		FIG. 7.
Cognitive and metabolic findings among mice lacking ST8Sia-II. A, ST8Sia-II-deficient mice were not impaired in the water maze relative to wild-type littermates. A repeated measures ANOVA for the acquisition trials revealed no significant effect of genotype (F(1, 37) = .99, p > 0.05). Both groups improved their performance over trials as exhibited by a significant effect of block of trials (F(7, 259) = 39, p < 0.001). In addition there was a significant genotype x block interaction (F(7, 259) = 2.3, p < 0.001). A subsequent test of significant main effects revealed that mutant mice took significantly more distance to find the platform on the third block of trials than the control mice (F(1, 37) = 10.5, p < 0.005). The third block was the only different data point, and overall there was no difference in acquisition trials. In addition, there was no significant difference in the percent distance spent in the quadrant that previously contained the platform (probe trial, t(37) = 0.22, p > 0.05). B, automated analyses with metabolic cages and repeated ANOVA measures of the data revealed normal motor activity, food and water consumption, oxygen consumption, carbon dioxide production, respiration, and caloric (heat) values (wt/wt, white circles; {Delta}/{Delta}, black circles). No evidence of basal hyperactivity is observed. C, ST8Sia-II-deficient mice were not different from wild-type littermates in either sensorimotor gating, as assessed by startle response to acoustic stimuli or prepulse inhibition (PPI). A repeated measure ANOVA for Startle response revealed no significant effect of genotype (F(1, 37) = .4, p > 0.05), or a genotype x block interaction (F(12, 444) = .12, p > 0.05). Startle response did significantly increase in both groups as a function of increasing startle decibel levels (F(12, 444) = 0.12, p < .001). A repeated measure ANOVA for PPI revealed no significant effect of genotype (F(1, 17) = .02, p > 0.05) or a genotype x block interaction (F(4, 68) = 0.48, p > 0.05). PPI did significantly increase in both groups as a function of increasing prepulse decibel level (F(4, 68) = 10.9, p < 0.001).

 
The open field test measures exploration and anxiety behaviors in a novel environment. ST8Sia-II-deficient mice traveled significantly more total distance in the open field and more distance in the center of the arena (Table II). Normalizing the distance traveled in the center to the total distance traveled revealed a trend of increased proportional time in the typically less preferred center area. Another indicator of exploratory behavior in the open field, termed rearing activity, was significantly increased among ST8Sia-II-deficient mice.


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  		TABLE II
Behavioral alterations resulting from ST8Sia-II deficiency

All mice analyzed included littermates between the ages of 12 and 16 weeks. Two separate cohorts totaling 24-26 mice of each genotype were analyzed as described under "Experimental Procedures."

 
In the passive avoidance task, the ST8Sia-II-deficient mice were also impaired, failing to increase their latency to enter the dark chamber (Table II). Performance in passive avoidance and fear-conditioning tasks requires that the animals be able to control their movement. If ST8Sia-II-deficient mice were inherently hyperactive, they would perform poorly in these tests without having impairments in fear memory. We therefore quantitatively analyzed motor activity and physiologic parameters over multiple light and dark cycles in a habituated environment. ST8Sia-II-deficient mice continued to exhibit circadian rhythm and yielded normal measurements of motor activity, carbon dioxide production, caloric heat value, and consumption of food and oxygen (Fig. 7B).

Behavioral responses to standard fear-conditioning tasks were also abnormal in ST8Sia-II-deficient mice (Table II). The association of a single cue (i.e. a tone) with an unpleasant electrical shock is considered unimodal and critically depends upon neural processing in the amygdala but not the hippocampus. In contrast, associating context (spatial and environmental cues making up the test chamber) with the unpleasant stimulus is considered multimodal and requires neural processing in both amygdala and hippocampus (47). ST8Sia-II-deficient mice were significantly impaired in both the cued and contextual versions of the fear-conditioning test. As well as normal nociception, ST8Sia-II-deficient mice did not score poorly in the acoustic startle test (Fig. 7C), indicating that the deficit in fear conditioning to cue was not due to an inability to experience discomfort or to hear the tone.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The ST8Sia-II polysialyltransferase contributes uniquely to PSA formation in vivo and thereby participates in distinct neurological processes that modulate hippocampal axonal targeting, exploratory behavior, and responses to fear conditioning. PSA expression in some regions of neurogenesis was found to require ST8Sia-II; nevertheless, mitotic neuronal precursor cell frequencies were unaffected. Instead, ST8Sia-II deficiency resulted in an alteration of axonal targeting involving hippocampal infrapyramidal mossy fibers in association with ectopic synapses where these fibers abnormally terminate at CA3a. These results indicate different contributions by ST8Sia-II and ST8Sia-IV in PSA formation and control over discrete neurological functions that can now be attributed to these two polysialyltransferases.

ST8Sia-II and PSA Involvement in Infrapyramidal Axonal Targeting and Fear Behaviors—Alterations of infrapyramidal mossy fiber targeting have been associated with anxiety, exploration, and fear behaviors (42, 48). In the open field test, inbred mouse strains with larger intra- and infrapyramidal mossy fiber fascicles habituate faster to a novel environment and have reduced fear behavior (49). Additionally, mice engineered bearing a defective Dcx gene, a cause of classical type 1 lissencephaly in human, have a similar defect in mossy fiber projection and exhibit reduced freezing to conditioned fear assays in both cue- and context-associated tasks (37). These behavioral changes are similar in CHL1 mutant mice that also have larger intra- and infrapyramidal mossy fiber projections (50). Moreover, the DA rat strain exhibits significantly larger infrapyramidal mossy fibers with reduced freezing and high rearing activities, whereas BDE rats with shorter infrapyramidal mossy fibers show freezing and low rearing activity (51).

The amygdala is also involved in fear behavior and normally expresses PSA (52-54). We detected normal PSA expression in the amygdala, hypothalamus, and piriform cortex of adult ST8Sia-II-deficient mice, and retrograde labeling with BDA injected into the amygdala demonstrated a normal trace pattern in the hippocampus (data not shown). Fear associating with a single cue (a tone) paired with electrical shock requires the amygdala but not the hippocampus. However, fear associating the environment (the context) with shock requires both the amygdala and dorsal hippocampus (52, 53, 55). The passive avoidance task requires multiple neurotransmitter systems throughout the brain including the amygdala and hippocampus (56). ST8Sia-II deficiency alters fear responses normally processed by both the hippocampus and amygdala.

Semaphorins and neuropilins have been implicated in axon guidance and branch elimination similar to the netrin-DCC, slit-robo, and ephrin-Eph interactions involving other processes in the hippocampus (57, 58). Recently, the receptors for the semaphorin 3-neuropilin-plexin complex were shown to mediate mossy fiber pruning (59, 60). It is not known whether PSA is relevant to semaphorin signaling. Neither polysialylation of the semaphorin-neuropilin-plexin complex nor a PSANCAM interaction with this complex has been reported.

The Genetic Bases of PSA and NCAM Function—With the characterization of mice deficient in ST8Sia-II, ST8Sia-IV, or NCAM, we can now assign the differential contributions of each to PSA expression and neurological functions (Table III). ST8Sia-II and ST8Sia-IV involvement in PSA formation is complementary in some brain regions. However, migrating cells in the rostral migratory stream express PSA in the absence of either ST8Sia-II or ST8Sia-IV suggesting that in some contexts both polysialyltransferases provide overlapping functions. Among studied forms of hippocampal synaptic plasticity, only LTP and LTD in the hippocampal CA1 region are dependent upon PSA, and specifically PSA produced by ST8Sia-IV. CA3 LTP remains a function of the NCAM protein backbone independent of PSA expression.


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  		TABLE III
Comparison of polysialic acid and NCAM function in neural development and synaptic plasticity

Comparison includes this work and is revised from Ref. 29. - indicates impairment of function; + indicates normal function.

 
NCAM deficiency leads to multiple neurological abnormalities (4-11). We find that the majority of phenotypes observed are present in mice with either ST8Sia-II or ST8Sia-IV deficiencies. For example, NCAM180-deficient mice exhibited a similar alteration in infrapyramidal mossy fiber axonal targeting (10, 61). This was not observed among ST8Sia-IV-deficient mice (29) indicating that correct targeting of infrapyramidal mossy fiber axons involves a function of PSA that is dependent upon both NCAM and ST8Sia-II. NCAM deficiency also impaired LTP in Schaffer collaterals of the CA1 region, which indicates a PSA-dependent function involving ST8Sia-IV (6, 29). However, NCAM-deficient mice, as well as mice treated with endo-N to remove PSA, have a thicker rostral migratory stream and a smaller olfactory bulb, perhaps due to slower tangential migration of precursor cells (8), whereas mice lacking either ST8Sia-II or ST8Sia-IV have a normal rostral migratory stream and olfactory bulb. This suggests functional overlap involving the two known polysialyltransferses. Alternatively, an unidentified polysialyltransferase may operate among these cells. Deriving and studying mice deficient in both ST8Sia-II and ST8Sia-IV will address this possibility.

Although our understanding of PSA formation and function in vivo is enhanced, the molecular basis for the phenotypes observed in PSA deficiency states remains obscure. Our studies of ST8Sia-II deficiency cannot discount the possibility of qualitative modifications in PSA structure. A change in size of this polymer might play a modulating role in cellular recognition among mossy fibers and CA3 pyramidal cells. We also cannot eliminate the possibility that PSA on CA3 pyramidal cells normally prompts infrapyramidal mossy fibers to fuse with suprapyramidal mossy fibers at CA3c, before reaching CA3b in the wild-type hippocampus. In fact, ST8Sia-II transcripts are heavily expressed in this region (62). A reduced level of PSA on pyramidal cells in ST8Sia-II-deficient mice may thereby evoke the mistargeting of infrapyramidal mossy fibers to CA3b and CA3a.

Neural plasticity mediated by PSA may also be influenced by the strength of axon-axon and axon-environment interactions in adult life (63). In the adult hippocampus, chronic stress as well as treatment with glucocorticoid stress hormones alters hippocampal PSA and NCAM expression with associated changes in neural plasticity involving mossy fiber terminals and CA3 pyramidal cells (64-66). The ST8Sia-II polysialyltransferase contributes to exploratory and fear behaviors that are influenced by stress, in contrast to the neurological processes mediated by ST8Sia-IV that participate in cognitive performance. Together these findings reveal that PSA is a post-translational modification formed by multiple gene products that engender discrete functions in vivo and represent the molecular basis for the majority of the neurological roles attributed to the NCAM glycoprotein.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants DK48247, PO1-HL57345 (to J. D. M.), and CA33895 (to M. F). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence may be addressed. E-mail: minoru{at}burnham-inst.org.

{ddagger}{ddagger} Supported as an Investigator of the Howard Hughes Medical Institute. To whom correspondence may be addressed. E-mail: jmarth{at}ucsd.edu.

1 The abbreviations used are: PSA, polysialic acid; NCAM, the neural cell adhesion molecule; endo-N, endoneuraminidase; LTP, long term potentiation; LTD, long term depression; ES, embryonic stem; RT, reverse transcriptase; BrdUrd, 5-bromo-2'-deoxyuridine; ACSF, artificial cerebrospinal fluid; fEPSPs, field excitatory postsynaptic potentials; TBS, {theta}-burst stimulation; STP, short term potentiation; HFS, high frequency stimulation; PTP, post-tetanic potentiation; CS, conditioned stimulus; ANOVA, analysis of variance; NMDA, N-methyl-D-aspartic acid; BDA, biotinylated dextran amine; wt, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Robert M. Campbell for derivation of mice bearing the ST8Sia-II mutation and Drs. Keith Murai and Barbara Ranscht for helpful discussions. We acknowledge the Consortium for Functional Glycomics (recipient of National Institutes of Health Grant GM62116) for access to metabolic and behavioral testing equipment.



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