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Originally published In Press as doi:10.1074/jbc.M708463200 on November 28, 2007

J. Biol. Chem., Vol. 283, Issue 3, 1463-1471, January 18, 2008
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Impact of the Polysialyltransferases ST8SiaII and ST8SiaIV on Polysialic Acid Synthesis during Postnatal Mouse Brain Development*Formula

Imke Oltmann-Norden{ddagger}, Sebastian P. Galuska§, Herbert Hildebrandt{ddagger}, Rudolf Geyer§, Rita Gerardy-Schahn{ddagger}, Hildegard Geyer§, and Martina Mühlenhoff{ddagger}1

From the {ddagger}Institute of Cellular Chemistry, Medical School Hannover, D-30625 Hannover, Germany and the §Institute of Biochemistry, Medical Faculty, University of Giessen, D-35392 Giessen, Germany

Received for publication, October 11, 2007 , and in revised form, November 28, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polysialic acid (polySia), a post-translational modification of the neural cell adhesion molecule (NCAM), is the key regulator of NCAM-mediated functions and crucial for normal brain development, postnatal growth, and survival. Two polysialyltransferases, ST8SiaII and ST8SiaIV, mediate polySia biosynthesis. To dissect the impact of each enzyme during postnatal brain development, we monitored the developmental changes in NCAM polysialylation in wild-type, ST8SiaII-, and ST8SiaIV-deficient mice using whole brain lysates obtained at 10 time points from postnatal days 1 to 21 and from adult mice. In wild-type and ST8SiaIV-null brain, polySia biosynthesis kept pace with the rapid increase in brain weight until day 9, and nearly all NCAM was polysialylated. Thereafter, polySia dropped by ~70% within 1 week, accompanied by the first occurrence of polySia-free NCAM-140 and NCAM-180. In ST8SiaII-null brain, polySia declined immediately after birth, leading to 60% less polySia at day 9 combined with the untimely appearance of polySia-free NCAM. Polysialyltransferase deficiency did not alter NCAM expression level or isoform pattern. In all three genotypes, NCAM-140 and NCAM-180 were expressed at constant levels from days 1 to 21 and provided the major polySia acceptors. By contrast, NCAM-120 first appeared at day 5, followed by a strong up-regulation inverse to the decrease in polySia. Together, we provide a comprehensive quantitative analysis of the developmental changes in polySia level, NCAM polysialylation status, and polysialyltransferase transcript levels and show that the predominant role of ST8SiaII during postnatal brain development is restricted to the first 15 days.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polysialic acid (polySia)2 is a unique post-translational modification primarily of the neural cell adhesion molecule NCAM (1–3). Composed of {alpha}2,8-linked N-acetylneuraminic acid, polySia forms a large negatively charged and highly hydrated glycan structure that can extend beyond the protein core. Attachment of polySia to NCAM doubles the hydrodynamic radius of the extracellular part of NCAM, thereby increasing the intermembrane space and disrupting the adhesive properties of NCAM and other cell adhesion molecules (4–6). Removal of polySia by treatment with endoneuraminidase, a bacteriophage-derived enzyme that specifically cleaves polySia (7), demonstrated intervention of polySia in dynamic cellular processes as different as migration of neuronal precursor cells, axonal outgrowth, synaptogenesis, physiological and morphological synaptic plasticity, and control of circadian rhythm (8–15). Although polySia levels are high during embryonic development, expression in the adult is restricted to brain regions of persistent neural plasticity such as the subventricular zone, the rostral migratory stream toward the olfactory bulb, the hippocampus, and the hypothalamo-neurohypophyseal system (16–18).

The outstanding role of polySia in controlling NCAM interactions became apparent by the lethal phenotype of mice lacking polySia while retaining normal NCAM expression (19, 20). Born overtly normal and at a Mendelian frequency, these mice are characterized by postnatal growth retardation, specific brain wiring defects, progressive hydrocephalus, and premature death during the early postnatal period. Strikingly, the severe phenotype was rescued by additional ablation of NCAM (19), demonstrating that the untimely expression of "naked", i.e. nonpolysialylated, NCAM has fatal consequences during early postnatal development.

The biosynthesis of polySia depends on the Golgi-resident polysialyltransferases ST8SiaII and ST8SiaIV, which share 59% identity on the amino acid sequence level. Each enzyme independently is capable of synthesizing polySia on NCAM (21, 22), starting on complex N-glycans in the fifth Ig-like domain (23–25). During development, the enzymes are differentially expressed in a tissue- and cell type-specific manner with overlapping expression pattern (26–30). Mice lacking either ST8SiaII (31) or ST8SiaIV (32) showed only a partial loss of polySia combined with mild but clearly distinct phenotypes. Whereas ST8SiaIV deficiency caused selective impairments of hippocampal synaptic plasticity, learning, and memory in adult mice (32, 33), loss of ST8SiaII resulted in abnormal lamination of hippocampal mossy fiber projections associated with ectopic synapse formation, impaired basal synaptic transmission in the dentate gyrus, and altered fear conditioning (31, 34). Ablation of ST8SiaIV resulted in a partial loss of polySia in the adult brain (32), whereas ST8SiaII deficiency caused a reduction in the level of polySia during the perinatal phase (35). At postnatal day 1 (P1), when under normal conditions the complete NCAM pool is polysialylated, a loss of ST8SiaII resulted in a decrease in polySia level by ~40%. The observed drop in polySia expression was combined with marked changes of the NCAM polysialylation status, and about half of the NCAM pool was found in the polySia-free state (35). This finding indicated striking differences in the impact of the two enzymes during postnatal brain development.

It is well known that polySia decreases rapidly after birth (36, 37). However, no information is available on the exact time course, changes in polysialyltransferase mRNA level, and the impact of the two polysialyltransferases on the developmental changes in NCAM polysialylation. In the present study, we have used biochemical analyses to quantitatively evaluate developmental changes in the polySia-NCAM pattern on a whole brain basis. PolySia level, NCAM isoform patterns, and the degree of NCAM polysialylation were studied on transcript, protein, and carbohydrate level in wild-type, ST8SiaII-/-, and ST8SiaIV-/- mice.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies, Enzymes, and Reagent—NCAM-specific mouse monoclonal antibody (mAb) H28 (38) and polySia-specific mAb 735 (39) were used after affinity purification on protein G- and protein A-Sepharose (GE Healthcare), respectively. Endosialidase of bacteriophage K1F was purified as described previously (7) and 1,2-diamino-4,5-methylenedioxybenzene (DMB) was purchased from Dojindo.

Breeding of Knock-out Mice—ST8SiaII (31) and ST8SiaIV knock-out mice (32) were back-crossed to C57BL/6J mice for six generations. Genotyping was performed as described (19).

Protein Extraction and Western Blot Analysis—Mouse brains were homogenized (19), and one aliquot of each lysate was treated with 25 ng/µl endosialidase for 45 min on ice. The proteins were separated by 7% SDS-PAGE under reducing conditions, loading 20 µg of total protein/lane. The proteins were transferred to nitrocellulose and subjected to immunostaining using 2.5 µg/ml anti-polySia antibody 735 or anti-NCAM antibody H28 and enhanced chemiluminescence for detection. The intensity of protein bands was analyzed on appropriately exposed Lumi-Film chemiluminescenct detection films (Roche Applied Science) as mean gray value by computerized densitometric scanning and Kodak 1D 3.5 Network software (Kodak).

RNA Extraction and Quantitative Real Time RT-PCR—RNA extraction and quantitative RT-PCR was performed as described (35) using a SDS7700 real time PCR system (Applied Biosystems) and the following gene-specific primers that span one intron: ST8SiaII, 5'-GGCTGTGGCCAGGAGATTG-3' and 5'-GGCATACTCCTGAACTGGAGCC-3'; ST8SiaIV, 5'-GCACCAAGAGACGCAACTCATC-3' and 5'-CAGAGCTGTTGACAAGTGATCTGC-3'; total NCAM, 5'-GGATGCCTCCATCCACCTC-3' and 5'-GGCCGTCTGATTCTCTACATAGG-3'; and NCAM-120, 5'-ACTTTGTGTTCAGGACCTCAGCC-3' and 5'-GAGGTGGAGCTTCCGCCC-3'. For normalization, primers specific for the gene encoding the TATA box-binding protein were used (5'-CACTTCGTGCAAGAAATGCTG-3' and 5'-AATCAACGCAGTTGTCCGTG-3'). For all primer pairs the amplification efficiencies were determined by analyzing the slope of a Ct/log(template concentration) plot (40).

DMB-HPLC Analysis—To analyze the total amount and the chain length pattern of polySia, the DMB-HPLC method (41) was used as described previously (35). Briefly, delipidated whole brain homogenates were dried, dissolved in 300 µl of DMB reaction buffer, and incubated for 24 h at 4 °C with shaking. After removal of insoluble material, the reaction was stopped by adding 70 µl of 1 M NaOH to 280 µl of supernatant. Released polySia chains were separated by HPLC on a DNAPac PA-100 column (Dionex), and DMB-labeled polySia chains were detected with a fluorescent detector. MilliQ water, and 1 M NaNO3 (E2) were used as eluents at a flow rate of 1 ml/min. Elution was performed by the following gradient: T0 min = 0% (v/v) E2; T5 min = 1% (v/v) E2; T15 min = 10% (v/v) E2; and T60 min = 50% (v/v) E2. The column was washed with 100% (v/v) E2 for 10 min. For quantification of the amount of individual polymer sizes, peak areas corresponding to the relative amount of each polySia chain with DP ≥8 were calculated. To determine the total amount of polySia, these peak areas were summarized.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Quantitative Assessment of PolySia Level during Postnatal Brain Development of Wild-type, ST8SiaII-/-, and ST8SiaIV-/- Mice—To evaluate the impact of ST8SiaII and ST8SiaIV on polySia biosynthesis during early postnatal brain development, a detailed quantitative analysis of polySia levels was performed in total brain homogenate of wild-type, ST8SiaII-/-, and ST8SiaIV-/- mice. For each genotype, brain samples were isolated at ten time points from P1 to P21 and from adult mice older than 6 month of age. Using delipidated whole brain homogenate, we determined the total amount of polySia by DMB-HPLC analysis as described previously (35), and the results were displayed as relative polySia concentrations with the value obtained for P1 wild-type brain set to 100% (Fig. 1A). In the wild-type situation, the polySia level remained almost constant during the first postnatal week, and a significant down-regulation was not observed before P9. Notably, the dramatic developmental decrease, which was described in previous studies (36, 37, 42), was found to occur in a narrow time window between P9 and P17. Within 8 days, the polySia level declined by almost 70% before a low but constant level was reached from P17 to P21. Toward adulthood, the amount of polySia further declined, and in brain isolated from mice older than 6 month of age, only 10% of the perinatal level was found.

The comparison of polySia concentrations in the two knock-out animals impressively highlighted how the individual polysialyltransferase impacts polySia production. Whereas a lack of ST8SiaIV had no effect on the polySia level during the first 3 weeks, striking differences were observed in ST8SiaII-deficient brain. Starting on a 40% lower level on P1, polySia declined immediately after birth and remained rather constant from P9 to P21. Notably, the dramatic alterations observed in ST8SiaII-null mice were transient, being apparent from P1 to P15 but no longer visible at P17. From P17 to P19, almost identical polySia levels were found in all three genotypes, and 30% of the P1 wild-type level was reached. In adult brain, the relative polySia levels were 10, 5, and 2% in wild-type, ST8SiaII-/-, and ST8SiaIV-/- mice, respectively. Therefore, the loss of either enzyme resulted in decreased polySia level in adult brain. The reduction was, however, more prominent in the case of ST8SiaIV deficiency.


Figure 1
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  		FIGURE 1.
Changes in polySia level and brain weight during postnatal brain development. A, whole brains of wild-type, ST8SiaII-/-, and ST8SiaIV-/- mice were obtained at the indicated postnatal stages from P1 to P21 and from the adult stage. PolySia concentrations were determined in brain homogenates by means of DMB-HPLC analysis. The values are the means ± S.D. of three independent experiments, and the values obtained for P1 wild-type brain were set to 100%. B, polySia concentrations determined in A were used to calculate the total amount of polySia per brain. The values obtained for P1 wild-type brain were set to 100%. C, weights of freshly dissected brains obtained at the indicated time points (n = 3 for each genotype and time point).

 
To acknowledge the fact that early postnatal brain development is accompanied by a rapid increase in brain size, developmental changes in polySia level were also displayed per brain (Fig. 1B). In wild-type and ST8SiaIV-/- brain, the total amount of polySia increased 3-fold after birth, and peak expression was reached at P9. By clear contrast, no such peak expression was found in ST8SiaII-deficient brain, and the amount of polySia stayed remarkably constant from P5 to P21. To ensure that ST8SiaII deficiency did not affect brain growth, brain weights were determined and plotted over age (Fig. 1C). In particular during the first 15 days, when striking differences in polySia level were found between the two knock-out mice, a similar gain in brain weight was observed for all three genotypes. Thus, the selective reduction in polySia observed in ST8SiaII-deficent brain was not caused by growth retardation but is obviously due to a lower biosynthetic capacity.

Analysis of the PolySia Chain Length Pattern during Postnatal Brain Development—To evaluate whether the developmental decrease in polySia level is paralleled by progressive shortening of polySia chains and whether the loss of either polysialyltransferase affects the chain length pattern, we analyzed the DP of polySia by DMB-HPLC analysis (41, 43). Examples of representative chromatographic profiles obtained with brain samples from wild-type brain at different time points are shown in supplemental Fig. S1. Each peak represents a polySia chain of defined DP, and determination of peak areas allows quantification of individual species resulting in a foot print of the chain length pattern. For each genotype, six time points were selected (P1, P5, P9, P13, P21, and adult), and the results were displayed in relation to the data obtained for wild-type brain at P1 and were set to 100% (Fig. 2). Importantly, long polySia chains were detected in all three genotypes and at all time points investigated, including adult brain. However, during development, the decrease in the amount of long chains was more pronounced than the reduction of short chains. In wild-type brain, this is most evident from P9 on. At P13 for example, polymers with DP 8–22 were uniformly decreased by 40%, whereas the abundance of longer chains declined increasingly with length leading to a 60% reduction of polymers with DP > 36. After P13, increased reduction with polymer length is visible over the whole DP range. These results demonstrate that during normal brain development, down-regulation of polySia is accompanied by alterations in the chain length profile.

Comparison of the curves obtained for wild-type and ST8SiaII-/- brain (Fig. 2) revealed that loss of ST8SiaII had a dual effect on the polysialylation pattern. First, ST8SiaII deficiency resulted in an overall decrease in polySia level as indicated by a shift of the corresponding curves along the y axis. Second, an altered chain length distribution was observed as indicated by different curve slopes. The latter effect affected all polymer sizes and was restricted to time points before P9. In ST8SiaIV-/- mice, by contrast, deviations from the wild-type pattern were most prominent for long polymers, and the number of affected polymer sizes increased with age (reduction in polymers with DP > 36 at P1, DP > 28 at P9, and DP > 12 at P13). At P21, almost similar chain length patterns and total amounts of polySia were observed for all three genotypes. In summary, these results demonstrate that from P1 to P13 loss of either enzyme results in subtle but distinct alterations in the chain length pattern, although only ST8SiaII deficiency causes a severe reduction in the total amount of polySia.

Developmental Changes in NCAM Polysialylation and NCAM Isoform Pattern in Wild-type, ST8SiaII-/-, and ST8SiaIV-/- Mice—Early postnatal brain development is characterized by a switch from polysialylated to nonpolysialylated NCAM (36, 42). To investigate whether polysialyltransferase deficiency affects the time course of this process, we performed a detailed Western blot analysis using whole brain homogenate from wild-type, ST8SiaII-/-, and ST8SiaIV-/- mice. The brains were isolated at subsequent days after birth (P1 to P21 and adult), and for each time point equal amounts of homogenate were subjected to SDS-PAGE and Western blotting (Fig. 3). Staining was performed either with mAb 735 (top panel) specific for {alpha}2,8-linked polySia with DP ≥ 8 (39, 44) or mAb H28 (middle and bottom panels) directed against a protein epitope common to all murine NCAM isoforms (38). Because each polySia chain provides multiple binding sites for an anti-polySia antibody, much stronger signals were obtained with mAb 735. However, staining with mAb H28 revealed a more detailed picture. This antibody binds preferentially the polySia-free form of NCAM and provides an ideal reagent to detect the appearance of nonpolysialylated NCAM in parallel to polySia-NCAM (Fig. 3, middle panel). Polysialylated NCAM of all isoforms migrates as a microheterogeneous band above 250 kDa, whereas focused bands at 120, 140, and 180 kDa indicate the expression of nonpolysialylated NCAM-120, -140, and -180, respectively. The analysis of wild-type brain revealed that polySia-free NCAM was barely discernible before P9, demonstrating that during the first postnatal week almost the complete brain NCAM pool is kept in the polysialylated form. After P9, the intensity of the polySia-NCAM signal decreased rapidly, paralleled by the appearance of nonpolysialylated NCAM of all three major isoforms. In ST8SiaIV-deficient brain, a very similar pattern was found, whereas the loss of ST8SiaII was accompanied by an untimely appearance of polySia-free NCAM. During the first postnatal days, when nonpolysialylated NCAM was not yet visible in wild-type and ST8SiaIV-/- brain, strong signals for polySia-free NCAM-140 and NCAM-180 were observed in ST8SiaII-deficient brain.


Figure 2
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  		FIGURE 2.
Developmental changes in the polySia chain length pattern. Whole brains of the indicated genotypes were obtained at P1, P5, P9, P13, P21, and from the adult stage and brain homogenates were analyzed by DMB-HPLC. In the obtained HPLC profiles, peak areas corresponding to the amount of each polySia chain in the range of 8–41 residues were determined, and the results are given as the mean values ± S.D. of three independent experiments. The amount of respective wild-type chain length species obtained at P1 was set to 100%.

 


Figure 3
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  		FIGURE 3.
Developmental changes in the polysialylation status of NCAM. Whole brains of wild-type, ST8SiaIV-/-, and ST8SiaII-/- mice were obtained at the indicated postnatal stages from P1 to P21 and from adult stage. Brain samples were homogenated, and lysates were separated by 7% SDS-PAGE using 20 µg of total protein/lane. After transfer to nitrocellulose membrane, the proteins were stained with either anti-polySia mAb 735 (upper panel) or anti-NCAM mAb H28 (middle and lower panels). In parallel experiments, aliquots of each brain lysate were pretreated with endosialidase (endo N) to remove polySia and to investigate the total NCAM expression levels and isoform pattern (lower panel). Bands representing polysialylated forms of NCAM (polySia-NCAM) and nonpolysialylated NCAM-120, NCAM-140, and NCAM-180 are indicated on the right.

 
To investigate whether these elevated levels of polySia-free NCAM were caused by an up-regulation of NCAM or a shift in the NCAM-isoform pattern, we analyzed NCAM expression in parallel brain samples that were pretreated with endosialidase to remove polySia (Fig. 3, bottom panel). In wild-type brain, NCAM-140 and NCAM-180 are the predominant isoforms during early postnatal development. Quantification of the corresponding bands revealed fairly constant expression levels during the first 3 weeks, whereas a decrease by almost 50% was observed toward adulthood (Fig. 4A). In clear contrast to the transmembrane isoforms, marked developmental changes were seen for NCAM-120. Whereas NCAM-120 was barely detectable during the first 4 days, expression levels increased continuously from P5 on, reaching maximal expression around P17 (see Figs. 3 and 4B for quantification). Thereafter, expression levels remained constant, and NCAM-120 became the dominant isoform in adult brain. Thus, up-regulation of NCAM-120 occurred in parallel to the down-regulation of polySia. Accordingly, the majority of NCAM-120 was expressed in the nonpolysialylated form, as demonstrated by the fact that the intensity of NCAM-120 signals was similar before and after endosialidase treatment (compare middle and bottom panels in Fig. 3). By contrast, endosialidase treatment resulted in the appearance of strong NCAM-140 and NCAM-180 signals, which were not apparent in untreated samples. These results clearly demonstrate that in mouse brain, polySia is predominantly attached to the transmembrane isoforms NCAM-140 and NCAM-180.


Figure 4
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  		FIGURE 4.
NCAM protein and transcript level during postnatal brain development. Developmental changes in the NCAM protein level in brain of wild-type, ST8SiaII-/-, and ST8SiaIV-/- mice were determined by densitometric evaluation of Western blots with endosialidase-treated brain lysates as shown in the lower panel of Fig. 3. For each of the indicated time points the intensities of the bands corresponding to the isoforms NCAM-120, NCAM-140, and NCAM-180 were determined. The results are shown separately for the total amount of NCAM-140 and NCAM-180 (A) and for NCAM-120 (B). For each genotype, three independent blots were evaluated and for each blot, the values obtained for P1 brain were used as a reference for NCAM-140 and NCAM-180 (A) and were set to 100%. In the case of NCAM-120 (B), values obtained for P21 were used as a reference and were set to 100%. C, to allow direct comparison of NCAM expression levels between individual genotypes, endosialidase-treated brain lysates of all three genotypes obtained at four selected time points (P1, P3, P9, and P15) were analyzed on the same blot. Four independent experiments were evaluated, and the means ± S.D. are shown. The values obtained for P1 (NCAM-140 and NCAM-180) or P15 (NCAM-120) wild-type brain were set to 100%. D and E, the relative mRNA level of total NCAM (all isoforms) and NCAM-120 were determined in brains of wild-type, ST8SiaIV-/-, and ST8SiaII-/- mice from P1 to P21 by quantitative real time RT-PCR. For each time point two independently generated cDNAs were measured in triplicate. The data are given as the means ± S.D., and for each genotype values obtained for P21 were set to 100%.

 
Quantification of NCAM signals obtained after endosialidase treatment revealed that in all three genotypes, similar developmental changes in the NCAM isoform pattern occurred (Fig. 4, A and B). In both knock-out mice, NCAM-140 and NCAM-180 were expressed at rather constant levels from P1 to P21 (Fig. 4A), whereas NCAM-120 was up-regulated with the same time course as in wild-type animals (Fig. 4B). Moreover, to directly compare total NCAM expression in the different genotypes, four time points (P1, P3, P9, and P15) were selected, and endosialidase-treated brain homogenates from all genotypes were analyzed on the same blot. Quantification of the NCAM signals (Fig. 4C) revealed very similar NCAM expression levels in all three genotypes, excluding the possibility that the untimely expression of polySia-free NCAM-140 and NCAM-180 observed in ST8SiaII-/- mice was caused by increased NCAM expression.

In line with the results obtained by Western blot analysis, almost identical total NCAM mRNA levels were determined in all three genotypes (Fig. 4D). In addition, NCAM-120 mRNA levels were monitored by quantitative RT-PCR performed with primers specific for the glycosylphosphatidylinositol-linked isoform (Fig. 4D). Similar to the increase observed on protein level (Fig. 4B), a strong up-regulation of NCAM-120 mRNA was seen from P1 to P21, demonstrating that NCAM-120 expression is tightly regulated on the transcriptional level.

ST8SiaII and ST8SiaIV Expression during Postnatal Brain Development—To evaluate the correlation between polySia down-regulation and changes in the expression level of the two polysialyltransferases, relative transcript levels of ST8SiaII and ST8SiaIV were determined simultaneously in wild-type brains isolated at eight postnatal stages from P1 to P21 (Fig. 5A). In agreement with our earlier study (35), ST8SiaII was the predominant polysialyltransferase at P1, although the difference in transcript level between ST8SiaII and ST8SiaIV was less than factor 2. After birth, the mRNA levels of both enzymes decline. However, clear differences in the rate of decrease were observed. From P1 to P21, a reduction by 60% was found for ST8SiaIV transcripts, whereas in the same time period the ST8SiaII level dropped by more than 95% with the strongest reduction in a short time window from P5 to P11. At P9, both polysialyltransferases reached similar transcript levels, and thereafter, ST8SiaIV became the prominent enzyme with 5-fold higher transcript levels at P21.


Figure 5
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  		FIGURE 5.
Developmental changes in transcript levels of ST8SiaII and ST8SiaIV. A, relative mRNA levels of ST8SiaII and ST8SiaIV were determined in the brains of wild-type mice obtained at the indicated time points by real time RT-PCR. For each time point, two independently generated cDNAs were measured in triplicate. The data are the means ± S.D., and the ST8SiaIV transcript level at P1 was set to 100%. B, comparison of the ST8SiaII transcript level in brain of wild-type and ST8SiaIV-/- mice. C, comparison of the ST8SiaIV transcript level in brain of wild-type and ST8SiaII-/- mice. For each genotype and time point, two independently generated cDNAs were measured in triplicate. The data are the means ± S.D., and the values obtained for P1 wild-type brain were set to 100%.

 
To investigate whether the loss of one enzyme was compensated by increased expression of the second enzyme, polysialyltransferase mRNA levels were determined in ST8SiaII- and ST8SiaIV-deficient brain. At six time points from P1 to P21, ST8SiaII mRNA levels were determined in ST8SiaIV-/- and wild-type mice (Fig. 5B), whereas ST8SiaIV mRNA levels were analyzed in ST8SiaII-/- and wild-type mice (Fig. 5C). Remarkably, in both knock-out mice, transcript levels of the remaining polysialyltransferase were unaltered compared with the wild-type level. Thus, the loss of one enzyme did not affect the expression level of the second enzyme, indicating that both enzymes are independently regulated throughout postnatal development.

Correlation between Polysialyltransferase mRNA Level and Product Formation—Comparison of Fig. 1A (polySia down-regulation during postnatal development) and Fig. 5A (changes in transcript levels of ST8SiaII and IV) suggested that product formation is tightly connected to polysialyltransferase mRNA levels. To evaluate this point in more detail, polySia levels determined at P1, P7, P9, P11, P15, and P21 were plotted versus the corresponding polysialyltransferase mRNA concentration (Fig. 6). In all three genotypes product formation increased linearly with increasing transcript level. However, a steeper slope was observed for ST8SiaII-catalyzed compared with ST8SiaIV-catalyzed polySia formation (see curves for ST8SiaIV- and ST8SiaII-null mice, respectively). This result indicates that ST8SiaII is either more efficient than ST8SiaIV or that ST8SiaII mRNA is translated with a much higher efficiency.


Figure 6
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  		FIGURE 6.
Correlation between polysialyltransferase mRNA level and polySia formation. Total polysialyltransferase transcript levels in brain of wild-type, ST8SiaII-/-, and ST8SiaIV-/- mice were determined by real time RT-PCR (see Fig. 5, B and C), and the values of six selected time points (P1, P7, P9, P11, P15, and P21) were plotted over the corresponding polySia level determined by DMP-HPLC analysis (see Fig. 1A). The total polysialyltransferase mRNA level of wild-type brains was obtained by summing the transcript levels of ST8SiaII and ST8SiaIV. Transcript and polySia levels in P1 wild-type brain were set to 100%.

 
In the plot depicted in Fig. 6, only the curves for wild-type and ST8SiaIV-deficient mice reached a saturation plateau at high transcript levels, i.e. at the first postnatal days. At this time point, the complete NCAM pool of both genotypes was found in the polysialylated state (Fig. 3), indicating that the saturation plateau is reached because of the fact that the acceptor NCAM became a limiting factor. By contrast, in ST8SiaII-null mice, nonpolysialylated NCAM was found at all postnatal stages (Fig. 3), and consequently, no saturation plateau was reached even at the highest ST8SiaIV transcript levels. Therefore, in ST8SiaII-deficient brain, the rate of polySia biosynthesis but not the availability of NCAM limited polySia formation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The first 3 weeks of postnatal mouse brain development are characterized by rapid brain growth, a second wave of neurogenesis producing huge numbers of interneurons, glial proliferation, directed migration, and neuronal maturation including axonal and dendritic growth, synaptogenesis, and myelination (45, 46). Coincident with the completion of the major morphogenetic events, polySia decreases postnatally, resulting in a conversion of polysialylated to nonpolysialylated NCAM, a process that was first described 25 years ago (36, 37). By making use of single knock-out mice lacking either ST8SiaII (31) or ST8SiaIV (32), we now analyzed the impact of the two polysialyltransferases on polySia biosynthesis during postnatal brain development. Therefore, precise information on the normal developmental changes in (i) polySia level and chain length profile, (ii) polysialyltransferase transcript level, and (iii) NCAM expression, isoform pattern, and polysialylation status was indispensable.

Using DMB-HPLC analysis (41, 43) for quantitative assessment of polySia level in whole brain homogenates, we show that in wild-type brain, the 3-fold increase in brain weight observed from P1 to P9 is fully matched by a parallel increase in polySia. Thus, during the first 9 days, polySia biosynthesis keeps up with brain growth, and all expressed NCAM is maintained in the polysialylated state. PolySia is mainly attached to the two transmembrane isoforms NCAM-140 and NCAM-180, which are expressed at rather constant levels from P1 to P21. The dramatic postnatal decrease in polySia by about 70% was found to be restricted to the time period from P9 to P17. Although polySia down-regulation was shown to be accompanied by a slight decrease in the amount of long polymers, polySia chains with up to 40 residues were detectable throughout postnatal development. Similar observations were made during embryonic chick development, where polymers with 40–50 residues were observed in all of the investigated stages (47). The lack of major alterations in the chain length pattern is also consistent with results obtained for the hypothalamic region of rat brain. Analysis of polySia at P7 and at the adult stage revealed a 9-fold decrease in polySia but only minor differences in DP (48).

Previous studies have demonstrated that polySia-NCAM acts as a negative regulator of myelination and that axonal expression is down-regulated to allow myelin deposition on polySia-free axons (49, 50). In line with this, the time window identified for the major loss of polySia coincides with the onset of myelination (51, 52). Inverse to polySia down-regulation, massive up-regulation of NCAM-120, the characteristic isoform of oligodendrocytes and myelin sheaths (53, 54), was observed. The majority of brain NCAM-120 was found in the polySia-free form, which is in agreement with the finding that migrating oligodendrocyte precursor cells but not mature myelinating oligodendrocytes express polySia (55–57).

Comparison of the developmental changes in wild-type and polysialyltransferase-deficient brain revealed that the loss of ST8SiaII had no significant impact on NCAM expression but markedly affected the time course of polySia down-regulation. In ST8SiaII-null brain, polySia declined immediately after birth reaching the polySia level of wild-type brain at P17 already at P9. Because only subtle changes in the chain length pattern were observed, the premature decrease in polySia is mainly due to an earlier shift from polysialylated to nonpolysialylated NCAM. Consequently, large amounts of polySia-free NCAM were found during the first postnatal week, when in wild-type brain nearly all NCAM is polysialylated. Notably, the striking alterations observed on the molecular level are of a temporary nature, apparent from P1 to P15. The time period of reduced polySia levels coincides with a time window critical for the development of the hippocampal formation. This brain structure undergoes many phenotypic changes during the first postnatal week (58) and is affected in ST8SiaII-null mice (31). Thus, transient alterations in NCAM polysialylation caused by ST8SiaII deficiency result in permanent morphological defects, demonstrating that the precise timing of polySia down-regulation is essential for normal brain development.

In contrast to ST8SiaII deficiency, the loss of ST8SiaIV had no significant impact on the polySia level from P1 to P21. Reduced polySia levels were observed only in the adult stage. This is in perfect agreement with the absence of morphologic defects and the adult expression of impaired synaptic plasticity in the hippocampus (32). Nevertheless, ST8SiaIV deficiency causes a slightly altered chain length pattern during the first postnatal weeks. Notably, this pattern is distinct not only from wild-type brain but also from the chain length distribution found in ST8SiaII-null brain. Thus, during postnatal brain development, ST8SiaIV is dispensable for the maintenance of normal polySia levels but is necessary to ensure the correct chain length profile. The importance of a concerted action of both enzymes on the chain length profile was highlighted by our recent study on the detailed analysis of polysialylated N-glycans of NCAM isolated from P1 brains of wild-type, ST8SiaII-deficient, and ST8SiaIV-deficient mice (59). Although no differences with regard to the number of polySia chains attached to N-glycans were observed in wild-type and mutant mice, the quality of the assembled polySia chains clearly depended on the enzyme setting. At the fifth N-glycosylation site of NCAM, an increased amount of long polySia chains was found when ST8SiaII and ST8SiaIV worked together, suggesting a synergistic action of the two enzymes (59).

Based on in situ hybridization and Northern blot analyses, several studies indicated that ST8SiaII and ST8SiaIV are prevalently expressed during development, starting from E8–E9 in rodents, whereas they are down-regulated in the adult (26–28, 30). However, depending on the method used, different time courses were observed, and direct quantitative comparison of transcript levels was hampered by the different G/C content of ST8SiaII and ST8SiaIV probes (28), leading to an overestimation of ST8SiaII signals. To circumvent this problem, we used quantitative real time RT-PCR to monitor developmental changes in polysialyltransferase transcript level. In line with our previous study for neonatal brain (35), we found that on P1 the ST8SiaII level was less than 2-fold higher than ST8SiaIV. On P9, both enzymes reached identical transcript levels, and thereafter, ST8SiaIV became the prevalent enzyme, reaching a 5-fold higher transcript level than ST8SiaII at P21. As expected, down-regulation of both enzymes preceded the postnatal drop in polySia. A linear correlation between polySia formation and polysialyltransferase transcript level was found only at low transcript level, i.e. from P11 to P21. Before this time period, enzyme levels are kept sufficiently high so that the acceptor NCAM is the limiting factor in polySia biosynthesis. This might be crucial to guarantee that all NCAM is fully polysialylated, thereby preventing untimely interactions mediated by polySia-free NCAM. As demonstrated by the severe phenotype of ST8SiaII/ST8SiaIV double-deficient mice, untimely expression of polySia-free NCAM has deleterious effects on brain development (19, 20). In the present study, we showed that even though ST8SiaII-deficient mice are not completely devoid of polySia, they are also characterized by premature expression of polySia-free NCAM. Consistent with this, reinvestigation of ST8SiaII-deficient mice revealed partial recapitulation of the brain wiring defects found in double knock-out mice,3 highlighting the important role of ST8SiaII for normal brain development.

Perturbations arising during brain development can lead to a range of pathologies in later life. In this respect, it is of particular interest that recent studies implicated a link between altered ST8SiaII expression and schizophrenia. In the human genome, the ST8SiaII gene (SIAT8B) is located on chromosome 15q26, a susceptibility region for both schizophrenia and bipolar disorder (60). In two independent studies a positive association between particular polymorphisms in the SIAT8B promoter region and schizophrenia (61, 62) was found, and postmortem analyses of schizophrenic brains revealed a reduction in the number of polySia-positive hilar cells in the hippocampus (63). Disturbances of the anatomical organization and hippocampal function have long been implicated in the etiology of the disease (64, 65), and the importance of the developmental phase of polySia-NCAM expression in the emergence of schizophrenia has been discussed (65). Therefore, this study, providing precise knowledge on the impact of the two polysialyltransferases during brain development, will contribute to further understanding of the molecular pathology of neurological disorders.


   FOOTNOTES
 
* This work was supported by grants from the Deutsche Forschungsgemeinschaft (to H. G., R. G., R. G.-S., and M. M.), Deutsche Krebshilfe (to H. H. and M. M.), and Promemoria EC FP6 (to R. G.-S.). 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed: Abteilung Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel.: 49-511-532-9807; Fax: 49-511-532-3956; E-mail: muehlenhoff.martina{at}mh-hannover.de.

2 The abbreviations used are: polySia, polysialic acid; NCAM, neural cell adhesion molecule; mAb, monoclonal antibody; DMB, 1,2-diamino-4,5-methylenedioxybenzene; DP, degree of polymerization; RT, reverse transcription; Pn, postnatal day n; HPLC, high pressure liquid chromatography. Back

3 H. Hildebrandt, I. Oltmann-Norden, I. Röckle, M. Mühlenhoff, B. Weinhold, and R. Gerardy-Schahn, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jamey Marth for kindly providing ST8SiaII knock-out mice, Dr. Birgit Weinhold for help in breeding mice and maintaining the mouse colonies, Dr. Ulrich Lehmann for expert help with the real time RT-PCR assays, and Ulrike Bernard, Daniela Wittenberg, and Kerstin Leib for excellent technical assistance.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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