The FASEB Journal article fj.15-270983. Published online April 6, 2015.

The FASEB Journal • Research Communication

Sialylation regulates brain structure and function Seung-Wan Yoo,* Mary G. Motari,* Keiichiro Susuki,†,1 Jillian Prendergast,* Andrea Mountney,‡ Andres Hurtado,§,{ and Ronald L. Schnaar*,2 Departments of *Pharmacology and Molecular Sciences, §Neurology, and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; †Department of Neuroscience, Baylor College of Medicine, Houston, Texas, USA; ‡Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA; and {International Center for Spinal Cord Injury, Hugo W. Moser Research Institute, Kennedy Krieger, Baltimore, Maryland, USA Every cell expresses a molecularly diverse surface glycan coat (glycocalyx) comprising its interface with its cellular environment. In vertebrates, the terminal sugars of the glycocalyx are often sialic acids, 9-carbon backbone anionic sugars implicated in intermolecular and intercellular interactions. The vertebrate brain is particularly enriched in sialic acid-containing glycolipids termed gangliosides. Human congenital disorders of ganglioside biosynthesis result in paraplegia, epilepsy, and intellectual disability. To better understand sialoglycan functions in the nervous system, we studied brain anatomy, histology, biochemistry, and behavior in mice with engineered mutations in St3gal2 and St3gal3, sialyltransferase genes responsible for terminal sialylation of gangliosides and some glycoproteins. St3gal2/3 double-null mice displayed dysmyelination marked by a 40% reduction in major myelin proteins, 30% fewer myelinated axons, a 33% decrease in myelin thickness, and molecular disruptions at nodes of Ranvier. In part, these changes may be due to dysregulation of ganglioside-mediated oligodendroglial precursor cell proliferation. Neuronal markers were also reduced up to 40%, and hippocampal neurons had smaller dendritic arbors. Young adult St3gal2/3 double-null mice displayed impaired motor coordination, disturbed gait, and profound cognitive disability. Comparisons among sialyltransferase mutant mice provide insights into the functional roles of brain gangliosides and sialoglycoproteins consistent with related human congenital disorders.—Yoo, S.-W., Motari, M. G., Susuki, K., Prendergast, J., Mountney, A., Hurtado, A., Schnaar, R. L. Sialylation regulates brain structure and function. FASEB J. 29, 000–000 (2015). www.fasebj.org ABSTRACT

Key Words: animal models • behavior • gangliosides • myelin oligodendrocyte precursor cells

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NEURONS AND GLIA CARRY DIVERSE surface coats of glycolipids and glycoproteins constituting their interface with their environment (1). Cell surface glycans regulate the activities Abbreviations: BrdU, bromodeoxyuridine; Caspr, contactinassociated protein; CNPase, 29,39-cyclic-nucleotide 39-phosphodiesterase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium binding adapter molecule 1; Kv1.2, potassium voltage-gated channel subfamily A member 2; MAG, myelin-associated (continued on next page)

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of membrane proteins, mediate cell-cell recognition, and regulate interactions between cells and their environment (2, 3). In vertebrates, glycolipids and glycoproteins are often terminated with sialic acids, 9-carbon backbone sugars with chemical properties well suited for molecular recognition (4). Brain sialoglycans are dominated by gangliosides, sialic acid-bearing glycosphingolipids that constitute ;75% of the brain’s sialic acid with sialoglycoproteins constituting the remaining ;25% (1, 5). In mammals, the same 4 gangliosides (GM1, GD1a, GD1b, and GT1b, nomenclature of Svennerholm (6); see Fig. 1C) comprise the vast majority of brain sialoglycans, whereas sialoglycoproteins are more diverse. Sialoglycan biosynthesis occurs via the stepwise action of glycosyltransferases (8, 9). Human mutations in the ganglioside-specific sialyltransferase gene ST3GAL5 result in severe infantile-onset seizures, developmental delay, and blindness (10–12), whereas human mutations in the brain ganglioside biosynthetic gene B4GALNT1 result in hereditary spastic paraplegia with cognitive impairment (13, 14). Mutations in the glycoprotein/ganglioside sialyltransferase gene ST3GAL3 result in nonsyndromic intellectual disability (15) and West syndrome (16) characterized by intellectual disability and infantile seizures. These findings identify sialoglycans as key components of human nervous system function. To increase understanding of the physiologic roles of nervous system sialoglycans, we studied mice lacking sialyltransferases ST3Gal-II and ST3Gal-III, coded by the genes St3gal2 and St3gal3. These enzymes cooperate in the terminal sialylation of brain gangliosides (7), together being responsible for biosynthesis of GD1a, GT1b, and GQ1b (Fig. 1A, C) that comprise half the gangliosides in mammalian brain (5). Ganglioside terminal sialylation is blocked 50% in St3gal2-null mice, unaffected in St3gal3null mice, and nearly fully blocked in St3gal2/3 1 Current affiliation: Boonshoft School of Medicine, Wright State University, Dayton, OH, USA. 2 Correspondence: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205-2105, USA. E-mail: [email protected] doi: 10.1096/fj.15-270983 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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Figure 1. Ganglioside and sialoglycoprotein expression in St3gal-mutant mice. A) TLC of brain gangliosides extracted from WT, St3gal2-null, St3gal3-null, and double-null mice (7). Migration positions of brain ganglioside standards are shown. The band migrating with GD1a in double-null mice (§) is an O-acetylated form of GD1b (7). This figure has been modified from Sturgill, E. R., Aoki, K., Lopez, P. H., Colacurcio, D., Vajn, K., Lorenzini, I., Maji’c, S., Yang, W.H., Heffer, M., Tiemeyer, M., Marth, J. D., and Schnaar, R. L. (2012) Biosynthesis of the major brain gangliosides GD1a and GT1b. Glycobiology 22, 1289–1301 (7), with permission from Oxford University Press. B) Structure of (trisialo)ganglioside GT1b with the sialic acid added by St3gal2 and St3gal3 gene products circled. C) Biosynthetic pathways of major brain gangliosides. Genes coding the glycosyltransferases are boxed. Genes mutated in human congenital diseases of sialoglycan biosynthesis are in red. D) Total lipid sialylation (total ganglioside), ganglioside terminal sialylation (GD1a/GT1b/GQ1b), and protein sialylation in the brain, expressed as a percentage of WT (mean 6 SEM). For terminal sialylation (center) and protein sialic acid (right), genotypes were significantly different by 1-way ANOVA (P , 0.001; n.s., not significant). Intergenotype differences were by post hoc pairwise analysis (Tukey). §Terminally sialylated gangliosides are overestimated in TLC analysis due to comigration of O-acetyl-GD1b with GD1a in double-null mice only. Independent mass spectrometric analysis (7) indicates that GD1a/GT1b/GQ1b comprise 2.2% of total gangliosides in double-null mice.

double-null mice [Fig. 1D (7)]. In contrast, sialoglycoprotein sialylation is blocked by half in St3gal3-null and doublenull mice, but not in St3gal2-null mice. Here, we report (continued from previous page) glycoprotein; MBP, myelin basic protein; NCAM, neural cell adhesion molecule; NeuN, neuronal nuclear antigen; NF, neurofascin; NG2, neural/glial antigen 2 proteoglycan; Olig2, oligodendrocyte transcription factor 2; OPC, oligodendrocyte precursor cell; P4, (1R,2R)-1-phenyl-2-hexadecanoylamino-3pyrrolidino-1-propanol; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PSA, polysialic acid; TLC, thin-layer chromatography; WT, wild-type

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cellular, biochemical, neuroanatomic, and behavioral phenotypes of St3gal2 and St3gal3 single and double-null mice, providing knowledge about the roles of gangliosides and sialoglycoproteins in brain development and function. Because double-null mice have the same quantitative sialoglycoprotein deficit as St3gal3 singlenull mice, but lack ganglioside terminal sialylation, we infer that deficits that are particularly severe or exclusively found in double-null mice are likely due to terminally sialylated gangliosides. In contrast, deficits shared by St3gal3-null and St3gal2/3 double-null are possibly driven by quantitative or qualitative losses of sialylated glycoproteins.

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MATERIALS AND METHODS Mice St3gal2-null and St3gal3-null mice were produced by targeted gene disruption and backcrossed onto a C57Bl/6 background (17). Mice used in this study arose from breeding pairs of mice null at the St3gal2 locus and heterozygote at the St3gal3 locus or wild-type (WT) at the St3gal2 locus and heterozygote at the St3gal3 locus. Genotyping was by real-time PCR (Transnetyx, Cordova, TN, USA). Behavior studies were performed at 7 weeks of age and animals sacrificed for biochemical and anatomic studies at 8 weeks of age. The Johns Hopkins University Animal Care and Use Committee approved these studies; procedures were consistent with federal law and U.S. Public Health Service Policy.

Histochemistry and immunohistochemistry Isoflurane-anesthetized mice were intracardially perfused with Dulbecco’s PBS followed by neutralized 4% paraformaldehyde in PBS. Brains were embedded in paraffin and 5 mm sections prepared. Nissl staining on deparaffinized sections used 0.5% (w/v) cresyl violet in 0.25% aqueous acetic acid. For Gallyas silver stain, deparaffinized sections were incubated for 5 minutes in 5% aqueous periodic acid and then in 10% potassium iodide, 0.035% silver nitrate in aqueous 4% NaOH. After washing (0.5% aqueous acetic acid), sections were treated with aqueous 2.5% sodium carbonate, 0.1% ammonium nitrate, 0.1% silver nitrate, 0.5% tungstosilicic acid, and 0.07% formaldehyde. Golgi-Cox staining of 150-mm-thick sections used the Hito Golgi-Cox Optimstain Kit (Hitobiotec, Wilmington, DE, USA). For immunohistochemistry, deparaffinized sections (above) were heated to boiling in 10 mM sodium citrate (pH 6.0) for antigen retrieval, treated with 0.3% hydrogen peroxide to inactivate endogenous peroxidase, then blocked with 10% normal serum in PBS. Sections were probed with antibodies recognizing the neuronal nuclear antigen (NeuN; 1:200; EMD Millipore, Billerica, MA, USA), glial fibrillary acidic protein (GFAP; 1:200; EMD Millipore), ionized calcium binding adapter molecule 1 (Iba-1; 1:500; Wako BioProducts, Richmond, VA, USA), myelin basic protein (MBP; 1:200; QED Bioscience, San Diego, CA, USA), or neural/glial antigen 2 proteoglycan (NG2; 1:200; EMD Millipore). Washed sections were then incubated with biotinconjugated secondary antibody (1:500) and avidin-biotin complex using a Vector ABC Kit with diaminobenzidine substrate (Vector Laboratories, Burlingame, CA, USA). Microscopic images were acquired using an automated Eclipse 90i microscope (Nikon Instruments, Melville, NY, USA). For analyses, multiple sequential sections were prepared; for corpus callosum, 4 sections from each mouse, spaced 0.8 mm apart (anterior to posterior +0.7 to 21.7 mm based on bregma), were stained, digital images captured, and stained areas (pixels) or cell numbers determined using NIS-Elements software (Nikon Instruments). Nodes of Ranvier were immunostained as described previously (18). Freshly dissected optic and sciatic nerves were placed in icecold neutralized 4% paraformaldehyde in PBS for 30 minutes, immersed in 20% sucrose in PBS at 4°C overnight, then frozen in Shandon M-1 Embedding Matrix (Thermo Fisher Scientific, Waltham, MA, USA). Sections (16 mm sciatic and 10 mm optic nerves) were cut by cryostat and mounted on gelatin-coated coverslips. Immunostaining was performed as described (19) using chicken antibodies to neurofascin (NF) for nodes (R&D Systems, Minneapolis, MN, USA), rabbit polyclonal antibodies to contactin-associated protein (Caspr) for paranodes (19), and mouse mAb to potassium voltage-gated channel subfamily A member 2 (Kv1.2) for juxtaparanodes (K14/16; NeuroMab,

SIALYLATION REGULATES BRAIN STRUCTURE AND FUNCTION

Davis, CA, USA). Secondary antibodies were from Life Technologies (Grand Island, NY, USA) and Jackson ImmunoResearch Laboratories (West Grove, PA, USA). After permeabilization in 0.1 M sodium phosphate (pH 7.4) containing 0.3% Triton X-100 and 10% goat serum for 1 h, sections were incubated overnight at 4°C with primary antibodies in the same buffer. Washed sections were treated with fluorescently labeled secondary antibodies for 1 hour at ambient temperature. Digital images were collected on an Axiovert 200 M fluorescence microscope fitted with an ApoTome for optical sectioning (Carl Zeiss Gesellschaft mit beschr¨ankter Haftung, Jena, Germany). Electron microscopy Mice were intracardially perfused with PBS then neutralized 4% paraformaldehyde, 2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) containing 3 mM MgCl2 and 2.5% sucrose. Brains and sciatic nerves were postfixed with 2% osmium tetroxide in 0.1 M sodium phosphate buffer (pH 7.4). Fixed tissues were dehydrated in graded ethanol, embedded in EMBed-812 resin (Electron Microscopy Sciences, Hatfield, PA, USA), and thin sections (90 nm) cut with an ultramicrotome. The sections were stained with uranyl acetate and lead citrate then viewed using a Zeiss Libra 120 transmission electron microscope with a Veleta camera (Olympus, M¨unster, Germany). For quantification of unmyelinated axons, g ratio and axon diameter, .350 axons per animal were analyzed using NIS-Elements software. Protein immunoblotting Brains were homogenized in CelLytic MT (Sigma-Aldrich, St. Louis, MO, USA) and solubilized proteins subjected to immunoblot analyses using antibodies against MBP (1:500), NG2 (1:1000), b-III-tubulin (1:2000; Covance, Princeton, NJ, USA), GFAP (1:1000), oligodendrocyte transcription factor 2 (Olig2; 1:1000; Abcam Incorporated, Cambridge, MA, USA), 29,39-cyclicnucleotide 39-phosphodiesterase (CNPase; 1:1000; Covance), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000; Sigma-Aldrich), polysialic acid (PSA)-neural cell adhesion molecule (NCAM; 1:2000; EMD Millipore), synaptophysin (1:2000; Sigma-Aldrich), or myelin-associated glycoprotein (MAG; 1:500; kind gift of Dr. Norman Latov, Cornell University, Ithaca, NY, USA). Bound antibody was detected with horseradish peroxidaseconjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) and visualized using ECL (GE Healthcare, Pittsburgh, PA, USA). Images were captured and gel bands quantified using a Gel Logic 4000 PRO imaging system (Carestream, Rochester, NY, USA). Cultured oligodendrocyte precursor cells Oligodendrocyte precursor cells (OPCs) were prepared from 5day-old Sprague-Dawley rat cortices as described previously (20). Dissociated cells were plated on culture dishes coated with poly-Dlysine (10 mg/ml; Sigma-Aldrich) in DMEM containing 15% fetal bovine serum, 2 mM glutamate, 100 mM nonessential amino acids (Life Technologies), 1 mM sodium pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10 ng/ml platelet-derived growth factor (PDGF; R&D Systems). After 7–10 days in culture, OPCs were collected by shaking (200 rpm overnight) and replated in DMEM containing 0.5% fetal bovine serum, 1% N2 supplement (Life Technologies), 2 mM glutamate, 100 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.5 mM N-acetylcysteine, and 10 ng/ml PDGF. For proliferation assays, OPCs were treated with 0–20 ng/ml PDGF along with 10 mM GM1 (Sigma-Aldrich), 10 mM GT1b (Sigma-Aldrich), or 1 mM

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(1R,2R)-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (P4) synthesized in house (21). Viable cells were quantified using CellTiter MTS (Promega, Madison, WI, USA). Alternatively, cells were labeled with 5 mg/ml bromodeoxyuridine (BrdU; SigmaAldrich), fixed (4% neutralized paraformaldehyde), stained with rabbit polyclonal anti-NG2 antibody (EMD Millipore) and mouse monoclonal anti-BrdU antibody (Sigma-Aldrich), and then with fluorescent secondary antibodies. For immunoblotting, OPCs were pretreated with GM1 (0–50 mM) for 2 hours, then stimulated with PDGF (20 ng/ml) for 10 minutes prior to harvest. A total of 50 mg proteins was resolved by SDS-PAGE and probed with antiPDGFreceptor(PDGFR)-a (CellSignalingTechnology),anti-phosphoPDGFR-a (Y849; Cell Signaling Technology), or anti-GAPDH (loading control).

Behavior Behavior was analyzed at standardized times of day for all genotypes using 7-wk-old mice. Open-field locomotion was measured using a photobeam system (San Diego Instruments, San Diego, CA, USA) consisting of a 16 3 16 inch acrylic box fitted with 16 1inch-spaced infrared beams in the x and y dimensions. Total beam breaks were recorded over 30 minutes then differentiated as central (central 8 3 8 beams) or peripheral (marginal 8 3 8 beams). Motor coordination was quantified by Rotarod (Rotamex; Columbus Instruments, Columbus, OH, USA) starting at 4 rpm and accelerating 4 rpm every 30 seconds until the mouse fell (up to 5 minutes maximum). Latency to fall was tested in 3 consecutive trials separated by 5-minute resting periods. Gait analysis used the Noldus CatWalk XT (version 9.0; Noldus Information Technology, Wageningen, The Netherlands). The data from 3–5 runs were combined and segmented to exclude portions of each run with low speed, excessive speed variation, or diminished step detection. Automated static and dynamic gait parameters were determined using CatWalk software (22, 23). Exploratory activity was measured using an Elevated Plus Maze (San Diego Instruments) consisting of 4 stainless steel runways (48 3 10 cm) connected at right angles to a central 10 3 10 cm platform. There are 2 apposing runways enclosed on 3 sides (open to the central platform) by a stainless steel wall 38 cm high, whereas the other apposing runways are open to the floor 50 cm below. Mice were placed on the center platform and the time spent in open versus closed arms recorded for 5 min. Cognitive function was assessed by passive avoidance. An enclosure was divided into one dark chamber with an underlying electrified grid (0.5 mA) and a bright chamber that was not electrified. A mouse was placed in the bright chamber, and latency to enter the dark chamber was measured. After entering the dark chamber and receiving an electric shock, the mouse was returned to its home cage. On the following day, the mouse was returned to the bright chamber and latency to enter the dark chamber measured (up to 5 minutes). The difference in latency between the 2 days reflects learning and memory of the shock event.

Statistics Results were analyzed using 1-way or repeated measures ANOVA followed by Tukey or Fisher’s least significant difference post hoc analyses when group differences were significant. For nonparametric analyses, Kruskal-Wallis 1-way ANOVA was performed followed by Dunn’s pairwise multiple comparisons if group differences were significant. Results are expressed as the means 6 SEM.

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RESULTS Ganglioside and sialoglycoprotein expression in St3gal-mutant mice Ganglioside and sialoglycoprotein expression in WT, St3gal2-null, St3gal3-null, and double-null mice is presented in Fig. 1. WT mice, like all mammals, express the same 4 major gangliosides, GM1, GD1a, GD1b, and GT1b, which comprise $94% of total gangliosides in mice and men (5, 24). Gangliosides with a sialic acid on the terminal galactose (circled structure in Fig. 1B; gangliosides GD1a, GT1b, and GQ1b) represent more than half of all ganglioside sialic acid in the brain (61% in mice and 54% in humans). Whereas total ganglioside expression (lipidlinked sialic acid) was equal in all genotypes tested, expression of terminally sialylated gangliosides varied (Fig. 1A, D). Terminal sialylation was blocked by half in St3gal2-null mice and nearly completely blocked in St3gal2/ 3 double-null mice. Quantification by thin-layer chromatography (TLC) overestimates GD1a, which comigrates with O-acetylated GD1b in mutant mice only (Fig. 1A). Mass spectrometry revealed a .97% decrease in terminally sialylated gangliosides in St3gal2/3 double-null mice (7). Decreases in terminally sialylated gangliosides were matched by increases in their precursors, GM1 and GD1b. The ganglioside pattern of St3gal3-null mice was the same as WT, whereas brain sialoglycoprotein expression was reduced by half (Fig. 1D). Sialoglycoprotein expression in double-null mice was not different from that in St3gal3single null mice. Together, these findings indicate that St3gal2-null mice are partially depleted in terminally sialylated gangliosides, St3gal3-null partially depleted in glycoprotein sialylation, and double-null mice nearly completely depleted in terminally sialylated gangliosides with protein sialylation reduced as in St3gal3-null mice (7). To evaluate whether St3gal1, St3gal4, St3gal5, or St3gal6 gene expression compensates for loss of St3gal2 and St3gal3 in double null mice, mRNA was extracted from brains of WT and St3gal2/3 double-null mice (in triplicate), and transcript profiling was performed by the National Center for Biomedical Glycomics, Complex Carbohydrate Research Center, University of Georgia (Athens, GA, USA) (25). In WT mouse brain, St3gal1 and -6 transcripts were at equally low levels, St3gal2, -3, and -4 transcripts were expressed at 2.4- to 5-fold higher levels, and St3gal5 transcripts at the highest level (28-fold that of St3gal1). In St3gal2/3 double-null mouse brains, transcript levels of St3gal1, -4, -5, and -6 were not different from those in WT brains, indicating no compensation at the transcriptional level. Brain gross anatomy and histology St3gal2/3 double-null mice are growth retarded; at weaning (21 days), they were less than half the weight of WT littermates (Fig. 2A). St3gal3-null mice were also significantly (28%) smaller than WT at weaning, whereas St3gal2-null mice were equivalent to WT. Among the genotypes tested, only St3gal2/3 double-null mice had notably smaller brains than their WT counterparts. Gross brain structures and cortical lamination appeared normal (Fig. 2B, C), the only notable difference being a thinner cortex in the double-null

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Figure 2. Growth and brain histology of sialyltransferase mutant mice. A) Representative mice and mouse brains at 4 weeks. Weights at breeding are shown for 58 WT, 36 St3gal2-null, 45 St3gal3-null, and 8 double-null mice. Genotypes were significantly different by 1-way ANOVA (P , 0.001). Pairwise groupings were significantly different as indicated (*P , 0.001, Tukey post hoc analysis). B) Nissl staining of coronal sections (upper, midlevel; lower, caudal) of 4-week-old mice. Scale bar, 1 mm. C) Neuronal distribution in the brain cortex (left) and hippocampus (right) as detected by anti-NeuN immunohistochemistry. Scale bars, 0.5 mm (cortex) and 0.2 mm (hippocampi).

mouse brain. These findings indicate global growth retardation of the brain without major anatomic deficits in selected brain regions in St3gal2/3 double-null mice. Subsequent histology, biochemistry, and behavioral studies were performed using active young adult (7–8-week-old) mice. Hypomyelination and dysmyelination The most notable anatomic deficit observed in any of the mutant mice was CNS hypomyelination in St3gal2/3 double-null mice (Fig. 3A, B). Gallyas staining revealed 40% less myelin in the corpus callosum of St3gal2/3-doublenull mice compared to WT but not St3gal2 or St3gal2/3 double-null mice. Hypomyelination was confirmed using Eriochrome and Luxol fast blue histochemistry and antiMBP immunohistochemistry, each of which demonstrated comparable decreases (data not shown). In all cases, the double-null corpus callosum was notably diminished, whereas single-null and WT mice were similar. Immunoblotting of whole-brain homogenates confirmed decreased expression of major myelin proteins in double-null mice (Fig. 3C, D). Molecular markers used in these studies are listed in Supplemental Table 1. CNPase was reduced (44%) only in the double-null mice. MBP was also reduced (34%) in double-null mice and less so (16%) in St3gal3-null mice. Remarkably, MAG expression was SIALYLATION REGULATES BRAIN STRUCTURE AND FUNCTION

reduced .90% in brain homogenates from double-null mice, whereas it was increased (;3-fold) in each of the single-null mice (Fig. 3C, E). This marked increase was MAG selective, in that other myelin proteins were not significantly increased in any of the mice. For comparison with the St3gal mutant mice, myelin proteins from age-matched Mag-null and B4galnt1-null mice were also subjected to immunoblot analyses (Fig. 3C). By comparison, all myelin markers (except MAG in Mag-null mice) were normal in these young adult mice [and see (24, 26)]. Ultrastructural analysis of the corpus callosum confirmed dysmyelination in St3gal2/3 double-null mice (Fig. 4A). The proportion of myelinated axons in the corpus callosum of WT C57Bl/6 mice was high (;90%), as reported previously (27, 28). This value was not reduced in St3gal2-null mice, significantly reduced (,10% compared to WT) in St3gal3-null mice, and markedly reduced (;30%) in double-null mice (Fig. 4B). In the double-null, myelination ranged from normal to thin to absent. Among myelinated axons, the thickness of the myelin sheath was significantly less in double-null mice, with the g ratio increased by ;10% (Fig. 4C). By comparison, St3gal2-null mice had slightly but significantly thicker myelin (6% lower g ratio), and St3gal3-null mice were the same as WT. Ultrastructural data indicate that a large proportion of axon myelination in the corpus callosum of double-null mice is either missing or diminished. 5

Figure 3. Hypomyelination in St3gal2/3 double-null mice. A) Myelin density (Gallyas stain) of midlevel coronal brain sections from WT and St3gal mutants at 7 weeks. Dotted boxes indicate areas of corpus callosum enlarged in insets. Scale bar, 1 mm. B) Intensity of Gallyas staining in the corpus callosum (n = 3–4 mice). Genotypes were significantly different by 1-way ANOVA (P , 0.02). Post hoc analyses indicated significant hypomyelination of St3gal2/3 double-null mice compared to WT (*P , 0.02, Tukey). C) Immunoblot of myelin proteins in whole-brain extracts. Complex ganglioside-deficient (B4galnt1-null, see Fig. 1C) and Mag-null mice are shown for comparison. D) Immunoblot quantification of major myelin proteins normalized to GAPDH (n = 3). Genotypes were significantly different by 1-way ANOVA (P , 0.001 for both proteins). Post hoc analyses indicated that CNPase was significantly diminished (compared to WT) in St3gal2/3 double-null mice (**P , 0.005, Tukey), whereas MBP was reduced in both double-null (**P , 0.001) and St3gal3-single null (*P , 0.02) mice. E) Immunoblot quantification of MAG (n = 3). Genotypes were significantly different by 1-way ANOVA (P , 0.01). Pairwise comparisons (t test) indicate that MAG was significantly decreased in double-null mice (**P , 0.01) and increased in the single-null mice (*P , 0.05) compared to WT.

To probe the mechanism for diminished myelination in double-null mice, OPCs were quantified using Olig2 transcription factor and NG2 proteoglycan as molecular markers. Both markers were significantly reduced in the brains of double-null mice (Fig. 5A). A reduction in NG2 was noted in single-null mice but did not reach statistical significance, whereas single-null mice displayed significantly reduced Olig2 levels that were intermediate between WT and double-null. To address whether the biochemical reduction in NG2 in double-null mice was due to a decrease in precursor cell numbers, expression of NG2-positive cell area in the corpus callosum was determined, revealing a sharp and statistically significant decline in the double-null mice (Fig. 5B), with intermediate levels in single-null mice that did not reach statistical significance. These data imply that lower OPC numbers are responsible for diminished myelination in St3gal2/3 double-null mice. To investigate one possible molecular mechanism for decreased OPCs, we tested the potential of increased or decreased gangliosides to modulate OPC proliferation in vitro using primary rat OPC cultures (Fig. 6). PDGF acting through PDGFR-a is key to OPC proliferation and the timing of OPC differentiation (20, 29, 30). Gangliosides modulate receptor tyrosine 6

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kinases (including PDGFR) via lateral association and either activation or inhibition (31, 32). To test the potential function of gangliosides in OPC proliferation, exogenous GT1b or GM1 was added to OPC cultures under conditions that lead to spontaneous incorporation into cell membranes (33). Some cultures were separately treated with P4, an inhibitor of glycosphingolipid biosynthesis that depletes all gangliosides (34). GM1 and P4 reduced PDGF-induced OPC proliferation, whereas GT1b had no effect (Fig. 6A). Labeling proliferating OPCs with BrdU revealed that the lower cell number correlated with significantly reduced DNA replication (Fig. 6B). Consistent with its effects in fibroblasts (35), GM1 inhibits PDGF-stimulated PDGFR-a tyrosine phosphorylation (at tyrosine 849, Fig. 6C). These data imply that blocking ganglioside biosynthesis or selectively increasing GM1 expression diminishes OPC proliferation leading to reduced myelination. The structure of myelin is especially well regulated at nodes of Ranvier, where adhesion molecules orchestrate the firm attachment of paranodal loops to the axon surface, physically separating the node and juxtaparanode into functional domains dominated by specific ion channels: sodium channels at the node, and potassium channels at the juxtaparanode. The structures and molecular

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Figure 4. Electron microscopy (EM) of WT and St3gal-mutant corpus callosum. A) Low-power EM images (upper row). Red arrowheads point to unmyelinated axons in St3gal2/3-double-null mice. Dotted boxes indicate areas enlarged in the row below, with selected axons marked with an asterisk. Representative images display the general state of myelination in the corpus callosum of each genotype (variations in axon number per field are not statistically significant). Scale bar, 500 nm. B) Quantification of myelinated axons. Genotypes were significantly different by Kruskal-Wallis 1-way ANOVA on ranks (P , 0.001). Pairwise groupings are significantly different as indicated (*P , 0.05, Tukey post hoc analysis). C) Myelin thickness (g ratio) as a function of axon diameter. Higher g ratios indicate thinner myelin. Each data point (n . 1000 for each genotype) is plotted, with embedded box and whisker plots (for nonnormally distributed data) indicating the median, 25–75% (box) and 10–90% (whiskers) for both g ratio and axon diameter. Genotypes were significantly different by Kruskal-Wallis 1-way ANOVA on ranks (P , 0.001). For g ratio, pairwise significance vs. WT is indicated (*P , 0.05, Dunn’s test).

distributions at peripheral (sciatic) nerve and central (optic) nerve nodes of Ranvier were determined by immunohistochemistry (Fig. 7). WT mice displayed wellregulated molecular distributions, with the node, paranode, and juxtaparanode well demarcated. Peripheral (sciatic) nerve nodes from double-null mice displayed structural abnormalities, including physical protrusion of the paranode and displaced potassium channels from the juxtaparanode into the paranode. No significant differences were noted in sciatic nerve nodes from St3gal2 or St3gal3 singlenull mice. Central (optic) nerve nodal molecular distributions were well demarcated in all mice. However, double-null mice displayed reduced paranodal length (238%) along with increased nodal length (+52%). St3gal3-null mice also displayed modestly increased nodal length (+23%). Sciatic nerve nodal lengths were not different among the genotypes, and paranodal length was slightly but significantly smaller (6%, 20.21 mm) in double-null mice only. SIALYLATION REGULATES BRAIN STRUCTURE AND FUNCTION

Neurons, astrocytes, and microglia The markers b-III-tubulin and synaptophysin in wholebrain extracts were used as indirect indicators of neuronal processes and synapses, respectively (Fig. 8A). Both markers were significantly decreased (;40%) in double-null mice. Smaller decreases in St3gal3 single-null mice were not statistically significant, and expression in St3gal2-null mice was normal. Golgi staining of hippocampal neurons was consistent with decreases in b-III-tubulin and synaptophysin, in that the dendritic arbors of hippocampal neurons were noticeably diminished in double-null mice (Fig. 8B). PSA expression was significantly diminished in all mutant genotypes tested (Fig. 8A). Decreases in PSA expression in St3gal3 single-null and St3gal2/3 double-null mice were 50–60% and not statistically different from each other. Expression in St3gal2-null mice was reduced, but less so (30%). 7

astrogliosis (anti-GFAP) was found in double-null hippocampi (Supplemental Fig. 1B). Immunoblot analyses of whole-brain homogenates confirmed the increase in astrocytes in double-null mice (2-fold increase in GFAP) with no change in either single-null genotype (Supplemental Fig. 1C). Behavior

Figure 5. Fewer OPCs in St3gal2/3 double-null mice. A) Immunoblots of OPC markers Olig2 and NG2 (and GAPDH loading control) in whole-brain extracts. Complex ganglioside-deficient (B4galnt1-null) and Mag-null mice are shown for comparison. Quantified immunoblot data for OPC marker proteins normalized to GAPDH are shown. Genotypes were significantly different by 1-way ANOVA (n = 3; P , 0.02 for NG2; P , 0.001 for Olig2). Post hoc pairwise analyses compared to WT are indicated (*P , 0.05 and **P , 0.01, Tukey). Olig2 was also reduced in double-null mice compared to each of the single-null genotypes (P , 0.05, Tukey). B) OPCs in the corpus callosum were quantified by immunohistochemistry. NG2-positive pixel areas within a well-defined area of corpus callosum were combined for 5 brain coronal sections from each of 4–6 mice of each genotype. Genotypes were significantly different by 1-way ANOVA (P # 0.005). Post hoc pairwise analyses indicated significantly reduced OPCs in double-null mice compared to WT (*P , 0.002, Tukey).

GFAP and Iba-1 were used as markers to quantify astrocytes and microglia, respectively. Immunohistochemical staining of the corpus callosum (Supplemental Fig. 1A) revealed increased infiltration of astrocytes (3-fold) and microglia (2-fold) in double-null mice, whereas staining in St3gal2 and St3gal3 single-null mice was equivalent to WT. A comparably increased level of 8

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Rotarod tests found that all mutant genotypes had significantly reduced motor coordination, with double-null mice significantly more affected than single-null strains (Fig. 9A). Detailed gait analyses revealed additional alterations in motor coordination, including a marked change in relative hindlimb/forelimb foot placement in double-null mice (Fig. 9B). Whereas WT and single-null mice consistently placed their hindlimb behind the last placement of their forelimb on the same side, double-null mice overstepped and placed their hindlimb in front of the last placement of the forelimb. This altered gait was matched by a change in step sequence (Fig. 9C) in which double-null mice avoided following a forelimb placement with a hindlimb placement on the same side (Aa) in favor of bringing the hindlimb on the opposite side forward (Ab). By comparison, WT and single-null mice had similar quantitative step sequence patterns. A listing of all gait parameters that were different only in St3gal2/3-double null mice is provided in Supplemental Table 2. A dozen gait parameters are significantly altered in double-null mice only. Some of these are related to their smaller size (e.g., mean step intensity), but others relate to speed and coordination. Despite their diminished motor skills, double-null mice push off faster (Max Contact At %, Supplemental Table 2), spend less time with 4 paws in contact with the surface, and more time with only 1 paw in contact. For each step, they spend more time swinging and less time in contact with the surface (Duty cycle, Supplemental Table 2). Consistent with the gait parameters, and in contrast to their motor deficits, St3gal3 single-null and St3gal2/3 doublenull mice were hyperactive when placed in an open-field test box (Fig. 9D). All of the increased activity was on the box periphery, associated with enhanced exploratory behavior. This tendency was confirmed using an Elevated Plus Maze, in which mice were given the opportunity to walk along enclosed or exposed pathways well above the floor. Whereas WT and single-null mice spend much more time in enclosed pathways (5:1–10:1), that ratio is sharply reduced to ,2:1 in double-null mice (Fig. 9E). A passive avoidance test was used to evaluate learning and memory (Fig. 9F). Given the choice, WT mice move quickly from a light to a dark chamber (average ;10 s). When they receive an electric shock in the dark chamber on day 1, they either fail to enter or delay entering when retested on day 2 (average .214 seconds, with a 300-second cutoff). By comparison, each mutant mouse strain took longer to enter the dark chamber on the first day (average 25–50 s), with St3gal3-null and double-null mice taking the longest, consistent with their enhanced exploratory behavior. Notably, reentry on day 2 was not significantly delayed for any of the mutants, with double-null mice actually entering more quickly on day 2.

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Figure 6. Ganglioside modulation of OPC proliferation. A) Rat OPC proliferation was induced with PDGF at the indicated concentrations. Cells were cotreated with GM1, GT1b, or P4 (glycosphingolipid biosynthesis inhibitor) as indicated. Viable cells after 6 days in culture were quantified, and PDGF enhancement is presented as the fold increase compared to cells cultured in the absence of PDGF or other effectors (mean 6 SEM). GM1 or P4 significantly diminished PDGF-mediated OPC proliferation (n = 6; P , 0.01 by 1-way ANOVA; *P , 0.05 by Tukey post hoc analysis). B) OPCs cultured in the presence of PDGF (10 ng/ml) were labeled with BrdU, fixed, and stained with antiNG2 (green) to identify OPCs and anti-BrdU (red) to identify proliferating cells. Scale bar, 50 mm. Arrowheads indicate NG2/BrdU doublepositive cells. GM1 and P4 significantly reduced the number of proliferating cells (n = 3; P , 0.01 by 1-way ANOVA; *P , 0.05 by Tukey post hoc analysis). C) OPCs cultured in the presence of the indicated concentrations of GM1 for 2 hours were then treated with PDGF (20 ng/ml) for 10 minutes. Solubilized cell proteins were resolved by SDS-PAGE and immunoblotted using anti-PDGFR-a-phosphoY849, anti-PDGFR-a, and GAPDH (loading control).

To rule out visual abnormalities as a major contributing factor, depth perception was evaluated (36). Mice were held by the tail and slowly lowered to a surface, and the tendency of the mice to extend their forelimbs as they reached the surface was assessed. This task was accomplished, and no difference was noted between genotypes. DISCUSSION Glycosylation is a defining characteristic of cell surfaces (37). Although glycosylation is structurally diverse, all mammalian brains express 4 closely related structures, gangliosides GM1, GD1a, GD1b, and GT1b, as prominent cell surface glycans (1). The functions of the major brain gangliosides, and the reasons for their evolutionary maintenance, are not well understood. However, human congenital disorders of ganglioside biosynthesis and mouse genetic models provide insights. Human mutations in the ganglioside-selective biosynthetic genes ST3GAL5 and B4GALNT1 result in seizure disorders and progressive motor neuropathy, respectively, both accompanied by cognitive deficits (11, 12, 14, 38). These phenotypes are reflected in mouse models; B4galnt1 mutant mice display progressive motor deficits (39, 40), and mice expressing SIALYLATION REGULATES BRAIN STRUCTURE AND FUNCTION

only GM3 suffer fatal audiogenic seizures (41). Because these mutations are early in the ganglioside biosynthetic pathway (Fig. 1C) and disrupt the expression of all complex gangliosides, the functions of individual major gangliosides cannot be inferred. Recently, we discovered that 2 of the 20 sialyltransferases common to mice and humans, St3gal2 and St3gal3, are responsible for addition of sialic acid to the terminal galactose of the gangliotetraose core (circled in Fig. 1B; referred to here as “terminal sialylation”) and are directly responsible for biosynthesis of 2 of the 4 major gangliosides, GD1a and GT1b (7). Because St3gal2-null mice express half the normal amount of GD1a/GT1b and St3gal3-null mice express half the normal amount of brain sialoglycoproteins (Fig. 1C), comparing the 2 single-null and the double-null mice was required to infer the functions of terminally sialylated gangliosides and sialoglycoproteins. Deficits that are particularly severe in double-null mice are likely due to changes in terminally sialylated gangliosides, whereas those shared by St3gal3-null and St3gal2/3 doublenull are possibly driven by altered sialoglycoproteins. Because other St3gal isoform transcripts are unchanged in the double-null mice, we infer that there is no compensation at the transcriptional level despite clear evidence in other studies of function-dependent regulation of St3gal isoform gene expression (42). In terms of ganglioside expression, it 9

Figure 7. Immunohistochemical analyses of nodes of Ranvier. A) The molecular structure of nodes is revealed by immunohistochemistry using NF (blue) for the node, Caspr (green) for the paranode, and potassium channel (Kv1.2, red) for the juxtaparanode. Arrows indicate paranodal protrusion, and arrowheads indicate paranodal invasion by potassium channels. B) Sciatic nerve paranodal abnormalities (left) were quantified in 2 sciatic nerve sections from each of 3–4 animals of each genotype. Optic nerve nodal (distance between adjacent Caspr clusters; n ; 70) and paranodal (Caspr cluster length; n ; 70) length was quantified in optic nerves from 3 animals of each genotype (right). Data are shown as box-andwhisker plots for nonnormally distributed data: median (line); 25th and 75th percentiles (lower and upper box edges) and minimum and maximum (whiskers) are shown. Statistical analyses used the Mann-Whitney U test with Bonferroni’s correction (*P , 0.01; **P , 0.001; ns, not significant).

should be noted that near-quantitative loss of GD1a and GT1b in St3gal2/3 double-null mice is matched by quantitative increases in GM1 and GD1b. Pathogenic loss of function (GD1a and GT1b) as well as pathogenic gain of function (GM1 and GD1b) must be equally considered. The clearest deficit observed in St3gal2/3 double-null mice was dysmyelination (Figs. 3 and 4). Histochemical and biochemical measures of myelination were exclusively or selectively diminished in double-null mice. Mechanistic studies indicated that these effects may be due to a decrease in OPC proliferation downstream of reduced sensitivity to PDGF, a regulator of OPC proliferation (20, 30). Expression of PDGFR-a identifies OPCs in the developing CNS and regulate the timing of their conversion from proliferation to differentiation. OPCs from PDGF-a receptor-null mice have proliferation defects and rapidly differentiate into postmitotic oligodendrocytes (29). Because gangliosides regulate receptor tyrosine kinases, including PDGFRs (35), we hypothesized that altered ganglioside expression resulted in altered OPC proliferation. Data support this hypothesis, in that St3gal2/3 10

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double-null mice had fewer OPCs, and treatment of cultured OPCs with GM1 (which is increased in the double-null mice) or a general inhibitor of glycosphingolipids reduced their proliferation in response to PDGF. The direct inhibition of oligodendrocyte proliferation by GM1 is consistent with data from patients with GM1 gangliosidosis, who build up GM1 in the brain and have decreased oligodendrocyte numbers and less myelin (43). Broader analyses of the structure-function relationships of gangliosides in PDGF-a regulation may be of value in interpreting genetic faults in ganglioside expression. Gangliosides detected by the mAb A2B5 are a marker of proliferative OPCs, disappearing upon differentiation to oligodendrocytes (44). A2B5 recognizes gangliosides GT3 and acetylated GT3 (45), which are unlikely to be affected by St3gal2/3 mutations because oligodendrocytes normally express high concentrations of their precursors GM3 and GD3 that are unaffected by these mutations (46). Nevertheless, transcript analyses of A2B5-expressing cells and their differentiated (O4-positive) counterparts from rat

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Figure 8. Neuronal markers in St3gal-mutant mice. A) Immunoblots of neuronal markers in whole-brain extracts from 3 individuals of each genotype. Genotypes were significantly different by 1-way ANOVA (n = 3; b-III-tubulin and synaptophysin, P , 0.02; PSA-NCAM, P , 0.001). Post hoc pairwise analyses compared to WT are indicated (* P # 0.02 and **P , 0.001, Tukey). B) Golgi-stained hippocampal CA1 pyramidal neurons. Scale bar, 100 mm.

brain demonstrated that St3gal2 transcripts were downregulated 3.8-fold, whereas St3gal3 transcripts were up-regulated 3-fold [(47) and related Gene Expression Omnibus database submissions http://www.ncbi.nlm.nih. gov/geoprofiles/31224781 and http://www.ncbi.nlm.nih.gov/ geoprofiles/31228656]. Defective nodes of Ranvier in St3gal2/3 double-null mice (Fig. 7) indicate that gangliosides mediate axon-myelin structure as well as OPC proliferation. B4galnt1-null mice, bearing a mutation earlier in the ganglioside biosynthetic pathway, showed similarly defective nodes of Ranvier (48), and genetic studies in mice with altered ganglioside expression revealed widespread progressive myelin instability (40, 48–51). These data, together with biochemical studies demonstrating that MAG binds selectively to the terminal sialic acid on GD1a and GT1b (52, 53), led to the hypothesis that stable axon-myelin interactions require the binding of MAG, on myelin, to GD1a and GT1b on axons (50). A remarkable finding of the current study was the .90% reduction in MAG expression exclusively in doublenull mice. These data are consistent with the hypothesis that MAG is destabilized in the absence of its ganglioside binding partners on axons in B4galnt1-null mice (24). In that study, loss of MAG was less extensive and only occurred over 6–12 months, perhaps due to the overexpression of GM3 (a MAG ligand, albeit a poor one) in B4galnt1-null mice compared with the overexpression of GM1 and GD1b (not MAG ligands) in St3gal2/3 double-null mice. Nevertheless, our data imply functions of gangliosides in myelination beyond their interaction with MAG (regulation of OPC proliferation) because young Mag-null mice do not display the same myelination deficits as young St3gal2/3 double-null mice (26, 54). Consistent with altered myelination, double-null mice displayed diminished motor coordination and altered gait, as do mice with mutations earlier in the ganglioside biosynthetic pathway (40). Despite their diminished motor abilities, St3ga/3 single-null and St3gal2/3 double-null mice were hyperactive, implicating glycoprotein sialylation as SIALYLATION REGULATES BRAIN STRUCTURE AND FUNCTION

a contributing factor. All 3 sialyltransferase mutant mouse lines displayed marked cognitive deficits as measured using a passive avoidance learning task at which WT siblings were adept (Fig. 9F). This measure of a more complex cognitive behavior appears to require quantitative ganglioside terminal sialylation, in that St3gal2-null mice are affected even though they have normal glycoprotein sialylation levels and express all 4 major brain gangliosides, albeit with altered concentrations compared to WT mice (Fig. 1A, D). Likewise, St3gal3-null mice, which have normal gangliosides but altered glycoprotein sialylation, are similarly affected. We conclude that both glycoprotein and glycolipid sialylations are required for the proper development and function of circuits required for acquisition of passive avoidance learning. PSA was diminished in the brains of all 3 mutant lines but more so and equally in St3gal3 single-null and St3gal2/3 double-null mice (Fig. 8A). PSA is a highly directed form of glycosylation, primarily on NCAM, in which polymers of a-2-8-linked sialic acid extend from terminal glycoprotein sialic acids. Based on our data, over half of PSA biosynthesis in the brain depends on a-2,3-sialylation by the St3gal3 gene product, which is highly expressed in the brain and generates N-linked glycan sialylation known to be required for PSA addition (55). Because many of the phenotypes noted in the current studies are exclusive or exacerbated in the double-null mice, they are unlikely to be related to PSA alterations, although those phenotypes shared by St3gal3-null and double-null mice may. Our data support the conclusion that both ganglioside and sialoglycoprotein terminal sialylations are required for development of optimal structure and function in the mammalian brain, and provide the context for consideration of the effects of sialylation in human genetic diseases. Human mutations in ST3GAL5 result in early seizures with motor and cognitive dysfunction (10–12, 38). This mutation early in ganglioside biosynthesis likely affects all complex brain gangliosides. Interestingly, St3gal5-null mice have relatively normal brain anatomy and function (56), 11

Figure 9. Behaviors of St3gal-mutant mice. A) Motor coordination and balance were evaluated using an increasing rate Rotarod test. Data from 3 consecutive trials are shown (n = 20 WT, 20 St3gal2-null, 21 St3gal3-null, and 13 double-null mice). Genotypes were significantly different by 1-way ANOVA (P , 0.001). Post hoc pairwise analyses (Tukey) indicated that all genotypes performed worse than WT (*P , 0.05; **P , 0.001). B) Print position (CatWalk) was determined by measuring the average placement of the hindlimb in reference to the prior placement of the forelimb on the same side (n = 7 for each genotype). Zero indicates that the hindlimb was placed at the same position as the prior forelimb, with positive values indicating placement behind and negative values indicating placement ahead of the prior forelimb placement. Genotypes were significantly different by 1-way ANOVA (P , 0.001). Post hoc pairwise analyses indicated that double-null mice were significantly different than all other genotypes (**P , 0.001, Tukey). C) Step sequences (CatWalk) categorized as 1 of 6 regular patterns (22). The 4 shown (Ca, Cb, Aa, and Ab) comprise 97–99% of the step patterns recorded for each genotype (n = 7 for each genotype). Genotype vs. step sequence was significantly different by 2-way repeated measures ANOVA (P , 0.01). Post hoc analyses indicated that doublenull mice were the only genotype that differed significantly from WT mice, with a decrease in Aa and increase in Ab patterns (*P , 0.02 and **P , 0.001, Tukey). This change indicates fewer sequential hindlimb steps following a forelimb step on the same side (Aa) and more following a forelimb step on the alternate side (Ab). D) Open-field activity was determined over a 30 min test period (n = 19 WT, 18 St3gal2-null, 22 St3gal3-null, and 16 double-null mice). Genotypes were significantly different by ANOVA on ranks (P , 0.001) for total beam breaks and peripheral beam breaks but did not vary in central beam breaks. Post hoc pairwise analyses demonstrate that St3gal3-null and double-null mice were significantly hyperactive in total and in the peripheral field compared to WT (*P , 0.05, Dunn’s test). E) Exploratory behavior was determined using an Elevated Plus Maze, in which WT mice keep largely to the enclosed arms (n = 19 WT, 19 St3gal2-null, 20 St3gal3-null, and 16 double-null mice). Genotypes were significantly different by ANOVA on ranks (P , 0.001). Pairwise post hoc analyses indicate that double-null mice were significantly different from WT (*P , 0.05, Dunn’s test). F) Learning/memory was measured using a passive avoidance task, in which learning increases the latency to enter an electrified chamber after a 1 day interval. Whereas WT mice were significantly delayed in their reentry (*P = 0.002; ns, not significant), none of the mutant mice was significantly delayed (n = 5 for each genotype). The difference in latency (day 2 2 1) was significantly different among genotypes by 1-way ANOVA (P = 0.001), with post hoc pairwise analyses indicating reduced differences between WT mice and each of the mutant genotypes (P , 0.01).

perhaps due to increased expression of the normally rare gangliosides, GM1b and GD1a, which maintain the terminal a-2-3 ganglioside sialic acid on an alternately sialylated core structure in mice (56). It is not known whether the same biosynthetic compensation occurs in humans. However, St8sia1-B4galnt1-double null (GM3 only) mice, in which GM3 builds up behind the double biosynthetic block (41), display early severe audiogenic seizure, a deficit modified by the strain background (57). Humans with mutations in B4GALNT1 have a milder disease characterized by hereditary spastic paraplegia including progressive loss of motor coordination accompanied by mild-to-moderate cognitive impairment in all 12

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subjects and treatable seizure activity in some (13, 14). Similarly, B4galnt1-null mice have a dysmyelination phenotype with progressive gait and motor coordination disorders, accompanied by hyperactivity (40, 49). Consistent with human genetic disorders of ganglioside biosynthesis (10–14, 38), as well as human mutations in ST3GAL3 (15, 16), which likely affect glycoprotein sialylation, mice deficient in either St3gal2 or St3gal3 display marked cognitive disability (Fig. 9F). Because St3gal2-null mice have quantitatively (but not qualitatively) altered gangliosides and little change in glycoprotein sialylation, whereas St3gal3-null mice have the opposite (Fig. 1D), human genetic disorders and mouse genetic data imply

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that quantitative deficits in either ganglioside or glycoprotein terminal sialylation result in cognitive dysfunction. Questions remain concerning the cellular bases for the various anatomic and behavioral deficits reported. Conditional null mice in St3gal gene isoforms will help resolve these questions, as they did in the case of B4galnt1-null mice, in which neuronal-specific transgenic expression of B4galnt1 in knockouts was sufficient to counter the deficits of animal-wide knockout, whereas glial-specific transgenic expression was not (58). The specific structures and mechanisms responsible for dysmyelination, hyperactivity, and cognitive dysfunction in mice and humans with altered terminal sialylation have yet to be fully determined. Nonetheless, it is clear that terminal sialylation is required for optimal brain structure and function, that humans and mice share many aspects of sialic acid neuroglycobiology, and that genetically altered mice are relevant models for further study of sialoglycan functions in the brain. The authors thank Jamey D. Marth (University of California, Santa Barbara, Santa Barbara, CA, USA) for providing founder mice for these studies and Alison Nairn (University of Georgia, Athens, GA, USA) for expert transcript analyses. This work was supported by U.S. National Institutes of Health, National Institute of Neurological Disorders and Stroke Grants NS037096 and NS057338 (to R.L.S.) and a U.S. National Multiple Sclerosis Society grant (to Dr. Matthew N. Rasband, Department of Neuroscience, Baylor College of Medicine in support of K.S.). The views of the authors do not purport or reflect the position of the U.S. Department of the Army or the U.S. Department of Defense (para 4-3, AR 360-5). All research was conducted in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals (U.S. National Research Council), and other federal statutes and regulations relating to animals and experiments involving animals.

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Received for publication January 29, 2015. Accepted for publication March 11, 2015.

YOO ET AL.