Physiol Rev 94: 461–518, 2014
doi:10.1152/physrev.00033.2013
SIALIC ACIDS IN THE BRAIN: GANGLIOSIDES AND
POLYSIALIC ACID IN NERVOUS SYSTEM
DEVELOPMENT, STABILITY, DISEASE, AND
REGENERATION
Ronald L. Schnaar, Rita Gerardy-Schahn, and Herbert Hildebrandt
Departments of Pharmacology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore,
Maryland; and Institute for Cellular Chemistry, Hannover Medical School, Hannover, Germany
Schnaar RL, Gerardy-Schahn R, Hildebrandt H. Sialic Acids in the Brain: Gangliosides
and Polysialic Acid in Nervous System Development, Stability, Disease, and Regeneration.
Physiol Rev 94: 461–518, 2014; doi:10.1152/physrev.00033.2013.—Every cell in
nature carries a rich surface coat of glycans, its glycocalyx, which constitutes the cell’s
interface with its environment. In eukaryotes, the glycocalyx is composed of glycolipids,
glycoproteins, and proteoglycans, the compositions of which vary among different tissues and cell
types. Many of the linear and branched glycans on cell surface glycoproteins and glycolipids of
vertebrates are terminated with sialic acids, nine-carbon sugars with a carboxylic acid, a glycerol
side-chain, and an N-acyl group that, along with their display at the outmost end of cell surface
glycans, provide for varied molecular interactions. Among their functions, sialic acids regulate
cell-cell interactions, modulate the activities of their glycoprotein and glycolipid scaffolds as well as
other cell surface molecules, and are receptors for pathogens and toxins. In the brain, two families
of sialoglycans are of particular interest: gangliosides and polysialic acid. Gangliosides, sialylated
glycosphingolipids, are the most abundant sialoglycans of nerve cells. Mouse genetic studies and
human disorders of ganglioside metabolism implicate gangliosides in axon-myelin interactions, axon
stability, axon regeneration, and the modulation of nerve cell excitability. Polysialic acid is a unique
homopolymer that reaches ⬎90 sialic acid residues attached to select glycoproteins, especially the
neural cell adhesion molecule in the brain. Molecular, cellular, and genetic studies implicate
polysialic acid in the control of cell-cell and cell-matrix interactions, intermolecular interactions at
cell surfaces, and interactions with other molecules in the cellular environment. Polysialic acid is
essential for appropriate brain development, and polymorphisms in the human genes responsible
for polysialic acid biosynthesis are associated with psychiatric disorders including schizophrenia,
autism, and bipolar disorder. Polysialic acid also appears to play a role in adult brain plasticity,
including regeneration. Together, vertebrate brain sialoglycans are key regulatory components that
contribute to proper development, maintenance, and health of the nervous system.
L
I.
II.
III.
IV.
V.
VI.
INTRODUCTION
SIALIC ACIDS: PROMINENT...
BRAIN-ENRICHED SIALOGLYCANS
BRAIN GANGLIOSIDE FUNCTIONS
POLYSIALIC ACID FUNCTIONS
SIALOGLYCANS AND DISEASES...
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462
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483
491
498
I. INTRODUCTION
Evolution has yet to generate a living cell without a dense
and complex coating of sugars (513). In eukaryotes, cell
surface sugars exist as glycoproteins, glycolipids, and proteoglycans, previously called complex carbohydrates but
now commonly referred to as glycans (to distinguish them
from dietary sugars). Invisible using standard microscopic
techniques, glycans are dominant chemical and physical
features of the extracellular surface of all cells, forming a
deep, rich, and diverse glycocalyx (FIGURE 1). Glycans are
the cell’s interface with their outside world (159, 186, 481,
515); cell surface glycans (among other functions) mediate
cell-cell recognition and regulate interactions between cells
and other components in their local environment (e.g., pathogens, toxins, hormones, etc.). Evolution selected glycans to
serve these roles based on their physical characteristics and
structural diversity. Because they are predominantly hydrophilic and often negatively charged, glycans are hydrated and
spread out in space (395). Compared with proteins, which fold
in on themselves, glycans occupy much more space per unit
mass and their conformations are very sensitive to even minor
changes in chemical structure. Because each sugar-sugar bond
can exist in two configurations (␣ and ␤), at any of multiple
hydroxyls and in branched arrays, a relatively small number of
fundamental building blocks (monosaccharides) combine to
create a wide diversity of oligosaccharide structures. As the
0031-9333/14 Copyright © 2014 the American Physiological Society
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BRAIN SIALOGLYCANS
FIGURE 1. Glycocalyx. All cells carry a surface glycan coat which
varies in size and composition. In the electron micrograph shown
(magnification ⫻120,000), the glycan coat of a fibroblast in culture
(BHK-21 cell) appears black after staining with ruthenium red. The
underlying lipid bilayer of the plasma membrane is distinctly visible as
a gray strand. The glycocalyx extends outward ⬃40 nm, dwarfing
the ⬃6-nm-thick bilayer. [From Martinez-Palomo et al. (286). Reprinted by permission of the American Association for Cancer Research.]
technologies required to determine glycan structure, expression, and function have improved, glycobiology has grown in
its contributions to an enhanced understanding of physiology
and pathology.
Cell surface glycans modulate cell functions by several distinct mechanisms (508). Because they are often large, hydrated, and anionic, glycans dominate the architecture and
physical properties of cell surfaces. Proteoglycans (such as
heparan and chondroitin sulfates) with their long glycosaminoglycan chains and mucins (high-molecular-weight
heavily glycosylated anionic glycoproteins) are common examples of glycans that define the physical milieu of the cell
surfaces on which they reside. Regardless of their size, addition of glycans to proteins as cotranslational or posttranslational modifications regulates the protein’s folding,
steady-state cellular distribution, stability, and functions.
Alterations of glycoprotein glycosylation can result in
changes as dramatic as misfolding and delivery to the proteasome, to altered cellular or tissue distributions, or loss of
function (e.g., for enzymes and ion channels). Such changes
are the basis for a class of human diseases called congenital
disorders of glycosylation (CDG) that result in severe developmental defects (150). Another major way in which cell
surface glycans regulate cell function is by specific interactions with complementary glycan binding proteins (518).
There are several families of glycan binding proteins,
termed lectins, each of which carries a distinct carbohydrate
recognition polypeptide domain evolved to bind specific
target glycans. In humans there are ⬎80 lectins in at least 12
distinct families (127). They mediate events as diverse as
protein sorting and cell-cell recognition. The physical and
biochemical functions of glycans are not mutually exclusive; glycans of large mucins or defined sequences on large
glycosaminoglycans can act as receptors for lectins, for example. Finally, as shown for the role of polysialic acid in
brain development, glycosylation can serve as a modifier of
protein function (195) (see sect. VB3). The diversity in glycan structures and their various mechanisms of action have
provided a wealth of evolutionary opportunities to engineer
molecular events and interactions.
462
Evolution favored glycans as cell surface molecules, at least
in part, because they have the ability to encode diverse
structures with very few building blocks (37). This structural and functional diversity and complexity is a challenge
to those in and out of the field, and in the past limited the
advancement of glycomics compared with genomics and
proteomics. Unlike nucleic acids and proteins, which are
template-based and linearly constructed, glycans are built
by suites of biosynthetic enzymes termed glycosyltransferases (59, 253), each of which adds a saccharide building
block to the growing glycan chain. The glycosidic linkage
formed between two sugars can, in theory, involve any of a
number of hydroxyls on the penultimate sugar giving either
linear or branched arrays. Whereas three different amino
acids or nucleic acids can combine to form six distinct structures, three different hexoses can combine to form over a
thousand distinct structures. With many glycans having a
dozen or more saccharides, the potential structural diversity
becomes truly daunting. Luckily for glycoscientists working
to functionally decode the glycome, there are biosynthetic
limitations and rules that confine the number of possible
structures, and the analytical power of mass spectrometry
and NMR have greatly enhanced our ability to sequence
glycans (40). Advances in the biochemistry, cell biology,
and genetics of glycobiology have provided the means to
relate glycan structures to physiology and pathology (351).
The result is that glycoscience is emerging as a major contributor to the understanding of biology and medicine.
The functional specificity of glycans is often dominated by
the sugars at the outermost ends of glycan chains. In mammals, glycoproteins and glycolipids are often terminated
with sialic acids, nine-carbon backbone sugars with distinctive chemical properties that determine their functions (86).
Sialic acids play a particularly important role in cell and
molecular interactions of glycans in mammals. Interruption
of sialic acid biosynthesis is embryonic lethal in mice, and
more subtle mutations in sialic acid metabolism result in a
variety of diseases in humans (196). In this review, we discuss the structures and roles of sialic acids, the diverse family of sialic acid-bearing glycans (the “sialome”), and draw
particular attention to two subclasses of sialoglycans, gangliosides and polysialic acids, that play decisive roles in
nervous system physiology and pathology.
II. SIALIC ACIDS: PROMINENT
DETERMINANTS OF THE CELL
SURFACE
A. Sialic Acid Structure and Diversity
Sialic acids (Sia) are nine-carbon backbone sugars bearing
chemical moieties well suited for their functions at the cell
surface (77, 413) (FIGURE 2). The most common Sia in
humans by far is N-acetylneuraminic acid (NeuAc). It is a
relatively strong acid (pKa 2.6) due to the C-1 carboxylate
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SCHNAAR ET AL.
FIGURE 2. Sialic acids. The sialic acids of animals are based on
neuraminic acid (Neu), which is typically found in its N-acetyl (NeuAc)
or N-glycolyl (NeuGc) forms, or on 2-keto-3-deoxy-nonulosonic acid
(Kdn). Other modifications (O-acylation, sulfation, phosphorylation,
cyclization) result in ⬎50 naturally occurring structures (412).
vicinal to the C-2 anomeric carbon. The exocyclic glycerol
side chain (C-7, C-8, C-9) provides the opportunity for
extensive hydrogen bonding. The N-acetyl group provides
opportunities for hydrophobic interactions. Each of these
moieties plays important roles in the binding specificities
and functions of sialoglycans.
The nomenclature of the sialic acids is complicated because
of their concomitant discovery as components of bovine
salivary mucins by Blix et al. (44) and human brain gangliosides by Klenk and Lauenstein (237). Blix named his sub´ ı␱), and Klenk
stance “sialic acid” after saliva (Greek ␴␣␭
named his substance “neuraminic acid” after the neurons of
brain gray matter. To further complicate matters, Blix isolated an N,O-diacetylated substance, whereas Klenk characterized the unacetylated free amine (174). By the time the
relationship between these substances was sorted out, there
were two names that have persisted to this day. Sialic acid
(Sia) now refers to any member of the inclusive nonulosonic
acid family (FIGURE 2). Neuraminic acid (Neu, 5-amino3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid) refers to the C-5 free amine form, which is rarely encountered
in nature. The most abundant sialic acids in nature are the
N-acetyl and N-glycolyl derivatives of Neu, NeuAc, and
NeuGc, respectively. An additional family member, ketodeoxynonulosonic acid (Kdn, 3-deoxy-D-glycero-D-galactonon-2-ulosonic acid), has no amine, instead bearing a hydroxyl at C-5. Although the term sialic acid is properly used
to refer to any member of this family, it is also commonly
used to refer to NeuAc, which is far and away the most
abundant sialic acid in humans.
Sialic acids have an interesting natural history. Only in the
deuterostome lineage (vertebrates and a few higher invertebrates) are sialic acids expressed abundantly (15). In other
lineages (bacteria, archaea, plants, fungi, protozoa, and
protostomes), Sia expression is detected in only a small
minority of species (525). Gram-negative bacteria that are
associated with human or animal hosts are by far the majority in this latter group and often the causative agent for
serious illness (510). Some neuroinvasive bacteria (e.g.,
Escherichia coli K1 and Neisseria meningitidis serogroup B)
express high-molecular-weight homopolymers of ␣2– 8and/or ␣2–9-linked NeuAc called polysialic acid (polySia)
(525). The size and charge of these Sia polymers support
bacterial dissemination by downmodulation of complement activity (6, 425). Moreover, the structural similarity
of the ␣2– 8-linked polySia capsule produced by Neisseria
meningitidis serogroup B to polySia synthesized in the human host hampers the initiation of an immune response
(589). A few other pathogenic bacteria (e.g., Campylobacter jejuni) express remarkably complex sialoglycans on lipooligosaccharides of their outer membranes (575). Some
of these glycans are so similar to vertebrate brain sialoglycans that they induce autoimmune neuropathies, as will be
discussed later in this review (577).
Sialic acids are rare in invertebrates, so much so that they
were long considered to be absent except in a few higher
species (15). This changed with the sequencing of the Drosophila genome, which revealed sequences similar to mammalian sialoglycan biosynthetic genes (244), including a
single sialyltransferase (DsiaT). Glycan analysis confirmed
the presence of NeuAc on Drosophila glycoproteins (17,
243). Notably, DsiaT is expressed only in a subset of differentiated central nervous system (CNS) neurons (381).
Disruption of the DsiaT gene results in pronounced locomotor defects, progressive loss of coordination, and progressive temperature-sensitive paralysis. These phenotypes
may be related to apparent defects in the development and
physiology of neuromuscular junctions and alterations in
the function of voltage-gated sodium channels. These data
reveal perhaps the most ancient functions of sialic acid, and
focus evolutionary attention on sialylation in the nervous
system.
More recent events in evolution hold hints as to sialic acid’s
roles in physiology and pathology inside and outside of the
nervous system. Whereas most vertebrates, including our
closest evolutionary relatives (bonobos and chimps), express
significant amounts of NeuGc as well as NeuAc (see FIGURE
2), humans only synthesize NeuAc (81). The inability of
humans to synthesize NeuGc is due to an exon deletion in
the gene responsible for converting the N-acetyl to the Nglycolyl form, CMP-N-acetylneuraminic acid hydroxylase
(CMAH). Molecular clock comparison of the disrupted human and intact chimpanzee genes places the insertion at ⬃3
million years ago. What caused the species to diverge? Since
sialic acid recognition is a virulence factor for some pathogens, it has been speculated that evolutionary selection
against expression of functional CMAH in the human lineage was due to a catastrophic pandemic by a pathogen that
targeted NeuGc (512). Varki (511) has termed this theoretical event the “sialoquake.” His hypothesis is consistent
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BRAIN SIALOGLYCANS
with the glycan binding specificities of related human and
non-human pathogens, and with evolutionary changes in
immune system sialic acid binding proteins that postdate
the loss of CMAH. Notably, even among species in which
NeuGc is the major sialic acid of most tissues, it is conspicuously diminished in the brain, where NeuAc greatly predominates. The Cmah gene in mice, for example, is expressed robustly in liver, kidney, thymus, and spleen, but
transcripts are undetectable in brain. The reason that
NeuAc is selected over NeuGc in the brains of non-human
mammals remains a mystery, but may relate to specific
functions of NeuAc in molecular recognition in the brain
(106).
Whereas variations at C-5 define core sialic acid structures
(FIGURE 2), O-substitutions at C-4, C-7, C-8, and/or C-9
(including acetyl, methyl, lactyl, sulfate, and phosphate
groups) generate further diversity in the Sia family (15,
412). In addition, the anionic C-1 carboxylate can form
lactones and lactams, neutralizing its charge. Together,
over 50 naturally occurring sialic acid structures have been
identified. In some cases, Sia diversity has been tied to Sia
function. For example, the influenza C virus hemagglutinin
binds only to NeuAc bearing a 9-O-acetyl group (387). The
budding virus releases itself from cells using a Sia-specific
O-acetylesterase (190). In non-human primates, Siglec-9, a
sialic acid-binding immune regulatory protein, binds preferentially or exclusively to sialoglycans terminated with
NeuGc, the most abundant Sia in those species. In contrast,
human Siglec-9 has a broader specificity, binding both
NeuAc and NeuGc. One can infer that after the “sialoquake,” human-lineage Siglec-9 variants that had the ability to bind NeuAc as well as NeuGc provided a selective
advantage (455).
The functions of sialic acids at the cell surface depend
on sialoglycan structures at several levels, from the modifications on each sialic acid carbon to their context within
oligosaccharides, larger glycans, and multiglycan complexes. Cohen and Varki (86) introduced an insightful conceptualization of the cell surface “sialome” on five hierarchical levels, making an analogy to the forest canopy
(FIGURE 3). At the outermost level are the sialic acid residues
(the leaves), which are in glycosidic linkage to underlying
linear and branched oligosaccharides (the stems and
branches), which in turn are components of glycoproteins
and glycolipids (the trees). At the cell surface, glycolipids
and glycoproteins organize into lateral domains (forests).
Just as the forest terrain varies greatly from place to place,
from boreal to jungle, depending on the types of vegetation,
the sialome varies among cell types and among lateral domains on the cell surface. Probing the structures and functions of the glycocalyx forests that dominate the chemical
and physical landscape of all cell surfaces is integral to a full
understanding of cell physiology and pathology.
464
B. Sialoglycans
Sialic acids are enzymatically added to the growing end of
glycan chains in ␣-anomeric configuration from the C-2 carbon of the Sia, typically in one of five linkages: ␣2–3- or ␣2–
6-linked to galactose (Gal), ␣2– 6-linked to N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc), or
␣2– 8-linked to another sialic acid (185). Whereas addition
of a single sialic acid often terminates a glycan chain, some
major sialoglycans carry short (NeuAc2), medium
(NeuAc3–7), or long (NeuAc8 –90⫹) sialic acid oligomers or
polymers (408). The functions of sialic acids depend on the
linkage to the underlying sugars of oligosaccharide chains
(the “branches,” FIGURE 3) and to the glycan class on which
they are carried (the “trees”; glycoproteins or glycolipids).
Sialic acids occupy the outermost termini of many N-linked
(asparagine-linked) and O-linked (serine- or threoninelinked) glycoprotein glycans in mammals, some examples
of which are shown in (FIGURE 3). The multiple antennae of
classical “complex type” N-linked glycans are often terminated with “NeuAc ␣2–3 (or ␣2– 6) Gal ␤1– 4 GlcNAc”
sequences, which may be clustered on multiple adjacent
branches of the same N-linked glycan (459). Many Olinked glycans are also sialylated, typically on short linear
or branched glycans. The most abundant O-glycans are
bound to proteins via a GalNAc-Ser/Thr linkage (62). Hundreds of densely packed sialylated O-linked glycans can be
found on cell surface mucins, large proteins with extracellular domains rich in glycosylated serine and threonine residues. Mucins provide a thick lawn of terminal Sia residues
that have profound physical and biochemical effects. Other
classes of sialylated O-glycans on glycoproteins are structurally less diverse, but no less important (151). For example, the sialylated trisaccharide “Sia ␣2–3(6) Gal ␤1– 4 GlcNAc ␤” is found in linkage to fucose (Fuc) or mannose
(Man) residues that are themselves O-linked to serines
and/or threonines on selected glycoproteins. O-Fuc glycans
function on Notch and Cripto family proteins involved in
development and cell differentiation, whereas O-Man glycans are relatively abundant in the brain (and at neuromuscular junctions), where they are found on dystroglycan
(among other proteins) and regulate cell-cell interactions
key to proper brain development (120). Disruption of protein O-fucosylation or protein O-mannosylation results in
severe developmental abnormalities.
Sialic acids are rarely extended by further glycosylation except with another sialic acid. Structures containing Sia-Sia
linkages are denoted as diSia [degree of polymerization
(DP) ⫽ 2], oligoSia (DP ⫽ 3–7) and polySia (DP ⫽8) (408).
DiSia structures are abundant on brain glycolipids (gangliosides) and less so on brain glycoproteins. Notably, polySia
is a prominent structural feature on a highly select group of
proteins that are modified by extension of a terminal ␣2–3linked or ␣2– 6-linked Sia with additional ␣2– 8-linked Sia
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SCHNAAR ET AL.
FIGURE 3. Hierarchical levels of sialome complexity. The sialome can be analyzed at the following complexity
levels. A: sialic acid core and core modifications: esterification (with various groups), O-methylation, lactonization, or lactamization yielding ⬎50 different structures. B: linkage to the underlying sugar (four major and many
minor linkages). C: identity and arrangement of the underlying sugars that can also be further modified by
fucosylation or sulfation. D: glycan class (N-linked, O-linked, or glycosphingolipids). E: spatial organization of the
Sia in sialylated microdomains, which have been referred to as “clustered saccharide patches” (509) or “the
glycosynapse” (182). Gal, galactose (Gal), GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose; Sia, sialic acid; Fuc, fucose; Asn, asparagine; Ser, serine; Thr, threonine. [Adapted
from Cohen and Varki (86), with permission from Mary Ann Liebert, Inc.]
residues, resulting in long chains that can reach DP ⬎90
(193) (FIGURE 4). The expression of polySia provides exceptional physical and biochemical properties to the carrier
protein and the cell surface on which it is expressed. This
topic is discussed in detail below.
Whereas sialic acids in many tissues are most abundant on
N- and O-linked glycoproteins, this is not true of the vertebrate brain, where sialoglycolipids dominate (420). These
invariably occur in the brain in the form of sialylated glycosphingolipids, for which the term gangliosides was
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BRAIN SIALOGLYCANS
Ig1
Sia
Ig-like domain
Gal
GlcNAc
Ig2
Fibronectin
type III repeat
Man
Fuc
Ig3
Ig4
N-linked glycan
Ig5
α2,3/
α2,6 α2,8 α2,8 α2,8 α2,8 α2,8
FnIII-2
FnIII-1
Asn
Polysialic acid (polySia)
NCAM
OH
O
AcHN
HO
COO–
O
OH
O
OH
AcHN
HO
COO–
O
OH
O
OH
AcHN
HO
COO–
O
OH
O
OH
AcHN
HO
COO–
O
O
OH
FIGURE 4. Schematic representation of polysialylated NCAM. NCAM is a prototypic member of the immunoglobulin family of cell adhesion molecules with five immunoglobulin-like modules (Ig1 to Ig5) and two fibronectin type III repeats (FnIII-1 and FnIII-2) in the extracellular domain. Of the six N-glycosylation sites (indicated by
black arrowheads on Ig3, Ig4, and Ig5), only two, located in the 5th Ig-like domain, can be extended by the
addition of polySia, a homopolymer of ␣2– 8-linked sialic acid residues. One of several possible core glycans is
depicted (see text for details). PolySia chains are linked to complex glycans with the first sialic acid in ␣2–3 or
␣2– 6 linkage. PolySia chains can comprise ⬎90 monomer units, leading to polyanions (negatively charged
carboxylate groups are highlighted with gray spheres) with high water binding capacity. The large hydration
shell formed by polySia markedly increases the hydrodynamic volume of its carrier molecule and thus interferes
with its natural binding capabilities. Bound to NCAM, polySia is believed to invert an adhesive into a repelling
molecule.
coined by Klenk in 1942 (236). Although gangliosides are
found on all vertebrate cells and tissues, they are much more
abundant and more complex in the brain, as will be detailed
later in this review.
At the level of the cell surface “forest,” glycans associate
into sialoglycan-rich lateral membrane microdomains (FIGURE 3) with distinct characteristics and functions. In some
cases, extracellular lectins crosslink cell surface glycoproteins via carbohydrate recognition (53, 60). Disrupting
these interactions leads to changes in cell surface residency,
lifetime, and function of sialoglycans (352). Another example of lateral association is the spontaneous formation of
membrane rafts (448, 458). Sphingolipids, including gangliosides, have long saturated carbohydrate chains on their
ceramide (lipid) moieties that associate with each other,
with cholesterol and/or with selected intracellular and
transmembrane proteins to create small (nanometer scale)
and dynamic lateral membrane microdomains. These domains concentrate and enrich selected membrane compo-
466
nents. They provide interaction sites (landing pads) for extracellular ganglioside-interacting proteins and enhance the
functions of interacting cell surface signaling molecules.
The structure, formation, dynamics, and functions of cell
surface microdomains remain one of the least well understood levels of the sialome, despite broad appreciation of
their functional significance.
C. Sialyltransferases and Sialidases
Sialoglycans are biosynthesized by the enzymatic transfer of
sialic acid from its activated nucleotide sugar donor (CMPSia) to the terminus of an oligosaccharide chain of a glycoprotein or glycolipid (FIGURE 5). The enzymes responsible,
sialyltransferases, have been cloned from organisms ranging from bacteria to human (185). There are 20 mammalian
sialyltransferases, each of which has high sequence similarity between human and mouse. Animal sialyltransferases
are characterized by two major and two minor conserved
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SCHNAAR ET AL.
NH2
A
N
O
OH
O
N
O
P
OH
HO
O
AcHN
CO2–
O
+
O–
OH
O
CO2–
OH
HO
OH
O
OH
OH
B
HO
OH
OH
HO
O
O
OH
HO
R
OH
OH
HO
O
AcHN
OH
OH
HO
AcHN
HO
O
HO
O
AcHN
O
OH
O
CO2
–
R
R
OH
CO2–
HO
CO2–
O
AcHN
OH
OH
O
HO
OH
O
HO
HO
NeuAc α2-6 Gal
ST6GAL genes (2)
O
HO
HO
OH
CO2–
CO2–
NeuAc α2-8 NeuAc
ST8SIA genes (6)
O
O
HO
O
O
OH
OH
OH
O
O
AcHN
HO
O
O
HO
OH
OH
OH
AcHN
OH
HO
OH
NeuAc α2-3 Gal
ST3GAL genes (6)
OH
HO
O
NeuAc α2-6 GalNAc
ST6GALNAC genes (6)
O
HO
O
O
HO
HO
R
NHAc
C
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467
BRAIN SIALOGLYCANS
amino acid sequences (sialylmotifs; see sect. IIIB2) involved
in donor (CMP-Sia) binding (22). Their discovery aided the
search for sialyltransferases in a wide variety of species.
Bacterial sialyltransferases appear to have evolved separately, but also exhibit conserved motifs, “bacterial sialylmotifs,” which appear to serve functions similar to those in
eukaryotic sialyltransferases (152, 224, 340, 557).
The 20 mammalian sialyltransferases (STs) are categorized
into four families (ST3Gal, ST6Gal, ST6GalNAc, or
ST8Sia) based on the linkage they generate (␣2–3, ␣2– 6, or
␣2– 8) and their primary saccharide acceptor (Gal, GalNAc,
or Sia) (FIGURE 5). Family members are arbitrarily denoted
by Roman numerals (e.g., ST3Gal-I through ST3Gal-VI),
and the genes that encode them by numbers (e.g.,
ST3GAL1-ST3GAL6 in human, St3gal1-St3gal6 in mice)
(495). There are six vertebrate ST3Gal enzymes, two
ST6Gal enzymes, six ST6GalNAc enzymes, and six ST8Sia
enzymes. The reasons for evolving multiple enzymes for
each linkage are not fully understood but are presumed to
account for fine specificities based on the underlying sugars
(branches) and glycan class (trees) of their acceptor glycans.
Since sialyltransferases coinhabit the Golgi apparatus, they
may have evolved multiple distinct acceptor specificities to
allow independent regulation of different sialoglycans. Genetic observations in human diseases and studies of engineered mouse mutants are helping to reveal the in vivo
specificities and functions of sialyltransferases and their
products (517).
Enzymatic modification of sialic acid residues to generate
diverse Sia species occurs at both the CMP-Sia and sialoglycan level (15). Hydroxylation of the C5 N-acetyl group
to convert NeuAc to NeuGc (by CMAH) occurs only at the
CMP-Sia level, prior to transfer of Sia to the glycan. In
contrast, O-acetylation and other Sia modifications may
occur at either the CMP-Sia level or after the Sia is transferred depending on the specific modification and enzyme
involved. The genetics and enzymology of Sia diversity and
its functions remain active areas of inquiry.
Sialic acids are removed from sialoglycans by the enzymatic
actions of sialidases, of which there are four in humans
generated by the genes NEU1-NEU4 (164, 309). In contrast
to sialyltransferases, the NEU enzymes are not strictly linkage specific and have a variety of subcellular distributions.
NEU1, which is the most abundant mammalian sialidase, is
primarily intralysosomal and is active on glycopeptides and
oligosaccharides but not on gangliosides. Mutations in the
human NEU1 gene are responsible for sialidosis, a congenital lysosomal storage disorder that disproportionally affects the nervous system (445). NEU2 is cytoplasmic, is
active at near-neutral pH, and has the ability to remove
sialic acids from different glycan classes. NEU3 is found on
the plasma membrane and is ganglioside-selective (requires
a hydrophobic aglycone), whereas NEU4 is predominantly
on intracellular membranes and has broad glycan class
specificity. Beyond their role in catabolism, sialidases have
recently been recognized as active regulators of sialoglycan
functions (304). NEU3, for example, has been implicated in
hippocampal neuron outgrowth (102), whereas NEU1 is
upregulated and targeted to the plasma membrane in differentiating monocytes (266) and activated T-cells (333) and
positively impacts insulin receptor signaling in peripheral
tissues (128). Moreover, NEU1-NEU3 are implicated in
skeletal muscle differentiation (136). More studies are
needed to better understand the different roles of these four
enzymes.
The term sialidase is synonymous with the term neuraminidase. Since “neuraminic acid” refers to the C-5 free amine
form (which is rarely found in nature) whereas “sialic acid”
refers to the entire family of nonulosonic acids (FIGURE 2),
the term sialidase is strictly more accurate. However, since
the term neuraminidase is firmly established as the nomenclature used to describe the sialidases of influenza viruses
and other pathogens, both terms are widely used.
D. Sialic Acid Binding Proteins
There are several mechanisms by which the sialome regulates cells and tissues (412, 516). Based on their relatively
strong anionic charge, sialic acids regulate glycoprotein secondary and tertiary structure and contribute to the hydration of proteins (such as mucins) at the cell surface. They
shield glycoproteins from interactions with enzymes (e.g.,
proteases) as well as with protein receptors on other cells,
regulating intracellular communication. In addition to
these general mechanisms, sialic acids are specific receptors
for complementary sialic acid binding proteins.
Their prominence on cell surfaces and their selective expression on the outermost branches of glycoprotein and glycolipid glycans make sialic acids well suited as specific receptors for complementary binding proteins that can “read”
FIGURE 5. Sialyltransferases. A: sialoglycans are synthesized by the enzymatic transfer of sialic acid from the activated nucleotide sugar donor
CMP-sialic acid, to an acceptor (e.g., the 3-carbon hydroxyl of the galactose of the disaccharide lactose, circled) to form a glycosidic linkage (e.g.,
NeuAc ␣2–3 Gal). B: there are 20 human sialyltransferase genes in four families designated by the major linkages they form. C: an example of
the structural differences between ␣2–3 and ␣2– 6 linked sialic acids. Each structure contains the acceptor disaccharide Gal ␤1– 4 GlcNAc in
the same orientation with a terminal sialic acid linked to the 3-carbon hydroxyl (left) or 6-carbon hydroxyl (right) of the terminal galactose. The
position of the sialic acid carboxylate is designated by an arrow to assist in visualization. Three-dimensional structures were generated using
Glycam-Web (www.glycam.org).
468
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SCHNAAR ET AL.
Selectins and siglecs are two important families of Sia-binding proteins in mammals. The three selectins (E-, L-, and
P-selectin) mediate various types of leukocyte and platelet
trafficking (292). Most of the 14 human siglecs (sialic acidbinding immunoglobulin-like lectins) are also involved in
immune cell regulation (98, 362). However, the family includes one nervous system member (Siglec-4, myelin-associated glycoprotein, MAG) that is described in detail later in
this review (422).
The three selectins each recognize ␣2–3-linked Sia on a
fucosylated core structure, such as sialyl Lewis x [NeuAc
␣2–3 Gal ␤1– 4 (Fuc ␣1–3) GlcNAc] and related structures
in the context of larger glycoproteins and glycolipids (292).
In contrast, siglecs have diversified to take advantage of the
many linkages and contexts in which sialic acids are found
on tissues (98). For example, Siglec-2 binds exclusively to
␣2– 6-linked Sia, whereas Siglec-8 binds to a Sia that is
␣2–3-linked to a galactose that also carries a 6-position
sulfate group. Siglec-7 and Siglec-11 prefer ␣2– 8-linked
Sia, with the latter functionally binding polysialic acid (14,
24; see sect. VA). Detailed structure-function studies reveal
that selectins primarily recognize the appropriately positioned anionic moiety of sialic acid, which can be replaced
with other anionic groups (73), whereas siglecs typically
require the entire sialic acid (92, 514). In both the selectin
and siglec families, sialic acid binding proteins have evolved
to recognize and initiate responses to particular sialoglycans that are recognized at several levels of molecular organization: the “leaves, stems, branches, and trees” of the cell
surface sialome forest.
III. BRAIN-ENRICHED SIALOGLYCANS
A. Gangliosides: Dominant Molecular
Determinants in the Brain
The glycome of each animal tissue is comprised of glycoproteins, glycolipids, and proteoglycans in varying proportions. The brain is unusual in that its glycome is dominated
by glycolipids (421) (FIGURE 6). These include galactosylceramide and its 3-O-sulfated form sulfatide, which are
major myelin lipids, and gangliosides, which are enriched
on nerve cells. Together, glycosphingolipids represent
⬃80% of the total glycan mass in the brain, and gangliosides carry ⬃75% of the brain’s sialic acid (FIGURE 7). In
addition to expressing a much higher proportion of its sialome as gangliosides, the adult brain expresses gangliosides
of higher complexity than those typical in other tissues
(420). This section describes the structures, biosynthesis,
and expression of brain gangliosides; their functions are
discussed later in this review.
Glycan mass
(µmoles of monosaccharide/g fresh weight)
the sialome code. This was first discovered in the study of
pathogens that evolved to bind to these abundant and readily accessible cell surface targets (259), of which influenza
viruses are the best studied (338, 450, 462). Influenza virions bind to terminal sialic acids on target cells (e.g., upper
airway epithelial cells in humans) via their surface hemagglutinin. After entering target cells and proliferating, the
daughter virions bud from the host cell surface, then release
themselves via surface sialidases (viral neuraminidases) that
act as “receptor destroying enzymes” (529). The viral hemagglutinin is specific for the Sia linkage (␣2–3 versus ␣2– 6),
and the strain specificity dictates species tropism (462).
Thus ␣2– 6-NeuAc-targeting strains are typically transmissible between humans because they bind to the major sialoglycans of the human upper airway, whereas ␣2–3-targeting strains infect birds, which prominently express that
linkage on their gut epithelial cells. Other pathogens and
pathogen protein toxins also take advantage of cell surface
sialoglycans for binding and entry, including Helicobacter
pylori, Escherichia coli K99, Vibrio cholerae (toxin), Clostridium tetani (toxin), Plasmodium falciparum, and others
(259). The discovery of pathogen Sia-binding proteins predated and anticipated the discovery of vertebrate counterparts.
Gangliosides
35
35
30
30
25
25
Galactosylceramides
20
20
15
15
O-linked
10
10
N-linked
5
5
0
0
Glycolipids
Glycoproteins Proteoglycans
FIGURE 6. Glycans in the adult rat brain. The mass of constituent
monosaccharides (␮mol/g fresh brain wt) for each glycan class and
subclass were calculated from published data as follows. 1) Galactosylceramide and sulfatide together represent 19.4 mg/g brain
fresh wt, with 76% (by weight) being galactosylceramide (346). This
represents ⬃20.3 ␮mol galactosylceramide and ⬃5.8 ␮mol sulfatide/g fresh wt. 2) Ganglioside sialic acid is expressed at 1.15 mg
(3.9 ␮mol)/g fresh wt. Since there is an average of 2.9 ganglioside
monosaccharides per ganglioside sialic acid, gangliosides represent
11.2 ␮mol monosaccharide/g fresh wt (482). 3) Glycoproteins
represent 71 ␮mol monosaccharide/g lipid-free dry wt. Since lipidfree dry weight is ⬃11% of brain fresh weight, glycoproteins represent ⬃7.8 ␮mol monosaccharide/g fresh wt (281). Of these, Olinked GalNAc is expressed at 0.28 ␮mol/g fresh wt. Since there is
an average of 3.2 O-linked monosaccharides per O-linked GalNAc,
O-linked glycoproteins represent 0.88 ␮mol/g fresh wt (139). Proteoglycan hexosamine is expressed at 4.1 ␮mol/g lipid-free dry wt in
the adult rat brain (⬃8.1 ␮mol total monosaccharide/g dry wt).
Based on lipid-free dry weight being 11% of fresh weight, proteoglycans represent 0.89 ␮mol/g fresh wt (282). [From Schnaar (421),
by permission of Oxford University Press.]
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469
BRAIN SIALOGLYCANS
µmol NeuAc/g fresh weight
A
3
ily members), and selected transmembrane scaffold and/or
signaling proteins (448, 458). Together, these lateral associations within the plasma membrane have been called lipid
rafts, glycolipid-enriched microdomains, or detergent-resistant membranes. For perspective, gangliosides constitute
⬃0.6% of total brain lipids, but are more highly enriched in
neurons. The lipid composition of the whole brain is as
follows (in ␮mol/g wet wt, adult rat): phosphoglycerolipids,
89; cholesterol, 69; galactosylceramides, 23; sphingomyelin, 7; gangliosides, 1.1 (347, 573).
Lipid-bound
Protein-bound
2
1
Liver
B
Gangliosides reside primarily in the outer leaflet of the
plasma membrane, with the ceramide and much of the first
sugar (glucose) engaged with the lipid leaflet, and the glycan
extending out into the surrounding milieu (110) (FIGURE 9).
The dual properties of intraleaflet lipid associations and
glycan extension outward from the membrane bilayer provide the ability of gangliosides to act as cell surface recognition and regulation molecules.
Brain
Brain
90
mole percent
Liver
30
20
10
0
GM3
GD3
GM2
GM1
GD1a GD1b
GT1b
GQ1b
increasing ganglioside complexity
FIGURE 7. Sialoglycoproteins and gangliosides in liver and brain.
A: protein-bound sialic acid is equivalent in the two tissues, whereas
expression of lipid-bound sialic acids (gangliosides) is ⬎10-fold
higher in the brain. B: ganglioside patterns (mol%) in human brain
and liver. Data compiled from References 5, 342, 482. [Adapted
from Schnaar (420), with permission from Elsevier.]
B. Brain Ganglioside Structures and
Metabolism
Gangliosides are members of the larger glycosphingolipid
family, consisting of glycans attached to a ceramide lipid
(FIGURE 8). Ceramides are composed of a long-chain amino
alcohol, sphingosine, with a C2 primary amine that is Nacylated with a long-chain fatty acid amide. Brain ganglioside ceramides typically have C1- and C3-hydroxyl groups
and a C4-C5 double bond, with the C1-hydroxyl linked via
glycosidic linkage to the glycan chain (181). The properties
of sphingolipids are unusual due to the nature of their lipid
moieties. The sphingosine chain of mammalian brain gangliosides is long (18 or 20 carbons), and the fatty acid amide
is saturated (typically C18:0). This results in a relatively
rigid structure within the plane of the outer leaflet of the
plasma membrane, and thereby results in enhanced lateral
self-association (e.g., see FIGURE 3, spatial organization).
Ganglioside-enriched domains may also be enriched in
other sphingolipids (notably sphingomyelin), cholesterol,
glycosylphosphatidylinositol-linked cell surface proteins,
fatty acylated intracellular signaling proteins (e.g., Src fam-
470
Although many ganglioside structures have been identified
in a variety of tissues and organisms, adult mammalian
brain gangliosides are dominated by just four closely related
structures (GM1, GD1a, GD1b, and GT1b) that together
represent the vast majority (97%) of gangliosides in the
adult human brain (482) (FIGURE 10). The same four structures comprise the major sialoglycans in the brains of all
adult mammals and birds, indicating a selective advantage
for expression of these particular structures. These four
gangliosides share the same neutral glycan core (Gal ␤1–3
GalNAc ␤1– 4 Gal ␤1– 4 Glc ␤1–1 Cer) with varying numbers of sialic acids attached to the internal and terminal
galactose residues. The four major brain gangliosides are
synthesized stepwise (FIGURE 11) by glycosyltransferases
that transfer each monosaccharide from its activated form
(UDP-Glc, UDP-Gal, UDP-GalNAc, or CMP-NeuAc) to the
growing glycan chain (TABLE 1). A key branch point in the
biosynthetic chain is GM3, which can be extended by addition of an ␣2– 8-linked NeuAc to form GD3 or by a ␤1–
4-linked GalNAc to form GM2. Once the GalNAc is transferred, internal ␣2– 8-linked NeuAc residues cannot be
added, dedicating this pathway to biosynthesis of GM1 and
GD1a, so-called “a-series” gangliosides. GD3, in contrast,
can be extended by addition of GalNAc and subsequent
sugars to form GD1b and GT1b, “b-series” gangliosides.
There are many less abundant gangliosides in the brain that
may have important functions in physiology and pathology.
Among these are the “0-series” gangliosides that arise from
transfer of GalNAc to lactosylceramide (no Sia on the internal Gal), the “c-series” gangliosides (not shown) that
have a trisialyl moiety (NeuAc ␣2– 8 NeuAc ␣2– 8 NeuAc
␣2–3) on the internal Gal. GQ1b carries a diSia moiety on
the outermost Gal residue, whereas the rare “␣-series” gangliosides carry an ␣2– 6-linked Sia on the GalNAc residue
(e.g., GD1␣; FIGURE 11). Very rare gangliosides may carry
up to seven total Sia residues (403), including linear oligom-
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SCHNAAR ET AL.
Gal
NeuAc
HO
OH
HO
OH
O
OH
NHAc
HO
–
CO2
O
OH
HO
AcHN
HO
OH
O
O
OH
OH
fatty acid amide
HN
O
O
OH
CO2–
OH
NeuAc
O
OH
O
HO
O
Glc
OH
O
AcHN
OH
HO
Gal
O
O
O
O
GalNAc
OH
O
AcHN
HO
HO
Glycan
sphingosine
ceramide
CO2–
NeuAc
FIGURE 8. Ganglioside GT1b. The glycan (shaded yellow) is in glycosidic linkage to the ceramide lipid which
is comprised of a long-chain base (sphingosine, pink) bearing a fatty acid amide (blue). Within the glycan, the
main binding site for myelin-associated glycoprotein is shaded (92, 565).
ers of up to five sialic acids (460). Gangliosides bearing
longer oligo- or polysialic acid chains have not been reported.
Ganglioside catabolism occurs stepwise by the actions of
glycan-specific exoglycosidases (406). Removal of specific
terminal glycans must proceed prior to further ganglioside
degradation. Mutations in specific ganglioside glycosidases
result in devastating lysosomal storage diseases. For example, mutation of the ␤-galactosidase gene GLB1 results
in GM1 gangliosidosis, whereas mutation of either of the
two genes coding the ␤-N-acetylhexosaminidase subunits
(HEXA or HEXB) results in GM2 gangliosidosis (406).
C. Brain Ganglioside Development and
Expression
FIGURE 9. Atomic-resolution conformational analysis of ganglioside GM3 in a lipid bilayer. The image represents a 20-ns snapshot
taken perpendicular to the plane of the bilayer near the head group
of GM3. The ganglioside is shown as a ball-and-stick model and the
membrane as a transparent space-filling model with the membrane
hydrophilic region in blue and membrane hydrophobic region in
white. [From DeMarco and Woods (110), by permission from Oxford University Press.]
The simple ganglioside structures GM3 and GD3 (see structures; FIGURE 11) dominate the glycome of the brain early in
development (573, 574). At embryonic day 14 (E14) in the
rat, GM3 and GD3 comprise nearly all of the brain gangliosides (FIGURE 12). Soon thereafter, more complex gangliosides arise such that by E20 they dominate the glycome. At
birth, GM3 and GD3 are minor components, with GD1a
and GT1b taking their place as the major sialoglycans in the
brain. As the brain matures further to adulthood, GM1 and
GD1b expression increase until the four major brain gangliosides (GM1, GD1a, GD1b, and GT1b) are at comparable levels and together represent the vast majority of total
brain gangliosides. Over this same time period, the total
brain ganglioside concentration is increasing sharply (FIGURE 12). Expressed as the amount of total ganglioside sialic
acid per gram fresh brain weight, ganglioside density increases fivefold between E14 and birth, and another threefold between newborn and adult in the rat. Likewise, ganglioside density in the human cerebral cortex increases
sharply during the last trimester of fetal development and
continues to increase during the first 2 years of life (470).
Thus both ganglioside density and structural complexity
increase as the brain develops and matures.
Although gangliosides are major cell surface determinants
throughout the nervous system, the four major gangliosides
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471
BRAIN SIALOGLYCANS
Gal
HO
GalNAc
OH
HO
OH
Gal
O
O
O
O
HO
OH
O
AcHN
O
OH
OH
HO
OH
HO
AcHN
HO
O
CO2
O
O
OH
OH
HO
HO
HO
O
OH
O
O
OH
HO
ceramide
OH
GD1b
[26%]
CO2
O
CO2
OH
HO
HO
OH
O
O
O
O
O
OH
CO2
O
OH
HO
AcHN
OH
OH
O
O
AcHN
HO
NHAc
HO
OH
OH
O
HO
O
O
ceramide
OH
GT1b
[18%]
CO2
O
O
OH
HO
O
OH
HO
HO
O
O
HO
O
OH
OH
OH
O
O
OH
HO
OH
NHAc
HO
AcHN
O
ceramide
OH
OH
O
HO
AcHN
O
CO2
O
AcHN
GD1a
[25%]
O
O
HO
OH
OH
HO
OH
O
O
OH
ceramide
OH
OH
NHAc
HO
AcHN
O
OH
O
OH
O
O
HO
NeuAc
O
O
GM1
[28%]
CO2
O
OH
HO
OH
Glc
OH
O
NHAc
HO
HO
OH
CO2
OH
FIGURE 10. The four major brain gangliosides of mammals and birds share the same neutral tetrasaccharide core (Gal ␤1–3 GalNAc ␤1– 4 Gal ␤1– 4 Glc) attached to ceramide, with varying numbers and linkage
positions of sialic acids. The sugars are color-coded: yellow, Gal/GalNAc; blue, Glc; and purple, NeuAc. Molar
percentages are for total human brain gangliosides.
472
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SCHNAAR ET AL.
FIGURE 11. Brain ganglioside biosynthesis. Complex brain gangliosides are biosynthesized stepwise by the
action of a suite of glycosyltransferases. The genes responsible for the expression of each glycosyltransferase
are boxed (see TABLE 1).
distribute nonuniformly in different brain structures (248,
467). In the adult mouse brain, for example, GM1 is concentrated in white matter tracts throughout the brain,
GD1a distributes in a largely complementary pattern in
gray matter (FIGURE 13), whereas GT1b and GD1b are
found in both white and gray matter (467). Certain scarce
gangliosides have remarkable cell-type restricted expression. The ␣-series gangliosides, which have an additional
sialic acid in ␣2– 6-linkage to the ganglioside GalNAc residue, are found selectively on cholinergic neurons (198,
212). They were discovered as the antigen of an antiserum
raised against the cholinergic electroplax organ of the electric fish Torpedo marmorata, and cross-react selectively
Table 1. Brain ganglioside biosynthetic gene nomenclature
Official Gene
Name
Ganglioside(s)
Synthesized*
Also Known As†
St3gal5
St8sia1
B4galnt1
B3galt4
St3gal2
St3gal3
St8sia5
GM3
GD3
GM2, GD2
GM1, GD1b
GD1a, GT1b
GD1a, GT1b
GQ1b
Siat9, SAT I
Siat8a, GD3S, SAT II
Galgt1, GalNAc T
GALT2, GALT4, GalT II
Siat4b, SAT IV
Siat6, ST3N
Siat8e, SAT V
*Only major brain gangliosides (or their synthetic intermediates)
listed (see FIGURE 11). †Each enzyme (or gene) has also been
referred to as the “synthase” associated with that ganglioside (e.g.,
“GM3 synthase” for St3gal5).
with cholinergic neurons in the mammalian brain (112,
384). The functional relationship between rare ganglioside
structures and cholinergic neurotransmission is as yet unknown.
An even more subtle ganglioside structural variation has
been linked to migrating cells in the developing brain. The
simple ganglioside GD3 (see structure; FIGURE 11) is the
major ganglioside in the early fetal brain, where it is widely
distributed. GD3 modified with an O-acetyl group on the
C9 hydroxyl of the terminal sialic acid, however, has highly
restricted expression that has been linked, for example, to
migrating neuroblasts of the rostral migratory stream that
arise from the subventricular zone during brain development (305, 418). O-Acetylated and lactone forms of gangliosides may constitute several percent of total adult brain
gangliosides (306, 467), and their expression may be underestimated due to loss of acetyl groups under commonly used
(alkaline) conditions for brain ganglioside purification
(419). How ganglioside acetylation affects cell function is
an area for further research (393).
D. Polysialic Acid and Polysialyltransferases
A major protein-bound sialoglycan in the brain is polysialic
acid (polySia), a linear homopolymer of ␣2– 8-linked sialic
acid residues. Predominantly linked to the neural cell adhesion molecule (NCAM) (FIGURE 4), polySia provides an
ideal example to highlight how versatile glycans expand the
information content of their protein scaffolds.
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473
1000
800
600
400
200
t
ul
rn
bo
ew
N
Ad
2
E2
0
E2
8
E1
6
E1
4
0
E1
Ganglioside sialic acid
(mg/kg wet weight)
BRAIN SIALOGLYCANS
GM4
GM3
8 –10 Sia residues seem to be the preferred structures (558)
and have been co-crystallized with interacting proteins
(360). Crystallization of polySia in complex with an enzymatically inactive form of a bacteriophage-derived polySia
degradase endosialidaseNF (endo-N-acetylneuraminidaseF; endoNF), demonstrated the existence of conformational freedom in polySia chains (426, 427). EndoNF possesses an extended interface (path of positively charged
amino acids) for the interaction with polySia (427), which
at two sites converts into high binding sites (FIGURE 14).
Electron density obtained at these positions demonstrated
two different helical conformations for polySia. The distance between the C2 atoms of the first and fourth sialic
GM2
GM1
s
t
da
rd
ul
an
St
bo
Ad
rn
2
E2
ew
N
0
E2
8
E1
6
E1
4
E1
St
an
da
rd
s
GD3
GD1a
GT1a
GD2
GD1b
GT2b
GQ1b
GM1
FIGURE 12. Ganglioside expression during brain development.
Top: rat brain ganglioside expression (ganglioside sialic acid density
as mg/kg wet wt) during brain development. Bottom: equal
amounts of extracted brain gangliosides from each developmental
age indicated were resolved by thin-layer chromatography and
stained using a sialic acid-specific colorimetric reagent (resorcinolHCl). Gangliosides GM3, GM2, and GD3 migrate as double bands
due to ceramide lipid diversity. [Adapted from Yu et al. (573), with
permission from John Wiley and Sons, Inc.]
GD1a
1. Polysialic acid
The term polysialic acid, when used in vertebrates, describes ␣2– 8-linked chains with DP ⱖ8 (FIGURE 4). This
terminology is based on the immunological properties of
polySia chains. Whereas a single antibody binding site can
typically encompass at most six saccharide units (222), a
polySia epitope comprises between 8 and 10 (140, 218).
Based on biophysical analyses, polySia chains form extended helices with eight to nine residues per turn, the conformation recognized by polySia-specific antibodies (30,
61, 135). With development of the first monoclonal antibody (mAb) directed against polySia (mAb735; Ref. 154)
ligand binding studies in solution confirmed Sia8 (a linear
octamer of ␣2,8-linked Sia residues) as the minimal epitope
(187, 188). Considering that polySia recognition by antibodies is temperature sensitive (114) and, as recently shown
by NMR, even the tetramer (Sia4) attains helical conformation at low temperature (263°K) in solution (29), it is understandable that helical structures in the polymer are transient and form dynamically along the chain (299). With
increasing polymer length (the threshold seems to be DP8)
and under physiological conditions, extended helices of
474
GD1b
GT1b
FIGURE 13. Ganglioside immunohistochemistry on adult wild-type
C57Bl/6 mouse mid-sagittal brain sections. [Adapted from Sturgill
et al. (467), by permission of Oxford University Press.]
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SCHNAAR ET AL.
A
C
12.4 Å - Sia5
14.7 Å - Sia4
B
FIGURE 14. The conformational freedom inherent in polySia chains has been instructively displayed by
cocrystallization with an inactive from of the polySia degrading endosialidaseNF. A: endosialidases are homotrimeric enzymes with an overall mushroomlike outline (466). In addition to the active site (not shown), each
monomer contains two peripheral high-affinity binding sites for polySia in the head (B) and the stalk (C) of the
mushroom (431). The polySia structures cocrystallized with the enzyme clearly indicated two different conformations of the polymer. The Sia5 molecule bound to the head domain attains a compressed conformation with
the distance between the C2 carbons of the most distant sugars being only 12.4 Å. In contrast, in the Sia4
fragment at the stalk domain, the distance from the C2 carbon at the reducing end sugar to the C2 carbon at
the nonreducing end sugar is 14.7 Å. This relaxed helical pitch is similar to the soluble state structure
described for polySia (558). These data demonstrate that the binding partner imposes structural information
onto polySia chains.
acid of a Sia4 fragment bound to the stalk domain of endoNF was 14.7 Å, corresponding to a translation of 4.9
Å/residue and thus highly reminiscent of the extended helical conformation described in solution (558). In contrast, a
distance of only 12.4 Å was determined between C2 of the
first and the fifth sialic acid of a Sia5 fragment bound to the
head domain of the protein, indicating a compressed helical
conformation with a translation of 3.1 Å per residue. These
data indicate that the binding partner constrains the conformation of the secondary structure organization in polySia chains, implying that no significant energy barriers exist
between the different polymer conformations. This flexibility, in turn, may be a key feature that explains polySia
binding to different proteins (see sect. VA).
In discussing the physiological features of polySia, most
studies focus on the size and charge of the molecule (for
review, see Refs. 316, 397). In biological samples, polySia
chains were shown to reach DPs ⬎90 (161, 330, 354). Their
polyanionic nature and, most probably, other yet unidentified chemical properties equip polySia with an unusual capacity to bind water. The resulting large hydration volume
increases the size of the carrier molecule and by stereochemical means confers repulsive forces to polysialylated surface
molecules, as shown for the major polySia carrier, the
NCAM (FIGURE 4). Consistent with this model, physicochemical measurements carried out in cell culture demonstrated an increased distance between two cell membranes
expressing polySia-NCAM (566, 567). The presence of
polySia on the cell surface is thus believed to generally
weaken contacts between neighboring cells. The model
agrees with a variety of polySia functions but fails to fully
describe the many pathophysiological aspects that have
been identified in mouse models with impaired polySia biosynthesis. This issue will be discussed in detail in section VA.
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BRAIN SIALOGLYCANS
2. Polysialyltransferases, polySia acceptors, and the
structural basis of polysialylation
In 1995 the genes encoding the enzymes responsible for
polySia biosynthesis, the polysialyltransferases (polyST),
were independently characterized by four groups (130, 242,
332, 415). Initially named STX and PST-1, the enzymes
were later designated ST8Sia-II and ST8Sia-IV, respectively,
according to a systematic nomenclature (495). The official
gene names are ST8SIA2 and ST8SIA4 in humans and
St8sia2 and St8sia4 in mice.
In mice (as well as in other mammals), the two enzymes
share ⬃60% identity at the amino acid level and are typical
representatives of the mammalian sialyltransferase family
with the conserved sialylmotifs L, S, III, and VS (105, 168)
(FIGURE 15C). In addition, polySTs are characterized by two
additional motifs termed the polysialyltransferase domain
(PSTD) and the polybasic region (PBR). The PSTD motif
consists of 32 amino acids and is essential to establish polysialylation capacity. The finding that arginine residues in
this stretch are indispensable for enzymatic function led to
the postulation that this region provides an extended binding site for the growing polySia chain and thereby confers
processivity to polySTs (104, 331). The PBR motif in contrast is part of the stem regions of the enzymes, and data
obtained for ST8Sia-IV revealed that amino acids located in
this area make specific contacts with NCAM the major
polySia acceptor protein (144, 581). NCAM in turn seems
to possess a complementary binding site (488) and an additional polyST recognition site in its 5th Ig-like domain
(487). The biological significance of protein-protein interactions between polySTs and NCAM is not fully understood. However, because completeness of NCAM polysialylation is of utmost importance during brain morphogenesis (see sect. V), it is tempting to speculate that these
contacts favor NCAM over potential concurrent polySia
acceptors like the synaptic cell adhesion molecule 1
(SynCAM 1). Of note, SynCAM 1 shows a broadly overlapping expression with NCAM during brain development,
but receives polySia only in a distinct cellular subset (162).
Another issue of intensive investigation is the selectivity
with which polySTs transfer polySia onto specific N-glycans on their acceptors. As illustrated in FIGURE 15A,
NCAM is a prototypic member of the immunoglobulin (Ig)
family of adhesion molecules, with six N-glycan attachment
sites, all modified with complex core structures (4, 267).
PolySia is selectively added to N-glycans 5 and 6, both of
which are in the fifth Ig-like domain (160, 528, 555). Extensive work has been carried out to determine if this selectivity is conferred by specific structural features of the Nglycan core structures at the two positions. However, the
isolation of polysialylated glycopeptides from NCAM expressed in cultured cells and from natural sources (i.e., postnatal mouse and bovine brain) and their detailed structural
analysis ruled out the theory that site selectivity for polySia
476
addition is determined by region-specific N-glycan core glycosylation (160, 528, 555). The N-linked structures at these
polySia addition sites included neutral, partially truncated
di-, tri-, and tetra-antennary fucosylated and nonfucosylated cores as well as acidic species and the glycopeptide
analysis clearly demonstrated roughly equal frequency of
all identified core structures at both polySia attachment
sites (528, 555). Considering that a distinct type of glycan
structure is not required to prime the polyST reaction, Angata and Fukuda (8) suggested that polyST activity requires
a proper spacing between acceptor glycans and the polyST
active sites. Subsequent studies searched for the “minimal
NCAM” that serves as polySia acceptor. Deletion and replacement studies carried out in different laboratories identified a variant encompassing the tandem domains Ig5 and
FnIII-1 to be efficiently polysialylated, while no polysialylation was seen when the FnIII-1 was deleted from this
construct (89, 337, 488).
For more than 20 years, the prominence of polySia on
NCAM (in the nervous system ⬎95% of polySia are linked
to NCAM) obscured the fact that other proteins are also
polysialylated inside and outside the nervous system (for a
recent review, see Ref. 316). Initially, evidence for the existence of additional polySia acceptors in the CNS was obtained in a biochemical study carried out in synaptosomal
fractions of adult rat brain. Using the polySia specific
mAb735 in NCAM-depleted brain lysates, Roth and Zuber
(590) obtained evidence for sodium channel ␣-subunit
polysialylation. Although this finding has not been confirmed by other methods, it is notable that the sodium channel ␣-subunit in Electrophorus electricus carries polySia
(215) and that mAb735 has been successfully used to enrich
for this molecule. More recent approaches used perinatal
brain from Ncam knockout mice, which clearly express
residual amounts of polySia (95) to find additional polySia
carriers. Application of high-resolution glycoproteomic approaches to these specimens identified SynCAM 1 (162).
Like NCAM, SynCAM 1 is a member of the Ig superfamily
with a short cytosolic domain, a single membrane span and
extracellular Ig-like domains containing potential N-glycosylation sites (39) (FIGURE 15B). N-glycans in SynCAM 1
Ig1 are essential for its function as a synapse inducer (142).
Polysialylation of SynCAM 1 under in vivo conditions fully
depends on the activity of ST8Sia-II (392). As shown in a
bead aggregation assay in vitro, polysialylation completely
abrogates homophilic adhesion of SynCAM 1 (162), indicating that polySia serves as a regulator of SynCAM 1 interactions, as it does for NCAM (see sect. VA). Although
broadly expressed in the developing rodent brain with patterns overlapping NCAM expression, only a small subfraction of SynCAM 1 is polysialylated when analyzed at postnatal day 1. Moreover, polySia-SynCAM 1 is confined to
few brain areas, mainly the pontomedullary hindbrain,
where it is found on so-called NG2 cells (162). NG2 cells
are multifunctional progenitor cells that form unique syn-
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SCHNAAR ET AL.
aptic associations with neurons through which they receive
excitatory synaptic inputs (343, 361, 494). It is, however,
not known if polySia impacts the functions of these cells.
The transfer of polySia onto SynCAM 1 occurs selectively
A
to the third N-glycan in the first Ig-module. As in NCAM,
the next downstream module, in this case the Ig2 domain, is
indispensable to make SynCAM 1 an acceptor for ST8SiaII, suggesting an important role of this module as a docking
B
NCAM
SynCAM 1
Ig1
Ig2
Ig3
ia
-II
FnIII-2
Golgi
lumen
NCAM 120
Ig2
NCAM 140
Ig1
NCAM 180
Ig5
FnIII-1
ST
-II
ia
8S
8S
-IV
ia
8S
ST
ST
Ig4
Ig3
Cytosol
C
Sialylmotifs
TMD
ST8Sia-II
L
S
III VS
N
ST8Sia-IV
C
N
C
PBR
PSTD
PolyST-motifs
D
rel. mRNA level
20
15
10
5
ST8Sia-IV
ST8Sia-II
0
E9
E11
E13
E15
P5
P10
P15
P20
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BRAIN SIALOGLYCANS
site for the enzyme (392). Together with proper spacing
between the membrane and the particular glycosylation
site, this provides a reasonable basis to explain the selective
transfer of polySia onto SynCAM 1 (FIGURE 15B).
The above example indicates that polySTs have developed
individual protein substrate specificities and have the capability to recognize acceptors in a context-dependent manner
(392). This general conclusion is supported by the finding
that ST8Sia-IV also has a specific protein substrate (in addition to NCAM), neuropilin-2 (NRP2) (316). Although
highly expressed in the developing brain in patterns widely
overlapping with the expression of ST8Sia-IV, polysialylation of NRP2 has thus far only been seen in mature dendritic cells of the immune system (31, 100, 382, 383).
It remains an open question whether specializations in substrate recognition provided the basis for the evolution of
two polySTs. Compelling evidence, however, exists that
proper time and tissue specific NCAM polysialylation
requires the concerted activity of both ST8Sia-II and
ST8Sia-IV (315). Analyses carried out in isogenic mouse
strains, differing only in the number of active polysialyltransferase alleles, highlighted differences in the contributions of the two enzymes. The in vivo studies corrected
previous in vitro data that ST8Sia-IV was the polyST with
higher activity (13, 234, 241). The animal models unequivocally showed that ST8Sia-II is the dominant enzyme during brain development. If analyzed at postnatal day 1,
⬃50% of the NCAM pool remained polySia-free in St8sia2
knockout mice, while no change was detected in St8sia4
knockouts compared with the wild type, indicating compensation and a high degree of redundancy between the two
enzymes in vivo. In contrast, the length of polySia chains
produced by ST8Sia-IV (in St8sia2 knockouts) was identical to wild type, whereas a significant drop in the amount of
long polySia chains (DP ⬎36) was found in mice lacking
ST8Sia-IV (160). These findings reveal one level of polySia
chain-length control. Another plausible mechanism of polySia chain-length control may be destabilization of the catalytically active ternary complex after the polyanionic chains
have reached a critical size (DP 80 –100). However, direct
evidence for this is not yet available.
To start building polySia chains on their glycoprotein acceptors, polySTs do not require the help of a “priming enzyme.” The sole prerequisite for the enzymes to start is the
presence of appropriate core glycans with a single terminal
sialic acid linked ␣2–3 or ␣2– 6 to the penultimate sugar
(160, 313). As shown for ST8Sia-II- and ST8Sia-IV-catalyzed NCAM polysialylation in vitro, this terminal sialic
acid can be linked either ␣2–3 or ␣2– 6 to the penultimate
sugar (12, 313) (FIGURE 4). Whereas one study found that
the type of linkage had no measured effect on the subsequent polysialylation step (313), a second study indicated
that the ␣2–3-linked sialic acid may be the preferred acceptor, at least in vitro (12). The finding that polySTs build
polySia on themselves has been referred to as auto-polysialylation (84, 312), which led to the hypothesis that preformed chains are transferred en bloc to acceptors. Supporting experimental evidence for this possibility, however, has
not been obtained (312). PolyST auto-polysialylation occurs on specific N-glycans that are highly conserved in all
␣2– 8-STs and is a prerequisite for enzyme function (314,
546). Much more work is needed to comprehend the catalytic mechanism of mammalian polySTs, for which crystal
structures do not yet exist.
3. Regulation of polysialic acid expression
The expression of polySia on the cell surface primarily represents the balance between synthesis of polysialylated
structures and their degradation by internalization and lysosomal degradation (115, 161, 417). Moreover, the chemical stability of polySia is pH dependent. Under mildly
acidic conditions, accelerated fragmentation of polySia has
been observed, which has been explained by an intramolecular self-cleavage mechanism and might facilitate lysosomal
degradation of polySia (279). On the other hand, acidic
conditions promote the formation of intramolecular lactones which are formed by condensation of the carboxyl
group of one sialic acid residue with the hydroxyl group in
position C9 of the adjacent residue to yield a six-membered
FIGURE 15. A: the predominant polySia acceptor is the neural cell adhesion molecule (NCAM). The three major isoforms NCAM-180, -140,
and -120 share identical extracellular domain structures but vary with respect to the size of the intracellular domain. In cell culture, all isoforms
are polysialylated, and polySia is selectively transferred onto two N-glycans in the 5th immunoglobulin like module (Ig5). The absence of
region-specific core glycans in these positions (see text) prompted the suggestion that membrane-bound polysialyltransferases require specific
spacing between acceptor glycans and their active sites (8). Later studies confirmed the model by demonstrating that the minimal polySia
acceptor encompasses the tandem domains Ig5 and FnIII-1. Black triangles indicate N-glycosylation sites. B: the spacing model is also suited to
explain the transfer of polySia onto selective N-glycans in other acceptors shown here SynCAM 1. Of note, both polySTs are independently able
to polysialylate NCAM, while SynCAM 1 is specifically recognized by ST8Sia-II. The molecular basis of specific acceptor recognition is not known.
C: schematic representation of the polysialyltransferases ST8Sia-II and ST8Sia-IV. Both polySTs consist of a short NH2-terminal cytosolic domain,
a transmembrane domain (TMD), a stem region, and a COOH-terminal catalytic domain. The sialyl motifs large (L), short (S), motif III (III), and very
short (VS) are conserved in all mammalian sialyltransferases (476) and are depicted as black boxes. The two polyST specific domains, termed
polybasic region (PBR) and polyST specific domain (PSTD), are shown as white boxes. Black triangles indicate N-glycosylation sites. D: overview
of ST8Sia-II and ST8Sia-IV expression during mouse brain development as determined by quantitative real-time RT-PCR from whole brain mRNA
extracts (354, 417).
478
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SCHNAAR ET AL.
ring (268). Lactonization reduces charge and flexibility of
polySia and increases the resistance to both enzyme- and
acid-catalyzed glycosidic bond cleavage (586). At neutral
pH, however, almost all polySia is lactone free (25), and the
role of lactone formation for chemical stability and physiological properties of polySia in vivo has not been established.
Enzymes that specifically and robustly degrade polySia at
the cell surface have not been convincingly demonstrated in
vertebrates. The observation that Neu1-Neu4 each has limited activity on polySia corresponds well with the long halflife of polySia in circulation (270, 530). However, extracellular functions of vertebrate sialidases are largely unexplored, which may be partly due to their instability and low
expression (304). It therefore remains possible that some of
these enzymes impact expression of polySia on intact cells.
In this light, evidence that Neu4 may be functional in polySia degradation warrants further exploration (474).
355). Combined with the observed differences that polySTs
have in terms of recognizing protein substrates (391, 392)
and in their capacity to elongate polySia chains (160, 230),
these data stress the advantage of having two polySTs.
Experimentally, it is worth noting that native phage-borne
endosialidases (endoN) coded by bacteriophages that penetrate bacterial polysialic acid capsules are key tools for
polySia removal (see below) and that enzymatically inactive
forms of these enzymes, which retain polySia binding activity, are useful artificial lectins to identify and/or enrich polySia and polysialylated proteins (214).
4. Polysialylation during brain development: overview
Another way to release polySia from the cell surface is shedding of the extracellular domain of the polySia carrier protein. In fact, elevated levels of shed NCAM fragments have
been detected in cerebrospinal fluid and post-mortem brain
samples of schizophrenic patients (468, 519) as discussed in
section VIE. Shedding of polysialylated NCAM has been
confirmed in cell culture, and the responsible enzymes were
identified as members of the ADAM (A Disintegrin and
metalloproteinase) family of metalloproteases (205, 225).
Since the shed fragment is stable and may retain biological
functions, shedding by itself cannot be considered a form of
polySia degradation. As shown recently, cleavage of polySia-NCAM by ADAM10 is a prerequisite for ephrinA5induced growth cone collapse (58). In this case the loss of
steric hindrance after removal of the polySia-NCAM ectodomain, and not the shed fragment itself, appears functionally important (194). A biological function for shed
polySia-NCAM, per se, has yet to be established.
In the mouse embryo, the first expression of the two polyST
mRNAs is detected at E8.5, at the time of neural tube closure (355, 374), and polySia immunoreactivity appears half
a day later at E9 (355, 374). PolySia is expressed throughout embryonic brain development and reaches peak expression levels perinatally (250, 355). Recent progress made in
HPLC-based detection of polySia (160, 161, 330, 354) allowed the profiling of their patterns both quantitatively and
qualitatively. During postnatal mouse brain development
(354), the amounts of polySia remain high during the first
postnatal week, before they rapidly decline between postnatal days 9 –17 to ⬃30% of the level at birth. This is
followed by a further decrease to ⬃10% in adulthood (⬎6
mo of age) (354). Perinatally, ⬎95% of polySia is added to
the isoforms NCAM-140 and -180, leading to the quantitative concealing of these proteins under a polySia shell.
As these isoforms stay constantly expressed during the first
three postnatal weeks, polySia reduction correlates with an
increase in the appearance of polySia-free (“naked”)
NCAM-140 and -180 (354). A decline of polySia and constancy of NCAM expression was also found in the prefrontal and visual cortices (34, 56, 113). Of note, a similar
developmental profile has been described for the human
prefrontal cortex (94).
The major control level of polySia expression appears to be
transcription and translation of the polySTs and NCAM
(11, 191, 192, 250, 355, 369). The mRNAs of the three
genes peak in the late embryonic and early postnatal mouse
brain together with the maximal levels of polySia immunoreactivity (354, 417) (FIGURE 15D). Quantitative analyses in
brains from mice lacking either St8Sia2 or St8sia4 indicated
that the two genes are independently expressed and, as discussed above, are capable of at least partial cross-compensation (161). Remarkably, the data indicate a perinatal
overcapacity of the polysialylation machinery (354). This
excess may be necessary to guarantee the completeness of
NCAM polysialylation during the critical phases of neural
development and wiring (further discussed in sect. VB3).
Despite their overlap during brain development, the mRNA
levels of the polySTs show considerable differences in their
tissue patterns if determined at later times (11, 192, 354,
Northern blotting (250, 355) and quantitative real-time
RT-PCR (qPCR) (354, 417) were used to assess the expression of the polyST-genes during embryonic and postnatal
mouse brain development. In accordance with the earlier
Northern blot results, qPCR indicates a steep increase of
St8sia2 and St8sia4 mRNA from E10.5 to E13.5 when plateau levels were reached and maintained until birth (417)
(FIGURE 15D). The same method applied to the mesencephalic dopaminergic system gave identical results and in
addition showed that upregulation of the two polySTs is
paralleled by a comparable increase in Ncam transcript levels (417). Postnatally, downregulation of the polySTs preceded the decline of polySia; however, in this developmental
window the two mRNAs showed different profiles. The
St8sia2 mRNA dropped sharply between postnatal days
5–11 on whole brain level, and between postnatal days
9 –12 in the visual cortex, while the decrease of St8sia4
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BRAIN SIALOGLYCANS
mRNA proceeded more slowly (34, 354) (FIGURE 15D). The
results were highly consistent with previous Northern blot
and in situ hybridization studies that identified ST8Sia-IV as
the prevailing polyST of the adult rodent brain (11, 192,
355). In humans, microarray data indicated an age-dependent decline of ST8SIA2 expression in the dorsolateral prefrontal cortex. This could be validated and quantified by
qPCR. Between the group of neonates and infants up to the
age of 1 yr and the age group of toddlers and children up to
13 yr of age, mRNA levels dropped by ⬃50%. As in mice,
this steep decline was followed by a gradual decrease from
teenagers to adults, who retained ⬃8% of the neonatal
ST8SIA2 mRNA level in this cortical region (291).
natal week (367). Thus the chain-like migration pattern of
polySia-positive neuroblasts at earlier stages is independent
from the glial scaffold, and this is consistent with in vitro
data showing that chain migration of SVZ explants from
5-day-old mice, i.e., the sliding of neuroblasts along each
other, occurs in the absence of glial guidance (542). As
further discussed in section VB, the efficiency of this migration process, but not chain migration per se, depends on the
presence of polySia. In contradiction to the prevailing view
of polySia as just a steric inhibitor of cell-cell interactions,
loss of polySia delays the formation and causes reduced
compaction or even a dispersal of isolated migratory chains
in vitro (76, 203).
5. Polysialylation at different stages of neurogenesis
Based on the knowledge that polySia is a marker of adult
neurogenesis in the rodent SVZ, immunostaining for polySia has been used to identify the presence of the SVZ/RMS
system in rabbits (274, 373), non-human primates (165,
246, 365, 410), and humans (36, 101, 379, 404, 405, 540).
While the organization of the SVZ-RMS in adult rabbits
and monkeys seems largely comparable to rodents, results
on the human brain differ. First reports described clusters or
chains of polySia-positive cells in the SVZ of adult humans
(36, 540). Work by the Alvarez-Buylla group also identified
polySia-positive cells in the SVZ, but these were not arranged in chains (379, 405). A later study then made the
heavily disputed claim that significant numbers of cells generated in the SVZ of adult humans migrate towards the
olfactory bulb in a rostral migratory stream (101). More
recently, an RMS with massive chain migration was found
in infants, but in contrast to the situation in rodents, only a
subfraction of the migrating neuroblasts seems to express
polySia (404). In newborns, a high number of polySia-positive immature neurons was detected in the RMS at the level
of the olfactory tract, which declines rapidly, and in a 7-yrold child, chains of migrating cells were no longer observed
(404). In specimens obtained from 4- to 6-mo-old humans,
polySia immunoreactivity was additionally detected in clusters of immature neurons forming a previously unknown
medial migratory stream that deviates from the RMS and
moves towards the ventromedial prefrontal cortex (404).
During formation of the mammalian brain, neuronal and
glial cells are derived from stem cells in the neuroepithelium. These stem cells give rise to radial glia, the proliferating precursors that generate neurons by asymmetric and
symmetric divisions and serve as a scaffold for radial migration of newborn neurons (370). The majority of polySiapositive cells in the embryonic brain belong to the neuronal
lineage (41) and most, if not all, neurons seem to be positive
for polySia at some stage of differentiation (48). To lay the
ground for discussing the major developmental functions of
polySia in section VB, we review the occurrence of polySia
during neurogenesis from the radial glia stage to migration
of polySia-positive precursors to axon growth and synaptogenesis.
PolySia has been detected on radial glia of the developing
cortex and mesencephalon (264, 265, 417). Likewise,
polySia is found on Bergmann and Müller glia, the radial
glia of cerebellum and retina, respectively (27, 189, 251).
The pattern is consistent with a role of polySia in radial
migration as suggested by Angata et al. (9), who observed
aberrantly localized pyramidal cells in the neocortex of
polyST-negative mice. At the next step of neurogenesis,
polySia is associated with long-distance cell migration of
various types of neuroblasts. The most prominent example
is the migration of olfactory interneuron precursors, which
are generated throughout life from dividing astrocytic precursors in the anterior subventricular zone of the lateral
ventricle (SVZ) and migrate tangentially in a process called
chain migration to the olfactory bulb, where they differentiate into granular and periglomerular interneurons (51,
122, 123, 394) (FIGURE 16). The neurogenic niche of the
adult SVZ is derived from the embryonic lateral ganglionic
eminence and a chain-like pattern of polySia-positive neuroblasts, the hallmark of the postnatal rostral migratory
stream (RMS), has also been described in the rat embryonic
forebrain (366). However, tangential migration in the embryonic and early postnatal RMS differs from chain migration in the mature brain. In the adult, the polySia-positive
chains move through longitudinally oriented glial tubes,
which develop from astrocytic networks in the third post-
480
Further types of migrating polySia-positive neuroblasts
comprise precursors of cortical interneurons (111, 437,
439), which originate from the embryonic medial ganglionic eminence and migrate tangentially into the pallium
(283). During their tangential migration, cortical interneurons are not in close contact with each other and move
independent of interactions with glia through the polySiapositive environment of the developing cortex (111, 264,
283, 284).
One of the most intricate patterns of long distance migration through the brain is performed by the gonadotropinreleasing hormone (GnRH; also known as luteinizing hormone-releasing hormone, LHRH) neurons originating from
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SCHNAAR ET AL.
A
ependymal cell
SVZ/RMS astrocyte
rapidly dividing precursor
polySia-positive migrating neuroblast
OB interneuron
Hp
NCx
SVZ
Cb
Th
Str
OB
RMS
Hy
SGZ
CA3
GCL
B
DG
III
mf
C
II
Pir
I
D
FIGURE 16. Hotspots of polySia immunoreactivity in the adult mouse brain. A: Nissl-stained sagittal section
with a schematic representation of the neurogenic system of the anterior subventricular zone (SVZ) of the
lateral ventricle (LV, lined by ependymal cells) and the rostral migratory stream (RMS) producing new interneurons of the olfactory bulb (OB). The neurogenic niche consists of slowly dividing astrocytes giving rise to rapidly
dividing precursors, which in turn generate the migratory, polySia-positive neuroblasts. [Based on Doetsch et
al. (121–123).] B: polySia expression on mossy fibers (mf) projecting from the granule cell layer (GCL) towards
the CA3 region of the hippocampus (Hp) and in the subgranular zone (SGZ, inset), the neurogenic niche of the
dentate gyrus (DG). C: pattern of polySia immunoreactivity in the piriform cortex (Pir; layer I-III). D: example of
a polySia-positive interneuron in the prefrontal part of the neocortex (NCx). [B and C from Natcher et al. (327).]
To facilitate orientation the approximate positions are indicated in A, but note that the micrographs shown in
B and C were obtained from coronal sections. Cb, cerebellum; Hy, hypothalamus; Str, striatum; TH, thalamus.
the olfactory placode, migrating along the olfactory or
vomeronasal nerve to the olfactory bulb and from there into
the developing forebrain (430, 547). Migration of GnRH
neurons during embryonic states is closely related to the
presence of polySia (148). In chickens (321, 323), rodents
(571), and humans (429), the GnRH neurons migrate along
polySia-positive fascicles of the embryonic vomeronasal
nerve (olfactory nerve in chicken) into the olfactory bulb,
but it remains a matter of debate whether the migrating cells
themselves are polySia positive or if migration is assisted by
polySia-positive olfactory ensheathing cells. These latter
cells represent a unique glial cell type associated with the
olfactory nerve and are known to express polySia (148). In
mouse (571) and chicken (322), injection of the polySia
degrading enzyme endoN inhibits migration of GnRH neurons. After polySia removal by endoN, only half of the cells
crossed the cribriform plate and entered the olfactory bulb
in the mouse model. Most interestingly, genetic deletion of
NCAM has no such effect (571), alluding to the existence of
an alternative polySia carrier in these cells.
RMS, polySia-positive neuroblasts are generated from astrocytic stem cells in this second major neurogenic niche of
the adult brain (121, 229). The newly generated cells of the
subgranular zone arrange in clusters from which they migrate tangentially before extending neurites and dispersing
into the granule cell layer (436, 441). Early, tangentially
oriented processes of immature granule cells stay in touch
with the proliferating clusters and provide a polySia-positive microenvironment which is believed to support the neurogenic niche (436). Adult neurogenesis in the dentate gyrus
has been observed across mammalian species including
non-human primates and humans (134, 175, 238, 246),
and polySia provides a useful, though not definitive, marker
for assessing changes in hippocampal neurogenesis associated with, e.g., exercise (453) or pathological conditions
(96, 219, 539). As judged by expression of polySia and
other markers, the pattern of dentate gyrus neurogenesis in
humans (238) is comparable to the situation in rodents
(435, 444) and undergoes similar qualitative and quantitative age-related changes.
Prominent polySia expression is associated with neurogenesis of granule cells in the dentate gyrus of the adult hippocampus (438, 440) (FIGURE 16B). Similar to the SVZ-
In the process of neurogenesis, polySia is associated with
neurite outgrowth, axon pathfinding, and fasciculation.
First described in chickens, polySia was shown to be present
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on axons and growth cones of retina ganglion cells and
motoneurons (124, 255, 478, 479). In rodents, developing
axons of Cajal Retzius cells, and axon tracts including the
optic nerve, corticospinal tract, and thalamocortical projections display strong polySia immunoreactivity (27, 83, 103,
416, 437).
Finally, polySia has been implicated in positive and negative
regulation of synaptogenesis (113, 116, 117, 442). How
these still controversial functions are realized is a matter of
intensive research and needs a much better understanding
of the time course and spatial localization of polySia during
synapse formation. An immunoelectron microscopic study
of the topology of polySia in rat striatum during postnatal
development demonstrated considerable dynamics. Intense
polySia labeling of the entire synapse was followed by progressive restriction of polySia to the pre- and postsynaptic
membranes and subsequent loss of polySia (496). In contrast, a study on the formation of oculomotor synapses in
the chick embryo indicated that polySia is absent from the
actual sites of synaptic contact formation, although it is
expressed on ciliary neurons (64). Similarly, polySia was
shown to be absent from mossy fiber synapses, although the
presynaptic axonal membrane was immunoreactive (428).
Some mature synapses, however, such as a subset of spine
synapses in the outer molecular layer of the dentate gyrus,
are polySia positive (428). During synaptogenesis of cultured hippocampal neurons, NCAM accumulates rapidly at
sites of contact (472). Although these cells are known to
express polySia (117, 126), the membrane distribution of
polySia has not been addressed in this system.
6. Polysialic acid on immature and mature neurons
of the adult brain
Apart from the neurogenic niches of the adult brain, three
types of neurons display prominent polySia immunoreactivity. The first population of polySia-positive cells is found
mainly in layer II of the paleocortex. These cells coexpress
doublecortin, a marker of immature neurons and are mostly
devoid of neuronal nuclear antigen (NeuN), a marker for
mature nerve cells (171, 273, 325, 503). In rodents, these
immature neurons are particularly abundant in the piriform
cortex (FIGURE 16C), but in other mammals, including nonhuman primates, the same cell type has also been detected in
the neocortex (273, 503). Although expressing immature
features in the adult brain, the polySia-positive layer II neurons are born prenatally (171, 273) and their numbers decline significantly with age (2, 505). Thus the increase of
polySia-positive cells after administration of an N-methylD-aspartate (NMDA) receptor antagonist suggests an activity-dependent modulation of glycosylation in existing neurons (325). Furthermore, a potential for maturation in the
context of olfactory processing is implicated by the increase
of differentiated, NeuN-expressing neurons at the expense
of polySia- and doublecortin-positive cells in the piriform
cortex of bulbectomized rats (173).
482
Second, mature (NeuN-positive) polySia-positive neurons
are present in different cortical areas, including the prefrontal cortex (504, 507) (FIGURE 16D), piriform cortex (325),
and hippocampus (326) as well as in the septum (143) and
amygdala (166, 328). These cells are inhibitory interneurons characterized by the presence of the GABAergic
marker glutamic acid decarboxylase. Depending on the
area under consideration, they show divergent expression
of markers indicative of different interneuron subtypes,
such as calbindin, somatostatin, parvalbumin or the vasoactive intestinal peptide (143, 172). PolySia-positive interneurons receive less synaptic input than their polySianegative counterparts and show reduced dendritic arborization and spine numbers. These morphological features are
consistent with the notion that polySia is a negative regulator of interneuron connectivity and may support plasticity
of inhibitory cortical networks (172). Corroborating this
hypothesis a recent report showing a transient increase in
dendritic spines of interneurons after removal of polySia
(178).
Third, some differentiated neurons of the mature brain are
characterized by polySia-negative somata while displaying
polySia on their neurites. Hippocampal mossy fibers are
unmyelinated axons of dentate gyrus granule cells and
most, if not all, mossy fibers show intense polySia staining,
but their somata in the granule cell layer are polySia negative (439) (FIGURE 16B). Similarly, polySia immunoreactivity is detected on axons and dendrites of pyramidal cells in
the hippocampal CA1 region (32, 349), whereas the cell
layer itself is polySia negative (50).
As reviewed in great detail elsewhere (48, 485), wide areas
of the adult brain retain a diffuse pattern of polySia staining, which as yet has not been assigned to defined cell populations. In the hypothalamo-neurohypophysial system,
some of the diffuse polySia immunoreactivity is associated
with neurons of hypothalamic magnocellular nuclei, but
fine perineuronal processes of astrocytes are the major
source of polySia (201, 307, 348, 454, 484, 486). Remodeling of glia and synapses during physiological regulation of
neurohormone release is associated with changes of polySia
patterns, and enzymatic removal of polySia prevents the
rearrangement of synapses and astrocytic processes indicating that polySia is a prerequisite for these changes (201,
307, 484).
7. Polysialic acid on nonneuronal brain cells
In addition to the astrocytes of the hypothalamus, polySia is
expressed on other astrocytic cells of the adult brain.
Among these are the pituicytes of the neurohypophysis
(232) and the radial glia-like tanycytes in the ependymal
layer of the third ventricular wall, which send processes into
the mediobasal hypothalamus (47). Furthermore, polySia is
found on reactive astrocytes, activated in response to vari-
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SCHNAAR ET AL.
Table 2. Ganglioside expression in genetically engineered mice
Gene(s) D
Major Brain Gangliosides
Major Nervous System Phenotype(s)
None (wild type)
B4galnt1
GM1, GD1a, GD1b, GT1b
GM3, GD3 (“complex
ganglioside knockout”)
St8sia1
GM1, GD1a
St3gal2/St3gal3
St3gal5
B4galnt1/St8sia1
B4galnt1/St3gal5
GM1, GD1b
GM1b, GD1␣
GM3 (“GM3 only”)
None (“ganglioside
knockout”)
No brain development
Uniform ⱖ75% decrease
Normal
Normal
Axon degeneration, dysmyelination, progressive
motor behavioral deficits, hyperactivity,
seizure susceptibility
Mostly normal (selective reduction in axon
regeneration)
Short lived, early dysreflexia
Mostly normal
Motor and sensory deficits, audiogenic seizure
Short lived, severe early neurodegeneration,
perturbed axon-myelin interactions
Death at gastrulation
Short lived, ataxic, progressive dysreflexia and
motor deficits, dysmyelination, loss of
Purkinje neurons
Normal
Not quantitatively
determined
Axon degeneration, dysmyelination, progressive
loss of Purkinje neurons
Ugcg
Ugcg in neurons
(Nes-Cre)
Ugcg in oligodendrocytes
(Cnp-Cre)
Ugcg in Purkinje neurons
(L7-Cre)
ous insults in brain and spinal cord (21, 49, 69, 256, 329,
345).
Most recently, a subpopulation of NG2 cells, the pluripotent early stage of the oligodendrocyte lineage, was shown
to be positive for polySia and SynCAM 1 in the perinatal
brain (162). While the further characterization of polySia
expressing NG2 cells is pending, it is known that oligodendrocyte precursor cells are polySia positive during their migratory phase. However, in this stage polySia is linked to
the NCAM isoforms NCAM-180 and -140. During maturation into myelinating oligodendrocytes polySia as well as
NCAM-140 and -180 are downregulated (27, 329, 493,
535). Inversely, NCAM-120 is upregulated in mature myelinating oligodendrocytes (38). In contrast to in vitro data
(147) NCAM-120 is not polysialylated during myelination
in vivo (354). Questions remain open as to whether the
switch in the NCAM isoform expression or the downregulation of polySTs causes the disappearance of polySia during oligodendrocyte maturation, and the functional consequences of these changes.
IV. BRAIN GANGLIOSIDE FUNCTIONS
A. Genetic Models of Ganglioside Expression
Because gangliosides are major molecular determinants at
the nerve cell surface and their structures undergo major
changes during brain development, gangliosides were hypothesized to be regulators of intracellular interactions in
the brain. Genetic models in mice address this hypothesis
and provide a nuanced and more accurate understanding of
brain ganglioside functions (216, 375, 421).
Reference Nos.
78, 358, 447, 475,
552
227, 353
467
560
227, 473
563
562
559
400
538
Several genetically engineered mouse lines targeting ganglioside biosynthetic genes have been created and studied
(TABLE 2). These fall into two major categories, mutations
that block all ganglioside biosynthesis (Ugcg-null,
B4galnt1/St3gal5-double null) and those that block within
the ganglioside biosynthetic pathway but retain ganglioside
expression (St3gal5, St8sia1, B4galnt1, St3gal2, St3gal3).
In the latter set of mutants, gangliosides build up behind the
block (see FIGURE 11) such that the total ganglioside density
in the adult mouse brain remains essentially unchanged.
Excellent examples of this phenomenon are found in
B4galnt1-null, St8sia1-null, and B4galnt1/St8sia1-double
null mice (227) (FIGURE 17). Wild-type mouse brains express GM1, GD1a, GD1b, and GT1b. Mice with disrupted
wt
St8sia1
B4gaint1
St8sia1/
B4gaint1
GM3
GM1
GD1a
GD1b
GT1b
GD3
origin
FIGURE 17. Brain ganglioside patterns from wild-type and ganglioside mutant mice. Brain gangliosides were extracted, and an
amount equivalent to 10 mg brain fresh wt was resolved by thin-layer
chromatography. Glycolipids were detected colorimetrically. Migration positions of standard brain gangliosides are indicated on the
sides of the panel. [Adapted from Kawai et al. (227), with permission
from American Society for Biochemistry and Molecular Biology.]
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BRAIN SIALOGLYCANS
GD3 synthesis (St8sia1-null) lack b-series gangliosides and
express commensurately increased a-series gangliosides
(GM1 and GD1a). Mice lacking the enzyme that extends
simple gangliosides by addition of GalNAc (B4galnt1-null)
express commensurately increased amounts of GM3 and
GD3. Finally, mice with both genes disrupted are biosynthetically “stuck” at GM3, which is overexpressed to levels
comparable to total brain ganglioside concentration in
wild-type mice. The phenotypes of these mutant mice reflect
specifically on the functions of gangliosides, since these glycosyltransferases are glycosphingolipid-specific.
1. Complex ganglioside knockout mice
The developmental switch from simple (GM3, GD3) to
complex gangliosides (GM1, GD1a, GD1b, GT1b) late in
fetal brain development (FIGURE 12) starts with the addition of GalNAc to the simple gangliosides GM3 and GD3 to
synthesize GM2 and GD2, respectively (see FIGURE 11).
A single glycolipid-specific N-acetylgalactosaminyltransferase (GM2/GD2 synthase), coded by the B4galnt1 gene, is
responsible. B4galnt1-null mice express GM3 and GD3 as
major brain gangliosides throughout life, never making the
switch to complex gangliosides (271, 475). On that basis,
B4galnt1-null mice can be considered “complex ganglioside knockout” mice. Given the observations that all adult
mammals and birds express the same four major complex
brain ganglioside structures, that gangliosides are among
the most abundant cell surface molecular determinants in
the brain, and that ganglioside expression undergoes a remarkable simple-to-complex conversion during brain development, it was notable that the first published study of
B4galnt1-null mice reported only subtle phenotypic defects
(475). Mutant pups were born at expected frequencies and
developed grossly normal nervous system anatomy and behavior, at least into young adulthood. Fine-structural analyses indicate that mice lacking complex gangliosides have
normal nerve cell morphology including dendritic length,
complexity, and spine density (119). These data demonstrate that complex gangliosides are not required for basic
nerve cell differentiation, migration, or synapse formation.
Nevertheless, B4galnt1-null mice display distinctive nervous system deficits that support a role for complex gangliosides in axon-myelin interactions and neuronal excitability.
The earliest deficit noted in B4galnt1-null mice (at 10 wk of
age) was a modest decrease in nerve conduction velocity in
somatosensory evoked potentials measured from the tibial
nerve near the Achilles tendon to the somatosensory cortex,
the longest distance measured (475). Subsequent studies
provided a mechanistic basis for this deficit. Complex gangliosides are required for optimal myelin formation, axonmyelin interactions, and peripheral and central axon stability (276, 358, 447, 469). In young adult mice, dysmyelination was responsible for reduced long-distance nerve
484
conduction velocity, whereas in older mice more serious
phenotypic deficits were noted (see below).
Myelin, elaborated by oligodendrocytes in the CNS and
Schwann cells in the peripheral nervous system (PNS),
wraps long segments of axons in a multilayered membrane
sheath interrupted only by well-sealed and exquisitely organized periodic gaps, the nodes of Ranvier (334). Myelin
insulates axons and directs action potentials to nodes of
Ranvier, allowing rapid (saltatory) nerve conduction and
greatly enhancing the speed and density of information processing in the brain. Although myelin function was once
thought to be limited to insulation and conduction velocity,
it is now appreciated that axon-myelin interactions are required to establish and maintain appropriate axon cytoarchitecture and to support long- and short-term axon stability and integrity. Myelin nurtures the axons it enwraps,
and when axon-myelin interactions are compromised, axons suffer (492). Functional interactions between axons
and myelin are choreographed by complementary cell surface recognition molecules on the innermost myelin wrap
and the apposing axon surface. Complex gangliosides act as
receptors in axon-myelin interactions, and mice with altered gangliosides display phenotypes consistent with this
function.
Although normal myelin is abundant in complex ganglioside knockout mice, they display progressive central and
peripheral axon degeneration characteristic of a failure of
myelin support of axons (FIGURE 18) and also display uncharacteristically large unmyelinated fibers in the CNS, indicative of a myelination defect (276, 447). Axons in complex ganglioside knockout mice have decreased intra-axonal neurofilament spacing, leading to significantly reduced
axon calibers (358, 447) that are consistent with a failure in
axon-myelin interactions (202). Defects at nodes of Ranvier
(FIGURE 19) provide additional evidence of a failure in axon-myelin interactions (469). In contrast to the uniform
regularly spaced paranodal loops of myelin with well-defined transverse bands found in wild-type mice, B4galnt1null mice display enlarged and disorganized loops, some of
which fail to form axo-glial junctions. A significant number
of nodes also display disorganization of the well-defined
molecular expression of adhesion molecules and ion channels at or near the nodes, changes consistent with reduced
nerve conduction velocities.
As complex ganglioside knockout mice age, progressive
neuropathy results in motor behavioral deficits, including
defective hindlimb reflexes, coordination, and gait (78,
358). By 1 year of age, the mice uniformly display severe
loss of hindlimb function, along with other behavioral
changes that are less readily ascribed to axonal loss or
changes in conduction velocity. Notably, the mice display
whole-body tremor and significant hyperactivity, despite
their motor deficits (358).
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An additional test of this hypothesis came with evaluation
of double null mice in this pathway. B4galnt1/St8sia1-double null mice (so called “GM3 only” mice; TABLE 2) lack
GD3 as well as all complex brain gangliosides. Like
B4galnt1-null mice, they display nerve degeneration and
progressive motor behavioral and sensory deficits (211,
473). Notably, GM3-only mice also were found to suffer a
seizure disorder, rendering them susceptible to lethal audiogenic seizures (227). B4galnt1/St3gal5-double null mice
FIGURE 18. Axon degeneration in complex ganglioside knockout
(B4galnt1-null) mice. Top: pathological features in PNS (sciatic
nerve). Low-power electron microscopic image showing axon degeneration with collapsed myelin (red asterisks) and a thinly myelinated
fiber surrounded by supernumerary Schwann cell process (red arrow). Normal myelin is denoted with a blue asterisk for comparison.
Scale bar ⫽ 2.5 ␮m. Bottom: pathological features in the CNS (optic
nerve). Electron microscopic image showing axonal degeneration
and myelin collapse (red arrow), and a large unmyelinated axon (red
asterisk). Normal myelin is denoted with a blue asterisk for comparison. Scale bar ⫽ 200 nm. [Adapted from Sheikh et al. (447).]
2. a-Series only, 0-series only, GM3 only, and total
ganglioside knockout mice
Mice lacking other ganglioside biosynthetic enzymes reveal
additional details about the functions of complex gangliosides in the nervous system. St8sia1-null mice, which lack
all b-series gangliosides but overexpress a-series gangliosides (TABLE 2), are nearly normal in that they do not display the axon-myelin deficits of B4galnt1-null mice (227,
353). Mice with a disrupted St3gal5 gene (lacking GM3
synthase; see FIGURE 11) were expected to lack all ganglioseries brain gangliosides. Surprisingly, these mice instead
expressed high concentrations of the normally rare 0-series
gangliosides GM1b and GD1␣ (563, 572) and displayed
none of the axon-myelin deficits of complex ganglioside
knockout mice (although they were deaf due to loss of the
organ of Corti; Ref. 572). Taken together, these studies
imply that complex brain gangliosides are required for axon-myelin interactions and long-term axon stability and
that complex 0-series and a-series gangliosides alone are
equally efficient in supporting axon-myelin interactions as
are the normal complement of a- and b-series gangliosides.
FIGURE 19. Node of Ranvier defects in complex ganglioside
knockout (B4galnt1-null) mice. A and B: electron micrographs of
12-wk-old paranodal loops from wild-type (A) and B4galnt1-null (B)
mouse optic nerves. Some paranodal loops face away from the axon
in the mutant mice (arrowheads). Scale bars ⫽ 0.5 ␮m. C and
D: immunostained molecules at nodes of Ranvier from wild-type (C)
and B4galnt1-null (D) mouse ventral roots. Sodium channel immunostaining (Nav, red) delineates the node, Caspr (green) delineates
the paranode, and potassium channels (Kv1.2, green) delineate the
juxtaparanode. In mutant mice, potassium channels invade the paranode, extending toward the node (arrowheads), and there is abnormal protrusion of sodium channels (arrow). [Adapted from Susuki et
al. (469), with permission from John Wiley and Sons, Inc.]
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BRAIN SIALOGLYCANS
were particularly instructive (563). As expected from the
biosynthetic block (FIGURE 11), these mice lacked all ganglio-series gangliosides and had increased LacCer expression. What little sialylated glycosphingolipid was found in
the brain had a different neutral glycan core than normal
brain gangliosides. These “ganglioside null” mice were
born with an intact nervous system, consistent with the
conclusion that gangliosides are not required for neuronal
development or gross brain morphogenesis. However, they
were short-lived, had small brains, vacuolization of the spinal and cerebellar white matter, marked degeneration of
myelinated axons, malformed nodes of Ranvier, and severe
hindlimb weakness. Their phenotype indicates defects in
axon-myelin interactions, along with other nervous system
deficits, that are similar but more severe than those in complex ganglioside (B4galnt1-null) knockout mice.
3. Glucosylceramide knockout mice
Deletion of the single gene responsible for glucosylceramide
synthesis (FIGURE 11), Ugcg, results in interruption of embryonic development during gastrulation (562). Therefore,
conditional Ugcg mutants have been used to test the roles of
GlcCer-based glycosphingolipids (primarily gangliosides)
in the nervous system. Ugcg deficiency driven by the nestin
(neural) promoter resulted in nearly complete loss of gangliosides in the nervous system (but not other major tissues)
in one model (217), and a 75% decrease in brain ganglioside expression in a second model (559). Mice lacking brain
gangliosides were born and appeared normal, but within
days displayed severe ataxia and died within the month.
Affected mice displayed deficient hindlimb reflexes and lack
of coordination. Their peripheral nerves displayed axon
degeneration and dysmyelination, and central neurons
showed excessive axonal pruning, although the length of
the remaining axons was maintained. Mice with a 75%
decrease in total brain gangliosides had a progressive neuropathy with dysreflexia by 3 mo of age, gait disorders, and
progressive loss of Purkinje neurons. A more subtle conditional mutation was created by knockout of Ugcg selectively in mature Purkinje neurons using Cre under the control of the L7 (Pcp2) promoter (538). These mice showed
progressive Purkinje cell loss, but more notably this was
preceded by axon degeneration in the absence of dendritic
changes. This was accompanied by abnormalities at nodes
of Ranvier, including disrupted paranodal loops and altered
distributions of nodal ion channels and adhesion molecules.
In comparison, knockout of Ugcg in myelinating cells (using Cre driven by a Cnp promoter) did not result in notable
changes in brain ganglioside expression, axonal/myelin histology, or phenotype (400). Together, these data are consistent with the interpretation that gangliosides are not required for nerve cell differentiation or nervous system anatomic development, but their expression on nerve cells is
required to maintain healthy intracellular interactions essential to nervous system stability and function, especially
axon-myelin interactions.
486
4. GD1a/GT1b knockout mice
As described above, mice lacking complex gangliosides, or
neurons lacking all gangliosides, display varying degrees
and onset of axon-myelin dysregulation, among other nervous system disorders. In contrast, mice expressing only
a-series or 0-series gangliosides do not display such severe
phenotypes, indicating that different complex gangliosides
might compensate for each other to support axon-myelin
stabilization. One test of this hypothesis was to block the
sialyltransferase(s) that add(s) the terminal ␣2–3 sialic acids
to GM1 (GD1a synthase) and GD1b (GT1b synthase). Experiments using single- and double-null mice revealed the
sialyltransferases involved (467). Of the six ␣2–3 sialyltransferases in mice (and humans), four (St3gal1 through
St3gal4) can synthesize GD1a/GT1b from GM1/GD1b in
vitro. In the study of each of these knockout mouse strains,
all of which are viable, only St3gal2-null mice had altered
brain ganglioside expression. They expressed half the
amount of brain GD1a and GT1b with GM1 and GD1b
building up commensurately behind the partial block.
St3gal2-null mice had no evident nervous system deficits.
Testing of double-null mice revealed that the St3gal3 gene
product was compensating for the absence of St3gal2, in
that GD1a or GT1b were nearly absent in St3gal2/3-double
null mice, with GM1 and GD1b proportionally increased.
Notably, these mice were short-lived and displayed early
dysreflexia. Although supportive of a role for GD1a and
GT1b, per se, in nervous system stability, the St3gal2/3double-null mice also had reduced total glycoprotein sialylation, complicating the molecular interpretation of their
phenotype. Nevertheless, they support the essential functions of ␣2–3 sialic acid-terminated glycans, which include
GD1a and GT1b, in the nervous system.
B. Molecular Mechanisms of Ganglioside
Function
Membrane gangliosides at the cell surface function in two
distinct modes, cis and trans (182). In the cis mode, gangliosides associate laterally with other membrane molecules,
including receptors and ion channels, to modulate their activities. In the trans mode, ganglioside glycans, which extend into the extracellular milieu, interact with complementary glycan binding proteins to mediate recognition, including cell-cell recognition. Separately, these cis and trans
modes of ganglioside molecular interactions are regulators
of cell recognition and cell signaling; together they can
translate cell surface recognition into changes in cell physiology.
1. Cis-regulation
Gangliosides associate laterally in the plane of the external
leaflet of the plasma membrane with each other and with
selected lipids and proteins in structures referred to as lipid
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SCHNAAR ET AL.
Laminin
self assembly
binding
NGF
TrkA
binding
GM1
Integrin
Lyn
P
Lyn
Neurite outgrowth
FIGURE 20. Model for laminin-1-induced clustering of GM-1, TrkA, ␤1 integrin, and signaling molecules in
lipid rafts to stimulate neurite outgrowth. Laminin-1 directly binds to GM1 and induces its focal aggregation,
enhancing the relocation of TrkA in lipid rafts and the subsequent activation of signaling molecules downstream
of TrkA, such as Lyn. Clustering of GM1 with laminin-1, along with laminin-1 self-assembly, also promotes
relocation and enrichment of ␤1 integrin in lipid rafts and enhances combined laminin-integrin signaling to
trigger neurite outgrowth. [From Ichikawa et al. (208), with permission from The Company of Biologists, Ltd.]
rafts (458). Lipid rafts are thought to be small (nanometer
scale) highly dynamic associations with a variety of lipid
and protein substructures (448). Ganglioside-containing
lipid rafts are enriched in other raft lipids such as sphingomyelin, cholesterol, and phosphatidylinositols; in lipidmodified proteins such as glycophosphatidylinositol-linked
surface proteins and lipid-modified signaling molecules
(such as those of the Src family); and in selected transmembrane proteins. Either in or out of rafts, gangliosides may
associate laterally with signaling molecules at the cell surface, regulate their lateral distribution and associations
with other molecules, and regulate their signaling capacity
(489). In this way, the expression and distribution of gangliosides provides an additional layer of control in cell surface signaling. An example of this is ganglioside-mediated
regulation of receptor tyrosine kinases (RTKs).
Ganglioside GM3, a common ganglioside in many cell types
outside of the nervous system, associates laterally with the
EGF receptor (55) and with the insulin receptor (344) to
downregulate their protein tyrosine kinase activities in response to agonist binding. Evidence for the physiological
significance of ganglioside-RTK regulation comes from
St3gal5-null mice, which lack GM3 and its downstream
metabolites (560). These mice displayed enhanced insulin
sensitivity characterized by increased insulin-induced insulin receptor phosphorylation in skeletal muscle. Remarkably, St3gal5-null mice were protected from high-fat dietinduced insulin resistance, suggesting that GM3 plays an
important role in metabolic control via this specific RTK
association. A model for GM3 regulation of differential
insulin receptor lateral distribution provides a framework
for understanding its control of insulin sensitivity. Insulin
receptors associate with caveolin-1, an interaction required
for their responsiveness (85), and separately with ganglioside GM3. Immunoprecipitation, colocalization, and fluorescence recovery after photobleaching studies support a
model in which these interactions are functionally competitive (223). In the absence of GM3, insulin receptors associate with caveolin-1 and are more insulin sensitive. When
GM3 is increased, insulin receptors associate selectively
with GM3, are released from their interaction with caveolin-1, and are more insulin resistant.
A CNS correlate to the above model is the enhancement of
high-affinity nerve growth factor receptor (TrkA) activity
by GM1 (125). Immunoprecipitates of TrkA contained
GM1, and GM1 addition activated TrkA’s protein tyrosine
kinase activity (324). A multireceptor system linking the
neurite-outgrowth activating activity of laminin-1 to GM1
and TrkA was recently described (208). Treatment of dorsal
root ganglion neurons with laminin clustered GM1, TrkA,
and ␤1-integrin, presumably via direct binding of laminin
to GM1. Knockdown of individual components via siRNA
suggested a model in which GM1 facilitates the association
of laminin, ␤1-integrin, TrkA, and the intracellular Src family member Lyn to activate neurite outgrowth (FIGURE 20).
Although mice lacking GM1 and other complex gangliosides (B4galnt1-null mice) elaborate a well-functioning nervous system, evidence for loss of regenerative capacity in
selected nerve pathways in these mice may relate to cis-
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BRAIN SIALOGLYCANS
regulation of neurotrophic factor receptors by gangliosides
(235).
2. Gangliosides as receptors for neurotropic
bacterial toxins
Brain gangliosides were first identified as receptor molecules in the context of bacterial toxins (500). The family of
potent protein neurotoxins secreted by certain clostridial
strains includes several types of botulinum neurotoxins and
the structurally related tetanus neurotoxin (471). Each
toxin is comprised of a heavy chain that binds to the neuronal cell surface and an enzymatic light chain that is inserted into the cytoplasm, proteolyzes key vesicle fusion
proteins (SNARE proteins), and thereby disrupts synaptic
transmission. Given the neuromuscular symptoms of tetanus, brain tissue extracts were tested for their ability to bind
and remove the toxin from solution. The “toxin-fixing”
component was extracted from brain tissue using ethanol
and was present primarily in brain gray matter. Its identification as ganglioside was confirmed by van Heyningen using brain gangliosides purified by leaders in the early ganglioside field, E. Klenk of Cologne and J. Folch-Pi of Harvard (500). Subsequently, van Heyningen and Miller (501)
purified and fractionated gangliosides from bovine brain
and found a major brain ganglioside species with a ratio of
sialic acid to hexosamine of 3:1 (now known to be GT1b) to
be particularly potent at “fixing” tetanus toxin. Subsequent
studies demonstrated that tetanus toxin binding to brain
synaptosomal membranes was most potently reversed by
GT1b followed by GD1b, GD1a, and GM1, which was
100-fold less potent than GT1b (388). Subsequent studies
indicate that all of the clostridial neurotoxins bind to gangliosides with differing specificities that depend on multiple
potential binding sites near the COOH terminus of the
heavy (binding) subunit (35, 471). Crystallography reveals
that the clostridial toxins evolved patches of amino acid
residues that form a network of hydrogen bonds and hydrophobic stacking interactions that specify ganglioside
glycan binding (461) (FIGURE 21). Proof of the role of gangliosides in clostridial toxicity comes from studies using
complex ganglioside knockout (B4galnt1-null) mice. Despite retaining simple gangliosides, these mice display reduced sensitivity to tetanus and botulinum toxins (233); the
potency of the clostridial toxins in inhibiting neuromuscular activity is reduced 100-fold in the absence of complex
gangliosides (65, 396).
In addition to the neurotropic toxins, gangliosides are receptors for other multi-subunit protein toxins. Cholera
toxin and E. coli enterotoxin LT-I bind selectively to GM1,
whereas E. coli enterotoxin LT-IIb binds to GD1a (93,
158). As with the clostridial neurotoxins, each toxin is
multi-subunit, with its binding subunit mediating toxin
binding to gangliosides at the cell surface and an enzymatic
subunit then entering the cytoplasm. These toxins target the
intestinal epithelium and dysregulate cAMP signaling,
488
FIGURE 21. Crystal structure of botulinum neurotoxin type A in
complex with the cell surface co-receptor GT1b. Top: close-up of the
GT1b binding site. GT1b represented as sticks with yellow carbons.
The GT1b coordinating residues are shown as sticks with gray carbons. Bottom: schematic picture of GT1b and its hydrogen bonds to
botulinum neurotoxin type A. The hydrogen bonds between the protein (blue) and GT1b (black) are shown as dotted red lines and the
GT1b internal hydrogen bonds as dotted black lines. Distances of key
hydrogen bonds are displayed in Ångstroms. The ␣2,8-linked sialic
acid (Sia7) that is disordered in the complex is shaded gray. Numbered monosaccharide names are shown; Glc, glucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Sia, sialic acid. [Adapted from
Stenmark et al. (461).]
resulting in severe diarrhea. As with the clostridial toxins,
ganglioside-binding enterotoxins have evolved protein
binding sites that provide a network of hydrogen bond and
hydrophobic stacking interactions engineered to specifically
bind ganglioside glycans (200).
3. Gangliosides as receptors in axon-myelin
interactions
The evolution of bacterial toxins that specifically bind to
brain gangliosides suggested that endogenous vertebrate
lectins may have evolved to do the same. A search for ganglioside binding proteins in the brain led to myelin-associated glycoprotein (MAG), and to an enhanced understanding of ganglioside functions in axon-myelin interactions
(228, 423).
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MAG is a single-pass transmembrane protein that is selectively expressed by myelinating cells: oligodendrocytes in
the CNS and Schwann cells in the PNS (377). In the CNS,
MAG is found only on the innermost (periaxonal) wrap of
myelin, directly apposed to the axon surface. In the PNS it is
also found on periaxonal myelin, as well as on SchmidtLanterman incisures, lateral loops, and the inner and outer
mesaxons (491). Based on its expression, MAG was long
proposed to be involved in axon-myelin interactions. Although gene deletion of MAG in mice did not result in
severe demyelination as some had expected (263, 308), the
study of Mag-null mice reveals important roles for MAG in
axon-myelin interactions relating to axon cytoarchitecture,
axon stability, and axon regeneration. Mag-null mice myelinate axons throughout the nervous system, and most myelin in Mag-null mice appears grossly normal. However,
myelination is delayed, and there is a higher proportion of
myelin structural abnormalities (155, 368). Mag-null mice
also have increased numbers of poorly formed nodes of
Ranvier and disruption of the spatial patterns of nodal ion
channels and adhesion molecules (280, 368). Normally,
myelination induces local increases in neurofilament phosphorylation in ensheathed axons, resulting in increased
axon diameter and increased conduction velocity (202).
Mag-null mice display reduced neurofilament phosphorylation and spacing and reduced axon diameter, even in myelinated axons (568). Myelination also nurtures axons; dysmyelination results in axon degeneration (335). MAG is
part of this nurturing effect in that Mag-null mice display
axon degeneration in both central and peripheral nerves,
resulting in progressive axonal loss over their lifetimes
(339). Notably, MAG also protects axons from short-term
toxic insults; Mag-null mice are more sensitive to axonopathic neurotoxins, and soluble forms of MAG can protect
axons from neurotoxic insults (339). In addition to its
broad role in supporting healthy long- and short-term axon-myelin interactions, MAG also inhibits axon regeneration (293, 317). MAG is one of a suite of molecules, including those on residual myelin, that limit recovery from traumatic nervous system injury by signaling axons to halt
outgrowth (258, 407, 570). Although genetic deletion of
these factors has, thus far, failed to generate broad and
robust regeneration after CNS injury, Mag-null mice display increased axon sprouting after injury in some studies
(257, 411). From long-term axon-myelin stabilization to
inhibition of regeneration, all of the physiological roles of
MAG have, at least in part, been linked to its recognition of
brain gangliosides (422).
MAG was a founding member of the siglec family of sialic
acid binding proteins (97, 228), with an NH2-terminal (outermost) V-type Ig domain that has distinctive structural
elements shared among the 14-member human siglec family
(514). Different siglecs have evolved to take advantage of
the various linkages and subterminal structures of sialoglycans to provide a high degree of binding specificity (278).
FIGURE 22. MAG binding to gangliosides: structural specificity
(91, 565). COS cells (fibroblasts) transfected to express full-length
MAG were placed in microwells adsorbed with the indicated concentrations of the indicated gangliosides. MAG-mediated cell adhesion is
expressed as a percent of the MAG-transfected cells added to each
well. Ganglioside structures are shown schematically using the key in
FIGURE 11. [Adapted from Collins et al. (91), with permission from
American Society for Biochemistry and Molecular Biology, and Yang
et al. (565).]
Initial specificity studies discovered that MAG bound preferentially to the trisaccharide “NeuAc ␣2–3 Gal ␤1–3 GalNAc” (228), the shared terminal sequence of gangliosides
GD1a and GT1b (see FIGURE 8). Direct testing of gangliosides for their ability to support MAG binding confirmed
GD1a/GT1b specificity among the major brain gangliosides
(FIGURE 22) and revealed enhanced binding to the rare ␣-series gangliosides (90 –92, 565). These data provide a context to consider the relationship between the physiological
functions of MAG and those of complex gangliosides, particularly GD1a and GT1b.
The phenotypes of mutant mice with altered ganglioside
expression, as described above, are consistent with a role
for GD1a and GT1b in the functions of MAG (422). Mice
lacking complex gangliosides (B4galnt1-null) display many
of the same phenotypic traits as Mag-null mice, including
myelin anomalies, progressive axon degeneration in the
central and peripheral nervous systems, reduced neurofilament spacing, reduced axon diameters of myelinated axons,
and disrupted nodes of Ranvier (358, 447, 469). Behaviorally, Mag-null and B4galnt1-null mice display similar disruptions in hindlimb reflexes and coordination, as well as
similar patterns of whole-body tremor and hyperactivity.
Double-null mice (B4galnt1/Mag) have similar defects as
each single null strain (358). In contrast to the B4galnt1null mice, St8sia1-null mice do not display altered axonmyelin interactions, nor do St3sia5-null mice. Since these
mouse strains express high amounts of GD1a (St8sia1) and
GD1␣ (St3sia5), respectively, and both of these gangliosides are high-affinity MAG receptors (409), the lack of
axon-myelin deficits is consistent with a phenotype based
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on MAG-ganglioside recognition. B4galnt1/St8sia1 double
null (GM3 only) and B4galnt1/St3gal5 double null (ganglioside knockout) mice are consistent with this hypothesis, in
that they lack high-affinity MAG receptor gangliosides and
display significant defects in axon-myelin interactions.
In addition to a role in stabilizing long-term axon-myelin
interactions, complex gangliosides mediate the ability of
MAG to protect axons from short-term toxic insults (339).
For example, addition of soluble expressed forms of MAG
to neurons in culture protects them from the effects of vincristine, an axonopathic neurotoxin. A mutant form of
MAG lacking the shared arginine in siglecs required for
sialic acid binding fails to protect neurons, and neurons
from complex ganglioside knockout mice (B4galnt1-null)
are not protected by wild-type MAG. Treatment of nerve
cells with sialidase or with an inhibitor of the ganglioside
biosynthetic pathway reverses the ability of MAG to protect
neurons (298). Together, the data imply that, among other
potential ganglioside functions, the major brain gangliosides GD1a and GT1b are essential to healthy axon-myelin
interactions due to their actions as receptors for MAG.
However, the stabilizing effects of MAG are not always
beneficial, in that MAG also inhibits axon regeneration.
4. Gangliosides as regulators of neuronal survival
and axon regeneration
It has long been observed that exogenous addition of gangliosides to nerve cells in culture results in neurite sprouting
and outgrowth (67, 389), and enhances survival in the face
of specific toxic insults (137). The mechanisms of these
well-established observations have been elusive and range
from specific interactions with growth factor receptors such
as TrkA (208, 324), to modification of calcium flux (107,
553), to generalized and structurally nonspecific effects on
the membrane milieu (457). Regardless of the mechanism(s), the results were sufficiently consistent to warrant
human trials of injectable GM1 in a variety of human diseases including spinal cord injury (163), Parkinson’s disease
(424), and stroke (70). The results have been variable, with
effects on recovery from spinal cord injury and stroke limited, and those in Parkinson’s disease more encouraging.
Animal models of ganglioside deficiency display some Parkinson-like symptoms that are alleviated by administration
of L-DOPA or cell-permeable ganglioside mimetics (550),
and gangliosides may be reduced in Parkinson’s disease patients (551). Rare reports of sporadic treatment-related anti-ganglioside immune responses resulting in Guillain-Barré
syndrome have raised concerns (209). With ganglioside delivery involving intravenous and/or intramuscular delivery,
the pharmacokinetics and pharmacodynamics of gangliosides as drugs, and their potential to enhance outcomes in
human disease, are a matter for further investigation.
490
MAG inhibition of axon regeneration (see above) is also
mediated, at least in part, by complex gangliosides. MAG
has several receptors on axons that act cooperatively or
independently to generate intracellular signals that halt
axon outgrowth (71, 423). When studied in vitro, MAG
inhibition of axon outgrowth from some nerve cell types
(but not others) was reversed by cleaving the terminal sialic
acids from gangliosides with sialidase, by inhibiting ganglioside biosynthesis, by exogenously added GT1b and/or
GD1a, or by sialoglycan inhibitors of MAG binding (297,
523, 526, 533, 534). MAG inhibition of axon outgrowth
from cerebellar granule neurons was found to be completely
ganglioside dependent, from hippocampal neurons was
partially ganglioside dependent, and from dorsal root ganglion neurons and retinal ganglion cells was ganglioside
independent, emphasizing the diverse pathways MAG uses
in this context (297, 523). Ganglioside-mediated MAG inhibition of axon outgrowth, like other modes of axon inhibition, is dependent on activation of the small intracellular
GTPase RhoA. The data are consistent with the hypothesis
that MAG binding to gangliosides initiates a signaling pathway that results in RhoA activation, at least in some types of
nerve cells. This hypothesis is supported by the observation
that clustering GD1a or GT1b with anti-ganglioside antibodies mimics MAG-mediated axon inhibition in a RhoAdependent manner (526, 534, 543), a mechanism that may
have implications in autoimmune neuropathologies involving anti-ganglioside antibodies as described in a subsequent
section of this review (446, 582).
Since gangliosides are on the outer leaflet of the axon
plasma membrane, recruitment of other membrane molecules is likely to be involved in MAG signaling. Although
such transducing molecule(s) have yet to be fully established, the precedent of cis-regulation by gangliosides is
instructive. One cis-interaction that may influence MAG/
ganglioside-mediated axon inhibition is the interaction of
gangliosides with another MAG receptor on axons, NgR1
(543). However, in other experiments ganglioside-mediated
and NgR-mediated MAG inhibition are clearly independent (297, 523). Gangliosides have also been reported to
interact with a transmembrane inhibitory coreceptor,
p75NTR (157, 561), although axon outgrowth inhibition
from some neurons is insensitive to loss of p75 (and the
related TROY), indicating other ganglioside-related transducers must be present (523). Together, the data support a
role for gangliosides as receptors for MAG on some nerve
cell types, transducing signals that inhibit axon outgrowth
after injury (423). The extent to which regeneration of any
particular nerve pathway is sialoglycan dependent requires
direct testing.
Treatment of cultured nerve cells with sialidases, which
modify sialoglycans including gangliosides, modulates neurite/axon outgrowth either positively or negatively depending on the neurons and sialidase (102, 474, 549). A role for
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endogenous sialoglycans in restricting axon regeneration in
vivo was demonstrated by testing sialidase as a treatment to
enhance axon outgrowth after CNS injury in animal models
(310, 311, 564). Delivery of bacterial sialidases to the site of
spinal cord injuries enhanced axon regeneration as well as
behavioral and physiological outcomes. In one model of
brachial plexus injury, nerve roots were cut flush to the
spinal cord in rats, a peripheral nerve graft was inserted into
the cord, and sialidase pumped to the injury site using an
implanted osmotic pump (564). Motoneuron outgrowth
into the grafts was enhanced 2.5-fold by sialidase treatment. In another injury model, rats were subjected to a
thoracic spinal cord contusion by blunt impact (310, 311).
Delivery of sialidase to the injury site by osmotic pump
increased axon sprouting distal to the lesion, and enhanced
recovery of hindlimb motor functions, coordination, and
spinal-mediated physiological function. Consistent with the
well-established specificity of bacterial sialidases, the treatments (primarily with exogenous V. cholerae sialidase) resulted in conversion of complex gangliosides, including
GD1a and GT1b, to GM1 (414). The data are consistent
with gangliosides being mechanistic targets of therapeutic
sialidase, either by loss of inhibitory gangliosides (GD1a
and GT1b), generation of stimulatory ganglioside GM1, or
both. Since sialidases act on sialoglycoproteins as well as
gangliosides, roles for other sialoglycans are not ruled out.
Regardless of the underlying mechanism(s), the ability of
sialidase to enhance outcomes from nerve injury identifies
nervous system sialoglycans as important factors controlling axon regeneration.
V. POLYSIALIC ACID FUNCTIONS
A. Polysialic Acid as Modulator of Cell
Interactions
After the initial in vitro finding that polysialylation decreases NCAM binding (99, 401), the prevailing “steric
hindrance model” was shaped mainly by work in the Rutishauser laboratory (397, 399). According to this model,
depicted in FIGURE 23A, polySia affects not only specific
functions of its carrier proteins but generally increases the
intercellular space (566, 567) and abrogates adhesive binding of other cell surface molecules such as cadherins and
integrins (156, 220, 221). Although consistent with some of
the biological functions ascribed to polySia, the model is not
sufficient to explain many of the defects identified in animals with genetically induced polySia deficiency, and falls
short of explaining the results of other cellular and molecular studies. Additional models of polySia function are summarized in FIGURE 23, B–H, and are discussed below.
In a series of in vitro studies, polySia was shown to control
NCAM signaling (132, 191, 386, 433, 434). EndoN-mediated removal of polySia from human neuroblastoma cells
initiated NCAM interactions at cell-cell contacts and was
followed by reduced cell proliferation and a parallel activation of the extracellular signal-regulated kinases 1 and 2
(ERK1/2) (FIGURE 23B). Activated ERK1/2, in turn, promoted survival and neuronal differentiation with enhanced
formation of neuritelike processes (191, 433, 434). These
same responses were obtained in a coculture system of
NCAM-negative neuroblasts exposed to polySia-negative,
NCAM-positive membranes, demonstrating that the polySia-free NCAM acts as ligand of a heterophilic interaction
partner (434). Similarly, neuronal precursors isolated from
the subventricular zone of early postnatal mice responded
with enhanced differentiation to the removal of polySia or
to exposure with polySia-negative NCAM. A higher degree
of differentiation was obtained by adding polySia-negative
NCAM to precursors from Ncam knockout mice, demonstrating that this NCAM function is also mediated via heterophilic binding (386).
NCAM-induced signaling is believed to involve its association with fibroblast growth factor (FGF) receptors, causing
receptor activation and downstream signaling through the
mitogen-activated protein kinase ERK1/2 pathway (72,
146, 197, 245, 341, 402). Most data are compatible with
the idea that homophilic trans-interactions of NCAM are a
prerequisite for the association with the FGF receptor in the
plane of the membrane (in cis). However, one study demonstrates FGF receptor-mediated signaling induced by application of soluble NCAM to NCAM-negative cells (146).
Consistent with this mode of heterophilic NCAM binding,
the activation of ERK1/2 in response to a loss of polySia
could be linked to FGF receptor activity (132) (FIGURE
23B). The activation could be reproduced by the stimulation of NCAM-negative cells with a NCAM-mimetic peptide representing the binding site of the FGF receptor (231,
336) and by contacting these cells with soluble NCAMforms containing at least one of the two FGF receptor binding sites localized in the FnIII-1 and FnIII-2 domains (see
FIGURE 15) (82, 231).
Notably, other cellular responses evoked by the removal of
polySia occur independent of FGF receptor activation
(132). The loss of polySia causes an increase in the number
of focal adhesions and promotes association of the Src family kinase p59Fyn with the focal adhesion scaffolding protein
paxillin and the focal adhesion kinase (FAK). Again, this
effect is mimicked by soluble forms of NCAM applied to
NCAM-negative cells, but cannot be induced with the FNIII
domains or the mimetic peptide. Instead, a fragment comprising the third and fourth Ig-like domain of NCAM is
necessary and sufficient to enhance focal adhesion without
a detectable activation of ERK1/2. Because polySia and
NCAM are enriched at sites of cell-cell contact and NCAM
is never detected in association with the focal adhesions,
these data imply that loss of polySia triggers an FGF receptor-independent mechanism by which NCAM-induced sig-
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BRAIN SIALOGLYCANS
A
B
C
P
ERK1/2
P
FAK
P
D
E
H
G
F
FIGURE 23. PolySia and cell interactions. A: polySia as a negative regulator of cell-cell apposition. Loss of
polySia allows for trans-interactions of NCAM and other cell surface proteins. B and C: loss of polySia induces
heterophilic trans-interactions and activates the protein kinase ERK1/2 via FGF receptor signaling (B), or
promotes focal adhesion at the cell-substrate interface by interactions with a yet unknown heterophilic binding
partner and signaling to the focal adhesion kinase (FAK) (C). D: polySia may interfere with cis-interactions of
NCAM and signaling complex formation. E and F: loss of polySia enables interactions of NCAM with proteoglycans of the ECM (E), or polySia-NCAM itself interacts with proteoglycans of the ECM (F). G: polySia as a
scavenger of soluble factors facilitating receptor activation. H: polySia modulates activation of ionotropic
glutamate receptors. See text for details.
naling at cell-cell contacts leads to enhanced cell-matrix
interactions by focal adhesions (132) (FIGURE 23C).
NCAM-dependent signaling via cis interactions with the
FGF receptor may occur in a cell autonomous way. In
492
␤-cells from pancreatic tumors, NCAM is essential for the
assembly of a signaling complex including N-cadherin and
FGF receptor 4, and activation of the FGF receptor in this
complex promotes integrin-mediated matrix adhesion (72).
As for other possible cis interactions of NCAM, the role of
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SCHNAAR ET AL.
polySia has not yet been considered in this model. However,
␤-cell tumors are known to express polySia (252), and it is
tempting to speculate that its presence affects the NCAMdependent signaling complex (FIGURE 23D).
Last in this series of indirect polySia functions is the modulation of NCAM binding to components of the extracellular matrix (ECM) (FIGURE 23, E AND F). “Naked” NCAM
binds with high affinity (dissociation constants ⬃0.1 nM) to
the prominent chondroitin sulfate proteoglycans neurocan
and phosphacan (153, 177, 303), two prominent constituents of the brain ECM (118), and it can be assumed that the
presence of polySia inhibits this interaction. In sharp contrast, NCAM binding to heparan sulfate proteoglycans (87,
88) is promoted by polySia (464). Together with cellular
assays showing that polySia-driven synapse formation is
abolished by heparinase treatment (117), the data indicate
that polySia itself or combined interactions of polySia and
NCAM may bind to the heparan sulfate proteoglycans in
the ECM or at the cell surface.
It is not clear how NCAM-expressing cells regulate the
establishment of homo- versus heterophilic NCAM binding. Apparently, both types of interactions coexist and are
integrated on the cellular level (433). Possibly, as shown for
the high-affinity interactions with proteoglycans (177), heterophilic interactions outcompete homophilic NCAMNCAM binding.
In the above examples, polySia acts to block interactions. In
contrast, models of direct polySia function are shown in
FIGURE 23, F–H. Among these, the best studied are interactions of polySia with soluble ligands. Interactions with the
brain-derived growth factor BDNF were initially proposed
based on the observation that sensitivity towards the brainderived growth factor BDNF requires polySia in two models: 1) long-term potentiation of synaptic transmission in
hippocampal slice cultures and 2) survival and differentiation of cultured cortical neurons (318, 531). Increased
BDNF responsiveness may be explained by polySia capturing the neurotrophic factor from the extracellular milieu,
enhancing its local concentration, increasing its availability,
and facilitating its interactions with BDNF receptors (FIGURE 23G). In cholinergic neurons of the septum, however,
polySia limits the binding of BDNF and reduces BDNFinduced activation of acetylcholine synthesis, suggesting
that polySia has a shielding function, which hampers interactions between receptor and ligand (66). Similar to the
responsiveness of cortical neurons, hypothalamic neurons
were more sensitive to ciliary neurotrophic factor in the
presence of polySia (532), and oligodendrocyte precursor
cells need polySia to sense a gradient of platelet-derived
growth factor (583). Indeed, evidence for direct interactions
of polySia with the neurotrophins BDNF, NT3, and NT4 as
well as with FGF2 and the neurotransmitter dopamine was
obtained by biochemical approaches using electrophoretic
and chromatographic methods (213, 226, 357).
Finally, polySia may interact directly with cell surface proteins (FIGURE 23H). Of high interest in this field, soluble
polySia as well as soluble NCAM-fragment carrying polySia were capable of potentiating the opening of AMPA-type
glutamate receptors and inhibiting activation of GluN2Bcontaining NMDA receptors (184, 497). Consistent with
the latter, two recent studies provide evidence that polySia
regulates synaptic plasticity by setting a balance in signaling
via extrasynaptic GluN2B and synaptic GluN2A receptors
(239, 240). In addition, polySia binds directly to human
Siglec-11 expressed by human brain microglia (537). Siglec11-mediated interaction with polySia reduced inflammatory and neurotoxic responses in an experimental system of
ectopic human Siglec-11 expression in mouse microglia
(537).
B. Lessons From Mouse Genetic Models
The first polySia-deficient mouse models were obtained by
complete inactivation of Ncam, deleting all isoforms (95),
or by ablation of exon 19 to generate a NCAM-180 knockout (490). In fact, the resulting NCAM-deficient mice were
almost completely negative for polySia, but in marked contrast to the predicted central roles of NCAM and polySia in
neurodevelopment and plasticity of the mature nervous system, Ncam knockouts showed a surprisingly mild phenotype, appeared healthy and fertile, and had an overall fairly
normal brain architecture. Although these data indicated
that the complex and energetically “expensive” polysialylation machinery had evolved primarily to produce polySia
on NCAM, grossly normal brain development of Ncam
knockout mice was paradoxically taken as evidence that
polySia has no vital function. Only later, with the identification of the polySTs, were mouse models generated that
allowed the biological functions of NCAM and its polySia
modification to be addressed separately. As detailed below,
these mouse models impressively document the importance
of a strictly controlled NCAM homeostasis and open up
exciting new vistas towards understanding the relevance of
protein glycosylation for the complex process of building
the vertebrate brain.
1. St8sia4 and St8sia2 single knockout mice
St8sia4 knockout mice were the first polyST-deficient animal model to be created (129). Consistent with the potential
of ST8Sia-II to almost entirely compensate for loss of
ST8Sia-IV during development (see sect. IIIB3), St8sia4
knockouts had no overt neuroanatomical defects. However, based on its developmental expression pattern,
St8sia4 knockout mice had markedly reduced polySia levels
in selected parts of the postnatal and adult brain (129, 327).
Hippocampal mossy fibers were devoid of polySia by im-
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munohistochemistry, but their lamination was normal
(129). Conversely, St8sia2 knockouts expressed normal
polySia levels on mature mossy fibers but, similar to Ncam
knockouts, axons showed guidance errors and formed ectopic synapses in the hippocampus (10). Taken together,
these data show that malformations in the mossy fiber tract
originate from a lack of polySia during development. Since
both polySTs are colocalized in the developing dentate
gyrus (192), the absence of ST8Sia-II is likely to cause only
a partial reduction of polySia (161, 354), which is, however, sufficient to damage mossy fiber formation as severely
as the loss of NCAM.
Additional evidence for the importance of polySia homeostasis during mossy fiber tract formation was obtained in a
hippocampal slice culture model (527). When slices derived
from wild-type brains were treated with N-propanoyl-Dmannosamine (ManNProp), a chemically modified sialic
acid precursor, polysialylation of NCAM was reduced, resulting in aberrant mossy fiber projections and formation of
ectopic synapses similar to those seen in the Ncam and
St8sia2 knockouts.
In young rats St8sia2 mRNA was localized to polySia-positive cells in the subgranular zone of the dentate gyrus (192).
Consistent with this, St8sia2 knockouts possess a diminished number of polySia-positive cells in this neurogenic
niche (10). The frequency of mitotic neural progenitor cells
was not altered (10), but many of the immature granule
neurons showed aberrant location and altered morphology,
suggesting a role of ST8Sia-II in their differentiation (327).
In St8sia2-null (⫺/⫺) mice, a reduction of polySia-positive
cells was also observed in the posterior part of the SVZ (10).
In both polyST single knockout lines, however, the levels of
polySia on the neuroblasts of the anterior SVZ and RMS
were unaffected (10, 129, 327).
2. St8sia2, St8sia4 double-knockout mice
The simultaneous deletion of both polySTs generates mice
that are polySia-negative, but retain normal levels of
NCAM isoform expression (9, 541). Since Ncam⫺/⫺ mice
are almost completely devoid of polySia, it is not surprising
that their two major neurodevelopmental defects, delamination of mossy fibers and the migration deficit of RMS
neuroblasts with smaller olfactory bulbs (FIGURE 24, A AND
B), are recapitulated in St8sia2, St8sia4 double-knockout
mice (2⫺/⫺4⫺/⫺) (9, 541). This is in good agreement with
previous findings showing that both defects can also be
induced by endosialidase treatment removing the sugar
polymer and leaving the NCAM protein backbone unaltered (356, 442). The phenotype of 2⫺/⫺4⫺/⫺ mice, therefore, corroborates that the loss of polySia causes these defects independent of the presence or absence of NCAM
(FIGURE 24B). The migration deficit of neuroblasts is linked
to marked changes of cellular arrangement within the RMS
494
(FIGURE 24, C AND D). As discussed in section IIIB5, a reduced compaction or even dispersal of the migratory chains
is induced by removal of polySia in vitro (76, 203). In addition, in Ncam knockouts and after endosialidase injection, the glial tubes, which serve as a scaffold for migratory
neuroblast chains, disintegrate and a massive astrogliosis
develops (28, 76). As illustrated in FIGURE 24C, similar
changes are also evident in the RMS of 2⫺/⫺4⫺/⫺ mice (H.
Hildebrandt, unpublished observations). In addition to reduced homotypic contacts between the migratory neuroblasts, ultrastructural analyses in Ncam knockouts revealed
the formation of zona-adherens-like heterotypic contacts of
precursors with astrocytes and an accumulation of axons in
the RMS (76). Thus altered neuroblast migration in the
absence of polySia or NCAM involves direct perturbations
of cell-cell interactions as well as an extensive rearrangement of cytoarchitecture.
In marked contrast to single knockout mice (Ncam⫺/⫺,
St8sia2⫺/⫺, or St8sia4⫺/⫺), double-knockout 2⫺/⫺4⫺/⫺
mice have severe developmental deficits. Although born at
Mendelian ratio and without overt morphological defects,
2⫺/⫺4⫺/⫺ mice fail to thrive and more than 80% die within
4 wk after birth (541). With regard to brain development,
many, but not all, of the 2⫺/⫺4⫺/⫺ mice develop a progressive, noncommunicating form of hydrocephalus associated
with cortical thinning, corpus callosum hypoplasia, and deformation of the hippocampal formation and fimbria (541).
This was confirmed in an independent study, which in addition described defects of cortical organization associated
with apoptotic cell death and reduced neuron numbers in
1-mo-old mice (9). At least some of the changes in the
postnatal cortex are associated with ventricular dilatation
and therefore may be secondary to a developing hydrocephalus. Another cause of cortical derangement may be altered
migration of neural precursors observed during embryonic
development in 2⫺/⫺4⫺/⫺ mice (9), but lineage identity of
these cells and underlying mechanisms await further analyses.
Prominent among the neurodevelopmental defects of 2⫺/⫺
4⫺/⫺ mice are malformations of a specific set of commissural and noncommissural brain axon tracts. Most pronounced is the complete absence of the anterior commissure, which connects the olfactory nuclei and lateral parts of
the temporal lobe. The anterior and posterior limbs of the
commissure rarely approach and never cross the midline.
Tracing of the anterior limb disclosed fasciculation defects
and early deviation of fibers from their normal trajectory.
These deficits were never observed in Ncam-null mice but
occurred with 100% penetrance in the 2⫺/⫺4⫺/⫺ doubleknockout, including 2⫺/⫺4⫺/⫺ mice that escape from hydrocephalus and have normal-sized ventricles. It is therefore
safe to say that these axonal defects develop independent
from ventricular dilatation (541).
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FIGURE 24. Two categories of neurodevelopmental defects in polySia-negative mice. A–D: shared features
of 2⫺/⫺4⫺/⫺ and Ncam knockout (N⫺/⫺) mice are caused by loss of polySia. A and B: migration deficits of
olfactory interneuron precursors in the rostral migratory stream (RMS) and smaller olfactory bulbs (OB) in
polysialyltransferase-negative (polyST⫺) 2⫺/⫺4⫺/⫺ mice and polySia-NCAM-negative Ncam knockout mice.
Wt, wild type. C: altered cytoarchitecture of the RMS in polyST⫺ mice. The ordered arrangement of GFAPpositive astrocyte tunnels around migratory chains of NCAM-positive neuroblasts as observed in the wild-type
(wt) is severely disturbed (H. Hildebrandt, unpublished data). D: schematic representation of the situation in the
presence or absence of polySia, based on the work of Chazal et al. (76). E–I: defects that manifest exclusively
in 2⫺/⫺4⫺/⫺ and are absent from Ncam knockout and 2⫺/⫺4⫺/⫺ N⫺/⫺ triple-knockout mice are caused by a
gain of polySia-free NCAM. E–G: misrouting of thalamocortical (green) and corticofugal fibers (red) in polySTbut not in 2⫺/⫺4⫺/⫺ N⫺/⫺ triple-knockout mice (NCAM⫺). Only in the polyST⫺ situation, thalamocortical axons
fail to pass through the internal capsule (ic) at embryonic day 14.5 (E and G) and fibers in the ventral thalamus
(boxed in E) are reduced and highly disorganized at postnatal day 1 (F; high magnification views of the ventral
thalamus at postnatal day 1 corresponding to the site boxed in E). Immunofluorescence with TAG1-, L1-, and
neurofilament-specific antibodies and nuclear counterstain with DAPI as indicated. Th, thalamus; ctx, cortex.
H: polySia on growth cones (arrowheads) and at the interface between fasciculated axons (arrows) in explants
of embryonic mammillary body grown on laminin. Endosialidase treatment (endo) causes defasciculation
(bottom right). Immunofluorescence with polySia- and ␤-III-tubulin-specific antibodies as indicated (H. Hildebrandt, unpublished data). I: schematic representation of the situation in the presence or absence of polySia on
NCAM. [Micrographs in A, E, and F from Schiff et al. (416) and Weinhold et al. (541), with permission from
American Society for Biochemistry and Molecular Biology.]
Other fiber tract deficits involve the internal capsule and, as
detailed below, are at least partially caused by defects of
thalamocortical and corticothalamic pathfinding (195, 416,
541). In addition, 2⫺/⫺4⫺/⫺ mice show a marked hypoplasia of the corticospinal tract at the level of the medullary
hindbrain but normal midline crossing of the remaining
fibers in the pyramidal decussation. The rostrocaudal
length of the corpus callosum is reduced with a smaller
splenium and a loss of fibers crossing the midline in the
posterior part. Moreover, the diameter of the mammillothalamic tract is reduced (195, 541). This tract projects
from the mammillary body of the hypothalamus to nuclei of
the dorsal thalamus. It closes a circuit involving the thalamus, cortex, hippocampus, and mammillary body (Papez’
circuit) and is essential for spatial working memory in the
mouse (380). Importantly, other ipsilaterally projecting and
commissural tracts such as the lateral olfactory tract, the
fasciculus retroflexus, the optic tract, and the posterior
commissure are regularly shaped and properly positioned.
Thus, contrasting with the widespread expression of polySia, the wiring defects are specific and it remains open why
only a selected set of axon tracts is affected by the loss of
polysialylation.
3. Polysialic acid as the key regulator of NCAM
The differences in the phenotype of 2⫺/⫺4⫺/⫺ and Ncam
knockout mice may be caused by loss of NCAM polysialy-
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BRAIN SIALOGLYCANS
lation, i.e., the untimely appearance of polySia-negative
NCAM. Another possibility is the lack of polySia on other
proteins, which is largely neglected but has received new
impetus from recent discoveries of alternative polySia carriers with potential neurodevelopmental impact (316). In
addition, the absence of polySia synthesis may affect the
cells’ sialic acid metabolism causing changes in other sialoglycoconjugates with unforeseen consequences. To address these uncertainties, it was of paramount importance
to determine whether the specific phenotype of the polySianegative but NCAM-positive 2⫺/⫺4⫺/⫺ mice could be rescued in triple-knockout mice with additional ablation of
NCAM (2⫺/⫺4⫺/⫺N⫺/⫺) (541). The 2⫺/⫺4⫺/⫺N⫺/⫺ tripleknockout mice recapitulate the phenotype of the Ncam
knockout with normal viability and body weight development, a slight and constant enlargement of the lateral ventricles, delaminated mossy fibers, expanded SVZ-RMS and
smaller olfactory bulbs (541). In contrast, the anterior commissure, internal capsule, corpus callosum, and mammillothalamic tract are completely normal in the mature brain of
2⫺/⫺4⫺/⫺N⫺/⫺ mice and the severe hypoplasia of the corticospinal tract is reverted to a milder deficit as seen in the
Ncam knockout mouse brain (390). This provides strong
evidence that a gain of polySia-free NCAM causes developmental deficits in 2⫺/⫺4⫺/⫺ mice.
A developmental study was performed to explore the cause
of the internal capsule deficit (416). As the major gateway
of fibers to and from the cerebral cortex, the internal capsule consists of a large collection of different fiber bundles.
These include corticofugal fibers, such as corticospinal axons, which pass into the cerebral peduncle, fibers projecting
to nuclei of the extrapyramidal system, as well as corticothalamic and thalamocortical axons. During normal embryogenesis, these two fiber systems grow towards each
other and intermingle to form the reciprocal connections
between cortex and thalamus (FIGURE 24, E–G). In the wildtype embryo, thalamocortical axons cross the primordium
of the reticular thalamic nucleus in the ventral thalamus in
a highly ordered fashion, before making a sharp turn to
enter into the internal capsule. In the polyST-negative 2⫺/
⫺/⫺
embryos, the fibers are present but highly disorga⫺4
nized, and they fail to turn into the internal capsule (FIGURE
⫺/⫺ ⫺/⫺ ⫺/⫺
4 N
mice, the additional dele24, E AND F). In 2
tion of NCAM restored the normal growth pattern and,
despite a minor developmental delay, the thalamocortical
axons turn correctly into the internal capsule giving rise to
a completely normal-sized fiber structure in the postnatal
brain. Although less prominent, the growth of corticofugal
axons is also affected, and deficiencies of corticothalamic
connections contribute to the hypoplasia of the internal
capsule in polysialylation-deficient mice (416) (TAG1 immunoreactive fibers in FIGURE 24E).
Taken together, the aberrant growth patterns of polySiadeficient, NCAM-positive axons in 2⫺/⫺4⫺/⫺ mice are as-
496
sociated with altered fasciculation and caused by the untimely appearance of polySia-negative NCAM. The impact
of polySia on axon outgrowth and fasciculation is a longstanding observation. In the motor plexus of the chicken
hindlimb, endosialidase, but also treatment with NCAM
antibodies, prevents correct nerve branching by increasing
axon-axon contacts (254, 255, 479). In contrast, in spinal
cord explant cultures and in the chicken optic tract in vivo,
endosialidase treatment causes defasciculation (3, 569).
Both of these phenomena have been explained by an altered
balance between axon-axon and axon-environment interactions, based on the model of polySia as an unspecific
steric inhibitor (398, 399) (FIGURE 23A). For the case of
altered axon tract development in 2⫺/⫺4⫺/⫺ mice, it is also
reasonable to assume that the pathfinding defects involve
altered interactions between growing axons and their environment. In this context, however, these interactions must
be specifically caused by the gain of polySia-free NCAM.
Similar to prior studies with chick spinal cord (3), mammillary body explants of embryonic mice grow out fascicles
when cultured on laminin. They show high levels of polySia
immunoreactivity on their axons and growth cones, and
defasciculate upon removal of polySia by endosialidase
treatment (FIGURE 24H). Remarkably, polySia is highly enriched between the fasciculated fibers, i.e., at axon-axon
rather than axon-matrix contacts. Thus, reminiscent of the
cellular model described above (see FIGURE 23C), loss of
polySia may result in NCAM signaling at axon-axon contacts that increases adhesion to the laminin matrix in preference to axon-axon interactions. In support of this model,
it is of note that focal adhesion kinase-dependent point
contacts regulate growth cone motility (385). It is currently
not clear whether this model, based on in vitro data, applies
to gain of NCAM function causing altered axon tract development in vivo. As indicated in the schematic representation (FIGURE 24I), NCAM signaling as well as direct,
high-affinity interactions of polySia-negative NCAM with
ECM compounds (see sect. VA and FIGURE 23E) may contribute to the defects of axon fasciculation and guidance
observed in the 2⫺/⫺4⫺/⫺ embryo.
Deficits of axon connectivity may cause secondary effects
due to reduced synaptic supply or innervation of inappropriate targets. As an example, the striking degeneration of
the reticular thalamic nucleus (Rt) in 2⫺/⫺4⫺/⫺ mice seems
to be caused by defects in thalamocortical and corticothalamic axon development (416). Rt neurons develop in normal numbers before dying by apoptosis in a narrow timewindow right after birth, which, under healthy conditions,
matches the onset of glutamatergic innervation by thalamocortical and corticothalamic fibers. Apoptotic death of Rt
neurons could also be induced by lesioning corticothalamic
fibers on whole brain slice cultures, suggesting that the degeneration of the Rt in 2⫺/⫺4⫺/⫺ mice is a consequence of
defective afferent innervation.
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4. Polysialic acid and synaptic plasticity
FIGURE 25. Gain of polySia-free NCAM determines axon tract
deficits. A: compared with the wild-type situation (wt), 2⫺/⫺4⫺/⫺
mice without functional polysialyltransferases (polyST⫺) show severe malformations of the internal capsule (ic, arrow) affecting fiber
connections between thalamus (th) and cortex (ctx). B: the degree of
the defect, based on morphometric evaluation at postnatal day 30
(P30), correlates with the relative levels of polySia-deficient NCAM
determined during the developmental phase at postnatal day 1 (P1).
Each of the data points stands for one of the nine mouse lines
investigated (see text for details). The polyST-negative 2⫺/⫺4⫺/⫺
mice show the strongest defect and the highest levels of polySianegative NCAM, set to 100%, respectively. [Adapted from
Hildebrandt et al. (195), by permission from Oxford University
Press.]
To substantiate the assumption that the malformation of
brain axon tracts are a direct consequence of the untimely
appearance of polySia-negative NCAM, the available
Ncam, St8sia2, and St8sia4 knockout mouse models were
used to breed mice with different levels of polySia-negative
NCAM during brain development (195). Nine allelic combinations with different levels of polySia, NCAM, and polySia-free NCAM at postnatal day one were selected for morphometric evaluation at the age of 4 wk. As shown in (FIGURE 25) for the example of the internal capsule, the degree
of the axon tract defects correlates precisely with the
amounts of polySia-free NCAM and not with the overall
polySia or NCAM level at postnatal day one (195). Similar
results were obtained for the other axon tracts, anterior
commissure, internal capsule, and corpus callosum, for
which morphological deficits have been identified in
polyST-negative mice. This strengthens the view that concealing NCAM is the key regulatory mechanism that makes
polySia essential for appropriate brain development. Considering a possible role of particularly ST8Sia-II in genetic
predisposition to psychiatric disorders (see sect. VI), the
stronger impact of ST8Sia-II in the developing brain together with the strictly dose-dependent relation between
polySia-deprived NCAM and neurodevelopmental defects
has the important implication that even minor quantitative
imbalances of NCAM polysialylation during brain development lead to defects in connectivity.
Several studies address the role of polySia in long-term potentiation (LTP) in the hippocampus, the primary experimental model to study synaptic plasticity as the basis of
learning and memory (43). First, a role of NCAM in LTP
was deduced from the observation that NCAM antibodies
block LTP in the CA1 region (272). Shortly afterwards, this
was corroborated by experiments with Ncam knockout
mice (320), and at the same time the involvement of polySia
was demonstrated (32, 320). Removal of polySia inhibits
LTP and the related paradigm of long-term depression in
the CA1 region (32, 320). In addition, local injections of
endosialidase into the brain reduce spatial memory, which
depends on plasticity in the hippocampus (32). Analyses of
St8sia2 and St8sia4 knockout mice provide evidence that
LTP in the CA1 region requires polySia synthesis by ST8SiaIV, whereas the loss of ST8Sia-II has no effect (10, 129).
Despite abnormal lamination and ectopic synapse formation of mossy fibers, LTP at the mossy fiber synapses in the
CA3 region is normal in the St8sia2 knockout (10). CA3
LTP is also not affected in St8sia4 knockout mice, which
lack polySia on their mossy fibers (129). As measured in
vivo, LTP at synapses of the perforant path in the dentate
gyrus is impaired in the Ncam knockout but not in either of
the polyST single knockout mice (239, 463). This implies
that this form of LTP requires the presence of NCAM,
either acutely or during development, while the role of polySia remains open; polySia is either not required, or the remaining enzyme in each of the two polyST knockout lines
produces sufficient polySia to maintain LTP in the dentate
gyrus. It therefore would be interesting to study dentate
gyrus LTP in 2⫺/⫺4⫺/⫺ mice, but their severely disturbed
development prevents a meaningful analysis. The role of
polySia in CA1 LTP may be linked to effects of polySia on
glutamate receptors of the AMPA and NMDA type, which
are independent from NCAM and possibly caused by direct
protein-binding of polySia (as in FIGURE 23H) (184, 497).
Consistent with this idea, impaired LTP in Ncam knockout
mice can be rescued by application of soluble polySia (443).
As a potential mechanism, polySia may inhibit NMDA receptors containing the GluN2B subunit changing the balance between extrasynaptic and synaptic NMDA receptor
signaling (239, 240).
5. Overexpression of ST8Sia-IV
The role of polySia on migrating oligodendrocyte precursor
cells and its downregulation during maturation (see sect.
IIIB7) suggest a role for polySia in myelination. In vitro data
indicate that polySia promotes oligodendrocyte precursor
cell migration in response to chemoattractive guidance cues
(169, 535, 583), while downregulation of polySia enhances
their differentiation into mature oligodendrocytes (108,
109). A transgenic mouse model has been established to
address the question of whether downregulation of polySia
is required for myelination in vivo (138). The expression of
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BRAIN SIALOGLYCANS
Table 3. Human diseases of sialic acid metabolism
Enzyme/Protein Deficit
Gene Mutation
Biochemical Outcome
Notes
Loss of function results in hereditary
inclusion body myopathy (HIBM),
loss of feedback inhibition results
in sialouria
Salla disease and infantile free sialic
acid storage disease (ISSD)
Associated with intellectual disability
Associated with schizophrenia
UDP-N-acetylglucosamine-2epimerase/N-acetylmannosamine
kinase
GNE
Sialic acid production is reduced (loss
of function) or increased (loss of
feedback inhibition)
Solute carrier family 17 member 5
(sialin)
␤-Galactoside ␣-2,3-sialyltransferase 3
␣-N-acetylneuraminide ␣-2,8sialyltransferase 2
Lysosomal sialidase
SLC17A5
Lysosomal accumulation of sialic acid
ST3GAL3
ST8SIA2
Undetermined in humans
Undetermined in humans
NEU1
Lysosomal accumulation of
sialoglycans
Sialidosis
Only disorders mentioned in the text are listed. For human diseases of ganglioside metabolism, see TABLE 4.
ST8Sia-IV under an oligodendrocyte-specific promoter prevents the postnatal reduction of polySia in oligodendrocytes
and causes a delay of myelin formation and defects of myelin structure. In another approach, ST8Sia-IV-overexpressing neural precursors were transplanted into the brain
of hypomyelinated shiverer mice (149). The engineered cells
displayed widespread integration and myelination in the
host, but differentiated more slowly than controls. Thus
downregulation of polysialylation during oligodendrocyte
differentiation is a prerequisite for optimal myelin formation and maintenance.
VI. SIALOGLYCANS AND DISEASES OF
THE NERVOUS SYSTEM
A. Human Diseases of Sialic Acid
Metabolism
Rare but serious human congenital disorders of sialic acid
metabolism lead to sialic acid deficiency and sialic acid excess (207, 465) (TABLE 3). The first and rate-limiting step of
sialic acid synthesis is conversion of UDP-N-acetylglucosamine to N-acetylmannosamine-6-phosphate by the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/
N-acetylmannosamine kinase, encoded by the GNE gene in
humans (207). Loss-of-function mutations in this gene are
the basis of Hereditary Inclusion Body Myopathy (HIBM),
a serious progressive muscle wasting disease. More than 60
mutations in the epimerase or kinase domains of GNE result in sialic acid deficiency and HIBM. Most mutations are
autosomal recessive and retain reduced but significant enzyme activity, whereas a few have no activity. Mutations
with no activity are found as heterozygotes only, consistent
the finding that complete disruption of the Gne gene (in
mice) results in early embryonic lethality (432). The basis
for the muscle wasting associated with reduced sialic acid
biosynthesis has not been established. Although it is reasonable to propose that hyposialylation, either in general or of
498
a specific sialoglycan, is the basis for HIBM, data have yet to
establish that relationship. Mutations in the allosteric site of
the GNE gene product result in sialouria, a very rare disorder in which overproduction of sialic acid occurs due to loss
of allosteric feedback control of the epimerase/kinase by a
downstream product, CMP-sialic acid (260). This disease
course is variable but typically lacks severe nervous system
defects, although delayed neuromuscular development has
been noted.
In contrast to HIBM and sialuria, Salla disease and the
allelic but more severe infantile free sialic acid storage disease (ISSD) are lysosomal sialic acid storage disorders that
result in debilitating nervous system pathology (548). The
milder Salla disease, named after the Finnish district in
which it arose (23), is marked by hypotonia, ataxia, and
delayed motor development that may be accompanied by
seizures. Severe CNS hypomyelination is a consistent finding in Salla disease, with thin corpus callosum observed by
magnetic resonance imaging in all patients (456). ISSD is
much more severe, with multiorgan system failure and early
death. Both are caused by mutations in a lysosomal membrane sialic acid exporter called sialin, which is encoded by
the SLC17A5 gene (524), and disease severity is inversely
correlated with residual sialic acid transport activity (548).
SLC17A5 mutation results in lysosomal sequestration of
sialic acid. It remains unresolved whether lysosomal sialic
acid storage, lack of cytoplasmic (recycled) sialic acid, or
loss of other transport activities of sialin are causative
in Salla and ISSD. A mouse model of sialin deficiency
(Slc17a5-null) phenocopied the human disorders. The mutant mice displayed poor coordination, tremor, seizures,
and early death (376). Analysis of one brain sialoglycan,
polySia-NCAM, revealed delayed developmental downregulation. It will be interesting to evaluate the effect of
lysosomal sialic acid buildup on other members of the brain
sialome, since Slc17a5-null mice and ganglioside-deficient
mice share some phenotypic characteristics (227, 376, 563).
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SCHNAAR ET AL.
Table 4. Congenital diseases of brain ganglioside metabolism
Enzyme/Protein Deficit
Gene Mutation
Major Brain Gangliosides
GM2/GD2 synthase
B4GALNT1
GM3 synthase
Hexosaminidase A
Hexosaminidase A and B
GM2 activator protein
␤-Galactosidase
Niemann-Pick C1 protein
Niemann-Pick C2 protein
ST3GAL5
HEXA
HEXB
GM2A
GLB1
NPC1
NPC2
Loss of GM1, GD1a, GD1b, GT1b with partial
replacement by GM3, GD3*
Undetermined
GM2 storage
GM2 storage
GM2 storage
GM1 storage
GM2, GM3 storage
GM2, GM3 storage
Notes
10 independent families
4 independent families
Tay Sachs disease
Sandhoff disease
GM1 gangliosidosis
UniProtKB/Swiss-Prot: O15118.2
UniProtKB/Swiss-Prot: P61916.1
*Postmortem brain ganglioside analysis from a single case study lacking genetic confirmation (289).
B. Human Diseases of Ganglioside
Metabolism
Human diseases of ganglioside metabolism (TABLE 4) are
rare, with congenital errors in biosynthesis reported in only
a few families and errors in catabolism occurring in 1 of
⬃100,000 live births. Both anabolic and catabolic disorders
are dominated by severe neurological deficits (52).
In one case study, an infant presented with severe mental
impairment from birth and failure to thrive, and died at 3
mo of age (289). Postmortem analysis revealed a complete
lack of complex brain gangliosides, and instead expression
of GM3 and GD3. Enzyme assays indicated sharply diminished GM2/GD2 synthase activity (UDP-GalNAc:GM3 Nacetylgalactosaminyl-transferase) in brain homogenates
(141). Although the genetic basis was not determined, the
biosynthetic deficiency was proposed to be lack of GM2/
GD2 synthase (the B4GALNT1 gene product), and patient
history indicated a familial disorder. Histological and ultrastructural studies of the brain noted major abnormalities
in myelin (477), supporting a role for gangliosides in
axon-myelin interactions in humans. The patient, however,
had much more severe pathology than B4galnt1-null mice,
with mental impairment evident from birth including hypotonia, lack of responsiveness, generalized seizures, and
death in infancy (277). In contrast to that very severe neurological phenotype, more recent genetic studies linked several independent mutations in human B4GALNT1 to hereditary spastic paraplegia in 10 families (54). Analysis of
⬃30 affected individuals revealed early-onset progressive
spasticity of the lower extremities resulting from axonal
degeneration. Unlike the earlier infant case study, these patients were relatively long-lived and typically ambulatory,
although some became confined to a wheelchair later in life.
Mild to moderate cognitive impairment and developmental
delay was noted, as well as white matter hyperintensities by
MRI (dysmyelination) and low testosterone levels in men.
These deficits are phenocopied by B4galnt1-null mice, in-
cluding the male testosterone deficit. Understanding the differences between different forms of B4GALNT1 deficiency
in humans, and the relationship between brain ganglioside
expression and neural deficits in these cases, awaits further
biochemical and histological analyses.
Recently, deficiencies of GM3 synthase (ST3GAL5 gene
mutations) were also discovered by genome-wide linkage
analyses (46, 145, 449, 536). Affected individuals in several
families presented with infantile seizures that were resistant
to treatment accompanied by hypotonia and neurological
deterioration. These deficits are not phenocopied in
St3gal5-null mice, but are similar to those in “GM3 only”
(St8Sia1/B4galnt1-doulbe null) and ganglioside-knockout
(St3gal5/B4galnt1-double null) mice. St3gal5-single null
mice express alternate complex brain gangliosides (GM1b
and GD1␣) that may compensate for the loss of other complex gangliosides. It has not been determined whether the
human loss of GM3 synthase results in comparable biosynthetic compensation in the brains of ST3GAL5 patients,
although studies on patient fibroblasts reveal lower rather
than higher GM1b expression (46). Regardless, seizure susceptibility appears to be a common feature of human diseases and mouse genetic models of altered brain ganglioside
expression.
Disorders of ganglioside catabolism result in lysosomal
storage diseases marked by progressive neurodegeneration
(52, 556). Normally, exoglycosidases sequentially remove
the outer-most (nonreducing terminal) ganglioside saccharides. Genetic defects in exoglycosidases result in lysosomal
buildup of the precursors, leading to severe neuronal pathology. Among these disorders, GM2 gangliosidoses have
been studied most extensively. GM2 catabolism in humans
requires removal of the terminal GalNAc by hexosaminidase A, an enzyme comprised of two subunits (␣ and ␤)
coded by separate genes, HEXA and HEXB, respectively.
Defects in either of the two gene products result in GM2
gangliosidosis, the extensive lysosomal buildup of GM2 in
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BRAIN SIALOGLYCANS
neurons. HEXA mutations are designated as Tay-Sachs disease and HEXB mutations as Sandhoff disease. The natural
history of the two diseases varies with the amount of residual enzyme activity, with the course of disease indistinguishable between Tay-Sachs and Sandhoff diseases (42). In
addition to hexosaminidase, GM2 catabolism requires an
activator protein that facilitates access of the enzyme to the
lipid substrate. Rare mutations in the human activator protein gene (GM2A) result in GM2 gangliosidosis without
loss of hexosaminidase enzyme activity (measured using
synthetic substrate), and patients display a disease course
similar to Tay Sachs and Sandhoff diseases (452). Affected
infants develop and progress normally for several weeks,
then are subject to profound progressive neurodegeneration
marked by increased startle response, hypotonia, and seizures. Mean survival is 4 years, with few individuals living
beyond 8 years. Although the mechanisms of neurodegeneration are not fully understood, a role for microglia, macrophages, and neuroinflammation has been proposed. Genetic deletion of the immune mediators MIP-1␣ (554) or
TNF-␣ (1) in Sandhoff disease mice (Hexb-null) reduced
neurodegeneration and increased lifespan.
Human gangliosidosis can also result from defects in the
␤-galactosidase responsible for hydrolysis of the terminal
galactose of GM1, coded by the GLB1 gene in humans (63,
68). Neurological deficits are invariably present in GM1
gangliosidosis patients. Different GLB1 mutations result in
infantile (type I), juvenile (type II), and adult (type III) forms
of the disease that are associated with severe psychomotor
regression by 6 mo of age (type I), progressive locomotor
deficits and seizures by 3 years of age (type II), and cerebellar dysfunction and dystonia later in childhood or into
adulthood (type III). Mouse models of GM1 gangliosidosis
have been useful for both pathophysiological and preclinical therapeutic studies (287, 288).
C. Anti-ganglioside Immunity
Guillain-Barré syndromes (GBS) are the leading cause of
acute neuromuscular paralysis, with ⬎75,000 cases per
year worldwide (206, 499, 577). Although GBS is multifaceted, a large subset of cases involve anti-ganglioside antibodies (545).
GBS is an acquired disease characterized by rapidly progressive bilateral and relatively symmetrical limb weakness
(499). Disease severity typically worsens for days or weeks
until most patients cannot walk and may require artificial
ventilation as respiratory muscles weaken. Although many
patients recover, ⬃5% of GBS patients die of the disease
and 20% remain unable to walk 6 mo after disease onset.
Many patients report ongoing symptoms including functionally significant fatigue lasting years after acute disease
resolution. The efficacy of anti-immune interventions
(plasma exchange and IVIG) (371, 483, 498), as well as
500
histopathology (179, 180, 544), support the conclusion
that peripheral nerves are subject to acute complementmediated antibody attack.
Based on the affected nerves and conductance pathophysiology, GBS is classified into three major subtypes (294, 499,
577): acute inflammatory demyelinating polyneuropathy
(AIDP), acute motor (or motor/sensory) axonal neuropathy
(AMAN or AMSAN), and Miller-Fisher Syndrome (MFS),
the last of which displays cranial nerve involvement resulting in opthalmoplegia and ataxia. In AIDP, the immune
attack is on Schwann cells (180) and the antigens are not
well established. In AMAN and MFS, the immune attack is
on axons (179) and/or nerve terminals (183), and the antigens are often gangliosides. The study of anti-ganglioside
antibodies in GBS was prompted by the discovery that some
patients with an otherwise unrelated disorder, IgM paraproteinemia associated with peripheral neuropathy, expressed anti-ganglioside IgM antibodies (378). Subsequent
studies discovered that a subset of North American GBS
patients (⬃20%) also expressed anti-ganglioside antibodies
(210). These findings set the stage for the discovery of the
pathophysiology of AMAN and MFS, both of which are
caused by immune crossover between ganglioside-like
epitopes on pathogens and endogenous gangliosides on axons and nerve terminals.
The onset of GBS often follows recovery from an infectious
disease, typically an upper respiratory tract infection or
diarrhea (206). Epidemiology, histology, and molecular
studies support the conclusion that an infection-induced
humoral immune response results in production of antibodies that cross react with endogenous epitopes on Schwann
cells or axons, recruit the immune membrane attack complex and macrophages, disrupt peripheral myelin and axons, and cause paralysis (499, 577) (TABLE 5). This type of
immune crossover attack, termed “molecular mimicry,” is
well established in AMAN and MFS (FIGURE 26). The
AMAN form of GBS was first recognized as a summertime
disease of primarily rural children and young adults in
northern China with acute axonal loss rather than the demyelinating electrophysiological hallmarks of the GBS
most common in Europe and North America (294, 295).
Although AMAN patients were afebrile at the time of admission, many reported recent illness, and over 90% had
robust serum antibodies against Campylobacter jejuni, a
common cause of human gastroenteritis. Histological stud-
Table 5. Anti-ganglioside antibodies in GBS subtypes
Subtype
Anti-ganglioside Antibodies (Minor)
AIDP
AMAN and AMSAN
Unknown
GM1, GD1a (GM1b, GalNAc-GD1a,
GT1a)
GQ1b, GT1a, GD3
MFS
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SCHNAAR ET AL.
FIGURE 26. Immunopathogenesis of the AMAN form of Guillain-Barré syndrome. Gangliosides GM1 and
GD1a are strongly expressed at nodes of Ranvier, where the voltage-gated sodium (Nav) channels are
localized. Anti-GM1 or anti-GD1a antibodies bind to the nodal axolemma, leading to formation of the complement membrane attack complex (MAC). This results in the disappearance of Nav clusters and the detachment
of paranodal myelin, which can lead to nerve-conduction failure and muscle weakness. Axonal degeneration
may follow at a later stage. Macrophages subsequently invade from the nodes into the periaxonal space,
scavenging the injured axons. [Adapted from Yuki and Hartung (577), with permission from Massachusetts
Medical Society.]
ies indicated immune attack, with complement, immune
membrane attack complexes, and macrophages at the axonal surface (176). Remarkably, chemical studies of C. jejuni strains associated with GBS revealed that bacterial surface lipooligosaccharides mimicked the structures of
human gangliosides associated with anti-ganglioside antibodies in GBS, notably GM1 and GD1a (19, 170, 199, 575,
576, 579) (FIGURE 27). Campylobacter sialyltransferase
polymorphisms control which ganglioside mimics are expressed (170, 575). Further evidence that GBS pathology
involves ganglioside molecular mimicry comes from the observation that rabbits immunized with purified C. jejuni
lipooligosaccharide (from a GBS-related strain) produced
circulating anti-ganglioside antibodies and experience flaccid paralysis along with the histochemical hallmarks of GBS
(578). With appropriate immune stimulation, an induced
immune response to purified gangliosides also resulted in
flaccid paralysis (580). These data establish ganglioside molecular mimicry in the pathophysiology of post-C. jejuni
AMAN, with anti-GM1 and anti-GD1a immunity of special note.
A similar association was established for MFS, but specifically with the complex ganglioside GQ1b (350) (FIGURE
27). Studies of patients suffering from typical MFS (opthal-
moplegia and ataxia), atypical MFS (opthalmoplegia without ataxia), and GBS with opthalmoplegia revealed that 28
of 30 had serum antibodies against GQ1b, whereas none of
the GBS patients without opthalmoplegia (or normal controls) had anti-GQ1b antibodies (79, 80). Most of these
patients reported antecedent infections, either upper respiratory or diarrhea. Whereas lipooligosaccharides from C.
jejuni strains isolated from GBS patients (without opthalmoplegia) expressed mimics of GM1 and GD1a (see above),
strains from MFS and related patients expressed terminal
disialylated structures akin to GQ1b that invoked antiGQ1b antibodies (170). GQ1b is expressed on the oculomotor, trochlear, and abducens nerves that control eye
movement, but rarely on other peripheral nerves, in part
explaining the characteristic neuropathy of MFS. MFS-associated C. jejuni strains express sialyltransferase polymorphisms that promote disialyl extensions of ganglioside-like
lipooligosaccharides (170, 575). Patients with opthalmoplegia associated with anti-GQ1b antibodies have been
grouped into a related spectrum of disorders including
MFS, Bickerstaff’s brain stem encephalitis, and acute ophthalmoparesis (350).
Ganglioside molecular mimicry has been established as a
pathogen-induced, complement-mediated humoral im-
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BRAIN SIALOGLYCANS
D. ST3GAL3 and Cognition
Genome-wide linkage analysis implicates sialylation, particularly via the enzyme ST3Gal-III (the product of the
ST3GAL3 gene), as a factor in higher cognitive functions
(131, 204). Nonsyndromic autosomal recessive intellectual
disability is a familial disorder characterized by a low intelligence quotient (IQ) associated with difficulty in learning
basic language and motor skills during childhood and significantly diminished intellectual capacity as an adult. Two
Iranian pedigrees including 12 affected individuals whose
average IQ was ⬍40 (⬍0.1 percentile) from four sets of
consanguineous parents were subjected to genetic linkage
analyses (204). This led to identification of two mutations
responsible (one in each pedigree), both in the ST3GAL3
gene. One mutation was in the catalytic region and resulted
in loss of sialyltransferase activity, whereas another was in
the transmembrane domain, did not affect enzyme activity,
but appeared to result in mistargeting of the enzyme to the
endoplasmic reticulum where the donors and acceptors are
absent.
West Syndrome is a severe infantile seizure disorder associated with developmental regression and intellectual disability that can occur as an autosomal recessive familial disorder. Members of a Palestinian pedigree including four affected individuals (three from consanguineous parents)
were subjected to genetic linkage analysis (131). A single
mutation in the “small sialylmotif” of the ST3GAL3 gene,
a domain required for enzyme activity, was found to be
responsible for the disorder.
FIGURE 27. Campylobacter jejuni lipooligosaccharides and molecular mimicry in GBS. Top: carbohydrate mimicry between a ganglioside (GM1) and a Campylobacter jejuni lipooligosaccharide (LOS).
The terminal tetrasaccharides are identical (dashed lines). Bottom:
Campylobacter jejuni gene polymorphism as a determinant of clinical
neuropathies after infection. C. jejuni that carries cst-II (Thr51) can
express GM1-like and GD1a-like LOS on its cell surface, which upon
infection may induce anti-GM1, anti-GD1a, or anti-GM1/GD1a complex IgG production. Since these products are expressed on motor
nerves of the four limbs, binding induces acute motor axonal neuropathy. In contrast, C. jejuni that carries cst-II (Asn51) expresses
GT1a-like or GD1c-like LOS on its cell surface, which upon infection
may induce anti-GQ1b IgG production. Anti-GQ1b IgG antibody binds
to GQ1b on oculomotor nerves and primary sensory neurons, inducing Fisher syndrome and related conditions. [Adapted from Yuki
(575), with permission from John Wiley and Sons, Inc., and (576),
with permission from Proceedings of the Japan Academy, Ser. B.]
mune response leading to peripheral nerve attack and resulting in motor deficits. Whereas many cases can be traced
to specific pathogen structures (notably lipooligosaccharides on C. jejuni), the causative agent in other cases remains unknown. Enhanced knowledge of these pathogens
and mechanisms of nerve damage may provide ways to
lessen the worldwide burden of GBS.
502
The above studies do not identify which classes of brain
sialoglycans are altered in human ST3GAL3 deficiency syndromes. The enzyme ST3Gal-III has broad acceptor specificity, transferring sialic acids to terminal Gal residues in
␤1–3 or ␤1– 4 linkage to GlcNAc or in ␤1–3 linkage to
GalNAc characteristic of O- and N-linked glycoproteins
and gangliosides, respectively (45). Although evoked in the
sialylation of Lewis epitopes, the specificity of ST3Gal-III is
much broader. In the mouse brain, ST3Gal-III synthesizes
both sialoglycoproteins and gangliosides (467). Further
studies will be required to understand the brain sialoglycans
affected in ST3GAL3 deficiency disorders and why these
disorders present with and without severe infantile seizures.
Enhanced genetic linkage analyses bring a new focus to
research on brain sialoglycans in higher cognitive function
in humans.
E. Polysialic Acid and Psychiatric Disorders
Dysregulation of polySia and NCAM have been frequently
observed in association with psychiatric disorders, mainly
schizophrenia, bipolar disorder, and autism (57, 519). In
schizophrenia, the described changes in NCAM range from
altered concentrations of NCAM fragments in either serum
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SCHNAAR ET AL.
or cerebrospinal fluid (275, 372, 502, 521, 522) to increased levels of a roughly 110-kDa band in Western blot
analyses of hippocampus and prefrontal cortex from postmortem brains of patients (520). A specific reduction of
polySia-positive cells was found in the hilus region of the
hippocampus (26) and, most recently, lower levels of polySia immunoreactivity, probably associated with interneuron changes, were detected in layers IV and V of the dorsolateral prefrontal cortex (PFC) of schizophrenics, while levels were unchanged in samples from patients with major
depression and bipolar disorder (167). In contrast, polySia
immunoreactivity was not altered in the amygdala of
schizophrenics, but was reduced in depressed patients and
increased in bipolar disorder (506).
null mice displayed reduced prepulse inhibition of the startle response as an operational measure of sensorimotor gating and significant working memory deficits, indicating cognitive impairments. Both traits resemble characteristic
features of schizophrenic patients and may be linked to the
neurodevelopmental disturbance of thalamocortical connectivity leading to reduced glutamatergic input of thalamic
afferents to the frontal and prefrontal cortex of St8sia2knockout mice (249).
In addition to changes of NCAM protein and polySia, important genetic associations have been discovered. NCAM1
and both polysialyltransferase genes map to chromosomal
regions that harbor susceptibility loci for schizophrenia
(11q23.1, 15q26, and 5q21 for NCAM1, ST8SIA2, and
ST8SIA4, respectively) (261, 269, 290). Analyses of genetic
variation in NCAM1 indicate associations of five contiguous single nucleotide polymorphisms (SNPs) with schizophrenia and different risk haplotypes in the NCAM1 gene
are linked to schizophrenia or bipolar disorder (20, 468).
Two independent studies performed with schizophrenic patients from Japan or China found significant associations
of different polymorphisms in the promoter region of
ST8SIA2, but not ST8SIA4, and a recent study with Australian patients describes a risk haplotype in the ST8SIA2
gene (18, 480). ST8SIA2 variants have also been associated
with autism spectrum and bipolar disorder (16, 291). In
vitro promoter assays were performed with a risk haplotype
of ST8SIA2 in the Japanese sample of schizophrenic patients, and the results point towards enhanced transcriptional activity (18). In contrast, the risk haplotype identified
in the Australian cohort is associated with reduced
ST8SIA2 mRNA expression (291). Moreover, a point mutation near the sialylmotif L, detected heterozygously in one
of the Japanese patients (18), leads to reduced ST8SIA2
activity and production of shorter polySia chains (213).
Together, the available data provide strong evidence that
variation in the ST8SIA2 gene is linked to increased risk of
mental illness.
Structural changes observed in epilepsy and neurodegenerative disease may be part of a pathological cascade or indicate attempts of repair. With regard to the role of polySia in
developmental and adult plasticity, it is assumed that the
increased expression of polySia as observed in the hippocampus and entorhinal cortex of patients suffering from
temporal lobe epilepsy is part of such disease-related plasticity (301). The same holds true for the higher polySia
levels detected in the molecular layer of the dentate gyrus in
patients with Alzheimer’s disease (300, 302). There is experimental evidence for polySia as a positive modulator of
hippocampal neurogenesis in the amygdala kindling model
of chronic temporal lobe epilepsy in the rat, which leads to
ectopic neurons in the hilus (364). Endosialidase treatment
has no beneficial effect on epilepsy development, but counteracts the increase of newborn neurons during the kindling
process. These findings suggest that enhanced neurogenesis
reflects an attempt of repair and implicates that stimulation
of polySia synthesis may reinforce endogenous mechanisms
of brain repair (363, 364).
Schizophrenia has a high heritability. Additionally, disruptions in neural development resulting in altered brain connectivity is increasingly recognized as a risk factor (33,
262). In light of the strong neurodevelopmental phenotype
of polyST-deficient mice and the clear dose-response relationship between loss of polySia and defects of brain connectivity (see sect. VB3), St8sia2-knockout mice are particularly well-suited to explore potential links between polysialylation deficits and pathophysiological features of
schizophrenia. Indeed, a recent study demonstrates a
schizophrenia-like phenotype of these mice (249). St8sia2-
F. Polysialic Acid in Epilepsy,
Neurodegeneration, and Nervous
System Repair
Beyond neurogenesis, an increase of polySia may promote
axon regeneration. Following lesions, reexpression of polySia is found on sprouting and regenerating CNS axons (7,
21, 319, 584). The presence of polySia on these axons may
play a permissive role. However, even more important as a
permissive factor may be the presence of polySia in reactive
gliosis associated with a CNS lesion (21, 69). Support of
regeneration by a polySia-positive environment is firmly
established by the results of several studies showing that
engineered overexpression of polySTs in astrocytes or
grafted Schwann cells promotes axonal regeneration in the
brain and spinal cord (133, 359, 585, 587, 588). Complementary, application of polySia mimetic peptides improves
functional recovery after spinal cord injury (285, 296).
In contrast to these favorable effects of polySia overproduction, a reduction of polySia may be a strategy to promote
myelin repair. The presence of polySia on axons hampers
myelin formation in vitro (74), and chronically demyelinated axons in multiple sclerosis lesions reexpress polySia
(75). In contrast, polySia is absent from so-called shadow
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BRAIN SIALOGLYCANS
plaques with partial repair, suggesting that the presence of
polySia could act as an inhibitor of remyelination. In a first
attempt to address this experimentally, remyelination after
cuprizone-induced demyelination (451) was studied in
St8sia4 knockout mice. A slight but significant acceleration
in the reexpression of several myelin markers indicates improved remyelination in the absence of ST8Sia-IV (247).
The mechanisms responsible for this improvement are yet
to be explored. Nevertheless, the data indicate that targeting polysialyltransferases has the potential to support remyelination.
In this review, we have considered the significant contributions of two classes of brain sialoglycans, gangliosides and
polysialic acid, to brain development, function, health, and
disease. The rapid development of new methods and new
knowledge in the field of glycobiology promises to bring
enhanced understanding of how glycans help to manage the
massive flow of information that underpins brain morphogenesis, maintenance, and functions, with the potential of
meaningfully improving our understanding of human physiology and pathology.
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ACKNOWLEDGMENTS
We are grateful to Martina Mühlenhoff, Beate Schwinzer,
Tim Kröcher, and Johannes Cramer for help with figure
preparation and critical reading of the manuscript.
Address for reprint requests and other correspondence:
R. L. Schnaar, Dept. of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725
N. Wolfe St., Baltimore, MD 21205 (e-mail: schnaar@jhu.
edu).
GRANTS
We acknowledge the support of National Institute of Neurological Disorders and Stroke Grants NS037096 and
NS057338 (to R. L. Schnaar). Work at Hannover Medical
School was supported by funds from the Deutsche Forschungsgemeinschaft, Deutsche Krebshilfe, the Sixth Framework Program of the European Commission (FP6 PROMEMORIA), and the German Ministry for Research and Education in the frame of ERA-NET NEURON (NeuConnect).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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