Glycosphingolipids in congenital disorders of glycosylation (CDG).

Congenital disorders of glycosylation (CDG) are a large family of rare disorders affecting the different glycosylation pathways. Defective glycosylation can affect any organ, with varying symptoms among the different CDG. Even between individuals with the same CDG there is quite variable severity. Associating specific symptoms to deficiencies of certain glycoproteins or glycolipids is thus a challenging task. In this review, we focus on the glycosphingolipid (GSL) synthesis pathway, which is still rather unexplored in the context of CDG, and outline the functions of the main GSLs, including gangliosides, and their role in the central nervous system. We provide an overview of GSL studies that have been performed in CDG and show that abnormal GSL levels are not only observed in CDG directly affecting GSL synthesis, but also in better known CDG, such as PMM2-CDG. We highlight the importance of studying GSLs in CDG in order to better understand the pathophysiology of these disorders.

Ganglioside deficiency in hypothalamic POMC neurons promotes body weight gain.

BACKGROUND: Glucosylceramide synthase (GCS; gene: UDP-glucose:ceramide glucosyltransferase (Ugcg))-derived gangliosides comprise a specific class of lipids in the plasma membrane that modulate the activity of transmembrane receptors. GCS deletion in hypothalamic arcuate nucleus (Arc) neurons leads to prominent obesity. However, it has not yet been studied how ganglioside depletion affects individual Arc neuronal subpopulations. The current study investigates the effects of GCS deletion specifically in anorexigenic pro-opiomelanocortin (POMC) neurons. Additionally, we investigate insulin receptor (IR) signaling and phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) binding to ATP-dependent K+ (KATP) channels of GCS-deficient POMC neurons. MATERIALS AND METHODS: We generated Ugcgf/f-Pomc-Cre mice with ganglioside deficiency in POMC neurons. Moreover, the CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas9 technology was used to inhibit GCS-dependent ganglioside biosynthesis in cultured mouse POMC neurons, yielding UgcgΔ-mHypoA-POMC cells that were used to study mechanistic aspects in further detail. Proximity ligation assays (PLAs) visualized interactions between gangliosides, IR, and KATP channel subunit sulfonylurea receptor-1 (SUR-1), as well as intracellular IR substrate 2 (IRS-2) phosphorylation and PIP3. RESULTS: Chow-fed Ugcgf/f-Pomc-Cre mice showed a moderate but significant increase in body weight gain and they failed to display an increase of anorexigenic neuropeptide expression during the fasting-to-re-feeding transition. IR, IRS-2, p85, and overall insulin-evoked IR and IRS-2 phosphorylation were elevated in ganglioside-depleted UgcgΔ-mHypoA-POMC neurons. A PLA demonstrated that more insulin-evoked complex formation occurred between PIP3 and SUR-1 in ganglioside-deficient POMC neurons in vitro and in vivo. CONCLUSION: Our work suggests that GCS deletion in POMC neurons promotes body weight gain. Gangliosides are required for an appropriate adaptation of anorexigenic neuropeptide expression in the Arc during the fasting-to-re-feeding transition. Moreover, gangliosides might modulate KATP channel activity by restraining PIP3 binding to the KATP channel subunit SUR-1. Increased PIP3/SUR-1 interactions in ganglioside-deficient neurons could in turn potentially lead to electrical silencing. This work highlights that gangliosides in POMC neurons of the hypothalamic Arc are important regulators of body weight.

Intracerebroventricular Infusion of Gangliosides Augments the Adult Neural Stem Cell Pool in Mouse Brain.

We previously reported that ganglioside GD3 is the predominant species in neural stem cells (NSCs) and reduced postnatal NSC pools are observed in both the subventricular zone and dentate gyrus (DG) of GD3-synthase knockout (GD3S-KO) mouse brains. Specifically, deficiency of GD3 in GD3S-KO animals revealed a dramatic reduction in cellularity in the DG of the hippocampus of the developing mouse brain, resulting in severe behavioral deficits in these animals. To further evaluate the functional role of GD3 in postnatal brain, we performed rescue experiments by intracerebroventricular infusion of ganglioside GD3 in adult GD3S-KO animals and found that it could restore the NSC pools and enhance the NSCs for self-renewal. Furthermore, 5xFAD mouse model was utilized, and GD3 restored NSC numbers and GM1 promoted neuronal differentiation. Our results thus demonstrate that exogenously administered gangliosides are capable to restore the function of postnatal NSCs. Since ganglioside expression profiles are associated not only with normal brain development but also with pathogenic mechanisms of diseases, such as Alzheimer's disease, we anticipate that the administration of exogenous gangliosides, such as GD3 and GM1, may represent a novel and effective strategy for promoting adult neurogenesis in damaged brain for disease treatment.

Fresh evidence for major brain gangliosides as a target for the treatment of Alzheimer's disease.

Although it was suggested that gangliosides play an important role in the binding of amyloid fragments to neuronal cells, the exact role of gangliosides in Alzheimer's disease (AD) pathology remains unclear. To understand the role of gangliosides in AD pathology in vivo, we crossed st3gal5-deficient (ST3-/-) mice that lack major brain gangliosides GM1, GD1a, GD3, GT1b, and GQ1b with 5XFAD transgenic mice that overexpress 3 mutant human amyloid proteins AP695 and 2 presenilin PS1 genes. We found that ST3-/- 5XFAD mice have a significantly reduced burden of amyloid depositions, low level of neuroinflammation, and did not exhibit neuronal loss or synaptic dysfunction. ST3-/- 5XFAD mice performed significantly better in a cognitive test than wild-type (WT) 5XFAD mice, which was comparable with WT nontransgenic mice. Treatment of WT 5XFAD mice with the sialic acid-specific Limax flavus agglutinin resulted in substantial improvement of AD pathology to a level of ST3-/- 5XFAD mice. Thus, our findings highlight an important role for gangliosides as a target for the treatment of AD.

Parkinsonian GM2 synthase knockout mice lacking mature gangliosides develop urinary dysfunction and neurogenic bladder.

Parkinson's disease is a neurodegenerative disorder that reduces a patients' quality of life by the relentless progression of motor and non-motor symptoms. Among the non-motor symptoms is a condition called neurogenic bladder that is associated with detrusor muscle underactivity or overactivity occurring from neurologic damage. In Parkinson's disease, Lewy-body-like protein aggregation inside neurons typically contributes to pathology. This is associated with dopaminergic neuron loss in substantia nigra pars compacta (SNc) and in ventral tegmental area (VTA), both of which play a role in micturition. GM1 gangliosides are mature glycosphingolipids that enhance normal myelination and are reduced in Parkinson's brain. To explore the role of mature gangliosides in vivo, we obtained GM2 Synthase knockout (KO) mice, which develop parkinsonian pathology including a loss of SNc dopaminergic neurons, which we reconfirmed. However, bladder function and innervation have never been assessed in this model. We compared GM2 Synthase KO and wild type (WT) littermates' urination patterns from 9 to 19 months of age by counting small and large void spots produced during 1 h tests. Because male and female mice had different patterns, we evaluated data by sex and genotype. Small void spots were significantly increased in 12-16 month GM2 Synthase KO females, consistent with overactive bladder. Similarly, at 9-12 month GM2 KO males tended to have more small void spots than WT males. As GM2 Synthase KO mice aged, both females and males had fewer small and large void spots, consistent with detrusor muscle underactivity. Ultrasounds confirmed bladder enlargement in GM2 Synthase KO mice compared to WT mice. Tyrosine hydroxylase (TH) immunohistochemistry revealed significant dopaminergic loss in GM2 Synthase KO VTA and SNc, and a trend toward TH loss in the GM2 KO periaqueductal gray (PAG) micturition centers. Levels of the nerve growth factor precursor, proNGF, were significantly increased in GM2 Synthase KO bladders and transmission electron micrographs showed atypical myelination of pelvic ganglion innervation in GM2 Synthase KO bladders. Cumulatively, our findings provide the first evidence that mature ganglioside loss affects micturition center TH neurons as well as proNGF dysregulation and abnormal innervation of the bladder. Thus, identifying therapies that will counteract these effects should be beneficial for those suffering from Parkinson's disease and related disorders.

Coordinated existence of multiple gangliosides is required for cartilage metabolism.

OBJECTIVE: Gangliosides, ubiquitously existing membrane components that modulate transmembrane signaling and mediate cell-to-cell and cell-to-matrix interactions, are key molecules of inflammatory and neurological disorders. However, the functions of gangliosides in the cartilage degradation process remain unclear. We investigated the functional role of gangliosides in cartilage metabolism related to osteoarthritis (OA) pathogenesis. DESIGN: We generated knockout (KO) mice by targeting the ß1, 4-N-acetylgalactosaminyltransferase (GalNAcT) gene, which encodes an enzyme of major gangliosides synthesis, and the GD3 synthase (GD3S) gene, which encodes an enzyme of partial gangliosides synthesis. In vivo OA and in vitro cartilage degradation models were used to evaluate the effect of gangliosides on the cartilage degradation process. RESULTS: The GalNAcT and GD3S KO mice developed and grew normally; nevertheless, OA changes in these mice were enhanced with aging. The GalNAcT KO mice showed significantly enhanced OA progression compared to GD3S mice in vivo. Both GalNAcT and GD3S KO mice showed severe IL-1α-induced cartilage degradation ex vivo. Phosphorylation of MAPKs was enhanced in both GalNAcT and GD3S KOs after IL-1α stimulation. Gangliosides modulated by GalNAcT or GD3S rescued an increase of MMP-13 induced by IL-1α in mice lacking GalNAcT or GD3S after exogenous replenishment in vitro. CONCLUSION: These data show that the deletion of gangliosides in mice enhanced OA development. Moreover, the gangliosides modulated by GalNAcT are important for cartilage metabolism, suggesting that GalNAcT is a potential target molecule for the development of novel OA treatments.

Ganglioside deficiency causes inflammation and neurodegeneration via the activation of complement system in the spinal cord.

BACKGROUND: Gangliosides, sialic acid-containing glycosphingolipids, are highly expressed in nervous systems of vertebrates and have been considered to be involved in the development, differentiation, and function of nervous tissues. Recent studies with gene-engineered animals have revealed that they play roles in the maintenance and repair of nervous tissues. In particular, knockout (KO) mice of various ganglioside synthase genes have exhibited progressive neurodegeneration with aging. However, neurological disorders and pathological changes in the spinal cord of these KO mice have not been reported to date. Therefore, we examined neurodegeneration in double knockout (DKO) mice of ganglioside GM2/GD2 synthase (B4GANLT1) and GD3 synthase (ST8SIA1) genes to clarify roles of gangliosides in the spinal cord. METHODS: Motor neuron function was examined by gait analysis, and sensory function was analyzed by von Frey test. Pathological changes were analyzed by staining tissue sections with Klüver-Barrera staining and by immunohistochemistry with F4/80 and glial fibrillary acidic protein (GFAP). Gene expression profiles were examined by using DNA micro-array of RNAs from the spinal cord of mice. Triple knockout mice were generated by mating DKO and complement component 3 (C3)-KO mice. Gene expression of the complement system and cytokines was examined by reverse transcription-polymerase chain reaction (RT-PCR) as a function of age. RESULTS: DKO mice showed progressive deterioration with aging. Correspondingly, they exhibited shrunk spinal cord, reduced thickness of spinal lamina II and III, and reduced neuronal numbers in spinal lamina IX, spinal lamina II, and spinal lamina I. Complement-related genes were upregulated in DKO spinal cord. Moreover, complement activation and inflammatory reactions were detected by GFAP-active astrocyte, microglial accumulation, and increased inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) and interleukin-1-beta (IL-1ß). Triple knockout mice showed restoration of reduced neuron numbers in the spinal cord of DKO mice, getting close to levels of wild-type mice. CONCLUSIONS: Disruption in the architecture of lipid rafts in the spinal cord was not so prominent, suggesting that mechanisms distinct from those reported might be involved in the complement activation in the spinal cord of DKO mice. Gene profiling revealed that inflammation and neurodegeneration in the spinal cord of DKO mice are, at least partly, dependent on complement activation.

Neuronal expression of GalNAc transferase is sufficient to prevent the age-related neurodegenerative phenotype of complex ganglioside-deficient mice.

Gangliosides are widely expressed sialylated glycosphingolipids with multifunctional properties in different cell types and organs. In the nervous system, they are highly enriched in both glial and neuronal membranes. Mice lacking complex gangliosides attributable to targeted ablation of the B4galnt1 gene that encodes ß-1,4-N-acetylegalactosaminyltransferase 1 (GalNAc-transferase; GalNAcT(-/-)) develop normally before exhibiting an age-dependent neurodegenerative phenotype characterized by marked behavioral abnormalities, central and peripheral axonal degeneration, reduced myelin volume, and loss of axo-glial junction integrity. The cell biological substrates underlying this neurodegeneration and the relative contribution of either glial or neuronal gangliosides to the process are unknown. To address this, we generated neuron-specific and glial-specific GalNAcT rescue mice crossed on the global GalNAcT(-/-) background [GalNAcT(-/-)-Tg(neuronal) and GalNAcT(-/-)-Tg(glial)] and analyzed their behavioral, morphological, and electrophysiological phenotype. Complex gangliosides, as assessed by thin-layer chromatography, mass spectrometry, GalNAcT enzyme activity, and anti-ganglioside antibody (AgAb) immunohistology, were restored in both neuronal and glial GalNAcT rescue mice. Behaviorally, GalNAcT(-/-)-Tg(neuronal) retained a normal "wild-type" (WT) phenotype throughout life, whereas GalNAcT(-/-)-Tg(glial) resembled GalNAcT(-/-) mice, exhibiting progressive tremor, weakness, and ataxia with aging. Quantitative electron microscopy demonstrated that GalNAcT(-/-) and GalNAcT(-/-)-Tg(glial) nerves had significantly increased rates of axon degeneration and reduced myelin volume, whereas GalNAcT(-/-)-Tg(neuronal) and WT appeared normal. The increased invasion of the paranode with juxtaparanodal Kv1.1, characteristically seen in GalNAcT(-/-) and attributed to a breakdown of the axo-glial junction, was normalized in GalNAcT(-/-)-Tg(neuronal) but remained present in GalNAcT(-/-)-Tg(glial) mice. These results indicate that neuronal rather than glial gangliosides are critical to the age-related maintenance of nervous system integrity.

Depletion of gangliosides enhances cartilage degradation in mice.

OBJECTIVE: Glycosphingolipids (GSLs) are ubiquitous membrane components that play a functional role in maintaining chondrocyte homeostasis. We investigated the potential role of gangliosides, one of the major components of GSLs, in osteoarthritis (OA) pathogenesis. DESIGN: Both age-associated and instability-induced OA models were generated using GM3 synthase knockout (GM3S(-/-)) mice. A cartilage degradation model and transiently GM3S-transfected chondrocytes were analyzed to evaluate the function of gangliosides in OA development. The amount of each series of GSLs in chondrocytes after IL-1α stimulation was profiled using mass spectrometry (MS). RESULTS: OA changes in GM3S(-/-) mice were dramatically enhanced with aging compared to those in wild-type (WT) mice. GM3S(-/-) mice showed more severe instability-induced pathologic OA in vivo. Ganglioside deficiency also led to the induction of matrix metalloproteinase (MMP)-13 and ADAMTS-5 secretion and chondrocyte apoptosis in vitro. In contrast, transient GM3S transfection of chondrocytes suppressed MMP-13 and ADAMTS-5 expression after interleukin (IL)-1α stimulation. GSL profiling revealed the presence of abundant gangliosides in chondrocytes after IL-1α stimulation. CONCLUSION: Gangliosides play a critical role in OA pathogenesis by regulating the expression of MMP-13 and ADAMTS-5 and chondrocyte apoptosis. Based on the obtained results, we propose that gangliosides are potential target molecules for the development of novel OA treatments.

Neuroblast migration and P2Y(1) receptor mediated calcium signalling depend on 9-O-acetyl GD3 ganglioside.

Previous studies indicated that a ganglioside 9acGD3 (9-O-acetyl GD3) antibody [the J-Ab (Jones antibody)] reduces GCP (granule cell progenitor) migration in vitro and in vivo. We here investigated, using cerebellar explants of post-natal day (P) 6 mice, the mechanism by which 9acGD3 reduces GCP migration. We found that immunoblockade of the ganglioside with the J-Ab or the lack of GD3 synthase reduced GCP in vitro migration and the frequency of Ca(2+) oscillations. Immunocytochemistry and pharmacological assays indicated that GCPs expressed P2Y(1)Rs (P2Y(1) receptors) and that deletion or blockade of these receptors decreased the migration rate of GCPs and the frequency of Ca(2+) oscillations. The reduction in P2Y(1)-mediated calcium signals seen in Jones-treated and GD3 synthase-null GCPs were paralleled by P2Y(1)R internalization. We conclude that 9acGD3 controls GCP migration by influencing P2Y(1)R cellular distribution and function.

Cholesterogenic genes expression in brain and liver of ganglioside-deficient mice.

The aim of this study was to determine the effect of changed ganglioside profile on transcription of selected genes involved in cholesterol homeostasis. For that purpose, the expression of 11 genes related to cholesterol synthesis, regulation, and cholesterol transport was investigated in selected brain regions (frontal cortex, hippocampus, brain stem, cerebellum) and liver of St8sia1 knockout (KO) mice characterized by deficient synthesis of b- and c-series gangliosides and accumulation of a-series gangliosides. The expression of majority of the analyzed genes, as determined using quantitative real time PCR, was slightly higher in St8sia1 KO compared to wild-type (wt) controls. More prominent changes were observed in Hmgr, Cyp51, and Cyp46 expression in brain (hippocampus and brain stem) and Srebp1a, Insig2a, and Ldlr in liver. In addition, the expression of master transcriptional regulators, Srebp1a, Srebp1c, and Insig2a, as well as transporters Ldlr and Vldlr differed between liver and brain, and within brain regions in wt animals. Cyp46 expression was expectedly brain-specific, with brain region difference in both wt and St8sia1 KO. The established change in transcriptome of cholesterogenic genes is associated to specific alteration of ganglioside composition which indicates relationship between gangliosides and regulation of cholesterol metabolism.

Neuroplastin expression in the hippocampus of mice lacking complex gangliosides.

We report changes in neuroplastin gene and protein expression in the hippocampus of B4galnt1 null mice, which lacks complex ganglioside structures, compared with that of wild-type mice. Neuroplastin mRNA expression was significantly higher in the hippocampi of B4galnt1 null mice than in wild-type mice. Moreover, Western blot analysis shows increased neuroplastin protein levels of neuroplastin-55 isoform in B4galnt1 null hippocampal homogenates. Immunohistochemistry revealed a substantially different distribution of neuroplastin immunoreactivity in sagittal sections of the hippocampi derived from B4galnt1 null in comparison with those from wild-type mice. Most strikingly, B4galnt1 null mice had relatively little neuroplastin immunoreactivity in the pyramidal layer of CA1 and CA3, whereas wild-type mice had strong neuroplastin staining of pyramidal cells. Results of this study support the hypothesis that alterations of brain ganglioside expression influence the expression of neuroplastin. As both neuroplastin and gangliosides have important roles in synaptic transmission, synaptic plasticity, and neurite outgrowth, it will be of particular interest to unravel the molecular mechanisms underlying the relationship between ganglioside composition and neuroplastin transcript and protein expression in the mammalian nervous system.

Essential roles of gangliosides in the formation and maintenance of membrane microdomains in brain tissues.

Gangliosides are considered to be involved in the maintenance and repair of nervous tissues. Recently, novel roles of gangliosides in the regulation of complement system were reported. Here we summarized roles of gangliosides in the formation and maintenance of membrane microdomains in brain tissues by comparing complement activation, inflammatory reaction and disruption of glycolipid-enriched microdomain (GEM)/rafts among several mutant mice of ganglioside synthases. Depending on the defects in ganglioside compositions, corresponding up-regulation of complement-related genes, proliferation of astrocytes and infiltration of microglia were found with gradual severity. Immunoblotting of fractions separated by sucrose density gradient ultracentrifugation revealed that DAF and NCAM having GPI-anchors tended to disappear from the raft fraction with intensities of DKO > GM2/GD2 synthase KO > GD3 synthase KO > WT. The lipid raft markers tended to disperse from the raft fractions with similar intensities. Phospholipids and cholesterol also tended to decrease in GEM/rafts in GM2/GD2 synthase KO and DKO, although total amounts were almost equivalent. All these results indicate that GEM/rafts architecture is destroyed by ganglioside deficiency with gradual intensity depending on the degree of defects of their compositions. Implication of inflammation caused by deficiency of gangliosides in various neurodegenerative diseases was discussed.

Effect of lipid raft disruption on TRPV1 receptor activation of trigeminal sensory neurons and transfected cell line.

The transient receptor potential vanilloid 1 (TRPV1) is a noxious heat-sensitive, chemonociceptive cation channel which is expressed in primary sensory neurons of polymodal nociceptors. The present study is devoted to analyse the role of lipid raft constituents in calcium influx evoked by various TRPV1 agonists on sensory neurons and on rTRPV1-transfected CHO cell line. Depletion of cholesterol by methyl beta-cyclodextrin (MCD, 1-10mM) diminished the percent of the calcium uptake response of cultured trigeminal neurons to capsaicin (100nM) or resiniferatoxin (RTX, 3nM). In contrast, in TRPV1-transfected cells the inhibition was observed only when capsaicin or N-oleoyldopamine (OLDA, 10microM) was applied, but not when RTX, anandamide (AEA, 10microM) or pH 5.5 was used for gating. The magnitude of Ca(2+)-transients evoked by capsaicin (330nM) was also inhibited in both cell types. Treatment of rTRPV1-expressing cells with sphinomyelinase inhibited the capsaicin-evoked (45)Ca-uptake leaving the RTX-induced response unchanged. On the other hand, in trigeminal neurons the effect of both compounds was inhibited by sphingomyelinase treatment. Inhibition of ganglioside biosynthesis by d-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP, 10-20microM) or myriocyn (5-50nM) diminished similarly capsaicin- or RTX-evoked calcium uptake in both cultured trigeminal neurons and rTRPV1-expressing cells. The present study revealed that depletion of different constituents of lipid raft inhibited gating the TRPV1 cation channel by various vanilloid and non-vanilloid agents. Evidence for a supporting role of cholesterol, sphingomyelin and gangliosides were obtained both in native and TRPV1-transfected cells. Differential modulation of responses to capsaicin and RTX was often observed.

Gangliosides are important for the preservation of the structure and organization of RBL-2H3 mast cells.

Gangliosides are known to be important in many biological processes. However, details concerning the exact function of these glycosphingolipids in cell physiology are poorly understood. In this study, the role of gangliosides present on the surface of rodent mast cells in maintaining cell structure was examined using RBL-2H3 mast cells and two mutant cell lines (E5 and D1) deficient in the gangliosides, GM(1) and the alpha-galactosyl derivatives of the ganglioside GD(1b). The two deficient cell lines were morphologically different from each other as well as from the parental RBL-2H3 cells. Actin filaments in RBL-2H3 and E5 cells were under the plasma membrane following the spindle shape of the cells, whereas in D1 cells, they were concentrated in large membrane ruffles. Microtubules in RBL-2H3 and E5 cells radiated from the centrosome and were organized into long, straight bundles. The bundles in D1 cells were thicker and organized circumferentially under the plasma membrane. The endoplasmic reticulum, the Golgi complex, and the secretory granule matrix were also altered in the mutant cell lines. These results suggest that the mast cell-specific alpha-galactosyl derivatives of ganglioside GD(1b) and GM(1) are important in maintaining normal cell morphology.

Total ganglioside ablation at mouse motor nerve terminals alters neurotransmitter release level.

Neuronal membrane gangliosides, forming a large family of sialylated glycosphingolipids, have been hypothesized to play important roles in synaptic transmission. We studied the ex vivo electrophysiological function of neuromuscular junctions of GM2/GD2-synthase*GD3-synthase compound null-mutant mice after acute removal of GM3, the only remaining ganglioside in this mouse, by in vitro treatment with neuraminidase. We found 16% enhancement of the acetylcholine release per nerve impulse at low-rate (0.3 Hz) nerve stimulation. Conversely, the treatment reduced the acetylcholine release evoked by high-rate (40 Hz) nerve stimulation. Also, 25 ms paired-pulse facilitation of endplate potentials was reduced by the neuraminidase-treatment. These effects may indicate a modest modulatory influence of the negative electrical charges carried by the sialic acid molecules of gangliosides on the function of presynaptic Ca(v)2.1 channels, affecting the magnitude and kinetics of the Ca(2+) influx that induces neurotransmitter release from the motor nerve terminal. Our results show that gangliosides are to some extent involved in neurotransmission at the neuromuscular junction, but that their presence is not an absolute requirement in this process.

Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor.

The high toxicity of clostridial neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven botulinum neurotoxin serotypes A-G (BoNT/A-G) inhibit acetylcholine release, leading to flaccid paralysis, while tetanus neurotoxin blocks neurotransmitter release in inhibitory neurons, resulting in spastic paralysis. Uptake of BoNT/A, B, E and G requires a dual interaction with gangliosides and the synaptic vesicle (SV) proteins synaptotagmin or SV2, whereas little is known about the entry mechanisms of the remaining serotypes. Here, we demonstrate that BoNT/F as wells depends on the presence of gangliosides, by employing phrenic nerve hemidiaphragm preparations derived from mice expressing GM3, GM2, GM1 and GD1a or only GM3. Subsequent site-directed mutagenesis based on homology models identified the ganglioside binding site at a conserved location in BoNT/E and F. Using the mice phrenic nerve hemidiaphragm assay as a physiological model system, cross-competition of full-length neurotoxin binding by recombinant binding fragments, plus accelerated neurotoxin uptake upon increased electrical stimulation, indicate that BoNT/F employs SV2 as protein receptor, whereas BoNT/C and D utilise different SV receptor structures. The co-precipitation of SV2A, B and C from Triton-solubilised SVs by BoNT/F underlines this conclusion.

In vitro analysis of glial cell function in ganglioside-deficient mice.

Gangliosides are a family of sialic acid-containing glycosphingolipids highly enriched in neuronal and glial membranes, where they play pleiotropic roles in nervous system function. In this glial cell biological study, we used mice deficient in glycosyltransferases involved in ganglioside biosynthesis to gain insights into the possible role of ganglioside overexpression or deficiency on glial cell proliferation, migration, and differentiation in vitro. Primary cultures of olfactory ensheathing cells, oligodendrocyte lineage cells, and Schwann cells isolated from beta1,4-N-acetylgalactosaminyl (beta1,4-GalNAc) transferase- and alpha-2,8-sialyltransferase-deficient mice demonstrated subtle differences in their behavior when compared with wild-type glia. Oligodendrocyte-axonal interactions were investigated in dissociated embryonic mixed spinal cord cultures in which axonal ensheathment with myelin internodes and organized nodes of Ranvier form. In these myelinating cultures, deficiency of complex gangliosides, as found in beta1,4-GalNAc T(-/-) mice, resulted in the temporal disorganization of K(v) and Na(+) channels at the nodes of Ranvier, similar to that seen in beta1,4-GalNAc T(-/-) mice in vivo. These data show that glycosyltransferase deficiency and the consequent ganglioside imbalance has subtle effects on a range of glial cell functions and that in vitro systems can be used to explore these in ways that complement whole animal physiology. Our results are also consistent with the absence of gross neurodevelopmental dysfunction in mice lacking a variety of different gangliosides, suggesting that ganglioside redundancy and substitution are mechanisms that compensate for the lack of a full complement of complex gangliosides.

Ganglioside depletion and EGF responses of human GM3 synthase-deficient fibroblasts.

Recognition of important roles of gangliosides in normal and abnormal cell function has motivated pharmacological modification of cellular ganglioside content. However, constitutive depletion of gangliosides in untransformed human cells has not been reported. In this context, the recent identification of a kindred carrying a point mutation in the GM3 synthase [ST3Gal5, Siat9] gene (Simpson MA, Cross H, Proukakis C, Priestman DA, Neville DC, Reinkensmeier G, Wang H, Wiznitzer M, Gurtz K, Verganelaki A, Pryde A, Patton MA, Dwek RA, Butters TD, Platt FM, Crosby AH. 2004. Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase. Nat Genet. 36:1225-1229) provided an opportunity to explore this possibility. We established primary cultures of skin fibroblasts of three patients homozygous for this autosomal recessive defect. They exhibited a 93% reduction in ganglioside content (0.8 +/- 0.2 nmol lipid-bound sialic acid per 10(7) cells versus 12.7 +/- 1.3 nmol per 10(7) normal fibroblasts). Importantly, this marked reduction was not compensated by the activation of an alternate pathway of ganglioside synthesis, as occurs in murine GM3 synthase knockout fibroblasts. Cell morphology appeared unaffected, but under stringent conditions EGF-induced proliferation and migration of the mutant fibroblasts were reduced by 80% and 60%, respectively. Probing potential explanations, we found that EGF binding (effective membrane EGF receptor (EGFR) number) was reduced by 52% (to 6.2 +/- 1.9 from 12.8 +/- 2.0 pmol/10(8) normal fibroblasts, P < 0.01), despite normal total EGFR protein. EGFR activation was likewise reduced as was EGF-induced Rho/Rac1 phosphorylation, which is associated with cell migration. We conclude that this GM3 synthase point mutation almost completely depletes human fibroblast cellular gangliosides, dampens membrane EGFR activation, and modulates related critical cell functions such as proliferation and migration. These cells offer a valuable model for the study of ganglioside modulation of cell function.