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Robert Ledeen

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A professor in neuroscience at the Rutgers University Medical School, I have devoted many years to the study of gangliosides and other glycoliipids, including their role in modulating neurochemical mechanisms of the nervous system. Our current interest is their role in Parkinson's disease, especially the potential therapeutic benefit of GM1 ganglioside.

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Papers by Robert Ledeen

Bidirectional transport of gangliosides, glycoproteins and neutral glycosphingolipids in the sensory neurons of rat sciatic nerve

Neuroscience, Mar 1, 1987

Bidirectional axonal transport of glycoconjugates was studied in the sensory axons of rat sciatic... more Bidirectional axonal transport of glycoconjugates was studied in the sensory axons of rat sciatic nerve following injection of radiolabelled precursors into L4 and L5 dorsal root ganglia. After varying time intervals, gangliosides and neutral glycosphingolipids were isolated from anterograde and retrograde accumulation segments and radioactivity determined. Radiolabelled glycoproteins were measured in delipidated residues. These glycoconjugates were shown to undergo both anterograde and retrograde transport, accumulation occurring in roughly parallel manner for the three classes. The velocity of anterograde transport was collectively estimated at approximately 360 mm/day. Neutral glycosphingolipids, previously unknown to be axonally trasported, were present in sensory axons and transported in roughly equivalent amounts as gangliosides-as judged by levels of transported radioactivity. TLC-radioautography revealed a number of molecular species in the general region of tetra-and larger glycosylceramides. Fractionation of gangliosides according to sialic acid content demonstrated the presence of mono-, di-and polysialo species at the anterograde site.

Axon-myelin transfer of phospholipids and phospholipid precursors

Molecular Neurobiology, Jun 1, 1992

Ascorbate-induced changes in gangliosides of calf aortic smooth muscle cells in culture: Possible influence of extracellular matrix

In vitro cellular & developmental biology, May 1, 1990

The ganglioside composition of calf aortic smooth muscle cells, cultured in the presence and abse... more The ganglioside composition of calf aortic smooth muscle cells, cultured in the presence and absence of ascorbate, was analyzed. Previous work has shown that ascorbate supplementation leads to the formation of an extracellular matrix consisting primarily of collagen and that this matrix influences the biosynthetic capabilities of the cell. Cell cultures were supplemented with ascorbate for 3 wk and labeled with [14C]glucosamine for 3 d before harvesting. Ascorbate supplementation resulted in increased ganglioside sialic acid levels and a change in chromatographic profile involving both absolute and relative increases in GD1a. The latter, along with polysialo species, showed increased incorporation of [14C]glucosamine. These findings are interpreted in relation to the proposed role of gangliosides as mediators in the interaction of various cells with extracellular matrix.

Ganglioside accumulation in activated glia in the developing brain: comparison between WT and GalNAcT KO mice

Journal of Lipid Research, Aug 1, 2015

Binge-like ethanol exposure in neonatal rodents, equivalent to the third trimester of human fetus... more Binge-like ethanol exposure in neonatal rodents, equivalent to the third trimester of human fetuses, induces robust neurodegeneration (1, 2) associated with reactive gliosis (3), activation of microglia (4, 5), and elevation of pro-and anti-infl ammatory cytokines (6), resulting in long-lasting physiological and neurobehavioral defi cits (7-10). This animal model of fetal alcohol spectrum disorders has been often used to clarify mechanisms behind ethanol toxicity during the developmental period, when neurons are particularly vulnerable to environmental toxins (11). We previously demonstrated that ethanol-induced apoptotic neurodegeneration and microglial activation in postnatal day 7 (P7) mice (12-14) are associated with increases in brain lipids: ceramide, TG, cholesterol ester (ChE), N-acylphosphatidylethanolamine (NAPE) (15), and GM2 ganglioside (16). Specifi cally, enhanced formation of sphingolipids, such as ceramide and GM2, appears to be involved in ethanol-induced neuroapoptosis (16-18). Gangliosides (sialic acid-containing glycosphingolipids), which have many biological functions as antigens, mediators of cell adhesion, and modulators of signal transduction (19, 20), are particularly abundant in the nervous system. In the P7 brain exposed to ethanol, GM2 ganglioside, which is a minor ganglioside in the brain and contains a relatively simple oligosaccharide moiety, increased mainly in lysosomes/late endosomes of activated microglia, suggesting that GM2 may be derived from more complex gangliosides of degenerating neurons (16) engulfed by activated microglia (14). Increased GM2 by ethanol exposure is also Abstract Our previous studies have shown accumulation of GM2 ganglioside during ethanol-induced neurodegeneration in the developing brain, and GM2 elevation has also been reported in other brain injuries and neurodegenerative diseases. Using GM2/GD2 synthase KO mice lacking GM2/GD2 and downstream gangliosides, the current study explored the signifi cance of GM2 elevation in WT mice. Immunohistochemical studies indicated that ethanol-induced acute neurodegeneration in postnatal day 7 (P7) WT mice was associated with GM2 accumulation in the late endosomes/ lysosomes of both phagocytic microglia and increased glial fibrillary acidic protein (GFAP)-positive astrocytes. However, in KO mice, although ethanol induced robust neurodegeneration and accumulation of GD3 and GM3 in the late endosomes/lysosomes of phagocytic microglia, it did not increase the number of GFAP-positive astrocytes, and the accumulation of GD3/GM3 in astrocytes was minimal. Not only ethanol, but also DMSO, induced GM2 elevation in activated microglia and astrocytes along with neurodegeneration in P7 WT mice, while lipopolysaccharide, which did not induce signifi cant neurodegeneration, caused GM2 accumulation mainly in lysosomes of activated astrocytes. Thus, GM2 elevation is associated with activation of microglia and astrocytes in the injured developing brain, and GM2, GD2, or other downstream gangliosides may regulate astroglial responses in ethanol-induced neurodegeneration.

Modulation by GM1 ganglioside of β1-adrenergic receptor-induced cyclic AMP formation in Sf9 cells

Biochimica et biophysica acta. Molecular cell research, May 1, 1995

The effect of ganglioside GM1 on isoproterenol-induced cAMP accumulation was studied in insect Sf... more The effect of ganglioside GM1 on isoproterenol-induced cAMP accumulation was studied in insect Sf9 cells expressing the human /3t-adrenergic receptor by infection with recombinant baculovirus. When such Sf9 cells were treated with isoproterenol plus IBMX, intracellular cAMP formation increased approximately 10-fold over the basal level. Preincubation of the baculovirus-infected cells with GM1 for 1 h caused a concentration-dependent inhibition of the isoproterenol-induced cAMP accumulation. Phosphatidylserine, GM3, GTlb and a bovine brain ganglioside preparation lacking GM1 did not cause significant inhibition. Forskolin-induced cAMP accumulation was not affected by the GM1 treatment. Inhibition of isoproterenol-induced cAMP formation by GM1 was not observed in Sf9 cells expressing fl2-adrenergic receptor instead of the flt-adrenergic receptor. Binding studies with (-)-[3H]CGP12177 showed that preincubation with GM1 significantly reduced the affinity of antagonist binding to the /3t-adrenergic receptor. These results suggest that GM1 or related ganglioside structure(s) may function as natural modulator(s) of the /3t-adrenergic receptor.

Introductory Comments

Annals of the New York Academy of Sciences, Jun 1, 1998

Gangliosides of cultured astroglia

Brain Research, Sep 1, 1988

Cultured astrocytes prepared from newborn rat brain and 13-day-old chick embryonic brain were ana... more Cultured astrocytes prepared from newborn rat brain and 13-day-old chick embryonic brain were analyzed qualitatively and quantitatively for ganglioside content. All preparations contained approximately the same total level: 2.4-3.4 micrograms N-acetylneuraminic acid (NeuAc)/mg protein. In contrast, the value for primary cultures of neurons from chick embryonic brain was 5.9. The non-hexosamine-containing species, GM3 and GD3, comprised 75-85% of the total in astroglial cultures, the remainder consisting mainly of structural types other than the gangliotetraose series; choleragenoid assay revealed the latter to be virtually absent or to comprise at most a few percent. Deficiency of gangliotetraose synthesizing ability was indicated by the very low level of UDP-GalNac:GM3 N-acetylgalactosaminyltransferase detected in the cells. Treatment of cultured astrocytes with astroglial growth factor 2 or dibutyryl cyclic AMP caused little if any change in quantity or pattern of gangliosides. The large majority of cells stained in a manner characteristic of astrocytes: positive for glial fibrillary acidic protein, negative for galactosyl ceramides. Staining with cholera toxin and anti-GM1 antibody was essentially negative, as was that with tetanus toxin, A2B5 monoclonal antibody, and antibody to GD3. All evidence thus points to cultured astrocytes of rat and chick brain containing appreciable gangliosides, most of which are GM3 and GD3 with the majority of the remainder comprising structures other than the gangliotetraose type.

Procedure for isolation of gangliosides in high yield and purity: Simultaneous isolation of neutral glycosphingolipids

Analytical Biochemistry, Jul 1, 1985

While several methods for ganglioside extraction and isolation have been described, relatively li... more While several methods for ganglioside extraction and isolation have been described, relatively little attention has been given to the effectiveness of separation from peptides, phospholipids, and various low-molecular-weight contaminants. A procedure is described for isolation of gangliosides in high purity and good yield from 1- to 400-mg samples (wet wt). A key step was mild acidification following homogenization, designed to dissociate gangliosides from lipophilic peptides which coextracted into organic solvents. This has proved particularly helpful for myelin and myelin-containing tissues (e.g., white matter, nerve) whose proteins have presented special problems in ganglioside isolation. In this study isolation was effected by consecutive chromatographies on Sephadex LH-20, DEAE-Sephadex, and silica gel following the initial acidification. The method applied to bovine white matter gave tissue concentrations (calculated from yields and radiolabeled tracer recoveries) that were similar to those obtained with three previously described procedures; however, peptide contaminants were an order of magnitude lower. Removal of low-molecular-weight contaminants, including nucleotide sugars, was virtually complete. In addition to ganglioside isolation the method can be used to obtain neutral glycosphingolipids as well. It is believed to have broad applicability to a diversity of tissues.

GDNF signaling implemented by GM1 ganglioside; failure in Parkinson's disease and GM1-deficient murine model

Experimental Neurology, 2015

GDNF is indispensible for adult catecholaminergic neuron survival, and failure of GDNF signaling ... more GDNF is indispensible for adult catecholaminergic neuron survival, and failure of GDNF signaling has been linked to loss of dopaminergic neurons in Parkinson's disease (PD). This study demonstrates attenuated GDNF signaling in neurons deficient in ganglio-series gangliosides, and restoration of such signaling with LIGA20, a membrane permeable analog of GM1. GM1 is shown to associate in situ with GFRα1 and RET, the protein components of the GDNF receptor, this being necessary for assembly of the tripartite receptor complex. Mice wholly or partially deficient in GM1 due to disruption of the B4galnt1 gene developed PD symptoms based on behavioral and neuropathological criteria which were largely ameliorated by gene therapy with AAV2-GDNF and also with LIGA20 treatment. The nigral neurons of PD subjects that were severely deficient in GM1 showed subnormal levels of tyrosine phosphorylated RET. Also in PD brain, GM1 levels in the occipital cortex, a region of limited PD pathology, were significantly below age-matched controls, suggesting the possibility of systemic GM1 deficiency as a risk factor in PD. This would accord with our finding that mice with partial GM1 deficiency represent a faithful recapitulation of the human disease. Together with the previously demonstrated age-related decline of GM1 in human brain, this points to gradual development of subthreshold levels of GM1 in the brain of PD subjects below that required for effective GDNF signaling. This hypothesis offers a dramatically different explanation for the etiology of sporadic PD as a manifestation of acquired resistance to GDNF.

Sodium-calcium exchanger complexed with GM1 ganglioside in nuclear membrane transfers calcium from nucleoplasm to endoplasmic reticulum

Proceedings of the National Academy of Sciences of the United States of America, Jun 30, 2009

The inner membrane of the nuclear envelope (NE) was previously shown to contain a Na/Ca exchanger... more The inner membrane of the nuclear envelope (NE) was previously shown to contain a Na/Ca exchanger (NCX) tightly linked to GM1 ganglioside that mediates transfer of nucleoplasmic Ca 2؉ to the NE lumen and constitutes a cytoprotective mechanism. This transfer was initially observed with isolated nuclei and is now demonstrated in living cells in relation to subcellular Ca 2؉ dynamics. Four cell lines with varying expression of NCX and GM1 in the NE were transfected with cameleon-fluorescent Ca 2؉ indicators genetically targeted to NE/ endoplasmic reticulum (ER) and nucleoplasm to monitor [Ca 2؉ ]ne/er and [Ca 2؉ ]n respectively. Cytosolic Ca 2؉ ([Ca 2؉ ]cyt) was indicated with fura-2. Thapsigargin caused progressive loss of [Ca 2؉ ]ne/er, which was rapidly replaced on addition of extrinsic Ca 2؉ to those cells containing fully functional NCX/GM1: differentiated NG108-15 and C6 cells. Reduced elevation of [Ca 2؉ ]ne/er following thapsigargin depletion occurred in cells containing little or no GM1 in the NE: undifferentiated NG108-15 and NG-CR72 cells. No change in [Ca 2؉ ]ne/er due to applied Ca 2؉ was seen in Jurkat cells, which entirely lack NCX. Ca 2؉ entry to NE/ER was also blocked by KB-R7943, inhibitor of NCX. [Ca 2؉ ]n and [Ca 2؉ ]cyt were elevated independent of [Ca 2؉ ]ne/er and remained in approximate equilibrium with each other. Ca 2؉ rise in the ER originated in the NE region and extended to the entire ER network. These results indicate the nuclear NCX/GM1 complex acts to gate Ca 2؉ transfer from cytosol to ER, an alternate route to the sarcoplasmic/ endoplasmic reticulum calcium ATPase pump. They also suggest a possible contributory mechanism for independent regulation of nuclear Ca 2؉. cameleon calcium indicator ͉ nuclear calcium ͉ nuclear envelope ͉ ER calcium ͉ ganglioside function

Axonogenesis in neuro‐2a cells correlates with GM1 upregulation in the nuclear and plasma membranes

Journal of Neuroscience Research, Aug 15, 1999

GM1 ganglioside was previously shown to occur in the nuclear membrane, as well as the plasma memb... more GM1 ganglioside was previously shown to occur in the nuclear membrane, as well as the plasma membrane, of central nervous system (CNS) and peripheral nervous system (PNS) neurons undergoing morphological differentiation in culture. NG108-15 neuroblastoma cells showed the same phenomenon when induced to extend axon-like but not dendrite-like processes, although in both cases terminal differentiation was evidenced by failure of extended neurites to retract following washout of neuritogenic agent. The present study of Neuro-2a neuroblastoma cells subjected to similar treatments has revealed both similarities and differences compared to NG108-15 cells. Similar to the latter, Neuro-2a cells responded to neuraminidase and ionomycin with axon-like outgrowth together with upregulation of nuclear GM1, and to three other agents (retinoic acid, dibutyryl cyclic AMP, exogenous GM1) with dendrite-like outgrowth that was unaccompanied by nuclear GM1 increase. Although both cell types responded to low serum by extending neurites of mixed axonal-dendritic properties, Neuro-2a, in keeping with its greater tendency to extend some neurites of axonal character in low serum, showed elevated nuclear GM1 in a significant number of such differentiated cells. All three axonogenic agents induced parallel upregulation of GM1 in plasma-, nuclear-, and Golgi membranes, and these increases were stable to washout. Neurites generated in Neuro-2a cells by the three dendritogenic agents lacked stability, unlike those produced by the same agents in NG108-15 cells. This study also amplified the differences in response triggered by exogenous GM1 compared to that resulting from enzyme-mediated elevation of endogenous GM1.

Neurological Diseases with Abnormal Ganglioside Patterns

Elsevier eBooks, 1966

Axon-Myelin transfer of phospholipids and phospholipid precursors:Labeling of Myelin phosphoinositides through axonal transport

Molecular Neurobiology, Dec 1, 1992

Galectin-1 Cross-Linking of GM1 Ganglioside in Autoimmune Suppression

Acs Symposium Series, 2012

Gangliosides in Neurodegenerative Diseases

Advances in neurobiology, Oct 19, 2022

Glycoproteins carrying O-linked N-acetylgalactosamine, N-acetylgluco-samine, mannose, fucose, glu... more Glycoproteins carrying O-linked N-acetylgalactosamine, N-acetylgluco-samine, mannose, fucose, glucose, and xylose are found in the nervous system. Lipids can be glycosylated as well. Membrane lipid, ceramide, is modified by the addition of either glucose or galactose to form glycosphingolipid, galactosylceramide, or glucosylceramide. Recent analyses have identified glucosylated lipids of cholesterol and phosphatidic acid. These O-linked carbohydrate residues are found primarily on the outer surface of the plasma membrane or in the extracellular space. Their expression is cell or tissue specific and developmentally regulated. Due to their structural diversity, they play important roles in a variety of biological processes such as membrane transport and cell-cell interactions.

Exogenous Gangliosides Stimulate Breakdown of Neuro-2A Phosphoinositides in a Manner Unrelated to Neurite Outgrowth

Journal of Neurochemistry, Aug 1, 1990

Gangliosides administered exogenously are wellknown effectors of differentiation in many neurobla... more Gangliosides administered exogenously are wellknown effectors of differentiation in many neuroblastoma lines and primary neuronal cultures. Previous studies suggested the phosphoinositide signaling mechanism could be a contributing factor. We have found that treatment of Neuro-2A cells with bovine brain ganglioside mixture (BEG) causes breakdown of phosphoinositides, as measured by increased levels of inositol phosphates. The effect was optimal at 60 min and required a minimal BEG concentration of 25 p M. However, addition of neomycin, which blocked phosphoinositide breakdown, had no observable effect on gangliosidestimulated neurite outgrowth. A similar result was obtained with +tectorigenin, which also inhibited phosphoinositide hydrolysis. When cells were treated with maitotoxin, an agent that promotes phosphoinositide breakdown, there was no enhancement of neurite outgrowth. These findings indicate that although exogenous gangliosides elevate inositol phosphate formation over a prolonged period in neuro-2A cells, this reaction is not integral to the differentiation of these cells. The possibility of secondary effects influencing neurite type and structure cannot be excluded. Key Words: Gangliosides-Phosphoinositide breakdown-Neuro-2A cells. Vaswani K. K. et al. Exogenous gangliosides stimulate breakdown of Neuro-2A phosphoinositides in a manner unrelated to neurite outgrowth.

Gangliosides and proteins in developing chicken brain myelin

Developmental Brain Research, Dec 1, 1982

Ganglioside and protein content of chicken brain myelin have been studied as a function of develo... more Ganglioside and protein content of chicken brain myelin have been studied as a function of development and maturation. Ganglioside concentration remained relatively constant at approximately 225-266 pg sialic acid per 100 mg myelin, from 17-dayold embryos to 540-day-old adults. The ganglioside distributional pattern was also constant, GMI and GM4 each accounting for approximately one-third of total sialic acid. These properties are contrasted with those of mouse and rat brain myelin which previously showed enrichment of GM1 at early stages of myelination and appearance of Gr~4 at a later stage of maturation. In an earlier study of the mouse total myelin gangliosides doubled in concentration between 3 and 5 weeks of age and full maturity. Myelin proteins of the chicken also remained relatively constant in concentration and distributional pattern during development, although the percentage of basic protein increased somewhat during the first few weeks after hatching. Correlations were observed between molar concentrations of basic protein and various gangliosides, particularly the monosialo types.

Gangliosides Stimulate Calcium Flux in Neuro-2A Cells and Require Exogenous Calcium for Neuritogenesis

Journal of Neurochemistry, Aug 1, 1990

The neuritogenic effect of exogenous ganglioside has been documented with a variety of neuronal a... more The neuritogenic effect of exogenous ganglioside has been documented with a variety of neuronal and neuroblastoma systems, but the mechanism is not understood. Involvement of Ca2+ is suggested by this study which demonstrates that treatment of Neuro-2A cells with bovine brain gangliosides (BBG) in Ca2+-depleted medium failed to produce neurite outgrowth. This was in contrast to treatment with retinoic acid or dibutyryl cyclic AMP which induced differentiation under the same conditions. Addition of BBG

Mice Lacking Major Brain Gangliosides Develop Parkinsonism

Neurochemical Research, Mar 12, 2011

Parkinson's disease (PD) is the second most prevalent late-onset neurodegenerative disorder that ... more Parkinson's disease (PD) is the second most prevalent late-onset neurodegenerative disorder that affects nearly 1% of the global population aged 65 and older. Whereas palliative treatments are in use, the goal of blocking progression of motor and cognitive disability remains unfulfilled. A better understanding of the basic pathophysiological mechanisms underlying PD would help to advance that goal. The present study provides evidence that brain ganglioside abnormality, in particular GM1, may be involved. This is based on use of the genetically altered mice with disrupted gene Galgt1 for GM2/GD2 synthase which depletes GM2/GD2 and all the gangliotetraose gangliosides that constitute the major molecular species of brain. These knockout mice show overt motor disability on aging and clear indications of motor impairment with appropriate testing at an earlier age. This disability was rectified by L-dopa administration. These mice show other characteristic symptoms of PD, including depletion of striatal dopamine (DA), loss of DA neurons of the substantia nigra pars compacta, and aggregation of alpha synuclein. These manifestations of parkinsonism were largely attenuated by administration of LIGA-20, a membrane permeable analog of GM1 that penetrates the blood brain barrier and enters living neurons. These results suggest that perturbation of intracellular mechanisms mediated by intracellular GM1 may be a contributing factor to PD. Keywords Parkinson's disease Á GM1 ganglioside Á LIGA-20 Á Dopaminergic neurons Á Alpha synuclein Abbreviations Ab antibody a-syn alpha-synuclein BSS balanced salt solution CtxB B subunit of cholera toxin DA dopamine or dopaminergic DOA days of age DOPAC dihydroxyphenyl acetic acid ER endoplasmic reticulum HRP horseradish peroxidase 5-HT 5-hydroxytryptamine (serotonin) 5-HIAA 5-hydroxyindole acetic acid IP intraperitoneally KO knockout MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine PBS phosphate buffered saline PD Parkinson's disease SNpc substantia nigra pars compacta TH tyrosine hydroxylase VTA ventral tegmental area WT wild type

C6 cells express a sodium-calcium exchanger/GM1 complex in the nuclear envelope but have no exchanger in the plasma membrane: Comparison to astrocytes

Journal of Neuroscience Research, 2004

Previous work demonstrated the presence of an isoform of Na(+)/Ca(2+) exchanger in the nuclear en... more Previous work demonstrated the presence of an isoform of Na(+)/Ca(2+) exchanger in the nuclear envelope of neurons and NG108-15 cells that is tightly associated with GM1 ganglioside and potentiated by the latter. This contrasted with the Na(+)/Ca(2+) exchanger(s) in the plasma membrane, which were suggested to associate more loosely with GM1. To study these aspects of Na(+)/Ca(2+) exchanger expression in nonneuronal neural cells, we have examined nuclear and plasma membrane exchanger patterns in astrocytes and C6 cells, a glia-derived line. We find both cell types contain the tightly associated exchanger/GM1 complex in the nuclear envelope but, surprisingly, only astrocytes possess Na(+)/Ca(2+) exchanger activity in the plasma membrane. This is the first reported example of a cell (C6) with Na(+)/Ca(2+) exchangers in the nuclear envelope but not in the plasma membrane. RT-PCR established the presence of the NCX1 subtype in C6 cells and both NCX1 and NCX2 in astrocytes. Comparison was made with NG108-15 cells, which have Na(+)/Ca(2+) exchangers in both nuclear and plasma membranes, and Jurkat cells, which have no Na(+)/Ca(2+) exchanger in either membrane. Culturing of C6 cells in the presence dibutyryl-cAMP caused upregulation of a high molecular weight isoform of the exchanger together with GM1 in the nuclear envelope, resulting in significant elevation of Na(+)/Ca(2+) exchanger activity in the latter. Application of exogenous GM1 to nuclei from non-treated cells also potentiated exchanger activity, although to a lesser degree. The Na(+)/Ca(2+) exchanger/GM1 complex occurs in the inner membrane of the nuclear envelope, suggesting a functional role in transferring Ca(2+) between nucleoplasm and the envelope lumen.

Bidirectional transport of gangliosides, glycoproteins and neutral glycosphingolipids in the sensory neurons of rat sciatic nerve

Neuroscience, Mar 1, 1987

Bidirectional axonal transport of glycoconjugates was studied in the sensory axons of rat sciatic... more Bidirectional axonal transport of glycoconjugates was studied in the sensory axons of rat sciatic nerve following injection of radiolabelled precursors into L4 and L5 dorsal root ganglia. After varying time intervals, gangliosides and neutral glycosphingolipids were isolated from anterograde and retrograde accumulation segments and radioactivity determined. Radiolabelled glycoproteins were measured in delipidated residues. These glycoconjugates were shown to undergo both anterograde and retrograde transport, accumulation occurring in roughly parallel manner for the three classes. The velocity of anterograde transport was collectively estimated at approximately 360 mm/day. Neutral glycosphingolipids, previously unknown to be axonally trasported, were present in sensory axons and transported in roughly equivalent amounts as gangliosides-as judged by levels of transported radioactivity. TLC-radioautography revealed a number of molecular species in the general region of tetra-and larger glycosylceramides. Fractionation of gangliosides according to sialic acid content demonstrated the presence of mono-, di-and polysialo species at the anterograde site.

Axon-myelin transfer of phospholipids and phospholipid precursors

Molecular Neurobiology, Jun 1, 1992

Ascorbate-induced changes in gangliosides of calf aortic smooth muscle cells in culture: Possible influence of extracellular matrix

In vitro cellular & developmental biology, May 1, 1990

The ganglioside composition of calf aortic smooth muscle cells, cultured in the presence and abse... more The ganglioside composition of calf aortic smooth muscle cells, cultured in the presence and absence of ascorbate, was analyzed. Previous work has shown that ascorbate supplementation leads to the formation of an extracellular matrix consisting primarily of collagen and that this matrix influences the biosynthetic capabilities of the cell. Cell cultures were supplemented with ascorbate for 3 wk and labeled with [14C]glucosamine for 3 d before harvesting. Ascorbate supplementation resulted in increased ganglioside sialic acid levels and a change in chromatographic profile involving both absolute and relative increases in GD1a. The latter, along with polysialo species, showed increased incorporation of [14C]glucosamine. These findings are interpreted in relation to the proposed role of gangliosides as mediators in the interaction of various cells with extracellular matrix.

Ganglioside accumulation in activated glia in the developing brain: comparison between WT and GalNAcT KO mice

Journal of Lipid Research, Aug 1, 2015

Binge-like ethanol exposure in neonatal rodents, equivalent to the third trimester of human fetus... more Binge-like ethanol exposure in neonatal rodents, equivalent to the third trimester of human fetuses, induces robust neurodegeneration (1, 2) associated with reactive gliosis (3), activation of microglia (4, 5), and elevation of pro-and anti-infl ammatory cytokines (6), resulting in long-lasting physiological and neurobehavioral defi cits (7-10). This animal model of fetal alcohol spectrum disorders has been often used to clarify mechanisms behind ethanol toxicity during the developmental period, when neurons are particularly vulnerable to environmental toxins (11). We previously demonstrated that ethanol-induced apoptotic neurodegeneration and microglial activation in postnatal day 7 (P7) mice (12-14) are associated with increases in brain lipids: ceramide, TG, cholesterol ester (ChE), N-acylphosphatidylethanolamine (NAPE) (15), and GM2 ganglioside (16). Specifi cally, enhanced formation of sphingolipids, such as ceramide and GM2, appears to be involved in ethanol-induced neuroapoptosis (16-18). Gangliosides (sialic acid-containing glycosphingolipids), which have many biological functions as antigens, mediators of cell adhesion, and modulators of signal transduction (19, 20), are particularly abundant in the nervous system. In the P7 brain exposed to ethanol, GM2 ganglioside, which is a minor ganglioside in the brain and contains a relatively simple oligosaccharide moiety, increased mainly in lysosomes/late endosomes of activated microglia, suggesting that GM2 may be derived from more complex gangliosides of degenerating neurons (16) engulfed by activated microglia (14). Increased GM2 by ethanol exposure is also Abstract Our previous studies have shown accumulation of GM2 ganglioside during ethanol-induced neurodegeneration in the developing brain, and GM2 elevation has also been reported in other brain injuries and neurodegenerative diseases. Using GM2/GD2 synthase KO mice lacking GM2/GD2 and downstream gangliosides, the current study explored the signifi cance of GM2 elevation in WT mice. Immunohistochemical studies indicated that ethanol-induced acute neurodegeneration in postnatal day 7 (P7) WT mice was associated with GM2 accumulation in the late endosomes/ lysosomes of both phagocytic microglia and increased glial fibrillary acidic protein (GFAP)-positive astrocytes. However, in KO mice, although ethanol induced robust neurodegeneration and accumulation of GD3 and GM3 in the late endosomes/lysosomes of phagocytic microglia, it did not increase the number of GFAP-positive astrocytes, and the accumulation of GD3/GM3 in astrocytes was minimal. Not only ethanol, but also DMSO, induced GM2 elevation in activated microglia and astrocytes along with neurodegeneration in P7 WT mice, while lipopolysaccharide, which did not induce signifi cant neurodegeneration, caused GM2 accumulation mainly in lysosomes of activated astrocytes. Thus, GM2 elevation is associated with activation of microglia and astrocytes in the injured developing brain, and GM2, GD2, or other downstream gangliosides may regulate astroglial responses in ethanol-induced neurodegeneration.

Modulation by GM1 ganglioside of β1-adrenergic receptor-induced cyclic AMP formation in Sf9 cells

Biochimica et biophysica acta. Molecular cell research, May 1, 1995

The effect of ganglioside GM1 on isoproterenol-induced cAMP accumulation was studied in insect Sf... more The effect of ganglioside GM1 on isoproterenol-induced cAMP accumulation was studied in insect Sf9 cells expressing the human /3t-adrenergic receptor by infection with recombinant baculovirus. When such Sf9 cells were treated with isoproterenol plus IBMX, intracellular cAMP formation increased approximately 10-fold over the basal level. Preincubation of the baculovirus-infected cells with GM1 for 1 h caused a concentration-dependent inhibition of the isoproterenol-induced cAMP accumulation. Phosphatidylserine, GM3, GTlb and a bovine brain ganglioside preparation lacking GM1 did not cause significant inhibition. Forskolin-induced cAMP accumulation was not affected by the GM1 treatment. Inhibition of isoproterenol-induced cAMP formation by GM1 was not observed in Sf9 cells expressing fl2-adrenergic receptor instead of the flt-adrenergic receptor. Binding studies with (-)-[3H]CGP12177 showed that preincubation with GM1 significantly reduced the affinity of antagonist binding to the /3t-adrenergic receptor. These results suggest that GM1 or related ganglioside structure(s) may function as natural modulator(s) of the /3t-adrenergic receptor.

Introductory Comments

Annals of the New York Academy of Sciences, Jun 1, 1998

Gangliosides of cultured astroglia

Brain Research, Sep 1, 1988

Cultured astrocytes prepared from newborn rat brain and 13-day-old chick embryonic brain were ana... more Cultured astrocytes prepared from newborn rat brain and 13-day-old chick embryonic brain were analyzed qualitatively and quantitatively for ganglioside content. All preparations contained approximately the same total level: 2.4-3.4 micrograms N-acetylneuraminic acid (NeuAc)/mg protein. In contrast, the value for primary cultures of neurons from chick embryonic brain was 5.9. The non-hexosamine-containing species, GM3 and GD3, comprised 75-85% of the total in astroglial cultures, the remainder consisting mainly of structural types other than the gangliotetraose series; choleragenoid assay revealed the latter to be virtually absent or to comprise at most a few percent. Deficiency of gangliotetraose synthesizing ability was indicated by the very low level of UDP-GalNac:GM3 N-acetylgalactosaminyltransferase detected in the cells. Treatment of cultured astrocytes with astroglial growth factor 2 or dibutyryl cyclic AMP caused little if any change in quantity or pattern of gangliosides. The large majority of cells stained in a manner characteristic of astrocytes: positive for glial fibrillary acidic protein, negative for galactosyl ceramides. Staining with cholera toxin and anti-GM1 antibody was essentially negative, as was that with tetanus toxin, A2B5 monoclonal antibody, and antibody to GD3. All evidence thus points to cultured astrocytes of rat and chick brain containing appreciable gangliosides, most of which are GM3 and GD3 with the majority of the remainder comprising structures other than the gangliotetraose type.

Procedure for isolation of gangliosides in high yield and purity: Simultaneous isolation of neutral glycosphingolipids

Analytical Biochemistry, Jul 1, 1985

While several methods for ganglioside extraction and isolation have been described, relatively li... more While several methods for ganglioside extraction and isolation have been described, relatively little attention has been given to the effectiveness of separation from peptides, phospholipids, and various low-molecular-weight contaminants. A procedure is described for isolation of gangliosides in high purity and good yield from 1- to 400-mg samples (wet wt). A key step was mild acidification following homogenization, designed to dissociate gangliosides from lipophilic peptides which coextracted into organic solvents. This has proved particularly helpful for myelin and myelin-containing tissues (e.g., white matter, nerve) whose proteins have presented special problems in ganglioside isolation. In this study isolation was effected by consecutive chromatographies on Sephadex LH-20, DEAE-Sephadex, and silica gel following the initial acidification. The method applied to bovine white matter gave tissue concentrations (calculated from yields and radiolabeled tracer recoveries) that were similar to those obtained with three previously described procedures; however, peptide contaminants were an order of magnitude lower. Removal of low-molecular-weight contaminants, including nucleotide sugars, was virtually complete. In addition to ganglioside isolation the method can be used to obtain neutral glycosphingolipids as well. It is believed to have broad applicability to a diversity of tissues.

GDNF signaling implemented by GM1 ganglioside; failure in Parkinson's disease and GM1-deficient murine model

Experimental Neurology, 2015

GDNF is indispensible for adult catecholaminergic neuron survival, and failure of GDNF signaling ... more GDNF is indispensible for adult catecholaminergic neuron survival, and failure of GDNF signaling has been linked to loss of dopaminergic neurons in Parkinson's disease (PD). This study demonstrates attenuated GDNF signaling in neurons deficient in ganglio-series gangliosides, and restoration of such signaling with LIGA20, a membrane permeable analog of GM1. GM1 is shown to associate in situ with GFRα1 and RET, the protein components of the GDNF receptor, this being necessary for assembly of the tripartite receptor complex. Mice wholly or partially deficient in GM1 due to disruption of the B4galnt1 gene developed PD symptoms based on behavioral and neuropathological criteria which were largely ameliorated by gene therapy with AAV2-GDNF and also with LIGA20 treatment. The nigral neurons of PD subjects that were severely deficient in GM1 showed subnormal levels of tyrosine phosphorylated RET. Also in PD brain, GM1 levels in the occipital cortex, a region of limited PD pathology, were significantly below age-matched controls, suggesting the possibility of systemic GM1 deficiency as a risk factor in PD. This would accord with our finding that mice with partial GM1 deficiency represent a faithful recapitulation of the human disease. Together with the previously demonstrated age-related decline of GM1 in human brain, this points to gradual development of subthreshold levels of GM1 in the brain of PD subjects below that required for effective GDNF signaling. This hypothesis offers a dramatically different explanation for the etiology of sporadic PD as a manifestation of acquired resistance to GDNF.

Sodium-calcium exchanger complexed with GM1 ganglioside in nuclear membrane transfers calcium from nucleoplasm to endoplasmic reticulum

Proceedings of the National Academy of Sciences of the United States of America, Jun 30, 2009

The inner membrane of the nuclear envelope (NE) was previously shown to contain a Na/Ca exchanger... more The inner membrane of the nuclear envelope (NE) was previously shown to contain a Na/Ca exchanger (NCX) tightly linked to GM1 ganglioside that mediates transfer of nucleoplasmic Ca 2؉ to the NE lumen and constitutes a cytoprotective mechanism. This transfer was initially observed with isolated nuclei and is now demonstrated in living cells in relation to subcellular Ca 2؉ dynamics. Four cell lines with varying expression of NCX and GM1 in the NE were transfected with cameleon-fluorescent Ca 2؉ indicators genetically targeted to NE/ endoplasmic reticulum (ER) and nucleoplasm to monitor [Ca 2؉ ]ne/er and [Ca 2؉ ]n respectively. Cytosolic Ca 2؉ ([Ca 2؉ ]cyt) was indicated with fura-2. Thapsigargin caused progressive loss of [Ca 2؉ ]ne/er, which was rapidly replaced on addition of extrinsic Ca 2؉ to those cells containing fully functional NCX/GM1: differentiated NG108-15 and C6 cells. Reduced elevation of [Ca 2؉ ]ne/er following thapsigargin depletion occurred in cells containing little or no GM1 in the NE: undifferentiated NG108-15 and NG-CR72 cells. No change in [Ca 2؉ ]ne/er due to applied Ca 2؉ was seen in Jurkat cells, which entirely lack NCX. Ca 2؉ entry to NE/ER was also blocked by KB-R7943, inhibitor of NCX. [Ca 2؉ ]n and [Ca 2؉ ]cyt were elevated independent of [Ca 2؉ ]ne/er and remained in approximate equilibrium with each other. Ca 2؉ rise in the ER originated in the NE region and extended to the entire ER network. These results indicate the nuclear NCX/GM1 complex acts to gate Ca 2؉ transfer from cytosol to ER, an alternate route to the sarcoplasmic/ endoplasmic reticulum calcium ATPase pump. They also suggest a possible contributory mechanism for independent regulation of nuclear Ca 2؉. cameleon calcium indicator ͉ nuclear calcium ͉ nuclear envelope ͉ ER calcium ͉ ganglioside function

Axonogenesis in neuro‐2a cells correlates with GM1 upregulation in the nuclear and plasma membranes

Journal of Neuroscience Research, Aug 15, 1999

GM1 ganglioside was previously shown to occur in the nuclear membrane, as well as the plasma memb... more GM1 ganglioside was previously shown to occur in the nuclear membrane, as well as the plasma membrane, of central nervous system (CNS) and peripheral nervous system (PNS) neurons undergoing morphological differentiation in culture. NG108-15 neuroblastoma cells showed the same phenomenon when induced to extend axon-like but not dendrite-like processes, although in both cases terminal differentiation was evidenced by failure of extended neurites to retract following washout of neuritogenic agent. The present study of Neuro-2a neuroblastoma cells subjected to similar treatments has revealed both similarities and differences compared to NG108-15 cells. Similar to the latter, Neuro-2a cells responded to neuraminidase and ionomycin with axon-like outgrowth together with upregulation of nuclear GM1, and to three other agents (retinoic acid, dibutyryl cyclic AMP, exogenous GM1) with dendrite-like outgrowth that was unaccompanied by nuclear GM1 increase. Although both cell types responded to low serum by extending neurites of mixed axonal-dendritic properties, Neuro-2a, in keeping with its greater tendency to extend some neurites of axonal character in low serum, showed elevated nuclear GM1 in a significant number of such differentiated cells. All three axonogenic agents induced parallel upregulation of GM1 in plasma-, nuclear-, and Golgi membranes, and these increases were stable to washout. Neurites generated in Neuro-2a cells by the three dendritogenic agents lacked stability, unlike those produced by the same agents in NG108-15 cells. This study also amplified the differences in response triggered by exogenous GM1 compared to that resulting from enzyme-mediated elevation of endogenous GM1.

Neurological Diseases with Abnormal Ganglioside Patterns

Elsevier eBooks, 1966

Axon-Myelin transfer of phospholipids and phospholipid precursors:Labeling of Myelin phosphoinositides through axonal transport

Molecular Neurobiology, Dec 1, 1992

Galectin-1 Cross-Linking of GM1 Ganglioside in Autoimmune Suppression

Acs Symposium Series, 2012

Gangliosides in Neurodegenerative Diseases

Advances in neurobiology, Oct 19, 2022

Glycoproteins carrying O-linked N-acetylgalactosamine, N-acetylgluco-samine, mannose, fucose, glu... more Glycoproteins carrying O-linked N-acetylgalactosamine, N-acetylgluco-samine, mannose, fucose, glucose, and xylose are found in the nervous system. Lipids can be glycosylated as well. Membrane lipid, ceramide, is modified by the addition of either glucose or galactose to form glycosphingolipid, galactosylceramide, or glucosylceramide. Recent analyses have identified glucosylated lipids of cholesterol and phosphatidic acid. These O-linked carbohydrate residues are found primarily on the outer surface of the plasma membrane or in the extracellular space. Their expression is cell or tissue specific and developmentally regulated. Due to their structural diversity, they play important roles in a variety of biological processes such as membrane transport and cell-cell interactions.

Exogenous Gangliosides Stimulate Breakdown of Neuro-2A Phosphoinositides in a Manner Unrelated to Neurite Outgrowth

Journal of Neurochemistry, Aug 1, 1990

Gangliosides administered exogenously are wellknown effectors of differentiation in many neurobla... more Gangliosides administered exogenously are wellknown effectors of differentiation in many neuroblastoma lines and primary neuronal cultures. Previous studies suggested the phosphoinositide signaling mechanism could be a contributing factor. We have found that treatment of Neuro-2A cells with bovine brain ganglioside mixture (BEG) causes breakdown of phosphoinositides, as measured by increased levels of inositol phosphates. The effect was optimal at 60 min and required a minimal BEG concentration of 25 p M. However, addition of neomycin, which blocked phosphoinositide breakdown, had no observable effect on gangliosidestimulated neurite outgrowth. A similar result was obtained with +tectorigenin, which also inhibited phosphoinositide hydrolysis. When cells were treated with maitotoxin, an agent that promotes phosphoinositide breakdown, there was no enhancement of neurite outgrowth. These findings indicate that although exogenous gangliosides elevate inositol phosphate formation over a prolonged period in neuro-2A cells, this reaction is not integral to the differentiation of these cells. The possibility of secondary effects influencing neurite type and structure cannot be excluded. Key Words: Gangliosides-Phosphoinositide breakdown-Neuro-2A cells. Vaswani K. K. et al. Exogenous gangliosides stimulate breakdown of Neuro-2A phosphoinositides in a manner unrelated to neurite outgrowth.

Gangliosides and proteins in developing chicken brain myelin

Developmental Brain Research, Dec 1, 1982

Ganglioside and protein content of chicken brain myelin have been studied as a function of develo... more Ganglioside and protein content of chicken brain myelin have been studied as a function of development and maturation. Ganglioside concentration remained relatively constant at approximately 225-266 pg sialic acid per 100 mg myelin, from 17-dayold embryos to 540-day-old adults. The ganglioside distributional pattern was also constant, GMI and GM4 each accounting for approximately one-third of total sialic acid. These properties are contrasted with those of mouse and rat brain myelin which previously showed enrichment of GM1 at early stages of myelination and appearance of Gr~4 at a later stage of maturation. In an earlier study of the mouse total myelin gangliosides doubled in concentration between 3 and 5 weeks of age and full maturity. Myelin proteins of the chicken also remained relatively constant in concentration and distributional pattern during development, although the percentage of basic protein increased somewhat during the first few weeks after hatching. Correlations were observed between molar concentrations of basic protein and various gangliosides, particularly the monosialo types.

Gangliosides Stimulate Calcium Flux in Neuro-2A Cells and Require Exogenous Calcium for Neuritogenesis

Journal of Neurochemistry, Aug 1, 1990

The neuritogenic effect of exogenous ganglioside has been documented with a variety of neuronal a... more The neuritogenic effect of exogenous ganglioside has been documented with a variety of neuronal and neuroblastoma systems, but the mechanism is not understood. Involvement of Ca2+ is suggested by this study which demonstrates that treatment of Neuro-2A cells with bovine brain gangliosides (BBG) in Ca2+-depleted medium failed to produce neurite outgrowth. This was in contrast to treatment with retinoic acid or dibutyryl cyclic AMP which induced differentiation under the same conditions. Addition of BBG

Mice Lacking Major Brain Gangliosides Develop Parkinsonism

Neurochemical Research, Mar 12, 2011

Parkinson's disease (PD) is the second most prevalent late-onset neurodegenerative disorder that ... more Parkinson's disease (PD) is the second most prevalent late-onset neurodegenerative disorder that affects nearly 1% of the global population aged 65 and older. Whereas palliative treatments are in use, the goal of blocking progression of motor and cognitive disability remains unfulfilled. A better understanding of the basic pathophysiological mechanisms underlying PD would help to advance that goal. The present study provides evidence that brain ganglioside abnormality, in particular GM1, may be involved. This is based on use of the genetically altered mice with disrupted gene Galgt1 for GM2/GD2 synthase which depletes GM2/GD2 and all the gangliotetraose gangliosides that constitute the major molecular species of brain. These knockout mice show overt motor disability on aging and clear indications of motor impairment with appropriate testing at an earlier age. This disability was rectified by L-dopa administration. These mice show other characteristic symptoms of PD, including depletion of striatal dopamine (DA), loss of DA neurons of the substantia nigra pars compacta, and aggregation of alpha synuclein. These manifestations of parkinsonism were largely attenuated by administration of LIGA-20, a membrane permeable analog of GM1 that penetrates the blood brain barrier and enters living neurons. These results suggest that perturbation of intracellular mechanisms mediated by intracellular GM1 may be a contributing factor to PD. Keywords Parkinson's disease Á GM1 ganglioside Á LIGA-20 Á Dopaminergic neurons Á Alpha synuclein Abbreviations Ab antibody a-syn alpha-synuclein BSS balanced salt solution CtxB B subunit of cholera toxin DA dopamine or dopaminergic DOA days of age DOPAC dihydroxyphenyl acetic acid ER endoplasmic reticulum HRP horseradish peroxidase 5-HT 5-hydroxytryptamine (serotonin) 5-HIAA 5-hydroxyindole acetic acid IP intraperitoneally KO knockout MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine PBS phosphate buffered saline PD Parkinson's disease SNpc substantia nigra pars compacta TH tyrosine hydroxylase VTA ventral tegmental area WT wild type

C6 cells express a sodium-calcium exchanger/GM1 complex in the nuclear envelope but have no exchanger in the plasma membrane: Comparison to astrocytes

Journal of Neuroscience Research, 2004

Previous work demonstrated the presence of an isoform of Na(+)/Ca(2+) exchanger in the nuclear en... more Previous work demonstrated the presence of an isoform of Na(+)/Ca(2+) exchanger in the nuclear envelope of neurons and NG108-15 cells that is tightly associated with GM1 ganglioside and potentiated by the latter. This contrasted with the Na(+)/Ca(2+) exchanger(s) in the plasma membrane, which were suggested to associate more loosely with GM1. To study these aspects of Na(+)/Ca(2+) exchanger expression in nonneuronal neural cells, we have examined nuclear and plasma membrane exchanger patterns in astrocytes and C6 cells, a glia-derived line. We find both cell types contain the tightly associated exchanger/GM1 complex in the nuclear envelope but, surprisingly, only astrocytes possess Na(+)/Ca(2+) exchanger activity in the plasma membrane. This is the first reported example of a cell (C6) with Na(+)/Ca(2+) exchangers in the nuclear envelope but not in the plasma membrane. RT-PCR established the presence of the NCX1 subtype in C6 cells and both NCX1 and NCX2 in astrocytes. Comparison was made with NG108-15 cells, which have Na(+)/Ca(2+) exchangers in both nuclear and plasma membranes, and Jurkat cells, which have no Na(+)/Ca(2+) exchanger in either membrane. Culturing of C6 cells in the presence dibutyryl-cAMP caused upregulation of a high molecular weight isoform of the exchanger together with GM1 in the nuclear envelope, resulting in significant elevation of Na(+)/Ca(2+) exchanger activity in the latter. Application of exogenous GM1 to nuclei from non-treated cells also potentiated exchanger activity, although to a lesser degree. The Na(+)/Ca(2+) exchanger/GM1 complex occurs in the inner membrane of the nuclear envelope, suggesting a functional role in transferring Ca(2+) between nucleoplasm and the envelope lumen.