In Search of a Solution to the Sphinx-Like Riddle of GM1
- First Online:
- Cite this article as:
- Ledeen, R.W. & Wu, G. Neurochem Res (2010) 35: 1867. doi:10.1007/s11064-010-0286-0
Among the many glycoconjugates contributing to the sugar code, gangliosides have drawn special attention owing to their predominance as the major sialoglycoconjugate category within the nervous system. However, their occurrence, albeit at lower levels, appears ubiquitous in vertebrate cells and even some invertebrate tissues. Now that over 100 gangliosides have been structurally characterized, their diverse physiological functions constitute a remaining enigma. This has been especially true of GM1, for which a surprising array of functions has already been revealed. Our current research has focused on two areas of GM1 function: (a) signaling induced in neural and immune cells by cross-linking of GM1 in the plasma membrane that leads to activation of TRPC5 (transient receptor potiential, canonical form 5) channels, a process important in neuritogenesis and autoimmune suppression; (b) activation by GM1 of a sodium-calcium exchanger (NCX) in the inner membrane of the nuclear envelope (NE) with resulting modulation of nuclear and cellular calcium. The latter has a role in maintaining neuronal viability, loss of which renders neurons vulnerable to Ca2+ overload. Pathological manifestations in mutant mice and their cultured neurons lacking GM1 have shown dramatic rescue with a membrane permeable derivative of GM1 that enters the nucleus and restores NCX activity. Nuclear function of GM1 is related to the presence of neuraminidase in the NE, an enzyme that generates GM1 through hydrolysis of GD1a. A different isoform of this enzyme was found in each of the two membranes of the NE.
KeywordsGM1GangliosideNeuraminidaseCalcium regulationCross-linking of GM1TRPC5 channelsGM1 in the nuclear envelopeSodium-calcium exchanger
B subunit of cholera toxin
Effector T cell
Regulatory T cell
Transient receptor potential, isoform 5 of canonical subgroup
Acknowledging the special significance of the 35th anniversary of this journal is an occasion for reflection by those of us who were privileged to be active in the field at that time. It was a landmark event in that it contributed significantly to emergence of neurochemistry as a mature and recognized discipline. Neurochemical Research soon came to provide an important vehicle for publication of new findings in this burgeoning field, and will likely remain so in the future.
Whereas sphingosine posed an enigma to Thudichum in relation to isolation and characterization, gangliosides today present a somewhat different sphinx-like challenge to neurochemists in relation to physiological function. The efforts of many laboratories in recent years have registered significant progress toward unraveling that mystery. Our studies have focused on the function of GM1 and in recent years we have become impressed with the need to consider intracellular loci in addition to the plasma membrane to obtain a more comprehensive picture. The variety of roles revealed for GM1 has been noteworthy, prominent among these being Ca2+ regulation by a diversity of regulatory mechanisms [9, 10]. As outlined below, these pertain to such processes as neuronal differentiation and immune cell regulation.
GM1 and Neuronal Development
Studies on neuroblastoma cell differentiation, generally an induced process, revealed that elevation of cell surface GM1 could cause significant neurite outgrowth. This could be achieved by exogenous application of GM1 (or other gangliosides)  but in more robust manner by application of exogenous neuraminidase (N’ase), an enzyme that converts oligosialogangliosides to GM1 . Similar outgrowth was achieved by Miyagi and coworkers through upregulation of a ganglioside-specific N’ase in Neuro2a cells . This kind of induced differentiation was accompanied by Ca2+ influx and indeed depended on elevation of intracellular Ca2+ . A specific effect for GM1, as opposed to alteration of other sialoglycoconjugates by this enzyme, was suggested in the observation that both neuritogenesis and Ca2+ influx could be blocked by the B subunit of cholera toxin (CtxB) , a ligand of relatively high affinity and specificity for GM1. A point of some interest was that not all neuroblastoma cells behaved like Neuro2a, some experiencing enhanced Ca2+ influx and neuritogenesis when exposed to CtxB following N’ase treatment . Although such influx was initially suggested to occur via L-type voltage regulated Ca2+ channels, subsequent work revealed such channels to be voltage independent  and to possess the properties of TRPC5 , isoform 5 of the canonical subgroup of mammalian genes homologous to the transient receptor potencial family in Drosophila [17, 18].
GM1 Cross-Linking by Galectin-1 in Autoimmune Suppression
It has been recognized for some time that GM1 is present in cells of the immune system and is intimately involved in regulatory mechanisms, but mechanistic details have been lacking. Some aspects of such involvement are now coming to light with revelation of their role in Treg-Teff interaction . This relates to their presence in autoreactive T cells that escaped thymic deletion and that lead to autoimmune disorders when not effectively suppressed. Such suppression is known to depend to a large extent on the Treg population that constitutively expresses CD4, CD25, and the Foxp3 forkhead box transcription factor [22–24]. A characteristic of Tregs is dramatic elevation and release of Gal-1 upon T cell receptor activation [21, 24] and, as mentioned, the homodimeric property of Gal-1 enables it to induce cross-linking of GM1 in the membrane of Teffs. It is such GM1-Gal-1 interaction that suppresses Teff proliferation, such suppression being further enhanced by significant upregulation of GM1 in Teffs following T cell receptor activation [21, 25]. Galectin-1 is known to react with glycoproteins as well as GM1 in various cell types [26, 27], whereas our evidence indicated preferential interaction with GM1 in the case of Teffs .
In vivo support for the above mechanism involving GM1 cross-linking came in the demonstration that experimental autoimmune encephalitis was strongly inhibited by both CtxB and Gal-1 , confirming earlier studies (in the case of Gal-1) [28, 29]. This parallels similar findings with other autoimmune conditions such as type 1 diabetes, which was inhibited in the NOD mouse by both Gal-1  and CtxB . Those findings are consistent with the general phenomenon of immune modulation by the cholera-like enterotoxins . The proposed role for GM1 provides a rationale for its potency in suppressing disease onset in a variety of animal models of autoimmunity and explains why GM1-null mice proved highly susceptible to EAE . The latter study thus provided evidence suggesting a similar signaling sequence as revealed for neuroblastroma cells, based on intimate association of GM1 with α5β1 (as well as α4β1) integrin as prelude to TRPC5 Ca2+ channel activation.
Nuclear GM1 as Regulator of Calcium Homeostasis
Considering the plethora of information acquired over many years on the structure, metabolism and function of gangliosides in the plasma membrane, it is perhaps surprising that we are only at an early stage of similar understanding about gangliosides of the nucleus. Earlier studies indicated they are indeed present in this organelle, the nuclear envelope (NE) being the primary locus [33–35]. GM1 was clearly demonstrated at this site in our study employing CtxB linked to horseradish peroxidase as cytochemical indicator . Its presence in the nucleus of neural cells was confirmed and amplified in a developmental study of rat brain . Since the NE consists of a double membrane, we attempted a more specific localization utilizing a procedure involving mild treatment with sodium citrate solution to selectively remove and isolate the outer membrane . The inner membrane was obtained from the resulting nucleus remnant. Both membranes were found to contain GM1 along with its ganglioside precursor, GD1a (Fig. 1 for structure) as the principle gangliosides . A concurrent finding was that GM1 existed in tight association with a Na+/Ca2+ exchanger (NCX), a protein not previously reported as a NE constituent. Such tight binding was not seen between GM1 and plasma membrane NCX, although there was an indication of looser association. Further probing of this high affinity interaction revealed it as essential for efficient functioning of NCX .
Further localization of nuclear NCX, employing the above mentioned procedure , established the inner membrane of the NE as the locus of the NCX/GM1 complex. Thus it is strategically situated to mediate Ca2+ transfer from nucleoplasm to NE lumen, and such transfer was demonstrated in vitro with 45Ca2+ and isolated nuclei . Since Ca2+ transfer by this NCX is driven by a Na+ gradient, this required elevation of Na+ in the NE which was accomplished by pre-incubating the nuclei in Na+-containing medium in the presence of appropriate ionophores. Such Na+ transfer is believed to occur naturally by a Na+/K+-ATPase reported to occur in the inner membrane of the NE and to create high intra-lumenal Na+ concentration . Additional evidence for NCX-mediated Ca2+ transfer to the NE was obtained with living cells employing cameleon-fluorescent Ca2+ indicators genetically targeted to the NE/endoplasmic reticulum (ER) and nucleoplasm [41, 42]. Using various cell lines we demonstrated that cells containing both GM1 and NCX in the NE transferred Ca2+ readily from nucleoplasm to the NE and contiguous ER network, in contrast to cells lacking either molecule which transferred little or no Ca2+ . Because of facile Ca2+ movement from cytosol to nucleoplasm via nuclear pore complexes, those results suggested nuclear NCX/GM1 as alternative mechanism to the SERCA pump for cytosol to ER transfer of Ca2+. Several isoforms of NCX are known to exist [44, 45] and we have speculated that specific forms with greater abundance of positively-charged amino acids are the ones that predominate in the NE .
Immunoprecipitation of NCX indicated that in addition to GM1, GD1a also occurs in association, although this ganglioside did not potentiate the exchanger. We now have evidence that GD1a can serve as metabolic reserve for GM1 in the NE, undergoing conversion to the latter by N’ase present in the NE. This enzyme was originally detected in the intact NE with activity toward gangliosides , and was subsequently shown by our group to occur in both membranes of the NE . Immunohistochemical analysis of the separated membranes indicated occurrence of Neu3 in the inner- and Neu1 in the outer membrane.
Cytoprotective function of nuclear NCX/GM1 complex
Understanding the sugar code, purportedly more complex in some respects than the genetic code, is today a major undertaking in biology . GM1 is a miniscule part of nature’s carbohydrate complexity but is already known to mediate a surprising array of functional roles in numerous cell types. Analogous to the nuclear NCX/GM1 complex, GM1 in the plasma membrane binds tightly to the NGF receptor (Trk A) and regulates receptor function . Another plasma membrane role is that of opioid receptor modulator, GM1 effecting conversion from inhibitory to excitatory mode . Intracellular roles for GM1 are drawing more attention, as in the discovery of its association with α-synuclein that promotes α-helical structure and prevents pathological fibrillation , a hallmark of Parkinson’s disease. GM1 present in mitochondria-associated ER membranes was described as influencing Ca2+ flux between the ER and mitochondria , consistent with an earlier study showing GM1 inhibition of Ca2+-ATPase in sarcoplasmic reticulum membrane . Several other enzymes are known to be inhibited by GM1 but non-specifically and at relatively high concentrations, thus questioning physiological significance in those cases. GM1 is one of the very few sialoglycoconjugates in nature whose sialic acid is resistant to co-localized N’ase, thereby providing a mechanism for its elevation through hydrolysis of associated oligosialogangliosides (principally GD1a). The fact that this relatively small molecule can fulfill a large and growing list of regulatory functions poses a question as to its general attributes that facilitate such functional diversity. While much progress has been made, some miles would seem to remain on the road to more complete solution of this sphinx-like riddle.
Supported by grants from the NIH and NMSS.