Gusheng Wu

Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase.

Despite its growing use as a radiological indicator of neuronal viability, the biological function of N‐acetylaspartate (NAA) has remained elusive. This is due in part to its unusual metabolic compartmentalization wherein the synthetic enzyme occurs in neuronal mitochondria whereas the principal metabolizing enzyme, N‐acetyl‐l‐aspartate amidohydrolase (aspartoacylase), is located primarily in white matter elements. This study demonstrates that within white matter, aspartoacylase is an integral component of the myelin sheath where it is ideally situated to produce acetyl groups for synthesis of myelin lipids. That it functions in this manner is suggested by the fact that myelin lipids of the rat optic system are well labeled following intraocular injection of [14C‐acetyl]NAA. This is attributed to uptake of radiolabeled NAA by retinal ganglion cells followed by axonal transport and transaxonal transfer of NAA into myelin, a membrane previously shown to contain many lipid synthesizing enzymes. This study identifies a group of myelin lipids that are so labeled by neuronal [14C]NAA, and demonstrates a different labeling pattern from that produced by neuronal [14C]acetate. High performance liquid chromatographic analysis of the deproteinated soluble materials from the optic system following intraocular injection of [14C]NAA revealed only the latter substance and no radiolabeled acetate, suggesting little or no hydrolysis of NAA within mature neurons of the optic system. These results suggest a rationale for the unusual compartmentalization of NAA metabolism and point to NAA as a neuronal constituent that is essential for the formation and/or maintenance of myelin. The relevance of these findings to Canavan disease is discussed.

Cross-Linking of GM1 Ganglioside by Galectin-1 Mediates Regulatory T Cell Activity Involving TRPC5 Channel Activation: Possible Role in Suppressing Experimental Autoimmune Encephalomyelitis

Several animal autoimmune disorders are suppressed by treatment with the GM1 cross-linking units of certain toxins such as B subunit of cholera toxin (CtxB). Due to the recent observation of GM1 being a binding partner for the endogenous lectin galectin-1 (Gal-1), which is known to ameliorate symptoms in certain animal models of autoimmune disorders, we tested the hypothesis that an operative Gal-1/GM1 interplay induces immunosuppression in a manner evidenced by both in vivo and in vitro systems. Our study of murine experimental autoimmune encephalomyelitis (EAE) indicated suppressive effects by both CtxB and Gal-1 and further highlighted the role of GM1 in demonstrating enhanced susceptibility to EAE in mice lacking this ganglioside. At the in vitro level, polyclonal activation of murine regulatory T (Treg) cells caused up-regulation of Gal-1 that was both cell bound and released to the medium. Similar activation of murine CD4+ and CD8+ effector T (Teff) cells resulted in significant elevation of GM1 and GD1a, the neuraminidase-reactive precursor to GM1. Activation of Teff cells also up-regulated TRPC5 channels which mediated Ca2+ influx upon GM1 cross-linking by Gal-1 or CtxB. This involved co-cross-linking of heterodimeric integrin due to close association of these α4β1 and α5β1 glycoproteins with GM1. Short hairpin RNA (shRNA) knockdown of TRPC5 in Teff cells blocked contact-dependent proliferation inhibition by Treg cells as well as Gal-1/CtxB-triggered Ca2+ influx. Our results thus indicate GM1 in Teff cells to be the primary target of Gal-1 expressed by Treg cells, the resulting co-cross-linking and TRPC5 channel activation contributing importantly to the mechanism of autoimmune suppression.

Induction of axon-like and dendrite-like processes in neuroblastoma cells

Neuroblastoma cells are widely utilized models for the study of the neuritic outgrowth phase of neuronal differentiation, but relatively few such studies have attempted to identify the nature of the process outgrowths. This identification will be necessary in developing strategies for utilizing these models to distinguish the underlying mechanisms involved in axonogenesis vs dendritogenesis. In an effort to identify procedures for inducing specific types of neurite outgrowth, and for distinguishing axon- from dendrite-like processes, we have subjected two neuroblastoma cell lines to a variety of stimuli previously shown to induce neurite outgrowth in these cells. These include neuraminidase, ionomycin, KCl+dibutyryl cAMP, cholera toxin B subunit, retinoic acid, dibutyryl cAMP (alone), GM1 ganglioside, and low serum. The first four of these (group 1) gave rise to neurites with axon-like characteristics, including immunostaining that was positive for phosphorylated high molecular weight neurofilament protein (NF-H) and synaptic vesicle protein-2 (SV2), but negative for microtubule-associated protein-2 (MAP2). The next three treatments (group 2) resulted in dendrite-like processes, as evidenced in immunostaining that was positive for MAP2 and negative for NF-H and SV2. Neurites produced by low serum had mixed properties. These cytoskeletal differences were supported by immunoblot analysis with antisera to the above cytoskeletal proteins. Striking morphological differences were also noted, group 2-induced neurites being significantly shorter with more branch points than those generated by group 1 stimulants. Time of exposure to stimulatory agent was crucial in determining expression of the neuritic phenotype. Correlation with previous studies suggests that axon-like neurites result from stimulants which elevate intracellular Ca2+, a dependence not previously reported to our knowledge. Dendrite-like process outgrowth, on the other hand, does not appear to depend on altered intracellular Ca2+.

Induction of Calcium Influx through TRPC5 Channels by Cross-Linking of GM1 Ganglioside Associated with α5β1 Integrin Initiates Neurite Outgrowth

Previous studies demonstrated that cross-linking of GM1 ganglioside with multivalent ligands, such as B subunit of cholera toxin (CtxB), induced Ca2+ influx through an unidentified, voltage-independent channel in several cell types. Application of CtxB to undifferentiated NG108-15 cells resulted in outgrowth of axon-like neurites in a Ca2+ influx-dependent manner. In this study, we demonstrate that CtxB-induced Ca2+ influx is mediated by TRPC5 channels, naturally expressed in these cells and primary neurons. Both Ca2+ influx and neurite induction were blocked by TRPC5 small interfering RNA (siRNA). Pretreatment of NG108-15 cells with neuraminidase increased cell-surface GM1 and greatly enhanced the signal. GM1 was not directly associated with TRPC5 but rather with α5β1 integrin, which opened the channel through a signaling sequence after cross-linking of the GM1/integrin complex. This cascade included autophosphorylation of focal adhesion kinase and subsequent activation of phospholipase Cγ (PLCγ) and phosphoinositide-3 kinase [PI(3)K]. Pharmacological blockers that inhibited tyrosine kinase, PLC, and PI(3)K suppressed both CtxB-induced Ca2+ influx and neurite outgrowth. These were also suppressed by SK&F96365, a nonspecific transient receptor potential channel blocker. Confocal immunocytochemistry revealed that GM1 cross-linking induced colocalization of GM1 with these signaling elements in sprouting regions of plasma membrane. In primary cerebellar granular neurons (CGNs), TRPC5 was detected at 2 d in vitro (2 DIV), a stage corresponding to CtxB-stimulated Ca2+ influx. Neurite outgrowth in CGNs, determined at 3 DIV, was accelerated by CtxB and suppressed by TRPC5 siRNA and the above blockers. The crucial role of GM1 was indicated with CGNs from ganglio-series null mice, in which growth of axons was significantly retarded.

Ganglioside function in calcium homeostasis and signaling.

Ganglioside function in eukaryotic cells encompasses a variety of modulatory interactions related to both development and mature cellular behavior. In relation to the nervous system this includes induction of neurite outgrowth and trophic/neuroprotective phenomena; more generally this applies to ganglioside effects on receptor function, adhesion reactions, and signal transduction mechanisms in neural and extraneural systems. Underlying many of these trophic effects are ganglioside-induced changes in cellular calcium, accomplished through modulation of Ca2+ influx channels, Ca2+ exchange proteins, and various Ca2+-dependent enzymes that are altered through association with gangliosides. A clear distinction needs to be drawn between intrinsic functions of gangliosides as naturally expressed by the cell and activities created by application of exogenous ganglioside(s) that may or may not reflect natural function. This review attempts to summarize findings in this area and point to possible future directions of research.

Potentiation of a sodium–calcium exchanger in the nuclear envelope by nuclear GM1 ganglioside

Calcium is recognized as an important intracellular messenger with a pivotal role in the regulation of many cytosolic and nuclear processes. Gangliosides of various types, especially GM1, are known to have a role in some aspects of Ca2+ regulation, operating through a variety of mechanisms that are gradually coming to light. The present study provides evidence for a sodium–calcium exchanger in the nuclear envelope of NG108‐15 neuroblastoma cells that is potently and specifically activated by GM1. Immunoblot analysis revealed an unusually tight association of GM1 with the exchanger in the nuclear envelope but not with that in the plasma membrane. Exchanger and associated GM1 were located in the inner membrane of the nuclear envelope, suggesting this system could function to transfer Ca2+ between nucleoplasm and the envelope lumen. The GM1‐enhanced exchange was blocked by cholera toxin B subunit while C2‐ceramide, a recently discovered inhibitor of the exchanger, blocked all transfer. Exchanger activity was significantly elevated in nuclei isolated from cells that were induced to differentiate by KCl + dibutyryl‐cAMP, a treatment previously shown to promote up‐regulation of nuclear GM1 in conjunction with axonogenesis. Similar enhancement was achieved by addition of exogenous GM1 to nuclei from undifferentiated cells. These results suggest a prominent role for nuclear GM1 in regulation of nuclear Ca2+ homeostasis.

Enhanced susceptibility to kainate-induced seizures, neuronal apoptosis, and death in mice lacking gangliotetraose gangliosides: protection with LIGA 20, a membrane-permeant analog of GM1.

Knock-out (KO) mice lacking gangliotetraose gangliosides attributable to disruption of the gene for GM2/GD2 synthase [GalNAcT (UDP-N-acetylgalactosamine:GM3/GD3 β-1,4-N-acetylgalactosaminyltransferase; EC 2.4.1.92)] are revealing key neural functions for the complex gangliosides of brain. This study has found such animals to be highly susceptible to kainic acid (KA)-induced seizures in terms of both seizure severity and duration. Intraperitoneal injection of 25 mg/kg KA produced status epilepticus for ∼200 min in normal mice or heterozygotes and more than four times longer in the KO mice. The latter group suffered ∼30% mortality, which increased to ∼75% at dosage of 30 mg/kg KA, compared with 10-14% for the other two genotypes at the latter dosage. Nissl staining and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assay revealed substantial deterioration of pyramidal neurons attributable to apoptosis in the KO hippocampus, especially the CA3 region. Seizure activity in the KO mouse was only moderately diminished by intraperitoneal injection of GM1 ganglioside, whereas LIGA 20, a semisynthetic analog of GM1, substantially reduced both seizure severity and cell damage. The potency of LIGA 20 was correlated with its enhanced membrane permeability (compared with GM1), as seen in the increased uptake of [3H]LIGA 20 into the subcellular fractions of brain including cell nuclei. The latter finding is consonant with LIGA 20-induced restoration of the Na+/Ca2+ exchanger located at the inner membrane of the nuclear envelope in KO mice, an exchanger dependent on tight association with GM1 or its analog for optimal activity. These results point to a neuroprotective role for GM1 and its associated exchanger in the nucleus, based on regulation of Ca2+ flux between nucleoplasm and nuclear envelope.

Nuclear sphingolipids: metabolism and signaling

Sphingolipids are most prominently expressed in the plasma membrane, but recent studies have pointed to important signaling and regulatory roles in the nucleus. The most abundant nuclear sphingolipid is sphingomyelin (SM), which occurs in the nuclear envelope (NE) as well as intranuclear sites. The major metabolic product of SM is ceramide, which is generated by nuclear sphingomyelinase and triggers apoptosis and other metabolic changes. Ceramide is further hydrolyzed to free fatty acid and sphingosine, the latter undergoing conversion to sphingosine phosphate by action of a specific nuclear kinase. Gangliosides are another type of sphingolipid found in the nucleus, members of the a-series of gangliotetraose gangliosides (GM1, GD1a) occurring in the NE and endonuclear compartments. GM1 in the inner membrane of the NE is tightly associated with a Na(+)/Ca(2+) exchanger whose activity it potentiates, thereby contributing to regulation of Ca(2+) homeostasis in the nucleus. This was shown to exert a cytoprotective role as absence or inactivation of this nuclear complex rendered cells vulnerable to apoptosis. This was demonstrated in the greatly enhanced kainite-induced seizure activity in knockout mice lacking gangliotetraose gangliosides. The pathology included apoptotic destruction of neurons in the CA3 region of the hippocampus. Ca(2+) homeostasis was restored in these animals with LIGA-20, a membrane-permeant derivative of GM1 that entered the NE and activated the nuclear Na(+)/Ca(2+) exchanger. Some evidence suggests the presence of uncharged glycosphingolipids in the nucleus.

The multi-tasked life of GM1 ganglioside, a true factotum of nature

GM1 ganglioside occurs widely in vertebrate tissues, where it exhibits many essential functions, both in the plasma membrane and intracellular loci. Its essentiality is revealed in the dire consequences resulting from genetic deletion. This derives from its key roles in several signalosome systems, characteristically located in membrane rafts, where it associates with specific proteins that have glycolipid-binding domains. Thus, GM1 interacts with proteins that modulate mechanisms such as ion transport, neuronal differentiation, G protein-coupled receptors (GPCRs), immune system reactivities, and neuroprotective signaling. The latter occurs through intimate association with neurotrophin receptors, which has relevance to the etiopathogenesis of neurodegenerative diseases and potential therapies. Here, we review the current state of knowledge of these GM1-associated mechanisms.

Chapter 7 Gangliosides as modulators of neuronal calcium

Publisher Summary This chapter describes some recent experiments of the authors laboratory that attempted to explore the connection between neuronal gangliosides and calcium flux. It is believed that these and related findings in other laboratories can explain at least some of the neuronotrophic properties of these molecules, including neuritogenic as well as neuroprotective effects. These studies entailed spectrofluorimetric measurement of Ca2+ during manipulation of endogenous or exogenous gangliosides as well as observation of resulting morphological changes. In an attempt to correlate the findings of many laboratories, the principal generality that emerges in regard to ganglioside function is that of protein modulation—encompassing a variety of enzymes, receptors, ion channels, and cell adhesion molecules residing in the plasma membrane. Additionally, the possibility of interaction with intracellular constituents must be considered in view of the occurrence of gangliosides and other glycosphingolipids in certain intracellular organelles.