S. Harth

Gangliosides in cultured neurons

Abstract Ganglioside patterns of separate cultures of neuronal and glial cells from 8-day-old chick embryo hemispheres were investigated. Significantly higher amounts of tri- and tetra-sialogangliosides and lower amounts of mono-sialogangliosides were found in neuronal cells. The increase of ganglioside levels and their patterns during maturation have been investigated in neurons. Two periods of ganglioside accumulation were observed: a first phase in which the ganglioside content increased slightly, corresponding to a phase of cell division, and a second period characterized by a marked increase in the amounts of gangliosides during the phase of cell maturation. During this latter period, the cells accumulated all usual central nervous system gangliosides (mono- to tetra-sialogangliosides, especially GD3, GD1a, GT1b, GQ1) and also gangliosides like GT1L and GP (probably pentasialoganglioside). During synapse formation (3rd–4th day) there was a decrease of GD3 and a high accretion of GD1a, which remained the major ganglioside species until the end of the neuronal culture (7 days). These striking variations are similar to those observed during in vivo brain ontogenesis and could be related to the passage of an embryonic to a mature cell.

Neuronal and Glial Cell Cultures, a Tool for Investigation of Ganglioside Function

Gangliosides are membrane constituents that are partly embedded in the bilayer structure of the membrane and partly exposed to the external environment by negative-charged polysaccharide chains. One may expect that they are involved in structural plasticity and in the functional activity of the plasma membrane. Great interest has been devoted to ganglioside structure and function during cell differentiation, maturation and ageing (see for review HAKOMORI, 1973). Changes associated with cell transformation, reduction of the levels of the most complex sphingolipids, a decrease in the activity of glycosyl- and/or sialyltransferases have raised the question of involvement of gangliosides in contact inhibition (HAKOMORI, 1973). Binding properties of gangliosides to exotoxins of Vibrio cholerae and Clostridium tetani drew attention to a function of sialoglycolipids as cell surface receptors and to their interactions with proteins and glycoproteins localized on cell surfaces or close to cell surfaces (SHAROM and GRANT, 1978; YAKAMAWA and NAGAI, 1978). Finally, an increasing interest in gangliosides of the nervous system arose when alterations of gangliosides in some genetic diseases was discovered (for review see SUZUKI, 1976) and when the abundance of gangliosides in plasma and synaptic membrane was established (MORGAN et al., 1971; LEDEEN, 1978). Moreover, attention became focused on the role of gangliosides in cation binding, transport and release (ABRAMSON, YU and ZABY, 1972; BEHR and LEHN, 1973; HAYASHI and KATAGIRI, 1974) and in neurotransmission (SVENNERHOLM, this book).

Effect of Exogenous Gangliosides on the Morphology and Biochemistry of Cultured Neurons

The role of sialoglycoconjugates in various processes essential for the life and development of the neuron is becoming more frequently apparent. In particular, gangliosides have been involved in a large variety of phenomena ranging from cell to cell recognition and adhesion,1,2 to differentiation 1,3 and from possible receptors for neurotransmitters and toxins1,4 to modulators of the movements of solutes across the nerve membranes.5

Biosynthesis of Galactocerebrosides and Glucocerebrosides in Glial Cell Lines

Abstract: UDP‐galactose:ceramide galactosyltransferase (CGalT, EC 2.4.1.45) and UDP‐glucose:ceramide glucosyltransferase (CGlcT, EC 2.4.1.80) were determined in the glial cell lines G26‐20, G26‐24, C6, and C6TK−. The enzymatic assay for CGalT in cultured glial cells was complicated by a rapid conversion of UDP‐galactose to UDP‐glucose, due to the elevated UDP‐galactose‐4′‐epi‐merase activity in certain glial cell clones. It seems that mechanisms regulating UDP‐galactose‐4′‐epimerase activity and levels of UDP sugars in the glial cell lines differ from those in brain tissue. Compared with the maximum activity of CGalT in the myelinating rat brain, the enzyme activities in the oligodendroglioma clonal cell lines G26–20 and G26–24 were 16–30 times lower. On the other hand, CGalT levels in G26‐20 and G26‐24 cells were comparable to the values found in young rat brain before myelination starts. No CGalT activity could be detected in C6 or C6TK− cells by the method used in this study, whereas CGlcT activity was found in all glial cell lines tested and its levels were close to the values observed in the young rat brain.

Ectoglycosyltransferase activities at the surface of cultured neurons

Glycosyltransferase activities (ectogalactosyl, ectofucosyl and ectosialyl) were studied at the external surface of exclusively neuronal cultures. An appropriate methodology gave the possibility to eliminate sources of errors due to the hydrolysis of nucleotide sugar substrates or due to cellular uptake of free sugars. Ovomucoid and asialofetuin coupled to Sepharose and Ultrogel beads were used as exogenous substrate to circumvent possible substrates pinocytosis. Ectoglycosyltransferase activities were studied as function of protein concentration, incubation time and amount of bead coupled exogenous acceptors. The data show that these enzymes are present at the external surface of the neuronal membrane; their possible role in cell - cell interactions is suggested.

Sialyltransferase Activities in Two Neuronal Models : Retina and Cultures of Isolated Neurons

Gangliosides have been found in various organs and tissues, however they are localized in a rather high amount and with a great diversity in the central nervous system (see for review LEDEEN and YU, 1976). The synthesis of gangliosides occurs by different pathways, requiring glycosyltransferases and sialyltransferases. Activities of these enzymes were studied both in non-nervous and in nervous tissues, with respect to their subcellular localization and kinetic properties. It was postulated that sialic acid residues on cell surfaces are involved in membrane-related cellular phenomena such as cellular recognition and adhesion, malignant transformation, contact inhibition and cellular migration (see for review SCHAUER, 1973). Further sialosyl groups seem to be involved in the receptor function of gangliosides (YAMAKAWA and NAGAI, 1978). Moreover, it was hypothesized that the formation of an enzyme-substrate complex between a glycosyltransferase from one cell and one acceptor from another could be responsible for cellular recognition (ROSEMAN, 1970).

Neuraminidase in Calf Retinal Outer Segment Membranes

Abstract: An enzyme catalyzing the hydrolysis of sialic acid (N‐acetylneuraminic acid: NeuNAc)‐containing glycoconjugates has been found in bovine retinal rod outer segment (ROS) membranes. The enzymatic activity is optimal at pH 4.0 and is stimulated by 0.15% Triton X‐100. Total activity was determined by the release of NeuNAc from endogenous and exogenous substrates (GDla). The ROS enzyme preferentially hydrolyses the ROS gangliosides, possibly because they are more accessible than the glycoproteins as substrates for the neuraminidase. Release of NeuNAc from gangliosides leads to important changes in the ganglioside patterns; whereas the amounts of GM1 increased throughout the incubation, the levels of polysialogangliosides GTlb and GD3 diminished owing to their rapid hydrolysis. The finding that gangliosides are hydrolysed more extensively than glycoproteins suggests that endogenous ROS gangliosides may be the principal source of metabolically available sialic acid in ROS. It was also observed that the activity of ROS neuraminidase is not affected by illumination of the membranes.

Lipids of Chick Retina during Ontogenesis

Biochemical studies of the ontogenesis of the brain encounter many difficulties due to morphologic and functional heterogeneity in the central nervous system (CNS) and due to differences in the developmental timing of the various brain regions when studying the whole brain. It is also difficult to establish which is the initial response to the factor under analysis and which are the secondary responses. The retina offers a rather simple developmental system which nevertheless is applicable to the comprehension of CNS. In the retina, the number of parameters is smaller, and it is possible to investigate specific structures with relatively little interference from neighbouring structures. The retina has a relatively simple morphology consisting roughly of alternating layers of cell bodies and synaptic regions, ordered from the scleral to the vitreal side as follows: photoreceptor region (outer and inner segment of rods and cones), outer synaptic layer (outer plexiform layer), region of horizontal, bipolar and amacrine cell bodies, zone of inner synapses (inner plexiform layer), ganglion cells. Non-neuronal cells, the so-called Muller cells, span the full thickness of the retina, and constitute the chief glial component of the retina. Microdissection of fresh or frozen tissue allows one to pool enriched fractions of a given cell type. Due to the dimensions of the whole intact retina, it has been termed an “instant” tissue slice which may be immersed in an appropriate medium, and maintained in physiological state by perfusion. The versatility of this isolated system allows one to study various parameters of metabolism, to analyse the bioelectrical response (electroretinogram: ERG) to its natural physiologic stimulus: light, under various adaptation conditions, and to investigate the influence of important biochemical compounds (putative neurotransmitters, their antagonists and agonists, as well as pharmacologic drugs) on the functional behavior of the retina. Such a perfusion system of an isolated retina allows one to correlate the effects due to compounds introduced in the perfusion medium, the electroretinogram modifications, and the biochemical changes at various levels in the layers of the tissue.

Effects of exogenous gangliosides on proliferation and maturation of neurons in primary cultures

The changes of some enzymes related to energy transductJon in rat brain subcellular fractions at 5, i0, 15, 20 and 30 days of postnatal development was investigated. The following enzyme activities in free and synaptic mitochondrJa obtained from cerebral hemispheres and cerebellum were evaluated: citrate synthase (EC 4.1.3.7); succ~nate dehydrogenase (EC 1.3.99.1); rotenone insensitive NADH-cytochrome c reductase (EC 1.6.99.3) cytochrome oxJdase (EC 1.9.3.1); glutamate dehydrogenase (EC 1.4.1.3). The activities of lactate dehydrogenase (EC 1.1.1.27), acetylcholJne esterase (EC 3.1.1.7) and cytochrome oxidase were measured on synaptosomes before lysis. The specific activities of c~trate synthase, glutamate dehydrogenase, cytochrome oxJdase Jn free mJtochondrJa increased during development. A different pattern of these enzymatic activities in fraction enriched Jn synaptic mJtochondrJa was observed.