Distribution of glycosphingolipids in nervous tissue. Immunohistochemical and biochemical studies

Sammanfattning: Glycosphingolipids (GSLs) are highly enriched in cell membranes of the mammalian nervous system. Their biological functions are not clarified although recent findings have supported their existence in plasma membrane domains, enriched in cholesterol, sphingomyelin and GSLs. These membrane domains, also named lipid rafts, are thought to be involved in many cellular events, where modulation of functional membrane proteins (receptors, kinases, ion channels) and signal transduction are the most studied. Animals, devoid of certain GSLs, display severe neurological symptoms and disturbed metabolism of GSLs in humans cause diseases often associated with death during childhood, which illustrates the vital biological importance of GSLs. However, knowledge regarding cell-type- specific distributions of GSLs in the nervous system is limited and the lipid composition of rafts has mainly been studied in vitro while brain tissues are not well explored. The aim of this thesis was to investigate both the cellular and raft distribution of glycosphingolipids with special focus on gangliosides, i.e sialic acid-containing GSLs. Immunostainings were performed with monoclonal anti-GSL antibodies on rat tissue of both the central and peripheral nervous system (cerebellum and spinal nerves respectively). Biochemical analyses were performed on twenty specimens of human frontal and temporal cortex tissue and corresponding detergent-resistant membranes (DRMs), reflecting lipid rafts.Immunohistochemical analysis of spinal nerves focused on ganglioside GM1 and on sulfatide, both being antigens involved in human peripheral neuropathies. Immunostaining was conspicuous in the paranodal myelin, the site of myelin attachment onto the axon at the nodes of Ranvier. This indicates the importance of these antigens in axon-glia interactions, also supported by malformed paranodal regions in knockout mice lacking sulfatide and most gangliosides, including GM1. In addition, GM1 immunostaining was noticed in specific sites of Schwann-cell cytoplasms of rat peripheral nerve, the Schmidt-Lanterman incisures.Immunohistochemical analysis of rat cerebellum focused on gangliosides GM1 and GD1b, two of the dominating gangliosides in the mammalian brain, and on GD3, a major ganglioside in the immature brain that is also implicated in signal transduction. Sulfatide, a myelin associated GSL, was studied with special focus on the distribution in non-myelin- forming cells. Immunostaining of GM1 was observed in cells interpreted as stellate and basket cell neurons. GD1b was distributed in astrocytic Bergmann glia fibres and GD3 in Purkinje and granule cell neurons. In addition, GD1b and GD3 immunostaining was associated with synaptic sites. Sulfatide immunostaining was observed in some astrocytic golgi epithelial cells and in the granule cell neurons, providing support for a distribution of sulfatide in both astrocytes and neurons and not just selectively in myelin.Immunostaining was to some extent located in patches, possibly reflecting raft-association. The biochemical analyses of DRMs supported this reflection as only part of GSLs were recovered in rafts. Major brain gangliosides (GM1, GD1a, GD1b, GT1b) were raft associated to a similar extent (18-26 %). However, a lower proportion (9%) was found for ganglioside GM2, a minor GSL in the adult brain but important for dendritogenesis. Thus, all major brain gangliosides are found in rafts in similar proportions and the lower proportion of GM2 might reflect a pool of non-raft-associated gangliosides which might be associated during certain events such as dendritogenesis, i.e. during development and regeneration.These results demonstrate cell-type-specific, synaptic and raft-associated distributions for GSLs and present additional evidence for different cellular distributions in nervous tissue.