Abstract
Sulfoglucuronyl carbohydrate (SGC) linked to the terminal moiety of neolacto-oligosaccharides is expressed in several glycoproteins of the immunoglobulin superfamily involved in neural cell-cell recognition as well as in two sulfoglucuronylglycolipids (SGGLs) of the nervous system. SGGLs and SGC-containing glycoproteins are temporally and spatially regulated during development of the nervous system. In the cerebellum, the expression of SGC, particularly that of SGGLs, is biphasic. Several studies have suggested that the initial rise and decline in the levels of SGGLs and SGC-containing proteins correlated with the migration of granule neurons from the external granule cell layer to the internal granule cell layer and their subsequent maturation, whereas the later rise and continued expression of SGGLs in the adult was associated with their localization in the Purkinje neurons and their dendrites in the molecular layer. Here it is shown by immunocytochemical methods that the expression of SGC declined progressively in granule neurons isolated from cerebella of increasing age. The decline in the expression of SGC in granule neurons was also shown with time in culture. These results correlated with the previously shown declining activity of the regulatory enzyme lactosylceramide N-acetylglucosaminyltransferase (GlcNAc-Tr) with age in vivo and in isolated granule neurons in culture. GlcNAc-Tr synthesizes a key precursor, lactotriosylceramide, involved in the biosynthesis of SGGL-1. The down-regulated synthesis of SGGLs in the mature granule neurons was shown by immunocytochemical and biochemical methods to be restored when a precursor, glucuronylneolactotetraosylceramide (GGL-1), which is beyond the GlcNAc-Tr step, was exogenously provided to these cells. The biological effect of such restoration of the synthesis of SGGLs in the mature granule neurons leads to cell aggregation and enhanced proliferation of neurites, amounting to dedifferentiation.
Highlights
Sulfoglucuronyl carbohydrate (SGC)1 is expressed in several glycoproteins of the immunoglobulin superfamily, such as neural-cell adhesion molecules, L1, J1, cytotactin, TAG-1, myelinassociated glycoprotein, and P0, involved in neural cell recognition, and in two neolactoglycolipids
Biochemical [15] and immunocytochemical (16 –18) analyses and studies with murine cerebellar mutants (19 –21) with a characteristic loss of specific cell types have suggested that the initial decline in the levels of SGGLs correlated with the migration of immature granule neurons from the germinal zone in the external granule cell layer (EGCL) to the internal granule cell layer (IGCL), guided by Bergmann glial fibers
The activity of GlcNAc-Tr was down-regulated from a maximum at embryonic day 15 to an undetectable level by PD10, whereas the other enzymes involved in the subsequent steps for the synthesis of SGGLs were not as severely affected [1, 22,23,24,25,26]
Summary
Sprague-Dawley albino rats were purchased from Zivic-Miller Laboratories, Inc. (Pittsburgh, PA). Tissue culture media and substrates were from Life Technologies, Inc. All fine chemicals were purchased from Sigma or Fisher. The hybridoma clone producing the HNK-1 IgM monoclonal antibody was from American Type Culture Collection (Rockville, MD). Anti-glial fibrillary acidic protein monoclonal antibodies and FITC-conjugated affinity-purified IgG secondary antibodies were from Boehringer Mannheim. Antibodies to microtubule-associated proteins (MAPs) raised in rabbit were from Sigma. Rhodamine-conjugated goat anti-mouse IgM secondary antibodies were from Cappel (Westchester, PA). The dilutions of the antibodies used were based upon the original hybridoma supernatant, serum from animals, or manufacturers’ supplied samples. The supernatant of the HNK-1 hybridoma clone was mixed with an equal volume of EZ-SepA solution (Amersham Pharmacia Biotech) at pH 7.5 at room temperature. After 30 min, the precipitate was collected by centrifugation as partially purified IgM protein
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