Immunocytochemical localization of G_D3 ganglioside to astrocytes in murine cerebellar mutants

Biochemical analysis of the murine mutants, Purkinje cell degeneration (pcd/pcd), staggerer (sg/sg) and lurcher (Lc/+), which are characterized by neuronal degeneration in the cerebellar cortex, have revealed substantially elevated levels of G_D3 ganglioside (ceramide-Glu-Gal-NANA-NANA). Ultrastructural studies on pcd/pcd and sg/sg have shown astrocytes elaborating slender sheet-like processes which wrap around neuronal processes. Seyfried et al.²³ hypothesized that the elevation in G_D3 seen in these mutants is attributed to its expression by altered astrocytes. Using a monoclonal antibody to G_D3 and a polyclonal antibody to GFAP we have explored the cellular localization of G_D3. Positive immunofluorescence was observed in sg/sg, pcd/pcd and Lc/+ cerebella, but not in age-matched normal littermates or in weaver (wv/wv) a fourth cerebellar murine mutation which destroys granule cells prior to their migration across the molecular layer. In wv/wv cerebella, astrocytes do not elaborate sheets of processes and no significant elevations of G_D3 are observed biochemically. The positive G_D3 staining in pcd/pcd and Lc/+ was confined to the granule cell layer and appeared as many punctate or short, fine profiles, suggestive of binding to thin cytoplasmic processes. No G_D3 positive staining was seen in the Bergmann glia or astrocytes of the white matter. G_D3-positive staining was seen throughout the cortex in sg/sg which displayed severe disruption of its histoarchitecture with no clear delineation between the molecular and granule cell layers. Ultrastructural localization of G_D3 was performed using pre-embedding immunocytochemistry with a PAP technique in sg/sg mice. The cytoplasmic processes and cell bodies of astrocytes displayed positive membrane staining. Our results suggest that astrocytes undergo important changes in membrane composition during pathological reaction caused by neuronal degeneration.