James E. Goldman

GD3 ganglioside is a glycolipid characteristic of immature neuroectodermal cells

Biochemical studies have indicated that the disialoganglioside, GD3, is a major glycolipid component of the immature vertebrate CNS, but a minor element within the mature CNS. We have investigated its cellular localization in rat CNS by immunofluorescence using a mouse monoclonal antibody that recognizes GD3. In tissue sections of postnatal CNS, the antibody bound to cells in several areas known to contain immature neuroectodermal populations: the subventricular zone beneath the lateral ventricle, the external germinative layer of the cerebellar cortex, and the dentate gyrus of the hippocampus. GD3-containing cells were also found in developing white matter of the forebrain and cerebellar folia. Using a double-label immunofluorescence method, we found that the GD3-positive white matter cells did not express the astrocytic marker, glial fibrillary acidic protein. In adult rat CNS, we could not detect antibody binding to neurons or glia. Scattered GD3-positive cells were apparent in the adult subventricular zone. Our results indicate that GD3 ganglioside is a membrane component characteristically expressed in the rat CNS by neuroectodermal stem cells, both neuronal and glial precursors.

Synthesis and Turnover of Cytoskeletal Proteins in Cultured Astrocytes

Abstract: We previously reported that the cytoskeleton of rat astrocytes in primary culture contains vimentin, glial fibrillary acidic protein (GFAP), and actin. These proteins were found in a fraction insoluble in Triton X‐100 and thought to be assembled in filamentous structures. We now used primary astrocyte cultures to study the kinetics of synthesis and turnover of these cytoskeletal proteins. The intermediate filament proteins were among the most actively synthesized by astrocytes. High levels of synthesis were detectable by the third day of culture in the early log phase of growth, and the pattern of labeling at day 3 was similar to that at 14 days when the cultures had reached confluency. In short‐term incorporation experiments vimentin, GFAP, and actin in the Triton‐insoluble fraction were labeled within 5 min after exposure of the cultures to radioactive leucine. We did not detect any saturation of labeling for up to 6 h of incubation. The turnover of filament proteins studied by following the decay of radioactivity from prelabeled vimentin, GFAP, and cytoskeletal actin displayed biphasic decay kinetics for all three proteins. In the initial phase a fast‐decaying pool with a half‐life of 12–18 h contributed about 40% of the total activity in each protein. A major portion, about 60%, of each protein, however, decayed much more slowly, exhibiting a half‐life of about 8 days.

Dibutyryl cyclic AMP causes intermediate filament accumulation and actin reorganization in astrocytes

We have examined the effects of dibutyryl-cyclic AMP (dBcAMP) on the organization and expression of filamentous proteins in astroglia. The drug produced several effects on astrocytes grown in primary cultures. Cultures ceased to grow, and cells changed shape to a contracted form, displaying thin cytoplasmic processes. Cellular levels of the intermediate filament (IF) proteins, vimentin and glial fibrillary acidic protein (GFAP), and actin, insoluble in Triton X-100, were examined by polyacrylamide gel electrophoretic analysis. The cellular content of both of the IF proteins increased concurrently, approximately doubling during a 2-week course of treatment. The content of actin associated with the Triton residue decreased, however, a biochemical alteration which correlated with a loss of stress fibers in treated cells. Treatment with sodium butyrate did not change either cell shape or cytoskeletal protein content. Filament protein expression in astrocytes can, therefore, be modulated via cAMP-dependent mechanisms. The effects do not, however, appear specific for the GFAP-type of intermediate filament.

The association of actin with Hirano bodies.

Ultrastructural studies of the rod-shaped cosinophilic inclusions of the hippocampus (Hirano bodies) have demonstrated thin, filamentous components. The size of the filaments suggests that actin polymers (microfilaments) might form part of the Hirano body. To investigate this possibility, sections of human hippocampus and neocortex were stained, using a peroxidase-antiperoxidase (PAP) technique, with an antiserum against actin. Dense reaction product was seen over rod-shaped bodies, the location and morphology of which were typical of Hirano bodies. Immunocytochemical reactions on tissue previously stained with hematoxylin and eosin allowed a direct comparison between Hirano bodies, identified by their shape and eosinophilia, and the PAP reaction product. These results suggest an abnormal organization of a major cytoskeletal protein in hippocampal neurons, especially those of aged brains.

Regulation of glial fibrillary acidic protein (GFAP) expression in CNS development and in pathological states

Studies that have described changes in glial fibrillary acidic protein (GFAP) expression during CNS development and during pathological reactions are summarized. We describe our own studies examining GFAP expression in vitro. In primary cultures established from newborn rat forebrain and maintained in the presence of serum, GFAP expression is first observed in cells from the subventricular germinal zone. Glial precursor cells, at an uncommitted stage, can be induced by serum to begin GFAP synthesis. In astrocytes already established in culture, GFAP synthesis can be further increased by dibutyryl cyclic AMP (dbcAMP). By labelling astrocytes with radioactive methionine, we demonstrate increases of GFAP and vimentin synthesis within 48 h of exposure to dbcAMP. Continuous exposure results in a gradual rise in the cellular level of GFAP.

Immunocytochemical localization of GD3 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 GD3 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. hypothesized that the elevation in GD3 seen in these mutants is attributed to its expression by altered astrocytes. Using a monoclonal antibody to GD3 and a polyclonal antibody to GFAP we have explored the cellular localization of GD3. 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 GD3 are observed biochemically. The positive GD3 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 GD3 positive staining was seen in the Bergmann glia or astrocytes of the white matter. GD3-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 GD3 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.

The AB-variant of GM2-gangliosidosis

SummaryClinical, neuropathological, and biochemical studies are reported in two children with the aB-variant of GM2-gangliosidosis. One patient had become symptomatic by 1–1.5 years, initially showing cerebellar signs, and then progressive psychomotor retardation, with hypotonia, spasticity, dementia, and macular cherry red spots, until death at the age of 4.5 years. The second patient showed an earlier onset of retardation and a more rapidly progressive course. At postmortem, the brains were of normal or near normal weights and displayed grossly only mild cerebral cortical and cerebellar atrophy, and mild pallor or attenuation of the white matter. Neuronal storage was widespread throughout the CNS, and both neurons and glia contained a variety of abnormal, membranous inclusions. Visceral organs were not involved. Ganglioside sialic acid was increased several fold in gray matter, with GM2 the predominant ganglioside species. N-acetyl-β-glucosaminidase activities in serum, leukocytes, fibroblasts, and postmortem gray matter, assayed with an artificial, fluorogenic substrate, were normal, as were activities of other lysosomal hydrolases.

High molecular weight microtubule-associated proteins bind to actin lattices (Hirano bodies)

SummaryHirano bodies are filamentous, paracrystalline inclusions that are found in dendrites and cell bodies of neurons in Alzheimers and other neurodegenerative diseases. Actin appears to be a major component of these structures. We present evidence that tropomyosin and high molecular weight microtubule-associated proteins (MAPs) are also components of Hirano bodies. Although an association betwen actin and MAPs has been noted in vitro, interactions in vivo have not heretofore been demonstrated. Since microtubules are not present in Hirano bodies, and anti-tubulin and anti-neurofilament antibodies do not bind to Hirano bodies, the association between MAPs and these inclusions is likely a result of interactions between MAPs and actin, and not MAPs and microtubules or neurofilaments.

Rapid increases in glial fibrillary acidic protein mRNA and protein levels in the copper-deficient, brindled mouse

Abstract: The brindled mouse (Mobr/y) carries an X‐linked mutation that produces severe copper deficiency. Affected males suffer profound deficits in oxidative metabolism. We have examined astrocyte pathology in Mobr/y during development and have found marked changes in the metabolism of glial fibrillary acidic protein (GFAP). Immunocytochemistry with anti‐GFAP antisera revealed a marked increase in staining at postnatal day 12 (P12), compared to heterozygous female and unaffected male littermates, particularly in neocortex and thalamus. Septum, hypothalamus, and striatum showed little change. Western blot analysis revealed increased levels of GFAP in Mobr/y forebrain and cerebellum. Levels of GFAP mRNA were determined by Northern blotting with a mouse GFAP cDNA probe. At P10, mRNA levels were normal, but increased to 8–10 times normal by P12. Levels at P15 remained similarly elevated. Thus, immunostaining and protein determinations correlate with mRNA elevations. Astrocytes can alter GFAP mRNA and protein levels over a relatively short time. Counts of neocortical cells did not reveal differences in cell numbers between Mobr/y and controls, indicating that the observed changes reflect increased cellular levels and not a large increase in the numbers of astrocytes.

Granulofilamentous inclusions in a meningioma

We have studied the histology, immunocytochemistry, and ultrastructure of a syncytial meningioma which contained the unusual feature of large eosinophilic intracytoplasmic inclusions. Electron microscopic examination revealed that these bodies were granular, osmiophilic masses, closely associated with cytoplasmic filaments of intermediate size. Desmosomal junctions with a somewhat abnormal morphology were also observed, and the possible relationship between the inclusions and the dense components of desmosomes is discussed. This tumor is also compared to other tumors of the central nervous system with hyaline and granulofilamentous inclusions. Cancer 46:156–161, 1980.