Thomas N. Seyfried

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.

A Review of Mouse Mutants as Genetic Models of Epilepsy

Summary: Here we review the major inherited convulsive disorders found in mice and discuss their possible relationship to specific clinical seizure disorders in humans. These mouse disorders include audiogenic seizures, the epilepsy (El) mutation, spontaneous seizures, the tottering/learner syndrome, cerebellar abnormalities, myelin disorders, and alcohol withdrawal seizures. Some of these disorders are symptomatic and others are idiopathic. We find that for many major types of epilepsy in humans there exists a similar counterpart in mice. Because the genetic constitution of the mouse is better known and more easily manipulated than that of other mammalian species, the mouse may serve as an excellent animal model for genetic and biochemical studies of epilepsy.

Cerebral, cerebellar, and brain stem gangliosides in mice susceptible to audiogenic seizures.

Abstract— The quantitative and qualitative distribution of gangliosides was investigated in the cerebrum, cerebellum and brain stem of audiogenic seizure resistant (C57BL/6J) and susceptible (DBA/2J) mice at 21 days of age. The concentration of gangliosides (μg/unit weight) was higher in the DBA cerebrum and brain stem, but lower in the DBA cerebellum compared to the concentration in C57 mice. In general, the brain water content was lower in DBA mice than in C57 mice. The distributions of a number of gangliosides were found to be different between the two strains and the differences were often in the same direction across the three brain regions. The most consistant and significant difference in ganglioside pattern observed between the strains was the higher concentration of GM1 in all three regions of the DBA brain. These results suggest that DBA mice have a more heavily myelinated CNS than C57 mice. The relationship of these observations to inherent audiogenic seizure susceptibility is discussed.

Differential Cellular Enrichment of Gangliosides in the Mouse Cerebellum: Analysis Using Neurological Mutants

Abstract: The cellular distribution of gangliosides in the cerebellum was studied in a series of adult mouse mutants that lose specific populations of neurons. The weaver (wv) mutation destroys the vast majority of granule cells, whereas the Purkinje cell degeneration mutation (pcd) destroys the vast majority of Purkinje cells. The staggerer (sg) and lurcher (Lc) mutations, on the other hand, destroy the vast majority of both granule and Purkinje cells. A proliferation of reactive glial cells, which occurs as a consequence of neuronal loss, has been reported in the sg/sg and pcd/pcd mutants, but not in the wv/wv mutant. Compared with the normal (+/+) mice, the concentration (μg/100 mg dry weight) of GD1a was significantly reduced in those mutants that lost granule cells, but was not reduced in the pcd/pcd mutant. The concentration of GTIa, on the other hand, was significantly reduced in those mutants that lost Purkinje cells, but was not reduced in the wv/wv mutant. A significant elevation in the concentration of GD3, which may be related to the proliferation of reactive glial cells, was observed in the pcd/pcd, sglsg, and Lc/+ mutants, but was not observed in the wv/wv mutant. Because these ganglioside abnormalities were confined to the cerebellum, they cannot result from genetic defects in ganglioside metabolism. Instead, these abnormalities result from a differential enrichment of gangliosides in neural membranes. Our findings suggest that GDT1a is more heavily concentrated in granule cells than Purkinje cells, whereas the opposite appears true for GTla. It also appears that GD3 is enriched in reactive glial cells and may play an important role during the morphological transformation of neural membranes.

Spontaneous polyspike discharges in an epileptic mutant mouse (tottering)

Abstract Electroencephalographic (EEG) recordings were made from unanesthetized, freely moving tottering mice. This neurological mutant exhibits spontaneous motor seizures. Although the intention of the present study was to determine an electrographic correlate of the motor seizures, no epileptiform discharge was found which was reliably associated with this type of ictal event. The present study revealed the occurrence of a paroxysmal pattern which appeared independently of the motor seizures. The polyspike pattern was a 6 s burst resembling the 3 s spike-wave complex often recorded in human epileptics. The waking behavior associated with the polyspiking in the tottering mice consisted of immobility and staring. In addition, polyspike bursts occurred during drowsiness. It is possible that the cerebellar defects found in tottering mice are related to their gait disturbance and to their motor seizures. The polyspike discharge appears to represent an independent ictal event, suggesting that tottering mice are susceptible to both motor and “absence” seizures. Previously documented examples of naturally occurring epilepsy consisted primarily of stimulus-evoked or “reflex” seizures. Tottering mice show promise for research of spontaneous, recurrent paroxysmal activity in a hereditary type of epilepsy.

Cellular Localization of Gangliosides in the Developing Mouse Cerebellum: Analysis Using the Weaver Mutant

Abstract: The distribution of gangliosides was studied in the weaver (wv/wv) mutant mouse, where the vast majority of postmitotic granule cell neurons die prior to their differentiation. The wv mutation also shows a dosage effect, as granule cell migration is slowed or retarded in the +/wv heterozygotes. By correlating changes in ganglioside composition with the well‐documented histological events that occur during cerebellar development in the normal (+/+), heterozygous (+/wv), and weaver (wv/ wv) mutant mice, information was obtained on the cellular localization and function of gangliosides. Ganglioside GM1 may be enriched in granule cell growth cones and play an important role in neurite outgrowth. A striking accumulation of GM1, which may result from altered metabolism, occurred in the adult wvlwv mice. GD3 was heavily concentrated in undifferentiated granule cells, but was rapidly displaced by the more complex gangliosides during differentiation. GD1a became enriched in granule cells during formation of synaptic and dendritic membranes, whereas GT1a appeared enriched in Purkinje cell synaptic spines. A possible fucose‐containing ganglioside was quantitated only in the wvlwv mice. Ganglioside GT1b became enriched in granule cells during synaptogenesis, whereas GQ1b became enriched in these cells after synaptogenesis. The concentrations of GT1b and especially GQ1b increased continuously with age. Our results provide further evidence for a differential cellular enrichment of gangliosides in the mouse cerebellum and also suggest that certain gangliosides may be differentially distributed within the membranes of these cells at various stages of development.

Cellular Distribution of Gangliosides in the Developing Mouse Cerebellum: Analysis Using the Staggerer Mutant

Abstract: The distribution of cerebellar gangliosides was studied in staggerer (sg/sg) mutant mice, where the majority of granule cells die after completing their migration across the molecular layer. In addition, the external granule cell layer in (sg/sg) mice persists longer than in normal mice. Moreover, in the sg/sg cerebellum, Purkinje cells are significantly reduced in number, and almost none have tertiary branchlet spines. The loss of Purkinje cells and granule cells in sg/sg mice is accompanied by an early‐onset reactive gliosis that continues through adulthood. By correlating changes in ganglioside composition with the well‐documented histological events of cerebellar development in normal and sg/sg mice, we obtained strong evidence for a nonrandom cellular distribution of gangliosides. The sharpest reduction in the GD1a content of sg/sg cerebellum occurred after 15 days of age, coincident with granule cell loss. GT1a, on the other hand, was significantly reduced from 15 through 150 days in the sg/sg mice. GD3 is a major ganglioside of the undifferentiated granule cell, but it becomes rapidly displaced by the more complex gangliosides with the onset of granule cell maturation. In the sg/sg mice, GD3 persisted at abnormally high levels from 15 to 28 days and then accumulated through adulthood. These findings, and those from other cerebellar mouse mutants, suggest that GD1a is enriched in granule cells and that GT1a is enriched in Purkinje cells. Our findings also suggest that GT1a is more concentrated in branchlet spines than in other regions of the Purkinje cell membrane. GT1b appears to be enriched in both granule cells and Purkinje cells, whereas GM1 appears to be enriched in myelin. Furthermore, the apparent persistence of the embryonic ganglioside GD3 in sg/sg mice results from an early‐onset reactive gliosis, together with a partial retardation in granule cell maturation. The accumulation of GD3 beyond 28 days reflects the continued accretion of GD3 in reactive glia.

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.

Genetic variability for regional brain gangliosides in five strains of young mice

The quantitative and qualitative distributions of gangliosides were determined in the cerebrum, cerebellum, and brain stem of five inbred strains (C57BL/6J, DBA/2J, LG/J, C3H/HeJ, BALB/cJ) of mice at 21 days of age. Genetic differences were found among the strains for wet weight, absolute amount of gangliosides per region, and concentration of ganglioside (expressed on both a wet and a dry weight basis) in all three regions of the brain. The water content of the various brain regions showed the least amount of genetic variability. Coefficients of genetic determination were used to estimate the magnitude of genetic influence on these traits in each brain region. Significant differences were also found among the five strains for the distribution of certain gangliosides. The DBA strain, which is susceptible to audiogenic seizure at this age, had the highest level of the myelin-enriched ganglioside GM1 in all brain regions. Most of the genetic variation that influences the content and distribution of gangliosides among neurologically normal mice can be considered polygenic. Several possible sources of this genetic variation that may contribute to the differences observed among the strains are discussed.

Ganglioside abnormalities associated with failed neural differentiation in a T-locus mutant mouse embryo

The T-locus on mouse chromosome 17 contains a number of mutations that disrupt cellular differentiation and embryonic development. Because of their purported role in neuronal differentiation and brain development, gangliosides were studied in mouse embryos homozygous for two T-locus mutations: T and twl. Mice homozygous for the dominant T mutation die from failed mesodermal differentiation in the notochord, whereas mice homozygous for the recessive twl mutation die from failed neural differentiation in the ventral portion of the neural tube. No major ganglioside abnormalities were found in T/T mutant embryos at Embryonic Day 10 (E-10). In contrast, E-11 twl/twl mutants expressed a marked deficiency of the tetrasialoganglioside GQ1. Since this ganglioside migrates with GQ1b in three different thin-layer solvent systems, it may have the same structure as GQ1b. To gain insight into regional distribution, gangliosides were examined in head regions and body regions of normal (+/+) E-11 embryos. The ganglioside composition of these regions was the same as that of the whole embryo, with GM3 and GD3 comprising about 75% of the total ganglioside distribution. Moreover, N-acetylneuraminic acid was the only sialic acid species detectable in the E-10 and the E-11 embryos. These findings indicate that N-acetylneuraminic acid-containing gangliosides are synthesized actively in E-10 and E-11 mouse embryos and also suggest that the GQ1 deficiency in the twl/twl mutants is closely associated with failed neural differentiation.