Thomas N. Seyfried - US grants

gangliosides; gangliosidosis; lipid biosynthesis; high performance liquid chromatography; mutant; monoclonal antibody; gas chromatography;

The purpose of this research is to study the neurochemical, physiological and genetic mechanisms of convulsive behavior in two different types of seizure prone mice: those susceptible to audiogenic seizures and those susceptible to spontaneous temporal lobe seizures. A great deal of knowledge is surfacing regarding seizures and we are now better able to put this knowledge into a theoretical framework. It is now possible to study seizures using genetic approaches. The information gleaned from the projects to be undertaken will have great bearing on how epileptic brain activity is researched in the future.

The purpose of this research is to study the neurochemical, physiological and genetic mechanisms of epilepsy in two different genetic mouse models of epilepsy. These mice include the audiogenic seizure (AGS) susceptible DBA/2 (D2) mouse (a model for generalized absence seizures), and the E1 (epilepsy) mouse (a model for temporal lobe epilepsy). Previous evidence indicates that AGS susceptibility in D2 mice is genetically associated with a deficiency of a brain ecto-Ca2+-ATPase activity. High AGS susceptibility and low brain ecto-Ca2+- ATPase activity are inherited together in Fl hybrids produced from crossing various strains. The biochemical basis for this ecto-Ca2+. ATPase deficiency is presently unknown. To determine if the reduced activity in D2 mice results from a qualitative defect in the enzyme, kinetic properties of the ecto-Ca2+-ATPase will be studied in microsomes and synaptic plasma membranes of the D2 mice and the seizure resistant C57BL/6 (B6) and Fl hybrid mice. We suggest that the ecto-Ca2+. ATPase is essential for the rapid hydrolysis of released ATP and its deficiency in D2 mice contributes to seizure susceptibility by allowing ATP to remain active in the synaptic cleft. This hypothesis is supported from recent studies in B6 and D2 hippocampal slices and will be studied further in B6 x D2 recombinant inbred strains; a powerful analytical tool in mammalian genetics. Preliminary studies also indicate that the major gene for AGS, aspl, is linked to the Ah locus and to a novel restriction fragment length DNA polymorphism on chromosome 12. The relative location of these genes will be mapped using mendelian backcross generations. Since this is the first genetic linkage known between a DNA polymorphism and an epilepsy gene, insight can be obtained on the molecular genetics of epilepsy. An important problem in epilepsy research that has been difficult to resolve concerns the distinction between neurochemical defects associated with the cause of seizures and defects associated with the effects of repeated seizure activity. A paradigm has been developed in E1 mice to deal with this problem. Since our preliminary results show that E1 mice express a reactive gliosis in hippocampus, and elevated content of ganglioside GD3 in cerebellum, and elevated aspartic acid release in hippocampal slices, this paradigm will be used to determine if these defects are elevated aspartic acid release in hippocampal slices, this paradigm will be used to determine if these defects are associated with the cause or effect of seizures. Because the seizures in the D2 and E1 mice occur naturally, the neurochemical defects associated with these seizures will be relevant basic mechanism of epilepsy.

The goal of this research is to clarify the nature of ganglioside abnormalities in mutant mouse embryos. Gangliosides are a family of sialic acid-containing glycosphingolipids that are enriched in the central nervous system (CNS) and are thought to play an important role in neuronal differentiation. The tW1 mouse mutation is located within the T/t complex on chromosome 17 and causes embryonic lethality from failed neuronal differentiation within the neural tube. Recent findings indicate the tW1/tW1 embryos express reductions of gangliosides in the "b" metabolic pathway (GD3, GD1b, GT1b, and GQ1b) and elevations of ganglioside in the "a" metabolic pathway (GM3, GM2, GM1, and GD1a). This shift in ganglioside distribution strongly suggests that the mutants have a partial deficiency in the sialyltransferase activity that catalyzes the synthesis of GD3 from GM3: a key enzyme in the synthesis of the complex gangliosides. Moreover, gangliosides of the "b" pathway have been implicated in neuronal differentiation and neuritogenesis. This hypothesis will be tested through a developmental analysis of ganglioside composition and metabolism in normal and tW1/tW1 mutants. The gangliosides will be studied from embryonic day 13 (E-13) to E-17 in whole embryos and in embryonic CNS tissues. The composition of neutral glycolipids will also be examined. Total ganglioside content will be analyzed using gas-liquid chromatography and the individual ganglioside species will be analyzed using high performance thin-layer chromatography and densitometry. A specific objective will be to determine if the ganglioside abnormalities are a primary or secondary effect of the tW1 mutation. This will be approached through in vivo and in vitro studies of ganglioside biosynthesis in the +/+ and tW1/tW1 embryos and in the phenotypically normal +/tW1 heterozygotes. The sialyltransferase activities that catalyze the synthesis of GM3, GD3, GD1a, and GQ1b will be analyzed in enzyme enriched membrane preparations from the +/+, +/tW1, and tW1/tW1 embryos. The N- acetylgalactosaminyltransferase activity that catalyzes the synthesis of GM2 from GM3 (a key enzyme in the synthesis of "a" pathway gangliosides) will also be analyzed in the normal and mutant embryos. The activity of GM1 beta-galactosidase will also be studied using GM1 as a natural substrate in embryos of inbred DBA/2 and C57BL/6 mice and their F1 hybrids. Because DBA mice have elevated levels of GM1 at both embryonic and juvenile ages, they may express a mild form of GM1 gangliosidosis and serve as an important animal model for this disorder. Since this is the first study of ganglioside abnormalities associated with inherited embryonic lethality and failed CNS development in mammals, new insight can be obtained on ganglioside function and on the genetic regulation of ganglioside metabolism.

The long term objective of this research is to better define the genetic and neurochemical basis of seizure susceptibility in the epileptic EL mouse. The EL mouse is considered a genetic model for temporal lobe epilepsy. Although seizure susceptibility in EL mice is inherited as a multifactorial trait, most of the genetic variability for this susceptibility is regulated by the major El-1 gene. El-1 was recently mapped to the distal region of chromosome 9 between the dilute/short ear and B-galactosidase loci. A novel system of polymorphic DNA markers, called simple sequence repeat polymorphisms (SSLPs) will be used to more precisely map El-1 within this interval. These SSLPs are rapidly typed from DNA preparations using the polymerase chain reaction (PCR). El-1 mapping will be performed in unique interval congenic strains and in F2 and backcross generations from crosses of EL with the exotic wild mouse strain, CAST. The interval congenic strains are constructed to contain either the dominant El-1 seizure gene in nonseizure genetic backgrounds or the recessive normal gene in the EL seizure background. The interspecific cross with CAST will generate numerous DNA polymorphisms for precisely mapping El-1. The region of chromosome 9 containing El-1 is highly conserved with a region on human chromosome 3q containing the ceruloplasmin (Cp) and transferrin (Trf) genes. Recent preliminary results show the coinheritance of EL seizures with Cp and Trf polymorphisms. These polymorphisms can serve as new genetic markers for more precisely mapping El-1. Neurochemical studies will involve analysis of endogenous neurotransmitter release and GFAP content in hippocampal slices from seizure susceptible and resistant mouse strains. Recent findings suggest that enhanced aspartate release is genetically associated with EL seizures and that hippocampal GFAP content is associated with the effects, rather than cause, of seizure activity. Neurotransmitter release and GFAP content will be analyzed further using HPLC and Western blot/immunostaining, respectively. These proposed studies can provide a better understanding of basic epilepsy mechanisms in both the mouse and man.

DESCRIPTION: (adapted from Applicant's Abstract) The broad objective of this research is to characterize the composition of glycosphingolipids (gangliosides and neutral glycolipids) in macrophages that infiltrate experimental mouse brain tumors. Although glycosphingolipids (GSLs) were studied previously in peritoneal macrophages, there have been no previous studies in either man or mouse on the GSL composition of macrophages that infiltrate brain tumors. Our preliminary studies in mouse brain tumors suggest that host infiltrating macrophages contribute significantly to the total GSL composition of solid brain tumors growing in vivo. The proposed research will be conducted on two experimental mouse brain tumors, ependymoblastoma and CT-2A. The two tumors differ in growth behavior, ganglioside composition and in the number of infiltrating macrophages. Glass bead columns will be used to separate macrophages from tumor cells and non- macrophage host-infiltrating cells after enzymatic tumor dissociation. The proposed research will involve the following specific aims: 1) A comparative analysis of GSLs in infiltrating macrophages isolated from the two brain tumors growing in both the brain and subcutaneously in the flank of the C57BL/6 mouse; 2) A similar analysis in these tumors growing in the flank of the SCID (severe combined immune deficiency) mouse. This second aim will determine the influence of host genetic background on the GSLs of tumor-infiltrating macrophages. The qualitative and quantitative analysis of the GSLs will involve high performance thin-layer chromatography and gas-liquid chromatography, respectively. TLC immunostaining will be used to quanitate the macrophage enriched GSLs, asialo-GM1 (GA1) and GM1b. Although GSLs have been considered for the classification, diagnosis and therapy of brain tumors, it has not been established whether the GSLs are expressed in the neoplastic tumor cells or in tumor-infiltrating host cells. The proposed research can provide new insight on the cellular origin of brain tumor-associated GSLs and on the role of environmental factors in regulating the GSL composition of tumor-infiltrating macrophages.

DESCRIPTION (provided by applicant): The research goal is to develop an effective life-long therapy for ganglioside storage diseases. The gangliosidoses are a group of incurable autosomal recessive inborn errors of metabolism involving storage of either ganglioside GM1 or GM2 in CNS lysosomes. Accumulation of GM1 or GM2 causes wide spread inflammation and neurodegeneration. GM1 gangliosidosis arises from a genetic deficiency of the acid ¿-galactosidase that catabolizes ganglioside GM1, whereas Sandhoff disease (SD) arises from genetic deficiency in the ¿-hexosaminidase ¿ subunit that catabolizes ganglioside GM2. Our studies will involve diverse and complimentary approaches for disease management primarily involving substrate reduction therapy and gene therapy. The studies will be largely conducted in ¿-gal -/-, and Hex¿ -/- mice that accumulate GM1 and GM2, respectively. Inhibited synthesis counterbalances impaired rate of catabolism and is referred to as substrate reduction therapy. The imino sugars, NB-DNJ, and NB-DGJ, as well as the novel PDMP analogue 3h inhibit the rate of glycosphingolipid (GSL) biosynthesis. Our recent findings show that CNS delivery and therapeutic efficacy of NB-DNJ is significantly increased when the inhibitor is administered together with the restricted high-fat, low carbohydrate ketogenic diet (KD-R). Adeno-Associated Virus (AAV) gene therapy provides the missing lysosomal enzyme thereby reducing GSL storage throughout the CNS. The proposed studies will be an extension of those conducted over the previous funding period and will involve the following specific aims. Aim 1 will examine active and passive transport mechanisms by which the restricted ketogenic diet (KD-R) facilitates brain delivery of imino sugar and 3h to the CNS. Aim 2 will determine the degree to which the ketogenic diet can facilitate delivery of imino sugar and 3h to neonatal mouse brain through the dam's milk. Aim 3 will evaluate the degree to which AAV gene therapy corrects lipid abnormalities and inflammation in purified myelin, optic nerve, and retina in storage disease mice. Our preliminary studies show for the first time elevated levels of the unusual phospholipid, bis(monoacylglycerol)phosphate in the brains of human SD and in the ¿-gal -/-, and Hex¿ -/- mice. This lipid will be used as a novel biomarker for correction of ganglioside storage and brain inflammation. The proposed research will provide insight on novel therapeutic strategies for managing human ganglioside storage diseases.

DESCRIPTION (provided by applicant): The objective of this research is to define the role of glycosphingolipids in brain tumor growth and vascularity (i.e., angiogenesis). Gangliosides are sialic acid-containing glycolipids that are enriched in plasma membranes and are shed from tumor cells into the extracellular matrix. Gangliosides may influence brain tumor angiogenesis through multiple effects on the tumor cells and tumor associated host cells, e.g., endothelial cells and macrophages. Preliminary findings from this laboratory suggest that the simple monosialoganglioside GM3 is antiangiogenic and that more complex gangliosides (GM2, GM1, GDla and GTlb) are proangiogenic in experimental mouse brain tumors. We propose that altering tumor ganglioside composition will influence tumor growth and vascularity. We will test this hypothesis by manipulating the genes for GalNAc transferase (GalNac-T) and sialyltransferase 2 (SAT-2), two essential enzymes required for the synthesis of complex gangliosides. These genes will be up-regulated or down-regulated in stable transfectants of three well-established experimental brain tumors, i.e., the mouse EPEN and CT-2A tumors and the human glioma U87. These tumors are good models for the proposed studies because they differ in ganglioside composition, vascularity, and growth rate. Control untransfected or vector transfected tumors will be compared with their stable transfectants and will be studied as solid tumors grown in vivo and as cell lines grown in vitro. The degree of tumor angiogenesis in vivo will be assessed from analysis of microvessel density, tumor growth rate, vascular endothelial growth factor (VEGF) expression, and macrophage infiltration. The mechanisms by which gangliosides influence angiogenesis will be examined using cultured endothelial cells and the Matrigel plug model of angiogenesis. The proposed research will better define the relationship between gangliosides and tumor angiogenesis and can lead to new experimental strategies for managing brain tumors.