Investigating Immune Responses to the scAAV9-HEXM Gene Therapy Treatment in Tay–Sachs Disease and Sandhoff Disease Mouse Models

GM2 gangliosidosis disorders are a group of neurodegenerative diseases that result from a functional deficiency of the enzyme β-hexosaminidase A (HexA). HexA consists of an α- and β-subunit; a deficiency in either subunit results in Tay–Sachs Disease (TSD) or Sandhoff Disease (SD), respectively. Viral vector gene transfer is viewed as a potential method of treating these diseases. A recently constructed isoenzyme to HexA, called HexM, has the ability to effectively catabolize GM2 gangliosides in vivo. Previous gene transfer studies have revealed that the scAAV9-HEXM treatment can improve survival in the murine SD model. However, it is speculated that this treatment could elicit an immune response to the carrier capsid and “non-self”-expressed transgene. This study was designed to assess the immunocompetence of TSD and SD mice, and test the immune response to the scAAV9-HEXM gene transfer. HexM vector-treated mice developed a significant anti-HexM T cell response and antibody response. This study confirms that TSD and SD mouse models are immunocompetent, and that gene transfer expression can create an immune response in these mice. These mouse models could be utilized for investigating methods of mitigating immune responses to gene transfer-expressed “non-self” proteins, and potentially improve treatment efficacy.     

Plasma Membrane Calcium ATPase-Neuroplastin Complexes Are Selectively Stabilized in GM1-Containing Lipid Rafts

The recent identification of plasma membrane (Ca²⁺)-ATPase (PMCA)-Neuroplastin (Np) complexes has renewed attention on cell regulation of cytosolic calcium extrusion, which is of particular relevance in neurons. Here, we tested the hypothesis that PMCA-Neuroplastin complexes exist in specific ganglioside-containing rafts, which could affect calcium homeostasis. We analyzed the abundance of all four PMCA paralogs (PMCA1-4) and Neuroplastin isoforms (Np65 and Np55) in lipid rafts and bulk membrane fractions from GM2/GD2 synthase-deficient mouse brains. In these fractions, we found altered distribution of Np65/Np55 and selected PMCA isoforms, namely PMCA1 and 2. Cell surface staining and confocal microscopy identified GM1 as the main complex ganglioside co-localizing with Neuroplastin in cultured hippocampal neurons. Furthermore, blocking GM1 with a specific antibody resulted in delayed calcium restoration of electrically evoked calcium transients in the soma of hippocampal neurons. The content and composition of all ganglioside species were unchanged in Neuroplastin-deficient mouse brains. Therefore, we conclude that altered composition or disorganization of ganglioside-containing rafts results in changed regulation of calcium signals in neurons. We propose that GM1 could be a key sphingolipid for ensuring proper location of the PMCA-Neuroplastin complexes into rafts in order to participate in the regulation of neuronal calcium homeostasis.     

A comparative study of gangliosides from the brains of various species

A comparison is made of the sialic acid and hexose content of crude gangliosides from the brains of 13 species, including mammals, a bird, and amphibian, a reptile and fish. The sialic acid content is relatively constant from species to species. Gangliosides are less abundant in tissues other than the brain. Four species (cat, dog, pig and sheep) were selected for the determination of their major ganglioside subfractions. The ganglioside subfractions were isolated (after extraction and partition dialysis) using descending thin layer chromatography. The population of ganglioside molecules varied from species to species. From dog and sheep a mono-, a di- and a trisialoganglioside were obtained; from cat a mono-, a di- and two trisialogangliosides; and from pig a mono- and two disialogangliosides. Each ganglioside subfraction was found to contain glucose, galactose and galactosamine in the ratio of approximately 1∶2∶1. The fatty acid moieties consisted of more than 80% stearic acid with lesser amounts of arachidic, palmitic and behenic acid. Sphingosine analyses indicated ratios of sphingosine to icosisphingosine of 7∶3 for the monosialo-, 1∶1 for the disialo- and 3∶7 for the trisialogangliosides.     

Effect of prenatal and postnatal exposure to ethanol on rat central nervous system gangliosides and glycosidases

We investigated the effect of maternal alcohol consumption on cell number, gangliosides and ganglioside catabolizing enzymes in the central nervous system (CNS) of the offspring. Virgin female rats of the Charles Foster strain were given 15% (v/v) ethanol in drinking water one month prior to conception and during gestation and lactation. At 21 days postnatal age, the offspring were sacrificed and the brains were separated into cerebrum, cerebellum and brain stem to investigate possible regional variations. Compared to controls, wet weight of cerebrum, cerebellum and brain stem, and of spinal cord was decreased in the pups exposed to alcohol. DNA and protein contents were also found to be lowered in all the CNS regions of the pups exposed to alcohol. Conversely, maternal alcohol consumption was found to increase the concentration and the content of total gangliosideN-acetylneuraminic (NANA) in CNS of the pups. In addition, alcohol treatment was found to induce alterations in the proportions of individual ganglioside fractions. Interestingly, these alterations are somewhat different than those observed in the neonatal brain and spinal cord of the pups subjected to prenatal alcohol exposure. The alterations in the proportions of ganglioside fractions were shown to be region-specific. Maternal alcohol consumption resulted in decreased activities of sialidase, β-galactosidase, β-glucosidase and β-hexosaminidase. The results suggest that the alcohol-associated increases in ganglioside concentration may be at least partly due to the decreased activities of ganglioside catabolizing enzymes.     

Lactational changes in the fatty acid composition of human milk gangliosides

The objectives of this work were to study the FA composition of milk gangliosides, as well as to gain further insight into the characterization of human milk gangliosides. The potential capacity of human milk gangliosides to adhere to human enterotoxigenic Escherichia coli (ETEC-strains) was also studied. Human milk gangliosides were isolated and identified by high-performance TLC or immunoassay. The latter also was used to assay bacterial adhesion. The FA composition of gangliosides was studied by GC. The presence of O-acetyl GD3 (Neu5,9Ac₂α2–8 NeuAcα2–3Galβ1–4GlcCer) and trace amounts of GM1 [Galβ1–3GalNAcβ1,−3(NeuAcα2–3)Galβ1–4GlcCer] in human milk was confirmed. Medium-chain FA were almost absent in colostrum, whereas in the subsequent stages they rose to 20%. The levels of long-chain FA decreased after colostrum. With respect to the degree of saturation, gangliosides from colostrum were richer in monounsaturated FA than gangliosides synthesized during the rest of the lactation period, opposite to the pattern for PUFA. A human-ETEC colonization factor antigen II-expressing strain showed binding capacity to human milk GM3 (NeuAcα2–3Galβ1–4GlcCer). New data on human milk gangliosides have been gathered. A thorough knowledge of their composition is needed since they may have important biological implications in regard to newborns' defense against infection. The ganglioside nomenclature of Svennerholm (34) is followed.     

Isolation and determination of cholesterol glucuronide in human liver

Separation of the acidic lipid fraction from human liver led to the identification of cholesterol-β-glucuronide for the first time from this organ. Cholesterol glucuronide was purified by DEAE-Sephadex column chromatography and preparative silica gel thin-layer chromatography. The content in normal human liver was about 33 nmol/g wet tissue. It must be emphasized that cholesterol glucuronide cannot be distinguished readily from ganglioside GM4 by thin-layer chromatography.     

Lipid class composition of normal human brain and variations in metachromatic leucodystrophy,Tay-Sachs,Niemann-Pick,chronic gaucher’s and Alzheimer’s diseases

Procedures suitable for obtaining representative samples of whole brain and of total grey and white matter of brain are presented and discussed. A procedure is described for the quantitative determination of lipid class distribution of human brain specimens utilizing in sequence : a cellulose column to separate gangliosides and nonlipid material from the remaining lipids, diethylaminoethyl (DEAE) cellulose column chromatography to separate the lipid classes into manageable groups, and finally quantitation of the lipid classes by thin-layer chromatography (TLC). TLC is made quantitative by correlating the amt of charring of spots on chromatograms with the amt of lipid present by means of transmission densitometry. The use of two-dimensional TLC for the analysis of brain lipids and its application to the study of pathological brain specimens is also described. The application of these procedures to the study of metachromatic leucodystrophy, Tay-Sachs, Niemann-Pick, and Alzheimer’s diseases and senile cerebral cortical atrophy is described and data are presented. In two cases of Alzheimer’s disease, a large reduction in fresh weight and total lipid of brain were found; the lipid class distribution of whole brain in one case and of total grey and total white matter in another were essentially normal. The lipid class distributions of the brain in metachromatic leucodystrophy, Tay-Sachs disease, and Niemann-Pick disease were shown to be similar to that of normal infant brain except that one sphingolipid was greatly increased in each disease (sulfatide in metachromatic leucodystrophy, one ganglioside in Tay-Sachs disease, and sphingomyelin in Niemann-Pick disease).     

Analytical separation of nonlipid water soluble substances and gangliosides from other lipids by dextran gel column chromatography

A column chromatographic procedure is reported utilizing a dextran gel (Sephadex) for the complete separation of the major lipid classes from water-soluble nonlipids. Lipids other than gangliosides are eluted first with chloroform/methanol 19/1 saturated with water, gangliosides with chloroform/methanol/water containing acetic acid, and water-soluble nonlipids with methanol/water 1/1. Results for adult human whole brain, grey and white matter, and normal infant whole brain lipids are presented. With beef brain lipid as sample the ganglioside fraction is essentially pure, but with human brain lipid samples only about 70% of the second fraction is ganglioside. All ganglioside and water soluble nonlipid of a human spleen chloroform/methanol extract was separated from lipids with the procedure. Control studies with P³²O₄≡ and C₁₄ labelled glucose showed that all counts were present in fraction 3. Similar studies with C₁₄ labelled amino acids (glycine, serine, alanine, phenylalanine) showed that only phenylalanine counts were eluted in fraction 2 along with the gangliosides. The procedure was applied for removal of large amounts of ammonium acetate from DEAE cellulose column fractions and for complete removal of adsorbent and salts from lipids eluted from thin-layer chromatograms. After passage through the dextran gel columns, lipids eluted from thin-layer chromatograms were found to give infrared spectra identical to those of pure samples obtained by other procedures.     

GM3 Ganglioside Linked to Neurofibrillary Pathology in a Transgenic Rat Model for Tauopathy

Glycosphingolipids (GSLs) are amphipathic lipids composed of a sphingoid base and a fatty acyl attached to a saccharide moiety. GSLs play an important role in signal transduction, directing proteins within the membrane, cell recognition, and modulation of cell adhesion. Gangliosides and sulfatides belong to a group of acidic GSLs, and numerous studies report their involvement in neurodevelopment, aging, and neurodegeneration. In this study, we used an approach based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (HRMS/MS) to characterize the glycosphingolipid profile in rat brain tissue. Then, we screened characterized lipids aiming to identify changes in glycosphingolipid profiles in the normal aging process and tau pathology. Thorough screening of acidic glycosphingolipids in rat brain tissue revealed 117 ganglioside and 36 sulfatide species. Moreover, we found two ganglioside subclasses that were not previously characterized—GT1b-Ac2 and GQ1b-Ac2. The semi-targeted screening revealed significant changes in the levels of sulfatides and GM1a gangliosides during the aging process. In the transgenic SHR24 rat model for tauopathies, we found elevated levels of GM3 gangliosides which may indicate a higher rate of apoptotic processes.     

Modulation of antioxidant enzymes and programmed cell death by n-3 fatty acids

Studies from our laboratory indicate that n-3 (fish oil, FO) lipids at 10% (w/w) in a nutritionally adequate, semipurified diet, and supplemented with equal levels of antioxidants, extended the life span of lupus-prone (NZB/NZW)F₁ (B/W) female mice as compared to n-6 (corn oil, CO) lipids. The early rise of autoimmune disease in CO-fed mice was closely linked to the loss of T-cell function. Both IL-2 production and IL-2 receptor expression were reduced due to the loss of naive T-Cells and a rise in memory T-cells. Proliferative response to both mitogens and superatigens (staphylococcal enterotoxins A and B) was higher in FO-fed 6.5-mon-old mice. These changes paralleled decreased PGE₂ production by splenic cells from FO-fed mice. Analysis of mRNA expression in different organs revealed differential effects of dietary lipids. In FO-fed, mice, transforming growth factor β1 (TGF β1) expression was decreased in kidneys, but splenic tissues had higher expression of TGF β mRNA. As TGF β promotes programmed cell death (PCD), we studied the effects of CO and FO on PCD rates in lymphocytes. Both propidium iodide staining and DNA fragmentation were elevated in lymphocytes of FO-fed mice when compared to CO-fed mice of similar age. Also, increased PCD correlated closely with increased Fas gene expression. Thus, in addition to various other antiinflammatory effects, dietary FO appears to increase PCD and prevent accumulation of self-reactive immune cells in lymphoid organs. Further studies are required to dissect the proand antiinflammatory mechanisms associated with dietary n-3 and n-6 lipids in modulating autoimmune disorders or malignancy during aging.     

Investigation of the biogenetic reaction sequence of cholesterol in rat tissues,through inhibition with AY-9944

An inhibitor of Δ⁷-reductase, AY-9944 (trans-1,4-bis(2-dichlorobenzylaminomethyl cyclohexane dihydrochloride), was used to investigate the last steps of cholesterol formation in brain and liver of adult and newborn rats. The accumulation of different sterols in the two tissues of the same animals was observed. Δ^5,7-Cholestadien-3β-ol, Δ^7,24-cholestadien-3β-ol and Δ^5,7,24-cholestatrien-3β-ol, which are not present in detectable amounts in control brains, were identified in brains of growing rats treated with AY-9944. An accumulation of Δ^5,7-cholestadien-3β-ol only was found in adult rat tissues. These differences in sterol accumulation are discussed in relation with the possible in vivo pathways of cholesterol biosynthesis.     

Concentrated oat β-glucan,a fermentable fiber,lowers serum cholesterol in hypercholesterolemic adults in a randomized controlled trial

Background  

Soluble fibers lower serum lipids, but are difficult to incorporate into products acceptable to consumers. We investigated the physiological effects of a concentrated oat β-glucan on cardiovascular disease (CVD) endpoints in human subjects. We also compared the fermentability of concentrated oat β-glucan with inulin and guar gum in a model intestinal fermentation system.     

Maternal protein deficiency in rat: Effects on central nervous system gangliosides and their catabolizing enzymes in the offspring

Maternal protein deficiency imposed on rats a month prior to conception, and during gestation and lactation, resulted in a significant cell loss in cerebrum, cerebellum, brain stem and spinal cord of pups at weaning. The cerebellum was the most affected central nervous system (CNS) region; it contained only 25% of the normal cell number. Undernourished pups were also found to have a lower concentration of total gangliosides in cerebrum as compared to that of controls. However, the total ganglioside concentration was unaffected in the cerebellum, brain stem and spinal cord by maternal undernutrition. In all regions, undernutrition caused significant changes in the proportions of individual gangliosides; these alterations were region-specific. Sialidase, β-galactosidase, β-glucosidase, and β-hexosaminidase, which are involved in the catabolism of gangliosides, showed higher activities in all the regions of undernourished pups, suggesting that these enzymes may play a role in maintaining the porportions of various ganglioside fractions.     

Gangliosides in membranes fromTorpedo electric organ

The electric organ membrane has been the subject of many studies, due principally to its rich content of nicotinic acetylcholine receptor (AChR). Knowing its lipid composition is clearly important. Although its major membrane lipids have been characterized, its ganglioside composition has not been as well-described. In this study, gangliosides were characterized in membranes prepared from two species of electric organ,Torpedo californica andT. nobiliana. The ganglioside content of total electric organ membranes and AChR-enriched membranes was similar in both species, accounting for from 0.9 to 1.5% of membrane lipid by weight. However, the AChR-enriched membranes contained significantly less ganglioside relative to AChR than did the total membrane preparations. Five major gangliosides were purified fromT. californica and identified as II³NeuNAc-GgOse₃ (GM2); II³(NeuNAc)₂-GgOse₃ (GD2), IV³NeuNAc, II³NeuNAc-GgOse₄ (GD1a), IV³NeuNAc, II³(NeuNAc)₂−GgOse₅ (GT1b), and IV³(NeuNAc)₂, II³(NeuNAc)₂−GgOse₄ (GQ1b). Together these five gangliosides accounted for over 90% of the total ganglioside present in the two membrane preparations from both species. The most abundant ganglioside by far was GM2, which accounted for about one-half of the ganglioside content, followed by GD2. Determination of the N-fatty acid composition was performed on gangliosides purified fromT. nobiliana. The lower-order gangliosides, GM2, GD2, and GD1a, contained substantial amounts of very long chain fatty acids (>20 carbons), including α-hydroxynervonic acid (15–21% of total). In contrast, unsubstituted, 14–18 carbon chains accounted for about 90% of the fatty acids on the two higher-order gangliosides, GT1b and GQ1b.     

Ketone bodies serve as important precursors of brain lipids in the developing rat

Ketone bodies are readily oxidized for energy by extrahepatic tissues. Since oxidation of ketone bodies produces acetyl coenzyme A (AcCoA), and hence could be an important source of immediate precursors for fatty acid synthesis, we investigated, in whole-brain homogenates of developing rats, the preferential utilization of [3-¹⁴C]acetoacetate (AcAc), [3-¹⁴C]β-hydroxybutyrate (β-OHB), and [U-¹⁴C]glucose for production of CO₂ and lipids, including phospholipids, glycerides, cholesterol, and free fatty acids. Throughout the postnatal period, the rate of AcAc oxidation was 2–3 and 2–6 times the rate for β-OHB and glucose, respectively. The eynthesis of lipids from AcAc was 7- to 11-fold higher than from glucose. The brain’s capacity for lipid synthesis from β-OHB was similar to that from AcAc during the first 8 days of life; however, during the next 10 days, the synthesis of lipids from β-OHB decreased to 60% of AcAc-dependent synthesis. The high rate of lipid synthesis from ketone bodies was accompanied by increased activities of cytoplasmic acetoacetyl CoA synthetase and acetoacetyl CoA thiolase in the developing brain. During the entire postnatal development, the proportion of radioactivity claimed by lipids vs. CO₂ from [3-¹⁴C]AcAc was 44–62% vs. 38–56%; from [3-¹⁴C]β-OHB, 50–81% vs. 19–50%; and from [U-¹⁴C]glucose, 14–43% vs. 57–86%. Phospholipids accounted for more than two-thirds of total lipids synthesized from either ketone bodies or glucose, while diglycerides plus cholesterol and free fatty acids accounted for most of the remainder. Addition of glucose to the incubation medium did not alter lipid production from AcAc throughout the suckling period, but moderately depressed energy production in the brain of 16- to 20-day-old rats. It is clear that in cell-free preparations from the brain of developing rats, ketone bodies are preferred over glucose as precursors for both energy and lipids, mainly phospholipids. These results suggest that ketone bodies are important for the growth and development of the brain.     

Characterization of gangliosides of porcine erythrocyte membranes: Occurrence of ganglioside GD3 as major ganglioside

Four major ganglioside species were isolated from porcine erythrocyte membranes by DEAE-Sephadex and Iatrobeads column chromatography. Treatment of the lipids with graded neuraminidase and β-galactosidase, gas chromatographic analysis of their carboyhydrates, sphingosine bases and molecular species of sialic acid revealed that the structure of these gangliosides were G_M3(NeuAc), G_M3(NeuGc), G_D3(NeuAc) and G_D3(NeuGc), each of which was 16±2 μg, 304±42 μg, 30±3 μg and 240±26 μg, respectively, per gram of the dry erythrocyte stroma. The amount of G_M3 and G_D3 accounted for more than 95% of total gangliosides of the erythrocytes. Porcine erythrocytes may provide a good source for large scale preparation of ganglioside G_D3 which recently was identified as a human melanoma-associated antigen. Gangliosides are named according to Svennerholm (1) and the recommendation of the IUPAC-IUB Commission on Biochemical Nomenclature (2).     

A comparison of the ganglioside distributions of fat tissues in various animals by two-dimensional thin layer chromatography

The ganglioside distributions of various fat tissues from human, rabbit, rat, mouse, chicken and frog were compared with pig adipose gangliosides by two-dimensional thin layers chromatography. It was found that there is a remarkable species variation in ganglioside distribution, especially in the composition and relative concentration of complex gangliosides. Differing from pig adipose tissues, those of human, rabbit, rat, mouse, chicken, but not frog, contained GM3 as a most abundant ganglioside. The data for human, rabbit and chicken indicated a simple distribution of only NeuActype gangliosides, while those for rat and mouse indicated a rather complicated pattern containing both NeuAc- and NeuGc-type gangliosides. The ganglioside pattern of the frog fat body differed markedly from those of mammalian fat tissues because of the presence of three different, unsual monosialosylgangliosides as major components. In other respects, a substantial amount of disialosylgangliosides was commonly found in all animal fat tissues.     

Lipid Classes and Fatty Acid Patterns are Altered in the Brain of γ-Synuclein Null Mutant Mice

The well-documented link between α-synuclein and the pathology of common human neurodegenerative diseases has increased attention to the synuclein protein family. The involvement of α-synuclein in lipid metabolism in both normal and diseased nervous system has been shown by many research groups. However, the possible involvement of γ-synuclein, a closely-related member of the synuclein family, in these processes has hardly been addressed. In this study, the effect of γ-synuclein deficiency on the lipid composition and fatty acid patterns of individual lipids from two brain regions has been studied using a mouse model. The level of phosphatidylserine (PtdSer) was increased in the midbrain whereas no changes in the relative proportions of membrane polar lipids were observed in the cortex of γ-synuclein-deficient compared to wild-type (WT) mice. In addition, higher levels of docosahexaenoic acid were found in PtdSer and phosphatidylethanolamine (PtdEtn) from the cerebral cortex of γ-synuclein null mutant mice. These findings show that γ-synuclein deficiency leads to alterations in the lipid profile in brain tissues and suggest that this protein, like α-synuclein, might affect neuronal function via modulation of lipid metabolism.     

A rapid method for the preparation of ganglioside Glac2 (GD3)

A method is described for the preparation of ganglioside G_lac2 [(II³(NeuAc)₂-LacCer, GD3] from cream of bovine milk using liquid-phase extraction with methanol or ethanol followed by anion exchange chromatography. The method is rapid and inexpensive; 1 kg cream, centrifuged from 14–15 L of bovine milk, yields approximately 70 mg of pure ganglioside G_lac2. The sialic acid constituent of ganglioside G_lac2 isolated from bovine milk cream consists solely of theN-acetylneuraminic acid derivative. The major components of its ceramide consist of octadecasphing-4-enine and the 22∶0 (behenic acid) and 23∶0 fatty acids. Short hand notations for gangliosides are according to Wiegandt (Ref. 1). G_lac2, (II³(NeuAc)₂-LacCer) is NeuAcα,8NeuAcα,-3Galβ,4Glcβ,1Cer; G_tet1, (II³NeuAc-Gg₄Cer) is Gal\,3GalNAcβ,-4(NeuAcα,3)Galβ,4Glcβ,1Cer.