Widespread correction of central nervous system disease after intracranial gene therapy in a feline model of Sandhoff disease.

Sandhoff disease (SD) is caused by deficiency of N-acetyl-ß-hexosaminidase (Hex) resulting in pathological accumulation of GM2 ganglioside in lysosomes of the central nervous system (CNS) and progressive neurodegeneration. Currently, there is no treatment for SD, which often results in death by the age of five years. Adeno-associated virus (AAV) gene therapy achieved global CNS Hex restoration and widespread normalization of storage in the SD mouse model. Using a similar treatment approach, we sought to translate the outcome in mice to the feline SD model as an important step toward human clinical trials. Sixteen weeks after four intracranial injections of AAVrh8 vectors, Hex activity was restored to above normal levels throughout the entire CNS and in cerebrospinal fluid, despite a humoral immune response to the vector. In accordance with significant normalization of a secondary lysosomal biomarker, ganglioside storage was substantially improved, but not completely cleared. At the study endpoint, 5-month-old AAV-treated SD cats had preserved neurological function and gait compared with untreated animals (humane endpoint, 4.4±0.6 months) demonstrating clinical benefit from AAV treatment. Translation of widespread biochemical disease correction from the mouse to the feline SD model provides optimism for treatment of the larger human CNS with minimal modification of approach.

Ketone supplementation decreases tumor cell viability and prolongs survival of mice with metastatic cancer.

Cancer cells express an abnormal metabolism characterized by increased glucose consumption owing to genetic mutations and mitochondrial dysfunction. Previous studies indicate that unlike healthy tissues, cancer cells are unable to effectively use ketone bodies for energy. Furthermore, ketones inhibit the proliferation and viability of cultured tumor cells. As the Warburg effect is especially prominent in metastatic cells, we hypothesized that dietary ketone supplementation would inhibit metastatic cancer progression in vivo. Proliferation and viability were measured in the highly metastatic VM-M3 cells cultured in the presence and absence of ß-hydroxybutyrate (ßHB). Adult male inbred VM mice were implanted subcutaneously with firefly luciferase-tagged syngeneic VM-M3 cells. Mice were fed a standard diet supplemented with either 1,3-butanediol (BD) or a ketone ester (KE), which are metabolized to the ketone bodies ßHB and acetoacetate. Tumor growth was monitored by in vivo bioluminescent imaging. Survival time, tumor growth rate, blood glucose, blood ßHB and body weight were measured throughout the survival study. Ketone supplementation decreased proliferation and viability of the VM-M3 cells grown in vitro, even in the presence of high glucose. Dietary ketone supplementation with BD and KE prolonged survival in VM-M3 mice with systemic metastatic cancer by 51 and 69%, respectively (p < 0.05). Ketone administration elicited anticancer effects in vitro and in vivo independent of glucose levels or calorie restriction. The use of supplemental ketone precursors as a cancer treatment should be further investigated in animal models to determine potential for future clinical use.

Ceruloplasmin gene defect associated with epilepsy in EL mice.

Epilepsy is a dominant trait in EL mice, a model for human complex partial seizures. We recently mapped the major gene, El-1, to chromosome 9 near the predicted location for the ceruloplasmin (Cp) gene. We now present evidence for a partial duplication in the Cp gene in EL mice. This Cp duplication is coinherited with seizures in backcross generations and is associated with enhanced expression of Cp mRNA and increased Cp oxidase activity. Moreover, the duplication is associated with an enhanced frequency of double recombinants, simulating negative interference. The findings are relevant to the basic mechanisms of epilepsy and to theories of genetic recombination and gene mapping.

Therapeutic effects of stem cells and substrate reduction in juvenile Sandhoff mice.

Sandhoff Disease (SD) involves the CNS accumulation of ganglioside GM2 and asialo-GM2 (GA2) due to inherited defects in the ß-subunit gene of ß-hexosaminidase A and B (Hexb gene). Substrate reduction therapy, utilizing imino sugar N-butyldeoxygalactonojirimycin (NB-DGJ), reduces ganglioside biosynthesis and levels of stored GM2 in SD mice. Intracranial transplantation of Neural Stem Cells (NSCs) can provide enzymatic cross correction, to help reduce ganglioside storage and extend life. Here we tested the effect of NSCs and NB-DGJ, alone and together, on brain ß-hexosaminidase activity, GM2, and GA2 content in juvenile SD mice. The SD mice received either cerebral NSC transplantation at post-natal day 0 (p-0), intraperitoneal injection of NB-DGJ (500 mg/kg/day) from p-9 to p-15, or received dual treatments. The brains were analyzed at p-15. ß-galactosidase staining confirmed engraftment of lacZ-expressing NSCs in the cerebral cortex. Compared to untreated and sham-treated SD controls, NSC treatment alone provided a slight increase in Hex activity and significantly decreased GA2 content. However, NSCs had no effect on GM2 content when analyzed at p-15. NB-DGJ alone had no effect on Hex activity, but significantly reduced GM2 and GA2 content. Hex activity was slightly elevated in the NSC + drug-treated mice. GM2 and GA2 content in the dual treated mice were similar to that of the NB-DGJ treated mice. These data indicate that NB-DGJ alone was more effective in targeting storage in juvenile SD mice than were NSCs alone. No additive or synergistic effect between NSC and drug was found in these juvenile SD mice.

Thyroid hormone influence on the susceptibility of mice to audiogenic seizures.

Serum thyroxine levels peak earlier and are significantly higher in audiogenic seizure-susceptible DBA/2J mice than in seizure-resistant C57BL/6J mice during early postnatal life. The seizure susceptibility of DBA/2J mice is suppressed by administration of an antithyroid drug or by radiothyroidectomy, while the seizure susceptibility of C57BL/6J mice is enhanced by treatment with excess thyroxine.

Genes for epilepsy mapped in the mouse.

The neurological mutant mouse strain E1 is a model for complex partial seizures in humans. The inheritance of epileptic seizures with seven conventional chromosomal markers and over 60 endogenous proviral markers was studied by means of back-crosses of E1 with two seizure-resistant strains, DBA/2J and ABP/LeJ. The major gene responsible for this epileptic phenotype (El-1) was localized to a region distal with respect to the centromere on chromosome 9. At least one other gene, El-2, linked to proviral markers on chromosome 2, also influences the seizure phenotype. In addition, a potential modifier of seizures was detected in the DBA/2J background. The location of El-1 on distal chromosome 9 may allow identification of an epilepsy candidate gene in humans on the basis of conserved synteny with human chromosome 3.

Complete correction of enzymatic deficiency and neurochemistry in the GM1-gangliosidosis mouse brain by neonatal adeno-associated virus-mediated gene delivery.

GM1-gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by autosomal recessive deficiency of lysosomal acid beta-galactosidase (betagal), and characterized by accumulation of GM1-ganglioside and GA1 in the brain. Here we examined the effect of neonatal intracerebroventricular (i.c.v.) injection of an adeno-associated virus (AAV) vector encoding mouse betagal on enzyme activity and brain GSL content in GM1-gangliosidosis (betagal(-/-)) mice. Histological analysis of betagal distribution in 3-month-old AAV-treated betagal(-/-) mice showed that enzyme was present at high levels throughout the brain. Biochemical quantification showed that betagal activity in AAV-treated brains was 7- to 65-fold higher than in wild-type controls and that brain GSL levels were normalized. Cerebrosides and sulfatides, which were reduced in untreated betagal(-/-) mice, were restored to normal levels by AAV treatment. In untreated betagal(-/-) brains, cholesterol was present at normal levels but showed abnormal cellular distribution consistent with endosomal/lysosomal localization. This feature was also corrected in AAV-treated mice. The biochemical and histological parameters analyzed in this study showed that normal brain neurochemistry was achieved in AAV-treated betagal(-/-) mice. Therefore we show for the first time that neonatal AAV-mediated gene delivery of lysosomal betagal to the brain may be an effective approach for treatment of GM1-gangliosidosis.

Sex-Related Abnormalities in Substantia Nigra Lipids in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative movement disorder involving the selective loss of dopamine-producing neurons in the substantia nigra (SN). Differences in disease presentation, prevalence, and age of onset have been reported between males and females with PD. The content and composition of the major glycosphingolipids, phospholipids, and cholesterol were evaluated in the SN from 12 PD subjects and in 18 age-matched, neurologically normal controls. Total SN ganglioside sialic acid content and water content (%) were significantly lower in the male PD subjects than in the male controls. The content of all major gangliosides were reduced in the male PD subjects to some degree, but the neuronal-enriched gangliosides, GD1a and GT1b, were most significantly reduced. The distribution of phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol was also significantly lower in the male PD subjects than in the male controls. However, the distribution of myelin-enriched cerebrosides and sulfatides was significantly higher in the male PD subjects than in the male controls suggesting myelin sparing in the male PD subjects. No elevation was detected for astrocytosis-linked GD3. These neurochemical changes provide evidence of selective neuronal loss in SN of the males with PD without robust astrocytosis. In contrast to the SN lipid abnormalities found in the male PD subjects, no significant abnormalities were found in the female PD subjects for SN water content or for any major SN lipids. These data indicate sex-related differences in SN lipid abnormalities in PD.

Genetic and environmental interactions determine seizure susceptibility in epileptic EL mice.

Gene identification has progressed rapidly for monogenic epilepsies, but complex gene-environmental interactions have hindered progress in gene identification for multifactorial epilepsies. We analyzed the role of environmental risk factors in the inheritance of multifactorial idiopathic generalized epilepsy in the EL mouse. Seizure susceptibility was evaluated in the EL (E) and seizure-resistant ABP/LeJ (A) parental mouse strains and in their AEF1 and AEF2 hybrid offspring using a handling-induced seizure test. The seizure test was administered in three environments (environments I, II and III) that differed with respect to the number of seizure tests administered (one test or four tests) and the age of the mice when tested (young or old). The inheritance of seizure susceptibility appeared dominant after repetitive seizure testing in young or old mice, but recessive after a single test in old mice. Heritability was high (0.67-0.77) in each environment. Significant quantitative trait loci (QTL) that were associated with environments I and III (repetitive testing) were found on chromosomes 2 and 9 and colocalized with previously mapped El2 and El4, respectively. The El2 QTL found in environment I associated only with female susceptibility. A novel QTL, El-N, for age-dependent predisposition to seizures was found on proximal chromosome 9 only in environment II. The findings indicate that environmental risk factors determine the genetic architecture of seizure susceptibility in EL mice and suggest that QTL for complex epilepsies should be defined in terms of the environment in which they are expressed.

Ganglioside composition of an experimental mouse brain tumor.

The ganglioside composition of an experimental ependymoblastoma was examined in C57BL/6 mice. This tumor was produced by Dr. H. Zimmerman in 1949 from methylcholanthrene implantation in the brain and has been maintained in serial transplants through many generations. The influence of tumor environment on ganglioside composition was determined by studying the tumor growing intracerebrally and s.c. (over the skull and in the flank). The ganglioside composition of this tumor is markedly different from that of adult mouse brain. The total ganglioside sialic acid content (micrograms/100 mg dry weight) of the tumor growing in the cerebrum, s.c. over the skull, and in the flank was 70.4 +/- 3.8 (N = 3), 66.8 (N = 2), and 41.7 +/- 0.7 (N = 3), respectively. These values are about 10-fold lower than the ganglioside content of normal mouse cerebrum. This tumor contained a significant amount of N-glycolyneuraminic acid (NGNA). Histological analysis revealed two basically different cell types. The predominant cell type is densely packed and poorly defined in shape, whereas the minor cell type is less densely packed and fibroblastlike in shape. GM3, which migrates as double bands on thin-layer chromatography, is the predominant ganglioside of this tumor in all three regions of growth. Also present in all regions are gangliosides NGNA-GM3 and GM1. Significant amounts of GD1a, GD1b, GT1b, and GQ1b are present only in the cerebral tumor. These gangliosides therefore represent contaminants from normal brain tissue surrounding the tumor and are not native to the tumor. Ganglioside GD3, however, is a minor component of the tumor. Using a thin-layer chromatography-immunostaining method with anti-GA1 antibody, we found significant amounts of ganglioside with a GA1 oligosaccharide backbone migrating near GD3 and GD2. This tumor is similar to other neural tumors in having elevated amounts of GM3 and reduced amounts of total ganglioside and polysialogangliosides but is unique in having a high content of NGNA-containing gangliosides. The possible origin of the NGNA-containing gangliosides is discussed.

Ganglioside biosynthetic gene expression in experimental mouse brain tumors.

The genes for cytidine monophospho-N-acetylneuraminic acid hydroxylase (NeuAc-H) and beta-1,4-N-acetylgalactosaminyl transferase (GalNAc-T) were examined using reverse transcription-PCR in two experimental mouse brain tumors, EPEN and CT-2A. NeuAc-H is required for the synthesis of gangliosides containing N-glycolylneuraminic acid, whereas GalNAc-T is required for the synthesis of ganglioside GM2. The genes were analyzed in solid tumors grown in vivo and in tumor cells grown in vitro. NeuGc-containing gangliosides are abundant in cells of the mouse immune system, including macrophages, but are undetectable in normal mouse brain. GM2 is expressed in both neural and nonneural mouse cells and tissues. The EPEN tumor cells synthesize only ganglioside GM3, whereas the CT-2A tumor cells synthesize GM3, GM2, GM1, and GD1a. NeuAc-H gene expression was detected in both solid tumors grown in vivo but was undetectable in either tumor cell line. The presence or absence of NeuAc-H gene expression in the tumor tissues and cells correlates with the presence or absence, respectively, of NeuGc-containing gangliosides. Differences in GalNAc-T gene expression between the solid tumors and the cultured tumor cells correlate with the expression of ganglioside GM2. The findings suggest that the differences in ganglioside biosynthetic gene expression between brain tumors grown in vivo and in vitro are associated with the presence or absence, respectively, of tumor-infiltrating host cells.

Gangliosides influence angiogenesis in an experimental mouse brain tumor.

Gangliosides are sialated glycosphingolipids present on the plasma membranes of all vertebrate cells. Tumors shed gangliosides into the extracellular microenvironment, which may influence tumor-host cell interactions. We have investigated the role of gangliosides on the growth and angiogenesis of the EPEN experimental mouse brain tumor. EPEN cells express only ganglioside G(M3), and the solid tumors formed in vivo are sparsely vascularized with extensive necrosis. We stably transfected the EPEN cells with the cDNA for N-acetylgalactosaminyl transferase, a key enzyme for the synthesis of complex gangliosides. In addition to G(M3), the transfected cell line (EPEN-GNT) expressed complex gangliosides G(M2), G(M1), and G(D1a). The EPEN-GNT tumor was more densely vascularized with less necrosis and grew more rapidly than the nontransfected EPEN or mock-transfected (EPEN-V) control tumors. Also, VEGF gene expression was higher in the EPEN-GNT tumor than in the control tumors. The synthesis of complex gangliosides in the EPEN-GNT tumor cells also stimulated vascularization in an in vivo Matrigel assay for angiogenesis. These results indicate that the ratio of G(M3) to complex gangliosides can influence the growth and angiogenic properties of the EPEN experimental brain tumor and are consistent with previous findings in other systems. We conclude that gangliosides may be important modulators of brain tumor angiogenesis.

Altered ganglioside composition in virally transformed rat embryo fibroblasts.

The composition of gangliosides was examined in a normal rat embryo fibroblast cell line (REF52) and in two viral transformants: a polyoma transformant (REF52-PyMLV) and a simian viral 40 transformant (REF52-SV40). The distribution of gangliosides in the cell lines was determined using gas-liquid chromatography and high-performance thin-layer chromatography. N-acetylneuraminic acid was the predominant sialic acid species detected in the three cell lines. The total ganglioside concentration (microgram/100 mg dry weight of cells) in the normal, PyMLV, and SV40 lines was 144.7 +/- 10.4, 153.8 +/- 9.2, and 86.1 +/- 6.8, respectively. Gangliosides GM3, GM2, GM1, and GD1a were the major species in the normal and transformed lines. The distribution of these gangliosides, however, differed markedly between the normal and the transformed lines and also between the transformed lines themselves. The transformed cells also differed from the normal cells in growth rate, morphology, and social behavior. The cell line with highest GM3 content (PyMLV) formed islands, whereas the normal and SV40 cell lines, which had lower GM3 levels, grew as monolayers. The findings suggest that PyMLV and SV40 transformation can have multiple and different effects on cellular ganglioside distribution and growth behavior.

Genetic linkage between the AH locus and a major gene that inhibits susceptibility to audiogenic seizures in mice.

Mice of the DBA/2 (D2) strain are highly susceptible to sound-induced seizures at 21 days of age; whereas, mice of the C57BL/6 (B6) strain are resistant to these seizures. Although the difference in susceptibility to audiogenic seizures (ASs) between these two strains is inherited as a multiple-factor trait, an association was observed between susceptibility to ASs and the Ah locus. The Ah locus controls the inducibility of aryl hydrocarbon hydroxylase (AHH) activity by a number of aromatic hydrocarbons. B6 mice carry the Ahb allele and have inducible AHH activity; whereas, D2 mice carry the Ahd allele and have noninducible activity. Inducibility is inherited as a Mendelian dominant trait in crosses between these strains. Mice carrying the Ahb allele are generally less susceptible to ASs sat 21 days of age than are mice carrying the Ahd allele. The combined results from B6 X D2 recombinant inbred strains, congenic strains (where the Ahb allele was placed into the D2 genome and the Ahd allele placed into the B6 genome), the B6D2F1 X D2 backcross generation, and a random survey of various inbred strains, suggest that the association between these two traits is due to genetic linkage, rather than to pleiotrophy or to chance. A major gene that inhibits susceptibility to ASs appears to be closely linked to the Ah locus. This gene has been designated Ias, for inhibition of ASs. A large portion of the genetic variability of AS susceptibility may be due to the segregation of Ias.

Genetic analysis of nucleotide triphosphatase activity in the mouse brain.

A Ca(2+)- or Mg(2+)-stimulated ecto-ATPase is thought to regulate the hydrolysis of extracellular ATP in nervous tissues. The hydrolysis of nucleotide triphosphates (NTPs) was analyzed in brain microsomal fractions from crosses of DBA/2J (D2) and C57BL/6J (B6) mice. The nucleotide triphosphatase (NTPase) activity was significantly reduced in D2 mice as compared to B6 mice, and B6D2F1 hybrids had activities intermediate to the parentals. A significant positive correlation was found between the hydrolysis of four NTPs (ATP, CTP, GTP and UTP) in 24 B6 x D2 (BXD) recombinant inbred (RI) strains of mice and in 80 B6D2F1 x D2 backcross mice. The RI strains and backcross mice fell into two distinct groups with respect to the NTPase activity. Linkage of NTPase activity was suggested with the chromosome 2 markers, D2Mit6 and Ass-1, in the RI strains, and was confirmed by analysis of other markers in the backcross population. These data suggest that the Ca(2+)- or Mg(2+)-stimulated hydrolysis of NTPs, designated Ntp, is regulated by a single gene located on proximal chromosome 2. Although an association was observed previously between Ca(2+)-ATPase activity and susceptibility to audiogenic seizures (AGS), no significant association was observed for the expression of Ntp and AGS susceptibility.

Genetic analysis of audiogenic seizure susceptibility in C57BL/6J X DBA/2J recombinant inbred strains of mice.

The inheritance of susceptibility to audiogenic seizures (ASs) was studied in the C57BL/6J (B6) and DBA/2J (D2) progenitor strains, their reciprocal F(1) hybrids, backcross generations and in 21 B6 x D2 recombinant inbred (RI) strains of mice at 21 days of age. All of the D2 mice tested experienced ASs, whereas none of the B6 mice responded to the sound. Although 23% of the F(1) mice experienced wild running, they were generally as resistant to ASs as their B6 parents. Mice of the F(1)x B6 backcross generation were also resistant to ASs. In the F(1)x D2 backcross generation, however, a significant preponderance (72%) of AS-susceptible mice was found. No significant association was observed between any of the four coat-color phenotypes that were segregating in this generation and susceptibility to ASs. A continuous distribution of mean seizure severity scores and several new audiogenic response phenotypes, distinctly different from the phenotypes of either progenitor strain, were found among the 21 RI strains. These and the results from the F(1)x D2 backcross generation suggest that the difference in AS susceptibility between 21-day-old B6 and D2 mice cannot be under the control of a single locus. In addition, no association was found between AS susceptibility and the chromosome 4 markers Lyb-2, Mup-1 and b among the 21 RI strains. An association was observed, however, between AS susceptibility and the Ah locus. Several of the RI strains that were AS resistant at 21 days of age became AS susceptible as adults. One RI strain was susceptible to ASs at both young and adult ages. The B6, D2 and F(1) mice were completely resistant to ASs at adult ages. Genetic differences were found among the RI strains for the incidence, onset, duration, and type of severity of ASs. A remarkable amount of phenotypic variability in the audiogenic response, which can be attributed only to the influence of environmental factors, occurred within several of the RI strains. A multiple-factor mode of inheritance involving a physiological threshold can account for our observations.

Non-Toxic Metabolic Management of Metastatic Cancer in VM Mice: Novel Combination of Ketogenic Diet, Ketone Supplementation, and Hyperbaric Oxygen Therapy.

The Warburg effect and tumor hypoxia underlie a unique cancer metabolic phenotype characterized by glucose dependency and aerobic fermentation. We previously showed that two non-toxic metabolic therapies - the ketogenic diet with concurrent hyperbaric oxygen (KD+HBOT) and dietary ketone supplementation - could increase survival time in the VM-M3 mouse model of metastatic cancer. We hypothesized that combining these therapies could provide an even greater therapeutic benefit in this model. Mice receiving the combination therapy demonstrated a marked reduction in tumor growth rate and metastatic spread, and lived twice as long as control animals. To further understand the effects of these metabolic therapies, we characterized the effects of high glucose (control), low glucose (LG), ketone supplementation (ßHB), hyperbaric oxygen (HBOT), or combination therapy (LG+ßHB+HBOT) on VM-M3 cells. Individually and combined, these metabolic therapies significantly decreased VM-M3 cell proliferation and viability. HBOT, alone or in combination with LG and ßHB, increased ROS production in VM-M3 cells. This study strongly supports further investigation into this metabolic therapy as a potential non-toxic treatment for late-stage metastatic cancers.

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.

Genes differentially expressed in the kindled mouse brain.

Kindling involves long-term changes in brain excitability and is considered a model of epilepsy and neuroplasticity. Differentially expressed genes in the kindled mouse brain were screened using an reverse transcription-polymerase chain reaction (RT-PCR) differential display (DD) method. C3H male mice were kindled with 40 stimuli in the hippocampus at 5-min intervals. Hippocampal RNA was isolated for DD from mice at 0.5 h, 1 day, 1 week, and 1 month after kindling and from sham-operated controls. About 30,000 bands were screened and of these, 50 were displayed differentially. Northern blot analysis confirmed that 26 of the 50 bands were differentially expressed following rapid kindling. Further sequence analysis revealed that 14 of the genes were previously identified and 12 were novel. The novel genes are referred to as King (1-12) genes because of their association with kindling. According to their temporal and quantitative pattern of expression in forebrain, the 26 genes were grouped into five types. Expression of five of the DD genes, one from each expression type, was further analyzed in hippocampus, forebrain, brainstem, and cerebellum of the kindled mice. Differential expression of these genes was observed in hippocampus and forebrain, but not in brainstem or cerebellum. Only one gene, a regulator of G-protein signaling 4 (RGS4), showed prolonged changes in expression in response to kindling. Our results show that rapid kindling produces spatial and temporal changes in gene expression that may influence kindling-associated neuroplasticity.