Dramatically different phenotypes in mouse models of human Tay-Sachs and Sandhoff diseases

We have generated mouse models of human Tay-Sachs and Sandhoff diseases by targeted disruption of the Hexa (alpha subunit) or Hexb (beta subunit) genes, respectively, encoding lysosomal beta-hexosaminidase A (structure, alpha) and B (structure, beta beta). Both mutant mice accumulate GM2 ganglioside in brain, much more so in Hexb -/- mice, and the latter also accumulate glycolipid GA2. Hexa -/- mice suffer no obvious behavioral or neurological deficit, while Hexb -/- mice develop a fatal neurodegenerative disease, with spasticity, muscle weakness, rigidity, tremor and ataxia. The Hexb -/- but not the Hexa -/- mice have massive depletion of spinal cord axons as an apparent consequence of neuronal storage of GM2. We propose that Hexa -/- mice escape disease through partial catabolism of accumulated GM2 via GA2 (asialo- GM2) through the combined action of sialidase and beta-hexosaminidase B.      

Molecular pathophysiology in Tay-Sachs and Sandhoff diseases as revealed by gene expression profiling

Tay-Sachs and Sandhoff diseases are lysosomal storage disorders characterized by the absence of beta-hexosaminidase activity and the accumulation of GM2 ganglioside in neurons. In each disorder, a virtually identical course of neurodegeneration begins in infancy and leads to demise generally by 4-6 years of age. Through serial analysis of gene expression (SAGE), we determined gene expression profiles in cerebral cortex from a Tay-Sachs patient, a Sandhoff disease patient and a pediatric control. Examination of genes that showed altered expression in both patients revealed molecular details of the pathophysiology of the disorders relating to neuronal dysfunction and loss. A large fraction of the elevated genes in the patients could be attributed to activated macrophages/microglia and astrocytes, and included class II histocompatability antigens, the pro-inflammatory cytokine osteopontin, complement components, proteinases and inhibitors, galectins, osteonectin/SPARC, and prostaglandin D2 synthase. The results are consistent with a model of neurodegeneration that includes inflammation as a factor leading to the precipitous loss of neurons in individuals with these disorders.     

A direct gene transfer strategy via brain internal capsule reverses the biochemical defect in Tay-Sachs disease

Therapy for neurodegenerative lysosomal Tay-Sachs (TS) disease requires active hexosaminidase (Hex) A production in the central nervous system and an efficient therapeutic approach that can act faster than human disease progression. We combined the efficacy of a non-replicating Herpes simplex vector encoding for the Hex A alpha-subunit (HSV-T0alphaHex) and the anatomic structure of the brain internal capsule to distribute the missing enzyme optimally. With this gene transfer strategy, for the first time, we re-established the Hex A activity and totally removed the GM2 ganglioside storage in both injected and controlateral hemispheres, in the cerebellum and spinal cord of TS animal model in the span of one month's treatment. In our studies, no adverse effects were observed due to the viral vector, injection site or gene expression and on the basis of these results, we feel confident that the same approach could be applied to similar diseases involving an enzyme defect.     

Plasmid-based gene transfer ameliorates visceral storage in a mouse model of Sandhoff disease

Sandhoff disease is a severe neurodegenerative disorder with visceral involvement caused by mutations in the HEXB gene coding for the beta subunit of the lysosomal hexosaminidases A and B. HEXB mutations result in the accumulation of undegraded substrates such as GM2 and GA2 in lysosomes. We evaluated the efficacy of cationic liposome-mediated plasmid gene therapy using the Sandhoff disease mouse, an animal model of a human lysosomal storage disease. The mice received a single intravenous injection of two plasmids, encoding the human alpha and beta subunits of hexosaminidase cDNAs. As a result, 10-35% of normal levels of hexosaminidase expression, theoretically therapeutic levels, were achieved in most visceral organs, but not in the brain, 3 days after injection with decreased levels by day 7. Histochemical staining confirmed widespread enzyme activity in visceral organs. Both GA2 and GM2 were reduced by almost 10% and 50%, respectively, on day 3, and by 60% and 70% on day 7 compared with untreated age-matched Sandhoff disease mice. Consistent with the biochemical results, a reduction in GM2 was observed in liver cells histologically as well. These initial findings support further development of the plasmid gene therapy against lysosomal diseases with visceral pathology.     

Peripheral nervous system manifestations in a Sandhoff disease mouse model: nerve conduction,myelin structure,lipid analysis

Background  

Sandhoff disease is an inherited lysosomal storage disease caused by a mutation in the gene for the β-subunit (Hexb gene) of β-hexosaminidase A (αβ) and B (ββ). The β-subunit together with the GM2 activator protein catabolize ganglioside GM2. This enzyme deficiency results in GM2 accumulation primarily in the central nervous system. To investigate how abnormal GM2 catabolism affects the peripheral nervous system in a mouse model of Sandhoff disease (Hexb-/-), we examined the electrophysiology of dissected sciatic nerves, structure of central and peripheral myelin, and lipid composition of the peripheral nervous system.     

The mutation mechanism causing juvenile-onset Tay-Sachs disease among Lebanese

Expression of the hexosaminidase isozymes was evaluated in fibroblast cell lines obtained from two sibs of Lebanese-Christian origin who presented with juvenile-onset Tay-Sachs disease. In the normal control fibroblasts the alpha subunit of hexosaminidase A (hex A) is synthesized as a 67 KD precursor which is cleaved in lysosomes to a mature 54 KD peptide. The patients' fibroblasts were capable of synthesizing the 67 KD precursor but failed to convert it to the mature subunit. The alpha subunit precursor synthesized by patients' cells could not be phosphorylated, nor was the patients' alpha subunit precursor secreted into the medium in response to NH4Cl, which caused accumulation of both alpha and beta subunit precursor in the medium of the normal control fibroblasts. The measurement of residual enzyme activity in the fibroblasts of patients which best correlated with the onset of the illness was the ion exchange chromatographic separation of Hex A-associated hydrolysis of the synthetic substrate 4-methylumbelliferyl N-acetyl-beta-D-glucosamine-6-sulfate (4MUGS). The patients had 0.32% and 0.36% of Hex A-associated 4MUGS cleaving activity compared to normal control fibroblasts as compared to less than 0.016% for infantile Tay-Sachs disease fibroblasts. The residual Hex A activity in patients' cells had a pH optimum identical with normal enzyme (pH 3.9-4.0), a reduced specific activity for 4MUGS (relative to hydrolysis of unsulfated synthetic substrate), and a greatly enhanced thermal stability. The occurrence of this form of Tay-Sachs disease in Lebanon, the fact that the condition has been described in three unrelated Lebanese immigrant families in Canada, together with the fact that the grandparents of the unrelated probands come from villages in both the northern and southern regions of Lebanon, leads us to speculate that a gene causing juvenile-onset Tay-Sachs disease may not be infrequent in Lebanon.     

Establishment of immortalized Schwann cells from Sandhoff mice and corrective effect of recombinant human β-hexosaminidase A on the accumulated GM2 ganglioside

We have established spontaneously immortalized Schwann cell lines from dorsal root ganglia and peripheral nerves of Sandhoff mice. One of the cell lines exhibited genetically and biochemically distinct features of Sandhoff Schwann cells. The enzyme activities toward 4-methylumbelliferyl N-acetyl--D-glucosamine (-hexosaminidases A, B, and S) and 4-methylumbelliferyl N-acetyl--D-glucosamine-6-sulfate (-hexosaminidases A and S) were decreased, and GM2 ganglioside accumulated in lysosomes of the cells. Incorporation of recombinant human -hexosaminidase isozymes expressed in Chinese hamster ovary cells into the cultured Sandhoff Schwann cells via cation-independent mannose 6-phosphate receptors was found, and the incorporated -hexosaminidase A degraded the accumulated GM2 ganglioside. The established Sandhoff Schwann cell line is useful for investigation and development of therapies for Sandhoff disease.     

GM2神经节苷脂沉积症发病的分子机理研究

目的：探讨GM2神经节苷脂沉积症发病的分子机理。方法：培养患者皮肤成纤维细胞，进行酶学、Western印迹杂交和免疫细胞化学分析。结果：4例GM2神经节苷脂沉积症患者成纤维细胞内β-氨基已糖苷酶（hexosaminidase,Hex）活性均显著降低，分别为正常人的12％、3％、15％和6％；两例Sandhoff病Hex成熟的α、β-亚基（αm、βm）明显减少，但婴儿型与成年型减少的程度不同，1例Tay-Sachs病只有αm明显减少，1例GM2激活蛋白缺陷症αm、βm与正常人无差异；4例患者成纤维细胞内均有不同程度的GM2神经节苷酯的堆积。结论：GM2神经节苷脂沉积症的发病与HEXA、HEXB、GM2A基因突变引起相应蛋白表达、成熟障碍有关。     

Comparison of enzyme and DNA analysis in a Tay-Sachs disease carrier screening program.

Tay-Sachs disease (GM2 gangliosidosis, type 1; TSD) is an autosomal recessive GM2 gangliosidosis resulting from the deficient activity of the lysosomal hydrolase beta-hexosaminidase A (Hex A). With a carrier frequency estimated at 1 in 25, it is a common lysosomal disorder in the Ashkenazi Jewish population. Tay-Sachs disease has provided the prototype for the prevention of severe recessive genetic diseases. Molecular analysis of the Hex A gene (HEXA) of Ashkenazi Jewish individuals affected with Tay-Sachs disease revealed that three common mutations cause the infantile and adult onset forms of the disease; a four base insertion in exon 11, a splice junction mutation in intron 12 and a point mutation in exon 7 (G269S). A study was undertaken to determine whether mutation analysis would be useful in TSD screening programs in identifying carriers and clarifying the status of individuals whose enzyme assays are inconclusive. Ashkenazi Jewish individuals who had been diagnosed as carriers, inconclusives by enzyme assay and non-carriers with low normal enzyme levels in the Mount Sinai Tay-Sachs Disease Prevention Program were examined for the presence of the three mutations using polymerase chain reaction (PCR) and allele specific oligonucleotide (ASO) hybridization. The insertion mutation was present in 29 of 34 carriers and 2 of 36 inconclusive individuals, the splice junction mutation was found in 4 of 34 carriers and the G269S mutation was found in 1 of 34 carriers. Of the 313 non-carrier individuals with normal enzyme activity in the lower normal range, one was positive for the splice junction mutation.(ABSTRACT TRUNCATED AT 250 WORDS)     

GM2 gangliosidosis in Saudi Arabia: multiple mutations and considerations for future carrier screening

The GM2 gangliosidose, Tay-Sachs and Sandhoff diseases, are a class of lysosomal storage diseases in which relentless neurodegeneration results in devastating neurological disability and premature death. Primary prevention is the most effective intervention since no effective therapy is currently available. An extremely successful model for the prevention of GM2 gangliosidosis in the Ashkenazi Jewish community is largely attributable to the very limited number of founder mutations in that population. Consistent with our previous observation of allelic heterogeneity in consanguineous populations, we show here that these diseases are largely caused by private mutations which present a major obstacle in replicating the Ashkenazi success story. Alternative solutions are proposed which can also be implemented for other autosomal recessive diseases in our population.     

Beta-N-acetylhexosaminidase activity in human oocytes and preimplantation embryos.

beta-N-acetylhexosaminidase is a lysosomal enzyme, which has two isoenzymes: beta-Hex A, a trimer consisting of one alpha-chain and two beta-chains (alpha beta 2) and beta-Hex B, a tetramer formed of four beta-chains (beta 2 beta 2). Genetic defects in the alpha-chain lead to Tay-Sachs disease, whereas mutations in the beta-chain gene lead to Sandhoff disease. In a previous study we developed a microassay for total beta-N-acetylhexosaminidase and used this for measuring activities in mouse oocytes and preimplantation embryos. In this study, to assess the feasibility of transferring this technique to the human for the purposes of preimplantation diagnosis for Tay-Sachs and Sandhoff disease, beta-Hex activity was assayed in human oocytes and embryos and in the medium in which they had been cultured. We showed that although the activity of beta-N-acetylhexosaminidase in human oocytes and embryos was > 500 times higher than in the mouse, it was not detectable in the culture medium and the activity in oocytes and embryos remained virtually constant throughout human preimplantation development, making it difficult to distinguish embryonic from maternal enzyme activity. In the absence of this distinction it would be inappropriate to use beta-N-acetylhexosaminidase activity for the purposes of preimplantation diagnosis of Sandhoff or Tay-Sachs disease. These experiments demonstrate that measuring the beta-N-acetylhexosaminidase activity in human embryos cannot be used at present for preimplantation diagnosis.     

Conditional expression of human ß-hexosaminidase in the neurons of Sandhoff disease rescues mice from neurodegeneration but not neuroinflammation

ABSTRACT: Background Neuroinflammation develops in neuronal storage diseases, further exacerbating the clinical phenotype as well as the underlying neurodegeneration. The purpose of this study was to examine the role of neuronal function in the development of neuroinflammation and the clinical phenotype in storage disorders. Methods The Sandhoff (HexB-/- knockout) mouse was employed as a representative model of neuronal storage disorders, whereby beta-hexosaminidase deficiency results in GM2 storage characterized by neuroinflammation, neurodegeneration, behavioral deficits and early death. We dissected the role of neuronal function in disease development by restoring beta-hexosaminidase selectively in the neurons of HexB-/- mice through conditional expression of the human HEXB gene selectively in neurons. Results Although beta-hexosaminidase restoration in the neurons of HexB-/- mice was sufficient to resolve GM2 storage, reverse behavioral deficits and rescue the mice from death, brain inflammation persisted, including the presence of reactive astrocytes and microglia/macrophages. Conclusions Taken together, this and previous studies by our lab and others show that restoration of neuronal function is necessary and sufficient for the successful treatment of neuronal storage diseases. Moreover, neuroinflammation appears to depend upon other brain cell types as well, and resolution of neuroinflammation is not sufficient disease therapy.     

Sialidase-mediated depletion of GM2 ganglioside in Tay-Sachs neuroglia cells.

Tay-Sachs disease is a severe, inherited disease of the nervous system caused by accumulation of the brain lipid GM2 ganglioside. Mouse models of Tay-Sachs disease have revealed a metabolic bypass of the genetic defect based on the more potent activity of the enzyme sialidase towards GM2. To determine whether increasing the level of sialidase would produce a similar effect in human Tay-Sachs cells, we introduced a human sialidase cDNA into neuroglia cells derived from a Tay-Sachs fetus and demonstrated a dramatic reduction in the accumulated GM2. This outcome confirmed the reversibility of GM2 accumulation and opens the way to pharmacological induction or activation of sialidase for the treatment of human Tay-Sachs disease.     

Antibodies to ganglioside GM1 in patients with Alzheimer's disease

Gangliosides are thought to have a role in neuronal development and regeneration while anti-ganglioside antibodies have been shown to impair these processes. In the present work we examined whether the neuronal degeneration in Alzheimer's disease is associated with the presence of anti-ganglioside antibodies. A significant level of antibodies specific to ganglioside GM1 but not to other gangliosides (GD1a, GD1b, GT1b and GQ1b) was found in patients with Alzheimer's disease as compared to normal age matched controls. A high level of antibodies to GM1 was also found in patients with multi-infarct dementia and Parkinson's disease with dementia but not in non-demented patients with other neurodegenerative diseases. These results may reflect a specific change in ganglioside metabolism which is associated with the neurodegenerative processes underlying Alzheimer's disease and other causes of dementia.     

Mice deficient in Neu4 sialidase exhibit abnormal ganglioside catabolism and lysosomal storage

Mammalian sialidase Neu4, ubiquitously expressed in human tissues, is located in the lysosomal and mitochondrial lumen and has broad substrate specificity against sialylated glycoconjugates. To investigate whether Neu4 is involved in ganglioside catabolism, we transfected beta-hexosaminidase-deficient neuroglia cells from a Tay-Sachs patient with a Neu4-expressing plasmid and demonstrated the correction of storage due to the clearance of accumulated GM2 ganglioside. To further clarify the biological role of Neu4, we have generated a stable loss-of-function phenotype in cultured HeLa cells and in mice with targeted disruption of the Neu4 gene. The silenced HeLa cells showed reduced activity against gangliosides and had large heterogeneous lysosomes containing lamellar structures. Neu4(-/-) mice were viable, fertile and lacked gross morphological abnormalities, but showed a marked vacuolization and lysosomal storage in lung and spleen cells. Lysosomal storage bodies were also present in cultured macrophages preloaded with gangliosides. Thin-layer chromatography showed increased relative level of GD1a ganglioside and a markedly decreased level of GM1 ganglioside in brain of Neu4(-/-) mice suggesting that Neu4 may be important for desialylation of brain gangliosides and consistent with the in situ hybridization data. Increased levels of cholesterol, ceramide and polyunsaturated fatty acids were also detected in the lungs and spleen of Neu4(-/-) mice by high-resolution NMR spectroscopy. Together, our data suggest that Neu4 is a functional component of the ganglioside-metabolizing system, contributing to the postnatal development of the brain and other vital organs.     

Ganglioside GM2 N-acetyl-beta-D-galactosaminidase and asialo GM2 (GA2) N-acetyl-beta-D-galactosaminidase; studies in human skin fibroblasts.

Ganglioside GM2 and its asialo-derivative, GA2 were radiolabeled in their N-acetyl-D-galactosaminyl moieties by oxidation with galactose oxidase and reduction with tritiated sodium borohydride. Specific activities of 6 X 10(4) dpm/nmol (GM2) and 1.8 X 10(6) dpm/nmol (GA2) were achieved. About 98% of the label was in N-acetyl-D-galactosamine. Using these substrates, an assay was developed for GM2-N-acetyl-beta-D-galactosaminidase (E.C.3.2.1.30) and GA2-N-acetyl-beta-D-galactosaminidase (E.C.3.2.1.30) activities in human cultured skin fibroblasts. The products of the GM2 cleaving reaction were identified as N-acetylgalactosamine and ganglioside GM3. Both GM2 and GA2 cleaving activities were stimulated about 5-fold by purified sodium taurocholate, and this stimulation was inhibited by neutral detergents, lipids and albumin at low concentrations. Addition of various salts, reducing agents and a protein activator factor from human liver of Li et al. (1973) did not stimulate GM2-N-acetyl-beta-D-galactosaminidase activity beyond that found with sodium taurocholate. Under optimal conditions, control fibroblast supernates cleaved ganglioside GM2 at a rate of 3.7 nmol/mg protein/h compared to 1100 for GA2-N-acetyl-beta-D-galactosaminidase and 4700 for 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminidase. Supernates from two patients with Tay-Sachs disease had markedly reduced activity levels for GM2-N-acetyl-beta-D-galactosaminidase but not for the other two substrates. Supernates from two patients with Sandhoff's disease had reduced activities for all three substrates. A supernate from one patient with juvenile GM2 gangliosidosis cleaved GM2 at a somewhat faster rate than those from Tay-Sachs or Sandhoff's patients. Two healthy adult women with markedly reduced hexosaminidase A activities using 4MU-N-acetyl-beta-D-glucosaminide as substrate had approximately half-normal activities using GM2 as substrate. A patient with the Tay-Sachs phenotype but with a partial deficiency of hexosaminidase A using the 4-MU substrate had a profound deficiency using GM2 as substrate. In such unusual hexosaminidase mutants, assays using GM2 as substrate are better indicators of phenotype than those using synthetic substrates.     

Tay-Sachs disease with conspicuous cranial computerized tomographic appearances

An autopsy case of a 3-year-old female infant with Tay-Sachs disease was presented. A cherry red spot in the fundus and a deficiency of N-acetyl-beta-hexosaminidase A in the white blood cells were revealed soon after admission at the age of one year. Her parents and sister were found to be healthy carriers. The patient showed a typical clinical course with marked cranial swelling. In addition to the marked ballooning of neurons on light microscope, membranous cytoplasmic body (MCB) on electron microscope and abnormal accumulation of GM2 ganglioside in the cerebral cortex by thin layer chromatography were confirmed in the autopsy specimens. In the late stage of her clinical course, the cranial computerized tomography (CT) demonstrated symmetric and deep-wavy hyperdense cerebral cortical zones, diffuse hypodensity and diminished volume of cerebral white matter, mild to moderate ventricular dilatation, and a small cerebellum and brainstem. These conspicuous appearances of the cranial CT seem to be characteristic of Tay-Sachs disease in the late stage, and they are derived from abnormal accumulation of GM2 ganglioside in the cerebral cortex, and diffuse intense demyelination (dysmyelinating demyelination) of the cerebral white matter.     

Alleviation of neuronal ganglioside storage does not improve the clinical course of the Niemann-Pick C disease mouse

Niemann-Pick disease Type C (NP-C) is a progressive neurodegenerative disorder caused by mutations in the NPC1 gene and characterized by intracellular accumulation of cholesterol and sphingo-lipids. The major neuronal storage material in NP-C consists of gangliosides and other glycolipids, raising the possibility that the accumulation of these lipids may participate in the neurodegenerative process. To determine if ganglioside accumulation is a crucial factor in neuropathogenesis, we bred NP-C model mice with mice carrying a targeted mutation in GalNAcT, the gene encoding the beta-1-4GalNAc transferase responsible for the synthesis of GM2 and complex gangliosides. Unlike the NP-C model mice, these double mutant mice did not exhibit central nervous system (CNS) accumulation of gangliosides GM2 or of glycolipids GA1 and GA2. Histological analysis revealed that the characteristic neuronal storage pathology of NP-C disease was substantially reduced in the double mutant mice. By contrast, visceral pathology was similar in the NP-C and double mutant mice. Most notably, the clinical phenotype of the double mutant mice, in the absence of CNS ganglioside accumulation and associated neuronal pathology, did not improve. The results demonstrate that complex ganglioside storage, while responsible for much of the neuronal pathology, does not significantly influence the clinical phenotype of the NP-C model.     

Thermodynamic determination of plasma and leukocyte beta-hexosaminidase isoenzymes in homozygote and heterozygote carriers for the GM2 gangliosidosis B1 variant

In the GM2 gangliosidosis B1 variant, the mutated isoenzyme A of beta-hexosaminidase (Hex) is incapable of hydrolyzing ganglioside GM2 and negatively charged substrates. Biochemical characterization of this lysosomal disease is carried out using synthetic alpha-subunit-specific sulfated substrates, as heat-inactivation assays are not applicable. The apparent enzyme activation energy of Hex using the chromogenic substrate 3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-beta-D-glucosaminide is related directly to the relative proportions of Hex A and Hex B isoenzymes. This thermodynamic variable was used for the study of Hex enzyme heterogeneity in 3 patients with the GM2 gangliosidosis B1 variant and 6 heterozygote carriers. Hex activity was determined at 25 degrees C, 30 degrees C, 35 degrees C, and 37 degrees C in a Cobas Bio analyzer (Roche Diagnostics, Basel, Switzerland), and Arrhenius plot slopes and apparent activation energies were calculated in plasma samples and mononuclear and polymorphonuclear leukocyte lysates. The determination of the Hex isoenzymes in plasma presented a high discrimination power for B1 variant patients but not for heterozygote carriers, in whom false-negative results may be obtained. However, thermodynamic evaluation of the isoenzyme composition of Hex in leukocyte lysates permits the biochemical identification of patients with the GM2 gangliosidosis B1 variant and of heterozygote carriers.