Ocular pathology of bovine GM1 gangliosidosis

Summary Late-onset of disturbed vision is a clinical feature of bovine GM₁ gangliosidosis. Studies on eight affected calves showed that ocular lesions were confined to the retinae and optic nerves. Myriad tiny white spots were visible by ophthalmic examination of the fundus. These spots were related to protuberances on the vitreal surface caused by distended retinal ganglion cells. The perikaryons of these cells were packed with dense aggregates of membranous cytoplasmic bodies (MCBs). Similar MCBs were present in the perikaryons of amacrine cells in the inner nuclear layer of the retina. Wallerian degeneration was present in the optic nerves. The lesions were compared with those described in gangliosidosis in man, cats, pigs and dogs. It is suggested that examination of the fundus is a useful clinical procedure in the diagnosis of bovine GM₁ gangliosidosis.     

A case of chronic GM1 gangliosidosis presenting as dystonia: clinical and biochemical studies

Summary Clinical and biochemical studies are reported on a 32-year-old man with GM₁ gangliosidosis who presented with a slowly progressive dystonia that began when he was aged 7 years and eventually became almost totally incapacitating at the age of 35. There was only mild intellectual deterioration, but myoclonus, seizures and macular cherry-red spots were never observed. Proton-density and T2-weighted MRI scans showed symmetrical hyperintense lesions of both putamina. No increase of GM₁ ganglioside was found in plasma or cerebrospinal fluid, and the metabolism of GM₁ ganglioside in cultured skin fibroblasts from the patient was also almost normal, although the residual activity of GM₁ ganglioside -galactosidase activity was only 10% of normal. These findings suggest that impaired GM₁ ganglioside metabolism is not present systemically as it is in the infantile and juvenile types of the disorder, but is mainly confined to the central nervous system in chronic GM₁ gangliosidosis.     

Monosialoganglioside GM1 blood levels in maternal and newborn umbilical cord blood at birth

Blood levels of the monosialoganglioside GM₁ (nomenclature according to Svennerholm) were tested at birth in the umbilical cord of 37 neonates and their mothers. Comparisons were made based on gestational age and modality of delivery. GM₁ blood levels at birth were significantly higher in mothers than in newborns (373.66±56.83 ng/ml vs 217.95±21.24 ng/ml; P<0.01). Newborns delivered by cesarian section showed levels of GM₁ significantly higher than those delivered vaginally (298.97±38.55 ng/ml vs 169.62±12.62 ng/ml; P<0.01). and preterm newborns had significantly higher levels of GM₁ than full-term newborns (253.50±40.83 ng/ml vs 193.71±21.74 ng/ml; P<0.01). No differences in blood levels of GM₁ were observed in the mothers in relation to length of pregnancy or modality of delivery. The higher levels of GM₁ observed in preterm newborns indicate an increased turnover and/or enhanced bioavailability of the monosialoganglioside GM₁ for the developing central nervous system. Further data are required to evaluate the significance of the increased cord levels of GM₁ in neonates after cesarian section.     

GM2 gangliosidosis in a Japanese Spaniel

Summary A storage disease in a 2-year-old Japanese Spaniel resembled a GM₂ gangliosidosis previously identified in a now extinct line of German Shorthaired Pointers. Despite a later appearance of signs in the Japanese Spaniel, the distribution, staining, and ultrastructure of the stored material were similar in the two breeds. Golgi studies of cerebral cortical neurons revealed the formation of spiny and aspiny enlargements at the axon hillock region (meganeurites) and the growth of secondary neurites from this region. As in the German Shorthaired Pointer model, there was massive storage of GM₂ ganglioside as well as a seemingly paradoxical increase in total -hexosaminidase activity measured in vitro.Supported in part by NIH grants NS-10967, RR-07003, and RR-00463     

The effect of GM1 ganglioside on coerulospinal, noradrenergic, adult neurons and on fetal monoaminergic neurons transplanted into the transected spinal cord of the adult rat

GM₁ ganglioside, thyroxine and hydrocortisone were tested for their ability to improve the survival and growth of fetal focus coeruleus noradrenergic neurons in the transected, adult spinal cord. GM₁ alone was also tested for its effect on fetal mesencephalic dopaminergic neurons implanted into a small dorsolateral cavity at the L2 region of the cord previously transected at the T9–T10 region. None of the substances tested had any measurable effect on either of the fetal implants. However, in the GM₁- and thyroxine-treated animals the somatic dendrites of the axotomized, noradrenergic, coerulospinal neurons appeared more robust, and more intensely fluorescent, compared to their appropriate controls. GM₁ also caused a pronounced sprouting of the axotomized monoaminergic (catecholaminergic and serotonergic) fibres in the rostral region of the cord adjacent to the transection site. All of the mesencephalic dopaminergic implants survived in both the GM₁-treated animals and their saline-injected controls. However, their development was apparently not influenced by GM₁. The results indicate that GM₁ and thyroxine can enhance those aspects of the reactive mechanisms of mature, axotomized, noradrenergic coerulospinal neurons that promote their regeneration. As such, GM₁ could become a useful tool in current attempts to foster the regeneration of damaged monoaminergic neurons in the mammalian CNS.     

Neurophysiological studies in GM1, gangliosidosis

Neurophysiological studies(EEG, ERG, VEP) have been carried out on 8 children with proven GM₁ gangliosidosis (3 of Type I and 5 of Type II). All the EEGs were abnormal showing an increasing amount of irregular slow activity as the disease progressed. Around 2 to 3 years of age, Type II patients often showed a fluctuating 4–5 c/s rhythmic activity especially prominent in the temporal regions. Paroxysmal activity was not a conspicuous feature in any of the patients. The ERG was normal in all cases but the VEP was variably altered. The EEG / ERG/ VEP findings in GM₁ gangliosidosis differ from those seen in most other neurometabolic disorders of childhood.

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Sommario Sono stati condotti studi neurofisiologici(EEG, ERG, PEV) in 8 bambini con accertato GM₁ gangliosidiosi (3 di tipo I e 5 di tipo II). Tutti gli EEG sono risultati anormali dimostrando un aumento di attività lenta e irregolare col procedere della malattia. I pazienti del gruppo II presentano, attorno ai 2–3 anni di età, un'attività fluttuante e ritmica a 4–5 c/s prevalente soprattutto nelle regioni temporali. L'attività parossistica non risultava continua in alcun caso. L'ERG era normale in tutti i casi e la PEV era variamente alterata. In sintesi i rilievi EEG, ERG, PEV nella GM₁ gangliosidiosi differiscono da quelli che si riscontrano nella maggioranza delle malattie neurometaboliche infantili.

     

Enzyme replacement treatment for Tay-Sachs disease brain cells in culture utilizing Concanavalin A-mediated hexosaminidase A uptake: Biochemical and morphological evidence of GM2 mobilization

Summary When Concanavalin A (Con A) is bound to the cell membrane, it functions as an artificial enzyme receptor, mediating the binding and intracellular incorporation of significant amounts of exogenous hexosaminidase A (Hex A) into Tay-Sachs disease (TSD) glial cells. The treated cells retained almost 50% of incorporated Hex A activity after 3 days incubation in Hex A free medium. Hex A was released from Con A within the cell and was available as free enzyme. Biochemical analysis of gangliosides in Con A and Hex A treated cells depicted a greater than 50% reduction in stored GM₂ ganglioside and a fourfold reduction in GM₂label (¹⁴C) when compared to controls. Ultrastructural evidence of GM₂ breakdown is presented which supports the biochemical and labeling studies.     

Interaction between nerve growth factor and GM1 monosialoganglioside in preventing cortical choline acetyltransferase and high affinity choline uptake decrease after lesion of the nucleus basalis

Monosialoganglioside GM₁ and nerve growth factor (NGF) were administered alone or concomitantly to adult male rats with a unilateral ibotenic acid lesion of the nucleus basalis magnocellularis (NBM). High-affinity choline uptake (HACU) rate and choline acetyltransferase (ChAT) activity were measured, 4 and 21 days after surgery, respectively, in the frontal and parietal cortices of both hemispheres. A 33–34% decrease in HACU rate and a 43-39% decrease in ChAT activity was found in the ipsilateral cortices 4 and 21 days, respectively, after the lesion. If the lesioned rats received NGF (10 μg i.c.v.) twice a week or daily administrations of GM₁ (30 mg/kg, i.p.), beginning immediately after surgery the decrease in HACU rate and ChAT activity was smaller. If NGF and GM, were given concomitantly no decrease in HACU rate and ChAT activity was detected in the lesioned hemisphere and a slight increase occurred in the contralateral hemisphere. However, after the concurrent administration of NGF (10 μg i.c.v.) and the inactive dose of GM₁ 10 mg/kg i.p. no decrease in HACU and ChAT activity was also found in the lesioned rats. The latter finding indicates a potentiation by GM₁ of NGF effects on the cholinergic neurons of the IBM. The two drugs may either antagonize the neurotoxic effects of ibotenic acid or stimulate a compensatory activity in the remaining neurons.     

Neuropathology of mice with targeted disruption of Hexa gene,a model of Tay-Sachs disease

A murine model of Tay-Sachs disease, the prototype of the GM₂ gangliosidoses, was produced through the targeted disruption of the Hexa gene encoding the subunit of -hexosaminidase A. The mice were completely devoid of -hexosaminidase A activity and accumulated GM₂ ganglioside in the CNS in an age-dependent manner. Neurons with membranous cytoplasmic bodies (MCBs), identical to those described in Tay-Sachs disease, were identified in the brain of these mice. The neurons with MCBs were periodic acid-Schiff-positive on frozen sections and immunostained with anti-GM₂ ganglioside antibody. However, unlike Tay-Sachs disease in which neurons throughout the brain are affected, the localization of storage neurons in these mice appeared to be limited to certain regions, i.e., cerebral cortex, the hippocampus, amygdala, hypothalamus, mammillary nucleus, etc. Storage neurons were absent in the olfactory bulb, cerebellar cortex and spinal anterior horns. The difference in the distribution of storage neurons suggests a difference of ganglioside metabolism between humans and mice. This model is useful for the study of the pathogenic mechanisms of neuronal storage in Tay-Sachs disease and for the evaluation of therapeutic strategies.     

α2-adrenoceptor-GTP binding regulatory protein-adenylate cyclase system in cerebral cortical membranes of adult and senescent rats

The characterization of [³H]clonidine binding and effects of GTP, forskolin, islet-activating protein (IAP) and cholera toxin on adenylate cyclase activity were investigated in cerebral cortical membranes from 70-day-old and 2-year-old rats. Neither K_d nor B_max values in [³H]clonidine binding were changed between day 70 and year 2. The activation of adenylate cyclase by forskolin was significantly higher in senescent than in adult animals. The inhibitory effect of adrenaline, which was completely abolished by the pretreatment with IAP/NAD on forskolin/GTP-stimulated cyclase activity, was low in senescent rats compared to that in adult ones. The stimulatory effect of cholera toxin/NAD was also low at the senescent stage compared to that at the adult stage. It is suggested that ligand binding affinity and the density in α₂-adrenoceptors do not change between day 70 and year 2 but that GTP binding and/or coupling activity of inhibitory as well as stimulatory GTP binding regulatory protein to catylytic unit decrease in synaptic membranes of 2-year-old compared to those of 70-day-old rat brain.     

Do GM1 antibodies induce demyelination?

We review clinical, neurophysiological, immunological, and experimental data concerning multifocal motor neuropathy (MMN), a newly recognized disorder that mimics MND. It is separated from MND by the presence of multifocal conduction block (CB) demonstrated electrophysiologically, and in some instances by the association of high liters of GM₁ antibodies. The possible immunopathogenetic effect of GM₁ antibodies is discussed. However, 70% of patients with MMNCB do not have elevated titers of GM₁ antibodies, but may respond nevertheless to immunosuppressive treatment. Thus, so far unrecognized antibodies may react against some other epitopes in the paranodal region than those attacked by GM₁ antibodies to cause CB. © 1994 John Wiley & Sons, Inc.     

Effect of an NMDA receptor antagonist and a ganglioside GM1 derivative upon ethanol-induced modification of parameters of oxidative stress in several brain regions

Dietary administration of ethanol to rats for 2 weeks was able to depress levels of glutathione (GSH) and Cu/Zn superoxide dismutase (SOD) in several brain regions. This was indicative of the generation of excess levels of reactive oxygen in treated animals. The potentially protective effect of both an NMDA receptor blocker (MK-801) and an internally esterified derivative of ganglioside GM₁ (AGF₂) upon ethanol-induced changes in these indices of oxidative stress, was studied. Both of these agents are reported to have neuroprotective properties, but neither was able to prevent ethanol-induced reduction of GSH and SOD levels in any brain area studied. In fact, both agents depressed SOD and GSH levels in midbrain independently of ethanol. MK-801 had a pronounced pro-oxidant potential, and when administered in combination with ethanol, GSH and SOD were reduced in midbrain and striatum to levels below those obtained with either agent alone. The pro-oxidant properties of ethanol may thus act independently of its actions upon the NMDA receptor. The protective properties of NMDA receptor inhibitors or gangliosides cannot be attributed to any antioxidant effect.     

GM1 monosialoganglioside pretreatment protects against soman-induced seizure-related brain damage

The effects of GM₁ monosialoganglioside pretreatment on brain damage resulting from soman-induced seizure activity were examined in this study. Male Sprague-Dawley rats were infused with GM₁ via an osmotic minipump connected through a permanent cannula implanted intracerebroventricularly and challenged with soman (83 μg/kg, i.e., 1.25 × LD₅₀) 4 d after initiation of GM₁ infusion. Electrocorticographic recordings were monitored via indwelling cortical electrodes. Twenty-seven hours after soman administration, anesthetized rats were euthanized via transcardial perfusion with buffered paraformaldehyde. Brains were processed for hematoxylin and eosin (H&E), cresyl violet (CV), and acetylcholinesterase (AChE) histochemistry, and glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) immunohistochemistry. All soman-challenged rats not infused with GM₁ (n=14) developed status epilepticus (SE). GM₁-infused, soman-challenged rats (n=11) showed initial signs of seizures; however, only five developed SE. The remaining six recovered and had no brain damage. In addition, the latter group showed a significantly higher residual AChE reactivity in the basolateral amygdala compared to rats that developed SE. Quantitative image analysis of MAP2-immunostained brain sections from the five GM₁-infused rats that developed SE showed a 85.9±14.1% reduction in cross-sectional area of necrosis in the piriform cortex when compared to the somanchallenged rats that were not infused with GM₁. This was paralleled by a pronounced decrease in morphological evidence of damage on H&E and CV-stained serial sections. Considerable brain region/treatment group variability was seen with GFAP immunostaining. The present findings demonstrate that GM₁ pretreatment interferes with the development of SE and significantly alleviates brain damage resulting from soman-induced seizures.     

β-adrenoceptor stimulation-induced increase in the number of α2-adrenoceptors of cerebral cortical membranes in rats

Effects of pre-treatment of synaptic membranes with β-adrenoceptor agonists and cholera toxin on [³H]clonidine and [³H]yohimbine binding were examined in rat cerebral cortex. Pre-incubation of cerebral cortical membranes with isoproterenol (10 or 200 μM) or dobutamine (1, 10 or 200 μM) at 37 °C for 40 min caused a significant elevation of specific [³H]clonidine binding but treatment with salbutamol (10 or 200 μM) did not. Scatchard analysis showed that 200 μM isoproterenol treatment resulted in a significant elevation of high affinity component of [³H]clonidine binding which was significantly decreased by the addition of 10μM GTP. A significant elevation in high affinity [³H]clonidine binding was observed by treatment with 100 μg/ml cholera toxin, while a significant decrease in low affinity one was by the treatment. Specific [³H]yohimbine binding was also elevated by 10 or 200 μM isoproterenol treatment. It is suggested that stimulation of β-receptors, presumably β₁-subtype could elevate the number of agonist and antagonist binding sites in α₂-receptors in synaptic membranes by partially mediated by stimulatory and/or inhibitory GTP binding regulatory proteins.     

Immunocytochemical study on cholera toxin binding sites by monoclonal anti-cholera toxin antibody in neuronal tissue culture

An indirect method of immunocytochemistry showed that cholera toxin and its B-subunit served as specific neuronal surface markers in conjunction with monoclonal anti-cholera toxin and FITC labeled anti-mouse Fab. The cell types which get specifically stained in culture were peripheral neurons from dorsal root ganglion cells, superior cervical ganglion cells, and cerebral neurons, all of which were rat tissue, and NGF-treated PC12 cells. Non-neuronal cells, i.e. Schwann cells, fibroblasts and glia cells, were not stained. This method can, therefore, be used to distinguish neurons from non-neuronal cells in neuronal tissue cultures, as in the case of tetanus toxin described in the literature^{5,10,23,25,28,29}.     

The formation and regional distribution of prostaglandins D2 and F2α in the brain of spontaneously convulsing gerbils

The distribution of the two major cycloxygenase products prostaglandin D₂ (PGD₂) and prostaglandin F_2α (PGF_2α) in 7 different regions of the brain (medulla, cerebellum, hypothalamus, striatum, midbrain, hippocampus and cerebral cortex) was studied. Basal levels were highest in hypothalamus and cortex. Following convulsions elicited by environmental stress prostaglandin concentrations increased in all areas, with largest increases (10–20-fold) in hippocampus and cortex, reaching 70 ng/g PGD₂ in hippocampus and 115 ng/g PGD₂ in cortex. These results demonstrate that, during spontaneous seizures, there is a greater increase in prostanoid production in those areas involved in the convulsive process.     

Autoradiographic mapping of 5-HT1B and 5-HT1D receptors in the post mortem human brain using [H]GR 125743

The distribution of 5-HT_1B and 5-HT_1D receptors in the human post mortem brain was examined using whole hemisphere autoradiography and the radioligand [³H]GR 125743. [³H]GR 125743 binding was highest in the substantia nigra and the globus pallidus. Lower levels were detected in the striatum, with the highest densities in the ventromedial parts. In the amygdala, the hippocampus, the septal region and the hypothalamus, lower [³H]GR 125743 binding was observed, reflecting low densities of 5-HT_1B/1D receptors. In the cerebral cortex, binding was similar in most regions, although restricted parts of the medial occipital cortex were markedly more densely labeled. Binding densities were very low in the cerebellar cortex and in the thalamus. Two methods were used to distinguish between the two receptor subtypes, the first using ketanserin to block 5-HT_1D receptors and the second using SB 224289 to inhibit 5-HT_1B receptor binding. The autoradiograms indicated that in the human brain, the 5-HT_1B receptor is much more abundant than the 5-HT_1D receptor, which seemed to occur only in low amounts mainly in the ventral pallidum. Although [³H]GR 125743 is a suitable radioligand to examine the distribution of 5-HT_1B receptors in the human brain in vitro, the selectivities of ketanserin and SB 224289 are not sufficiently high to give definite evidence for the occurrence of the 5-HT_1D receptor in the human brain.     

Effects of GM1-ganglioside and α-sialyl cholesterol on amino acid uptake,protein synthesis,and Na+, K+-ATPase activity in superior cervical and nodose ganglia excised from adult rats

We examined the effect of GM₁-ganglioside in combination with cholera toxin B, and synthetic α-sialyl cholesterol (α-SC) on neutral amino acid (tritiated α-aminoisobutyric acid, [³H]AIB) uptake, protein synthesis ([³H]leucine incorporation), and Na⁺, K⁺-ATPase activity in isolated superior cervical ganglia (SCG) and nodose ganglia (NG) from adult rats after aerobic incubation, usually for 2 h at 37°C in vitro. Cholera toxin B, that specifically masks the oligosaccharide chain of GM₁-ganglioside, antagonized the GM₁-induced changes in [³H]AIB uptake, [³H]leucine incorporation, and Na⁺, K⁺-ATPase activity almost completely in SCG, but partially in NG. Although cholesterol itself had little effect on either [³H]AIB uptake and Na⁺, K⁺-ATPase activity both in SCG and NG, α-SC caused considerable reduction of both amino acid uptake and the transport enzyme activity in each ganglia. However, cholesterol was more effective than α-SC in decreasing [³H]leucine incorporation in either ganglia. Whereas addition of EGTA markedly reduced either GM₁-induced or α-SC-induced change in [³H]leucine incorporation into acid-insoluble fraction in both SCG and NG, application of Ca²⁺ ionophore produced considerable recovery of the protein synthesis from the inhibited level by Ca²⁺-deprivation. ATP and creatine phosphate contents in SCG were elevated by the presence of GM₁ or α-SC, whereas [³H]AIB uptake and Na⁺, K⁺-ATPase activity were inhibited, suggesting that utilization for membrane transport was diminished as a result of GM₁- or α-SC-induced decrease of ATPase activity.     

β-Galactosidase deficiency in a Korat cat: a new form of feline GM1-gangliosidosis

A 7-month-old Korat cat was referred for a slowly progressive neurological disease. Circulating monocytes and lymphocytes showed the presence of single or multiple empty vacuoles and blood leukocytes enzyme assay revealed a very low β-galactosidase activity level (4.7 nmol/mg per h) as compared to unaffected parents and relatives. Histologically, the cat, euthanized at the owner request at 21 months of age, presented diffuse vacuolization and enlargement of neurons throughout the brain, spinal cord and peripheral ganglia, severe cerebellar neuronal cell loss, and moderate astrocytosis. Stored material was stained with periodic acid-Schiff on frozen sections and with the lectins Ricinus communis agglutinin-I, concanavalin A and wheat germ agglutinin on paraffin-embedded sections. Ultrastructurally, neuronal vacuoles were filled with concentrically whorled lamellae and small membrane-bound vesicles. In the affected cat, β-galactosidase activity was markedly reduced in brain (18.9%) and liver (33.25%), while total β-hexosaminidase activity showed a remarkable increase. Quantitation of total gangliosides revealed a 3-fold increase in brain and 1.7-fold in liver of affected cat. High-performance thin layer chromatography (HPTLC) detected a striking increase of G_M1-ganglioside. On densitometric analysis of HPTLC bands, the absorption of G_M1-ganglioside band was 98.52% of all stained bands (G_D1a ,G_D1b, G_T1b). Based on clinical onset, morphological and histochemical features, and biochemical findings, the Korat cat G_M1-gangliosidosis is comparable with the human type II (juvenile) form. However, clinical progression, survival time and level of β-galactosidase deficiency do not completely fit with those of human type II G_M1-gangliosidosis. The disease in the Korat cat is also different from other reported forms of feline G_M1-gangliosidosis. Received: 27 January 1998 / Revised, accepted: 18 March 1998     

Measurement of 5-HT(1A) receptor density and in-vivo binding parameters of [(18)F]mefway in the nonhuman primate

The goal of this work was to characterize the in-vivo behavior of [¹⁸F]mefway as a suitable positron emission tomography (PET) radiotracer for the assay of 5-hydroxytryptamine_1A (5-HT_1A) receptor density (B_max). Six rhesus monkeys were studied using a multiple-injection (M-I) protocol consisting of three sequential bolus injections of [¹⁸F]mefway. Injection times and amounts of unlabeled mefway were optimized for the precise measurement of B_max and specific binding parameters k_off and k_on for estimation of apparent K_D. The PET time series were acquired for 180 minutes with arterial sampling performed throughout. Compartmental analysis using the arterial input function was performed to obtain estimates for K₁, k₂, k_off, B_max, and K_Dapp in the cerebral cortex and raphe nuclei (RN) using a model that accounted for nontracer doses of mefway. Averaged over subjects, highest binding was seen in the mesial temporal and dorsal anterior cingulate cortices with B_max values of 42±8 and 36±8 pmol/mL, respectively, and lower values in the superior temporal cortex, RN, and parietal cortex of 24±4, 19±4, and 13±2 pmol/mL, respectively. The K_Dapp of mefway for the 5-HT_1A receptor sites was 4.3±1.3 nmol/L. In conclusion, these results show that M-I [¹⁸F]mefway PET experiments can be used for the in-vivo measurement of 5-HT_1A receptor density.