Mouse models of Tay-Sachs and Sandhoff diseases differ in neurologic phenotype and ganglioside metabolism.

Tay-Sachs and Sandhoff diseases are clinically similar neurodegenerative disorders. These two sphingolipidoses are characterized by a heritable absence of beta-hexosaminidase A resulting in defective GM2 ganglioside degradation. Through disruption of the Hexa and Hexb genes in embryonic stem cells, we have established mouse models corresponding to each disease. Unlike the two human disorders, the two mouse models show very different neurologic phenotypes. Although exhibiting biochemical and pathologic features of the disease, the Tay-Sachs model showed no neurological abnormalities. In contrast, the Sandhoff model was severely affected. The phenotypic difference between the two mouse models is the result of differences in the ganglioside degradation pathway between mice and humans.

Mice lacking both subunits of lysosomal beta-hexosaminidase display gangliosidosis and mucopolysaccharidosis.

The GM2 gangliosidoses, Tay-Sachs and Sandhoff diseases, are caused by mutations in the HEXA (alpha-subunit) and HEXB (beta-subunit) genes, respectively. Each gene encodes a subunit for the heterodimeric lysosomal enzyme, beta-hexosaminidase A (alpha beta), as well as for the homodimers beta-hexosaminidase B (beta beta) and S (alpha alpha). In this study, we have produced mice that have both Hexa and Hexb genes disrupted through interbreeding Tay-Sachs (Hexa-/-) and Sandhoff (Hexb-/-) disease model mice. Lacking both the alpha and beta-subunits these 'double knockout' mice displayed a total deficiency of all forms of lysosomal beta-hexosaminidase including the small amount of beta-hexosaminidase S present in the Sandhoff disease model mice. More surprisingly, these mice showed the phenotypic, pathologic and biochemical features of the mucopolysaccharidoses, lysosomal storage diseases caused by the accumulation of glycosaminoglycans. The mucopolysaccharidosis phenotype is not seen in the Tay-Sachs or Sandhoff disease model mice or in the corresponding human patients. This result demonstrates that glycosaminoglycans are crucial substrates for beta-hexosaminidase and that their lack of storage in Tay-Sachs and Sandhoff diseases is due to functional redundancy in the beta-hexosaminidase enzyme system.

A genetic model of substrate deprivation therapy for a glycosphingolipid storage disorder.

Inherited defects in the degradation of glycosphingolipids (GSLs) cause a group of severe diseases known as GSL storage disorders. There are currently no effective treatments for the majority of these disorders. We have explored a new treatment paradigm, substrate deprivation therapy, by constructing a genetic model in mice. Sandhoff's disease mice, which abnormally accumulate GSLs, were bred with mice that were blocked in their synthesis of GSLs. The mice with simultaneous defects in GSL synthesis and degradation no longer accumulated GSLs, had improved neurologic function, and had a much longer life span. However, these mice eventually developed a late-onset neurologic disease because of accumulation of another class of substrate, oligosaccharides. The results support the validity of the substrate deprivation therapy and also highlight some limitations.

Mice deficient in all forms of lysosomal beta-hexosaminidase show mucopolysaccharidosis-like pathology.

Lysosomal beta-hexosaminidase consists of 2 subunits, alpha and beta. Mutations in the alpha-subunit gene cause Tay-Sachs disease, while mutations in the beta-subunit gene cause Sandhoff disease. Mice generated by targeted disruption of either the alpha- or beta-subunit genes displayed the pathological features of Tay-Sachs disease or Sandhoff disease, respectively. In this report we describe the pathologic features of mice that carry both disrupted genes and that are deficient in all forms of beta-hexosaminidase activity. These mice displayed physical dysmorphia and extensive neuro-visceral storage. Neurons in the CNS and PNS contained pleomorphic inclusions in addition to membranous cytoplasmic bodies characteristic of gangliosidosis. Diffuse hypomyelination was also apparent in the CNS. Vacuolated cytoplasm was a conspicuous feature of chondrocytes, osteocytes and renal tubular epithelium on routine hematoxylin and eosin (H&E) -stained sections. Numerous vacuolated cells were also noted in the connective tissue, cornea, heart valves, arterial walls, liver, spleen, skin and throughout other visceral organs. These vacuolated cells stained positive with PAS, colloidal iron and alcian blue, indicating an accumulation of glycosaminoglycans. Furthermore, cultured fibroblasts showed a defect in the degradation of glycosaminoglycans, and glycosaminoglycans were excreted in the urine of these mice (1). Thus, morphological and biochemical features in these mice are consistent with those of mucopolysaccharidosis and demonstrate an essential role of beta-hexosaminidase in the degradation of glycosaminoglycans.

Distribution of the galectin-1 mRNA in the rat nervous system: its transient upregulation in rat facial motor neurons after facial nerve axotomy.

Galectin-1 is a member of the animal lectin family that displays conserved consensus sequences and similar carbohydrate binding specificities. Recent analyses revealed that galectin-1 plays an important role in the process of nerve regeneration. We analyzed the topological expression of galectin-1 mRNA in adult rat nervous system. Galectin-1 mRNA was predominantly observed in the cell bodies of neurons such as oculomotor nucleus (III), trochlear nucleus (IV), trigeminal motor nucleus (V), abducens nucleus (VI), facial nucleus (VII), hypoglossal nucleus (XII), red nucleus, and locus ceruleus. Neurons in pineal gland and dorsal root ganglia expressed galectin-1 mRNA. We next tested whether the axotomy of facial nerve altered the expression of galectin-1 mRNA in motor neurons. In the adult rats, the axotomy of facial nerve induced transient upregulation of galectin-1 mRNA around 6 h after axotomy. These results indicate that galectin-1 may play roles in the early event of the nerve injury and regeneration through the transient change of its expression level.

Increase in intracellular Ca(2+) and calcitonin gene-related peptide release through metabotropic P2Y receptors in rat dorsal root ganglion neurons.

We examined the effects of the activation of metabotropic P2Y receptors on the intracellular Ca(2+) concentration and the release of neuropeptide calcitonin gene-related peptide (CGRP) in isolated adult rat dorsal root ganglion neurons. In small-sized dorsal root ganglion neurons (soma diameter<30 microm) loaded with fura-2, a bath application of ATP (100 microM) evoked an increase in intracellular Ca(2+) concentration, while the removal of extracellular Ca(2+) partly depressed the response to ATP, thus suggesting that the ATP-induced increase in intracellular Ca(2+) concentration is due to both the release of Ca(2+) from intracellular stores and the influx of extracellular Ca(2+). Bath application of uridine 5'-triphosphate (UTP; 100 microM) also caused an increase in intracellular Ca(2+) concentration in small-sized dorsal root ganglion neurons and the P2 receptor antagonists suramin (100 microM) and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 10 microM) virtually abolished the response, indicating that the intracellular Ca(2+) elevation in response to UTP is mediated through metabotropic P2Y receptors. This intracellular Ca(2+) increase was abolished by pretreating the neurons with thapsigargin (100 nM), suggesting that the UTP-induced increase in intracellular Ca(2+) is primarily due to the release of Ca(2+) from endoplasmic reticulum Ca(2+) stores. An enzyme-linked immunosorbent assay showed that an application of UTP (100 microM) significantly stimulated the release of CGRP and that suramin (100 microM) totally abolished the response, suggesting that P2Y receptor-mediated increase in intracellular Ca(2+) is accompanied by CGRP release from dorsal root ganglion neurons. These results suggest that metabotropic P2Y receptors contribute to extracellular ATP-induced increase in intracellular Ca(2+) concentration and subsequent release of neuropeptide CGRP in rat dorsal root ganglion neurons.

Immunohistochemical localization of calbindin D-28k in the migratory pathway from the rat olfactory placode.

The spatiotemporal localization of calbindin D-28k (Calb), a calcium-binding protein, was examined immunohistochemically in the developing rat olfactory system with special reference to cell migration from the olfactory placode. Calb immunoreactivity was first detected at embryonic day 12 (E12) in a few cells just outside the olfactory epithelium, and at E13, Calb-immunoreactive cells were found scattered in the laminin-rich mesenchyme. By E14, Calb-immunoreactive cells had increased in number and were seen along the entire migratory route between the vomeronasal organ, a derivative of the medial olfactory pit, and the ventromedial surface of the telencephalic vesicle. Calb neurones were not seen in the olfactory epithelium, a derivative of the lateral olfactory pit. Although the distribution pattern of Calb-immunoreactive cells was similar to that of luteinizing hormone releasing hormone (LHRH)-producing neurones, which are known to originate in the vomeronasal organ and migrate into the forebrain, Calb and LHRH immunoreactivities were contained in separate neuronal populations. Calb-immunoreactive cells were localized along the vomeronasal nerves, identified by labelling the vomeronasal organ with the lipophilic dye, DiI, and strongly immunoreactive for neural cell adhesion molecule (NCAM). These data strongly suggest that, in addition to LHRH neurones, the rat vomeronasal organ generates Calb-immunoreactive neurones which migrate along the vomeronasal nerves to enter the forebrain. The final fate and functional importance of these cells remains to be determined.

Age-related changes of DRG neuronal attachment to extracellular matrix proteins in vitro.

Attachment of dorsal root ganglion (DRG) neurones from 3-month-old (young adult) and 24-month-old (aged) mice to the following substrates was evaluated after 6 h in culture: (1) poly-L-lysine (PL), (2) PL + type I collagen (CL-I), (3) PL + type IV collagen (CL-IV), (4) PL + laminin (LM) and (5) PL + fibronectin (FN). In the young adult mice, each substrate coated onto PL, significantly increased the ratio of attachment, compared with PL alone. In the aged mice, CL-I and LM, but not CL-N or FN improved the attachment. There was no difference in cell survival or neurite extension after 48 h in culture between the two groups of mice. These results suggest that ageing causes selective changes of the neuronal cell-surface receptors respective to extracellular matrix proteins such as CL-IV and FN.

Enhanced neural regeneration from transected vagus nerve terminal in diabetic mice in vitro.

This study examined the effect of diabetes on neural regeneration in vitro. Nodose ganglia (NG) with vagal nerve fibers, dissected from streptozotocin-induced diabetic and normal C57BL/6J mice were embedded in collagen gel. After 3 and 7 days in culture, the numbers of regenerating neurites from transected nerve terminals of NG in diabetic mice were significantly greater than those in controls. Although many studies have revealed diabetes-associated impairment in neural regeneration, the results in the present study suggest that experimental diabetes could induce the potential to enhance regenerative capability of vagal sensory nerves after axotomy.

IL-6 up-regulates CNTF mRNA expression and enhances neurite regeneration.

Interleukin-6 (IL-6) is a neurotrophic cytokine, however, its direct effect on nerve regeneration has not been well characterized. We therefore examined the effect of IL-6 on neurite regeneration using the rat dorsal root ganglion. IL-6 significantly enhanced neurite regeneration from transected nerve terminals. We also examined the mRNA expression of IL-6 family cytokines and their receptors during the regeneration. The mRNA expressions of IL-6, IL-6 receptor, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) receptor alpha, and LIF receptor beta showed no significant differences by the addition of IL-6. In contrast, IL-6 enhanced the mRNA expression of gp130 and CNTF. In addition, CNTF significantly increased neurite regeneration when added exogenously. Our data suggest that IL-6 enhanced regeneration via up-regulating CNTF expression.

Administration of an aldose reductase inhibitor, ONO-2235, to streptozotocin-diabetic mice restores reductions of DRG neuronal attachment to extracellular matrix proteins in vitro.

Attachments of cultured dorsal root ganglion (DRG) neurons to the extracellular matrix (ECM) proteins (type I and IV collagens, laminin and fibronectin) and the adhesion ligand arginine-glycine-aspartic acid (RGD) were impaired in mice 2 weeks after the induction of diabetes by streptozotocin (STZ). However, administration of the aldose reductase inhibitor, ONO-2235, to the STZ-diabetic mice for 1 week restored DRG neuronal attachment to the ECM proteins and RGD to a level close to normal mice. These results suggest that activation of the aldose reductase and subsequent metabolic disorders in diabetic animals may play an important role in detrimental alterations of the neuronal cell-surface receptors for the ECM proteins.

A high glucose environment improves survival of diabetic neurons in culture.

Dorsal root ganglia neurons from streptozotocin-induced diabetic and normal C57BL mice were cultured in serum-containing and serum-free media. The ratio of dead cells was higher in diabetic neurons than in controls in the early stages of culture. The effect of glucose concentration on survival in the culture medium was also measured for 1 week. Treatment with high glucose concentrations improved the survival of diabetic neurons, which was enhanced by duration of diabetes in the animal. These results indicate that exposure to hyperglycemia in vivo might adapt neurons to a high glucose environment in vitro.

An in vitro model to study diabetic neuropathy.

Adult dorsal root ganglion (DRG) neurons from diabetic and normal C57BL mice were cultured and compared for survival and neurite extension. Neurons from diabetic mice have improved their survival and neurite extension when the insulin concentration was increased but it was never as good as that of controls. The increase of glucose concentration in serum-free media to ten times higher than normal improved both survival and neurite extension of neurons from diabetic animals.

Enantio-selective occurrence of (S)-tetrahydropapaveroline in human brain.

Tetrahydropapaveroline is an endogenous complex alkaloid derived from dopamine through the oxidation by monoamine oxidase. This alkaloid is considered to be involved in the pathogenesis of alcoholism and to act as a false neurotransmitter. Recently the (S) enantiomer was proposed to be a precursor of morphine biosynthesis in the opium poppy. In this paper stereo-chemical characteristic of tetrahydropapaveroline in human brains was examined. In all four control human brains examined, only the (S)-tetrahydropapaveroline was detected. The concentrations were 0.12-0.22 pmol/g wet weight of brain tissue, and the presence of alcohol in blood did not affect the concentration. The results suggest that (S)-tetrahydropapaveroline may be enantio-selectively synthesized in human brain and it may be an intermediate of the de novo synthesis of morphine analogues.

Dopaminergic neurotoxins, 6,7-dihydroxy-1-(3', 4'-dihydroxybenzyl)-isoquinolines, cause different types of cell death in SH-SY5Y cells: apoptosis was induced by oxidized papaverolines and necrosis by reduced tetrahydropapaverolines.

Dopamine-derived 6,7-dihydroxy-1-(3', 4'-dihydroxybenzyl)-isoquinolines, papaverolines and tetrahydropapaverolines, have been proposed to be neurotoxin candidates related to the pathogenesis of Parkinson's disease. In this paper, the cytotoxicity of papaverolines and their N-methyl derivatives was examined using human dopaminergic neuroblastoma SH-SY5Y cells as a model of dopamine neurons. Apoptotic and necrotic cell death were assessed by morphological observation of cells after staining with propidium iodide and Hoechst 33342. Papaveroline and N-methyl-papaveroline induced apoptosis in almost all the cells with typical features of condensed and fragmented nuclei. On the other hand, (R)- and (S)-tetrahydropapaveroline caused necrosis in cells. Tetrahydropapaverolines markedly reduced adenosine triphosphate (ATP) level, whereas papaverolines did not, suggesting that the types of cell death induced by these isoquinolines, necrosis and apoptosis, depend on ATP concentrations in the cells.

Diabetes alters neurite regeneration from mouse retinal explants in culture.

We examined the effect of experimental diabetes on neurite regeneration from adult mouse retinal explants cultured in the presence of different concentrations of glucose. The numbers of regenerating neurites at 3, 6 and 10 days in culture at normal glucose concentration (7 mM) were significantly smaller in streptozotocin-induced diabetic C57BL/6 mice than in normal control mice. In contrast, treatment of retinal explants with high glucose concentration (57 mM) significantly diminished the number of regenerating neurites in the control mice, but not in the diabetic mice. These results suggest that retina in diabetic mice has impaired capability of neurite regeneration in a normal glucose environment, but is adaptable to a high glucose environment in vitro.

Nerve growth factor (NGF) restores depletions of calcitonin gene-related peptide and substance P in sensory neurons from diabetic mice in vitro.

Dorsal root ganglion neurons from streptozotocin (STZ)-induced diabetic, genetic diabetic and normal mice were cultured in serum-containing media with or without nerve growth factor (NGF). The immunocytochemical analysis carried out after 1 week in culture revealed that the ratios of neurons immunoreactive to calcitonin gene-related peptide (CGRP) in NGF-free medium in the STZ-diabetic mice (average 23.2%) were significantly lower than those in the normal mice (45.1%). The ratios of neurons immunoreactive to CGRP and substance P (SP) in the NGF-free medium were also lower in the genetic diabetic mice (23.6% and 21.8%) than those in the normal ones (40.7% and 34.2%). However, treatment with NGF restored these reduced immunoreactivities in the diabetic groups in a dose-dependent manner. These results show that NGF can be effective for the diabetes-induced depletion of CGRP and SP in sensory neurons, and suggest its possible role in the prevention and improvement of diabetic sensory neuropathy.

Diabetes-induced reduction of neuronal survival in hypotonic environments in culture.

Dorsal root ganglion (DRG) neurons from streptozotocin (STZ)-induced diabetic and normal C57BL mice were exposed to three different hypotonic environments (1/2, 1/4, and 1/8 osmolar solutions). After rapid applications of these hypotonic solutions to the neurons, the cell volume autoregulatory mechanism operated in 1/2 osmolar solution but was disrupted in superhypotonic solutions below 1/4 osmolar in both kinds of mice. None of the neurons could survive 12 h after treatment with superhypotonic solutions. On the other hand, a gradual reduction of osmolarity of the culture medium enabled neurons in the normal mice to survive in 1/2 and 1/4 osmolar solutions as well as in an isotonic solution. However, this reduction of osmolarity increased neuronal cell death in the diabetic mice. These results suggest that the ability of DRG neurons to survive in hypotonic environments in culture may be lost in diabetes.

Diabetes impairs DRG neuronal attachment to extracellular matrix proteins in vitro.

Attachments of dorsal root ganglion (DRG) neurons from streptozotocin (STZ)-induced diabetic and normal C57BL mice to the following substrates were evaluated in vitro: a) poly L-lysine (PL), b) PL + type I collagen (CL-I), c) PL + type IV collagen (CL-IV), d) PL + laminin (LM) and e) PL + fibronectin (FN). After 6 h in culture, there was no significant difference in the average ratio of cells adhesive to PL between the diabetic (74.9%) and normal group of mice (75.6%). In the normal group, the addition of extracellular matrix (ECM) proteins such as CL-I, CL-IV, LM and FN to PL increased the ratios of cell attachment from 75.6% to nearly 90%. In the diabetic group, however, none of these proteins improved the attachment (the ratio changed from 74.9% to nearly 70%). Survival and neurite extension of attached cells after 48 h in culture were not different between the two groups. These results suggest that the cell-surface receptors, which enable DRG neurons to bind to the extracellular matrix proteins, are impaired by diabetes, resulting in being one of the causes of diabetic neuropathy.

Response of aged neurons to super-hypotonic environment.

Cultured sensory neurons from aged mice (24 months old) lost capability of cell volume auto-regulatory mechanism after the rapid treatment with a hypotonic solution of 1/4 the normal osmolarity or lower. However, they could survive and extend neurites in super-hypotonic media such as 1/4 osmolar and even 1/8 osmolar culture media when the osmolarity was reduced gradually to such a low level.