Experimental diabetic neuropathy. Effect of ganglioside treatment on axonal transport of cytoskeletal proteins.

Abnormalities in axonal transport of proteins are thought to play an important role in the pathogenesis of diabetic neuropathy. Gangliosides exert a positive action on numerous alterations in biochemistry and physiology of diabetic nerves. This study was undertaken to assess the effects of exogenous gangliosides on the axonal transport of structural proteins such as actin and tubulin in the sensory fibers of short-term (9-wk) and long-term (6-mo) diabetic rats. Adult Sprague-Dawley rats were made diabetic with a single injection of 70 mg/kg streptozocin i.p. Subgroups were injected daily with either highly purified ganglioside mixture (10 mg/kg i.p.) or saline for 1 mo, beginning either 2 or 17 wk after streptozocin injection. Age-matched rats were used as controls. Axonal transport was studied by the pulse-labeling technique. Three weeks after labeling, sciatic nerves were dissected out and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. In diabetic rats of both experimental designs, the transport rate of tubulin and actin was decreased by approximately 30% compared with control rats. Ganglioside treatment counteracted such alterations in both 9-wk and 6-mo diabetic rats. These data suggest a pharmacological effect that could be correlated with molecular interactions between integral membrane glycolipids and cytoskeletal elements.

Inner ester derivatives of gangliosides protect autonomic nerves of alloxan-diabetic rats against Na+, K(+)-ATPase activity defects.

Bovine brain gangliosides have been shown to prevent decay in Na+,K(+)-ATPase activity in sciatic and optic nerves of alloxan- and streptozotocin-diabetic rats. In the search for a drug with greater bioavailability and increased incorporation into neural tissue, ganglioside inner ester derivatives (AGF1) were recently developed. We evaluated the effect of AGF1 treatment on Na+,K(+)-ATPase activity in homogenates of vagus nerve from alloxan-diabetic rats (100 mg/kg s.c.). Animals were treated with AGF1: 10 mg/kg 6 days/week i.p., or 30 mg/kg biweekly i.p. Treatment began 10 d post-alloxan and continued for 8 consecutive weeks. Normal age- and sex-matched rats were used as controls. Alloxan intoxication produced a 39% decrease in Na+,K(+)-ATPase activity of the vagus nerve, which was completely restored (96-97% recovery) by both AGF1 regimes. Results suggest that ganglioside inner ester derivatives may be used in the clinical setting for the management of diabetic autonomic neuropathy.

Proteins of slow axonal transport in sciatic motoneurones of rats with streptozotocin-induced diabetes or galactosaemia.

This study examined the distribution of axonally transported tubulin and a 68 kDa polypeptide in the sciatic nerve 34 days after injection of labelled methionine into the ventral horn of the spinal cord of control rats, rats with streptozotocin-induced diabetes mellitus and rats fed a diet containing 40% galactose. The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of pellets produced by treatment of nerve extracts with Triton X-100 followed by differential ultra-centrifugation. The most marked effect of both diabetes and galactosaemia was to reduce the amount of activity present in tubulin transported at a rate of 1.4 to 2.1 mm/day. The distribution of activity in the 68 kDa polypeptide band was not markedly affected by either of the experimental conditions. These findings, taken together with those of other studies, indicate that the polyol pathway may contribute to the development of some defects of nerve function in diabetic rats, but is uninvolved in others.

Slow axonal transport impairment of cytoskeletal proteins in streptozocin-induced diabetic neuropathy.

The impairment of slow axonal transport of cytoskeletal proteins was studied in the sciatic nerves of streptozocin-diabetic rats. [35S]Methionine was unilaterally injected into the fourth lumbar ganglion and spinal cord, to label the sensory and motor axons, respectively, and then the polymerized elements of the cytoskeleton and the corresponding soluble proteins were analyzed separately. In addition, the pellet/supernatant ratio for tubulin and actin was also assessed. Our results indicate that the velocity of slow component a (SCa) of axonal transport, particularly that of neurofilaments, was strongly reduced (by 60%) in sensory axons. At the same time, a decreased pellet/supernatant ratio of tubulin, possibly owing to a depolymerization of stable microtubules, was also observed. The transport of slow component b (SCb) of axonal transport was also impaired, but the extent of this impairment could not be precisely evaluated. In contrast, motor axons showed little or no impairment of both SCa and SCb at the time studied, a result suggesting a delayed development of the neuropathy in motor axons.

Postnatal development of bulbospinal serotoninergic system. effects of GMI ganglioside following neonatal 5,7-dihydroxytryptamine treatment.

The postnatal development of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the pons medulla and spinal cord segments of rats treated with 5,7-dihydroxytryptamine (5,7-HT) and/or GM1 ganglioside has been investigated. Animals have been sacrificed at 4, 7, 28 and 56 days of age. In control rats, 5-HT and 5-HIAA increase in all areas during the first postnatal week. Thereafter, 5-HT and 5-HIAA remain constant in the thoracic and lumbar segments while a further increment takes place in the cervical portion. In the pons medulla, 5-HT reaches a plateau at 28 days, while 5-HIAA reaches a peak at 7 days and then declines to the adult value at 28 days. Neonatal administration of 5,7-HT produces mixed-type alterations in the developing bulbospinal serotoninergic system. Whilst 5-HT and 5-HIAA markedly decrease in the most distal 5-HT nerve terminal projections (thoracic and lumbar cord) they increase in the pons medulla ("pruning effect"). These alterations are accompanied by regional variations of the 5-HIAA/5-HT ratio, an index of 5-HT turnover. In particular, a prominent decrease of 5-HIAA/5-HT occurs in the lumbar segment of 1- and 2-month-old rats. In this area, where the effect of 5,7-HT is the most severe, an "up-regulation" of 5-HT, receptors is observed in 2-month-old rats. GM1 administration does not modify the development of the bulbospinal serotoninergic system. However, GM1 treatment has a counteracting effect on the alterations induced by 5,7-HT. Recovery of 5-HT and 5-HIAA levels occurs in the thoracic and lumbar cord of 1- and 2-month-old rats and is paralleled by a reduction of the "pruning effect" in the pons medulla. Furthermore, in the lumbar cord of 2-month-old rats, GM1 prevents the decrease of the 5-HIAA/5-HT ratio and the "up-regulation" of 5-HT(l) receptors induced by the neurotoxin. It is suggested that the GM1 effect is due to a prevention of the retrograde axonal degeneration occurring after the lesion and/or a growth stimulation of injured axons.

Tetanus toxin affects the K+-stimulated release of catecholamines from nerve growth factor-treated PC12 cells.

Tetanus toxin specifically binds to neuronal surfaces and interferes with the release of transmitters. The effect of tetanus toxin pretreatment of PC12 cell line, taken as a model of neuronal cells in culture, was studied and found that it depresses depolarization-dependent catecholamines secretion. This effect is limited to PC12 cells fully differentiated by the action of Nerve Growth Factor (NGF) and is indicative of the expression of specific binding sites for tetanus toxin during transition from the undifferentiated state. Specific binding of [125I] tetanus toxin to NGF-treated PC12 was demonstrable. The toxin has no effect on the 45Ca accumulation coupled with the depolarization dependent release of catecholamines.